Fluorescent reversible probe for detecting superoxide anion free radicals as well as synthesis method and application of fluorescent reversible probe

Abstract

The invention belongs to the field of superoxide anion free radical detection, and particularly relates to a reversible fluorescent probe for detecting superoxide anion free radicals as well as a synthesis method and application of the reversible fluorescent probe. The fluorescent reversible probe is 3-(benzothiazole-2-yl)-[1, 1 '-biphenyl]-2, 3', 4 '-triphenol. The preparation method comprises the following steps: dissolving 3-bromo-2-hydroxybenzaldehyde in absolute ethyl alcohol, adding sodium hydrogen sulfite and o-aminothiophenol, and carrying out heating reflux reaction; dissolving a reaction product and 3, 4-dimethoxyphenylboronic acid in tetrahydrofuran, adding a potassium carbonate solution and tetrakis (triphenylphosphine) palladium, and carrying out heating reflux reaction; and dissolving the product obtained by the reaction in dichloromethane to completely dissolve the solid, adding boron tribromide, and reacting under the ice salt bath condition to obtain the target probe. The fluorescent reversible probe can be used for superoxide anion free radical detection, and the detection method is simple, convenient to operate, good in selectivity, high in sensitivity, long in emission wavelength, large in Stokes shift and high in signal-to-noise ratio.

Description

Technical Field

[0001] The invention belongs to the field of superoxide anion free radical detection, and specifically relates to a fluorescent reversible probe for detecting superoxide anion free radicals, a synthesis method and application thereof. Background Art

[0002] Superoxide anion free radical (O 2 .- ), plays an important role in organic synthesis, and specific detection in cells and organisms is of great significance for clarifying its various effects in biological and pharmacological processes. However, it has the characteristics of short life, strong activity and low concentration in biological tissues. Superoxide anion free radical (O 2 .- ) and its secondary products can lead to local or global redox imbalance, thus inducing inflammation.

[0003] Currently, a variety of highly selective and sensitive fluorescent probes have been developed to monitor superoxide anion (O 2 · - However, many existing probes are ineffective in responding to 2 · - The probes rely on irreversible chemical reactions, which makes it impossible to distinguish between short-term ROS bursts that occur during normal physiological processes and sustained ROS levels caused by oxidative stress. In addition, these probes often produce biologically toxic byproducts such as trifluoromethanesulfonate or phenylphosphonic acid after activation, which further limits their practical applications.

[0004] In view of this, superoxide anion radical (O 2 .- ) and catechol were used to design their new reversible probe. Although some photoacoustic probes have been reported, their detection sensitivity is relatively low; Tang Bo's team also reported a series of superoxide anion radicals (O 2 .- ) fluorescent probes, but the detection wavelengths of these probes are generally in the blue light region, which are particularly affected by biological background fluorescence. In addition, most of these fluorescent probes rely on weak intramolecular charge transfer (ICT) caused by the conversion of phenol to quinone to release fluorescent signals, resulting in a small Stokes shift and a low signal-to-noise ratio. Therefore, how to ensure that the fluorescent probe has a large Stokes shift and a high signal-to-noise ratio is a key issue. 2 .- )Important issues that need to be urgently addressed in fluorescent probes. Summary of the invention

[0005] The purpose of the present invention is to provide a fluorescent reversible probe for detecting superoxide anion free radicals, a synthesis method thereof and an application thereof. The fluorescent reversible probe synthesized by the present invention can be used for superoxide anion free radical detection, has a simple detection method, is easy to operate, has good selectivity, high sensitivity, has a large Stokes shift and a high signal-to-noise ratio, and has a detection wavelength in the green light band, avoiding blue light interference of biological background fluorescence.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A fluorescent reversible probe, which is 3-(benzothiazol-2-yl)-[1,1'-biphenyl]-2,3',4'-triphenol, has the following structural formula:

[0008]

[0009] A method for synthesizing the fluorescent reversible probe as described above comprises the following steps:

[0010] Step 1: dissolving 3-bromo-2-hydroxybenzaldehyde in anhydrous ethanol, adding sodium bisulfite and o-aminothiophenol, heating under reflux to react, and obtaining 2-benzothiazol-2-yl-6-bromophenol;

[0011] Step 2: Dissolve 2-benzothiazol-2-yl-6-bromophenol and 3,4-dimethoxyphenylboronic acid in tetrahydrofuran, add potassium carbonate solution and tetrakistriphenylphosphine palladium, and heat under reflux to react to obtain 3-(benzothiazol-2-yl)-3 ， , 4 ， -dimethoxy-[1,1 ， -biphenyl]-2-phenol;

[0012] Step 3: 3-(Benzothiazol-2-yl)-3 ， , 4 ， -dimethoxy-[1,1 ， -biphenyl]-2-phenol is dissolved in dichloromethane, and boron tribromide is added, and the mixture is reacted in an ice-salt bath to obtain 3-(benzothiazol-2-yl)-[1,1'-biphenyl]-2,3',4'-triphenol.

