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

Abstract Saxagliptin 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 ( Ti PDHm) 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 Ti PDHm 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 Ti PDHm 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.