A methoxynaphthalene fluorescent compound and application thereof

Abstract

The application discloses a methoxynaphthalene fluorescent compound and application thereof. The fluorescent compound can be used as a fluorescent probe and can detect pH value changes in a solution and cells. Compared with a probe for qualitatively researching the strength of pH value, the probe can quantize the pH value of the solution and can research the pH value changes in the cells. Therefore, the fluorescent compound can be used for preparing a pH value sensitive tumor diagnosis reagent, a fluorescent imaging reagent or a radioactive imaging reagent of the tumor.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a methoxynaphthalene fluorescent compound and application thereof. BACKGROUND

[0002] As an important indicator in the field of chemical industry, pH value reflects the concentration of hydrogen ions (H + ) in the solution, and is a key physical quantity for determining the acidity and alkalinity of the solution. By detecting the pH value of the solution, we can determine its chemical properties, thereby more effectively controlling and adjusting the chemical reaction process. In addition, pH value also plays an irreplaceable role in the biological field, which reflects the changes in the microenvironment of cells, and is closely related to the interaction of biological molecules, the diffusion of metabolic products, and the process of signal transduction. Therefore, the detection of pH value has a wide range of applications in environmental protection, food industry, biological medicine and other fields.

[0003] Traditional pH value detection methods mainly include acid-base indicators, pH meters and pH test papers. However, in order to further improve the accuracy and convenience of measurement, researchers continue to develop new detection instruments and methods, such as glass electrode method, ion selective electrode method and conductivity method.

[0004] The glass electrode method is favored for its high precision, fast response and wide measurement range. However, since the electrode is easily contaminated and damaged, it needs to be calibrated regularly to ensure the accuracy of the measurement results.

[0005] The ion selective electrode method is popular for its accurate and reliable measurement results and wide application range. However, the preparation process of ion selective electrode is relatively complex and the cost is also high, which to some extent limits its application.

[0006] The conductivity method is a relatively simple and easy method, which does not require special pH detection instruments, but only needs to use general conductivity instruments. However, for some specific solutions such as weak acid and weak base solutions, the measurement results of the conductivity method may not be accurate enough, and it cannot detect the pH value of micro samples.

[0007] In recent years, organic fluorescent probes based on fluorescence imaging technology have shown great potential in the field of pH value detection. This technology can visualize the changes of pH value in situ, non-destructively and individually. At present, pH value responsive fluorescent compounds mainly study the relative strength of pH value qualitatively through the change of fluorescence intensity, and its detection range is usually between 2.0-7.0. However, probes that can detect the pH value of alkaline environment and quantify the pH value are still relatively few.

[0008] Therefore, it is particularly important to develop a probe capable of quantifying the pH value of a solution and observing the pH value change in cells. This will provide more accurate and effective tools for research and application in the fields of chemical industry, biological medicine, etc. SUMMARY

[0009] The present application aims to provide a methoxynaphthalene fluorescent compound and its application.

[0010] The technical scheme of the present application is as follows:

[0011] The present application provides a methoxynaphthalene fluorescent compound, which has a chemical name of (E)-3-(2-hydroxyethyl)-2-(2-(6-methoxynaphthalene-2) vinyl) benzothiazole-3-iodine salt and a structural formula of:

[0012]

[0013] The fluorescent compound is composed of thiazole salt and methoxynaphthalene.

[0014] Since the pH value in cells is related to various diseases (cancer, etc.), the fluorescent compound of the present application can be used for preparing a pH value sensitive tumor diagnostic reagent, a fluorescent imaging reagent or a radioactive imaging reagent for tumors.

[0015] The present application also provides an application of the fluorescent compound in preparing a pH value sensitive tumor diagnostic reagent.

[0016] The present application also provides an application of the fluorescent compound in preparing a fluorescent imaging reagent or a radioactive imaging reagent for tumors.

[0017] The present application also provides an application of the fluorescent compound in detecting the pH value change in a solution and cells.

[0018] The fluorescent compound can be used to observe the pH value change in cells after staining the cells without washing the cells.

[0019] In order to balance the toxicity of the probe to cells and the staining effect, preferably, the concentration of the fluorescent compound for staining cells is 1-15 mu M.

[0020] In order to balance the cost and the staining effect, preferably, the concentration of the fluorescent compound in a solution is 1-15 mu M.

[0021] Further preferably, the concentration of the fluorescent compound is 10 mu M.

