Application of methoxynaphthalene fluorescent compound in preparation of reagent for detecting mitochondria and / or cell nucleus and reagent for observing apoptosis
By developing methoxynaphthalene fluorescent compounds, red light channel imaging of mitochondria in living cells and green light channel imaging of cell nuclei in fixed cells, solving the problem of insufficient fluorescence response of existing fluorescence probes, and providing accurate cell apoptosis observation tools, which are applied to the chemical industry and biomedicine fields.
Patent Information
- Application Number
- CN202510452122.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-11
AI Technical Summary
The existing fluorescence probes have no obvious fluorescence response during the observation of cell apoptosis, making it difficult to visualize the structure of tiny organelles and may damage biological samples, and lack efficient fluorescence probe tools.
A methoxynaphthalene fluorescent compound was developed to prepare reagents for detection of mitochondria and nucleus, and to achieve green light channel imaging of mitochondria and red light channel imaging of cell nucleus through excitation wavelength of 488 nm, and to combine mitochondria tracking deep red fluorescent probe MTDR and nucleus blue fluorescent probe Hoechst 33342 for colocalization.
The red light channel imaging of mitochondria in living cells and green light channel imaging of cell nuclei in fixed cells is realized, providing a more accurate cell apoptosis observation tool, suitable for research in the chemical industry and biomedicine field.
Smart Images
Figure CN120290169A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the application of a methoxynaphthalene fluorescent compound in the preparation of reagents for detecting mitochondria and / or cell nuclei and reagents for observing apoptosis, belonging to the technical field of fluorescent probes. Background Art
[0002] Apoptosis is a form of programmed cell death, which is an orderly and gene-controlled cell death process. Apoptosis plays an important role in the development of organisms, maintaining tissue homeostasis, and the occurrence of diseases. During embryonic development, apoptosis helps remove redundant cells and form the correct morphology of organs and tissues; in adult organisms, apoptosis helps remove damaged or aged cells and maintain tissue balance and function; apoptosis can remove abnormal cells and prevent the formation and development of tumors. Therefore, the study of the apoptosis mechanism is of great significance for understanding diseases and developing new treatment methods.
[0003] Currently, the methods for observing apoptosis mainly include optical microscopy, transmission electron microscopy, and scanning electron microscopy. However, optical microscopy cannot visualize the tiny organelle structures related to autophagy and apoptosis, while transmission electron microscopy or scanning electron microscopy cannot visualize living biological samples. In addition, these methods usually involve complex procedures and may cause damage to biological samples. In contrast, fluorescent probes have high advantages, including rapid response, excellent selectivity and sensitivity, and in-situ monitoring of biological processes. Therefore, a fluorescent probe can be developed to visualize the apoptosis process.
[0004] Currently, there are few fluorescent probes capable of observing apoptosis. For example, (Z)-4-(6-methoxynaphthaleneacetamide)-2-(pyridine)-4-acrylonitrile disclosed in Patent Application 202311782485X stains lysosomes to identify living or dead cells. However, most probes have the problem of unclear fluorescence response. Therefore, it is particularly important to develop a fluorescent probe capable of observing apoptosis. This will provide more accurate and effective tools for research and applications in fields such as the chemical industry and biomedicine. Summary of the Invention
[0005] Object of the Invention: The object of the present invention is to provide the application of a methoxynaphthalene fluorescent compound in the preparation of reagents for detecting mitochondria and / or cell nuclei and reagents for observing apoptosis.
[0006] Technical Solution: The present invention provides the application of a methoxynaphthalene fluorescent compound in the preparation of reagents for detecting mitochondria and / or cell nuclei and reagents for observing apoptosis, and the structural formula of the compound is:
[0007]
[0008] Further, the preparation route of the methoxynaphthalene fluorescent compound is as follows:
[0009]
[0010] Further, it specifically includes the following steps: Dissolve compound 1 and iodoethanol in ethanol, stir, reflux at 100 °C, cool and filter, wash with anhydrous EtOH, dry to obtain compound 2; dissolve compound 2 and 3 in methanol, stir, add piperidine, reflux at 90 °C, cool to room temperature and wash with petroleum ether; use a CH2Cl2 / CH3OH mixture as the eluent for column chromatography separation and purification to obtain the product.
[0011] Further, the volume ratio of the CH2Cl2 / CH3OH mixture is 12:1 to 7:1.
