A ratio-type fluorescent probe for simultaneously distinguishing hypochlorous acid and hydrogen peroxide

Abstract

The application discloses a ratio type fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and hydrogen peroxide, and belongs to the technical field of chemical analysis and detection, and a molecular structural formula of the probe is as follows: In the detection process, the probe has the advantages of good selectivity, high sensitivity and the like. The probe can simultaneously distinguish and detect hypochlorous acid and hydrogen peroxide in cells and in vivo, and has the function of detecting the levels of hydrogen peroxide and hypochlorous acid in alcoholic liver injury. The excellent performances show that the fluorescent probe has important application values in the fields of environment and biology.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and detection technology, specifically relating to a ratiometric fluorescent probe that can simultaneously distinguish and detect hypochlorous acid and hydrogen peroxide, and its application in environmental and biological systems. Background Technology

[0002] Reactive oxygen species (ROS), as essential intracellular and intercellular substances, play a crucial role in cell signaling, differentiation, migration, cellular immunity, and the body's defense against pathogens. ROS species include singlet oxygen, hydrogen peroxide, superoxide radicals, and hypochlorous acid. Among them, hydrogen peroxide (H₂O₂) is the most frequently reported ROS signaling molecule, possessing multiple indispensable functions in cell signal transduction and homeostasis. Excessive hydrogen peroxide can cause oxidative damage, associated with various diseases such as cancer, neurodegenerative diseases, inflammation, and cardiovascular diseases. Hypochlorous acid (HClO) is a strong oxidant with bactericidal properties, capable of combating microbial invasion by the immune system. However, abnormal accumulation of hypochlorous acid in the body can cause severe tissue damage, leading to a series of diseases, including neurodegenerative diseases, ischemia, rheumatoid arthritis, and cancer. In organisms, the levels of H₂O₂ and HClO maintain a defined normal range and a dynamic equilibrium. Disruption of this equilibrium can trigger a series of diseases. Therefore, tools capable of simultaneously detecting hydrogen peroxide and hypochlorous acid are urgently needed.

[0003] Fluorescent probe technology is a non-invasive bioluminescent imaging and detection method, and its advantages of high sensitivity and high spatiotemporal resolution have attracted the attention of researchers. Although many types of probes can specifically detect hydrogen peroxide or hypochlorous acid, those that can simultaneously distinguish between the two are very rare. Furthermore, ratiometric fluorescent probes, through self-calibration of the two fluorescence signals, can reduce interference from probe concentration, instrument factors, and the environment, thereby improving their detection accuracy. To date, ratiometric fluorescent probes capable of simultaneously distinguishing between hypochlorous acid and hydrogen peroxide are very rare, and their application in disease models has not been extensively explored. Therefore, developing ratiometric fluorescent probes capable of simultaneously distinguishing between hypochlorous acid and hydrogen peroxide, and applicable to exogenous and endogenous sources in cells / living tissues and disease models, is of great significance. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention aims to provide a ratiometric fluorescent probe that can simultaneously distinguish and detect hypochlorous acid and hydrogen peroxide.

[0005] The fluorescent probe of this invention has the following molecular structure:

[0006] The fluorescent probe in this invention is prepared via the following synthetic route:

[0007]

[0008] The fluorescent probe detection mechanism in this invention is as follows:

[0009] The fluorescent probe itself emits red fluorescence. After reacting with hypochlorous acid, the S atom on the phenothiazine is oxidized to -S=O, and the fluorescence of the probe changes from red to green. Therefore, the probe can achieve ratiometric detection of hypochlorous acid. When reacting with hydrogen peroxide, the borate ester is released, the coumarin part emits blue fluorescence, while the red fluorescence of the phenothiazine coumarin part does not change. Using the red fluorescence as an internal standard, ratiometric detection of hydrogen peroxide can be achieved.

