Involves the synthesis of a large collection of
molecules (Library), and the whole collection
is tested for biological activity. When, the
active compound is identified, it’s made in
quantity.
Therefore, Combinatorial Chemistry involves two
phases.
-1. Making a Library
-2. Finding the biologically active compound
Involves rapid efficient synthesis, and screening
respectively.
Bioassays of Natural Products- If a mixture of
compounds showed no biological activity, then all the
compounds in the mixture could be regarded as being
inactive. This assumes that Compound A in the mixture
does not effect the assay of Compound B or even react
with compound B.
Origins in Peptide Synthesis
Unprotected Coupling Three Competing Nucleophiles
O
X
NH2
O
X
OH
H2N
NH2
O
OH
H2N
O
O
O
R
NH
2 X H2N
X
R
If X= F, Cl, Br, I
HN
R
O
Diketopiperazine
Good protecting Group and Suitable Activating Group X
DCC is a Dehydrating Agent
CH3
t
Boc
OH
N
H
+
OR
H2N
O
O
N
C
N
Dicyclohexylcarbodiimide
(DCC)
CH3
t
Boc
H
O
N
N
H
OR
O
H
N
H
C
N
O
Dicyclohexylurea (DCU)
What is the mechanism for DCC mediated peptide
bond formation?
The Merrifield Synthesis
CH2Cl
CH2Cl
tBoc
N
H
1984 Noble Prize
COO
SN 2
CH2
CH2
O
C
t
O
t
N
H
NH
C
Boc
Boc
O
O
mild acid -hydrolysis
CH2
CH2
O
C
O
NH2
O
C
O
N
H2
O
O
O
O
NH2
NH2
DCC-mediated amino acid coupling
1. DCC, 2. t-Boc N
H
O
O
O
t-Boc
N
H
NH
O
COOH
O
N
H
O
N
H
t-Boc
125 Amino Acid - PEPTIDE (17%)
369 chemical reactions - 11,931 automated steps
Purification is carried out by simply washing the Resin beads
(with the attached peptide) with the appropriate solvent.
Impuries are carried away with the solvent.
To get an overall yield of 17%, Merrifield had to have an
average yield greater than 99% for each individual step.
Synthetic transformations on solid phase need to
be driven to completion, because purification of
intermediates is not possible. Thus, for long
synthetic transformations (such as the
polymerisation of amino acids), high yields are
essential.
Yields of final products as a function of yield per step
and number of steps
Yield per step
99%
95%
90%
85%
80%
2
98%
90%
81%
72%
64%
3
4
97% 96%
86% 82%
73% 66%
61% 52%
51% 41%
5
95%
77%
59%
44%
33%
6
94%
74%
53%
38%
26%
Solid Phase Reaction
Solid Support
Linker
Starting Material
Solid Support = Polymer Resin
7
93%
70%
48%
32%
21%
The Linker must have similar properties to a Protecting Group
in Organic Synthesis
R
O
Cl
O
HF, pyridine
1963 Merrifield Cleavage Procedure
HO
R
O
1973 Wang Cleavage Procedure
O
R
O
O
50% TFA solution
The enzyme synthesized by Merrifield was bovine pancreatic
ribonuclease A.
Therefore, to get this enzyme, precise control over the
sequence of AA additions is essential.
Today, an entire industry has been developed to serve the
peptide synthesis field.
There are companies providing automated peptide
synthesizers, as well as peptide building blocks, reagents and
resins.
Man is therefore moving closer to the accomplishments of
nature.
Polymer Support
Ph
Ph
n
polystyrene
styrene
divinylbenzene
Ph
Ph
Ph
Ph
Ph
cross-linked polystyrene
The Merrifield resin is now commercially available
(polymer beads 0.04-0.15 mm), and is a co-polymer of
styrene-divinyl benzene with 1-2% cross-links.
Polystyrene resins swells in DMF, DCM, and toluene.
This swelling of the resin beads is important as it
increases the size of the beads allowing greater
compound loading, and more thorough mixing of
reagents.
