WO2021169762A1 - 基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用 - Google Patents
基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用 Download PDFInfo
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- WO2021169762A1 WO2021169762A1 PCT/CN2021/075160 CN2021075160W WO2021169762A1 WO 2021169762 A1 WO2021169762 A1 WO 2021169762A1 CN 2021075160 W CN2021075160 W CN 2021075160W WO 2021169762 A1 WO2021169762 A1 WO 2021169762A1
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/10—Non-macromolecular compounds
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- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Definitions
- the invention belongs to the fluorescent labeling technology, and specifically relates to a novel neutral mitochondrial fluorescent label based on a nitrogen-containing heterocyclic ring.
- Mitochondria are one of the most basic organelles in cells. In addition to providing energy for cells as the main place for aerobic respiration, it also participates in the transfer of cytogenetic material and cell differentiation (see: Levenson, R.; Macara, IG; Smith, RL; Cantley, L.; Housman, D. Cell) 1982, 28, 855.) and other important physiological activities. Therefore, in scientific research, real-time monitoring of mitochondria is particularly important. Among various technical means, fluorescent labeling technology stands out due to its simple operation and low preparation cost. A variety of fluorescent probes and dyes with mitochondrial targeting functions have also been conceived.
- the present invention discloses a new type of neutral mitochondrial fluorescent markers based on nitrogen-containing heterocycles, which can be used as mitochondrial fluorescent markers.
- the present invention solves for the first time that the targeting ability of fluorescent dye organelles with neutral structures is random and uncertain It also avoids the problem that neutral fluorophores are commercial markers for lipid droplets in cells. While improving the good optical properties of fluorophores, the present invention modulates the organelle targeting ability of the original fluorophores through creative modification of their structure. Moreover, the markers improve the biological properties of the fluorophore, and nitrogen-containing heterocyclic building blocks are cheap and easy to obtain, which is beneficial to control the cost of new dyes.
- a fluorescent marker for neutral mitochondria based on a nitrogen-containing heterocyclic ring which is one of the following chemical formulas:
- X 1 and X 2 are independently selected from CH or heteroatoms; M, E, E 1 , and B 1 are independently selected from alkyl groups with less than 6 carbon atoms; the present invention is based on the neutral mitochondrial fluorescence of nitrogen-containing heterocycles
- the label contains an NH bond.
- the neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocyclic ring is one of the following chemical formulas:
- X 1 is selected from CH or N; X 2 is selected from CH or N.
- the present invention discloses the application of the above-mentioned neutral mitochondrial fluorescent marker based on nitrogen-containing heterocyclic ring in mitochondrial fluorescent labeling; or the application of the above-mentioned neutral mitochondrial fluorescent marker based on nitrogen-containing heterocyclic ring in preparing mitochondrial fluorescent labeling reagent.
- the present invention discloses a preparation method of the above-mentioned neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocyclic ring, which is characterized in that it is one of the following preparation methods:
- the invention discloses a cell imaging method, which comprises the following steps:
- the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle prepared in step (3) is co-cultured with the cells and then the mitochondrial green marker is added, and after the culture is continued, cell imaging is performed.
- the cells include normal cells and cancer cells.
- the deprotection is carried out in the presence of hydrochloric acid; the reaction of compound 6 and compound 7 is carried out in the presence of a precious metal salt catalyst, preferably under alkaline conditions; the reaction of compound 9 and compound 7 is carried out in the presence of a precious metal salt catalyst, The reaction is preferably under alkaline conditions; the reaction of compound 13 and compound 7 is carried out in the presence of a precious metal salt catalyst, preferably under alkaline conditions.
- the noble metal salt catalyst includes a palladium salt catalyst.
- these heterocycles contain NH bonds
- X 1 and X 2 are independently selected from CH or heteroatoms
- M, E, E 1 , and B 1 are substituents, independently selected from alkyl groups with less than 6 carbon atoms.
- the alkyl group in the present invention means a saturated branched or straight chain monovalent hydrocarbon group with 1 to 6 carbon atoms, such as methyl (Me), n-butyl (Bu), ethyl (Et) and the like.
- a laser confocal microscope is used for cell imaging; the blue channel uses 405nm excitation to collect fluorescence signals in the range of 410 ⁇ 500nm; the red channel uses 561nm excitation to collect fluorescence signals in the range of 570 ⁇ 750nm; the green channel uses Excite at 488nm and collect fluorescence signals in the range of 500-550nm.
- the present invention discloses for the first time a fluorescent marker of neutral mitochondria based on a nitrogen-containing heterocycle for fluorescent labeling of cell neutrophils, which can realize cell imaging after co-cultivation with cells. While improving the good optical performance of the fluorophore, the present invention modulates the organelle targeting ability of the original fluorophore through creative modification of its structure, and has low cytotoxicity during cell imaging, little damage to biological samples, and is not affected by other organelles. , Cell samples can be observed for a long time, and the markers can improve the biological performance of the fluorophore.
- the nitrogen-containing heterocyclic building blocks are cheap and easy to obtain, which is beneficial to control the cost of new dyes.
- Figure 1 is the synthetic route of the dye involved in the present invention.
- Figure 2 is a proton nuclear magnetic resonance spectrum of dye 1a
- Figure 3 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 1a in chloroform
- Figure 4 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 1b in chloroform
- Figure 5 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 1c in chloroform
- Figure 6 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2a in chloroform
- Figure 7 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2b in chloroform
- Figure 8 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2c in chloroform
- Figure 9 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3a in chloroform
- Figure 10 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3b in chloroform
- Figure 11 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3c in chloroform
- Figure 12 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of the dye 3d in chloroform
- Figure 13 shows the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 4 in chloroform
- Figure 14 is a cell imaging diagram of dye 1a in L929 cells and HeLa cells;
- Figure 15 is a cell imaging diagram of dye 1b in L929 cells and HeLa cells;
- Figure 16 is a cell imaging diagram of dye 1c in L929 cells and HeLa cells;
- Figure 17 is a cell imaging diagram of dye 2a in L929 cells and HeLa cells;
- Figure 18 is a cell imaging diagram of dye 2b in L929 cells and HeLa cells;
- Figure 19 is a cell imaging diagram of dye 2c in L929 cells and HeLa cells;
- Figure 20 is a cell imaging diagram of dye 3a in L929 cells and HeLa cells;
- Figure 21 is a cell imaging diagram of dye 3b in L929 cells and HeLa cells;
- Figure 22 is a cell imaging diagram of dye 3c in L929 cells and HeLa cells;
- Figure 23 is a cell imaging diagram of dye 3d in L929 cells
- Figure 24 is a cell imaging diagram of dye 3d in HeLa cells
- Figure 25 is a cell imaging image of dye 4 in HeLa cells.
- the dye 1a, dye 1b, dye 1c, dye 2a, dye 2b, dye 2c, dye 3a, dye 3b, and dye 3c prepared above are the neutral mitochondrial fluorescent markers based on nitrogen-containing heterocycles according to embodiments of the present invention.
- dye 1a In the ultraviolet-visible absorption spectrum, dye 1a has the maximum absorption at 378nm; in the fluorescence spectrum, dye 1a has the highest fluorescence intensity at 452nm, the excitation wavelength is 370nm, and the slit width is 3nm/1.5 nm.
- the maximum absorption wavelength of dye 1b In the ultraviolet-visible absorption spectrum, the maximum absorption wavelength of dye 1b is 382nm; in the fluorescence spectrum, the maximum emission wavelength of dye 1b is 485nm, the excitation wavelength at this time is 374nm, and the slit width is 3nm/1.5nm.
- the maximum absorption wavelength of dye 1c is 385nm; in the fluorescence spectrum, the maximum emission wavelength of dye 1c is 454nm, the excitation wavelength at this time is 380nm, and the slit width is 3nm/1.5nm.
- dye 2a has the maximum absorption at 364nm; in the fluorescence spectrum, dye 2a has the highest fluorescence intensity at 480nm, the excitation wavelength at this time is 374nm, and the slit width is 3nm/1.5 nm.
- the maximum absorption wavelength of dye 2b is 360nm; in the fluorescence spectrum, the maximum emission wavelength of dye 2b is 458nm, the excitation wavelength at this time is 370nm, and the slit width is 3nm/1.5nm.
