Combinatorial biocatalysis

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.     

组合生物催化

自然界最有效的分子是由酶催化的反应所产生,并对这些产物进行自然选择,使其具有优化的生理活性,组合生物催化（Combinatorial Biocatalysis)利用酶反应的多样性,完成有机库（Organic Library)的反复合成,这些反复的反应,可以用分离的酶或全细胞,在天然或非天然的环境中、在溶液或固相中与底物进行反应。组合生物催化是组合方法的在药物发现和发展中产生和优化先导化合物（LeadCompound)的一个有力补充。     

Biocatalytic combinatorial synthesis

Combinatorial biocatalysis, based on a principle of the combinatorial use of biosynthetic steps rather than the combinatorial use of reagents, offers a complementary approach to combinatorial chemistry, which, used individually or in connection with synthetic organic transformations, provides access to analogues not readily accessible by chemical synthetic means alone. The issues and strategies particular to this approach are discussed. Examples are given demonstrating these principles as well as the unique advantages of achieving chemo-, regio- and stereoselectivity under mild reaction conditions that biocatalytic methods offer.     

Diversity assessment.

Several themes have been highlighted recently in both conferences and publications: the availability of product-focused and pharmacophore-based methods for the analysis and design of combinatorial libraries; the power of cell-based methods for molecular similarity, diversity and library design applications; methods for 'rational' diverse subset selection (with applicability to library design); the need for specialized optimization programs for the design of combinatorial libraries that maximize the use of common reagents; and the concept of 'drug-likeness' and its importance in the design of combinatorial libraries.     

Peptide and peptidomimetic libraries

Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.     

Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.     

Host-guest chemistry: combinatorial receptors.

A combinatorial approach to receptor design provides an expedient method to discover the most effective host-guest complexes from within a library. Recent advances focus on generation of larger libraries, facile detection, combinatorial catalysis and the formation of dynamic receptor libraries.     

Solid phase synthesis of mixture-based acyclic and heterocyclic small molecule combinatorial libraries from resin-bound polyamides.

The development of soluble mixture-based heterocyclic combinatorial libraries derived from amino acids and peptides is described. Starting with a "toolbox" of various chemical transformations, including alkylations, reductions, acylations, and the use of a variety of bifunctional reagents, the "libraries from libraries" concept has been expanded to encompass the development of more than fifty positional scanning combinatorial libraries each composed of tens of thousands of low molecular weight acyclic and heterocyclic compounds.     

Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.     

Combinatorial chemistry,automation and molecular diversity: new trends in the pharmaceutical industry

Combinatorial chemistry has emerged as a set of novel strategies for the synthesis of large sets of compounds (combinatorial libraries) for biological evaluation. Within a few years combinatorial chemistry has undergone a series of changes in trends, which are closely related to two important factors in libraries: numbers and quality. While the number of compounds in a library may be easily expressed, it is a lot more difficult to indicate the degree of quality of a library. This degree of quality can be split into two aspects : purity and diversity. The changing trends in combinatorial chemistry with respect to the strategies, the technologies, the libraries themselves (numbers and purity aspects) and the molecular diversity are outlined in this paper.     

The evolution of biotransformation technologies

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.     

Green fluorescent protein as a scaffold for intracellular presentation of peptides.

Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence.     

Directed Evolution of Enzymes for Biocatalytic Applications

Directed molecular evolution is a rapidly growing field revolutionizing the development of biocatalysts with improved properties. This review describes methods to create mutant libraries and assays for rapid screening or selection of desired variants. Selected examples emphasizing the evolution of enzymes for applications in biocatalysis show that it is possible to alter substrate specificity, modulate enantioselectivity and increase enzyme performance under process conditions.     

Combinatorial carbohydrate chemistry

The application of combinatorial chemistry to the synthesis of carbohydrate-based compound collections has received increased attention in recent years. New strategies for the solution-phase synthesis of oligosaccharide libraries have been reported, and the use of monosaccharides as scaffolds in the generation of combinatorial libraries has been described. Novel approaches to the assembly of carbohydrate-based antibiotics, such as aminoglycoside analogs and vancomycin derivatives, have also been disclosed.     

Dissimilarity-based algorithms for selecting structurally diverse sets of compounds.

This paper commences with a brief introduction to modern techniques for the computational analysis of molecular diversity and the design of combinatorial libraries. It then reviews dissimilarity-based algorithms for the selection of structurally diverse sets of compounds in chemical databases. Procedures are described for selecting a diverse subset of an entire database, and for selecting diverse combinatorial libraries using both reagent-based and product-based selection.     

Structure-based library design: molecular modelling merges with combinatorial chemistry

Recent advances in both computational and experimental techniques now allow a very fruitful interplay of computational and combinatorial chemistry in the structure-based design of combinatorial libraries.     

Structure‐based design of combinatorial mutagenesis libraries

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high‐throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general‐purpose method, called “Structure‐based Optimization of Combinatorial Mutagenesis ” (SOCoM ), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library‐averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β‐lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure‐based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large‐scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure‐based assessments, such as the energy gap between alternative conformational or bound states.     

Combinatorial search of substituted beta-cyclodextrins for phosphatase-like activity.

Thirteen per-6-akylamino-6-deoxy-beta-cyclodextrin libraries (beta-CD libraries) were generated by a solution-phase combinatorial synthesis starting from per-6-iodo-6-deoxy-beta-CD and different combinations of eleven individual amine nucleophiles. Certain libraries showed the ability to hydrolyzep-nitrophenyl phosphate in the presence of Zn2+.     

Symmetric building blocks and combinatorial functional group transformation as versatile strategies in combinatorial chemistry

A combination of symmetric building blocks and combinatorial functional group transformation for synthesis of pyrimidines was investigated. The purpose of the study was to maximize the return on invested synthetic efforts of reaction development for libraries. A representative set of symmetric diacids was coupled onto deprotected TentaGel Rink Amide resin. The amino function served as a model of a chemical process providing a functional group for additional synthetic steps, while the symmetric building blocks served as a model to connect synthesis protocols and to switch to a different synthesis paradigm consecutively. The reaction sequence was continued in a noncombinatorial step by coupling a bifunctional reagent (3-aminoacetophenone) to the remaining carboxy function of the symmetric diacid. The ketone served as a model of a reagent prepared for combinatorial functional group transformation. The arylmethylketone was reacted with a set of aryl- and heteroarylaldehydes to give alpha,beta-unsaturated ketones. Subsequently, guanidine, alkyl-, and arylcarboxamidines were introduced in combinatorial synthesis of substituted pyrimidines by reaction with the alpha, beta-unsaturated ketone functionality. The combination of symmetric building blocks and combinatorial functional group transformation created a versatile reaction sequence ideally suited for production of libraries from libraries with added diversity.