Methods in Enzymology, Vol. 369: Combinatorial Chemistry, Part B

Preface

Combinatorial chemistry has matured from a field where efforts initially

focused on peptide-based research to become an indispensable research tool

for molecular recognition, chemical-property optimization, and drug discovery.

Originally used as a method to primarily generate large numbers of molecules,

combinatorial chemistry has been significantly influenced and integrated with

other important fields such as medicinal chemistry, analytical chemistry, syn-

thetic chemistry, robotics, and computational chemistry.

Even though the initial focus of attention was providing larger numbers of

molecules with a ‘‘diversity’’ goal in mind, other factors came into play

depending upon the problem scientists were trying to solve, such as bioactivity,

solubility, permeability properties, PK, ADME, toxicity, and patentability.

One can think of combinatorial chemistry and compound screening as an

iterative Darwinian process of divergence and selection. Particularly in drug

discovery, where time is a critical factor to success, combinatorial chemistry

offers the means to test more molecule hypotheses in parallel.

We will always be limited to a finite number of molecules that we can

economically synthesize and evaluate. Even with all the advances in automa-

tion technologies, combinatorial chemistry, and higher-throughput screens that

improve our ability to rapidly confirm or disprove hypotheses, the synthesis

and screening cycle remains the rate-determining process. Fortunately, we

continue to make great strides forward in the quality and refinement of pre-

dictive algorithms and in the breadth of the training sets amassed to aid in the

drug discovery/compound optimization iterative process.

Anyone who has optimized chemical reactions for combinatorial libraries

or process chemistry knows first hand how much experimentation is required to

identify optimal conditions. Chemical feasibility is at the heart of small mol-

ecule discovery and chemotype prioritization since it essentially defines what

can and cannot be analoged (i.e., analogability). Although analogability is not

the only driving factor, quite often it is overlooked. For example, when com-

mercially-available compounds or complex natural products are screened, the

leads generated are often dropped because of the difficulty to rapidly analog

them in the lead optimization stage.

The desirability of a chemotype is a function of drug-likeness, potency,

novelty, and analogability. A particularly attractive feature of combinatorial

chemistry is that when desirable properties are identified, they can often be

xiii

xiv preface

optimized through second-generation libraries following optimized synthetic

protocols. If this process of exploring truly synthetically accessible chemical

spaces could be automated, then it would open up the exciting possibility of

modeling the iterative synthesis and screening cycle.

Predicting, or even just mapping, synthetic feasibility is a sleeping giant;

few people are looking into it, and the ramifications of a breakthrough would

be revolutionary for both chemistry and drug discovery. In-roads to predicting

(or even just mapping) chemical feasibility have the potential to have as large

an impact on drug discovery as computational models of bioavailability and

drugability. These are important questions where scientists are now starting to

generate a large-enough body of information on high-throughput synthetic

chemistry to begin to more globally understand what is cost-effectively pos-

sible. Within the biopharmaceutical industry, significant investments in new

technologies have been made in molecular biology, genomics, and proteomics.

However, with the exception of combinatorial chemistry, relatively little has

been done to advance the fundamental nature of chemistry in drug discovery

from a conceptual perspective.

Now, after having gone through the molecule-generating period where

research institutions have a large historical compound collection and the pro-

liferation of combinatorial chemistry services, the trend is now after making

more targeted-oriented molecular entities also known as ‘‘focused libraries.’’

An important emerging question is: How can one most effectively make the

best possible ‘‘focused libraries’’ to answer very specific research questions,

given all the possible molecules one could theoretically synthesize?

The first installment in this series (Volume 267, 1996) mostly covered

peptide and peptidomimetic based research with just a few examples of small

molecule libraries. In this volume we have compiled cutting-edge research in

combinatorial chemistry, including divergent areas such as novel analytical

techniques, microwave-assisted synthesis, novel linkers, and synthetic ap-

proaches in both solid-phase and polymer-assisted synthesis of peptides, small

molecules, and heterocyclic systems, as well as the application of these tech-

nologies to optimize molecular properties of scientific and commercial interest.

Guillermo A. Morales

Barry A. Bunin

METHODS IN ENZYMOLOGY

EDITORS-IN-CHIEF

John N. Abelson Melvin I. Simon

DIVISION OF BIOLOGY

CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P. Colowick and Nathan O. Kaplan

Contributors to Volume 369

Article numbers are in parentheses and following the names of contributors.Affiliations listed are current.

Fernando Albericio (2), University

of Barcelona, Barcelona Biomedical

Research Institute, Barcelona Science

Park, Josep Samitier 1, Barcelona,

08028, Spain

Alessandra Bartolozzi (19), Surface

Logix, Inc., 50 Soldiers Field Place,

Brighton, Massachusetts, 02135

Hugues Bienayme (24), Chrysalon Mo-

lecular Research, IRC, 11 Albert Einstein

Avenue, Villeurbannem, 69100, France

Sylvie E. Blondelle (18), Torrey Pines

Institute for Molecular Studies, 3550

General Atomics Court, San Diego,

California, 92121

Cesar Boggiano (18), Torrey Pines

Institute for Molecular Studies, 3550

General Atomics Court, San Diego,

California, 92121

Stefan Brase (7), Institut fur Organische

Chemie, Universitat Karlsruhe (TH),

Fritz-Haber-Weg 6, Karlsruhe, D-76131,

Germany

Andrew M. Bray (3), Mimotopes Pty

Ltd., 11 Duerdin Street, Clayton, Vic-

toria, 3168, Australia

Wolfgang K.-D. Brill (23), Discovery

Research Oncology, Pharmacia Italy

S.p.A, Viale Pasteur 10, Nerviano (MI),

I-20014, Italy

Max Broadhurst (14), Alchemia Pty Ltd.,

Eight Mile Plains, Queensland 4113, Aus-

tralia

ix

Balan Chenera (24), Amgen Inc., Depart-

ment of Small Molecule Drug Discovery,

One Amgen Center Drive, Thousand

Oaks, California, 91320

James W. Christensen (5), Advanced

ChemTech Inc., 5609 Fern Valley Road,

Louisville, Kentucky, 40228

Andrew P. Combs (12), Incyte Corpo-

ration,Wilmington,Delaware,19880-0500

Scott M. Cowell (16), Department of

Chemistry, University of Arizona,

Tucson, Arizona, 85721

Stefan Dahmen (7), Institut fur Orga-

nische Chemie, RWTH Aachen, Pirlet-

Str. 1, Aachen, 52074, Germany

Ninh Doan (17), Division of Hematology

and Oncology, Department of Internal

Medicine, UC Davis Cancer Center, Uni-

versity of California Davis, Sacramento,

California, 95817

Roland E. Dolle (8), Senior Director of

Chemistry, Department of Chemistry,

Adolor Corporation, 700 Pennsylvania

Drive, Exton, Pennsylvania, 19345

Nicholas Drinnan (14), Alchemia Pty

Ltd., Eight Mile Plains, Queensland

4113, Australia

Amanda M. Enstrom (17), Division of

Hematology and Oncology, Department

of Internal Medicine, UC Davis Cancer

Center, University of California Davis,

Sacramento, California, 95817

x contributors to volume 369

Liling Fang (1), ChemRx Division, Dis-

covery Partners International, 385 Oyster

Point Boulevard, Suite 1, South San

Francisco, California, 94080

Eduard R. Felder (23), Discovery Re-

search Oncology, Pharmacia Italy

S.p.A., Viale Pasteur 10, Nerviano

(MI), I-20014, Italy

Arpad Furka (5), Eotvos Lorand Univer-

sity, Department of Organic Chemistry,

P.O. Box 32, Budapest 112, H-1518,

Hungary

A. Ganesan (22), University of Southamp-

ton, Department of Chemistry, Highfield,

Southampton, SO17 1BJ,United Kingdom

J. Gabriel Garcia (20), 4SC AG, Am

Klopferspitz 19A, 82152, Martinsried,

Germany

Brian Glass (13), Incyte Corporation,

Wilmington, Delaware, 19880-0500

Matthias Grathwohl (14), Alchemia Pty

Ltd., Eight Mile Plains, Queenland 4113,

Australia

Michael J. Grogan (19), Surface Logix,

Inc., 50 Soldiers Field Place, Brighton,

Massachusetts, 02135

Xuyuan Gu (16), Department of


Tuscon, Arizona, 85721

Eric Healy (5), Advanced ChemTech Inc.,

5609 Fern Valley Road, Louisville,

Kentucky, 40228

Timothy F. Herpin (4), Rhone-Poulenec

Rorer, 500 Arcola Road, Collegeville,

Pennsylvania, 19426

Cornelia E. Hoesl (25), Torrey Pines In-

stitute, Room 2-136, 3550 General Atom-

ics Court, San Diego, California, 92121

Christopher P. Holmes (9), Affymax Inc.,

4001 Miranda Avenue, Palo Alto,

California, 94304

Richard Houghten (25), Torrey Pines In-

stitute for Molecular Studies, 3550 Gen-

eral Atomics Court, Room 2-136, San

Diego, California, 92121

Victor J. Hruby (16), Department of


Tucson, Arizona, 85721

Christopher Hulme (24), Amgen Inc., De-

partment of Small Molecule Drug Discov-

ery, One Amgen Center Drive, 29-1-B,

Thousand Oaks, California, 91320

Sharon A. Jackson (12), Aventis Pharma-

ceuticals, 202-206, Bridgewater, New

Jersey, 08807-0800

Ian W. James (3), Mimotopes Pty Ltd., 11

Duerdin Street, Clayton, Victoria, 3168,

Australia

Wyeth Jones (24), Amgen Inc., Depart-


One Amgen Center Drive, 29-1-B, Thou-

sand Oaks, California, 91320

Patrick Jouin (10), CNRS UPR 9023,

CCIPE, 141, rue de la Cardonille, Mont-

pellier Cedex 05, 34094, France

C. Oliver Kappe (11), Institute of Chemis-

try, Karl-Franzens-University Graz,

Heinrichstrasse 28, Graz, A-8010, Austria

Steven A. Kates (19), Surface Logix, Inc.,

50 Soldiers Field Place, Brighton, Massa-

chusetts, 02135

Viktor Krchnak (6), Torviq, 3251 West

Lambert Lane, Tuscon, Arizona, 85742

Kit S. Lam (15, 17), Division of Hematol-

ogy and Oncology, Department of In-

ternal Medicine, UC Davis Cancer



Alan L. Lehman (17), Division of Hema-

tology and Oncology, Department of In-




contributors to volume 369 xi

Ruiwu Liu (15, 17), Division of Hematol-

ogy and Oncology, Department of In-




Matthias Lormann (7), Kekule-Institut fur

Organische Chemie und Biochemie der

Rheinischen, Friedrich Wilhelms Univer-

sitat Bonn, Gerhard-Domagk-Strasse 1,

Bonn, D-53121, Germany

Jan Marik (15), Division of Hematology

and Oncology, Department of Internal

Medicine, UC Davis Cancer Center, Uni-

versity of California Davis, Sacramento,

California, 95817

Katia Martina (23), Discovery Research

Oncology, Pharmacia Italy S.p.A., Viale

Pasteur 10, Nerviano (MI), I-20014, Italy

Joeseph Maxwell (17), Division of Hema-

tology and Oncology, Department of In-




Wim Meutermans (14), Alchemia Pty Ltd.,

3 Hi-Tech Court, Brisbane Technology

Park, Eight Mile Plains, QLD 4113, Aus-

tralia

George C. Morton (4), Rhone-Poulenc

Rorer, 500 Arcola Road, Collegeville,

Pennsylvania, 19426

Adel Nefzi (25), Torrey Pines Institute for

Molecular Studies, 3550 General Atomics

Court, San Diego, California, 92121

Thomas Nixey (24), Amgen Inc., Depart-




John M. Ostresh (25), Torrey Pines Insti-

tute, Room 2-136, 3550 General Atomics

Court, San Diego, California 92121

Vitecek Padera (6), Torvic, 3251 W Lam-

bert Lane, Tucson, Arizona, 84742

E.R. Palmacci (13), 77 Massachusetts

Avenue, T18-209, Cambridge, Massachu-

setts, 02139

Yijun Pan (9), Affymax Inc., 4001 Mi-

randa Avenue, Palo Alto, California,

94304

Jack G. Parsons (3), Mimotopes Pty Ltd.,

11 Duerdin Street, Clayton, Victoria,

3168, Australia

Robert Pascal (10), UMR 5073, Univer-

site de Montpellier 2, CC017, place

Eugene Bataillon, Montpellier Cedex 05,

F-34094, France

Clemencia Pinilla (18), Torrey Pines In-

stitute for Molecular Studies and Mixture

Sciences, Inc., 3550 General Atomics


Obadiah J. Plante (13), Massachusetts

Institute of Technology, Department of

Chemistry, 77 Massachusetts Avenue,

Cambridge, Massachusetts, 02139-4307

Gregory Qushair (2), University

of Barcelona, Barcelona Biomedical

Research Institute, Barcelona Science

Park, Josep Samitier 1, Barcelona,

08028, Spain

Jorg Rademann (21), Eberhard-Karls-Uni-

versity, Tubingen, Institute of Organic

Chemistry, Auf der Morgenstelle 18, Tu-

bingen, 72076, Germany

Joseph M. Salvino (8), Director of Com-

binational Chemistry, Adolor Corpor-

ation, 700 Pennsylvania Drive, Exton,

Pennsylvania, 19345

Peter H. Seeberger (13), Laboratorium

fuer Organische Chemie, HCI F 315,

Wolfgang-Pauli-Str. 10, ETH-Hoengger-

berg, CH-8093 Zurich, Switzerland

Craig S. Sheehan (3), Mimotopes Pty

Ltd., 11 Duerdin Street, Clayton, Vic-

toria, 3168, Australia

xii contributors to volume 369

Adrian L. Smith (24), Amgen Inc., Depart-


One Amgen Center Drive, Thousand

Oaks, California, 91320

Regine Sola (10), UMR 5076, Ecole

Nationale Superieure de Chimie de

Montpellier, 8, rue Delaware l’Ecole

Normale, Montpellier Cedex 05, F-

34296, France

Aimin Song (17), University of California,

UC Davis Cancer Center, Division of

Hematology and Oncology, 4501 X

Street, Sacramento, California, 95817

Alexander Stadler (11), Institute of

Chemistry, Karl-Franzens-University

Graz, Heinrichstrasse 28, Graz, A-8010,

Austria

Paul Tempest (24), Amgen Inc., Depart-




David Tumelty (9), Affymax, Inc.,

4001 Miranda Avenue, Palo Alto,

California, 94304

Josef Vagner (16), Department of Chem-

istry, University of Arizona, Tuscon, Ari-

zona, 85741

Jesus Vazquez (2), University of Barce-

lona, Barcelona Biomedical Research

Institute, Barcelona Science Park, Josep

Samitier 1, Barcelona, 08028, Spain

Michael L. West (14), Alchemia Pty Ltd.,

Eight Mile Plains, Queensland 4113,

Australia

Zemin Wu (3), Mimotopes Pty Ltd., 11

Duerdin Street, Clayton, Victoria, 3168,

Australia

Bing Yan (1), ChemRx Division, Discovery

Partners International, 385 Oyster Point,

Boulevard, Suite 1, South San Francisco,

California, 94080

Yongping Yu (25), Torrey Pines Institute,

Room 2-136, 3550 General Atomics


Florencio Zaragoza (26), Medicinal

Chemistry, Novo Nordisk A/S, Novo Nor-

disk Park, Malov, 2760, Denmark

Jiang Zhao (1), ChemRx Division, Discov-

ery Partners International, 385 Oyster

Point Boulevard, Suite 1, South San

Francisco, California, 94080

[1] high-throughput LC/UV/MS analysis of libraries 3

[1] High-Throughput Parallel LC/UV/MS Analysis ofCombinatorial Libraries

By Liling Fang, Jiang Zhao, and Bing Yan

Introduction

Combinatorial chemistry and high-throughput organic synthesis allowthe preparation of a large number of diverse compounds in a relative shortperiod of time in order to accelerate discovery efforts in the pharmaceut-ical and other industries. A library can comprise hundreds to thousandsof compounds with the need to rapidly analyze those compounds for theiridentity and purity. Different compound separation and mass spectrometry(MS) techniques have been applied for the characterization of combinator-ial libraries. These include separation techniques such as liquid chromatog-raphy (LC) and capillary electrophoresis and different ionization methodsand mass analyzers.1–3 LC/MS* is the most popular technique used in com-binatorial library analysis because it combines separation, molecularweight determination, and relative purity evaluation in a single sample in-jection. However, the throughput of conventional LC/MS could not meetthe need to analyze every member in a large combinatorial library in atimely fashion.

Higher-throughput analysis was achieved by utilizing shorter columnsat higher flow rates.4 Supercritical fluid chromatography (SFC)/MS has

1 A. Hauser-Fang and P. Vouros, ‘‘Analytical Techniques in Combinatorial Chemistry’’

(M. E. Swartz, ed.). Marcel Dekker, New York, 2000.2 B. Yan, ‘‘Analytical Methods in Combinatorial Chemistry.’’ Technomic, Lancaster, 2000.3 D. G. Schmid, P. Grosche, H. Bandel, and G. Jung, Biotechnol. Bioeng. Comb. Chem. 71,

149 (2001).

*Abbreviations: CLND, chemiluminescence nitrogen detection; C log P, calculated partition

coefficient; ELSD, evaporative light scattering detection; ESI-MS, electrospray ionization

mass spectrometry; FWHM, full width at half maximum; i.d., inner diameter; LC, HPLC,

liquid chromatography, high-performance liquid chromatography; LC/MS, liquid chroma-

tography – mass spectrometry; LC/MS/MS, liquid chromatography – mass spectrometry –

mass spectrometry; LC/UV/MS, liquid chromatography mass spectrometry with a UV

detector; LIB, compound library; log P, water/octanol partition coefficient; MUX,

multiplexed; RSD, relative standard deviation; SFC, supercritical fluid chromatography;

TFA, trifluoroacetic acid; TIC, total ion current; TOF, time of flight; TOFMS, time of flight

mass spectrometry.4 H. Lee, L. Li, and J. Kyranos, Proceedings of the 47th ASMS Conference on Mass

Spectrometry and Allied Topics, Dallas, Texas, June 13–17, 1999.

Copyright 2003, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 369 0076-6879/03 $35.00

4 analytical techniques [1]

been used to achieve desirable high speed taking advantage of the low vis-cosity of CO2.5 However, the serial LC/MS approach by its nature does notmatch the speed of parallel synthesis. Parallel LC/MS is the method ofchoice to increase throughput while maintaining the separation efficiency.

An eight-probe Gilson 215/889 autosampler was incorporated into aquadruple mass spectrometer.6 This arrangement enabled the injection ofeight samples (a column from a 96-well microtiter plate) simultaneouslyfor flow-injection analysis/MS (FIA-MS) analysis to achieve a throughputof 8 samples/min. A novel multiplexed electrospray interface (MUX)7

was developed in 1999 and became commercially available for parallelhigh-throughput LC/UV/MS analysis. The eight-way MUX consists ofeight nebulization-assisted electrospray ionization sprayers, a desolvationgas heater probe, and a rotating aperture. It can accommodate all eighthigh-performance liquid chromatograph (HPLC) streams at a reduced flowrate of <100 �l/min per stream and conduct electrospray ionization for alleight streams simultaneously. Ions are continuously formed at the tip ofeach sprayer and the MUX interface allows sprayers to be sampled sequen-tially using the rotating aperture driven by a programmable stepper motor.At any given time, only ions from one stream are admitted into the ionsampling cone, while ions from the other seven sprayers are shielded. Eachliquid stream is sampled for a preset time with mass spectra acquired in fullmass range into eight simultaneously open data files synchronized with thespray being sampled. With a 0.1-s acquisition time per sprayer and 0.05-sintersprayer delay time, the time-of-flight (TOF) mass analyzer can acquirea discrete data file of electrospray ion current sampled from each streamover the entire HPLC separation with a cycle time of 1.2 s. Therefore,this eight-way MUX-LCT was like having eight individual electrosprayionization (ESI)-MS systems working simultaneously.

The MUX interface enables the coupling of parallel liquid chromatog-raphy to a single mass spectrometer. This technology has had a greatimpact in high-throughput LC/MS analysis. In drug development, a four-way MUX interface was used on a triple quadrupole mass spectrometerto simultaneously validate LC/MS/MS methods for the quantitation ofloratadine and its metabolite in four different biological matrixes8 and of

5 M. C. Ventura, W. P. Farrell, C. M. Aurigemma, and M. J. Greig, Anal. Chem. 71, 2410

(1999).6 T. Wang, L. Zeng, T. Strader, L. Burton, and D. B. Kassel, Rapid Commun. Mass Spectrom.

12, 1123 (1998).7 V. De Biasil, N. Haskins, A. Organ, R. Bateman, K. Giles, and S. Jarvis, Rapid Commun.

Mass Spectrom. 13, 1165 (1999).8 M. K. Bayliss, D. Little, D. M. Mallett, and R. S. Plumb, Rapid Commun. Mass Spectrom.

14, 2039 (2000).


diazepam in rat liver microsomes for in vitro metabolic stability.9 The four-channel LC/MS/MS system was also reported for the quantification of adrug in plasma on both the narrow-bore and capillary scales.10 By incorpor-ating divert valves into this system, aliquots of plasma could be directlyanalyzed without sample preparation. The four-channel LC/MS/MS has re-duced method validation time, increased sample throughput by 4-fold, andafforded adequate sensitivity, precision, and negligible intersprayer cross-talk.8,9 In protein analysis, an eight-way MUX coupled with a TOFMSanalyzer has proved to be a powerful tool to monitor the protein purifica-tion process by screening fractions from preparative ion-exchange chroma-tography with a throughput of 50 protein-containing fractions in less thanan hour.11

A high-pressure gradient parallel pumping system (JASCO PAR-1500)has been developed to conduct high-throughput parallel liquid chromatog-raphy.12 It is a 10-pump system where two pumps are used to generate abinary gradient and eight pumps to deliver the mixed solvent to eight LCcolumns. Comparing this system to a conventional system with two pumpsor a binary pump for LC gradient and a simple splitter to divide the gradi-ent to eight LC columns, this system can ensure uniform flow rates througheach LC column. This system has been used for peptides and combinatorialsample,12 protein analysis,13 and bioanalysis.9

We have optimized an eight-way MUX coupled to a TOFMS analyzerto carry out eight-channel parallel LC/UV/MS analysis of combinatoriallibraries14 in the past 2 years. This system has not only provided thecapacity needed for library analysis, but also enabled simultaneous evalu-ation of experimental parameters to expedite the method developmentprocess. In this chapter, we discuss the optimization of this system andpresent a high-throughput protocol for combinatorial library analysis. Wealso compare the eight-channel parallel LC/UV/MS system to a conven-tional single channel LC/UV/MS system in terms of performance andoperation.

9 D. Morrison, A. E. Davis, and A. P. Watt, Anal. Chem. 74, 1896 (2002).10 L. Yang, T. D. Mann, D. Little, N. Wu, R. P. Clement, and P. J. Rudewicz, Anal. Chem. 73,

1740 (2001).11 B. Feng, A. Patel, P. M. Keller, and J. R. Slemmon, Rapid Commun. Mass Spectrom. 15,

821 (2001).12 D. Tolson, A. Organ, and A. Shah, Rapid Commun. Mass Spectrom. 15, 1244 (2001).13 B. Feng, M. S. McQueney, T. M. Mezzasalma, and J. R. Slemmon, Anal. Chem. 73,

5691 (2001).14 J. Zhao, D. Liu, J. Wheatley, L. Fang, and B. Yan, Proceedings of the 49th ASMS

Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May, 27–31, 2001.

N

N N

HN

O

O

H3C

CH3

N

N N

NH

D Fmoc-Asp(OtBu)-OH

O

OCH3

CH3

OO

H3C

H3C

NH

N

H

H

HH3COOC

H

H OCH3

HO

OOCH3

OCH3

OCH3H3CO

O NH

O OHO O

O

B 5-phenyl-1h-tetrazoleA Theophylline

E Dioctyl phthalate

C Reserpine

C log P = −0.39log P = 0.05

C log P = 3.85

log P = 4.43

C log P = 8.39

C log P = 0.18log P = 2.41

C log P = 3.32

Fig. 1. The structures of the five commercial compounds (A to E) used to monitor

performance.


System Optimization

The high-throughput parallel LC/UV/MS system consists of an auto-sampler with eight injection probes, two pumps for generating binary gra-dient, eight UV detectors, and an eight-way MUX with a TOF massspectrometer. This two-pump arrangement keeps the system simple andcost efficient. However, it does not provide pressure regulation for each LCchannel. To ensure flow consistency across each channel, we paid specialattention to the selection of tubing, joints, and columns. Columns are fromthe same manufacturer and the same batch. The tubing is the same lengthinitially for each channel and is further adjusted by checking the flow at theend. With these precautions, the flow from this two-pump system could besplit evenly among the eight channels. In addition, a standard mixture isanalyzed every 24 injections, and the retention times of these standardsare closely monitored to ensure an even flow across the eight channels.

Standards and Flow Monitoring

Five commercial compounds are chosen as standards in system opti-mization and quality control (Fig. 1). Theophylline (log P 0.05), 5-phenyl-1H-tetrazole (log P 2.41), reserpine (C log P 3.32), Fmoc-Asp(OtBu)-OH(log P 4.43), and dioctyl phthalate (C log P 8.39)15 were selected for our

15 L. Tang, W. Fitch, M. Alexander, and J. Dolan, Anal. Chem. 72, 5211 (2000).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25 30

Time (day)

Ret

ention

tim

e (m

in)

1.00

Retention time (min)

0.5 1.0 1.5 2.0 2.5 3.0

1.451.95 2.40

1 d

26 d

31 d

7 d

12 d

17 d

22 d

0.99 1.44 1.95 2.40

1.03 1.49 1.99 2.45

1.01 1.46 1.96 2.42

1.03 1.48 2.00 2.44

1.01 1.46 1.97 2.42

1.00 1.45 1.97 2.42

A

B

C

D

A B

Fig. 2. A selection of UV214 chromatograms from channel 5 on different days and the

retention times from the eight-channel system on every day for standard compounds (A to D)

monitored over a 1-month period.


standard mixture. Although only three compounds have experimentallydetermined log P values (see above) their C log P values range from�0.4 to 8.4, which covers most of the elution range for combinatoriallibrary compounds.

Without backpressure regulation for each channel, it is necessary tominimize the flow rate fluctuation over time. The relative standard devi-ation (RSD%) in retention time variation among the eight channels over1 month for compounds A and B was less than 2% and for C and D itwas less than 1%. The RSD% for all channels over a 1-month period forcompounds A to D was 3.2, 2.4, 1.6, and 1.5%, respectively. Therefore, thissystem is well suited for combinatorial library analysis. The UV chromato-grams from channel 5 from different days are shown as an example inFig. 2A. The retention times of the four compounds (compounds A to D)from all eight channels during a 1-month period are shown in Fig. 2B.

The throughput of this eight parallel LC/UV/MS system is 3200 com-pounds per day for a 3.5-min cycle time per injection of eight samplesunder current optimized conditions. It could be further increased by in-creasing the gradient slope and flow rate. We have also determined thatfive compounds in the standard mixture gave a linear response from 0.01to 0.4 mg/ml.16

16 L. Fang, M. Wang, M. Pennacchio, and J. Pan, J. Comb. Chem. 2, 254 (2000).


The T-Joint Position

A zero dead volume T-joint is used after each UV detector to split theLC eluent to the MS analyzer and the waste to ensure a flow of 100 �l/minentering each channel. The position of the T-joint affected the separationin the total ion chromatogram (TIC). When the T-joint was placed closeto the UV cell (320 mm from the UV cell), the distance between theT-joint and the eight-way MUX interface is 780 mm. A sample had totravel 12 s to reach the eight-way MUX inlet after the UV detector at aflow rate of 50 �l/min. Fmoc-Asp(OtBu)-OH had a full width at half max-imum (FWHM) of 0.05 min when detected at the UV detector (Fig. 3C),but the peak width was doubled at the position of the MUX inlet(Fig. 3D). Such a peak broadening in TIC could have jeopardized productidentification. To minimize these effects, the T-joint was moved as close aspossible to the eight-way MUX inlet. With this modification the samplereached the MUX inlet 2 s after leaving the UV detector. The FWHM inthe UV and TIC chromatograms were both 0.05 min (Fig. 3A and B). Thuspeak delay and broadening were all eliminated.

LC Conditions

Unlike a single LC/UV/MS system, reducing solvent consumption is im-portant in this eight-channel LC/UV/MS system. A flow rate of 24 ml/minon eight 4.6 � 50-mm columns was used initially. This operation resulted ina solvent consumption of 34.5 liters/day. To maintain the same separation

A


2.2 2.4 2.6 2.8

B

C

D

UV214

UV214

TIC

TIC

2.31

2.31

2.44

2.52

Configuration B

Configuration B

Configuration A

Configuration A

Fig. 3. UV214 and TIC chromatograms of compound D obtained from configurations A

(A and B) and B (C and D).


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

A

B

C

D

0.52 1.07

1.72

2.213.40

0.360.92

1.70

2.14

3.34

0.28 0.72

1.48

1.91

3.08

0.24 0.601.35

1.742.91

Fig. 4. UV chromatograms of the standard mixture at one of the eight channels separated

using a 4.6-mm-i.d. column at 24 ml/min (A), and a 2.1-mm-i.d. column, at 8 (B), 12 (C), and

16 ml/min (D).


efficiency and minimize solvent consumption, columns with a smaller innerdiameter (i.d.), such as 2.1 mm, were evaluated. The standard mixture wasanalyzed at flow rates of 6, 8, 10, 12, 14, and 16 ml/min. The LC gradientwas 10–100% B in 3.0 min and 100% B for 0.5 min. The chromatogramsin Fig. 4 show the separation results obtained from the original 4.6–mm-i.d.column at 24 ml/min (A) and 2.1-mm-i.d. column at 8 ml/min (B), 12 ml/min (C), and 16 ml/min (D). Below 8 ml/min, the very lipophilic dioctylphthalate did not elute. The separation was good at 12 ml/min and evenbetter at 16 ml/min. A flow rate of 12 ml/min with the 2.1-mm-i.d. columnwas sufficient to maintain the separation efficiency, and consumed only halfthe solvent.

For a particular library, an optimal LC column needs to be selected.This parallel LC/UV/MS system could evaluate eight different columns in4 min. Five C18 columns of 2 � 50 mm packed with 5-�m particles made bydifferent manufacturers were evaluated simultaneously based on the separ-ation efficiency of the standard mixture using trifluoroacetic acid (TFA) oracetic acid as modifier. Chromatograms at 12 ml/min using 0.05% TFAfrom each column are shown in Fig. 5. The Aqua column gave poor peakshape for the early-eluting compound A. The Aqua and Luna columnsdid not separate compounds B and C (data not shown) using 0.1% aceticacid. The remaining three columns separated the five compounds well.

A


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

B

C

D

E

Fig. 5. UV chromatograms of the standard mixture separated using 2.1-mm-i.d. columns

at 12 ml/min: Polaris (A), Zorbax (B), Omnisphere (C), Luna (D), and Aqua (E).


An Efficient Rerun Protocol

In a high-throughput analysis mode, variable sample concentrationsometimes leads to inadequate or saturated signals and at times blockingof the injector ports. Therefore, reanalysis of selected samples became anecessity. However, due to the rigid design of the Gilson 215 Multiprobeliquid handler, MUX-LCT cannot handle reanalysis efficiently. Forexample, when an injector is blocked during an overnight queue, theremay be 24 failed samples on two 96-well plates (Fig. 6). Since the 8-probeinjector has to inject an entire column of eight samples for each run, it willtake 24 runs or 108 min to complete the analysis.

We have developed a new process to improve the efficiency of samplereanalysis. This process includes four steps: data review, replating, reanaly-sis, and data alignment. We have also generated an Excel template, aGilson’s Unipoint protocol, and an in-house visual basic program toautomate the process.

Raw data processed by the OpenLynx program was first reviewed forconsistency. Since the full scale of our analog channel is 2.1 � 106, sampleswith a UV peak height over that limit saturate the UV detector. Besidesthe detector saturation, samples can also overload the HPLC column andgenerate broad peaks (FWHM > 0.1 min) in HPLC chromatogram. Bothtypes of signal saturation were identified, and a dilution factor was esti-mated for each sample. Low sample concentration was another reasonfor rerun. Failed external standards and sample carryover are indicationsof an injector blockage. Samples with hydrophilic diversity eluted withthe solvent front using the generic method. These samples needed to be

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

A A

B B

C C

D D

E

F F

G G

H H

Reformat

Alignment

Plate BPlate A Target plate

E

A

B

C

D

F

G

H

E

1 9 17

2 10 18

3 11 19

4 12 20

5 13 21

6 14 22

7 15 23

8 16 24

1314 15 6 17 18 19 20 21 22 23241

Fig. 6. Reformatting and realigning processes in reanalysis.


dissolved in water instead of methanol and should be analyzed using a shal-lower gradient. All the above samples were entered into an Excel templatewith a plate view. There are three output lists generated automatically bythe template: output to liquid handler for plating, output to MassLynx forreanalyzing, and output for realigning the final data.

A Gilson liquid handler was programmed with the Unipoint software todilute and reformat failed samples. A volume of 120 �l of solvent (metha-nol unless specified otherwise) was added to each failed vial. The solutionwas taken up and released by the sampling needle three times to ensure ef-ficient mixing. A fraction of the liquid was then transferred to the targetplate. The fraction volume was determined on the Excel template by thedilution factor, for example 40 �l for 2:1 dilution. Solvent was allowed toevaporate at ambient temperature from the new plate, and 200 �l of solventwas then added to each well in the plate. The new plate, reformatted andcompressed, was analyzed with the MUX-LCT system using the sample listfrom the template. Using this new format, the 24 samples in the exampleabove (Fig. 6) were reanalyzed in three runs (13.5 min) instead of 24 runs(108 min). This represents an 8-fold improvement in efficiency.

We have also created a visual basic program to modify sample locationinformation. Since sample location was hard-coded in the data file, sampleson the reformatted plate were of a different location from their original.Once processed by OpenLynx, these samples will cause a ‘‘multiple injec-tion conflict’’ with the samples that were originally in these locations. Thevisual basic program used Microsoft scripting runtime objects to locateeach sample on the reformatted plate. It opened the header file, searchedfor the sample location, and replaced it with the original location. This pro-gram can also append customized information, such as a new identity after‘‘cherry picking,’’ into the sample file. All added operations, such as platingand alignment, were performed offline of the MUX-LCT. With this newprocess, sample reanalysis became much more efficient.


Combinatorial Library Analysis

In LC/MS analysis of combinatorial libraries, the MS determines theproduct identity and its purity is determined by other on-line detection tech-niques such as UV, evaporative light scattering detection (ELSD), andchemiluminescent nitrogen detection (CLND).17–20 UV detection is usedhere to assess product purity based on the assumption of similar absorptioncoefficients at 214 nm for the desired product and the side-products.

To develop a method for combinatorial library analysis, we first ana-lyzed six to eight representative compounds from each library under gen-eric LC/UV/MS conditions. These conditions would be used for libraryanalysis unless adjustments had to be made based on the study of theserepresentative compounds.

Evaluation of Representative Library Compounds

Five to eight representative compounds were evaluated simultaneouslydue to the parallel nature of the system. Depending on the structure of alibrary, this analysis was performed using acetic acid or TFA as modifier.We found that the general LC gradient worked well for most of the libraryexcept in a few cases in which very polar compounds eluted early. In thesecases, the sample solvent, solvent gradient, or LC column was varied to op-timize the retention time. However, we had to adjust ion optics settings formost libraries to ensure that the MHþ ion was the predominant ion to makeproduct identification simple. We found that sample cone voltage was acritical parameter when all other ion optics parameters were kept constant.This was reasonable because the sample cone separates the ionizationchamber with a pressure near atmospheric pressure from the vacuumregion with a pressure of a few Torr. Ions could be fragmented due to col-lision with the gas molecules in this region. A higher sample cone voltagewould produce more energetic ions to undergo collision-induced dissoci-ation. This eight parallel LC/MS system has dramatically accelerated thisprocess because up to eight compounds can be evaluated simultaneouslyunder the same experimental conditions.

Six compounds from library 1 (LIB1) have been analyzed simultan-eously at sample cone voltages of 10, 20, 30, and 40 V. The mass spectraof two compounds (LIB1-1 and LIB1-2) are shown in Fig. 7. Only MHþ

17 L. Fang, M. Demee, T. Sierra, J. Zhao, D. Tokushige, and B. Yan, Rapid Commun. Mass

Spectrom. 16, 1440 (2002).18 L. Fang, J. Pan, and B. Yan, Biotechnol. Bioeng. Comb. Chem. 71, 162 (2001).19 D. A. Yurek, D. L. Branch, and M. Kuo, J. Comb. Chem. 4, 138 (2002).20 E. W. Taylor, M. G. Qian, and G. D. Dollinger, Anal. Chem. 70, 3339 (1998).

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

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100

%

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100

%

0

100

%

PFF115-299-45-A3-40V 86 (1.717) Cm (85:87) TOF MS ES+368287.1265.1 386.2

313.1

314.1

771.4

387.2772.5

773.5

PFF115-299-45-A3-30V 87 (1.737) Cm (86:88) TOF MS ES+438771.4386.2265.1

261.2 287.1387.2

772.5

773.5

PFF115-299-45-A3-20V 84 (1.677) Cm (83:85) TOF MS ES+464386.2

265.2

771.4

387.2772.4

773.5

PFF115-299-45-A3-10V 91 (1.817) Cm (91:93) TOF MS ES+416386.2

771.5

387.2 772.5

773.5

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

0

100

%

0

100

%

PFF115-299-40-3-40V 125 (2.487) Cm (124:126) TOF MS ES+499423.2

316.1

290.1317.2

424.2

845.4

PFF115-299-40-3-30V 125 (2.487) Cm (124:126) TOF MS ES+ 480423.2

316.1

424.2

845.4

PFF115-299-40-3-20V 122 (2.427) Cm (121:123) TOF MS ES+383423.2

845.4424.2

PFF115-299-40-3-10V 127 (2.527) Cm (126:128) TOF MS ES+165423.2

845.4424.2846.4

20V

10V

30V

40V

20V

10V

30V

40V

A B

Fig. 7. Mass spectra of LIB1-1 (A) and LIB1-2 (B) at sample cone voltage of 10, 20, 30, and 40 V.

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[100% relative abundance (RA)] and 2MHþ (dimer, 50% RA) can befound at 10 V for these compounds. Parent ions have been broken apartas the sample cone voltage increases from 10 to 40 V. A major fragment(m/z ¼ 316.1) with 70% RA could be detected in addition to MHþ (m/z¼ 423.2, 100% RA) at 40 V for LIB1-1 (Fig. 7A). However, more extensivefragmentation was observed for LIB1-2 (Fig. 7B). Four fragment ionscould be encountered along with MHþ (m/z ¼ 386.2, 90% RA) and2MHþ (m/z ¼ 771.4, 85% RA) at 40 V. In terms of sensitivity, the totalion counts for both of the compounds are lowest at 10 V and highest at30 V for LIB1-1 and at 20 V for LIB1-2. In general, the higher cone voltageproduces the stronger ion intensity. However, higher cone voltage alsocauses fragmentation, which in turn leads to uncertainty in product identi-fication. As a compromise for six compounds, the sample cone voltage wasset to 20 V. The LC/MS chromatogram and mass spectra of all fivecompounds under optimized conditions are shown in Fig. 8.

Six representative compounds from library 2 (LIB2) have also been ana-lyzed to optimize the sample cone voltage. Mass spectra of two compounds(LIB2-1 and LIB2-2) at sample cone voltages of 20, 30, and 40 V are shownin Fig. 9. MHþ ions are shown as the predominant ions only at 40 V. Frag-ment ions (m/z¼ 378.3) could be observed with an RA of 100% and 80%for LIB2-1 and LIB2-2 at 30 V. MHþ with 30% RA could be found as aminor ion at 20 V while doubly charged ions with 100% RA were the majorion. With a resolution around 5000, TOFMS made it easy to assign chargestates to each ion in the spectrum. Three ions with m/z of 234.6, 378.3, and468.3 found from LIB2-1 at 30 V are displayed in the 3 amu window in Fig.10A, B, and C, respectively. Charge states could be easily assigned based onthe mass difference between C12 and C13 for each ion observed in the massspectrum. A mass difference of a half unit indicated that the ion with m/z of234.6 (Fig. 10A) has a charge state of 2 while ions of 378.3 and 468.3have a charge state of 1 since a mass difference of one unit was observed.It is concluded that product from LIB2 could be easily identified by adoubly charged ion using a sample cone voltage of 20 V or identified bya singly charged ion at 40 V. Detection sensitivity is higher for the doublycharged ion at 20 V than that of the singly charged ion at 40 V. Aftermethod development, a set of optimized ion optics settings was saved andused for future analysis of the library along with the suitable LC conditions.

Library Analysis

Libraries were analyzed in 10 96-well plate batches. Each QC plate con-tained 88 sample compounds. The last column of each plate was reservedfor sampling blank and standard controls. Standards were analyzed in

100 200 300 400 500 600 700 800 900 1000m/z0

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%

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%

0

100

%

0

100

%

PFF115-63-2-D4-MUX2 67 (1.595) Cm (67:69) 1: TOF MS ES+483457.1

399.1

913.2

458.1

855. 2

914.2

PFF115-63-1-A5-MUX2 58 (1.387) Cm (57:59) 1: TOF MS ES+988349.1

697.2350.1 698.2

PFF115-45-C3-MUX2 46 (1.094) Cm (46:48) 1: TOF MS ES+1.11e3362.1

363.1723.3

PFF115-45-A3-MUX2 70 (1.673) Cm (69:71) 1: TOF MS ES+1.17e3386.1

265.1

771.3387.2

427.2772.3

PFF115-40-3-MUX2 74 (1.761) Cm (73:75) 1: TOF MS ES+917423.1

845.3424.2 846.3

0.50 1.00 1.50 2.00 2.50 3.00Time0

100

%

0

100

%

0

100

%

PFF115-63-2-D4-MUX2 1: TOF MS ES+ TIC

5.55e31.60

1.04

PFF115-63-1-A5-MUX2 1: TOF MS ES+TIC

7.41e31.39

PFF115-45-C3-MUX2 1: TOF MS ES+TIC

6.06e31.09

PFF115-45-A3-MUX2 1: TOF MS ES+TIC

1.11e41.67

1.12 1.50

PFF115-40-3-MUX2 1: TOF MS ES+TIC

7.41e31.76

0

100

%

0

100

%

0

100

%

Fig. 8. UV214 chromatogram and mass spectra of LIB1-1 to LIB1-5 under optimized conditions.

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0

100

%

PFF107-3-B4-a-1 48 (1.142) Cm (47:49) 1: TOF MS ES+ 209468.3

333.2232.1

209.2 290.2 378.3469.3

470.3

PFF107-3-B4-a 45 (1.070) Cm(44:47) 1: TOF MS ES+ 968378.3

234.7

235.2

468.4

379.3469.4

PFF107-3-B4-a 45 (1.070) Cm(44:47) 1: TOF MS ES+ 1.28e3234.6

235.2

468.3235.7

40V

30V

20V

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

0

100

%

PFF107-3-D4-a-1 44(1.056) Cm (43:45) 1: TOF MS ES+ 450484.3

333.2232.1 378.3485.3

PFF107-3-D4-a 41 (0.984) Cm(40:42) 1: TOF MS ES+ 647484.4

378.3242.7

243.2394.3

485.4

PFF107-3-D4-a 40 (0.960) Cm(40:42) 1: TOF MS ES+ 1.17e3242.6

243.2

484.3

243.7 485.3

40V

30V

20V

A B

0

100

%

0

100

%

Fig. 9. Mass spectra of LIB2-1 (A) and LIB2-2 (B) at sample cone voltage of 20, 30, and 40 V.

16

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]

234 235 236 237m/z0

100

%

PFF107-3-B4-a 45 (1.070) Cm (44:48) 1: TOF MS ES+

370234.7

235.2

235.7

378 379 380 381m/z0

100

%

PFF107-3-B4-a 45 (1.070) Cm (44:48) 1: TOF MS ES+

1.09e3378.3

379.3

468 469 470 471m/z0

100

%

PFF107-3-B4-a 45 (1.070) Cm (44:48) 1: TOF MS ES+

459468.4

469.4

A B C

Fig. 10. Isotope of three ions found from LIB2-1 at a sample cone voltage of 30 V. (A) Charge state of 2; (B, C) charge state of 1.

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Average purity 80.6%

0

500

1000

1500

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10 20 30 40 50 60 70 80 90 100

Purity by UV214 (%)

Num

ber

of co

mpounds

Fig. 11. Library LIB2 purity distribution of 5280 compounds measured at UV214.


every 24 injections during analysis to monitor the performance consistencyof all eight channels. The analysis queue was constructed from an Excelspreadsheet and imported into the MassLynx software for execution. Afteracquisition, the data were processed using MassLynx in batches. Processeddata could be reviewed in OpenLynx by selecting a plate and clicking onthe desired well. The UV chromatogram and mass spectrum of the desiredproduct in LIB2, plate26, well D1, are shown as an example in Fig. 9. Wegenerated an Excel report that included filename, expected molecularweight, purity of desired products at 214 nm, and a plate view with purityindicated for all compounds in the 10-plate batch. Library LIB2 wascomposed of 60 plates; it was analyzed in positive ion mode and processedin six batches. The purity distribution of library LIB2 is shown in Fig. 11with an average of 80.6% for 5280 compounds measured at 214 nm.Figure 11 shows the plate view of all 60 plates. According to this protocol,we have completed more than a half million LC/UV/MS analyses in aperiod of 15 months with two eight-channel MUX-LCT systems.

Comparison of the Eight-Channel LC/UV/MS (MUX-LCT) Systemwith a Conventional Single-Channel LC/UV/MS System

The significant advantage of the parallel LC/MS system is its through-put. Because eight LC/UV/MS analyses can be conducted simultaneously,the total analysis time is decreased by a factor of eight. To analyze everycompound in a library of 2500 compounds at 3.5 min cycle time requires146 h using a single channel LC/UV/MS system. However, it requires only


18.2 h to complete this task using an eight-channel parallel LC/UV/MSsystem, and this makes it possible to perform LC/UV/MS analysis on everycompound for all of our libraries. In addition, this system also speedsup method development because it simultaneously evaluates up to eightparameters or variables such as the performance of eight different columns.

UV and TIC Chromatograms

An important concern in using an eight-way MUX interface is that theacquisition cycle time (the time required to acquire one data point for eachchannel) is longer, and the data acquisition time per channel is shorter,than for a single-spray system. Therefore, the sensitivity might be lowerand the peak shape could be distorted. In our current system with a time-of-flight mass spectrometer, the minimum time required for each acquisi-tion cycle is 1.2 s with 0.1 s for data acquisition and 0.05 s for intersprayerdelay. The chromatographic baseline peak width was between 5 and 6 s inthe UV chromatograms and between 6 and 7 s in the TICs under generalLC/UV/MS conditions. A maximum of five MS data points could be ac-quired to define a peak, which resulted in slightly distorted peak shapesin the TICs. On the other hand, peak shapes were much better defined ina single-channel system because more than 10 data points could be easilyobtained. For combinatorial library analysis, lower sensitivity is not a prob-lem because the parallel synthesis method always produces enough com-pound for analysis. The limited number of data points across an LC peakwas usually not a problem because the MS data were used only to identifythe peak of interest. In theory, one or two data points (TOF mass spectra)should be sufficient to confirm the expected molecular weight. The productpurity was obtained from the UV chromatogram, where the number ofdata points was sufficient to ensure excellent peak shape and precision.

Data Acquisition Using Positive and Negative Ionization

In a single-spray system, it is common to analyze samples in both posi-tive and negative ion modes by switching polarity during a single data ac-quisition. This practice makes the best use of precious MS time andidentifies products by their presence in both positive and negative ionforms. Both positive and negative ESI modes are available for the eight-channel MUX-LCT system. However, the polarity change within a singledata acquisition would make the cycle time much longer. Therefore, weprefer to analyze samples using a single polarity, and conduct a separateexperiment with the other polarity if necessary. With this arrangement,high-throughput LC/MS analysis with both positive and negative modeis available.


Sample Rerun

For a conventional single-channel LC/UV/MS system, a single un-satisfied well could be easily reanalyzed. In the eight parallel LC/UV/MSsystem, the rerun procedure was different from that of the single-spraysystem. If problems were found in a single channel, such as retention timeshift or channel blockage, 12 wells in a row would fail and the whole platehad to be reanalyzed. We have developed a rerun protocol that made theparallel LC/MS analysis as efficient as the single-channel system.

Operation and Maintenance

In the eight-channel parallel LC/UV/MS system, a standard mixturewas analyzed every 24 injections. This was indispensable for the operation.The variation of the retention time across eight channels was monitoredclosely to ensure consistency for the eight channels. A significant retentiontime shift indicated problems that usually could be overcome by replacingthe frit in the precolumn filter. A diminished peak area or a change in peakshape of standards indicated column deterioration. We started with eightcolumns from the same batch for sample analysis. Deteriorated columnswere replaced individually. This practice gave us satisfactory analysis datafor combinatorial library analysis with minimal cost.

We anticipated difficulty in maintaining and troubleshooting an eight-channel parallel system because the problems in the autosampler, LCcolumns, UV detectors, and MS interface would be multiplied by eight.In fact, with the convenience of simultaneous analysis of the other sevenchannels, the diagnosis and troubleshooting were made easier. The com-plete system was easily divided into four functions: injection, separation,UV detection, and MS detection. By running the standard mixture on eightchannels then switching channels at different function sites and rerunningthe standard mixture, problems were easily isolated. Fixing the problemswas exactly the same as for the single-spray system.

Conclusion

We have optimized an eight-channel parallel LC/UV/MS (MUX-LCT)system for high-throughput LC/UV/MS analysis of large combinatorial lib-raries. Since the LC gradient is divided into eight LC columns by a simplesplitter, the flow fluctuation has been continuously monitored and minim-ized using a standard mixture during analysis to ensure performance con-sistency among the eight channels. To preserve the separation integrity inthe total ion chromatogram, the zero dead volume T-joint used to split theflow (after UV detection) should be best placed as close to the eight-way

[2] qualitative colorimetric tests for sps 21

MUX inlet as possible. A flow rate of 12 ml/min on eight 2.1 � 50 mm Po-laris C18 columns was optimal for general purposes in our study. Thissystem could analyze more than 3000 compounds per day for a gradientseparation with a cycle time of 3.5 min.

We have carried out more than half a million LC/UV/MS analyses in 15months using two eight-channel parallel LC/UV/MS systems. We foundthat it was beneficial to evaluate a few representative compounds fromeach library and optimize ion optics to make product identification simpleand reliable. This parallel system has enabled simultaneous evaluation ofeight compounds and significantly improved the speed of optimization.The identity and purity of every single product could be obtained fromOpenLynx in 10 96-well plate per batch process and transferred into anExcel spreadsheet for the entire library. Compared with a single-channelLC/UV/MS system, the parallel LC/UV/MS system has the advantages ofhigh throughput and simultaneous evaluation of eight parameters.

Acknowledgments

We thank Jason Cournoyer, Michael Demee, Duayne Tokushige, Melody Wen, and

Teresa Sierra for their assistance throughout this work.

[2] Qualitative Colorimetric Tests for SolidPhase Synthesis

By Jesus Vazquez, Gregory Qushair, and Fernando Albericio

Introduction

Solid-phase synthesis (SPS)* is limited by a shortage of simple and rapidtechniques for reaction monitoring, specifically for functional group trans-formations. The traditional preparation and subsequent analysis (HPLC,

*Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane;

DIEA, N,N-diisopropylethylamine; DME, N,N’-dimethylformamide; DTNB, 5,51-dithio(2-

nitrobenzoic acid) or Ellman’s reagent; Et3N, triethylamine; EtOH, ethanol; HOAc, acetic

acid; EtOAc, ethyl acetate; Hex, mixture of hexane isomers plus methylcyclopentane; HPLC,

high-pressure liquid chromatography; MeOH, methanol; MG, malachite green; MS, mass

spectrometry; NMM, N-methylmorpholine; NMP, N-methylpyrrolidinone; Purpald, 4-amino-3-

hydrazino-5-mercapto-1,2,4-triazole; SPS, solid-phase synthesis; TCT, trichlorotriazine;

THF, tetrahydrofuran; TLC, thin-layer chromatography; TNBSA, trinitrobenzenesulfonic

acid; TosCl-PNBP, p-tosylchloride p-nitrobenzylpyridine; TRIS, tris(hydroxymethyl)

aminomethane.



MUX inlet as possible. A flow rate of 12 ml/min on eight 2.1 � 50 mm Po-laris C18 columns was optimal for general purposes in our study. Thissystem could analyze more than 3000 compounds per day for a gradientseparation with a cycle time of 3.5 min.

We have carried out more than half a million LC/UV/MS analyses in 15months using two eight-channel parallel LC/UV/MS systems. We foundthat it was beneficial to evaluate a few representative compounds fromeach library and optimize ion optics to make product identification simpleand reliable. This parallel system has enabled simultaneous evaluation ofeight compounds and significantly improved the speed of optimization.The identity and purity of every single product could be obtained fromOpenLynx in 10 96-well plate per batch process and transferred into anExcel spreadsheet for the entire library. Compared with a single-channelLC/UV/MS system, the parallel LC/UV/MS system has the advantages ofhigh throughput and simultaneous evaluation of eight parameters.

Acknowledgments

We thank Jason Cournoyer, Michael Demee, Duayne Tokushige, Melody Wen, and

Teresa Sierra for their assistance throughout this work.


[2] Qualitative Colorimetric Tests for SolidPhase Synthesis

By Jesus Vazquez, Gregory Qushair, and Fernando Albericio

Introduction

Solid-phase synthesis (SPS)* is limited by a shortage of simple and rapidtechniques for reaction monitoring, specifically for functional group trans-formations. The traditional preparation and subsequent analysis (HPLC,

*Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane;

DIEA, N,N-diisopropylethylamine; DME, N,N’-dimethylformamide; DTNB, 5,51-dithio(2-

nitrobenzoic acid) or Ellman’s reagent; Et3N, triethylamine; EtOH, ethanol; HOAc, acetic

acid; EtOAc, ethyl acetate; Hex, mixture of hexane isomers plus methylcyclopentane; HPLC,

high-pressure liquid chromatography; MeOH, methanol; MG, malachite green; MS, mass

spectrometry; NMM, N-methylmorpholine; NMP, N-methylpyrrolidinone; Purpald, 4-amino-3-

hydrazino-5-mercapto-1,2,4-triazole; SPS, solid-phase synthesis; TCT, trichlorotriazine;

THF, tetrahydrofuran; TLC, thin-layer chromatography; TNBSA, trinitrobenzenesulfonic

acid; TosCl-PNBP, p-tosylchloride p-nitrobenzylpyridine; TRIS, tris(hydroxymethyl)

aminomethane.




MS, etc.) of resin cleavage products are time-consuming processes, hencealternative methods are desirable. The use of colorimetric functional grouptests, wherein aliquots of resin are mixed with stock solutions and changesin solution/resin color are used to indicate the presence or absence of func-tional groups on the resin, was initiated by Kaiser et al.1 in the use of nin-hydrin to test for primary amines in the SPS of peptides. Today organicchemists have at their disposal an ever-broadening array of tests, bothqualitative and quantitative, for alcohols, aldehydes, amines, carboxylicacids, and thiols. We present a literature overview of the most widely usedqualitative tests including instructions on reagent preparation and storage,experimental protocol, and the scope and limitations of each test.2 Themajority of these tests can be performed in less than 10 min with simplelaboratory equipment and minimal reagent preparation. We also reportthe results of experiments to determine the functional group response ofeach test using amino acids as representative organic compounds.

It was our intention to provide a central reference for the most commonqualitative tests with a special emphasis on substrate compatibility, namelyfunctional group interference. For example, it is known that certain aminoacids may give unusual results for a given test (such as cysteine with theninhydrin test) and some of the original colorimetric test publications in-clude brief reports on the potential for functional group interference (falsepositives) for a given test. To determine the utility of each test in the pres-ence of multiple functional groups, each of the summarized colorimetrictests was applied against a broad range of amino acids. The aim of this ex-ercise was to determine the universality of each test, and to identify andreport those cases with unusual results. The amino acids were tested forthe presence of each functional group under all possible levels of amineand lateral chain protection, thus enabling us to determine the extent towhich chemical interference by other functional groups could affect eachtest (see Scheme 1). Table I summarizes qualitative colorimetric testsreported in the literature for various organic functional groups.

We also investigated the use of each test at medium (approximately0.5 mmol/g) and low (approximately 0.025 mmol/g) resin loading.

General Experimental Procedures

Resin (polystyrene-based resin, 1% divinylbenzene, 100–200 mesh)substitution and amino acid deprotection were carried out in disposable

1 E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. Cook, Anal. Biochem. 34, 595 (1970).2 To the best of our knowledge, only one other review covering some of the methods

described herein exists in the literature: C. Kay, O. E. Lorthioir, N. J. Parr, M. Congreve,

S. C. McKeown, J. J. Scicinski, and S. V. Ley, Biotechnol. Bioeng. 71, 110 (2001).

Scheme 1. Amino acids were tested at all possible levels of protection. This enabled us to

differentiate between test results caused by a given free functional group and results that may

have been caused by other chemical moieties in the molecule.


syringes fitted with polypropylene filter discs using standard solid-phasepeptide synthesis procedures. For the majority of the tests described, theexperimental protocols were adapted with minor changes from the originalpublications, none of which we feel jeopardizes the essence of each test(i.e., the underlying chemistry). In the majority of cases, tests were per-formed immediately after preparation of each resin by aliquoting the resininto equivalent portions using the following technique: to the syringe con-taining the master quantity of resin is added dichloromethane (DCM, ca.1 ml/100 mg resin), the resin is agitated with a pipetter by continuouslytaking up and ejecting a small volume in order to create a uniform suspen-sion, and the desired volume of suspension is quickly removed and trans-ferred to an Eppendorf tube or glass vial and allowed to air dry. Thistechnique is more effective than dispensing dry resin with a spatula sinceit is faster and more precise for tiny aliquots (1–5 mg) of resin. Eppendorftubes (2 ml) were used for all tests except the TosCl-PNBS, Kaiser,Vazquez, and Purpald tests. Disposable glass tubes (800 �l) were usedfor the Kaiser and Vazquez tests, a disposable syringe (1 ml) fitted with apolypropylene filter disc was used for the TCT, Methyl red, and Purpald

TABLE I

Summary of Qualitative Colorimetric Test

Functional group Tests

Primary aliphatic

amine

Kaiser (ninhydrin),a–c trinitrobenzenesulfonic acid (TNBSA),d

NF-31,e chloranil,f–h bromophenol blue,i,j

nitrophenylisothiocyanate-O-trityl(NPIT),j,k

Malachite green isothiocyanate (MGI),j,l Traut’s reagents,j,m

and Ellman’s reagents,j,m

Secondary aliphatic amine TNBS,d NF-31,e chloranil,f–h bromophenol blue,i,j MGIi,j

Primary alcohol TosCl-PNBP,n (1,3,5)-trichlorotriazine (TCT) with fluorescein,

Alazarin R, or fuchsin,o,p

Secondary alcohol TosCl-PNBP,n TCT-fluorescein, Alizarin R, or fuchsino,p

Tertiary alcohol Diphenyldichlorosilane-methyl redq

Phenol TosCl-PNBP,n,r TCT-fluorescein, Alizarin R, or fuchsin,o,p

diphenyldichlorosilane-methyl redq

Thiol Ellman’s reagents,t

Carboxylic acid Malachite green,u Cystamine-Ellman’s reagentj,v

Aldehydew Vazquez ( p-anisaldehyde),x Purpaldy

a E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. Cook, Anal. Biochem. 34, 595 (1970).b V. K. Sarin, S. B. H. Kent, J. P. Tam, and R. B. Merrifield, Anal. Biochem. 117, 147 (1981).c W. Troll and R. K. Cannan, J. Biol. Chem. 200, 803 (1953).d W. S. Hancock and J. E. Battersby, Anal. Biochem. 71, 260 (1976).e A. Madder, N. Farcy, N. G. C. Hosten, H. De Muynck, P. J. De Clercq, J. Barry, and A. P.

Davis, Eur. J. Org. Chem. 2787 (1999).f T. Christensen, Acta Chem. Scand. B 33, 763 (1979).g T. Vojkovsky, Peptide Res. 8, 236 (1995).h The chloranil test can also be used to selectively react with primary amines (see

experimental section).i V. Krchnak, J. Vagner, P. Safar, and M. Lebl, Collect. Czech. Chem. Commun. 53, 2542

(1988).j This test was not reviewed for this publication.k S. S. Chu and S. H. Reich, Bioorg. Med. Chem. Lett. 5, 1053 (1995).l A. Shah, S. S. Rahman, V. de Biasi, and P. Camillero, Anal. Commun. 34, 325 (1997).m T. T. Ngo, Appl. Biochem. Biotechnol. 13, 213 (1986).n O. Kuisle, M. Lolo, E. Quinoa, and R. Riguera, Tetrahedron 55, 14807 (1999).o M. E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 41, 7395 (2000).p M. E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 42, 2927 (2001).q B. A. Burkett, R. C. D. Brown, and M. M. Meloni, Tetrahedron Lett. 42, 5773 (2001).r There are conflicting reports in the literature on the utility of this test for phenols.n,q

s G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).t J. P. Baydal, A. M. Cameron, N. R. Cameron, D. M. Coe, R. Cox, B. G. Davis, L. J. Oates,

G. Oye, and P. G. Steel, Tetrahedron Lett. 42, 8531 (2001).u M. E. Attardi, G. Porcu, and M. Taddei, Tetrahedron Lett. 41, 7391 (2000).v T. T. Ngo, Appl. Biochem. Biotechnol. 13, 207 (1986).w An aldehyde (BAL) linker was used as a model for this functional group.x J. Vazquez and F. Albericio, Tetrahedron Lett. 42, 6691 (2001).y J. J. Cournoyer, T. Kshirsagar, P. P. Fantauzzi, G. M. Figliozzi, T. Makdessian, and

B. J. Yan, J. Comb. Chem. 4, 120 (2002).



tests, and the TosCl-PNBP test was performed on TLC plates (silica gel,aluminum backed). NF-31 test sample tubes were heated directly in apreheated, multiwell aluminum block. Kaiser and Vazquez test sampletubes were heated in a preheated sand bath inside of a laboratory oven.Heating of the silica plates for the TosCl-PNBP test was performed usinga laboratory heat gun on high setting.

Aliphatic Amines

3 V.4 W.

Test: Kaiser (Ninhydrin)1,3,4

Application: detection of primary aminesTest time: 4 minReagent preparation time: 1 dayRecommended storage time: 1 month at room temperature in

light-proof containers (such as amber bottles)

Required Reagents

Ninhydrin dissolved in ethanolPhenol dissolved in ethanolaq. KCN dissolved in pyridine

Preparation of Reagent Solutions

Reagent Solution A. Phenol (40 g) in added to EtOH (10 ml) and themixture is heated until complete dissolution of the phenol. A solution ofKCN (65 mg) in water (100 ml) is added to pyridine (freshly distilled overninhydrin, 100 ml). Both solutions are stirred for 45 min with AmberliteMB-3 (Merck), filtered, and mixed.

Reagent Solution B. A solution of ninhydrin (2.5 g) in absolute EtOH(50 ml) is prepared and maintained in a light-proof container, preferablyunder inert atmosphere.

Experimental Procedure. The resin is washed with appropriate solventsand a small portion (ca. 1–5 mg) is transferred to a small glass tube. Tothis tube are added three drops of each of the reagent solutions A and B.The tube is then heated at 100

�for 3 min. A negative test, indicating the

absence of free primary amines, is communicated by a yellow or orange-pink solution and naturally colored beads. A positive test is indicated bya dark blue or purple solution and beads. Variations in the darkness ofthe solution reflect variations in amine concentration while variations in

K. Sarin, S. B. H. Kent, J. P. Tam, and R. B. Merrifield, Anal. Biochem. 117, 147 (1981).

Troll and R. K. Cannan, J. Biol. Chem. 200, 803 (1953).


the color observed (red, green, etc.) are particular to certain substrates andmay represent false positives.

Notes. The Kaiser test is generally reliable; however, when used to teststerically hindered amines such as aminoisobutyric acid (Aib), results maybe difficult to interpret.

We found that the color yielded for primary alkyl amine positive tests isstrongly influenced by the presence of other functional groups. Specificallywe could not establish a clear positive for arginine, asparagine, and cysteinewhen their lateral chains were deprotected. A similar effect was observedin the case of secondary amines or sterically hindered amines such as Aib.

We found that at lower levels of resin functionalization a clear positivewas difficult to observe.

5 W.6 A.

A.

Test: TNBSA (trinitrobenzenesulfonic acid)5

Application: detection of primary/secondary aminesTest time: 5 minReagent preparation time: minutesRecommended storage time: up to 1 month refrigerated storage

Required Reagents

A 1% (w/v) solution of TNBSA in DMFA 10% solution of N,N-diisopropylethylamine (DIEA) in DMF

Experimental Procedure. The resin is washed with MeOH and a smallportion (1–3 mg) is transferred to an Eppendorf tube and suspended inDMF. To this tube is added 1 drop of each of the above solutions. The solu-tion is left for 5 min at room temperature. The resin is washed extensivelywith DMF. The presence of free amines is indicated by orange or red beads.

Notes. The TNBSA test was found to be efficient for primary amines,including sterically hindered amines as seen in our probes with the tertiaryamine of Aib.

We also found that at lower levels of resin functionalization a clearpositive was difficult to observe.

Test: NF-316

Application: detection of primary/secondary aminesTest time: 10 minReagent preparation time: 1 week preparationRecommended storage time: up to 1 month at 4

�

S. Hancock and J. E. Battersby, Anal. Biochem. 71, 260 (1976).

Madder, N. Farcy, N. G. C. Hosten, H. De Muynck, P. J. De Clercq, J. Barry, and

P. Davis, Eur. J. Org. Chem. 2787 (1999).


Required Reagents

Disperse Red 1Ethyl diazoacetatePhosphorous oxychloridep-Nitrophenol

Solution Preparation. NF-31 is obtained via a three-step procedurestarting from Disperse Red 1.

Ether Formation with Ethyldiazoacetate. Disperse Red 1 (2.0 g,1.0 eq.) and rhodium tetraacetate [Rh2(OAc)4] (47 mg, 0.016 eq.) are dis-solved in DCM–toluene (1:1, 50 ml) in a dry two-neck round-bottom flaskand stirred at 40

�. A solution of ethyldiazoacetate (2.64 ml, 4.0 eq.) in tolu-

ene (13 ml) is added dropwise (Caution) and the solution is left to reactovernight. The solution is concentrated to dryness by rotary evaporation.The reaction flask is placed on an ice bath and a solution of 10% aq. HOAc(50 ml) is added to the crude product. The mixture is adsorbed onto silicagel, and the product is purified by column chromatography [silica gel,EtOAc-Hex (1:9)].

Saponification. The purified product (1g, 1.0 eq.) from step 1 andKOH (870 mg, 5 eq.) are dissolved in MeOH–toluene [(5:1), 60 ml]. Thesolution is stirred and brought to reflux (ca. 85

�) for 90 min. The reaction

is allowed to cool and a red precipitate is observed. The cooled reactionmixture is concentrated by rotary evaporation to a volume of 10 ml. Then10% aq. HCl (10 ml) is added, followed by water (25 ml). The precipitateis extracted with DCM and the organic phase is washed with water anddried on anhydrous MgSO4. The mixture is filtered, and the filtrate isconcentrated by rotary evaporation and the product purified by columnchromatography.

Condensation with p-Nitrophenol. The product (500 mg, 1.0 eq.)from step 2 and p-nitrophenol (178 mg, 1.0 eq.) are dissolved in DCM(26 ml) and pyridine (22 ml) is then added. The aforementioned solutionis maintained at �15

�, a solution of POCl3 (222 �l, 1.8 eq.) in DCM

(2 ml) is added dropwise, and the solution is then left to react overnightat room temperature. The crude reaction mixture is then washed with aq.saturated NaHCO3 and brine and dried over anhydrous MgSO4. The crudeproduct is concentrated by rotary evaporation and purified by columnchromatography [silica gel, EtOAc-Hex (1:9)].

Experimental Procedure. The resin is washed with methanol and a smallportion (1–3 mg) is transferred to an Eppendorf tube. To this tube is addedNF-31 solution (0.002 M in acetonitrile, 200 �l). The tube is heated in analuminium dry heating block at 70

�for 8 min. The resin is washed exten-

sively with MeOH (3�), DMF (3�), and DCM (3�). The presence of free


amines is indicated by red-colored beads whereas a negative test yieldsnaturally colored beads.

Notes. The NF-31 test was found to be highly sensitivite for primary andsecondary amines.

During our probes of this test we found that false positives are given ifthe resin is not washed thoroughly with the appropriate solvents.

7 T.8 T.9 O.

Test: Chloranil7,8

Application: detection of primary/secondary aminesTest time: 5 minReagent preparation time: minutesRecommended storage time: up to 1 month refrigerated storage

Required Reagents

Acetaldehyde (for detection of primary or secondary amines) oracetone (for detection of secondary amines)

Saturated solution of chloranil in toluene

Experimental Procedure. The resin is washed with MeOH and a smallportion (1–3 mg) is transferred to a small glass tube. To this tube is addedacetaldehyde (primary or secondary amines) or acetone (secondaryamines) (200 �l) followed by the chloranil solution (50 �l). The solutionis shaken at room temperature for 5 min. The presence of free amines is in-dicated by a green- or blue-colored solution. Negative samples register asyellow, amber, or brown.

Notes. The presence of a secondary amine should be confirmed by apositive result obtained for the secondary test and a simultaneouslyobtained negative result for the primary test. Likewise the Kaiser test canbe used in place of the primary amine version of the chloranil.

This test gave excellent results in both of its forms (testing for primaryand for secondary amines); a clear positive was observed even at low levelsof resin functionalization for the sterically hindered Aib.

Alcohols

Test: TosCl-PNBP9

Application: detection of alcohols and phenolsTest time: ca. 5 minReagent preparation time: 5 minRecommended storage time: no more than 2 weeks at 4

�

Christensen, Acta Chem. Scand. B 33, 763 (1979).

Vojkovsky, Peptide Res. 8, 236 (1995).

Kuisle, M. Lolo, E. Quinoa, and R. Riguera, Tetrahedron 55, 14807 (1999).


Required Reagents

10 B.11 M.12 M.

A solution of p-toluenesulfonyl chloride (0.12 M) in toluene(solution 1)

A solution of p-nitrobenzylpyridine (0.30 M) in toluene (solution 2)A 10% (v/v) solution of piperidine in CHCl3 (solution 3)

Experimental Procedure. The resin is washed with DCM. A small por-tion (3–5 mg) of resin is deposited onto a silica plate by pipette as a DCMsuspension. The suspension should be pipetted quickly so that it forms adisperse disc (not a mound). Once dry, the resin is treated with one dropof solution 1 and one drop of solution 2. The plate is then heated with aheat gun by swaying the plate in front of the gun from a distance of ap-proximately 5 cm for approximately 1 min. A yellow color should appearand then disappear within the heating time, leaving the resin similar to orslightly darker than its natural color. At this point a drop of solution 3 isadded to the resin sample on the plate. Purple coloration of the beadsindicates the presence of free hydroxyl groups (light pink or purple atlow concentration, dark purple at high concentration).

Notes. To get reliable results, concentrations of reagents should beapproximately four times higher than that reported in the original paper.

To perform several tests on one silica plate, the resin spots should bedeposited approximately 1–2 cm from each other in each direction.

It is always advisable to carry out control tests, both a positive (a resinbearing either a free alcohol or phenol) and a negative (ideally, an acety-lated hydroxy resin). In this case, heating of the plate should be carriedout until the positive resin control takes an orange-red color.

We found this test to be highly dependent on the quality of the solutionsused. Solutions stored at room temperature for prolonged periods of timegave almost 100% false positives.

Although there are conflicting reports in the literature on the utility ofthis test for phenols,9,10 in our hands the tests for the phenol of Tyr gave theexpected positive results.

Test: TCT-(Fluoresceine, Alizarin R, or Fuchsin)11,12

Application: Detection of alcoholsTest time: ca. 30 minReagent preparation time: minutesRecommended storage time: No more than 2 weeks at 4

�

A. Burkett, R. C. D. Brown, and M. M. Meloni, Tetrahedron Lett. 42, 5773 (2001).

E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 41, 7395 (2000).

E. Attardi, A. Falchi, and M. Taddei, Tetrahedron Lett. 42, 2927 (2001).


Reagents

1,3,5-Trichlorotriazine (TCT)N-Methylmorpholine (NMM)Fluoresceine, Alizarin R, or Fuchsin

Experimental Procedure. The procedure is composed of six steps:

1. A few milligrams of resin is placed in a small glass tube and washedwith DMF.

2. DMF (3 ml) is added followed by NMM (1 ml) and TCT (5 mg).3. The tube is heated at 70

�for 20 min.

4. The solution is pipetted off and the resin is washed thoroughly withDMF.

5. DMF (3 ml) is then added to the resin followed by AliR (5 mg) (or3 ml of a 0.025% solution of fuchsin or fluoresceine in NMP) andNMP (1 ml).

6. After 5 min the resin is washed thoroughly with DMF until a clearsolution is obtained, then washed with THF and finally with DCM.

A positive test is communicated by red beads for the AliR test andgreen or fluorescent beads in the case of the fluoresceine test.

Notes. A drawback found in the use of this test is the formation of awhite precipitate during the activation of alcohol with the triazine.

If fluoresceine is used, the resin beads must be viewed with ultravioletlight as visible light is not sufficient to determine results.

Ambiguous results were often obtained with this test, above all withphenols such as the one of tyrosine.

This test also yields positive results in the presence of other nucleo-philes such as primary and secondary amines, carboxylic acids, and thiols.

Test: Diphenyldichlorosilane-Methyl red10

Application: Detection of alcoholsTest time: ca. 25 minReagent preparation time: minutesRecommended storage time: Up to 1 month at room temperature

Required Reagents

10% triethylamine (Et3N) in dry DCMDiphenyldichlorosilane0.75% (w/v) of methyl red in DMF

Experimental Procedure. A few milligrams of resin are moistened witha solution of 10% Et3N in anhydrous DCM (200 �l) and treated with di-phenyldichlorosilane (100 �l) for 10 min. The resin is then filtered, washed


twice with 10% Et3N in anhydrous DCM, at which point a 0.75% (w/w) so-lution of methyl red in DMF (300 �l) is added and the resin allowed toshake for 10 min. The resin is filtered, washed with DMF (5 � 1 min),and then with DCM (5 � 1 min).

A positive test is indicated by orange beads, which become more red-dish with time. In the case of ambiguous results, the beads can be treatedwith formic acid and will take on a purple color in the case of a positiveresult.

Notes. This test proved to be satisfactory not only for phenols but forprimary and secondary alcohols as well.

In some cases, light purple beads may be observed even without theaddition of formic acid as a true positive.

We strongly recommend the use of a blank control run in parallel withthe sample to be tested as the colorant used in this test readily stays trappedwithin resins.

This test also yields positive results in the presence of othernucleophiles such as primary and secondary amines, carboxylic acids, andthiols.

Thiols

13 G.14 J. P

G.15 M.

Test: Ellman’s reagent13–15

Application: Detection of thiolsTest time: 4 minReagent preaparation time: 5 minRecommended storage time: up to 1 month at 4

�

Required Reagent

5,50-Dithio(2-nitrobenzoic acid), ‘‘DTNB,’’ or ‘‘Ellman’s reagent’’dissolved in aq. TRIS solution (1 M, pH 8)

Experimental Procedure. To a suspension of an aliquot of resin in DMFis added three or four drops of the DTNB solution. The solution is shakenat room temperature for 3 min. The presence of free thiols is indicated by ayellow-orange color.

Notes. This test, an adaptation of the original Ellman’s reagent test,worked well for the detection of free thiol groups.

L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).

. Baydal, A. M. Cameron, N. R. Cameron, D. M. Coe, R. Cox, B. G. Davis, L. J. Oates,

Oye, and P. G. Steel, Tetrahedron Lett. 42, 8531 (2001).

Royo, unpublished results (1991).


Carboxylic Acids

16 M.17 J. V

Test: Malachite green (MG)16

Application: Detection of carboxylic acidsTest time: ca. 5 minReagent preparation time: minutesRecommended storage time: up to 1 month at 4

�

Required Reagents

A 0.25% (w/v) solution of malachite green dissolved in ethanol Et3N

Experimental Procedure. A small portion (1–3 mg) of resin is trans-ferred to an Eppendorf tube and washed with MeOH. To this tube is addedthe malachite green solution (1 ml) followed by two drops of Et3N. The so-lution is left to stand at room temperature for 3 min and the resin is washedextensively with MeOH. The presence of free carboxylic acids is indicatedby green beads.

Notes. We found this test to be highly reproducible, but the test time iscrucial to obtaining accurate results. We observed false positives whenresin samples were left in the malachite green solution for more than15 min, and a loss of color when green beads are left in MeOH for morethan 15 min.

Aldehydes

Test: Vazquez17

Application: Detection of aldehydesTest time: 5 minReagent preparation time: ca. 5 minRecommended storage time: up to 1 month at room temperature

Required Reagents

A solution of EtOH (88 ml), H2SO4 (9 ml), and HOAc (1 ml)p-Anisaldehyde

Experimental Procedure. A solution of p-anisaldehyde (26 �l) in thefirst reagent (1 ml) is made. A small portion (1–3 mg) of resin is transferredto a small glass tube and washed with MeOH. To this tube is added the pre-vious EtOH/H2SO4/HOAc solution (500 �l). The solution is heated in asand bath at 110

�for 4 min. The presence of free aldehydes is indicated

by orange- to burgundy-colored beads.

E. Attardi, G. Porcu, and M. Taddei, Tetrahedron Lett. 41, 7391 (2000).

azquez and F. Albericio, Tetrahedron Lett. 42, 6691 (2001).


Notes. This test is very reliable. This test is compatible with acid-labileresins such as the Wang and chlorotrityl resins. While in the case of theWang resin similar results were obtained, in the case of the chlorotritylresin solutions also became colored, indicating cleavage of the aldehyde(BAL handle) from the resin.

18 J. J

Ya

Test: Purpald18

Application: Detection of aldehydesTest time: 25 minReagent preparation time: minutesRecommended storage time: N/A (see Notes)

Required Reagents

4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald)1 M NaOH

Experimental Procedure. The resin is washed with MeOH and a smallportion (ca. 5 mg) is transferred to a disposable syringe (1 ml) fitted witha polypropylene filter disc. To the resin is added of DMF (1 ml) and thesyringe is capped and shaken for 5 min. The DMF is then drained and afreshly prepared solution of Purpald dissolved in 1 M NaOH (250 �l) isadded. The syringe is capped and shaken for 5 min. The solution is drainedand the resin is washed with DCM (3 � 1 ml). The resin is then leftuncapped for 10 min. The presence of free aldehydes is indicated by brownor purple beads. At lower values of resin loading, a longer air oxidationtime may be required for color to develop (up to 20 min).

Notes. Due to the instability of Purpald in solution, it is imperative thatonly freshly prepared reagent solution be used.

Conclusions and Summary

Results obtained in the application of these tests are summarized inTable II. We have encountered some variations in the reproducibility andaccuracy of some tests. Due to the numerous factors that can influence col-orimetric test results (e.g., test reagent stability, resin type, functionalgroup interference, and lability of protecting group) we highly recommendperforming a positive and a negative control for any test applied to a newsynthesis. We also emphasize the importance of reagent solution purity onthe outcome of test results, hence we strongly encourage the use of cor-rectly prepared and carefully stored reactants. To minimize false results

. Cournoyer, T. Kshirsagar, P. P. Fantauzzi, G. M. Figliozzi, T. Makdessian, and B. J.

n, J. Comb. Chem. 4, 120 (2002).

TABLE II

Summary of Results Obtained in the Application of the Testsa

Colorimetric test

Functional group Kaiser TNBS NF-31

Chloranil

first amine

Chloranil

second amine

TosCl-

PNBP TCT

Methyl

red Ellman MG Vazquez Purpald

Primary alcohol �/� �/� �/� �/� �/� þþ/þ þþ/þ þþ/þ �/� �/� �/� �/�Secondary alcohol �/� �/� �/� �/� �/� þþ/þ þþ/þ þþ/þ �/� �/� �/� �/�Phenol �/� �/� �/� �/� �/� þ/� þþ/þ þþ/þ �/� �/� �/� �/�Aldehyde �/� �/� �/� �/� �/� �/� �/� �/� �/� �/� þþ/þ þþ/þPrimary amine þþ/þ þþ/þ þþ/þþ þþ/þ þ/� �/� þþ/þ þ/þ� �/� �/� �/� �/�Hindered primary

amine

þ�/� þþ/þ þþ/þ þ/þ� þ�/� �/� þ/þ� þ�/� �/� �/� �/� �/�

Secondary amine þ/� þþ/þ þþ/þþ þþ/þ þþ/þ �/� þ/þ þ/þ� �/� �/� �/� �/�Guanidine �/� �/� �/� �/� �/� �/� �/� �/� �/� �/� �/� �/�Carboxylic acid �/� �/� �/� �/� þ�/� �/� þ/þ� þ�/� �/� þþ/þþ �/� �/�Thiol �/� �/� �/� �/� �/� �/� þþ/þ þþ/þ þ/þ �/� �/� �/�

a A/B, A is the result obtained for a 100% loading resin and B is the result for a 5% loading resin; þþ, intense; þ, less intense; þ�, not clear;

�, no difference with a blank control.

34

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s[2

]


due to lability of the resin–product bond or the product itself (such asFmoc-protected amino acids), colorimetric tests should be performed withthe utmost immediacy in regards to completion of the step to be monitored.Hence the storage of resin over long periods of time (more than 24 h)before testing is not advisable. When the result of a colorimetric test is indoubt we advise repeating the test a few times until a reproducible result isobtained. The use of multiple tests for the same functional group mayelucidate ambiguous or otherwise challenging cases.

Acknowledgments

The authors’ laboratory work was supported by CICYT (BQU2000–0235) and the

Generalitat de Catalunya (Grup Consolidat and Centre de Referencia en Biotecnologia). The

enthusiastic collaboration of Dr. Miriam Royo, Gloria Sanclimens, Aida Martinez, and

Meritxell Teixido at the Barcelona Biomedical Research Institute, Barcelona Science Park,

University of Barcelona in the experimental portion of the work and the preparation of the

manuscript is greatly appreciated.

[3] solid-phase synthesis on synphase lanterns 39

[3] A Review of Solid-Phase Organic Synthesis onSynPhase‘ Lanterns and SynPhase‘ Crowns

By Jack G. Parsons, Craig S. Sheehan, Zemin Wu,Ian W. James, and Andrew M. Bray

Introduction

A decade ago there were few examples of solid-phase organic synthesisoutside of the specialized areas of peptide synthesis and oligonucleotidesynthesis. It is not hard to understand why solid-phase synthesis is attract-ive for these target classes. The synthesis comprises a small range of highlyoptimized repetitive reactions that lend themselves to automation, hencegreatly increasing the access to these important biomolecules.1 Early workon small molecule solid-phase synthesis by Leznoff and co-workers2,3 andFrechet and co-workers4,5 illustrated the potential of the approach butdid not spark broad interest. Effective solid-phase synthesis required thatsolution phase chemists develop and become familiar with the new hand-ling methods. It should also be noted that the solid supports used for pep-tide synthesis were not optimized for nonpeptide synthesis, which requiresa far broader range of reaction conditions. Furthermore, unless each reac-tion step in a solid-phase synthesis is highly optimized, poor quality prod-ucts are generated.6 Strong interest in the synthesis of small moleculecompounds was finally kindled by the benzodiazepine synthesis describedby Ellman and Bunin in 1992, which illustrated the potential for the rapidparallel synthesis of compounds of pharmaceutical interest.7 At this time,pharmaceutical and biotechnology companies had an increasing demandfor biologically relevant compounds due to the strong growth in high-throughput screening. Through the 1990s, solid-phase synthesis became awell-established tool in the pharmaceutical industry for the generation oflarge sets of small molecule compounds for lead finding and optimization.8,9

1 W. C. Chan and P. D. White, ‘‘Fmoc Solid-phase Peptide Synthesis: A Practical Approach.’’

Oxford University Press, Oxford, 2000.2 J. Y. Wong, C. Manning, and C. C. Leznoff, Angew. Chem. Int. Ed. Engl. 13, 666 (1974).3 C. C. Leznoff, T. M. Fyles, and J. Weatherston, Can. J. Chem. 55, 1143 (1977).4 M. J. Farrall and J. M. J. Frechet, J. Org. Chem. 41, 3877 (1976).5 J. M. J. Frechet and E. Seymour, Isr. J. Chem. 17, 253 (1978).6 A. M. Bray, D. S. Cheifari, R. M. Valerio, and N. J. Maeji, Tetrahedron Lett. 36, 5081

(1995).7 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992).8 R. E. Dolle, J. Comb. Chem. 3, 477 (2000).9 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273 (2001).



Fig. 1. Graft polymer devices manufactured and sold by Mimotopes Pty Ltd for solid-

phase synthesis.

40 combinatorial synthesis [3]

Mimotopes has been involved in the development, use, and commer-cialization of radiation-grafted polymer surfaces for multiple parallel syn-thesis since the late 1980s.10–12 Although other workers have reported theuse of radiation-graft polymers in solid-phase synthesis,13,14 as far as we areaware, the graft polymer devices manufactured and sold by Mimotopes(SynPhase Crowns, SynPhase Lanterns) are the only current commercialproducts of this type. These products are presented in Fig. 1. The SynPhaseLanterns are the current design for small molecule synthesis. The initial

10 H. M. Geysen, S. J. Barteling, and R. H. Meloen, Proc. Natl. Acad. Sci. USA 82, 178 (1985).11 F. Rasoul, F. Ercole, Y. Pham, C. T. Bui, Z. Wu, S. N. James, R. W. Trainor, G. Wickham,

and N. J. Maeji, Biopolymers 55, 207 (2000).12 N. J. Maeji, R. M. Valerio, A. M. Bray, R. A. Campbell, and H. M. Geysen, Reactive Polym.

22, 203 (1994).13 R. H. Berg, K. Almdal, W. B. Pedersen, A. Holm, J. P. Tam, and R. B. Merrifield, J. Am.

Chem. Soc. 111, 8024 (1989).14 R. Li, X.-Y. Xiao, and A. W. Czarnik, Tetrahedron Lett. 39, 8581 (1998).

Fig. 2. The 8 � 12 Multipin format.


purpose of the technology, which was referred to as Multipin Technologyin the late 1980s, was to prepare large sets of very small amounts ofsupport-bound peptide oligomers for use in screening antibodies and othersoluble receptors.15 At that time, a suitably functionalized polyacrylic acidgraft polymer was used as the support for peptide synthesis. Peptide quan-tities were small and purities were low, and the peptides were not cleavedfrom the solid phase. Nevertheless, the support-bound peptide librarieswere very effective in rapid epitope determination. To perform multiplepeptide synthesis on the micromole scale, it was necessary to developnew graft polymers and new shapes with higher surface area to volumeratios.16

In the mid to late 1990s, Mimotopes was producing rigid injectionmolded polypropylene devices that were surface grafted with either ahydrophilic copolymer of methacrylic acid/dimethyl acrylamide or the rela-tively hydrophobic polystyrene.12 The polymer was then suitably deriva-tized to allow the incorporation of a linker system. In contrast to thevarious commercial resins available at the time, the Crown was a macro-scopic, quantized solid phase. As shown in Fig. 2, the Crowns were typi-cally fitted to a polypropylene stem, which in turn could be fitted into a

15 H. M. Geysen, S. J. Rodda, T. J. Mason, G. Tribbick, and P. G. Schoofs, J. Immunol.

Methods 102, 259 (1987).16 N. J. Maeji, A. M. Bray, and R. M. Valerio, Peptide Res. 8, 33 (1995).


polypropylene holder that presented the Crowns in an 8 � 12 matrix thatmatched the ubiquitous microtiter plate format. Both the Crown and theLantern have been fitted with individual tags for use in synthesis via thedirected ‘‘split and pool’’ approach. Tagging has been achieved using inertcolored tags or radiofrequency (RF) tags.17,18 This technology is welldescribed elsewhere.19

A number of successful small molecule library syntheses have beenreported on the SynPhase Crown surface. A review summarizes thesepapers to 1997.19 Nevertheless, we recognized the deficiencies of thisdesign for solid-phase organic synthesis, especially its low loading/volumeratio, and a new high surface area device was developed. The SynPhaseLantern was introduced into the market in 2000.11 The L-series and thelarger D-series Lanterns have replaced the earlier designs (see Fig. 1).The Lantern is made up of a series of uniformly spaced flat rings. It resem-bles a Chinese lantern, hence the name. Although the D-series Lanternsare smaller than the I-series SynPhase Crowns, they have a larger surfacearea/volume ratio. This is of benefit when using expensive reagents wherevolumes need to be reduced. The D-series Lantern has a higher loading of35 �mol/unit. A second improvement is that the polystyrene graft surfacehas been optimized for use in small molecule synthesis.20–22 This wasachieved over time by using a comparative combinatorial approach.Method refinement also enabled tight control of loading variation betweenbatches and within batches. In large-scale library synthesis, loading vari-ation results in yield variation, which has been cited as a potential issuewith the earlier Crown products.23 Lanterns are now being used primarilyby chemists in pharmaceutical companies for the synthesis of libraries ofsmall molecule compounds. Consequently, much of the synthetic work thathas been performed has not yet been published, and much of it may neverbe published. Nevertheless, there is a steady stream of publications on theuse of Lanterns in solid-phase synthesis. This review aims to summarize

17 E. J. Moran, S. Sarshar, J. F. Cargill, M. M. Shahbaz, A. Lio, A. M. M. Mjalla, and R. W.

Armstrong, J. Am. Chem. Soc. 117, 10787 (1995).18 R. Giger, Pin sort-and-combine method for HTP synthesis of individual crowns. Paper

presented at the Cambridge Healthtec Institute Conference, Barcelona, Spain, 1997.19 I. W. James, G. Wickham, N. J. Ede, and A. M. Bray, in ‘‘Solid-Phase Organic Synthesis’’

(K. Burgess, ed.), p. 195. Wiley-Interscience, New York, 2000.20 Z. Wu, F. Ercole, M. FitzGerald, S. Perera, P. Riley, R. Campbell, Y. Pham, P. Rea,

S. Sandanayake, M. N. Mathieu, A. M. Bray, and N. J. Ede, J. Comb. Chem. 5, 166 (2003).21 A. R. Vaino and K. D. Janda, J. Comb. Chem. 2, 579 (2000).22 S. W. Gerritz, Curr. Drug Discov. 19, (2002).23 C. G. Boojamra, K. M. Burow, L. A. Thompson, and J. A. Ellman, J. Org. Chem. 62, 1240

(1997).

SynPhase Crown SynPhase Lantern

Fig. 3. SynPhase Lantern and SynPhase Crown symbols used in this chapter.


work that has been published between 1997 and early 2002, and builds onour earlier review that covered solid-phase organic synthesis on radiation-grafted polymers.19 As summarized in Fig. 3, the Crown is depicted by astar and the Lantern is represented by a rectangular symbol.

Heterocycles

Benzodiazepines

Benzodiazepines were the first class of heterocyclic compounds to besynthesized on the SynPhase surface. In 1994, Ellman and co-workers24

reported a 192 member library of structurally diverse 1,4-benzodiazepines.These compounds were prepared on Mimotopes pins that were graftedwith polyacrylic acid, the surface originally used for antibody epitope elu-cidation.10 Ellman and co-workers25 subsequently synthesized a 1680-member 1,4-benzodiazepine library on SynPhase Crowns that were graftedwith a methacrylic acid/dimethylacrylamide copolymer, one of the firstSynPhase surfaces designed for solid-phase synthesis. The synthesis wasperformed on a preformed linker-template system in order to avoid lowaminobenzophenone incorporation; in this case the HMP

*

acid-labile linker

24 B. A. Bunin, M. J. Plunkett, and J. A. Ellman, Proc. Natl. Acad. Sci. USA 91, 4708 (1994).25 B. A. Bunin, M. J. Plunkett, J. A. Ellman, and A. M. Bray, New J. Chem. 21, 125 (1997).* Abbreviations: AcOH, acetic acid; Ar, general aryl group; BAL, backbone amide linker;

BEMP, 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine; Bn,

benzyl; Boc, tert-butoxycarbonyl; BOP, benzotriazol-1-yloxytris(dimethylamino)phospho-

nium hexafluorophosphate; CDI, carbonyldiimidazole; DEAD, diethyl azodicarboxylate;

DIC, N,N-diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMAP, N,N-dimethyl-

aminopyridine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU,

O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, O-

(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEMA, 2-hydroxy-

ethyl methacrylamide; HMP, 4-(hydroxymethyl)phenoxyacetyl (linker system); HOAt, 3-

hydroxy-7-azabenzotriazole; HOBt, 3-hydroxybenzotriazole; HPLC, high-performance

liquid chromatography; LCMS, liquid chromatography mass spectrometry; LHP, ‘‘long chain

HMP’’: 4-(hydroxymethyl) phenoxypentanoyl (linker system); mCPBA, m-chloroperbenzoic

O

NH2

R1

R2

HMP

O

NH

OR3

NHfmoc

R1

R2

HMP

R2

HMP

N

N

R3

OR4

R1

O O O

HMP OH NH

OO

OH

1 2 3

Scheme 1. General solid-phase route to benzodiazepines.


was employed. It should be noted that using the trichloroacetimidatemethod described by Hanessian and Xie26 phenols can now be readilyloaded onto this linker system on the solid-phase.27 Coupling of Fmoc-protected amino acids onto 1 was achieved via preformed acid fluoridesto afford support-bound amides 2 (Scheme 1). Following Fmoc deprotec-tion, cyclization to the 1,4-benzodiazepine resulted via treatment of thesupport-bound intermediate with 5% AcOH in NMP. Alkylation wasachieved by subsequent treatment with excess lithiated 5-phenylmethyl-2-oxazolidinone and the respective alkyl halide to give densely functionalizedbenzodiazepine compounds 3. Cleavage was achieved with trifluoroaceticacid. A modified version of this synthesis on a polystyrene graft surface hasbeen described (Scheme 2).28 A preformed 2-aminobenzophenone linker-template system 5 was coupled to aminomethylated polystyrene Lanterns 4by the action of DIC and HOBt in DMF. The resulting support-bound an-iline 6 was acylated with an in situ generated Fmoc amino acid fluoride toafford the amide 7. Deprotection followed by acid-catalyzed intramolecularcyclization generated the functionalized 1,4-benzodiazepines 8.

acid; MD, methacrylic acid/dimethylacrylamide copolymer MA-DMA; Mmt, 4-methoxyl-

trityl; NMM, N-methylmorpholine; NMP, 1-methyl-2-pyrrolidinone; Py, Pyridine; PyBOP,

benzotriazol-1-yloxytris(pyrrolidino) phosphonium hexafluorophosphate; RINK, rink amide

forming linker; TFA, trifluoroacetic acid; TFFA, trifluoroacetic anhydride; TFFH, fluoro-

N,N,N0,N0-tetramethylformadinium hexafluorophosphate; Wang, hydroxymethylphenoxy

linker on polystyrene.26 S. Hanessian and F. Xie, Tetrahedron Lett. 39, 733–736 (1998).27 Mimotopes SynPhase‘ Application Note SCN005, www.synphase.com28 Mimotopes SynPhase‘ Application Notes SCN011 and SCN012, www.synphase.com

NH2.TFA NH2

TEA

DMF/CH2Cl2

OO

HOO

O NH2

DIC, HOBt, DMF

O NH2

OI

NH O

HO

fmoc

TFFH/DIEA/DMF/40 �C

20% piperidine/DMF

5% AcOH/NMP, 60 �C

I

NH

O

N

O

95% TFA/H2O

I

NH

O

N

HO

HMP OH NH

OO

OH

HMP

HMPO

I

O HN O

NH

fmoc

HMP

4

5

6

7

8

Scheme 2. Assembly of benzodiazepines using in situ-generated acyl fluorides.


Purines

The group of Schultz has synthesized a 406-member purine libraryon MD grafted SynPhase Crowns with varying substituents at the C2and C6 positions.29 A common 2-chloropurine derivative was coupled onto

2

6RINK

NH

N

N

N

N

Cl

NH

O

NH

OO

NH2

MeO

OMe

2

6RINK

NH

N

N

N

N

Cl

N

OOR1

2

6RINK

NH

N

N

N

N

NH

N

OOR1

R2

RINK NH2

9 10

11

Scheme 3. Solid-phase synthesis of 2,6-disubstituted purines.


support-bound Rink amide forming linker 9. The first combinatorial stepinvolved acylation of an exocyclic nitrogen attached to C6 to give amides10 (Scheme 3). An SNAr reaction with diverse amines afforded the final2,6-substituted purine derivatives 11. Cleavage was achieved with TFA.Compounds from this class were found to inhibit glucose synthase kinase.30

Schultz and co-workers31 also described the preparation of a 2,6,9-trisubstituted purine library. A preformed 2-fluoro-6-(4-aminobenzylamino)purine was reductively aminated onto the BAL linker 12. Mitsunobuchemistry was employed to alkylate the C9 position on the support-boundintermediate (Scheme 4). Subsequently, SNAr chemistry was used to in-corporate amines at C6. The newly introduced primary and secondaryamines bear diverse functional groups and the Mitsunobu reaction allowsfor incorporation of primary and secondary alcohols lacking acidic hydro-gens. The support-bound product 13 was cleaved with 90% TFA/10% H2Oto give a library with HPLC purities ranging between 51 and 85%.

In a more recent paper, Schultz and co-workers32 extended the technol-ogy on SynPhase Crowns to synthesize more diverse 2,6,9-trisubstituted

29 T. C. Norman, N. C. Gray, J. T. Kohand, and P. G. Schultz, J. Am. Chem. Soc. 118, 7430 (1996).30 S. Rosenberg, K. L. Spear, R. Valerio, and A. Bray, PCT Int. Appl. WO 9705257 (1997).31 N. S. Gray, S. Kwon, and P. G. Schultz, Tetrahedron Lett. 38, 1161 (1997).32 N. S. Gray, L. Wodicka, A.-M. Thunnissen, T. C. Norman, S. Kwon, F. H. Espinoza, D. O.

Morgan, G. Barnes, S. LeClerc, L. Meijer, S.-H. Kim, D. J. Lockhart, and P. G. Schultz,

Science 281, 533 (1998).

N9

N

N

N

NHNH

FH

2

6

SNAr

Mitsunobu

BAL

N9

N

N

N

NHNH

NH

R2

R1

2

6

BAL

O

OO

OMeO

OMeBAL

12 13

Scheme 4. Solid-phase synthesis of 6,9-disubstituted purines.

NH

N

N

N

Cl

F

1. Amination at C2 (R1NH2)and/oralkylation at N9 (R2OH)

and /or amination at C6 (R3NH2)2. Acidic cleavage

N

N

N

N

NH

NH

R3

R1

R2

O

2

69

NHNH

NH

NHO

Scheme 5. General route to diverse purine libraries.


purine libraries. As summarized in Scheme 5, a number of synthetic routeswere used, where the core purine was linked to the solid-phase via the 2, 6,or 9 positions. The synthetic methodologies were essentially as describedabove. Potent inhibitors of human cyclin-dependent kinase 2 (CDK2) wereidentified using these trisubstituted purine libraries.

Quinazolines

Polystyrene grafted SynPhase Lanterns were used by Makino et al.33 atAjinomoto to synthesize a diverse quinazoline-2-thioxo-4-one library. Lan-terns functionalized with long-chain HMP (hydroxymethylphenoxyvalericamide) linker were coupled with nitrophenols by the action of DIC/HOAt/DMAP to give nitrobenzenes 14 (Scheme 6). The nitro group was

33 S. Makino, E. Nakanishi, and T. Tsuji, Tetrahedron Lett. 42, 1749 (2001).

NO2-Ar-OH

DIC/HOAt/DMAP/NMP

SnCl2/NMP/EtOHN S

CO2Me

NMP

BrDIEA/NMP1.

2. 95% TFA/H2O

95% TFA/H2O

NNH

Ar

S

O

HO +

NN

Ar

O

HO

S

HO

LHP OH LHP O Ar NO2

LHP O Ar NH2

LHP NNHS

O

O Ar

S

NNH

O

HO Ar

LHP OH NH

O

O

OH

14

15

16

17

18

Scheme 6. Diverse quinazoline-2-thioxo-4-one libraries.


reduced with SnCl2 to efficiently give support-bound amines 15. Subse-quent reaction with 2-methoxycarbonylphenyl isothiocyanate gave atransient thiourea that readily cyclized to the required quinazoline-2-thioxo-4-ones 16. Products were obtained in high purity, typically greaterthan 95%. It is interesting to note that Makino observed a number of S-alkylated by-products 17 when the synthesis was performed on Wang resin.They stem from reaction with the benzylic linker cations generated bycleavage with 95% TFA/H2O. This side reaction was reported to be far lesspronounced in the case of long-chain HMP derivatized Lanterns. Makinoinvestigated the derivatization of quinazoline-2-thioxo-4-ones with alkylhalides. Reaction with DIEA in NMP gave the best results with the add-ition of allyl bromide giving products 18 in greater than 95% purity. Excel-lent yields and purities were obtained using a variety of alkyl and arylhalides.

NH2

O

O

F

O2N CO2H

DIC/HOAt/NMP

R-NH2

NMP

NN N

O

N

decalin, 95 �C

95% TFA/H2OHO

ON

ONO2

N

RO

HMP OH NH

OO

OH

HMP HMPNH

O

O

O

F

NO2

HMPNH

O

O

O

HN

NO2

R

HMP O

O

O

N

NO2

R

N

O

19

20

21 22

Scheme 7. Synthesis of diverse 1,3-disubstituted quinazoline-2,4-diones.


Makino et al.34 also published a synthesis of a diverse 1,3-disubstitutedquinazoline-2,4-dione library on HMP-derived polystyrene SynPhase Lan-terns bearing a 4-aminobenzoic ester. As outlined in Scheme 7, 2-fluoro-5-nitrobenzoic acid was coupled to the linker-template system to give amide19. Nucleophilic substitutions with a range of alkyl amines proceeded at 25

�

to give products 20 with excellent purity. Aryl amines had to be heated to80

�. Next, carbonylation with carbonyl diimidazole in decalin at 95

�for

16 h afforded the quinzoline-2,4-diones 21 with 100% conversion. Productswere cleaved from the solid support with 95% TFA/H2O to give 22; in mostcases purities greater than 95% were obtained. Makino et al. examined sev-eral solid supported amines. Quinazoline-2,4-diones were obtained in highpurity from 3-aminobenzoic acid ester. Aminocinnamic esters gave signifi-cant amounts of a Michael addition product resulting from the additionof imidazole to the cinnamoyl moiety, though �-elimination with BEMPcatalysis prior to cleavage gave the required products in high purity.

Quinazoline-2,4-dione oligomers were synthesized on SynPhase Lan-terns by reducing the nitro group to the aniline with SnCl2, and subsequentacylation of the amine followed by SNAr reaction with primary amines togive 23 (Scheme 8). Carbonylation with CDI in decalin at elevated tem-perature gave the quinazoline-2,4-dione oligomer (4-mer) 24 followingcleavage from the solid phase.

34 S. Makino, N. Suzuki, E. Nakanishi, and T. Tsuji, Synlett, 333 (2001).

O

O

NH

O

HN

NH

O

HN

NH

O

HN

NH

O

NO2

HN

HO

O

N

O

N

N

O

N

NH

O

HN

N

O

NO2

NO O

O

1. CDI, decalin95 �C2. 95% TFA/H2O

O

O

NH

O

F

NO22. F

HO2C

NO2

DIC/HOAt

O

O

NH

O

HN

NH

O

F NO2

1. SnCl2

3. SNAr reactioncyclobutylamine

2.

HO2C

F NO2

DIC/HOAt

O

O

NH

O

HN

NH

O

NO2HN

repeat reduction,substitution, acylationcycles

1. SnCl2

3. SNAr reaction, propylamine

24

19

23

Scheme 8. Stepwise assembly of quinazoline-2,4-dione oligomers.


Makino et al.35 developed a solid-phase synthesis of 1,3-disubstituted 2-thioxoquinazoline-4-ones using HMP Lanterns that were derivatized witha 4-aminobenzoate ester. Following the assembly of amide 25 (Scheme 9),SNAr reaction with alkyl amines gave support-bound products 26 with highconversion. The key thiocarbonylation step was achieved with thiocarbo-nyldiimidazole in decalin at 95

�for 16 h in the presence of DMAP to afford

1,3-disubstituted 2-thioxoquinazoline-4-ones 27. The target compounds 28

35 S. Makino, N. Suzuki, E. Nakanashi, and T. Tsuji, Tetrahedron Lett. 41, 8333 (2000).

O

ONH2

F

CO2HO2N

DIC/HOAt/NMP

N NN N

S

DMAP/decalin95 �C

95% TFA/H2O NN

HO

O

O

S R

NO2

HMP HMP O

ONH

O

F

NO2

RNH2, NMP HMP O

ONH

O

HN

NO2

R

HMPN

N

O

S R

NO2

O

O

HMP OH NH

OO

OH

25

26

2728

Scheme 9. Synthesis of 2-thioxoquinazoline-4-ones.


were cleaved from the Lanterns using 95% TFA/H2O. The synthesis gavetargets with purities generally greater than 95% and yields ranging from59 to 100%.

Arylaminobenzimidazoles

Krchnak and co-workers36 at Selectide devised a novel tracking strategythat was demonstrated in the synthesis of a benzimidazole library. Rinkamide-derived Crowns were acylated with 4-fluoro-3-nitrobenzoic acid togive 29 and further functionalized via SNAr substitution with eight amines(Scheme 10). The o-nitroaniline-derivatized Crowns 30 were threaded ontoa steel string and tied to form a ‘‘necklace.’’ The eight respective R1 groupswere defined by the order of placement onto each necklace. Twelve identi-cal necklaces were prepared. The support-bound nitroanilines were re-duced with SnCl2 in NMP in bulk and each necklace was treated with a

36 J. M. Smith, J. Gard, W. Cummings, A. Kanizsai, and V. Krchnak, J. Comb. Chem. 1, 368

(1999).

NH2-R1

SnCl2, NMP R2NCSDIC

HCl (g), 2 hR2

NH2

RINK

RINK

RINK

RINK

RINKNH

F

NO2

O

NH

NH

NO2

O

R1

NH

NH

NH2

O

R1

N

NNH

O

NH

R1

R2H2N

N

O

NNH

R1

29 30

31 32

NH

OO

NH2MeO

OMe

Scheme 10. Synthesis of 2-aminobenzimidazole libraries.


different isothiocyanate and 1 M DIC solution at ambient temperatureovernight to form the respective support-bound arylaminobenzimidazoles31. The 96-member library was cleaved by treatment with gaseous HClfor 2 h and subsequent extraction from the solid phase. Quality of theproducts 32 ranged from 56 to 99% purity with the average being 83%.

Wu et al.37 at Mimotopes developed a one-pot synthesis of benzimida-zoles using chloromethylated SynPhase Lanterns functionalized with Wanglinker (Scheme 11). The support-bound ester 33 was treated with aliphaticand aromatic amines to afford o-nitroanilines 34 with little, if any, prema-ture cleavage. After some experimentation, one-pot reduction/cyclizationto the desired benzimidazole 35 was achieved using two equivalents ofaldehyde and 0.75 M SnCl2�2H2O (10 equivalents) in DMF and heating at60

�for 3 h. The support-bound products were readily cleaved from the Lan-

terns with 20% TFA/CH2Cl2 to afford the desired disubstituted benzimida-zoles 36. A 25-member library was synthesized using five aldehydes and fiveamines. A benzimidazolium by-product 37 was observed for aliphatic

37 Z. Wu, P. Rea, and G. Wickham, Tetrahedron Lett. 41, 9871 (2000).

CO2H

FNO2

DIC/DMAP R1NH3

SnCl2.2H2O, R2CHO

DMF, 60 �C

20% TFA/CH2Cl2N

NR2

R1

O

H2N

Wang OH Wang O

O

F

NO2

Wang O

O

NHR1

NO2

Wang OH OOH

Wang N

N

O

O

R2

R1

33

34 35

36

Scheme 11. Synthesis of 5-carboxybenzimidazole libraries.

N

N

O

HO

R2

R2

R1

+

37

Fig. 4. Benzimidazolium by-product.


aldehydes but not for benzaldehydes (Fig. 4). Target compound puritiesranged from 56 to 93% and yields were typically 85%.

Pyridin-2-ones

SynPhase Crowns with a Rink amide linker were used by Linn et al.38

at Glaxo-Wellcome to synthesize a library of 1,3,5-trisubstituted pyridin-2-ones (Scheme 12). The solid bound amide 38 was treated with 3-amino-5-methoxycarbonyl-1H-pyridin-2-one with Cs2CO3 in DMF to affordpyridine-2-one 39. Coupling of diphenylacetic acid HATU and DIEA in

38 J. A. Linn, S. W. Gerritz, A. L. Handlon, C. E. Hyman, and D. Heyer, Tetrahedron Lett. 40,

2227 (1999).

O

HO Br( )4

NHNH2

O

CO2Me

Cs2CO3, DMF16 h, rt

Ph2CH

O

OH

HATU, DIEA,DMF, rt, 18 h

LiOH

F

F

F

F

F

OH1.

py, TFAA,DMF, 4 h

1. BnNH2, DMF, 18 h, rt

NH2N

O

O

NH

O

CHPh2

O NHCH2Ph

( )5

RINK NH2 RINK NH

O

Br( )4

RINK NNH

O

O

NH2

CO2Me

( ) RINKN

NH

O

O

NH

CO2Me

O

CHPh2( )

55

RINKN

NH

O

O

NH

O

CHPh2

CO2H

( )5

RINK NH2NH

OO

NH2

MeO

OMe

2. 95% TFA(aq), 1 hRINK ( )5N

NH

O

O

NH

O

CHPh2

CO2C6F5

38

39 40

41

42 43

Scheme 12. Synthesis of pyridin-2-one libraries.


DMF gave the required amide 40. Saponification of the ester with 1 MLiOH in 1,4-dioxane gave solid-bound acid 41. The support-bound penta-fluorophenol ester 42 was then generated, allowing efficient coupling witha range of benzylamines. Generally, ‘‘reversed’’ couplings are far less effi-cient than conventional couplings where the carboxilic acid componentreacts with a support-bound amine. Cleavage with 95% TFA/H2O affordeda library of trisubstituted pyridin-2-ones 43.

Oxazoles

Quibell and co-workers39 at Peptide Therapeutics used a 5-(hydroxy-methyl)oxazole scaffold 44 to prepare a range of oxazole libraries onHEMA-grafted, glycine-derivatized Gears (a small Crown design). The

O

O

N

OH

NH

fmoc

R O

HO

BOP, HOBt, NMM

1.

2. Piperidine, DMF

fmocAA-OC6F5

HOBt, DMF,o/nO

OC6F5

O

HOBt, DMF, o/n

1. Piperidine, DMF

2.

Gly-RINK-NH2

RINKGly

Gly-RINK , HEMA graftNH

OO

NH2

OMe

MeO

NH

O

N

O

O

NH NH2

R OOH

RINKGlyN

O

O

NH N

H

R OOH O

NH

R

fmoc

RINKGly

O

N

O

O

NH N

H

R OOH O

NH

RO

44

45

Scheme 13. Preparation of libraries based on a 5-(hydroxymethyl)oxazole scaffold.


scaffold was built up on the solid phase as outlined in Scheme 13 on a Rinklinker. The scaffold was used to synthesize aryl ethers, thioethers, sulfones,sulfonamides, and carboxamides via the free hydroxymethyl group.

Alkyl/aryl ethers 46 were synthesized by a Mitsunobu reaction withDEAD, PPh3, NEt3, and a wide variety of alkyl and aryl alcohols(Scheme 14). Products were obtained in greater than 90% purity. Reactionof 5-(hydroxymethyl)oxazole 45 with CBr4, PPh3 gave bromides 47 thatwere efficiently converted into thioethers 48 by overnight treatment withthiols in NMP. The synthesis of sulfones 49 was achieved by oxidation ofthe thioethers precursors with mCPBA in CH2Cl2. Carboxamides 50 weresynthesized by converting the free bromides to azides with NaN3, DIEAin NMP/H2O. The azides were reduced to amines with dithiothreitoland DIEA. The amine analogs were acylated with a variety of alkyl, aryl

39 U. Grabowska, A. Rizzo, K. Farnell, and M. Quibell, J. Comb. Chem. 2, 475 (2000).

Gly

O

N

O

O

NH N

H

R OOH O

NH

RO

Gly

O

N

O

O

NH N

H

R OBr O

NH

RO

Gly

O

N

O

O

NH N

H

R ONH2 O

NH

RO

45 46

47 48

49

51

50

RINK

RINK

RINK

1. NaN3

2. DTT

1. PPh3,DEAD, ArOH

5% Et3SiH1. TFA

2. TFA5% Et3SiH

3. TFA/5% Et3SiH2. mCPBA

5% Et3SiH2. TFA

2. TFA5% Et3SiH

O

O

O

N

O

NH

R OH

H2N

O

NH

RO

R

CBr4,PPh3

1.NMP/thiolO

O

O

NNH

R OH

H2N

O

NH

RO

S( )n

1. NMP/thiol reagent

O

O

O

NNH

R OH

H2N

O

NH

RO

SO2

( )n

1. RCO2HHBTU, HOBtNMM, DMF

O

O

O

NNH

R OH

H2N

O

NH

RO

N

R

O

1. RSO2ClDMAP, DMF

O

O

O

N

NH

NH

RR

OH

H2N

O

NH

RO

SO

O

Scheme 14. An alternative approach to oxazole-based libraries.


carboxylic acids and anhydrides to give high-quality carboxamides 50. Theamines were also treated with sulfonyl chlorides to give sulfonamides 51 ingreater than 90% purity.

Hydantoin/Isoxazolines

Kurth and co-workers40 (Novartis) prepared a 990-member compoundlibrary of hydantoin and isoxazoline containing heterocycles on amine-derived SynPhase Crowns. As shown in Scheme 15, the synthesis was

40 K. Park, J. Ehrler, H. Spoerri, and M. J. Kurth, J. Comb. Chem. 3, 171 (2001).

NH

O

OH

NHBoc

O

HO

DIC, DMAPDMF/CH2Cl2

NH

O

O

ONH2

1.

2. TFA, NEt3

DioxanePhNCO

nPrNO2

PhNCONEt3Dioxane

ON

NH

O

O

ONH

O

NH

NEt3, CH3CN

ON

NH

N

O

O

NH

O

O

ONH

O

NH

Major diastereomer

52

53

54

Scheme 15. Synthesis of a hydantoin library via 1,3-dipolar cycloaddition.


performed using simultaneous cleavage and cyclization on a base labilelinker. Boc-protected amino acids were coupled onto the free hydroxylgroup with DIC/DMAP. Treatment with TFA, followed by neutralization,afforded the amine 52. Reaction with phenylisocyanate gave the solidbound urea 53. Subsequent 1,3-dipolar cycloaddition with a Mukaiyama-generated nitrile oxide afforded the isoxazoline 54. Treatment withtriethylamine produced hydantoins as an 8:1 diastereomeric mixture.

An alternative route to the same 990-compound library was developedusing 5 amino acids, 9 nitroalkanes, and 22 isocyanates (Scheme 16). Thehydroxymethyl function was coupled with the 5 alkene-containing Bocamino acids. Reaction of the resulting esters 55 with 5 nitroalkanes gavethe isoxalines 56 after Boc deprotection. Coupling of the amine with theisocyanates afforded ureas 57. Cleavage with triethylamine produced theisoxazolinoimidazolidione final products 58. HPLC purities of greater than70% were obtained for 90% of the samples in the library. Reaction yieldswere typically between 40 and 60%. Diversity is further introduced ontothe hydantoin and isoxazoline heterocycles with a variety of connectinggroups between the two heterocycles.

NH

O

O

O

X

NH2

ON R1

NH

O

OH

DIC, DMAPDMF/CH2Cl2

1. PhNCO, NEt3

NH

O

O

ONHBoc

X

R1CH2NO2

2. TFA, NEt3

Dioxane, R3NCO

NH

O

O

O

X

NH

O

NH

R2

ON R1

NEt3, CH3CN

ON R1

X

N

O NH

OR2

HO

ONHBoc

X

55

56

57

58

Scheme 16. Alternative route to hydantoin libraries.


Quinoxalines

Wu and Ede41 (Mimotopes) described the first synthesis of quinoxa-lines on the solid support using polystyrene-grafted SynPhase Lanterns(Scheme 17). Following coupling of 4-fluoro-3-nitrobenzoic acid to Syn-Phase Rink Lanterns to give 59, amino substitution of the aryl fluoridewas achieved by the action of aqueous ammonia solution in 5% DIEA/DMF at 60

�for 5 h. The o-nitroaniline 60 was reduced to the o-phenylene-

diamine 61 by SnCl2�2H2O. One-pot N-alkylation with 10 �-bromoketonesfollowed by in situ cyclization and oxidation gave regioisomeric quinoxa-lines 62 and 63. In all cases, good yields were obtained with product puritiesranging between 65 and 86% for the sum of quinoxaline isomers.

Perhydro-1,4-diazepine-2,5-diones

Amblard and co-workers42 from Montpellier University optimizedthe synthesis of a 3,7-disubstituted perhydro-1,4-diazepine-2,5-dione 64on polystyrene Crowns (Scheme 18). Seven-membered heterocyclic

41 Z. Wu and N. J. Ede, Tetrahedron Lett. 42, 8115 (2001).42 J. Giovannoni, G. Subra, M. Amblard, and J. Martinez, Tetrahedron Lett. 42, 5389 (2001).

1. 20% piperidine, DMF

2. 4-fluoro-3-nitrobenzoic acidDIC, HOBt, DMF

NH3 (aq), DIEA/DMF SnCl2.2H2O, NMP.

a -bromoketones

DMF, 60 �C

H2N

O

N

NH2N

O

N

N

+

20% TFA/CH2Cl2

60 �C, 5 h

RINK NH

O

F

NO2

RINK NH

O

NH2

NO2

RINK NH

O

NH2

NH2 RINK

RINK NH2

RINK NH2 NH

OO

NH2

MeO

OMe

59

60

61

62 63

NH

O

N

N

Scheme 17. Solid-phase quinoxaline synthesis.


compounds lacking a fused aromatic nucleus are difficult to prepare. How-ever, it had been shown that the formation of cyclic compounds such as di-ketopiperazines was favored in the presence of a secondary amide adoptinga cis-configuration. Amblard and co-workers42 used a backbone amide lin-ker (BAL) as both the cis-configuration inductor and to anchor the growingcompound to the solid support. The Multipin method was used to rapidlyexplore a large range of reaction parameters in parallel experiments anddetermine the optimum reaction conditions. To rapidly optimize the reac-tion conditions for each step, a Phe-Ala spacer connected to a BAL linkermimic 65 was used to enable evaluation of each step by a single LCMS an-alysis. This useful technique has been described previously.6 The BAL lin-ker mimic 65, a carboxybenzaldehyde moiety, allowed reductive aminationbut was stable to acidic treatment. The best conversion to the free diazepin66 was 94%. The conditions optimized for 65 were directly transferred to

NH

OO

NH2

MeO

OMe

RINK NH2

65 66

H

O

Phe-Ala

O

RINK Phe-Ala

O

N

N

O

O

RINK

SpacerBAL linkermimic

NH

HN

O

O

64

Scheme 18. Solid-phase synthesis of a 1,4-diazepine-2,5-dione.


polystyrene Crowns derivatized with the BAL linker (28 �mol loading).The benzodiazepine 64 was obtained with 98% purity without further opti-mization. Although the synthesis of a single diazepinone was described,diversity on the diazepinone core could be introduced by use of variousamino acids and their �-homologs.

Carbohydrates

The synthesis of polymer supported oligosaccharides via n-pentenylglycosides by Fraser-Reid and co-workers43 was described in a previousreview.19 Solid-phase synthesis was used to simplify the synthesis of a libraryof 14 oligosaccharide-conjugated enediynes 69 (Scheme 19).44 Takahashiand co-workers44 (Tokyo Institute of Technology) synthesized thesecompounds to study sequence-selective cleavage of DNA. The putative

43 R. Rodebaugh, S. Joshi, B. Fraser-Reid, and H. M. Geysen, J. Org. Chem. 62, 5660 (1997).44 A. Matsuda, T. Doi, H. Tanaka, and T. Takahashi, Synlett, 1101 (2001).

S O

OHO O

O

Si

Et

EtO

OO

O

NH

(OR)n

A

DNA recognition site

B

DNA cleaving site

Linear or branchedOligosaccharide

S O

OHO O

O

OO

HO

(OR)n

NH

SiO

OSH

O

Et

Et

O

(OR)n

67

68

69

Scheme 19. Solid-phase synthesis of oligosaccharide-conjugated enediynes as putative

DNA-binding and cleaving agents.


DNA-binding systems 69 consist of an oligosaccharide (A) selected torecognize a DNA sequence and a labile nine-membered enediyne moiety(B) that can generate a reactive diradical. These compounds were foundto be difficult to handle in a previous solution phase synthesis due to thepresence of both the water-soluble sugar (A) and the hydrophobic ene-diyne moiety (B). The oligosaccharide-trialkylsilyl linker system was pre-pared by solution phase synthesis and loaded onto SynPhase polystyreneCrowns to give 67 (Scheme 19). As the Crowns were fitted with radiofre-quency transponders to allow unambiguous identification, the remaininglibrary synthesis could be performed in a single vial. The enediyne moietywas then conjugated to the support-bound oligosaccharides to give the con-jugate 68. The site isolation inherent in solid-phase synthesis preventedthe formation of dimeric by-products that are obtained in an analogoussolution-phase synthesis. As the assembly proceeded with high efficiency,

NH

OO

NH2

MeO

OMe

RINK NH2

OX

OMeBnO

BnOBnO

70

X−

O

O

O SO2

O

OMeBnO

BnOBnO

NH

( )9RINK

717273

X = N3

X = IX = OAc

Scheme 20. Nucleophilic cleavage of monosaccharides from the solid phase to introduce

C6 functionality.


high-purity target compounds 69 were obtained following washing of thesolid-phase and subsequent cleavage with AcOH/THF/H2O. In contrast,the purification of the target compounds prepared via solution phaseconjugation was difficult.

Takahashi et al.45 used a 4-hydroxybenzenesulfonate linker on Syn-Phase polystyrene Crowns to prepare glucose derivatives and macrocycles(Scheme 20). Displacement of the monosaccharide-supported Crowns 70with nucleophiles such as azide, iodide, and acetate gave the respective 6-substituted glucose derivatives 71, 72, and 73 in excellent purities andyields. The authors suggest that this methodology could be utilized forthe preparation of oligosaccharide libraries.

A similar method was used for the synthesis of a cyclic cyanohydrinether 75. The cyclization took place by the intramolecular displacementof the polymer-supported cyanohydrin 74 by treatment with lithium hexa-methyldisilazide (Scheme 21). Target 75 was obtained in 46% yield. Whilethe yield is moderate, a pure product was obtained after a simple workupprocedure. The cyanohydrin ether 75 can be readily converted to a cyclicketone.46

45 T. Takahashi, S. Tomida, H. Inoue, and T. Doi, Synlett, 1261 (1998).46 T. Takahashi, H. Nemoto, and J. Tsuji, Tetrahedron Lett. 24, 2005 (1983).

74

O

EtO

CN

75

LiN(SiMe3)2

O2S O

O

O

EtO

CN

TRT

TRT OH OHNH

O

Scheme 21. Macrocyclization on the SynPhase surface.


Functionalized Peptide Libraries

Dragovich et al.47 from Agouron Pharmaceuticals synthesized a seriesof irreversible human rhinovirus 3C protease (HRV 3CP) inhibitors 77.The inhibitors 77 contain a tripeptide-binding domain that provides affinityfor the target protein and a Michael acceptor that irreversibly forms acovalent adduct with the active cysteine residue of the 3C enzyme. TheN-terminal amides 77 were prepared by coupling of the tripeptide 76 witha variety of carboxylic acids and acid chlorides (Scheme 22). Approxi-mately 500 unique compounds were prepared and their affinity againstHRV-14 3CP determined with a high-throughput assay. Eight of the mostactive compounds were resynthesized by solution-phase techniques for amore accurate determination of their binding constants. The most potentreported compound gave an EC50 value of 250 nM.

Liu et al.48 (Academy of Military Medical Science, Beijing) developeda method for the combinatorial synthesis of muramyl dipeptide derivatives79 (Scheme 23). Two important building blocks, protected muramic acidand Fmoc-d-isoGln, were prepared by solution-phase synthesis. The pep-tide 78 was then prepared on MD grafted I-series Crowns using standardcoupling and Fmoc deprotection methodologies. The peptides werecoupled to acids and cleaved from the solid support to give a library of60 muramyl dipeptide derivatives 79. All compounds in the library werereported to have purities greater than 75%.

47 S. Dragovich, R. Zhou, D. J. Skalitzky, S. A. Fuhrman, A. K. Patick, C. E. Ford, J. W.

Meador, III, and S. T. Worland, Bioorg. Med. Chem. 7, 589 (1999).48 G. Liu, S.-D. Zhang, S.-Q. Xia, and Z.-K. Ding, Bioorg. Med. Chem. Lett. 10, 1361 (2000).

NH

OO

NH2

MeO

OMe

RINK NH2

P4

NH

NH

NH

O

O

O

CO2Et

NH2O

76 77

RINK

FmocHNNH

NH

O

O

CO2Et

NHO

Scheme 22. Synthesis of human rhinovirus 3C protease inhibitors.


Cyclic Peptides

Lambert and co-workers49 (University of Melbourne) synthesized a li-brary of cyclic thioether peptides with a pendant 9-aminoacridine moiety asa DNA-binding agent 81. Diversity in the library was achieved by assem-bling every permutation of four amino acid residues within the cyclic pep-tide (Scheme 24). The linear peptides 80 were synthesized in parallel withstandard Fmoc chemistry on SynPhase Crowns functionalized with a Rinklinker. The acridine moiety was incorporated onto the C-terminal lysineside chain using 9-phenoxyacridine. Cysteine deprotection and peptide cy-clization also took place under the acidic conditions used for the cleavageof 80 from the solid support. The library of cyclic thioether peptides 81 wasobtained in high yields and purity (11 of 12 members had purities >95%).

Lambert and co-workers50 also synthesized a pair of cyclic octapeptides82 and 83 and studied their propensity to form nanotubular aggregates(Fig. 5). A linear peptide was initially synthesized on SynPhase Crownswith a Rink amide handle by standard Fmoc chemistry. The first residueused was aspartic acid protected as the �-allyl ester. The support-boundlinear peptide was cyclized in a head-to-tail manner by deprotection of

49 K. D. Roberts, J. N. Lambert, N. J. Ede, and A. M. Bray, Tetrahedron Lett. 39, 8357 (1998).50 M. E. Polaskova, N. J. Ede, and J. N. Lambert, Aust. J. Chem. 51, 535 (1998).

NH2

O

NHAc

OCH2Ph

O

O

NH

O NH

NH2

O

NH

O

O

NHCOR

OH

OH

78

79

RINKNH

O

NHAc

OCH2PhO

OPh

O

O

NH

O NH

NH2

O

NH

O

O

NHDde

NH

OO

NH2

MeO

OMe

RINK NH2

Scheme 23. Synthesis of muramyl dipeptide derivatives.


the terminal and aspartic acid residues followed by activation with HATU/HOAt/DIEA. Acidification of aqueous solutions of 82 initiated forma-tion of needle-like crystals. The morphology and infrared absorption char-acteristics of these crystals suggested that they were hydrogen-bondednanotubular aggregates.

Ellman and co-workers51,52 synthesized a library of 1152 �-turn mi-metics 84 (Scheme 25) and screened these compounds for activity againstthe N-formyl-Mer-Leu-Phe (fMLF) receptor. Antagonists to the fMLF re-ceptor could potentially be used as therapeutic agents for the treatment ofinflammatory and infectious diseases. The �-turn mimetics 84 consisted of9- and 10-member rings with an aminoalkylthiol serving as the constrainingbackbone. A �-bromo acid and an Fmoc-protected �-amino acid provided

51 A. A. Virgilio, A. M. Bray, W. Zhang, L. Trinh, M. Snyder, M. M. Morrissey, and J. A.

Ellman, Tetrahedron 53, 6635 (1997).52 A. Virgilio and J. A. Ellman, J. Am. Chem. Soc. 116, 11580 (1994).

80

81

AA1, AA2, AA3 and AA4 = permutations of 2 × Asp, Ile and Lys

NH

OO

NH2

MeO

OMe

RINK NH2

RINK

HN N

NH

CO NH

CO-NH

Mmt-S

Br-CH2CO-AA1-AA2-AA3-AA4

NH RINKCO-AA1-AA2-AA3-AA4

CH2

HN N

NH

CO NH

CO-

S

Scheme 24. Synthesis of acridinyl cyclic peptides as potential DNA-binding agents.


the functionalized i þ 1 and i þ 2 side chains, respectively. The synthesis of11 of the mimetics 84 was optimized on Rink amide-derivatized Rapp Ten-tagel. The complete library, comprising all combinations of two backbonecomponents, 32 �-amino acids and 18 �-bromo acids, was synthesized onMD SynPhase Crowns using the Multipin methodology. The reaction se-quence optimized on Tentagel resin performed equally well on the Syn-Phase Crowns. The mimetics on each Crown were synthesized with amixture of the two aminoalkylthiol backbone components to give boththe 9- and 10-membered rings systems.

The library of mimetics 84 was screened in a radioligand-bindingassay against a cloned fMLF receptor. Four compounds correspondingto the contents from the two most active wells were prepared on a largerscale and purified. In both instances, the inhibitory activity was due

NH

NH

NH

NH

NH

HN

NH

NH

O

O

O

OO

O

O

O

H2N H2N

NH2 NH2

O

O

OHO

O

OH

82

N

NH

N

NH

N

HN

N

NH

O

O

O

OO

O

O

OMe

Me

MeMe

O

O

OHO

O

OH

Me

Me

Me

Me

83

Fig. 5. Cyclic peptides with the potential to form nanotubular aggregates.


predominantly to one of the two compounds from each well. The IC50

values of compounds 85 and 86 were 10 and 13 �M, respectively (Fig. 6).Basso and Ernst53 (University of Basel) synthesized a library of cyclic

hexapeptides for screening as potential Selectin antagonists. Details ofthe screening results were not given. The peptides were synthesized onSynPhase Lanterns that were derivatized with a tetrahydropyranyl linker.As outlined in Scheme 26, a series of preformed hydroxyproline dipeptides87 was coupled onto the solid phase via the hydroproline side chain to give88. The linear peptide systems 89 were assembled on the solid phase usingstandard Fmoc chemistry. Head-to-tail cyclization of the peptides was per-formed on the solid phase using PyBOP/HOAt/DIEA prior to cleavage.The cyclic peptides 90 were obtained in 42–82% purity. The authorsreported that the desired cyclic peptide was the major product in all cases.

Other Small Molecules

Amines

The solution method for the synthesis of polyamines of Bergeron wasadapted to the solid-phase by Uriac and co-workers54 as outlined inScheme 27. Polyamines are of biological interest due to their essential role

53 A. Basso and B. Ernst, Tetrahedron Lett. 42, 6687 (2001).54 S. Tomasi, M. Le Roch, J. Renault, J. C. Corbel, and P. Uriac, Pharm. Pharmacol.

Commun. 6, 155 (2000).

NH

OO

NH2

MeO

OMe

RINK NH2

NSS-t-Bu

HNO

O

ONHFmoc

Ri+2

NH

( )n

RINK

HATUDIEAOH

NHFmoc

Ri+2

O

DIC

HOBr

O

H2N

ONH

RINK

Br

HNO

ONH

RINK

NH

SS-t-Bu

HNO

ONH

( )n

RINK

H2N SS-tBu( )n

S

NH

N

HN

NH2

O

O

O

ORi+2

Ri+1

( )n1. Bu3P, H2O2. TMG

3. TFA/Me2S/H2O (18:1:1)

1. 20% piperidine

2. DIC (0.5eq)

HOBr

Ri+1

O

NSS-t-Bu

HNO

O

ONH

Ri+1

Br

ORi+2

NH

( )n

RINK

84 n = 1, 2

Scheme 25. Solid-phase synthesis of macrocyclic �-turn mimetics.


in cell growth and the fact that some polyamine analogs are potent anti-cancer and antiparasitic agents. The classic solution-phase synthesis ofBergeron, which involves the N-alkylation of sulfonamides, is often compli-cated by the purification of the hydrophilic products. By performing thesereactions on the solid phase, Uriac’s group found that purification was sim-plified and hence the preparation of libraries of these compounds becomesviable.

S

NH

N

HN

NH2

O

O

O

O

OH

N

NH

S

O

O

NH

NH

NH2

O

O

85 86

Fig. 6. Macrocyclic �-turn mimetics.

X1 = L-Asp, L-Ser, L-LysX2 = L-Glu, D-GluX3 = L-Lys, D-Lys

NH NH

X2 X1X3

N

O

O

HO

O

90 89

N

OX1

OX2X3

NH

O

O

OO

88

N

OX1

O

NH

OO

OO

87

N

O X1

O

OH

Fmoc

Fmoc-Gly-Phe

Fmoc

Scheme 26. Solid-phase synthesis of cyclic peptides using hydroxyproline as a linking site.


HMP OH NH

OO

OH

H2N NN

O

OSO2Mes· CF3CO2H

91

H2N NNH2

SO2Mes· 2CF3CO2H

92

O O

O

NO2HMP

O NH

NN

O

OSO2MesO

HMP

Scheme 27. Solid-phase synthesis of polyamines.


Synthesis of the orthogonally diprotected homospermidine 91 and themonoprotected homospermidine 92 was performed on SynPhase Crownswith a carbonate handle. The optimization of the reactions was aided byrapid handling of Crowns and performing reactions in parallel.

Handlon and Hyman55 from Glaxo Wellcome prepared three librariestotaling 2880 members based around the diphenylmethylamine scaffolds93, 94, and 95 for use in screening against 7-transmembrane domain recep-tors. The key step of each of these libraries involved the reductive amina-tion of benzophenones (Scheme 28). The first library (scaffold 93) wasprepared on BAL linker Crowns fitted with RF encoding tags.17 The keyreductive amination of the solid supported benzophenone intermediates

55 A. L. Handlon and C. E. Hyman, Design and synthesis of a 7TM targeted library:

Titanium(IV)-chloride-mediated reductive amination of benzophenones on solid support.

Poster presented at 219th ACS National Meeting, San Francisco, CA, March 2000.

X

YNH

M3

ONH

O

M1

z

93

1. M3NH2, TiCl4CH2Cl2

2. NaCNBH3/MeOH3. 95% TFA/H2O

M1NH2

NaBH(OAc)3

DMF/AcOH

HOBr

O

DIC/DMF

OH X

YO

Ar

Cs2CO3

BrN

O

M1

BALNHM1

BALBALH

O

X

Y

ON

O

M1

O

z

BAL

BALH

ONH

O

O OMe

O

H

OMe

Scheme 28. Synthesis of a diphenylmethylamine library targeted to 7-transmembrane

domain receptors.


(40 examples) was performed in two steps by treatment with an amine (40examples) in the presence of titanium(IV) chloride followed by sodium cy-anoborohydride. It was found that this reductive amination step did nottake place under standard conditions (amine, catalytic acetic acid, reducingagent) or when boron trifluoride or TiClx(OiPr)y was used in place of tita-nium(IV) chloride. The use of RF encoding tags allowed the pooling ofCrowns into 40 reaction vessels (one vessel for each amine). To performthis reaction in 96-well plates would have required pipetting titanium(IV)chloride into 1600 reaction wells. The two remaining libraries (scaffolds 94and 95) were prepared on Rink linker-derivatized Crowns. Two librarieswere generated after the reductive amination step by either direct cleavage(scaffold 94) or oxidation of the sulfide to a sulfone with hydrogen peroxidefollowed by cleavage (scaffold 95) (Fig. 7).

Ureas and Their Chalogen Analogs

Series of ureas 96, thioureas 97, carbamates 98, and semicarbazides 99were prepared on resin or SynPhase Lanterns by Phoon and Sim56 fromSingapore’s Institute of Molecular and Cell Biology. These derivatives

56 C. W. Phoon and M. M. Sim, Synlett, 697 (2001).

94 95

X

YNH

M3

SM1H2N

O

Z

X

YNH

M3

SM1H2N

OO

O

Z

Fig. 7. Diphenylmethylamine scaffolds.


were prepared by the attachment of amines or hydrazines onto bromo-Wang resin followed by treatment with isocyanates, isothiocyanates, orchloroformates (Scheme 29). Ureas 96 were obtained in excellent yieldsand purity. Thioureas 97 were obtained in lower purity due to the presenceof hydroxybenzylated side products, carbamates 98 in good yields and pur-ities, and semicarbazides 99 in good yields but modest purities. A selectionof the derivatives was also prepared on SynPhase Lanterns. In comparisonto resin, the products from the Lanterns were obtained in higher puritythough in reduced overall yields. It should be noted that the reactions onLanterns employed shorter reaction times than those on resins.

Future Perspectives

Solid-phase organic synthesis is now a well-established approach togenerating lead finding and lead optimization libraries. Golbiowski et al.9

recently published a review of lead compounds discovered using combina-torial libraries. Many of the examples were generated using solid-phasesynthesis. As illustrated throughout this chapter, solid-phase organic syn-thesis on SynPhase Crowns and SynPhase Lanterns has been adopted bymany laboratories for the synthesis of libraries of small molecule com-pounds. The authors are aware of many other interesting syntheses thatmay never be published, which are focused on discovering new lead com-pounds. The main emphasis of this chapter has been the preparation ofcompounds for screening in the drug discovery process. It should be notedthat the applications of combinatorial solid-phase synthesis extend beyondthis one field. For example, Gilbertson and co-workers57,58 have made use

S Br Bromo-Wang polystyrene resin orBromo-Wang SynPhase Lantern

S Br

S NNH2

R2

S NHR1

S N

R1

NH

X

R3

S N

R1

O

O

R4

S NNH

R2

NH

O

R3

NH

NH

X

R3R1

NH

O

O

R4R1

R2HNNH

NH

O

R3

98

99

9697

X = OX = S

Scheme 29. General solid-phase route to ureas, thioureas, carbamates, and semicarbazides.


of SynPhase Crowns in the development of new support-bound ligands forasymmetric synthesis. The use of the solid phase to ‘‘scavenge’’ reagentsand by-products in parallel solution-phase synthesis is a growing field ofinterest to the practitioner of combinatorial chemistry.59 It should be notedthat suitably functionalized SynPhase Lanterns could be used to removeexcess reagents from multiple parallel solution-phase reactions. This is a lo-gical extension of the technology, which is now available.

Although polystyrene is a useful solid support for a broad range ofchemistries, it is not ideally suited to syntheses that require the use of aque-ous solvents. To expand the application of SynPhase Lanterns in solid-phase synthesis, Mimotopes has developed a new hydrophilic polyamidesurface.20 One of the goals of manufacturers of polymeric supports forsolid-phase synthesis is to generate surfaces that allow reaction ratesand reaction conversions on the solid phase to approach those obtainedin solution-phase synthesis. In solid-phase synthesis, reactions are generallydriven to completion by using excess reagent, where this is possible. Des-pite the growing use of solid-phase synthesis in the drug discovery process,there is strong need for more thorough physical chemistry investigations ofprocess on any solid phase, including the SynPhase surfaces, with onlylimited studies being reported so far. Gerritz22 has undertaken a compari-son on the efficiency of small molecule solid-phase synthesis on a range of

57 S. R. Gilbertson and X. Wang, Tetrahedron 55, 11609 (1999).58 S. R. Gilbertson, S. E. Collibee, and A. Agarkov, J. Am. Chem. Soc. 122, 6522 (2000).59 S. V. Ley and I. R. Baxendale, Nat. Rev. Drug Discov. 1, 573 (2002).

NH

NH

O

O

Si

NH

O

O O

ONHBoc

NH

HN NH

OHO

H

100

Scheme 30. Solid-phase synthesis of 3,9-diazabicyclo[3.3.1]non-6-en-2-one scaffold.


solid phases and found that SynPhase Lanterns gave very favorable results.In a separate study of interest, Gerritz et al.60 reported the high-throughputdetermination of Hammett relationships for the displacement of a solidsupported active ester with a range of anilines on a range of solid supports.It is of interest to note that the observed Hammett reaction constants weredependent on the solid phase.

Addendum

Several more publications describing the use of SynPhase Lanterns insolid-phase organic synthesis have recently appeared. Fukase and co-workers61 described the solid-phase synthesis of oligosaccharides using anovel alkyne-based linker. The Sonogashira reaction was used in loadingthe substrate onto the solid-phase. Workers at Novartis described thesolid-phase synthesis of the bicyclic compound 100 via sequential Dakin-West and Pectit-Spengler reactions (Scheme 30).62 Ede63 recently pub-lished a history of the development of the SynPhase grafted supports withan emphasis on peptide synthesis. Gerritz et al.64 have summarized severalyears of library production work undertaken at GlaxoSmithKline.

60 S. W. Gerritz, R. P. Trump, and W. J. Zuercher, J. Am. Chem. Soc. 122, 6357 (2000).61 M. Izumi, K. Fukase, and S. Kusumoto, Synlett, 1409 (2002).62 D. Orain, R. Canova, M. Dattilo, E. Kloeppner, R. Denay, G. Koch, and R. Giger, Synlett,

1443 (2002).63 N. J. Ede, J. Immunol. Methods 267, 3 (2002).64 S. W. Gerritz, M. H. Norman, L. A. Barger, J. Berman, E. C. Bidham, M. J. Bishop, D. H.

Drewry, D. T. Garrison, D. Heyer, S. J. Hodson, J. A. Kakel, J. A. Linn, B. E. Marron, S. S.

Nanthakumar, and F. J. Navas, III, J. Comb. Chem. 5, 110 (2003).

[4] directed sorting approach for large libraries 75

[4] Directed Sorting Approach for the Synthesis of LargeCombinatorial Libraries of Discrete Compounds

By Timothy F. Herpin and George C. Morton

Introduction

One of the very first steps in the drug discovery process is to find an ini-tial molecule—a hit or a lead—eliciting a desired biological activity. Overthe years, many approaches have been used to identify a lead molecule, allof them with some degree of success. The testing of natural product ex-tracts either directly in vivo or in vitro has been a successful source ofleads1 in many examples. The rational design of lead compounds basedon the knowledge of the natural ligand, the structure of the receptor, orthe mechanism of action of the receptor has also been successfully used.2

Recently, methods that screen for low-affinity molecular fragments to besubsequently combined into a lead molecule have been reported to suc-cessfully provide lead structures.3 However, the approach that is still con-sidered the most promising is the high-throughput in vitro screening ofsynthetic compounds and combinatorial libraries.4 It is the most generalapproach—it can be applied to most types of targets—and when successful,it allows the drug discovery process to start with an easy-to-synthesize smallmolecule.

With the rapid progress of high-throughput screening there has beena growing need for large collections of compounds. In the early 1990s,two main technologies emerged for the rapid synthesis of compound librar-ies. In 1991, Furka et al.5 described the ‘‘split-and-pool’’ concept for the

1 (a) J. Singh, G. D. Bagchi, A. Singh, and S. Kumar, J. Med. Aromatic Plant Sci. 22/4A-23/1A, 554 (2001). (b) J. Josephson, Mod. Drug Discov. 3, 45 (2000). (c) D. G. I. Kingston,

Pract. Med. Chem. 101 (1996).2 (a) D. L. Kirkpatrick, S. Watson, and S. Ulhaq, Comb. Chem. High Throughput Screening 2,

211 (1999). (b) M. Iino, T. Furugori, T. Mori, S. Moriyama, A. Fukuzawa, and T. Shibano,

J. Med. Chem. 45, 2150 (2002). (c) S. Flohr, M. Kurz, E. Kostenis, A. Brkovich, A. Fournier,

and T. Klabunde, J. Med. Chem. 45, 1799 (2002). (d) G. S. Chen, C. S. Chang, W. M. Kan,

C. L. Chang, K. C. Wang, and J. W. Chern, J. Med. Chem. 44, 3759 (2001).3 (a) J. Fejzo, C. A. Lepre, J. W. Peng, G. W. Bemis, Ajay, and M. A. Murcko, Chem. Biol. 6,

755 (1999). (b) P. J. Hajduk, A. Gomtsyan, S. Didomenico, M. Cowart, E. K. Bayburt,

L. Solomon, J. Severin, R. Smith, K. Walter, T. F. Holxman, A. Stewart, S. McGaraughty,

M. F. Jarvis, E. A. Kowaluk, and S. W. Fesik, J. Med. Chem. 43, 4781 (2000). (c) D. A.

Erlanson, A. C. Braisted, D. R. Raphael, M. Randal, R. M. Stroud, E. M. Gordon, and J. A.

Wells, Proc. Natl. Acad. Sci. USA 97, 9367 (2000).4 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273 (2001).




generation of large peptide libraries. In 1993, workers at Parke-Davis de-scribed the parallel synthesis of small molecules on solid support using aparallel synthesizer.6 For a few years, each technology evolved separately.The split-and-pool approach allowed very large numbers of compounds(100,000s) to be generated but had complications for the screening andthe identification of active compounds. Some techniques were invented toimprove the identification of active compounds such as tagging methodsand fast deconvolution techniques.7 Although solid-phase parallel synthe-sis offered discrete compounds that were convenient to test, the throughputwas limited to a few hundred compounds. When directed sorting was intro-duced in 1995, it allowed the gap between these two technologies to bebridged.8 The new approach allowed the preparation of combinatorial lib-raries with the same efficiency as the split-and-pool method, but provideddiscrete and identified compounds like parallel synthesis. This new methodcan be used to routinely prepare libraries of several thousand compounds,and it has been quickly adopted by the pharmaceutical industry as one ofthe techniques of choice. This chapter will review the principle of directedsorting, and it will also present two detailed examples of its application indrug discovery: (1) the synthesis of piperazine-2-carboxamide derivatives,and (2) the synthesis of benzothiazepine derivatives.

Directed Sorting Approach

Principle

The directed sorting approach (or mix-and-sort approach) uses smallsynthetic objects, usually called microreactors, which are equipped with areadable encoding system. Typically the synthetic objects can be resin

5 (a) A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37, 487

(1991). (b) A. Furka, L. K. Hamaker, and M. L. Peterson, in ‘‘Combinatorial Chemistry’’

(H. Fenniri, ed.), p. 1. Oxford University Press, Oxford, UK, 2000. (c) A. Furka, Comb.

Chem. High Throughput Screening 3, 197 (2000).6 (a) S. H. DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. R. Cody, and M. R.

Pavia, Proc. Natl. Acad. Sci. USA 90, 6909 (1993). (b) S. H. DeWitt, A. W. Czarnik, in

‘‘Combinatorial Chemistry Synthesis and Application’’ (S. R. Wilson and A. W. Czarnik,

eds.), p. 25. John Wiley & Sons, New York, 1997.7 (a) K. S. Lam, M. Lebl, and V. Krchnak, Chem. Rev. 97, 411 (1997). (b) A. W. Czarnik,

Curr. Opin. Chem. Biol. 1, 60 (1997). (c) J. J. Baldwin, Mol. Diversity 2, 81 (1996). (d) Z.-J.

Ni, D. Maclean, C. P. Holmes, and M. A. Gallop, Methods Enzymol. 267, 261 (1996).8 (a) K. C. Nicolaou, X.-Y. Xiao, Z. Parandoosh, A. Senyei, and M. P. Nova, Angew. Chem.

Int. Ed. Engl. 34, 2289 (1995). (b) X.-Y. Xiao and M. P. Nova, in ‘‘Combinatorial Chemistry

Synthesis and Application’’ (S. R. Wilson and A. W. Czarnik, eds.), p. 135. John Wiley &

Sons, New York, 1997.


beads placed in a semi-porous container (MicroKans), a grafted polymersurface, or large plugs of resin beads.9 The coding system can be a radiofre-quency tag (RF-Tag), a two-dimensional (2D) optical bar code, a colorscheme, or a spatial array.

An example of how the directed sorting method works is described inFig. 1. Imagine a synthesis involving three chemical steps to construct mol-ecules with a common central scaffold incorporating three substituents. Ateach step three possible reagents can be used. A total of 27 (3 � 3 � 3) dif-ferent compounds can be made through the combination of all of the re-agents. At the beginning of the synthesis, the synthetic objects are taggedwith unique identifiers for the 27 compounds that can be made. The tagsare read in the first sorting step, and the synthetic objects are sortedaccording to which first reagent they should be reacted with. They are thenreacted in batch for the first chemical step. The synthetic objects arethen pooled, and sorted again, this time for the second reagent. After a re-action in batch, the process is repeated for the third combinatorial step. Atthe end of the synthesis, all 27 compounds have been prepared and the tagsencode their identity. The compounds can then be released from the syn-thetic objects and collected in vials or a multiwell plate. The end result ofthe process affords 27 discrete compounds prepared through 9 chemicalreactions and 27 cleavage steps.

Equipment

Several types of equipment can be used to prepare combinatorial lib-aries through the directed sorting technique. Irori9b has two types ofsystems available, one based on the use of RF tags (Accutag9c system)and another one based on the use of 2D optical tags (Nanokan system).The Accutag system is well suited to prepare libraries of 10–10,000 com-pounds, whereas the Nanokan system is useful for 10,000–100,000 memberlibraries. Both systems are composed of synthetic objects (MicroKans, Na-nokans), tags (RF, 2D optical), an automated sorter, software to track thesynthesis (Synthesis Manager), as well as a cleavage system. An alternativecleavage system is available from Bohdan.10 For small libraries, the use ofcolor-coded MicroKans has been reported.11

9 (a) B. Atrash, M. Bradley, R. Kobylecki, D. Cowell, and J. Reader, Angew. Chem. Int. Ed.

Engl. 40, 938 (2001). (b) http://www.irori.com. (c) MicroKan, MacroKan, NanoKan,

Accutag, Accusort are trademarks of Irori.10 C. J. Andres, R. T. Swann, K. Grant-Young, S. V. D’Andrea, and M. S. Deshpande, Comb.

Chem. High Throughput Screening 2, 29 (1999).11 J. W. Guiles, C. L. Lanter, and R. A. Rivero, Angew. Chem. Int. Ed. Engl. 37, 926 (1998).

ABC ACA ACB ACC BAA

AAA AAB AAC ABA ABB

BCA BCB BCC CAA CAB

BAB BAC BBA BBB BBC

CCB CCC

CAC CBA CBB CCA

ABCACA

ACB

ACC BAA

AAA

AAB

AAC

ABA

ABB BCA BCB BCC CAA CAB

BAB BAC BBA

BBB BBC

CCB CCC

CAC CBA CBB

CBC CCA

Reagent A Reagent B Reagent C

CBC

ABC

ACA ACB ACC

BAA

AAA

AAB

AAC

ABA

ABB BCA BCB BCCCAA CAB

BAB BAC BBA

BBB BBC CCB

CCCCAC CBA CBB

CBC

CCA


ABCACA

ACB

ACC

BAA

AAA

AAB

ABA ABB

BCA

BCB

BCCCAA CAB

BABBBA

BBB BBC

CCB CCC

CBA

CBB

CBCCCA


AAC

BAC

CAC

ABC ACA ACB ACC BAA

AAA AAB AAC ABA ABB

BCA BCB BCC CAA CAB

BAB BAC BBA BBB BBC

CCB CCC

CAC CBA CBB

CBC

CCA

Fig. 1. Example of directed sorting method.



A system based on grafted polymer synthetic objects (lanterns) is avail-able from Mimotopes Pyt. Ltd.12 The system is composed of the syntheticobjects (lanterns), RF-Tags (Transtems), software to track the synthesis(TranSort), and a cleavage system. Additional systems that are not com-mercially available have been reported. Workers at Aventis have de-veloped a highly automated system based on the Mimotope lanterns.13

Recently workers at Millenium Pharmaceuticals reported the StAC system(Stratified Adressable System), a three-dimensional spatial array thatallows discrete compounds to be synthesized using the same efficiency asthe directed sorting approach.14

Application

The directed sorting approach has been widely applied both in industryand in academia. Two examples are detailed in this chapter. Some other re-cently published examples include a benzopyran library synthesized usingthe Nanokan system,15 libraries derived from phenolic amino acid scaffoldsprepared with the Accutag system,16 and libraries of 1,5-benzodiazepine-2-one derivatives.17

Piperazine Carboxamide Libraries

The directed sorting method will first be illustrated by the preparationof libraries of piperazine-2-carboxamide derivatives.18 The piperazine-2-carboxylic acid scaffold is a pharmacologically important19 center core

12 http://www.mimotopes.com/.13 J. A. Connelly, New automation technology for the efficient production of large

combinatorial libraries of small molecules by ‘‘directed sort and combine’’ methods.

Abstracts of Papers, American Chemical Society 221st BTEC-041 (2001).14 (a) J. C. Reader, C. D. Boden, D. Cowell, C. M. Grant, M. Jones, V. A. Reader, C. C.

