Artificial Friedel–Crafts Alkylase Design With Genetic Incorporated Unnatural S‐(3‐aminophenyl)‐Cysteine

ABSTRACT The design of artificial enzymes with non‐canonical amino acids (ncAAs) capable of catalyzing non‐natural reactions represents a promising frontier in biocatalysis. However, the synthetic challenges and high cost associated with ncAAs bearing catalytic residues have limited progress in this area. Here, we report the development of artificial Friedel–Crafts alkylases using a one‐pot strategy that seamlessly integrates the biosynthesis of ncAAs with their site‐specific incorporation into proteins via genetic code expansion. This innovative approach enables the functionalization of enzymes with novel catalytic properties tailored for stereoselective Friedel–Crafts alkylation. The artificial Friedel–Crafts alkylase exhibited efficient catalytic activity for the enantioselective Friedel–Crafts alkylation of enals and indoles via iminium activation, yielding a range of chiral indole alcohols with up to 98% enantiomeric excess (e.e.) and 99% yield following directed evolution. By demonstrating the feasibility and advantages of this one‐pot strategy, we aim to establish a versatile platform for the design of artificial enzymes and to pave the way for broader applications in enzyme engineering and synthetic biology with in situ biosynthesized ncAAs.