WO2023087503A1 - Preparation of novel isophorone derivatives and use thereof in fields of organelle labeling and viscosity detection - Google Patents

Preparation of novel isophorone derivatives and use thereof in fields of organelle labeling and viscosity detection Download PDF

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WO2023087503A1
WO2023087503A1 PCT/CN2021/143128 CN2021143128W WO2023087503A1 WO 2023087503 A1 WO2023087503 A1 WO 2023087503A1 CN 2021143128 W CN2021143128 W CN 2021143128W WO 2023087503 A1 WO2023087503 A1 WO 2023087503A1
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isophorone
dye
derivatives
fluorescence
imaging
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葛健锋
喻情
倪靖阳
孙如
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苏州大学
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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Definitions

  • the invention belongs to the technical field of fluorescent labeling, in particular to the preparation of novel isophorone derivatives and their application in the fields of cell organelle labeling and viscosity detection.
  • a variety of metabolic diseases such as obesity, fatty liver, cardiovascular disease and diabetes, and neutral lipid storage diseases are often accompanied by abnormal lipid droplets (see: ACS Sens. 2021, 6, (1), 22–26 ).
  • a suitable viscosity environment is an important factor to maintain the smooth operation of each organelle, and the viscosity change of an organelle is also considered to be an important indicator of its working state.
  • the present invention provides the preparation of novel isophorone derivatives and their application in the fields of organelle labeling and viscosity detection.
  • a kind of isophorone derivative, described derivative structural formula is:
  • R is selected from the following structural formulas (1)-(4):
  • the preparation method of the isophorone derivative described in claim 1, comprises the following steps:
  • compound M is 1H-indazole-5-carbaldehyde, 4-(4-hydroxypiperidin-1-yl)benzaldehyde, 4-hydroxy-1-naphthaldehyde or 1-pyrenecarbaldehyde.
  • the molar ratio of (3,5,5-trimethylcyclohex-2-enylidene)malononitrile to compound M is 1:0.7 ⁇ 1:2.0.
  • the organic solvent is ethanol or DMF.
  • the catalyst includes a basic catalyst and an acidic catalyst for aldol condensation
  • the acidic catalyst is piperidine.
  • the basic catalyst is trimethylchlorosilane.
  • the organelle is lysosome, mitochondria, endoplasmic reticulum or lipid droplet.
  • the present invention also provides a method for non-diagnostic and non-therapeutic cell fluorescence imaging, comprising the following steps: co-cultivating cells with the isophorone derivative in a culture medium, and then performing cell imaging to complete cell fluorescence imaging .
  • cells and medium are conventional products, which have no influence on the technical effect of the invention.
  • the concentration of the isophorone derivative in the culture medium is 10-20 ⁇ M.
  • the present invention also provides a method for non-diagnostic and non-therapeutic cell fluorescence imaging, comprising the following steps: co-cultivating cells with the isophorone derivatives and ionophores in a medium, and then performing cell imaging to complete cell imaging.
  • the isophorone derivatives can be used for fluorescent labeling of different organelles.
  • fluorescent markers for mitochondria, lysosomes, endoplasmic reticulum, and lipid droplets can be added to the culture medium respectively to obtain Mitochondria, lysosomes, endoplasmic reticulum, lipid droplet fluorescent markers and ionophores (monensin, dexamethasone, nystatin) can be added to the medium respectively to obtain substances and ionophores; preferably, in the medium containing mitochondria, lysosomes, endoplasmic reticulum, and lipid droplet fluorescent markers, the concentrations of the fluorescent markers in the medium are 10 ⁇ M, 14 ⁇ M, and 10 ⁇ M, respectively , 20 ⁇ M; in the medium containing the fluorescent marker and the ionophore, the concentration of the fluorescent marker in the medium is 2 ⁇ M, and the concentration of the ionophore is 10 ⁇ M.
  • cell imaging can be performed using a laser confocal microscope.
  • the green light channel can be excited at a wavelength of 488nm to collect fluorescent signals in the wavelength range of 468-550nm
  • the red light channel can be excited at a wavelength of 561nm to collect 570- Fluorescent signal in the wavelength range of 800nm
  • cell imaging results show that the four isophorone derivative fluorescent dyes contained in the present invention can well label mitochondria, lysosomes, endoplasmic reticulum or lipid droplets respectively, and can be used as mitochondrial green markers Red markers for lysosomes, red markers for endoplasmic reticulum, red markers for lipid droplets.
  • the fluorescence of the fluorescent dyes in their corresponding organelles was significantly enhanced.
  • the co-cultivation is at saturated humidity, 37° C., and 5% CO 2 incubator for 5 minutes; After incubation for 5 min, cells were imaged using confocal microscopy after washing with PBS buffer.
  • the ionophore is monensin, dexamethasone or nystatin.
  • the concentration of the isophorone derivative in the culture medium is 1-2 ⁇ M; the concentration of the ionophore is 5-20 ⁇ M.
  • the invention not only has the ability to fluorescently mark organelles at high concentrations, but also has the ability to detect the viscosity of organelles through fluorescence intensity at low concentrations.
  • the present invention also provides the application of the isophorone derivative in detecting viscosity.
  • the organelle viscosity is detected by the fluorescence intensity of the isophorone derivative.
  • the invention discloses a series of isophorone derivative fluorescent dyes, which not only have different cell organelle targeting capabilities, but also can detect viscosity at the same time. Due to the strong intramolecular charge transfer (ICT) effect, most of these four neutral fluorescent dyes are still able to reach the near-infrared (NIR) light region. Thanks to the neutral fluorochrome, cytotoxicity is significantly reduced compared to salt-based fluorochromes.
  • the invention firstly solves the problem of unclear targeting ability of the isophorone derivative fluorescent dye to the organelle, and can monitor the viscosity at the same time.
  • the present invention uses cheap building blocks to participate in the synthesis, which makes it possible to design viscosity probes with different organelle targeting capabilities using a single basic skeleton structure, and significantly reduces the cost and difficulty, which has important scientific significance and commercial value.
  • the invention discloses four fluorescent dyes derived from isophorone, which can mark different organelles respectively after being co-cultured with cells; at the same time, the four fluorescent dyes are all sensitive to viscosity.
  • the fluorescence of the fluorescent dye in the corresponding organelle is significantly enhanced, and the viscosity of the corresponding organelle can be detected.
  • the invention uses a basic structure to improve the design, participate in the synthesis through different cheap building blocks, improve the optical properties and biological activities of fluorescent markers, and can regulate the ability of organelle targeting, which has important scientific significance and commercial value .
  • Fig. 1 is the synthetic route of the dyestuff designed by the present invention.
  • Fig. 2 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dyes 1a-1d of the present invention in glycerol and dimethyl sulfoxide respectively.
  • (a) is the UV-visible absorption spectrum and fluorescence spectrum of 1a in glycerol
  • (b) is the UV-visible absorption spectrum and fluorescence spectrum of 1a in dimethyl sulfoxide
  • (c) is the UV-visible absorption spectrum and fluorescence spectrum of 1b in glycerol UV-visible absorption spectrum and fluorescence spectrum
  • (d) is the UV-visible absorption spectrum and fluorescence spectrum of 1b in dimethyl sulfoxide
  • (e) is the UV-visible absorption spectrum and fluorescence spectrum of 1c in glycerin
  • ( f) is the UV-visible absorption spectrum and fluorescence spectrum of 1c in dimethyl sulfoxide
  • (g) is the UV-visible absorption spectrum and fluorescence spectrum of 1d in glycerol
  • Fig. 3 is a cell imaging diagram of dyes 1a-1d of the present invention in HeLa cells respectively.
  • (a1-a6) is the cell imaging figure of 1a in HeLa cells
  • (b1-b6) is the cell imaging figure of 1b in HeLa cells
  • (c1-c6) is the cell imaging figure of 1c in HeLa cells
  • ( d1-d6) are cell imaging images of 1d in HeLa cells.
  • Fig. 4 is a cell imaging diagram of dyes 1a-1d of the present invention before and after adding ionophores to HeLa cells.
  • A is the cell imaging image of 1a before and after adding ionophore in HeLa cells
  • B is the cell imaging image of 1b before and after adding ionophore in HeLa cells
  • C is the cell imaging image of 1c before and after adding ionophore in HeLa cells
  • D is the cell imaging diagram of 1d before and after adding ionophores in HeLa cells.