Renou, and M. Stirling, Multi-dimensional parallel solid-phase synthesis of libraries using

StAC technology. Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, August

26–30, 2001. (b) V. A. Reader, C. J. Boden, D. Cowell, C. M. Grant, M. Jones, J. C. Reader,

C. C. Renou, and M. Stirling, Library synthesis using StAC technology: An efficient and

inexpensive way of making compounds with 3 points of diversity or more on solid support.

Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, August 26–30, 2001.15 K. C. Nicolaou, J. A. Pfefferkorn, H. J. Mitchell, A. J. Roecker, S. Barluenga, G.-Q. Cao,

R. L. Affleck, and J. E. Lillig, J. Am. Chem. Soc. 122, 9954 (2000).16 A. D. Morley, Tetrahedron Lett. 41, 7405 (2000).17 T. F. Herpin, K. G. Van Kirk, J. M. Salvino, S. T. Yu, and R. F. Labaudiniere, J. Comb.

Chem. 2, 513 (2000).18 Work originally published in T. F. Herpin, G. C. Morton, A. K. Dunn, C. Fillon, P. R.

Menard, S. Y. Tang, J. M. Salvino, and R. F. Labaudiniere, Mol. Diversity 4, 221 (2000).19 (a) J. P. Vacca, B. D. Dorsey, W. A. Schleif, R. B. Levin, S. L. McDaniel, P. L. Darke,

J. Zugay, J. C. Quintero, and O. M. Blahy, Proc. Natl. Acad. Sci. USA 91, 4096 (1994).

N

O

NN

HOO

O

N N

O

N

N O

N

O

O

N

NH

N

N N

NH

O

ONH

HO

HO

Ph

1 2

3

Fig. 2. Examples of piperazine-2-carboxamide derivatives pharmacologically active.


found in angiotensin II antagonist20 1, substance P antagonist21 2, and theaspartyl protease inhibitor indinavir 322 (Fig. 2). Libraries built around thiscore scaffold are expected to be of general interest for high-throughputscreening campaigns.

The piperazine-2-carboxylic acid scaffold 4 is well suited for a combina-torial approach as it is a small, constrained structure with three functionalgroups (one carboxylic acid and two amines) that may be conveniently sub-stituted by solid-phase chemistry. Orthogonal protection of the two aminogroups could easily be carried out on a large scale by solution-phasechemistry18 (Scheme 1).

(b) E. Mishani, C. S. Dence, T. J. McCarthy, and M. J. Welch, Tetrahedron Lett. 37, 319

(1996). (c) I. A. Cliffe, C. I. Brightwell, A. Fletcher, E. A. Forster, H. L. Mansell, Y. Reilly,

C. Routledge, and A. C. White, J. Med. Chem. 36, 1509 (1993). (d) B. M. Kim, B. E. Evans,

K. F. Gilbert, C. M. Hanifin, J. P. Vacca, S. R. Michelson, P. L. Darke, J. A. Zugay, E. A.

Emini, W. Schleif, J. H. Lin, I.-W. Chen, K. Vastag, P. S. Anderson, and J. R. Huff, Bioorg.

Med. Chem. Lett. 5, 2707 (1995).20 (a) M. T. Wu, T. J. Ikeler, W. T. Ashton, R. S. L. Chang, V. J. Lotti, and W. J. Greenlee,

Bioorg. Med. Chem. Lett. 3, 2023 (1993). (b) W. T. Ashton, W. J. Greenlee, M. T. Wu, C. P.

Dorn, M. MacCoss, and S. G. Mills, World Patent WO 9220661 (1992).21 S. G. Mills, R. J. Budhu, C. P. Dorn, W. J. Greenlee, M. Maccoss, and M. T. Wu, World

Patent WO 9413646 (1994).22 (a) K. Rossen, S. A. Weissman, J. Sager, R. A. Reamer, D. Askin, R. P. Volante, and P. J.

Reider, Tetrahedron Lett. 36, 6419 (1995). (b) D. S. Stein, D. G. Fish, J. A. Bilello, S. L.

Preston, G. L. Martineau, and G. L. Drusano, AIDS 10, 485 (1996).

NH

CO2HNH

N

CO2HN

Boc

Alloc

N

CO2HNAlloc

Fmoc

a, b c, d

4 5 6

Scheme 1. Synthesis of the protected scaffold. (a) Boc-ON, dioxane:water (1:1), pH 11;

(b) Allyl chloroformate, pH 9.5; (c) 50% TFA-DCM; (d) Fmoc-Cl, Na2CO3, dioxane:water.


Two distinct libraries were synthesized and combined to produce 15,000discrete compounds. Both libraries were prepared with the Irori Accutagsystem using resin-filled MicroKans as synthetic objects. The first librarywas prepared according to the chemistry outlined in Scheme 2. Thus theMicroKans were equipped with an RF-tag and resin containing the di-methoxy-benzaldehyde linker 7 (BAL).23* The first combinatorial stepwas attachment of a primary amine by reductive amination. The Micro-Kans were then pooled and the resulting secondary amines were acylatedwith the orthogonally protected piperazine-2-carboxylic acid scaffold 6 ina single batch to yield 9. The fluorenylmethyloxycarbonyl (Fmoc) groupwas removed with piperidine, and the MicroKans were sorted for thesecond combinatorial step. The 4-amine was reacted with sulfonyl chlor-ides, isocyanates, chloroformates, and carboxylic acids. The MicroKanswere then pooled, the allyloxycarbonyl (Alloc) group was removed withPd(PPh3)4, and the MicroKans were sorted for the third combinatorialstep. The 1-amine was functionalized similarly to afford sulfonamides,ureas, carbamates, and amides. This chemistry was used to produce a10,000-member library. See Table I for a representative sample of thereagents used.

The second library built around the piperazine-2-carboxylic acid scaf-fold was designed to fill some of the diversity gaps left by the first library.

23 (a) K. J. Jensen, J. Alsina, M. F. Songster, J. Vagner, F. Albericio, and G. Barany, J. Am.

Chem. Soc. 120, 5441 (1998). (b) C. G. Boojamra, K. Burow, L. A. Thompson, and J. A.

Ellman, J. Org. Chem. 62, 1240 (1997).*Abbreviations: BAL, backbone amide linker; BSA, bis(trimethylsilyl)acetamide; DBU, 1,8-

diazabicyclo[5.4.0]undec-7-ene; DCE, dichloroethane; DCM, dichloromethane; DIC, 2-

diisopropylcarbodiimide; DIEA, diisopropylethyl amine; DMAP, N,N-dimethylaminopyr-

idine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDC, 1-(3-dimethylaminopro-

pyl)-3-ethylcarbodiimide hydrochloride; HBTU, [O-(7-azabenzotriazol-1-yl)-1,1,3,3-

tetramethyluronium hexafluorophosphate; MCPBA, m-chloroperoxybenzoic acid; NMP, N-

methylpyrrolidinone; NMM, N-methylmorpholine; PfP, pentafluorophenol; RT, room

temperature; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

OO

O

O

R1 NH2O

O

ONHR1

N

NO

O

ONR1

O Alloc

Fmoc

N

NO

O

ONR1

O

R2

Alloc

N

NO

O

ONR1

O

R2

R3f

N

NNH

R1O

R2

R3c, d

e, d

a b

7 8

9

10 1112

6

Scheme 2. A 10,000-member combinatorial library: (a) NaBH(OAc)3, DMF, AcOH; (b) HBTU, DIEA, DMF; (c) piperidine:DMF (1:1);

(d) RSO2Cl, NMM, DCM, or RNCO, DCM or ROCOCl, DIEA, DCM, or RCOOH, EDC, NMP; (e) Pd(PPh3)4, morpholine, THF,

DMSO, aq. HCl; (f) 50% TFA-DCM.

82

co

mb

in

ato

ria

lsy

nth

esis

[4]

TABLE I

Representative Sample of the Reagents Used in the 10,000-Member

Piperazine-2-carboxamide Library

R1

NH2 N NNH2 NH2

O

O

NH2 NHBocNH2N

O

NH2

R2

O

OCl O

O

Cl OH

O F

FFN

ON

O

NHBoc

OH

O

NOH

O N

NH

OH

ON

OH

OS

O

OCl

NHBocOH

O

O

O

R3

O

O

Cl OH

O OOH

O OH

OH

OS

O

O Cl

O

O

Cl O

CF3

CF3

S

OO

ClN

OON

H

O

OH

O

NHBoc

OH

ON

NH

N

NO


In the first library, all of the compounds contained the 2-secondary amidethat was the point of attachment to the resin. Also, none of the compoundscontained a basic tertiary amine in the piperazine ring.

The synthetic scheme for the second library is outlined in Scheme 3.The scaffold 17 was synthesized on a large scale in solution phase. Bifunc-tional reagents containing a handle (carboxylic acid or amine) and a halidewere loaded onto Wang resin. Bromo- or chlorocarboxylic acids werereacted with Wang resin using either the Yamaguchi method (2,6-dichloro-benzoyl chloride)24 or by conversion to the acid chloride. Alternatively,Fmoc amino acids were loaded onto Wang resin with diisopropylcarbodii-mide (DIC) – dimethylaminopyridine (DMAP). The Fmoc group wasremoved and the free amine was then acylated with bromo- or chlorocar-boxylic acids. Symmetric diamines were also loaded on the nitrophenolcarbonate derivative of Wang resin and acylated with bromo- or chlorocar-boxylic acids. The bromides or chlorides were converted in situ to the cor-responding iodide and then reacted with the unprotected amine of 17. Siteisolation on the resin ensured clean monoalkylation. The carboxylic acid

24 P. Sieber, Tetrahedron Lett. 28, 6147 (1987).

OO R

Br

NH

N

O Alloc

OH

N N

O

R Alloc

HO

O

R2 N

N N

O

R AllocO

R2 N

N N

O

RO Y R3

Y= CO, SO2, COO

R2 N

N N

O

R Y R3

Y= CO, SO2, COO

OOH

OO

O

O NO2

OO

O

RNHFmoc

a

b

c

d

e, f

gi

, h

16

17

18

192021

R2'R2'R2'

13

14

15

Scheme 3. A 5000-member library. (a) Br-R-COOH, dichlorobenzoyl chloride, pyridine, DCM, or Br-R-COCl; (b) (i) piperidine, DMF; (ii)

Br-R-COOH, DIC, HOBt, DMF; (c) (i) symmetrical diamine, DMF; (ii) Br-R-COOH, DIC, HOBt; (d) KI, DMF, 80C; (e) DIC, PfP, DMF;

(f) R2-NH-R20, DMF; (g) Pd(PPh3)4, morpholine, THF, DMSO, aq. HCl; (h) RSO2Cl, NMM, DCM, or ROCOCl, DIEA, DCM, or RCOOH,

EDC, NMP; (i) 50% TFA-DCM.

84

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[4]


was then converted to the pentafluorophenyl ester and reacted with aminesin a second combinatorial step. After the removal of the alloc group, thethird combinatorial step consisted of acylation, sulfonylation, and carba-mate formation, all of which were performed under the same conditionsas in the first library. Table II lists a representative sample of the reagentsused in the 5100-member library. A full matrix of 17 � 25 � 10 was con-structed for this library.

Both libraries were constructed as a combination of a small number ofreagents, therefore analysis of a small random portion of the library wasexpected to be representative of the overall purity. The quality of bothlibraries was assessed by analyzing 5% of the compounds by high-pressureliquid chromatography (HPLC) with a combination of mass spectrometry(MS) and evaporative light scattering (ELS) detection. The MS detectorwas used to confirm the identity of the compounds and purity was basedon ELS detection. Figure 3 shows the purity distribution for the two librar-ies. In both cases, the majority of the compounds were present in highpurity. For the 10,000-member library, a significant portion of the librarywas affected by an unexpected side reaction. Compounds containing

TABLE II

Representative Sample of the Reagents Used in the 5000-Member 4-Alkyl-piperazine-

2-carboxamide Combinatorial Librarya

R1

HOH

OBr

Br

HO2C N

OOH

OCl N

OBr

HN

O

OH NH

O Br

d a a b c b

R2

N

O

NH2

NH

N

NH2 NH2N N NNH2

SNH2

OO

NH2 O

O

NH2

O NH2NHN NHBoc

NH2

R3

HO

O

Cl OH

O

OH

O

Cl

ClS

O

O Cl NHBocOH

O

a Notes: (a) Loaded with the Yamaguchi method. (b) Fmoc-amino acid loaded with DIC/

DMAP, deprotected and reacted with bromo-acid. (c) Symmetric diamine loaded on the

nitrophenyl carbonate derivative of Wang resin and subsequently reacted with bromo-

acid. (d) Piperazine carboxamide scaffold reacted with the nitrophenyl carbonate

derivative of Wang resin.

0

20

40

60

80

100

120

140

160

180

10 40 70 100

Purity

Num

ber

of c

ompo

unds

ana

lyze

d

0

10

20

30

40

50

60

5 25 45 65 85Purity

Num

ber

of c

ompo

unds

ana

lyze

d

Purity range % of library Purity range % of library

76-100% 53% 76-100% 62%

51-75% 12% 51-75% 28%

26-50% 6% 26-50% 5%

0-25% 29% 0-25% 5%

A B

Fig. 3. Purity distribution for (A) the 10,000-member library and (B) the 5000-member

library.


aromatic ureas at the R3 position cyclized to form hydantoins in high purity(Scheme 4).25 In the 5000-member library, 95% of the expected com-pounds were detected and no significant side reaction was observed.

Combining Directed Sorting with Parallel Synthesis—BenzothiazepineLibrary

The directed sorting method has also been used to produce a largeamount of compound (up to 100 mg).26 In the following example, themethod was used to generate 100 mg of 3-aminobenzothiazepine deriva-tives. Each derivative was then acylated with 20 different carboxylic acidsusing the tetrafluorophenol resin (TFP-resin).27 Overall this allowed alarge library of 16,000 discrete compounds to be produced.

25 J. Kavalek, V. Machacek, G. Svobodova, and V. Sterba, Collect. Czech. Chem. Commun. 8,

1999 (1987).26 Work originally published in G. C. Morton, J. M. Salvino, R. F. Labaudiniere, and T. F.

Herpin, Tetrahedron Lett. 41, 3029 (2000).27 (a) J. M. Salvino, N. V. Kumar, E. Orton, J. Airey, T. Kiesow, K. Crawford, R. Mathew,

P. Krolikowski, M. Drew, D. Engers, D. Krolinkowski, T. Herpin, M. Gardyan,

G. McGeehan, and R. Labaudiniere, J. Comb. Chem. 2, 691 (2000). (b) R. E. Dolle,

Methods Enzymol. (2003).

TFA

DCMN

N

O OMe

OMe

N

ONH

Ar

O

R2

R1NR2

N

NO

O

Ar

22 23

Scheme 4. Cyclization of aromatic ureas to hydantoins.


1,5-Benzothiazepine derivatives are of particular interest for lead dis-covery because they have been shown to have activity against differentfamilies of targets. The 1,5-benzothiazepine scaffold has not only been usedas a constrained dipeptide mimic in protease inhibitors such as interleukin-1�-converting enzyme inhibitors,28 elastase,29 and angiotensin-convertingenzyme inhibitors,30 but also in antagonists of several G-protein-coupledreceptors such as the cholecystokinin31 receptor or the angiotensin II re-ceptor.32 Our synthetic route, depicted in Scheme 5, used the 3-aminogroup as a point of attachment, and exploited the wide variety of ortho-ha-lonitrobenzene derivatives commercially available and the facile substitu-tion of the benzothiazepine amide nitrogen.

The library was made with the Irori Accutag system, using MacroKanscontaining 400 mg of resin and an RF tag as synthetic object. Cysteine 25was reacted with the nitrophenyl carbonate derivative of Wang resin 24by first using bis-(trimethylsilyl)-acetamide33 (BSA) to dissolve the aminoacid (Scheme 5). The resin was then loaded into MacroKans, and sorted forthe first combinatorial step. The thiol 26 was then reacted with a variety ofhalonitrobenzene derivatives 27. Formation of up to 25% of cystine by di-merization of the resin-bound cysteine was observed during this step. Thiscould be suppressed by using a strictly inert atmosphere and DBU as base.The MacroKans were combined and reacted in a single batch with tin-dichloride dihydrate to reduce the nitro group, and then with EDC to cy-clize the aniline to the benzothiazepine derivatives 30. The MacroKanswere sorted into two batches and further diversity could be obtained by

28 J. M. C. Golec, D. J. Lauffer, D. J. Livingston, M. D. Mullican, P. L. Nyce, A. L. C.

Robidoux, and M. W. Wannamaker, World Patent WO 9824804 (1998).29 J. W. Skiles, R. Sorcek, S. Jacober, C. Miao, P. W. Mui, D. McNeil, and A. S. Rosenthal,

Bioorg. Med. Chem. Lett. 3, 773 (1993).30 J. Slade, J. J. Stanton, D. Ben-David, and G. Mazzenga, J. Med. Chem. 28, 1517 (1985).31 A. Nagel, World Patent WO 9401421 (1994).32 P. Buhlmayer and P. Furet, World Patent WO 9413651 (1994).33 B. A. Dressma, L. A. Spangle, and S. W. Kaldor, Tetrahedron Lett. 37, 937 (1996).

O

O NO2O NH2

O

OH

SH

+

X

NO2

R1

24 25

27

28 2930

a

b

c d

e

R1

R1

R1 S

N

NH2

OR2

R1

32a Z=:32b Z=O

30 Z=:31b Z=O

33a Z=:33b Z=O

Z Z

ZZ

ZZ

f g

R1R1

26

NH

O

OH

SHO

O

O NH

O

OH

S

O2N

O

NH

O

OH

S

NH2

O

OO

S

NH

NH

OO

OS

NH

NH

OO

O

S

N

NH

OO R2

Scheme 5. (a)(1) BSA, DMF, argon, RT; (2) 10% AcOH in DMF; (b) X = Cl, Br, or F, DBU, DMF, argon, RT; (c) SnCl2-2H2O, DMF, RT;

(d) EDC, NMP, RT; (e) m-CPBA, DCM, RT; (f) R2-X (X = Br, Cl), KI, DMF, DBU, RT; (g) 50% TFA-DCM, RT, 1 h.

88

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nth

esis

[4]


oxidation of one batch of the sulfide 30 to the sulfone 31b with m-CPBA.The MacroKans were pooled and sorted for the last combinatorial step. Al-kylation of the amide nitrogen could be accomplished with benzamidebases as reported in the solid-phase synthesis of benzodiazepine derivati-ves,23b but we found that DBU gave cleaner results with a simpler protocol.Cleavage from the resin afforded benzothiazepine derivatives 33a and 33bin high yield and excellent purity (see Table III for a selection).

Once released from the resin, the 3-aminobenzothiazepine derivativescould be further reacted with resin-bound tetrafluorophenyl ester ofcarboxylic acids to form amides (Scheme 6). Carboxylic acids were loaded

TABLE III

Examples of 1,5-Benzothiazepin-2-one Derivatives

T S

N

NH2

OR2

R13

467

8

ZZ

Entry R1 T R2 Z Puritya Yieldb

1 H CH H — 82 78

2 H CH H O 100 59

3 7-CF3 CH CH2-Phc — 100 63

4 7-OMe CH CH2-Phc — 95 68

5 7-C(O)-Ph CH CH2-Phc — 100 60

6 7-C(O)-(2-methoxymethyl-

pyrrolidine)

CH CH2-Phc — 94 61

7 7-NH-C(O)-NH-CH2-Ph CH CH2-Phc — 93 65

8 7-C(O)-(2-methoxymethyl-

pyrrolidine)

CH CH2-Phc O 67 49

9 7-NH-C(O)-NH-CH2-Ph CH CH2-Phc O 94 57

10 8-OMe N CH2-Phc — 100 58

11 9-Me CH CH2-Phc — 94 62

12 H CH (CH2)3-Phd — 100 60

13 H CH CH2-C(O)-N(Et)2c — 83 70

14 H CH CH2-4-fluorophenylc — 71 53

15 H CH CH2-3-chlorothiophenec — 70 54

16 H CH CH2-3-phenyloxadiazolec — 69 51

17 H CH (CH2)2 CH3e — 89 60

18 H CH (CH2)3-O-CH2-Phd — 84 62

a Purity measured by HPLC/ELSD (evaporative light scattering detector).b Yield based on weight of crude extract from the resin.c The alkyl chloride was used for alkylation.d The alkyl bromide and potassium iodide were used for alkylation.e The alkyl iodide was used for alkylation.

S

N

NH

OR2

O

R3 R1

ZZ

33O OH

F F

FF

O O

O

R3

F

F

F

F

ba

R3CO2H

34 35 36

Scheme 6. (a) DIC, DMAP, RT; (b) DMF, RT.


onto tetrafluorophenol resin 34 using the standard protocol developed bySalvino et al.27 19F NMR of the resin was used to quantify the loading ofeach carboxylic acid. These resin bound esters 35 were then reacted withthe 3-amino group of the benzothiazepine derivatives in DMF for a fewhours. After filtration and evaporation, 3-amido-1,5-benzothiazepine de-rivatives 36 were obtained in high yields and excellent purity. Table IVillustrates the generality of this reaction.

A 16,000-member library was synthesized using this procedure. Twenty-one halonitrobenzenes were combined with 20 halides and half wereconverted to the sulfone, resulting in 840 3-amino-1,5-benzothiazepinederivatives. These were then reacted with 19 carboxylic acids. Table Vshows a representative example of the reagents used. A small randomsample of 480 compounds from the library was analyzed by HPLC/MS.Figure 4 shows the purity distribution for the library. Most of thecompounds were prepared in high purity.

Conclusion

The directed sorting approach is a convenient technology to producelarge combinatorial libraries of discrete compounds. As can be seen withthe two examples presented, it is a very versatile and practical method toproduce compounds in a variety of formats.

Experimental Section

General Information

Chloromethyl polystyrene beads of 150–300 �M (loading 2 mmol/g)and Wang resin beads 150–300 �M (loading 1.7 mmol/g) were purchasedfrom Polymer Laboratories. 4-Hydroxy-2,6-dimethoxybenzaldehyde waspurchased from Perseptive Biosystems. BAL resin was prepared accordingto the published procedure.23 A loading of 0.8 mmol/g was determined byloading 4-bromobenzylamine to the resin and using elemental analysis.Rink amide resin was purchased from Irori. All other reagents were


purchased from standard commercial sources and used without furtherpurification. Solvents used were from EM Science of OmniSolv distilledgrade unless specified otherwise. 1H NMR and 13C NMR spectra wereobtained in 5-mm tubes on a 300-MHz Bruker ARX spectrometer inCDCl3 unless otherwise stated. Mass spectra were recorded on Finnigan4500 EI and Sciex API 3 IS spectrometers.

The libraries were constructed using the Irori system. For the 10,000-member library, MicroKans were filled with BAL resin by suspending theresin in an isobuoyant suspension (DMF:DCE 2:1) and dispensing it with aPackard Multiprobe liquid handler. For the 5000-member library, resinbound scaffold 18 was prepared in bulk and loaded into the MicroKans

TABLE IV

Example of Reaction of 1-Benzyl-3-amino-1,5-benzothiazepine with Resin-Bound

Tetrafluorophenyl Esters

S

N

NH2

OF

F F

F

O

O

R3 +

RT

DMF

R3S

N

NH

OO

35 37 38

R

Ester resin

loading (%)aPurity of

product (%)b R

Ester resin

loading (%)aPurity of

product (%)b

O CH3 40 90N

H3C 100 84

90 75O

100 66

NCH3

CH3

100 79O

O 100 78

100 80N

80 77

O

OCH3

95 79 NH

CH3

O60 92

100 71Cl

Cl100 78

a Tetrafluorophenol resin loading was measured using 19F NMR.b Purity of the crude benzothiazepine derivative measured by HPLC with ELS detection.


using the method described above. All reactions involving MicroKanswere performed in round-bottom flasks equipped with overhead stirrers.The Autosort10K was used to sort the MicroKans between combi-natorial steps and cleavage of the library compounds was effected in theAccucleave 96.

TABLE V

Representative Sample of the Reagents Used in the 16,000-Member 1,5-Benzothiazepine

Combinatorial Library

R1

N+

O−

O

F

N+

NH

O−

ONH

O

O

F

N+

O−

O

Cl

ON+

O−

OO

Cl

N+

NHO−

O

OCl

R2

Cl Br N

O

ClN

O

Cl O

OCl

R3

HO

O

HO

O

Cl

Cl NHO

O O

O

HO

O NOH

O

O

0

10

20

30

40

50

60

70

5 15 25 35 45 55 65 75 85 95

Purity

Num

ber

of c

ompo

unds

ana

lyze

d

Fig. 4. Purity distribution for the 16,000-member 1,5-benzothiazepine library. Purity based

on HPLC with ELS detection.


Resin Bound Amines 8

For each amine, 250 MicroKans (each MicroKan contained 12 mg of0.8 mmol/g loaded BAL resin 7) were placed into a 1-liter three-neckedround-bottom flask fitted with an overhead stirrer. The resin in the Micro-Kans was swelled in a solution of 1% acetic acid in DMF (300 ml). The airbubbles in the MicroKans were removed by placing the round-bottom flaskunder house vacuum. The amine (20.0 mmol) and sodium triacetoxyboro-hydride (4.24 g, 20.0 mmol) were added sequentially. The reaction wasstirred at RT for 6.5 h. For workup, each reaction was individually drainedand washed with DMF. All of the MicroKans were then combined andwashed with 1:1 DMF/MeOH, DMF, DCM, and Et2O. The MicroKanswere then dried overnight with a stream of nitrogen gas.

Resin Bound Amides 9

The 4500 MicroKans containing resin bound amines 8 were placedinto a 12-liter three-necked round-bottom flask fitted with an overheadstirrer. Dimethylformamide (4.5 liters) was added to swell the resin in theMicroKans. 1-Alloc-4-fmoc-piperazine-2-carboxylic acid scaffold 6 (78.6 g,180.0 mmol) was dissolved into DMF (500 ml) and added to the Micro-Kans. HBTU (68.3 g, 180.0 mmol) and DIEA (62.7 ml, 360.0 mmol) werethen added sequentially. The reaction was stirred at RT for 6.5 h. Thesolution was drained and the MicroKans were washed with DMF,DCM, and Et2O. The MicroKans were dried overnight with a stream ofnitrogen gas.

Removal of Fmoc Protecting Group from 9

The MicroKans containing 9 were placed into a 12-liter three-neckedround-bottom flask fitted with an overhead stirrer. Dimethylformamide(2.5 liters) and piperidine (2.5 liters) were added to the MicroKans. The re-action was stirred at RT for 3.5 h. The reaction solution was drained andthe MicroKans were washed with DMF, DCM, and Et2O. The MicroKanswere then dried overnight with a stream of nitrogen gas.

Acylation with Carboxylic Acids to Produce 10 or 11 or 20

For each carboxylic acid, 200 MicroKans were placed into a 1-literthree-necked round-bottom flask fitted with an overhead stirrer. The resinin the MicroKans was swelled in NMP (300 ml). The carboxylic acid(20.0 mmol) and EDC (3.83 g, 20.0 mmol) were added sequentially. Thereaction was stirred overnight at RT. For workup, each reaction was indi-vidually drained and washed once with DMF. All of the MicroKans from


each acid were then combined and washed with DMF, DCM, and Et2O.The MicroKans were then dried overnight with a stream of nitrogen gas.

Acylation with Chloroformates to Produce 10 or 11 or 20

For each chloroformate, 200 MicroKans were placed into a 1-literthree-necked round-bottom flask fitted with an overhead stirrer. The resinin the MicroKans was swelled in anhydrous DCM (300 ml). DIEA (3.5 ml,20.0 mmol) and the chloroformate (20.0 mmol) were then added sequen-tially. The reaction was stirred overnight at RT. For workup, each reactionwas individually drained and washed once with DMF. All of the Micro-Kans were then combined and washed with DMF, DCM, and Et2O. TheMicroKans were then dried overnight with a stream of nitrogen gas.

Urea Formation with Isocyanates to Produce 10 or 11

For each isocyanate, 500 MicroKans were placed into either a 2- or 3-liter three-necked round-bottom flask fitted with an overhead stirrer. Theresin was swelled in anhydrous DCM (600 ml). The isocyanate(50.0 mmol) was then added neat. The reaction was stirred overnight atRT. For workup, each reaction was individually drained and washed withDMF. All of the MicroKans were then combined and washed with DMF,THF, DCM, and Et2O. The MicroKans were then dried overnight with astream of nitrogen gas.

Sulfonamide Formation to Produce 10 or 11 or 20

For each sulfonyl chloride, 500 MicroKans were placed into either a 2- or3-liter three-necked round-bottom flask fitted with an overhead stirrer. Theresin was swelled in anhydrous DCM (600 ml). NMM (5.5 ml, 50.0 mmol)and the sulfonyl chloride (50.0 mmol) were then added sequentially. The re-action was stirred overnight at RT. For workup, each reaction was individu-ally drained and washed with DMF. All of the MicroKans were thencombined and washed with DMF, THF, DCM, and Et2O. The MicroKanswere then dried overnight with a stream of nitrogen gas.

Removal of Alloc Protecting Group from Resin 10 or 19

The 5000 MicroKans containing resin 10 or 19 were placed into a 12-liter three-necked round-bottom flask fitted with an overhead stirrer.THF (2 liters), DMSO (2 liters), and 0.5 N HCl (1 liter) were added tothe MicroKans. The reaction flask was then flushed with nitrogen.Pd(Ph3P)4 (8.06 g, 6.98 mmol) and morpholine (218 ml, 2500 mmol) wereadded sequentially. The reaction was stirred overnight under a flow of


nitrogen gas. For workup, the reaction was drained and the MicroKanswere washed with THF, sodium diethyldithiocarbamate (0.02 M inDMF), DMF, 0.5% DIEA in DCM, DCM, and Et2O. The MicroKans werethen dried overnight with a stream of nitrogen gas.

Preparation of Resin 16 by the Yamaguchi Method

4-(Bromomethyl)phenylacetic acid (529 mg, 2.34 mmol) was pouredinto 5 ml of DMF, followed by pyridine (170 �l, 2.125 mmol) and 2,6-dichlorobenzoylchloride (301 �l, 2.125 mmol). The mixture was shakenfor 1 h and then the Wang resin (loading 1.7 mmol/g) was added to themixture. The mixture was shaken overnight, drained, and the resin waswashed with DMF, THF, DCM, and ether. The resin was dried overnightunder reduced pressure.

Preparation of Resin 16 from Acid Chlorides

In a flask flushed with nitrogen, the acid (5.5 eq, 2.34 mmol) and oxalylchloride (3 ml, 6.02 mmol) were mixed in DCM (10 ml). One drop of DMFwas added. The mixture was stirred for 1 h. The solvent and oxalyl chloridewere then removed by evaporation. The acid choride was dissolved in 9 mlof a mixture of DCM and pyridine (9:1). Wang resin (loading 1.7 mmol/g)was added and the mixture was shaken overnight. The mixture was drainedand the resin was washed with DMF, THF, DCM, and ether. The resin wasdried overnight under reduced pressure.

Preparation of Resin 16 from Amino Acids

Wang resin (150 mg, 0.255 mmol) was suspended in DCM (2 ml). TheFmoc amino acid (1.25 mmol) was added, followed by DIC (200 �l,1.25 mmol) and DMAP (6.4 mg, 0.052 mmol). The mixture was shaken over-night. The mixture was drained and the resin was washed with DCM, DMF,THF, DCM, and ether. The resin was then suspended in a mixture of piperi-dineandDMF(1:1).Themixturewasshakenovernight,drained,andtheresinwas washed with DCM, DMF, THF, DCM, and ether. The resin (100 mg,0.17 mmol) was suspended in DCM (15 ml). The bromo-acid (2.55 mmol)was added, followed by DIC (400 �l, 2.55 mmol). The mixture was shakenovernight, drained, and the resin was washed with DCM, DMF, THF,DCM, and ether. The resin was dried overnight under reduced pressure.

Preparation of Resin 16 from Symmetrical Diamines

Nitrophenol carbonate resin (100 mg, 0.16 mmol) was suspended in2 ml of DMF. The diamine (1.6 mmol) was added and the mixture was


shaken overnight at room temperature. The mixture was drained and theresin was washed with DMF, THF, DCM, and ether. The resin was sus-pended in DCM (10 ml). The bromo-acid (1.7 mmol) was added, followedby DIC (267 �l, 1.7 mmol). The mixture was shaken overnight, drained,and the resin was washed with DCM, DMF, THF, DCM, and ether. Theresin was dried overnight under reduced pressure.

Alkylation with Piperazine-2-Carboxamide Scaffold to Produce 18

The resin 16 (4.6 g, 7.82 mmol) was suspended in DMF (15 ml).1-Alloc-2-carboxypiperidine trifluoroacetic acid salt (2.71 g, 23.4 mmol)was added followed by potassium iodide (458 mg, 23.4 mmol) and DIEA(2.89 ml, 45 mmol). The reaction mixture was heated overnight at 80

�, then

drained, and the resin was washed with DMF, THF, DCM, and ether.

Amide Bond Formation to Produce 19

For each amine, 402 MicroKans (each MicroKan contained 6 mg of1.7 mmol/g loaded resin 18) were placed into a 1-liter three-neckedround-bottom flask fitted with an overhead stirrer. The resin in the Micro-Kans was swelled in DMF (300 ml). Diisopropylcarbodiimide (4.79 ml)and pentafluorophenol (5.63 g) were added and the resulting mixture wasstirred at RT for 2 h. Each reaction was individually drained and washedwith DMF. The MicroKans in each round-bottom flask were again sus-pended in DMF (300 ml) and the corresponding amine (20.4 mmol) wasadded to each vessel. In the case of hydrochloride salts, DIEA (10 eq)was added. This reaction was stirred at room temperature overnight. Eachreaction was individually drained. All of the MicroKans were then com-bined and washed with DMF, THF, DCM, and ether. The MicroKans weredried overnight with a stream of nitrogen.

Cleavage

The MicroKans containing BAL resin were cleaved with 50% TFA inDCM for 1 h.

Cysteine Carbamate Resin 26

Nitrophenol carbonate resin 24 (320 g, 544.0 mmol) was swelled inanhydrous DMF (5 liters) in a 12-liter three-necked round-bottomflask. Argon gas was bubbled through this slurry for 1 h while stirringwith an overhead stirrer. In a second 3-liter three-necked round-bottomflask, anhydrous DMF (500 ml) and BSA (1.5 liter) were added. Thissolution was degassed for 1 h by bubbling argon gas through the solution.


dl-Cysteine was then introduced into the BSA solution. After stirring for30 min, all of the cysteine had dissolved up into solution. This cysteine so-lution was then canulated into the 12-liter flask containing the resin slurry.The reaction was stirred overnight at room temperature under argon. Forthe workup, the reaction solution was drained under argon. The resin wasthen washed with DMF, 10% AcOH in DMF, DMF, THF, DCM, and di-ethyl ether under argon. The resin was dried overnight with a stream ofargon gas. This resin was then dry loaded into Irori MacroKans using apowder dispensing gun (Perry Industries). The MacroKans were manuallysorted.

Halo-Nitrobenzene Coupling 28

For each halonitrobenzene 27, 42 MacroKans (each containing 300 mgof cysteine resin with a loading of 1.2 mmol/g) were placed into a 1-literthree-necked flask fitted with an overhead stirrer and argon line. The flaskwas flushed with argon for 30 min. Degassed anhydrous DMF (250 ml) wasadded to the flask. While stirring the MacroKans under argon, DBU(22.6 ml, 151.2 mmol) was added. After stirring for approximately 5 min,the halonitrobenzene 27 (151.2 mmol) was then added. The reaction wasstirred under argon for several hours. The argon lines were then removed,the reaction capped tightly, and stirred overnight at room temperature. Forthe workup, the reaction solution was drained under argon. The Macro-Kans were then washed with DMF, 10% HOAc in DMF, 20% aqueousTHF, THF, DCM, and diethyl ether. The MacroKans were dried overnightwith a stream of nitrogen gas.

Reduction of Nitro Group to Prepare 29

A total of 924 MacroKans (each containing 300 mg of resin 28 with aloading of 1.2 mmol/g) was placed into a 12-liter three-necked round-bottom flask fitted with an overhead stirrer and a heating mantle. DMF(6 liters) and tin dichloride dihydrate (750.5 g, 3326.4 mmol) were addedto the flask. The reaction was stirred at 50

�overnight. For workup, the re-

action solution was drained. The resin was then washed with DMF, aque-ous THF, THF, DCM, and diethyl ether. The MacroKans were driedovernight with a stream of nitrogen gas.

Cyclization to Benzothiazepine 30

A total of 924 MacroKans (each containing 300 mg of resin 29 with aloading of 1.2 mmol/g) was placed into a 12-liter three-necked round-bottom flask fitted with an overhead stirrer and a heating mantle. The resin


in the MacroKans was swelled in anhydrous NMP (6 liters). EDC (318.9 g,1663.2 mmol) was added and the reaction was stirred overnight. For theworkup, the MacroKans were washed with DMF, aqueous DMF, DMF,aqueous THF, THF, DCM, and diethyl ether. The MacroKans were driedovernight with a stream of nitrogen gas.

Oxidation to Sulfone 31b

The resin in 420 MacroKans (each containing 300 mg of resin 30 with aloading of 1.2 mmole/g) was swelled in DCM (4 liters). MCPBA (208.7 g of50% pure reagent, 604.8 mmol) was added and the reaction was stirredat room temperature for 5.5 h. For the workup, the reaction solution wasdrained and the resin was washed with DCM, aqueous THF, THF, anddiethyl ether. The Microkans were dried overnight with a stream ofnitrogen gas.

Alkylation Reaction to 32

For each alkyl halide, 44 MacroKans (each containing 300 mg of resin30 or 31b with a loading of 1.2 mmol/g) were placed into a 1-liter three-necked round-bottom flask fitted with an overhead stirrer. The resin inthe MacroKans was swelled with anhydrous DMF (350 ml). The DBU(23.7 ml, 158.4 mmol) was added and the reaction was stirred for 15 min.The alkyl halide (158.4 mmol) was then introduced. KI (158.4 mmol) ifneeded was added last. The reaction was stirred overnight at room tem-perature. For the workup, the reaction solution was drained and theMacroKans were washed with DMF, 10% HOAc in DMF, 20% aqueousTHF, THF, DCM, and diethyl ether. The MacroKans were dried overnightwith a stream of nitrogen gas.

Archiving, Cleaving, and Free Basing of Resin 33

For each MacroKan, the cap was removed and the resin and tag werepoured into a 16 � 100-mm glass test tube. Twenty-two racks of 40tubes each were archived in the Irori system. For each tube, the resinwas cleaved with a 50% TFA/DCM solvent mixture for 1 h and then con-centrated down. The resulting residue was azeotroped with DCM toremove the remaining traces of TFA. MP-Carbonate resin (411 mg,1.08 mmol) and DMF (4 ml) were added to the residue (approximately0.360 mmol of product per MacroKan) in each test tube. After vortexingfor several seconds to dissolve the cleaved compounds, the reaction wasallowed to sit at room temperature overnight. The liquid above the resinwas transferred to another test tube through a filter tube using a Packard

[5] string synthesis 99

liquid handler. The resin was then washed twice with DMF (3 ml). Each ofthese washings was transferred to the collection tube giving approximately9 ml total of a benzothiazepine free amine stock solution.

TFP Resin Loading 35

For each carboxylic acid, TFP resin 34 (20 g with a loading of1.25 mmol/g) was introduced into a 250-ml glass peptide vessel. The resinwas swelled in DMF (120 ml). The carboxylic acid (250 mmol), DMAP(3.05 g, 25 mmol), and DIC (39.14 ml, 250 mmol) were then added sequen-tially. The reaction was mixed on a wrist shaker overnight. For the workup,the reaction solution was drained and the resin was washed with DMF,THF, DCM, and diethyl ether. The resin was dried in a vacuum oven atroom temperature overnight.

Reaction with TFP Resin to Prepare 36

Each set of 80 benzothiazepine 33 free amine stock solutions (two racksof 40 tubes each) was reacted with 20 acylated TFP resins 35. Of each stocksolution 400 �l was added to approximately 15 mg of acylated TFP resinper well. (Note: Each 80-well plate contained the same TFP-activated acidresin in each well but a different amine in each well.) The reactions weremixed on an orbital shaker at room temperature for 3 days. The productsuspension was filtered through filter plates into two daughter plates usinga Tomtec. The products were concentrated at 65

�.

[5] Split-Mix Synthesis Using MacroscopicSolid Support Units

By Arpad Furka, James W. Christensen, and Eric Healy

Introduction

Split-mix synthesis1–3 made it possible to prepare new compounds inpractically unlimited numbers. That procedure was based on the solid-phase method4 in which each coupling cycle was replaced by the followingoperations:

1 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, in ‘‘Highlights of Modern

Biochemistry,’’ Proceedings of the 14th International Congress of Biochemistry, Vol. 5,

p. 47. VSP, Utrecht, The Netherlands, 1988.



liquid handler. The resin was then washed twice with DMF (3 ml). Each ofthese washings was transferred to the collection tube giving approximately9 ml total of a benzothiazepine free amine stock solution.

TFP Resin Loading 35

For each carboxylic acid, TFP resin 34 (20 g with a loading of1.25 mmol/g) was introduced into a 250-ml glass peptide vessel. The resinwas swelled in DMF (120 ml). The carboxylic acid (250 mmol), DMAP(3.05 g, 25 mmol), and DIC (39.14 ml, 250 mmol) were then added sequen-tially. The reaction was mixed on a wrist shaker overnight. For the workup,the reaction solution was drained and the resin was washed with DMF,THF, DCM, and diethyl ether. The resin was dried in a vacuum oven atroom temperature overnight.

Reaction with TFP Resin to Prepare 36

Each set of 80 benzothiazepine 33 free amine stock solutions (two racksof 40 tubes each) was reacted with 20 acylated TFP resins 35. Of each stocksolution 400 �l was added to approximately 15 mg of acylated TFP resinper well. (Note: Each 80-well plate contained the same TFP-activated acidresin in each well but a different amine in each well.) The reactions weremixed on an orbital shaker at room temperature for 3 days. The productsuspension was filtered through filter plates into two daughter plates usinga Tomtec. The products were concentrated at 65

�.


[5] Split-Mix Synthesis Using MacroscopicSolid Support Units

By Arpad Furka, James W. Christensen, and Eric Healy

Introduction

Split-mix synthesis1–3 made it possible to prepare new compounds inpractically unlimited numbers. That procedure was based on the solid-phase method4 in which each coupling cycle was replaced by the followingoperations:

1 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, in ‘‘Highlights of Modern

Biochemistry,’’ Proceedings of the 14th International Congress of Biochemistry, Vol. 5,

p. 47. VSP, Utrecht, The Netherlands, 1988.




1. dividing the solid support into equal portions;2. coupling each portion individually with a different building block; and3. mixing the portions.

Repetition of these simple operation steps resulted in an exponentialincrease in the number of synthesized compounds. The products could beused either as mixtures or as unidentified individual compounds formedon microscopic polymer beads. The parallel synthesis developed by Geysenand his colleagues,5 although less productive than the split-mix method,yielded known products, each in several milligram quantities. To offerthe advantages of this parallel synthetic methodology while preserving highproductivity, split-mix synthesis was modified by applying macroscopicsolid support units instead of the conventional bead form support. Theresin was enclosed in permeable capsules. The number of capsules wasequal to the number of compounds to be prepared and, after each reactionstep, the capsules were redistributed among the reaction vessels. To makethis possible, the capsules were encoded by electronic chips also enclosed inthe capsules.6,7 The code written into the chip by radiofrequency radiationallows the history of the synthetic transformations to which the capsule wasexposed to be tracked.

The string synthesis8,9 described below also applies to macroscopic solidsupport units with the upside that these units are uncoded providing amethodology that is cheaper and faster.

Principle

Any combinatorial synthetic method carried out with macroscopic solidsupport units, even if coding is omitted, has to ensure that the route ofevery unit in the entire multistep synthetic process can be traced. In stringsynthesis this can be achieved by

2 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Abstract of the 10th International

Symposium on Medicinal Chemistry, Budapest, Hungary, p. 288, 1988.3 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37, 487

(1991).4 R. B. J. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).5 H. M. Geysen, R. H. Meloen, and S. J. Barteling, Proc. Natl. Acad. Sci. USA 81, 3998 (1984).6 E. J. Moran, S. Sarshar, J. F. Cargill, M. Shahbaz, A. Lio, A. M. M. Mjalli, and R. W.

Armstrong, J. Am. Chem. Soc. 117, 10787 (1995).7 K. C. Nicolaou, X.-Y. Xiao, Z. Parandoosh, A. Senyei, and M. P. Nova, Angew. Chem. Int.

Ed. Engl. 36, 2289 (1995).8 A. Furka, J. W. Christensen, E. Healy, H. R. Tanner, and H. Saneii, J. Comb. Chem. 2, 220

(2000).9 A. Furka, Comb. Chem. High Throughput Screening 3, 197 (2000).


1. arranging the units into spatially defined groups: one group isformed for each building block of the first reaction step and therelative position of the units need to be maintained during thechemical reactions;

2. redistribution of the units among the groups of the next syntheticstep has to be carried out according to a predetermined pattern thatcan be reflected in a computer; and

3. the redistribution pattern has to ensure formation of all expectedmembers of a combinatorial library.

In this case, the products formed on the support units can be identifiedby computer prediction based on the relative positions of the unitsoccupied in the final groups.

There are different possibilities for practical realization of the prin-ciples and the string synthesis described below represents only one of them.

String Synthesis: Support Units and Strings

The support units used in the string synthesis were Chiron MimotopesCrowns shown in Fig. 1. They were used together with different colorstems, also purchased from Chiron Mimotopes. Spatially ordered groupsare formed with the crowns (Fig. 1A) by stringing them together with athread. The stems serve to string the crowns together and to facilitateredistribution. The stems were modified as shown in Fig. 1.

First, a hole was drilled to allow the string to be passed through, andthen the stem was carved to keep the holes parallel and facilitate threadingthem while in the sorting device (see Fig. 4). The modified stems can be

A B C D E F G

2 mm 4−5 mm

Fig. 1. Chiron Mimotopes Crowns. (A) Crown; (B) stem; (C) carved stem with hole; (D)

support unit; (E) full-length stem labeling the head of the string. The scratches represent the

string number; (F) half stem that marks the tail of the string; (G) Mimotopes SynPhase

Lantern with stem.

Tail

25201 5 1015

Head

Fig. 2. Stringed crowns. Full and half-length stems mark the head and the tail end of the

string. Positions of the crowns are numbered from the head.


used repeatedly. SynPhase Lanterns (Fig. 1G), available at Mimotopes Pty.Ltd. (http://www.mimotopes.com), may also be used in place of the crowns.

The string itself must be resistant to solvents and other reaction condi-tions involved in the synthesis. In the illustrated example, a polyethylenefishing line was used. The stringed crowns are shown in Fig. 2.

To unequivocally define the position of the crowns, the two ends of thestring must be distinguishable. The head is marked by a full-length stem(Fig. 1E) and the tail of the string (Fig. 1F) is labeled by a stem cut in half(Figs. 1 and 2). The numbering position of the crowns start at the head.Depending on the number of building blocks used in each consecutive re-action step, a separate string of crowns is incorporated. As a consequence,the strings themselves must be numbered or otherwise labeled. The sim-plest way to label the strings is by making visible scratches on the stemmarking the head. Using colored stems is also a possibility. If lanterns areused as support units, it is worth taking into account that they have a holein their center, so they can be threaded without the need for stems.

The chemical reactions described later in this chapter were carried outon crowns, by coupling amino acids onto the respective strings. Thenumber of crowns used in the synthesis is equal to the number of expectedproducts. Therefor, if five amino acids are used in a coupling step, five dif-ferent strings are made, each containing the same number of crowns. Afterthreading the crowns, each string was placed into a reaction vessel andcoupled with a different amino acid. For the present work, all couplingswere carried out in five reaction vessels (Fig. 3).

Manual Device for Redistribution

The strings coming from the reaction vessels after completing the first(and any further) coupling reactions are called source strings. Theircrowns, which are then rearranged into their strings for the next reactionstep, are denoted as destination strings. Redistribution of the crowns is

1 2 3 4 5

Fig. 3. Strings in reaction vessels and their numbers represented below by the scratches on

the stems.

Source tray Destination tray

Fig. 4. Manual sorting device.


carried out using a very simple device that can be easily made by a machineshop. The device contains two identical pieces shown in Fig. 4.

Both pieces are metal plates with several numbered parallel slotsand bent at the two edges. The crowns are first positioned in the slots ofthe source tray (Fig. 5) then sorted by pushing them into the slots of thedestination tray (Fig. 6).

It is important to place each string into the slot carrying the samenumber, and position the heads and tails of the source strings into the slotsof the source tray as indicated in the figure, otherwise the software usedto track the crowns (see Software section) cannot be used. It is also im-portant to number the destination strings according to the numbers of thedestination slots and render their heads and tails to the heads and tails ofthe destination slots. The crowns were loaded into the slots of the sourcetray while still attached to the string (Fig. 7A), then the string was cutand removed. The crowns were then sorted in string free form (Fig. 7B)and then restrung (Fig. 7C).

A device design to sort up to 750 crowns is demonstrated in Fig. 8.The two trays are identical except for the direction of the numbering of

the slots. Changing the position of the numbers in any of the trays wouldmake the software inapplicable. If lanterns are used with stems as supportunits, the width of the slots have to be smaller since the stems of the lanterns

54321

54321

Source tray Destination tray

Head Tail

Head Tail

Fig. 6. Position of the source and destination trays at the beginning of sorting (top view).

Pushing over crowns from source slot No. 5 into destination slot No. 1.

Fig. 5. Crowns hanging in the slots of the sorting device.


are smaller in size. If the lanterns are used without their stems a differentdesign should be applied. The slots can be replaced by small troughs.

Redistribution Pattern

The redistribution sequence made in the course of a synthetic processmust ensure formation of all the components expected at the end of a com-binatorial synthesis. In the conventional split-mix synthesis this is achievedby pooling the content of all reaction vessels of a given reaction step,thoroughly mixing them, then transferring equal portions of the combinedresin into the reaction vessels for the next reaction step. As a result, allproducts formed in a previous reaction vessel are evenly distributed amongthe reaction vessels assigned to the next reaction step. This can be con-sidered as the combinatorial redistribution rule. This rule can be translatedto string synthesis in the following way: the crowns of a string containingthe same product have to be evenly distributed among the strings of thenext reaction step. Obeying this rule, there are still many ways to carryout solid support redistributions.9 The semiparallel sorting processdescribed below is designed to ensure a fast redistribution of solid supports.

The first column in Fig. 9 depicts redistribution of 125 crowns in a singlesorting cycle. In the first redistribution cycle the crowns are delivered fromeach source slot in groups of five. Relative position 1 (RP 1) illustratesthe starting orientation of both source and destination trays. Note thatsource slot No. 5 is in alignment with destination slot No. 1. From this

40 cm

33 cm

34 cm

48 cm

BendingBending

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

A B

Fig. 8. Proposed sorting device. (A) Design of the source tray with 15 slots (width 3 mm);

(B) top view of the final form of destination tray.

A

B

C

Fig. 7. Crowns in the slot of the sorter. (A) Before cutting the string; (B) string free form

before and after sorting; (C) stringing the crowns after sorting.


position five crowns are pushed into destination slot No. 1, then the destin-ation tray is repositioned. In RP 2, source slots No. 4 and No 5 are facing des-tination slots No. 1 and No. 2, respectively. This new position makes itpossible to push five crowns into both destination slots No. 1 and No. 2.

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

1

2

3

4

5

6

7

8

9

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

54321

RP Sorting step 1 Sorting step 2

Fig. 9. Semiparallel sorting of 125 crowns from five source slots into five destination slots.

First column: sorting step 1, executed in the nine positions of one distribution cycle; five

crowns are delivered from each source slot. Second column: first distribution cycle of sorting

step 2: one crown is delivered from each slot. The numbers at the left side of the figure mark

the nine different relative positions (RPs) occupied by the source and the destination trays in

a redistribution cycle. The figure shows the top views of the two trays. The original and the

destination positions of the repositioned crowns are indicated by black circles.



The destination tray is moved further step by step all the way down to RP 9pushing groups of five crowns into all destination slots that are in alignmentwith the slots of the source trays. Thus, in RP 3, RP 4, RP 5, RP 6, RP 7, RP 8,and RP 9, 15, 20, 25, 20, 15, 10, and 5 crowns are pushed into destination slots,respectively. The nine RPs complete a full redistribution cycle since thereare no more possible relative positions of the source and destination slots.

The semiparallel redistribution follows two simple rules:

1. The destination tray is gradually moved from the start position—when only single slots of both trays are in alignment—to the endposition where the other two single slots are facing each other.

2. Delivery of crowns is executed in all slots that are in alignment.

Since the delivery of crowns mostly occurs in groups, the redistributionprocess is fast.

Software

The software for the string synthesis technique is written in visual basicand the data appear in Microsoft Excel sheets. This software can be down-loaded via the Internet from http://szerves.chem.elte.hu/furka by clickingon the title ExcelBook appearing on the lower part of the main page. Thissoftware is compatible with only those PC systems that have Excel installed.

The software was created to handle a maximum of 1000 crowns, 20 re-agents (building blocks), and 9 reaction steps. The Excel datasheet wherethe starting data have to be entered is illustrated in Fig. 10.

The starting data consist of the number and symbols of the buildingblocks (monomers) to be used in the coupling steps. The symbols aresingle-letter abbreviations. In the case of peptide synthesis the symbols cor-respond to their respective amino acids. When using the software, the areaswhere data are to be entered appear yellow on the screen. Row 26 (the rowfollowing MONOMERS IN COUPLINGS) shows the string numbers.Each of the monomers entered in a column are assigned to successivelyundergo coupling with the string appearing in the same column. Once allthe required data are entered, the software executes the calculation pre-senting the resulting calculated data in the blue regions of the Excel sheet.The calculated data include the total number of crowns needed in thesynthesis as well as the number of coupling steps (Fig. 10, column B).The number of source and destination slots used in the first and subsequentsorting steps (Fig. 10D and E) and the number of crowns occupying theseslots (Fig. 10F and G) also appear along with the number of crowns thatcontain the same product (Fig. 10H). The number of crowns in a group thatwill have to be moved in every sorting cycle from a source to a destinationslot is indicated in Fig. 10, column I.

Starting Data for Semi-Parallel Sorting SORTING: in every cycle delivery starts from the highest number (rightmost) source slot into the first (leftmost) destination slot. Run: Ctrl + S The number of reagents and the symbols of monomers (A,B,C etc.) have to be entered The number attached to the sequences only show the original position of the units The number of units in a block to be delivered is indicated by red numbers. Do not delete blue cells! Enter data only into yellow cells! Number of building blocks Sort

numberNumber of slotes Crowns in tubes Identical

crownsCrownsto move(maximum number is 52) Source Destin. Source Destin.

CP 1 5 1 5 5 25 25 25 5 CP 2 5 2 5 5 25 25 5 1 CP 3 5 3 0 1 0 CP 4 4 0 0 CP 5 5 0 0 CP 6 6 0 0 CP 7 7 0 0 CP 8 8 0 0 CP 9 9 0

0000000

0000000

0000000

000000 0

CP 10Maximum number of reagents 20

Total number of crowns

125 Maximum number of sorter slots 20

Number of coupling steps:

3 Maximum number of crowns 1,000

Pause (in seconds): MONOMERS IN COUPLINGS

1 2 3 4 5 6 7 8CP 1 I F L V G CP 2 E F W Y S CP 3 E F W Y S CP 4CP 5CP 6CP 7CP 8CP 9CP 10

A B C D E F G H I

Fig. 10. Data sheet of the Excel Book where the starting data can be entered. The symbols

of the columns are presented in the last row.


The program can be started by pressing together the ‘‘Ctrl’’ and ‘‘S’’(‘‘Ctrl S’’) keys of the keyboard. The results of calculations appear insheets Sort #1 through Sort #9. The sheets show a block of products presentin the crowns of the source slots and, below these, a block of productssorted into the destination slots. The positions of the crowns are counteddownward from the top. The number of sheets showing the results ofcouplings and sortings is equal to the number of sortings plus one. The lastsheet contains the predicted product distribution on the final strings.

Coupling 1

Sort No. 1

Coupling 2

Sort No. 2

Coupling 3

Products

Str 1 Str 2 Str 3 Str 4 Str 5



I F L V G

String 1 String 2 String 3 String 4 String 5

E F W Y S

E F W Y S

Fig. 11. Flow diagram of the synthesis.


Synthesis of a Library of 125 Tripeptides

The synthesis was carried out using 125 Chiron Mimotopes Crowns(capacity 5.3 �mol each) derivatized with an Fmoc*-Rink amide linker.The procedure was started with the formation of five strings by threading25 crown units on Berkley Fire Line fishing line. Five Fmoc-protectedamino acids were used in each coupling position as demonstrated in theflow diagram of the synthesis (Fig. 11).

* Abbreviations: Fmoc, 9-fluorenylmethoxycarbonyl; DMF, N,N-dimethylformamide; DCM,

dichloromethane; HOBt, 1-hydroxybenztriazole; DIC, diisopropylcarbodiimide; NMP,

N-methylpyrrolidinone; TFA, trifluoroacetic acid.


Coupling

Couplings were carried out with strings placed in 100-ml flasks. Toremove the Fmoc-protecting group 10 ml 1:1 v/v DMF-piperidine wasadded to each flask and then the flasks were placed on an orbital mixerand shaken for 30 min at room temperature. The solutions were decantedfrom strings and the strings were then washed with 3 � 15 ml DMF, 15 mlDCM, 15 ml DMF, 15 ml DCM, and 2 � 15 ml DMF. The deprotectionand washing operation was repeated one more time and finally the stringsand the crowns were washed with additional 2 � 15 ml DCM. After dryingall the strings and crowns, individual solutions were prepared with 10 mmolof an Fmoc amino acid, 10 mmol of HOBt, and 15 mmol of DIC in 10 mlNMP. These solutions were added to the flasks placing them on an orbitalmixer and shaking them for 2 h at room temperature. The final solutionswere decanted and the strings and crowns were washed with 3 � 40 mlDMF, 40 ml DCM, and 2 � 40 ml DMF. The above coupling and washingoperation was repeated one more time washing at the end the strings andcrowns with an additional 2 � 40 ml DCM. The crowns were dried in anoven and then the strings were removed for sorting.

Sorting of the Crowns

In the three-step synthesis of the tripeptide library, the crowns are re-distributed in two different sorting steps. Sorting step 1 follows the attach-ment of the amino acids to the solid support at coupling position 1 (CP1).Sorting step 2 is carried out after the second coupling cycle at coupling pos-ition 2 (CP2). Before beginning the sorting operations, the starting datawere entered into computer (Fig. 10). As showing column I of the data-sheet (row CP1), in the first sorting procedure the crowns must be movedfrom each slot in groups of five. In the second sorting procedure the crownsare moved one at a time (column I, row CP2).

Sorting step 1 is demonstrated in the left column of Fig. 9. The sortingof the 125 crowns was finished on the ninth position of a single redistribu-tion cycle. Sorting step 2 is demonstrated in the second column of Fig. 9. Inthis case only a single crown was moved from slot to slot. On the ninth pos-ition of the first distribution cycle shown in Fig. 9, a total of 25 crowns weredelivered to the destination tray. The rest of the crowns were redistributedin four additional cycles not shown here.

Cleavage

To cleave the products from the supports, the crowns were separatelyplaced in test tubes, and the compounds deprotected by adding 1 ml 1:1v/v piperidine-DMF to each tube. After allowing the mixtures to stand


for 30 min the crowns were filtered and then washed with 3 � 2 ml DMF,2 ml DCM, 2 ml DMF, and 2 � 2 ml DCM. The crowns were placed backinto test tubes, 1 ml 95% TFA/H2O was added to each tube, and the mix-tures were allowed to stand for 30 min at room temperature. The final so-lutions were decanted into vials. The crowns were washed with 1 ml 95%TFA/H2O, the solutions transferred to the same vials, and the solventswere removed using a rotavap.

Product Distribution

Location of products and intermediates on the strings appear in sheetsSort #1, Sort #2, and Sort #3. Some of the predictions are summarized inTable I. It can be seen that after the first coupling on string 1, as expected,

TABLE I

Content of No. 1 Strings (Str.) After Couplings (Cpl.) and Sortings (Sort) and Position

of Products on the Final Stringsa

Position

Cpl. 1

Str. 1

Sort 1

Str. 1

Cpl. 2

Str. 1

Sort 2

Str. 1

Str. 1

Products

Str. 2

Products

Str. 3

Products

Str. 4

Products

Str. 5

Products

1 I I EI EI EEI FEI WEI YEI SEI

2 I I EI FI EFI FFI WFI YFI SFI

3 I I EI WI EWI FWI WWI YWI SWI

4 I I EI YI EYI FYI WYI YYI SYI

5 I I EI SI ESI FSI WSI YSI SSI

6 I F EF EF EEF FEF WEF YEF SEF

7 I F EF FF EFF FFF WFF YFF SFF

8 I F EF WF EWF FWF WWF YWF SWF

9 I F EF YF EYF FYF WYF YYF SYF

10 I F EF SF ESF FSF WSF YSF SSF

11 I L EL EL EEL FEL WEL YEL SEL

12 I L EL FL EFL FFL WFL YFL SFL

13 I L EL WL EWL FWL WWL YWL SWL

14 I L EL YL EYL FYL WYL YYL SYL

15 I L EL SL ESL FSL WSL YSL SSL

16 I V EV EV EEV FEV WEV YEV SEV

17 I V EV FV EFV FFV WFV YFV SFV

18 I V EV WV EWV FWV WWV YWV SWV

19 I V EV YV EYV FYV WYV YYV SYV

20 I V EV SV ESV FSV WSV YSV SSV

21 I G EG EG EEG FEG WEG YEG SEG

22 I G EG FG EFG FFG WFG YFG SFG

23 I G EG WG EWG FWG WWG YWG SWG

24 I G EG YG EYG FYG WYG YYG SYG

25 I G EG SG ESG FSG WSG YSG SSG

a Amino acids are indicated with one-letter symbols.


all units contained amino acid I. After the first sorting step, string 1 con-tained five products in groups of five crowns. The product distribution inthe rest of the strings was exactly the same. After the second coupling,string 1 contained five dipeptides in groups of five crowns. After the secondsorting step, as Table I shows, all products in string 1 were different. Theproduct distribution in the rest of the strings, not shown in the Table I,was exactly the same. Positions of the formed tripeptides on the five stringsafter the third coupling are also shown in Table I.

Verification of Product Distribution

Randomly selected sequences were independently synthesized andcompared to those cleaved from the crowns. The data gathered by high-performance liquid chromatography and mass spectrometry unequivocallyand positively confirmed the predicted product distribution.

Acknowledgments

The authors thank the Hungarian government for Grants FKFP/0149/2000, OTKA

T34868, and NKFP 1/047.

[6] The Encore Technique: A Novel Approach toDirected Split-and-Pool Combinatorial Synthesis

By Viktor Krchnak and Vitecek Padera

Introduction

The traditional and established way of conducting chemical transform-ations employs one reaction vessel for each compound synthesized. To pre-pare 50 different acetamides, 50 reaction vessels are needed, one for eachamide. When synthesis of 50 amides is performed at the same time, this ap-proach is referred to as a parallel synthesis. It is, of course, always desirableto simplify the process by reducing the number of vessels used in the syn-thesis. However, it is not feasible just to mix 50 amines into one reactionvessel and acetylate the mixture of amines because the isolation of 50amides from the resultant reaction mixture could be complicated and timeconsuming. Simple separation of individual components was made possibleby Merrifield’s solid-phase synthesis.1 Solid support-bound substrates can

1 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).



all units contained amino acid I. After the first sorting step, string 1 con-tained five products in groups of five crowns. The product distribution inthe rest of the strings was exactly the same. After the second coupling,string 1 contained five dipeptides in groups of five crowns. After the secondsorting step, as Table I shows, all products in string 1 were different. Theproduct distribution in the rest of the strings, not shown in the Table I,was exactly the same. Positions of the formed tripeptides on the five stringsafter the third coupling are also shown in Table I.

Verification of Product Distribution

Randomly selected sequences were independently synthesized andcompared to those cleaved from the crowns. The data gathered by high-performance liquid chromatography and mass spectrometry unequivocallyand positively confirmed the predicted product distribution.

Acknowledgments

The authors thank the Hungarian government for Grants FKFP/0149/2000, OTKA

T34868, and NKFP 1/047.


[6] The Encore Technique: A Novel Approach toDirected Split-and-Pool Combinatorial Synthesis

By Viktor Krchnak and Vitecek Padera

Introduction

The traditional and established way of conducting chemical transform-ations employs one reaction vessel for each compound synthesized. To pre-pare 50 different acetamides, 50 reaction vessels are needed, one for eachamide. When synthesis of 50 amides is performed at the same time, this ap-proach is referred to as a parallel synthesis. It is, of course, always desirableto simplify the process by reducing the number of vessels used in the syn-thesis. However, it is not feasible just to mix 50 amines into one reactionvessel and acetylate the mixture of amines because the isolation of 50amides from the resultant reaction mixture could be complicated and timeconsuming. Simple separation of individual components was made possibleby Merrifield’s solid-phase synthesis.1 Solid support-bound substrates can

1 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).



[6] encore technique 113

be pooled into one reaction vessel and the individual substrates are thenphysically separated by covalent attachment to the carrier.

Frank et al.2 recognized the potential of combing a number of differentsolid-phase bound substrates for a reaction with a single reagent usingcellulose-based paper discs as solid-phase support for the synthesis of oligo-nucleotides. Each disc contained a different substrate (oligonucleotide)and only four reaction vessels were required for the synthesis of anynumber of oligonucleotides. ‘‘Whenever growing chains on different en-tities have to be elongated with the same building block these entities aregathered in the same reaction vessel.’’2 Later, Frank and Doring3 appliedthe pooling strategy to peptide synthesis.

Houghten4 expanded this methodology to resin beads, a common sup-port for solid-phase peptide synthesis. He placed resin beads into polypro-pylene meshed packets similar in appearance to tea (T-)bags. Before theaddition of the next amino acid to the resin-bound growing peptide chain,the T-bags were distributed into different reaction vessels, each vessel con-taining resin-bound intermediates receiving the same amino acid. To carryout reaction transformations common to all peptides (e.g., cleavage of theamino-protecting group) all bags were pooled into one reaction vessel.

The concept of reducing the number of reaction vessels and exponen-tially increasing the number of synthesized compounds was brought to anext level of simplicity by the split-and-pool method of Furka et al.5 Thesplit-and-pool method was independently applied by Lam et al.6 in a one-bead–one-compound concept for the combinatorial synthesis of largecompound arrays (libraries) and by Houghten et al.7 for the iterative librar-ies. Now several millions peptides could be synthesized in a few days. InFurka’s method the resin beads receiving the same amino acid were con-tained in one reaction vessel—identical to Frank’s method—however, thebeads were pooled and then split randomly before each combinatorialstep. Thus the method is referred to as the random split-and-pool methodto differentiate it from Frank’s method in which each solid-phase particlewas directed into a particular reaction vessel (the directed split-and-poolmethod).

2 R. Frank, W. Heikens, G. Heisterberg-Moutsis, and H. Blocker, Nucleic Acids Res. 11, 4365

(1983).3 R. Frank and R. Doring, Tetrahedron 44, 6031 (1988).4 R. A. Houghten, Proc. Natl. Acad. Sci. USA 82, 5131 (1985).5 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37, 487

(1991).6 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp,

Nature 354, 82 (1991).7 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo,

Nature 354, 84 (1991).


These two methods have two key common features:

1. A limited number of reaction vessels is needed for the synthesis. Thenumber of reaction vessel equals the number of building blocks inany particular reaction step. The number of compounds synthesizedexponentially exceeds the number of vessels.

2. The amount of material synthesized depends on the yield from onesolid-phase particle (disc) or container (T-bag).

The key differences are as follows. In the directed split-and-poolmethod

1. The chemist controls the distribution of compounds in a library. Anycombination of building blocks can be excluded from the synthesis.

2. The chemical history of the particles is recorded (e.g., each T-bag islabeled by an alphanumeric code).

In the random split-and-pool method

1. The distribution of compounds in a library is driven by statisticalprobabilities due to the random split process. Each compound issynthesized numerous times when the number of beads exceedsseveral times the number of compounds, or only a subset ofcompounds is produced when the number of beads is lower than thenumber of possible combinations of building blocks.

2. The chemical history of the beads is lost. After each combinatorialstep, the resin beads from all reaction vessels are pooled andrandomly split into reaction vessels for the next combinatorial step.

The principal differences between those two methods are reflected intheir applications. The random split-and-pool method is suited for the syn-thesis of smaller quantities of large sizable libraries (i.e., million com-pounds), whereas the directed split-and-pool technique is suited for thesynthesis of larger quantities of smaller compound collections (i.e., severalhundred to several thousand compounds). In this chapter, a simpletechnique for directed split-and-pool technique is described.

Directed Split-and-Pool Method

Three technical issues have to be solved in order to make the directedsplit-and-pool method attractive for routine synthesis:

1. One entity of solid-phase support (particle or container) has toprovide a sufficient amount of compound.

2. The chemical history of individual entities needs to be recorded.


3. The process of distribution of entities, particularly for relativelylarger libraries, needs to be integrated and/or automated.

Solid Support

A single resin bead does not usually provide the required micromolaramount of target material. The sufficient yield from one entity has beensolved in two different ways. The first solution is to enclose a sufficientamount of resin beads into a container. Houghten introduced the meshedpolypropylene packets (T-bags).4 IRORI changed the shape of a T-baginto a meshed can (MacroKan, MicroKan, and NanoKan for 100, 30, and8 mg of resin, respectively) in order to allow robot handling.8,9 Recently,very promising resin plugs have been developed10 and commercialized byPolymer Laboratories (StratoSpheres Plugs). Resin beads are mixed withfine powdered high-density polyethylene (HDPE) and heated to melt theHDPE. After cooling, plugs are formed and used as a solid-phase support.

An alternative solution is to produce a solid-phase particle that yieldsmicromolar amount of product. Mimotopes Pty. Ltd. has developed andcommercialized the SynPhase Crown and later Lanterns, a rigid polypro-pylene mold with a grafted layer of polystyrene. The solid-phase synthesistakes place on the derivatized graft.11

Encoding

The simplest solution for tracking the chemical history is labeling of in-dividual entities (paper discs, T-bags) by an alphanumeric code thata chemist can read. Alternatively, the reaction containers can also becolor coded. Radiofrequency tagging8 and optical encoding12 of containersenabled computer-assisted reading of the tag and automation of thedirected split-and-pool process. A radiofrequency tag is inserted into eachcontainer with resin beads and, before a combinatorial step, the individualtags are read and the containers distributed into corresponding reaction

8 X. Y. Xiao, R. Li, H. Zhuang, B. Ewing, K. Karunaratne, J. Lillig, R. Brown, and K. C.

Nicolaou, Biotechnol. Bioeng. 71, 44 (2000).9 K. C. Nicolaou, J. A. Pfefferkorn, H. J. Mitchell, A. J. Roecker, S. Barluenga, G. Q. Cao,

R. L. Affleck, and J. E. Lillig, J. Am. Chem. Soc. 122, 9954 (2000).10 B. Atrash, M. Bradley, R. Kobylecki, D. Cowell, and J. Reader, Angew. Chem. Int. Ed.

Engl. 40, 938 (2001).11 F. Rasoul, F. Ercole, Y. Pham, C. T. Bui, Z. Wu, S. N. James, R. W. Trainor, G. Wickham,

and N. J. Maeji, Biopolymers 55, 207 (2000).12 C. Y. Xiao, C. F. Zhao, H. Potash, and M. P. Nova, Angew. Chem. Int. Ed. Engl. 36, 780

(1997).

Fig. 1. A necklace of SynPhase Lantern.


vessels. The process is referred to as directed sorting and it has beenfully automated and commercialized by IRORI. The Mimotope SynPhaseLanterns have been used in an analogous way.

An alternative method for tracking the chemical history is encoding byspatial address. The identity of each entity is defined by its spatial address.A one-dimensional directed split-and-pool procedure, referred to as neck-lace coding, has been developed for synthesis carried out on SynPhaseCrowns and Lanterns.13 Individual supports are strung on a Teflon threadand the position of a particle on the thread (necklace) encoded the previ-ous chemical history (Fig. 1). A similar concept was later reported by Furkaand co-workers14,15 Two-and three-dimensional encoding of the directedsplit-and-pool synthesis platform has been patented by Selectide Corp.16

Process Integration/Automation

The third challenge was to integrate and/or automate the handling ofindividual particles between combinatorial steps. During the early yearsof directed split-and-pool methodology the entire process was done

13 J. Smith, J. Gard, W. Cummings, A. Kaniszai, and V. Krchnak, J. Comb. Chem. 1, 368

(1999).14 A. Furka, Comb. Chem. High Throughput Screening 3, 197 (2000).15 A. Furka, J. W. Christensen, E. Healy, H. R. Tanner, and H. Saneii, J. Comb. Chem. 2, 220

(2000).16 M. Patek, P. Safar, M. Smrcina, E. Wegrzyniak, P. Strop, G. Flynn, and S. A. Baum, World

Patent WO 0138268 (2001).


manually. Manual splitting is still useful for handling small libraries. How-ever, the manual process is tedious and prone to human error. Highly auto-mated and sophisticated robotics systems have been designed to executeunattended directed split-and-pool combinatorial synthesis. IRORI’s fullyautomated directed sorting robot is based on optical encoding of Nano-Kans.8,12 An alternative robotic system, based on a sophisticated and com-plex two- and three-dimensional spatial encoded directed split-and-poolmethod, has been custom built by Selectide scientists.16

An intermediate level of sophistication was achieved by semiautomatedsorting of radiofrequency tagged containers (e.g., MicroKans,8 Lanterns11).A simple instrument that integrates the process of one-dimensional neck-lace encoding by creating a linear sequences of solid-phase particles isthe Encore synthesizer.

Description of the Encore Method

The Encore method is based on one-dimensional spatial encoding ofthe synthesis chemical history, referred to as necklace coding, and appliedfor the synthesis on SynPhase Crowns and Lanterns.13 One-dimensionalspatial encoding eliminates the need to label each solid-phase particle;the chemical history of a particle is encoded by its sequential position ona linear string, a necklace. To preserve the linear sequence during synthe-sis, Lanterns are strung together using a chemical-resistant Teflon rope. Inprinciple, it is possible to create a single string (necklace) of Lanterns foreach reaction vessel. However, this approach would require reshufflingthe Lanterns between combinatorial steps. A more effective approach isto create numerous short necklaces after the first combinatorial step, andreshuffle the necklaces after the second combinatorial step (there is noneed to distinguish among Lanterns during the first combinatorial stepsince all Lanterns in one reaction vessel are identical).

The current version of the Encore synthesizer has been designed for thesynthesis of up to 960 compounds using an algorithm to handle 10, 8, and 12building blocks in the first, second, and third combinatorial steps, respect-ively (Fig. 2). The total number of combinatorial compounds produced thisway is n ¼ 10 � 8 � 12 ¼ 960. For the first combinatorial step, 10 reactionvessels are charged with 96 (8 � 12) solid-phase supports and the first set ofbuilding blocks is chemically attached to solid-phase supports. After thisstep, all 96 particles per reaction vessel are identical and particles fromdifferent reaction vessels differ only by the kind of first building blockattached to solid-phase particles.

After the first combinatorial step, 96 (8 � 12) necklaces are formed,each necklace containing 10 particles, one from each of the original 10

Fig. 2. The principle of the Encore technique. (A) The first combinatorial step is

performed in 10 reaction vessels, each charged with 96 scattered Lanterns. (B) Twelve

necklaces with 10 stringed Lanterns are placed into each of eight reaction vessels of the

second step. (C) The third combinatorial step is performed in 12 reaction vessels with eight

necklaces per vessel, each tagged with a different color.


reaction vessels. The position of a particle on a necklace encodes the firstbuilding block. At this stage, all 96 necklaces are identical.

The second combinatorial step is performed with eight building blocksfor which eight reaction vessels are used with each vessel containing 12necklaces. To record (encode) the second building block, all necklaces inone reaction vessel are labeled by the same color. Consequently, eight dif-ferent colors are needed to label necklaces for eight reaction vessels. Obvi-ously, any other suitable ways of labeling could be applicable, includingalphanumeric labeling.

After finishing the second combinatorial step, one necklace from eachof the eight reaction vessels is placed into a reaction vessel for the thirdcombinatorial step. There are 12 new reaction vessels for the third com-binatorial step, each vessel containing eight necklaces of different color.After finishing the synthesis, the necklaces are disassembled and theindividual particles are placed into a 96-well plate for the cleavage andcollection of target compounds.

The Encore technique combines three different coding methods: se-quential position on a necklace for the first combinatorial step, color codingof individual necklaces for the second combinatorial step, and reactionvessel coding to identify the last building block. Accordingly, we termedthis technique Encore for Encoding by a Necklace, Color, and Reactionvessel.

The algorithm described above is for a three-step combinatorial synthe-sis. However, the method is not limited to only three-step combinatoriallibraries; the solid-phase support can be derivatized before the directedsplit-and-pool synthesis on the Encore synthesizer. The necklace codingcan also be a very useful tool during the chemistry development process.


The reactivity of a building block with different polymer-supportedsubstrates can be evaluated in one reaction vessel.

Description of the Encore Instrument

The Encore synthesizer has been designed to integrate the process ofhandling of solid-phase particles in combinatorial synthesis. The currentversion of the synthesizer enables synthesis in batches of up to 960 com-pounds on SynPhase Lanterns. The Encore synthesizer facilitates thesorting of Lanterns into sequences (necklace coding) and the plating ofthe Lanterns after completion of the synthesis for final release of com-pounds from Lanterns. The Encore synthesizer consists of the followingfive tools.

Arraying Tool

The Arraying Tool (Fig. 3) arranges 96 random Lanterns from one re-action vessel after the first combinatorial step (all Lanterns are chemicallyidentical) into a two-dimensional array of 8 rows and 12 columns. TheArraying Tool has a standard 96-well plate footprint.

Magazine

The Magazine (Fig. 4) is a polyethylene block with 96 shafts. Each shaftaccommodates up to 10 Lanterns. The Magazine enables Lanterns to be

Fig. 3. The Arraying Tool.

Fig. 4. Magazine (white block at the top) with the dispensing manifold (black block).


collected into 96 shafts in a predetermined sequence and serves two func-tions: (1) arrangement of Lanterns for transfer into reaction vessels by theLapis Tool and (2) arrangement of Lanterns for final distribution intocleavage plates.

Lapis Tool

The Lantern Picking and Stringing (Lapis) Tool (Fig. 5) is used tosecure and pick Lanterns from the Magazine while preserving the sequenceof Lanterns in a shaft (necklace coding). Loaded Lapis tools are placedinto reaction vessels. The Lapis Tool is made of stainless steel and poly-ethylene and it is compatible with a wide variety of solvents, reagents,and reaction conditions.

Lantern Dispensing Tool

After finishing the combinatorial synthesis, Lanterns are distributedinto 96-well plates (one Lantern per well) using the Lantern DispensingTool (Fig. 6). The Lantern dispensing tool enables the transfer ofone ‘‘layer’’ of Lanterns (e.g., 96 Lanterns) from the Magazine into a96-well plate.

Lantern Leveling Tool

To ensure a reliable distribution of Lanterns into cleavage plates,Lanterns are stacked tightly in shafts using the Leveling Tool (Fig. 7).

Fig. 5. The Lapis tools with color tags and 10 Lanterns.

Fig. 6. Lantern Dispensing Tool.


Synthetic Protocol

Synthesis of a three-step combinatorial library of 960 compounds using10, 8, and 12 building blocks in the first, second, and third combinatorialsteps, respectively, consists of the following steps:

1. Ten reaction vessels are charged with 96 Lanterns each and the firstcombinatorial synthetic step is performed. A suitable reactionvessel is a 50-ml syringe and the synthesis can be performed on theDomino Block synthesizer (Torviq, Tucson, AZ). At this point, allthe Lanterns in a reaction vessel are identical. There is no need todistinguish among Lanterns in one reaction vessel.

Fig. 7. Lantern Leveling Tool.


2. All 96 Lanterns from the first reaction vessel are emptied onto theArraying Tool and the Arraying Tool is gently shaken in an orbitalmotion until all Lanterns are sequestered into the openings of theArraying Tool. This operation typically takes less than 15 sec.

3. The Arraying Tool is placed on top of the Magazine and thestainless-steel plate is removed from the Arraying Tool. This stepenables the Lanterns to drop into the shafts of the Magazine.

4. The Leveling Tool is inserted into 96 openings of the ArrayingTool and any stacked Lantern is gently pushed into the shafts of theMagazine.

5. Steps (2) to (4) are repeated nine times to accommodate all theLanterns from all 10 reaction vessels. When all Lanterns aredistributed, the full Magazine contains 960 Lanterns in 96 shafts,each shaft containing 10 Lanterns and all shafts having exactly thesame sequence of Lanterns. The position of a Lantern in thesequence codes for the first building block.

6. Ninety-six Lapis Tools are labeled by color tags with eight differentcolors (or color combinations) to achieve 12 sets of Lapis Toolsfrom each color (or color combination).

7. The Lapis Tool labeled by the first color (or color combination) ispushed through the openings in all 10 Lanterns of a shaft. Thesequence of Lanterns from this shaft is stringed on a Lapis Tool.The Lapis Tool carrying a sequence of 10 Lanterns is placed intoreaction vessel #1 for the second combinatorial step.


8. Step (7) is repeated with the remaining Lapis Tools tagged with thefirst color. As a result, reaction vessel #1 is loaded with 12 LapisTools labeled and identified by the same color.

9. The same sequence of steps (7 and 8) is repeated using the next setof 12 Lapis Tools labeled with the second color (or colorcombination) and reaction vessel #2 is loaded with 12 Lapis Tools.

10. The rest of the Lanterns are strung in an analogous way resulting ineight reaction vessels charged with Lanterns strung on Lapis Tools.Each reaction vessel containing Lapis Tools is labeled with thesame color (or color combination) and this color code correspondsto the second building blocks.

11. The second combinatorial step is performed in eight reaction vessels.12. The Lapis Tools are rearranged before the third combinatorial step

in such a way that each of 12 new reaction vessels contains eightnecklaces with a different color tag.

13. The third combinatorial step is performed.14. Lapis Tools with Lanterns are placed into the shafts of the

Magazine as described above. Eight Lapis Tools from reactionvessel #1 are moved into the first column of the Magazine in such away that the sequence of Lapis color tags corresponds to thesequence of second building blocks.

15. Lapis Tools with Lanterns from the remaining reaction vessels aremoved into the Magazine in an analogous way.

16. The Lapis Tool from each shaft is removed leaving the sequence of10 Lanterns in each shaft.

17. Lanterns are distributed for compound release into wells of 96-wellplates, one Lantern per well. The Magazine is placed on top of thefirst 96-well plate. The Magazine is positioned on the right-handside of the Lantern dispenser and it is secured in this position bytwo springs. The Lantern Leveling Tool is placed on top of theLanterns.

18. The Magazine is pushed by hand to the left side. Once the endposition is reached, a metal handle located on the side of theMagazine locks it in position.

19. With this motion the bottom layer of Lanterns drops into theLantern dispensing manifold (a black anodized aluminum piece,see Fig. 4). Completion of the Lantern drop can be visuallyinspected by observing the Lantern Leveling Tool. The tool has todrop by 5 mm (the height of a Lantern). If a single Lantern doesnot drop into the Dispensing Tool, the Leveling Tool will not moveor will be lopsided. In such a case a gentle push on the LevelingTool usually moves the Lantern into the Dispensing manifold.


20. The metal handle is lifted. The Magazine moves back to its originalposition. At this time, 96 Lanterns are released into the 96-wellplate. The Dispensing Tool is lifted and the cleavage plate isvisually inspected for the presence of Lanterns.

21. Steps 18 to 21 are repeated.22. The target compounds are cleaved from the Lanterns in the 96-well

plates.

Conclusion

Combinatorial chemistry has moved from specially centralized labora-tories, often equipped with multimillion-dollar robots, onto the bench ofindividual medicinal chemists. This change in direction requires the avail-ability of personal chemistry tools that are simple to operate, easy to ar-range in the laboratory, and reasonably priced. Such instruments are nowavailable for the effective synthesis of combinatorial libraries. The Encoresynthesizer represents a simple and efficient personal chemistry tool thatallows the execution of directed split-and-pool combinatorial synthesis.The current version of the Encore synthesizer is designed for solid-phasesynthesis on SynPhase Lanterns; however, it can be modified for synthesison alternative solid supports such as resin plugs from Polymer Laboratories(e.g., StratoSpheres Plugs).

The split-and-pool synthesis not only simplifies the complexity of thecombinatorial synthetic process, but also offers additional important bene-fits. To undertake a full range of solid-phase chemical reactions, elaboratereaction conditions are needed for some chemical transformations. Theseinclude, but are not limited to, low temperature and inert atmosphere con-ditions. Parallel synthesis of a thousand compounds requires handling of athousand reaction vessels. The timely addition of sensitive reagents (e.g.,butyl lithium) at low temperature (�78

�) under inert atmosphere during

parallel synthesis is not a trivial task. It can be done if sophisticated auto-mated synthesizer equipment is designed to handle and tolerate such reac-tion conditions. Such a synthesis can alternatively be performed easily in amanual fashion using a split-and-pool method that requires only a limitednumber of reaction vessels. Examples from Nicolaou’s17 and Schrei-ber’s18,19 laboratories have shown that the split-and-pool method is themethodology of choice for the synthesis of complex and diversity-orientedcombinatorial libraries.

17 K. C. Nicolaou and J. A. Pfefferkorn, Biopolymers 60, 171 (2001).18 S. L. Schreiber, Science 287, 1964 (2000).19 H. Kubota, J. Lim, K. M. Depew, and S. L. Schreiber, Chem. Biol. 9, 265 (2002).

[7] multifunctional linkers for synthesis of libraries 127

[7] Multifunctional Linkers as an Efficient Tool for theSynthesis of Diverse Small Molecule Libraries: The

Triazene Anchors

By Stefan Brase, Stefan Dahmen, and Matthias E. P. Lormann

Introduction

Over the past decade, the drug discovery paradigm has undergoneextraordinary changes. With the rapid exploration of potential drug candi-dates via high-throughput screening now a current challenge is the ever-increasing demand for novel small compounds to satisfy and sustain suchscreening campaigns. As a result, innovative combinatorial approachestoward novel drug-like compounds have become key tools for successfuldrug discovery programs.

One of the major tools in combinatorial chemistry has been rooted inthe ingenious solid-phase synthesis of peptides by Merrifield.1

After extending the use of polymer supports into the realm of organicsynthesis, the access to small molecule libraries has been accelerated dueto the availability of carrying out suitable chemical reactions with the aidof automated synthesis equipment.2,3

While the transformation of chemical functionalities and the assemblyof building blocks on solid support are similar to conventional solution-phase chemistry, linkers and their associated strategies play a pivotal rolein the successful implementation of solid-phase organic chemistry and theirapplication to combinatorial chemistry.4–6 In general, linkers are bifunc-tional molecules that act as spacers between the resin and the attachedbuilding block. The functional group on the solid support that serves asthe point of origin for a synthetic sequence is generally unchanged uponcleavage conditions. However, the bond between the linker and the immo-bilized compound is sensitive to certain reaction conditions leading to bondbreakage freeing the final compound from the immobilized linker. Trad-itionally, linkers were designed to release one functional group acting more

1 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).2 L. A. Thompson and J. A. Ellman, Chem. Rev. 96, 555 (1996).3 S. L. Schreiber, Science 287, 1964 (2000).4 F. Zaragoza Dorwald, ‘‘Organic Synthesis on Solid-Phase: Supports, Linkers, Reactions.’’

Wiley-VCH, Weinheim, 2000.5 S. Dahmen and S. Brase, in ‘‘Handbook of Combinatorial Chemistry’’ (K. C. Nicolaou, R.

Hanko, and W. Hartwig, eds.), Chapter 4. VCH, Weinheim, 2002.6 I. W. James, Tetrahedron 55, 4855 (1999).



Fig. 1. Linker types for solid-phase synthesis.

128 linkers and their applications [7]

or less like bulky protecting groups. These types of linkers could be definedas monofunctional linkers (Fig. 1). Although the release of carboxylic acidsand amines, which are essential for peptide synthesis, has been extensivelystudied, the synthesis of small-molecular-weight compound librariesrequires more versatile linkers and cleavage strategies.7

One limitation of monofunctional linkers is that they provide only onetype of compound in a library. However, the so-called multifunctional lin-kers offer the important opportunity to incorporate additional diversityupon cleavage. In this case, the number of new functionalities (Fig. 1,type I) can multiply the number of compounds produced (Fig. 2).

7 B. J. Backes and J. Ellman, Curr. Opin. Chem. Biol. 1, 86 (1997).

Fig. 2. Diversity through multifunctional cleavage.


If the linker is susceptible to cleavage upon treatment with differentbuilding blocks [e.g., nucleophile (A) and electrophiles (B) in Fig. 1,type II], a substantial library of novel molecules could be prepared fromone linked compound (Fig. 2).

Literature precedence for multifunctional linkers can be found amongvarious types of anchoring groups (Fig. 3).

When considering using a multifunctional linker, one must take intoaccount the nature of the cleavage reagent and the cleaving step. Forexample, cleavage of an immobilized compound anchored via an ester link-age with excess of a Grignard reagent will require an aqueous workup withthe potential of losing valuable material, as well as having to develop atedious workup and product-isolation procedures. Thus, supplementarybuilding blocks need to be easily removed (i.e., must be volatile, solublein certain solvents, amenable to react with scavenger resins, etc.) andshould not interfere with the characteristics of the whole library such astheir biological properties.

Triazenes as Linkers

The chemistry of diazonium salts provides tremendous opportunitiesfor the construction of a wide range of aromatic compounds. Triazenesnot only provide interesting new possibilities for activation of the

Fig. 3. Examples for multifunctional linkers.4


ortho-position of arenes by coordination of metal ions and/or by loweringthe electron density of the arene ring, they are also ideal synthons for dia-zonium salts. Inspired by the use of triazenes in the total synthesis of van-comycin8 and the pioneering work of Moore and co-workers9 and Tour andco-workers10 in the synthesis of triazenes on a solid support to produce io-doarenes, a whole set of triazene-based linkers has been developed11

(Fig. 4). Triazenes are stable toward daylight, oxygen (air), moisture, redu-cing agents, oxidizing reagents, and alkyl lithium reagents under certainconditions.12,13 However, triazenes are labile toward Brønsted acids andcertain Lewis acids producing diazonium salts and amines.

Two linkers based on the triazene chemistry have been developed.While the T1 linker system consists of 3,3-dialkyl-1-aryl triazene bound to

8 K. C. Nicolaou, C. N. C. Boddy, S. Brase, and N. Winssinger, Angew. Chem. 111, 2230

(1999).9 J. K. Young, J. C. Nelson, and J. S. Moore, J. Am. Chem. Soc. 116, 10841 (1994).

10 L. Jones, J. S. Schumm, and J. M. Tour, J. Org. Chem. 62, 1388 (1997).11 S. Brase, D. Enders, J. Kobberling, and F. Avemaria, Angew. Chem. Int. Ed. 37, 3413

(1998).12 M. Lormann, S. Dahmen, and S. Brase, Tetrahedron Lett. 41, 3813 (2000).13 M. Lormann, S. Dahmen, F. Avemaria, F. Lauterwasser, and S. Brase, Synlett, 917 (2002).

Fig. 4. The triazene linkers.


the support via the alkyl chain (Scheme 1), the T2 linker family is based onimmobilized aryl diazonium salts (compounds 56, 66, and 71).14

The triazene T1 linker has been successfully used as a linker for arenes.Up to now, approximately 100 different anilines 7 have been immobil-ized.15 In general, the synthesis starts with diazotation of an aniline in anorganic solvent using alkyl nitrite reagents. The immobilization on solidsupport has been successfully carried out via the use of a benzylamino-polystyrene or piperazinylmethylpolystyrene resin, each accessible fromMerrifield resin in only one step with loadings generally around 1 mmol/g(1–2% cross-linked with divinylbenzene) (see Experimental section).Although both linkers are equally suitable, the benzylamino resin is more

14 S. Brase, J. Kobberling, D. Enders, M. Wang, R. Lazny, and S. Brandtner, Tetrahedron Lett.

40, 2105 (1999).15 S. Brase, unpublished results (1998–2003).

Scheme 1. Concept of the T1 linker.16


sensitive toward strong bases (e.g., BuLi)12 while the piperazine resin is notavailable with a high loading capacity due to possible cross-linking duringits preparation from Merrifield resin. Both resins are now commerciallyavailable (Novabiochem).

Functionalization on the polymer bead has been demonstrated ex-tensively. Acidic cleavage of the triazene resin yields the amine resin 10,which can be recycled, and the modified aryl diazonium salts 8-R0 whichcan be further transformed directly at the cleavage step in high yields(>90%) and purities (>90–95% according to GC, NMR, HPLC* analyses)(Scheme 2).

Traceless Linkers

One prominent class of monofunctional anchor that provides access tomolecules having ‘‘no attachment memory’’ for solid-phase synthesis iscalled traceless or ‘‘clean break’’ linkers.7,16–18 Although this definitioncould be used for the classification of linkers, one would advantageously

*Abbreviations: DMF, N,N-dimethylformamide; DMA, N,N-dimethylacetamide; GC, gas

chromatography; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic

resonance; RCM, ring closing metathesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran.16 S. Brase and S. Dahmen, Chem. Eur. J. 6, 1899 (2000).17 A. B. Reitz, Curr. Opin. Drug. Disc. Dev. 2, 358 (1999).18 A. C. Comely and S. E. Gibson, Angew. Chem. Int. Ed. Engl. 40, 1012 (2001).

Scheme 2. Possibilities of the T1 triazene linker.16


define a traceless linker as being able to generate new C—H bonds or tokeep all functionalities during cleavage intact/unchanged upon cleavage.Thus, this linker type allows the formation of arenes, alkanes, alkenes,and alkynes bearing no chemical evidence of attachment to a support.For that reason, this anchoring mode has no potentially undesirable con-straints on the structure of the products. Therefore, the triazene linkershave been envisaged as traceless linkers.

As pointed out above, acidic media (below pH 3) cleave the triazenes togive the diazonium salts. The diazonium salts can be further functionalizedas exemplified in the case of the reduction to the hydrocarbon 17-H in THFwith the aid of ultrasound11 through a radical pathway. A new reagent

Scheme 3. The T1 linker for traceless cleavage.


found for this reduction was trichlorosilane (see Experimental section),12

which not only serves as a source of trace-quantity hydrochloric acid tocleave the triazene moiety, but also as a hydride donor reducing the diazo-nium ions cleanly (Scheme 3). The synthetic utility of the T1 linker hasbeen demonstrated in short reaction sequences. Thus, cinnamic esters weresynthesized in a sequence starting from the iodoarene resin 26. A Heckcoupling19 with acrylates using palladium catalysis affords immobilized cin-namates, which in turn could be detached either directly with trichlorosi-lane to afford cinnamate derivative 25, or after further transformationscleaved with an HCl/THF mixture to give 27 in high yields without theneed of further purification or workup procedures (Scheme 3).

Other traceless linkers have been developed based on silyl linkers,20

acylhydrazines,21 dialkylsulfones,22 olefin metathesis cleavage (RCM), ar-ylsulfonates,23 phosphonium salts,24 or phosphine–chromium complexes.25

In general, the chemistry of traceless linkers is a fast emerging field in theintensive investigated area of solid-phase organic synthesis. Although therewas initially some confusion about the definition or classification of thetraceless linker, it is now clear that this anchoring mode will play an import-ant role in the design and synthesis of drug-like molecules. With the tria-zene linker a new chemistry field is now set up for new developments andthe investigation of some older ideas that had been overlooked until now.

19 S. Brase, J. Kobberling N. Griebenow, in ‘‘Handbook of Organo-Palladium Chemistry’’

(E.-i. Negishi, ed.), Chapter X. 3. Wiley, New York, 2002.20 M. J. Plunkett and J. A. Ellman, J. Org. Chem. 60, 6006 (1995).21 F. Stieber, U. Grether, and H. Waldmann, Angew. Chem. Int. Ed. Engl. 38, 1073 (1999).22 K. W. Jung, X. Y. Zhao, and K. D. Janda, Tetrahedron 53, 6645 (1997).23 S. J. Jin, D. P. Holub, and D. J. Wustrow, Tetrahedron Lett. 39, 3651 (1998).24 I. Hughes, Tetrahedron Lett. 37, 7595 (1996).25 S. E. Gibson, N. J. Hales, and M. A. Peplow, Tetrahedron Lett. 40, 1417 (1999).


Multifunctional Cleavage

Functionalization during the cleavage process is an attractive objectivefor the generation of diverse compound libraries.

As already shown by Moore and co-workers9 and Tour and co-workers,10 addition of methyl iodide to a triazene resin at elevated tem-perature (110

�) gives rise to aryliodides 17-I (Nu ¼ I) in excellent yields.

We have shown that aryl halides 17-X (X ¼ Cl, Br, I) are readily availableby the action of lithium halides in the presence of an acidic ion-exchangeresin or with the corresponding trimethylsilyl halide at room tempera-ture.26 A mixture of acetic anhydride and acetic acid produces phenolacetates 17-OAc.26

Aryl diazonium salts and azide transfer reagents react directly withoutthe need of a catalyst to afford aryl azides. In contrast to the general Sand-meyer reaction, this reaction does not proceed through the cleavage of thecarbon–heteroatom bond. In this particular case an open-chain pentazeneor a cyclic pentazole is formed, which in turn loses nitrogen to give the de-sired aryl azide.27 With this information in hand, a solid-phase syntheticprotocol was developed for the synthesis of aryl azides. This synthesiswas achieved via the cleavage of the triazene resin with 10% TFA in di-chloromethane at room temperature in the presence of trimethylsilyl azide,a commercially available azide derivative with no explosive properties.28

After a couple of minutes the mixture was filtered, the solvent was re-moved, and the aryl azides 17-N3 were isolated in good yields (mostly>90%) and high purity (>95%) without any further purification. The re-quired mild cleavage conditions allow the synthesis of various functiona-lized arenes. Only small amounts of silyl residues (<5%) were detectedby NMR spectroscopy.

While the range of electrophiles that could be employed is quite broad,the most versatile benefit was the development of a cleavage cross-couplingstrategy.29 Starting from modified triazene resins, a one-pot cleavage cross-coupling reaction was conducted with two equivalents of trifluoroaceticacid in MeOH at 0

�to give a diazonium intermediate 8-R0. In situ coupling

with both electron-deficient or electron-rich alkenes 19 in the presence ofcatalytic amounts (5% mol) of palladium(II) acetate furnished the corres-ponding products 20 in excellent yield and purities. This one-pot cleavagecross-coupling reaction affords salt-free products since the resin 10 also

26 S. Brase, M. Lormann, and J. Heuts, Tetrahedron Lett. In preparation (2003).27 R. N. Butler, A. Fox, S. Collier, and L. A. Burke, J. Chem. Soc. Perkin. Trans. 2, 2243

(1998).28 W. C. Groutas and D. Felker, Synthesis 861 (1980).29 S. Brase and M. Schroen, Angew. Chem. Int. Ed. 38, 1071 (1999).


participates as a ‘‘scavenger resin’’ trapping the trifluoroacetic acid inexcess. Using palladium on charcoal as the catalyst in the cross-coupling re-action has the advantage of decreasing palladium contamination as well asproviding the conditions for a subsequent hydrogenation reaction. Thissalt-free cleavage cross-coupling strategy allows the clean synthesis of sub-stituted (cyclo)alkenyl- and (cyclo)alkyl (hetero)arene derivatives and it isespecially suitable for automated synthesis since there is no need for puri-fication of the final compounds. This modular protocol is amenable to vir-tually any amino arene as well as alkenes and alkynes tolerating mostfunctional groups providing a synthetic pathway for the formation of highlylipophilic molecules. Multicomponent Heck reactions (e.g., domino HeckDiels–Alder reaction) are possible in this context and lead to furtherdiversification.30

Concept for Heterocycle Synthesis

Heterocycles play a pivotal role in drug discovery.31 It was shown thatcompounds with biological activity are often derived from heterocyclicstructures based on natural and synthetic products possessing variouspharmacological properties. Substituted heterocyclic compounds offer ahigh degree of structural diversity and have proven to be broadly usefulas therapeutic agents. With the disclosure of the benzodiazepine librariesby Ellman’s group in 1992,32 the development of strategies for the gen-eration of heterocyclic libraries33 on solid support has become of greatinterest in combinatorial chemistry.34

Therefore, we envisaged using the triazene T1 linker for the synthesis ofdiverse heterocycle libraries. While in concept I (Scheme 4) the diazoniumion was directly incorporated into the heterocycle core, the flexibility of theazide functionality has been advantageously used in concept II (Scheme 4).In concept III the heterocyclic core is generated on the bead using(classic) ring-formation reactions followed by a subsequent multifunctionalcleavage to give rise to the library members.

Concept I in Scheme 4 represents a straightforward synthesis of ben-zoannelated heterocycles (e.g., cinnolines, benzotriazoles) via a diazoniumintermediate and a nucleophilic ortho-substituent. While in the manyexamples, the diazonium group, upon cleavage from the resin, is lost as

30 A. de Meijere, H. Nuske, M. Es-Sayed, T. Labahn, M. Schroen, and S. Brase, Angew. Chem.

Int. Ed. 38, 3669 (1999).31 S. Brase, C. Gil, and K. Knepper, Bioorg. Med. Chem. 10, 2415 (2002).32 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992).33 R. G. Franzen, J. Comb. Chem. 2, 195 (2000).34 R. E. Dolle, J. Comb. Chem. 2, 383 (2000).

Scheme 4. Concept for cleavage/heterocyclization with the T1 system.


nitrogen gas, a suitable nucleophilic ortho-substituent favors cyclizationto give heterocyclic structures (Scheme 5). Using this approach, variousheterocyclic structures are conceivable.

Benzotriazoles, for example, are accessible from o-aminoaryl-substitutedtriazenes after a two-step reaction sequence: a nucleophilic displacementfollowed by cleavage/heterocyclization.35 The nucleophilic halide displace-ment of activated haloarenes is an indispensable tool for the synthesis ofhighly substituted arenes. Fluoronitroarenes in particular have served asexcellent precursors in this transformation. Thus, it was appealing to com-bine this SNAr reaction with the flexibility of diazonium chemistry. In thiscase, an immobilized fluoronitrophenyl triazene would be the equivalent ofthe Sanger reagent.

Starting from commercially available 2-fluoro-5-nitroaniline, this anil-ine was diazotized and coupled to benzylaminomethyl polystyrene to givethe immobilized triazene 36 (see Experimental section). At this point, itwas crucial to avoid using hydroxylic solvents such as methanol in thewashing steps since these solvents are known to react with fluorotriazenes.Acetone was found to be a good substitute for methanol.

35 M. E. P. Lormann, C. H. Walker, and S. Brase, Chem. Commun. 1296 (2002).

Scheme 5. Concept I for cleavage/heterocyclization using the diazonium functionality.


Although 1-alkyl and aryl benzotriazoles have been prepared fromsymmetrical and unsymmetrical diamines by diazotation in moderate togood yields,36 a synthesis starting from triazenes has not been disclosed.Benzotriazoles are important intermediates, protecting groups, activatinggroups, and final products in organic synthesis.37 Furthermore, benzotria-zoles are indispensable tools for the synthesis of various functional groups.Various 1-alkyl benzotriazoles are biologically active and show nanomolarbinding affinities to various proteins despite the fact that this structuralunit is not found in nature.38 The antiemetic and neuroleptic compound ali-zapride (Vergentan) is a 3-unsubstituted 3H-benzotriazole used for thetreatment of side effects in chemotherapy where cisplatin (Platinol), anantitumor agent, is used.39 Recently, benzotriazoles have served as linkersand auxiliaries in solid-phase synthesis.40–42 Therefore, the conversion ofthe polymer-bound fluoronitroarenes to 1-alkyl benzotriazoles was investi-gated. After nucleophilic displacement with primary amines to furnish theaniline resins 37, the cleavage with trifluoroacetic acid in dichloromethaneproceeded smoothly at room temperature within minutes giving the de-sired 1-alkyl-5-nitro-1H-benzotriazoles 38 in excellent yields and purities(Scheme 6). This route was successfully adopted for the synthesis of a

43
200-member library by means of automated synthesis.
36 J. C. Muir, G. Pattenden, and T. Ye, Tetrahedron Lett. 39, 2861 (1998).37 A. R. Katritzky, X. F. Lan, J. Z. Yang, and O. V. Denisko, Chem. Rev. 98, 409 (1998).38 C. E. Augelli-Szafran, T. S. Purchase, B. D. Roth, B. Tait, B. K. Trivedi, M. Wilson,

N. Suman-Chauhan, and L. Webdale, Bioorg. Med. Chem. Lett. 7, 2009 (1997).39 K. Munstedt, I. Wunderlich, E. Blauth-Eckmeyer, M. Zygmunt, and H. Vahrson, Oncology

55, 293 (1998).40 A. R. Katritzky, S. A. Belyakov, and D. O. Tymoshenko, J. Comb. Chem. 1, 173 (1999).41 A. Paio, A. Zaramella, R. Ferritto, N. Conti, C. Marchioro, and P. Seneci, J. Comb. Chem.

1, 317 (1999).42 K. Schiemann and H. D. H. Showalter, J. Org. Chem. 64, 4972 (1999).43 M. E. P. Lormann, M. Es-Sayed, and S. Brase, unpublished results (2002).

Scheme 6. Synthesis of benzotriazoles.


Other heterocyclic systems that can be prepared include cinnolines41, which are available from o-alkynylaryl triazenes via a von Richtercleavage-cyclization strategy. Cyclizations of diazonium salts having anortho-positioned electron-rich double or triple bond have been known forover a century through the von Richter (ortho-alkynyl),44 Widman–Stoer-mer (ortho-alkenyl),45,46 or Borsche–Koelsch (ortho-acetyl)47,48 reactions,all of which yield cinnoline derivatives, an interesting set of building blocksfor biologically active compounds. In particular, 4-halocinnolines can serveas valuable starting materials due to the ease of halogen substitution. Thefirst solid-phase von Richter reaction was reported with the triazene T1

linker.49

Starting with the immobilization of diverse ortho-haloaryl diazoniumcompounds, the palladium-catalyzed cross-couplings were performedunder standard conditions [Pd(OAc)2, NEt3, DMF, 80

�] with different

alkynes affording ortho-alkynylarene resins 40. The von Richter cleavagereactions were conducted with aqueous hydrogen chloride or hydrogenbromide in acetone or dioxane for 1 h at room temperature. Filtrationand dilution of the filtrates with water afforded the expected cinnoline li-brary format (146 member) in a 47–95% yield range and with 60–95%purity without any further purification (NMR, GC, GC-MS) (Scheme 7).

Concept II is based on the flexibility of the azide functionality.Upon thermolysis or photolysis, appropriately substituted aryl azides losenitrogen gas to give intermediate nitrenes, which in turn cyclize with suit-able ortho-substituents to give benzoannelated heterocycles. An unsatur-ated ortho-substituent consisting of double bonds including carbon,

44 V. von Richter, Ber. Dtsch. Chem. Ges. 16, 677 (1883).45 O. Widman, Ber. Dtsch. Chem. Ges. 17, 722 (1884).46 R. Stoermer and H. Fincke, Ber. Dtsch. Chem. Ges. 42, 3115 (1909).47 W. Borsche and A. Herbert, Liebigs Ann. Chem. 546, 293 (1941).48 C. F. Koelsch, J. Org. Chem. 8, 295 (1943).49 S. Brase, J. Heuts, and S. Dahmen, Tetrahedron Lett. 40, 6201 (1999).

Scheme 7. Synthesis of cinnolines.


oxygen, or nitrogen atoms then gives indoles/carbazoles (starting fromC——C),50 indazoles (C——N),50,51 benzoisozazoles (C——O), benzooxadia-zoles (benzofurazanes) (N——O),52 benzimidazoles (N——C), or benzotria-zoles (N——N) (Scheme 8). Alternatively, the intra- or intermolecularaza-Wittig reaction might also be envisaged,53 in which case larger ringsizes (six- and seven-membered rings) are also achievable.

One of the first examples of azide thermolysis cyclization reactions wasdemonstrated in the synthesis of biologically active benzofuroxanes.49 Thecyclization of aryl azides 51 based on the ortho-nitro resins 50 was achievedat ca. 70

�to give the benzofuroxanes 52 (Scheme 9).

Triazene T2 Linker

Whereas the T1 linker involves an immobilization of a diazonium salton an amine resin, the T2 linker represents the reversal of this concept.As outlined in Scheme 10, m-aminophenol (54) was attached to Merrifieldresin 53 by displacing the chloride group via the phenolic group with theaid of sodium hydride in dimethylformamide (DMF) (see Experimental

50 V. V. Rozhkov, A. M. Kuvshinov, V. I. Gulevskaya, I. I. Chervin, and S. A. Shevelev,

Synthesis 2065 (1999).51 B. J. Clark and R. J. Grayshan, Chem. Res. Miniprint 3786 (1981).52 M. R. Kamal, M. M. El-Abadelah, and A. A. Mohammad, Heterocycles 50, 819 (1999).53 S. Brase, C. Gil, K. Kuepper, Bioorg. Med. Chem. 10, 2415–2437 (2002).

Scheme 8. Concept II for cleavage/heterocyclization using the azide functionality.

Scheme 9. Synthesis of benzofuroxanes.

Scheme 10. Synthesis of the T2 linker.



section).14,54 m-Aminophenol was used as the linker molecule because it isnot as easily oxidized as other aminophenols. Diazotation of the anilineresin 55 at �10

�in THF with tBuONO and boron trifluoride etherate

yielded resin 56, which is stable several hours at this temperature.Various diazonium salts have been prepared using this approach55 and

thermoanalytically characterized.56 Both the structure of the diazoniummoiety and the counterion clearly influence the stability of the diazoniummoiety. The thermally stable diazonium ion 71 (Z ¼ Cl, Y ¼ CH2O) [t1/2

(25�) > 100 days] is also capable of scavenging various nucleophiles

(amines, phenols, and anilines).57 This resin with a tetrafluoroborate coun-terion (resin 66) is called T2* diazonium resin and it is now commerciallyavailable from Novabiochem.

Coupling of the diazonium resins such as the T2 diazonium resin 56with various primary or secondary amines at �10

�to room temperature

led to the formation of a series of new triazene resins (Scheme 10) (seeExperimental section).

In addition to primary or secondary amines, attachment of hydro-xylamine, hydrazines, sulfoximines, or phenols proceeds equally well(Scheme 10). Secondary amines can be cleaved directly from the resin,while primary amines give rise to a different reaction pathway (see below).

Primary amines can be derivatized on the free N–H functionality andtherefore can be modified to an array of products. Thus, ureas 75,54 thiour-eas 74,58 guanidines 62,58 and carboxamides 7654 were prepared inexcellent yields (see Scheme 14).

Solid-phase peptide synthesis (SPPS) has provided various solutions forthe linking, chemical transformation, and detachment of amide structuresto the chemistry community. In general, these protocols involve the attach-ment of amine derivatives using a carbon linkage or, in case of amino acids,by their carboxy functionality. Linking by the N–H functionality of theamide bond has been developed through the so-called backbone amide lin-kers (BAL). Originally designed for the N–H protection of amide bonds tocircumvent �-turns and other problems during peptide synthesis, theseamide-protecting groups can also serve as linkers for SPPS. Barany andco-workers59 described an application of a backbone amide linker for the

54 S. Brase, S. Dahmen, and M. Pfefferkorn, J. Comb. Chem. 2, 710 (2000).55 S. Brase, S. Dahmen, and M. Schroen, unpublished results (2000).56 S. Brase, S. Dahmen, C. Popescu, M. Schroen, and F.-J. Wortmann, Polym. Degr. Stab. 75,

329 (2002).57 S. Dahmen and S. Brase, Angew. Chem. Int. Ed. 39, 3681 (2000).58 S. Dahmen and S. Brase, Org. Lett. 2, 3563 (2000).59 K. J. Jensen, J. Alsina, M. F. Songster, J. Vagner, F. Albericio, and G. Barany, J. Am. Chem.

Soc. 120, 5441 (1998).


synthesis of oligopeptides based on the peptide amide linker (PAL) con-cept. Recently, a new backbone amide linker using indole chemistry hasalso been devised.60

The T2 linker has recently been shown to be a versatile backbone amideanchor. Immobilized disubstituted triazenes were acylated with carboxylicacid anhydrides or chlorides to give amide derivatives. These amides werecleaved under very mild conditions using trimethyl chlorosilane. This se-quence thus employs the T2 system as backbone amine linker and wasdemonstrated in the automated library synthesis of substituted amidederivatives.54

Urea derivatives, which are important biologically active compoundsand building blocks for organic syntheses, have been previously synthe-sized on solid support by various strategies.4,6 In our case, starting withthe immobilization of alkene-amines on the T2 linker, the resulting tria-zenes were treated with isocyanates in THF at room temperature with acatalytic amount of triethylamine. Dihydroxylation of the alkene groupwas carried out with commercially available AD-mix � (asymmetric dihy-droxylation mix) in various solvent mixtures without the use of furtheradditives. The optimum mixture was found to be THF and water (v:v5:1), which apparently favors the swelling properties as well as the dihy-droxylation mechanism. The cleavage was conducted using trimethylsilylchloride (TMSCI) in dichloromethane (10%) at room temperature. Theresulting ureas 58 were isolated in fair to good yields and excellent purities,both of which were consistently greater than 92% (Scheme 11). This syn-thesis was successfully transferred to a Bohdan Neptune station using aprototype solid-phase unit (see Experimental section) as well as to theBohdan Miniblock system to yield small libraries of urea derivatives.54

Guanidines are basic molecules with the capacity to form H-bondinginteractions. They are a promising class of potentially useful pharmaco-logically active compounds61 and their liquid phase synthesis has foundwidespread applications in organic chemistry.62

The solid-phase synthesis of guanidines, however, focuses mainly onthree different approaches: the formation of resin bound carbodiimides63

and their reaction with amines, the solid-phase synthesis involving electro-philes in solution,64 and the reaction of supported thioureas with amines.65

60 K. G. Estep, C. E. Neipp, L. M. S. Stramiello, M. D. Adam, M. P. Allen, S. Robinson, and

E. J. Roskamp, J. Org. Chem. 63, 5300 (1998).61 R. G. S. Berlinck, Nat. Prod. Rep. 16, 339 (1999).62 J. Chen, M. Pattarawarapan, A. J. Zhang, and K. Burgess, J. Comb. Chem. 2, 276 (2000).63 D. H. Drewry, S. W. Gerritz, and J. A. Linn, Tetrahedron Lett. 38, 3377 (1997).64 S. Robinson and E. J. Roskamp, Tetrahedron 53, 6697 (1997).65 P. C. Kearney, M. Fernandez, and J. A. Flygare, Tetrahedron Lett. 39, 2663 (1998).

Scheme 11. Synthesis of ureas using the T2 linker.

Scheme 12. Synthesis of guanidine libraries.


The triazene T2 linker14 and the improved T2* linker57 systems offer aunique approach to the formation of guanidines in which all three substitu-ents can be varied to a wide extent. Starting from disubstituted triazene onthe T2* linker, deprotonation using NaH/DMF and subsequently acylationby the addition of isothiocyanates (Scheme 12) yielded a library of resin-bound thioureas. For the reaction of the thioureas with amines, the use


of mercury(II) oxide (caution: very toxic) proved to be superior over awhole variety of coupling reagents that were envisaged. Traces of theformed insoluble black mercury(II) sulfide could be efficiently removedby simple filtration of the cleavage solution over a short pad of silica. Inthe final diversification step before cleavage, the reaction of the diaze-nylthiourea resins 63 with ammonia, primary and secondary amines, wasconducted under optimized reaction conditions. Cleavage of polymer-supported diazenylguanidines with 10% TFA in dichloromethane yieldedguanidines 62 as their trifluoroacetate salts with high purities (>90%). Elu-tion of the TFA salts in methanol over a short column of basic anion-exchange resin [strong anion-exchange resin (Lewatit MP 5080, MerckDarmstadt)] efficiently produced the nonprotonated guanidines 62 in highyield (Scheme 12) (see Experimental section).

While the cleavage of trisubstituted triazenes gives rise to the formationof secondary amines in excellent yields, the cleavage of disubstituted tria-zene 69 gives rise to aliphatic diazonium salts. The newly formed diazoniumion undergoes substitution with the nucleophile present in the reaction mix-ture. Therefore, alkyl halides 79-X,66 alcohols 78-OH,67 ethers 78-OR1,67 aswell as alkyl carboxylic 78-OCOR, sulfonic esters 78-OSO2R68 phosphoricesters 78-OPO(OR)2,69 and phosphinic esters 78-OPO(H)(OR)67 can beformed by cleavage with trimethylsilyl halides (X ¼ I, Br, Cl), aqueous tri-fluoroacetic acid,67 carboxylic acids,70 sulfonic acids, phosphoric acids, andphosphoric acids, respectively. The regioselectivity of the cleavage can beexplained by the presence of one tautomer of the triazene in which thehydrogen atom is on the triazene-nitrogen linked to the arene ring. Overall,this reaction sequence provides the means for substituting an amino groupfor an oxygen or a halogen (Cl, Br, I) atom (Scheme 13).

In summary, the triazene T2 linker system displays an original anchoringgroup with ample possibilities for variations (Scheme 14).

Summary and Conclusion

Over the past years, various new types of linkers have emerged. Espe-cially for the synthesis of small molecules on solid support, the design of anew anchoring group might be essential for the success of the synthesis.

66 C. Pilot, S. Dahmen, F. Lauterwasser, and S. Brase, Tetrahedron Lett. 42, 9179 (2001).67 S. Brase and C. Pilot, unpublished results (1998).68 N. Vignola, S. Dahmen, D. Enders, and S. Brase, Tetrahedron Lett. 42, 7833 (2001).69 N. Vignola, S. Dahmen, D. Enders, and S. Brase, J. Comb. Chem. 5, 138 (2003).70 J. Rademann, J. Smerdka, G. Jung, P. Grosche, and D. Schmid, Angew. Chem. Int. Ed. 40,

381 (2001).

Scheme 13. The multifunctional cleavage of the T2* linker.

Scheme 14. Possibilities with the T2 linker.


Linker, cleavage conditions, and functional groups are appointed to eachother. Therefore, the decision to use a specific linker has to be balancedwith the nature of the library to be synthesized.



Chemicals, Solvents, Reagents

Chemicals were purchased from Aldrich, Fluka, Janssen, and Merck.Merrifield resin (1–2% cross-linked, 200–400 mesh) was obtained fromNovabiochem or Polymer Laboratories. To obtain the molecular mass ofthe resin and to calculate the elemental analysis the following calculationhas to be performed:

molar massnew ¼ 1000

Loadingold

� ðmolar massSub � molar massAddÞ (1)

Solvents (benzene, ether, tetrahydrofuran, dichloromethane) for reac-tions involving organometallic and other sensitive materials were distilledunder argon prior to use. All resins were washed sequentially by using avacuum reservoir connected to a sintered glass frit. Cleavage was con-ducted using Teflon tubes with a frit connected to a vacuum line or witha glass pipette filled with glass wool. Evaporation of the solvent wasachieved using a rota-evaporator and/or high vacuum (ca. 0.1 mbar).

T1 Linker: Synthesis of Benzylaminomethyl Polystyrene

To a suspension of 10 g of Merrifield resin in 100 ml of dry DMF wereadded 5 equivalents of benzylamine (20 equivalents for the synthesis ofpiperazinomethyl polystyrene) and 1 equivalent of potassium iodide. Thereaction mixture was agitated with an overhead stirrer at 80

�for 72 h. After

cooling the mixture to room temperature, the resin was filtered on asintered frit and washed with the following solvents (50 ml portions):DMF, methanol, DMF, methanol, water, DMF, methanol, DMF, diethy-lether, dichloromethane, diethylether, dichloromethane, diethylether, di-chloromethane, and diethylether. The resin was then dried under vacuum.

Representative Procedure for the Synthesis of Triazene T1 Resins

With continuous stirring, 2.5 equivalents of 4-fluoro-3-nitroaniline(4.02 g, 26 mmol) was dissolved in 50 ml of dry dichloromethane and20 ml of CH3CN. After cooling this mixture to �100, 2.5 equivalents of tri-fluoroacetic acid were added dropwise followed by the slow addition (twoportions with a 30-min interval) of 2.5 equivalents of i-C5H11ONO. The re-action mixture was then stirred for 1 h between �10

�and �5

�, cooled with

an acetone/dry ice bath to approx. �78�, and then quenched under stirring

with ether (50 ml). The solvent was decanted and another 50 ml of etherwas added. This washing process was repeated three times. The resulting


suspension was warmed to �15�

and 1 equivalent of benzylaminomethylpolystyrene (10 g) and approximately 20 ml of pyridine were added. The re-action was stirred for a few minutes until all of the resin turned red. Theresin was then washed with a 3:1 ratio mixture of absolute DMA and pyri-dine, then washed once with diethyl ether, and then with distilled pentaneto shrink the resin. The resin 36 was then washed with alternate cycles ofdiethyl ether and dichloromethane until the resin was clean and nocolorization could be seen in the solvents after washing. To finish preparingthe resin, it was washed with distilled pentane and then freeze-driedovernight.

General Procedure for the Traceless Cleavage of the T1 Linker

A suspension of the resin (200 mg) prepared as above in dichloro-methane was treated with 4 equivalents of trichlorosilane (Caution: corro-sive and low boiling material), heated to 40

�under stirring for 15 min or at

room temperature for 60 min. After cooling the mixture to room tempera-ture, silica gel (100 mg) was added, the mixture was filtered, and the solventremoved.

The T2 Linker: 3-Aminophenyl-1-oxymethylpolystyrene (55)

A dry 500-ml, three-necked round-bottom flask was fitted with a mech-anical stirrer, gas inlet, and addition funnel. The apparatus was purged withargon and charged with 250 ml of dry DMF and 2.52 g (63.0 mmol, 1.51 g,60% in paraffin) of sodium hydride. After adding 20.0 g (12.8 mmol,loading ¼ 0.64 mmol/g) of Merrifield resin (53), 6.9 g (63 mmol) of m-aminophenol was added portion wise (caution: H2 evolution). After areaction time of 20 h, the resin was then washed on an inert gas frit withsolvents (three times with each approx. 200 ml): THF, Et2O, and MeOH.Subsequently the resin was dried in vacuo. –IR (KBr): v ¼ 3390 cm�1,3200, 3080, 3060, 3020, 2910, 2840, 2640, 2600, 2310, 2340, 2380, 2260,2110, 1940, 1870, 1800, 1710, 1670, 1620, 1590, 740, 690. –C125H125ON(1655.0): calcd C 90.63, H 7.55, N 0.85; found C 89.90, H 8.03, N 0.89.

Preparation of Diazonium Salt 56 on the Resin

The above amine resin 55 (10.0 g, 6.4 mmol) was suspended in dry THFand cooled by means of a cold bath (EtOH/dry ice) to �20

�. After 20 min,

BF3�Et2O (6.9 ml, 7.7 g, 54 mmol) was added and then after 5 min tert-BuONO (5.7 ml, 5.0 g, 49 mmol) was added. After a reaction time of30 min, the mixture was collected in an inert gas frit, filtered, and washedwith chilled THF (4 � 15 ml/g resin).


Preparation of Triazene Resin 4

Resin 56 was swelled in THF (15 ml/g resin) at �10�

and treated with anamine (5 equivalents). After a reaction time of 1 h, a solution of MeOH inTHF (1:1 v/v) was added to quench the reaction. The resin was then washedon an inert gas frit with the following solvents (three times with eachapprox. 20 ml for each solvent per 1.00 g resin): THF, Et2O, and MeOH.Subsequently the resin was dried in vacuo.

Manual Preparation of Ureas

Resin 4 obtained as described above was suspended in dry THF(10 ml/g resin) under argon for 10 min and treated with triethylamine(1 equivalent) and an isocyanate (4 equivalents). After a reaction time of1 h, a solution of MeOH in THF (50%) was added to quench the reaction.The resin was then washed on an inert gas frit with the following solvents(three times with each approx. 20 ml for each solvent per 1.00 g resin):THF, Et2O, and MeOH. Subsequently the resin was dried in vacuo. A fil-tration setup, consisting of a glass pipette filled with a plug of glass wool,was filled with 100 mg of the resin and treated three times with 1.5 ml cleav-age solution (5% TFA in CH2Cl2) for 5 min at room temperature, by whichthe resin turns red. The combined filtrates were concentrated in vacuo.

Automated Preparation of Amides

Resin 4 (1.5 g) was suspended in 7.5 ml CH2Cl2 and 7.5 ml DMF. Ineach reaction vessel was distributed 2.5 ml (250 mg, 0.1575 mmol, loading0.64 mmol/g) of this isopycnic resin suspension. After being filtered, theresin was washed twice with 2.0 ml THF and finally suspended in 1 mlTHF. The reaction chambers used for the reaction with the acid chlorideswere filled with 0.3 ml triethylamine in THF (0.36 mol/liter, 2 ml, 4-foldexcess). The reactants were added and the reaction chambers were agitatedin a parallel compartment overnight at room temperature. The resin waswashed (two cycles with each 4.0 ml CH2Cl2 and MeOH, then 4.0 mlCH2Cl2) and the solvent was removed in vacuo. The cleavage was con-ducted with 10 ml of a 10% TMSCl solution in CH2Cl2 under mechanicalagitation for 1 h. Subsequently, after filtering and washing the resin with1.0 ml of CH2Cl2, the combined filtrates were concentrated on a parallelevaporator. The yields were measured automatically. Except for the sus-pending and the weighing process as well as the transport of the shakerunit, all processes have been fully automated.


Synthesis of Guanidines Using the T2* Linker

In a three-necked round-bottom flask equipped with a mechanical stir-rer was suspended 10.0 g (5.3 mmol, loading 0.53 mmol/g) of T2* diazoniumresin in THF. The mixture was cooled to �5 to �10

�and a primary amine

(2.65–5.3 mmol, 5–10 equivalents) was added under stirring. After stirringfor 0.5–1 h, the ice bath was removed and the mixture allowed to reachroom temperature. The resin was filtered off, washed sequentially withTHF and methanol, and dried under high vacuum. The resulting resin(2.0 g, 0.96–0.90 mmol, loading 0.48–0.45 mmol/g) was then suspended in25 ml of anhydrous DMF under argon. NaH (490 mg, 12 mmol, 60% in min-eral oil) was added and the mixture was agitated under a stream of argon.After 2 min, an isothiocyanate (6 mmol, 6.25–6.7 equivalents) was addedand the mixture was agitated for another 2 h at room temperature. The re-action was quenched by the addition of methanol, the resin filtered off, se-quentially washed with THF and methanol, and then dried under highvacuum. A 15-ml vial was charged with 250 mg (0.11 mmol, loading0.45 mmol/g) of resin and 100 mg (0.46 mmol, 4.2 equivalents) of orangeHgO (caution: very toxic). A magnetic stirring bar was added and the vialwas closed. THF (4 ml) and aqueous ammonia solution (2 ml, 25% inwater) were added through the septum and the mixture was agitated for12–24 h at 45

�. After being cooled to room temperature, the vials were

opened and the resin filtered off and sequentially washed with THF andMeOH. The resin (100–300 mg, 0.14–0.4 mmol) was placed in a 10-ml tubeequipped with a frit and 4–6 ml of TFA solution (10% in dichloromethane,v:v) was eluted over the resin. The filtrate was evaporated under reducedpressure.

Acknowledgments

The chemistry described has been conducted by a young and enthusiastic team, whose

names appear in the appropriate references. We thank our academic and industrial partners

for fruitful collaborations and comments. This work was supported by the DFG (Deutsche

Forschungsgemeinschaft). Bayer, BASF, Novabiochem, and Grunenthal are also gratefully

acknowledged.

[8] tfp-activated resins for amine derivatization 151

[8] The Development and Applicationof Tetrafluorophenol-Activated Resins for Rapid

Amine Derivatization

By Joseph M. Salvino and Roland E. Dolle

Introduction

Tetrafluorophenol (TFP)*-activated resins1–3 are reactive polymericacylating and sulfonylating reagents useful for synthesizing pure single-compound arrays of amides and sulfonamides. TFP-activated resins maybe regarded as resin-bound equivalents to acid chlorides. They are easyto prepare in a single step from commercially available polymer-supportedTFP, are stable to prolonged storage, and react with a wide range of N-nucleophiles including anilines. This chapter provides detailed protocolsfor synthesizing the reagents, establishing reagent loading, and reactionwith amines.

Background

Amines and their amide and sulfonamide congeners are undoubtedlythe most prevalent functionality found in drug leads and clinical therapeut-ics. It is therefore a rather common occurrence that once an amine-bearinglead structure is identified, a derivatization campaign is initiated to estab-lish a structure–activity relationship (SAR). Such a campaign generallymakes use of reductive amination, acylation, sulfonylation, and ureidochemistries to create a set or library of amine analogs for biological evalu-ation. With the advent of combinatorial chemical technology, a number ofhigh-speed synthesis protocols for amine derivatization have been de-veloped, suitable for both parallel (discrete compound) and split- and-pool(mixture synthesis) formats. In cases where the amine species is attached to

* Abbreviations: DCM, dichloromethane; DIC, 1,3-diisopropylcarbodiimide; DIEA, diiso-

propylethylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide;

ELSD, evaporative light scattering detection; HOBt, hydroxybenzotriazole; IR, infrared;

LC/MS, high-pressure liquid chromatography/mass spectrometry; NMM, N-methylmorpho-

line; NMR, nuclear magnetic resonance; PyBop, benzotriazol-1-yloxytripyrrolidino-

phosphonium hexafluorophosphate; SAR, structure–activity relationship; TFP,

tetrafluorophenol; THF, tetrahydrofuran.1 J. S. Salvino, N. V. Kumar, E. Orton, J. Airey, T. Kiesow, K. Crawford, R. Mathew,

P. Krolikowski, M. Drew, D. Engers, D. Krolikowski, T. Herpin, M. Gardyan, G. McGeehan,

and R. Labaudiniere, J. Comb. Chem. 2, 691 (2000).




solid support, it may be reacted in a straightforward fashion with a diverseset of derivatizing reagents/conditions and cleaved from resin to providematerial for biological testing. However, in cases in which the aminespecies may not be conveniently attached to a solid support or it is not de-sired to do so, it is necessary to conduct derivatization directly in solution.One of the more convenient protocols to facilitate the latter process is thereaction of amines with polymeric-activated resins (Table I).4–20

The use of polymeric supported reagents for amine acylation was con-ceived over 30 years ago primarily for application in peptide synthesis.4,5

When used in excess, activated esters derived from polystyrene-bound nitro-phenol6,7 and N-hydroxybenzotriazole (HOBt)8 were shown to effect fastand quantitative acylations with primary amines including amino acids andpeptide fragments. Product isolation required simple filtration. Several vari-ations of these reagents have since been reported in the literature,9–11 in-cluding HOBt resin (a variant of Patchornik’s original HOBt resin),9

pyrimidine esters,10 dihydropyridino[2,3-d]pyrimidine amides,11 selenoesters,12 phenol esters on macroporous polymer disks,13 activated acyltria-zoles,14 hydroxamic esters,15 esters of pyrimidine N-oxide and azoles,16 acy-loxyquinolines,17 thiol esters,18 oxime esters,19 and Kenner’s ‘‘safety-catch’’resin.20 One or more technical deficiencies, however, have prevented their

2 Y. Gong, M. Becker, Y. M. Choi-Sledeski, R. S. Davis, J. M. Salvino, V. Chu, K. D. Brown,

and H. W. Pauls, Bioorg. Med. Chem. Lett. 10, 1033 (2000).3 J. M. Salvino, B. Gerard, H.-F. Ye, B. Sauvagnat, and R. E. Dolle, J. Comb. Chem. 5,

260 (2003).4 A. Patchornik, M. Fridkin, and E. Katchalsky, German Patent 1913486 (1969). Chem.

Abstr. 72, 66932y (1970).5 D. L. Marshall and I. E. Liener, J. Org. Chem. 35, 867 (1970).6 R. Kalir, M. Fridkin, and A. Patchornik, Eur. J. Biochem. 42, 151 (1974).7 B. J. Cohen, H. Karoly-Hafeli, and A. Patchornik, J. Org. Chem. 49, 922 (1982).8 R. Kalir, A. Warshawsky, M. Fridkin, and A. Patchornik, Eur. J. Biochem. 59, 55 (1975).9 I. E. Pop, B. P. Deprez, and A. L. Tartar, J. Org. Chem. 62, 2594 (1997).

10 M. Botta, F. Corelli, E. Petricci, and C. Seri, Heterocycles 56, 369 (2002).11 R. B. Nicewonger, L. Ditto, D. Kerr, and L. Varady, Bioorg. Med. Chem. Lett. 12, 1799 (2002).12 H. Qian, L.-X. Shao, and X. Huang, Synlett 1571 (2001).13 J. A. Tripp, F. Svec, and J. M. J. Frechet, J. Comb. Chem. 3, 604 (2001).14 A. R. Katritzky, A. Pastor, M. Voronkov, and D. Tymoshenko, J. Comb. Chem. 3, 167 (2001).15 P. N. Sophiamma and K. Sreekumar, Reactive Funct. Polym. 35, 169 (1997).16 I. Kakobsone, M. Klavins, and A. Zicmanis, Latv. PSR Zinat. Akad. Vestis, Kim. Ser. 483

(1990); 481 (1989).17 X. Huang, C. C. Chan, and Q. S. Zhou, Makromol. Chem. Rapid Commun. 6, 397 (1985).18 M. Stern, M. Fridkin, and A. Warshawsky, J. Polym. Chem. Ed. 20, 1569 (1982).19 W. F. Degrado and T. E. Kaiser, J. Org. Chem. 47, 3258 (1982).20 G. W. Kenner, J. R. McDermott, and R. C. Sheppard, J. Chem. Soc. Chem. Commun.

636 (1971).

TABLE I

Selected Examples of Known Linkers for Generating Activated Resins for Amine

Derivatization

Entry Linkera Transfer group(s) (reference)

1 –C(O)R7

2 –C(O)R5

3 –C(O)R8

4 –C(O)R20

5 –C(O)R,–SO2R, –CH2CO2R1

6 –C(O)R10

7 –C(O)R11

a The asterick (*) indicates the atom to which the transfer group is attached.



widespread use in the production of single compound arrays. These defi-ciencies include the following: the inherent reactivity of the polymeric acti-vated resin can limit the scope of N-nucleophiles that may react with thereagents; the instability of the activated resin makes resin manipulationand storage inconvenient; the method of activation can be a potential issue,e.g., side products are observed using hydrogen peroxide5 or alkylatingagents20 in activation protocols; it may be difficult to establish the absoluteloading of the activated resins to ensure that a limiting amount of amine isused during derivatization; and known resin linkers are largely limited toamide bond formation and are not generally suitable for expansion to sul-fonylations, carbamoylations, urea synthesis, and carbon–carbon bondformation.

Recently, Salvino et al.1 developed TFP-activated resins as novel re-agents to specifically address many of the liabilities listed above. PolymericTFP-activated carboxylate esters react with amines with broad chemical re-activity, analogous to the well-known reactivity of pentafluorophenol carb-oxylate esters commonly used for solution amide coupling. Due to theelectron-withdrawing carbonyl group in the 4-position of the TFP linker,polymeric TFP sulfonate esters are sufficiently activated to react smoothlywith amines yielding sulfonamides. By virtue of the fluorine atoms presenton the linker, the loading (esterification) to the resin and its subsequent re-lease (amide/sulfonamide formation) can be quantitatively measured using19F NMR.1,21,22

Synthesis of Polymeric TFP

The synthesis of polymeric TFP-activated resins is performed withsimple equipment and apparatus. Commercially available aminomethylpolystyrene (Polymer Labs) is acylated with 4-hydroxy-2,3,5,6-tetrafluoro-benzoic acid hydrate (Aldrich) using HOBt and 1,3-diisopropylcarbodii-mide (DIC) as coupling reagents in dimethylformamide (DMF; Fig. 1) togive the TFP-linked resin. During the coupling procedure, ca. 10% TFPtetrafluorobenzoic acid ester is formed by reaction of the unprotectedphenol oxygen with the activated fluoroinated benzoic acid. Ester forma-tion is clearly evident by the ester carbonyl stretch at 1765 cm�1 in the IRspectra of the resin and by the two extra F signals in the 19F NMR. Treatingthe resin with a slight excess of piperidine in DMF liberates the phenol. It is

21 M. Drew, E. Orton, P. Krolikowski, J. M. Salvino, and N. V. Kumar, J. Comb. Chem. 2,

8 (2000).22 A. Svensson, K.-E. Bergquist, T. Fex, and J. Kihlberg, Tetrahedron Lett. 39, 7193 (1998) and

references therein.

Fig. 1. Preparation of TFP resin from aminomethyl polystyrene and 4-hydroxy-2,3,5,6-

tetrafluorobenzoic acid.


essential that the piperidine treatment be carried out because residualphenol ester generates a corresponding 4-hydroxy-2,3,5,6-tetrafluorobeza-mide or -sulfonamide contaminant upon amine derivatization. Followingacidification of the resin, washing, and drying, the TFP resin is ready for ac-tivation with carboxylic and sulfonic acids. The loading of TFP to methyla-minopolystyrene resin is determined by elemental analysis by F ionselective chromatography. Typical loading values are within �70% of thestarting polystyrene loading.

Synthesis of TFP-Activated Resins

Acylation of the TFP resin to furnish carboxylate-activated resins maybe carried out by using three complementary protocols (Fig. 2). One opti-mized protocol is TFP resin esterification with a carboxylic acid and DICin dichloromethane (DCM).3 TFP resin is first swelled with DCM for 10 minmin with mild agitation. The acid (3.5 equiv.) is added and agitated gentlyuntil it dissolves. In cases where the acid is poorly soluble in DCM, it mayfirst be dissolved in DMF. Hydroxybenzotriazole (HOBt, 0.1 equiv.) is thenadded to the reaction mixture followed by the addition of DIC (3.5 equiv.).The acylation reaction is agitated for 12 h at room temperature after whichtime the resin is collected by filtration and washed with DMF, THF (tetrahy-drofuran), and DCM (dichloromethane), and dried in vacuo. In an earlierprotocol,1 DMAP [4-(N,N-dimethylamino)pyridine] was used in place ofHOBt, but it was found that loadings were irreproducible (unpublished ob-servation). Alternative protocols for phenol acylation employ either acidchlorides [N-methylmorpholine (NMM) as base in DCM] or carboxylic acidsodium salts and PyBop in DMF. The activated TFP reagents are stablewhen dry and no special handling is required. Although no detailed stability

Fig. 2. Preparation of polymeric TFP-activated carboxylate and sulfonate ester resins.


studies have been carried out, many TFP reagents in our laboratorieshave been stored at room temperature for >2 years without loss of activity.

The reaction of TFP resin with sulfonyl chlorides (3 equiv.) in the pres-ence of diisopropylamine (DIEA; 3 equiv.) provides the corresponding sul-fonate-activated resins (Fig. 2). Alternatively, sulfonic acids may be loadeddirectly onto TFP using DIC (via the intermediate sulfonic acid anhydride)as the coupling reagent. Analogous to the carboxylate esters, the sulfonateester resins are stable when dry and may be kept at room temperature foran extended period of time without decomposition. This is one of the clearadvantages of the polymeric TFP reagents versus acid chlorides; the latterdecompose once exposed to air and moisture.

Quality Control of TFP-Activated Resins

Activated TFP resins may be created from a wide selection of structuralclasses (Table II). The coupling yield or loading of carboxylic or sulfonicacids to TFP resin varies as a function of acid. For carboxylic acids, typicalloadings achieved are on the order of 70–80% while sulfonic acid loadingapproaches 100%. However, loadings from 100% to <10% are observeddepending on the reactivity of the acid and the presence of other functionalgroups and, hence, it must be empirically determined. Examples of acidsthat do not load well include phenylacetic acids (likely forming ketenesupon addition of amine), acids containing amine functionality, acids con-taining a pendent functionality that may assist in ester bond cleavage,and certain oxazole carboxylic acids. Determination of loading is criticalto library purity and, therefore, it is essential to reliably establish couplingyield. There are two options available to do this: on-resin 19F NMR analysisor by reaction of a standard amine with resin and measuring the conversion

TABLE II

Examples of Diverse Building Blocks Used in the Preparation and Reactions of

TFP-Activated Resins


to amide off-resin via 1H NMR. Each custom prepared activated TFP resinis subjected to one of these options as a rigorous quality control step.

Both the quality of TFP resin and the resulting polymeric-activatedresins may be quantitatively determined by 19F NMR spectroscopy.1 Thisnondestructive technique takes advantage of the high sensitivity, naturalabundance (the 1/2 spin nucleus 19F is 100% abundant), and large chemicalshift dispersion (�200 ppm) of the 19F nucleus. The 19F NMR spectrum ofTFP resin shows two resonances at �148 and �165 ppm (Fig. 3), each cor-responding to 2 equiv. fluorine nuclei. The 19F NMR spectrum of TFP-activated carboxylate and sulfonate resins shows two resonances at ca.�145 and �154. Gel phase 19F NMR spectra are easy to interpret. Thereis only one resonance line per nonequivalent F and nonfluoroinated re-siduals are transparent to the method. Relative resin loading23 is quantified

23 Absolute loading is established by elemental analysis (F ion chromatography).

Fig. 3. 19F NMR illustration indicating resonances for the two equivalent F nuclei in

nonactivated and activated TFP resins.


by simply integrating the F signals resulting from free and activated formsof polymeric TFP. The analytical method can be done in a high-throughputmode with 100 samples processed overnight using 50 mg of resin suspendedin deuterated DMF in a standard 5-mm NMR tube.

In lieu of 19F NMR spectroscopy, a second method for determiningloading involves the reaction of an accurately weighed sample of resin witha known quantity of a reactive amine, e.g., 2-methylbenzylamine. The reac-tion product is evaluated by 1H NMR and LC/MS. Loading is determinedby integration and comparison of the aromatic methyl protons and the�-methylene protons of the product amide relative to the starting amine.

Reaction of TFP Reagents with N-Nucleophiles

The reaction of a TFP reagent with an amine is performed by first sus-pending a 20% excess of activated resin (loading established) in DMF. Alimiting amount of amine, 0.8 equiv. based on the resin loading, is addedto the resin as a dilute solution in a solvent such as DMF.24 The reaction

Fig. 4. Reaction of polymeric TFP-activated reagents with amines.


mixture is incubated for a minimum of 3 h, but typically is allowed to runovernight as a matter of convenience. Workup of the reaction requires fil-tration of the excess TFP-containing resin and collecting the filtrate. Asmall sample of the filtrate is analyzed by LC/MS and, optionally, evapora-tive light scattering (ELSD), to assess product purity (generally >95%)and to confirm compound identity. The remaining sample is concentratedto dryness to afford the derivatized amine that may be submitted forbiological evaluation (Fig. 4).

The process of library synthesis is outlined in Table III. For large librarycampaigns, 80 wells of a 96-deepwell plate (2 ml/well) are formatted insuch a fashion that one-half of the plate (40 wells) is charged with oneTFP resin, and the remaining half plate charged with a second TFP resin.Formatting is facilitated via a 96-well pipetting robot, such as the TomtecQuadra 3, or an eight-tip liquid handling robot, such as the PackardMultiprobe equipped with wide-bore needles. Resin can be accurately dis-pensed into the plate via a gently agitating resin slurry in THF–DCM (4:1).The solvent is evaporated by allowing the plate to stand in a ventilatedfume hood or concentrated using a plate evaporator, such as the GenevacHT-1 centrifuge evaporator. Upon dispensing 2 � 40 different amines asdilute solutions into the plate, 80 different reaction mixtures are created.After standing for 12 h, the mixtures are aspirated and dispensed into a96-well filter plate fitted with a 30-�m filter. A 96-deepwell plate is placeddirectly underneath the filter plate and the filtrate is collected. Dry prod-ucts are obtained upon concentrating the deepwell plate in a Genevac

24 The N-nucleophile may be a primary or secondary amine, or an aniline. N-Nucleophiles

that react poorly with TFP reagents include very hindered amines (e.g., o-substituted

anilines and 1-aminonaphthalene), deactivated anilines, and aminothiazoles.

TABLE III

Process for Library Synthesis Using Polymeric TFP-Activated Reagents

Step Task Activity

1 Procure building

blocks

Purchase or synthesize amines, acids, acid chlorides, sulfonic

acids, and sulfonyl chlorides

2 Generate TFP

reagents

Load carboxylic acid and sulfonyl chloride building blocks to

TFP resin

3 Quality control

reagents

Use 19F NMR spectroscopy or react each resin with

2-methylbenzylamine followed by 1H NMR analysis

4 Distribute resins Measure portions of resins into vials or plates and swell with

DMF and calculate reagent equivalents

5 Add amines Accurately weigh out amines and prepare dilute (10�M) stock

solutions in DMF; dispense 0.8 equivalent of each amine

into vials or plates

6 Incubate reaction

mixtures

Reaction mixtures agitated at room temperature for 12 h

7 Filter reaction

mixtures

Collect filtrates and discard spent resin

8 Product analysis Submit small portions of filtrates for LC/MS analysis; in case of

plates, create daughter plate(s) and submit for same;

establish purity and confirm identity of new derivatives

9 Final sample

preparation

Remove solvent from filtrates and dry sample in vacuo;

register compounds or plates and submit products for

biological testing


centrifuge.25 Analytical data are collected on approximately 20% of thecompounds in libraries with >1000 members.

For focused libraries, where one amine is converted to ca. 80 analogs, 80polypropylene fritted vessels or wells of a 96-deepwell plate are chargedwith 80 different TFP reagents. The focused library is obtained followingincubation of the dispensed resins with one amine and workup as describedabove. Analytical data are collected on all the members of the library.

Adolor Corporation currently possesses a 300-member custom TFP re-agent kit that continues to increase in number. Focused libraries are pro-duced in as little as 2–3 days by derivatizing an amine with a subset ofthe reagents. Analytical data are collected on all the members of the li-brary. Focused libraries find utility in SAR exploration and patent protec-tion.2 For larger library campaigns, which require 3–4 weeks to complete,

25 It is convenient to produce ca. 10 �M of product per well by dispensing ca. 12–15 mg of

TFP-activated resin per well, assuming a loading of ca. 0.95 mmol/g and an 80–90% reaction

yield. This quantity of product is sufficient for evaluation in multiple biological assays.


the entire reagent kit is coupled to a set of 40–50 commercially availableor custom prepared amines3 to furnish 12,000–15,000 compounds. Largearrays, requiring 3–4 weeks to complete, are screened across a broadrange of molecular targets for new lead discovery.26

Experimental ProtocolsPreparation of TFP Resin (Scheme 1)

ReagentsDMF (6 liter total)Aminomethyl polystyrene (100 g; 1.6 mmol/g)TFP in DMF (57 g TFP dissolved in 240 ml DMF)HOBt hydrate in DMF (32.3 g dissolved in 60 ml DMF)DIC (37.5 ml)Piperidine (17.3 ml)2 M aqueous HCl–DMF (150 ml of 2 M HCl added portion-wise to 1.3

liters of DMF with stirring)THF (2 liter total)DCM (2 liter total)

Procedure. A 2-liter three-necked flask fitted with an overhead stirrerand thermometer was charged with DMF (1 liter) and aminomethylpolystyrene resin (Polymer Labs). Freshly prepared solutions of TFP inDMF and HOBt in DMF were sequentially added followed by DIC.The reaction mixture was stirred at room temperature for 16 h andthen filtered and washed well with DMF, THF, and DCM to give theTFP resin containing ca. 10% of the undesired TFP-tetrafluorobenzoicacid ester.

Hydrolysis of the undesired ester was carried out as follows. Thewashed resin cake obtained above was placed into a 2-liter three-neckedround-bottom flask, again fitted with an overhead stirrer and thermometer.DMF was added (1 liter) and gentle stirring was initiated. Piperidinewas added and the reaction mixture was stirred for 2 h, after which timethe reaction mixture was filtered and washed thoroughly with DMF(1 liter). The resin cake of TFP piperidine salt was transferred into a 2-liter three-necked round-bottom flask, again fitted with an overheadstirrer and thermometer. The resin was resuspended (gentle stirring) in asolution of 2 M aqueous HCl–DMF. The reaction mixture was stirred for

26 Screening hits from lead finding campaigns are resynthesized in 10 mg amounts using either

TFP technology or solution-phase chemistry to confirm their biological activity and to

provide accurate primary and functional in vitro data (Ki, IC50, EC50, etc.)


1.5 h and then filtered using a Buchner funnel. The resin cake was washedwith DMF (0.6 liter), THF (1 liter), and DCM (0.6 liter). The resin wasdried in vacuo at 45

�to give a beige solid (140 g). FT-IR 1650 cm�1 (ab-

sence of the 1765 cm�1 absorption indicating complete hydrolysis of the un-wanted ester). 19F NMR � �148 and �165 ppm. Loading of resin wasdetermined by elemental analysis by F ion selective chromatography.Found %F ¼ 7.2 corresponding to 0.95 mmol/g. Note that several vendorssell TFP resin including Argonaut Technologies and Polymer Laboratories.

Preparation of TFP-Activated Carboxylic Acid Esters (Scheme 2)

ReagentsDCM (150 ml total)TFP resin (2 g; 1.9 mmol, 0.95 mmol/g)Carboxylic acid building block (3.8 mmol)HOBt hydrate (51.3 mg)DIC (0.6 ml)DMF (150 ml total)

Procedure. TFP resin was added to a 100-ml polypropylene reactionvial. The resin was swelled in DCM (30 ml) for 15 min followed by theaddition of a carboxylic acid building block. Upon dissolution of theacid, HOBt, DIC, and DCM (20 ml) were added. The reaction mixturewas gently agitated for 3 h and filtered, and the resin was washedwith DMF (3 � 30 ml) and DCM (3 � 30 ml), and dried in vacuo for 12 hat 25

�.

Preparation of TFP-Activated Sulfonic Acid Esters (Scheme 2)

ReagentsDMF (120 ml total)TFP resin (2 g; 1.9 mmol, 0.95 mmol/g)Sulfonyl chloride building block in DMF (2.85 mmol dissolved in

10 ml)DIEA (1 ml)DCM (90 ml)

Procedure. TFP resin was added to a 100-ml polypropylene reactionvial. The resin was swelled with DMF (20 ml) for 15 min. Diisopropylamine(DIEA) was added followed by a solution of a sulfonyl chloride buildingblock previously dissolved in DMF (10 ml). The reaction mixture wasgently agitated for 3 h and filtered, and the resin was washed with DMF(3 � 30 ml) and DCM (3 � 30 ml), and dried in vacuo for 12 h at 25

�.


Loading Determination (Chemical Type)

ReagentsTFP-activated carboxylic or sulfonate ester (50 mg; ca. 0.05 mmol, ca.

0.95 mmol/g)2-Methylbenzylamine in DMF (5.74 mg, 0.05 mmol dissolved in 2.5 ml

DMF)

Procedure. A TFP-activated resin was placed in a small reaction vesselto which was added a DMF solution of 2-methylbenzylamine. The reactionmixture was gently agitated for 12 h and filtered. The filtrate was evapor-ated to dryness and the residue dissolved in CDCl3 or other deuteratedsolvent. The sample was analyzed by 1H NMR. Loading was deter-mined by integration and comparison of the aromatic methyl protons andthe �-methylene protons of the product amide relative to the startingamine.

Amine Derivatization with TFP-Activated Esters

Reagents(Reaction in vials)DMF (150 ml total)TFP resin (90 mg; 0.08 mmol, 0.95 mmol/g)Amine (0.065 mmol)DCM (6 ml total)

Procedure. A 3-ml polypropylene reaction vial was charged with a TFP-activated resin, either the carboxylate or sulfonate ester type, and 1 ml ofDMF. The reaction mixture was gentle agitated for 10 min and an amine(0.065 mmol) was added. The reaction mixture was further agitated for16 h and filtered. The resin was washed with DCM (3 � 2 ml) and the fil-trate and washings were combined. The solvent was removed in vacuoand the amide or sulfonamide (>85% yield) so obtained was analyzed byLC/MS for purity and identity.


[9] The Traceless Solid-Phase Synthesis ofOrganic Molecules

By David Tumelty, Yijun Pan, and Christopher P. Holmes

Introduction

As the field of solid-phase organic synthesis continues to progress andexpand, one aim of practitioners is to synthesize molecules that do not signaltheir solid-phase ‘‘origins’’ by the presence of extraneous remnants left aftercleavage from the resin. These often appeared in earlier work as primary orsecondary carboxamides or carboxyl groups appended to the target mol-ecules and conveniently overlooked. These additional functionalities couldhinder or obscure biological activity in an otherwise promising targetcompound or scaffold, as well as being chemically rather unaesthetic.

