Introduction
Combinatorial chemistry is a new technique found by scientists mainly for the process of discovering drugs. It is a technology for carrying out simultaneous synthesis as well as testing of hundreds and thousands of compounds of related structure in a single container. Traditional chemistry is usually marked by the use of several synthetic steps to produce one compound (Terrett, 1998). Combinatorial chemistry, on the other hand, is based on efficient, parallel synthesis, in that many chemical compounds can be simultaneously produced in a library than the number of steps used in the synthesis (Terrett, 1998).
This method reduces the time taken to synthesis compounds and hence is very valuable to drug discovery processes. Initially, the field of combinatorial chemistry focused primarily on the synthesis of peptide and oligonucleotide libraries (Borman, 2002). In the 1990s, the focus shifted to the synthesis of small drug like organic compounds and since then, it is used mainly in pharmaceutical companies and biotechnology firms in drug discovery efforts (Borman, 2002). It has evolved in recent years into broad fields of applications as diverse as material science, catalyst development and biochemistry to identify the substrates of novel enzymes (Schrader and Anslyn, 2007).
Background
The objective of combinatorial chemistry is to provide techniques for the systematic creation of large and structurally diverse libraries. From a technical perspective, libraries can be created some ways: libraries of oligomers of naturally occurring monomers such as oligopeptide libraries and oligonucleotide libraries; libraries of oligomers of non-naturally occurring monomers such as oligocarbamate libraries, oligourea libraries, oligosulfone libraries and oligosulfoxide libraries; libraries of monomers in which the central moiety is a small organic molecule attached to multiple substituent sites (Nogrady and Weaver, 2005).
These include those with synthetic ease privileged structure (dioxapiperazine), pharmacological activity privileged structures (Benzodiazepines, dihydropyridines, hydantoins) and novel template structures (dihydrobenzopyrans.). Preparation of these libraries is done through automated methods such as parallel synthesis or solid phase synthetic methods (Nogrady and Weaver, 2005)
Combinatorial Chemistry and Drug Synthesis
Parallel Synthesis
During the process of parallel synthesis a large number of products are produced at the same time. For example, alcohols react with acid chlorides to form esters. When 12 alcohols and eight acid chlorides are made to react together, reaction of each alcohol with each acid chloride produces 12 x 8 = 96 esters (Lister, 2009). The automated process is carried out using computer controlled syringes called robots. Multiple parallel syntheses can be considered as combinatorial chemistry on a smaller scale. It is a related group of methodologies used to prepare a selected small subset of molecules, whose preparation is theoretically possible. The content of libraries prepared in multiple parallel syntheses is more focused and less diverse than those with authentic combinatorial technology (Mitscher and Dutta, 2003).
Solid Phase Techniques
Another approach used in combinatorial chemistry is to carry out reactions on compounds while they are bonded to polymer beads and this method is referred to as ‘solid phase techniques’. In solid phase organic chemistry, the starting material for a reaction is bonded covalently to beads of a plastic resin, generally polystyrene-based – via a linking group (Swartz, 2000). When the reaction is started on the material X which is attached to the resin, the product is formed in such a way that the product molecule remains bonded to the resin and can be released by breaking the link, or retained on the bead for further reaction. The link must be weak enough to be broken to release the product under conditions that will not break bonds in the new molecule which have been formed (Swartz, 2000).
Solid Phase Peptide Synthesis
Combinatorial chemistry is said to have its origins in solid phase peptide synthesis. This technique was originally developed by the American chemist Bruce Merrifield (1963) for making peptides (polyamino acids a few amino acids long) but the principle can be applied to all kinds of chemical reactions. Amino acids have the general formula H2NCH(R)CO2H and can link together by the reaction of an –NH2 group on one amino acid with the –CO2H on the next (Fig 2).making polypeptides. The –CO2H group of the first amino acid, A, is bonded to a –CH2Cl group attached to the polystyrene resin. This is the link.
The second amino acid has its –NH2 group ‘protected’ – it is represented by HO–B–P, where P is the protecting group (Lister, 2009). This prevents this amino acid from reacting with other molecules of the same amino acid rather than the ones bonded to the resin. A solution of the second amino acid is then added to the resin and a dipeptide, A–B, is formed attached to the resin. The protecting group is then removed. A solution of a third amino acid (with its –NH2 group protected) is added to the resin and a tripeptide, A–B–C, is formed, attached to the resin.
The process of adding amino acids to the resin can be carried out up to 100 times to produce a polypeptide with different amino acids in any required sequence, attached to the resin. Finally the polypeptide can be released from the resin by breaking the link. The final product can be obtained in a pure form by washing off excess reagents from the polymer (Lister, 2009). The solid phase techniques advanced over the years and in the mid 1980s, it was possible, using this approach, to make many peptides simultaneously in the same reaction vessel.
