WO2022147872A1 - 酰胺衍生物中性线粒体荧光标记物及其制备方法和应用 - Google Patents

酰胺衍生物中性线粒体荧光标记物及其制备方法和应用 Download PDF

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WO2022147872A1
WO2022147872A1 PCT/CN2021/075159 CN2021075159W WO2022147872A1 WO 2022147872 A1 WO2022147872 A1 WO 2022147872A1 CN 2021075159 W CN2021075159 W CN 2021075159W WO 2022147872 A1 WO2022147872 A1 WO 2022147872A1
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dye
formula
mitochondrial
neutral
mitochondrial fluorescent
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葛健锋
马威
孙如
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苏州大学
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Definitions

  • the invention relates to a fluorescent marker technology, in particular to an amide derivative neutral mitochondrial fluorescent marker and a preparation method and application thereof.
  • Mitochondria are organelles that exist in most cells and are covered by two membranes. They are the energy-producing structures in cells and the main place for cells to perform aerobic respiration. In addition to providing energy for cells as the main site of aerobic respiration, it also participates in important physiological activities such as cell genetic material transfer and cell differentiation, and has the ability to regulate cell growth and cell cycle. Therefore, in scientific research, it is particularly important to monitor mitochondria in real time.
  • fluorescent labeling technology stands out due to its advantages of simple operation and low preparation cost.
  • Various fluorescent probes and dyes with mitochondrial targeting functions have also been developed.
  • Detection of Sens Actuators B2018, 259, 299. enables imaging of mitochondria .
  • mitochondrial-targeting fluorescent probes and dyes it is not difficult to find that most of their main structures contain triphenylphosphine salts, pyridinium salts and indole salts. This is true of even the most commonly used commercial mitochondrial red and green markers. This is because the presence of proton pumps on the inner mitochondrial membrane makes it easier for these cationic dyes to penetrate the mitochondrial membrane and accumulate in the mitochondria. But the problem also follows. After these cations enter the mitochondria, they will change the membrane potential of the mitochondria, causing changes in the cellular microenvironment and leading to apoptosis. Therefore, fluorescent dyes with neutral structures with mitochondrial targeting ability are urgently needed.
  • the purpose of the present invention is to provide an amide derivative neutral mitochondrial fluorescent marker and a preparation method and application thereof.
  • the present invention provides an amide derivative with excellent mitochondrial targeting ability. Mitochondrial targeting by sex dyes does not rely on the negative charge on the inner mitochondrial membrane, addressing the deficiencies of existing cationic mitochondrial dyes.
  • the first object of the present invention is to disclose an amide derivative neutral mitochondrial fluorescent marker, and the neutral mitochondrial fluorescent marker of the amide derivative is represented by one of formulas (I)-(IV):
  • R 1 and R 2 are independently selected from hydrogen or an alkyl group with 1 to 6 carbon atoms; M, E 1 and E 2 are independently selected from an alkyl group with 1 to 6 carbon atoms; n is Any integer from 1 to 3.
  • the alkyl group in the present invention represents 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.
  • R 1 and R 2 are independently selected from hydrogen or methyl; M is methyl; E 1 and E 2 are both ethyl; and n is 1 or 2.
  • the above-mentioned compounds of formulae (I)-(IV) all include fluorescent dyes and amide compounds connected with them through chemical bonds.
  • the coumarin boron lipid derivatives and the Nile red boron lipid derivatives with lipid droplet targeting are connected with the amide compound, so that the mitochondrial targeting ability is successfully achieved.
  • the invention not only improves the optical properties of the fluorescent dye, but also regulates the organelle targeting ability of the original dye by modifying its structure.
  • the biological properties of the dyes were significantly improved after the dyes were linked with the amide compounds. Amide compounds are cheap and easy to obtain, which is beneficial to control the cost of new dyes, and has great scientific significance and commercial value.
  • the second object of the present invention is to provide a method for preparing the above-mentioned amide derivative neutral mitochondrial fluorescent marker, comprising the following steps:
  • R 1 and R 2 are independently selected from hydrogen or an alkyl group with 1 to 6 carbon atoms; M, E 1 and E 2 are independently selected from an alkyl group with 1 to 6 carbon atoms; n is Any integer from 1 to 3.
  • R 1 and R 2 are independently selected from hydrogen or methyl; M is methyl; E 1 and E 2 are both ethyl; and n is 1 or 2.
  • the structural formula of the neutral mitochondrial fluorescent marker of the amide derivative is as follows:
  • the noble metal salt catalyst includes a palladium salt catalyst.
  • reaction temperature 85-110°C.
  • reaction time is 8-12h.
  • the third object of the present invention is to disclose the application of the above-mentioned neutral mitochondrial fluorescent marker of amide derivatives in the preparation of mitochondrial fluorescent labeling reagent.
  • mitochondrial fluorescent labeling reagents target the mitochondria of cancer cells.
  • the method for cell imaging using mitochondrial fluorescent labeling reagents includes the following steps:
  • the mitochondrial fluorescent labeling reagent and cells were co-incubated for more than 10 min at 37°C and 5% CO 2 , and then the cells were imaged using a confocal laser microscope to collect fluorescent signals.
  • the mitochondrial fluorescent labeling reagent includes an amide derivative neutral mitochondrial fluorescent label represented by formula (I) or formula (II)
  • the blue light channel is used to excite, specifically, a 405 nm light source is used to excite, and the fluorescence in the range of 410-500 nm is collected.
  • Fluorescence signal; the neutral mitochondrial fluorescent marker of the amide derivative represented by formula (I) or formula (II) is a mitochondrial blue marker.
  • the mitochondrial fluorescent labeling reagent includes an amide derivative neutral mitochondrial fluorescent marker represented by formula (III) or formula (IV)
  • use the red light channel to excite specifically, use a 561 nm light source to excite, and collect fluorescent signals in the range of 570-750 nm .
  • the neutral mitochondrial fluorescent marker of the amide derivative represented by formula (III) or formula (IV) is a red mitochondrial marker.
  • the present invention has at least the following advantages:
  • the invention discloses the neutral mitochondrial fluorescent marker of amide derivatives for the first time, which can control the organelle targeting ability of the original dye by creatively modifying its structure while ensuring that the optical properties of the fluorophore are not changed.
  • the biological properties of the dye and the amide compound are obviously improved, and the amide compound is cheap and easy to obtain, which is beneficial to control the cost of the new dye. Due to the electrically neutral structure, these compounds do not change the mitochondrial membrane potential when targeting mitochondria, have good biodescriptivity and low cytotoxicity.
  • the imaging of mitochondria in cells can be realized.
  • the compounds of the present invention have low cytotoxicity when performing cell imaging, little damage to biological samples, are not affected by other organelles, can observe cell samples for a long time, and have great scientific significance and commercial value. This solves the problems of uncertainty in the targeting ability of existing neutral structural fluorescent dyes to organelles and the fact that neutral dyes are commercial markers of lipid droplets in cells.