As solid-phase routes and linkers have become increasingly sophisti-cated in recent years, many workers in the field have forwarded novelchemical methods to attempt to overcome some of these previously men-tioned limitations.1 This chapter describes two such approaches, whichillustrate different tactics used in the goal of synthesizing organic com-pounds in a traceless manner. In the first strategy, a linker and scaffoldcombine synergistically to achieve a traceless synthesis of diverse substi-tuted benzimidazole compounds and libraries.2 Second, a novel linker isused in a more global fashion to synthesize target compounds by activationof chemically diverse phenols.3,4

Traceless Solid-Phase Synthesis of Benzimidazoles

Background

Several solid-phase syntheses of benzimidazoles have been reported inrecent years.5,6,7 Recently some have been described in which the finalproducts could be regarded as traceless.2,8–10 Our initial goal was to design

1 V. Krchnak and M. W. Holladay, Chem. Rev. 102, 61 (2002).2 D. Tumelty, K. Cao, and C. P. Holmes, Org. Lett. 3, 83 (2001).3 Y. Pan and C. P. Holmes, Org. Lett. 3, 2769 (2001).4 Y. Pan, B. Ruhland, and C. P. Holmes, Angew. Chem. Int. Ed. Engl. 40, 4488 (2001).5 D. Tumelty, M. K. Schwarz, K. Cao, and M. C. Needels, Tetrahedron Lett. 40, 6185 (1999).6 D. Tumelty, M. K. Schwarz, and M. C. Needels, Tetrahedron Lett. 39, 7467 (1998).7 J. P. Mayer, G. S. Lewis, C. McGee, and D. Bankaitis-Davis, Tetrahedron Lett. 39, 6655 (1998).



[9] traceless solid-phase organic synthesis 165

a synthetic route that would permit the traceless release of benzimidazolesfrom single beads to support Affymax’s encoded combinatorial libraryscreening technologies.11 We required that the final compounds would bereleased without the benefit of further solution-phase reactions to com-plete the synthesis and that purification steps would not be carried out(in this particular format). This necessitated the development of a novelsynthetic strategy in which the required products were synthesized on thesolid support in a quaternary salt form, and treatment with base releasedthe products in a Hofmann elimination reaction. The basic concept hasbeen previously reported for the synthesis of simple amines on the REMlinker.12 Our modified plan to synthesize and release the benzimidazolecompounds is shown in Fig. 1.

Here, we envisage building the benzimidazole scaffold directly ontothe linker and, by analogy with a regular tertiary amine synthesis on theREM linker, we can quaternize the resin-bound benzimidazole compoundsby reaction with reactive bromides. The quaternary salt can then beliberated by a Hofmann elimination reaction upon treatment with base.

Development of the Traceless Route

The uncertain part of the synthesis scheme (prior to testing it experi-mentally) was whether quaternization on the ring nitrogen that was notdirectly attached to the resin would provide a sufficiently strong electron-withdrawing force to permit the Hofmann elimination under mild condi-tions. This idea was initially tested in a double-linker scheme as shown inFig. 2. This construct, although not the final chemical route, was extremelyvaluable in proving that the concept was valid and enabling various por-tions of the scheme to be optimized more effectively. This construct hasalso proven useful in the development of related chemistries using a similarHofmann elimination strategy.

In this scheme, Fmoc*-�-alanine is coupled to ArgoGel-Wang resinusing standard methodology to give resin 1, and then the Fmoc group is

8 V. Krchnak, J. Smith, and J. Vagner, Tetrahedron Lett. 42, 1627 (2001).9 A. Mazurov, Bioorg. Med. Chem. Lett. 10, 67 (2000).

10 W. Huang and R. M. Scarborough, Tetrahedron Lett. 40, 2665 (1999).11 Z. J. Ni, D. Maclean, C. P. Holmes, and M. A. Gallop, Methods Enzymol. 267, 261 (1996).12 J. R. Morphy, Z. Rankovic, and D. C. Rees, Tetrahedron Lett. 37, 3209 (1996).* Abbreviations: AcOH, acetic acid; Alloc, allyloxycarbonyl; ArB(OH)2, a generalized

aromatic boronic acid; BINAP, (R)-(þ)-2,20-Bis(diphenylphosphino)-1,10-binaphthyl, a

chiral chelating ligand useful in palladium-mediated reactions; DCM, 1,2-dichloromethane,

a common organic solvent (caution: suspected carcinogen); DIEA, N,N0-diisopropylethy-

lamine, a hindered organic base; DMF, N,N0-dimethylformamide, a polar organic solvent;

R1

R1

O N

O

NR2

O NH2

O

O

OHN

R2

R1

O N

O

R2

R1

O N+

OR1

R3R2

NR3R2

R1

R1

O N

O

NR2

R3

N

NR2

R3

+

+

Fig. 1. Comparison of a regular tertiary amine synthesis on REM resin (left) with the

planned traceless benzimidazole route.

DMSO, dimethylsulfoxide, a polar organic solvent; dppp, 1,3-bis(diphenyl-phosphino)pro-

pane; Fmoc, 9-fluorenylmethyloxycarbonyl, a protecting group of amines; GC-MS,

combined gas chromatography and mass spectrometry instrumentation; HPLC, high-

pressure (performance) liquid chromatography; LC-MS, combined liquid chromatography

and mass spectrometry instrumentation; MeOH, methanol; Na(CN)BH3, sodium cyano-

borohydride, a reducing agent often used for the reduction of imines to amines; NMP, N-

methylpyrrolidinone, an organic solvent; Na2S2O4, sodium hyposulfite (sodium dithionite),

a mild, water-soluble reducing agent; Oxone, potassium peroxymonosulfate, an oxidizing

agent (Dupont); Pd(dppf)Cl2, [1,10-bis(diphenylphosphino)ferrocene]dichloropalladiu-

m(II); Pd(OAc)2, palladium(II) acetate; PEG-PS, a resin composed of low-cross-linked

polystyrene linked with polyethylene glycol of various lengths; PFS linker/resin,

perfluoroalkylsulfonyl linker attached to resin; SnCl2�2H2O, tin(II) dichloride dihydrate,

often used for reduction of aromatic nitro groups to corresponding anilines; tBoc, tert-

butyloxycarbonyl, a common, acid-labile protecting group for amines; TEA, triethylamine,

an organic base; TFA, 1,1,1-trifluoroacetic acid, a strong organic acid; THF, tetrahydrofur-

an, an organic solvent; TLC, thin-layer chromatography.


O

O NH

O

O

O

O

O NH

O

N+O O

R1

O

O N

O

NR2

R1

O

O NH

O

NH2

R1

R1

O

O N

O

NR2

R3

R1

HO N

O

NR2

Br

R1

CF3CO2-

HO N

O

NR2

R3O

O

O

R1

N

NR2

R3

+

+

+ TEA.HBr

+

1 2

3

4

4a

5

5a

6

a,b

c

d

e

e

f

g

Fig. 2. Double-linker route used in development: (a) 20% piperidine/NMP; (b) ortho-

nitrofluoro/-chloro-R1-arene, DIEA, NMP, 12 h, 60�; (c) SnCl2�2H2O, NMP, 12 h; (d) R2-

CHO, NMP, 12 h, 50�; (e) TFA, DCM, 30 min; (f) R3-bromide, NMP, 18 h, 60

�; (g) TEA,

DCM, 18 h, 25�.


removed with piperidine. The exposed resin-bound amine acts as the startinganchor for building diverse benzimidazole compounds by the sequentialreaction of three components, namely ortho-fluoro- or ortho-chloronitroar-enes, aldehydes, and alkyl- or benzylbromides. The fluoronitroarenes areadded to give resin 2, with the expected steric and electronic considerationsdictating the kinetics of the nucleophilic substitution reaction. We compen-sate for the differing kinetics to a large extent by using as high a concentra-tion of the amine as possible in a polar organic solvent, such as NMP orDMSO. Several chloroarenes are useful in the scheme, although they tendto require heating and the reactions are often difficult to force to comple-tion. The extent of the reaction is monitored by the ninhydrin test and incases where an incomplete reaction occurs, the unreacted resin-bound


amine is capped by reaction with acetic anhydride/pyridine/DMF for20 min. Fortunately, this capping step does not acetylate the resin-boundaniline under these conditions, presumably due to the low nucleophilicityof the resin-bound ortho-nitroaniline.

The resin-bound phenylene diamine intermediates 3 are then generatedby nitro group reduction with tin(II) chloride in NMP and cyclization/aromatization with a wide variety of aldehydes gave the resin-bound benzi-midazole intermediates 4. The treatment of this intermediate with 50%TFA/DCM liberates the substituted 3-(benzoimidazol-1-yl)-propionic acidderivative 4a. Analysis of this intermediate by HPLC and LC-MS gavea measure of the purity of the resin-bound product and enabled the opti-mization of conditions for the incorporation of the R1-nitroarenes andR2-aldehydes by an iterative process.

Incorporation of the third and final diversity element to give resin 5 isachieved via quaternization of the resin-bound benzimidazole intermedi-ates using a large excess of a primary alkyl or benzyl bromide. A high con-centration solution of the desired bromide in NMP or DMSO (at least1.5 M, with at least 30 equivalents over the estimated resin loading) provedeffective in most cases, and heating this reaction to around a maximum of55

�helps to achieve a higher final yield in this model system. We observed

some premature release of the product from the resin when heating at 60�

and above. For the final scheme (see below), modifications to the linker en-abled a higher temperature to be used for the reaction with bromides,which increases the yields of the final products by an average of about20%. Despite the slight temperature sensitivity of this two-linker modelsystem, it was valuable in determining the optimal conditions for quaterni-zation using different classes of alkylating agent. Treatment of 5 with TFAreleases the quaternary salt derivative 5a, which enables assessment of thesuccess of the alkylation conditions. Again, evaluating different conditionsenabled us to optimize this reaction for a range of alkylating agents.

Despite the fact that the quaternization event occurs on a nitrogenatom that is not directly attached to the resin, we were gratified to observethat the products 6 are indeed released by base treatment. Release of thedesired compounds from the resin was carried out in the developmentstages with various proportions of TEA or DIEA in DCM. We later ob-served that release could be carried out in several different solvents usingone of a variety of organic and inorganic bases.13 For single compound

13 For example, ammonia in dioxane or methanol and inorganic bases, such as sodium

hydroxide or ammonium hydroxide solutions. Such cleavages gave satisfactory results in a

variety of solvents, such as N-methylpyrrolidinone, dimethyl sulfoxide, acetonitrile,

methanol, dioxane, and acetone.


work at the development stage, a single treatment with 5% TEA in DCM for16 h gives the highest yields of compound recovery. The excess TEA saltscan be removed by extraction, prior to purification of the desired compound(if necessary), to recover the product 6 as a white solid in most cases. Thedouble-linker construct again proved valuable in determining the end pointand yield of the cleavage reaction under different reaction conditions. Aftertreatment with base to release the product, the resin is thoroughly washedwith DCM then subjected to TFA treatment. This allows us to examinethe products remaining on the resin. If there was incomplete Hofmann elim-ination of the desired product, compound 5a is observed. By optimization ofthe basic conditions, the presence of 5a can be reduced to low levels or oftencompletely eliminated. This procedure is allied with standard gravimetricanalysis of the expected compound to determine the cleavage yields for arange of compounds. In our hands, the cleavage yields (i.e., target compoundremoved from the resin) is typically >80%, with generally less than 10% ofthe desired material remaining bound to the resin.14

Library Rehearsal

Despite the variable yields of the products, we observe that they arevery pure, almost without exception, directly after cleavage from the resin(usually >90% of target compound by HPLC trace integral). Interestingly,combinations of the R1, R2, and R3 monomers that interact unfavorably toproduce a low yield of target compound (as judged by both the resin andgravimetric analyses as outlined above) always give pure products uponcleavage.

It became obvious that optimization of the R1 and R2 monomer com-binations (that lead to high-purity resin-bound intermediate 4) would givethe best chance for eventual product formation. Most commercially avail-able aromatic and some aliphatic aldehydes work well in the syntheticscheme. Some examples of the R1 monomers used for subsequent libraryformation are shown in Fig. 3.

Having determined the most successful R1/R2 combinations, wescreened a wide variety of alkylating agents for their ability to quaternizeresin intermediate 4. For several types of resin intermediate 4 (chosen

14 To further elucidate this point, we were able to remove from the resin about four-fifths of

the quaternized target compound that had been synthesized (this was judged using the

double-linker approach mentioned above). The variability in the actual amount of target

material synthesized on (and competent to be released from) the resin was dictated by the

reaction (in)compatibility of the three monomers comprising each individual benzimidazole

compound.

Cl

O

O

O2N

O

O

Cl

O2N

F

O2N

Cl

NO2N

F

BrO2N

F Cl

O2N

Cl

O

O

O2N

F

O2N

F

FO2N

F

F

F

FO2N

F

F

O2N

F Br

FO2N

F

O

O

O2N

Cl

OO2N

F

SO

OO2N

Fig. 3. A selection of nitroarenes used in library production.


to have differing steric and electronic properties), a certain number of al-kylating species give acceptable formation of resin 5 under common condi-tions (2 M in NMP, 18 h, 60

�). The yield of this reaction largely determines

the final yield of the reaction, almost independently of the conditions usedfor the final base-promoted elimination. Several of the alkylating speciesused in subsequent library production are shown in Fig. 4.

Final Improved Reaction Route

Several modifications were made to the scheme to improve the stabilityof some of the resin intermediates and provide improved overall yields ofthe cleaved products. The final scheme used to synthesize tagged libraries isshown in Fig. 5.

Two major changes to the double-linker resin used in developmentwere introduced: a modification to the REM-type linker itself and the in-clusion of tags for chemical encoding. The new resin/linker is easily madefrom commercially available reagents. In our hands, the use of PEG-PS-based resins works best for the scheme, which does call for both organicand aqueous reaction conditions. We have previously reported the use ofan unencoded version of this route.2

A halogenated PEG-PS resin (TentaGel-Br or ArgoGel-Cl) is reactedwith tert-butyl-N-(2-mercaptoethyl)carbamate to give resin 7. Coupling ofthis reagent to the resin introduces both the amine function that serves asan anchor for benzimidazole synthesis, as well as a sulfur group. The sulfuris later oxidized to a sulfoxide providing the driving force for the eliminationreaction that ultimately releases the final products. For library synthesis,

Br Br Br Br

F

Br

F

Br

F

F

Br

F F

Br

Cl

Br

O2N

Br

O

CF3

Br

CF3

Br

F CF3

Br

SCF3

Br Br

Br

NC

Br

N

Cl

CF3

OO2NBr

Br

OBr

NH2BrO Br Br

N

O

O Br

Fig. 4. A selection of alkyl and benzyl bromides used in library production.

SNH

tBoc

R1

N

NR2

R3

HNAlloc

SNH

NO2

HNTag1

Alloc

SN

NR2

HNTag1

Tag2

R1R1

SN

NR2

O

O

HNTag1

Tag2

R1S

N

NR2

O

O

R3

HNTag1

Tag2

R1

Br

S

O

O

HNTag1

Tag2 + TEA.HBr

7 8 9

10 11

+

f g

kh, i, b j, d, h

6

+

12

Fig. 5. Final library scheme. Conditions as in Fig. 2 and (h) Alloc removal and tag coupling;

(i) TFA, dimethylsulfide, DCM, 2 � 30 min, then DIEA, DCM, 2 � 30 min, 25�; (j) Na2S2O4,

water, MeOH, 16 h, 25�; (k) aqueous Oxone, 12 h, 25

�.


we have devised a method for differentiating the resin, such that approxi-mately one-tenth of the available functionality was reserved for encodingprocedures. Prior to acidolysis to remove the tBoc protecting group, theresin is divided and each resin pool is encoded using the tagging strategy.Each tagged resin pool is then treated with the nitroarenes as before togive resin 8 and the resins are pooled prior to nitro group reduction.


The nitro group reduction is carried out on the pooled resin usingsodium hydrosulfite in water to form the resin-bound substituted pheny-lene diamine precursor.15 The resin is again split into smaller pools andthe second diversity elements (various aliphatic and aromatic aldehydes)are added. As previously observed, no exogenous oxidants are necessaryto form the required resin-bound benzimidazoles. After the final round oftagging, the resin 9 is once again pooled and treated with a chemical oxi-dant to convert the sulfur group to a sulfoxide. Aqueous Oxone provedmost effective, with complete conversion observed after overnight treat-ment. Prewashing of the resin with methanol aids in its subsequent solv-ation by the Oxone solution. The resin is then split into pools foraddition of the final diversity elements, the alkyl and benzyl bromides. Thisproved to be the step in the synthesis scheme that largely determines thefinal yield of product, as noted before.

Quaternization of the resin-bound intermediates 10 is carried out with ahigh concentration solution of the desired alkylating agent in NMP orDMSO to give resin 11. Heating this reaction to around a maximum of70

�helps to achieve higher final yield. We had previously determined that

this sulfur-based linker has increased temperature stability compared tothat used in the development stage. In model studies, we observe some pre-mature release of the product from this resin only when heating at 90

�and

above.These final diversity elements were ‘‘spatially encoded,’’ i.e., the resin

pools are kept separated and assayed separately, to allow for identificationof the final monomers without the need for a further chemical encodingstep. As a result no further pooling was necessary after the quaternizationreaction and we were able to tailor the most favorable reaction conditionsfor incorporation of the required alkylating agents in each reaction.

The release of the final products from the beads can be achieved usingseveral different procedures, as previously determined at the developmentstage. For library production, we are able to release sufficient compound bysolvating the beads with DMSO and subjecting them to treatment with am-monia gas.16 After release of the desired compound 6, we recover the en-coding bead 12 and carry out the decoding procedures to determine theidentity of the R1 and R2 monomers from beads of interest (after variousassays have been carried out on the released compounds).

15 R. A. Scheuerman and D. Tumelty, Tetrahedron Lett. 41, 6531 (2000).16 R. Brown, J. Comb. Chem. 1, 283 (1999).


Conclusion

This section described the successful development and implemen-tation of a traceless synthetic route to create libraries of chemically diversebenzimidazole compounds. The chemical route delivers compounds inmoderate yields but in high purity directly after cleavage from the solidsupport. The basic concept of this traceless approach has been applied toseveral other related heterocyclic systems that will be reported in duecourse.17

Experimental

Reagents and General Methods

We have previously described the basic resin handling and washingprocedures, as well as nitro group reduction, cyclization with aldehydesto form the benzimidazole ring, and chemical encoding procedures fora related benzimidazole system.18 Reagents and solvents used are avail-able from Aldrich (Milwaukee, WI) and Calbiochem-Novabiochem (SanDiego, CA).

General Procedure for Coupling of o-Fluoro/Chloro-Nitroarenes

The same procedure is used for the formation of resins 2 and 8 byreaction of the nitroarenes with the resin-bound �-alanine or mercaptan/amine linker, respectively. The o-fluoronitroarenes (Fig. 3) are dissolvedin DMSO or NMP (at a concentration between 1.5 and 2 M) and addedto the resin, followed by diisopropylethylamine (10 equivalents) andadditional DMSO or NMP (if required) to ensure resin solvation. Althoughmany nitroarenes react rapidly at room temperature, in a library formatthe resin/nitroarene mixture is heated overnight at 50

�to help achieve

equivalent reaction kinetics between different monomers. Under thesame conditions, some o-chloronitroarenes are synthetically useful. Theextent of the reaction can be assessed qualitatively (or quantitatively, ifdesired) by carrying out a ninhydrin test to check for the presence offree amine. In any case, the resins are acetylated with five equivalents ofacetic anhydride/pyridine/DMF (1:1:10) for 20 min to cap any unreactedamine.

17 D. Tumelty, unpublished results (2000).18 D. Tumelty, L.-C. Dong, K. Cao, L. Le, and M. C. Needels, in ‘‘High Throughput

Synthesis’’ (I. Sucholeiki, ed.), p. 93. Marcel Dekker, New York, 2001.


Procedures for Reduction of the Aromatic Nitro Group

For formation of resin 3, the resin is washed in NMP (20 ml/g of resin),filtered, and left solvated. Separately, tin(II) chloride dihydrate (approxi-mately 40 equivalents with respect to resin-bound nitro groups) is dissolvedin NMP with vigorous stirring, then the solution is added to the resin andmixed by nitrogen bubbling for 12 h at room temperature. The resin isfiltered, washed, and left solvated prior to the next synthetic step.

For reduction of resin 8, concerns about the possibility of traces of tinby-products contaminating subsequent assays led to the adoption of a dif-ferent reduction procedure for library production.15 The tagged resins 8 arecombined into one pool in a large peptide synthesis vessel, washed withmethanol, filtered, and the resin left solvated. Separately, an aqueous solu-tion of 0.5 M aqueous sodium hydrosulfite/0.5 M potassium carbonate isprepared and added to the resin (40 ml/g of resin) and then the resin/solu-tion is bubbled with nitrogen at room temperature for 16 h. The resultingresin is washed with water, water/MeOH (1:1), MeOH, MeOH/NMP(1:1), NMP, DCM, MeOH, and ether, then filtered and dried overnightin vacuo prior to the next step.

General Procedure for Quaternization with Alkyl/benzyl Bromides

Resin 4 or 10 is solvated by washing in NMP. For quaternization, abenzyl or alkyl bromide (50 equivalents) is dissolved in NMP to give afinal solution with a concentration of 2 M. This solution is added to theresin in a glass vial and stirred at 50–70

�for 18 h. After this time, the dark

brown resin is transferred to a polypropylene tube and washed with NMP,DCM, MeOH, then finally diethyl ether and dried overnight in vacuo.

Preparation of Resin 7

A PEG-PS resin is subjected to a proprietary procedure where about90% of the initial amine functionality is loaded with a moiety bearing a re-active bromide, while the remainder has an amine function protected by anallyloxycarbonyl group. The resin (40 g, approximately 15 mmol with re-spect to the bromine group) is solvated with NMP (200 ml) in a 1-literpear-shaped flask, fitted with a nitrogen-bubbler. t-Butyl-N-(2-mercap-toethyl)carbamate (Aldrich, 20 ml, 7 equivalents; caution: Stench!) isadded, followed by solid potassium carbonate (9 g, 4 equivalents), and theresulting resin/solution is stirred with an overhead paddle-stirrer at 60

�for

12 h in a thermostatically controlled oil bath. After this time, the slurry istransferred to a 2-liter peptide synthesis vessel and the resin is subsequentlywashed under vigorous nitrogen bubbling using NMP, NMP/water, MeOH/water, water, MeOH/water, NMP/water, NMP, DCM, MeOH, and ether


(3 � 250 ml each), and finally dried overnight in vacuo. A pale yellow resinis obtained (43 g). A small resin sample is taken and, after removal of thetBoc group, the loading of the resin is assessed using two complementarymethods: either a quantitative ninhydrin test19 or coupling an Fmoc groupto the exposed amine, deprotecting with piperidine in DMF (1:4), andquantitatively assessing the concentration of the dibenzofulvene adductformed at 302 nm.20 Either method usually gives loading values between0.30 and 0.33 mmol/g for the amine linker.

Preparation of Resin 10

The tagged resins are pooled into one large batch in a 2-liter peptidevessel, washed with methanol, and then left solvated. Separately, solid Ox-one is dissolved in water (to a final concentration of 0.4 M), sonicating for5 min to aid in solvation. The aqueous Oxone solution (10 equivalents withrespect to the nitro group loading of the resin) is added to the methanol-solvated resin and stirred/bubbled for 16 h at room temperature. Theresulting resin 10 was then washed with water, MeOH/water (1:1), MeOH,MeOH/NMP, NMP, DCM, and ether, then dried in vacuo overnight priorto the next step.

Traceless Syntheses Using a Novel Triflate-Type Linker

Background

Our goal for this work was somewhat different from the precedingtraceless benzimidazole syntheses. Here we aimed to develop a novelsolid-phase linker that would serve as an activating group for a wide varietyof phenols, permitting subsequent transformations to occur between theresin-bound phenol and a variety of different classes of input molecules.The strategy is based on the well-known activation properties of triflates,which are widely used as precursors for aryl and vinyl cations due to theirexcellent leaving group properties.21 Once an oxygen atom on the phenolmoiety is activated by the triflate (trifluoromethanesulfonyl) group, it be-comes possible to carry out a reductive cleavage (essentially deoxygenatingthe phenol) or cross-coupling reactions, e.g., through palladium-catalyzedSuzuki, Stille, and Heck reactions.22 This gives rise to a variety of

19 V. K. Sarin, S. B. H. Kent, J. P. Tam, and R. B. Merrifield, Anal. Biochem. 117, 147 (1981).20 M. K. Schwarz, D. Tumelty, and M. A. Gallop, J. Org. Chem. 64, 2219 (1999).21 J. F. Hartwig, Angew. Chem. Int. Ed. Engl. 37, 2046 (1998).22 B. A. Lorsbach and M. J. Kurth, Chem. Rev. 99, 1549 (1999).


substituted aromatics or olefins at the ‘‘inert’’ phenolic or vinyl oxygen pos-ition. Our goal therefore was to design a triflate-like linker upon which wecan conduct such triflate-directed transformations on solid-phase resin andthis section describes the successful implementation of this strategy.

Perfluoroalkylsulfonyl (PFS) Linker/Resin

We recently reported the synthesis of a perfluoroalkylsulfonyl linker at-tached to TentaGel resin 13 (Fig. 6), which proves to act in a fashion similarto triflates as we had hoped, and demonstrated its application for the trace-less cleavage of phenols using palladium-catalyzed reduction and Suzukicross-coupling reactions.3,4

The new polymer-supported linker allows the attachment of phenolicrings to the solid phase through the formation of aryl nonflates and subse-quent traceless cleavage of the hydroxyl groups on aryl rings. A variety ofphenols can be attached to this resin through the formation of the polymer-supported perfluoroalkylsulfonates 14 in DMF at room temperature usingpotassium carbonate as base (Fig. 7). The attachment of phenols to thislinker is especially attractive since the mild reaction conditions allow manyuseful functional groups (such as aldehydes, nitro, carboxylic acids, ketones,and alcohols) to be incorporated without additional protection, and these

SF

F FO

SF

F F F F

F F F F O O O ONH

O13

Fig. 6. Structure of the perfluoroalkylsulfonyl (PFS) linker/resin.

FS

OO

F FHO

OSOO

F F

NR2

R3

R1

R1 R1

R2

R1R1

13 14

l n

m o

Fig. 7. Divergent syntheses from a PFS-derivatized linker. (l) Substituted phenol,

potassium carbonate, DMF; (m) Suzuki reaction; (n) reduction; (o) amine displacement.


groups can themselves serve as combinatorial sites for the synthesis of largelibraries. We have found that the resin-bound perfluoroalkylsulfonatespecies have similar reactivities to aryl triflates, such that most of theknown palladium-catalyzed reactions involving aryl triflates were possibleon the support (Fig. 7). Thus, a cleavage/cross-coupling strategy of simul-taneously introducing diversity while liberating the desired molecule fromthe support provides a powerful technique for the traceless synthesis ofmolecules. We have initially targeted the Suzuki and Buchwald aminationreactions as methods for generating biaryls and anilines, respectively.

Cleavage of the Resin-Bound Phenols Using the SuzukiCoupling Reaction

The Suzuki coupling reaction is a powerful tool for carbon–carbon bondformation in combinatorial library production.23 Many different reactionconditions and catalyst systems have been reported for the cross-couplingof aryl triflates and aromatic halides with boronic acids in solution. Aftersome experimentation, we found that the ‘‘Suzuki cleavage’’ of the resin-bound perfluoroalkylsulfonates proceeded smoothly by using [1,10-bis(diphenylphosphino)ferrocene]dichloropalladium(II), triethylamine, andboronic acids in dimethylformamide at 80

�within 8 h afforded the desired

biaryl compounds in good yields.24 The desired products are easily isolatedby a simple two-phase extraction process and purified by preparative TLCto give the biaryl compounds in high purity, as determined by HPLC,GC-MS, and LC-MS analysis.

A small library of biaryl compounds was synthesized in order to exam-ine the scope and generality of the resin-bound PFS linker and the traceless‘‘Suzuki cleavage’’ strategy, as shown in Fig. 8. The aryl perfluoroalkylsul-fonate resin 15 is prepared by attaching 4-hydroxybenzaldehyde to resin.Resin 16 is prepared by a reductive amination of 15 with primary aminesusing sodium cyanoborohydride as the reducing agent. The presence ofsome acetic acid is also important in this step to promote the reductive ami-nation reaction. The secondary amines generated in this step are used asanother diversity site through functionalization of this amine. Biaryls 18are produced in a traceless fashion from resin 17 in yields ranging from65 to 90% upon exposure to the Suzuki conditions. We have observed thatmost boronic acids are suitable for this cleavage/cross-coupling procedureto generate a wide variety of molecules.

23 J. Hassan, M. Sevignon, C. Gozzi, E. Schulz, and M. Lemaire, Chem. Rev. 102, 1359 (2002).24 B. Ruhland, A. Bombrun, and M. A. Gallop, J. Org. Chem. 62, 7820 (1997).

OSOO

F F

O

OSOO

F F

NH

R1

OSOO

F F

N

R1

R2

O

N

R1

R2

O

Ar

15 16

17 18

p q

r

Fig. 8. A four-step synthesis on the PFS linker. (p) R1-NH2, Na(CN)BH3, AcOH, THF;

(q) R2-NH2, TEA, CH2Cl2; (r) ArB(OH)2, Pd(dppf)Cl2, TEA, DMF.


Cleavage of the Resin-Bound Phenols Using CatalyticReductive Elimination

The deoxygenation of phenols by a palladium-mediated reduction insolution phase is well established.25 Reductive cleavage of the polymer-supported aryl triflate-type species allows phenols to be cleaved from theresin without any trace of the phenolic hydroxyl group. This strategy isvaluable for combinatorial syntheses since the structure of the final productcleaved from the resin is independent of the position of the hydroxyl groupon the starting phenol and enables greater flexibility in choosing the build-ing blocks for library syntheses. The deoxygenation of the polymer-supported aryl nonaflate species was studied through a palladium-mediatedreduction reaction. We discovered that the polymer-bound aryl nonaflateswere efficiently cleaved with a mixture of triethylamine and formic acid inthe presence of a catalytic amount of palladium(II) acetate and 1,3-bis(di-phenylphosphino)propane to afford high yields of the reduced arenesunder mild conditions. The desired products are again isolated by a two-phase extraction and the trace metal catalyst is removed by eluting the or-ganic solution through a thin pad of silica gel. The resulting products areobtained in good yields as determined by HPLC, GC-MS, and LC-MS.Figure 9 shows two examples of the traceless cleavage of the resin-boundphenols using this palladium-catalyzed reductive elimination. It is notablethat the cleaved product 20 does not have any remnant of the anchoring hy-droxyl group and also that the polymer-bound perfluoroalkylsulfonateserved here as a linker, a protecting group, and an activating group forthe phenols. The aryl perfluoroalkylsulfonyl resin also permits monoattach-ment of a symmetric bisphenol to form resin 21 and only the attached

25 S. Cacchi, P. G. Ciattini, E. Morera, and G. Ortar, Tetrahedron Lett. 27, 5541 (1986).

OSOO

F FN N

OSOO

F FOH

OH

19 20

21 22

s

s

Fig. 9. Reductive cleavage from the PFS linker. (s) Pd(OAc)2, dppp, TEA-HCO2H, DMF.


phenol group is deoxygenated, leading to a nonsymmetrical phenol 22upon reductive cleavage.

The application of resin 13 to the solid-phase synthesis of other usefultarget compounds was also explored and an example of this is the multistepsynthesis of Meclizine (Fig. 10).26 The starting material, 3-methyl-4-hydro-xybenzaldehyde, is attached to the PFS linker, and a polymer-bound amineintermediate is prepared by a reductive amination of resin 23 withamine 24. The resulting resin 25 is subjected to a palladium-mediated re-ductive cleavage to give Meclizine 26 in 80% yield, based on the originalresin loading.

Cleavage of the Resin-Bound Phenols Using Catalytic Amination

Substituted anilines often appear as a key element in biologicallyactive compounds. The palladium-catalyzed amination of aryl triflates hasdrawn increasing interest as a synthetic route to a wide variety of arylamines.27 The diversity of phenols and amines that is available, along withthe simple attachment of phenols to the PFS linker, suggested to us that thecatalytic amination of resin-bound aryl triflate species would provide an-other useful synthetic route to aryl amines. Figure 11 shows a generalsolid-phase protocol for the traceless cleavage of phenols from the PFS lin-ker using catalytic amination. The reaction is carried out with phenol-loaded resins 19 or 28, palladium(II) acetate, BINAP, cesium carbonate,and the corresponding amines in THF at 80

�for 16 h. The desired products

27 and 29 are obtained in 70–80% yields based on the actual loading of the

26 Meclizine is an oral antiemetic used to treat nausea, vomiting, and dizziness associated with

motion sickness. Compound 26, synthesized by our route, was spectroscopically identical

with a commercially obtained sample (Sigma).27 J. Ahman and S. L. Buchwald, Tetrahedron Lett. 38, 6363 (1997).

OSOO

F F

O

HN

N

ClO

SOO

F F

NN

Cl

NN

Cl

23

24

25

26 Meclizine

p

s

Fig. 10. Synthesis of Meclizine on the PFS linker.

OSOO

F FN

NN

NNHN Et

OSOO

F F N

N

O N

OHN

1927

28 29

t

t

Fig. 11. Cleavage from the PFS linker with amines. (t) Pd(OAc)2, BINAP, Cs2CO3, THF.


phenols. The traceless ‘‘amination cleavage’’ approach permits the intro-duction of a new aromatic amine functionality at the phenolic oxygen pos-ition during cleavage and provides a powerful method to synthesizelibraries with rich synthetic diversity.

Conclusion

The resin-bound perfluoroalkylsulfonyl linker is compatible with manycommon solid-phase reactions, such as tin dichloride-mediated aromaticnitro group reduction, trifluoroacetic acid-mediated tBoc deprotection, re-ductive amination reactions, acylation, and sulfonation. It is possible toperform several sequential synthetic reactions on the nonflate resin so thatmultistep syntheses can be carried out. The solid-phase approach providesan operationally simple, inexpensive, and general protocol for the cleavage


of the aryl-oxygen bond. We anticipate that the ease of preparation, excel-lent stability, and synthetic versatility of the polymer-supported linker willprove useful in solid-phase and combinatorial chemistry. Since a largenumber of phenols with a variety of functional groups are commerciallyavailable, and a variety of palladium-mediated reactions can be used forthe resin-bound aryl triflates, our novel resin-bound perfluoroalkylsulfonyllinker will provide a powerful method to synthesize structurally diverselibraries.

Experimental


All starting materials were obtained from Aldrich (Milwaukee, WI).TentaGel resin was obtained from Rapp Polymere (Tubingen, Germany).Procedures for the synthesis of the PFS linker and its attachment to resin toform 13 have been previous described.3

General Procedure for Attachment of Phenols to Resin 13 (to form 14, 15,19, 21, 23, or 28)

The phenol (20 equivalents), potassium carbonate (22 equivalents), andresin 13 (1 equivalent with respect to the sulfonyl fluoride group) are mixedwith DMF (10 ml/g of resin) and shaken overnight at room temperature.The resin is filtered and washed with water, DMF, and DCM, and then isdried under vacuum overnight to give the required resin-bound phenol.

General Procedure for Cleavage of Phenols Using the Suzuki CouplingReaction: Preparation of Resins 15–17 and Compounds 18

A mixture of 4-hydroxybenzadehyde (5.0 mmol), potassium carbonate(6.6 mmol), and resin-bound linker 13 (3.0 g, 1.0 mmol) is added to DMF(8.0 ml) and the mixture was shaken at room temperature overnight. Theresin was filtered and washed with water, DMF, and DCM, and then driedunder vacuum overnight to give resin 15. A portion of the dried resin(0.50 g, 0.16 mmol) is then mixed with a primary amine (R1-NH2, 2.0mmol), THF (2.0 ml), Na(CN)BH3 (1 N solution in THF, 2.0 ml, 2.0 mmol),mmol), and acetic acid (0.11 ml, 1.95 mmol) and the mixture is shaken over-night at room temperature. The beads are filtered and washed with water,DMF, and DCM, and dried under vacuum overnight to give resin 16.

To a portion of the dried amine resin 15 (0.20 g, 0.066 mmol) is addedTEA (2.3 mmol), DCM (4.0 ml), and an acid chloride (R2-COCl, 1.6 mmol)mmol) at 0

�. The mixture was allowed to warm-up and then shaken at room


temperature overnight. The resin is filtered, washed with DMF and DCM,and dried under vacuum overnight, as before, to give resin 17.

For the Suzuki cleavage reaction, a portion of the dry resin 17 (0.20 g,0.07 mmol) is mixed with Pd(dppf)Cl2 (7.2 mg), an arylboronic acid(0.25 mmol), TEA (0.1 ml, 0.60 mmol), and DMF (2.0 ml) in a glass vialunder nitrogen. The mixture is then shaken at 90

�overnight. The polymer

beads are next filtered and washed several times with Et2O and the com-bined organic phase is washed with aqueous 2% sodium carbonate andwater and then evaporated to dryness. The crude products are purified bypreparative TLC (or other suitable methods) to give the desired products18 in 65–90% yields, with >98% purity as determined by HPLC.

General Procedure for Cleavage of Phenols by a Reductive EliminationReaction: Preparation of Compounds 20 and 22

To dried resins 19 and 21 (0.1 g resin, approximately 0.04 mmol with re-spect to the loading of the phenol) are added Pd(OAc)2 (8.0 mg), 1,3-bis(diphenyl-phosphino)propane (dppp, 17.0 mg), DMF (1.4 ml), and a mix-ture of HCO2H (0.2 ml) and TEA (0.8 ml). The mixture is shaken at 85

�

for 2 h, and then the resin is filtered and washed several times with diethylether. The combined organic phase is washed with aqueous sodium carbon-ate solution then water and evaporated to dryness. The residue obtained isdissolved in diethyl ether and eluted through a short column of alumina toremove any remaining inorganic residues. The crude products are purifiedby preparative TLC (or other suitable methods) to give the desiredproducts 20 and 22 in >95% purity.

[10] Unnatural Diamino Acid Derivatives as Scaffoldsfor Creating Diversity and as Linkers for Simplifying

Screening in Chemical Libraries

By Robert Pascal, Regine Sola, and Patrick Jouin

Introduction

The introduction of conformational restrictions into flexible active mol-ecules is a well-known strategy for trying to increase their potency and/orselectivity toward their biological targets.1 Several methods have been usedfor constraining flexible molecules. Cyclic derivatives of linear peptides orpeptidomimetics can thus be prepared by reactions involving side-chain



temperature overnight. The resin is filtered, washed with DMF and DCM,and dried under vacuum overnight, as before, to give resin 17.

For the Suzuki cleavage reaction, a portion of the dry resin 17 (0.20 g,0.07 mmol) is mixed with Pd(dppf)Cl2 (7.2 mg), an arylboronic acid(0.25 mmol), TEA (0.1 ml, 0.60 mmol), and DMF (2.0 ml) in a glass vialunder nitrogen. The mixture is then shaken at 90

�overnight. The polymer

beads are next filtered and washed several times with Et2O and the com-bined organic phase is washed with aqueous 2% sodium carbonate andwater and then evaporated to dryness. The crude products are purified bypreparative TLC (or other suitable methods) to give the desired products18 in 65–90% yields, with >98% purity as determined by HPLC.

General Procedure for Cleavage of Phenols by a Reductive EliminationReaction: Preparation of Compounds 20 and 22

To dried resins 19 and 21 (0.1 g resin, approximately 0.04 mmol with re-spect to the loading of the phenol) are added Pd(OAc)2 (8.0 mg), 1,3-bis(diphenyl-phosphino)propane (dppp, 17.0 mg), DMF (1.4 ml), and a mix-ture of HCO2H (0.2 ml) and TEA (0.8 ml). The mixture is shaken at 85

�

for 2 h, and then the resin is filtered and washed several times with diethylether. The combined organic phase is washed with aqueous sodium carbon-ate solution then water and evaporated to dryness. The residue obtained isdissolved in diethyl ether and eluted through a short column of alumina toremove any remaining inorganic residues. The crude products are purifiedby preparative TLC (or other suitable methods) to give the desiredproducts 20 and 22 in >95% purity.


[10] Unnatural Diamino Acid Derivatives as Scaffoldsfor Creating Diversity and as Linkers for Simplifying

Screening in Chemical Libraries

By Robert Pascal, Regine Sola, and Patrick Jouin

Introduction

The introduction of conformational restrictions into flexible active mol-ecules is a well-known strategy for trying to increase their potency and/orselectivity toward their biological targets.1 Several methods have been usedfor constraining flexible molecules. Cyclic derivatives of linear peptides orpeptidomimetics can thus be prepared by reactions involving side-chain



H2N COOH

H2N

H2N COOH

NH2

R

HN COOH(CH2)nH2N

21 3 (n = 2,3)

Fig. 1. Unnatural aliphatic diamino acids.

[10] unnatural diamino acids as scaffolds and handles 183

functional groups and/or C- or N-termini.2 For this purpose, lactam bridgeslinking Lys and Asp residues have been introduced in peptides.3 However,selecting linear oligomers prior to introducing conformational restrictionsis not essential and combinatorial chemistry has indeed been successful inthe direct production of lead compounds from chemical libraries.4 Apowerful method is to build libraries starting from suitable scaffolds thatdisplay several pendant functional groups to introduce diversity.1,5–7

In this context, amino acids bearing an extra amino functionality arepotentially very attractive either for peptide cyclization or as central scaf-fold structures displaying three points of diversity. A major advantage ofcarboxyl and amino groups is their compatibility with standard protocolsof peptide synthesis. Moreover, hydrophilic amide linkages present in theproducts are likely to increase their bioavailability as potential drugs.Finally, constructions based on amide bonds are usually chemically stableand their stability toward proteases is likely to be increased if unnaturaldiamino acids (Fig. 1) are involved. In spite of these useful features, few un-natural diamino acids with appropriate protecting groups have beenreported and still fewer are commercially available. A survey of such struc-tures is presented here and some of their possible applications in combina-torial chemistry are mentioned or illustrated by methodologicaldevelopments carried out in our research group: the use of derivatives ofbenzoic acid as scaffolds for creating diversity and a procedure for handlinga linker for solid-phase synthesis derived from l-2,3-diaminopropionic acid

1 E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, and M. A. Gallop, J. Med.

Chem. 37, 1385 (1994).2 J. N. Lambert, J. P. Mitchell, and K. D. Roberts, J. Chem. Soc. Perkin Trans. 1, 471 (2001).3 W. Zhang and J. W. Taylor, Tetrahedron Lett. 37, 2173 (1996) and references cited therein.4 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273

(2001).5 A. J. Souers and J. A. Ellman, Tetrahedron 57, 7431 (2001).6 J. A. Ellman, Acc. Chem. Res. 29, 132 (1996).7 M. Royo, M. del Fresno, A. Frieden, W. Van Den Nest, M. Sanseverino, J. Alsina,

S. A. Kates, G. Barany, and F. Albericio, React. Funct. Polym. 41, 103 (1999).

H2NCOOH

NH2

COOH

NH2H2N

COOH

NH2H2N

COOH

H2N

NH

NH

NH

COOH

H2N

HN

COOHNH

HN

COOH

4 5 6

7 8

10 11

**

** HN

COOH

NH2

9

Fig. 2. Unnatural cyclic diamino acids.


(1) (Dpr) and allowing the mild release of molecules in media that can bereadily made compatible with biological assays.

Two lower homologues of lysine, Dpr3,8–10 and l-2,4-diaminobutyricacid (2) (Dab), have often been used to introduce conformational restric-tions on peptides by lactam bridges. The Dpr to Asp linkage can also beused as a stable surrogate of disulfide bonds for stabilizing loops.11,12

Except for small cyclic systems, the residual conformational flexibility isnot likely to provide an appropriate rigidity to structures based on Dpror Dab residues. A similar finding can be applied to the backbone-to-back-bone cyclization strategy based on the use of N-aminoalkyl amino acidresidues 3.13

In our opinion, better rigidity may be expected from cyclic buildingblocks (Fig. 2). Compounds 4–6 containing three- or four-membered ringshave been prepared under conveniently protected forms.14,15 They have

8 J. Rizo, S. C. Koerber, R. J. Bienstock, J. Rivier, A. T. Hagler, and L. M. Gierasch, J. Am.

Chem. Soc. 114, 2852 (1992).9 C. H. Hassall, R. G. Tyson, and K. K. Chexal, J. Chem. Soc. Perkin Trans. 1, 2010 (1976).

10 P. Wipf and H.-Y. Kim, Tetrahedron Lett. 33, 4275 (1992).11 D. Limal, J.-P. Briand, P. Dalbon, and M. Jolivet, J. Peptide Res. 52, 121 (1998).12 C. Mendre, R. Pascal, and B. Calas, Tetrahedron Lett. 35, 5429 (1994).13 B. Muller, D. Besser, P. Kleinwachter, O. Arad, and S. Reissmann, J. Peptide Res. 54, 383

(1999).14 T. Wakamiya, Y. Oda, H. Fujita, and T. Shiba, Tetrahedron Lett. 27, 2143 (1986).15 E. Gershonov, R. Granoth, E. Tzehoval, Y. Gaoni, and M. Fridkin, J. Med. Chem. 39, 4833

(1996).


been mainly used as constrained analogues of lysine or ornithine or asintermediates in the preparation of analogues of arginine. Synthetic routesgiving access to building blocks derived from all the stereoisomers of4-aminoproline (7) and its homologue 8 have also been devised.16–18 Resi-due 9 has been used to promote helix formation in peptides.19 Carboxylicacids 10 and 11, derived from piperazine and imidazolidine, respectively,have been synthesized with orthogonal amino-protecting groups.20–22 Thestructure of residue 10 is also found in several �-turn mimetics.5 Althoughmany residues in Fig. 2 might be used as scaffolds for combinatorial synthe-sis, it is interesting to point out that only cis-aminoprolines 7 have beenconsidered for such applications.7

Several other protected diamino acids involve aromatic rings(Fig. 3).23–27 Their structures may also be suitable as scaffolds for buildingsynthetic libraries, but this application has been proposed only for com-pounds 12, 15, and 16.24,25 Whereas most of the structures displayed inFig. 2 require stereoselective syntheses, the preparation of diamino acids12–17 is facilitated by the absence of an asymmetric center.

One of the most important limitations in the use of these diamino acidsis the need of an additional orthogonal protection for the amino groups.Using the t-butoxycarbonyl/benzyl (Boc/Bzl) strategy of solid-phase pep-tide synthesis, this additional orthogonality can be easily provided by abase-labile protecting group such as the 9-fluorenylmethyloxycarbonyl

16 T. R. Webb and C. Eigenbrot, J. Org. Chem. 56, 3009 (1991).17 Z. Zhang, A. Van Aerschot, C. Hendrix, R. Busson, F. David, P. Sandra, and P. Herdewijn,

Tetrahedron 56, 2513 (2000).18 M. Tamaki, G. Han, and V. J. Hruby, J. Org. Chem. 66, 1038 (2001).19 C. L. Wysong, T. S. Yokum, G. A. Morales, R. L. Gundry, M. L. McLaughlin, and

R. P. Hammer, J. Org. Chem. 61, 7650 (1996).20 B. D. Dorsey, R. B. Levin, S. L. McDaniel, J. P. Vacca, J. P. Guare, P. L. Darke, J. A. Zugay,

E. A. Emini, W. A. Schleif, J. C. Quintero, J. H. Lin, I.-W. Chen, M. K. Holloway,

P. M. D. Fitzgerald, M. G. Axel, D. Ostovic, P. S. Anderson, and J. R. Huff, J. Med. Chem.

37, 3443 (1994).21 A. M. Warshawsky, M. V. Patel, and T.-M. Chen, J. Org. Chem. 62, 6439 (1997).22 L. Rene, L. Yaouancq, and B. Badet, Tetrahedron Lett. 39, 2569 (1998).23 R. M. Keenan, J. F. Callahan, J. M. Samanen, W. E. Bondinell, R. R. Calvo, L. Chen,

C. DeBrosse, D. S. Eggleston, R. C. Haltiwanger, S. M. Hwang, D. R. Jakas, T. W. Ku,

W. H. Miller, K. A. Newlander, A. Nichols, M. F. Parker, L. S. Southhall, I. Uzinskas,

J. A. Vasko-Moser, J. W. Venslavsky, A. S. Wong, and W. F. Huffman, J. Med. Chem. 42,545 (1999).

24 B. R. Neustadt, E. M. Smith, T. Nechuta, and Y. Zhang, Tetrahedron Lett. 39, 5317 (1998).25 R. Pascal, R. Sola, F. Labeguere, and P. Jouin, Eur. J. Org. Chem. 3755 (2000).26 M. H. Gelb and R. H. Abeles, J. Med. Chem. 29, 585 (1986).27 V. Santagada, F. Fiorino, B. Severino, S. Salvadori, L. H. Lazarus, S. D. Bryant, and

G. Caliendo, Tetrahedron Lett. 42, 3507 (2001).

NHNH2

COOHBoc

NH

NH

COOH

Boc

H2N COOH

NH

Boc

H2N COOH

NH

Fmoc

HN COOH

NH

Boc

Fmoc

NH2N Fmoc

COOH

COOH

NH

H2N

Fmoc

12 13 14

15 16 17

18

Z

Fig. 3. Unnatural arylamino amino acid building blocks.


(Fmoc) group. Using the Fmoc/t-butyl strategy, three main orthogonalclasses of protecting groups are available and have been recentlyreviewed.28 The first class consists of highly acid-labile groups such as2-(4-biphenyl)isopropoxycarbonyl (Bpoc), trityl (Trt), or derivatives, and�, �-dimethyl -3,5-dimethoxybenzyloxycarbonyl (Ddz), which can be re-moved in the presence of the t-butyl group with practically complete select-ivity. The second one involves groups removed by hydrazine such as the1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group or prefer-entially its isovaleryl analogue, Ddiv, which may avoid the intramolecularmigration observed with the Dde group.29 The third one involves the palla-dium-labile allyloxycarbonyl group (Alloc). However, in the case of thearomatic protected scaffolds 12, 15, and 16, the protection of the arylaminogroup was shown to be useless because it can be replaced by the choice ofselective coupling conditions.24,25

Our interest in diamino acid building blocks or scaffolds is connectedwith the studies of a new type of safety-catch linkers based on a dia-mino acid residue that we have carried out.30–32 Safety-catch linkers33 for

28 F. Albericio, Biopolymers 55, 123 (2000).29 S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W. Bycroft, and W. C. Chan,

Tetrahedron Lett. 39, 1603 (1998).30 R. Sola, P. Saguer, M.-L. David, and R. Pascal, J. Chem. Soc. Chem. Commun. 1786 (1993).

H2N COOH H2N COOH

H2N

15 or 16i, ii, iii iv

AmAbz

Fig. 4. Preparation of the AmAbz scaffold and of its protected derivatives 15 and 16. (i)

MeOCOCl, Na2SO4, dioxane; (ii) N-hydroxymethylphthalimide, 96% H2SO4–H2O (9:1, v/v);

(iii) NaOH, H2O; (iv) Boc2O or Fmoc–OSu, NaOH, dioxane-H2O.


solid-phase synthesis are designed to be cleaved by performing two differ-ent reactions with the advantage of providing an increased stability duringthe synthesis. Indeed, the Dpr(Phoc) linker30–32 (Phoc ¼ phenyloxycarbo-nyl) is incorporated by formation of amide bonds as stable as any peptidebond, but it can be activated as a cyclic N-acylurea and, thereby, made sen-sitive to nucleophiles at the end of the synthesis. It is well suited for the syn-thesis of peptides or peptidomimetics on hydrophilic supports and it iscompatible with Boc and Fmoc strategies. Moreover, due to the stabilityof this linker in strongly acidic media, side-chain-protecting groups canbe removed in an independent step preceding the cleavage in weakly alka-line aqueous solution. This linker could then become a powerful tool forthe preparation of libraries of peptides or peptidomimetics free of depro-tection contaminants and suitable for direct biological assays after additionof an appropriate buffer. The Dpr(Phoc) linker is therefore fully compat-ible with the high-throughput screening of libraries synthesized via solidphase, which requires ready purification procedures. Indications that re-lated aromatic structures might improve the cleavage rate have also beenreported.34

Methodology for the Use of the Protected Aromatic Scaffold 16

4-Amino-3-(aminomethyl)benzoic acid (AmAbz) can be easily pre-pared in three steps by amidomethylation of aminobenzoic acid (Fig. 4).25

Then the benzylamino group can be selectively protected by reaction withmild reagents such as Boc2O or Fmoc–OSu capable of discriminating be-tween the two amino groups to give the building blocks AmAbz(Boc)(15) and AmAbz(Fmoc) (16), respectively (by convention, the 4-aminogroup is defined here as the main chain and the 3-aminomethyl group as

31 R. Sola, J. Mery, and R. Pascal, Tetrahedron Lett. 37, 9195 (1996).32 R. Pascal and R. Sola, Tetrahedron Lett. 38, 4549 (1997).33 M. Patek and M. Lebl, Biopolymers 47, 353 (1998).34 R. Pascal, D. Chauvey, and R. Sola, Tetrahedron Lett. 35, 6291 (1994).

Fig. 5. Solid-phase synthesis of the AmAbz scaffold-containing heptapeptide 19. (i)

Piperidine–DMF (1:4);(ii) amino acid building block, BOP/HOBt/DIEA, DMF;(iii) Fmoc-

Phe, DIC, CH2Cl2; (iv) TFA, H2O, TIS (95:2.5:2.5, v/v/v).


the side chain for building abbreviations). The potential application of thisscaffold to the preparation of a library containing the 6.4 � 107 heptapep-tides obtained by combination of the 20 natural amino acids is illustratedby the solid-phase synthesis of the branched heptapeptide 19 (Fig. 5). Ami-nomethylpolystyrene resin (0.56 mmol/g) is derivatized with the Rinkamide linker and the two residues (Gly and Val) at the C-terminus canbe introduced by standard Fmoc-based solid-phase methods of peptide syn-thesis. At that time, AmAbz(Fmoc), which is now commercially available,can be introduced using BOP* activation in the presence of HOBt. With

* Abbreviations: BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluoro-

phosphate; DCC, N,N0-dicyclohexylcarbodiimide; DIC, N,N0-diisopropylcarbodiimide;

DME, dimethoxyethane; DMF, N,N-dimethylformamide; DIEA, N-ethyldiisopropyla-

mine; Et2O, diethyl ether; HOBt, 1-hydroxybenzotriazole; HOSu, N-hydroxysuccinimide;

t-BuOMe, tert-butylmethylether; TFA, trifluoroacetic acid; TIS, triisopropylsilane.


this acylation method, involving hydroxybenzotriazole (HOBt) activeesters, the free arylamino group is unaffected.25 The next residues can beselectively attached to the benzylamino group by using the same method.Then, more powerful conditions are required to acylate the arylaminogroup. Although PyBroP with diisopropylethylamine (DIEA) in CH2Cl2has been reported to be efficient,24 we have preferred a coupling procedureinvolving diisopropylcarbodiimide (DIC) activation in CH2Cl2 becausebasic conditions are likely to promote the racemization of the activatedamino acid. As a matter of fact, the arylamino group is not protonated inthe medium owing to its low basicity and can thus be acylated even in theabsence of base. The next residues can be introduced using standardcoupling conditions. The side-chain-protecting groups can be removedand the product released by acidolysis.

Methodology for the Use of Dpr(Phoc) Linker

The attachment of the Dpr(Phoc) residue to the support can be made,as previously reported,30–32 by coupling the Boc-Dpr(Phoc) building block.It is worth mentioning that this building block could not be obtained as acrystalline solid, a practical consideration to keep in mind when using thislinker. An improvement based on the preparation of the crystalline acti-vated ester Boc-Dpr(Phoc)–OSu is presented here as a convenient alterna-tive route as well as the procedures for applying it to Boc or Fmoc methodsof solid-phase synthesis (Fig. 6). Before the cleavage by mild alkaline hy-drolysis into carboxylic acid, the stable C-terminal amide linkage must beconverted into the labile acylurea via an intramolecular reaction inducedby the breakdown of the phenyl carbamate moiety under mild alkaline con-ditions. At this activation stage, high selectivity is needed. To prevent anyside reaction of the preceding amide group at this stage, the linker must beattached to solid supports bearing secondary amino groups. Thus TentagelS-NH2 resin can be modified with Boc-Sar (Sar ¼ N-methylglycine). Afterthe deprotection step, Boc-Dpr(Phoc)–OSu is reacted with the resin in thepresence of HOBt and DIEA. The reaction generally proceeds to comple-tion within 15–20 h as indicated by the chloranil test.35 The resin can thenbe used for solid-phase synthesis. Using Fmoc strategy, the linker must becyclized into the Imc (Imc ¼ 2-oxoimidazoline-5-carboxylic acid residue)form, which is resistant to piperidine treatment.31 This operation can bebetter carried out after the attachment of the C-terminal residue exceptfor sequences that are prone to diketopiperazine formation, which then

35 T. Vojkovsky, Peptide Res. 8, 236 (1995).

Fig. 6. Use of Dpr(Phoc) linker in Boc (left) or Fmoc (right) chemistries. PG, Boc or

Fmoc; scPGs, side-chain-protecting groups.


would require special treatment.31 After peptide chain elongation, the side-chain-protecting groups can be removed with the usual reagents for Boc orFmoc strategies prior to the alkaline cleavage of the linker; purificationsteps are not essential when using this procedure since the deprotection


contaminants are removed by simply washing the resin. Finally, crudeproducts of acceptable purity can generally be released in solutionfrom the support by a 1–3 h treatment with 0.01 N NaOH in water or2-propanol–water (7:3, v/v) at room temperature.

Experimental Protocols

General

A reaction vessel equipped with a fritted disc, a solvent inlet, and adevice to transfer in and out liquids under low nitrogen pressure was usedto carry out solid-phase syntheses. This manual apparatus enables suspen-sions of resin to be stirred either by gently rocking the vessel on a shaker orby bubbling nitrogen through the fritted plate.

Chemicals

Aminomethylpolystyrene resin (0.56 mmol/g, NovaBiochem, Laufel-fingen, Switzerland)

TentaGel S-NH2 resin (0.28 mmol/g, RappPolymere, Tubingen,Germany)

Fmoc-Rink amide linker (NovaBiochem, Laufelfingen, Switzerland)AmAbz(Fmoc) (16) (prepared according to Pascal et al.25 or

commercially available from Senn Chemical AG, Dielsdorf,Switzerland)

Use of AmAbz Building Blocks in the Solid-Phase Synthesis ofPeptidomimetics: The Typical Example of Heptapeptide 1925

Elongation. Aminomethylpolystyrene resin (1 g, 0.56 mmol) is firstwashed with DMF, CH2Cl2, TFA/CH2Cl2 (1:1, v/v), CH2Cl2, DIEA/CH2Cl2 (1:20, v/v), CH2Cl2, and DMF four times each. Then, Fmoc-Rinklinker (0.45 g, 0.84 mmol) is added to the resin with DMF (3 ml), BOP(0.37 g, 0.84 mmol), and DIEA (0.22 ml, 1.26 mmol) and the mixture isshaken for 90 min. The resin is filtered and washed with DMF, and thenDIEA (0.29 ml, 1.68 mmol) and acetic anhydride (0.53 ml, 5.6 mmol) areadded to cap unreacted amino groups. Except for Fmoc-Phe, the incorpor-ation of the next amino acids is carried out as follows: (1) the Fmoc group isremoved with piperidine/DMF (1:4, v/v) (1 � 1 min þ 3 � 3 min) and theresin is filtered and washed with DMF (4 � 1 min); (2) the N-protectedamino acid building block (1.85 mmol) and HOBt�H2O (0.26 g, 1.68 mmol)mmol) are then added to the resin using a minimum volume of DMF, thenDIEA (0.44 ml, 2.52 mmol) and BOP (0.743 g, 1.68 mmol) are added and


the mixture is shaken for 60 min. The resin is filtered and washed withDMF (4 � 1 min) and subjected to the next deprotection step or, at theend of the synthesis, it is filtered, washed with DMF, CH2Cl2, MeOH,100% EtOH, and Et2O, and then dried.

Acylation of the Arylamino Group. The resin is washed with DMF(4 � 1 min) and CH2Cl2 (4 � 1 min). A solution of Fmoc-Phe (0.716 g,1.85 mmol) in CH2Cl2 (10 ml) is added to the resin then DIC (0.263 ml,1.68 mmol) is added to the mixture. CH2Cl2 (10 ml) is added 5 min later be-cause of the precipitation of a Fmoc-Phe-activated species and the reactionmixture is shaken for 3 h. Fmoc-Phe coupling is then repeated using a similarprocedure except that DMF (6 ml) is added after 5 min and CH2Cl2 isevaporated off by nitrogen bubbling during the reaction.

Release and Recovery of Peptide 19. The dried resin (0.21 g) is sus-pended in TFA/H2O/TIS (95:2.5:2.5, v/v/v, 8.4 ml) and the mixture isshaken for 3 h. The suspension is filtered and the resin is washed withTFA (2 � 2 ml). The filtrate is concentrated, diluted with Et2O (100 ml),and then extracted with water (2 � 20 ml). The combined aqueous layersare concentrated and freeze-dried to give peptide 19 as a white solid(20 mg).

Preparation and Use of Dpr(Phoc) Linker

Preparation of Boc-Dpr.30,36 A mixture of diacetoxyiodobenzene(24.16 g, 75 mmol), acetonitrile (100 ml), and water (100 ml) is stirreduntil almost complete dissolution. Acetic acid (8.6 ml, 150 mmol) is thenadded and solid Boc-Asn (11.61 g, 50 mmol) is introduced into the flaskwith acetonitrile (25 ml) and water (25 ml). The mixture is stirred at roomtemperature for 24 h. The phenyl iodide by-product is removed by extrac-tion with t-BuOMe (2 � 100 ml). The aqueous layer is concentrated underreduced pressure and the solid residue is suspended in cold EtOH (100 ml),collected by filtration, and washed with cold EtOH then with Et2O, anddried under vacuum to give crude Boc-Dpr as a white crystalline solid(7.62 g, 75%).

Preparation of Boc-Dpr(Phoc).30 Crude Boc-Dpr (4.52 g, 22.1 mmol) isdissolved in water (110 ml) with sodium bicarbonate (4.65 g, 55.3 mmol).The mixture is stirred vigorously at room temperature, while phenylchloroformate (3.35 ml, 26.7 mmol) is added in five portions over 30 min.Stirring is continued for 4 h then the mixture is transferred into a separating

36 L.-h. Zhang, G. S. Kauffman, J. A. Pesti, and J. Yin, J. Org. Chem. 62, 6918 (1997); 63,

10085 (1998).


funnel and washed with t-BuOMe (2 � 100 ml). The t-BuOMe layer is sep-arated and the aqueous layer is acidified with 1 M NaHSO4 (33 ml) and ex-tracted with ethyl acetate (2 � 75 ml). The combined extracts are washedwith water and brine, then dried (Na2SO4) and concentrated under reducedpressure. The oily residue is diluted with CH2Cl2 (5 ml), then the solvent isevaporated under reduced pressure to give Boc-Dpr(Phoc) as a white solidfoam (5.53 g, 77%).

Preparation of Boc-Dpr(Phoc)–OSu. A solution of Boc-Dpr(Phoc)(11.06 g, 34.1 mmol) and HOSu (4.12 g, 35.8 mmol) in DME (20 ml) iscooled to 0

�then a solution of DCC in DME (15 ml) is added. The mixture

is stirred at 0�

for 60 min and allowed to stand overnight at 4�. The precipi-

tate is filtered off, washed with cold DME, and the filtrate is concentratedunder reduced pressure. Toluene (100 ml) is added to the residue and crys-tallization is initiated by ultrasonic irradiation (15 s) and continued over-night at 4

�. The solid is collected by filtration and washed with pentane.

Recrystallization from toluene gives Boc-Dpr(Phoc)–OSu as a white solid(11.2 g, 78%).

Preparation of the Resin Carrying Boc-Dpr(Phoc). The Tentagel resin(1 g, 0.28 mmol) is swollen in DMF (10 ml) for 30 min and then filtered andwashed with DMF four times. A mixture of Boc-Sar (0.175 g, 0.93 mmol)HOBt�H2O (0.129 g, 0.84 mmol) and DIC (0.132 ml, 0.84 mmol) is stirredfor 5 min in CH2Cl2 (1 ml) then added to the resin with a minimum volumeof DMF to allow stirring. DIEA (0.146 ml, 0.84 mmol) is added and the sus-pension is shaken for 60 min. The resin is filtered and washed with DMFand CH2Cl2 four times each and Boc-protecting groups are removed withTFA/CH2Cl2 (1:1, v/v) (5 ml, 1 � 1 min þ 1 � 30 min). Then the resin isfiltered and washed with CH2Cl2 (4 � 1 min), neutralized with DIEA/CH2Cl2 (1:20, v/v) (5 ml, 3 � 2 min), and washed with CH2Cl2 and DMFfour times each. Boc-Dpr(Phoc)–OSu, 0.177 g (0.42 mmol) and HOBt�H2O(0.064 g, 0.42 mmol) are added with DMF (2 ml). The suspension is shaken,then DIEA (0.073 ml, 0.42 mmol) is added and shaking is continued for24 h at room temperature, then the resin is filtered and washed withDMF four times. Acetic anhydride (0.26 ml, 2.8 mmol) and pyridine/CH2Cl2 (1:19, v/v) (5 ml) are added and allowed to react for 15 min tocap unreacted amino groups. The resin is filtered and washed with DMF,CH2Cl2, MeOH, EtOH, and Et2O four times each and dried undervacuum. Boc-protecting groups are removed with TFA/CH2Cl2 and theC-terminal residue of the target peptide is coupled to the resin (as an N-Boc- or N-Fmoc-protected building block) using the Boc protocol de-scribed below. Then, elongation can be continued using either the Boc orthe Fmoc protocols as follows.


Boc Protocol

Deprotection and Coupling. The resin (0.28 mmol) is treated withTFA/CH2Cl2 (1:1, v/v) (5 ml, 1 � 1 min þ 1 � 30 min), then filtered andwashed (1 min) with CH2Cl2, MeOH, and DMF four times each. Theamino acid building block (0.84 mmol) is activated with HOBt�H2O(0.129 g, 0.84 mmol) and DIC (0.132 ml, 0.84 mmol) by stirring for 5 min inCH2Cl2 (1 ml). Then the mixture is added to the resin with a minimumvolume of DMF to allow stirring. DIEA (0.122 ml, 0.70 mmol) is added andthe suspension is shaken for 30 min. Further DIEA is added (0.073 ml,0.42 mmol) and shaking is continued for 30 min. The resin is filtered andwashed with DMF then CH2Cl2 four times each.

Fmoc Protocol

Cyclization of the Linker. After the N-Fmoc-protected C-terminalresidue has been coupled, the resin (0.28 mmol) is filtered, washed withDMF, and then repeatedly treated with a solution of PhONa-PhOH inDMF (7 ml, 10 � 20 min) prepared as follows: a mixture of phenol(0.264 g, 2.80 mmol) and 1 N NaOH (1.4 ml, 1.4 mmol) is concentratedunder reduced pressure without heating; DMF (10 ml) is added to the resi-due then the solvent is evaporated under reduced pressure; phenol (0.033 g,0.35 mmol) is added to the residue and the final volume of the solution isadjusted to 70 ml with DMF. The resin is filtered and washed with DMFfour times.

Deprotection and Coupling. The resin (0.28 mmol) is treated with pi-peridine/DMF (1:4, v/v) (5 ml, 1 � 1 min þ 3 � 3 min), then washed withDMF (4 � 1 min). A mixture of the N-Fmoc amino acid building block(0.84 mmol), HOBt�H2O (0.129 g, 0.84 mmol), and DIC (0.132 ml,0.84 mmol) is stirred for 5 min in CH2Cl2 (1 ml) then added to the resin witha minimum volume of DMF to allow stirring. DIEA (0.073 ml, 0.42 mmol)is added and the suspension is shaken for 30 min. Further DIEA is added(0.073 ml, 0.42 mmol) and shaking is continued for 30 min. The resin isfiltered and washed with DMF four times.

[11] generation of dihydropyrimidine libraries 197

[11] Building Dihydropyrimidine Libraries viaMicrowave-Assisted Biginelli Multicomponent Reactions

By C. Oliver Kappe and Alexander Stadler

Introduction

Multicomponent reactions (MCRs) are of increasing importance inorganic and medicinal chemistry.1–5 In times where a premium is put onspeed, diversity, and efficiency in the drug discovery process,6 MCR strat-egies offer significant advantages over conventional linear-type synthe-ses.1–5 In such MCR reactions three or more reactants come together in asingle reaction vessel to form novel products that contain portions of all thecomponents.1–5 In an ideal case, the individual building blocks are commer-cially available or easily prepared covering a broad range of structural vari-ations. MCRs can rapidly provide products with the diversity needed forthe discovery of new lead compounds or lead optimization employing com-binatorial chemistry techniques.2–6 The search and discovery for newMCRs on one hand,7 and the full exploitation of already known multicom-ponent reactions on the other, are of considerable current interest. OneMCR that belongs in the latter category is the venerable Biginelli dihydro-pyrimidine synthesis. In 1893 Italian chemist P. Biginelli reported the acid-catalyzed cyclocondensation reaction of ethyl acetoacetate, benzaldehyde,and urea, as shown in Eq. (1).8

OMe

EtO2C

+Ph

O OH+

NH2

H2N

EtOH/HCl

NH

NH

Me

EtO2C

O

Ph

Surprisingly, the synthetic potential of this heterocycle synthesisremained unexplored for quite some time. In the 1970s and 1980s interestfor the original Biginelli cyclocondensation reaction slowly increased and

1 I. Ugi, A. Domling, and W. Horl, Endeavour 18, 115 (1994).2 R. W. Armstrong, A. P. Combs, P. A. Tempest, S. D. Brown, and T. A. Keating, Acc. Chem.

Res. 29, 123 (1996).3 L. F. Tietze and M. E. Lieb, Curr. Opin. Chem. Biol. 2, 363 (1998).4 S. L. Dax, J. J. McNally, and M. A. Youngman, Curr. Med. Chem. 6, 255 (1999).5 A. Domling, Comb. Chem. High Throughput Screen. 1, 1 (1998).6 M. Plunkett and J. A. Ellman, Sci. Am. 276, 68 (1997).7 L. Weber, K. Illgen, and M. Almstetter, Synlett 366 (1999).8 P. Biginelli, Gazz. Chim. Ital. 23, 360 (1893).



E

OR1

R2

HO

NH2

ZHN

R2

N

NHE

ZR1

R3

H+

R3

+

E = ester, amide, acyl, nitro

Z = O, S, NR

R1-R3 = H, alkyl, (het)aryl

1, DHPM

Fig. 1. Building blocks and points of diversity in the Biginelli three-component

dihydropyrimidine synthesis.

198 microwave-assisted synthesis [11]

its scope was gradually extended by variation of all three building blocksallowing access to a large number of multifunctionalized dihydropyrimi-dines (DHMP) of type 1.9 Today all three-component condensationsinvolving suitable CH-acidic-containing carbonyl compounds, aldehydes,and urea-type building blocks are considered Biginelli condensations asindicated in Fig. 1.10 A high degree of diversity can therefore be introducedby variation of all three components.9 Of the three building blocks in theBiginelli reaction, it is the aldehyde component that can be varied to thelargest extent introducing a point of diversity at the C4 position ofthe DHPM scaffold 1 (R2 in Fig. 1). In general, it has been found thatthe Biginelli reaction works best with aromatic aldehydes carrying eitherelectron-withdrawing or electron-donating groups at the o, m, or ppositions. Interestingly, good yields are usually obtained with m- orp-electron-withdrawing substituted aromatic aldehydes. Heterocyclic alde-hydes derived from furan, thiophene, and pyridine rings also generally fur-nish acceptable DHPM yields. Traditionally, simple alkyl acetoacetates areemployed as CH-acidic-containing carbonyl building block (E, R1 in Fig.1), but other types such as 3-oxoalkanoic esters or thioesters can also beused successfully in the Biginelli reaction. Benzoylacetic esters can alsobe used, but yields are usually significantly lower and the overall condensa-tion process is more sluggish. Primary, secondary, and tertiary acetoaceta-mides can be used in place of esters to produce pyrimidine-5-carboxamides.In addition, �-diketones serve as viable substrates in Biginelli reactions andthe condensation can also be applied to cyclic �-diketones. Furthermore,nitroacetone is a good building block leading to 5-nitro-substituted DHPMderivatives generally with very high yields. The urea analog is the compon-ent in the Biginelli reaction that faces the most restrictions in terms ofallowed structural diversity (Z, R3 in Fig. 1). Therefore, most of the pub-lished Biginelli examples involve urea itself as building block. However,

9 C. O. Kappe, Tetrahedron 49, 6937 (1993).10 C. O. Kappe, Acc. Chem. Res. 33, 879 (2000).


simple monosubstituted alkyl ureas in general react equally well in a re-giospecific manner and provide good yields of exclusively N1-substitutedDHPMs. Although thiourea and substituted thioureas follow the same gen-eral reactivity pattern as in ureas, they require longer reaction times toachieve good conversions. Yields are typically lower as compared to thecorresponding urea derivatives.

This flexibility in building block selection allows for considerable diver-sity to be introduced in the DHPM products.10 In the context of libraryproduction and screening, the Biginelli multicomponent protocol is par-ticularly attractive since the resulting DHPM scaffold covers a wide rangeof biological targets. In the past decades, a broad range of biologicaleffects, such as antiviral, antitumor, antibacterial, and antiinflammatoryactivities, has been ascribed to these partly reduced pyrimidine deriva-tives.11 More recently, appropriately functionalized DHPMs have emergedas orally active antihypertensive agents12–14 or �1a-adrenoceptor-selectiveantagonists.15 Furthermore, the structurally rather simple DHPM deriva-tive monastrol has been identified as a novel cell-permeable molecule thatblocks normal bipolar spindle assembly in mammalian cells causing cell-cycle arrest.16 Monastrol specifically inhibits the mitotic kinesin Eg5 motorprotein and can be considered as a new lead for the development of antic-ancer drugs.16 Apart from synthetic DHPM derivatives, several marinenatural products containing the dihydropyrimidine-5-carboxylate corehave recently been isolated exhibiting interesting biological activities.17

Most notably among these natural products are the batzelladine alkaloidsA and B, which inhibit the binding of HIV envelope protein gp120 tohuman CD4 cells serving as potential new leads for AIDS therapy.18

11 C. O. Kappe, Eur. J. Med. Chem. 35, 1043 (2000).12 K. S. Atwal, B. N. Swanson, S. E. Unger, D. M. Floyd, S. Moreland, A. Hedberg, and

B. C. O’Reilly, J. Med. Chem. 34, 806 (1991).13 G. C. Rovnyak, K. S. Atwal, A. Hedberg, S. D. Kimball, S. Moreland, J. Z. Gougoutas,

B. C. O’Reilly, J. Schwartz, and M. F. Malley, J. Med. Chem. 35, 3254 (1992).14 G. J. Grover, S. Dzwonczyk, D. M. McMullen, D. E. Normandin, C. S. Parham, P. G. Sleph,

and S. Moreland, J. Cardiovasc. Pharm. 26, 289 (1995).15 J. C. Barrow, P. G. Nantermet, H. G. Selnick, K. L. Glass, K. E. Rittle, K. F. Gilbert, T. G.

Steele, C. F. Homnick, R. M. Freidinger, R. W. Ransom, P. Kling, D. Reiss, T. P. Broten,

T. W. Schorn, R. S. L. Chang, S. S. O’Malley, T. V. Olah, J. D. Ellis, A. Barrish,

K. Kassahun, P. Leppert, D. Nagarathnam, and C. Forray, J. Med. Chem. 43, 2703 (2000).16 T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King, S. L. Schreiber, and T. J. Mitchison,

Science 286, 971 (1999).17 L. Heys, C. G. Moore, and P. J. Murphy, Chem. Soc. Rev. 29, 57 (2000).18 A. D. Patil, N. V. Kumar, W. Kokke, M. F. Bean, A. J. Freyer, C. De Brosse, S. Mai,

A. Truneh, D. J. Faulkner, B. Carte, A. L. Breen, R. P. Hertzberg, R. K. Johnson,

J. W. Westley, and B. C. M. Potts, J. Org. Chem. 60, 1182 (1995).

Fig. 2. Solid-phase Biginelli condensation using �-aminobutyric acid -derived urea on

Wang resin.


Previous Dihydropyrimidine Libraries

Since the experimental conditions for the traditional Biginelli reactionare rather straightforward, small libraries of DHPMs are readily accessibleby parallel synthesis. Along these lines the generation of a 140-membersingle-compound DHPM library by combination of 25 aldehydes, 6 ureas/thioureas, and 7 acetoacetates or acetoamides under standard reaction con-ditions has been reported.19,20 Apart from these conventional solution-phase methods to prepare DHPM libraries, it is also possible to employpolymer-supported reagents to aid in the purification and workup protocol.Polymer-assisted solution-phase chemistry using polymer-supported Lewisacid [Yb-(III)-reagent supported on Amberlyst 15] in combination withpolymer-supported urea scavenging resins (Amberlyst 15 and Ambersep900 OH) permits a rapid parallel Biginelli synthesis with a simple andefficient purification strategy.21

Solid-phase protocols allow an even higher degree of throughput andautomation as shown in the example in Fig. 2.22 In this example, �-amino-butyric acid-derived urea was attached to Wang resin using standard pro-cedures. The resulting polymer-bound urea was then condensed with anexcess of a �-ketoester and aromatic aldehydes (such as benzaldehyde,Fig. 2) in the presence of a catalytic amount of hydrochloric acid to affordthe corresponding immobilized DHPMs. Subsequent cleavage of the prod-uct from the polystyrene resin with trifluoroacetic acid provided DHPMsin high yields and excellent purities.

In a variation of the above protocol, the Biginelli synthesis was easilyadapted to fluorous-phase conditions.23,24 Here a fluorous urea derivative

19 K. Lewandowski, P. Murer, F. Svec, and J. M. J. Frechet, Chem. Commun. 2237 (1998).20 K. Lewandowski, P. Murer, F. Svec, and J. M. J. Frechet, J. Comb. Chem. 1, 105 (1999).21 A. Dondoni and A. Massi, Tetrahedron Lett. 42, 7975 (2001).22 P. Wipf and A. Cunningham, Tetrahedron Lett. 36, 7819 (1995).

Fig. 3. Solid-phase Biginelli condensations using Wang resin-bound acetoacetates.


was prepared by attaching a suitable fluorous tag to hydroxyethylurea. Thefluorous urea was then condensed with excess of acetoacetates and alde-hydes in a suitable solvent containing hydrochloric acid. After extraction ofthe fluorous DHPMs with fluorous solvent, desilylation with tetrabuty-lammonium fluoride followed by extractive purification provided the‘‘organic’’ Biginelli products in good overall yields. Considering the simpleexperimental techniques used in this fluorous chemistry, automationshould be feasible, thus allowing the preparation of DHPM libraries.

In addition to the methods described above where the urea componentis linked to a solid (or fluorous) support, it is also possible to link insteadthe acetoacetate building block to the solid support as shown in theexample in Fig. 3.25 Thus, Biginelli condensation of Wang-bound acetoace-tates with excess aldehydes (such as 2-trifluoromethylbenzaldehyde, Fig. 3)and urea/thiourea provides the desired DHPMs on solid support. Subse-quent cleavage with trifluoroacetic acid furnishes the free carboxylic acidsin high overall yields.

There are alternative solid-phase protocols described in the literaturefor the generation of DHPMs, not via the classic three-component Biginelliapproach but through related modifications.26,27 By employing any of thesolid-phase synthesis methods described above, large libraries of DHPMscan potentially be generated in a relatively straightforward fashion. Be-cause of the inherent benefits of solution-phase protocols over solid-phasestrategies, we herein describe an automated solution-phase method using

23 A. Studer, S. Hadida, R. Ferritto, S.-Y. Kim, P. Jeger, P. Wipf, and D. P. Curran, Science

275, 823 (1997).24 A. Studer, P. Jeger, P. Wipf, and D. P. Curran, J. Org. Chem. 62, 2917 (1997).25 M. G. Valverde, D. Dallinger, and C. O. Kappe, Synlett 741 (2001).26 L. D. Robinett, K. M. Yager, and J. C. Phelan, 211th National Meeting of the American

Chemical Society, New Orleans, 1996, ORGN 122. American Chemical Society,

Washington, DC, 1996.27 C. O. Kappe, Bioorg. Med. Chem. Lett. 10, 49 (2000).


microwave irradiation to prepare small focused DHPM libraries. The keyelement in our strategy relies on reaction enhancement by microwaveirradiation.

Microwave-Assisted Organic Synthesis

Parallel to the recent developments in combinatorial chemistry, micro-wave-enhanced synthesis has attracted a substantial amount of attention inrecent years. During the past decade an ever-increasing number of reportshave been published that advocate the advantages and the use of micro-wave irradiation to carry out organic synthesis.28 Significant increases inrates, yields, and purities of final products have frequently been observedwhen employing this nonconventional and energy-efficient heatingmethod. Using microwave dielectric heating, the microwave radiationpasses through the walls of the reaction vessel heating only the reactantsand solvent, but not the reaction vessel. The energy transfer is not pro-duced by conduction or convection, but by dielectric loss. Thus, the degreeto which a sample could undergo microwave heating depends on the dielec-tric properties that are represented by the so-called loss tangent (tan �).29

Materials dissipate microwave energy by two main mechanisms: dipole ro-tation and ionic conduction. When molecules with a permanent dipole aresubmitted to an electric field, they become aligned. If this field oscillates,the orientation changes with each alternation. The strong agitation, pro-vided by the reorientation of molecules, in phase with the electrical fieldexcitation, causes an intense internal heating. During ionic conduction, asthe dissolved charged particles in a sample (usually ions) oscillate backand forth under the influence of the microwave field, they collide with theirneighboring molecules or atoms. This collision causes agitation or motion,creating heat.

The main benefits of performing reactions under microwave irradiationconditions are the significant rate enhancements and the higher productyields that can frequently be observed. This method has been success-fully applied in various fields of synthetic organic chemistry28–41 such as

28 P. Lidstrom, J. Tierney, B. Wathey, and J. Westman, Tetrahedron 57, 9225 (2001).29 C. Gabriel, S. Gabriel, E. H. Grant, B. S. J. Halstead, and D. M. P. Mingos, Chem. Soc. Rev.

27, 213 (1998).30 A. de la Hoz, A. Diaz-Ortis, A. Moreno, and F. Langa, Eur. J. Org. Chem. 3659 (2000).31 R. S. Varma, J. Heterocycl. Chem. 36, 1565 (1999).32 N. Elander, J. R. Jones, S.-Y. Lu, and S. Stone-Elander, Chem. Soc. Rev. 29, 239 (2000).33 M. Larhed, C. Moberg, and A. Hallberg, Acc. Chem. Res. 35, 717 (2002).34 A. Loupy, A. Petit, J. Hamelin, F. Texier-Boullet, P. Jacquault, and D. Mathe, Synthesis

1213 (1998).


cycloaddition reactions,30 heterocycle synthesis,31 radiolabeled materials,32

transition metal-catalyzed processes,33 solvent-free reactions,34 and phase-transfer catalysis.35 In fact, it is becoming evident that microwave-assistedapproaches could be developed for many chemical transformations requir-ing heat. Not surprisingly, these features have recently started to attractthe interest of the drug discovery/medicinal chemistry communitieswhere reaction speed is of great importance.36–39 The combination ofmicrowave heating technology and combinatorial chemistry applicationstherefore seems a logical consequence of the increased speed and effective-ness offered by using microwave irradiation instead of conventionalheating methods. While different hypotheses have been proposed toaccount for the observed rate enhancements under microwave irradiation,a generally accepted rationalization remains elusive.40 Regardless of theorigin/existence of a special microwave effect, this novel heating source isextremely efficient and applicable to a broad range of practical synthesis.

Although many of the early experiments in microwave-assisted organicsynthesis have been carried out in domestic microwave ovens, the currenttrend clearly is to use specialized instruments for this type of chemical syn-thesis. Experiments carried out in domestic ovens have been found to bedifficult to reproduce, owing to the lack of temperature and pressure con-trol, pulsed irradiation, uneven electromagnetic field distributions, and theunpredictable formation of hotspots.

For several years a number of commercial microwave systems havebeen available offering either batch or continuous flow-type reactors forchemical synthesis. Most microwave reactors commercially available todayfeature built-in magnetic stirrers, direct temperature control of the reactionmixture with the aid of fiberoptic probes or infrared (IR) sensors, and soft-ware that enables on-line temperature/pressure control by regulation ofmicrowave power output. In recent years all suppliers of microwave instru-mentation for organic synthesis have moved toward combinatorial/high-throughput platforms.41 Currently two different philosophies with respectto microwave reactor design are emerging: multimode and monomode(single-mode) reactors. In the so-called multimode instruments (conceptu-ally similar to a domestic oven), the microwaves that enter the cavity arebeing reflected by the walls and the load over the typically large cavity.

35 S. Deshayes, M. Liagre, A. Loupy, J.-L. Luche, and A. Petit, Tetrahedron 55, 10851 (1999).36 M. Larhed and A. Hallberg, Drug Discov. Today 6, 406 (2001).37 J. L. Krstenansky and I. Cotterill, Curr. Opin. Drug Discov. Dev. 4, 454 (2000).38 A. Lew, P. O. Krutzik, M. E. Hart, and A. R. Chamberlin, J. Comb. Chem. 4, 95 (2002).39 C. O. Kappe, Curr. Opin. Chem. Biol. 6, 314 (2002).40 L. Perreux and A. Loupy, Tetrahedron 57, 9199 (2001).41 A. Loupy, ‘‘Microwaves in Organic Synthesis.’’ Wiley-VCH, Weinheim, 2002.


A mode stirrer ensures that the electromagnetic field distribution is ashomogeneous as possible. In the much smaller mono- or single-mode cav-ities, only one mode is present and the electromagnetic irradiation, throughan accurately designed wave guide, is focused directly onto the reactionvessel mounted at a fixed distance from the radiation source. For applica-tions of combinatorial chemistry the key difference between these twotypes of reactor systems is that whereas in multimode cavities several reac-tion vessels can be irradiated simultaneously in multivessel rotors (parallelsynthesis), in monomode systems only one vessel can be irradiated at thetime. In the latter case high throughput can be achieved by integrated ro-botics that move individual reaction vessels in and out of the microwavecavity (automated sequential synthesis). For the current preparation of aDHPM library, the latter concept has been employed.

Microwave-Assisted Biginelli Reactions

The concept of speeding up the Biginelli dihydropyrimidine synthesisby microwave irradiation has already been reported several times in theliterature. Those procedures involve microwave heating open vessels con-taining a solution of the building blocks in a suitable solvent (i.e., ethanol,acetic acid) with an acidic catalyst (HCl or montmorillonite clay).42–46

Alternatively, several authors have exploited the inherent benefits of carry-ing out microwave-heated Biginelli reactions in the absence of solvent(‘‘dry-media synthesis’’).34 In this case the building blocks are eitheradsorbed on an inorganic support or admixed with a suitable nonvolatilecatalyst.46–50 In most cases, significant rate enhancements have beenreported in those studies where reaction times are reduced from severalhours when using conventional reflux conditions to a few minutes. More-over, the final yields were also improved as compared to the outcome ofstandard thermal protocols. Both procedures, however, have employed do-mestic microwave ovens that have severe limitations when used to carryout chemical transformations. In fact, in some instances it was proven that

42 R. Gupta, A. K. Gupta, S. Paul, and P. L. Kachroo, Ind. J. Chem. 34B, 151 (1995).43 A. Dandia, M. Saha, and H. Taneja, J. Fluorine Chem. 90, 17 (1998).44 J. S. Yaday, B. V. Subba Reddy, E. Jagan Reddy, and T. Ramalingam, J. Chem. Res.

354 (2000).45 S. J. Tu, J. F. Zhou, P. J. Cai, H. Wang, and J. C. Feng, Synth. Commun. 32, 147 (2002).46 A. Stadler and C. O. Kappe, J. Chem. Soc. Perkin Trans. 1, 1363 (2000).47 R. Gupta, S. Paul, and A. K. Gupta, Ind. J. Chem. Technol. 5, 340 (1998).48 J. L. Krstenansky and Y. Khmelnitsky, Bioorg. Med. Chem. 7, 2157 (1999).49 H. A. Stefani and P. M. Gatti, Synth. Commun. 30, 2165 (2000).50 C. O. Kappe, D. Kumar, and R. S. Varma, Synthesis 1799 (1999).


experimental artifacts were responsible for the observed enhancements.46

In contrast, very few publications have reported Biginelli condensationsusing dedicated microwave reactors for chemical syntheses. In those casesethanol was used as solvent and the reactions were carried out in sealedvessels under fully controlled conditions (temperature/pressure) showingthe expected rate enhancements.46,51,52 However, so far what has beensomewhat neglected in microwave-assisted processes is throughput andautomation. In one case a dedicated parallel reactor with 36 compartmentswas used for the preparation of multiple DHPM derivatives in one singleirradiation experiment in a dedicated multimode microwave cavity.51

One limitation of this approach, however, is that all reaction vessels duringlibrary production are exposed to the same irradiation conditions in termsof reaction time and microwave power. An alternative way to achieve highthroughput in microwave-assisted synthesis would be to perform reactionssequentially in an automated fashion. The benefit of this approach is thatapart from the achievable throughput in the library production, fast iter-ations in protocol development and in optimization of reaction conditionscan be realized. Here we report the generation of a small library of 48dihydropyrimidine derivatives via the three-component Biginelli con-densation via automated sequential microwave-assisted synthesis using acommercially available microwave synthesizer.

Microwave Chemistry Utilizing the Emrys Synthesizer

The microwave synthesizer used in this work (Fig. 4) is composed of amonomode (also referred to as single-mode) microwave cavity that oper-ates at a frequency of 2.45 GHz with continuous microwave irradiationpower from 0 to 300 W. The reaction vials are glass-made 10-ml closedtubes, sealed with Teflon septa and an aluminum crimp top. The vials areavailable in two different designs to carry out reactions in 0.5–2.0 or 2.0–5.0 ml scale. Magnetic stirring bars are available for both types of vials.The vials are moved in and out of the microwave cavity in an automatedfashion by a gripper incorporated into the platform. Inside the microwavecavity these vessels can be exposed to up to 20 bars of pressure and 250

�.

The reaction temperature is measured with an IR sensor (infrared therm-ometry) on the outer surface of the reaction vial. Specialized software regu-lates the microwave output power so that the preselected maximumtemperature is maintained for the desired reaction/irradiation time. Re-agents can be added manually into the vials before capping or through

51 M. Nuchter, W. Lautenschlager, B. Ondruschka, and A. Tied, LaborPraxis 25, 28 (2001).52 A. Stadler and C. O. Kappe, J. Comb. Chem. 3, 624 (2001).

Fig. 4. Monomode microwave reactor with integrated robotic platform for automated

use (left). A liquid handler allows dispensing of reagents into Teflon-sealed reaction vials,

while a gripper moves each vial in and out of the microwave cavity after irradiation. The

instrument processes up to 120 reactions per run with a maximum throughput of 12–15

reactions/h. The temperature is measured by an IR sensor on the outside of the reaction

vessel. Details of the cavity/gripper (top right) and reaction vials (bottom right) are also

displayed (Emrys Synthesizer, Personal Chemistry AB). Reprinted with permission from

Wiley-VCH.41


the Teflon septum via the platform’s liquid handler. When using the liquidhandler, the system can be programmed to deliver a sequence of reagentsfrom different stock solutions into different process vials. Upon completionof the irradiation period process, the reaction vessels are cooled down rap-idly (20–80 s) to ambient temperature by using a stream of compressed air(gas jet cooling).

Synthesis Criteria for Dihydropyrimidine Library

To construct a small library of DHPMs via microwave-enhanced Bigi-nelli condensation, we felt that several criteria should be met.

1. The DHPMs should be synthesized in solution using standardBiginelli three-component condensation reaction mixtures. Al-though microwave-assisted Biginelli reactions have been publishedunder solvent-free conditions,46–50 we felt that such methods would


not allow the production of high-quality libraries in an automatedfashion.

2. The solvent system employed should be able to dissolve most of thebuilding blocks at room temperature so the liquid handler could beused to dispense all building blocks into the appropriate reactionvials.

3. The resulting DHPM products should be only sparingly soluble inthe solvent system used at room temperature to facilitate theirisolation.

4. All building blocks used for the construction of the DHPM libraryshould be commercially available covering a reasonable diversityrange.

5. Since the reaction conditions will be run in a sequential format,individual reaction times need to be kept at a minimum (10–20 min)to achieve a reasonable throughput.

6. Reaction scale must deliver multi-100 mg quantities of material forfurther scaffold decoration work.

Reaction Optimization in the Emrys Synthesizer

Today there is a great variety of suitable reaction conditions for carry-ing out Biginelli condensations. For the originally published condensationof ethyl acetoacetate with benzaldehyde and urea more than 40 differentexperimental protocols are known.53 Traditionally, Biginelli condensationsare carried out in a solvent such as ethanol or methanol, but more recentlyaprotic solvents such as tetrahydrofuran, dioxane, or acetonitrile have alsobeen used successfully. In some cases it is necessary to use acetic acid assolvent. This is particularly important in those cases in which condensationof an aromatic aldehyde and urea will lead to precipitation of an insolublebisurea derivative, i.e., ArCH(NHCONH2)2,54 which may not react anyfurther along the desired pathway when ethanol alone is used as a solvent.Biginelli reactions in water and ionic liquids are also reported. Althoughthese methods are for solution-phase synthesis, a recent trend involvesthe condensation without any solvent using the components eitheradsorbed on an inorganic support or in the presence of a suitable catalyst.

The Biginelli condensation strongly depends on the amount of acidiccatalyst present in the reaction medium. Traditionally, strong Brønstedacids such as hydrochloric or sulfuric acid have been employed, but todaythe use Lewis acids such as BF3�OEt2, LaCl3, FeCl3, Yb(OTf)3, InCl3,

53 C. O. Kappe and A. Stadler, Org. React. 63, (2003).54 C. O. Kappe, J. Org. Chem. 62, 7201 (1997).


BiCl3, LiClO4, Mn(OAc)3, or ZrCl4 is prefered.53 It is worth noting that itis also possible to use a solid acid catalyst such as an acidic clay, zeolite, orAmberlyst material.

As far as the molar ratio of building blocks is concerned, Biginelli reac-tions generally employ an excess of the CH-acidic carbonyl or urea com-ponents, and not of the aldehyde. Since the resulting DHPM products ofa Biginelli-type condensation are usually only sparingly soluble in solventssuch as methanol or ethanol at room temperature, in many cases workupinvolves only isolation of the formed product by a simple filtration. Alter-natively, it is also possible to precipitate the products by addition of water.

For our microwave-promoted high-throughput library synthesis wedecided to use a protocol that utilizes a combination of Lewis acids andBrønsted acids as catalysts.

Step 1: Choice of Solvent

To make full use of the liquid handler incorporated in the microwavereactor platform (see Fig. 4) we attempted to dissolve all three types ofbuilding blocks (Fig. 1) in solvents that are compatible with the reactionconditions. Disappointingly, most urea components, in particular ureaitself, were not soluble in sufficient amounts at room temperature in anyof the following standard solvent systems: ethanol, acetic acid, tetrahydro-furan, dioxane, acetonitrile, N-methylpyrrolidone, and N,N-dimethylfor-mamide. Water could not be used as a solvent due to its incompatibilitywith the high-temperature microwave conditions. Since many of the pub-lished protocols employ either ethanol or acetic acid as solvents in Biginel-li-type condensations, we decided to explore the use of a 3:1 mixture ofacetic acid (AcOH) and ethanol (EtOH) to develop a microwave-assistedsolution phase protocol. Both solvents effectively couple with microwaveirradiation (tan � at 2.45 GHz/25

�: EtOH ¼ 0.941, AcOH ¼ 0.174).29 This

allows the reaction mixture to heat up very rapidly under microwave irradi-ation conditions leading to so-called microwave flash heating conditions.Attempts using other solvents, such as dioxane or THF, proved far less ef-fective both in terms of heating rates and product yields. Not only did theAcOH/EtOH solvent combination have the advantage that all buildingblocks are soluble under the reaction conditions at elevated temperatures,but the resulting DHPM products were also comparatively insoluble atroom temperature facilitating product isolation. Thus, for the automated li-brary synthesis protocols, the CH-acidic carbonyl components were dis-solved in AcOH and the aldehyde building blocks in EtOH, whereas theurea components and the catalyst had to be dispensed as solids into theprocess vials.

Fig. 5. The influence of the catalyst on the Biginelli condensation involving ethyl

acetoacetate, benzaldehyde, and urea [see Eq. (1)] in a 3:1 AcOH/EtOH solvent mixture

under microwave irradiation (120�, 10 min).


Step 2: Selection of Catalyst

The next parameter involved the selection of a suitable catalyst. Fromour previous experience with microwave-assisted Biginelli condensationsat high temperature it became evident that hydrochloric acid was not themost suitable reaction promoter due to its role in the then explored decom-position of the urea components to ammonia, leading to unwanted by-products.46 We then explored the use of more tolerable Lewis acids suchas Yb(OTf)3,55 InCl3,56 FeCl3,57 and LaCl3,58 all recently reported to bevery effective catalysts for Biginelli condensations, presumably by stabiliz-ing the key N-acyliminium ion intermediates.54 An initial screen of allthese catalysts for the model system ethyl acetoacetate, benzaldehyde,and urea revealed that 10 mol% Yb(OTf)3 was the most effective catalystwith the AcOH/EtOH 3:1 solvent system providing the desired DHPM in92% isolated yield (Fig. 5). The use of only 5 mol% of the same catalystfurnished a significantly lower yield (68%). In the conventionally heatedBiginelli reactions a period of 2–3 h of refluxing conditions (in ethanol,concentrated hydrochloric acid as catalyst) is typically required to achievea 60–70% yield. For all experiments displayed in Fig. 5 an initial set ofconditions involving 10-min microwave flash heating at 120

�was chosen.

55 Y. Ma, C. Qian, L. Wang, and M. Yang, J. Org. Chem. 65, 3864 (2000).56 B. C. Ranu, A. Hajra, and U. Jana, J. Org. Chem. 65, 6270 (2000).57 J. Lu, Y. Bai, Z. Wang, B. Yang, and H. Ma, Tetrahedron Lett. 41, 9075 (2000).58 J. Lu and H. Ma, Synlett 63 (2000).


Step 3: Optimization of Time and Temperature

Having identified an efficient solvent/catalyst combination [AcOH/EtOH 3:1, 10 mol% Yb(OTf)3] we next dealt with the issue of reactiontime and temperature. One of the benefits of using microwave flash heatingin sealed vessels is the fact that one is not limited by the boiling point of thesolvents or reagents as in conventional synthesis using refluxing conditions.After a few optimization cycles we discovered that 120

�proved to be an op-

timum reaction temperature (Fig. 6). While higher temperatures wouldlead to decreased yields due to the formation of undesired by-products,lower reaction temperatures on the other hand required longer reactiontimes for complete conversion. For the model system displayed in Eq. (1)a total irradiation time of 10 min at 120

�resulted in 92% isolated yield of

pure product. The final DHPM product precipitated directly after theactive cooling period (Fig. 7) and showed no traces of impurities as deter-mined by 1H NMR analysis. Although an increase in reaction time to15 min would further increase the yield for this example to 94%, we havedecided generally not to use longer reaction times unless warranted by thespecific building block combinations (see Step 4). All reactions were run inthe larger 2.0–5.0 ml process vials using 4.0 mmol of each building block ina total of 1.6–2.0 ml of solvent mixture. At a temperature of 120

�this led to

a pressure of ca. 3–4 bar in the vial, well below the accessible limit of 20bar. Although it has been shown that higher yields of DHPMs can be

Fig. 6. Optimization of reaction time and temperature for the Biginelli condensation

involving ethyl acetoacetate, benzaldehyde, and urea [see Eq. (1)] in a 3:1 AcOH/EtOH

solvent mixture with 10 mol% Yb(OTf)3 as a catalyst.

0

20

40

60

80

100

120

140

0 60 120 180 240 300 360 420 480 540 600 660

Time (sec)

Tem

pera

ture

(8C

)

0

1

2

3

4

5

6

Pre

ssur

e (b

ar)

Fig. 7. Temperature (bold) and pressure profiles for the Biginelli condensation

involving ethyl acetoacetate, benzaldehyde, and urea [Eq. (1)] in AcOH/EtOH (3:1) under

sealed vessel/microwave irradiation conditions. Microwave flash heating (300 W, 0–40 s),

temperature control using the feedback from IR thermography (constant 120�, 40–600 s), and

active cooling (600–660 s).


obtained by employing either the carbonyl or urea building blocks inexcess, we have not optimized for molar ratios of reagents using equimolaramounts of building blocks in all experiments.

Step 4: Optimization for Troublesome Building Blocks

Having identified an optimized set of reaction conditions (conditions A,Fig. 8) for the model substrate of the planned DHPM library, we nextlooked at potentially troublesome reagents and reagent combinations inour selection of building blocks. Thioureas, for example, are known to givesignificantly lower yields when employed in the Biginelli condensation.9

We have discovered that for thioureas LaCl3 is usually the preferred cata-lyst in a microwave-assisted protocol (conditions B, Fig. 8). In the case ofacid-sensitive aldehydes such as furane-2-carbaldehyde, we devised amodified protocol in which neat ethanol was used as a solvent at 100

�(con-

ditions C, Fig. 8). Other examples of reaction conditions fine-tuned to spe-cific building block combinations are also presented in Fig. 8. The yields forthe optimized microwave-assisted Biginelli condensations are in generalcomparable or higher than the yields obtained using the standard refluxconditions. More importantly, however, reaction times have been brought

NH

NH

S

EtO

Me

O

B: AcOH/EtOH 3:1, LaCl3 (10 mol%) 120 �C, 10 min, 56% yield

NH

NH

O

EtO

Me

OO

C: EtOH, Yb(OTf)3 (10 mol%) 120 �C, 20 min, 50% yield

N

NH

S

EtO

Me

O

Me

D: EtOH, LaCl3 (10 mol%) 120 �C, 10 min, 41% yield

NH

NH

O

H2N

Me

O

E: EtOH, HCl (10 mol%) 120 �C, 15 min, 59% yield

NH

NH

O

EtO

Me

O

A: AcOH/EtOH 3:1, Yb(OTf)3 (10 mol%)120 �C, 10 min, 56% yield

NO2

Fig. 8. Optimized reaction conditions A–E for selected examples of DHPM products.


down from several hours (4–12 h) under reflux conditions to 10–20 minusing superheated solvents and direct in-core microwave-flash heating.The optimization cycles described above can be carried out within a fewhours, providing optimized sets of conditions (conditions A–E, Fig. 8)useful for synthesizing a larger library.

Automated Sequential Library Production

With a set of five optimized reaction conditions (solvent, catalyst, time)in hand for a variety of representative Biginelli condensations (Fig. 8), wenext turned our attention toward the production of a small library ofDHPMs. A set of structurally diverse representative building blocks waschosen: 17 CH-acidic carbonyl compounds, 25 aldehydes, and 8 urea/thioureas (the structures of the building blocks can be determined fromthe structures of the final DHPM products displayed in Fig. 9). A combin-ation of all these building blocks in a Biginelli-type fashion would lead to alibrary of 3400 unique DHPMs. To demonstrate the practicality of our syn-thetic protocol we decided to generate a representative subset library of 48DHPM analogs involving all building blocks mentioned above. While theoptimization experiments described previously were carried out manually(i.e., adding individual reagents, catalysts, and solvents into the processvials before capping of the vial) we now attempted to automate this

NH

NHEtO

O

O

HN

HN OEt

O

O

NH

NHEtO

O

O N

NHEtO

O

O

Et

N

NHEtO

O

O

Ph

NH

NHEtO

O

S N

NHEtO

O

S

Me

NH

NHEtO

O

O

NO2

NH

NHEtO

O

S

OH

NH

NHEtO

O

O

OMe

OMe

NH

NHEtO

O

O

Cl

NH

NHEtO

O

O

F

F

NH

NHEtO

O

O

CF3

N

NHEtO

O

O NH

NHEtO

O

O

N

NH

NHEtO

O

O

O

NH

NHEtO

O

O

S

NH

NHEtO

O

O NH

NHEtO

O

O

NH

NHMeO

O

O

F

NH

NHMeO

O

S

NH

NHMeO

O

O

MeO

OMe

OMe

NH

NHMeO

O

O

OH

OMe

N

NHO

O

O

NO2

Me

NH

NHMeO

O

O

O

O2N

A/120/10/92 A/120/10/18 A/120/10/43 B/120/10/56 D/120/10/41

A/120/10/54 C/120/20/45 C/120/10/52 A/120/10/68

A/120/20/49 C/120/10/78 C/120/10/41 A/120/10/30 A/120/10/89

NH

NHMeO

O

O

C/100/20/50 A/120/10/35 C/120/20/67 C/100/15/46 A/120/10/62

A/120/10/81 D/120/20/58 C/120/10/73 C/100/10/34 A/120/10/51

C/120/10/61

Fig. 9. (continued)


NH

NHO

O

O NH

NHO

O

O

NO2

N

NHO

O

O

NO2

NH

NHO

O

O N

NHO

O

O

Ph

Me

Cl

Cl

NH

NHH2N

O

O

NO2

N

NHH2N

O

S

Ph

NH

NHNH

O

O

NO2

Me

NH

NHEt2N

O

O

Br

NH

NHNH

O

O

Ph

NH

NHNH

O

O

Ph

OH

OMe

NH

NHNH

O

S

Ph

Cl

NH

NH

O

O NH

NH

O

O NH

NHMeO

O

O NH

NHMeO

O

O

F

F

NH

NHMeO

O

O

OMe

OMeMeO

NH

NHMeO

O

O

NO2

MeONH

NHMeO

O

O

F

F

N

NHMeO

O

O

Cl

Cl

Me

NH

NHEtO

O

O

NO2

NH

NHEtO

O

OPh

NO2

NH

NHO2N

O

C/120/10/50 C/120/15/73 A/120/10/34 C/120/10/49 A/120/10/25

C/120/10/53 C/120/10/68 C/120/20/31 E/120/20/31 C/120/20/35

A/120/10/35 C/120/10/26 A/120/10/64 C/120/10/41 A/120/20/40

E/120/15/59E/120/15/21 A/120/10/66 A/120/10/61 A/120/10/55

B/120/10/89E/100/15/28 B/100/15/83

Fig. 9. Structural representation of a 48-member DHPM library. Given are the reaction

parameters in the format: conditions/temperature (�)/reaction time (min)/yield (%).

Conditions refers to the solvent/catalyst systems A–E specified in Fig. 8.



process as far as possible using of the liquid handler/gripper devices of themicrowave synthesizer. For that purpose, stock solutions of all CH-acidiccarbonyl components in acetic acid were prepared and stored in designatedrack positions [for experiments employing methods C–E (Fig. 8) EtOH wasused as solvent]. Similarly, all solid aldehydes were dissolved in absoluteethanol, the only exception being aldehydes that would not be soluble inthe required concentration in ethanol. These were weighed directly in thereaction vials. Liquid CH-acidic carbonyl compounds and aldehydes werenot used as stock solutions, they were dispensed neat. All urea/thioureaderivatives and the catalysts Yb(OTf)3 and LaCl3 were weighed directlyinto the process vials before capping. After all building blocks and reactionconditions were entered into the software, the sequential irradiation of all48 process vials was programmed identifying the rack position of the cor-responding stock solutions. The appropriate building blocks were first dis-pensed into the corresponding vials through the Teflon septa, then eachvial was sequentially moved in and out of the microwave cavity by the grip-per. Irradiation using the conditions specified in Fig. 9 produced the de-sired DHPMs in 18–92% isolated yield. It can be determined from thedata presented in Fig. 9 that an ample degree of diversity in all the threebuilding blocks is well tolerated. Thus all five variable substituents (R1–R3, E, Z, see Fig. 1) around the DHPM scaffold can be modified, increasingthe structural diversity of DHPM analogs that can be synthesized. In themajority of cases, the solid DHPM derivatives would precipitate directlyfrom the reaction mixture after active cooling. The examples that wouldnot crystallize directly upon cooling were crystallized after the crude reac-tion mixture was poured onto ice water. 1H NMR spectra were obtainedfrom all samples to determine their chemical identity and purity. All prod-ucts had at least >90% purity; in most cases no foreigner signals other thanthe ones for the products could be identified from their 1H NMR spectrum.While the 48 examples of DHPM analogs presented in Fig. 9 are a repre-sentative subset of the full 3400-member library, it should be stressed thatnot all combinations of building blocks may lead to DHPMs in such a highstate of purity. For some of the examples given in Fig. 9 the yields were notas high as previously reported using different (e.g., solvent-free) Biginelliprotocols. Undoubtedly, these yields could be further optimized by fine-tuning the microwave-assisted reaction conditions, i.e., varying the molarratios of reagents or using different solvent/catalyst combinations. No at-tempts were made along these lines since the protocols described abovedeliver quantities of several 100 mg of pure DHPM products. Recent evi-dence from this laboratory also indicates that these reactions could becarried out to a significantly smaller or larger scale without reoptimizationof reaction conditions.59 The high reaction speed and ease of product


generation/isolation far outweigh the higher yields that may be obtainableby other protocols, such as solvent-free procedures, where the protocolscannot be easily automated.

With an average irradiation time of ca. 15 min (including the timeneeded for dispensing reagents and moving vials in and out of the cavity)the generation of the 48-member library could be achieved within 12 h.Since the instrument is designed for fully automated unattended operation,a library of this size can conveniently be prepared overnight.

Concluding Remarks

Very recently, a report was published51 covering high-speed parallel Bi-ginelli reactions carried out in specifically designed multimode microwavereactors. While this method allows for a considerable throughput due tothe relatively short timeframe of a microwave-enhanced chemical reaction,the individual control over each reaction vessel in terms of reaction tem-perature/pressure is limited. As an alternative to parallel synthesis we havereported the automated sequential synthesis of DHPM libraries. Contraryto the parallel mode where all reaction vessels are exposed to the same ir-radiation conditions, irradiating each individual reaction vessel separatelynot only gives better control over the reaction parameters, but also allowsfor the rapid optimization of reaction conditions. To ensure similar tem-peratures in a parallel set-up, the same amount of the identical solventhas to be used in each reaction vessel due to the dielectric properties in-volved. For the preparation of relatively small libraries where delicatechemistries are to be performed, a sequential synthetic format is the modeof choice.

Microwave-assisted synthesis in general is likely to have a tremendousimpact in the medicinal/combinatorial chemistry communities because itshortens reaction times, improves final yields and purities, and can carryout reactions that previously were thought impossible to do. It should bestressed that in general the rate enhancements seen in microwave-assistedsynthesis can be attributed to the very rapid heating of the reaction mixture(flash heating) and the high temperatures that can be reached, rather thanto any other specific or nonthermal microwave effect.40

Moreover, the short reaction times open up new approaches for rapidtesting of ideas and fast iterations in protocol development as demon-strated here successfully for the Biginelli reaction. While microwaveheating is today still considered by some as a laboratory curiosity, we be-lieve that this technology will be used extensively in the future for many

59 A. Stadler, S. Pichler, G. Horeis, and C. O. Kappe, Tetrahedron 58, 3177 (2002).


chemical processes requiring heat. For this, new developments in micro-wave reactor and vessel design are still warranted to address issues suchas scale-up, higher throughput in monomode cavities, more reliable paral-lel reactors with improved temperature control, and specialized vesseldesign for solid-phase organic synthesis. Despite these current limitationsit is clear that the use of microwave heating in combinatorial chemistry willcontinue to grow and is likely to become a standard procedure in the nextfew years.



All building blocks were purchased from commercial sources and usedwithout further purification. Lewis acid catalysts Yb(OTf)3�H2O (Aldrich40,532-9), LaCl3�7 H2O (Aldrich 26,207-2), InCl3 (Aldrich 42,941-4), andFeCl3�6 H2O (Aldrich 20,792-6) were purchased from Aldrich ChemicalCo. in the specifications given. 1H NMR spectra were recorded on a BrukerAMX360 or AMX500 instrument in CDCl3 or DMSO-d6, operating at 360or 500 MHz, respectively.

Microwave Irradiation Experiments

The Emrys synthesizer (PersonalChemistry AB)60 was used in thestandard configuration as delivered including proprietary Workflow Man-ager software (version 1.1). Optimization experiments were performed in‘‘single-run’’ mode (i.e., by manual filling of reaction vials) and by specify-ing the irradiation time and maximum temperature. The DHPM librarygeneration (Fig. 9) was done in an automated fashion using the liquid hand-ler capabilities of the instrument/software. For that purpose, each of therequired 48 2.0–5.0 ml process vials was filled with 4.0 mmol of the corres-ponding urea/thiourea building blocks and 10 mol% (0.4 mmol) of Lewisacid catalyst [for method E 100 �l of 4 M HCl in dioxane (Aldrich34,554-7) was employed]. The vials were sealed with the Teflon septumand aluminum crimp using an appropriate crimping tool. Each vial wasthen placed in its correct position in the rack of the Smith synthesizer asspecified by the software. Then 3.3 M stock solutions of CH-acidic car-bonyl compounds in AcOH and 10.0 M stock solutions of aldehydes wereprepared and similarly placed in designated rack positions. If solutionscould not be prepared because of insufficient solubility, the insoluble

60 PersonalChemistry AB, Kungsgatan 76, SE-753 18 Uppsala, Sweden; phone: (internat.)

þ46-18-4899000; fax: (internat.) 46-18-4899100; http://www.personalchemistry.com.


building block was added manually to the urea/thiourea and catalyst com-ponents directly into the vial. In cases where the building block was aliquid, dispensing was done using the reagent neat in which case additionalsolvent mixture was added to reach the minimum filling volume of 2.0 ml(ca. 1.5 ml). For conditions C–E stock solutions of the correspondingCH-acidic carbonyl compounds in EtOH were additionally prepared.Using the liquid handler, 4.0-mmol aliquots of the corresponding aldehydes(400 �l) and carbonyl compounds (1200 �l) were dispensed to the processvials containing the appropriate urea/thiourea and catalyst (an additional400 �l of solvent was added to reach the minimum volume of 2.0 ml). Aftercharging all the reagents in an individual vessel, the vial was moved in andout of the microwave cavity where irradiation for 10–20 min at 100–120

�

(Figs. 8 and 9) was performed. After the full irradiation sequence was com-pleted, racks containing the processed vials were stored at 4

�for 8 h. In

case of precipitation, the solid products were filtered, washed with cold(4

�) EtOH, and dried. Where no precipitation was experienced, the crude

reaction mixture was treated with 10 ml of ice water and allowed to standfor 12 h at 4

�. The solid DHPM products were filtered and treated as

above. The purity of all DHPM products was >90% according to 1HNMR measurements (360 MHz). 1H NMR spectral data for all 48 DHPMs,including melting points (mp), are given below.

Spectral Data for DHPM Library

Ethyl 6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carbox-ylate: yield 978 mg (92%); mp 204

�; 1H NMR (DMSO-d6) � 1.12 (t,

J¼ 7.5 Hz, 3H), 2.28 (s, 3H), 4.03 (q, J¼ 7.5 Hz, 2H), 5.17 (d, J¼ 3 Hz,1H), 7.22–7.41 (m, 5H), 7.78 (br s, 1H), 9.22 (br s, 1H).

Ethyl 6-methyl-4-phenyl-1-ethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 208 mg (18%); mp 124–125

�; 1H NMR (DMSO-d6) �

1.08 (m, 6H), 2.31 (s, 3H), 3.83 (m, 2H) 4.03 (q, J¼ 7.5 Hz, 2H), 5.14 (d,J¼ 3 Hz, 1H), 7.20–7.34 (m, 5H), 7.87 (br s, 1H).

Ethyl 6-methyl-4-phenyl-1-benzyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 608 mg (43%); mp 155–56


1.11 (t, J¼ 7.5 Hz, 3H), 2.39 (s, 3H), 4.03 (q, J¼ 7.5 Hz, 2H), 4.88, 5.11(2d, J¼ 17.5 Hz, 2H), 5.25 (d, J¼ 3.0 Hz, 1H), 7.05–7.39 (m, 10H), 8.13(d, J¼ 3.0 Hz, 1H).

Ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carb-oxylate: yield 620 mg (56%); mp 205

�; 1H NMR (DMSO-d6) � 1.12 (t,

J¼ 7.5 Hz, 3H), 2.31 (s, 3H), 4.02 (q, J¼ 7.5 Hz, 2H), 5.20 (d, J¼ 3.0 Hz,1H), 7.20–7.41 (m, 5H), 9.68 (br s, 1H), 10.31 (br s, 1H).


Ethyl 1,6-dimethyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 480 mg (41%); mp 144–147


1.18 (t, J¼ 7.5 Hz, 3H), 2.55 (s, 3H), 3.51 (s, 3H), 4.12 (q, J¼ 7.5 Hz, 2H),5.22 (d, J¼ 3.5 Hz, 1H), 7.18–7.41 (m, 5H), 9.88 (br s, 1H).

Ethyl 6-methyl-4-(2-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 662 mg (54%); mp 215–216


0.94 (t, J¼ 7.5 Hz, 3H), 2.30 (s, 3H), 3.88 (q, J¼ 7.5 Hz, 2H), 5.81 (d,J¼ 3.0 Hz, 1H), 7.49–7.98 (m, 5H), 9.39 (br s, 1H).

Ethyl 6-methyl-4-(3-hydroxyphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 531 mg (45%); mp 179


1.07 (t, J¼ 7.5 Hz, 3H), 2.28 (s, 3H), 4.03 (q, J¼ 7.5 Hz, 2H), 5.09 (d, J¼ 4Hz, 1H), 6.63 (m, 3H), 7.11 (m, 1H), 9.43 (br s, 1H), 9.58 (s, 1H), 10.27 (brs, 1H).

Ethyl 6-methyl-4-(3,4-dimethoxyphenyl)-2-oxo-1,2,3,4-tetrahydropyri-midine-5-carboxylate: yield 670 mg (52%); mp 175

�; 1H NMR (DMSO-d6)

� 1.15 (t, J¼ 7.5 Hz, 3H), 2.28 (s, 3H), 3.73 (s, 6H), 4.12 (q, J¼ 7.5 Hz, 2H),5.11 (d, J¼ 3 Hz, 1H), 6.70–7.95 (m, 3H), 7.69 (br s, 1H), 9.17 (br s, 1H).

Ethyl 6-methyl-4-(2-chlorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 805 mg (68%); mp 210–212


1.08 (t, J¼ 7.5 Hz, 3H), 2.32 (s, 3H), 3.91 (q, J¼ 7.5 Hz, 2H), 5.67 (d,J¼ 2.5 Hz, 1H), 7.22–7.46 (m, 4H), 7.72 (br s, 1H), 9.30 (br s, 1H).

Ethyl 6-methyl-4-(3,4-difluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 723 mg (61%); mp 188


1.11 (t, J¼ 7.5 Hz, 3H), 2.29 (s, 3H), 4.02 (q, J¼ 7.5 Hz, 2H), 5.17 (d,J¼ 3.0 Hz, 1H), 7.05–7.50 (m, 3H), 7.80 (br s, 1H), 9.28 (br s, 1H).

Ethyl 6-methyl-4-(2-trifluoromethylphenyl)-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate: yield 644 mg (49%); mp 202–204

�; 1H NMR

(DMSO-d6) � 0.97 (t, J¼ 7.5 Hz, 3H), 2.45 (s, 3H), 3.97 (q, J¼ 7.5 Hz,2H), 5.37 (br s, 1H), 5.82 (br s, 1H), 7.32–7.70 (m, 4H), 8.46 (br s, 1H).

Diethyl 1,4-phenylen-di-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 692 mg (78%); mp >330

�(dec.); 1H NMR (DMSO-

d6) � 1.09 (t, J¼ 7.5 Hz, 6H), 2.19 (s, 6H), 3.96 (q, J¼ 7.5 Hz, 4H), 5.11(d, J¼ 3 Hz, 2H), 7.17 (s, 4H), 7.67 (br s, 2H), 9.14 (br s, 2H).

Ethyl 6-methyl-4-(1-naphthyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 575 mg (41%); mp 104–106


0.79 (t, J¼ 2.5 Hz, 3H), 2.56 (s, 3H), 3.80 (q, J¼ 3.0 Hz, 2H), 4.32 and4.42 (2d, J¼ 4 Hz, 2H), 5.13 (d, J¼ 3.0 Hz, 1H), 5.17 (m, 1H), 5.89 (m,1H), 6.07 (s, 1H), 7.40–7.97 (m, 7H), 8.30 (s, 1H).

Ethyl 6-methyl-4-(3-pyridinyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 325 mg (31%); mp 209–211


1.08 (t, J¼ 7.5 Hz), 2.01 (s, 3H), 3.97 (q, J¼ 7.0 Hz), 5.19 (d, J¼ 3.0 Hz,1H), 7.35–7.62 (m, 2H), 7.78 (br s, 1H), 8.45 (s, 2H), 9.28 (br s, 1H).


Ethyl 6-methyl-4-(2-thienyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 951 mg (89%); mp 214–217


1.19 (t, J¼ 7.5 Hz, 3H), 2.28 (s, 3H), 4.10 (q, J¼ 7.5 Hz, 2H), 5.43 (d,J¼ 3.0 Hz, 1H), 6.89–7.11 (m, 2H), 7.33–7.42 (d, J¼ 6.0 Hz, 1H), 7.92 (brs, 1H), 9.33 (br s, 1H).

Ethyl 6-methyl-4-(2-furanyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 503 mg (50%); mp 193–194


1.21 (t, J¼ 7.5 Hz, 3H), 2.36 (s, 3H), 4.13 (q, J¼ 7.5 Hz, 2H), 5.48 (d,J¼ 3.0 Hz, 1H), 5.83 (br s, 1H), 6.12 (d, J¼ 3 Hz, 1H), 6.27 (s, 1H), 7.32(s, 1H), 7.79 (br s, 1H).

Ethyl 6-methyl-4-(5-pentyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carb-oxylate: yield 357 mg (35%); mp 148–150

�; 1H NMR (DMSO-d6) � 0.86 (t,

J¼ 7.5 Hz, 3H), 1.18 (t, J¼ 7.5 Hz, 3H), 1.20–1.37 (m, 6H), 2.15 (s, 3H),3.99–4.13 (m, 4H), 5.39 (m, 1H), 7.30 (br s, 1H), 8.90 (br s, 1H).

Ethyl 4,6-dimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate:yield 534 mg (67%); mp 194

�; 1H NMR (DMSO-d6) � 1.08 (d, J¼ 5.5 Hz,

3H), 1.20 (t, J¼ 7.5 Hz, 3H), 2.15 (s, 3H), 4.00–4.14 (m, 3H), 7.19 (br s,1H), 8.96 (br s, 1H).

Methyl 6-methyl-4-(3-methoxy-4-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahy-dropyrimidine-5-carboxylate: yield 539 mg (46%); mp 245–247

�; 1H NMR

(DMSO-d6) � 2.24 (s, 3H), 3.54 (s, 3H), 3.72 (s, 3H), 5.06 (d, J¼ 3.0 Hz,1H), 6.58–6.80 (m, 3H), 7.62 (br s, 1H), 8.89 (s, 1H), 9.10 (br s, 1H).

Methyl 6-methyl-4-(3,4,5-trimethoxyphenyl)-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate: yield 834 mg (62%); mp 203–204

�; 1H NMR

(DMSO-d6) � 2.25 (s, 3H), 3.57 (s, 3H), 3.63 (s, 3H), 3.73 (s, 6H), 5.11 (d,J¼ 3.0 Hz, 1H), 6.65 (d, J¼ 3.0 Hz, 2H), 7.70 (br s, 1H), 9.19 (br s, 1H).

Methyl 6-methyl-4-(4-fluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 860 mg (81%); mp 188–190

�; 1H NMR (DMSO-

d6) � 2.25 (s, 3H), 3.97 (s, 3H), 5.38 (d, J¼ 3.0 Hz, 1H), 7.12–7.27 (m,4H), 7.75 (br s, 1H), 9.22 (br s, 1H).

Methyl 6-methyl-4-(4-methylphenyl)-2-thioxo-1,2,3,4-tetrahydropyri-midine-5-carboxylate: yield 643 mg (58%); mp 153–155

�; 1H NMR

(DMSO-d6) � 2.26 (s, 3H), 2.28 (s, 3H), 3.63 (s, 3H), 5.13 (d, J¼ 3.5 Hz,1H), 7.08–7.15 (m, 4H), 9.61 (br s, 1H), 10.26 (br s, 1H).

Methyl 6-methyl-4-(2-methylphenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 760 mg (73%); mp 235–237

�; 1H NMR (DMSO-

d6) � 2.31 (s, 3H), 2.43 (s, 3H), 3.48 (s, 3H), 5.41 (d, J¼ 4.0 Hz, 1H),7.11–7.22 (m, 4H), 7.62 (br s, 1H), 9.19 (br s, 1H).

Methyl 6-methyl-4-(5-nitro-2-furyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 385 mg (34%); mp 237–239

�; 1H NMR (DMSO-

d6) � 2.26 (s, 3H), 3.60 (s, 3H), 5.31 (d, J¼ 3.0 Hz, 1H), 6.59, 7.60 (2d,J¼ 4.5 Hz, 2H), 8.000 (br s, 1H), 9.48 (br s, 1H).


Allyl 1,6-dimethyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 677 mg (51%); mp 122–123

�; 1H NMR (DMSO-

d6) � 2.51 (s, 3H), 3.20 (s, 3H), 4.52 (m, 2H), 5.10–5.20 (m, 2H), 5.45 (d,J¼ 3.5 Hz, 1H), 5.70–5.90 (m, 1H), 6.82 (d, J¼ 3.5 Hz, 1H), 7.30–7.60 (m,2H), 8.00–8.10 (m, 2H).

Isopropyl 6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 550 mg (50%); mp 192–193


1.00, 1.19 (2d, J¼ 7.5 Hz, 6H), 2.28 (s, 3H), 4.82 (m, 1H), 5.13 (d,J¼ 3.0 Hz, 1H), 7.20–7.31 (m, 5H), 7.71 (br s, 1H), 9.17 (br s, 1H).

Isopropyl 6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 654 mg (73%); mp 199–201

�; 1H NMR (DMSO-

d6) � 1.00, 1.18 (2d, J¼ 6.0 Hz, 6H), 2.28 (s, 3H), 4.84 (m, 1H), 5.31 (d,J¼ 3.0 Hz, 1H), 7.65–7.77 (m, 2H), 7.91 (br s, 1H), 8.09–8.19 (m, 2H),9.38 (br s, 1H).

Isopropyl 6-methyl-4-(3-nitrophenyl)-1-allyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate: yield 490 mg (34%); mp 115–118

�; 1H NMR

(DMSO-d6) � 1.00, 1.18 (2d, J¼ 7.0 Hz, 6H), 2.47 (s, 3H), 4.03, 4.46 (2d,J¼ 7.5 Hz, 2H), 4.86 (m, 1H), 4.95 (d, J¼ 7.5 Hz, 1H), 5.10 (d, J¼ 5.5 Hz,1H), 5.32 (d, J¼ 3.0 Hz, 1H), 5.83 (m, 1H), 7.64–7.69 (m, 2H), 8.10 (br s,1H), 8.14 (d, 2H).

tert-Butyl 6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 566 mg (49%); mp 200–202


1.30 (s, 9H), 2.24 (s, 3H), 5.12 (d, J¼ 3.0 Hz, 1H), 7.20–7.39 (m, 5H), 7.69(br s, 1H), 9.09 (br s, 1H).

Benzyl 1,6-dimethyl-4-(2,3-dichlorophenyl)-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate: yield 412 mg (25%); mp 125–126

�; 1H NMR

(DMSO-d6) � 2.57 (s, 3H), 3.14 (s, 3H), 4.52 (m, 2H), 4.96–5.16 (m, 2H),5.74 (d, J¼ 3.5 Hz, 1H), 6.90–7.60 (m, 8H), 8.10 (d, J¼ 3.5 Hz, 1H).

6-Methyl-5-acetyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine: yield490 mg (53%); mp 240–242

�; 1H NMR (DMSO-d6) � 2.01 (s, 3H), 2.29 (s,

3H), 5.25 (d, J¼ 3 Hz, 1H), 7.26–7.32 (m, 5H), 7.80 (br s, 1H), 9.16 (br s, 1H).6-Methyl-5-acetyl-4-(3-methylphenyl)-2-oxo-1,2,3,4-tetrahydropyri-

midine: yield 666 mg (68%); mp 250–252�; 1H NMR (DMSO-d6) � 2.09 (s,

3H), 2.28 (s, 6H), 5.21 (d, J¼ 3 Hz, 1H), 7.05–7.20 (m, 4H), 7.75 (br s, 1H),9.12 (br s, 1H).

Methyl 4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate:yield 290 mg (31%); mp 226–227

�; 1H NMR (DMSO-d6) � 3.55 (s, 3H),

5.11 (d, J¼ 3.0 Hz, 1H), 7.25–7.35 (m, 6H), 7.69 (s, 1H), 9.20 (br s, 1H).Methyl 4-(3,4,5-trimethoxyphenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-

5-carboxylate: yield 401 mg (31%); mp 216–219�; 1H NMR (DMSO-d6) �

3.56 (s, 3H), 3.63 (s, 3H), 3.73 (s, 6H), 5.10 (d, J¼ 3.0 Hz, 1H), 7.32 (d,J¼ 7 Hz, 1H), 6.55 (s, 2H), 7.64 (s, 1H), 9.18 (d, J¼ 3.0 Hz, 1H).


Methyl 4-(3,4-difluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 377 mg (35%); mp 229–231


3.56 (s, 3H), 5.15 (d, J¼ 3.0 Hz, 1H), 7.11 (br s, 1H), 7.22–7.45 (m, 3H),7.75 (s, 1H), 9.28 (br s, 1H).

Methyl 6-methoxymethyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate: yield 450 mg (35%); mp 182–184

�; 1H NMR

(CDCl3) � 3.48 (s, 3H), 3.65 (s, 3H), 4.66 (m, 2H), 5.50 (d, J¼ 3.0 Hz, 1H),6.33 (br s, 1H), 7.49–7.68 (m, 3H), 8.14 (d, J¼ 7 Hz, 1H), 8.17 (br s, 1H).

Methyl 6-ethyl-4-(2,3-dichlorophenyl)-1-methyl-2-oxo-1,2,3,4-tetrahy-dropyrimidine-5-carboxylate: yield 360 mg (26%); mp 163

�; 1H NMR

(DMSO-d6) � 1.17 (t, J¼ 7.5 Hz, 3H), 2.98 (m, 2H), 3.17 (s, 3H), 3.49 (s,3H), 5.60 (d, J¼ 3.0 Hz, 1H), 7.25–7.55 (m, 3H), 8.02 (br s, 1H).

Methyl 6-ethyl-4-(3,4-difluorophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxylate: yield 762 mg (64%); mp 186


1.13 (t, J¼ 7.5 Hz, 3H), 2.69 (m, 2H), 3.57 (s, 3H), 5.18 (d, J¼ 3.0 Hz, 1H),7.03–7.49 (m, 3H), 7.82 (br s, 1H), 9.32 (br s, 1H).

Ethyl 6-propyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 549 mg (41%); mp 173–175


0.90 (t, J¼ 7.5 Hz, 3H), 1.10 (t, J¼ 7.5 Hz, 3H), 1.57 (q, J¼ 7.5 Hz, 2H),2.60 (m, 2H), 4.00 (q, J¼ 7.5 Hz, 3H), 5.27 (d, J¼ 3.5 Hz, 1H), 7.49 (d,J¼ 8.5 Hz, 2H), 7.86 (br s, 1H), 8.21 (d, J¼ 8.5 Hz, 2H), 9.30 (br s, 1H).

Ethyl 6-phenyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: yield 591 mg (40%); mp 233–235

�; 1H NMR (DMSO-d6)

0.74 (t, J¼ 7.5 Hz, 3H), 3.74 (q, J¼ 7.5 Hz, 2H), 5.39 (d, J¼ 3.0 Hz, 1H),7.32–8.35 (m, 9H), 8.02 (br s, 1H), 9.48 (br s, 1H).

6-Methyl-1,4-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbox-amide: yield 277 mg (21%); mp 198–200

�; 1H NMR (DMSO-d6) � 1.73 (s,

3H), 5.28 (d, J¼ 3 Hz, 1H), 7.18, 7.21 (2 br s, 2H), 7.33–7.45 (m, 10H), 9.62(br s, 1H).

6-Methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-car-boxamide: yield 652 mg (59%); mp 281

�(dec.); 1H NMR (DMSO-d6) �

2.07 (s, 3H), 5.36 (d, J¼ 3 Hz, 1H), 6.97 (br m, 2H), 7.49 (d, J¼ 8.0 Hz,2H), 7.66 (br s, 1H), 8.21 (d, J¼ 8.0 Hz, 2H), 8.72 (br s, 1H).

Methyl 6-methyl-4-(2-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide: yield 766 mg (66%); mp 142–144


1.76 (s, 3H), 2.45 (s, 3H), 5.37 (d, J¼ 3 Hz, 1H), 7.24 (br s, 1H), 7.47–7.90(m, 4H), 8.72 (br s, 1H).

Diethyl 6-methyl-4-(4-bromophenyl)-2-oxo-1,2,3,4-tetrahydropyrimi-dine-5-carboxamide: yield 895 mg (61%); mp 227–230

�; 1H NMR

(DMSO-d6) � 1.05 (t, J¼ 7.0 Hz, 6H), 1.64 (s, 3H), 3.05 (q, J¼ 7.0 Hz,4H), 4.99 (d, J¼ 3 Hz, 1H), 7.15 (d, J¼ 8.0 Hz, 2H), 7.32 (br s, 1H), 7.51(d, J¼ 8.0 Hz, 2H), 8.48 (br s, 1H).

[12] MA-SPOS of oxazolidinones 223

6-Methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxanil-lide: yield 680 mg (55%); mp 245–247

�; 1H NMR (DMSO-d6) � 2.04 (s,

3H), 5.40 (d, J¼ 3 Hz, 1H), 6.99 (br s, 1H), 7.24–7.71 (m, 10H), 8.69(s, 1H), 9.54 (s, 1H).

6-Methyl-4-(3-methoxy-4-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahydropyr-imidine-5-carboxanillide: yield 400 mg (28%); mp 238–241

�; 1H NMR

(DMSO-d6) � 2.03 (s, 3H), 3.66 (s, 3H), 5.32 (d, J¼ 3 Hz, 1H), 6.69–7.55(m, 9H), 8.62 (s, 1H), 8.89 (s, 1H), 9.49 (s, 1H).

6-Methyl-4-(2-chlorophenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylanillide: yield 1278 mg (89%); mp 195–197

�; 1H NMR (DMSO-

d6) � 2.02 (s, 3H), 5.74 (d, J¼ 3.0 Hz, 1H), 6.99–7.87 (m, 9H), 9.49 (br s,1H), 9.63 (br s, 1H), 10.04 (br s, 1H).

6-Methyl-5-nitro-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine: yield775 mg (83%); mp 188–190

�; 1H NMR (DMSO-d6) � 1.58 (s, 3H), 5.65 (d,

J¼ 3.0 Hz, 1H), 7.26–7.47 (m, 6H), 8.07 (br s, 1H).

[12] Microwave-Assisted Solid-Phase Organic Synthesis(MA-SPOS) of Oxazolidinone Antimicrobials

By Andrew P. Combs, Brian M. Glass, and Sharon A. Jackson

Introduction

Microwave-assisted organic synthesis (MAOS)* has dramaticallyevolved since its first use in the mid-1980s. Recent advances in microwaveequipment have provided homogeneous microwave heating in safe and re-liable instruments for use in the chemistry laboratory. Microwave-assistedheating has been utilized with many solution-phase reactions, typically re-ducing reaction times from days or hours to minutes or even seconds. Theobserved rapid microwave dielectric heating is primarily due to the con-tinuous realignment of polar molecules with the paramagnetic oscillatingfield. The microwave radiation is increasingly more absorbed by moleculeswith larger dipole moments and thus reactions are often performed in

*Abbreviations: Bal, 4-formyl-3,5-dimethoxyphenoxy linker; DCM, dichloromethane; DIEA,

diisopropylethylamine; DMF, dimethylformamide; ELSD, evaporative light scattering

detector; ESI MS, electrospray ionization mass spectroscopy; EtOAc, ethylacetate; HPLC,

high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectros-

copy; MA, microwave assisted; MAOS, microwave-assisted organic synthesis; MeOH,

methanol; PEG-PS, polyethyleneglycol grafted on 1% cross-linked polystyrene; SPOS,

solid-phase organic synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran.



6-Methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxanil-lide: yield 680 mg (55%); mp 245–247

�; 1H NMR (DMSO-d6) � 2.04 (s,

3H), 5.40 (d, J¼ 3 Hz, 1H), 6.99 (br s, 1H), 7.24–7.71 (m, 10H), 8.69(s, 1H), 9.54 (s, 1H).

6-Methyl-4-(3-methoxy-4-hydroxyphenyl)-2-oxo-1,2,3,4-tetrahydropyr-imidine-5-carboxanillide: yield 400 mg (28%); mp 238–241

�; 1H NMR

(DMSO-d6) � 2.03 (s, 3H), 3.66 (s, 3H), 5.32 (d, J¼ 3 Hz, 1H), 6.69–7.55(m, 9H), 8.62 (s, 1H), 8.89 (s, 1H), 9.49 (s, 1H).

6-Methyl-4-(2-chlorophenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylanillide: yield 1278 mg (89%); mp 195–197

�; 1H NMR (DMSO-

d6) � 2.02 (s, 3H), 5.74 (d, J¼ 3.0 Hz, 1H), 6.99–7.87 (m, 9H), 9.49 (br s,1H), 9.63 (br s, 1H), 10.04 (br s, 1H).

6-Methyl-5-nitro-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine: yield775 mg (83%); mp 188–190

�; 1H NMR (DMSO-d6) � 1.58 (s, 3H), 5.65 (d,

J¼ 3.0 Hz, 1H), 7.26–7.47 (m, 6H), 8.07 (br s, 1H).


[12] Microwave-Assisted Solid-Phase Organic Synthesis(MA-SPOS) of Oxazolidinone Antimicrobials

By Andrew P. Combs, Brian M. Glass, and Sharon A. Jackson

Introduction

Microwave-assisted organic synthesis (MAOS)* has dramaticallyevolved since its first use in the mid-1980s. Recent advances in microwaveequipment have provided homogeneous microwave heating in safe and re-liable instruments for use in the chemistry laboratory. Microwave-assistedheating has been utilized with many solution-phase reactions, typically re-ducing reaction times from days or hours to minutes or even seconds. Theobserved rapid microwave dielectric heating is primarily due to the con-tinuous realignment of polar molecules with the paramagnetic oscillatingfield. The microwave radiation is increasingly more absorbed by moleculeswith larger dipole moments and thus reactions are often performed in

*Abbreviations: Bal, 4-formyl-3,5-dimethoxyphenoxy linker; DCM, dichloromethane; DIEA,

diisopropylethylamine; DMF, dimethylformamide; ELSD, evaporative light scattering

detector; ESI MS, electrospray ionization mass spectroscopy; EtOAc, ethylacetate; HPLC,

high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectros-

copy; MA, microwave assisted; MAOS, microwave-assisted organic synthesis; MeOH,

methanol; PEG-PS, polyethyleneglycol grafted on 1% cross-linked polystyrene; SPOS,

solid-phase organic synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran.




solvents with high dielectric constants. Several in-depth reviews are avail-able on the general principles and recent advances in the microwave-assistedorganic synthesis field.1

The successful application of microwave dielectric heating to solid-phase organic reactions has also been reported recently.2 Microwave-assisted solid-phase organic synthesis (MA-SPOS) provides an additionalmeans by which solid-phase reactions can be driven to completion in muchshorter reaction times. MA-SPOS has proven to be an invaluable optimiza-tion method, since optimization of solution-phase or solid-phase chemis-tries is by far the most time-consuming portion of developing reliable andgeneral chemical library syntheses. Many reaction parameters can betested in just a few hours or days due to the shortened reaction times andthus solid-phase chemistries can be optimized in dramatically reducedtimes. Once a solid-phase synthesis is derived, a library of compoundscan be efficiently prepared. Several commercially available microwave in-struments are currently available for performing up to 50 reactions in par-allel or robotic sequential microwave heating of hundreds of individualreactions at specified temperatures, pressures, and times.

Instrumentation

In 1996, we initiated our microwave-assisted organic synthesis researchusing a domestic Sharp carousel microwave oven. We soon demonstratedthe effectiveness of this synthetic tool for solution-phase and solid-phasesynthesis of libraries of compounds directed toward our drug discoveryefforts.3 Though we were excited by the potential of this technology, thereremained many limitations and safety concerns associated with the use of adomestic microwave oven. The most prominent limitations were the lack oftemperature and pressure control. Nonhomogeneous heating of laboratorysamples within these microwave sources can result in an explosion. Whileseveral multimode laboratory safe microwave instruments were commer-cially available at the time we opted for the relatively new single-modeSmith synthesizer from Personal Chemistry, shown in Fig. 1. The Smithsynthesizer features an enclosed reaction chamber with temperature,

1 Several reviews are available: (a) B. Wathey, J. Teirney, P. Lidstrom, and J. Westman, Drug

Discov. Today 7, 373 (2002). (b) P. Lidstrom, J. Tierney, B. Wathey, and B. Westman,

Tetrahedron 57, 9225 (2001). (c) M. Larhed and A. Hallberg, Drug Discov. Today 6, 406

(2001). (d) S. Caddick, Tetrahedron 51, 10403 (1995).2 A. Lew, P. O. Krutzik, M. E. Hart, and A. R. Chamberlin, J. Comb. Chem. 4, 95 (2002).3 (a) A. P. Combs, S. Saubern, M. Rafalski, and P. Y. S. Lam, Tetrahedron Lett. 40, 1623

(1999). (b) B. M. Glass and A. P. Combs, ‘‘High-Throughput Synthesis,’’ p. 123. Marcel

Dekker Inc., New York, 2001.

Fig. 1. Personal Chemistry Smith synthesizer. (Photograph compliments of Personal

Chemistry Inc.)


pressure, and time control for microwave-heating sealed-glass vessels. Re-action pressures up to 300 psi are tolerated by this system. The integratedGilson robotic platform has proven robust for automated sequential reac-tion optimization and/or library production. The system is also easy to useand has superb safety features.

Solid-Phase Biaryloxazolidinones

A microwave-assisted solid-phase synthesis of the antimicrobial oxazo-lidinone pharmacophore is described herein as a demonstration of the util-ity of this emerging technology toward drug discovery.4 The optimizationprocess and full experimental details for the synthesis of a small libraryof oxazolidinones are exemplified.

The oxazolidinones comprise a unique class of potent antibacterialagents.5 This antibacterial pharmacophore structure (1) was discoverednearly two decades ago by scientists at E. I. Du Pont De Nemours andCompany.6 Several compounds advanced to preclinical safety studies, in-cluding Dup 721 and E3656, are shown in Fig. 2. Unfortunately, these com-pounds were dropped from development due to dose-limiting toxicities. Inthe late 1990s, scientists at the Upjohn Company reported the discoveryand phase I clinical trials of two oxazolidinone antibacterial agents, linezo-lid and eperezolid,7 displaying diminished toxicities. Pharmacia-Upjohn’spersistence resulted in the recent approval of linezolid as the first new classof antimicrobials in over a decade.

4 C. D. Dzierba and A. P. Combs, Annu. Rep. Med. Chem. 37, 247, (2002).5 S. J. Brickner, Curr. Pharm. Design 2, 175 (1996).6 A. M. Slee, M. A. Wuonola, and R. J. McRipley, Antimicrob. Agents Chemother. 31, 1791 (1987).

ON

O

NH

R2

ON

O

NHAcON

O

NHAc

N

E3656

ON

O

NHAcDup 721

O

N

O

F

Linezolid

OxazolidinonePharmacophore 1

R1

Fig. 2. Oxazolidinone antimicrobial pharmacophore.


Renewed interest at DuPont-Merck Pharmaceuticals Company in thisclass of antibacterial compounds led to a second discovery chemistry effort.Hundreds of compounds had been previously synthesized over the decadelong program at DuPont, but a systematic variation of many functionalgroups had not been explored thoroughly. Oxazolidinones, such asE3656, containing the customary acetamide at the R1 position and the biar-yl functionality at the R2 position were targeted for extensive analoging,due to the excellent potency for the series and the incomplete SAR avail-able. A method for the rapid parallel synthesis of this class of compoundswas thus desired to enable thorough analoging around this core in hopesof identifying new potent clinical candidates lacking the toxicologicalliabilities of the previous candidates.

Retrosynthetic analysis of the oxazolidinone pharmacophore 1 shownin Fig. 3 revealed that the oxazolidinone aminomethyl group could serveas an attachment site to the solid support during the elaboration of a suit-ably substituted scaffold. We envisioned amine scaffold 2 could be readilycoupled to a resin-bound aldehyde. Attachment of two points of diversityvia derivatization of the solid-supported secondary amine 3 with a variety

7 (a) G. E. Zurenko, C. W. Ford, D. K. Hutchinson, S. J. Brickner, and M. R. Barbachyn,

Exp. Opin. Invest. Drugs 6, 151 (1997). (b) S. J. Brickner, Curr. Pharm. Design 2, 175 (1996).

(c) S. J. Brickner, D. K. Hutchinson, M. R. Barbachyn, P. R. Manninen, D. A. Ulanowicz,

S. A. Garmon, K. C. Grega, S. K. Hendges, D. S. Toops, C. W. Ford, and G. E. Zurenko,

J. Med. Chem. 39, 673 (1996).

Fig. 3. Retrosynthetic analysis of the oxazolidinone pharmacophore.


of acylating agents and palladium-mediated couplings of arylboronic acidsto the solid-supported aryl-iodide 4 would provide arrays of the desiredfully functionalized oxazolidinones 5.

Oxazolidinone scaffold 2 was synthesized in six steps by literaturemethods.7c Coupling of only 1.5 equivalents of the valuable scaffold amine2 to Bal-resin under reductive amination conditions afforded the resin-bound secondary amine 3, as shown in Fig. 4. The resulting resin-boundsecondary amine 3 was acylated with several acylating reagents, includingacetic anhydride, to afford the N-acyl-aryliodide 4 with a resin loading of0.27 mmol/g.

The Suzuki coupling of arylboronic acids to the solid-supported arylha-lide 4 was initially performed by conventional heating methods, DMF at85

�in an oil bath for 16 h, to provide the desired products 6 after TFA

cleavage. Due to the extended heating times necessary for the Suzuki reac-tion, full optimization of the reaction conditions were not investigatedprior to compound library synthesis. Reaction conditions sufficient forthe library synthesis were determined within a couple of weeks affordingmoderate yields of the desired oxazolidinones. Libraries of hundreds of

Fig. 4. Solid-phase synthesis of oxazolidinones.


oxazolidinones were synthesized using a variety of acylating reagents andarylboronic acids using this methodology. HPLC purification of the desiredproducts was typically necessary due to the modest yields and purities ofthe various library members. Attempts to perform these same Suzuki coup-lings in a domestic microwave afforded the desired product, but yields andpurities were inconsistent, presumably due to the nonhomogeneity of theheating and lack of sufficient temperature and pressure controls.

We have subsequently revisited this reaction and successfully optimizedthe Suzuki microwave-assisted coupling conditions using the Smith syn-thesizer. Several parameters were investigated, including the palladiumcatalysts, the reaction temperatures, and the reaction times (Table I).Optimization reactions were run in the Smith synthesizer using 50 mg ofresin 7 and 6 equivalents of 4-methoxyphenylboronic acid to afford oxa-zolidinone 8. In just a few days, optimized conditions were identified thatafforded the desired product in excellent yields and purities with reactionstimes of only 5–10 min.8

A small library of oxazolidinones was then synthesized using the robot-ics of the Smith synthesizer to run sequentially each new boronic acid in theSuzuki reaction. Cleavage of the products and filtration through a smallplug of silica provided excellent yields and purities of the desired oxazoli-dinones, including compound 12, the previous clinical candidate E3656, in96% yield and 96% purity (Table II).

This MA-SPOS of the oxazolidinone class of antimicrobial/antibioticsallows for the rapid synthesis of libraries of compounds that simultaneously

8 M. Larhed, G. Lindeberg, and A. Hallberg, Tetrahedron Lett. 37, 8219 (1996).

TABLE II

MA-SPOS of Oxazolidinones

N O

O

NH

O

R

Compound R-Group Purity (%)a Yield (%)b

9 3-MeO 89 72

10 4-MeO 95 78

11 3-F 95 91

12 4-F 95 92

13 3-pyridyl 96 96

14 4-Me 85 94

a Purity assessed by LC/MS with quantitation by ELSD.b Crude yields based on gravimetric analysis.

TABLE I

Optimization of Suzuki Reaction Conditions

Entry Temp (�C) Time (min) Catalyst % conversiona

1 140 5 Pd(PPh3)4 74

2 160 5b Pd(PPh3)4 83

3 180 10 Pd(PPh3)4 66

4 140 5 PdCl2(PPh3)2 66

5 180 5 PdCl2(PPh3)2 88

a Conversion based on HPLC UV analysis of cleaved product vs. unreacted aryl iodide.b Resin was subjected to Suzuki coupling conditions with 4-methylarylboronic acid twice.


vary both the N-acyl functionality and the biarylsubstituent of the oxazoli-dinone pharmacophore. Many potent antibacterial compounds were iden-tified from the compound libraries produced using variations of this rapidparallel synthesis strategy. Compound structures and associated biological


activities of these antimicrobial compound libraries will be reported indue course.

Conclusion

MA-SPOS is a valuable methodology for the rapid synthesis of noveland diverse chemical entities. While solid-phase technologies have previ-ously allowed for the rapid purification of reaction products by simplywashing excess reagents from the resin-bound products, recent advancesin microwave technologies now enable the chemist to drive chemical reac-tions to completion via microwave dielectric heating in unprecedentedtimeframes. The combinatorial chemist is now able to break through thesolid-phase synthetic chemistry optimization bottleneck and develop newreaction conditions in hours or days rather than weeks or months. Therapid production of libraries of novel compounds has thus been enabledby synergizing the best aspects of MA chemistry and SPOS.


Bal-PEG-PS (HL) resin (loading of 0.45 mmol/g) was purchased fromPerceptive Biosystems. Commercially available starting materials and re-agents were purchased from Aldrich and used without further purification.Microwave-assisted chemistry was performed in a Personal ChemistrySmith synthesizer. All other reactions were performed in capped poly-propylene-fritted tubes manufactured by Jones Chromatography. Thepolypropylene tubes were mixed using a Labquake tube rotor/rockermanufactured by Thermolyne. LC/MS analyses were performed on aMicromass ZMD Electrospray spectrometer equipped with a Gilson 215liquid handler, a Sedere Sedex 75 ELS detector, and a Waters Symmetrychromatography column (C18, 5 �M, 2.1 � 50 mm). The HPLC gradientran from 0% acetonitrile/water containing 0.1% TFA to 100% over 8 minat 1.5 ml/min flow rate. Peaks areas were integrated by evaporative lightscattering.


Coupling of Iodoaryloxazolidinone to BAL Resin

Bal resin (0.4 g, 0.45 mmol/g, 0.18 mmol, Perseptive Biosystems) wasweighed into a fritted polypropylene tube and swelled with 4 ml of DCM.To the suspension was added oxazolidinone HCl salt 2 (96 mg, 0.27 mmol),sodium triacetoxyborohydride (190 mg, 0.90 mmol), and acetic acid (80 �l,


2% final concentration). The resin was mixed for 16 h, and then washedwith DCM, THF, and MeOH (3� each).

Acylation of Solid-Supported Iodoaryloxazolidinone

Resin 3 was swelled with 4 ml of DCM followed by treatment withacetic anhydride (85 �l, 0.90 mmol) and DIEA (160 �l, 0.90 mmol). Theresin was mixed for 30 min, then washed with DCM, THF, and MeOH(3� each). A portion of the resin (50 mg) was cleaved with 50% TFA inDCM and dried in vacuo. The loading of resin 4 (R1 ¼ CH3) was determinedto be 0.27 mmol/g by gravimetric analysis of the cleaved product.

Microwave-Assisted Solid-Phase Suzuki Coupling

Resin 4 (R1 ¼ CH3) (50 mg, 0.014 mmol) was weighed into a conicalPersonal Chemistry Smith process vial equipped with a conical-shapedstir bar. To the resin was added 4-fluorophenylboronic acid (11 mg,0.08 mmol), DMF (1 ml), 2 M aqueous sodium carbonate (80 �l), and di-chlorobis(triphenylphosphine)palladium(II) (1–2 mg). The heavy walledglass vial was crimp sealed and placed on the Gilson platform. The micro-wave program was set to a 5 min duration at 180

�on the normal absorption

setting and the vial was then processed. After completion, the vial wasdecrimped and resin 5 (R1 ¼ CH3, R2 ¼ 4-F-phenyl) was transferred to afritted tube and washed with DMF, H2O, MeOH, and DCM (3� each).

Cleavage of Biaryloxazolidinone from the Solid Support

Resin 5 (R1¼CH3, R2¼ 4-F-phenyl) was mixed with 2 ml of 50% TFAin DCM for 30 min. The resin was washed with DCM and the filtrates werecollected and concentrated in vacuo. The residue was dissolved in EtOAcand filtered through a short plug of silica (75 mg) in a thin fritted polypro-pylene tube to remove residual palladium. The product was dried undervacuum to give the desired product 12 in good yield (4.1 mg, 92%) andpurity (95%). ESI MS: Theor: 329.1 (M þ H)þ. Found: 329.2 (M þ H)þ.

