Combinatorial synthesis

Combinatorial synthesis is a means of producing a large number of compounds in

a short time period using a defined reaction route and a large variety of starting
materials and reagents.
Basic concept of combinatorial chemistry:
Combinatorial chemistry differs from traditional synthesis in that it involves the
simultaneous reaction of a set of compounds to produce a set of products known
as combinatorial library.
Consider, for example the reaction a set of three compounds (A 1,A2,A3) with a set
of three building blocks(B1,B2,B3),In combinational synthesis A1 would
simultaneously undergo separate reaction with compounds B 1,B2 and B3
respectively (Figure 1). At the same time compounds A 1 and A3 would also be
undergoing reactions with compounds B1, B2 and B3. These simultaneous
reactions would produce a library of nine products. If this process is repeated by
reacting these nine products with three new building block (C 1, C2, C3), a
combinatory library of 27 new products would be obtained.
 Fig: Combinatorial library.

Design of combinatorial synthesis

One of the two general strategies are followed when designing a combinatorial
synthesis

1. Sequential attachment of building blocks: in this case the building

blocks are successively added to the preceding structure so that it grows
in only one direction. It usually relies on the medicinal chemist finding
suitable protecting groups so that the reactions are selective.

2. Non – sequential attachment of building block: In this case the

synthesis can proceed in different direction from an initial building block
known as a template, provided that the template has either the necessary
functional groups or they can be generated during the course of the
synthesis. This process may also require the use of suitable protecting
groups.

General techniques used in combinatorial synthesis: There are two general

techniques used in combinatorial synthesis:
A. Combinatorial synthesis carried out on a solid support (solid phase
technique /solid support method)
B. Combinatorial synthesis carried out in solution.
Solid phase technique /solid support method: The majority of combinatorial
experiments have been achieved using solid phase technique where the reaction
is carried out on a solid support such as a bead.
Combinatorial synthesis carried out in solution: Combinatorial synthesis in
solution can be used to produce libraries that consist of single compounds or
mixtures using traditional organic chemistry. Solution phase syntheis is
accomplished in a single vessel or different vessel where the substrates are not
bound to solid support and usually lead to a mixture of compounds. For example
10 amines and 10 acid chlorides react to each other to give 100 products in a
single vessel as a mixture.

In both solid support and solution synthetic methods synthesis usually precedes
using one of the strategies for designing a combinatorial synthesis. Each type of
synthesis method has its own district advantages and disadvantages.

A comparison of the advantages and disadvantages of the solid support and

solution techniques of combinatorial chemistry.

Solid support In solution

 Reagents can be used in excess in  Reagents can not be used in excess
order to drive the reaction in unless additional purification is
completion carried out
 Purification is easy, simply wash  Pueification can be difficult.
the support 
 Automation is easy  Automation is difficult
 Fewer suitable reactions  In theory any organic reactions can
be used
 Scale up relatively expensive  Scale up is easy and releatively
 Excess reagents and by products inexpensive
can be casily removed  Separation of reagents and by
 Using a low reagent loading products are difficult
undesired side reactions can be  Minimization of undesired side
minimized reactions is difficult
 It takes time and resourses to  Only requires time to develop the
develop and optimize the reaction chemistry
conditions

Solid phase technique /solid support method:

Advantages of Solid supported synthesis:

1. Specific reactants can be bound to specific beads
2. Beads can be mixed and reacted in the same reaction vessel
3. Products formed are distinctive for each bead and physically distinct
4. Excess reagents can be used to drive reactions to completion
5. Excess reagents and by products are easily removed
6. Using of low loadings of reagent (0.8 mmol/g support) undesired side
reactions such as cross coupling can be supressed.
7. Reaction intermediates are attached to bead and do not need to be isolated
and purified
8. Individual beads can be separated to isolate individual products
9. Polymeric support can be regenerated and re-used after cleaving the product
10. Automation is possible
The essential requirements for solid phase synthesis are:
 A cross-linked in soluble polymeric support which is inert to the synthetic
conditions (e.g. a resin bead )
 An anchor or linker covalently linked to the resin. The anchor will have a
reactive functional group such that substrates can be attached to it.
 A bond linking the substrate to the linker which will be stable to the
reaction conditions used in the synthesis.
 A means of cleaving the product or the intermediates from the linker.
 Chemical protecting groups for functional groups not involved in the
synthetic a route.

