Structure-dependent mitochondria or lysosome-targeting styryl fluorophores bearing remarkable Stokes shift

A series of 9 monocationic styryl dyes was prepared in moderate to good yields via Knoevenagel condensation between various CH active N -quaternary heterocycles and 4-(4-methylpiperazin-1-yl)benzaldehyde. Structural elucidation of the newly synthesized fluorophores was achieved by 1 H NMR, 13 C NMR, 77 Se-NMR and high-resolution mass spectrometry (HRMS) in ESI mode. Novel dyes are characterised by very pronounced Stokes shift (94–203 nm), while fluorescence quantum yields (0.18–2.38 × 10 −2 ) were strongly dependent on the selection of N -quaternary heterocycle. New dyes bind to DNA/RNA by micromolar affinity, yielding strong fluorescence increase. Among the studied series the strongest enhancement was observed for the two quinolinium derivatives and the benzo[ e ]indole analogue. The dye emission response selectivity between ds-DNA and ds-RNA was opposite for para -quinolinium and ortho -quinolinium analogue, stressing the importance of fine tuning of fluorophore by means of electronic properties and positioning within the DNA/RNA binding site. Combined results of several methods support binding of dyes within DNA minor or RNA major groove, with exception of indole and benzo[ e ]indole analogues, which show partial intercalation. Majority of dyes investigated in this work showed negligible cytotoxic activity , with an exception of the benzo[ e ]indole-dye micromolar cytotoxicity. The majority of the studied dyes exhibit localization within mitochondria, with two exceptions. The benzo[ d] [1,3]selenazole-derived dye preferentially localizes in lysosomes, while benzo[ e ]indole-dye is equally distributed between mitochondria and lysosomes at variance to indole-dye highly selective for mitochondria, suggesting different intracellular mechanisms of these two related dyes. Observed bioactivity of benzo[ e ]indole-dye combined with easily monitored localization by strong fluorescence makes this dye potential theranostic agent. • Very pronounced Stokes shift (94–203 nm). • Dye emission response selectivity between ds-DNA and ds-RNA. • DNA minor or RNA major groove binding. • Structure-dependent cytotoxic activity. • Fluorimetrically monitored localization - potential theranostic agent.