A PRIMER ON
COMBINATORIAL
CHEMISTRY
Derek S. Tan, PhD
© 2005
Combinatorial Chemistry
Solid phase synthesis is rapid and efficient
amino
acid
AA2
AA3
AA3
coupling &
deprotection
AA1
AA2
coupling &
deprotection
AA1
AA3
AA2
coupling &
deprotection
AA3
cleavage
polystyrene
bead
AA1
AA2
AA3
• Reactions can be driven to completion using
excess reagents and multiple couplings
• Intermediates do not need to be purified
• Final compounds are cleaved from the beads
and purified by HPLC as necessary
• Some procedures can be machine automated
Professor R. Bruce Merrifield
The Rockefeller University
Nobel Prize, 1984
2
Combinatorial Chemistry
Any modular structure can be combinatorialized
R3
R2
O
+H N
3
R1
N
H
O
H
N
O
R3
NH H
R4
N
H
O
R4
O
R2
O
O O
O P
–O O
HO
R3
O O
O P
–O O
O H
N
O
H
R1
O
O
R1
R2
N
O–
O
R4
O
N
R4
R3
R1
O O
N
O P
–O O
OH
R2
• A fully combinatorial library includes all possible combinations of building blocks
Each position can be 1 of 20
possible amino acids
Gly
Ala
Val
Leu
Ile
Ser
Thr
Cys
Met
Lys
Phe
Tyr
His
Trp
Arg
Asp
Glu
Asn
Gln
Pro
AA2
AA1
AA2
AA3
AA1
AA2
AA3
AA1
…
= 202 =
400 combinations
20 x 20 x 20 = 203 =
8,000 combinations
= 204 =
160,000 combinations
20 x 20
AA1
…
AA4
AA20
= 2020 = 1.0 x 1026 combinations
3
Combinatorial Chemistry
Mixture synthesis is rapid but products must be deconvoluted
R2
O
+H N
3
R1
N
H
Peptide Library Example:
Limit to 3 possibilities at 2 positions
 3 x 3 = 9 possible dipeptides
O
O
Mixture Synthesis
• Must compensate for different rates of coupling for different amino acids due to steric effects – [Val] > [Gly]
Gly
usually many beads
per product instead of
just one as shown
Ile
Gly
Gly
Gly
Gly
Ile
Gly
Val
Gly
Gly
Ile
Ile
Ile
Ile
Ile
Val
Ile
Val
Gly
Val
Val
Ile
Val
Val
Val
Val
Gly
Ile
Val
Gly
Ile
Val
2 positions
x 2 rxns each
= 4 reactions
mixture of
9 products
4
Combinatorial Chemistry
Parallel synthesis provides individual compounds but is laborious for large
libraries
Parallel Synthesis
• Only feasible for small libraries, otherwise requires automated synthesis
Gly
Gly
Gly
Gly
Gly
Ile
Gly
Val
Gly
Gly
Ile
Ile
Ile
Val
Ile
Gly
Val
Ile
Val
Val
Val
Ile
Gly
Val
Gly
Gly
Ile
Gly
Ile
many beads in
each of 9 individual
reaction vessels
Gly
Ile
Ile
Ile
9 products
x 4 rxns each
= 36 reactions
Val
Ile
Ile
Val
Gly
Val
Val
Ile
Val
Val
Val
Val
5
Combinatorial Chemistry
Split–Pool synthesis combines the advantages of mixture and parallel synthesis
Split–Pool Synthesis
• Large libraries of individual compounds can be synthesized efficiently
Gly
SPLIT
Gly
Gly
Ile
Gly
Ile
Gly
Val
Gly
Val
Ile
Gly
Ile
Gly
Ile
Ile
Ile
Ile
Ile
Val
Ile
Val
Val
Gly
Val
Gly
Val
Ile
Val
Ile
Val
Val
Val
Gly
Gly
Gly
POOL
SPLIT
Gly
Ile
many beads
Ile
Val
Val
Val
3 reaction vessels
3 possibilities
x 2 couplings
+ 2 deprotections
= 8 reactions
Each bead exposed to only a single reaction sequence, thus each has only a single product!
• Furka, Á.; Sebestyén, F.; Asgedom, M.; Dibó, G. Int. J. Pept. Protein Res. 1991, 37, 487-493.
• Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84
6
Combinatorial Chemistry
Comparison of techniques
R2
O
+H
3N
R1
N
H
O
R4
O
H
N
R3
N
H
O
O
Larger Example:
All 20 possibilities at 4 positions
 20 x 20 x 20 x 20 = 160,000 possible tetrapeptides
• Mixture Synthesis
4 positions x (1 coupling + 1 deprotection) = 8 reactions
1 mixture of 160,000 products
• Parallel Synthesis
160,000 products x (4 couplings + 4 deprotections) = 1,280,000 reactions
160,000 individual products
• Split–Pool Synthesis
split–pool
reaction arrow
(20 possibilities x 4 couplings) + 4 deprotections = 84 reactions
160,000 individual products
7
Combinatorial Chemistry
Parallel synthesis provides individual compounds but is laborious for large
libraries
Parallel Synthesis
• Only feasible for small libraries, otherwise requires automated synthesis
Gly
Gly
Gly
*
Gly
Gly
Ile
many beads in
9 individual
reaction vessels
Ile
Gly
Gly
Ile
Ile
Ile
Val
Ile
Gly
Val
9 products
x 4 rxns each
= 36 reactions
Val
Ile
Ile
Val
Gly
Val
Val
Ile
Val
Ile
Val
Val
Val
Val
Val
*
Val
Ile
Ile
*
Gly
Gly
Ile
*
Ile
Val
Gly
*
Gly
Ile
Gly
*
Gly
Val
*some savings are
possible by pooling
early steps
(3 x 2) + (9 x 2)
= 24 reactions
8
Combinatorial Chemistry
Comparison of techniques
R2
O
+H
3N
R1
N
H
O
R4
O
H
N
R3
N
H
O
O
Larger Example:
All 20 possibilities at 4 positions
 20 x 20 x 20 x 20 = 160,000 possible tetrapeptides
• Mixture Synthesis
4 positions x (1 coupling + 1 deprotection) = 8 reactions
1 mixture of 160,000 products
• Parallel Synthesis
160,000 products x (4 couplings + 4 deprotections) = 1,280,000 reactions
If pooling is used: (20 x 2) + (202 x 2) + (203 x 2) + (204 x 2) = 336,840 reactions
160,000 individual products
• Split–Pool Synthesis
split–pool
reaction arrow
(20 possibilities x 4 couplings) + 4 deprotections = 84 reactions
160,000 individual products but recursive deconvolution or encoding is required
9
Identification of Hits from Split–Pool Libraries
Recursive deconvolution involves successive resynthesis and rescreening
R2
O
+H
3N
R1
Trp
N
H
R3
O
Met
R4
O
H
N
Cys
N
H
O
O
Pro
Positional information is lost during the pooling steps:
each bead has only a single peptide sequence attached…
…but which sequence is it?
Edman sequencing is only useful for natural peptides
Assay product from each bead separately
1) Do not repool at 4th step
R1 = Trp
*
*
(R1
2) Fixed 4th step
= Trp)
Do not repool at 3rd step
160,000
assays
R2 = Met
8,000
assays
Trp
3) Fixed 4th step (R1 = Trp)
Fixed 3rd step (R2 = Met)
Do not repool at 2nd step
4) Fixed 4th step (R1 = Trp)
Fixed 3rd step (R2 = Met)
Fixed 2nd step (R3 = Cys)
Do not repool at 1st step
*
Met
Cys
Met
R3 = Cys
400
assays
Trp
Trp
*
R4 = Pro
20
assays
10
Identification of Hits from Split–Pool Libraries
Radiofrequency encoding allows single copy libraries to be made
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
beads are placed in a
polypropylene capsule
with a radiofrequency tag
computer directs
SPLIT process
based on tags
POOL
building block
coupling
compounds
are tested
individually
repeat 2
more times
radiofrequency tags
indicate reaction
sequence used
computer directs
SPLIT process
based on tags
POOL
building block
coupling
computer directs
SPLIT process
based on tags
• Nicolaou, K. C.; Xiao, X.-Y.; Parandoosh, Z.; Senyei, A.; Nova, M. P. “Radiofrequency
encoded combinatorial chemistry.” Angew. Chem., Intl. Ed. 1995, 34, 2289–91.
Trp-Met-Cys-Pro
Limitation:
compounds
103
11
Identification of Hits from Split–Pool Libraries
Covalent chemical encoding has been proposed using oligonucleotide tags
• Brenner, S.; Lerner, R. A. “Encoded combinatorial chemistry.” Proc. Natl. Acad. Sci. USA 1992, 89, 5381–5383.
