Enzymatic production of Cilastatin intermediate via highly enantioselective hydrolysis of methyl (±)-2,2-dimethylcyclopropane carboxylate using newly isolated Rhodococcus sp. ECU1013

( S )-(+)-2,2-Dimethylcyclopropane carboxylic acid [( S )-(+)-DMCPA] is a key chiral intermediate for production of Cilastatin, an excellent renal dehydropeptidase-I inhibitor. In this study, a new method for preparation of ( S )-(+)-DMCPA with microbial esterases was investigated. A microbial screening program obtained six esterase-producing isolates that could display relatively high activities and enantioselectivities using racemic ethyl 2,2-dimethylcyclopropane carboxylate (DMCPE) as screening substrate, aiming at forming optically pure ( S )-(+)-DMCPA. Further selection was carried out with substrates having different alcohol moieties, including methyl, ethyl, and 2-chloroethyl esters. Finally, one of these strains, numbered ECU1013, with high enantioselectivity toward the hydrolytic resolution of methyl 2,2-dimethylcyclopropane carboxylate (DMCPM), afforded the ( S )-product in 92 % ee , and was later identified as Rhodococcus sp. According to our research, there were several active esterases to DMCPM in cells of Rhodococcus sp. ECU1013; however, ( S )-preferential esterase was selectively enriched based on the time-dependent profile of esterases biosynthesis, thereby the enantiomeric excess of biotransformation product ( ee p ) was constantly increased, finally maintained at 95 % ( S ). To improve the yield, various organic solvents were employed for better dispersion of the hydrophobic substrate. As a result, (±)-DMCPM of up to 400 mM in the organic phase of isooctane was enantioselectively hydrolyzed into ( S )-(+)-DMCPA, with an isolation yield of 38 % and a further increase of ee p to 99 %.