反应型荧光探针的设计、合成及性能研究
发布时间：2018-09-12 17:43
【摘要】：有机荧光探针具有检测限低、操作简单、能够实时检测和便于在生物体内应用等优点,被广泛用于污染物监测、生物成像等诸多领域。长期以来,在有机荧光探针研究中,人们更多关注重金属离子的检测,该类探针发展得较为成熟。而涉及阴离子、氨基酸和有机小分子的文献不是很多,并且大都存在选择性较差,灵敏度不高等问题。本论文围绕次氯酸、生物硫醇和水合联氨的荧光检测展开研究,设计合成了四种反应型荧光探针,并在对其充分表征基础上,深入研究了探针的识别机理,并将其用于细胞成像。1、基于菲并咪唑衍生物的比率型次氯酸探针的合成及应用。针对以往菲并咪唑类荧光探针虽然荧光量子产率高、光化学稳定性好,但水溶性较差问题,设计合成了以菲并咪唑为荧光团,酰基苯肼为识别基团的比率型次氯酸探针PIPH,该探针在有机溶剂用量为50%情况下,对次氯酸表现出较好的检测能力,加入次氯酸后荧光强度增大了约20倍。为了详细研究荧光探针的检测机理,我们通过核磁、质谱等检测手段,将检测后的产物与PIPH进行了对比,发现检测过程中,PIPH被次氯酸氧化,转化为PIBA;同时利用Gaussian 09软件、密度泛函/含时密度泛函理论(DFT/TD-DFT)对PIPH和PIBA的基态与激发态的空间结构和外层电子云排布进行了优化。理论计算证实,由于激发态的PIPH荧光团与识别基团近似于共平面,出现分子内电荷效应(ICT效应)并导致荧光淬灭;与次氯酸反应后,PIPH的酰基苯肼被氧化分解,ICT效应消失,荧光增强;He La细胞的荧光成像结果表明,PIPH具有很好的生物相容性,能够定性检测细胞外源引入的次氯酸。2、基于萘酰亚胺衍生物的比率型生物硫醇探针合成及应用。设计合成了以萘酰亚胺为荧光团,2,4-二硝基苯磺酰氧基为识别基团的生物硫醇探针DNBS,该探针对半胱氨酸、高半胱氨酸和谷胱甘肽三种生物硫醇均呈现良好的荧光检测能力,其中对半胱氨酸的检测限达28.9 n M。同时,质谱研究发现,检测过程中DNBS与生物硫醇反应后生成了新的化合物NIOH,使得探针分子原本存在的d-Pe T效应被打断,释放出萘酰亚胺特有的荧光信号,并随检测物浓度增加荧光信号逐渐增强;细胞成像实验表明,DNBS具有良好的生物相容性,并对细胞外源引入的生物硫醇具有良好特异识别能力。3、基于1,4-二羟基蒽醌衍生物的半胱氨酸荧光探针的合成及性能研究。针对以往生物硫醇类荧光探针大多选择性较差,容易彼此干扰问题,设计合成了以1,4-二羟基蒽醌为荧光团,丙烯酸酯为识别基团的探针AQDA。该探针基于光诱导电子转移机理(PET效应),仅对半胱氨酸有特异性荧光响应,包括高半胱氨酸和谷胱甘肽在内的其他天然氨基酸均不会对其造成干扰,而且与以往大多数同类探针相比,AQDA水溶性得到很大改善,在实际检测中仅需添加20%的有机溶剂。同时,通过核磁滴定和质谱分析等手段,对比研究了检测过程中AQDA的变化情况,明确了反应机理,并通过Gaussian 09软件进行了理论研究,发现模拟得到的吸收光谱与荧光发射光谱与实际测试结果高度一致,进一步从理论上解释了AQDA的荧光检测机制;最后,在生物硫醇抑制(NEM)与谷胱甘肽抑制剂(BSO)存在下,研究了AQDA对细胞自身产生的半胱氨酸的成像情况,证实了其对细胞内源性的半胱氨酸具有良好的检测能力。4、基于荧光素的水合联氨探针的合成及其性能研究。设计合成了以荧光素为荧光团,4-溴丁酰氧基为识别基团的比率型水合联氨探针FLB。通过质谱、核磁滴定等检测手段确定了探针的识别机理,发现探针FLB中的荧光素片段在强吸电子基团4-溴丁酰基的作用下,五元螺环处于关闭状态,荧光淬灭,加入水合肼后,4-溴丁酸酯与水合肼发生取代-加成消除反应,FLB被转化为荧光素,放出明显的绿色荧光。而且,FLB对水合肼的检测限达到0.92 ppb,远低于美国环保署(EPA)关于饮用水中肼的最高允许含量(10 ppb);而且FLB的水溶性较好,实际检测中只需添加10%的有机溶剂;同时FLB具有非常好的选择性,包括15种阴离子、10种阳离子以及9种小分子在内的34种干扰物质均没有对水合肼的检测造成明显干扰;最后,通过细胞成像实验发现,FLB具有在生物体内检测水合肼的能力。
[Abstract]:Organic fluorescent probes have been widely used in many fields, such as pollutant monitoring, bioimaging and so on. For a long time, more attention has been paid to the detection of heavy metal ions in the organic fluorescent probes, and the development of these probes is relatively mature. In this paper, four kinds of reactive fluorescent probes were designed and synthesized based on the fluorescence detection of hypochlorite, biomercaptan and hydrazine, and their full characterization was studied. Recognition mechanism and its application in cell imaging. 1. Synthesis and application of ratio-type hypochlorite probes based on phenamidazole derivatives. Aiming at the problems of high fluorescence quantum yield and good photochemical stability, but poor water-solubility of phenamidazole fluorescent probes, phenylhydrazine was designed and synthesized. The ratio type hypochlorite probe PIPH showed good detection ability for hypochlorite when the amount of organic solvent was 50%. The fluorescence intensity increased about 20 times after adding hypochlorite. It was found that PIPH was oxidized by hypochlorite and converted to PIBA during the detection process, and the spatial structure of ground state and excited state of PIPH and PIBA was optimized by DFT / TD-DFT using Gaussian 09 software. Similar to the coplanar, the intramolecular charge effect (ICT effect) occurs and results in fluorescence quenching; after reaction with hypochlorite, the acylphenylhydrazine of PIPH is oxidized and decomposed, the ICT effect disappears, and the fluorescence is enhanced; the fluorescence imaging results of He La cells show that PIPH has good biocompatibility and can qualitatively detect the hypochlorite introduced by exogenous cells, based on naphthalene. Synthesis and application of ratio biothiol probes of imide derivatives. A novel biothiol probe DNBS was designed and synthesized. The probe has good fluorescence detection ability for cysteine, homocysteine and glutathione, among which p-cysteine is the fluorescent group and 2,4-dinitrobenzenesulfonyl oxy group is the recognition group. The detection limit of cystine was 28.9 n M. Meanwhile, mass spectrometry showed that the reaction of DNBS with biomercaptan resulted in the formation of a new compound NIOH, which interrupted the d-Pe T effect of the probe molecule and released the fluorescence signal specific to naphthalimide. The fluorescence signal was enhanced gradually with the increase of the concentration of the probe. The results showed that DNBS had good biocompatibility and good specific recognition ability for the biothiols introduced from exogenous cells. 