Combinatorial Chemistry – Solid Phase Synthesis | High Throughput Screening

Combinatorial Chemistry Introduction

Combinatorial chemistry has its earliest origin in solid phase peptide in 1960 by Bruce Merrifield. In 1980’s researchers H.Hario Grysen developed this technique further, creating libraries of different peptide on separate supports. From 1990 onwards, there was an upsurge in combinatorial synthesis and small molecules were also synthesized as multi component mixture.

Since then combinational chemistry has expanded from peptide to organic, organometallic, inorganic and polymer chemistry. Drug discovery is a lengthy and expensive process, within which the synthesis of exploratory compounds be the slowest step. But in combinatorial synthesis a large number of chemical compounds can be synthesized quickly.

Combinatorial chemistry can be viewed as a tool which allows large number of compounds to be synthesized simultaneously in a time taken to prepare only handful of compounds by traditional synthetic methods.

The number of compounds produced far exceeds the number of chemical steps required to make them. A chosen set of building blocks are reacted together to make every available product and collection of these product is referred as “library” or an “array”. This collection of compounds can be synthesized as a mixture or individuals using a variety of techniques.

Combinatorial synthesis is a simultaneous synthesis of a large number of possible compounds that could be formed from a number of building blocks. The product of such process is called “Combinatorial Library”.

For example, in orthodox synthesis compound A would react with compound B to give product AB, which would be isolated after working up and purification. In contrast to this approach, combinatorial chemistry offers the potential to make every combination of compound A1 to An with compound B1 to Bn.

The compounds are present as a mixture and not as individual characterized structures. The structure of compound in a mixture is not known with certainty and the compounds are not separated or purified instead each mixture is tested for biological activity as a whole. If there is no activity then there is no need to study the mixture any more. If the activity is observed then the active component in the mixture is identified and separated.

This process is:

• Faster, more efficient, cheaper and give rise to million of compounds in the same time, as it takes to make one compound.
• If we want to find a lead compound quickly and efficiently combinatorial chemistry provides a mean for producing this quantity of compounds.
  Combinational Chemistry can be applied to:
  • Solution phase synthesis
  • Solid phase synthesis.

In solution phase synthesis, the library members are typically synthesized as individual compounds, so called parallel synthesis. On solid support, the split and mix techniques as well as parallel synthesis can be applied.

Combinatorial Chemistry – Solid Phase Synthesis

In this method, the reaction is carried out on a solid support such as resin beads. The bead is treated with different starting materials, which bound together. Then it is mixed with another reagent to get the product. Since the products are bound to solid support, excess reagent or by-product can be easily removed by washing with appropriate solvent.

Large excess of reagent solvent can be used to drive the reaction sequence are bound to the bead and need not be purified individual beads can be separated at the end of the experiment to get individual products. The polymeric support can be regenerated and reused if appropriate cleavage conduction and suitable anchor / linker groups are chosen.

Solid phase synthesis was pioneered by Merrifield for the synthesis of peptides. As a result most early work carried out on combinational synthesis was performed on peptides. But large amount of research has been carried out in solid phase synthetic method to synthesize small non-peptide molecules.

The essential requirement for solid phase synthesis is:

• Cross linked insoluble polymeric support which is inert to synthetic condition eg. Resin.
• Anchor or linker covalently linked to the resin.
• Bond linking the substrate to the linker, which will be stable to the reaction condition.
• Chemical protecting groups for protecting the functional group not involved in the synthesis.

The Solid Support

The choice of solid support depends on the type of reaction. In addition resin, is the stable and commonly used solid support. Some examples are

• Polystyrene.
  The polystyrene support is normally cross linked by the addition of 1% Divinyl benzene (DVB) to the polymerization mixture. This degree of cross linking offers the best compromise between mechanical stability and swelling performance. Highly cross linked polystyrene have a higher mechanical and thermal stability, but their swelling performance and loading capacity are clearly reduced.
• Tentagel is polystyrene – polyoxa ethylene graft co-polymer.
• Pepsin is a polyamide support especially developed for peptide synthesis.
• PEGA [Poly (acrylic amide – ethylene glycol) copolymers].

Due to its amphiphilic properties, these supports are mainly used for solid phase organic synthesis (SPOP) of polar compounds.

Linkers

The linker is the molecule that sits between compound and the solid support. The linkers move the point of substrate attachment away from the surface of the bead. This has the effect of reducing steric hindrance, thereby making the reaction easier and allows the final product to cleave off in high yield.

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 functional product once it is released. Resin having different linkers has different names.

For example

• The Wang resin: It has linkers which is suitable for the attachment and release of carboxylic acids.
• The Rink resin: Suitable for the attachment of carboxylic acid and the release of Carboxamide.
• The Dihydropyran derivative resin: Suitable for the attachment of alcohol.

Protecting groups

Protecting groups are important for blocking and regenerating certain functional group in a reaction sequence. Some examples of protecting groups are FMOC (Fluoro methoxy carbonyl benzyl ester) and TBOC (Tertiary butyloxy carbonyl).

Combinational synthesis on solid support is usually carried out by using either parallel synthesis or Furka split and mix procedures.

