Fluorescent probe based on methoxybenzothiophene derivative as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to a fluorescent probe based on a methoxybenzothiophene derivative and a preparation method and application thereof. The fluorescent probe is composed of an energy donor naphthalimide, an energy receptor methoxy benzothiophene derivative and a connector (piperazine). The fluorescent probe can be used for detecting SO2 derivatives and viscosity.

Description

Technical Field

[0001] The invention belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to a fluorescent probe based on a methoxybenzothiophene derivative and a preparation method and application thereof. Background Art

[0002] Sulfur dioxide (SO 2 ) is a common environmental pollutant, with bisulfite / sulfite (HSO 3 - / SO 3 2- ) is widely present in the environment, food and organisms. When the concentration of sulfur dioxide in the environment is too high, acid rain is easily formed, posing a serious threat to the ecosystem. 2 Excessive levels can lead to metabolic abnormalities and even diseases, such as cardiovascular disease, endocrine abnormalities, cancer, etc.

[0003] The viscosity of the cell microenvironment not only reflects the physical properties of the microenvironment, but is also closely related to the physiological and pathological states of the cells. For example, a microenvironment with high viscosity may restrict the movement of cells and affect the migration path and speed of cells in tissues, which is of great significance in the study of embryonic development, tissue repair, and cancer metastasis. In addition, the tumor microenvironment usually has abnormal viscosity, which is related to the growth, invasion, and metastasis of cancer cells. By detecting the viscosity in the tumor microenvironment, it can help identify the aggressiveness of the tumor and predict its possibility of metastasis, thereby providing a basis for clinical diagnosis and treatment. Therefore, by performing viscosity testing in the cell microenvironment, researchers can have a deeper understanding of cell behavior, the impact of the microenvironment on cells, and how to use this information for applications and innovations in the fields of medicine and bioengineering.

[0004] Small molecule fluorescent probe method has many advantages such as good selectivity, strong anti-interference ability, high sensitivity and low detection limit, and has become a hot topic in current research; the ratio fluorescent probe based on fluorescence resonance energy transfer (FRET) / twisted intramolecular charge transfer (TICT) mechanism is generally composed of three parts: energy donor, linker and energy acceptor. The ratio fluorescent probe based on the FRET mechanism absorbs energy from the energy donor and transfers the energy to the acceptor through the intramolecular resonance process, and the probe exhibits enhanced acceptor fluorescence. The TICT process mainly occurs in the acceptor part. As the concentration of the detected object changes, the FRET process is gradually blocked, the acceptor cannot accept the energy of the donor, and the fluorescence emission intensity of the donor and the acceptor gradually changes.

[0005] Compared with traditional single-response fluorescent probes, dual-response fluorescent probes have the effect of "killing two birds with one stone". The currently reported dual-response fluorescent probes based on FRET / TBET mechanism for HSO 3- / SO 3 2- There are relatively few mitochondrial-targeting fluorescent probes with dual responses to mitochondria-sensing activity and viscosity. Summary of the invention

[0006] The first object of the present invention is to provide a fluorescent probe based on methoxybenzothiophene derivatives, wherein the fluorescent probe is composed of three parts: an energy donor naphthalimide, an energy acceptor methoxybenzothiophene derivative, and a linker (piperazine); the chemical structure is shown in formula (I): Formula (I).

[0007] The second object of the present invention is to provide a method for preparing the fluorescent probe based on methoxybenzothiophene derivatives, comprising the following steps: S1. Synthesis of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)benzoic acid: 4-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (1 mmol) and pyrrolidine (1.5 mol) were dissolved in DMF, and triethylamine was added as a catalyst. The mixture was reacted at 60°C for 12 h, and the reaction was stopped. The organic solvent was removed by rotary evaporation under reduced pressure, and the target product 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid was separated by column chromatography using silica gel powder (200-300 mesh); S2. Synthesis of (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt: 6-methoxy-2,3-dimethylbenzo[d]thiazol-3-ium salt (1.0 mmol) and 4-(piperazin-1-yl)benzaldehyde (1.2 mmol) were dissolved in anhydrous ethanol, heated under reflux for 12 hours, the reaction was stopped, the solvent was removed by rotary evaporation under reduced pressure, and then separated and purified by column chromatography, silica gel powder 200-300 mesh, and the developing solvent was a mixed solution of dichloromethane and methanol in a volume ratio of 1:25, to obtain the target product (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt; S3. Synthesis of target probe: Dissolve 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt in dry dichloromethane, add 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as catalysts, stir at room temperature for 24 hours, and stop the reaction; remove the solvent by rotary evaporation under reduced pressure, purify and separate the crude product by column chromatography, silica gel powder 200-300 mesh, and a mixed solution of dichloromethane and methanol in a volume ratio of 1:40 as a developing solvent to obtain the target product; Schematic diagram of the synthesis of the probe: .