Houghten’s (1985) use of “tea bags” as porous containers for solid phase resin beads, allowed the same peptide coupling step to be applied to many beads simultaneously irrespective of the sequence already attached to the bead. The application of the mix and split procedure to this process in 1991 allowed huge numbers of peptides to be made in a very few number of chemical steps and this marked the birth of combinatorial synthesis.
Libraries of peptides and oligonucleotides were relatively easy to handle both in the mixture and in the individual one-bead-one compound format. In the case of solid phase synthesis of organic molecules, pioneering work was done by Bunin ad Ellman, who synthesized small benzodiazepine library. This preliminary report was the first widely recognized solid supported synthesis of nonoligomeric compounds and set the stage for rapid development of combinatorial synthesis methods for small organic molecules (Swartz, 2000).
Split and Mix Procedure
The split and mix technique was developed by Furka and colleagues (1988). In this method a single vessel containing a mixture of closely related peptides is used. The technique requires the repetition of three operations: dividing the polymer on which the peptide is growing into equal portions, coupling each portion with a different amino acid and homogenous mixing of these portions (Foye et al, 2007). If one starts with only three amino acids bound to a polymer, the number of peptides obtained would triple after each coupling step, according to the formula 3n. If all 20 of the natural amino acids were bound to a polymer, however, one could generate 3.2 million different peptide after only five couplings according to the formula 20n (40) (Foye et al, 2007).
These techniques have been developed to help in screening large number of drugs for potential drug activity. The ability of a compound to affect enzymes and bind to receptors on cells can be studied as a measure of its drug activity. Thus, combinatorial chemistry helps to test chemicals for potential drug activity in a test-tube (in vitro) rather than on living things (in vivo) (Lister, 2009). Only those drugs that are found promising are developed further.
Solution Phase approaches
The initial focus of combinatorial chemistry has been on solid phase approaches and solution chemistry was not thought to be ideal for its application, as few reactions produced high yields reliably when equimolar amounts of reactants were used. Thus, reactions in solution were accompanied by tedious methods of isolation and purification procedures. Moreover, reactivity of building blocks was more obvious in solution compared to solid phase reactions (Bannwarth et al, 2000).
However, in recent years, a new area is that of solid phase supported solution chemistry, which allows for the application of excesses of reagents to drive reactions to completion. The excess can then be reacted with solid phase bound functionalities and removed by an ensuing filtration step. It is also possible for intermediate and final products to be trapped by suitably modified support materials. Solid phase-bound reagents are becoming increasingly important as another field of polymer supported solution chemistry.
An example is the use of solid phase bound triphenylphosphine in Wittig-type reactions to avoid the cumbersome separation of the product from the triphenylphosphine oxide that is formed as a byproduct (Bannwarth et al, 2000). Perfluorinated “pony tails” are a recent approach for the efficient solution synthesis of combinatorial libraries. The compound carrying the fluorous tag can be extracted into a perfluorinated solvent and thus easily separated from other components (Bannwarth et al, 2000).
Parallel solid phase synthesis of peptides has been made commercially viable through automated equipment. However, in the case of broadly based organic synthesis being attempted on solid phase, products of high purity are not easily obtained from resin cleavage unless the chemist has spent considerable time optimizing reaction conditions for each step before the start of the synthesis process. There is a lack of suitable methods for analysis of resin-bound reaction products (Swartz, 2000).
Once automated solid phase combinatorial techniques are used, the chemist can use conventional methods of assaying purity (chromatography) or structure (eg. spectroscopy) only when the product is cleaved from resin for analysis. Solid phase nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) methods now provide the most useful information about structures bound to resin (Swartz, 2000).
Biochemical Application Case Study
Combinatorial chemistry finds wide application in the field of biochemistry. In this laboratory experiment different substrates for the enzyme, mushroom tyrosinase is synthesized using the combinatorial chemistry technique and the different substrates are tested whether they could serve as substrates for the enzyme: The enzyme catalyzes two reactions that are essential for the biosynthesis of the phenolic pigment, melanin and results in two different products – two kinds of melanin pigments – the red to yellow pheomelanin and the brown to black eumelanin. In this reaction, Melanin biosynthesis is initiated by the enzyme, tyrosinase.
Inhibiting the activity of this enzyme is important to produce drugs that can serve as insecticides, cosmetics and preservatives whereas acceleration of these reactions can provide skin tan materials. Tyrosine shows very broad substrate specificity and acts on some compounds. In the laboratory, using combinatorial chemistry technique, some cysteinyl catechol adducts on this enzyme were prepared and tested. A microtitre plate is taken with marked wells. Eight different catecholic compounds are added to the wells. All the catechols are oxidized except those in the last row using sodium periodate solution and thiol.
