Studies On Synthesis And Application Of New Near-infrared Fluorescent Reagents Based On BODIPY Dyes For Thiols

Low-molecular-weight thiols, such as glutathione (GSH) and cysteine (Cys), are widely distributed in the organisms, and play crucial roles in metabolism activities. As the main reductants, thiols can remove active oxygen and free radicals to maintain the redox balance. They also react with NO to form S-nitrosothiols (RSNO) to regulate NO levels and are involved in the signal transduction. The alteration of thiol results in some diseases like liver damage, Alzheimer's disease, cancers, AIDS, etc. Owing to the unique physiological and pathological functions of thiols in vivo, it is very important to monitor their contents and distributions in the organisms.Fluorescence analysis has the advantages of high sensitivity, good selectivity and easy visualization and has been extensively applied to the fields of chemistry, life science, medicine and environmental science. Near-infrared (NIR,> 600 nm) fluorescence, characterized by low matrix interferences, deep penetration and little biological damage, is the focus in fluorescence analysis. Small molecule fluorescent reagents are mostly used in the NIR region. However, ideal NIR small molecule fluorescent reagents are still limited. Boron dipyrromethene (BODIPY) dyes have excellent properties including large molar absorption coefficient, high fluorescence quantum yield, and good stability to solvents, pH and light. In this dissertation, by the review of recent analytical progress of NIR fluorescent reagents and their applications, two novel NIR fluorescent reagents for thiols, l,7-dimethyl-3,5-distyryI-8-phenyl-(2'-maleimide)difluoroboradiaza-s-indacene (DMDSPAB-o-M) and l,7-dimethyl-3,5-diphenyl-8-distyryl-(4'-iodoacetamide)difluoroboradiaza-s-indacene (DMDSPAB-I), have been developed, which are suitable for spectrofluorometry, fluorescence imaging, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), respectively. Based on DMDSPAB-o-M and DMDSPAB-I, some new analytical methods have been established for thiols determination in blood, cells and tissues from animal or plant samples and a simultaneous fluorescence imaging of thiols and NO has been obtained.The major work in the dissertation is summarized as follows:(1) Styryls have been introduced to 3 and 5-positions of BODIPY core to enlarge the conjugated system and red-shift the fluorescence to NIR region. Using maleimide or iodoacetyl groups as the reactive group, DMDSPAB-o-M and DMDSPAB-I have been designed, synthesized and characterized. DMDSPAB-o-M (?= 0.557) displays a rapid response to thiols with 43-fold turn-on fluorescence at 645 nm which is excited at 632 nm. The fluorescence quantum yield of its thiol derivative reaches 0.215. Their fluorescence is almost unchanged with continuous irradiation by a laser light source for 120 min or variation of pH value from 2.0 to 11.0. Both DMDSPAB-I and its thiol derivatives fluoresce strongly with the fluorescence quantum yields of 0.557 and 0.560, respectively., The excitation/emission wavelengths of DMDSPAB-I and its derivatives are both at 620/630 nm. Their photostability is as good as that of DMDSPAB-o-M and its derivatives. Moreover, their fluorescence is more insensitive to solvents and pH.(2) Based on "turn-on" NIR reagent, DMDSPAB-o-M, a simple spectrofluorometric method has been established for direct determination of GSH. In pH 9.0 PBS buffer, DMDSPAB-o-M reacts with GSH at 37 ? for 5 min. The linear range of this method is 6-200 nM and the detection limit is 0.44 nM (S/N= 3). There is no interference from Cys (Cys/GSH molar ratio? 1/2). This method has been applied to the direct determination of GSH in the whole blood and liver of mice, with the recoveries of 95.2%-102.6%(RSDs? 4.0%). The proposed method is suitable to detect GSH in mammal and plant samples directly, which is sensitive, selective, simple and rapid.(3) As a hydrophobic reagent, DMDSPAB-o-M readily permeates into cells and tissues. Therefore, DMDSPAB-o-M has been applied to thiols imaging in ECV-304 cells and liver tissues of mice. Incubated with DMDSPAB-o-M only for 5 min, ECV-304 cells emit bright red fluorescence, which is generated by the reaction of DMDSPAB-o-M with intracellular thiols. After DMDSPAB-o-M is injected into the body of mice for 30 min, the fluorescence from livers of normal mice is high and that from liver injured mice is weaker, showing the decrease of thiols level in the liver of injured mice. This fluorescence imaging method has merits of low toxicity, no biological matrix interference, and can intuitively observe the contents and distribution of thiols quickly and sensitively, which presents a hopeful potential in the diagnosis and treatment of diseases related to thiols.(4) The simultaneous two-color fluorescence imaging of thiols and NO in cells and tissues has been achieved by a dual-labeling strategy. Two trun-on fluorescent reagents, DMDSPAB-o-M and 1,3,5,7-dimethyl-8-(3',4'-diaminophenyl)difluoroboradiaza-5-indacene (DAMBO), are used to label thiols and NO, respectively. After incubated with DMDSPAB-o-M and DAMBO, the two-color imaging of Raw 264.7 cells, HeLa cells and onion tissues are obtained by exciting at wavelengths of 475 nm and 590 nm, respectively. Compared with the single-labeling imaging for thiols or NO, the dual-labeling imaging reflects the distribution and relationship of thiols and NO in the same sample simultaneously, which is more practical and accurate. The proposed method provides a new technological platform to study the relationship between thiols and NO intuitively and effectively.(5) DMDSPAB-I and its thiol derivative are highly fluorescent and relatively insensitive to solvents and pH. Taking GSH, Cys, homocysteine (Hey), N-acetylcysteine (NAC), cysteinylglycine (CysGly) and penicillamine (PA) as the targets, the HPLC method for thiol analysis has been developed based on DMDSPAB-I derivatization. The derivatization is carried out in pH 11.0 Na2B4O7-NaOH buffers at 45 ? for 25 min. The six derivatives are separated within 25 min on C18 column with the mobile phase of methanol/tetrahydrofuran/water/pH 2.0 H3Cit-NaOH buffer solution (77:6:12:5, v/v/v/v). The lowest detection limit is 0.24 nM (S/N= 3). The proposed method has been applied to the analysis of the blood samples of mice with the recoveries of 94.7-104.3%and RSD less than 4.8%. This NIR fluorescence reagent-based HPLC method has smoother and cleaner baseline, less interfering peaks and lower detection limits.(6) The excitation wavelengths of DMDSPAB-I and its thiol derivatives are 620 nm, which match the 635 nm semiconductor diode laser. An effective CE-LIF approach has been proposed for thiols including GSH, Cys, Hey, y-glutarnylcysteine (y-GluCys), CysGly and NAC using DMDSPAB-I as the labeling reagent. The separation complete within 14 min in the running buffer composed of 16 mM pH 7.0 sodium citrate solution and 60%(v/v) acetonitrile. The detection limits are between 0.11 and 0.31 nM (S/N= 3), which are better than or comparable to those reported with other derivatizaiong-based CE-LIF methods. As the first trial of NIR CE-LIF method for thiols determination, the proposed method has been applied to the detection of thiols in cucumber, tomato and arabidopsis samples with the recoveries of 96.5-104.3%(RSDs?4.4%).