Two Subtle Amino Acid Changes in a Transaminase Substantially Enhance or Invert Enantiopreference in Cascade Syntheses

Abstract Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3‐alkyl‐substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de ) in the amination of ( S )‐3‐methylcyclohexanone, the product of the enoate‐reductase‐catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de ) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S ‐configured diastereomer (70 % de ). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1 R ,3 S )‐1‐amino‐3‐methylcyclohexane with 89 % de at 87 % conversion.