Benzothiadiazole fluorescent probe as well as preparation and application thereof

Abstract

The invention discloses a benzothiadiazole fluorescent probe as well as preparation and application thereof. According to the present invention, through the two-step Suzuki coupling reaction, the 4-pyridyl group and the N-methyl piperazine group are sequentially introduced, such that the benzothiadiazole fluorescent probe for lysosome polarity detection is simply, conveniently and efficiently synthesized; the probe disclosed by the invention has a remarkable positive solvent color effect, and the fluorescence intensity of the probe is reduced along with the increase of the polarity of a medium, so that specific fluorescence enhancement can be realized in a low-polarity lysosome microenvironment. The probe has excellent light stability and is suitable for long-time dynamic fluorescence imaging monitoring.

Description

Technical Field

[0001] This invention relates to an organic small molecule fluorescent probe, and more particularly to a benzothiadiazole fluorescent probe and its preparation and application. Background Technology

[0002] Polarity, as a crucial parameter characterizing changes in the microenvironment of organisms, participates in most cellular processes, including enzyme catalysis, protein activation, and lipid composition. Cell polarity is a characteristic feature of cellular function and regulation; when physiological and pathological activities of cells change, their polarity also alters accordingly. Therefore, detecting cell polarity is significant for monitoring different cellular states and contributes to a deeper understanding of related physiological and pathological processes. Furthermore, different organelles within the cell perform specific biological functions, and their local microenvironment polarity levels also differ. Therefore, detecting the polarity of different organelles is of great value for accurately interpreting changes in cellular state.

[0003] Lysosomes, as important organelles in eukaryotic cells, play crucial roles in physiological processes including the degradation of biomolecules, the recycling of cellular components, and metabolic regulation. Impaired lysosomal function can lead to pathological conditions such as lysosomal storage diseases, neurodegenerative diseases, cardiovascular diseases, and cancer. As vital intracellular degradation organelles, the dynamic regulation of lysosomal polarity characteristics, such as membrane potential, pH, and ion homeostasis, is essential for maintaining cellular function. Recent studies have shown that abnormal lysosomal polarity is closely related to the pathological progression of neurodegenerative diseases, cancer, and metabolic diseases (such as lysosomal storage diseases and diabetes). Therefore, establishing precise techniques for detecting dynamic changes in lysosomal polarity is of great significance for elucidating its regulatory mechanisms in life activities.

[0004] Fluorescence imaging technology, due to its non-invasiveness, high sensitivity, and excellent spatiotemporal resolution, has been widely used in cell biology. Fluorescent probes, as the core tool of this technology, are widely applied in areas such as dynamic monitoring of the cellular microenvironment, sensing of bioactive substances, disease diagnosis, and in vivo imaging, thanks to their high specificity, real-time visualization, and non-invasive detection. In the research and application of fluorescent probes, classic fluorescent cores such as coumarin, bodily oil, rhodamine, and fluorescein are often used as the core framework. Benzothiadiazole, as a novel hybrid fluorophore, possesses photophysical properties such as good photostability, high fluorescence intensity, and easy structural modification, and has been applied in the design and synthesis of fluorescent probes. Currently, although there are reports on fluorescent probes for detecting lysosomal polarity, existing research mainly focuses on obesity and inflammation models, and studies on the level and dynamic changes of lysosomal polarity in neural cells are yet to be reported; how to achieve quantitative determination of lysosomal polarity in neural cells based on small molecule fluorescent probes is an urgent technical problem to be solved. Summary of the Invention

[0005] Objectives of this invention: The first objective is to provide a benzothiadiazole fluorescent probe capable of targeting lysosomes and sensitive to dynamic changes in lysosomal polarity. The second objective is to propose a benzothiadiazole fluorescent probe, addressing the problem of how to prepare such probes. The third objective is to propose the application of benzothiadiazole fluorescent probes in targeting and labeling lysosomes in cells and / or detecting changes in lysosomal polarity, addressing the problem of how to target and label lysosomes in cells and / or detect changes in lysosomal polarity.

[0006] Technical solution: The present invention provides a benzothiadiazole fluorescent probe with the following structural formula:

[0007] Wherein, the X group is a substituted or unsubstituted piperazine group, and the Y group is a substituted or unsubstituted pyridinyl group.