[0010] The fluorescent probe of this invention has high sensitivity. The fluorescence response of the fluorescent probe to hydrogen peroxide and hypochlorous acid was tested at 25°C in PBS buffer (V... PBSbuffer / V 乙腈 The test was conducted in a solution of 7 / 3 (pH = 7.4). The probe itself emits red fluorescence at 605 nm. When hypochlorous acid is added, green fluorescence is produced at 500 nm (excitation wavelength 440 nm). Furthermore, the fluorescence intensity at 500 nm (Ig) increases with increasing hypochlorous acid concentration. 500nm ) and fluorescence intensity at 605 nm (I 605nm The ratio of I to 3 increases significantly. When three times the amount of hypochlorous acid is added, I... 500nm / I 605nm The fluorescence intensity reached its maximum, showing a 352-fold increase compared to the blank probe solution. Simultaneously, the probe exhibited a strong linear relationship with hypochlorous acid concentrations (20-30 μM), with a linear correlation coefficient of 0.99153. Based on a signal-to-noise ratio (S / N) of 3, the detection limit for hypochlorous acid was calculated to be 0.064 μM. When the probe responded to different concentrations of hydrogen peroxide, the peak intensity at 605 nm remained almost unchanged with the addition of hydrogen peroxide, but a new peak appeared at 450 nm (excitation wavelength 360 nm) with increasing hydrogen peroxide concentration, gradually increasing with further increases in concentration. When 15 times the amount of hydrogen peroxide was added, the fluorescence intensity of the probe reached equilibrium. 450nm / I 605nm The detection limit increased from 0.15 to 1.17, a 7.8-fold improvement. Simultaneously, the probe exhibited a strong linear relationship with hydrogen peroxide concentrations (0-80 μM), with a linear correlation coefficient of 0.99564. Based on a signal-to-noise ratio (S / N) of 3, the detection limit for hypochlorous acid was calculated to be 2.90 μM.

[0011] The fluorescent probe of this invention exhibits good selectivity. It selects anions (HS-H+). - SO3 2- S2O3 2- ), cations (Na) + K +Mg 2+ Ca 2+ ), biothiols (GSH, Hcy, Cys) and reactive oxygen species ( 1 O2, ·OH, ·O t Bu, ROO, ONOO - When chloroform (NO) is added to the probe solution as an interfering substance, the fluorescence spectrum shows almost no change. However, when hypochlorous acid and hydrogen peroxide are added, the fluorescence changes significantly.

[0012] The fluorescent probe of this invention has a fast response speed. Upon reaction with hypochlorous acid, the fluorescence intensity changes immediately. 500nm / I 605nm Equilibrium is reached within 3 minutes. The fluorescence intensity of the blank probe solution remains unchanged for 30 minutes. After the addition of hydrogen peroxide, I... 450nm / I 605nm The fluorescence intensity ratio of the blank probe solution gradually increases and reaches equilibrium within 20 minutes, while the fluorescence intensity ratio of the blank probe solution remains unchanged within 90 minutes.

[0013] The fluorescent probe of this invention exhibits low cytotoxicity. After incubation at 37°C for 24 hours with the probe (concentration less than 15 μM), the cell survival rate is greater than 90%.

