Directed Evolution of an (<i>R</i>)‐Selective Transaminase Toward Higher Efficiency of Sitagliptin Analog Biosynthesis

Transaminase (TA)-catalyzed asymmetric amination is considered as a green chemistry approach to synthesize pharmaceutical analogs, but their ability to accept substrate for catalyzing sterically hindered ketones remains a challenge. Sitagliptin is an antihyperglycemic drug to treat type II diabetes. Herein, we exploited an efficient (R)-selective TA to biosynthesize sitagliptin analog (R)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)butan-1-one. Starting from a previously constructed (R)-ATA5, two rounds of directed evolution were performed through combining error-prone PCR, site-directed saturation and combinatorial mutagenesis. The resultant variant ATA5/F189H/S236T/M121H showed a 10.2-fold higher activity and a 4-fold improved half-life at 45°C. Crucially, the variant was able to either catalyze the amination of 700 mM substrate with a conversion up to 93.1% and product e.e.> 99% in a cosolvent reaction system, or biotransform 200 mM substrate with a conversion of 97.6% and product e.e.> 99% in a cosolvent-free system. Furthermore, the structural analysis gave insight into how the mutations affected enzymatic activity and thermostability. This study, which consists of constructing a robust (R)-selective TA and the new synthesis route with the highest conversion ever reported, provides a reference for industrial manufacturing sitagliptin analog.