Encoded Combinatorial Libraries Encoded Combinatorial Libraries

Gilbert WasongaGilbert Wasonga

Michigan StateMichigan State

UniversityUniversity

02/11/200902/11/2009

Combinatorial Chemistry

� Combinatorial chemistry is the systematic and repetitive

covalent assembly of reactant molecules to create a diverse

array of molecules with a common scaffold

� Benefits of combinatorial chemistry� Benefits of combinatorial chemistry

� Economics

� Speed

2

Lead Compounds from Combinatorial Chemistry

N

N

O

HN

Cl

N

N

O

HN

Cl

Ki = 4 6nM Ki = 21nM

Bayer's CB-1 Anagonist

3

Dolle, R. E. et al, W. J. Comb. Chem. 2008, 10, 753

Ki = 4.6nM Ki = 21nM

NH

O

N

N

NCN

NH

O

N

N

N

OHKi = 9nM

Ki = 14nM

Pfizer's mGluR1 Antagonist

Tools for Combinatorial Synthesis

� Solid phase synthesis

� Resins

� Linkers� Linkers

� Screening methods

4

Solid Phase Synthesis

Bruce Merrifield

5

Kimmerlin, J.; et al. J. Peptide Res. 2005, 65 229

http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/

Cl

O

OHNH2

OMeMeOO

Merrifield resinEster

Wang resinEster Rink resin

Amide

Mix and Split Synthesis

A1 A2 A3

A1A2 A3

Beads

Split

Divesity

A1A1 A2 A2 A3A3

6Houghten, R. A. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 5131

B1 B2 B3

A2B1 A3B1 A1B2 A2B2 A1B3 A2B3

Mix

Split

Diversity

A1B1 A3B3A3B2

A1 A2 A3 A2 A1 A3 A1 A3 A2

Challenges in Split Synthesis

� Time consuming deconvolution process

� The amount of material on one bead is insufficient for

structural determination

7

� The chemical history of the compounds synthesized is lost

Encoding Strategies

� Positional encoding

� Graphical encoding

� Radiofrequency encoding

� Spectrometric encoding

� Chemical encoding

Combinatorial Chemistry Bannwarth W.; Hinzen B., Eds 2nd Wiley-VCH: Weinhem, 20068

Chemical Encoding Strategy

� One bead two compound approach

�Peptide Nucleic Acid encoding

�DNA-encoding

9

One bead One Compound Encoding

10Liu, R.; et al. J. Am. Chem. Soc. 2002, 124, 7678

Edman Sequencing

11

Peptidomimetics

� Peptides do not make good drug candidate because they are

rapidly proteolyzed and have poor bioavailability

� Peptidomimetics allows access to compounds without

undesirable characteristics of linear peptides

� Cyclic peptoid provide ideal scaffolds as protein ligands

� Abiotic

� Conformationally order

Rezai, T. et al J. Am. Chem. Soc. 2006, 128, 2510

12

Conformational Basis for Permeability

13Rezai, T. et al J. Am. Chem. Soc. 2006, 128, 2510

Challenges In Peptoid Libraries Screening

� Peptoid sequence cannot be determined by Edman

sequencing

� Cyclic peptoids fragment at multiple positions complicating

interpretation of MS/MS spectrum interpretation of MS/MS spectrum

14

Kwon, Y-U. et al Chem. Commun. 2008, 5704

Chemical Encoding Strategy

� One bead two compound approach

�DNA-encoding

�Peptide Nucleic Acid encoding

15

One Bead Two Compound Encoding

Kwon, Y-U.; Kodadek, T.; Chem. Commun. 2008, 5704

16

Synthesis of Encoded Cyclic Peptoid Libraries

HN NH

NHFmoc

O O

17

NH

NH

NHivDde

O

O

H

TrtS

7 : 1

Kwon, Y-U.; et al. Chem. Commun. 2008, 5704

Coupling Reaction

18

Alluri, P. G. et al J. Am. Chem.Soc. 1999, 121, 7967

Encoded Cyclic Peptoid libraries

Cyclic strand

19Kwon, Y-U.; et al. Chem. Commun. 2008, 5704

Cyclic strand

Encoding strand

Cyclization Reaction

20

Shin ,S. B.; et al. J. Am.Chem. Soc. 2007, 129,3218

Mass Spectrum

21Kwon, Y-U.; et al. Chem. Commun. 2008, 5704

Sequence Analysis

22Kwon, Y-U.; et al. Chem. Commun. 2008, 5704

Chemical Encoding Strategy

� One bead two compound approach

�DNA-encoding

�Peptide Nucleic Acid encoding

23

DNA Encoded Libraries

TCT GGA-NH2- 5'