[0014] The fluorescent probe of this invention can simultaneously detect endogenous and exogenous hydrogen peroxide and hypochlorous acid levels in cells. Fluorescence imaging of endogenous and exogenous HClO and H₂O₂ by the probe: Cells incubated with the probe for 20 min as a control group showed obvious red fluorescence; when cells were incubated with hypochlorous acid for 20 min, followed by 20 min of incubation with the probe, the cells showed obvious green fluorescence; when cells were incubated with hydrogen peroxide for 20 min, followed by 20 min of incubation with the probe, the cells showed both blue and red fluorescence; when cells were incubated with both hypochlorous acid and hydrogen peroxide for 20 min, followed by 20 min of incubation with the probe, the cells showed strong blue and green fluorescence. The results indicate that this probe can simultaneously identify and detect exogenous hypochlorous acid and hydrogen peroxide within cells. Fluorescence imaging of endogenous HClO and H2O2 in cells by the probe: In the control group, cells incubated with the probe (10.0 μM) for 20 min showed red fluorescence; cells incubated with hypochlorous acid for 20 min, followed by incubation with the probe for 20 min, showed green fluorescence; cells cultured with LPS and PMA for 1 h produced endogenous HClO and H2O2, and then incubated with the probe for 20 min, showing green and blue fluorescence; in the control group, cells cultured with LPS and PMA for 1 h, followed by incubation with NAC (intracellular reactive oxygen species scavenger) for 1 h, and finally incubated with the probe for 20 min, showed red fluorescence. The results indicate that the fluorescent probe of this invention can detect endogenous HClO and H2O2 in cells.

[0015] The fluorescent probe of this invention can simultaneously detect exogenous and endogenous hydrogen peroxide and hypochlorous acid levels in zebrafish. Fluorescence imaging of exogenous HClO and H₂O₂ in zebrafish was performed as follows: Zebrafish incubated with the probe for 20 min as a control group showed obvious red fluorescence; when zebrafish were incubated with hypochlorous acid for 20 min, followed by 20 min with the probe, they showed obvious green fluorescence; when zebrafish were first incubated with hydrogen peroxide for 20 min, followed by 20 min with the probe, they showed both blue and red fluorescence; when zebrafish were first incubated with hypochlorous acid and hydrogen peroxide for 20 min, followed by 20 min with the probe, they showed strong blue and green fluorescence. The results indicate that this probe can simultaneously identify and detect exogenous hypochlorous acid and hydrogen peroxide in zebrafish. Fluorescence imaging of endogenous HClO and H2O2 in zebrafish using the probe: In the control group, zebrafish incubated with the probe (10.0 μM) for 20 min showed red fluorescence; zebrafish incubated with hypochlorous acid for 20 min, followed by incubation with the probe for 20 min, showed green fluorescence; zebrafish cultured with LPS and PMA for 1 h produced endogenous HClO and H2O2, and after incubation with the probe for 20 min, showed green and blue fluorescence; in the control group, zebrafish cultured with LPS and PMA for 1 h, followed by incubation with NAC (a reactive oxygen species scavenger in zebrafish) for 1 h, and finally incubated with the probe for 20 min, showed red fluorescence. The results indicate that the fluorescent probe of this invention can detect endogenous HClO and H2O2 in zebrafish.

[0016] The fluorescent probe of this invention can detect the levels of hydrogen peroxide and hypochlorous acid in alcoholic liver injury. A mouse model of alcoholic liver injury was established. After the model was established, mice were sacrificed, liver tissue sections were collected, incubated with the probe, and observed through imaging. Imaging results showed that the liver tissues of mice in the experimental groups exhibited strong blue and green fluorescence, with almost no red fluorescence. The fluorescence intensity of liver tissue sections from experimental group 2 (fed 50% ethanol aqueous solution every 12 hours, dosage: 10 g / kg, for a total of 5 times) was higher than that from experimental group 1 (fed 50% ethanol aqueous solution every 12 hours, dosage: 5 g / kg, for a total of 5 times), which was due to the larger amount of alcohol perfused in experimental group 2. Only red fluorescence was observed in the control group (fed water every 12 hours). This indicates that alcoholic liver injury is accompanied by the production of large amounts of hydrogen peroxide and hypochlorous acid. H&E staining of sections showed that cells in alcoholic liver injury sections were significantly edematous, with mild staining of the cytoplasm, and some hepatocytes showed fatty deformation. Control liver tissue sections showed no obvious lesions. This indicates that alcohol causes liver tissue inflammation, and excessive alcohol consumption leads to liver tissue damage. This is consistent with the results of fluorescence imaging, indicating that the probe can detect hydrogen peroxide and hypochlorous acid levels in an alcoholic liver injury model. Attached Figure Description