[0008] Further, in step S3, the molar ratio of the 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt is 1:1; the molar ratio of the 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:10.

[0009] Furthermore, the amount of dry dichloromethane added is limited to 0.5 mmol of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid corresponding to 40 mL of dry dichloromethane.

[0010] The present invention also provides the fluorescent probe based on the methoxybenzothiophene derivative in HSO 3 - / SO 3 2- Application in detection.

[0011] The present invention also provides application of the fluorescent probe based on methoxybenzothiophene derivatives in viscosity detection.

[0012] Beneficial technical effects of the present invention: (1) The fluorescent probe of the methoxybenzothiophene derivative described in the present invention is in SO 2 When the derivative is absent, the probe is excited, the energy donor transfers energy to the energy acceptor, and the probe emits enhanced acceptor fluorescence; in SO 2 In the presence of derivatives, the double bond recognition site in the energy acceptor is bound to SO 2The derivative reacts, the energy acceptor structure changes, and the FRET process within the molecule is blocked. At this time, the probe is excited to emit fluorescence as an energy donor. 2 The concentration of the derivatives was different, and the probe and SO 2 The degree of reaction of the derivatives is different, which is manifested by changes in the fluorescence intensity of the two emission wavelengths of the probe.

[0013] (2) In addition, the energy acceptor part of the probe has an intramolecular TICT process. When the viscosity in the detection system increases, the TICT effect in the energy acceptor part weakens, and the probe emits enhanced fluorescence of the energy acceptor when excited. Based on the above changes, the detection of SO using ratio probes has been realized. 2 Derivatives and viscosity purposes.

[0014] (3) SO based on FRET / TICT mechanism described in the present invention 2 The derivative and viscosity dual-response ratiometric probe can not only quantitatively detect SO 2 The concentration and viscosity of the derivatives can be used to analyze SO in living cells. 2 The probe can be used to detect SO in food and water samples. 2 The probe is a good mitochondrial-targeted SO probe based on fluorescence resonance energy transfer and twisted intramolecular charge transfer mechanism. 2 Derivatives and viscosity dual-response fluorescent probes are expected to play an important role in environmental protection, food safety and clinical medicine, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the fluorescent probe prepared in Example 1 ( 1 1H NMR).

[0016] Figure 2 The fluorescent probe prepared in Example 1 selectively detects SO 2 Fluorescence spectra of the derivatives.

[0017] Figure 3 The fluorescent probe prepared in Example 1 detects SO 2 Fluorescence spectra of the derivatives were used to characterize their anti-interference ability.

[0018] Figure 4 The fluorescence intensity changes of the fluorescence spectrum of the fluorescent probe prepared in Example 1 at 530nm and 582nm, and their ratio (I 530 / I 582 ) and SO 2 Plot of the linear relationship between the concentrations of the derivatives.

[0019] Figure 5 The fluorescence intensity change of the fluorescence spectrum of the fluorescent probe prepared in Example 1 at 582 nm, and Ln(I 582 ) and viscosity.

[0020] Figure 6 The fluorescent probe prepared in Example 1 was used to detect endogenous SO in AGS cells incubated under different conditions. 2 Confocal fluorescence imaging of derivatives, green fluorescence channel (490-550nm) and red fluorescence channel (550-700nm); wherein: (a) is a cell imaging image under the conditions shown; (b) is a bar graph of the ratio of green channel fluorescence intensity to red channel fluorescence intensity under the conditions corresponding to (a); Figure 7 The confocal fluorescence imaging diagram of the viscosity change of AGS cells by the probe described in the present invention, with an excitation wavelength of 405 nm and a red channel of 550-700 nm, wherein: (a) is a cell imaging diagram under the conditions shown; (b) is a red channel relative fluorescence intensity ratio diagram under the conditions corresponding to (a).

