Design,Synthesis And Application Of Rhodamine Derivative-based Fluorescent Probes

As one of the most important reactive oxygen spexies, hypochlorous acid was generated from H2O2 and Cl- by catalysis of enzyme myeloperoxidase (MPO) in living organisms. It plays an essential role in many biological processes, and is natural barrier for biological against bacterial invasion. However, uncontrolled generarion of hypochlous acid may be closely associated with some diseases. So far, the specific role of hypochlorous acid in theses diseases is not quite clear owing to the limitation of detection technology. Therefore, in order to clearly understand the role of HOCl in physiological activity, prevent and treat various diseases, the development of highly sensitive, real-time detection methods for HOCl is particularly important, but also is a challenge faced by the world’s scientists. Fluorescent probe technology is undoubtedly an effective means of solving this problem due to its many advantages including excellent selevtivity, high sensitivity, simple operation, fast response and small tissue damage. Thus, the detection methods based on fluorescent probe for HOCl develop fast, and the related reports increase rapidly.Most of these HOCl probes are single fluorescence emission. They are very easily affected by the variations in the environment, concentration and excitation intensity. Especially, small Stokes shift will reduce seriously on cell imaging resolution. Nevertheless, the ratiometric fluorescent probe with dual fluorescence emission at different wavelength can afford a built-in correction, and can eliminate the interference from the environment, concentration and excitation intensity. Importantly, the ratiometric fluorescence probe has a large pseudo-Stokes shift, which will greatly enhance cell imaging resolution, so the ratiometric fluorescence probe is undoubtedly a good choice to solve the above problems.Rhodamine fluorophores have many excellent photochemical properties, such as high molar extinction coefficient, high quantum yield, absorption and emission in visible light region and excellent photostability, so they are widely used in design of many probes. The main design principle is controlling xanthene conjudated system by spiro-ring to regulate fluorescence emission. When the spiro-ring of a probe is closing state, xanthene structure is in a non-conjugated system and no fluorescence emission is shown. When the probe reacts with analyte, the spiro-ring opens to induce xanthene structure changing into conjugated system, so the probe gives strong fluorescence emission. The probe which transforms from no fluorescence to strong fluorescence is called "turn on" probe. Conversely, when spiro-ring changes from opening to closing, the xanthene structure converts from the conjugated state to non-conjugated state, so the fluorescence is quenched. We call it as "turn off" probe. Thus, the clear fluorescence emission mechanism provides a clear idea for design of probes based on xanthene fluorophore.According to the reports, myeloperoxidase (MPO) which catalyzes the reaction of H2O2 and Cl" to form HOCl in vivo has higher activity under acid condition than alkaline condition. Therefore, HOCl in vivo is most likely generated under acidic conditions. Thus, developing HOCl fuorescence probes which can be used in acid condition is of signicance for the detection of HOCl origin in living cells.In light of the importance of developing HOCl fluorescence probe and the unique advantages of the ratiometric fluorescence probe, we focus mainly on the research of the ratiometric fluorescence probes for HOCl in this dissertation. We designed and synthesized a series of xanthene-based HOCl ratiometric fluorescence probes CRH, CRA, Naph-Rh, CRSH and DRSH, and an Hg2+ fluorescent probe RDH. Their spectra properties and applications were studied systematically.Chapter 1, we outlined the importance of the development of HOCl fluorescent probes, introduced simply the recognition mechanisms of HOCl fluorescent probes and the design principle of rhodamine-based fluorescence probes, and summarized the progress of hypochlorous acid probes.Chapter 2, based on a coumarin-rhodamine dyad, we designed two ratiometric fluorescence probes CRH and CRA. CRH uses diacylhydrazine as a detection group, which can respond quickly to -OCl under alkaline condition. CRA uses directly rhodamine acid as recognition moiety, which can respond fast to HOCl under acidic condition. The results show that both probes CRH and CRA exhibit high selectivity and sensitivity to -OC1/HOCl.Chapter 3, we designed a ratiometric fluorescence probe Naph-Rh based on TBET for HOCl. In this probe, rhodamine thiohydrazide and naphthalene formyl are connected by a simple synthesis method to construct a structure of monothio-bishydrazide. When Naph-Rh reacts with HOCl, monothio-bishydrazide can be converted into electronically conjugated bonds. Thus, the spiro-ring of rhodamine is in opening state and the TBET process is setup simultaneously. The study results indicate that probe Naph-Rh shows excellent selectivity, high sensitivity and fast response to HOCl, and can be suitable for the detection of endogenous HOCl in cells.Chapter 4, we designed a ratiometric probe CRSH for HOCl based on FRET system. Rhodamine thiohydrazide is used as the detection group in probe CRSH. It can respond fast to HOCl under acidic condition to cause the fluorescence change, so as to achieve the purpose of detecting HOCl. The results show that probe CRSH has good selectivity, high sensitivity, excellent sensitivity, strong membrane permeability and good stability, and can better be applied to the detection of intracellular HOCl.Chapter 5, In order to increase substantial spectral overlap between the donor emission and acceptor absorption peaks, and improve the energy transfer efficiency, we designed and synthesized two HOCl ratiometric probes CARSH and DRSH on the basis of probe CRSH. In both probes, two longer emission wavelength fluorophores as energy donors match with rhodamine to establish two novel FRET platforms. The calculated results show that the novel templates can improve the efficiency of energy transfer.Chapter 6, based on xanthene fluorophore, we designed an Hg2+ fluorescent probe with a simple structure and studied its spectra properties and applications. The results show that the probe RDH has high selectivity, excellent sensitivity, good anti-interference ability and low cytotoxicity. RDH is used suitably in the detection of intracellular Hg2+.