C–N Bond Forming Radical Rebound Is the Enantioselectivity-Determining Step in P411-Catalyzed Enantioselective C(sp3)–H Amination: A Combined Computational and Experimental Investigation

Engineered metalloenzymes represent promising catalysts for stereoselective C-H functionalization reactions. Recently, P450 enzymes have been evolved to allow for new-to-nature intramolecular C(sp3)-H amination reactions via a nitrene transfer mechanism, giving rise to diamine derivatives with excellent enantiocontrol. To shed light on the origin of enantioselectivity, a combined computational and experimental study was carried out. Hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the activation energies and enantioselectivities of both the hydrogen atom transfer (HAT) and the subsequent C-N bond forming radical rebound steps. Contrary to previously hypothesized enantioinduction mechanisms, our calculations show that the radical rebound step is enantioselectivity-determining, whereas the preceding HAT step is only moderately stereoselective. Furthermore, the selectivity in the initial HAT is ablated by rapid conformational change of the radical intermediate prior to C-N bond formation. This finding is corroborated by our experimental study using a set of enantiomerically pure, monodeuterated substrates. Furthermore, classical and ab initio molecular dynamics simulations were carried out to investigate the conformational flexibility of the carbon-centered radical intermediate. This key radical species undergoes a facile conformational change in the enzyme active site from the pro-(R) to the pro-(S) configuration, whereas the radical rebound is slower due to the spin-state change and ring strain of the cyclization process, thereby allowing stereoablative C-N bond formation. Together, these studies revealed an underappreciated enantioinduction mechanism in biocatalytic C(sp3)-H functionalizations involving radical intermediates, opening up new avenues for the development of other challenging asymmetric C(sp3)-H functionalizations.