WO2024108656A1 - 一种线粒体靶向水溶性聚集诱导发光染料及其制备方法 - Google Patents

一种线粒体靶向水溶性聚集诱导发光染料及其制备方法 Download PDF

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WO2024108656A1
WO2024108656A1 PCT/CN2022/137075 CN2022137075W WO2024108656A1 WO 2024108656 A1 WO2024108656 A1 WO 2024108656A1 CN 2022137075 W CN2022137075 W CN 2022137075W WO 2024108656 A1 WO2024108656 A1 WO 2024108656A1
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dye
aggregation
rho
induced
luminescent dye
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张鹏飞
罗媛
谢样梓
蔡林涛
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/90—Xanthenes with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
    • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom

Definitions

  • the invention relates to the field of luminescent materials, and in particular to an aggregation-induced luminescence fluorescent dye with a targeting function and a preparation method thereof.
  • Aggregation-Caused Quenching refers to the phenomenon that the fluorescence of traditional organic light-emitting materials will be weakened or even disappear when they are in high concentration or solidified, that is, the material is in an aggregated state and fluorescence quenching occurs. In practical applications, this effect will bring some negative effects. For example, when hydrophobic organic light-emitting materials are added to the commonly used detection medium - water, due to the low solubility, the light-emitting molecules form an aggregated state in water, resulting in a decrease in the light intensity.
  • AIE has become a research hotspot in the fields of luminescent materials and photophysics, and was listed as the second of the top 10 research frontiers in the field of chemistry in the "2015 Research Frontiers” report jointly released by the Documentation and Information Center of the Chinese Academy of Sciences and Thomson Reuters.
  • the 2016 "Nature” News Feature column, titled “The nanolight revolution is coming” focused on introducing AIE materials and evaluated that AIE materials provide solutions to the problems of currently commonly used quantum dots and luminescent polymer dots, and are a new generation of nanoluminescent materials. Therefore, it is of great significance to develop water-soluble AIE molecules with long-wavelength emission, aggregation-induced emission properties and low phototoxicity, but at the same time, the way forward remains extremely challenging.
  • fluorescence bio-imaging as a powerful non-invasive imaging technology, has shown unique advantages, especially the development and application of super-resolution fluorescence microscopy technology.
  • super-resolution fluorescence imaging is based on larger-scale photon statistics, which in principle breaks the original optical far-field diffraction limit on the limit resolution of the optical system, surpassing the optical resolution limit and reaching nanometer-level resolution.
  • fluorescent chromophores used is organic small molecule fluorescent chromophore compounds, such as rhodamine dyes, which are now widely used in super-resolution imaging. Such compounds are easy to synthesize, easy to modify, and have low toxicity, and have received widespread attention in the development and application of organic small molecule fluorescent probes.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and solve the problem of poor water solubility of traditional rhodamine dyes mentioned in the above background technology.
  • the present invention provides the following technical solutions:
  • an aggregation-induced emission dye is provided, and its characteristic structure is shown in FIG1 .
  • the second aspect of the present invention provides a method for preparing an aggregation-induced emission dye, 2.
  • the method comprises: S1, dissolving potassium carbonate, MG-B, and Br-Rho-2 in N,N-dimethylformamide, and adding tetrakis(triphenylphosphine)palladium to react and purify after nitrogen protection; S2, dissolving the purified product in dichloromethane, adding tetrachloro-p-benzoquinone and reacting, and obtaining the dye after purification.
  • S1 dissolving potassium carbonate, MG-B, and Br-Rho-2 in N,N-dimethylformamide, and adding tetrakis(triphenylphosphine)palladium to react and purify after nitrogen protection
  • S2 dissolving the purified product in dichloromethane, adding tetrachloro-p-benzoquinone and reacting, and obtaining the dye after purification.
  • the molar ratio of the MG-B to the Br-Rho-2 is 12:10.
  • the molar ratio of the potassium carbonate, the tetrakis(triphenylphosphine)palladium and the tetrachlorobenzoquinone is 1000:0.8:15.
  • the reaction time is 12-24 hours.
  • the reaction time is 30 min.
  • the purification in step S1 comprises: extracting, washing, drying and column chromatography the reaction mixture.
  • the adsorbent is silica gel and the eluent is a mixed solution of ethyl acetate/hexane in a volume ratio of 1:20-1:10.