Conjugation

5'-NH2(CH2)6PO4-GCA GCT TCT XXX XXX CGA CCA TGG-3'

X denotes different combinations of 6 bases for individual compounds

General structure 48merOligonucleotide

3'-GGT CCA

24Dumelin, E. C. et al. Bioconjugate Chem. 2006, 17, 366

TCT GGA-NH23'-GGT CCA

TCT GGA-NH23'-GGT CCA

TCT GGA-NH23'-GGT CCA

619 compounds libary each with a specific coding sequence

Synthesis of DNA-Compound Conjugate

HO OA C G A T PO

OOH

EDC, Imidazole

H2NNH2

HOO

A C G A TP

O

HN

OH

NH2

HO OA C G A T PO

OOH N C N

NH

HO A C G A T O HO

3' 5'

25Ghosh, S. S.; et al. Bioconjugate Chem. 1990, 1, 71

HOO

A C G A TP

O

O

OHHN

N

NH

N

HN HO OA C G A T P

NOH

N

NH

NH

HN

O

isourea by-product

H2NNH2

N

HN

HOO

A C G A TP

O

HN

OH

NH2Phosphoramidate

Phosphorimidazole

Activated esterintermediate

Synthesis of Oligonucleotide-Compound

Conjugates

Dumelin, E. C.; et al. Bioconjugate Chem. 2006, 17, 36626

Library of Potential Albumin Binders

ONH-DNA

O

NH-DNA

OI

O

NH-DNA

O

NH-DNA

O

NH-DNA

O

B

N

N S

N

NH-DNA

O

O

O

259326

539

428535

536

Cl

27

NH-DNA

O

NH-DNA

OBr

533

624

NH-DNA

O

622

O

NH-DNA

O

O

NH-DNA

O

NH

Cl

NN

NH-DNA

O

Br

NH-DNA

O453 454

64

619

313

S NH-DNA

Dumelin, E. C.; et al. Angew. Chem., Int. Ed. 2008, 47,3196

Human Serum Albumin (HSA)

