CN104673278A - Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe - Google Patents
Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe Download PDFInfo
- Publication number
- CN104673278A CN104673278A CN201510083494.9A CN201510083494A CN104673278A CN 104673278 A CN104673278 A CN 104673278A CN 201510083494 A CN201510083494 A CN 201510083494A CN 104673278 A CN104673278 A CN 104673278A
- Authority
- CN
- China
- Prior art keywords
- formula
- glutathione
- naphthalimide
- bromo
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种检测谷胱甘肽的荧光探针及其制备方法与使用方法。本发明利用1,8-萘酰亚胺构筑经典的ICT体系,并在4-位引入苯亚砜部分,调控探针分子的ICT效应。当无谷胱甘肽的存在下,由于4-位苯亚砜的强吸电子效应导致探针分子无荧光发射;而当存在谷胱甘肽的条件下,苯亚砜部分能被谷胱甘肽取代,进而发生从4-位硫原子(来自谷胱甘肽)到1,8-萘酰亚胺的分子内电子转移,而该ICT效应使得探针分子发射出强荧光。本发明能够实现对细胞内谷胱甘肽的检测,具有操作简便,成本低廉,响应灵敏,易于推广和应用等优点。
The invention discloses a fluorescent probe for detecting glutathione, a preparation method and an application method thereof. The invention utilizes 1,8-naphthalimide to construct a classic ICT system, and introduces a phenylsulfoxide part at the 4-position to regulate the ICT effect of the probe molecule. In the absence of glutathione, the probe molecule has no fluorescence emission due to the strong electron-withdrawing effect of the 4-position phenylsulfoxide; while in the presence of glutathione, the phenylsulfoxide part can be absorbed by glutathione Peptide substitution, and then intramolecular electron transfer from the 4-position sulfur atom (from glutathione) to 1,8-naphthoimide, and this ICT effect makes the probe molecule emit strong fluorescence. The invention can realize the detection of intracellular glutathione, and has the advantages of simple operation, low cost, sensitive response, easy popularization and application, and the like.
Description
技术领域technical field
本发明属于生物检测技术领域,具体涉及一种作为谷胱甘肽荧光探针材料使用的萘酰亚胺-苯亚砜衍生物及其制备方法与使用方法。The invention belongs to the technical field of biological detection, and in particular relates to a naphthalimide-phenylsulfoxide derivative used as a glutathione fluorescent probe material and a preparation method and use method thereof.
背景技术Background technique
谷胱甘肽(Glutathione,GSH)是由谷氨酸、半胱氨酸和甘氨酸组成的三肽分子,是人体中含量最多的含巯基氨基酸。在生命体内,谷胱甘肽对维持细胞内氧化还原平衡、外源物质的新陈代谢、细胞内信号转换和基因调节等起着重要的作用。细胞内谷胱甘肽浓度的异常将导致一系列生理疾病,例如白细胞缺失、银屑病、肝损伤、癌症和艾滋病等(参见L.A.Herzenberg,S.C.De Rosa,J.G.Dubs等,Glutathione deficiency is associated withimpaired survival in HIV disease,Proc.Natl.Acad.Sci.USA.,1997,94:1967-1972)。因此,定量、实时地监测生物体内谷胱甘肽的含量一直是生物化学和化学生物学的研究热点。Glutathione (GSH) is a tripeptide molecule composed of glutamic acid, cysteine and glycine, and is the most abundant thiol-containing amino acid in the human body. In the living body, glutathione plays an important role in maintaining intracellular redox balance, metabolism of exogenous substances, intracellular signal conversion and gene regulation. Abnormality of intracellular glutathione concentration will lead to a series of physiological diseases, such as leukocyte loss, psoriasis, liver damage, cancer and AIDS, etc. in HIV disease, Proc. Natl. Acad. Sci. USA., 1997, 94: 1967-1972). Therefore, quantitative and real-time monitoring of glutathione content in organisms has always been a research hotspot in biochemistry and chemical biology.
荧光检测法由于其优秀的检测灵敏度和选择性,并能实现对生物样品的实时、在线检测而受到研究者的广泛关注。萘酰亚胺类荧光分子因其具有良好的光稳定性、高摩尔消光系数和量子产率等独特优点而成为该方法最重要的荧光母体之一,在多种待测分子的荧光检测中得到了广泛的应用。Due to its excellent detection sensitivity and selectivity, and the ability to realize real-time and on-line detection of biological samples, fluorescence detection has attracted extensive attention from researchers. Naphthalimide fluorescent molecules have become one of the most important fluorescent precursors for this method because of their unique advantages such as good photostability, high molar extinction coefficient, and quantum yield. a wide range of applications.
目前已开发的用于检测谷胱甘肽的小分子荧光探针主要基于巯基与2,4-二硝基苯磺酰氨基或是2,4-二硝基苯璜酰酯基之间的特异性化学反应而设计的。当存在谷胱甘肽的条件下,探针分子中的2,4-二硝基苯磺酰氨基或是2,4-二硝基苯璜酰酯基能被谷胱甘肽中的巯基取代,原2,4-二硝基苯磺酰氨基或是2,4-二硝基苯璜酰酯基发生离去而导致探针分子的荧光性质发生变化,从而实现对谷胱甘肽的特异性设别。The currently developed small molecule fluorescent probes for the detection of glutathione are mainly based on the specificity between sulfhydryl and 2,4-dinitrobenzenesulfonylamino or 2,4-dinitrobenzenesulfonyl ester groups. Designed for sexual chemistry. In the presence of glutathione, the 2,4-dinitrobenzenesulfonylamino or 2,4-dinitrobenzenesulfonyl ester group in the probe molecule can be replaced by the sulfhydryl group in glutathione , the original 2,4-dinitrobenzenesulfonylamino group or 2,4-dinitrobenzenesulfonyl ester group leaves, resulting in a change in the fluorescence properties of the probe molecule, thereby achieving specificity for glutathione Gender.
然而,已报道的谷胱甘肽探针大多数受到生物体内同样含有巯基的氨基酸,如:半胱氨酸(Cysteine,Cys)和高半胱氨酸(Homocysteine,Hcy)的干扰(参见J.Bouffard,Y.Kim,T.M.Swager等,A Highly SelectiveFluorescent Probe for Thiol Bioimaging,Org.Lett.,2008,10:37-40;X.-D.Jiang,J.Zhang,X.Shao等,A selective fluorescent turn-on NIRprobe for cysteine,Org.Biomol.Chem.,2012,10:1966-1968),难以实现其在复杂的生物体内进行谷胱甘肽的特异性检测。因此亟须一种新颖的、具有良好生物稳定性的且能实现优秀特异性响应的用于生物体内谷胱甘肽检测的荧光探针。However, most of the reported glutathione probes are interfered by amino acids that also contain sulfhydryl groups in organisms, such as cysteine (Cysteine, Cys) and homocysteine (Homocysteine, Hcy) (see J. Bouffard, Y.Kim, T.M.Swager, etc., A Highly Selective Fluorescent Probe for Thiol Bioimaging, Org.Lett., 2008, 10: 37-40; X.-D.Jiang, J.Zhang, X.Shao, etc., A selective fluorescent turn-on NIRprobe for cysteine, Org. Biomol. Chem., 2012, 10: 1966-1968), it is difficult to realize the specific detection of glutathione in complex organisms. Therefore, there is an urgent need for a novel fluorescent probe for the detection of glutathione in vivo that has good biological stability and can achieve excellent specific response.
