雙功能熒光探針的合成及其對(duì)汞離子和硫醇的識(shí)別性能研究
發(fā)布時(shí)間：2019-04-09 19:54
【摘要】：香豆素類熒光基團(tuán)具有熒光量子產(chǎn)率高、Stokes位移大、光物理和光化學(xué)性質(zhì)可調(diào)以及光穩(wěn)定性好等優(yōu)點(diǎn),是熒光探針分子設(shè)計(jì)中的優(yōu)秀候選熒光團(tuán)。小分子量的硫醇類化合物如半胱氨酸(Cys),同型半胱氨酸(Hcy)和谷胱甘肽(GSH)涉及至關(guān)重要的細(xì)胞過程,包括氧化還原平衡和細(xì)胞的成長。汞在自然界中分布量極小卻又分布廣泛,環(huán)境中的無機(jī)汞轉(zhuǎn)化為有機(jī)汞被生物體吸收后,又會(huì)通過食物鏈富集在較高階層的動(dòng)物體內(nèi),繼而對(duì)人體健康造成一定的危害。因此,對(duì)于硫醇類化合物和汞離子的識(shí)別大大增加了人類的健康的保障。為此,本文選擇合成香豆素為母體的識(shí)別硫醇類化合物和汞離子的雙功能香豆素作為研究課題。本論文總結(jié)概括了熒光探針的研究進(jìn)展和機(jī)理,并進(jìn)一步介紹了香豆素類汞離子熒光探針和硫醇熒光探針的應(yīng)用進(jìn)展以及它們的合成方法。本文設(shè)計(jì)合成了多種以4-甲基-7-羥基香豆素、3-乙�；�-7-羥基香豆素和3-羥基-6H-苯并[b,d]吡喃-6-酮為母體的ABCDE五個(gè)系列的香豆素?zé)晒馓结槨系列是以硫羰基作為汞離子識(shí)別基團(tuán),苯磺酸酯類衍生物作為硫醇識(shí)別基團(tuán);B系列利用苯肼反應(yīng)生成酰腙類香豆素作為識(shí)別汞離子的基團(tuán),苯磺酸酯類衍生物作為硫醇識(shí)別基團(tuán);由于汞離子可以與雙鍵、三鍵發(fā)生作用,硫醇可以與甲酰基,共軛烯烴發(fā)生作用,基于此原理設(shè)計(jì)合成了C系列、D系列和E系列,利用不飽和鍵,比如烯烴、炔烴、共軛烯烴、甲�；鶃韺�(shí)現(xiàn)對(duì)汞離子和硫醇分子的識(shí)別。本文對(duì)以上五個(gè)系列的熒光探針分子的合成條件進(jìn)行了探索,找到了最佳反應(yīng)條件。利用IR、1H NMR、13C NMR和MS對(duì)合成的新化合物的結(jié)構(gòu)進(jìn)行了表征。本文選出了2種典型的熒光探針A2和E1,以紫外可見吸收光譜和熒光發(fā)射光譜為測(cè)試手段,對(duì)其檢測(cè)汞離子和硫醇分子的選擇性、靈敏度、響應(yīng)時(shí)間、檢測(cè)機(jī)理以及探針的酸堿耐受性進(jìn)行了研究。測(cè)試結(jié)果表明在激發(fā)波長為320 nm,溶液中溶液中乙腈與水的比例為8:2時(shí),探針A2(20μM)的熒光強(qiáng)度達(dá)到最大,最大熒光發(fā)射波長為451 nm,并且加入的Hg2+為0.6當(dāng)量時(shí),熒光發(fā)射波長增強(qiáng)2倍。在激發(fā)波長為445 nm時(shí),甲硫酚使探針A2(20μM)熒光強(qiáng)度增加了14倍,半胱氨酸(Cys)使探針A2(20μM)熒光強(qiáng)度增加了4.5倍,并且探針A2與甲硫酚響應(yīng)時(shí)間非常短,1 min內(nèi)就可以響應(yīng)完全。探針A2對(duì)甲硫酚的靈敏度非常高,當(dāng)加入甲硫酚只有0.125當(dāng)量時(shí),探針A2(20μM)的熒光強(qiáng)度就增強(qiáng)了16倍。與組氨酸(His)、甘氨酸(Gly)、苯丙氨酸(Phe)、纈氨酸(Val)、天冬酰胺(Asn)、谷氨酸(Glu)選擇性對(duì)照實(shí)驗(yàn)中,只有Cys能夠明顯增強(qiáng)探針A2的熒光強(qiáng)度,其他小分子化合物對(duì)其不受影響。在激發(fā)波長為272 nm時(shí),溶液中乙醇與水的比例為1:9時(shí),探針E1(20μM)熒光強(qiáng)度達(dá)到最大,并且探針E1在pH=4-11的溶液中能夠穩(wěn)定存在。Hg2+加入后,1 min內(nèi)探針E1的最大熒光強(qiáng)度峰由458 nm處藍(lán)移到400 nm處,并且加入0.6當(dāng)量的Hg2+就可以發(fā)生明顯的藍(lán)移,而其他離子,比如:Ca2+、Cu2+、Zn2+、Ce3+、Co2+、Sr2+、Mn2+、Bi3+、Ni2+、Mg2+、Na+、Li+則不能使探針E1的熒光發(fā)生藍(lán)移。當(dāng)激發(fā)波長為375nm時(shí),Cys能夠使探針E1(20μM)的熒光強(qiáng)度降低至原來的0.07倍,當(dāng)激發(fā)波長為233nm時(shí),甲硫酚能夠使探針E1(20 nM)的熒光強(qiáng)度降低至原來的0.01倍。并且探針E1對(duì)Cys具有非常高的靈敏性,0.4當(dāng)量的Cys就能使其熒光強(qiáng)度降低一倍達(dá)到識(shí)別的目的。
[Abstract]:The coumarin-based fluorescent group has the advantages of high fluorescence quantum yield, large Stokes shift, light physical and optical properties, good light stability and the like, and is an excellent candidate fluorophore in the fluorescent probe molecular design. Small-molecular-weight thiol compounds, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are involved in vital cell processes, including redox balance and cell growth. The distribution of mercury in nature is very small and widely distributed, and the conversion of the inorganic mercury in the environment into the organic mercury is absorbed by the living body, and the mercury can be enriched in the high-class animal body through the food chain, and then a certain harm to the health of the human body is caused. Therefore, the identification of the thiol compound and the mercury ion greatly increases the human health protection. To this end, the two-functional coumarin, which is used to identify the thiol compound and the mercury ion, is used as the research subject. The research progress and mechanism of the fluorescent probe are summarized, and the application progress of the coumarin mercury ion fluorescent probe and the thiol fluorescent probe and their synthesis methods are also introduced. A variety of 4-methyl-7-hydroxycoumarin,3-ethanediyl-7-hydroxycoumarin and 3-hydroxy-6H-benzo[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d] the A-series is a sulfur-based group as a mercury ion-identifying group, and the benzenesulfonic acid ester derivative is used as a thiol recognition group; the B-series is used for generating a p-type coumarin as a group for identifying a mercury ion and a benzenesulfonic acid ester derivative as a thiol recognition group by using a benzene-benzene reaction; since the mercury ions can interact with the