Abstract
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.
Title
A Single Bioorthogonal Reaction for Multiplex Cell Surface Protein Labeling
Authors
Yang Huang, Chengyang Wu, Anjing Lu, Jingzhe Wang, Jian Liang, Han Sun, Liqing Yang, Shixiang Duan, Andrey A. Berezin, Chuanliu Wu, Bo Zhang, Yi-Lin Wu*, Yu-Hsuan Tsai*
Journal Information
JACS (2025)