3. Synthesis and properties of cysteine fluorescent probes based on 1,4-Dihydroxyanthraquinone derivatives were studied. In view of the poor selectivity of most of the biothiols fluorescent probes in the past, they were easy to interfere with each other. The probe AQDA, which uses 1,4-Dihydroxyanthraquinone as fluorescent group and acrylate as recognition group, is based on photo-induced electron transfer mechanism (PET effect) and has only specific fluorescence response to cysteine. Other natural amino acids, including homocysteine and glutathione, do not interfere with the probe and are similar to most previous homocysteines. The water-solubility of AQDA was greatly improved compared with that of probes, and only 20% organic solvent was added in the actual detection. At the same time, the changes of AQDA in the detection process were studied by means of NMR titration and mass spectrometry, the reaction mechanism was clarified, and the theoretical study was carried out by Gaussian 09 software. The simulated absorption light was found. The spectra and fluorescence emission spectra were in good agreement with the experimental results, which further explained the fluorescence detection mechanism of AQDA in theory. Finally, in the presence of biothiol inhibition (NEM) and glutathione inhibitor (BSO), the imaging of cysteine produced by AQDA on cells was studied, and it was confirmed that AQDA had the ability to detect intracellular endogenous cysteine. 4. Based on the synthesis of fluorescein-based hydrazine probe and its properties, a ratio-type hydrazine probe FLB was designed and synthesized with fluorescein as the fluorescent group and 4-bromobutyroxy as the recognition group. The recognition mechanism of the probe was determined by mass spectrometry, nuclear magnetic resonance titration and the fluorescein in the probe FLB was found. Under the action of 4-bromobutyryl group, the fragment was shut down and fluorescence quenched. After hydrazine hydrate was added, 4-bromobutyrate reacted with hydrazine hydrate by substitution-addition elimination. FLB was converted into fluorescein and emitted obvious green fluorescence. Moreover, the detection limit of FLB for hydrazine hydrate was 0.92 ppb, which was far lower than that of environmental protection in the United States. The maximum permissible content of hydrazine in drinking water (10 ppb) of the EPA, and the water solubility of FLB is good, only 10% of organic solvents are added in the actual detection; at the same time, FLB has very good selectivity, including 15 anions, 10 cations and 9 small molecules of 34 interfering substances, including no obvious interference in the detection of hydrazine hydrate; Finally, cell imaging experiments show that FLB has the ability to detect hydrazine hydrate in organism.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3
[Abstract]:Organic fluorescent probes have been widely used in many fields, such as pollutant monitoring, bioimaging and so on. For a long time, more attention has been paid to the detection of heavy metal ions in the organic fluorescent probes, and the development of these probes is relatively mature. In this paper, four kinds of reactive fluorescent probes were designed and synthesized based on the fluorescence detection of hypochlorite, biomercaptan and hydrazine, and their full characterization was studied. Recognition mechanism and its application in cell imaging. 1. Synthesis and application of ratio-type hypochlorite probes based on phenamidazole derivatives. Aiming at the problems of high fluorescence quantum yield and good photochemical stability, but poor water-solubility of phenamidazole fluorescent probes, phenylhydrazine was designed and synthesized. The ratio type hypochlorite probe PIPH showed good detection ability for hypochlorite when the amount of organic solvent was 50%. The fluorescence intensity increased about 20 times after adding hypochlorite. It was found that PIPH was oxidized by hypochlorite and converted to PIBA during the detection process, and the spatial structure of ground state and excited state of PIPH and PIBA was optimized