Metabolism of N‐ methyl‐amide by cytochrome P450s

Abstract

We report unambiguous proof of the stability of a carbinol intermediate in the case of P450 metabolism of an N ‐methylated natural cyclo ‐peptide, namely tentoxin. Under mild acidic or neutral conditions, the lifetime of carbinol‐amide is long enough to be fully characterized. This metabolite has been characterized using specifically labeled 14 C‐methyl tentoxin isotopomers, HPLC, HPLC‐MS, MS‐MS and NMR. Under stronger acidic conditions, the stability of this metabolite vanishes through deformylation. A theoretical mechanistic investigation reveals that the stability is governed by the accessibility of the nitrogen lone pair and its protonation state. For carbinol‐amines, even in neutral conditions, the energy barrier for deformylation is low enough to allow rapid deformylation. Carbinol‐amide behaves differently. Under neutral conditions, delocalization of the nitrogen lone pair increases the energy barrier of deformylation that is a slow process under such conditions. After protonation, we were able to optimize a deformylation transition that is lower in energy and thus accounts for the lower stability of carbinol‐amides observed experimentally in acidic conditions. Finally, by considering the protocol usually used for extraction and analysis of this type of metabolite, carbinol‐amide may thus be frequently ignored in drug metabolism pathways.