  • Fig. 1 is the synthetic route schematic diagram of dyestuff of the present invention
  • Figure 2 shows the results of the photostability test of dyes 2a-e
  • FIG. 3 shows the results of the photostability test of dyes 3a-e
  • Fig. 4 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2a in chloroform
  • Fig. 5 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2b in chloroform
  • Fig. 6 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2c in chloroform
  • Fig. 7 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2d in chloroform
  • Fig. 8 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 2e in chloroform
  • Fig. 9 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3a in chloroform
  • Fig. 10 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3b in chloroform
  • Figure 11 is the UV-Vis absorption spectrum and fluorescence spectrum of dye 3c in chloroform
  • Fig. 12 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3d in chloroform
  • Figure 13 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dye 3e in chloroform
  • Figure 14 is a cell imaging diagram of compound 2 in HeLa cells
  • Figure 15 is a cell imaging diagram of dye 2a in HeLa cells
  • Figure 16 is a cell imaging diagram of dye 2b in HeLa cells
  • Figure 17 is a cell imaging diagram of dye 2c in HeLa cells
  • Figure 18 is a cell imaging diagram of dye 2d in HeLa cells
  • Figure 19 is a cell imaging diagram of dye 2e in HeLa cells
  • Figure 20 is a cell imaging diagram of compound 3 in HeLa cells
  • Figure 21 is a cell imaging diagram of dye 3a in HeLa cells
  • Figure 22 is a cell imaging diagram of dye 3b in HeLa cells
  • Figure 23 is a cell imaging diagram of dye 3c in HeLa cells
  • Figure 24 is a cell imaging diagram of dye 3d in HeLa cells
  • Figure 25 is a cell imaging diagram of dye 3e in HeLa cells
  • Figure 26 is the cytotoxicity test results of dyes 2a-e and 3a-e;
  • Figure 27 is the photobleaching resistance test result of dye 2a
  • Figure 28 shows the test results of the anti-photobleaching performance of dye 3a.
  • laser confocal microscope was used for cell imaging; the blue light channel was excited at 405 nm, and the fluorescence signal in the range of 410-500 nm was collected; the green light channel was excited at 488 nm, and the fluorescence signal in the range of 500-550 nm was collected; the red light channel Using excitation at 561 nm, the fluorescence signal was collected in the range of 570-750 nm.
  • the synthetic route of the embodiment of the present invention is shown in FIG. 1 , and the numbers below the chemical formula represent the compounds.
  • the ratio of raw materials and the purification method are conventional ratios or conventional purification methods, the examples are schematic representations, and the correctness of the product structure is verified by nuclear magnetic resonance in the present invention.
  • Dye 2a (262.0 mg) was prepared from compound 2 (1 mmol, 357.21 mg) and compound 1a (1 mmol, 200.0 mg) in 75.0% yield as a yellow solid. Characterization of dye 2a: mp 195.4–200.5°C. IR ⁇ (KBr, cm -1 ): 3404, 3128, 2974, 2928, 2868, 1672, 1605, 1575, 1163, 1305, 1092, 856, 802, 770, 658.
  • Dye 2b (262.1 mg) was prepared from compound 2 (1 mmol, 357.21 mg) and compound 1b (1 mmol, 214.1 mg) in 72.1% yield as a pale yellow solid. Characterization of dye 2b: mp 180.5–185.0°C. IR ⁇ (KBr, cm -1 ): 3299, 3055, 2976, 2936, 1711, 1620, 1609, 1523, 1406, 1316, 1213, 1165, 935, 793, 669.
  • Dye 2c (302.6 mg) was prepared from compound 2 (1 mmol, 357.21 mg) and compound 1c (1 mmol, 228.0 mg) in 80.0% yield as a yellow solid. Characterization of dye 2c: mp 175.5–180.0°C. IR ⁇ (KBr, cm -1 ): 2971, 2928, 1702, 1628, 1617, 1508, 1442, 1357, 1213, 1164, 1080, 983, 918, 876, 782, 679, 632.
  • Dye 2d (307.7 mg) was prepared from compound 2 (1 mmol, 357.21 mg) and compound 1d (1 mmol, 212.0 mg) in 85.0% yield as a yellow solid. Characterization of dye 2d: mp 168.5–173.0°C. IR ⁇ (KBr, cm -1 ): 3143, 2975, 2916, 1701, 1617, 1584, 1522, 1409, 1354, 1263, 1211, 1162, 990, 867, 798, 689.
  • Dye 2e (308.3 mg) was prepared from compound 2 (1 mmol, 357.21 mg) and compound 1e (1 mmol, 226.0 mg) in 82.0% yield as a yellow solid. Characterization of dye 2e: mp 173.5–178.0°C. IR ⁇ (KBr, cm -1 ): 3177, 3041, 2967, 1703, 1674, 1618, 1545, 1443, 1356, 1282, 1183, 1145, 1006, 949, 904, 865, 738, 697.
  • Dye 3a (65.5 mg) was prepared from compound 3 (0.5 mmol, 222.1 mg) and compound 1a (0.6 mmol, 120.0 mg) in a dark green solid yield of 30.0%. Characterization of dye 3a: mp 212.5–215.0°C. IR ⁇ (KBr, cm -1 ): 3327, 3144, 2965, 2923, 1701, 1682, 1636, 1619, 1520, 1409, 1323, 1230, 1147, 1009, 946, 845, 774, 669, 644 .
  • Dye 3b (85.7 mg) was prepared from compound 3 (0.5 mmol, 222.1 mg) and compound 1b (0.6 mmol, 128.0 mg) in 38.0% yield as a black solid. Characterization of dye 3b: mp 198.5–203.0°C. IR ⁇ (KBr, cm -1 ): 3295, 2970, 1639, 1578, 1563, 1548, 1463, 1372, 1278, 1181, 1076, 950, 866, 768, 689.
  • Dye 3c (151.1 mg) was prepared from compound 3 (0.5 mmol, 222.1 mg) and compound 1c (0.6 mmol, 130.8 mg) in 65.0% yield as a black solid. Characterization of dye 3c: mp 200.0–205.5°C. IR ⁇ (KBr, cm -1 ): 2965, 2925, 1623, 1579, 1518, 1406, 1324, 1280, 1146, 1078, 1000, 950, 875, 799, 726, 680, 634.
  • Dye 3d (75.3 mg) was prepared from compound 3 (0.5 mmol, 222.1 mg) and compound 1d (0.6 mmol, 127.2 mg) in 32.0% yield as a black solid. Characterization of dye 3d: mp 220.5–225.0 °C.
  • Dye 3e (97.2 mg) was prepared from compound 3 (0.5 mmol, 222.1 mg) and compound 1e (0.6 mmol, 135.6 mg) in 42.0% yield as a black solid. Characterization of dye 3e: mp 230.0–235.0 °C.
  • the above-prepared dyes 2a-2e, 3a-e were tested for photostability.
  • the corresponding quality dyes 2a-2e, 3a-e and reference substances coumarin and Nile red were weighed, and the Each of the samples was dissolved in acetonitrile (concentration: 10 ⁇ M), and all samples were irradiated with a Philips iodine tungsten lamp (500 W), and the distance between the lamp and the sample was set to 25 cm.
  • An 8 cm thick NaNO 2 (60 g.L -1 ) cold trap was placed between the lamp and the sample to remove heat and short wavelength light.
  • dyes 2a-e, 3a-e have high photostability.
  • dye 2a In the UV-Vis absorption spectrum, dye 2a has the maximum absorption at 388nm; in the fluorescence spectrum, dye 2a has the highest fluorescence intensity at 457nm, the excitation wavelength at this time is 380nm, and the slit width is 3nm/1.5 nm.
  • the maximum absorption wavelength of dye 2b In the UV-Vis absorption spectrum, the maximum absorption wavelength of dye 2b is 386 nm; in the fluorescence spectrum, the maximum emission wavelength of dye 2b is 459 nm, the excitation wavelength at this time is 385 nm, and the slit width is 3 nm/1.5 nm.