by DFT / TD-DFT using Gaussian 09 software. Similar to the coplanar, the intramolecular charge effect (ICT effect) occurs and results in fluorescence quenching; after reaction with hypochlorite, the acylphenylhydrazine of PIPH is oxidized and decomposed, the ICT effect disappears, and the fluorescence is enhanced; the fluorescence imaging results of He La cells show that PIPH has good biocompatibility and can qualitatively detect the hypochlorite introduced by exogenous cells, based on naphthalene. Synthesis and application of ratio biothiol probes of imide derivatives. A novel biothiol probe DNBS was designed and synthesized. The probe has good fluorescence detection ability for cysteine, homocysteine and glutathione, among which p-cysteine is the fluorescent group and 2,4-dinitrobenzenesulfonyl oxy group is the recognition group. The detection limit of cystine was 28.9 n M. Meanwhile, mass spectrometry showed that the reaction of DNBS with biomercaptan resulted in the formation of a new compound NIOH, which interrupted the d-Pe T effect of the probe molecule and released the fluorescence signal specific to naphthalimide. The fluorescence signal was enhanced gradually with the increase of the concentration of the probe. The results showed that DNBS had good biocompatibility and good specific recognition ability for the biothiols introduced from exogenous cells. 3. Synthesis and properties of cysteine fluorescent probes based on 1,4-Dihydroxyanthraquinone derivatives were studied. In view of the poor selectivity of most of the biothiols fluorescent probes in the past, they were easy to interfere with each other. The probe AQDA, which uses 1,4-Dihydroxyanthraquinone as fluorescent group and acrylate as recognition group, is based on photo-induced electron transfer mechanism (PET effect) and has only specific fluorescence response to cysteine. Other natural amino acids, including homocysteine and glutathione, do not interfere with the probe and are similar to most previous homocysteines. The water-solubility of AQDA was greatly improved compared with that of probes, and only 20% organic solvent was added in the actual detection. At the same time, the changes of AQDA in the detection process were studied by means of NMR titration and mass spectrometry, the reaction mechanism was clarified, and the theoretical study was carried out by Gaussian 09 software. The simulated absorption light was found. The spectra and fluorescence emission spectra were in good agreement with the experimental results, which further explained the fluorescence detection mechanism of AQDA in theory. Finally, in the presence of biothiol inhibition (NEM) and glutathione inhibitor (BSO), the imaging of cysteine produced by AQDA on cells was studied, and it was confirmed that AQDA had the ability to detect intracellular endogenous cysteine. 4. Based on the synthesis of fluorescein-based hydrazine probe and its properties, a ratio-type hydrazine probe FLB was designed and synthesized with fluorescein as the fluorescent group and 4-bromobutyroxy as the recognition group. The recognition mechanism of the probe was determined by mass spectrometry, nuclear magnetic resonance titration and the fluorescein in the probe FLB was found. Under the action of 4-bromobutyryl group, the fragment was shut down and fluorescence quenched. After hydrazine hydrate was added, 4-bromobutyrate reacted with hydrazine hydrate by substitution-addition elimination. FLB was converted into fluorescein and emitted obvious green fluorescence. Moreover, the detection limit of FLB for hydrazine hydrate was 0.92 ppb, which was far lower than that of environmental protection in the United States. The maximum permissible content of hydrazine in drinking water (10 ppb) of the EPA, and the water solubility of FLB is good, only 10% of organic solvents are added in the actual detection; at the same time, FLB has very good selectivity, including 15 anions, 10 cations and 9 small molecules of 34 interfering substances, including no obvious interference in the detection of hydrazine hydrate; Finally, cell imaging experiments show that FLB has the ability to detect hydrazine hydrate in organism.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3
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