Conversion of the 2,2,6,6-tetramethylpiperidine moiety to a 2,2-dimethylpyrrolidine by cytochrome P450: evidence for a mechanism involving nitroxide radicals and heme iron.
Earlier we described a novel cytochrome P450 (CYP) catalyzed metabolism of the 2,2,6,6-tetramethylpiperidine (2,2,6,6-TMPi) moiety in human liver microsomes to a ring-contracted 2,2-dimethylpyrrolidine (2,2-DMPy) [Yin, W., et al. (2003) Drug Metab. Dispos. 31, 215-223]. In the current report, evidence is provided for the involvement of 2,2,6,6-TMPi hydroxylamines and their one-electron oxidation products, the nitroxide radicals, as intermediates in this pathway. Nitroxide radicals could be converted to their corresponding 2,2-DMPy metabolites by "inactivated CYP3A4", as well as by a number of other heme proteins and hemin, suggesting that this is a heme-catalyzed process. The conversion of nitroxide radicals to the 2,2-DMPy products by CYP3A4 or hemin was accompanied by the generation of acetone in incubations, providing evidence that the three-carbon unit from 2,2,6,6-TMPi was lost as acetone. With one model 2,2,6,6-TMPi nitroxide radical, evidence for an alternate pathway, which resulted in the formation of an intermediate that incorporated two oxygen atoms from water of the incubation medium before collapsing to the 2,2-DMPy product, was also obtained. To account for both pathways, a mechanism involving interaction of the nitroxide radicals with heme iron (Fe(III)), followed by a homolytic scission of the N-O bond and transfer of the nitroxide oxygen to heme iron to form a perferryl-oxygen complex, is proposed. The nitrogen-centered 2,2,6,6-TMPi radical thus formed then precipitates the contraction of the piperidine ring via C2-C3 bond cleavage, and the resulting product further oxidizes to an exocyclic iminium ion (by the perferryl-oxygen complex); the latter may undergo capture by water from the incubation medium and eliminate the three-carbon unit via N-dealkylation. It remains to be determined whether this novel interaction of nitroxide radicals with heme iron has any relevance in regard to the known biological properties of these stable radical species.
Yin
Wenji
W
Department of Drug Metabolism, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, USA.
Mitra
Kaushik
K
Stearns
Ralph A
RA
Baillie
Thomas A
TA
Kumar
Sanjeev
S
D000332:Aerobiosis; D000693:Anaerobiosis; D002248:Carbon Monoxide / Q000737:chemistry; D003497:Cyclic N-Oxides / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003676:Deferoxamine / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry; D006801:Humans; D007501:Iron / Q000737:chemistry*; D007502:Iron Chelating Agents / Q000737:chemistry; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D009589:Nitrogen Oxides / Q000378:metabolism*; D010103:Oxygen Isotopes; D010881:Piperidones / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014216:Triacetoneamine-N-Oxyl / Q000031:analogs & derivatives* / Q000378:metabolism*
D003497:Cyclic N-Oxides; D005609:Free Radicals; D007502:Iron Chelating Agents; D009589:Nitrogen Oxides; D010103:Oxygen Isotopes; D010881:Piperidones; D011994:Recombinant Proteins; D014216:Triacetoneamine-N-Oxyl; C002273:tempidon; D006418:Heme; D009249:NADP; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006427:Hemin; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C039900:nitroxyl; D003676:Deferoxamine