技术领域technical field
本发明属于荧光染料技术领域，特别是涉及一种具有荧光开启性质的荧光染料、制备方法及其应用。The invention belongs to the technical field of fluorescent dyes, and in particular relates to a fluorescent dye with fluorescence opening properties, a preparation method and an application thereof.
背景技术Background technique
8-氨基喹啉是一种常见的药物和化学中间体，而8-氨基喹啉类药物是最早被发现，且至今仍在用于临床合成的抗疟疾药。早在1926年，8-氨基喹啉类药物-扑疟喹啉已被用于临床。近年来，随着荧光探针的发展，发现8-氨基喹啉基于带有共轭的喹啉结构所产生的荧光在小分子检测和生物活性检测中具有优良的性质。更令人惊喜的是，近些年来研究者们对其药物价值的不断深入研究发现，功能化的8-氨基喹啉具有良好的抗肿瘤和抗阿尔海默兹症性质。因此，功能化喹啉特别是8-氨基喹啉，在生物化学、材料化学、以及药物化学领域具有重要和广泛的意义。8-Aminoquinoline is a common drug and chemical intermediate, and 8-aminoquinoline drugs are the first to be discovered and are still used in clinical synthesis of antimalarial drugs. As early as 1926, the 8-aminoquinoline drug - paramalaria quinoline has been used clinically. In recent years, with the development of fluorescent probes, it has been found that the fluorescence generated by 8-aminoquinoline based on the conjugated quinoline structure has excellent properties in small molecule detection and biological activity detection. What is even more surprising is that in recent years, researchers have continuously in-depth research on its drug value and found that functionalized 8-aminoquinoline has good anti-tumor and anti-Alheimer's properties. Therefore, functionalized quinolines, especially 8-aminoquinolines, have important and extensive significance in the fields of biochemistry, materials chemistry, and medicinal chemistry.
叠氮基和四氮杂萘被广泛用作LFBR的猝灭剂，可以通过铜催化的 (CuAAc)或“Diels-Alde”环加成进行生物正交反应。而叠氮基的荧光探针由于体积小，猝灭效率高广受研究者们的青睐。叠氮基可以开启覆盖广泛的发射范围点击反应，在过去的几年中，许多研究人员在活细胞中报道了用叠氮化荧光探针进行细胞外或细胞内生物分子标记。然而，尽管该部分工作已经取得了丰富的研究成果，但目前关于叠氮化荧光探针的报道很少，甚至没有可以形成一个新型且具有优异荧光性能的基于叠氮基的生物正交turn-on探针的相关报道。Azido and tetraazine are widely used as quenchers for LFBR, which can be bioorthogonal via copper-catalyzed (CuAAc) or “Diels-Alde” cycloaddition. Azido-based fluorescent probes are widely favored by researchers due to their small size and high quenching efficiency. The azide group can initiate click reactions covering a broad emission range, and in the past few years, many researchers have reported extracellular or intracellular biomolecular labeling with azide fluorescent probes in living cells. However, although this part of the work has achieved rich research results, there are few reports on azide fluorescent probes, and there is even no azido-based bioorthogonal turn- Related reports on the on probe.
目前，直接将叠氮基引入相对复杂的分子仍相当困难。虽然研究人员已尝试了从硝基到氨基再到叠氮基，通过这三个陡度引入叠氮基团，但优异的荧光探针应该具备点击反应后具有高度荧光，具有快速的反应动力学 (以允许有效的成像和时间分辨率)，并且易于修饰(以引入叠氮基团)。而显然现有的利用基于叠氮基的LFBR的方法无法完全达到以上标准。因此，尽管叠氮化物具有潜在的应用价值，但是当下一个主要的局限性在于缺乏复杂的合成方法，这阻碍了新的荧光叠氮化物探针的发展。同样也意味着探索便捷地引入叠氮基的新的合成方法已然迫在眉睫。At present, it is still quite difficult to directly introduce azido groups into relatively complex molecules. Although researchers have tried to introduce azide groups from nitro to amino groups to azide groups through these three steepnesses, excellent fluorescent probes should be highly fluorescent after click reactions and have fast reaction kinetics (to allow efficient imaging and temporal resolution), and are easily modified (to introduce azide groups). Obviously, the existing methods using azido-based LFBR cannot fully meet the above standards. Therefore, despite the potential applications of azides, the next major limitation is the lack of complex synthetic methods, which hinders the development of new fluorescent azide probes. It also means that it is imminent to explore new synthetic methods for conveniently introducing azido groups.
综上，本发明针对上述现有技术存在的问题，开发出一种具有荧光开启性质的荧光染料、制备方法及其应用。To sum up, the present invention aims at the problems existing in the above-mentioned prior art, and develops a fluorescent dye with fluorescence-on property, a preparation method and its application.
发明内容Contents of the invention
本发明的目的在于提供一种具有荧光开启性质的荧光染料、制备方法及其应用。The object of the present invention is to provide a kind of fluorescent dye with fluorescence opening property, preparation method and application thereof.
为了达到上述目的，本发明采取以下技术方案：In order to achieve the above object, the present invention takes the following technical solutions:
一种具有荧光开启性质的荧光染料，为5-叠氮-8-氨基喹啉类荧光染料，该荧光染料在其结构式如式(I)所示：A kind of fluorescent dye with fluorescence opening property is 5-azido-8-aminoquinoline fluorescent dye, and this fluorescent dye is shown in formula (I) in its structural formula:
其中，式(I)中的R1为H、苯乙烯、丁-1,3-二烯-1-基苯、或4-乙烯基苯酚；R2为H或苯乙烯；R3为H或苯乙烯。Wherein, R in formula (I)1 is H, styrene, but-1,3-dien-1-ylbenzene, or 4-vinylphenol; R2 is H or styrene; R3 is H or styrene.
优选的，所述荧光染料在5-叠氮-8-氨基喹啉的R1、R2或R3位为共轭基团。本发明通过在式(I)所示化合物的C-2、C-3、C-6位引入共轭基团，以此来增加喹啉共轭体系的大小，从而可以提高荧光强度和发射波长。Preferably, the fluorescent dye is a conjugated group at the R1 , R2 or R3 position of 5-azido-8-aminoquinoline. The present invention increases the size of the quinoline conjugated system by introducing conjugated groups at the C-2, C-3, and C-6 positions of the compound shown in formula (I), thereby increasing the fluorescence intensity and emission wavelength .
优选的，所述荧光染料的结构如式(DA)、(DA-1)、(DA-2)、 (DA-3)、(DA-4)或(DA-5)所示：Preferably, the structure of the fluorescent dye is shown in formula (DA), (DA-1), (DA-2), (DA-3), (DA-4) or (DA-5):
式(DA)所示的荧光染料的制备方法，制备路线如下所示：The preparation method of the fluorescent dye shown in formula (DA), the preparation route is as follows:
制备方法包括如下步骤：The preparation method comprises the following steps:
(1)化合物1与三乙胺溶于二氯甲烷中，在室温下搅拌，反应液冷却后，滴加酰氯搅拌过夜，得到化合物2；(1) Compound 1 and triethylamine were dissolved in dichloromethane, stirred at room temperature, after the reaction solution was cooled, acid chloride was added dropwise and stirred overnight to obtain Compound 2;
(2)选择NaN3作为叠氮化试剂，K2S2O8作为氧化剂，铜(II)化合物催化化合物2的叠氮化反应，得到化合物DQ；(2) NaN3 is selected as the azidation reagent, K2 S2 O8 is used as the oxidant, and the copper (II) compound catalyzes the azidation reaction of compound 2 to obtain compound DQ;
(3)在80-85℃甲醇溶液中用KOH水解化合物DQ得到所述式(I) 所示的荧光染料。(3) Hydrolyzing the compound DQ with KOH in methanol solution at 80-85° C. to obtain the fluorescent dye represented by the formula (I).