  • the purification comprises: performing column chromatography.
  • the adsorbent is silica gel
  • the eluent is a methanol/dichloromethane mixed solution with a volume ratio of 1:10-1:5.
  • the third aspect of the present invention provides an application of an aggregation-induced luminescence dye in living cell imaging and an application of an aggregation-induced luminescence dye in mitochondrial targeted staining.
  • the present invention has the following beneficial effects:
  • the present invention is the first to develop and synthesize a mitochondrial-targeted water-soluble organic small molecule fluorescent dye MG-Rho-2, which has aggregation-induced emission properties.
  • the MG-Rho-2 molecule provided by the present invention has good biocompatibility and complete water solubility, and can be applied to ultrafast, wash-free, low-phototoxicity imaging of living cells, which greatly facilitates biological research related to living cells.
  • the dye MG-Rho-2 provided by the present invention exhibits a better signal-to-noise ratio than commercial mitochondrial targeting dyes.
  • the present invention adopts a one-pot method to prepare the dye MG-Rho-2, which is simple to operate and is conducive to laboratory and industrial applications.
  • FIG1 is the structural formula of the fluorescent probe material MG-Rho-2;
  • Figure 2 is the structural formula of Br-Rho-2
  • FIG3 is the structural formula of MG-B
  • FIG4 is a synthetic route diagram of MG-Rho-2
  • FIG5 is a hydrogen nuclear magnetic resonance spectrum of MG-Rho-2
  • FIG6 is a carbon NMR spectrum of MG-Rho-2
  • FIG7 is a high-resolution mass spectrum of MG-Rho-2
  • FIG8 is a graph of the optical properties of MG-Rho-2, wherein FIG8A is a photoluminescence spectrum of MG-Rho-2 in methanol, FIG8B is a fluorescence spectrum of MG-Rho-2 in tetrahydrofuran/water mixtures of different ratios, and FIG8C is a graph showing the variation of the luminescence intensity of MG-Rho-2 with the water content;
  • CLSM confocal laser scanning microscope
  • Rhodamine dye is one of the most widely used dye platforms in the field of fluorescent probes.
  • the fluorescent probe built on this platform is usually a rhodamine spirolactam derivative, which is non-fluorescent by itself. After interacting with the guest, it can cause the spirolactam structure to open, thereby obtaining strong fluorescence emission and obvious color change. Since Czarnik's group first reported the "rhodamine-hydrazine" Cu 2+ fluorescent probe using the above properties in 1997, various fluorescent probes based on the rhodamine dye platform have been developed one after another.
  • rhodamine fluorescent dyes easily form dimers, which cause intermolecular quenching, thereby reducing the fluorescence brightness of the dye.
  • the medium for most biochemical reactions and biological detection is water, and the high hydrophobicity of rhodamine causes it to be insoluble in water.
  • the above shortcomings limit the further application of rhodamine fluorescent dyes. Introducing sulfonic acid functional groups on traditional rhodamine dyes is the most common method to increase the water solubility of fluorescent dyes and prevent fluorescence quenching, but the synthesis is relatively complicated.
  • the present invention has successfully developed a small organic molecule water-soluble probe with aggregation-induced emission properties based on a rhodamine skeleton, hereinafter referred to as MG-Rho-2. Thanks to the two quaternary ammonium groups of the MG-Rho-2 molecule, it is completely water-soluble and can be applied to ultrafast, wash-free, low-phototoxic imaging of living cells, which will greatly facilitate biological research related to living cells.
  • Example 1 Preparation of MG-Rho-2 and characterization by hydrogen spectrum, carbon spectrum and mass spectrum
  • the synthetic route of MG-Rho-2 is shown in FIG4 , and specifically comprises the following steps:
  • the reaction mixture was extracted with dichloromethane, washed three times with a saturated sodium chloride solution, and the organic phases were combined and dried over anhydrous sodium sulfate.
  • the crude product was concentrated and purified by column chromatography, wherein silica gel was selected as the adsorbent and a mixed solution of ethyl acetate/hexane with a volume ratio of 1:20-1:10 was selected as the eluent.
  • the purified product was dissolved in dichloromethane, 0.15 mmol of tetrachlorobenzoquinone was added, and the mixture was stirred at room temperature for 30 min in one pot.