  • the maximum absorption wavelength of dye 2c is 385nm; in the fluorescence spectrum, the maximum emission wavelength of dye 2c is 455nm, the excitation wavelength at this time is 380nm, and the slit width is 3nm/1.5nm.
  • dye 2d has the maximum absorption at 380nm; in the fluorescence spectrum, dye 2d has the highest fluorescence intensity at 457nm, the excitation wavelength at this time is 380nm, and the slit width is 3nm/1.5 nm.
  • the maximum absorption wavelength of dye 2e is 385 nm; in the fluorescence spectrum, the maximum emission wavelength of dye 2e is 458 nm, the excitation wavelength at this time is 385 nm, and the slit width is 3 nm/1.5 nm.
  • dye 3a has the maximum absorption at 554nm; in the fluorescence spectrum, dye 3a has the highest fluorescence intensity at 604nm, the excitation wavelength at this time is 550nm, and the slit width is 1.5nm/ 3nm.
  • the maximum absorption wavelength of dye 3b is 552nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3b is 597nm, the excitation wavelength at this time is 550nm, and the slit width is 1.5nm/3nm.
  • the maximum absorption wavelength of dye 3c is 552nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3c is 621nm, the excitation wavelength at this time is 550nm, and the slit width is 1.5nm/3nm.
  • the maximum absorption wavelength of dye 3d is 551 nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3d is 607 nm, the excitation wavelength at this time is 545 nm, and the slit width is 1.5 nm/3 nm.
  • the maximum absorption wavelength of dye 3e is 553nm; in the fluorescence spectrum, the maximum emission wavelength of dye 3e is 585nm, the excitation wavelength at this time is 550nm, and the slit width is 1.5nm/3nm.
  • the above UV absorption and fluorescence emission testing methods are conventional methods.
  • Compounds 2 and 3 were formulated into stock solutions using DMSO (dimethyl sulfoxide), and then added to conventional cell culture media to make Compounds 2 and 3 3
  • the concentration of 3 in the cell culture medium is 2 ⁇ M, and then co-incubated with HeLa cells in a saturated humidity, 37 °C, 5% CO 2 incubator (same as in the following experiments) for 10 minutes, and then washed three times with PBS buffer, using laser confocal Microscope for cell imaging.
  • the blue light channel is excited at 405 nm to collect fluorescence signals in the range of 410-500 nm
  • the green light channel is excited at 488 nm, and the fluorescence signals are collected in the range of 500-550 nm
  • the red light channel is excited at 561 nm
  • the fluorescence signals are collected in the range of 570-750 nm.
  • dye 2a was formulated as a stock solution using DMSO (dimethyl sulfoxide) and then added to regular cell culture medium to bring the concentration of dye 2a in the cell culture medium 2 ⁇ M, and then co-incubated with HeLa cells in a saturated humidity, 37°C, 5% CO 2 incubator (same for the following experiments) for 10 minutes, and then added the mitochondrial red marker Mito respectively.
  • Red CMXRos 100 nm were incubated for an additional 10 minutes; then after three washes in PBS buffer, cells were imaged by confocal microscopy.
  • the blue light channel is excited at 405 nm, and the fluorescence signal in the range of 410-500 nm is collected, and the red light channel is excited at 561 nm, and the fluorescence signal in the range of 570-750 nm is collected.
  • the results are shown in Figure 15, where (a) is the bright field, (b) is the cell imaging image of dye 2a, (c) is the cell imaging image of the mitochondrial red marker, (d) is the blue light channel and the red light channel.
  • Overlay graph (e) is the fluorescence intensity of the ROI line in the overlay graph, (f) is the co-localization experiment, and their co-localization coefficient is 0.88.
  • dye 2b (2 ⁇ M), dye 2c (2 ⁇ M), dye 2d (2 ⁇ M), and dye 2e (2 ⁇ M) are the same as the above dye 2a, only the dye 2a can be replaced, and the rest remain unchanged.
  • (a) is the bright field
  • (b) is the image of the cell with dye 2b
  • (c) is the image of the cell with the mitochondrial red marker
  • (d) is the superposition of the blue and red channels
  • (e) is the fluorescence intensity of the ROI line in the overlay
  • (f) is the co-localization experiment, and their co-localization coefficients are 0.94, respectively.
  • (a) is the bright field
  • (b) is the cell imaging image of dye 2d
  • (c) is the cell imaging image of the mitochondrial red marker
  • (d) is the superposition of the blue light channel and the red light channel Figure
  • (e) is the fluorescence intensity of the ROI line in the overlay
  • (f) is the co-localization experiment, and their co-localization coefficients are 0.92, respectively.
  • the dye 3a was prepared into a stock solution using DMSO (dimethyl sulfoxide), and then added to the regular cell culture medium, so that the concentration of dye 3a in the cell culture medium was 2 ⁇ M. 2 incubators (the same for the following experiments) for 10 minutes, and then add the mitochondrial green marker Mito respectively Green FM (100 nm) was incubated for another 10 minutes; after three washes in PBS buffer, cells were imaged by confocal microscopy.
  • the red channel uses excitation at 561 nm and collects fluorescence signals in the range of 570-750 nm.
  • the green light channel uses excitation at 488nm and collects fluorescence signals in the range of 500-550nm.
  • dye 3b (2 ⁇ M), dye 3c (2 ⁇ M), dye 3d (2 ⁇ M), and dye 3e (2 ⁇ M) are the same as the above dye 3a, only the dye 3a can be replaced, and the rest remain unchanged.
  • (a) is the bright field
  • (b) is the cell imaging image of dye 3b
  • (c) is the cell imaging image of the mitochondrial green marker
  • (d) is the red light channel and the green light channel.
  • Overlay graph (e) is the fluorescence intensity of the ROI line in the overlay graph
  • (f) is the co-localization experiment, and their co-localization coefficient is 0.88.
  • Overlay graph (e) is the fluorescence intensity of the ROI line in the overlay graph, (f) is the co-localization experiment, and their co-localization coefficient is 0.96.
  • (a) is the bright field
  • (b) is the cell imaging image of dye 3e
  • (c) is the cell imaging image of the mitochondrial green marker
  • (d) is the red light channel and the green light channel.
  • Overlay graph (e) is the fluorescence intensity of the ROI line in the overlay graph
  • (f) is the co-localization experiment, and their co-localization coefficient is 0.92.
  • the present invention also tests the cytotoxicity of dyes 2a-e or 3a-e.
  • the viability of HeLa cells in the presence of these dyes was measured by using the CCK-8 method. HeLa cells were incubated with different concentrations of dye (2, 4, 6, 8 and 10 ⁇ M) for 6 hours, respectively.
  • Figure 26a shows the cytotoxicity test results of dyes 2a-e
  • Figure 26b shows the cytotoxicity test results of dyes 3a-e. The results show that they have good cell viability and are suitable for live cell imaging.
  • HeLa cells were more than 100% viable after 6 h incubation with dyes 3a-c, suggesting that dyes 3a-c are not only non-toxic but can also promote cell growth.
  • cell viability (%) (A sample -A b )/(A c -A b ), where A c : negative control (including culture medium and cells, no test dye added), A b : Blank (including the dye to be tested and medium, no cells added), A sample : test group (including the medium, cells and dye to be tested).
  • Fig. 27(a) is a brightfield image
  • Fig. 27(b-h) are successively irradiated with excitation light source (561nm) in HeLa cells containing dye 2a for 1, 5, 10, 15, 20, 25, Fluorescence image after 30 minutes.
