A dual-site benzothiazole fluorescent probe, a preparation method and application thereof

Abstract

This invention provides a two-site benzothiazole fluorescent probe, its preparation method, and its application, belonging to the field of organic synthesis technology. This invention provides a two-site benzothiazole fluorescent probe, constructed by modifying the benzothiophene fluorophore. This two-site benzothiazole fluorescent probe can chemically react with H₂O₂ and hypochlorite ions separately under the same detection conditions to generate different fluorescent substances, achieving the distinguishing detection of H₂O₂ and hypochlorite ions under the same excitation. It can simultaneously detect H₂O₂ and hypochlorite ions, exhibiting advantages such as high detection sensitivity, good selectivity, and high discrimination, and has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a two-site benzothiazole fluorescent probe, its preparation method, and its application. Background Technology

[0002] Hydrogen peroxide (H2O2) is one of the most abundant reactive oxygen species in cells and a metabolic byproduct. Excessive production of H2O2 can lead to severe oxidative stress, thus being associated with various diseases such as cardiovascular disease, neurodegenerative diseases, inflammation, diabetes, Alzheimer's disease, and cancer. Hypochlorous acid (HClO), as another strong oxidant, can effectively eliminate invading microorganisms and pathogens. HClO is closely related to immune regulation, and its abnormal production can cause a series of health problems such as rheumatoid arthritis, reperfusion injury, acute lung injury, ischemia, and atherosclerosis. Therefore, accurate detection of H2O2 and HClO in biological systems is of great significance. In most cases, reactive oxygen species are interdependent, participating in similar physiological and pathological processes, but each plays a differentiated role. For example, in neutrophils, myeloperoxidase can catalyze the reaction of chloride ions with H2O2 to produce HClO; simultaneously, H2O2, as a signal transduction molecule, can regulate macrophages to produce HClO with bactericidal effects. It is noteworthy that H2O2 and HClO coexist in some cells and are simultaneously involved in the development of cardiovascular diseases, Parkinson's disease, and neurodegenerative diseases. However, due to their similar chemical properties, distinguishing between them remains a significant challenge. Therefore, developing analytical methods capable of simultaneously monitoring H2O2 and HClO is urgently needed to elucidate their interaction mechanisms. Currently, fluorescent probes for detecting both have been reported, but most only enable the identification of a single analyte. While simultaneously using two chemical sensors can monitor H2O2 and HClO, detection difficulties arise due to differences in photobleaching rates, different cellular localization, mutual interference, and spectral crosstalk. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a dual-site benzothiazole fluorescent probe, its preparation method, and its application. The dual-site benzothiazole fluorescent probe provided by this invention can simultaneously detect H2O2 and hypochlorite, and has the advantages of high detection sensitivity, good selectivity, and high discrimination, and has good application prospects.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a two-site benzothiazole fluorescent probe (SBBT) having the structure shown in Formula I: Formula I.

[0005] This invention also provides a method for preparing the dual-site benzothiazole fluorescent probe described in the above technical solution, comprising the following steps: Compound 1 was subjected to a cyclization reaction with compound 2 (aminothiophenol) to obtain compound 3; Compound 3 and compound 4 (4-(bromomethyl)phenylboronic acid pinacol ester) were subjected to a Williamson synthesis reaction to obtain the two-site benzothiazole fluorescent probe; The structures of compounds 1-4 are shown in the following formulas: , , , .

[0006] Preferably, the solvent for the cyclization reaction includes aminosulfonic acid (NH2SO3H), acetonitrile (CH3CN), and water.

[0007] Preferably, the molar ratio of compound 1 to aminosulfonic acid is 1:0.1.

[0008] Preferably, the cyclization reaction takes 24 hours.

[0009] Preferably, the molar ratio of compound 3 to compound 4 is 1:1.5.

[0010] Preferably, the Williamson synthesis reaction is carried out in an alkaline environment provided by K2CO3.

[0011] Preferably, the Williamson synthesis reaction is carried out at a temperature of 90-100°C for 12-24 hours.

[0012] This invention also provides the application of the dual-site benzothiazole fluorescent probe described in the above technical solution in the simultaneous detection of H2O2 and hypochlorite.

[0013] The present invention also provides the application of the dual-site benzothiazole fluorescent probe described in the above technical solution in the preparation of detection reagents, which are used to simultaneously detect H2O2 and hypochlorite.

