COMBINATORIAL CHEMISTRY AND DYNAMIC COMBINATORIAL CHEMISTRY

Sonya Balduzzi, PhD

From DNA To Proteins…

Polymer-Supported Peptide Synthesis

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

Combinatorial Synthesis vs Parallel Synthesis

Combinatorial Synthesis “Split synthesis” method Each well or reaction vessel contains many types of support-

bound compounds Biological assays are performed with mixtures of compounds Analysis of compound mixtures requires a deconvolution

procedure in order to identify an individual compound of interest

Parallel Synthesis “One-bead-one-compound” method Each well or reaction vessel contains one type of support-bound

compound Biological assays are performed with individual compounds Analysis of compounds which are either support-bound or free in

solution

Polymer-Supported Synthesis

ResinMoleculeSpacer Linker

Polymeric Resins

Resin - the polymeric matrix which forms the solid support and is inert to the reaction conditions employed during library synthesis

Resin Types:

Merrifield (chloromethylpolystyrene)

Hydroxymethylpolystyrene

Aminomethylpolystyrene

TentaGel (polystyrene-poly(ethylene glycol) copolymer)

ArgoGel (polystyrene-poly(ethylene glycol) copolymer)

PEGA (poly(ethylene glycol)-polyacrylamide copolymer)

Cl

Linkers

Linker – serves to attach the molecule of interest to the solid support

Integral Linkers – the linker is part of the polymeric resin (eg. Merrifield)

Nonintegral Linkers – the linker is attached to the polymeric resin in a separate step, typically via ether, amide or C-C connections

– provide a more uniform degree of loading of the molecule of interest than integral linkers

Electrophilic Linker Cleavage

Cl O

OH

O

OHMeO

Merrifield Wang Sasrin

NH

O

OHTrityl

OH

Hindered handle

O

NH2 OMe

OMe

RinkNH2

Me

p-Methylbenzhydrylamine

Nucleophilic Linker Cleavage

Nucleophilic addition to linker functionality

Base-catalyzed elimination

Richard Morphy, J.; Rankovic, Z.; Rees, D. C. Tetrahedron Lett.1996, 37, 3209.

Photolytic Linker Cleavage

PiUai, V. N. R. Synthesis 1980, 1-26.

Cyclative Linker Cleavage

R4HN

O

O

N

R2R1

R3

TFA

N

NO

R1

R2

R4

R3

Benzodiazepines

DeWitt, S. H.; Kiely, J. S.; Stankovic, C. J~; Schroeder, M. C.; Cody, D. M. R.; Pavia, M. R. Proc. Natl. Acad Sci. U. S. A. 1993, 90, 6909-6913.

Reductive Linker Cleavage

S R

O

LiBH4

HO R

Oxidative Linker Cleavage

SeR1

O

R2

R1

O

R2

H2O2

Kobayashi, S.; Hachiya, I.; Yasuda, M. Tetrahedron Lett. 1996,37, 5569.

Michels, R.; Kato, M.; Heitz, W. Makromol. Chem. 1976, 177, 2311.

“Traceless” Linker

N N

NO

CH3

Si

Me Me

SiCl

Me Me

H3C O

N

N

N

O

H3C

H3C

O

Bu4NF

THF

Plunkett, M. J.; Ellman, J, A. J. Org. Chem. 1997, 62, 2885-2893.

“Safety-Catch” Linker

Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. Chem. Commun. 1971, 636.

Spacer Molecules

Introduced between polymeric resin and linker molecule

Spacers serve to distance the chemistry from the solid support (immobilized catalysts)

Spacers alter both the solubility and swelling properties of the resin (eg. PEG increases the solubility of Merrifield resin in polar organic solvents)

Spacers alter the cleavage properties of the linker

Page, P.; Bradley, M.; Walters, I.; Teague, S. J. Org. Chem. 1999, 64, 794.

Combinatorial Synthesis vs Parallel Synthesis

Combinatorial Synthesis “Split synthesis” method Each well or reaction vessel contains many types of support-

bound compounds Biological assays are performed with mixtures of compounds Analysis of compound mixtures requires a deconvolution

procedure in order to identify an individual compound of interest

Parallel Synthesis “One-bead-one-compound” method Each well or reaction vessel contains one type of support-bound

compound Biological assays are performed with individual compounds Analysis of compounds which are either support-bound or free in

solution

Combinatorial Synthesis Parallel Synthesis

A1 A2

resin

A1 A2

mix and split resin

A2

A1

A2

A1

B1 B2

A2B1

A1B1

A2B2

A1B2

A2B2

A1B2

A2B1

A1B1

A2B2

A1B2

A2B1

A1B1

mix and split resin

C1 C2

A2B2C1

A1B2C1

A2B1C1

A1B1C1

A2B2C2

A1B2C2

A2B1C2

A1B1C2

resin

A1 A1B1 A1B1 B1 A2B2C1

resin= resin + linker

Deconvolution of Combinatorial Libraries of Compounds

The biologically active compound in a mixture of many compounds is determined

Iterative deconvolution method

Positional scanning deconvolution method

Molecular tags

Iterative Deconvolution Method

Method developed for analysis of peptide mixtures

The library is resynthesized in a different format

Each well or reaction vessel contains a mixture of compounds that differ at only one position