- the maximum absorption wavelength of dye 2c is 356nm; in the fluorescence spectrum, the maximum emission wavelength of dye 1c is 441nm, the excitation wavelength at this time is 360nm, and the slit width is 3nm/3nm.
- dye 3a In the ultraviolet-visible absorption spectrum, dye 3a has the maximum absorption at 548nm; in the fluorescence spectrum, dye 3a has the highest fluorescence intensity at 606nm, the excitation wavelength at this time is 560nm, and the slit width is 1.5nm/ 1.5nm.
- the maximum absorption wavelength of dye 3b In the ultraviolet-visible absorption spectrum, the maximum absorption wavelength of dye 3b is 549nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3b is 608nm, the excitation wavelength at this time is 540nm, and the slit width is 1.5nm/1.5nm .
- the maximum absorption wavelength of dye 3c is 554nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3c is 611nm, the excitation wavelength at this time is 540nm, and the slit width is 1.5nm/1.5nm .
- the maximum absorption wavelength of dye 3c is 556nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3d is 619nm, the excitation wavelength at this time is 570nm, and the slit width is 1.5nm/1.5nm .
- the maximum absorption wavelength of dye 4 is 501nm; in the fluorescence spectrum, the maximum emission wavelength of dye 4 is 515nm, the excitation wavelength at this time is 495nm, and the slit width is 1.5nm/1.5nm .
- the above UV absorption and fluorescence emission test methods are conventional methods.
- DMSO dimethyl sulfoxide
- the blue channel uses 405nm excitation to collect the fluorescence signal in the range of 410-500nm, and the red channel uses 561nm excitation to collect the fluorescence signal in the range of 570-750nm.
- the results show that dye 1a has mitochondrial labeling ability in both normal cells and cancer cells, and can be used as a blue mitochondrial marker.
- DMSO dimethyl sulfoxide
- the green channel uses 488nm excitation to collect fluorescence signals in the range of 500-550nm.
- dye 3a has mitochondrial labeling ability in both normal cells and cancer cells, and can be used as a red marker for mitochondria.
- the results are shown in Figure 20, where (a), (g) are bright field, (b), (h) are cell imaging images of dye 3a, (c), (i) are cell imaging images of mitochondrial green markers , (D), (j) are the superimposed images of the red light channel and the green light channel, (e), (k) are the fluorescence intensity of the ROI line in the superimposed image, (f), (l) are the co-localization experiments, they The colocalization coefficients are 0.91 (L929) and 0.90 (HeLa), respectively.
- (a) and (g) are bright field
- (b) and (h) are cell imaging images of dye 3b
- (c) are cell imaging images of mitochondrial green markers
- (d) is cell imaging images of mitochondrial green markers
- (d) is the superimposed image of the red light channel and the green light channel
- (e) is the superimposed image of the red light channel and the green light channel
- (e) is the fluorescence intensity of the ROI line in the superimposed image
- (f) are the colocalization experiments, and their colocalization coefficients They are 0.88 (L929) and 0.90 (HeLa) respectively.
- (a), (g) are bright field, (b), (h) are cell imaging images of dye 3c, (c), (i) are cell imaging images of mitochondrial green markers, (d) , (j) is the superimposed image of the red light channel and the green light channel, (e), (k) are the fluorescence intensity of the ROI line in the superimposed image, (f), (l) are the colocalization experiments, and their colocalization coefficients They are 0.89 (L929) and 0.87 (HeLa) respectively.
- DMSO dimethyl sulfoxide
- the red channel uses 561nm excitation to collect fluorescence signals in the range of 570-750nm.
- the green channel uses 488nm excitation to collect fluorescence signals in the range of 500-550nm.
- the results showed that the dye 3d marked both mitochondria and lipid droplets at the same time, which is not suitable for cell imaging as a mitochondrial marker.
- (a), (f) are bright field
- (b) are cell imaging images of dye 3d
- (c) are cell imaging images of lipid droplet green marker
- (h) The cell imaging images of the green markers of mitochondria
- (d) are the superimposed images of the red light channel and the green light channel
- (e) are the fluorescence intensity of the ROI line in the superimposed image.
- (a), (f) are bright field
- (b) are cell imaging images of dye 3d
- (c) are cell imaging images of lipid droplet green marker
- (h) The cell imaging images of the green markers of mitochondria
- (d) are the superimposed images of the red light channel and the green light channel
- (e) are the fluorescence intensity of the ROI line in the superimposed image.
- DMSO DMSO to prepare dye 4 into mother liquor, then add it to conventional cell culture medium to make the concentration of dye 4 in cell culture medium 1 ⁇ M, and then co-culture with HeLa cells in a saturated humidity, 37°C, 5% CO 2 incubator 10 Minutes, then add Mito, a red marker for mitochondria Red CMXRos (100nm) was incubated for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope.
- the red channel uses 561nm excitation to collect fluorescence signals in the range of 570-750nm.
- the green channel uses 488nm excitation to collect fluorescence signals in the range of 500-550nm.
