技术领域technical field
本发明属于氟硼荧衍生物技术领域，具体为一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物及其制备方法和应用。The invention belongs to the technical field of borofluorene derivatives, and specifically relates to a 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivative and a preparation method and application thereof.
背景技术Background technique
内质网是真核细胞内非常重要的多功能细胞器，细胞内蛋白质的合成、折叠、修饰，脂类和固醇合成是以内质网为场所而进行的。在完成这些基本生理功能的同时，内质网凭借其庞大的膜结构成为协调信号转导的枢纽平台，维持细胞内环境的稳定。当遗传或环境损伤引起内质网应激，内质网应激过强或持续时间过久可引起细胞凋亡，从而引发一些列的疾病[参见：J.E.Vance,Traffic,2015,16,1.]，专一性跟踪检测细胞内内质网的形态及分布有利于深入了解和研究许多相关的生理活动[参见：Z.Yang,Y.He,J.H.Lee,W.-S.Chae,W.X.Ren,J.H.Lee,C.Kang and J.S.Kim,Chem.Commun.,2014,50,11672.]。The endoplasmic reticulum is a very important multifunctional organelle in eukaryotic cells. The synthesis, folding and modification of intracellular proteins, the synthesis of lipids and sterols take place in the endoplasmic reticulum. While completing these basic physiological functions, the endoplasmic reticulum has become a hub platform for coordinating signal transduction by virtue of its huge membrane structure, maintaining the stability of the intracellular environment. When genetic or environmental damage causes endoplasmic reticulum stress, excessive endoplasmic reticulum stress or prolonged endoplasmic reticulum stress can cause apoptosis, which can lead to a series of diseases [see: J.E.Vance, Traffic, 2015, 16, 1. ], specifically tracking and detecting the morphology and distribution of endoplasmic reticulum in cells is conducive to in-depth understanding and research on many related physiological activities [see: Z.Yang, Y.He, J.H.Lee, W.-S.Chae, W.X.Ren , J.H. Lee, C. Kang and J.S. Kim, Chem. Commun., 2014, 50, 11672.].
近年来，对于标记内质网的靶向试剂有一些报道，但具体靶向定位标记的机理并不十分明确。我们需要更多的研究来设计可靠的内质网标记靶向试剂，目前常用的是将靶向基团与荧光团相结合来构筑新型的内质网靶向试剂[参见：W.Xu,Z.Zeng,J.-H.Jiang,Y.-T.Chang and L.Yuan,Angew.Chem.,Int.Ed.2016,55,13658]。In recent years, there have been some reports on targeting reagents for labeling the endoplasmic reticulum, but the mechanism of the specific targeting and localization of the labeling is not very clear. We need more research to design reliable ER-labeled targeting reagents. Currently, combining targeting groups with fluorophores is commonly used to construct novel ER-targeting reagents [see: W.Xu,Z . Zeng, J.-H. Jiang, Y.-T. Chang and L. Yuan, Angew. Chem., Int. Ed. 2016, 55, 13658].
在众多荧光染料中，氟硼络合的二吡咯甲烷类荧光染料又称氟硼荧具有较高的摩尔消光系数、荧光量子产率高、稳定的光谱性质、高的光热及化学稳定性、分子量小和较低的细胞毒性等优点，作为生物分子、离子等荧光探针和细胞器成像荧光试剂等已被广泛应用[参见：(a)S.Arai,S.-C.Lee,D.Zhai,M.Suzuki and Chang,Y.T.Sci.Rep.2014,4,6701；(b)X.Kong,F.Su,L.Zhang,J.Yaron,F.Lee,Z.Shi,Y.Tian and Meldrum,D.R.Angew.Chem.,Int.Ed.2015,54,12053；(c)L.Yang,Y.-J.Ji,J.-F.Yin,Y.Wu,H.Fan,Y.Zhang and G.-C.Kuang,Soft Matter,2016,12,8581.]。有很多基于氟硼荧骨架的标记分子已经被广泛做为细胞器的标准标记试剂在市场上流通，如LysoTrackerTM Red、LysoTrackerTM Green、ER-TrackerTM Red和ER-TrackerTM Green[参见：I.Johnson,M.T.Z.Spence,The Molecular Probes Handbook,A Guide to Fluorescent Probes andLabeling Technologies,11th Ed.