  • the dyestuff (concentration is 10 ⁇ M) prepared in embodiment 1-embodiment 4 is tested its ultraviolet absorption and fluorescence emission spectrum in glycerol and dimethyl sulfoxide, and abscissa is wavelength, and ordinate is respectively absorbance or fluorescence intensity, result Figure 2.
  • the dye 1a In the ultraviolet-visible absorption spectrum, the dye 1a has the maximum absorption at 424nm; in the fluorescence emission spectrum, the dye 1a has the highest fluorescence intensity at 565nm and is the highest in glycerol, at this time the excitation wavelength is 424nm, and the slit width is 5nm/5nm, see Figure 2-(a)(b) respectively.
  • the dye 1b In the ultraviolet-visible absorption spectrum, the dye 1b has the maximum absorption at 493nm; in the fluorescence emission spectrum, the dye 1b has the highest fluorescence intensity at 685nm and is the highest in glycerin, at this time the excitation wavelength is 545nm, and the slit width is 10nm/1.5nm, see Figure 2-(c)(d) respectively.
  • the dye 1c In the ultraviolet-visible absorption spectrum, the dye 1c has the maximum absorption at 473nm; in the fluorescence emission spectrum, the dye 1c has the highest fluorescence intensity at 659nm and is the highest in glycerol. At this time, the excitation wavelength is 506nm, and the slit width is 10nm/3nm, see Figure 2-(e)(f) respectively.
  • the dye 1d In the ultraviolet-visible absorption spectrum, the dye 1d has the maximum absorption at 476nm; in the fluorescence emission spectrum, the dye 1d has the highest fluorescence intensity at 660nm and is the highest in glycerol, at this time the excitation wavelength is 507nm, and the slit width is 5nm/5nm, see Figure 2(g)(h) respectively.
  • DMSO dimethyl methoxide
  • the red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-750nm is collected.
  • Cell imaging shows that dye 1a can well label mitochondria in HeLa cells and can be used as a green mitochondrial marker.
  • (a1) is the bright field imaging image
  • (a2) is the cell imaging image of dye 1a
  • (a3) is the cell imaging image of the commercial mitochondrial red marker
  • (a4) is the green light channel and
  • the overlay of the red channel (a5) is the colocalization experiment, the colocalization coefficient is 0.92
  • (a6) is the fluorescence intensity of the ROI line in the overlay.
  • the concentration of dye 1b in the cell culture medium was 14 ⁇ M, and then the commercial lysosome green marker Lyso Tracker Green (2 ⁇ M) was added and incubated for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope.
  • the red light channel is excited with a wavelength of 561nm to collect fluorescent signals in the range of 600-750nm, and the green light channel is excited with a wavelength of 488nm to collect fluorescent signals in the range of 468-550nm.
  • Cell imaging shows that dye 1b can well label lysosomes in HeLa cells and can be used as a lysosomal red marker.
  • the concentration of dye 1c in the cell culture medium was 10 ⁇ M, and then the commercial endoplasmic reticulum green marker ER Tracker Green (6 ⁇ M) was added to incubate for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope.
  • the red light channel is excited with a wavelength of 561nm to collect fluorescent signals in the range of 600-750nm, and the green light channel is excited with a wavelength of 488nm to collect fluorescent signals in the range of 468-550nm.
  • Cell imaging shows that dye 1c can well label the endoplasmic reticulum in HeLa cells and can be used as a red marker of the endoplasmic reticulum.
  • the concentration of dye 1d in the cell culture medium was 20 ⁇ M, then the commercial lipid droplet green marker LDs Tracker Green (4 ⁇ M) was added and incubated for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope.
  • the red light channel is excited with a wavelength of 561nm to collect fluorescent signals in the range of 600-750nm, and the green light channel is excited with a wavelength of 488nm to collect fluorescent signals in the range of 468-550nm.
  • Cell imaging shows that the dye 1d can well label lipid droplets in HeLa cells and can be used as a red marker for lipid droplets.
  • the concentration of dye 1a in the cell culture medium was 2 ⁇ M, the concentration of the ionophore monensin was 10 ⁇ M, co-cultured for 30 minutes, washed three times with PBS buffer, and the cells were imaged by laser confocal microscope.
  • the green light channel is excited with a wavelength of 488nm, and the fluorescent signal in the range of 500-550nm is collected. Imaging of cells revealed that the fluorescent signal of dye 1a was significantly enhanced after mitochondrial viscosity was increased by addition of monensin.
  • the concentration of dye 1b in the cell culture medium was 2 ⁇ M, the concentration of the ionophore dexamethasone was 10 ⁇ M, co-incubated for 30 minutes, washed three times with PBS buffer, and the cells were imaged using a laser confocal microscope. The red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-800nm is collected. Imaging of cells revealed that the fluorescent signal of dye 1b was significantly enhanced after lysosomal viscosity was increased by adding dexamethasone.
  • the concentration of dye 1c in the cell culture medium was 2 ⁇ M, and the concentration of the ionophore monensin was 10 ⁇ M, co-incubated for 30 minutes, washed three times with PBS buffer, and the cells were imaged using a laser confocal microscope.
  • the red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-800nm is collected. Imaging of the cells revealed that the fluorescent signal of dye 1c was significantly enhanced after the viscosity of the ER was increased by adding monensin.
  • the concentration of the dye 1d in the cell culture medium was 2 ⁇ M, and the concentration of the ionophore nystatin was 10 ⁇ M, co-incubated for 30 minutes, washed three times with PBS buffer, and the cells were imaged using a laser confocal microscope.
  • the red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-800nm is collected. Imaging of cells revealed that the fluorescent signal of dye 1d was significantly enhanced after lipid droplet viscosity was increased by adding nystatin.

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Abstract

Compound (3,5,5-trimethylcyclohex-2-enylidene)malononitrile reacts with different building blocks containing aldehyde groups to obtain four isophorone derivatives. The derivatives can mark mitochondria, lysosomes, endoplasmic reticula or lipid droplets, are sensitive to viscosity, and are remarkably enhanced in fluorescence in a viscous environment. The derivatives can perform organelle targeting capability adjustment, so that it is possible to design fluorescent markers having different organelle targeting capabilities by using a single basic framework structure.

Description

新型异佛尔酮衍生物的制备及其在细胞器标记和粘度检测领域的应用Preparation of novel isophorone derivatives and their application in the field of organelle labeling and viscosity detection 技术领域technical field
本发明属于荧光标记技术领域，尤其是指新型异佛尔酮衍生物的制备及其在细胞器标记和粘度检测领域的应用。The invention belongs to the technical field of fluorescent labeling, in particular to the preparation of novel isophorone derivatives and their application in the fields of cell organelle labeling and viscosity detection.
背景技术Background technique
细胞内微环境(包括pH、温度、极性、粘度等)的变化会破坏细胞器的稳态和功能(参见:Chem.Sci.,2020,11,596–601)。特别的是，有研究已经证明线粒体粘度异常与神经退行性疾病、动脉硬化和糖尿病有关(参见:Chem.Commun.,2019,55,7410-7413)。溶酶体生理状态异常会导致溶酶体贮积病、肺结核、代谢紊乱、炎症，神经退行性疾病(参见:J.Mater.Chem.B,2018,6,6592-6598)。内质网出现紊乱很容易引起记忆力衰退，心脏病以及肾病(参见:J.Mater.Chem.B,2021,9,5664-5669)。多种代谢性疾病，如肥胖、脂肪肝、心血管疾病及糖尿病、中性脂贮存性疾病往往都伴随着脂滴的功能异常(参见:ACS Sens.2021,6,(1),22–26)。合适的粘度环境是维持各个细胞器平稳运行的重要因素，细胞器的粘度变化也被认为是反应其工作状态的重要指标。Changes in the intracellular microenvironment (including pH, temperature, polarity, viscosity, etc.) can disrupt organelle homeostasis and function (see: Chem. Sci., 2020, 11, 596–601). In particular, studies have demonstrated that abnormal mitochondrial viscosity is associated with neurodegenerative diseases, arteriosclerosis, and diabetes (see: Chem. Commun., 2019, 55, 7410-7413). Abnormal lysosomal physiological status can lead to lysosomal storage diseases, tuberculosis, metabolic disorders, inflammation, neurodegenerative diseases (see: J. Mater. Chem. B, 2018, 6, 6592-6598). Disorders of the endoplasmic reticulum can easily cause memory loss, heart disease and kidney disease (see: J. Mater. Chem. B, 2021, 9, 5664-5669). A variety of metabolic diseases, such as obesity, fatty liver, cardiovascular disease and diabetes, and neutral lipid storage diseases are often accompanied by abnormal lipid droplets (see: ACS Sens. 2021, 6, (1), 22–26 ). A suitable viscosity environment is an important factor to maintain the smooth operation of each organelle, and the viscosity change of an organelle is also considered to be an important indicator of its working state.