- the conventional CCK-8 method was used to test the cytotoxicity of the dye prepared in the example, the test time was 6 hours, and the Meilun CCK-8 cell proliferation toxicity detection kit was used. The results showed that when the dye concentration is 2 ⁇ M to 10 ⁇ M (DMSO is the solvent), The survival rates of L929 cells and HeLa cells are both greater than 95%.
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Abstract
本发明公开了基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用,本发明首次公开的中性荧光团为含有N-H键的杂环,靶向线粒体解决了现有中性结构的荧光染料细胞器靶向能力是随机的且不确定的问题,也避免中性荧光团是细胞中脂滴的商业标记物的问题,本发明改善荧光团良好的光学性能的同时,通过对其结构创造性的修饰来调控原荧光团的细胞器靶向能力,而且标记物改善荧光团的生物学性能,含氮杂环砌块廉价易得,有利于控制新染料的成本,具有重大的科学意义和商业价值。
Description
本发明属于荧光标记技术,具体涉及基于含氮杂环的新型中性线粒体荧光标记物。
线粒体是细胞中最基础的细胞器之一。它除了作为有氧呼吸的主要场所为细胞提供能量外,也参与细胞遗传物质传递和细胞分化(参见:Levenson,R.;Macara,I.G.;Smith,R.L.;Cantley,L.;Housman,D.Cell 1982,28,855.)等重要的生理活动。因此在科学研究中,对线粒体进行实时监控显得尤为重要。在各种技术手段中,荧光标记技术因其操作简便、制备成本低等优势脱颖而出。各种具有线粒体靶向功能的荧光探针和染料也随之孕育而生。纵观已经被报道的线粒体靶向荧光探针和染料,不难发现他们的主体结构中绝大部分都包含三苯基膦盐、吡啶盐和吲哚盐(参见:Angew Chem Int Ed2016,55,13658.)。即便是最常用的商业用线粒体红色和绿色标记物亦是如此。这是因为线粒体内膜上存在质子泵使得的这些阳离子染料更容易穿透线粒体膜而在线粒体中聚集。但问题也随之而来,这些阳离子进入线粒体后会改变线粒体的膜电位,造成细胞凋亡(参见:Sens Actuators B2019,292,16.)。
发明内容
本发明公开了基于含氮杂环的一类新型中性线粒体荧光标记物,可以作为线粒体荧光标记物,本发明首次解决了现有中性结构的荧光染料细胞器靶向能力是随机的且不确定的问题,也避免中性荧光团是细胞中脂滴的商业标记物的问题,本发明改善荧光团良好的光学性能的同时,通过对其结构创造性的修饰来调控原荧光团的细胞器靶向能力,而且标记物改善荧光团的生物学性能,含氮杂环砌块廉价易得,有利于控制新染料的成本。
本发明采用如下技术方案:
基于含氮杂环的中性线粒体荧光标记物,为以下化学式中的一种:
其中,X
1、X
2独立的选自CH或者杂原子;M、E、E
1、B
1独立的选自碳原子数小于6的烷基;本发明基于含氮杂环的中性线粒体荧光标记物含有N-H键。
优选的,所述基于含氮杂环的中性线粒体荧光标记物为以下化学式中的一种:
X
1选自CH或N;X
2选自CH或N。
本发明公开了上述基于含氮杂环的中性线粒体荧光标记物在线粒体荧光标记中的应用;或者上述基于含氮杂环的中性线粒体荧光标记物在制备线粒体荧光标记试剂中的应用。
本发明公开了上述基于含氮杂环的中性线粒体荧光标记物的制备方法,其特征在于,为以下制备方法中的一种:
(1)化合物6与化合物7反应得到化合物8;化合物8脱保护,得到基于含氮杂环的中性线粒体荧光标记物;
(2)化合物9与化合物7反应得到化合物10;化合物10脱保护,得到基于含氮杂环的中性线粒体荧光标记物;
(3)化合物13与化合物7反应得到化合物14;化合物14脱保护,得到基于含氮杂环的中性线粒体荧光标记物。
本发明公开了一种细胞成像方法,包括以下步骤:
(1)化合物6与化合物7反应得到化合物8;化合物8脱保护,得到基于含氮杂环的中性线粒体荧光标记物;
(2)化合物9与化合物7反应得到化合物10;化合物10脱保护,得到基于含氮杂环的中性线粒体荧光标记物;
(3)化合物13与化合物7反应得到化合物14;化合物14脱保护,得到基于含氮杂环的中性线粒体荧光标记物;
(4)将步骤(1)或者步骤(2)制备的基于含氮杂环的中性线粒体荧光标记物与细胞共培养后加入线粒体红色标记物,继续培养后,进行细胞成像;
或者将步骤(3)制备的基于含氮杂环的中性线粒体荧光标记物与细胞共培养后加入线粒体绿色标记物,继续培养后,进行细胞成像。所述细胞包括正常细胞、癌细胞。
本发明中,脱保护在盐酸存在下进行;化合物6与化合物7的反应在贵金属盐催化剂存在下进行,优选在碱性条件下反应;化合物9与化合物7的反应在贵金属盐催化剂存在下进行,优选在碱性条件下反应;化合物13与化合物7的反应在贵金属盐催化剂存在下进行,优选在碱性条件下反应。优选的,贵金属盐催化剂包括钯盐催化剂。
本发明中,化合物的化学结构式如下:
化合物14的化学结构式如下:
其中,这些杂环含有N-H键,X
1、X
2独立的选自CH或者杂原子;M、E、E
1、B
1为取代基,独立的选自碳原子数小于6的烷基。本发明所述的烷基表示碳原子为1~6的饱和支链或者直链单价烃基,比如甲基(Me)、正丁基(Bu)、乙基(Et)等。
本发明中,利用激光共聚焦显微镜进行细胞成像;蓝光通道使用405nm激发,收集410~500nm范围内的荧光信号;红光通道使用561nm激发,收集570~750nm范围内的荧光信号;绿光通道使用488nm激发,收集500~550nm范围内的荧光信号。
本发明首次公开了用于细胞中性粒荧光标记的基于含氮杂环的中性线粒体荧光标记物,与细胞共培养后,可以实现细胞成像。本发明改善荧光团良好的光学性能的同时,通过对其结构创造性的修饰来调控原荧光团的细胞器靶向能力,进行细胞成像时细胞毒性低、对生物样品损坏小、不受其他细胞器的影响、可以对细胞样品进行长时间观测,而且标记物改善荧光团的生物学性能,含氮杂环砌块廉价易得,有利于控制新染料的成本。
图1为本发明涉及的染料的合成路线;
图2为染料1a的核磁共振氢谱图;
图3为染料1a在氯仿中的紫外-可见吸收光谱和荧光光谱;
图4为染料1b在氯仿中的紫外-可见吸收光谱和荧光光谱;
图5为染料1c在氯仿中的紫外-可见吸收光谱和荧光光谱;
图6为染料2a在氯仿中的紫外-可见吸收光谱和荧光光谱;
图7为染料2b在氯仿中的紫外-可见吸收光谱和荧光光谱;
图8为染料2c在氯仿中的紫外-可见吸收光谱和荧光光谱;
图9为染料3a在氯仿中的紫外-可见吸收光谱和荧光光谱;
图10为染料3b在氯仿中的紫外-可见吸收光谱和荧光光谱;
图11为染料3c在氯仿中的紫外-可见吸收光谱和荧光光谱;
图12为染料3d在氯仿中的紫外-可见吸收光谱和荧光光谱;
图13为染料4在氯仿中的紫外-可见吸收光谱和荧光光谱;
图14为染料1a在L929细胞和HeLa细胞中的细胞成像图;
图15为染料1b在L929细胞和HeLa细胞中的细胞成像图;
图16为染料1c在L929细胞和HeLa细胞中的细胞成像图;
图17为染料2a在L929细胞和HeLa细胞中的细胞成像图;
图18为染料2b在L929细胞和HeLa细胞中的细胞成像图;
图19为染料2c在L929细胞和HeLa细胞中的细胞成像图;
图20为染料3a在L929细胞和HeLa细胞中的细胞成像图;
图21为染料3b在L929细胞和HeLa细胞中的细胞成像图;
图22为染料3c在L929细胞和HeLa细胞中的细胞成像图;
图23为染料3d在L929细胞中的细胞成像图;
图24为染料3d在HeLa细胞中的细胞成像图;
图25为染料4在HeLa细胞中的细胞成像图。
本发明实施例的合成路线参见附图1,化学式下方的数字表示化合物。本发明化合物合成中,原料比例以及纯化方法采用常规比例或者常规纯化方法,实施例为示意性表述。
实施例
取化合物5(2.0毫摩尔,618.1毫克)、联硼酸频那醇酯(2.5毫摩尔,634.8毫克)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.2毫摩尔,146.3毫克)和磷酸钾(4.0毫克,849.1毫克)溶解于25.0毫升1,4-二氧六环中;氮气置换三次,于100℃反应12小时;降至室温后,将反应后的混合物抽滤,滤液通过旋转蒸发仪除去溶剂,经柱层析(洗脱剂:石油醚/乙酸乙酯(5/1,v/v))分离后得到淡黄色中间体6,244.9毫克,产率35%;核磁测试:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)7.52(d,1H,J=9.0,Ar-H),6.67(d,1H,J=8.9,Ar-H),6.48(s,1H,Ar-H),3.43(q,J=6.9Hz,4H,2×CH
2),2.37(s,3H,CH
3),1.30(s,12H,4×CH
3),1.12(t,J=6.1Hz,6H,2×CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)163.4,159.1,156.4,150.8,125.9,109.5,108.1,97.4,84.0,44.7,24.8,18.0,12.5.
取中间体6(1.0mmol,357.2mg)、化合物7a(叔丁基5-溴-1H-吲唑-1-羧酸酯,1.2mmol,355.2mg)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1mmol,73.1mg)和磷酸钾(2.0mmol,424.5mg)溶于15.0mL1,4-二氧六环中,反应体系由氮气置换三次,随后在回流条件下反应12小时;降至室温后,将反应后的混合物进行抽滤,滤液通过旋转蒸发仪除去溶剂;纯净中间体8a经柱层析分离后得到,洗脱剂:二氯甲烷/甲醇(100/1,v/v),淡黄色固体,192.3mg,产率43%。中间体8a的核磁测试:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.23(d,J=8.6Hz,1H,Ar-H),8.19(s,1H,Ar-H),7.68(s,1H,Ar-H),7.48 (d,J=8.8Hz,2H,Ar-H),6.64(d,J=9.0Hz,2H,Ar-H),6.57(s,1H,Ar-H),3.44(q,J=7.0Hz,4H,2×CH
2),1.74(s,9H,3×CH
3)2.24(s,3H,CH
3),1.22(t,J=6.0Hz,6H,2×CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)162.2,155.1,150.4,149.1,148.8,139.6,139.0,131.7,131.0,126.1,126.0,123.0,120.2,114.4,109.4,108.7,97.5,84.9,44.8,28.2,24.8,16.4,12.4.
将中间体8a(0.3mmol,134.2mg)溶解于1.0mL浓盐酸和3.0mL1,4-二氧六环的混合溶液中,在室温下搅拌,利用薄层色谱监测反应,当原料反应完全后,加入饱和碳酸氢钠溶液,然后用三氯甲烷(3×30.0mL)萃取,收集有机层,加入无水Na
2SO
4干燥后,蒸干溶剂;粗产物经柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(30/1,v/v),得到98.9mg纯产物,淡黄色固体,产率95%,称为染料1a。图2为染料1a的核磁共振氢谱图(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)13.13(s,1H,N-H),8.09(s,1H,Ar-H),7.65(s,1H,Ar-H),7.59(d,J=5.3Hz,1H,Ar-H),7.57(d,J=4.9Hz,1H,Ar-H),7.24(d,J=8.3Hz,1H,Ar-H),6.74(d,J=8.5,1H,Ar-H),6.57(s,1H,Ar-H),3.46(q,J=7.3Hz,4H,2×CH
2),2.20(s,3H,CH
3),1.14(t,J=6.1Hz,6H,2×CH
3);染料1a的核磁共振碳谱图(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)162.6,155.1,150.2,148.8,139.5,135.0,129.4,128.0,126.1,123.3,122.6,121.1,109.7,109.5,108.6,97.5,44.7,16.4,12.5.