；Molecular Probes:Eugene,OR,2010.]。但是，这些常见的市售内质网靶向试剂结构复杂，合成不方便，价格昂贵。Among many fluorescent dyes, the dipyrromethane fluorescent dyes complexed with fluoroboron, also known as fluoroboronfluorescein, have a high molar extinction coefficient, high fluorescence quantum yield, stable spectral properties, high photothermal and chemical stability, With the advantages of small molecular weight and low cytotoxicity, it has been widely used as fluorescent probes such as biomolecules and ions and fluorescent reagents for organelle imaging [see: (a) S.Arai, S.-C.Lee, D.Zhai , M.Suzuki and Chang, YTSci.Rep.2014, 4, 6701; (b) X.Kong, F.Su, L.Zhang, J.Yaron, F.Lee, Z.Shi, Y.Tian and Meldrum, DR Angew. Chem., Int. Ed. 2015, 54, 12053; (c) L. Yang, Y.-J. Ji, J.-F. Yin, Y. Wu, H. Fan, Y. Zhang and G. -C. Kuang, Soft Matter, 2016, 12, 8581.]. There are many labeling molecules based on the fluoroborate skeleton that have been widely circulated in the market as standard labeling reagents for organelles, such as LysoTrackerTM Red, LysoTrackerTM Green, ER-TrackerTM Red and ER-TrackerTM Green [see: I. Johnson, MTZ Spence, The Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11th Ed.; Molecular Probes: Eugene, OR, 2010.]. However, these common commercially available endoplasmic reticulum targeting reagents are complex in structure, inconvenient to synthesize, and expensive.
因此，提供一种以用于内质网标记的荧光分子，结构简单，合成简便，生产成本低，成为了本领域技术人员亟待解决的问题。Therefore, providing a fluorescent molecule for endoplasmic reticulum labeling, which has a simple structure, easy synthesis, and low production cost has become an urgent problem to be solved by those skilled in the art.
发明内容Contents of the invention
本发明的目的在于提供一种可以用于内质网标记的氟硼荧类衍生物小分子，将本发明氟硼荧类衍生物用于内质网荧光标记，可以有效解决现有技术中内质网靶向试剂结构复杂，合成不方便，价格昂贵的问题。The purpose of the present invention is to provide a small molecule of fluorine-boron derivatives that can be used for endoplasmic reticulum labeling. Using the fluorine-boron derivatives of the present invention for fluorescent labeling of endoplasmic reticulum can effectively solve the internal problems in the prior art. Plasma reticulum targeting reagents have complex structures, inconvenient synthesis, and high prices.
本发明还提供该氟硼荧类衍生物的制备方法及应用。The invention also provides the preparation method and application of the fluorine boron derivatives.
本发明目的通过以下技术方案来实现：The object of the invention is achieved through the following technical solutions:
一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物，具有下述式Ⅰ所示结构：A 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroboron derivative, which has the structure shown in the following formula I:
其中：R1,R2,R3为氢、氘、烷基、烷氧基、羰基、酯基、卤素、取代芳基或取代杂芳基中的一种或两种。进一步，所述烷基、烷氧基、酯基或羰基的碳链为碳个数为0～40的直链、支链或环链。Wherein: R1 , R2 , R3 are one or two of hydrogen, deuterium, alkyl, alkoxy, carbonyl, ester, halogen, substituted aryl or substituted heteroaryl. Further, the carbon chain of the alkyl group, alkoxy group, ester group or carbonyl group is a straight chain, branched chain or cyclic chain with 0-40 carbons.