迄今为止，基于扭曲的分子内电荷转移(TICT)机理，已经有许多小分子粘度探针被开发出来，在粘性环境中，分子内旋转受到抑制，非辐射衰变减弱，荧光显著增强(参见:Anal.Chem.2020,92,(5),3517–3521)。但是，它们仍然有很多改进的空间。第一，许多粘度探针容易受到亲核试剂的干扰，选择性并不理想。第二，大多数粘度探针发射波长较短，无法达到近红外区域。几乎所有近红外粘度探针均为盐类化合物，虽然发射波长达到了近红外光区，但是盐类化合物的高毒性仍然无法很好地解决。第三，没有明确的细胞器靶向功能。目前报道具有明确细胞器靶向的粘度探针大多为阳离子粘度探针，它们大多进入线粒体，这是由于线粒体的负电荷膜电位，使得阳离子探针容 易进入线粒体，然而这不可避免地破坏了生物体的微环境(参见:Anal.Chem.2019,91,(13),8415–8421)。虽然粘度探针近些年发展迅速，但是关于其它细胞器粘度探针的报道仍然十分有限。To date, many small-molecule viscosity probes have been developed based on the twisted intramolecular charge transfer (TICT) mechanism, in which intramolecular rotation is suppressed, nonradiative decay is attenuated, and fluorescence is significantly enhanced in a viscous environment (see: Anal . Chem. 2020, 92, (5), 3517–3521). However, they still have a lot of room for improvement. First, many viscosity probes are susceptible to interference from nucleophiles and are not very selective. Second, most viscosity probes emit at shorter wavelengths and cannot reach the near-infrared region. Almost all near-infrared viscosity probes are salt compounds. Although the emission wavelength reaches the near-infrared region, the high toxicity of salt compounds still cannot be solved well. Third, there is no clear organelle-targeting function. The currently reported viscosity probes with clear organelle targeting are mostly cationic viscosity probes, and they mostly enter mitochondria, which is due to the negatively charged membrane potential of mitochondria, making it easy for cationic probes to enter mitochondria, however this inevitably damages the organism microenvironment (see: Anal. Chem. 2019, 91, (13), 8415–8421). Although viscosity probes have developed rapidly in recent years, reports on other organelle viscosity probes are still very limited.
发明内容Contents of the invention
为解决上述技术问题，本发明提供了新型异佛尔酮衍生物的制备及其在细胞器标记和粘度检测领域的应用。In order to solve the above technical problems, the present invention provides the preparation of novel isophorone derivatives and their application in the fields of organelle labeling and viscosity detection.
一种异佛尔酮衍生物，所述衍生物结构式为：A kind of isophorone derivative, described derivative structural formula is:
Figure PCTCN2021143128-appb-000001
Figure PCTCN2021143128-appb-000001
其中，R选自以下结构式(1)-(4)：Wherein, R is selected from the following structural formulas (1)-(4):
Figure PCTCN2021143128-appb-000002
Figure PCTCN2021143128-appb-000002
权利要求1所述的异佛尔酮衍生物的制备方法，包括以下步骤：The preparation method of the isophorone derivative described in claim 1, comprises the following steps:
取(3,5,5-三甲基环己-2-烯亚基)丙二腈与化合物M溶解于有机溶剂中，在催化剂作用下，在80℃-120℃反应12小时得到所述异佛尔酮衍生物；Dissolve (3,5,5-trimethylcyclohex-2-enylidene)malononitrile and compound M in an organic solvent, and react at 80°C-120°C for 12 hours under the action of a catalyst to obtain the iso Phorone derivatives;
其中，化合物M为1H-吲唑-5-甲醛、4-(4-羟基哌啶-1-基)苯甲醛、4-羟基-1-萘甲醛或1-芘甲醛。Wherein, compound M is 1H-indazole-5-carbaldehyde, 4-(4-hydroxypiperidin-1-yl)benzaldehyde, 4-hydroxy-1-naphthaldehyde or 1-pyrenecarbaldehyde.
在本发明的一个实施例中，所述(3,5,5-三甲基环己-2-烯亚基)丙二腈与化合物M的摩尔比为1:0.7～1:2.0。In one embodiment of the present invention, the molar ratio of (3,5,5-trimethylcyclohex-2-enylidene)malononitrile to compound M is 1:0.7˜1:2.0.
在本发明的一个实施例中，所述有机溶剂为乙醇或DMF。In one embodiment of the present invention, the organic solvent is ethanol or DMF.
在本发明的一个实施例中，所述催化剂包括羟醛缩合的碱性催化剂和酸性催化剂；In one embodiment of the invention, the catalyst includes a basic catalyst and an acidic catalyst for aldol condensation;
在本发明的一个实施例中，所述酸性催化剂为哌啶。In one embodiment of the present invention, the acidic catalyst is piperidine.
在本发明的一个实施例中，所述碱性催化剂为三甲基氯硅烷。In one embodiment of the present invention, the basic catalyst is trimethylchlorosilane.
所述的异佛尔酮衍生物在制备细胞器荧光试剂中的应用。Application of the isophorone derivatives in the preparation of organelle fluorescent reagents.
所述的异佛尔酮衍生物在非诊断治疗目的的细胞器荧光成像中的应用。The application of the isophorone derivatives in organelle fluorescence imaging for non-diagnostic and therapeutic purposes.
在本发明的一个实施例中，所述细胞器为溶酶体、线粒体、内质网或脂滴。In one embodiment of the present invention, the organelle is lysosome, mitochondria, endoplasmic reticulum or lipid droplet.
本发明还提供了一种非诊断非治疗目的的细胞荧光成像的方法，包括以下步骤：将细胞与含有所述异佛尔酮衍生物在培养基中共培养，再进行细胞成像，完成细胞荧光成像。本发明中，细胞、培养基都为常规产品，对发明技术效果没有影响。The present invention also provides a method for non-diagnostic and non-therapeutic cell fluorescence imaging, comprising the following steps: co-cultivating cells with the isophorone derivative in a culture medium, and then performing cell imaging to complete cell fluorescence imaging . In the present invention, cells and medium are conventional products, which have no influence on the technical effect of the invention.
在本发明的一个实施例中，所述所述异佛尔酮衍生物在培养基中的浓度为10～20μM。In one embodiment of the present invention, the concentration of the isophorone derivative in the culture medium is 10-20 μM.
本发明还提供了一种非诊断非治疗目的的细胞荧光成像的方法，包括以下步骤：将细胞与所述异佛尔酮衍生物和离子载体在培养基中共培养，再进行细胞成像，完成细胞荧光成像，所述异佛尔酮衍生物能进行不同细胞器的荧光标记。The present invention also provides a method for non-diagnostic and non-therapeutic cell fluorescence imaging, comprising the following steps: co-cultivating cells with the isophorone derivatives and ionophores in a medium, and then performing cell imaging to complete cell imaging. For fluorescence imaging, the isophorone derivatives can be used for fluorescent labeling of different organelles.
在本发明的一个实施例中，可以分别将线粒体、溶酶体、内质网、脂滴荧光标记物加入培养基中，分别得到含有线粒体、溶酶体、内质网、脂滴荧光标记物的培养基；可以分别将线粒体、溶酶体、内质网、脂滴荧光标记物和离子载体(莫能霉素、***、制霉菌素)加入培养基中，得到含有所述荧光标记物和离子载体的培养基；优选的，含有线粒体、溶酶体、内质网、脂滴荧光标记物的培养基中，所述荧光标记物在培养基中的浓度分别为10μM，14μM，10μM，20μM；含有所述荧光标记物和离子载体的培养基中，所述荧光标记物在培养基中的浓度均为2μM，离子载体的浓度均为10μM。In one embodiment of the present invention, fluorescent markers for mitochondria, lysosomes, endoplasmic reticulum, and lipid droplets can be added to the culture medium respectively to obtain Mitochondria, lysosomes, endoplasmic reticulum, lipid droplet fluorescent markers and ionophores (monensin, dexamethasone, nystatin) can be added to the medium respectively to obtain substances and ionophores; preferably, in the medium containing mitochondria, lysosomes, endoplasmic reticulum, and lipid droplet fluorescent markers, the concentrations of the fluorescent markers in the medium are 10 μM, 14 μM, and 10 μM, respectively , 20 μM; in the medium containing the fluorescent marker and the ionophore, the concentration of the fluorescent marker in the medium is 2 μM, and the concentration of the ionophore is 10 μM.
在本发明的一个实施例中，细胞成像可以利用激光共聚焦显微镜进行，比如绿光通道选用488nm波长激发，收集468-550nm波长范围内的荧光信号，红光通道可用561nm波长激发，收集570-800nm波长范围内的荧光信号；细胞成像结果显示本发明包含的四种异佛尔酮衍生物荧光染料分别能够很好地标记线粒体、溶酶体、内质网或脂滴，可作为线粒体绿色标记物，溶酶体红色标记物，内质网红色标记物，脂滴红色标记物。通过加入离子载体使细胞粘度增加后，所述荧光染料在其对应细胞器中荧光显著增强。In one embodiment of the present invention, cell imaging can be performed using a laser confocal microscope. For example, the green light channel can be excited at a wavelength of 488nm to collect fluorescent signals in the wavelength range of 468-550nm, and the red light channel can be excited at a wavelength of 561nm to collect 570- Fluorescent signal in the wavelength range of 800nm; cell imaging results show that the four isophorone derivative fluorescent dyes contained in the present invention can well label mitochondria, lysosomes, endoplasmic reticulum or lipid droplets respectively, and can be used as mitochondrial green markers Red markers for lysosomes, red markers for endoplasmic reticulum, red markers for lipid droplets. After the cell viscosity was increased by the addition of ionophores, the fluorescence of the fluorescent dyes in their corresponding organelles was significantly enhanced.
在本发明的一个实施例中，共培养为在饱和湿度，37℃，5％CO 2培养箱共同培养5分钟；优选的分别加入线粒体、溶酶体、内质网、脂滴荧光标记物再培养5分钟，然后用PBS缓冲液洗涤，利用激光共聚焦显微镜进行细胞成像。 In one embodiment of the present invention, the co-cultivation is at saturated humidity, 37° C., and 5% CO 2 incubator for 5 minutes; After incubation for 5 min, cells were imaged using confocal microscopy after washing with PBS buffer.
在本发明的一个实施例中，所述离子载体为莫能霉素、***或制霉菌素。In one embodiment of the present invention, the ionophore is monensin, dexamethasone or nystatin.
在本发明的一个实施例中，所述异佛尔酮衍生物在培养基中的浓度为1～2μM；所述离子载体浓度均为5～20μM。In one embodiment of the present invention, the concentration of the isophorone derivative in the culture medium is 1-2 μM; the concentration of the ionophore is 5-20 μM.
本发明根据异佛尔酮衍生物结构的改变，既具有高浓度下荧光标记细胞器的能力；又具有在低浓度下，通过荧光强度对细胞器粘度进行检测。According to the structure change of the isophorone derivative, the invention not only has the ability to fluorescently mark organelles at high concentrations, but also has the ability to detect the viscosity of organelles through fluorescence intensity at low concentrations.
本发明还提供了所述的异佛尔酮衍生物在检测粘度中的应用。The present invention also provides the application of the isophorone derivative in detecting viscosity.
在本发明的一个实施例中，通过异佛尔酮衍生物的荧光强度对细胞器粘度进行检测。In one embodiment of the present invention, the organelle viscosity is detected by the fluorescence intensity of the isophorone derivative.
本发明公开了一系列异佛尔酮衍生物荧光染料，它们不仅具有不同的细胞器靶向能力，并且同时能够检测粘度。由于强分子内电荷转移(ICT)效应，这四种中性荧光染料大部分仍能够达到近红外(NIR)光区。得益于中性荧光染料，细胞毒性与盐类荧光染料相比显著降低。本发明首次解决了异佛尔酮衍生物荧光染料细胞器靶向能力不明确的问题，并且同时能够监测粘度。本发明通过价格低廉的砌块参与合成，使得使用单一基本骨架结构设计具有不同细胞器靶向能力的粘度探针成为可能，并且将成本以及难度显著降低，具 有重要的科学意义和商业价值。The invention discloses a series of isophorone derivative fluorescent dyes, which not only have different cell organelle targeting capabilities, but also can detect viscosity at the same time. Due to the strong intramolecular charge transfer (ICT) effect, most of these four neutral fluorescent dyes are still able to reach the near-infrared (NIR) light region. Thanks to the neutral fluorochrome, cytotoxicity is significantly reduced compared to salt-based fluorochromes. The invention firstly solves the problem of unclear targeting ability of the isophorone derivative fluorescent dye to the organelle, and can monitor the viscosity at the same time. The present invention uses cheap building blocks to participate in the synthesis, which makes it possible to design viscosity probes with different organelle targeting capabilities using a single basic skeleton structure, and significantly reduces the cost and difficulty, which has important scientific significance and commercial value.
本发明的上述技术方案相比现有技术具有以下优点：The above technical solution of the present invention has the following advantages compared with the prior art:
本发明公开了四种异佛尔酮衍生物荧光染料，与细胞共培养后，可以分别标记不同的细胞器；同时，四种荧光染料都对粘度敏感，离子载体和荧光染料与细胞共同培养后，荧光染料在对应细胞器中的荧光显著增强，可对对应的细胞器粘度进行检测。本发明使用一种基础结构进行改进设计，通过廉价的不同砌块参与合成，改善了荧光标记物的光学性质和生物活性，并且能够调节细胞器靶向的能力，这具有重要的科学意义和商业价值。The invention discloses four fluorescent dyes derived from isophorone, which can mark different organelles respectively after being co-cultured with cells; at the same time, the four fluorescent dyes are all sensitive to viscosity. The fluorescence of the fluorescent dye in the corresponding organelle is significantly enhanced, and the viscosity of the corresponding organelle can be detected. The invention uses a basic structure to improve the design, participate in the synthesis through different cheap building blocks, improve the optical properties and biological activities of fluorescent markers, and can regulate the ability of organelle targeting, which has important scientific significance and commercial value .
附图说明Description of drawings
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中In order to make the content of the present invention more easily understood, the present invention will be described in further detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein
图1为本发明设计的染料的合成路线。Fig. 1 is the synthetic route of the dyestuff designed by the present invention.
图2为本发明染料1a-1d分别在甘油和二甲基亚砜中的紫外-可见吸收光谱和荧光光谱。其中(a)为1a在甘油中的紫外-可见吸收光谱和荧光光谱，(b)为1a在二甲基亚砜中的紫外-可见吸收光谱和荧光光谱，(c)为1b在甘油中的紫外-可见吸收光谱和荧光光谱，(d)为1b在二甲基亚砜中的紫外-可见吸收光谱和荧光光谱，(e)为1c在甘油中的紫外-可见吸收光谱和荧光光谱，(f)为1c在二甲基亚砜中的紫外-可见吸收光谱和荧光光谱，(g)为1d在甘油中的紫外-可见吸收光谱和荧光光谱，(f)为1d在二甲基亚砜中的紫外-可见吸收光谱和荧光光谱；Fig. 2 is the ultraviolet-visible absorption spectrum and fluorescence spectrum of dyes 1a-1d of the present invention in glycerol and dimethyl sulfoxide respectively. Wherein (a) is the UV-visible absorption spectrum and fluorescence spectrum of 1a in glycerol, (b) is the UV-visible absorption spectrum and fluorescence spectrum of 1a in dimethyl sulfoxide, (c) is the UV-visible absorption spectrum and fluorescence spectrum of 1b in glycerol UV-visible absorption spectrum and fluorescence spectrum, (d) is the UV-visible absorption spectrum and fluorescence spectrum of 1b in dimethyl sulfoxide, (e) is the UV-visible absorption spectrum and fluorescence spectrum of 1c in glycerin, ( f) is the UV-visible absorption spectrum and fluorescence spectrum of 1c in dimethyl sulfoxide, (g) is the UV-visible absorption spectrum and fluorescence spectrum of 1d in glycerol, (f) is 1d in dimethyl sulfoxide UV-Vis absorption and fluorescence spectra in
图3为本发明染料1a-1d分别在HeLa细胞中的细胞成像图。其中(a1-a6)为1a在HeLa细胞中的细胞成像图，(b1-b6)为1b在HeLa细胞中的细胞成像图，(c1-c6)为1c在HeLa细胞中的细胞成像图，(d1-d6)为1d在HeLa细胞中的细胞成像图。Fig. 3 is a cell imaging diagram of dyes 1a-1d of the present invention in HeLa cells respectively. Wherein (a1-a6) is the cell imaging figure of 1a in HeLa cells, (b1-b6) is the cell imaging figure of 1b in HeLa cells, (c1-c6) is the cell imaging figure of 1c in HeLa cells, ( d1-d6) are cell imaging images of 1d in HeLa cells.