[0017] Figure 1 (A1) The fluorescence spectrum changes of the fluorescent probe (10.0 μM) of the present invention after reacting with different concentrations of hypochlorous acid in PBS buffer solution (containing 30% acetonitrile, pH = 7.4). The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. (A2) After the fluorescent probe (10.0 μM) reacts with different concentrations of hypochlorous acid, I 500 / I 605 The value changes, with the horizontal axis representing concentration and the vertical axis representing the fluorescence intensity ratio I. 500 / I 605 (B1) The fluorescence spectrum changes of the fluorescent probe (10.0 μM) after interaction with different concentrations of hydrogen peroxide, with the horizontal axis representing wavelength and the vertical axis representing fluorescence intensity; (B2) The fluorescence spectrum changes of the fluorescent probe (10.0 μM) after interaction with different concentrations of hydrogen peroxide, I 450 / I 605 The value changes, with the horizontal axis representing concentration and the vertical axis representing the fluorescence intensity ratio I. 450 / I 605 .

[0018] Figure 2 The fluorescent probe (10.0 μM) of the present invention was reacted with 3 equivalents of hypochlorous acid and 15 equivalents of hydrogen peroxide, as well as other interfering substances, in PBS buffer solution (containing 30% acetonitrile, pH = 7.4). 1 O2,·OH,·O t Bu,ROO·,ONOO - NO; GSH, Cys and Hcy; Na + ,K + Ca 2+ Mg 2+ ,HS - SO3 2- and S2O3 2- The graph shows the change in fluorescence intensity after treatment, with wavelength on the horizontal axis and fluorescence intensity on the vertical axis.

[0019] Figure 3 The fluorescent probe (10.0 μM) of the present invention was reacted with hypochlorous acid (Al) (3.0 equiv.) and hydrogen peroxide (B1) (15.0 equiv.) in PBS buffer solution (containing 30% acetonitrile, pH = 7.4). 500 / I 605 ,I 450 / I 605 The values ​​change over time. The horizontal axis represents time, and the vertical axis represents the fluorescence intensity ratio.

[0020] Figure 4 This is a cell toxicity test of the fluorescent probe of the present invention. The horizontal axis represents the probe concentration, and the vertical axis represents the cell viability.

[0021] Figure 5 This invention provides confocal cell imaging for detecting exogenous hypochlorous acid and hydrogen peroxide in cells using fluorescent probes. Row 1: Cells incubated with the probe for 20 min; Row 2: Cells incubated with hypochlorous acid for 20 min, then with the probe for 20 min; Row 3: The probe incubated with hypochlorous acid for 20 min, then with the probe for 20 min; Row 4: The probe incubated with hypochlorous acid and hydrogen peroxide for 20 min, then with the probe for 20 min. The concentrations of the probe, hypochlorous acid, and hydrogen peroxide were 10.0 μM, 30.0 μM, and 150 μM, respectively. (Bluetooth channel: E) x =405nm,E m =450±25nm; green channel:E x =488nm,E m =525±25nm; red channel:E x =488nm,E m =595±25nm.

[0022] Figure 6 This invention provides confocal cell imaging for detecting endogenous hypochlorous acid and hydrogen peroxide using fluorescent probes. Row 1: Cells were incubated with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 h, then with the probe for 20 min. Row 2: Cells were incubated with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 h, then with NAC (1.0 mM) for 1 h, and finally with the probe for 20 min. The concentrations of the probe, hypochlorous acid, and hydrogen peroxide were 10.0 μM, 30.0 μM, and 150 μM, respectively. (Block channel: E) x =405nm,E m =450±25nm; green channel:E x =488nm,E m =525±25nm; red channel:E x =488nm,E m =595±25nm.