[0013] Preferably, in step 1, the molar ratio of 3-bromo-2-hydroxybenzaldehyde, sodium bisulfite and o-aminothiophenol is 14.9:29.9:17.9.

[0014] Preferably, the temperature of the heating reflux reaction in step 1 is 70° C. and the time is 12 h.

[0015] Preferably, in step 2, the molar ratio of 2-benzothiazol-2-yl-6-bromophenol to 3,4-dimethoxyphenylboronic acid is 1.6:2.5.

[0016] Preferably, the temperature of the heating reflux reaction in step 2 is 75° C. and the time is 12 h.

[0017] Preferably, the reaction time in step 3 under ice-salt bath conditions is 10 h.

[0018] A reagent for detecting superoxide anion free radicals, comprising the fluorescent reversible probe as described above.

[0019] An application of the fluorescent reversible probe as described above is used to detect superoxide anion free radicals; the application does not involve the diagnosis and treatment of diseases.

[0020] A method for detecting superoxide anion free radicals comprises the following steps: mixing the fluorescent reversible probe as described above with a sample to be detected, and performing fluorescence detection; the method does not involve the diagnosis and treatment of diseases.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention constructs a superoxide anion radical (O 2 .- ) probe, which is characterized by releasing a strong fluorescence response signal in the green light band under dual mechanism regulation (ESIPT, PET), with a large Stokes shift and a high signal-to-noise ratio. This innovative design not only significantly enhances the sensing performance of the probe, but also demonstrates excellent biocompatibility, thus providing a powerful tool for accurately monitoring superoxide anions in complex biological environments.

[0023] 2. The fluorescent reversible probe of the present invention is simple to synthesize and has low cost.

[0024] 3. The fluorescent reversible probe of the present invention can realize superoxide anion free radical (O 2 .- ) differentiation detection, the detection result has high sensitivity, short response time and good selectivity.

[0025] 4. The detection method of the present invention is simple and can be realized with the help of a fluorescence spectrometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Proton spectrum of the fluorescent reversible probe XZBU-BDT;

[0027] Figure 2 Carbon spectrum of the fluorescent reversible probe XZBU-BDT;

[0028] Figure 3 Mass spectrum of the fluorescent reversible probe XZBU-BDT;

[0029] Figure 4 Fluorescence emission diagram of superoxide anion radical sample measured by fluorescent reversible probe XZBU-BDT;

[0030] Figure 5 Working curve of fluorescent reversible probe XZBU-BDT to superoxide anion radical;

[0031] Figure 6 The response results of the fluorescent reversible probe XZBU-BDT in PBS (pH=7.4) to possible interferences in the physiological environment;

[0032] Figure 7 Cytotoxicity test results of the fluorescent reversible probe XZBU-BDT. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Example 1

[0035] Synthesis and characterization of probes

[0036] The synthetic route is:

[0037]

[0038] Step 1: In a 100mL eggplant-shaped bottle, add 3-bromo-2-hydroxybenzaldehyde (14.9mmol, 3g) and 50mL anhydrous ethanol to completely dissolve 3-bromo-2-hydroxybenzaldehyde, and then add sodium bisulfite (29.9mmol, 2.1g); fix the eggplant-shaped bottle on an oil bath, install a condensing reflux device, set the oil bath temperature to 70°C, start timing at this temperature and heat reflux reaction for 1 hour; weigh o-aminothiophenol (17.9mmol, 2.24g) and slowly pour it into the eggplant-shaped bottle, continue to heat reflux reaction at 70°C for 12h; use thin layer chromatography to confirm the reaction progress, after the reaction is completed, cool it and let it stand for suction filtration, and wash it with anhydrous ethanol in small amounts several times, finally place the filter cake in a watch glass, adjust the oven temperature to 70°C, and dry it to obtain a light yellow solid (2.36g, 77.5%), which is 2-benzothiazol-2-yl-6-bromophenol (compound 1).

