Muhammad Jbara (@jbaram.bsky.social)

First post @bsky.app Late-Stage Minimal Labeling of Peptides and Proteins for Real-Time Imaging of Cellular Trafficking, published in ACS Central Science. Amazing collaboration with the Marc Vendrell group—congratulations to Ferran, Nithun and all authors! pubs.acs.org/doi/10.1021/acscentsci.4c01249

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Late-Stage Minimal Labeling of Peptides and Proteins for Real-Time Imaging of Cellular Trafficking The cellular uptake routes of peptides and proteins are complex and diverse, often handicapping therapeutic success. Understanding their mechanisms of internalization requires chemical derivatization with approaches that are compatible with wash-free and real-time imaging. In this work, we developed a new late-stage labeling strategy for unprotected peptides and proteins, which retains their biological activity while enabling live-cell imaging of uptake and intracellular trafficking. Benzo-2,1,3-thiadiazoles were selectively incorporated into Cys residues of both linear and cyclic peptides via Pd-mediated arylation with good yields and high purities. The resulting labeled peptides are chemically stable under physiological conditions and display strong fluorogenic character for wash-free imaging studies. We utilized this approach to prepare native-like analogues of cell-penetrating peptides and performed time-course analysis of their internalization routes in live cells by fluorescence lifetime imaging. Furthermore, we applied our strategy to label the chemokine protein mCCL2 and monitor its internalization via receptor-mediated endocytosis in live macrophages. This study provides a straightforward strategy for late-stage fluorogenic labeling of intact peptides and small proteins and direct visualization of dynamic intracellular events. https://pubs.acs.org/doi/10.1021/acscentsci.4c01249