  • Fig. 28(a) is a brightfield image
  • Fig. 28(a) is a brightfield image
  • the present invention discloses for the first time that after the neutral mitochondrial fluorescent marker of amide derivatives is co-cultured with cells, mitochondrial imaging in cells can be realized. While improving the optical properties of the dye, the invention modulates the organelle targeting ability of the dye by creatively modifying its structure, and has low cytotoxicity during cell imaging, little damage to biological samples, is not affected by other organelles, and can be used for cell imaging. Cell samples can be observed for a long time without permeabilization or fixation of cells, so cells can be monitored in real time without being affected by other organelles, and the experimental results show that the dye of the invention has excellent targeting to mitochondria sex.

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Abstract

本发明涉及一系列酰胺衍生物中性线粒体荧光标记物及其制备方法和应用，本发明首次公开中性染料与酰胺化合物相连后具有了优秀的线粒体靶向能力。此发明解决了现有中性结构荧光染料细胞器靶向能力不确定性和中性染料是细胞中脂滴的商业标记物的问题。本发明在改善荧光染料的光学性能的同时，通过对其结构创造性的修饰来调控原染料的细胞器靶向能力，而且与酰胺化合物连接后染料的生物学性能有了明显的改善。酰胺类化合物廉价易得，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

酰胺衍生物中性线粒体荧光标记物及其制备方法和应用 技术领域
本发明涉及荧光标记物技术，尤其涉及一种酰胺衍生物中性线粒体荧光标记物及其制备方法和应用。
背景技术
线粒体是一种存在于大多数细胞中的由两层膜包被的细胞器，是细胞中制造能量的结构，是细胞进行有氧呼吸的主要场所。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等重要的生理活动，并拥有调控细胞生长和细胞周期的能力。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。
在过去的二十年中，科研工作者合成了各种各样的线粒体荧光探针，通过对线粒体中金属离子(参见：Sreenath,K.；Allen,J.R.；Davidson,M.W.；Zhu,L.ChemCommun(Camb)2011,47,11730.)、有机小分子(参见：Ji,A.；Fan,Y.；Ren,W.；Zhang,S.；Ai,H.W.ACS Sens2018,3,992.)和大分子(参见：Yang,L.；Niu,J.-Y.；Sun,R.；Xu,Y.-J.；Ge,J.-F.Sens Actuators B2018,259,299.)的检测实现了对线粒体的成像。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。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，造成细胞微环境的变化并导致细胞凋亡，所以亟需中性结构具有线粒体靶向能力的荧光染料。
发明内容
为解决上述技术问题，本发明的目的是提供一种酰胺衍生物中性线粒体荧光标记物及其制备方法和应用，本发明提供了一种具有优秀线粒体靶向能力的酰胺衍生物，这类中性染料靶向线粒体并不依赖于线粒体内膜上的负电荷，解决了现有阳离子线粒体染料的缺陷。
本发明的第一个目的是公开一种酰胺衍生物中性线粒体荧光标记物，酰胺衍生物的中性线粒体荧光标记物为式(I)-(IV)之一所示：
Figure PCTCN2021075159-appb-000001
其中，R 1、R 2分别独立地选自氢或碳原子数为1～6的烷基；M、E 1、E 2分别独立地选自碳原子数为1～6的烷基；n为1～3中任一整数。
本发明中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。
进一步地，R 1、R 2分别独立地选自氢或甲基；M为甲基；E 1和E 2均为乙基；n为1或2。
上述式(I)-(IV)化合物均包括荧光染料及与其通过化学键相连的酰胺类化合物。本发明将具有脂滴靶向的香豆素硼脂衍生物和尼罗红硼脂衍生物与酰胺化合物相连后使其成功的具有了线粒体靶向能力。本发明在改善荧光染料的光学性能的同时，并通过对其结构的修饰来调控原染料的细胞器靶向能力。而且染料与酰胺化合物相连后其生物学性能得到了明显的改善。酰胺类化合物廉价易得，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。
本发明的第二个目的是提供一种上述酰胺衍生物中性线粒体荧光标记物的制备方法，包括以下步骤：
在弱碱性条件和有机溶剂中，利用式(2)所示的化合物与式(1a-c)或式(1d-e)所示的化合物反应得到式(I)或式(II)所示的酰胺衍生物中性线粒体荧光标记物；或者
在弱碱性条件和有机溶剂中，利用式(3)所示的化合物与式(1a-c)或式(1d-e)所示的化合物反应得到式(III)或式(IV)所示的酰胺衍生物中性线粒体荧光标记物；
其中式(1a-c)、(1d-e)、(2)或(3)结构式分别如下：
Figure PCTCN2021075159-appb-000002
其中，R 1、R 2分别独立地选自氢或碳原子数为1～6的烷基；M、E 1、E 2分别独立地选自碳原子数为1～6的烷基；n为1～3中任一整数。
优选地，R 1、R 2分别独立地选自氢或甲基；M为甲基；E 1和E 2均为乙基；n为1或2。具体地，酰胺衍生物的中性线粒体荧光标记物的结构式如下：
Figure PCTCN2021075159-appb-000003
进一步地，反应均在贵金属盐催化剂存在下进行。
进一步地，贵金属盐催化剂包括钯盐催化剂。
进一步地，反应温度为85-110℃。
进一步地，反应时间为8-12h。
本发明的第三个目的是公开上述酰胺衍生物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。
进一步地，线粒体荧光标记试剂靶向癌细胞的线粒体。
进一步地，利用线粒体荧光标记试剂进行细胞成像的方法包括以下步骤：
将线粒体荧光标记试剂与细胞在37℃及5％CO 2条件下共培养10min以上，然后利用激光共聚焦显微镜进行细胞成像，收集荧光信号。
进一步地，当线粒体荧光标记试剂包括式(I)或式(II)所示的酰胺衍生物中性线粒体荧光标记物时，使用蓝光通道激发，具体使用405nm光源激发，收集410～500nm范围内的荧光信号；式(I)或式(II)所示的酰胺衍生物中性线粒体荧光标记物为线粒体蓝色标记物。
当线粒体荧光标记试剂包括式(III)或式(IV)所示的酰胺衍生物中性线粒体荧光标记物时，使用红光通道激发，具体使用561nm光源激发，收集570～750nm范围内的荧光信号。式(III)或式(IV)所示的酰胺衍生物中性线粒体荧光标记物为线粒体红色标记物。
借由上述方案，本发明至少具有以下优点：
本发明首次公开了酰胺衍生物中性线粒体荧光标记物，在保证不改变荧光团光学性能的同时，通过对其结构创造性的修饰来调控原染料的细胞器靶向能力。而且染料和酰胺化合物相连后其生物学性能得到了明显的盖改善，酰胺类化合物廉价易得，有利于控制新染料的成本。由于是电中性结构，此类化合物靶向线粒体时不会改变线粒体的膜电位，具有良好的生物形容性，细胞毒性较低。
本发明的酰胺衍生物中性线粒体荧光标记物与细胞共培养后，可以实现细胞中线粒体成像。本发明此类化合物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，具有重大的科学意义和商业价值。解决了现有中性结构荧光染料细胞器靶向能力不确定性和中性染料是细胞中脂滴的商业标记物的问题。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1为本发明的染料的合成路线示意图；