� Most abundant protein in the circulatory system

� Principle function is transport of fatty acids

� It is also capable of binding a great variety of metabolites and drugs

Curry, S.; et al. Nat. Struct. Mol. Biol. 1998, 5, 82728

Identification of HSA Binders

� Dimerization of DNA encoded compounds to cDNA

� Incubation of with Albumin immobilized on resin

� Washing with buffer solution� Washing with buffer solution

� PCR amplification using 5’-labeled primer

� Hybridization to DNA microarray

29

Dumelin, E. C.; et al. Angew. Chem., Int. Ed. 2008, 47,3196

Selection HSA Binders

30Dumelin, E. C.; et al. Angew. Chem., Int. Ed. 2008, 47,3196

Potential HSA Binders

31

Dumelin, E. C.; et al. Angew. Chem., Int. Ed. 2008, 47,3196

Enrichment Profile

32Dumelin, E. C.; et al. Angew. Chem., Int. Ed. 2008, 47,3196

Promiscuous Binders for HSA

33Dumelin, E. C.; et al. Angew. Chem., Int. Ed. 2008, 47,3196

Consensus Structure of Identified Binders

OH

OX

Y

�X must be a hydrophobic substituent

�Y can stand for a hydrophobic substituent

�Butanoyl moiety

�Hyrdophobic groups in para position

34Dumelin, E. C.; et al Angew. Chem., Int. Ed. 2008, 47,3196

Chromatographic Albumin Binding Assay

35Dumelin, E. C.; et al Angew. Chem., Int. Ed. 2008, 47,3196

Chromatographic Albumin Binding Assay

36

Dumelin, E. C.; et al Angew. Chem., Int. Ed. 2008, 47,3196

Mass Analysis of Oligonucleotide-Compound

Conjugate

37

Dumelin, E. C.; et al Angew. Chem., Int. Ed. 2008, 47,3196

Chemical Encoding Strategy

� One bead two compound approach

�DNA-encoding

�Peptide Nucleic Acid encoding

38

Peptide Nucleic Acid (PNA) Encoding

A A APNA

Harris, J. L.; et al. Chem. Eur. J. 2005, 11,6792

39

DNA

DNA

PN

PNA Encoded Split Synthesis

A1 A2 A3

A1A2 A3

Beads

Split

Divesity , PNA encoding

A1A1 A2 A2 A3A3

40

B1 B2 B3

A2B1 A3B1 A1B2 A2B2 A1B3 A2B3

Mix

Split

Diversity, PNA encoding

A1B1 A3B3A3B2

A1 A2 A3 A2 A1 A3 A1 A3 A2

Harris, J. L.; et al. Chem. Eur. J. 2005, 11,6792

Chemical Structure of PNA and DNA Oligomers

NH

O

OP

O

O

41

N

OO

Base

O

OBase

PNA DNA

Nielsen, P. E.; et al. Science, 1991, 254

Peptide Nucleic Acid (PNA)

Nielsen, P. E.; et al. Science,1991, 254 42

Synthesis of Peptide Nucleic Acid Monomers

N

NH

O

NH2

Pyridine, 00C

2eq, CbZ-ClN

NH

O

NHCbz

56%

N

NO

NHCbz

CO2CH3

BrCH2CO2CH3/ K2CO3 NaOH (aq.)

40 %

N

NO

NHCbz

CO2H

DMF

43Dueholm. K. L. et al J. Org. Chem. 1994, 59, 5767

N

N NH

N

NH2

CO2CH3

BrCH2CO2Et / NaH

DMF73%

Pyridine, 00C

2eq, CbZ-ClN

N N

N

NH2

CO2Et

N

N N

N

NHCbZ

CO2Et

+ BzCl + CO2

Rapoport’s Reagent

44

Watkins, B. E.; et al. J. Org. Chem. 1982, 47, 4471

Dueholm. K. L. et al. J. Org. Chem. 1994, 59, 5767

Guanine Synthesis

45

Dueholm. K. L. et al J. Org. Chem. 1994, 59, 5767

Breipohl, G. et al. Tetrahedron 1997, 53, 14671

Guanine Synthesis

46

Breipohl, G. et al. Tetrahedron 1997, 53, 14671

Peptide Nucleic Acid Synthesis

Debaene, F.; et al. Org. Lett. 2003, 5, 4445 47

Coupling Reaction

Debaene, F.; et al. Org. Lett. 2003, 5, 444548

PNA Encoding

HN

N

N

N

HN

N N

N

O

O O

O

PG2N

N

N N

N

NPG2

T C A G

PG2 : Boc, Mmt

NHBoc

NPG2

49

NH

NNH

NNH

N

O O O

O

B

O

B

O

B

NH

NO

ONH

O

O

R1

R2

R3Fluorous Tag

NHBoc

Debaene, F.; et al. Tetrahedron 2007, 63, 6577

Modified PNA Oligomers

Debaene, F.; et al. Tetrahedron 2007, 63, 6577

Menchise, V.; et al. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 12021

50

Solubility of Modified Oligomer

pH 5.5 Buffer pH 7.0 Buffer H2O

Unmodified PNA 23% 19% 21%

PNA-Monomer 60% 51% 47%

PNA-Monomer 100% 100% 100%

Debaene, F.; et al. Tetrahedron, 2007, 63, 6577 51

% of PNA remaining in solution after sonication and centrifugation

Encoding Protease Libraries

PNA codon elements for diversityPNA codon elements for diversity

R4 R3 R2 R1

Asp GGA A CCG GCA GAGA

Lys AAGG CGA CGA GACG

Phe CGGC GCA ACG CAGG

Ala GCCG TGC GTG CGAA

Leu GAAC GGC AGC AGGC

52Debaene, et al. Tetrahedron 2007, 63, 6577

Summary

� PNA encoding strategies are being as means of tracking chemical history of library members

� By combining genetics with chemical synthesis new chemical entities can be unambiguously identified from combinatorial libraries

� For combinatorial chemistry to have maximum impact, a large range of bond-forming reaction need to be developed on solid phase

53

Acknowledgement

Dr. Xuefei Huang Dino

Jason Vivian

Bin BoBin Bo

Medha Hovig

Gopi Phil

Steve

54

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