A photo-enhanced oxidative coupling for site-specific protein Labeling via noncanonical amino acid incorporation

Genetically encoded noncanonical amino acids (ncAAs) enable site-specific installation of chemical functionalities into proteins, expanding the scope of protein engineering and bioconjugation. Here we report a photo-enhanced oxidative coupling strategy that leverages a vinyl sulfide-containing ncAA for selective protein labeling. An ortho-naphthoquinone methide (oNQM) intermediate is generated in situ from a stable precursor under mild oxidative conditions using ferricyanide, and labeling efficiency is markedly enhanced upon 365 nm irradiation. Ethyl vinyl sulfide was identified as a compact, electronically suitable dienophile that can be incorporated into proteins either through lysine modification or via genetic encoding of N6-((2-(vinylthio)ethoxy)carbonyl)-L-lysine (VtK). Under photo-enhanced oxidative conditions, robust labeling was observed at pH 6-7 with minimal background modification of wild-type proteins. Site-specific incorporation of VtK into the outer membrane protein OmpX enabled selective labeling in cell lysates and in live Escherichia coli cells, demonstrating compatibility with complex biological environments. This work establishes a genetically encodable, photo-enhanced oxidative coupling modality that complements existing ncAA-based bioconjugation strategies and expands the protein engineering toolbox.