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

Mark R Petchey,Gideon Grogan,Ian J S Fairlamb,Richard C Lloyd,Benjamin Rowlinson

doi:10.1021/acscatal.0c00929

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 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 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-(2-aminoethyl)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.

Highlights

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
Similar chemistry is observed in nonribosomal peptide synthases (NRPSs),[11] which first catalyze the adenylation of a carboxylic acid, followed by the formation of thioester linkage to a phosphopantetheine on an acyl carrier protein, after which attack of an amine nucleophile catalyzed by a ligase domain forms the amide bond
Letter formed by a carboxylic acid reductase (CAR) adenylation domain could be recruited for the milligram-scale synthesis of the anticonvulsant compound ilepcimide, when presented with a large excess of the amine precursor, piperidine.[18]

Summary

■ ACKNOWLEDGMENTS

Jennifer Andexer of the University of Freiburg for the gift of plasmids containing genes encoding the polyphosphate kinases AjPPK2-II and SmPPK2-I. ■ ABBREVIATIONS (20) Ji, C.; Chen, Q.; Li, Q.; Huang, H.; Song, Y.; Ma, J.; Ju, J. Chemoenzymatic Synthesis of -Carboline Derivatives Using McbA, a ATP, adenosine triphosphate; NRPS, nonribosomal peptide. 2014, 55, synthase; ABS, amide bond synthetase

■ REFERENCES

Findings

Recent Advances in Engineering Nonribosomal Peptide Assembly