WO2024103461A1 - 一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用 - Google Patents

一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用 Download PDF

Info

Publication number
WO2024103461A1
WO2024103461A1 PCT/CN2022/137700 CN2022137700W WO2024103461A1 WO 2024103461 A1 WO2024103461 A1 WO 2024103461A1 CN 2022137700 W CN2022137700 W CN 2022137700W WO 2024103461 A1 WO2024103461 A1 WO 2024103461A1
Authority
WO
WIPO (PCT)
Prior art keywords
rha
lysosome
fluorescent dye
rho
aggregation
Prior art date
Application number
PCT/CN2022/137700
Other languages
English (en)
French (fr)
Inventor
张鹏飞
罗媛
蔡林涛
Original Assignee
深圳先进技术研究院
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院 filed Critical 深圳先进技术研究院
Publication of WO2024103461A1 publication Critical patent/WO2024103461A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • 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
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/12—Amino derivatives of triarylmethanes without any OH group bound to an aryl nucleus
    • 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, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • 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

Definitions

  • the invention belongs to the technical field of luminescent materials, and in particular relates to a lysosome-targeted acid-responsive aggregation-induced luminescent fluorescent dye MG-RhA and a preparation method and application thereof.
  • AIE Aggregation-induced emission
  • 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 News Feature column of Nature in 2016, titled “The nanolight revolution is coming” focused on introducing AIE materials and evaluated that the discovery of AIE materials provides solutions to the problems of currently commonly used quantum dots and luminescent polymer dots, and is a new generation of nanoluminescent materials.
  • fluorescence bioimaging As a powerful non-invasive imaging technology, fluorescence bioimaging has unique advantages in biological applications. In particular, the development and application of super-resolution microscopy technology has made great progress in recent years, but it has been limited by the brightness and stability of fluorescent dyes. Compared with traditional optical imaging, super-resolution fluorescence imaging is based on larger-scale photon statistics. In order to further improve imaging resolution and improve imaging quality, people hope to design fluorescent dyes with higher brightness and better photostability. As one of the main types of fluorescent chromophores used, organic small molecule fluorescent chromophore compounds have also made great progress. Thanks to the advantages of simple synthesis, easy modification, and low toxicity, the development and application of organic small molecule fluorescent probes have attracted particular attention. The development of acid-responsive AIEgen (molecules with AIE properties) with long-wavelength emission, aggregation-induced emission (AIE) characteristics and low phototoxicity has always been a goal pursued by people, but the road ahead is still very challenging.
  • rhodamine dyes are widely used in super-resolution imaging, their fluorescence brightness is usually affected by the non-radiative transition process of intramolecular torsional charge transfer (TICT) after excitation, and their imaging potential has not been fully released.
  • TCT intramolecular torsional charge transfer
  • rhodamine dyes have weak biocompatibility and complex synthesis routes. More importantly, most of the existing rhodamine skeleton dyes are aggregation-induced quenching (ACQ) dyes.
  • the present invention aims to transform traditional rhodamine into a dye with aggregation-induced emission (AIE) properties through reasonable molecular design.
  • the present invention aims to provide a lysosome-targeted acid-responsive aggregation-induced emission (AIE) fluorescent dye MG-RhA and a preparation method and application thereof.
  • AIE acid-responsive aggregation-induced emission
  • a lysosome-targeted acid-responsive aggregation-induced luminescent fluorescent dye MG-RhA the structural formula of the fluorescent dye is shown in Formula I:
  • the present invention also provides a method for preparing the lysosome-targeted acid-responsive aggregation-induced luminescence fluorescent dye MG-RhA, comprising the following steps:
  • a mixture of potassium carbonate, MG-B and Br-Rho-COOH is added to N,N-dimethylformamide, tetrakis(triphenylphosphine)palladium is added under nitrogen protection, reflux reaction is carried out, extraction is carried out with dichloromethane, washing is carried out with a saturated sodium chloride solution, the organic phases are combined and dried, concentrated, and purified by silica gel column chromatography, and then the first crude product is dissolved in dichloromethane, tetrachloro-p-benzoquinone is added, and stirred at room temperature by a one-pot method, and the obtained second crude product is purified by silica gel column chromatography to obtain a dark green MG-Rho-COOH solid;