图4为本发明染料1a-1d分别在HeLa细胞中加离子载体前后的细胞成像图。其中(A)为1a在HeLa细胞中加离子载体前后的细胞成像图，(B)为 1b在HeLa细胞中加离子载体前后的细胞成像图，(C)为1c在HeLa细胞中加离子载体前后的细胞成像图，(D)为1d在HeLa细胞中加离子载体前后的细胞成像图。Fig. 4 is a cell imaging diagram of dyes 1a-1d of the present invention before and after adding ionophores to HeLa cells. (A) is the cell imaging image of 1a before and after adding ionophore in HeLa cells, (B) is the cell imaging image of 1b before and after adding ionophore in HeLa cells, (C) is the cell imaging image of 1c before and after adding ionophore in HeLa cells (D) is the cell imaging diagram of 1d before and after adding ionophores in HeLa cells.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.
实施例1Example 1
取化合物1((3,5,5-三甲基环己-2-烯亚基)丙二腈，1.7毫摩尔，254毫克)，化合物a(1H-吲唑-5-甲醛，1.7毫摩尔，200毫克)，溶解于10毫升无水乙醇中，向其中加入100微升的哌啶。氮气保护下在80℃反应回流12小时。反应结束降至室温后，通过旋转蒸发仪除去有机溶剂。纯净产物经柱层析分离后得到，洗脱剂：二氯甲烷，橙黄色固体染料1a，317.83毫克，产率57％。Take compound 1 ((3,5,5-trimethylcyclohex-2-enylidene) malononitrile, 1.7 mmol, 254 mg), compound a (1H-indazole-5-carbaldehyde, 1.7 mmol , 200 mg), was dissolved in 10 ml of absolute ethanol, and 100 microliters of piperidine was added thereto. Under the protection of nitrogen, the reaction was refluxed at 80°C for 12 hours. After the reaction was cooled down to room temperature, the organic solvent was removed by a rotary evaporator. The pure product was separated by column chromatography, eluent: dichloromethane, orange-yellow solid dye 1a, 317.83 mg, yield 57%.
染料1a的核磁共振氢谱(300MHz,DMSO-d 6) 1H NMR(400MHz,DMSO-d 6)δ(ppm)13.24(s,1H,NH),8.13(d,J＝20.77Hz 2H,CH),7.82(d,J＝8.48Hz,1H,Ar-H),7.58(d,J＝7.77Hz,1H,Ar-H),7.43(s,1H,Ar-H),5.76(s,1H,CH),2.62(d,J＝11.74Hz,CH 3),1.04(s,1H,CH 2)。染料1a的核磁共振碳谱(600MHz,DMSO-d 6), 13C NMR(600MHz,DMSO-d 6)δ(ppm)143.24,129.33,113.13,111.70,107.41,101.66,100.60,98.05,96.23,94.87,86.94,86.12,83.67,48.28,27.81,15.22,11.12,4.61.HRMS(ESI +):m/z计算值：C 20H 20N 4 +[M+H] +:315.1604,实测值：315.1707。 H NMR spectrum of dye 1a (300MHz, DMSO-d 6 ) 1 H NMR (400MHz, DMSO-d 6 )δ(ppm) 13.24(s, 1H, NH), 8.13(d, J=20.77Hz 2H, CH ),7.82(d,J=8.48Hz,1H,Ar-H),7.58(d,J=7.77Hz,1H,Ar-H),7.43(s,1H,Ar-H),5.76(s,1H , CH), 2.62 (d, J = 11.74 Hz, CH 3 ), 1.04 (s, 1H, CH 2 ). C NMR spectrum of dye 1a (600MHz, DMSO-d 6 ), 13 C NMR (600MHz, DMSO-d 6 ) δ (ppm) 143.24, 129.33, 113.13, 111.70, 107.41, 101.66, 100.60, 98.05, 96.23, 94.87 , 86.94, 86.12, 83.67, 48.28, 27.81, 15.22, 11.12, 4.61. HRMS (ESI + ): m/z calculated: C 20 H 20 N 4 + [M+H] + : 315.1604, found: 315.1707.
实施例2Example 2
取化合物1((3,5,5-三甲基环己-2-烯亚基)丙二腈，1.0毫摩尔，186毫克)，化合物b(4-(4-羟基哌啶-1-基)苯甲醛，1.0毫摩尔，205毫克)，溶解于10毫升无水乙醇中，向其中加入100微升哌啶，氮气保护下反应回流12小时。反应结束降至室温后，通过旋转蒸发仪除去有机溶剂。纯净产物经柱层析分离后得到，洗脱剂：二氯甲烷，棕红色固体染料1b，194.40毫克，产率52％。Take compound 1 ((3,5,5-trimethylcyclohex-2-enylidene) malononitrile, 1.0 mmol, 186 mg), compound b (4-(4-hydroxypiperidin-1-yl ) benzaldehyde, 1.0 mmol, 205 mg), was dissolved in 10 milliliters of absolute ethanol, 100 microliters of piperidine was added thereto, and the reaction was refluxed for 12 hours under nitrogen protection. After the reaction was cooled down to room temperature, the organic solvent was removed by a rotary evaporator. The pure product was separated by column chromatography, eluent: dichloromethane, brown-red solid dye 1b, 194.40 mg, yield 52%.
染料1b的核磁共振氢谱(300MHz,DMSO-d 6) 1H NMR(400MHz,DMSO-d 6)δ(ppm)7.56(d,J＝8.36Hz,2H,Ar-H),7.21(m,J＝16.02Hz,2H,CH),6.96(d,J＝8.50Hz,2H,Ar-H),6.77(s,1H,CH),4.72(d,J＝3.75Hz,1H,OH),3.71(d,J＝9.77Hz,2H,CH 2),3.03(t,J＝10.44Hz,2H,CH 2),2.58(d,4H,CH 2),1.81(d,J＝9.48Hz,2H,CH 2),1.43(m,J＝9.50Hz,2H,CH 2),1.01(s,3H,CH 3)。染料1b的核磁共振碳谱(600MHz,DMSO-d 6), 13C NMR(600MHz,DMSO-d 6)δ(ppm)142.57,129.70,124.08,111.33,102.26,97.59,97.52,93.21,86.93,86.11,46.11,38.40,17.56,14.87,10.76,6.08,4.18.HRMS(ESI +):m/z计算值：C 24H 28N 3O +[M+H] +:374.2227,实测值：374.2498。 H NMR spectrum of dye 1b (300MHz, DMSO-d 6 ) 1 H NMR (400MHz, DMSO-d 6 )δ(ppm) 7.56(d, J=8.36Hz, 2H, Ar-H), 7.21(m, J=16.02Hz, 2H, CH), 6.96(d, J=8.50Hz, 2H, Ar-H), 6.77(s, 1H, CH), 4.72(d, J=3.75Hz, 1H, OH), 3.71 (d, J=9.77Hz, 2H, CH 2 ), 3.03(t, J=10.44Hz, 2H, CH 2 ), 2.58(d, 4H, CH 2 ), 1.81(d, J=9.48Hz, 2H, CH 2 ), 1.43 (m, J=9.50 Hz, 2H, CH 2 ), 1.01 (s, 3H, CH 3 ). C NMR spectrum of dye 1b (600MHz, DMSO-d 6 ), 13 C NMR (600MHz, DMSO-d 6 ) δ(ppm) 142.57, 129.70, 124.08, 111.33, 102.26, 97.59, 97.52, 93.21, 86.93, 86.11 , 46.11, 38.40, 17.56, 14.87, 10.76, 6.08, 4.18. HRMS (ESI + ): m/z calculated: C 24 H 28 N 3 O + [M+H] + : 374.2227, found: 374.2498.
