Combinatorial Chemistry

Medicinal Chemistry & Drug Discovery II (MSB)

The concept of combinatorial synthesis
 Combinatorial chemistry is an approach of synthetic chemistry
that allows simultaneous synthesis of a large number of the possible
compounds.
 Simultaneous synthesis is achieved by addition of a range of closely
related building blocks and optimal environment.
 The products resulting from combinatorial synthesis are together
called combinatorial library.
 Libraries may be a collection of individual compounds or mixtures
of compounds.
 Libraries are then subjected to high throughput screening to identify
potentially bioactive compounds.
An example of combinatorial approach
Approaches/ Strategies in combinatorial
chemistry
 Generally two types of approaches are followed in combinatorial chemistry:
 (a) Linear synthesis: Sequential attachment of building blocks in one direction
 (b) Template method: Multidirectional attachments on a single templates
Linear synthesis
 Building blocks are successively added to the preceding structure.
 The structure grows only in one direction.
 Suitable protecting groups are employed to ensure selectivity and
unidirectional growth.
 Molecular diversity is absent or limited.
 Structure of the resultant compound is easily identified.
 Suitable for synthesis of polymers or oligomers from multiple
monomeric units e.g. protein, peptide, oligonucleotides etc.
Template method
 Starts with a single template molecule.
 Multiple building blocks are applied at once to the original
template.
 Generally, no functional groups are protected on the original
template, to allow multidirectional growth.
 Extensive molecular can be obtained.
 A mixture of compounds is obtained as product.
 Structures of the products can be predictable but must be
determined with further analysis.
 Reactions in template method
The reactions used when designing a combinatorial sequence should ideally satisfy the
following criteria:
 The reactions should be specific, relatively easy to carry out and high yielding.
 The reactions used in the sequence should allow for the formation of as wide a range of
structures for the final products as possible, including all the possible stereoisomers.
 The reactions should be suitable for use in automated equipment.
 The building blocks should be as diverse as possible so that the range of final products
includes structures that utilizes all the types of bonding to bind to or react with the target.
 The building blocks should be readily available.
 It must be possible to accurately determine the structures of the final products.
 Techniques/ Methods in
combinatorial chemistry
 There are two general techniques employed in combinatorial synthesis-
 Synthesis on solid support: The first building block or template is
attached to a solid support and the reaction takes place as such. The
complete products are detached at the end of synthesis.
 Synthesis in solution: All the building blocks are dissolved in the solvent
and reaction takes place within the solution. Products are obtained by
altering the solubility.
 Both solid support and solution synthetic methods may be used to produce
libraries that consist of either individual compounds or mixtures of
compounds.
 Comparison between the techniques
Synthesis on solid support Synthesis in solution
Reagents can be used in excess in order to Reagents cannot be used in excess, unless
drive the reaction to completion. addition purification is carried out.

Purification is easy, simply wash the support. Purification can be difficult.

Automation is easy. Automation may be difficult.

Fewer suitable reactions. In theory any organic reaction can be used.

Scale up is relatively expensive. Scale up is relatively easy and inexpensive.

Not well documented and time will be Only requires time for the development of the
required to find a suitable support and linker chemistry.
for a specific synthesis.
Merrifields solid support peptide synthesis

 A linear synthetic approach where one amino acid is attached at each step.
 Uses polystyrene–divinylbenzene resin beads as a solid support.
 Each bead has a large number of monochlorinated methyl side chains.
 The C-terminal of the first amino acid in the peptide chain is attached to these chloro
groups.
 The large number of chlorinated side chains on the bead means that one bead acts as
the solid support for the formation of a large number of peptides.
 Additional amino acids are added to the growing peptide chain.
Merrifields solid support peptide synthesis
 The peptide synthesis takes place from carbonyl end (C-terminal) to amino end (N-
terminal) i.e. the carbonyl group of one amino acid is attached to amino group of
previous amino acid.
 All amino acids are protected at N-terminal to ensure unidirectional reaction.
Protecting groups such as t-butyloxycarbonyl (Boc) are used.
 The C-terminal is converted to more active acylated derivative of
dicyclohexylcarbodiimide (DCC).
Merrifields solid support peptide synthesis
Merrifields solid support peptide synthesis
Merrifields solid support peptide synthesis
Parallel synthesis
 A form of linear synthesis where a large number of similar reactions
take place side by side, but in separation.
 Generally grid shaped reactions vessels are used which contain a
large number of small reaction chambers arranged in grid e.g. 96-
well plate.
 Within each well, there is solid support.
 A specific building block is added to each well and attached to the
support with the help of a linker.
 All the reactions takes place in one direction.
Parallel synthesis
 Let us consider the preparation of a combinatorial library of hydantoins by the
reaction of isocyanates with amino acids using a 96-well array.
 Eight N-protected amino acids (X1, X2 X8) are placed in the well array so that
only one type of amino acid occupies a column, that is, column A will only
contain amino acid X1, column B will only contain amino acid X2, and so on.
 12 isocyanates (Y1, Y2 Y8) added to the wells so that each numbered row
contains only one type of isocyanate. In other words, compound Y1 is only added
to row one, compound Y2 is only added to row two, and so on.
Parallel synthesis
Fodor’s method for parallel synthesis

