McLoughlin, B. H.; Williams, C.; McKenna, S. M.; Besermenji, K.; Drienovska, I.; Westerveld, M.; Rabadan Gonzalez, I.; Calvert, S. H.; McGouran, J. F.; Scanlan, E. M.; Nolan, M. D.; Petracca, R.

Fluorescent labelling of biomolecules enables the visualization and tracking of molecular processes in cells, providing insights into cellular function and disease mechanisms. Despite recent advances in bioorthogonal labelling, efficient and site-selective strategies for targeting alkene functionalities remain limited. Here, we report AbzSH, a compact aminobenzamide-based fluorescent thiol probe for selective alkene labelling through thiol-ene ligation. Photoinduced ligation proceeds under mild, radical-mediated conditions in aqueous media and exhibits broad substrate compatibility, including carbohydrate, amino acid, and steroid scaffolds. The method is readily extended to protein labelling, enabling selective modification of both chemically functionalized proteins and engineered proteins containing genetically encoded alkene motifs. Efficient and chemoselective labelling was further demonstrated in complex cell lysates, highlighting the robustness of the approach in biologically relevant environments. Collectively, these results establish AbzSH as a versatile fluorescent probe for alkene-directed biomolecular labelling and provide a broadly applicable platform for chemical biology, molecular imaging, proteomics, and drug discovery.