Photoactivatable Carborhodol and Carborhodamine Dyes with One Cleavable Group: Synthesis, Spectra, and Fluorescence Nanoscopy Applications

Photoactivatable (PA) dyes with symmetric structures and two caging groups, rhodamines and carbo- and silicon-rhodamines, have been widely applied in super-resolution microscopy of subcellular structures with optical resolution far below the diffraction limit. The presence of two "heavy" caging groups reduces the solubility of a probe and, eventually, makes it less biocompatible. The photocleavage in two steps prolongs the photolysis time required for complete PA; it may cause excessive bleaching and secondary photoreactions. Here, we introduce "monocaged" PA fluorescent dyes based on xanthene cores with two heteroatoms (N, O, or N, N). In contrast to standard approaches, we protected only one heteroatom (either N or O) with a photocleavable (4,5-dimethoxy-2-nitrobenzyl) group and demonstrate that it is sufficient to mask the fluorescence of carbo-rhodol and carbo-rhodamine dyes. The monocaged PA probes have significantly lower molecular masses than their analogs with two bulky caging groups. The probes with a reactive group (COOH) provide facile labeling and undergo irreversible single-step photoactivation toward products emitting yellow or orange light. For carborhodol with a free hydroxyl and the protected <i>N</i>-methyl group, the reversible increase in emission was found at pH > 7 (as an activation tool, orthogonal to photolysis). Carboxamides incorporating the HaloTag amine (O2) ligand were applied for targeting and imaging of the HaloTag self-labeling enzyme fused with a protein of interest (vimentin). The utility and imaging performance of the probes with two heteroatoms belonging to a fluorophore, but only one caging group, were demonstrated in live cell labeling, conventional (confocal) microscopy, Minimal Photon Fluxes (MINFLUX) nanoscopy, and single-molecule localization methods (SMLM).