Glucose-Modified Viscosity-Responsive Turn-On Fluorescence Molecular Rotors for Wash-Free Live-Cell Imaging

The overexpression of glucose transporters (GLUTs) in cancer cells represents a promising diagnostic target. While 2-NBDG, a fluorescent glucose analog, enables GLUT-mediated imaging, it suffers from low brightness and requires high concentrations and washing. Herein, we report the development of turn-on fluorescent probes for GLUTs based on the viscosity-sensitive fluorophore in GFP for a wash-free imaging of prostate cancer cell, PC-3. A series of fluorescent molecular rotors (FMRs) were synthesized by modifying the 2-methyl-4-(p-dimethylaminobenzylidene)-5-imidazolinone (DMAB) core. Structural modifications, such as cyclic amine substitution and julolidine incorporation, enhanced fluorescence quantum yields (Φ_F up to 0.17 in glycerol) and viscosity sensitivity (χ up to 0.87). Among them, the julolidine-containing fluorophore Julo-Ph showed the best performance. The DMAB-based fluorophores were conjugated to glucose or glucosamine to create GLUT-targeted probes. A glucosamine-bound conjugate, GlcN-Julo-Ph, was found to enable a wash-free bright fluorescence imaging of PC-3 cancer cells. Inhibition studies and docking simulations strongly suggested a GLUT-mediated uptake of the fluorophore. GlcN-Julo-Ph outperformed 2-NBDG in emitting a brighter intracellular fluorescence at a lower concentration, notably, under wash-free conditions. Our findings shed light on the utility of viscosity-sensitive FMRs for the design of turn-on imaging probes and offer a promising platform for a rapid, low-background cancer cell detection.