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"Kombinatorische Chemie und Festphasensynthese“"

Ulrich JordisInst. Für Angewandte Synthesechemie

2006

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Historical Landmarks• 1963: Merrifield: peptide on polystyrene

resin• 1965: Letsinger and Khorana:

Oligonucleotides• 1974: F. Camps: Synthesis of

Benzodiazepines on sold support• 1996: Ugi four component reaction in

combination with 1,3-dipolar cycloaddition

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Literatur: Bücher• D. Olbrecht, J.M. Villalgordo: „Solid-Supported

Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries“ Pergamon 1998

• Barry A. Bunin: The Combinatorial Index Academic Press 1998, 322 S.

• S. R. Wilson, A. W. Czarnik: Combinatorial Chemistry, J. Wiley 1997, 269 S.

• A. W. Czarnik, S. H. DeWitt: A Practical Guide to Combinatorial Chemistry, ACS 1997, 450 S.

• N. K. Terret: Combinatorial Chemistry, Oxford Univ. Press 1998, 186 S.

Literatur: Bücher II• Günther Jung, Ed.: Combinatorial Chemistry Synthesis,

Analysis, ScreeningWiley-VCh 1999• M. R. Pavia and W. H. Moos: Annual Reports in

Combinatorial Chemistry and Molecular Diversity Vol. 2, Kluwer Academic Publishers, 1999

• Advanced ChemTech Handbook of Combinatorial & Solid Phase Organic Chemistry

• NOVABIOCHEM: Solid Phase Organic Chemistry Handbook

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Literatur: Bücher III• Combinatorial Chemistry: A Practical

Approachby Willi Bannwarth, Eduard Felder, Raimund Mannhold, Hugo Kubinyi, Hendrik Timmerman - Wiley-VCH

Literatur: Datenbanken• Solid Phase Organic Reactions (SPORE)

ISIS/Host (MDL) >6.000 reactions containsextensive data from solid-phase organic chemistry, including polymeric materials, linkers, solid supports, and protecting groups.

• http://www.mdli.com/dats/synthdb.html

• Synopsys’ Solid-Phase Synthesis (SPS)ca. 9.000 reactions from more than 1,673 citationsincluding patentshttp://www.synopsys.co.uk/

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The Integrated ApproachBiochemistry

Genomic Science Molecular Biology

Target Identification and Selection:•Enzymes•Receptors

•Transcription Factors

Protein Expression and Purification

Rational and Random DesignProtein Expression and Purification

CombinatorialCombinatorialCompoundCompoundLibrariesLibraries

Structure Determination(X-Ray, 2D- and 3D-NMR)Structure Determination(X-Ray, 2D- and 3D-NMR)

Assay DevelopmentAssay Development

Cocrystallization of Ligand and Target ProteinCocrystallization of Ligand and Target Protein

Structure Based"de novo" DesignStructure Based"de novo" Design

Lead / LigandOptimization

High Throughput Screening (HTS)High Throughput Screening (HTS)

Lead / LigandIdentificationLead / LigandIdentification

Development Compound

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Types of Libraries

• Catalyst Development• Ligand Design• Drug Discovery

Lead FindingLead Optimization

• Peptides • Proteins • Nucleotides• Olicosaccharides• Antibodies • High Temp.

Supraconductors• Inks & Dyes

Productivity

traditional

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Combinatorial Libraries

Combinations (1)A1-B1-C1

-C2-C3

A1-B2-C1-C2-C3

A1-B3-C1-C2-C3

A2-B1-C1-C2-C3

A2-B2-C1-C2-C3

A2-B3-C1-C2-C3

A3-B1-C1-C2-C3

A3-B2-C1-C2-C3

A3-B3-C1-C2-C3

A ≠ B ≠ C,

only one linear assembly possible:

