Lysosome selective bright orange-red emitting flavonoid (2) was synthesized by attaching a strong donor (NPh2) group into flavonoid skeleton. As a result of efficient intra molecular charge transfer due to the strong donor group, a significant bathochromic shift was observed from the emission of 2b (with a -NPh2 group, λem ≈ 590 nm), in comparison that of 1b (with a -NMe2 group, λem ≈ 519 nm). The role of the substituent effect towards ICT was further studied by low temperature spectral analysis. Fluorescence spectra at low temperature confirmed that large Stokes shift for probe 2 (Δλ ≈ 150 nm) was due to strong ICT. Probe 2b exhibited exceptional selectivity towards cellular lysosomes in live cells studies thus generating bright orange-red emission upon localization. Intra-cellular pH analysis results confirmed that probe 2b did not participate in the elevation of lysosomal pH upon staining with different probe concentrations (0.5 μM - 2.0 μM) which is a potential advantage compared to acidotropic commercial LysoTracker® probes. This study further illustrated that the substituents in probe 2 play a significant role towards probe's organelle selectivity since probe 2a (R = OH) did not show any lysosomal localization compared with 2b. In addition, the calculated cytotoxicity data further revealed that this new probe design is highly biocompatible (LC50 > 50 μM) and suitable for long term imaging. Graphical Abstract.
Keywords: Excited state intra-molecular proton transfer (ESIPT); Flavonoid; Intra-molecular charge transfer (ICT); Lysosome probes; Stokes shift; Substituent effect.