title: Metabolism of N-methyl-amide by cytochrome P450s: formation and characterization of highly stable carbinol-amide intermediate.
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.
authors.affiliation: Université de Toulouse, Toulouse, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Perrin
authors.forename: Nicolas
authors.initials: N
authors.lastname: Loiseau
authors.forename: François
authors.initials: F
authors.lastname: André
authors.forename: Marcel
authors.initials: M
authors.lastname: Delaforge
mesh_terms: D000577:Amides / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D000432:Methanol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D008862:Microsomes, Liver / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry; D013816:Thermodynamics
chemical_list: D000577:Amides; D002250:Carbon Radioisotopes; D010456:Peptides, Cyclic; D003577:Cytochrome P-450 Enzyme System; C003384:tentoxin; D000432:Methanol