[0023] Figure 7This invention provides confocal fluorescence imaging for detecting hypochlorous acid and hydrogen peroxide in zebrafish using a fluorescent probe. Row 1: Zebrafish incubated with the probe for 20 min; Row 2: Zebrafish incubated with hypochlorous acid for 20 min, then with the probe for 20 min; Row 3: Zebrafish incubated with hypochlorous acid for 20 min, then with the probe for 20 min; Row 4: The probe is incubated with hypochlorous acid for 20 min, then with hydrogen peroxide for 20 min, and finally with the probe for 20 min. The concentrations of the probe, hypochlorous acid, and hydrogen peroxide are 10.0 μM, 30.0 μM, and 150 μM, respectively. Black channel: E x =405nm,E m =450±25nm; greenchannel:E x =488nm,E m =525±25nm; red channel:E x =488nm,E m =595±25nm.

[0024] Figure 8 Confocal cell imaging was performed to detect endogenous hypochlorous acid and hydrogen peroxide in zebrafish using the fluorescent probe of this invention. Row 1: Zebrafish were incubated with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 h, then incubated with the probe for 20 min. Row 2: Zebrafish were incubated with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 h, then incubated with NAC (1.0 mM) for 1 h, and finally incubated with the probe for 20 min. The concentrations of the probe, hypochlorous acid, and hydrogen peroxide were 10.0 μM, 30.0 μM, and 150 μM, respectively. (Bluechannel:E) x =405nm,E m =450±25nm; green channel:E x =488nm,E m =525±25nm; redchannel:E x =488nm,E m =595±25nm.

[0025] Figure 9 This study investigates the imaging effects of alcoholic liver injury in mice. (A) Fluorescence imaging of liver tissue from mice in the control group (fed water every 12 hours) (ad), liver tissue from mice in experimental group 1 (fed 50% ethanol aqueous solution every 12 hours, dosage: 5 g / kg) (eh), and liver tissue sections from mice in experimental group 2 (fed 10 g / kg of 50% ethanol aqueous solution every 12 hours) (il). (B) The process of establishing the alcoholic liver injury model. (C) Fluorescence intensity corresponding to the tissue sections.

[0026] Specific implementation examples

[0027] Example 1: Synthesis of Compound 2

[0028] Compound 1 (2.306 g, 10.0 mmol) and n-butyl bromide (2.765 g, 20 mmol) were dissolved in 25 mL of anhydrous dimethyl sulfoxide. Sodium hydroxide (0.800 g, 20.0 mmol) and a catalytic amount of potassium iodide were then added. The mixture was refluxed at 100 °C for 6 h. After the reaction was complete, the mixture was cooled to room temperature and extracted three times with dichloromethane. The solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether / dichloromethane = 2 / 1, v / v) to give product 2, a colorless oily liquid. Yield: 2.5935 g; Yield: 95.0%.

[0029] Example 2: Synthesis of Compound 3

[0030] Under argon protection, N,N-dimethylformamide (0.51 mL, 7.2 mmol) was slowly added to phosphorus oxychloride (0.6 mL, 7.2 mmol), and the mixture was stirred in an ice-water bath for 15 min. Intermediate 2 (1.6767 g, 6 mmol) was dissolved in anhydrous N,N-dimethylformamide and then added to the above solution. The mixture was refluxed at 60 °C for 4 h. After the reaction was complete, the mixture was poured into 50 mL of ice water, neutralized with 10% sodium hydroxide solution, and extracted three times with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a yellow solid product 3. Yield: 1.3146 g; Yield: 70.1%.

[0031] Example 3: Synthesis of Compound 4

[0032] Aluminum powder (0.2025 g, 3.6 mmol) was added to anhydrous acetonitrile (15 mL) and stirred at room temperature for 5 min. Iodine (0.0306 g, 2.55 mmol) was added to the mixture in small amounts several times, and stirred under nitrogen protection. Compound 3 (0.9530 g, 3.0 mmol) was dissolved in anhydrous acetonitrile (15 mL) and added dropwise to the mixture. The resulting suspension was refluxed for 6 h. After cooling to room temperature, the mixture was extracted three times with 100 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, v / v) to give a yellow oily liquid product 4. Yield: 0.7891 g; Proportion: 87.3%.