发明内容Contents of the invention
为了克服现有技术中的上述缺陷,本发明旨在提供一种来自萘酰亚胺和苯亚砜的用于检测谷胱甘肽的荧光探针及其制备方法和使用方法。In order to overcome the above-mentioned defects in the prior art, the present invention aims to provide a fluorescent probe for detecting glutathione derived from naphthalene imide and phenyl sulfoxide and its preparation method and use method.
本发明的核心在于利用1,8-萘酰亚胺构筑经典的ICT体系,并在4-位引入苯亚砜部分,调控探针分子的ICT效应。当无谷胱甘肽的存在下,由于4-位苯亚砜的强吸电子效应导致探针分子无荧光发射;而当存在谷胱甘肽的条件下,苯亚砜部分能被谷胱甘肽取代,进而发生从4-位硫原子(来自谷胱甘肽)到1,8-萘酰亚胺的分子内电子转移,而该ICT效应使得探针分子发射出强荧光。通过上述方案,获得了高度谷胱甘肽特异性的“关-开”型的荧光响应,大大提高了检测的选择性和灵敏度。The core of the invention is to use 1,8-naphthoimide to construct a classic ICT system, and introduce a phenylsulfoxide moiety at the 4-position to regulate the ICT effect of the probe molecule. In the absence of glutathione, the probe molecule has no fluorescence emission due to the strong electron-withdrawing effect of the 4-position phenylsulfoxide; while in the presence of glutathione, the phenylsulfoxide part can be absorbed by glutathione Peptide substitution, and then intramolecular electron transfer from the 4-position sulfur atom (from glutathione) to 1,8-naphthoimide, and this ICT effect makes the probe molecule emit strong fluorescence. Through the above scheme, a highly glutathione-specific "off-on" type fluorescence response is obtained, which greatly improves the selectivity and sensitivity of detection.
首先,为了达到上述目的,本发明提供了一种如式(I)所示的萘酰亚胺-苯亚砜衍生物。First, in order to achieve the above object, the present invention provides a naphthalimide-phenylsulfoxide derivative as shown in formula (I).
式(I)中,n为0~18的原子数;R1为氢,或甲基,或乙基,或异丙基,或氟;R2为甲基,或羟基,或羧基,或磺酸基,或(三苯基)磷基,或中的任何一种。In formula (I), n is the atomic number of 0~18; R 1 is hydrogen, or methyl, or ethyl, or isopropyl, or fluorine; R 2 is methyl, or hydroxyl, or carboxyl, or sulfonate Acid group, or (triphenyl) phosphorus group, or any of any kind.
式(I)所示化合物具体为式(II)所示化合物(Nap-G),The compound shown in formula (I) is specifically the compound (Nap-G) shown in formula (II),
上述探针的制备方法包括下列步骤:The preparation method of above-mentioned probe comprises the following steps:
步骤一:在惰性气氛下,式(III)所示4-溴-1,8-萘二甲酸酐与氨基化合物在醇中反应得到式(IV)所示取代的4-溴-1,8-萘酰亚胺。Step 1: Under an inert atmosphere, react 4-bromo-1,8-naphthalic anhydride shown in formula (III) with an amino compound in alcohol to obtain substituted 4-bromo-1,8- Naphthalimide.
式(IV)中,n为0~18的原子数,R2为甲基,或羟基,或羧基,或磺酸基,或(三苯基)磷基,或中的任何一种。In formula (IV), n is the number of atoms from 0 to 18, and R is methyl , or hydroxyl, or carboxyl, or sulfonic acid, or (triphenyl)phosphoryl, or any of the.
步骤二:在惰性气氛下,在碱存在下,式(IV)所示化合物与式(V)所示化合物在醇中反应得到式(VI)所示取代的4-苯硫酚基-1,8-萘酰亚胺。Step 2: Under an inert atmosphere, in the presence of a base, react the compound shown in formula (IV) with the compound shown in formula (V) in alcohol to obtain substituted 4-thiophenol-1 shown in formula (VI), 8-naphthalimide.
式(VI)中,n为0~18的原子数;R1为氢,或甲基,或乙基,或异丙基,或氟;R2为甲基,或羟基,或羧基,或磺酸基,或(三苯基)磷基,或中的任何一种。In formula (VI), n is the number of atoms from 0 to 18; R 1 is hydrogen, or methyl, or ethyl, or isopropyl, or fluorine; R 2 is methyl, or hydroxyl, or carboxyl, or sulfonate acid group, or (triphenyl)phosphoryl group, or any of the.
步骤三:在惰性气氛下,式(VI)所示化合物与间氯过氧苯甲酸在有机溶剂中反应即得式(I)所示化合物。Step 3: Under an inert atmosphere, react the compound represented by formula (VI) with m-chloroperoxybenzoic acid in an organic solvent to obtain the compound represented by formula (I).
步骤一中所述氨基化合物具体为正丁胺;式(III)所示4-溴-1,8-萘二甲酸酐和所述氨基化合物的摩尔比为1~50:1,;所述醇为甲醇、乙醇,正丙醇、异丙醇、正丁醇或乙二醇单甲醚;所述反应温度为50~120度,反应时间为1~20小时;The amino compound described in step 1 is specifically n-butylamine; the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride represented by formula (III) to the amino compound is 1 to 50:1; the alcohol It is methanol, ethanol, n-propanol, isopropanol, n-butanol or ethylene glycol monomethyl ether; the reaction temperature is 50-120 degrees, and the reaction time is 1-20 hours;
作为优选,式(III)所示4-溴-1,8-萘二甲酸酐和所述氨基化合物的摩尔比为10:1;所述醇为乙醇;所述反应温度为80度;反应时间为5小时。As preferably, the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride shown in formula (III) and the amino compound is 10:1; the alcohol is ethanol; the reaction temperature is 80 degrees; the reaction time for 5 hours.
上述制备方法,步骤二中所述醇为甲醇、乙醇、丙醇、丁醇、乙二醇单甲醚或二乙二醇单甲醚;式(IV)所示4-溴-1,8-萘酰亚胺和式(V)所示苯硫酚化合物的摩尔比为1~10:1;In the above preparation method, the alcohol described in step 2 is methanol, ethanol, propanol, butanol, ethylene glycol monomethyl ether or diethylene glycol monomethyl ether; 4-bromo-1,8- The molar ratio of naphthalimide and thiophenol compound shown in formula (V) is 1~10:1;
所述碱为有机碱或无机碱;The base is an organic base or an inorganic base;
所述有机碱为三乙胺、吡啶或二异丙基乙基胺中;所述无机碱为碳酸钾、碳酸钠、氢氧化钠、氢氧化钾、碳酸氢钠或碳酸氢钾;The organic base is triethylamine, pyridine or diisopropylethylamine; the inorganic base is potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or potassium bicarbonate;
反应温度为0~140度;反应时间为1~48小时。The reaction temperature is 0-140 degrees; the reaction time is 1-48 hours.
