Liquid-phase combinatorial synthesis: In search of small-molecule enzyme mimics

Summary The applications, advantages and recent advances in liquid-phase combinatorial chemistry using poly(ethyleneglycol) as a soluble polymer support are reviewed. Our recent efforts towards the synthesis of peptide-based catalysts on polyethyleneglycol are reported. The screening of libraries of peptides for catalysis is discussed.     

Application of computer assisted combinatorial chemistry in antivirial,antimalarial and anticancer agents design

Combinatorial chemistry and technologies have been developed to a stage where synthetic schemes are available for generation of a large variety of organic molecules. The innovative concept of combinatorial design assumes that screening of a large and diverse library of compounds will increase the probability of finding an active analogue among the compounds tested. Since the rate at which libraries are screened for activity currently constitutes a limitation to the use of combinatorial technologies, it is important to be selective about the number of compounds to be synthesized. Early experience with combinatorial chemistry indicated that chemical diversity alone did not result in a significant increase in the number of generated lead compounds. Emphasis has therefore been increasingly put on the use of computer assisted combinatorial chemical techniques. Computational methods are valuable in the design of virtual libraries of molecular models. Selection strategies based on computed physicochemical properties of the models or of a target compound are introduced to reduce the time and costs of library synthesis and screening. In addition, computational structure-based library focusing methods can be used to perform in silico screening of the activity of Compounds against a target receptor by docking the ligands into the receptor model. Three case studies are discussed dealing with the design of targeted combinatorial libraries of inhibitors of HIV-1 protease, P. falciparum plasmepsin and human urokinase as potential antivirial, antimalarial and anti-cancer drugs. These illustrate library focusing strategies.     

Combinatorial chemistry of β-hairpins

Combinatorial chemistry is expanding rapidly both in terms of chemistry development and application to the synthesis of compound libraries for lead discovery and optimization. Combinatorial technologies continue evolving and developing, in fact they are being used as basic research tools in different fields that include peptide/protein folding. This review examines the use of combinatorial chemistry in the design of peptides and protein domains that adopt beta-sheet conformations. In particular, the use of conformationally restricted peptide libraries has allowed the identification of linear peptides that are folded in a beta-hairpin structure in plain aqueous solutions.     

Automating combinatorial chemistry: A primer on benchtop robotic systems

Summary Benchtop robotic systems are inexpensive, flexible automation tools with potential applications in a wide array of disciplines such as combinatorial chemistry, high-throughput screening, and genomics. We explain the basic components of a benchtop system and explore factors to consider when purchasing or customizing a robot, such as automation benefits, vendor selection, and current system limitations. Issues involving system specification, software design, and hardware customization are then discussed. Additionally, system optimization, validation, and support are detailed. Given a properly designed and implemented system, the combinatorial laboratory can markedly increase compound synthesis and purification.     

Synthetic methods for polyamine linkers and their application to combinatorial chemistry

Summary Polyamines and polyamine conjugates display a diverse range of important biological functions, ranging from antibiotics to immunosuppressants and glutamate receptor antagonists. For these reasons, polyamines provide an excellent template/scaffold for combinatorial chemistry. In this paper we present methods for the solid-phase immobilisation of polyamines for use in synthetic and combinatorial chemistry and describe how they have been employed in the preparation of a number of important polyamine conjugates and polyamine libraries. Thus, we have designed, synthesised and utilised a number of polyamine linkers for both solution and resin screening combinatorial application.     

Xanthines as a scaffold for molecular diversity

Summary Xanthines represent a new, versatile scaffold for combinatorial chemistry. A five-step solid-phase synthesis of xanthine derivatives is described which includes alkylations, a nucleophilic displacement reaction at a heterocycle and a ring closure reaction by condensation of a nitroso function with an activated methylene group. The selected reaction sequence allows the production of a highly diverse small-molecule combinatorial compound library.     

Advances in Microwave-Assisted Combinatorial Chemistry Without Polymer-Supported Reagents

Summary Combinatorial methodologies have dramatically changed the chemical research and discovery process, offering an unlimited source of new molecule entities to be screened for activity. The application of microwave irradiation in Combinatorial Chemistry and high-throughput synthesis has become increasingly popular. By taking advantage of this energy source, compound libraries for lead generation can be assembled in a fraction of time required by conventional thermal heating. This review focuses on the advances in developing synthetic methodologies in microwave without polymer-supported reagents suitable for combinatorial chemistry, including the advances in microwave-assisted fluorous synthesis technology.     

