Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

Abstract In situ quantification of the conjugation efficiency of azide‐terminated synthetic polymers/imaging probes and thiol‐functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self‐sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide‐ and thiol‐functionalized precursors led to highly fluorescent bioconjugates, whereas single‐click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn‐on of C1 , whereas prominent changes in FRET ratios occurred for antibody–imaging‐probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.