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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 - PowerPoint PPT Presentation
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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
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