[13] automated synthesis of polysaccharides 235

[13] Automated Synthesis of Polysaccharides

By Obadiah J. Plante, Emma R. Palmacci, and Peter H. Seeberger

Introduction

Carbohydrates are the most structurally diverse class of biopolymers. Inaddition to branching and anomeric configuration, structural diversity isfurther complicated when considering that natural structures are oftenfound attached to proteins, lipids, or both. The properties attributed tocarbohydrates and glycoconjugates are as varied as their structure, rangingfrom sources of bioenergetics to critical markers for cancer metastasis.1,2

A better understanding of the biological roles of oligosaccharides andglycoconjugates is needed to advance the field of glycobiology. To meetthis challenge and create new approaches to carbohydrate production,an automated synthesis method was developed recently and is the focusof this chapter.

Background

The major challenges in carbohydrate synthesis are two-fold: (1) how tocontrol the stereochemistry of each newly formed glycosidic linkage and(2) how to incorporate various degrees of branching (Fig. 1). Unlike pep-tide and nucleic acid synthesis, carbohydrate synthesis requires the installa-tion of a new stereocenter during each elongation event. Furthermore,many degrees of branching are common in carbohydrates whereas theother biopolymers are strictly linear in sequence. The current state of theart in peptide and DNA synthesis is a line of fully automated synthesizersthat allows for the production of oligomers by nonspecialists.3,4 The same isnot true for carbohydrate synthesis and no general method has emergeddespite years of research.5

The most widely adopted methods for carbohydrate synthesis utilizeeither enzymatic or chemical approaches. Enzymatic carbohydrate synthe-sis is a vibrant area of research that has been reviewed thoroughly and will

1 C.-H. Wong, S. L. Haynie, and G. M. Whitesides, J. Org. Chem. 47, 5418 (1983).2 S. Hakomori and Y. Zhang, Chem. Biol. 4, 97104 (1997).3 M. H. Caruthers, G. Beaton, J. V. Wu, and W. Wiesler, Methods Enzymol. 211, 3 (1992).4 G. B. Fields, Z. Tian, and G. Barany, in ‘‘Synthetic Peptides: A User’s Guide’’ (H. Grant,

ed.). Freeman, New York, 1992.5 K. Toshima and K. Tatsuta, Chem. Rev. 93, 1503 (1983).



OO

AcHN

OH

O ORO

HOHO

OHHO

O

HOOHOH New stereocenter at each

glycosidic linkage

Branching can occurat any position

Fig. 1. Stereochemistry and regiochemistry in carbohydrates.

236 oligosaccharide chemistry [13]

not be discussed in detail here.6 Enzymatic methods offer exquisite stereo-chemical and regiochemical control although the limitations with regardsto substrate specificity preclude their widespread use. Chemical methods,on the other hand, are used frequently to produce highly branched complexcarbohydrates.7 By incorporating unique protecting groups within a carbo-hydrate monomer, many patterns of complex structures can be accessed.Furthermore, several strategies are available for ensuring the appropriatestereochemical outcome of the coupling reaction. The requirement ofmultiple synthetic steps limits the scope of this method and necessitatesan expertise in carbohydrate synthesis in order to be successful.

Despite the almost limitless diversity that chemical synthesis offers, nogeneral method for the synthesis of carbohydrates exists. Carbohydratesynthesis has been considered an art form rather than a science based onthe finding that subtle changes in solvent, reagent, temperature, concentra-tion, and/or substrate may alter the stereo- and regiochemical outcome ofan elongation event. The lack of predictive tools for carbohydrate synthesishas precluded all previous efforts to develop a general method. Com-pounding the intricacies of carbohydrate synthesis further is the tediousnature of the synthetic process. A typical heptasaccharide synthesisrequires at least 14 distinct synthetic steps and purification events withoverall yields commonly in the 0.1–5% range after months of manuallabor. To address all of the variables in carbohydrate synthesis, researchershave attempted to generalize the glycosylation conditions for a particularset of building blocks.8 This technique has proven useful for small librariesof carbohydrates but has yet to offer a universal solution for complexcarbohydrate synthesis.

To address the need for new synthetic methods we developed a proced-ure that utilizes solid-phase methods to screen for appropriate buildingblock reactivity and that assembles complex carbohydrates with minimalmanual labor.9 Rather than confine a group of building blocks to a given

6 K. Koeller and C.-H. Wong, Chem. Rev. 100, 4465 (2000).7 K. C. Nicolaou and H. J. Mitchell, Angew. Chem. Int. Ed. Engl. 40, 1576 (2001).8 L. Yan, C. M. Taylor, R. Goodnow, and D. Kahne, J. Am. Chem. Soc. 116, 6953 (1994).


set of reaction conditions, the automated method described belowprovides a framework for exploring potential reaction conditions while atthe same time minimizing the amount of effort required to create complexcarbohydrates. It is anticipated that further advances in automatedcarbohydrate synthesis will shape the field of glycobiology in muchthe same manner that gene and peptide synthesis machines have shapedthe nature of biotechnology over the past two decades.

Overview

The general strategy for automated carbohydrate synthesis is based onsolid-phase techniques.10 For carbohydrate synthesis, the acceptor-boundmethod involves the sequential addition of carbohydrate building blocks(glycosyl donors) to an insoluble polystyrene support (Scheme 1).11 Sideproducts, reagents, and unreacted starting material are washed away andthe carbohydrate chain is covalently attached to the polymer support. Aunique protecting group is removed to provide another nucleophilicposition for subsequent elongation. At the conclusion of the synthesis,the carbohydrate is liberated from the support, deprotected, and purified.The solid-phase platform is ideal for automation due to the repetitivenature of the process. The successful application of automated methodsfor carbohydrate synthesis requires the appropriate choice of polymersupport, linker, building blocks, reagents, temperature control, andcleavage conditions. We investigated each of the aforementioned vari-ables and selected the most promising set of conditions compatible withautomation.12

9 O. J. Plante, E. R. Palmacci, and P. H. Seeberger, Science 291, 1523 (2001).10 W.-C. Haase and P. H. Seeberger, Chem. Rev. 100, 4349 (2000).11 P. H. Seeberger and S. J. Danishefsky, Acc. Chem. Res. 31, 685 (1998).12 O. J. Plante, E. R. Palmacci, and P. H. Seeberger, Adv. Carbohydr. Chem. Biochem. 58, 35

(2003).

Scheme 1. Solid-phase synthesis using the acceptor-bound method.

Fig. 2. Schematic description of the automated carbohydrate synthesis machine.


The instrument chosen for the evaluation of carbohydrate synthesis wasan ABI-433 peptide synthesizer (Fig. 2). The instrument was adapted forcarbohydrate synthesis and customized coupling cycles were developed.A specially designed low-temperature reaction vessel was installed andinterfaced with a commercially available cooling device.13 The necessaryreagents were loaded onto the instrument ports and reaction conditionswere programmed on the computer, in a fashion similar to the automatedsynthesis of peptides.

Detailed Description

An automated solid-phase method necessitates a polymer support anda linker that are compatible with the reagents used in carbohydrate synthe-sis. Several strategies have been developed that address functional groupcompatibility and swelling of the polymer support. Of these strategies, afamily of olefinic linkers has proven to be readily cleaved under neutral

13 A Julabo circulating cooler was used with an ethanol/ethylene glycol mixture.

Scheme 2. Automated carbohydrate synthesis using glycosyl trichloroacetimidates.


conditions at the end of a synthesis.14,15 Olefin-based linkers are also stableto a variety of acidic glycosylation conditions when attached to polystyreneresins. Both lightly cross-linked, swellable resins such as Merrifield’s (1%cross-linked) resin and rigid macroreticular resins (Argopore) are amen-able to functionalization with olefinic linkers. Initial studies determinedthat the polystyrene resins were compatible with common glycosylationconditions and our olefinic linker.14

When initiating a synthesis, the choice of glycosylating agent and pro-tecting groups governs the selection of activating and deblocking reagents.The automated method described here has proven useful with glycosyl tri-chloroacetimidate and glycosyl phosphate building blocks. Temporary pro-tecting groups such as levulinate esters, silyl ethers, and acetate esters alsoare compatible with automation. Using this set of reagents we anticipatethat the majority of natural carbohydrate linkages can be accessed.

A typical coupling cycle (Scheme 2) for glycosyl trichloroacetimidatesis outlined in Table I. A polystyrene support, functionalized with an olefi-nic linker, is loaded into a reaction vessel in the instrument.16,17 The acti-vating reagent (trimethylsilyl trifluoromethanesulfonate (TMSOTf*/

14 R. B. Andrade, O. J. Plante, L. G. Melean, and P. H. Seeberger, Org. Lett. 1, 1811 (1999).15 L. Knerr and R. R. Schmidt, Eur. J. Org. Chem. 2803 (2000).16 One percent cross-linked polystyrene and Argopore resin performed equally well. The

compatibility with polar solvents makes Argopore the most versatile resin when

investigating new reaction conditions.17 An Applied Biosystems 433A was adapted for carbohydrate synthesis.* Abbreviations: DMF, N,N-dimethylformamide; DMT, 4,40-dimethoxytrityl; HPLC, high-

pressure liquid chromatography; NPG, n-pentenyl glycoside; TEA, triethylamine; TCA,

trichloroacetic acid; THF, tetrahydrofuran; TMSOTf, trimethysilyl trifluoromethanesulfonate.

TABLE I

Automated Coupling Cycle Using Glycosyl Trichloroacetimidatesa

Step Function Reagent Time (min)

1 Couple 10 equiv. donor and 0.5 equiv. TMSOTf 30

2 Wash Dichloromethane 6

3 Couple 10 equiv. donor and 0.5 equiv. TMSOTf 30


5 Wash Methanol:dichloromethane (1:9) 6

6 Deprotection 2 � 10 equiv. NaOMe (methanol:

dichloromethane) (1:9)

80


8 Wash 0.2 M acetic acid in tetrahydrofuran 4

9 Wash Tetrahydrofuran 6


a Scale: 25 �mol.


CH2Cl2) and deblocking reagent (e.g., NaOMe) are inserted into the in-strument along with the glycosyl imidate building blocks. The synthesis isperformed in an iterative manner according to the programmed couplingcycle. In general, double glycosylations (95–98%) result in (�5%) greateryield per coupling when compared to single glycosylations (90–95%).

In addition to glycosyl trichloroacetimidate building blocks, we de-veloped a similar coupling cycle for glycosyl phosphate building blocks(Table II). Glycosyl phosphates are versatile building blocks that are acti-vated under mild conditions to form glycosidic linkages in high yield.18 Theoverall synthesizer configuration is consistent with the imidate method,however, the reagent solutions are modified to accommodate phosphatereactivity and protecting group removal. A reaction vessel designed forlow temperature and a cooling apparatus are required to enable the �15

�

temperature necessary for productive phosphate couplings. In this cycle,the deprotection events [N2H4/Pyr:AcOH (3:2)] were quantitative and allcoupling steps were >90% yield. An example of automated carbohydratesynthesis using glycosyl phosphates is shown in Scheme 3.

The coupling protocols described in the two previous examples can becombined to allow for the synthesis of branched carbohydrates.19 The auto-mated solid-phase synthesis of a tetrasaccharide is illustrated in Scheme 4.Both glycosyl phosphate and glycosyl imidate building blocks were usedalong with acetate and levulinate esters as temporary protecting groups.

18 O. J. Plante, R. B. Andrade, and P. H. Seeberger, Org. Lett. 1, 211 (1999).19 M. C. Hewitt and P. H. Seeberger, Org. Lett. 3, 3699 (2001).

TABLE II

Automated Coupling Cycle Used with Glycosyl Phosphatesa

Step Function Reagent Time (min)

1 Couple 5 equiv. donor and 5 equiv. TMSOTf 15


3 Couple 5 equiv. donor and 5 equiv. TMSOTf 15



6 Wash Pyridine:acetic acid (3:2) 3

7 Deprotection 2 � 20 equiv. hydrazine (pyridine:

acetic acid) (3:2)

30

8 Wash Pyridine:acetic acid (3:2) 3


10 Wash 0.2 M acetic acid in tetrahydrofuran 4



a Scale: 25 �mol.

Scheme 3. Automated carbohydrate synthesis using glycosyl phosphates.


After a successful synthesis, the resin is transferred to a round-bottomflask for cleavage. The most convenient method of cleavage involves reac-tion of the resin with Grubbs’ catalyst in CH2Cl2 under an atmosphere ofethylene. The product is liberated to afford an n-pentenyl glycoside (NPG).NPGs serve as versatile intermediates in carbohydrate synthesis and arereadily converted into various functionalities useful for immobilization toa surface, conjugation to proteins, or fluorescent labeling.20

20 T. Buskas, E. Soderberg, P. Konradsson, and B. Fraser-Reid, J. Org. Chem. 65, 958 (2000).

Scheme 4. Synthesis of a tetrasaccharide using a variety of reaction conditions.


Purification of the synthetic carbohydrate is accomplished usingeither flash silica gel chromatography or HPLC depending on the purityof the sample. The most common side-products in automated solid-phasesynthesis are deletion sequences (n�1, n�2, etc.). The prevalence ofdeletion sequences complicates purification of the final product. To aidin the purification process, a capping procedure was developed thatallows for the facile removal of deletion sequences.21 Following eachcoupling event, unreacted hydroxyl groups that may give rise to deletionsequences are subjected to a capping reagent that renders these sitessilent in subsequent couplings (Fig. 3). The caps also function as a handleto readily separate all unwanted capped sequences from the desireduncapped products. For example, installation of a polyfluorinated silylether F-tag onto unreacted hydroxyl groups after glycosylation pre-cludes further elongation of the deletion sequence. Following cleavagefrom the resin all of the fluorinated intermediates are easily removed bypassing through a pad of fluorinated silica gel. This modification of theautomated coupling cycles greatly facilitates the purification of syntheticcarbohydrates.

21 E. R. Palmacci, M. C. Hewitt, and P. H. Seeberger, Angew. Chem. Int. Ed. Engl. 40, 4433

(2001).

Fig. 3. Schematic description of the use of an F-tag reagent for the purification of

compounds synthesized on a solid support.


State of the Art

Automated solid-phase carbohydrate synthesis utilizes instrumentationand reaction design in order to remove the most time-consuming aspectof carbohydrate production. This process reduces the challenge ofcarbohydrate synthesis to the production of simple building blocks, forwhich there is ample synthetic precedence. Although a full set of buildingblocks has yet to be established, the variety of possible building blockswill enable the production of diverse libraries of complex carbohydrates.Carbohydrate libraries have been sought for many years and technologiesfor their production will find widespread application in academic andindustrial laboratories. A representative sample of the carbohydratesequences prepared using automated solid-phase synthesis is shown inFig. 4.

Automated carbohydrate synthesis allows for the production ofcomplex carbohydrates orders of magnitude faster than other approaches.This advance has the potential to parallel the breakthroughs achievedby researchers in the peptide and DNA fields that opened up the pro-teomic and genomic eras in biotechnology. By increasing the scope of thecarbohydrate building block library and streamlining the reactionconditions further we anticipate that automated solid-phase carbohydratesynthesis will become the method of choice for carbohydrate production.

OBnO

BnOOPiv

OBn

OOBnO

OBnO

O

OBnOBnO

OBnO

OBnOBnO

OAcBnO

O

O

OAcBnO

BnO

O

O

BnOBnO

O

O

BnOBnO

n = 3

BnO OBnO

BnO OAc

BnO OBnO

BnO O

O

BnO OBnO

BnO On = 3, 5, 8

O OBnO

OBn

NPhth

O

BnO OBnO

BnO

O

BnO

OBnO

PivO

OBn

BnO OBnO

PivO

BnO OBnO

PivO

OBnO OO

PivO

O

BnO OBnO

BnO

OBn

OBnO OO

PivOBnO OBnO

BnO

OBn3 n=3LevO

O

n = 1, 3

7 89 n =3

12 13

10 n = 5

11 n = 8

Fig. 4. Representative sequences prepared by automation.


Experimental Procedures

Materials and Methods

All reactions were performed in oven-dried glassware under an atmos-phere of argon unless noted otherwise. Reagent grade chemicals were usedas supplied except where noted. TMSOTf was purchased from AcrosChemicals. N,N-Dimethylformamide (DMF) was obtained from AldrichChemical Co. (Sure-Seal Grade) and used without further purification.Merrifield’s resin (1% cross-linked) was obtained from Novabiochem.Argopore resin was purchased from Argonaut Technologies. Dichloro-methane (CH2Cl2) and tetrahydrofuraran (THF) were purchased fromJ. T. Baker (Cycletainer) and passed through neutral alumina columnsprior to use. Toluene was purchased from J. T. Baker (Cycletainer) andpassed through a neutral alumina column and a copper(II) oxide columnprior to use. Pyridine, triethylamine, and acetonitrile were refluxed overcalcium hydride and distilled prior to use. Analytical thin-layer chromatog-raphy was performed on E. Merck silica column 60 F254 plates (0.25 mm).Compounds were visualized by dipping the plates in a cerium sulfate–ammonium molybdate solution followed by heating. Liquid columnchromatography was performed using forced flow of the indicated solventon Silicycle 230–400 mesh (60 A pore diameter) silica gel.


HPLC analysis was performed on a Waters Model 600E Multisolventdelivery system using analytical (Nova-Pak 60-A, 4-�m, 3.6 � 150-mm)and preparative (Nova-Pak 60-A, 6-�m, 7.8 � 300-mm) silica columns.

Synthesis of Linker-Functionalized Resin 1. 8-(4,40-Dimethoxytrityl)-4-(Z)-octenol (4.6 g, 11 mmol, 4.0 equiv.) was dissolved in DMF (20 ml) andcooled to 0

�. NaH (60% dispersion in mineral oil, 0.53 g, 11 mmol, 4.0

equiv.) was added and the solution was stirred for 1 h. Merrifield’s resin[1% cross-linked (1.2 mmol/g): 2.21 g, 2.65 mmol, 1.0 equiv.] was loadedinto a solid-phase flask and swollen with DMF (20 ml). The linker solutionwas transferred to the resin suspension via cannula and tetrabutylammo-nium iodide (98 mg, 0.27 mmol, 0.1 equiv.) was added. After shaking for1 h at 0

�in the dark, the reaction mixture was warmed to room tempera-

ture and shaken for 12 h. Capping of unreacted sites was accomplishedby reaction with methanol (1.0 ml) and NaH (60% dispersion in mineraloil, 0.10 g) for 16 h. Methanol (5 ml) was added and the resin was trans-ferred to a fritted funnel. The resin was washed with 3 � 50 ml of each ofthe following: MeOH:DMF (1:1), DMF, MeOH:THF (1:9), THF,MeOH:CH2Cl2 (1:9), and CH2Cl2. Drying under vacuum over P2O5

afforded 3.38 g resin. Analysis of a small sample of resin (10 mg) via adimethoxytrityl cation assay revealed the loading to be 0.57 mmol/g (seebelow). Deprotection of the DMT functionalized resin was accomplishedby washing the resin with 3 � 50 ml of 3% dichloroacetic acid/CH2Cl2.Further washing with 3 � 50 ml of CH2Cl2, 1% TEA/CH2Cl2, THF, andCH2Cl2 and drying under vacuum afforded 2.39 g linker-functionalizedresin (0.78 mmol/g).

Trityl Cation Assay.22 DMT-functionalized resin (10–20 mg) wasweighed into a 10-ml volumetric flask. Trichloroacetic acid (3% solution ¼1.50 g TCA in 50 ml dichloromethane) in CH2Cl2 was added and a 200- to300-�l aliquot was transferred to another 10-ml volumetric flask anddiluted with 3% TCA/ CH2Cl2. Analysis was done by UV-VIS absorptionat 504 nm (A504). Loading calculation:

Calculation:

[(A504)(10 ml)]/76 ¼ X micromole ¼ 0.00 X millimole in final solution

[0.00X mmol/(vol. aliquot in ml)](10 ml) ¼ number of millimoles ininitial solution

Loading:

Number of millimoles in initial solution/(mass resin in g) ¼ number ofmillimoles/g

22 R. T. Pon, in, ‘‘Methods in Molecular Biology 20: Protocols for Oligonucleotides and

Analogs’’ (S. Agrawal, ed.), p. 467. Humana Press, Totowa, New Jersey, 1993.


Example: 12.9 mg resin was loaded into a 10-ml volumetric flask.Trichloroacetic acid (3%) in CH2Cl2 was added and a 200-�l aliquot wastransferred and diluted to 10 ml. Analysis by UV-VIS gave A504 ¼ 1.05.

Calculation:

[(1.05)(10 ml)]/(76 ml/�mol) ¼ 0.138 �mol ¼ 0.000138 mmol(0.000138 mmol/0.20 ml)(10 ml) ¼ 0.00692 mmol

0.00692 mmol/0.0129 g ¼ 0.54 mmol/g

Synthesizer Configuration. An Applied Biosystems peptide synthesizermodel 433A (ABI-433A) was adapted for carbohydrate synthesis. Thenecessary reagents were attached to reagent ports 1–7 and CH2Cl2 andTHF were installed as the bulk solvents on ports 9 and 10. Glycosyl donorbuilding blocks were weighed and loaded into cartridges as were thecapping reagents. The coupling cycles were programmed and all synthesissteps were performed by the instrument. Low-temperature applicationswere achieved by attachment of a Julabo circulating bath to a customizedreaction vessel. The temperature of the reaction vessel was manuallycontrolled on the Julabo.

Automated Synthesis Using Glycosyl Trichloroacetimidates. Octenediolfunctionalized resin (25 �mol, 83 mg, 0.30 mmol/g loading) was loadedinto a reaction vessel and inserted into a modified ABI-433A peptide syn-thesizer. The resin was glycosylated with donor 2 (10 equiv., 0.25 mmol,160 mg) delivered in CH2Cl2 (4 ml) and TMSOTf (0.5 equiv., 1 ml,0.0125 M TMSOTf in CH2Cl2). Mixing of the suspension was performed(10 s vortex, 50 s rest) for 30 min. The resin was washed with CH2Cl2(6 � 4 ml each) and glycosylated a second time. Upon completion of thedouble glycosylation the resin was washed with CH2Cl2 (6 � 4 ml each)and MeOH:CH2Cl2 (1:9) (4 � 4 ml each). The glycosylated resin wassubjected to the deprotection protocol for acetyl esters (see below) or tothe standard cleavage conditions.

General Deprotection Conditions (Acetate Ester/NaOMe). Deprotec-tion of the acetyl ester was carried out by treatment of the glycosylatedresin with sodium methoxide (10 equiv., 0.5 ml, 0.5 M NaOMe in MeOH)in CH2Cl2 (5 ml) for 30 min. The resin was then washed withMeOH:CH2Cl2 (1:9) (1 � 4 ml) and subjected to the deprotection condi-tions a second time for 30 min. Removal of any soluble impurities wasaccomplished by washing the resin with MeOH:CH2Cl2 (1:9) (4 � 4 mleach), 0.2 M AcOH in THF (4 � 4 ml each), THF (4 � 4 ml each), andCH2Cl2 (6 � 4 ml each). The deprotected polymer-bound glycoside wasthen elongated by reiteration of the above glycosylation/deprotectionprotocol or subjected to the standard cleavage protocol.


Automated Synthesis Using Glycosyl Phosphates. Octenediol functio-nalized resin (25 �mol, 83 mg, 0.30 mmol/g loading) was loaded into a re-action vessel equipped with a cooling jacket and inserted into a modifiedABI-433A peptide synthesizer. The resin was glycosylated using donor 4(5 equiv., 0.125 mmol, 90 mg) delivered in CH2Cl2 (4 ml) and TMSOTf(5 equiv., 1 ml, 0.125 M TMSOTf in CH2Cl2) at �15

�. Mixing of the sus-

pension was performed (10 s vortex, 50 s rest) for 15 min. The resin wasthen washed with CH2Cl2 (6 � 4 ml each) and glycosylated a second time.Upon completion of the double glycosylation the resin was washed withMeOH:CH2Cl2 (1:9) (4 � 4 ml each), THF (4 � 4 ml), and pyridine:aceticacid (3:2) (3 � 4 ml) and warmed to 15

�. The glycosylated resin was

subjected to the deprotection protocol for levulinate esters (see below) orto the standard cleavage conditions.

General Deprotection Conditions (Levulinate:N2H4). Deprotection ofthe levulinate ester was carried out by treating the glycosylated resin withhydrazine acetate [40 equiv., 4 ml, 0.25 M N2H4-HOAc in pyridine:aceticacid (3:2)] for 15 min. The resin was subjected to the deprotection condi-tions a second time for 15 min. Removal of any soluble impurities was ac-complished by washing the resin with pyridine:acetic acid (3:2) (3 � 4 ml),0.2 M AcOH in THF (4 � 4 ml each), THF (4 � 4 ml each), and CH2Cl2(6 � 4 ml each). The deprotected polymer-bound glycoside was thenelongated by reiteration of the above glycosylation/deprotection protocolor subjected to the standard cleavage protocol.

Oligosaccharide Cleavage from the Polymer Support. The glycosylatedresin (25 �mol) was dried in vacuo over phosphorous pentoxide for 12 hand transferred to a 10-ml flask. The flask was purged with ethylene andGrubb’s catalyst [bis(tricyclohexylphosphine)benzylidine ruthenium(IV)dichloride, 4.1 mg, 0.005 mmol, 20 mol%] was added. The reactionmixture was diluted with CH2Cl2 (3 ml) and stirred under 1 atm ethylenefor 36 h. Triethylamine (111 ml, 0.80 mmol, 160 equiv.) and tris hydroxy-methylphosphine (50 mg, 0.40 mmol, 80 equiv.) were added and theresulting solution was stirred at room temperature for 1 h.23 The paleyellow reaction mixture was diluted with CH2Cl2 (25 ml) and washedwith water (3 � 25 ml), saturated aqueous NaHCO3 (3 � 25 ml), and brine(3 � 25 ml). The aqueous phase was extracted with CH2Cl2 (3 � 25 ml)and the combined organics were dried over Na2SO4, filtered, and concen-trated. The resulting oligosaccharides were purified either by flash columnchromatography on silica gel or high-pressure liquid chromatography(HPLC).

23 H. D. Maynard and R. H. Grubbs, Tetrahedron Lett. 40, 4137 (1999).


F-Tag Protocols

Incorporation of the F-Tag Cap into the Automated Solid-PhaseSynthesis Cycle. The resin (50 �mol) was swelled in a 0.1 M solution of2,6-lutidine in CH2Cl2 (4 ml, 8.0 equiv.). After vortexing for 5 s, a 0.1 Msolution of the F-Tag triflate in CH2Cl2 (2.5 ml, 5.0 equiv., loaded into cart-ridges) was delivered to the reaction vessel. Mixing of the suspension wasperformed (10 s vortex, 50 s rest) for 15 min.

Oligosaccharide Cleavage from the Polymer Support and PurificationUsing the F-Tag Method. The glycosylated resin was cleaved as describedabove. The crude mixture was dissolved in CH2Cl2/MeOH (1:1) (1 ml) andadded to a column of tridecafluoro [Si-(CH2)2-(CF2)5-CF3)3] functionalizedsilica gel (Silicycle) equilibrated in 80% MeOH/20% H2O. One columnlength of 80% MeOH/20% H2O was eluted, and the solvent was changedto 100% MeOH. Nonfluorinated material typically eluted in fractions 1–4,while fluorinated material remained on the column until the gradient wasincreased to 100% MeOH. The desired nonfluorinated fractions were con-centrated and analyzed by HPLC. Recycling of the fluorous silica gel waspossible after washing with three column lengths MeOH, four columnlengths CH2Cl2, and drying with nitrogen.

[14] Solid-Phase Oligosaccharide Chemistryand Its Application to Library Synthesis

By Matthias Grathwohl, Nicholas Drinnan, Max Broadhurst,Michael L. West, and Wim Meutermans

Introduction

Biological processes are controlled at the molecular level through net-works and cascades of molecular interactions, primarily involving threeclasses of biomolecules: peptides, oligonucleotides, and oligosaccharides.Oligosaccharides play a major role in cell recognition and cell-signalingevents through the involvement of carbohydrate-recognizing proteins suchas lectins and selectins.1,2 The information of affinity and selectivity of acarbohydrate substrate to its target is stored in the nature of its constituentsand the three-dimensional structure, or connectivity profile. A simplecomparison illustrates the virtually endless size of structural diversity that

1 A. Varki, Glycobiology 3, 97 (1993).2 R. A. Dwek, Chem. Rev. 96, 683 (1996).



F-Tag Protocols

Incorporation of the F-Tag Cap into the Automated Solid-PhaseSynthesis Cycle. The resin (50 �mol) was swelled in a 0.1 M solution of2,6-lutidine in CH2Cl2 (4 ml, 8.0 equiv.). After vortexing for 5 s, a 0.1 Msolution of the F-Tag triflate in CH2Cl2 (2.5 ml, 5.0 equiv., loaded into cart-ridges) was delivered to the reaction vessel. Mixing of the suspension wasperformed (10 s vortex, 50 s rest) for 15 min.

Oligosaccharide Cleavage from the Polymer Support and PurificationUsing the F-Tag Method. The glycosylated resin was cleaved as describedabove. The crude mixture was dissolved in CH2Cl2/MeOH (1:1) (1 ml) andadded to a column of tridecafluoro [Si-(CH2)2-(CF2)5-CF3)3] functionalizedsilica gel (Silicycle) equilibrated in 80% MeOH/20% H2O. One columnlength of 80% MeOH/20% H2O was eluted, and the solvent was changedto 100% MeOH. Nonfluorinated material typically eluted in fractions 1–4,while fluorinated material remained on the column until the gradient wasincreased to 100% MeOH. The desired nonfluorinated fractions were con-centrated and analyzed by HPLC. Recycling of the fluorous silica gel waspossible after washing with three column lengths MeOH, four columnlengths CH2Cl2, and drying with nitrogen.


[14] Solid-Phase Oligosaccharide Chemistryand Its Application to Library Synthesis

By Matthias Grathwohl, Nicholas Drinnan, Max Broadhurst,Michael L. West, and Wim Meutermans

Introduction

Biological processes are controlled at the molecular level through net-works and cascades of molecular interactions, primarily involving threeclasses of biomolecules: peptides, oligonucleotides, and oligosaccharides.Oligosaccharides play a major role in cell recognition and cell-signalingevents through the involvement of carbohydrate-recognizing proteins suchas lectins and selectins.1,2 The information of affinity and selectivity of acarbohydrate substrate to its target is stored in the nature of its constituentsand the three-dimensional structure, or connectivity profile. A simplecomparison illustrates the virtually endless size of structural diversity that

1 A. Varki, Glycobiology 3, 97 (1993).2 R. A. Dwek, Chem. Rev. 96, 683 (1996).



O

O

OO

O

O

O

O

O

NH

HN

O

O

B

O

O

PO

O

O

B

O

O

O

PO O

64 trinucleotides 64 tripeptides ? (>10,000 trisaccharides)

O

O

Fig. 1. Number of trimers that can be generated from four monomer building blocks for

nucleotides, peptides, and carbohydrates. The number of trisaccharides that can be generated

in theory using four monosaccharide building blocks depends on what one wants to include. If

) symbolizes the direction of the donor to the acceptor then two types of accessible

trisaccharides can be envisaged: (1) A)B)C and (2) A)B(C. The number of possible

combinations, in the absence of trehaloses, but including the possibility of � and � anomers is

83 � (4 � 4) for option 1 and 83 � (4 � 3) for option 2. Total number of possibilities is 14,336.

[14] solid-phase oligosaccharide chemistry 249

can be theoretically accessed through linking carbohydrate monomers,especially when compared to peptides and oligonucleotides (Fig. 1).

Most of the carbohydrate-based processes, however, are poorly under-stood largely due to the fact that determining the structures and connectiv-ities of natural substrates remains difficult and identification of novelcarbohydrate-based substrates is impeded by a general lack of access tocarbohydrates. Combinatorial peptide and oligonucleotide chemistry isnow commonplace and enables large libraries of diverse sequences tobe readily generated in a short time frame. This has not only drasticallyaccelerated the process of hit discovery in many drug discovery projects,but has also provided essential tools to validate targets and determineprotein function in the world of peptides and oligonucleotides. Thus, thereis a clear need for combinatorial oligosaccharide approaches to enableaccess to libraries of structurally diverse oligosaccharides.3 With suchdiverse libraries at hand, the chances of identifying biologically activeoligosaccharides should become far greater, which should help unravelkey carbohydrate entities involving biological processes, and thereforeassist the development of new therapeutic drugs. In the following sectionswe will first address some recent developments at Alchemia in solid-phase

3 L. A. Marcaurelle and P. H. Seeberger, Curr. Opin. Chem. Biol. 6, 289 (2002).


carbohydrate chemistry, then we will cover suggested methods to developstructurally diverse oligosaccharide libraries.

Solid-Phase Oligosaccharide Synthesis (SPOS)

The synthesis of carbohydrates on solid support has recently receivedrenewed interest with some concomitant successes.4,5 Although not yetcommonplace, the solid-phase synthesis of carbohydrates is becomingmore prevalent and indeed more accepted as an alternative to the estab-lished solution-phase methods of oligosaccharide synthesis. A wide varietyof solid supports, including controlled pore glass,6 soluble mpeg/dox-basedresins,7,8 and more classic polystyrene and grafted polystyrene-based resins9

have been employed for the synthesis of carbohydrates with variousdegrees of success. Critical to a successful solid-phase synthesis method-ology is the linker that connects the growing oligosaccharide to a solidsupport. The linker is generally recognized as a modified protecting groupand, as such, it must display complete orthogonality to any other protectinggroups employed during the synthesis. This is particularly pertinent inoligosaccharide synthesis, which typically requires a number of orthogonalprotecting groups to distinguish between different hydroxyl functions anddifferent saccharide linkages on the carbohydrate rings.

A novel N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde)* lin-ker resin 1 has recently demonstrated potential for solid-phase synthesis

4 M. I. Osborn and T. H. Khan, Tetrahedron 55, 1807 (1999).5 P. H. Seeberger and W.-C. Haase, Chem. Rev. 100, 4349 (2000).6 A. Heckel, E. Mross, K. H. Jung, J. Rademann, and R. R. Schmidt, Synlett 171 (1998).7 G. Hodosi and J. J. Krepinsky, Synlett 159 (1996).8 S. P. Douglas, D. M. Whitfield, and J. J. Krepinsky, J. Am. Chem. Soc. 117, 2116 (1995).9 L. G. Melean, W.-C. Haase, and P. H. Seeberger, Tetrahedron Lett. 41, 4329 (2000).* Abbreviations: Ac, acetyl; Ac2O, acetic anhydride; AcOH, acetic acid; Bz, benzoyl,

C6H5C——O; ClAc, monochloroacetyl, ClCH2C(——O); ClBz, p-chlorobenzoyl, ClC6H4C(——O); CSPOS, combinatorial solid-phase oligosaccharide synthesis; 1,2-DCE, 1,2-dichlor-

oethane; DCM, dichloromethane; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl;

DMAP, 4-dimethylaminopyridine, p-(CH3)2N-C5H4N; DMF, N,N-dimethylformamide;

�,�-DMT, �,�-dimethoxytoluene; DMTST, dimethyl(methylthio)sulfonium triflate; DVB,

divinylbenzene; ELSD, evaporative light scattering detection; Fmoc-Cl, 9-fluorenylmethy-

loxycarbonyl chloride; Lev, levulinoyl, CH3C(——O)CH2 CH2C(——O); MBHA, methylbenz-

hydrylamine; MeCN, acetonitrile; MeOH, methanol; min, minute(s); MS, molecular sieves;

NaOMe, sodium methoxide; NEt3, triethylamine; Piv, pivaloyl, (CH3)3CC——O; Pn,

protecting group P in hydroxyl position n; PMB, p-methoxybenzyl, MeOC6H4CH2; PS,

polystyrene; Py, pyridine; SPOS, solid-phase oligosaccharide synthesis; Rf, retention factor;

Rt, retention time; TBDPS, tert-butyldiphenylsilyl; TBDMS, tert-butyldimethylsilyl; THF,

tetrahydrofurane; TsOH, p-toluenesulfonic acid.

Fig. 2. Different attaching strategies for the solid-phase synthesis with the Dde linker

system 1 leading to sugar loaded resins 2 and 3.


(Fig. 2).10,11 Dde-based linkers have proved stable to most of the chemicalconditions commonly employed in carbohydrate synthesis and are readilycleaved by treatment with ammonia, hydrazine, or primary aliphaticamines. Solid-phase synthesis with the Dde linker can be undertaken eitherby directly attaching a carbohydrate amino group to the linker resin 1 asin construct 2, or alternatively by immobilizing a primary sugar residuevia a spacer to the linker resin 1 to form construct 3. This chapter reportis concerned with the former method, including potential applications thissolid-phase methodology might have for the creation of a combinatorialoligosaccharide system (Fig. 2).

The initial experiments were designed with the intent to pursue a highloading and high yielding solid-phase synthesis of a biologically active carbo-hydrate. To this end MBHA resin (0.7 mmol/g) was employed with theaim of preparing tens to hundreds of milligrams of desired product. Trisac-charide 4, a mammalian cell surface epitope known to elicit a high antibodyresponse in humans as a result of the high titer of the anti-Gal antibody inhuman sera,12,13 was chosen as the target oligosaccharide (Fig. 3).

10 I. Toth, G. Dekany, and B. Kellam, PCT/AU98/00808 (1996).11 N. Drinnan, M. West, M. Broadhurst, B. Kellam, and I. Toth, Tetrahedron Lett. 42, 1159 (2001).12 U. Galili, B. A. Macher, J. Buehler, and S. B. Shohet, J. Exp. Med. 165, 573 (1985).13 U. Galili, S. B. Shohet, E. Kobrin, C. L. Stults, and B. A. Macher, J. Biol. Chem. 263, 17755

(1988).

Fig. 3. Target �-Gal trisaccharide 4 and resin-bound glucosamine 5.


This decision was based on the critical roles 4 plays in both the bindingof Clostridium difficile associated toxin A, an etiological agent of antibiotic-associated diarrhea and Pseudomembranus colitis, and the role that trisac-charide 4 plays in xenograft rejection. The resin bound glucosamine 5 hasbeen previously described and is the ideal construct from which to com-mence the present synthesis.12 A sugar linker conjugate was initially syn-thesized in solution phase and then coupled to a relatively high loadingMBHA resin. The cleavage of the carbohydrate products from the resin iseffected with a 5% hydrazine hydrate/DMF solution. The critical buildingblock in the synthetic sequence to trisaccharide 4 is intermediary galactoseblock 6, which required differential protection (Fig. 4).

The 3-hydroxyl group of the galactosyl ring (protected by temporarygroup P1) needed to be accessed to provide for further chain elongation,and the 2-hydroxyl group needed to be protected with a participating groupP2 so as to provide 1,2-trans stereochemistry. The terminal galactosyl build-ing block 7 should enable generation of a highly stereoselective � linkageleading to the target trisaccharide 4. Accordingly, a set of galactose build-ing blocks 6 containing different acyl substituents in the 2-position wassynthesized (Fig. 5). There was a spectrum of reactivities anticipated withthe chosen range of acyl subsituents but, as expected,14,15 the donor sugar6a protected by the 2-O-pivaloyl group provided the best stereochemicaloutcome (�/�; 1:16). It is pertinent to note at this stage that the 2-O-benzoyl block 6b provided a very serviceable outcome (�/�; 1:10). Thetemporary protecting group (P1) chosen was 9-fluorenylmethoxycarbonyl(Fmoc) due to its high base lability but excellent acid stability; it resultedin a group that could be cleaved easily but would demonstrate excellentstability to glycosylation conditions (Fig. 5).

14 T. Nukada, A. Berces, and D. M. Whitfield, J. Org. Chem. 64, 9030 (1999).15 T. Nishimura, T. Takano, F. Nakatubo, and K. Murakami, Mokuzai Gakkaishi 31, 40

(1993).

OHO SMe

OR

OO

Ph

OOHOH

HO SMeOR

OOTBDMSO

O SMeOR

OFmocO SMe

OR

OO

Ph

OOTBDMSO

O SMeOH

d)

a) b)

c)

6a R = Piv6b R = Bz6c R = 4-ClBz6d R = Ac

Fig. 5. Sequence to core galactosyl donors 6a–6d, bearing different stereodirecting groups

in position 2. Reagents and conditions: (a) DMAP, 1,2-DCE, R-Cl; (b) MeCN/MeOH, TsOH;

(c) �,�-DMT, MeCN, TsOH; (d) Fmoc-Cl, DMAP, 1,2-DCE.

Fig. 4. Synthetic strategy with resin-bound glucosamine acceptor 5 and galactosyl donors 6

and 7 for the assembly of human antigen trisaccharide 4. 7a, X ¼ Cl; 7b, X ¼ H; 6a P2 ¼ Piv;

6b P2 ¼ Bz.


Resin bound monosaccharide 5 was glycosylated with pivaloate donor6a in the presence of dimethyl(methylthio)trifluoromethanesulfonate(DMTST, Fig. 6).

Due to the effectiveness of DMTST in solution-phase chemistry, it wasused as the sole promoter or leaving group activator in all glycosylationsduring our studies. The resin was then subjected to 20% NEt3/DMFto effectively cleave the Fmoc group yielding disaccharide acceptorresin 8. The final glycosylation was effected with donor sugar 7a and the

Fig. 6. Solid-phase synthesis sequence of human antigen trisaccharide 4. Reagents and

conditions: (a) (i) DMTST, DCM, MS 4 A, (ii) 20% NEt3/DMF; (b) 7a, DMTST, DCM, MS 4

A; (c) 5% hydrazine hydrate/DMF; (d) (i) Ac2O, Py, (ii) NaOMe/MeOH, reflux, (iii) H2, Pd/

C, THF, AcOH, MeOH/THF.


resin-bound trisaccharide 9 was subjected to cleavage conditions using5% hydrazine hydrate/DMF. Trisaccharide 10 was treated with aceticanhydride/pyridine to acetylate the amino function generated from thecleavage, followed by treatment with NaOMe/methanol under reflux tocleave the pivaloate ester. All benzyl groups were then removed by hy-drogenolysis. The overall yield for the synthesis of trisaccharide 4 afterpurification by column chromatography was 76%. The fully deprotectedtrisaccharide was compared with a sample prepared by solution-phasemethods and showed matching physical properties.16 After the success ofthis initial synthesis it was noted that by introducing minor modificationsto the synthesis, an extended human blood group determinant could beeasily synthesized and that in fact with these modifications there wouldbe quite a high degree of structural diversity accessible. To demonstratethe growing versatility of the system, tetrasaccharide 11, which is con-sidered as either an extended H or B type blood group determinant, waschosen as the next target (Fig. 7).

To achieve the synthesis of the branched tetrasaccharide 11, a furtherbuilding block fucosyl donor 12 was chosen, which promised a highlystereoselective glycosylation outcome. By using the benzoyl galactosyl

16 G. Dekany, L. Bornaghi, J. Pappageorgiou, M. West, and N. Drinnan, PCT AU01/00028

(2000).

OOO

O

ONHAc

HO

OH

OH OH

HOHO

O

O

OHCH3

HOOH

OH OH

OHOCH3

BzOOBz

OBnSMe

11 12

Fig. 7. Blood group determinant tetrasaccharide 11 and fucosyl donor 12.


building block 6b instead of the pivaloyl-protected building block 6a,deprotection of the 2-hydroxyl group could be effected due to the fargreater lability of the benzoate ester to base cleavage (Fig. 5). The nextcritical modification required was the substitution of the chlorobenzylatedgalactose donor 7a for a benzylated donor 7b (Fig. 4). Terminal donor 7awas originally chosen for its highly crystalline characteristics, but underthe conditions of hydrogenolysis the chlorobenzyl groups liberate HCl, inthis case four molar equivalents of it. Considering the known sensitivityof fucosyl glycosidic linkages it was deemed expedient to go with simplebenzyl groups. Resin sugar conjugate 5 was glycosylated with donor sugar6b in a fashion similar to that described for the synthesis of 4 (Fig. 6) toform resin-bound disaccharide 13 (Fig. 8). Similarly, Fmoc deprotectionfollowed by glycosylation with donor sugar 7b produced resin-bound trisac-charide 14. This resin was then subjected to a mixture of NaOMe inMeOH/THF to provide resin-bound trisaccharide 15. After the resin waswashed and dried it was glycosylated with the fucose building block 12 tofurnish resin-bound tetrasaccharide 16. The resin was cleaved with a 5%hydrazine hydrate/DMF solution, followed by the addition of an Ac2O/Py mixture. After workup the residue was treated with a solution ofNaOMe/MeOH and then chromatographed to give the anomerically puretetrasaccharide 17 in 46% yield over nine steps (Fig. 8).

The synthesis of the tetrasaccharide 17 proved to be a high yieldingand facile synthesis. Analyses of cleavage solutions indicate that the ‘‘onresin’’ chemistry proceeds extremely clean. Accumulation of alternativeanomers appears to account for the majority of reaction by-products.Similarly, the results for the solid-phase synthesis of 4 compare veryfavorably with those achieved via comparable solution-phase synthesis.17

Overall, 50 mg of the free trisaccharide 4 was prepared. While this systemdemonstrates a certain level of versatility, there are several criteria tobe met to achieve fully generalized combinatorial methodology based on

17 P. J. Garegg and S. Oscarson, Carbohydr. Res. 136, 207 (1985).

Fig. 8. Solid-phase synthesis sequence to fully protected blood group determinant

tetrasaccharide 17 starting from disaccharide acceptor resin 13. Reagents and conditions:

(a) 7b, DMTST, DCM, MS 4 A (b) NaOMe, MeOH, THF; (c) 12, DMTST, DCM, MS 4 A (d)

(i) 5% hydrazine hydrate, DMF, (ii) Ac2O, Py; (e) NaOMe/MeOH.


this kind of technology. The primary issues to address involve selection ofappropriate protecting groups (Fig. 9).

In this system, although it is possible to synthesize a range of lactosa-mine-based tri- and tetrasaccharides employing a variety of differentmonosaccharide donors (Fig. 9), the protecting groups presently used arenot fully orthogonal and therefore not amenable to a generalized combina-torial methodology. In this system, the Fmoc group would be cleaved underthe same conditions as the benzoyl-protecting group and so is invalidateddue to lack of orthogonality. A silyl-protecting group instead of Fmocwould provide the appropriate stability and orthogonality and, therefore,advances itself as a good alternative. The benzylidene ring opening

Fig. 9. General methodology for the preparation of lactosamine-based libraries.


reaction is well established as a typical solution-phase carbohydrate syn-thetic technique, but its use in solid-phase techniques is rarely mentioned.This reaction could allow discriminate access to either the 4- and 6-hydroxyl groups of a pyranose ring while at the same time successfully cap-ping the other hydroxyl group. It is conceivable that a fully orthogonaloligosaccharide synthetic strategy could be accessed by the developmentof such building blocks. In the following section we will describe the useof a silyl-protected building block in the synthesis of structurally diversegalactosyl oligosaccharides.

Combinatorial Solid-Phase Oligosaccharide Synthesis (CSPOS)

A combinatorial approach should provide the ability to generate arraysof compounds instead of single molecules by linear syntheses, and providethe chemist with the opportunity to fully exploit different aspects ofdiversity, whether it be structural, functional, or otherwise.18,19 This isparticularly relevant when considering monosaccharides and carbohy-drates due to the high functional density and stereoisomeric forms foundwith these molecules. Consequently, a juxtaposition of traditional solid-phase synthetic carbohydrate techniques with combinatorial synthesistechniques to provide a combinatorial solid-phase oligosaccharide synthe-sis (CSPOS) platform is an ideal approach for the production of carbohy-drate structures to enable the investigation of the biological roles ofcarbohydrates and to further drug discovery. However, compared to

18 N. K. Terrett, M. Gardner, D. Gordon, R. J. Kobylecki, and J. Steele, Tetrahedron 51, 8135

(1995).19 F. Balkenhohl, C. von dem Bussche-Huennefeld, A. Lansky, and C. Zechel, Angew. Chem.

108, 2436 (1996); Angew. Chem. Int. Ed. Engl. 35, 2288 (1996).

Fig. 10. Issues and criteria in SPOS and CSPOS.


conventional solid-phase oligosaccharide synthesis,4,5 the CSPOS method-ology is still in its infancy.20–23 One of the major problems to overcome inorder to fully develop an efficient system is the requirement to generatesuitably protected building blocks for each monomer type (e.g., glucose,galactose, mannose) as well as for each linkage position per monomertype (i.e., for the 1!2, 1!3, 1!4, and 1!6 connections), orthogonal pro-tected to each other.24,25 Further basic considerations and additionalrequirements of both, SPOS and CSPOS, include (Fig. 10) the following:

20 R.

C.21 T.

18922 D.

(1923 T.

Ch24 G.25 M.

En

Support, linker, and cleavage compatible with target structure?Orthogonal protecting groups and elongation method?Common, flexible, and automation-friendly protocol?Hydroxyl and donor reactivity and regiochemistry?Defined stereochemistry at the anomeric carbon?

In the synthesis of diverse oligosaccharide libraries, a conventionalSPOS methodology would involve laborious solution-phase preparationof many building blocks and that number would increase if increased diver-sity of the library is to be obtained. To develop a versatile and practical ap-proach, it is necessary to reduce the number of building blocks ideally

Liang, L. Yan, J. Loebacg, Y. Uozumi, K. Sekanina, N. Horan, J. Gildersleeve,

Thompson, A. Smith, K. Biswas, W. C. Still, and D. Kahne, Science 274, 1520, (1996).

Zhu and G.-J. Boons, Angew. Chem. 110, 2000 (1998); Angew. Chem. Int. Ed. Engl. 37,

8 (1998).

J. Silva, H. Wang, N. M. Allanson, R. K. Jain, and M. J. Sofia, J. Org. Chem. 64, 5926

99).

Takahashi, H. Inoue, Y. Yamamura, and T. Doi, Angew. Chem. 113, 3330 (2001); Angew.

em. Int. Ed. Engl. 40, 3230 (2001).

Baranay and R. B. Merrifield, J. Am. Chem. Soc. 116, 7363 (1977).

Schelhaas and H. Waldmann, Angew. Chem. 108, 2192 (1996); Angew. Chem. Int. Ed.

gl. 35, 2056 (1996).

O

LevO OTBDPS

PMBO

OClAc

O

O

OMe

DisaccharidesTrisaccharidesTetrasaccharidesPentasaccharides

n. Selective deprotection

Combinatorial approachin solution

n + 1. Glycosylation

Fig. 11. Combinatorial oligosaccharide library approach in solution with an orthogonal

protecting group strategy. [See C.-H. Wong, X.-S. Ye, Z. Zhang, J. Am. Chem. Soc. 120, 7137

(1998).]


employing only one single, orthogonal protected universal building blockper monomer type.26 This block should allow us to access each linkageposition on the carbohydrate residue independently and with no preferencein order of the removal of protecting groups (i.e., a completely permutableorder of all protecting group manipulations).

Wong et al.27 described a similar method and the first combinatorialapplication thereof in solution phase in 1998. As part of this methodology,the use of a methyl 6-hydroxyhexanate galactoside building block ispresented (Fig. 11).

This strategy employs individual deprotection steps for every hydroxylgroup prior to the sequential glycosylation with different glycosyl donors,linking the new residue to each of the four galactose hydroxyl groups. Bythis means a library of di-, tri-, tetra-, and pentasaccharides was prepared.The limitation of this method is that the building block cannot be usediteratively to form linear or branched linear oligosaccharides. After eachdeprotection there is essentially a capping step with the next glycosylation.The incumbent building block is not differentially protected and there-fore does not allow for any further chain elongation. Essentially, sub-stitutions may occur only around one monosaccharide scaffold. Inaddition, the high variation in donor/acceptor reactivities leads to a highlevel of uncertainty in predicting both the extent and the stereochemicaloutcome of each reaction.28,29

In our search for more versatile approaches toward linear oligosacchar-ides we developed a new solid-phase method combining the use of an orth-ogonal25,26 protected universal building block with powerful perbenzylatedacceptors bound on the resin. After initial attachment of the first building

26 G. Dekany, L. Bornaghi, and J. Pappageorgiou, US PCT 09/889687 (2000).27 C.-H. Wong, X.-S. Ye, and Z. Zhang, J. Am. Chem. Soc. 120, 7137 (1998).28 Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, T. Baasov, and C.-H. Wong, J. Am. Chem.

Soc. 121, 6527 (1999).29 X.-S. Ye and C.-H. Wong, J. Org. Chem. 65, 2410 (2000).

Fig. 12. Iterative CSPOS sequence.


block to the resin, the synthesis follows an iterative, combinatorial processas stepwise outlined below (Fig. 12):

1. Removal of protecting groups at nonlinking positions [step (1), P2,P4, P6)]. These positions will be free hydroxyl functionalities in thefinal product.

2. Temporarily cap these hydroxyl positions (e.g., benzylation)generating a highly electron-rich carbohydrate residue [step (2),capping].

3. Removal of the temporary protecting group at the selected linkingposition [step (3), P3 deprotection].

4. Stereoselective solid-phase glycosylation of the highly reactive, fullybenzylated acceptor using the orthogonal protected building blockbearing a neighbor participating group (P2) [step(4), glycosylation].

Branched oligosaccharides might be approached by this method in asimilar fashion using benzyl-protected building blocks in the cappingstep. This new method offers rapid access to a whole variety of glycostruc-tures in a simple manner. A maximum in structural diversity can be gainedby using only a minimum number of building blocks. Compared to SPOSapproaches, this CSPOS method should be able to generate more valuethrough a much higher efficiency in terms of building blocks. In our firststudy on the combinatorial assembly of galactose oligomers, we designedthiomethyl glycoside 1830 as universal building block (Fig. 13).27 Thio-methyl galactoside 18 is readily available via a high yielding six-stepsynthesis starting from methyl 1-thio-�-d-galactoside.17,31 Thiodonorshave already been applied successfully in the combinatorial synthesis of

30 M. Grathwohl, M. Broadhurst, and W. Meutermans, Oral and Poster Presentation, 21th

International Carbohydrate Symposium, Cairns, Australia, 2002, OP 051 and PP 204.31 P. Fugedi, P. J. Garegg, H. Lonn, and T. Norberg, Glycoconjugate J. 4, 97 (1987).

Fig. 13. Universal building block 18, photo linker resin 19, and CSPOS sequence to

the fully benzylated, resin-bound monosaccharide acceptors 21, 22, 23, and 24. Reactions:

(a) loading, (b) transesterification, (c) TBDPS cleavage, (d) alkylation, (e) ring opening [! 6-

OH], (f) ring opening [! 4-OH], (g) acidic TBDPS cleavage, (h) neutral/acidic benzylation.


oligosaccharides22,24 and offer the additional advantage of an easy tohandle donor type. In these types of donors, the leaving group at theanomeric center may be introduced stereoselectively at an early stageand serves throughout the whole synthesis as a protecting group at C1,being stable toward most conditions used in oligosaccharide construction.31

On position C2 of our proposed universal building block 18, we havechosen the ClBz group (p-chlorobenzoyl) as a hydroxyl protection withstereodirecting properties, on C3 the O-TBDPS group, and in positionsC4 and C6 a cyclic benzylidene acetal protection (Fig. 13).

All protecting groups have proven to fulfill the given requirementsof orthogonality for a permutable, combinatorial access to each linkageposition of the core residue (Fig. 12). In our initial experiments on Wang


resin and on Rink resins with a carbamate type linkage, the regioselectivering opening of the 4,6-O-benzylidene acetal to the corresponding benzylethers catalyzed by Lewis acids, was accompanied by the loss of productfrom the resin. To avoid these problems and to increase the stability ofthe linkage against acids, we choose the 6-nitroveratryl-based photo linkersupport 19 with an amino methyl-PS-core resin.32,33 Photo-labile resin ap-proaches were already successful applied in SPOS,34–38 as well as in com-binatorial approaches.39 Loading of the universal building block 18 ontoresin 19 was performed via treatment with DMTST [dimethyl(methylthio)-sulfonium triflate] under anhydrous conditions (MS 4 A) yielding poly-meric support 20 in a quantitative manner. Loading in this sequence wasdetermined by mass gain as well as by preparative cleavage of the corres-ponding hemiacetals (h�, 365 nm, THF) from the resin. The sequences tofully benzylated acceptor resins 21 and 22, bearing unprotected hydroxylgroups at C4 and C6, start both with cleavage of the acyl group in position2 via transesterification under Zemplen conditions40 [reaction (b), NaOMe,THF/MeOH (5:1)], followed by removal of the 3-O-TBDPS group [reac-tion (c), TBAF, DMF/THF (2:1), 65

�]. Independent results have shown

that this sequence may be shortened by one step through a simultaneouscleavage of both groups under conditions c). Alternatively, selective accessto both groups proved to be possible using milder and slightly acidicconditions as described for the synthesis of acceptor 24. Finally, doublealkylation led to the common intermediate for both sequences, e.g., 2,3-di-O-benzyl-4,6-O-benzylidene galactosyl resin [reaction(d), KOtBu,BnBr, DMF].41 Initial experiments to generate the 4-hydroxy derivative

32 C. P. Holmes and D. G. Jones, J. Org. Chem. 60, 2318 (1995).33 C. P. Holmes, J. Org. Chem. 62, 2370 (1997).34 N. Winssinger, J. Pastor, F. DeRoose, and K. C. Nicolaou, J. Am. Chem. Soc. 119, 449

(1997).35 R. Rodebaugh, S. Joshi, B. Fraser-Reid, M. H. Geysen, and G. M. Paul, J. Org. Chem. 62,

5660 (1997).36 R. Rodebaugh, S. Joshi, B. Fraser-Reid, and M. H. Geysen, Tetrahedron Lett. 38, 7653

(1997).37 K. C. Nicolaou, N. Watanabe, J. Li, J. Pastor, and N. Winssinger, Angew. Chem. 110, 1636

(1998); Angew. Chem. Int. Ed. Engl. 37, 1559 (1998).38 A. B. Kantchev and J. R. Parquette, Tetrahedron Lett. 40, 8049 (1999).39 M. J. Sofia, N. Allanson, N. T. Hatzenbuhler, R. Jain, R. Kakarla, N. Kogan, R. Liang,

D. Lui, D. J. Silva, H. Wang, D. Gange, J. Anderson, A. Chen, F. Chi, R. Dulina, B. Huang,

M. Kamau, C. Wang, E. Baizman, A. Branstrom, N. Bristol, R. Goldman, K. Han,

C. Longley, S. Midha, and H. R. Axelrod, J. Med. Chem. 42, 3194 (1999).40 G. Zemplen, Ber. Dtsch. Chem. Ges. 60, 1555 (1927).41 T. Wunberg, C. Kallus, T. Opatz, S. Henke, W. Schmidt, and H. Kunz, Angew. Chem. 110,

2620 (1998); Angew. Chem. Int. Ed. Engl. 37, 2503 (1998).


22 via the benzylidene ring opening system Et3SiH/TFA did not lead to thedesired product. Finally, both regioselective ring opening reactions weresuccessfully achieved and furnished 6-hydroxy derivative 21 [reaction (e),BH3�THF, Bu2BOTf, CH2Cl2]42–44 and the corresponding 4-hydroxy resin22 [reaction (f), NaCNBH3, HCl, MS 4 A, CH2Cl2].45 It is worth mention-ing that both regioisomers showed remarkable differences in their physicalproperties [6-hydroxy derivative 21: Rf (Tol/Ac, 2:1) ¼ 0.34, Rt (LCMS)46

¼ 5.25; 4-hydroxy derivative 22: Rf (Tol/Ac, 2:1) ¼ 0.54, Rt (LCMS)46 ¼5.31]. Permutation of the protecting group manipulation steps, i.e., ringopening [reaction (e)], transesterification [reaction (b)], followed by adouble benzylation of positions 2 and 6 and cleavage of the TBDPS-protecting group at the desired linking position furnished the perbenzy-lated regioisomer 23, ready for linkage through its 3 position. The sequenceto fully benzylated, 2-OH acceptor resin 24 starts with a slightly acidiccleavage of the TBDPS group in position 3 [reaction (g), HF�Py, Py]47 leav-ing the ester protection in position 2 unaffected.48 Reductive ring openingof the 4,6-O-benzylidene acetal to the corresponding 4-O-Bn derivative[reaction (e), BH3�THF, Bu2BOTf] furnished the 3,6-diol resin in a cleanand regioselective manner. To generate the 2-hydroxy acceptor resin 24,the nonlinking sites have to be capped via benzylation prior to deprotec-tion of the 2-O-C1Bz group. Preferentially, this alkylation step is to be per-formed under acidic or neutral conditions in order to avoid acyl groupmigration. Unfortunately, the conditions for the acidic double benzylationin positions 3 and 6 [reaction (h), BnOC(NH)CCl3, BF3�OEt2], thoughsuccessfully employed in solution-phase synthesis,49,50 have so far notbeen transferable to resin chemistries. We are currently investigating alter-native alkylation reagents and conditions. Having access to acceptor resins21, 22, and 23, solid-phase glycosylation to the corresponding, �-linkeddisaccharides 25, 26, and 27 was performed using again donor 18 (Fig. 14).

42 L. Liang and T.-H. Chan, Tetrahedron Lett. 39, 355 (1998).43 X. Wu, M. Grathwohl, and R. R. Schmidt, Org. Lett. 3, 747 (2001).44 X. Wu, M. Grathwohl, and R. R. Schmidt, Angew. Chem. 114, 4664 (2002); Angew. Chem.

Int. Ed. Engl. 41, 4489 (2002).45 P. J. Garegg, Pure Appl. Chem. 56, 845 (1984).46 System parameters: All LCMS spectra were recorded on a Micromass LCZ/LCT system

Mux 4 [gradient, MeCN/H2O (5:95) to (100:0); time, 12 min; flow rate, 2 ml/min; column,

Zorbax, SB-C18 (4.6 � 50 mm); pore size, 5 �m] after analytical cleavage from the

corresponding resin.47 B. M. Trost, C. G. Caldwell, E. Murayama, and D. Heissler, J. Org. Chem. 48, 3252 (1983).48 M. Grathwohl and R. R. Schmidt, Synthesis 2263 (2001).49 H.-P. Wessel, T. Iversen, and D. R. Bundle, J. Chem. Soc. Perkin Trans. I 2247 (1985).50 H.-P. Wessel and D. R. Bundle, J. Chem. Soc. Perkin Trans. I 2251 (1985).

Fig. 14. CSPOS sequence to disaccharides 25, 26, and 27 via the fully benzylated, resin-

bound monosaccharide acceptors 21, 22, and 23. Reagents and conditions: (a) DMTST, 18 (2.0

equiv.), MS 4 A, CH2Cl2, two runs.


In this elongation cycle, using 2.0 equivalents of donor 18 and repeatingthe glycosylation reaction once to ensure complete conversion, achievedbest results. The use of molecular sieves proved to be crucial; removal ofthe finely grounded powder after reaction was done via simple washingsthrough a polyethylene porous disc. To prove the method and to extendit to the next generation of linkages, the trisaccharides, we decided to syn-thesize an example of the Gal�(1!6)Gal�(1!3)Gal trisaccharide resin 29following the sequence outlined below (Fig. 15).

Starting from fully protected disaccharide resin 27, the primary resin-bound acceptor 28 was synthesized according to the conditions describedabove for the monosaccharide cases. All conditions could be successfullytransferred to the disaccharide stage and the final elongation to resin-bound trisaccharide 29 was performed under standard conditions [condi-tions (e), DMTST, 18 (2.0 equiv.), MS 4 A, CH2Cl2, two runs] to generatean anomeric mixture of hemiacetals 30 after preparative cleavage fromthe resin (Fig. 16). Performing the elongation via repeated glycosyla-tion cycles led to a virtually complete consumption of disaccharide ac-ceptor 28 (starting material <2%) and afforded the target trisaccharide

Fig. 15. CSPOS sequence to the final Gal�(1!6)Gal�(1!3)Gal trisaccharide resin 29,

starting from fully protected disaccharide resin 27. Reagents and conditions: (a) HF�Py, Py;

(b) NaOMe, THF/MeOH (5:1); (c) KOtBu, BnBr, DMF; (d) BH3�THF, Bu2BOTf, CH2Cl2;

(e) DMTST, 18 (2.0 equiv.), MS 4 A, CH2Cl2, two runs.


O-(4,6-O-benzylidene-3-O-tert-butyldiphenylsilyl-2-O-para-chlorobenzoyl-�-d-galactopyranosyl)-(1!6)-(2,3,4-tri-O-benzyl-�-d-galactopyranosyl)-(1!3)-2,4,6-tri-O-benzyl-�/�-d-galactopyranose 30 in over 88% purity(LCMS trace of the crude cleavage mixture, Rt (LCMS)46 ¼ 10.42, Rf

(Tol/Ac, 2:1) ¼ 0.86; C90H93ClO7Si, calcd: 1526.59 [MNH4þ], found:

1526.69 [MNH4þ]) after a 12-step solid-phase synthesis (Fig. 16).

Conclusion

The overwhelming potential for creating diversity in oligosaccharidelibraries, at least from a theoretical point of view, is inevitably linked withgrowing complexity in the required synthetic routes. A truly versatilemethod would provide parallel access to structurally diverse oligosacchar-ide libraries but is currently not at hand. The solid-phase concept that weproposed here of selective deprotection and capping nonlinking positionsprior to glycosylating perbenzylated acceptors enables access to combina-torial libraries of structurally different oligosaccharides with a minimalnumber of building blocks. Our initial results indicate that this is a realisticapproach, though much more development work needs to be done beforeits full potential can be explored. Here we have demonstrated that byusing one universal building block we can access variously linked di- andtrisaccharides in a regio- and stereoselective manner on solid-phase,thereby opening the possibilities of generating combinatorial libraries ofstructurally diverse oligosaccharides.

Mw=1508

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00Time5

100

%

AL098-67_AN#2609 ELSDAn1

2.36e5Area%

9.481.80

88.72

Area3283.00623.34

30717.55

Height232796057

218744

Time7.428.11

10.42

10.42

7.42

8.11

Mw=1508

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z0

100

%

AL098-67_AN#2609127 (10.361) Cm (127:130) 1: Scan ES+4.07e31528.6877

1526.6973

1492.7949

1059.38021061.6107

1581.6956

1582.7329

1583.6792

1584.80551831.66601649.7124

PR

SM

OBnO

BnOO

BnO

OO

BnOOBn

OBnOH

OTBDPSO

OO

Ph

ClBzO

C90H93ClO17Si

Exact Mass: 1508.59

30

Fig. 16. ELSD chromatogram and mass spectrum from CSPOS sequence of the crude trisaccharide product 30 after cleavage from resin 29

(12 steps). Glycosylation to 29 was performed in two runs using 2.0 equivalents of donor 18 per run.

26

6o

lig

osa

cc

ha

rid

ec

he

mistr

y[1

4]


Acknowledgments

This work was supported in part by the Australian Research Grants Council and the

Government of Queensland. The authors thank all Alchemia core facilities that participated

in these study, especially Mr. Hoan The Vu for his support in analytical tasks and the entire

VAST� team for their support of this research.

[15] encoded peptidomimetic and small molecule libraries 271

[15] Design, Synthesis, Screening, and Decoding ofEncoded One-Bead One-Compound Peptidomimetic and

Small Molecule Combinatorial Libraries

By Ruiwu Liu, Jan Marik, and Kit S. Lam

Introduction

In 1991, we first introduced the ‘‘one-bead one-compound’’ (OBOC*)combinatorial library method.1 Since then, it has been successfully appliedto the identification of ligands for a large number of biological targets.2,3

Using well-established on-bead binding or functional assays, the OBOCmethod is highly efficient and practical. A random library of millions ofbeads can be rapidly screened in parallel for a specific acceptor molecule(receptor, antibody, enzyme, virus, etc.). The amount of acceptor neededis minute compared to solution phase assay in microtiter plates. The posi-tive beads with active compounds are easily isolated and subjected to struc-tural determination. For peptides that contain natural amino acids andhave a free N-terminus, we routinely use an automatic protein sequencerwith Edman chemistry, which converts each �-amino acid sequentially toits phenylthiohydantoin (PTH) derivatives, to determine the structure ofpeptide on the positive beads.

We have recently reported methods for sequencing peptides containingmany unnatural �-amino acids.4,5 However, there is no simple way to

* Abbreviations: AllocOSu, allyloxycarbonyl-N-hydroxysuccinimide; BCIP, 5-bromo-4-

chloro-3-indolyl; Boc, tert-butyloxycarbonyl; tBu, tert-butyl; DCM, dichloromethane; Dde,

1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; Ddz, �;�-dimethyl-3,5-dimethoxyben-

zyloxycarbonyl; DIC, N,N0-diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine;

DMF, N,N-dimethylformamide; FmocOSu, 9-fluorenylmethyloxycarbonyl-N-hydroxysucci-

nimide; OBOC, one-bead one-compound; PBS, phosphate-buffered saline; Pmc, 2,2,5,7,8-

pentamethylchroman-6-sulfonyl; PTH, phenylthiohydantoin; RR, relative reactivities;

Teoc, 2-(trimethylsilyl)ethoxycarbonyl; TFA, trifluoroacetic acid; Trt, trityl.1 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp,

Nature 354, 82 (1991).2 K. S. Lam, T. Sroka, M. L. Chen, Y. Zhao, Q. Lou, J. Wu, and Z. G. Zhao, Life Sci. 62,

1577 (1998).3 K. S. Lam, M. Lebl, and V. Krchnak, Chem. Rev. 97, 411 (1997).4 R. Liu and K. S. Lam, Anal. Biochem. 295, 9 (2001).5 R. Liu and K. S. Lam, in ‘‘Peptides: The Wave of the Future. Proceedings of the Second

International and the Seventeen American Peptide Symposium’’ (M. Lebl and R. A.

Houghten, eds.), p. 299. American Peptide Society and Kluwer Academic Publishers, San

Diego, 2001.



272 peptide synthesis and screening [15]

directly determine the structure of peptidomimetic and small moleculecompounds on a single bead. Typical spectroscopic methods such as massspectrometry (MS), nuclear magnetic resonance (NMR), and infrared(IR) will not provide sufficient information for complete structure elucida-tion of compounds contained in a single bead. Another approach is to usean encoding strategy. Various physical and chemical encoding methodshave been reported by several groups and the subject has been recentlyreviewed.3,6–9 These encoding methods are successful to a varying degree,but are far from ideal. Physical encoding methods are unable to encode alarge number of compounds, such as more than 100,000. Furthermore, someof these methods are not applicable to OBOC bead library technology.

While currently available chemical encoding methods are useful forOBOC bead library, they have some limitations. First, the coding moleculeis randomly present at the surface, which may interfere with the binding ofthe target protein to the testing compound resulting in a false-positiveresult. A spatial segregation of the ligand and the encoding tag is necessaryto minimize the unwanted interaction. Second, the current chemical encod-ing method requires that the synthetic chemistry of the coding tag and thetesting compound be orthogonal, which doubles the number of requiredsynthetic steps.

To overcome these limitations, we have recently reported a novel,highly efficient, and robust encoding system for OBOC peptidomimeticand small molecule combinatorial libraries.10 In this method, only thetesting compounds are present on the exterior of the bead (i.e., exposedto the target proteins), and the coding peptide molecules are placed inthe core of the bead. A novel strategy has also been employed to eliminatethe extra synthetic steps and minimize the amount of undesirable sideproducts by combining the synthetic steps of both coding molecules andtesting compounds.

The use of polyglutamic acid11 or proteases11,12 to prepare topologicallysegregated bifunctional beads is successful to a certain degree, but far fromideal since they are technically difficult to control, laborious, and not repro-ducible from batch to batch. Recently we have developed a highly efficient

6 A. W. Czarnik, Curr. Opin. Chem. Biol. 1, 60 (1997).7 X. Y. Xiao, Front. Biotechnol. Pharm. 1, 114 (2000).8 C. Barnes and S. Balasubramanian, Curr. Opin. Chem. Biol. 4, 346 (2000).9 R. L. Affleck, Curr. Opin. Chem. Biol. 5, 257 (2001).

10 R. Liu, J. Marik, and K. S. Lam, J. Am. Chem. Soc. 124, 7678 (2002).11 M. Lebl, K. S. Lam, S. E. Salmon, V. Krchnak, N. Sepetov, and P. Kocis, U. S. Patent 5 840

485 (1998).12 J. Vagner, G. Barany, K. S. Lam, V. Krchnak, N. F. Sepetov, J. A. Ostrem, P. Strop, and

M. Lebl, Proc. Natl. Acad. Sci. USA 93, 8194 (1996).


method termed the ‘‘biphasic solvent approach’’ for spatial segregation onindividual TentaGel beads (Rapp Polymere). This approach makes use ofthe two immiscible solvents system in which TentaGel resin has approxi-mately equal swelling volumes in both phases. The crucial requirement isto keep the first solvent inside the bead and the second solvent outsidethe bead while the reaction on the outer layer is occurring. The derivatizingreagents that modify the surface of the bead should be soluble in the outersolvent only. Water and a mixture of dichloromethane (DCM)/diethylether do meet these requirement. The ratio of the DCM/diethyl ethermay be calculated from the swelling volumes and then adjusted experimen-tally for the given reaction. For TentaGel beads, the resin is swollen in thefirst solvent (water) and then the coupling reaction to the surface of the wetbead is carried out in the second solvent (DCM/diethyl ether). We have dis-covered that the optimized ratio of DCM/diethyl ether is 55/45. The deriva-tizing reagent, e.g., 9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide(FmocOSu) or allyloxycarbonyl-N-hydroxysuccinimide (AllocOSu), in thesolvent mixture reacts with the amino group on the surface of the TentaGelbead rapidly (normally less than 30 min), whereas the amino group in theinterior of the bead in water remains intact. Thus, only the outer layer ofthe bead is derivatized. By adjusting the amount of derivatizing reagentused, the thickness of the outer layer can be easily controlled.

Encoded libraries are synthesized on topologically segregated bifunc-tional beads described above. To efficiently generate an encoded chemicallibrary, the appropriate orthogonal protecting groups and syntheticschemes must be chosen so that the number of necessary chemical reac-tions can be minimized by coupling the building blocks to the testing andcoding arms simultaneously. For example, the orthogonal protectinggroups for amine can be Fmoc, Alloc, tert-butyloxycarbonyl (Boc), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), �,�-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz), and 2-(trimethylsilyl)ethoxycarbonyl(Teoc) groups. In our encoding system, the testing compound is a peptido-mimetic or small molecule, and the coding molecule is a peptide containinga large number of unnatural �-amino acids derived from different build-ing blocks that are used for generating the peptidomimetic and smallmolecules. The retention time of these unnatural �-amino acids must bepredetermined by a protein sequencer.

The general synthetic and decoding scheme of an encoded library isshown in Scheme 1. The amino group on the outer layer of the bead is firsttopologically protected with a protecting group P, such as Fmoc, Alloc, anda free amino group in the interior. A coding arm precursor, (P3)A0-(P2)Y -(P1)X, is then constructed in the interior of the bead. (P1)X and (P2)Y rep-resent trifunctional amino acids, which are preprotected by orthogonal

Edman degradation

Identify D, E, F Chemical structure

_

S D_

_ _ _ X

_

_ _ _Y X

__PTH X

D

H2N H2N

H2N

H2N

_ _ _Y X

Y

D_P

_ _ _Y X_ _ _Y X

P3

P3

P2 P1

P3

P2 P1

P3

P2

P2

P1

P1

P2

P2

PTH

A9 A9A9

A9 A9

A9

S SS

D

EF

E

E

E E

FF

F

E

D

_P NH

_P NH

_ YPTH

Scheme 1. General synthetic and encoding/decoding scheme of encoded library.


groups, P1 and P2, respectively. D represents a bifunctional building blockthat links the scaffold to the bead, and it can be coded by amino acidA0, which represents a single amino acid or a combination of two aminoacids. S stands for the scaffold of the testing arm with preprotectedmultifunctional groups onto which building blocks can be covalentlyattached.

To minimize the synthetic steps, the protecting groups on the scaffoldare carefully chosen so that they are identical or very similar to the types(e.g., Fmoc and Dde both can be removed by 2% hydrazine) used onthe coding arm. During the peptidomimetic and small molecule library syn-thesis, both the deprotection of protecting groups on both the coding armand testing arm, and the coupling of each building block (E or F) to boththe testing arm and coding arm are achieved simultaneously. Therefore,each step of the peptidomimetic or small molecule synthesis can proceedas if there is no coding arm. The decoding process is straightforward sincethere is no need of coding tag cleavage and retrieval prior to Edman micro-sequencing. The testing molecule is stable to the Edman condition, andthe coding peptide consists of �-amino acids that can be readily sequenced(Scheme1). The signal of the amino acid (A0) in the first cycle correspondsto D of the testing compound. The signal of PTH-(F)Y in the secondcycle corresponds to the building block F. The signal of PTH-(E)X in thethird cycle corresponds to the last coupling moiety (E). By combiningall the decoded structural information, the complete chemical structureof the testing molecule is easily determined. Alternatively, the codingtag can be released (if a cleavable linker is used) and decoded unambigu-ously by mass spectrometry, if each of the coding subunit(s) has a uniquemolecular mass.

TABLE I

Coding Units and Corresponding Reactions

Coding Units Building Blocks Chemical Reactions

Lysine, 4-amino

phenylalanine

Carboxylic acids, acyl halides,

sulfonyl halides, acid anhydride

Amide formation

Lysine, 4-amino

phenylalanine

Isocyanates, isothiocyanates Urea, thiourea formation

Lysine, 4-amino

phenylalanine

Aldehydes (aliphatic, aromatic,

heterocyclic)

Reductive alkylation

Lysine, 4-amino

phenylalanine

Alkyl halides Nucleophilic substitution

(N-alkylation)

4-Bromophenylalanine Aryl boronic acids Suzuki coupling

4-Bromophenylalanine Amines Nucleophilic substitution

Glutamic acid Amines Amide formation

Serine Carboxylic acids, acyl halides,

sulfonyl halides, acid anhydride

Acylation

Tyrosine Alcohols Mitsunobu reaction

Cysteine Alkyl halides S-Alkylation

1-Amino-4-ketocyclohexyl

carboxylic acid

Ph3P ¼ CHR Wittig reaction

2-Aminopent-4-enoic

acid

RCH2NO2 1,3-Dipolar cycloaddition

reaction

2-Amino-3-acryloyl-

oxypropionic acid

Secondary amines Michael addition

2-Amino-3-(4-formyl-

benzoylamino)propionic

acid

Ketones Aldol condensation


In principle, various types of encoded peptidomimetic and small mol-ecule libraries can be generated using this method via different chemicalreactions (Table I). The trifunctional amino acids listed in Table I areeither commercially available or can be easily synthesized. These aminoacids with orthogonal protecting groups are used as encoding units. Theside chains of these �-amino acids are sites to which building blocks areattached via a large number of organic reactions, including C–Cbond-forming reactions (e.g., Suzuki coupling and Wittig reaction).

In summary, the biphasic approach to derivatize a resin bead topologic-ally and bifunctionally is highly efficient, inexpensive, and easy to control.The advantages of this encoding method include (1) the chemical stabilityof the peptide tag under many organic reaction conditions, (2) the ease ofsimultaneous coupling of common building blocks to the scaffold of thetesting compounds and the side chains of the �-amino acids on the codingpeptide, and (3) straightforward and unambiguous decoding with auto-matic microsequencing or mass spectrometry. One added advantage is


the potential reduced usage of expensive scaffold if the majority of substi-tution of beads is coding tag, which is relatively cheap in general. Besideseliminating a number of synthetic steps by combining the synthesis on thetesting and encoding arms in many of the coupling steps, this method alsoenables the coding arm precursor to be prepared in bulk prior to the syn-thesis of the libraries. Consequently, encoded peptidomimetic and smallmolecule libraries may be prepared as if there is no coding arm.

General Synthetic Procedures for Encoded Peptidomimetic and SmallMolecule Libraries

In general, an Fmoc/Boc orthogonal protecting strategy is most oftenused in library synthesis. Therefore, the protecting groups P and P3 inScheme 1 are Fmoc and Boc, respectively.

1. The coding-precursor strand is first constructed in the interior ofthe topologically segregated bifunctional TentaGel beads describedabove.

a. Couple the first coding unit, Boc-protected amino acid Boc-(P1)X,to the beads. Boc-(P1)X (3 equiv. to the free NH2 of the total resin)and 1-hydroxybenzotriazole (HOBt) (3 equiv.) are dissolved inN,N-dimethylformamide (DMF), then added to the resinfollowed by addition of N,N0-diisopropylcarbodiimide (DIC)(3 equiv.). The beads are shaken at room temperature for 2 h.

b. Wash the beads with DMF, MeOH, and DCM, three timeseach, respectively.

c. Remove Boc with 50% trifluoroacetic acid (TFA) in DCM at roomtemperature for 30 min; after filtration, the bead is neutralizedwith 2% N,N-diisopropylethylamine (DIEA) in DMF.

d. Couple the second coding unit, Boc-protected amino acidBoc-(P2)Y, in the same manner as in step (a).

e. Repeat steps (b) and (c).f. Split the peptide-resin into n equal portions (n equal to the

number of the first testing blocks).g. Couple the third coding units, n different Boc-A0 (single Boc-

amino acids or a combination of two Boc-amino acids, one ineach well, 3 equiv.), in the same manner as in step (1). Usually,the third coding unit can be a large number of amino acids (upto 253). After coupling, keep it in a spatially separated format.

h. Remove Fmoc with 20% piperidine in DMF (twice, 5 min, 15 min).i. Wash the beads with DMF, MeOH, and DMF, three times each,

respectively.


2. Couple the first testing building block, e.g., n different Fmoc-aminoacids in the same manner as above. Dissolve all Fmoc-amino acidsin equal volumes of HOBt solution in DMF and add the solutionsto the corresponding wells (i.e., one amino acid in each well),followed by the addition of DIC. The coupling proceeds at roomtemperature for 2 h.

3. Combine and mix all the beads, and then deprotect the ‘‘Fmoc’’ asstep (1 h).

4. Couple the scaffold (P1)S(P2) using compatible chemistry.5. Remove the protecting group P1 of both the coding arm and

scaffold by means of a compatible reaction.6. The beads are split into m equal portions (m is equal to the number

of the second testing building blocks).7. Couple common building block E to both the coding and testing

arms of individual beads by means of compatible chemistry.8. Combine and mix all the beads and wash with different solvents.9. Remove the protecting group P2 of both the coding arm and

scaffold by means of a compatible reaction.10. Split the beads into I equal portions (I is equal to the number of the

third testing building blocks).11. Couple common building block F to both the coding and testing

arms of individual beads by means of compatible chemistry.12. Combine and mix all the beads, and wash with DMF, MeOH, and

DCM, three times each, respectively; dry the beads under vacuumfor 1–3 h.

13. Remove the Boc-protecting group of the coding molecule and allother protecting groups of any testing and coding molecule withTFA-containing reagent, e.g., reagent K (TFA:phenol:thioanisole:-water:1,2-ethanedithiol/82.5:5:5:5:2.5, v/v/v/v/v); the deprotectionproceeds at room temperature for 2 h.

14. Neutralize the beads with 2% DIEA in DMF twice, then washsequentially with DMF, MeOH, DCM, DMF, DMF/water, water,and finally PBS buffer. The library is ready for screening.

On-Bead Screening of Encoded Peptidomimetic and SmallMolecule Libraries

Various on-bead binding or functional screening methods for OBOCcombinatorial libraries are described in Chapter 17 in this volume and willnot be repeated here. The positive beads are physically isolated under amicroscope for structure determination.


Decoding of Peptidomimetic and Small Molecule Libraries

Decoding of peptide-encoded peptidomimetic and small moleculelibrary can be accomplished by two methods: (1) direct microsequencingof a bead-bound peptide tag on an automatic protein sequencer,10 and(2) mass spectroscopic analysis of a released peptide tag retrieved from abead.13 The latter approach requires a cleavable linker. Sample processingfor decoding by the first method is easy and straightforward as there is noneed for cleavage and retrieval of the coding tag. Since the testing moleculeis unsequencable, only the signals from the coding peptide are observed.Comparing the coding signals (retention time) with the external reference(predetermined retention time of PTH amino acid derivatives), the struc-ture of the building blocks is determined. By combining the structure infor-mation of scaffold (known) and building blocks, the entire chemicalstructure of the peptidomimetic or small molecule is easily determined.Peptide sequencing is performed on a protein sequencer following themanufacturer’s instructions but with a slightly modified gradient programand method as described in chapter 17 in this volume.

Materials and Methods

All solvents and chemicals were purchased from Aldrich and were ana-lytical grade if not given otherwise. FmocOSu and protected l-amino acidswere purchased from ChemImpex Inc, Wood Dale, IL. Perkin-Elmer/Applied Biosystems protein sequencer (model 494) was used for thesequencing of the peptides.

Preparation of Topologically Segregated Bifunctional Beads with 70%Fmoc Outside and 30% Free NH2 Inside

TentaGel S NH2 beads (2.0 g, loading 0.52 mmol) were swollen inwater for 24 h. Water was removed by filtration, and the solution ofFmocOSu (122.8 mg, 0.364 mmol) in DCM/diethyl ether (100 ml, 55/45)mixture was added to the wet beads, followed by addition of DIEA(126 �l, 0.728 mmol). The mixture was shaken vigorously at room tem-perature for 30 min. The beads were washed three times with DCM/diethylether mixture and six times with DMF to remove water from the beads.

Synthesis of an Encoded Peptidomimetic Library, an Example

The synthesis of a 158,400-membered encoded peptidomimetic library(96 � 50 � 33) is given in Scheme 2, using �-Phe(4-NH2) as a trifunctionalscaffold for the testing compound and Phe(4-NH2) as a peptide backbone

13 A. H. Franz, R. Liu, A. Song, K. S. Lam, and C. B. Lebrilla, J. Comb. Chem. 5, 125 (2003).

NH2

Fmoc-NH

1) Couple coding block Boc-Lys(Dde)2) Deprotect Boc with 50% TFA/DCM3) Couple coding block Boc-Phe(NO2)4) Deprotect Boc with 50% TFA/DCM

5) Couple 96 coding blocks Boc-Aa1(P)6) Remove Fmoc with 20% piperidine7) Couple 96 testing blocks Fmoc-Aa2(P)

Fmoc-Aa2-NH

1) Remove Fmoc with 20% piperidine2) Couple Fmoc-beta-Phe(4-NO2)

Aa2 NH

1) Reduce NO2 with SnCl22) Couple 33 R1COOH, R1COCl or R1SO2Cl

Aa2 NH

P 5) Remove Boc and side chain protectinggroups of 96 amino acids

Boc-Aa1(P)-Phe(4-NO2)-Lys(Dde)

NH2-Aa1-Phe(4-NHCOR1)-Lys(COR2)

CONHR2CONH

NHCOR1

CONHFmocNH

NO2

Coding peptide

Testing molecule

3) Remove Dde and Fmoc with 20% piperidine4) Couple 50 R2COOH or (R2CO)2O

Boc-Aa1(P)-Phe(4-NO2)-Lys(Dde)

NH2

H2NFmoc-OSu

P

Scheme 2. Synthesis of encoded model library.

[15]

en

co

de

dpe

ptid

om

im

etic

an

dsm

al

lm

ol

ec

ul

el

ib

ra

rie

s2

79


for the coding tag. Their corresponding precursors �-Phe(4-NO2) andPhe(4-NO2) have been selected for the attachment of the building blocksbecause the NO2 group can be treated like a protected amino group, andit can be easily converted to amino groups by reduction with SnCl2. Theside chain amino groups of these �-amino acids are acylated by a largenumber of carboxylic acids, acid anhydrides, acyl chlorides, or sulfonylchlorides. The elution profiles of many PTH derivatives of lysine andPhe(4-NH2) have been predetermined using an improved sequencing gra-dient program.10 Forty-eight carboxylic acids and two acid anhydrides havebeen selected as building blocks to couple to the side chain of lysine.Twenty-seven carboxylic acids, three acyl chlorides, and three sulfonylchlorides have been chosen to acylate the amino group of aniline. Toobtain an unambiguous sequence, the principle of choosing a buildingblock is that two amino acid derivatives must have a retention time differ-ence greater than 0.10 min. In the encoded library shown in Scheme 2, thefirst testing building block is 96 amino acids. They are encoded with 22single amino acids (19 natural amino acids plus norvaline, norleucine,and cyclohexylglycine) and 74 combinations of two amino acids from the22 amino acids. To ensure that a signal from each of the doublet aminoacids is detectable, preferably in ‘‘equipeak height’’ ratio, the ratio of thetwo encoding Boc-amino acids has to be adjusted according to their rela-tive reactivities (RR) (see Table II).10

In the encoding strategy, the reduction of the NO2 group, the removalof protecting groups Fmoc and Dde of NH2 (using 2% hydrazine in DMF),and the acylation of both coding and testing arms are achieved simultan-eously. This greatly reduces the number of synthetic steps. The experiment

TABLE II

Relative Reactivities (RR) of 22 Boc-amino Acidsa

Amino Acids RR Amino Acids RR Amino Acids RR

Boc-Asp(OtBu)-OH 8.38 Boc-Nva-OH 5.18 Boc-Arg(Pmc)-OH 1.73

Boc-Asn(Trt)-OH 6.40 Boc-Trp(Boc)-OH 3.08 Boc-Tyr(tBu)-OH 5.19

Boc-Ser(tBu)-OH 1.47 Boc-Phe-OH 5.44 Boc-Pro-OH 3.98

Boc-Gln(Trt)-OH 2.44 Boc-Ile-OH 1.28 Boc-Lys(Boc)-OH 3.00

Boc-Thr(tBu)-OH 2.29 Boc-Glu(OtBu)-OH 7.28 Boc-Leu-OH 4.09

Boc-Gly-OH 3.50 Boc-His(Trt)-OH 1.00 Boc-Nle-OH 4.98

Boc-Met-OH 4.77 Boc-Ala-OH 6.08 Boc-Chg-OH 2.56

Boc-Val-OH 2.98

a Natural amino acids are designated by the standard three-letter code. Nva, norvaline;

Nle, norleucine; Chg, cyclohexylglycine; tBu, tert-butyl; Trt, trityl; Pmc, 2,2,5,7,8-

pentamethylchroman-6-sulfonyl.


detail of the synthesis of an encoded model peptidomimetic library is asfollows.

Topologically segregated bifunctional TentaGel resin (2.0 g, 0.52 mmol)(30% substitution of the beads for the coding tag) as prepared above is usedfor the library synthesis. The coding-precursor strand is first built in the in-terior of the bead: a solution of Boc-Lys(Dde) (250 mg, 0.468 mmol), HOBt(71.6 mg, 0.468 mmol), and DIC (73.2 �l, 0.468 mmol) in DMF (20 ml) isadded to the beads. After coupling at room temperature for 2 h, the ninhy-drin test is negative, i.e., the coupling reaction is finished. The beads are thenwashed with DMF, MeOH, and DCM, three times each. Then 50% TFA inDCM (20 ml) is added to the beads. After 30 min, the beads are washed withDCM, neutralized with 2% DIEA in DMF twice, and washed with DMFthree times. A solution of Boc-Phe(4-NO2) (145.2 mg, 0.468 mmol) andHOBt (71.6 mg, 0.468 mmol) in DMF (20 ml) is added to the beadsfollowed by addition of DIC (73.2 �l, 0.468 mmol). The amino acid couplingand the subsequent Boc group deprotection are achieved in the samemanner as above. The peptide-resin is split into 96 equal portions in a 96-well plate. Ninety-six different ‘‘coding molecules’’ (22 single Boc-aminoacids and 74 combinations of two Boc-amino acids, one in each well, 3equiv.) are dissolved in a solution of HOBt (3 equiv.) and DIC (3 equiv.)in DMF, and are added into 96 wells (each well receives only one aminoacid). The coupling is carried out at room temperature for 2 h. After filtra-tion, the beads are washed with DMF, MeOH, and DMF, respectively, threetimes each.

Twenty percent piperidine in DMF (20 ml, twice, 5 min, 15 min) is usedto remove the Fmoc group in a 96-well plate. The beads are washed withDMF, MeOH, and DMF, respectively, three times each. Ninety-sixFmoc-amino acids (0.0114 mmol) are dissolved in 96 equal volumes of a so-lution of HOBt (total 167.2 mg HOBt, 1.092 mmol) and DIC (total 171 �l,1.092 mmol) in DMF (57.6 ml). The solutions are added to the correspond-ing wells (i.e., one amino acid in each well). The coupling reaction proceedsat room temperature for 2 h. After filtration, all resin beads in 96 wells arecombined, mixed, and washed with DMF, MeOH, and DMF, respectively,three times each. The ‘‘Fmoc’’ deprotection is achieved with 20% piperi-dine in the same manner. A solution of Fmoc-�-Phe(4-NO2) (472 mg,1.092 mmol), HOBt (167.2 mg, 1.092 mmol), and DIC (171 �l,1.092 mmol) in DMF is added to the resin. The coupling is conducted atroom temperature for 2 h. The beads are washed with DMF, MeOH, andDMF, respectively. NO2 groups in both coding and testing arms are re-duced with 2 M SnCl2�2H2O in DMF (30 ml) at room temperature for2 h, twice. The resin is split into 33 equal portions and transferred to 33 dis-posable polypropylene columns with a polyethylene frit. Acylation of the


amino group in aniline is as follows: 27 individual carboxylic acids (20equiv. each) in a solution of DIC and DIEA in DCM are added to thebeads of 27 columns (each column receives one carboxylic acid). Three acylchlorides and three sulfonyl chlorides in a solution of DIEA in DCM areadded to the beads of six columns separately. The coupling reaction iscarried out at room temperature overnight. After filtration, all 33 portionsof beads are combined and randomized, and then washed with DMF,MeOH, and DMF again. Two percent hydrazine in DMF (30 ml) is addedto the beads. The reaction proceeds at room temperature for 5 min, andrepeats for another 10 min. Fmoc in the testing arm and Dde in the codingarm are removed simultaneously. The resin is then distributed into 50 por-tions to 50 columns, and 48 different carboxylic acids (20 equiv. each) arepreactivated by BOP in DMF/DIEA and added to the correspondingcolumns. Two acid anhydrides, i.e., diglycolic anhydride and glutaric an-hydride, are dissolved in a solution of DIEA in DMF (0.5 ml) and addedto two separate columns. The acylation is conducted at room temperaturefor 2 h and repeated once. After filtration, all 50 portions of resin are com-bined and washed with DMF, methanol, and DCM. The beads are thendried under vacuum for 1 h. Side chain deprotection is carried out with re-agent K (40 ml) at room temperature for 2 h. After neutralization with 2%DIEA/DMF twice, the resin is washed sequentially with DMF, MeOH,DCM, DMF, DMF/water, water, and finally PBS. The library is now readyfor screening.

Fmoc-amino acids used as building blocks of testing compounds are asfollows: Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-His(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH,Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-d-Ala-OH,Fmoc-d-Arg(Pmc)-OH, Fmoc-d-Trp(Boc)-OH, Fmoc-d-Cys(Trt)-OH,Fmoc-d-Asp(OtBu)-OH, Fmoc-d-Glu(OtBu)-OH, Fmoc-d-His(Trt)-OH,Fmoc-d-Gln(Trt)-OH, Fmoc-d-Leu-OH, Fmoc-d-Met-OH, Fmoc-d-Pro-OH, Fmoc-d-Ser(tBu)-OH, Fmoc-d-Lys(Boc)-OH, Fmoc-d-Tyr(tBu)-OH, Fmoc-d-Thr(tBu)-OH, Fmoc-d-Phe-OH, Fmoc-d-Asn(Trt)-OH,Fmoc-3-(4-pyridyl)alanine, Fmoc-d-3-(3-pyridyl)alanine, Fmoc-4-tert-butoxyproline, Fmoc-3-chlorophenylalanine, Fmoc-norleucine, Fmoc-2-cyclohexylglycine, Fmoc-2-aminoisobutyric acid, Fmoc-tranexamic acid,Fmoc-(R,S)-3-amino-3-(2-furyl)propionic acid, Fmoc-(R,S)-(6,7-di-methoxy)-1,2,3,4-tetrahydroquinoline-3-carboxylic acid, Fmoc-(R,S)-3-amino-3-(4-hydroxyphenyl)propionic acid, Fmoc-(R,S)-3-aminovalericacid, Fmoc-(R,S)-3-amino-3-(3,4-dichlorophenyl)propionic acid, Fmoc-isonipecotic acid, Fmoc-(R,S)-3-amino-3-(3,4-methylenedioxyphenyl)


propionic acid, Fmoc-(R,S)-3-amino-3-(4-tert-butylphenyl)propionic acid,Fmoc-d-3-(2-thienyl)-l-alanine, Fmoc-(R,S)-3-amino-3-(4-methoxyphe-nyl)propionic acid, Fmoc-3,5-diiodotyrosine, Fmoc-(R,S)-3-amino-3-(4-N,N-dimethylaminophenyl)propionic acid, Fmoc-cis-3-amino-2-cyclohex-ane carboxylic acid, Fmoc-(R,S)-3-amino-3-(4-bromophenyl)propionicacid, Fmoc-(R,S)-3-amino-3-(2-thienyl)propionic acid, Fmoc-(R,S)-3-amino-3-(1-naphthyl)propionic acid, Fmoc-(R,S)-3-amino-3-(3-pyridyl)-propionic acid, Fmoc-trans-3-amino-2-cyclohexane carboxylic acid, 4-N-Fmoc-amino-4-carboxytetrahydropyran, 2-N-Fmoc-amino-3-(4-N-Boc-pi-peridinyl)propionic acid, 4-N-Fmoc-amino-4-carboxy-1,1-dioxo-tetrahy-drothiopyran, N-Fmoc-(R,S)-amino-(3-N-Boc-piperidinyl)carboxylic acid,N-Fmoc-amino-(4-tert-butoxycyclohexyl)carboxylic acid, N-Fmoc-(R,S)-amino-2-naphthylacetic acid, N-Fmoc-amino-(4-N-Boc-piperidinyl)car-boxylic acid, N-Fmoc-amino-4-ketocyclohexyl carboxylic acid, Fmoc-(2S,5R)-5-phenylpyrrolidine-2-carboxylic acid, Fmoc-4-(3-carboxymethyl-2-keto-one-benzimidazolyl)piperidine, Fmoc-cis-3-amino-1-cyclohexanecarboxylic acid, Fmoc-4-phenylpiperidine-4-carboxylic acid, Fmoc-d-thia-zolidine-4-carboxylic acid, Fmoc-(R)-(þ)-2-piperidinecarboxylic acid,Fmoc-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid, Fmoc-3-Boc-2, 3-dia-minopropionic acid, Fmoc-3-carboxymethyl-1-phenyl-1,3,8-triazaspir-o[4,5]decan-4-one, Fmoc-2-aminoindane-2-carboxylic acid, Fmoc-3-benzothienylalanine, Fmoc-3,3-diphenylalanine, Fmoc-2-amino-5-ureido-valeric acid, Fmoc-d-3,4-dichlorophenylalanine, Fmoc-(3-aminomethyl)-benzoic acid, Fmoc-2-amino-4-tert-butoxybutanoic acid, Fmoc-O-benzyl-hydroxyproline, Fmoc-O-methyltyrosine, Fmoc-3,5-dibromotyrosine,Fmoc-4-tert-butyloxycarbonylaminophenylalanine, Fmoc-4-benzoylpheny-lalanine, Fmoc-d-tetrahydroisoquinoline-3-COOH, Fmoc-thiazolidine-4-carboxylic acid, Fmoc-d-propargylglycine, Fmoc-3-(2-naphthyl)alanine,Fmoc-d-3-(2-naphthyl)alanine, Fmoc-3-aminobenzoic acid, (2S,4S) Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic acid. (d stands for d configuration.)

Carboxylic acids and anhydrides used for the derivatization of Lys areas follows: diglycolic anhydride, 2,5-thiophenedicarboxylic acid, (S)-(�)-2-pyrrolidone-5-carboxylic acid, glutaric anhydride, malonic acid, trans-4-co-tininecarboxylic acid, levulinic acid, propionic acid, 3,5-dihydroxybenzoicacid, Boc-1-aminocyclopropane-1-carboxlic acid, 2-pyrazinecarboxylicacid, 4-acetamidobenzoic acid, 3-pyridinepropionic acid, hydroxyphenyla-cetic acid, trans-3-(3-pyridyl)acrylic acid, 3-hydroxy-2-quinoxaline carbox-ylic acid, isobutyric acid, butyric acid, 4-bromo-3,5-dihydroxybenzoicacid, 3-oxo-1-indancarboxylic acid, 3-thiophenecarboxylic acid, 2-thiophe-necarboxylic acid, (S)-(þ)-oxo-4-phenyl-3-oxazolidineacetic acid, 2,6-dichloronicotic acid, 3-nitrophenylacetic acid, 4-(dimethylamino) phenyla-cetic acid, 4-(dimethylamino)benzoic acid, 2,5-dimethoxybenzoic acid,


phenoxyacetic acid, 3-(dimethylamino)benzoic acid, 3-benzoyl-2-pyridinecarboxylic acid, hexanoic acid, 3,4-difluorobenzoic acid, indole-2-carbox-ylic acid, phenylpropionic acid, 4-chlorophenylacetic acid, 4-bromobenzoicacid, bromophenylacetic acid, 3,5-dimethylbenzoic acid, 1-naphthylaceticacid, 2-biphenylcarboxylic acid, 2-phenoxybutyric acid, 2,4-dichloropheny-lacetic acid, 3,4-dichlorophenylacetic acid, (2-naphthoxy)acetic acid, (1-naphthoxy)acetic acid, 4-biphenylcarboxylic acid, 4-phenoxybenzoic acid,2-phenyl-4-quinoline carboxylic acid, 5-(4-chlorophenyl)-2-furoic acid.

Carboxylic acids, acyl chlorides, and sulfonyl chlorides used for deri-vatization of 4-aminophenylalanine and �-4-aminophenylalanine are asfollows: 5-hydantoinacetic acid, trans-4-cotininecarboxylic acid, isonicotinicacid, 3-pyridinepropionic acid, 4-hydroxyphenylacetic acid, 2-butynoic acid,2-pyrazinecarboxylic acid, cyclopropanecarboxylic acid, 3-hydroxy-2-qui-noxaline carboxylic acid, 5-bromovaleric acid, propargyl chloroformate,3,4-dimethoxybenzoyl chloride, 2-thiophenesulfonyl chloride, 3-thiophene-carboxylic acid, 2-thiophenecarboxylic acid, 2-methylbutyric acid, 2-thio-pheneacetyl chloride, benzoic acid, furylacrylic acid, 4-nitrophenyl aceticacid, 2,5-dimethoxyphenylacetic acid, p-toluenesulfonyl chloride, 4-(di-methylamino)phenylacetic acid, 3-indolepropionic acid, phenoxyaceticacid, 3-(dimethylamino)benzoic acid, cyclohexanecarboxylic acid, naphtha-lenesulfonyl chloride, 4-bromophenylacetic acid, 4-bromobenzoic acid, 2-phenoxybutyric acid, 3,4-dichlorophenylacetic acid, (1-naphthoxy)aceticacid.

Screening of an Encoded Peptidomimetic Library withStreptavidin-Alkaline Phosphatase Conjugate

An enzyme-linked colorimetric assay14,15 is used to screen for ligandsthat bind to streptavidin (also see Chapter 17 in this volume). To find thestrongest ligands, an extremely dilute streptavidin-alkaline phosphataseconjugate concentration (1:100,000) is used. Library beads (0.1 ml) aretransferred into a disposable polypropylene column with a polyethylenefrit. The beads are washed with phosphate-buffered saline (PBS) (10 �4 ml), and then with 0.1% Tween/0.1% gelatin/0.05% sodium azide/PBS(PBSTG-azide, 3� 4 ml). The bead-supported library is filtered andblocked with PBSTG-azide (4 ml) at room temperature for 1 h. The beadsare filtered and washed with 0.1% Tween-20/0.1% gelatin/0.05% sodiumazide/PBS (3 � 4 ml). Streptavidin-alkaline phosphatase conjugate

14 G. Liu and K. S. Lam, in ‘‘Combinatorial Chemistry, A Practical Approach Series’’

(H. Fenniri, ed.), p. 33. Oxford University Press, New York, 2000.15 K. S. Lam and M. Lebl, ImmunoMethods 1, 11 (1992).

TABLE III

Peptidomimetic Ligands for Streptavidin

Aa2

R1

NH

O

HN

NH

O

O

R2

Entry Aa2 Structure R1COOH Structure R2COOH Structure

01 d-Arg H2N COOH

NH

NH2

NH

Isonicotinic acidN

COOH

3-Thiophene carboxylic

acidS

COOH

02 d-Arg

H2N COOH

NH

NH2

NH

Isonicotinic acidN

COOH2-Thiophene carboxylic acid

S COOH

03 CkbpaHN N N

O

COOH Isonicotinic acidN

COOH3-Thiophene carboxylic acid

S

COOH

04 d-Arg

H2N COOH

NH

NH2

NH

Isonicotinic acidN

COOH3-Benzoyl-2-pyridine carboxylic

acid N

O

COOH

05 d-ThiS

H2N COOH

Isonicotinic acidN

COOH4-(Dimethylamino)phenylacetic

acidN

COOH

(continues)

[15]

en

co

de

dpe

ptid

om

im

etic

an

dsm

al

lm

ol

ec

ul

el

ib

ra

rie

s2

85

06 CptdbN

N

OHN COOH Isonicotinic acid

N


acidN

COOH

07 Cptd NN

OHN

COOH Isonicotinic acidN


acidN

COOH

08 Cptd NN

OHN

COOH 2-Pyrazine carboxylic acidN

N

COOH4-(Dimethylamino)phenylacetic acid N

COOH

09 �-Phe(4-tBu) COOHNH2

2-Pyrazine carboxylic acidN

N

COOH(S)-(+)-2-Oxo-4-phenyl-3-

oxazolidineacetic acid

N

O O

COOH

10 �-Phe(4-tBu) COOHNH2

2-Pyrazine carboxylic acidN

N

COOH(S)-(+)-2-Oxo-4-phenyl-3-

oxazolidineacetic acid

N

O O

COOH

a Ckbp, 4-(3-carboxymethyl-2-keto-one-benzimidazolyl)-piperidine.b Cptd, 3-Carboxymethyl-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one.

TABLE III (Continued)

Entry Aa2 Structure R1COOH Structure R2COOH Structure

28

6pe

ptid

esy

nth

esis

an

dsc

re

en

in

g[1

5]


solution is added at a dilution of 1:100,000 (original concentration is 1 mg/ml) in 0.1% Tween-20/0.1% gelatin/0.05% sodium azide/PBS (4.0 ml). Thebead library is incubated at room temperature for 1 h. The beads arefiltered and washed with 0.1% Tween-20/PBS (3 � 4 ml), followed bywashing with Tris buffer saline (TBS, 4 ml). The beads are placed intotwo polystyrene Petri dishes (15 cm diameter). Each dish then receivesthe alkaline phosphatase substrate 5-bromo-4-chloro-3-indoyl phosphatep-toluidine salt (BCIP) in BCIP buffer (0.5 ml) to develop color at roomtemperature for 1 h. The enzymatic reaction is stopped with 1.0 M HCl(1.0 ml). The dark turquoise colored positive beads are retrieved andtreated with 6.0 M guanidine–HCl pH 1.0 solution to strip the protein offthe beads. The positive beads are then washed with water and sequenced.

The following chemical composition of buffers was used. PBS: 8.0 mMNa2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4; TBS:2.5 mM Tris–HCl, 13.7 mM NaCl, 0.27 mM KCl, pH 8.0; BCIP substratein BCIP buffer: 1.65 mg BCIP in 10 ml of 0.1 M Tris–HCl, 0.1 M NaCl,and 2.34 mM MgCl2, pH 8.8.

Ten dark beads have been picked up and sequenced. The result isshown in Table III. Ligands with high consensus have been identified. Allthe ligands have an aromatic N-heterocyclic structure (R1) attached to theamino group of aniline. Although there is some variation in the first build-ing units (Aa2) and the last building blocks (R2), they can be cataloged intoseveral different groups, respectively. Some ligands even have an identicalstructure, e.g., compounds 6 and 7 and compounds 9 and 10. The screeningresult is very similar to our previous data with the library using Phe(4-NH2)as a scaffold.6 It validates the fact that the encoding system for the OBOCpeptidomimetic and small molecule libraries is rapid, direct, and reliable.

Very recently, we reported on a novel chemical encoding method thatuses separate coding tags with a cleavable linker in the bead interior. Thesecoding tags can be released for decoding with mass spectrometry.16

Acknowledgments

The author would like to thank Ms. Amanda M. Enstrom for editorial support. This work

was supported by NIH R33CA86364. Ruiwu Liu is supported in part by the University of

California Systemwide Biotechnology Research Program, Grant 2001-07.

16 A. Song, J. Zhang, C. B. Lebrilla, K. S. Lam, J. Am. Chem. Soc. 125, 6180 (2003).


[16] Intelligent Design in Combinatorial Chemistry:Use of Designed Peptide Libraries to Explore Secondary

and Tertiary Structures in Peptides and Proteins

By Scott M. Cowell, Xuyuan Gu, Josef Vagner, andVictor J. Hruby

Introduction

It is hard to imagine all the possible roles of combinatorial chemistry inmodern science. Combinatorial chemistry became a major tool in medi-cinal chemistry by solving two major problems simultaneously. A needexisted to dramatically increase the number of new chemical entities and,at the same time, reduce the assay time of these entities to find novelligands. Because of its ability to very quickly (a few days) synthesize mil-lions of compounds that can be tested for possible lead compounds in drugdiscovery, combinatorial chemistry has become a critical component ofdrug design in modern pharmaceutical science. It subsequently spurredresearch to reduce assay times to screen these millions of compounds.Combinatorial chemistry could be seen as the ‘‘engineering’’ of the scienceof discovery of novel ligands. The future of combinatorial science seemsendless as other areas of science adapt the methodology.

Although it has developed rapidly in other areas of chemistry, theorigins of combinatorial chemistry were in the area of peptide design.While the methodology has embraced many different facets of synthetic or-ganic chemistry, the role of combinatorial chemistry in peptide science isfar from over, mainly because it still is one of the few areas of organicchemistry in which true combinatorial science can be pursued. Peptide syn-thesis in combinatorial chemistry is a viable field in finding lead compoundsand in the evaluation of other aspects of protein research. These cannot befully duplicated by most other areas of synthetic organic chemistry, be-cause of the very high fidelity of organic peptide synthesis, and becauseof the central significance of the proteins and peptides in living systems.1

Peptide libraries can be used to better understand everything from proteinfolding and evaluation of secondary structures of proteins, to the abilityto fully examine which key amino acid residues are needed to impartbiological functions of these molecules.

1 V. J. Hruby, J.-M. Ahn, and S. Liao, Curr. Opin. Chem. Bio. 1, 114 (1997).



[16] intelligent design in combinatorial chemistry 289

With today’s technology, one can theoretically prepare virtually limit-less numbers of peptide compounds. While this can be accomplished usingautomated methods, it still requires intelligent design by the scientist. Intel-ligent design formulates specific questions to be answered without the needto synthesize libraries with millions of different compounds. Intelligentdesign also endeavors to evaluate the library for a particular questionquickly. This chapter will discuss the evolution of intelligent design in com-binatorial peptide synthesis and its use in the evaluation of secondary andtertiary structures in proteins.

Background

The roots of combinatorial chemistry are in Merrifield’s2 work on solid-phase peptide synthesis, a technique that has revolutionized peptide chem-istry. However, the limiting factor of traditional solid-phase peptidesynthesis is the linearity of the method. Perhaps the first example of a pep-tide library (not combinatorial) was accomplished by Geysen et al.3 Thisinvolved the synthesis of a library of peptides on pins to identify an im-munologically important coat protein of foot-and-mouth disease virus.The study centered on a particular 7-mer sequence of the coat protein,and scanning the amino acids on this peptide to determine the significanceof each amino acid. Similar peptide libraries have been produced usingphage display.4,5 Another very early example of a solid-phase peptide li-brary is the early studies by Houghten,6 who made peptides using ‘‘tea-bags’’ to synthesize 248 different 13-mer peptides in less than 4 weeks,and evaluated their binding using a particular antibody–antigen reaction.

A significant advance in the field came with the design of a solid-phasepeptide synthesis for the evaluation of ligand-binding activity using a truecombinatorial approach.7 Here literally millions of individual peptideswere produced using standard peptide synthesis on ‘‘resin’’ beads, whichwere then evaluated directly using a marker attached to a monoclonal anti-body against �-endorphin. This method succeeded in pinpointing singlebeads that contained the proper sequence to bind to the antibody. Theparticular bead was then isolated and microsequenced to find the leadpeptide for further evaluation. The concept of ‘‘one bead-one peptide’’

2 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).3 H. M. Geysen, R. H. Meloen, and S. J. Barteling, Proc. Natl. Acad. Sci. USA 81, 3998 (1984).4 T. Clackson, H. R. Hoogenboom, A. D. Griffiths, and G. Winter, Nature 352, 624 (1991).5 J. K. Scott and G. P. Smith, Science 249, 386 (1990).6 R. A. Houghten, Proc. Nat. Acad. Sci. USA 82, 5131 (1985).7 K. S. Lam. S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knap,

Nature 354, 82 (1991).


was coined.8 A very large library (tens of millions or more) could be pro-duced in much less time than previously reported. One of the advantagesof this process is that a variety of unnatural amino acids could now be in-corporated into the libraries. For example, an all d-amino acid-containingligand was discovered for the �-endorphin receptor.8 This could not be ac-complished using phage display libraries. Furthermore, since the synthesisof the peptides was direct and did not contain the natural bias of phagelibraries, one had direct control of the combinatorial conditions in whichthe libraries were synthesized. More recently, a variation of this ‘‘onebead-one peptide’’ method was used to determine the molecular recogni-tion of a synthetic receptor to a peptide library designed to be similar toa vancomycin-resistant peptide.9

A question of how to properly screen peptide mixtures that are releasedfrom solid support was being asked. This involved the idea that the fast re-lease of all peptides into a mixture might cause interference with the test.For example, in screening for novel ligands for G-protein-coupled recep-tors, done by releasing literally thousands of peptides into solution, thereis a chance that multiple peptides will interact with the receptor. Some ofthese peptides might act as agonists while others act as antagonists. Thiscan cause problems in finding which peptides are responsible for an ob-served receptor interaction. One group designed a system that allows forthe slow release of peptides utilizing gaseous trifluoroacetic acid (TFA)to the TFA-resistant linker methylbenzhydrylamine (MBHA).10 This con-trolled release approach allows for the screening of one library using thesame bioassay multiple times and, under the proper circumstances, allowsfor the isolation of the one bead that is responsible for an agonist responsethat was coupled to a color change.

At the same time that combinatorial libraries were examined on solidsupport, scientists were able to evaluate combinatorial libraries in solutionwith success.11–14 It was suggested that the attached peptides might give

8 K. S. Lam, M. Lebl, V. Krchnak, S. Wade, F. Abdul-Latif, R. Ferguson, C. Cuzzocrea, and

K. Wertman, Gene 137, 13 (1993).9 R. Xu, G. Greiveldinger, L E. Marenus, A. Cooper, and J. A. Ellman, J. Am. Chem. Soc.

121, 4898 (1999).10 C. K. Jayawickreme, G. F. Graminski, J. M. Quillan, and M. R. Lerner, Proc. Natl. Acad.

Sci. USA 91, 1614 (1994).11 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo,

Nature 354, 84 (1991).12 H. Han, M. M. Wolfe, S. Brenner, and K. D. Janda, Proc. Natl. Acad. Sci. USA 92, 6419 (1995).13 R. N. Zuckermann, J. M. Kerr, M. A. Siani, S. C. Banville, and D. V. Santi, Proc. Natl.

Acad. Sci. USA 89, 4505 (1992).14 C. Pinilla, J. R. Appel, and R. A. Houghton, Gene 128, 71 (1993).


different data as compared with peptides in solution. However, similaritybetween the binding of ligands on solid supports and in solution are ob-served. For example, a � opioid receptor antagonist with nanomolar bind-ing was found by screening a library of 52,128,400 hexapeptides.15 This wasfollowed up in the same group by doing positional scanning on each residueof the hexapeptide16 as well as a tetrapeptide17 library to further examinethe effects of individual amino acids.

Positional scanning with phage libraries were used to discover theamino acid residues in peptides responsible for binding to the calcium-binding protein calmodulin.18 In this case, the binding polypeptide neededTrp as a key residue located in the fourteenth position from the N-terminusfor strong binding.

Positional scanning on peptides is one of the strengths of combinatorialchemistry and it was used successfully to find antithrombic activity in atetrapeptide series.19 Another form of positional scanning was used to de-termine the optimal hexapeptide for prostate-specific antigen. This was ac-complished by varying residues 1 through 4. This has led to novelcompounds that are being further defined.20 An additional example of pos-itional scanning was the isolation of pentapeptides responsible for erythro-mycin resistance in Escherichia coli.21 In this case, the pentapeptidesinterfere with the binding of the erythromycin to the ribosome. This inter-action was also studied using a pentapeptide library in vivo to determinethe properties of the pentapeptide that cause resistivity to occur. It wasshown that hydrophobic residues on the peptide in the 1, 3, and 5 positionswere responsible for the major resistance of the drugs.

It should be noted that it is not always libraries with large peptides thatgive lead compounds. In one case, a dipeptide library used for conforma-tional studies led to peptoid ligands. In this case, the conformation of thedipeptides held the key to binding activity. Therefore, it is the conform-ation of the peptides that will give lead compounds to peptidomimetics.A good antagonist for the human tachykinin NK3 was found using thisapproach.22

15 C. T. Dooley, N. N. Chung, P. W. Schiller, and R. A. Houghten, Proc. Natl. Acad. Sci. USA

90, 10811 (1993).16 C. T. Dooley and R. A. Houghten, Life Sci. 52, 1509 (1993).17 C. T. Dooley, P. Ny, J. M. Bidlack, and R. A. Houghten, J. Biol. Chem. 273, 18848 (1998).18 B. K. Kay and N. B. Adey, Gene 169, 133 (1996).19 B. Kundu, M. Bauser, J. Betschinger, W. Kraas, and G. Jung, Bioorg. Med. Chem. Lett. 8,

1669 (1998).20 C. F. Yang, E. S. Porter, J. Boths, D. Kanyi, M. Hsieh, and B. S. Cooperman, J. Peptide Res.

54, 444 (1999).21 T. Tenson, L. Xiong, P. Kloss, and A. S. Mankin, J. Biol. Chem. 272, 17425 (1997).


Exploration of Antimicrobial Peptides Using Combinatorial Chemistry

Another use for combinatorial libraries has been the screening of pep-tides for antimicrobrial properties. In this case, the design of the library isbased on antimicrobial peptides found in nature. A combinatorial synthesisis used to find alternative unnatural amino acids expected to mimic theantimicrobial properties.23 Peptide libraries also have been used to findcompounds that could bind the lytic peptide mellitin.24 The library was syn-thesized in solution phase, purified, and evaluated using time-of-flight massspectrometry (TOF-MS). The sequences determined to bind to mellitincontained hydrophobic pairs. By binding to mellitin, they were able to pre-vent the cell-surface mellitin interaction. This is an example of a peptide li-brary able to afford compounds that interact with other small peptideswithout having to find an interacting protein first.

The exploration of antimicrobial peptides was taken a step furtherwhen the major consideration of the target molecule is the secondary struc-ture it can adopt. Blondelle et al. designed a library in which the goal was toincrease the amphipathic characteristic of the peptide. However, it soonbecame clear from early results that the presence of the helix-breakingPro in the molecule causes a large increase in the lytic properties of themolecule.25 This demonstrates the power of libraries for determining path-ways that otherwise would not have been explored. Moreover, the second-ary structures of the amphipathic peptides have been studied using peptidelibraries in which it was observed that the smaller the amphipathicity, thegreater the antimicrobial activity.26 The use of these libraries for the studyof secondary structures in the peptide has been of great value to derivebetter antimicrobial drugs. The assay method relies on the deconvolutionof the mixture of peptides in order to elucidate the structure of biologicallyactive compounds.

Amphipathic peptides have been used as a starting point for the intelli-gent design of tertiary structures of proteins.27 In this case, the residuesresponsible for the amphipathicity were held constant while the restwere varied to try to overcome the limitations necessary to stabilize the

22 P. Boden, J. M. Eden, J. Hodgson, D. C. Horwell, J. Hughes, A. T. McKnight, R. A.

Lewthwaite, M. C. Pritchard, J. Raphy, K. Meecham, G. S. Ratcliffe, N. Suman-Chauhan,

and G. N. Woodruff, J. Med. Chem. 39, 1664 (1996).23 S. E. Blondelle, E. Takhashi, P. A. Weber, and R. A. Houghten, Antimicrob. Agents

Chemother. 38, 2280 (1994).24 S. E. Blondelle, R. A. Houghten, and E. Perez-Paya, J. Biol. Chem. 271, 4093 (1996).25 S. E. Blondelle, E. Takahashi, R. A. Houghten, and E. Perez-Paya, Biochem. J. 313,

141 (1996).26 S. E. Blondelle and K. Lohner, Biopolymers 55, 74 (2000).27 E. Perez-Paya, R. A. Houghton, and S. E. Blondelle, J. Biol. Chem. 271, 4120 (1996).


formation of the tertiary structures. These factors included van der Waalsinteractions, electrostatic interactions, etc. This study determined the opti-mal amphipathic peptides for aggregation to occur and proved that simpleprotein design can be achieved using combinatorial peptide synthesis.

Design Strategies in Combinatorial Libraries of Peptides

A paper published in the 1990s represents a breakthrough in the designof peptide libraries. Sepetov et al.28 approached the design of the libraryfrom the standpoint of the functionality of the final peptide. The premisefrom which they started was that not all residues in a peptide are neededfor biological activity. These ‘‘noncritical’’ residues are needed merely scaf-folding and contribute to the proper orientation for the other residuesneeded for interaction. Therefore, the proper design of peptide librariesshould be the identification and isolation of critical residues with the focusof design being only to modify these residues necessary for optimal inter-actions (the ‘‘pharmacophores’’ elements). With this approach, the size ofthe library could be greatly reduced. Pharmacophores are identified usuallythrough the literature for a particular ligand. The pharmacophore moietiesare strategically placed at certain positions in the peptide chain varying the‘‘scaffolding’’ residues. The pharmacophores are then varied in location inthe peptide chain iteratively. One can view this as a modified alanine scanwhere certain residues are held constant and other residues are varied. Thisstrategy greatly streamlines the process of building libraries with the know-ledge as to which residues are key to desired interactions with target recep-tors. An example of this methodology is reported by de Koster et al.29 Thisresearch group used a 15-mer template generated from a phage library as astarting point for further optimization of the peptide. Once they deter-mined that two of the residues were inconsequential to the antibody inter-actions, they generated a limited 12-residue library via amino acid scanningto optimize antibody interactions. This study led to the identification ofnovel compounds with high binding affinity for which it is speculated thata second-generation library could provide greater success with a smaller li-brary. Combinatorial libraries have been used to find the binding affinitiesto a transporter associated with antigen processing (TAP).30 Uebel et al.30

28 N. F. Sepetov, V. Krchnak, M. Stankova, S. Wade, K. S. Lam, and M. Lebl, Proc. Natl.

Acad. Sci. USA 92, 5436 (1995).29 H. S. de Koster, R. Amons, W. E. Benckhuijsen, M. Feijlbrief, G. A. Schellekens, and J. W.

Drijfhout, J. Immunol. Methods 187, 179 (1995).30 S. Uebel, W. Kraas, S. Kienle, K. H. Wiesmuller, G. Jung, and R. Tampe, Proc. Natl. Acad.

Sci. USA 94, 8976 (1997).


found that the residues at the termini of the peptides are key for bindingto TAP, while the other portions were needed only as a backbone for themolecule.

A variation of the pharmacophore library was seen in an approach usedto determine CD4þ T cell epitopes.31 In a 14-mer peptide, the researcherskeep residues in positions 1, 4, and 6 constant since they functioned as‘‘anchor positions’’ for binding receptor, and proceeded to vary the otherresidues in the peptide. This allowed the use of a shorter synthetic routecompared to one that would be needed if all positions were to be varied.Limiting the size of the library allowed the authors to obtain better assays.By doing a partial release of the peptides, the authors were able to find thefinal peptide that represented the epitope of the T cell receptor. In anotherexample, the epitope to inhibit stimulation of the thyrotropin receptor alsowas found via combinatorial libraries.32 Since the synthesis of a totallyrandom hexapeptide library was deemed impractical, the authors optedto hold one position constant while the other five residues were random-ized. This method was repeated for each residue in the peptide. The resi-dues that were determined to be the most active were used as a basis fora second-generation library. The only limitation of the library was notthe quantity of product synthesized, but to properly pinpoint the peptidesin an assay.

Another method for designing libraries is to formulate target moleculesbased on physicochemical properties such as the ability to cross the blood–brain barrier. Based on literature or other sources, data are obtained thatshow certain residues or peptide sequences could be developed to studyand predict a particular task in question. With this information, an algo-rithm, commonly known as a ‘‘filter,’’ is created to screen a virtual library.Using quantitative structure–activity relationships (QSAR), the computeris essentially predicting which moiety in the virtual library may lead to anovel ligand. The practicality of this method lies in the time savings for li-brary design, thus decreasing the size of the library to a more manageablenumber.33

In a similar method, computer models can also be developed to screenvirtual libraries based on the binding pockets of receptors.34 In this case,the computational models directly probe the binding sites in receptors

31 H. S. Hiemstra, G. Duinkerken, W. E. Benckhuijesen, R. Amons, R. R. P. de Vries, B. O.

Roep, and J. W. Drijfhout, Proc. Natl. Acad. Sci. USA 94, 10313 (1997).32 J. Y. Park, I. J. Kim, M. H. Lee, J. K. Seo, P. G. Suh, B. Y. Cho, S. H. Ryu, and C. B. Chae,

Endocrinology 138, 617 (1997).33 Ajay, G. W. Bemis, and M. A. Murcko, J. Med. Chem. 42, 4942 (1999).34 C. M. Murray and S. J. Cato, J. Chem. Inf. Comput. Sci. 39, 46 (1999).


and screening libraries to find peptides with a high probability of binding tothe pocket. This approach, now referred to as ‘‘combinatorial docking,’’gives researchers a more educated starting point to design compoundlibraries.35

Protein Design

Combinatorial libraries have been used to design a four-helix bundle.36

The libraries were designed to cover the packing of the ligand in the librar-ies around a heme group after being bound to a template attached to acellulose backbone. The library was varied to determine the affects ofdifferent core amino acids on the packing of the heme in the bundle. Thiswork demonstrates the use of peptide libraries to design tertiary structuressuch as a heme bundle.

How are stable proteins separated from those that are forming mutantglobular proteins? One research group was able to separate de novo pro-teins using a histidine tagging method on a phage-generated combinatoriallibrary and then using proteases to cut the proteins that were not packedin a hydrophobic core.37 The experiments were monitored using surfaceplasmon resonance, which demonstrated the presence of protein after theexposure to the protease.

The design of tertiary structures using combinatorial libraries led onegroup to propose that the evolution of tertiary structures occurs similarlyin vivo. Riechmann and Winter were able to show that comparable tertiarystructures could occur by combinatorial shuffling of polypeptide fragments.This group was able to elicit the formation of a folding similar to the�-barrel domain of a CspA fragment using a phage display library with apolypeptide not possessing the same sequence homology as the in vivoCspA molecule. It is possible that Nature might have used combinatorylibraries in evolution but this has not been proved at this time.38

Cyclization

Tertiary structures also have been explored by modifying the flexibilityof peptides in libraries. One of the first papers that introduced the head-to-tail cyclization of a library was by Spatola et al.39 The researchers relied on

35 H.-J. Bohm and M. Stahl, Curr. Opin. Chem. Biol. 2, 282 (2000).36 H. K. Rau, N. DeIong, and W. Haehnel, Angew. Chem. Int. Ed. Engl. 39, 250 (2000).37 M. D. Finucane, M. Tuna, J. H. Lees, and D. N. Woolfson, Biochemistry 38, 11604 (1999).38 L. Riechmann and G. Winter, Proc Natl. Acad. Sci. USA 97, 10068 (2000).39 A. F. Spatola, Y. Crozet, D. de Wit, and M. Yanagisawa, J. Med. Chem. 39, 3842 (1996).


a side chain attachment of the first amino acid residue to the resin. Al-though no novel hits were obtained for the antagonist in question, thepaper validated the method and acted as a proof of concept. Positionalscanning of a cyclic peptide was used to identify chymotrypsin inhibitors.40

The libraries were cyclized on the resin via oxidation of the cysteine resi-dues. Residues at positions 1, 2, and 4 were scanned. In other studies, amethod for ring closing metathesis of a portion of a growing peptide wasdeveloped.41 Though not attempted in an actual library, this method doesoffer some unique advantages in the design of tertiary structures in a li-brary. The cyclization of peptides on the resin has always been importantsince the decrease in the degrees of freedom of a peptide increases thechance to induce selectivity. Therefore, there is a better chance of havingselectivity of the molecules. One example of this relies on the cyclizationon resin using an Asp resin.42

One of the many examples of how a library can be used to explore therational for a particular binding is the use of a cyclic minilibrary where anNK-1 peptide was converted into a somatostatin receptor ligand.43 Whilethe cyclization did not occur on the resin, the authors used the library togive them a lead compound that they might not have found without theuse of the small library.

Hinges in Peptide Libraries

A drawback of combinatorial libraries is the large number of com-pounds needed to explore all structural possibilities as the length of peptidechains grows. Intelligent design is needed in order to decrease the size ofthe libraries and obtain relevant information. This concept was shown inthe incorporation of various structural elements to enhance turn param-eters in a library.44 Novel �-turns play an important role in nature, a factthat has prompted some investigators to build libraries to find novel�-turns.45 To potentially have greater ligand affinities and explore second-ary structures of peptides, a �-turn mimetic has been incorporated into

40 J. D. McBride, H. N. Freeman, and R. J. Letherbarrow, J. Peptide Sci. 6, 446 (2000).41 J. F. Reichwein, B. Wels, J. A. W. Kruijtzer, C. Versluis, and R. M. J. Liskamp, Angew.

Chem. Int. Ed. Engl. 38, 3684 (1999).42 A. F. Spatola, K. Darlak, and P. Romanovskis, Tetrahedron Lett. 37, 591 (1996).43 R. Hirshmann, W. Yao, M. A. Cascieri, C. D. Strader, L. Meaechler, M. A. Cichy-Knight,

J. Hynes, Jr., R. D. van Rihn, P. A. Sprengeler, and A. B. Smith III, J. Med. Chem. 39, 2441

(1996).44 G. J. Moore, Drug Dev. Res. 42, 157 (1997).45 A. J. Souers, A. A. Virgilio, A. Rosenquist, W. Fenuik, and J. A. Ellman, J. Am. Chem. Soc.

121, 1817 (1999).


peptide libraries. This has proven to be very effective in providing leads fornovel ligands.46 Becker et al.,46 coining the term ‘‘turn-scan,’’ discovered apossible inverse agonist for the � opioid receptor and an agonist for theorphan receptor ORL1 using this method. The use of the �-turn in librariesprovides an approach to determine the impact on conformation individualresidues have on ligands.

The incorporation of mimetic units into a peptide to change the proper-ties of the peptide also has been used to obtain better protease inhibitors.47

A cyclohexanone was incorporated in the middle of a tetramer in order togenerate strong binding interaction with chosen protease inhibitors. Byconformationally constraining the tetramer using cyclohexanone, theywere able to gain insight into the nature of the binding pocket of the prote-ases. �-Amino acids have been incorporated into peptidomimetics to facili-tate cyclization of the libraries.48 Similarly, peptidomimetics have beensynthesized using �-methylated amino acids. The difference between pep-tide and peptidomimetics is further blurred as different compounds aresubstituted in peptides in place of amino acid residues to influence thestructures of the resultant libraries.49

Conclusion

Although combinatorial chemistry has shown much promise for theevaluation of molecules, combinatorial chemistry is a tool, and successgreatly depends on the ingenuity and intuition of the scientists. Tradition-ally, insight into the natural world came slowly with each molecule synthe-sized. This approach allows us to obtain systematic chemical insight intobioactivity. Combinatorial chemistry, with its ability to synthesize manycompounds with fewer synthetic steps, has caused this level of insight tobe sacrificed for the ability to produce thousands of compounds at onetime. Now thousands of chemical entities are designed to answer one par-ticular question. Combinatorial chemistry gives rise to a higher level ofinsight with proper library design. Whether a shift from traditional solu-tion-phase chemistry to solid-phase combinatorial chemistry is causing aloss of insight at a more fundamental level is a debate for the future. At thispoint, combinatorial chemistry is a part of chemistry and its proper use andthe application of intelligent design from the chemists and engineers whoaccomplish this are providing us with new insights into the physical world.

46 J. A. J. Becker, A. Wallace, A. Garzon, P. Ingallinella, E. Bianchi, R. Cortese, F. Simonin,

B. L. Kieffer, and A. Pessi, J. Biol. Chem. 274, 27513 (1999).47 P. A. Abato, J. L. Conroy, and C. T. Seto, J. Med. Chem. 42, 4001 (1999).48 E. A. Jefferson and E. E. Swayze, Tetrahedron Lett. 40, 7757 (1999).49 S. W. Kim, Y. S. Shimn, and S. Ro, Bioorg. Med. Chem. Lett. 8, 1665 (1998).


[17] Synthesis and Screening of ‘‘One-BeadOne-Compound’’ Combinatorial Peptide Libraries

By Kit S. Lam, Alan L. Lehman, Aimin Song, Ninh Doan,Amanda M. Enstrom, Joeseph Maxwell, and Ruiwu Liu

Introduction

We first reported the ‘‘one-bead one-compound’’ (OBOC)* combina-torial library method in 1991.1 In this method, compound beads are pre-pared by a ‘‘split-mix’’ synthesis approach1–3 (Fig. 1) that results in thedisplay of many copies of the same compound on one single bead.1,4 Tensof thousands to millions of these compound beads can easily be prepared.The bead library is then mixed with a target molecule, such as a protein, an

*Abbreviations: Acm, acetamidomethyl; All, allyl; ATP, adenosine triphosphate; BCIP,

5-bromo-4-chloro-3-indolyl-phosphate; Bn, benzyl; t-Boc, tert-butyloxycarbonyl; BOP, benzo-

triazol-1-yl-oxy-tris(dimethylamine)-phosphonium hexafluorophosphate; t-But, tert-butyl;

coupling DMF, HPLC grade DMF; DABCYL, 4-[ [40-(dimethylamino)phenyl]azo]-benzoic

acid; DCC, N,N0-dicyclohexylcarbodiimide; DCM, dichloromethane; DCU, dicyclohexylurea;

Dde, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DIC, N,N0-diisopropylcarbodiimide;

DIEA, N,N-diisopropylethylamine; Dmab, 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-

3-methylbutyl]-amino}benzyl; DMEM, Dulbecco’s modified Eagle’s medium; DMF, N,

N-dimethylformamide; DMSO, Dimethyl sulfoxide; Dnp, 2,4-dinitrophenyl; Dns, dansyl;

DPTU, diphenylthiourea; EDANS, 5-[(2-aminoethyl)-amino]naphthelensulfonic acid; EDT,

1,2-ethanedithiol; FCS, fetal calf serum; Fm, 9-fluorenylmethyl; Fmoc, 9-fluorenylmethox-

ycarbonyl; N�-Fmoc-AAs, N-fluorenylmethoxycarbonyl �-amino acids; HBTU, 2-(1H-

benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HF, hydrofluoric acid;

HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, N-hydroxybenzotriazole; HOPfp, pentafluor-

ophenol; HPLC, high-performance liquid chromatography; NMM, N-methylmorpholine;

OBOC, one-bead one-compound; PBSTG-azide, phosphate-buffered saline/0.1% Tween-20/

0.1% gelatin/0.05% sodium azide; PEG, polyethyleneglycol; PEGA beads, [bis(2-acrylamido-

prop-1-yl) poly(ethylene glycol) cross-linked dimethyl acrylamide and mono-2-acrylamidoprop-

1-yl [2-aminoprop-1-yl] poly(ethylene glycol)]; PITC, phenylisothiocyanate; Pmc, 2,2,5,7,8-

pentamethylchroman-6-sulfonyl; PMTC, phenylmethylthiocarbamate; PTH, phenylthiohydan-

toin; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; Py-

BroP, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; RT, retention time; TBS,

Tris buffer saline; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-

borate; TEA, triethylamine; TFA, trifluoroacetic acid; Tis, tiisopropylsilane; Trt, trityl.1 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp,


Nature 354, 84 (1991).3 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Int. J. Peptide Protein Res. 37,

487 (1991).4 K. S. Lam, M. Lebl, and V. Krchnak, Chem. Rev. 97, 449 (1997).



Split

L M N

L L M L N LL ML N

M MM N

N MN N

Mix/Split

L M N

Mix/Split

L M N

I

II

III(27 tripeptides)

(9 dipeptides)

(coupling step) L M N

L M N

L L ML L L

L M LL L N

L M M

L N N

L N LL N M

M M N

M L MM L L

M M LM L N

M M M

M N N

M N LM N M

N M N

N L MN L L

N M LN L N

N M M

N N N

N N LN N M

Fig. 1. Split/mix synthesis approach to generate an OBOC combinatorial library.

[17] oboc combinatorial peptide libraries 299

enzyme, or other biomolecule, or whole cells and intact microorganismssuch as viruses, bacteria, and yeasts. Compound beads that interact withthese target molecules are identified by an appropriate reporter system.These positive beads are then physically isolated for structural determin-ation by instruments such as a protein sequencer. In our initial work, pep-tide libraries and enzyme-linked colorimetric assays were used.1 Laterother screening methods were developed to identify peptide ligands andenzyme substrates against a number of target molecules.4 Small moleculesand peptidomimetic libraries that require special encoding systems todecode these unsequencable compounds were also developed.4–9 In thischapter, we shall focus on methods for preparing peptide libraries, andthe various screening methods that can be applied to both peptide and

5 A. Borchardt and W. C. Still, J. Am. Chem. Soc. 116, 373 (1994).6 R. Liu, J. Marik, and K. S. Lam, J. Am. Chem. Soc. 124, 7678 (2002).7 A. W. Czarnik, Curr. Opin. Chem. Biol. 1, 60 (1997).8 C. Barnes and S. Balasubramanian, Curr. Opin. Chem. Biol. 4, 346 (2000).9 R. L. Affleck, Curr. Opin. Chem. Biol. 5, 257 (2001).


small molecule libraries. In Chapter 15, this volume, we describe a novelencoding method for OBOC small molecule and peptidomimetic libraries.

Synthesis of Linear and Cyclic Peptide Libraries

Lam et al.4 reviewed the choice of solid supports for the OBOC com-binatorial chemistry methodology. TentaGel resin (Rapp Polymere, Tubin-gen, Germany) is a good choice for OBOC peptide libraries due to itsuniformity in size as well as its nonstickiness. It consists of hydroxyethyl-polystyrene beads onto which have been grafted 3000- to 4000-Da poly-ethyleneglycol (PEG) linkers. A functional group, e.g., hydroxyl or amino,is located at the end of the linkers. This resin can be swollen in a wide rangeof solvents from water to toluene. The PEG linker is advantageous formany biological assays. For protease substrate screening, porous beadssuch as PEGA beads {bis(2-acrylamidoprop-1-yl) poly(ethylene glycol)cross-linked dimethyl acrylamide and mono-2-acrylamidoprop-1-yl [2-aminoprop-1-yl] poly(ethylene glycol)} (Calbiochem-Novabiochem, SanDiego) are preferable.

In principle, either the Fmoc/t-But (9-fluorenylmethoxycarbonyl/tert-butyl) or t-Boc/Bn (tert-butyloxycarbonyl/benzyl) amino acid protectionstrategies could be used for library synthesis on TentaGel. However,hydrofluoric acid (HF), which is highly toxic and requires a special appar-atus, is needed to cleave off the side-chain-protecting groups for the t-Boc/Bn chemistry. Since it partially degrades the PEG chain on the resin, theFmoc/t-But strategy is preferable for the synthesis of peptide libraries onTentaGel.

Efficient and unambiguous peptide-bond formation requires chemicalactivation of the carboxyl group of the N�-protected amino acid. Manyof the commercially available activating reagents can be used for solid-phase peptide library synthesis, including DIC (N,N0-diisopropylcarbo-diimide), BOP (benzotriazol-1-yl-oxy-tris(dimethylamine)-phosphoniumhexafluorophosphate), HBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrame-thyluronium hexafluorophosphate], TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate], PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), and PyBroP(bromo-tris-pyrrolidino-phosphonium hexafluorophosphate). The classiccondensation reagent, DCC (N,N0-dicyclohexylcarbodiimide), is not suit-able for peptide library synthesis because it will produce an insolubleDCU (dicyclohexylurea) byproduct. An additive reagent to generate anactive ester can significantly increase the rate of the coupling reac-tion and reduce the racemization of N�-protected amino acids. HOBt(1-hydroxybenzotriazole) and HOPfp (pentafluorophenol) are usually


used. When BOP, HBTU, TBTU, PyBOP, or PyBroP is used as the acti-vating reagent, a base is needed to neutralize the acidic side-product, whichmay induce undesired side-reactions. DIEA (N,N-diisopropylethylamine),NMM (N-methylmorpholine), and TEA (triethylamine) are recommendedfor this purpose. In this chapter, we describe a procedure that uses DIC as anactivating reagent and HOBt as an additive to synthesize peptide libraries.

Preparation of the Amino Acid Solutions

Freshly made amino acid solutions are recommended for peptide syn-thesis, although stock solutions of N�-Fmoc-AAs (N-fluorenylmethoxycar-bonyl �-amino acids) with HOBt in DMF (N,N-dimethylformamide) canbe stored at 4

�for a week. Since the synthesis of a hexapeptide or octapep-

tide library usually takes only 2–3 days, all the amino acid solutions may beprepared before the synthesis. A 3-fold excess of N�-Fmoc-AAs is neededin each coupling step to ensure completion of the coupling reaction.We have been using the following equations to calculate the amount ofnecessary reagents.

Waa ¼ MWaað Þ 3Lð Þ gð Þ Nð Þ=X (1)

WHOBt ¼ 153:2ð Þ 3Lð Þ gð Þ Nð Þ (2)

VHOBt ¼ 0:7ð Þ V 0ð Þ Xð Þ Nð Þ (3)

VDIC ¼ 126:2ð Þ 3Lð Þ gð Þ= 0:806ð Þ Xð Þ (4)

Waa ¼ mass of each protected amino acid required for librarysynthesis

WHOBt ¼ total amount of HOBt necessary for the whole libraryVHOBt ¼ total volume of HOBt solution needed

VDIC ¼ volume of DIC required for each amino acid couplingreaction

MWaa ¼ molecular weight of the protected amino acidL ¼ loading of the resin in mmol/gg ¼ mass of the resin

N ¼ total number of amino acid residues in the peptide sequenceX ¼ total number of amino acids used in each coupling stepV0 ¼ volume of amino acid/HOBt solution to be added to each

reaction vessel (for a reaction vial containing 100 mg TentaGelresin, we generally use 1.0 ml amino acid/HOBt solution)

153.2 ¼ molecular weight of HOBt�H2O (HOBt monohydrate)126.2 ¼ molecular weight of DIC0.806 ¼ density of DIC


Reagents. N�-Fmoc-AAs with the appropriate side-chain-protectinggroups are used in our library synthesis, e.g., N�-Fmoc-Arg(Pmc)-OH, N�-Fmoc-Asn(Trt)-OH, N�-Fmoc-Asp(Ot-Bu)-OH, N�-Fmoc-Cys(Trt)-OH,N�-Fmoc-Gln(Trt)-OH, N�-Fmoc-Glu(Ot-Bu)-OH, N�-Fmoc-His(Trt)-OH, N�-Fmoc-Lys(Boc)-OH, N�-Fmoc-Ser(t-Bu)-OH, N�-Fmoc-Thr(t-Bu)-OH, N�-Fmoc-Trp(Boc)-OH, and N�-Fmoc-Tyr(t-Bu)-OH. Otheramino acids without side-chain-protecting groups are N�-Fmoc-Ala-OH,N�-Fmoc-Gly-OH, N�-Fmoc-Ile-OH, N�-Fmoc-Leu-OH, N�-Fmoc-Met-OH, N�-Fmoc-Phe-OH, N�-Fmoc-Pro-OH, and N�-Fmoc-Val-OH. Manyother Fmoc unnatural amino acids are commercially available. HPLC (high-performance liquid chromatography) grade DMF is used for coupling andanalytical reagent grade DMF is used for washing of the resins. TentaGel SNH2 resin (e.g., 0.27 mmol/g) is used for library preparation. (Natural aminoacids are designated by the standard three-letter code.)

Procedure. To prepare a linear heptapeptide library of 20 amino acidsand 2 g of resins, we use Eqs. (1)–(4) to calculate the amount of reagents:Waa ¼ 3 � 0.27 � 2.0 � 7 � MWaa/20 ¼ 0.567 � MWaa mg; WHOBt ¼ 153.2� 3 � 0.27 � 2.0 � 7 ¼ 1737.3 mg; VHOBt ¼ 0.7 � 1.0 � 20 � 7 ¼ 98 ml;VDIC ¼ 126.2 � 3 � 0.27 � 2.0/(0.806 � 20) ¼ 12.7 �l. Then 1737.3 mgHOBt�H2O in DMF is dissolved until the final volume is 98 ml and4.9 ml of HOBt solution is added to each of the 20 amino acid containersand then coupling DMF is added until the final volume is (V0) � (N) ¼1.0 � 7 ¼ 7.0 ml. The amino acid should be dissolved by vortexing; how-ever, sonication can be used if dissolution is incomplete. One milliliter ofamino acid and HOBt solution (V0) is added into each reaction vessel ateach coupling step. Prior to transfer into the reaction vessel the suspensionmust be completely dissolved.

Monitor Coupling Reaction

The most popular test for the presence or absence of free amino groupsis the Kaiser test.10 The test is simple and quick; however it should be notedthat some deprotected amino acids do not show the expected dark bluecolor typical of free primary amino groups (e.g., serine, asparagine, asparticacid).11 Furthermore, for secondary amines such as proline, the resin willturn brown instead of blue. For secondary amines and aromatic amines,the chloranil test is recommended.12 In this volume, Albericio’s research

10 E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. I. Cook, Anal. Biochem. 34, 595 (1970).11 J. D. Fontenot, J. M. Ball, M. A. Miller, C. M. David, and R. C. Montelaro, Peptide Res. 4,

19 (1991).12 T. Vojkovsky, Peptide Res. 8, 236 (1995).


group has included a comprehensive study on colorimetric tests forsolid-phase synthesis.

The Kaiser test uses three solutions: ninhydrin (5 g in 100 ml of etha-nol), phenol (80 g crystalline phenol in 20 ml ethanol), and pyridine/KCN[dilute 2 ml of 0.001 mol/liter aqueous solution of KCN (potassium cyan-ide) with 98 ml of pyridine]. The test is performed by transferring a fewresin beads to a 6 � 50-mm glass test tube. The beads are washed with etha-nol and a drop of each of the three testing solutions is added. The tubeis then heated to 100

�for 5 min in a heating block. Blue (brown for

proline)-colored beads and solution (positive test) indicate the presenceof free amino groups on the resin. Colorless beads (negative test) indicatecomplete coupling. The quality of phenol is important. Impure phenol mayresult in a false-positive Kaiser test.

Although the order in which the three reagents are added does notaffect the test result, we recommend adding the KCN/pyridine solutionfirst, because ethanol makes the beads adhere to the wall of tube.

The chloranil test uses two reagents: 2% acetaldehyde in DMF (v/v)and 2% p-chloranil in DMF (w/v). A few resin beads are transferred to asmall glass test tube, washed with coupling DMF, and one drop of eachof the two reagents is added. After standing at room temperature for 5 min,min, the beads will turn blue or dark green indicating the presence of freeamino groups on the resin.

Synthesis of a Linear Heptapeptide Library with 19 EukaryoticAmino Acids (Cysteine Excluded)

For linear peptide library synthesis, cysteine is usually excluded toavoid undesired formation of disulfide bonds.

Reagents. Reagents are TentaGel S NH2 resin (80–90 �m), loading:0.27 mmol/g, protected amino acid stock solutions, 25% piperidine/DMF(v/v), Kaiser test reagents, DIC, analytical reagent grade DMF (washingDMF), methanol, HPLC grade DMF (coupling DMF), dichloromethane(DCM), cleavage solution: phenol/thioanisole/water/1,2-ethanedithiol(EDT)/trifluoroacetic acid (TFA) (7.5:5:5:2.5:82.5, w/v/v/v/v), and PBS(phosphate-buffered saline): 137 mmol/liter NaCl, 2.68 mmol/liter KCl,8.0 mmol/liter Na2HPO4, and 1.47 mmol/liter KH2PO4, pH 7.4.

Procedure. TentaGel S NH2 resin (1.9 g, 0.27 mmol/g) is swollen inDMF overnight. The resin is then equally distributed into 19 polypropylenecolumns each with a tight-sealing cap, a frit, and a stopper. The solvent isdrained under reduced pressure. One milliliter of amino acid/HOBt solu-tion and 12.7 �l of DIC is added to each column. The reaction mixturesare agitated for 1 h at room temperature. Complete coupling is confirmed


with the Kaiser test. For those columns with incomplete coupling reactions,prolonged reaction time or a ‘‘repeat coupling’’ by adding fresh amino acid/HOBt solution and DIC is applied until the Kaiser test is negative. After allthe coupling reactions are completed, the resin is combined and transferredto a siliconized glass column with a frit at the bottom. The resin is washedwith washing DMF twice (15 ml each wash) and coupling DMF (15 ml �3). The N�-Fmoc-protecting group is removed by incubation twice in10 ml of 25% piperidine/DMF for 15 min each. The resin is then washedwith washing DMF (15 ml � 3), methanol (15 ml � 3), and couplingDMF (15 ml � 4). The Kaiser test is performed to confirm the Fmoc depro-tection. The cycle of resin distribution, amino acid coupling, Kaiser test,resin mixing, resin washing, Fmoc deprotection, resin washing, and Kaisertest again is repeated until amino acid assembly is completed. The N�-Fmoc-protecting group on the last amino acid should be removed priorto side-chain deprotection. The resin is washed with DCM (15 ml � 3)and is dried under vacuum for 2 h. The side-chain-protecting groups areremoved by agitating the resin with 30 ml cleavage solution for 4 h atroom temperature. The bead-supported library is then washed withwashing DMF (15 ml � 5), methanol (15 ml � 3), DCM (15 ml � 3),coupling DMF (15 ml � 3), 30% water/DMF (15 ml � 1), 60% water/DMF (15 ml � 1), water (15 ml � 1), and PBS buffer (15 ml � 10), andis stored in 0.05% sodium azide/PBS at 4

�.

Synthesis of a Disulfide Cyclic Peptide Library

Efficient disulfide formation requires a careful choice of oxidationmethods and protecting groups for cysteine. There are several methods tocyclize a peptide via disulfide bond formation. We recommend the DMSOoxidation method13,14 for free thiol oxidation and the iodine oxidationmethod15 for the protected Cys(S-Trt)/Cys(S-Acm) oxidation. A proced-ure using the dimethyl sulfoxide (DMSO) oxidation method for the synthe-sis of a disulfide cyclic peptide library is described in this section. Cys(S-Trt) is placed at the first and last residue position of the library duringlibrary synthesis. After library synthesis is finished, all the side-chain-protecting groups including the Trt group on cysteines are removed withTFA. DMSO is used to oxidize the two cysteines to form a disulfide bond. TheEllman test is carried out to monitor the completion of disulfide bond

13 A. Otaka, T. Koide, A. Shide, and N. Fujii, Tetrahedron Lett. 32, 1223 (1991).14 J. P. Tam, C. R. Wu, W. Liu, and J. W. Zhang, J. Am. Chem. Soc. 113, 6657 (1991).15 M. Pohl, D. Ambrosius, J. Groetzinger, T. Kretzschmar, D. Saunders, A. Wollmer, D.

Brandenburg, D. Bitter-Suermann, and H. Hoecker, Int. J. Peptide Protein Res. 41, 362

(1993).


formation.16,17 Triisopropylsilane (TIS) is used as the scavenger in thecleavage solution instead of EDT, since the latter can reduce disulfidebonds during the oxidation step.

Reagents. N�-Fmoc-Cys(S-Trt)-OH, N�-Boc-Cys(S-Trt)-OH, reagentsfor peptide synthesis, Ellman’s reagent: 4.0 mg 5,50-dithiobis(2-nitroben-zoic acid) in 1.0 ml of 20 mmol/liter sodium phosphate buffer pH 8.0; oxi-dation solution: water/acetic acid/DMSO (75:5:20, v/v/v), the pH isadjusted with ammonium hydroxide to 6.0 before DMSO is added; cleavagesolution: phenol/thioanisole/water/TIS/TFA (7.5:5:5:2.5:82.5, w/v/v/v/v).

