Biocatalytic Synthesis of Moclobemide Using the Amide Bond Synthetase McbA Coupled with an ATP Recycling System

The biocatalytic synthesis of amides from carboxylic acids and primary amines in aqueous media can be achieved using the ATP-dependent amide bond synthetase McbA, via an adenylate intermediate, using only 1.5 equivalents of the amine nucleophile. Following earlier studies that characterized the broad carboxylic acid specificity of McbA, we now show that, in addition to the natural amine substrate 2-phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine and hexylamine and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9H-β-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its ami-dation conformation, coupled with modeling and mutational studies, reveal an amide access tunnel and a possible role for residue D201 in amide activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piper-idine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobe-mide, through the reaction of 4-chlorobenzoic acid with 1.5 equivalents of 4-(2-aminoethyl)morpholine, and utilizing polyphos-phate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equivalents of ATP, required for recycling of the cofactor.