实施例3Example 3
取化合物1((3,5,5-三甲基环己-2-烯亚基)丙二腈，1.0毫摩尔，186毫克)，化合物c(4-羟基-1-萘甲醛，1.0毫摩尔，172毫克)溶解于10毫升无水乙醇中，向其中加入100微升哌啶，氮气保护下反应回流12小时。反应结束降至室温后，通过旋转蒸发仪除去有机溶剂。纯净产物经柱层析分离后得到，洗脱剂：二氯甲烷，棕红色固体染料1c，270.4毫克，产率61％。Take compound 1 ((3,5,5-trimethylcyclohex-2-enylidene) malononitrile, 1.0 mmol, 186 mg), compound c (4-hydroxyl-1-naphthaldehyde, 1.0 mmol , 172 mg) was dissolved in 10 ml of absolute ethanol, 100 microliters of piperidine was added thereto, and the reaction was refluxed for 12 hours under nitrogen protection. After the reaction was cooled down to room temperature, the organic solvent was removed by a rotary evaporator. The pure product was separated by column chromatography, eluent: dichloromethane, brown-red solid dye 1c, 270.4 mg, yield 61%.
染料1c的核磁共振氢谱(300MHz,CDCl 3) 1H NMR(400MHz,CDCl 3)δ(ppm)8.29(d,J＝7.82Hz,1H,Ar-H),8.14(d,J＝8.14Hz,1H,Ar-H),7.82(d,J＝15.95Hz,2H,CH),7.72(d,J＝7.88Hz,1H,Ar-H),7.62(m,J＝8.00Hz,2H,Ar-H),7.02(d,J＝15.72Hz,1H,Ar-H),6.90(m,J＝7.99Hz,2H,Ar-H),5.93(s,1H,OH),2.62(d,J＝11.42Hz,4H,CH 2),1.12(s,6H,CH 3)。染料1c的核磁共振碳谱(600MHz,DMSO-d 6), 13C NMR(600MHz,DMSO-d 6)δ(ppm)164.73,142.78,129.43,128.21,106.49,104.90,101.96,100.88,99.72,98.78,97.47,96.94,95.71,95.16,94.26,86.69,85.86,81.17,80.13,47.34,14.92,10.66,4.23.HRMS(ESI +):m/z计算值：C 23H 20N 2NaO +[M+Na] +:363.1468,实测值：363.1662。 H NMR spectrum of dye 1c (300MHz, CDCl 3 ) 1 H NMR (400MHz, CDCl 3 ) δ (ppm) 8.29 (d, J = 7.82Hz, 1H, Ar-H), 8.14 (d, J = 8.14Hz ,1H,Ar-H),7.82(d,J=15.95Hz,2H,CH),7.72(d,J=7.88Hz,1H,Ar-H),7.62(m,J=8.00Hz,2H,Ar -H),7.02(d,J=15.72Hz,1H,Ar-H),6.90(m,J=7.99Hz,2H,Ar-H),5.93(s,1H,OH),2.62(d,J = 11.42 Hz, 4H, CH 2 ), 1.12 (s, 6H, CH 3 ). Carbon NMR spectrum of dye 1c (600MHz, DMSO-d 6 ), 13 C NMR (600MHz, DMSO-d 6 ) δ(ppm) 164.73, 142.78, 129.43, 128.21, 106.49, 104.90, 101.96, 100.88, 99.72, 98.78 , 97.47, 96.94, 95.71, 95.16, 94.26, 86.69, 85.86, 81.17, 80.13, 47.34, 14.92, 10.66, 4.23. HRMS (ESI + ): m/z calculated: C 23 H 20 N 2 NaO + [M+ Na] + : 363.1468, measured value: 363.1662.
实施例4Example 4
取化合物1((3,5,5-三甲基环己-2-烯亚基)丙二腈，0.86毫摩尔，161毫克)，化合物d(1-芘甲醛，0.86毫摩尔，200毫克)溶解于10毫升无水乙醇中，向其中加入100微升哌啶，氮气保护下反应回流12小时。反应结束降至室温后，有大量沉淀析出，抽滤出沉淀，并用无水乙醇洗涤，即得最终纯净产物，红色固体染料1d，178.0毫克，产率52％。Take compound 1 ((3,5,5-trimethylcyclohex-2-enylidene) malononitrile, 0.86 mmol, 161 mg), compound d (1-pyrene formaldehyde, 0.86 mmol, 200 mg) Dissolve in 10 ml of absolute ethanol, add 100 microliters of piperidine therein, and react under nitrogen protection and reflux for 12 hours. After the reaction was completed and cooled down to room temperature, a large amount of precipitates precipitated out. The precipitates were filtered out and washed with absolute ethanol to obtain the final pure product, red solid dye 1d, 178.0 mg, with a yield of 52%.
染料1d的核磁共振氢谱(300MHz,DMSO-d 6) 1H NMR(400MHz,DMSO-d 6)δ(ppm)8.80(d,J＝9.17Hz,1H,Ar-H),8.66(d,J＝8.27Hz,1H,Ar-H),8.39(m,J＝8.14Hz,4H,Ar-H),8.24(J＝5.04Hz,2H,Ar-H),8.14(t,J＝7.95Hz,1H,Ar-H),7.78(d,J＝16.41Hz,2H,CH),7.02(s,1H,CH),2.81(2H,CH 2),2.62(2H,CH 2),1.09(6H,CH 3。因1d溶解性较差，核磁共振碳谱未能得到。HRMS(ESI +):m/z计算值：C 29H 22N 2Na +[M+Na] +:421.1675,实测值：421.2039。 H NMR spectrum of dye 1d (300MHz, DMSO-d 6 ) 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm) 8.80(d, J=9.17Hz, 1H, Ar-H), 8.66(d, J＝8.27Hz, 1H, Ar-H), 8.39(m, J＝8.14Hz, 4H, Ar-H), 8.24(J＝5.04Hz, 2H, Ar-H), 8.14(t, J＝7.95Hz ,1H,Ar-H),7.78(d,J=16.41Hz,2H,CH),7.02(s,1H,CH),2.81(2H,CH 2 ),2.62(2H,CH 2 ),1.09(6H , CH 3 . Due to the poor solubility of 1d, C NMR spectrum could not be obtained. HRMS (ESI + ): m/z calculated value: C 29 H 22 N 2 Na + [M+Na] + : 421.1675, measured value : 421.2039.
测试例1test case 1
对实施例1-实施例4制备的染料(浓度均为10μM)在甘油和二甲基亚砜中测试其紫外吸收和荧光发射光谱，横坐标为波长，纵坐标分别为吸光度或荧光强度，结果如图2。The dyestuff (concentration is 10 μ M) prepared in embodiment 1-embodiment 4 is tested its ultraviolet absorption and fluorescence emission spectrum in glycerol and dimethyl sulfoxide, and abscissa is wavelength, and ordinate is respectively absorbance or fluorescence intensity, result Figure 2.
在紫外-可见吸收光谱中，染料1a在424nm处有最大吸收；在荧光发射光谱中，染料1a在565nm处有最高的荧光强度且在甘油中最高，此时激发波长为424nm，狭缝宽度为5nm/5nm，分别见图2-(a)(b)。In the ultraviolet-visible absorption spectrum, the dye 1a has the maximum absorption at 424nm; in the fluorescence emission spectrum, the dye 1a has the highest fluorescence intensity at 565nm and is the highest in glycerol, at this time the excitation wavelength is 424nm, and the slit width is 5nm/5nm, see Figure 2-(a)(b) respectively.
在紫外-可见吸收光谱中，染料1b在493nm处有最大吸收；在荧光发射光谱中，染料1b在685nm处有最高的荧光强度且在甘油中最高，此时激发波长为545nm,狭缝宽度为10nm/1.5nm，分别见图2-(c)(d)。In the ultraviolet-visible absorption spectrum, the dye 1b has the maximum absorption at 493nm; in the fluorescence emission spectrum, the dye 1b has the highest fluorescence intensity at 685nm and is the highest in glycerin, at this time the excitation wavelength is 545nm, and the slit width is 10nm/1.5nm, see Figure 2-(c)(d) respectively.
在紫外-可见吸收光谱中，染料1c在473nm处有最大吸收；在荧光发射光谱中，染料1c在659nm处有最高的荧光强度且在甘油中最高，此时激发波长为506nm，狭缝宽度为10nm/3nm，分别见图2-(e)(f)。In the ultraviolet-visible absorption spectrum, the dye 1c has the maximum absorption at 473nm; in the fluorescence emission spectrum, the dye 1c has the highest fluorescence intensity at 659nm and is the highest in glycerol. At this time, the excitation wavelength is 506nm, and the slit width is 10nm/3nm, see Figure 2-(e)(f) respectively.
