Chemoselective restoration of para-azido-phenylalanine at multiple sites in proteins.

The site-specific incorporation of nonstandard amino acids (nsAAs) during translation has expanded the chemistry and function of proteins. The nsAA para -azido-phenylalanine (pAzF) encodes a biorthogonal chemical moiety that facilitates "click" reactions to attach diverse chemical groups for protein functionalization. However, the azide moiety is unstable in physiological conditions and is reduced to para -amino-phenylalanine (pAF). Azide reduction decreases the yield of pAzF residues in proteins to 50%–60% per azide and limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein without introducing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins by restoring azides for multi-site functionalization with diverse chemical moieties, setting the stage for the production of genetically encoded biomaterials with broad applications in biotherapeutics, materials science, and biotechnology. [Display omitted] • In vivo reduction of para -azido-phenylalanine (pAzF) hinders protein functionalization • Reaction with imidazole-1-sulfonyl azide selectively recovers pAzF • Recovery of pAzF in proteins enables robust multi-site functionalization Protein expression with para -azidophenylalanine (pAzF) enables a site-specific modification with synthetic modalities to endow proteins and biopolymers with new functionalities. The azide can be intracellularly reduced to an amine, preventing homogeneous functionalization of these proteins. Here, Arranz-Gibert et al. develop a strategy to selectively restore pAzF in proteins for robust functionalization. [ABSTRACT FROM AUTHOR]