[0014] This invention provides a two-site benzothiazole fluorescent probe, and compared with the prior art, the advantages of this invention are as follows: This invention constructs a dual-site fluorescent probe by modifying the benzothiophene fluorophore, which can simultaneously detect H2O2 and hypochlorite ions. It has the advantages of high detection sensitivity, good selectivity and high discrimination, and has good application prospects.

[0015] This invention also provides a method for preparing the two-site benzothiazole fluorescent probe described in the above technical solution. The preparation method of this invention is simple to operate and suitable for industrial application. Attached Figure Description

[0016] Figure 1 The reaction principle of cyclization reaction; Figure 2 The reaction principle of the Williamson synthesis reaction; Figure 3 The image shows the proton NMR spectrum of the dual-site fluorescent probe. Figure 4 The images show the spectral response of a dual-site fluorescent probe to H2O2 and HClO. In the images, A is the fluorescence spectrum of the probe after interacting with different concentrations of H2O2, B is the linear relationship curve between the probe fluorescence intensity and the H2O2 concentration, C is the fluorescence spectrum of the probe after interacting with different concentrations of HClO, and D is the linear relationship curve between the probe fluorescence intensity and the HClO concentration. Figure 5 The effect of different analytes on the response of a two-site fluorescent probe; Figure 6 This is a fluorescence imaging image of endogenous H2O2 and HClO in HeLa cells using a two-site benzothiazole fluorescent probe. Detailed Implementation

[0017] This invention provides a two-site benzothiazole fluorescent probe having the structure shown in Formula I: Formula I.

[0018] The dual-site benzothiazole fluorescent probe of this invention can react chemically with H2O2 and hypochlorite under the same detection conditions to generate different fluorescent substances, thus enabling the differentiation and detection of H2O2 and hypochlorite under the same excitation. After reacting with H2O2, the dual-site benzothiazole fluorescent probe exhibits significant fluorescence emission at 501 nm under an excitation wavelength of 375 nm, and after reacting with hypochlorite, it exhibits significant fluorescence emission at 450 nm under an excitation wavelength of 375 nm. The probe itself does not exhibit obvious fluorescence. Therefore, it enables the detection of specific analytes based on specific excitation and fluorescence emission signals. When both reactive oxygen species are present, H2O2 and hypochlorite can be distinguished based on the fluorescence emission signals.

[0019] This invention also provides a method for preparing the dual-site benzothiazole fluorescent probe described in the above technical solution, comprising the following steps: Compound 1 and compound 2 were subjected to a cyclization reaction to obtain compound 3; Compound 3 and compound 4 were subjected to a Williamson synthesis reaction to obtain the dual-site benzothiazole fluorescent probe. The structures of compounds 1-4 are shown in the following formulas: , , , .

[0020] Unless otherwise specified, all raw materials used in this invention are commercially available products in the field.

[0021] This invention involves a cyclization reaction between compound 1 and compound 2 to obtain compound 3. The reaction principle of the cyclization reaction is as follows: Figure 1 As shown.

[0022] In this invention, the solvent for the cyclization reaction preferably includes aminosulfonic acid, acetonitrile, and water.

[0023] In this invention, the molar ratio of compound 1 to aminosulfonic acid is preferably 1:0.1.

[0024] In this invention, the preferred ratio of compound 1 to acetonitrile is 1 mmol: 5 mL.

[0025] In this invention, the cyclization reaction time is preferably 24 hours, and the temperature is preferably room temperature, i.e., no additional heating or cooling is required.

[0026] After the cyclization reaction is completed, the present invention preferably filters the resulting system, and the white solid obtained is compound 3.

[0027] In this invention, compound 1, aminothiophenol, and aminosulfonic acid are preferably added to acetonitrile, followed by the addition of water. The reaction system is stirred at room temperature, and a precipitate appears in the solution. After the cyclization reaction is completed, the resulting system is filtered to obtain a white solid, namely compound 3.

[0028] After obtaining compound 3, the present invention performs a Williamson synthesis reaction with compound 4 to obtain the dual-site benzothiazole fluorescent probe. The reaction principle of the Williamson synthesis reaction is as follows: Figure 2 As shown.

[0029] In this invention, the molar ratio of compound 3 to compound 4 is preferably 1:1.5.

[0030] In this invention, the Williamson synthesis reaction is preferably carried out in an alkaline environment, which is preferably provided by K2CO3.