进一步，所述取代芳基和取代杂芳基中的取代基团为烷基、烷氧基或羰基中的一种或几种。更进一步，所述烷基、烷氧基或羰基的碳链为碳个数为0～40的直链、支链或环链。Further, the substituents in the substituted aryl and substituted heteroaryl are one or more of alkyl, alkoxy or carbonyl. Furthermore, the carbon chain of the alkyl group, alkoxy group or carbonyl group is a straight chain, branched chain or cyclic chain with 0-40 carbons.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的一个具体实施例，具有下述式Ⅱ所示结构：As a specific example of a 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroboron derivative described in the present invention, it has the structure shown in the following formula II:
一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的制备方法，包括：A preparation method of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives, comprising:
1)将8-对氰基苯基氟硼荧与苯并咪唑类衍生物，氧化剂和溶剂混合均匀，随后在无水无氧条件下反应,反应式如下：1) Mix 8-p-cyanophenylfluoroborin with benzimidazole derivatives, oxidant and solvent evenly, and then react under anhydrous and oxygen-free conditions. The reaction formula is as follows:
2)反应完成后，冷却至室温，移除溶剂后加入二氯甲烷将反应体系溶解，再经硅藻土过滤，并用二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析分离纯化，真空干燥即可制得式Ⅰ化合物。2) After the reaction is completed, cool to room temperature, remove the solvent and add dichloromethane to dissolve the reaction system, then filter through diatomaceous earth, and wash with dichloromethane, combine the filtrates, remove the solvent under reduced pressure, and use a silica gel column for the residue The compound of formula I can be obtained by separation and purification by chromatography and vacuum drying.
进一步，上述步骤1)中,所述无水无氧条件可以采取惰性气体保护的方式，更进一步可以优选为氮气气氛下反应。Further, in the above step 1), the anhydrous and oxygen-free conditions can be protected by an inert gas, and further preferably, the reaction can be performed under a nitrogen atmosphere.
本发明制备方法所用合成路线为C–H/N–H直接氧化偶联反应，与传统的氟硼荧氨化制备技术相比较，缩短了较为冗长的有机合成步骤，避免了底物预活化的繁琐过程，提高了反应的兼容性，增加了合成反应总产率。The synthetic route used in the preparation method of the present invention is C-H/N-H direct oxidative coupling reaction, compared with the traditional preparation technology of fluoborofluoresceinization, it shortens the comparatively tedious organic synthesis steps and avoids the problem of substrate pre-activation The cumbersome process improves the compatibility of the reaction and increases the total yield of the synthesis reaction.
作为本分发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的制备方法的一个具体实施例，所述氧化剂为一水合醋酸铜、醋酸铜、氯化铜、溴化铜、三氟乙酸酮、三氟甲烷磺酸铜(Ⅱ)、乙酰丙酮铜、碳酸银、氧化银、醋酸银、硝酸银、六氟锑酸银、氧气、醋酸碘苯、苯醌、二氯二氰苯醌、过二硫酸钠、过二硫酸铵、过二硫酸钾、二叔丁基过氧化物中的一种或多种。As a specific embodiment of the preparation method of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives described in this invention, the oxidizing agent is copper acetate monohydrate, acetic acid Copper, copper chloride, copper bromide, ketone trifluoroacetate, copper (II) trifluoromethanesulfonate, copper acetylacetonate, silver carbonate, silver oxide, silver acetate, silver nitrate, silver hexafluoroantimonate, oxygen, acetic acid One or more of iodobenzene, benzoquinone, dichlorodicyanoquinone, sodium peroxodisulfate, ammonium peroxodisulfate, potassium peroxodisulfate, and di-tert-butyl peroxide.
作为本分发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的制备方法的一个具体实施例，所述溶剂为甲醇、乙醇、四氢呋喃、二氯甲烷、三氯甲烷、乙醚、二甲基亚砜、苯、邻二氯苯、氯苯、甲苯、二甲苯、均三甲苯、环己烷、石油醚、叔戊醇、1,4-二氧六环、1,2-二氯乙烷、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺中的一种或多种。As a specific embodiment of the preparation method of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluoro derivatives described in this invention, the solvent is methanol, ethanol, tetrahydrofuran, Dichloromethane, chloroform, ether, dimethyl sulfoxide, benzene, o-dichlorobenzene, chlorobenzene, toluene, xylene, mesitylene, cyclohexane, petroleum ether, tert-amyl alcohol, 1,4- One or more of dioxane, 1,2-dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide.
作为本分发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的制备方法的一个具体实施例，所述8-对氰基苯基氟硼荧的反应浓度为0.0001～10mol/L。进一步优选为0.1～8mol/L,0.5～5mol/L,1～3mol/L。As a specific example of the preparation method of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives described in this invention, the 8-p-cyanophenyl fluorine The reaction concentration of boron fluorescent is 0.0001～10mol/L. More preferably, it is 0.1-8 mol/L, 0.5-5 mol/L, 1-3 mol/L.