[0021] Figure 8 Schematic diagram of the imaging of the organelle localization of the probe of the present invention in living AGS cells, wherein: (a) is the imaging diagram of the probe of the present invention, λ ex =405nm, green channel: 490-550nm; (b) is the imaging image of the commercial dye Mito-Tracker™ Red targeting mitochondria ex =647nm, red channel: 647-700nm; (c) is the overlapping image of (a) and (b); (d) is a schematic diagram of the colocalization coefficient. DETAILED DESCRIPTION

[0022] The following is a detailed description of the present invention in conjunction with specific embodiments. The examples described below are only the best implementation of the present invention and are not intended to limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the implementation according to the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0023] The drugs, reagents, consumables and cells used in the present invention are all commercially available products.

[0024] Example 1 S1. Synthesis of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)benzoic acid: 4-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (1 mmol) and pyrrolidine (1.5 mol) were dissolved in DMF, and triethylamine was added as a catalyst. The mixture was reacted at 60°C for 12 h, and the reaction was stopped. The organic solvent was removed by rotary evaporation under reduced pressure, and the target product 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid was separated by column chromatography using silica gel powder (200-300 mesh); S2. Synthesis of (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt: 6-methoxy-2,3-dimethylbenzo[d]thiazol-3-ium salt (1.0 mmol) and 4-(piperazin-1-yl)benzaldehyde (1.2 mmol) were dissolved in anhydrous ethanol, heated under reflux for 12 hours, the reaction was stopped, the solvent was removed by rotary evaporation under reduced pressure, and then separated and purified by column chromatography, silica gel powder 200-300 mesh, and the developing solvent was a mixed solution of dichloromethane and methanol in a volume ratio of 1:25, to obtain the target product (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt; S3. Dissolve 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid II (0.5 mmol, 193.1 mg) and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt III (0.5 mmol, 246.5 mg) in dry dichloromethane (40 mL), add 4-dimethylaminopyridine (DMAP, 0.1 mmol, 12.5 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 1 mmol, 191.0 mg) as catalysts, stir at room temperature for 24 hours, and stop the reaction. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified and separated by column chromatography using silica gel powder 200-300 mesh and a mixed solution of dichloromethane and methanol (MeOH / DCM, 1 / 40, V / V) as the developing solvent. The yield of the target product was 36%.

[0025] The composite image of step 3 is as follows: .

[0026] Spectrum data for confirmation of the probe molecular structure prepared in Example 1: HRMS (ESI): m / z, calculated [C 44 H 40 O 4 N 5 S]+ It is 734.2796, and the actual measured value is 734.2805.

[0027] 1 HNMR (400 MHz, DMSO- d 6 ): δ=2.041(s, 4H), 3.590(s, 6H), 3.810(s, 6H), 3.906(s, 3H), 4.242(s, 3H), 6.948(d, J=8.8H z, 1H), 7.101 (d, J=8.8Hz), 7.400 (dd, J=9.2and2.4Hz, 1H), 7.445 (d, J=8.8Hz, 2H), 7.601 (d , J=8.0Hz, 2H), 7.652-7.718 (m, 2H), 7.935 (d, J=8.0Hz, 3H), 8.013 (d, J=15.6Hz, 1H), 8.055 7(d, J=9.6Hz, 1H), 8.270(d, J=8.4Hz, 1H), 8.463(d, J=7.2Hz, 1H), 8.820(d, J=8.8Hz, 1H), see Figure 1 .

[0028] 13 CNMR (100 MHz, DMSO- d 6 ): δ=26.06, 36.37, 53.43, 56.00, 56.70, 107.06, 109.02, 109.36, 114.45, 115.82, 117.68, 122.37, 123.77, 124.09, 128.09, 1 29.44, 129.93, 131.31, 131.63, 132.58, 133.58, 136.60, 153.03, 153.52, 159.52, 163.04, 163.49, 164.52, 169.31, 169.80.