作为优选,步骤二中所述醇乙二醇单甲醚;式(IV)所示4-溴-1,8-萘酰亚胺和式(V)所示苯硫酚化合物的摩尔比为5:1;反应温度为为100度;反应时间24小时。As preferably, alcohol ethylene glycol monomethyl ether described in step 2; 4-bromo-1,8-naphthalimide shown in formula (IV) and the mol ratio of thiophenol compound shown in formula (V) are 5 : 1; the reaction temperature is 100 degrees; the reaction time is 24 hours.
步骤(3)中所述间氯过氧苯甲酸与式(VI)所示取代的4-苯硫酚基-1,8-萘酰亚胺的摩尔比为0.5~1.5:1;The molar ratio of m-chloroperoxybenzoic acid described in step (3) to substituted 4-thiophenol-1,8-naphthalimide represented by formula (VI) is 0.5 to 1.5:1;
所述有机溶剂为氯仿、二氯甲烷、乙腈、DMF、DMSO或1,2-二氯乙烷;The organic solvent is chloroform, dichloromethane, acetonitrile, DMF, DMSO or 1,2-dichloroethane;
反应温度为0~100度;反应时间为1~24小时。The reaction temperature is 0-100 degrees; the reaction time is 1-24 hours.
作为优选,间氯过氧苯甲酸与式(VI)所示取代的4-苯硫酚基-1,8-萘酰亚胺的摩尔比1:1;所述有机溶剂为二氯甲烷;反应温度为25度;反应时间为5小时。As preferably, the mol ratio of m-chloroperoxybenzoic acid and substituted 4-thiophenol-1,8-naphthalimide shown in formula (VI) is 1:1; the organic solvent is dichloromethane; the reaction The temperature is 25 degrees; the reaction time is 5 hours.
一种检测谷胱甘肽的荧光探针的使用方法;该方法具体包括以下步骤:A method for using a fluorescent probe for detecting glutathione; the method specifically includes the following steps:
步骤1:向不同浓度谷胱甘肽的缓冲盐中加入相同浓度的式(I)所示化合物,配置至少3种不同谷胱甘肽含量的含有式(I)所示化合物的标准溶液。Step 1: adding the same concentration of the compound represented by the formula (I) to buffer salts with different concentrations of glutathione, and preparing at least three standard solutions containing the compound represented by the formula (I) with different glutathione contents.
所示缓冲溶液以是磷酸盐缓冲溶液、Tris-HCl缓冲溶液、HEPES缓冲溶液、硼酸-硼酸钠缓冲溶液中的任何一种,具体以是磷酸盐缓冲溶液;The buffer solution shown is any one of phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, boric acid-sodium borate buffer solution, specifically phosphate buffer solution;
所示标准溶液的pH值为6~11,具体以是7.2;The pH value of the standard solution shown is 6-11, specifically 7.2;
所示标准溶液中式(I)所示化合物的浓度为1nM~10μM;The concentration of the compound shown in the formula (I) in the shown standard solution is 1nM~10μM;
所示标准溶液中谷胱甘肽的含量为0.1nM~1mM;The content of glutathione in the standard solution shown is 0.1nM~1mM;
步骤2:分别测定所述标准溶液的荧光发射光谱,激发波长为366nm,以谷胱甘肽浓度为横坐标,以I482为纵坐标,建立标准曲线。Step 2: Measure the fluorescence emission spectrum of the standard solution respectively, the excitation wavelength is 366nm, the glutathione concentration is taken as the abscissa, and the I482 is taken as the ordinate to establish a standard curve.
I482表示所述标准溶液在波长为482nm处的荧光发射峰强度值;I 482 represents the fluorescent emission peak intensity value of the standard solution at a wavelength of 482nm;
步骤3:向待测样品中加入式(I)所示化合物,控制其浓度与所述标准溶液中式(I)所示化合物的浓度相等;测定其在激发波长为366nm的激发光下的荧光发射谱,即根据标准曲线计算得出待测样品的谷胱甘肽含量。Step 3: Add the compound shown in formula (I) to the sample to be tested, control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission under the excitation light with excitation wavelength of 366nm Spectrum, that is, calculate the glutathione content of the sample to be tested according to the standard curve.
上述步骤:2或步骤3中荧光强度在荧光仪上进行检测。The above steps: the fluorescence intensity in step 2 or step 3 is detected on a fluorometer.
本发明具有如下特点:The present invention has following characteristics:
1)本发明提供的荧光探针是白色固体,萘酰亚胺和亚砜的分子结构特征保证了探针的结构和光学稳定性。1) The fluorescent probe provided by the present invention is a white solid, and the molecular structure characteristics of naphthalimide and sulfoxide ensure the structure and optical stability of the probe.
2)本发明提供的荧光探针,其溶液对谷胱甘肽的浓度敏感,随着谷胱甘肽浓度的增加,紫外灯下观察到其水溶液的荧光由无色变为亮绿色。2) The solution of the fluorescent probe provided by the present invention is sensitive to the concentration of glutathione. As the concentration of glutathione increases, the fluorescence of its aqueous solution changes from colorless to bright green under ultraviolet light.
3)本发明提供的荧光探针,其发射波长为482nm,属于“关-开”型荧光响应,能大大消除检测时背景差异对结果的影响,提高检测的灵敏度。3) The fluorescent probe provided by the present invention has an emission wavelength of 482nm and belongs to the "off-on" type of fluorescence response, which can greatly eliminate the influence of background differences on the results during detection and improve the sensitivity of detection.
4)本发明提供的荧光探针对谷胱甘肽具有优秀的响应灵敏性,常见共存含巯基的氨基酸,如半胱氨酸、高半胱氨酸等对检测无干扰。4) The fluorescent probe provided by the present invention has excellent response sensitivity to glutathione, and commonly coexisting amino acids containing sulfhydryl groups, such as cysteine and homocysteine, do not interfere with the detection.
5)本发明提供的荧光探针对谷胱甘肽浓度呈线性关系,以用于谷胱甘肽的精确测量。5) The fluorescent probe provided by the present invention has a linear relationship with the concentration of glutathione, so as to be used for accurate measurement of glutathione.
本发明提供的萘酰亚胺类染料的“关-开”型谷胱甘肽探针及其试剂盒对谷胱甘肽溶液具有良好的响应,能够实现对细胞内谷胱甘肽的检测,具有操作简便,成本低廉,响应灵敏,易于推广和应用等优点。The "off-on" glutathione probe of naphthalimide dyes provided by the present invention and its kit have good response to glutathione solution, and can realize the detection of intracellular glutathione, It has the advantages of simple operation, low cost, sensitive response, easy popularization and application, and the like.
附图说明Description of drawings
图1为实施例1制备的荧光探针Nap-G的合成路线。Fig. 1 is the synthetic route of fluorescent probe Nap-G prepared in Example 1.
图2为实施例6制备的Nap-G试剂盒对谷胱甘肽水溶液的颜色响应图。Fig. 2 is the color response diagram of the Nap-G kit prepared in Example 6 to glutathione aqueous solution.
