1. Combinatorial Chemistry
PHR 401
Medicinal Chemistry III
Prepared by NZK

2. Combinatorial chemistry was developed to produce the large numbers of compounds required for high- throughput screening. It allows the simultaneous synthesis of a large number of the possible compounds that could be formed from a number of building blocks.
The products of such a process are known as a combinatorial library.

4. The basic concept of combinatorial chemistry is best illustrated by an example. Consider the reaction of a set of three compounds (A1–A3) with a set of three building blocks (B1–B3). In combinatorial synthesis, A1 would simultaneously undergo separate reactions with compounds B1, B2 and B3, respectively (Fig. 5.2). At the same time compounds A2 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 blocks (C1–C3), a combinatorial library of 27 new products would be obtained.

6. The combinatorial approach does mean that normally large libraries of many thousands of compounds can be formed rapidly in the same time that it takes to produce one product using the traditional approach to synthesis.

7. The design of combinatorial syntheses: One of two general strategies may be followed when designing a combinatorial synthesis (Fig.5.3a). In the first case the building blocks are successively added to the preceding structure so that it grows in only one direction (linear synthesis, see section ). It usually relies on the medicinal chemist finding suitable protecting groups so that the reactions are selective. This design approach is useful if the product is a polymer (oligomer) formed from a small number of monomeric units. Alternatively, the synthesis can proceed in different directions 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 (Fig. 5.3b). Both routes may require the use of protecting groups (see section ).

9. The reactions used when designing a combinatorial sequence should ideally satisfy the following criteria:
1. The reactions should be specific, relatively easy to carry out and give a high yield.
2. 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.
3. The reactions should be suitable for use in automated equipment.
4. The building blocks should be readily available.
5. The building blocks should be as diverse as possible so that the range of final products includes structures that utilise all the types of bonding (see section 8.2) to bind to or react with the target.
6. It must be possible to accurately determine the structures of the final products.

10. Techniques used in combinatorial synthesis
The solid support method:
General methods in solid support combinatorial chemistry
Parallel synthesis
Furka’s mix and split technique

11. 2. Encoding methods:
Sequential chemical tagging
Still’s binary code tag system
Computerised tagging

12. 3. Combinatorial synthesis in solution:
Parallel synthesis in solution
The formation of libraries of mixtures
Libraries formed using monomethyl polyethylene glycol (OMe- PEG)
Libraries produced using dendrimers as soluble supports
Libraries formed using fluorocarbon reagents
Libraries produced using resin-bound scavenging agents
Libraries produced using resin-bound reagents
Resin capture of products

13. This method used polystyrene–divinylbenzene resin beads as a solid support for the product of each stage of the synthesis. Each bead had a large number of monochlorinated methyl side chains. The C-terminal of the first amino acid in the peptide chain was attached to the bead by an SN2 displacement reaction of these chloro groups by a suitable amino acid (Fig. 5.4).

14. Additional amino acids were added to the growing peptide chain using the reaction

16. Solid support combinatorial chemistry has been carried out on a variety of supports that include:
polymer beads,
arrays of wells,
arrays of pins,
glass plates,
spatial arrays on
microchips and
cellulose sheets.

17. General methods in solid support combinatorial chemistry
The group that anchors the compound being synthesised to the bead is known either as a handle or a linker

18. As well as modifying the properties of the bead they move the point of substrate attachment further from the bead, making reaction easier. The choice of linker will depend on the nature of the reactions used in the proposed synthetic pathway.
For example, an acid-labile linker, such as HMP (hydroxymethylphenoxy), would not be suitable if the reaction pathway contained reactions that were conducted under strongly
acidic conditions.
Consideration must also be given to the ease of detaching the product from the linker at the end of the synthesis. The method employed must not damage therequired product but must also lend itself to automation.

20. Parallel synthesis
This technique is normally used to prepare combinatorial libraries that consist of separate compounds. It is not suitable for the production of libraries containing thousands to millions of compounds. In parallel synthesis the compounds are prepared in separate reaction vessels but at the same time, that is, in parallel. The array of individual reaction vessels often takes the form of either a grid of wells in a plastic plate or a grid of plastic rods called pins attached to a plastic base plate (Fig. 5.7) that fits into a corresponding set of wells. In the former case the synthesis is carried out on beads placed in the wells whilst in the latter case it takes place on so-called plastic ‘crowns’ pushed on to the tops of the pins, the building blocks being attached to these crowns by linkers similar to those found on the resin beads. Both the well and pin arrays are used in the same general manner; the position of each synthetic pathway in the array and hence the structure of the product of that pathway is usually identified by a grid code.

23. Furka’s mix and split technique
The Furka method was developed by Furka and co-workers from 1988 to 1991.
It uses resin beads
used to make both large (thousands) and small (hundreds) combinatorial libraries.
Large libraries are possible because the technique produces one type of compound on each bead, that is, all the molecules formed on one bead are the same but different from those formed on all the other beads. Each bead will yield up to product molecules, which is sufficient to carry out high-throughput screening procedures.
Advantage
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.

25. Encoding methods
A wide variety of encoding methods have been developed to record the history of beads used in the Furka mix and split technique. This section outlines a selection of these methods

26. 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 (Fig. 5.12).
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. The tag compound must be produced in a sufficient amount to enable it to be decoded to give the history and hence the possible structure of the library compound.

27. Compounds used for tagging must satisfy a number of criteria: (1) 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. (2) The tagging reaction must take place under conditions that are compatible with those used for the synthesis of the library compound. (3) It must be possible to separate the tag from the library compound. (4) Analysis of the tag should be rapid and accurate using methods that could be automated.

29. Combinatorial synthesis in solution
Solid phase combinatorial synthesis has a number of inbuilt disadvantages: 1. All the libraries have a common functional group at the position corresponding to the one used to link the initial building block to the linker or bead. 2. Syntheses are usually carried out using the linear approach. 3. Requires especially modified reactions with high yields (>98 per cent) if multistep syntheses are attempted. 4. Requires additional synthesis steps to attach the initial building block to and remove the product from the support. 5. The final product is contaminated with fragments (truncated intermediates) of the product formed by incomplete reaction at different stages and often needs additional purification steps.

30. Many of these disadvantages are eliminated or reduced when combinatorial syntheses are carried out in solution. For example, solution phase combinatorial chemistry does not have to have a common functional group at the position corresponding to the one used to link the synthesis substrate to the linker or bead. Both the linear, template and convergent synthesis routes (see sections and ) can be followed. Unmodified traditional organic reactions may be used but multistep syntheses will still require very efficient reactions. It does not require additional synthetic steps to attach the initial building block to and remove the product from a solid support. The product is not likely to be contaminated with truncated intermediates but unwanted impurities will still need to be removed at each stage in a synthesis.