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Abstract

Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme which catalyses the conversion of nitriles to amides. The high efficiency and broad substrate range of NHase have led to the successful application of this enzyme as a biocatalyst in the industrial syntheses of acrylamide and nicotinamide and in the bioremediation of nitrile waste. Crystal structures of both cobalt(III)- and iron(III)-dependent NHases reveal an unusual metal binding motif made up from six sequential amino acids and comprising two amide nitrogens from the peptide backbone and three cysteine-derived sulfur ligands, each at a different oxidation state (thiolate, sulfenate and sulfinate). Based on the active site geometry revealed by these crystal structures, we have designed a series of small-molecule ligands which integrate essential features of the NHase metal binding motif into a readily accessible peptide environment. We report the synthesis of ligands based on a pyridine-2,6-dicarboxylic acid scaffold and L-cysteine, L-S-methylcysteine, L-methionine or L-penicillamine. These ligands have been combined with cobalt(III) and iron(III) and tested as catalysts for biomimetic nitrile hydration. The highest levels of activity are observed with the L-penicillamine ligand which, in combination with cobalt(III), converts acetonitrile to acetamide at 1.25 turnovers and benzonitrile to benzamide at 1.20 turnovers.

2.2. Nitrile Hydration Experiments

Ligand 4 returned the best turnover results found with these systems, when combined with Na3[Co(NO2)6] and stirred for 12 h at 25 °C and pH 9 ( shows the reaction outcome, shows a representative chromatogram for this level of turnover). The geminal dimethyl unit adjacent to the thiolate enhances the sub-stoichiometric turnover seen with 1 to a gently catalytic level of 1.25 turnovers. The addition of peroxide diminishes catalytic activity with this ligand (down to 0.7 turnovers), while increasing reaction temperature to 50 °C also lowers activity to 0.7 turnovers.

2.2. Nitrile Hydration Experiments

To investigate the scope of this ligand, a further set of experiments was carried out using a second substrate, the aromatic nitrile benzonitrile 19. Under these conditions the combination of ligand 4 and Na3[Co(NO2)6 also turned over benzonitrile 19 as a substrate, with a similar level of conversion to benzamide 20 (1.2 turnovers). (To aid the miscibility of the benzonitrile in water, a small amount of DMF was added to these reactions: 200 µL in a total reaction volume of 7.2 mL).