在DA的C-2、C-3、C-6位引入共轭基团的合成路线如下：The synthetic route for introducing conjugated groups at the C-2, C-3, and C-6 positions of DA is as follows:
一、在C-2位引入共轭基团合成路线：1. The synthetic route of introducing a conjugated group at the C-2 position:
式4-11所示的荧光染料的制备方法包括如下步骤：The preparation method of the fluorescent dye shown in formula 4-11 comprises the steps:
(1)将化合物4-5(2-甲基-8-硝基喹啉)与N-溴代丁二酰亚胺溶于四氯化碳溶剂，AIBN作为偶联试剂，在80℃条件下回流5小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-6；(1) Dissolve compound 4-5 (2-methyl-8-nitroquinoline) and N-bromosuccinimide in carbon tetrachloride solvent, AIBN as coupling reagent, at 80°C After refluxing for 5 hours, cool to room temperature, extract with dichloromethane, and rotary evaporate under reduced pressure to obtain compound 4-6;
(2)将化合物4-6溶于N,N-二甲基乙二胺(DMF)，向其中加入适量的三苯基磷，在100℃下反应12小时后，冷却至室温，向其中加入50 mL的甲苯，使产物析出，得到化合物4-7；(2) Dissolve compound 4-6 in N,N-dimethylethylenediamine (DMF), add an appropriate amount of triphenylphosphine to it, react at 100°C for 12 hours, cool to room temperature, add 50 mL of toluene, the product was precipitated to obtain compound 4-7;
(3)将化合物4-7溶于N,N-二甲基乙二胺，向其中加入适量的烯烃苯基(如苯乙烯、1,3-苯丁烯、对羟基苯乙烯)，在100℃下反应12小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-8；(3) Dissolving compound 4-7 in N,N-dimethylethylenediamine, adding an appropriate amount of olefin phenyl (such as styrene, 1,3-phenylbutene, p-hydroxystyrene), at 100 After reacting at ℃ for 12 hours, cooled to room temperature, extracted with dichloromethane, and rotary evaporated under reduced pressure to obtain compound 4-8;
(4)将化合物4-8溶于乙醇，向其中加入适量的还原铁粉和乙酸，在80℃下回流反应12小时后，趁热过滤，收集有机相，冷却后用二氯甲烷萃取，减压旋蒸，得到化合物4-9。(4) Compound 4-8 was dissolved in ethanol, an appropriate amount of reduced iron powder and acetic acid were added thereto, and after reflux reaction at 80°C for 12 hours, it was filtered while it was hot, and the organic phase was collected, extracted with dichloromethane after cooling, and reduced Rotary steaming under pressure to obtain compound 4-9.
(5)化合物4-9与三乙胺溶于二氯甲烷中，在室温下搅拌，反应液冷却后，滴加酰氯搅拌过夜，得到化合物4-10；(5) Compound 4-9 and triethylamine were dissolved in dichloromethane, stirred at room temperature, after the reaction solution was cooled, acid chloride was added dropwise and stirred overnight to obtain compound 4-10;
(6)选择NaN3作为叠氮化试剂，K2S2O8作为氧化剂，铜(II)化合物催化化合物4-10的叠氮化反应，得到化合物4-11；(6) NaN3 is selected as the azidation reagent, K2 S2 O8 is used as the oxidant, and the copper (II) compound catalyzes the azidation reaction of compound 4-10 to obtain compound 4-11;
二、在C-3位引入共轭基团合成路线2. Introducing a conjugated group synthesis route at the C-3 position
式4-27所示的荧光染料的制备方法包括如下步骤：The preparation method of the fluorescent dye shown in formula 4-27 comprises the steps:
(1)将化合物4-21(8-硝基喹啉)与适量的TBHP和碘溶于乙醇，在80℃条件下回流12小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-22；(1) Dissolve compound 4-21 (8-nitroquinoline) with appropriate amount of TBHP and iodine in ethanol, reflux at 80°C for 12 hours, cool to room temperature, extract with dichloromethane, spin under reduced pressure Steam to obtain compound 4-22;
(2)将化合物4-22(3-碘-8-氨基喹啉化合物)溶于N,N-二甲基乙二胺(DMF)，向其中加入适量的醋酸钯和烯烃苯基(如：苯乙烯)，在80℃下反应12小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-23；(2) Compound 4-22 (3-iodo-8-aminoquinoline compound) is dissolved in N,N-dimethylethylenediamine (DMF), and an appropriate amount of palladium acetate and olefin phenyl (such as: Styrene), reacted at 80°C for 12 hours, cooled to room temperature, extracted with dichloromethane, and rotary evaporated under reduced pressure to obtain compound 4-23;
(3)将化合物4-23溶于乙醇，向其中加入适量的还原铁粉和三倍当量的乙酸，在100℃下反应12小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-24；(3) Dissolve compound 4-23 in ethanol, add an appropriate amount of reduced iron powder and three times the equivalent of acetic acid, react at 100°C for 12 hours, cool to room temperature, extract with dichloromethane, spin under reduced pressure Steam to obtain compound 4-24;
(4)将化合物4-24与三乙胺溶于二氯甲烷中，在室温下搅拌，反应液冷却后，滴加酰氯搅拌过夜，得到化合物4-25；(4) Compound 4-24 and triethylamine were dissolved in dichloromethane, stirred at room temperature, after the reaction solution was cooled, acid chloride was added dropwise and stirred overnight to obtain compound 4-25;
(6)选择NaN3作为叠氮化试剂，K2S2O8作为氧化剂，铜(II)化合物催化化合物4-10的叠氮化反应，得到化合物4-26；(6) NaN3 is selected as the azidation reagent, K2 S2 O8 is used as the oxidant, and the copper (II) compound catalyzes the azidation reaction of compound 4-10 to obtain compound 4-26;
(7)在80-85℃甲醇溶液中用KOH水解化合物4-26得到所述4-27 的荧光染料。(7) Hydrolyzing compound 4-26 with KOH in methanol solution at 80-85° C. to obtain the fluorescent dye of 4-27.
三、在C-6位引入共轭基团合成路线3. Introducing a conjugated group synthesis route at the C-6 position
式4-20所示的荧光染料的制备方法包括如下步骤：The preparation method of the fluorescent dye shown in formula 4-20 comprises the steps:
(1)将化合物4-13(2-甲基-8-硝基喹啉)与适量的N-溴代丁二酰亚胺和AIBN溶于四氯化碳，在80℃条件下回流5小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-14；(1) Dissolve compound 4-13 (2-methyl-8-nitroquinoline) with an appropriate amount of N-bromosuccinimide and AIBN in carbon tetrachloride, and reflux at 80°C for 5 hours Then, cooled to room temperature, extracted with dichloromethane, and rotary evaporated under reduced pressure to obtain compound 4-14;
(2)将化合物4-14(2-溴甲基-8-硝基喹啉)溶于四氯化碳，向其中加入适量的N-溴代丁二酰亚胺和AIBN，在80℃下反应5小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-15；(2) Dissolve compound 4-14 (2-bromomethyl-8-nitroquinoline) in carbon tetrachloride, add appropriate amount of N-bromosuccinimide and AIBN to it, at 80°C After reacting for 5 hours, cool to room temperature, extract with dichloromethane, and rotate under reduced pressure to obtain compound 4-15;
(3)将化合物4-15溶于N,N-二甲基乙二胺，向其中加入适量的烯烃苯基(如：苯乙烯)，在100℃下反应12小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-16；(3) Dissolve compound 4-15 in N,N-dimethylethylenediamine, add an appropriate amount of olefin phenyl (such as: styrene) to it, react at 100°C for 12 hours, cool to room temperature, and After extraction with dichloromethane, rotary evaporation under reduced pressure gave compound 4-16;
(4)将化合物4-16溶于乙醇，向其中加入适量的还原铁粉和乙酸，在80℃下反应12小时后，冷却至室温，经二氯甲烷萃取后，减压旋蒸，得到化合物4-17；(4) Dissolve compound 4-16 in ethanol, add an appropriate amount of reduced iron powder and acetic acid to it, react at 80°C for 12 hours, cool to room temperature, extract with dichloromethane, and spin evaporate under reduced pressure to obtain compound 4-17;
(5)将化合物4-17与三乙胺溶于二氯甲烷中，在室温下搅拌，反应液冷却后，滴加酰氯搅拌过夜，得到化合物4-18；(5) Dissolving compound 4-17 and triethylamine in dichloromethane, stirring at room temperature, after cooling the reaction solution, adding acid chloride dropwise and stirring overnight, to obtain compound 4-18;
(6)选择NaN3作为叠氮化试剂，K2S2O8作为氧化剂，铜(II)化合物催化化合物4-10的叠氮化反应，得到化合物4-19；(6) NaN3 is selected as the azidation reagent, K2 S2 O8 is used as the oxidant, and the copper (II) compound catalyzes the azidation reaction of compound 4-10 to obtain compound 4-19;
(7)在80-85℃甲醇溶液中用KOH水解化合物4-19得到所述4-20 的荧光染料。(7) Hydrolyzing compound 4-19 with KOH in methanol solution at 80-85° C. to obtain the fluorescent dye of 4-20.
