Study And Application Of New Longer-wavelength Fluorescent Probes With Large Stokes Shifts Based On BODIPY For Nitric Oxide

Fluorescent probes have good specificity, high selectivity and rapid response to the targets, meanwhile they are easy to be chemically designed, synthesized, and modified to meet the different needs of detection. Fluorescence probes combined with fluorescence spectrophotometer, high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and fluorescence microscopy, not only improves the sensitivity and accuracy of the detection, also can provide fluorescence imaging and the spatial and temporal distribution of the targets, which greatly widen the application range of fluorescent probes. Fluorescent probes have been widely used in analytical chemistry, clinical medicine and life science, and involved in various ions, small molecule, and biological macromolecules such as proteins and DNA.Boron dipyrromethene (BODIPY) as a member of small organic molecular fluorescent probes, not only inherits the low biological toxicity and good membrane permeability of organic small molecules, but also has high fluorescence quantum yield and molar absorption coefficient, good photostability and good resistance to different solvents and pH value. However, most of fluorescent probes derived from BODIPY lie in the visible region (500-550nm) with small Stokes shifts (less than10nm). Their biological applications are limited because of photo damage by the excitation light source and autofluorescence from the living organisms.Fluorescent probes with larger Stokes shifts in the far-visible and near-infrared spectra (600-900nm) are more superior for imaging in living cells and tissues, since fluorescence emission in the longer-wavelength region can avoid visible light absorption, reduce autofluorescence from biological matrix, encourage deeper tissue penetration in vivo and provide a non-invasive fluorescence imaging. Meanwhile, large Stokes shifts (the difference in wavelength between the excitation and emission maxima for a fluorescent substance) diminish self-quenching and light scattering interferences, and thus enhance the detection sensitivity.Nitric oxide (NO) as an indispensable biological mediator, is endogenously produced by nitric oxide synthase (NOS) and involved in a series of physiological and pathophysiological functions in the nervous, cardiovascular, and immune systems. It also participates in the physiological functions of plants including the normal metabolism and the response to biotic and abiotic stresses.In this paper, we have designed new fluorophores based on BODIPY core to be linked with3,4-diamino aromatic moiety commonly used as trapping domain for NO, to develop new NO fluorescent probes with red fluorescence (590-643nm) and large Stokes shifts (21-38nm) in the far-visible/NIR spectral region. We use new fluorescent probes to establish a series of good sensitive and selective methods for detecting NO, including fluorescence photometry, high performance liquid chromatography with fluorescence detection (HPLC-FD), ionic liquid monolithic column solid-phase microextraction with HPLC-FD, capillary electrophoresis with laser induced fluorescence (CE-LIF), fluorescent microscope imaging. The major in the thesis are summarized as follows:1. For the purpose of achieving both red-emitting fluorescence and larger Stokes shift, methoxyphenyl groups have been introduced into3,5-positions of the BODIPY core to provide3,4-diamino compounds as the new fluorescent probes:4,4-difluoro-8-(3',4'-diaminophenyl)-3,5-bis(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indancene (p-MOPB) and4,4-difluoro-8-(3',4'-diaminophenyl)-3,5-bis(2-methoxyphenyl)-4-bora-3a,4a-diaza-s-indancene (o-MOPB). By one step reaction, demethylation of o-methoxyphenyl group on o-MOPB with BBr3has been carried out to yield the B,O-chelated fluorescent probe, boron chelated8-(3',4'-diaminophenyl)-3,5-bis(2-hydroxyphenyl)-4-bora-3a,4a-diaza-s-indancene (BOPB). Structures of fluorescent probes were characterized by use of1H-NMR spectra,13C-NMR spectra and high-resolution mass spectrum.2. We have comprehensively and systematically investigated fluorescent properties of new fluorescent probes and their nitric oxide derivative products. On one hand,p-MOPB, o-MOPB and BOPB almost have no fluorescence and very low fluorescence quantum yield (0.001). On the other hand, p-MOPB-T, o-MOPB-T and BOPB-T, the corresponding triazoles from the reaction of p-MOPB and o-MOPB with NO, immediately turn on high red-emitting fluorescence. Maximum excitation/emission wavelength of p-MOPB-T is582/620nm, Stokes shift is38nm, and fluorescence quantum yield is0.25. Maximum excitation/emission wavelength of o-MOPB-T is552/590nm, Stokes shift is38nm, and fluorescence quantum yield is0.07. Maximum excitation/emission wavelength of BOPB-T is622/643nm, Stokes shift is38nm, and fluorescence quantum yield is0.21. The change of fluorescence intensity with pH is small when pH<4or pH>6, the change at pH4-6is bigger, and stronger fluorescence appears in alkaline environment. Fluorescent probes have good photostability, and their fluorescence intensities are not affected by different solvents.3. After