  • step (1) the molar ratio of MG-Br, pinacol borate, potassium acetate and [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride is 0.1:0.3:0.125:0.012.
  • the reflux temperature is 120-150° C.
  • the reflux time is 10-25 h.
  • the molar ratio of the potassium carbonate, the MG-B, the Br-Rho-COOH, the tetrakis(triphenylphosphine)palladium, and the tetrachlorobenzoquinone is (10:0.12:0.1:0.008:0.15)-(20:0.24:0.2:0.016:0.3).
  • the reflux temperature is 100-120° C.
  • the reflux time is 10-12 h.
  • the molar ratio of hydrazine hydrate to MG-Rho-COOH is (10:1)-(20:1).
  • the reflux temperature is 50°C-70°C, and the reflux time is 3h-12h.
  • the extraction process comprises: firstly adding saturated saline and 0.1 M NaOH in a volume ratio of 1:1 to the mixed solution after the reaction is completed, and then adding dichloromethane for extraction.
  • the present invention also provides the use of the lysosome-targeted acid-responsive aggregation-induced luminescence fluorescent dye MG-RhA or the lysosome-targeted acid-responsive aggregation-induced luminescence fluorescent dye MG-RhA prepared by the preparation method as a fluorescent probe in intelligent sensing, functional imaging, biological detection, and immunotherapy.
  • the MG-RhA designed and synthesized by the present invention is a new structure and, as a new type of AIE dye, has very broad application prospects.
  • the present invention successfully developed an organic small molecule acid-responsive probe MG-RhA with aggregation-induced emission (AIE) properties based on a rhodamine skeleton.
  • the probe is purposeful and directional, reduces the number of reaction steps and by-products, and is easy to purify. It transforms traditional rhodamine B into a dye with aggregation-induced emission properties. Thanks to the two quaternary ammonium groups in the molecule, the complete acid response of the molecule enables it to be successfully applied to ultrafast, wash-free, low-phototoxicity imaging of living cells, which will greatly facilitate biological research related to living cells.
  • the fluorescent dye provided by the present invention can be used for wash-free staining of living cells and has a better signal-to-noise ratio than commercial lysosome-targeted dyes.
  • the dye has a novel structure and can target lysosomes well under acidic conditions. It has broad application prospects in the fields of biological stains, cell staining imaging, and organic semiconductor materials.
  • Figure 1 is the H-NMR spectrum of MG-RhA
  • FIG2 is a carbon NMR spectrum of MG-RhA
  • FIG3 is a high-resolution mass spectrum of MG-RhA
  • Figure 4 shows the co-localization analysis of MG-RhA targeting lysosomes
  • FIG5 is a graph showing the fluorescence intensity of MG-RhA acid response
  • FIG6 is a graph showing the AIE fluorescence characteristics of MG-RhA
  • FIG7 is a high-resolution mass spectrum of MG-Rho-COOH
  • FIG8 is a high-resolution mass spectrum of MG-B.
  • the technical problem to be solved by the present invention is to provide a lysosome-targeted acid-responsive aggregation-induced emission (AIE) fluorescent dye MG-RhA and a preparation method thereof.
  • AIE aggregation-induced emission
  • the present invention develops and synthesizes a lysosome-targeted acid-responsive organic small molecule fluorescent dye for the first time
  • MG-RhA is a dye with aggregation-induced emission (AIE) characteristics, which can be used for wash-free staining of living cells and has a better signal-to-noise ratio than commercial lysosome-targeted dyes.
  • the dye of the present invention has a novel structure and can well target lysosomes under acidic conditions. This method is innovative and has broad application prospects.
  • AIE acid-responsive aggregation-induced emission
  • a method for synthesizing a lysosome-targeted acid-responsive aggregation-induced emission (AIE) fluorescent dye MG-RhA comprises the following steps, as shown in Formula II to Formula V:
  • the intermediate MG-B was characterized by high-resolution mass spectrometry: the high-resolution mass spectrum of MG-B is shown in Figure 8, and the mass spectrum data are: HRMS (ESI) calcd. for C 29 H 38 BN 2 O 2 [M + H] + 457.30263, found: 457.30215.
  • reaction mixture was refluxed at 65 °C for 6 h. After the reaction was completed, it was cooled to room temperature and mixed with saturated brine and 0.1 M NaOH at a ratio of 1:1 (v:v). DCM was then added for extraction. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a crude product.
  • the present invention systematically characterizes the target compound MG-RhA during the synthesis process, including nuclear magnetic resonance hydrogen spectrum, carbon spectrum, and high-resolution mass spectrum, proving that the scheme is feasible and the target compound is correct.
  • the hydrogen spectrum of MG-RhA is shown in Figure 2.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Materials Engineering (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用，属于发光材料技术领域，该荧光染料MG-RhA以罗丹明B为骨架，将传统的罗丹明B改造成为具有聚集诱导发光性质的染料。该荧光染料结构新颖，能在酸性条件下很好地靶向溶酶体，可用于活细胞的免洗染色，具有比商业化的溶酶体靶向染料更好的信噪比，在生物染色剂、细胞染色成像和有机半导体材料等领域具有广泛的应用前景。