图3为实施例6制备的Nap-G试剂盒对不同谷胱甘肽水溶液的荧光响应图。Fig. 3 is the fluorescence response graph of the Nap-G kit prepared in Example 6 to different glutathione aqueous solutions.
图4为实施例6制备的Nap-G试剂盒在波长482nm下的荧光发射强度的比值I482与谷胱甘肽浓度关系曲线。Fig. 4 is the ratio I 482 of the fluorescence emission intensity of the Nap-G kit prepared in Example 6 at a wavelength of 482nm and the glutathione concentration curve.
图5为实施例6制备的Nap-G试剂盒对常见共存离子或生物小分子的荧光响应图。Fig. 5 is a graph showing the fluorescence response of the Nap-G kit prepared in Example 6 to common coexisting ions or small biomolecules.
图6为实施例6制备的Nap-G试剂盒对细胞内谷胱甘肽的荧光成像图;其中,(a)为未加Nap-G之前的细胞荧光成像图;(b)为加入Nap-G后的细胞荧光成像图;(c)为加入Nap-G和谷胱甘肽后细胞荧光成像图。Fig. 6 is the fluorescence imaging figure of the Nap-G kit prepared in embodiment 6 to intracellular glutathione; Wherein, (a) is the cell fluorescence imaging figure before adding Nap-G; (b) is adding Nap- Cell fluorescence imaging after G; (c) is cell fluorescence imaging after adding Nap-G and glutathione.
具体实施方式Detailed ways
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等,如无特殊说明,均从商业途径得到。The materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
如图1所示,实施例1、荧光探针Nap-G的制备As shown in Figure 1, the preparation of embodiment 1, fluorescent probe Nap-G
步骤a):在惰性气氛下,将5.00g 4-溴-1,8-萘二甲酸酐加入到100mL无水乙醇中,再注入2.5mL正丁胺,在50℃的温度下,回流反应6小时。反应完全后,放置过夜有针状结晶析出,过滤,冷乙醇洗涤三次,得到中间体N-正丁基-4-溴-1,8-萘酰亚胺4.20g(产率为80%)。Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 100mL of absolute ethanol, then inject 2.5mL of n-butylamine, and reflux at a temperature of 50°C for 6 Hour. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.20 g of intermediate N-n-butyl-4-bromo-1,8-naphthalimide (80% yield).
步骤b):在惰性气氛下,将1.00g N-正丁基-4-溴-1,8-萘酰亚胺和1.87g对甲基苯硫酚加入50mL三颈瓶中,注入20mL乙二醇单甲醚和2.1mL三乙胺,在0℃的温度下,回流反应5小时。反应完全后,将反应液倒入200mL冰水中,有大量固体析出,过滤,洗涤,真空干燥得,N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺0.967g(产率85%),黄色固体粉末。Step b): Under an inert atmosphere, add 1.00g of N-n-butyl-4-bromo-1,8-naphthalimide and 1.87g of p-methylthiophenol into a 50mL three-necked bottle, inject 20mL of ethylene di Alcohol monomethyl ether and 2.1 mL of triethylamine were reacted under reflux for 5 hours at a temperature of 0°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.967g (yield 85%), yellow solid powder.
1H NMR(400MHz,CDCl3)δ8.63(d,J=7.9Hz,2H),8.32(d,J=7.9Hz,1H),7.77(t,J=7.9Hz,1H),7.45(d,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J=15.2,7.6Hz,3H),1.44(dq,J=14.8,7.3Hz,3H),0.97(t,J=7.3Hz,4H)。 1 H NMR (400MHz, CDCl 3 ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).
步骤c):在惰性气氛下,将0.9g N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺溶于20mL二氯甲烷中,分三批向体系中加入0.45g间氯过氧苯甲酸,室温反应1小时。待反应完全后,旋干反应液,经柱层析分离纯化得到最后产物Nap-G 0.6g(产率62%),白色固体。Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalimide in 20mL of dichloromethane, and add to the system in three batches Add 0.45g m-chloroperoxybenzoic acid to it, and react at room temperature for 1 hour. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.6 g of the final product Nap-G (62% yield), a white solid.
1H NMR(400MHz,CDCl3)δ8.65(d,J=7.7Hz,1H),8.51(d,J=7.3Hz,1H),8.40(dd,J=8.1,2.7Hz,2H),7.68(t,J=7.9Hz,1H),7.49(d,J=8.1Hz,2H),7.13(d,J=8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H);13C NMR(100MHz,CDCl3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82;HRMS(ESI-TOF):m/z 391.1245[M+H]+,calc’d.391.1242. 1 H NMR (400MHz, CDCl 3 ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); 13 C NMR (100MHz, CDCl 3 ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] + ,calc'd.
实施例2、荧光探针Nap-G的制备Embodiment 2, the preparation of fluorescent probe Nap-G
步骤a):在惰性气氛下,将5.00g 4-溴-1,8-萘二甲酸酐加入到100mL无水甲醇中,再注入5.0mL正丁胺,在80℃的温度下,回流反应1小时。反应完全后,放置过夜有针状结晶析出,过滤,冷乙醇洗涤三次,得到中间体N-正丁基-4-溴-1,8-萘酰亚胺4.40g(产率为84%)。Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalic anhydride to 100mL of anhydrous methanol, then inject 5.0mL of n-butylamine, and reflux at a temperature of 80°C for 1 Hour. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.40 g of intermediate N-n-butyl-4-bromo-1,8-naphthalimide (yield 84%).
步骤b):在惰性气氛下,将1.00g N-正丁基-4-溴-1,8-萘酰亚胺和3.94g对甲基苯硫酚加入50mL三颈瓶中,注入20mL丁醇和2.5mL二异丙基乙基胺,在100℃的温度下,回流反应1小时。反应完全后,将反应液倒入200mL冰水中,有大量固体析出,过滤,洗涤,真空干燥得,N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺1.0g(产率88%),黄色固体粉末。Step b): Under an inert atmosphere, add 1.00g N-n-butyl-4-bromo-1,8-naphthalimide and 3.94g p-methylthiophenol into a 50mL three-necked bottle, inject 20mL butanol and 2.5 mL of diisopropylethylamine was refluxed at 100°C for 1 hour. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 1.0 g (yield 88%), yellow solid powder.
1H NMR(400MHz,CDCl3)δ8.63(d,J=7.9Hz,2H),8.32(d,J=7.9Hz,1H),7.77(t,J=7.9Hz,1H),7.45(d,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J=15.2,7.6Hz,3H),1.44(dq,J=14.8,7.3Hz,3H),0.97(t,J=7.3Hz,4H)。 1 H NMR (400MHz, CDCl 3 ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).
步骤c):在惰性气氛下,将0.9g N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺溶于20mL氯仿中,分三批向体系中加入0.45g间氯过氧苯甲酸,在0℃的温度下,反应5小时。待反应完全后,旋干反应液,经柱层析分离纯化得到最后产物Nap-G 0.56g(产率57%),白色固体。Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylthio)-1,8-naphthalimide in 20mL of chloroform, and add to the system in three batches 0.45g m-chloroperoxybenzoic acid was reacted for 5 hours at a temperature of 0°C. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.56 g (57% yield) of the final product Nap-G, a white solid.
