Biocatalytic resolution of substituted styrene oxides
Yeates, Charl Alan
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Stereochemistry and chirality are arguably two of the most important subjects pertaining to the development of new pharmaceutical drugs. Since enantiomers have the potential to encompass different pharmacological effects in biological systems, both enantiomers have to be tested for pharmacological activity. Not only has obtaining these single enantiomers become crucial, but formulation of the pure enantiomer of a drug also has the potential to contain advantages for both pharmaceutical formulation and therapeutic effect. Epoxide hydrolase is an enzyme commonly found in nature that catalyses the hydrolysis of epoxides, resulting in the formation of the corresponding vicinal diol. Over the last few years a large amount of research has been completed on these enzymes from sources such as mammals, insects, bacteria and fungi. Micro-organisms especially have enjoyed ample attention because of their abundant supply. Recently it was found that certain yeasts contain this enzyme and have the ability to enantioselectively catalyse certain hydrolysis reactions. Styrene oxides are terminal epoxides that are, due to the reactivity of the epoxide ring, useful synthons in the organic synthesis of pharmaceutical products. The first objective of this project was to synthesize three nitro derivatives of styrene oxide namely para-, meta-, and ortho-nitrostyrene oxide. Al three products were obtained from the corresponding nitrophenacyl bromide in yields of 52%, 90% and 57% respectively. The second objective was lo find a suitable yeast slrain containing the epoxide hydrolase enzyme to enantioselectively hydrolyse the synthesised products and unsubstituted styrene oxide. A screening was completed during which 410 yeast strains from more than 44 genera were tested. Epoxide hydrolase activity was found to be widespread throughout the screened yeast domain, while the genera Candida, Debaryomyces, Pichia, Rhodosporidium, Rhodotorula and Trichosporon specifically were very successful in catalysing the hydrolysis of the substrates. Rhodosporidium toruloides UOFS Y-0471 and Rhodotorula glutinis UOFS Y-0653 were chosen for further studies because of their superior enantioselectivity. The final objective was to optimise these reactions in terms of pH, temperature and substrate concentration. It was found that a pH value of 7.2 and a temperature of 45’C yielded optimal enzyme activity. Increased temperatures (45’C), however, lead to a decrease in enantioselectivity and, in the case of R. toruloides together with the substrate puranitrostyrene oxide, reversed enantioselectivity. Lower temperatures (15’C) increased enantioselectivity, resulting in a remarkable improvement from a 10% yield of the single enantiomer (45’C) to a 35% yield. Surprisingly this temperature decrease had a very small affect upon the reaction time.
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