  • the obtained crude product was purified by silica gel column chromatography, wherein a methanol/dichloromethane mixed solution with a volume ratio of 1:10-1:5 was selected as the eluent to finally obtain the target compound as a dark green solid.
  • the target compound was characterized by carbon NMR spectrum. The result is shown in Figure 6.
  • the specific data are: 13 C NMR (100 MHz, MeOD-d 4 ) ⁇ (ppm) 176.2, 157.7, 157.4, 157.1, 157.1, 144.1, 143.7, 141.2, 140.5, 139.7, 139.2, 135.5, 135.4, 132.1, 131.4, 130.6, 127.4, 127.0, 127.0, 127.0, 127.0, 114.3, 113.5, 112.9, 96.3, 39.7.
  • the target compound was characterized by high resolution mass spectrometry (HRMS), and electrospray ionization (ESI) was selected as the ionization mode.
  • HRMS high resolution mass spectrometry
  • ESI electrospray ionization
  • Figure 7 The molecular formula is C 46 H 46 N 4 O 2+ , the calculated molecular weight is 335.18303, and the experimental molecular weight is 335.18307.
  • FIG. 8 the aggregation-induced emission properties of MG-Rho-2 were studied.
  • the results show that the absorption peak of MG-Rho-2 in methanol is 550 nm and the emission peak is 600 nm.
  • Figures 8B and 8C show the luminescence intensity of MG-Rho-2 in aqueous solutions of tetrahydrofuran at different proportions.
  • the fluorescence intensity is very weak.
  • the emission intensity of MG-Rho-2 also increases.
  • the fluorescence intensity is the largest, which is increased by 132 times, proving that the MG-Rho-2 synthesized in this scheme has good aggregation-induced emission properties.
  • FIG. 9 the mitochondrial targeting properties of MG-Rho-2 were studied.
  • FIG 9A the mitochondrial region of Hela cells was specifically stained with MG-Rho-2, and the structure of mitochondria can be clearly observed, indicating that MG-Rho-2 can be localized in the mitochondria of living cells.
  • Figure 9B was stained with the mitochondrial green fluorescent probe Mito Tracker Green, and it was superimposed with the MG-Rho-2 staining result to obtain Figure 9C, and it can be observed that the two are almost completely overlapped.
