A Single Bioorthogonal Reaction for Multiplex Cell Surface Protein Labeling

Small-molecule fluorophores are invaluable tools for fluorescence imaging. However, means for their covalent conjugation to the target proteins limit applications in multicolor imaging. Here, we identify 2-[(alkylthio)(aryl)methylene]malononitrile (TAMM) molecules reacting with 1,2-aminothiol at a labeling rate over 10⁴ M^–1 s^–1 through detailed mechanistic investigation. The fast TAMM molecules and mild reaction conditions enable site-specific labeling of surface proteins in not only cell lines but also primary neurons and living mice. The combination of genetic code expansion and sequence-specific proteolytic cleavage enables selective modification of three different cell surface proteins through iterative TAMM condensation. TAMM condensation is also compatible with Cu-catalyzed azide–alkyne cycloaddition and tetrazine ligation for four-color fluorescent labeling, reaching the maximum available colors of conventional confocal microscopes. Thus, bioconjugation chemistry is no longer the limiting factor for multiplex cell surface protein imaging.