Design And Synthesis Of Molecular Fluorescent Probes For Bioimaging

Thiols, which are components of many proteins and simple molecules, play animportant role in the cellular antioxidant defense system. A wide range of intracellular andextracellular events, including redox, methyl transfer, carbon dioxide fixation, and CoAparticipating reactions, are considered to be relative to cellular thiols. Intracellular pHplays vital roles in physiological and pathological processes including receptor-mediatedsignal transduction, enzymatic activity, cell growth and apoptosis, ion transport andhomeostasis, calcium regulation, endocytosis, chemotaxis, cell adhesion and tumor growth.Endogenous H2S in living systems, recently recognized as a gaseous transmitter, exhibitsbenefciary effects in a number of pathophysiological conditions, e.g. vasodilation,antioxidant and anti-infammation. SO2is a toxic gas present in polluted air and isdetrimental to many organs, but it can be generated endogenously in the cardiovascularsystem in vivo. There is evidence that gaseous SO2can serve as a gasotransmitter in bloodpressure regulation and vascular remodeling. The roles of these analytes in variousphysiological events have recently spurred signifcant efforts in the development offluorescent probes for quantitative determination of thiols, pH value, H2S and SO2inbiological settings, which is important in biochemistry and clinical medicine.This thesis includes six parts, and the contents are listed as follows:1. Probe1was reported for the quick detection of biothiol with143nm and109nmlarge blue-shifts in absorption and emission spectra, respectively. The probe can quicklydetect biothiols in a wide pH range with high selectivity and sensitivity over other aminoacids. The detection mechanism, which was induced by the addition of mercaptan towardthe activated double bond in the probe, was verifed using1H NMR and mass spectrometryanalysis. In addition, fuorescence imaging of intracellular thiols with the water-solubleprobe was also examined using KB cells.2. A novel fluorescent turn-on red-emitting chemosensor2has been successfullydesigned and synthesized by incorporating asquaraine derivative with a 2,4-dinitrobenzenesulfonyl group. Its optical properties towards cysteine and homocysteinecan be achieved in acetonitrile-PBS (1:9, v/v) solution with an emission enhancement of33and22-fold, respectively, and its properties of high selectivity, high sensitivity and rapidresponse (within20minutes), make it a potential probe for real applications. Furthermore,the in vitro assays show the probe can easily penetrate cell membranes to performfluorescence imaging detection for thiol species in living cells.3. Three D-π-A fluorescent pH probes have been synthesized by linking the coumarinfluorophore to either benzothiazole or indole. Based on an intramolecular charge transfer(ICT) mechanism, the sensors exhibited significant dual-emission changes in differentspectral windows upon pH changes. Probes with benzothiazole (3) and indole (4,5) unitshave opposite responses to changes in pH, in terms of shifts in absorption and emission.Probe5is a pH-responsive, NIR ratiometric fluorescence imaging chemosensor anddisplayed a signifcant122nm red shift of the emission maximum under acid conditions.The excellent cell permeability, sensitivity, specificity, photostability, reversibility and fastresponse speed of probe5make it an effective intracellular pH-imaging agent forquantitative fuorescence imaging of acidic microenvironments in KB and HeLa cells. Thisstudy reveals that the conjugation of coumarin with benzothiazole or indole to form an ICTplatform is an effective method to construct new NIR fuorescent pH probes for acidicmicroenvironments.4. A one-step process has been developed for the synthesis of a rosamine derivative(probe6) from m-diethylaminophenol, trimethyl orthoformate and PPTS. This compoundbehaved as an OFF-ON pH probe (pKa=4.73) which exhibited a400-fold enhacement inthe fluorescent signal as well as being highly selective, rapidly responsive, temperatureindependent, cell permeable, photostable and reversible. The pH probe could therefore beused as a red-emitting lysosome-specific probe for in cellulo imaging.5. Three new fluorescent probes,7,8and9, for the detection of H2S based on thebenzo[a]phenoxazine scaffold have been reported. Especially, by introducing adinitrophenyl ether group into the3-position of9-(diethylamino)-5H-benzo[a]phenoxazin-5-one, probe7was developed as a unique NIR fuorescence turn-on H2S probe. Afteraddition of5equiv. H2S to the solution of probe7, the fluorescence intensity of theemission band centered at666nm increased significantly (18-fold). The formation ofcompound8was confirmed by MS and1H NMR analysis. Probe7can efficiently differentiate H2S from biothiols and other biologically related species based on theselective thiolysis of dinitrophenyl ether ofthe benzopyrylium moiety. Furthermore, in vitroassays demonstrate that probe7is suitable for fuorescent turn-on imaging in living HeLacells, while probe8displays opposite responses to changes of H2S concentration.6. Based on benzo[a]phenoxazin and benzo[a]xanthenium platforms, we havepresented three novel water-soluble probes10-12. Probe10and11could be used ascolorimetric and fluorescence turn-on sensors for SO2derivatives SO32-and HSO3-in PBSbuffer due to the nucleophilic attack of SO32-or HSO3-to the5-position of the moieties.While upon addition of SO32, the strong fuorescence of probe12was severely quenchedand the color of the solution changed from pink to colorless due to the interruption ofπ-conjugated system. The probes displays advantages such as relatively fast reaction time,high reactivity physiological pH range, good specificity and sensitivity. Preliminarybiological experiments indicate their potential to detect SO2derivatives in living cells withexcellent cell permeability and low cytotoxicity. Fluorescent Probes based on this detectionmechanism have been the first to report the unique detection characteris in this field.