[0008] Preferably, the X group is In this context, the R1, R2, R3, R4, and R5 groups are each independently selected from H, methyl, and ethyl.

[0009] Specifically, the chemical structural formula of the benzothiadiazole fluorescent probe is as follows: .

[0010] This invention synthesizes a fluorescent probe, BTDNN, for lysosomal polarity detection via a two-step Suzuki coupling reaction, sequentially introducing 4-pyridinyl and N-methylpiperazine groups. The probe uses benzothiadiazole as the fluorophore and modifies its ends with 4-pyridinyl and N-methylpiperazine as protonating groups; under acidic conditions, these groups undergo protonation, thereby achieving specific targeting of lysosomes. Co-localization experiments further confirm the specific targeting ability of BTDNN to lysosomes.

[0011] Secondly, the probe structure contains benzothiadiazole as a strong electron-withdrawing group and N-methylpiperazine as an electron-donating group, resulting in a significant intramolecular charge transfer effect. This effect causes the probe to exhibit a pronounced solvation-induced color change behavior. In highly polar media, a strong dipole-dipole interaction occurs between the probe and the solvent, and the excited-state energy is mainly dissipated non-radiatively, leading to a red shift in emission wavelength and a decrease in fluorescence intensity. In low-polarity media, the dipole-dipole interaction weakens, and the excited-state energy is mainly released through radiative transitions, resulting in a corresponding blue shift in emission wavelength. Based on this mechanism, the probe can achieve sensitive monitoring of lysosomal polarity.

[0012] Furthermore, this probe possesses excellent photophysical properties, such as large Stokes shift and good biocompatibility. More importantly, it can track the dynamic changes in lysosomal polarity of BV2 neurons in real time and achieve quantitative detection of lysosomal polarity. Therefore, this probe provides a novel and effective tool for studying the role of lysosomal polarity regulation in the development and progression of nervous system diseases.

[0013] The second aspect of this invention discloses a method for preparing the above-mentioned benzothiadiazole fluorescent probe, comprising the following steps:

[0014] Where Z is a halogen, and the R1, R2, R3, R4, and R5 groups are all independently selected from H or methyl.

[0015] Preferably, the phase transfer catalyst comprises at least one of tetrabutylammonium bromide, tetrabutylammonium tribromide, tetramethylammonium bromide, tetrapropylammonium chloride, and tetrabutylammonium iodide; and the palladium catalyst comprises at least one of tetra(triphenylphosphine)palladium, bis(triphenylphosphine)palladium dichloride, tris(dibenzylacetone)palladium, and bis(acetonitrile)palladium dichloride. The R1 group is methyl, the R2, R3, R4, and R5 groups are all H, and Z is Br or Cl.

[0016] Preferably, the molar ratio of compound I to compound II is 0.5-1.0:1.0-2.0.

[0017] Preferably, the heating reaction conditions are as follows: compound I, compound II, phase transfer catalyst, palladium catalyst, organic solvent and water are mixed to obtain a mixture, and the mixture is heated to 80-100℃ and stirred for 12-36 h; the organic solvent includes at least one of toluene, xylene, dichloromethane and dioxane.

[0018] Preferably, the preparation method of compound I is as follows:

[0019] Wherein, Z is Br or Cl; the palladium catalyst includes at least one of tetra(triphenylphosphine)palladium, bis(triphenylphosphine)palladium dichloride, tris(dibenzylacetone)palladium, and bis(acetonitrile)palladium dichloride.

[0020] Furthermore, in the preparation method of compound I, the heating conditions are reflux at 80-100℃ for 12-36 h; the molar ratio of 4-pyridineboronic acid to compound IV is 3-4:3-4; and the reaction solvent is a mixture of at least one of toluene, xylene, dichloromethane, and dioxane with water.

[0021] The third aspect of this invention discloses the application of the above-mentioned benzothiadiazole fluorescent probes in targeting and labeling lysosomes in cells and / or detecting changes in the polarity level of lysosomes in cells.

[0022] In some embodiments, the cell is one of a nerve cell, a microglia, or an astrocyte.

