Biosynthesis and genetic encoding of activated nitriles for fast protein conjugation and tunable fluorogenic labeling

Few chemistries are suitable for in-cell protein labeling, and the required reagents are costly. We present an approach for the coupled biosynthesis and genetic encoding of activated nitriles, delivering a facile way to furnish proteins with biocompatible reactive handles suitable for subsequent site-specific modifications both in cell and in vitro. The strategy utilizes the endogenous bacterial cysteine biosynthetic machinery to produce the nitrile-bearing non-canonical amino acids (ncAAs) in situ and then perform genetic encoding through an engineered orthogonal translation system. We demonstrate the utility of our system for rapid site-specific bioconjugation and macrocyclization through the nitrile-aminothiol (NAT) click reaction. In addition, we introduce the aromatic condensation NAT (arcNAT) click reaction as a tool for generating a diverse array of turn-on fluorophores. arcNAT achieves fluorogenic labeling of proteins for live-cell microscopy without requiring washing steps. Our approach provides a uniquely convenient, versatile, and cost-effective platform for the post-translational diversification of proteins.