Cytochrome P450s were recently identified as viable systems for catalyzing C-H amination reactions via nitrene transfer, a synthetically valuable transformation for forging new C-N bonds that is not found in nature. Despite this progress, the catalytic efficiency of P450s in these non-native reactions is limited and significantly inferior to that exhibited in their native monooxygenase function. Guided by mechanistic hypotheses concerning non-productive pathways operating during P450-catalyzed C-H amination, rationally designed mutations were introduced to targeting the native proton shuttle pathway and thus disfavour byproduct formation during the C-H amination reaction. This strategy led to the development of engineered P450 variants with dramatically enhanced C-H amination activity (&gt;5,000 turnovers) as well as improved chemoselectivity, corresponding to the most efficient C-H amination biocatalyst reported to date. Kinetic isotope effect experiments revealed that the mechanism proceeds with C-H bond activation as the rate limiting step and provided insights into the competition between  productive versus non-productive nitrene transfer pathways. The substrate scope of these improved C-H amination biocatalysts could be extended to azide-based substrates not efficiently processed by the parent enzyme. These studies demonstrate the value of mechanism-guided rational design for the development of efficient P450 catalysts for non-native group transfer reactions.

Mechanism-guided design of efficient P450 catalysts for C-H amination via nitrene transfer

255th National Meeting (2018)

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