  • the present invention provides an aggregation-induced luminescence dye MG-Rho-2, which has excellent biocompatibility, completely water-soluble mitochondrial targeting effect, and can be applied to ultrafast, wash-free, low-phototoxic imaging of living cells.
  • the present invention provides a preparation method of MG-Rho-2, which is innovative and easy to operate, and the structure of the prepared product is confirmed to be correct after characterization by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high-resolution mass spectrum.

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Abstract

发光材料领域，具体涉及一种具有靶向功能的聚集诱导发光荧光染料及其制备方法。所提供的染料具有良好的生物相容性和完全水溶性，可应用于活细胞的超快免洗低光毒性成像，极大地助力了活细胞相关的生物研究。

Description

一种线粒体靶向水溶性聚集诱导发光染料及其制备方法 技术领域
本发明涉及发光材料领域，具体涉及一种具有靶向功能的聚集诱导发光荧光染料及其制备方法。
 背景技术
聚集荧光淬灭（Aggregation-Caused Quenching，ACQ）是指传统有机发光材料在高浓度或被制成固态时，荧光会减弱甚至不发光，即材料处于聚集态时出现了荧光淬灭的现象。在实际应用中，该效应会带来一些负面影响，比如将疏水性的有机发光物加入至常用的检测介质——水中，由于溶解度较低，发光分子在水中形成聚集态，导致了发光强度降低。
2001年，唐本忠院士首次提出了在聚集态下高效发光的聚集诱导发光（Aggregation-Induced Emission，AIE）概念，受到了国内外同行的广泛关注。现已有数十个国家的上百个课题组开展了有关研究，涉及新AIE分子、AIE机理、AIE材料等多个方向，也在光电器件、生物探针与成像、化学传感、智能材料应用等诸多领域取得了显著成果。目前AIE已经成为发光材料和光物理等领域的研究热点，并被中国科学院文献情报中心和汤森路透联合发布的《2015研究前沿》报告列为化学领域的10大研究前沿的第二位。此外，2016年《自然》News Feature专栏以“纳米光革命正在来临（The nanolight revolution is coming）”为题重点介绍了AIE材料，并评价AIE材料为当前常用的量子点与发光聚合物点存在的问题提供了解决方案，是新一代的纳米发光材料。因此，开发具有长波长发射的聚集诱导发光特性和低光毒性的水溶性AIE分子具有重要意义，但同时，前进的道路依然极具挑战性。
在生物光学成像领域中，荧光生物成像作为一种强大的非创伤性成像技术展现出了独特的优势，特别是超分辨率荧光显微成像技术的开发和应用。相较于传统光学成像，超分辨荧光成像建立在更大规模的光子统计数据基础上，从原理上打破了原有的光学远场衍射极限对光学系统极限分辨率的限制，超越光学分辨率极限，达到了纳米级分辨率。
为了进一步提升成像分辨率，改善成像质量，人们期望设计出具有更高亮度以及光稳定性更好的荧光染料。其中一类主要使用的荧光生色团是有机小分子荧光生色团化合物，比如现已被广泛应用于超分辨成像的罗丹明染料。此类化合物合成简便、易于修饰、毒性较小，在有机小分子荧光探针的开发及应用中受到了广泛关注。
但是，传统罗丹明染料均为聚集诱导淬灭染料，水溶性较差，生物相容性较弱且合成路线复杂，同时，其荧光亮度受激发后分子内扭转电荷转移的非辐射跃迁过程的影响，成像潜力并未得到完全的释放，不利于进一步地应用。
 技术问题
本发明的目的在于克服现有技术不足，解决上述背景技术中提到的传统罗丹明染料水溶性差的问题。
 技术解决方案
为实现上述目的，本发明提供以下技术方案：
本发明的第一方面，提供一种聚集诱导发光染料，其特征结构如图1所示。
本发明的第二方面，提供一种制备聚集诱导发光染料的方法，2.所述方法包括：S1、将碳酸钾、MG-B、Br-Rho-2溶于N,N-二甲基甲酰胺，通氮气保护后，加入四(三苯基膦)钯反应并进行纯化；S2、将纯化后产物溶于二氯甲烷，加入四氯对苯醌并反应，经纯化后得到所述染料。所述Br-Rho-2的结构式如图2所示；所述MG-B的结构式如图3所示。
