双功能荧光探针的合成及其对汞离子和硫醇的识别性能研究
发布时间：2019-04-09 19:54
【摘要】：香豆素类荧光基团具有荧光量子产率高、Stokes位移大、光物理和光化学性质可调以及光稳定性好等优点,是荧光探针分子设计中的优秀候选荧光团。小分子量的硫醇类化合物如半胱氨酸(Cys),同型半胱氨酸(Hcy)和谷胱甘肽(GSH)涉及至关重要的细胞过程,包括氧化还原平衡和细胞的成长。汞在自然界中分布量极小却又分布广泛,环境中的无机汞转化为有机汞被生物体吸收后,又会通过食物链富集在较高阶层的动物体内,继而对人体健康造成一定的危害。因此,对于硫醇类化合物和汞离子的识别大大增加了人类的健康的保障。为此,本文选择合成香豆素为母体的识别硫醇类化合物和汞离子的双功能香豆素作为研究课题。本论文总结概括了荧光探针的研究进展和机理,并进一步介绍了香豆素类汞离子荧光探针和硫醇荧光探针的应用进展以及它们的合成方法。本文设计合成了多种以4-甲基-7-羟基香豆素、3-乙酰基-7-羟基香豆素和3-羟基-6H-苯并[b,d]吡喃-6-酮为母体的ABCDE五个系列的香豆素荧光探针。A系列是以硫羰基作为汞离子识别基团,苯磺酸酯类衍生物作为硫醇识别基团;B系列利用苯肼反应生成酰腙类香豆素作为识别汞离子的基团,苯磺酸酯类衍生物作为硫醇识别基团;由于汞离子可以与双键、三键发生作用,硫醇可以与甲酰基,共轭烯烃发生作用,基于此原理设计合成了C系列、D系列和E系列,利用不饱和键,比如烯烃、炔烃、共轭烯烃、甲酰基来实现对汞离子和硫醇分子的识别。本文对以上五个系列的荧光探针分子的合成条件进行了探索,找到了最佳反应条件。利用IR、1H NMR、13C NMR和MS对合成的新化合物的结构进行了表征。本文选出了2种典型的荧光探针A2和E1,以紫外可见吸收光谱和荧光发射光谱为测试手段,对其检测汞离子和硫醇分子的选择性、灵敏度、响应时间、检测机理以及探针的酸碱耐受性进行了研究。测试结果表明在激发波长为320 nm,溶液中溶液中乙腈与水的比例为8:2时,探针A2(20μM)的荧光强度达到最大,最大荧光发射波长为451 nm,并且加入的Hg2+为0.6当量时,荧光发射波长增强2倍。在激发波长为445 nm时,甲硫酚使探针A2(20μM)荧光强度增加了14倍,半胱氨酸(Cys)使探针A2(20μM)荧光强度增加了4.5倍,并且探针A2与甲硫酚响应时间非常短,1 min内就可以响应完全。探针A2对甲硫酚的灵敏度非常高,当加入甲硫酚只有0.125当量时,探针A2(20μM)的荧光强度就增强了16倍。与组氨酸(His)、甘氨酸(Gly)、苯丙氨酸(Phe)、缬氨酸(Val)、天冬酰胺(Asn)、谷氨酸(Glu)选择性对照实验中,只有Cys能够明显增强探针A2的荧光强度,其他小分子化合物对其不受影响。在激发波长为272 nm时,溶液中乙醇与水的比例为1:9时,探针E1(20μM)荧光强度达到最大,并且探针E1在pH=4-11的溶液中能够稳定存在。Hg2+加入后,1 min内探针E1的最大荧光强度峰由458 nm处蓝移到400 nm处,并且加入0.6当量的Hg2+就可以发生明显的蓝移,而其他离子,比如:Ca2+、Cu2+、Zn2+、Ce3+、Co2+、Sr2+、Mn2+、Bi3+、Ni2+、Mg2+、Na+、Li+则不能使探针E1的荧光发生蓝移。当激发波长为375nm时,Cys能够使探针E1(20μM)的荧光强度降低至原来的0.07倍,当激发波长为233nm时,甲硫酚能够使探针E1(20 nM)的荧光强度降低至原来的0.01倍。并且探针E1对Cys具有非常高的灵敏性,0.4当量的Cys就能使其荧光强度降低一倍达到识别的目的。
[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.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：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.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
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