Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labeling in live cells

Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamages, while allowing to image about 10 times deeper than with a confocal. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. Among our series of 6 new probes, our best candidate possesses a turn-on ratio of 22 and an impressive kinetic rate constant of 1.1x103M-1s-1, about 10 times higher than that reported for the fluorogenic iEDDA reaction with bicyclo[6.1.0]no-4-yn-9yl)methanol (BCN). Live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe with high-contrast images. The high two-photon brightness of the clicked adduct (362 GM) allows the use of a weak laser power compatible with in vivo imaging.