Novel Enzymatic Method for Imine Synthesis via the Oxidation of Primary Amines Using D-Amino Acid Oxidase from Porcine Kidney

Abstract

During studies on the oxidative cyanation reaction catalyzed by a variant of D-amino acid oxidase from porcine kidney (pkDAO) (Y228L/R283G), an unexpected formation of 1-phenyl-N-(1-phenylethylidene)ethanamine (PPEA) was detected. The optimal reaction conditions for the synthesis of PPEA and the reaction mechanism were investigated using the pkDAO variant. The highest PPEA synthesis was obtained in the reaction with 150 mM (R)-MBA at pH 9.0 and at 20 °C. Since PPEA synthesis proceeded by trapping the intermediate 1-phenylethanimine (1-PEI) by 15N-labeled n-hexylamine, which is not a substrate for the pKDAO variant, it was deduced that PPEA would be synthesized by a nucleophilic substitution of 1-PEI by another molecule of (R)-MBA. PPEA was further identified by its conversion to bis(1-phenylethyl)amine (BPEA) through reduction with NaBH4. Thus, a new enzymatic method of imine synthesis by oxidation of primary amine by the variant pkDAO was achieved for the first time.