Harnessing Cyanine-like Properties to Develop Bright Fluorogenic Probes Based on Viscosity-Sensitive Molecular Rotors

Dipolar fluorescent molecular rotors (FMRs) are environmentally-sensitive fluorophores that can be used in bioimaging applications to sense local viscosity and polarity. Their sensitivity to viscosity can also be used for the fluorogenic labeling of biomolecules such as DNA or proteins. In particular, we have previously used FMRs to develop a series of tunable fluorogens targeting the self-labeling protein tag Halotag for wash-free protein imaging in live cells. Despite these very useful properties, FMRs typically display moderate molar absorption coefficients that limits their overall fluorescence brightness. Herein, we synthesized a series of three model hemicyanines based on a styrylindolenium scaffold and performed a detailed study of their photophysical properties in solvents with various polarity and viscosity. We show that with a strong julolidine electron-donating group it is possible to combine intense cyanine-like absorption with the high sensitivity to viscosity of FMRs. We use this property to develop a lysosomal pH sensor and two bright cell-impermeant fluorogens targeting HaloTag for imaging membrane proteins. We believe that this bright fluorogenic scaffold based on a simple chemical structure can be used in the future to build up a variety of probes and sensors with efficient photophysical properties.