本发明还提供具有荧光开启性质的一种荧光染料作为荧光探针的应用。The present invention also provides the application of a fluorescent dye with the property of opening fluorescence as a fluorescent probe.
进一步的，荧光染料的应用方法为：将式(1)所示荧光染料在五水合硫酸铜和维生素C的存在下，与炔基化合物在四氢呋喃和水(体积比为5:5)的混合溶剂中，在室温下进行反应得到环加成反应DZ。Further, the application method of the fluorescent dye is: the fluorescent dye shown in the formula (1) is mixed with the mixed solvent of the alkynyl compound in tetrahydrofuran and water (volume ratio is 5:5) in the presence of copper sulfate pentahydrate and vitamin C. In , the reaction was carried out at room temperature to give the cycloaddition reaction DZ.
化合物DZ在手持式紫外灯下便可以发生荧光。反应路线如下：Compound DZ can fluoresce under a hand-held UV lamp. The reaction scheme is as follows:
其中，R为苯、甲苯、乙苯、丙烷基、呋喃、甲氧基、氯苯、或者苯胺。Wherein, R is benzene, toluene, ethylbenzene, propyl group, furan, methoxy group, chlorobenzene, or aniline.
本发明具有以下技术特点：The present invention has the following technical characteristics:
本发明利用8-氨基喹啉类化合物为底物，通过C-H活化合成了非氟化合物5-叠氮-8-氨基喹啉类荧光染料。在与炔烃进行1,3-偶极环加成之后，三唑的形成提供具有高量子产率的荧光团。进而通过在氨基喹啉的 C-2,3,5或8位上使用不同的取代基，可以将发射波长从430nm调整到 595nm。通过利用这种“开启”特性，为活细胞生物成像用提供了有力工具。The invention uses 8-aminoquinoline compounds as substrates to synthesize non-fluorine compound 5-azido-8-aminoquinoline fluorescent dyes through C-H activation. After 1,3-dipolar cycloaddition with alkynes, the formation of triazoles affords fluorophores with high quantum yields. Furthermore, the emission wavelength can be adjusted from 430nm to 595nm by using different substituents on the C-2, 3, 5 or 8 positions of aminoquinoline. By exploiting this "turn-on" property, a powerful tool for live-cell bioimaging is provided.
附图说明Description of drawings
图1为实施例8-15制备的荧光团DZ-a～DZ-h的紫外吸收光谱。Fig. 1 is the ultraviolet absorption spectrum of the fluorophores DZ-a to DZ-h prepared in Examples 8-15.
图2为通过点击反应来研究荧光染料的荧光turn-on响应，检测实施例1、Fig. 2 is to research the fluorescent turn-on response of fluorescent dye by click reaction, detection embodiment 1,
实施例2、实施例4制备的DQ、DA以及DA-2在反应前后的荧光强度。图3为荧光染料在细胞中的turn-on的性能。选择了DQ，DZ和DZ-2作为例子.a)，b)，c)为10μM DQ和10μM乙炔基苯处理后的细胞成像；d)， e)，f)为加入10μM CuSO4和Vc的10μM DQ和10μM乙炔基苯处理后的细胞成像；g)，h)，i)为加入10μM CuSO4和Vc的10μM DZ和10μM乙炔基苯处理后的细胞成像；j)，k)，l)为加入10μM CuSO4和Vc处理10μM DZ-2和10μM乙炔基苯处理后的细胞成像。其中a)，d)，g)，j)为UV通道；b)，e)，h)，k)为白光通道；c)，f)，i)，l)为全覆盖。Fluorescence intensity of DQ, DA and DA-2 prepared in Example 2 and Example 4 before and after the reaction. Figure 3 shows the turn-on performance of fluorescent dyes in cells. DQ, DZ and DZ-2 are selected as examples. a), b), c) are images of cells treated with 10 μM DQ and 10 μM ethynylbenzene; d), e), f) are cells treated with 10 μM CuSO4 and Vc Image of cells treated with 10 μM DQ and 10 μM ethynylbenzene; g), h), i) is image of cells treated with 10 μM DZ and 10 μM ethynylbenzene added with 10 μM CuSO4 and Vc; j), k), l) Cells treated with 10 μM DZ-2 and 10 μM ethynylbenzene were imaged for the addition of 10 μM CuSO4 and Vc. Among them, a), d), g), j) are UV channels; b), e), h), k) are white light channels; c), f), i), l) are full coverage.
图4为荧光染料用于直接跟踪活细胞中的各种细胞器的研究。a)，b)，c) 为加入10μMCuSO4和Vc的5.0μM DZ和5.0μM N,N-二甲基丙-2-炔-1-胺 处理后的细胞成像；e)，f)，g)为加入10μMCuSO4和Vc的5.0μM DQ和 5.0μM N,N-二甲基丙-2-炔-1-胺处理后的细胞成像；i)，j)，k)为加入10μM CuSO4和Vc的5.0μM DA和5.0μM三苯基膦-炔基处理后的细胞成像。Figure 4 shows the use of fluorescent dyes to directly track various organelles in living cells. a), b), c) are images of cells treated with 5.0 μM DZ and 5.0 μM N,N-dimethylprop-2-yn-1-amine added with 10 μM CuSO4 and Vc; e), f), g ) is imaging of cells treated with 5.0 μM DQ and 5.0 μM N,N-dimethylprop-2-yn-1-amine with addition of 10 μM CuSO4 and Vc; i), j), k) are addition of 10 μM CuSO4 and Imaging of cells after Vc treatment with 5.0 μM DA and 5.0 μM triphenylphosphine-alkynyl.
具体实施方式Detailed ways
以下具体实施例是对本发明提供的方法与技术方案的进一步说明，但不应理解成对本发明的限制。The following specific examples are further descriptions of the methods and technical solutions provided by the present invention, but should not be construed as limiting the present invention.
实施例1：5-叠氮-8-氨基喹啉(DA)的合成Embodiment 1: the synthesis of 5-azido-8-aminoquinoline (DA)
在100mL单颈烧瓶中加入8-苯甲酰胺喹啉(10mmol)和KOH (20mmol)溶于适量甲醇，将所得混合物在80℃加热12小时，并冷却至室温，反应液用CH2Cl2洗涤三次，收集的CH2Cl2溶液用饱和NaCl水溶液洗涤三次后用Na2SO4干燥，并通过旋转蒸发除去溶剂，得到粗产物。粗产物进一步通过硅胶柱用PE/EtOAc(v/v；2:1)的混合溶剂作为洗脱液进行纯化，得产品DA(产率75％)的白色固体。Add 8-benzamide quinoline (10mmol) and KOH (20mmol) in an appropriate amount of methanol to a 100mL single-necked flask, heat the resulting mixture at 80°C for 12 hours, and cool to room temperature, and wash the reaction solution with CH2 Cl2 Three times, the collectedCH2Cl2 solution was washedthree times with saturated aqueous NaCl solution, driedoverNa2SO4 , and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was further purified by a silica gel column using a mixed solvent of PE/EtOAc (v/v; 2:1) as the eluent to obtain the product DA (yield 75%) as a white solid.
1H NMR(500MHz,CDCl3)δ8.11(s,1H),8.05(d,J＝8.6Hz,1H), 7.70(d,J＝16.3Hz,2H),7.64(dd,J＝13.1,8.0Hz,3H),7.44–7.40(m,2H), 7.36–7.28(m,2H),7.14(d,J＝8.0Hz,1H),6.94(dd,J＝7.5,0.9Hz,1H), 5.08(s,2H)；1H NMR (500MHz, CDCl3 ) δ8.11(s, 1H), 8.05(d, J=8.6Hz, 1H), 7.70(d, J=16.3Hz, 2H), 7.64(dd, J=13.1, 8.0 Hz,3H),7.44–7.40(m,2H), 7.36–7.28(m,2H),7.14(d,J＝8.0Hz,1H),6.94(dd,J＝7.5,0.9Hz,1H), 5.08 (s,2H);
13C NMR(126MHz,CDCl3)δ154.23,148.06,135.51,132.81,131.11.125.96.123.96.106.25.13C NMR (126MHz, CDCl3 ) δ154.23, 148.06, 135.51, 132.81, 131.11.125.96.123.96.106.25.
