Automated synthesis and combinatorial chemistry

The advent of combinatorial chemistry for the high-throughput synthesis of compounds has driven the advancement of new and emerging technologies for synthetic chemistry laboratories. Automated methods for reaction design, information management, chemical synthesis, compound analysis, and biological testing are necessary to realize the full potential of combinatorial chemistry efforts.     

'One bead two compound libraries' for detecting chemical and biochemical conversions

When combinatorial chemistry was introduced 13 years ago, the expectations were high for the delivery of results, particularly in the pharmaceutical industry. However, combinatorial chemistry was implemented independently of the application for which the products were going to be used. Resins developed only for efficient solid-phase synthesis were used and products were employed in existing assays developed for traditional solution studies. There was almost no assay or technology development and the use of real combinatorial methods soon had to give way to high-throughput synthesis and traditional screening. However, during recent years more sophisticated resins and assay techniques have been developed that may result in a second and more successful implementation of real integrated combinatorial chemistry. The first in this line of new developments is the 'one bead two compound' assay, in which the resin bead in addition to a combinatorial library member contains a reporter compound that can act as a beacon to monitor the activity of the library member. This powerful concept can be generally applied in all fields of combinatorial chemistry including drug, catalysts and material development.     

Combinatorial synthesis of natural products

Combinatorial syntheses allow production of compound libraries in an expeditious and organized manner immediately applicable for high-throughput screening. Natural products possess a pedigree to justify quality and appreciation in drug discovery and development. Currently, we are seeing a rapid increase in application of natural products in combinatorial chemistry and vice versa. The therapeutic areas of infectious disease and oncology still dominate but many new areas are emerging. Several complex natural products have now been synthesised by solid-phase methods and have created the foundation for preparation of combinatorial libraries. In other examples, natural products or intermediates have served as building blocks or scaffolds in the synthesis of complex natural products, bioactive analogues or designed hybrid molecules. Finally, structural motifs from the biologically active parent molecule have been identified and have served for design of natural product mimicry, which facilitates the creation of combinatorial libraries.     

Single-bead analysis in combinatorial chemistry

Notable limitations have previously prevented the wide application of split synthesis. However, recent developments in highly condensed and miniaturized biological screening and single-bead analysis methods have argued for a revival of split combinatorial synthesis. Although there are still many challenges, we are now in a much better position to accomplish high-throughput analysis and screening of one-bead-one-compound libraries.     

Combinatorial search for advanced luminescence materials.

Phosphors are key materials in fluorescent lighting, displays, x-ray scintillation, etc. The rapid development of modern photonic technologies, e.g., mercury-free lamps, flat panel displays, CT-detector array, etc., demands timely discovery of advanced phosphors. To this end, a combinatorial approach has been developed and applied to accelerated experimental search of advanced phosphors and scintillators. Phosphor libraries can be made in both thin film and powder form, using masking strategies and liquid dispensing systems, respectively. High-density libraries with 100 to 1000 discrete phosphor compositions on a 1"-square substrate can be made routinely. Both compositions and synthesis temperatures can be screened in a high-throughput mode. In this article, details on the existing methods of combinatorial synthesis and screening of phosphors will be reported with examples. These methods are generic tools for application of combinatorial chemistry in the discovery of other solid state materials. A few highly efficient phosphors discovered with combinatorial methods have been reproduced in bulk form and their luminescent properties measured.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

A multidimensional overpressured layer chromatographic method for the characterization of tetrazine libraries

The recent combinatorial approach in synthetic organic chemistry started a new age in drug discovery. The generation of compound libraries in combination with high-throughput screening has become the method of choice for the production of new pharmacological leads for chemical optimization. Characterization and separation of such pool of compounds have been lagging behind the synthetic and screening methodologies. Overpressured layer chromatography (OPLC) is an instrumentalized planar liquid chromatographic technique associated with the use of optimized layers prepared from particles of narrow particle size distribution and small diameter. On one hand, uni-directional OPLC allows the simultaneous separation of large number of samples in minutes. On the other hand, two-dimensional OPLC offers multidimensional separation on a single layer. This paper shows the complete multidimensional separation of a tetrazine library prepared by parallel combinatorial synthesis. In general, this approach may become the method of choice for the characterization of compound libraries.     