[0012] The present invention also provides a reagent for detecting mitochondria and / or nuclei, and the fluorescent probe in the reagent is a methoxynaphthalene compound, and the structural formula of the compound is:
[0013]
[0014] The present invention also provides a reagent for observing apoptosis, and the fluorescent probe in the reagent is a methoxynaphthalene compound, and the structural formula of the compound is:
[0015]
[0016] Further, the concentration of the fluorescent probe is 5 - 20 μM.
[0017] Further, the solvent in the reagent includes one of EtOH, MeCN, DMSO, H2O, 1,4-dioxane, DMF, and Gly.
[0018] Further, the reagent also includes a mitochondrial tracking deep red fluorescent probe MTDR and / or a nuclear blue fluorescent probe Hoechset 33342.
[0019] Further, when the fluorescent compound is used as a mitochondrial green fluorescent probe, the excitation wavelength is 488 nm, and the collection wavelength is 500 - 560 nm; when used as a nuclear red fluorescent probe, the excitation wavelength is 488 nm, and the red light collection wavelength is 560 - 660 nm.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention firstly discloses a methoxynaphthalene fluorescent compound as a fluorescent probe for imaging mitochondria in the red light channel in living cells and imaging nuclei in the green light channel in fixed cells. Since mitochondria and nuclei are closely related to the diagnosis of various diseases (such as cancer) and drug research and development. Therefore, the fluorescent compound of the present invention can provide a more accurate and effective tool for research and application in the fields of chemical industry, biomedicine, etc. Brief description of the drawings
[0021] Figure 1 Absorption spectra (A) and fluorescence spectra (B) of TB (10 μM) in different solvents.
[0022] Figure 2 Fluorescence spectra (A) of TB (10 μM) in aqueous solution and solid and CIE diagram (B).
[0023] Figure 3 Confocal fluorescence images of normal and fixed HeLa cells stained with TB (5 μM, 30 min).
[0024] Figure 4 Confocal fluorescence images of active HeLa cells co-stained with TB (5 μM, 30 min) and MTDR (2 μM, 30 min) or fixed HeLa cells co-stained with TB (5 μM, 30 min) and Hoechst 33342 (5 μM, 30 min).
[0025] Figure 5 Confocal fluorescence images of normal or rotenone-treated HeLa cells stained with TB (5 μM, 30 min). Detailed implementation manners
[0026] The technical solution of the present invention will be further described below in conjunction with the drawings.
[0027] The test instrument for absorption spectra used in the examples is Hitachi U-2910 spectrophotometer; the fluorescence spectrum instrument is Hitachi F-2700 spectrophotometer, and the cell imaging instrument is lecia confocal microscope.
[0028] Example 1 Synthesis of fluorescent compound
[0029] (E)-3-(2-Hydroxyethyl)-2-(2-(6-methoxynaphthalen-2-yl)vinyl)benzothiazolium-3-iodide (i.e., compound TB)
[0030] The reaction route is as follows:
[0031]
[0032] 1) Synthesis of benzothiazole iodide (Compound 2)
[0033] Dissolve Compound 1 (2-methylbenzothiazole, 1.49 g, 10 mmol) and iodoethanol (1.76 mL, 10 mmol) in 25 mL of absolute ethanol. Stir in a flask at room temperature for 1 hour. Then reflux at 100 °C for 8 h. After cooling and filtration, wash three times with absolute EtOH. After drying, a white solid is obtained, which is Compound 2 (mass: 3.24 g, yield: 92%).
[0034] 2) Synthesis of Compound TB
[0035] Dissolve Compound 2 (1 mmol, 0.32 g) and Compound 3 (1 mmol, 0.186 g) in 25 mL of methanol. Stir in a flask for 1 h, and add 2 drops of piperidine. After stirring, reflux at 90 °C for 10 h. After cooling to room temperature, wash with petroleum ether. Perform column chromatography purification using a CH2Cl2 / CH3OH mixture (12:1 - 7:1, v / v) as the eluent to obtain a yellow solid, which is Compound TB (mass: 0.26 g, yield: 60%).
[0036] 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.46 (d, J = 8.0 Hz, 2H), 8.34 (q, J = 12.0 Hz, 2H), 8.14 (q, J = 16.0 Hz, 2H), 7.98 (t, J = 10.0 Hz, 2H), 7.78 - 7.89 (m, 2H), 7.45 (d, J = 4.0 Hz, 1H), 7.29 (t, J = 2.6 Hz, 1H), 5.08 - 5.23 (m, 3H), 3.94 (s, 5H).