图2为染料2a-e光稳定性测试结果；
图3为染料3a-e光稳定测试结果；
图4为染料2a在氯仿中的紫外-可见吸收光谱和荧光光谱；
图5为染料2b在氯仿中的紫外-可见吸收光谱和荧光光谱；
图6为染料2c在氯仿中的紫外-可见吸收光谱和荧光光谱；
图7为染料2d在氯仿中的紫外-可见吸收光谱和荧光光谱；
图8为染料2e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3e在氯仿中的紫外-可见吸收光谱和荧光光谱；
图14为化合物2在HeLa细胞中的细胞成像图；
图15为染料2a在HeLa细胞中的细胞成像图；
图16为染料2b在HeLa细胞中的细胞成像图；
图17为染料2c在HeLa细胞中的细胞成像图；
图18为染料2d在HeLa细胞中的细胞成像图；
图19为染料2e在HeLa细胞中的细胞成像图；
图20为化合物3在HeLa细胞中的细胞成像图；
图21为染料3a在HeLa细胞中的细胞成像图；
图22为染料3b在HeLa细胞中的细胞成像图；
图23为染料3c在HeLa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3e在HeLa细胞中的细胞成像图；
图26为染料2a-e和3a-e的细胞毒性测试结果；
图27为染料2a的抗光漂白性能测试结果；
图28为染料3a的抗光漂白性能测试结果。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中，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；绿光通道使用488nm激发，收集500～550nm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。
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，且本发明通过核磁验证产物结构的正确性。
实施例一
染料2a-e合成的一般步骤如下:
取化合物2(1.0毫摩尔，357.2毫克)、酰胺化合物1a-e中的一种(1.0毫摩尔)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(Pd(dppf)Cl 2，0.03毫摩尔，22毫克)和磷酸钾(K 3PO 4，2.5毫摩尔，557.5毫克)溶解于15.0毫升1,4-二氧六环(1,4-dioxane)溶剂中；然后反应体系通过氮气置换三次，然后回流8到10小时，TLC检测反应进度；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，经柱层析(洗脱剂：二氯甲烷/甲醇(100/1，v/v))分离后得到纯净染料2a-e。
具体地，各产物具体合成原料及产率、结构表征结果分别如下：
染料2a(262.0毫克)由化合物2(1毫摩尔，357.21毫克)和化合物1a(1毫摩尔，200.0毫克)制备得到，黄色固体产率75.0％。染料2a的表征：mp 195.4–200.5℃.IR ν(KBr,cm -1):3404,3128,2974,2928,2868,1672,1605,1575,1163,1305,1092,856,802,770,658. 1H NMR(400MHz,CDCl 3):δ(ppm)7.87(d,J＝7.9Hz,2H,2×Ar-H),7.47(d,J＝8.9Hz,1H,Ar-H),7.40(d,J＝7.8Hz,2H,2×Ar-H),6.64(d,J＝10.2Hz,1H,Ar-H),6.55(s,1H,Ar-H),6.23(s,1H,N-H),5.72(s,1H,N-H),3.44(q,J＝7.0Hz 4H,2×CH 2),2.23(s,3H,CH 3),1.23(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,DMSO-d 6):δ(ppm)167.5,160.3,154.6,150.1,148.6,138.2,133.1,130.3,127.0,126.7,118.9,108.7,108.3,96.4,43.9,16.0,12.2.HRMS(ESI +):m/z calcd C 21H 23N 2O 3 +for[M+H] +351.1703,found:351.1741.
染料2b(262.1毫克)由化合物2(1毫摩尔，357.21毫克)和化合物1b(1毫摩尔，214.1毫克)制备得到，淡黄色固体产率72.1％。染料2b的表征：mp 180.5–185.0℃.IR ν(KBr,cm -1):3299,3055,2976,2936,1711,1620,1609,1523,1406,1316,1213,1165,935,793,669. 1H NMR(400MHz,CDCl 3):δ(ppm)7.80(d,J＝7.9Hz,2H,2×Ar-H),7.46(d,J＝8.9Hz,1H,Ar-H),7.36(d,J＝7.9Hz,2H,2×Ar-H),6.63(d,J＝10.2Hz,1H,Ar-H),6.55(s,1H,Ar-H),6.29(s,1H,N-H),3.44(q,J＝7.1Hz,4H,2×CH 2),3.02(d,J＝4.63Hz,3H,CH 3),2.23(s,3H,CH 3),1.23(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,CDCl 3):δ(ppm)168.1,161.8,155.1,150.4,148.9,138.5,133.8,130.8,126.8,126.2,120.0,109.3,108.7,97.4,44.7,26.8,16.3,12.4.HRMS(ESI +): m/z calcd C 22H 25N 2O 3 +for[M+H] +365.1860,found:365.1992.
染料2c(302.6毫克)由化合物2(1毫摩尔，357.21毫克)和化合物1c(1毫摩尔，228.0毫克)制备得到，黄色固体产率80.0％。染料2c的表征:mp 175.5–180.0℃.IR ν(KBr,cm -1):2971,2928,1702,1628,1617,1508,1442,1357,1213,1164,1080,983,918,876,782,679,632. 1H NMR(400MHz,CDCl 3):δ(ppm)7.49(d,J＝7.5Hz,2H,2×Ar-H),7.46(d,J＝9.1Hz,1H,Ar-H),7.35(d,J＝7.4Hz,2H,2×Ar-H),6.63(d,J＝8.9Hz,1H,Ar-H),6.56(s,1H,Ar-H),3.43(q,J＝7.0Hz,4H,2×CH 2),3.14(s,3H,CH 3),3.05(s,3H,CH 3),2.23(s,3H,CH 3),1.23(t,J＝6.7Hz,6H,2×CH 3). 13C NMR(151MHz,CDCl 3):δ(ppm)171.5,161.8,155.1,150.4,148.9,136.8,135.4,130.6,127.0,126.1,120.1,109.3,108.7,97.4,44.7,39.7,35.4,16.3,12.4.HRMS(ESI +):m/z calcd C 23H 27N 2O 3 +for[M+H] +379.2016,found:379.2020.
染料2d(307.7毫克)由化合物2(1毫摩尔，357.21毫克)和化合物1d(1毫摩尔，212.0毫克)制备得到，黄色固体产率85.0％。染料2d的表征:mp 168.5–173.0℃.IR ν(KBr,cm -1):3143,2975,2916,1701,1617,1584,1522,1409,1354,1263,1211,1162,990,867,798,689. 1H NMR(400MHz,CDCl 3):δ(ppm)8.39(s,1H,N-H),7.46(d,J＝8.9Hz,1H,Ar-H),7.18(s,1H,Ar-H),7.13(d,J＝7.8Hz,1H,Ar-H),6.93(d,J＝7.1Hz,1H,Ar-H),6.63(d,J＝7.8Hz,1H,Ar-H),6.55(s,1H,Ar-H),3.59(s,2H,CH 2),3.44(q,J＝7.1Hz,4H,2×CH 2),2.25(s,3H,CH 3),1.22(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,CDCl 3):δ(ppm)177.4,162.3,154.9,150.1,148.4,141.8,130.1,129.3,126.7,126.0,125.2,120.5,109.4,109.3,108.5,97.3,44.6,36.1,16.2,12.3.HRMS(ESI +):m/z calcd C 22H 23N 2O 3 +for[M+H] +363.1703,found:363.1700.
