ENANTIOSELECTIVE MICROBIAL REDUCTION OF 3,5-DIOXO-6-(BENZYLOXY) HEXANOIC ACID, ETHYL-ESTER

The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction o f 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoa ceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optica l purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l-1 and cells were used at 20% (w/v, wet cells) conce ntrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32-degrees-C. Cell extracts of A. calcoacetic us SC 13876 in the presence of NAD+, glucose, and glucose dehydrogenas e reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydr oxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-o xo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were furth er reduced to 2a by cell extracts. Reaction yield of 92% and optical p urity of 99% were obtained when the reaction was carried out in a 1-1 batch using cell extracts. The substrate was used at 10 g l-1. Product 2a was isolated from the reaction mixture in 72% overall yield. The G C and HPLC area % purity of the isolated product was 99% and the optic al purity was 99.5%. The reductase which converted 1 to 2a was purifie d about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding t o 35,000 daltons.