A ⇒ B ⇒ C

3 X 3 X 3 = 27 combinations

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Combinations (2)A1-B1-C1

-C2-C3

A1-B2-C1-C2-C3

A1-B3-C1-C2-C3

A2-B1-C1-C2-C3

A2-B2-C1-C2-C3

A2-B3-C1-C2-C3

A3-B1-C1-C2-C3

A3-B2-C1-C2-C3

A3-B3-C1-C2-C3

A ≠ B ≠ C,

six linear permutations possible:

A ⇒ B ⇒ CA ⇒ C ⇒ BB ⇒ A ⇒ CB ⇒ C ⇒ AC ⇒ A ⇒ BC ⇒ B ⇒ A

3 X 3 X 3 X 6 = 162 combinations

Combinations (3)

One directed sequence:

3 X 3 X 3 = 27 combinations

AB

CA

B

CA

B

C

All cyclic permutations:

3 X 3 X 3 X 2 = 54 combinations

A ≠ B ≠ C A ≠ B ≠ C

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Number of different peptides increases exponentially with the length of the molecule

Number of amino acid residues Peptide

Number of distinct peptides

2 NH2-X1X2-COOH 4003 NH2-X1X2X3-COOH 8.0004 NH2-X1X2X3X4-COOH 160.0005 NH2-X1X2X3X4X5-COOH 3.200.0006 NH2-X1X2X3X4X5X6-COOH 64.000.0007 NH2-X1X2X3X4X5X6X7-COOH 1.280.000.0008 NH2-X1X2X3X4X5X6X7X8- 25.600.000.000

X = 20 natural amino acids

Numbers

Traditional- / Array- / One-pot simultaneous- / Indexed Comb. Lib. Synthesis

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Indexed, Orthogonal Combinatorial Libraries

Komponenten der Bibliothek

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Test der Mischungen

Indexed (=Orthogonal) Combinatorial Libraries

Pirrung, M.C. et.al., Chem. Biol. 1995, 2, 621-626

1A-1H

2A-2H

3A-3H

4A-4H

5A-5H

6A-6H

A1-A6 B1-B6 C1-C6 D1-D6 E1-E6 F1-F6 G1-G6 H1-H6

2C

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Orthogonal LibrariesA1B1A1B2A1B3

A2B1A2B2A2B3

A3B1A3B2A3B3

A4B1A4B2A4B3

A1B1A2B1A3B1A4B1

A1B1 A2B1 A3B1 A4B1

A1B2A2B2A3B2A4B2

A1B2 A2B2 A3B2 A4B2

A1B3A2B3A3B3A4B3

A1B3 A2B3 A3B3 A4B4

Deconvolution

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Scaffold Survey

N

NH

Cl

O

NH

Antiviral (AIDS) (Roche)

NH

O

SCH3

OH

N

NO

N

CH3

NH2O

SH

O

Farnesyltransferase Inhibitor; Antineoplastic (Genentech)

N

NO

CH3

NH

O

O

O

O

CH3

CH3

Alcohol Deterrent (Biomeasure)

N

N

Cl

O

Cl

S

CH3O

O

Anxiolytic; Anticonvulsant

(Gerardo Ramon)

N

NOCH3

NH

NH

O

S

OH

O

CCK Antagonist; Non-Opioid Analgesic;Anxiolytic

(Merck & Co.)

N

NO

NH

O NH

Antiulcerative; Pancreas Disorders; CCK Antagonist

(Jouveinal)

N

NO

NH

NH

OS N

CH3

CH3

NH

NNN

Antiarthritic; Gastrin Antagonist; Anxiolytic; CCK Antagonist

(Merck & Co.)

N

NO

CH3

NH

NH

O

Uterine Relaxant; Oxytocin Antagonist (Merck & Co.)