Disadvantages of the Merrifield Resin
1. Solvent Compatibility
Doesn’t swell in aqueous solutions
2. Not Compatible with certain reagents
Alternative is TentaGel resin, which consists of 1% crosslinked polystyrene on to which 70% polyethyleneglycol (PEG)
has been grafted.
By polymerisation of Oxirane onto a hydroxylated polystyrene
support.
CH2
HC
(OCH2CH2) OCH2 CH2 X
n
PS-DVB copolymer
PEG
Spacer
n = 70
Functional Group
PEG
HO
O
O
OH
n
Advantages of TentaGel
1. Swells in both protic and aprotic solvents.
2. The attached reacting groups project into solution
rather than being anchored close to the polymer
backbone. Conditions similar to solution phase
chemistry.
There are Two types of Parallel Solid Phase Synthesis.
1. Multiple parallel synthesis of individual
compounds. In this case, only one compound
is prepared per reaction vessel. This is
especially suitable for the rapid optimisation
of previously identified lead compounds –
Non-Combinatorial
2. The multiple parallel synthesis of compound
libraries. Here, many different compounds
having a common backbone are synthesised
simultaneously in each reaction vessel.
Combinatorial libraries are often prepared by
the ‘mix-split’ method.
In these combinatorial mixtures the number of
compounds is seen to increase exponentially
N X N possibilities
Combinatorial chemistry is an approach to synthetically
produce molecular diversity.
P
P
P
A3
A2
A1
coupling
P
P
P
A1
A2
A3
mixing
'mix and split synthesis'
P
P
P
A1
A2
A3
dividing
P
P
P
P
P
P
P
P
P
Ax
Ax
Ax
Ax
Ax
Ax
Ax
Ax
Ax
B
coupling
1
B
2
B
3
P
P
P
P
P
P
P
P
P
AxB1
AxB1
AxB1
AxB2
AxB2
AxB2
AxB3
AxB3
AxB3
mixing
Combinatorial peptide chemistry. The number of
different peptides increases exponentially with length.
Number
Peptide
Number of
of
Distinct
amino
peptides
acid
residues
H2N X1X2 COOH
2
400
H2N X1X2X3 COOH
3
8000
H2N X1X2X3X4 COOH
4
160,000
H2N X1X2X3X4X5 COOH
5
3,200,000
H2N X1X2X3X4X5X6 COOH
6
64,000,000
H2N X1X2X3X4X5X6X7 COOH 1,280,000,000
7
H2N X1X2X3X4X5X6X7X8 COOH 25,600,000,000
8
Xn represents individual aa residues. Number of
distinct peptides are based on 20 residues at each
position.
Therefore, generally there is a need to drive reactions
to completion, because the purification of
intermediates is difficult. For, the development of
new reaction sequences for solid-phase synthesis, the
optimum conditions for each step must be identified.
In 1992 Ellman became the first chemist to report
combinatorial libraries for non-oligiomeric compounds.
Reported-
Synthesis of 1,4-Benzodiazepine Library
RD
O
RB
N
RC
N
RA
As part of a drug discovery program
Ph
Cl
Ph
O
Cl
N
N
N
NHCH3
Librium
N
O
CH3
Valium (diazepam)
RA, RB, RC, RD building blocked that can be permuted.
Three components 2-aminobenzophenones, amino acids
and alkylating agents.
FMOC was chosen for NH2 protection, as base cleavage
is required for its removal, since acid-labile linker was
used.
RB
RB
NH2
NHFMOC
1. Fmoc-Cl
O
O
2. Apply to support
Wang-type resin
RA
RA
3. weak base hydrolysis
4. N-Fmoc-amino acid coupling
H
O
RB
HN
N
cyclisation
O
C
R
N
5. weak base hydrolysis
RB
FMOC
RC
NH
6. 5% acetic acid wash
O
RA
alkylation
RA
7. Bu-Li
8. RD-X
RD
RD
O
RB
RC
N
RA
O
RB
N
N
RC
aqueous TFA hydrolyis
(CH3)2S
N
RA