Procedure. A solution of N�-Fmoc-Cys(S-Trt)-OH (949 mg, 3.0 equiv.)and HOBt�H2O (248 mg, 3.0 equiv.) in 15 ml DMF is added to 2.0 g Ten-taGel S NH2 resin (0.27 mmol/g) preswollen in DMF. DIC (255 �l, 3.0equiv.) is added. The reaction mixture is shaken overnight at room tem-perature. After the coupling is completed, the resin is washed with washingDMF (15 ml � 2) and coupling DMF (15 ml � 3). The N�-Fmoc-protectinggroup is removed by incubation twice in 10 ml of 25% piperidine/DMF for15 min each. The resin is then washed with washing DMF (15 ml � 3),methanol (15 ml � 3), and coupling DMF (15 ml � 4). The Kaiser test isperformed to confirm the Fmoc deprotection. The peptide library is thenassembled following the method described above. After the peptide libraryassembly is completed, N�-Boc-Cys(S-Trt)-OH is coupled to the lastamino acid using the same method as for N�-Fmoc-Cys(S-Trt)-OH. Theresin is washed with DCM (15 ml � 3) and is dried under vacuum for2 h. The side-chain-protecting groups are removed by adding 30 ml ofthe cleavage solution to the resin for 4 h at room temperature with gentleshaking. The cleavage solution is drained, and the bead-supported library iswashed with DMF (15 ml � 5), methanol (15 ml � 3), DCM (15 ml � 3),DMF (15 ml � 3), 30% water/DMF (15 ml � 1), 60% water/DMF (15 ml �1), and water (15 ml � 5). A few resin beads are transferred to a glass testtube and a few drops of Ellman’s reagent are added. In this step, the Ell-man test must be positive. The yellow color of the solution indicates thepresence of free thiol group. The bead-supported library is then oxidizedin 1 liter of the oxidation solution with gentle shaking in a 2-liter bottlefor 48 h until the Ellman test is negative (colorless solution). Magneticstirring is not recommend in this step as the stirring bar may break thebeads. The resin is then thoroughly washed with water, followed byPBS. The bead-supported cyclic library is stored in 0.05% sodium azide/PBS at 4

�.

16 G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).17 G. L. Ellman, K. D. Courtney, V. Andres, Jr., and R. M. Featherstone, Biochem.

Pharmacol. 7, 88 (1961).


On-Resin Synthesis of a Cyclic Peptide Library Using Lys andGlu Side-Chains

The amide bond formation between the side-chain carboxyl and aminogroups of glutamic acid (or aspartic acid) and lysine, respectively, providesan excellent alternative approach to cyclize peptides on solid support.18,19

Various protection strategies can be used for this purpose. Many side-chain-protected derivatives of Glu, Asp, and Lys, e.g., N�-Boc-Asp(OFm)-OH, N�-Boc-Glu(OFm)-OH, N�-Fmoc-Asp(OAll), N�-Fmoc-Glu(OAll)-OH, N�-Boc-Asp(ODmab)-OH, N�-Boc-Glu(ODmab)-OH,N�-Fmoc-Lys(Dde)-OH, and N�-Boc-Lys(Fmoc)-OH, are commerciallyavailable. Similar to the N�-Fmoc-protecting group, the Fm group is labileto 20% piperidine/DMF, while the allyl group can be selectively removedby Pd(PPh3)4 in CHCl3:AcOH:N-methylmorpholine (37:2:1) withoutaffecting the other protecting groups.20 Both the Dmab and Dde groupsare removed by treating the resin with 2% hydrazine/DMF. In this section,we describe a procedure for the synthesis of a cyclic peptide library usingN�-Fmoc-Lys(Dde)-OH and N�-Boc-Glu(ODmab)-OH as building blocksfor the cyclization. The peptides are cyclized with an amine bond betweenthe �-carboxyl group of Glu and the e-amino group of Lys.

Reagents. Reagents for peptide synthesis are N�-Fmoc-Lys(Dde)-OH and N�-Boc-Glu(ODmab)-OH, acetic anhydride, PyBOP, DIEA,1-hydroxy-7-azabenzotriazole (HOAt), and 2% hydrazine/DMF.

Procedure. Two grams TentaGel S NH2 resin (0.27 mmol/g) is swollenin DMF overnight. The loading of the resin is decreased using the followingsteps: (1) mix 0.30 mmol of N�-Fmoc-Gly-OH (89 mg), 0.31 mmol ofHOBt�H2O (47 mg), and 0.31 mmol of DIC (48.5 �l) in 15 ml of couplingDMF; (2) add the mixture to the TentaGel S NH2 resin and shake gentlyovernight at room temperature; (3) after washing the resin with DMFand DCM, block the exposed amino group with 15% acetic anhydride inDCM for 30 min. In this case, the final loading of the down-substitutedresin will be 0.15 mmol/g. After Fmoc deprotection and resin washingas above, a solution of N�-Fmoc-Lys(Dde)-OH (320 mg, 2.0 equiv.),HOBt�H2O (92 mg, 2.0 equiv.), and DIC (94 �l, 2.0 equiv.) in 15 ml ofcoupling DMF is added to the resin. The mixture is shaken overnight atroom temperature. The Kaiser test is performed to confirm completecoupling reaction. After Fmoc deprotection and resin washing, the peptidelibrary is assembled as described above, until the desired cyclization

18 A. F. Spatola and P. Romanovskis, ‘‘Combinatorial Peptide and Nonpeptide Libraries: A

Handbook,’’ p. 327. VCH Verlagsgesellschaft mbH, Weinheim, 1996.19 J. S. McMurray, Peptide Res. 7, 195 (1994).20 ‘‘Novabiochem Catalog and Peptide Synthesis Handbook (1997/1998),’’ p. S33.


position (Glu) is reached. The N�-Boc-Glu(ODmab)-OH is coupled to thelast amino acid by adding a solution of N�-Boc-Glu(ODmab)-OH(735 mg, 2.0 equiv.), HOBt�H2O (92 mg), and DIC (94 �l) in 15 ml DMFto the resin. The reaction mixture is shaken overnight or until the Kaisertest is negative. The resin is then washed with DMF (15 ml � 5), and incu-bated twice in 80 ml of 2% hydrazine/DMF for 3 min each. After washingwith DMF (15 ml � 6), the cyclization step is performed by adding 5.0equiv. of PyBOP and HOAt and 10 equiv. of DIEA in DMF. The reactionmixture is shaken at room temperature overnight, until the Kaiser test isnegative. Finally, the side-chain-protecting groups are removed using thesame method as for the linear peptide libraries. The bead-supported li-brary is then washed with DMF (15 ml � 5), methanol (15 ml � 3),DCM (15 ml � 3), DMF (15 ml � 3), 30% water/DMF (15 ml � 1), 60%water/DMF (15 ml � 1), water (15 ml � 1), and PBS buffer (15 ml � 10),and is stored in 0.05% sodium azide/PBS at 4

�.

The Synthesis of ‘‘Fluorescence-Quench’’ Combinatorial Library forProtease Substrate Determination

We have found that porous PEGA bead resin is more suited for prote-ase substrate screening than TentaGel resin. The porosity of the PEGAbeads allows more protease to reach the interior of the beads, which pro-duces a favorable fluorescent signal-to-noise ratio. The peptide synthesisfollows the Fmoc chemistry scheme and mix-split synthesis method exceptthat the first amino acid in this library is lysine or any other amino acid witha free nitrogen on the side chain that can be coupled to a fluorescent(donor) molecule. The last amino acid in this library should be a quencher(acceptor). The following possible fluorescence-quencher pairs can beused: (1) ortho-aminobenzoic acid as the donor with Phe(4-NO2), nitroben-zylamide, 2,4-dinitrophenyl (Dnp), Tyr(3-NO2), or 4-nitroaniline as thereceptor; (2) aminocoumarin as the donor and aminoquinolinone typeflourophores and nitroaromatic derivatives as the acceptor; (3) 5-[(2-aminoethyl)-amino]naphthelensulfonic acid (EDANS) as the donorand 4-[[40-(dimethylamino)phenyl]azo]-benzoic acid (DABCYL) as thequencher; and (4) tryptophan as the donor and the dansyl (Dns) as the ac-ceptor. If a significant number of false-positive fluorescent beads remainafter library synthesis, the library may be capped with a different quencherthat utilizes a different coupling chemistry. This capping step should beperformed prior to side-chain deprotection. Therefore, before the final de-protection step, we routinely sample the library for inspection under thefluorescent microscope. We describe below a procedure for the synthesisof a fluorescence quench combinatorial library using ortho-aminobenzoicacid as the fluorophore and 3-nitrotyrosine as the quencher.


Reagents. Reagents include amino PEGA resin (0.30 mmol/g), N�-Fmoc-Lys(Boc)-OH, N-Boc-Abz-OH (N-Boc-2-aminobenzoic acid), N�-Fmoc-Tyr(3-NO2)-OH, 50% TFA/DCM, 2.5% DIEA/DCM, and reagentsfor peptide synthesis.

Procedure. A solution of N�-Fmoc-Lys(Boc)-OH (422 mg, 3.0 equiv.)and HOBt�H2O (138 mg, 3.0 equiv.) in 10 ml DMF is added to 1.0 g aminoPEGA resin (0.3 mmol/g) preswollen in DMF. DIC (141 �l, 3.0 equiv.) isadded. The reaction mixture is shaken at room temperature until theKaiser test is negative (about 2 h). The resin is washed with washingDMF (10 ml � 3) and coupling DCM (10 ml � 5). The Ne-Boc-protectinggroup is removed by incubation in 15 ml of 50% TFA/DCM for 20 min atroom temperature. The resin is immediately washed with DCM (10 ml � 2),2.5% DIEA/DCM (10 ml � 3), DCM (10 ml � 2), methanol (10 ml � 3),and DMF (10 ml � 4). The Kaiser test is performed to confirm Boc depro-tection. A solution of N-Boc-Abz-OH (356 mg, 5.0 equiv.), HOBt�H2O(230 mg, 5.0 equiv.), and DIC (235 �l, 3.0 equiv.) in 10 ml DMF is addedto the resin. The mixture is shaken at room temperature overnight, untilthe Kaiser test is negative. The peptide library is then assembled on the�-amino group of lysine after Fmoc deprotection and resin washing usingthe method described above. At the end of the peptide library construc-tion, N�-Fmoc-Tyr(3-NO2)-OH is coupled to the N-terminus by adding asolution of N�-Fmoc-Tyr(3-NO2)-OH (673 mg, 5.0 equiv.), HOBt�H2O(230 mg, 5.0 equiv.), and DIC (235 �l, 3.0 equiv.) in 10 ml of DMF to theresin. The reaction mixture is shaken overnight, until the Kaiser test isnegative. After Fmoc deprotection and resin washing, the side-chain-protecting groups are removed using the same method as for normal linearpeptide libraries. The ‘‘fluorescence-quench’’ bead library is then washedwith washing DMF (10 ml � 5), methanol (10 ml � 3), DCM (10 ml � 3),DMF (10 ml � 3), and methanol (10 ml � 5), and is stored in methanol inthe dark at 4

�.

Library Screening

To screen OBOC combinatorial libraries, tens of thousands to millionsof compound beads are first mixed with a target molecule. The beads thatinteract with the target molecule will be identified and then isolatedfor structure determination. For target molecules that cannot be visualizeddirectly through a microscope, a reporter group such as an enzyme, fluores-cent probe, or radionuclide is conjugated to the target molecule. If an anti-body to the target molecule is readily available, an alternative method usesthe antibody with a reporter group. Otherwise, the target molecule can bebiotinylated and probed with streptavidin with the reporter group. Protein


kinase, protease substrates determination, and other functional assays use[�-32P]ATP (adenosine triphosphate) and fluorescence-quench peptides,respectively, to identify the positive beads. For identification of ligands thatbind to the surface of whole microorganisms or eukaryotic cells, the easiestapproach is to directly inspect the library under a microscope for organismor cell-bound beads. The screening method described below can be used toscreen both peptide and small molecule OBOC libraries.4

Enzyme-Linked Colorimetric Assay

This method is suitable for a broad range of experimental sizes. Between2 � 103 and 2 � 106 beads can easily and rapidly be screened with thismethod.1,21 Colorimetric screening relies on tight binding between thetarget molecule and specific beads in the combinatorial library, which mustbe maintained throughout subsequent washing and color developmentsteps. The enzyme-linked colorimetric screening method is divided into fourbasic steps: (1) preblocking, (2) marker molecule prescreening and removal,(3) target molecule incubation, and (4) color development. The preblockingstep utilizes blocking protein, typically gelatin or bovine serum albumin, tocoat surfaces on the combinatorial bead that bind protein nonspecifically.Subsequent bead incubation steps are also done in the presence of blockingprotein to minimize nonspecific binding of the marker and target molecules.Marker molecule prescreening is done to identify and remove compoundbeads that interact directly with the marker molecules. The target moleculeincubation step incubates the bead library and the target molecule. Color de-velopment methods vary with the type of marker molecules used in previoussteps. The protocol presented herein will use a biotin label on the target mol-ecule and a streptavidin-alkaline phosphatase conjugate (Zymed, South SanFrancisco, CA) to mark the positive beads.

Reagents. Unless noted, all reagents are commonly available from anumber of sources. The following buffers and solutions are needed for thecolorimetric screening: phosphate-buffered saline/0.1% Tween-20/0.1%gelatin/0.05% sodium azide (PBSTG-azide): 8 mM, Na2HPO4, 1.5 mMKH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2, 0.1% Tween-20 (v/v,Sigma), 0.1% Gelatin (Sigma), and 0.05% sodium azide (Sigma); 5-bro-mo-4-chloro-3-indolyl-phosphate (BCIP) buffer: 0.1 M Tris–HCl, pH 8.8,0.1 M NaCl, and 2.34 mM MgCl2; BCIP stock: 50 mg/ml BCIP (Amresco,Solon, OH or Biosynth AG, Staad, Switzerland) in DMF; Tris buffersaline (TBS): 2.5 mM Tris–HCl, 13.7 mM NaCl, and 2.7 mM KCl, pH8.0; guanidine: 8 M guanidine–HCl in H O.
2
21 K. S. Lam, Methods Mol. Biol. 87, 7 (1998).


Methods. A number of suitable labeling strategies are available forcolorimetric screening of OBOC combinatorial libraries. In general thesemethods utilize alkaline phosphatase to dephosphorylate BCIP. The result-ing dephosphorylated BCIP molecules dimerize to form indigo, which isturquoise colored and precipitates on the surface of the TentaGel bead,thereby marking the bead containing the interacting peptide (Fig. 2A).Other marker strategies include target molecule-directed antibodies withalkaline phosphatase-conjugated secondary antibodies, as well as taggingstrategies such as penta-His tags and nickel-alkaline phosphatase.

Biotinylation of Target Protein. Protein samples are biotinylated essen-tially as described by the manufacturer (Pierce, Milwaukee, WI). Briefly,EZ-Link Sulfo-NHS-LC-Biotin is employed to attach a biotin moleculevia a linker to primary amines on the surface of the target protein. Themolar ratio of biotin linker to protein is adjusted so that each target proteinmolecule carries approximately one biotin. Overbiotinylation of thetarget protein is discouraged as this may alter the structure or function ofthe protein.

Colorimetric Screening. All incubations are performed at room tem-perature or at 4

�on a bench top rotator (Barnstead Thermolyne—

Labquake) with incubation times as noted. After incubation, the solutionis removed either by gravity or by vacuum applied to the bottom of thecolumn and followed by addition and removal of the wash solution twomore times. The level of the wash solution is never allowed to fall belowthe level of settled beads in the column during solution removal. Thereare approximately 750,000 TentaGel beads (80–90 �m in diameter) perml of settled beads in solution. Between 5000 and 50,000 beads are trans-ferred to a 1-ml microcolumn (Perkin Elmer Life Sciences, Boston, MA).Larger columns or containers can be used when larger numbers of beadsare to be screened. The column is drained and the beads are washed threetimes with PBSTG-azide. PBSTG-azide (1 ml) is added and the column iscapped with a top closure. The beads are incubated for 1 h on a rotator atroom temperature or at 4

�. The gelatin in the buffer will coat nonspecific

protein-binding sites on the TentaGel beads. Following the gelatin pre-block, the column is washed three times with PBSTG-azide and incubatedwith streptavidin-alkaline phosphatase conjugate (Zymed, 1.5 mg/ml)diluted in PBSTG-azide for 1 h. The streptavidin-alkaline phosphataseprescreen always employs higher concentrations of streptavidin-alkalinephosphatase than the subsequent marking steps. Dilutions typically rangebetween 1:3000 and 1:10,000. The streptavidin-alkaline phosphatase con-centration can be adjusted up or down if the color intensity of the prescreenneeds to be adjusted. In preparation for color development, the column iswashed once with TBS and twice with BCIP buffer. Special care is taken to

Fig. 2. Screening methods: (A) enzyme-linked colorimetric method for ligands that bind a

to soluble protein target; photomicrograph of a BCIP-stained bead in a background of many

negative beads; (B) whole-cell binding assay for cell surface ligands; photomicrograph of a

peptide bead surrounded by a monolayer of Jurkat T-lymphoma cells; (C) protease substrate

determination; ‘‘fluorescence-quench’’ library cleaved by a protease and the brightly blue

fluorescent beads are positives; (D) protein kinase substrate determination; autoradiograph

showing positive beads labeled with [�-32P]ATP in the presence of a purified protein kinase.



wash away all traces of streptavidin-alkaline phosphatase from the previ-ous step. Unbound streptavidin-alkaline phosphatase in the colorimetricreaction will dephosphorylate BCIP nonspecifically, resulting in high back-ground coloration. After washing with BCIP buffer, a solution of BCIP inBCIP buffer is prepared by the addition of 3.3 �l of BCIP stock solutionper ml of BCIP buffer. The BCIP solution is added to the column, whichis then incubated for 1–2 h. Columns that have retained a large amountof streptavidin-alkaline phosphatase conjugate will begin to show a faintblue color after approximately 15 min. The column is washed three timeswith PBSTG-azide to stop the reaction and remove residual BCIP. Blue-colored beads are removed manually with the aid of a gel loading pipettetip and a dissecting stereomicroscope. Depending on the combinatorial li-brary being screened, upward of 1 in 1000 beads will be stained darkly. Allbeads showing color should be removed from the library at this point.

Following the prescreening and washing, the beads are incubated withbiotinylated target protein in PBSTG-azide for 1–2 h. Different target pro-tein samples will require titration of amounts and incubation times to showadequate signal in subsequent color development steps. Typically, incuba-tion of 1 �g of protein per 1 ml column volume for 1 h is an adequate titra-tion starting point. To mark bound target proteins, the column is washedthree times with PBSTG-azide and is incubated with a reduced concentra-tion of streptavidin-alkaline phosphatase a second time. Typical dilutionsare 2- to 3-fold lower than the first streptavidin-alkaline phosphatase incu-bation, e.g., 1:3000 followed by 1:10,000 or 1:10,000 followed by 1:20,000.Incubation times can be adjusted to alter sensitivity. Typical incubationtimes are 1–2 h. For resolution of color on beads containing bound targetand marker molecules, the column is washed as above, first with TBSand then twice with BCIP buffer solutions. The color is developed asabove and the reaction is stopped after 1 h. The newly colored beads areisolated and their ligands characterized. In some experiments, one maywant to decolorize these positive beads with 100% DMF, and restain themwith the molecular target using an orthogonal reporting system, or in thepresence and absence of a known competing ligand. These latter stepscould greatly increase the chance that the selected compound beads aretrue positives.

Whole Cell Binding Assay

Screening combinatorial peptide libraries with intact cells is a rapid wayof discovering cell surface-binding ligands.22–25 The procedure is dividedinto three sections: (1) preparation and sterilization of the bead library,


(2) incubation with the target cells, and (3) recovery and characterizationof the positive beads.

Reagents. The following buffers and solutions are needed for whole cellscreening: growth media: Dulbecco’s modified Eagle’s medium (DMEM),10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 �g/ml strepto-mycin, PBS: 8 mM Na2HPO4, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mMKCl, pH 7.2.

Methods. Whenever possible, all work is done under sterile conditions ina laminar flow hood. Whole-cell binding assays can incubate for as short as5 min or as long as days. To ensure that the reaction is not contaminated,the washing column and the combinatorial library are sterilized as follows:200 �l of settled beads (�150,000 beads) are placed in a 1-ml disposablecolumn (Perkin Elmer Life Sciences). The column and beads are washedfive times with PBS and five times with H2O. The final wash step is with70% ethanol, which is added to the column but not removed until the libraryis needed for incubation with the target cells. When the cells are ready, theethanol is removed from the column and the beads are rinsed once with cul-ture media and resuspended in 0.6 ml of culture media. Using a sterile six-well tissue culture plate, three wells are prepared with 3 � 105 target suspen-sion cells, such as lymphoma cells, in 3 ml of culture media. Then 0.2 ml ofbeads (containing 50 K beads) is added to each of the three wells. The beadsand cells are incubated on a platform shaker at 70–110 rpm for 15 min to48 h at 37

�with 5% CO2. Some cells may bind beads rapidly (i.e., several

minutes) and other cells may take longer. After gentle mixing and settlingof the beads, free cells are removed with a sterile pipette.

If the target cell type is adherent, this protocol can be modified. Forloosely adherent cells it may be adequate to manually remove the cellsusing a cold PBS wash and centrifugation followed by removal of PBSand resuspension in culture media. This allows for preservation of thecell surface adhesion molecules, which may result in more rapid binding.However, care should be taken to avoid unnecessary cell aggregation. Forthese cells, the shaking step may be omitted and the cells incubated withbeads for 30 min to 1 day at 37

�with 5% CO2. For more strongly bound ad-

herent cells, trypsinizing the cells will allow the cells to revert to suspensioncells rapidly with minimal mechanical damage to the cells. The trypsin re-action is stopped with an equal volume of media containing FBS as soon as

22 M. E. Pennington, K. S. Lam, and A. E. Cress, Mol. Diversity 2, 19 (1996).23 I. B. DeRoock, M. E. Pennington, T. C. Sroka, K. S. Lam, T. Bowden, E. L. Bair, and A. E.

Cress, Cancer Res. 61, 3308 (2001).24 S. Park, M. Renil, B. Vikstrom, N. Amro, L.-W. Song, B. Xu, and K. S. Lam, Lett. Peptide

Sci. 8, 171 (2002).25 D. H. Lau, L. Guo, R. Liu, A. Song, C. Shao, and K. S. Lam, Biotechnol. Lett. 24, 497 (2002).


the cells release from the tissue culture plastic. Following centrifugation,the trypsin is removed and the cells are resuspended in culture media andcounted. The cells can be plated into six-well plates as above or in varioustissue culture dishes at a final concentration of about 70% confluency assuspension cells. Beads are then added as above, and for the case of trypsi-nized cells the plates can either be placed on a shaker at 70 rpm to facilitatecell–bead contact or incubated without shaking. In the case of trypsinizedcells, bead binding may be delayed due to the action of the trypsin on thesurface receptors. The dishes should be examined under a microscope atvarious time points to identify any beads that are covered in bound cells.

Under an inverted microscope, beads with bound cells are retrieved.The estimated number of bound cells on each bead is recorded and eachretrieved bead is placed in an individual well of a 96- or 24-well plate orgroups of similarly binding beads from the same library and cell type canbe placed into a small Petri dish. Strongly bound beads should be coveredwith cells (Fig. 2B). Subsequent wash steps may be performed on the benchtop under nonsterile conditions. The retrieved beads in the 96-well plateare washed by carefully removing each bead in series and placing it in aPetri dish filled with 8 M guanidine–HCl. Incubate until no cells are visibleon the surface of the bead (approximately 15 min). At this time, the beadis removed and placed in a second Petri dish containing H2O. Each bead iswashed no fewer than three times in H2O to ensure protein removal fromthe bead surface. In some experiments, the cleaned positive beads can beretested for binding against the same or a different cell line in the samefashion or in the presence of a blocking antibody to the suspected target.If bead sequencing is desired, each individual bead is carefully placed onglass filter paper and submitted for sequencing.

Screening for Protease Substrates

Meldal and co-workers26,27 first described the fluorescence-quenchingapproach to screen the OBOC combinatorial libraries for protease sub-strates. A ‘‘fluorescence-quench’’ combinatorial library prepared fromthe porous PEGA bead resin (see above) is first inspected under a fluores-cent microscope and the fluorescent beads are removed and discarded.After all of the false-positive beads have been removed, the peptide beadsare transferred into an Eppendorf tube and washed 5� with water, followedby a 5� wash with the appropriate protease buffer. Protease is then added

26 M. Meldal, I. Svendsen, K. Breddam, and F. I. Auzanneau, Proc. Natl. Acad. Sci. USA 91,

3314 (1994).27 M. Meldal and I. Svendsen, J. Chem. Soc. Perkin Trans. I 1591 (1995).


to the Eppendorf tube and incubated at room temperature or 37�

for 1–3 h.The beads are then washed with PBSTG, transferred to a Petri dish, andexamined under a fluorescent microscope (excitation: 254–310 nm,emission: 420 nm). The positive beads appear bright blue (Fig. 2C).

The fluorescent beads are manually isolated, washed with 8 Mguanidine–HCl and water, and then submitted for sequence analysis. Weexpect that some of the peptides on the positive beads will be cleaved atthe proteolytic site. As a result, the PTH-amino acids detected during eachcycle of Edman degradation will have been generated from both of thesepeptides. This complicates the sequence analysis somewhat. However, italso allows us to determine the proteolytic cleavage site of the peptide, inaddition to its uncleaved sequence.

COPAS Screening

Recently we have employed the COPAS Biobead bead sorter (UnionBiometrica, Somerville, MA) to isolate positive beads from combinatoriallibraries. Combinatorial libraries are incubated as described above with afluorescently tagged protein. The beads are washed to remove unboundproteins and then sorted based on the fluorescence intensity of the proteinbound to individual beads. Combinatorial beads can be sorted at a rate of50–100 thousand beads per hour and the positive beads dispensed into 96-well plates. If necessary, the combinatorial beads can be prescreened toremove beads with excessive fluorescence background.

Protein Kinase Substrate Assay

Radionuclide-based screening for protein kinase substrates is a quickand effective method for determining peptide substrates for protein ki-nases.28–31 The method presented here is based on the screening of randomsynthetic combinatorial peptide libraries with a protein kinase and isdivided into two main parts: (1) the phosphorylation of the peptide beadlibrary and (2) the recovery and characterization of positive beads.

Reagents. Unless noted, all reagents are commonly available from anumber of sources. The following buffers and solutions are needed for ki-nase screening: MES buffer: 30 mM 2-(N-morpholino)ethanesulfonic acid(MES), 10 mM MgCl2, and 0.4 mg/ml bovine serum albumin (BSA) pH

28 J. Wu, Q. N. Ma, and K. S. Lam, Biochemistry 49, 14825 (1994).29 K. S. Lam, J. J. Wu, and Q. Lou, Intl. J. Protein Peptide Res. 45, 587 (1995).30 Q. Lou, M. E. Leftwich, and K. S. Lam, Bioorg. Med. Chem. 5, 677 (1996).31 J. J. Wu, D. E. H. Afar, H. Phan, O. N. Witte, and K. S. Lam, Comb. Chem. High

Throughput Screen. 5, 83 (2002).


6.8; HCl: 1 N HCl; 5� PBS: 40 mM Na2HPO4, 7 mM KH2PO4, 680 mMNaCl, 13 mM KCl, with the addition of 0.05% Tween-20, pH 7.2.

Procedure. The kinase screening reaction is performed in a 1-ml polypro-pylene column (Perkin Elmer Life Sciences, Boston, MA). [�-32P]ATP(25 Ci/mmol) is used as substrate for the kinase labeling reaction. The typeof protein kinase being screened determines the choice of combinatorial li-brary. Typically, the combinatorial libraries will consist of a serine, threo-nine, or tyrosine residue flanked by three to five additional random aminoacids on both the N- and C-terminal sides and can be constructed to satisfythe needs of individual screening experiments. We have found that back-ground phosphorylation can be greatly reduced by using PEGA beads in-stead of TenteGel beads. Approximately 0.5 ml settled volumes of beadsare placed in the column and washed five times with MES. The level of bufferis allowed to drop to the level of the combinatorial beads before additionalwashings are performed. At no time is the level of the buffer allowed to fallbelow the level of the beads. The reaction is assembled inside the column ina volume equal to twice the volume of the settled beads. A typical reactioncontains 500 �l of settled beads, 0.1 �M [�32P]ATP, 0.4 mg/ml BSA,10 mM MgCl2, and 30 mM MES buffer pH 6.8 in a final volume of 1 ml.The kinase reaction is initiated by the addition of 1–2 �g/ml of purified pro-tein kinase and is incubated at room temperature for 1 h on a bench toprotator (Barnstead Thermolyne—Labquake). The reaction is stopped bywashing the column 5 times with 5� PBS containing 0.05% Tween-20. Add-itional wash steps can be performed as necessary if unbound ATP is presentin the eluted PBS wash. To further reduce nonspecific binding of ATP to thebead library, 1 ml of 1 N HCl is added to the column, which is heated to 95

�

for 10 min to hydrolyze the ATP, and then washed 10 times with 5� PBS.Recovery of Positive Beads. After washing, the combinatorial beads are

suspended in 1% agarose (SeaPlaque, FMC BioProducts) heated to 70–75�

and poured onto a 8.5 � 11-in. sheet of transparency film on a flat and levelsurface. Two to three milliliters of agarose should be thinly spread over anarea of �16 in2 ( �100 cm2). The beads and the agarose are evenly spreadacross the surface, allowed to cool, and air dried overnight. A rectangularbox marked by a wax pencil on the transparency helps to keep the agarosesolution on the transparency prior to gelling. Thickly spread agarose willcrack as it dries. Autoradiogram markers or other forms of marking mater-ial are placed on three locations on the transparency film, which is thencovered with Saran wrap and then exposed to X-ray film (Kodak, doublesided) for 24–48 h at room temperature. The film is developed and themarkers are used to precisely align the radiolabeled bead image map onthe developed film with the beads on the transparency (Fig. 2D). Retrievalof beads carrying a radiolabel from the agarose on the transparency is


accomplished using a hollow punch to precisely excise the region corres-ponding to the positive signal on the aligned film from the agarose on thetransparency. The excised beads and agarose are swollen in 200 �l of H2Oand heated to 70–75

�to dissolve the agarose. After dissolving the agarose,

the beads are washed five times in 5� PBS with 0.05% Tween-20. One per-cent agarose is used to resuspend the beads and they are then exposed tofilm as above for secondary screening. The bead density on the second tran-sparency film should be low enough that individual beads can be alignedwith positive signals on the film. If necessary, tertiary screening can be per-formed. Individual beads are excised by adding 20 �l of H2O to the regioncorresponding to the positive signal to swell and soften the agarose. Thepositive bead can then be retrieved from the agarose with a 25-gauge needleor gel loading pipette tip. Individual recovered beads are washed twice with8 M guanidine–HCl and twice with H2O prior to microsequencing.

Microsequencing of Peptide Beads

The structure of peptides containing 20 eukaryotic natural aminoacids is now routinely determined by the use of automatic protein micro-sequencer, which uses Edman chemistry to convert each �-amino acidsequentially to its phenylthiohydantoin (PTH) derivative. The formedPTH-amino acids can be identified by their retention times on HPLCsystems by comparison with reference standards derived from the 20 nat-ural amino acids. For an OBOC peptide library composed of natural aminoacids, the sequencing protocols of the automatic sequencer are well de-veloped and standardized. However, structure determination of peptidescomposed of unnatural �-amino acids requires modification of the standardsequencing program.32 For peptides composed of non-�-amino acids, onecan use an encoding strategy or mass spectrometry if a cleavable linker isemployed. In this chapter, we shall focus on the new sequencing methodwe have developed for unnatural �-amino acids.

A large number of unnatural �-amino acids are commercially available.Many of them are more hydrophobic than natural amino acids. To detectand clearly separate hydrophobic PTH-amino acids using the sequencer,we have extended the elution time from a total of 22 min to 28 min priorto washing the column with 90% solvent B (88% acetonitrile/12% isopropa-nol, v/v) (Table I). To use the existing software in the protein sequencer toread all 20 PTH-natural amino acids automatically, the first 18 min of thegradient profile is identical to that recommended by the manufacturer. Thisensures that the elution profile of natural amino acids remains unchanged.

32 R. Liu and K. S. Lam, Anal. Biochem. 295, 9 (2001).

TABLE I

Normal and Modified Gradient Program for PTH-Amino Acid Elutiona

Normal 0.0 0.3 0.4 18.0 18.5 21.5 22.0

Time (min) % B 6 6 16 46 90 90 50

Modified 0.0 0.3 0.4 18.0 24.5 27.5 28.0

Time (min) % B 6 6 16 46 90 90 50

a Solution B: 88% acetonitrile/12% isopropanol v/v (solvent B2).

TABLE II

Cycles and Gradients of Normal and Modified Methods

Normal

(PL PVDF

peptide)

Cartridge

cycle

Default Cart

PL PVDF

peptide

None None Cart begin

Flask cycle

Gradient

Flask Normal

Fast

Normal I

Prep Pump

Prep Pump

Flask Blank

Fast

Normal I

Flask Standard

Fast

Normal I

Modified Cartridge

cycle

Default Cart

PL PVDF

peptide

None None Cart Begin

Flask cycle

Gradient

Flask Normal

Modified

Prep Pump

Prep Pump

Flask Blank

Fast

Normal I

Flask Standard

Fast

Normal I


To reduce the sequencing time for each peptide containing unnaturalamino acids, the normal gradient (Fast Normal I) is used in Flask Blank andFlask Standard cycles, except during Flask Normal when the modified gra-dient is used (Table II). This results in 12 min in total being saved in eachpeptide sequence (6 min in Flask Blank and 6 min in Flask Standard). Byincreasing the buffer concentration from 20 ml to 25 ml of Premix bufferconcentrate per liter of solvent A3 (3.5% tetrahydrofuran/water), a betterseparation of PTH-amino acids near the region of PTH-His and PTH-Arghas been achieved, which increases the ability to use more unnatural aminoacids. To eliminate the phenylmethylthiocarbamate (PMTC) peak [anadduct of the original coupling reagent phenylisothiocyanate (PITC) andmethanol], a new coupling base R2C (methyl piperidine/water/butanol/isopropanol) is used to replace R2B (methyl piperidine/water/methanol).PMTC usually is the largest peak in the chromatogram, and it elutes rightafter the Edman chemistry by-product diphenylthiourea (DPTU), whichmay interfere with integration of hydrophobic PTH-amino acids. In


addition, it mislabels the reference peak causing the misidentification ofthe PTH-amino acids, resulting in incorrect sequence calling.

DPTU is an excellent internal reference peak because DPTU appearson each amino acid residue cycle during the process of sequencing. Underthe same sequencing conditions (i.e., the same column, gradient, columntemperature, and mobile phase), the shift of a certain PTH-amino acidpeak itself and the relative retention time (RT) difference to DPTU is lessthan 0.05 min. Therefore, it is not difficult to distinguish two amino acidswith an RT difference greater than 0.10 min. It is not necessary to run ablank and a standard cycle prior to sequencing each peptide if the DPTUpeak is eluted at the same position as that of the standard. In this case, apreviously established standard can be used as a reference standard foramino acid assignment. By setting the cycle number to the number of pep-tide residues plus 1 and using a further-modified method (the same asTable II except no Flask Blank and Flask Standard cycles), 44 min (22 minmin for the blank cycle and 22 min for the standard cycle) is saved for eachpeptide. However, when the column is changed or a new bottle of mobilephase is used, larger shifts of the PTH-amino acid elution positions mayoccur. It is then necessary to run an external reference standard containingboth natural and unnatural amino acids. While the existing program in the se-quencer can read the natural amino acids, unnatural amino acids have to bemanually identified by comparing their RT with the external reference. Wegenerate the external reference standard of PTH-amino acids in situ byplacing amino acids mixtures that are covalently coupled to beads in the se-quencing cartridge and letting the sequencer run through one sequencingcycle. Amino acid beads can be easily prepared by coupling a mixture ofequal-molar Fmoc-unnatural and Fmoc-natural amino acids to the TentaGelNH2 beads in a one-pot reaction using standard HOBt/DIC coupling (seesynthesis of peptide in this chapter), with subsequent Fmoc and side-chaindeprotection, resulting in all amino acids covalently attached to each bead.

We have selected many unnatural �-amino acids to study their sequen-cing profiles. Table III summarizes the RT of 74 PTH derivatives of bothnatural and unnatural amino acids. Through Table III it is easy to select40–45 amino acids with �RT greater than 0.10 min as building blocksto construct peptide libraries. This greatly increases the diversity of thepeptide libraries that can be generated.

For OBOC small peptide libraries or libraries with a fixed position, it isnot always necessary to sequence individual beads. In cases in which struc-tural consensus can be expected, it is sometimes much faster to sequence amultiplicity of beads. From the sequencing data, the information about thestructure–activity relationships can be obtained. In the cycles with a require-ment for high specificity only one particular amino acid can be found, while

TABLE III

RT of PTH-Amino Acids (Natural and Unnatural) on an ABI Protein Sequencera

Amino Acid RT (min) Amino Acid RT (min) Amino Acid RT (min)

Aspartic acid 4.17 �-Aminoisobutyric

acid

10.90 Leucine 18.36

1-Aminocyclopropane-

1-carboxylic acid

4.59 �-Aminobutyric

acid

10.99 3,5-Dibromotyrosine 18.43

Asparagine 4.70 Proline 13.02 Norleucine 18.88

Serine 5.34 Methionine 13.70 4-Aminophenylalanine 18.97

Glutamine 5.61 Valine 14.03 1-Amino-1-(3-piperidinyl)-

carboxylic acid

19.19

Threonine 5.85 1-Amino-1-cyclopentane

carboxylic acid

14.78 1-Amino-1-(4-piperidinyl)-

carboxylic acid

19.22

Homoserine 5.95 4-Cyanophenylalanine 14.81 2-Amino-3-(4-piperidinyl)-

propionic acid

20.05

Citrulline 6.07 Norvaline 15.32 4-Methylphenylalanine 20.38

Glycine 6.11 3-(2-Thienyl)alanine 15.61 3,5-Diiodotyrosine 20.67

Glutamic acid 6.49 3-Nitrotyrosine 15.62 4-Azidophenylalanine 20.83

Homocitrulline 6.98 �,�-Diaminopropionic

acid

15.75 2-Aminoindane-2-

carboxylic acid

20.89

Hydroxyproline 7.57,8.75 "-Benzoyllysine 15.79 3-Chlorophenylalanine 21.13

�-Aminohexanedioic

acid

7.80 �, �-Diaminopropionic

acid

16.32 Homophenylalanine 21.66

32

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7]

Histidine 8.08 Thiazolidine-4-

carboxylic acid

16.46 �-Cyclohexylglycine 21.86

"-Acetyl lysine 8.13 Tryptophan 16.48 3-Benzothienylalanine 22.01

Alanine 8.37 O-Methyltyrosine 16.66 4-Benzoylphenylalanine 22.11

3-(4-Pyridyl)alanine 8.59 Ornithine 16.83 3-(2-Naphthyl)alanine 22.41

3-(3-Pyridyl)alanine 8.71 Phenylglycine 17.09 3-(1-Naphthyl)alanine 22.65

4-Amino-4-carboxy-1,

1-dioxotetrahydrothiopyran

8.81 Phenylalanine 17.24 2-Amino-2-naphthyl-

acetic acid

22.72

1-Amino-1-(4-hydroxycyclo-

hexyl)carboxylic acid

9.23 4-Nitrophenylalanine 17.27 3,4-Dichlorophenyl-

alanine

23.23

1-Amino-1-(4-ketocyclo-

hexyl)carboxylic acid

9.95 �-tert-Butylglycine 17.65 O-Benzyl-hydroxyproline 23.31

Propargylglycine 10.01 �-Benzyloxycarbonyl 17.72 Cyclohexylalanine 23.98

4-Amino-4-carboxytetra-

hydropyran

10.03 Isoleucine 17.76 3,3-Diphenylalanine 24.02

Arginine 10.13 Lysine 18.05 Di-n-propylglycine 27.17

Tyrosine 10.50 1-Amino-1-cyclohexane

carboxylic acid

18.13

a Internal reference, DPTU, 14.82 min.

[17]

ob

oc

co

mb

in

ato

ria

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21


in positions not essential for the activity numerous amino acids can be found.To prevent the loss of sequencing information of a unique bead (e.g., whereonly one positive bead was found in the screening and verified to be specific)caused by unforeseeable problems such as malfunctioning instruments ora power failure, the bead can be cut in half under a microscope with aninjection needle and half of the bead can be submitted for sequencing.

Edman Sequencing

Peptide sequencing is performed on a Perkin-Elmer/Applied Biosys-tems Protein Sequencer (Procise 494). The equipment parameters are asfollows: column, Spheri-5 C18 PTH column, 5 �m, 2.1 � 220 mm; columntemperature, 55

�; cartridge temperature, 48

�; flask temperature, 64

�.

Premix buffer concentrate (concentration: 17.5 mM sodium acetate,33 mM acetic acid, 10 mM hexanesulfonic acid sodium salt) is purchasedfrom the manufacturer. Reservoir A: 25 ml premix buffer added to 1 litersolvent A3 (3.5% v/v tetrahydrofuran in water). Reservoir B: 88%acetonitrile/12% isopropanol, v/v (solvent B2).

For sequencing peptides containing natural amino acids, the manufac-turers instructions are followed. For sequencing peptides consisting of bothnatural and unnatural amino acids, a slightly modified gradient programand modified method are employed (refer to Tables I and II).

Acknowledgments

This work was supported by NIH R01-CA78868, R33-CA86364, R33-CA89706, the

California Cacner Research Program, Contract No. 00-00764V-20133. Ruiwu Liu is supported

in part by the University of California Systemwide Biotechnology Research Program, Grant

2001–07.

[18] Synthetic Combinatorial Libraries as anAlternative Strategy for the Development of Novel

Treatments for Infectious Diseases

By Sylvie E. Blondelle, Clemencia Pinilla, and Cesar Boggiano

Background

Infectious diseases are now the leading cause of death worldwide (25%of all world death is caused by infectious diseases and 45% in low-incomecountries), the main cause of death among children (65%), and the thirdleading cause of death in the United States. In particular, diarrhea, human



in positions not essential for the activity numerous amino acids can be found.To prevent the loss of sequencing information of a unique bead (e.g., whereonly one positive bead was found in the screening and verified to be specific)caused by unforeseeable problems such as malfunctioning instruments ora power failure, the bead can be cut in half under a microscope with aninjection needle and half of the bead can be submitted for sequencing.

Edman Sequencing

Peptide sequencing is performed on a Perkin-Elmer/Applied Biosys-tems Protein Sequencer (Procise 494). The equipment parameters are asfollows: column, Spheri-5 C18 PTH column, 5 �m, 2.1 � 220 mm; columntemperature, 55

�; cartridge temperature, 48

�; flask temperature, 64

�.

Premix buffer concentrate (concentration: 17.5 mM sodium acetate,33 mM acetic acid, 10 mM hexanesulfonic acid sodium salt) is purchasedfrom the manufacturer. Reservoir A: 25 ml premix buffer added to 1 litersolvent A3 (3.5% v/v tetrahydrofuran in water). Reservoir B: 88%acetonitrile/12% isopropanol, v/v (solvent B2).

For sequencing peptides containing natural amino acids, the manufac-turers instructions are followed. For sequencing peptides consisting of bothnatural and unnatural amino acids, a slightly modified gradient programand modified method are employed (refer to Tables I and II).

Acknowledgments

This work was supported by NIH R01-CA78868, R33-CA86364, R33-CA89706, the

California Cacner Research Program, Contract No. 00-00764V-20133. Ruiwu Liu is supported

in part by the University of California Systemwide Biotechnology Research Program, Grant

2001–07.


[18] Synthetic Combinatorial Libraries as anAlternative Strategy for the Development of Novel

Treatments for Infectious Diseases

By Sylvie E. Blondelle, Clemencia Pinilla, and Cesar Boggiano

Background

Infectious diseases are now the leading cause of death worldwide (25%of all world death is caused by infectious diseases and 45% in low-incomecountries), the main cause of death among children (65%), and the thirdleading cause of death in the United States. In particular, diarrhea, human



[18] combinatorial libraries in infectious diseases 323

immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), malaria, measles, pneumonia, and tuberculosis cause 90% of infec-tious disease-related deaths. Antimicrobial drug resistance is a majorconcern in infectious diseases such as acute respiratory infections, tubercu-losis, and diarrheal diseases, while the arsenal of drugs available to treat in-fectious diseases is being progressively depleted. For example, in the area ofhospital infection control, there is an increasing concern regarding the rapidemergence of drug-resistant strains with high epidemic potential.1,2 Of evengreater concern is the fact that a number of bacteria, such as Staphylococcusaureus, the most common cause of nosocomial wound infections, are be-coming resistant to vancomycin, a drug of last resort.3 Although there arereports of about a dozen new drugs that show promising antibacterial activ-ity by interfering with protein synthesis, cell wall formation, or DNA repli-cation, a new generation of antibiotics remains urgently needed to combatthe ever increasing spread of antibiotic-resistant bacterial strains.

Life-threatening infections caused by pathogenic fungi are also becomingincreasingly common, especially in individuals with suppressed immunesystems such as a number of cancer patients and individuals with AIDS.4

Unfortunately, there are only a limited number of antifungal compoundsavailable to counter such infections, which implies a strong need for develop-ment of new compounds with antifungal activities. The main groups ofantifungals currently available are polyenes (e.g., amphotericin B, nystatin,and pimaricin), imidazoles (e.g., ketoconazole, fluconazole, miconazole),antimetabolites (e.g., 5-fluorocytosine), echinocandins, and pneumocandins.

While over 33 million people are living with HIV/AIDS, the impact ofHIV therapies has been shown by a nationwide decline in AIDS-relateddeaths. The vast majority of anti-HIV therapeutic agents have been limitedto reverse transcriptase and protease inhibitors.5,6 However, the develop-ment of resistance to these inhibitors7 and the toxic side effects associatedwith these compounds8 indicate a major need for further discovery anddevelopment of alternative strategies such as those targeting other stages

1 J. T. Cross, Jr. and G. D. Campbell, Jr., Clin. Chest Med. 20, 499 (1999).2 C. O. Solberg, Scand. J. Infect. Dis. 32, 587 (2000).3 K. Hiramatsu, Lancet Infect. Dis. 1, 147 (2001).4 J. Ponton, R. Ruchel, K. V. Clemons, D. C. Coleman, R. Grillot, J. Guarro, D. Aldebert,

P. Ambroise-Thomas, J. Cano, A. J. Carrillo-Munoz, J. Gene, C. Pinel, D. A. Stevens, and

D. J. Sullivan, Med. Mycol. 38, 225 (2000).5 Z. Temesgen, Expert Opin. Pharmacother. 2, 1239 (2001).6 R. P. van Heeswijk, A. Vedkamp, J. W. Mulder, P. L. Meenhorst, J. M. Lange, J. H.

Beijnen, and R. M. Hoetelmans, Antiviral Ther. 6, 201 (2001).7 D. Pillay, Antiviral Ther. 6, 15 (2001).8 C. Vigouroux, S. Gharakhanian, Y. Salhi, T. H. Nguyen, N. Adda, W. Rozenbaum, and

J. Capeau, Diabetes Metab. 25, 383 (1999).


of the HIV life cycle to prevent transmission and spread of HIV as well asto improve the quality of life of HIV-infected patients.

Synthetic combinatorial chemistry approaches have created a vast newsource of molecular diversity for the potential identification of lead com-pounds. This revolutionary field enables hundreds to thousands of timesmore compounds to be synthesized and screened in shorter periods of timerelative to traditional approaches. In particular, novel antibacterials and/orantifungals9,10 as well as inhibitory peptides of either HIV proteases11,12 orHIV integrase proteins13 were identified following the screening of syntheticcombinatorial libraries (SCLs). As described below, SCLs have been usedby this laboratory to develop antimicrobial and antifungal compounds, aswell as novel treatments for HIV infection.

Mixture-Based Combinatorial Libraries

Mixture-based SCLs are built around a given pharmacophore by gener-ating all possible combinations of selected sets of functional groups at a de-fined number of positions, which are readily screened as mixtures ofsoluble compounds.9,14 Each SCL differs from the others by its chemicalclass (i.e., from peptides to organic molecules), size (i.e., total number ofindividual compounds present in the library), number and location of thediversity positions, and format driving the deconvolution process towardthe identification of the active components. Although the majority of com-binatorial chemistry programs prefer the preparation of large numbers ofindividual compounds by parallel synthesis methods, more than 100 separ-ate studies in which active compounds have been identified from mixture-based libraries of various classes have now been reported. These studieshave been carried out by nearly 50 separate research groups in assaysranging from binding assays to cell-based assays.9 The number of com-pounds present within different mixture-based libraries tested ranged fromless than 10 to greater than 1014, and the number of individual compounds

9 R. A. Houghten, C. Pinilla, J. R. Appel, S. E. Blondelle, C. T. Dooley, J. Eichler, A. Nefzi,

and J. M. Ostresh, J. Med. Chem. 42, 3743 (1999).10 S. E. Blondelle and K. Lohner, Biopolymers (Peptide Sci.) 55, 74 (2000).11 R. A. Owens, P. D. Gesellchen, B. J. Houchins, and R. D. DiMarchi, Biochem. Biophys.

Res. Commun. 181, 402 (1991).12 T. A. Rano, Y. Cheng, T. T. Huening, F. Zhang, W. A. Schleif, L. Gabryelski, D. B. Olsen,

L. C. Kuo, J. H. Lin, X. Xu, T. V. Olah, D. A. McLoughlin, R. King, K. T. Chapman, and

J. R. Tata, Bioorg. Med. Chem. Lett. 10, 1527 (2000).13 R. A. P. Lutzke, N. A. Eppens, P. A. Weber, R. A. Houghten, and R. H. Plasterk, Proc.

Natl. Acad. Sci. USA 92, 11456 (1995).14 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, and J. H. Cuervo,

Nature 354, 84 (1991).


per mixture ranged from less than 10 to more than 2 � 1010. It should benoted, however, that the levels in activity (micromolar to fentomolar) ofthe individual compounds identified from these libraries are independentof the complexity and chemical nature of the library tested. Indeed, themost active compounds derived from a given library may have micro-molar range activity against a specific target system, while other indivi-dual compounds derived from the same library may have nanomolar orsubnanomolar activity in a different bioassay system.

Deconvolution procedures are an essential element in the identificationof active individual compounds from mixture-based libraries. In particular,the positional scanning approach15 uses a positional scanning SCL (PS-SCL), in which the key functionality(ies) at each position of the activecompound(s) can be determined directly from the library screening data.Thus, a PS-SCL is composed of sublibraries, each addressing a single pos-ition of the sequence while representing the same collection of individualcompounds (Table I). When used in concert, the data derived from eachsublibrary yield information about the key functionalities for each position.If the key functionalities at each position are found to be connected to eachother (i.e., represent the same active compound(s) present in the corres-ponding mixtures), then combinations of these functionalities will lead toactive individual compounds. Individual compounds that represent all pos-sible combinations of the selected functionalities at each position are thensynthesized in order to confirm the screening data and determine their rela-tive activities. Mixture-based PS-SCLs therefore have the advantage ofgreatly decreasing the economics and time constraints of single compoundarray systems. Screening of millions of compounds can be accomplishedeven in assays that are not formatted for conventional high throughput.

Development of Antifungal Agents Using the PS-SCL Approach

Candida albicans and Cryptococcus neoformans are two of the mostcommon fungi responsible for opportunistic infections. For instance, oro-pharyngeal candidiasis is the fungal infection most frequently associatedwith HIV-positive patients.16 Cr. neoformans is the causative agent of cryp-tococcosis, which is the leading cause of morbidity and mortality due tofungi in patients with AIDS.17 In a continuing effort to generate and screen

15 C. Pinilla, J. R. Appel, P. Blanc, and R. A. Houghten, Biotechniques 13, 901 (1992).16 W. G. Powderly, K. H. Mayer, and J. R. Perfect, AIDS Res. Hum. Retroviruses 15, 1405

(1999).17 K. J. Kwon-Chung, T. C. Sorrell, F. Dromer, E. Fung, and S. M. Levitz, Med. Mycol. 38, 205

(2000).

TABLE I

Illustration of a Tripeptide PS-SCL Structurea

Sublibrary 1: OXXb Sublibrary 2: XOX Sublibrary 3: XXO

AXX . . . LXX . . . YXX XAX . . . XLX . . . XYX XXA . . . XXL . . . XXY

AAA LAA YAA AAA ALA AYA AAA AAL AAY

AAC LAC YAC AAC ALC AYC ACA ACL ACY

. . . . . . . . . . . . . . . . . . . . . . . . . . .

AKA LKA YKA KAA KLA KYA KAA KAL KAY

AKC LKC YKC KAC KLC KYC KCA KCL KCY

. . . . . . . . . . . . . . . . . . . . . . . . . . .AYW LYW YYW YAW YLW YYW YWA YWL YWY

AYY LYY YYY YAY YLY YYY YYA YYL YYY

a The tripeptide PS-SCL has three diversity positions and thus consists of three sublibraries, each of which has a single defined amino acid at

one position and an equimolar mixture of amino acids at each of the other two positions. Twenty l-amino acids are used at each defined

position, and there are 20 mixtures per sublibrary, as well as at each mixture position. Thus there are 202 ¼ 400 peptides per mixture and 203

¼ 8,000 peptides in the entire library. The same 8,000 peptides are present in each sublibrary, but are arranged differently within the

mixtures of each sublibrary.b ‘‘O’’ (referred as ‘‘the defined position’’) represents the l-amino acid common to every peptide present at this position in a given mixture;

‘‘X’’ (referred as ‘‘the mixture position’’) represents all 20 l-amino acids that can be present at this position in a peptide contained in a

given mixture. All peptides are present in a close to equimolar concentration.

32

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g[1

8]


various classes of antifungal compounds against C. albicans and Cr. neo-formans, a number of mixture-based PS-SCLs made up of heterocycliccompounds were evaluated for the identification of novel antifungal candi-date drugs.18,19 Heterocyclic compounds offer a high degree of structuraldiversity and have proven to be broadly and economically useful as thera-peutic agents, particularly nitro-carbon heterocyclic structures.20 To illus-trate the PS-SCL approach, we report the generation and identificationof N-benzyl aminocyclic ureas having anticandidal activities.18

Screening of a PS-SCL for Antifungal Activity

An N-benzyl aminocyclic urea PS-SCL was prepared as described else-where.21,22 This library was built with a diversity of 40, 37, and 80 separatefunctionalities at position 1, 2, and 3, respectively. As shown in Table II, thecomplete PS-SCL contains 118,400 compounds (40 � 37 � 80) and consistsof three sublibraries, each of which has a single defined functionality at oneposition and a mixture of functionalities at each of the other two positions.The number of individual compounds per mixture varied from one subli-brary to the others since this is based on the diversity at the defined pos-ition of that sublibrary. Thus, each sublibrary contains the same 118,400compounds and differs from the others by how the compounds are groupedwithin each mixture.

Each mixture was screened at 250 �g/ml against 1–5 � 105 colony-forming unit (CFU)/ml blastoconidial C. albicans prepared by inoculatingtwo colonies of a newly grown C. albicans culture in yeast media (YM)broth (Difco Laboratories, Detroit, MI), vortexed, and diluted 10-fold inYM. The assay was carried out in 96-well flat-bottom tissue culture plates(Costar, Pleasanton, CA). An equal volume (100 �l) of yeast suspension in2� YM broth was added to the mixtures and the plates incubated for 48 hat 30

�. The relative percent growth of C. albicans found in the presence of

each mixture was determined by the optical density at 620 nm (OD620)using a Spectra Max 250 microplate spectrophotometer. Duplicate samplesof each mixture were used in each assay, and the assays were repeated atleast twice. As shown in Fig. 1, a small number of mixtures exhibited

18 S. E. Blondelle, A. Nefzi, J. M. Ostresh, and R. A. Houghten, Pure Appl. Chem. 70, 2141

(1998).19 S. E. Blondelle, E. Crooks, J. M. Ostresh, and R. A. Houghten, Antimicrob. Agents

Chemother. 43, 106 (1999).20 H. J. Roth and A. Kleeman, ‘‘Pharmaceutical Chemistry.’’ John Wiley & Sons, New York,

1988.21 A. Nefzi, J. M. Ostresh, J.-P. Meyer, and R. A. Houghten, Tetrahedron Lett. 38, 931 (1997).22 C. E. Hoesl, A. Nefzi, J. M. Ostresh, Y. Yu, and R. A. Houghten, Chapter 25, this volume.

TABLE II

N-benzyl Aminocyclic Urea PS-SCL Descriptiona

Sublibrary 1 Sublibrary 2 Sublibrary 3

NH

RO BZ

NN

O

RX

RX

NH

RX BZ

NN

O

RX

RO

NH

RX BZ

NN

O

RO

RX

Number of mixtures 40 37 80

Number of compounds

per mixture

37 � 80 ¼ 2960 40 � 80 ¼ 3200 40 � 37 ¼ 1480

Total number of compounds

in sublibrary

40 � 2960 ¼ 118; 400 37 � 3200 ¼ 118; 400 80 � 1480 ¼ 118; 400

a Ro represents a defined functionality, common to all compounds present in a given mixture; Rx represents a close to equimolar mixture of

all functionalities used to build the library at a given position.

32

8pe

ptid

esy

nth

esis

an

dsc

re

en

in

g[1

8]

Sublibrary 3

0%

20%

40%

60%

80%

100%

Sublibrary 1

0%

20%

40%

60%

80%

100%

Sublibrary 2

0%

20%

40%

60%

80%

100%

Fig. 1. Anticandidal activity of N-benzyl aminocyclic urea PS-SCL. Each graph represents

the activity of a sublibrary, and each bar within a graph represents the percent inhibition of

the growth of C. albicans ATCC 10231 by a separate mixture. The line represents the average

activity for all of the mixtures making up the corresponding sublibrary.


TABLE III

Anticandidal Activity of Most Active N-benzyl Aminocyclic Urea Mixtures

RO1a RO2 RO3 IC50 (�g/ml)b

(S)-Cyclohexylmethyl X X 173

(R)-Cyclohexylmethyl X X 182

(R)-Isobutyl X X 188

(S)-Dibenzylaminoethyl X X 219

X (S)-N-Methylindole-3-

methyl

X 93

X (S)-n-Butyl X 140

X (S)-Cyclohexylmethyl X 151

X X 4-Dimethylaminobenzyl 149

X X 3,3-Dicyclohexylpropyl 180

X X 4-Cyclohexylbutyl 210

a Functionality defining the mixture.b The IC50 values are determined against C. albicans ATCC 10231.


significant antifungal activity. To better differentiate the most active mix-tures, the concentration necessary to inhibit 50% growth (IC50) was deter-mined for those mixtures that exhibited more than 50% inhibition at250 �g/ml by testing them at concentrations varying from 250 to 2 �g/mlin H2O derived from serial 2-fold dilutions (Table III). These IC50 valueswere calculated using a sigmoidal curve fitting software (Graphpad—ISI,San Diego, CA).

Deconvolution Process

Based on the screening results of the PS-SCL, a limited number ofmixtures were selected to carry out a first deconvolution process accord-ing to criteria of high antifungal activity and significant differences inthe chemical nature of the defined functionalities within each sublibrary.Thus, 12 N-benzyl aminocyclic ureas, which corresponded to all possiblecombinations of the functionalities defining selected mixtures at eachposition (two mixtures from sublibraries 1 and 3, and three mixtures fromsublibrary 2, 2 � 3 � 2 ¼ 12), were generated in order to confirm thescreening data and determine their relative activities. The IC50 values ofthese compounds varied from 10 �M to greater than 500 �M. Similaractivities were observed when tested against Cr. neoformans. The struc-tures of the two most active N-benzyl aminocyclic ureas are shown inTable IV.

TABLE IV

Antifungal Activity of the Two Most Active N-benzyl Aminocyclic Ureasa

NHNN

O

NNHNN

ON

IC50 (�M) MIC (�M)b IC50 (�M) MIC (�M)b

C. albicans 48 60–120 36 40–60

Cr. neoformans 16 60–120 9 30–60

a The fungi strains used in these studies were C. albicans ATCC 10231 and Cr. neoformans

ATCC 32045. Each compound was incubated for 48 h at 30�

or 72 h at 26�

for C. albicans

or Cr. neoformans, respectively.b The minimum inhibitory concentrations (MICs) were defined as the lowest concentration

of compound at which less than 2% growth was detected.


Development of Antibacterial Agents Using the PS-SCL Approach

Continued development of novel therapeutics for the treatment ofbacterial infection has become an important clinical imperative. This isespecially true due to the emergence and dissemination of new opportunis-tic pathogens in a growing immune system-debilitated host population, aswell as to the emerging resistance of common or resurgent pathogens tostandard antibiotics in community-acquired infection.2,4,23 In particular,colonization with methicillin-resistant S. aureus (MRSA) is a growing clin-ical challenge in hospitals and community care centers and has been recog-nized as a major nosocomial pathogen in hospitals.2,24 Vancomycin and anumber of fluoroquinolones are currently the agents of choice againstMRSA.25 However, fluoroquinolone-resistant MRSA strains26 as well asvancomycin-resistant MRSA strains3 have now been clinically isolated.

Pseudomonas aeruginosa is another major human pathogen that causesdisease in patients with impaired host defenses.27 Ps. aeruginosa is primar-ily associated with ultimately fatal chronic respiratory infection in cystic fi-brosis and other forms of bronchiectasis.28 Despite strong inflammatory

23 J. P. Metlay, Curr. Opin. Infect. Dis. 15, 163 (2002).24 F. M. Hussain, S. Boyle-Vavra, and R. S. Daum, Pediatr. Infect. Dis. J. 20, 763 (2001).25 J. M. Boyce, J. Hosp. Infect. 48, S9 (2001).26 J. F. Acar and F. W. Goldstein, Clin. Infect. Dis. 24, S67 (1997).27 R. Wilson and R. B. Dowling, Thorax 53, 213 (1998).28 A. L. Brennan and D. M. Geddes, Curr. Opin. Infect. Dis. 15, 175 (2002).


antibody response and intensive antibiotic therapies, Ps. aeruginosa israrely eliminated once colonization of the airways is established. To reachthe sites of infection, antibiotics must be able to penetrate into bronchialmucosa, which renders Ps. aeruginosa resistant to a large number of antibi-otics.27 Among existing antibiotics, quinolone derivatives such as ciproflox-acin have the greatest ability to penetrate the mucosal barrier. However,resistance to quinolone derivatives has also emerged.29 The SCL approachwas then undertaken to identify novel structures having activity againstMRSA and/or Ps. aeruginosa using standard microdilution susceptibilityassays as described above for antifungal activity screening.9,10 Examplesof the ranges of activities and chemical structures of antimicrobial com-pounds identified from SCLs are shown in Table V. The activities were de-termined following overnight incubation at 37

�in Mueller Hinton (MH)

broth against MRSA and Ps. aeruginosa culture prepared by inoculating100 �l of an overnight grown bacterial cultures in 10 ml of 2� MH broth,incubated for 2 h at 37

�, and diluted 40-fold in 2� MH broth, for an

approximate final assay concentration of 1–5 � 105 CFU/ml.

Development of HIV-1 Antagonists Using the SCL Approach

Although the introduction of highly active antiretroviral therapy(HAART) significantly improved the quality of life of HIV-1-infected indi-viduals, the reverse transcriptase and protease inhibitors used in HAARTcannot completely eradicate HIV-1 from infected people,30 and the emer-ging transmission of resistant variants and long-term side effects of theseinhibitors31,32 raise the need for novel strategies to fight HIV-1 infection.The discovery in the early 1990s that peptides corresponding to segmentsof HIV-1 gp41 env potently inhibit virus entry into target cells,33,34 com-bined by the recent emergence of structural and functional informationon HIV-1 envs and membrane fusion process,35–38 opened a new strategy

29 T. Kohler, M. Michea-Hamzehpour, P. Plesiat, A.-L. Kahr, and J.-C. Pechere, Antimicrob.

Agents Chemother. 41, 2540 (1997).30 D. Finzi, J. Blankson, J. D. Siliciano, J. B. Margolick, K. Chadwick, T. Pierson, K. Smith,

J. Lisziewicz, F. Lori, C. Flexner, T. C. Quinn, R. E. Chaisson, E. Rosenberg, B. D. Walker,

S. Gange, J. Gallant, and R. F. Siliciano, Nat. Med. 5, 512 (1999).31 N. L. Michael and J. P. Moore, Nat. Med. 5, 740 (1999).32 J. Martinez-Picado, M. P. DePasquale, N. Kartsonis, G. J. Hanna, J. Wong, D. Finzi,

E. Rosenberg, H. F. Gunthard, L. Sutton, A. Savara, C. J. Petropoulos, N. Hellmann, B. D.

Walker, D. D. Richman, R. Siliciano, and R. T. D’Aquila, Proc. Natl. Acad. Sci. USA 97,

10948 (2000).33 C. Wild, T. Oas, V. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA

89, 10537 (1992).34 S. Jiang, K. Lin, N. Strick, and A. R. Neurath, Nature 365, 113 (1993).

TABLE V

Example of Acyclic and Heterocyclic Antimicrobial Structures Identified

from PS-SCLs

Structure

Number of

compounds in SCL

MIC (�g/ml) of Most Active

Compoundsa

MRSA Ps. aeruginosa

H2N

HN

NH

NH2

R

R

R

4

49,521,980 2–4 16–32

R

HN

HN

R

R

O

O

125,934 2–4 nd

NN

R

O

O

CH

R

C

O

N

R

H

32,400 8–16 nd

HN N

OR

S

R

CONH2

6,525 2–4 nd

N

N

N

R

RR

102,459 4–8 62–125

a The bacterial strains used in these studies are MRSA ATCC 33591 and Ps. aeruginosa

ATCC 27853.


for the development of potentially useful therapeutics. Thus, therapeuticapproaches involving the inhibition of the membrane fusion are gaining im-portance. The major issues when considering peptides as potential drugs

35 W. A. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387,

426 (1997).36 D. C. Chan and P. S. Kim, Cell 93, 681 (1998).37 P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson,

Nature 393, 648 (1998).38 E. A. Berger, P. M. Murphy, and J. M. Farber, Annu. Rev. Immunol. 17, 657 (1999).


are the peptides’ susceptibility to proteolysis, immune clearance, and lackof bioavailability. Compounds less than 1000 Da, which represent the vastmajority of clinically useful drugs, are indeed more desirable for masstreatment. Furthermore, the crystal structure of the gp41 core39 showedthe presence of three prominent pockets that could accommodate mol-ecules with a molecular weight of approximately 500 Da. The SCL ap-proach was then used to develop small molecules having inhibitoryactivity for HIV-1-mediated membrane fusion as novel lead anti-HIVcompounds.

Determination of Inhibition of HIV-1 Mediated Fusion

The intrinsic fusion selectivity of the viral envelope glycoprotein plays amajor role for infection of CD4þ T cell lines versus primary macrophages.40

This selectivity is determined by ‘‘fusion cofactors’’ (CXCR4 for T celllines and CCR5 for macrophages) that belong to the chemokine receptorfamily.38 To address the two processes and enable a rapid screening ofvarious PS-SCls, a 96-well plate-based assay was adapted from the env-mediated membrane fusion assay described by Berger’s group.41 Thissystem uses two different cell populations, effector cells (expressing HIV-1 env and T7 RNA pol using vaccinia virus) and target cells (expressingconstitutively CD4 and the required coreceptor, and �-galactosidase genedownstream of a T7 promotor also using a vaccinia construct). Thus, ef-fector cells (HeLa) and target cells (HeLa CD4þ for the X4 system andHOS CD4þ CCR5þ for the R5 system) were diluted to a final density of2 � 106 cells/ml for the R5 system and 6 � 105 cells/ml for the X4 system.Effector and target cells (25 �l each) were dispensed into individual wellsof a 96-well, polystyrene, half-area flat-bottom, high-binding plate contain-ing 50 �l of test samples at varying concentrations derived from serial2-fold dilutions or media in the control wells. The positive controls con-tained effector and target cells and the negative control contained un-cleaved control cells and target cells. Plates were centrifuged at 400 g for2 min and then incubated for 3 h to allow fusion.

To determine the level of fusion between effector and target cells, 25 �lof 2% (v/v) Tween-20 was added to each well, mixed by pipetting, frozen,and thawed twice to facilitate cell lysis. Then 25 �l of lysate was transfer-red to a 96-well half-area plate and 25 �l of substrate solution-�-gal(8 mM chlorophenol red-�-d-galactopiranoside, 60 mM Na2HPO4�H2O,40 mM NaH2PO4�H2O, 10 mM KCl, 1 mM MgSO4�H2O, 50 mM

39 D. C. Chan, C. T. Chutkowski, and P. S. Kim, Proc. Natl. Acad. Sci. USA 95, 15613 (1998).40 C. C. Broder and E. A. Berger, Proc. Natl. Acad. Sci. USA 92, 9004 (1995).41 O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68, 5411 (1994).


2-mercaptoethanol) was added. The OD570 was measured and the percentinhibition was calculated relative to the positive controls.

Identification of Inhibitors of HIV-1-Mediated Fusion from PS-SCLs

An example of PS-SCL screening for anti-HIV activity is shown in Fig. 2with the screening of a PS-SCL of diethylenetriamines and a PS-SCL of N-methyl-1,4,5-trisubstituted 2,3-diketopiperazines in the X4 system. The twolibraries were generated using the same building blocks and the diethylene-triamine PS-SCL served as starting material for the synthesis of theN-methyl-1,4,5-trisubstituted 2,3-diketopiperazine library following the‘‘library from library’’ concept.42–44 Thus, N-methyl-1,4,5-trisubstituted2,3-diketopiperazines were obtained by overnight treatment with oxalyldii-midazole of resin-bound reduced acylated dipeptides. These reduced acy-lated dipeptides led to diethylenetriamines following cleavage from theresin with anhydrous HF, extraction, and lyophilization. Interestingly, asignificantly different profile of activity was observed between the two lib-raries (i.e., mixtures defined with the same functionality showed a differentrelative activity in the two libraries). These results indicate that thepresence of the cyclic structure plays a role in the activity of the individualcompounds.

Based on the screening results, 27 diethylenetriamines and 24 N-methyl-1,4,5-trisubstituted 2,3-diketopiperazines were generated andassayed in the X4 and R5 systems. The most active diethylenetriamineshad IC50 values varying from 30 to 500 nM and 840 nM to 1.1 �M in theR5 and X4 systems, respectively, while the most active diketopiperazineshad IC50 values varying from 5 to 36 �M in both systems.

Inhibition of HIV-1 Replication

The fusion assays described above combined with the SCL approachesrepresent a straightforward and rapid means for the development of novelcompounds that inhibit HIV-1 env-mediated membrane fusion. Althoughbased on recombinant glycoproteins, the process of cell-to-cell fusion isgenerally thought to be analogous to the processes involved in fusion ofviral membrane during virus penetration.40 To further investigate the inhibi-tory activity of the newly identified antagonists, their ability to inhibit HIV-1infectivity was evaluated by carrying out replication assays. Thus, twowell-characterized HIV-1 isolates were selected for this assay: HIV-1 IIIb

42 J. M. Ostresh, G. M. Husar, S. E. Blondelle, B. Dorner, P. A. Weber, and R. A. Houghten,

Proc. Natl. Acad. Sci. USA 91, 11138 (1994).43 A. Nefzi, J. M. Ostresh, and R. A. Houghten, Tetrahedron 55, 335 (1999).44 A. Nefzi, M. A. Giulianotti, and R. A. Houghten, Tetrahedron 56, 3319 (2000).

Diethylenetriamine PS-SCL

N-methyl-1,4,5-trisubstituted-2,3-diketopiperazine PS-SCL

Sublibrary 1

0

20

40

60

80Sublibrary 2

0

20

40

60

80Sublibrary 3

0

20

40

60

80

Sublibray 1

0

20

40

60

80Sublibrary 2

0

20

40

60

80Sublibrary 3

0

20

40

60

80

Fig. 2. Inhibitory activity of HIV-1-mediated fusion by diethylenetriamine and N-methyl-1,4,5-trisubstituted 2,3-diketopiperazine PS-SCLs.

Each bar within a graph represents the percent inhibition as measured in the X4 fusion system by a separate mixture at 2.5 �g/ml for the

diethylenetriamines and at 25 �g/ml for the diketopiperazines. The line represents the average activity for all of the mixtures making up the

corresponding sublibrary.

33

6pe

ptid

esy

nth

esis

an

dsc

re

en

in

g[1

8]

NH

HN N

H

Fig. 3. Chemical structure of the diethylenetriamine identified from the PS-SCL showing

the greatest inhibition of HIV-1 replication.


(X4 virus) and HIV-1 SF162 (R5 virus). These viruses were propagatedand titrated in peripheral blood monocellular cells (PBMCs) from HIV-1-negative donors. Preactivated PBMCs with phytohemagglutinin (PHA)and interleukin (IL)-2 were infected in a 15-ml tube with 0.03–0.01 tissue cul-ture infectious dose (TCID) virus and incubated overnight at 37

�under a 5%

CO2 atmosphere. Following three washes with RPMI containing 10% fetalcalf serum, the cells were resuspended in 96-well plates at 30,000 cells/well inthe presence or absence of different dilutions of test compounds, and theplates were incubated for 7 days at 37

�under 5% CO2 atmosphere. The pres-

ence of HIV-1 p24 was tested using commercial ELISA kits (Perkin Elmer,Life Sciences). For the most active compounds (structure shown in Fig. 3)44% and 62% inhibition of the replication of the X4 and R5 viruses, respect-ively, were observed at 3 �M. Although the mechanism of inhibitionremains to be determined, due to the small size of the identified inhibitors,they might interact with the gp41 N36 binding pocket.39

Novel Strategies toward the Development of Vaccines againstInfectious Diseases

The worldwide significance of infectious diseases has led to great inter-est in generating vaccines to cure or prevent such diseases. Potent and uni-versally applicable vaccines must be recognized as nonself by the immunesystems of many individuals of a wide patient population. T lymphocyteswith the help of activated B lymphocytes regulate both the cellular andhumoral responses of the immune system. By their T cell receptor (TCR)T cells identify antigens (short peptides) bound to major histocompatibilitycomplex (MHC) molecules on the antigen-presenting cells (APC), andbind, recognize, and are activated by these antigens. The two major T cellsubpopulations, CD8þ cytolytic T lymphocytes (CTL) and CD4þ helperT lymphocytes, which act as effectors during infection, or as inducers ofT and B cell functions, are involved in both physiological and pathologicalimmune responses. CD8þ T cells typically recognize 9 amino acid-long


peptides in the context of MHC class I molecules, whereas CD4þ T cellsrecognize 11–15 amino acid-long peptides in the context of MHC class IImolecules. An approach directed toward the identification of antigens thatstimulate T cell responses important to clear infection, i.e., through theidentification of immunodominant epitopes and epitope mimics, is antici-pated to lead to a successful development of prophylactic and therapeuticvaccines to combat infectious diseases.

Identification of Epitope Mimics

The use of peptide libraries to study T cell specificity was first demon-strated by Gundlach et al.45 Since then studies have focused on T cell spe-cificity with emphasis on the identification of immunodominant epitopes ininfectious diseases and autoimmune disorders, and on the determination oftumor antigens. The PS-SCL approach, as described above, was success-fully used to study T cell recognition and to identify and optimize peptideshaving a range of activities in stimulating proliferative responses, cytokineproduction, and/or lysis by CD4þ and CD8þ murine and human T cellclones and T cell lines of known and unknown specificity.46–53

In the cases of clones of known specificity, approximately 25% of theidentified epitope mimics were found to be superagonists, having EC50

values (concentration of peptide that causes a half-maximal response by aclone or a line) that are 1 to 3 orders of magnitude more effective than thenative ligand.48,49,51 Importantly, most of these agonists are effective im-munogens capable of inducing T cell-mediated potent immune responses

45 B. R. Gundlach, K.-H. Wiesmuller, T. Junt, S. Kienle, G. Jung, and P. Walden, J. Immunol.

Methods 192, 149 (1996).46 C. Pinilla, R. Martin, B. Gran, J. R. Appel, C. Boggiano, D. B. Wilson, and R. A. Houghten,

Curr. Opin. Immunol. 11, 193 (1999).47 B. Hemmer, B. Gran, Y. Zhao, A. Marques, J. Pascal, A. Tzou, T. Kondo, I. Cortese,

B. Bielekova, S. Straus, H. F. McFarland, R. A. Houghten, R. Simon, C. Pinilla, and

R. Martin, Nat. Med. 5, 1375 (1999).48 D. B. Wilson, C. Pinilla, D. H. Wilson, K. Schroder, C. Boggiano, V. Judkowski, J. Kaye,

B. Hemmer, R. Martin, and R. A. Houghten, J. Immunol. 163, 6424 (1999).49 B. Hemmer, C. Pinilla, B. Gran, M. Vergelli, N. Ling, P. Conlon, H. F. McFarland, R. A.

Houghten, and R. Martin, J. Immunol. 164, 861 (2000).50 R. Martin, B. Gran, Y. Zhao, S. Markovic-Plese, B. Bielekova, A. Marques, M. H. Sung,

B. Hemmer, R. Simon, H. F. McFarland, and C. Pinilla, J. Autoimmun. 16, 187 (2001).51 C. Pinilla, V. Rubio-Godoy, V. Dutoit, P. Guillaume, R. Simon, Y. Zhao, R. A. Houghten,

J.-C. Cerottini, P. Romero, and D. Valmori, Cancer Res. 61, 5153 (2001).52 C. La Rosa, R. Krishnan, S. Markel, J. P. Schneck, R. Houghten, C. Pinilla, and D. J.

Diamond, Blood 97, 1776 (2001).53 V. Rubio-Godoy, C. Pinilla, V. Dutoit, E. Borras, R. Simon, Y. Zhao, J.-C. Cerottini,

P. Romero, R. A. Houghten, and D. Valmori, Cancer Res. 62, 2058 (2002).


to the native peptide ligand in vivo. An additional 25% are effectiveantagonists, and are able to inhibit responses to prepulsed agonists. It isnoteworthy that most of these identified epitope mimics have multiple non-conservative changes of sequence that would have been impossible topredict by traditional and restricted analoging methods.

An example of the identification of epitope mimics for CD8þ T cellclones in infectious diseases is the generation of nonapeptide analogs ofthe pp65495–503 CTL epitope for cytomegalovirus (CMV).52 CMV is an im-portant human pathogen affecting immunosuppressed patients.54 Thepp65495–503 CTL epitope is universally recognized among CMV-seroposi-tive individuals who express HLA-A*0201.55,56 However, the relative bind-ing affinity of this epitope to HLA-A*0201 is moderate, and an increasedimmunogenicity might prove beneficial in its use as a CTL epitope vaccine.The different steps involved in the identification of pp65495–503 epitopemimics from the screening of the library are described in Fig. 4, pathwayA. Thus, each mixture of a C-terminal amidated nonapeptide PS-SCLwas incubated at 50 �g/ml with cloned T cells (3-3F4—a CMV-specificCD8þ HLA-A*0201-restricted T cell clone) and 51Cr-labeled T2 cells(HLA-A2þ, TAP-deficient human lymphoblastoid hybridoma often usedfor peptide presentation protocols)57 for 4 h and the levels of chromiumrelease were determined using a Cobra II gamma counter.

Upon screening the nonapeptide PS-SCL against the 3-3F4 CMV-spe-cific T cell clone, one to three mixtures exhibited significant activities ineach sublibrary, and five of nine amino acids defining the most active mix-tures corresponded to the residues of the native peptide epitope. In par-ticular, the mixtures defined by the known anchor residues at positions 2and 9 (Leu and Val, respectively) were among the highest responders atthose positions.58 Based on the screening results, a series of 16 individualnonapeptides was generated. The two most active peptides, despite havingfour amino acid substitutions, were 1000- and 10,000-fold more active thanthe native epitope. To verify whether the increases seen in recognition by theHLA-A*0201-restricted T cell clone 3-3F4 would be common to all T cellclones that recognize the native epitope, a chromium release assay was alsoperformed using a cohort of four additional T cell clones derived from fourdifferent CMV-seropositive volunteers. The two most active peptides

54 D. Salmon-Ceron, HIV Med. 2, 255 (2001).55 M. P. Weekes, M. R. Wills, K. Mynard, A. J. Carmichael, and J. G. Sissons, J. Virol. 73, 2099

(1999).56 A. Solache, C. L. Morgan, A. I. Dodi, C. Morte, I. Scott, C. Baboonian, B. Zal, J. Goldman,

J. E. Grundy, and J. A. Madrigal, J. Immunol. 163, 5512 (1999).57 R. D. Salter, D. N. Howell, and P. Cresswell, Immunogenetics 21, 235 (1985).58 H.-G. Rammensee, T. Friede, and S. Stevanovic, Immunogenetics 41, 178 (1995).

Screen PS-SCL

Generate a matrix based on the activityof each mixture of the PS-SCL

Test individual peptides for T cellstimulatory activity

Score and rank all peptides fromproteins contained in database(s)

Few mixtures areclearly active

Synthesize allcombinations of aminoacids defining the most

active mixtures

Many mixtures showsimilar activity

Generate and screen a biasedlibrary with reducedcomplexity based on

screening results

B: Biometrical analysis:Identification of natural epitopes in

protein databases

A: Identification of epitope mimics that donot necessarily correspond to natural

epitopes

Synthesize top ranked peptides

Fig. 4. Flowchart of the successive steps involved in the optimization and identification of

T cell epitopes using (A) PS-SCLs and (B) PS-SCL-based biometrical analysis.


were not recognized by any of these clones. The disparity in recognition be-tween native pp65495–503 and these peptides may result from the peptides’ in-ability of interact with many different TCRs, which might be due to the highamino acid substitutions relative to the native sequence. These results there-fore suggest that the native sequence is recognized by a diverse group ofTCRs, and that its moderate immunogenicity is likely a compromise tomaintain broad recognition and protective immunity against CMV. In con-trast, the two epitope mimics identified from the library are highly antigenicbut recognized by a highly restricted number of TCRs, thus at the expense oflosing universal recognition. To maintain ‘‘broad recognition’’ while pre-serving improved activity, a series of p65405–503 peptide analogs weredesigned by substituting native amino acids with library-directed changes.Using this approach three peptides were identified that were 100-fold moreactive than the native ligand and, at the same time, conserved ‘‘broadrecognition.’’ Furthermore, all three analogs expanded peripheral bloodCTLs from CMV-seropositive individuals, which not only recognizedpeptide-coated targets, but were able to lyse CMV-infected targets.

The extent of CD8þ T cell activation can alternatively be assessed byquantitative evaluation of cytokine (e.g., �-interferon) production andrelease measured by standard sandwich ELISA. The assay to be usedfor evaluating the extent of T cell activation needs to be selected and


optimized for each T cell clone and T cell line prior to testing a PS-SCL. Itis indeed essential to optimize the T cell assay conditions in order toachieve the best level of sensitivity and reproducibility and to successfullyselect the most active mixtures. The basic protocols as well as optimizationstrategies have been described in detail.59 If a limited number of mixturesshow significant activity in each sublibrary, then a series of individual pep-tides representing all combinations of amino acids defining those mixturesare generated as described in the examples above. Alternatively, in thosecases in which too many mixtures show similar high activity in several sub-libraries, and in turn, the number of individual peptides to be synthesized istoo high, a biased PS-SCL is generated by fixing the positions where aminoacids having high specificity could be identified following the original li-brary screening, and including a limited number of amino acids at the otherpositions selected based on the results of the primary screening. This strat-egy was successfully applied for the identification of peptide ligands specificfor a diabetogenic T cell clone.60

Identification of Natural Epitopes in Protein Databases

With the inclusion of computational methods in T cell immunology, anumber of strategies have emerged that use computer algorithms to predictwhich peptide fragments from an untested protein will fit into an MHCmolecule and trigger an immunological response.61 Although there is an in-creasing number of different computer algorithms, they all are based on theavailable databases for the MHC anchors, together with the assumption ofan independent contribution of each amino acid in the peptide sequence toMHC binding.62 The recent development of a large database of the se-quences of natural epitopes eluted from both MHC class I and class II mol-ecules has provided a new basis for computer models to predict epitopes.63

PS-SCL-based biometrical analysis is a novel strategy that uses the powerof computers and the information available in the databases to systematic-ally compare the data derived from a PS-SCL screening with all peptidefragments present in protein databases.64 Unlike other strategies using

59 C. Pinilla, J. R. Appel, and R. A. Houghten, in ‘‘Current Protocols in Immunology’’ (J. E.

Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds.), p. 9.5.1.

John Wiley & Sons, New York, 2001.60 V. Judkowski, C. Pinilla, K. Schroder, L. Tucker, N. Sarvetnick, and D. B. Wilson, J.

Immunol. 166, 908 (2001).61 M. Hagmann, Science 290, 80 (2000).62 T. Sturniolo, E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler,

F. Gallazzi, M. P. Protti, F. Sinigaglia, and J. Hammer, Nat. Biotechnol. 17, 555 (1999).63 V. Brusic, J. Zeleznikow, and N. Petrovsky, J. Immunol. Methods 238, 17 (2000).64 Y. Zhao, B. Gran, C. Pinilla, S. Markovic-Plese, B. Hemmer, A. Tzou, L. W. Whitney, W. E.

Biddison, R. Martin, and R. Simon, J. Immunol. 167, 2130 (2001).


mathematical algorithms, the biometrical analysis is not based on the de-scribed MHC motifs nor is any previous knowledge of the restriction elem-ent for a given T cell clone required. The different steps of the biometricalanalyses are described in Fig. 4, pathway B.

The first example of the PS-SCL-based biometrical approach was thestudy of the specificity of GP5F11,64 a CD4þ T cell clone that was establishedfrom peripheral blood of a patient with multiple sclerosis using influenzavirus hemagglutinin (HA) peptide 309–318 as an antigen. The biometricalanalysis based on the screening of a decapeptide PS-SCL predicted thenative peptide (HA 309–318) as the sixth peptide out of over 20 million dec-apeptides analyzed having the highest stimulatory index for this clone, andtwo analogs of the same HA sequence ranked third and eighth. Synthesis ofthese peptides confirmed their activity for GP5F11. Similarly, the nativeligand was predicted among peptides having high stimulatory activity fromthe biometrical analysis based on the screening of a nonapeptide PS-SCLusing a CD8þ T cell clone that recognized HIV-1 Gag p17-derived epitopeSL9.65 Thus, the SL9 epitope was ranked 116 out of over 22 million nonapep-tides with high predicted stimulatory activity and the nonapeptides thatranked first and second were naturally occurring double substitutions of thisepitope. Other examples with T cell clones of known specificity have beenreported for a Melan-A (a melanoma associated antigen)-specific CD8þ

T cell clone, with a well known epitope (Melan-A 26–35)51 and for a tyrosi-nase (another melanoma-associated antigen)-specific CD8þ T cell clone,with a known epitope (tyrosinase peptide 368–376).53 Melan-A 26–35 waspredicted as the fourth decapeptide with high stimulatory activity, while tyr-osinase peptide 368–376 ranked tenth following the biometrical analysesbased on the screening of a nonapeptide PS-SCL and twenty-ninth whenthe analysis was based on the screening of a decapeptide PS-SCL. Theseexamples using T cell clones of known specificity clearly demonstrate thestrength of the PS-SCL-based biometrical analysis approach for the identifi-cation and optimization of native ligands from T cell clones of both knownand unknown specificity. This is further supported in the following examplein which the clone specificity was unknown.

Thus, the PS-SCL-based biometrical analysis was applied for the identi-fication of T cell ligands for CSF-3, a CD4þ clone of unknown specificity thatwas isolated from the cerebrospinal fluid of a patient with chronic Lyme dis-ease of the central nervous system.47 Each mixture of a decapeptide PS-SCL

65 C. Boggiano, C. Pinilla, B. D. Walker, and S. E. Blondelle, in ‘‘Peptides: The Wave of the

Future. Proceedings of the 2nd International/17th American Peptide Symposium’’ (R. A.

Houghten and M. Lebl, eds.), p. 1023. Kluwer Academic Publishers, Norwell and

Dordrecht, 2001.


was assayed for activation of CSF-3 in a standard proliferation assay thatmeasure the incorporation of [3H]thymidine into newly synthesized DNA(using irradiated MHC matching PBMCs as APCs). A matrix was then gener-ated based on the screening results. The entry for a given amino acid in a spe-cific position was based on the stimulation index obtained for the mixturefrom the PS-SCL corresponding to that amino acid defined in that position.The biometrical analysis then used this matrix to score all decapeptides inthe entire Borrelia burgdorferi (the agent causing Lyme disease) genome aswell as in databases compiling all known human or viral proteins by movinga scoring window across the known protein sequences in increments of oneamino acid and adding the individual stimulatory values relevant to eachamino acid and its position of each decamer sequence. This way, peptides pre-sent in databases can be identified in a complete unbiased fashion and with un-precedented efficiency, and ranked according to a score that is predictive oftheir stimulatory potency. Each peptide was then ranked based on their pre-dicted stimulatory score. The percentage of sequences with high scores wassubstantially greater in the B. burgdorferi genome than in the human and viraldatabases, suggesting a higher probability of the T cell clone recognizingB. burgdorferi antigens. The best ranking sequences derived from B. burgdor-feri, human, or viral proteins were synthesized and tested to confirm theirstimulatory capacity. As anticipated, the most active peptides derived fromB. burgdorferi. The primary amino acid sequences of these peptides differedsubstantially, indicating that little or no sequence homology was requiredfor cross-recognition. These studies demonstrate the effectiveness of thePS-SCL-based biometrical approach for the identification of specific targetepitopes for an organ-infiltrating T cell population that had been stimulatedwith a crude lysate from an infectious organism.

Conclusions

One of the fundamental objectives in organic and medicinal chemistry isthe design, synthesis, and production of molecules having value as humantherapeutic agents. The successes of organic and medicinal chemistry com-bined with the rapid expansion of combinatorial chemistry have fundamen-tally changed human therapeutics over the past 30–40 years. It can beexpected from the current successes that the ability to synthesize anddensely search the molecular space of therapeutically important receptorswill permit highly active analogs of existing pharmacophores to be identi-fied. Furthermore, the use of combinatorial variance of synthetic reactionconditions will allow the identification of entirely new synthetic approachesto novel pharmacophores. Combinatorial chemistry and synthetic ap-proaches can then be expected to rapidly lead to more active, more specific,


safer, and less expensive therapeutics. Mixture-based SCL approaches asdescribed may be considered an ‘‘optimized approach’’ of the identificationof novel pharmacophores from natural sources. Indeed, both SCLs and nat-ural product extracts are composed of mixtures of compounds that are dir-ectly screened without intensive initial purification. In both cases, a largenumber of active individual compounds were identified from such mixtures.

Although not thoroughly studied in the area of infectious disease, thereported successes over the past 12 years of combinatorial library ap-proaches show the ability to synthesize and densely search the molecularspace against therapeutically important targets such as drug-resistant mi-croorganisms and HIV, permitting highly active antimicrobial, antifungal,and antiviral compounds to be identified. Compounds such as those de-scribed here can serve as starting points for the development of therapeuticagents that inhibit or treat bacterial, fungal, or viral infection. In particular,the identified small molecules have the desired physicochemical propertiesfor oral therapeutic application. Thus, they are small in size (molecularweight less than 1000 Da) and are unlikely to be subject to proteolysis.

The efficiency of PS-SCLs combined with biometrical analyses to identifyhighly active B cell and T cell ligands has now been clearly demonstrated.These studies have also led to the identification of immunologically relevantpeptides for use as potential preventive and therapeutic vaccines, provided in-sight into the requirements for TCR interactions with peptide/MHC com-plexes in immunogenicity, and established new principles regarding thelevel of cross-reactivity in immunological recognition. As shown in theexamples described above, the use of PS-SCLs in studies of T cell specificitycan be directed toward the identification of two types of T cell ligands: (1)those that do not necessarily correspond to sequences in reported proteins(epitope mimics), and (2) when combined with biometrical analyses, thosecorresponding to proteins present in a database (optimal native epitopes).Such strategies are then expected to greatly increase the potential to developsequences with multiple clonal recognition and cross-reactivity ability and tobe of great value for the development of vaccines against infectious diseases.

Acknowledgments

The authors thank Dr. Houghten, Dr. Nefzi, and John Ostresh at Torrey Pines Institute

for Molecular Studies for their scientific discussion and Marc Giulianotti, Ed Brehm, and Ema

Crooks for their technical assistance. The authors also thank Dr. Berger at the National

Institute of Allergy and Infectious Diseases for providing the vaccinia virus constructs, and

Dr. Mosier at the Scripps Research Institute for providing the HIV-1 isolates. This research

was supported in part by Universitywide AIDS Research Program Grant R-98-TPI-064,

National Institute of Dental and Craniofacial Research Grant RO1-DE12923, and Mixture

Sciences, Inc., San Diego, California.

[19] applications of polymer-supported reagents 347

[19] Advances in the Applications of Polymer-SupportedReagents for Organic Synthesis

By Alessandra Bartolozzi, Michael J. Grogan, and Steven A. Kates

Introduction

In the last half of the twentieth century organic chemistry evolved to alevel of extreme sophistication in which complex molecules could be pre-pared from basic building blocks. The process typically involves perform-ing a reaction in an organic solvent followed by isolation of a desiredproduct from excess reagents and reaction by-products. Subject to chemicalproperties unique to the individual components, these reactions and purifi-cations are tedious and time consuming, and require considerable expert-ise. Bruce Merrifield was the first to apply an alternative approach for theiterative synthesis of polypeptides, for which he was awarded the NobelPrize in 1984.1 By covalently anchoring the starting amino acid to a solidsupport, he was able to perform synthetic steps with known solution-phasechemistry, and purification of bound intermediates could be effected withquick solvent washes. Following chain elongation, the bound polypeptidewas cleaved from the solid support with concomitant release of the side-chain-protecting groups. The physical differences between compounds thatare so pronounced for solution-phase synthesis are leveled for solid-phasesynthesis by the common denominator for each reaction, the solid support.Key advantages to the solid-phase technique include simple filtration andwashing without manipulative losses as well as lending itself easily to auto-mation. Subsequent to Merrifield’s discovery, other repetitive processessuch as oligonucleotide synthesis2 and protein sequencing3 have beenadapted to automated solid-phase methods.

Beginning in the early 1990s, solid-phase techniques were combinedwith modern synthetic methods to construct small molecules for drug dis-covery. With a starting material on solid support, sets of reactants arecoupled in a combinatorial fashion. Various strategies of solid-phaseorganic synthesis, with or without automation, are used to prepare librariesof low-molecular-weight structures. Common to all approaches is thesimple ability to wash the solid support in order to add reagents or remove

1 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).2 R. L. Letsinger and M. J. Kornet, J. Am. Chem. Soc. 85, 3045 (1963).3 R. A. Laursen, Eur. J. Chem. 20, 89 (1971).



348 polymer-assisted approaches [19]

impurities, enabling the rapid execution of parallel reactions to generatesets of compounds. As an alternative to building compound libraries onresin, solid-supported synthetic reagents have been developed. This hasthe advantage that two additional steps, compound attachment and cleav-age associated with solid-phase synthesis, are not required, and yet the keyadvantage of purification by filtration remains possible. Supported reagentsare therefore particularly useful for quick reaction execution and com-pound optimization necessary for drug discovery. Functionalized polymershave also been used as scavengers to purify solution-phase reactions,whereby one reactant may be used in large excess and the polymer is addedto capture either the product or impurities.

Supported scavengers and reagents for a wide range of synthetic con-versions have been developed and are becoming commercially available.Compared to the linkers incorporated in solid-phase synthesis, these re-agents are more generally accessible. This chapter discusses new develop-ments in polymer-supported reagents for 2001–2002, with reagents groupedby different classes of chemical transformations. The features and charac-teristics of the common solid supports, polystyrene (PS),* polyethyleneglycol (PEG), and silica, have been extensively reviewed and will notbe discussed in this work.4 For other discussions of solid-supportedreagents and solid-phase synthesis, a number of excellent reviews havebeen written.5,6

* Abbreviations: AIBN, 2,20-azobisisobutyronitrile; Alk, alkyl; Ar, aryl; Bn, benzyl; Boc, tert-

butyloxycarbonyl; t-But, tert-butyl; Cbz, benzyloxycarbonyl; DBU, 1,8-diazabicy-

clo[5.4.0]undec-7-ene; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide;

DMSO, dimethylsulfoxide; Fmoc, 9-fluorenylmethoxycarbonyl; GC, gas chromatography;

Hal, halogen; HOSu, N-hydroxysuccinimide; Me, methyl; PEG, polyethylene glycol; PS,

polystyrene; PS-TEMPO, 4-(polystyrylmethyloxy)-2,2,6,6-tetramethylpiperidin-1-yloxy;

RCM, ring-closing metathesis; ROMP, ring-opening metathesis polymerization; RP-HPLC,

reverse-phase high-performance liquid chromatography; Sar, sarcosine; Suc, succinoyl;

TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin-layer chromatography; UV,

ultraviolet.4 S. A. Kates and F. Albericio, Eds., ‘‘Solid-Phase Synthesis: A Practical Guide.’’ Marcel

Dekker, New York, 2000.5 For previous reviews, see (a) S. V. Ley, I. R. Baxendale, R. N. Bream, P. S. Jackson, A. G.

Leach, D. A. Longbottom, M. Nesi, J. S. Scott, R. L. Storer, and S. J. Taylor, J. Chem. Soc.

Perkin Trans I 3815 (2000). (b) A. Kirschning, H. Monenschein, and R. Wittenberg, Angew.

Chem. Int. Ed. Engl. 40, 650 (2001). (c) Y. R. de Miguel, E. Brule, and R. G. Margue, J. Chem.

Soc. Perkin Trans I 3085 (2001). (d) J. Eames and M. Watkinson, Eur. J. Org. Chem. 1213

(2001). (e) B. Clapham, T. S. Reger, and K. D. Janda, Tetrahedron 57, 4637 (2001).6 (a) C. Blackburn, F. Albericio, and S. A. Kates, Recent Res. Dev. Org. Chem. 1, 477 (1997).

(b) C. Blackburn, F. Albericio, and S. A. Kates, Drugs Future 22, 1007 (1997). (c) S. A.

Kates, Biopolymers 47, 309 (1998).


Acids and Bases

Polymer-supported acids and bases have been used extensively as ion-exchange resins for purifications and reagents for synthetic transform-ations. Their handling properties guarantee easy manipulations allowingrapid reaction optimization and parallel synthesis development. Using vari-ous polystyrene-amine resins in three different synthetic steps, Yadav-Bhatnagar et al.7 performed the parallel synthesis of cysteine trap proteaseinhibitors. A set of benzyloxycarbonyl (Cbz)-protected amino acids is con-verted with diazomethane to �-diazoketones by activation with a polystyr-ene-supported amine (Table I, entry 1). The �-diazoketones are convertedto the corresponding �-bromoketones by treatment with polystyrene-basedammonium bromide (entry 2). Nucleophilic displacements of these brom-ides by carboxylic acids, thiols, and amines are promoted with polystyr-ene-supported dimethylamine affording a library of 80 highly purepeptide-based compounds (entry 3).

Kirschning et al.8 succeeded in promoting glycosylations of alcoholswith a supported silylsulfonate as a Lewis acid (entry 4). Treatment of aglycosyl acetate with the polymer-bound silyl triflate efficiently transfersthe carbohydrate to a range of alcohols, affording the corresponding2-iodo-�-glycosides in high yields. Subsequent reductions provide 2-deoxysugars.

Dondoni and Massi9 executed a Bignelli synthesis to prepare dihydro-pyrimidinones starting from aldehydes, 1,3-dicarbonyl compounds, andurea with ytterbium(III) supported on Amberlyst 15 resin as a Lewis acid(entry 5). After condensation, the reaction mixture is treated with bothstrongly basic and acid resins to sequester reaction by-products. For morethan 30 reported examples, dihydropyrimidones obtained directly fromconcentration of the filtrates are produced in high yields and in excellentpurities.

To recycle a valuable amine acylation catalyst, Janda and co-workers10

attached a proline-based catalyst to a polymeric support for the enan-tioselective kinetic resolution of alcohols (entry 6). The resin-bound cata-lyst behaves similarly to the soluble catalyst, providing good yields ofsecondary alcohols and their corresponding esters with good to excellentenantioselectivities for various substrates.

7 N. Yadav-Bhatnagar, N. Desjonqueres, and J. Mauger, J. Comb. Chem. 4, 49 (2002).8 A. Kirschning, M. Jesberger, and A. Schonberger, Org. Lett. 3, 3623 (2001).9 A. Dondoni and A. Massi, Tetrahedron Lett. 42, 7975 (2001).

10 B. Clapham, C.-W. Cho, and K. D. Janda, J. Org. Chem. 66, 868 (2001).

TABLE I

Acids and Bases

Entry

number

Supported

reagent Transformation Reference

1NH

Cbz

O

OH

R

O

O

Cl1)

PS-amine, DMF

2) CH2N2, CH2Cl2

NH

Cbz

O

R

N2

R = Bn, Alk, Indolyl

7

2CH2Cl2

ON2

NH O

R

BrPS-HBrNH

Cbz

R

Cbz 7

3 NH

Cbz

O

RRCOOH or RSH

or R1R2NH

O

R

NH

NH

Cbz

Cbz

O

R

NH

Cbz

O

R

Br

OCOR

SR

NR1R2

7

4

OI

BzOTBSO

OAc

OI

BzOTBSO

OR

R = Alk, Bn, sugar, steroid

CH2Cl2 or Et2O

R OH8

5 O

R2 R3

O

H H2N

O

NH2

O

R1

+ +

NH

NH

O

R3

R1

O

R2Toluene 9

6 R1 R2

OHBzCl, NEt3

R1 R2

OBz

R1 R2

OH+

R1, R2 = Alk, Bn

10


Synthesis of Dihydropyrimidones (Entry 5)

A screw-capped vial containing a magnetic stir bar was charged withYb(III) resin (160 mg), urea (1.50 mmol), aldehyde (0.50 mmol), and �-dicarbonyl (0.50 mmol) and heated at 120

� for 5 min. Yb(III) resin

(170 mg) was added and the mixture was heated at 120 �

for 48 h withgentle stirring. After cooling to 60

�, methanol (1 ml) was added. The sus-

pension was stirred for an additional 30 min then the resin was filteredand washed thoroughly with ethyl acetate. Amberlyst 15 (400 mg) and

TABLE II

Oxidations–Reductions

Entry

number

Supported


7 R

OH

R1

R1 = H, Alk

R

O

R1

KBr, NaOCl

CH2Cl2 11

8

KBr, NaOCl

CH3CN

R = Alk, Bn

R OHR OH

O

12

9

O

H

OH

HCH2Cl2 13

10R

R1RhCl(PPh3)3

DMSO

RR1

R = Et, Ph

R1 = COOH, COOMe,CN, COMe, CHO

14


Ambersep 900 �OH (400 mg) were added to the combined filtrates. Thesuspension was shaken for 2 h and then the resins were filtered and washedthoroughly with methanol. The combined filtrates were concentrated togive dihydropyrimidinones.

Oxidations and Reductions

The Anelli oxidation of alcohols to aldehydes and ketones has beenaccomplished using polymer-supported nitroxyl radical catalysts. The pra-cticality of removing polymer-supported reagents by filtration to simplifyproduct purification is highlighted by these examples. Bolm and co-workers11 demonstrated that a silica-supported nitroxyl catalyst is easilyfiltrated after use from the reaction solution, recovered and recycled, andthe residual inorganic salts present in the reaction mixture are separatedfrom the organic product by aqueous extraction (Table II, entry 7).

11 T. Fey, H. Fischer, S. Bachmann, K. Albert, and C. Bolm, J. Org. Chem. 66, 8154 (2001).


As an extension to this synthetic method, Yasuda and Ley12 performedthe Anelli oxidation with a polystyrene-supported nitroxyl radical and apolymer-supported chlorite (entry 8). The latter is incorporated in the reac-tion to oxidize the intermediate aldehydes to carboxylic acids. The choiceof reagents and reaction design affords pure carboxylic acids in high yields.

Various polymer-supported hydrides have been applied successfully toreductions of both carbonyl and olefin groups. Rajasree and Devaky13 de-scribe a cross-linked polystyrene-supported ethylenediamine borane re-agent for the selective reduction of aldehydes in the presence of ketones(entry 9). This borane reagent is easily prepared and can be recycledafter completion of the reaction. This is a practical alternative to stand-ard borane reagents such as diborane, borane-amine, or borane-sulfidecomplexes.

Desai and Danks14 reported the use of a polymer-supported formatefor transfer hydrogenation to alkenes with Wilkinson’s rhodium catalyst(entry 10). A range of unsaturated carbonyl compounds is reduced to thecorresponding alcohols by the formate resin under microwave irradiation.Reactions occur rapidly and afford the desired product in high yields. Typ-ical reagents for solution-phase transfer hydrogenations such as ammo-nium formate salts have the propensity to sublime and be hygroscopic.The polymer-supported formate circumvents these drawbacks providinggreater reproducibility, particularly when optimizing and scaling reactionconditions.

Synthesis of Carboxylic Acids (Entry 8)

Prior to use, 4-(polystyrylmethyloxy)-2,2,6,6-tetramethylpiperidin-1-yloxy (PS-TEMPO) (18.8 mg, 0.03 mmol) was washed with acetonitrile(2 � 2 ml). A solution of NaOCl (8.5 mg, 0.01 mmol) in water (0.5 ml)was added to a suspension of washed PS-TEMPO, PS-chlorite (0.70 g,1.50 mmol), PS-dihydrogen phosphate (154 mg, 0.30 mmol), and KBr(3.6 mg, 0.03 mmol) in acetonitrile (1 ml). After 10 min at room tempera-ture, a solution of the alcohol (0.30 mmol) in acetonitrile (1 ml) was added.The mixture was stirred at room temperature for 24–48 h, then passedthrough a short plug of Celite (1 g). After removal of the solvent underreduced pressure, the residue was dissolved in acetonitrile (0.5 ml) andpassed through a short plug of silica (0.3 g) eluting with 25% methanolin chloroform (10 ml). The filtrate was concentrated to give the desiredcarboxylic acid derivative.

12 K. Yasuda and S. V. Ley, J. Chem. Soc. Perkin Trans. I 1024 (2002).13 K. Rajasree and K. S. Devaky, J. Appl. Polym. Sci. 82, 593 (2001).14 B. Desai and T. N. Danks, Tetrahedron Lett. 42, 5963 (2001).


Hydrogenation of Alkenes (Entry 10)

E-3-Phenylpropenoic acid (0.025 g, 0.16 mmol), Amberlite IRA 938-supported formate (0.2 g) and Wilkinson’s catalyst (0.04 g, 0.0043 mmol)were suspended in dimethylsulfoxide (0.5 ml) in a reaction tube. The mix-ture was irradiated with microwaves at 100 W for 30 s. On cooling, themixture was diluted with dichloromethane (5 ml) and filtered. The filtratewas washed with water (2 � 5 ml). The organic layer was dried over anhyd-rous magnesium sulfate, the solvent removed under reduced pressure, andthe residue was purified by filtration through a plug of alumina to give3-phenylpropanoic acid (0.024 g, 95% yield).

Sulfur and Phosphorous Transfer Reagents

New polymer-supported reagents for sulfide transfer have been de-veloped to avoid exposure to malodorous and toxic sulfur reagents. Leyet al.15 prepared a stable aminothiophosphate polystyrene resin for the con-version of secondary and tertiary amides to thioamides in high conversionand purity (Table III, entry 11). This procedure is extremely clean andaffords the desired product with short reaction times in comparison to Law-esson’s reagent. In addition, the aminothiophosphate resin dehydratesprimary amides to nitriles.

A supported sulfide source has been demonstrated by Zhang et al.16 tosynthesize oligonucleotide phosphorothioates (entry 12). Aminodithiazole-thione attached to a methacrylate-ethyleneglycol copolymer is used to effi-ciently convert a nucleotide phosphite to a phosphorothioate. The productnucleotide possesses protecting groups suitable for solid-phase oligonu-cleotide synthesis and thus is a valuable building block for nucleic acidtherapeutics.

Ley and Taylor17 reported a polymer-supported oxazaphospholidine toconvert isothiocyanides to isonitriles for subsequent application in Ugithree-component coupling reactions (entry 13). This method affords cleanisonitriles and facilitates the handling of a toxic and unstable reactant.

Glycosyl phosphates are important building blocks in carbohydrate syn-thesis. Hindsgaul and co-workers18 applied a capture-phosphorylationmethod with a polystyrene-supported phosphoramidite to simplify theirsynthesis (entry 14). Unreacted alcohols are removed by washing the resinand the desired glcyophosphates are released in high purity by a standard

15 S. V. Ley, A. G. Leach, and R. I. Storer, J. Chem. Soc. Perkin Trans I 358 (2001).16 Z. Zhang, Y. Han, J. X. Tang, and J.-Y. Tang, Tetrahedron Lett. 43, 4347 (2002).17 S. V. Ley and S. J. Taylor, Bioorg. Med. Chem. Lett. 12, 1813 (2002).18 K. Parang, E. J.-L. Fournier, and O. Hindsgaul, Org. Lett. 3, 307 (2001).

TABLE III

Sulfur -Phosphorous Transfer Reagents

Entry

number

Supported


11 R

O

NMe2

R = Alk, Ar

R

S

NMe2

200� C, microwave

NNMe

MePF6

Ionic solvent:

15

12 PR1O OR2

OR3

PR1O OR2

OR3

S

CH2Cl216

13Toluene

MicrowaveR N C S R N C 17

14

1) THF, 1H -tetrazole, PS-phosphoamidite2) t -BuOOH, THF

3) DBU, THF4) NaOMe, MeOH, dioxane

P

O

O−O

O−

R = sugar, nucleoside

R OH R18


three-step procedure. This method avoids using excess phosphoramiditesand reduces polyphosphorylation of diols and thus is a flexible tool forthe synthesis of carbohydrate and nucleotide monophosphates.

Carbon Transfers

To provide a source of allyl nucleophile for additions to aldehydes,Barrett and co-workers19 prepared an allylboronate nucleophile attachedto a norbornene diol gel generated by ring-opening metathesis polymeriza-tion (ROMP) (Table IV, entry 15). This ROMP gel reagent has physicalproperties more desirable than the commonly used low-weight boranesand boronic acid reagents. The homoallylic alcohols are produced in highyield starting from a wide range of aldehydes.

To avoid the introduction of soluble phosphines into a reaction mixture,Westman20 performed a one-pot phosphorous ylide formation and Wittig

19 T. Arnauld, A. G. M. Barrett, and R. Seifried, Tetrahedron Lett. 42, 7899 (2001).20 J. Westman, Org. Lett. 3, 3745 (2001).

TABLE IV

Carbon Nucleophiles

Entry

number

Supported


15 OOB

n

R = H, MeR

R1

O

H R1

OH R

R1 = Alk, Ar

19

16R1

O

HBr R2

K2CO3

MeOH+ R1R2

R1 = Ar, Heterocycle

R2 = COOMe, Ph, CN, COPh

20

17R = Alk, Bn

R1 = Alk, Ar, Bn

OS

OHR1

O O

OS

OR1

O OR

21


reaction on solid support (entry 16). Microwave irradiation of variousaldehydes and alkyl halides in the presence of a polymer-supported triphe-nylphosphine produced the desired olefins in high purity with reactiontimes <5 min.

To prepare alkyl sulfonate esters, sulfonic acids were treated withsupported alkyl triazenes (entry 17).21 The acids decompose the triazenesto diazoalkanes, which subsequently alkylate the sulfonates in situ. Thesesupported triazenes avoid the dangerous handling of pure diazoalkanes.

Synthesis of Olefins via Wittig Reaction (Entry 17)

An aldehyde was mixed with solid supported triphenylphosphine oxide(3 equiv.), alkyl halide (4 equiv.), and potassium carbonate (4 equiv.) inmethanol (2 ml). The mixture was heated at 150

� for 5 min. The residue

was filtered through a short plug of silica gel and washed. The solutionwas concentrated and purified by reverse-phase high-performance liquidchromatography (RP-HPLC).

21 N. Vignola, S. Dahmen, D. Enders, and S. Brase, Tetrahedron Lett. 42, 7833 (2001).


Electrophilic Reagents and Radical Reaction Reagents

Polystyrene-bound selenium bromide has made organic transform-ations involving electrophilic selenium readily accessible.22 Compared tosolution phase, the supported reagent simplifies the purification step redu-cing the exposure to toxic materials. Huang and Sheng23 have used thisversatile reagent to capture phosphorous carbon ylides on the support(Table V, entry 18). These selenoalkylidenes react with aldehydes to formvinylic selenides that, upon acidic hydrolysis, are cleaved to releasealdehydes or ketones from the resin.

With a polymer-bound chiral selenium bromide reagent, the stereose-lective activation of olefins has been accomplished (entry 19).24 Uehlinand Wirth24 found that both mesoporous silica and polystyrene-supported

TABLE V

Electrophilic and Radical Reactions

Entry

number

Supported


18

1) Supported ylide

2) H+R2

O

HR2

O

R1

R2 = Alk, Ar

23

19

PhR

R = H, Alk, OAlk

R1

PhR

OR1

R1 = Alk, Bn

OH

1) PS-SeBr

2) Bu3SnH24

20 Activated carbon

R = Alk, Ar

+1) Heat

2) Activated carbonR I R H

SnMe2

H

25

21 26

22 K. C. Nicolaou, J. Pastor, S. Barluenga, and N. Winssinger, Chem. Commun. 1947 (1998).23 X. Huang and S.-R. Sheng, Tetrahedron Lett. 42, 9035 (2001).24 L. Uehlin and T. Wirth, Org. Lett. 3, 2931 (2001).


reagents gave clean products with moderate to good enantiopurities afterreductive, nucleophilic, or oxidative cleavage of the organoselenium inter-mediates. For all these examples, the recovered selenium bromide reagentcan be regenerated for repeated use.

Trialkyltin hydrides are common reagents in organic radical chemistry,but the toxic by-products are extremely difficult to remove from reactionproducts. To facilitate their removal, a pyrene-functionalized tin hydridehas been prepared (entry 20).25 After standard solution-phase radical reac-tions with the stannane, filtration through activated carbon traps the tinspecies to afford pure products.

As a general platform for converting carboxylic acids or alcohols intoreactive radicals, a supported N-hydroxythiazole-thione has been de-veloped (entry 21).26 The intermediate heterocyclic carboxylate esters aretransformed into pure alkyl bromides by photolysis in the presence ofBrCCl3. In a second application, the support contained an alkyl ether asopposed to a hydroxyl group. Cleavage under similar conditions provideda furan by the cyclization of a released oxygen radical. The heterocyclenecessary for this radical process remains attached to the Wang resin,presenting an alternative to commonly used thiohydroxamates.27

Reaction of Pyrene-Functionalized Tin Hydride with an Alkyl Halide(Entry 20)

A solution of substrate (0.20 mmol), pyrene-dimethyltinhydride (1.2equiv.), and 2,20-azobisisobutyronitrile (AIBN, 0.1 equiv.) in dry degassedbenzene (1–2 ml) was stirred under reflux for 1 h [thin-layer chromatog-raphy (TLC) monitoring] under inert atmosphere. The mixture was cooledto room temperature and evaporated. Methanol/dichloromethane (3:2,4 ml) was added followed by the addition of activated carbon (800 mg).The suspension was stirred for 30 min [the adsorption of the pyrene corewas monitored by ultraviolet (UV)]. After filtration, the activated carbonwas washed with methanol and the combined filtrates were concentratedto yield the product in pure form.

Supported Transition Metal Catalysts

Organometallic complex catalysts have been removed from reactionmixtures with solid-supported phosphines as well as other ligands to facili-tate reagent handling and product purification. Alternatively, Leadbeater28

25 S. Gastaldi and D. Stien, Tetrahedron Lett. 43, 4309 (2002).26 L. De Luca, G. Giacomelli, G. Porcu, and M. Taddei, Org. Lett. 3, 855 (2001).27 D. H. R. Barton, D. Crich, and W. B. Motherwell, Tetrahedron 41, 3901 (1985).28 N. E. Leadbeater, J. Org. Chem. 66, 2168 (2001).


used polystyrene-supported triphenylphosphine as a ligand for an oxida-tion ruthenium(II) complex (Table VI, entry 22). The utility of this com-plex for the hydrogenation of ketones to alcohols as well as for theoxidation of methylenes or alcohols to ketones is demonstrated. For theexamples described, the yields of the reactions are similar or higher thanthose reported for the soluble ruthenium complex.

TABLE VI

Transition Metal Catalysts

Entry

number

Supported


22 R R1 R

O

R1Peracetic acid

1,2-dichloroethane

R,R1 = Alk, Ar

28

23

EtOOC COOEt COOEtEtOOCCH2Cl2 29

24Ar +

R, R1 = H, Ar, Alk

X ArR

HNR1

RN

R1

X = Hal

Pd(OAc)230

25+ B Ar1Ar X Ar1 Ar

X = Hal

OH

OHPd2(dba)3 30

26

OO O

11

Pd

SPh

SPh

Cl

50 R

I

+

T = 1208C

DMF, NEt3

R = COOMe, Ph,COO-t-Bu, CN

R

31

27

O

OBu

X

R

Pd(OAc)2Bu4NOAcscCO2

R

COOBu

+

X = Br, I

32


A variant of the widely used Grubbs ruthenium alkylidene catalyst forthe ring-closing metathesis (RCM) and ROMP of olefins was constructedon a polystyrene resin by Mol and co-workers29 (entry 23). The phosphineligands in the ruthenium complex are potential points of attachment tosolid phase; however, they must disassociate for olefin metathesis to occur.Thus, a supported carboxylate ligand is chosen to replace one of the an-ionic chloride ligands of the Grubbs complex to anchor to the resin. Thisstrategy ensures catalyst activity without loss of metal complexes in solu-tion phase. This supported catalyst is highly effective for both RCM andself-methathesis of olefins, and can be regenerated and stored withoutsignificant loss of activity.

Parrish and Buchwald30 performed couplings with a polystyrene-supported biphenyl-phosphine palladium complex between aryl halidesand either amines (entry 24) or boronic acids (entry 25). The resin-boundcomplex is analogous to the corresponding homogeneous compound and iseffective for couplings to unactivated aryl halides, including aryl chlorides.The complex is air-stable and retains activity after recovery withoutapparent loss of palladium.

Pollino and Weck31 executed Heck olefinations with a palladium com-plex containing an unusual sulfur–carbon–sulfur trivalent ligand supportedon a ROMP polymer backbone (entry 26). This is a very efficient cata-lyst and can be recycled with good retention of activity. The controlledspacing of the metal complexes along the ROMP polymer backbonesuggests the potential of supported complexes with designed intermetaldistances.

Gordon and Holmes32 used a supported triphenylphosphine–Pd(II)complex as an effective catalyst for Heck and Suzuki couplings in supercrit-ical carbon dioxide (entry 27). After optimization of the amine base for thereaction, the final products have been isolated in good yields and highpurity with no traces of metal.

Synthesis of Arylamines (Entry 24)

A solution of Pd(OAc)2 in toluene (1.00 ml of a 7.50 mM solution,7.50 �mol) was added to an oven-dried test tube (16 � 100 mm) containingdialkylarylphosphine resin (9.8 �mol) and sealed with a septum. The mix-ture was stirred at 2

�for 30–75 min. Sodium tert-butoxide (108 mg,

29 P. Nieczypor, W. Buchowicz, W. J. N. Meester, F. P. J. T. Rutjes, and J. C. Mol, Tetrahedron

Lett. 42, 7103 (2001).30 C. A. Parrish and S. L. Buchwald, J. Org. Chem. 66, 3820 (2001).31 J. M. Pollino and M. Weck, Org. Lett. 4, 753 (2002).32 R. S. Gordon and A. B. Holmes, Chem. Commun. 640 (2002).


1.12 mmol) was added to the flask, and the system was purged with Argon.An aryl halide (0.75 mmol), an amine (0.975 mmol), and toluene (0.50 ml)were added sequentially via syringe. The flask was placed into an 80

�oil

bath, and the reaction mixture was stirred for 15–20 h. After cooling,the mixture was diluted with ether (5 ml), filtered through Celite, andrinsed with ether (2 � 5 ml). The combined organic layers were dilutedwith ether (50 ml) and washed with a saturated aqueous bicarbonate solu-tion (3 � 50 ml). The organic phase was dried over anhydrous sodium sul-fate and removal of the solvents under reduced pressure provided purefinal products.

Synthesis of Biphenyls Via Suzuki Coupling (Entry 25)

Tetrahydrofuran (1.00 ml) was added to an oven-dried test tube (16 �100 mm) sealed with a septum containing dialkylarylphosphine resin(5.2 �mol) and Pd2(dba)3 (1.7 mg, 1.9 �mol). The mixture was stirred atroom temperature for 30–75 min. An aryl halide (0.75 mmol) was addedvia syringe. Cesium carbonate (489 mg, 1.50 mmol) and a boronic acid(0.90 mmol) were added simultaneously to the flask, and the system waspurged with Argon Tetrahydrofuran (0.50 ml) was added via syringe. Theflask was placed into a 65

�oil bath, and the reaction mixture was stirred for

17–21 h. After cooling, the mixture was diluted with ether (5 ml), filteredthrough Celite, and rinsed with ether (2 � 5 ml). The combined organiclayers were transferred to a separatory funnel and diluted with ether(50 ml). The organic layer was washed with 1 N aqueous NaOH solution(3 � 50 ml), dried over sodium sulfate, and concentrated in vacuo toprovide a pure product.

Esterification and Amide-Coupling Reagents

Carboxylic acids are activated by a wide variety of reagents for conden-sations with amines, alcohols, and other nucleophiles. Many condensationreactions generate by-products that are difficult to remove, thus supportedforms of the reagents can greatly improve the purification step. As anexample, Zander and Frank33 used a polystyrylsulfonyl chloride resin to ac-tivate carboxylic acids for esterification with a range of alcohols, includingphenols and tert-butanol (Table VII, entry 28). Also, Keck et al.34 improveda macrolactonization with a polymer-bound carbodiimide (entry 29). Com-pared to the solution-phase reagent, fewer equivalents of the supported

33 N. Zander and R. Frank, Tetrahedron Lett. 42, 7783 (2001).34 G. E. Keck, C. Sanchez, and C. A. Wager, Tetrahedron Lett. 41, 8673 (2000).

TABLE VII

Activation and Condensation of Carbonyl Compounds

Entry

number

Supported


28

NHFmocHO

R1

O

ONHFmoc

O

OHFmocNH

R2OHO

OR2

FmocNH

R1, R2 = Alk, Ar

R1OH

O

33

29O

HO OHHCl, CHCl3

n O

O

n

n = 8, 9, 11, 12

NN

34

30

O

N

Ph

n

OO

O O

OMeCF3

NH2R

R = Alk, Bn

RNH

O

F3COMe 35

31 NO

OO

Ph

n

O

O

R

RR = H, t-But

NH2

R1

O

R1O NH

R2

O

R1OK2CO3

O

O

R

R

36,37

32

1) Supported phosgene equivalent

2)

R1 N NR4

O

R2 R3

R1 NH

R2

R3

HNR4

38

33 39


carbodiimide are necessary to give the macrolactones in equivalent orhigher yields. The isolation of reactive sites on the solid support suppressesundesired oligomerizations of the hydroxy acids.

Depending on their stability, activated esters can be generated andstored as pure reagents on solid supports. The by-products of carbodiimides


and similar reagents for carboxylic acid activation can be removed beforecouplings with amines or other nucleophiles. This greatly simplifies purifi-cations and reduces reagent consumption. As an example, the widely usedMosher’s acid has been activated and incorporated as a polymer-supported N-hydroxysuccinimide ester (entry 30).35 Treatment of amineswith this reagent produces Mosher amides with high purities allowing easyderivatization of chiral amines for stereochemical determinations.

Group Transfer Reagents

For the 9-fluorenylmethoxycarbonyl (Fmoc) protection of amino acids,Chinchilla et al.36,37 prepared a similar ROMP-polymer that supports anactivated N-hydroxysuccinimide Fmoc-carbonate (Table VII, entry 31).Various Fmoc-amino acids are prepared in pure form after removal ofthe polymer reagent by filtration and aqueous phase separation.

Analogous to these amide-forming reactions, polymer-supported ben-zotriazole has been used to facilitate urea syntheses by capturing and acti-vating phosgene for sequential additions of amines (entry 32).38 The firstamine is reacted with the resin at room temperature to form an acylamineintermediate to the triazole support. The second amine is reacted with thesupported intermediate at higher temperature. The discrete temperaturedifference between the first and second amine additions ensure high purityof the asymmetric ureas. These coupling conditions have been optimizedfor automated parallel synthesis, and over 150 ureas have been rapidlyproduced and with good purities.

To synthesize guanidines, a carbamate-linked electrophilic reagentwas prepared on polystyrene (entry 33).39 Displacement of the reagent’ssulfonamide leaving group with different amines generates resin-boundintermediates that yield pure guanidines after treatment with trifluoroace-tic acid. The reagent is effective even for less reactive secondary aminesand simplifies the purification and handling of the highly polar guanidineproducts.

35 T. Arnauld, A. G. M. Barrett, B. T. Hopkins, and F. J. Zecri, Tetrahedron Lett. 42, 8215

(2001).36 R. Chinchilla, D. J. Dodsworth, C. Najera, and J. M. Soriano, Tetrahedron Lett. 42, 7579

(2001).37 R. Chinchilla, D. J. Dodsworth, C. Najera, and J. M. Soriano, Bioorg. Med. Chem. Lett. 12,

1817 (2002).38 A. Paio, R. F. Crespo, P. Seneci, and M. Ciraco, J. Comb. Chem. 3, 354 (2001).39 C. W. Zapf, C. J. Creighton, M. Tomioka, and M. Goodman, Org. Lett. 3, 1133 (2001).


Synthesis of Esters (Entry 28)

N-Methylimidazole (22.5 �l) was added to a mixture of Fmoc-glycinol(20.0 mg, 0.07 mmol), Fmoc-glycine (27.3 mg, 1.3 equiv.), and polystyryl-sulfonyl chloride resin (61.6 mg, 1.3 equiv., 1.49 mmol/g) in dichloro-methane (1 ml). After 2 h, aminomethylated PS-resin (138 mg, 2 equiv.)was added and the suspension shaken for 30 min. After filtration througha fritted filter, the resin was rinsed thoroughly with dichloromethane, andthe combined solvents were concentrated to �3 ml. Amberlyte 15(176 mg) was added and the suspension was shaken again for 30 min andfiltered as described above. After evaporation of the solvent, the pureproduct was obtained as a colorless foam 93% yield.

Synthesis of Fmoc-Amino Acids (Entry 31)

A solution of the amino acid (0.40 mmol) and potassium carbonate(39 mg, 0.40 mmol) in water (15 ml) was added to a suspension of Fmoc-P-OSu (270 mg, 0.40 mmol) in acetone (20 ml). The suspension was stirredat room temperature for 24 h and the solvents were removed under re-duced pressure. Toluene was added (20 ml) and the resulting solid wasfiltered. The solid was suspended in water (20 ml) and the P-HOSu (N-hydroxysuccinimide) was removed by filtration. The filtrate was acidifiedwith concentrated HCl (2 ml) and extracted with ethyl acetate (3 �20 ml). The combined organic layers were dried over sodium sulfate andevaporated to give the pure Fmoc-protected amino acids.

Synthesis of Guanidines (Entry 33)

Guanidine hydrochloride (7.5 g, 78.5 mmol) and di-tert-butyldicarbo-nate (Boc2O, 13.71 g, 62.9 mmol) were stirred for 15 h at room tempera-ture in a mixture of 4 N aqueous sodium hydroxide and dioxane (1:2,120 ml). This intermediate N-Boc (tert-butyloxycarbonyl) guanidine(2.39 g, 15 mmol) was treated with p-nitrophenyl carbonate Wangresin (5.0 g, 3 mmol) in N,N-dimethylformamide (DMF) for 15 h inthe presence of triethylamine (2.1 ml, 5 equiv.) and a catalytic amount of4-dimethylaminopyridine (DMAP). Trifluoromethanesulfonic anhydride(2.52 ml, 5 equiv.) was added at �78

�to thoroughly rinsed and dried resin.

The reaction mixture was allowed to warm to 0�, filtered, and thoroughly

rinsed with methanol and dichloromethane. The resin (87 mg, 42 �mol)was treated with H-Sar-OMe (sarcosine methyl ester, 43 mg, 10 equiv.)for 20 h and subsequently washed with methanol and dichloromethane.Treatment with dichloromethane-trifluoroacetic acid (1:1, 2 ml) for 3 hliberated the pure guanidine from the solid support.


Scavengers

In addition to supported reagents, extensive use of polymers for purifi-cation of solution-phase reactions has been reported. For example, triaryl-phosphines are common reagents for organic and organometallic reactions,but the chromatographic separation of phosphines and phosphine oxidesfrom the desired products can be difficult. To simplify this step, Lipshutzand Blomgren40 applied a Merrifield chloromethyl polystyrene supportfor the electrophilic capture of triphenylphosphine and the correspondingoxide (Table VIII, entry 34). Following Pd(PPh3)4-mediated cross-couplings or after reduction of an azide with PPh3, the concentrated reac-tion mixtures were heated in acetone with sodium iodide and the captureresin. This method is highly effective for a variety of compounds, includingamines and alcohols, and does not impair product yields.

Combination of amines and dendrimers functionalized proton sca-venger resins efficiently removes excess reagents from amide-formationreactions. Marsh et al.41 used a supported branched polyamine and

40 B. H. Lipshutz and P. A. Blomgren, Org. Lett. 3, 1869 (2001).41 A. Marsh, S. J. Carlisle, and S. C. Smith, Tetrahedron Lett. 42, 493 (2001).

TABLE VIII

Scavengers

Entry

number

Supported


34 40

35 ClCl

OPh NH2

Cl

NH

O

Ph−HCl 41

36 42


dichlorotriazene as acid and amine scavengers, respectively, for amide for-mation reactions (entry 35). The highly branched structures have highloading capacities for efficient purification of amides and sulfonamides.

Similarly, a monolithic polymer of PolyHIPE functionalized withtris(aminoethyl)amine captures acid chlorides in solution with high effi-ciency (entry 36).42 Contrary to suspensions of polymer beads, the porouspolymer monolith is used in a flow-through reaction format.

Scavenging of Phosphines and Phosphinoxides (Entry 34)

The crude reaction was filtered, and the filtrate [containing triphenyl-phosphine (75 mg, 0.28 mmol)] was concentrated in vacuo. Acetone(1.5 ml), sodium iodide (84 mg, 0.56 mmol), and high loading Merrifieldresin (140 mg, 4.38 mmol of Cl/g) were added, and the slurry wasallowed to stir at room temperature. After 18 h, the mixture was filteredand washed with tetrahydrofuran (3 � 3 ml), water (3 � 3 ml), acetone(3 � 3 ml), and methanol. Gas chromatography (GC) analysis of com-bined filtrates and weight gain of the resin indicated complete removal oftriphenylphosine from the reaction mixture.

Fig. 1. Multistep synthesis of nicotines.

42 P. Krajnc, J. F. Brown, and N. R. Cameron, Org. Lett. 4, 2497 (2002).


Multistep Synthesis Using Polymer-Supported Reagents

With the many types of supported reagents available, efficient multistepsyntheses of complex organic structures are being performed in solutionphase. A recent synthesis of nicotine alkaloids employs several classes ofthe reagents reviewed above (Fig. 1). Starting from pyridine-3-aldehyde(1), a Grignard addition was quenched with Amberlite carboxylic acid resin.The product alcohol 2 was cleanly transformed with a supported triarylpho-sphine to alkyl bromide 3. Nucleophilic displacement with a supported azideto prepare 4 and subsequent reductive amination/cyclization produced,following ion-exchange purification, the nornicotine 5 in>90% purity. Fromthis route, the advantages of supported reagents for simplifying purificationsare obvious and also enable rapid diversity-oriented parallel syntheses forpreparation of compound libraries. Using different supported reagents,the amine 5 was converted in high purity to a set of compounds includingalkyl amines, amides, ureas, and sulfonamides.

[20] Advanced Polymer Reagents Based on ActivatedReactants and Reactive Intermediates: Powerful Novel

Tools in Diversity-Oriented Synthesis

By Jorg Rademann

Introduction: Current Challenges in CombinatorialChemistry Research

Combinatorial chemistry provides an array of concepts and methods tosolve molecular optimization problems—in drug research and beyond—more rapidly and efficiently than classic synthetic approaches.1 Since spe-cific molecular interactions between proteins and their ligands have beenrecognized as the molecular basis of most biological processes including dis-ease, it became possible to study and optimize the interactions betweendrugs and their target proteins on a molecular level. Thus, drug developmenthas been turned into a rational and systematic process of optimization.

The establishment of combinatorial chemistry has created noveldemands for basic as well as for applied research. Among the prominentchallenges is the development of an efficient synthetic methodology fordiversity-oriented synthesis2 as well as the creation of powerful interfaces

1 J. Rademann, in ‘‘Molecular Pharmacology—An Encyclopedic Reference’’ (S. Offermanns

and W. Rosenthal, eds.). Springer, Heidelberg, in press, 2003.



Multistep Synthesis Using Polymer-Supported Reagents

With the many types of supported reagents available, efficient multistepsyntheses of complex organic structures are being performed in solutionphase. A recent synthesis of nicotine alkaloids employs several classes ofthe reagents reviewed above (Fig. 1). Starting from pyridine-3-aldehyde(1), a Grignard addition was quenched with Amberlite carboxylic acid resin.The product alcohol 2 was cleanly transformed with a supported triarylpho-sphine to alkyl bromide 3. Nucleophilic displacement with a supported azideto prepare 4 and subsequent reductive amination/cyclization produced,following ion-exchange purification, the nornicotine 5 in>90% purity. Fromthis route, the advantages of supported reagents for simplifying purificationsare obvious and also enable rapid diversity-oriented parallel syntheses forpreparation of compound libraries. Using different supported reagents,the amine 5 was converted in high purity to a set of compounds includingalkyl amines, amides, ureas, and sulfonamides.


[20] Advanced Polymer Reagents Based on ActivatedReactants and Reactive Intermediates: Powerful Novel

Tools in Diversity-Oriented Synthesis

By Jorg Rademann

Introduction: Current Challenges in CombinatorialChemistry Research

Combinatorial chemistry provides an array of concepts and methods tosolve molecular optimization problems—in drug research and beyond—more rapidly and efficiently than classic synthetic approaches.1 Since spe-cific molecular interactions between proteins and their ligands have beenrecognized as the molecular basis of most biological processes including dis-ease, it became possible to study and optimize the interactions betweendrugs and their target proteins on a molecular level. Thus, drug developmenthas been turned into a rational and systematic process of optimization.

The establishment of combinatorial chemistry has created noveldemands for basic as well as for applied research. Among the prominentchallenges is the development of an efficient synthetic methodology fordiversity-oriented synthesis2 as well as the creation of powerful interfaces

1 J. Rademann, in ‘‘Molecular Pharmacology—An Encyclopedic Reference’’ (S. Offermanns

and W. Rosenthal, eds.). Springer, Heidelberg, in press, 2003.



[20] advanced polymer reagents 367

between synthesis and screening steps.3 For diversity-oriented synthesis,reagents and reactions have to be devised allowing for reliable and robusttransformations of simple, chemically diverse starting materials to diverse,complex products. These efforts have to be supplemented by the further re-finement of the phase systems devised for synthetic purposes as well as forbioassays.

One of the major innovations in combinatorial and medicinal chemistryin recent years aiming at efficient diversity-oriented synthesis has been theimplementation of polymer reagents in polymer-assisted solution phase(PASP)* synthesis. This contribution will present—following an introduc-tion to the field—a concept of advanced polymer reagents based on react-ive intermediates and active reactants that should extend the scope PASPsynthesis significantly. Experimental procedures describing preparationand use of the novel polymer reagents are included.

Polymer-Assisted Solution Phase (PASP) Synthesis

Complex organic molecules can be constructed in a homogeneous solu-tion. However, for diversity-oriented purposes it is often advantageous toemploy a multiple phase system to greatly facilitate isolation and separ-ation procedures as well as the removal of excess reagents and the comple-tion of reactions. Solid-phase synthesis is the most widely applied examplefor multiple phase systems in combinatorial chemistry, possessing signifi-cant advantages in comparison to homogeneous single phases. Surfaces,soluble polymers, fluorous biphasic systems, or supercritical carbon dioxideare alternative examples of multiple phase systems employed in synthesis,compound purification, or compound screening.

Synthesis in solution, however, continues to possess indisputable advan-tages in respect to the versatility and reliability of applicable reactions, theease of analytical monitoring, and the accumulated knowledge of synthetic

2 S. L. Schreiber, Science 287, 1964 (2000).3 J. Rademann and G. Jung, Science 287, 1946 (2000).* Abbreviations: ATR IR, Fourier transform attenuated reflection infrared spectroscopy;

DIPEA, diisopropylethylamine; DCM, dichloromethane; DMF, dimethylformamide;

DMSO, dimethylsulfoxide; ENDOR, electron-nuclear double resonance spectroscopy;

ESI, electrospray ionization; ESR, electron spin resonance; Et2O, diethylether; FT, Fourier

transform; GC, gas chromatography; HPLC, high-performance liquid chromatography; HR

MAS, high-resolution magic angle spinning; IBX, 1-hydroxy-(1H)-benzo-1,2-iodoxol-3-one-

1-oxide, 2-iodoxybenzoic acid; ICR, ion cyclotron resonance; MS, mass spectrometry; NBS,

N-bromosuccinimide; NMO, N-methylmorpholino-N-oxide; NMR, nuclear magnetic

resonance; PASP, polymer-assisted solution phase; PEI, polyethylene imine; RT, room

temperature; TEMPO, 2,2,6,6-tetramethylpiperidinoxyl radical; THF, tetrahydrofuran;

TPAP, tetrapropylammonium perruthenate.

Fig. 1. Polymer-assisted solution phase (PASP) synthesis combines the merits of solution-

phase chemistry with the advantages of facilitated phase separation by using polymer reagents

(a) or scavenger resins (b).


protocols. Thus, an ideal synthetic strategy would be to combine the meritsof solution-phase with the advantages of solid-phase synthesis protocols,specifically the ability to use reagents in high excess, to remove them by fil-tration, and to employ automated multiple synthesizers. This combinationis realized by PASP synthesis using functional polymers either as scaven-gers for purification or as reactants involved directly in a chemical trans-formation (Fig. 1).4–6 Polymer reagents can be used in high excess andthen removed by filtration, facilitating product purification for analysisand for further chemical transformations. They are especially suitable forparallel and split-and-pool combinatorial synthesis. They allow the prepar-ation of complex libraries by multistep syntheses in solution, they can beutilized in automated and in flow-through systems, and they can beemployed to transform single compounds as well as complex mixtures.

The first polymer-supported reagents were derived from ion-exchangeresins immobilizing ionic reagents on macroporous polystyrene resins.7

This approach grants easy access to many reagents. For preparation, a so-lution of a salt is added in excess to the resin, the mixture is allowed toequilibrate, and then the resin is washed with nonionic solvents. Leachingof the reactive ions, however, is a general problem of this type of support.In principle, the immobilized ions can be exchanged by other competingions available in solution. Several important polymer reagents are

4 A. Akelah and D. C. Sherrington, Chem. Rev. 81, 557 (1981).5 A. Kirschning, H. Monenschein, and R. Wittenberg, Angew. Chem. 113, 670 (2001); Angew.

Chem. Int. Ed. Engl. 40, 650 (2001).6 S. V. Ley, I. R. Baxendale, R. M. Bream, P. S. Jackson, A. G. Leach, D. A. Longbottom,

M. Nesi, J. S. Scott, R. I. Storer, and S. J. Taylor, J. Chem. Soc. Perkin Trans. I, 3815 (2000).7 F. Helfferich, ‘‘Ion Exchange.’’ McGraw-Hill, New York, 1962.

Fig. 2. First polymer reagents, either in the form of ion-exchange resins or covalently

attached, were employed for various simple transformations in the 1960s and 1970s.


still based on ion-exchange resins, such as the borohydride resins8,9 forreductive amination and the perruthenate resin10 for oxidation.

The next generation of polymer-supported reagents was based on cova-lently linked reactants. The concept was initiated in the 1960s and 1970swith the introduction of peptide coupling reagents such as supported carbo-diimides,5,11 active esters,12 phosphines,13 and bases (Fig. 2). A majoradvantage of covalent linking is avoiding leaching of the immobilized re-agents from the resin. At that time polymer-supported chemistry was stilllimited to a few fundamental chemical transformations and was not widelyaccepted as a useful synthetic method beyond the areas of peptide and oli-gonucleotide chemistry. It was only when solid-phase chemistry became apowerful tool for combinatorial chemistry in the 1990s that polymer-supported reagents gained acceptance for the generation of compoundlibraries and multistep syntheses of natural products.

8 B. Sansoni and O. Sigmund, Naturwissenschaften 48, 598 (1961).9 H. W. Gibson and F. C. Bailey, J. Chem. Soc. Chem. Commun. 815 (1977).

10 B. Hinzen and S. V. Ley, J. Chem. Soc. Perkin Trans. I 1907 (1997).11 Y. Wolman, S. Kivity, and M. Frankel, J. Chem. Soc. Chem. Commun. 629 (1967).12 R. Kalir, A. Warshawsky, M. Fridkin, and A. Patchornik, Eur. J. Biochem. 59, 55 (1975).13 W. Heitz and R. Michels, Angew. Chem. 84, 296 (1972); Angew. Chem. Int. Ed. Engl. 12, 298

(1972).


A Concept for Advanced Polymer Reagents

Despite the current success and popularity of polymer reagents, theavailability of functional resins has been a severe limitation in recent years.For many synthetically important transformations, reliable reagents werenot available. Moreover, polymer-assisted synthesis was usually restrictedto small scale applications, and also suffered from the inherent limitationsof the standard support material (e.g., cross-linked polystyrene) such assolvent incompatibility, adsorption of reagents,14 or the chemical reactivityof the resin backbone.

Better designed polymer reagents should extend the scope of polymer-assisted transformations to more challenging chemistries. Novel reagentsshould be able to replace hazardous, toxic, and other undesirable reactantswith clean and reliable polymer-supported alternatives. Ideally, these poly-mer reagents ought to be recyclable and efficient in catalytic amounts.Novel, high-loading polymer supports should enable applications ofadvanced polymer reagents on a large scale as well.

The generation and investigation of activated reactants and intermedi-ates in polymer gels have extended the field of polymer-assisted conver-sions considerably. In this chapter we will discuss four selected examplesof advanced polymer-supported reagents including reactive oxidantsemployed on alcohols and in single electron oxidations involving radicalion intermediates (example 1), generation and release of reactive inter-mediates and activated reactants on polymer supports including the releaseof carbenium ions (example 2, alkylating resins), the synthesis and applica-tions of supported carbanion equivalents (example 3), and release of rad-icals (example 4). It will be demonstrated as well that generation orrelease of reactive intermediates from polymer gels allows for the exploit-ation of solid-phase specific reactivities. Investigation of these specificsolid-phase effects will contribute to improved understanding and advance-ment in polymer-supported organic chemistry.15 As a result, in example 5the development of high-loading ULTRA resins that were especiallydesigned for polymer reagents will be demonstrated.

Example 1: Oxidizing Polymers

The oxidation of alcohols to carbonyl compounds is one of the mostrelevant transformations in organic synthesis, due to the large diversityof products that can be obtained from aldehyde and ketone precursors.

14 J. Rademann, M. Barth, R. Brock, H.-J, Egelhaaf, and G. Jung, Chem. Eur. J. 7, 3884

(2001).15 J. Rademann, Angew. Chem., submitted (2003).


A variety of methods have been described to solve the task in solu-tion.16 Common oxidative agents for this transformation include variousheavy-metal reagents such as chromium-or ruthenium-based oxides, pyri-dine-SO3, and dimethylsulfoxide (DMSO) in combination with acetic an-hydride, carbodiimide, or oxalyl chloride for activation. One of the mostprominent methods for the reliable conversion of sensitive compounds isthe Dess–Martin reagent or its nonacetylated equivalent, 1-hydroxy-(1H)-benzo-1,2-iodoxol-3-one-1-oxide (2-iodoxybenzoic acid, IBX).

Polymer-Supported Heavy-Metal Oxides

There are several examples of polymer-assisted oxidation reagents, in-cluding heavy-metal oxides bound to ion-exchange resins. Perruthenateresin,10 the immobilized analog of tetrapropylammonium perruthenate(TPAP), can be employed stochiometrically as well as catalytically. Inthe latter case, additional cooxidants [e.g., N-methylmorpholino-N-oxide(NMO)] are required. The use of elemental oxygen has been described aswell. Perruthenate resin has been recently employed in a reaction sequenceleading to heterocycles (Fig. 3),17 although its usage is limited to benzylicalcohols. In general, ion-exchange resins suffer from the potential leachingof heavy metals into the solution mixture, as heavy metals can beexchanged for other anions present in the solution.

Oxidations with Immobilized Oxoammonium Salts

The 2,2,6,6-tetramethylpiperidinoxyl radical (TEMPO) was first pre-pared in 1960 by Lebedev and Kazarnovskii by oxidation of its piperidineprecursor.18 The steric hindrance of the NO bond in TEMPO makes it ahighly stable radical species, resistant to air and moisture. ParamagneticTEMPO radicals can be employed as powerful spin probes for elucidatingthe structure and dynamics of both synthetic and biopolymers (e.g., pro-teins and DNA) by ESR spectroscopy.19 Unlike solid-phase 1H-NMRwhere magic angle spinning is required in order to reduce the anisotropiceffects in the solid-phase environment, solid-phase ESR spectroscopy canbe conducted without specialized equipment. Thus, we conducted com-parative ESR studies of various polymers with persistent radical labels,and we also determined rotational correlation times as a function of

16 L. A. Paquette, ed. ‘‘Encyclopedia of Reagents for Organic Synthesis.’’ Wiley, Chichester,

1995.17 F. Haunert, M. H. Bolli, B. Hinzen, and S. V. Ley, J. Chem. Soc. Perkin Trans. I 2235

(1998).18 O. L. Lebedev and S. N. Kazarnovskii, Zhur. Obshch. Khim. 30, 1631 (1960).19 S. L. Regen, J. Am. Chem. Soc. 96, 5175 (1974).

Fig. 3. Perruthenate resin, an oxidizing resin based on ion exchange of heavy metal oxides,

has been successfully employed in the preparation of heterocycle libraries. In this example,

benzaldehydes were generated and reacted in aldol reactions with Nafion-TMS as Lewis acid.


solvent, temperature, and resin type. For TEMPO radicals a versatileredox chemistry was reported in which radical species can be transformedby a two-electron reduction to the respective hydroxylamine or by a two-electron oxidation to the oxoammonium salt.20 One-electron oxidationsinvolving oxoammonium salts have been postulated as well.21 Commonly,TEMPO is employed under phase-transfer conditions with, for example,sodium hypochlorite as the activating oxidant in the aqueous phase. Inoxidations of primary alcohols, carboxylic acids are often formed by over-oxidation in addition to the desired aldehydes. For catalytic oxidations theoxoammonium salt was postulated as the active intermediate.22

Thus, isolation of oxoammonium salts on insoluble, cross-linked poly-mer supports was investigated along with their implementation in poly-mer-assisted solution-phase synthesis.23 These isolated oxoammoniumsalts could be employed in a water-free system to generate highly reactiveoxidation agents without the overoxidation problems normally seen in thepresence of water.

20 A. E. J. de Nooy, A. C. Besemer, and H. van Bekkum, Synthesis 1153 (1996).21 M. F. Semmelhack, C. R. Schmid, and D. A. Cortes, Tetrahedron Lett. 27, 1119 (1986).22 P. L. Anelli, C. Biffi, F. Montanari, and S. Quici, J. Org. Chem. 52, 2559 (1987).23 S. Weik, G. Nicholson, G. Jung, and J. Rademann, Angew. Chem. 113, 1489 (2001); Angew.

Chem. Int. Ed. Engl. 40, 1436 (2001).


The 4-hydroxy-TEMPO radical was coupled to Merrifield resin (chlor-omethyl polystyrene resin cross-linked with 1% divinylbenzene) employingsodium hydride as base yielding resin 1 with a loading of 0.93 mmol/g(Fig. 4). ESR spectroscopy detected the presence of the free radical elec-tron displaying the characteristic triplet signal coming from the couplingof the 14N nucleus. Likewise, the HR-MAS NMR spectrum displayed sig-nificant line broadening that can be attributed to the enhanced relaxationof the nuclei due to interaction with the persistent electron spins. Oxidationof the radical resin 1 to the oxoammonium resin 2 was best performed withN-chlorosuccinimide. Following oxidation, resin 2 displays strong absorp-tion at 1700 cm�1 in the FT-ATR-IR spectrum, a characteristic of theN——O double bond of the reactive species. This oxidation is accompaniedby a distinct color change from colorless to either a bright orange-red whenchloride or brown-red when bromide is the counterion. Chloride provedto be superior as a counterion being more reactive and leading to fewerby-products.

Protocol for Oxidations Employing Oxoammonium Resins22

Preparation of Oxoammonium Resin. 2. N-Chlorosuccinimide (6equiv.) was dissolved in DCM and then 4 M HCl in dioxane was added(5 equiv.). After 5 min the solution was added to resin 1 (1 equiv.) swollenin dry DCM. Agitation for 15 min was followed by filtration of the resinand washing with dry DCM. The Half-life (t1/2) of the activated form wasca. 1 week when stored in vacuo at 4

�.

Oxidation of Alcohols. Alcohols (1 equiv.) were dissolved in dry DCM.Freshly prepared oxoammonium resin 2 (5 equiv. as calculated from theloading of resin 1) was added and agitated at RT for 1 h for primary alco-hols and 2 h for secondary alcohols. The resin was filtered and washed withDCM, and the combined filtrates were analyzed by GC and GC-MS. Yields

Fig. 4. Polymer-supported oxoammonium salts; resins 2 are highly reactive oxidants

generated in situ by oxidation of TEMPO radical resin 1 with N-chlorosuccinimide.


were determined for 10 mg of starting alcohol. After a 1-h reaction, theresin was washed four times with 3 ml DCM and the solvent was evapor-ated at RT. Representative examples are the oxidation of piperinyl alcoholto piperonal (8.9 mg, 90% yield), cinnamic alcohol to cinnamic aldehyde(8.7 mg, 88% yield), and (þ)-borneol to (þ)-campher (9.2 mg, 91% yield).The identity of the isolated products was also confirmed by NMR analysis(250 MHz, CDCl3).

The versatility of the novel reagent was investigated with a diverse se-lection of alcohols at RT with 3 equiv. of the reagent for 1 h. Results of theoxidations can be summarized as follows. Clean, fast, and quantitative con-version to the respective aldehyde or ketone product was observed for allbenzylic, allylic, and primary aliphatic as well as for most secondary ali-phatic alcohols in yields around 90%. As expected, diols yielded lactonesin the secondary oxidation step. Easily enolizable primary ketonesobtained from cyclohexanol, 1-phenylpropan-2-ol, and cholesterol couldbe further converted to the respective 1,2-diones. In this reaction the pri-mary oxidation product (e.g., cyclohexanone) is transformed to the final di-ketone product via an enolized intermediate. Interesting cascade-likereactions were observed with (�-e)-unsaturated terpene alcohols such asgeraniol and citronellol. Monitoring by GC-MS revealed that with anexcess of resin 2, the primary oxidation products, the open-chain terpenealdehydes were cyclized in an acid-catalyzed ene reaction (Prins reaction),to furnish (secondary) cyclohexyl alcohols. For example, starting from �-citronellol, the intermediary citronellal is cyclized yielding the secondaryalcohol isopulegol. In the case of geraniol, the secondary alcohol obtainedwas even further oxidized to yield the respective cyclohexanone in goodpurity. In addition, oxidating resin 2 was effective in the conversion of acompound collection of 15 chemically diverse alcohols. Under the de-scribed nonaqueous conditions the oxoammonium resin, however, failedwith nitrogen-containing substrates such as protected amino alcohols,presumably due to a single electron oxidation reported earlier.20

In summary, the polymer-bound oxoammonium reagent was highly ef-ficient in polymer-supported oxidations of various alcohols and wascapable of cleanly converting chemically diverse compound collections.No overoxidation to carboxylic acids was observed. It is obvious that thisreagent shall be of great value in polymer-supported transformations insolution, in automated parallel synthesis operations, and in flow-throughreactors in up-scaled production processes.

Catalytic applications employing TEMPO resin 1 are particularly desir-able for preparations on larger scale, increasing the efficiency of the poly-mer-supported reagent. As additional work-up is required for the removalof cooxidants or mediators, catalytic applications do not fit well into

Fig. 5. Reactive oxoammonium resins 2 can be regenerated catalytically in a three-phase

system. With only 6% of TEMPO resin 1, gram amounts of alcohols can be converted efficiently.


parallel and high-throughput synthetic formats. Polymer-supportedTEMPO can be employed with sodium hypochlorite in stochiometricamounts for the oxidation of alcohols. An interesting alternative is theuse of oxone as an insoluble cooxidant together with tetrabutylammoniumbromide as a transfer reagent.24 This reagent system can be easily extendedto a three-phase system, employing immobilized TEMPO, dissolved trans-fer reagent, and insoluble oxone as cooxidant (Fig. 5).25 Elemental oxygencan also be employed as cooxidant for oxidations mediated by TEMPOresin 1. Soluble copper(II) salts can be employed as mediators. However,the rate of oxidation is considerably slower than with oxone as cooxidant.

