A Short Review on Concept and Types of Combinatorial Chemistry

 

Arvid Kumar Jha, Achal Mishra*, Bhavna Yadav, Shekhar Verma, Yashwant Swarnakar, Permanand Verma, Devendra Sahu

Faculty of Pharmaceutical Sciences, Shri Shankaracharya Group of Institution, Bhilai

*Corresponding Author E-mail:

 

ABSTRACT:

Combinatorial chemistry is a technique involves synthesis of compounds in mass instead of a single compound, which are screened as a whole for particular biological activity. This brief review article includes combinatorial strategies, and types of combinatorial technique.

 

KEY WORDS: Solid phase, Solid support, Linkers, Solution Phase, One bead one compound.

 

INTRODUCTION:

Combinatorial Chemistry and Combinatorial Technology (CC/CT) are a new interdisciplinary field joining computer assisted combinatorial chemistry with automated parallel synthesis of chemical libraries followed by automated screening. This nascent technology already produced more new compounds in just a few years than the pharmaceutical industry did in its entire history. Combinatorial chemistry has turned traditional chemistry upside down. It required chemists not to think in terms of synthesizing single, well-characterized compounds but in terms of simultaneously synthesizing large populations of compounds.1

 

Combinatorial approaches were originally based on the premise that the probability of finding a molecule in a random screening process is proportional to the number of molecules subjected to the screening process. In its earliest expression, the primary objective of combinatorial chemistry focused on the simultaneous generation of large numbers of molecules and on the simultaneous screening of their activity. Following this approach, the success rate to identify new leads is greatly enhanced, while the time required is considerably reduced.2

 

The development of new processes for the generation of collection of structurally related compounds (libraries) with the introduction of combinatorial approaches has revitalized random screening as a paradigm for drug discovery and has raised enormous excitement about the possibility of finding new and valuable drugs in short times and at reasonable costs.2

 

The Need for Combinatorial Technologies

Drug discovery in the past has been based traditionally about the random screening of collections of chemically synthesized compounds or extracts derived from natural sources, such as microorganisms, bacteria, fungi, plants, of terrestrial or marine origin or by modifications of chemicals with known physiological activities.2

 

Sources of molecular diversity

·         Plant extracts

·         Microbial extracts

·         Collection of chemical compounds (synthetic)

·         Oligonucleotide libraries (biological or synthetic)

·         Oligosaccharide libraries

·         Chemical compounds libraries (synthetic)

·         Peptide libraries (biological or synthetic)

 

Collection of structurally related compounds (peptides, oligonucleotides, oligosaccharides, organic molecules) obtainable by chemical or biological means simultaneously as a mixture and screened for activity as a mixture of compounds, without any isolation protocol step. Identification of active compounds derives from the synthesis/production protocol used to generate the library. Great acceleration of leads identification since millions of different compounds can be screened simultaneously.3

 

Principle of combinatorial chemistry

Illustrated in figure 2. Classically, chemists perform reactions using one molecular species of each reagent (A and B) and expect to obtain if not a single, at least a major, product (A-B). In the case of combinatorial chemistry, instead of a single molecular species, groups of building blocks are reacted together. Using simultaneously a group of IZ building blocks (A, to A,) with another group of n’ building blocks (B, to IS,,.) leads to a mixture of all combinations (A,B, to A,B,.).


 

Fig. 1. Principal characteristics of conventional vs. combinatorial strategy of drug discovery

 

Classical Synthesis

Basic idea of combinatorial chemistry:

·         Preparation of a large number of different compounds at the same time

·         High throughput-screening provides the most promising substances

 

Fig-2 Principle of combinatorial chemistry

 

Fig.3- Principal of solid phase synthesis

 

Figure-4

 

Type of Combinatorial Chemistry

Combinatorial chemistry is of two types: first is solid phase combinatorial chemistry and second is solution phase combinatorial chemistry.

 

1.  Solid phase combinatorial chemistry

In solid phase combinatorial chemistry, reagents or products are attached to solid supports such as polystyrene beads is the most traditional form of phase trafficking. In solid-phase organic synthesis, it’s easy to purify products by filtration, it’s possible to do mix-and-split synthesis (a technique used to make very large libraries), excess reagents can be used to drive reactions to completion, and syntheses can be automated easily.1 Solid phase chemistry has some advantages over the solution-phase. First, in solid-phase synthesis, large excesses of reagents can be used to drive reactions to completion; these excess reagents can then be removed at the end of the reactions by filtration and washing. Second, because of easy separation of reagents and products, solid phase chemistry can be automated more easily than solution chemistry. Separation of compounds bound to the solid support from those in solution is accomplished by simple filtration. 4, 5

 

Since Merrifield pioneered solid phase synthesis back in 1963, work, which earns him a Nobel Prize, the subject, has changed radically. Merrifield’s Solid Phase synthesis concept, first developed for biopolymer, has spread in every field where organic synthesis is involved. Many laboratories and companies focused on the development of technologies and chemistry suitable to SPS. This resulted in the spectacular outburst of combinatorial chemistry, which profoundly changed the approach for new drugs, new catalyst or new natural discovery.

