SuFEx-Enabled Tyrosine Specific Bioconjugation of Near-Infrared Fluorophores

Abstract Sulfur (VI) fluoride exchange (SuFEx) chemistry has been widely applied in small-molecule medicinal chemistry, but its translation to near-infrared (NIR) fluorophores and application to antibody labeling remain largely unexplored. Here, we report a general synthetic approach for the preparation of aryl sulfonyl fluoride-functionalized indolenines and their incorporation into heptamethine cyanine (Cy7) fluorophores. The strategy is compatible with a broad range of indolenine substrates bearing diverse electronic and solubilizing substituents, enabling the synthesis of a library of water-soluble, SuFEx-enabled Cy7 fluorophores. Using SAT-NIR-746-SF as a lead probe, we demonstrate that the aryl sulfonyl fluoride handle is stable under physiologically relevant conditions (t1/2 > 18 h at pH 7.4) and enables efficient antibody labeling (degree of labeling up to 6.1) under mild aqueous conditions. The degree of labeling can be systematically controlled by varying reaction pH, incubation time, and reagent stoichiometry. The strategy was successfully applied to therapeutic monoclonal antibodies, including trastuzumab, panitumumab, and cetuximab, and the resulting conjugates were stable under acidic, physiological, and serum conditions. Amino acid selectivity assays and tandem mass spectrometry-based proteomics of labeled trastuzumab confirmed modification exclusively at tyrosine residues, establishing the tyrosine-selective nature of this conjugation platform. SAT-NIR-746-SF-conjugated secondary antibodies provided specific labeling of phospho-histone H3 (H3S10P) in confocal immunofluorescence microscopy. These results establish SuFEx-enabled Cy7 fluorophores as a practical platform for tyrosine-selective antibody bioconjugation and demonstrate their utility in fluorescence imaging applications.