The asymmetric enzymatic ketone reduction of 4,4-dimethoxytetrahydro-2H-pyran-3-one provided the (R)-r-hydroxyketal, an important chiral precursor for a pharmaceutical intermediate, with high enantioselectivity (>99% ee). An economical process including in situ NADPH-cofactor regeneration using glucose dehydrogenase has been developed to produce the desired material in high yield (96-98%). The two-enzyme process was employed at pilotplant scale to produce 80 kg of (R)-4,4-dimethoxytetrahydro-2Hpyran-3-ol. Critical factors for scale-up were found to be pH control and agitation speed.

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.