Ascorbate-enabled C-H bond amination catalyzed by myoglobin reconstituted with a trifluoromethyl-substituted Iron porphyrin.

Nitrogen-containing organic molecules are essential structural motifs in bioactive compounds, pharmaceuticals and functional materials. Direct C-H amination via nitrene transfer provides an efficient and atom-economical route for C-N bond formation. However, most hemoprotein-based catalysts require strong reductants such as dithionite to generate the metal-nitrene intermediate. Here, we report myoglobin reconstituted with an iron complex (FePor(CF₃)₂) bearing two trifluoromethyl groups at the pyrrole β-positions of the porphyrin framework. This arrangement promotes intramolecular benzylic C-H bond amination under mild conditions using sodium l-ascorbate as the sole reductant. The FePor(CF₃)₂ cofactor exhibits a positively shifted Fe(III)/Fe(II) redox potential that allows efficient reduction to occur in the protein scaffold and suppresses noncatalytic substrate reduction, leading to improved chemoselectivity for secondary C-H bond amination. The H64A mutant of myoglobin reconstituted with FePor(CF₃)₂ achieved a turnover number (TON) of 133 for tertiary benzylic C-H amination. Kinetic studies revealed that the reaction rate is inversely correlated with the C-H bond dissociation energy, with a smaller negative slope in the bond dissociation energies (BDEs) plot compared to a synthetic cofactor with a negative redox potential. This suggests that there is a mechanistic shift in the rate-determining step from hydrogen atom transfer to nitrene formation. These findings highlight the potential of cofactor redox tuning to control reactivity and selectivity in artificial heme enzymes for abiological C-H bond functionalization.