Description

一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用 技术领域
本发明属于发光材料技术领域，具体涉及一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用。
 背景技术
聚集诱导发光（AIE）现象是唐本忠院士2001年首次提出的概念。由于AIE分子(AIEgens)独特的聚集态高效发光特性,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有关AIE的研究，在发展新AIE分子、AIE机理、AIE材料在光电器件、生物探针与成像、化学传感、智能材料应用等领域均取得了显著的成果。AIE已经成为发光材料和光物理等领域的一个研究热点，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Nature的News Feature专栏以“The nanolight revolution is coming”(纳米光革命正在来临)为题重点介绍了AIE材料,并评价AIE材料的发现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。
荧光生物成像作为一种强大的非创伤性成像技术在生物应用方面具有独特的优势，特别是超分辨显微技术的开发和应用近年来取得长足的发展，但一直受限于荧光染料的亮度和稳定性。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的基础上。为了进一步提升成像分辨率、改善成像质量，人们希望设计出具有更高亮度以及更好光稳定性的荧光染料。作为其中主要使用的一类荧光生色团，有机小分子荧光生色团化合物也获得了很大的发展。得益于合成简便，易于修饰，毒性较小等优势，有机小分子荧光探针的开发及应用尤其受到关注。开发具有长波长发射的聚集诱导发光(AIE)特性和低光毒性的酸响应AIEgen（具有AIE性质的分子）一直是人们追求的目标，但前进的道路依然极具挑战性。
罗丹明染料虽然被广泛应用于超分辨成像，但其荧光亮度通常受激发后分子内扭转电荷转移（TICT）的非辐射跃迁过程的影响，其成像潜力并未得到完全的释放。
 技术问题
传统的罗丹明染料生物相容性较弱且合成路线复杂，更重要的是现有的罗丹明骨架染料绝大部分都是聚集诱导淬灭（ACQ）染料。本发明旨在通过合理的分子设计将传统的罗丹明改造成为具有聚集诱导发光（AIE）性质的染料。
 技术解决方案
针对现有技术不足，本发明旨在提供一种溶酶体靶向的酸响应聚集诱导发光（AIE）荧光染料MG-RhA及其制备方法与应用。
为达到上述目的，本发明的技术方案是这样实现的：
一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA，荧光染料的结构式如式I所示：
式I。
本发明还提供了该溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，包括以下步骤：
(1)制备MG-B：
将摩尔比为0.1:0.2:0.3的4-溴苯甲醛、无水氯化锌、N,N-二甲基苯胺与无水乙醇混合并于100℃搅拌过夜，冷却后浓缩以除去剩余N,N-二甲基苯胺，再用水和丙烯酸乙酯萃取，合并有机相后干燥、硅胶柱纯化，得到白色的MG-Br粉末；再将MG-Br、频哪硼酸酯和醋酸钾溶于N,N-二甲基甲酰胺中，加入已溶于N,N-二甲基甲酰胺内的[1,1'-双(二苯基膦)二茂铁]二氯化钯(II)作为铃木偶联反应的催化剂，80℃搅拌12h后冷却，用二氯甲烷和水萃取，收集有机相并干燥，浓缩、硅胶柱层析纯化、干燥后得到白色的MG-B固体；
(2)制备Br-Rho-COOH：
将52.8 mmol N,N-二乙氨基苯酚、21.1 mmol 4-溴邻苯二甲酸酐、90 mL丙酸和0.5 mL甲磺酸混合并回流加热，得到的深色粗产物溶于二氯甲烷，用饱和食盐水萃取，收集有机相并浓缩、硅胶柱层析纯化，用甲醇/二氯甲烷梯度重复硅胶层析分离异构体，干燥后得到深紫色的Br-Rho-COOH固体；
