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

[摘要] English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasinglyimportant in the last few years. This same trend is now found in the synthesis ofagrochemicals. Epoxides, due to their high reactivity with a large number of reagents,and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactiveintermediates, are versatile chiral synthons in the synthesis of many bioactivecompounds. Extensive research efforts have thus been directed towards the synthesis ofoptically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxidehydrolases has recently emerged as a very attractive strategy for the synthesis ofenantiopure epoxides.Both chemical and biological catalysts that may be employed to obtain enantiopureepoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1).The potential use of microbial epoxide hydrolases, including those from yeasts aselucidated during this study, was emphasised in this review.At the onset of this study, epoxide hydrolase activity had been identified in only oneyeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed withexcellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases mightbe very interesting catalysts. This had indeed been found to be true during the course ofthis study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2-epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (DivisionIndustrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxidehydrolases have been found that display this unique enantioselectivity for epoxideslacking other substituents (Chapter 2).Extensive screening of yeasts from the renowned UOFS Yeast Culture Collectionrevealed that epoxide hydrolase activity was constitutively present in about 20% of theyeasts 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 activityin bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us toinvestigate the epoxide hydrolase activity of the yellow pigmented bacteriumChryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxidehydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by abacterial 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 toC-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases fromall the enantioselective yeasts were found to be membrane-associated (Chapter 5).The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in anelegant one-step protocol from the microsomal fraction, using affinity chromatography(Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu ofinformation about the primary structure of yeast epoxide hydrolases, inactivation of theenzyme by modification of specific amino acids was studied. Asp/Glu and His residueswere found to be essential for catalytic activity. In addition, it was found that one or moreSer residues in the catalytic site are indispensible for catalytic activity. These resultsindicate 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 bypurified epoxide hydrolase. Hydrolysis was characterised by a strong dependence ofenantioselectivity on the presence of the substrate as a second (Iypophilic) phase. Thepurified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours(Chapter 7).

[发布机构] University of the Free State