 Synthesis of peptide libraries by parallel synthesis

 A glass plate acts as solid support

 The plate is treated so that its surface is coated with hydrocarbon

chains containing a terminal amino group
 These amino groups are protected by the UV-labile 6-
nitroveratryloxycarbonyl (NVOC) group
Fodor’s method for parallel synthesis
Fodor’s method for parallel synthesis

 A photolithography mask (M1) is placed over the plate so that only a

specific area of the plate can be irradiated with UV light
 Irradiation results in removal of protecting group

 So the incoming building blocks will bind with the amino groups in the
irradiated area
 Use of multiple masks (M2, M3, M4……. Mn) in different orders will
result in simultaneous synthesis of large number of compounds
Fodor’s method for parallel synthesis
Furka’s mix and split technique

 Reaction takes place on resin beads as solid support.

 Firstly, all the resin beads are grouped equally and each of the individual
initial building blocks are attached to each group by suitable reactions.
 All the beads are mixed together and distributed again into equal groups
such as each group now contains all the different types of starting material.
 Different building blocks are added to each individual groups and reaction
takes place.
 The products are mixed again, divided into equal groups and added with
separate building blocks for each group.
Furka’s mix and split technique
Furka’s mix and split technique

 The technique has the advantage that it reduces the number of reactions
required to produce a large library.

For example, if the synthetic pathway required three steps, it would

require 30,000 separate reaction vessels to produce a library of 10,000
compounds if the reactions were carried out in separate reaction
vessels using orthodox chemical methods. The Furka mix and split
method reduces this to about 22 reactions.
 Unlike in parallel synthesis the history of the bead cannot be traced
from a grid reference, it has to be traced using either a suitable
encoding method or deconvolution.
Encoding methods: Sequential chemical tagging

 Sequential chemical tagging uses specific compounds (tags) as a code

for the individual steps in the synthesis
 These tag compounds are sequentially attached in the form of a
polymer-like molecule to the same bead as the library compound at
each step in the synthesis, usually by the use of a branched linker
 One branch is used for the library synthesis and the other for the
encoding
 At the end of the synthesis both the library compound and the tag
compound are liberated from the bead
Ideal criteria for tagging compounds

 The concentration of the tag should be just sufficient for its analysis,
that is, the majority of the linkers should be occupied by the
combinatorial synthesis
 The tagging reaction must take place under conditions that are
compatible with those used for the synthesis of the library compound
 It must be possible to separate the tag from the library compound
 Analysis of the tag should be rapid and accurate using methods that
could be automated
Encoding in peptide synthesis
 Use of oligonucleotides to encode amino acids in peptide synthesis

 At each stage in the peptide synthesis a second parallel synthesis is

carried out on the same bead to attach the oligonucleotide tag
 On completion of the peptide synthesis the oligonucleotide tag is isolated
from the bead and its base sequence is determined and decoded to give
the sequence of amino acid residues in the peptide
 Syntheses are usually carried out using a branched linker so that the
synthesis of the encoding molecule and the combinatorial library
molecule it encodes runs in parallel
Encoding in peptide synthesis

 For example, the Zuckermann approach uses a diamine linker protected

at one end by an Fmoc group and at the other end by a Moz group. The
Fmoc group was cleaved under basic conditions and the suitably
protected building block was joined to the linker. The Moz group was
removed under acid conditions and a suitably protected peptide was
attached.
Encoding in peptide synthesis
Combinatorial synthesis in solution

 Can be applied to any known reaction

 Main problem lies in the purification

 A number of strategies have been developed to solve this problem

 Use of soluble supports with different solubility in different type of
solvents
 Use of scavenging reagents
Libraries formed using
Monomethyl polyethylene glycol (OMe-PEG)

 Polyethylene glycols polymers are soluble in both water and organic solvents, the degree
of solubility depending on the length of the polymer chain

 Combinatorial syntheses in solution are carried out using monomethyl polyethylene

glycol (OMe-PEG-OH), which tends to precipitate in diethyl ether

 The synthesis is started by reacting the acid group of an acidic building block to the
hydroxy group of OMe-PEG

 The product is precipitated by adding diethyl ether and the excess reagent and other
impurities are removed by washing

 The solid product is redissolved in fresh solvent and the second stage of the synthesis is
carried out using a similar reaction and washing procedure
Libraries formed using
Monomethyl polyethylene glycol (OMe-PEG)
Libraries produced using
Resin-bound scavenging agents

 This approach depends on the removal of excess reagents and byproducts

by the use of so called solid phase scavenging or sequestering agents
 They consist of resin beads to which is permanently attached a suitable
residue with an organic functional group that will readily react with the
relevant reactant or byproduct
 This reaction results in the formation of a solid complex containing the
excess reagent/byproduct, etc., which can be removed from the reaction
mixture by filtration through a cartridge containing an appropriate resin
Libraries produced using
Resin-bound scavenging agents