优选地，所述步骤S1中，所述MG-B、所述Br-Rho-2的摩尔比为12:10。
优选地，所述步骤S1和S2中，所述碳酸钾、所述四(三苯基膦)钯、所述四氯对苯醌的摩尔比为1000:0.8:15。
优选地，所述步骤S1中，所述反应时间为12-24h。
优选地，所述步骤S2中，所述反应时间为30min。
在一些实施例中，所述步骤S1中所述纯化包括：将反应后混合物萃取、洗涤、干燥并进行柱层析。优选地，所述步骤S1中所述柱层析中，吸附剂是硅胶，洗脱剂是体积比为1:20-1:10的乙酸乙酯/己烷混合溶液。
在一些实施例中，所述步骤S2中，所述纯化包括：进行柱层析。优选地，所述步骤S2中所述柱层析中，吸附剂是硅胶，洗脱剂是体积比为1:10-1:5的甲醇/二氯甲烷混合溶液。
本发明的第三方面，提供一种聚集诱导发光染料在活细胞成像中的应用以及一种聚集诱导发光染料在线粒体靶向染色中的应用。
 有益效果
与现有技术相比，本发明的有益效果是，
（1）本发明首次开发合成了一种具有线粒体靶向的水溶性有机小分子荧光染料MG-Rho-2，该染料具有聚集诱导发光特性。
（2）本发明提供的MG-Rho-2分子具有良好的生物相容性和完全水溶性，可应用于活细胞的超快免洗低光毒性成像，极大地助力了活细胞相关的生物研究。
（3）本发明提供的染料MG-Rho-2相比商业化的线粒体靶向染料表现出了更好的信噪比。
（4）本发明采用一锅法制备染料MG-Rho-2，操作简单，利于实验室和工业化的应用。
 附图说明
图1为荧光探针材料MG-Rho-2的结构式；
图2为Br-Rho-2的结构式；
图3为MG-B的结构式；
图4为MG-Rho-2的合成路线图；
图5为MG-Rho-2的核磁共振氢谱图；
图6为MG-Rho-2的核磁共振碳谱图；
图7为MG-Rho-2的高分辨质谱图；
图8为MG-Rho-2光学性质图，其中图8A为MG-Rho-2在甲醇中的光致发光光谱，图8B为MG-Rho-2在不同比例四氢呋喃/水混合物中的荧光光谱，图8C为MG-Rho-2发光强度随水含量变化图；
图9为hela细胞的激光扫描共聚焦显微镜（CLSM）图，其中，图9A为5μMMG-Rho-2孵育图（lex=560 nm，lem=570-750 nm），图9B为Mito Tracker Green孵育图（lex=488 nm，lem=500-560 nm），图9C为叠加图，图9D为共定位系数拟合图。
 本发明的实施方式
下面将结合具体实施方式对本专利的技术方案作进一步详细地说明，应该指出，以下详细说明都是示例性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
罗丹明染料（Rhodamine）是荧光探针领域应用最为广泛的染料平台之一。以该平台为基础构建的荧光探针通常是一个罗丹明螺环内酰胺衍生物，该螺环内酰胺衍生物自身是非荧光的，其与客体相互作用后，可导致螺环内酰胺结构打开，从而获得强的荧光发射以及明显的颜色变化。Czarnik小组于1997年首次利用上述性质报道了“罗丹明-肼”Cu 2+荧光探针以来，基于罗丹明染料平台的各种荧光探针相继被开发。
常规罗丹明荧光染料容易形成二聚体，发生分子间淬灭，从而导致染料荧光亮度的减弱。此外，大部分的生化反应和生物检测的介质为水，而罗丹明的高度疏水性导致其难溶于水。上述缺点限制了罗丹明类荧光染料的进一步的应用。在传统的罗丹明染料上引入磺酸官能团是最常见的增加荧光染料水溶性和防止荧光淬灭的方法，但合成较为复杂。
本发明成功开发了基于罗丹明骨架的具有聚集诱导发光特性的有机小分子水溶性探针，后文以MG-Rho-2表示。得益于MG-Rho-2分子的两个季铵盐基团，使其具有完全水溶性，能应用于活细胞的超快免洗低光毒性成像，这将极大地助力活细胞相关的生物研究。
实施例1：MG-Rho-2的制备及氢谱、碳谱、质谱表征
（1）目标化合物的制备
MG-Rho-2合成路线如图4所示，具体包括以下步骤：
将组成为10 mmol碳酸钾、0.12 mmol MG-B和0.1 mmol Br-Rho-2的混合物溶于3 mL N,N-二甲基甲酰胺。通氮气保护后，加入0.008 mmol四(三苯基膦)钯，回流反应12h。
将反应后混合物用二氯甲烷萃取，再用饱和氯化钠溶液洗涤3次后，合并有机相，并用无水硫酸钠干燥。粗产物经浓缩后，利用柱层析法纯化，其中，吸附剂选择硅胶，洗脱剂选择体积比为1:20-1:10的乙酸乙酯/己烷混合溶液。
将纯化后产物溶解在二氯甲烷中，加入0.15 mmol四氯对苯醌，一锅法室温搅拌30 min。
所得粗产物利用硅胶柱层析以纯化，其中洗脱剂选择体积比为1:10-1:5的甲醇/二氯甲烷混合溶液，最终得到呈墨绿色固体的目标化合物。
（2）氢谱表征
对目标化合物进行核磁共振氢谱表征，结果如图5所示，具体数据为： 1H NMR（400 MHz，MeOD-d 4）δ（ppm） 1H NMR（400 MHz，MeOD）δ 8.16-8.02（m，4H），7.65-7.42（m，10H），7.16-7.04（m，6H），7.06-6.89（m，2H），3.36（t，J=18.4 Hz，24H）。
