Rapid identification of enantioselective ketone reductions using targeted microbial libraries

A collection of about 300 microbes was surveyed for the ability to generate chiral secondary alcohols by enantioselective reduction of a series of alkyl aryl ketones. Microbial cultures demonstrating utility in reducing model ketones were arrayed in multi-well plates and used to rapidly identify specific organisms capable of producing chiral alcohols used as intermediates in the synthesis of several drug candidates. Approximately 60 cultures were shown to selectively reduce various ketones providing both the R and S enantiomers of the corresponding alcohols in 92-99% ee with yields up to 95% at 1-4 g/L. An alternative approach to chiral alcohols based on selective microbial oxidation of racemic alcohols is also reported. This study provides a useful reference for generating chiral alcohols by selective microbial bioconversion.     

Rapid identification of substrates for novel proteases using a combinatorial peptide library

Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology. The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library. The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A). We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library. The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies.     

Quality assessment of combinatorial pooled libraries using mass spectrometry

Parallel synthesis techniques aim to prepare collections of single compounds which, once tested, can easily be identified by their sole location in the synthesic array. On the other hand, true combinatorial chemistry produces libraries of compounds as mixtures of variable size which require a deconvolution procedure for identification of the active hits or leads. In the latter case, analytical methods are crucial for the success of the strategy and mass spectrometry plays a major role. If the goal is to identify all the library components, including expected products as well as by-products, various mass spectrometric techniques may be necessary. Library components can be separated according to their mass by increasing mass resolution or by their elution time by coupling liquid chromatography and mass spectrometry. The efficiency of such separation techniques are discussed as a function of the size and the degeneracy of the library. Library members possess common structural features which impart similar fragmentation patterns after ionization in the gas phase. This feature can be exploited by tandem mass spectrometry to specifically detect subfamilies of products. Examples of precursor ion scans, product ion scans and constant neutral loss scans will be shown that facilitate partial characterization of libraries. To solve the difficult problem of the quantitative analysis of libraries, i.e., to evaluate their equimolarity, the use of an evaporative light scattering detector (ELSD) or a chemiluminescent nitrogen detector (CLND) is suggested as more appropriate.     

How to synthesize macrocycles efficiently by using virtual combinatorial libraries

The selection of different diimines 4 a-c by alkaline earth ions from a virtual combinatorial library (VCL) is described. The products were stabilized by reduction to the diamines 6 a-c; this allowed easy analysis. The library can be directed toward different target molecules 6 a-c upon addition of alkaline earth ions with different radii. Competition experiments show the possibility of synthesizing the macrocycles 6 a, 6 b, and 6 c simultaneously when using Mg(2+), Ca(2+), and Ba(2+) as template ions. The scope of this thermodynamically controlled, reversible approach for macrocycle syntheses is illustrated.     

Multiobjective optimization of combinatorial libraries

Combinatorial chemistry and high-throughput screening have caused a fundamental shift in the way chemists contemplate experiments. Designing a combinatorial library is a controversial art that involves a heterogeneous mix of chemistry, mathematics, economics, experience, and intuition. Although there seems to be little agreement as to what constitutes an ideal library, one thing is certain: only one property or measure seldom defines the quality of the design. In most real-world applications, a good experiment requires the simultaneous optimization of several, often conflicting, design objectives, some of which may be vague and uncertain. In this paper, we discuss a class of algorithms for subset selection rooted in the principles of multiobjective optimization. Our approach is to employ an objective function that encodes all of the desired selection criteria, and then use a simulated annealing or evolutionary approach to identify the optimal (or a nearly optimal) subset from among the vast number of possibilities. Many design criteria can be accommodated, including diversity, similarity to known actives, predicted activity and/or selectivity determined by quantitative structure-activity relationship (QSAR) models or receptor binding models, enforcement of certain property distributions, reagent cost and availability, and many others. The method is robust, convergent, and extensible, offers the user full control over the relative significance of the various objectives in the final design, and permits the simultaneous selection of compounds from multiple libraries in full- or sparse-array format.     

Design of diverse and focused combinatorial libraries using an alternating algorithm

There is considerable research in chemistry to develop reaction conditions so that any of a very large number of reactants will successfully form new compounds, e.g. for two components, A(i) + B(j) --> A-B(ij). The numbers of A's and B's usually make it impossible to make all the possible products; with multicomponent reactions, there could easily be millions to billions of possible products. There is a need to identify subsets of reagents so that the resulting products have desirable predicted properties. Our idea is to select reactants sequentially and iteratively to optimize the evolving candidate library. The new Alternating Algorithm, AA, can be used for diversity, a space-filling design, or for a focused design, using either a near neighborhood or structure-activity relationship, SAR. A diversity design seeks to select compounds different from one another; a focused design seeks to find compounds similar to an active compound or compounds that follow a structure activity relationship. The benefit of the method is rapid computation of diversity or focused combinatorial chemical libraries.     

Solid-phase extraction for combinatorial libraries

Solid-phase extraction (SPE) has during the last three years emerged as a convenient method for the purification of compound libraries prepared by solution synthesis. The widespread use of SPE in combinatorial chemistry can be explained by straightforward SPE method development facilitated by the availability of numerous commercial SPE resins. High-speed automated SPE is readily accomplished by taking advantage of commercial laboratory robot systems. The present review summarizes and discusses advancements made in the use of different SPE resins and molecule tagging techniques for optimization of ion-exchange, reversed-phase, normal-phase and fluorous-phase SPE in combinatorial chemistry.     