取中间体6(1.0毫摩尔,357.2毫克)、化合物7b(叔丁基5-溴-1H-吡咯并[2,3-b]吡啶-1-羧酸酯,1.2毫摩尔,355.2毫克)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和磷酸钾(2.0毫摩尔,424.5毫克)溶于15.0毫升1,4-二氧六环中,氮气置换三次后在回流条件下反应8小时,降至室温后,将反应后的混合物进行抽滤,滤液通过旋转蒸发仪除去溶剂,中间体8b经柱层析分离后得到纯净产物,洗脱剂:二氯甲烷/甲醇(100/1,v/v),淡黄色固体,176.8毫克,产率40%。中间体8b的核磁测试:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.38(s,1H,Ar-H),7.93(s,1H,Ar-H),7.66(d,J=3.3Hz,1H,Ar-H),7.47(d,J=8.9Hz,1H,Ar-H),6.64(d,J=8.9Hz,1H,Ar-H),6.56(s,1H,Ar-H),6.54(d,J=3.3Hz,1H,Ar-H),3.44(q,J=7.0Hz,4H,2×CH
2),2.27(s,3H,CH
3),1.69(s,9H,3×CH
3),1.23(t,J=6.7Hz,6H,2×CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)162.1,155.2,150.4,149.3,147.9,147.5,146.7,131.4,126.9,126.2,126.1,122.7,117.9,109.4,108.7,104.7,97.5,84.1,44.8,28.1,16.5,12.4.
将中间体8b(0.3毫摩尔,134.2毫克)溶解于1.0毫升浓盐酸和3.0毫升1,4-二氧六环的混合溶液中,在室温下搅拌1.5小时,利用薄层色谱监测反应完全后,加入饱和碳酸氢钠溶液中和反应体系,然后用三氯甲烷(3×30.0毫升)萃取,收集有机层,加入无水Na
2SO
4干燥后,蒸干溶剂,粗产物经随后进行柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(30/1,v/v),得到97.9毫克纯产物,淡黄色固体,产率94%,称为染料1b;核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)11.73(s,1H,N-H),8.09(s,1H,Ar-H),7.86(s,1H,Ar-H),7.60(d,J=9.0Hz,1H,Ar-H),7.51(s,1H,Ar-H),6.75(d,J=8.6Hz,1H,Ar-H),6.58(s,1H,Ar-H),6.48(s,1H,Ar-H),3.46(q,J=6.9 Hz,4H,2×CH
2),2.22(s,3H,CH
3),1.15(t,J=6.7Hz,6H,2×CH
3);(151MHz,DMSO-d
6)
13C NMR(151MHz,DMSO-d
6)δ(ppm)161.7,155.1,150.5,149.4,148.0,144.6,130.4,127.2,126.9,123.2,119.5,118.5,109.2,109.1,100.3,97.0,44.4,16.7,12.8.
取中间体6(1.0毫摩尔,357.2毫克),化合物7c(叔丁基5-溴-1H-吡唑并[3,4-b]吡啶-1-羧酸酯,1.2毫摩尔,356.4毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和磷酸钾(2.0毫摩尔,424.5毫克)溶于15.0毫升1,4-二氧六环中。氮气置换三次后在回流条件下反应8小时。降至室温后,将反应后的混合物进行抽滤,滤液通过旋转蒸发仪除去溶剂。中间体8c经柱层析分离后得到纯净产物,洗脱剂:二氯甲烷/甲醇(100/1,v/v),淡黄色固体,147.9毫克,产率33%。中间体8c的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.65(s,1H,Ar-H),8.20(s,1H,Ar-H),8.15(s,1H,Ar-H),7.49(d,J=9.0Hz,1H,Ar-H),6.67(d,J=8.5Hz,1H,Ar-H),6.57(s,1H,Ar-H),3.45(q,J=7.0Hz,4H,2×CH
2),2.29(s,3H,CH
3),1.75(s,9H,3×CH
3),1.24(t,J=7.0Hz,6H,2×CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)162.2,156.1,152.9,151.6,150.5,150.3,147.5,136.3,132.2,125.5,119.3,115.4,109.1,108.8,108.4,97.7,85.9,44.7,28.1,18.4,12.5.
将中间体8c(0.3毫摩尔,134.5毫克)溶解于1.0毫升浓盐酸和3.0毫升1,4-二氧六环的混合液中,室温搅拌1.5小时。利用薄层色谱监测反应完全后加入饱和碳酸氢钠溶液中和反应体系。然后用三氯甲烷(3×30.0毫升)萃取,收集有机层。加入无水Na
2SO
4干燥后,蒸干溶剂后进行柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(30/1,v/v)。得到100.3毫克淡黄色固体,为染料1c。核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)13.74(s,1H,N-H),8.42(s,1H,Ar-H),8.18(s,1H,Ar-H),8.16(s,1H,Ar-H),7.62(d,J=8.6Hz,1H,Ar-H),6.76(d,J=8.7Hz,1H,Ar-H),6.59(s,1H,Ar-H),3.46(q,J=6.1Hz,4H,2×CH
2),2.23(s,3H,CH
3),1.15(t,J=6.9Hz,6H,2×CH
3);(151MHz,DMSO-d
6)
13CNMR(151MHz,DMSO-d
6)δ(ppm)161.6,155.2,151.2,151.1,150.78,150.1,133.8,132.1,127.3,124.5,117.4,114.4,109.3,109.0,97.0,44.4,16.7,12.8。
取化合物9(1.0毫摩尔,379.2毫克),化合物7a(1.2毫摩尔,355.2毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于15.0毫升1,4-二氧六环中。氮气置换三次后回流6小时,反应完成后,冷却至室温抽滤,滤液在旋转蒸发仪上蒸干溶剂。通过柱层析提出产物,洗脱剂为二氯甲烷。得到白色固体300.3毫克中间体10a,产率64%。核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.66(t,J=8.0Hz,2H,Ar-H),,8.37(d,J=8.5Hz,1H,Ar-H),8.29(s,1H,Ar-H),8.21(d,J=8.5Hz,1H,Ar-H),7.88(s,1H,Ar-H),7.75(d,J=7.6Hz,1H,Ar-H),7.72(d,J=8.8Hz,1H,Ar-H),7.68(d,J=7.8Hz,1H,Ar-H),4.22(t,J=7.4Hz,2H,CH
2),1.78(s,9H,3×CH
3),1.76-1.71(m,2H,CH
2),1.50-1.44(m,2H,CH
2),1.00(t,J=7.2Hz,3H,CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)164.2,164.0,149.1,146.0,139.5, 139.5,134.4,132.2,131.2,130.8,130.7,130.2,128.7,128.2,127.0,126.1,123.0,122.2,122.1,114.8,85.4,40.3,30.2,28.2,20.4,13.8.
取中间体10a(0.5毫摩尔,234.6毫克)溶于2.0毫升浓盐酸和6.0毫升1,4-二氧六环的混合溶液中。室温搅拌过夜,将析出的白色固体抽滤,随后用饱和碳酸氢钠溶液洗涤,即得到162.4毫克纯产物2a,白色固体,产率88%,称为染料2a。核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)8.56-8.54(m,2H,Ar-H),8.28(d,J=8.4Hz,1H,Ar-H),,8.22(s,1H,Ar-H),,7.94(s,1H,Ar-H),7.82(t,J=7.3Hz,2H,Ar-H),7.76(d,J=8.4Hz,1H,Ar-H),7.51(d,J=8.5Hz,1H,Ar-H),4.07(t,J=7.3Hz,2H,CH
2),1.67-1.63(m,2H,CH
2),1.41-1.35(m,2H,CH
2),0.94(t,J=7.2Hz,3H,CH
3);(151MHz,DMSO-d
6)
13C NMR(151MHz,DMSO-d
6)δ(ppm)163.9,163.7,147.2,140.1,134.5,132.9,131.1,130.8,130.8,130.1,128.7,128.4,127.8,123.5,122.8,122.4,121.3,111.0,40.5,30.1,20.2,14.2.
