Substrate Profiling of the Cobalt Nitrile Hydratase from Rhodococcus rhodochrous ATCC BAA 870

Abstract

The aromatic substrate profile of the cobalt nitrile hydratase from Rhodococcus rhodochrous ATCC BAA 870 was evaluated against a wide range of nitrile containing compounds (>60). To determine the substrate limits of this enzyme, compounds ranging in size from small (90 Da) to large (325 Da) were evaluated. Larger compounds included those with a bi-aryl axis, prepared by the Suzuki coupling reaction, Morita–Baylis–Hillman adducts, heteroatom-linked diarylpyridines prepared by Buchwald–Hartwig cross-coupling reactions and imidazo[1,2-a]pyridines prepared by the Groebke–Blackburn–Bienaymé multicomponent reaction. The enzyme active site was moderately accommodating, accepting almost all of the small aromatic nitriles, the diarylpyridines and most of the bi-aryl compounds and Morita–Baylis–Hillman products but not the Groebke–Blackburn–Bienaymé products. Nitrile conversion was influenced by steric hindrance around the cyano group, the presence of electron donating groups (e.g., methoxy) on the aromatic ring, and the overall size of the compound.

Synthesis of novel pyridine and pyrimidine derivatives as potential inhibitors of HIV-1 reverse transcriptase using palladium-catalysed C-N cross-coupling and nucleophilic aromatic substitution reactions

“…2,6-Bis(4-chlorophenoxy)nicotinonitrile (3a). 22 Reaction of 2,6-dichloronicotinonitrile (9) (173 mg, 1.0 mmol) with 4-chlorophenol (256 mg, 2.0 mmol) gave product 3a (276 mg, 78%) as a white oil. IR (cm - General procedure for the preparation of compounds 3b and 4b.…”

“…(3b). 22 Reaction of 2,6dichloronicotinonitrile 8 (346 mg, 2.0 mmol) gave 6-(4-chlorophenoxy)nicotinonitrile 9 (257 mg, 87% with respect to the phenol) as a white oil. 1 (3-5 ml) and a magnetic stirrer bar were added to an oven-dried 10 ml round-bottomed flask and purged with nitrogen.…”

“…As a result, compound 3c was obtained indirectly from the nitrile derivative 3b using biocatalysis as a key step for selectively hydrolysing the pyridylcarbonitrile (Scheme 1). Employing methodology developed in our laboratory for the hydrolysis of β-aminonitriles, 21 purified nitrile hydratase enzyme from the bacterial species Rhodococcus rhodochrous ATCC BAA-870 22 was successfully used to selectively hydrolyse the pyridylnitrile group, leaving the benzonitrile group intact. As confirmatory evidence for successful enzymatic conversion and formation of compound 3c, two additional amide protons were observed as a broad singlet which overlapped with the aryl proton doublet signal at 7.49 ppm (J 8.0 ppm, H-3') in the range 7.51-7.45 ppm of the 1 H NMR spectrum.…”

Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases

“…1 Activity relates to total protein content of the cell free extract (CFE). 2 Specific activity calculated on basis of estimated NHase content in CFE. 3 Addition of 1 mM of CoCl 2 at induction.…”

“…Nitrile hydratases (NHase; EC 4.2.1.84) catalyze the hydration of nitriles to the corresponding amides ( Figure 1a) [1]. Due to the broad substrate acceptance of NHases, numerous amides are accessible from their respective nitrile precursors [2]. The first example of an industrial bioconversion process for the manufacturing of the commodity chemical acrylamide involved NHase in a microbial host [3].…”