Benzyne Formation in the Mechanism-Based Inactivation of Cytochrome P450 by 1-Aminobenzotriazole and N-Benzyl-1-Aminobenzotriazole: Computational Insights

Abstract

Cytochrome P450 enzymes (P450s) are ubiquitously distributed heme enzymes that play catalytic roles in the essential oxidative biotransformation of a wide range of exogenous and endogenous organic compounds. Strong inhibition of P450s through mechanism-based inactivation (MBI) essentially should not occur, because it would affect important metabolic processes adversely. However, accumulated evidence shows that the MBI of a P450 is not a rare event. MBI can also be exploited for useful applications such as reaction phenotyping. Thus, MBI is clearly one of the major problems concerning P450s, but the reaction mechanisms underlying MBI are not very clear in many cases. In this work, we used density functional theory (DFT) calculations to understand how a metabolite (benzyne) is formed from two mechanism-based inactivators of P450s: 1-aminobenzotriazole (ABT) and N-benzyl-1-aminobenzotriazole (BBT). ABT has been widely used for reaction phenotyping. Our DFT calculations show that the formation of benzyne from ABT occurs via two sequential H-abstraction reactions from the exocyclic N–H bonds, similar to the reaction of 1,1-dimethylhydrazine (Hirao, H.; Chuanprasit, P.; Cheong, Y. Y.; Wang, X. Chem. Eur. J. 2013, 19, 7361–7369). The transition states for these H-abstractions are stabilized by a proton-coupled electron transfer effect. The formation of benzyne from BBT is also triggered by H-abstraction from the N–H bond. However, in this case, the second step is H-abstraction from a benzylic C–H bond. In addition, for the formation of benzyne from BBT, another catalytic cycle should be necessary. Our computational study therefore elucidates the difference in reaction mechanisms between ABT and BBT, providing new insights into the processes involved in the MBI caused by these compounds.