Construction of Coumarin-Based Bioorthogonal Macromolecular Probes for Photoactivation.

Photoactivatable fluorescence imaging is one of the most valuable methods for visualizing protein localization, trafficking, and interactions. Here, we designed four bioorthogonal fluorescent probes - by installing photoactive cages and HaloTag ligands onto the different positions of the coumarin fluorophore. Although - all exhibited rapid photostimulated responses in aqueous solution, only was found to have an obvious aggregation-induced emission (AIE). Next, macromolecular fluorescent probes _ were obtained by covalently attaching - to HaloTag-fused proteins of interest (POIs). _ exhibited a higher fluorescence increase than that of _ upon photoactivation in both liquid and solid phases. Moreover, __ and __ presented the fluorescence resonance energy transfer (FRET) from photocleaved and to GFP in the protein complex. We further examined the fluorescence labeling ability of - to intracellular IRE1_Halo protein and found that and containing the HaloTag ligand on the C4 position of coumarin could be retained in cells for long-term tracking of the IRE1_Halo protein. Hence, we established a platform of novel bioorthogonal fluorescent probes conjugating onto Halo-tagged POIs for rapid photoactivation in vitro and in cells.