实施例2：染料前体化合物2(DQ)的合成Embodiment 2: the synthesis of dye precursor compound 2 (DQ)
在100mL单颈烧瓶中加入8-氨基喹啉(10mmol)和三乙胺(15mmol)，溶于CH2Cl2(30mL)。在室温下搅拌5分钟后，反应溶液在冰浴中冷却。滴加酰氯(11mmol)。然后将反应溶液搅拌过夜，随后将混合物通过硅藻土垫过滤，将残余物用CH2Cl2(25mL)洗涤收集的CH2Cl2溶液用1M NaHCO3水溶液洗涤三次。收集有机层并用无水Na2SO4干燥。通过旋转蒸发除去溶剂，得到的粗产物通过硅胶柱用PE/EtOAc(v/v；10:1)的混合溶剂纯化，得到纯产物2(产率80％)。Add 8-aminoquinoline (10 mmol) and triethylamine (15 mmol) into a 100 mL single-necked flask, and dissolve in CH2 Cl2 (30 mL). After stirring at room temperature for 5 minutes, the reaction solution was cooled in an ice bath. Acid chloride (11 mmol) was added dropwise. The reaction solution was then stirred overnight, then the mixture was filtered through a pad of celite, the residue was washed withCH2Cl2 (25 mL) and the collectedCH2Cl2 solution was washedthree times with 1M aqueousNaHCO3 . The organic layer was collected and driedover anhydrousNa2SO4 . The solvent was removed by rotary evaporation, and the obtained crude product was purified by a silica gel column with a mixed solvent of PE/EtOAc (v/v; 10:1) to obtain pure product 2 (yield 80%).
1H NMR(500MHz,CDCl3)δ10.61(s,1H),8.90(dd,J＝7.6,1.1Hz, 1H),8.68(dd,J＝4.2,1.6Hz,1H),8.03(dd,J＝3.7,2.1Hz,2H),7.95(dd,J ＝8.2,1.6Hz,1H),7.48(m,4H),7.36(dd,J＝8.2,1.1Hz,1H),7.26(dd,J＝ 8.2,4.2Hz,1H)；1H NMR (500MHz, CDCl3 ) δ10.61(s, 1H), 8.90(dd, J=7.6, 1.1Hz, 1H), 8.68(dd, J=4.2, 1.6Hz, 1H), 8.03(dd, J =3.7,2.1Hz,2H),7.95(dd,J=8.2,1.6Hz,1H),7.48(m,4H),7.36(dd,J=8.2,1.1Hz,1H),7.26(dd,J= 8.2, 4.2Hz, 1H);
13C NMR(126MHz,CDCl3)δ164.74,147.81,138.20,135.83,134.64, 134.09,131.42,128.37,127.47,126.84,126.80,121.26,121.18,116.00.13C NMR (126MHz, CDCl3 ) δ164.74, 147.81, 138.20, 135.83, 134.64, 134.09, 131.42, 128.37, 127.47, 126.84, 126.80, 121.26, 121.18, 116.00.
实施例3：染料DA-1的合成Embodiment 3: the synthesis of dyestuff DA-1
在250mL的圆底烧瓶中加入6-苯乙烯-5叠氮-8氨基喹啉(10mmol)， KOH(20mmol)，溶于100mL甲醇，将所得混合物在80℃加热12小时，并冷却至室温，通过旋转蒸发除去混合物，并将残余物用CH2Cl2洗涤三次。收集的CH2Cl2溶液用NaCl水溶液洗涤三次。之后，收集有机层并用 Na2SO4干燥。通过旋转蒸发除去溶剂。然后将粗产物通过硅胶柱用 PE/EtOAc(v/v：2:1)混合溶剂作为洗脱液进行纯化。并提供产品DA-1 (产率75％)的黄色固体。Add 6-styrene-5azido-8 aminoquinoline (10mmol) and KOH (20mmol) in a 250mL round bottom flask, dissolve in 100mL methanol, heat the resulting mixture at 80°C for 12 hours, and cool to room temperature, The mixture was removed by rotary evaporation, and the residue was washedthree times withCH2Cl2 .The collectedCH2Cl2 solution was washed three times with aqueous NaCl. After that, the organic layer was collected and dried withNa2SO4. Solvent was removed by rotary evaporation. Then the crude product was purified by silica gel column with PE/EtOAc (v/v: 2:1) mixed solvent as eluent. And afforded the product DA-1 (75% yield) as a yellow solid.
1H NMR(500MHz,CDCl3):δ＝5.92(s,2H),7.05(d,J＝2.5Hz, 1H),7.29-7.41(m,5H),7.60-7.63(m,4H),7.99(dd,J1＝14.0Hz,J2＝8.5Hz, 2H)；1H NMR (500MHz, CDCl3 ): δ=5.92(s, 2H), 7.05(d, J=2.5Hz, 1H), 7.29-7.41(m, 5H), 7.60-7.63(m, 4H), 7.99( dd, J1=14.0Hz, J2=8.5Hz, 2H);
13C NMR(125MHz,CDCl3):δ＝105.2,119.5,122.2,127.0,128.2, 128.3,128.7,129.0,130.5,133.1,134.9,136.6,144.2,153.6,157.5。13C NMR (125MHz, CDCl3 ): δ=105.2, 119.5, 122.2, 127.0, 128.2, 128.3, 128.7, 129.0, 130.5, 133.1, 134.9, 136.6, 144.2, 153.6, 157.5.
实施例4：染料DA-2的合成Embodiment 4: the synthesis of dyestuff DA-2
在250mL的圆底烧瓶中加入2-苯乙烯-5叠氮-8氨基喹啉(10mmol)， KOH(20mmol)，溶于100mL甲醇，将所得混合物在80℃加热12小时，并冷却至室温，通过旋转蒸发除去混合物，并将残余物用CH2Cl2洗涤三次。收集的CH2Cl2溶液用NaCl水溶液洗涤三次。之后，收集有机层并用 Na2SO4干燥。通过旋转蒸发除去溶剂。然后将粗产物通过硅胶柱用 PE/EtOAc(v/v：2:1)混合溶剂作为洗脱液进行纯化。并提供产品DA-2 (产率77％)的黄色固体。In a 250 mL round bottom flask, 2-styrene-5 azide-8 aminoquinoline (10 mmol), KOH (20 mmol) were added, dissolved in 100 mL of methanol, and the resulting mixture was heated at 80° C. for 12 hours, and cooled to room temperature, The mixture was removed by rotary evaporation, and the residue was washedthree times withCH2Cl2 .The collectedCH2Cl2 solution was washed three times with aqueous NaCl. After that, the organic layer was collected and dried withNa2SO4. Solvent was removed by rotary evaporation. Then the crude product was purified by silica gel column with PE/EtOAc (v/v: 2:1) mixed solvent as eluent. And afforded the product DA-2 (77% yield) as a yellow solid.
1H NMR(500MHz,CDCl3):δ＝5.11(s,2H,),7.05(d,J＝8.0Hz,1H,), 7.29-7.49(m,5H,),7.52-7.64(m,4H,),7.76(d,J＝8.5Hz,1H,),8.09(d,J＝ 8.5Hz,1H)；1H NMR (500MHz, CDCl3 ): δ=5.11(s,2H,),7.05(d,J=8.0Hz,1H,), 7.29-7.49(m,5H,),7.52-7.64(m,4H, ),7.76(d,J=8.5Hz,1H,),8.09(d,J=8.5Hz,1H);
13C NMR(125MHz,CDCl3):δ＝107.9,119.0,119.3,126.2,127.1, 128.2,128.3,128.4,128.6,129.5,133.9,136.2,136.5,139.9,155.0.13C NMR (125MHz, CDCl3 ): δ＝107.9, 119.0, 119.3, 126.2, 127.1, 128.2, 128.3, 128.4, 128.6, 129.5, 133.9, 136.2, 136.5, 139.9, 155.0.
