Benzothiadiazole type fluorescent probe for detecting hclo and preparation method thereof

Abstract

The application discloses a benzothiadiazole type fluorescent probe for detecting HClO and a preparation method thereof, and belongs to the technical field of organic synthesis and HClO analysis. An intermediate B-CHO is obtained through Suzuki coupling with 4,7-dibromo-2,1,3 benzothiadiazole and p-formylphenylboronic acid as raw materials, and then a high-selectivity hypochlorous acid fluorescent probe is prepared through condensation reaction of the intermediate B-CHO with hydroxylamine hydrochloride. The probe can rapidly and specifically recognize HClO under the interference of common cations, anions, H2O2 and biological thiols. The fluorescent organic small molecule BTD-OH has the advantages of simple synthesis method, good thermal stability, strong fluorescence emission, large Stokes shift (119nm) and good stability, and can be stored as a pure solid or a solution at room temperature. The probe has the advantages of simple preparation, high yield, fast response speed, small toxicity, strong stability and specificity, and can be used as an effective hypochlorous acid detection tool in actual application.

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis and HClO analysis technology, specifically to a benzothiadiazole-type fluorescent probe for detecting HClO and its preparation method. Background Technology

[0002] The detection of hypochlorous acid using small organic molecule probes is an ideal tool for biological systems and aquatic environment analysis due to its advantages such as simple synthesis, high sensitivity, rapid response, high selectivity, and high spatiotemporal resolution. Benzothiadiazole (BTD) based on π-extension structures is relatively stable, and its electron-deficient structure is crucial for designing efficient and stable structures. BTD derivatives typically have large Stokes shifts, avoiding unnecessary background interference; they generally do not fade under prolonged irradiation; most fluorescent small-molecule BTD derivatives can transpose on cell membranes, making them suitable for detection in biological cells; they often exhibit bright emission without noticeable flicker to the naked eye, resulting in a good signal-to-noise ratio; most fluorescent small-molecule BTD derivatives can be excited in the visible light region; and BTD derivatives can be stored as pure solids or solutions at room temperature.

[0003] More and more people are paying attention to the use of disinfectants, and the selection and use of disinfectants is an important issue of concern nationwide. Hypochlorous acid is a reactive oxygen species (ROS) and a powerful bleaching agent in daily life. It is also a major antibacterial agent in many biological and pathological processes, with the advantages of rapid and strong bactericidal action. However, abnormal changes in its concentration levels can cause many serious diseases, such as Parkinson's syndrome, atherosclerosis, cardiovascular disease, lung injury, rheumatoid arthritis, and even some cancers. Excessive use of disinfectants containing hypochlorous acid may lead to an increase in chlorine compounds in the aquatic environment, posing a potential threat to aquatic life. Therefore, the detection of hypochlorous acid is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a benzothiadiazole-type fluorescent small molecule that is simple to synthesize, has mild reaction conditions, and is low in cost. It can be used for the highly sensitive and selective detection of HClO.

[0005] To achieve the above objectives, the present invention adopts the following solution.

[0006] A benzothiadiazole-type fluorescent probe for detecting HClO has the following structural formula:

[0007]

[0008] The preparation method of the BTD-OH is as follows:

[0009]

[0010] Specifically, the preparation method of the probe molecule for detecting HClO includes the following steps:

[0011] (1) 4,7-dibromo-2,1,3-benzothiadiazole, 4-formylphenylboronic acid, tetra(triphenylphosphine)palladium and potassium phosphate were dissolved in the reaction solvent of ethanol and water. The mixture was heated under N2 protection and refluxed. After the reaction was completed, the system was cooled to room temperature, deionized water was added to quench the reaction, and the mixture was filtered, dried and purified by column chromatography to obtain a pale yellow solid B-CHO.

[0012] (2) Add intermediate B-CHO and hydroxylamine hydrochloride to the solvent, add two drops of triethylamine and heat to reflux. After the reaction is complete, cool the system thoroughly and filter to obtain a pale yellow solid. Recrystallize with dichloromethane to obtain a bright yellow solid BTD-OH.

[0013] In step (1), the molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole and 4-formylphenylboronic acid is 1:2.5-1:3. The molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole, tetra(triphenylphosphine)palladium and potassium phosphate is 1:0.02:3.

[0014] The volume ratio of ethanol to water in the reaction solvent in step (1) is 3:1.

[0015] In step (1), the mixture is refluxed at 80-120℃ for 5-12 hours under N2 protection.

[0016] The molar ratio of intermediate B-CHO and hydroxylamine hydrochloride in step (2) is 1:2.5-1:3. The solvent in step (2) is anhydrous ethanol. Two drops of triethylamine are added in step (2), and the mixture is heated under reflux at 30-80°C for 8-12 hours.

[0017] The method of using the benzothiadiazole-type fluorescent probe for detecting HClO is as follows:

[0018] (1) The probe molecule was added to anhydrous ethanol to prepare a 20 μM standard solution. PBS buffer was added to the standard solution to adjust the pH to 7.4. Then, different concentrations of HClO / ClO were added. - The solution was allowed to stand for 1 minute, and then fluorescence testing was performed to obtain a standard curve of hypochlorous acid concentration and fluorescence intensity.

