Nitrile Hydration by Thiolate- and Alkoxide-Ligated Co-NHase Analogues. Isolation of Co(III)-Amidate and Co(III)-Iminol Intermediates

Nitrile hydratases (NHases) are thiolate-ligated Fe(III)- or Co(III)- containing enzymes, which convert nitriles to the corresponding amide under mild conditions. Proposed NHase mechanisms involve M(III)-NCR, M(III)-OH, M(III)-iminol, and M(III)-amide intermediates. There have been no reported crystallographically characterized examples of these key intermediates. Spectroscopic and kinetic data support the involvement of a M(III)-NCR intermediate. A H-bonding network facilitates this enzymatic reaction. Herein we describe two biomimetic Co(III)−NHase analogues that hydrate MeCN, and four crystallographically characterized NHase intermediate analogues, [Co^III(S^Me2N₄(tren))(MeCN)]²⁺ (1), [Co^III(S^Me2N₄(tren))(OH)]⁺ (3), [Co^III(S^Me2N₄(tren))(NHC(O)CH₃)]⁺ (2), and [Co^III(O^Me2N₄(tren))(NHC(OH)CH₃)]²⁺ (5). Iminol−bound 5 represents the first example of a Co(III)-iminol compound in any ligand environment. Kinetic parameters (k₁(298 K)= 2.98(5) M⁻¹ s⁻¹, ΔH^‡ = 12.65(3) kcal/mol, ΔS^‡ = −14(7) e.u.) for nitrile hydration by 1 are reported, and the activation energy E_a = 13.2 kcal/mol is compared with that (E_a= 5.5 kcal/mol) of the NHase enzyme. A mechanism involving initial exchange of the bound MeCN for OH− is ruled out by the fact that nitrile exchange from 1 (k_ex(300 K) = 7.3(1) × 10⁻³ s⁻¹) is 2 orders of magnitude slower than nitrile hydration, and that hydroxide bound 3 does not promote nitrile hydration. Reactivity of an analogue that incorporates an alkoxide as a mimic of the highly conserved NHase serine residue shows that this moiety facilitates nitrile hydration under milder conditions. Hydrogen-bonding to the alkoxide stabilizes a Co(III)-iminol intermediate. Comparison of the thiolate versus alkoxide intermediate structures shows that CN bond activation and CO bond formation proceed further along the reaction coordinate when a thiolate is incorporated into the coordination sphere.