Benzothiadiazole type fluorescent probe for detecting HClO and preparation method of benzothiadiazole type fluorescent probe

Abstract

The invention discloses a benzothiadiazole type fluorescent probe for detecting HClO and a preparation method of the benzothiadiazole type fluorescent probe, and belongs to the technical field of organic synthesis and HClO analysis. The high-selectivity hypochlorous acid fluorescent probe is prepared by taking 4, 7-dibromo-2, 1, 3-benzothiadiazole and p-formyl phenylboronic acid as raw materials, carrying out Suzuki coupling to obtain an intermediate B-CHO, and then carrying out condensation reaction on the intermediate B-CHO and hydroxylamine hydrochloride. The probe can be used for rapidly and specifically recognizing HClO under the interference of common cations, anions, H2O2 and biological mercaptan. The fluorescent small organic molecule BTD-OH is simple and convenient in synthesis method, has good thermal stability, strong fluorescence emission, large Stokes shift (119nm) and good stability, and can be stored as a pure solid or solution at room temperature. The probe is simple to prepare, high in yield, high in response speed, low in toxicity and high in stability and specificity, and can be used as an effective hypochlorous acid detection tool in practical application.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis and HClO analysis, and in particular to a benzothiadiazole fluorescent probe for detecting HClO and a preparation method thereof. Background Art

[0002] The detection of hypochlorous acid using organic small molecule probes has the advantages of simple synthesis, high sensitivity, rapid response, high selectivity and high spatiotemporal resolution, making it an ideal tool for detecting analytes in biological systems and water environments. The structure of benzothiadiazole (BTD) based on π extension is relatively stable, and its electron-deficient structure is very important for planning a highly efficient and stable structure. BTD derivatives usually have a large Stokes shift to avoid unnecessary background interference, and usually do not fade under long-term irradiation. Most fluorescent small molecule BTD derivatives can transpose cell membranes and can be used for the detection of biological cells. They often emit brightly, but there is no obvious flickering to the naked eye, and a good signal-to-noise ratio can be obtained. Most fluorescent small molecule BTD derivatives can be excited in the visible light region. BTD derivatives can also be stored as pure solids or solutions at room temperature.

[0003] More and more people are beginning to pay attention to the use of disinfectants, and the selection and use of disinfectants are also important issues of concern to people across the country. Hypochlorous acid is a reactive oxygen species (ROS). It is a powerful bleaching agent in daily life and the main antibacterial agent in many biological and pathological processes. It has the advantages of fast sterilization speed and strong sterilization power. However, abnormal changes in its concentration level may cause many more serious diseases, such as Parkinson's syndrome, atherosclerosis, cardiovascular disease, lung damage, rheumatoid arthritis and even some cancers. Excessive use of disinfectants containing hypochlorous acid may lead to an increase in chlorine compounds in the water environment, thereby causing potential harm to organisms in the aquatic environment. Therefore, the detection of hypochlorous acid is of great significance. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a benzothiadiazole-type fluorescent small molecule with simple synthesis, mild reaction conditions and low cost, which can be used for highly sensitive and highly selective detection of HClO.

[0005] In order to achieve the above purpose, the present invention adopts the following scheme

[0006] A benzothiadiazole fluorescent probe for detecting HClO, the structural formula of which is shown below:

[0007]

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

[0009]

[0010] Specifically, the method for preparing the probe molecule for detecting HClO comprises the following steps:

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

[0012] (2) The intermediate B-CHO and hydroxylamine hydrochloride are added to a solvent, two drops of triethylamine are added, and the mixture is heated to reflux. After the reaction is completed, the system is fully cooled and filtered to obtain a light yellow solid, which is then recrystallized from dichloromethane to obtain a bright yellow solid BTD-OH.

[0013] In the step (1), the molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole to 4-formylphenylboronic acid is 1:2.5-1:3. The molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole, tetrakis(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), N 2 Reflux at 80-120°C for 5-12h under protection.

