Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation.
Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.
Korzekwa
K R
KR
Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
Swinney
D C
DC
Trager
W F
WF
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002722:Chlorobenzenes; D002849:Chromatography, Gas; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D004852:Epoxy Compounds / Q000493:pharmacokinetics; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D015394:Molecular Structure; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013696:Temperature
D002722:Chlorobenzenes; D004852:Epoxy Compounds; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium