Biocatalytic Radical C(sp³)-N Coupling via Active Site Templating

Area of Science:

• Biocatalysis
• Organic Chemistry
• Enzyme Engineering

Background:

• Stereoselective nucleophilic substitution to form tertiary amines often uses copper-catalyzed radical reactions.
• These methods are limited by competing arene radical alkylation, reducing efficiency and selectivity.

Purpose of the Study:

• To develop a novel, metal-free photoenzymatic mechanism for enantioselective nucleophilic substitution.
• To engineer an enzyme capable of catalyzing C(sp³)-N coupling with high stereo- and chemoselectivity.

Main Methods:

• Protein engineering of a flavin-dependent oxidoreductase through six rounds of optimization.
• Utilizing a photoenzymatic approach for C(sp³)-N bond formation between tertiary alkyl halides and anilines.
• Employing multivariate statistical analysis, density functional theory, and mechanistic experiments to elucidate the reaction pathway.

Main Results:

• A highly engineered enzyme variant achieved good yields for C(sp³)-N coupling.
• Demonstrated high chemoselectivity for N-alkylation over C-alkylation.
• Achieved broad substrate scope with high enantioselectivity in the nucleophilic substitution reactions.

Conclusions:

• A distinct, copper-free photoenzymatic mechanism for enantioconvergent nucleophilic substitution was discovered.
• The engineered enzyme suppresses competing pathways by templating interactions within its active site.
• This work expands enzymatic catalysis for challenging C(sp³)-N bond formation with precise stereochemical control.