作为本分发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的制备方法的一个具体实施例，所述8-对氰基苯基氟硼荧，苯并咪唑类衍生物，氧化剂的摩尔比为1:(0.01～50):(0.01～100)。进一步优选为1:(0.1～40):(10～80),1:(5～30):(20～60)。As a specific example of the preparation method of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives described in this invention, the 8-p-cyanophenyl fluorine Boron fluorescein, benzimidazole derivatives, the molar ratio of the oxidizing agent is 1:(0.01-50):(0.01-100). More preferably, it is 1:(0.1-40):(10-80), 1:(5-30):(20-60).
作为本分发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的制备方法的一个具体实施例，所述步骤1)的反应温度为-40～160℃，进一步优选为20～100℃,40～80℃；时间为0.1～720h，进一步优选为10～30h。As a specific example of the preparation method of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives described in this invention, the reaction temperature of the step 1) is- 40-160°C, more preferably 20-100°C, 40-80°C; time is 0.1-720h, more preferably 10-30h.
本发明还提供所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用，其在细胞内质网专一性荧光显影和荧光标记中的应用。The present invention also provides the application of the 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluoro derivatives, which can be used in specific fluorescence imaging and fluorescent labeling of endoplasmic reticulum. application.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用的一个具体实施例，包括以下步骤：As a specific embodiment of the application of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives described in the present invention, the following steps are included:
步骤1：将细胞于培养基中培养；Step 1: culturing cells in culture medium;
步骤2：将培养后的细胞去除培养基，加入3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的缓冲液，培养；Step 2: removing the culture medium from the cultured cells, adding a buffer solution of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborophore derivatives, and culturing;
步骤3：步骤2培养结束后，将培养玻底皿经荧光共聚焦显微镜成像。Step 3: After the culture in step 2 is completed, image the cultured glass-bottom dish through a fluorescent confocal microscope.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用的一个具体实施例，所述细胞为HepG2细胞。As a specific example of the application of a 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborophore derivative in the present invention, the cells are HepG2 cells.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用的一个具体实施例，所述培养基为含有10％胎牛血清的DMEM(H)培养基。As a specific example of the application of a 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorene derivative of the present invention, the medium is a medium containing 10% fetal bovine serum DMEM (H) medium.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用的一个具体实施例，所述3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的缓冲液浓度为2.5μM的磷酸盐缓冲液。As a specific example of the application of a 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivative described in the present invention, the 3,5-dibenzimidazolyl- The concentration of the buffer solution for the 8-p-cyanophenylfluoroborate derivative is 2.5 μM phosphate buffer solution.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用的一个具体实施例，步骤1中的培养条件为37℃下培养24小时。As a specific example of the application of a 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborophore derivative in the present invention, the culture condition in step 1 is to cultivate at 37°C for 24 Hour.
作为本发明所述一种3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的应用的一个具体实施例，包括以下步骤：As a specific embodiment of the application of a kind of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives described in the present invention, the following steps are included:
向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时；将培养基去除，加入2.5μM化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的磷酸盐缓冲液，培养30分钟；待培养结束后，取出培养玻底皿，用磷酸盐缓冲液清洗2～3次后，将培养玻底皿经荧光共聚焦显微镜成像。Add 5% CO2 to the DMEM (H) medium containing 10% fetal bovine serum, culture HepG2 cells at 37°C for 24 hours; remove the medium, add 2.5 μM compound 3,5-dibenzimidazole Base-8-p-cyanophenylfluoroborate in phosphate buffer, incubate for 30 minutes; after the incubation, take out the culture glass-bottom dish, wash it with phosphate buffer for 2 to 3 times, and then remove the culture glass-bottom dish Imaged by fluorescent confocal microscopy.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
与现有的市售内质网荧光标记试剂相比，本发明3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物，合成路线更加简洁、高效、环境友好，产物价格低廉、容易大量获取。具体表现为:Compared with the existing commercially available endoplasmic reticulum fluorescent labeling reagents, the synthetic route of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluorine derivatives of the present invention is more concise, efficient and environment-friendly , the product is cheap and easy to obtain in large quantities. The specific performance is:
1、本发明所用合成路线为C–H/N–H直接氧化偶联反应，与传统的氟硼荧氨化制备技术相比较，缩短了较为冗长的有机合成步骤，避免了底物预活化的繁琐过程，提高了反应的兼容性，增加了合成反应总产率。1. The synthetic route used in the present invention is C-H/N-H direct oxidative coupling reaction. Compared with the traditional preparation technology of fluoroborate fluoresceinization, it shortens the comparatively tedious organic synthesis steps and avoids the problem of substrate preactivation. The cumbersome process improves the compatibility of the reaction and increases the total yield of the synthesis reaction.