Immobilized TEMPO has been used for the one-pot oxidation of alco-hols to carboxylic acids as well.26 For this purpose TEMPO resin 1 wascombined with two ion-exchange resins loaded with chlorite anions andhydrogen phosphate in the presence of catalytic amounts of potassiumbromide and sodium hypochlorite in solution. The reaction requiredwork-up for the removal of salts, but tolerated several protecting schemesand afforded pure products in good to excellent yields. The reaction is ini-tiated by catalytic TEMPO oxidation of alcohols to aldehydes driven bydissolved hypochlorite followed by oxidation to the carboxylic acidseffected by chlorite.

24 C. Bolm, A. S. Magnus, and J. P. Hildebrand, Org. Lett. 2, 1173 (2000).25 S. Barthelemy, S. Weik, and J. Rademann, unpublished results (2001).26 K. Yasuda and S. V. Ley, J. Chem. Soc. Perkin Trans. I 1024 (2002).


Oxidations with Immobilized Periodinanes27

In recent years hypervalent iodine compounds have been extensivelyinvestigated yielding many results of practical synthetic importance. Iodi-nane reagents [e.g., iodoso or iodine(III)] have been prepared in asupported fashion by several groups, mainly as the bis-acetoxy-iodoso de-rivative28–30 or as the respective dihalogeno compounds.31 Iodoso reagentsare employed in the oxidation of hydroquinones as well as phenols, andhave been exploited in the formation of spiroketals from various tyrosines.

On the contrary, periodinanes [e.g., iodoxo or iodine(V) reagents] arewidely employed in the oxidation of sensitive and complex alcohols, prefer-ably as IBX32,33 or its acetylation product, the Dess–Martin reagent.34 Per-iodinanes have not been prepared on a polymer support so far, although asilica-supported IBX has been reported recently.35

The limitations discovered for oxoammonium resins in respect to theoxidation of nitrogen-containing moieties prompted the investigation ofpolymer-supported periodinanes as potential alternatives.26 To obtain afunctional iodine(V) reagent, a derivative of 2-iodobenzoic acid was re-quired suitable for immobilization and still retaining oxidation propertiessimilar to the parent compound. 4-Hydroxy-2-iodo-benzoic acid esterspermit efficient immobilization to chloromethyl polystyrene via the phen-oxide. Methyl 5-hydroxy-2-iodobenzoate was obtained in two steps from3-hydroxyanthranilic acid by a Sandmeyer reaction followed by esterifica-tion with thionylchloride in methanol. It was coupled to chloromethyl poly-styrene cross-linked with 1% divinylbenzene (1.20 mmol/g) by usingcesium carbonate as base (Fig. 6). Loading of the resin was determinedby elemental analysis finding it close to the theoretical loading (98%). Sa-ponification was effected by treatment with potassium trimethylsilanoxidein THF yielding resin 4.

27 G. Sorg, A. Mengel, G. Jung, and J. Rademann, Angew. Chem. 113, 4532 (2001); Angew.

Chem. Int. Ed. Engl. 40, 4395 (2001).28 M. L. Hallensleben, Angew. Makromol. Chem. 27, 223 (1972).29 S. V. Ley, A. W. Thomas, and H. Finch, J. Chem. Soc. Perkin Trans. I 669 (1999).30 G.-P. Wang and Z.-C. Chen, Synthetic Commun. 29, 2859 (1999).31 M. Zupan and A. Pollak, J. Chem. Soc. Chem. Commun. 715 (1975).32 M. Frigerio and M. Santagostino, Tetrahedron Lett. 35, 8019 (1994).33 C. Hartmann and V. Meyer, Chem. Ber. 26, 1727 (1893).34 D. B. Dess and C. Martin, J. Org. Chem. 48, 4156 (1983).35 M. Mulbaier and Giannis, Angew. Chem. 113, 4530 (2001); Angew. Chem. Int. Ed. Engl. 40,

4393 (2001).

Fig. 6. Polymer-supported IBX (resin 5) can be activated and recycled with monoperoxy

sulfonic acid (Caro’s acid). The polymer reagent is capable of alcohol oxidations,

dehydrogenations, and radical cyclization reactions.


Protocol for Oxidation with and Reactivation of Polymer-SupportedPeriodinane26

(Re)activation of Resin 4. Resin 4 (100 mg, 0.092 mmol) was treatedwith a solution of tetrabutylammonium oxone (460 mg, 0.46 mmol, activeoxygen �1.6%) and methylsulfonic acid (30 �l, 0.46 mmol) in dry DCM(1.2 ml) and agitated for 3 h at RT. The product was washed thoroughlywith DCM, Et2O, DCM, Et2O, DCM, Et2O (seven times each) and driedyielding resin 4. IR: � = 1578, 1602, 1655 cm�1. Iodine content: 10.8%.Loading: 0.85 mmol/g. Taking into account the mass increase, this corres-ponds to 93% conversion of the chloromethyl groups over three steps. Theoxidative activity of resin 5 (0.8 mmol/g) was determined by converting anexcess of piperonyl alcohol as test substrate.

Oxidation of Alcohols. Alcohols (1 equiv.) were dissolved in dry DCM(15 mmol/liter) and treated with resin 5 (1.75 equiv.) for 3 h at RT. Theresin was filtered off and washed with dry DCM. From the filtrate the vola-tile compounds were analyzed by GC-MS. The nonvolatile compounds wereanalyzed by HPLC-MS. For product isolation the collected filtrates fromseveral washings (DCM, 3� 2 ml) were evaporated yielding 5 mg of startingalcohols: piperonal 4.2 mg (84% yield) and Fmoc-l-phenylalaninal 4.1 mg(82% yield). Purity and identity of the products were determined by GCor HPLC and by NMR spectroscopy.


Oxidation of resin 4 to resin 5 was investigated under various conditions.Initial screening for oxidizing activity was conducted by HPLC analysis ofthe reaction with piperonylalcohol as test substrate. Potassium bromateand potassium hydromonoperoxosulfate triple salt (Caroate, Oxone) failedin aqueous solvent mixtures. By employing the phase transfer catalyst 18-crown-6 together with the caroate in a triphase system, traces of piperonalwere detected. To avoid the presence of water and to ensure proper swellingof the resin, tetrabutylammonium oxone in dichloromethane (DCM) wasselected, yielding a low resin activity of 0.1–0.2 mmol/g. Monoperoxysulfo-nic acid (Caro’s acid) is a stronger oxidant than its anion, which is present inOxone and was therefore examined in further oxidation experiments. Anequimolar mixture of tetrabutylammonium oxone with methyl sulfonic acid(DCM, RT, 3 h) furnished resin 5 with a high activity of 0.8 mmol/g. Resin5 was characterized by IR spectroscopy, elemental analysis, and MAS-NMR. Elemental analysis indicated a loading of 0.84 mmol/g, correspond-ing to a yield of 94% in respect to the initial loading, taking into accountthe mass increase of the resin. No loss of iodine was observed under thestrongly acidic reaction conditions. The oxidizing polymer 5 was stabletoward air and moisture and it could be stored without loss of activity.

The oxidation properties of periodinane resin 5 (1.75 equiv., DCM, RT,3 h) were investigated by reaction with a collection of diverse alcohols in-cluding benzylic, allylic, primary aliphatic alcohols including the unsatur-ated terpene alcohols citronellol and geraniol, secondary aliphaticalcohols, and the carbamate-protected aminoalcohols Fmoc-Phe-ol andFmoc-Ile-ol. All reactions were followed by GC-MS or by HPLC (UV at215 and 280 nm). Products were identified by NMR spectroscopy and bymass spectrometry (EI, 70 eV); isolated yields were determined by weight.Most alcohols were converted to the respective aldehyde or ketone prod-ucts in good to excellent yields and purities. Following extensive washings,resin 5, which had not been exposed to temperatures higher than RT, couldbe recycled by repeated oxidation. In addition to the oxidation of alcohols,further important transformations effected by IBX were investigated withresin 5. Cyclohexanol reacted with resin 5 in a closed vessel (2.3 equiv.,DCM, 2 h, 65

�) yielding �,�-unsaturated cyclohexenone via cyclohexanone

and a postulated iodine-enol ether intermediate.36 The unsaturated carba-mate 6 was treated with resin 5 (4 equiv., THF/DMSO 10:1, 90

�, 16 h) in

order to undergo radical cyclization affording product 7 with 30% yield.37

It should be noted that IBX at elevated temperatures can oxidize benzylic

36 K. C. Nicolaou, Y.-L. Zhong, and P. S. Baran, J. Am. Chem. Soc. 122, 7596 (2000).37 K. C. Nicolaou, Y.-L. Zhong, and P. S. Baran, Angew. Chem. 112, 639 (2000); Angew.

Chem. Int. Ed. Engl. 39, 625 (2000).


positions, which are abundant in the polystyrene backbone of resin 4 andmight account for a competing reaction pathway.

Resin 5 was prepared as the first polymer-supported periodinane re-agent. This resin was obtained with high loading (0.8 mmol/g) and wascapable of converting a collection of diverse alcohols, including complexand sensitive structures, efficiently in good to excellent yields to the re-spective carbonyl compounds. In addition, the �,�-desaturation of carbonylcompounds and the radical cylization of an unsaturated carbamate weredemonstrated. This novel reagent is likely to find broad application in poly-mer-assisted solution-phase synthesis. Furthermore, this new oxidizingresin should be well suited for integration into parallel polymer-supportedreaction sequences for the production of novel compound libraries. This re-agent has also been efficient in the oxidation of a selection of medium tolarge-sized peptide alcohols. With 5 equiv. of resin 5 the respective peptidealdehydes were obtained in good to excellent purity. For example, the nat-ural peptide alcohol alamethicin F-30 containing as many as 19 aminoacids was efficiently oxidized to its aldehyde derivative as verified byESI-FT-ICR MS.

Example 2. Alkylating Polymers38

Many alkylating agents, such as diazoalkanes, sulfates, sulfonatesesters, and alkyl halogens, are highly toxic, mutagenic, or explosive com-pounds making a safer resin-bound alternative an attractive substitution.Solid-supported sulfonate esters have been employed in alkylations ofamines and thiols at elevated temperatures. Sulfonate alkylations of carb-oxylic acids were reported following our work, requiring the addition of abase that can be removed with a scavenger resin.39

Because elemental nitrogen is an excellent leaving group in alkylations,alkylating polymers ideally work best when releasing carbenium ions and ni-trogen from precursors bound to insoluble polystyrene gels.38 The alkylatingspecies were generated from solid-phase bound 1-aryl-3-alkyltriazenesunder acidic conditions and were demonstrated as very reactive, mild, andversatile alkylation reagents.

As higher reactivity of electron-rich triazenes was observed, p-alkoxy-substituted anilines were selected as an efficient starting material. Solid-supported triazenes were prepared via a solid-phase bound nitroaryl, whichwas reduced to the polymeric ammonium hydrochloride resin 8 (Fig. 7).

38 J. Rademann, J. Smerdka, G. Jung, P. Grosche, and D. Schmid, Angew. Chem. 113, 390

(2001); Angew. Chem. Int. Ed. Engl. 40, 381 (2001).39 N. Zander and R. Frank, Tetrahedron Lett. 42, 7783 (2001).

Fig. 7. Triazenes as versatile polymer-supported diazoalkane analogues (resins 10) were

obtained from polymeric diazonium salts (resins 9) and releasing carbenium ions upon acidic

activation. The reaction can be employed for the alkylation of carboxylic acids with a reaction

half life of ca. 5 min.


The latter resin was treated with tert-butyl nitrite in DCM at �18�

resultingin diazotation. The best results in the diazotation reaction were obtained bydirectly employing the ammonium hydrochloride salt resin 8. The diazo-nium salt resin 9 was reacted with various primary amines including methy-lamine, n-butylamine, n-dodecylamine, several allyl-and benzylamines,20-amino-2-ethylpyridine, and diamines including 2-morpholinoethyl amineand 1,13-diamino-4,7,10-tridecane (diamino-PEG-200), to furnish thesupported triazene resins 10. Starting from polystyrene-containing chloro-methyl groups (2.0 mmol/g) methyl resin 10 was obtained with a loading of1.54 mmol/g of reactive sites. Taking into account the mass increase duringthe reaction sequence, this loading corresponds to an excellent conversion(94%) of chloromethyl groups into triazenes. Completion of aryl ether for-mation, reduction, and diazotation as well as triazene formation could bemonitored by following characteristic vibrational bands in the attenuatedtotal reflection IR spectrum (FT-ATR-IR) obtained directly from washedand dried resin samples. The methyl triazene resin 10 was stable at roomtemperature for at least several months when kept in the dark.

Various reactions were investigated using the novel polymer reagentresin 10. First, esterifications were studied with acids representing a broad


pKa range, molecular weight, steric constraints, as well as diverse function-alities. As the reaction requires acidic conditions to activate the triazenemoiety by protonation, the pKa of the substrate plays a crucial role in thisreaction. Furthermore, there is evidence pointing at carbenium ion inter-mediates either as ion pairs in the case of benzyl ions or via a concertedmechanism in case of primary alkyl ions.

Protocol for the Alkylation of Carboxylic Acids by Use ofAlkylating Resins37

Representative Example. A carboxylic acid (5 mg) was dissolved inDCM (5 ml) (or DCM/MeOH 9:1, or THF, or dioxane) and treated withn-butyl triazene resin 10 (R1 = n-butyl, 5 equiv.) for 6 h. The resin waswashed with DCM, MeOH, and DCM (2 � 2.5 ml) and the solvent was re-moved by evaporation furnishing 5.3 mg of n-butyl product in the case ofbenzilic acid (79.5% yield). HPLC analysis was conducted to determinethe purity of the products.

Sterically demanding benzilic acid was used to optimize the reactionconditions in respect to reagent excess and reaction time. Complete con-version (98%) of benzilic acid to the corresponding methyl ester wasobtained with two equivalents of the methyl triazene resin 10 after 6 h;96% conversion was obtained when using the n-butyl triazene resin 10.The reaction between p-nitrophenylacetic acid (1 equiv., 2 mg/ml) andthe polymer-supported triazene (2 equiv.) in DCM was monitored byHPLC. A 53% conversion from the acid to the ester product was observedafter 5 min; data analysis indicated a second-order reaction as observed inhomogeneous solution.

A diverse selection of acids was converted to highly pure ester productswhen treated with 5 equiv. of the alkylating polymers 10 for 6 h. Represen-tative yields were in the range of 80%. In case of the esterification of ben-zilic acid with n-butyl triazene resin, NMR analysis was employed tovalidate the structure of the expected n-butyl ester. The NMR spectrumdid not display any signals corresponding to an isobutyl ester by-productformed by rearrangement. In contrast, alkylation to the tert-butyl estersfailed, though the generation of gaseous products indicated decompositionof the triazene moiety. Likely, the intermediary tert-butyl cation undergoesproton abstraction yielding isobutene. Functional groups that can be toler-ated by resin 10 include aliphatic hydroxy groups, enolizable carbonyl func-tions, and nitrogen heterocycles with limited basicity as in pyridine (pKa

5.25) and pyrazoles. The conversion of acid-sensitive structures was exem-plified with penicillin V. Esterification of this especially labile structurefailed under acidic and basic conditions as well as with diazomethane.


The efficiency of this polymer-supported alkylation was investigated usingvarious protected amino acids and peptides including a decapeptide bear-ing various acid-labile protecting groups and a mass of 2924.5 Da. Thelatter was reacted with methyltriazene resin 10 (R1 ¼ CH3, 5 equiv.). After6 h the starting peptide was consumed and the product was confirmed byHPLC and ESI-MS. Uncatalyzed etherification was successful only withstrongly acidic phenols such as pentafluorophenol (70% purity). Triazeneresins based on diamines were also found to be efficient in the alkylationreaction.

In addition, the conversion of a compound collection of drug-likeheterocycles as might be used in a medicinal chemistry program was inves-tigated. An equimolar mixture of 20 pyrazole acids, synthesized by a split-and-mix approach, was treated with methyl resin 10 (R1 ¼ CH3, 5 equiv.)for 6 h to yield the respective pyrazole esters. All 20 pyrazole acids in thestarting reaction mixture and all their corresponding 20 pyrazole methylesters in the product mixture could be identified by FT-ICR MS coupledto micro-HPLC with a relative mass error <2.2 ppm.

In summary, alkylating resins 10 have been demonstrated to be very ef-ficient and versatile esterification reagents that allow the conversion of di-verse, highly functionalized substrates under extremely mild reactionconditions. These novel reagents might be valuable especially in the paral-lel conversion of mixtures of highly functionalized compound collectionssuch as those used in lead optimization efforts. Alkylating polymers shouldalso be useful in flow-through applications allowing in situ conversions(e.g., in gas chromatography). Further potential of the presented alkylationreagents can be expected for other nucleophiles such as alcohols, phenols,amines, thiols, and various C-nucleophiles including cyanide ion.

Example 3. Polymer-Supported Carbanion Equivalents

To establish more complex reaction sequences with polymer reagents,generation of reactive electrophiles either by oxidizing polymers or by therelease of carbenium ions is supplemented well by polymer-supported car-banion equivalents. For example, the combination of an oxidizing resin witha support carrying carbanion equivalents will be especially rewarding,allowing reaction sequences with C–C coupling steps and therefore accessto a wealth of potentially relevant products. Classically, polymer-supportedcarbanion equivalents are obtained from polymeric phosphines,13 which areused for the generation of polymeric Wittig salts, ylides, and finally olefines.

The �-hydroxy carbonyl moiety 11 (Fig. 8 is an important structuralmotif found in many natural products including carbohydrates, fatty acids,and secondary metabolites. Potent protease inhibitors and antitumor

Fig. 8. Resins 12 and 13 as examples of polymeric acyl anion equivalents.


agents contain this motif as well. A general polymer-supported access tothese compounds has not been established so far. In nature, this motif isprovided via acyl anion equivalents (‘‘active aldehydes’’) derived fromthiamine pyrophosphate (TPP, vitamin B1) as enzymatic cofactor. To intro-duce diversity-oriented access to this important structural motif, polymer-supported acyl anion equivalents were selected as starting points.

Various thiamine derivatives were prepared on polymer support andwere tested in C–C coupling reactions.40 The most straightforward ap-proach was the alkylation of substituted 1,3-thiazoles with chloromethylpolystyrene resin yielding supported thiazolium chloride salts. Alterna-tively, resin-supported thiamine derivatives were constructed in a two-stepprocess by alkylation of the primary alcohol with chloromethyl polystyrenefollowed by alkylation of the ring nitrogen yielding resin 12 (Fig. 8). Notonly does this pathway introduce a spacer between the active thiamine

40 S. Weik and J. Rademann, Angew. Chem. 115, 2595 (2003); Angew. Chem. Int. Ed. Engl. 42,

2491 (2003).


and the polymer backbone, it allows for the diverse substitution of the ringnitrogen for variation of steric demand at the nucleophilic 2-position or forthe introduction of a chiral thiamine as the C–C bond-forming catalyst.

Resin 12 can be efficiently employed for benzoin condensations withcatalytic amounts of the supported thiamine (10 mol%). The reaction hasto be performed under exclusion of air to avoid formation of benzylic orbenzoic acids. Ethanol equilibrium is reached after 6 h (60

�) with a

maximum of 40% conversion.Thiamine-catalyzed transformations are reversible, thus N,N-dialkyl

hydrazones were selected as alternative acyl anion equivalents that werereported to react with electrophiles without acidic activation.41 One espe-cially reactive example, formaldehyde hydrazone resin 13, was constructedfrom polymer-supported hydrazines and was employed in the first polymer-supported, uncatalyzed acyl anion additions (Fig. 8).38 As test substrates,nitroalkenes (as Michael acceptors) and activated aldehydes were selected.Reactivity of these acyl anion equivalents depended critically not only onthe nature of the starting hydrazine, but also on the protocol for hydrazineformation.

Example 4. Radical Release from Polymer Gels42

The generation of radical intermediates inside of polymer gels isappealing not only for synthetic purposes but as well for understandingpolymer-supported reactivity. The relative isolation or hindered diffusionof radicals inside of polymer gels might influence the lifetime of radicalsso that reactivities are specific for solid-supported reaction systems (spe-cific solid-phase effects). For example, the recombination of radicals couldbe disfavored in the solid-supported reaction. In addition, reactivity of rad-icals with the polymer backbone also has to be considered. To investigatesolid-supported radical chemistry, analytical techniques for the observationof reactive intermediates in polymer gels and product distribution arerequired.

Until now, the chemistry of radicals on solid supports has been investi-gated mostly in respect to intramolecular radical cyclizations and radicalchain reactions. One reason for refraining from free radical transformationsis the chemical nature of the polystyrene with its abundance of benzylicpositions that are prone to H-radical abstraction and oxidation.

Symmetric azo compounds are useful initiators of radical chain reac-tions. However, in many instances, the radicals are released from the initial

41 R. Fernandez, E. Martin-Zamora, C. Pareja, and J. M. Lassaletta, J. Org. Chem. 66, 5201

(2001).42 J. Rademann, P. Schuler, and G. Nicholson, unpublished results (2002).


radical pair only to a small extent and react preferably by recombination ordisproportion. In contrast, two radicals of very different reactivity gener-ated from an asymmetric precursor molecule can be released from theinitial radical pair to a high percentage, if alternative reaction pathwaysare accessible.43

Trityl-azo-aryl resins 14 were obtained from trityl chloride resin(0.96 mmol/g) in two steps. Aryl hydrazines (3 equiv.) were coupled inthe presence of diisopropylethyl amine (DIPEA) at RT, followed by oxida-tion with N-bromosuccinimide (NBS) in DCM/pyridine (9:1). The reactionwas analyzed by elemental analysis and FT-ATR IR, respectively. Radicalthermolysis was monitored by ESR spectroscopy in order to determine theonset of radical generation, to characterize and study the persistence of theformed radicals. A sample of resin 14 swollen in CHCl3 at room tempera-ture did not contain any detectable radicals, but heating to 40

�generated a

distinct ESR signal. Signal intensity was further increased by warming to60

�, resulting in vigorous gas evolution. ENDOR (electron-nuclear double

resonance) spectroscopy of the sample at 60�

was used to characterize theradical structure yielding coupling constants that could be fully assignedthe protons of the trityl-type radical. These radicals are highly persistent,being detected for hours at room temperature.

To investigate putative matrix effects, thermolytic radical fragmenta-tion of 4-bromophenyl-azo-triphenylmethyl was conducted at variousconcentrations with a suspension of resin 14 in an H-donating solvent(CHCl3, 60

�, 2 h). The product distribution for solution and solid-phase ex-

periments was analyzed by GC. In solution experiments even at high con-centrations (0.1 M) only a few recombined products between aryl and tritylradicals were detected (2.7%). Low recombination, however, is essentialfor good recovery of fragmentation products from the solid support. Insolution and on solid support, the main product formed from the arylradicals was bromobenzene, while 4,40-dibromo biphenyl was formed as aby-product from the recombination of two bromophenyl radicals. Forma-tion of the recombined product was concentration dependent. A minorby-product was bromo-4-chlorobenzene. Benzophenone was detectedto a significant extent in the presence of oxygen, probably formed viadisproportion of the intermediate trityl peroxide.

Comparing the results from radical release in solution and on solid sup-port, it was determined that the amounts of recombined product were re-duced significantly in the matrix-supported reaction even at higherconcentrations (0.1 M solution: 2.6%, 0.16 M polymer support: 0.4%).On the other hand, in both solution and polymer gel experiments, the

43 T. B. Patrick, R. P. Willaredt, and D. J. DeGonia, J. Org. Chem. 50, 2232 (1985).


amount of bromo-4-chlorobenzene was not concentration dependent.These findings clearly indicate a matrix effect that can be attributed tothe relative isolation and/or hindered diffusion of released radicals insidethe polymer. This matrix effect was found in both released aryl radicalsand polymer-attached trityl radicals (Fig. 9).

The high purity (>95%) of the released product from resin 14 promptedus to investigate the potential of radical release as a traceless linker con-cept. To our knowledge, thermolytic radical fragmentation of covalentbonds is a mechanism of bond dissociation that has not been exploited sofar for linker chemistry and for solid-phase transformations. Cleavageyields, determined by releasing nonvolatile products, were found to behigher than 90%.

Radical fragmentation of 2-nitrophenyl-azo-trityl resin was studied inthe presence of various radical acceptor solvents to elucidate possible rad-ical reaction pathways. When using benzene as solvent, only 2-nitro-bi-phenyl was formed as the product of radical substitution reaction (SNR)in 67% yield. Hydrogen-radical abstraction from the polymer backbone(e.g., from the benzylic units of polystyrene) was completely suppressed.When toluene was used as solvent, a mixture of the following productswas obtained: nitrobenzene, 4-methyl-20-nitrobisphenyl, 2-methyl-20-nitro-bisphenyl, and 3-methyl-20-nitrobisphenyl (9:9:1:1). In the case of toluene,the nitro-aryl radicals undergo H-abstraction with radical substitution as acompeting reaction pathway. These results indicate that H-abstraction

Fig. 9. Thermolytic radical release from polymer-supported trityl-azo-arenes resins 14.


occurs on the toluene side chain rather from the benzylic positions ofpolystyrene. With pyridine as solvent, no product was detected in solution,despite the fact that gas evolution indicated radical formation. Residual ni-tro groups detected in the IR spectrum suggest that radical scavenging bythe resin backbone was the predominant reaction pathway.

Aryl radicals can be generated inside of polystyrene gels by thermolysisof trityl-azo-aryl compounds. Radical formation and the long lifetime of thetrityl radicals can be monitored by ESR spectroscopy. Matrix effect studiesof radicals inside of polymer gels indicated a significant reduction of radicalrecombination as well as a prolonged lifetime of radicals inside of polymergels. In addition, we have shown that radical quenching by the polystyrenebackbone is strongly disfavored in comparison to H-abstraction from thesolvent. In the absence of efficient radical reaction pathways involving solv-ent interactions, radical quenching by the trityl polystyrene resin became themajor reaction pathway. These findings will be helpful for the further ex-ploitation of matrix-supported or matrix-released radicals and could be ofparticular interest for the investigation of radicals of biological importancestudied using the presented methodology.

Example 5. Optimization of the Polymer Support: Highly LoadingResins for Polymer-Supported Reagents44

To date the majority of polymer-supported chemistry is conducted onlyon a few solid support materials. Recently, it has been documented thatspecific solid-phase effects have significant impact on the success or failureof polymer-supported reactions.15 Considering the limitations of polystyr-ene, which is the standard material for most applications today, it becomeseven more evident that innovations in the area of support materials willopen the door to novel opportunities for polymer-supported chemistries.

One major drawback of the current methods is the low atom economy45

of solid-supported chemistry with conventional resins in comparison to so-lution-phase synthesis. The low loadings are one important reason for ex-cluding solid-supported methods from many resource-and cost-sensitiveapplications such as scale-up projects. Furthermore, polystyrene-basedresins are restricted by solvent compatibility, thermal and chemicalstability, and extensive adsorption of reagents.

Large-scale polymer-supported chemistries could significantly acceler-ate the drug discovery process, particularly for the generation of lead

44 J. Rademann and M. Barth, Angew. Chem. 114, 3087 (2002); Angew. Chem. Int. Ed. Engl.

41, 2975 (2002).45 B. Trost, Angew. Chem. 107, 285 (1991); Angew. Chem. Int. Ed. Engl. 30, 214 (1991).


compounds in larger quantities. Moreover, matrix-supported synthesis is ofstrong interest for ‘‘green chemistry’’ providing environmentally friendlyproduction processes. Efficient scale-up requires higher yields per reactionvolume and has to be more time efficient than can be provided with thecurrent generation of support materials. Increasing the loading of polymersupports, i.e., the millimoles of reactive sites per gram of polymer, is oneimportant prerequisite for a scale-up resin.

High-loading resins have been prepared using several approaches. Den-drimer synthesis on polystyrene resins is one possibility.46 However, thesynthesis is relatively tedious and only the loading per bead (not per gram)is increased significantly due to the large weight increase. Unfortunately,loading per gram is not much increased by dendrimer synthesis. An alter-native approach is grafting on polystyrene by living radical polymeriza-tion.47 The O-TEMPO group is attached to the benzylic units inpolystyrene resin. Heating to 140

�generates free benzyl radicals that initi-

ate the radical polymerization of olefines in the solution (e.g., acrylamides).High loadings of grafted functional styrene monomers are obtained by thisroute leading to so-called RASTA resins.

Resins constructed from low-molecular-weight monomers with efficientfunctionalization sites at each monomer would possess greatly enhancedresin capacity. The maximum theoretical loading of poly-(4-chloromethyl)styrene is 6 mmol/g. In commerically available polystyrene supports,however, only a small fraction of this can be obtained due to methylenecross-linking under Friedel–Crafts conditions. Polyvinyl alcohol as well aspolyethylene imines (PEI) possess significantly higher loadings up to23 mmol/g functional groups. Since polyvinyl alcohols or esters are proneto elimination or acid hydrolysis, polyethylene imine was preferred as thelead polymer for resins with significantly higher loading and atom economythan conventional polymers. To control the degree of cross-linking and thedegree of amine substitution in the resin product, linear polyethylene iminewas selected as the polymeric starting material.42 Cross-linking of poly(ethylene imine) was investigated with polyaldehydes, polyacids, andmultivalent alkylating agents. Dialdehydes furnishing the resin structurevia a thermodynamically controlled equilibrium reaction were found tobe superior allowing the preparation of resins with robust protocols frominexpensive and easily available precursors. The dialdehyde was crucialfor the success of the reaction and for the mechanical properties of thepolymer formed. The rigid terephthalic dialdehyde afforded the chem-ically, mechanically, and thermally robust ULTRA resin 15. Whereas

46 V. Swali, N. J. Wells, G. J. Langley, and M. Bradley, J. Org. Chem. 66, 4902 (1997).47 L. C. Hodges, L. S. Harikrishnan, and S. Ault-Justus, J. Comb. Chem. 2, 80 (2000).


glutaric dialdehyde yielded a softer resin, glyoxal delivered no resin atall. Presumably, under the described conditions only a rigid dialdehydecross-linking between two polymer chains was favored relative to theintramolecular reaction within one chain.

ULTRA resins were prepared from varying molar ratios between dia-ldehydes and the linear PEI and with PEI of varying length (Fig. 10).In all cases resin micropellets of a defined size range were generated bypolymer extrusion in the swollen state, followed by sieving.

ULTRA resins were employed for the preparation of polymer reagentsas well as for solid-phase synthesis. ULTRA resin 15 can be directlyemployed as a polymeric base with a loading of 15.2 mmol/g. Following re-ductive amination with formaldehyde, a resin containing 13.2 mmol/g oftertiary amines was obtained. An ULTRA resin for ion exchange was pre-pared by alkylation of the tertiary amine resin with methyl iodide; the max-imum loading of the resin with chloride was 8 mmol/g corresponding to achlorine content of 28%. Starting from ULTRA resin 15, 4-chloropyridi-nium hydrochloride and triethylamine under microwave-assisted condi-tions at 220

�afforded a resin analogous to the acylation catalyst

dimethylamino pyridine.For solid-phase synthesis various linker molecules were constructed on

the ULTRA resin 15. To determine the optimal spacer length, ULTRAresins were coupled with spacers of variable length. With 4-(40-acetoxy-methyl-30-methoxy-phenoxy) butyrate and a loading of 2.5 mmol/g thesynthesis of heterocycles and peptides was investigated. A pyrazole carbox-ylic acid was prepared using a procedure established for Wang polystyrene

Fig. 10. High-loading ULTRA resins 15 based on the reductive cross-linking of linear

polyethylene imines.


without modification. Starting with 5 mg of the ULTRA resin 15, thisthree-step process produced 8.8 mg of the heterocycle (80% purity in theraw product, 65% yield following chromatography).

Limitations in product size were investigated by preparing severalpeptides of various lengths. Peptides containing 7, 9, 13, and 19 amino acidresidues were prepared on a synthesis robot employing Fmoc-protectedamino acids and carbodiimide-hydroxybenzotriazol activation (0.25 M,1 h) without detection of deletion products. To illustrate the remarkableeconomy of the ULTRA resin, 3.4 mg of the starting resin 15 sufficed forthe synthesis of 42 mg resin with fully protected tridecapeptide. The rawproduct was obtained by ethereal trituration in excellent purity and yield(90% purity in the raw product, 78% yield, 13.1 mg after preparativeHPLC).

In summary, ULTRA resins can be prepared with extremely highloading compared to standard resins in use today. Secondary amine groupsof the resin were very accessible to various derivatizations and even largerproduct molecules could be assembled successfully in the resin interior.Thus, these resins allowed solid-supported chemistry that was greatly im-proved in atom economy and provide a significant contribution to theefficient scale-up of polymer-supported syntheses.

Conclusions

The generation and release of polymer-supported reactive intermedi-ates or activated reactants have been demonstrated to be a powerful con-cept leading to novel insights of matrix-assisted reactivity. Following thisconcept, several novel advanced polymer reagents for important trans-formations have been introduced. The reagents are capable of cleanlyconverting sensitive single compounds as well as complex mixtures insolution, and additionally, they are recyclable and/or can be employedcatalytically.

Despite the significant advances reached in recent years, further re-search and innovations are required to overcome the most significant limi-tations in polymer-supported chemistry. Still too little is understood aboutthe influence of the physical and chemical properties of support materialson solid-supported reactivity. The impact of polymer reagents will rise con-siderably, if innovative support materials become available that allow thescale-up of polymer-supported reaction sequences. In addition, biocompat-ible supports will extend the opportunities of solid-supported chemistrytoward biochemical transformations and screening applications. ULTRAresins, as a novel resin concept with extreme high loading and enormousswelling in water, will contribute significantly to these future developments.

[21] scavenger resins in solution-phase combichem 391

[21] Scavenger Resins in Solution-Phase CombiChem

By J. Gabriel Garcia

Introduction

Combinational chemistry jointly with high-throughput biologicalscreening has dramatically increased the number of potential drug candi-dates that need to be synthesized.1 As a result, they are being widely usedin medicinal chemistry as a tool for accelerating synthesis and drug discov-ery. Combinatorial chemistry is a highly effective method for the gener-ation of multiple small molecule libraries of drug-like compounds,2–6 andhas the ability to generate a large set of structurally related analogues.Therefore, it has become a legitimate tool for increasing productivity inthe functional assessment of compound libraries and the rapid develop-ment of structure–activity relationships.7 To keep up with the fast-pacedprogress of both solution- and solid-phase combinatorial chemistry, an in-creased number of commercially available materials with a multitude ofapplications has been generated.3,4,8–10

The most time-consuming factor in organic synthesis is the purificationof the desired product, thus the bottleneck in combinatorial chemistry isthe rapid purification of library compounds. Traditional purificationmethods such as aqueous extraction and chromatography become timeconsuming as the performed number of simultaneous (parallel) reaction in-creases. Novel methodologies are thus being developed to overcome thisproblem with the aim of implementing automated work-up proceduresfor crude solution-phase reactions. The most popular methodology gaining

1 G. M. Coppola, Tetrahedron Lett. 39, 8233 (1998).2 S. W. Kaldor, M. G. Siegel, J. E. Fritz, B. A. Dressman, and P. J. Hahn, Tetrahedron Lett.

37, 7193 (1996).3 L. A. Thompson and J. A. Ellman, Chem. Rev. 96, 555 (1996).4 E. M. Gordon, M. A. Gallop, and D. V. Patel, Acc. Chem. Res. 29, 144 (1996).5 S. H. DeWitt and A. W. Czarnik, Acc. Chem. Res. 29, 114 (1996).6 L. M. Gayo and M. J. Suto, Tetrahedron Lett. 38, 513 (1997).7 R. E. Dolle and K. H. Nelson, Jr., J. Comb. Chem. 1, 235 (1999).8 R. Storer, Drug Discov. Today 1, 248 (1996). A. Chuckolowski, T. Masquelin, D. Obrecht,

J. Atdlweiser, and J. M. Villagordo, Chimica 50, 525, (1996). D. M. Coe and R. Storer,

Annu. Rep. Comb. Chem. Mol. Divers. 1, 50 (1997). A. T. Merritt, Comb. Chem. High

Throughput Screen. 1, 57 (1998).9 A. Chesney, P. Barnwell, D. F. Stonehouse, and P. G. Steel, Green Chem. 57 (2000).

10 F. Balkenhohl, C. v.d. Bussche-Huennefeld, A. Lansky, and C. Zechel, Angew. Chem. Int.

Ed. Engl. 35, 2288 (1996). J. C. Hogan, Jr., Nat. Biotechnol. 15, 328 (1997).




widespread acceptance for solution-phase combinatorial chemistry11 is theuse of scavenger resins, which are also known as polymer-supportedquenching/scavenging reagents.2,12 Additional methodologies applied forthe same purpose of rapid purification include chemical tagging of re-agents, solid-phase extraction, and fluorous-phase extraction.13,14 Recently,a number of methods have been developed for the aqueous extraction of alarge number of organic reactions in parallel. However, most of these tech-niques involve expensive robotic systems and are unable to deal with emul-sions, and thus these techniques are restricted to using solvents denser thanwater.14

Solution-phase combinatorial synthesis provides a homogeneous reac-tion medium and overcomes the drawbacks of a solid-phase strategy. Aneasy and reliable purification method is required in solution-phase com-binatorial (parallel) synthesis to facilitate automation. The throughputin solution-phase automated synthesis is directly related to the facility ofperforming a purification process (work-up), compound separation, etc.15

This chapter will cover an overview of recent advances in solid-phase-assisted solution-phase combinatorial synthesis, specifically the use ofscavenger resins in assisting in the isolation of pure product without theneed for chromatography. In addition, an experimental section has beenincluded.

During the past few years, polymer-assisted solution-phase synthesishas become the prevalent method for the parallel synthesis of chemical lib-raries as confirmed by the increasing number of publications and reviewson the subject.16–29 A key step in the parallel solution-phase combinatorial

11 R. J. Booth and J. C. Hodges, Acc. Chem. Res. 32, 18 (1999).12 R. J. Booth and J. C. Hodges, J. Am. Chem. Soc. 119, 4882 (1997).13 D. L. Flynn, R. V. Devraj, and J. J. Parlow, Curr. Opin. Drug Discov. Dev. 1, 41 (1998). J. J.

Parlow, R. V. Devraj, and M. S. South, Curr. Opin. Chem. Biol. 3, 320 (1999).14 E. Maslana, R. Schmitt, and J. Pan, J. Autom. Methods Manage. Chem. 22, 187 (2000).

N. Bailey, W. J. Cooper, M. J. Deal, A. W. Dean, A. L. Gore, M. C. Hawes, D. B. Judd,

A. T. Merritt, R. Storer, S. Travers, and S. P. Watson, Chimica 51, 832 (1997).

M. Rabinowitz, P. Seneci, T. Rossi, M. DalCin, M. Deal, and G. Terstappen, Bioorg.

Med. Chem. Lett. 10, 1007 (2000).15 R. Ferritto and P. Seneci, Drugs Future 23, 643 (1998).16 A comprehensive review article: S. V. Ley, I. R. Baxendale, R. N. Bream, P. S. Jackson,

A. G. Leach, D. A. Longbottom, M. Nesi, J. S. Scott, R. I. Storer, and S. J. Taylor, J. Chem.

Soc. Perkin Trans. I 3815 (2000).17 H. N. Weller, Mol. Divers. 4, 47 (1999).18 R. Ferritto and P. Sensci, Drugs Future 23, 643 (1998).19 H. Y. An and P. D. Cook, Chem. Rev. 100, 3311 (2000).20 J. G. Breitenbucher, K. L. Arienti, and K. J. McClure, J. Comb. Chem. 3, 528 (2001).21 B. A. Bunin, ‘‘The Combinatorial Index.’’ Academic Press, New York, 1998. G. Yung,

‘‘Combinatorial Chemistry: Synthesis, Analysis, Screening.’’ Wiley-VCH, Weinheim, 1999.


synthesis of compound collections involves the purification of eachmember of the library. The possibility of avoiding aqueous work-up, crys-tallization, and chromatographic procedures makes the whole processmore suitable for automation and enhances its efficiency from economicand ecological viewpoints. Alternatively, this rationale can be used in solu-tion-phase synthesis where reactive scavenger resins are added to the com-pleted reaction to remove any excess reagents. The advantage of thistechnique is that the reaction can be monitored, the product remains insolution, and product purity can be determined by standard methods.

Combinatorial chemistry, now a widely practiced technique in thepharmaceutical and biotechnology industry, is regarded as an importantcomponent of the drug discovery process.30 While traditional combinator-ial chemistry is carried out on solid supports, the use of solution-phasetechniques for library generation has gained momentum since the late1990s.31 The introduction of solid-supported scavengers facilitated thistrend12,32 and a diverse set of scavengers is now commercially available.Reactive groups (e.g., amines, aldehydes, thiols, hydrazines, isocyanates)linked to poly(styrene-divinylbenzene) beads (typically 1 or 3% cross-linked) are used to quench excess or to selectively react with starting ma-terials of complementary reactivity in solution. These quenching reagentsare particularly useful in solution-phase combinatorial chemistry, wherethe purification of large numbers of compounds is difficult to achieve usingtraditional methods such as crystallization or flash chromatography. How-ever, the use of quenching reagents does have some drawbacks. First, theymust be used in solvents with good polymer swelling properties such as

22 J. A. Ellman and L. A. Thompson, Chem. Rev. 96, 555 (1996).23 J. J. Parlow and J. E. Normansell, Mol. Divers. 1, 266 (1995). D. C. Sherrington and

P. Hodge, ‘‘Syntheses and Separations Using Functional Polymers.’’ Wiley, Chichester, UK,

1988. A. Akelah and D. C. Sherrington, Chem. Rev. 81, 557 (1981).24 A. Studer, S. Hadida, R. Ferritto, S. Kim, P. Jeger, P. Wipf, and D. P. Curran, Science 275,

823 (1997).25 J. S. Fruchtel and G. Jung, Angew. Chem. Int. Ed. Engl. 35, 17 (1996).26 A. Kirschning, H. Monenschein, and R. Wittenberg, Angew. Chem. Int. Ed. Engl. 40, 650

(2001).27 A. Dondoni and A. Massi, Tetrahedron Lett. 42, 7975 (2001).28 G. L. Bolton, R. J. Booth, M. W. Creswell, J. C. Hodges, J. S. Warmus, M. W. Wilson, and

R. M. Kennedy, U. S. Patent 9742230 (1997).29 J. Eames and M. Watkinson, Eur. J. Org. Chem. 1213 (2001).30 R. B. Nicewonger, L. Ditto, and L. Varady, Tetrahedron Lett. 41, 2323 (2000).31 R. E. Dolle and K. H. Nelson, Jr., J. Comb. Chem. 1, 235 (1999).32 D. L. Flynn, J. Z. Crich, R. V. Devraj, S. L. Hockerman, J. J. Parlow, M. S. South, and

S. Woodard, J. Am. Chem. Soc. 119, 4874 (1997).


DMF,* methylene chloride, or THF. Second, these solvents are undesirablewhen running thousands of reactions (DMF is difficult to remove from thefinal product, methylene chloride is toxic, and THF may contain perox-ides). Third, according to most manufacturer’s instructions,16 an excess ofquenching reagent is required for a few hours to overnight to completelyremove impurities, which translates into longer synthesis times whenrunning large number of reactions.

A goal to overcoming the latter is to find alternative base matrices forsolid-phase quenching reagents that would rapidly remove impurities(15 min or less) in a broader range of solvents. Macroporous support, ahighly cross-linked polystyrene material class, is the support of choice forsolid-phase organic synthesis in acetonitrile because it swells equally inpolar and nonpolar solvents as long as the solvent wets the surface.33 Inaddition, macroporous resins are widely used as chromatography matriceswhere effective mass transfer between the pores and the bulk solvents canovercome the slow diffusion kinetics experienced when 1–2% cross-linkedgels are used.34

The combination of solution-phase with solid-phase reagents allows forthe selective removal of excess reagents and/or by-products within a certainreaction containing a wide variety of functional groups.2,12,32,35–44 These

* Abbreviations: Amberlite IRA 400 borohydride resin, commercially available resin for

selective reduction of �,�-unsaturated aldehydes and ketones; DBU, 1,8-diazabicyclo

[5.4.0]undec-7-en; DCM, dichloromethane; DMF, dimethylformamide; EtOAc, ethyl

acetate; Et2NH, diethylamine; Et3N, triethylamine; HBr, hydrobromic acid; HCl,

hydrochloric acid; MS, mass spectrometry; MeOH, methyl alcohol; NH4OH, ammonium

hydroxide; NaBH4, sodium borohydride; NaOH, sodium hydroxide; NaOMe, sodium

methanolate; PAMAM, polyaminoamide resin (commercially available); RT, room

temperature; THF, tetrahydrofuran.33 A. K. Ghosh, P. Mathivanan, and J. Cappiello, Tetrahedron Lett. 38, 2427 (1997).34 C. A. Doyle and J. G. Dorsey, in ‘‘Handbook of HPLC’’ (E. Katz, R. Eksteen,

P. Schoenmakers, and N. Miller, eds.), p. 293. Marcel Dekker, New York, 1999.35 S. W. Kaldor, J. E. Fritz, J. Tang, and E. R. McKinney, Bioorg. Med. Chem. Lett. 6, 3041

(1996).36 S. D. Brown and R. W. Armstrong, J. Am. Chem. Soc. 118, 6331 (1996).37 T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 118, 2574 (1996).38 S. D. Brown and R. W. Armstrong, J. Org. Chem. 62, 7076 (1997).39 A. Cheminat, C. Benezra, M. J. Farral, and J. M. J. Frechet, Tetrahedron Lett. 21, 617

(1980).40 A. Cheminat, C. Benezra, M. J. Farral, and J. M. J. Frechet, Can. J. Chem. 59, 1405 (1981).41 J. M. J. Frechet, M. J. Farral, C. Benezra, and A. Cheminat, Polym. Prep. Am. Chem. Soc.

Div. Polym. Chem. 21, 101 (1980).42 J. J. Parlow, D. A. Mischke, and S. S. Woodard, J. Org. Chem. 62, 5908 (1997).43 J. J. Parlow, W. Naing, M. S. South, and D. L. Flynn, Tetrahedron Lett. 38, 7959 (1997).44 A. J. Shuker, M. G. Siegel, D. P. Matthews, and L. O. Weigel, Tetrahedron Lett. 38, 6149

(1997).

Fig. 1. General approaches to the removal of excess reagents using nucleophilic and

electrophilic scavenger resins.


compounds are anchored onto the resin leaving the desired material in so-lution so a simple filtration renders pure product without the need for fur-ther chromatographic purification as seen in Fig. 1. This principle makesuse of innovative resins with a variety of functional groups backed by morethan 20 years of research on the subject.11,12,32,38–47 A recent descriptivereview summarizes a great amount of data.16 A considerable amount ofscavenger resins is commercially available for these specific work-up pur-poses depending upon their applicability.48 In addition, specific scavengerresins can easily be custom prepared by simple chemical transformationson the available resins.37

When reacting two substrates in solution (solution phase) to form a de-sired product (R1-El-Nu-R2 in Fig. 1), a resin with the desired characteris-tics (solid phase) is utilized to trap undesired material. A scavenging resin,usually added upon reacting of the substrates, interacts with the undesiredreagent, thus forming a chemically modified new resin. Upon simple filtra-tion, this resin is separated from the reaction mixture providing (in somecases clean) product without further purification being necessary.

For a solution-phase parallel synthesis to be efficient, a complete con-version of reactants to product with little or no formation of by-productsor impurities is required. Here the concept of solid-phase-assisted solu-tion-phase organic synthesis comes into play. The important characteristicto have in mind about polymer-assisted solution-phase organic synthesis

45 D. C. Sherrington and P. Hodge, ‘‘Synthesis and Separations Using Functional Polymers.’’

John Wiley & Sons, New York, 1988.46 P. Lazlo, ‘‘Preparative Chemistry Using Supported Reagent.’’ Academic Press, New York,

1987.47 D. L. Flynn, Med. Res. Rev. 19, 408 (1999).48 Some representative examples can be found at www.sigma-aldrich.com, www.argotech.com,

www.albmolecular.com, www.polymerlabs.com, www.glycopep.com, www.huric.com,

www.novabiochem.com, and www.silicycle.com.


should be the added simplicity in manipulating both reaction work-ups andpurifications. This characteristic centers on simple resin filtration to separ-ate pure product from starting material, thus allowing the chemist to useexcess reagents to drive reactions to completion. Additional characteristicsshould be the cost efficiency factor and the ability to obtain larger amountsof final products.49

The use of scavenger resins in manual or automated parallel synthesiswork-ups is intended to reduce purification times. Due to the commercialavailability of a great number of scavenger resins, large amounts of syn-thetic organic reactions are benefited. One of the potential drawbacksthese resins may encounter in organic synthesis is that in some instancesa large quantity of resin is necessary to clean up a typical product, thus pre-senting physical difficulties since the beads themselves swell in solvent,49 aparticularly difficult task when the reaction is performed on a small scale.To broaden the scope of applications for scavenger resins, the use of a com-bination of different resins50 is advised when the extraction of a variety ofundesired products and/or excess starting materials is desired.

Several polymer-supported quenching reagents have been designed toremove a wide variety of functionalities. These resins vary in applicationsdepending upon their inherent chemical nature. Some of the most widelyused scavenger supports include resins specific for trapping HCl,51 acidhalides,28 alcohols,28 aldehydes,52,53 alkyl halides,28 amine scaven-gers,2,12,26,28,29 boronic acids,54,55 carboxylic acids,56,57 isocyanates,12,28,58

isothiocyanates,12,28,58 and sulfonyl chloride.2,12,28,30,32,51,55,59 In addition,some of these resins have been expanded into dendrimer-type resins withapplications in combinatorial chemistry45,60 as high loading proton, nucleo-phile, or electrophile scavengers. Two creative examples are shown in

49 R. Santini, M. C. Griffith, and M. Qi, Tetrahedron Lett. 39, 8951 (1998).50 D. Cork and N. Hird, Drug Discov. Today 7, 56 (2002).51 C. Gennari, S. Ceccarelli, U. Piarulli, C. Montalbetti, and R. F. W. Jackson, J. Org. Chem.

63, 5312 (1998).52 N. Bicak and B. F. s,enkal, J. Polym. Sci. 35, 2857 (1997).53 M. Panunzio, M. Villa, A. Missio, T. Rossi, and P. Seneci, Tetrahedron Lett. 39, 6585 (1998).54 P. Hodge and J. Waterhouse, J. Chem. Soc. Perkin Trans. I 2319 (1983).55 A. G. M. Barrett, M. L. Smith, and F. J. Zecri, J. Chem. Soc. Chem. Commun. 2317 (1998).56 S. Affrossman and J. P. Murray, J. Chem. Soc. B 1015 (1966).57 I. C. Chisem, J. Rafelt, M. T. Shieh, J. Chisem, J. H. Clark, R. Jachuck, D. Macquarrie,

C. Ramshaw, and K. Scott, J. Chem. Soc. Chem. Commun. 1949 (1998).58 J. Habermann, S. V. Ley, and J. S. Scott, J. Chem. Soc. Perkin Trans. I 3127 (1998).59 M. Caldarelli, J. Habermann, and S. V. Ley, Bioorg. Med. Chem. Lett. 9, 2049 (1999).60 L. Williams and S. M. Neset, 4th Int. Elect. Conf. Synth. Org. Chem. (ECSOC-4), B0011

(2000). R. M. Kim, M. Manna, S. M. Hutchins, P. R. Griffin, N. A. Yates, A. M. Bernick,

and K. T. Chapman, Proc. Natl. Acad. Sci. USA 93, 10012 (1996). A. B. Kantchev and J. R.

Parquette, Tetrahedron Lett. 40, 8049 (1999).


Fig. 2. Resin 1 is a [1,3,5]triazin-2-oxy-based resin with morpholine endtips. Resin 2 is a simple starbust polyaminoamide (PAMAM) commerciallyavailable resin with 64 surface primary amino groups.54

Commercially available basic resins that are available employed forneutralizing HCl51 contain a tertiary amine or pyridinyl functionality,which readily traps proton species. Some examples are amine-basedmorpholinomethyl (3) and piperidinemethyl (4) resins (Fig. 3).

Commercially available acidic resins, as well as ion exchangers, that areused for scavenging amines or other basic compounds contain an acidicfunctionality, which interacts with the counter basic moiety, thus formingan easily removable salt. Some representative examples are those of ben-zoic acid (5) and sulfonic acid (6) resins (Fig. 4).

Carbonate resins are generally used to neutralize strong mineral acids(e.g., HBr) when generated in situ as a by-product on certain reactions.These resins normally contain a quaternary ammonium carbonate saltfunctionality. Specific examples are triethylammonium carbonate (7) andtrimethylammonium bicarbonate (8) resins (Fig. 5) ion exchanging the an-ionic halogen for carbonate.

To capture electrophilic substrates such as acid halides, aldehydes, alkylhalides, isocyanates, and isothiocyanates, a variety of nucleophilic resins arecommonly used. Some commercially available and representative examplesare tris-(2-aminoethyl)amine (9), thiophenol (10), sulfonylhydrazide (11),triphenylphosphine (12), and methylthiourea (13) polymer resins (Fig. 6).

To capture nucleophilic substrates such as alcohols, amines, triphenyl-phosphine, carboxylic acids, and the like, a variety of electrophilic resinshave been developed. Some interesting examples are benzaldehyde (14),methylisocyanate (15), methylisothiocyanate (16), chloromethystyrene(17, Merrifield resin),61 benzenesulfonyl chloride (18), and N-methylisatoicanhydride (19) resins (Fig. 7).

Among the custom made resins that have been developed recently forscavenging electrophilic substrates, a few examples are worth mentioning:oligo(ethyleneimine) (20), morpholinodiethanolamine (21), amine/ami-noalcohol (22), guanidine (23), and 4-phenol-substituted (24) (Fig. 8).

Some rather interesting custom made electrophilic resins that havebeen developed lately as nucleophile scavenging materials are the acidchloride (25) and arenesulfonyl chloride (26) resin shown in Fig. 9. Resin26 is stable to various reaction conditions over resin 18 and can be usedwhile conducting subsequent chemical steps.62

61 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).62 J. K. Rueter, S. O. Nortey, E. W. Baxter, G. C. Leo, and A. B. Reitz, Tetrahedron Lett. 39,

975 (1998). H. M. Zhong, M. N. Greco, and B. E. Maryanoff, J. Org Chem. 62, 9326 (1997).

Fig. 2. [1,3,5]Triazin-2-oxy-based resin with morpholine end tips (1) and starbust

polyaminoamide (2) commercially available resins.


Fig. 3. Morpholino and piperidinemethyl-based commercially available basic resins.

Fig. 4. Benzoic and sulfonic acid-based commercially available acidic resins.

Fig. 5. Triethylammonium carbonate (7) and trimethylammonium bicarbonate (8)-based

commercially available neutralizing resins.

Fig. 6. Tris-(2-aminoethyl)amine (9), thiophenol (10), sulfonylhydrazide (11), triphenyl-

phosphine (12), and methylthiourea (13)-based commercially available nucleophilic resins.


Scavenger Resins Synthetic Applications

The synthesis of compound 2712,63 is of special interest since it showsthe application of different types of resins (basic and electrophilic) beingused sequentially at different stages during the synthesis for the removal

63 W. Murray, M. Wachter, D. Barton, and Y. Forero-Kelly, Synthesis 18 (1991).

Fig. 7. Benzaldehyde (14), methylisocyanate (15), methylisothiocyanate (16) chloro-

methystyrene (17, Merrifield resin), benzenesulfonyl chloride (18), and N-methylisatoic

anhydride (19)-based commercially available electrophilic resins.

Fig. 8. Oligo(ethyleneimine) (20), morpholinodiethanolamine (21), amine/aminoalcohol

(22), guanidine (23), and 4-phenolsubstituted (24)-based custom made electrophilic

scavenging resins.

Fig. 9. Acid chloride (25) and arenesulfonyl chloride (26)-based custom made nucleophilic

scavenging resins.


of hydrochloric acid and excess of 4-hydrazinobenzoic acid, respectively. Inthe first step of the synthesis the reaction mixture is treated withcommercially available morpholine resin 3 to trap hydrochloric acid,


then after filtration of protonatated resin 3 the isocyanate resin 15 isadded. Thus, an aminourea resin is being generated that can also beextracted from the reaction mixture by simple filtration affording clean4-(3-methyl-5-phenyl-pyrazol-1-yl)-benzoic acid 27 (Fig. 10).

Another interesting example encountering numerous applications wasreported by Bolton et al.28 It comprises the implementation of a commer-cially available high-loading nucleophilic resin 9 for the removal of excessstarting material 2-bromobenzoyl chloride (28) during the synthesis of asimple amide such as that of N-benzyl-2-bromo-N-methylbenzamide (29),as indicated in Fig. 11.

Fig. 10. Synthesis of 4-(3-methyl-5-phenyl-pyrazol-1-yl)-benzoic acid (27).

Fig. 11. Synthesis of N-benzyl-2-bromo-N-methylbenzamide (29).


Rueter et al.62 described an efficient and clean synthesis of diethyl-(2-p-tolyl-ethyl)amine (30) from 2-p-tolylethanol and diethylamine by makinguse of the benzenesulfonyl chloride resin (26) to ‘‘catch’’ the intermediateO-alkylated substrate (31) followed by the ‘‘release’’ (from the intermedi-ate resin) of the final product (30) upon treatment with diethylamine(Fig. 12).

When benzylbromide is allowed to react with 4-(3-mercapto-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (32) in order to synthesize thecorresponding thiobenzyl adduct 4-(3-benzylsulfanyl-5-phenyl-[1,2,4]tria-zol-4-ylmethyl)-benzamide (33), a nucleophilic sulfur-based resin (34) isemployed to trap excess benzylbromide from the reaction mixture affordinga final clean product (33).28 In addition, a basic Amberlite (OH�) is added toassist in the efficient deprotonation of the mercapto functionality in (34) asdepicted in Fig. 13.

A very interesting reported application of a resin combination (specif-ically for the synthesis of a wide variety of amides)12,63 is depicted by thesynthesis of N-benzyl-2-bromo-N-methy-benzamide (35), shown in Fig. 14,from simple starting materials. At first, benzylmethylamine is reacted with

Fig. 12. Synthesis of diethyl-(2-p-tolyl-ethyl)amine (30).

Fig. 13. Synthesis of 4-(3-benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide

(33).

Fig. 14. Synthesis of N-benzyl-2-bromo-N-methyl-benzamide (35).

Fig. 15. Synthesis of 1-benzyl-3-phenyl-thiourea (36).


2-bromobenzoyl chloride in the presence of the basic morpholine resin (3),which acts as a proton scavenger for hydrochloric acid generated in situ.Second, addition of the electrophilic (isocyanate) resin (15) yields a newchemically modified resin in the form of a urea when reacting with excessbenzylmethylamine. Finally, upon filtration of the final urea resin, pureN-benzyl-2-bromo-N-methyl-benzamide (35) is isolated.

The synthesis 1-benzyl-3-phenyl-thiourea (36) requires the applicationof a recently described and commercialized isatoic resin (19).1 Benzyla-mine readily reacts with isothiocyanato-benzene to afford 1-benzyl-3-phenyl-thiourea (36). However, excess benzylamine needs to be removedby adding resin (19), thus providing pure product (36) upon simplefiltration of the amide resin formed as depicted in Fig. 15.

Recently, a novel commercially available acetoacetoxyethyl metacry-late resin (37) is finding wide applications as a selective electrophilic scav-enger resin. This resin has the ability to differentiate primary amines from amixture where secondary amines are present.64,65 An illustrative example isdepicted in the synthesis of dibenzylamine (38, Fig. 16) from benzaldehydeand benzylamine. Unreacted benzylamine is selectively removed from thereaction mixture upon treatment with the ketoacetate resin (37).

64 Z. Yu, S. Alesso, D. Pears, P. A. Worthington, R. W. A. Luke, and M. Bradley, Tetrahedron

Lett. 41, 8963 (2000).65 Z. Yu, S. Alesso, D. Pears, P. A. Worthington, R. W. A. Luke, and M. Bradley, J. Chem.

Soc. Perkin Trans. I 1947 (2001).

Fig. 16. Synthesis of dibenzylamine (38).

Fig. 17. Synthesis of a boronic acid-based resin.


The newly developed boronic acid scavenger diethanolamine resin(39)66 (Fig. 17) has found applications in a wide variety of importantcoupling reactions, such as the Suzuki couplings.67 This resin is largely usedin the pharmaceutical industry for drug development. A representativeexample published by Hall et al.66 shows the relatively fast immobilizationof 4-carboxyboronic acid at room temperature. This resin undoubtedly willfind extensive applications in modern synthetic organic chemistry as well,as demonstrated by the increased number of carbon–carbon,68 carbon–nitrogen,69,70 and carbon–oxygen71 bond formation, in addition to urea72

66 D. G. Hall, J. Tailor, and M. Gravel, Angew. Chem. Int. Ed. Engl. 38, 3064 (1999).67 N. Miyaura and A. Suzuki, Chem. Rev. 95, 2457 (1995).68 A. Suzuki, J. Organomet. Chem. 576, 147 (1999).69 J. F. Hartwig, Angew. Chem. Int. Ed. Engl. 37, 2046 (1998). J. P. Wolfe, S. Wagaw, J.-F.

Marcoux, and S. L. Buchwald, Acc. Chem. Res. 31, 805 (1998). B. H. Yang and S. L.

Buchwald, J. Organomet. Chem. 576, 125 (1999).70 G. Mann, J. F. Hartwig, M. S. Driver, and C. Fernandez-Rivas, J. Am. Chem. Soc. 120, 827

(1998). B. C. Hamann and J. F. Hartwig, J. Am. Chem. Soc. 120, 7369 (1998). D. W. Old,

J. P. Wolfe, and S. L. Buchwald, J. Am. Chem. Soc. 120, 9722 (1998). J. F. Hartwig,

M. Kawatsure, S. I. Hauck, K. H. Shaughnessy, and L. M. Alcazar-Roman, J. Org. Chem.

Fig. 18. Synthesis of tetra-substituted pyrazoles (40).


and ketone73 formation reactions in which boronic acids are beingemployed.

Some useful resins that have found their way into quite substantial ap-plications are the carbonate resins. For instance, trimethylammonium bi-carbonate resin (8) is used to neutralize carboxylic acids or strongmineral acids formed in situ in specific reactions. An interesting exampleis the quenching/scavenging of acid bromide reported by Staufer and Kat-zenellenbogen74 during the synthesis of tetra-substituted pyrazoles (40)from immobilized starting materials as seen in Fig. 18.

A nucleophilic resin with the potential for being transformed into ahigh-loading or dendrimer-type of resin is the triethylenetetraamine resin(9) employed in the scavenging of excess electrophiles. Representativeand useful reactions for this resin are in the preparations of ureas andthioureas. For example, when 2-(2-isocyanato-ethyl)thiophene is reactedwith butylamine during the preparation of 1-butyl-3-(2-thiophen-2-yl-ethyl)urea (41), triethylenetetraamine resin (9) readily scavenges any excess of2-(2-isocyanato-ethyl)thiophene. This excess material is easily removedfrom the reaction mixture by simple filtration as the urea adduct resin(42).12 Similarly, in the synthesis of 1-(3-isopropoxy-propyl)-3-phen-ethylthiourea (43), triethylenetetraamine resin (9) readily scavenges excess(2-isothiocyanato-ethyl)benzene by forming a bis-isothiourea resin (44)easily removable by filtration from the reaction mixture,12 as indicated inFig. 19.

A quite useful resin in nucleophilic carbon–carbon bond formation re-actions is the carboxylic resin (5). The soft metal from the nucleophile is

64, 5575 (1999). J. P. Wolfe and S. L. Buchwald, J. Org. Chem. 65, 1144 (2000). J. P. Wolfe,

H. Tomori, J. P. Sadighi, J. Yin, and S. L. Buchwald, J. Org. Chem. 64, 1158 (2000).71 D. A. Evans, J. L. Katz, and T. R. West, Tetrahedron Lett. 39, 2937 (1998).72 G. A. Artamkina, A. G. Sergeev, and I. P. Beletskaya, Tetrahedron Lett. 42, 4381 (2001).73 M. Haddach and J. R. McCarthy, Tetrahedron Lett. 40, 3109 (1999).74 S. R. Stauffer and J. A. Katzenellenbogen, J. Comb. Chem. 2, 318 (2000).


trapped in the form of a metal carboxylic salt resin (45) as depicted inFig. 20. Upon completion of the carbon-carbon bond formation reaction,the resin is then added to the mixture. The resin addition insures both carb-oxylic salt formation as well as protonation of the hydroxy anion formedduring the reaction. An illustrative example is the alkylation of benzalde-hyde with buthyllithium.32 The reaction between these two reactants takesplace from �78

�C to room temperature over a 2.5 h time period. The reac-

tion mixture is then treated with carboxylic acid resin (5). Upon simplefiltration, clean 1-phenylpentan-1-ol (46) is obtained.

The synthesis of 3-benzyl-2-phenylthiazolidin-4-one (47, Fig. 21) dem-onstrates the mercaptane scavenging applicability of the aminoethanethiolresin (48). Benzaldehyde is allowed to react with benzylamine in the pres-ence of mercapto acetic acid (49) in toluene under refluxing conditions.

Fig. 19. Synthesis of 1-butyl-3-(2-thiophen-2-yl-ethyl)urea (41) and 1-(3-isopropoxy-

propyl)-3-phenethylthiourea (43).

Fig. 20. Synthesis of 1-phenylpentan-1-ol (46).

Fig. 21. Synthesis of 3-benzyl-2-phenylthiazolidin-4-one (47).

Fig. 22. Synthesis of benzylamine.


The addition of the aminoethanethiol resin (48) follows, and an aminoetha-nesulfanyl acetic acid resin (50) is being formed alongside the desired prod-uct. Upon filtration of the resin, clean 3-benzyl-2-phenylthiazolidin-4-one(47) is obtained.75

One of the most used resins in solid-phase combinatorial organic syn-thesis, which has found a myriad of applications, is the Merrifield resin(17).61 This resin is also the building block for a tremendous amount ofnovel resins being developed in combinatorial chemistry with applicationsin both solid-phase as well as solid-phase-assisted solution-phase combina-torial chemistry. A recent, useful, and novel example is the report of itsbeing employed as a triphenylphosphine scavenging resin.76 During theconversion of azidomethylbenzene (51) into benzylamine, excess triphenyl-phosphine is allowed to react with Merrifield resin (17) in the presence ofsodium iodide in acetone. A phosphonium-substituted resin (52) is thusformed. Upon simple filtration, pure benzylamine is isolated as shown inFig. 22.

75 S. E. Ault-Justus, J. C. Hodges, and M. W. Wilson, Biotechnol. Bioeng. 61, 17 (1998).76 B. H. Lipshutz and P. A. Blomgren, Org. Lett. 3, 1869 (2001).


Concluding Remarks

Described are varied resins available for wide application in solid-phase-assisted solution-phase combinatorial chemistry during the processof the purification of reaction mixtures. Therefore, what was once a com-plex work-up procedure was transformed into a simple reaction mixture fil-tration with the possibility of avoiding liquid-phase extraction protocols forreaction quenching and work-ups. Hence, this process has some remark-able advantages. Primarily, there is minimization or avoidance of chroma-tographic purification of the desired product. This results in the feasibilityof being implemented in high-throughput parallel automated synthesis. Asa consequence, the reaction protocols allow for excess reagents to be addedto drive solution-phase reactions to completion. In conclusion, solid-phase-assisted solution-phase combinatorial chemistry processes result in reducedtime and costs in overall synthetic development.



Unless otherwise indicated, all reactions are run in capped glass vials,without the use of an inert atmosphere, and were shaken on an orbitalshaker. THF was purchased distilled in a sure-seal bottle. Other reagentsand anhydrous solvents were commercially available and used withoutfurther purification. Scavenger resins are available from a wide variety ofsuppliers48 and used without further purification.

Resin Preparation

Oligo(ethyleneimine) Resin (20). A cold (0�) solution of the chlorosul-

fonylated resin (18)77 (15 g, 0.12 mol) in dimethoxyethane (40 ml) wastreated with a dimethoxyethane solution (20 ml) of triethylenetetraamine(0.596 mol) dropwise. The resulting reaction mixture was allowed to stirfor 24 h at RT. Upon filtration, the hydrochloride salt form of the resinformed was washed with ethanol (20 ml) followed by water in excessamounts. The resin was further washed with a 5% NaOH solution(100 ml) under stirring for 30 min. Upon decantation, water (100 ml) wasadded and the resulting mixture was boiled for 30 min. The reaction mix-ture was filtered and washed successively with water in excess followedby ethanol (20 ml). The resin was then dried under vacuum at 40

�for 24 h.

77 N. Bicak and B. F. s,enkal, React. Funct. Polym. 29, 123 (1996).


Aminodiol/morpholine Resin (21). A suspension of Merrifield resin(2 g, 4.3 mmol Cl/g resin, 8.6 mmol) in DMF (20 ml) was treated withdiethanolamine (1.5 g, 14.3 mmol) and morpholine (1.2 g, 14.3 mmol) byshaking the resulting reaction mixture at 65

�for 6 h under a nitrogen at-

mosphere. Upon cooling to RT, the resin was filtered and washed withMeOH, DMF, Et3N, MeOH, DCM, Et3N, MeOH, DCM, MeOH, DCM,and MeOH. The resulting resin was then dried at 45

�under vacuum.

Amine/aminoalcohol Resin (22). A suspension of Merifield resin (5 g,4.3 mmol Cl/g resin, 21.5 mmol) and morpholine (5.2 ml, 60 mmol) inDMF (35 ml) was treated with piperidin-3-yl-methanol (2.3 g, 20 mmol) at65

�for 6 h under a nitrogen atmosphere. Upon cooling to RT, the resin was

filtered and washed with DMF, MeOH, Et3N, DMF, MeOH, Et3N, MeOH,DCM, MeOH, DCM, and EtOAc. The resulting amine/aminoalcohol resinwas dried at 45

�under vacuum overnight.

Guanidine Resin (23). A suspension of Merrifield resin (5 g, 1.7 mmolCl/g resin) in DMF (100 ml) was treated with guanidine hydrochloride(5 g) and a solution (1 M) of potassium tert-butoxide in THF (50 ml).The resulting reaction mixture was heated at 90

�for 24 h. Upon cooling,

the resulting resin was filtered and washed with DMF/DBU (7:3), DMF, di-oxane, water, THF, and ethyl ether. The resin thus produced was driedunder vacuum at RT overnight.

Phenol Resin (24). A solution of 3-(4-hydroxyphenyl)propionic acid(2.2 g, 13.3 mmole), N-ethyl-N0-dimethylaminopropylcarbodiimide (2.5 g,13 mmol) and 1-hydroxybenzotriazole monohydrate (1.8 g, 12 mmol) inDMF (20 ml) was treated with aminomethylpolystyrene resin (1.0 g,4.5 mmol N/g resin). The resulting reaction mixture was stirred for 36 hat room temperature. The product resin was obtained upon filtration andwashings with DCM, MeOH, a solution of NH4OH:MeOH (1:1), DMF,DCM, MeOH, DCM, and hexanes (2�) followed by drying under vacuumat 40

�.

Acid Chloride Resin (25). A suspension of 1-substituted benzylamine-HCl resin (2 g, 0.83 mmol N/g resin, 1.66 mmol) in DCM (20 ml) andN-methylmorpholine (1.2 ml, 10.9 mmol) was treated with benzene-1,3,5-tricarboxylic acid chloride (0.93 g, 3.5 mmol) by shaking the reaction mix-ture for a few minutes. The resulting suspension was stirred at RT for 1 h,diluted with DCM (200 ml), and filtered. The resin was then washed withDCM (2�) followed by EtOAc (2�) and dried at 35

�under vacuum.

Arenesulfonyl Chloride Resin (26). To a suspension of chlorobenzylMerrifield resin (5.0 g, 4.9 mmole) and anhydrous 4-hydroxybenzenesulfo-nic acid sodium salt (2.94 g, 15 mmole; commercial material was dehy-drated at 110

�in vacuo for 8 h) in N,N-dimethylacetamide (50 ml) was

added NaOMe (0.81 g, 15 mmol), and the mixture was stirred at 90�

for 2


days. The resin product (phenylsulfonic acid) was collected by filtration,washed (sequentially with DMF, 1.0 N HCl, MeOH, and DCM), and driedunder vacuum at 50

�for 24 h. The resin was treated with 1:1 Et2NH–DMF

(50 ml) for 1 h and washed with DMF. The reaction was suspended inDMF (50 ml) followed by the addition of PCl5 (5.2 g, 25 mmol) in severalportions, and the resulting suspension was stirred at 23

�for 4 h. After

washing the mixture with DMF and DCM, the resin was dried in a vacuumoven at 50

�overnight to afford the arenesulfonyl chloride resin suitable for

further applications.

Solid-Phase Assisted Solution-Phase Synthesis

4-(3-Methyl-5-phenyl-pyrazol-1-yl)-benzoic Acid (27). A mixture of4-hydrazino-benzoic acid hydrochloride (113 mg, 0.6 mmol) and morpho-line-based resin (3) in MeOH (2 ml) was treated with 1-phenyl-butane-1,3-dione (81.5 mg, 0.5 mmol) and shaken for 2.5 h. The MeOH wasallowed to evaporate under a stream of N2. DCM (4 ml) was then addedto the reaction mixture followed by the addition of isocyanate resin (15)(350 mg). The resulting reaction mixture was shaken for 16 h at which timeadditional amounts of isocyanate resin (15) (120 mg) were added. The mix-ture was shaken for 4 h followed by filtration. The filtered resin was washedwith DCM (2 � 1.5 ml). Upon concentration of the organic filtrate, cleanproduct was obtained. MS: 278.11 (M + 1).

N-Benzyl-2-bromo-N-methyl-benzamide (29). A solution of benzyl-N-methylamine (0.4 mmol), triethylamine (3 mmol), and 2-bromoben-zoylchloride (0.6 mmol) in DCM (1 ml) was shaken for 4 h. Amine-basedresin (9) (100 mg) was added and the reaction mixture was shaken over-night. Upon filtration and concentration, the residue was partitioned be-tween aqueous NaOH and EtOAc. Concentration of the organic layerafforded the purified product. MS: 304, 306 (M + 1).

Diethyl-(2-p-tolyl-ethyl)-amine (30). A solution of 2-(p-tolyl)-ethanol(3–5 mole-equiv.), triethylamine (3–5 mole-equiv.), and arenesulfonylchloride resin (26) in DCM (10 ml/mmol) was allowed to react at RT for48 h. The resulting resin (31) was then filtered and washed with DCM(2�), MeOH (2�), and DCM (2�). Upon treatment of resin (31) withexcess diethylamine at 60

�for 6 h, diethyl-(2-p-tolyl-ethyl)-amine (30)

was obtained.4-(3-Benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide

(33). A solution of 4-(3-mercapto-5-phenyl-[1,2,4]triazol-4-ylmethyl)-ben-zamide (32, 0.1 mmol) in THF (6 ml) was treated with Amberlite resin(OH� form, 0.1 mmol OH�) and benzyl bromide (0.15 mmol). The result-ing mixture was shaken at room temperature until complete consumption


of the starting thiol. Aminothiol resin (34, 100 mg) was added and the mix-ture was shaken at RT for 1 h. TLC showed that the excess benzyl bromidewas consumed. The resulting resins were removed by filtration and washedwith DCM. Upon concentration of the combined filtrates, 4-(3-benzylsulfa-nyl-5-phenyl-[1,2,4]triazol-4-ylmethyl)-benzamide (33) was obtained.

N-Benzyl-2-bromo-N-methyl-benzamide (35). A suspension of mor-pholine resin (3, 0.63 mmol) in DCM (2 ml) was treated with N-methyl-benzylamine (0.23 mmol) and 2-bromobenzoyl chloride (0.146 mmol).The reaction mixture was shaken for 5 h. Isocyanate resin (15, 0.2 g) wasadded followed by DCM (1 ml). The reaction mixture was then shakenfor 16 h. Filtration of the resin followed by concentration of the filtrategave the purified product.

1-Benzyl-3-phenylthiourea (36). A solution of isothiocyanatobenzene(1 mmol) in DCM (7 ml) was treated with benzylamine (1.2 mmol). Theresulting reaction mixture was allowed to stir at room temperature over-night. Then isatoic anhydride resin (19, 100 mg, 3.2 mmol anhydride/g)was introduced into the reaction vessel. The contents were shaken at RTfor 1.5 h followed by resin filtration. Upon evaporation of the filtrate solv-ent, pure benzylamine-free 1-benzyl-3-phenyl-thiourea (36) was obtained.

Dibenzylamine (38). A solution of benzaldehyde (0.5 mmol) in isopro-panol (1.5 ml) was treated with benzylamine (0.8 mmol). The resulting re-action mixture was shaken at RT for 2 h. Then Amberlite IRA 400borohydride resin (2.5 mmol NaBH4/g resin) was added and the mixturewas shaken at RT for 24 h. DCM (1.5 ml) was introduced followed bythe addition of the acetoacetoxyethyl metacrylate resin (37). The contentswere shaken at RT for 36 h. Upon filtration of the resin followed by solventevaporation of the filtrate, pure benzylamine-free dibenzylamine (38) wasobtained.

1-Butyl-3-(2-thiophen-2-yl-ethyl)urea (41). A solution of 2-(thienyl-2-yl)ethyl isocyanate (47 mg, 0.3 mmol) in DCM (2 ml) was treated with n-butylamine (25 �l, 0.25 mmol) at RT. The reaction mixture was shakenfor 1 h followed by addition of resin (9) (50 mg, 4.3 mmol N/g). After 2 hthe resin was filtered and washed with DCM (2 � 1.5 ml). The combinedorganic filtrates, when concentrated to dryness, gave the desired urea(44 mg, 99% yield) as an oil that crystallizes upon prolonged standing.

1-(3-Isopropoxy-propyl)-3-phenethyl-thiourea (43). A solution of 3-isopropoxypropyl amine (25 mg, 0.25 mmol) and 2-phenylethylisothiocya-nate (44 �l, 0.3 mmol) in DCM (2 ml) was shaken for 1.5 h at RT. Thetriethylenetetraamine resin (9, 50 mg) was then added and the resultingreaction mixture was shaken for 2 h. Upon filtration, the resin waswashed with DCM (2 � 1.5 ml). Evaporation of the filtrates gave thestarting-material-free thiourea.


1-Phenylpentan-1-ol (46). A solution of benzaldehyde (0.5 mmol) inTHF (2 ml) was treated at �78

�with a solution of n-buthyllithium

(0.36 ml, 1.6 M solution in hexanes, 0.57 mmol). The resulting reactionmixture was allowed to slowly reach RT and stirred for 2.5 h. Then carbox-ylic acid resin (5, 0.80 g, 8 mmol, �10.0 mequiv./g) was added and thesuspension was stirred for 4 h. The resin was filtered and rinsed withTHF a few times until complete washing of the product. Upon evaporationof the filtrate, lithium-free product was obtained, which was then dissolvedin DCM followed by the addition of polyamine resin (0.50 g, 1.49 mmol).This suspension was stirred at room temperature for 5 h. The reactionmixture was then filtered and the resin rinsed with DCM until com-plete washing of the product. Upon evaporation of the solvent, pure(benzaldehyde-free) 1-phenylpentan-1-ol was obtained.

3-Benzyl-2-phenylthiazolidin-4-one (47). A solution of benzylamine(0.2 ml, 0.2 mmol) in toluene (0.2 ml) was treated with a benzaldehyde(1.2 ml, 0.30 mmol) solution in toluene (1.2 ml), followed by the additionof a mecaptoacetic acid (49, 1.2 ml, 0.6 mmol) solution in toluene(1.2 ml) and molecular sieves (3 A). The reaction mixture was heated to80

�for 1.5 h. Then 2-aminoethanethiol resin (48, 0.3 g, 1.0 mmol) was

added and the mixture was allowed to cool to RT overnight. The mixturewas then treated with basic alumina (0.5 g) and shaken for 1 h. Additionaltoluene (5 ml) was added. Upon filtration and evaporation of the solvent,the product (47) was thus obtained.

Benzylamine. A solution of azidomethylbenzene (51) in THF wastreated with water followed by excess addition of triphenylphosphine(75 mg, 0.28 mmol). The reaction mixture was allowed to react at RT.Upon concentration under vacuum, acetone (1.5 ml) was added followedby the addition of sodium iodide (84 mg, 0.56 mmol) and high loadingMerrifield resin (17, 140 mg, 4.38 mmol of Cl/g). The resulting mixturewas allowed to stir at RT overnight. The resin was filtered and washed withTHF (3 � 3 ml), water (3 � 3 ml), acetone (3 � 3 ml), and finally methanolaffording triphenylphosphine-free benzylamine.

[22] cyclative cleavage strategies 415

[22] Cyclative Cleavage Strategies for the Solid-PhaseSynthesis of Heterocycles and Natural Products

By A. Ganesan

Introduction

For many years, solid-phase organic synthesis was predominantlyemployed in the stepwise assembly of peptides and nucleotides, using theC-terminal carboxylic acid and the 30-terminal alcohol, respectively, as attach-ment points to the resin. Because these functional groups are part of the finaloligomer, their unmasking by resin cleavage at the end of the synthesis is notan issue. More recently, solid-phase techniques have become tremendouslypopular for combinatorial chemistry, particularly in the areas of drug andcatalyst discovery. In these applications, the functional group used for immo-bilization may serve no other purpose. Consequently, the release of thisdangling functionality (colloquially referred to as a ‘‘navel’’) upon cleavagecan be an undesirable ‘‘memory’’ or ‘‘trace’’ of the point of attachment. Sinceresin attachment usually involves nucleophilic and often ionizable hetero-atom functional groups such as carboxylic acids, amines, alcohols, and thiols,this can seriously perturb the structural properties of the target molecule.

One solution that circumvents the above problem is to design a solid-phasesynthesis in which the last step is an intramolecular cyclization reaction involv-ing the point of attachment (Fig. 1). This strategy was first termed ‘‘cyclativecleavage’’ in a review by DeWitt and Czarnik1 describing the Diversomereffort at Parke-Davis. Others have used ‘‘cyclitive cleavage,’’ ‘‘cyclizationcleavage,’’ ‘‘cyclorelease,’’ ‘‘cycle-elimination,’’ or ‘‘traceless cleavage’’ inter-changeably, although the original definition2 reserves ‘‘traceless’’ synthesisstrictly for the formation of a C–H bond upon cleavage.

Compared to conventional resin cleavage methods, the cyclative strat-egy has two principal advantages. First, the resulting cyclic molecule doesnot have a free vestigial functional group at the point of resin attachment.Second, only the final intermediate on solid phase has the necessary func-tionality for the cyclization reaction. This is of great value when carryingout multiple reaction steps on solid phase, as any by-products or failedintermediates of earlier reactions are unable to undergo the final cycliza-tion. Thus, the overall yield of a cyclative cleavage sequence may be lowor high depending on the efficiency of each step. However, even in those

1 S. H. DeWitt and A. W. Czarnik, Acc. Chem. Res. 29, 114 (1996).2 M. J. Plunkett and J. A. Ellman, J. Org. Chem. 60, 6006 (1995).



Fig. 1. Conventional versus cyclative solid-phase cleavage.

416 small molecule and heterocycle synthesis [22]

cases in which the yield is low, the purity of cyclatively cleaved materialshould still be high. Often, compounds can be submitted to biologicalassays without further purification.

In the following reaction schemes, the yields quoted are usually overall forthe total sequence on solid phase. For simplicity, the exact nature of the solid-phase linker is not detailed. While some of these cyclizations are likely to beefficient regardless of the linker used, others can be highly susceptible to stericand electronic effects. When in doubt, the original literature should be con-sulted, bearing in mind that it is the exception rather than the rule for authorsto explain why a particular resin or linker was chosen. To facilitate an appre-ciation of the underlying chemistry, the examples are classified according tocyclization reaction rather than type of molecule produced. The literaturecoverage3 extends to mid-2002. The focus is on small molecules, and thesynthesis of cyclic peptides4 is deliberately omitted.

Nitrogen Nucleophile Attacking sp2 or sp3 Carbon: Five-MemberedRing Formation

This category represents the most popular subclass of cyclative cleavagereactions. There are many solid-phase sequences with resin attachment viacarboxylic acids, followed by elaboration to a free amine five centers away

3 For other recent reviews on solid-phase cleavage strategies, see (a) A. C. Comely and

S. E. Gibson, Angew. Chem. Int. Ed. Engl. 40, 1012 (2001). (b) V. Krchnak and

M. W. Holladay, Chem. Rev. 102, 61 (2002). (c) P. Blaney, R. Grigg, and V. Sridharan,

Chem. Rev. 102, 2607 (2002).4 For a review, see J. N. Lambert, J. P. Mitchell, and K. D. Roberts, J. Chem. Soc., Perkin

Trans. I 471 (2001).

Fig. 2. Examples of hydantoin synthesis by cyclative cleavage.


and cleavage by intramolecular cyclization. A pioneering example5 is theacid-catalyzed cyclization reported by the Parke-Davis group (Fig. 2).Using the Diversomer apparatus for parallel synthesis, 39/40 hydantoinswere successfully prepared. Later, the hydantoin cyclization was accom-plished6 by a Lilly group under basic conditions by heating with excesstriethylamine. An array of 800 hydantoins was prepared, and a randomsampling of 15% of the library showed product formation in 90% of thecases. This was soon followed by a report by Kim et al.7 of similar cleavageat room temperature using neat diisopropylamine.

In the hydantoin synthesis by Hanessian and Yang,8 the initial cycliza-tion product undergoes solution-phase loss of benzyl alcohol and nucleo-philic attack to give 5-alkoxyhydantoins, while that by Wilson et al.9

provides access to 1-aminohydantoins (Fig. 3). Cyclization in a more com-plex setting is illustrated by the Affymax10 transformation of a bicyclic scaf-fold resulting from 1,3-dipolar cycloaddition to a tricyclic hydantoin. Dueto its importance in drug discovery, there are numerous other examples11

of hydantoin and thiohydantoin synthesis via cyclative cleavage.

5 S. H. DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. Reynolds Cody, and

M. R. Pavia, Proc. Natl. Acad. Sci. USA 90, 6909 (1993).6 B. A. Dressman, L. A. Spangle, and S. W. Kaldor, Tetrahedron Lett. 37, 937 (1996).7 S. W. Kim, S. Y. Anh, J. S. Koh, J. H. Lee, S. Ro, and H. Y. Cho, Tetrahedron Lett. 38, 4603

(1997).8 S. Hanessian and R.-Y. Yang, Tetrahedron Lett. 37, 5835 (1996).9 L. Wilson, M. Li, and D. E. Portlock, Tetrahedron Lett. 39, 5135 (1998).

10 G. Peng, A. Sohn, and M. A. Gallop, J. Org. Chem. 64, 8342 (1999).11 (a) J. Matthews and R. A. Rivero, J. Org. Chem. 62, 6090 (1997). (b) S. W. Kim, J. S. Koh,

S. Ro, and E. J. Lee, Mol. Divers. 3, 129 (1997). (c) A. Boeijen, J. A. W. Kruitzer, and

R. M. J. Liskamp, Bioorg. Med. Chem. Lett. 8, 2375 (1998). (d) J. Stadlwieser, E. P. Ellmerer-

Muller, A. Tako, N. Maslouh, and W. Bannwarth, Angew. Chem. Int. Ed. Engl. 37, 1402

(1998). (e) W. Karnbrock, M. Deeg, J. Gerhardt, and W. Rapp, Mol. Divers. 4, 165 (1998).

(f) Y.-D. Gong, S. Najdi, M. M. Olmstead, and M. J. Kurth, J. Org. Chem. 63, 3081 (1998).

(g) K.-H. Park, M. M. Olmstead, and M. J. Kurth, J. Org. Chem. 63, 6579 (1998).

(h) S.-H. Lee, S.-H. Chung, and Y.-S. Lee, Tetrahedron Lett. 39, 9469 (1998). (i) Y. Hamuro,

W. J. Marshall, and M. A. Scialdone, J. Comb. Chem. 1, 163 (1999). (j) K.-H. Park and

M. J. Kurth, Tetrahedron Lett. 40, 5841 (1999). (k) K.-H. Park and M. J. Kurth, J. Org.

Chem. 64, 9297 (1999). (l) K.-H. Park and M. J. Kurth, Tetrahedron Lett. 41, 7409 (2000).

(m) F. Albericio, J. Garcia, E. L. Michelotti, E. Nicolas, and C. M. Tice, Tetrahedron Lett.

41, 3161 (2000). (n) M. Lamothe, M. Lannuzel, and M. Perez, J. Comb. Chem. 4, 73 (2002).

Fig. 3. Further examples of hydantoin synthesis by cyclative cleavage.



A number of other five-membered ring nitrogen heterocycles have beenprepared by cyclative cleavage. The illustrative examples (Fig. 4) depict thesynthesis of pyrazolones,12 succinimides and phthalimides,13 pyrrolo[3,4-b]pyridines,14 2-aminoimidazolones,15 imidazo[4,5-b]pyridin-2-ones,16 and1,2,4-triazoline-3,5-diones.17

The final example in this section features a rare instance where the elec-trophilic center is sp3-hybridized carbon, as most cyclative cleavages in-volve the attack of carbonyl derivatives. Oxazolidinones are formedcyclatively18 by the displacement of a sulfonate ester by an acylsulfonamide(Fig. 5). In a variant19 of this cyclization, a quasi-meso bis-sulfonatepartitions into a pair of quasi-enantiomeric sulfonates, one resin boundand the other cleaved, depending on the direction of intramolecular cycli-zation. The resin-bound enantiomer can then be displaced by an externalnucleophile.

Nitrogen Nucleophile Attacking sp2 Carbonyl: Six-MemberedRing Formation

Among the six-membered ring heterocycles, diketopiperazines aremost commonly prepared by cyclative cleavage. Indeed, diketopiperazineformation is often observed as an undesirable by-product during peptidesynthesis20 and the facile nature of this cyclization makes it an obviouschoice for library generation. In an early example from Pfizer,21 a set of10 immobilized �-amino acids was reductively alkylated with 10 aldehydes,followed by acylation with 10 �-amino acids and cyclization (Fig. 6). By

12 (a) L. Tietze and A. Steinmetz, Synlett 667 (1996). (b) L. Tietze, A. Steinmetz, and

F. Balkenhohl, Bioorg. Med. Chem. Lett. 7, 1303 (1997). (c) O. Attanasi, P. Filippone,

B. Guidi, T. Hippe, F. Mantellini, and L. F. Tietze, Tetrahedron Lett. 40, 9277 (1999).

(d) O. A. Attanasi, L. De Crescentini, P. Filippone, F. Mantellini, and L. F. Tietze,

Tetrahedron 57, 5855 (2001).13 (a) D. R. Barn and J. R. Morphy, J. Comb. Chem. 1, 151 (1999). (b) Z. Xiao, K. Schaefer,

S. Firestone, and P.-K. Li, J. Comb. Chem. 4, 149 (2002).14 A. Bhandari, B. Li, and M. A. Gallop, Synthesis 1951 (1999).15 (a) D. H. Drewry and C. Ghiron, Tetrahedron Lett. 41, 6989 (2000). (b) M. Li and

L. J. Wilson, Tetrahedron Lett. 42, 1455 (2001).16 M. Ermann, N. M. Simkovsky, S. M. Roberts, D. M. Parry, and A. D. Baxter, J. Comb.

Chem. 4, 352 (2002).17 K.-H. Park and L. J. Cox, Tetrahedron Lett. 43, 3899 (2002).18 (a) P. ten Holte, L. Thijs, and B. Zwanenburg, Tetrahedron Lett. 39, 7407 (1998). (b) P. ten

Holte, B. C. J. van Esseveldt, L. Thijs, and B. Zwanenburg, Eur. J. Org. Chem. 2965 (2001).19 P. ten Holte, L. Thijs, and B. Zwanenburg, Org. Lett. 3, 1093 (2001).20 E. Pedoroso, A. Grandas, X. de las Heras, R. Eritja, and E. Giralt, Tetrahedron Lett. 27, 743

(1986).21 D. W. Gordon and J. Steele, Bioorg. Med. Chem. Lett. 5, 47 (1995).

Fig. 4. Five-membered azole heterocycles prepared by cyclative cleavage.


Fig. 5. Cyclative cleavage at sp3 hybridized carbon.

Fig. 6. An early example of diketopiperazine library synthesis by Pfizer.


mix-and-split synthesis, a library of 1000 diketopiperazines was prepared as10 mixtures, each potentially containing 100 components.

Many groups have subsequently reported11i,22 syntheses of diketopiper-azines and related scaffolds such as monoketopiperazines and diketomor-pholines by cyclative cleavage. Among examples that are more complex

22 (a) J. Kowalski and M. A. Lipton, Tetrahedron Lett. 37, 5839 (1996). (b) A. Chucholowski,

T. Masquelin, D. Obrecht, J. Stadlwieser, and J. M. Villalgordo, Chimia 50, 525 (1996).

(c) A. K. Szardenings, T. S. Burkoth, H. H. Lu, D. W. Tien, and D. A. Campbell, Tetrahedron

53, 6573 (1997). (d) B. O. Scott, A. C. Siegmund, C. K. Marlowe, Y. Pei, and K. L. Spear, Mol.

Divers. 1, 125 (1996). (e) A. K. Szardenings, D. Harris, S. Lam, L. Shi, D. Tien, Y. Wang,

D. V. Patel, M. Navre, and D. A. Campbell, J. Med. Chem. 41, 2194 (1998). (f) R. A. Smith,

Fig. 7. Examples of bi- and polycyclic diketopiperazine synthesis.


are the �-turn mimetics prepared by Golebiowski et al.,23 and the solid-phase total synthesis of demethoxyfumitremorgin C reported by Wangand Ganesan24 (Fig. 7). The research groups of Ganesan24 and Koomen25

have both utilized this procedure for the synthesis of analogues of fumitre-morgin alkaloids, which have attracted attention as neuroactive agents,mammalian cell cycle inhibitors, and antagonists of the breast cancerresistance protein.

M. A. Bobko, and W. Lee, Bioorg. Med. Chem. Lett. 8, 2369 (1998). (g) P. P. Fantauzzi and

K. M. Yager, Tetrahedron Lett. 39, 1291 (1998). (h) M. del Fresno, J. Alsina, M. Royo,

G. Barany, and F. Albericio, Tetrahedron Lett. 39, 2639 (1998). (i) W.-R. Li and S.-Z. Peng,

Tetrahedron Lett. 39, 7373 (1998). (j) K. Shreder, L. Zhang, J.-P. Gleeson, J. A. Ericsson,

V. V. Yalamouri, and M. Goodman, J. Comb. Chem. 1, 383 (1999). (k) F. Berst, A. B. Holmes,

M. Ladlow, and P. J. Murray, Tetrahedron Lett. 41, 6649 (2000). (l) J. C. Gonzalez-Gomez,

E. Uriarte-Villares, and S. Figueroa-Perez, Synlett 1085 (2002).23 A. Golebiowski, S. R. Klopfenstein, J. J. Chen, and X. Shao, Tetrahedron Lett. 41, 4841

(2000).24 H. Wang and A. Ganesan, Org. Lett. 1, 1647 (1999).25 (a) A. van Loevezijn, J. H. van Maarseveen, K. Stegman, G. M. Visser, and G. J. Koomen,

Tetrahedron Lett. 39, 4737 (1998). (b) A. van Loevezijn, J. D. Allen, A. H. Schinkel, and

G. J. Koomen, Bioorg. Med. Chem. Lett. 11, 29 (2001).

Fig. 8. Cyclative cleavage to give dihydropyrimidinediones, quinazolinediones, and

quinolinones.


The reaction of immobilized �-amino acids with isocyanates providesureas that can be cyclatively cleaved26 to dihydropyrimidinediones, whilethe analogous reaction27 with anthranilic acids affords 2,4-quinazoline-diones (Fig. 8). A similar process is the cyclization28 of �-keto esters de-rived from substituted anthranilates to furnish 4-hydroxyquinolinones,while the hydrazones of immobilized �-keto esters are cyclatively cleaved29

to dihydropyridazinones.

26 S. A. Kolodziej and B. C. Hamper, Tetrahedron Lett. 37, 5277 (1996).27 (a) L. Gouilleux, J.-A. Fehrentz, F. Winternitz, and J. Martinez, Tetrahedron Lett. 37, 7031

(1996). (b) A. L. Smith, C. G. Thomson, and P. D. Leeson, Bioorg. Med. Chem. Lett. 6, 1483

(1996). (c) H. Shao, M. Colucci, S. Tong, H. Zhang, and A. L. Castelhano, Tetrahedron Lett.

39, 7235 (1998). (d) H.-Y. P. Choo, M. Kim, S. K. Lee, S. W. Kim, and I. K. Chung, Bioorg.

Med. Chem. 10, 517 (2002).28 M. M. Sim, C. L. Lee, and A. Ganesan, Tetrahedron Lett. 39, 6399 (1998).29 N. Gouault, J.-F. Cupif, S. Picard, A. Lecat, and M. David, J. Pharm. Pharmacol. 53, 981

(2001).

Fig. 9. Cyclative cleavage syntheses of quinazolinones.


A Roche synthesis22b,30 (Fig. 9) illustrates the basic principle behind thecyclization of substituted anthranilates to quinazolinones, a route similarto other31 examples. The concise solid-phase total synthesis32 of the fumi-quinazoline alkaloid glyantrypine features a different approach wherebya piperidine amidine undergoes thermal reorganization to the naturalproduct via two six-membered ring closures. The paper also describes thepreparation of unnatural analogues.

Nitrogen Nucleophile Attacking sp2 Carbonyl: Seven-Membered andLarger Ring Formation

Cyclative cleavage to five- and six-membered ring heterocycles is dom-inated by the synthesis of hydantoins and diketopiperazines, respectively.In the seven-membered ring case, it is the benzodiazepine nucleusthat has attracted attention, due to its status as a ‘‘privileged structure’’in drug discovery. In the precombinatorial days, Camps et al.33 reported a

30 J. M. Villalgordo, D. Obrecht, and A. Chucholowsky, Synlett 1405 (1998).31 (a) R.-Y. Yang and A. Kaplan, Tetrahedron Lett. 41, 7005 (2000). (b) A. P. Kesarwani,

G. K. Srivastava, S. K. Rastogi, and B. Kundu, Tetrahedron Lett. 43, 5579 (2002). (c) Y. Yu,

J. M. Ostresh, and R. A. Houghten, J. Org. Chem. 67, 5831 (2002).32 H. Wang and A. Ganesan, J. Comb. Chem. 2, 186 (2000).33 F. Camps, J. Cartells, and J. Pi, An. Quim. 70, 848 (1974).

Fig. 10. Cyclative cleavage syntheses of benzodiazepines.


solid-phase route to benzodiazepinones that was later adapted to parallelsynthesis5 by the Parke-Davis group (Fig. 10). A related approach34 pro-vides access to 1,4-benzodiazepine-2,5-diones.

Due to the extra degrees of freedom and entropic cost, cyclization reac-tions that are suitable for five- and six-membered ring formation can be alot slower and proceed in lower yields for the seven-membered ring case.While the cyclization22g of a tetrahydro-�-carboline proceeded reasonably(Fig. 11), the cleavage35 of an acyclic sulfonamide needed extendedreaction times and a variation of the fumiquinazoline total synthesis32 forseven-membered ring formation proceeded in poor yield. A cyclization of!-amino acids is the sole report36 of even larger ring sizes being formed. Inthis case, the acid was activated as an HOBt ester on solid phase.

Oxygen Nucleophiles

Although cyclization using an amine is more common, there areexamples with oxygen nucleophiles. An alcohol was employed37 to giveboth five- and six-membered lactones (Fig. 12). In a similar vein are the

34 J. P. Mayer, Z. Jingwen, K. Bjergarde, D. M. Lenz, and J. J. Gaudino, Tetrahedron Lett. 37,8081 (1996).

35 D. B. A. de Bont, W. J. Moree, and R. M. J. Liskamp, Bioorg. Med. Chem. 4, 667 (1996).36 W. Huang and A. G. Kalivretenos, Tetrahedron Lett. 36, 9113 (1995).

Fig. 11. Other cyclative cleavages forming seven-membered or larger sized rings.


syntheses of oxazolidinones38 and phthalides39 by cyclative cleavage. Acoumarin synthesis40 relied on photochemical isomerization as the triggerfor cleavage, and the method was demonstrated for quinolone synthesiswhen the phenol was replaced with an aniline.

37 (a) C. Le Hetet, M. David, F. Carreaux, B. Carboni, and A. Sauleau, Tetrahedron Lett. 38,

5153 (1997). (b) S. Kobayashi, T. Wakabayashi, and M. Yasuda, J. Org. Chem. 63, 4868

(1998). (c) N. Gouault, J.-F. Cupif, A. Sauleau, and M. David, Tetrahedron Lett. 41, 7293

(2000).38 H.-P. Buchstaller, Tetrahedron 54, 3465 (1998).39 (a) P. Garibay, P. H. Toy, T. Hoeg-Jensen, and K. D. Janda, Synlett 1438 (1999).

(b) P. Garibay, P. Vedsø, M. Begtrup, and T. Hoeg-Jensen, J. Comb. Chem. 3, 332 (2001).40 Y. Kondo, K. Inamoto, and T. Sakamoto, J. Comb. Chem. 2, 232 (2000).

Fig. 12. Examples of cyclative cleavage by oxygen nucleophiles.


Carbon Nucleophiles

The first cyclative cleavage of a small molecule was Crowley and Rapo-port’s study41 of intramolecular Dieckmann condensations on solid phase(Fig. 13), which was complicated by the reversible nature of the cyclization.Despite the unique opportunities afforded by C–C rather than C–X bondformation during cleavage, there are few examples from the combinatorialage. A modern version of the Claisen-type condensation by Kulkarni andGanesan42 uses a strongly acidic active methylene group to ensure unidirec-tional cyclization, and furnishes tetramic acids with three points of diversity.

41 (a) H. Rapoport and J. I. Crowley, J. Am. Chem. Soc. 92, 6363 (1970). (b) J. I. Crowley and

H. Rapoport, J. Org. Chem. 45, 3215 (1980).42 B. A. Kulkarni and A. Ganesan, Tetrahedron Lett. 39, 4369 (1998).

Fig. 13. Cyclative cleavages based on Claisen-type condensations.


Soon thereafter, the same route was published43 by three industrial groupswith slight variations in substrate and cleavage conditions.

Organometallic Reactions

Many transition metal-catalyzed cross-coupling reactions have poten-tial for effecting cyclative cleavage, and exhibit a wide tolerance of func-tional groups. The Nicolaou group demonstrated44 the feasibility ofmacrocyclizations via Stille coupling in a total synthesis of zearalenone(Fig. 14). The cyclative cleavage yielded a bis-MEM ether, which upondeprotection afforded the natural product.

The application of ring closing metathesis for the synthesis ofFriedinger lactams was studied by Piscopio et al.45 (Fig. 15), as well as othergroups,46 while the formation of macrocycles was accomplished byBlechert’s group.47

43 (a) J. Mathews and R. A. Rivero, J. Org. Chem. 63, 4808 (1998). (b) L. Weber, P. Iaiza,

G. Biringer, and P. T. Barbier, Synlett 1156 (1998). (c) T. Romoff, L. Ma, Y. Wang, and

D. A. Campbell, Synlett 1341 (1998).44 K. C. Nicolaou, N. Winssinger, J. Pastor, and F. Murphy, Angew. Chem. Int. Ed. Engl. 37,

2534 (1998).45 (a) A. D. Piscopio, J. F. Miller, and K. Koch, Tetrahedron Lett. 38, 7143 (1997).

(b) A. D. Piscopio, J. F. Miller, and K. Koch, Tetrahedron Lett. 39, 2667 (1998).

(c) A. D. Piscopio, J. F. Miller, and K. Koch, Tetrahedron 55, 8189 (1999).46 (a) J. H. van Maarseveen, J. A. J. den Hartog, V. Engelen, E. Finner, G. Visser, and

C. G. Kruse, Tetrahedron Lett. 37, 8249 (1996). (b) J. J. N. Veerman, J. H. van Maarseveen,

G. M. Visser, C. G. Kruse, H. E. Schoemaker, H. Hiemstra, and F. P. J. T. Rutjes, Eur.

J. Org. Chem. 2583 (1998). (c) R. C. D. Brown, J. L. Castro, and J.-D. Moriggi, Tetrahedron

Lett. 41, 3681 (2000).47 J. Pernerfoster, M. Schuster, and S. Blechert, Chem. Commun. 1949 (1997).

Fig. 14. Cyclative cleavage via Stille macrocyclization.

Fig. 15. Cyclative cleavage via ring closing metathesis.


The most impressive solid-phase sequence leading to a nonoligomericmolecule is the Nicolaou group’s total synthesis48 of the epothilone mitoticspindle poisons. Here, ring closing metathesis resulted in a mixture of fourseparable diastereomeric macrocylic olefins (Fig. 16). These were depro-tected and epoxidized in solution phase, and one of the diastereomers thusconverted to synthetic epothilone A. The method was also applied to thesynthesis of diverse analogues for biological screening.

48 (a) K. C. Nicolaou, N. Winssinger, J. Pastor, S. Ninkovic, F. Sarabia, Y. He, D. Vourloumis,

Z. Yang, T. Li, P. Giannakakou, and E. Hamel, Nature 387, 268 (1997). (b) K. C. Nicolaou,

D. Vourloumis, T. H. Li, J. Pastor, N. Winssinger, Y. He, S. Ninkovic, F. Sarabia,

H. Vallberg, F. Roschangar, N. P. King, M. R. V. Finlay, P. Giannakakou, P. VerdierPinard,

and E. Hamel, Angew. Chem. Int. Ed. Engl. 36, 2097 (1997).

Fig. 16. Cyclative cleavage as part of an epothilone A total synthesis.


‘‘Reverse’’ Cyclative Cleavage

In polar cyclative cleavages, it is invariably the terminal functionalgroup that is a nucleophile in attacking the point of resin attachment. How-ever, the reverse disconnection whereby the functional group at the pointof immobilization serves as the nucleophile is possible. This can also resultin cyclization and cleavage from the resin, although it strictly falls outsidethe original definition of cyclative cleavage.

Haloetherification49 and halolactonization50 reactions, for example,were studied by Kurth’s group, while cyanogen bromide-mediated cleav-age51 of methionine residues was another route to lactones (Fig. 17). Theintramolecular ring opening of epoxides,37a trapping of N-acyliminium ionsto give bicyclic �-lactams,52 and a synthesis of benzisoxazoles53 are otherexamples of heterocycles prepared in this manner.

To date, ‘‘reverse’’ cyclative cleavage with C–C bond formationhas relied on ylid chemistry. A single example was reported54 of the intra-molecular Wittig condensation of an immobilized amide to give a two-substituted indole (Fig. 18). Macrocyclization through an immobilized

49 (a) X. Beebe, N. E. Schore, and M. J. Kurth, J. Am. Chem. Soc. 114, 10061 (1992).

(b) X. Beebe, N. E. Schore, and M. J. Kurth, J. Org. Chem. 60, 4196 (1995).50 (a) H.-S. Moon, N. E. Schore, and M. J. Kurth, J. Org. Chem. 57, 6088 (1992).

(b) H.-S. Moon, N. E. Schore, and M. J. Kurth, Tetrahedron Lett. 35, 8915 (1994).

(c) D. A. Ockey, D. R. Lane, J. A. Seeley, and N. E. Schore, Tetrahedron 56, 711 (2000).51 D.-H. Ko, D. J. Kim, C. S. Lyu, I. K. Min, and H.-S. Moon, Tetrahedron Lett. 39, 297 (1998).52 (a) B. Furman, R. Thurmer, Z. Kałuza, W. Voelter, and M. Chmielewski, Tetrahedron Lett.

40, 5909 (1999). (b) B. Furman, R. Thurmer, Z. Kałuza, R. Lysek, W. Voelter, and

M. Chmielewski, Angew. Chem. Int. Ed. Engl. 38, 1121 (1999).53 (a) S. D. Lepore and M. R. Wiley, J. Org. Chem. 64, 4547 (1999). (b) S. D. Lepore and

M. R. Wiley, J. Org. Chem. 65, 2924 (2000).54 I. Hughes, Tetrahedron Lett. 37, 7595 (1996).

Fig. 17. ‘‘Reverse’’ cyclative cleavage by oxygen and nitrogen nucleophiles.


�-ketophosphonate was the pivotal step in Nicolaou’s approach55 tomuscone. Conjugate addition of dimethyl lithiocuprate completed the totalsynthesis. Another recent example56 involved macrocyclization through asulfur ylid and ejection by cyclopropane formation.

55 K. C. Nicolaou, J. Pastor, N. Winssinger, and F. Murphy, J. Am. Chem. Soc. 120, 5132

(1998).56 E. La Porta, U. Piarulli, F. Cardullo, A. Paio, S. Provera, P. Seneci, and C. Gennari,

Tetrahedron Lett. 43, 761 (2002).

Fig. 18. ‘‘Reverse’’ cyclative cleavage involving phosphorus and sulfur chemistry.


Summary

In solid-phase synthesis, cyclative cleavage can be achieved by any of abroad range of reaction chemistries. It is a powerful strategy for effectingresin cleavage that often proceeds under mild reaction conditions, some-times requiring only heating in the absence of acids or bases. Furtheradvances can be anticipated in both the types of reactions used for cyclativecleavage as well as the classes of target molecules released.

Experimental


Fmoc*-amino acids loaded on Wang resin are purchased from CNBiosciences (San Diego, CA). All other reagents and solvents are fromSigma-Aldrich (Milwaukee, WI).

* Abbreviations: DMF, N,N-dimethylformamide; Fmoc, (9H-fluoren-9-ylmethoxy)carbonyl;

Phe, phenylalanine; THF, tetrahydrofuran; Trp, tryptophan; Val, valine.


Solid-Phase Synthesis Terminated by Diketopiperazine CyclativeCleavage (Scheme 1). The Fmoc-l-Trp resin (loading �0.5 mmol/g) is firstdeprotected with 20% piperidine in DMF for 20 min, and the resin washedand dried. The resin (�1 g, 0.62 mmol/g) is shaken with benzaldehyde(0.63 ml, 10 molar equiv.) and trimethyl orthoformate (1.36 ml, 20 molarequiv.) in 7 ml CH2Cl2 overnight, followed by washing and drying to giveresin 1.

The resin is swollen in CH2Cl2, and solutions of Fmoc-l-Phe-Cl (0.5 Min CH2Cl2, 10 molar equiv.) and pyridine (4 M in CH2Cl2, 15 molar equiv.)were added. After agitation for 40 h, the resin 2 is filtered, washed, anddried. Cyclative cleavage is achieved by the addition of 20% piperidine inCH2Cl2 for 20 min. The supernatant is filtered off, and the resin washed.The combined filtrates are concentrated and triturated with hexanes toremove the nonpolar dibenzofulvene-piperidine adduct. The residue ispurified by preparative thin-layer chromatography on silica. Two majorbands, corresponding to the cis and trans diastereomers of the tetrahy-dro-�-carboline 3, are collected to furnish the desired material in a totalof 85% yield (based on the loading of the Trp-Wang resin), cis–trans ratio57/43.

Solid-Phase Synthesis Terminated by Tetramic Acid Cyclative Cleavage(Scheme 2). The Fmoc-l-Val resin (loading �0.7 mmol/g) is first depro-tected with 20% piperidine in DMF for 20 min, and the resin washed anddried. The resin (�0.3 g, 0.67 mmol/g) is shaken with p-anisaldehyde(0.25 ml, 10 molar equiv.) and sodium triacetoxyborohydride (0.65 g, 15molar equiv.) in 10 ml CH2Cl2 for 8 h, followed by washing and drying togive resin 4.

Scheme 1. Synthesis of a tetrahydro-�-carboline-diketopiperazine.

Scheme 2. Synthesis of a tetramic acid.


The resin is swollen in CH2Cl2, and hydroxybenzotriazole hydrate(0.41 g, 15 molar equiv.) and cyanoacetic acid (0.26 g, 15 molar equiv.)were added. After cooling to 0

�, diisopropylcarbodiimide (0.64 ml, 20

molar equiv.) is added. The reaction mixture is warmed to room tempera-ture and agitated for 18 h, after which the resin 5 is filtered, washed, anddried. Cyclative cleavage is achieved by the addition of 1 M tetrabutylam-monium hydroxide in MeOH (0.82 ml) and THF (10 ml) for 6 h. Thesupernatant is filtered off, the resin washed, and the filtrates combined toafford the tetramic acid 6 as its tetrabutylammonium salt. The filtrate isacidified with Amberlyst A-15 sulfonic acid ion-exchange resin (0.71 g)and the resin washed. The ion-exchange resin treatment is repeated, andthe combined filtrates concentrated. After trituration with hexanes, thefree tetramic acid is obtained in 91% yield (based on the loading of theVal-Wang resin) as a colorless solid.

Acknowledgments

The work described in the experimental section was carried out at the Institute of

Molecular and Cell Biology, National University of Singapore, and funded by the National

Science and Technology Board of Singapore.

[23] derivatization reactions of heterocyclic scaffolds 435

[23] Derivatization Reactions of HeterocyclicScaffolds on Solid Phase: Tools for the Synthesis of

Drug-Like Molecule Libraries

By Eduard R. Felder, Wolfgang K.-D. Brill, and Katia Martina

Introduction

The quest to identify new molecular entities with useful, novel, or en-hanced property profiles has intensified over the past years, among otherreasons because of the incessant rise of new assays having high-throughputscreening capacity. The pharmaceutical industry is particularly committedto the generation of large numbers of patentable compounds to choosefrom, in view of the high attrition rate experienced in selecting most effect-ive and well-tolerated new drugs. In the earliest research phases the com-pounds must match a precisely defined property profile in order to confirmtheir potential to progress rapidly toward market application.

Combinatorial chemistry is a rich source for the rapid generation ofnew compounds, but its value as a drug discovery tool goes beyond the pro-lific delivery of molecules and resides also in its inherent flexibility to adaptto specific problem solving. The first applications underscored the powerfulnumeric productivity resulting from the basic principles of combinatorialchemistry formulated by Furka et al.1 For years the chemical nature ofcombinatorial libraries was centered around oligomeric compounds suchas peptides and analogs thereof, i.e., chemical classes with serious limitsin the role of drug candidates or lead structures. The potentially muchbroader scope of the technology became evident once the combinatorialprinciple was shown to be applicable not only by linking monomeric build-ing blocks to chain-like molecules, but also by combining chemical trans-formations, cyclizations, or, for that matter, any type of derivatization.2

Combinatorial chemistry is now used broadly throughout the drug discov-ery process,3 not only for the production of large libraries for lead finding,but also for the optimization of leads with focused libraries, designed withmaximum input from existing structural information.

The drug industry pays great attention to ensuring that newly generatedmolecules are as ‘‘drug-like’’ as possible, thus possessing the kind of

1 A. Furka, F. Sebestyen, M. Asgedom, and G. Dibo, Abstr. 14th Int. Cong. Biochem. Prague

5 (Abstr. FR:013), 47 (1988).2 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992).3 R. E. Dolle, J. Comb. Chem. 3, 1 (2001).




physicochemical properties compatible with bioavailability and favorablepharmacokinetics. A seminal paper by Lipinski et al.4 has contributed toraising consciousness about applying simple, but statistically validated,relevant predictor rules as filters on large compound lists when planningsyntheses or compound acquisitions. The aim is to limit the appearanceof compound-associated problems (e.g., lack of oral absorption) in drug de-velopment. Lipinski’s caution concerning poor absorption or permeation iscommonly referred to as the ‘‘rule-of-five,’’ because the cutoffs for each ofits four parameters are all close to five or a multiple of five. Unless specificbiological transport mechanisms exist, compounds matching more than oneof the following descriptions are likely to be poorly absorbed: (1) there aremore than five H-bond donors, (2) the molecular weight is over 500, (3) thelog P is over 5, and (4) there are more than 10 H-bond acceptors. Computa-tional methods have been designed to estimate these and other predictiveproperties in silico prior to the production of compound libraries.5

Heterocycles turn out to be an excellent chemical platform for the gener-ation of drug-like molecules, taking into account the criteria just described.In retrospect, this is confirmed by the observation that heterocycles arevery common among drugs. According to the CMC2001.1 database,56.8% of the current drugs contain heterocyclic entities.6

A few simple additional considerations further substantiate the excellentprerequisites of heterocyclic compounds in terms of ‘‘drug-likeness.’’ Low-molecular-weight compounds predominantly bind to hydrophobic pocketson proteins. Although hydrogen bonds or electrostatic interactions withina hydrophobic environment enhance binding dramatically, the hydrophobiccontacts between drug and receptor have to be maximized as a consequenceof the Lennard–Jones potential. This can be the case only if the drug mol-ecule has a shape complementing that of its binding site on a protein. Polardrugs often have liabilities, which ultimately originate from the energybarrier associated with dismantling the solvation shield (dehydration) inprocesses critical for bioavailability (transport across membranes). A tightlybound drug molecule is likely to be buried deeply in a hole or a fold of itsreceptor. The geometries of optimal hydrogen bonding between polar resi-dues have to be fulfilled. The interacting functionalities have to be preor-iented so that binding results in minimal conformational strain on drug

4 C. A. Lipinski, F. Lombardo, B. W. Dominy, and P. J. Feeney, Adv. Drug. Delivery Rev. 23,

3 (1997).5 H. Matter, K.-H. Baringhaus, T. Naumann, T. Klabunde, and B. Pirard, Comb. Chem. High

Throughput Screen. 4, 453 (2001).6 The CMC 2001.1 database is a product of MDL Information Systems Inc. (San Leandro,

CA, USA).


and target. Electric fields within the binding pocket should be compensated.The conformational flexibility should be as low as possible. In narrow pro-tein folds the optimal orientation of clustered functional groups may beachieved upon fixation onto or integration into cyclic structures.7 Aromaticheterocycles allow hydrophobic interactions and dipole interactions to befine tuned by electronic alterations of their �-systems.

The chemistry of many types of heterocycles is well known and versatilemethodologies exist for attaching a wide variety of functional groups.These features are very favorable when large numbers of analogs of acertain scaffold type have to be synthesized using multiparallel synthesis.Solid-phase synthesis being a key preparative methodology in the indus-trial context of high-throughput processes and laboratory automation, het-erocyclic chemistry has been adapted to this format over recent years. Adetailed protocol about the solid supported synthesis of 1,4-benzodiazepinelibraries has been published.8 A vast number of publications related tosolid-phase heterocyclic chemistry have appeared and reviews on this sub-ject reflect this intense activity.9,10 We noticed, however, that this literaturemostly focuses on the formation of heterocycles from noncyclic precursorbuilding blocks. Derivatizations of already preformed heterocyclic scaffoldswith straightforward standard reactions are covered only occasionally.

Here we illustrate selected examples of common derivatization reac-tions on heterocycles grafted on the solid phase. The aim is to provide asense of the relevant factors and experimental conditions that allow appli-cation of known chemical reactions on solid supported heterocyclic sub-strates for the preparation of novel compounds. In industry, it is notuncommon to seek and secure novelty in a whole chemical class rather thanin single derivatives. A direct route to broad coverage aims at the develop-ment of a novel heterocyclic scaffold, which in turn is derivatized withstandard reactions in order to create a thematic library. The novel scaffoldmaterial may be prepared in bulk, typically in solution. Subsequently it isloaded onto a solid support, which is then appropriately portioned for mul-tiple derivatizations in parallel. The latter may involve common reactions,but the resulting products are novel.

Examples of such reactions, ordinary in nature, but powerful whenapplied to innovative heterocyclic scaffolds, will be discussed. Attentionis focused on specific steps of interest, which stand out as representatives

7 O. Brummer, B. Clapham, and K. D. Janda, Curr. Opin. Drug Discov. Dev. 3, 462 (2000).8 B. A. Bunin, M. J. Plunkett, and J. A. Ellman, Methods Enzymol. 267, 448 (1996).9 P. M. S. Chauhan and S. K. Srivastava, Comb. Chem. High Throughput Screen. 4, 35 (2001).

10 E. R. Felder and A. L. Marzinzik, in ‘‘Combinatorial Chemistry—A Practical Approach’’

(W. Bannwarth and E. R. Felder, eds.), p. 157. Wiley, Weinheim, 2000.


of broader significance. Lengthy descriptions of complete pathways (andthe related information overlap on repetitive steps) are beyond the scopeof this chapter. Particular emphasis is put on nucleophilic aromatic dis-placements (SNAr), a type of reaction that is potentially useful on a largevariety of appropriate precursors, for example, on halogenated cores.

In solid-phase synthesis intermediates and products are bound to a solidsupport via a covalent linker. The linker must allow selective removal ofthe final product from the support, but must be stable under the reactionconditions throughout the synthesis. The advantage of a solid-phaseapproach is that reagents can be used in large excess to drive reactions tocompletion and most side products are just washed off from the solid sup-port. However, the solid-phase implies steric constraints onto the reactionsperformed. The choice of method depends on the synthetic problem; it isoften not obvious and usually results from a reaction optimization study.

Nucleophilic Aromatic Displacements

SNAr reactions can be used to bind scaffolds onto linkers, to performring closure reactions to afford a scaffold, or to diversify the scaffold withvarious groups or functionalities.

Grafting a scaffold to a polymeric support sometimes involves hetero-cycles having more than one atom susceptible to SNAr reactions. Charac-teristic examples for syntheses involving SNAr resin capture reactions arederivatizations of pyrazines,11 pyrimidines,12 and triazines.13,14

Ding et al.15 captured a number of dichloroheterocyclic scaffolds, whereone chloro atom is prone to nucleophilic aromatic substitution onto resin-bound amine nucleophiles, as shown in Fig. 1. They found the PAL* linker

11 I. Parrot, C. G. Wermuth, and M. Hilbert, Tetrahedron Lett. 40, 7975 (1999).12 F. Guillier, P. Roussel, H. Moser, P. Kane, and M. Bradley, Chem. Eur. J. 5, 3450 (1999).13 M. Stankova and M. Lebl, Mol. Divers. 2, 75 (1996).14 T. Masquelin, N. Meunier, F. Gerber, and G. Rosse, Heterocycles 48, 2489 (1998).15 S. Ding, N. S. Gray, X. Wu, Q. Ding, and P. G. Schultz, J. Am. Chem. Soc. 124, 1594 (2002).* Abbreviations: BuOH, n-butanol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloro-

methane; DEAD, diethylazodicarboxylate; DiAD, diisopropylazodicarboxylate; DIC,

diisopropylcarbodiimide; DIPEA, diisopropylethylamine; DMA, N,N-dimethylacetamide;

DME, dimethoxyethane; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide;

Dppp, 1,3-bis-(diphenylphosphino)-propane; EtOAc, ethylacetate; EtOH, ethanol; HATU,

(N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanami-

nium hexafluorophosphate N-oxide; HOAc, acetic acid; HOAt, 1-hydroxy-7-azabenzotria-

zole; KOtBu, potassium tert-butoxide; mCPBA, meta-chloroperbenzoic acid; MeOH,

methanol; MMMP, magnesium monoperoxyphthalate; NMP, N-methylpyrrolidone; NaB-

H(OAc)3, sodium triacetoxyborohydride; NaOMe, sodium methylate; nBu4NBr, tetra-

butylammoniumbromide; PAL, peptide amide linker; P(Ph)3, triphenylphosphine; P(tBu)3,

tri-tert-butylphosphine; Pd(OAc)2, palladiumacetate; Pd2(dba)3, tris(dibenzylideneacetone)


advantageous to immobilize various amines onto the solid support using re-ductive amination. The polymer-bound amines were then acting as nucleo-philes in the reaction with various electron-deficient dichloro heterocycles.The heterocycles in Fig. 1 participated in nucleophilic displacement inn-butanol-containing diisopropylethylamine (Hunig’s base, DIPEA).Choosing the most appropriate solvent is beneficial for the reaction rateand the swelling of the solid support, which renders the polymer-boundfunctionalities accessible. Electron-deficient dichloro heterocycles (e.g.,pyrimidines) can be captured at room temperature, while electron-richerheterocycles, such as pyrazines, phthalazines, and pyridazines, requireelevated temperatures. In turn, indole and pyridine derivatives failed toreact with PAL-resin-bound primary bound amines. Nucleophilic dis-placement or the Pd-mediated coupling reaction on the remaining chlorosubstituent has been investigated. It turns out that only a small subset ofthe polymer-bound heterocycles could be modified by SNAr reactionsunder standardized conditions. The less reactive C2 chloro group of pyrimi-dine and quinazoline (see Fig. 2) was found to react with various aminesquantitatively at high concentration (>2 M for 12 h at 100

�).

Brill et al.16 confirmed earlier observations that for the substitution onthe C2 of pyrimidines the less nucleophilic anilines are actually more react-ive than aliphatic amines, presumably because of an auto-acid-catalyzedmechanism. In turn, less reactive centers like the C6 chloro group of pyr-imidines could be displaced with anilines in the presence of tert-butoxideas a base (see the experimental section).

Instead, palladium-catalyzed reactions offer the most versatility interms of substrate structure. Since they usually require inert conditions,their use in a parallel synthesis setting is more demanding with regards tolaboratory equipment and logistics.

To expand the diversity of their libraries Brill et al.16 also modified vari-ous heterocycles by alkylation, acylation, or metal-mediated coupling reac-tion prior to resin capture. A remaining chloro substituent was stillavailable for nucleophilic displacement or a palladium-mediated couplingreaction with anilines, phenols, and boronic acids on solid phase [seeFig. 10 for the preparation of purine derivative (62)].

Guillier et al.12 avoided the problem of regioselectivity in the substitutionof chloro atoms in polyhalogenated pyrimidines by capturing a symmetrical

dipalladium(0); PrOH, n-propanol; pTSOH, p-toluenesulfonic acid; Py, pyridine; RT, room

temperature; TBAN, tetrabutylammoniumnitrate; TEAA, triethylammoniumacetate; TFA,

trifluoroacetic acid; TFAA, trifluoroacetic acid anhydride; THF, tetrahydrofuran; TMSCl,

trimethylsilyl chloride.16 W. K.-D. Brill, C. Riva-Toniolo, and S. Mueller, Synlett 1097 (2001).

O

OMe

OMe

O

P O P NH P

N Cl

PO

Cl Cl

N

N

NH

Cl

Cl

N

9

Purine:

1

23

6

7

8

3e

N

NCl

Cl

N

NCl

ClPyrimidines:

3a 3b

12

34

5

6

N

N

Cl

Cl

N

N

Cl

ClH2N

3c 3dN

N

Cl

Cl

N

N ClCl

3j 3k

Pyrazine

N

Cl

Cl

N N

N

Cl Cl

Quinazolines:

12

3 4

56

7

8

3f 3g

N

N

Cl

Cl

N

N

Cl

Cl3h 3i

N3

N2

Cl

Cl

7

8

14

5

6

Phthalazine:

3n

N3

N2

Cl

Cl

N

N

Cl

Cl

Quinoxaline:

12

34

56

Pyridazine:

1

45

68

7

3l 3m

R1i

PAL-resin

R1

ii

1

1

2

3

4

Fig. 1. Capture of diverse dichloroheterocycles on solid phase by nucleophilic aromatic substitution.

44

0sm

al

lm

ol

ec

ul

ea

nd

he

te

ro

cy

cl

esy

nth

esis

[23]

O

OMe

OMe

OO

OMe

OMeHN R1

ClN

N

Cl

ClN

NN

R1

O

OMe

OMe

ClN

N

Cl

ClN

NN

R1

O

OMe

OMe

R2

R1

NH

N

N

HN

R2

R1

NH

N

N

HN

R1-NH2

a

R2-NH2

c (if R2 = aliphatic)d (if R2 = aromatic)

e

R2-NH2

c (if R2 = aliphatic)d (if R2 = aromatic)

e

b

b

5a

5b

6a

6b

Fig. 2. Derivatizing the C2 chloro group of resin-bound pyrimidines and quinazolines. (a)

NaBH(OAc)3, 1% HOAc, THF; (b) DIPEA (3 equiv.), heterocycle (2 equiv.), BuOH, 90�,

24 h; (c) amine (2 M), 100�, 12 h; (d) arylamine (0.2 M), 80

�, 12 h; (e) 45% TFA/DCM, 2 h.


4,6-dichloro-2-(methylthio)pyrimidine (8) (Fig. 3). They generally focusedon heterocycles bearing unsubstituted amino functions and thus used bulkyRink-amine resin 7 as the solid-phase capture component, as shown inFig. 3. The latter offers on the one hand the advantage of mild cleavageconditions, but on the other hand it also limits the choice of amines thatcan be immobilized onto the support and still remain nucleophilic enoughfor subsequent resin capture.

The capture of 4,6-dichloro-2-(methylthio)pyrimidine (8) was per-formed in DMF with diisopropylethylamine (DIPEA, Huenig’s base) as abase and tetrabutylammonium bromide as a catalyst at 90

�. The substitution

of the remaining chlorine atom on the polymer-bound scaffold requiresharsher conditions. Thus the immobilized 6-chlorothiomethylpyrimidine(9) could be substituted with aliphatic amines in neat amine at 140

�. The

coupling with anilines could be afforded consistently only by using KOtBuas base and [18]crown-6. Also, the use of Pd catalysts gave positive results,but failures were observed occasionally. Finally, the substitution of thethiomethyl group in resin-bound 2-(methylthio)pyrimidine-4,6-diamines

N N

Cl Cl

SMe

N N

NH

Cl

SMe

N N

NH

Cl

SO2Me

N N

NH

Cl

N

N N

NH

N

N N

NH

SMe

SMe

N N

NH

SO2Me

SO2Me

N N

NH

N

SMe

N N

NH

N

N

N N

NH

SO2Me

N

ii

PNH2

P P

P

O

NH2

O O

PP P

PNH2

P P P

:

7

7

8

9

10

+

R1

R1�

R1�R1

11a

n = 1: 11b

i

ii

iii

13 14

R2

R2�

16

11b,c

R2

R2�

12

R1 R1�

15

R1 R1�

12

iv v

vii

vi iv

S(On)Me

n = 2: 11c11b

vi

Rink-resin:

Fig. 3. Capturing symmetrically functionalized pyrimidine on Rink resin for subsequent derivatization. (i) DIPEA, nBu4NBr, DMF, 90�;

(ii) mCPBA, dioxane, 1 M NaOH, RT; (iii) R1R10 NH, 15 h, RT; (iv) R2R20 NH, 15 h, 140�; (v) MMPP, EtOH/DMF 1:4, 2 h, 0

�; (vi) R1R10 NH, 15 h,

140�; (vii)NaSMe (15)crown-5, EtOH/DMF (1/4), 15 h, 130

�.

44

2sm

al

lm

ol

ec

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ea

nd

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cl

esy

nth

esis

[23]


(16) was investigated. For the oxidation of the thiomethyl group it wasfound that magnesium monoperoxyphthalate (MMPP) gives the best results.Although the sulfoxide form (11b) was predominant, contamination withsulfone 11c was unavoidable. Nonetheless, these two functional groupswould be displaced during the next reaction. Interestingly, there was noreport of any formation of N-oxidized products. The final displacementwas then achieved again with a set of neat amines including aniline at 140

�.

In a separate report, the regioselectivity and reactivity problems in thesubstitution of pyrimidines were avoided using 4,6-dichloro-5-nitropyrimi-dine as starting material,17 a very electron-poor heterocycle, which is highlyreactive in nucleophilic aromatic substitutions. It reacts readily with thefree amino group of the (trialkoxybenzhydrylamine) Rink linker on solidphase. This heterocycle could serve as a scaffold by itself and could alsobe used as a building block (precursor) to make other heterocycles suchas purines.

Triazine is another scaffold of interest not only for herbicides andpharmaceuticals, but also for the development of novel catalysts18 andthe construction of affinity chromatography matrices. As a result, severaltriazine libraries have been synthesized on various matrices such as trad-itional polystyrene Wang-type resins, polypropylene membranes, agarose,and glass surfaces, including the microscale. A general approach to synthe-size substituted 2,4,6-triamino-s-triazines begins with a polymer-boundamine, which is used to capture symmetrical trichlorotriazine. Furtherselective sequential substitution of the two remaining chloro atoms canbe performed using amines incrementing the reaction temperature.19 How-ever, the selectivity of the monosubstitution is often variable,20 and partialsolution-phase approaches are being reported. Thus, Masquelin et al.14

began by reacting cyanuric chloride (17) with an amine in solution(Fig. 4), a very convenient approach since many products can be isolatedby crystallization or precipitation without difficult chromatographic pro-cedures. The resulting dichloro-s-triazines (18) are then captured by a thiolresin 19. Finally, the remaining chloro function is substituted with variousamines, typically using elevated temperatures. The resin attachment can becleaved after first oxidizing the thioether function with N-phenylsulfonyl-3-phenoxaziridine21 followed by treatment with very nucleophilic and prefer-ably volatile amines. Although this method provides clean final products,

17 R. Di Lucrezia, I. H. Gilbert, and C. D. Floyd, J. Comb. Chem. 2, 249 (2000).18 S. Masala and M. Taddei, Org. Lett. 9, 1355 (1999).19 M. Stankova and M. Lebl, Mol. Divers. 2, 75 (1996).20 D. Scharn, H. Wenschuh, U. Reinecke, J. Schneider-Mergener, and L. Germeroth, J. Comb.

Chem. 2, 361 (2000).21 F. A. Davis and O. D. Stringer, J. Org. Chem. 47, 1774 (1982).

N

N N N N N

N

Cl

Cl

Cl

N N

Cl

HN

Cl

N N

S

HN

Cl

N N

S

HN

S

O O

N

O

N

N

N

S

HN

N

O

NH

N

N

N

HN

NN

SH R1 R1

R2

R1

R2

R1

R2

17

i

18

19ii

20

iii

21

22

iv

v

2324

R2'

R2�R2´

R1

Fig. 4. Triazine derivatization in a combined solution/solid-phase approach. (i) For R1¼H:

aq. NH3, Et2O, �20�–0

�; for R1NH2: acetone, 2 N NaOH, �20

�–0

�; (ii) dioxane, DIPEA, 40

�;

(iii) R2R20 NH, DMA, 45�; (iv) CH2Cl2; (v) pyrrolidine, dioxane, 60

�.


there is a limit to how nucleophilic and volatile amines can be for use in thefinal step.

Alternative activation of chloro atoms in triazines is the substitution of thechloro atom by a ‘‘transient nucleophile’’ such as N-methylmorpholine.22,23

Several solid-phase approaches to afford purines were performed.Some may involve selective substitution of the purine directly; othersbegin by construction of a pyrimidine scaffold followed by closure of theimidazole ring.

One type of synthesis was described by Ding et al.15 (Fig. 1). A draw-back to this approach is the low reactivity of a polymer-bound amine. Thisproblem may be alleviated to some extent by activating the heterocyclewith ammonium salts16 and by the choice of linker. Mainly acid-labileindole24 (28) and PAL linkers25 (26) have been employed. The possibilityof activating C2 by acylation of N626 of the purine scaffold was not

22 Z. J. Kaminiski, P. Paneth, and J. Rudzinski, J. Org. Chem. 63, 4248 (1998).23 S. Masala and M. Taddei, Org. Lett. 9, 1355 (1999).24 K. G. Estep, C. E. Neipp, L. M. S. Stramiello, M. D. Adam, M. P. Allen, S. Robinson, and

E. J. Roskamp, J. Org. Chem. 63, 5300 (1998).25 F. Albericio, N. Kneib-Cordonier, S. Biancolana, L. Gera, R. I. Masada, D. Hudson, and

G. Barany, J. Org. Chem. 55, 3730 (1990).26 Y.-T. Chang, N. S. Gray, G. R. Rosania, D. P. Sutherlin, S. Kwon, T. C. Norman, R. Sarohia,

M. Leost, L. Meijer, and P. G. Schultz, Chem. Biol. 6, 361 (1999).

O

OMe

OMe

O

NH

N

O O

P O P NH

N

NN

NH

N

X N

NN

N

N

XN

NN

NH X

Cl P P

R1R1

ii iii

30 31R329

N

NN

N Y

N

N

NN

N Y

HNP

iv v

32 33R3

R1 R1

R3

NoR1i

AMS resin

25

27

26

28

Crowns orPS-resin

Y=OAr, NHR2, NR2R2�, Ar

Fig. 5. Derivatization of resin-bound purines. (i) R1NH2, [Me4N]þ [HB(OAc)3]�, then

NaBH3CN; (ii) 26 or 28, DIPEA, THF, 60�, 16 h; (iii) R2OH (10 equiv.) P(Ph)3 (10 equiv.),

DEAD (10 equiv.), THF; (iv) for X ¼ F: R2NH2, n-BuOH/DMSO, 120�; for X ¼ Cl: ArOH,

ArB(OH)2, R2NH2, or R2NR20 H Pd cat.; (v) 5% TFA/DCM.


exploited in the example described here (Fig. 5). However, the use ofPd-catalyzed reactions, as described later, allowed the substitution of theC2–C1 atom by amino, aryloxy, and aryl groups. In fact, a way to overcomethe lack of reactivity of chlorine at the purine C2 position and poorly react-ive halides on other heterocycles is the use of Pd-catalyzed C–N and C–Cformations, as illustrated in more detail in Fig. 10 and related procedures inthe experimental section.

An interesting synthetic approach has been used in the synthesis ofadenosine analogs. Rodenko et al.27 generated a leaving group at C2 of6-chloropurinylribofuranoside (34) via electrophilic nitration (Fig. 6). Thereplacement 6-chloro function was very facile in the electron-poor purine(35). In turn, the 2-nitro group of the 2-nitro-9-ribosylpurin-6- amines(36) could be displaced by aliphatic amines at 80–90

�.

27 B. Rodenko, M. J. Wanner, and G.-J. Koomen, J. Chem. Soc. Perkin Trans I 1247 (2002).

O O

NOO

N

N

N

Cl

O

O O

NOO

N

N

N

HN

O

NO2

R1

O O

NOO

N

N

N

HN

O

NH

R1

R2

H2NR1

H2NR2

O O

NOO

N

N

N

Cl

O

NO2

OH OH

NHOO

N

N

N

HN

NH

R1

R2

i ii

iii

iv, v

34 3536

3738

Fig. 6. Adenosine analogs via electrophilic nitration for the activation of the purine

scaffold. (i) Bu4Nþ NO3� (TBAN), DCM, TFAA, 0

�, 2.5 h; (ii) amine, DCM, DIPEA, 4 h,

RT; (iii) amine, NMP, DIPEA, 80–90�, 24 h; (iv) pTsOH�H2O, DCM/MeOH 97:3; (v)

NaOMe, THF, MeOH, 1 h, RT.


Quinazolines are of great interest in the pharmaceutical industry asprotein tyrosine kinase inhibitors. Dener et al.28 described a synthesisstarting from 2-methoxybenzaldehyde, Wang, or Rink resins. With thealdehyde resin reductive aminations were undertaken to yield polymer-bound secondary amines (Fig. 7). The latter were subjected to 2,4-dichloro-6,7-dimethoxyquinazoline to give the 4-amino-substituted derivatives.These were then allowed to react with primary or secondary amines at135–140

�in the presence of DBU in DMA. As a result of a detailed scope

and limitation study, Dener et al.28 note that some bifunctional amines,such as piperazine, give to some extent dimeric derivatives.

Dener et al.28 also indicated a list of amine functionalities that fail todisplace the 2-chloro group. In turn, Wu et al.29 performed similar chemis-try displacing the chloro group at the C2 position under milder conditions(lower temperature), but this methodology is restricted to more electron-deficient and more reactive quinazolines bearing further chloro groups atC6, C7, or C8.

28 J. M. Dener, T. G. Lease, A. R. Novack, M. J. Plunkett, M. D. Hocker, and P. P. Fantauzzi,

J. Comb. Chem. 3, 590 (2001).29 Z. Wu, J. Kim, R. M. Soll, and D. S. Dhamoa, Biotechnol. Bioeng. Comb. Chem. 71, 88

(2000/2001).

N

N

Cl

Cl

N

N

NCl

R2

N

N

NN

R2

O

O

NH

P P

R2

NHP

R1

R1 R1

R3

40

i

41 42

ii

R1

39:

R3'

Fig. 7. Immobilization and subsequent derivatization of quinazolines. (i) DIPEA, THF,

60�; (ii) R3R30 NH, DMA, 135–140

�.


Cobb et al.30 constructed 4-amino-2-carboxy-6-chloroquinazolin-4-oneon a solid support using the 2-carboxylic ester linkage as the resin pointof attachment. The quinazolinone (43) was converted to the resin-bound4-chloroquinazoline (44) with SOCl2. The chloro group was then displacedeven with anilines under acid-catalyzed conditions at room temperature.Finally, the resin-bound quinazoline-2-carboxylic ester (45) was cleavedfrom the resin and decarboxylated with TMSCl/NaI (Fig. 8) (yield 69%,purity 95%).

There are a few examples for arylations of aliphatic amines, which areSNAr reactions. However, most of these examples are restricted to a highlynucleophilic piperazine scaffold. A broader approach, which may havemore general value since it does not require the presence of an activatingadditional nitro or chloro substituent, has been developed by Ruhlandet al.31 (Fig. 9). The authors formed an iron-�-complex upon reaction of adichloroarene (52) with ferrocene. The resulting ferrocene-hybrid complex(53) is capable of undergoing SNAr reactions much more readily than the‘‘electron-richer’’ parent chloroarene (52). Using this methodology poly-mer-bound piperazine and 1,4-diazepane resins 51a and 51b (Fig. 9) couldbe readily arylated. Ruhland et al.31 showed that in the case of dihaloar-enes, the second halogen group on the iron-complexed aromatic couldundergo further aminations, etherifications, thiolations, and phosphineand seleno-ether formations. Unfortunately, the scission of iron-aryl

30 J. M. Cobb, M. T. Fiorini, M.-E. T. Goddard, and C. Abell, Tetrahedron Lett. 40, 1045

(1999).31 T. Ruhland, K. S. Bang, and K. Andersen, J. Org. Chem. 67, 5257 (2002).

ClCl

NNCl

R X

N N

NNX

RNNO

O

XR

Cl

O Cl

O

nn

N

NH

ClCl

Fe+

PF−6

FeFe+

NNX

R

Fe+

Al, AlCl3

Na+

hn (visible light)

n=1 or 2Merrifield-resin

i

50

51a or 51b

5253

54

ii

iii

iv

vvi

n

5657a,b55

+

n

n

Fig. 9. Nucleophilic aromatic substitution promoted by iron-�-complex formation on solid

phase. Piperazine or diazepane, THF/DMF 1: (i) 50�, 18 h; (ii) (1) 95

�, 4 h; (2) NH4PF6; (iii)

K2CO3, THF, 60�, 16 h; (iv) various nucleophiles, 45–90

�; (v) CH3CN/H2O, visible light; (vi)

1,2-dichloroethane, 0�, RT, 16 h.

O

O

N

HN

OCl

O

O

N

N

ClCl Br

NH2

O

O

N

N

HNCl

Br

N

N

HN

Cl

Br

O

O

N

N

HN

Cl

Br

Si

I

I

O

O

N

N

HN

Cl

Br

Si

O

O

N

N

HNCl

Br

H

CO2

Mechanism of cleavage

43 44 45

46

47

48

49

i ii

iii

Fig. 8. Quinazolines via decarboxylation of resin-bound carboxylic esters. (i) SOCl2, DMF,

reflux; (ii) HCl/PrOH/DMF, RT, 18 h; (iii) TMSCl, NaI, CH3CN/dioxane, 75�, 72 h.



�-bonds is not very facile. A photochemical reaction was used to liberatethe polymer-bound arene (56) after the SNAr substitution. The latter wasthen cleaved from the resin with methyl chloroformate to give 57a and57b (Fig. 9). This approach provides a robust alternative to Pd-, Ni-, orCu-catalyzed aminations and etherifications, which often require rigorousexclusion of oxygen.

In conclusion, SNAr reactions are often facilitated by polar protic solv-ents such as butanol or by polar solvents in the presence of Brønsted orLewis acids. When displacements of halides with amines are used, bothpolar and protic solvents can solubilize the ammonium halides resultingfrom the reactions, and lead to the formation of Brønsted acids. Fluorideor chloride ions are preferred leaving groups for this reaction. Sulfinyland sulfenyl groups require somewhat harsher conditions.

If SNAr reactions are to be performed, it is advisable to render the scaf-fold as electron deficient as possible. Thus, thioethers may be oxidized tosulfoxide or sulfonyl groups, and nitrogen-containing functionalities shouldbe amides or nitro groups prior to a substitution. Of course, these derivati-zations are limited by the reactivity of the corresponding heterocycle, sincemore than one functionality may be displaced, if multiple leaving groupsare present.

The following sections highlight some additional reaction types, whichcan be used proficiently for the derivatization of heterocyclic scaffolds.

Palladium-Catalyzed Reactions

As mentioned earlier, Ding et al.15 captured a number of dichlorohetero-cyclic scaffolds where one chloro atom is prone to nucleophilic aromaticsubstitution onto resin-bound amine nucleophiles (Fig. 1). Even thoughit was demonstrated that in many cases the second chlorine may be substi-tuted with SNAr reactions, it was pointed out that palladium-catalyzedreactions offer the most versatility in terms of substrate structure. Whenintroducing amino, aryloxy, and aryl groups, Ding et al.15 reported Pd-catalyzed reactions as a way to overcome the lack of reactivity of chlorineat the purine C2 position and poorly reactive halides on other heterocycles(Fig. 10).

The reaction sequence starts by anchoring 2,6-dichloropurine ontothe solid-phase PAL-amine at the more reactive C6 position with exclusiveregioselectivity. A multitude of PAL-amine resins 59 can be preparedahead by reductive amination of commercial (4-formyl-3,5-dimethoxyphe-noxy)-methylpolystyrene. The N9 position of the purine (60) may be modi-fied by Mitsunobu alkylation. The final derivatization step involves apalladium-catalyzed cross-coupling reaction in position 2. This reaction

O

OMe

OMe

O

NH

R1

N R1

NH

N

N

N

Cl

R2

N R1

N

N

N

N

ClR2

N

N R1

N

N

N

X NH

R3

R4

R3

N OR3

R3

58 PAL

R1-NH2

a

d, e, f, or g

PAL

b

5960

60c

Mitsunobu

61

choice ofnucleophile

Pd-chemistry

62

X =

Fig. 10. Multiple derivatization of purines including palladium-catalyzed reaction at

the poorly reactive C2 position. (a) NaBH(OAc)3, 1% HOAc, THF; (b) 59 (0.5 equiv.),

2,6-dichloropurine (1 equiv.), DIEA (1.5 equiv.), BuOH 80�; (c) R2OH, PPh3, DiAD

(1.5:2:1.3) in excess, THF, RT; (d) boronic acids (5 equiv.), 7% Pd2(dba)3, 14% carbene

ligand, Cs2CO3 (6 equiv.), 1,4-dioxane, 90�, 12 h; (e) anilines (5 equiv.), 7% Pd2(dba)3, 14%

carbene ligand, KOtBu (6 equiv.), 1,4-dioxane, 90�, 12 h; (f) phenols (5 equiv.), 7% Pd2(dba)3,

28% phosphine ligand, K3PO4 (7 equiv.), toluene, 90�, 12 h; (g) primary or secondary amines

(5 equiv.), 90�, 12 h.


requires 5 equivalents of the coupling partner (arylboronic acids, anilines,or phenols), 7 mol% Pd2(dba)3, 14 mol% of a suitable ligand, and 6equivalents of the base at 80

�in dioxane (for C–C and C–N formation)

or toluene (for C–O formation).Over the past 5 years the number of reports on the use of palladium-

catalyzed reactions for solid-phase derivatizations has greatly increased.In this section (and throughout this chapter), we limit our scope to repre-sentative applications for the modification of solid-supported heterocyclicscaffolds. A more general overview of the versatility of Suzuki, Heck,and Stille reactions on solid supports was recently provided by Franzen.32

Extensive use of Pd-catalyzed reactions was included in the synthesis of2,6,8-trisubstituted purines (Fig. 11).33 The synthesis started by anchoringdichloropurine to Rink resin via N9 linkage. Polymer-bound 2,6-dichloro-purine (63) was selectively substituted at C6 via acid-catalyzed SNAr sub-stitutions. In the absence of Pd catalysis, the substitution on C2 could beperformed only with strongly nucleophilic amines. To expand the scopeof C2 substitution, catalytic amounts of Pd were used. Under these reactionconditions arylboronic acids and amines successfully substituted the chloroatom on C2 to afford C2–C and C2–N bonds. Subsequently, the C8 positionwas brominated with a bromine–lutidine complex33 (66) to give resin 67.

32 R. Franzen, Can. J. Chem. 78, 957 (2000).33 W. K.-D. Brill and C. Riva-Toniolo, Tetrahedron Lett. 42, 6515 (2001).

N

NN

N

Cl

Cl N

NN

N

NR1

R2

R3

N

NN

N

N

Br

R1R2

R3N

NN

N

NR1

R2

R3

R4

N

NN

NH

NR1

R2

R3

R4

O O O

OH

P P

P P

P

N

NN

N Cl

NR1

R2

P

N

BrBr

i iii

63 65

6768

v

ii

69

66polystyrene/DVB

:

64

iv

vi

Rink resin:

R2 = Aryl or prim./sec. alkyl- or (aryl-)amino

Fig. 11. Bromination and subsequent Stille coupling at the C8 position of purines on solid

phase. (i) TFAA, 2,6-lutidine, then 2,6-dichloropurine, NMP, 2,6-lutidine; (ii) primary or

secondary alkylamine (4 equiv.), TFA (0.4 equiv.) in NMP, 55�, 90 h; (iii) ArB(OH)2 or

RNH2 or RNHR0 (8.3 equiv.), K3PO4 (31.4 equiv.), Pd2(dba)3, P(tBu)3, NMP; (iv) 66 (5�)

NMP, 3 h, RT; (v) Stille coupling: Pd(OAc)2 dppp, stannane (8.3 equiv.), NMP, 100�, 20 h;

(vi) 20% TFA in CH2Cl2.


The latter intermediate could undergo Stille couplings to afford trisubsti-tuted purine (68).

In many cases, high yields (>75% based on polymer loading) of thefinal product were obtained. Some drawbacks to this approach include par-tial dehalogenation of (67) back to (65) during the Stille coupling, the pres-ence of Pd impurities in some products, and structural and chemicalconstraints imposed by the bromination reaction.

Acylations, Alkylations, Reductive Alkylations(Aminations, Alkaminations)

Following Ellman’s pioneering work on benzodiazepines,2 anotherstudy from the same laboratory illustrated the vast potential of combininga variety of chemical reactions on drug-like heterocycles. In this case a pre-formed heterocycle was grafted as a whole onto the solid phase to be sub-sequently derivatized via a three-component palladium-mediated couplingreaction followed by a reductive alkylation or an acylation (Fig. 12).34

34 J. S. Koh and J. A. Ellman, J. Org. Chem. 61, 4494 (1996).

ON

O

Teoc

N

OO

SiMe3

OH

N

O

Teoc

Pd Br

PPh3

N

O

Teoc

R1

R2 R2R1

HN

O

R2R1N

O

XR3

R2R1

X

N

OH

R3

THP

+a

THP

b

70 71

THP THP

R1: arylR2: aryl, H, alkinyl X: CO, CH2

f

THP R1: arylR2: aryl, H, alkinylX: CO, CH2

g or h ic, or d, or e

72 73 74 75

Fig. 12. Versatile derivatization scheme of a tropane template, including palladium-

mediated coupling. (a) TsOH, CH2Cl2; (b) aryl bromide, Pd(PPh3)4, THF, 66�; (c) arylboronic

acid, 2 N Na2CO3, PPh3, THF, or anisole, 66�; (d) formic acid, Et3N, PPh3, DMF, 66

�; (e)

phenylacetylene, CuI, Bu4NCl, DMF, 66�; (f) Bu4NF, THF, 50

�; (g) aldehyde, NaBH(OAc)3,

DMF; (h) HATU, Et3N, benzoic acid, HOAt, DMF; (i) TFA/H2O (20:1).


The derivatization of the secondary amine functionality of a tropanetemplate is described in more detail in the experimental section.

The acylation is carried out with a classic protocol employing a moderncoupling reagent of the uronium type, namely the highly effectiveHATU,35,36 which contains an azabenzotriazolyl moiety, having demon-strated advantages over the formerly used benzotriazolyl derivatives. Acy-lations on solid phase have been studied intensely for decades in thecontext of peptide chemistry. Experience from innumerable optimizationstudies reported in this field benefits the combinatorial chemist by provid-ing a rich choice of protocols with well-documented scope and limitations.Optimal results cannot be obtained with any single protocol in all cases.The observation is that the relative performances of distinct combinationsof activating agents and reaction conditions often vary depending on thesubstrates. Informative overviews on coupling methodologies for amideand ester bond formation are available.37,38 Much of the effort in these

35 L. A. Carpino, J. Am. Chem. Soc. 115, 4397 (1993).36 L. A. Carpino and A. El-Faham, J. Org. Chem. 60, 3561 (1995).37 F. Albericio and L. Carpino, Methods Enzymol. 289, 104 (1997).38 F. Albericio and S. A. Kates, in ‘‘Solid-Phase Synthesis’’ (S. A. Kates and F. Albericio,

eds.), p. 275. Marcel Dekker Inc., New York, 2000.


studies has been devoted to preventing racemization during the coupling ofchiral amino acids. When working with nonchiral building blocks, thisaspect can be neglected and more aggressive approaches can be appliedas well. Such methods may involve acyl halogenides or, for the acylationof unreactive alcohols, the use of n-butyllithium in the presence of an acidchloride39 or superbase-promoted acylations, for example, with the com-mercially available nonionic superbase P(MeNCH2CH2)3N.40 Acylationson amines usually do not need the presence of excess base, unless theamino group must be liberated from a protonated salt form or if thecoupling agents or adjuvants are of an acidic nature (e.g., hydroxybenzo-triazol derivatives). If carbodiimide-based coupling reagents are used inrelatively polar solvents such as DMF, a preactivation of the carboxylicacid to form an activated ester [which can be done in situ, e.g., with (aza)-benzotriazolyl derivatives] prevents formation of acylurea side products.The solid-phase reaction format has the advantage that these side productscan be easily washed away, if the activated ester is in solution. Any partialdepletion of the acyl component has negligible effects on yields if a suffi-cient molar excess is applied. A caveat for long preactivation times is thetendency toward racemization of the activated species, although this isirrelevant when nonchiral building blocks are used. With CH2Cl2 as a solv-ent, the straight use of carbodiimides without active ester preformation ispossible and the acylurea side reaction is not observed. On the other hand,if the amine component is exposed to a coupling reagent of the uroniumtype before the carboxylic acid is added, there is risk of guanidino sideproduct formation. Particularly in parallel synthesis the optimal timing ofreagent addition cannot always be maintained for logistical reasons andthe consequences of a prolonged temporary absence of one of the reactioncomponents must be assessed in advance.

