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

Abstract

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 equiv 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 2phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine, and hexylamine, and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9Hβ-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its amidation conformation, coupled with modeling and mutational studies, reveal an amine access tunnel and a possible role for residue D201 in amine activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piperidine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobemide, through the reaction of 4-chlorobenzoic acid with 1.5 equiv of 4-(2aminoethyl)morpholine, and utilizing polyphosphate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equiv of ATP, required for recycling of the cofactor.

Biocatalysis: Enzymatic Synthesis for Industrial Applications

“…It was applied to synthesize Moclobemide ( 98 ) from the corresponding acid 96 (4 m m ) and amine 97 (6 m m ) with a facile ATP recycling system (Scheme 34). [146b] 98 was produced at 70 % conversion and 64 % isolated yield. Although these cascades are not efficient yet, enzyme engineering and optimized ATP recycling (Chapter 7.1) may enhance these concepts for environmentally friendly amide syntheses, particularly if the final driving energy is pulled from cheap inorganic polyphosphate.…”

Synthetic Enzyme‐Catalyzed CO2 Fixation Reactions

“…This process however resulted in lower conversion, generating the corresponding amide in up to 15 % (Scheme 13d,e), perhaps owing to the low efficiency of the CAR‐mediated amidation reaction. Recently, amide bond‐forming biocatalysts have attracted significant attention, and more efficient and substrate promiscuous amide bond‐forming enzymes are emerging [120–122] . These amidases may be explored as alternative amidation catalysts in the synthesis of amides by CO 2 fixation.…”

Biocatalytic routes to anti-viral agents and their synthetic intermediates

“…Exemplary is the use of amide bond synthetase McbA for the synthesis of the monoamine oxidase A inhibitor moclobemide. 468 The amidation could even be performed at near stoichiometric amounts of amine. The potential, but also the challenges, of biocatalytic amide bond formation have recently been reviewed by Petchey and Grogan.…”