实施例5：染料DA-3的合成Embodiment 5: the synthesis of dyestuff DA-3
在250mL的圆底烧瓶中加入3-苯乙烯-5叠氮-8氨基喹啉(10mmol)， KOH(20mmol)，溶于100mL甲醇，将所得混合物在80℃加热12小时，并冷却至室温，通过旋转蒸发除去混合物，并将残余物用CH2Cl2洗涤三次。收集的CH2Cl2溶液用NaCl水溶液洗涤三次。之后，收集有机层并用 Na2SO4干燥。通过旋转蒸发除去溶剂。然后将粗产物通过硅胶柱用 PE/EtOAc(v/v：2:1)混合溶剂作为洗脱液进行纯化。并提供产品DA-3 (70％)的黄色固体。In a 250 mL round bottom flask, 3-styrene-5 azide-8 aminoquinoline (10 mmol), KOH (20 mmol) were added, dissolved in 100 mL of methanol, the resulting mixture was heated at 80° C. for 12 hours, and cooled to room temperature, The mixture was removed by rotary evaporation, and the residue was washedthree times withCH2Cl2 .The collectedCH2Cl2 solution was washed three times with aqueous NaCl. After that, the organic layer was collected and dried withNa2SO4. Solvent was removed by rotary evaporation. The crude product was then purified by silica gel column with PE/EtOAc (v/v: 2:1) mixed solvent as eluent. And afforded the product DA-3 (70%) as a yellow solid.
1H NMR(500MHz,CDCl3):δ＝5.42(s,2H)，7.34-7.43(m,4H), 7.48-7.55(m,1H),7.64-7.65(m,2H),7.72(d,J＝8.5Hz,1H),7.83(d,J＝ 16.5Hz,1H),7.96-8.01(m,1H),8.19(d,J＝8.5Hz,1H)；1H NMR (500MHz, CDCl3 ): δ=5.42(s, 2H), 7.34-7.43(m, 4H), 7.48-7.55(m, 1H), 7.64-7.65(m, 2H), 7.72(d, J =8.5Hz,1H),7.83(d,J=16.5Hz,1H),7.96-8.01(m,1H),8.19(d,J=8.5Hz,1H);
13C NMR(125MHz,CDCl3):δ＝121.4,123.7,124.3,127.6,127.7, 128.0,128.5,128.8,129.1,131.5,133.7,136.0,136.2,136.6,139.5,148.0, 157.9.13C NMR (125MHz, CDCl3 ): δ＝121.4, 123.7, 124.3, 127.6, 127.7, 128.0, 128.5, 128.8, 129.1, 131.5, 133.7, 136.0, 136.2, 136.6, 139.5, 148.0, 157.9.
实施例6：染料DA-4的合成Embodiment 6: the synthesis of dyestuff DA-4
在250mL的圆底烧瓶中加入5叠氮-2-(1,3-苯丁二烯)-8氨基喹啉 (10mmol)，KOH(20mmol)，溶于100mL甲醇，将所得混合物在80℃加热12小时，并冷却至室温，通过旋转蒸发除去混合物，并将残余物用 CH2Cl2洗涤三次。收集的CH2Cl2溶液用NaCl水溶液洗涤三次。之后，收集有机层并用Na2SO4干燥。通过旋转蒸发除去溶剂。然后将粗产物通过硅胶柱用PE/EtOAc(v/v：2:1)混合溶剂作为洗脱液进行纯化。并提供产品DA-4(72％)的黄色固体。In a 250 mL round bottom flask, add 5-azido-2-(1,3-phenylbutadiene)-8 aminoquinoline (10 mmol), KOH (20 mmol), dissolve in 100 mL methanol, and heat the resulting mixture at 80 °C After 12 hours, and cooled to room temperature, the mixture was removed by rotary evaporation, and the residue was washedthree times withCH2Cl2 .The collectedCH2Cl2 solution was washed three times with aqueous NaCl. After that, the organic layer was collected and dried withNa2SO4. Solvent was removed by rotary evaporation. The crude product was then purified by silica gel column with PE/EtOAc (v/v: 2:1) mixed solvent as eluent. And afforded the product DA-4 (72%) as a yellow solid.
1H NMR(500MHz,CDCl3):δ＝6.32(s,2H)，7.37(d,J＝16.5Hz,1H), 7.47-7.52(m,5H),7.60-7.63(m,2H),7.69-7.77(m,2H),7.77(d,J＝7.5Hz, 2H),8.07(d,J＝8.5Hz,1H),8.11(d,J＝8.5Hz,1H)；1H NMR (500MHz, CDCl3 ): δ=6.32(s, 2H), 7.37(d, J=16.5Hz, 1H), 7.47-7.52(m, 5H), 7.60-7.63(m, 2H), 7.69- 7.77(m, 2H), 7.77(d, J=7.5Hz, 2H), 8.07(d, J=8.5Hz, 1H), 8.11(d, J=8.5Hz, 1H);
13C NMR(125MHz,CDCl3):δ＝119.4,122.5,126.3,127.4,127.5, 128.7,129.2,129.6,129.8,132.0,133.0,133.1,133.4，135.5,136.4,148.3, 155.6.13C NMR (125MHz, CDCl3 ): δ＝119.4, 122.5, 126.3, 127.4, 127.5, 128.7, 129.2, 129.6, 129.8, 132.0, 133.0, 133.1, 133.4, 135.5, 136.4, 148.3, 155.6.
实施例7：染料DA-5的合成Embodiment 7: the synthesis of dyestuff DA-5
在250mL的圆底烧瓶中加入2-对羟基苯乙烯-5叠氮-8氨基喹啉 (10mmol)，KOH(20mmol)，溶于100mL甲醇，将所得混合物在80℃加热12小时，并冷却至室温，通过旋转蒸发除去混合物，并将残余物用 CH2Cl2洗涤三次。收集的CH2Cl2溶液用NaCl水溶液洗涤三次。之后，收集有机层并用Na2SO4干燥。通过旋转蒸发除去溶剂。然后将粗产物通过硅胶柱用PE/EtOAc(v/v：2:1)混合溶剂作为洗脱液进行纯化。并提供产品DA-4(产率79％)的黄色固体。Add 2-p-hydroxystyrene-5 azide-8 aminoquinoline (10mmol), KOH (20mmol) in a 250mL round bottom flask, dissolve in 100mL methanol, heat the resulting mixture at 80°C for 12 hours, and cool to At room temperature, the mixture was removed by rotary evaporation, and the residue was washedthree times withCH2Cl2 .The collectedCH2Cl2 solution was washed three times with aqueous NaCl. After that, the organic layer was collected and dried withNa2SO4. Solvent was removed by rotary evaporation. Then the crude product was purified by silica gel column with PE/EtOAc (v/v: 2:1) mixed solvent as eluent. And afforded the product DA-4 (79% yield) as a yellow solid.
1H NMR(500MHz,CDCl3):δ＝5.32(s,2H)，7.45-7.49(m,2H,),7.59(t, J＝8.0Hz,1H),7.64(d,J＝9.0Hz,1H),7.74(d,J＝8.5Hz,1H),7.81(d,J＝ 16.5Hz,1H),7.85(d,J＝7.5Hz,1H),7.90(d,J＝7.5Hz,1H),8.10-8.15(m, 3H),10.08(s,1H)；1H NMR (500MHz, CDCl3 ): δ=5.32(s, 2H), 7.45-7.49(m, 2H,), 7.59(t, J=8.0Hz, 1H), 7.64(d, J=9.0Hz, 1H ),7.74(d,J=8.5Hz,1H),7.81(d,J=16.5Hz,1H),7.85(d,J=7.5Hz,1H),7.90(d,J=7.5Hz,1H), 8.10-8.15(m, 3H), 10.08(s, 1H);
13C NMR(125MHz,CDCl3):δ＝125.8,127.3,128.1,128.2, 128.7,129.5,129.7,130.1,132.9,133.4,135.7,136.3,136.9,137.3,148.6, 156.1,192.0.13C NMR (125MHz, CDCl3 ): δ＝125.8, 127.3, 128.1, 128.2, 128.7, 129.5, 129.7, 130.1, 132.9, 133.4, 135.7, 136.3, 136.9, 137.3, 148.6, 156.1, 192.0.
