Dual-function enzyme catalysis for enantioselective carbon-nitrogen bond formation

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Chemistry

Background:

• Chiral amines are crucial building blocks in pharmaceuticals.
• Traditional synthesis of chiral amines often involves multi-step processes and transition metal catalysts.
• Enzymatic approaches offer a sustainable alternative but face challenges in controlling stereochemistry for carbene insertion reactions.

Purpose of the Study:

• To engineer cytochrome P450 enzymes capable of catalyzing carbene N-H insertion.
• To achieve high activity and enantioselectivity in the synthesis of biologically relevant alpha-amino lactones.
• To elucidate the mechanism of this novel enzymatic transformation.

Main Methods:

• Directed evolution and site-directed mutagenesis of cytochrome P450 enzymes.
• In vitro enzymatic assays to measure catalytic activity and enantioselectivity.
• Computational modeling (e.g., DFT) to investigate reaction mechanisms.

Main Results:

• Engineered P450 enzymes demonstrated high catalytic activity for carbene N-H insertion.
• The biocatalysts produced alpha-amino lactones with excellent enantioselectivity (up to 98% e.e.).
• Total turnovers reached up to 32,100 with >99% yield, indicating high efficiency and stability.
• Computational studies revealed the dual-function nature of the active site, facilitating both carbene transfer and stereoselective protonation.

Conclusions:

• Engineered cytochrome P450 enzymes can act as efficient dual-function catalysts for carbene N-H insertion.
• This work presents a new-to-nature enzymatic route for synthesizing valuable chiral amines.
• The findings pave the way for developing novel biocatalytic tools for asymmetric synthesis.