Pergamon Tetrahedron: Asymmetry, Vol. 8, No. 1, pp. 65-71, 1997 (~) 1997, Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0957-4166(96)00493-4 0957-4166/97 $17.00 + 0.00 Biocatalytic resolution of 2-methyl-2-(aryl)alkyloxiranes using novel bacterial epoxide hydrolases I. Osprian, W. Kroutil, M. Mischitz and K. Faber * Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, A-8010 Graz, Austria Abstract: Biocatalytic resolution of alkyl- and (arylalkyl)-oxiranes was accomplished by employing the epoxide hydrolase activity of lyophilized whole cells of seven bacterial strains belonging to the genera Rhodococcus, Mycobacterium and Nocardia. Whereas no suitable biocatalyst was found for 1-octene oxide, excellent selectivities were obtained with 2-methyl-2-substituted epoxides bearing an alkyl- or (aryl)alkyl side chain. (~) 1997, Elsevier Science Ltd. All rights reserved. Introduction Recently we have shown that whole bacterial cells ofRhodococcus sp. NCIMB 11216 can be used as a convenient source for epoxide hydrolases for the biocatalytic resolution of oxiranes on a preparative scale 12. The products obtained - optically active epoxides and vicinal diols - can be employed as high-value intermediates for the synthesis of bioactive target molecules such as flavor and fragrance compounds 3 as well as pheromones 4. For 2,2-disubstituted oxiranes, the reaction was shown to proceed via a classic kinetic resolution pattern, i.e. it involves an attack of a formal [OH-] at the less hindered carbon atom while the absolute configuration at the chiral carbon center at C-2 is retained. As a consequence, the selectivity of the reaction can be accurately described by using the Enantiomeric Ratio 5 (E-value) 6,7. In order to broaden the applicability of this method, we extended our study in two directions: (i) a search for other bacteria possessing highly selective epoxide hydrolase activity, and (ii) the investigation of substrates bearing an additional functional group. Since we knew from our study 8 on Rhodococcus sp. NCIMB 11216, that more polar functional groups are not tolerated by the enzyme 9, we envisaged that an aryl group might serve as a lipophilic masked carboxylate functionality l° which would be tolerated by the enzyme(s). In eukaryotes, epoxide hydrolases play a key role in the metabolism of xenobiotics, in particular of aromatic systems 11,12. On the contrary, the natural substrates for these enzymes in procaryotes (bacteria) are unknown and it can only be speculated that they are involved in the utilization of alkenes as carbon-sources (Scheme 1). Thus, an alkene is epoxidized (by a mono-oxygenase) to yield an epoxide, which can be degraded in two ways: (i) hydrolysis by an epoxide hydrolase yields an innocuous vicinal diol 1, or alternatively, (ii) rearrangement of the oxirane catalyzed by an epoxide isomeraset 3 furnishes an aldehyde or ketone. In presence of CO2, carboxylation leads to the formation of a 13-keto acid 35. As may be deduced from our extensive screening for bacterial epoxide hydrolase activity, epoxide-hydrolysis seems to be more associated within the genus Rhodococcus, Nocardia, Mycobacterium and Arthrobacter, whereas epoxide-isomerization is associated with the Pseudomonas family 14. Results and discussion We initiated our screening along the following considerations. Due to the fact that enzymatic epoxide hydrolysis constitutes not only an important step in the provision of carbon (catabolism) but is also a detoxification reaction required for survival of the cell, the chance of finding highly enantioselective * Corresponding author. Email: faber@orgc.tu-graz.ac.at 65