[0022] The present application also provides a preparation method of the fluorescent compound.

[0023] 2-methylbenzothiazole reacts with iodoethanol to obtain compound 2; the fluorescent compound is synthesized by Knoevenagel reaction of compound 2 and compound 3, and the reaction route is as follows:

[0024]

[0025] Beneficial effects:

[0026] When the methoxynaphthalene fluorescent compound is used as a fluorescent probe, the change of pH value in solution and cell can be detected. Compared with the probe for qualitatively studying the strength of pH value, the probe of the present application can quantize the pH value of solution and study the change of pH value in cell. Therefore, the fluorescent compound of the present application can be used for preparing a pH value sensitive tumor diagnosis reagent. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The figure is the absorption spectrum and fluorescence spectrum of TB (10 μM) in different solvents.

[0028] Figure 2 The figure is the absorption spectrum and fluorescence spectrum of TB (10 μM) in different pH value solutions.

[0029] Figure 3 The figure is the fitting curve of the fluorescence intensity of TB (10 μM) at 560 nm and pH value.

[0030] Figure 4 The figure is the confocal fluorescence image of TB (5 μM, 15 min) staining normal and chloroquine treated HeLa cells respectively. DETAILED DESCRIPTION

[0031] The technical scheme of the present application will be described in detail below through specific embodiments, but the protection scope of the present application is not limited to the embodiments.

[0032] The testing instrument of absorption spectrum is Hitachi U-2910 spectrophotometer; the fluorescence spectrum instrument is Hitachi F-2700 spectrophotometer, and the cell imaging instrument is lecia confocal microscope.

[0033] In the following embodiments, the materials, reagents and the like are obtained from commercial channels if not specially specified.

[0034] Example 1 Synthesis of fluorescent compound

[0035] (E)-3-(2-hydroxyethyl)-2-(2-(6-methoxynaphthalen-2)vinyl)benzothiazole-3-iodine salt (i.e. compound TB)

[0036] The reaction route is as follows:

[0037]

[0038] 1) Synthesis of benzothiazole iodide (compound 2)

[0039] Compound 1 (2-methylbenzothiazole, 10 mmol) and iodine ethanol (1.76 mL, 10 mmol) were dissolved in 25 mL of anhydrous ethanol, stirred in a flask at room temperature for 1 h. Then refluxed at 100 °C for 8 h, cooled and filtered, and washed with anhydrous EtOH for 3 times. After drying, white solid was obtained, which was compound 2 (mass: 3.24 g, yield: 92%).

[0040] 2) Synthesis of compound TB

[0041] Compound 2 (1 mmol) and compound 3 (1 mmol) were dissolved in 25 mL of methanol, stirred in a flask for 1 h, and 2 drops of piperidine were added. After stirring, refluxed at 90 °C for 10 h, cooled to room temperature, and washed with petroleum ether. Purified by column chromatography with CH2Cl2 / CH3OH mixture (12:1-7:1, v / v) as eluent to obtain yellow solid, which was compound TB (mass: 0.26 g, yield: 60%).

[0042] 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).

[0043] Example 2 Test experiment of photophysical properties

[0044] Test solution containing 10 μM TB was prepared with different solvents (the solvents used are shown in Table 1) Figure 1 , and the above solution was tested for its absorption spectrum with ultraviolet-visible spectrophotometer and for its fluorescence emission spectrum with fluorescence spectrometer, and the results are shown in Figure 1 .

[0045] As can be seen from the figure, the fluorescent compound TB has an absorption peak at 430 nm, and the absorption peak ranges from 350-500 nm (see Fig. A in Figure 1 ). It has a fluorescence peak in the range of 490-690 nm (see Fig. B in Figure 1 ). The above results show that the fluorescent compound can be excited with light of 350-500 nm, and its emission spectrum ranges from 490-690 nm.

[0046] Fluorescent test experiment of fluorescent compound TB in different pH value solutions

[0047] Experimental method:

[0048] (1) Take the organic fluorescent compound TB prepared in Example 1, and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0049] (2) Prepare PBS solutions with different pH values (pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0);

[0050] (3) Add the probe stock solution prepared in step (1) to the different pH value solutions prepared in step (2) to prepare test solutions, so that the final concentration of fluorescent probe TB in each solution is 10 μM;

[0051] (4) Test the absorption spectrum and fluorescence emission spectrum of the above solution using an ultraviolet-visible spectrophotometer and a fluorescence spectrometer to obtain the corresponding curves. TB has an absorption in the range of 355-500 nm, and the fluorescence emission spectrum ranges from 500-650 nm.

