1. Design, Synthesis And Application Of PH, Mecury Ion Fluorescent Probes2. A Novel Approach For5-Methylcytosine Detection

In this thesis, we have designed two kinds of probes:one is protonation-based pH probes and the other is desulfurization-based mercury (II) probe. And we have also established a novel combined bisulfite-UDG approach for selective5-methylcytosine detection.Intracellular pH plays a key role in many physiological and pathological processes, such as receptor-mediated signal transduction, enzymatic activity, ion transport and homeostasis, inflammation and tumor growth. As minor pH variations may induce cellular dysfunction, quantitatively detecting proton is of great importance in cellular analysis or diagnosis. In the first part, we designed and synthesized a series of pH activable probes (compound1-4), which contain4-nitro-benzo[1,2,5]oxadiazole as fluorophore and piperazine moiety as proton acceptor. Upon protonation, photo-induced electron transfer (PET) processes in these compounds are blocked, then strong fluorescence can be observed. These probes have high sensitivity, good selectivity and photo stability. Through photochemical property assessments and live cell imaging screening, we selected compound1with relatively strong fluorescence and pKa value of5.7as optimal probe. After incubation with Hela cells, compound1exhibited strongly green fluorescence that co-localized with commercial marker LysoTracker Red dyes, which confirmed that the distinctive acidic environment of the lysosome can activate the probe to emit fluorescence. In addition, confocal microscopy and flow cytometry assay show that compound1could visualize intracellular pH change in a rapid and real-time way. Furthermore, as folate receptors on many cancer cells are overexpressed, the folate group of compound1facilitates its binding and internalization into those cancer cells.Mercury, which is a typical contaminant, is enormously toxic to the human body and other organisms, even at low concentrations. They also accumulate through the food chain. Various diseases and biofunctional disorders related to the brain, lung, kidneys and other organs are caused by Mercury (II) contamination, which makes necessary the detection of trace amounts of mercury ions in environmental and biological samples. In the second part, we designed and synthesized a naphthalene derivate (compound6) as turn-on two-photon fluorescent probe for mercury(II) detection. The strong thiophilicity of mercury ion promotes the deprotection of the thioketal group in compound6and finally form highly fluorescent product compound7. During the detection, both of one-photon and two-photon fluorescent emission increased dramatically upon mercury ion addition and showed a linear relationship within the range of26nM-3μM. Compound6exhibits stable and specific fluorescent response toward mercury ion without the interference of pH variation and other competing metal cations. Besides, this probe can avoid cellular auto-fluorescence and detect mercury ion in live cell environments through two-photon confocal microscopy.DNA modification, a significant epigenetic event, largely affects genes’ binding with the transcription factors and some other DNA binding proteins. Among DNA modifications, methylation, especially cytosine methylation is of great importance and attracts extensive studies as it leads to the silence of tumor-suppressor gene expression. In the third part, a novel combined bisulfite uracil-DNA glycosylase (UDG) assay has been developed on the basis of bisulfite modification to generate uracil from cytosine, subsequent UDG-mediated uracil elimination and ultimate DNA cleavage in alkaline condition. This strategy can be used to selectively detect exact number and loci of5-methylcytosine residues regardless of sequence context. Moreover, it provides straightforward and quantitative detection of DNA methylation level across a wide range.