Substituent-Driven ESIPT in Chromeno-Pyrroloquinoline Probe for Differentiating Normal and Cancer Cells

Despite significant advancements in the structural flexibility and functional diversity of fluorescent molecular sensors, the chromophores often require complex synthetic processes and are typically designed to perform only a specific function. In this work, we have demonstrated the unique features of fluorophores based on a fused coumarin-indole scaffold, which are synthetically available via a one-step reaction. Four fluorophores (ICH, ICEst, ICOMe, and ICNMe₂) with varying substituents were synthesized and characterized. Subsequently, their response towards aggregation, solvent polarity, and viscosity was studied. Probe ICNMe₂ exhibited aggregation-induced emission (AIE), while others displayed aggregation-caused quenching. The viscosity-sensitive nature of these fluorophores was evaluated using the Förster-Hoffman equation. ICNMe₂ displayed the highest sensitivity towards polarity and polarity-independent viscosity. The plausible mechanism involved is intramolecular charge transfer (ICT) in probes ICH, ICEst, and ICOMe, whereas excited state intramolecular proton transfer (ESIPT) coupled ICT in the case of ICNMe₂. Based on the distinct AIE-viscosity responses and large Stokes shift (~175 nm), ICNMe₂ was utilized for distinguishing normal (RAW 264.7) cells and cancer (A549) cells using confocal microscopy. Results demonstrated that ICNMe₂ could effectively extend its photophysical activity in the cellular milieu with an enhanced emission in channel-1 (λ_em=460-530 nm) for A549 compared to RAW 264.7 cells.