实施例8：荧光团DZ-a的合成Example 8: Synthesis of Fluorophore DZ-a
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL 二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率 65％。Add 0.01mol 5-azide 8-aminoquinoline, 0.01mol phenylacetylene, 0.01mol copper sulfate pentahydrate and 0.01mol ascorbic acid into a 50mL round bottom flask, dissolve in a mixed solution of tetrahydrofuran and water, React at room temperature for 12 hours to obtain the corresponding triazole compound, and use a rotary evaporator to evaporate the organic solvent under reduced pressure. 30 mL of saturated brine was added, 30 mL of dichloromethane was added thereto for extraction three times, and the organic phase was collected and dried over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 65%.
1H NMR(500MHz,DMSO)δ9.02(s,1H),8.85(dd,J＝4.1,1.6Hz, 1H),8.01–7.99(m,2H),7.90(d,J＝8.6Hz,1H),7.86(dd,J＝8.6,1.6Hz, 1H),7.59(m,J＝6.5,5.2,3.2Hz,5H),7.53(d,J＝8.6Hz,1H),6.96(d,J＝ 8.2Hz,1H),6.52(s,2H).1H NMR(500MHz,DMSO)δ9.02(s,1H),8.85(dd,J=4.1,1.6Hz,1H),8.01–7.99(m,2H),7.90(d,J=8.6Hz,1H) ,7.86(dd,J=8.6,1.6Hz,1H),7.59(m,J=6.5,5.2,3.2Hz,5H),7.53(d,J=8.6Hz,1H),6.96(d,J=8.2 Hz,1H),6.52(s,2H).
13C NMR(126MHz,DMSO)δ147.80(s),147.32(s),145.28(s), 136.14(s),130.61(s),129.50(s),129.03(s),127.01(s),125.68(s),124.55(s), 124.10(s),123.12(s),119.62(s),118.09(s),106.39(s).13C NMR (126MHz, DMSO) δ147.80(s), 147.32(s), 145.28(s), 136.14(s), 130.61(s), 129.50(s), 129.03(s), 127.01(s), 125.68 (s), 124.55(s), 124.10(s), 123.12(s), 119.62(s), 118.09(s), 106.39(s).
实施例9：荧光团DZ-b的合成Example 9: Synthesis of Fluorophore DZ-b
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的对甲基苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率70％。Add 0.01mol 5-azido-8-aminoquinoline, 0.01mol p-methylphenylacetylene, 0.01mol copper sulfate pentahydrate and 0.01mol ascorbic acid into a 50mL round bottom flask, dissolve in tetrahydrofuran and water The solution was reacted at room temperature for 12 hours to obtain the corresponding triazole compound, and the organic solvent was evaporated under reduced pressure with a rotary evaporator. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 70%.
1H NMR(500MHz,DMSO)δ8.92(s,1H),8.85(dd,J＝3.9,1.3Hz, 1H),7.86(m,J＝12.4,4.7Hz,3H),7.58(dd,J＝8.4,4.0Hz,2H),7.31(d,J＝ 7.9Hz,2H),6.96(d,J＝8.2Hz,1H),6.51(s,2H),2.36(s,3H)；1H NMR (500MHz, DMSO) δ8.92(s, 1H), 8.85(dd, J=3.9, 1.3Hz, 1H), 7.86(m, J=12.4, 4.7Hz, 3H), 7.58(dd, J= 8.4,4.0Hz,2H),7.31(d,J=7.9Hz,2H),6.96(d,J=8.2Hz,1H),6.51(s,2H),2.36(s,3H);
13C NMR(126MHz,DMSO)δ147.74(s),147.19(s),146.40(s), 137.33(s),136.16(s),130.64(s),129.46(s),127.79(s),125.59(s),125.26(s), 124.13(s),123.74(s),123.05(s),106.40(s),20.85(s).13C NMR (126MHz, DMSO) δ147.74(s), 147.19(s), 146.40(s), 137.33(s), 136.16(s), 130.64(s), 129.46(s), 127.79(s), 125.59 (s), 125.26(s), 124.13(s), 123.74(s), 123.05(s), 106.40(s), 20.85(s).
实施例10：荧光团DZ-c的合成Example 10: Synthesis of Fluorophore DZ-c
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的对乙基苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率72％。Add 0.01mol of 5-azido-8-aminoquinoline, 0.01mol of p-ethylphenylacetylene, 0.01mol of copper sulfate pentahydrate and 0.01mol of ascorbic acid into a 50mL round-bottomed flask, dissolve in a mixture of tetrahydrofuran and water The solution was reacted at room temperature for 12 hours to obtain the corresponding triazole compound, and the organic solvent was evaporated under reduced pressure with a rotary evaporator. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 72%.
1H NMR(500MHz,DMSO)δ8.92(s,1H),8.85(dd,J＝4.0,1.3Hz, 1H),7.89(d,J＝8.0Hz,2H),7.85(dd,J＝8.6,1.3Hz,1H),7.58(dd,J＝8.4, 4.4Hz,2H),7.34(d,J＝8.1Hz,2H),6.96(d,J＝8.2Hz,1H),6.51(s,2H), 2.66(q,J＝7.6Hz,2H),1.22(d,J＝7.6Hz,3H)；1H NMR (500MHz, DMSO) δ8.92(s, 1H), 8.85(dd, J=4.0, 1.3Hz, 1H), 7.89(d, J=8.0Hz, 2H), 7.85(dd, J=8.6, 1.3Hz, 1H), 7.58(dd, J＝8.4, 4.4Hz, 2H), 7.34(d, J＝8.1Hz, 2H), 6.96(d, J＝8.2Hz, 1H), 6.51(s, 2H) , 2.66(q,J=7.6Hz,2H),1.22(d,J=7.6Hz,3H);
13C NMR(126MHz,DMSO)δ148.25(s),147.68(s),146.92(s), 144.18(s),136.66(s),131.12(s),128.77(s),128.53(s),126.08(s),125.85(s), 124.61(s),124.27(s),123.55(s),120.30(s),106.91(s),28.43(s),15.97(s).13C NMR (126MHz, DMSO) δ148.25(s), 147.68(s), 146.92(s), 144.18(s), 136.66(s), 131.12(s), 128.77(s), 128.53(s), 126.08 (s), 125.85(s), 124.61(s), 124.27(s), 123.55(s), 120.30(s), 106.91(s), 28.43(s), 15.97(s).
实施例11：荧光团DZ-d的合成Example 11: Synthesis of Fluorophore DZ-d
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的己-1-炔化合物和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率77％。Add 0.01mol 5-azide 8-aminoquinoline, 0.01mol hex-1-yne compound, 0.01mol copper sulfate pentahydrate and 0.01mol ascorbic acid into a 50mL round bottom flask, dissolve in tetrahydrofuran and water The mixed solution was reacted at room temperature for 12 hours to obtain the corresponding triazole compound, and the organic solvent was evaporated under reduced pressure with a rotary evaporator. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 77%.
1H NMR(500MHz,DMSO)δ8.83(dd,J＝4.1,1.6Hz,1H),8.21(s, 1H),7.72(dd,J＝8.6,1.6Hz,1H),7.56(dd,J＝8.6,4.1Hz,1H),7.47(d,J＝ 8.2Hz,1H),6.93(d,J＝8.2Hz,1H),6.44(s,2H),2.76–2.73(m,2H),1.71– 1.66(m,2H),1.41(dd,J＝14.9,7.4Hz,2H),0.97–0.93(m,3H)；1H NMR (500MHz, DMSO) δ8.83(dd, J=4.1, 1.6Hz, 1H), 8.21(s, 1H), 7.72(dd, J=8.6, 1.6Hz, 1H), 7.56(dd, J= 8.6,4.1Hz,1H),7.47(d,J＝8.2Hz,1H),6.93(d,J＝8.2Hz,1H),6.44(s,2H),2.76–2.73(m,2H),1.71– 1.66(m,2H),1.41(dd,J＝14.9,7.4Hz,2H),0.97–0.93(m,3H);
13C NMR(126MHz,DMSO)δ147.66(s),146.93(s),140.10(s), 136.17(s),130.56(s),125.42(s),124.56(s),124.19(s),122.93(s),120.12(s), 106.43(s),31.04(s),24.69(s),21.73(s),13.70(s).13C NMR (126MHz, DMSO) δ147.66(s), 146.93(s), 140.10(s), 136.17(s), 130.56(s), 125.42(s), 124.56(s), 124.19(s), 122.93 (s), 120.12(s), 106.43(s), 31.04(s), 24.69(s), 21.73(s), 13.70(s).
