Process Study on the Enzyme-Catalyzed Preparation of Key Chiral Intermediates for Saxagliptin

Abstract

AbstractSaxagliptin is a therapeutic drug for diabetes. The key synthesis process of the drug involves catalyzing 2-(3-hydroxy-1-adamantyl)-2-oxoacetic acid (A) into (S)-3-hydroxyadamantane glycine (B), during which enzymes phenylalanine dehydrogenase mutant from Thermoactinomyces intermedius (TiPDHm) and formate dehydrogenase (FDH) were most often used for biocatalysis. However, the process was limited due to difficulty in enzyme preparation and a low conversion rate. This study focuses on co-expression of TiPDHm and FDH in recombinant Escherichia coli, cell homogenate clarification, enzyme concentration as well as the optimized conditions of enzyme-catalyzed reaction. Our data showed that the wet weight density of bacteria reached 300 g/L, and the yields of TiPDHm and FDH were 7674.24 and 2042.52 U/L, respectively. The combination of ammonium formate and polyethyleneimine favors the clarification of the bacteria homogenate. The clarified enzyme solution obtained can be concentrated by ultrafiltration and directly used in a reductive amination reaction in a high concentration of keto acid A. The reaction time was only 12 hours and the conversion rate reached 95%. Therefore, this process could provide a reference for enzyme-catalyzed preparation of saxagliptin on an industrial scale.