The use of solid support for organic synthesis relies on three interconnected requirements:

 

1) A cross linked, insoluble polymeric material that is inert to the condition of synthesis;

2) Some means of linking the substrate to this solid phase that permits selective cleavage of some or all of the product from the solid support during synthesis for analysis of the extent of reaction(s),and ultimately to give the final product of interest;

3) A chemical protection strategy to allow selective protection and deprotection of reactive groups.

 

Merrifield developed a series of chemical reactions that can be used to synthesise proteins. The direction of synthesis is opposite to that used in the cell. The intended carboxy terminal amino acid is anchored to a solid support. Then, the next amino acid is coupled to the first one. In order to prevent further chain growth at this point, the amino acid, which is added, has its amino group blocked. After the coupling step, the block is removed from the primary amino group and the coupling reaction is repeated with the next amino acid. The process continues until the peptide or protein is completed. Then, the molecule is cleaved from the solid support and any groups protecting amino acid side chains are removed. Finally, the peptide or protein is purified to remove partial products and products containing errors.6

 

1.1 Solid support used in Solid phase synthesis

Most solid state combinatorial chemistry is conducted by using polymer beads ranging from 10 to 750 μm in diameter. The solid support must have the following characteristics for an efficient solid-phase synthesis: 5- 7

1) Physical stability and of the right dimensions to allow for liquid handling and filtration;

2) Chemical inertness to all reagents involved in the synthesis;

3) An ability to swell while under reaction conditions to allow permeation of solvents and reagents to the reactive sites within the resin;

4) Derivatization with functional groups to allow for the covalent attachment of an appropriate linker or first monomeric unit.8

The compounds to be synthesized are not attached directly to the polymer molecules. They are usually attached by using a linker moiety that enables attachment in a way that can be easily reversed without destroying the molecule that is being synthesized and allow some room for rotational freedom of the molecules attach to the polymer.

 

1.1.1 Types of solid that are used:

Polystyrene resins in this Polystyrene is cross linked with divinyl benzene (about 1% crosslinking).polystyrene resin are suitable for nonpolar solvents.

 

Tenta Gel resins Polystyrene in which some of the phenyl groups have polyethylene glycol (PEG) groups attached in the para position. The free OH groups of the PEG allow the attachment of compounds to be synthesized. PEG containing resins are suitable for use in polar solvents.

 

Polyacrylamide resins like super blue these resin swell better in polar solvent, since the contain amide bonds, more closely resemble biological materials.

 

Glass and ceramic beads these type of solid supports are used when high temperature and high pressure reaction are carried out. 2, 9

 

1.2 Linkers used in solid phase synthesis

To support the attachment of a synthetic target, the polymer is usually modified by equipping it with a linker. Linker must be stable under the reaction conditions, but they must be susceptible to a cleavage. Some specialized linker have been developed to meet particular reaction or product conditions this type of linker is known as traceless linkers, it can be cleaved from the resin with no residual functionality left. This type of linkers allows the attachment of aryl and alkyl products that do not have OH or NH functionality example of these linker include silyl group (-Si(CH3)2) that is sensitive to acid and can be cleaved to give unsubstituted phenyl or alkyl product. 2

Inert to synthetic condition and chemically transformed to allow final liberation of the product from the resin. Now a ultraviolet light sensitive protecting groups are used, like affymax group is used in the synthesis of carboxylic acid and carboxamide products. Some groups have used linkers that can only be cleaved by enzymes. 9-10A novel linker possessing selenocyanate and masked carboxylic acid was developed for the solid-phase synthesis of dehydropeptides. This linker was used to demonstrate the synthesis of the model compound of RGD-conjugated dehydropeptide. 11 Oxabicyclo norbornenes constitute a convenient and readily cleaved linker for solid-phase organic synthesis. A simple and inexpensive furfuryl-substituted resin has been shown to capture and release maleimide dienophiles under conditions compatible with intermediate synthetic steps.12A new linker based on a chroman system is developed for the side-chain anchoring of Arg and other guanidine-containing molecules. The system is compatible with the Fmoc/tBu solid-phase strategy, because the release of the final product is achieved by treatment with TFA in the presence of scavengers. 13-14

 

Merrifield resin.