Parallel Synthesis

In this method, the compounds are prepared in separate vessel but at the same time that is in parallel. The array of reaction vessel are taken either in a grid well in a plastic plate (if the synthesis in carried out in a bead) or pins (grid of plastic rods) called crowns. The building blocks are attached to these beads or crowns. The structure of product formed is usually identified by the grid code.

Finally, the products are liberated from the resin by appropriate linked cleavage reaction and the product is isolated. The structure of these products are usually determined by following the history of the synthesis using the grid references of the wells and confirmed by instrumental methods.

Mix and Split Technique

Mix and split technique may be used to make both large and small combinatorial libraries using relatively few reaction steps.

A bath of resin is divided into equal portion in different reaction vessel, for example three. Each portion of resin is treated with different derivatives of the first building block (A, B and C) as the first step. After washing to remove the excess reagent and by – products the beads are pooled together in one pot and mixed thoroughly before being split into an equal portion again for coupling to the next building block.

This gives nine different compounds in statistically equal amount. Thus the beads are split into portion, mixed together and re-split depending upon the number of different building blocks be used. This process is continued until the required library is synthesized.

If same building block is used at each step the maximum possible numbers of compounds that can be synthesized for the given number of different building blocks (b) are given by

Number of compounds = bx

Unlike in parallel synthesis, the history of the bead cannot be traced from the grid reference; it is traced by using suitable encoding method or deconvolution.

Deconvolution

Assuming that a compound mixture proves to be biologically active, the tricky job of identifying the active component now needs to be carried out. Isolation and identification of the most active compound in mixture is known as deconvolution. There are several methods of doing this. eg. Micromanipulation, Sequential release.

Encoding Methods

Encoding methods use a code to indicate what has happened at each step in the synthesis and identify the structure of the most active library member. Two popular techniques are (i) Chemical tagging method and (ii) Radio frequency tagging.

Chemical tagging method

Chemical tagging method uses specific compounds (tags) as a code for the individual step in the synthesis. These tags are sequentially attached in the form of polymer-like molecule to the same linker or bead as the library compound at each step in the synthesis.

At the end of the synthesis both the library compound and tag compound are liberated from the bead.

Branched linkers, with one side for attaching the library compound and another side for attaching the tag are often used for encoding.

Radio Frequency (RF) Tagging

It employs a microchip tag like a bar code to label each library member. Tags are robust, encapsulated in glass casing, stable to chemical and synthetic conditions. Each individual tag code is associated with the identity of its library member – a set of building blocks used in a data base computer and accompanied a particular portion of resin throughout its synthetic journey in a porous ‘tea bag’ vessel.

Solution Phase Synthesis

The main problem with preparing libraries using solution chemistry is the difficulty of removing unwanted impurities at each step in the synthesis. Consequently, many of the strategies used for the preparation of libraries using solution chemistry are directed to the purification of the products of each steps of the synthesis. The other practical problem has usually restricted the use of solution combinatorial chemistry to synthetic pathways consisting of two or three steps.

Combinatorial synthesis in solution can be used to produce libraries that consist of single compound or mixtures using traditional organic chemistry. Single compound libraries are prepared using the parallel synthesis technique. Libraries of mixtures are formed by separately reacting each of the members of a set of similar compounds with the same mixture of all the member of the second set of compounds.

Consider, for example, a combinatorial library of amides formed by reacting a set of five acid chlorides (A’ – A3) with ten amines (B1 – B1o). Each of the five acid chlorides is reacted separately with an equimolar mixture of all ten amine and each of the amine is reacted with an equimolar mixture of all the acid chlorides. This produces a library consisting of a set of five mixtures based on individual acid halides and ten mixtures based on individual amines.

This means that, each compound in the library is prepared twice, once from the acid chloride set and another from the amine set. Consequently, determining the most biologically active of the mixtures from the acid halide set will define the acyl part of the most active amide and similarly identifying the most biologically active of the amine based set of mixtures will identify the amine residue of that amide. Libraries used in this manner are often referred as indexed libraries.

 High throughput screening (HTS) or Screening

The success of library depends not only on it containing the right compounds but also on the efficacy of screening procedure. Since combinatorial synthesis produces a large quantity of structure in a very short time period, biological testing should be carried out quickly and automatically. The technique used by this system is known as High throughput screening (HTS).

Traditionally, compounds are automatically tested and analyzed on a plate containing 96 small wells with the capacity of 0.1 ml. But it HTS 1536 well with capacity of 1-10 μl only used. Moreover methods such as fluorescence and chemiluminescence are being developed, which will allow the simultaneous identification of active compound.

Combinatorial Chemistry  Applications

From the prospective of the pharmaceutical industry, the ultimate test for combinatorial chemistry is its application to the discovery of commercially successful drugs. The overall aim is to identify a chemical lead with in vitro activity in a particular screening assay that is amenable to further chemical and biological optimization.

The synthesis analogues of existing lead structure, elucidate the structure activity relationships and to improve biological activity in vitro (eg, affinity, efficacy, selectivity) and in vivo (eg. behavioral pharmacology, metabolism, kinetics).

In short, this technique is applied to both lead identification and lead optimization. The integration of these two has established combinatorial chemistry as a useful tool for drug discovery and development.