[0037] Example 2 Photophysical Property Test Experiment
[0038] Prepare test solutions containing 10 μM TB using EtOH, MeCN, DMSO, H2O, 1,4-dioxane, DMF, and Gly as solvents respectively. Measure the absorption spectra of the above solutions using a UV-visible spectrophotometer and the fluorescence emission spectra using a fluorescence spectrometer. The results are shown in Figure 1 .
[0039] It can be seen from the figure that: The fluorescent compound TB has an absorption peak at 430 nm, and the absorption peak range is 350 - 500 nm (see Figure 1 A). It has fluorescence peaks in the range of 490 - 690 nm (see Figure 1B). The above results show that the fluorescent compound can be excited by light with a wavelength of 350 - 500 nm, and the emission spectrum ranges from 490 - 690 nm.
[0040] The fluorescence spectra of the solid probe and the aqueous solution of 10 μM TB were measured using a fluorescence spectrometer, and the results are shown in Figure 2 .
[0041] From Figure 2 A, it can be seen that the fluorescence peak of the probe in the aqueous solution is 550 nm, and the fluorescence peak of the probe in the solid state is 600 nm. By making CIE pictures of the two spectra, it can be seen that the probe changes from yellow - green light to red light ( Figure 2 B), indicating that the probe can perform fluorescence imaging in two channels.
[0042] Example 3 Cell Imaging Experiment of Probe TB
[0043] 1. HeLa cells were cultured adherently in high - glucose H - DMEM medium containing 10% fetal bovine serum in an incubator at 37 °C and 5% CO₂ with saturated humidity. The culture medium was changed every 2 - 3 days, and sub - culture was carried out.
[0044] 2. When the cells grew to the logarithmic phase, slide culture was carried out:
[0045] ① The cover glass was soaked in absolute ethanol for 30 min, dried with an alcohol lamp, and then placed in a disposable 35 - mm culture dish for later use.
[0046] ② The HeLa cells grown to confluence in a 100 - mL cell flask were washed three times with PBS, digested with 1 mL of 0.25% trypsin for 5 minutes, the trypsin was carefully poured out, fresh culture medium was added, and the cells were pipetted evenly and counted. The cell density was controlled by the addition amount of the culture medium so that the final cell concentration was 1×10 5 cells per milliliter. Then, the cells were inoculated into the above - mentioned culture dish containing the cover glass and cultured in a 5% CO₂ incubator to make the cells adhere tightly to the culture dish. After the HeLa cells grew on the cover glass and reached confluence, they were used for cell experiments.
[0047] 3. A stock solution of the probe with a concentration of 1 mM was prepared with DMSO. The cultured active HeLa cells were incubated in a culture medium containing 5 μM TB for 30 min, then washed with PBS and imaged using a laser confocal microscope. The HeLa cells fixed with paraformaldehyde were stained with a culture medium containing 5 μM TB for 30 min. Then, they were washed with PBS, and the colored parts, fluorescence distribution, and brightness changes in the cells were recorded. The results are shown in Figure 3 .
[0048] Among them, Figure 3Confocal microscopy images of normal (live cells) or fixed (dead cells) HeLa cells stained with probe TB (5 μM, 30 min). The excitation wavelengths of TB in both the green and red channels are 488 nm, the green fluorescence collection wavelength is 500 - 560 nm, and the red channel light collection range is 560 - 660 nm. It can be seen from the figure that when stained with 5 μM, in normal cells, the probe can stain mitochondria-like organelles in the red channel, and in fixed cells, the probe can stain nucleus-like organelles in the green channel.
[0049] Example 4: Cellular co-localization experiment of probe TB
[0050] The steps of culturing and seeding HeLa cells are the same as in Example 3. Prepare a stock solution of TB probe with a concentration of 1 mM using DMSO. Incubate the cultured active HeLa cells in a culture medium containing 5 μM TB for 30 min, then stain the cells with 2 μM MTDR (mitochondrial tracking deep red fluorescent probe) for 30 min, wash the cells twice with PBS, and then image them with a confocal microscope. Staining fixed cells is similar to staining live cells. First, fix the cells with paraformaldehyde for 30 min, then stain the HeLa cells in a culture medium containing 5 μM TB for 30 min, and then stain the cells with 5 μM Hoechst 33342 (nucleus blue fluorescent probe) for 30 min.