[0016] The molar ratio of the 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 for using the benzothiadiazole 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 solution was added to the standard solution to adjust the pH to 7.4, and then different concentrations of HC10 / C10 were added. - The solution was allowed to stand for 1 min, and a fluorescence test 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 it stand for 1 min, perform fluorescence test, and bring the fluorescence intensity of the test solution into the standard curve to obtain the hypochlorous acid concentration in the test solution.

[0020] Compared with the prior art, the present invention has the following effects: 4,7-dibromo-2,1,3-benzothiadiazole and p-formylphenylboronic acid are used as raw materials, an intermediate B-CHO is obtained through Suzuki coupling, and then a condensation reaction is carried out with hydroxylamine hydrochloride to prepare a highly selective hypochlorous acid fluorescent probe BTD-OH, the probe has a simple synthesis method, a large Stokes shift (119nm), a strong fluorescence intensity, a short response time, good selectivity for HClO, low toxicity, and better thermal stability than other organic fluorescent small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum (1H NMR) of compound B-CHO.

[0022] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum (1H NMR) of compound BTD-OH.

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

[0024] Figure 4 The fluorescence spectra of the probe BTD-OH and HClO, where BTD-OH is the blank sample, BTD-OH+ClO - Add 10 μM HClO / ClO - Sample of solution.

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

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

[0027] Figure 7 Schematic diagram of the detection of HClO / ClO by probe BTD-OH.

[0028] Figure 8 This is a diagram showing the selection of probe BTD-OH for interfering substances. DETAILED DESCRIPTION

[0029] The present invention is described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0030] Embodiment 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, and a mixed solvent of ethanol and water with a volume ratio of 3:1 was used as the reaction solvent. 2 The reaction mixture was refluxed at 100°C for 6 h under protection, and the reaction endpoint was monitored by thin layer chromatography. After the reaction was completed, the system was cooled to room temperature, and distilled water was added to quench the reaction. After filtration and drying, the mixture was purified by column chromatography using a PE:EA=4:1 system to obtain 204.6 mg of light yellow solid B-CHO (yield: 59.4%).

[0033] B-CHO H NMR spectrum Figure 1 As shown: 1 H NMR (400 MHz, CDCl 3 ): δ10.14(s,2H),8.18(d,J=8.2Hz,4H),8.08(d,J=8.4Hz,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, two drops of triethylamine were added, and the mixture was heated under reflux at 50°C for 10 h. After the reaction system was fully cooled, a light yellow solid was obtained by filtration, and the mixture was recrystallized from dichloromethane to obtain 101.1 mg of bright yellow solid BTD-OH (yield: 54.1%).

[0036] BTD-OH H NMR spectrum Figure 2 As shown: 1 H NMR (400 MHz, DMSO-d 6 ): 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 for detecting HClO / ClO -

[0038] (301) Solvent Optimization

[0039] The probe molecules were added to different solvents to prepare a 20 μM standard solution. After standing for 1 min, fluorescence testing was performed. The results were as follows: Figure 3 shown.

[0040] from Figure 3It can be seen that the fluorescence emission intensity of the probe molecule is good in most solvents, so we chose ethanol with less toxicity as the best solvent for subsequent detection.

[0041] (302) Fluorescence performance analysis

[0042] The fluorescent probe molecule is used in the HClO / ClO buffer system - The specific method for determining the content is as follows:

[0043] The probe molecule was added to anhydrous ethanol to prepare a 20 μM standard solution, PBS buffer solution was added to the standard solution to adjust the pH to 7.4, and then different concentrations (0.1 μM, 0.3 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 7 μM, 10 μM) of HC10 / C10 were added. - , let it stand for 1 minute, and then conduct fluorescence test. Set blank in the process. The result is as follows Figure 4-6 shown.

[0044] from Figure 4 It can be seen that the maximum excitation wavelength of the probe molecule is 404nm, and the maximum emission wavelength is 525nm. - The fluorescence then undergoes an obvious blue shift, and the color changes from bright yellow to blue-green under 365nm ultraviolet light, thereby achieving the identification of HClO / ClO - purpose. Figure 5 It can be seen that the probe BTD-OH has a strong effect on HClO / ClO - It has a fast response time and can be used for HClO / ClO - Rapid detection is achieved. Figure 6 It can be seen that with HC10 / C10 - As the concentration increases, the fluorescence intensity increases. The probe BTD-OH reacts with HClO / ClO - It has a wide detection range and can detect HClO / ClO - Achieve quantitative detection.

