Development of Blue‐to‐Near‐Infrared rTCO–Caged Fluorogenic Probes for Click‐to‐Release Monitoring

ABSTRACT Bioorthogonal click‐to‐release reactions provide a powerful strategy for chemically controlled activation of functional molecules in biological systems. Here, we report the development of a series of 12 trans ‐cyclooctene‐caged fluorogenic probes, including 11 new compounds, covering the blue‐to‐near‐infrared spectral region. Using different probe designs, including direct caging, self‐immolative linkers, and covalent assembly, we evaluated their fluorescence turn‐on, release kinetics, and performance in live cells upon activation with a sulfonated tetrazine trigger. The best‐performing probes exhibited a 44‐fold fluorescence turn‐on and a second‐order rate constant of 1027 M − 1 s − 1 , while selected probes produced detectable intracellular fluorescence within 2 min and reached signal saturation within approximately 10 min. The results reveal important differences in probe behavior between solution and cells, highlighting the need to evaluate fluorogenic click‐to‐release probes in their intended biological context. This study expands the available toolbox for monitoring tetrazine‐triggered bioorthogonal release reactions and identifies promising candidates for cellular applications.