2、与市售的内质网标记试剂ER-Tracker Red和ER-Tracker Green相比，本发明化合物合成简单，价格低廉。ER-Tracker Red和ER-Tracker Green的价格高达4863元/100μg(Thermo Fisher Scientific公司)，而我们的产物则相对的便宜很多，合成原料中，即使是氧化剂用最为昂贵的AgOAc，其成本价格也才289元/25g(安耐吉公司)，而其它合成原料都是市场上便宜易得的化合物，本申请氟硼荧类衍生物价格大概在3000～5000元/1g。同时在本发明的反应条件下，产率可以达到60％以上。所以采用本发明氟硼荧类衍生物作为内质网荧光标记的成本将远低于市售的标记试剂。2. Compared with the commercially available endoplasmic reticulum labeling reagents ER-Tracker Red and ER-Tracker Green, the compound of the present invention is simple to synthesize and inexpensive. The price of ER-Tracker Red and ER-Tracker Green is as high as 4863 yuan/100μg (Thermo Fisher Scientific Company), while our products are relatively much cheaper. Among the synthetic raw materials, even the most expensive AgOAc for oxidants has a cost price of Only 289 yuan/25g (Annaiji Company), and other synthetic raw materials are cheap and easy-to-obtain compounds on the market. The price of fluorine boron derivatives in this application is about 3000-5000 yuan/1g. At the same time, under the reaction conditions of the present invention, the yield can reach more than 60%. Therefore, the cost of using the fluoroborate derivatives of the present invention as endoplasmic reticulum fluorescent markers will be much lower than that of commercially available labeling reagents.
附图说明Description of drawings
图1为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物的结构通式。Fig. 1 is the general structural formula of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborophore derivatives.
图2为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧核磁图谱H谱。Fig. 2 is the NMR spectrum H spectrum of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate.
图3为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧核磁图谱C谱。Fig. 3 is the NMR spectrum C of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate.
图4为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的紫外可见光吸收光谱和荧光发射光谱。Fig. 4 is the ultraviolet-visible light absorption spectrum and the fluorescence emission spectrum of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate.
图5为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧标记细胞内质网的细胞成像图。Fig. 5 is a cell imaging diagram of endoplasmic reticulum labeled with 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate.
图6为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧在HepG2细胞中的CCK8细胞毒性实验结果。Fig. 6 is the result of CCK8 cytotoxicity experiment of 3,5-dibenzimidazolyl-8-p-cyanophenylbororfluorescein in HepG2 cells.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
本发明实施例中，HepG2细胞株采购于ATCC(American Type CultureCollection)公司，10％胎牛血清采购于Hyclone公司，DMEM(H)(Dulbecco’s modifiedessential medium)培养基采购于美国Gibco。内质网染料ER-TrackerTM Green采购于Thermo Fisher Scientific公司。In the embodiment of the present invention, HepG2 cell line was purchased from ATCC (American Type Culture Collection), 10% fetal bovine serum was purchased from Hyclone Company, and DMEM (H) (Dulbecco's modifiedessential medium) medium was purchased from Gibco, USA. The endoplasmic reticulum dye ER-TrackerTM Green was purchased from Thermo Fisher Scientific.