[0023] In some embodiments, the cells are living cells, and the polarity level change is a polarity change of lysosomes under physiological or pathological conditions.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Molecular design strategy: The fluorescent probe in this invention uses benzothiadiazole as the parent nucleus and introduces proton-responsive groups 4-pyridine and N-methylpiperazine in sequence through a two-step Suzuki coupling reaction. This structure can be protonated in an acidic environment, thereby achieving specific targeting of lysosomes.

[0025] (2) Spectral performance advantages: The fluorescent probe in this invention exhibits a significant positive solvochromic effect, and its fluorescence intensity is negatively correlated with the polarity of the medium, which can achieve specific fluorescence enhancement detection of low polarity lysosomal microenvironment.

[0026] (3) Imaging application characteristics: The fluorescent probe in this invention has excellent anti-photobleaching properties and is suitable for long-term dynamic fluorescence imaging monitoring. Based on the probe's excellent response to lysosomal polarity, this invention further applies it to assess the changes in polarity levels of BV2 lysosomes in nerve cells and quantify lysosomal polarity in BV2 nerve cells. Attached Figure Description

[0027] Figure 1 The hydrogen NMR spectrum of the probe BTDNN; Figure 2 The carbon NMR spectrum of the probe BTDNN; Figure 3 High-resolution mass spectrum of probe BTDNN; Figure 4 The results show the detection of the polar response optical properties of the probe BTDNN; Figure 5 The results show the polar response characteristics of the probe BTDNN. Figure 6 The results of photostability testing of the BTDNN probe; Figure 7 The results show the anti-interference capability test results of the BTDNN probe; Figure 8 The image shows the BV2 cytotoxicity test results of the probe BTDNN. Figure 9This is a colocalization map of the probe BTDNN and a commercially available lysosomal dye. Figure 10 This is a fluorescence imaging image of the probe BTDNN on the changes in lysosomal polarity of nerve cells BV2; Figure 11 This is a diagram showing the quantitative determination of the polarity of BV2 lysosomes in nerve cells by the probe BTDNN. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] Example 1: The structural formula of a benzothiadiazole fluorescent probe is as follows:

[0030] The probe is prepared as follows: (1) Synthesis of compound I:

[0031] Weigh 1.0 g (3.40 mmol) of 4,7-dibromo-2,1,3-benzothiadiazole, 0.42 g (3.42 mmol) of 4-pyridineboronic acid, 0.0476 g (0.04 mmol) of tetra(triphenylphosphine)palladium, and 2.35 g (17.03 mmol) of anhydrous potassium carbonate into a 100 mL round-bottom flask. Add 30 mL of a mixed solution of 1,4-dioxane and 30 mL of ultrapure water. Place the reaction system under a nitrogen atmosphere and stir, then reflux at 90 °C for 24 h. After the reaction is complete, allow it to cool naturally to room temperature and quench it quickly in 100 mL of water. Then transfer the mixed solution to a 250 mL separatory funnel and extract with dichloromethane (35 mL × 3). Combine the organic phases and wash successively with water (35 mL × 3) and saturated brine (35 mL × 3). The organic phase was dried over anhydrous magnesium sulfate, followed by rotary evaporation to remove dichloromethane. The crude product was purified by silica gel column chromatography using ethyl acetate:dichloromethane in a 1:2 (V:V) solvent system. Vacuum drying yielded compound I. The 1H NMR data of compound I are 1 ¹H NMR (400MHz, Chloroform-d) δ 8.79 (d, J=6.2Hz, 2H), 7.99 (d, J=7.6Hz, 1H), 7.92 (d, J=4.6Hz, 2H), 7.71 (d, J=7.6Hz, 1H). Its carbon NMR spectrum is as follows: 13CNMR (100MHz, Chloroform-d) δ 154.34, 152.86, 150.67, 144.26, 132.52, 129.31, 123.87, 115.78. Its high-resolution mass spectra [M+H] are also available. + The theoretical value is 291.9539, and the measured value is 291.9535.