[0033] Example 4: Synthesis of Compound 5

[0034] Compound 4 (0.6130 g, 2.1 mmol) and diethyl malonate (0.8011 g, 5.0 mmol) were dissolved in 15 mL of anhydrous ethanol. Piperidine (400 μL, 4.1 mmol) and acetic acid (200 μL, 3.5 mmol) were then added. The mixture was stirred at room temperature for 7 h. The solvent was evaporated under reduced pressure to dryness, yielding the crude product. The crude product was purified by column chromatography to obtain a yellow solid, product 5. Yield: 0.4859 g; Yield: 60.5%.

[0035] Example 5: Synthesis of Compound 6

[0036] Compound 5 (0.2504 g, 0.6 mmol) and sodium hydroxide (0.0760 g, 2.0 mmol) were weighed into 15 mL of anhydrous methanol and refluxed for 0.5 h. The solvent in the reaction solution was evaporated to dryness under reduced pressure by rotary evaporation. The solution was then dissolved in 50 mL of dichloromethane, neutralized with 10% HCl solution, washed with water, and the organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure by rotary evaporation and further dried under vacuum to obtain oxblood red solid product 6. Yield: 0.2663 g; Yield: 90.1%.

[0037] Example 6: Synthesis of Compound 7

[0038] Compound 6 (0.5061 g, 1.37 mmol), piperazine-1-carboxylic acid tert-butyl ester (0.4019 g, 1.63 mmol), 4-dimethylaminopyridine (0.3012 g, 2.47 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in 10 mL of dichloromethane. The mixture was stirred at room temperature for 4 h under argon protection. The solvent was evaporated under reduced pressure to obtain a crude product, which was purified by column chromatography (ethanol / dichloromethane = 1:100, v / v) to give an orange solid product, the probe molecule (0.5836 g, 80%). HRMS (ESI) m / z:calcd for C 29 H 33 N3NaO5S[M+Na] + ,558.2039;found,558.2046. 1H NMR (400MHz, CDCl3) δ (ppm): 7.79 (s, 1H), 7.20 (d, J = 7.3Hz, 1H), 7.16 (s, 1H), 7.11 (d, J = 7.6, 1.5Hz, 1H), 6.97 (s, 1H), 6.92 (d, J = 8.2Hz, 1H ),6.72(s,1H),3.90(m,2H),3.72(s,2H),3.54(m,4H),3.35(s,2H),1.80(q,J=7.6Hz,2H),1.47(s,9H),0.97(t,J=7.4Hz,3H),0.88(s,2H). 13 C NMR (100MHz, CDCl3) δ (ppm): 164.13, 158.28, 155.30, 154.53, 143.32, 142.73, 127.76, 127.50, 125.36, 123.77, 121.57, 120.53, 1 16.10,113.09,102.41,80.32,63.56,56.49,56.49,48.12,47.15,47.15,42.20,28.47,28.37,26.91,26.91,26.91,20.06,13.73.