实施例1Example 1
3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的合成Synthesis of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborin
将8-对氰基苯基氟硼荧(14.6mg,0.05mmol)，苯并咪唑(23.6mg,0.20mmol)，AgOAc(33.4mg,4.0equiv)，二甲基亚砜(1.0mL)加入反应管，在氮气条件下搅拌均匀后加热到80℃，反应12小时；具体反应式如下：8-p-Cyanophenylfluoroborate (14.6mg, 0.05mmol), benzimidazole (23.6mg, 0.20mmol), AgOAc (33.4mg, 4.0equiv), dimethyl sulfoxide (1.0mL) were added to the reaction tube, stirred evenly under nitrogen, heated to 80°C, and reacted for 12 hours; the specific reaction formula is as follows:
反应完成后，将反应管冷却至室温，移除溶剂后加入10mL二氯甲烷将反应体系溶解，再经硅藻土过滤并用10～20mL的二氯甲烷洗涤，合并滤液，减压移去溶剂，剩余物用硅胶柱层析(二氯甲烷/石油醚/乙酸乙酯＝2:2:1,v/v/v)分离纯化，真空干燥后得到黑色固体目标产物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧15.7mg，产率60％。After the reaction is complete, cool the reaction tube to room temperature, remove the solvent and add 10 mL of dichloromethane to dissolve the reaction system, then filter through diatomaceous earth and wash with 10-20 mL of dichloromethane, combine the filtrates, remove the solvent under reduced pressure, The residue was separated and purified by silica gel column chromatography (dichloromethane/petroleum ether/ethyl acetate=2:2:1, v/v/v), and after vacuum drying, the target product 3,5-dibenzimidazole was obtained as a black solid 15.7 mg of 1-8-p-cyanophenylfluoroborate, the yield was 60%.
本实施例产物二苯并咪唑基-8-对氰基苯基氟硼荧核磁图谱H谱和C谱分别如图2及图3所示，结构数据表征如下：The H-spectrum and C-spectrum of the product dibenzimidazolyl-8-p-cyanophenylfluoroborate in this example are shown in Figure 2 and Figure 3, respectively, and the structural data are characterized as follows:
1H NMR(400MHz,CDCl3)δ＝6.81(d,J＝4.4Hz,2H),7.02(d,J＝4.8Hz,2H),7.32-7.39(m,4H),7.46-7.48(m,2H),7.78(d,J＝8.4Hz,2H),7.87(dd,J＝6.0Hz,2.4Hz,2H),7.93(d,J＝8.4Hz,2H),8.62(s,2H).13C NMR(100MHz,CDCl3)δ＝111.2,114.97,115.03,117.9,121.3,124.2,124.7,131.3,131.9,132.55,132.64,133.7,137.3,142.2,143.3,143.9,147.6.HRMS(ESI+):计算值C30H19BF2N7[M+H]+:526.1758，实测值526.1761。1 H NMR (400MHz, CDCl3) δ = 6.81 (d, J = 4.4Hz, 2H), 7.02 (d, J = 4.8Hz, 2H), 7.32-7.39 (m, 4H), 7.46-7.48 (m, 2H ), 7.78(d, J=8.4Hz, 2H), 7.87(dd, J=6.0Hz, 2.4Hz, 2H), 7.93(d, J=8.4Hz, 2H), 8.62(s, 2H).13 C NMR (100MHz, CDCl3) δ = 111.2, 114.97, 115.03, 117.9, 121.3, 124.2, 124.7, 131.3, 131.9, 132.55, 132.64, 133.7, 137.3, 142.2, 143.3, 143.9, 147.6. HRMS: calculated value C30 H19 BF2 N7 [M+H]+ : 526.1758, found 526.1761.
实施例2Example 2
实施例1制备得到的化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的紫外-可见-近红外吸收光谱图和荧光发射谱图The ultraviolet-visible-near-infrared absorption spectrum and fluorescence emission spectrum of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate prepared in Example 1
将化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧溶于二氯甲烷中，配成1×10-5mol/L，取2.5mL放入比色皿中，测定紫外-可见-近红外吸收以及荧光发射光谱。Dissolve the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborin in dichloromethane to make 1×10-5 mol/L, take 2.5mL into a cuvette, The UV-Vis-NIR absorption and fluorescence emission spectra were measured.
图4为化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的紫外可见光吸收光谱和荧光发射光谱，其中黑色实线表示紫外可见光吸收光谱，黑色虚线表示荧光发射光谱。从图4中可以看出，化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的吸收光谱最大吸收峰位于561nm；荧光发射光谱最大吸收峰位于592nm，斯托克斯位移为31nm。Figure 4 is the UV-visible absorption spectrum and fluorescence emission spectrum of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroboron, where the black solid line represents the UV-vis absorption spectrum, and the black dotted line represents the fluorescence emission spectrum . As can be seen from Figure 4, the maximum absorption peak of the absorption spectrum of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate is located at 561nm; the maximum absorption peak of the fluorescence emission spectrum is located at 592nm, Stoke The Stein shift is 31nm.