[0032] (2) Synthesis of the fluorescent probe BTDNN:

[0033] Compound I (0.24 g, 0.84 mmol), 4-(4-methylpiperazine-1-phenyl)boronic acid pinacol ester (0.40 g, 1.33 mmol), tetrabutylammonium bromide (0.16 g, 0.50 mmol), tetra(triphenylphosphine)palladium (0.08 g, 0.07 mmol), and anhydrous potassium carbonate (2.20 g, 15.94 mmol) were weighed and placed in a 50 mL round-bottom flask. Toluene (10 mL) and ultrapure water (8 mL) were added, and the mixture was stirred under nitrogen protection and refluxed at 90 °C for 24 h. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, and the reaction solution was quenched in 100 mL of ultrapure water. The mixture was then transferred to a 250 mL separatory funnel and extracted with dichloromethane (35 mL × 3). The organic phases were combined and washed successively with water (35 mL × 3) and saturated brine (35 mL × 3). The organic phase was dried over anhydrous magnesium sulfate, followed by rotary evaporation to remove dichloromethane. The crude product was purified by silica gel column chromatography with dichloromethane:methanol = 50:1 (V:V) as the developing solvent. After vacuum drying, the target fluorescent probe BTDNN was obtained.

[0034] BTDNN's 1H NMR spectrum is as follows Figure 1 As shown, 1 ¹H NMR (400 MHz, Chloroform-d) δ 8.77 (d, J=6.0 Hz, 2H), 7.99–7.90 (m, 4H), 7.87 (d, J=7.4 Hz, 1H), 7.77 (d, J=7.4 Hz, 1H), 7.09 (d, J=8.8 Hz, 2H), 3.43–3.31 (m, 4H), 2.66–2.61 (m, 4H), 2.39 (s, 3H). Its carbon NMR spectrum is as follows: Figure 2 As shown, 13CNMR (100MHz, Chloroform-d) δ 153.97, 153.39, 151.16, 144.63, 134.75, 129.98, 128.80, 127.44, 126.21, 123.27, 115.33, 67.77, 54.69, 48.09, 45.85, 25.40. Its high-resolution mass spectra are as follows: Figure 3 As shown, [M+H] + The theoretical value is 388.1590, and the measured value is 388.1588.

[0035] Example 2: Everything else is the same as in Example 1, except that: In step (1), the amount of 4,7-dibromo-2,1,3-benzothiadiazole is 3 mmol and the amount of 4-pyridineboronic acid is 4 mmol; reflux at 80 °C for 36 h; 1,4-dioxane is replaced with dichloromethane; In step (2), 0.5 mmol of compound I and 1.0 mmol of 4-(4-methylpiperazine-1-phenyl)boronic acid pinacol ester were weighed; the mixture was refluxed at 80 °C for 36 h; tetrabutylammonium bromide was replaced with tetrabutylammonium tribromide, and toluene was replaced with xylene; Replace the tetra(triphenylphosphine)palladium in steps (1) and (2) with bis(triphenylphosphine)palladium dichloride.

[0036] Example 3: Everything else is the same as in Example 1, except that: In step (1), the amount of 4,7-dibromo-2,1,3-benzothiadiazole is 4 mmol and the amount of 4-pyridineboronic acid is 3 mmol; reflux at 100 °C for 12 h; 1,4-dioxane is replaced with toluene; In step (2), 1.0 mmol of compound I and 2.0 mmol of 4-(4-methylpiperazine-1-phenyl)boronic acid pinacol ester were weighed; the mixture was refluxed at 100 °C for 12 h; tetrabutylammonium bromide was replaced with tetrapropylammonium chloride, and toluene was replaced with dioxane; Replace the tetra(triphenylphosphine)palladium in steps (1) and (2) with tri(dibenzylideneacetone)dipalladium.

[0037] Comparative Example 1: Everything else is the same as in Example 1, except that: Replace compound II with the following compound: .

[0038] The final product obtained is: .

[0039] Comparative Example 2: The following existing compounds were used as fluorescent probe samples: .

[0040] Comparative Example 3: Everything else is the same as in Example 1, except that: Replace compound II with the following compound: .

[0041] The final product obtained is: .

[0042] Comparative Example 4: Everything else is the same as in Example 1, except that: Replace compound II with the following compound: .