取化合物9(1.0毫摩尔,379.2毫克),化合物7b(1.2毫摩尔,355.2毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于15.0毫升1,4-二氧六环中。反应在氮气氛围下回流6小时。反应结束后冷却至室温,抽滤得到滤液。滤液蒸干后,通过柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(100/1,v/v)。得到白色固体,243.9毫克,产率52%。中间体10b的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.68(d,J=7.6Hz,1H,Ar-H),8.66-8.64(m,2H,Ar-H),8.22(d,J=8.4Hz,1H,Ar-H),8.04(s,1H,Ar-H),7.78(d,J=3.8Hz,1H,Ar-H),7.75(d,J=7.6Hz,1H,Ar-H),7.71(d,J=8.1Hz,1H,Ar-H),4.23(t,J=7.5Hz,2H,CH
2),1.79-1.75(m,2H,CH
2),1.72(s,9H,3×CH
3),1.50-1.45(m,2H,CH
2),1.00(t,J=7.2Hz,3H,CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)164.2,164.0,148.1,147.8,145.7,143.7,132.1,131.3,130.7,130.4,130.1,129.55,128.6,128.5,127.9,127.1,123.0,122.8,122.3,104.5,84.6,40.3,30.2,28.1,20.4,13.8.
取中间体10b(0.5mmol,234.6mg)溶于2.0ml浓盐酸和6.0ml1,4-二氧六环的混合液中。室温搅拌过夜,有固体析出。抽滤,滤饼用饱和碳酸氢钠溶液洗涤,得164.3mg白色固体,产率89%,称为染料2b。核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)11.95(s,1H,N-H),8.57(d,J=4.1Hz,1H,Ar-H),8.55(d,J=3.5Hz,1H,Ar-H),8.37(s,1H,Ar-H),8.31(d,J=8.4Hz,1H,Ar-H),8.16(s,1H,Ar-H),7.88-7.83(m,2H,Ar-H),7.63(s,1H,Ar-H),6.59(s,1H,Ar-H),4.08(t,J=7.3Hz,2H,CH
2),1.69-1.61(m,2H,CH
2),1.42-1.35(m,2H,CH
2),0.95(t,J=7.3Hz,3H,CH
3);(151MHz,DMSO-d
6)
13C NMR(151MHz,DMSO-d
6)δ(ppm)163.8,163.6,148.7,145.0,143.6,132.8,131.2,130.7,130.3,129.7,129.1,128.4,127.9,127.9,126.3,122.8,121.4,119.8,100.8,40.5,30.1,20.214.2.
取化合物9(1.0毫摩尔,379.2毫克),化合物7c(1.2毫摩尔,356.4毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于15.0毫升1,4-二氧六环中。在氮气保护下,回流4小时。待反应结束冷却至室温后,通过抽滤得到滤液。将滤液旋转蒸发后,通过柱层析分离提纯,展开剂:二氯甲烷/甲醇(100/1,v/v). 得到白色固体,150.5毫克,产率32%。中间体10c的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.91(s,1H,Ar-H),8.70(d,J=7.6Hz,1H,Ar-H),8.68(d,J=7.4Hz,1H,Ar-H),8.31(s,1H,Ar-H),8.26(s,1H,Ar-H),8.13(d,J=8.4Hz,1H,Ar-H),7.77-7.73(m,2H,Ar-H),4.23(t,J=7.5Hz,2H,CH
2),1.78(s,9H,3×CH
3),1.74-1.71(m,2H,CH
2),1.52-1.45(m,2H,CH
2),1.00(t,J=7.3Hz,3H,CH
3);(151MHz,CDCl
3,)
13C NMR(151MHz,CDCl
3)δ(ppm)164.0,163.8,151.6,151.5,147.7,142.1,137.4,131.4,131.4,131.0,130.6,130.6,130.2,128.7,127.5,123.2,122.9,117.6,85.9,40.4,30.2,28.1,20.4,13.8.
取中间体10c(0.3毫摩尔,141.1毫克)溶于1.0毫升浓盐酸和3.0毫升1,4-二氧六环的混合溶液中。室温搅拌过夜,有固体析出。抽滤,滤饼用饱和碳酸氢钠溶液洗涤,得到119.9毫克白色固体,产率85%,称为染料2c。核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)13.94(s,1H,N-H),8.69(s,1H,Ar-H),8.59-8.56(m,2H,Ar-H)8.48(s,1H,Ar-H),8.29-8.27(m,2H,Ar-H),7.92(d,J=7.3Hz,1H,Ar-H),7.87(t,J=7.6Hz,1H,Ar-H),4.09(t,J=6.2Hz,2H,CH
2),1.67-1.64(m,2H,CH
2),1.41-1.35(m,2H,CH
2),0.95(t,J=6.8Hz,3H,CH
3);(151MHz,DMSO-d
6)
13C NMR(151MHz,DMSO-d
6)δ(ppm)163.8,163.6,151.8,149.9,143.8,134.3,132.5,131.6,131.3,130.7,130.2,129.3,128.4,128.1,127.5,122.9,121.9,114.6,40.5,30.1,20.2,14.2。
取化合物11(2.0毫摩尔,668.3毫克)溶解于30.0毫升无水四氢呋喃溶液中,随后加入N-苯基双(三氟甲烷磺酰)亚胺(4.0毫摩尔,1.4克)和三乙胺(4.0毫摩尔,0.6毫升)。在氮气下室温搅拌24h。反应结束后,真空蒸发溶剂,柱层析分离,洗脱剂:二氯甲烷/甲醇(50/1,v/v)。得到559.3毫克绿色固体,产率60%。中间体12的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.70(d,J=8.8Hz,1H,Ar-H),8.15(s,1H,Ar-H),7.58(d,J=5.6Hz,1H,Ar-H),7.55(d,J=5.8Hz,1H,Ar-H),6.66(d,J=9.1Hz,1H,Ar-H),6.43(s,1H,Ar-H),6.38(s,1H,Ar-H),3.48(q,J=7.1Hz,4H,2×CH
2),1.26(t,J=7.1Hz,6H,2×CH
3);(151MHz,DMSO-d
6)
13C NMR(151MHz,DMSO-d
6)δ(ppm)181.3,152.3,151.4,150.5,147.0,137.6,133.4,131.7,131.5,126.5,125.2,124.0,118.1,110.3,105.5,96.1,45.2,12.6.
取中间体12(2.0毫摩尔,932.2毫克),联硼酸频那醇酯(2.5毫摩尔,634.8毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.2毫摩尔,146.3毫克)和乙酸钾(4.0毫摩尔,392.6毫克)溶解于40.0毫升1,4-二氧六环中。混合物在氮气氛围下,回流8小时。反应结束后,降至室温,抽滤,所得滤液旋转蒸发除去溶剂。通过柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(100/1,v/v)。得到绿色固体,790.7毫克,产率89%。中间体13的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.78(s,1H,Ar-H),8.61(d,J=7.6Hz,1H,Ar-H),8.10(d,J=7.6Hz,1H,Ar-H),7.58(d,J=8.8Hz,1H,Ar-H),6.63(d,J=8.5Hz,1H,Ar-H),6.43(s,1H,Ar-H),6.37(s,1H,Ar-H),3.45(q,J=6.7Hz,4H,2×CH
2),1.38(s,12H,4×CH3),1.25(t,J=7.1Hz,6H,2× CH
3);(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)183.8,152.2,150.8,146.7,139.9,136.9,134.1,132.7,131.2,130.8,125.0,122.8,109.7,105.8,96.2,84.1,83.1,45.1,24.9,24.5,12.6.
