Комбинаторлық химия: Құрамалардың жылдам синтезі мен іздеуі

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 outside chemistry, too.

Combinatorial chemistry was invented by Furka Á (from Eötvös Loránd University in Budapest, Hungary) who described the principle of it, the combinatorial synthesis and a deconvolution procedure in a document that was notarized in 1982. The principle of the combinatorial method is: synthesize a multi component compound mixture (combinatorial library) in a single stepwise procedure and screen it to find drug candidates or other kinds of useful compounds also in a single process. The most important innovation of the combinatorial method is to use mixtures in the synthesis and screening that ensures the high productivity of the process. Motivations that led to the invention had been published in 2002.

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. 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. 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).

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. The single purification step at the end of a synthesis allows one or more impurities to be removed, assuming the chemical structure of the offending impurity is known. While the use of solid supported reagents greatly simplifies the synthesis of compounds, many combinatorial syntheses require multiple steps, each of which still requires some form of purification. Armstrong, et al. describe a one pot method for generating combinatorial libraries, called multiple component condensations (MCCs). In this scheme, three or more reagents react such that each reagent is incorporated into the final product in a single step, eliminating the need for a multi step synthesis that involves many purification steps. In MCCs, there is no deconvolution required to determine which compounds are biologically active because each synthesis in an array has only a single product, thus the identity of the compound should be unequivocally known. In another array synthesis, Still generated a large library of oligopeptides by split synthesis. The drawback to making many thousands of compounds is that it is difficult to determine the structure of the formed compounds. Their solution is to use molecular tags, where a tiny amount (1 pmol/bead) of a dye is attached to the beads, and the identity of a certain bead can be determined by analyzing which tags are present on the bead. Despite how easy attaching tags makes identification of receptors, it would be quite impossible to individually screen each compound for its receptor binding ability, so a dye was attached to each receptor, such that only those receptors that bind to their substrate produce a color change. When many reactions need to be run in an array (such as the 96 reactions described in one of Armstrong's MCC arrays), some of the more tedious aspects of synthesis can be automated to improve efficiency. This work, the "DIVERSOMER method" was pioneered at Parke Davis in the early 1990s to run up to 40 chemical reactions in parallel. These efforts led to the first commercially available equipment for combinatorial chemistry (Diversomer synthesizer which was sold by Chemglass) and the first use of liquid handling robotics within a chemistry labortory. This method uses a device that automates the resin loading and wash cycles, as well as the reaction cycle monitoring and purification, and demonstrates the feasibility of their method and apparatus by using it to synthesize a variety of molecule classes, such as hydantoins and benzodiazepines, running 8 or 40 individual reactions in parallel. This and several other pioneering efforts in combinatorial chemistry were featured as "classical" papers in the field in 1999. Oftentimes, it is not possible to use expensive equipment, and Schwabacher, et al. describe a simple method of combining parallel synthesis of library members and evaluation of entire libraries of compounds. In their method, a thread that is partitioned into different regions is wrapped around a cylinder, where a different reagent is then coupled to each region which bears only a single species. The thread is then re divided and wrapped around a cylinder of a different size, and this process is then repeated. The beauty of this method is that the identity of each product can be known simply by its location along the thread, and the corresponding biological activity is identified by Fourier transformation of fluorescence signals. In most of the syntheses described here, it is necessary to attach and remove the starting reagent to/from a solid support. This can lead to the generation of a hydroxyl group, which can potentially affect the biological activity of a target compound. Ellman uses solid phase supports in a multi step synthesis scheme to obtain 192 individual 1,4 benzodiazepine derivatives, which are well known therapeutic agents. To eliminate the possibility of potential hydroxyl group interference, a novel method using silyl aryl chemistry is used to link the molecules to the solid support which cleaves from the support and leaves no trace of the linker. When anchoring a molecule to a solid support, intermediates cannot be isolated from one another without cleaving the molecule from the resin. Since many of the traditional characterization techniques used to track reaction progress and confirm product structure are solution based,
different techniques must be used. Gel phase 13 C NMR spectroscopy, MALDI mass spectrometry, and IR spectroscopy have been used to confirm structure and monitor the progress of solid phase reactions. Gordon et al., describe several case studies that utilize imines and peptidyl phosphonates to generate combinatorial libraries of small molecules.

If the synthesized molecules of a combinatorial library are cleaved from the solid support a soluble mixture forms. In such solution, millions of different compounds may be found. When this synthetic method was developed, it first seemed impossible to identify the molecules, and to find molecules with useful properties. Strategies for identification of the useful components had been developed, however, to solve the problem. All these strategies are based on synthesis and testing of partial libraries. The earliest iterative strategy is described in the above mentioned document of Furka notarized in 1982 and.

