Biocatalytic cascade reactions for asymmetric synthesis of aliphatic amino acids in a biphasic reaction system

Abstract

Enantiopure aliphatic amino acids, including l-3-hydroxyadamantylglycine (l-Hag), l-tert-leucine (l-Tle) and l-norvaline, are essential chiral building blocks for a number of pharmaceutical drugs. Here, we developed cascade enzyme reactions in an extractive biphasic system using a branched-chain amino acid transaminase (BCTA) and an (S)-selective ω-transaminase (ω-TA) for asymmetric synthesis of the aliphatic amino acids from achiral α-keto acid precursors. The extractive cascade reactions enabled equilibrium shift of the BCTA reaction by recycling an amino acid cosubstrate as well as acceleration of the ω-TA reaction by removing an inhibitory ketone product from an aqueous phase. Starting with 20mM α-keto acid, 4mM rac-homoalanine and 50mM rac-α-methylbenzylamine (rac-α-MBA), the biphasic cascade reactions afforded synthesis of four unnatural amino acids (i.e., l-Tle, l-Hag, l-norvaline and l-norleucine) and two natural amino acids (i.e., l-valine and l-Leucine) with >92% conversion yield and >99.9% ee. To demonstrate the industrial feasibility of the extractive cascade reaction, preparative-scale synthesis of l-Hag was performed in a reaction mixture consisting of 300mL hexane and 50mL aqueous solution (50mM phosphate buffer, pH 7.0) charged with 50mM keto acid substrate, 5mM l-homoalanine, 120mM rac-α-MBA, 2U/mL BCTA and 16U/mL ω-TA. Conversion yield of l-Hag reached 92% with >99.9% ee at 70h. Product isolation led to 0.32g white solid of l-Hag (62 % isolation yield).