Solid phase synthesis of complex natural products and libraries thereof.

Natural products have served as an important source of medicinal compounds and pharmaceutical leads over the last century. Within the last 10 years, significant interest has developed in applying combinatorial chemistry techniques to the study of natural products and their biological activities. In this review, we examine several representative efforts wherein natural product skeletons have been constructed or immobilized on solid support and subsequently derivatized, giving rise to analog libraries useful in understanding the structure-activity relationships of the parent natural product. Issues such as target selection, library design, linker development, automation, and library characterization are addressed.     

Natural products as a screening resource

Natural products have been the most productive source of leads for new drugs, but they are currently out of fashion with the pharmaceutical industry. New approaches to sourcing novel compounds from untapped areas of biodiversity coupled with the technical advances in analytical techniques (such as microcoil NMR and linked LC-MS-NMR) have removed many of the difficulties in using natural products in screening campaigns. As the 'chemical space' occupied by natural products is both more varied and more drug-like than that of combinatorial chemical collections, synthetic and biosynthetic methods are being developed to produce screening libraries of natural product-like compounds. A renaissance of drug discovery inspired by natural products can be predicted.     

Wanted: new multicomponent reactions for generating libraries of polycyclic natural products

The amalgamation of two of combinatorial chemistry's most attractive concepts--natural product libraries and multicomponent reactions (MCRs)--should provide a powerful tactic for generating libraries of bioactive compounds. Yet, despite many recent advances in this area, only a few MCRs can deliver functionalized products whose structures closely resemble that of complex polycyclic natural products. A large proportion of recently developed MCRs are based on [4+2] or [3+2] cycloadditions, and isocyanide-based processes. Because of substrate limitations, however, they are not always ideally suitable for applications in diversity-oriented synthesis of natural product-like compounds. A promising area awaiting further development is the use of transition metal-catalyzed cascade reactions.     

High-diversity combinatorial libraries

The synthesis of complex chemical structures by combinatorial chemistry has gained considerable interest. New chemical methods have been developed that enable the synthesis of compound libraries exhibiting structural diversities similar to those of natural products. The concept of 'chemical genomics' has been introduced, reflecting a new quality of understanding and creating the relationship between diverse artificial chemical structures with the space of biological responses and possible protein ligands.     

Combinatorial biocatalysis: taking the lead from nature

Combinatorial biocatalysis is an emerging technology in the field of drug discovery. The biocatalytic approach to combinatorial chemistry uses enzymatic, chemoenzymatic, and microbial transformations to generate libraries from lead compounds. Important recent advances in combinatorial biocatalysis include iterative derivatization of small molecules and complex natural products, regioselectively controlled libraries, novel one-pot library syntheses, process automation, and biocatalyst enhancements.     

Combinatorial biosynthesis--potential and problems

Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author's laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations.     

Characterization of Sphingomonas aldehyde dehydrogenase catalyzing the conversion of various aromatic aldehydes to their carboxylic acids

Natural products represent an important source of drugs in a number of therapeutic fields, e.g. antiinfectives and cancer therapy. Natural products are considered as biologically validated lead structures, and evolution of compounds with novel or enhanced biological properties is expected from the generation of structural diversity in natural product libraries. However, natural products are often structurally complex, thus precluding reasonable synthetic access for further structure-activity relationship studies. As a consequence, natural product research involves semisynthetic or biotechnological approaches. Among the latter are mutasynthesis (also known as mutational biosynthesis) and precursor-directed biosynthesis, which are based on the cellular uptake and incorporation into complex antibiotics of relatively simple biosynthetic building blocks. This appealing idea, which has been applied almost exclusively to bacteria and fungi as producing organisms, elegantly circumvents labourious total chemical synthesis approaches and exploits the biosynthetic machinery of the microorganism. The recent revitalization of mutasynthesis is based on advancements in both chemical syntheses and molecular biology, which have provided a broader available substrate range combined with the generation of directed biosynthesis mutants. As an important tool in supporting combinatorial biosynthesis, mutasynthesis will further impact the future development of novel secondary metabolite structures.     

Diversifying microbial natural products for drug discovery

Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.V. Knight and J.-J. Sanglier contributed equally to this work     

Synthesis of natural-product-based compound libraries

Natural products cover a diversity space not yet available from synthetic libraries, with an unrivalled success rate as drug leads. The combinatorial synthesis of non-oligomeric natural-product-based libraries, however, is still limited to few examples because access to easily modified units strongly depends on the availability of a core structure either from a natural source, or through a suitable synthetic route. Only a few resourceful groups have managed the latter approach for more demanding multifunctional natural drug leads, such as epothilones.     

