Computational Study of Engineered Cytochrome P450-Catalyzed C–H Amination: The Origin of the Regio- and Stereoselectivity

Cytochrome P450 enzymes were recently engineered to catalyze the C–H amination reaction of aryl sulfonyl azides with excellent regio- and stereoselectivity (Arnold and co-workers J. Am. Chem. Soc. 2014, 136, 15505). The mechanism of this reaction was studied by quantum mechanical (QM)/molecular mechanical (MM) calculations in this work. The C–H activation is found to be a stepwise process consisting of hydrogen abstraction (H-abstraction) of the reactive C–H bond by an iron nitrenoid cofactor to produce the biradical intermediate and subsequent radical rebinding to form the final product. The rate of rotation of the carbon radical center was estimated to be much faster than that of radical rebinding, which implies that the H-abstraction does not determine the stereoselectivity. For mutant A, the H-abstraction step has a barrier of 16.7 kcal/mol, which is 3.0 kcal/mol higher than that of the following radical rebinding step. The H-abstraction step determines the regioselectivity, but the radical rebinding step determines the stereoselectivity. Barriers of these two steps are 16.1 and 27.5 kcal/mol, respectively, for mutant B. It is different from mutant A in that the radical rebinding step has the higher barrier and determines both the regio- and stereoselectivity. The initial distances between the hydrogens of reactive C–H bonds and the iron nitrenoid were found to not correlate with their reactivities. The calculated barriers are qualitatively consistent with the experimentally observed regio- and stereoselectivity with the exception of the stereoselectivity of mutant B. The lower barriers of mutant A presumably come from the stabilization effect of the H-bond between G265 and the sulfone O. This H-bond does not exist in mutant B. The conformation of the protein backbone, with the exception of the active site, does not change much (RMSD < 0.05) along the reaction pathway