[0051] Among them, Figure 4 Confocal pictures of active HeLa cells co-stained with TB and MTDR or fixed cells stained with Hoechst 33342 and TB. Among them, the excitation wavelength of TB in the green channel is 488 nm, and the fluorescence collection wavelength is 500 - 560 nm; the excitation wavelength of MTDR in the red channel is 635 nm, and the fluorescence collection wavelength is 640 - 740 nm. It can be seen from the figure that the part of mitochondria stained by TB overlaps greatly with that stained by the commercial mitochondrial fluorescent probe, and its co-localization coefficient is 0.87. The excitation wavelength of TB in the green channel is 488 nm, and the fluorescence collection wavelength is 560 - 660 nm; the excitation wavelength of Hoechst 33342 in the blue channel is 405 nm, and the fluorescence collection wavelength is 410 - 460 nm. It can be seen from the figure that the part of the nucleus stained by TB overlaps greatly with that stained by the commercial nucleus fluorescent probe, and its co-localization coefficient is 0.85, indicating that the probe can image the nucleus in fixed cells. Therefore, this probe can be used to distinguish between live and dead cells.
[0052] Example 5: Experiment on observing cell apoptosis with probe TB
[0053] The procedures for culturing HeLa cells and preparing specimens were the same as those in Example 3. A stock solution of the TB probe with a concentration of 1 mM was prepared using DMSO. The cultured viable HeLa cells were incubated in a culture medium containing 5 μM TB for 30 min as the control group; the experimental group was treated with 5 μM rotenone for 10 h to induce apoptosis. Finally, the cells in the control group and the experimental group were stained with a culture medium containing 5 μM TB for 30 min, and then washed with PBS. The colored regions, fluorescence distribution, and brightness changes in the cells were recorded. The results are shown in Figure 5 .
[0054] Among them, Figure 5 are confocal images of normal and rotenone-induced apoptotic cells stained with TB. As can be seen from the figure, under normal incubation conditions, TB can image mitochondria in the red light channel. In apoptotic cells, the fluorescence intensity in the red light channel decreases, and the morphology of the cell nucleus appears in the green light channel, and the fluorescence intensity ratio of the green light channel to the red light channel increases ( Figure 5 B), indicating that the probe can distinguish normal cells from apoptotic cells.
[0055] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims
1. Use of a methoxynaphthalene fluorescent compound in preparing a reagent for detecting mitochondria and / or cell nuclei and / or a reagent for observing apoptosis, characterized in that, The structural formula of the said compound is as follows:
2. The application according to claim 1, characterized in that, The preparation route of the said methoxynaphthalene fluorescent compound is as follows:
3. The application according to claim 1, characterized in that, The preparation method of the said methoxynaphthalene fluorescent compound specifically includes the following steps: Dissolve compound 1 and iodoethanol in ethanol, stir, reflux at 100 °C, cool and filter, then wash with anhydrous EtOH, dry to obtain compound 2; Dissolve compound 2 and 3 in methanol, stir, add piperidine, reflux at 90 °C, cool to room temperature and wash with petroleum ether; Use the CH2Cl2 / CH3OH mixture as the eluent for column chromatography separation and purification to obtain it.
4. The preparation method according to claim 3, wherein The volume ratio of the said CH2Cl2 / CH3OH mixture is 12:1 to 7:
1.
5. A reagent for detecting mitochondria and / or cell nuclei, characterized in that, The fluorescent probe in the said reagent is a methoxynaphthalene compound, and its structural formula is as follows:
6. An apoptosis observation reagent, characterized in that, The fluorescent probe in the said reagent is a methoxynaphthalene compound, and its structural formula is as follows:
7. The reagent according to claim 5 or 6, characterized in that, The concentration of the said fluorescent probe is 5 - 20 μM.
8. The application according to claim 5 or 6, characterized in that, The solvent in the said reagent includes one of EtOH, MeCN, DMSO, H2O, 1,4-dioxane, DMF, and Gly.
9. The application according to claim 5 or 6, characterized in that, The said reagent also includes a mitochondrial tracking deep red fluorescent probe MTDR and / or a nuclear blue fluorescent probe Hoechset 33342.
10. The application according to claim 5 or 6, characterized in that, When the said fluorescent compound is used as a mitochondrial green fluorescent probe, the excitation wavelength is 488 nm, and the collection wavelength is 500 - 560 nm; When used as a nuclear red fluorescent probe, the excitation wavelength is 488 nm, and the red light collection wavelength is 560 - 660 nm.