Investigation On The TICT Mechanism Of Rhodamine Fluorescent Dyes And Development Of Fluorescence Probes

Abstract:Organic fluorescent dyes have been widely used for labeling of proteins, nucleic acids, lipids, carbohydrates, toxins, hormones and other biological molecules. More than85%of fluorophores are "Donor-acceptor" fluorescent dyes, having one or two donors and an accepter. However, the fluorescent quantum yield (0) of most fluorescent dyes is far below100%, which is mainly ascribed to the formation of the twisted intermolecular charge transfer at excited state (TICT theory). However, the TICT theory can not make a full explanation to the molecular structures of a variety of fluorescent dyes and is challenged to some extent. Furthermore, many aspects influencing the TICT mechanism need to be improved, such as the volume effect of amino donors, acceptor and the existence of two donors. Therefore, it is of great significance to further investigate the TICT mechanism, which is dedicated to the design of improved fluorescent dyes with excellent spectral properties and high quantum efficiency.Rhodamine dyes, a typical kinds of donor-acceptor fluorophores, exhibit good photophysical and photochemical properties. In this thesis, a series of Rhodamines having different acceptor or donors were synthesized and their photophyical photochemical properties were investigated. The relationship between the structures and optical properties in Rhodamines is dicussed. The discovery in this study can contribute to the TICT theory, be applied to many kinds of donor-acceptor fluorescent dyes, and useful to develop more efficient fluorescent dyes.The main contents are as follows:1. Seven novel symmetrical and unsymmetrical Rhodamine fluorescent dyes, having rigid/flexible alkyl and different electronegativities amino groups, were synthesized and studied by UV-vis, fluorescence spectra. The fluorescent quantum yield and lifetimes of the obtained Rhodamines were measured in different solvent at different temperatures. The results indicate that the rigidity of the donors and amino groups can effectively prevent the formation of TICT state and increase fluorescence lifetime. In TICT state, positive chares are mainly located on the two amino groups, and the one with better electron donating ability can sustain more positive charge. For unsymmetrical Rhodamines, there is a close relationship between lifetime and the rigid-flexible characteristics of stronger electron donationg amino group. The rigidity can prevent the TICT formation and make the dye higly fluorescent, whereas the flexibility can favor the TICT formation and decrease the fluorescence lifetime of Rhodamines.2. Using naphthalene sulfonic anhydride as raw material, seven naphthalenesulfonic acid Rhodamine fluorescent dyes have been successfully synthesized. The UV-vis absorbance spectra, fluorescence spectra, and the fluorescence lifetime under different solvents and different temperatures were determined. The difference in the fluorescence properties between the Rhodamine fluorescent dyes prepared using naphthalene sulfonic anhydride and benzene sulfonic anhydride as raw materials were discussed. Owing to the steric hindrance effect and the electronic effect of naphthalene sulfonic anhydride, it is not easy for the TICT excited state to form, which results in the improvement in the fluorescence properties. In comparison with the traditional Rhodamine fluorescent dyes in which benzene was used as the accepter, the seven Rhodamine fluorescent dyes synthesized exhibited a promoted fluorescence lifetime of38%.3. Nine rhodamines with different-sized dialkylamino groups were synthesized and their fluorescence lifetimes were measured in different solvents and temperatures. The results show that the volume of the amino groups can affect the formation of TICT state, thereby make the difference in the fluorescence lifetimes of fluorescent dyes. In the forming process of TICT excited state, Rhodamines with larger donor exhibit longer lifetimes than the smaller one, indicating the volume of the amino groups can affect the formation of TICT state. This result proved "volume effect in rotation activity" in TICT state, which is question by many researchers.4. Based on the TICT mechamism, Rhodamine dyes (AZESR, TMSR and PYRSR) with azetidine, dimethylamine and pyrrolidine as the electron donors were synthesized. Their absorption and emission spectra, ? and fluorescence lifetimes were measured. The results showed that the AZESR possessed higher0and longer lifetime which were nearly independent of temperature and solvent.5. Based on ESIPT, a novel ratiometric fluorescent probe was synthesized. The probe is suitable for quantitative and qualitative detection of sulfite. With the addition of sodium sulfite, the fluorescence spectra exhibited a95-nm red-shift. By taking advantage of the ICT process inherent in4-hydroxynaphthalimide, a novel ratiometric fluorescence probe was designed. Owing to the hydrolysis of ester catalyzed by Cu2+, the probe showed good specificity and quick response to Cu2+. Most importantly, the probe has been successfully used for monitoring of Cu2+in living cells.