Lysine-reactive tag system for cell-surface protein labeling via N-acyl-N-alkyl sulfonamide chemistry

Target-selective and site-specific incorporation of a desired functionality into a membrane protein endogenously expressed in cells is a great challenge in chemical biology. Here, we describe a versatile strategy for covalent chemical modification of short peptide tag-fused membrane proteins expressed in cells. This method is based on the recognition-driven reaction of a lysine-containing short histidine tag (KH6 or H6K sequence) and a binuclear nickel (II)-nitrilotriacetic acid (BisNi²⁺-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA). Our reactive peptide tag system enabled the selective modification of cell-surface proteins with affinity tags or fluorescent probes through a simple one-step protocol. In this study, we fused the KH6 or H6K tag to cell-surface proteins such as Neuroligin-1, epidermal growth factor receptor (EGFR), and Neurexin-1β, and achieved selective chemical modification of these proteins in live cells. Furthermore, we demonstrated that our short peptide tag allows efficient labeling not only when introduced at the protein termini but also when incorporated internally.