Enzymatic Preparation Of An R-Amino Acid Intermediate For A Gamma-Secretase Inhibitor

(R)-5,5,5-Trifluoronorvaline, an intermediate for a gamma-secretase inhibitor (BMS-708163) under development, was initially prepared from the corresponding keto acid using a commercially available D-amino acid dehydrogenase for reductive amination and glucose dehydrogenase for cofactor recycling. This amino acid could also be prepared using a D-amino acid transaminase with alanine as the amino donor, but the transamination also requires lactate dehydrogenase, NAD, formate, and formate dehydrogenase to remove pyruvate in order to bring the reaction to completion. An effective proprietary D-amino acid dehydrogenase was constructed by modification of the D-diaminopimelic acid dehydrogenase gene from Bacillus sphaericus, and a glucose dehydrogenase gene was cloned from Gluconobacter oxidans. Both genes were expressed in the same strain of Escherichia coli, and the glutamate dehydrogenase gene was inactivated in the expression strain to eliminate background production of the S-amino acid and improve the ee of the product to 100%. The amino acid could be isolated or converted without isolation to a p-chlorophenylsulfonamide carboxamide intermediate needed for the synthetic route to the gamma-secretase inhibitor development candidate.