[0039] Example 7: Synthesis of Compound 9

[0040] 2,4-Dihydroxybenzaldehyde (0.8301 g, 6.01 mmol), pinacol ester of 4-bromomethylphenylboronic acid (1.754 g, 5.93 mmol), K₂CO₃ (0.8453 g, 6.13 mmol), and KI (0.1053 g, 0.64 mmol) were weighed into 10 mL of acetonitrile and refluxed for 4 h. The solvent was evaporated under reduced pressure to obtain the crude product, which was purified by column chromatography (dichloromethane) to give a white solid product 9. Yield: 1.9781 g; Yield: 81.3%. HRMS(ESI) m / z: calcd for C 20 H 24 BO5S[M+H] + ,355.1717;found,355.1716. 1 HNMR (400MHz, CDCl3) δ (ppm): 11.45 (s, 1H), 9.71 (s, 1H), 7.84 (d, J = 8.0Hz, 2H), 7.42 (t, J =7.8Hz, 3H), 6.60 (d, J = 8.7, 2.3Hz, 1H), 6.49 (d, J = 2.3Hz, 1H), 5.13 (s, 2H), 1.35 (s, 12H). 13C NMR (100MHz, CDCl3) δ (ppm): 194.40, 165.81, 164.42, 138.70, 135.30, 135.17, 132.48, 132.4 8,126.56,126.56,115.35,108.91,101.73,83.91,83.91,70.27,24.87,24.87,24.87,24.87.

[0041] Example 8: Synthesis of Compound 10

[0042] An ethanol solution of compound 9 (0.8981 g, 2.54 mmol) and cycloisopropyl malonate (0.4012 g, 2.79 mmol) was weighed into a 25 mL flask, and catalytic amounts of piperidine and acetic acid were added. The solvent was refluxed at 80 °C for 3 h under argon protection, cooled to room temperature, filtered, and dried to give yellow compound 10. Yield: 0.5256 g; yield: 49.3%. HRMS(ESI) m / z: calcd for C 23 H 24 BO7[M+H] + ,423.1615; found,423.1613.1HNMR(400MHz,CDCl3)δ(ppm):8.85(s,1H),7.86(d,J=8.0Hz,2H),7.64( d,J=8.8Hz,1H),7.43(d,J=8.0Hz,2H),7.08(d,J=11.1Hz,1H),6.97(s,1H),5.23(s,2H),1.35(s,12H). 13 C NMR(100MHz, CDCl3)δ(ppm):165.17,164.48,163.02,156.91,151.17,137.83,135.31,135.31,131.75,1 26.59,126.59,115.69,112.50,111.07,101.92,83.99,83.99,70.99,53.44,24.87,24.87,24.87,24.87.

[0043] Example 9: Probe Synthesis

[0044] Compound 7 (0.0835 g, 0.16 mmol) was weighed and dissolved in 6 mL of dichloromethane, and trifluoroacetic acid (3 mL) was added dropwise. Under argon protection, the mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure to obtain the crude product, which was purified by column chromatography (dichloromethane / ethanol, v / v = 100:1) to give an orange solid. Yield: 0.0421 g; Yield: 32.1%. HRMS(ESI) m / z:calcd for C 47 H 47 BN3O9S[M+H] + ,840.3126;found,840.3116. 1 H NMR (400MHz, CDCl3) δ (ppm): 7.93 (s, 1H), 7.86 (m, 3H), 7.43 (t, J = 8.0Hz, 3H), 7.18 (d, J = 6.3Hz, 2H), 7.11 (d, J = 7.3Hz, 1H), 7.01 (m, 4H), 6.72(d,J=14.3Hz,1H),5.17(d,J=5.9Hz,2H),3.86(s,6H),3.48(s,4H),1.86(m,2H),1.34(s,12H),1.25(d,J=4.7Hz,2H),0.96(m,3H). 13 CNMR(100MHz, CDCl3)δ(ppm):162.93,158.34,156.89,156.15,155.33,151.13,149.89,142.68,138 .45,137.84,135.20,135.20,131.74,129.81,127.76,127.50,127.50,126.59,125.43,123.79,123 .16,121.61,120.74,120.17,116.11,115.66,113.07,112.48,112.10,101.89,83.93,83.93,70.97,53.45,53.45,48.12,46.97,46.97,42.36,31.43,28.47,24.86,24.86,24.86,20.05,13.72.

