The Optimized Design, Synthesis Of Novel Rhodamine, Pyrene Derivatives And Their Application In Fluorescent Probes

Due to its high sensitivity, fast analysis with spatial resolution for providing insitu and real^-time information, and nondestructive sample preparation, the fluorescentprobe method seems to be an ideal candidate for both sensing and bioimaging metalions, anions and small molecules in various samples. As a consequence, in the pasttwo decades, a greatly increasing interest was focus on the development ofsmall^-molecule fluorescent probes for metal ions, anions and small molecules. Amongthem, probes which exhibit analyte-triggered fluorescence enhancement have attractedparticular attention. Such probes afford ‘turn-on’ responses toward analyte, which aremore suitable for fluorescence detection and bioimaging applications than thoseshowing analyte-induced fluorescence quenching responses. On the other hand, thoseprobes based on single emission intensity changes tend to be affected by a variety offactors such as instrumental efficiency and environmental conditions, as well as theconcentration of probe molecule. These interferences can be eliminated by employingratiometric fluorescent probes, which allow the measurement of changes of theintensity ratio at two emission bands induced by analytes and provide built-incorrection for the above-mentioned environmental effects. Therefore, Searching fornovel molecular recognition systems with the ability to applied in real samples andbiological samples, remains task of theoretical and practical significance.Taking the above-mentioned reasonings as research objectives, the contents ofthis thesis are presented as follows:1. In Chapter2, a simple and convenient probe of pyrene formohydrazide （PZ）was designed and synthesized. The new probe exhibits high selectivity and sensitivityfor Cu²⁺based upon that the ion is known reactivity toward carboxylate derivatives.While the unique propensity of Cu²⁺to promote hydrolysis has been known forseveral decades, this information has been used previously for Cu²⁺quantitation infew cases. In this work, increase in fluorescence emission intensity was observedupon adding Cu²⁺in EtOH/HEPES （1:1, v/v） solution at pH7.4. The linear responserange covers a concentration range of Cu²⁺from5.0×10^-8to1.0×10^-6M and thedetection limit is1.4×10^-8M.2. In Chapter3, a novel probe of dibenzoylhydrazine-linked dipyrene xanthene（DP） was designed and synthesized. In this compound, two pyrene monomers is fixed in a small molecule probe by using a rigid structure of xanthene to form a pyreneexcimer, this kind of pyrene excimer affords a long wavelength, wide shapefluorescence emission from pyrene excimer. It’s noted that OCl^-can selectivelyoxidize dibenzoylhydrazine into dibenzoyl diimide, which can further undergo adecomposition in some nucleophilic solvents water for example. According to thisreaction, decrease in495nm and increase in about400nm were observed upon addingOCl^-in EtOH/HEPES （1:1, v/v） solution at pH7.4, which due to the pyrene excimerwas separated into two pyrene monomers by OCl^-. The proposed chemodosimeterexhibits high selectivity over other common anions, and excellent ratio-metricdetection for OCl^-.3. In Chapter4, we synthesized and obtained thio-spirolactam rhodamine Bderivative （RS） by a mild synthetic method rather than the traditional demandingsynthesis methods. Compared with the general literature reported rhodaminespirolactam structure, we only use one S atom to form a stable spirocyclic ring, whichis not sensitive to acid while general spirolactam ring is easily opened by acid. Likecommon reported rhodamine spirolactam derivatives, the anticipated thio-spirolactamrhodamine B derivative （RS） was colorless, non absorption and non fluorescence.When S atom was coordinated with Hg²⁺, the original rhodamine structure wasdestroyed and reduced to a conjugated system, which emitted strong fluorescence andachieved the detection of Hg²⁺. Furthermore, as a recognition group, the one S atom isintroduced into our designed probe RS. Therefore, our new probe exhibits highselectivity for Hg²⁺over other metal ions and high sensitivity with a detection limit of2.5nM. The proposed chemosensor has been used for imaging of Hg²⁺in living cellswith satisfied results, further demonstrating its value of the practical applications inenvironmental and biological systems.4. In Chapter5, we described the optimized design, synthesis and application ofa novel rhodamine thiospirolactam derivative RT-3as an ‘off-on’ fluorescent probefor the detection of Hg²⁺in aqueous samples. The ‘off-on’ fluorescence and colorsignal change of the probe are based on an Hg²⁺-triggered domino reaction whichbrings on the opened-ring form of the rhodamine spirolactam to regain the conjugatedsystem of the rhodamine skeleton. In the well designed probe, the thiospirolactamserves as both Hg²⁺binding unit and electron-defect carbon center, a phenolichydroxyl with very strong nucleophilicity after deprotonation is chosen as theattacking unit, and a benzene ring is introduced on the linker to afford steric effects,which benefits an efficient nucleophilic reaction, with a high sensitivity towards Hg²⁺. It exhibits a stable response for Hg²⁺from1.0×10^-8to1.0×10^-6M, with a detectionlimit of3.0nM. The response of the probe to Hg²⁺is highly selective andpH-insensitive, with a fast response time. All these unique features make itparticularly favorable for cellular Hg²⁺imaging applications. It has been preliminarilyused for highly sensitive monitoring of Hg²⁺levels in living cells with satisfyingresolution.5. In Chapter6, we designed and synthesized a novel fluorescein-appendedrhodamine spirolactam derivative FR, and its preliminary application as a ratiometricfluorescent cellular imaging probe for Zn²⁺had been investigated. Its ratiometricfluorescent signal change is based on an intramolecular fluorescence resonance energytransfer （FRET） mechanism modulated by a specific metal ion induced ring-openingprocess of the rhodamine spirolactam （acting as a trigger）. In the new developedsensing system, the emission peaks of the two fluorophores are well^-resolved, whichcan avoid the emission spectra overlap problem generally met by spectra-shift typeprobes and benefits for observation of fluorescence signal change at two differentemission wavelengths with high resolution. It also benefits for a large range ofemission ratios, thereby a high sensitivity for Zn²⁺detection. Under optimizedexperimental conditions, the probe exhibits a stable response for Zn²⁺over aconcentration range from2.0×10-7to2.0×10-5M, with a detection limit of4.0×10^-8M.Most importantly, the novel probe has well solved the problem of seriousinterferences from other transition metal ions generally met by previously reportedtypical fluorescent probes for Zn²⁺with the di（2-picolyl）amine moiety as the receptorand shows a reversible and fast response toward Zn²⁺.6. In Chapter7, we designed and synthesized a novel a coumarin-rhodamine （CR）through bond energy transfer （TBET） system and demonstrated that TBET was aconvenient strategy to design an efficient ratiometric fluorescent bioimaging probe formetal ions. Such TBET strategy is also universal, since no spectral overlap betweenthe donor and the acceptor is necessary, and many more dye pairs than that of FRETcould be chosen for probe design. As a proof-of-concept, Hg²⁺was chosen as a modelmetal ion. By combining TBET strategy with dual^-switch design, the proposedsensing platform shows two well^-separated emission peaks with a wavelengthdifference of110nm, high energy transfer efficiency of97.4%, and a largesignal^-to-background ratio of697.7, which affords a high sensitivity for the probewith a detection limit of7.0nM for Hg²⁺. The proposed chemosensor has been usedfor direct measurement of Hg²⁺content in river water samples and imaging of Hg²⁺in living cells with satisfied results.