double bonds and the triple bonds, the mercaptans can interact with the silyl groups and the co-olefins, and the C-series, the D-series and the E-series are synthesized based on the principle, and the unsaturated bonds, such as olefins, alkynes, copolyalkenes, The silyl group is used to realize the identification of the mercury ions and the thiol molecules. The synthesis conditions of the above five series of fluorescent probe molecules are explored, and the optimum reaction conditions are found. The structure of the new compound was characterized by IR, 1H NMR, 13C NMR and MS. Two typical fluorescent probes, A2 and E1, were selected to test the selectivity, the sensitivity, the response time, the detection mechanism and the acid-base resistance of the probe. The results show that when the excitation wavelength is 320 nm, the ratio of acetonitrile to water in the solution is 8:2, the fluorescence intensity of the probe A2 (20. mu.M) reaches the maximum, the maximum fluorescence emission wavelength is 451 nm, and when the added Hg2 + is 0.6 equivalent, the fluorescence emission wavelength is enhanced by 2 times. When the excitation wavelength was 445 nm, the fluorophenol increased the fluorescence intensity of the probe A2 (20. mu.M) by 14 times, and the cysteine (Cys) increased the fluorescence intensity of the probe A2 (20. mu.M) by 4.5 times, and the probe A2 and the thiophenol response time were very short and the response time was within 1 min. The sensitivity of the probe A2 to the thiophenol is very high, and the fluorescence intensity of the probe A2 (20. mu.M) is increased by 16 times when the addition of the fluorophenol is only 0.125 equivalent. In a selective control experiment with histidine (His), glycine (Gly), phenylalanine (Phe), amino acid (Val), galanthamine (Asn), and glutamic acid (Glu), only Cys can enhance the fluorescence intensity of probe A2, and other small molecule compounds are not affected. When the excitation wavelength is 272 nm, the ratio of ethanol to water in the solution is 1:9, the fluorescence intensity of the probe E1 (20. mu.M) reaches the maximum, and the probe E1 can be stably present in the solution of pH = 4-11. After Hg2 + is added, the maximum fluorescence intensity peak of the probe E1 is blue shifted to 400 nm from 458 nm, and the addition of 0.6 equivalent of Hg2 + can cause significant blue shift, while other ions, such as Ca2 +, Cu2 +, Zn2 +, Ce3 +, Co2 +, Sr2 +, Mn2 +, Bi3 +, Ni2 +, Mg2 +, Na + and Li + do not shift the fluorescence of the probe E1. When the excitation wavelength is 375nm, the fluorescence intensity of the probe E1 (20. mu.M) can be reduced to 0.07 times that of the original, and when the excitation wavelength is 233 nm, the fluorescent intensity of the probe E1 (20 nM) can be reduced to 0.01 times that of the original. And the probe E1 has a very high sensitivity to the Cys, and the 0.4 equivalent of Cys can double the fluorescence intensity of the probe E1 to the target of identification.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657.3
[Abstract]:The coumarin-based fluorescent group has the advantages of high fluorescence quantum yield, large Stokes shift, light physical and optical properties, good light stability and the like, and is an excellent candidate fluorophore in the fluorescent probe molecular design. Small-molecular-weight thiol compounds, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are involved in vital cell processes, including redox balance and cell growth. The distribution of mercury in nature is very small and widely distributed, and the conversion of the inorganic mercury in the environment into the organic mercury is absorbed by the living body, and the mercury can be enriched in the high-class animal body through the food chain, and then a certain harm to the health of the human body is caused. Therefore, the identification of the thiol compound and the mercury ion greatly increases the human health protection. To this end, the two-functional coumarin, which is used to identify the thiol compound and the