[0031] In this invention, the molar ratio of compound 3 to K2CO3 is preferably 1:1.5.

[0032] In this invention, the solvent for the Williamson synthesis reaction preferably includes acetonitrile.

[0033] In this invention, the preferred ratio of compound 3 to acetonitrile is 1 mmol: 10 mL.

[0034] In this invention, the temperature of the Williamson synthesis reaction is preferably 90~100℃, and the time is preferably 12~24h.

[0035] After the Williamson synthesis reaction is completed, the solvent is preferably removed from the obtained system, and then separated by column chromatography to obtain the two-site benzothiazole fluorescent probe.

[0036] In this invention, the solvent removal is preferably achieved by rotary drying.

[0037] In this invention, the eluent for column chromatography separation is preferably a mixed solvent of ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is preferably 1:8.

[0038] This invention also provides the application of the dual-site benzothiazole fluorescent probe described in the above-mentioned technical solution in the simultaneous detection of H2O2 and hypochlorite, for non-disease diagnosis and treatment purposes. In biological cells, high concentrations of H2O2 and hypochlorite can lead to oxidative stress, causing cell damage and various diseases. The real-time and accurate detection and monitoring of H2O2 and hypochlorite levels using the probe of this invention is of great significance for understanding their biological properties, revealing disease mechanisms, and developing treatment strategies. The probe itself does not carry a fluorescent signal; however, after crossing the cell membrane and entering the cell, it reacts with H2O2 and hypochlorite within the cell, generating a fluorescent signal. The level of H2O2 and hypochlorite within the cell can be determined by detecting the fluorescence intensity.

[0039] In this invention, the application is preferably used for the simultaneous detection of H2O2 and HClO.

[0040] In this invention, when used, the dual-site benzothiazole fluorescent probe is preferably used in solution form. The solvent of the solution includes an organic phase and an aqueous phase, and the volume ratio of the organic phase to the aqueous phase is preferably 1:9. The organic phase is preferably N,N-dimethylformamide (DMF), and the aqueous phase is preferably phosphate buffered saline (PBS) with pH=7.4.

[0041] In this invention, the concentration of the dual-site benzothiazole fluorescent probe in the solution is preferably 1 mM.

[0042] The present invention also provides the application of the dual-site benzothiazole fluorescent probe described in the above technical solution in the preparation of detection reagents, which are used to simultaneously detect H2O2 and hypochlorite.

[0043] In this invention, the detection reagent is preferably used to simultaneously detect H2O2 and HClO.

[0044] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] Example 1 The preparation of a two-site benzothiazole fluorescent probe includes the following steps: Synthetic compound 3 a. Compound 1 (1.0 mmol), aminothiophenol (1.0 mmol) and aminosulfonic acid (0.1 mmol) were added to 5 mL of acetonitrile, followed by 10 drops of water. The reaction system was stirred at room temperature for 24 h, and a precipitate appeared in the solution.

[0046] b. Filter the solution from step a to obtain a white solid, namely compound 3; Synthetic two-site benzothiazole fluorescent probes a. Compound 3 (1 mmol), 4-(bromomethyl)phenylboronic acid pinacol ester (1.5 mmol) and K2CO3 (1.5 mmol) were mixed in 10 mL of acetonitrile and refluxed at 90 °C for 12 h.

[0047] b. The solvent in the reaction solution from step a is evaporated to dryness, and the resulting product is purified by column chromatography using a mixed solvent of ethyl acetate / petroleum ether (volume ratio of ethyl acetate to petroleum ether is 1:8) to finally obtain the target product SBBT.

[0048] Figure 3 This is the proton NMR spectrum of a dual-site fluorescent probe.