1H NMR(400MHz,CDCl3)δ8.65(d,J=7.7Hz,1H),8.51(d,J=7.3Hz,1H),8.40(dd,J=8.1,2.7Hz,2H),7.68(t,J=7.9Hz,1H),7.49(d,J=8.1Hz,2H),7.13(d,J=8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H);13C NMR(100MHz,CDCl3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82;HRMS(ESI-TOF):m/z 391.1245[M+H]+,calc’d.391.1242. 1 H NMR (400MHz, CDCl 3 ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); 13 C NMR (100MHz, CDCl 3 ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] + ,calc'd.
实施例3、荧光探针Nap-G的制备Embodiment 3, the preparation of fluorescent probe Nap-G
步骤a):在惰性气氛下,将5.00g 4-溴-1,8-萘二甲酸酐加入到100mL无水正丙醇中,再注入7.5mL正丁胺,在120℃的温度下,回流反应5小时。反应完全后,放置过夜有针状结晶析出,过滤,冷乙醇洗涤三次,得到中间体N-正丁基-4-溴-1,8-萘酰亚胺4.60g(产率为87%)。Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalic anhydride to 100mL of anhydrous n-propanol, then inject 7.5mL of n-butylamine, and reflux at a temperature of 120°C React for 5 hours. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.60 g of intermediate N-n-butyl-4-bromo-1,8-naphthoimide (yield 87%).
步骤b):在惰性气氛下,将1.00g N-正丁基-4-溴-1,8-萘酰亚胺和0.38g对甲基苯硫酚加入50mL三颈瓶中,注入20mL二乙二醇单甲醚和2.9g碳酸钾,在140℃的温度下,回流反应48小时。反应完全后,将反应液倒入200mL冰水中,有大量固体析出,过滤,洗涤,真空干燥得,N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺0.67g(产率59%),黄色固体粉末。Step b): Under an inert atmosphere, add 1.00g of N-n-butyl-4-bromo-1,8-naphthalimide and 0.38g of p-methylthiophenol into a 50mL three-necked bottle, inject 20mL of diethyl Glycol monomethyl ether and 2.9 g of potassium carbonate were refluxed for 48 hours at a temperature of 140°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.67g (yield 59%), yellow solid powder.
1H NMR(400MHz,CDCl3)δ8.63(d,J=7.9Hz,2H),8.32(d,J=7.9Hz,1H),7.77(t,J=7.9Hz,1H),7.45(d,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J=15.2,7.6Hz,3H),1.44(dq,J=14.8,7.3Hz,3H),0.97(t,J=7.3Hz,4H)。 1 H NMR (400MHz, CDCl 3 ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).
步骤c):在惰性气氛下,将0.9g N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺溶于20mL 1,2-二氯乙烷中,分三批向体系中加入0.23g间氯过氧苯甲酸,0度下反应24小时。待反应完全后,旋干反应液,经柱层析分离纯化得到最后产物Nap-G 0.32g(产率34%),白色固体。Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylthio)-1,8-naphthalimide in 20mL of 1,2-dichloroethane, Add 0.23g m-chloroperoxybenzoic acid to the system in three batches, and react at 0°C for 24 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain the final product Nap-G 0.32g (yield 34%), white solid.
1H NMR(400MHz,CDCl3)δ8.65(d,J=7.7Hz,1H),8.51(d,J=7.3Hz,1H),8.40(dd,J=8.1,2.7Hz,2H),7.68(t,J=7.9Hz,1H),7.49(d,J=8.1Hz,2H),7.13(d,J=8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H);13C NMR(100MHz,CDCl3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82;HRMS(ESI-TOF):m/z 391.1245[M+H]+,calc’d.391.1242. 1 H NMR (400MHz, CDCl 3 ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); 13 C NMR (100MHz, CDCl 3 ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] + ,calc'd.
实施例4、荧光探针Nap-G的制备Embodiment 4, the preparation of fluorescent probe Nap-G
步骤a):在惰性气氛下,将5.00g 4-溴-1,8-萘二甲酸酐加入到100mL无水正丁醇中,再注入10.0mL正丁胺,在50℃的温度下,回流反应20小时。反应完全后,放置过夜有针状结晶析出,过滤,冷乙醇洗涤三次,得到中间体N-正丁基-4-溴-1,8-萘酰亚胺4.84g(产率为88%)。Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 100mL of anhydrous n-butanol, then inject 10.0mL of n-butylamine, and reflux at a temperature of 50°C React for 20 hours. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.84 g of intermediate N-n-butyl-4-bromo-1,8-naphthoimide (yield 88%).
步骤b):在惰性气氛下,将1.00g N-正丁基-4-溴-1,8-萘酰亚胺和2.87g对甲基苯硫酚加入50mL三颈瓶中,注入20mL乙醇和3.5g氢氧化钾,在100℃的温度下,回流反应12小时。反应完全后,将反应液倒入200mL冰水中,有大量固体析出,过滤,洗涤,真空干燥得,N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺0.84g(产率74%),黄色固体粉末。Step b): Under an inert atmosphere, add 1.00g of N-n-butyl-4-bromo-1,8-naphthalimide and 2.87g of p-methylthiophenol into a 50mL three-necked bottle, inject 20mL of ethanol and 3.5g of potassium hydroxide was refluxed for 12 hours at a temperature of 100°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.84g (yield 74%), yellow solid powder.
1H NMR(400MHz,CDCl3)δ8.63(d,J=7.9Hz,2H),8.32(d,J=7.9Hz,1H),7.77(t,J=7.9Hz,1H),7.45(d,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J=15.2,7.6Hz,3H),1.44(dq,J=14.8,7.3Hz,3H),0.97(t,J=7.3Hz,4H)。 1 H NMR (400MHz, CDCl 3 ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).
步骤c):在惰性气氛下,将0.9g N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺溶于20mL DMSO中,分三批向体系中加入0.30g间氯过氧苯甲酸,于100度下反应1小时。待反应完全后,旋干反应液,经柱层析分离纯化得到最后产物Nap-G 0.23g(产率24%),白色固体。Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalimide in 20mL of DMSO, and add to the system in three batches 0.30g m-chloroperoxybenzoic acid was reacted at 100°C for 1 hour. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.23 g (yield 24%) of the final product Nap-G, a white solid.
1H NMR(400MHz,CDCl3)δ8.65(d,J=7.7Hz,1H),8.51(d,J=7.3Hz,1H),8.40(dd,J=8.1,2.7Hz,2H),7.68(t,J=7.9Hz,1H),7.49(d,J=8.1Hz,2H),7.13(d,J=8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H);13C NMR(100MHz,CDCl3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82;HRMS(ESI-TOF):m/z 391.1245[M+H]+,calc’d.391.1242. 1 H NMR (400MHz, CDCl 3 ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); 13 C NMR (100MHz, CDCl 3 ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] + ,calc'd.