A1 A2

resin

A1 A2

B1C1

A1B1C2

A1B1C1

A1B2C2

A1B2C1

B2C1

B2C2B1C2

A2B1C2

A2B1C1

A2B2C2

A2B2C1

B1C1

B1C2

B2C1

B2C2

Combinatorial Synthesis Iterative Deconvolution

A1 A2

resin

A1 A2

mix and split resin

A2

A1

A2

A1

B1 B2

A2B1

A1B1

A2B2

A1B2

A2B2

A1B2

A2B1

A1B1

A2B2

A1B2

A2B1

A1B1

mix and split resin

C1 C2

A2B2C1

A1B2C1

A2B1C1

A1B1C1

A2B2C2

A1B2C2

A2B1C2

A1B1C2

A1 A2

resin

A1 A2

B1C1

A1B1C2

A1B1C1

A1B2C2

A1B2C1

B2C1

B2C2B1C2

A2B1C2

A2B1C1

A2B2C2

A2B2C1

B1C1

B1C2

B2C1

B2C2

*

Molecular Tags

Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.; Reader, J. C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C.

Sci. U.S.A. 1993, 90, 10922.Proc. Natl. Acad.

Diazoketones as Molecular Tags

40 Different diazoketones can be prepared from:

10 different diols 4 different chlorophenols vanillic acid

Encoding requires predictable reactions !

Characterization of Compounds

On solid support IR Enzyme-linked colorimetric assay (for peptides)

(Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82)

In solution NMR, MS, IR

DYNAMIC COMBINATORIAL CHEMISTRY

Static vs Dynamic Combinatorial Chemistry

Lehn, J.-M.Chem. Eur. J. 5, 2455–2463 (1999).

Dynamic Combinatorial Chemistry

Definition

All possible combinations of compounds are generated through reversible connection processes, either covalent or noncovalent (virtual combinatorial library)

Upon introduction of a target molecule (such as a receptor), a binding event can occur between the target and individual compounds

The compound which binds most strongly to the target will be produced in greatest quantity (amplification phenomenon) through changes in the composition of the equilibrium (adaptive phenomenon)

Olof Ramström and Jean-Marie LehnNature Reviews Drug Discovery2002, 1, 26

Emil Fischer's Lock and Key Analogy

Dynamic Combinatorial Chemistry

Advantages

DCC provides a more efficient method to produce large quantities of compounds for biological screening since individual members of the virtual combinatorial library are not actually isolated and purified prior to screening

Detection and characterization of compounds is facilitated by an increased signal strength of the strongest binding compound as a result of its amplification

Isolation of “amplified” compounds is facilitated through physical separation of the target-compound complex (dialysis)

The ability to analyze target-bound compounds would eliminate a few synthetic steps

Dynamic Combinatorial Chemistry

Requirements

Reversible connection processes must occur between components of a library

Reversible connection processes can be covalent or noncovalent

Equilibrium between the components of a library must be reached on the same time scale as the reaction between a target molecule and the components of a library

The components of a library must have similar reactivity in order to attain thermodynamic control

Generation of a Virtual Combinatorial Library

Casting: Receptor-induced self assembly of a complementary substrate

Lehn, J.-M.Chem. Eur. J. 5, 2455–2463 (1999).

Molding: Substrate-induced self assembly of a complementary receptor

Generation of a Virtual Combinatorial Library - Casting

I. Huc, J.-M. Lehn, Proc. Natl. Acad. Sci. USA 1997, 94, 2106

Generation of a Virtual Combinatorial Library - Casting

I. Huc, J.-M. Lehn, Proc. Natl. Acad. Sci. USA 1997, 94, 2106

HPLC Trace of Reaction Mixtures

Target-Accelerated Combinatorial Synthesis of Vancomycin Analogues

Vancomycin An antibiotic (last line of defence against gram-positive bacteria) which

inhibits bacterial growth by interfering with bacterial cell wall peptidoglycan biosynthesis

Vancomycin forms 5 hydrogen bonds to the terminal Lys-D-Ala-D-Ala residue of bacterial peptidoglycan

Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst, C.; Labischinski, H.;Endermann, R. Angew. Chem. Int. Ed. 2000, 39, 3823.

Dimerization of Vancomycin

Monomer vancomycin bound to a peptide substrate has a greater dimerization constant than free monomeric vancomycin

The dimeric form of vancomycin has greater biological activity than the monomeric form (increased binding affinity to peptidoglycan through extended hydrogen-bonding)

Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst, C.; Labischinski, H.;Endermann, R. Angew. Chem. Int. Ed. 2000, 39, 3823.

Combinatorial Synthesis of Dimeric Vancomycin Analogues

Combinatorial Synthesis of Dimeric Vancomycin Analogues

Ac2-L-Lys-D-Ala-D-Ala

Mass Spectrometric Analysis of the Vancomycin Dimer Mixture

m = 2, 3 ,4

C2-C2

C2-C3 C3-C2

C2-C4 C4-C2 C3-C3

C3-C4 C4-C3

C4-C4

Correlation Between Biological Activity and Tether Length

n = number of atoms between the two nitrogens

MIC = minimum inhibitionconcentration

n = 16 corresponds to the C2-C2 homodimer

Dynamic Combinatorial Chemistry

Limitations

Only reversible reactions can be employed

Equilibrium must be established under reaction conditions in which the target is stable (biological molecules have solvent and pH requirements)

There are limits on the potential size of a combinatorial library since the concentration of all the components must be sufficient for binding to the target