（3）碳谱表征
对目标化合物进行核磁共振碳谱表征，结果如图6所示，具体数据为： 13C NMR（100 MHz，MeOD-d 4）δ（ppm）176.2，157.7，157.4，157.1，157.1，144.1，143.7，141.2，140.5，139.7，139.2，135.5，135.4，132.1，131.4，130.6，127.4，127.0，127.0，127.0，127.0，114.3，113.5，112.9，96.3，39.7。
（4）高分辨质谱表征
对目标化合物进行高分辨质谱（High Resolution Mass Spectrum，HRMS）表征，选择电喷雾离子源（Electron Spray Ionization，ESI）作为离子化方式，结果如图7所示，分子式为C 46H 46N 4O 2+，分子量计算值为335.18303，分子量试验值为335.18307。
以上氢谱、碳谱和质谱结果均证明了目标化学物为MG-Rho-2，即本方案的制备方法可行。
实施例2：MG-Rho-2聚集诱导发光性质表征
如图8所示，研究了MG-Rho-2的聚集诱导发光性质。结果表明，MG-Rho-2在甲醇中的吸收峰为550 nm，发射峰为600 nm。图8B和8C显示MG-Rho-2在不同比例四氢呋喃水溶液中的发光强度，当水含量为100%时荧光强度很微弱，随着四氢呋喃比例的增加，MG-Rho-2的发射强度也增强了。其中，四氢呋喃含量为90%时，荧光强度最大，提高了132倍，证明了本方案所合成的MG-Rho-2具有良好的聚集诱导发光性质。
实施例3：MG-Rho-2线粒体靶向性质表征
如图9所示，研究了MG-Rho-2的线粒体靶向性质。图9A中，用MG-Rho-2特异性染色了hela细胞的线粒体区域，可清晰地观察到线粒体的结构，表明MG-Rho-2能够定位于活细胞的线粒体。图9B用线粒体绿色荧光探针Mito Tracker Green进行了染色，并将其与MG-Rho-2染色结果叠加，得到图9C，可观察到两者几乎完全重叠。此外，用皮尔逊相关系数Rr表示两变量间的线性依赖程度，以量化MG-Rho-2与Mito Tracker Green之间的染色区域重叠情况。结果显示，Rr=0.94，表明MG-Rho-2对线粒体具有特异性靶向作用。
本发明提供了一种聚集诱导发光染料MG-Rho-2，该染料具有优良的生物相容性、完全水溶性的线粒体靶向作用，能够应用于活细胞的超快免洗低光毒性成像。此外，本发明提供一种MG-Rho-2的制备方法，该方法具有创新性且操作简单，所制得产品经核磁共振氢谱、碳谱和高分辨质谱表征后确认结构正确。
以上所述仅是本发明的一些实施方式。对于本领域技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

Claims (10)

  1. 一种聚集诱导发光染料，其特征在于，所述染料的特征结构如下式所示：
    。
  2. 一种制备根据权利要求1所述的染料的方法，其特征在于，所述方法包括：
    S1、将碳酸钾、Br-Rho-2、MG-B溶于N,N-二甲基甲酰胺，通氮气保护后，加入四(三苯基膦)钯反应并进行纯化；
    其中所述Br-Rho-2的结构式如下式所示：
    ；
    所述MG-B的结构式如下式所示：
    ；
    S2、将纯化后产物溶于二氯甲烷，加入四氯对苯醌并反应，经纯化后得到所述染料。
  3. 根据权利要求2所述的方法，其特征在于，所述步骤S1中，所述MG-B、所述Br-Rho-2的摩尔比为12:10。
  4. 根据权利要求3所述的方法，其特征在于，所述步骤S1和S2中，所述碳酸钾、所述四(三苯基膦)钯、所述四氯对苯醌的摩尔比为1000:0.8:15。
  5. 根据权利要求4所述的方法，其特征在于，所述步骤S1中，所述反应时间为12-24h。
  6. 根据权利要求5所述的方法，其特征在于，所述步骤S2中，所述反应时间为30min。
  7. 根据权利要求2-6中任一项所述的方法，其特征在于，所述步骤S1中所述纯化包括：将反应后混合物萃取、洗涤、干燥并进行柱层析。
  8. 根据权利要求2-6中任一项所述的方法，其特征在于，所述步骤S2中，所述纯化包括：进行柱层析。
  9. 一种聚集诱导发光染料在活细胞成像中的应用，其特征在于，所述聚集诱导发光染料为权利要求1所述聚集诱导发光染料或权利要求2-8中任一项所述方法制得的聚集诱导发光染料。
  10. 一种聚集诱导发光染料在线粒体靶向染色显色中的应用，其特征在于，所述聚集诱导发光染料为权利要求1所述聚集诱导发光染料或权利要求2-8中任一项所述方法制得的聚集诱导发光染料。
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