Detection method for specifically detecting hypochlorous acid

Abstract

The invention discloses a detection method for specific detection of hypochlorous acid, a fluorescence probe for detecting hypochlorous acid is adopted for detection, and the fluorescence probe is a hypochlorous acid (HClO) fluorescence probe with high sensitivity and high selectivity and can be used for monitoring the dynamic generation process of hypochlorous acid in living cells in a stress state. The probe is especially suitable for real-time and visual monitoring of the change of the hypochlorous acid level in living cells under the stimulation of lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) and the like. And a potential tool is provided for immunology and inflammation research, disease mechanism research, drug development and screening, environmental monitoring and food safety.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, and specifically to a method for the specific detection of hypochlorous acid. Background Technology

[0002] HClO, a major component of household bleach and disinfectants for swimming pools and drinking water, is widely encountered in daily life. More importantly, it plays a crucial role in the physiological and pathological processes of various organisms. Within organisms, especially living cells, HClO, within a certain concentration range, can participate in immune defense through inflammatory responses, eliminating invading bacteria and pathogens. However, HClO exceeding a certain concentration range can alter nucleic acids, proteins, and lipids through oxidation or chlorination, causing tissue damage and inducing various diseases ranging from inflammatory disorders to cancer, particularly immune and neurological disorders such as Alzheimer's disease, Parkinson's disease, stroke, cerebral ischemia, multiple sclerosis, depression, and schizophrenia. Therefore, precise and real-time monitoring of the content, distribution, and dynamic movement of HClO in organisms is of paramount clinical and research significance for a deeper understanding of its biological mechanisms and for the early diagnosis and treatment of related diseases.

[0003] Currently, there are many methods for detecting HClO, including traditional methods such as colorimetry, electrochemical methods, and chromatography. Relatively speaking, fluorescence sensing technology has advantages such as high selectivity, high sensitivity, low detection limit, simple operation, and the ability to perform real-time in-situ imaging, making it an indispensable tool in biomedical research. Fluorescent probe molecules can reveal the dynamic changes in their local microenvironment, making them excellent for monitoring the dynamic changes of HClO in vivo, especially in cells. Currently, some fluorescent probes have been applied to the detection of HClO in living cells. However, their relatively small Stokes shift and long response time limit their performance in imaging applications. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the specific detection of hypochlorous acid. Through molecular design, a fluorescent probe structure is obtained that can specifically recognize HClO in living cells, so that the fluorescent probe can recognize HClO in living cells and has the advantages of specificity, high sensitivity, short response time and large Stokes shift.

[0005] According to the purpose of this invention, a method for specifically detecting hypochlorous acid is provided, which uses a fluorescent probe for detecting hypochlorous acid. The specific chemical structural formula of the probe is shown below: ; The detection method includes the following steps: S1. The fluorescent probe is incubated with the cell or tissue sample to be detected, so that the probe can specifically target HClO in the cell or tissue sample; S2. Excite the sample at an appropriate excitation wavelength and collect the fluorescence signal emitted by the probe in the cells specifically recognizing HClO; S3. Detect, identify, quantify, or analyze HClO in the sample based on the fluorescence signal.

[0006] Furthermore, the method for preparing the probe includes the following steps: S1. Weigh out phosphorus pentoxide and dissolve it in an appropriate amount of phosphoric acid. Stir thoroughly to dissolve. Transfer an appropriate amount of ethyl acetoacetate and p-toluenethiophenol and mix them. Stir thoroughly to prepare a mixed solution. Slowly add the mixed solution to the phosphoric acid solution. Heat under reflux at 90°C for 1 hour. After the reaction is complete, return to room temperature, quench with water, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a white solid crude product. Purify by column chromatography to obtain white product 1. S2. Weigh intermediate 1 and malononitrile and dissolve them in an appropriate amount of acetic anhydride. Stir thoroughly to dissolve the raw materials and heat under reflux at 140°C for 4 hours. After the reaction is complete, restore to room temperature and add an appropriate amount of anhydrous methanol to the reaction mixture. Heat under reflux at 55°C for 2 hours and remove the reaction solvent by rotary evaporation. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a yellow solid crude product by rotary evaporation. Purify by column chromatography to obtain yellow product 1b. S3. Weigh 4-hydroxy-3,5-diiodobenzaldehyde and dissolve it in an appropriate amount of acetonitrile. Add anhydrous K2CO3 and stir thoroughly. Then add 4-bromomethylphenylboronic acid pinacol ester and heat under reflux at 80℃ for 12 hours. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain white product 2. S4. Weigh intermediate 1b and intermediate 2 and dissolve them in an appropriate amount of acetonitrile. Add piperidine and heat under reflux at 80°C for 8 hours. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain the orange-red product XL-TI.