Virtual Library

R1=

R2=

R3=

N

NO

R2

OH

R3

R1

*

*

O

O*

*

C

**

*

*S

*

O

F

F

F

*

SN S *

SO

OCl

*

S

Cl

C

**

O N *O

O*

N

*

O O **

O

*

H

**

*

N

*

O*

N*

F

*

H

*

C

* ** *

* *

N

N *

O

*

O*

O

** *

*

# Specific Structures: 2520

MWT Range: 189.2 - 751.3

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Manual Synthesizer

Robotics: A Robotics: A ZymarkZymark RobotRobot

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Zymark Robot

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Solid SupportsCrosslinked OrganicPolymers

• insoluble in organic solvents• microporous, gels• macroporous

Linear OrganicPolymers

• usually soluble in organic solvents

Dendrimers • solubility depends on size andshape

Inorganic Supports • porous glass, SiO2, Al2O3, clays,graphite

Solid Supports: Linkers

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Dendrites as new types of Polymers

Dendrites

http://acsinfo.acs.org/hotartcl/cenear/960603/dend.html

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Measuring yields ⇒ loading

• Loading: mmol functional group / g resin(typisch: 0.2 - 1.1 mmol/g)

• Yield: loading of product/loading of starting material

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Solid Phase Synthesis: Analytical Techniques

• Produkt an Polymer gebunden:ElementaranalyseTitration reaktiver Gruppen

• Analyse nach Abspaltung:HPCL, GC, CE, IR, NMR, MS....

• Erfahrung lehrt, daß jeder Reaktionsschritt analytisch verifiziert werden muß!

Single bead Analysis• 0.1-0.2 μg mit ~400 pmol Verbindung• Matrix assisted laser desorption (MALDI)• Molecular weight imaging using time-of-

flight secondary ion MS• FTIR microscopy: ester carbonyl detected

from 130 fmol of material (~0.025% of total bead loading)

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Encoding of Beads

• Deconvolution problem! • Encoding: synthesis history is

recorded on the bead - one compound per bead

• Molecular tags (chemisches Etikettieren)• Radiofrequency encoding• Laser optical synthesis chip (LOSC)• Isotope encoding• Peptide tags

Oligon cleotide tags

Encoding / Molecular Tags• Analyse durch electron capture gas

chromatography (ECCO)

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Encoding / Molecular Tags

Encoding / Radiofrequency

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Encoding / Radiofrequency

Encoding / Laser Optical Synthesis Chip (LOSC)

PS Copolymer alsTräger für Festphasen-Synthese

lasergeritzter 2DStrichcode aufkeramischemGrundkörper

Chip: 10 x 10 x 2 mm, Beladungsdichte 5-8 μmol / Chip

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PS-PEG Tantacle Resin

POE=Polyoxyethylen

PS-PEG Tantacle Resin: Gelphase

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Single-Bead Gelphase 1H-NMR

HPLC and GC/MS of single beads

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Photoacoustic FT-IR (two beads)

Photoacoustic FT-IR

• Attenuated total reflection (ATR) IR:IR beam can penetrate bead 1-2 μm

• Photoacoustic spectroscopy (PAS):IR beam can penetrate bead 30-40 μm

Ion Cyclotron Resonance (ICR) MS• Allows detection of masses

with high accuracy, e.g. m/z calc.: 267.113, found: 267.107

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Ion Cyclotron Resonance (ICR) MSIon Cyclotron Resonance (ICR) MS

MAS NMR• spinning at ~2 kHz• Varian Nano-NMR probe:

< 40 μl• 3,5-dimethoxy-13C-benzoic acid:

~800 pmol / sample• direct 1H NMR from 10-65 nmol /

sample

MAS: Magic Angle Spinning

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Gel Phase 13C-NMR 13C enriched) of single 740 μm Boc-Ala-Val-Ala bead

Solid Phase Synthesis: Scheme

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Solid Phase Synthesis: FT-IR of Polymer

Solid Phase Synthesis: GC analysis of library

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Solid Phase Synthesis: Single-bead GC/MS

Swelling Capacity

Solvent crosslinked PS(1% DVB)

Merrifield resin(2% DVB)

MeOH/EtOH 0.95-1.05 -

AcOH - 1MeCN 2 -Pyridine - 3DMF 3.5 2

THF, Dioxane 4.9-5.5 2.5Ether 2.6

CH2Cl2 5.2Toluene 5.3 2.8

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Chloromethylated Polystyrenes

