A benzothiazole fluorescent compound and its application in observing pH value

Abstract

The application discloses a benzothiazole fluorescent compound and application thereof in observing pH value. The fluorescent compound can detect pH value changes in solution and cells. When the fluorescent compound is used as a fluorescent probe, compared with existing probes for qualitatively studying relative strength and weakness of pH value, the probe can quantize solution pH value and study pH value changes in cells. In addition, the fluorescent compound can also be used for preparing a pH value sensitive tumor diagnosis reagent, a fluorescent imaging reagent or a radioactive imaging reagent for tumors.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a benzothiazole fluorescent compound and its application in pH observation. Background Technology

[0002] pH value is an important indicator in the chemical industry. It is a physical quantity that reflects the acidity or alkalinity of a solution and the concentration of hydrogen ions (H+) in the solution. + The pH value of a solution determines its chemical properties, allowing for better control and regulation of chemical reactions. pH also reflects changes in the intracellular microenvironment and is inextricably linked to biomolecular interactions, metabolite diffusion, and signal transduction. Therefore, pH is widely used in environmental protection, the food industry, and biomedicine.

[0003] Traditional methods for detecting pH values ​​mainly include acid-base indicators, pH meters, and pH test strips. To measure pH values ​​accurately and conveniently, researchers have continuously developed various detection instruments and methods, such as the glass electrode method, the ion-selective electrode method, and the conductivity method.

[0004] The advantages of the glass electrode method are high accuracy, fast response, and wide measurement range. However, because the electrodes are easily contaminated and damaged, this method requires periodic calibration.

[0005] The advantages of ion-selective electrode methods are accurate and reliable measurement results and wide applicability. However, the preparation of ion-selective electrodes is relatively complex and costly.

[0006] The advantage of the conductivity method is its simplicity and ease of use; it does not require specialized pH measuring instruments, only a general conductivity instrument. However, this method is not accurate enough for certain solutions, such as weak acid and weak base solutions.

[0007] Recently, organic fluorescent probes based on fluorescence imaging technology have become a powerful technique for in-situ, non-destructive, and independent visualization of pH changes. Currently, pH-responsive fluorescent compounds mainly qualitatively study the relative strength of pH values ​​through changes in fluorescence intensity, with a detection range generally between 2.0 and 7.0. Probes capable of detecting and quantifying pH values ​​in alkaline environments are relatively few.

[0008] Therefore, it is of great significance to develop a probe that can quantify the pH value of a solution and observe changes in pH value in cells. Summary of the Invention

[0009] Purpose of the invention: The purpose of this invention is to provide a benzothiazole fluorescent compound and its application in pH observation.

[0010] Technical solution: The objective of this invention is achieved through the following technical solution:

[0011] This invention provides a fluorescent compound with the following chemical structural formula:

[0012]

[0013] The fluorescent compound consists of two parts: a thiazole salt and N,N-diethylbenzene.

[0014] The present invention also provides the application of the fluorescent compound in pH detection for non-disease diagnostic and therapeutic purposes.

[0015] Furthermore, after the cells are stained with the fluorescent compound, changes in intracellular pH can be observed.

[0016] Furthermore, the fluorescent compound can stain cells without washing them to observe changes in intracellular pH.

[0017] To balance the toxicity of the probe to cells and the staining effect, the concentration of the fluorescent compound used to stain cells is 1-15 μM.

[0018] Furthermore, the fluorescent compound can detect changes in pH value in the solution.

[0019] To balance cost and staining effect, the concentration of the fluorescent compound in solution is 1-15 μM.

[0020] Furthermore, the concentration of the fluorescent compound is 10 μM.

[0021] The preparation method of the fluorescent compound is as follows:

[0022] 2-Methylbenzothiazole reacts with iodoethanol to give compound 2; compound 2 and compound 3 are then reacted with Knoevenagel to synthesize the fluorescent compound, the reaction route being as follows:

[0023]

[0024] The present invention also provides the application of the fluorescent compound in the preparation of pH-sensitive tumor diagnostic reagents.

[0025] Since the pH value in cells is related to a variety of diseases (such as cancer), the fluorescent compounds of the present invention can be used to prepare pH-sensitive tumor diagnostic reagents, and to prepare fluorescent imaging reagents or radioactive imaging reagents for tumors.

