Bioorthogonally Applicable Fluorogenic Cyanine-Tetrazines for No-Wash Super-Resolution Imaging

The synthesis, fluorogenic characterization, and labeling application of four tetrazine-quenched cyanine probes with emission maxima in the red-far red range is reported. Fluorescence of the cyanine-cores is quenched via through-bond-energy-transfer (TBET) exerted by a bioorthogonal tetrazine unit. Upon bioorthogonal labeling reaction with cyclooctyne tagged proteins, the quenching effect ceases, and thus the fluorescence reinstates, resulting in an increase in fluorescence intensity. As a rare example among indocyanines, one of our new probes was found suitable in STED-based super-resolution imaging. The applicability of this fluorogenic Tet-Cy3 probe was therefore further demonstrated in the bioorthogonal labeling of cytoskeletal protein, actin, with subsequent super-resolution microscopy (STED) imaging even under no-wash conditions.