Computational screening of combinatorial catalyst libraries

A catalyst design methodology, utilizing combinatorial synthesis in parallel with chemometric analysis, is presented, which considers the 3D steric and electrostatic properties of substituents about a constant core structure.     

Toward dynamic combinatorial libraries of cryptands

The first examples of dynamic combinatorial libraries of cryptands, created using the chemistry of imines is presented. Experiments, in which two trialdehydes compete for one triamine in a small library, show the full reversibility of cryptand formation, since the library composition is the same irrespective of the sequence of introducing the building blocks to the mixture.     

Rapid parallel synthesis of combinatorial libraries of substituted 3-thio-1,2,4-triazoles and 2-thioimidazoles

Combinatorial libraries of substituted 3-thio-1,2,4-triazoles and 2-thioimidazoles were synthesized in good yield by alkylation of the products via the reaction of isothiocyanates and carboxylic acid hydrazides or beta-aminoketones, respectively. A total of 275 3-thio-1,2,4-triazoles and 283 2-thioimidazoles were synthesized out of the attempted 288 in each case. Most the yields were between 45% and 98%, and all the compounds synthesized were purified to >85% purity. Variations in yields revealed no definitive trends and were mainly attributed to bulky alkylating agents used and the plate well location of the reaction mixtures.     

Exploring the relation between amplification and binding in dynamic combinatorial libraries of macrocyclic synthetic receptors in water

Herein we describe an extensive study of the response of a set of closely related dynamic combinatorial libraries (DCLs) of macrocyclic receptors to the introduction of a focused range of guest molecules. We have determined the amplification of two sets of diastereomeric receptors induced by a series of neutral and cationic guests, including biologically relevant compounds such as acetylcholine and morphine. The host–guest binding affinities were investigated using isothermal titration calorimetry. The resulting dataset enabled a detailed analysis of the relationship between the amplification of selected receptors and host–guest Gibbs binding energies, giving insight into the factors affecting the design, simulation and interpretation of DCL experiments. In particular, two questions were addressed: Is amplification by a given guest selective for the best receptor? And does the best guest induce the largest amplification of a given receptor? Our experimental results and computer simulations showed that the relative levels of amplification of hosts by a guest are well‐correlated with their relative affinities, and simulations have confirmed previous observations that amplification can be selective for the best receptor when only modest amounts of guest are used. In contrast, the correlation between guest binding and the extent of amplification of a given receptor across a wide range of guests tends to be poorer, because every guest has its own unique set of affinities for competing receptors in the DCL. This implies that the results of screening a DCL for selective receptors by comparing the response of the mixture to two different guests should be interpreted with caution. DCLs are complex mixtures in which all compounds are connected through a set of equilibria. Obtaining quantitative information about all host–guest binding constants from such systems will require the explicit and simultaneous consideration of all of the main equilibria within a DCL.     

Tailored polymer-supported templates in dynamic combinatorial libraries: simultaneous selection, amplification and isolation of synthetic receptors

The thermodynamically controlled synthesis and isolation of macrocyclic receptors from dynamic combinatorial libraries has been achieved in a single step using a polymer-supported template. The templates were cinchona alkaloids which show interesting enantio- and diastereoselective molecular recognition events in libraries based on pseudo-dipeptide building blocks. The synthetic routes used to derivatise the alkaloids and attach them to polymer supports minimised any influence of the tethering linkage on the templating activity. Systematic studies have been carried out to probe how the polymer morphology and the template loading affect the selectivity and isolation yield of the macrocyclic receptors. Molecular recognition between solid-phase bound templates and selected receptors also enabled their affinity-type chromatographic separation.     

Dynamic combinatorial libraries constructed on polymer scaffolds

Functionalized polymers were prepared by grafting acylhydrazides onto a polyvinylbenzaldehyde scaffold through reversible hydrazone linkages. The dynamic nature of these linkages allows the functionalized polymers to exchange and reshuffle their appendages, and the resultant mixture of polymers can be considered as a dynamic combinatorial library constructed upon a polymer scaffold. The dynamic nature of these functionalized polymers was demonstrated.     

Artificial aldolases from peptide dendrimer combinatorial libraries

Peptide dendrimers were investigated as synthetic models for aldolase enzymes. Combinatorial libraries were prepared with aldolase active residues such as lysine and proline placed at the dendrimer core or near the surface. On-bead selection for aldolase activity was carried out using the dye-labelled 1,3-diketone 1a, suitable for covalent trapping of enamine-reactive side-chains, and the fluorogenic enolization probe 6. Aldolase dendrimers catalyzed the aldol reaction of acetone, dihydroxyacetone and cyclohexanone with nitrobenzaldehyde. Much like enzymes, the dendrimers exhibited strong aldolase activity in aqueous medium, but were also active in organic solvent. Dendrimer-catalyzed aldol reactions reached complete conversion in 3 h at 25 degrees C with 1 mol% catalyst and gave aldol products with up to 65% ee. A positive dendritic effect in catalysis was observed with both lysine and proline based aldolase dendrimer catalysts.     

A combinatorial approach toward smart libraries of discontinuous epitopes of HIV gp120 on a TAC synthetic scaffold

We describe rapid and convenient access to smart libraries of protein surface discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a triazacyclophane scaffold molecule via CuAAC. In this way protein mimics for use as synthetic vaccines and beyond will become available.