Design And Synthesis Of Molecular Fluorescent Probes For The Visualization And Imaging Of Small Bio-Molecules

In living systems, large numbers of biological molecules play important roles in the processes of metabolism, such as glucose, amino acid, hydrogen sulfide (H2S) and reactive oxygen species (ROS). Among them, glucose, cysteine and H2S has been proven with strong reductibility, and ROS as the byproducts of respiratory action have oxidizability. Therefore, the levels of biological substances can represent the cell/organism status. On the other hand, fluorescence probes have provided powerful tools to investigate the concentrations of bio-molecules and can be used to observe transformations of various species. Here, we have designed and synthesized two non-luminescent probes to detect ROS and H2S. Upon the addition of analytes, the luminescences of probes were specially turned on through the reactions of probes with analytes. In the present dissertation, the mainly results were briefly summarized as following:(1) A single fluorescent probe was designed and synthesized to achieve the effective discrimination hydroxyl radicals (·OH) and hypochlorous acid (HCIO) in vitro. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethyleneglycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with ·OH and HCIO simultaneously result in cyan and green emissions, respectively.(2) Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multi-parallel assays of several ROS with multiple probes are often limited by the available number of spectrally non-overlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. In this chapter, we use the as-synthesized probe and provide the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway (from intestine to blood to organ/tissue to kidney to excretion) clearly present the profiling of spontaneous ·OH and HCIO in these metabolic organs. In particular, the stress generation of OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.(3) Luminescent chemosensors for H2S have been paid more and more attention due to its close association with our health. However, the current reported probes for H2S detection have still faced with problems, such as low sensitivity/selectivity, poor aqueous-solubility or the interference of background fluorescence. In this chapter, we report an ultrasensitive and time-gated "switch on" probe for the detection of H2S, and achieve its application in the test paper for the visualization of exhaled H2S. The complex probe is synthesized with a luminescent Tb3+center and three ligands of azido (-N3) substituted pyridine-2,6-dicarboxylic acid, endowing the probe high hydrophilicity and relatively fast reaction dynamics with H2S due to three-N3 groups in each molecule. The introduced-N3 group as a strong electron-withdrawing moiety effectively changes the energy level of ligand via intramolecular charge transfer (ICT), and thus breaks the energy transferring from ligand to lanthanide ion, resulting in the quenching of Tb3+luminescence. Upon the addition of H2S, the —N3 group can be reduced into amine group to break the process of ICT, and the luminescence of Tb3+ is recovered with the sensitivity of nanomolar level. With a long lifetime of luminescence of Tb3+center (1.9 ms), the usage of time-gated technique effectively eliminates the background fluorescence by the fluorescence collection delayed for 0.1 ms. Interestingly, the test paper imprinted by the complex probe ink can clearly visualize the trace H2S gas exhaled by mice.