[0007] Further, in step S1, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 15:1, the molar ratio of phosphorus pentoxide to phosphoric acid is 1:1, and the molar ratio of ethyl acetoacetate to p-toluenethiophenol is 1:1. In step S2, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: dichloromethane = 3:1, and the molar ratio of 1: malononitrile is 1:10. In step S3, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 8:1, and the molar ratio of 4-hydroxy-3,5-diiodobenzaldehyde: 4-bromomethylphenylboronic acid pinacol ester is 1:3. In step S4, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of 1b:2 is 1.2:1. The reactions in steps S3-S4 were all carried out under nitrogen protection.

[0008] Furthermore, the probe exhibits high sensitivity, good selectivity, and low cytotoxicity, and is capable of specifically recognizing intracellular HClO.

[0009] Furthermore, the method is used to monitor changes in HClO content within living cells.

[0010] Furthermore, the monitoring involves real-time monitoring of the dynamic processes of HClO in living cells under specific physiological or pathological stimuli.

[0011] Furthermore, the specific stimuli include, but are not limited to, lipopolysaccharide (LPS) and phorbol 12-tetradecanoate 13-acetate (PMA).

[0012] The beneficial effects of this invention are: The fluorescent probe XL-TI of this invention possesses high sensitivity, high selectivity, and low cytotoxicity. It can specifically recognize hypochlorous acid (HClO) in living cells, exhibiting a short response time (reaching a plateau within 30 seconds) and a large Stokes shift (167 nm), effectively reducing spectral crosstalk and improving detection accuracy and sensitivity. The probe demonstrates good stability and adaptability under different pH conditions, enabling real-time monitoring of the dynamic changes in intracellular HClO under stimulation by lipopolysaccharide (LPS) and phorbol 12-tetradecanoate 13-acetate (PMA). This method is simple to operate, uses inexpensive solvents, and involves convenient post-processing. The probe preparation process is also simple, providing an efficient and reliable detection tool for immunological research, disease mechanism exploration, drug screening, and environmental monitoring. Attached Figure Description

[0013] Figure 1 The synthetic route of probe XL-TI in this embodiment of the invention; Figure 2 The proton NMR spectrum of probe XL-TI in an embodiment of the present invention; Figure 3 The Stokes displacement of probe XL-TI in an embodiment of the present invention; Figure 4The response time of probe XL-TI to HClO in an embodiment of the present invention; Figure 5 The fluorescence spectra of the probe XL-TI for recognizing different substances are shown in the embodiments of the present invention. Figure 6 The fluorescence intensity of probe XL-TI under different concentrations of HClO in embodiments of the present invention; Figure 7 The fluorescence intensity of probe XL-TI against HClO at different pH values ​​is shown in the embodiments of the present invention. Figure 8 Cell imaging of probe XL-TI in an embodiment of the present invention; Figure 9 This is another cell imaging of the probe XL-TI in an embodiment of the present invention. Detailed Implementation

[0014] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0015] Example 1 like Figure 1 As shown, A specific detection method for hypochlorous acid is disclosed, which employs a fluorescent probe for detecting hypochlorous acid. The specific chemical structural formula of the probe is shown below: ; The detection method includes the following steps: S1. The fluorescent probe is incubated with the cell or tissue sample to be detected, so that the probe can specifically target HClO in the cell or tissue sample; S2. Excite the sample at an appropriate excitation wavelength and collect the fluorescence signal emitted by the probe in the cells specifically recognizing HClO; S3. Detect, identify, quantify, or analyze HClO in the sample based on the fluorescence signal.

[0016] Furthermore, the method for preparing the probe includes the following steps: S1. Weigh out phosphorus pentoxide and dissolve it in an appropriate amount of phosphoric acid. Stir thoroughly to dissolve. Transfer an appropriate amount of ethyl acetoacetate and p-toluenethiophenol and mix them. Stir thoroughly to prepare a mixed solution. Slowly add the mixed solution to the phosphoric acid solution. Heat under reflux at 90°C for 1 hour. After the reaction is complete, return to room temperature, quench with water, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a white solid crude product. Purify by column chromatography to obtain white product 1. S2. Weigh intermediate 1 and malononitrile and dissolve them in an appropriate amount of acetic anhydride. Stir thoroughly to dissolve the raw materials and heat under reflux at 140°C for 4 hours. After the reaction is complete, restore to room temperature and add an appropriate amount of anhydrous methanol to the reaction mixture. Heat under reflux at 55°C for 2 hours and remove the reaction solvent by rotary evaporation. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a yellow solid crude product by rotary evaporation. Purify by column chromatography to obtain yellow product 1b. S3. Weigh 4-hydroxy-3,5-diiodobenzaldehyde and dissolve it in an appropriate amount of acetonitrile. Add anhydrous K2CO3 and stir thoroughly. Then add 4-bromomethylphenylboronic acid pinacol ester and heat under reflux at 80℃ for 12 hours. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain white product 2. S4. Weigh intermediate 1b and intermediate 2 and dissolve them in an appropriate amount of acetonitrile. Add piperidine and heat under reflux at 80°C for 8 hours. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain the orange-red product XL-TI.

