We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths...

Donor-acceptor Stenhouse adducts (DASAs) are reversibly photoswitchable dyes, which are able to interconvert between a red/NIR absorbing triene-like state and a colorless cyclic state. Although optically attractive for multiple applications, their low solubility and lack of photoswitching in water impede their use in aqueous environments. We developed water-soluble DASAs based on indoline as donor and methyl, or trifluoromethyl, pyrazolone-based acceptors. In acetonitrile, photophysical analysis and photochemical studies, accounted with a three-state kinetic model, con-firmed the reversible photoswitching mechanism previously proposed. In water, the colorless cyclic state is a thermodynamic sink at neutral pH values. In contrast, in acidic conditions, we observed a fast scrambling of DASAs' endgroup resulting in the in situ formation of Stenhouse salts (StS), which are in turn capable of reversible photoswitching. We believe that this unexpected result is of interest not only for the future design of DASAs with improved stability, but also for further development and applications of StS as photoswitchable probes.

We report on new fluorogenic HaloTag probes based on a molecular rotor design. Thanks to their viscosity-sensitive emission, the probes light-up upon reaction with the protein self-labeling tag HaloTag. The palette of probes cover an emission range from green to red and exhibit remarkably low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast in live Hela cells.

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range. The probes were studied in glycerol and in presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast.