Combinatorial chemistry comprises chemical synthetic methods that make it possible to prepare a large number (tens to thousands or even millions) of compounds in a single process. These compound libraries can be made as mixtures, sets of individual compounds or chemical structures generated by computer software. Combinatorial chemistry can be used for the synthesis of small molecules and for peptides.
Strategies that allow identification of useful components of the libraries are also part of combinatorial chemistry. The methods used in combinatorial chemistry are applied of outside chemistry as well.
Introduction The basic principle of combinatorial chemistry is to prepare libraries of a very large number of compounds and identify those which are useful as potential drugs or agrochemicals. This relies on high-throughput screening which is capable of assessing the output at sufficient scale.
Although combinatorial chemistry has only really been taken up by industry since the 1990s, its roots can be seen as far back as the 1960s, when a researcher at Rockefeller University, Bruce Merrifield, started investigating the solid-phase synthesis of peptides. Synthesis of peptides in a combinatorial fashion quickly leads to large numbers of molecules. Using the twenty natural amino acids, for example, in a tripeptide creates 8,000 (203) possibilities. Solid-phase methods for small molecules were later introduced and Furka devised a "split and mix" approach
In its modern form, combinatorial chemistry has probably had its biggest impact in the pharmaceutical industry. Researchers attempting to optimize the activity profile of a compound create a 'library' of many different but related compounds. Advances in robotics have led to an industrial approach to combinatorial synthesis, enabling companies to routinely produce over 100,000 new and unique compounds per year.Jeffrey W. Noonan et al. "Advancing Parallel Solution Phase Library Synthesis through Efficient Purification, Quantitation, and Characterization Techniques" Journal of Laboratory Automation, 48 (1992) 3789].
In order to handle the vast number of structural possibilities, researchers often create a 'virtual library', a computational enumeration of all possible structures of a given pharmacophore with all available reactants.E. V.Gordeeva et al. "COMPASS program - an original semi-empirical approach to computer-assisted synthesis" Tetrahedron, 48 (1992) 3789]. Such a library can consist of thousands to millions of 'virtual' compounds. The researcher will select a subset of the 'virtual library' for actual synthesis, based upon various calculations and criteria (see ADME, computational chemistry, and QSAR).
In 1996, at Parke-Davis Pharmaceutical Research, scientist Anthony Czarnik directed research into and reported the first use of automation in synthesizing compound libraries. As the founding editor of the American Chemical Society's Journal of Combinatorial Chemistry, he also led research into RFID tags for targeted sorting in compound library synthesis.
Polymers (peptides and oligonucleotides)
Combinatorial split-mix (split and pool) synthesis
Combinatorial split-mix (split and pool) synthesis Furka Á, Sebestyén F, Asgedom M, Dibó G. Cornucopia of peptides by synthesis. In Highlights of Modern Biochemistry, Proceedings of the 14th International Congress of Biochemistry. VSP.Utrecht.1988; 5; p. 47.Á. Furka, F. Sebestyen, M. Asgedom, G. Dibo, General method for rapid synthesis of multicomponent peptide mixtures. Int. J. Peptide Protein Res., 1991, 37, 487-493. is based on the solid-phase synthesis developed by Merrifield.Merrifield RB, 1963 J. Am. Chem. Soc. 85, 2149. If a combinatorial peptide library is synthesized using 20 amino acids (or other kinds of building blocks), the bead form solid support is divided into 20 equal portions. This is followed by coupling a different amino acid to each portion. The third step is the mixing of all portions. These three steps comprise a cycle. Elongation of the peptide chains can be realized by simply repeating the steps of the cycle.
The procedure is illustrated by the synthesis of a dipeptide library using the same three amino acids as building blocks in both cycles. Each component of this library contains two amino acids arranged in different orders. The amino acids used in couplings are represented by yellow, blue and red circles in the figure. Divergent arrows show the division of solid support resin (green circles) into equal portions, vertical arrows mean coupling, and convergent arrows represent the mixing and homogenizing of the portions of the support.
The figure shows that, in the two synthetic cycles, 9 dipeptides are formed. In the third and fourth cycles, 27 tripeptides and 81 tetrapeptides would form respectively.
The "split-mix synthesis" has several outstanding features:
It is highly efficient. As the figure demonstrates, the number of peptides formed in the synthetic process (3, 9, 27, 81) increases exponentially with the number of executed cycles. Using 20 amino acids in each synthetic cycle, the number of formed peptides are 400, 8,000, 160,000 and 3,200,000, respectively. This means that the number of peptides increases exponentially with the number of the executed cycles. All peptide sequences that are formed in the process can be deduced by a combination of the amino acids used in the cycles. Portioning of the support into equal samples assures formation of the components of the library in nearly equal molar quantities. Only a single peptide forms on each bead of the support. This is the consequence of using only one amino acid in the coupling steps. It is completely unknown, however, which is the peptide that occupies a selected bead. The split-mix method can be used for the synthesis of organic or any other kind of library that can be prepared from its building blocks in a stepwise process.
