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

Enantiopure aliphatic amino acids, including 1-3-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 omega-transaminase (omega-TA) for asymmetric synthesis of the aliphatic amino acids from achiral alpha-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 omega-TA reaction by removing an inhibitory ketone product from an aqueous phase. Starting with 20 mM alpha-keto acid, 4 mM rac-homoalanine and 50 mM rac-alpha-methylbenzylamine (rac-alpha-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 300 mL hexane and 50 mL aqueous solution (50 mM phosphate buffer, pH 7.0) charged with 50 mM keto acid substrate, 5 mM L-homoalanine, 120 mM rac-alpha-MBA, 2 U/mL BCTA and 16 U/mL omega-TA. Conversion yield of L-Hag reached 92% with >99.9% ee at 70 h. Product isolation led to 0.32 g white solid of L-Hag (62 % isolation yield). (C) 2015 Elsevier B.V. All rights reserved.