Korzekwa K R, Swinney D C, Trager W F
Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
Biochemistry. 1989 Nov 14;28(23):9019-27. doi: 10.1021/bi00449a010.
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.
对一系列选择性氘代氯苯进行细胞色素P - 450催化羟基化反应，测定了非竞争性和竞争性分子间氘同位素效应。通过两种完全独立的方法（电化学 - 液相色谱法和气相色谱 - 质谱法）测定，氯苯 - 2H5间位羟基化反应伴随有1.27的同位素效应。与氯苯 - 3,5 - 2H2和 - 2,4,6 - 2H3间位羟基化反应相关的所有同位素效应约为1.1。相比之下，对氯苯 - 4 - 2H1、 - 3,5 - 2H2和 - 2,4,6 - 2H3邻位和对位羟基化反应的竞争性同位素研究表明，当在氧化位点取代氘时，会产生显著的反同位素效应（约0.95），而对于质子化位点的氧化未观察到同位素效应。这些结果排除了初始环氧化物形成和初始电子提取（电荷转移）作为细胞色素P - 450催化氯苯羟基化反应的可行机制。然而，这些结果可以用一种机制来解释，即一个活性的类三线态氧原子以类似于烯烃氧化的方式加到π体系上。生成的四面体中间体然后可以直接重排为苯酚，或通过环氧化物或酮中间体重排。