The Merrifield resin can be used to attach carboxylic acids to the resin. The product can be cleaved from the resin in carboxylic acid form using HF.

 

Trityl chloride resin.

 

The trityl chloride resin is much more reactive than the Merrifield resin. It can be used for attachment of a vide variety of compounds like carboxylic acids, alcohols, phenols, amines, thiols. The products can be cleaved under mild conditions using a solution of trifluoroacetic acid (TFA) in varying concentrations (2-50%). 9-14

 

1.2.1 Protecting groups

If a chemist wants to carry out a reaction on only one functional group of a multifunctional group compound, the reactivity of the rest of the functional groups needs to be suppressed. This can be achieved by application of protecting groups. A protecting group is reversibly attached to the functional group to convert it to a less reactive form. When the protection is no longer needed, the protecting group is cleaved and the original functionality is restored. A large number of protecting groups were developed for use in peptide synthesis since the amino acids are multi-functional compounds. It is an important requirement for a protecting group to be stable under the expected reaction conditions and to be cleavable - if possible – at mild reaction conditions. The stability/cleavage conditions of a protecting group are considered relative to those of the others. Two protecting groups are said to be orthogonal if either of them can be removed without affecting the stability of the other one. Some of the protecting groups most widely used in peptide synthesis are described below. 13-14

Protection of amino groups

The benzylcarbonyl (Z) group. Bergmann and Zervas suggested the benzyloxycarbonyl group for amino-protection in peptide synthesis in 1932 and this important protection type is still in use. The Z group can be introduced by the reaction of the amino group containing compound with benzylchloroformate under Schotten-Bauman conditions. The Z protection is stable under mildly basic conditions and nucleophilic reagents at ambient temperature. Cleavage can be brought about by HBr/AcOH, HBr/TFA or catalytic hydrogenolysis.

 

2. Solution Phase Combinatorial Synthesis

Before introduction by Merreifield 20 of solid phase synthesis, the organic compounds were generally prepared in solution. The use of the solution phase synthesis in combinatorial chemistry has some advantages and also serious disadvantages. The main advantage is that the overwhelming majority of synthetic procedures recorded in the literature are realized in solution phase. The disadvantage, on the other hand, is that in a multi-step reaction the products need to be isolated and purified in each step that is often tedious and time consuming. Nevertheless, solution phase synthetic methods are applied in combinatorial chemistry, too. There approaches, however, that make possible to reduce the disadvantages and so to make the solution phase procedures competitive and applicable besides the solid phase methods. Dendrimer supported synthesis. Dendrimers are branching oligomers. They are built up in stepwise manner from monomers that result in branching at every coupling position. These oligomers are soluble, relatively large molecules their size considerable exceeds those of the building blocks and reagents used in combinatorial syntheses. To the ends of their branches linkers can be attached so they can serve as soluble supports for combinatorial synthesis

 

Separations using fluorous tags and fluorous solvents.

The fluorous solvents are immiscible with most organic solvents and water. This fact is exploited in using fluorous-organic liquid-liquid extraction for separation of products of solution phase combinatorial syntheses from reagents.This separation works if fluorous tags are attached to the reagents or to the products. The attachment of the fluorous tags may occur before the combinatorial reaction step or after it. A Stille coupling is carried out using a fluorous reactant and a fluorous solvent, the commercially available FC-72, consisting mainly C6F14 isomers. At the end of coupling, the product was extracted into dichloro methane and the by product (Cl-Sn(Ch2CH2C6F13)3) was found in CF-72.8

 

Other Method of Combinatorial Chemistry

One bead one compound technique:

With this strategy, a specific quantity of beads is allocated for each possible structure in the library; those beads contain only molecules of the given library member. The beads may be tagged in various ways to help identify the synthetic compound. The advantage of the one bead one compound strategy is the simplicity of analysis and screening. The disadvantage is keeping the beads separate and having to deal with a large number of synthesis in parallel. It is otherwise called as Split and Mix technique. 5

 

Iterative deconvulution:

This is the strategy first described 20 yrs ago when combinatorial chemistry was started. Each group has beads bearing a variety of compounds, but a given structure only appears in one of the groups. Suppose the active structure is ABC in the 3rd group. Since it is in the 3rd group, we know a C in position 3 is needed for activity. We synthesize a smaller library of the structures, in 3 groups.

 

(AAC+BAC+CAC,ABC+BBC+CBC, and ACC+BCC+ CCC.)