Chloromethylated PS (2) CH2

OCH3

CH2

Cl

CH2

CH3

CH2

OCH3 CH3

Cl

CH2

CH3

styrene, DVBstyrene, DVB

DVB

BCl3, CCl4, O°, 2h NaOCl, CHCl3 or ClCH2CH2Cl, BnN+Et3Cl-;SO2Cl2, AIBN, 60°

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Functionilised PS Resins• -CH2Cl

– further reactions (nucleophilic displacement)• -CH2NH2• -from Polystyrene (PS)

– Friedel-Crafts Alkylation– Sulfonation, Nitration– Bromination– Lithiation

From Merrifield-Type Resins (1)

CH3

Cl

CH3

O CH3

O

CH3

OH

LiAlH4, Et2O, r.t., 4 horNH2NH2, DMF, r.t., 76 h

1% DVB, 0.73 - 3.9 mmol Cl/g:KOAc, DMA, 85°, 24 h

2% DVB, 2.8 mmol/g:KOH, 1-pentanol, reflux, 24 h

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From Merrifield-Type Resins (2)

KOH, EtOH/dioxane

1% DVB, 3.5 mmol Cl/g:thiourea, dioxane/EtOH (4:1), 85°, 15 h

1% DVB, 1.15 mmol/g:KSAc, DMF

CH3

SH

CH3

O NH2

NHH Br

CH3

Cl

S CH3

OLiBH4 / Et2O

S

From Merrifield-Type Resins (3)

1,2-dimethoxyethane,m-CPBA, 55°, 19 h

2% DVB, 1.36 mmol Cl/g:NaCN, DMF, H2O

2% DVB, 6.7 mmol/g:ClPPh2, Li, THF

CH3

Cl

H2SO4, AcOH, H2O120°, 20 H

O

H

N

P

O

OH

2% DVB, 1.05 mmol/g:DMSO, NaHCO3,155°, 6 h

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Aminomethylated PS

Friedel-Crafts Acylation of PS

Photolabile o-nitrobenzhydryl

Oxim

Trityl

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Bromination / Lithiation

Bromination:PS 1% DVB, Br2, Tl(OAc)3or FeCl3 CCl4. Br2, or Br2, BF3, nitromethane, r.t. 18 h

N-BuLi, cyclohexane, TMEDA, 65°, 4.5 H

nBuLi, hexane or toluene, 60°, 3 h

Nitration, Sulfonation

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From PS-Lithium (1)

From PS-Lithium (2)

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Polyacrylamide Resins

CH2

O

N CH3

CH3

CH2

O

NH

NH

OCH2

CH2

O

NH

NH

OCH2

CH2

O

N

CH3

O

OCH3

TentaGel® Resinsethylene oxide

Propylene oxide,SnCl4, CH2Cl2

Ethylene oxide,KOH, dioxane, 110°

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Novel Polymeric Supports: PEGA

Novel Polymeric Supports (2): CLEAR

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Solid Supported Reagents: Side Reactions

Solid Supported Reagents: Amberlyst - anionic nucleophiles, oxidants or

reducing agents

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Synthesis Robot

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Synthesis Robot

•Combination of Software and Hardware

•Reaction Block: -40 to 150 C, Filtration, Washing

•Elevated, programmed stirring

•Inert Gas / Vacuum / Pressure

•Fluid Handling

•Software

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MultiSynTech SyRo

Chemfile Editor

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Von der klassischen Vorschrift...

... zum Chemfile

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Reagent Library

Building Blocks

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http://phoenix.http://phoenix.tuwien.ac.attuwien.ac.at//combichemcombichem/index.htm/index.htm

http://phoenix.http://phoenix.tuwien.ac.attuwien.ac.at//combichemcombichem/references.htm/references.htm

ujordis@pop.tuwien.ac.atujordis@pop.tuwien.ac.at