[0043] The final product obtained is: .

[0044] Comparative Example 5: Everything else is the same as in Example 1, except that: Replace compound I with the following compound:

[0045] The final product obtained is: .

[0046] Comparative Example 6: Everything else is the same as in Example 1, except that: Replace compound I with the following compound:

[0047] The final product obtained is: .

[0048] Comparative Example 7: The following existing compounds were used as fluorescent probe samples: .

[0049] Comparative Example 8: Everything else is the same as in Example 1, except that: Replace compound I with the following compound:

[0050] The final product obtained is: .

[0051] Comparative Example 9: The following existing compounds were used as fluorescent probe samples: .

[0052] Comparative Example 10: Everything else is the same as in Example 1, except that: Replace compound II with the following compound:

[0053] The final product obtained is:

[0054] Comparative Example 11: The following existing compounds were used as fluorescent probe samples: .

[0055] The polar response optical properties of the fluorescent probe BTDNN prepared in Example 1 were tested using the following method: First, the probe BTDNN was dissolved in anhydrous toluene, ethyl acetate, 1,4-dioxane, N,N-dimethylformamide, and dimethyl sulfoxide to prepare 1 mM stock solutions. During testing, the stock solutions were diluted to a working concentration of 10 μM with the corresponding solvents, and its response behavior in different polar solvents was investigated using UV absorption and fluorescence spectroscopy.

[0056] The results are as follows Figure 4 As shown, Figure 4 Figure (A) shows the UV-Vis absorption spectra of the probe BTDNN in solvents of different polarities. Figure 4 Figure (B) shows the normalized fluorescence spectra of the probe BTDNN in different solvents. Figure 4 Figure (C) shows the relationship between the maximum fluorescence emission wavelength and the solvent polarity parameter E. T The relationship between (30); like Figure 4 As shown in Figure A, the probe exhibits a characteristic absorption peak around 430 nm in all the above solvents, and the maximum absorption wavelength changes little with solvent polarity, indicating that its dipole moment in the ground state is less affected by solvent polarity. Figure 4 The normalized fluorescence emission spectrum in Figure B shows that as the solvent polarity increases, the maximum emission peak of the probe gradually red-shifts, indicating that its fluorescence emission has a significant solvent dependence. Figure 4 Figure C further illustrates the relationship between the maximum fluorescence emission wavelength and the solvent polarity parameter E. T Quantitative standard curve between (30) and (30).

[0057] To further investigate the polar response characteristics of the probe BTDNN prepared in Example 1, this invention further selected a 1,4-dioxane / water mixed solvent system for research, and the polarity of the system was quantified by the polarity parameter Δf. The probe BTDNN was dissolved in anhydrous 1,4-dioxane to prepare a 1 mM stock solution, and during testing, it was diluted with different proportions of anhydrous 1,4-dioxane and water to a working concentration of 10 μM.

[0058] result Figure 5 As shown, Figure 5 Figure (A) shows the UV-Vis absorption spectra of the probe BTDNN in different proportions of water and 1,4-dioxane. Figure 5 Figure (B) shows the fluorescence spectra of the probe BTDNN in different proportions of water and 1,4-dioxane. Figure 5 Figure (C) shows the maximum emission wavelength (λ) of the probe BTDNN. em The relationship between (max) and solvent polarity parameter (Δf); like Figure 5 As shown in Figure A, the maximum absorption wavelength of the probe changes relatively little with solvent polarity. Figure 5 As shown in Figure B, when the proportion of 1,4-dioxane in the system increases from 40% (Δf = 0.301) to 91% (Δf = 0.223), the fluorescence intensity of the probe increases by about 11.6 times. Figure 5 The results in Figure C show that the maximum fluorescence intensity of the system exhibits a good linear relationship with Δf (0.223 - 0.301). Figure 7 The results confirmed that the BTDNN probe is a polarity-sensitive fluorescent probe.