[0019] (2) Add PBS buffer solution to the standard solution to adjust the pH to 7.4, then add the test solution, let stand for 1 min, perform fluorescence test, and input the fluorescence intensity of the test solution into the standard curve to obtain the concentration of hypochlorous acid in the test solution.

[0020] Compared with the prior art, the present invention has the following advantages: using 4,7-dibromo-2,1,3-benzothiadiazole and p-formylphenylboronic acid as raw materials, the intermediate B-CHO was obtained by Suzuki coupling, and then a highly selective hypochlorous acid fluorescent probe BTD-OH was prepared by condensation reaction with hydroxylamine hydrochloride. The probe synthesis method is simple, with a large Stokes shift (119 nm), strong fluorescence intensity, short response time, good selectivity for HClO, low toxicity, and better thermal stability compared with other organic fluorescent small molecules. Attached Figure Description

[0021] Figure 1 The image shows the 1H NMR spectrum of compound B-CHO.

[0022] Figure 2 The image shows the 1H NMR spectrum of compound BTD-OH.

[0023] Figure 3 The fluorescence spectra of the probe BTD-OH in different solvents are shown.

[0024] Figure 4 The image shows the fluorescence spectra of the probe BTD-OH and its reaction with HClO, where BTD-OH is the blank sample, and BTD-OH + ClO - To add 10 μM HClO / ClO - A sample of the solution.

[0025] Figure 5 The time response diagram of the probe BTD-OH to HClO is shown.

[0026] Figure 6 Different concentrations (0-10 μM) of HClO / ClO were added to the PBS buffer system to detect the probe BTD-OH. - Fluorescence intensity changes (left) and linear relationship (right).

[0027] Figure 7 This is a schematic diagram illustrating the principle of BTD-OH probe for detecting HClO / ClO.

[0028] Figure 8 This is a selection diagram of the interfering substances by the probe BTD-OH. Detailed Implementation

[0029] The present invention will be described below with reference to the accompanying drawings and specific embodiments. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0030] Example 1:

[0031] (1) Preparation of compound B-CHO

[0032] 4,7-Dibromo-2,1,3-benzothiadiazole (1 mmol), 4-formylphenylboronic acid (3 mmol), tetrakis(triphenylphosphine)palladium (0.02 mmol), and potassium phosphate (3 mmol) were added to the reaction solvent. A mixture of ethanol and water in a volume ratio of 3:1 was used as the reaction solvent. The mixture was refluxed at 100 °C for 6 h under N2 protection. The reaction endpoint was monitored by thin-layer chromatography. After the reaction was completed, the system was cooled to room temperature, and the reaction was quenched by adding distilled water. After filtration and drying, the mixture was purified by column chromatography in a PE:EA ratio of 4:1 system to obtain a pale yellow solid B-CHO 204.6 mg (yield: 59.4%).

[0033] The hydrogen NMR spectrum of B-CHO is as follows: Figure 1 As shown: 1 H NMR (400MHz, CDCl3): δ10.14 (s, 2H), 8.18 (d, J = 8.2 Hz, 4H), 8.08 (d, J = 8.4 Hz, 4H), 7.92 (s, 2H).

[0034] (2) Preparation of fluorescent probe molecule BTD-OH

[0035] The intermediate B-CHO (1 mmol) and hydroxylamine hydrochloride (3 mmol) were dissolved in anhydrous ethanol, and two drops of triethylamine were added. The mixture was heated under reflux at 50 °C for 10 h. After the reaction system was fully cooled, it was filtered to obtain a pale yellow solid. The solid was recrystallized from dichloromethane to obtain a bright yellow solid BTD-OH 101.1 mg (yield: 54.1%).

[0036] The hydrogen NMR spectrum of BTD-OH is shown below. Figure 2 As shown: 1 H NMR (400MHz, DMSO-d6): δ11.35(s,2H),8.21(s,2H),8.05(d,J=8.3Hz,4H),7.98(s,2H),7.75(d,J=8.3Hz,4H).

[0037] (3) Probe molecules are used to detect HClO / ClO -

[0038] (301) Solvent optimization

[0039] The probe molecule was added to different solvents to prepare 20 μM standard solutions. After standing for 1 min, fluorescence testing was performed, and the results are as follows. Figure 3 As shown.

[0040] from Figure 3 As can be seen, the probe molecules exhibit good fluorescence emission intensity in most solvents. Therefore, we chose ethanol, which has relatively low toxicity, as the optimal solvent for subsequent detection.

[0041] (302) Fluorescence performance analysis

[0042] The fluorescent probe molecules are used to buffer HClO / ClO in the system. - The specific methods for determining the content are as follows:

[0043] The probe molecule was added to anhydrous ethanol to prepare a 20 μM standard solution. PBS buffer was added to the standard solution to adjust the pH to 7.4. Then, different concentrations (0.1 μM, 0.3 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 7 μM, 10 μM) of HClO / ClO were added. - Let stand for 1 minute, then perform fluorescence testing. A blank was set up during the process. The results are as follows: Figure 4-6 As shown.