[0017] In step S1, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 15:1, the molar ratio of phosphorus pentoxide to phosphoric acid is 1:1, and the molar ratio of ethyl acetoacetate to p-toluenethiophenol is 1:1. In step S2, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: dichloromethane = 3:1, and the molar ratio of 1: malononitrile is 1:10. In step S3, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 8:1, and the molar ratio of 4-hydroxy-3,5-diiodobenzaldehyde: 4-bromomethylphenylboronic acid pinacol ester is 1:3. In step S4, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of 1b:2 is 1.2:1. The reactions in steps S3-S4 were all carried out under nitrogen protection.

[0018] The probe has high sensitivity, good selectivity and low cytotoxicity, and can specifically recognize HClO in cells.

[0019] The method described above is used to monitor changes in HClO content within living cells.

[0020] The monitoring refers to the real-time monitoring of the dynamic process of HClO in living cells under specific physiological or pathological stimuli.

[0021] The specific stimuli include, but are not limited to, lipopolysaccharide (LPS) and phorbol 12-tetradecanoate 13-acetate (PMA).

[0022] Example 2 like Figure 1 As shown, this embodiment provides a specific detection method for hypochlorous acid, which uses a fluorescent probe for detecting hypochlorous acid. The specific chemical structure of the probe is shown below: ; The preparation method of the fluorescent probe for detecting HClO: S1. Weigh out 72 g (0.5 mol) of phosphoric acid and dissolve it in 50 g (0.5 mol). Stir thoroughly to dissolve. Transfer 6.51 g (50.0 mmol) of ethyl acetoacetate and 6.21 g (50.0 mmol) of p-toluene to mix thoroughly to prepare a mixed solution. Slowly add the mixed solution to the phosphoric acid solution and heat under reflux at 90 °C for 1 h. After the reaction is complete, return to room temperature, quench with water, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a white solid crude product. Purify by column chromatography to obtain white product 1. S2. Weigh intermediate 1 (1.4 g, 8.0 mmol) and malononitrile (5.28 g, 80.0 mmol) and dissolve them in acetic anhydride (20 mL). Stir thoroughly to dissolve the raw materials and heat under reflux at 140 °C for 4 h. After the reaction is complete, return to room temperature and add anhydrous methanol (30 mL) to the reaction mixture. Heat under reflux at 55 °C for 2 h. Remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a yellow solid crude product by rotary evaporation. Purify by column chromatography to obtain yellow product 1b. S3. Weigh 0.373 g (1 mmol) of 4-hydroxy-3,5-diiodobenzaldehyde and dissolve it in acetonitrile (10 mL). Add anhydrous K2CO3 (0.207 g (1.5 mmol) and stir thoroughly. Then add 0.297 g (1 mmol) of 4-bromomethylphenylboronic acid pinacol ester and heat under reflux at 80 °C for 12 h. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain white product 2. S4. Weigh intermediate 1b (0.142 g, 0.6 mmol) and intermediate 2 (0.295 g, 0.5 mmol) and dissolve them in acetonitrile (10 mL). Add piperidine (0.5 mL, 5 mmol) and heat under reflux at 80 °C for 8 h. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain the orange-red product XL-TI. MRI results as follows Figure 2 As shown, XL-TI: 1 H NMR (400 MHz, Chloroform- d ) δ: 8.74 (s, 1H), 7.98 (s, 2H), 7.87 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.60 –7.53 (m, 2H), 7.47 (d, J = 8.3 Hz, 1H), 7.03 (s, 2H), 5.05 (s, 2H), 2.51 (s, 3H), 1.36 (s, 12H). Example 3 This embodiment provides a general fluorescence testing method: The fluorescent probe XL-TI of this invention was accurately weighed and dissolved in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a 1 mmol / L probe solution. Subsequently, 20 µL of the 1 mmol / L probe stock solution was added sequentially to a 5 mL centrifuge tube, followed by 1980 µL of the test solvent solution (such as DMSO:PBS buffer solution = 9 / 1 (v / v)). The mixture was then thoroughly mixed to obtain the working solution. The prepared working solution was subjected to fluorescence testing, with the following parameters: excitation slit width 10 nm, emission slit width 10 nm, and excitation wavelength 470 nm.

