反應(yīng)型熒光探針的設(shè)計(jì)、合成及性能研究
發(fā)布時(shí)間：2018-09-12 17:43
【摘要】：有機(jī)熒光探針具有檢測(cè)限低、操作簡(jiǎn)單、能夠?qū)崟r(shí)檢測(cè)和便于在生物體內(nèi)應(yīng)用等優(yōu)點(diǎn),被廣泛用于污染物監(jiān)測(cè)、生物成像等諸多領(lǐng)域。長(zhǎng)期以來,在有機(jī)熒光探針研究中,人們更多關(guān)注重金屬離子的檢測(cè),該類探針發(fā)展得較為成熟。而涉及陰離子、氨基酸和有機(jī)小分子的文獻(xiàn)不是很多,并且大都存在選擇性較差,靈敏度不高等問題。本論文圍繞次氯酸、生物硫醇和水合聯(lián)氨的熒光檢測(cè)展開研究,設(shè)計(jì)合成了四種反應(yīng)型熒光探針,并在對(duì)其充分表征基礎(chǔ)上,深入研究了探針的識(shí)別機(jī)理,并將其用于細(xì)胞成像。1、基于菲并咪唑衍生物的比率型次氯酸探針的合成及應(yīng)用。針對(duì)以往菲并咪唑類熒光探針雖然熒光量子產(chǎn)率高、光化學(xué)穩(wěn)定性好,但水溶性較差問題,設(shè)計(jì)合成了以菲并咪唑?yàn)闊晒鈭F(tuán),酰基苯肼為識(shí)別基團(tuán)的比率型次氯酸探針PIPH,該探針在有機(jī)溶劑用量為50%情況下,對(duì)次氯酸表現(xiàn)出較好的檢測(cè)能力,加入次氯酸后熒光強(qiáng)度增大了約20倍。為了詳細(xì)研究熒光探針的檢測(cè)機(jī)理,我們通過核磁、質(zhì)譜等檢測(cè)手段,將檢測(cè)后的產(chǎn)物與PIPH進(jìn)行了對(duì)比,發(fā)現(xiàn)檢測(cè)過程中,PIPH被次氯酸氧化,轉(zhuǎn)化為PIBA;同時(shí)利用Gaussian 09軟件、密度泛函/含時(shí)密度泛函理論(DFT/TD-DFT)對(duì)PIPH和PIBA的基態(tài)與激發(fā)態(tài)的空間結(jié)構(gòu)和外層電子云排布進(jìn)行了優(yōu)化。理論計(jì)算證實(shí),由于激發(fā)態(tài)的PIPH熒光團(tuán)與識(shí)別基團(tuán)近似于共平面,出現(xiàn)分子內(nèi)電荷效應(yīng)(ICT效應(yīng))并導(dǎo)致熒光淬滅;與次氯酸反應(yīng)后,PIPH的�；诫卤谎趸纸�,ICT效應(yīng)消失,熒光增強(qiáng);He La細(xì)胞的熒光成像結(jié)果表明,PIPH具有很好的生物相容性,能夠定性檢測(cè)細(xì)胞外源引入的次氯酸。2、基于萘酰亞胺衍生物的比率型生物硫醇探針合成及應(yīng)用。設(shè)計(jì)合成了以萘酰亞胺為熒光團(tuán),2,4-二硝基苯磺酰氧基為識(shí)別基團(tuán)的生物硫醇探針DNBS,該探針對(duì)半胱氨酸、高半胱氨酸和谷胱甘肽三種生物硫醇均呈現(xiàn)良好的熒光檢測(cè)能力,其中對(duì)半胱氨酸的檢測(cè)限達(dá)28.9 n M。同時(shí),質(zhì)譜研究發(fā)現(xiàn),檢測(cè)過程中DNBS與生物硫醇反應(yīng)后生成了新的化合物NIOH,使得探針分子原本存在的d-Pe T效應(yīng)被打斷,釋放出萘酰亞胺特有的熒光信號(hào),并隨檢測(cè)物濃度增加熒光信號(hào)逐漸增強(qiáng);細(xì)胞成像實(shí)驗(yàn)表明,DNBS具有良好的生物相容性,并對(duì)細(xì)胞外源引入的生物硫醇具有良好特異識(shí)別能力。3、基于1,4-二羥基蒽醌衍生物的半胱氨酸熒光探針的合成及性能研究。針對(duì)以往生物硫醇類熒光探針大多選擇性較差,容易彼此干擾問題,設(shè)計(jì)合成了以1,4-二羥基蒽醌為熒光團(tuán),丙烯酸酯為識(shí)別基團(tuán)的探針AQDA。該探針基于光誘導(dǎo)電子轉(zhuǎn)移機(jī)理(PET效應(yīng)),僅對(duì)半胱氨酸有特異性熒光響應(yīng),包括高半胱氨酸和谷胱甘肽在內(nèi)的其他天然氨基酸均不會(huì)對(duì)其造成干擾,而且與以往大多數(shù)同類探針相比,AQDA水溶性得到很大改善,在實(shí)際檢測(cè)中僅需添加20%的有機(jī)溶劑。同時(shí),通過核磁滴定和質(zhì)譜分析等手段,對(duì)比研究了檢測(cè)過程中AQDA的變化情況,明確了反應(yīng)機(jī)理,并通過Gaussian 09軟件進(jìn)行了理論研究,發(fā)現(xiàn)模擬得到的吸收光譜與熒光發(fā)射光譜與實(shí)際測(cè)試結(jié)果高度一致,進(jìn)一步從理論上解釋了AQDA的熒光檢測(cè)機(jī)制;最后,在生物硫醇抑制(NEM)與谷胱甘肽抑制劑(BSO)存在下,研究了AQDA對(duì)細(xì)胞自身產(chǎn)生的半胱氨酸的成像情況,證實(shí)了其對(duì)細(xì)胞內(nèi)源性的半胱氨酸具有良好的檢測(cè)能力。4、基于熒光素的水合聯(lián)氨探針的合成及其性能研究。設(shè)計(jì)合成了以熒光素為熒光團(tuán),4-溴丁酰氧基為識(shí)別基團(tuán)的比率型水合聯(lián)氨探針FLB。通過質(zhì)譜、核磁滴定等檢測(cè)手段確定了探針的識(shí)別機(jī)理,發(fā)現(xiàn)探針FLB中的熒光素片段在強(qiáng)吸電子基團(tuán)4-溴丁�；淖饔孟�,五元螺環(huán)處于關(guān)閉狀態(tài),熒光淬滅,加入水合肼后,4-溴丁酸酯與水合肼發(fā)生取代-加成消除反應(yīng),FLB被轉(zhuǎn)化為熒光素,放出明顯的綠色熒光。而且,FLB對(duì)水合肼的檢測(cè)限達(dá)到0.92 ppb,遠(yuǎn)低于美國(guó)環(huán)保署(EPA)關(guān)于飲用水中肼的最高允許含量(10 ppb);而且FLB的水溶性較好,實(shí)際檢測(cè)中只需添加10%的有機(jī)溶劑;同時(shí)FLB具有非常好的選擇性,包括15種陰離子、10種陽(yáng)離子以及9種小分子在內(nèi)的34種干擾物質(zhì)均沒有對(duì)水合肼的檢測(cè)造成明顯干擾;最后,通過細(xì)胞成像實(shí)驗(yàn)發(fā)現(xiàn),FLB具有在生物體內(nèi)檢測(cè)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3
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2 王姍姍;;小分子熒光探針在硫醇檢測(cè)中的最新研究進(jìn)展[J];科技信息;2010年23期
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