在紫外-可见吸收光谱中，染料1d在476nm处有最大吸收；在荧光发射光谱中，染料1d在660nm处有最高的荧光强度且在甘油中最高，此时激发波长为507nm，狭缝宽度为5nm/5nm，分别见图2(g)(h)。In the ultraviolet-visible absorption spectrum, the dye 1d has the maximum absorption at 476nm; in the fluorescence emission spectrum, the dye 1d has the highest fluorescence intensity at 660nm and is the highest in glycerol, at this time the excitation wavelength is 507nm, and the slit width is 5nm/5nm, see Figure 2(g)(h) respectively.
测试例2 test case 2
使用DMSO(二甲基亚)将染料1a配置成母液，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10μM，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，随后加入商业线粒体红色标记物Mito Tracker Red CMXRos(2μM)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。绿光通道选用488nm波长激发，收集468-550nm范围内的荧光信号。红光通道使用561nm波长激发，收集600-750nm范围内的荧光信号。细胞成像显示染料1a在HeLa细胞中能够很好地标记线粒体，可做线粒体绿色标记物。结果如图3所示，其中(a1)为明场成像图，(a2)为染料1a的细胞成像图，(a3)为商业线粒体红色标记物的细胞成像图，(a4)为绿光通道和红光通道的叠加图，(a5)为共定位实验，共定位系数为0.92，(a6)为叠加图中ROI线的荧光强度。 Use DMSO (dimethyl methoxide) to prepare the dye 1a into a mother solution, and then add it to a regular cell culture medium, so that the concentration of the dye 1a in the cell culture medium is 10 μM, and then mix with HeLa cells in saturated humidity, 37 ° C, 5% CO 2 incubators for co-cultivation (the following experiments are the same) for 10 minutes, then add the commercial mitochondrial red marker Mito Tracker Red CMXRos (2μM) and incubate for another 10 minutes; then wash three times with PBS buffer, and use a laser confocal microscope for cell imaging. The green light channel is excited with a wavelength of 488nm, and the fluorescent signal in the range of 468-550nm is collected. The red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-750nm is collected. Cell imaging shows that dye 1a can well label mitochondria in HeLa cells and can be used as a green mitochondrial marker. The results are shown in Figure 3, where (a1) is the bright field imaging image, (a2) is the cell imaging image of dye 1a, (a3) is the cell imaging image of the commercial mitochondrial red marker, (a4) is the green light channel and The overlay of the red channel, (a5) is the colocalization experiment, the colocalization coefficient is 0.92, (a6) is the fluorescence intensity of the ROI line in the overlay.
测试例3Test case 3
染料1b在细胞培养基中的浓度为14μM，随后加入商业溶酶体绿色标记物Lyso Tracker Green(2μM)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波长激发，收集600-750nm范围内的荧光信号，绿光通道选用488nm波长激发，收集468-550nm范围内的荧光信号。细胞成像显示染料1b在HeLa细胞中能够很好地标记溶酶体，可做溶酶体红色标记物。结果如图3所示，其中(b1)为明场成像图，(b2)为商业溶酶体绿色标记物的细胞成像图，(b3)为染料1b的细胞成像图，(b4)为绿光通道和红光通道的叠加图，(b5)为共定位实验，共定位系数为0.94，(b6)为叠加图中ROI线的荧光强度。The concentration of dye 1b in the cell culture medium was 14 μM, and then the commercial lysosome green marker Lyso Tracker Green (2 μM) was added and incubated for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope. The red light channel is excited with a wavelength of 561nm to collect fluorescent signals in the range of 600-750nm, and the green light channel is excited with a wavelength of 488nm to collect fluorescent signals in the range of 468-550nm. Cell imaging shows that dye 1b can well label lysosomes in HeLa cells and can be used as a lysosomal red marker. The results are shown in Figure 3, where (b1) is the bright field imaging image, (b2) is the cell imaging image of the commercial lysosome green marker, (b3) is the cell imaging image of dye 1b, and (b4) is the green light Overlay of channel and red channel, (b5) is the colocalization experiment, the colocalization coefficient is 0.94, (b6) is the fluorescence intensity of the ROI line in the overlay.
测试例4Test case 4
染料1c在细胞培养基中的浓度为10μM，随后加入商业内质网绿色标记物ER Tracker Green(6μM)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波长激发，收集600-750nm范围内的荧光信号，绿光通道选用488nm波长激发，收集468-550nm范围内的荧光信号。细胞成像显示染料1c在HeLa细胞中能够很好地标记内质网，可做内质网红色标记物。结果如图3所示，其中(c1)为明场成像图，(c2)为商业内质网绿色标记物的细胞成像图，(c3)为染料1c的细胞成像图，(c4)为绿光通道和红光通道的叠加图，(c5)为共定位实验，共定位系数为0.96，(c6)为叠加图中ROI线的荧光强度。The concentration of dye 1c in the cell culture medium was 10 μM, and then the commercial endoplasmic reticulum green marker ER Tracker Green (6 μM) was added to incubate for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope. The red light channel is excited with a wavelength of 561nm to collect fluorescent signals in the range of 600-750nm, and the green light channel is excited with a wavelength of 488nm to collect fluorescent signals in the range of 468-550nm. Cell imaging shows that dye 1c can well label the endoplasmic reticulum in HeLa cells and can be used as a red marker of the endoplasmic reticulum. The results are shown in Figure 3, where (c1) is the bright field imaging image, (c2) is the cell imaging image of the commercial endoplasmic reticulum green marker, (c3) is the cell imaging image of dye 1c, (c4) is the green light Overlay of channel and red channel, (c5) is the colocalization experiment, the colocalization coefficient is 0.96, (c6) is the fluorescence intensity of the ROI line in the overlay.
测试例5 Test case 5
染料1d在细胞培养基中的浓度为20μM，随后加入商业脂滴绿色标记物LDs Tracker Green(4μM)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波长激发，收集600-750nm范围内的荧光信号，绿光通道选用488nm波长激发，收集468-550nm范围内的荧光信号。细胞成像显示染料1d在HeLa细胞中能够很好地标记脂滴，可做脂滴红色标记物。结果如图3所示，其中(d1)为明场成像图，(d2)为商业脂滴绿色标记物的细胞成像图，(d3)为染料1d的细胞成像图，(d4)为绿光通道和红光通道的叠加图，(d5)为共定位实验，共定位系数为0.90，(d6)为叠加图中ROI线的荧光强度。The concentration of dye 1d in the cell culture medium was 20 μM, then the commercial lipid droplet green marker LDs Tracker Green (4 μM) was added and incubated for another 10 minutes; after washing three times with PBS buffer, the cells were imaged using a laser confocal microscope. The red light channel is excited with a wavelength of 561nm to collect fluorescent signals in the range of 600-750nm, and the green light channel is excited with a wavelength of 488nm to collect fluorescent signals in the range of 468-550nm. Cell imaging shows that the dye 1d can well label lipid droplets in HeLa cells and can be used as a red marker for lipid droplets. The results are shown in Figure 3, where (d1) is the bright field imaging image, (d2) is the cell imaging image of commercial lipid droplet green marker, (d3) is the cell imaging image of dye 1d, (d4) is the green light channel and the overlay of the red channel, (d5) is the colocalization experiment, the colocalization coefficient is 0.90, (d6) is the fluorescence intensity of the ROI line in the overlay.
测试例6Test case 6
染料1a在细胞培养基中的浓度为2μM，离子载体莫能霉素的浓度为10μM，共同培养30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。绿光通道选用488nm波长激发，收集500-550nm范围内的荧光信号。细胞成像显示，通过加入莫能霉素使线粒体粘度增加后，染料1a的荧光信号显著增强。结果如图4(A)所示，其中(a)为明场成像图，(b)为染料1a 的细胞成像图，(c)为(a)和(b)的叠加图，(d)为明场成像图，(e)为染料1a和莫能霉素的细胞成像图，(f)为(d)和(e)的叠加图，(g)为平均荧光强度。The concentration of dye 1a in the cell culture medium was 2 μM, the concentration of the ionophore monensin was 10 μM, co-cultured for 30 minutes, washed three times with PBS buffer, and the cells were imaged by laser confocal microscope. The green light channel is excited with a wavelength of 488nm, and the fluorescent signal in the range of 500-550nm is collected. Imaging of cells revealed that the fluorescent signal of dye 1a was significantly enhanced after mitochondrial viscosity was increased by addition of monensin. The results are shown in Figure 4(A), where (a) is the bright field imaging image, (b) is the cell imaging image of dye 1a, (c) is the overlay of (a) and (b), (d) is Bright field imaging, (e) is the cell imaging of dye 1a and monensin, (f) is the overlay of (d) and (e), (g) is the average fluorescence intensity.
