Bioconjugation is an applied technology that has attracted much attention, but the variation in its methodology is limited. Strategies are often used to form covalent bonds between amino acid residues in protein structures and artificial small molecule compounds. However, among the 20 naturally occurring amino acid residues, only nucleophilic residues such as lysine and cysteine can be modified, and the development of methods targeting tyrosine residues (Tyr) is eagerly demanded as a next-generation bioconjugation.Tyrosine residues (Tyr) are involved in signal transduction through phosphorylation and are abundant at the interfaces of protein-protein interactions. From the viewpoint of elucidating biological phenomena, developing Tyr-selective and efficient chemical modification reactions is desirable. Various approaches have been developed to label Tyrs (tyrosine click, Y-click).1-3
In this study, we investigated various urazole derivatives and oxidation conditions and found that stable urazole radical reagents can be prepared under certain conditions. The urazole radicals rapidly labeled tyrosine residues. This modification is more efficient and Tyr-selective than conventional methods. It was possible to use urazole derivatives with the azide group, and by combining them with the click reaction, arbitrary functions such as biotin and fluorescent groups can be introduced to Tyrs.
It was found that tyrosine residues on the surface of proteins tended to be labeled when several purified proteins were used as substrates. In addition, we comprehensively labeled Tyr on proteins in cell lysate. Tyrs were labeled with cleavable linker-biotin, and trypsin-digested peptide fragments were enriched using avidin beads. We successfully identified more than 4,000 labeling sites by nanoLC-MS/MS analysis. Motif searches of the labeling sites characterized the structures around the reactive Tyrs.