染料2e(308.3毫克)由化合物2(1毫摩尔，357.21毫克)和化合物1e(1毫摩尔，226.0毫克)制备得到，黄色固体产率82.0％。染料2e的表征:mp 173.5–178.0℃.IR ν(KBr,cm -1):3177,3041,2967,1703,1674,1618,1545,1443,1356,1282,1183,1145,1006,949,904,865,738,697. 1H NMR(400MHz,CDCl 3):δ(ppm)8.79(s,1H,N-H),7.46(d,J＝8.9Hz,1H,Ar-H),7.13(s,1H,Ar-H),7.09(d,J＝8.0Hz,1H,Ar-H),6.87(d,J＝7.8Hz,1H,Ar-H),6.63(d,J＝7.8Hz,1H,Ar-H),6.55(s,1H,Ar-H),3.42(q,J＝7.1Hz,4H,2×CH 2),2.99(t,J＝7.1Hz,2H,CH 2),2.68(t,J＝7.2,2H,CH 2),2.25(s,3H,CH 3),1.22(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,CDCl 3):δ(ppm)171.6,162.2,155.0,150.2,148.4,136.6,130.1,129.7,126.7,126.0,123.5,120.3,115.1,109.4,108.6,97.4,44.7,30.6,25.4,16.3,12.4.HRMS(ESI +):m/z calcd C 23H 25N 2O 3 +for[M+H] +377.1860,found:377.1967.
实施例二
染料3a-e合成的一般步骤如下:
取化合物3(0.5毫摩尔，222.1毫克)、酰胺化合物1a-e中的一种(0.6毫摩尔)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(Pd(dppf)Cl 2，0.015毫摩尔，12毫克)和醋酸钾(AcOK，1.5毫摩尔，334.5毫克)溶解于15.0毫升1,4-二氧六环(1,4-dioxane)溶剂中；然后反应体系通过氮气置换三次，95℃下加热10-12小时，TLC检测反应进度；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，经柱层析(洗脱剂：二氯甲烷/甲醇(50/1，v/v))分离后得到纯净染料。
具体地，各产物具体合成原料及产率、结构表征结果分别如下：
染料3a(65.5毫克)由化合物3(0.5毫摩尔，222.1毫克)和化合物1a(0.6毫摩尔，120.0毫克)制备得到，墨绿色固体产率30.0％。染料3a的表征:mp 212.5–215.0℃.IR ν(KBr,cm -1):3327,3144,2965,2923,1701,1682,1636,1619,1520,1409,1323,1230,1147,1009,946,845,774,669,644. 1H NMR(400MHz,DMSO-d 6):δ(ppm)8.61(d,J＝8.3Hz,1H,Ar-H),8.38(s,1H,Ar-H),8.16(d,J＝8.9Hz,1H,Ar-H),8.06(s,1H,Ar-H),8.0(d,J＝8.1Hz,2H,2×Ar-H),7.88(d,J＝7.9Hz,2H,2×Ar-H),7.63(d,J＝8.9Hz,1H,Ar-H),7.41(s,1H,Ar-H),6.85(d,J＝7.4Hz,1H,Ar-H),6.67(s,1H,N-H),6.32(s,1H,N-H),3.48(q,J＝7.0Hz,4H,2×CH 2),1.14(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,DMSO-d 6):δ(ppm)181.7,166.7,159.5 151.2,149.9,145.8,138.6,133.3,132.8,131.1,130.2,129.4,125.6,124.8,123.9,123.2,109.8,109.7,104.4,95.9,44.2,12.3.HRMS(ESI +):m/z calcd C 27H 24N 3O 3 +for[M+H] +438.1812,found:438.1825.
染料3b(85.7毫克)由化合物3(0.5毫摩尔，222.1毫克)和化合物1b(0.6毫摩尔，128.0毫克)制备得到，黑色固体产率38.0％。染料3b的表征:mp 198.5–203.0℃.IR ν(KBr,cm -1):3295,2970,1639,1578,1563,1548,1463,1372,1278,1181,1076,950,866,768,689. 1H NMR(400MHz,DMSO-d 6):δ(ppm)8.70(d,J＝8.3Hz,1H,Ar-H),8.59(s,1H,Ar-H),7.96(d,J＝8.3Hz,1H,Ar-H),7.88(d,2H,J＝7.6Hz,2×Ar-H),7.82(d,J＝8.0Hz,2H,2×Ar-H),7.65(d,J＝8.9Hz,1H,Ar-H),6.73(d,J＝8.9Hz,1H,Ar-H),6.53(s,1H,Ar-H),6.21(s,1H,N-H),5.47(d,J＝8.9Hz,1H,Ar-H),3.52(q,J＝7.0Hz,4H,2×CH 2),3.05(d,J＝3.7Hz,CH 3),1.28(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,TFA-d):δ(ppm)170.5,150.7,148.9 143.0,140.1,135.7,134.6,132.2,130.5,130.4,127.8,127.6,125.6,124.5,122.5,119.2,117.4,115.5,113.6,111.7,100.1,97.3,47.8,29.7,27.5,12.4.HRMS(ESI +):m/z calcd C 28H 26N 3O 3 +for[M+H] +452.1969,found:452.2001.
染料3c(151.1毫克)由化合物3(0.5毫摩尔，222.1毫克)和化合物1c(0.6毫摩尔，130.8毫克)制备得到，黑色固体产率65.0％。染料3c的表征:mp 200.0–205.5℃.IR ν(KBr,cm -1):2965,2925,1623,1579,1518,1406,1324,1280,1146,1078,1000,950,875,799,726,680,634. 1H NMR(400MHz,CDCl 3):δ(ppm)8.70(d,J＝8.3Hz,1H,Ar-H),8.55(s,1H,Ar-H),7.95(d,J＝8.2Hz,1H,Ar-H),7.80(d,J＝7.4Hz,2H,2×Ar-H),7.61(d,J＝8.9Hz,1H,Ar-H),7.55(d,J＝7.8Hz,2H,2×Ar-H),6.67(d,J＝8.7Hz,1H,Ar-H),6.46(s,1H,Ar-H),6.41(s,1H,Ar-H),3.45(q,J＝7.1Hz,4H,2×CH 2),3.15(s,3H,CH 3),3.05(s,3H,CH 3),1.26(t,J＝6.9Hz,6H,2×CH 3). 13C NMR(151MHz,DMSO-d 6):δ(ppm)183.4,171.3,152.2,150.8,146.7,141.3,141.1,139.3,135.6,132.0,131.1,129.6,127.8,127.1,125.1,124.5,123.9,109.8,105.7,96.2,45.1,39.6,35.4,12.6.HRMS(ESI +):m/z calcd C 29H 28N 3O 3 +for[M+H] +466.2125,found:466.2123.
染料3d(75.3毫克)由化合物3(0.5毫摩尔，222.1毫克)和化合物1d(0.6毫摩尔，127.2毫克)制备得到，黑色固体产率32.0％。染料3d的表征:mp 220.5–225.0℃.IR ν(KBr,cm -1):3252,2971,1713,1641,1619,1520,1467,1375,1353,1271,1177,1094,999,887,842,828,804,719,692,674,647,636. 1H NMR(400MHz,DMSO-d 6):δ(ppm)10.52(s,1H,N-H),8.56(d,J＝7.9Hz,1H,Ar-H),8.28(s,1H,Ar-H),8.05(d,J＝8.7Hz,1H,Ar-H),7.66(s,1H,Ar-H),7.62(d,J＝8.4Hz,2H,2×Ar-H),6.93(d,J＝7.9Hz,1H,Ar-H),6.83(d,J＝7.8Hz,1H,Ar-H),6.66(s,1H,Ar-H),6.30(s,1H,Ar-H),3.55(s,2H,CH 2),3.47(q,J＝7.0Hz,4H,2×CH 2),1.13(t,J＝6.8Hz,6H,2×CH 3). 13C NMR(151MHz,TFA-d):δ(ppm)180.2,171.5,150.8,143.5,141.1,135.9,135.7,133.9,131.4,129.8,128.0,126.0,125.3,123.7,123.0,119.6,115.3,114.7,113.5,112.8,112.0,50.6,10.8.HRMS(ESI +):m/z calcd C 28H 24N 3O 3 +for[M+H] +450.1812,found:450.1833.
