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Combinatorial chemistry comprises chemical synthetic methods that make possible to prepare 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 in computer. Strategies that allow identification of useful components of the libraries are also part of combinatorial chemistry. The methods used in combinatorial chemistry are applied outside chemistry, too.
Synthesis of molecules in a combinatorial fashion can quickly lead to large numbers of molecules. For example, a molecule with three points of diversity (R1, R2, and R3) can generate possible structures, where , , and are the numbers of different substituents utilized.
The basic principle of combinatorial chemistry is to prepare libraries of very large number of compounds then identify the useful components of the libraries.
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. The principles were developed by Árpád Furka of Eötvös Loránd University and described in a proposal in 1982 which describes a way to synthesize a combinatorial peptide library and a strategy of identification of the biologically active component of the peptide mixture. Professor Pieczenik, a colleague of Nobel Laureate Merrifield, synthesized the first combinatorial library. In the 1980s researcher H. Mario Geysen developed this technique further, creating arrays of different peptides on separate supports, but not a combinatorial library based on random synthesis.
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.
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. 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).
The combinatorial “split-mix synthesis” is based on the solid phase synthesis developed by Merrifield. 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 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 dividing solid support resin (green circles) into equal portions, vertical arrows mean coupling and convergent arrows represent mixing and homogenizing the portions of the support.
The figure shows that in the two synthetic cycles 9 dipeptides are formed. In a third and fourth cycles 27 tripeptides and 81 tetrapeptides would form, respectively.
The “split-mix synthesis” has several outstanding features:
In 1990 three groups described methods for preparing peptide libraries by biological methods and one year later Fodor et al. published a remarkable method for synthesis of peptide arrays on small glass slides.
A “parallel synthesis” method was developed by Mario Geysen and his colleagues for preparation of peptide arrays. 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 the parallel synthesis. In the method described by two groups 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. is named “string synthesis”. In this method the capsules carried no code. They are stringed like the 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.
Dynamic combinatorial chemistry uses a different approach for the synthesis of compound libraries. „Dynamic combinatorial libraries” are generated from an array of reactive building blocks that can react with one another through reversible reactions. In this case, the product distribution in the library is determined by the relative stability of the members. A component of the library can be stabilized by adding a template than can form non-covalent bonds with it. This alters the product distribution and favors the formation of the stabilized library member at the expense of the components.
If a combinatorial library is cleaved from the solid support, in the resulting solution a multi component mixture is found. In such solution, millions of different compounds may be found. At the beginning, it seemed impossible to identify and find among them those components that show useful properties. Strategies were developed, however, to solve the problem if proper screening methods are available. All these strategies are based on synthesis and testing of partial libraries.
The earliest strategy, the “Iteration method” is described in the above mentioned document of Furka notarized in 1982. Identification of the sequence of the active peptide involves removal of samples after each coupling step of the synthesis before mixing. These samples are used in the step-by-step identification by testing and coupling starting at the N-terminus.
The “Positional scanning” method is based on synthesis and testing of a series of sub-libraries in which a certain sequence position in all components is occupied by the same amino acid.
“Omission libraries” in which a certain amino acid is missing from all peptides of the mixture as well as “amino acid tester libraries”  that contain those peptides that are missing from the omission libraries can also be used in the deconvolution process.
If the peptides are not cleaved from the solid support we deal with a mixture of beads, each bead containing a single peptide. Smith and his colleagues showed earlier that peptides could be tested in tethered form, too. This approach was also used in screening peptide libraries. The tethered peptide library was tested with a dissolved target protein. The beads to which the protein was attached were picked out, removed the protein from the bead then the tethered peptide was identified by sequencing.
A somewhat different approach was followed by Taylor and Morken. They used infrared thermography to identify catalysts in non-peptide tethered libraries. When the beads were immersed into a solution of a substrate. the catalyst containing beads were glowing because of the heat evolved in them and could be picked out.
The components of combinatorial libraries can also be tested one by one after cleaving them from the individual beads.
If we deal with a non-peptide organic libraries library it is not as simple to determine of the identity of the content of a bead as in the case of a peptide one. In order to circumvent this difficulty methods were developed to attach to the beads, in parallel with the synthesis of the library, molecules that encode the identity of the compound formed in the bead. The attached molecules may form peptide or nucleotide sequences or a binary code.
Materials science has applied the techniques of combinatorial chemistry to the discovery of new materials. This work was pioneered by P.G. Schultz et al. in the mid nineties in the context of luminescent materials obtained by co-deposition of elements on a silicon substrate. His work was preceded by J. J. Hanak in 1970 but the computer and robotics tools were not available for the method to spread at the time. Work has been continued by several academic groups as well as companies with large research and development programs (Symyx Technologies, GE, Dow Chemical etc.). The technique has been used extensively for catalysis, coatings, electronics, and many other fields. The application of appropriate informatics tools is critical to handle, administer, and store the vast volumes of data produced. New types of Design of experiments methods have also been developed to efficiently address the large experimental spaces that can be tackled using combinatorial methods.
Even though combinatorial chemistry has been an essential part of early drug discovery for more than two decades, so far only one de novo combinatorial chemistry-synthesized chemical has been approved for clinical use by FDA (sorafenib, a multikinase inhibitor indicated for advanced renal cancer). The analysis of poor success rate of the approach has been suggested to connect with the rather limited chemical space covered by products of combinatorial chemistry. When comparing the properties of compounds in combinatorial chemistry libraries to those of approved drugs and natural products, Feher and Schmidt noted that combinatorial chemistry libraries suffer particularly from the lack of chirality, as well as structure rigidity, both of which are widely regarded as drug-like properties. Even though natural product drug discovery has not probably been the most fashionable trend in pharmaceutical industry in recent times, a large proportion of new chemical entities still are nature-derived compounds, and thus, it has been suggested that effectiveness of combinatorial chemistry could be improved by enhancing the chemical diversity of screening libraries. As chirality and rigidity are the two most important features distinguishing approved drugs and natural products from compounds in combinatorial chemistry libraries, these are the two issues emphasized in so-called diversity oriented libraries, i.e. compound collections that aim at coverage of the chemical space, instead of just huge numbers of compounds.
In the 8th edition of the International Patent Classification (IPC), which entered into force on January 1, 2006, a special subclass has been created for patent applications and patents related to inventions in the domain of combinatorial chemistry: "C40B".