mercury ion, is used as the research subject. The research progress and mechanism of the fluorescent probe are summarized, and the application progress of the coumarin mercury ion fluorescent probe and the thiol fluorescent probe and their synthesis methods are also introduced. A variety of 4-methyl-7-hydroxycoumarin,3-ethanediyl-7-hydroxycoumarin and 3-hydroxy-6H-benzo[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d]-[b, d] the A-series is a sulfur-based group as a mercury ion-identifying group, and the benzenesulfonic acid ester derivative is used as a thiol recognition group; the B-series is used for generating a p-type coumarin as a group for identifying a mercury ion and a benzenesulfonic acid ester derivative as a thiol recognition group by using a benzene-benzene reaction; since the mercury ions can interact with the double bonds and the triple bonds, the mercaptans can interact with the silyl groups and the co-olefins, and the C-series, the D-series and the E-series are synthesized based on the principle, and the unsaturated bonds, such as olefins, alkynes, copolyalkenes, The silyl group is used to realize the identification of the mercury ions and the thiol molecules. The synthesis conditions of the above five series of fluorescent probe molecules are explored, and the optimum reaction conditions are found. The structure of the new compound was characterized by IR, 1H NMR, 13C NMR and MS. Two typical fluorescent probes, A2 and E1, were selected to test the selectivity, the sensitivity, the response time, the detection mechanism and the acid-base resistance of the probe. The results show that when the excitation wavelength is 320 nm, the ratio of acetonitrile to water in the solution is 8:2, the fluorescence intensity of the probe A2 (20. mu.M) reaches the maximum, the maximum fluorescence emission wavelength is 451 nm, and when the added Hg2 + is 0.6 equivalent, the fluorescence emission wavelength is enhanced by 2 times. When the excitation wavelength was 445 nm, the fluorophenol increased the fluorescence intensity of the probe A2 (20. mu.M) by 14 times, and the cysteine (Cys) increased the fluorescence intensity of the probe A2 (20. mu.M) by 4.5 times, and the probe A2 and the thiophenol response time were very short and the response time was within 1 min. The sensitivity of the probe A2 to the thiophenol is very high, and the fluorescence intensity of the probe A2 (20. mu.M) is increased by 16 times when the addition of the fluorophenol is only 0.125 equivalent. In a selective control experiment with histidine (His), glycine (Gly), phenylalanine (Phe), amino acid (Val), galanthamine (Asn), and glutamic acid (Glu), only Cys can enhance the fluorescence intensity of probe A2, and other small molecule compounds are not affected. When the excitation wavelength is 272 nm, the ratio of ethanol to water in the solution is 1:9, the fluorescence intensity of the probe E1 (20. mu.M) reaches the maximum, and the probe E1 can be stably present in the solution of pH = 4-11. After Hg2 + is added, the maximum fluorescence intensity peak of the probe E1 is blue shifted to 400 nm from 458 nm, and the addition of 0.6 equivalent of Hg2 + can cause significant blue shift, while other ions, such as Ca2 +, Cu2 +, Zn2 +, Ce3 +, Co2 +, Sr2 +, Mn2 +, Bi3 +, Ni2 +, Mg2 +, Na + and Li + do not shift the fluorescence of the probe E1. When the excitation wavelength is 375nm, the fluorescence intensity of the probe E1 (20. mu.M) can be reduced to 0.07 times that of the original, and when the excitation wavelength is 233 nm, the fluorescent intensity of the probe E1 (20 nM) can be reduced to 0.01 times that of the original. And the probe E1 has a very high sensitivity to the Cys, and the 0.4 equivalent of Cys can double the fluorescence intensity of the probe E1 to the target of identification.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657.3
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1 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
2 向雨秘;龍少波;朱R，
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