实施例12：荧光团DZ-e的合成Example 12: Synthesis of Fluorophore DZ-e
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的对甲氧基苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率80％。Add 0.01mol of 5-azido-8-aminoquinoline, 0.01mol of p-methoxyphenylacetylene, 0.01mol of copper sulfate pentahydrate and 0.01mol of ascorbic acid into a 50mL round bottom flask, dissolve in tetrahydrofuran and water The mixed solution was reacted at room temperature for 12 hours to obtain the corresponding triazole compound, and the organic solvent was evaporated under reduced pressure with a rotary evaporator. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 80%.
1H NMR(500MHz,DMSO)δ8.86(s,1H),8.85(dd,J＝5.5,1.2Hz, 1H),7.90(d,J＝8.7Hz,2H),7.85(dd,J＝8.6,1.5Hz,1H),7.60–7.55(m, 2H),7.06(dd,J＝8.9,2.4Hz,3H),6.97(d,J＝8.2Hz,1H),6.50(s,2H),3.82 (s,3H)；1H NMR (500MHz, DMSO) δ8.86(s, 1H), 8.85(dd, J=5.5, 1.2Hz, 1H), 7.90(d, J=8.7Hz, 2H), 7.85(dd, J=8.6, 1.5Hz,1H),7.60–7.55(m,2H),7.06(dd,J=8.9,2.4Hz,3H),6.97(d,J=8.2Hz,1H),6.50(s,2H),3.82 ( s,3H);
13C NMR(126MHz,DMSO)δ147.74(s),147.19(s),146.40(s), 137.33(s),136.16(s),130.64(s),129.46(s),127.79(s),125.59(s),125.26(s), 124.13(s),123.74(s),123.05(s),106.40(s),20.85(s).13C NMR (126MHz, DMSO) δ147.74(s), 147.19(s), 146.40(s), 137.33(s), 136.16(s), 130.64(s), 129.46(s), 127.79(s), 125.59 (s), 125.26(s), 124.13(s), 123.74(s), 123.05(s), 106.40(s), 20.85(s).
实施例13：荧光团DZ-f的合成Example 13: Synthesis of Fluorophore DZ-f
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的对二乙炔噻吩苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率83％。Add 0.01 mol of 5-azido 8-aminoquinoline, 0.01 mol of p-diacetylene thiophene phenylacetylene, 0.01 mol of copper sulfate pentahydrate and 0.01 mol of ascorbic acid in a 50 mL round bottom flask, dissolve in tetrahydrofuran and water The mixed solution was reacted at room temperature for 12 hours to obtain the corresponding triazole compound, and the organic solvent was evaporated under reduced pressure with a rotary evaporator. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 83%.
1H NMR(500MHz,DMSO)δ8.85(dd,J＝4.1,1.5Hz,1H),8.83(s, 1H),7.96(dd,J＝2.9,1.1Hz,1H),7.84(dd,J＝8.6,1.5Hz,1H),7.71(dd,J ＝5.0,2.9Hz,1H),7.62(dd,J＝5.0,1.1Hz,1H),7.60–7.55(m,2H),6.96(d, J＝8.2Hz,1H),6.51(s,2H)；1H NMR (500MHz, DMSO) δ8.85(dd, J=4.1, 1.5Hz, 1H), 8.83(s, 1H), 7.96(dd, J=2.9, 1.1Hz, 1H), 7.84(dd, J= 8.6,1.5Hz,1H),7.71(dd,J＝5.0,2.9Hz,1H),7.62(dd,J＝5.0,1.1Hz,1H),7.60–7.55(m,2H),6.96(d,J =8.2Hz, 1H), 6.51(s, 2H);
13C NMR(126MHz,DMSO)δ147.75(s),147.21(s),142.91(s), 136.15(s),131.83(s),130.57(s),127.20(s),125.88(s),125.59(s),124.12(s), 123.91(s),123.06(s),121.09(s),119.67(s),106.40(s).13C NMR (126MHz, DMSO) δ147.75(s), 147.21(s), 142.91(s), 136.15(s), 131.83(s), 130.57(s), 127.20(s), 125.88(s), 125.59 (s), 124.12(s), 123.91(s), 123.06(s), 121.09(s), 119.67(s), 106.40(s).
实施例14：荧光团DZ-g的合成Example 14: Synthesis of Fluorophore DZ-g
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的对氯苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入 30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率78％。Add 0.01mol of 5-azido-8-aminoquinoline, 0.01mol of p-chlorophenylacetylene, 0.01mol of copper sulfate pentahydrate and 0.01mol of ascorbic acid into a 50mL round bottom flask, and dissolve in a mixed solution of tetrahydrofuran and water , react at room temperature for 12 hours to obtain the corresponding triazole compound, and use a rotary evaporator to distill off the organic solvent under reduced pressure. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 78%.
1H NMR(500MHz,DMSO)δ8.98(s,1H),8.85(dd,J＝4.1,1.6Hz, 1H),7.98(dd,J＝5.1,3.3Hz,2H),7.85(dd,J＝8.6,1.6Hz,1H),7.59(dt,J＝4.1,2.2Hz,2H),7.51(dd,J＝10.6,4.9Hz,2H),7.41–7.37(m,1H),6.97(d, J＝8.2Hz,1H),6.51(s,2H)；1 H NMR (500MHz, DMSO) δ8.98(s, 1H), 8.85(dd, J＝4.1, 1.6Hz, 1H), 7.98(dd, J＝5.1, 3.3Hz, 2H), 7.85(dd, J =8.6,1.6Hz,1H),7.59(dt,J=4.1,2.2Hz,2H),7.51(dd,J=10.6,4.9Hz,2H),7.41–7.37(m,1H),6.97(d, J＝8.2Hz, 1H), 6.51(s, 2H);
13C NMR(126MHz,DMSO)δ158.23(s),157.55(s),147.79(s), 147.26(s),146.36(s),136.17(s),130.64(s),128.96(s),128.05(s),125.66(s), 125.34(s),124.21(s),123.11(s),119.76(s),106.42(s).13C NMR (126MHz, DMSO) δ158.23(s), 157.55(s), 147.79(s), 147.26(s), 146.36(s), 136.17(s), 130.64(s), 128.96(s), 128.05 (s), 125.66(s), 125.34(s), 124.21(s), 123.11(s), 119.76(s), 106.42(s).
实施例15：荧光团DZ-h的合成Example 15: Synthesis of Fluorophore DZ-h
在50mL的圆底烧瓶中加入0.01mol 5-叠氮8-氨基喹啉、0.01mol的对氨基苯乙炔和0.01mol的五水硫酸铜水合物和0.01mol抗坏血酸，溶于四氢呋喃和水的混合溶液中，常温反应12小时，得到对应得三氮唑化合物，用旋转蒸发仪减压蒸去有机溶剂。加入30mL饱和食盐水，向其中加入30mL二氯甲烷萃取三次，收集有机相，用无水硫酸钠干燥。用旋转蒸发仪减压抽去有机溶剂，产物利用硅胶板层析分离得到纯品为淡黄色固体，产率80％。Add 0.01mol of 5-azido-8-aminoquinoline, 0.01mol of p-aminophenylacetylene, 0.01mol of copper sulfate pentahydrate and 0.01mol of ascorbic acid into a 50mL round bottom flask, and dissolve in a mixed solution of tetrahydrofuran and water , react at room temperature for 12 hours to obtain the corresponding triazole compound, and use a rotary evaporator to distill off the organic solvent under reduced pressure. Add 30 mL of saturated brine, add 30 mL of dichloromethane to extract three times, collect the organic phase, and dry over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure with a rotary evaporator, and the product was separated by silica gel plate chromatography to obtain a pure product as a light yellow solid with a yield of 80%.