[0044] from Figure 4 It can be seen that the maximum excitation wavelength of the probe molecule is 404 nm, and the maximum emission wavelength is 525 nm. The addition of HClO / ClO... - The fluorescence then underwent a significant blue shift, changing from bright yellow to blue-green under 365nm ultraviolet light, thus enabling the identification of HClO / ClO. - The purpose. From Figure 5 It can be seen from the probe BTD-OH that it affects HClO / ClO - It has a fast response time and can handle HClO / ClO - Achieve rapid detection. From Figure 6 It can be seen that with HC10 / C10 - As the concentration increases, the fluorescence intensity increases, and the probe BTD-OH reacts with HClO / ClO. - It has a wide detection range and can detect HClO / ClO - To achieve quantitative detection.

[0045] The probe molecules were reacted with HClO / ClO - Mass spectrometry analysis was performed on the extracted solution after the reaction, revealing a mass peak (345.07123) corresponding to the compound after the reaction, but not the mass peak corresponding to the probe BTD-OH. Therefore, a possible reaction mechanism was speculated, such as... Figure 7 As shown. From Figure 7 It can be seen that the probe BTD-OH is a type of Schiff base fluorescent probe obtained by introducing hydroxylamine. When HClO is present, ClO… - Nucleophilic attack causes the C=N group to break. Under the influence of water molecules, the hydroxylamine leaves, HClO is eliminated and reduced back to an aldehyde group, the conjugated system decreases, the maximum absorption wavelength blue shifts, and the molecular fluorescence color changes to blue.

[0046] (4) Specific detection

[0047] The above 20 μM probe molecule standard solution was followed by the addition of 10 μM of different cations (Na+). + Mg 2+ ,Bi 3+ ,Cr 3+ ,Ba 2+ Hg 2+ Al 3+ ,Fe 3+ Ca 2+ Cd 2+ ,Pb 2+ ,K + Cu 2+ ,Zn 2+ Co 2+ ); anion (F - ,Cl - ,Br - ,I - SO3 2- SO4 2- NO3 - HCO3 - CO3 2- ,PO4 3- After reacting with common oxides H2O2 and other biothiols Cys and GSH for 1 min, the fluorescence changes in this system were measured, and the results are as follows: Figure 8 As shown. From Figure 8 It can be seen from the probe BTD-OH that it affects HClO / ClO - It exhibits good specificity, with the maximum absorption wavelength remaining essentially unchanged around 525 nm and the fluorescence intensity also being relatively stable, indicating that the probe molecule BTD-OH is effective against HClO / ClO. - It has good specificity and can identify HClO / ClO - Achieve accurate detection.

[0048] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO, characterized in that: 。 2. The method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO according to claim 1, characterized in that, Includes the following steps: (1) 4,7-dibromo-2,1,3-benzothiadiazole, 4-formylphenylboronic acid, tetra(triphenylphosphine)palladium and potassium phosphate were dissolved in the reaction solvent of ethanol and water. The mixture was heated under N2 protection and refluxed. After the reaction was completed, the system was cooled to room temperature, deionized water was added to quench the reaction, and the mixture was filtered, dried and purified by column chromatography to obtain a pale yellow solid B-CHO. (2) Add intermediate B-CHO and hydroxylamine hydrochloride to the solvent, add two drops of triethylamine and heat to reflux. After the reaction is complete, cool the system thoroughly and filter to obtain a pale yellow solid. Recrystallize with dichloromethane to obtain a bright yellow solid BTD-OH.

3. The method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO according to claim 2, characterized in that, In step (1), the molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole and 4-formylphenylboronic acid is 1:2.5-1:

3.

4. The method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO according to claim 2, characterized in that, The volume ratio of ethanol to water in the reaction solvent in step (1) is 3:

1.

5. The method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO according to claim 2, characterized in that, In step (1), the mixture is refluxed at 80-120 °C for 5-12 h under N2 protection.

6. The method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO according to claim 2, characterized in that, In step (2), the molar ratio of intermediate B-CHO and hydroxylamine hydrochloride is 1:2.5-1:3, and the solvent is anhydrous ethanol.

7. The method for preparing a benzothiadiazole-type fluorescent probe for detecting HClO according to claim 2, characterized in that, Add two drops of triethylamine in step (2) and heat under reflux at 30-80 °C for 8-12 h.

8. The method of using the benzothiadiazole-type fluorescent probe for detecting HClO prepared by the preparation method according to any one of claims 1-7 is as follows: (1) The probe molecule was added to anhydrous ethanol to prepare a solution of 20 μ A standard solution of M was prepared by adding PBS buffer to the standard solution to adjust the pH to 7.4, followed by the addition of different concentrations of HClO / ClO. - The solution was allowed to stand for 1 minute, and then fluorescence testing was performed to obtain a standard curve of hypochlorous acid concentration and fluorescence intensity. (2) Add PBS buffer solution to the standard solution to adjust the pH to 7.4, then add the test solution, let stand for 1 min, perform fluorescence test, and input the fluorescence intensity of the test solution into the standard curve to obtain the concentration of hypochlorous acid in the test solution.

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