Peter C. Michels

Microwave assisted combinatorial chemistry synthesis of substituted pyridines

Abstract A new highly efficient MICROCOS technology (Microwave-assisted Combinatorial Synthesis) for generating combinatorial libraries is described. The technology is applied to the high throughput, automated, one-step, parallel synthesis of diverse substituted pyridines using the Hantzsch synthesis. The advantages of microwave-assisted chemistry for combinatorial synthesis include a broad range of available chemistries, simple reaction setup and product recovery readily amenable to automation, extremely short reaction times, and high product yields.

Combinatorial biocatalysis: a natural approach to drug discovery

Natures most potent molecules are produced by enzyme-catalysed reactions, coupled with the natural selection of those products that possess optimal biological activity. Combinatorial biocatalysis harnesses the natural diversity of enzymatic reactions for the iterative synthesis of organic libraries. Iterative reactions can be performed using isolated enzymes or whole cells, in natural and unnatural environments, and on substrates in solution or on a solid phase. Combinatorial biocatalysis is a powerful addition to the expanding array of combinatorial methods for the generation and optimization of lead compounds in drug discovery and development.

Pressure Effects on Enzyme Activity and Stability at High Temperatures

Publisher Summary This chapter describes a promising strategy for obtaining new stable enzymes— that is, to isolate them from micro-organisms that grow under extreme conditions, the so-called extremophiles. The application of enzymes in industrial processes are limited by the inability of the enzymes to withstand harsh operating conditions, for example, high temperatures, pH extremes, high detergent concentrations, and high organic solvent concentrations. A long-standing goal of biotechnology has therefore been to expand the operating range of the enzymes beyond typical intracellular conditions. A general framework for analyzing pressure effects on enzyme stability are developed by first considering one of the simplest, yet most widely applicable, two-step models for enzyme inactivation. This model involves an initial equilibrium step, where the enzyme undergoes cooperative unfolding to give a reversibly denatured state. The reversibly denatured state is then subject to an irreversible inactivation step(s) yielding inactive enzyme. This model is summarized in the chapter. Case studies on pressure effects and protein stability are also given.

Regioselective enzymatic acylation as a tool for producing solution-phase combinatorial libraries

Abstract A simple combinatorial strategy for sequential regioselective enzymatic acylation of multifunctional lead compounds has been developed and demonstrated using a polyhydroxylated flavonoid, bergenin, as a model. The approach is based on the ability of different enzymes to regioselectively acylate different sites on a lead molecule without affecting other similar functional groups. In sharp contrast to enzymatic acylation, conventional chemical acylation methods showed almost complete lack of regioselectivity. The enzymatic strategy was applied successfully to produce a solution phase combinatorial library of 167 distinct selectively acylated derivatives of bergenin on a robotic workstation in a 96-well plate format. General applicability of the automated combinatorial biocatalysis strategy is discussed.

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone.

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.

Reversible derivatization to enhance enzymatic synthesis: chemoenzymatic synthesis of doxorubicin-14-O-esters.

An efficient three‐step, chemoenzymatic synthesis of unprotected doxorubicin‐14‐O‐esters from doxorubicin hydrochloride salt is described. The key step is a lipase‐catalyzed regioselective transesterification/esterification using commercially available acyl donors and doxorubicin reversibly derivatized with N‐alloc to improve substrate loadings. The overall yield is ca. 60% and chromatographic purification is not required, thereby making the process more amenable to scale‐up. Biotechnol. Bioeng. 2008;101: 435–440.

Extractive Biotransformation for Production of Metabolites of Poorly Soluble Compounds: Synthesis of 32-Hydroxy-rifalazil

A novel reaction system was developed for the production of metabolites of poorly water-soluble parent compounds using mammalian liver microsomes. The system includes the selection and use of an appropriate hydrophobic polymeric resin as a reservoir for the hydrophobic parent compounds and its metabolites. The utility of the extractive biotransformation approach was shown for the production of a low-yielding, synthetically challenging 32-hydroxylated metabolite of the antibiotic rifalazil using mouse liver microsomes. To address the low solubility and reactivity of rifalazil in the predominantly aqueous microsomal catalytic system, a variety of strategies were tested for the enhanced delivery of hydrophobic substrates, including the addition of mild detergents, polyvinylpyrrolidone, glycerol, bovine serum albumin, and hydrophobic polymeric resins. The latter strategy was identified as the most suitable for the production of 32-hydroxy-rifalazil, resulting in up to 13-fold enhancement of the volumetric productivity compared with the standard aqueous system operating at the solubility limit of rifalazil. The production process was optimized for a wide range of reaction parameters; the most important for improving volumetric productivity included the type and amount of the polymeric resin, cofactor recycling system, concentrations of the biocatalyst and rifalazil, reaction temperature, and agitation rate. The optimized extractive biotransformation system was used to synthesize 32-hydroxy-rifalazil on a multimilligram scale.