实施例5、荧光探针Nap-G的制备Embodiment 5, the preparation of fluorescent probe Nap-G
步骤a):在惰性气氛下,将5.00g 4-溴-1,8-萘二甲酸酐加入到100mL无水乙二醇单甲醚中,再注入0.5mL正丁胺,在80℃的温度下,回流反应20小时。反应完全后,放置过夜有针状结晶析出,过滤,冷乙醇洗涤三次,得到中间体N-正丁基-4-溴-1,8-萘酰亚胺3.2g(产率为60%)。Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 100mL of anhydrous ethylene glycol monomethyl ether, then inject 0.5mL of n-butylamine, at a temperature of 80°C Down, reflux reaction for 20 hours. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 3.2 g of intermediate N-n-butyl-4-bromo-1,8-naphthalimide (yield 60%).
步骤b):在惰性气氛下,将1.00g N-正丁基-4-溴-1,8-萘酰亚胺和2.15g对甲基苯硫酚加入50mL三颈瓶中,注入20mL丙醇和2.0mL吡啶,在50℃的温度下,回流反应8小时。反应完全后,将反应液倒入200mL冰水中,有大量固体析出,过滤,洗涤,真空干燥得,N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺0.76g(产率67%),黄色固体粉末。Step b): Under an inert atmosphere, add 1.00g N-n-butyl-4-bromo-1,8-naphthalimide and 2.15g p-methylthiophenol into a 50mL three-necked flask, inject 20mL propanol and 2.0 mL of pyridine was refluxed for 8 hours at a temperature of 50°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.76g (67% yield), yellow solid powder.
1H NMR(400MHz,CDCl3)δ8.63(d,J=7.9Hz,2H),8.32(d,J=7.9Hz,1H),7.77(t,J=7.9Hz,1H),7.45(d,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J=15.2,7.6Hz,3H),1.44(dq,J=14.8,7.3Hz,3H),0.97(t,J=7.3Hz,4H)。 1 H NMR (400MHz, CDCl 3 ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).
步骤c):在惰性气氛下,将0.9g N-正丁基-4-(对甲基苯硫基)-1,8-萘酰亚胺溶于20mL乙腈中,分三批向体系中加入0.45g间氯过氧苯甲酸,室温反应2小时。待反应完全后,旋干反应液,经柱层析分离纯化得到最后产物Nap-G 0.55g(产率57%),白色固体。Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylthio)-1,8-naphthalimide in 20mL of acetonitrile, and add to the system in three batches 0.45g m-chloroperoxybenzoic acid was reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.55 g (57% yield) of the final product Nap-G as a white solid.
1H NMR(400MHz,CDCl3)δ8.65(d,J=7.7Hz,1H),8.51(d,J=7.3Hz,1H),8.40(dd,J=8.1,2.7Hz,2H),7.68(t,J=7.9Hz,1H),7.49(d,J=8.1Hz,2H),7.13(d,J=8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H);13CNMR(100MHz,CDCl3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82;HRMS(ESI-TOF):m/z 391.1245[M+H]+,calc’d.391.1242. 1 H NMR (400MHz, CDCl 3 ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H); 13 CNMR(100MHz,CDCl 3 )δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33, 127.96, 127.49, 125.64, 124.99, 123.70, 123.50, 40.41, 30.12, 21.41, 20.34, 13.82; HRMS (ESI-TOF): m/z 391.1245[M+H] + , calc'd.391.1242.
实施例6、式(I)所示化合物的光谱性质Spectral properties of the compound shown in embodiment 6, formula (I)
称取3.9mg Nap-G,溶于10mL DMSO,配成母液(1mM),即得到Nap-G试剂盒。将100μL的此母液滴加到不同浓度谷胱甘肽的磷酸盐缓冲液中,并用相应的磷酸盐缓冲液定容到10mL。测量其荧光发射光谱。荧光发射光谱测定时以366nm进行激发,发射峰的强度为I482;激发和发射的狭缝宽度分别为5/5。Weigh 3.9mg of Nap-G, dissolve it in 10mL DMSO, and prepare the mother solution (1mM) to obtain the Nap-G kit. Add 100 μL of this mother solution dropwise to phosphate buffer saline with different concentrations of glutathione, and dilute to 10 mL with the corresponding phosphate buffer. Measure its fluorescence emission spectrum. When measuring the fluorescence emission spectrum, excitation was performed at 366 nm, and the intensity of the emission peak was I 482 ; the slit widths of excitation and emission were 5/5, respectively.
图2为Nap-G试剂盒对谷胱甘肽水溶液的颜色响应图。由图2知,当加入谷胱甘肽水溶液后,肉眼观察到溶液的颜色变化不明显,但溶液的荧光由未加谷胱甘肽时的无荧光变为加后的亮绿色荧光。证明本发明试剂盒对谷胱甘肽具有直观的显色响应。Figure 2 is the color response diagram of Nap-G kit to glutathione aqueous solution. As can be seen from Figure 2, after the glutathione aqueous solution was added, the naked eye observed that the color of the solution did not change significantly, but the fluorescence of the solution changed from no fluorescence when no glutathione was added to bright green fluorescence after addition. It is proved that the kit of the present invention has an intuitive color response to glutathione.
图3为Nap-G试剂盒对不同谷胱甘肽水溶液的荧光响应图。由图3知,随着谷胱甘肽浓度的增加,波长为482nm处的发射峰的荧光强度逐渐增大,证明本发明试剂盒对谷胱甘肽具有灵敏的“关-开”型响应。Fig. 3 is the fluorescence response graph of Nap-G kit to different glutathione aqueous solutions. As can be seen from Figure 3, with the increase of glutathione concentration, the fluorescence intensity of the emission peak at a wavelength of 482nm gradually increases, which proves that the kit of the present invention has a sensitive "off-on" type response to glutathione.
图4为Nap-G试剂盒在波长482nm下的荧光发射强度I482与谷胱甘肽浓度关系曲线。由图4知,随着水溶液中谷胱甘肽浓度的增加,荧光发射强度I482逐渐增大。在谷胱甘肽浓度为0~1mM的范围内,发射峰的荧光发射强度I482与水溶液中谷胱甘肽的浓度呈良好的线性关系(R2=0.995)。证明本发明试剂盒以对谷胱甘肽进行准确测量。Fig. 4 is the curve of the relationship between the fluorescence emission intensity I 482 and the glutathione concentration of the Nap-G kit at a wavelength of 482nm. It can be seen from Figure 4 that with the increase of glutathione concentration in the aqueous solution, the fluorescence emission intensity I 482 increases gradually. When the glutathione concentration is in the range of 0-1 mM, the fluorescence emission intensity I 482 of the emission peak has a good linear relationship with the concentration of glutathione in the aqueous solution (R 2 =0.995). The kit of the invention is proven to perform accurate measurements of glutathione.
图5为Nap-G试剂盒对常见共存离子或生物小分子的荧光响应图。由图5知,常见共存阳离子、阴离子、生物小分子的加入并不能使溶液的荧光发射强度I482发生改变。证明本发明试剂盒对谷胱甘肽具有优秀的选择性。Figure 5 is the fluorescence response graph of the Nap-G kit to common coexisting ions or small biological molecules. It can be known from Figure 5 that the addition of common coexisting cations, anions, and small biomolecules cannot change the fluorescence emission intensity I 482 of the solution. It is proved that the kit of the present invention has excellent selectivity to glutathione.