The method is made understandable by the figure. A 27 member peptide library is synthesized from three amino acids. After the first (A) and second (B) cycles samples were set aside before mixing them. The products of the third cycle (C) are cleaved down before mixing then are tested for activity. Suppose the group labeled by + sign is active. All members have the red amino acid at the last coupling position (CP). Consequently, the active member also has the red amino acid at the last CP. Then the red amino acid is coupled to the three samples set aside after the second cycle (B) to get samples D. After cleaving, the three E samples are formed. If after testing the sample marked by + is the active one it shows that the blue amino acid occupies the second CP in the active component. Then to the three A samples first the blue then the red amino acid is coupled (F) then tested again after cleaving (G). If the + component proves to be active, the sequence of the active component is determined and shown in H.

Positional scanning was introduced independently by Furka et al. and Pinilla et al. The method is based on the synthesis and testing of series of sublibraries. in which a certain sequence position is occupied by the same amino acid. The figure shows the nine sublibraries (B1 D3) of a full peptide trimer library (A) made from three amino acids. In sublibraries there is a position which is occupied by the same amino acid in all components. In the synthesis of a sublibrary the support is not divided and only one amino acid is coupled to the whole sample. As a result, one position is really occupied by the same amino acid in all components. For example, in the B2 sublibrary position 2 is occupied by the "yellow" amino acid in all the nine components. If in a screening test this sublibrary gives positive answer it means that position 2 in the active peptide is also occupied by the "yellow" amino acid. The amino acid sequence can be determined by testing all the nine (or sometime less) sublibraries.

In omission libraries a certain amino acid is missing from all peptides of the mixture. The figure shows the full library and the three omission libraries. At the top the omitted amino acids are shown. If the omission library gives a negative test the omitted amino acid is present in the active component.

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. The method is based on the heat that is evolved in the beads that contain a catalyst when the tethered library immersed into a solution of a substrate. When the beads are examined through an infrared microscope the catalyst containing beads appear as bright spots and can be picked out.

If we deal with a non peptide organic libraries library it is not as simple to determine the identity of the content of a bead as in the case of a peptide one. In order to circumvent this difficulty methods had been developed to attach to the beads, in parallel with the synthesis of the library, molecules that encode the structure of the compound formed in the bead. Ohlmeyer and his colleagues published a binary encoding method They used mixtures of 18 tagging molecules that after cleaving them from the beads could be identified by Electron Capture Gas Chromatography. Sarkar et al. described chiral oligomers of pentenoic amides (COPAs) that can be used to construct mass encoded OBOC libraries. Kerr et al. introduced an innovative encoding method An orthogonally protected removable bifunctional linker was attached to the beads. One end of the linker was used to attach the non natural building blocks of the library while to the other end encoding amino acid triplets were linked. The building blocks were non natural amino acids and the series of their encoding amino acid triplets could be determined by Edman degradation. The important aspect of this kind of encoding was the possibility to cleave down from the beads the library members together with their attached encoding tags forming a soluble library. The same approach was used by Nikolajev et al. for encoding with peptides. In 1992 by Brenner and Lerner introduced DNA sequences to encode the beads of the solid support that proved to be the most successful encoding method. Nielsen, Brenner and Janda also used the Kerr approach for implementing the DNA encoding
In the latest period of time there were important advancements in DNA sequencing. The next generation techniques make it possible to sequence large number of samples in parallel that is very important in screening of DNA encoded libraries. There was another innovation that contributed to the success of DNA encoding. In 2000 Halpin and Harbury omitted the solid support in the split mix synthesis of the DNA encoded combinatorial libraries and replaced it by the encoding DNA oligomers. In solid phase split and pool synthesis the number of components of libraries can't exceed the number of the beads of the support. By the novel approach of the authors, this restraint was eliminated and made it possible to prepare new compounds in practically unlimited number. The Danish company Nuevolution for example synthesized a DNA encoded library containing 40 trillion! components
The DNA encoded libraries are soluble that makes possible to apply the efficient affinity binding in screening. Some authors apply the DEL for acromim of DNA encoded combinatorial libraries others are using DECL. The latter seems better since in this name the combinatorial nature of these libraries is clearly expressed. Several types of DNA encoded combinatorial libraries had been introduced and described in the first decade of the present millennium. These libraries are very successfully applied in drug research. DNA templated synthesis of combinatorial libraries described in 2001 by Gartner et al. Dual pharmacophore DNA encoded combinatorial libraries invented in 2004 by Mlecco et al. Sequence encoded routing published by Harbury Halpin and Harbury in 2004. Single pharmacophore DNA encoded combinatorial libraries introduced in 2008 by Manocci et al. DNA encoded combinatorial libraries formed by using yoctoliter scale reactor published by Hansen et al. in 2009
Details are found about their synthesis and application in the page DNA encoded chemical library. The DNA encoded soluble combinatorial libraries have drawbacks, too. First of all the advantage coming from the use of solid support is completely lost. In addition, the polyionic character of DNA encoding chains limits the utility of non aqueous solvents in the synthesis. For this reason many laboratories choose to develop DNA compatible reactions for use in the synthesis of DECLs. Quite a few of available ones are already described

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 the 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 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".