Now when we screen those mixtures, we find activity in the middle group of beads. This tells us that a B in position 2 is required for activity. The final step is to synthesize ABC, BBC, and CBC, keeping them separate, and screen each to find ABC as the active structure. 6

 

Subtractive deconvulution:

This is the strategy similar to iterative deconvulution but uses negative logic, namely, leave out a functional group, and if activity is absent, the functional group that is missing must be needed for activity. This is particularly useful for QSAR-type studies in which, say, a cl group is placed at several positions on a phenyl ring. The entire library is screened as a mixture to get the baseline activity level. If activity is detected, a set of sub libraries is prepared, with each missing one building block (subtraction of a functional groups from the active compounds) will be less active than the parent library. The Least active sub libraries identify the most important functional groups. A reduced library containing only these functional groups is then prepared, and the most active compounds are identified by either one compound synthesis or iterative deconvulution. 7

 

Bogus-coin detection:

This begins with generating and screening the entire library as a single mixture. If activity is detected, the building blocks are divided into 3 groups (alpha, beta, gamma)and additional sub libraries are prepared. In these subsets, the number of building blocks from the alpha group is decreased, the number from the beta group is increased, and the number from the gamma group is unchanged. The resulting effect on activity (up. down, unchanged) suggests which group of building blocks was contributing most to activity. This approach is applied iteratively to zoom in one of the groups that are most active. 8

 

Orthogonal pooling:

The orthogonal pooling means perpendicular or uncorrelated. In this type of pooling, we distribute the functional groups to be considered into sets of libraries A,B,C etc., which can contain mixtures of the same compounds. However, the functional groups are distributed such that any subset in A,B shares only one functional group, For example, if we have a very small library of structures aa, ab and ac .We might put aa and ab into group A, aa and ac into group B, ab and ac into group C. If ab is the active structure, screening A,B,C would show activity in A and C , but not in B, telling us that ab is the active one. 9

 

Positional scanning:

This is a noninterative deconvulution screening strategy in which a subset library is created with a single building block fixed at one position and all building blocks in the other positions. In principle, by selecting the functional group from the most active subset at each position, the most active compound overall is discovered. This ignores interaction between building blocks, which may complicate the results. 10

 

CONCLUSIONS:

Combinatorial approaches have been introduced from the beginning in the drug discovery field, given their tremendous impact of the identification of new leads. Many active compounds have been selected to-date, following combinatorial methodologies, and a considerable number of those have progressed into clinical trials. However, combinatorial chemistry and related technologies for producing and screening large numbers of molecules also find useful applications.

 

REFERENCES:

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4.        http.www.google.com.

5.        Wilson and Griswold Textbook of Pharmaceutical and medicinal chemistry VIth edition.2007.

6.        Schafer RW, Mersereau RM, and Richards MA. Constrained iterative deconvulution algorithms, Proceedings of the IEEE. 69(4) ; 1981: 432–450.

7.        www.mprg.org/people/buehrer/ultra/pdfs/UWB_Deconvolution.pdf.pubs.acs.org/cgibin/jtext?jmcmar/40/i26/abs/jm970503.

8.        Harris CJ, Hong X, and Gan Q. Estimation and Fusion from Data: A Neurofuzzy Approach. Adaptive Modeling , New York: Springer- Verlag., 2002.

9.        Pinilla C, et. al . Exploring immunological specificity using synthetic peptide combinatorial libraries. Current Opinion in Immunology, 11(5); 1999:193- 202.

10.     Sasa M, et.al . Journal of Pharmaceutical and Biomedical Analysis.15(3); 1996: 393-401.

11.     Maier WF. Schleyer P, and Von R.  Evaluation and Prediction of the Stability of Bridgehead Olefins. Journal of the American Chemical Society. 103(8); 1981;  1891.

12.     Normanskii VE, Raikhlin NT, Primary biological screening of photosensitizers for photodynamic activity on a nontumor model in vivo. Bulletin of Experimental Biology and Medicine. 114(6); 1992;  1873-1877.

13.     Teodoro ML, Kavraki LE, Conformational flexibility models for the receptor in structure based drug design. Current Pharmaceutical Design 23(9); 2003:1635.

14.     Kumar P. Jitendra, Shivannarayana P, and Saikishore V., An Overview on combinatorial Chemistry. Research journal of pharmacy and technology. 5 (5); 2012: 570-579.

 

 

 

 

 

 

Received on 06.12.2013         Modified on 25.12.2013

Accepted on 10.01.2014         © AJRC All right reserved

Asian J. Research Chem. 7(2): February 2014; Page 243-248