Abstract
Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.
Highlights
Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes
Kinetic Deuterium Isotope Effects in NADPH-supported P450 N-Demethylations—The isotope effects were measured with a series of 4-substituted N,N-dimethylanilines, using several approaches (Table I)
Kinetic Deuterium Isotope Effects in horseradish peroxidase (HRP)-catalyzed N-Demethylations—HRP was considered as a model peroxidase because of its known high isotope effects in N-dealkylations [14, 44] and the strong evidence that this hemoprotein catalyzes N-dealkylation reactions by sequential electron transfer (9 –13) but cannot catalyze oxygenation or hydrogen abstraction from unactivated carbon atoms [12, 49, 50]
Summary
P450, cytochrome P450 (EC 1.14.14.1, termed heme-thiolate protein P450 by the Enzyme Commission [4]); PhIO, iodosylbenzene; TPP, 5,10,15,20-tetraphenyl-21H,23H-porphine; HPLC, high performance liquid chromatography; CuOOH, cumene hydroperoxide; GC, gas (liquid) chromatography; D or d, deuterium; E0, oxidation-reduction potential. Reaction IV is a variant of Reactions II and III and has been our own preference (Scheme 1) It incorporates the oxygen labeling results with the known tendency of high-valent states of hemoproteins to accept a single electron from amines and other chemicals of low E0 and has been used to rationalize observed rearrangements [16], radicals trapped from 1,4-dihydropyridine oxidation [17], linear free energy relationships [18, 19], several similarities with the electrochemical oxidation of amines [20], and low kinetic deuterium isotope effects [14, 21]. The basis of mechanistic conclusions in favor of Reaction IV over III [12, 22, 23] has been questioned because of a reported trend in the kinetic deuterium isotope effects of a series of four 4-substituted N,N-dimethylanilines (24 –26). Oxygen surrogates (chemicals that directly activate P450s to high valent forms) [27] have been used in studies on isotope effects and in production of visible aminium radicals with P450
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