Where possible, it is usually preferable to have the activated species(e.g., the active ester) in solution and the substrate (e.g., the amine com-ponent) on the solid phase. If the case requires an inverted situation, asfor the synthesis of a thiazolidinone library (see Fig. 13),41 identificationof the best reaction conditions may be more challenging, considering therelative scarcity of literature examples.

The example previously described in Fig. 12 involves a reductive alky-lation, a widely used derivatization reaction in combinatorial chemistry.The use of sodium triacetoxyborohydride has been thoroughly validatedfor the solid-phase reaction format. With this reagent the pH is maintained

39 E. Mc Kaiser and R. A. Woodruff, J. Org. Chem. 35, 1198 (1970).40 B. A. D’Sa and J. G. Verkade, J. Org. Chem. 61, 2963 (1996).41 M. C. Munson, A. W. Cook, J. A. Josey, and C. Rao, Tetrahedron Lett. 39, 7223 (1998).

R1 R1

O

R2

N SNH

O

O

COOH

O

R2

N SH2N

O

NH

R3

H2N R3

c

76 77

a then b

Wang

Fig. 13. Acylation via activation of a resin-bound carboxylic acid. (a) Pentafluorophenyl

trifluoroacetate (6 equiv.) in Py/DMF (1:10), 1 h; (b) amine (6 equiv.) CH2Cl2/DMF; (c) TFA/

DCM (1:1) 1 h.

R1

R2HN

O

N

HN

OR1

R3

R2H2N

O

N

N

O

HN

O

NH

HN

OR1

79 8078

a b

cArgoGelRink

Fig. 14. Stepwise selective amine and amide alkylation. (a) R2-CH2-X (2 M), DMF, 50�,

24–48 h; (b) lithiated 4-Bz-2-oxazolidinone, R3-CH2-X (15 equiv.), THF/DMF, 1 h; (c) 90%

TFA/DCM, 0.5 h.


in the desired, slightly acidic range. Secondary amine functions (like thedescribed tropane scaffold) are unaffected by the potential bisalkylationside-reactions, which may occur between aliphatic aldehydes and primaryamines. One way of minimizing the risk of bis-alkylations is the preform-ation of the imine intermediate in the absence of the reducing agent42

and, subsequent, time-delayed (>20 min) addition of the borohydride.Straight N-alkylations with alkyl halogenides were commonly used at

the inception of combinatorial chemistry, when large oligo-N-substitutedglycine libraries were prepared for lead finding.43 The same reaction typecan be conveniently used on heterocyclic scaffolds, since selectivity is themost critical aspect if multiple alkylation sites are present. The example il-lustrated in Fig. 1444 indicates that stepwise selective alkylation withoutdifferential protection is also feasible on solid phase, if the nucleophilicitydifference of the targeted nitrogen atoms is sufficiently high.

A discussion on protecting group chemistry and the related strategies tomask the reactivity of one functional group while allowing the modificationof another group in the same molecule is beyond the scope of this chapter.

42 D. W. Gordon and J. Steele, Bioorg. Med. Chem. Lett. 5, 47 (1995).43 R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, and W. H. Moos, J. Am. Chem. Soc. 114,

10646 (1992).44 M. K. Schwarz, D. Tumelty, and M. A. Gallop, Tetrahedron Lett. 39, 8397 (1998).

HN

fmocNH

OH

N

OH

O

F

O2N

NH2

N

O

O

N

O

O

NO2

HN

O

O

HN

O

O81

82

a b c

d e, f

Fig. 15. Reduction of an aromatic nitro group on solid phase. (a) Twenty percent

piperidine, DMF, 20 min; salicylaldehyde, HC(OMe)3 then NaBH(OAc)3, DMF, 12 h, RT;

(b) 2-fluoro-5-nitrobenzoic acid, HOAt, DIC, DMF, 24 h, RT; (c) 5% DBU, DMF, 24 h, RT;

(d) SnCl2�2H2O, DMF, 12 h, RT; (e) furfuryl chloride, Hunig’s base, DMF, 8 h, RT; (f) 20%

TFA, CH2Cl2, 40 min.


Protocols of peptide chemistry and, to some extent, biooligomer synthesis(e.g., nucleotides, saccharides) are valuable sources of information on thistopic with regards to solid-phase synthesis peculiarities. Here we focus on aparticular functional group transformation, which takes the role of ‘‘depro-tecting’’ a masked functionality, namely the nitro-to-amine reduction. Thisapproach provides a versatile tool for planning multistep derivatizations ofheterocycles, as exemplified in Fig. 15.45

A key step in tin-based reductions on the solid phase is the washingprocedure, which is used for the removal of residual tin salts. To be onthe safe side, numerous washes with a very broad spectrum of solventsare recommended. In our experience good results are obtained withpolystyrene-based resins when washes with warm DMF/water (1:1) areincluded.

Name Reactions

As of today, the variety of chemical reactions already applied in thesolid-phase synthesis format is very rich and comprises the majority ofthe organic synthesis reactions with significant preparative scope. How-ever, the number of examples describing the use of classic reactions forthe ‘‘decoration’’ of solid supported scaffolds, i.e., aiming at standardizedderivatization of already formed heterocycles, is still surprisingly low.The reaction types previously mentioned in this chapter seem to be themost frequently applied for this specific purpose. In this section we cover

45 X. Ouyang and A. S. Kiselyov, Tetrahedron 55, 8295 (1999).


some name reactions, which were used in this context. Again, we realizedthat the overall pool of examples to choose from is rather limited.

The Mitsunobu reaction is a very powerful and versatile tool in solid-phase chemistry. This was also recognized early for the preparation of com-binatorial libraries.46 It effectively leads to the nucleophilic substitution ofan alcohol by the conjugate base of an acidic reactant with sterical inver-sion at the alcohol carbon. This reaction is mediated by the redox combin-ation of a phosphine with a dialkyl azodicarboxylate. The sequence ofreagent addition has little effect on the yield of the reaction if the timedelays are kept minimal (the adduct forming the redox system has limitedstability). In general, the adduct is prepared first in order to reduce poten-tial side reactions related to the triphenylphosphine nucleophilicity. Incombinatorial chemistry this reaction is often used for N-alkylations andether formations. Esterifications are an attractive option as well since theactivation process is reversed (compared to the classic activation of the acylcomponent). This provides more flexibility in avoiding activation of the im-mobilized species on the solid phase. Due to the instability of the redoxsystem it makes little sense to prolong reaction times beyond 2 h.Repeating the reaction with fresh reagents is more effective and easy todo in the solid-phase format. As washes with methanol or isopropanolare not uncommon in working with polystyrene-based supports, suchsolvents should be avoided previous to a Mitsunobu reaction or utmostcare has to be applied in removing residual alcohol traces prior to thereaction. Arylsulfonamides are acidic enough to be substrates in Mitsuno-bu reactions. Electron-withdrawing substituents on the arylsulfonyl groupfacilitate the reaction, but they also render the moiety more sensitive tocleavage with nucleophiles. As illustrated in Fig. 16 (step b)47 this factcan be exploited for the monoalkylation of an amino group, where tri-fluoroacetylation activates the group making it accessible for Mitsunobureaction. The trifluoroacetyl residue can be removed subsequently bytreatment with a nucleophile. Purines were also derivatized by Mitsunobureaction at the endocyclic N9 as already illustrated in Fig. 1015 and reportedby Dorff and Garigipati.48

The base facilitated Knoevenagel reaction has been used on anoxindole scaffold as indicated in Fig. 17.49 Position 3 of this heterocycleconsists of an activated methylene function, which lends itself to classic

46 V. Krchnak, J. Vagner, Z. Flegelova, A. S. Weichsel, G. Barany, and M. Lebl, in ‘‘Peptides:

Chemistry, Structure and Biology, Proceedings of the 14th American Peptide Symposium,’’

p. 307, Columbus, OH, 1996.47 T. C. Norman, N. S. Gray, J. T. Koh, and P. G. Schultz, J. Am. Chem. Soc. 118, 7430 (1996).48 P. H. Dorff and R. S. Garigipati, Tetrahedron Lett. 42, 2771 (2001).49 Y.-L. Chou, M. M. Morrissey, and R. Mohan, Tetrahedron Lett. 39, 757 (1998).

N

N N

N

Cl

H2NO O

NH

OH H

N

N N

N

Cl

NH O

F3C

O

N

N N

N

Cl

N

O

F3C

O

R1

N

N N

N

HN

HNOHR1

R2

Linker

Linker

8384

85 86

a

b c, d

Fig. 16. Mitsunobu reaction for exocyclic N2 alkylation on a purine scaffold. (a)

Trifluoroacetic anhydride (0.2 M), 0.3 M 4-methyl-2,6-di-tert-butylpyridine, CH2Cl2, 37�, 4 h;

(b) 0.2 M R1-OH, 0.4 M PPh3, 0.2 M DEAD, THF, �10�

to RT, 6 h; (c) 0.25 M amine,

DMSO, 70�, 12 h; (d) TFA/H2O, 90:10 (v/v), RT, 1 h.

NH

O

TIPSO

HN O

O N

OHO

NO

tBu

tBu

OH

a

87 88

b

Fig. 17. Knoevenagel reaction on an immobilized oxindole scaffold. (a) 4-Hydroxy-3,5-di-

tert-butylbenzaldehyde (3 equiv.), pyrrolidine (3 equiv.), CH2Cl2/MeOH (4:1), 24 h; (b) 1 M

Bu4NF/THF, 1 h.


formation of C–C bonds with carbonyl components. The analogousposition of the fully aromatized indole scaffold can be addressed with aMannich reaction as described by Zhang et al.50 (see Fig. 18). These twomethods are quite valuable for the generation of biologically interestingindole-based chemical libraries and for the solid-phase synthesis of indolealkaloids.

Conclusion

Substituted heterocyclic compounds offer a high degree of structuraldiversity and have proven to be broadly useful as therapeutic agents. The

50 H.-C. Zhang, K. K. Brumfield, L. Jaroskova, and B. E. Maryanoff, Tetrahedron Lett. 39,

4449 (1998).

NH

NH

O

NH

O

H2N

OR2

NH

N

R1

R2

R1

NH

N

a

89 90

b

Rink amide resin

Fig. 18. Mannich reaction on a resin-bound indole scaffold. (a) Amine (1.5 equiv.), HCHO

(1.5 equiv.), HOAc/1,4-dioxane (1:4), 1.5 h RT; (b) 30% TFA, CH2Cl2, 1 h, RT.


derivatization on the solid phase of preassembled heterocyclic core scaf-folds (which may be previously prepared in large scale in solution) is anattractive approach for the high-throughput synthesis of new chemicalentities for activity screens. Validated protocols of standard reactionsapplicable to a variety of solid-supported substrates are useful tools,which can be used repeatedly as long as the supply of novel scaffolds ismaintained.

In this chapter we have exemplified this type of derivatization strategyon a number of cases, all of which were reported in the literature with awell-documented experimental protocol. We expect that a growing numberof examples will be published in the coming years, both in the patent litera-ture (because of the industrial focus of the field) and in scientific journals.The added value of parallel and combinatorial chemistry will be furthersubstantiated by these examples.

Experimental

Substitution of Remaining Chloro Group with Amines viaNon-Palladium-Catalyzed Amination Reaction without Base (Fig. 2)15

A resin of type 5 (0.05 mmol) is suspended in the solution of an amine(2 M in n-butanol, 0.2 ml). After 12 h of heating at 80

�in a sealed reaction

vessel under argon, the resin is washed with methanol (4 � 1 ml) anddichloromethane (4 � 1 ml) and dried under vacuum. The product (6) isobtained by cleavage in DCM:TFA:Me2S:H2O 45:45:5:5 (0.3 ml) for 2 h.

Substitution of Remaining Chloro Group with Amines via Non-Palladium-Catalyzed Amination Reaction with KOtBu as Base (Fig. 2)15

To a suspension of a resin of type 5 (0.05 mmol) in THF (anhydrous,0.25 mmol) an amine is added (0.25 mmol), followed by addition of KOtBusolution (in THF, 1.0 M, 0.25 ml, 0.25 mmol). After 12 h of heating at 70

�


in a sealed reaction vessel under argon, the resin is washed with methanol(4 � 1 ml) and CH2Cl2 (4 � 1 ml) and dried under vacuum. The product issubsequently cleaved using CH2Cl2:TFA:Me2S: H2O 45:45:5:5 (0.3 ml) for2 h to afford the desired final product of type 6.

General Reaction Conditions for the Conversions in Fig. 312

Small scale reactions (<250 mg of resin) and resin washing and dryingprocedures are performed at room temperature in polypropylene filtrationtubes (1,3, or 6 ml) with polyethylene frits and a Visprep solid-phase ex-traction vacuum manifold supplied by Supelco. Cleavage reactions forhigh-performance liquid chromatography (HPLC) analysis are performedusing approximately 5 mg of resin in 1.9-ml polypropylene microcentrifugetubes (Eppendorf) while agitating on a tube rotator. Otherwise the reac-tions are performed in a round-bottom flask with magnetic stirring set atthe lowest possible speed to avoid grinding of the resin.

General Procedure for the Synthesis of Rink Resin-Bound6-Chloro-2-methane sulfenyl pyrimidine (9) (Fig. 3)12

Rink amide resin 7 (4.01 g, loading: 0.73 mmol/ g, 2.93 mmol) is swollenin DMF (75 ml) for 30 min. Then 4,6-dichloro-2-methylthiopyrimidine (8)(2.84 g, 14.6 mmol), tetrabutylammonium bromide (1.84 g, 5.7 mmol), anddiisopropylethylamine (5.1 ml, 29.3 mmol) are added and the mixturegently stirred and heated overnight at 90

�. The resin is filtered and washed

thoroughly with DMF (2 � 40 ml), CH2Cl2 (2 � 40 ml), MeOH (2 �40 ml), and Et2O (2 � 40 ml). The resin is first predried by passing airthrough the filter tube, and then placed under high vacuum. A negative nin-hydrine test of the resin indicates full substitution. Of the monochlorinatedpyrimidine resin 4.33 g is obtained (9).

Cleavage of 4-Amino-6-chloro-2-methanesulfenyl Pyridinefrom (9) (Fig. 3)12

An aliquot of resin 9 (50 mg) is swollen in CH2Cl2 (250 �l). A solutionof trifluoroacetic acid in water 95:5 (4.75 ml) is added and the mixture isstirred for 3 h at room temperature. The suspension is filtered and theresin is washed with CH2Cl2(2 � 5 ml), CH3CN (2 � 5 ml), and CH2Cl2(2 � 5 ml). The filtrates are combined and the solvents evaporatedunder reduced pressure. The residue is then taken up in a CH3CN/ watersolution (2 ml, 1:1) and freeze-dried overnight. The resulting residue ispurified by silica gel chromatography or semipreparative reverse phase(RP)-HPLC.


Resin-Bound 2-Methylthio-6-piperidinopyrimidine (16)(R2NR20 ¼ Piperidine) (Fig. 3)12

Resin 9 (130 mg, 0.51 mmol/g, loading 0.066 mmol) is swollen in theminimal amount of N-methylpyrrolidone (1.5 ml) for 30 min. Piperidine(4 ml) is added and the mixture is stirred gently (or better shaken) and re-fluxed overnight. The resin is then filtered, washed thoroughly with DMF(2 � 5 ml), CH2Cl2 (2 � 5 ml), MeOH (2 � 5 ml), and DMF (2 � 5 ml).The resin is predried by passing air through the filter tube, which is thenplaced under high vacuum. Resin 16 (R2NR20 ¼ piperidine) is obtainedwith a theoretical loading of 0.50 mmol/g.

Cleavage of 2-Methylthio-6-piperidinopyrimidine from Resin 16 (Fig. 3)12

The cleavage is performed according to the previously describedmethod. The crude product is further subjected to flash column chromatog-raphy on silica gel using ethyl acetate/petroleum ether giving 4-amino-2-methansulfenyl-6-piperidinopyrimidine.

Oxidation of the Methylthio Group (Fig. 3)12

Resin-Bound 2-Methanesulfinyl-6-piperidinopyrimidine (9b) (R2NR20 ¼Piperidine) (11b). Resin 16 (500 mg, 0.50 mmol/ g loading, 0.25 mmol) isswollen in DMF (20 ml) for 30 min. The mixture is cooled to 0

�and a solu-

tion of magnesium monoperoxyphthalate (MMPP, 212 mg, 0.34 mmol) inDMF (5 ml) is added dropwise and shaking continues for 2 h at 0

�. After

the resin is washed and dried in the usual manner, an aliquot of the resin(5 mg) is taken and cleaved off. RP-HPLC analysis indicates that approxi-mately 6% starting material remained unreacted and a new oxidation cycleis performed with MMPP (0.14 mmol) for 1 h at 0

�. Analysis confirmed the

total conversion of resin 16 into a mixture of resin-bound sulfoxide (11b)(�79%) and sulfone (11c) (�15%). A theoretical loading of 0.50 mmol/gis assumed for subsequent work.

Displacement of Methanesulfinyl (11b) or Methanesulfonyl (11c)Groups (Fig. 3)12

4-Amino-2-benzylamino-6-piperidin-1-yl Pyrimidine (12) (R1NR10H ¼

Benzylamine, R2NR20H ¼ Piperidine). Resin mixture 11b, 11c (130 mg,

0.50 mmol/g loading, 0.065 mmol) is swollen in the minimal amount ofN-methylpyrrolidone (1.5 ml) for 30 min. Benzylamine (4 ml) is addedand the mixture is shaken overnight at 140

�. The resin is filtered and

washed thoroughly with DMF (2 � 5 ml), MeOH (2 � 5 ml), then Et2O(2 � 5 ml). The resin is predried by passing air through the filter tube,which is then subjected to high vacuum.


Cleavage of the Resin 12 (Fig. 3)12

Resin 12 (110 mg, 0.054 mmol) is treated with TFA as previously de-scribed for resin 9. The crude product (purity �88%) is subjected to chro-matography on silica using EtOAc/ petroleum ether as the mobile phase.The purified product 4-amino-2-benzylamino-6-piperidin-1-yl pyrimidineis obtained in >70% yield.

Synthesis of 2,4,6-Amino-Substituted s-Triazines Using a Partial SolutionPhase Approach (Fig. 4)14

Example: 4,6-Dichloro-N-(3, 5-dichlorophenyl)-s-triazin-2-amine (R1 ¼3,5-Dichlorophenyl) (18) (Fig. 4)14. A finely dispersed suspension of (17) isprepared by adding water (175 ml) into a stirred solution of 2,4,6-trichloro-1,3,5-triazine (17) (15 g, 79.7 mmol) in acetone (150 ml). After cooling to�20

�a solution of 3,5-dichloroaniline (13.3 g, 79.7 mmol) in acetone is

added, followed by the dropwise addition of NaOH (2 N, 40 ml). The mix-ture is stirred for 2 h at 0

�. The acetone is evaporated. The precipitate is

filtered off, washed with water, and dried over MgSO4. Approximately23 g of product (18) (R1 ¼ 3,5-dichlorophenyl) is obtained in >95% yield.

Polymer-Bound Thiol (19) (Fig. 4)14

A mixture of Merrifield resin (chloromethylpolystyrene 0.5–2% cross-linked with DVB) (100 g, 1.8 mmol/g), thiourea (68.5 g, 900 mmol), andDMA (1 liter) is shaken at 85

�for 20 h. Washes with isopropyl alcohol

(1 � 5 min), dioxane (2 � 4 min), dioxane/H2O (1:1) (6 � 4 min), DMA(3 � 4 min), and isopropyl alcohol (5 � 4 min) at RT are performed onan automated washing station using about 1 liter of the appropriate solventper wash. Drying under high vacuum for 20 h affords polymer-bound thiur-onium salt with >90% yield based on elemental analysis. Of this resin 91 gis suspended in dioxane/pyrrolidine (4:1, 900 ml) and stirred gently at 110

�

for 2 h, preferably using an overhead stirrer. Resin 19 is washed and driedas described for the thiuronium salt. It can be used for the resin capturestrategy. The thiol content is �88% of the initial loading.

Polymer-Bound Triazine Attached via a Thiol Linker (20) (Fig. 4)14

The resin bearing a thiol function (19) (4 g, 11.2 mmol) is washed withDMA (3 � 15 ml) at 40

�. 6-(3,5-Dichloroanilino)-2,4-dichloro-s-triazine

(5.49 g, 14.0 mmol), DIPEA (2.45 ml, 14 mmol), and DMA (15 ml) areadded and the mixture is shaken at 40

�for 18 h. The polymer-bound tria-

zine is washed successively at 40�

with DMA (15 ml), isopropyl alcohol(15 ml), and hexane (15 ml). Resin 20 is dried at 50

�under high vacuum


and is analyzed by elemental analysis. The same general procedure can beapplied for 2-amino-4,6-dichloro-1,3,5-triazine (R1 ¼ NH2).

Displacement of the Remaining ChloroAtom to (21) (Fig. 4)14

To dry resin 20 (0.92 g, 0.25 mmol) are added an amine (such as 3,5-dichloroaniline) (0.625 mmol), DIPEA (0.215 ml, 1.25 mmol), and DMA(1.5 ml). After shaking the mixture at 45

�for 17 h, the resin is washed

successively with DMA (2 � 5 ml), isopropyl alcohol (5 ml), and DCM(3 � 5 ml).

Oxidation of the Thio Group (Fig. 4)14,51

The resin is treated with N-phenyloxaziridine (0.075 g, 0.288 mmol)and DCM (2.5 ml) for 14 h at RT. The oxidized resin is washed withDCM (5 ml), isopropyl alcohol (5 ml), and dioxane (2 � 5 ml).

Cleavage and Release of the 2,4-Bis-(3,5-dichloroanilinyl)-6-pyrrolidinyl-1,3,5-triazine (24) (R1, R2 ¼ 3,5-Dichlorophenyl, R2 ¼ H) from Resin 23upon Substitution of the Oxidized Thioether Link (Fig. 4)14

A solution of pyrrolidine (0.041 ml, 0.50 mmol) in dioxane (2.5 ml) isadded to resin 23 and the mixture is shaken for 6 h at 60

�. The filtrate is ly-

ophilized to yield the substituted 2,4,6-triamino-s-triazine (24), such as 2-benzylamino-4-(3,5-dichloroanilinyl)-6-pyrrolidinyl-1,3,5-triazine (R1 ¼benzyl; R2 ¼ 3,5-dichlorophenyl, R20 ¼ H) in high purity and 31% yield.

Conversion of a Resin-Bound Quinazolin-4-one (43) into a4-Chloroquinazoline (44) (Fig. 8)30

To a resin bearing 2-carboxyquinazolin-4-one (43) (200 mg; �60 �mol)swollen in anhydrous DMF (5 ml) is added thionyl chloride (5 ml). Thesuspension is refluxed for 3 h, then the resin is filtered and reswollen inDMF for the subsequent substitution of the 4-chloro group.

Preparation of Amino-Substituted Quinazoline (45) and Cleavage fromthe Resin to Obtain the 2-Unsubstituted 4-Arylaminoquinazoline(46) (Fig. 8)30

Resin 44 (preswollen in 5 ml DMF) is exposed to 3-bromoaniline(150 �l, 5 equiv.) in propan-2-ol (5 ml) containing four drops of conc.HCl. The reaction is shaken at room temperature overnight. The resin 45is filtered, washed with DMF (5 � 20 ml), CH2Cl2 (5 � 20 ml), and diethyl

51 F. A. Davis and O. D. Stringer, J. Org. Chem. 47, 1774 (1982).


ether (5 � 20 ml) and dried in vacuo. Resin 45 (73 mg) is swollen in di-oxane (4 ml) and CH3CN (4 ml). NaI (100 mg) and trimethylsilylchloride(200 �l) are added. The suspension is heated at 75

�for 72 h and then

filtered. The filtrate is concentrated in vacuo and taken up in water. Theproduct is extracted with DCM (3 � 10 ml), washed with aq. Na2S2O4,dried over MgSO4, and concentrated in vacuo to give 4-(3-bromoanilino)-6-chloroquinazoline (46).

Facilitated Arylations via Iron-� Complex

�6-1,2-Dichlorobenzyne-�5-cyclopentadienyliron(II) Hexafluorophos-phate (53) (Fig. 9)31. 1,2-Dichlorobenzene (500 g, 3.4 mol), anhydrous alu-minum chloride (91.2 g, 68 mol), aluminum powder (9.2 g, 0.34 mol), andferrocene (63.2 g, 0.34 mol) are heated to 95

�for 4 h under vigorous stir-

ring. The reaction is cooled to 0�

and H2O (500 ml) is added slowly, whilekeeping the temperature below 50

�. (Caution: Very exothermic!) Et2O

(500 ml) is added and the reaction mixture is stirred for 30 min. Theaqueous phase is isolated, filtered, and extracted with Et2O (2 � 200 ml).The dark-green aqueous phase is filtered and saturated aqueous ammo-nium hexafluorophosphate solution is added in small portions until precipi-tation of the desired product is completed (38% yield). This procedurecan also be applied to 1,3-dichlorobenzene, 1,4-dichlorobenzene, and 1,3-dichloro-2-methylbenzene.

Representative Reaction with Piperazine Bound to Polymeric Supports(Fig. 9)31. Piperazin-1-yl methyl polystyrene resin 51a (24 g, 24.2 mmol),�6-1,2-dichlorobenzene-�5-cyclopentadienyl iron(II) hexafluorophosphate(53) (25 g, 69.6 mmol), and potassium carbonate (13.4 g, 97 mmol) aresuspended in dry THF (400 ml) at RT. (Caution: Exothermic reaction!)The reaction mixture is agitated overnight. Resin 54 is washed with dryTHF (2 � 100 ml), CH2Cl2 (3 � 100 ml), MeOH (2 � 100 ml), and CH2Cl2(3 � 100 ml), and then dried in vacuo at 40

�to afford a red resin.

Reaction of Resin-Bound Iron Complex (54) with Aliphatic Alkoxides(Fig. 9)31. A solution of 3-methoxyphenylmethoxide (0.50 M) is preparedby slowly adding 3-methoxyphenylmethanol to sodium hydride (60%suspension in mineral oil) in dry THF at room temperature. (Caution:Generation of heat and hydrogen.) The mixture is stirred for an additional30 min after gas generation ceased. The alcoholate (11 mmol) is trans-ferred to the resins bearing the aryl-iron complex (54) (3 g, 1.9–2.2 mmol)and THF (45 ml) is added. The reaction is stirred using an overheadstirrer at 60

�for 48 h. The resin 55 is filtered and washed with THF

(2 � 100 ml), H2O (2 � 100 ml), THF (2 � 100 ml), MeOH (2 � 100 ml),and CH2Cl2 (3 � 100 ml) and dried in vacuo at 40

�to give a red resin.


Reaction of Resin-Bound Iron Complex (54) with Alkyl Mercaptans,Thiophenols, and Phenols (Fig. 9)31. Sodium thiolates are prepared analo-gously to the alkoxides from thiol and sodium hydride, except that dryDMF is used as a solvent. The substitution on the polymer-bound arene(54) is performed at 70

�in DMF within 16 h. The resin is filtered and

washed with DMF (2 � 50 ml), MeOH (2 � 50 ml), H2O (2 � 50 ml),MeOH (2 � 50 ml), and CH2Cl2 (3 � 50 ml) and then dried in vacuoat 40

�to yield a red resin.

Representative Reaction of Resin Bound Iron Complex (54) withAlkylamines (Fig. 9)31. A resin bearing the aryl-iron complex (3 g, 1.9–2.2 mmol), 4,4-(10,10-dimethyl-9,10-dihydroanthracene-9-yl)-piperidine(3.2 g, 11 mmol), and diisopropylethylamine are suspended in dry DMF(40 ml) and stirred at 90

�for 96 h. Resin 55 (XR ¼ NHR) is filtered and

washed with DMF (2� 50 ml), MeOH (2� 50 ml), H2O (2� 50 ml), MeOH(2 � 50 ml), and CH2Cl2 (3 � 50 ml), and then dried in vacuo at 40

�.

Decomplexation on Solid Phase (Fig. 9)31. To prepare a 0.5 M 1,10-phenanthroline solution, to 1,10-phenanthroline (180 g, 1 mol) CH3CN(600 ml) and H2O (200 ml) are added in that order under stirring. Morewater is added (approximately 100 ml) until the solution becomes clear.

Resins bearing the aryl-iron complexes (55) are suspended in a light-transparent reactor tube with the phenanthroline solution (10 ml/g ofresin). The suspensions are agitated under irradiation with visible lightfor 12 h. The appearance of an intensively red color of the liquid phase in-dicates the progress of the decomplexation. The resulting resins 56 arefiltered and washed with MeOH (3 � 50 ml), THF (3 � 50 ml), and MeOH(3 � 50 ml) until the washing solutions are colorless (approximately fivecycles). The irradiation and washing procedure is repeated until the decom-plexation is complete. The resins are washed with MeOH (3 � 50 ml), THF(3 � 50 ml), a 1 M solution of triethylamine in THF (3 � 50 ml), MeOH (2� 50 ml), CH2Cl2 (3 � 50 ml), MeOH (2 � 50 ml), and CH2Cl2 (3 � 50 ml)and dried in vacuo at 50

�.

Standard Cleavage from the Polymeric Support (Fig. 9)31. To a suspen-sion of the decomplexed resin 56 (2.41 g, 2 mmol) in 1,2-dichloroethane(30 ml) at 0

�is added slowly methylchloroformate (25 mmol). After 1 h

of agitation at 0�, the suspension is allowed to come to RT and the reaction

to continue overnight. The resin is filtered and washed with H2O(1 � 30 ml), CH2Cl2 (3 � 30 ml), and H2O (1 � 30 ml). The liquid phaseis rendered alkaline using NaOH (1 M, 30 ml) and then separated.The aqueous layer is extracted with CH2Cl2 (3 � 30 ml). The combinedorganic phases are washed with brine and dried over MgSO4. Flash columnchromatography (silica, heptane/EtOAc) is used for the purification of (57)(up to 72% isolated yield).


Derivatization of Dichloroheterocyclic Scaffolds (Fig. 10)15

Reductive Amination for Synthesis of PAL-Resin-Bound Amines (59)(Fig. 10)15. To a suspension of 4-formyl-3,5-dimethoxyphennoxymethylfunctionalized polystyrene resin (PAL)25 (10.0 g, 11.3 mmol) in DMF(350 ml) a primary amine (56.5 mmol) is added followed by addition ofsodium triacetoxyborohydride (7.18 g, 33.9 mmol) and acetic acid (6.52 ml,113 mmol). The suspension is shaken gently at RT overnight. Resin 59 iswashed with methanol (4 � 300 ml) and CH2Cl2 (4 � 300 ml) and driedunder vacuum. Subsequent derivatization products can be cleaved fromthe resin by a 2 h treatment with CH2Cl2/TFA/Me2S/H2O 45:45:5:5.

Mitsunobu Reaction for Endocyclic N9-Alkylation on Purine Scaffold(60) (Fig. 10).15 This is described later in the section dedicated to Mitsunobureactions.

Suzuki Coupling to Displace the Remaining Chloro Atom on C2 withAryl Groups (Fig. 10).15 To a 10-ml flame-dried Schlenk flask containingthe solid supported intermediate 61 (0.10 mmol, 1.0 equiv.) are addedan arylboronic acid (0.50 mmol, 5.0 equiv.), Pd2(dba)3 (0.007 mmol,0.07 equiv.), 1,3-bis(2,6-diisopropylphenyl)-1H-imidazol-3-ium chloride(carbine ligand, 0.014 mmol, 0.14 equiv.), and Cs2CO3 (0.60 mmol,6.0 equiv.). The Schlenk flask is purged with Ar and charged with anhyd-rous 1,4-dioxane (1.0 ml). The reaction is heated to 80

�under Ar. After

12 h the resin is filtered, washed (4 � 1 ml) with a sodium diethyldithio-carbamate solution (0.05 M in DMF), CH2Cl2 (4 � 1 ml), and MeOH(4 � 1 ml). The resin is dried in vacuo.

Pd-Catalyzed Displacement of the Remaining Chloro Atom on C2 withAmines (Fig. 10).15 To a 10-ml flame-dried Schlenk flask with resin 61(0.10 mmol, 1.0 equiv.) are added an amine or aniline (0.50 mmol,5.0 equiv.), Pd2(dba)3 (0.007 mmol, 0.07 equiv.), 1,3-bis-(2,6-diisopropyl-phenyl)-1H-imidazol-3-ium chloride (carbine ligand, 0.014 mmol,0.14 equiv.), and KOtBu (0.60 mmol, 6.0 equiv.). The Schlenk flask ispurged with Ar and charged with anhydrous 1,4-dioxane (1.0 ml). Thereaction is heated to 80

�under Ar. After 12 h the resin is washed with a

sodium dithiocarbamate solution (0.05 M in DMF, 4 � 1 ml), CH2Cl2(4 � 1 ml), and MeOH (4 � 1 ml) and is subsequently dried in vacuo.

Pd-Catalyzed Displacement of the Remaining Chloro Atom on C2 byPhenols (Fig. 10).15 To a 10-ml flame-dried Schlenk flask with resin 61(0.10 mmol, 1.0 equiv.) are added a phenol (0.50 mmol, 5.0 equiv.),Pd2(dba)3 (0.007 mmol, 0.07 equiv.), 1,10-biphenyl-2-yl-[di-(tert-butyl)]-phosphine (phosphine ligand) (0.028 mmol, 0.28 equiv.), and K3PO4

(0.70 mmol, 7.0 equiv.). The Schlenk flask is purged with Ar and chargedwith anhydrous toluene (1.0 ml). The reaction is heated to 80

�under Ar.


After 12 h the resin is washed with a sodium diethyldithiocarbamate solu-tion (0.05 M in DMF, 4 � 1 ml), CH2Cl2 (4 � 1 ml) and MeOH (4 � 1 ml),and is subsequently dried in vacuo.

Preparation of 2,6-Disubstituted Purines (65) Using Pd-Mediated ReactionsInvolving Amines and Boronic Acids (Fig. 11)33

The following reactions are performed in a 96-well polypropylene reac-tion block with 1.8-ml wells. To a well with resin 64 (30 mg, 0.015 mmol)and an amine or boronic acid (R2 ¼ primary or secondary aliphatic or aro-matic amine, or aryl boronic acid) (0.125 mmol, 8.3 equiv.) the inorganicsalt K3PO4 [100 ( 10) mg, 0.471 mmol, 31.4 equiv.] is added as a dry solid.The reactor block is sealed and the wells purged with Ar by applyingvacuum and Ar in an alternating fashion three times successively.Subsequently 1 ml of a freshly prepared solution of Pd2dba3/CHCl3(0.00298 M, 0.2 equiv.) and P(tBu)3 (0.04 M, 2.7 equiv.) in previously de-gassed Ar-saturated NMP is added. The reaction wells are purged withAr another three times. The reaction block is then kept at 100

�for 40 h.

After draining all the wells, the resin is washed with 0.5 ml of aq. 0.25 MTEAA, in DMA/H2O 4:1 (10�), 5% sodium N,N-diethyl dithiocarbonatein DMA (10�), DMA (10�), DCM (5�), MeOH (5�), DCM (5�), andthen n-pentane (5�). Compounds of type (65) with C2–C and C2–N bondscan be obtained after cleavage. The main impurity was found to beunreacted starting material (64).

Stille Coupling on Support-Bound Compounds 67 (Fig. 11)33

NMP is distilled over CaH2 under Ar at 20 Torr. Resins containing bro-minated intermediates of type 67 (60 mg, 0.030 mmol) and Cu2O (36.0 mg,0.25 mmol, 8.3 equiv.) are placed in the reaction wells as solids. The reac-tion block is sealed and purged with Ar. Through the septa in the reactionblock are added 0.5 ml of a freshly prepared stock solution of Pd(OAc)2

(1.38 mg, 0.006125 mmol, 0.2 equiv.) and 1,3-bis-(diphenylphosphino)-propane (dppp) (5.15 mg, 0.01249 mmol, 0.41 equiv.) in previously de-gassed Ar-saturated NMP. Subsequently, 0.25 mmol (8.3 equiv.) of theappropriate stannanes is added as a 0.25 M solution in NMP. The reactionblock is kept at 100

�for 20 h. The reaction wells are washed with the

following solvents (10 � 0.5 ml):(1) aq. 0.25 M TEAA, in DMA/H2O(4:1), (2) acetonitrile/HOAc/DMA 2:1:2 (this wash solubilizes the excessCu2O, which may clot frits), and (3) 5% sodium N,N-diethyl dithiocarbo-nate in DMA. Alternating washes follow with MeOH (5�), CH2Cl2 (5�),again CH2Cl2 (5�), and n-pentane successively. The resin 68 is driedin vacuo for 2 h and product 69 is obtained by appropriate cleavage.


Acylations, Alkylations, Reductive Alkylations(Aminations, Alkaminations)

Reductive Alkylation of Resin-Bound Tropane 73 (Fig. 12).34 A solu-tion of an aldehyde (0.6 mmol) in 1% HOAc in DMF (9 ml) is added to0.4 g of dry resin 73 (0.3 mmol/g, 0.12 mmol). The suspension is gentlystirred for 1 h and then 127 mg of sodium triacetoxyborohydride is added(0.6 mmol). Stirring is maintained for 35 h at which point methanol isadded to quench the excess of the reducing agent and to dissolve the boratesalts. The solution is then removed from the resin via filtration cannula andrinsed with DMF (3 � 10 ml), DMF/H2O (1:1; 3 � 10 ml), THF (3 �10 ml), and DCM (3 � 10 ml). The resin 74 (X ¼ CH2) is dried undervacuum to constant weight.

Acylation of Resin-Bound Tropane (73) (Fig. 12).34 Intermediate 73(0.12 mmol) is added to the following solution: HATU (228 mg, 0.6 mmol,5 equiv.), triethylamine (120 mg, 1.2 mmol, 10 equiv.), benzoic acid(73 mg, 0.6 mmol, 5 equiv.), and HOAt (16 mg, 0.12 mmol, 1 equiv.).The suspension is gently stirred at RT overnight. The solution is removedvia filtration cannula and the resin (74, X ¼ CO) is washed with DMF (3 �10 ml), CH2Cl2 (3 � 10 ml) and dried in vacuo.

Acylation via Activation of Resin-Bound Carboxylic Acid (Fig. 13)41.Resin 76 is swelled in pyridine/DMF (1:10) and reacted with pentafluoro-phenyl trifluoroacetate (6 equiv.) in pyridine/DMF (1:10) for 1 h. Oncefiltered, the resin is washed with DMF, then subjected to an 18 h treatmentwith an appropriate amine building block (6 equiv.). Once filtered, theresin is washed with DMF (5�), CH2Cl2 (2�), and MeOH (3�) and driedin vacuo to provide the product grafted on solid support. Final products oftype (77) are obtained with an average purity of 65% by cleavage withTFA/DCM (1:1) for 1 h and subsequent rinsing with appropriate solvents.The cleavage solution and all organic washes are combined and evaporated(yields up to 85%).

Stepwise Selective Amine and Amide Alkylation (Fig. 14).44 A firstalkylation step is performed by suspending (78) in a 2 M solution of asuitable alkyl halide in DMF at 50

�for 24–48 h. After thorough washing

with DMF (3�), CH2Cl2 (3�), and THF (3�) intermediate (79) (usuallyformed with >85% purity) is subjected to the final alkylation. The reactionflask is sealed with a fresh rubber septum and flushed with nitrogenfollowed by cooling to 0

�. In a separate flame-dried 25-ml round-bottom

flask 12 equiv. (with respect to 79) of 5-phenylmethyl-2-oxazolidinone isadded. To the reaction flask freshly distilled THF is added (the appropriatevolume to provide a 0.2 M solution of the 5-phenylmethyl-2-oxazolidi-none). The resulting clear solution is then cooled to �78

�and 1.6 M n-butyl


lithium in hexanes is added dropwise by syringe under stirring (10 equiv.with respect to 79). The solution is stirred at �78

�for 15 min and trans-

ferred by cannula to the solid support (79) with stirring at 0�. The resulting

slurry is stirred at 0�

for 1.5 h, at which point 15 equiv. of the appropriatealkyl halide is added by syringe followed by addition of anhydrous DMF toreach a final solvent ratio of approx. 70:30 THF/DMF. The slurry is allowedto warm to ambient temperature under stirring. After 3–12 h at RT thesolvent is removed by filtration cannula. The support is then washed withTHF (1�), THF/H2O (1:1; 2�), THF (2�), and CH2 Cl2 (2�). To the fullyderivatized product on solid support an excess of TFA/CH2Cl2 (90:10) isadded and the cleavage reaction is allowed to continue for 0.5 h at RT.The cleavage solution is removed by filtration cannula, and the resin isrinsed with an appropriate solvent (e.g., MeOH/CH2Cl2). Concentrationof the combined filtrates provides the crude product (80) obtained in>80% yield and >80% purity. Purification by RP-HPLC leads to >95%purity and yields in the range of 56–69%.

Reduction of an Aromatic Nitro Group on Solid Phase (Fig. 15).45

Resin 81 (1 g, 0.47 mmol/g) is suspended in 20 ml of a 1.5 M solution ofSnCl2�2H2O in DMF. The mixture is shaken for 12–24 h and filtered. Theresin is washed with MeOH, CH2Cl2, DMF, dioxane, and Et2O and driedin vacuo to obtain (82).

Mitsunobu Reaction for Exocyclic N2-Alkylation on a Purine Scaffold(Fig. 16).47 2,6-Di-tert-butyl-4-methylpyridine (1.54 g, 7.5 mmol, 0.3 M) isadded to freshly distilled CH2Cl2 (25 ml) at 0

�under N2. To this solution is

added trifluoroacetic anhydride (706 �l, 5.0 mmol, 0.2 M) and the mixtureis stirred for 5–10 min. The solution is then transferred to the resin 83 andthe flask is vortexed and vented several times to relieve pressure. The flaskis shaken for 6–12 h after which the solvent is removed and the resinwashed with dry CH2Cl2 (5 � 25 ml) with vortexing between each rinse.The resulting resin 84 is dried.

Diethylazodicarboxylate (394 �l, 2.5 mmol) is added dropwise to tri-phenylphosphine (1.31 g, 5.0 mmol) dissolved in dry 1:1 THF/CH2Cl2(5 ml) at 0

�. After stirring at 0

�for 1 h, the solution is transferred by Teflon

cannula to the trifluoroacetylated resin 84 (180 mg) simultaneously withthe dropwise addition of the appropriate alcohol (2.0 mmol) in THF(100 �l). The flask is vortexed and vented several times and shaken for12 h. The solvent is removed and resin 85 washed with DMF (6 � 10 ml)followed by CH2Cl2 (6 � 10 ml). The rinsed resin 85 is dried.

Knoevenagel Reaction on an Oxindole Scaffold (87) (Fig. 17)49. To asuspension of resin 87 (1 g, 0.32 mmol/g, 0.32 mmol) in 10 ml of a mixtureCH2Cl2/MeOH (4:1), 4-hydroxy-di-tert-butylbenzaldehyde (243 mg,1 mmol) and pyrrolidine (83 �l, 1 mmol) are added. The suspension is

[24] isocyanide-based multicomponent reactions 469

stirred for 24 h, and the resin is filtered, washed with appropriated solvents,and dried under vacuum. The resin is subsequently subjected to cleavageconditions (1 h treatment with 1 M tetrabutylammonium fluoride) andthe product 88 is obtained by liquid–liquid extraction into organic solvent.

Mannich Reaction on a Resin-Bound Indole Scaffold (89) (Fig. 18).50

Commercially available indole 5- or 6-carboxylic acid supported on Rinkamide resin is suspended in HOAc/1,4-dioxane (1:4). A secondary amine(1.5 equiv.) and formaldehyde (1.5 equiv.) are added. The suspension isshaken for 1.5 h at RT. The resin is then filtered, washed with appropriatesolvents, and dried under vacuum to obtain (90).

[24] Library Generation via PostcondensationModifications of Isocyanide-Based

Multicomponent Reactions

By Christopher Hulme, Hugues Bienayme, Thomas Nixey,Balan Chenera, Wyeth Jones, Paul Tempest, and

Adrian L. Smith

Introduction

New developments in the search for novel pharmacological agents overthe past decade have focused on the preparation of chemical libraries assources for new leads for drug discovery. To aid this search a plethora ofpersonal synthesizers and new automation technologies have emerged tohelp fuel the lead discovery engines of drug discovery organizations. Infact, multistep solid-phase syntheses of diverse libraries in excess of10,000 products are now feasible via split and mix techniques. At the sametime, a multitude of more efficient, diverse, or target-oriented solution-phase chemical methodologies have appeared in the chemical literature,which have enabled the relatively facile construction of successful leadgeneration libraries with low FTE* input and little capital expenditure.

* Abbreviations: AcCl, acetyl chloride; BDP, benzodiazepine; Boc, tert-butyloxycarbonyl;

DCE, 1,2-dichloroethane; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine;

DMF, N,N-dimethylformamide; EtOAc, ethyl acetate; FTE, full-time employee; HPLC,

high-pressure liquid chromatography; HRMS, high-resolution mass spectroscopy; IMCR,

isocyanide multicomponent condensation reaction; LC/MS, liquid chromatography/mass

spectrometry; MCR, multicomponent condensation reaction; MeOH, methanol;

MP-carbonate, macroporous carbonate resin (Argonaut Technologies); NMR, nuclear

magnetic resonance; PS, polystyrene; PS-DIEA, polystyrene-bound diisopropylethylamine;



stirred for 24 h, and the resin is filtered, washed with appropriated solvents,and dried under vacuum. The resin is subsequently subjected to cleavageconditions (1 h treatment with 1 M tetrabutylammonium fluoride) andthe product 88 is obtained by liquid–liquid extraction into organic solvent.

Mannich Reaction on a Resin-Bound Indole Scaffold (89) (Fig. 18).50

Commercially available indole 5- or 6-carboxylic acid supported on Rinkamide resin is suspended in HOAc/1,4-dioxane (1:4). A secondary amine(1.5 equiv.) and formaldehyde (1.5 equiv.) are added. The suspension isshaken for 1.5 h at RT. The resin is then filtered, washed with appropriatesolvents, and dried under vacuum to obtain (90).


[24] Library Generation via PostcondensationModifications of Isocyanide-Based

Multicomponent Reactions

By Christopher Hulme, Hugues Bienayme, Thomas Nixey,Balan Chenera, Wyeth Jones, Paul Tempest, and

Adrian L. Smith

Introduction

New developments in the search for novel pharmacological agents overthe past decade have focused on the preparation of chemical libraries assources for new leads for drug discovery. To aid this search a plethora ofpersonal synthesizers and new automation technologies have emerged tohelp fuel the lead discovery engines of drug discovery organizations. Infact, multistep solid-phase syntheses of diverse libraries in excess of10,000 products are now feasible via split and mix techniques. At the sametime, a multitude of more efficient, diverse, or target-oriented solution-phase chemical methodologies have appeared in the chemical literature,which have enabled the relatively facile construction of successful leadgeneration libraries with low FTE* input and little capital expenditure.

* Abbreviations: AcCl, acetyl chloride; BDP, benzodiazepine; Boc, tert-butyloxycarbonyl;

DCE, 1,2-dichloroethane; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine;

DMF, N,N-dimethylformamide; EtOAc, ethyl acetate; FTE, full-time employee; HPLC,

high-pressure liquid chromatography; HRMS, high-resolution mass spectroscopy; IMCR,

isocyanide multicomponent condensation reaction; LC/MS, liquid chromatography/mass

spectrometry; MCR, multicomponent condensation reaction; MeOH, methanol;

MP-carbonate, macroporous carbonate resin (Argonaut Technologies); NMR, nuclear

magnetic resonance; PS, polystyrene; PS-DIEA, polystyrene-bound diisopropylethylamine;




Several groups have pioneered the use of such multicomponent condensa-tion reaction (MCR) technologies, including those led by Ugi, Bienayme,Domling, and Weber, spawning new chemically driven companies thathave rapidly built up their own internal corporate collections. One particu-lar branch of MCR methodologies, namely postcondensation modifications(or secondary reactions) of IMCRs (isocyanide – based multicomponentreactions), forms the basis of this chapter.

UDC (Ugi/De-Boc/Cyclize) Methodology

Benzodiazepines have been of pharmacological interest for decadesand represent one of the most intensely studied heterocyclic templatesknown in drug discovery circles. Reports of the biological utility of1,4-benzodiazepine-2,5-diones (BDPs), in particular, have appeared inmany areas, including applications as antagonists of the platelet glycopro-tein IIb-IIIa,1 anticonvulsant agents,2 antihypnotic agents,3 reverse tran-scriptase inhibitors,4 and selective cholecystokinin (CCK) receptorsubtype A or B antagonists.5 Many routes to libraries of this class of mol-ecule have been developed, including one of the early pioneering solid-phase methods reported by Bunin and Ellman.6 A shorter and moreefficient route to this class of molecule was initially proposed by Keatingand Armstrong7 employing the Ugi MCR8 with anthranilic acids and1-isocyanocyclohexene, 2 (Armstrong’s convertible isocyanide)9 as the acid

PS-TsNHNH2, polystyrene-bound tosylhydrazine; PS-NCO, polystyrene-bound isocyanate;

QC, quality control; RPR, Rhone-Poulenc Rorer; RT, room temperature; TBAF,

tetrabutylammonium fluoride; TFA, trifluoroacetic acid; TFP, tetrafluorophenol; THF,

tetrahydrofuran; TMSN3, trimethylsilyl azide; TOF, time of flight mass spectrometry;

TsNHNH2, p-toluenesulfonylhydrazine; UDC, Ugi/DeBoc/Cyclize; Wang, 4-(hydroxy-

methyl)phenoxy polystyrene.1 R. S. McDowell, B. K. Blackburn, T. R. Gadek, L. R. McGee, T. Rawson, M. E. Reynolds,

K. D. Robarge, T. C. Somers, E. D. Thorsett, M. Tischler, R. R. Webb, and M. C. Venuti,

J. Am. Chem. Soc. 116, 5077 (1994).2 N. S. Cho, K. Y. Song, and C. Parkanyi, J. Heterocycl. Chem. 26, 1807 (1989).3 L. H. Sternbach, J. Med. Chem. 22, 1 (1979).4 R. Pauwels, K. Andries, J. Desmyler, D. Schols, M. J. Kukla, H. J. Breslin,

A. Raeymaeckers, J. Van Gelder, R. Woostenborgles, J. Heykants, K. Schellekens, M. A.

C. Jansen, E. D. Clercq, and P. A. J. Janssen, Nature 343, 470 (1990).5 M. G. Bock, R. M. Dipardo, B. E. Evans, K. E. Rittle, W. L. Whitter, D. F. Veber, P. S.

Anderson, and R. M. Freidinger, J. Med. Chem. 32, 13 (1989).6 B. A. Bunin and J. A. Ellman, J. Am. Chem. Soc. 114, 10997 (1992).7 (a) T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 118, 2574 (1996). For earlier

syntheses of 1,4-benzodiazepines see (b) M. Gates, J. Org. Chem. 45, 1675 (1980). (c)

M. Uskokovic, J. Iacobelli, and W. Wenner, J. Org. Chem. 27, 3606 (1962).8 (a) I. Ugi, Angew. Chem. Int. Ed. Engl. 1, 8 (1962). (b) I. Ugi and C. Steinbruckner, Chem

Ber. 94, 734 (1961). (c) I. Ugi, A. Domling, and W. Horl, Endeavor 18, 115 (1994).

N

NO

O

R2

R1

N+O

O

R1

R2NH2

NH2

N

O R1

O

NH+

R2

NH2

N

O R1

O

O

R2N

OH

O

R3

BocR4

N

NO

R3Boc

R4

R1

O

HN

R1 CHOR2 NH2

NCR2

AcCl/MeOH

or 10% TFA/DCE

2 3

4

1

5

6

7

H+

R3

Scheme 1. Synthetic route for the preparation of 1,4-benzodiazepine libraries.


and isocyanide inputs, respectively. Subsequent postcondensation acid-catalyzed cyclization of the anthranilic amine (the so-called ‘‘internalnucleophile’’ proceeding via an intermediate munchnone) produces thedesired BDP, with reported isolated yields for the two-step procedureranging from 15 to 50%.10 Intrigued with this report and attracted bythe broad biological activity and desirable pharmacokinetics of 1,4-benzodiazepine derivatives, workers at Rhone-Poulenc Rorer (RPR;Aventis) developed an alternative protocol that employed N-Boc-protectedanthranilic acids, 1 (Scheme 1).11

It was thought that protection of the anthranilic nitrogen could preventcompeting and undesired participation in the Ugi reaction, a potential ex-planation for the moderate yields reported for the parent synthetic route.7

Boc removal and cyclization to BDP could be sequentially achieved ontreatment with acid in ‘‘one pot.’’ Cyclization may potentially occur uponacid treatment via any of the three intermediates 4, 5, and 6. Synthesesof five BDPs, designated 8, 9, 10, 11, and 12 (Fig. 1) were evaluated withisolated yields from 70 to 95%.

The chemistry was then successfully advanced to production stage andthe generation of two 96-well plates of 1,4-benzodiazepine-2,5-diones wasreported. The LC/MS A% (area%) yields taken from two 96-well platesare presented in Table I, along with the participating reagents (Fig. 2).

9 (a) T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 117, 7842 (1995). (b) F. K.

Rosendahl and I. Ugi, Ann. Chem. 666, 65 (1963).10 T. A. Keating and R. W. Armstrong, J. Org. Chem. 61, 8935 (1996).11 C. Hulme, S.-Y. Tang, C. J. Burns, I. Morize, and R. Labaudiniere, J. Org. Chem. 63, 8021

(1998).

NH

N

O

ONH

N

O

ONH

N

O

O NH

N

O

ONH

NO

O

O

N

10 12118 9

Fig. 1. Five fully characterized examples of 1,4-benzodiazepines.

TABLE I

LC/MS Area% Purities of a 96-Member Array of BDPs

15 16 17 18 19 20 21 22 23 24 25 26

27 40/16 40/29 40/27 54/15 40/25 39/40 26/16 18/21 41/15 0/0 39/31 47/30

28 85/87 82/72 77/64 79/72 82/69 84/67 81/73 78/67 82/74 43/10 88/74 84/73

29 88/84 85/73 89/68 92/81 92/78 88/68 90/75 82/73 84/79 39/8 76/77 85/72

30 87/80 72/52 69/43 79/70 70/41 80/51 87/63 81/64 81/70 51/25 75/60 80/62

31 45/10 37/24 39/22 36/12 34/20 33/12 41/7 28/26 44/10 9/0 37/16 39/20

32 79/49 74/61 63/51 75/12 66/53 69/54 59/10 74/61 83/67 6/0 71/49 67/64

33 89/87 86/69 88/66 85/82 89/63 85/70 90/74 83/69 86/84 38/8 84/74 88/78

34 85/64 86/63 80/67 85/82 84/75 85/64 82/69 84/67 86/75 27/11 84/69 83/61

NH2NH2 NH2

NH2NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

O

N

N SO

OH2N

NN

O

O O O

OO

OO

O

OO

O

O

CO2H CO2H

NHBoc NBoc

34

13 1415 16 17

18 19 20

21 22 23

24 25 26

27 28 29

30 31 32

33

Fig. 2. Diversity reagents employed in the preparation of a 96-member array of BDPs.



The first percentage corresponds to A% yields with N-Boc anthranilic acid13 and the second percentage is for the N-methylated analogue 14.

Note: For A% yields x/y: The first yield x represents that for reactionswith N-Boc anthranilic acid, 13. The second yield y represents that forN-Me-Boc anthranilic acid, 14. Row 27 represents yields of reactions withaldehyde 27. Column 15 represents yields of reactions with amine 15.

The reaction appeared general for both a range of commercially avail-able aldehydes (e.g., aldehydes with attached ester, heteroaryl, aryl, amido,thioalkyl, alkyl, and cycloalkyl functionality, such as aldehydes 27 through34) and primary amines (e.g., with attached alkyl, aryl, heteroaryl, acidic,and basic functionality such as amines 15 through 26). The process is alsoviable, albeit lower yielding, for N-methylated-Boc anthranilic acids. Assuch this synthetic route, coupled with a facile production protocol, hasnow been adopted throughout the pharmaceutical industry as a preferredmethodology to access arrays of 1,4-benzodiazepines-2,5-diones. Note-worthy is the fact that since the first report of this route, the number of com-mercially available N-Boc anthranilic acids in the ACD (available chemicalsdirectory) has increased from 2 to 35.12 The abbreviation UDC (Ugi/De-Boc/Cyclize) was thus introduced to describe the one-pot sequence ofevents, which was subsequently extended to several other pharmacologic-ally relevant templates utilizing the ‘‘universal isontrile’’ concept. Forexample, application of N-Boc-protected �-amino acids allows conversionto diketopiperazines13 35 with four potential diversity points. Similarly,Boc-protected ethylene diamines and mono-N-Boc-protected phenylenediamines afford ketopiperazines14 37 and dihydroquinoxalinones,15

12 N-Boc anthranilic acids are readily accessible in multigram quantities via the synthetic

route shown in the scheme below from the corresponding anthranilic acid or isatin. N-Boc

diamines are readily available as described in A. P. Krapcho, M. J. Maresh, and J. Lunn,

Synthetic Commun. 23, 2443 (1993). The majority of available N-Boc anthranilic acids may

be purchased from Anaspec AA.

THFH2O2

MeOHNH2

CO2H

NH2

CO2Me (Boc)2O

N(Boc)2

CO2Me

NHBoc

CO2H

HCl(g)

THF, reflux

K2CO3, MeOH

NH

O

O

NaOMe, MeOH

13 C. Hulme, M. Morrissette, F. Volz, and C. Burns, Tetrahedron Lett. 39, 1113 (1998).14 (a) C. Hulme, J. Peng, B. Louridas, P. Menard, P. Krolikowski, and N. V. Kumar,

Tetrahedron Lett. 39, 8047 (1998). (b) Note that MP-carbonate (Argonaut Technologies)

was used to facilitate cyclization in the ketopiperazine series. (c) I. Ugi and A. V.

Zychlinski, Heterocycles 49, 29 (1998).15 T. Nixey, P. Tempest, and C. Hulme, Tetrahedron Lett. 43, 1637 (2002).

N

N

O

O

R2

R1

NC

R4

R3

NN

O

O

R1

R3

R4

R2

NH

N

R3

O R2

O

R1

N

N

O R2

O

R1R3

R4

NO

N R1O

R2

R3

R4

NN

O O

R2R1

R3

UDC

UDC

UDC

UDCUDC

UDC

7

35

36

3738

39

NR1 R3

R5 R4

R240

Scheme 2. Examples of templates available from cyclohexenyl isonitrile and UDC

methodology.


respectively 36. Both monocyclic lactams 38 (from N-Boc-�-amino alde-hydes) and bicyclic-lactams 39, where postcondensation modification occursafter a tethered input has been used for the Ugi reaction, are readily access-ible.16 Additionally, if no internal nucleophile is present, the munchnonemay still be trapped by an external dipolarophile to yield polysubstitutedpyrroles, 4017 (Scheme 2).

The convertible isocyanide also enables transformation of the second-ary amide in the Ugi product to a carboxylic acid, ester, or thioester, whichis thus amenable to further functionalization. The aforementionedtemplates are all readily accessible via manufacture in 96-well plates using96-well plate liquid handlers. The initial condensations are optimal withexcess aldehyde (2 equiv.), which can be subsequently removed via asimple scavenging and filtration step with PS-TsNHNH2.18 Several ‘‘uni-versal’’ resin-bound isocyanides have also been developed to exploitUDC methodology for the generation of the above heterocyclic products.

16 C. Hulme, L. Ma, J. Romano, M. P. Cherrier, J. Salvino, and R. Labaudiniere, Tetrahedron

Lett. 41, 1889 (2000).17 (a) T. A. Keating and R. W. Armstrong, J. Am. Chem. Soc. 118, 2574 (1996). (b) A. M. M.

Mjalli, S. Sarshar, and T. J. Baiga, Tetrahedron Lett. 37, 2943 (1996).18 PS-TsNHNH2 was purchased from Argonaut Technologies, Foster City, CA.

Fig. 3. Examples of immobilized convertible isonitriles.


Piscopio and co-workers19 originally produced an immobilized version ofcyclohexenyl isocyanide, 41, and Ugi and co-workers20 utilized a solution-phase carbonate convertible isocyanide, recently extended to solid phaseby Kennedy et al. 43.21 A universal rink isocyanide resin has also beenreported by workers at Proctor and Gamble.22 Of particular note is thesafety-catch linker isocyanide resin 44, developed at Rhone-Poulenc Rorer(now Aventis), which releases the multicomponent adduct through N-Bocactivation (i.e., the safety catch) and subsequent hydrolysis, or esterifica-tion, of the amide carbonyl (Fig. 3).23 This allows the generation of amethyl ester, which can be further manipulated in solution to give the rangeof heterocycles accessible via cyclohexenyl isocyanide (Scheme 3). Themethoxide safety-catch clipping strategy and subsequent solution-phase cy-clization offer similar advantages to a traceless linker as no functionalityderived from clipping remains at the end of the synthetic protocol.24

The UDC concept can be further extended by application of ethylglyoxylate (a ‘‘convertible aldehyde’’). Simple reaction of 48 in the UgiMCR with N-Boc anthranilic acids, N-Boc-�-amino acids, mono-N-Bocdiamines, and mono-N-Boc phenylenediamines, followed by acid treat-ment and in some cases proton scavenging, affords 1,4-benzodiazepines49, diketopiperazines 50, ketopiperazines 51, and dihydroquinoxalinones52, respectively.25 Note that products differ from those obtained fromconvertible isocyanides in that they contain an additional exocyclic amide

19 J. F. Miller, K. Koch, and A. D. Piscopio, 214th ACS National Meeting, Las Vegas, Nevada,

ORGN-232 (1997).20 T. Lindhorst, H. Bock, and I. Ugi, Tetrahedron 55, 7411 (1999).21 A. L. Kennedy, A. M. Fryer, and J. A. Josey, Org. Lett. 4, 1167 (2002).22 J. J. Chen, A. Golebiowski, J. McClenaghan, S. R. Klopfenstein, and L. West, Tetrahedron

Lett. 42, 2269 (2001).23 (a) C. Hulme, J. Peng, G. Morton, J. M. Salvino, T. Herpin, and R. Labaudiniere,

Tetrahedron Lett. 39, 7227 (1998). (b) D. L. Flynn, R. E. Zelle, and P. A. Grieco, J. Org.

Chem. 48, 2424 (1983).24 M. J. Plunkett and J. A. Ellman, J. Org. Chem. 60, 6006 (1995).25 C. Hulme and M. P. Cherrier, Tetrahedron Lett. 40, 5295 (1999).

Scheme 3. Applications of a safety-catch isonitrile resin in UDC methodology.

N

N

O

O

R2

R4

R3

NN

O

OR3

R4

R2

NH

N

R3

O R2

ON

N

O R2

OR3

R4

O

O

O

O

HN R1

HN

O

R1

O

NH

R1

O

NH

R1

UDC UDC

UDCUDC

48

49 50

5152

Scheme 4. Accessible templates via use of ethyl glyoxalate.


group. Representative examples, 53 through 60, with A% purities as judgedby ELS (evaporative light scattering detection) are shown in Fig. 4.

Workers at RPR put several of these chemistries into full production.Of particular note was the preparation of a 12,480-member diketopipera-zine library via the aforementioned solution phase approach and producedas a 30 (RNH2) � 16 (RNC) � 26 (N-Boc-�-amino acid) full array. Detailsreport a 2-week production time (1 FTE) with four wells/plate analyzed(624 samples) using a selection algorithm that repeated every six platesto ensure a set of representative compounds from the full library. Theoverall purity distribution is excellent (Fig. 5). Reported physicochemicalproperties of the collection were also favorable, showing a 66% pass ratefor Lipinski’s ‘‘rule of five’’ and 80% of the library possessed a PSA (polarsurface area) value between 50 and 140 A2 suggesting good permeabilityproperties.26 Also of note from reagent selection is that 60% of the librarymembers contained at least one or more acid or basic functional groupsthrough R1–R4. These are of particular value for the discovery of

26 J. Kelder, P. D. J. Grootenhuis, D. M. Bayada, L. P. C. Delbressine, and J.-P. Ploemen,

Pharm. Res. 16, 1514 (1999).

0

20

40

60

80

% o

f sa

mpl

es

0-25% 26-50% 51-75% 76-100%

Purity ranges (ELS)

Fig. 5. Purity distribution for 12,480 diketopiperazines.

NH

N

O

O

HNN

O

O

NH

N

O

ONH

N

O

O

O

HN

HN

O

O

NH

O

NH

NH

NO

O

O

HNCl N

NO

O

O

HN

HNN

O

O

HN

O ONN

NH

N

O

O

O

NH

60% 63% 100%

70% 100% 100% 20%

53 54 55 56

57 58 59 60

82%

Fig. 4. Representative examples of BDPs and diketopiperazines from ethylglyoxalate

employing UDC methodology.


high-affinity ligands where charge-reinforced H-bonds often play pivotalroles.27 The ability to introduce acids and bases is compatible with theactual cyclization step, where simple selection of reagents, unable to com-pete with the Boc-protected internal nucleophile for cyclization, yet con-taining tethered Boc-protected amines or t-butyl esters, are unmaskedduring the synthetic protocol. Twelve of the amino acids used wererevealed and included tyrosine, histidine, proline, aspartic acid (t-Bu pro-tected), asparagine, lysine (Boc protected), methionine, phenylalanine,valine, glycine, and alanine.

Repositioning the electrophilic carbonyl tethered to the primary amine61 to use in the initial condensation also adds to the number of potential

27 A. M. Davis and S. J. Teague, Angew. Chem. Int. Ed. Engl. 38, 736 (1999).

Scheme 6. Preparation of diketomorpholines.

Scheme 5. BDPs and DKPs from immobilized �-amino acids. Reagents and conditions: (i)

R1CHO (3 equiv.), 61 (3 equiv., Wang resin), R3NC (3 equiv.), R4CO2H ¼ N-Boc anthranilic

acid (3 equiv.), all 0.5 M solutions (MeOH/CH2Cl2, 1:1), RT 24 h. Wash resin CH2Cl2 (�3),

MeOH (�3). (ii) R1CHO ¼ N-Boc-�-amino-aldehyde (3 equiv.), R3NC (3 equiv.), R4CO2H

(3 equiv.), 61 (3 equiv., hydroxymethyl resin) all 0.5 M solutions (MeOH/CH2Cl2, 1:1). Wash

resin CH2Cl2 (�3), MeOH (�3). (iii) Ten percent TFA in CH2Cl2, wash resin CH2Cl2 (�2).


sites for an appropriately positioned amino internal nucleophile to attackand has been reported by Hulme and co-workers (Scheme 5).28

Note that the benzodiazepines 62, are accessible via Wang resin, eitherby direct cyclocleavage or cyclization onto the clipped carboxylic acid.However, access to the ketopiperazines 63 is higher yielding utilizing hy-droxymethyl resin as opposed to Wang, and mechanistically results onlyfrom direct cyclocleavage. Interestingly, Szardenings et al.29 have alsoreported this methodology and successfully extended it to the synthesis ofdiketomorpholines 64 (Scheme 6). As such these heterocycles are versatilesynthetic precursors to optically active �-hydroxy acids,30 however, onlylimited reports of biological utility exist.31

28 C. Hulme, L. Ma, and R. Labaudiniere, Tetrahedron Lett. 41, 1509 (2000).29 A. K. Szardenings, T. S. Burkoth, H. H. Lu, D. W. Tien, and D. A. Campbell, Tetrahedron

53, 6573 (1997).30 G. Frater, U. Muller, and W. Gunther, Tetrahedron Lett. 22, 4221 (1981).31 M. S. Iyer, K. M. Gigstad, N. D. Namdev, and M. Lipton, J. Am. Chem. Soc. 118, 4910

(1996).

O NO

R3

HN

O

R2

HN R4

R5

O NO

R3

HN

O

R2

R1

OR3

HN

O

O N

R1

NH

R4

R5

R1 = NBoc

R5R4

R2 = NR5

Boc

R4

N

NO

O

R2

R1

R3N

NO

O

HN R3

R4

R2

R5

N N

HN

R3O

R1R5

R4

O

65

66 68 67

Scheme 7. Ureas and hydantoins from postcondensation modifications of the Ugi-5CR.

Reagents and conditions: (i) Ten percent trifluoroacetic acid in dichloroethane. (ii) Na2CO3

(sat.). (iii) 1 N KOH in MeOH/THF/H2O, 3 days; then conc. HCl.


Alcohols are in equilibrium with their carbamic acid in the presence ofCO2 and thus employing the CO2/MeOH reagent combination (MeO-CO2H), coupled with a UDC strategy, affords access to a variety of bio-logically important heterocycles.32 Named the Ugi-5CR, this modificationof the Ugi reaction was originally reported in 1961 and only two furtherreports of this reaction have appeared since that date.33 Recent investiga-tions by Keating and Armstrong34 have, however, extended the scope ofthis condensation via the use of CS2 and COS as oxidized carbon sources.Thus, the Ugi-5CR condensation product 65, derived from N-Boc-�-aminoaldehydes and N-Boc diamines, followed by acid deprotection and basetreatment, affords the two cyclic ureas, 66 and 67, respectively (Scheme 7).

During the course of this work it was also found that simple treat-ment of the carbamate condensation product 65 with 1 M KOH gave thecorresponding hydantoin, 68. Reports of the biological utility of cyclicureas and hydantoins have appeared in several areas, including applica-tions as inhibitors of integrins and kinases.35 Representative examples ofeach core, 69 through 76, with isolated yields are shown in Fig. 6.

N-Boc-�-aminoaldehyde condensation products 78 are also precursorsfor nucleophilic attack via an internal nucleophile onto the carbonyl de-rived from the carboxylic acid of the classical Ugi adduct in an acid-catalyzed process. Thus, reaction in the Ugi followed by TFA treatmentand prolonged evaporation in a Savant or GeneVac evaporator (8 h)affords imidazolines 79 containing four potential points of diversity in good

32 C. Hulme, L. Ma, J. Romano, G. Morton, S.-Y. Tang, M. P. Cherrier, S. Choi, and

R. Labaudiniere, Tetrahedron Lett. 41, 1883 (2000).33 I. Ugi and C. Steinbruckner, Chem. Ber. 94, 2802 (1961).34 T. A. Keating and R. W. Armstrong, J. Org. Chem. 63, 867 (1998).

N

NO

ON

HN

O

O

HN

N N

HN

O

O

N

O

75%

N

NO

O

60%

N N

NO

O

74%N

N

38%

N

HN

O

O

HN

NN

38%

N

HN

O

O

HN

50%

N

HN

O

O

HN

O

NN

25%

S

< 10%

69 70 71 72

73 74 75 76

Fig. 6. Representative examples of ureas and integrins.


yield (Scheme 8).36 Any uncyclized material may be removed by one-potscavenging with PS-trisamine and PS-NCO.37 This may be furtherextended by utilizing N-Boc-protected phenylene diamines 80 to givearrays of benzimidazoles 82 via a solution phase procedure in excellentoverall yield (Scheme 9).38 Both represent examples of UDC methodologyand have been extended to scale production runs (>10 K) by workers atboth Amgen and RPR.

Imidazolines have been shown to have biological utility as antidepres-sants, and imidazoline ligands are known for a number of receptorswidely distributed in both the peripheral and central nervous system.39

The imidazoline moiety has also been extensively studied as an amide bond

35 (a) K. Karabelas, M. Lepisto, and P. Sjo, World Patent WO9932483 (1999). (b) K. E. Miller,

J. F. Carpenter, and R. R. Brooks, Cardiovasc. Drugs Ther. 12, 83 (1998). (c) A. E. Busch,

B. Eigenberger, N. K. Jurkiewicz, J. J. Salata, A. Pica, H. Suessbrich, and F. B. Lang, Br.

J. Pharmacol. 123, 23 (1998). (d) J. J. Edmunds, S. Klutchko, J. M. Hamby, A. M. Bunker,

C. J. C. Connolly, R. T. Winters, J. Quin, I. Sircar, and J. C. Hodges, J. Med. Chem. 38,

3759 (1995). For previous syntheses of hydantoins see (e) S. Hanessian and R.-Y.

Yang, Tetrahedron Lett. 37, 5835 (1996). (f) K. M. Short, B. W. Ching, and A. M. M.

Mjalli, Tetrahedron Lett. 37, 7489 (1996). (g) B. A. Dressman, L. A. Spangle, and S. W.

Kaldor, Tetrahedron Lett. 37, 937 (1996). (h) S. W. Kim, S. Y. Ahn, J. S. Koh, J. H. Lee,

S. Ro, and H. Y. Cho, Tetrahedron Lett. 38, 4603 (1997).36 C. Hulme, M. Morrissette, and L. Ma, Tetrahedron Lett. 40, 7925 (1999).37 PS-NCO was purchased from Argonaut Technologies, Foster City, California.38 P. Tempest, M. Kelly, and C. Hulme, Tetrahedron Lett. 42, 4959 (2001).39 (a) M. Pigini, P. Bousquet, A. Carotti, M. Dontenwill, M. Gianella, R. Moriconi, A.

Piergentili, W. Quaglia, S. K. Tayebati, and L. Brasili, Bioorg. Med. Chem. 5, 833 (1997). (b)

M. Harfenist, D. J. Heuser, C. T. Joyner, J. F. Batchelor, and H. L. White, J. Med. Chem. 39,

1857 (1996). (c) H. C. Jackson, I. J. Griffin, and D. J. Nutt, Br. J. Pharmacol. 104, 258

(1991). (d) E. Tibirica, J. Feldman, C. Mermet, F. Gonon, and P. Bousquet, J. Pharmacol.

134, 1 (1987).

R2

NH2

CO2HR1

NCR4

MeOH

R2

N

OR1

O

HN

R4

TFA/DCM

77 78 79

U DC

CHO

R3

HN R3N

NO

HN

R4

R2

R1

R3

BocHN

Boc

Scheme 8. Formation of imidazolines via UDC methodology. Reagents and conditions: (i)

77 (2 equiv.), R1CO2H, R2NH2, R4NC, RT, 48 h. (ii) PS-tosylhydrazine (3 equiv.), PS-N-

methylmorpholine (3 equiv.). THF: CH2Cl2, 24 h. (iii) Thirty percent TFA/CH2Cl2, 12 h.

NH2 HN Boc

CHO

R3

CO2H

R1

R2

NC

R4

MeOHN

HNBoc

R2

O

R1

O

HN

R4

R3TFA/DCM

R2

N

NR1

R3

HN R4

O80 81 82

U DC

Scheme 9. Formation of benzimidazoles via UDC methodology. Reagents and conditions:

(i) R3CHO (2 equiv.), R1CO2H, 80, R4NC, RT, 48 h. (ii) PS-tosylhydrazine (3 equiv.), PS-N-

methylmorpholine (3 equiv.). THF: CH2Cl2, 24 h. (iii) Thirty percent TFA/CH2Cl2, 12 h.


replacement in biologically active peptides.40 Benzimidazoles have beenshown to exhibit a wide range of biological function, including utility asFactor Xa inhibitors, NPY 1 antagonists, and proton pump inhibitors.41

Clearly rapid access to large numbers of these classes of molecule is ofmajor significance for new lead generation in the pharmaceutical sector.In a recent report, Nixey et al.42 combined glyoxylic acids with N-Boc phe-nylenediamines 80 and supporting reagents to give the condensation prod-uct 83. TFA treatment gave the quinoxalinone, 84, containing fourdiversity inputs generally in good to excellent yield (Scheme 10). Qui-noxalinones have been shown to exhibit a wide range of biological func-tions, including utility as kinase inhibitors and GABAA receptor agonistsacting through the BDP-binding site.43 Four representative examples areshown in Fig. 7. The methodology was reported to be compatible with

40 (a) I. Gilbert, D. C. Rees, and R. S. Richardson, Tetrahedron Lett. 32, 2277 (1991). (b) R. C.

F. Jones and G. J. Ward, Tetrahedron Lett. 29, 3853 (1988).41 (a) Z. Zhao, D. Arnaiz, B. Griedel, S. Sakata, J. Dallas, M. Whitlow, L. Trinh, J. Post,

A. Liang, M. Morrissey, and K. Shaw, Bioorg. Med. Chem. Lett. 10, 963 (2000). (b)

H. Zarrinmayeh, A. Nunes, P. Ornstein, D. Zimmerman, B. Arnold, D. Schober,

S. Gackenheimer, R. Bruns, P. Hipskind, T. Britton, B. Cantrell, and D. Gehlert, J. Med.

Chem. 41, 2709 (1998). (c) J. Horn, Clin. Ther. 22, 266 (2000).42 T. Nixey, P. Tempest, and C. Hulme, Tetrahedron Lett. 43, 1637 (2002).43 K. Masumoto, 118th Annual Meeting of the Pharmacological Society of Japan, March

31–April 12, Kyoto, 1998, Abstr. 01(XD) 10–1.

0

20

40

60

80

% o

f sa

mpl

es

0-25% 26-50% 51-75% 76-100%

Purity ranges (ELS)

Fig. 8. Purity distribution after production of 96 quinoxalinones.

NH2 HN Boc

CHO

R3

CO2H

R2

NC

R4

MeOHNH

N

HN

Boc

R2

O

O

R4

R3TFA/DCM

80 83

U DC

O R1

R1

OR2

84

N

N

O

R1

R3HN

O

R4

(i) (ii)

Scheme 10. Formation of quinoxalinones via UDC methodology.

86 87 8885

N

N

HN

O

N

N

HN

O

N

N

HN

O

N

N

HN

ONO

O

O O O O

N

HO

Fig. 7. Representative examples of quinoxalinones.


96-well production and the purity distribution for a plate of compounds isshown in Fig. 8.44

Miscellaneous Postcondensation Modifications

TMSN3 Modified Ugi Reactions

Originally reported in 1961,8 the TMSN3-modified Ugi reaction in-volves condensation of an appropriately substituted aldehyde or ketonewith a primary or secondary amine. Reaction of the Schiff base with an

44 LC/MS analysis was performed using a C18 Hypersil BDS 3-�m 2.1 � 50 mm column (UV

220 nm) with a mobile phase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to

100% over 5 min.

R1 CHO R2 NH2

N

CNO

R3

O

TMSN3

NN

N

NR1

NHR2

CO2R3

N

N

R1

N

N

N

N

CO2R3

R2

HCl

89 90 91

Scheme 11. Bicyclic tetrazoles via the TMSN3-modified Ugi reaction.

O

O NN

NN

NR5

HN R6

R4

Boc

R3HN

R6

Boc

O

O

R3

NCR4R5NH

O

O NN

NN

NHR4 R4

R3R1 R1

R2R2

R3

R1 R2

O O

O N NR4

R3R1

R2

(ii), (iii), (iv)

TMSN3

(i)

(i)(v)

R1= R2 = H

R5 = H

+

N3−

R5U

DC

92

93

95

C

NN

N N

NR5

R6

R4

HN

R3

O

N NNNN

O

94

Scheme 12. Synthetic routes to fused 6,5-ketopiperazine-tetrazoles and 7,5-azepine-

tetrazoles. Reagents and conditions: (i) R1R2C ¼ O (1.5 equiv., 0.1 M in MeOH), TMSN3

(1 equiv., 0.1 M in MeOH), R3NH2 (1 equiv., 0.1 M in MeOH), 92 (1 equiv., 0.1 M in MeOH),

24 h, RT. (ii) 10% TFA in CH2Cl2. (iii) PS-DIEA, DMF/dioxane, 1:1, reflux. (iv) PS-NCO,

PS-TsNHNH2 THF/DCE, 1:1. (v) Reflux, MeOH.


isocyanide and trapping of the intermediate nitrilium ion with azide,affords monocyclic tetrazoles 90 in good overall yield. The initial tetra-zole-forming reaction is particularly well suited for the solution-phasepreparation of monocyclic tetrazoles, and efficient enough to generate lib-raries with three points of diversity in the 10,000-member range. Bienaymewas the original pioneer in utilizing this version of the Ugi for combinator-ial and lead generation purposes. As such, a combination of the TMSN3-modified Ugi reaction with a subsequent Michael 1,4-addition eliminationprocess yields bicyclic tetrazoles in a two-step one-pot protocol using anisocyanide that contains a �-dimethylaminoacrylate moiety 89.45 Thus,highly elaborate and rigid molecules can be prepared directly from simpleprecursors (Scheme 11) using this methodology.

Workers at Amgen further exploited this reaction in an attempt to access6,5-fused ketopiperazine-tetrazoles 96 and 7,5-fused azepine-tetrazoles 94,respectively (Scheme 12). Thus, reaction of methyl-isocyanoacetates 92,

45 For an early application of the TMSN3-modified Ugi reaction producing fused tetrazoles

see H. Bienayme, Tetrahedron Lett. 39, 2735 (1998).

N

HN

N NN

O

N

84%97

N

HN

N NN

O

NNN

N

80%98

91%

NNN N

N O

100 89%

NNN N

N O

NN

101 72%102

NNN N

N O

75%99

N

N

N NN

O

NNN

N

Fig. 9. Representative examples of 7,5-azepine tetrazoles and 6,5-ketopiperazinetetrazoles.


N-Boc-�-aminoaldehydes, TMSN3, and secondary amines was found toproceed with high yield and final product purity (in most cases >70%A% as judged by LC/MS at UV 215 nm), giving the Ugi adduct 93. Acidtreatment, proton scavenging, and PS-NCO clean-up afforded arrays ofazepine-tetrazoles 94 and, as such, represent an example of UDC method-ology.46 Switching to a primary amine resulted in an appropriately pos-itioned internal nucleophile 95 for subsequent cyclization to fusedketopiperazine-tetrazoles 96.47 Representative examples (A% purities asjudged by LC/MS UV 220 nm) for both cores are shown in Fig. 9, andisolated yields are reported to be similar.

Postcondensation Passerini Reactions

Utilizing a postcondensation modification of the Passerini reaction,48

both Banfi and workers at Amgen49 recognized the potential for a one-pot, two-step transformation to produce nor-statines with the general struc-ture 104 containing three points of potential diversity. Recognizing their

46 T. Nixey, M. Kelly, D. Semin, and C. Hulme, Tetrahedron Lett. 43, 3681 (2002).47 T. Nixey, M. Kelly, and C. Hulme, Tetrahedron Lett. 41, 8729 (2000).48 (a) M. Passerini, Gazz. Chim. Ital. 51, 126 (1921). (b) M. Passerini, Gazz. Chim. Ital. 51, 181

(1921).49 (a) L. Banfi, G. Guanti, and R. Riva, Chem. Commun. 985 (2000). (b) W. Jones, S. Tadesse,

B. Chenera, V. Viswanadhan, and C. Hulme, A.C.S, 223nd American Chemical Society

Meeting & Exposition, April 2002, New Orleans.

NH CHO

R1

R2 NC

HN

O

NH

R2

OR1

O

R3

NH OH

NH

R2

OR1

R3 CO2H

i) ii)

iii)

O

R3

104103

Boc Boc

Scheme 13. Application of the Passerini reaction for the preparation of nor-statines.

Reagents and conditions: (i) 0.1 M solutions in MeOH, 18 h, RT, then PS-TsNHNH2 in

CH2Cl2. (ii) Ten percent trifluoroacetic acid (TFA) in CH2Cl2. (iii) PS-N-methylmorpholine

in CH2Cl2.


utility as known transition state mimetics for the inhibitors of aspartyl pro-teases,50 the Amgen group advanced the methodology to full productionreadiness and has reported the preparation of a 9600-member hit gener-ation library. A full array was produced with N-Boc-�-aminoaldehydes(8), isocyanides (20), and carboxylic acids (60) being employed in conjunc-tion with a variety of immobilized scavenger resins. The key step wassimple TFA treatment of the Passerini product 103 followed by protonscavenging to promote full acyl transfer to 104 (Scheme 13).

Primary screening interest for the set was the aspartyl protease�-secretase,51 one of two proteases that cleave the �-amyloid precursorprotein (APP) to produce �-amyloid peptide (A�) in the human brain, akey event in the pathogenesis of Alzheimer’s disease.52 No biological datahave yet been reported. However, representative examples and a full QCdistribution taken from a 10% random selection of the final library areshown in Figs. 10 and 11.

The methodology appears amenable to a range of functionality andamply demonstrates the efficiency of Passerini MCR methodology whencompared to analogous linear syntheses. Also note that the overall purityof the library (judged by LC/MS at UV 215 nm) is excellent, a reflectionof the short route and compatibility with solution-phase protocols enablingresin-bound removal of excess reagents.

50 (a) B. M. Dunn, ‘‘Structure and Function of the Aspartic Proteases: Genetics, Structures,

and Mechanisms,’’ Vol. 306, p. xviii. Plenum Press, New York, 1991. (b) K. Takahashi,

‘‘Aspartic Proteinases: Structure, Function, Biology, and Biomedical Implications.’’ Plenum

Press, New York, 1995. (c) C. E. Lee, E. K. Kick, and J. A. Ellman, J. Am. Chem. Soc. 120,

9735 (1998) and references therein.51 R. Vassar, B. Bennett, and S. Babu-Khan, Science 286, 735 (1999).52 (a) D. Selkoe, Trends Cell Biol. 8, 447 (1998). (b) D. Selkoe, Nature 399A, 23 (1999). (c) S.

Sinha and I. Lieberburg, Proc. Natl. Acad. Sci. USA 96, 11049 (1999).

0

20

40

60

80

% o

f sa

mpl

es

0-25% 25-50% 50-75% 75-100%

Purity ranges (UV 215)

Fig. 11. Purity distribution after production of 9600 nor-statines.

NH

O

OH

NH

O

NH

O O

OH

NH

O

ONH

O

OH

NH

OO

O

105 106 107

Fig. 10. Representative examples of nor-statines.


TMSN3-Modified Passerini Reaction

Continuing the role of MCRs in the generation of transition statemimetics and potential aspartyl protease inhibitors, the underusedTMSN3-modified version of the Passerini condensation was applied to pro-duce tetrazole-based cis-constrained nor-statine isosteres, 110 (mimeticsof a mimetic).53 Thus, replacing the standard carboxylic acid input of thePasserini with TMSN3 and combining with N-Boc-�-aminoaldehydes andisocyanides, tetrazoles 108 and 109 were produced. Treatment of thesilylated product, 108, with TBAF afforded 109 and acid deprotectionfollowed by N-capping gave the desired cis-constrained nor-statine-tetrazole mimetic 110 (Scheme 14). Earlier work by Abell and Foulds54

clearly demonstrated the importance of tetrazole-based cis-constrainedhydroxyethylamine isosteres as a new class of HIV-1 protease inhibitor,although the linear synthesis was lengthy and not well suited to theproduction of arrays.

Several representative examples of the initial condensation togetherwith isolated yields were reported (111 through 115; Fig. 12) and the pro-tocol was advanced to production readiness employing 80 resin-bound

53 T. Nixey and C. Hulme, Tetrahedron Lett. 43, 6833 (2002).54 A. D. Abell and G. J. Foulds, J. Chem. Soc. Perkin Trans. I 2475 (1997).

Scheme 14. Synthetic route to tetrazole analogs of nor-statines using the TMSN3-modified

Passerini reaction.

HN

NN

N

NOH

111

O

83% 112

HN

N NN

NOH

O

NH

76% 114

HN

N NN

NOH

O

S

80% 115

HN

N NN

NOH

ON

80%113

HN

N NN

NOH

O

70%

CN

Fig. 12. Representative examples of tetrazole-based nor-statine isosteres.


TFP (tetrafluorophenol) esters55 in a 96-well filter plate, encapsulated in aCalypso frame assembly. Final compound purities were improved (13% onaverage) by the addition of PS-NCO.

Automation

One attractive feature of the multicomponent reaction is the relativeease of its automation. Discrete reactions may be run in parallel by eithersolution- or solid-phase protocols in a standard 96-well format. Reagentdispensing is facile and rapid and may be performed with a range of com-mercially available automated 96-well of X, Y dispensers (Quadra 96,Rapid Plate, Hydra 96, Tecan Genesis, Gilson 215 etc.). Figure 13 showsthe Quadra 96 with six plates on deck, consisting of one plate with 8 rows,one with 10 columns, two open plates, the wash station (in black), and thefinal 96-well plate. The typical cycle time for one plate with 20 �mol ofproduct targeted is a mere 6 min. Also noteworthy are the commerciallyavailable Calypso reaction frame assemblies (Charybdis technologies)(Fig. 14) that, combined with 96-well plate polyfiltronic filter plates, allowautomation of a range of solid-phase syntheses and parallel purification

55 (a) J. Salvino, V. N. Kumar, E. Orton, J. Airey, T. Kiesow, K. Crawford, M. Rose,

P. Krolikowski, M. Drew, D. Engers, D. Krolinkowski, T. Herpin, M. Gardyan,

G. McGeehan, and R. Labaudiniere, J. Comb. Chem. 2, 691 (2000). (b) M. Drew,

E. Orton, P. Krolikowski, J. Salvino, and N. V. Kumar, J. Comb. Chem. 2, 8 (2000). (c)

W. Jones, D. Overland, L. Poppe, J. Cardenas, M. Pate, and C. Hulme, LabAutomation

2002, T002.

Fig. 13. Production ready Quadra96 with six modified plates on deck.

Fig. 14. Zymark rapid plate fitted with Calpyso frame assembley.


with immobilized scavenger resins. The blocks are ideally suited for usewith the Zymark Rapid Plate. Filtration of the reaction mix from suchscavenger resins is straightforward, as shown in Fig. 15. The operationmay be performed in one step via use of the polyfiltronic filter plate loaded

Fig. 15. Production ready Quadra96 with scavenger resin filtration setup.


on top of a new collection plate. Note that the majority of these automatedtools are conceptually simple and the range of operational conditions theytolerate can be restricting. When optimizing the chemistry of a specificcompound class, these constraints must be kept in mind. Hence, powerfulMCR transformations, which are often operationally friendly, offer aspecial bonus and should be considered with extra attention.

Conclusion

The chemistry of isocyanides began in the 1850s and was largelyignored until the discovery of the classical Passerini (1921)47 and Ugireactions (1959).8 Forty years have elapsed since those early days ofpeptide-like Ugi products to the more recent highly elaborate heterocycles,exemplified by Bienayme, Domling, Weber, Schreiber, Armstrong, Bossio,and others.56 IMCR methodologies have in fact now touched most stages

56 (a) R. W. Armstrong, S. D. Brown, T. A. Keating, and P. A. Tempest, in ‘‘Combinatorial

Chemistry. Synthesis and Application’’ (S. R. Wilson and A. W. Czarnik, eds.), p. 153. John

Wiley, New York, 1997. (b) C. Blackburn, B. Guan, P. Fleming, K. Shiosaki, and S. Tsai,

Tetrahedron Lett. 39, 3635 (1998). (c) H. Bienayme and K. Bouzid, Angew, Chem. Int. Ed.

Engl. 37, 2234 (1998). (c) R. Bossio, S. Marcaccini, P. Paoli, and R. Pepino, Synthesis 672

(1994). (d) S.-J. Park, G. Keum, S.-B. Kang, H.-Y. Koh, and Y. Kim, Tetrahedron Lett. 39,

7109 (1998). (e) A. Domling, Comb. Chem. High Throughput Screen. 1, 1 (1998). (f) S. L.

Schreiber, Science 287, 1964 (2000). (g) L. Weber, S. Wallbaum, C. Broger, and

K. Gubernator, Angew. Chem. Int. Ed. Engl. 34, 2280 (1995).


of the drug discovery process spanning lead discovery, lead optimization,and final drug manufacture. Examples of the latter include the preparationXylocain and, more recently, the HIV protease inhibitor Crixivan.57 Add-itional utility is now emerging for the preparation of natural product-likediversity libraries targeting protein–protein interactions encompassedby the emerging field of chemical genomics.58 What does the futurehold for isocyanide-based multicomponent reactions? With the advent offunctional proteomics delivering hundreds of new targets to drug discov-ery, ultra-high-throughput screening, and a premium on novel biologicallyactive entities, it seems reasonable to speculate that the discovery of newIMCRs will continue, spawning multiple postcondensation possibilitiesvia secondary reactions. Unions of IMCRs may also continue to receiveattention56e and more elaborate preparations of heterocycles amenablefor further diversification will no doubt be found.

In summary, isocyanide-based multicomponent reactions and subse-quent secondary reactions have experienced a resurgence of interest inthe past decade and appear well positioned for growth as we enter thenew millenium.


Reagents were obtained from commercial sources and used as re-ceived. N-Boc anthranilic acid 13 was purchased from Advanced Chem-Tech and N-Boc-methyl anthranilic acid 14 was purchased from Aldrich.Proton nuclear magnetic resonance (1H NMR) spectra were run at500 MHz. LC/MS analysis was performed using a C18 Hypersil BDS3-�m 2.1 � 50-mm column (UV 220 nm) with a mobile phase of 0.1%TFA in CH3CN/H2O, gradient from 10% CH3CN to 100% over 5 or15 min and APcI ionization.

Typical Experimental Procedure (Plate Production)

Stoichiometric amounts (0.1 mL) of 0.1 M solutions of the four Ugicomponents in methanol were combined in order of their participation inthe Ugi reaction (aldehyde first, amine second, isocyanide third, and thecarboxylic acid fourth) and shaken at room temperature for 20 h. Thereagents were dispensed into the 96-well plate using a Quadra 96 (Tomtec)multidispensing system. The solvent was then evaporated in vacuo in a

57 (a) K. Rossen, P. J. Pye, L. M. DiMichele, R. P. Volante, and P. J. Reider, Tetrahedron

Lett. 39, 6823 (1998). (b) K. Rossen, J. Sager, and L. M. DiMichele, Tetrahedron Lett. 38,

3183 (1997).58 S. L. Schreiber, Bioorg. Med. Chem. 6, 1127 (1998).


Savant evaporator at 65�

for 2 h. The Ugi products were then treated with a10% AcCl/MeOH solution (400 �l/well) and shaken overnight at roomtemperature. The solvent was then removed in vacuo at 65

�with a Savant

evaporator for 2 h. LC/MS analyses were performed on every well and A%yields of the desired product are reported in Fig. 5.

Typical Experimental Procedure (Scale Up)

Stoichiometric amounts (1.75 mL) of 0.1 M solutions of the fourUgi components were combined in order of their participation in the Ugireaction and stirred at room temperature for 20 h. The solvent was evap-orated in vacuo at 40

�and dried under high vacuum. A 10% solution

of AcCl in MeOH (7 mL) was added to the crude material and stirredat room temperature for 15 h. The solvent was then evaporated in vacuoat 40

�. The crude material was preabsorbed onto flash silica and puri-

fied by flash column chromatography (EtOAc:hexane) to yield the desi-red product. The following benzodiazepines were prepared via thisprocedure.

(R,S)-3-Isopropyl-4-(4-methoxybenzyl)-1,4-benzodiazepine-2,5-dione(8). See scale-up general procedure, isolated yield 81%. For major dia-stereomer only: 1H NMR (500 MHz, CDCl3, ppm): 8.9 (s, 1H), 7.99–8.01 (m, 1H), 7.41–7.45 (m, 1H), 7.31–7.33 (m, 2H), 7.21–7.24 (m, 1H),6.85–6.89 (m, 1H), 6.82–6.84 (m, 2H), 5.14–5.17 (m, 1H), 4.44–4.47(m, 1H), 3.74 (s, 3H), 3.62–3.65 (m, 1H), 1.65–1.70 (m, 1H), 0.82–0.83(m, 3H), 0.66–0.67 (m, 3H). For both diastereomers: 13C (125 MHz,CDCl3, ppm): 172.2, 166.3, 159.5, 134.9, 132.8, 131.7, 130.4, 128.8, 127.0,125.0, 120.0, 114.2, 71.3, 55.4, 54.9, 27.8, 19.8, 19.6.

(R,S)-4-Benzyl-3-(2-pyridyl)-1,4-benzodiazepine-2,5-dione (9). Seescale-up general procedure, isolated yield 32% of a 10:1 mixture of con-formers. For major diastereomer only: 1H NMR (500 MHz, CDCl3,ppm): 8.68 (1H, br s), 8.19–8.20 (1H, m), 7.68–7.70 (1H, m), 7.53–7.55(2H, m), 7.26–7.36 (4H, m), 7.14–7.17 (1H, m), 6.93–6.96 (1H, m), 6.86–6.89 (1H, m), 6.73–6.77 (1H, m). For major diastereomer only: 13C(125 MHz, CDCl3, ppm): 171.5, 167.2, 154.0, 148.7, 136.4, 136.2, 134.7,132.1, 131.0, 129.2, 128.9, 128.2, 127.2, 124.5, 122.4, 120.2, 119.6.

(R,S)-3-Ethyl-4-hexyl-1,4-benzodiazepine-2,5-dione (10). See scale-upgeneral procedure, isolated yield 42% of a 2:1 mixture of conformers. Formajor diastereomer only: 1H NMR (500 MHz, CDCl3, ppm): 0.80–0.98(6H, m), 1.20–1.30 (6H, m), 1.50–1.70 (4H, m), 3.25–3.35 (1H, m), 3.90–3.94 (1H, m), 4.00–4.10 (1H, m), 6.90–6.95 (1H, m), 7.2–7.25 (1H, m),7.40–7.5 (1H, m), 7.90–7.96 (1H, m), 8.60 (1H, br s). For both diastereo-mers: 13C (125 MHz, CDCl3, ppm): 172.4, 172, 171.7, 168.2, 165.6, 135.6,


134.49, 132.3, 132.1, 131.7, 131.4, 128.0, 127.1, 125.2, 124.8, 120.3, 119.7,66.5, 57.0, 52.3, 51.7, 42.5, 31.5, 28.6, 28.0, 26.6, 26.4, 22.7, 22.5, 19.6, 14.0,11.2, 10.8.

(R,S)-4-Isobutyl-3-(2-phenylethyl)-1,4-benzodiazepine-2,5-dione (11).See scale-up general procedure, isolated yield 79% of a 2:1 mixture of con-formers. 1H NMR (500 MHz, CDCl3, ppm): 0.88–0.89 (3H, m), 0.93–0.94(3H, m), 1.70–1.78 (1H, m), 1.85–1.90 (1H, m), 1.91–2.00 (1H, m), 2.50–2.62 (2H, m), 2.90–2.93 (1H, m), 3.96–4.04(2H, m), 6.98–7.01 (2H, m),7.1–7.3 (5H, m), 7.4–7.45 (1H, m), 7.95–8.0 (1H, m), 8.78(1H, s). For bothdiastereomers: 13C (125 MHz, CDCl3, ppm): 172.3, 171.8, 168.5, 166.0,140.4, 139.7, 135.6, 134.5, 132.5, 132.2, 131.7, 131.6, 128.5, 128.5, 128.4,128.3, 128.3, 127.7, 127.0, 126.4, 126.4, 126.2, 125.2, 125.0, 120.3, 119.8,117.3.

(R,S)-3-Hexyl-4-decyl-1,4-benzodiazepine-2,5-dione (12). See scale-upgeneral procedure, isolated yield 65% of a 2:1 mixture of conformers. Formajor diastereomer only: 1H NMR (500 MHz, CDCl3, ppm): 8.84 (1H, brs), 7.93–7.96 (1H, m), 7.41–7.45 (1H, m), 7.21–7.27 (1H, m), 6.95–6.99 (1H,m), 3.98–4.04 (2H, 2 � m), 3.29–3.33 (1H, m), 1.09–1.32 (26H, m), 0.87–0.89(6H, m). For both diastereomers: 13C (125 MHz, CDCl3, ppm): 172.7,172.2, 171.9, 168.2, 165.7, 135.6, 134.6, 132.3, 132.1, 131.6, 131.4, 130.3,127.9, 127.1, 125.1, 124.8, 120.3, 119.7, 117.4, 116.3, 65.1, 55.6, 52.3, 51.7,42.6, 31.9, 31.6, 31.5, 31.3, 29.6, 29.5, 29.3, 29.2, 29.1, 29.0, 28.6, 28.5, 28.1,27.0, 26.8, 26.6, 26.3, 26.1, 22.7, 22.6, 22.5, 22.4, 14.1, 14.0, 14.0, 13.9.

Diketopiperazines (50)

Typical Experimental Procedure (Scale Up). Stoichiometric amounts(6.2 mL) of 0.1 M methanolic solutions of the three supporting Ugicomponents and ethyl glyoxalate (7.75 ml) were combined and stirredat reflux overnight. The solvent was evaporated in vacuo and crude Ugiproduct dried under high vacuum. A 10% solution of AcCl in MeOH(25 mL) or a 10% solution of TFA (trifluoroacetic acid) in dichloroethane(25 mL) was added to the crude material and stirred at room temperatureovernight. The solvent was evaporated in vacuo. The crude material waspreadsorbed onto flash silica and purified by flash column chromatography(EtOAc:hexane, 1:4) to yield the desired product 57 (192 mg, 71%) as awhite solid.

Typical Experimental Procedure (Plate Production). Equal amounts(0.1 mL) of 0.1 M solutions in methanol of the four components areemployed generating a theoretical 10 �mol of final product. Reagents weredispensed into a 96-well plate using either a Quadra 96 or Rapid Plate(Zymark) 96-well dispenser. The deprotection/cyclization steps were


performed using either a 10% solution of acetyl chloride in methanol or a10% solution of TFA in dichloroethane. Evaporations were performed at65

�in a Savant evaporator.1-Benzyl-5-(1-benzyl-1H-imidazol-4-ylmethyl)-3,6-dioxo-piperazine-

2-carboxylic Acid Cyclohexylamide (109). For major diastereomer: 1HNMR (500 MHz, CD3OD, ppm): 8.91 (s, 1H), 7.32–7.44, 7.23–7.32,7.11–7.24 (3 � m, 12H), 5.36–5.39 (m, 2H), 4.77–4.93 (m, 2H), 4.68–4.70,4.36–4.38 (m, 1H), 4.03–4.13 (m, 1H), 3.11–3.55 (m, 2H), 3.15–3.21 (m,1H), 1.60–1.79, 1.10–1.29 (2 � m, 10H). 13C (125 MHz, CDCl3, ppm):168.68, 167.61, 166.65, 166.32, 166.28, 164.93, 136.88, 136.49, 136.38,136.08, 135.50, 135.37, 131.53, 131.53, 130.58, 130.55, 130.52, 130.43,130.42, 129.98, 129.95, 129.84, 129.78, 129.69, 129.59, 129.51, 129.26. M.spec. (TOF MS ESþ) 500 (MHþ). For minor diastereomer. 1H NMR(500 MHz, CD3OD, ppm): 8.92 (s, 1H), 7.39–7.44, 7.23–7.34, 7.11–7.13 (3� m, 12H), 5.36–5.39 (m, 2H), 4.77–4.93 (m, 2H), 4.68–4.70, 4.35–4.38 (m,1H), 4.03–4.13 (m, 1H), 3.37–3.55 (m, 2H), 3.14–3.18 (m, 1H), 1.58–1.93,1.11–1.29 (2 � m, 10H). 13C (125 MHz, CDCl3, ppm): 168.49, 167.45,166.52, 166.14, 164.77 136.73, 136.34, 136.25, 135.38, 135.25, 131.35,130.44, 130.37, 130.29, 129.84, 129.81, 129.70, 129.55, 129.45, 129.36,129.12, 121.92, 121.65, 56.09, 54.84, 53.90, 53.85, 50.66, 50.59, 50.06, 50.00,33.47, 33.40, 33.12, 32.15, 28.28, 26.50, 25.89, 25.82. M. spec. (TOF MSESþ) 500 (MHþ).

5-Benzo[b]thiophen-3-ylmethyl-3,6-dioxo-1-propyl-piperazine-2-carbo-xylic Acid Benzylamide (1). For major diastereomer: 1H NMR (500 MHz,CDCl3, ppm): 7.65–7.80 (s, 2H), 7.10–7.40 (3 � m, 7H), 6.95 (s, 1H), 6.5 (s,1H), 4.35–4.55 (m, 2H), 4.48 (s, 2H), 4.05–4.2 (m, 2H), 3.7–3.9(m, 2H), 3.2–3.6 (m, 2H), 2.8–2.9 (m, 2H), 1.4–1.6 (m, 2H), 1.2–1.3 (m, 2H), 0.8–0.9 (m,3H). 13C (125 MHz, CDCl3, ppm): 165.8, 164.8, 163.9, 140.5, 138.2, 137.5,130.7, 128.7, 127.6, 124.7, 124.4, 124.3, 122.8, 121.6, 64.1, 55.9, 48.2, 44.1,34.4, 20.1, 11.2. For minor diastereomer: 1H NMR (500 MHz, CDCl3,ppm): 7.75–7.9 (m, 2H), 7.35–7.45 (m, 2H), 7.1–7.3 (m, 5H), 6.78 (s, 1H),6.05 (s, 1H), 4.4–4.5 (2 � m, 3H), 3.8–4.0, 4.2–4.3 (2 � m, 3H), 2.85–2.95,3.05–3.15 (2 � m, 2H), 1.4–1.6 (m, 2H), 0.85–0.95 (m, 3H) 13C (125 MHz,CDCl3, ppm): 166.5, 166.5, 164.4, 140.8, 138.1, 137.1, 130.5, 128.7, 127.6,124.6, 123.1, 121.7, 65.2, 53.0, 48.2, 44.2, 31.3, 20.4, 11.2.

Azepine-tetrazoles (94)

Typical Experimental Procedure (Scale Up). The following procedurewas followed for the large-scale preparation of 99: Solutions of N-(tert-butoxycarbonyl)-d-prolinal (0.1 M, 10 ml in MeOH), 1-(2-pyrimidyl)piperazine (0.1 M, 10 ml in MeOH), methyl isocyanoacetate (0.1 M,


10 mL in MeOH), and TMSN3 (0.1 M, 10 mL in MeOH) were added to around-bottom flask and stirred at room temperature for 18 h. The solutionwas concentrated and the resulting oil was redissolved in 10% TFA/DCM.After an additional 18 h the solution was concentrated and PS-DIEA(3.54 mmol/g, 0.85 g, 3 mmol) was added to the oil followed by a solutionof DMF/dioxane (50%, 60 ml). The slurry was heated in a shaker-ovenat 80

�for 96 h, followed by filtration and evaporation of the solvent. The

oil was purified by flash column chromatography (2% MeOH/chloroform)to yield an off-white solid, 99 (267 mg, 75%). 1H (400 MHz, CDCl3): 8.28(2H, d, J ¼ 4.5 Hz), 6.50 (1H, dd, J ¼ 4.5, 4.5 Hz), 5.41 (1H, d, J ¼ 15.5 Hz),5.13 (1H, d, J ¼ 15.5 Hz), 4.37 (1H, m), 4.01 (1H, d, J ¼ 11.5 Hz), 3.85 (4H,m), 3.64 (1H, m), 3.52 (1H, m), 2.99 (2H, m), 2.56 (1H, m), 2.41 (3H, m),2.05 (2H, m). 13C (100 MHz, CDCl3): 163.9, 161.3, 157.7, 151.5, 110.1,63.0, 56.9, 52.5, 50.0, 47.2, 43.8, 30.7, 22.3. FTIR: 3272, 1633, 1150,636 cm�1. HRMS: MHþ theoretical value 356.1947: Actual value356.1952. dM/M ¼ 1.4 ppm.

Typical Experimental Procedure (Plate Production). Production of an80-member array was successfully completed using a Charybdis 96-wellTeflon block, encapsulated in a Calypso reaction frame assembly. Reagentswere transferred into the 96-well plate using either a Quadra 96 or RapidPlate 96. The blocks were then heated at 65

�for 3 days and the solvent

evaporated in vacuo at 65�. Scavenging with PS-TsNHNH2 (6 equiv.) and

PS-NCO (1 equiv.) was performed at the plate level and the resins wereadded using a Millipore column loader. Evaporation was performed in aSavant evaporator for 2 h.

Ketopiperazine Tetrazoles (96)

Typical Experimental Procedure (Scale Up). A mixture of phenpropio-naldehyde (0.1 M, 15 mL in MeOH), phenpropylamine (0.1 M, 15 mL inMeOH), methyl isocyanoacetate (0.1 M, 15 mL in MeOH), and TMSN3

(0.1 M, 15 mL in MeOH) was stirred at reflux for 48 h. LC/MS analysisafter 2 days revealed 72% (product at UV 220 nm, 9% acyclic) and 30%(product at UV 254 nm, 18% acyclic). The solvent was evaporated in vacuoand material dried under high vacuum for 1 h. The crude material wasredissolved in THF:DCM (1:1, 20 ml) and PS-NCO (300 mg) was added.The suspension was shaken for 15 h at room temperature. The resin wasfiltered, crude product preabsorbed onto flash silica, and purified bycolumn chromatography (EtOAc:hexane, 1:2) to yield 102 (321 mg, 60%)as an oil. 1H (400 MHz, CDCl3): 7.07–7.11, 7.17–7.20, 7.23–7.27 (10H, 3 �m, 2 � C6H5), 4.71–4.97 (2H, m, CH2CO), 4.85–4.86 (1H, m, CH), 4.07–4.14, 2.99–3.03 (2H, 2 � m, NCH2), 2.60–2.65 (3H, m, CH2CH2), 2.36–2.41


(2H, m, CH2), 1.89–2.02, 2.10–2.12 (3H, 2 � m, CH2CH2); 13C(100 MHz,CDCl3): 161.37, 150.31, 140.88, 139.13, 129.14, 128.82, 128.68, 128.37,127.11, 126.59, 53.92, 52.26, 48.12, 45.28, 35.20, 33.52, 30.19, 28.08. HMBC(heteronuclear multibond correlation) revealed connectivities between thetwo ring methine protons and one methide proton, confirming the cyclicstructure. HRMS: theoretical value 362.1981; actual value 362.2000. dM/M ¼ 5.24 ppm.

Nor-statines (104)

Typical Experimental Procedure (Plate Production). Production ofa 9600-member array was successfully completed in a 96-well format.N-Boc-�-amino aldehydes (0.2 M, 200 �L in MeOH), isonitrile (0.1 M,200 �L in MeOH), and carboxylic acids (0.1 M, 200 �L in MeOH) weredispensed using a Quadra 96 liquid dispenser. Each plate represented a1 (acid) � 8 (aldehyde) � 10 (isonitrile) array with one unique acid perplate. Plates were capped and agitated gently for 36 h. Solvent was evapor-ated in a Savant evaporator for 3 h on the high setting (60

�). PS-TsNHNH2

(>3 equiv., Argonaut) was added with a top resin loader. Crude mixtureswere then dissolved in 1:1 THF:DCE via use of a Rapid Plate 96(600 �L) and plates agitated for 12 h. Filtration was performed by transferof solutions to 96-well filter plates sat above new collection vessels usingthe Quadra 96. Solvent was then evaporated on high for 2 h. Then600 �L of 10% TFA:DCM was added to each well via the Quadra 96 andplates agitated for a further 12 h. Solvent was evaporated in a Savant evap-orator on high setting for 2 h. PS-N-methylmorpholine (>3 equiv.) wasadded to each well with a top resin loader and the crude material dissolvedin dichloroethane (600 �L) and plates agitated for 12 h. Filtration was per-formed by transfer of solutions to 96-well filter plates sat above new collec-tion vessels using the Quadra 96. Solvent was then evaporated on highfor 2 h. LC/MS analysis was performed using a C18 Hypersil BDS 3-�m2.1 � 50-mm column with a mobile phase of 0.1% TFA in CH3CN/H2O,gradient from 10% CH3CN to 100% over 15 min using APcI ionization.QC of four samples from each plate (480 compounds) was performed usinga repeating algorithmn. A% purities are reported in Fig. 8.

Tetrazole-nor-statine Mimetics (110)

Typical Experimental Procedure (Scale Up). Compound 109 (12 mg,0.036 mmol) was treated with TFA (50% in DCM) for 5 min, evaporated,and the oil was dissolved in DCM. MP-carbonate (3.15 mmol/g, 50 mg)was added and the mixture was shaken overnight, filtered, and evaporated.


The resulting amine was dissolved in DMF (1.2 mL) and was added to poly-mer bound 3-cyanobenzoate TFP ester (101 mg, 85 mmol/g, 0.085 mmol).The slurry was heated at 60

�for 16 h, filtered, and the DMF was removed

in vacuo. The resulting oil was purified by preparative HPLC to give 113 asan off-white solid (12 mg, 86% yield). 1H (400 MHz, CDCl3): 9.85 (1H, s),9.35 (1H, s), 9.12 (1H, m), 8.40 (1H, d, J ¼ 8.5 Hz), 8.18 (1H, d, J ¼ 8.5 Hz),8.08 (1H, dd, J ¼ 8.5, 8.5 Hz), 7.89 (1H, dd, J ¼ 8.5, 8.5 Hz), 7.35 (1H, dd, J¼ 7.5, 7.5 Hz), 7.21 (1H, d, J ¼ 7.5 Hz), 7.19 (1H, d, J ¼ 7.5 Hz), 4.98 (1H,m), 4.00 (2H, m), 1.99 (3H, s), 1.90 (3H, s). 13C (100 MHz, CDCl3): 157.5,155.5, 136.4, 135.1, 132.2, 130.9, 128.8, 128.6, 80.6, 64.4, 44.6, 28.2(3C), 17.5,17.3. FTIR: 3272, 1633, 1150, 636 cm�1. HRMS: MHþ theoretical value363.1565; actual value 363.1569. dM/M ¼ 1.1 ppm.

Typical Experimental Procedure (Plate Production). Production ofan 80-member array was successfully completed using a 96-well filter plateencapsulated in a Calypso reaction frame assembly. The assembly washeated at 60

�for 18 h, cooled, and the slurry was then filtered into a

collection plate and the solvent was evaporated in vacuo at 65�. Scavenging

with PS-NCO (1 equiv.) was performed at the plate level and the resinswere added using a Millipore column loader. LC/MS analysis was per-formed using a C18 Hypersil BDS 3-�m 2.1 � 50-mm column with a mobilephase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to 100%over 15 min.

[25] Mixture-Based Combinatorial Libraries:From Peptides and Peptidomimetics to Small

Molecule Acyclic and Heterocyclic Compounds

By Cornelia E. Hoesl, Adel Nefzi, John M. Ostresh,Yongping Yu, and Richard A. Houghten

Introduction

The recent emergence of combinatorial libraries made up of millionsof chemically diverse compounds has revolutionized the drug discoveryprocess. In contrast to the expectations of ‘‘rational drug design,’’ whichenables compounds to be designed based on a detailed understandingof molecular interactions, chemical library diversity allows both directde novo discovery of lead compounds, as well as enhancement of the activ-ity of existing compounds. Combinatorial chemistry has proven to be a



The resulting amine was dissolved in DMF (1.2 mL) and was added to poly-mer bound 3-cyanobenzoate TFP ester (101 mg, 85 mmol/g, 0.085 mmol).The slurry was heated at 60

�for 16 h, filtered, and the DMF was removed

in vacuo. The resulting oil was purified by preparative HPLC to give 113 asan off-white solid (12 mg, 86% yield). 1H (400 MHz, CDCl3): 9.85 (1H, s),9.35 (1H, s), 9.12 (1H, m), 8.40 (1H, d, J ¼ 8.5 Hz), 8.18 (1H, d, J ¼ 8.5 Hz),8.08 (1H, dd, J ¼ 8.5, 8.5 Hz), 7.89 (1H, dd, J ¼ 8.5, 8.5 Hz), 7.35 (1H, dd, J¼ 7.5, 7.5 Hz), 7.21 (1H, d, J ¼ 7.5 Hz), 7.19 (1H, d, J ¼ 7.5 Hz), 4.98 (1H,m), 4.00 (2H, m), 1.99 (3H, s), 1.90 (3H, s). 13C (100 MHz, CDCl3): 157.5,155.5, 136.4, 135.1, 132.2, 130.9, 128.8, 128.6, 80.6, 64.4, 44.6, 28.2(3C), 17.5,17.3. FTIR: 3272, 1633, 1150, 636 cm�1. HRMS: MHþ theoretical value363.1565; actual value 363.1569. dM/M ¼ 1.1 ppm.

Typical Experimental Procedure (Plate Production). Production ofan 80-member array was successfully completed using a 96-well filter plateencapsulated in a Calypso reaction frame assembly. The assembly washeated at 60

�for 18 h, cooled, and the slurry was then filtered into a

collection plate and the solvent was evaporated in vacuo at 65�. Scavenging

with PS-NCO (1 equiv.) was performed at the plate level and the resinswere added using a Millipore column loader. LC/MS analysis was per-formed using a C18 Hypersil BDS 3-�m 2.1 � 50-mm column with a mobilephase of 0.1% TFA in CH3CN/H2O, gradient from 10% CH3CN to 100%over 15 min.


[25] Mixture-Based Combinatorial Libraries:From Peptides and Peptidomimetics to Small

Molecule Acyclic and Heterocyclic Compounds

By Cornelia E. Hoesl, Adel Nefzi, John M. Ostresh,Yongping Yu, and Richard A. Houghten

Introduction

The recent emergence of combinatorial libraries made up of millionsof chemically diverse compounds has revolutionized the drug discoveryprocess. In contrast to the expectations of ‘‘rational drug design,’’ whichenables compounds to be designed based on a detailed understandingof molecular interactions, chemical library diversity allows both directde novo discovery of lead compounds, as well as enhancement of the activ-ity of existing compounds. Combinatorial chemistry has proven to be a



[25] mixture-based combinatorial libraries 497

powerful tool for providing important information for the fundamentalunderstanding of molecular recognition.1 In the postgenomic era, thelibrary approach can be extended to identify a small molecule partnerfor every gene product and furthermore to determine the function andbiological role of the gene.2

The key feature of combinatorial chemistry is the simultaneous synthe-sis of a large number of analogs under similar reaction conditions. Histor-ically, the concept was first developed for the synthesis of peptide librariesdue primarily to the fact that robust solid-phase chemistry3 was availablefor peptides. In the 1980s, various multiple synthesis techniques for therapid parallel synthesis of large numbers of peptides emerged includingthe synthesis on plastic pins,4 on glass chips,5 on paper,6 and in ‘‘tea-bags.’’7

Inspired by the multiple-synthesis techniques, Lam et al.8 and Houghtenet al.9 published simultaneously in 1991 the first practical and broadlyapplicable validations of the combinatorial library approach. Since then,a wide range of bioactive peptides10 has been identified by employingcombinatorial chemistry methods, including novel antibacterials,11 potentagonists and antagonists of opioid receptors,12 inhibitors of melittin’s

1 (a) J. Eichler and R. A. Houghten, Biochemistry 32, 11035 (1993). (b) C. Pinilla, J. R.

Appel, and R. A. Houghten, Biochem. J. 301, 847 (1994). (c) J. R. Appel, J. Buencamino,

R. A. Houghten, and C. Pinilla, Mol. Divers. 2, 29 (1996). (d) B. Hemmer, M. Bergelli,

C. Pinilla, R. A. Houghten, and R. Martin, Immunol. Today 19, 163 (1998).2 G. Dorman, P. Krajcsi, and F. Darvas, Curr. Drug Discov. 21 (October 2001). http://

www.currentdrugdiscovery.com.3 (a) R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). (b) R. B. Merrifield, Science 232,

341 (1986).4 H. M. Geysen, R. H. Meloen, and S. J. Barteling, Proc. Natl. Acad. Sci. USA 81, 3998

(1984).5 S. P. A. Fodor, R. J. Leighton, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas, Science 251,

767 (1991).6 R. Frank and R. Doring, Tetrahedron 44, 6031 (1988).7 R. A. Houghten, Proc. Natl. Acad. Sci. USA 82, 5131 (1985).8 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp,


Nature 354, 84 (1991).10 (a) R. A. Houghten, C. Pinilla, J. R. Appel, S. E. Blondelle, C. T. Dooley, J. Eichler,

A. Nefzi, and J. M. Ostresh, J. Med. Chem. 42, 3743 (1999). (b) R. E. Dolle, Mol. Divers. 3,199 (1998).

11 (a) S. E. Blondelle, E. Perez-Paya, and R. A. Houghten, Antimicrob. Agents Chemother. 40,

1067 (1996). (b) S. E. Blondelle, E. Takahashi, R. A. Houghten, and E. Perez-Paya,

Biochem. J. 313, 141 (1996).12 C. T. Dooley, N. N. Chung, P. W. Schiller, and R. A. Houghten, Proc. Natl. Acad. Sci. USA

90, 10811 (1993).


hemolytic activity, antigenic peptides recognized by monoclonal anti-bodies,13 and potent endothelin antagonists.10 Highly active chymotrypsininhibitors have also been found upon screening of a cyclic peptide templatecombinatorial library.14

In general, the peptide library approaches presented to date fall intothree broad categories. The first category represents synthetic approaches,in which peptide mixtures are synthesized, cleaved from the solid support,and the non-support-bound synthetic combinatorial library is assayed in so-lution, which allows each compound within the mixture to freely interactwith a given receptor.15 The focus of the research in our laboratory isdirected to the preparation, screening, and deconvolution of non-support-bound combinatorial libraries. The second category involves the chemicalsynthesis of peptide libraries, which are still attached to the solid supportduring the screening.8 Alternatively to chemical techniques, recombinantDNA methods, as the third category of peptide library preparation, havebeen successfully used to generate millions of peptides expressed rand-omly in a fusion phage or other vector system.16 This method, however,remains restricted to the 20 proteinogenic amino acids, whereas chem-ical approaches allow the incorporation of nonproteinogenic and non-natural amino acids as well as chemically modified amino acids and theircarboxylic acids.

Two different approaches, the ‘‘divide, couple and recombine’’ (DCR)method9 (also known as ‘‘split-resin’’ method8) and the reagent mixturemethod17 are widely used for the chemical generation of immense mixturesand will be explained here in detail.

The low oral bioavailability, the susceptibility to proteolytic break-down, and the inability to pass through the blood–brain barrier make pep-tides of less general utility as pharmaceutical leads as compared tononpeptidic compounds. Substituted heterocyclic compounds offer a highdegree of structural diversity and have proven to be broadly useful astherapeutic agents. In the last decade, the design and development of

13 (a) D. R. Burton, C. F. Barbas III, M. A. A. Persson, S. Koenig, R. M. Chanock, and R. A.

Lerner, Proc. Natl. Acad. Sci. USA 88, 10134 (1991). (b) C. Motti, M. Nuzzo, A. Meola,

G. Galfre, F. Felici, R. Cortese, A. Nicosia, and P. Monaci, Gene 146, 191 (1994).14 J. Eichler, A. W. Lucka, and R. A. Houghten, Peptide Res. 7, 300 (1994).15 C. Pinilla, J. R. Appel, P. Blanc, and R. A. Houghten, BioTechniques 13, 901 (1992).16 (a) S. E. Cwirla, W. A. Peters, R. W. Barrett, and W. J. Dower, Proc. Natl. Acad. Sci. USA

87, 6378 (1990). (b) J. K. Scott and G. P. Smith, Science 249, 386 (1990). (c) J. J. Devlin, L. C.

Panganiban, and P. E. Devlin, Science 249, 404 (1990). (d) M. B. Zwick, J. Q. Shen, and

J. Scott, Curr. Opin. Biotechnol. 9, 427 (1998).17 J. M. Ostresh, J. H. Winkle, V. T. Hamashin, and R. A. Houghten, Biopolymers 34, 1681

(1994).


strategies for the synthesis of individual and combinatorial libraries ofsmall molecules have become an area of intense research. Many well-known reactions of organic chemistry have been successfully accomplishedon solid supports, including nucleophilic aromatic substitution, condensa-tion reactions, cycloaddition reactions, reduction and oxidation reactions,organometallic reactions, olefin formation reactions, and multicomponentreactions.18 The solid-phase reaction repertoire is continuously growing.

Taking advantage of the large diversity afforded by peptide librariesand the well-understood chemistry that provides peptides and peptidomi-metics in excellent synthetic purity, one research focus in our group hasbeen on the development of methods to chemically transform existingresin-bound peptide libraries to libraries of peptidomimetics and acyclicand heterocyclic compounds. Termed the ‘‘libraries from libraries’’ con-cept,19 the postsynthetic chemical modification of peptide libraries leadsto combinatorial peptidomimetic libraries as well as acyclic and heterocyc-lic low-molecular-weight organic compound libraries having different phys-ical, chemical, and biological properties compared to the peptide librariesused as starting materials. Initial examples of this approach include the per-alkylation and/or exhaustive reduction of the amide bonds in peptidesyielding peptidomimetics and polyamines.20 Case studies illustrating thesynthesis of a range of heterocyclic compounds from resin-bound aminoacids, peptides, and modified resin-bound peptides are presented here.

Preparation of Mixture-Based Synthetic Combinatorial Libraries

Two major techniques are generally employed in solid-phase chemistryto generate mixture-based synthetic combinatorial libraries (SCL) of mil-lions of compounds. Multiple functionalities at diverse positions withinthe library are incorporated either by mixing multiple resins or by usingmixtures of incoming reagents.

Resin Mixtures

The ‘‘divide-couple-recombine’’ (DCR) method,9 also known as ‘‘split-resin’’ synthesis,8 involves the coupling of reactants to individual aliquotsof resin followed by thorough mixing of the resin portions. The resin is then

18 W. D. Bennett, in ‘‘Combinatorial Chemistry’’ (H. Fenniri, ed.), p. 139. Oxford University

Press, Oxford and New York, 2000.19 J. M. Ostresh, G. M. Husar, S. E. Blondelle, B. Dorner, P. A. Weber, and R. A. Houghten,

Proc. Natl. Acad. Sci. USA 91, 11138 (1994).20 J. M. Ostresh, C. C. Schoner, V. T. Hamashin, A. Nefzi, J.-P. Meyer, and R. A. Houghten,

J. Org. Chem. 63, 8622 (1998).


divided again for the next building block incorporation. Thereby, a one-bead one-compound library is generated. The procedure ensures that anapproximately equimolar representation of all individual compoundswithin the library is obtained. Due to the statistical distribution of beadsat each step, a crucial step is the determination of the appropriate amountof resin to be used in order to achieve a complete inclusion of all com-pounds in the library. The number of beads should be at least 10 timesthe theoretical number of compounds in the library to ensure that alllibrary components are present with statistical probability. In contrastto the ‘‘reagent-mixture’’ method, described below, the DCR method ismore labor and cost intensive, because the amount of resin and work in-creases proportionally with the number of building blocks incorporatedat a particular position. A more detailed presentation of the DCR method,including the experimental procedure, can be found in Ostresh et al.21

Reagent Mixtures

The ‘‘reagent mixture method’’ is an alternative way for the introduc-tion of mixture positions using a mixture of incoming building blocks thatare reacted with the resin-bound compounds. To ensure an equal distribu-tion of individual compounds within the library, the proportion of eachbuilding block in the reaction mixture is varied inversely to its reactionrate, i.e., the higher the reaction rate of a particular building block, thelower is the concentration of this building block in the reaction mixture.To determine the reaction rates and thereby the ratio of every buildingblock in the reagents mixture, a competition experiment is performed in-volving the reaction of an equimolar reagents mixture with the resin-boundmaterial and determination of the relative composition of the products byHPLC.* Since in solid-phase synthesis typically a large excess of incomingreagents is used, the reaction can be considered a pseudo-first-order reac-tion (i.e., the reaction rates of the incoming building blocks are independ-ent of the resin-bound compound with which they react). This was shown

21 J. M. Ostresh, S. E. Blondelle, B. Dorner, and R. A. Houghten, Methods Enzymol. 267, 220

(1996).* Abbreviations: Boc, t-butyloxycarbonyl; (COIm)2, 1,10-oxalyldiimidazole; DBU, 1,8-

diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DIC, N,N0-diisopropylcarbodii-

mide; DIEA, N,N0-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO,

dimethylsulfoxide; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, N-[(1H-benzotriazol-1-

yl)dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide;

HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography;

RP-HPLC, reverse-phase high-performance liquid chromatography; IPA, isopropyl alcohol;

MBHA, p-methylbenzhydrylamine; SPPS, solid-phase peptide synthesis; TFA, trifluoro-

acetic acid; THF, tetrahydrofuran.


for mixtures of incoming amino acids,17 aldehydes,22 and carboxylic acids23

reacting with resin-bound amino groups. It is important that the relativeratios of the incoming reagents are similar equal and show similar reactivityin the reaction with the resin-bound reagent (e.g., similar nucleophilicity,no significant steric hindrance). The use of reagent mixtures requiresa thorough knowledge of the mechanism and kinetics involved in the spe-cific reactions carried out. In the synthesis of peptides with great length,conformational effects that can alter the relative rates must be taken intoaccount. However, once the isokinetic ratios are determined under con-trolled reaction conditions, the reagent mixture concept offers the advan-tage that both defined and mixture position can be incorporated into amolecule at any given position. This is a requirement for the synthesis ofpositional scanning combinatorial libraries. By simply establishing the iso-kinetic ratio of amino acids in the coupling reaction with a resin-boundamino function and using the ‘‘reagent mixture’’ method, we have madea wide range of peptide libraries accessible. Furthermore, those librarieswere used as starting materials in the synthesis of a variety ofpeptidomimetic and small molecule libraries.

Libraries from Libraries: Generation of Peptidomimetic Libraries

Using the ‘‘libraries from libraries’’ concept,19 the diversity of existingcombinatorial libraries is leveraged through successive transformations ofthese libraries. Entirely new chemical diversities are generated by thechemical modification of existing libraries providing different compoundswith physical, chemical, and biological properties. Two requirements haveto be fulfilled for the chemical modification of an existing library: (1) onemust begin with a well-defined library, and (ii) one must use a chemical re-agent that can effectively alter chemical moieties, while leaving either all ofthe compound mixture on the resin or alternatively removing all of themixture from the resin (in the latter case, the employed reagents must beselectively removed from the cleaved mixture). The ‘‘libraries from librar-ies’’ approach was first demonstrated by the peralkylation of an existinghexapeptide library,19 since the integrity of the peptide library had beenwell established in earlier work. Modification of peptide libraries is highlydesirable due to the limitations of peptides as potential drugs (lack oforal activity, rapid breakdown by proteolytic enzymes, rapid clearance

22 J. M. Ostresh, C. C. Schoner, M. A. Giulianotti, M. J. Kurth, and R. A. Houghten, in

‘‘Peptides, Frontiers of Peptide Science, Proceedings of the 15th American Peptide

Symposium’’ (J. Tam and P. T. P. Kaumaya, eds.), p. 57. Kluwer, Dordrecht, The

Netherlands, 1999.23 J. M. Ostresh and R. A. Houghten, U. S. Patent US5856107 (1999).

Fig. 1. Libraries from libraries. Generation of peptidomimetic libraries by chemical

modification of an existing dipeptide library. (The same transformations were applied to

longer peptides.)


from circulation, and typical inability to pass through the blood–brain bar-rier to effect central nervous system activity). Indeed, the enzymatic sus-ceptibility of the prepared permethylated peptide library was very low.19

Figure 1 summarizes the chemical transformations that have been success-fully performed in the past 10 years to yield linear peptidomimetic andsmall molecule libraries. A vital modification is the exhaustive reductionof the peptide backbone leading to polyamines.24 Polyamines have beenshown to be pharmacologically interesting compounds. They are ideallysuited to bind to and then condense DNA,25 and multiple amine func-tionalities are common in drugs active within the central nervous system.In addition, the polyamine libraries were further transformed yieldingpeptidomimetic libraries such as N-terminally acylated polyamine libraries,peracylated polyamine libraries, and polyurea libraries. Furthermore, theresin-bound polyamines served as templates for the generation of differentclasses of azaheterocyclic compounds.

Chemistry Optimization

Generally, when developing a mixture-based synthetic combinatoriallibrary, reaction conditions for all the steps in the reaction scheme needto be optimized to achieve the widest possible breadth of diversity and

24 A. Nefzi, J. M. Ostresh, and R. A. Houghten, Tetrahedron 55, 335 (1999).25 D. R. Morris and L. J. Marton, in ‘‘Polyamines in Biology and Medicine,’’ p. 183. Marcel

Dekker, Inc., New York, 1981.


reproducibility. Variation of the reaction conditions using control com-pounds derived from the most reactive and least reactive building blocksis required. We usually determine the purity of the product by RP-HPLC.Within a single class of compounds, physical properties like absorbance orionization can vary widely. Thus, the detection method used should bebased on the particular structures being synthesized. After establishing thebest reaction conditions, every proposed building block should be testedbefore inclusion in the library and only those yielding acceptable productsunder the criteria of >80% yield and purity are considered for inclusion inthe library synthesis. The scale-up of the reaction needs to be investigated,since the library synthesis involves a much larger amount of resin and re-agents compared to the initial experiments. To determine the breadth ofthe synthetic approach, we vary the building blocks for the first position ofdiversity, while keeping the other positions fixed, followed by the variationof the second position, and so on. If adverse interactions between certainbuilding blocks are expected, additional controls must be included. Con-currently with the mixture-based library synthesis, the selected controlcompounds incorporating the different building blocks have to be preparedto verify the completeness and the reproducibility of all individual reactionsteps. Finally, mass spectral analysis of mixtures within the library is usedto confirm that the expected range of masses is present.

Solid-Phase Synthesis of Heterocyclic Compounds from Resin-BoundAmino Acids, Short Peptides, and Polyamines

A large number of drugs feature a heterocyclic component. Thus, thedesign, synthesis, and evaluation of heterocyclic libraries have rapidlybecome a major field of organic chemistry. Over the past decade, we havedeveloped synthetic routes to a wide range of different heterocyclesstarting from resin-bound amino acids, short peptides, and polyamines.

As an example of libraries made starting from resin-bound aminoacids, a mixture-based combinatorial library containing 16,000 differ-ent 2,3,5-trisubstituted 4H-imidazolones 1 was prepared using 40 differentamino acids, 20 different isothiocyanates, and 20 different amines (Fig. 2).26

The synthetic strategy involves the conversion of resin-bound amino acidsto resin-bound thioureas using isothiocyanates. Reaction of the thioureaswith HgCl2 and a wide range of primary and secondary amines gaveresin-bound guanidine intermediates. Under acidic cleavage conditions,cyclization yielded the imidazolones 1.

Using the DCR method, a mixture-based library of 95 mixtures of48 benzothiazepine-5-ones 2 (Fig. 2) was produced in eight synthetic

26 Y. Yu, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 3, 521 (2001).

Fig. 2. Solid-phase synthesis of heterocyclic compounds from resin-bound amino acids.


steps.27 2-Fluoro-5-nitrobenzoic acid was coupled to the resin-bound aminoacid N-�-Fmoc-l-cysteine by nucleophilic substitution of the fluorogroup forming a thioether functionality. The Fmoc group was cleavedfollowed by the reductive alkylation of the amine. Cyclization to nitro-benzothiazepine-5-ones was achieved by intramolecular amide bondformation of the resulting secondary amine function and the carboxylicacid moiety of the aromatic ring. Reduction with SnCl2, N-acylation ofthe resulting amino group and cleavage from the resin using HF/anisoleyielded compounds 2.

Starting from N-�-Fmoc-aspartic acid �-t-butylester, a wide range of1,3,4,7-tetrasubstituted perhydro-1,4-diazepine-2,5-diones 3 were synthe-sized (Fig. 2).28 After deprotection of the aspartic acid amino functionand reductive alkylation, a second amino acid was coupled and a secondreductive alkylation was carried out. Following tBu cleavage, the thermo-dynamically favorable coupling of the resulting secondary amine to the sidechain of aspartic acid was readily accomplished.

A wide range of 1,2,5-trisubstituted 4-imidazolidinones 4 (Fig. 2)29 wasprepared by a synthetic approach based on the formation of a reactiveadduct of benzotriazole and aldehyde.30 The N-[1-(benzotriazol-1-yl)alkyl]derivative from the �-amino group of a resin-bound amino acid under-went spontaneous intermolecular nucleophilic substitution with the

27 A. Nefzi, N. A. Ong, M. A. Giulianotti, J. M. Ostresh, and R. A. Houghten, Tetrahedron

Lett. 40, 4939 (1999).28 A. Nefzi, J. M. Ostresh, and R. A. Houghten, Tetrahedron Lett. 38, 4943 (1997).29 M. Rinnova, A. Vidal, A. Nefzi, and R. A. Houghten, J. Comb. Chem. 4, 209 (2002).30 A. R. Katrizky, S. Rachwal, and B. Rachwal, J. Chem. Soc. Perkin Trans. I 799 (1987).

Fig. 3. Solid-phase synthesis of hydantoin and thiohydantoin libraries from resin-bound

dipeptides.


nearest amide to form the five-membered 4-imidazolidinone ring in anonstereospecific manner.

A variety of heterocyclic compounds was generated starting fromresin-bound dipeptides. The reaction of the N-terminal amino groupof resin-bound dipeptides with carbonyldiimidazole or thiocarbonyldiimi-dazole gave intermediate isocyanates or thioisocyanates that furtherreacted intramolecularly to form the hydantoins 5 or thiohydantoins 6having two positions of diversity (Fig. 3). We have also synthesizedbranched hydantoins 7 (or thiohydantoins 8) starting from resin-boundorthogonally protected diamino acids (amino acids having amino groupin the side chain) (Fig. 3). Following coupling of a second amino acid andhydantoin (or thiohydantoin) formation, the side chain of the diamino acidwas deprotected and the free amino group was then either N-acylated withvarious carboxylic acids or reacted with isocyanates to urea moieties.Cleavage from the solid support by HF/anisole yielded the correspondinghydantoins 7 or thiohydantoins 8.31

To increase the number of diversities, the hydantoin (or thiohydantoin)formation reaction was performed starting from N-alkylated dipeptides(Fig. 3). In the last synthesis step, the hydantoin (or thiohydantoin) ringwas alkylated followed by the cleavage from the resin. Using 54 differentamino acids for the first position of diversity (R1), 60 different amino acidsfor the second position of diversity (R2), and four different alkylating

31 A. Nefzi, J. M. Ostresh, M. Giulianotti, and R. A. Houghten, Tetrahedron Lett. 39, 8199

(1998).

Fig. 4. Solid-phase synthesis of an indolepyridoimidazole library and an imidazo-

imidazolone library from resin-bound acylated dipeptides.


reagents (R3), two libraries (38,880 hydantoins 9 and 38,880 thiohydantoins10) were prepared.32

Starting from resin-bound N-acylated dipeptides 11 having tryptophanor a tryptophan analog as the C-terminal amino acid, a mixture-based com-binatorial library of 46,750 indolepyridoimidazoles 12 was synthesized bydouble cyclodehydration under Bischler–Napieralski conditions (POCl3,dioxane, 100

�) (Fig. 4).23 Twenty-two tryptophan analogs, 25 amino acids,

and 85 carboxylic acids were used for the library synthesis.The synthetic strategy was extended to the parallel synthesis of [3,5,7]-

1H-imidazo[1,5-a]imidazol-2(3H)-ones 13 starting from resin-boundN-acylated dipeptide 11 having various nonaromatic amino acids as theC-terminal amino acid (Fig. 4).33 The chloriminium ion, which is generatedas an intermediate from the peptidic amide bond using POCl3, is reactedwith the second amide group instead of an aromatic ring to form the ami-dine structure. A second generated chloriminium ion induced the forma-tion of the second heterocycle and, thereby, led to compounds 13. Avariety of reaction conditions were tried and the best results were obtainedusing 15 equiv. of freshly distilled POCl3 at 100

�in dioxane for 18 h.

Dehydration of monoacylated diamines using POCl3 yielded substi-tuted imidazolines 16 and 19 (Fig. 5). Selective acylation of the primaryamino group of the diamines 14 and 17 was successfully achieved using a

32 A. Nefzi, C. Dooley, J. M. Ostresh, and R. A. Houghten, Bioorg. Med. Chem. Lett. 8, 2273

(1998).33 Y. Yu, H. M. El Abdellaoui, J. M. Ostresh, and R. A. Houghten, Tetrahedron Lett. 42, 623

(2001).

Fig. 5. Solid-phase synthesis of imidazolines from resin-bound acylated diamines.


moderate excess of a wide range of carboxylic acids (5 equiv, 0.1 M inDMF) in the presence of HBTU and DIEA. Treatment of the amides 15and 18 with POCl3 in dioxane led to cyclodehydration of the resultingin situ-formed chloriminium intermediates to generate the resin-boundimidazolines.34

When the diamines 14 were acylated by 4-fluoro-3-nitrobenzoic acid,following dihydroimidazole formation, the fluoro and nitro group couldbe successfully utilized to construct a 2-alkylthiobenzimidazole, dihydro-quinoxalin-2,3-dione, and 2-iminobenzimidazole ring (Fig. 6).35 The fluorogroup of 21 was displaced by nucleophilic substitution with a primaryamine. The nitro group was reduced and the resulting o-dianilino com-pounds were treated with three different reagents to yield the biheterocyc-lic dihydroimidazole analogs 22, 23, and 24. Cyclization with cyanogenbromide (CNBr) generated the disubstituted dihydroimidazolyl 2-iminodi-hydrobenzimidazoles 22. Treatment with 1,10-oxalyldiimidazole (COIm)2

followed by N-alkylation using DBU and alkyl halides led to the trisubsti-tuted dihydroimidazolyl dihydroquinoxalin-2,3-diones 23. The reactionwith thiocarbonyldiimidazole gave dihydroimidazolyl dihydrobenzimida-zol-2-thiones, which were subsequently S-alkylated with alkyl halides inthe presence of a weak base (1-methylimidazole) yielding, after cleavagefrom the resin, the dihydroimidazolyl 2-alkylthiobenzimidazoles 24. Com-pounds 24 were easily oxidized to trisubstituted dihydroimidazolyl 2-alkyl-sulfonylbenzimidazoles using hydrogen peroxide under weakly basicconditions [1 M (NH4)2CO3 in 50% acetonitrile in water].

34 A. N. Acharya, J. M. Ostresh, and R. A. Houghten, J. Org. Chem. 66, 8673 (2001).35 A. N. Acharya, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 4, 214 (2002).

Fig. 6. Solid-phase synthesis of biheterocyclic dihydroimidazole derivatives from resin-

bound diamines.

Fig. 7. Solid-phase synthesis of 1,7-disubstituted-1,3,5-triazepane-2,4-diones and a

2-aryliminoimidazolidine library from resin-bound diamines.


Starting from resin-bound diamines 25, the parallel synthesis of 1,7-disubstituted 1,3,5-triazepane-2,4-diones 26 was carried out using phenylisocyanatoformate (Fig. 7).36 To avoid regioselectivity problems in thecyclization step, amino acids that generate reactive functionalities afterreduction (asparagine, glutamine, lysine) were not included in the R1

position.In an efficient one-pot reaction, diamines 25 were converted to 1,5-

disubstituted 2-aryliminoimidazolidines 27 via mercury(II)-activated tri-substituted thioureas using arylisothiocyanates, HgCl2, and triethylaminein DMF at room temperature (Fig. 7).37 A mixture-based combinatorial

36 Y. Yu, J. M. Ostresh, and R. A. Houghten, Org. Lett. 3, 2797 (2001).

Fig. 8. Solid-phase synthesis of a trisubstituted bicyclic guanidine library.

Fig. 9. Solid-phase synthesis of an urea-tethered bicyclic guanidine library from resin-

bound polyamines.


library containing 16,000 (40 R1 � 20 R2 � 20 R3) 1,5-disubstituted2-aryliminoimidazolidines 27 was synthesized.

Starting from resin-bound reduced acylated dipeptides, a mixture-basedcombinatorial library of 102,459 protonated bicyclic guanidines 30 was syn-thesized in the positional scanning format. Following exhaustive reductionof the resin-bound N-acylated dipeptides 28, the resin-bound triamines 29were treated with thiocarbonyldiimidazole to generate after HF cleavagethe trisubstituted bicyclic guanidines (Fig. 8).20 Amino acids, such as argin-ine or lysine, which yield reactive funtionalities following amide reduction,were not included in the library. Forty-nine amino acids for the firstvariable position of diversity, 51 amino acids for the second position, and41 carboxylic acids for the third position were found to yield the productin a purity greater than 80% after cyclization, thereby meeting the librarycriteria imposed.

To increase the amount of diversity, the solid-phase synthesis of anurea-linked library containing 47,600 bicyclic guanidines 33 was achievedstarting from a glutamine-containing resin-bound N-acylated dipeptide li-brary (Fig. 9).38 Following exhaustive reduction, the selective protectionof the primary amine with trityl yielded the resin-bound polyamines 31.Treatment of the three secondary amines with thiocarbonyldiimidazolegenerated the resin-bound bicyclic guanidines 32. After trityl deprotection,coupling of an amino acid, N-deprotection of the just-coupled aminoacid, isocyanate treatment, and final HF cleavage, the urea-linked bicyclicguanidines 33 were obtained.

37 Y. Yu, J. M. Ostresh, and R. A. Houghten, J. Org. Chem. 67, 3138 (2002).38 A. N. Acharya, A. Nefzi, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 3, 189 (2001).


Solid-Phase Synthesis of Bis-Heterocyclic Compounds from Resin-BoundAcylated and Nonacylated Polyamines

An efficient, practical solid-phase synthesis of a variety of bis-hetero-cyclic compounds was developed starting from resin-bound orthogonallyprotected lysine (Fig. 10). Tetraamines 36 were synthesized by exhaustivereduction of resin-bound tetraamides 35. Cyclization with different com-mercially available bifunctional reagents such as cyanogen bromide, thio-carbonyldiimidazole, carbonyldiimidazole, and oxalyldiimidazole yieldedthe corresponding bis-heterocyclic compounds bis-cyclic guanidines 37,39

bis-cyclic thioureas 38, bis-cyclic ureas 39, and bis-diketopiperazines 40,respectively.40 Reduction of compounds 40 led to bis-piperazines 41.

Fig. 10. Solid-phase synthesis of bis-heterocyclic libraries from resin-bound orthogonally

protected lysine.

Fig. 11. Solid-phase synthesis of bis-cyclic thioureas and bis-cyclic guanidines from resin-

bound reduced tripeptides.


Extending the above-mentioned cyclization reactions to other poly-amines, resin-bound tetraamines 42 from resin-bound tripeptides containingthree secondary amines and one terminal primary amine were treated withthiocarbonyldiimidazole41 or cyanogen bromide42 (Fig. 11). Kinetically,the primary amine reacts first with thiocarbonyldiimidazole (or cyanogenbromide), followed immediately by cyclization to form the energeticallyfavorable five-membered ring. The two remaining secondary amines thenfurther react to form the second cyclic thiourea (or cyclic guanidine). It isimportant to work at lower concentrations with small excesses of the re-agents to minimize the formation of undesired impurities most likely dueto cyclization between the two internal secondary amines. In support ofthe kinetic hypothesis, it was found that the reaction of resin-bound polya-mines containing four secondary amines with thiocarbonyldiimidazole ledto the formation of multiple products.

Parallel Synthesis of Combinatorial Libraries

As continuation and extension of the parallel synthesis and ‘‘librariesfrom libraries’’ concept, there is the simultaneous synthesis of various lib-raries (i.e., a resin-bound library is converted in parallel into several newlibraries employing different reagents). Figure 12 illustrates the strategy.The initial step is the preparation of the dipeptide library 45. Introductionof the benzyl group was achieved by selective N-alkylation of N-terminallytrityl-protected resin-bound amino acids in the presence of lithium t-butox-ide and benzyl bromide. As expected, the alkylation of the amide nitrogen

39 A. N. Acharya, J. M. Ostresh, and R. A. Houghten, J. Comb. Chem. 3, 578 (2001).40 A. Nefzi, M. A. Giulianotti, and R. A. Houghten, J. Comb. Chem. 3, 68 (2001).41 A. Nefzi, M. A. Giulianotti, N. A. Ong, and R. A. Houghten, Org. Lett. 2, 3349 (2000).42 A. N. Acharya, J. M. Ostresh, and R. A. Houghten, Tetrahedron 57, 9911 (2001).

Fig. 12. Parallel synthesis of combinatorial libraries of heterocycles via resin-bound

triamines.


next to the resin dramatically increased the acid sensitivity of the MBHAresin-bound amino acids, excluding the use of Boc-amino acids in anyfurther coupling step. Therefore, Fmoc-amino acids were used to obtainthe second position of diversity. Following Fmoc removal and N-acylation,exhaustive reduction of the amide bonds yielded the triamine library 46,which served as starting material for the parallel synthesis of five differentheterocyclic libraries employing various cyclization reagents. The reactionof 46 with carbonyldiimidazole or (thiocarbonyldiimidazole) led to cyclicureas 47 or (thioureas 48).43 Diazepinediones 49 were formed by cycli-zation using malonyl chloride. Treatment of library 46 with oxalyl-diimidazole afforded the 2,3-diketopiperazines 50.44 Following cyclizationwith oxalyldiimidazole, piperazines 51 were obtained by reduction ofthe oxamide moiety on the solid support. Ninety-six different building

43 A. Nefzi, J. M. Ostresh, J. -P. Meyer, and R. A. Houghten, Tetrahedron Lett. 38, 931 (1997).44 A. Nefzi, M. A. Giulianotti, and R. A. Houghten, Tetrahedron 56, 3319 (2000).


blocks (29 Boc-protected amino acids for the first position of diversity, 27Fmoc-protected amino acids for the second position of diversity, and 40carboxylic acids for the third position of diversity) were included for theparallel synthesis of libraries 47–51. For the parallel cyclization using fivedifferent cyclization reagents, 480 individual control compounds (96 � 5)needed to be prepared concurrently to the synthesis of the 480 mixtures(96 � 5) to ensure the completeness and reproducibility of the performedcyclization reactions. Mixtures, containing in total 156,600 structurally dif-ferent heterocycles, were prepared in an efficient, reliable, and fast mannerusing 960 ‘‘teabags.’’ Along the same lines, in addition to the N-benzylatedcompounds, the corresponding N-methylated compounds were synthesizedemploying the parallel synthesis of libraries.

Experimental Procedure for the Parallel Synthesis of HeterocyclicPositional Scanning Libraries 47 to 51

General Requirements for the Synthesis

BOC-amino acid derivatives, Fmoc-amino acid derivatives, HOBt, andHBTU were purchased from Calbiochem-Novabiochem Corp. (San Diego,CA), Bachem Bioscience Inc. (Philadelphia, PA), and Bachem California(Torrance, CA). MBHA resin, 1% divinylbenzene, 100–200 mesh,0.81 mmol/g substitution, was purchased from Chem Impex Intl. (WoodDale, IL). DIC was purchased from Chem Impex Intl., trifluoroacetic acid(TFA) from Halocarbon (River Edge, NJ), and hydrogen fluoride from AirProducts (San Marcos, CA). All other reagents and anhydrous solvents(DMSO and THF) were purchased from Aldrich Chemical Co. (Milwau-kee, WI). Resin packets (teabags) are made with polypropylene mesh(74 �m) using an impulse sealer. Teabags are numbered, filled with100 mg of MBHA resin (1% divinylbenzene, 100–200 mesh, 1 mmol/g sub-stitution) to each bag and sealed. For standard reactions, the teabags wereput into a polypropylene bottle, containing the reagents solution, and thebottle was shaken using a reciprocating shaker. Air-sensitive reactionswere performed either within a glovebox or, when heating was required,in a Pyrex resin kettle. Completeness of the coupling reactions was verifiedby the ninhydrin test.45

45 E. T. Kaiser, R. L. Colescott, C. D. Blossinger, and P. I. Cook, Anal. Biochem. 34, 595

(1970).


Coupling of Boc Amino Acids to MBHA Resin to Introduce the FirstPosition of Diversity

Standard SPPS (solid-phase peptide synthesis) methodology was used(6 equiv. of Boc amino acid or Boc amino acid mixture, 6 equiv. DIC/HOBt, 1 h in DMF). Twenty-nine Boc amino acids were coupled individu-ally to 145 resin-filled teabags (numbered 471–29, 481–29, 491–29, 501–29,511–29). A mixture, containing 29 Boc amino acids in a predetermined iso-kinetic ratio,17 was coupled to 335 resin-filled teabags (numbered 4730–96,4830–96, 4930–96, 5030–96, 5130–96). The teabags were pooled together, theBoc group was removed (55% TFA in DCM, 30 min), and the resin wasneutralized (5% DIEA in DCM).

Trityl Protection of the Resin-Bound Amino Acids

The resin-bound amino acids (480 teabags) were treated with 15 equiv.of trityl chloride (0.1 M in 90% DMF/10% DCM) containing 20 equiv. ofDIEA for 3 h. Following washes with DMF, the resin-filled teabags weredried overnight under high vacuum.

Alkylation of Trityl-Protected Resin-Bound Amino Acids

All manipulations were carried out under nitrogen atmosphere in aglovebox. Lithium t-butoxide (0.5 M, 20 equiv.) in THF was added to the480 teabags. After shaking for 15 min, the base solution was removed. Sixtyequivalents of benzyl bromide in DMSO was added and the teabags wereshaken for 2 h. The alkylation solution was removed. Deprotonation andalkylation were repeated three times. Following washes with DMF, IPA,and DCM, the resin packets were dried under high vacuum.

Removal of the Trityl Group

The trityl-protecting group was removed by treatment of the 480teabags with 2% TFA in DCM (twice for 10 min).

Coupling of Fmoc Amino Acids to Introduce the Second Positionof Diversity

Standard SPPS methodology was used (6 equiv. of Fmoc amino acid orFmoc amino acid mixture, 6 equiv. DIC/HOBt, 1 h in DMF). Twenty-seven Fmoc amino acids were coupled individually to 135 resin-filledteabags (numbered 4730–56, 4830–56, 4930–56, 5030–56, 5130–56). A mixture,containing 27 Fmoc amino acids in a predetermined isokinetic ratio,46

was coupled to 345 resin-filled teabags (numbered 471–29, 4757–96, 481–29,


4857–96, 491–29, 4957–96, 501–29, 5057–96, 511–29, 5157–96). The teabags werepooled together and the Fmoc group was removed (20% piperidine inDMF, 2 � 10 min), followed by washes with DMF and DCM.

N-Acylation to Introduce the Third Position of Diversity

Standard SPPS methodology was used (10 equiv. of carboxylic acid orcarboxylic acid mixture, 10 equiv. DIC/HOBt, 12 h in DMF). Forty carbox-ylic acids were coupled individually to 200 resin-filled teabags (numbered4757–96, 4857–96, 4957–96, 5057–96, 5157–96). A mixture, containing 40 car-boxylic acids in a predetermined isokinetic ratio,23 was coupled to 280resin-filled teabags (numbered 471–56, 481–56, 491–56, 501–56, 511–56). Theresin-filled teabags were dried overnight under high vacuum.

Exhaustive Reduction of the Amide Bond

All the teabags were put into a resin kettle under nitrogen. Fifteenequivalents of boric acid and 15 equiv. of trimethylborate were addedfollowed by the slow addition of 45 equiv. of borane (1 M in THF). Afterhydrogen production ceased, the reaction was heated at 65

�for 72 h. The

reaction solution was decanted and quenched by the slow addition ofmethanol. The resin was washed with methanol, THF, and piperidine.The polyamine–borane complex was disproportionated by overnight treat-ment (16 h) with piperidine at 65

�followed by washes with DMF, DCM,

and methanol.

Parallel Synthesis of Heterocyclic Libraries 47 to 51

The cyclization reactions were performed under nitrogen atmospherein a glovebox. All teabags were prewashed with anhydrous DCM prior tocyclization reactions. Five equivalents of carbonyldiimidazole (0.1 M in an-hydrous DCM) was added to 96 teabags (471–96) and the reaction wasshaken 16 h to afford, following washes with DCM and IPA and HF cleav-age, the resin-bound cyclic urea library 47. Synthesis of the resin-boundcyclic thiourea library 48 was achieved by treatment of 96 teabags(numbered 481–96) with 5 equiv. of thiocarbonyldiimidazole (0.1 M in an-hydrous DCM) for 16 h followed by washes with DCM and IPA and HFcleavage. Cyclization of 96 teabags (numbered 491–96) using 5 equiv. ofmalonyl chloride in anhydrous DCM for 16 h led, following HF cleavage,

46 J. Eichler, C. Pinilla, S. Chendra, J. R. Appel, and R. A. Houghten, in ‘‘Innovation and

Perspectives in Solid Phase Synthesis’’ (R. Epton, ed.), p. 33. Mayflower Worldwide

Limited, Birmingham, 1994.


to the resin-bound diazepinedione library 49. Teabags (192) (numbered501–96, 511–96) were treated with 5 equiv. of oxalyldiimidazole (0.1 M in an-hydrous DCM) for 16 h. Following washes with DCM and IPA, two sets ofthe resin-bound 2,3-diketopiperazine library 50 were obtained. One set wascleaved to generate the 2,3-diketopiperazine library 50. The second set(teabags, numbered 511–96) was reduced in the presence of BH3-THF asdescribed for the amide reduction. Following HF cleavage the resin-boundpiperazine library 51 was obtained.

Cleavage of Resin-Bound Heterocyclic Libraries from the MBHA Resin

To obtain the non-support-bound libraries 47 to 51, the heterocycliccompounds, still contained within their teabags, were cleaved for 7 h withhydrogen fluoride/anisole using a multivessel apparatus47 for simultaneouscleavage.

Simultaneous Synthesis of Control Teabags

To ensure the completeness and reproducibility of all reaction steps,concurrently to the library synthesis, 480 control teabags (numbered47C1–C96, 48C1–C96, 49C1–C96, 50C1–C96, 51C1–C96) were treated under thesame reaction conditions described above. To 145 teabags (47C1–C29,48C1–C29, 49C1–C29, 50C1–C29, 51C1–C29), the 29 Boc-amino acids were indi-vidually coupled as the first amino acid. Following trityl protection, alkyla-tion was carried out as described above. Fmoc-phenylalanine was coupledfollowed by N-acylation with phenylacetic acid. Teabags (135) (47C30–C56,48C30–C56, 49C30–C56, 50C30–C56, 51C30–C56) were treated with Boc-phenyl-alanine, trityl protected, and alkylated, followed by the second couplingof 27 individual Fmoc-amino acids and N-acylation with phenylacetic acid.N-Acylation of 200 teabags (47C57–C96, 48C57–C96, 49C57–C96, 50C57–C96,51C57–C96) was performed with 40 individual carboxylic acids after couplingof Boc-phenylalanine, trityl protection, alkylation, and coupling withFmoc-phenylalanine.

Conclusion

Employing the teabag approach for parallel synthesis, the reagent mix-ture method, and the ‘‘libraries from libraries’’ concept, a wide range of dif-ferent small molecule compounds was successfully prepared in ourlaboratory during the past decade. Starting from amino acids and short

47 R. A. Houghten, M. K. Bray, S. T. DeGraw, and C. J. Kirby, Int. J. Peptide Protein Res. 27,

673 (1986).

[26] substitutions on polymer-bound polyelectrophiles 517

peptides, new reactions for the solid-phase synthesis of heterocycles weredeveloped and optimized for library synthesis. Using the positional scan-ning technique, screening and deconvolution of the libraries led to theidentification of various biologically active compounds. The synthesis ofcombinatorial libraries has found broad acceptance in the field of medicinalchemistry since it greatly increases the likelihood of finding useful thera-peutic and diagnostic agents. In the emerging field of proteomics, combina-torial library synthesis has reached new importance as a tool for theidentification of new targets for drug development. The scope and versatil-ity of synthetic combinatorial libraries can be expected to further expandtheir significance for basic research and drug discovery.

[26] New Strategies for the Solid-Phase Synthesisof Highly Functionalized, Small Molecules:Sequential Nucleophilic Substitutions on

Polymer-Bound Polyelectrophiles

By Florencio Zaragoza

Introduction

For the fast identification of lead compounds for novel, small moleculeenzyme inhibitors or other ligands for proteins, the screening of large anddiverse arrays of compounds prepared on insoluble supports is one of themost efficient approaches.1–8 Parallel solid-phase synthesis has been foundto be particularly well suited for the preparation of such arrays of diversecompounds since multistep synthetic sequences on insoluble supports canbe conducted on fully automated synthesizers.

1 R. E. Dolle, Mol. Divers. 3, 199 (1998).2 J. L. Conroy, P. Abato, M. Ghosh, M. I. Austermuhle, M. R. Kiefer, and C. T. Seto,

Tetrahedron Lett. 39, 8253 (1998).3 M. Renil, M. Ferreras, J. M. Delaisse, N. T. Foged, and M. Meldal, J. Peptide Sci. 4, 195

(1998).4 J. C. Spetzler, V. Westphal, J. R. Winther, and M. Meldal, J. Peptide Sci. 4, 128 (1998).5 T. S. Haque, A. G. Skillman, C. E. Lee, H. Habashita, I. Y. Gluzman, T. J. A. Ewing, D. E.

Goldberg, I. D. Kuntz, and J. A. Ellman, J. Med. Chem. 42, 1428 (1999).6 M. Steger and D. W. Young, Tetrahedron 55, 7935 (1999).7 A. K. Szardenings, V. Antonenko, D. A. Campbell, N. DeFrancisco, S. Ida, L. Shi,

N. Sharkov, D. Tien, Y. Wang, and M. Navre, J. Med. Chem. 42, 1348 (1999).8 D. S. Yamashita, X. Dong, H. J. Oh, C. S. Brook, T. A. Tomaszek, L. Szewczuk, D. G. Tew,

and D. F. Veber, J. Comb. Chem. 1, 207 (1999).



peptides, new reactions for the solid-phase synthesis of heterocycles weredeveloped and optimized for library synthesis. Using the positional scan-ning technique, screening and deconvolution of the libraries led to theidentification of various biologically active compounds. The synthesis ofcombinatorial libraries has found broad acceptance in the field of medicinalchemistry since it greatly increases the likelihood of finding useful thera-peutic and diagnostic agents. In the emerging field of proteomics, combina-torial library synthesis has reached new importance as a tool for theidentification of new targets for drug development. The scope and versatil-ity of synthetic combinatorial libraries can be expected to further expandtheir significance for basic research and drug discovery.


[26] New Strategies for the Solid-Phase Synthesisof Highly Functionalized, Small Molecules:Sequential Nucleophilic Substitutions on

Polymer-Bound Polyelectrophiles

By Florencio Zaragoza

Introduction

For the fast identification of lead compounds for novel, small moleculeenzyme inhibitors or other ligands for proteins, the screening of large anddiverse arrays of compounds prepared on insoluble supports is one of themost efficient approaches.1–8 Parallel solid-phase synthesis has been foundto be particularly well suited for the preparation of such arrays of diversecompounds since multistep synthetic sequences on insoluble supports canbe conducted on fully automated synthesizers.

1 R. E. Dolle, Mol. Divers. 3, 199 (1998).2 J. L. Conroy, P. Abato, M. Ghosh, M. I. Austermuhle, M. R. Kiefer, and C. T. Seto,

Tetrahedron Lett. 39, 8253 (1998).3 M. Renil, M. Ferreras, J. M. Delaisse, N. T. Foged, and M. Meldal, J. Peptide Sci. 4, 195

(1998).4 J. C. Spetzler, V. Westphal, J. R. Winther, and M. Meldal, J. Peptide Sci. 4, 128 (1998).5 T. S. Haque, A. G. Skillman, C. E. Lee, H. Habashita, I. Y. Gluzman, T. J. A. Ewing, D. E.

Goldberg, I. D. Kuntz, and J. A. Ellman, J. Med. Chem. 42, 1428 (1999).6 M. Steger and D. W. Young, Tetrahedron 55, 7935 (1999).7 A. K. Szardenings, V. Antonenko, D. A. Campbell, N. DeFrancisco, S. Ida, L. Shi,

N. Sharkov, D. Tien, Y. Wang, and M. Navre, J. Med. Chem. 42, 1348 (1999).8 D. S. Yamashita, X. Dong, H. J. Oh, C. S. Brook, T. A. Tomaszek, L. Szewczuk, D. G. Tew,

and D. F. Veber, J. Comb. Chem. 1, 207 (1999).




Each synthetic step of syntheses performed on insoluble supports mustgenerally be optimized to such an extent to yield intermediates of highpurity, because these polymer-bound intermediates cannot be purified.Once such an optimized solid-phase synthesis has been developed, finalproducts of high purity can often be obtained directly after cleavagefrom the insoluble support and evaporation of the cleavage reagent (e.g.,trifluoroacetic acid).

Such products may be screened directly without any further purifica-tion. Because no purification on the final products is required in such in-stances, parallel solid-phase synthesis can be used to prepare arrays ofcompounds with a broad range of lipophilicity, charge, and molecularweight.9 Such arrays would be difficult to prepare by parallel solution-phase chemistry not only because each compound would require a differ-ent work-up strategy, but also because the purification of small, hydrophilicmolecules is often a difficult task.

Large arrays of compounds are mostly used at the beginning of medi-cinal chemistry projects for the fast identification of new lead structures.To increase the odds of finding innovative ligands, such arrays should notcontain large numbers of closely related compounds (which are likely tohave similar biological properties), but highly diverse compounds withwidely different properties.

Moreover, because new leads and not finished drugs are initially sought,such arrays should consist of lead-like, not drug-like compounds. Leadcompounds tend to be smaller and more hydrophilic than drugs, and conse-quently compound arrays for the discovery of new leads should mainly con-tain low-molecular-weight (e.g., MW < 350 g mol�1) and hydrophilic (e.g.,log P < 3) molecules.10,11 Because an excessive increase in the complexityof a molecule severely reduces its chances of binding to a given protein,11

the total number of functional groups capable of strongly interacting withproteins (pharmacophores) per molecule should also have an upper limit.Finally, a high-quality lead structure should also enable the quick prepar-ation of a large number of closely related analogs.

It is crucial that synthetic chemistry procedures do not become thebottleneck in the optimization process since this process can move forwardswiftly only when a large set of compounds is available for the determinationof their pharmacological properties. Large compound arrays, however, willbe meaningful only for the fast optimization of those parameters that can

9 F. Zaragoza, ‘‘Organic Synthesis on Solid Phase.’’ Wiley-VCH, Weinheim, New York, 2000.10 S. J. Teague, A. M. Davis, P. D. Leeson, and T. Oprea, Angew. Chem. Int. Ed. Engl. 38,

3743 (1999).11 M. M. Hann, A. R. Leach, and G. Harper, J. Chem. Inf. Comput. Sci. 41, 856 (2001).


also be quickly determined, for instance, by in vitro assays obtaining thebinding affinity to an enzyme or receptor and selectivity. If the time re-quired for testing one compound in a biological assay becomes significantlylonger than the time required for synthesizing the compound, large arraysof compounds generally do not accelerate the optimization process. Thereason for this is that a large array of compounds is designed without anyknowledge about the biological properties of each individual compound.This results in the generation of redundant compounds that would not havebeen prepared if the test result of a selection of compounds of the array hadbeen known beforehand. In the case of slow and labor-intensive biologicalassays it is therefore more efficient to prepare and test small arrays of com-pounds. This is, for instance, the case when properties such as clearance,half-life, oral availability, or brain–plasma ratio are to be optimized. Thedetermination of these parameters requires in vivo pharmacology, whichhas a rather low throughput when compared to in vitro assays.

Compound arrays are usually prepared by performing the same reac-tion sequence in each reactor, but using different reagents. For this reasonall the compounds of such an array will generally contain a repetitive struc-tural element. Diverse libraries will be obtained only if this repetitiveelement is both small and pharmacophore poor, and if the synthesisenables the incorporation of highly variable, pharmacophore-rich sidechains. Synthetic routes for the preparation of lead-like arrays shouldtherefore be based on readily available, pharmacophore-rich reagents suchas amines, alcohols, carboxylic acids, and thiols. Less suitable chemicalswould include hard-to-find reagents (e.g., difunctional reagents, such ashaloketones, diols, diamines, dicarboxylic acids, and amino acids) or re-agents that are inherently pharmacophore poor (e.g., acyl halides, isocyan-ates, and strong alkylating agents). Chemistry leading to repetitivestructural elements displaying important pharmacophores (e.g., diketopi-perazines, benzodiazepines, and peptides) will lead to sparsely diversecompound arrays because the repetitive structural element will be thedominating feature of all the members of the array. Such chemistries aretherefore less suitable for the preparation of diverse compound librariesand should be avoided.

Sequential Nucleophilic Substitutions on Insoluble Supports

What kind of chemistry and reagents will then be capable of deliveringarrays of compounds suitable for the identification of high-quality leads asoutlined above? As discussed in the introduction, for the preparation ofhighly diverse, lead-like libraries the incorporation of pharmacophore-richreagents into the final products is required. The majority of important


pharmacophores, such as the residues of proteinogenic amino acids (hy-droxyl, amino, guanidino, mercapto, carboxyl, 4-imidazolyl, and 3-indolylgroups), are nucleophilic functional groups, and electrophilic reagents con-taining such pharmacophores will generally require protection of the latterto avoid polymerization (first equation, Fig. 1). Partially protected electro-philic reagents, however, are not available in large number and high diver-sity, and are often expensive. The use of protective groups also increasesthe number of synthetic operations and thereby the costs of library produc-tion. Because synthetic intermediates cannot be purified during solid-phasesynthesis, an increased number of synthetic operations will likely lead toless pure final products. For these reasons, syntheses based on the reactionof electrophilic reagents with polymer-bound nucleophiles are of limitedutility for the preparation of lead-like compound arrays.

Nucleophilic reagents containing additional nucleophilic functionalgroups, such as diamines or aminoalcohols, on the other hand, are oftencheap, easily available, and can be used without any protective groups.With this type of reagents the most nucleophilic group will react first usu-ally with no interference of any other nucleophilic group present (secondequation, Fig. 1). The reaction of nucleophilic reagent with polymer-boundelectrophiles is therefore a valuable strategy for the preparation oflead-like compound arrays since it allows the chemist to introduce highlyvariable pharmacophores into the final product.

PolCl

PolH2N PolNH

O

FGCOXFG

SHFG

PolSFG

E Pol

X1

X2

X3

E Pol

Nu1

Nu2

Nu3

FG = NR-PG, O-PG, S-PG; protective groups are required

+

+

FG = NR2, OH, SH; protective groups are not required

Sequential nucleophilic substitutions:

1. Nu1H2. Nu2H3. Nu3H

Electrophilic reagents + resin-bound nucleophile:

Nucleophilic reagents + resin-bound electrophile:

Fig. 1. Nucleophilic substitutions as strategy for the use of unprotected, pharmacophore-

rich reagents. FG, functional group; Nu, nucleophile; PG, protective group; Pol, polymeric

support; X, leaving group for nucleophilic displacement.


The advantages of using nucleophilic reagents for library productioncan be exploited even further by performing sequential nucleophilic substi-tutions on a polymer-bound polyelectrophile. A suitable polyelectrophilewould be a polymer-bound compound with two or more leaving groupsthat could be orthogonally displaced by different nucleophiles. Becausemany pharmacophore-rich nucleophilic reagents are commercially avail-able, this strategy represents a simple method for the preparation of highlydiverse, pharmacophore-rich compounds.

Various examples of 2-fold sequential nucleophilic substitutions on in-soluble supports have been reported in the literature. Suitable polyelectro-philes are polyhalo triazines,12,13 pyrimidines,12,14 and purines,12,15–17 andthe most common nucleophiles are amines, thiols, and phenols. The useof 2,3-dichloropropionic acid18 and 4,5-difluoro-2-nitrobenzoic acid19 is dis-cussed below to illustrate the scope and limitations of this strategy for thepreparation of lead-like compound arrays.

2,3-Dichloropropionic Acid Derivatives as Polyelectrophile

2,3-Dichloropropionic acid is a cheap reagent that can be linked to in-soluble polymers either as an ester or an amide (1, Fig. 2). Although bothchlorine atoms of this substrate were expected to show a slightly differentreactivity toward nucleophiles, all attempts to displace these two leavinggroups by two different nucleophiles failed. Regardless of the reaction con-ditions and the type of nucleophile used, both chlorine atoms were dis-placed by the first nucleophile.

On the other hand, further experiments revealed that treatment ofimmobilized 2,3-dichloropropionic acid derivatives 1 with thiols affordeddithioethers 2, which were found to be unstable in the presence ofbases, undergoing clean �-elimination when treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)* to yield acrylic acid derivatives 3. When this

12 D. Scharn, L. Germeroth, J. Schneider-Mergener, and H. Wenschuh, J. Org. Chem. 66, 507

(2001).13 D. Scharn, H. Wenschuh, U. Reineke, J. Schneider-Mergener, and L. Germeroth, J. Comb.

Chem. 2, 361 (2000).14 F. Guillier, P. Roussel, H. Moser, P. Kane, and M. Bradley, Chem. Eur. J. 5, 3450 (1999).15 W. K.-D. Brill and C. Riva-Toniolo, Tetrahedron Lett. 42, 6515 (2001).16 W. K.-D. Brill, C. Riva-Toniolo, and S. Muller, Synlett 1097 (2001).17 K. Kim and B. Wang, Chem. Commun. 2268 (2001).18 F. Zaragoza and H. Stephensen, Angew. Chem. Int. Ed. Engl. 39, 554 (2000).19 M. Grimstrup and F. Zaragoza, Eur. J. Org. Chem. 3233 (2001).* Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, N,N-dimethylformamide;

MCPBA, 3-chloroperbenzoic acid; NMP, N-methyl-2-pyrrolidinone; Pol, undefined poly-

meric support; PS, cross-linked polystyrene; Wang resin, 1–2% cross-linked polystyrene with

p-benzyloxybenzyl alcohol linker.

XPol

O

Cl

Cl XPol

O

Nu1

Nu2

XPol

O

Cl

Cl XPol

O

SR1

R1S

XPol

O

SR1

XPol

O

SR1

R2N

?

R1SH, DIPEA R2NH, DBU

1 2

3 4

NMP, 20−80 �C, 22 hNMP, 20 �C, 22 h

Fig. 2. Two-fold sequential nucleophilic substitution at support-bound 2,3-dichloropro-

pionic acid for the synthesis of 3-amino-2-thiopropionic acid derivatives. X ¼ O, NR.


elimination was performed in the presence of an aliphatic or aromaticamine, addition of the latter to the electrophilic double bond of intermedi-ates 3 took place to yield 3-amino-2-thiopropionic acid derivatives 4.18

The elimination/addition reaction already proceeded at room tempera-ture when the dichloropropionic acid had been linked as an ester to thesupport, but required heating when an amide linkage had been chosen.When amines with low nucleophilicity were used, such as aniline or �-amino acid esters, higher reaction temperatures were also beneficial. Occa-sional by-products for this reaction sequence were acrylic acid derivativesor the corresponding hydrogenated products (2-thiopropionic acidderivatives). These by-products were usually formed when a very smallexcess of amine was used in the elimination/addition step. Both the thiolsand the amines used in this reaction sequence could be polyfunctional, asillustrated by the examples sketched in Fig. 3.

4,5-Difluoro-2-nitrobenzamides as Polyelectrophile

With the aim of identifying a suitable polyelectrophile to enable threesequential nucleophilic substitutions, we investigated the reactivity ofsupport-bound 4,5-difluoro-2-nitrobenzoic acid toward nucleophilic re-agents.19 To minimize the risk of premature cleavage from the supportduring the substitution reactions it was decided to immobilize this acidvia an amide linkage with Wang-resin-bound piperazine. We anticipatedthat the optimized reaction conditions using this linker would be directlytransferable to other linkers for amides, such as backbone amide linkers.9

CO2H

SPh

N

CO2H

SPh

NH

CO2Me

Ph

N

O

NHS

NH

HO

Cl

N

O

NHS

NH

Cl

NH2

N

O

NHSPh

NH

Ph

N

O

NHSPh

NHO

Ph

90% pure75% yield

73% pure49% yield

82% pure79% yield

72% pure31% yield

91% pure94% yield

61% pure90% yield

Fig. 3. Examples of products prepared by sequential nucleophilic substitution at support-

bound 2,3-dichloropropionic acid derivatives. Purities were determined by HPLC monitoring

at 214 nm.


Sequential aromatic nucleophilic substitutions on resin-bound 4,5-difluoro-2-nitrobenzamides required a careful selection of the nucleophilesand optimization of each step (Fig. 4). We found that the use of amines asfirst nucleophile proceeded cleanly, but the resulting 5-amino-4-fluoro-2-nitrobenzamides were too unreactive toward the nucleophilic displacementof the nitro group. Because the reactivity of arenes toward nucleophiles isstrongly influenced by the electronic properties of its substituents, wefound that for our purposes the first substitution had to be done with thiolsand not with a more electron-donating nucleophile. Treatment of the inter-mediate thioethers 7 with amines led to a clean substitution of the secondfluoride, but again, the resulting 4-amino-5-thio-2-nitrobenzamides weretoo unreactive toward nucleophiles to undergo the desired substitution ofthe nitro group.

To facilitate the nitro group displacement reaction, the thioethers 7,obtained by treatment of difluoride 6 with thiols, were oxidized with3-chloroperbenzoic acid to afford the corresponding sulfones 8 (Fig. 4).The resulting sulfones 8 showed the expected high reactivity towardnucleophiles, as demonstrated by the efficient displacement of both thesecond fluoride and the nitro group with two different aliphatic amines toyield the highly substituted benzamides 10.

As illustrated by the examples sketched in Fig. 5, pharmacophore-richthiols and amines could be used in all three displacement reactions toyield substituted benzamides displaying highly variable arrangements ofdifferent pharmacophoric groups.

XPol

O

NO2F

F

NPS

ONO2

FF

R

ONO2

FO2S Ph

N

R

PS

NH

N

O2S Ph

O

N

R

PSH2N

XPol

ONu1

Nu2 Nu3

NH

O2S Ph

O

N

R

PS

NO2

H2N

ONO2

FS Ph

N

R

PS

?

BnSH (1 mole/liter, 13 eq)AcOH (13 eq)

NMP, 22 �C, 3 h

Ethylenediamine(0.5 mole/liter, 14 eq)NMP, 22 �C, 2 � 2 h

Piperidine(2 mole/liter, 100 eq)

NMP, 100 �C

5

6 7 8

9 10

2 � 3 h

MCPBA(0.5 mole/liter, 9 eq)DCM, 22 �C, 2 � 2 h

Fig. 4. Three-fold sequential nucleophilic substitution at support-bound 4,5-difluoro-2-

nitrobenzoic acid.

NH

O2S

O

N

NH

NH

N

Ph

NHHO

NH

O2S

O

N

NH

NHH2N

OHN O2S

O

N

NH

NH

N

N

O

N

81% pure (214 nm)46% yield



Fig. 5. Examples of substituted benzamides prepared by 3-fold sequential nucleophilic

substitution on an insoluble support.


Conclusion

As illustrated by the examples given in Figs. 3 and 5, sequential nucleo-philic substitutions on support-bound polyelectrophiles give quick access tohighly functionalized, pharmacophore-rich small molecules. Because noprotective groups are required, the scope of these syntheses is broad, anda multitude of different pharmacophoric patterns can be generated byusing exclusively simple, commercially available reagents. The broad scope


of this new synthetic methodology should enable the quick optimization ofhits by preparing further, more focused compound arrays using the samechemistry.



All reagents were used as supplied. p-Benzyloxybenzyl alcohol resin(Wang resin) with a loading of approx. 1 mmol g�1 was purchased fromBachem (Bubendorf, Switzerland). All reactions were performed in fritt-ed Teflon reactors, using a setup as described in Zaragoza.9 Furtherexperimental details are given in Grimstrup and Zaragoza.19

2-Phenylsulfanyl-3-(piperidin-1-yl)propionic Acid Trifluoroacetate

CO2H

S

NCF3CO2H

To Wang resin (0.60 g, approx. 0.60 mmol) was added a solution of2,3-dichloropropionic acid (0.87 g, 6.09 mmol) in dichloromethane (12 ml)followed by the addition of N,N0-diisopropylcarbodiimide (0.48 ml,3.07 mmol) and 4-dimethylaminopyridine (0.06 ml, 1 M in DMF, 0.06mmol). The mixture was shaken at room temperature for 20 h, filtered,and the resin was washed with dichloromethane (3 � 6 ml). This resin wassuspended in NMP (9.0 ml), treated with diisopropylethylamine (0.90 ml,5.17 mmol) and thiophenol (0.60 ml, 5.84 mmol), and the resulting mixturewas shaken at room temperature for 22 h. After filtration and washing withNMP (3 � 6 ml), the resin was suspended in NMP (9.0 ml), and piperidine(0.60 ml, 6.07 mmol) and DBU (0.90 ml, 6.02 mmol) were added. Themixture was shaken at room temperature for 22 h, filtered, and the resinwas extensively washed with NMP, dichloromethane, and methanol, andresuspended in 1,2-dichloropropane. After shaking overnight at room tem-perature to remove traces of NMP the mixture was filtered, and the resinwas suspended in a mixture of dichloromethane (4.5 ml) and trifluoroaceticacid (4.5 ml). After shaking at room temperature for 0.5 h the mixture wasfiltered, the resin was washed twice with dichloromethane, and the com-bined filtrates were concentrated. Crystallization of the residue from a mix-ture of ethyl acetate and heptane at �20

�yielded the title compound


(144 mg, 63% yield) as a colorless solid, mp 81–83�. LCMS m/z 266 (MHþ);

1H NMR (400 MHz, DMSO-d6) � 1.51 (s, br, 2H), 1.72 (s, br, 4H), 2.98–3.29 (m, 4H), 3.34 (dd, J ¼ 13 Hz, 5 Hz, 1H), 3.53 (dd, J ¼ 13 Hz, 8 Hz,1H), 4.29 (dd, J ¼ 5 Hz, 8 Hz, 1H), 7.38–7.43 (m, 3H), 7.50–7.55 (m, 2H).Anal. Calcd. for C14H19NO2S�C2HF3O2 (379.40): C, 50.65; H, 5.31; N,3.69. Found: C, 50.94; H, 5.38; N, 3.63.

1-[5-Benzenesulfonyl-2-(piperidin-1-yl)-4-(pyridin-4-ylmethylamino)benzoyl]piperazine Trifluoroacetate

N

O

NH

N

S

NH

O

O

N

2 CF3CO2H

Wang-resin-bound piperazine20 (5.9 g, approx. 6.4 mmol) was treatedwith a mixture of 4,5-difluoro-2-nitrobenzoic acid (3.3 g, 16 mmol), di-chloromethane (25 ml), NMP (25 ml), 1-hydroxybenzotriazole (2.5 g,19 mmol), N,N0-diisopropylcarbodiimide (2.5 ml, 16 mmol), and diisopro-pylethylamine (1.1 ml, 6.3 mmol) at room temperature for 2.5 h.

After washing and drying, part of the resulting resin-bound difluoroar-ene (2.0 g, approx. 1.6 mmol) was treated with a mixture of NMP (20 ml),thiophenol (2.0 ml, 20 mmol), and acetic acid (1.1 ml, 19 mmol) at roomtemperature for 3 h. After washing and drying, part of the resulting resin-bound thioether (1.5 g, approx. 1.1 mmol) was treated twice with a solutionof MCPBA (2.5 g, 70%, 10 mmol) in dichloromethane (20 ml) at roomtemperature for 2 h.

After washing and drying, part of the resulting resin-bound 2-fluoroar-ylsulfone (1.0 g, approx. 0.74 mmol) was treated twice with a solution of4-(aminomethyl)pyridine (1.0 ml, 10 mmol) in NMP (20 ml) at room tem-perature for 2 h. After washing and drying, part of the resulting resin-bound nitroaniline (0.5 g, approx. 0.35 mmol) was treated twice with asolution of piperidine (4.0 ml, 40 mmol) in NMP (20 ml) at 100

�for 3 h.

After extensive washing with dichloromethane and methanol, part of this

20 F. Zaragoza and S. V. Petersen, Tetrahedron 52, 10823 (1996).


resin (164 mg, approx. 0.111 mmol) was treated with a mixture of trifluoro-acetic acid and dichloromethane (1:1) at room temperature for 0.5 h.Filtration, washing with dichloromethane, and concentration of the com-bined filtrates yielded 0.081 mmol (determined by 1H NMR with internalstandard; 73% yield) of the title compound. LCMS m/z 520 (MHþ); 1HNMR (400 MHz, DMSO-d6) �1.45 (br s, 6H), 2.74 (br s, 2H), 2.87 (br s,2H), 3.02 (br s, 2H), 3.18 (br s, 3H), 3.46 (br s, 2H), 4.10 (br s, 1H), 4.74(d, J ¼ 6 Hz, 2H), 5.84 (s, 1H), 7.19 (t, J ¼ 6 Hz, 1H), 7.54 (d, J ¼ 6 Hz,2H), 7.58–7.76 (m, 4H), 8.02 (d, J ¼ 7 Hz, 2H), 8.70 (d, J ¼ 6 Hz, 2H),9.21 (br s, 2H).

Acknowledgments

The technical assistance of Henrik Stephensen during the development of sequential

nucleophilic substitutions at support-bound 2,3-dichloropropionic acid is gratefully acknow-

ledged. Thanks are also extended to Marie Grimstrup, who optimized the synthesis of

substituted benzamides.

Author Index

Numbers in parentheses are footnote reference numbers and indicate that an author’s work is

referred to although the name is not cited in the text.

A

Abato, P. A., 297, 517

Abdul-Latif, F., 290

Abeles, R. H., 185

Abell, A. D., 486

Abell, C., 447, 462(30)

Acar, J. F., 331

Acharya, A. N., 507, 509, 510(39), 511

Adam, M. D., 143, 444

Adda, N., 323

Adey, N. B., 291

Afar, D. E. H., 315

Affleck, R. L., 79, 115, 272, 299

Affrossman, S., 396

Agarkov, A., 72(58), 73

Ahman, J., 179

Ahn, J.-M., 288

Ahn, S. Y., 480

Airey, J., 86, 90(27), 151, 154(1), 487

Ajay, 75, 294

Akelah, A., 368

Albericio, F., 21, 24, 32, 81, 90(23), 142,

183, 185(7), 186, 348, 418, 422, 444,

452, 465(25)

Albert, K., 351

Alcazar-Roman, L. M., 404

Aldebert, D., 323, 331(4)

Alesso, S., 403

Alexander, M., 6

Allanson, N. M., 258, 261(22), 262

Allen, J. D., 422

Allen, M. P., 143, 444

Almdal, K., 40

Almstetter, M., 197

Alsina, J., 81, 90(23), 142, 183, 185(7), 422

Amblard, M., 58, 59(42)

Ambroise-Thomas, P., 323, 331(4)

Ambrosius, D., 304

529

Amons, R., 293, 294

Amro, N., 313

An, H. Y., 392

Andersen, K., 447, 463(31), 464(31)

Anderson, J., 262

Anderson, P. S., 80, 185, 470

Andrade, R. B., 239, 240(18), 241

Andres, C. J., 77

Andres, V., Jr., 305

Andries, K., 470

Anelli, P. L., 372, 373(22)

Anh, S. Y., 417

Antonenko, V., 517

Appel, J. R., 113, 290, 298, 324, 325, 338, 341,

497, 498, 498(9; 10), 499(9), 514(46), 515

Arad, O., 184

Arienti, K. L., 392

Armstrong, R. W., 42, 70(17), 100, 197, 394,

395(37; 38), 470, 470(9), 471, 471(7), 474,

479, 489

Arnaiz, D., 481

Arnauld, T., 354, 355(19), 361(35), 362

Arnold, B., 481

Artamkina, G. A., 404(72), 405

Asgedom, M., 75(5), 76, 99(2; 3), 100, 113,

298, 435

Ashton, W. T., 80

Askin, D., 80

Atdlweiser, J., 391

Atrash, B., 77, 115

Attanasi, O., 419

Attardi, M. E., 24, 29, 32

Atwal, K. S., 199

Augelli-Szafran, C. E., 138

Ault-Justus, S., 388, 407

Aurigemma, C. M., 4

Austermuhle, M. I., 517

Auzanneau, F. I., 314

Avemaria, F., 130, 133(11)

530 author index

Axel, M. G., 185

Axelrod, H. R., 262

B

Baasov, T., 259

Baboonian, C., 339

Babu-Khan, S., 485

Bachmann, S., 351

Backes, B. J., 128, 132(7)

Badet, B., 185

Bagchi, G. D., 75

Bai, Y., 209

Bailey, F. C., 369

Bailey, N., 392

Bair, E. L., 313

Baizman, E., 262

Balasubramanian, S., 272, 299

Balkenhohl, F., 257, 391, 419

Ball, J. M., 302

Bandel, H., 3

Banfi, L., 484

Bang, K. S., 447, 463(31), 464(31)

Bankaitis-Davis, D., 164

Bannwarth, W., 418

Banville, S. C., 290

Baran, P. S., 378

Barany, G., 81, 90(23), 142, 183, 185(7), 235,

258, 261(24), 272, 422, 444, 456, 465(25)

Barbachyn, M. R., 225(7), 226

Barbas, C. F. III, 498

Barbier, P. T., 428

Barger, L. A., 74

Baringhaus, K.-H., 436

Barluenga, S., 79, 115, 356

Barn, D. R., 419

Barnes, C., 272, 299

Barnes, G., 46

Barnwell, P., 391

Barrett, A. G. M., 354, 355(19), 361(35),

362, 396

Barrett, R. W., 183, 498

Barrish, A., 199

Barrow, J. C., 199

Barry, J., 24, 26

Barteling, S. J., 40, 43(10), 100, 289, 497

Barth, M., 370, 387

Barthelemy, S., 375

Bartolozzi, A., 347

Barton, D., 357, 399, 402(63)

Basso, A., 67

Batchelor, J. F., 480

Bateman, R., 4

Battersby, J. E., 24, 26

Baum, S. A., 116, 117(16)

Bauser, M., 291

Baxendale, I. R., 73, 348, 368, 392, 394(16)

Baxter, A. D., 419

Baxter, E. W., 397, 402(62)

Bayada, D. M., 476

Bayburt, E. K., 75

Baydal, J. P., 24, 31

Bayliss, M. K., 4, 5(8)

Bean, M. F., 199

Beaton, G., 235

Becker, J. A. J., 297

Becker, M., 151(2), 152, 160(2)

Beebe, X., 430

Befzi, A., 335

Begtrup, M., 426

Beijnen, J. H., 323

Beletskaya, I. P., 404(72), 405

Belyakov, S. A., 138

Bemis, G. W., 75, 294

Benckhuijsen, W. E., 293, 294

Ben-David, D., 87

Benezra, C., 394, 395(39–41)

Bennett, B., 485

Bennett, W. D., 499

Berces, A., 252

Berg, R. H., 40

Bergelli, M., 497

Berger, E. A., 332(38), 333, 334, 334(38),

335(40)

Bergquist, K.-E., 154

Berlinck, R. G. S., 143

Berman, J., 74

Bernick, A. M., 396

Berst, F., 422

Besemer, A. C., 372, 374(20)

Besser, D., 184

Betschinger, J., 291

Bhandari, A., 419

Biaga, T. J., 474

Bianchi, E., 297

Biancolana, S., 444, 465(25)

Bicak, N., 396, 408

Biddison, W. E., 341, 342(64)

Bidham, E. C., 74

Bidlack, J. M., 291

author index 531

Bielekova, B., 338, 342(47)

Bienayme, H., 469, 483, 489

Bienstock, R. J., 184

Biffi, C., 372, 373(22)

Biginelli, P., 197

Bilello, J. A., 80

Biringer, G., 428

Bishop, M. J., 74

Biswas, K., 258

Bitter-Suermann, D., 304

Bjergarde, K., 425

Blackburn, B. K., 470

Blackburn, C., 348, 489

Blahy, O. M., 79

Blanc, P., 325, 498

Blaney, P., 416

Blankson, J., 332

Blauth-Eckmeyer, E., 138

Blechert, S., 428

Blocker, H., 113

Blomgren, P. A., 364, 407

Blondelle, S. E., 113, 290, 292, 298, 322, 324,

327, 335, 342, 497, 498(9; 10), 499,

499(9), 500, 501(19), 502(19)

Blossinger, C. D., 513

Bobko, M. A., 422

Bock, H., 475

Bock, M. G., 470

Boddy, C. N. C., 130

Boden, C. D., 79

Boden, C. J., 79

Boden, P., 291(22), 292

Boeijen, A., 417

Boggiano, C., 322, 338, 342

Bohm, H.-J., 295

Bolli, M. H., 371

Bolm, C., 351, 375

Bolognesi, D., 332

Bolton, G. L., 392(28), 393, 396(28), 401(28),

402(28)

Bombrun, A., 177

Bondinell, W. E., 185

Bono, E., 341

Boojamra, C. G., 42, 81, 89(23b), 90(23)

Boons, G.-J., 258

Booth, R. J., 392, 392(28), 393, 393(12),

394(12), 395(11; 12), 396(12; 28),

399(12), 401(28), 402(12; 28), 405(12)

Borchardt, A., 299

Bornaghi, L., 254, 259

Borras, E., 338, 342(53)

Borsche, W., 139

Bossinger, C. D., 22, 24, 25(1), 302

Bossio, R., 489

Boths, J., 291

Botta, M., 152

Boule-Vavra, S., 331

Bousquet, P., 480

Bouzid, K., 489

Boyce, J. M., 331

Bradley, M., 77, 115, 388, 403, 438, 459(12),

460(12), 461(12), 521

Braisted, A. C., 75

Branch, D. L., 12

Brandenburg, D., 304

Brandtner, S., 131, 142(14), 144(14)

Branstrom, A., 262

Brase, S., 127, 130, 131, 132, 132(12), 133(11;

16), 134, 134(12), 135, 136, 137, 138, 139,

140, 140(49), 142, 142(14), 143(54),

144(14; 57), 145, 355

Brasili, L., 480

Braxenthaler, M., 341

Bray, A. M., 39, 40, 41, 42, 43, 43(19), 46,

59(6), 60(19), 64, 65, 73(20)

Bray, M. K., 516

Bream, R. M., 348, 368, 392, 394(16)

Breddam, K., 314

Breen, A. L., 199

Breitenbucher, J. G., 392

Brennan, A. L., 331

Brenner, S., 290

Breslin, H. J., 470

Briand, J.-P., 184

Brickner, S. J., 225, 225(7), 226

Brightwell, C. I., 80

Brill, W. K.-D., 435, 439, 444(16), 450,

466(33), 521

Bristol, N., 262

Britton, T., 481

Broadhurst, M., 248, 251, 260

Brock, R., 370

Broder, C. C., 334, 335(40)

Broger, C., 489

Brook, C. S., 517

Brooks, R. R., 480

Broten, T. P., 199

Brown, J. F., 364(42), 365

Brown, K. D., 151(2), 152, 160(2)

Brown, R., 172

532 author index

Brown, R. C. D., 24, 29, 30(10), 115,

117(8), 428

Brown, S. D., 197, 394, 395(38), 489

Brule, E., 348

Brumfield, K. K., 457

Brummer, O., 437

Bruns, R., 481

Brusic, V., 341

Bryant, S. D., 185

Buchowicz, W., 358(29), 359

Buchstaller, H.-P., 426

Buchwald, S. L., 179, 358(30), 359, 404, 405

Budhu, R. J., 80

Buehler, J., 251, 252(12)

Buencamino, J., 497

Buhlmayer, P., 87

Bui, C. T., 40, 115, 117(11)

Bundle, D. R., 263

Bunin, B. A., 39, 43, 136, 392, 435, 437,

451(2), 470

Bunker, A. M., 480

Burgess, K., 143

Burke, L. A., 135

Burkett, B. A., 24, 29, 30(10)

Burkoth, T. S., 421, 478

Burns, C. J., 471, 473

Burow, K. M., 42, 81, 89(23b), 90(23)

Burton, D. R., 498

Burton, L., 4

Busch, A. E., 480

Buskas, T., 241(20), 242

Bussche-Huennefeld, C. v. d., 391

Busson, R., 185

Butler, R. N., 135

Bycroft, B. W., 186

C

Cacchi, S., 178

Caddick, S., 224

Cai, P. J., 204

Calas, B., 184

Caldarelli, M., 396

Caldwell, C. G., 263

Caliendo, G., 185

Callahan, J. F., 185

Calvo, R. R., 185

Cameron, A. M., 24, 31

Cameron, N. R., 24, 31, 364(42), 365

Camillero, P., 24

Campbell, D. A., 421, 425(22g), 428, 478, 517

Campbell, G. D., Jr., 323

Campbell, R. A., 40, 42, 73(20)

Camps, F., 424

Cannan, R. K., 24, 25

Cano, J., 323, 331(4)

Canova, R., 74

Cantrell, B., 481

Cao, G.-Q., 79, 115

Cao, K., 164, 170(2), 173

Capeau, J., 323

Cappiello, J., 394

Carboni, B., 425(37), 426

Cardenas, J., 487

Cardullo, F., 431

Cargill, J. F., 42, 70(17), 100

Carlisle, S. J., 364

Carmichael, A. J., 339

Carotti, A., 480

Carpenter, J. F., 480

Carpino, L., 452

Carreaux, F., 425(37), 426

Carrillo-Munoz, A. J., 323, 331(4)

Carte, B., 199

Cartells, J., 424

Caruthers, M. H., 235

Cascieri, M. A., 296

Castelhano, A. L., 423

Castro, J. L., 428

Cato, S. J., 294

Ceccarelli, S., 396

Cerottini, J.-C., 338, 342(51; 53)

Chadwick, K., 332

Chae, C. B., 294

Chaisson, R. E., 332

Chamberlin, A. R., 202(38), 203, 224

Chan, C. C., 152

Chan, D. C., 332(36), 333, 334, 337(39)

Chan, T.-H., 263

Chan, W. C., 39, 186

Chang, C. L., 75

Chang, C. S., 75

Chang, R. S. L., 80, 199

Chang, Y.-T., 444

Chanock, R. M., 498

Chapman, K. T., 324, 396

Chauhan, P. M. S., 437

Chauvey, D., 187

Cheifari, D. S., 39, 59(6)

Cheminat, A., 394, 395(39–41)

author index 533

Chen, A., 262

Chen, G. S., 75

Chen, I.-W., 80, 185

Chen, J., 143

Chen, J. J., 422, 475

Chen, L., 185

Chen, M. L., 271

Chen, T.-M., 185

Chen, Z.-C., 376

Chendra, S., 514(46), 515

Chenera, B., 469, 484

Cheng, Y., 324

Chern, J. W., 75

Cherrier, M. P., 474, 475, 479

Chervin, I. I., 140

Chesney, A., 391

Chexal, K. K., 184

Chhabra, S. R., 186

Chi, A., 262

Chinchilla, R., 361(36; 37), 362

Ching, B. W., 480

Chisem, I. C., 396

Chisem, J., 396

Chmielewski, M., 430

Cho, B. Y., 294

Cho, C.-W., 349, 350(10)

Cho, H. Y., 417, 480

Cho, N. S., 470

Choi, S., 479

Choi-Sledeski, Y. M., 151(2), 152, 160(2)

Choo, H.-Y. P., 423

Chou, Y.-L., 456

Christensen, J. W., 99, 100, 116

Christensen, T., 24, 28

Chu, S. S., 24

Chu, V., 151(2), 152, 160(2)

Chucholowski, A., 391, 421, 424, 424(22b)

Chung, I. K., 423

Chung, N. N., 291, 497

Chung, S.-H., 418

Chutkowski, C. T., 334, 337(39)

Ciattini, P. G., 178

Cichy-Knight, M. A., 296

Ciraco, M., 361(38), 362

Clackson, T., 289

Clapham, B., 348, 349, 350(10), 437

Clark, B. J., 140

Clark, J. H., 396

Clement, R. P., 5

Clemons, K. V., 323, 331(4)

Clercq, E. D., 470

Cliffe, I. A., 80

Cobb, J. M., 447, 462(30)

Cody, D. M. R., 76

Coe, D. M., 24, 31, 391

Cohen, B. J., 152

Coleman, D. C., 323, 331(4)

Colescott, R. L., 22, 24, 25(1), 302, 513

Collibee, S. E., 72(58), 73

Collier, S., 135

Colucci, M., 423

Combs, A. P., 197, 223, 224, 225

Comely, A. C., 131, 416

Congreve, M., 22

Conlon, P., 338

Connelly, J. A., 79

Connolly, C. J. C., 480

Conroy, J. L., 297, 517

Conti, N., 138

Cook, A. W., 453

Cook, P., 22, 24, 25(1)

Cook, P. D., 392

Cook, P. I., 302, 513

Cooper, A., 290

Cooper, W. J., 392

Cooperman, B. S., 291

Coppola, G. M., 391

Corbel, J. C., 67

Corelli, F., 152

Cork, D., 396

Cortes, D. A., 372

Cortese, I., 338, 342(47)

Cortese, R., 297, 498

Cotterill, I., 202(37), 203

Cournoyer, J. J., 24, 33

Courtney, K. D., 305

Cowart, M., 75

Cowell, D., 77, 79, 115

Cowell, S. M., 288

Cox, L. J., 419

Cox, R., 24, 31

Crawford, K., 86, 90(27), 151,

154(1), 487

Creighton, C. J., 361(39), 362

Crespo, R. F., 361(38), 362

Cress, A. E., 313

Cresswell, P., 339

Creswell, M. W., 392(28), 393, 396(28),

401(28), 402(28)

Crich, D., 357

534 author index

Crich, J. Z., 393, 394(32), 395(32), 396(32),

406(32)

Crooks, E., 327

Cross, J. T., Jr., 323

Crowley, J. I., 427

Crozet, Y., 295

Cuervo, J. H., 113, 290, 298, 324, 497, 498(9),

499(9)

Cummings, W., 51, 116, 117(13)

Cunningham, A., 200

Cupif, J.-F., 423, 425(37), 426

Curran, D. P., 200(23; 24), 201, 392(24), 393

Cuzzocrea, C., 290

Cwirla, S. E., 498

Czarnik, A. W., 40, 76, 272, 299, 391, 415

D

Dahmen, S., 127, 130, 131, 132, 132(12),

133(16), 134(12), 139, 140(49), 142,

143(54), 144(57), 145, 355

Dalbon, P., 184

DalCin, M., 392

Dallas, J., 481

Dallinger, D., 201

Dandia, A., 204

D’Andrea, S. V., 77

Danishefsky, S. J., 237

Danks, T. N., 351(14), 352

D’Aquila, R. T., 332

Darke, P. L., 79, 80, 185

Darlak, K., 296

Darvas, F., 497

Dattilo, M., 74

Daum, R. S., 331

David, C. M., 302

David, F., 185

David, M., 423, 425(37), 426

David, M.-L., 186, 189(30), 192(30)

Davis, A. E., 5

Davis, A. M., 477, 518

Davis, A. P., 24, 26

Davis, B. G., 24, 31

Davis, F. A., 443, 462

Davis, R. S., 151(2), 152, 160(2)

Dax, S. L., 197

Deal, M. J., 392

Dean, A. W., 392

de Biasi, V., 4, 24

de Bont, D. B. A., 425

De Brosse, C., 185, 199

De Clercq, P. J., 24, 26

De Crescentini, L., 419

Deeg, M., 418

DeFrancisco, N., 517

DeGonia, D. J., 385

Degrado, W. F., 152

DeGraw, S. T., 516

DeIong, N., 295

Dekany, G., 251, 254, 259

de Koster, H. S., 293

de la Hoz, A., 202, 203(30)

Delaisse, J. M., 517

de las Heras, X., 419

Delbressine, L. P. C., 476

del Fresno, M., 183, 185(7), 422

De Luca, L., 356(26), 357

Demee, M., 12

de Meijere, A., 136

de Miguel, Y. R., 348

De Muynck, H., 24, 26

Denay, R., 74

Dence, C. S., 80

Dener, J. M., 446

den Hartog, J. A. J., 428

Denisko, O. V., 138

de Nooy, A. E. J., 372, 374(20)

DePasquale, M. P., 332

Depew, K. M., 124

Deprez, B. P., 152

DeRoock, I. B., 313

DeRoose, F., 262

Desai, B., 351(14), 352

Deshayes, S., 202(35), 203

Deshpande, M. S., 77

Desjonqueres, N., 349, 350(7)

Desmyler, J., 470

Dess, D. B., 376

Dessen, A., 332(35), 333

Devaky, K. S., 351(13), 352

Devlin, J. J., 498

Devlin, P. E., 498

Devraj, R. V., 392, 393, 394(32), 395(32),

396(32), 406(32)

de Vries, R. R. P., 294

de Wit, D., 295

DeWitt, S. H., 76, 391, 415, 417, 425(5)

Dhamoa, D. S., 446

Diamond, D. J., 338, 339(52)

Dıaz-Ortis, A., 202, 203(30)

author index 535

Dibo, G., 75(5), 76, 99(2; 3), 100, 113,

298, 435

Didomenico, S., 75

Di Lucrezia, R., 443

DiMarchi, R. D., 324

DiMichele, L. M., 490

Ding, J., 341

Ding, Q., 438, 444(15), 449(15), 458(15),

465(15)

Ding, S., 438, 444(15), 449(15), 458(15),

465(15)

Ding, Z.-K., 63

Dipardo, R. M., 470

Ditto, L., 152, 393, 396(30)

Doan, N., 298

Dodi, A. I., 339

Dodsworth, D. J., 361(36; 37), 362

Doi, T., 60, 62, 258

Dolan, J., 6

Dolle, R. E., 39, 136, 151, 151(3), 152, 391,

393, 435, 517

Dollinger, G. D., 12

Dominy, B. W., 436

Domling, A., 470, 482(8), 489, 489(8),

490(56e)

Domling, S., 197

Dondoni, A., 200, 349, 350(9), 392(27), 393

Dong, L.-C., 173

Dong, X., 517

Dontenwill, M., 480

Dooley, C. T., 113, 290, 291, 298, 324, 497,

498(9; 10), 499(9), 506

Dorff, P. H., 456

Doring, R., 113, 497

Dorman, G., 497

Dorn, C. P., 80

Dorner, B., 335, 499, 500, 501(19), 502(19)

Dorsey, B. D., 79, 185

Dorsey, J. G., 394

Douglas, S. P., 250

Dower, W. J., 183, 498

Dowling, R. B., 331

Doyle, C. A., 394

Dragovich, S., 63

Dressman, B. A., 87, 391, 392(2), 394(2),

396(2), 417, 480

Drew, M., 86, 90(27), 151, 154, 154(1), 487

Drewry, D. H., 74, 143, 419

Drijfhout, J. W., 293, 294

Drinnan, N., 248, 251, 254

Driver, M. S., 404

Dromer, F., 325

Drusano, G. L., 80

D’Sa, B. A., 453

Duinkerken, G., 294

Dulina, R., 262

Dunn, A. K., 18(80), 79

Dunn, B. M., 485

Dutoit, V., 338, 342(51; 53)

Dwek, R. A., 248

Dzierba, C. D., 225

Dzwonczyk, S., 199

E

Eames, J., 348, 392(29), 393, 396(29)

Ede, N. J., 42, 43(19), 58, 60(19), 64,

73(20), 74

Eden, J. M., 291(22), 292

Edmunds, J. J., 480

Egelhaaf, H.-J., 370

Eggleston, D. S., 185

Ehrler, J., 56

Eichler, J., 324, 497, 498, 498(10),

514(46), 515

Eigenberger, B., 480

Eigenbrot, C., 185

El-Abadelah, M. M., 140

El Abdellaoui, H. M., 506

Elander, N., 202, 203(32)

El-Faham, A., 452

Ellis, J. D., 199

Ellman, G. L., 24, 31, 305, 392(22), 393

Ellman, J. A., 39, 42, 43, 65, 81, 89(23b),

90(23), 128, 132(7), 134, 136, 183, 185(5),

197, 290, 296, 391, 415, 435, 437, 451,

451(2), 467(34), 470, 475, 485, 517

Ellmerer-Muller, E. P., 418

Emini, E. A., 80, 185

Enders, D., 130, 131, 133(11), 142(14),

144(14), 145, 355

Engelsen, V., 428

Engers, D., 86, 90(27), 151, 154(1), 487

Enstrom, A., 298

Eppens, N. A., 324

Ercole, F., 40, 42, 73(20), 115, 117(11)

Ericsson, J. A., 422

Eritja, R., 419

Erlanson, D. A., 75

Ermann, M., 419

536 author index

Ernst, B., 67

Espinoza, F. H., 46

Es-Sayed, M., 136, 138

Estep, K. G., 143, 444

Evans, B. E., 80, 470

Evans, D. A., 404(71), 405

Evans, D. J., 186

Ewing, B., 115, 117(8)

Ewing, T. J. A., 517

F

Falchi, A., 24, 29

Fang, L., 3, 5, 7, 12

Fantauzzi, P. P., 24, 33, 422, 446

Farber, J. M., 332(38), 333, 334(38)

Farcy, N., 24, 26

Farnell, K., 54(39), 55

Farrall, M. J., 39, 394, 395(39–41)

Farrell, W. P., 4

Faulkner, D. J., 199

Featherstone, R. M., 305

Feeney, P. J., 436

Fehrentz, J.-A., 423

Feijlbrief, M., 293

Fejzo, J., 75

Felder, E. R., 435, 437

Feldman, J., 480

Felici, F., 498

Felker, D., 135

Feng, B., 5

Feng, J. C., 204

Fenuik, W., 296

Ferguson, R., 290

Fernandez, M., 143

Fernandez, R., 384

Fernandez-Rivas, C., 404

Ferreras, M., 517

Ferritto, R., 138, 200(23), 201, 392,

392(24), 393

Fesik, S. W., 75

Fex, T., 154

Fey, T., 351

Fields, G. B., 235

Figliozzi, G. M., 24, 33

Figueroa-Perez, S., 422

Filippone, P., 419

Fillon, C., 18(80), 79

Finch, H., 376

Fincke, H., 139

Finlay, M. R. V., 429

Finner, E., 428

Finucane, M. D., 295

Finzi, D., 332

Fiorini, M. T., 447, 462(30)

Fiorino, F., 185

Firestone, S., 419

Fischer, H., 351

Fish, D. G., 80

Fitch, W., 6

FitzGerald, M., 42, 73(20)

Fitzgerald, P. M. D., 185

Flegelova, Z., 456

Fleming, P., 489

Fletcher, A., 80

Flexner, C., 332

Flohr, S., 75

Floyd, C. D., 443

Floyd, D. M., 199

Flygare, J. A., 143

Flynn, D. L., 392, 393, 394, 394(32), 395,

395(32; 43), 396(32), 406(32), 475

Flynn, G., 116, 117(16)

Fodor, S. P. A., 183, 497

Foged, N. T., 517

Fonon, F., 480

Fontenot, J. D., 302

Ford, C. E., 63

Ford, C. W., 225(7), 226

Forero-Kelly, Y., 399, 402(63)

Forray, C., 199

Forster, E. A., 80

Foulds, G. J., 486

Fournier, A., 75

Fournier, E. J.-L., 353, 354(18)

Fox, A., 135

Frank, R., 113, 360, 361(33), 379, 497

Frankel, M., 369

Franz, A. H., 278

Franzen, R. G., 136, 450

Fraser-Reid, B., 60, 241(20), 242, 262

Frater, G., 478

Frechet, J. M. J., 39, 152, 200, 394, 395(39–41)

Freeman, H. N., 296

Freidinger, R. M., 199, 470

Freyer, A. J., 199

Fridkin, M., 152, 184, 369

Friede, T., 339

Frieden, A., 183, 185(7)

Frigerio, M., 376

author index 537

Fritz, J. E., 391, 392(2), 394, 394(2), 396(2)

Fruchtel, J. S., 392(25), 393

Fryer, A. M., 475

Fugedi, P., 260, 261(31)

Fuhrman, S. A., 63

Fujii, N., 304

Fujita, H., 184

Fukase, K., 74

Fukuzawa, A., 75

Fung, E., 325

Furet, P., 87

Furka, A., 75(5), 76, 99, 99(2; 3), 100, 104(9),

113, 116, 298, 435

Furman, B., 430

Furugori, T., 75

Fyles, T. M., 39

G

Gabriel, C., 202, 208(29)

Gabriel, S., 202, 208(29)

Gabryelski, L., 324

Gackenheimer, S., 481

Gadek, T. R., 470

Galfre, G., 498

Galili, U., 251, 252(12)

Gallant, J., 332

Gallazzi, F., 341

Gallop, M. A., 76, 165, 175, 177, 183, 391, 417,

419, 454, 467(44)

Ganesan, A., 415, 422, 423, 424, 425(32), 427

Gange, D., 262

Gange, S., 332

Gaoni, Y., 184

Garcia, J. G., 391, 418

Gard, J., 51, 116, 117(13)

Gardner, M., 257

Gardyan, M., 86, 90(27), 151, 154(1), 487

Garegg, P. J., 255, 260, 260(17), 261(31), 263

Garibay, P., 426

Garigipati, R. S., 456

Garmon, S. A., 225(7), 226

Garrison, D. T., 74

Garzon, A., 297

Gastaldi, S., 356(25), 357

Gates, M., 470, 471(7)

Gatti, P. M., 204, 206(49)

Gaudino, J. J., 425

Gayo, L. M., 391

Geddes, D. M., 331

Gehlert, D., 481

Gelb, M. H., 185

Gene, J., 323, 331(4)

Gennari, C., 396, 431

Gera, L., 444, 465(25)

Gerard, B., 151(3), 152

Gerber, F., 438, 443(14), 461(14), 462(14)

Gerhardt, J., 418

Germeroth, L., 443, 521

Gerritz, S. W., 42, 53, 73(22), 74, 143

Gershonov, E., 184

Gesellchen, P. D., 324

Geysen, H. M., 40, 41, 43(10), 60, 100,

289, 497

Geysen, M. H., 262

Gharakhanian, S., 323

Ghiron, C., 419

Ghosh, A. K., 394

Ghosh, M., 517

Giacomelli, G., 356(26), 357

Gianella, M., 480

Giannakakou, P., 429

Giannis, 376

Gibson, H. W., 369

Gibson, S. E., 131, 134, 416

Gierasch, L. M., 184

Giger, R., 42, 74

Gigstad, K. M., 478

Gil, C., 136, 140

Gilbert, I. H., 443, 481

Gilbert, K. F., 80, 199

Gilbertson, S. R., 72(57; 58), 73

Gildersleeve, J., 258

Giles, K., 4

Giovannoni, J., 58, 59(42)

Giralt, E., 419

Giulianotti, M. A., 335, 501, 504, 505,

510(40), 511, 512

Glass, B. M., 223, 224

Glass, K. L., 199

Gleeson, J.-P., 422

Gluzman, I. Y., 517

Goddard, M.-E. T., 447, 462(30)

Goldberg, D. E., 517

Goldman, J., 339

Goldman, R., 262

Goldstein, F. W., 331

Golebiowski, A., 39(9), 40, 72(9), 75, 183,

422, 475

Golec, J. M. C., 87

538 author index

Gomtsyan, A., 75

Gong, Y., 151(2), 152, 160(2)

Gong, Y.-D., 418

Gonzalez-Gomez, J. C., 422

Goodman, M., 361(39), 362, 422

Goodnow, R., 236

Gordon, D. W., 257, 419, 454

Gordon, E. M., 75, 183, 391

Gordon, R. S., 358(32), 359

Gore, A. L., 392

Gouault, N., 423, 425(37), 426

Gougoutas, J. Z., 199

Gouilleux, L., 423

Gozzi, C., 177

Grabowska, U., 54(39), 55

Graminski, G. F., 290

Gran, B., 338, 341, 342(47; 64)

Grandas, A., 419

Granoth, R., 184

Grant, C. M., 79

Grant, E. H., 202, 208(29)

Grant-Young, K., 77

Grathwohl, M., 248, 260, 263

Gravel, M., 404

Gray, N. C., 46, 46(29)

Gray, N. S., 438, 444, 444(15), 449(15), 456,

458(15), 465(15)

Grayshan, R. J., 140

Greco, M. N., 397, 402(62)

Greenlee, W. J., 80

Grega, K. C., 225(7), 226

Greig, M. J., 4

Greivedinger, G., 290

Grether, U., 134

Griebenow, N., 134

Grieco, P. A., 475

Griedel, B., 481

Griffin, I. J., 480

Griffin, P. R., 396

Griffith, M. C., 396

Griffiths, A. D., 289

Grigg, R., 416

Grillot, R., 323, 331(4)

Grimstrup, M., 521, 522(19), 525(19)

Groetzinger, J., 304

Grogan, M. J., 347

Grootenhuis, P. D. J., 476

Grosche, P., 3, 145, 379

Groutas, W. C., 135

Grover, G. J., 199

Grundy, J. E., 339

Gu, X., 288

Guan, B., 489

Guanti, G., 484

Guare, J. P., 185

Guarro, J., 323, 331(4)

Gubernator, K., 489

Guidi, B., 419

Guiles, J. W., 77

Guillaume, P., 338, 342(51)

Guillier, F., 438, 459(12), 460(12),

461(12), 521

Gulevskaya, V. I., 140

Gundlach, B. R., 338

Gundry, R. L., 185

Gunthard, H. F., 332

Gunther, W., 478

Guo, L., 313

Gupta, A. K., 204, 206(47)

Gupta, R., 204, 206(47)

H

Haase, W.-C., 237, 250, 258(5)

Habashita, H., 517

Habermann, J., 396

Haddach, M., 405

Hadida, S., 200(23), 201, 392(24), 393

Haehnel, W., 295

Haggarty, S. J., 199

Hagler, A. T., 184

Hagmann, M., 341

Hahn, P. J., 391, 392(2), 394(2), 396(2)

Hajduk, P. J., 75

Hajra, A., 209

Hakomori, S., 235

Hales, N. J., 134

Hall, D. G., 404

Hallberg, A., 202, 202(36), 203, 203(33),

224, 228

Hallensleben, M. L., 376

Halstead, B. S. J., 202, 208(29)

Haltiwanger, R. C., 185

Hamaker, L. K., 75(5), 76

Hamann, B. C., 404

Hamashin, V. T., 499, 509(20)

Hamby, J. M., 480

Hamel, E., 429

Hamelin, J., 202, 203(34), 204(34)

Hammer, J., 341

author index 539

Hammer, R. P., 185

Hamper, B. C., 422

Hamuro, Y., 418, 421(11i)

Han, G., 185

Han, H., 290

Han, K., 262

Han, Y., 353, 354(16)

Hanashin, V. T., 498, 501(17)

Hancock, W. S., 24, 26

Handlon, A. L., 53, 70

Hanessian, S., 44, 417, 480

Hanifin, C. M., 80

Hann, M. M., 518

Hanna, G. J., 332

Haque, T. S., 517

Harfenist, M., 480

Harikrishnan, L. S., 388

Harper, G., 518

Harris, D., 421, 425(22g)

Harrison, S. C., 332(35), 333

Hart, M. E., 202(38), 203, 224

Hartmann, C., 376

Hartwig, J. F., 175, 404

Haskins, N., 4

Hassall, C. H., 184

Hassan, J., 177

Hatzenbuhler, N. T., 262

Hauck, S. I., 404

Haunert, F., 371

Hauser-Fang, A., 3

Hawes, M. C., 392

Haynie, S. L., 235

He, Y., 429

Healy, E., 99, 100, 116

Heckel, A., 250

Hedberg, A., 199

Heikens, W., 113

Heissler, D., 263

Heisterberg-Moutsis, G., 113

Heitz, W., 369, 382(13)

Hellmann, N., 332

Hemmer, B., 338, 341, 342(47; 67), 497

Hendges, S. K., 225(7), 226

Hendrickson, W. A., 332(37), 333

Hendrix, C., 185

Henke, S., 262

Herbert, A., 139

Herdewijn, P., 185

Herpin, T. F., 18(80), 75, 79, 86, 90(27), 151,

154(1), 475, 487

Hersh, E. M., 113, 271, 289, 298, 299(1),

309(1), 497, 498(8), 499(8)

Hertzberg, R. P., 199

Heuser, D. J., 480

Heuts, J., 135, 139, 140(49)

Hewitt, M. C., 240(19), 242, 242(21), 245

Heyer, D., 53, 74

Heykants, J., 470

Heys, L., 199

Hiemstra, H. S., 294, 428

Hilbert, M., 438

Hildebrand, J. P., 375

Hindsgaul, O., 353, 354(18)

Hinzen, B., 369, 371, 371(10)

Hippe, T., 419

Hipskind, P., 481

Hiramatsu, K., 323, 331(3)

Hird, N., 396

Hirshmann, R., 296

Hocker, M. D., 446

Hockerman, S. L., 393, 394(32), 395(32),

396(32), 406(32)

Hodge, P., 392(23), 393, 395, 396, 397(54)

Hodges, J. C., 392, 392(28), 393, 393(12),

394(12), 395(11; 12), 396(12; 28),

399(12), 401(28), 402(12; 28), 405(12),

407, 480

Hodges, L. C., 388

Hodgson, J., 291(22), 292

Hodosi, G., 250

Hodson, S. J., 74

Hoecker, H., 304

Hoeg-Jensen, T., 426

Hoesl, C. E., 327, 496

Hoetelmans, R. M., 323

Hogan, J. C., Jr., 391

Holladay, M. W., 164, 416

Holloway, M. K., 185

Holm, A., 40

Holmes, A. B., 358(32), 359, 422

Holmes, C. P., 76, 164, 165, 170(2), 176(3; 4),

181(3), 262

Holub, D. P., 134

Holxman, T. F., 75

Homnick, C. F., 199

Hoogenboom, H. R., 289

Hopkins, B. T., 361(35), 362

Horan, N., 258

Horeis, G., 215(59), 216

Horl, W., 197, 470, 482(8), 489(8)

540 author index

Horn, J., 481

Horwell, D. C., 291(22), 292

Hosten, N. G. C., 24, 26

Hothi, B., 186

Houchins, B. J., 324

Houghten, R. A., 113, 115(4), 289, 290, 291,

292, 298, 324, 325, 327, 335, 338, 339(52),

341, 342(47; 51; 53), 424, 496, 497, 498,

498(9; 10), 499, 499(9), 500, 501,

501(17; 19), 502, 502(19), 503, 504,

505, 506, 506(23), 508, 508(37), 509,

509(20), 510(39; 40), 511, 512, 514(46),

515, 515(23)

Howell, D. N., 339

Hruby, V. J., 113, 185, 271, 288, 289, 298,

299(1), 309(1), 497, 498(8), 499(8)

Hsieh, M., 291

Huang, B., 262

Huang, W., 164(10), 165, 425

Huang, X., 152, 356

Hudson, D., 444, 465(25)

Huening, T. T., 324

Huff, J. R., 80, 185

Huffman, W. F., 185

Hughes, I., 134, 430

Hughes, J., 291(22), 292

Hulme, C., 469, 471, 473, 473(15),

474, 475, 478, 479, 480, 481, 484, 486,

487, 489(47)

Husar, G. M., 335, 499, 501(19), 502(19)

Hussain, F. M., 331

Hussain, J. P., 331

Hutchins, S. M., 396

Hutchinson, D. K., 225(7), 226

Hwang, S. M., 185

Hyman, C. E., 53, 70

Hynes, J., Jr., 296

I

Iacobelli, J., 470, 471(7)

Iaiza, P., 428

Ida, S., 517

Iino, M., 75

Ikeler, T. J., 80

Illgen, K., 197

Inamoto, K., 426

Ingallinella, P., 297

Inoue, H., 60, 62, 258

Iversen, T., 263

Iyer, M. S., 478

Izumi, M., 74

J

Jachuck, R., 396

Jackson, H. C., 480

Jackson, P. S., 348, 368, 392, 394(16)

Jackson, R. F. W., 396

Jackson, S. A., 223

Jacober, S., 87

Jacquault, P., 202, 203(34), 204(34)

Jagan Reddy, E., 204

Jain, R. K., 258, 261(22), 262

Jakas, D. R., 185

James, I. W., 39, 42, 43(19), 60(19),

127, 143(6)

James, S. N., 40, 115, 117(11)

Jana, U., 209

Janda, K. D., 42, 134, 290, 348, 349, 350(10),

426, 437

Jansen, M. A., 470

Janssen, P. A. J., 470

Jaroskova, L., 457

Jarvis, M. F., 75

Jarvis, S., 4

Jayawickreme, C. K., 290

Jefferson, E. A., 297

Jeger, P., 200(23; 24), 201, 392(24), 393

Jensen, K. J., 81, 90(23), 142

Jesberger, M., 349, 350(8)

Jiang, S., 332

Jin, S. J., 134

Jingwen, Z., 425

Johnson, R. K., 199

Jolivet, M., 184

Jones, D. G., 262

Jones, J. R., 202, 203(32)

Jones, L., 130

Jones, M., 79

Jones, R. C. F., 481

Jones, W., 469, 484, 487

Josephson, J., 75

Josey, J. A., 453, 475

Joshi, S., 60, 262

Jouin, P., 182, 185, 186(25), 187(25), 189(21),

191(25)

Joyner, C. T., 480

Judd, D. B., 392

Judkowski, V., 338, 341

author index 541

Jung, G., 3, 145, 291, 293, 338, 367, 370, 372,

376, 379, 392(25), 393

Jung, K. H., 250

Jung, K. W., 134

Junt, T., 338

Jurkiewicz, N. K., 480

K

Kachroo, P. L., 204

Kahne, D., 236, 258

Kahr, A.-L., 332

Kaiser, E., 22, 24, 25(1), 302, 513

Kaiser, T. E., 152

Kakarla, R., 262

Kakel, J. A., 74

Kakobsone, I., 152

Kaldor, S. W., 87, 391, 392(2), 394, 394(2),

396(2), 417, 480

Kalir, R., 152, 369

Kalivretenos, A. G., 425

Kallus, C., 262

Kaluza, Z., 430

Kamal, M. R., 140

Kamau, M., 262

Kaminiski, Z. J., 444

Kan, W. M., 75

Kane, P., 438, 459(12), 460(12), 461(12), 521

Kang, S.-B., 489

Kaniszai, A., 51, 116, 117(13)

Kantchev, A. B., 262, 396

Kanyi, D., 291

Kaplan, A., 424

Kapoor, T. M., 199

Kappe, C. O., 197, 198, 199, 199(10), 201,

202(39), 203, 204, 205, 205(46),

206(46; 50), 207, 208(53), 209(46; 54),

215(59), 216

Karabelas, K., 480

Karnbrock, W., 418

Karoly-Hafeli, H., 152

Kartsonis, N., 332

Karunaratne, K., 115, 117(8)

Kassahun, K., 199

Kassel, D. B., 4

Katchalsky, E., 152

Kates, S. A., 183, 185(7), 347, 348, 452

Katritzky, A. R., 138, 152, 504

Katz, J. L., 404(71), 405

Katzenellenbogen, J. A., 405

Kauffman, G. S., 192

Kavalek, J., 86

Kawatsure, M., 404

Kay, B. K., 291

Kay, C., 22

Kaye, J., 338

Kazarnovskii, S. N., 371

Kazmierski, W. M., 113, 271, 289, 298, 299(1),

309(1), 497, 498(8), 499(8)

Kearney, P. C., 143

Keating, T. A., 197, 394, 395(37), 470, 470(9),

471, 471(7), 474, 479, 489

Keck, G. E., 360, 361(34)

Keenan, R. M., 185

Kelder, J., 476

Kellam, B., 251

Keller, P. M., 5

Kelly, M., 480, 484, 489(47)

Kennedy, A. L., 475

Kennedy, R. M., 392(28), 393, 396(28),

401(28), 402(28)

Kenner, G. W., 152, 154(20)

Kent, S. B. H., 24, 25, 175, 454

Kerr, D., 152

Kerr, J. M., 290, 454

Kesarwani, A. P., 424

Keum, G., 489

Khan, T. H., 250, 258(4)

Khmelnitsky, Y., 204, 206(48)

Kick, E. K., 485

Kiefer, M. R., 517

Kieffer, B. L., 297

Kiely, J. S., 76, 417, 425(5)

Kienle, S., 293, 338

Kiesow, T., 86, 90(27), 151, 154(1), 487

Kihlberg, J., 154

Kim, B. M., 80

Kim, D. J., 430

Kim, H.-Y., 184

Kim, I. J., 294

Kim, J., 446

Kim, K., 521

Kim, M., 423

Kim, P. S., 332(36), 333, 334, 337(39)

Kim, R. M., 396

Kim, S., 392(24), 393

Kim, S.-H., 46

Kim, S. W., 297, 417, 423, 480

Kim, S.-Y., 200(23), 201

Kim, Y., 489

542 author index

Kimball, S. D., 199

King, N. P., 429

King, R. W., 199, 324

Kingston, D. G. I., 75

Kirby, C. J., 516

Kirkpatrick, D. L., 75

Kirschning, A., 348, 349, 350(8), 368, 392(25),

393, 396(25)

Kiselyov, A. S., 455

Kivity, S., 369

Klabunde, T., 75, 436

Klavins, M., 152

Kleeman, A., 327

Kleinwachter, P., 184

Kling, P., 199

Kloeppner, E., 74

Klopfenstein, S. R., 39(9), 40, 72(9), 75, 183,

422, 475

Kloss, P., 291

Klutchko, S., 480

Knapp, R. J., 113, 271, 289, 298, 299(1),

309(1), 497, 498(8), 499(8)

Kneib-Cordonier, N., 444, 465(25)

Knepper, K., 136

Knerr, L., 239

Ko, D.-H., 430

Kobayashi, S., 425(37), 426

Kobberling, J., 130, 131, 133(11), 134,

142(14), 144(14)

Kobrin, E., 251

Kobylecki, R. J., 77, 115, 257

Koch, G., 74

Koch, K., 428, 475

Kocis, P., 272

Koeller, K., 236

Koelsch, C. F., 139

Koenig, S., 498

Koerber, S. C., 184

Kogan, N., 262

Koh, H.-Y., 489

Koh, J. S., 417, 451,

467(34), 480

Koh, J. T., 456

Kohand, J. T., 46, 46(29)

Kohler, T., 332

Koide, T., 304

Kokke, W., 199

Kolodziej, S. A., 422

Kondo, T., 338, 342(47)

Kondo, Y., 426

Konradsson, P., 241(20), 242

Koomen, G.-J., 422, 445

Kornet, M. J., 347

Kostenis, E., 75

Kowalski, J., 421

Kowaluk, E. A., 75

Kraas, W., 291, 293

Krajcsi, P., 497

Krajnc, P., 364(42), 365

Krapcho, A. P., 473

Krchnak, V., 24, 51, 76, 112, 116,

117(13), 164, 164(8), 165, 271, 272,

272(3), 290, 293, 298, 299(4), 300(4),

309(4), 416, 456

Krepinsky, J. J., 250

Kretzschmar, T., 304

Krishnan, R., 338, 339(52)

Krolikowski, D., 86, 90(27), 487

Krolikowski, P., 86, 90(27), 151, 154, 154(1),

473, 487

Krstenansky, J. L., 202(37), 203,

204, 206(48)

Kruijtzer, J. A. W., 296, 417

Kruse, C. G., 428

Krutzik, P. O., 202(38), 203, 224

Kshirsagar, T., 24, 33

Ku, T. W., 185

Kubota, H., 124

Kuisle, O., 24, 28

Kukla, M. J., 470

Kulkarni, B. A., 427

Kumar, D., 204, 206(50)

Kumar, N. V., 86, 90(27), 151, 154, 154(1),

199, 473, 487

Kumar, S., 75

Kumar, V. N., 487

Kundu, B., 291, 424

Kuntz, I. D., 517

Kunz, H., 262

Kuo, L. C., 324

Kuo, M., 12

Kurth, M. J., 56, 175, 418,

430, 501

Kurz, M., 75

Kusumoto, S., 74

Kuvshinov, A. M., 140

Kwon, S., 46, 444

Kwon-Chung, K. J., 325

Kwong, P. D., 332(37), 333

Kyranos, J., 3

author index 543

L

Labahn, T., 136

Labaudiniere, R. F., 18(80), 79, 86, 90(27),

151, 154(1), 471, 474, 475, 478, 479, 487

Labeguere, F., 185, 186(25), 187(25), 189(21),

191(25)

Ladlow, M., 422

Lam, K. S., 76, 113, 271, 272, 272(3), 278,

278(10), 280(10), 284, 289, 290, 293, 298,

299, 299(1; 4), 300(4), 307, 309(1; 4), 313,

315, 317, 497, 498(8), 499(8)

Lam, P. Y. S., 224

Lam, S., 421, 425(22 g)

Lambert, J. N., 64, 183, 416

Lamothe, M., 418

Lan, X. F., 138

Lane, D. R., 430

Lang, F. B., 480

Langa, F., 202, 203(30)

Lange, J. M., 323

Langley, G. J., 388

Lannuzel, M., 418

Lansky, A., 257, 391

Lanter, C. L., 77

La Porta, E., 431

Larhed, M., 202, 202(36), 203, 203(33),

224, 228

La Rosa, C., 338, 339(52)

Lassalette, J. M., 384

Lau, D. H., 313

Lauffer, D. J., 87

Laursen, R. A., 347

Lautenschlager, W., 205, 216(51)

Lauterwasser, F., 130, 145

Lazarus, L. H., 185

Lazlo, P., 395

Lazny, R., 131, 142(14), 144(14)

Le, L., 173

Leach, A. G., 348, 353, 354(15), 368,

392, 394(16)

Leach, A. R., 518

Leadbeater, N. E., 357, 358(28)

Lease, T. G., 446

Lebedev, O. L., 371

Lebl, M., 24, 76, 186(32), 187, 271, 272,

272(3), 284, 290, 293, 298, 299(4), 300(4),

309(4), 438, 443, 456

Lebrilla, C. B., 278

Lecat, A., 423

LeClerc, S., 46

Lee, C. E., 485, 517

Lee, C. L., 423

Lee, E. J., 417

Lee, H., 3

Lee, J. H., 417, 480

Lee, M. H., 294

Lee, S.-H., 418

Lee, S. K., 423

Lee, W., 422

Lee, Y.-S., 418

Lees, J. H., 295

Leeson, P. D., 423, 518

Leftwich, M. E., 315

LeHetet, C., 425(37), 426

Lehman, A. L., 298

Leighton, R. J., 497

Lemaire, M., 177

Lenz, D. M., 425

Leo, G. C., 397, 402(62)

Leost, M., 444

Lepisto, m., 480

Lepore, S. D., 430

Leppert, P., 199

Lepre, C. A., 75

Lerner, M. R., 290, 498

Lerner, R. A., 498

Le Roch, M., 67

Letherbarrow, R. J., 296

Letsinger, R. L., 347

Levin, R. B., 79, 185

Levitz, S. M., 325

Lew, A., 202(38), 203, 224

Lewandowski, K., 200

Lewis, G. S., 164

Lewthwaite, R. A., 291(22), 292

Ley, S. V., 22, 73, 348, 351(12), 352, 353,

354(15; 17), 368, 369, 371, 371(10), 375,

376, 376(26), 392, 394(16), 396

Leznoff, C. C., 39

Li, B., 419

Li, J., 262

Li, L., 3

Li, M., 417, 419

Li, P.-K., 419

Li, R., 40, 115, 117(8)

Li, T., 429

Li, T. H., 429

Li, W.-R., 422

Liagre, M., 202(35), 203

544 author index

Liang, A., 481

Liang, L., 263

Liang, R., 258, 262

Liao, S., 288

Lidstrom, P., 202, 224

Lieb, M. E., 197

Lieberburg, I., 485

Liener, I. E., 152, 154(5)

Lillig, J. E., 79, 115, 117(8)

Lim, J., 124

Limal, D., 184

Lin, J. H., 80, 185, 324

Lin, K., 332

Lindeberg, G., 228

Lindhorst, T., 475

Ling, N., 338

Linn, J. A., 53, 74, 143

Lio, A., 42, 70(17), 100

Lipinski, C. A., 436

Lipshutz, B. H., 364, 407

Lipton, M. A., 421, 478

Liskamp, R. M. J., 296, 418, 425

Lisziewicz, J., 332

Little, D., 4, 5, 5(8)

Liu, D., 5

Liu, G., 63, 284

Liu, R., 271, 272, 278, 278(10), 280(10), 298,

299, 313, 317

Liu, W., 304

Livingston, D. J., 87

Lockhart, D. J., 46

Loebacg, J., 258

Lohner, K., 292, 324

Lolo, M., 24, 28

Lombardo, F., 436

Longbottom, D. A., 348, 368, 392, 394(16)

Longley, C., 262

Lonn, H., 260, 261(31)

Lori, F., 332

Lormann, M., 127, 130, 132(12), 134(12), 135,

137, 138

Lorsbach, B. A., 175

Lorthioir, O. E., 22

Lotti, V. J., 80

Lou, Q., 271, 315

Loupy, A., 202, 202(35; 40; 41), 203, 203(34),

204(34), 216(40)

Louridas, B., 473

Lu, A. T., 497

Lu, H. H., 421, 478

Lu, J., 209

Lu, S.-Y., 202, 203(32)

Luche, J.-L., 202(35), 203

Lucka, A. W., 498

Lui, D., 262

Luke, R. W. A., 403

Lunn, J., 473

Lutzke, R. A. P., 324

Lysek, R., 430

Lyu, C. S., 430

M

Ma, H., 209

Ma, L., 428, 474, 478, 479, 480

Ma, Q. N., 315

Ma, Y., 209

MacCoss, M., 80

Machacek, V., 86

Macher, B. A., 251, 252(12)

Maclean, D., 76, 165

Macquarrie, D., 396

Madder, A., 24, 26

Madrigal, J. A., 339

Maeji, N. J., 39, 40, 41, 59(6), 115, 117(11)

Magnus, A. S., 375

Mai, S., 199

Makdessian, T., 24, 33

Makino, S., 47, 49, 50

Mallett, D. M., 4, 5(8)

Malley, M. F., 199

Mankin, A. S., 291

Mann, G., 404

Mann, M., 396

Mann, T. D., 5

Manninen, P. R., 225(7), 226

Manning, C., 39

Mansell, H. L., 80

Mantellini, F., 419

Marcaccini, S., 489

Marcaurelle, L. A., 249

Marchioro, C., 138

Marcoux, J.-F., 404

Marenus, L. E., 290

Maresh, M. J., 473

Margolick, J. B., 332

Margue, R. G., 348

Marik, J., 271, 272, 278(10), 280(10), 299

Markel, S., 338, 339(52)

Markovic-Plese, S., 338, 341, 342(64)

author index 545

Marlowe, C. K., 421

Marques, A., 338, 342(47)

Marron, B. E., 74

Marsh, A., 364

Marshall, D. L., 152, 154(5)

Marshall, W. J., 418, 421(11i)

Martin, C., 376

Martin, R., 338, 341, 342(47; 64), 497

Martina, K., 435

Martineau, G. L., 80

Martinez, J., 58, 59(42), 423

Martinez-Picado, J., 332

Martın-Zamora, E., 384

Marton, L. J., 502

Maryanoff, B. E., 397, 402(62), 457

Marzinzik, A. L., 437

Masada, R. I., 444, 465(25)

Masala, S., 443, 444

Masko-Moser, J. A., 185

Maslana, E., 392

Maslouh, N., 418

Mason, T. J., 41

Masquelin, T., 391, 421, 424(22b), 438,

443(14), 461(14), 462(14)

Massi, A., 200, 349, 350(9), 392(27), 393

Masumoto, K., 481

Mathe, D., 202, 203(34), 204(34)

Mathew, R., 86, 90(27), 151, 154(1)

Mathews, J., 428

Mathieu, M. N., 42, 73(20)

Mathivanan, P., 394

Matsuda, A., 60

Matter, H., 436

Matthews, D. P., 394, 395(44)

Matthews, J., 417

Matthews, T., 332

Mauger, J., 349, 350(7)

Mayer, J. P., 164, 425

Mayer, K. H., 325

Mayer, T. U., 199

Mazurov, A., 164(9), 165

Mazzenga, G., 87

McBride, J. D., 296

McCarthy, J. R., 405

McCarthy, T. J., 80

McClenaghan, J., 475

McClure, K. J., 392

McDanal, V., 332

McDaniel, S. L., 79, 185

McDermott, J. R., 152, 154(20)

McDowell, R. S., 470

McFarland, H. F., 338, 342(47)

McGaraughty, S., 75

McGee, C., 164

McGee, L. R., 470

McGeehan, G., 86, 90(27), 151, 154(1), 487

McKaiser, E., 453

McKeown, S. C., 22

McKinney, E. R., 394

McKnight, A. T., 291(22), 292

McLaughlin, M. L., 185

McLoughlin, D. A., 324

McMullen, D. M., 199

McMurray, J. S., 306

McNally, J. J., 197

McNeil, D., 87

McQueney, M. S., 5

McRipley, R. J., 225

Meador, J. W. III, 63

Meaechler, L., 296

Medal, M., 517

Meecham, K., 291(22), 292

Meenhorst, P. L., 323

Meester, W. J. N., 358(29), 359

Meijer, L., 46, 444

Meldal, M., 314, 314(27), 315, 517

Melean, L. G., 239, 250

Meloen, R. H., 40, 43(10), 100, 289, 497

Meloni, M. M., 24, 29, 30(10)

Menard, P. R., 18(80), 79, 473

Mendre, C., 184

Mengel, A., 376

Meola, A., 498

Mermet, C., 480

Merrifield, R. B., 24, 25, 40, 99(4), 100, 112,

127, 175, 258, 261(24), 289, 347, 397,

407(61), 497

Merritt, A. T., 391, 392

Metlay, J. P., 331

Meunier, N., 438, 443(14), 461(14), 462(14)

Meutermans, W., 248, 260

Meyer, J.-P., 327, 499, 509(20), 512

Meyer, V., 376

Mezzasalma, T. M., 5

Miao, C., 87

Michael, N. L., 332

Michea-Hamzehpour, M., 332

Michelotti, E. L., 418

Michels, R., 369, 382(13)

Michelson, S. R., 80

546 author index

Midha, S., 262

Miller, J. F., 428, 475

Miller, K. E., 480

Miller, M. A., 302

Miller, W. H., 185

Mills, S. G., 80

Min, I. K., 430

Mingos, D. M. P., 202, 208(29)

Mischke, D. A., 394, 395(42)

Mishani, E., 80

Missio, A., 396

Mitchell, H. J., 79, 115, 236

Mitchell, J. P., 183, 416

Mitchison, T. J., 199

Miyaura, N., 404

Mjalli, A. M. M., 42, 70(17), 100, 474, 480

Moberg, C., 202, 203(33)

Mohammad, A. A., 140

Mohan, R., 456

Mol, J. C., 358(29), 359

Monaci, P., 498

Monenschein, H., 348, 368, 392(25),

393, 396(25)

Montalbetti, C., 396

Montanari, F., 372, 373(22)

Montelaro, R. C., 302

Moon, H.-S., 430

Moore, C. G., 199

Moore, G. J., 296

Moore, J. P., 332

Moore, J. S., 130, 135(9)

Moos, W. H., 454

Morales, G. A., 185

Moran, E. J., 42, 70(17), 100

Moree, W. J., 425

Moreland, S., 199

Moreno, A., 202, 203(30)

Morera, E., 178

Morgan, C. L., 339

Morgan, D. O., 46

Mori, T., 75

Moriconi, R., 480

Moriggi, J.-D., 428

Moriyama, S., 75

Morize, I., 471

Morley, A. D., 79

Morphy, J. R., 165, 419

Morris, D. R., 502

Morrison, D., 5

Morrissette, M., 473, 480

Morrissey, M. M., 65, 456, 481

Morte, C., 339

Morton, G. C., 18(80), 75, 79, 86, 475, 479

Moser, H., 438, 459(12), 460(12), 461(12), 521

Motherwell, W. B., 357

Motti, C., 498

Mross, E., 250

Mueller, S., 439, 444(16)

Mui, P. W., 87

Muir, J. C., 138

Mulbaier, M., 376

Mulder, J. W., 323

Muller, B., 184

Muller, S., 521

Muller, U., 478

Mullican, M. D., 87

Munson, M. C., 453

Munstedt, K., 138

Murakami, K., 252

Murayama, E., 263

Murcko, M. A., 75, 294

Murer, P., 200

Murphy, F., 428, 431

Murphy, P. J., 199

Murphy, P. M., 332(38), 333, 334(38)

Murray, C. M., 294

Murray, J. P., 396

Murray, P. J., 422

Murray, W., 399, 402(63)

Mynard, K., 339

N

Nadji, S., 418

Nagarathnam, D., 199

Nagel, A., 87

Naing, W., 394, 395(43)

Najdi, S., 418

Najera, C., 361(36; 37), 362

Nakanishi, E., 47, 49, 50

Nakatubo, F., 252

Namdev, N. D., 478

Nantermet, P. G., 199

Nanthakumar, S. S., 74

Naumann, T., 436

Navas, F. J. III, 74

Navre, M., 421, 425(22 g), 517

Nechuta, T., 185, 186(24), 189(24)

Needels, M. C., 164, 173

author index 547

Nefzi, A., 324, 327, 496, 497, 498(10), 499,

502, 504, 505, 506, 509, 509(20), 510(40),

511, 512

Neipp, C. E., 143, 444

Nelson, J. C., 130, 135(9)

Nelson, K. H., Jr., 391, 393

Nemoto, H., 60, 62

Neset, S. M., 396

Nesi, M., 348, 368, 392, 394(16)

Neurath, A. R., 332

Neustadt, B. R., 185, 186(24), 189(24)

Newlander, K. A., 185

Ngo, T. T., 24

Nguyen, T. H., 323

Ni, Z.-J., 76, 165

Nicewonger, R. B., 152, 393, 396(30)

Nichols, A., 185

Nicholson, G., 372, 384, 388(42)

Nicolaou, K. C., 76, 79, 100, 115, 117(8), 124,

130, 236, 262, 356, 378, 428, 429, 431

Nicolas, E., 418

Nicosia, A., 498

Nieczypor, P., 358(29), 359

Ninkovic, S., 429

Nishimura, T., 252

Nixey, T., 469, 473(15), 474, 481, 484, 486,

489(47)

Norberg, T., 260, 261(31)

Norman, M. H., 74

Norman, T. C., 46, 46(29), 444, 456

Normandin, D. E., 199

Normansell, J. E., 392(23), 393

Nortey, S. O., 397, 402(62)

Nova, M. P., 76, 100, 115, 117(12)

Novack, A. R., 446

Nuchter, M., 205, 216(51)

Nukada, T., 252

Nunes, A., 481

Nuske, H., 136

Nussbaum, O., 334

Nutt, D. J., 480

Nuzzo, M., 498

Ny, P., 291

Nyce, P. L., 87

O

Oas, T., 332

Oates, L. J., 24, 31

Obrecht, D., 391, 421, 424, 424(22b)

Ockey, D. A., 430

Oda, Y., 184

Oh, H. J., 517

Olah, T. V., 199, 324

Old, D. W., 404

Ollmann, I. R., 259

Olmstead, M. M., 418

Olsen, D. B., 324

O’Malley, S. S., 199

Ondruschka, B., 205, 216(51)

Ong, N. A., 504, 511

Opatz, T., 262

Oprea, T., 518

Orain, D., 74

O’Reilly, B. C., 199

Organ, A., 4, 5

Ornstein, P., 481

Ortar, G., 178

Orton, E., 86, 90(27), 151, 154, 154(1), 487

Osborn, M. I., 250, 258(4)

Oscarson, S., 255, 260(17)

Ostovic, D., 185

Ostrem, J. A., 272

Ostresh, J. M., 324, 327, 335, 424, 496, 497,

498, 498(10), 499, 500, 501, 501(17; 19),

502, 502(19), 503, 504, 505, 506, 506(23),

508, 508(37), 509, 509(20), 510(39), 511,

512, 515(23)

Otaka, A., 304

Ouyang, X., 455

Overland, D., 487

Owens, R. A., 324

Oye, G., 24, 31

P

Padera, V., 112

Paio, A., 138, 361(38), 362, 431

Palmacci, E. R., 235, 236(9), 237, 242(21), 245

Pan, J., 7, 12, 392

Pan, Y., 164, 176(3; 4), 181(3)

Paneth, P., 444

Panganiban, L. C., 498

Panunzio, M., 396

Paoli, P., 489

Pappageorgiou, J., 254, 259

Paquette, L. A., 371

Parandoosh, Z., 76, 100

Parang, K., 353, 354(18)

Pareja, C., 384

548 author index

Parham, C. S., 199

Park, J. Y., 294

Park, K., 56

Park, K.-H., 418, 419

Park, S., 313

Park, S.-J., 489

Parkanyi, C., 470

Parker, M. F., 185

Parlow, J. J., 392, 392(23), 393, 394, 394(32),

395(32; 42; 43), 396(32), 406(32)

Parquette, J. R., 262, 396

Parr, N. J., 22

Parrish, C. A., 358(30), 359

Parrot, I., 438

Parry, D. M., 419

Parsons, J. G., 39

Pascal, J., 338, 342(47)

Pascal, R., 182, 184, 185, 186, 186(25; 31; 32),

187, 187(25), 189(21; 30–32), 190(31),

191(25), 192(30)

Passerini, M., 484

Pastor, A., 152

Pastor, J., 262, 356, 428, 429, 431

Patchornik, A., 152, 369

Pate, M., 487

Patek, M., 116, 117(16), 186(32), 187

Patel, A., 5

Patel, D. V., 391, 421, 425(22 g)

Patel, M. V., 185

Patick, A. K., 63

Patil, A. D., 199

Patrick, T. B., 385

Pattarawarapan, M., 143

Pattenden, G., 138

Paul, G. M., 262

Paul, S., 204, 206(47)

Pauls, H. W., 151(2), 152, 160(2)

Pauwels, R., 470

Pavia, M. R., 76, 417, 425(5)

Pears, D., 403

Pechere, J.-C., 332

Pedersen, W. B., 40

Pedoroso, E., 419

Pei, Y., 421

Peng, G., 417

Peng, J. W., 75, 473, 475

Peng, S.-Z., 422

Pennacchio, M., 7

Pennington, M. E., 313

Pepino, R., 489

Peplow, M. A., 134

Perera, S., 42, 73(20)

Perez, M., 418

Perez-Paya, E., 292, 497

Perfect, J. R., 325

Pernerfoster, J., 428

Perreux, L., 202(40), 203, 216(40)

Persson, M. A. A., 498

Pessi, A., 297

Pesti, J. A., 192

Peters, W. A., 498

Petersen, S. V., 526

Peterson, M. L., 75(5), 76

Petit, A., 202, 203, 203(34), 204(34; 35)

Petricci, E., 152

Petropoulos, C. J., 332

Petrovsky, N., 341

Pfefferkorn, J. A., 79, 115, 124

Pfefferkorn, M., 142, 143(54)

Pham, Y., 40, 42, 73(20), 115, 117(11)

Phan, H., 315

Phelan, J. C., 201

Phoon, C. W., 71

Pi, J., 424

Piarulli, U., 396, 431

Pica, A., 480

Picard, S., 423

Pichler, S., 215(59), 216

Piergentili, A., 480

Pierson, T., 332

Pigini, M., 480

Pillay, D., 323

Pilot, C., 145

Pinel, C., 323, 331(4)

Pinilla, C., 113, 290, 298, 322, 324, 325, 338,

339(52), 341, 342, 342(47; 51; 53; 64),

497, 498, 498(9; 10), 499(9), 514(46), 515

Pirard, B., 436

Pirrung, M. C., 497

Piscopio, A. D., 428, 475

Plante, O. J., 235, 236(9), 237, 239,

240(18), 241

Plasterk, R. H., 324

Plesiat, P., 332

Ploemen, J.-P., 476

Plumb, R. S., 4, 5(8)

Plunkett, M. J., 43, 134, 197, 415, 437,

446, 475

Pohl, M., 304

Polaskova, M. E., 64

author index 549

Pollak, A., 376

Pollino, J. M., 358(31), 359

Ponton, J., 323, 331(4)

Pop, I. E., 152

Popescu, C., 142

Poppe, L., 487

Porcu, G., 24, 32, 356(26), 357

Porter, E. S., 291

Portlock, D. E., 39(9), 40, 72(9), 75, 183, 417

Post, J., 481

Potash, H., 115, 117(12)

Potts, B. C. M., 199

Powderly, W. G., 325

Preston, S. L., 80

Pritchard, M. C., 291(22), 292

Protti, M. P., 341

Provera, S., 431

Purchase, T. S., 138

Pye, P. J., 490

Q

Qian, C., 209

Qian, H., 152

Qian, M. G., 12

Quaglia, W., 480

Quibell, M., 54(39), 55

Quici, S., 372, 373(22)

Quillan, J. M., 290

Quinn, T. C., 332

Quinoa, E., 24, 28

Quintero, J. C., 79, 185

Qushair, G., 21

R

Rabinowitz, M., 392

Rachwal, B., 504

Rachwal, S., 504

Raddrizzani, L., 341

Rademann, J., 145, 250, 366, 367, 370, 372,

375, 376, 379, 383, 384, 387, 388(42)

Raeymaeckers, A., 470

Rafalski, M., 224

Rafelt, J., 396

Rahman, S. S., 24

Rajasree, K., 351(13), 352

Ramalingam, T., 204

Rammensee, H.-G., 339

Ramshaw, C., 396

Randal, M., 75

Rankovic, Z., 165

Rano, T. A., 324

Ransom, R. W., 199

Ranu, B. C., 209

Rao, C., 453

Raphael, D. R., 75

Raphy, J., 291(22), 292

Rapoport, H., 427

Rapp, W., 418

Rasoul, F., 40, 115, 117(11)

Rastogi, S. K., 424

Ratcliffe, G. S., 291(22), 292

Rau, H. K., 295

Rawson, T., 470

Rea, P., 42, 52, 73(20)

Reader, J. C., 77, 79, 115

Reader, V. A., 79

Reamer, R. A., 80

Rees, D. C., 165, 481

Regen, S. L., 371

Reger, T. S., 348

Reich, S. H., 24

Reichwein, J. F., 296

Reider, P. J., 80, 490

Reilly, Y., 80

Reinecke, U., 443, 521

Reiss, D., 199

Reissmann, S., 184

Reitz, A. B., 131, 397, 402(62)

Renault, J., 67

Rene, L., 185

Renil, M., 313, 517

Renou, C. C., 79

Reynolds, M. E., 470

Reynolds Cody, D. M., 417, 425(5)

Richardson, R. S., 481

Richman, D. D., 332

Riechmann, L., 295

Riguera, R., 24, 28

Riley, P., 42, 73(20)

Rinnova, M., 504

Rittle, K. E., 199, 470

Riva, R., 484

Riva-Toniolo, C., 439, 444(16), 450,

466(33), 521

Rivero, R. A., 77, 417, 428

Rivier, J., 184

Rizo, J., 184

550 author index

Rizzo, A., 54(39), 55

Ro, S., 297, 417, 480

Robarge, K. D., 470

Roberts, K. D., 64, 183, 416

Roberts, S. M., 419

Robidoux, A. L. C., 87

Robinett, L. D., 201

Robinson, J., 332(37), 333

Robinson, S., 143, 444

Rodda, S. J., 41

Rodebaugh, R., 60, 262

Rodenko, B., 445

Roecker, A. J., 79, 115

Roep, B. O., 294

Romano, J., 474, 479

Romanovskis, P., 296, 306

Romero, P., 338, 342(51; 53)

Romoff, T., 428

Rosania, G. R., 444

Roschangar, F., 429

Rose, M., 487

Rosenberg, E., 332

Rosenberg, S., 46

Rosenquist, A., 296

Rosenthal, A. S., 87

Roskamp, E. J., 143, 444

Rosse, G., 438, 443(14), 461(14),

462(14)

Rossen, K., 80, 490

Rossi, T., 392, 396

Roth, B. D., 138

Roth, H. J., 327

Roussel, P., 438, 459(12), 460(12),

461(12), 521

Routledge, C., 80

Rovnyak, G. C., 199

Royo, M., 31, 183, 185(7), 422

Rozenbaum, W., 323

Rozhkov, V. V., 140

Rubio-Godoy, V., 338,

342(51; 53)

Ruchel, R., 323, 331(4)

Rudewicz, P. J., 5

Rudzinski, J., 444

Rueter, J. K., 397, 402(62)

Ruhland, B., 164, 176(4), 177

Ruhland, R., 447, 463(31), 464(31)

Rutjes, F. P. J. T., 358(29),

359, 428

Ryu, S. H., 294

S

Sadighi, J. P., 405

Safar, P., 24, 116, 117(16)

Sager, J., 80, 490

Saguer, P., 186, 189(30), 192(30)

Saha, M., 204

Sahin, U., 341

Sakamoto, T., 426

Sakata, S., 481

Salata, J. J., 480

Salhi, Y., 323

Salmon, S. E., 113, 271, 272, 289, 298, 299(1),

309(1), 497, 498(8), 499(8)

Salmon-Ceron, D., 339

Salter, R. D., 339

Salvadori, S., 185

Salvino, J. M., 18(80), 79, 86, 90(27), 151,

151(2; 3), 152, 154, 160(2), 475, 487

Salvino, J. S., 151, 154(1)

Samanen, J. M., 185

Sanchez, C., 360, 361(34)

Sandanayake, S., 42, 73(20)

Sandra, P., 185

Saneii, H., 100, 116

Sanseverino, M., 183, 185(7)

Sansoni, B., 369

Santagada, V., 185

Santagostino, M., 376

Santi, D. V., 290

Santini, R., 396

Sarabia, F., 429

Sarin, V. K., 24, 25, 175

Sarohia, R., 444

Sarshar, S., 42, 70(17), 100, 474

Sarvetnick, N., 341

Saubern, S., 224

Sauleau, A., 425(37), 426

Saunders, D., 304

Sauvagnat, B., 151(3), 152

Savara, A., 332

Scarborough, R. M., 164(10), 165

Schaefer, K., 419

Scharn, D., 443, 521

Schelhaas, M., 258, 259(25)

Schellekens, G. A., 293

Schellekens, K., 470

Scheuerman, R. A., 172, 174(15)

Schiemann, K., 138

Schiller, P. W., 291, 497

author index 551

Schinkel, A. H., 422

Schleif, W. A., 79, 80, 185, 324

Schmid, C. R., 372

Schmid, D., 379

Schmid, D. G., 3, 145

Schmidt, R. R., 239, 250, 263

Schmidt, W., 262

Schmitt, R., 392

Schneck, J. P., 338, 339(52)

Schneider-Mergener, J., 443, 521

Schober, D., 481

Schoemaker, H. E., 428

Schols, D., 470

Schonberger, A., 349, 350(8)

Schoner, C. C., 499, 501, 509(20)

Schoofs, P. G., 41

Schore, N. E., 430

Schorn, T. W., 199

Schreiber, S. L., 124, 127, 199, 366(2), 367,

489, 490

Schroder, K., 338, 341

Schroeder, M. C., 76, 417, 425(5)

Schroen, M., 135, 136, 142

Schuler, P., 384, 388(42)

Schultz, P. G., 46, 46(29), 438, 444, 444(15),

449(15), 456, 458(15), 465(15)

Schulz, E., 177

Schumm, J. S., 130

Schuster, M., 428

Schwartz, J., 199

Schwarz, M. K., 164, 175, 454, 467(44)

Scialdone, M. A., 418, 421(11i)

Scicinski, J. J., 22

Scott, B. O., 421

Scott, I., 339

Scott, J. K., 289, 498

Scott, J. S., 348, 368, 392, 394(16), 396

Scott, K., 396

Sebestyen, F., 75(5), 76, 99(2; 3), 100, 113,

298, 435

Seeberger, P. H., 235, 236(9), 237, 239,

240(18; 19), 241, 242, 242(21), 245, 249,

250, 258(5)

Seeley, J. A., 430

Seitfried, R., 354, 355(19)

Sekanina, K., 258

Selkoe, D., 485

Selnick, H. G., 199

Semin, D., 484

Semmelhack, M. F., 372

Seneci, P., 138, 361(38), 362, 392, 396, 431

Senkal, B. F., 396, 408

Sensci, P., 392

Senyei, A., 76, 100

Seo, J. K., 294

Sepetov, N., 272, 293

Sergeev, A. G., 404(72), 405

Seri, C., 152

Seto, C. T., 297, 517

Severin, J., 75

Severino, B., 185

Sevignon, M., 177

Seymour, E., 39

Shah, A., 5, 24

Shahbaz, M. M., 42, 70(17), 100

Shao, C., 313

Shao, H., 423

Shao, L.-X., 152

Shao, X., 422

Sharkov, N., 517

Shaughnessy, K. H., 404

Shaw, K., 481

Sheehan, C. S., 39

Shen, J. Q., 498

Sheng, S.-R., 356

Sheppard, R. C., 152, 154(20)

Sherrington, D. C., 368, 392(23), 393, 395

Shevelev, S. A., 140

Shi, L., 421, 425(22g), 517

Shiba, T., 184

Shibano, T., 75

Shide, A., 304

Shieh, M. T., 396

Shimn, Y. S., 297

Shiosaki, K., 489

Shohet, S. B., 251, 252(12)

Short, K. M., 480

Showalter, H. D. H., 138

Shreder, K., 422

Siani, M. A., 290

Sieber, P., 83

Siegel, M. G., 391, 392(2), 394, 394(2),

395(44), 396(2)

Siegmund, A. C., 421

Sierra, T., 12

Sigmund, O., 369

Siliciano, J. D., 332

Siliciano, R., 332

Sillivan, D. J., 323, 331(4)

Silva, D. J., 258, 261(22), 262

552 author index

Sim, M. M., 71, 423

Simkovsky, N. M., 419

Simon, R., 338, 341, 342(47; 51; 53; 64)

Simonin, F., 297

Singh, A., 75

Singh, J., 75

Sinha, S., 485

Sinigaglia, F., 341

Sircar, I., 480

Sissons, J. G., 339

Sjo, P., 480

Skalitzky, D. J., 63

Skehel, J. J., 332(35), 333

Skiles, J. W., 87

Skillman, A. G., 517

Slade, J., 87

Slee, A. M., 225

Slemmon, J. R., 5

Sleph, P. G., 199

Smerdka, J., 145, 379

Smith, A., 258

Smith, A. B. III, 296

Smith, A. L., 423, 469

Smith, E. M., 185, 186(24), 189(24)

Smith, G. P., 289, 498

Smith, J. M., 51, 116, 117(13), 164(8), 165

Smith, K., 332

Smith, M. L., 396

Smith, R., 75

Smith, R. A., 421

Smith, S. C., 364

Smrcina, M., 116, 117(16)

Snyder, M., 65

Soderberg, E., 241(20), 242

Sodroski, J., 332(37), 333

Sofia, M. J., 258, 261(22), 262

Sohn, A., 417

Sola, R., 182, 185, 186, 186(25; 31; 32), 187,

187(25), 189(21; 30–32), 190(31),

191(25), 192(30)

Solache, A., 339

Solas, D., 497

Solberg, C. O., 323, 331(2)

Soll, R. M., 446

Solomon, L., 75

Somers, T. C., 470

Song, A., 278, 298, 313

Song, K. Y., 470

Song, L.-W., 313

Songster, M. F., 81, 90(23), 142

Sophiamma, P. N., 152

Sorcek, R., 87

Sorg, G., 376

Soriano, J. M., 361(36; 37), 362

Sorrell, T. C., 325

Souers, A. J., 183, 185(5), 296

South, M. S., 392, 393, 394, 394(32),

395(32; 43), 396(32), 406(32)

Southhall, L. S., 185

Spangle, L. A., 87, 417, 480

Spatola, A. F., 295, 296, 306

Spear, K. L., 46, 421

Spetzler, J. C., 517

Spoerri, H., 56

Sprengeler, P. A., 296

Sreekumar, K., 152

Sridharan, V., 416

Srivastava, G. K., 424

Srivastava, S. K., 437

Sroka, T., 271, 313

Stadler, A., 197, 204, 205, 205(46), 206(46),

207, 208(53), 209(46), 215(59), 216

Stadlwiesser, J., 418, 421, 424(22b)

Stahl, M., 295

Stankova, M., 438, 443

Stankove, M., 293

Stankovic, C. J., 76, 417, 425(5)

Stanton, J. J., 87

Stauffer, S. R., 405

Steel, P. G., 24, 31, 391

Steele, J., 257, 419, 454

Steele, T. G., 199

Stefani, H. A., 204, 206(49)

Steger, M., 517

Stegman, K., 422

Stein, D. S., 80

Steinbruckner, C., 470, 479, 482(8), 489(8)

Steinmetz, A., 419

Stephensen, H., 521, 522(18)

Sterba, V., 86

Stern, M., 152

Sternbach, L. H., 470

Stevanovic, S., 339

Stevens, D. A., 323, 331(4)

Stewart, A., 75

Stieber, F., 134

Stien, D., 356(25), 357

Still, W. C., 258, 299

Stirling, M., 79

Stoermer, R., 139

author index 553

Stone-Elander, S., 202, 203(32)

Stonehouse, D. F., 391

Storer, R. I., 348, 353, 354(15), 368,

391, 392, 394(16)

Strader, C. D., 296

Strader, T., 4

Stramiello, L. M. S., 143, 444

Straus, S., 338, 342(47)

Strick, N., 332

Stringer, O. D., 443, 462

Strop, P., 116, 117(16), 272

Stroud, R. M., 75

Stryer, L., 497

Studer, A., 200(23; 24), 201, 392(24), 393

Stults, C. L., 251

Sturniolo, T., 341

Subba Reddy, B. V., 204

Subra, G., 58, 59(42)

Suessbrich, H., 480

Suh, P. G., 294

Suman-Chauhan, N., 138,

291(22), 292

Sung, M. H., 338

Sutherlin, D. P., 444

Suto, M. J., 391

Sutton, L., 332

Suzuki, A., 404

Suzuki, N., 49, 50

Svec, F., 152, 200

Svendsen, I., 314, 314(27), 315

Svensson, A., 154

Svobodova, G., 86

Swali, V., 388

Swann, R. T., 77

Swanson, B. N., 199

Swayze, E. E., 297

Sweet, R. W., 332(37), 333

Szardenings, A. K., 421, 425(22g),

478, 517

Szewczuk, L., 517

T

Taddei, M., 24, 29, 32, 356(26), 357, 443, 444

Tadesse, S., 484

Tailor, J., 404

Tait, B., 138

Takahashi, E., 292, 497

Takahashi, K., 485

Takahashi, T., 60, 62, 258

Takano, T., 252

Tako, A., 418

Tam, J. P., 24, 25, 40, 175, 304

Tamaki, M., 185

Tampe, R., 293

Tanaka, H., 60

Taneja, H., 204

Tang, J. X., 353, 354(16), 394

Tang, J.-Y., 353, 354(16)

Tang, L., 6

Tang, S.-Y., 18(80), 79, 471, 479

Tanner, H. R., 100, 116

Tartar, A. L., 152

Tata, J. R., 324

Tatsuta, K., 235

Tayebati, S. K., 480

Taylor, C. M., 236

Taylor, E. W., 12

Taylor, J. W., 183, 184(3)

Taylor, S. J., 348, 353, 354(17), 368,

392, 394(16)

Teague, S. J., 477, 518

Teirney, J., 224

Teitze, L. F., 419

Temesgen, Z., 323

Tempest, P. A., 197, 469, 473(15), 474, 480,

481, 489

ten Holte, P., 419

Tenson, T., 291

Terrett, N. K., 257

Terstappen, G., 392

Tew, D. G., 517

Texier-Boullet, F., 202, 203(34), 204(34)

Thijs, L., 419

Thomas, A. W., 376

Thompson, C., 258

Thompson, L. A., 42, 81, 89(23b), 90(23), 127,

391, 392(22), 393

Thomson, C. G., 423

Thorsett, E. D., 470

Thunnissen, A.-M., 46

Thurmer, R., 430

Tian, Z., 235

Tibirica, E., 480

Tice, C. M., 418

Tied, A., 205, 216(51)

Tien, D. W., 421, 425(22g), 478, 517

Tierney, J., 202

Tietze, L. F., 197, 419

Tischler, M., 470

554 author index

Tokushige, D., 12

Tolson, D., 5

Tomasi, S., 67

Tomaszek, T. A., 517

Tomida, S., 60, 62

Tomioka, M., 361(39), 362

Tomori, H., 405

Tong, S., 423

Toops, D. S., 225(7), 226

Toshima, K., 235

Toth, I., 251

Toy, P. H., 426

Trainor, R. W., 40, 115, 117(11)

Travers, S., 392

Tribbick, G., 41

Trinh, L., 65, 481

Tripp, J. A., 152

Trivedi, B. K., 138

Troll, W., 24, 25

Trost, B. M., 263, 387

Trump, R. P., 74

Truneh, A., 199

Tsai, S., 489

Tsuji, J., 60, 62

Tsuji, T., 47, 49, 50

Tu, S. J., 204

Tucker, L., 341

Tuereci, O., 341

Tumelty, D., 164, 170(2), 172, 173, 174(15),

175, 454, 467(44)

Tuna, M., 295

Tymoshenko, D. O., 138, 152

Tyson, R. G., 184

Tzehoval, E., 184

Tzou, A., 338, 341, 342(47; 64)

U

Uebel, S., 293

Uehlin, L., 356

Ugi, I., 197, 470, 473, 475, 479,

482(8), 489(8)

Ulanowicz, D. A., 225(7), 226

Ulhaq, S., 75

Unger, S. E., 199

Uozumi, Y., 258

Uriac, P., 67

Uriarte-Villares, E., 422

Uskokovic, M., 470, 471(7)

Uzinskas, I., 185

V

Vacca, J. P., 79, 80, 185

Vagner, J., 24, 81, 90(23), 142, 164(8), 165,

272, 288, 456

Vahrson, H., 138

Vaino, A. R., 42

Valerio, R. M., 39, 40, 41, 46, 59(6)

Vallberg, H., 429

Valmori, D., 338, 342(51; 53)

Valverde, M. G., 201

Van Aerschot, A., 185

van Bekkum, H., 372, 374(20)

Van Den Nest, W., 183, 185(7)

van Esseveldt, B. C. J., 419

Van Gelder, J., 470

van Heeswijk, R. P., 323

Van Kirk, K. G., 79

van Loevezijn, A., 422

van Maarseveen, J. H., 422, 428

van Rihn, R. D., 296

Varady, L., 152, 393, 396(30)

Varki, A., 248

Varma, R. S., 202, 203(31), 204, 206(50)

Vassar, R., 485

Vastag, K., 80

Vazquez, J., 21, 24, 32

Veber, D. F., 470, 517

Vedkamp, A., 323

Vedsø, P., 426

Veerman, J. J. N., 428

Venslavsky, J. W., 185

Ventura, M. C., 4

Venuti, M. C., 470

VerdierPinard, P., 429

Vergelli, M., 338

Verkade, J. G., 453

Versluis, C., 296

Vidal, A., 504

Vignola, N., 145, 355

Vigouroux, C., 323

Vikstrom, B., 313

Villa, M., 396

Villagordo, J. M., 391, 421, 424, 424(22b)

Virgilio, A. A., 65, 296

Visser, G. M., 422, 428

Viswanadhan, V., 484

Voelter, W., 430

Vojkovsky, T., 24, 28, 189, 303

Volante, R. P., 80, 490

author index 555

Volz, F., 473

von dem Bussche-Huennefeld, C., 257

von Richter, V., 139

Voronkov, M., 152

Vourloumis, D., 429

Vouros, P., 3

W

Wachter, M., 399, 402(63)

Wade, S., 290, 293

Wagaw, S., 404

Wager, C. A., 360, 361(34)

Wakabayashi, T., 425(37), 426

Wakamiya, T., 184

Walden, P., 338

Waldmann, H., 134, 258, 259(25)

Walker, B. D., 332, 342

Walker, C. H., 137

Wallace, A., 297

Wallbaum, S., 489

Walter, K., 75

Wang, B., 521

Wang, C., 262

Wang, G.-P., 376

Wang, H., 204, 258, 261(22), 262, 422,

424, 425(32)

Wang, K. C., 75

Wang, L., 209

Wang, M., 7, 131, 142(14), 144(14)

Wang, T., 4

Wang, X., 72(57), 73

Wang, Y., 421, 425(22g), 428

Wang, Z., 209

Wannamaker, M. W., 87

Wanner, M. J., 445

Ward, G. J., 481

Warmus, J. S., 392(28), 393, 396(28), 401(28),

402(28)

Warshawsky, A., 152, 185, 369

Watanabe, N., 262

Waterhouse, J., 396, 397(54)

Wathey, B., 202, 224

Watkinson, M., 348, 392(29), 393, 396(29)

Watson, S., 75

Watson, S. P., 392

Watt, A. P., 5

Weatherston, J., 39

Webb, R. R., 470

Webb, T. R., 185

Webdale, L., 138

Weber, L., 197, 428, 489

Weber, P. A., 292, 324, 335, 499,

501(19), 502(19)

Weck, M., 358(31), 359

Weekes, M. P., 339

Wegrzyniak, E., 116, 117(16)

Weichsel, A. S., 456

Weigel, L. O., 394, 395(44)

Weik, S., 372, 375, 383

Weissenhorn, W. A., 332(35), 333

Weissman, S. A., 80

Welch, M. J., 80

Weller, H. N., 392

Wells, J. A., 75

Wells, N. J., 388

Wels, B., 296

Wenner, W., 470, 471(7)

Wenschuh, H., 443, 521

Wermuth, C. G., 438

Wertman, K., 290

Wessel, H.-P., 263

West, L., 475

West, M. L., 248, 251, 254

West, T. R., 404(71), 405

Westley, J. W., 199

Westman, J., 202, 224, 354, 355(20)

Westphal, V., 517

Wheatley, J., 5

White, A. C., 80

White, H. L., 480

White, P. D., 39, 186

Whitesides, G. M., 235

Whitfield, D. M., 250, 252

Whitlow, M., 481

Whitney, L. W., 341, 342(64)

Whitter, W. L., 470

Wickham, G., 40, 42, 43(19), 52, 60(19),

115, 117(11)

Widman, O., 139

Wiesler, W., 235

Wiesmuller, K.-H., 293, 338

Wild, C., 332

Wiley, D. C., 332(35), 333

Wiley, M. R., 430

Willaredt, R. P., 385

Williams, L., 396

Wills, M. R., 339

Wilson, D. B., 338, 341

Wilson, D. H., 338

556 author index

Wilson, L. J., 417, 419

Wilson, M., 138

Wilson, M. W., 392(28), 393, 396(28),

401(28), 402(28), 407

Wilson, R., 331

Winkle, J. H., 498, 501(17)

Winssinger, N., 130, 262, 356, 428, 429, 431

Winter, G., 289, 295

Winternitz, F., 423

Winters, R. T., 480

Winther, J. R., 517

Wipf, P., 184, 200, 200(23; 24), 201,

392(24), 393

Wirth, T., 356

Wischnat, R., 259

Witte, O. N., 315

Wittenberg, R., 348, 368, 392(25),

393, 396(25)

Wodicka, L., 46

Wolfe, J. P., 404, 405

Wolfe, M. M., 290

Wollmer, A., 304

Wolman, Y., 369

Wong, A. S., 185

Wong, C.-H., 235, 236, 259, 260(27)

Wong, J., 332

Wong, J. Y., 39

Woodard, S., 393, 394, 394(32), 395(32; 42),

396(32), 406(32)

Woodruff, G. N., 291(22), 292

Woodruff, R. A., 453

Woolfson, D. N., 295

Woostenborgles, R., 470

Worland, S. T., 63

Worthington, P. A., 403

Wortmann, F.-J., 142

Wu, C. R., 304

Wu, J., 315

Wu, J. J., 315

Wu, J. V., 235, 271

Wu, M. T., 80

Wu, N., 5

Wu, X., 263, 438, 444(15), 449(15), 458(15),

465(15)

Wu, Z., 39, 40, 42, 52, 58, 73(20), 115,

117(11), 446

Wuin, J., 480

Wunberg, T., 262

Wunderlich, I., 138

Wuonola, M. A., 225

Wustrow, D. J., 134

Wyatt, R., 332(37), 333

Wysong, C. L., 185

X

Xia, S.-Q., 63

Xiao, C. Y., 115, 117(12)

Xiao, X.-Y., 40, 76, 100, 115, 117(8), 272

Xiao, Z., 419

Xie, F., 44

Xiong, L., 291

Xu, B., 313

Xu, R., 290

Xu, X., 324

Y

Yadav-Bhatnagar, N., 349, 350(7)

Yaday, J. S., 204

Yager, K. M., 201, 422

Yalamouri, V. V., 422

Yamamura, Y., 258

Yamashita, D. S., 517

Yan, B., 3, 5, 12

Yan, B. J., 24, 33

Yan, L., 236, 258

Yanagisawa, M., 295

Yang, B., 209

Yang, B. H., 404

Yang, C. F., 291

Yang, J. Z., 138

Yang, L., 5

Yang, M., 209

Yang, R.-Y., 417, 424, 480

Yang, Y., 517

Yang, Z., 429

Yao, W., 296

Yaouancq, L., 185

Yasuda, K., 351(12), 352, 375, 376(26)

Yasuda, M., 425(37), 426

Yates, N. A., 396

Ye, H.-F., 151(3), 152

Ye, T., 138

Ye, X.-S., 259, 260(27)

Yin, J., 192, 405

Yokum, T. S., 185

Young, D. W., 517

Young, J. K., 130, 135(9)

author index 557

Youngman, M. A., 197

Yu, S. T., 79

Yu, Y., 327, 424, 496, 503, 506, 508,

508(37), 509

Yu, Z., 403

Yung, G., 392

Yurek, D. A., 12

Z

Zal, B., 339

Zander, N., 360, 361(33), 379

Zapf, C. W., 361(39), 362

Zaragoza, F., 517, 518, 521, 522(9; 18; 19),

525(9; 19), 526

Zaragoza Dorwald, F., 127, 130(4), 143(4)

Zaramella, A., 138

Zarrinmayeh, H., 481

Zechel, C., 257, 391

Zecri, F. J., 361(35), 362, 396

Zeleznikow, J., 341

Zelle, R. E., 475

Zemplen, G., 262

Zeng, L., 4

Zhang, A. J., 143

Zhang, F., 324

Zhang, H., 423

Zhang, H.-C., 457

Zhang, J. W., 304

Zhang, L.-h., 192

Zhang, S.-D., 63

Zhang, W., 65, 183, 184(3)

Zhang, Y., 185, 186(24), 189(24), 235

Zhang, Z., 185, 259, 260(27), 353, 354(16)

Zhao, C. F., 115, 117(12)

Zhao, J., 3, 5, 12

Zhao, X. Y., 134

Zhao, Y., 271, 338, 341,

342(47; 51; 53; 64)

Zhao, Z. G., 271, 481

Zhong, H. M., 397, 402(62)

Zhong, Y.-L., 378

Zhou, J. F., 204

Zhou, Q. S., 152

Zhou, R., 63

Zhu, T., 258

Zhuang, H., 115, 117(8)

Zicmanis, A., 152

Zimmerman, D., 481

Zuckermann, R. N., 290, 454

Zuercher, W. J., 74

Zugay, J. A., 79, 80, 185

Zupan, M., 376

Zurenko, G. E., 225(7), 226

Zwanenburg, B., 419

Zwick, M. B., 498

Zychlinski, A. V., 473

Zygmunt, M., 138

Subject Index

A

4-Amino-3-hydrazino-5-mercapto-1,2,4-

triazole, aldehyde assay in solid-phase

synthesis, 33

p-Anisaldehyde, aldehyde assay in

solid-phase synthesis, 32–33

B

Benzimidazole library

isocyanide-based multicomponent

reactions, 481

synthesis using SynPhase Crowns and

Lanterns, 51–53

traceless synthesis

o-floro/chloro-nitroarene coupling, 173

overview of development and

optimization, 164–173

quaternization with alkyl/benzyl

bromides, 174

reagents, 173

reduction of aromatic nitro

group, 174

resin preparation, 174–175

Benzodiazepine library

applications, 470

cyclative cleavage in solid-phase synthesis,

424–425


reactions

nuclear magnetic resonance

characterization, 491–492

overview, 470–471, 473, 478

plate production, 490–491

scale-up, 491–492

synthesis using SynPhase Crowns and

Lanterns, 43–45

Benzofuroxane library, synthesis using T1

triazene linkers, 140–141, 147–148

Benzothiazepine library, synthesis with

directed sorting and parallel synthesis,

86–87, 89–90

559

Benzotriazole library, synthesis using T1

triazene linkers, 136–138, 147–148

Benzylamine, polymer-assisted solution

phase synthesis using scavenger resins,

407, 412

N-Benzyl-2-bromo-N-methylbenzamide,

polymer-assisted solution phase

synthesis using scavenger resins, 403,

403, 410–411

3-Benzyl-2-phenylthiazolidin-4-one,


synthesis using scavenger resins,

406–407, 412

1-Benzyl-3-phenyl-thiourea,


synthesis using scavenger resins, 403, 411

4-(3-Benzylsulfanyl-5-phenyl-[1,2,4]triazol-4-

ylmethyl)-benzamide, polymer-assisted

solution phase synthesis using scavenger

resins, 402, 410–411

1-Butyl-3-(2-thiophen-2-yl-ethyl)urea,



405–406, 411

C

Carbohydrate library

oligosaccharide synthesis, see

Oligosaccharide solid-phase synthesis

synthesis using SynPhase Crowns, 60–63

Chloranil, amine assay in solid-phase

synthesis, 28, 303

Cinnoline library, synthesis using T1 triazene

linkers, 136–137, 139–140

Claisen-type condensation, cyclative

cleavage in solid-phase synthesis,

427–428

Cyclative cleavage,

solid-phase synthesis

advantages in heterocycle synthesis,

415–416

carbon nucleophiles, 427–428

560 subject index

Cyclative cleavage,

solid-phase synthesis (cont.)

diketopiperazine cyclative cleavage, 433

five-membered ring formation by nitrogen

nucleophile attack of sp2 or sp3

carbon, 416–419

organometallic reactions, 428–429

overview, 415

oxygen nucleophiles, 425–427

prospects, 432

reverse cyclative cleavage, 430–431

seven-membered ring formation by

nitrogen nucleophile attack of sp2

carbon, 424–425

six-membered ring formation by nitrogen

nucleophile attack of sp2 carbon, 419,

421–424

tetramic acid cyclative cleavage,

433–434

D

Diamino acid linkers

4-amino-3-(aminomethyl)benzoic acid

scaffold preparation and peptide

synthesis, 187–189, 191–192

constructs

unnatural aliphatic amino acids, 183–184

unnatural arylamino amino acids,

185–186

unnatural cyclic amino acids, 184–185

2,4-diaminobutyric acid, 184

2,3-diaminoproprionic acid(Phoc) linker

attachment to support, 189–191, 193

deprotection and coupling in peptide

synthesis, 194

stability, 187

structure, 183–184

synthesis, 192–193

safety-catch linkers, 186–187

2,3-Diaminoproprionic acid(Phoc) linker, see


1,4-Diazepine-2,5-dione library, synthesis

using SynPhase Crowns, 58–60

Dibenzylamine, polymer-assisted solution

phase synthesis using scavenger resins,

404, 411

2,3-Dichloroproprionic acid, derivatives as

polyelectrophiles for sequential

nucleophilic substitutions, 521–522

Diethyl-(2-p-tolyl-ethyl)amine,


synthesis using scavenger resins, 402, 410

4,5-Difluoro-2-nitrobenzamine, derivatives

as polyelectrophiles for sequential

nucleophilic substitutions, 522–523

Dihydroimidazole library, mixture-based

synthetic combinatorial

libraries, 507–508

Dihydropyrimidine library

Biginelli synthesis, see Microwave-assisted

Biginelli multicomponent reactions

clinical applications, 199

parallel synthesis, 200

solid-phase synthesis approaches, 200–202

Diketomorpholine library, isocyanide-based

multicomponent reactions, 478

Diketopiperazine library

cyclative cleavage in solid-phase

synthesis, 421–422, 433


reactions


characterization, 493

overview, 476–478

plate production, 492

scale-up, 492

Diphenyldichlorosilane-methyl red, alcohol

assay in solid-phase synthesis, 30–31

Diphenylmethylamine library, synthesis

using SynPhase‘ Crowns, 70–71

Directed sorting, see Solid-phase synthesis

5,50-Dithio(2-nitrobenzoic acid), thiol assay

in solid-phase synthesis, 31

E

Ellman’s reagent, see 5,50-Dithio(2-

nitrobenzoic acid)

Emrys synthesizer, microwave-assisted

organic synthesis, 205–206

Encore synthesis

advantages of split-and-pool synthesis, 124

instrumentation

Arraying Tool, 119

Lantern Dispensing Tool, 120

Lantern Leveling Tool, 120

Lapis Tool, 120

Magazine, 119–120

overview, 117–119

subject index 561

steps in combinatorial library


G

Guanidine library

mixture-based synthetic combinatorial

libraries, 509


synthesis, 363

synthesis using T2 triazene

linkers, 143–145, 149–150

H

Heterocycle library synthesis, see also

specific compounds


libraries, see Mixture-based synthetic

combinatorial libraries

cyclative cleavage, see Cyclative cleavage,


derivatization reactions on solid-phase

acylation, 451–453, 467

alkylation, 451, 453–454, 467–468

aromatic nitro group reduction, 455, 468

derivatization of C2 chloro group of

resin-bound heterocycles, 439, 441,

458–459

facilitated arylations via iron-�

complex, 447–449, 463

Knoevenagel reaction, 456–457, 468–469

Mannich reaction, 457, 469

Mitsunobu reaction, 456, 468

nucleophilic aromatic

displacements, 438–449

overview, 437–438

palladium-catalyzed reactions, 449–451,

465–466

prospects, 457–458

purines and adenosine analog

synthesis, 445

quinazolines, 446–448, 462–463

Rink resin compound capture and

derivatization, 441–443, 459–461

Stille coupling, 450–451, 466

triazine derivatization, 443–444, 461–462

drug development applications,

435–437, 469

multicomponent condensation reactions,

see Isocyanide-based multicomponent

reactions

triazene T1 linkers, 136–140, 147–148

High-performance liquid chromatography,

combinatorial library analysis, see

Liquid chromatography/ultraviolet/mass

spectrometry, combinatorial library

analysis

HIV, see Human immunodeficiency virus

HPLC, see High-performance liquid

chromatography

Human immunodeficiency virus

synthetic combinatorial libraries for

drug development

membrane fusion inhibition

assay, 334–335

peptide considerations as drugs, 334

replication inhibition assay, 335, 337

therapeutic targets, 323–324, 332

Hydantoin library

cyclative cleavage in solid-phase



reactions, 479



synthesis using SynPhase‘ Crowns, 56–58

I

Imidazoline library


reactions, 480–481



Imidazolone library, mixture-based synthetic

combinatorial libraries, 503–505

Indolepyridoimidazole library, mixture-

based synthetic combinatorial

libraries, 506

Isocyanide-based multicomponent reactions

automation, 487–489

azepine-tetrazole preparation, 493–494

benzodiazepine library synthesis



overview, 470–471, 473, 478

plate production, 490–491

562 subject index

Isocyanide-based multicomponent reactions

(cont.)

scale-up, 491–492

diketopiperazine library synthesis



overview, 476–478

plate production, 492

scale-up, 492

ketopiperazine tetrazole

preparation, 494–495

nor-statine preparation, 495

postcondensation Passerini



tetrazole-nor-statine preparation, 495–496

trimethylsilyl azide-modified Passerini

reaction, 486–487

trimethylsilyl azide-modified Ugi


Ugi multicomponent condensation

reactions

benzimidazole library synthesis, 481

deprotection, 473–474

diketomorpholine library synthesis, 478

ethyl glyoxylate templates, 475–477

hydantoin library synthesis, 479

imidazoline library synthesis, 480–481

immobilized convertible isonitriles, 475

quinoxalinone library synthesis, 481–482

UDC concept, 473–475

urea library synthesis, 479–480

1-(3-Isopropoxypropyl)-3-phenylthiourea,



405–406, 411

K

Knoevenagel reaction, derivatization

reactions on solid-phase, 456–457,

468–469

L

Liquid chromatography/ultraviolet/mass

spectrometry, combinatorial library

analysis

comparison of eight-channel system with

single-channel system

data acquisition in positive and negative

ion modes, 19

operation and maintenance, 20

reanalysis of samples, 20

sensitivity, 19

time of analysis, 18–19

eight-way multiplexed electrospray

interface, 4–5

high-throughput modifications, 3–4

instrumentation, 6

library evaluation conditions, 14, 18

optimization

column selection, 9

flow monitoring, 7

flow rates, 8–9, 21

standards, 6–7

T-joint positioning, 8, 20–21

pumping system, 5

reanalysis of samples, 10–11

representative library compound

evaluation, 12, 14, 21

M

Malachite green, carboxylic acid assay in

solid-phase synthesis, 32

Mannich reaction, derivatization reactions

on solid-phase, 457, 469

Mass spectrometry, combinatorial library

analysis, see Liquid chromatography/

ultraviolet/mass spectrometry,

combinatorial library analysis

4-(3-Methyl-5-phenyl-pyrazol-1-yl)-benzoic

acid, polymer-assisted solution phase

synthesis using scavenger

resins, 399–401, 410

Microwave-assisted Biginelli

multicomponent reactions

advantages, 216–217

Biginelli dihydropyrimidine


dihydropyrimidine library synthesis

automated sequential library

production, 212, 215–216

criteria, 206–207

microwave irradiation, 217–218



reaction optimization

catalyst selection, 209

subject index 563

overview, 207–208

solvent selection, 208

time and temperature, 210–211

troublesome building blocks, 211–212

reagents, 217

Emrys synthesizer, 205–206

multicomponent reaction advantages, 197

overview, 204–205

Microwave-assisted organic synthesis

dihydropyrimidines, see



instrumentation, 203–206, 224–225

oxazolinidones, see Oxazolinidone library

principles, 202–203, 223–224


Mitsunobu reaction, derivatization reactions

on solid-phase, 456, 468

Mixture-based synthetic combinatorial

libraries

chemistry optimization, 502–503

heterocyclic compound synthesis from

resin-bound compounds

bis-heterocyclic compound

synthesis from resin-bound

polyamines, 510–511

dihydroimidazoles, 507–508

guanidines, 509

hydantoins, 505–506

imidazolines, 506–507

imidazolones, 503–505

indolepyridoimidazoles, 506

overview, 496–499

parallel synthesis of heterocyclic positional

scanning libraries

alkylation of trityl-protected amino

acids, 514

amide bond reduction, 515

Boc amino acid coupling to resin, 514

cleavage conditions, 516

control teabag synthesis, 516

Fmoc amino acid coupling, 514–515

N-acylation, 515

overview, 511–513

parallel synthesis, 515–516

requirements, 513

trityl group removal, 514

trityl protection of resin-bound amino

acids, 514

peptide library generation, 324–325

peptidomimetic library

generation, 501–502


reagent mixtures, 500–501

resin mixtures, 499–500

MS, see Mass spectrometry

Multicomponent reaction

advantages, 197

Biginelli dihydropyrimidine synthesis, see



multicomponent condensation reactions,


reactions

N

NF-31, amine assay in solid-phase

synthesis, 26–28

Ninhydrin, amine assay in solid-phase

synthesis, 25–26, 303

NMR, see Nuclear magnetic resonance

Nuclear magnetic resonance

benzodiazepine library


dihydropyrimidine library


diketopiperazine library


tetrafluorophenol-activated resin quality

control, 156–158

O

Oligosaccharide solid-phase synthesis

applications, 243, 248–249

automated synthesis

branching, 240

cleavage conditions, 241, 247

deprotection, 246–247

F-tag capping and purification of

products, 242, 248

glycosyl phosphate coupling,

240–241, 247

glycosyl trichlooacetimidate

coupling, 239–240, 246

instrumentation, 238, 243, 246

linkers, 238–239, 250–251

564 subject index

Oligosaccharide solid-phase synthesis (cont.)

materials, 244–245

overview, 236–238

resin synthesis, 245

trityl cation assay, 245–246

blood group determinant tetrasaccharide


branching problem, 235–236

combinatorial oligosaccharide library


human antigen trisaccharide


prospects, 265

stereochemistry problem, 235–236

One-bead one-compound library

Boc-amino acid relative reactivity, 280

decoding, 273–274, 278

encoding

advantages, 275–276

biphasic solvent approach for bead

segregation, 273, 275, 278, 281

coding units and reactions, 275

overview, 272

peptidomimetic and small molecule

library synthesis, 273–274, 276–278,

280–284

historical perspective, 289–290

peptide library synthesis

activating agents, 300–301

amino acid solution


coupling reaction monitoring, 302–303

cyclic peptide library synthesis

using lysine and glutamate

residues, 306–307

disulfide cyclic peptide library


fluorescence quench library

synthesis for protease substrate

determination, 307–308

linear hexapeptide synthesis, 303–304

solid supports, 300

screening

encoded libraries, 277, 284, 287, 299–300

enzyme-linked colorimetric

assay, 309–310, 312

protease substrates, 314–315

protein kinase substrate

assay, 315–317

whole cell binding assay, 313–314

sequencing

Edman sequencing, 322

microsequencing, 317–319, 322

retention time of derivatized amino

acids on protein sequencer, 320–321

split-mix synthesis, 298–299

structural characterization of

compounds, 271–272

Oxazole library, synthesis using SynPhase‘Crowns, 54–56

Oxazolinidone library

antimicrobial activity, 225–226

microwave-assisted synthesis

acylation of attached

iodoaryloxazolidinone, 231

cleavage of biaryloxazolidinone from

solid support, 231

iodoaryloxazolidinone coupling to

resin, 230–231

optimization of Suzuki reaction, 228–229

overview, 228–230

reagents, 230

Smith synthesizer, 228

solid-phase Suzuki coupling, 231

retrosynthetic analysis of oxazolinidone

pharmacophore, 226–228

P

Parallel synthesis, see Solid-phase synthesis

PASP synthesis, see Polymer-assisted

solution phase synthesis

Passerini reaction, see Isocyanide-based


Peptide combinatorial library

antibacterial agent development using

positional scanning synthetic


pathogens, 331–332

screening, 332

antifungal agent development using

positional scanning synthetic


deconvolution, 330

pathogens, 326–327

screening, 327, 330

antimicrobial peptide library

design, 292–293

applications, 288–289

combinatorial docking, 295

subject index 565

cyclization, 295–296

design strategies, 293–295, 297

diamino acid linkers in synthesis, see


hinges in libraries, 297–297


human immunodeficiency virus antagonist

development

membrane fusion inhibition

assay, 334–335

peptide considerations as drugs, 334

replication inhibition assay, 335, 337


libraries, see Mixture-based synthetic


one-bead one-compound libraries, see

One-bead one-compound library

positional scanning, 291, 325

screening approaches, 290–291

split-and-pool approach in

synthesis, 75–76

synthesis using SynPhase‘ Crowns

cyclic peptides, 64–67, 69

muramyl peptide derivatives, 63, 65

rhinovirus protease inhibitors, 63–64

tripeptide library string synthesis

cleavage, 110–111

coupling, 110

overview, 109

product distribution and

verification, 111–112

sorting of crowns, 110

synthesis using T2 triazene linkers, 142–143

tertiary structure design, 295

vaccine development

epitope mimic identification, 338–341

natural epitope identification in protein

databases, 341–343

overview, 337–338

prospects, 344

Perfluoroalkylsulfonyl linker

cleavage of resin-bound phenols

catalytic amination, 179–180

catalytic reductive

elimination, 178–179, 182

Suzuki coupling reaction, 177, 181–182

phenol attachment, 175–176, 181

prospects for traceless library



1-Phenylpentan-1-ol, polymer-assisted

solution phase synthesis using scavenger

resins, 406, 412

Piperazine-2-carboxamide library, synthesis

with directed sorting, 79–81, 83, 85–86

Polyamine library, synthesis using SynPhase

Crowns, 67–68, 70

Polymer-assisted solution phase synthesis,

see also Polymer-supported reagents

advantages over solid-phase

synthesis, 392

alkylating polymers

carboxylic acid alkylation, 381–382

overview, 379–381

carbanion equivalent reagents, 382–384

highly-loading resins for

polymer-supported reagents, 387–390

oxidizing polymers

heavy-metal oxide reagents, 371

overview, 370–371

oxoammonium salt resins

alcohol oxidation, 373–375

overview, 371–373

preparation, 373

periodinane resins

alcohol oxidation, 377–379

overview, 376

reactivation, 377

principles, 367–369

prospects, 370, 390

radical release from polymer

gels, 385–387

scavenger resins

benzylamine synthesis, 407, 412

N-benzyl-2-bromo-N-methylbenzamide

synthesis, 401, 403, 410–411

3-benzyl-2-phenylthiazolidin-4-one

synthesis, 406–407, 412

1-benzyl-3-phenyl-thiourea

synthesis, 403, 411

4-(3-benzylsulfanyl-5-phenyl-

[1,2,4]triazol-4-ylmethyl)-

benzamide synthesis, 402, 410–411,

399–401, 410

1-butyl-3-(2-thiophen-2-yl-ethyl)urea

synthesis, 405–406, 411

dibenzylamine synthesis, 404, 411

diethyl-(2-p-tolyl-ethyl)amine

synthesis, 402, 410

historial perspective, 393

566 subject index

Polymer-assisted solution phase synthesis

(cont.)

1-(3-isopropoxypropyl)-3-

phenylthiourea synthesis,

405–406, 411

4-(3-methyl-5-phenyl-pyrazol-1-yl)-

benzoic acid synthesis overview of

types, 396–397

1-phenylpentan-1-ol synthesis,

406, 412


quenching reagent criteria, 393–394

rationale, 391–393, 396

synthesis of resins

acid chloride resin, 409

amine/aminoalcohol resin, 409

aminodiol/morpholine resin, 409

arenesulfonyl chloride resin, 409–410

boronic acid diethanolamine

resin, 404–405

guanidine resin, 409

oligo(ethyleneimine) resin, 408

phenol resin, 409

Polymer-supported reagents

acids/bases and dihydropyrimidone


amide-coupling reagents, 360–362

carbon transfer reagents and olefin

synthesis via Wittig reaction, 354–355

electrophilic reagents, 356–357

esterification reagents, 360–362

group transfer reagents

ester synthesis, 363

Fmoc-amino acid synthesis, 363

guanidine synthesis, 363

overview, 362


multistep synthesis applications, 366

oxidations and reductions

alkene hydrogenation, 353

carboxylic acid synthesis, 352

overview, 351–352

polymer-assisted solution phase synthesis,

see Polymer-assisted solution phase

synthesis

radical reaction reagents, 356–357

scavengers

overview, 364–365

phosphine and phosphinoxide

scavenging, 365

sulfur and phosphorous transfer

reagents, 353–354

transition metal catalysts

arylamine synthesis, 359–360

biphenyl synthesis via Suzuki

coupling, 360

overview, 357–359

Purine library

cyclative cleavage, solid-phase

synthesis, 445

synthesis using SynPhase‘Crowns, 45–47

Purpald, see 4-Amino-3-hydrazino-5-

mercapto-1,2,4-triazole

Pyrin-2-one library, synthesis using

SynPhase‘ Crowns, 53–54

Q

Quinazoline library

derivatization reactions on

solid-phase, 446–448, 462–463

synthesis using SynPhase‘Lanterns, 47–51

Quinazolinone library, cyclative cleavage in


Quinoxaline library, synthesis using

SynPhase‘ Lanterns, 58–59

Quinoxalinone library, isocyanide-based

multicomponent reactions, 481–482

R

Radiation-graft polymers, see SynPhase‘Crown; SynPhase‘ Lantern

Rink resin, compound capture and

derivatization, 441–443, 459–461

S

Scavenger resins, see Polymer-assisted

solution phase synthesis

Solid-phase-assisted-solution-phase

combinatorial synthesis, see

Polymer-assisted solution phase

synthesis; Polymer-supported reagents

Solid-phase synthesis, see also

specific compounds

colorimetric tests

subject index 567

alcohols

diphenyldichlorosilane-methyl red

test, 30–31

p-toluenesulfonyl chloride/

p-nitrobenzylpyridine test, 28–29

1,3,5-trichlorotriazine-(fluorescein,

alizarin R, or fuchsin) test, 29–30

aldehydes

4-amino-3-hydrazino-5-mercapto-

1,2,4-triazole test, 33

p-anisaldehyde test, 32–33

aliphatic amines

chloranil test, 28

NF-31 test, 26–28

ninhydrin test, 25–26

trinitrobenzenesulfonic acid test, 26

carboxylic acids, malachite green test, 32

overview, 22–25

reproducibility, 35

specificity of tests, 33–34

thiols, 5,5’-dithio(2-nitrobenzoic acid)

test, 31

cyclative cleavage, see Cyclative cleavage,


directed sorting approach

acylation with carboxylic acids, 93–94

acylation with chloroformates, 94

alkylation reaction, 98

amide bond formation, 96

beads and resins, 90–91

benzothiazepine library synthesis with

directed sorting and parallel

synthesis, 86–87, 89–90

cleavage conditions, 96, 98–99

cyclization to benzothiazepine, 97–98

equipment, 77, 79

halo-nitrobenzene coupling, 97

nitro group reduction, 97

piperazine-2-carboxamide library

synthesis, 79–81, 83, 85–86

principles, 76–77

protecting group removal, 93–95

resin preparation, 95–97, 99

sulfonamide formation, 94

sulfone oxidation, 98

urea formation with isocyanates, 94

washing of beads, 93

historical perspective, 39, 112–113, 347

linkers, see also Diamino acid linkers;

Tetrafluorophenol-activated resins;

Triazene anchors

amine derivatization linkers, 153

monofunctional linker limitations, 128

multifunctional linker advantages, 129

oligosaccharide solid-phase synthesis,

238–239, 250–251

traceless linkers, see also

Perfluoroalkylsulfonyl linker;

Triazene anchors

definition, 132–133, 164

types, 134

types, 127–130

microwave-assisted synthesis, see

Microwave-assisted organic synthesis

oligosaccharides, see Oligosaccharide


parallel synthesis, 76

polymer-bound polyelectrophiles and

sequential nucleophilic substitutions

1-[5-benzenesulfonyl-2-(piperidin-1-yl)-

4-(pyridin-4-

ylmethylamino)benzoyl]piperazine

trifluoroacetate synthesis, 526–527

2,3-dichloroproprionic acid derivatives

as polyelectrophiles, 521–522

4,5-difluoro-2-nitrobenzamines as

polyelectrophiles, 522–523

2-phenylsulfanyl-3-(piperidin-1-

yl)propionic acid trifluoroacetate



radiation-graft polymers from Chiron

Mimitopes, see SynPhase‘ Crown;

SynPhase‘ Lantern

split-mix synthesis, see Split-mix synthesis

tea bag synthesis, 113

traceless synthesis of benzimidazole library

o-floro/chloro-nitroarene

coupling, 173

overview of development and


quaternization with alkyl/benzyl

bromides, 174

reagents, 173

reduction of aromatic nitro group, 174

resin preparation, 174–175

568 subject index

Split-and-pool synthesis

advantages, 124

directed synthesis, see also

Encore synthesis

encoding, 115–116

process integration and

automation, 116–117

random synthesis comparison, 113–114

solid supports, 115

peptide libraries, 75–76

Split-mix synthesis

one-bead one-compound

library, 298–299


string synthesis

manual redistribution of

crowns, 102–104

overview, 100–101

redistribution pattern, 104–105, 107

software, 107–108

stringing of support units, 101–102

SynPhase‘ Crowns as support

units, 101–102

tripeptide library synthesis

cleavage, 110–111

coupling, 110

overview, 109




SPS, see Solid-phase synthesis

String synthesis, see Split-mix synthesis

Suzuki reaction

biphenyl synthesis with transition metal

catalysts, 360

microwave-assisted synthesis

of oxazolinidones


solid-phase coupling, 231

perfluoroalkylsulfonyl linker, cleavage of

resin-bound phenols, 177, 181–182

SynPhase‘ Crown

benzimidazole synthesis, 51–53

benzodiazepine synthesis, 43–44

carbohydrate synthesis, 60–63

1,4-diazepine-2,5-dione synthesis, 58–60

diphenylmethylamine library, 70–71

directed synthesis, see Encore synthesis

hydantoin synthesis, 56–58

overview, 41–43

oxazole synthesis, 54–56

peptide synthesis

cyclic peptides, 64–67, 69

muramyl peptide derivatives, 63, 65

rhinovirus protease inhibitors, 63–64

tripeptide library string synthesis

cleavage, 110–111

coupling, 110

overview, 109




polyamine synthesis, 67–68, 70

prospects, 72–73

purine synthesis, 45–47

pyrin-2-one synthesis, 53–54

string synthesis, see Split-mix synthesis

urea synthesis, 72

SynPhase‘ Lantern

benzimidazole synthesis, 52–53

benzodiazepine synthesis, 44–45

directed synthesis, see Encore synthesis

overview, 40–44

prospects, 73–74

quinazoline synthesis, 47–51

quinoxaline synthesis, 58–59

urea synthesis, 71–72

T

Tetrafluorophenol-activated resins

amine derivatization

applications, 151–152, 154

library synthesis, 158–161, 163

carboxylic acid ester

preparation, 162

loading determination, 163

polymeric tetrafluorophenol


quality control, 156–158

sulfonic acid ester preparation, 162

synthesis of resins, 155–156, 161–162

TNBS, see Trinitrobenzenesulfonic acid

p-Toluenesulfonyl chloride/

p-nitrobenzylpyridine test, alcohol assay

in solid-phase synthesis, 28–29

Triazene anchors

acidic cleavage, 132–133

applications in solid-phase


subject index 569

U

Ugi multicomponent condensation reactions,


reactions

Urea library



synthesis using SynPhase‘ Crowns and

Lanterns, 71–72

synthesis using T2 triazene linkers, 143, 149

W

Wittig reaction, carbon transfer reagents and

olefin synthesis, 354–355

heterocycle derivatization, 443–444,

461–462

heterocycle library synthesis with T1

linkers, 136–140, 147–148

multifunctional cleavage, 135–136, 145

T2 linkers

guanidine library synthesis, 143–145,

149–150

peptide library


synthesis, 140–142, 148–149

urea library synthesis, 143, 149

traceless cleavage, 132–134, 148

types, 130–132

1,3,5-Trichlorotriazine-(fluorescein, alizarin

R, or fuchsin) test, alcohol assay in

solid-phase synthesis, 29–30

Trinitrobenzenesulfonic acid, amine assay in


Documents

Methods in Enzymology, Vol. 369: Combinatorial Chemistry, Part B