The Construction And Biosensing Study Of Organic Fluorescent Probes

Fluorescent probes have been recognized as the efficient molecular tools that can help monitor and visualize trace amounts of samples in live cells or tissues because of its high sensitivity and high spatiotemporal resolution. In particular, the exploitation of the reaction-based fluorescent probes have attracted increasing attention and become an active research field in recent years2 because of their higher selectivity with larger spectroscopic changes. In addition, the near-infrared (NIR) fluorescent probes are attractive for biological and clinical applications because of minimum photodamage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence of biomolecules in the living systems. In this thesis, we have designed and synthesized a series of coumarin-hemicyanine, thiopyronin, coumarin-flavylium and carboxylic acid-funtionalized NIR fluorescent probes. These probes have been successfully applied to detect and sense biothiols Cys and GSH, biological gasotransmitter H2S as well as toxic Hg2+ ion in invitro and in cells.1. A chlorinated coumarin-hemicyanine dye (2-1) with three potential reaction sites was exploited as fluorescent probe for biothiols. The Cys-induced substitution-rearrangement-cyclization cascade, Hcy-induced substitution-rearrangement cascade, and GSH-induced substitution-cyclizatioin cascade lead to the corresponding amino-coumarin, amino-coumarin-hemicyanine, thiol-coumarin with distinct photophysical properties, enabling Cys and GSH to be selectively detected from different emission channels at two different excitation wavelengths. We also demonstrated that probe 2-1 is able to simultaneously monitoring Cys and GSH in COS-7 cells in multicolor imaging.2. A 4-methoxythiophenol-substituted pyronin dye 3-1 was exploited as reaction-type fluorescent probe for biothiols Cys/Hcy and GSH. The probe itself is nonfluorescent due to the photoinduced electron transfer (PET) process. The Cys (or Hcy)-induced substitution-rearrangement cascade reaction and GSH-induced substitution reaction with the probe lead to the corresponding aminopyronin and thiopyronin dyes with distinct photophysical properties, enabling Cys/Hcy and GSH to be detected from visible and near-infrared (NIR) emission channels, respectively, in pure PB buffer with relatively fast kinetics and obvious fluorescence turn-on response. Assisted by laser scanning confocal microscope, we also demonstrated that the probe could simultaneously sense Cys/Hcy and GSH in COS-7 cells in multicolor imaging.3. We have developed a novel ratiometric fluorescent probe 4-1 to detect H2S with fast response and high selectivity under physiological conditions based on the novel nucleophilic addition reaction of H2S towards electrically positive benzopyrylium moiety of the probe. Moreover, the strategy also enables the determination of H2S with an attractive ratiometric fluorescence method, by which a huge H2S-induced change in the intensity ratio and a big emission shift could be obtained before and after addition of H2S. The obvious changes in the fluorescence color can be observed by eyes. Preliminary confocal laser scanning microscopy experiments show that the probe has potential for practical application in living cells.4. We have developed a NIR and ratiometric fluorescence platform based on a carboxylic acid-functionalized coumarin-flavylium cye 5-2. The ring-open form of 5-2 shows NIR absorption and emission; however, its ring-close form displays the visible absorption and emission because an intact 7-diethylaminocoumarin fluorophore was involved in the structure. The value of 5-2 as the platform was confirmed by its thiolactone derivative 5-1. Due to the high thiophilicity of Hg2+, the reaction of 5-1 with Hg2+ leads to the fast ring-opening of the corresponding spirocyclic moiety of 5-1 within several seconds, and, simultaneously, elicits excellent ratiometric fluorescent response with high selectivity and sensitivity. The cell imaging experiment reveals that 5-1 is cell permeable, and could be employed for ratiometric fluorescence imaging of Hg2+ in living cells.