In 1990, three groups described methods for preparing peptide libraries by biological methodsJ. J. Devlin, L. C. Panganiban and P. E. Devlin Science 1990, 249, 404. and one year later Fodor et al. published a remarkable method for synthesis of peptide arrays on small glass slides.Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D, 1991. Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767-73.
A "parallel synthesis" method was developed by Mario Geysen and his colleagues for preparation of peptide arrays.H. M. Geysen, R. H. Meloen, S. J. Barteling Proc. Natl. Acad. Sci. USA 1984, 81, 3998. They synthesized 96 peptides on plastic rods (pins) coated at their ends with the solid support. The pins were immersed into the solution of reagents placed in the wells of a microtiter plate. The method is widely applied, particularly by using automatic parallel synthesizers. Although the parallel method is much slower than the real combinatorial one, its advantage is that it is exactly known which peptide or other compound forms on each pin.
Further procedures were developed to combine the advantages of both split-mix and parallel synthesis. In a method described by two groups,E. J. Moran, S. Sarshar, J. F. Cargill, M. Shahbaz, A Lio, A. M. M. Mjalli, R. W. Armstrong J. Am. Chem. Soc. 1995, 117, 10787.K. C. Nicolaou, X –Y. Xiao, Z. Parandoosh, A. Senyei, M. P. Nova Angew. Chem. Int. Ed. Engl. 1995, 36, 2289. the solid support was enclosed into permeable plastic capsules together with a radiofrequency tag that carried the code of the compound to be formed in the capsule. The procedure was carried out similar to the split-mix method. In the split step, however, the capsules were distributed among the reaction vessels according to the codes read from the radiofrequency tags of the capsules.
A different method for the same purpose was developed by Furka et al. named "string synthesis".Á. Furka, J. W. Christensen, E. Healy, H. R. Tanner, H. Saneii J. Comb. Chem. 2000, 2, 220. In this method, the capsules carry no code. They are strung like pearls in a necklace and placed into the reaction vessels in stringed form. The identity of the capsules, as well as their contents, are stored by their position occupied on the strings. After each coupling step, the capsules are redistributed among new strings according to definite rules.
Small molecules
In the drug discovery process, the synthesis and biological evaluation of small molecules of interest have typically been a long and laborious process. Combinatorial chemistry has emerged in recent decades as an approach to quickly and efficiently synthesize large numbers of potential small molecule drug candidates. In a typical synthesis, only a single target molecule is produced at the end of a synthetic scheme, with each step in a synthesis producing only a single product. In a combinatorial synthesis, when using only single starting material, it is possible to synthesize a large library of molecules using identical reaction conditions that can then be screened for their biological activity. This pool of products is then split into three equal portions containing each of the three products, and then each of the three individual pools is then reacted with another unit of reagent B, C, or D, producing 9 unique compounds from the previous 3. This process is then repeated until the desired number of building blocks is added, generating many compounds. When synthesizing a library of compounds by a multi-step synthesis, efficient reaction methods must be employed and, if traditional purification methods are used after each reaction step, yields and efficiency will suffer.
Solid-phase synthesis offers potential solutions to obviate the need for typical quenching and purification steps often used in synthetic chemistry. In general, a starting molecule is adhered to a solid support (typically an insoluble polymer), then additional reactions are performed, and the final product is purified and then cleaved from the solid support. Since the molecules of interest are attached to a solid support, it is possible to reduce the purification after each reaction to a single filtration/wash step, eliminating the need for tedious liquid-liquid extraction and solvent evaporation steps that most synthetic chemistry involves. Furthermore, by using heterogeneous reactants, excess reagents can be used to drive sluggish reactions to completion, which can further improve yields. Excess reagents can simply be washed away without the need for additional purification steps such as chromatography.
Over the years, a variety of methods have been developed to refine the use of solid-phase organic synthesis in combinatorial chemistry, including efforts to increase the ease of synthesis and purification, as well as non-traditional methods to characterize intermediate products. Although the majority of the examples described here will employ heterogeneous reaction media in every reaction step, Booth and Hodges provide an early example of using solid-supported reagents only during the purification step of traditional solution-phase syntheses. In their view, solution-phase chemistry offers the advantages of avoiding attachment and cleavage reactions necessary to anchor and remove molecules to resins, as well as eliminating the need to recreate solid-phase analogues of established solution-phase reactions.