Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins

English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasingly important in the last few years. This same trend is now found in the synthesis of agrochemicals. Epoxides, due to their high reactivity with a large number of reagents, and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactive intermediates, are versatile chiral synthons in the synthesis of many bioactive compounds. Extensive research efforts have thus been directed towards the synthesis of optically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxide hydrolases has recently emerged as a very attractive strategy for the synthesis of enantiopure epoxides. Both chemical and biological catalysts that may be employed to obtain enantiopure epoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1). The potential use of microbial epoxide hydrolases, including those from yeasts as elucidated during this study, was emphasised in this review. At the onset of this study, epoxide hydrolase activity had been identified in only one yeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed with excellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases might be very interesting catalysts. This had indeed been found to be true during the course of this study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2- epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (Division Industrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxide hydrolases have been found that display this unique enantioselectivity for epoxides lacking other substituents (Chapter 2). Extensive screening of yeasts from the renowned UOFS Yeast Culture Collection revealed that epoxide hydrolase activity was constitutively present in about 20% of the yeasts screened, and that other basidiomycetous yeasts from the genera Rhodotorula, Rhodosporidium and Trichosporon shared this unique enantioselectivity for 1,2- epoxyoctane with Rhodotorula glutinis (Chapter 3). he apparent association between carotinoid production and epoxide hydrolase activity in bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us to investigate the epoxide hydrolase activity of the yellow pigmented bacterium Chryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxide hydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by a bacterial epoxide hydrolase was found for the first time (Chapter 4). A survey of the enantioselectivities of yeasts for a homologous range of 1,2- epoxyalkanes, 1,2-epoxyalkenes as well as the 2,2-disubstituted 2-methyl-1,2- epoxyheptane and benzyl glycidyl ether was conducted. Excellent biocatalysts for C-5 to C-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases from all the enantioselective yeasts were found to be membrane-associated (Chapter 5). The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in an elegant one-step protocol from the microsomal fraction, using affinity chromatography (Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu of information about the primary structure of yeast epoxide hydrolases, inactivation of the enzyme by modification of specific amino acids was studied. Asp/Glu and His residues were found to be essential for catalytic activity. In addition, it was found that one or more Ser residues in the catalytic site are indispensible for catalytic activity. These results indicate that yeast epoxide hydrolases probably belong to the same subfamily of a,l3- hydrolase fold enzymes as the microsomal epoxide hydrolases from other eukaryotes. Unusual kinetic behaviour was observed during the hydrolysis of 1,2-epoxyalkanes by purified epoxide hydrolase. Hydrolysis was characterised by a strong dependence of enantioselectivity on the presence of the substrate as a second (Iypophilic) phase. The purified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours (Chapter 7).