(3)制备MG-Rho-COOH：
将碳酸钾、MG-B和Br-Rho-COOH的混合物加到N,N-二甲基甲酰胺中，氮气保护下加入四(三苯基膦)钯，回流反应，用二氯甲烷萃取，饱和氯化钠溶液洗涤，合并有机相后干燥，浓缩，用硅胶柱层析纯化，再将第一粗产物溶解于二氯甲烷，加入四氯对苯醌通过一锅法室温搅拌，得到的第二粗产物经硅胶柱层析纯化，得到墨绿色的MG-Rho-COOH固体；
(4)制备MG-RhA：
将水合肼和MG-Rho-COOH分别溶于甲醇溶液后再混合得到混合液，将混合液加热回流进行反应；反应结束后冷却，萃取，收集有机相，再干燥，旋转蒸发浓缩得到粗产物；最后将粗产物通过硅胶柱层析纯化，即可得到墨绿色的荧光染料MG-RhA固体。
优选的，步骤(1)中，MG-Br、频哪硼酸酯、醋酸钾和[1,1'-双(二苯基膦)二茂铁]二氯化钯(II)的摩尔比为0.1:0.3:0.125:0.012。
优选的，步骤(2)中，回流温度为120-150℃，回流时间为10-25 h。
优选的，步骤(3)中，所述碳酸钾、所述MG-B、所述Br-Rho-COOH、所述四(三苯基膦)钯、所述四氯对苯醌的摩尔比为(10:0.12:0.1:0.008:0.15)-(20:0.24:0.2:0.016:0.3)。
优选的，步骤(3)中，回流温度为100-120℃，回流时间为10-12h。
优选的，步骤(4)中，水合肼与MG-Rho-COOH的摩尔比为(10:1)-(20:1)。
优选的，步骤(4)中，回流温度为50℃-70℃，回流时间为3h-12h。
优选的，步骤(4)中，萃取过程包括：先向反应结束后的混合液中加入体积比为1:1的饱和食盐水与0.1 M NaoH，再加入二氯甲烷进行萃取。
本发明还提供了该溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA或该制备方法制备得到的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA作为荧光探针在智能传感、功能成像、生物检测、免疫治疗中的应用。
 有益效果
(1)本发明设计合成的MG-RhA是新结构，作为一个新型的AIE染料，具有非常广阔的应用前景。
(2)本发明成功开发了基于罗丹明骨架的具有聚集诱导发光（AIE）特性的有机小分子酸响应探针MG-RhA，具有目的性和指向性，减少反应步数和副产物，纯化简单，将传统的罗丹明B改造成为具有聚集诱导发光性质的染料，得益于分子的两个季铵盐基团，分子的完全酸响应使之成功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。
(3)本发明提供的荧光染料可用于活细胞的免洗染色，具有比商业化的溶酶体靶向染料更好的信噪比，并且，此染料结构新颖，能在酸性条件下很好的靶向溶酶体，在生物染色剂、细胞染色成像和有机半导体材料等领域具有广泛的应用前景。
 附图说明
此处的附图被并入说明书中并构成说明书的一部分，示出了符合本发明的实施例，并与说明书一起用于解释本发明的原理，其中：
图1为MG-RhA的核磁氢谱图；
图2为MG-RhA的核磁碳谱图；
图3为MG-RhA的高分辨质谱图；
图4为MG-RhA靶向溶酶体的共定位分析；
图5为MG-RhA酸响应荧光强度图；
图6为MG-RhA的AIE荧光特性图；
图7为MG-Rho-COOH的高分辨质谱图；
图8为MG-B的高分辨质谱图。
 本发明的实施方式
下文的公开提供了许多不同的实施方式或例子用来实现本发明的不同结构。为了简化本发明的公开，下文中对特定例子的部件和设置进行描述。当然，它们仅仅为示例，并且目的不在于限制本发明。此外，本发明可以在不同例子中重复参考数字和/或参考字母，这种重复是为了简化和清楚的目的，其本身不指示所讨论各种实施方式和/或设置之间的关系。