1H NMR(500MHz,DMSO)δ8.86(s,1H),8.85(dd,J＝5.5,1.2Hz, 1H),7.90(d,J＝8.7Hz,2H),7.85(dd,J＝8.6,1.5Hz,1H),7.60–7.55(m, 2H),7.06(dd,J＝8.9,2.4Hz,3H),6.97(d,J＝8.2Hz,1H),6.50(s,2H)1H NMR (500MHz, DMSO) δ8.86(s, 1H), 8.85(dd, J=5.5, 1.2Hz, 1H), 7.90(d, J=8.7Hz, 2H), 7.85(dd, J=8.6, 1.5Hz, 1H), 7.60–7.55(m, 2H), 7.06(dd, J＝8.9, 2.4Hz, 3H), 6.97(d, J＝8.2Hz, 1H), 6.50(s, 2H)
13C NMR(126MHz,DMSO)δ158.23(s),157.55(s),147.79(s), 147.26(s),146.36(s),136.17(s),130.64(s),128.96(s),128.05(s),125.66(s), 125.34(s),124.21(s),123.11(s),119.76(s),106.42(s).13C NMR (126MHz, DMSO) δ158.23(s), 157.55(s), 147.79(s), 147.26(s), 146.36(s), 136.17(s), 130.64(s), 128.96(s), 128.05 (s), 125.66(s), 125.34(s), 124.21(s), 123.11(s), 119.76(s), 106.42(s).
实施例16：实施例8-15制备的荧光团DZ-a～DZ-h荧光性质的测定。Example 16: Determination of the fluorescence properties of the fluorophores DZ-a to DZ-h prepared in Examples 8-15.
准确称取一定量的荧光团化合物(DZ-a至DZ-h)，用二甲基亚砜配制成浓度为10mM的探针母液，用移液枪吸取1μL分别加入到999μL磷酸缓冲液中，37℃摇床上充分混匀10分钟后，将含有荧光团的液体吸取到比色皿中测定在不同溶液的紫外吸收光谱，以及发射波长。结果如图1 所示：Accurately weigh a certain amount of fluorophore compounds (DZ-a to DZ-h), prepare a probe stock solution with a concentration of 10 mM with dimethyl sulfoxide, and pipette 1 μL into 999 μL phosphate buffer respectively. After fully mixing on a shaker at 37°C for 10 minutes, the liquid containing the fluorophore was drawn into a cuvette to measure the ultraviolet absorption spectra and emission wavelengths of different solutions. The result is shown in Figure 1:
实施例17：通过点击反应来研究荧光染料的荧光turn-on响应，我们检测了染料在反应前后的荧光强度。对比了铜催化电击反应下DQ、DA 以及DA-2的荧光强度，结果如图2所示，检测发现DA的turn-on性能是756倍。Example 17: To study the fluorescent turn-on response of fluorescent dyes through click reaction, we detected the fluorescent intensity of the dyes before and after the reaction. The fluorescence intensities of DQ, DA and DA-2 under the copper-catalyzed electric shock reaction were compared, the results are shown in Figure 2, and the turn-on performance of DA was found to be 756 times higher.
实施例18：评估荧光染料在细胞中的turn-on的性能。Example 18: Evaluation of the turn-on performance of fluorescent dyes in cells.
选择了DQ，DZ和DZ-2作为例子。DQ, DZ and DZ-2 were chosen as examples.
(1)选取宫颈癌细胞(HeLa)作为研究对象，首先将细胞在37℃， 5％CO2的细胞培养箱中培养24小时，(培养基是含10％胎牛血清的DMEM高糖培养基)；(1) Select cervical cancer cells (HeLa) as the research object, first culture the cells at 37°C, 5% CO2 in a cell incubator for 24 hours, (the culture medium is DMEM high-glucose medium containing 10% fetal bovine serum );
(2)用胰酶消化细胞，将细胞转移到细胞成像皿中，继续在37℃， 5％CO2的细胞培养箱中培养12小时，待细胞贴壁以后，分别在成像皿中分别加入DQ，DZ，DZ-2和等价的CuSO4和Vc继续孵化30分钟；(2) Digest the cells with trypsin, transfer the cells to a cell imaging dish, and continue culturing for 12 hours in a cell culture incubator at 37°C with 5% CO2 . After the cells adhere to the wall, add DQ to the imaging dish respectively. , DZ, DZ-2 and equivalent CuSO4 and Vc continue to incubate for 30 minutes;
(3)用PBS缓冲液洗去成像皿里的培养基。样品分别用荧光成像仪进行荧光成像。(3) Wash away the medium in the imaging dish with PBS buffer. Samples were imaged separately using a fluorescence imager.
在固定细胞中清楚地观察到荧光turn-on，而当不添加CuSO4和Vc 时则没有观察到荧光，如图3所示。结果证明，颜色可调荧光探针非常适合于固定细胞成像研究。Fluorescence turn-on was clearly observed in fixed cells, while no fluorescence was observed when CuSO4 and Vc were not added, as shown in Figure 3. The results demonstrate that the color-tunable fluorescent probes are well suited for fixed-cell imaging studies.
实施例19：荧光染料用于直接跟踪活细胞中的各种细胞器的研究。Example 19: Fluorescent dyes are used in the study of direct tracking of various organelles in living cells.
使用吗啉炔基和三苯基膦炔基靶向点击反应性炔基溶酶体和线粒体。选择DND-99和Mito-Red-FM作为溶酶体和线粒体的tracker。Targeting click-reactive alkyneyl lysosomes and mitochondria using morpholynyl and triphenylphosphinoalkynyl groups. DND-99 and Mito-Red-FM were selected as trackers for lysosomes and mitochondria.
(1)选取肝细胞(HePG-2)作为研究对象，首先将细胞在37℃， 5％CO2的细胞培养箱中培养24小时，(培养基是含10％胎牛血清的 DMEM高糖培养基)；(1) Select hepatocytes (HePG-2) as the research object, first culture the cells at 37°C, 5% CO2 in a cell incubator for 24 hours, (the culture medium is DMEM containing 10% fetal bovine serum and cultured in high glucose base);
(2)用胰酶消化细胞，将细胞转移到细胞成像皿中，继续在37℃， 5％CO2的细胞培养箱中培养12小时，待细胞贴壁以后，分别在成像皿中分别加入N，N-二甲基丙-2-炔-1-胺和三苯基膦炔基孵化30分钟；(2) Digest the cells with trypsin, transfer the cells to the cell imaging dish, and continue culturing for 12 hours at 37°C in a 5%CO2 cell incubator. After the cells adhere to the wall, add N to the imaging dish respectively. , N-dimethylprop-2-yn-1-amine and triphenylphosphinoalkynyl were incubated for 30 minutes;
(3)随后在成像皿中分别加入用DZ(N，N-二甲基丙-2-炔-1-胺和三苯基膦-炔基)和DQ(三苯基膦-炔基)孵化，并进行共聚焦成像。(3) Then add DZ (N,N-dimethylprop-2-yn-1-amine and triphenylphosphine-alkynyl) and DQ (triphenylphosphine-alkynyl) to the imaging dish to incubate , and perform confocal imaging.
得到的结果如图4所示，证明该荧光染料可用于追踪活细胞中的细胞器(如线粒体，溶酶体)。The obtained results are shown in Figure 4, demonstrating that the fluorescent dye can be used to track organelles (such as mitochondria, lysosomes) in living cells.
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求保护范围内。The descriptions of the above embodiments are only used to help understand the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.
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| CN201810456814.4ACN108485307A (en) | 2018-05-14 | 2018-05-14 | A kind of fluorescent dye, preparation method and applications for opening property with fluorescence |
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| CN102115610B (en)* | 2010-01-05 | 2014-07-02 | 大连理工大学 | Fluorescent dye, preparation method and application thereof |
| CN106478594A (en)* | 2016-08-26 | 2017-03-08 | 浙江工业大学 | A kind of fluorescent dye and its synthesis and the application preparing fluorescent probe |
| CN106987152A (en)* | 2017-03-08 | 2017-07-28 | 三峡大学 | One class fluorine boron near infrared fluorescent dye and the application in non-protonic solvent in the detection of minor amount of water |
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| CN102115610B (en)* | 2010-01-05 | 2014-07-02 | 大连理工大学 | Fluorescent dye, preparation method and application thereof |
| CN106478594A (en)* | 2016-08-26 | 2017-03-08 | 浙江工业大学 | A kind of fluorescent dye and its synthesis and the application preparing fluorescent probe |
| CN106987152A (en)* | 2017-03-08 | 2017-07-28 | 三峡大学 | One class fluorine boron near infrared fluorescent dye and the application in non-protonic solvent in the detection of minor amount of water |
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| CN111607248B (en)* | 2020-06-04 | 2022-04-12 | 绍兴文理学院 | Quinoline type lyotropic color-changing fluorescent dye, preparation method thereof and application thereof in organic solvent water content measurement |
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