High-volume cellular screening for anticancer agents with combinatorial chemical libraries: A new methodology

Summary A single-step cancer cell cytotoxic assay system for anticancer drug discovery has been developed which facilitates rapid screening of large combinatorial chemical libraries synthesized using the one-bead-one-compound (OBOC) methodology. Each OBOC library bead incorporates two orthogonally cleavable linkers that release the bead-bound compound at a different pH. The assay utilizes high concentrations of tumor cells mixed directly with OBOC beads and plated in soft agarose containing tissue culture medium. One of the orthogonal linkers is cleaved at neutral pH in tissue culture releasing an aliquot of compound to diffuse at a relatively high local concentration into the soft agarose immediately surrounding the bead. Active compounds are identified visually from a clear ring of tumor cell lysis which forms within 48 h around just the rare bead releasing a cytotoxic compound. The bead releasing a cytotoxin is then plucked from the agar and the remaining compound still linked to the bead can be released for structural analysis, followed by compound resynthesis and confirmatory testing. This assay system has been successfully applied to identification of lead cytotoxic compounds from model peptidic and non-peptidic combinatorial chemical libraries. Use of this methodology may facilitate anticancer drug discovery.     

Analytical methods for quality control of combinatorial libraries

This literature review covers the applications of analytical techniques to solid phase organic chemistry and combinatorial chemistry published between June 96 and September 1997. Highlighted are mass spectrometry, NMR, IR and chromatographic analyses of solid phase synthesis reactions and combinatorial libraries.     

Quantitation of combinatorial libraries of small organic molecules by normal-phase HPLC with evaporative light-scattering detection

Summary The advantages of evaporative light-scattering detection over UV detection for the quantitation of combinatorial libraries composed of small organic compounds by HPLC are described. The detector's response is independent of the sample chromophore, which makes it well-suited to chromatographie analyses of mixtures of dissimilar solutes. Thus, HPLC with evaporative light-scattering detection offers the potential for reducing false positive or false negative results in screening assays, because of its ability to detect the presence of impurities that absorb poorly in the UV (e.g., those impurities originating from the polymeric support). Furthermore, the evaporative light-scattering detector exhibits a nearly equivalent response to compounds of similar structural class. Hence, rapid quantitation of compound libraries may be carried out with the use of a single external standard. For example, the quantitation errors, based on a single external standard, for a series of steroids, hydantoins, and BOC- and Fmoc-protected amino acids by normal-phase HPLC with evaporative light-scattering detection average approximately ± 10%. The application of the evaporative light-scattering detector to the quantitation of low-level sample impurities and the detector's compatibility with gradient elution are also described.     

Multidimensional chromatography coupled with mass spectrometry for target-based screening

Summary The synthesis of structural analogs and the process of drug discovery have evolved dramatically through recent advances in solid-phase synthesis reagents and automated screening systems. As molecular diversity strategies emerge, the need for automated target-based selection of lead candidates becomes equally important. Multidimensional automated chromatographic techniques coupled to electrospray ionization mass spectrometry facilitate the selection process and provide maximum characterization information in a single screening run. The capture of tightly bound affinity leads by target biomolecules, followed by subsequent release and high-resolution separation with sensitive detection, significantly reduces the time required to identify and characterize lead compounds. This automated multidimensional chromatographic approach coupled with mass spectrometry, Selectronics, was used with several organic and natural libraries to demonstrate an automated target-based screening technique to select for high-affinity binders as potential lead compounds.Abbreviations ESI electrospray ionization - HPLC high-performance liquid chromatography - HTS high-throughput screening - ESI-TOF electrospray ionization time-of-flight - SAR structure-activity relationship     

Chemoinformatics methods for systematic comparison of molecules from natural and synthetic sources and design of hybrid libraries

Until recently, the field of diversity and library design has more or less ignored natural products as a compound source. This is probably due to at least two reasons. First, combinatorial and reaction-based approaches have been major focal points in the early days of computational library design. In addition, a widespread view is that natural products are often highly complex and not amenable to medicinal chemistry efforts. This contribution introduces recent computational approaches to systematically analyze natural molecules and bridge the gap between natural products and synthetic chemistry programs. Large scale comparisons of natural and synthetic molecules are discussed as well as studies designed to identify 'synthetic mimics' of natural products with specific activity. In addition, a concept for the design of natural/synthetic hybrid libraries is introduced. Although research in this area is still in its early stages, an important lesson to be learned from computational analyses is that there is no need to a priori 'shy away' from natural products as a source for molecular design.     

