title: Asymmetric reduction of alpha, beta-unsaturated ketone to (R) allylic alcohol by Candida chilensis.
abstract: A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.
authors.affiliation: Bioprocess R and D, Merck Research Laboratories, Merck and Co., Inc., Rahway, New Jersey, USA. david_pollard@merck.com
authors.forename: D J
authors.initials: DJ
authors.lastname: Pollard
authors.forename: K
authors.initials: K
authors.lastname: Telari
authors.forename: J
authors.initials: J
authors.lastname: Lane
authors.forename: G
authors.initials: G
authors.lastname: Humphrey
authors.forename: C
authors.initials: C
authors.lastname: McWilliams
authors.forename: S
authors.initials: S
authors.lastname: Nidositko
authors.forename: P
authors.initials: P
authors.lastname: Salmon
authors.forename: J
authors.initials: J
authors.lastname: Moore
mesh_terms: D001711:Biotransformation; D002175:Candida / Q000378:metabolism*; D005285:Fermentation; D019850:Heptanol / Q000031:analogs & derivatives*; D007218:Industrial Microbiology / Q000379:methods; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors; D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010865:Pilot Projects; D020005:Propanols / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D039302:Integrin alphaVbeta3; D007659:Ketones; D020005:Propanols; C006463:allyl alcohol; D019850:Heptanol