实施例3Example 3
实施例1制备得到的化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧与市售内质网染色剂ER-TrackerTM Green在HepG2细胞中的荧光共聚焦共成像Fluorescent confocal confocal imaging of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate prepared in Example 1 and the commercially available endoplasmic reticulum stain ER-TrackerTM Green in HepG2 cells
首先，向含有10％胎牛血清的DMEM(H)培养基中通入5％CO2，将HepG2细胞于37℃下培养24小时。将培养基去除，加入2.5μM化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的磷酸盐缓冲液，随后加入1μM市售内质网染色剂ER-TrackerTM Green于37℃下共同培养30分钟。待培养结束后，取出培养玻底皿，用磷酸盐缓冲液清洗2～3次后，将培养玻底皿经荧光共聚焦显微镜成像。First, 5% CO2 was introduced into the DMEM (H) medium containing 10% fetal bovine serum, and HepG2 cells were cultured at 37°C for 24 hours. The culture medium was removed, and 2.5 μM of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate in phosphate buffer was added, followed by 1 μM of the commercially available endoplasmic reticulum stain ER-Tracker™ Green Co-incubation was carried out at 37°C for 30 minutes. After the culture is over, take out the culture glass-bottom dish, wash it with phosphate buffer saline for 2 to 3 times, and image the culture glass-bottom dish through a fluorescent confocal microscope.
图5为化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的荧光成像图(激发波长：553nm，发射波长收集范围：550-650nm)。通过与市售内质网染色剂ER-TrackerTM Green的荧光成像图(激发波长：488nm，发射波长收集范围：450-550nm)相对比可以直观地看出，化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧在细胞中的分布和市售内质网染色剂ER-TrackerTM Green基本一致，corresponding Pearson’s系数达到0.89，说明化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧具有优异的内质网示踪效果，能专一性标记细胞内的内质网。Fig. 5 is a fluorescence imaging diagram of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroboron (excitation wavelength: 553nm, emission wavelength collection range: 550-650nm). Itcan be seen visually that the compound 3,5-dibenzimidazole The distribution of 8-p-cyanophenylfluoroborin in cells is basically the same as that of the commercially available endoplasmic reticulum stain ER-TrackerTM Green, and the corresponding Pearson's coefficient reaches 0.89, indicating that the compound 3,5-dibenzimidazolyl -8-P-cyanophenylboroborofluorescein has an excellent endoplasmic reticulum tracing effect, and can specifically label the endoplasmic reticulum in cells.
实施例4Example 4
实施例1制备得到的化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的CCK8细胞毒性实验The CCK8 cytotoxicity test of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate prepared in Example 1
将处于对数生长期的HepG2细胞接种于96孔培养板中，每孔接种3000个细胞，在37℃下用通入5％CO2的含有10％胎牛血清的DMEM(H)培养基中培养过夜。待细胞完全贴壁后，向其中加入不同浓度的化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧，每组浓度另设3个复孔和空白对照孔。加样后继续培养细胞24小时，使用CCK8检测法检测细胞存活率。HepG2 cells in the logarithmic growth phase were inoculated into 96-well culture plates, with 3000 cells per well, at 37°C in DMEM (H) medium containing 10% fetal calf serum with 5%CO2 Incubate overnight. After the cells were completely adhered to the wall, different concentrations of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate were added thereto, and three replicate wells and blank control wells were set up for each concentration group. The cells were cultured for 24 hours after adding the sample, and the cell viability was detected by the CCK8 assay.
图6为3,5-二苯并咪唑基-8-对氰基苯基氟硼荧在HepG2细胞中的CCK8细胞毒性实验结果。如图6所示，在0.25～4μM的浓度范围内，化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧的细胞存活率均非常高(存活率超过90％)，在8μM时存活率才会明显降低(存活率小于60％)，表明化合物3,5-二苯并咪唑基-8-对氰基苯基氟硼荧在我们的标记工作浓度内毒性是很小的，可以忽略不计。Fig. 6 is the result of CCK8 cytotoxicity experiment of 3,5-dibenzimidazolyl-8-p-cyanophenylbororfluorescein in HepG2 cells. As shown in Figure 6, within the concentration range of 0.25 to 4 μM, the cell survival rate of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborate is very high (the survival rate exceeds 90%) , the survival rate was significantly reduced (survival rate was less than 60%) at 8 μM, indicating that the toxicity of the compound 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborfluorene was very low in our labeling working concentration. Small, can be ignored.
