The chiral intermediate (S)-1-(2'-bromo-4'-fluoro phenyl)ethanol 2 was prepared by the enantioselective microbial reduction of 2-bromo-4-fluoro acetophenone 1. Organisms from genus Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces, Sphingomonas and Baker's yeast reduced 1 to 2 in >90% yield and 99% enantiomeric excess (ee). In an alternative approach, the enantioselective microbial reductions of methyl, ethyl, and tert-butyl 4-(2'-acetyl-5'-fluorophenyl) butanoates 3, 5, and 7, respectively, were demonstrated using strains of Candida and Pichia. Reaction yields of 40-53% and ee's of 90-99% were obtained for the corresponding (S)-hydroxy esters 4, 6, and 8. The reductase, which catalyzed the enantioselective reduction of ketoesters was purified to homogeneity from cell extracts of Pichia methanolica SC 13825. It was cloned and expressed in Escherichia coli with recombinant cultures used for the enantioselective reduction of keto methyl ester 3 to the corresponding (S)-hydroxy methyl ester 4. On a preparative scale, a reaction yield of 98% and an ee of 99% was obtained. (C) 2004 Elsevier Ltd. All rights reserved.

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.