取中间体13(1.0毫摩尔,444.2毫克),化合物7a(1.2毫摩尔,355.2毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于35.0毫升1,4-二氧六环中。氮气置换3次后,加热回流12小时。反应结束后冷却至室温,抽滤,滤液蒸干溶剂后,通过柱层析分离,洗脱剂:二氯甲烷/甲醇(100/1,v/v)。得到192.3毫克红棕色固体,产率43%。中间体14a的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.72(d,J=8.3Hz,1H,Ar-H),8.59(s,1H,Ar-H),8.28(d,J=8.9Hz,1H,Ar-H),8.25(s,1H,Ar-H),8.10(s,1H,Ar-H),8.00(d,J=8.3Hz,1H,Ar-H),7.95(d,J=8.7Hz,1H,Ar-H),7.63(d,J=9.0Hz,1H,Ar-H),6.69(d,J=9.1Hz,1H,Ar-H),6.49(s,1H,Ar-H),6.43(s,1H,Ar-H),3.47(q,J=6.9Hz,4H,2×CH
2),1.76(s,9H,3×CH
3).1.27(t,J=7.0Hz,6H,2×CH
3);(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)183.5,152.2,150.7,149.1,146.7,141.7,139.8,139.4,139.3,135.9,132.1,131.1,130.9,129.8,128.6,126.68,125.1,124.6,124.1,119.5,114.9,109.8,105.7,96.2,85.1,45.1,28.2,12.6.
取中间体14a(0.3毫摩尔,160.3毫克)溶于2.0毫升浓盐酸和6.0毫升1,4-二氧六环的混合溶液中。在室温下搅拌。利用薄层色谱监测反应完全后,加入饱和碳酸氢钠溶液中和反应体系。然后用三氯甲烷(3×30.0毫升)萃取,收集有机层。加入无水Na
2SO
4干燥后,蒸干溶剂后进行柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(30/1,v/v)。得到121.1毫克墨绿色固体,产率93%,称为染料3a。核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)13.21(s,1H,N-H),8.57(d,J=8.3Hz,1H,Ar-H),8.35(s,1H,Ar-H),8.16-8.12(m,3H,Ar-H),7.76(d,J=8.7Hz,1H,Ar-H),7.63(d,J=8.7Hz,1H,Ar-H),7.60(d,J=9.2Hz,1H,Ar-H),6.81(d,J=8.7Hz,1H,Ar-H),6.64(s,1H,Ar-H),6.29(s,1H,Ar-H).3.51(q,J=6.9Hz,4H,2×CH
2),1.17(t,J=6.8Hz,6H,2×CH
3);(151MHz,DMSO-d
6)
13C NMR(151MHz,DMSO-d
6)δ(ppm)181.8,151.8,150.7,146.3,142.0,138.0,134.2,131.4,131.3,130.8,129.8,129.7,129.5,125.4,124.3,124.2,122.5,118.8,110.8,110.3,109.4,104.5,96.0,44.4,12.4.
取中间体13(1.0毫摩尔,444.2毫克),化合物7b(1.2毫摩尔,355.2毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于35.0毫升1,4-二氧六环中。在氮气氛围下回流7小时。反应结束后,冷却至室温,抽滤。所得滤液在旋转蒸发仪上除去溶剂。柱层析分离,洗脱剂:二氯甲烷/甲醇(100/1,v/v)得到棕色固体204.1毫克,产率47%。中间体14b的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.86(s,1H,Ar-H),8.74(d,J=8.3Hz,1H,Ar-H),8.57(s,1H,Ar-H),8.24(s,1H,Ar-H),7.98(d,J=8.2Hz,1H,Ar-H),7.70(d,J=3.8Hz,1H,Ar-H),7.63(d,J=9.0Hz,1H,Ar-H),6.68(d,J=8.9Hz,1H,Ar-H),6.60(d,J=3.9Hz,1H,Ar-H),6.43(s,1H,Ar-H),3.47(q,J=7.0Hz,4H, 2×CH
2),1.70(s,9H,3×CH
3),1.26(t,J=6.8Hz,6H,2×CH
3);(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)183.5,152.3,150.8,148.0,147.9,146.8,144.1,140.1,139.5,132.2,131.2,131.1,131.0,129.9,127.7,127.4,125.1,124.7,124.2,123.2,109.8,105.8,104.7,96.3,84.2,45.1,28.1,12.6.
取中间体14b(0.3mmol,160.3mg)溶于2.0ml浓盐酸和6.0ml1,4-二氧六环的混合溶液中。在室温下搅拌。利用薄层色谱监测反应完全后加入饱和碳酸氢钠溶液。然后用三氯甲烷(3×30.0ml)萃取,收集有机层。加入无水Na
2SO
4干燥后,蒸干溶剂后进行柱层析分离提纯,洗脱剂:二氯甲烷/甲醇(30/1,v/v)。得到118.5mg绿色固体,产率91%,称为染料3b。核磁:(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)11.82(s,1H,N-H),8.66-8.63(m,2H,Ar-H),8.40(d,J=5.6Hz,1H,Ar-H),8.21(d,J=7.0Hz,1H,Ar-H),7.67(d,J=9.0Hz,1H,Ar-H),7.56(s,1H,Ar-H),6.87(d,J=9.0Hz,1H,Ar-H),6.71(s,1H,Ar-H),6.56(s,1H,Ar-H),6.36(s,1H,Ar-H),3.52(q,J=7.0Hz,4H,2×CH
2),1.17(t,J=6.8Hz,6H,2×CH
3);(151MHz,TFA-d)
13C NMR(151MHz,TFA-d)δ(ppm)150.8,148.0,138.0,137.1,136.8,134.3,131.3,130.6,130.5,130.2,130.0,127.6,125.8,123.4,120.0,115.7,114.9,113.7,113.0,104.0,101.9,50.5,10.2.
取中间体13(1.0毫摩尔,444.2毫克),化合物7c(1.2毫摩尔,356.4毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于35.0毫升1,4-二氧六环中。在氮气氛围下回流7小时。反应结束后,冷却至室温,抽滤。所得滤液在旋转蒸发仪上蒸干溶剂。柱层析分离,洗脱剂:二氯甲烷/甲醇(100/1,v/v)得到棕色固体183.8mg,产率35%。中间体14c的核磁:(400MHz,CDCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)9.11(s,1H,Ar-H),8.75(d,J=7.0Hz,1H,Ar-H),8.56(s,1H,Ar-H),8.41(s,1H,Ar-H),8.26(s,1H,Ar-H),7.96(d,J=7.9Hz,1H,Ar-H),7.63(d,J=7.8Hz,1H,Ar-H),6.69(d,J=8.7Hz,1H,Ar-H),6.48(s,1H,Ar-H),6.43(s,1H,Ar-H),3.48(q,J=5.8Hz,4H,2×CH
2),1.77(s,9H,3×CH
3).1.27(t,J=5.4Hz,6H,2×CH
3);(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)183.2,152.4,151.5,151.0,150.1,147.8,146.9,139.2,138.7,137.6,132.3,132.1,131.6,131.3,129.8,128.4,125.2,125.0,124.4,118.0,110.0,105.7,96.3,85.5,45.1,28.1,12.6.
取中间体14c(0.3毫摩尔,166.0毫克)溶于2.0毫升浓盐酸和6.0毫升1,4-二氧六环的混合液中。室温下搅拌。利用薄层色谱监测至反应完全后加入饱和碳酸氢钠溶液。用三氯甲烷(3×30.0毫升)萃取,收集有机层。加入无水Na
2SO
4干燥后,蒸干溶剂后进行柱层析分离,洗脱剂:二氯甲烷/甲醇(30/1,v/v)。得到113.6毫克紫色固体,产率87%,称为染料3c。(400MHz,DMSO-d
6)
1H NMR(400MHz,DMSO-d
6)δ(ppm)13.81(s,1H,N-H),8.97(s,1H,Ar-H),8.66-8.64(m,2H,Ar-H),8.43(s,1H,Ar-H),8.24-8.22(m,2H,Ar-H),7.66(d,J=8.9Hz,1H,Ar-H),6.87(d,J=8.3Hz,1H,Ar-H),6.70(s,1H,Ar-H),6.36(s,1H,Ar-H),3.51(q,J=6.5Hz,4H,2×CH
2),1.17(t,J=6.0Hz,6H,2×CH
3);(151MHz,TFA-d)
13CNMR(151MHz,TFA-d)δ(ppm)180.9,150.5,143.5,143.2,142.9,137.3,134.9, 134.6,132.7,132.1,131.9,130.0,125.9,123.5,120.2,115.5,114.8,113.6,112.9,49.7,18.5,10.4。
上述制备的染料1a、染料1b、染料1c、染料2a、染料2b、染料2c、染料3a、染料3b、染料3c为本发明实施例的基于含氮杂环的中性线粒体荧光标记物。
对比例
取中间体13(1.0毫摩尔,444.2毫克),化合物15(1.2毫摩尔,249.6毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于35.0毫升1,4-二氧六环中。在氮气下回流4小时。反应结束后,冷却至室温,抽滤。所得滤液在旋转蒸发仪上蒸干溶剂。柱层析分离,洗脱剂:二氯甲烷/甲醇(100/1,v/v)得到棕色固体298.9毫克,产率67%,为染料3d。核磁共振氢谱(400MHz,CHCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.90(s,1H,Ar-H),8.86(s,1H,Ar-H),8.78(d,J=8.2Hz,1H,Ar-H),8.72(s,1H,Ar-H),8.47(s,1H,Ar-H),8.23(s,2H,Ar-H),8.13(d,J=8.1Hz,1H,Ar-H),7.64(d,J=8.9Hz,1H,Ar-H),6.69(d,J=8.8Hz,1H,Ar-H)6.49(s,1H,Ar-H),6.44(s,1H,Ar-H),3.48(q,J=6.9Hz,4H,2×CH
2),1.28(t,J=6.8Hz,6H,2×CH
3);碳谱:(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)183.4,152.3150.9,146.8,145.5,144.9,143.3,142.6,141.6,140.6,139.3,132.2,131.7,131.2,130.0,129.9,129.7,127.3,125.2,124.8,124.6,109.9,105.8,96.3,45.1,12.6.
