US EPA

1104666 

Journal Article 

Metabolism of N-methyl-amide by cytochrome P450s: formation and characterization of highly stable carbinol-amide intermediate 

Perrin, L; Loiseau, N; André, F; Delaforge, M 

2011 

1 

FEBS Journal
ISSN: 1742-464X
EISSN: 1742-4658 

278 

12 

2167-2178 

English 

21518254 

10.1111/j.1742-4658.2011.08133.x 

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.