Computational Mechanistic Investigation of Biocatalytic C(sp³)–H Insertions with Monosubstituted Carbenes via Engineered Heme Proteins

A recent experimental study by Arnold's research group (<i>Nature</i> <b>2019</b>, <i>565</i>, 67-72) showed the engineered cytochrome P450 enzyme (cytochrome P411), with acceptor-only carbenes, offers a new sustainable alternative to metal catalysts that have traditionally dominated C-H functionalization. However, there is still no detailed mechanistic understanding thus far. In this study, a series of DFT calculations were performed to uncover the basic reaction mechanism as well as insights into the observed carbene substituent effect behind this novel reaction. Computational results showed that the reaction follows the radical hydrogen atom transfer (HAT) mechanism, which is consistent with experimental work. The electron-withdrawing carbene substituent together with the negatively charged axial ligand plays an important role in steering the reaction toward this mechanism. The more favorable experimental reactivity with the carbene derived from ethyl diazoacetate than diazoacetone was also reproduced. It was found to originate from the additional hydrogen bonds with the carboxylate substituent of the carbene, which further stabilizes the transition state toward a more facile HAT reaction. This quantum chemical study provides useful molecular-level insights that help shed light on the future design of the biocatalysts to contribute to the development of efficient, sustainable C-H functionalization approaches.