[0029] Example 2 In a 10 ml volumetric flask containing 5 μM of the probe prepared in Example 1, the volume was fixed (EtOH / PBS, V / V, 5 / 5, pH = 7.4), and then 50 μl of Hcy, C 2 O 4 2- , CH 3 COO - , SO 4 2- , HCO 3 - , CO 32- , F - ,Br - , Cys, I, Gly, NO 3 - , S 2 O 3 2- , NH 4 + , Cl - , S 2- , K + , Zn 2+ ,Mg 2+ , Cu 2+ , Al 3+ Plasma, followed by fluorescence testing.

[0030] Test results such as Figure 2 As shown in the figure, the orange curve represents HSO 3 ⁻ / SO 3 ²⁻, which is obviously different from the fluorescence intensity curves of other ions, indicating that HSO 3 ⁻ / SO 3 ²⁻It has a significant inhibitory effect on the fluorescence intensity. For other anions and cations such as Hcy, C 2 O 4 ²⁻,CH 3 COO⁻,SO 4 ²⁻, HCO 3 ⁻, CO 3 ²⁻, F⁻, Br⁻, Cys, I⁻, Gly, NO 3 ⁻,S 2 O 3 ²⁻, NH 4 ⁺, Cl⁻, S²⁻, K⁺, Zn²⁺, Mg²⁺, Cu²⁺, Al³⁺, etc. did not significantly change the peak position or intensity of the fluorescence intensity, which indicates that the above ions have little interference with the intensity and peak value of the fluorescent probe prepared in Example 1. In summary, the fluorescent probe prepared in Example 1 has good performance in detecting HSO 3 ⁻ / SO 3 ² Highly selective.

[0031] Example 3 Into a 10 ml volumetric flask containing 5 μM of the probe prepared in Example 1, the volume was fixed (EtOH / PBS, V / V, 5 / 5, pH = 7.4), and then 50 μl of Hcy, C 2 O 4 2- , CH 3 COO - , SO 42- , HCO 3 - , CO 3 2- , F - ,Br - , Cys, I, Gly, NO 3 - , S 2 O 3 2- , NH 4 + , Cl - , S 2- , K + , Zn 2+ ,Mg 2+ , Cu 2+ , Al 3+ plasma, and then add 50 μl of HSO 3 - / SO 3 2- After the effect is complete, a fluorescence test is performed and the results are as follows Figure 3 As shown in the figure, it can be seen that even in the presence of other ions, the fluorescent probe prepared in Example 1 can still achieve the effect of HSO 3 ⁻ / SO 3 ²⁻, indicating that the fluorescent probe detects HSO 3 - / SO 3 2- Has good anti-interference ability.

[0032] Example 4 A 10 ml volumetric flask containing 5 μM of the fluorescent probe prepared in Example 1 was fixed to volume (EtOH / PBS, V / V, 5 / 5, pH = 7.4), and then different concentrations of HSO were added using a microinjector. 3 - / SO 3 2- , and then conduct fluorescence test. The results are as follows Figure 4 As shown, it shows that the fluorescent probe increases with SO 2 With the increase of derivative concentration, the fluorescence emission intensity of the probe at 530 nm gradually increased, and the fluorescence emission intensity at 582 nm gradually decreased; the fluorescence intensity ratio of the two emission peaks (I 530 / I 582 ) relative to SO 2 The concentration of the derivatives is linear within a certain range, that is, the fluorescent probe can quantitatively detect HSO 3 - / SO 32- concentration.

[0033] Example 5 To a 10 ml volumetric flask containing 5 μM of the fluorescent probe prepared in Example 1, a PBS / glycerol mixed solution with different PBS contents was added to the volume. Adding glycerol to the PBS buffer can increase the viscosity of the solution, thereby affecting the diffusion rate of molecules or particles in the solution. In this example, different proportions of PBS-glycerol mixed solutions were used to simulate the in vivo environment. The glycerol ratio was increased from 0% to 99.5%, and the fluorescence test was performed after mixing evenly. The results are shown in FIG. Figure 5 As shown in FIG. 1 , as the proportion of glycerol increases, the color of the fluorescence changes from purple to orange. The fluorescence intensity of the fluorescent probe at 582 nm increases with the increase of the proportion of glycerol, that is, with the increase of viscosity. In addition, Ln(I 582 ) value and the glycerol ratio, i.e., viscosity, are linearly related.