本发明所要解决的技术问题在于提供一种溶酶体靶向的酸响应聚集诱导发光（AIE）荧光染料MG-RhA及其制备方法。具体来讲，本发明首次开发合成了一个具有溶酶体靶向的酸响应有机小分子荧光染料，并且MG-RhA是一种具有聚集诱导发光（AIE）特性的染料，此染料可用于活细胞的免洗染色，具有比商业化的溶酶体靶向染料更好的信噪比。并且，本发明的染料结构新颖，能在酸性条件下很好的靶向溶酶体，此方法具有创新性，广泛的应用前景。
实施例
一种溶酶体靶向的酸响应聚集诱导发光（AIE）荧光染料MG-RhA的合成方法，荧光染料的结构式如下：
式I
一种溶酶体靶向的酸响应聚集诱导发光（AIE）荧光染料MG-RhA的合成方法包括以下步骤，如式II~式V所示：
式II
式III 式IV
式V
(1)制备MG-B：
①将4-溴苯甲醛(22.1 mg, 0.1 mmol)、无水氯化锌(28.1 mg, 0.2 mmol)、N, N-二甲基苯胺(36.2 mg, 0.3 mmol)和无水乙醇(1 mL)的混合物在100℃下搅拌过夜。冷却至室温后，将混合物浓缩以除去剩余的N, N-二甲基苯胺。用水(4 mL)和丙烯酸乙酯 (3-5 mL)提取粗产物。合并有机相后用无水硫酸钠干燥，粗产物经硅胶柱纯化(PE:EA= 10:1)，得到白色的溴代孔雀石绿（MG-Br）粉末。
②将MG-Br(40.8 mg, 0.1 mmol)、频哪硼酸酯(80 mg, 0.3 mmol)和醋酸钾(49 mg, 0.125 mmol)溶于N,N-二甲基甲酰胺(3 mL)中。将[1,1'-双(二苯基膦)二茂铁]二氯化钯(II)(10 mg, 0.012 mmol, 10 %)作为铃木偶联反应的催化剂溶于N,N-二甲基甲酰胺(1 mL)中加入反应物混合物中，在80℃下搅拌12小时。然后将混合物冷却，加入二氯甲烷 (10 mL)，用30 mL的水在分液漏斗中萃取，收集有机相，用无水硫酸钠干燥，旋转蒸发干燥。粗产品经硅胶柱层析纯化，去除溶剂，真空干燥后得到目标产物孔雀石绿硼酸酯（MG-B）为白色固体。
对中间体MG-B进行了高分辨质谱表征：MG-B的高分辨质谱如图8所示，质谱数据为：HRMS (ESI) calcd. for C 29H 38BN 2O 2 [M + H] + 457.30263, found: 457.30215。
(2)制备Br-Rho-COOH：
①将N,N-二乙氨基苯酚(7.2 g, 52.8 mmol)、4-溴邻苯二甲酸酐(4.8 g, 21.1 mmol)、丙酸(90 mL)和甲磺酸(0.5 mL)的混合物120℃-150℃回流加热10-25 h。
②得到深色粗产物溶解在二氯甲烷(1000 mL)中，饱和食盐水萃取。收集有机相浓缩。粗产品经硅胶柱层析纯化，用甲醇：二氯甲烷(20:1-10:1)梯度进行三次重复硅胶层析分离异构体，去除溶剂，真空干燥后得到中间体Br-Rho-COOH为深紫色固体。
(3)制备MG-Rho-COOH：
①将碳酸钾(10 mmol)、MG-B(0.12 mmol)和Br-Rho-COOH (0.1 mmol)的混合物中加到N,N-二甲基甲酰胺(3 mL)中。通氮气保护后，加入四(三苯基膦)钯(0.008 mmol)。
②反应混合液回流12 h，用二氯甲烷萃取，饱和氯化钠溶液洗涤3次。合并有机相无水硫酸钠干燥。粗产物浓缩后，以(乙酸乙酯/己烷=1:20-1:10)为洗脱剂，硅胶柱层析纯化。
③然后将纯化后的产物溶解在二氯甲烷里，加入四氯对苯醌(0.15 mmol)一锅法室温搅拌30 min。得到的粗产物经硅胶柱层析纯化(甲醇/二氯甲烷=1:10-1:5)得到最终目标产物MG-Rho-COOH为墨绿色固体。
如图7，对中间产物MG-Rho-COOH进行了高分辨质谱表征:HRMS (ESI) calcd. for C 51H 54N 4O 3 2+ [M] 2+ 385.20905, found: 385.20925。
(4)制备MG-RhA：
①将100 uL(1.89 mmol)水合肼溶于2 mL甲醇中，再将其加入到溶有100mg(0.12 mmol) MG-Rho-COOH的2 mL甲醇中。