[0045] The probe molecules were mixed with HClO / ClO - The solution after reaction was extracted and then analyzed by mass spectrometry. The mass spectrum peak (345.07123) of the corresponding compound after reaction was found, but the mass spectrum peak corresponding to the probe BTD-OH was not found. Therefore, the possible reaction mechanism was speculated, such as Figure 7 As shown. 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 exists, ClO -It will attack nucleophilically to break C=N. Under the action of water molecules, hydroxylamine leaves, HClO is eliminated and reduced to aldehyde group, the conjugated system is reduced, the maximum absorption wavelength is blue-shifted, and the molecular fluorescence color turns to blue.

[0046] (4) Specificity detection

[0047] The above 20 μM probe molecule standard solution was then added with 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 - ,SO 3 2- ,SO 4 2- ,NO 3 - ,HCO 3 - ,CO 3 2- ,PO 4 3- );Common oxides H 2 O 2 After reacting with other biothiols Cys and GSH for 1 min, the fluorescence changes in this system were measured. The results are as follows: Figure 8 As shown. Figure 8 It can be seen that the probe BTD-OH has a strong effect on HClO / ClO - It has good specificity, the maximum absorption wavelength is basically unchanged at about 525nm and the fluorescence intensity is relatively stable, indicating that the probe molecule BTD-OH has good specificity for HClO / ClO - It has good specific recognition and can identify HClO / ClO - Achieve accurate detection.

[0048] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A benzothiadiazole-based fluorescent probe for detecting HClO, characterized in that: The structural formula is as follows:

2. The benzothiadiazole fluorescent probe for detecting HClO according to claim 1, characterized in that: The preparation method of BTD-OH is:

3. The benzothiadiazole-based fluorescent probe for detecting HClO according to claim 1, characterized in that: The following steps are involved: (1) dissolving 4,7-dibromo-2,1,3-benzothiadiazole, 4-formylphenylboronic acid, tetrakis(triphenylphosphine)palladium and potassium phosphate in a reaction solvent of ethanol and water, heating under reflux under N2 protection, cooling the system to room temperature after the reaction is completed, adding deionized water to quench the reaction, filtering and drying, and then purifying by column chromatography to obtain a light yellow solid B-CHO; (2) The intermediate B-CHO and hydroxylamine hydrochloride are added to a solvent, two drops of triethylamine are added, and the mixture is heated to reflux. After the reaction is completed, the system is fully cooled and filtered to obtain a light yellow solid, which is then recrystallized from dichloromethane to obtain a bright yellow solid BTD-OH.

4. The benzothiadiazole 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 to 4-formylphenylboronic acid is 1:2.5-1:

3.

5. The benzothiadiazole-based 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.

6. The benzothiadiazole fluorescent probe for detecting HClO according to claim 2, characterized in that: In step (1), reflux at 80-120° C. for 5-12 h under N 2 protection.

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

8. The benzothiadiazole-based fluorescent probe for detecting HClO according to claim 1, characterized in that: In step (2), two drops of triethylamine were added and the mixture was heated to reflux at 30-80°C for 8-12h.

9. The benzothiadiazole-based fluorescent probe for detecting HClO according to claim 1, characterized in that: The method for using the benzothiadiazole fluorescent probe for detecting HClO is as follows: (1) The probe molecule was added to anhydrous ethanol to prepare a 20 μM standard solution, PBS buffer solution was added to the standard solution to adjust the pH to 7.4, and then different concentrations of HC10 / C10 were added. - The solution was allowed to stand for 1 min, and a fluorescence test 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 it stand for 1 min, perform fluorescence test, and bring the fluorescence intensity of the test solution into the standard curve to obtain the hypochlorous acid concentration in the test solution.

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