实施例7、细胞内谷胱甘肽含量的测定Embodiment 7, the mensuration of intracellular glutathione content
1)在37度和5%(v/v)CO2条件下,用含有10%(v/v)FBS(胎牛血清)、100U/mL盘尼西林、100μg/mL的链霉素的DMEM培养基培养HeLa细胞。细胞使用前用PBS缓冲液清洗。1) Under the conditions of 37 degrees and 5% (v/v) CO 2 , use DMEM medium containing 10% (v/v) FBS (fetal bovine serum), 100 U/mL penicillin, and 100 μg/mL streptomycin Culture HeLa cells. Cells were washed with PBS buffer before use.
2)在HeLa细胞中加入PBS(pH 7.4),再加入Nap-G(10μM)孵育30min,用PBS洗三遍后,进行共聚焦荧光成像,其中激发波长为366nm,收集波段为400-650nm。然后,向上述HeLa细胞中再加入GSH(1mM)的磷酸缓冲盐溶液,继续孵育30min后,在激光共聚焦显微镜上进行成像,其中激发波长为366nm,收集波段为400-650nm。2) Add PBS (pH 7.4) to HeLa cells, then add Nap-G (10 μM) and incubate for 30 minutes, wash with PBS three times, and perform confocal fluorescence imaging, where the excitation wavelength is 366nm and the collection wavelength is 400-650nm. Then, add GSH (1mM) phosphate-buffered saline solution to the above HeLa cells, continue to incubate for 30min, and perform imaging on a laser confocal microscope, wherein the excitation wavelength is 366nm, and the collection waveband is 400-650nm.
由图6知,载有Nap-G的细胞在未加谷胱甘肽之前呈现荧光“关”的状态,无荧光发射;而当加入谷胱甘肽以后,细胞呈现荧光“开”的状态,发射绿色荧光,表明Nap-G以很好地透过细胞膜,并且以在细胞内与谷胱甘肽发生特异性响应。证明本发明试剂盒以在细胞中对谷胱甘肽进行检测。As can be seen from Figure 6, before the addition of glutathione, the cells loaded with Nap-G were in the "off" state of fluorescence, without fluorescence emission; however, after adding glutathione, the cells were in the state of "on" fluorescence, It emits green fluorescence, indicating that Nap-G can penetrate the cell membrane well and respond specifically to glutathione in the cell. The kit of the present invention is demonstrated to detect glutathione in cells.
最后应说明的是,上述实施例仅举出以Nap-G化合物为荧光试剂,其余荧光试剂由于结构与性质相近,其浓度、实验激发波段选择不一一列出,然而其并非用于限定本发明。任何本领域的技术人员,在不脱离本发明的精神和范围的情况下,应当以作出各种修改和变更。Finally, it should be noted that the above examples only use the Nap-G compound as the fluorescent reagent, and the concentration and experimental excitation band selection of the remaining fluorescent reagents are not listed one by one due to their similar structures and properties. invention. Any person skilled in the art should make various modifications and changes without departing from the spirit and scope of the present invention.
Claims (7)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510083494.9A CN104673278A (en) | 2015-02-15 | 2015-02-15 | Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510083494.9A CN104673278A (en) | 2015-02-15 | 2015-02-15 | Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe |
Publications (1)
Publication Number | Publication Date |
---|---|
CN104673278A true CN104673278A (en) | 2015-06-03 |
Family
ID=53308883
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510083494.9A Pending CN104673278A (en) | 2015-02-15 | 2015-02-15 | Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104673278A (en) |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105001161A (en) * | 2015-06-08 | 2015-10-28 | 华中师范大学 | Sulfamide compound based on 1,8-naphthalimides as framework and preparation method and application thereof |
CN105601658A (en) * | 2016-01-15 | 2016-05-25 | 中南大学 | Application and preparation method of novel fluorescent probe capable of distinguishing biological mercaptans |
CN106478505A (en) * | 2016-08-28 | 2017-03-08 | 浙江工业大学 | A kind of two-photon GSH probe and its preparation and application |
CN107033879A (en) * | 2017-04-26 | 2017-08-11 | 许昌学院 | It is a kind of to be used to detect fluorescence probe of glutathione and its preparation method and application |
CN107235946A (en) * | 2017-06-20 | 2017-10-10 | 济南大学 | A kind of glutathione fluorescence probe and its preparation method and application |
CN107677649A (en) * | 2016-08-02 | 2018-02-09 | 天津师范大学 | The method of label-free indium sulphur fluorescence probe synchronous fluorescence standard measure detection glutathione |
CN108362673A (en) * | 2018-03-07 | 2018-08-03 | 济南大学 | A method of detection glutathione, histidine |
CN109400530A (en) * | 2018-12-05 | 2019-03-01 | 安庆北化大科技园有限公司 | Suitable for the naphthalimide aromatic sulfide type photoinitiator and preparation method of UV-LED photocuring and application |
CN109734647A (en) * | 2019-02-28 | 2019-05-10 | 浙江理工大学 | A kind of fluorescent probe for detecting cysteine and preparation method and use method thereof |
CN110041317A (en) * | 2019-05-16 | 2019-07-23 | 福州大学 | A kind of naphthalimide fluorescence probe and its preparation and application |
CN110357867A (en) * | 2019-07-26 | 2019-10-22 | 大连大学 | A kind of glutathione ratio fluorescent sensor and its preparation and application based on cystine linkage |
CN110590663A (en) * | 2019-09-23 | 2019-12-20 | 广西师范大学 | A kind of 1,8-naphthalimide derivative and application thereof |
CN110590664A (en) * | 2019-09-23 | 2019-12-20 | 广西师范大学 | A kind of preparation method of fluorescent probe and the application of this fluorescent probe |
CN115003678A (en) * | 2019-12-30 | 2022-09-02 | 塞尔吐温株式会社 | Real-time fluorescence imaging sensor for measuring glutathione in endoplasmic reticulum and method of using the same |
CN115322225A (en) * | 2022-07-12 | 2022-11-11 | 上海工程技术大学 | Naphthalimide derivative fluorescent dye and preparation method and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102532178A (en) * | 2011-12-23 | 2012-07-04 | 浙江理工大学 | Fluorescent probe for detecting biologic thiol and preparation method and usage method thereof |
WO2013126816A1 (en) * | 2012-02-23 | 2013-08-29 | Oregon State Board Of Higher Education On Behalf Of Portland State University | Selective detection of thiols |
-
2015
- 2015-02-15 CN CN201510083494.9A patent/CN104673278A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102532178A (en) * | 2011-12-23 | 2012-07-04 | 浙江理工大学 | Fluorescent probe for detecting biologic thiol and preparation method and usage method thereof |
WO2013126816A1 (en) * | 2012-02-23 | 2013-08-29 | Oregon State Board Of Higher Education On Behalf Of Portland State University | Selective detection of thiols |
Non-Patent Citations (2)
Title |
---|
R. CHEN ET AL.,: ""Novel chemosensors for detection of glutathione by reduction or substitution of naphthalimide derivatives containing sulfoxide or sulfone substituents"及其supporting information", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
S. CHATTERJEE ET AL.,: ""Synthesis and photoinduced intramolecular charge transfer of N-substituted 1,8-naphthalimide derivatives in homogeneous solvents and in presence of reduced glutathione"", 《JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A: CHEMISTRY》 * |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105001161B (en) * | 2015-06-08 | 2018-02-02 | 华中师范大学 | One kind is based on sulfonamide compounds of 1,8 naphthalimides for skeleton, preparation method and applications |