实施例5Example 5
在实施例1的制备方法中，分别采用氘、烷基、烷氧基、羰基、酯基、卤素、取代芳基或取代杂芳基代替R1，R2和R3中的氢。其它条件不变，成功制备出了一系列能实现细胞内质网专一性荧光显影和荧光标记的3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物。其中以实施例1中制备的产品在细胞内质网荧光显影和荧光标记效果最好。In the preparation method of Example 1, hydrogen in R1 , R2 and R3 is replaced by deuterium, alkyl, alkoxy, carbonyl, ester group, halogen, substituted aryl or substituted heteroaryl, respectively. With other conditions unchanged, a series of 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborofluoro derivatives capable of realizing specific fluorescence imaging and fluorescent labeling of the endoplasmic reticulum of cells were successfully prepared. Among them, the product prepared in Example 1 has the best effect in fluorescence imaging and fluorescent labeling of endoplasmic reticulum.
实施例6Example 6
在实施例1或5的制备方法中，调整相关参数进行系列实验：In the preparation method of embodiment 1 or 5, adjust relevant parameter and carry out series experiment:
在步骤1)中分别选择-40℃，-20℃，0℃、20℃、40℃、60℃、100℃、120℃、140℃、160℃代替80℃；反应时间控制在0.1～720h；In step 1), respectively select -40°C, -20°C, 0°C, 20°C, 40°C, 60°C, 100°C, 120°C, 140°C, 160°C instead of 80°C; the reaction time is controlled at 0.1-720h;
分别控制8-对氰基苯基氟硼荧类衍生物，苯并咪唑类衍生物，氧化剂的摩尔比为1:(0.01～50):(0.01～100)；Respectively control 8-p-cyanophenyl borofluorofluores derivatives and benzimidazole derivatives, and the molar ratio of the oxidizing agent is 1:(0.01～50):(0.01～100);
用一水合醋酸铜、醋酸铜、氯化铜、溴化铜、三氟乙酸酮、三氟甲烷磺酸铜(Ⅱ)、乙酰丙酮铜、碳酸银、氧化银、硝酸银、六氟锑酸银、氧气、醋酸碘苯、苯醌、二氯二氰苯醌、过二硫酸钠、过二硫酸铵、过二硫酸钾、二叔丁基过氧化物代替醋酸银；Copper acetate monohydrate, copper acetate, copper chloride, copper bromide, ketone trifluoroacetate, copper (II) trifluoromethanesulfonate, copper acetylacetonate, silver carbonate, silver oxide, silver nitrate, silver hexafluoroantimonate , oxygen, iodobenzene acetate, benzoquinone, dichlorodicyanoquinone, sodium peroxodisulfate, ammonium peroxodisulfate, potassium peroxodisulfate, di-tert-butyl peroxide instead of silver acetate;
用甲醇、乙醇、四氢呋喃、二氯甲烷、三氯甲烷、乙醚、苯、邻二氯苯、氯苯、甲苯、二甲苯、均三甲苯、环己烷、石油醚、叔戊醇、1,4-二氧六环、1,2-二氯乙烷、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺代替二甲基亚砜。Methanol, ethanol, tetrahydrofuran, dichloromethane, chloroform, ether, benzene, o-dichlorobenzene, chlorobenzene, toluene, xylene, mesitylene, cyclohexane, petroleum ether, tert-amyl alcohol, 1,4 -Dioxane, 1,2-dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide instead of dimethylsulfoxide.
通过实验参数筛选发现上述各条件下均可成功制备出能实现细胞内质网专一性荧光显影和荧光标记的3,5-二苯并咪唑基-8-对氰基苯基氟硼荧类衍生物。其中以实施例1中制备的产品在细胞内质网荧光显影和荧光标记效果最好。Through the screening of experimental parameters, it was found that under the above conditions, 3,5-dibenzimidazolyl-8-p-cyanophenylfluoroborophores, which can realize specific fluorescence imaging and fluorescent labeling of the endoplasmic reticulum of cells, can be successfully prepared derivative. Among them, the product prepared in Example 1 has the best effect in fluorescence imaging and fluorescent labeling of endoplasmic reticulum.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.
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