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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
THANK YOUTHANK YOUTHANK YOUTHANK YOU
55
THANK YOUTHANK YOUTHANK YOUTHANK YOU
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