②反应混合液65 ℃回流6 h，反应完成后冷却至室温，用饱和食盐水跟0.1 M NaoH按1:1（v:v）混合，后加入DCM进行萃取，收集有机相，用无水硫酸钠干燥，旋转蒸发浓缩得到粗产品。
③得到的粗产物以(乙酸乙酯/己烷=1:20-1:10)为洗脱剂，硅胶柱层析纯化。得到最终目标产物MG-RhA为墨绿色固体。
本发明在合成过程中对目标化合物MG-RhA进行了系统的表征，包括核磁氢谱，碳谱，和高分辨质谱。证明方案可行，目标化合物正确。
MG-RhA的氢谱如图2所示，氢谱数据为： 1H NMR (400 MHz, DMSO) δ (ppm) 8.03 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 9.6 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 9.0 Hz, 2H), 7.10 (d, J = 9.5 Hz, 3H), 7.01 (d, J = 8.4 Hz, 2H), 6.63 (d, J = 8.6 Hz, 2H), 6.42 – 6.34 (m, 7H), 4.33 (d, J = 24.9 Hz, 2H), 3.30 (s, 12H), 2.84 (d, J = 6.8 Hz, 8H), 1.08 (d, J = 6.6 Hz, 12H)。
MG-RhA的碳谱如图3所示，碳谱数据为： 13C NMR (100 MHz, MeOD-d4) δ (ppm) 176.6, 166.5, 157.2, 153.8, 152.2, 144.4, 140.5, 139.7, 139.0, 135.4, 131.7, 130.4, 127.5, 127.1, 126.9, 124.4, 120.8, 113.5, 108.2, 66.6, 48.2, 48.0, 47.8, 47.6, 47.4, 47.2, 47.0, 44.0, 39.6, 11.5。
MG-RhA的高分辨质谱如图4所示，质谱数据为：HRMS (ESI) calcd. for C 51H 55N 6O 2 + [M] + 783.43810, found: 783.43848。
性能检测：
(1)溶酶体靶向性：
分别将本发明的染料MG-RhA与市售的染料Lyso tracker用于孵育海拉细胞后，进行激光扫描共聚焦显微镜成像（CLSM），在MG-RhA的红色荧光区域，溶酶体的结构清晰可见（见图4），表明MG-RhA定位于活细胞的溶酶体。皮尔逊相关系数(Rr;从+1到1)高达0.905，表示两个变量之间的线性依赖程度，用于量化MG-RhA与市售染料之间的染色区域重叠。此实验证明了本发明所开发染料MG-RhA的溶酶体靶向性。
(2)酸响应性：检测MG-RhA在不同PH的缓冲液里光致发射（PL）光谱图，结果如图5所示，从图5中可以看出，MG-RhA的荧光强度随着溶液酸性的增加而增强，证明了其酸性响应的特性。
(3)荧光特性：研究MG-RhA在不同比例的水/四氢呋喃混合物中的AIE特征，参见图6，可以看到，随着水/四氢呋喃混合物中四氢呋喃浓度由0到90%的逐渐增加，MG-RhA的荧光强度也随着增加。由图6可以观察到，MG-RhA最强的荧光强度在四氢呋喃的比例为90%时，荧光强度最高。此实验证明了本发明所开发染料MG-RhA的荧光特性。
本领域技术人员在考虑说明书及实践这里的发明后，将容易想到本发明的其它实施方案。本发明旨在涵盖本发明的任何变型、用途或者适应性变化，这些变型、用途或者适应性变化遵循本发明的一般性原理并包括本发明的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的，本发明的真正范围和精神由权利要求指出。
应当理解的是，本发明并不局限于上面已经描述并在附图中示出的精确结构，并且可以在不脱离其范围进行各种修改和改变。本发明的范围仅由所附的权利要求来限制。

