Expanding the Genetic Code: Incorporation of Functional Secondary Amines via Stop Codon Suppression

Abstract Enzymes are attractive catalysts for chemical industries, and their use has become a mature alternative to conventional chemical methods. However, biocatalytic approaches are often restricted to metabolic and less complex reactivities, given the limited amount of functional groups present. This drawback can be addressed by incorporating non‐canonical amino acids (ncAAs) harboring new‐to‐nature chemical groups. Inspired by organocatalysis, we report the design, synthesis and characterization of a panel of ncAAs harboring functional secondary amines and their cellular incorporation into different protein scaffolds. D / L ‐pyrrolidine‐ and D / L ‐piperidine‐based ncAAs were successfully site‐specifically incorporated into proteins via stop codon suppression methodology. To demonstrate the utility of these ncAAs, the catalytic performance of the obtained artificial enzymes was investigated in a model Michael addition reaction. The incorporation of pyrrolidine‐ and piperidine‐ based ncAAs significantly expands the available toolbox for protein engineering and chemical biology applications.