[0023] Example 4 Measurement of Stokes displacement using the XL-TI probe: The probe working solution was prepared according to the method described in Example 3, and fluorescence spectroscopy was performed. The Stokes shift was calculated by analyzing its excitation and emission spectra. (Refer to...) Figure 3 As shown, the Stokes shift of probe XL-TI is 167 nm. The results indicate that probe XL-TI exhibits a large Stokes shift, which is beneficial for reducing spectral crosstalk and improving detection sensitivity.

[0024] Example 5 Measurement of the response time of the XL-TI probe: Following the method described in Example 3, a working solution of probe XL-TI was prepared in DMSO:PBS buffer solution = 9 / 1 (v / v). The fluorescent probe XL-TI was then reacted with HClO at different time points. (Refer to...) Figure 4 As shown, the results indicate that the fluorescence intensity reaches a plateau at 30 s, indicating that the response time of the probe XL-TI is very short and the reaction with HClO is very rapid.

[0025] Example 6 Fluorescence spectra of XL-TI for recognizing different ions: Following the method described in Example 3, a working solution of probe XL-TI was prepared in DMSO:PBS buffer solution = 9 / 1 (v / v). The fluorescent probe XL-TI was then reacted with 15 different substances. (Refer to...) Figure 5 As shown, the results indicate that the fluorescent probe exhibits significant fluorescence enhancement only when it interacts with HClO. Example 7 Fluorescence intensity of XL-TI after reaction with different concentrations of HClO: Following the method described in Example 3, a working solution of probe XL-TI was prepared in DMSO:PBS buffer solution = 9 / 1 (v / v). The fluorescent probe XL-TI was then reacted with HOC at different concentrations. (Refer to...) Figure 6 As shown, the results indicate that the fluorescence intensity reaches its highest value when the concentration of probe XL-TI is 10 μmol / L and the concentration of HClO is 300 μmol / L.

[0026] Example 8 pH stability assessment of probe XL-TI: Following the method described in Example 3, the working solution of probe XL-TI was adjusted to different pH values, and fluorescence intensity tests were performed. (Refer to...) Figure 7As shown, the results indicate that the fluorescence intensity of probe XL-TI remained relatively stable under different pH conditions, without significant impact from pH. This demonstrates that probe XL-TI exhibits strong adaptability and practicality to environments with varying pH levels, further enhancing its versatility in biological applications.

[0027] Example 9 Cell imaging of probe XL-TI: HeLa cells were first incubated with different concentrations of HClO (0, 25, 50, 75, 100 μM) for 30 minutes. An additional group of HeLa cells was incubated with 100 μM HClO for 30 minutes, followed by incubation with 1 mM N-acetylcysteine ​​(NAC), a reactive oxygen species scavenger, for 1 hour. Finally, all six groups were incubated with a 10 μM probe for 30 minutes, as per the reference. Figure 8 As shown. Further investigation was conducted on cell imaging using the XL-TI probe after stimulation with lipopolysaccharide (LPS) and phorbol 12-tetradecanoate 13-acetate (PMA) followed by N-acetylcysteine ​​(NAC) repair. The control group was incubated with XL-TI probe and HeLa cells for 30 minutes; the experimental group was stimulated with LPS (1.0 μg / mL) for 12 hours, followed by PMA (1.0 μg / mL) for 1 hour, and finally incubated with XL-TI probe for 30 minutes; the inhibition group was stimulated with LPS (1.0 μg / mL) for 12 hours and PMA (1.0 μg / mL) for 1 hour, followed by pre-incubation with NAC (1.0 mM) for 1 hour, and finally incubated with XL-TI probe for 30 minutes. (Refer to...) Figure 9 As shown in the figure, the results indicate that the probe XL-TI not only responds well to exogenous HClO in cells, but also to endogenous HClO produced under LPS and PMA stimulation. This demonstrates that the probe XL-TI has a good monitoring effect on intracellular HClO, which is of great significance for the diagnosis and treatment of HClO-related diseases.

[0028] The detection method provided by this invention is simple to operate, uses inexpensive solvents, and has convenient post-processing.