Discovery of a herbicidal lead using polymer-bound activated esters in generating a combinatorial library of amides and esters

Summary A combinatorial library containing mixtures of amides and esters was prepared through solid-phase chemistry. The advantages of using solid-phase chemistry over solution-phase chemistry to prepare this library are discussed. The library was screened through a high-throughput whole organism herbicidal assay upon which a mixture containing amides was found to have herbicidal activity. Deconvolution of the mixture providedN-(3-benzoylphenyl)-3-(1,1-dimethylethyl)-1-methyl)-1H-pyrazole-5-carboxamide as a herbicidal lead with broadleaf and narrowleaf pre-emergence herbicidal activity as low as 100 g/ha on some weed species. This study represents the first report of an agrochemical discovered using a combinatorial approach. Supplementary Material, comprising experimental procedures for the preparation of resin II and the reaction of II with nucleophiles, GC/MS and LC/MS data for the reaction ofII with morpholine, and a detailed experimental for the preparation of 2 with characterization data, is available upon request.     

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage

Summary We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 × 10⁹ members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of -endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v -ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.     

Phosphorus-based combinatorial libraries: Use of amino acid derivatives as synthons

Summary Phosphorus has been used as a scaffold to prepare combinatorial libraries of phosphoramidates in which one of the diversity elements resulted from derivatives of amino acids. A small library was prepared for analytical and characterization purposes, followed by a larger library of approximately 8800 compounds. Libraries were assembled on solid supports using the conventional pool-and-divide method, followed by cleavage from the supports at the end of the synthesis. Mass spectrometry was used to confirm that library synthesis had been successful. Individual compounds were also prepared to study the stability of compounds of this type.     

Advances in diversity profiling and combinatorial series design

Rapid advances in synthetic and screening technology have recently enabled the simultaneous synthesis and biological evaluation of large chemical libraries containing hundreds to tens of thousands of compounds, using molecular diversity as a means to design and prioritize experiments. This paper reviews some of the most important computational work in the field of diversity profiling and combinatorial library design, with particular emphasis on methodology and applications. It is divided into four sections that address issues related to molecular representation, dimensionality reduction, compound selection, and visualization.     

Chemoinformatics methods for systematic comparison of molecules from natural and synthetic sources and design of hybrid libraries

Until recently, the field of diversity and library design has more or less ignored naturalproducts as a compound source. This is probably due to at least two reasons. First,combinatorial and reaction-based approaches have been major focal points in the earlydays of computational library design. In addition, a widespread view is that naturalproducts are often highly complex and not amenable to medicinal chemistry efforts. Thiscontribution introduces recent computational approaches to systematically analyzenatural molecules and bridge the gap between natural products and synthetic chemistryprograms. Large scale comparisons of natural and synthetic molecules are discussed aswell as studies designed to identify `synthetic mimics' of natural products with specificactivity. In addition, a concept for the design of natural/synthetic hybrid libraries isintroduced. Although research in this area is still in its early stages, an important lesson tobe learned from computational analyses is that there is no need to a priori `shy away'from natural products as a source for molecular design.     

Libraries from libraries: Generation and comparison of screening profiles

Summary A positional scanning tetrapeptide library was chemically modified through alkylation and/or reduction of the amide bonds, thus generating three new combinatorial libraries with physico-chemical properties very different from the parent peptide library (libraries from libraries). Specific results were obtained with each of these libraries upon screening in -opioid receptor binding and microdilution antimicrobial assays, illustrating the potential of the libraries from libraries concept for the efficient generation of a variety of chemically diverse combinatorial libraries.     

A new combination of protecting groups and links for encoded synthetic libraries suited for consecutive tests on the solid phase and in solution

Summary A strategy for high-throughput evaluation of combinatorial compound libraries is reported, which circumvents the necessity to test complex mixtures. The method is based on a new combination of protecting groups, solid-phase linker and tags. The bulk of the library first undergoes a binding assay with the components grafted on beads. A selection of beads carrying strong ligands is stripped from the labelled target and distributed into microvessels. The ligands are cleaved and rinsed into microeluates. Subsequently, a more detailed characterization with a functional assay in solution determines the best performers, which are identified through the peptidic tag left behind on the corresponding mother bead.Abbreviations Boc tert-butyloxycarbonyl - CCL combinatorial compound libraries - Ddz ,-dimethyl-3,5-dimethoxybenzyloxycarbonyl - DICD N,N-diisopropylcarbodiimide - DIPEA diisopropylethylamine - DMA dimethylacetamide - ESL encoded synthetic libraries - FITC fluoresceinisothiocyanate - GABA -aminobutyric acid - HOBT N-hydroxybenzotriazole - MALDI matrix-assisted laser desorption ionization - Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl - Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl - TFA trifluoroacetic acid - Trt trityl