Enzyme-controlled nitrogen-atom transfer enables regiodivergent C-H amination

Area of Science:

• Biocatalysis
• Organic Chemistry
• Enzyme Engineering

Background:

• Cytochrome P450BM3 (CYP102A1) variants catalyze C-H amination, forming new C-N bonds.
• Achieving catalyst-controlled regioselectivity in C-H amination remains a significant challenge.

Purpose of the Study:

• To engineer P450BM3 variants with divergent regioselectivity for C-H amination.
• To investigate the mechanism of catalyst-controlled regioselectivity in C-H amination.

Main Methods:

• Protein engineering of P450BM3 variants.
• C-H amination assays.
• Kinetic isotope effect measurements.
• X-ray crystallography (2.66 Å resolution).

Main Results:

• Two engineered P450BM3 variants demonstrated divergent regioselectivity.
• One variant favored benzylic C-H amination, the other homo-benzylic C-H amination.
• Nearly identical kinetic isotope effect values (2.8-3.0) suggest rate-limiting C-H abstraction.
• Crystal structure indicates engineered active site preorganizes substrate for reactivity.

Conclusions:

• Engineered P450BM3 variants can control regioselectivity in C-H amination.
• Enzyme active site engineering is a viable strategy for achieving selective C-H functionalization.
• Regioselectivity is likely controlled by precise localization of a C-H bond near the iron nitrenoid.