The development of high-throughput screening approaches for stem cell engineering

It has become increasingly clear that both soluble factors, such as growth factors, and insoluble factors, including the surfaces on which cells grow, can have controlling effects on stem cell behavior and differentiation. While much progress has been made in biomaterial design and application, the rational design of biomaterial cues to direct stem cell behavior and differentiation remains challenging. Recent advances in automated, high-throughput methods for synthesizing and screening combinatorial biomaterial libraries and cellular microenvironments promise to accelerate the discovery of factors that control stem cell behavior. Specific examples include miniaturized, automated, combinatorial material synthesis and extracellular matrix screening methods as well microarrayed methods for creating local microenvironments of soluble factors, such as small molecules, siRNA, and other signaling molecules.     

Natural products and combinatorial chemistry: back to the future

The introduction of high-throughput synthesis and combinatorial chemistry has precipitated a global decline in the screening of natural products by the pharmaceutical industry. Some companies terminated their natural products program, despite the unproven success of the new technologies. This was a premature decision, as natural products have a long history of providing important medicinal agents. Furthermore, they occupy a complementary region of chemical space compared with the typical synthetic compound library. For these reasons, the interest in natural products has been rekindled. Various approaches have evolved that combine the power of natural products and organic chemistry, ranging from the combinatorial total synthesis of analogues to the exploration of natural product scaffolds and the design of completely unnatural molecules that resemble natural products in their molecular characteristics.     

High-throughput protein crystallization

The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.     

Impact of mass spectrometry on combinatorial chemistry

In the past few years, the emergence of combinatorial chemistry has drawn increasing attention and a great deal of analytical research has been centered around this new methodology. These new methods capable of producing vast numbers of samples, which are in many cases highly complex, demand fast and reliable analytical techniques able to provide high quality information concerning sample compositions. Mass spectrometry (MS) is the method of choice to face these analytical challenges. In particular, the introduction of electrospray ionization (ESI and matrix assisted laser desorption/ionization (MALDI)) have been the driving forces for many of the recent innovations, not only within the fields of the biosciences, but also in combinatorial chemistry. These ionization techniques are extremely versatile for the characterization of both single compound collections and compound mixture collections. The high-throughput capabilities, as well as many possible couplings with separation techniques (HPLC, CE) have been thus facilitated. However, mass spectrometry is not only limited to use as an instrument for synthesis control, but also plays an increasing role in the identification of active compounds from complex libraries. Recently, new initiatives for library analysis and screening have arisen from the application of the latest developments in mass spectrometry, Fourier transform ion cyclotron resonance (FTICR).     

AutoClickChem: click chemistry in silico

Academic researchers and many in industry often lack the financial resources available to scientists working in "big pharma." High costs include those associated with high-throughput screening and chemical synthesis. In order to address these challenges, many researchers have in part turned to alternate methodologies. Virtual screening, for example, often substitutes for high-throughput screening, and click chemistry ensures that chemical synthesis is fast, cheap, and comparatively easy. Though both in silico screening and click chemistry seek to make drug discovery more feasible, it is not yet routine to couple these two methodologies. We here present a novel computer algorithm, called AutoClickChem, capable of performing many click-chemistry reactions in silico. AutoClickChem can be used to produce large combinatorial libraries of compound models for use in virtual screens. As the compounds of these libraries are constructed according to the reactions of click chemistry, they can be easily synthesized for subsequent testing in biochemical assays. Additionally, in silico modeling of click-chemistry products may prove useful in rational drug design and drug optimization. AutoClickChem is based on the pymolecule toolbox, a framework that may facilitate the development of future python-based programs that require the manipulation of molecular models. Both the pymolecule toolbox and AutoClickChem are released under the GNU General Public License version 3 and are available for download from http://autoclickchem.ucsd.edu.     