optimizing the reaction conditions of p-MOVB with NO, a fluorescence spectrophotometric method has been developed to be high sensitive, good selective, simple and rapid response to the detection of NO. p-MOPB and NO take6.0minutes to finish the derivative reaction in pH9.0phosphate buffer under35?, and other coexisting metal ions and free radicals do not interfere with the reaction. The linear range of the method is6.5×10-9-4.0×10-6M, and the detection limit (S/N=3) is2.0nM. The spectrophotometric method has been used to measure NO of the roots of soybean and mungbean induced by IAA. The NO levels of soybean and mungbean roots without induction of IAA were5.39and3.62nmol/g, respectively. The NO levels of roots with induction of IAA were26.17and18.35nmol/g, respectively. Recoveries are from96.60%to98.52%, and R.S.D. is less than3.27%. The fluorescence spectrophotometric method can be applied directly to the analysis of real biological sample without complex pretreatment.4. p-MOPB has been used as the derivative reagent to combine with HPLC-FD to develop a precolumn derivation HPLC-FD method. The NO content of the whole blood and cerebral cortex tissue in mice has been determined by using the method. p-MOPB and NO reacts completely within9min in pH9.0phosphate buffe at30?. Mobile phase of HPLC consists of methanol and buffer (v/v,90/10, buffer:0.1M, pH4.5H3Cit-NaOH). It takes4.0min to finish the separation with the flow rate of0.7 mL/min. Linear range of HPLC-FD is5.0×10-9M-1.0×10-6M. When the signal-to-noise ratio is3, the detection limit (LOD) is0.3nM. Recoveries vary from96.5%-98.7%with R.S.D.(n=6) of1.92-3.17%. This method has high sensitivity, simplicity, short separation time, fast derivative reaction. It can effectively eliminate the interference of the scattered light and avoid background fluorescence of biological sample.Clean and flat chromatogram shows great potential in the analysis of complex biological samples.5. A sensitive method has been proposed for the determination of NO in tomato and orange fruits by SPME in conjunction with HPLC-FD. p-MO?B was used to be a precolumn derivative fluorescent probe. SPME monolithic capillary consisted of ionic liquid1-methyl-3-[3-(trimethoxysilyl)propyl]imidazolium chloride [TPMIM][Cl]. The sample solution (2.0mL) was extracted via the monolithic capillary at0.1mL/min.0.05mL methanol was passed through the monolithic capillary at0.05mL/min to finish the desorption, and the eluate was collected for the HPLC analysis. The linearity range is1.0×10-10M-1.0×10-7M. The detection limit (LOD) is1.5×10-11M. The method has better sensitivity than directly HPLC analysis without SPME, LOD of which is3.0×10-10M. The NO levels of tomato and orange fruits are236nM and175nM, respectively, with the recoveries of87%-87%. This high sensitive and good selective method is suitable for the trace analysis of complex biological samples.6. The excitation/emission wavelength of BOPB is622/643nm, and it matches the635nm line of the semiconductor diode laser, which can avoid biological autofluorescence and improve the sensitivity of analysis. BOPB was used as a precolumn derivative reagent to combine with capillary electrophoresis laser induced fluorescence detection (CE-LIF) to develop a new method for detection of NO in mice liver injury model. The reaction of BOPB and NO happened in pH7.4PBS buffer under35?and it was accomplished within12min. Unde the optimal conditions of CE-LIF, NO was separated by baseline within7min. Linear range of the method is1.0×10-9-5.0×10-7M with the detection limit of0.02nM. The result indicates that the induction of D-galactosamine and endotoxin significantly increases NO in liver injury model of mice. At present. BOPB is the first near-infrared probe to be combined with CE-LIF for the detection of NO with the longest wavelength, the fastest derivative reaction, the lowest detection limit.7. Fluorescent probe p-MOPB has longer emission (620nm) and good photostability, which is more superior for cellular imaging and biological analysis. Based on the evaluation results of MTT assay, the feasibility of p-MOPB in cell imaging has been assessed by fluorescent microscope. Meanwhile, lipopolysaccharide (LPS) and interferon-y (IFN-y) were used to induce the expression of inducible nitric oxide synthase (iNOS) and then stimulate the production of NO in raw264.7macrophages. When raw264.7macrophages were only incubated with5.0?M p-MOPB for1.0h, faint red fluorescence was observed. However, raw264.7macrophages exhibited a significant increase of red fluorescence on images, when pretreated with LPS (1?g mL-1) and IFN-y (2000U mL-1) for12h and then co-incubated with5.0?M p-MOPB for1.0h. In contrast, when raw264.7macrophages were pretreated with LPS and IFN-? for12h, and then a scavenger for NO, Carboxy-PTIO (200?M), was added before the addition of p-MOPB, the fluorescence was weakened remarkably. These phenomena demonstrate that a dramatic enhancement of red fluorescence on images is attributed to the NO production in raw264.7macrophages stimulated by LPS and IFN-y. Hence, it is proved that p-MOPB can penetrate the cell membrane readily, trap NO efficiently and generate "turn-on" red fluorescence.