取化合物15(1.2毫摩尔,355.2毫克),[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.1毫摩尔,73.1毫克)和乙酸钾(2.0毫摩尔,196.3毫克)溶于35.0毫升1,4-二氧六环中。氮气置换3次后,加热回流4小时。反应结束后冷却至室温,抽滤,滤液蒸干溶剂后,通过柱层析分离,洗脱剂:二氯甲烷。得到118.9毫克橙色固体,产率22%。中间体16的核磁:(400MHz,CHCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)8.29(d,J=8.5Hz,1H,Ar-H),8.24(s,1H,Ar-H),8.01(s,1H,Ar-H),7.86(d,J=8.6Hz,1H,Ar-H),7.79(d,J=7.6Hz,2H,Ar-H),7.39(d,J=7.6Hz,2H,Ar-H),6.01(s,2H,Ar-H),2.57(s,6H,2×CH
3),1.76(s,9H,3×CH
3),1.47(s,6H,2×CH
3);(151MHz,CDCl
3)
13C NMR(151MHz,CDCl
3)δ(ppm)155.6,149.1,143.0,141.3,141.2,139.7,139.3136.0134.1,131.4,128.7,128.5,127.8,126.5,121.3,119.2115.0,85.1,28.2,14.6.
取中间体16(0.2mmol,108.1mg)溶于1.0ml三氟乙酸和2.0ml二氯甲烷的溶液中。室温搅拌20分钟后用饱和碳酸钠溶液中和并用三氯甲烷(3×30.0毫升)萃取,收集有机层。加入无水Na
2SO
4干燥后,蒸干溶剂。进行柱层析分离,洗脱剂:二氯甲烷/甲醇(30/1,v/v)得到橙色固体20.1mg,为染料4。核磁共振氢谱(400MHz,CHCl
3)
1H NMR(400MHz,CDCl
3)δ(ppm)
1H NMR(400MHz,DMSO)δ13.12(s,1H,Ar-H),8.11(d,J=9.9Hz,2H,Ar-H),7.88(d,J=6.9Hz,2H,Ar-H),7.74(d,J=7.4Hz,2H,Ar-H),7.61(d,J=7.8Hz,2H,Ar-H),7.41(d,J=7.1Hz,2H,Ar-H),6.15(s,2H,Ar-H),2.42(s,6H,2×CH
3),1.40(s,6H,2×CH
3)。
对上述实施例以及对比例制备的染料在氯仿中(浓度10μM)的紫外吸收和荧光发射进行了测试,横坐标为波长,纵坐标分别为吸光度和荧光强度,结果如图3至13所示。
在紫外-可见吸收光谱图中,染料1a在378nm处有最大吸收;在荧光光谱图中,染料1a在452nm处有最高的荧光强度,此时的激发波长为370nm,狭缝宽度为3nm/1.5nm。在紫外-可见吸收光谱图中,染料1b的最大吸收波长为382nm;在荧光光谱图中,染料1b的最大发射波长为485nm,此时的激发波长为374nm,狭缝宽度为3nm/1.5nm。在紫外-可见吸收光谱图中,染料1c的最大吸收波长为385nm;在荧光光谱图中,染料1c的最大发射波长为454nm,此时的激发波长为380nm,狭缝宽度为3nm/1.5nm。在紫外-可见吸收光谱图中,染料2a在364nm处有最大吸收;在荧光光谱图中,染料2a在480nm处有最高的荧光强度,此时的激发波长为374nm,狭缝宽度为3nm/1.5nm。在紫外-可见吸收光谱图中,染料2b的最大吸收波长为360nm;在荧光光谱图中,染料2b的最大发射波长为458nm,此时的激发波长为370nm,狭缝宽度为3nm/1.5nm。在紫外-可见吸收光谱图中,染料2c的最大吸收波长为356nm;在荧光光谱图中,染料1c的最大发射波长为441nm,此时的激发波长为360nm,狭缝宽度为3nm/3nm。在紫外-可见吸收光谱图中,染料3a在548nm处有最大吸收;在荧光光谱图中,染料3a在606nm处有最高的荧光强度,此时的激发波长为560nm,狭缝宽度为1.5nm/1.5nm。在紫外-可见吸收光谱图中,染料3b的最大吸收波长为549nm;在荧光光谱图中,染料3b的最大发射波长为608nm,此时的激发波长为540nm,狭缝宽度为1.5nm/1.5nm。在紫外-可见吸收光谱图中,染料3c的最大吸收波长为554nm;在荧光光谱图中,染料3c的最大发射波长为611nm,此时的激发波长为540nm,狭缝宽度为1.5nm/1.5nm。在紫外-可见吸收光谱图中,染料3c的最大吸收波长为556nm;在荧光光谱图中,染料3d的最大发射波长为619nm,此时的激发波长为570nm,狭缝宽度为1.5nm/1.5nm。在紫外-可见吸收光谱图中,染料4的最大吸收波长为501nm;在荧光光谱图中,染料4的最大发射波长为515nm,此时的激发波长为495nm,狭缝宽度为1.5nm/1.5nm。以上紫外吸收和荧光发射测试方法为常规方法。
使用DMSO(二甲基亚砜)将染料1a配制成母液,随后加入常规细胞培养基中,使得染料1a在细胞培养基中的浓度为1μM,再分别与L929细胞和HeLa细胞在饱和湿度、37℃、5%CO
2培养箱共同培养(以下实验相同)10分钟,随后分别加入现有线粒体红色标记物Mito
Red CMXRos(100nm)再培养10分钟;然后经PBS缓冲液洗三次后,利用激光共聚焦显微镜进行细胞成像。蓝光通道选用405nm激发,收集410-500nm范围内的荧光信号,红光通道使用561nm激发,收集570-750nm范围内的荧光信号。结果表明染料1a在正常细胞和癌细胞中都具有线粒体标记能力,可作为线粒体蓝色标记物。结果如图14所示,其中(a),(g)为明场,(b),(h)为染料1a的细胞成像图,(c),(i)为线粒体红色标记物的细胞成像图,(d),(j)为蓝光通道和红光通道的叠加图,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,他们的共定位系数分别为0.90(L929)和0.84(HeLa)。
染料1b、染料1c、染料2a、染料2b、染料2c(都是1μM)的实验方法与上述染料1a一样,仅将染料1a更换,其余不变。如图15,(a),(g)为明场,(b),(h)为染料1b的细胞成像,(c),(i)为线粒体红色标记物的细胞成像,(d),(j)为蓝光通道和红光通道的叠加,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,共定位系数分别为0.81(L929)和0.83(HeLa)。如图16,(a),(g)为明场,(b),(h)为染料1c的细胞成像,(c),(i)为线粒体红色标记物的细胞成像,(d),(j)为蓝光通道和红光通道的叠加,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,共定位系数均为0.77。如图17,(a),(g)为明场,(b),(h)为 染料2a的细胞成像,(c),(i)为线粒体红色标记物的细胞成像,(d),(j)为蓝光通道和红光通道的叠加,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,共定位系数分别为0.79(L929)和0.80(HeLa)。如图18,(a),(g)为明场,(b),(h)为染料2b的细胞成像,(c),(i)为线粒体红色标记物的细胞成像,(d),(j)为蓝光通道和红光通道的叠加,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,共定位系数分别为0.85(L929)和0.79(HeLa)。如图19,(a),(g)为明场,(b),(h)为染料2c的细胞成像图,(c),(i)为线粒体红色标记物的细胞成像图,(d),(j)为蓝光通道和红光通道的叠加图,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,共定位系数分别为0.82(L929)和0.84(HeLa)。表明染料1b、染料1c、染料2a、染料2b、染料2c在正常细胞和癌细胞中都具有线粒体标记能力,可作为线粒体蓝色标记物。