Resin beads:

1) Polystyrene – divinylbenzene resin beads/cross-linked polystyrene:

The earliest form of resin used by Merrifield was partially cross-linked
polystyrene beads where the styrene was cross-linked with 1%
divinylbenzene .the beads are derivatized with a chloromethyl group (the
anchorl linker ),to which amino acids can be coupled via an ester group.
This ester group is stable to the reaction condition used in peptide
synthesis but can be cleaved at the end of the synthesis using vigorous
acidic conditions.
2) Polyamide resin: It is polyacrylamide polymers, usually formed by using
N, N-dimethylacrylamide as backbone monomer N, N-
bisacryloylethylenediamine as cross-linker.
3) Tentagel resin: It consists of a polyethylene glycol attached to cross –
linked polystyrene through ether link. It is obtained by polymerization of
ethylene oxide on cross – linked polystyrene.
This resin has been developed to be more suitable for the combinatorial
synthesis of no – peptides.
Although beads are the common shape for the solid support, a range of
other shapes has been designed (e.g. pins) to maximize the surface area
available for reaction and hence maximize the amount of compound linked
to the solid support.

Anchor/ linker:

The anchor/ linker is a molecular unit covalently attached to the solid support. It
contains a reactive functional group with which the first of the proposed synthesis
can react and hence become attached to the resin. The resulting link must be
stable to the reaction conditions used throughout the synthesis, but be easily
cleaved to release the final compound once the synthesis is complete.
Different linkers are used depending on the functional group which will be
present on the substrate, and the functional group which is desired on the final
product once it is released.

Resins having different linkers are given different names. For example:

1) Wang resin: The Wang resin has a linker (the linkage point is circled)
which is suited for the attachment and release of carboxylic acids.

2) Rink resin: The rink resin is suited for the attachment of carboxylic acids
and the release of carboxamides.

3) Dihydropyran functionalized resin/ dihydropyran derivatized

resin: The Dihydropyran functionalized resin is suitable for the
attachment and release of alcohols.

Solid phase technique /solid support method:

Combinatorial synthesis on solid supports usually carried out by the

following procedures:

1) Parallel synthesis
2) Mix and split technique (mixed combinatorial synthesis)
3) Sequential chemical tagging method
4) Still’s binary code tag system
 1) Parallel synthesis: Combinatorial synthesis can be carried out such that
a single product is obtained in each different reaction flask a process
known as parallel synthesis. This is useful for drug optimization.
To carry out drug optimization, the synthesis of a large number of
analogues is required to test structural activity relationships and or to
improve the activity of the lead compound. Parallel synthesis allows the
rapid synthesis of analogues which vary only slightly in structure from
the lead compound. The emphasis is on producing individual compounds
(one to each reaction flask) which can be isolated, identified and tested.

Methods of parallel synthesis:

a) Houghton’s tea bag procedure
b) Automated parallel synthesis
c) Fodor’s method for parallel synthesis

2) Mix and split technique (mixed combinatorial synthesis):

Combinatorial synthesis is often designed to produce a mixture of
products in each reaction vessel, starting materials and reagents a
process known as mixed combinatorial synthesis. This is frequently used
for drug discovery, i.e. finding a lead compound.

General principle of mixed combinatorial synthesis:

Mixed combinatorial synthesis does not mean that all possible starting materials
are thrown together in one reaction flask. Planning has to go into designing a
mixed combinatorial synthesis to minimize the effort involved and to maximize the
number of different structures obtained.
As an example, suppose you wish to synthesize all the possible dipeptides of five
different amino acids. Using orthodox chemistry, you would synthesize one at a
time. There are 25 possible dipeptides and so you would have to carry out 25
separate experiments.

Glycine (Gly) Gly- Gly Ala- Gly Phe-Gly Val- Gly Ser-Gly
Alanine (Ala) Gly-Ala Ala- Ala Phe-Ala Val- Ala Ser-Ala
Phenylalanine Gly-Phe Ala-Phe Phe-Phe Val-Phe Ser-Phe
Valine (Val) Gly- Val Ala- Val Phe- Val Val- Val Ser- Val
Serine (Ser) Gly- Ser Ala- Ser Phe-Ser Val- Ser Ser-Ser
Fig: Traditional synthesis of dipeptides

However, using combinatorial chemistry the same products could be obtained

with far less effort. If all five different amino acids are separately bound to resin
beads, the beads can then mixed together and treated with a second amino acid
to produce all possible dipeptides in five experiments. For example, in one
experiment the fivedifferent amino acids bound to resin beads could be combined
with glycine to produce five of the 25 possible dipeptides.
 Fig: Synthesis of five different dipeptides

This mixture could then be tested for actively. If the results were positive, the
emphasis would be on identifying which of the dipeptides was active. If there
was no activity present, then the mixture could be ignored.

Aims:
1. To use a standard synthetic route to produce a large variety of different
analogues where each reaction vessel or tube contains a mixture of products
2. The identities of the structures in each vessel are not known with certainty
3. Useful for finding a lead compound
4. Capable of synthesising large numbers of compounds quickly
5. Each mixture is tested for activity as the mixture
6. Inactive mixtures are stored in combinatorial libraries
7. Active mixtures are studied further to identify active component