[0034] Example 6 Control group (Ctr): AGS cells were incubated with the fluorescent probe (4 μM) solution prepared in Example 1 for 1 h, then rinsed three times with PBS, and then confocal imaging was performed; GSH / Na 2 S 2 O 3 Group: AGS cells were first incubated with the fluorescent probe (4 μM) solution for 1 h, and then with GSH (500 μM) and Na 2 S 2 O 3 (250 μM) were incubated for 1 h, washed three times with PBS, and subjected to confocal imaging; GSH group: AGS cells were first incubated with the probe (4 μM) solution for 1 h, then incubated with GSH (500 μM) for 1 h, washed three times with PBS, and then confocal imaging was performed; TNBS / GSH / Na 2 S 2 O 3 Group: AGS cells were first incubated with TNBS (500 μM) for 1 h, then incubated with the probe (4 μM) for 1 h, and finally incubated with GSH (500 μM) / Na 2 S 2 O 3 (250 μM) for 1 h, washed three times with PBS, and then confocal imaging was performed.

[0035] The results are as follows Figure 6 As shown: In GSH / Na 2 S 2 O 3 Under the co-existence conditions, AGS cells can produce endogenous SO2 Derivatives, the green fluorescence intensity of the probe in the cell is enhanced, the red fluorescence intensity is weakened, and the relative fluorescence intensity ratio I Green / I Red Significantly increased; in TNBS / GSH / Na 2 S 2 O 3 Under the same conditions, TNBS can inhibit endogenous SO 2 The above experiments show that the probe can effectively detect endogenous SO in living cells. 2 derivative.

[0036] Example 7 The AGS cells were incubated with the fluorescent probe (4 μM) prepared in Example 1 for 1 h, and then incubated with different concentrations of nystatin (0, 10, 30, 40 μM) for 0.5 h. After washing with PBS three times, confocal imaging was performed. The red fluorescence channel was 550-700 nm. Incubation of cells with nystatin resulted in an increase in intracellular viscosity. The results are shown in Figure 2. Figure 7 As shown, as the concentration of nystatin in the cultured cells increases, the intracellular viscosity gradually increases, and the red fluorescence of the probe in the cells gradually increases, indicating that the fluorescent probe can effectively detect the intracellular viscosity.

[0037] Example 8 (a) AGS cells were incubated with the fluorescent probe (4 μM) prepared in Example 1 for 1 h, washed 3 times with PBS, and then imaged with confocal microscopy. ex =405nm, green channel: 490-550nm; see the results Figure 8 a, the fluorescent probe has strong green fluorescence in mitochondria; (b) AGS cells were incubated with Mito-Tracker™ Red (0.2 μM), a commercial dye targeting mitochondria, for 1 h, washed three times with PBS, and then subjected to confocal imaging. ex =647nm, red channel: 647-700nm; Mito-Tracker™ Red is a fluorescent dye commonly used for mitochondrial staining. It can specifically target and label mitochondria in living cells. Mito-Tracker™ Red has strong red fluorescence in mitochondria. Figure 8 b; Figure 8 c is Figure 8 a and Figure 8 The overlap diagram of b shows that the two have achieved good overlap. Figure 8d is a schematic diagram of the co-localization coefficient of the two, and the coefficient is 0.93. The co-localization experimental results show that the fluorescent probe has a high mitochondrial localization targeting.

[0038] Example 9 Accurately weigh 20g of white sugar and prepare 1000ml of PBS buffer containing sucrose. Add 50μl of probe stock solution to a 10ml volumetric flask, then use EtOH / PBS (V / V, 5 / 5, pH=7.4) to make up the volume. Then measure the fluorescence and obtain the fluorescence intensity ratio I 530 / I 582 , substitute into the working curve obtained in Example 4, and calculate its SO 2 Derivative content. Then, different concentration gradients of HSO were added dropwise 3 - / SO 3 2- (8, 10, 15 μM), and measured the fluorescence spectrum to calculate the spike recovery rate of the probe. The determination method for white wine is shown above. The results are shown in Table 1. The calculated spike recovery rates are all in the range of 86%-108%, indicating that the probe can detect SO in actual samples. 2 The derivatives have high accuracy, anti-interference ability and reproducibility.