使用DMSO(二甲基亚砜)将染料3a配制成母液,随后加入常规细胞培养基中,使得染料3a在细胞培养基中的浓度为1μM,再分别与L929细胞和HeLa细胞在饱和湿度、37℃、5%CO
2培养箱共同培养(以下实验相同)10分钟,随后分别加入现有线粒体绿色标记物Mito
Green FM(100nm)再培养10分钟;PBS缓冲液洗三次后,利用激光共聚焦显微镜进行细胞成像。红光通道使用561nm激发,收集570-750nm范围内的荧光信号。绿光通道使用488nm激发,收集500-550nm范围内的荧光信号。结果表明染料3a在正常细胞和癌细胞中都具有线粒体标记能力,可作为线粒体红色标记物。结果如图20所示,其中(a),(g)为明场,(b),(h)为染料3a的细胞成像图,(c),(i)为线粒体绿色标记物的细胞成像图,(d),(j)为红光通道和绿光通道的叠加图,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,他们的共定位系数分别为0.91(L929)和0.90(HeLa)。
染料3b(1μM)、染料3c(1μM)的实验方法与上述染料3a一样,仅将染料3a更换,其余不变。结果表明染料3b、染料3c在正常细胞和癌细胞中都具有线粒体标记能力,可作为线粒体红色标记物。如图21,(a),(g)为明场,(b),(h)为染料3b的细胞成像图,(c),(i)为线粒体绿色标记物的细胞成像图,(d),(j)为红光通道和绿光通道的叠加图,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,他们的共定位系数分别为0.88(L929)和0.90(HeLa)。如图22,(a),(g)为明场,(b),(h)为染料3c的细胞成像图,(c),(i)为线粒体绿色标记物的细胞成像图,(d),(j)为红光通道和绿光通道的叠加图,(e),(k)为叠加图中ROI线的荧光强度,(f),(l)为共定位实验,他们的共定位系数分别为0.89(L929)和0.87(HeLa)。
使用DMSO(二甲基亚砜)将染料3d配制成母液,随后加入常规细胞培养基中,使得染料3d在细胞培养基中的浓度为1μM,再分别与L929细胞和HeLa细胞在饱和湿度、37℃、5%CO
2培养箱共同培养10分钟,随后分别加入线粒体绿色标记物Mito
Green FM(100nm)和脂滴绿色标记物(合成参见:Chen,Y.;Wei,X.R.;Sun,R.;Xu,Y.J.;Ge,J.F.Org Biomol Chem2018,16,7619.)再培养10分钟;PBS缓冲液洗三次后,利用激光共聚焦显微镜进行细胞成像。红光通道使用561nm激发,收集570-750nm范围内的荧光信号。绿光通道采用488nm激发,收集500-550nm范围内的荧光信号。结果表明,染料3d同时标记了线粒体和脂滴两个细胞器,不适合作为线粒体标记物进行细胞成像。如图23所示,其中(a),(f)为明场,(b),(g)为染料3d的细胞成像图,(c)为脂滴绿色标记物的细胞成像图,(h)为线粒体绿色标记物的细胞成像图,(d),(i)为红光通道和绿光通道的叠加图,(e),(j)为叠加图中ROI线的荧光强度。如图24所示,其中(a),(f)为明场,(b),(g)为染料3d的细 胞成像图,(c)为脂滴绿色标记物的细胞成像图,(h)为线粒体绿色标记物的细胞成像图,(d),(i)为红光通道和绿光通道的叠加图,(e),(j)为叠加图中ROI线的荧光强度。
使用DMSO将染料4配制成母液,随后加入常规细胞培养基中,使得染料4在细胞培养基中的浓度为1μM,再与HeLa细胞在饱和湿度、37℃、5%CO
2培养箱共同培养10分钟,随后加入线粒体红色标记物Mito
Red CMXRos(100nm)再培养10分钟;PBS缓冲液洗三次后,利用激光共聚焦显微镜进行细胞成像。红光通道使用561nm激发,收集570-750nm范围内的荧光信号。绿光通道采用488nm激发,收集500-550nm范围内的荧光信号。结果表明,染料4不能与线粒体红色标记物进行很好的叠加,不可作为线粒体标记物进行细胞成像。如图25所示,(a)为明场,(b)为染料4的细胞成像图,(c)为线粒体红色标记物的细胞成像图,(d)为红光通道和绿光通道的叠加图,(e)为叠加图中ROI线的荧光强度。
采用常规CCK-8法测试实施例制备的染料细胞毒性,测试时间为6小时,采用美仑CCK-8细胞增殖毒性检测试剂盒,结果显示,染料浓度为2μM至10μM时(DMSO为溶剂),L929细胞和HeLa细胞的存活率都大于95%。
Claims (10)
- 权利要求1所述基于含氮杂环的中性线粒体荧光标记物在线粒体荧光标记中的应用;或者权利要求1所述基于含氮杂环的中性线粒体荧光标记物在制备线粒体荧光标记试剂中的应用。
- 权利要求1所述基于含氮杂环的中性线粒体荧光标记物的制备方法,其特征在于,为以下制备方法中的一种:(1)化合物6与化合物7反应得到化合物8;化合物8脱保护,得到基于含氮杂环的中性线粒体荧光标记物;(2)化合物9与化合物7反应得到化合物10;化合物10脱保护,得到基于含氮杂环的中性线粒体荧光标记物;(3)化合物13与化合物7反应得到化合物14;化合物14脱保护,得到基于含氮杂环的中性线粒体荧光标记物。
- 根据权利要求4所述基于含氮杂环的中性线粒体荧光标记物的制备方法,其特征在于,脱保护在盐酸存在下进行;化合物6与化合物7的反应在贵金属盐催化剂存在下进行;化合物9与化合物7的反应在贵金属盐催化剂存在下进行;化合物13与化合物7的反应在贵金属盐催化剂存在下进行。
- 根据权利要求5所述基于含氮杂环的中性线粒体荧光标记物的制备方法,其特征在于,贵金属盐催化剂包括钯盐催化剂。
- 一种细胞成像方法,其特征在于,包括以下步骤:(1)化合物6与化合物7反应得到化合物8;化合物8脱保护,得到基于含氮杂环的中性线粒体荧光标记物;(2)化合物9与化合物7反应得到化合物10;化合物10脱保护,得到基于含氮杂环的中性线粒体荧光标记物;(3)化合物13与化合物7反应得到化合物14;化合物14脱保护,得到基于含氮杂环的中性线粒体荧光标记物;(4)将步骤(1)或者步骤(2)制备的基于含氮杂环的中性线粒体荧光标记物与细胞共培养后加入线粒体红色标记物,继续培养后,进行细胞成像;或者将步骤(3)制备的基于含氮杂环的中性线粒体荧光标记物与细胞共培养后加入线粒体绿色标记物,继续培养后,进行细胞成像。
- 根据权利要求7所述细胞成像方法,其特征在于,脱保护在盐酸存在下进行;化合物6与化合物7的反应在贵金属盐催化剂存在下进行;化合物9与化合物7的反应在贵金属盐催化剂存在下进行;化合物13与化合物7的反应在贵金属盐催化剂存在下进行。
- 根据权利要求7所述细胞成像方法,其特征在于,利用激光共聚焦显微镜进行细胞成像;蓝光通道使用405nm激发,收集410~500nm范围内的荧光信号;红光通道使用561nm激发,收集570~750nm范围内的荧光信号;绿光通道使用488nm激发,收集500~550nm范围内的荧光信号。
- 根据权利要求7所述细胞成像方法,其特征在于,所述细胞包括正常细胞、癌细胞。
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