[0026] Beneficial effects:

[0027] The fluorescent compound described in this invention can detect changes in pH in solutions and cells. When used as a fluorescent probe, compared to existing probes that qualitatively study the relative strength of pH values, this probe can quantify solution pH and study changes in intracellular pH. Furthermore, the fluorescent compound described in this invention can also be used to prepare pH-sensitive tumor diagnostic reagents. Attached Figure Description

[0028] Figure 1 The absorption and fluorescence spectra of TN (10 μM) in different solvents are shown.

[0029] Figure 2 The absorption and fluorescence spectra of TN (10 μM) in solutions at different pH values ​​are shown.

[0030] Figure 3 The curve showing the fluorescence intensity of TN (10 μM) at 587 nm versus pH value is shown.

[0031] Figure 4 Confocal fluorescence images of HeLa cells stained with TN (5 μM, 15 min) for normal cells and chloroquine-treated cells, respectively. Detailed Implementation

[0032] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0033] The absorption spectroscopy was performed using a Hitachi U-2910 spectrophotometer; the fluorescence spectroscopy was performed using a Hitachi F-2700 spectrophotometer; and the cell imaging was performed using a Lecia confocal microscope.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are obtained commercially.

[0035] Example 1 Synthesis of Fluorescent Compounds

[0036] (E)-2-(4-(dimethylamino)-2-hydroxyvinyl)-3-(2-hydroxyethyl)benzothiazole-3-iodide (abbreviated as TN)

[0037] The reaction route is as follows:

[0038]

[0039] 1) Synthesis of benzothiazole iodide (compound 2)

[0040] Compound 1 (10 mmol of 2-methylbenzothiazole) and iodoethanol (1.72 mL, 10 mmol) were dissolved in 20 mL of anhydrous ethanol and stirred in a flask at room temperature for 1 hour. The mixture was then refluxed at 100 °C for 8 hours, cooled, filtered, and washed three times with anhydrous EtOH. After drying, a white solid, compound 2 (mass: 3.23 g, yield: 90%), was obtained.

[0041] 2) Synthesis of compound TN

[0042] Compound 2 (1 mmol) and compound 3 (1 mmol) were dissolved in 20 mL of methanol and stirred in a flask for 1 h. Five drops of piperidine were added. After stirring, the mixture was refluxed at 85 °C for 8 h. After cooling to room temperature, the mixture was washed with petroleum ether. The solution was purified by column chromatography using a CH2Cl2 / CH3OH mixture (10:1–6:1, v / v) as the eluent to obtain a yellow solid, which was compound TN (mass: 0.25 g, yield: 62%).

[0043] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 10.76 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 16. 0Hz,1H),8.04(d,J=12.0Hz,1H),7.80(d,J=8.0Hz,1H),7.71(t,J=8.0Hz,1H),7.62 (t,J=6.0Hz,1H),7.51(t,J=16.0Hz,1H),6.45(d,J=8.0Hz,1H),6.19(s,1H),5.14 (s,1H),4.77(t,J=4.0Hz,2H),:3.87(s,1H),3.42-3.47(m,1H),1.14-1.23(m,6H).

[0044] 13 C NMR(400MHz,DMSO-d6)δ(ppm):180.14,143.01,142.54,139.82,136.16,13 2.13,128.20,120.49,114.21,113.36,105.76,51.35,34.73,33.70,26.97.

[0045] Example 2: Photophysical Property Testing Experiment

[0046] Use different solvents (see [link to solvent list]). Figure 1 Prepare a test solution containing 10 μM TN. Measure the absorption spectrum of the solution using a UV-Vis spectrophotometer and the fluorescence emission spectrum using a fluorescence spectrometer. The results are shown in the table below. Figure 1 .

[0047] from Figure 1 It can be seen that the fluorescent compound TN has an absorption peak at 540 nm, and the absorption peak range is in the range of 400-600 nm. Figure 1 (A). It exhibits a fluorescence peak in the 550-675nm range ( Figure 1 (B) This indicates that the fluorescent compound can be excited by light in the range of 400-600 nm, and its emission spectrum ranges from 550-675 nm.