[0029] The fluorescent probe provided by this invention is simple to prepare and low in cost; the fluorescent probe of this invention can recognize HClO in living cells and has a short response time and a large Stokes shift; the detection method provided by this invention is simple to operate, uses inexpensive solvents, and has convenient post-processing.

[0030] This invention provides a fluorescent probe with specific selectivity, high sensitivity, short response time, low cytotoxicity, and a large Stokes shift. The fluorescent probe disclosed in this invention has a large Stokes shift, which can significantly reduce spectral crosstalk and improve detection accuracy and sensitivity. Furthermore, the probe and related detection methods of this invention can be effectively used to monitor changes in HClO content in living cells, and are particularly suitable for real-time and accurate monitoring of the dynamic process of HClO content in living cells under various physiological stimuli such as lipopolysaccharide (LPS) and phorbol 12-tetradecanoate 13-acetate (PMA).

[0031] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for specifically detecting hypochlorous acid, characterized in that, A fluorescent probe for detecting hypochlorous acid was used for detection, and the specific chemical structure of the probe is shown below: ； The detection method includes the following steps: S1. The fluorescent probe is incubated with the cell or tissue sample to be detected, so that the probe can specifically target HClO in the cell or tissue sample; S2. Excite the sample at an appropriate excitation wavelength and collect the fluorescence signal emitted by the probe in the cells specifically recognizing HClO; S3. Detect, identify, quantify, or analyze HClO in the sample based on the fluorescence signal.

2. The method for specifically detecting hypochlorous acid according to claim 1, characterized in that, The method for preparing the probe includes the following steps: S1. Weigh out phosphorus pentoxide and dissolve it in an appropriate amount of phosphoric acid. Stir thoroughly to dissolve. Transfer an appropriate amount of ethyl acetoacetate and p-toluenethiophenol and mix them. Stir thoroughly to prepare a mixed solution. Slowly add the mixed solution to the phosphoric acid solution. Heat under reflux at 90°C for 1 hour. After the reaction is complete, return to room temperature, quench with water, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a white solid crude product. Purify by column chromatography to obtain white product 1. S2. Weigh intermediate 1 and malononitrile and dissolve them in an appropriate amount of acetic anhydride. Stir thoroughly to dissolve the raw materials and heat under reflux at 140°C for 4 hours. After the reaction is complete, restore to room temperature and add an appropriate amount of anhydrous methanol to the reaction mixture. Heat under reflux at 55°C for 2 hours and remove the reaction solvent by rotary evaporation. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a yellow solid crude product by rotary evaporation. Purify by column chromatography to obtain yellow product 1b. S3. Weigh 4-hydroxy-3,5-diiodobenzaldehyde and dissolve it in an appropriate amount of acetonitrile. Add anhydrous K2CO3 and stir thoroughly. Then add 4-bromomethylphenylboronic acid pinacol ester and heat under reflux at 80℃ for 12 hours. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain white product 2. S4. Weigh intermediate 1b and intermediate 2 and dissolve them in an appropriate amount of acetonitrile. Add piperidine and heat under reflux at 80°C for 8 hours. After the reaction is complete, return to room temperature, remove the reaction solvent by rotary evaporation, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and obtain a solid crude product by rotary evaporation. Purify by column chromatography to obtain the orange-red product XL-TI.

3. The method for specifically detecting hypochlorous acid according to claim 2, characterized in that, In step S1, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 15:1, the molar ratio of phosphorus pentoxide to phosphoric acid is 1:1, and the molar ratio of ethyl acetoacetate to p-toluenethiophenol is 1:

1. In step S2, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: dichloromethane = 3:1, and the molar ratio of 1: malononitrile is 1:

10. In step S3, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 8:1, and the molar ratio of 4-hydroxy-3,5-diiodobenzaldehyde: 4-bromomethylphenylboronic acid pinacol ester is 1:

3. In step S4, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of 1b:2 is 1.2:

1. The reactions in steps S3-S4 were all carried out under nitrogen protection.

4. The method for specifically detecting hypochlorous acid according to claim 1, characterized in that, The probe has high sensitivity, good selectivity and low cytotoxicity, and can specifically recognize HClO in cells.

5. The method for specifically detecting hypochlorous acid according to claim 1, characterized in that, The method described above is used to monitor changes in HClO content within living cells.

6. The detection method according to claim 5, characterized in that, The monitoring refers to the real-time monitoring of the dynamic process of HClO in living cells under specific physiological or pathological stimuli.

7. The detection method according to claim 6, characterized in that, The specific stimuli include, but are not limited to, lipopolysaccharide (LPS) and phorbol 12-tetradecanoate 13-acetate (PMA).

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