染料3e(97.2毫克)由化合物3(0.5毫摩尔，222.1毫克)和化合物1e(0.6毫摩尔，135.6毫克)制备得到，黑色固体产率42.0％。染料3e的表征:mp 230.0–235.0℃.IR ν(KBr,cm -1):3463,3213,2974,1656,1639,1620,1575,1488,1399,1354,1275,1178,1077,998,949,847,806,739,669,649. 1H NMR(400MHz,DMSO-d 6):δ(ppm)10.25(s,1H,N-H),8.59(d,J＝8.7Hz,1H,Ar-H),8.34(s,1H,Ar-H),8.10(d,J＝8.1Hz,1H,Ar-H),7.66(s,1H,Ar-H),7.62(d,J＝8.2Hz,2H,2×Ar-H),7.00(d,J＝7.9Hz,1H,Ar-H),6.86(d,J＝7.0Hz,1H,Ar-H),6.70(s,1H,Ar-H),6.34(s,1H,Ar-H),3.52(q,J＝7.0Hz,4H,2×CH 2),3.00(t,J＝6.9Hz,2H,CH 2),1.23(t,J＝7.1Hz,2H,CH 2),1.17(t,J＝6.9Hz,6H,2×CH 3). 13C NMR(151MHz,TFA-d):δ (ppm)177.0,150.7,147.6,142.9,136.8,135.7,134.53,133.8,131.2,129.8,127.1,127.0,125.2,124.5,122.9,119.6,117.4,115.4,114.7,113.5,112.8,102.2,50.5,28.9,23.7,10.8.HRMS(ESI +):m/z calcd C 29H 26N 3O 3 +for[M+H] +464.1969,found:464.1920.
实施例三
对上述制备的染料2a-2e、3a-e(浓度为10μM)进行光稳定性测试，首先称取相应质量的染料2a-2e、3a-e和参照物香豆素和尼罗红，将其分别溶解在乙腈中(浓度为10μM)，用飞利浦碘钨灯(500W)照射所有的样品，灯与样品间的距离设为25cm。在灯和样品之间放置一个8cm厚的NaNO 2(60g.L -1)冷阱，以消除热量和短波长光。连续照射6小时，其中每半小时进行一次紫外荧光测试，六小时后，光稳定性根据照射前后不同时间吸收强度的变化来计算剩余吸收率。如图2和图3所示，连续照射6小时香豆素剩余吸收为94％，尼罗红相对剩余吸收为97％。染料2a-e，3a-e的剩余吸收分别是2a：93％，2b：92％，2c：95％，2d：91％，2e：97％，3a：93％，3b：92％，3c：94％，3d：93％，3e：92％，可以看出染料2a-2e和3a-e有着较高的光稳定性。
实施例四
对上述制备的染料(浓度为10μM)在氯仿中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4至13所示。
在紫外-可见吸收光谱图中，染料2a在388nm处有最大吸收；在荧光光谱图中，染料2a在457nm处有最高的荧光强度，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2b的最大吸收波长为386nm；在荧光光谱图中，染料2b的最大发射波长为459nm，此时的激发波长为385nm，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385nm；在荧光光谱图中，染料2c的最大发射波长为455nm，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2d在380nm处有最大吸收；在荧光光谱图中，染料2d在457nm处有最高的荧光强度，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2e的最大吸收波长为385nm；在荧光光谱图中，染料2e的最大发射波长为458nm，此时的激发波长为385nm，狭缝宽度为3nm/1.5nm。在紫外-可见吸收光谱图中，染料3a在554nm处有最大吸收；在荧光光谱图中，染料3a在604nm处有最高的荧光强度，此时的激发波长为550nm，狭缝宽度为1.5nm/3nm。在紫外-可见吸收光谱图中，染料3b的最大吸收波长为552nm；在荧光光谱图中，染料3b的最大发射波长为597nm，此时的激发波长为550nm，狭缝宽度为1.5nm/3nm。在紫外-可见吸收光谱图中，染料3c的最大吸收波长为 552nm；在荧光光谱图中，染料3c的最大发射波长为621nm，此时的激发波长为550nm，狭缝宽度为1.5nm/3nm。在紫外-可见吸收光谱图中，染料3d的最大吸收波长为551nm；在荧光光谱图中，染料3d的最大发射波长为607nm，此时的激发波长为545nm，狭缝宽度为1.5nm/3nm。在紫外-可见吸收光谱图中，染料3e的最大吸收波长为553nm；在荧光光谱图中，染料3e的最大发射波长为585nm，此时的激发波长为550nm，狭缝宽度为1.5nm/3nm。以上紫外吸收和荧光发射测试方法为常规方法。
实施例五
为了测试化合物2和3在化学连接酰胺衍生物前的荧光标记能力，使用DMSO(二甲基亚砜)将化合物2和化合物3配制成母液，随后加入常规细胞培养基中，使得化合物2和化合物3在细胞培养基中的浓度为2μM，再与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，绿光通道使用488nm激发，收集500～550nm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。结果表明化合物2在HeLa细胞中对脂滴进行了染色。结果如图14所示，其中(a)为明场，(b)为染料2的细胞成像图，(c)为脂滴绿色色标记物的细胞成像图，(d)为蓝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，(f)为共定位实验，他们的共定位系数为0.78。化合物3在HeLa也具有脂滴标记能力，结果如图20所示，其中(a)为明场，(b)为化合物3的细胞成像图，(c)为脂滴绿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，(f)为共定位实验，他们的共定位系数为0.86
为了测试化合物2a与商用线粒体标记物相比的荧光标记能力，使用DMSO(二甲基亚砜)将染料2a配制成母液，随后加入常规细胞培养基中，使得染料2a在细胞培养基中的浓度为2μM，再与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，随后分别加入线粒体红色标记物Mito
Figure PCTCN2021075159-appb-000004
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。结果如图15所示，其中(a)为明场，(b)为染料2a的细胞成像图，(c)为线粒体红色标记物的细胞成像图，(d)为蓝光通道和红光通道的叠加图，(e)为叠加图中ROI线的荧光强度，(f)为共定位实验，他们的共定位系数为0.88。结果表明，染料2a的荧光图像与商用线粒体红色标记物Mito
Figure PCTCN2021075159-appb-000005
Red CMXRos的荧光图像在线粒体中的分布情况一致，且强度相近，表明染料2a在HeLa细胞中具有线粒体标记能力，可作为线粒体蓝色标记物。
染料2b(2μM)、染料2c(2μM)、染料2d(2μM)、染料2e(2μM)的实验方法与上述染料2a一样，仅将染料2a更换即可，其余不变。如图16所示，其中(a)为明场，(b)为染料2b的细胞成像图，(c)为线粒体红色标记物的细胞成像图，(d)为蓝光通道和红光通道的叠加图，(e)为叠加图中ROI线的荧光强度，(f)为共定位实验，他们的共定位系数分别为0.94。如图17所示，其中(a)为明场，(b)为染料2c的细胞成像图，(c)为线粒体红色标记物的细胞成像图，(d)为蓝光通道和红光通道的叠加图，(e)为叠加图中ROI线的荧光强度，(f)为共定位实验，他们的共定位系数分别为0.88。如图18所示，其中(a)为明场，(b)为染料2d的细胞成像图，(c)为线粒体红色标记物的细胞成像图，(d)为蓝光通道和红光通道的叠加图，(e)为叠加图中ROI线的荧光强度，(f)为共定位实验，他们的共定位系数分别为0.92。如图19所示，其中(a)为明场，(b)为染料2e的细胞成像图，(c)为线粒体红色标记物的细胞成像图，(d)为蓝光通道和红光通道的叠加图，(e)为叠加图中ROI线的荧光强度，(f)为共定位实验，他们的共定位系数分别为0.91。结果表明染料2b-e的荧光图像与商用线粒体红色标记物Mito