Combinatorial catalyst discovery.

There have been recent attempts to use the principles of combinatorial chemistry and high-throughput screening strategies for catalyst identification. With the technology available that allows the synthesis of large libraries, scientists of varied backgrounds have implemented screening efforts to identify active and selective catalysts. Within this context, several techniques have come to light in the past year: infrared thermography is used to identify optimal catalysts by monitoring the change in temperature for exothermic reactions; fluorescence and colored-dye assays, a familiar tool to biologists, is being applied to the identification of catalysts that exhibit the highest activity. Whereas none of these screening methods provide a general solution to the problem of screening large combinatorial libraries (there is likely to be no general solution), each advance represents an important intellectual and technological step forward.     

A pipeline for ligand discovery using small-molecule microarrays

Uncovering the functions of thousands of gene products, in various states of post-translational modification, is a key challenge in the post-genome era. To identify small-molecule probes for each protein function, high-throughput methods for ligand discovery are needed. In recent years, small-molecule microarrays (SMMs) have emerged as high-throughput and miniaturized screening tools for discovering protein-small-molecule interactions. Microarrays of small molecules from a variety of sources, including FDA-approved drugs, natural products and products of combinatorial chemistry and diversity-oriented synthesis, have been prepared and screened by several laboratories, leading to several newly discovered protein-ligand pairs.     

Recent advances in the discovery of organometallic catalysts using high-throughput screening assays

Combinatorial techniques were developed initially to accelerate the identification of molecules with biological activity. The successes of these techniques inspired the design of high-throughput methods to assist in the discovery of new catalysts. Over the past year, many groups in academia and industry have utilized high-throughput screening assays to reduce the time required to identify catalysts for asymmetric processes, cross-coupling reactions and other metal-catalyzed transformations. The continued success of combinatorial techniques in organometallic chemistry should propagate the development of new and improved methods to facilitate catalyst discovery.     

Techniques for high-throughput characterization of peptides, oligonucleotides and catalysis efficiency

Combinatorial processes have been widely applied to many disciplines in chemistry and biology. The vast numbers of unique entities generated by combinatorial synthesis have led to the development of high-throughput methods for characterizing samples, to avoid bottlenecks created by the application of conventional, serial-based analytical techniques. In recent years, high-throughput and novel methods utilizing mass spectrometry, multiplexed capillary electrophoresis, various forms of optical detection, and even sound waves have been investigated for a variety of applications.     

Genetic optimization of combinatorial libraries

Most agrochemical and pharmaceutical companies have set up high-throughput screening programs which require large numbers of compounds to screen. Combinatorial libraries provide an attractive way to deliver these compounds. A single combinatorial library with four variable positions can yield more than 10(12) potential compounds, if one assumes that about 1000 reagents are available for each position. This is far more than any high-throughput screening facility can afford to screen. We have proposed a method for iterative compound selection from large databases, which identifies the most active compounds by examining only a small fraction of the database. In this article, we describe the extension of this method to the problem of selecting compounds from large combinatorial libraries. Copyright 1998 John Wiley & Sons, Inc.     

Proton-activated fluorescence as a tool for simultaneous screening of combinatorial chemical reactions

In recent years, combinatorial chemistry has had a significant impact on catalyst discovery in diverse fields. Proton-activated fluorescence (PAF) has been successfully demonstrated as a technique for effective screening of catalysts for electro-oxidation, enzymatic ester hydrolysis and nonenzymatic acyl transfer reactions. Among the working prototypes are screens for high-throughput assays of arrayed solid-state catalysts, dissolved enzymatic and small-molecule catalysts, as well as catalysts immobilized in solid-phase synthesis beads or polymeric gels. Given the range of reactions that may be set up to provide a change in local pH, the potential of PAF to facilitate catalyst discovery and process development is significant.     

Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli

We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular L-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for L-tyrosine-overproducing strains.