High-yield enzymatic synthesis of mono– and trifluorinated alanine enantiomers

Abstract

Fluorinated amino acids are a promising entry point for incorporating new-to-Nature chemistries in biological systems. Hence, novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers. To this end, the alanine dehydrogenase fromVibrio proteolyticusand the diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwere applied to thein vitroproduction of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, an alanine racemase fromStreptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in the β-elimination of fluorine from the monosubstituted fluoroalanine. Thein vitroenzymatic cascade based on the dehydrogenases ofV.proteolyticusandS.thermophilumincluded a cofactor recycling system, whereby a formate dehydrogenase fromPseudomonassp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. Moreover, the selected dehydrogenases were also able to catalyze the conversion of trifluoropyruvate into trifluorinated alanine, as a first-case example of biocatalysis with amino acids carrying a trifluoromethyl group.