Natural product derivatives with bactericidal activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis

We have shown that the intentional engineering of a natural product biosynthesis pathway is a useful way to generate stereochemically complex scaffolds for use in the generation of combinatorial libraries that capture the structural features of both natural products and synthetic compounds. Analysis of a prototype library based upon nonactic acid lead to the discovery of triazole-containing nonactic acid analogs, a new structural class of antibiotic that exhibits bactericidal activity against drug resistant, Gram-positive pathogens including Staphylococcus aureus and Enterococcus faecalis.     

Natural product-like libraries based on non-aromatic, polycyclic motifs

Diversity-oriented synthesis is an intriguing approach for creating structurally diverse compounds that cover the pharmaceutically relevant chemical space in an optimal way. On the other hand, an over-proportionally large number of drugs or lead structures originate from compounds isolated from natural sources. Thus, not surprisingly, an increasing number of combinatorial libraries are based on motifs resembling natural products. A particular aspect of many natural products is the presence of non-aromatic, polycyclic core structures. The fusion of several rings leads to geometrically well-defined structures and, thus, holds the promise of a high functional specialisation. In this review we present several actual examples of natural product-like libraries with non-aromatic polycyclic motifs based on naturally occurring compounds.     

Use of combinatorial peptide libraries for T-cell epitope mapping

T lymphocytes play important roles not only in infectious diseases and autoimmunity, but also in immune responses against tumors. For many of these disorders, the relevant target antigens are not known. Designing effective methods that allow the search for T-cell epitopes is therefore an important goal in the areas of infectious diseases, oncology, vaccine development, and numerous other biomedical specialties. So far, the strategies used to examine T-cell recognition have been based largely on mapping T-cell epitopes with overlapping peptides from known proteins or with entire proteins, e.g., from a specific virus, bacterium, or human tissue. These approaches are tedious and have a number of limitations. It is, for example, almost impossible to isolate T cells that infiltrate an organ or infectious site and identify their specificity unless one already has a concept as to which antigens may be relevant. During recent years, a number of laboratories have developed less biased approaches that employ either the selection of putative T-cell epitopes based on the prediction of binding to certain major histocompatibilty complex (MHC) molecules and peptide or protein libraries that have been generated in expression systems, e.g. phage, or rely on combinatorial peptide chemistry. The latter technique has been refined by a number of laboratories including ours. Bead-bound or, preferably, positional scanning synthetic and soluble combinatorial peptide libraries allow the identification of T-cell epitopes within complex mixtures of proteins even for T cells that have been expanded from an organ infiltrate with a polyclonal stimulus. The practical steps that are involved in the latter method are described in this article.     

Plant natural products: Back to the future or into extinction?

Natural product substances have historically served as the most significant source of new leads for pharmaceutical development. However, with the advent of robotics, bioinformatics, high throughput screening (HTS), molecular biology-biotechnology, combinatorial chemistry, in silico (molecular modeling) and other methodologies, the pharmaceutical industry has largely moved away from plant derived natural products as a source for leads and prospective drug candidates. Can, or will, natural products ever recapture the preeminent position they once held as a foundation for drug discovery and development? The challenges associated with development of natural products as pharmaceuticals are illustrated by the Taxol^® story. Several misconceptions, which constrain utilization of plant natural products, for discovery and development of pharmaceuticals, are addressed to return natural products to the forefront.     

Natural products as a hunting ground for combinatorial chemistry

Natural products have a long history of success as biologically active leads for therapeutic agents. The ability to prepare analogues and to discover structure-activity relationships is necessary to truly harness the potential of natural products. Recently, combinatorial chemistry has risen to this challenge, and even fairly complex natural products can be targeted for parallel synthesis. Academic and industrial efforts have employed natural products from the peptide, alkaloid, polyketide, and terpenoid and steroid classes in combinatorial chemistry approaches for the production of medicinally important compounds.     

Design of compound libraries based on natural product scaffolds and protein structure similarity clustering (PSSC)

Recent advances in structural biology, bioinformatics and combinatorial chemistry have significantly impacted the discovery of small molecules that modulate protein functions. Natural products which have evolved to bind to proteins may serve as biologically validated starting points for the design of focused libraries that might provide protein ligands with enhanced quality and probability. The combined application of natural product derived scaffolds with a new approach that clusters proteins according to structural similarity of their ligand sensing cores provides a new principle for the design and synthesis of such libraries. This article discusses recent advances in the synthesis of natural product inspired compound collections and the application of protein structure similarity clustering for the development of such libraries.     

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。     

Combinatorial biosynthesis of polyketides--a perspective

Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make 'unnatural' natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.     

Diversifying carotenoid biosynthetic pathways by directed evolution.

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.     

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace? SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.     

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.