[0045] Example 10: Detection of exogenous and endogenous hypochlorous acid and hydrogen peroxide in cells using a probe. HepG2 cells were cultured in DMEM medium with 10% fetal bovine serum added. A cell growth environment consisting of 95% air and 5% CO2 was maintained. For imaging of exogenous hypochlorous acid and hydrogen peroxide, cells were first incubated with hypochlorous acid (30.0 μM) for 20 min, washed three times with PBS buffer, and then incubated with the probe for 20 min, showing green fluorescence. Cells were incubated with hydrogen peroxide (150.0 μM) for 20 min, washed three times with PBS buffer, and then incubated with the probe for 20 min, showing red and blue fluorescence. In the control group, cells were incubated with the probe (10.0 μM) for 20 min, showing red fluorescence. The results indicate that the probe of this invention can effectively detect exogenous HClO and H2O2 in cells.

[0046] To detect endogenous HClO and H2O2 in cells, cells were cultured for 1 h with LPS (1 μg / mL) and PMA (1 μg / mL), respectively, to generate endogenous HClO and H2O2. The cells were then washed three times with PBS buffer and incubated with the probe (10.0 μM) for 20 min, resulting in green and blue fluorescence imaging. In the control group, cells were cultured for 1 h with LPS (1 μg / mL) and PMA (1 μg / mL), washed three times with PBS buffer, incubated with NAC (1.0 mM) (an intracellular reactive oxygen species scavenger) for 1 h, washed three times with PBS buffer, and incubated with the probe for 20 min, resulting in red fluorescence. The results indicate that the fluorescent probe of this invention can detect endogenous HClO and H2O2 in cells.

[0047] Example 11: Detection of exogenous and endogenous hypochlorous acid and hydrogen peroxide in zebrafish using probe molecules

[0048] When detecting exogenous HClO and H2O2 in zebrafish, zebrafish were first incubated with hypochlorous acid (30.0 μM) for 20 min, followed by incubation with the probe for 20 min, resulting in green fluorescence. When zebrafish were incubated with hydrogen peroxide (150.0 μM) for 20 min, followed by incubation with the probe for 20 min, they exhibited red and blue fluorescence. In the control group, zebrafish incubated with the probe (10.0 μM) for 20 min showed red fluorescence. The results indicate that the probe of this invention can effectively detect exogenous HClO and H2O2 in zebrafish.

[0049] To detect endogenous HClO and H2O2 in zebrafish, zebrafish were cultured with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 h to generate endogenous HClO and H2O2, respectively. After incubation with the probe (10.0 μM) for 20 min, the zebrafish exhibited green and blue fluorescence. In the control group, zebrafish were cultured with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 h, then incubated with NAC (1.0 mM) (a reactive oxygen species scavenger) for 1 h, and finally incubated with the probe for 20 min, resulting in red fluorescence within the cells. The results indicate that the fluorescent probe of this invention can detect endogenous HClO and H2O2 in zebrafish.

[0050] Example 12: Detection of hypochlorous acid and hydrogen peroxide by probe in alcoholic liver injury

[0051] Mice of similar weight were divided into three groups. Mice were kept at 25±2℃ under a 12h / 12h light / dark cycle. The control group mice were given tap water every 12 hours. Group 1 mice were given 50% ethanol solution (5g / kg body weight) every 12 hours for a total of 5 times to establish an acute liver injury model. Group 2 mice were given 50% ethanol solution (10g / kg body weight) every 12 hours for a total of 5 times to establish an acute liver injury model. After establishing the acute liver injury model, all mice in the experimental groups were sacrificed, and liver tissue was cryopreserved, cultured, and sectioned. The lesions were observed using H&E staining. The tissue was incubated with a 10μM probe for 0.5h, washed three times with PBS, and imaged under a microscope. The results showed that the probe can detect hypochlorous acid and hydrogen peroxide in alcoholic liver injury.

Claims

1. A ratiometric fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and hydrogen peroxide, characterized in that, Its structural formula is:

Images