Claims (10)

  1. 一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA，其特征在于，所述荧光染料的结构式如式I所示：
    式I。
  2. 根据权利要求1所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，包括以下步骤：
    (1)制备MG-B：
    将摩尔比为0.1:0.2:0.3的4-溴苯甲醛、无水氯化锌、N,N-二甲基苯胺与无水乙醇混合并于100℃搅拌过夜，冷却后浓缩以除去剩余N,N-二甲基苯胺，再用水和丙烯酸乙酯萃取，合并有机相后干燥、硅胶柱纯化，得到白色的MG-Br粉末；再将所述MG-Br、频哪硼酸酯和醋酸钾溶于N,N-二甲基甲酰胺中，加入已溶于N,N-二甲基甲酰胺内的[1,1'-双(二苯基膦)二茂铁]二氯化钯(II)作为铃木偶联反应的催化剂，80℃搅拌12h后冷却，用二氯甲烷和水萃取，收集有机相并干燥，浓缩、硅胶柱层析纯化、干燥后得到白色的MG-B固体；
    (2)制备Br-Rho-COOH：
    将52.8 mmol N,N-二乙氨基苯酚、21.1 mmol 4-溴邻苯二甲酸酐、90 mL丙酸和0.5 mL甲磺酸混合并回流加热，得到的深色粗产物溶于二氯甲烷，用饱和食盐水萃取，收集有机相并浓缩、硅胶柱层析纯化，用甲醇/二氯甲烷梯度重复硅胶层析分离异构体，干燥后得到深紫色的Br-Rho-COOH固体；
    (3)制备MG-Rho-COOH：
    将碳酸钾、所述MG-B和所述Br-Rho-COOH的混合物加到N,N-二甲基甲酰胺中，氮气保护下加入四(三苯基膦)钯，回流反应，用二氯甲烷萃取，饱和氯化钠溶液洗涤，合并有机相后干燥，浓缩，用硅胶柱层析纯化，再将第一粗产物溶解于二氯甲烷，加入四氯对苯醌通过一锅法室温搅拌，得到的第二粗产物经硅胶柱层析纯化，得到墨绿色的MG-Rho-COOH固体；
    (4)制备MG-RhA：
    将水合肼和所述MG-Rho-COOH分别溶于甲醇溶液后再混合得到混合液，将所述混合液加热回流进行反应；反应结束后冷却，萃取，收集有机相，再干燥，旋转蒸发浓缩得到粗产物；最后将所述粗产物通过硅胶柱层析纯化，即可得到墨绿色的所述荧光染料MG-RhA固体。
  3. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(1)中，所述MG-Br、所述频哪硼酸酯、所述醋酸钾和所述[1,1'-双(二苯基膦)二茂铁]二氯化钯(II)的摩尔比为0.1:0.3:0.125:0.012。
  4. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(2)中，所述回流温度为120-150℃，所述回流时间为10-25 h。
  5. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(3)中，所述碳酸钾、所述MG-B、所述Br-Rho-COOH、所述四(三苯基膦)钯、所述四氯对苯醌的摩尔比为(10:0.12:0.1:0.008:0.15)-(20:0.24:0.2:0.016:0.3)。
  6. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(3)中，所述回流温度为100-120℃，所述回流时间为10-12h。
  7. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(4)中，所述水合肼与所述MG-Rho-COOH的摩尔比为(10:1)-(20:1)。
  8. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(4)中，所述回流温度为50℃-70℃，所述回流时间为3h-12h。
  9. 根据权利要求2所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA的制备方法，其特征在于，步骤(4)中，所述萃取过程包括：先向反应结束后的所述混合液中加入体积比为1:1的饱和食盐水与0.1 M NaoH，再加入二氯甲烷进行萃取。
  10. 根据权利要求1所述的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA或权利要求2~8任一项所述的制备方法制备得到的溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA作为荧光探针在智能传感、功能成像、生物检测、免疫治疗中的应用。
PCT/CN2022/137700 2022-11-14 2022-12-08 一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用 WO2024103461A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211422593.1A CN118027049A (zh) 2022-11-14 2022-11-14 一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用
CN202211422593.1 2022-11-14

