Biocatalytic asymmetric Michael addition reaction of l-arginine to fumarate for the green synthesis of N-(([(4S)-4-amino-4-carboxy-butyl]amino)iminomethyl)-l-aspartic acid lithium salt (l-argininosuccinic acid lithium salt)

Abstract

The basic natural amino acid L-argininosuccinate containing two chiral centers occurs in L-alanine, L-arginine, L-aspartate, L-glutamate and L-proline metabolic pathways and plays a role in the biosynthesis of secondary metabolites and other amino acids. It is a precursor for arginine in the urea cycle or the citrulline–NO cycle as well as a precursor to fumarate in the citric acid cycle via argininosuccinate lyase. We aimed to run part of the urea cycle in reverse by catalyzing not the elimination but the addition reaction of L-arginine to fumarate in order to synthesize L-argininosuccinate. Argininosuccinate lyase (ASL) from Saccharomyces cerevisiae has been chosen as the catalyst for this addition reaction. The selected ARG4 gene was synthesized and homogeneously expressed in E. coli leading to a highly active argininosuccinate lyase. The ASL-catalyzed addition reaction of L-arginine to fumarate has been successfully developed at gram scale. After a standard workup procedure the pure final product L-argininosuccinate has been isolated in good yield and high purity.