[0059] The photostability and anti-interference performance of the probe BTDNN prepared in Example 1 were tested using the following method: Photostability test: The probe BTDNN was dissolved in 1,4-dioxane and a mixed solvent of 1,4-dioxane and 30% water (v / v) to prepare 10 μM working solutions. The fluorescence intensity at 630 nm was continuously monitored over 1 h at an excitation wavelength of 430 nm. The results are as follows: Figure 6 As shown, the fluorescence kinetics curves of the probe remained stable in both solvents with different polarities, indicating that it has good photostability under the measured conditions. Figure 6 The upper curve corresponds to the 1,4-dioxane system, and the lower curve corresponds to the mixed solvent system containing 30% water.

[0060] Anti-interference performance test: First, the probe BTDNN was dissolved in 1,4-dioxane to prepare a 1 mM stock solution. Different interfering substances were added to 20 mM PBS buffer (pH=7.4) to make the working concentration of the probe 10 μM and the concentration of the interfering substances 100 μM. The interfering substances were H2O2 and Fe2O3. 2+ NO2 - , S 2- , ClO - , AA, Fe 3+ Zn 2+ Cys, GSH, and Hcy were used. Fluorescence emission of each solution was measured at an excitation wavelength of 430 nm. Simultaneously, the fluorescence emission spectra of the blank group (10 μM probe) and the control group (10 μM probe + 80% 1,4-dioxane) were measured. Results are as follows... Figure 7 As shown, the fluorescence intensity of the probe BTDNN did not change significantly in the presence of the listed interfering substances, indicating that it has good anti-interference ability against common interfering substances.

[0061] The cytotoxicity of the probe BTDNN prepared in Example 1 was evaluated as follows: Cell culture: BV2 neural cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin (100 U / mL), and streptomycin (100 μg / mL). Cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0062] Cytotoxicity assay: The cytotoxicity of the probe BTDNN was evaluated using the MTT assay. Logarithmic-phase BV2 cells were seeded in 96-well plates. After cell attachment, the cells were co-incubated with a series of concentrations of probe BTDNN for 24 h. Subsequently, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 h. After incubation, formazan dissolving solution was added, and incubation continued for 4 h to allow the formazan crystals to fully dissolve. Finally, the absorbance of each well was measured at 570 nm using a microplate reader, and cell viability was calculated. Figure 8 As shown, the survival rate of BV2 cells remained above 80% when the probe BTDNN concentration was 15 μM. These results indicate that the probe possesses good biocompatibility and low cytotoxicity, making it suitable for subsequent fluorescence imaging studies in biological systems.

[0063] The probe BTDNN prepared in Example 1 was used in a cell colocalization experiment, as follows: To investigate the lysosomal localization ability of the probe BTDNN, the commercial lysosomal fluorescent probe LysoGreen was used for co-localization verification. BV2 cells were seeded in single-well culture dishes and cultured for 24 h, followed by washing two to three times with PBS. The cells were then incubated with the probe BTDNN (5 μM) for 30 min, and then co-incubated with LysoGreen for 30 min. After incubation, the cells were washed two to three times with PBS. Imaging was performed using laser confocal microscopy with an excitation wavelength of 405 nm. The emitted light was collected in the green channel (490–540 nm) and the red channel (600–650 nm), respectively. Results are shown below. Figure 9 As shown, the fluorescence signals of the probe BTDNN and LysoGreen highly overlap, with a Pearson correlation coefficient of 0.97, indicating that the probe has good targeting ability.

[0064] The Pearson correlation coefficients of the fluorescent probes synthesized or purchased in Examples 2-3 and Comparative Examples 1-11 with the commercial lysosomal dye (LysoGreen) were further determined, and the results are as follows: Table 1. Pearson correlation coefficients between different fluorescent probes and LysoGreen.

[0065] As can be seen from the results in Table 1, in Comparative Examples 1-11, when the methylpiperazine group, phenyl or pyridyl group attached to the benzothiadiazole core is missing or replaced by other similar groups or the position of the group substitution is changed, the ability of the corresponding compound to specifically target lysosomes is much lower than that of the probe BTDNN, making it difficult to use for precise targeting of lysosomes and further quantitative detection of changes in the polarity level of lysosomes in living cells.

