Development of Blue-to-Near-Infrared rTCO-Caged Fluorogenic Probes for Click-to-Release Monitoring.

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.