Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins
| dc.contributor.advisor | Smit, M. S. | |
| dc.contributor.advisor | Litthauer, D. | |
| dc.contributor.author | Botes, Adriana Leonora | |
| dc.date.accessioned | 2017-05-08T06:40:01Z | |
| dc.date.available | 2017-05-08T06:40:01Z | |
| dc.date.issued | 1999-06 | |
| dc.description.abstract | 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). | en_ZA |
| dc.description.abstract | Afrikaans: Die sintese van farmaseutiese middels in enantiomeries suiwer vorm het die afgelope paar jaar toenemend belangrik geword. Dieselfde neiging word nou in die sintese van landbou chemikalië waargeneem. Epoksiede, as gevolg van hul reaktiwiteit met talle ander molekules, en visinale diole, in die vorm van hul sikliese sulfate of sulfiete as reaktiewe intermediêre, is veelsydige chirale sintone in die sintese van talle bioaktiewe verbindings. Intensiewe navorsing, gerig op die sintese van opties aktiewe epoksiede en visinale diole, is dus die afgelope tyd gedoen. Kinetiese resolusie van rasemiese epoksiede is 'n aantreklike strategie vir die sintese van enantiomeries suiwer epoksiede. In Hoofstuk 1 is 'n oorsig van beide chemiese en biologiese kataliste wat gebruik kan word om enantiomeries suiwer epoksiede vanaf relatiewe goedkoop rasemiese substrate te verkry, aangebied. Die moontlike toepassings van mikrobiese epoksied hidrolases, ingeslote dié van giste, soos uitgebeeld tydens hierdie studie, is beklemtooon in die oorsig. Met die aanvang van hierdie studie, is epoksied hidrolase aktiwiteit in slegs een gis, Rhodotorula glutinis, geïdentifiseer. Die wye reeks substrate wat met uitstekende enantioselektiwiteit deur hierdie gis gehidroliseer is, het daarop gedui dat gis epoksied hidrolases uiters interessante biokataliste mag blyk te wees. Dit is inderdaad bevind dat dit die geval is tydens hierdie studie. Enantioselektiewe hidrolise van 'n homoloë reeks alifatiese 1,2-epoksiealkane is verkry, in samewerking met die groep van Jan de Bont (Afdeling Industriële Mikrobiologie, Wageningen LU, Nederland). Geen ander mikrobiese epoksied hidrolases met hierdie unieke enantioselektiwiteit vir epoksiede sonder ander substituente, is voorheen gevind nie (Hoofstuk 2). Uitgebreide sifting van giste uit die welbekende UOVS Giskultuur Versameling het getoon dat epoksied hidrolase aktiwiteit in sowat 20% van die giste voorkom, en dat ander basidiomysete giste van die genera Rhodotorula, Rhodosporidium en Trichosporon ook die unieke enantioselektiwiteit van Rhodotorula glutinis vir 1,2- epoksieoktaan toon. Die klaarblyklike assosiasie tussen karotenoid produksie en epoksied hidrolase aktiwiteit in bakterieë en in die rooi giste Rhodotorula en Rhodospordium, het gelei tot die ondersoek van die epoksied hidrolase aktiwitiet van die geel gepigmenteerde bakterium Chryseomonas /uteo/a in ons versameling. Hierdie bakterium het inderdaad epoksied hidrolase aktiwiteit getoon, en matige enantioselektiwiteit (E =20) vir 1,2-epoksiealkane deur 'n bakteriese epoksied hidrolase is vir die eerste keer gevind (Hoofstuk 4). 'n Opname van die enantioselektiwiteite van giste vir 'n homoloë reeks 1,2- epoksiealkane, 1,2-epoksiealkene, sowel as die 2,2-digesubstitueerde 2-metiel-1,2- epoksieheptaan en bensiel glisidiel eter, is gedoen. Uitstekende biokataliste vir C-5 tot C-8 epoksiealkane en die C-8 epoksiealkeen is gevind. Die epoksied hidrolases van al die enantioselektiewe giste was membraan-geassosieerd (Hoofstuk 5). Die epoksied hidrolase van die gis Rhodosporidium toru/oides is in 'n elegante een-stap protokol uit die mikrosomale fraksie gesuiwer deur middel van affiniteitschromatografie (Hoofstuk 6). Aanvanklike pogings om die aminosuur volgorde te bepaal, was egter onsuksesvol. By gebrek aan inligting oor die primêre struktuur van gis epoksied hidrolases, is die deaktivering van die ensiem deur modifikasie van spesifieke aminosure bestudeer. Asp/Glu en His residue was noodsaaklik vir katalitiese aktiwiteit. Verder is daar ·ook gevind dat een of meer Ser residue onontbeerlik vir katalitiese aktiwiteit is. Hierdie resultate dui daarop dat gis epoksied hidrolases waarskynlik tot dieselfde subfamilie van a,~-hidrolase vou ensieme behoort as die mikrosomale epoksied hidrolases van ander eukariote. Ongewone kinetiese gedrag is waargeneem tydens die hidrolise van 1,2-epoksiealkane deur gesuiwerde epoksied hidrolase. Hidrolise is gekenmerk deur 'n sterk afhanklikheid van enantioselektiwiteit van die teenwoordigheid van die substraat as 'n tweede (Iipofiliese) fase. Die suiwer epoksied hidrolase was nie baie stabiel nie, met 'n halfleeftyd van 18 uur by 35°C (Hoofstuk 7). | af |
| dc.identifier.uri | http://hdl.handle.net/11660/6180 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Alkenes | en_ZA |
| dc.subject | Chiral drugs | en_ZA |
| dc.subject | Thesis (Ph.D. (Microbiology and Biochemistry))--University of the Free State, 1999 | en_ZA |
| dc.title | Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins | en_ZA |
| dc.type | Thesis | en_ZA |