[0048] Example 3: Fluorescence test of fluorescent compound TN in solutions with different pH values

[0049] Experimental methods:

[0050] (1) Take the organic fluorescent compound TN prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0051] (2) Prepare PBS solutions with different pH values ​​(pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0);

[0052] (3) Add the probe stock solution prepared in step (1) to the solutions with different pH values ​​prepared in step (2) respectively to prepare test solutions, so that the final concentration of fluorescent probe TN in each solution is 10 μM.

[0053] (4) The absorption spectrum and fluorescence emission spectrum of the above solution were tested using a UV-Vis spectrophotometer and a fluorescence spectrometer to obtain the corresponding curves. TN has absorption in the range of 400-600 nm and fluorescence emission spectrum in the range of 535-715 nm.

[0054] The absorption spectra of the above solutions were measured using a UV spectrophotometer. The absorption spectra of TN (10 μM) in solutions at different pH values ​​are shown in the figure. Figure 2 A. Its fluorescence emission spectrum was measured using a fluorescence spectrometer, and the corresponding curve was obtained by fitting its fluorescence spectrum, as shown in the figure. Figure 2 B.

[0055] As shown in the figure, the absorption peak changes from 530 nm to 560 nm with increasing pH, and the fluorescence intensity decreases with increasing pH. This indicates that the probe is sensitive to pH.

[0056] Then, the fluorescence intensity at 587 nm was fitted to the pH value to obtain the fitting curve (see...). Figure 3 The fitting formula is I = -141.04 × pH + 1948.48 (where I is the fluorescence intensity value), which shows that the probe can quantitatively measure the pH value in the solution.

[0057] Example 4: Observation of cell pH changes using the TN probe

[0058] HeLa cells were cultured in a high-glucose culture medium containing 10% fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator. The culture medium was changed every 2-3 days and the cells were passaged.

[0059] Once the cells have grown to the logarithmic growth phase, mount them onto a slide for culture.

[0060] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and then place it in a disposable 35mm petri dish for later use;

[0061] ② Wash the confluent cells in a 100mL cell culture flask three times with PBS, digest with 1mL of 0.25% trypsin for 5 minutes, carefully pour off the trypsin, add fresh culture medium, mix well by pipetting, and count the cells. Control the cell density by adding culture medium to achieve a final cell concentration of 1×10⁶ cells / mL. 5 Each cell was seeded and then inoculated into a culture dish containing a coverslip, and placed in a 5% CO2 incubator to allow the cells to grow in close contact with the culture dish. Once the HeLa cells have grown and completely covered the coverslip, they can be used for cell experiments.

[0062] A 1 mM probe stock solution was prepared using DMSO. Viable HeLa cells were incubated in culture medium containing 5 μM TN for 15 min, then treated with 10 μM chloroquine solution for 2 h. Cells were observed using a laser confocal microscope. The control group was directly stained without any treatment. The stained areas, fluorescence distribution and brightness changes, and co-localization information in the cells were recorded. Results are shown in [Figure number missing]. Figure 4 .

[0063] in, Figure 4 Image A shows confocal microscopy images of normal HeLa cells stained with probe TN (5 μM, 15 min) and viable HeLa cells treated with 10 μM chloroquine (which reduces the acidity of acidic substances in the cells). Figure 4 In the figure, B represents the relative fluorescence intensity of viable HeLa cells after 2 hours. The excitation wavelength of TN in the green channel is 488 nm, and the fluorescence collection wavelength is 500-600 nm. The fluorescence intensity of the probe decreases over time. This confirms that the probe can observe changes in intracellular pH.

[0064] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A fluorescent compound, characterized in that, Its chemical structural formula is:

2. The application of the fluorescent compound of claim 1 in pH detection for non-disease diagnosis and treatment purposes.

3. The application according to claim 2, characterized in that, After the cells are stained with the fluorescent compound, changes in intracellular pH can be observed.

4. The application according to claim 3, characterized in that, The concentration of the fluorescent compound used to stain cells was 1-15 μM.

5. The application according to claim 2, characterized in that, The fluorescent compound can detect changes in pH in a solution.

6. The application according to claim 5, characterized in that, The concentration of the fluorescent compound in solution is 1-15 μM.

7. The application according to claim 3 or 5, characterized in that, The concentration of the fluorescent compound is 10 μM.

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

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

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