Figure PCTCN2021075159-appb-000006
Red CMXRos的荧光图像在线粒体中的分布情况一致，且强度相近，表明染料2b、染料2c、染料2d、染料2e在HeLa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2μM，与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，随后分别加入线粒体绿色标记物Mito
Figure PCTCN2021075159-appb-000007
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。结果如图21所示，其中(a)为明场，(b)为染料3a的细胞成像图，(c)为线粒体绿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，(f)为共定位实验，他们的共定位系数为0.94。结果表明，染料3a的荧光图像与商用线粒体绿色标记物Mito
Figure PCTCN2021075159-appb-000008
Green FM的荧光图像在线粒体中的分布情况一致，且强度相近，表明染料3a在HeLa细胞中具有线粒体标记能力，可作为线粒体红色标记物。
染料3b(2μM)、染料3c(2μM)、染料3d(2μM)、染料3e(2μM)的实验方法与上述染料3a一样，仅将染料3a更换即可，其余不变。如图22所示，其中(a)为明场，(b)为染料3b的细胞成像图，(c)为线粒体绿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，(f)为共定位实验，他们的共定位系数为0.88。如图23所示，其中(a)为明场，(b)为染料3c的细胞成像图，(c)为线粒体绿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，(f)为共定位实验， 他们的共定位系数为0.94。如图24所示，其中(a)为明场，(b)为染料3d的细胞成像图，(c)为线粒体绿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，(f)为共定位实验，他们的共定位系数为0.96。如图25所示，其中(a)为明场，(b)为染料3e的细胞成像图，(c)为线粒体绿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，(f)为共定位实验，他们的共定位系数为0.92。结果表明，染料3b-e的荧光图像与商用线粒体绿色标记物Mito
Figure PCTCN2021075159-appb-000009
Green FM的荧光图像在线粒体中的分布情况一致，且强度相近，表明染料3b、染料3c、染料3d、染料3e在HeLa细胞中具有线粒体标记能力，可作为线粒体红色标记物。
实施例六
此外，本发明还测试染料2a-e或3a-e的细胞毒性。通过使用CCK-8方法测量在这些染料存在下HeLa细胞的活力。将HeLa细胞分别与不同浓度的染料(2、4、6、8和10μM)孵育6小时。如图26所示，图26a为染料2a-e的细胞毒性测试结果，图26b为染料3a-e的细胞毒性测试结果。结果表明它们具有良好的细胞生存力并且适合活细胞成像。而且，HeLa细胞与染料3a-c孵育6小时后存活率超过100％，这表明染料3a-c不仅无毒，而且可以促进细胞生长。
图26中，细胞存活率(％)＝(A sample–A b)/(A c–A b)，其中A c：阴性对照(包括培养基和细胞，无待测染料添加)，A b：空白(包括待测染料和培养基，无细胞添加)，A sample：测试组(包括培养基、细胞和待测染料)。
实施例七
众所周知，作为生物标志物，具有优异的抗光漂白性能是非常重要的。因此，选择染料2a(5μM)和3a(5μM)作为代表，以在活细胞中进行光漂白研究。如图27所示，图27(a)为明场图像；图27(b-h)依次为含染料2a的HeLa细胞中用激发光源(561nm)连续照射1、5、10、15、20、25、30分钟之后的荧光图像。如图28所示，图28(a)为明场图像；图28(b-h)依次为含染料3a的HHeLa细胞中被激发光源(405nm)连续照射1、5、10、15、20、25、30分钟后的的荧光图像。结果表明，连续照射1、5、10、15、20、25、30分钟之后，染料2a和3a仍然可进行线粒体成像，荧光强度仅稍微降低。
可以看出，本发明首次公开了酰胺衍生物中性线粒体荧光标记物与细胞共培养后，可以实现细胞中线粒体成像。本发明在改善染料的光学性能的同时，通过对其结构创造性的修饰来调控染料的细胞器靶向能力，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，而且不需要对细胞进行通透处理，也不需 要固定细胞，可以对细胞实时监测，不受其他细胞器的影响，而且实验结果说明本发明染料对线粒体具有优异的靶向性。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 一种酰胺衍生物中性线粒体荧光标记物，其特征在于：所述述酰胺衍生物的中性线粒体荧光标记物为式(I)-(IV)之一所示：
    Figure PCTCN2021075159-appb-100001
    其中，R 1、R 2分别独立地选自氢或碳原子数为1～6的烷基；M、E 1、E 2分别独立地选自碳原子数为1～6的烷基；n为1～3中任一整数。
  2. 根据权利要求1所述的酰胺衍生物中性线粒体荧光标记物，其特征在于：R 1、R 2分别独立地选自氢或甲基；M为甲基；E 1和E 2均为乙基；n为1或2。
  3. 一种权利要求1或2所述的酰胺衍生物中性线粒体荧光标记物的制备方法，其特征在于，包括以下步骤：
    在弱碱性条件和有机溶剂中，利用式(2)所示的化合物与式(1a-c)或式(1d-e)所示的化合物反应得到式(I)或式(II)所示的酰胺衍生物中性线粒体荧光标记物；或者
    在弱碱性条件和有机溶剂中，利用式(3)所示的化合物与式(1a-c)或式(1d-e)所示的化合物反应得到式(III)或式(IV)所示的酰胺衍生物中性线粒体荧光标记物；
    其中式(1a-c)、(1d-e)、(2)或(3)结构式分别如下：
    Figure PCTCN2021075159-appb-100002
    Figure PCTCN2021075159-appb-100003
  4. 根据权利要求3所述的制备方法，其特征在于：反应均在贵金属盐催化剂存在下进行。
  5. 根据权利要求4所述的制备方法，其特征在于：所述贵金属盐催化剂包括钯盐催化剂。
  6. 根据权利要求3所述的制备方法，其特征在于：反应温度为85-110℃。
  7. 权利要求1或2所述的酰胺衍生物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。
  8. 根据权利要求7所述的应用，其特征在于：所述线粒体荧光标记试剂靶向癌细胞的线粒体。
  9. 根据权利要求7所述的应用，其特征在于，利用所述线粒体荧光标记试剂进行细胞成像的方法包括以下步骤：
    将所述线粒体荧光标记试剂与细胞在37℃及5％CO 2条件下共培养10min以上，然后利用激光共聚焦显微镜进行细胞成像，收集荧光信号。
  10. 根据权利要求9所述的应用，其特征在于：当所述线粒体荧光标记试剂包括式(I)或式(II)所示的酰胺衍生物中性线粒体荧光标记物时，使用405nm光源激发，收集410～500nm范围内的荧光信号；
    当所述线粒体荧光标记试剂包括式(III)或式(IV)所示的酰胺衍生物中性线粒体荧光标记物时，使用561nm光源激发，收集570～750nm范围内的荧光信号。
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