[0066] The compound synthesized in Example 1 was used as a fluorescent probe sample for fluorescence imaging to detect changes in lysosomal polarity in BV2 cells, as follows: Dimethyl sulfoxide (DMSO) rapidly reduces lysosomal polarity, while dithiothreitol (DTT) increases it. To evaluate the ability of the probe BTDNN to monitor changes in lysosomal polarity, BV2 cells were treated with DMSO and DTT (1 mM and 5 mM), respectively, and then imaged using confocal laser scanning microscopy. Figure 10 As shown, the fluorescence intensity of cells treated with DMSO was significantly enhanced, while the fluorescence intensity of cells treated with DTT was significantly reduced. Figure 10 The results showed that the probe BTDNN was able to respond sensitively to changes in lysosomal polarity.

[0067] Based on the excellent ability of the probe BTDNN to monitor changes in lysosomal polarity, this invention further quantifies lysosomal polarity in BV2 cells. BTDNN-labeled cells are subjected to spectral scanning imaging to obtain the in-situ emission spectrum of the region of interest (ROI), and then... Figure 4 The standard curve of the C-plot is used to calculate the polarity parameter E. T (30). For example, Figure 11 As shown, the average E of the lysosomal microenvironment of BV2 cells was measured. T (30) The value is 35.8. Since the emission wavelength provided by spectral scanning imaging can effectively avoid the interference of fluorescence intensity by factors such as probe concentration and laser intensity, the results are more reliable.

Claims

1. A benzothiadiazole fluorescent probe, characterized in that, The structural formula is as follows: Wherein, the X group is a substituted or unsubstituted piperazine group, and the Y group is a substituted or unsubstituted pyridinyl group.

2. The benzothiadiazole fluorescent probe according to claim 1, characterized in that, The X group is In this context, the R1, R2, R3, R4, and R5 groups are each independently selected from H, methyl, and ethyl.

3. The benzothiadiazole fluorescent probe according to claim 2, characterized in that, The chemical structural formula is as follows: 。 4. The method for preparing the benzothiadiazole fluorescent probe according to claim 1, characterized in that, Includes the following steps: Where Z is a halogen, and the R1, R2, R3, R4, and R5 groups are all independently selected from H or methyl.

5. The method for preparing the benzothiadiazole fluorescent probe according to claim 4, characterized in that, The phase transfer catalyst includes at least one of tetrabutylammonium bromide, tetrabutylammonium tribromide, tetramethylammonium bromide, tetrapropylammonium chloride, and tetrabutylammonium iodide; the palladium catalyst includes at least one of tetra(triphenylphosphine)palladium, bis(triphenylphosphine)palladium dichloride, tris(dibenzylacetone)palladium, and bis(acetonitrile)palladium dichloride. The R1 group is methyl, the R2, R3, R4, and R5 groups are all H, and Z is Br or Cl.

6. The method for preparing the benzothiadiazole fluorescent probe according to claim 4, characterized in that, The molar ratio of compound I to compound II is 0.5-1.0:1.0-2.

0.

7. The method for preparing the benzothiadiazole fluorescent probe according to claim 4, characterized in that, The heating reaction conditions are as follows: Compound I, Compound II, phase transfer catalyst, palladium catalyst, organic solvent and water are mixed to obtain a mixture, and the mixture is heated to 80-100℃ and stirred for 12-36 h; the organic solvent includes at least one of toluene, xylene, dichloromethane and dioxane.

8. The method for preparing the benzothiadiazole fluorescent probe according to claim 4, characterized in that, The preparation method of compound I is as follows: Wherein, Z is Br or Cl; the palladium catalyst includes at least one of tetra(triphenylphosphine)palladium, bis(triphenylphosphine)palladium dichloride, tris(dibenzylacetone)palladium, and bis(acetonitrile)palladium dichloride.

9. The method for preparing the benzothiadiazole fluorescent probe according to claim 8, characterized in that, The heating conditions are reflux at 80-100℃ for 12-36 h; the molar ratio of 4-pyridineboronic acid to compound IV is 3-4:3-4; the reaction solvent is a mixture of at least one of toluene, xylene, dichloromethane, and dioxane with water.

10. The use of the benzothiadiazole fluorescent probe according to any one of claims 1-3 in targeting and labeling lysosomes in cells and / or detecting changes in the polarity level of lysosomes in cells.

Images