A (cysteine) and glutathione catechol adducts are formed. The last row has unmodified catechols. Water is added now to all the wells and 8 different quinones have been made from 8 different catecholic compounds and they are now reacted with four different thiols: cysteine, N-acetyl cysteine, dithiothreitol and glutathione. This gave 32 different compounds and along with unmodified originals, 40 different compounds have been synthesized.
Another microtitre plate is used to assay for enzyme activity using sodium phosphate buffer pH 6.5 and mushroom tyrosinase. Color change due to production of colored quinones indicates tyrosinase activity on a substrate. To determine the time course of the substrate specificity studies of tyrosinase, electronic 20 machines are used and by using graphic method to plot the absorbance value against time, the best and worst substrate for the enzyme can be determined.
To transform the one-bead-one-compound libraries to the arena of small organic molecules requires methods that allow simple and unequivocal determination of the structure of the individual bead containing pico-molar amounts of analyzable material. This problem was addressed by inclusion of tagging into the synthetic scheme.
The structure of the relevant molecule is determined by reading the tag. Clark Still found an elegant method of tagging in which the tagging is achieved by a small set of halogenated ethers attached to the bead as a defined mixture in each step of the synthesis, forming digital code (each molecule of the tagging substance is either present – digit 1- or absent – digit 0), evaluated after detachment from the bead by gas chromatography. The combinatorial techniques have been extended to the field of material science and the libraries are produced in a spatially addressable form and used to find new superconductive, photoluminescent or magnetoresistive materials.
Practical methods of synthesis using combinatorial chemistry
Irori introduced a technique for synthesizing numerous organic compounds in parallel. This was based on the idea that it is possible to label individual polymeric beads with the readable radiofrequency tag, which will be built during the split and mix synthesis of any type of molecule. The more recent adaptation of this technique is the labeling of small disks containing 2-10 mg. of synthetic substrate called “NanoKans” by a two dimensional bar code on a small ceramic chip (Mei and Czamik, 2005).
On the other hand, thousands of compounds may be relatively cheaply in polypropylene microtiter plates using either surface suction or tilted centrifugation. The simplest and most economical method has been introduced by Krchnak and involves the use of disposable polypropylene syringes equipped with polypropylene frits. The syringe is charged with the solid support of choice and all steps of the synthesis are performed by aspirating appropriate reagents using needles and septum closed bottles. The operation of syringes can be simplified by the use of domino blocks. (Mei and Czamik, 2005).
Biocatalysis
The technique of combinatorial biocatalysis is another approach to obtaining diverse libraries. In this approach, natural catalysis such as enzymes and whole cells, and recombinant and engineered enzymes are used to derive directly many different synthetic compounds and natural products. Many types of reactions are catalyzed by enzymes and microorganisms and these include reactions that can introduce functional groups or modify the existing functionalities, or addition onto functional groups. These biocatalysis reactions can take place in aqueous and non-aqueous solutions (Griffin and O’Grady, 2006).
Challenges in Combinatorial Chemistry
Though combinatorial chemistry increased the quantity of chemical entities, there was a problem regarding the quality of the products… The number of compounds that made it to human clinical testing has not increased much since the advent of industrialized R&D. According to a study by David Newman of the National Cancer Institute, combinatorial chemistry methods failed to produce a single FDA-approved drug by the end of 2002 (Pisano, 2006). This is because combinatorial chemistry does not make compounds with specific structural characteristics that will make suitable as a drug. For a compound to qualify as a drug, it should be biologically active, i.e. can bind to a target of interest and pharmacologically suitable, i.e. have a form that can be absorbed and metabolized appropriately.
Hence to find a good drug, it is important to include biological and pharmacological data. This was found to be lacking on combinatorial chemistry. The products of combinatorial chemistry were often biologically inert and pharmacologically intractable (Pisano, 2006). Over the yes, improvements have been made in the library design process and attention has shifted to smaller high quality libraries of discrete compounds, using various data mining technologies, new parallel and combinatorial approaches and filter for lead like and drug like properties as well as avoiding non selective or promiscuous inhibitors (Griffin and O’Grady, 2007).
A large number of natural products like libraries have been prepared and investigated in various screens. Moreover, recent advances in analytical chemistry have enhanced the purification of products using mass triggered preparative liquid chromatography. Although the quality of leads discovered directly from combinatorial libraries is steadily increasing, it has been found that parallel synthesis is even more powerful at the lead optimization phase, when a series of related compounds needs to be made and tested rapidly (Griffin and O’Grady, 2006). Another challenge is that presently, screening for biological activity requires large amounts of substance compared to what is present on a single bead.
There is a quest for getting more material on a single bead as well as increasing the sensitivity of the screening tests. Moreover, the high rate of discovery of new chemical entities using combinatorial chemistry has given rise to new problems in areas such as handling and storage of analytical and biological data (Lister, 2009). These problems need to be overcome in the future to widen the scope of combinatorial chemistry.
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