[0039] Step 2: In a 250 mL eggplant-shaped bottle, compound 1 (1.6 mmol, 0.5 g) and 3,4-dimethoxyphenylboronic acid (2.5 mmol, 0.45 g) were completely dissolved in 100 mL of tetrahydrofuran; 2 mL of 2 mol / L potassium carbonate solution was added dropwise to the eggplant-shaped bottle, and a catalyst of tetrakistriphenylphosphine palladium (0.08 mmol, 0.094 g) was added under nitrogen protection, and the mixture was heated to reflux at 75 ° C. and nitrogen protection for 12 h; the reaction progress was tracked by thin layer chromatography. After the reaction was completed, the solvent was evaporated, and the mixture was extracted with dichloromethane and water. All organic layers were combined, a small amount of anhydrous sodium sulfate was added thereto for drying, and a brown-yellow solid was obtained by rotary evaporation. The mixture was purified by column chromatography using pure dichloromethane as a developing solvent to obtain a brown-yellow solid (0.11 g, 18.52%), i.e. 3-(benzothiazol-2-yl)-3 ， , 4 ， -dimethoxy-[1,1 ， -biphenyl]-2-phenol (Compound 2).

[0040] Step 3: Compound 2 (2.8 mmol, 0.1 g) and dichloromethane were added to an eggplant-shaped flask to completely dissolve the solid and maintain the temperature below 0°C; 3 mL of boron tribromide was slowly added to the eggplant-shaped flask using a syringe and reacted for 10 h in an ice-salt bath; the reaction progress was tracked by thin layer chromatography. After the reaction was completed, 1 mL of methanol and a small amount of water were used to quench the mixture, and the mixture was extracted with dichloromethane and water. The organic layer was collected and dried over anhydrous sodium sulfate. A light yellow solid was obtained by rotary evaporation. 6 mL of anhydrous ethanol was added and ultrasonically oscillated to slightly dissolve the solid. The solid was then filtered and washed to obtain 3-(benzothiazol-2-yl)-[1,1'-biphenyl]-2,3',4'-triphenol, named XZBU-BDT (0.04 g, 43.29%).

[0041] The characterization data of XZBU-BDT are as follows:

[0042] 1 H NMR(500MHz,Chloroform-d)δ:13.05(s,1H),7.90–7.82(m,2H),7.62(dd,J=7.9,1.6Hz,1H),7.43(ddd,J=8.2,7.2,1.3Hz,1H), 7.35(dtd,J=7.3,3.6,1.2Hz,2H),7.17(d,J=2.0Hz,1H),7.04(dd,J=8.2,2.0Hz,1H),6.98–6.87(m,2H),5.27(d,J=28.3Hz,2H).

[0043] 13C NMR(126MHz,DMSO)δ:169.67,154.64,151.48,145.31,145.19,133.88,132.87,130.67,1 28.74,127.63,127.55,126.41,122.82,122.40,120.79,120.51,117.29,117.16,115.81.

[0044] HR-MS Cald: 336.0689 Found: 336.06863

[0045] Example 2

[0046] The probe is sensitive to superoxide anion radical (O 2 .- )'s fluorescence response spectrum

[0047] Prepare 2 mmol / L fluorescent reversible probe solution: take 1.0 mg of the target product (M=335.06 g / mol) and add 1500 μL of DMSO (dimethyl sulfoxide) to dissolve.

[0048] Use a pipette to transfer 2 mL of PBS solution into a quartz cuvette, then transfer 10 μL of probe solution into it, mix it several times with the help of a pipette, use 400 nm as the excitation wavelength to measure the fluorescence emission intensity at 500 nm, and then add superoxide anion radicals (O 2 .- ), mix well and then detect the response. See the fluorescence emission diagram Figure 4 .

[0049] Fluorescence detection was performed at an excitation wavelength of 400 nm. 2 .- ) concentration increases, the probe is oxidized to o-benzoquinone, the fluorescence emission intensity at 500 nm increases, and superoxide anion radicals (O 2 .- ) concentration is proportional to the XZBU-BDT fluorescence intensity it causes. The probe has a large Stokes shift of 100nm, which can effectively avoid self-absorption and inner filter effects, and has high sensitivity (LOD = 3.4μM). The working curve is shown in Figure 5 .