[0052] Measure the absorption spectrum of the above solution using an ultraviolet spectrophotometer, see Figure 2 A. Test the fluorescence emission spectrum using a fluorescence spectrometer, and fit the fluorescence spectrum to obtain the corresponding curve, see Figure 2 B.

[0053] As can be seen from the figure, as the pH value increases, the absorption peak is around 420 nm, and the fluorescence intensity increases with the increase of pH value. This indicates that the probe is sensitive to pH value.

[0054] Fit the fluorescence intensity at 560 nm with the pH value to obtain the fitting curve (see Figure 3 ), and the fitting formula is I = 535.92 x pH - 2348 (where I is the fluorescence intensity value, and the pH detection range is 6.0-9.0).

[0055] It can be seen that the probe can quantitatively measure the pH value in the solution.

[0056] Example 4: Observation of pH value change of probe TB in cells

[0057] HeLa cells were cultured in high glucose culture medium containing 10% fetal bovine serum, and incubated in a 37°C, 5% CO2 saturated humidity incubator. The culture medium was changed every 2-3 days, and the cells were subcultured.

[0058] When the cells grew to the logarithmic phase, the cells were cultured:

[0059] ① Cover glass was soaked in anhydrous ethanol for 30 min, dried by alcohol lamp and put into disposable 35 mm culture dish, ready for use;

[0060] ② The cells in 100 mL cell bottle were washed with PBS for three times, digested with 1 mL 0.25% trypsin for 5 min, and the trypsin was carefully poured out. Fresh culture solution was added and mixed evenly, and the cell density was controlled by adding culture solution to make the final concentration of cells 1 x 10 5 per mL. Then the cells were inoculated into the culture dish containing cover glass, and put into 5% CO2 incubator for culture, so that the cells grew tightly on the culture dish. After the HeLa cells grew and covered the cover glass, they were used for cell experiment.

[0061] The probe stock solution was prepared with DMSO at a concentration of 1 mM. The cultured active HeLa cells were incubated in culture solution containing 5 μM TB for 15 min, and 10 μM chloroquine solution was added to treat the cells for 2 h, and then observed by laser confocal microscope. The control group was directly dyed without any treatment. The colored parts in the cells, fluorescence distribution and brightness changes, colocalization information, etc. were recorded, and the results were shown in Figure 4 .

[0062] Among them, Figure 4 A in the formula is the confocal microscope image of normal and active HeLa cells treated with 10 μM chloroquine solution (acidic substances in cells were weakened) dyed with probe TB (5 μM, 15 min). Figure 4 B in the formula is the relative fluorescence intensity of active HeLa cells in 2 h. The excitation wavelength of TB in green channel is 488 nm, and the fluorescence collection wavelength is 500-600 nm. With the extension of time, the fluorescence intensity of the probe is enhanced. This confirms that the probe can observe the change of pH value in the cell.

[0063] Although the present application has been shown and described with reference to certain preferred embodiments, it is to be understood that such embodiments are merely illustrative of the present application and are not to be construed as limiting the application. Various changes in form and details can be made without departing from the spirit and scope of the present application as defined by the appended claims.

Claims

1. A methoxynaphthalene fluorescent compound, characterized in that, Its chemical name is: (E)-3-(2-hydroxyethyl)-2-(2-(6-methoxynaphthalene-2)vinyl)benzothiazole-3-iodide, and its structural formula is:

2. The use of the fluorescent compound of claim 1 in the preparation of pH-sensitive tumor diagnostic reagents.

3. The use of the fluorescent compound of claim 1 in the preparation of fluorescent imaging reagents or radioactive imaging reagents for tumors.

4. The use of the fluorescent compound of claim 1 in the preparation of detection solutions and test solutions for changes in intracellular pH.

5. The application according to claim 4, characterized in that, The fluorescent compound was used to stain cells without washing them, and changes in intracellular pH were observed.

6. The application according to claim 5, characterized in that, The concentration of the fluorescent compound used to stain cells was 1-15 μM.

7. The application according to claim 4, characterized in that, The concentration of the fluorescent compound in solution is 1-15 μM.

8. The application according to claim 6 or 7, characterized in that, The concentration of the fluorescent compound is 10 μM.

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