Publications (1)

Publication Number Publication Date
WO2024103461A1 true WO2024103461A1 (zh) 2024-05-23

Family

ID=90982818

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/137700 WO2024103461A1 (zh) 2022-11-14 2022-12-08 一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用

Country Status (2)

Country Link
CN (1) CN118027049A (zh)
WO (1) WO2024103461A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830315A (zh) * 2015-05-11 2015-08-12 济南大学 一种二价铜离子荧光探针及其制备方法和应用
CN105199713A (zh) * 2015-10-08 2015-12-30 上海交通大学 具有高效固态发光性质的罗丹明b衍生物及其制备和应用
CN111333668A (zh) * 2018-12-18 2020-06-26 中国科学院大连化学物理研究所 一类近红外孔雀绿染料及其合成方法和应用
CN115991717A (zh) * 2021-10-18 2023-04-21 深圳先进技术研究院 一种孔雀石绿硼酸酯及其衍生物、制备方法和应用
CN115991696A (zh) * 2021-10-18 2023-04-21 深圳先进技术研究院 一种聚集诱导发光荧光染料MG-Rho及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830315A (zh) * 2015-05-11 2015-08-12 济南大学 一种二价铜离子荧光探针及其制备方法和应用
CN105199713A (zh) * 2015-10-08 2015-12-30 上海交通大学 具有高效固态发光性质的罗丹明b衍生物及其制备和应用
CN111333668A (zh) * 2018-12-18 2020-06-26 中国科学院大连化学物理研究所 一类近红外孔雀绿染料及其合成方法和应用
CN115991717A (zh) * 2021-10-18 2023-04-21 深圳先进技术研究院 一种孔雀石绿硼酸酯及其衍生物、制备方法和应用
CN115991696A (zh) * 2021-10-18 2023-04-21 深圳先进技术研究院 一种聚集诱导发光荧光染料MG-Rho及其制备方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
REN MINGGUANG, DENG BEIBEI; WANG JIAN-YONG; LIU ZHAN-RONG; LIN WEIYING: "A dual-emission fluorescence-enhanced probe for imaging copper(II) ions in lysosomes† ", JOURNAL OF MATERIALS CHEMISTRY. B, ROYAL SOCIETY OF CHEMISTRY, GB, vol. 3, no. 33, 1 January 2015 (2015-01-01), GB , pages 6746 - 6752, XP093171332, ISSN: 2050-750X, DOI: 10.1039/C5TB01184A *
TATSUMI, YUTO ET AL.: "A Photo- and Halochromic Multicolor Switching System Consisting of Diarylethene and Malachite Green Moieties", NEW JOURNAL OF CHEMISTRY, vol. 38, no. 12, 24 June 2014 (2014-06-24), pages 5706 - 5714, XP093058512, DOI: 10.1039/C4NJ00434E *
ZHOU LIYI, ZHANG XIAOBING; WANG QIANQIAN; LV YIFAN; MAO GUOJIANG; LUO AILI; WU YONGXIANG; WU YUAN; ZHANG JING; TAN WEIHONG: "Molecular Engineering of a TBET-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Living Cells and Tissues", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 136, no. 28, 16 July 2014 (2014-07-16), pages 9838 - 9841, XP093171329, ISSN: 0002-7863, DOI: 10.1021/ja504015t *
ZHOU, LIYI ET AL.: "A New Ratiometric Two-photon Fluorescent Probe for Imaging of Lysosomes in Living Cells and Tissues", TETRAHEDRON, vol. 72, no. 31, 18 June 2016 (2016-06-18), pages 4637 - 4642, XP029635479, DOI: 10.1016/j.tet.2016.06.038 *

Also Published As

Publication number Publication date
CN118027049A (zh) 2024-05-14

Similar Documents

Publication Publication Date Title
Wang et al. Rational design of novel near-infrared fluorescent DCM derivatives and their application in bioimaging
WO2018192521A1 (en) Probe for dual-mode bio-imaging
CN111718365B (zh) 一种三聚茚基共轭三bodipy类近红外荧光染料及其制备方法
CN111978248A (zh) 一种多位点可修饰聚集诱导发光喹啉腈衍生物、制备方法及其应用
CN113278155B (zh) 一种近红外有机超分子组装体及其制备方法和应用
CN113461722B (zh) 一种具有aie效应的双bodipy类近红外荧光染料及其制备方法
CN114656493A (zh) 一种具有聚集诱导发光、近红外发射荧光染料吡咯并吡咯菁类化合物及其制备方法
CN113831287A (zh) 具有活性端的萘酰亚胺类化合物及其制备方法和应用
CN115991696B (zh) 一种聚集诱导发光荧光染料MG-Rho及其制备方法和应用
WO2024103461A1 (zh) 一种溶酶体靶向的酸响应聚集诱导发光荧光染料MG-RhA及其制备方法与应用
CN115819281B (zh) 一种氰基取代的对苯撑乙烯衍生物、制备方法及应用
CN114853656B (zh) 具有aee特性的咔唑类衍生物、制备方法及应用
CN116023392A (zh) 苯并[1,2-c:4,5-c`]双([1,2,5]噻二唑)类染料的合成及应用
CN110143907B (zh) 一种9,10-二吡咯基蒽荧光化合物及其制备方法和应用
CN114409587A (zh) 制备aie分子的方法、aie分子及其应用
CN113150017A (zh) 一种偶联双bodipy类近红外吸收染料及其制备方法
CN117164609B (zh) 一种基于氟化硼-三苯胺-苯乙腈的d-a型聚集诱导光电材料及其制备方法和应用
CN113501836A (zh) 一种星型bodipy近红外荧光染料及其制备方法
WO2024108656A1 (zh) 一种线粒体靶向水溶性聚集诱导发光染料及其制备方法
CN105061320A (zh) 一种咪唑三酰腙类化合物，其作为有机发光材料的应用及其制备方法
CN112724701B (zh) 一类红光香豆素类聚集诱导发光荧光染料
CN114853665B (zh) 一种荧光化合物及其制备方法和应用
CN116375726B (zh) 具有光致变色与力致变色性能的四苯乙烯环化化合物和合成方法及应用
CN114394924B (zh) 一种用于粘度检测的有机双态荧光分子探针及其制备方法与应用
CN115584142B (zh) 一种红色发光环状结构花菁染料及其合成方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22965639

Country of ref document: EP

Kind code of ref document: A1