1) SPLIT
2) couple R4
3) couple DNA
4) POOL
HO
1) SPLIT
2) couple R3
3) couple DNA
4) POOL
R4
+H
3N
O
1) SPLIT
2) couple R2
3) couple DNA
4) POOL
1) SPLIT
2) couple R1
3) couple DNA
4) POOL
4 x 4 oligonucleotides
can encode 16 amino acids
R2
O
+H N
3
R1
+H N
3
A T
O
N
H
O
R3
R3
N
H
O
A T C G
O
R4
O
H
N
R4
O
N
H
O
O
A T C G T C A A
screen, PCR amplify,
sequence DNA to determine
encoded reaction sequence
Trp-Met-Cys-Pro
• Similarly to phage display, this approach provides a physical link between the
reaction sequence and the encoding tags
• However, if small molecules other than peptides are desired, compatibility of the
ligand molecules with oligonucleotide synthesis and compatibility of the
oligonucleotide encoding tags with ligand synthesis are major limitations
12
Identification of Hits from Split–Pool Libraries
Covalent chemical encoding can be accomplished using binary haloaromatic
tags
• Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.; Reader, J. C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C.
“Complex synthetic chemical libraries indexed with molecular tags.” Proc. Natl. Acad. Sci. USA 1993, 90, 10922–10926.
• Nestler, H. P.; Bartlett, P. A.; Still, W. C. “A general method for molecular tagging of encoded combinatorial chemistry libraries.”
J. Org. Chem. 1994, 59, 4723–4724.
chemically inert
O
O
( )n
encoding tags
Xm
OMe
N2
1) SPLIT
4
O
2) couple R
POOL
Rh2(TFA)4
R4
R4
O
HO
+H
O
3N
+H
O
1) SPLIT
2) couple R3
3) couple tags
4) POOL
1) SPLIT
2) couple R2
3) couple tags
4) POOL
O
3N
R2
O
+H N
3
N
H
R1
O
R3
O
O
N
H
Binary Encoding
“n” tags encode 2n–1 bb’s
5 tags encode 25-1 = 31 bb’s
20 tags encode 220-1 = 106
TAGSn,m
TAGSn,m
R4
O
H
N
O
O
O
+H N
3
R1
N
H
O
H
N
O
R3
N
H
O
O–
O
Xm
O
O
TAGSn,m
Me3Si group makes tag more
volatile for GC analysis
ceric
ammonium
nitrate (CAN)
TAGSn,m
TAGSn,m
R4
O
( )n
OMe
screen on bead or
cleave and screen in solution
R2
Xm
OMe
couple tags directly to
polystyrene backbone
1) SPLIT
2) couple R1
3) couple tags
4) POOL
O
( )n
O
( )n
Xm
OMe
O
TAGSn,m
beads retain encoding tags
OTMS
HO
O
( )n
Me
Xm
N
TMS
TMSO
O
( )n
Xm
oxidant
Electron capture gas chromatography
(EC-GC) decoding in comparison to
standard sample with all tags 13
Identification of Hits from Split–Pool Libraries
Split–pool synthesis of a non-natural peptide library with binary encoding
1)
R3
OH
FmocHN
TAGS3
O
2 building blocks
R3
20% piperidine
in DMF
O
FmocHN
HO
TAGS3
R3
O
H2N
O
O
2) encode position R3
2 compounds
TAG 3A or 3B
O
1)
FmocHN
TAGS3
OH
R2
3 building blocks
O
FmocHN
R3
N
H
R2
TAGS3
O
R3
TAGS2
O
N
H
R2
O
2) encode position R2
O
20% pip
TAGS2 in DMF H2N
O
2 x 3 = 6 compounds
TAG 2A and/or 2B
Building blocks and binary codes
R1
1)
TAGS3
OH
FmocHN
R1
O
3 building blocks
O
H
N
FmocHN
O
R2
R3
N
H
O
FmocN
O
0-1
TAGS1
O
2 x 3 x 3 = 18 compounds
TAG 1A and/or 1B
20% piperidine
in DMF
O
H
N
H2N
O
R2
N
H
O
TAGS1
8 reactions, 18 compounds, 6 tags
FmocHN
OH
1-0
1-1
O
O
TAGS2
O
OH
OH
FmocN
R3
O
S
FmocHN
0-1
TAGS3
R1
1-0
O
FmocN
2) encode position R1
OH
FmocHN
TAGS2
O
O
OH
FmocHN
OH
O
OH
FmocHN
OH
Me3Si
0-1
1-0
= TAG 1B
= TAG 1A
1-1
= TAG 1A
+ TAG 1B
14