Fluorescent thiol labeling is a powerful technique for investigating peptide and protein functions, interactions, and cellular localization, offering minimal structural disruption due to the low natural abundance of cysteine residues. The high nucleophilicity of cysteine thiol groups further facilitates efficient and selective labeling. However, existing thiol-labeling strategies, most commonly involving iodoacetamides, maleimides, or methanethiosulfonates (MTS), often suffer from limited selectivity, undesirable side reactions, and the instability of the resulting conjugates. To overcome these limitations, we developed three spectrally orthogonal MTS-derived fluorescent labeling agents based on coumarin and BODIPY photoremovable protecting groups and a BODIPY thiosulfate water-soluble analogue. Using conventional MTS chemistry, these agents form disulfide-linked conjugates with thiols, which are subsequently converted into stable thioethers upon light-induced sulfur extrusion. This two-step approach significantly improves the stability of labeled conjugates, providing a robust and permanent method for fluorescent labeling of small molecules, peptides, and proteins. Our findings offer a promising strategy for precise fluorescent labeling in biological applications.

Department of Inorganic Chemistry Faculty of Science Charles University Hlavova 2030 Prague 2 128 40 Czech Republic

Department of Organic Chemistry Faculty of Science Charles University Hlavova 2030 Prague 2 128 40 Czech Republic

Department of Organic Chemistry University of Chemistry and Technology Prague Technická 5 Prague 6 166 28 Czech Republic

Institute of Organic Chemistry and Biochemistry of the CAS Flemingovo náměstí 542 Prague 160 00 Czech Republic

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