D-Amino acids (D-AAs) are increasingly recognized as valuable precursors for the synthesis of fine chemicals, including pharmaceuticals and pesticides. This study reports the construction of an enzyme cascade system specifically designed for the synthesis of D-AAs from their corresponding racemates and L-isomers. Employing this system, we successfully synthesized seven enantiomerically pure D-AAs at a preparative scale.

Abstract

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH₄Cl. Concurrently, any generated H₂O₂ is decomposed into O₂ and H₂O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.