Stereoselective synthesis of opine-type secondary amine car☐ylic acids by a new enzyme opine dehydrogenase use of recombinant enzymes

Abstract

The substrate specificity of the recently discovered enzyme, opine dehydrogenase (ODH) from Arthrobacter sp. strain 1C for amino donors in the reaction that forms secondary amines using pyruvate as a fixed amino acceptor is examined. The enzyme was active toward short-chain aliphatic ( S)-amino acids and those substituted with acyloxy, phosphonooxy, and halogen groups. The enzyme was named N-[1-( R)-(car☐yl)ethyl]-( S)-norvaline: NAD + oxidoreductase (L-norvaline forming). Other substrates for the enzyme were 3-aminobutyric acid and ( S)-phenylalaninol. Optically pure opine-type secondary amine car☐ylic acids were synthesized from amino acids and their analogs such as ( S)-methionine, ( S)-isoleucine, ( S)-leucine, ( S)-valine, ( S)-phenylalanine, ( S)-alanine, ( S)-threonine, ( S)-serine, and ( S)-phenylalaninol, and α-keto acids such as glyoxylate, pyruvate, and 2-oxobutyrate using the enzyme, with regeneration of NADH by formate dehydrogenase (FDH) from Moraxella sp. C-1. The absolute configuration of the nascent asymmetric center of the opines was of the ( R) stereochemistry with > 99.9% e.e. One-pot synthesis of N-[1-( R)-(car☐yl)ethyl]-( S)-phenylalanine from phenylpyruvate and pyruvate by using ODH, FDH, and phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus, is also described.