[0039] Table 1 The probes for SO in white sugar and white wine 2 Determination of derivative concentration and spike recovery .

Claims

1. A fluorescent probe based on a methoxybenzothiophene derivative, characterized in that: The fluorescent probe is composed of three parts: energy donor naphthalene imide, energy acceptor methoxybenzothiophene derivative, and linker (piperazine); the chemical structure is shown in formula (I): Formula (I).

2. A method for preparing a fluorescent probe based on a methoxybenzothiophene derivative as claimed in claim 1, characterized in that: The steps include: S1. Synthesis of 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)benzoic acid: 4-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and pyrrolidine were dissolved in DMF, and triethylamine was added as a catalyst. The reaction was carried out at 60° C. for 12 h, and the reaction was stopped. The organic solvent was removed by rotary evaporation under reduced pressure, and the target product 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid was separated by column chromatography using silica gel powder 200-300 mesh; S2. Synthesis of (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt: Dissolve 6-methoxy-2,3-dimethylbenzo[d]thiazol-3-ium salt and 4-(piperazin-1-yl)benzaldehyde in anhydrous ethanol, heat and reflux for 12 hours, stop the reaction, remove the solvent by vacuum rotary evaporation, and separate and purify by column chromatography, silica gel powder 200-300 mesh, and a mixed solution of dichloromethane and methanol in a volume ratio of 1:25 as a developing solvent to obtain the target product (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt; S3. Synthesis of target probe: Dissolve 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (supplementary preparation method) and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt in dry dichloromethane, add 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as catalysts, stir at room temperature for 24 hours, and stop the reaction; remove the solvent by rotary evaporation under reduced pressure, and purify and separate the crude product by column chromatography, using silica gel powder 200-300 mesh and a mixed solution of dichloromethane and methanol in a volume ratio of 1:40 as a developing solvent to obtain the target product; The schematic diagram of the synthesis of the target probe is as follows: 。 3. The method for preparing a fluorescent probe based on a methoxybenzothiophene derivative according to claim 2, characterized in that: In step S1, the molar ratio of 4-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid to pyrrolidine is 1:1.5; In step S2, the molar ratio of 6-methoxy-2,3-dimethylbenzo[d]thiazol-3-ium salt to 4-(piperazin-1-yl)benzaldehyde is 1:1.2; In step S3, the molar ratio of the 4-(1,3-dioxo-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid and (E)-6-methoxy-3-methyl-2-(4-(piperazin-1-yl)phenylvinyl)benzo[d]thiazol-3-ium salt is 1:1; the molar ratio of the 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:

10.

4. Use of the fluorescent probe based on methoxybenzothiophene derivatives as claimed in claim 1 in the detection of SO2 derivatives.

5. The use of the fluorescent probe based on methoxybenzothiophene derivatives in the detection of SO2 derivatives according to claim 4, characterized in that: The fluorescent probe is selective in detecting SO2 derivatives.

6. The use of the fluorescent probe based on methoxybenzothiophene derivatives in the detection of SO2 derivatives according to claim 4, characterized in that: The fluorescent probe is measured by the fluorescence intensity ratio I 530 / I 582 Realize the quantitative detection of the concentration of SO2 derivatives.

7. The use of the fluorescent probe based on methoxybenzothiophene derivatives in the detection of SO2 derivatives according to claim 4, characterized in that: The fluorescent probe is used for detecting endogenous SO2 derivatives in cells.

8. Use of the fluorescent probe based on methoxybenzothiophene derivatives as claimed in claim 1 in viscosity detection.

9. The use of the fluorescent probe based on methoxybenzothiophene derivatives in viscosity detection according to claim 8, characterized in that: The fluorescent probe is used for detecting intracellular viscosity.

10. Use of the fluorescent probe based on methoxybenzothiophene derivatives as claimed in claim 1 in mitochondrial localization and targeting.

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