测试例7Test case 7
染料1b在细胞培养基中的浓度为2μM，离子载体***的浓度为10μM，共同培养30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波长激发，收集600-800nm范围内的荧光信号。细胞成像显示，通过加入***使溶酶体粘度增加后，染料1b的荧光信号显著增强。结果如图4(B)所示，其中(a)为明场成像图，(b)为染料1b的细胞成像图，(c)为(a)和(b)的叠加图，(d)为明场成像图，(e)为染料1b和***的细胞成像图，(f)为(d)和(e)的叠加图，(g)为平均荧光强度。The concentration of dye 1b in the cell culture medium was 2 μM, the concentration of the ionophore dexamethasone was 10 μM, co-incubated for 30 minutes, washed three times with PBS buffer, and the cells were imaged using a laser confocal microscope. The red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-800nm is collected. Imaging of cells revealed that the fluorescent signal of dye 1b was significantly enhanced after lysosomal viscosity was increased by adding dexamethasone. The results are shown in Figure 4(B), where (a) is the bright field imaging image, (b) is the cell imaging image of dye 1b, (c) is the overlay of (a) and (b), (d) is Bright field imaging, (e) is the cell imaging of dye 1b and dexamethasone, (f) is the overlay of (d) and (e), (g) is the average fluorescence intensity.
测试例8Test case 8
染料1c在细胞培养基中的浓度为2μM，离子载体莫能霉素的浓度为10μM，共同培养30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波长激发，收集600-800nm范围内的荧光信号。细胞成像显示，通过加入莫能霉素使内质网粘度增加后，染料1c的荧光信号显著增强。结果如图4(C)所示，其中(a)为明场成像图，(b)为染料1c的细胞成像图，(c)为(a)和(b)的叠加图，(d)为明场成像图，(e)为染料1c和莫能霉素的细胞成像图，(f)为(d)和(e)的叠加图，(g)为平均荧光强度。The concentration of dye 1c in the cell culture medium was 2 μM, and the concentration of the ionophore monensin was 10 μM, co-incubated for 30 minutes, washed three times with PBS buffer, and the cells were imaged using a laser confocal microscope. The red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-800nm is collected. Imaging of the cells revealed that the fluorescent signal of dye 1c was significantly enhanced after the viscosity of the ER was increased by adding monensin. The results are shown in Figure 4(C), where (a) is the bright field imaging image, (b) is the cell imaging image of dye 1c, (c) is the overlay of (a) and (b), (d) is Bright field imaging, (e) is the cell imaging of dye 1c and monensin, (f) is the overlay of (d) and (e), (g) is the average fluorescence intensity.
测试例9Test case 9
染料1d在细胞培养基中的浓度为2μM，离子载体制霉菌素的浓度为10μM，共同培养30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波长激发，收集600-800nm范围内的荧光信号。细胞成像显示，通过加入制霉菌素使脂滴粘度增加后，染料1d 的荧光信号显著增强。结果如图4(D)所示，其中(a)为明场成像图，(b)为染料1d的细胞成像图，(c)为(a)和(b)的叠加图，(d)为明场成像图，(e)为染料1d和制霉菌素的细胞成像图，(f)为(d)和(e)的叠加图，(g)为平均荧光强度。The concentration of the dye 1d in the cell culture medium was 2 μM, and the concentration of the ionophore nystatin was 10 μM, co-incubated for 30 minutes, washed three times with PBS buffer, and the cells were imaged using a laser confocal microscope. The red channel is excited with a wavelength of 561nm, and the fluorescent signal in the range of 600-800nm is collected. Imaging of cells revealed that the fluorescent signal of dye 1d was significantly enhanced after lipid droplet viscosity was increased by adding nystatin. The results are shown in Figure 4(D), where (a) is the bright field imaging image, (b) is the cell imaging image of dye 1d, (c) is the overlay of (a) and (b), (d) is Bright field imaging, (e) is the cell imaging of dye 1d and nystatin, (f) is the overlay of (d) and (e), (g) is the average fluorescence intensity.
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (10)

  1. 一种异佛尔酮衍生物，其特征在于，所述衍生物结构式为：An isophorone derivative, characterized in that the derivative structural formula is:
    Figure PCTCN2021143128-appb-100001
    Figure PCTCN2021143128-appb-100001
    其中，R选自以下结构式(1)-(4)：Wherein, R is selected from the following structural formulas (1)-(4):
    Figure PCTCN2021143128-appb-100002
    Figure PCTCN2021143128-appb-100002
  2. 权利要求1所述的异佛尔酮衍生物的制备方法，其特征在于，包括以下步骤：The preparation method of the isophorone derivative described in claim 1, is characterized in that, comprises the following steps:
    取(3,5,5-三甲基环己-2-烯亚基)丙二腈与化合物M溶解于有机溶剂中，在催化剂作用下，加热反应得到所述异佛尔酮衍生物；Dissolving (3,5,5-trimethylcyclohex-2-enylidene)malononitrile and compound M in an organic solvent, under the action of a catalyst, heating and reacting to obtain the isophorone derivative;
    其中，所述化合物M为1H-吲唑-5-甲醛、4-(4-羟基哌啶-1-基)苯甲醛、4-羟基-1-萘甲醛或1-芘甲醛。Wherein, the compound M is 1H-indazole-5-carbaldehyde, 4-(4-hydroxypiperidin-1-yl)benzaldehyde, 4-hydroxy-1-naphthaldehyde or 1-pyrenecarbaldehyde.
  3. 根据权利要求2所述的制备方法，其特征在于，所述(3,5,5-三甲基环己-2-烯亚基)丙二腈与化合物M的摩尔比为1:0.7～1:2.0。The preparation method according to claim 2, characterized in that, the molar ratio of (3,5,5-trimethylcyclohex-2-enylidene) malononitrile to compound M is 1:0.7～1 :2.0.
  4. 权利要求1所述的异佛尔酮衍生物在制备细胞器荧光试剂中的应用。The application of the isophorone derivative described in claim 1 in the preparation of organelle fluorescence reagent.
  5. 权利要求1所述的异佛尔酮衍生物在非诊断治疗目的的细胞器荧光成像中的应用。The application of the isophorone derivative according to claim 1 in the fluorescence imaging of organelles for non-diagnostic and therapeutic purposes.
  6. 根据权利要求5所述的应用，其特征在于，所述细胞器为溶酶体、线粒体、内质网或脂滴。The use according to claim 5, characterized in that the organelle is lysosome, mitochondria, endoplasmic reticulum or lipid droplet.
  7. 一种非诊断非治疗目的的细胞荧光成像的方法，其特征在于，包括以下步骤：将细胞与含有权利要求1所述异佛尔酮衍生物在培养基中共培养，再进行细胞成像，完成细胞荧光成像，所述异佛尔酮衍生物能进行不同细胞器的荧光标记。A method for fluorescence imaging of cells for non-diagnostic and non-therapeutic purposes, characterized in that it comprises the following steps: co-cultivating cells with the isophorone derivatives described in claim 1 in a medium, and then performing cell imaging to complete cell imaging. For fluorescence imaging, the isophorone derivatives can be used for fluorescent labeling of different organelles.
  8. 一种非诊断非治疗目的的细胞荧光成像的方法，其特征在于，包括以下步骤：将细胞与含有权利要求1所述异佛尔酮衍生物和离子载体在培养基中共培养，再进行细胞成像，完成细胞荧光成像。A method for fluorescence imaging of cells for non-diagnostic and non-therapeutic purposes, characterized in that it comprises the following steps: co-cultivating cells with isophorone derivatives and ionophores described in claim 1 in a medium, and then performing cell imaging , complete cell fluorescence imaging.
  9. 根据权利要求8所述的方法，其特征在于，所述离子载体为莫能霉素、***或制霉菌素。The method according to claim 8, wherein the ionophore is monensin, dexamethasone or nystatin.
  10. 权利要求1所述的异佛尔酮衍生物在检测粘度中的应用。The application of the isophorone derivative described in claim 1 in detecting viscosity.
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