Late-Stage Minimal\nLabeling of Peptides and Proteins\nfor Real-Time Imaging of Cellular Trafficking

The cellular uptake\nroutes of peptides and proteins are\ncomplex\nand diverse, often handicapping therapeutic success. Understanding\ntheir mechanisms of internalization requires chemical derivatization\nwith approaches that are compatible with wash-free and real-time imaging.\nIn this work, we developed a new late-stage labeling strategy for\nunprotected peptides and proteins, which retains their biological\nactivity while enabling live-cell imaging of uptake and intracellular\ntrafficking. Benzo-2,1,3-thiadiazoles were selectively incorporated\ninto Cys residues of both linear and cyclic peptides via Pd-mediated\narylation with good yields and high purities. The resulting labeled\npeptides are chemically stable under physiological conditions and\ndisplay strong fluorogenic character for wash-free imaging studies.\nWe utilized this approach to prepare native-like analogues of cell-penetrating\npeptides and performed time-course analysis of their internalization\nroutes in live cells by fluorescence lifetime imaging. Furthermore,\nwe applied our strategy to label the chemokine protein mCCL2 and monitor\nits internalization via receptor-mediated endocytosis in live macrophages.\nThis study provides a straightforward strategy for late-stage fluorogenic\nlabeling of intact peptides and small proteins and direct visualization\nof dynamic intracellular events.