Solvent-Tunable Rotational Barrier-Based Polarity-Responsive Probe: From Molecular Design to Cellular Autophagy Applications

Fluorescent probes are extensively employed in biological imaging to monitor the physicochemical properties of cellular microenvironments. However, when organelles such as lysosomes experience simultaneous changes in polarity and viscosity, conventional probe designs often fail to accurately interpret microenvironmental alterations due to complex interactions in the fluorescence signals. To address this challenge, we have developed an innovative solvent-tunable rotational barrier (STRB)-based fluorescent probe, XZTU-Pola, which efficiently targets lysosomes through a morpholine group (Pearson coefficient = 0.94) and incorporates an acetylene-bridged phenylmorpholine and ADM structure for sensitive polarity response. Experimental results show that XZTU-Pola exhibits nearly identical absorption wavelengths in both methanol and tetrahydrofuran, indicating conformational changes upon excitation in these solvents. In fluorescence spectroscopy, XZTU-Pola is almost non-emissive in methanol, while it displays significant fluorescence emission in tetrahydrofuran (approximately 160-fold increase in fluorescence intensity), exhibiting an inverse correlation with solvent polarity. Theoretical calculations reveal that solvent polarity modulates the rotational barrier of XZTU-Pola, with a rotational barrier of 2.99 eV in non-polar solvents (local-excited state, f=0.466), which decreases to 2.08 eV in polar solvents (twisted intramolecular charge transfer state, f=0.0047), leading to substantial changes in fluorescence intensity. Further application in biological systems demonstrated that XZTU-Pola precisely monitors dynamic changes in lysosomal polarity during autophagy, exhibiting remarkable resistance to interference. These findings underscore the broad potential of XZTU-Pola for cellular polarity imaging, offering a novel technological approach and theoretical foundation for the study of related diseases.