[0050] Example 3

[0051] Probe selectivity test

[0052] In order to evaluate the detection efficiency of the probe under physiological conditions, this example evaluated various interfering substances (including Ca 2+ , Cu 2+ , Cu+ , Fe 3+ , Fe 2+ , K + 、Na + Mg 2+ 、Zn 2+ , CH 3 COO - , HSO 3 - 、SO 3 2- 、SO 4 2- 、Cys、GSH、ONOO - 、NO 2 - 、NO 2 , NO, HClO, H 2 S 2 , H 2 S.H. 2 O 2 ) on the fluorescence intensity of the probe. The results are shown in Figure 6 As shown, these potential interfering substances have little effect on the fluorescence intensity of the probe at 500 nm, indicating that the detection of superoxide ions by the probe is almost unaffected by these interfering substances and has good selectivity.

[0053] Example 4

[0054] Cytotoxicity testing of probes

[0055] In this example, the MTT method was used to evaluate the cytotoxicity of the probe. Figure 7 As shown, after BV-2 cells were incubated with the probe for 12 hours, the cell survival rate remained above 88%, confirming that the probe had good biocompatibility and low cytotoxicity.

[0056] The above description is only for better explaining the embodiments of the present invention, and is not intended to limit the present invention. Any modification or equivalent substitution that does not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims

1. A fluorescent reversible probe, characterized in that: The fluorescent reversible probe is 3-(benzothiazol-2-yl)-[1,1'-biphenyl]-2,3',4'-triphenol, and its structural formula is as follows:

2. A method for synthesizing the fluorescent reversible probe according to claim 1, characterized in that: Here are the steps: Step 1: dissolving 3-bromo-2-hydroxybenzaldehyde in anhydrous ethanol, adding sodium bisulfite and o-aminothiophenol, heating under reflux to react, and obtaining 2-benzothiazol-2-yl-6-bromophenol; Step 2: Dissolve 2-benzothiazol-2-yl-6-bromophenol and 3,4-dimethoxyphenylboronic acid in tetrahydrofuran, add potassium carbonate solution and tetrakistriphenylphosphine palladium, and heat under reflux to react to obtain 3-(benzothiazol-2-yl)-3 ， , 4 ， -dimethoxy-[1,1 ， -biphenyl]-2-phenol; Step 3: 3-(Benzothiazol-2-yl)-3 ， , 4 ， -dimethoxy-[1,1 ， -biphenyl]-2-phenol is dissolved in dichloromethane, and boron tribromide is added, and the mixture is reacted in an ice-salt bath to obtain 3-(benzothiazol-2-yl)-[1,1'-biphenyl]-2,3',4'-triphenol.

3. The method for synthesizing a fluorescent reversible probe according to claim 2, characterized in that: In the step 1, the molar ratio of 3-bromo-2-hydroxybenzaldehyde, sodium bisulfite and o-aminothiophenol is 14.9:29.9:17.

9.

4. The method for synthesizing a fluorescent reversible probe according to claim 2 or 3, characterized in that: The temperature of the heating reflux reaction in step 1 is 70° C. and the time is 12 h.

5. The method for synthesizing a fluorescent reversible probe according to claim 2, characterized in that: In the step 2, the molar ratio of 2-benzothiazol-2-yl-6-bromophenol to 3,4-dimethoxyphenylboronic acid is 1.6:2.

5.

6. The method for synthesizing a fluorescent reversible probe according to claim 2 or 5, characterized in that: The heating reflux reaction temperature in step 2 is 75° C. and the time is 12 h.

7. The method for synthesizing a fluorescent reversible probe according to claim 2, characterized in that: The reaction time in the step 3 under the ice-salt bath condition is 10 hours.

8. A reagent for detecting superoxide anion free radicals, characterized in that: The method comprises the fluorescent reversible probe according to claim 1.

9. A use of the fluorescent reversible probe according to claim 1, characterized in that: Used to detect superoxide anion free radicals; the application does not involve the diagnosis and treatment of diseases.

10. A method for detecting superoxide anion free radicals, characterized in that: The steps are as follows: mixing the fluorescent reversible probe described in claim 1 with a sample to be tested, and performing fluorescence detection; the method does not involve the diagnosis and treatment of diseases.

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