Synthesis of new fluorescent compounds of biological interest: application in the detection of toxic metals in cells of marine and terrestrial biomarkers

With the rapid development of industrialization, economic conditions and social employment, the problem of environmental pollution is growing and concerns about heavy metals (HMs) are rising. HMs selective detection has developed into an essential branch of research in chemistry, biology, environmental, agricultural science and food safety. In this thesis project, four fluorescent probes (FLY, AJ2NBD, AJ2DAN and HNBO-DEN) were evaluated as optical chemosensors for the detection of selected metal ions in both solution and biological environment. While FLY and AJ2NBD were previously synthesized and characterized, AJ2DAN and HNBO-DEN represent two new molecules. All compounds feature a fluorophore moiety (NBD, dansyl and HNBO) bound to a macrocycle or open chain polyamine scaffold. The ability to act as fluorescent sensors was assessed by spectroscopic studies in solution. AJ2DAN revealed as a pH probe in aqueous solution, being OFF at strong acidic pH and ON at pH > 4, based on a PCT fluorescence mechanism, and showing large Stokes shifts. HNBO-DEN revealed an ESIPT-based fluorescence mechanism for the free ligand in both aprotic (ACN and DMSO) and protic polar solvents (water and EtOH-water mixture). The study of the binding properties towards metal ions pointed instead at a different fluorescence mechanism, with the emission growing along with the deprotonation of HNBO group. The selectivity towards metal ions depends on the solvent, shifting from Mg2+, in DMSO and ACN, to Zn2+ and Cd2+, in THF, dioxane, water, methanol and ethanol. HNBO-DEN was also conjugated to NFR 700 NPs (DEN-NPs) and the binding yields were evaluated. Several functional studies of the four fluorescent dyes were performed for biologic applications. HT-29 and Caco-2 cell lines were labelled by AJ2DAN, highlighting absence of dye induced toxicity, indeed Chloroquine application, a known inhibitor of autophagic flux reducing autophagosome-lysosome fusion, causes the rise of autophagic intermediates and leads to the increase of AJ2DAN mean fluorescence intensity (MFI). These data suggest AJ2DAN as a potential probe to detect autophagic intermediates and intracellular vacuoles with not too low pH values. HNBO-DEN shown Cadmium-dependent fluorescence in both HT-29 cells and mussel haemocytes. In the former, the dye yielded better results after a 24-hour Cd(II) exposure, while in haemocytes it detected Cd(II) more effectively after 30 minutes of treatment. This difference seems to reside in the specific characteristics of the cell models to respond to Cd(II) pollution. The DEN-NPs showed similar results. FLY and AJ2NBD were employed in environmental studies, using hepatopancreas of Armadillidium vulgare (a bioindicator and bioaccumulator organism). These dyes can support biologic analyses on polluted samples and may provide the basis for their potential use in environmental diagnostics using flow cytometry.