Probing the mechanism of the cytochrome P-450 catalysed dealkylation of amides

Abstract

Cytochrome P-450 (P450) metabolises N,N-dialkylamides to their corresponding N-dealkylated products via formation of an N-hydroxyalkyl-N-alkylamide intermediate. The first-step of these oxidative dealkylations, has been studied both by the use of probe substrates able to trap intramolecularly the reactive intermediate, and also by the use of the intermolecular spin trapping agent PBN, together with ESR analysis. The results obtained indicate that oxidative dealkylation of amides occurs via hydrogen atom abstraction from the alkyl carbon atom α- to the amide nitrogen leading to the formation of a carbon-centred radical intermediate. Such an intermediate can subsequently undergo hydroxyl group insertion, from the activated haem-oxygen complex of P450, to give the N-hydroxyalkyl intermediate. No evidence for an iminium ion intermediate was obtained. Dealkylation of chiral substrates reveals little stereoselectivity in the dealkylation reactions. In contrast, by using fixed-conformation probe substrates, a regioselectivity for the oxidation of the Z- group was observed. Comparison of these results with those of a biomimetic system and with semi-empirical M.O. calculations suggests that these reactions are kinetically rather than thermodynamically driven. Finally, the effects of the generation of a carboncentred radical intermediate during amide metabolism on the P450 enzyme system were analysed. Oxidation of N,N-dimethylformamide and N,N-dimethylacetamide results in the inactivation of the metabolising enzyme. PBN, but not ascorbic acid or glutathione, protects against this inactivation. Such inactivation is due to the carbon-centred radical species attacking the prosthetic haem group of the enzyme, resulting in the formation of a modified haem that retains its tetrapyrrolic structure. The modified haem can be synthesised from haemin by reaction with a chemically-generated α-carbon-centred radical. The full characterisation of the structure of the modified haem requires further work, but preliminary studies reveal it to be the result of attack at the vinyl or meso positions of the haem rather than at the pyrrolic nitrogen atoms.