CN105001161A (en) * | 2015-06-08 | 2015-10-28 | 华中师范大学 | Sulfamide compound based on 1,8-naphthalimides as framework and preparation method and application thereof |
CN105601658A (en) * | 2016-01-15 | 2016-05-25 | 中南大学 | Application and preparation method of novel fluorescent probe capable of distinguishing biological mercaptans |
CN107677649B (en) * | 2016-08-02 | 2020-01-07 | 天津师范大学 | Method for quantitative detection of glutathione by simultaneous fluorescence assay with label-free copper indium sulfide fluorescent probe |
CN107677649A (en) * | 2016-08-02 | 2018-02-09 | 天津师范大学 | The method of label-free indium sulphur fluorescence probe synchronous fluorescence standard measure detection glutathione |
CN106478505A (en) * | 2016-08-28 | 2017-03-08 | 浙江工业大学 | A kind of two-photon GSH probe and its preparation and application |
CN106478505B (en) * | 2016-08-28 | 2019-04-09 | 浙江工业大学 | A two-photon GSH probe and its preparation and application |
CN107033879A (en) * | 2017-04-26 | 2017-08-11 | 许昌学院 | It is a kind of to be used to detect fluorescence probe of glutathione and its preparation method and application |
CN107235946A (en) * | 2017-06-20 | 2017-10-10 | 济南大学 | A kind of glutathione fluorescence probe and its preparation method and application |
CN108362673A (en) * | 2018-03-07 | 2018-08-03 | 济南大学 | A method of detection glutathione, histidine |
CN108362673B (en) * | 2018-03-07 | 2020-09-01 | 济南大学 | Method for detecting glutathione and histidine |
CN109400530B (en) * | 2018-12-05 | 2022-04-12 | 安庆北化大科技园有限公司 | Naphthalimide aryl thioether type photoinitiator suitable for UV-LED photocuring, and preparation method and application thereof |
CN109400530A (en) * | 2018-12-05 | 2019-03-01 | 安庆北化大科技园有限公司 | Suitable for the naphthalimide aromatic sulfide type photoinitiator and preparation method of UV-LED photocuring and application |
CN109734647A (en) * | 2019-02-28 | 2019-05-10 | 浙江理工大学 | A kind of fluorescent probe for detecting cysteine and preparation method and use method thereof |
CN110041317A (en) * | 2019-05-16 | 2019-07-23 | 福州大学 | A kind of naphthalimide fluorescence probe and its preparation and application |
CN110041317B (en) * | 2019-05-16 | 2022-01-04 | 福州大学 | Naphthalimide fluorescent probe and preparation and application thereof |
CN110357867A (en) * | 2019-07-26 | 2019-10-22 | 大连大学 | A kind of glutathione ratio fluorescent sensor and its preparation and application based on cystine linkage |
CN110357867B (en) * | 2019-07-26 | 2022-07-01 | 大连大学 | Glutathione ratio fluorescence sensor based on disulfide bond and preparation and application thereof |
CN110590663A (en) * | 2019-09-23 | 2019-12-20 | 广西师范大学 | A kind of 1,8-naphthalimide derivative and application thereof |
CN110590664A (en) * | 2019-09-23 | 2019-12-20 | 广西师范大学 | A kind of preparation method of fluorescent probe and the application of this fluorescent probe |
CN115003678A (en) * | 2019-12-30 | 2022-09-02 | 塞尔吐温株式会社 | Real-time fluorescence imaging sensor for measuring glutathione in endoplasmic reticulum and method of using the same |
CN115003678B (en) * | 2019-12-30 | 2024-05-10 | 塞尔吐温株式会社 | Real-time fluorescence imaging sensor for measuring glutathione in endoplasmic reticulum and method of using the same |
CN115322225A (en) * | 2022-07-12 | 2022-11-11 | 上海工程技术大学 | Naphthalimide derivative fluorescent dye and preparation method and application thereof |
CN115322225B (en) * | 2022-07-12 | 2024-10-29 | 上海工程技术大学 | Naphthalimide derivative fluorescent dye and preparation method and application thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104673278A (en) | Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe | |
CN104710979A (en) | Fluorescent probe used for detecting glutathione as well as preparation method and application thereof | |
CN105542756B (en) | A kind of fluorescence probe for detecting formaldehyde and preparation method and application | |
EP3489324A1 (en) | Fluorescent probe and preparation method and use thereof | |
CN104610959B (en) | Fluorescence probe for detecting hydrogen sulfide as well as preparation method and application method of fluorescence probe | |
CN103087545B (en) | A class of fluorescent dyes based on fluorescein, its preparation method and application | |
CN103382313B (en) | A kind of naphthalimide fluorescent dye and its preparation and application | |
CN108752331A (en) | Synthesis and application a kind of while that distinguish detection Cys, Hcy and GSH Multifunction fluorescent molecular probe | |
CN115650897B (en) | Fluorescent probe for simultaneously detecting Cys and mitochondrial viscosity and preparation method and application thereof | |
CN107098923A (en) | One class feux rouges targets fluorescent dye and preparation method thereof and purposes near infrared emission lysosome | |
CN105693736B (en) | A kind of light-operated fluorescent switch probe of the rhodamine containing glycine structure and its application | |
CN108329302A (en) | A kind of half flower cyanines class near infrared fluorescent probe compound of sulfide specificly-response and its preparation method and application | |
CN112409238B (en) | Benzindole compounds and preparation method and application in detection of cysteine | |
CN103194085A (en) | Novel BODIPY flourescent dye with adjustable emission wavelength and preparation method thereof | |
CN108440476A (en) | One kind is used for while detecting hydrazine hydrate and sulfurous acid(Hydrogen)Fluorescence probe of salt and its preparation method and application | |
CN104744453B (en) | Semicyanines for the detection of mitochondrial polarity | |
CN105062467B (en) | Rotor-type two-photon mitochondrion fluorescence probe and application thereof | |
CN103382189B (en) | A class of cyanine compound, its preparation method and application | |
CN104610960B (en) | A kind of fluorescent probe of detection cysteine and preparation method thereof and using method | |
Chen et al. | Rhodamine Fluorophores for STED Super‐Resolution Biological Imaging | |
CN106432312A (en) | Mitochondria target fluorescence probe, as well as preparation method and application thereof | |
CN102702769A (en) | A kind of green light fluorescent cyanine dye, preparation method and application thereof | |
CN110407865B (en) | The compound of formula (I) based on benzenesulfonamide structure and its preparation method and application | |
Gao et al. | Development of a highly selective two-photon probe for methylglyoxal and its applications in living cells, tissues, and zebrafish | |
CN103333211B (en) | A class of dual-wavelength emission, double heteronuclear metal complexes and their preparation methods and applications |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150603 |