[0049] Application of dual-site benzothiazole fluorescent probes in distinguishing and detecting H2O2 and HClO in the in vitro environment Spectral response of the probe to H2O2 and HClO: Accurately weigh the dual-site benzothiazole fluorescent probe and dissolve the probe molecules in an environment with an organic phase and aqueous phase volume ratio of 1:9 to obtain a probe molecule solution for analysis and detection. The organic phase was DMF, and the aqueous phase was phosphate buffer solution with pH=7.4. The concentration of the probe molecule solution was 1 mM. Prepare 1 mM aqueous solutions of H2O2 and HClO separately. Add 20 μL of the probe molecule solution and analyte stock solution (1 mM) to a 2 mL test tube, dilute to the mark with PBS / DMF (9:1, v / v) solution, and shake thoroughly for 10 min at room temperature before spectral testing. Measure the fluorescence emission intensity using an excitation wavelength of 375 nm. After reacting with H2O2, the dual-site benzothiazole fluorescent probe showed significant fluorescence emission at 501 nm under an excitation wavelength of 375 nm. After reacting with hypochlorite, it showed significant fluorescence emission at 450 nm under an excitation wavelength of 375 nm. Figure 4 As shown, Figure 4 Figure A shows the fluorescence spectra of the probe after interaction with different concentrations of H₂O₂; Figure B shows the linear relationship between the probe fluorescence intensity and the H₂O₂ concentration; Figure C shows the fluorescence spectra of the probe after interaction with different concentrations of HClO; and Figure D shows the linear relationship between the probe fluorescence intensity and the HClO concentration. The probe itself does not exhibit significant fluorescence. Therefore, specific excitation and fluorescence emission signals are used to detect specific analytes. When both reactive oxygen species are present, H₂O₂ and hypochlorite can be distinguished based on the fluorescence emission signals. This achieves high sensitivity by differentiating H₂O₂ and HClO using fluorescence emission signals at the same excitation wavelength. The detection limits for H₂O₂ and HClO are 3.6 × 10⁻⁶ and 3.6 × 10⁻⁶, respectively. -8 M and 9.1×10 -9 M is ideal for imaging and analyzing endogenous H2O2 and HClO in living cells.

[0050] The probe molecule solution is effective against interfering ions, including TBHP (tert-butanol peroxide) and O2. - , 1 O2, ∙OH, ONOO - NO, Na2S2, NO3 - NO2 - SCN - S2O3 2- SO3 2- GSH, Cys, Hcy, HS - S 2- F - Cl - , Br - I - Mg 2+ K + Ca 2+ Zn2+ Fe 3+ There was no obvious response, and the concentration of the interfering ions was 1 mM. Figure 5 As shown, Figure 5 The horizontal axis represents: 1 blank, 2 H2O2 / HClO, 3 TBHP, 4 O2 - 5 1 O2, 6∙OH, 7 ONOO - 8 NO, 9 Na2S2, 10 NO3 - , 11 NO2 - 12 SCN - 13S2O3 2- 14 SO3 2- , 15 GSH, 16 Cys, 17 Hcy, 18 HS - , 19 S 2- 20 F - ,21 Cl - ,22 Br - ,23 I - 24Mg 2+ 25 K + 26 Ca 2+ 27 Zn 2+ 28 Fe 3+ .

[0051] Dual-channel fluorescence imaging analysis of endogenous H2O2 and HClO in HeLa cells HeLa cells were seeded in confocal culture dishes and cultured at 37°C for 24 h. H2O2 detection: HeLa cells were first co-cultured with a probe (10 μM) for 30 min, then co-cultured with H2O2 (100 μM) for 1 h. After washing three times with phosphate buffer, fluorescence imaging was performed. Hypochlorous acid (HClO) detection: HeLa cells were co-cultured with a probe (10 μM) for 30 min, then co-cultured with HClO (200 μM) for 30 min. After washing three times with phosphate buffer, fluorescence imaging was performed. Figure 6 This image shows fluorescence imaging of endogenous H2O2 and HClO in HeLa cells using a dual-site benzothiazole fluorescent probe. When the probe-loaded cells were cultured with H2O2, fluorescence signals were observed in the green channel. After co-culturing with HClO, fluorescence signals were observed in the blue channel. This demonstrates that the dual-site benzothiazole fluorescent probe successfully achieved dual-channel fluorescence imaging analysis of both endogenous and exogenous H2O2 and HClO in cells.

[0052] This invention constructs a dual-site fluorescent probe by modifying the benzothiophene fluorophore, which can simultaneously detect H2O2 and HClO. It has advantages such as high detection sensitivity, good selectivity and high discrimination, and has good application prospects.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A two-site benzothiazole fluorescent probe having the structure shown in Formula I: Equation I.

2. The method for preparing the dual-site benzothiazole fluorescent probe according to claim 1, characterized in that, Includes the following steps: Compound 1 and compound 2 were subjected to a cyclization reaction to obtain compound 3; Compound 3 and compound 4 were subjected to a Williamson synthesis reaction to obtain the dual-site benzothiazole fluorescent probe. The structures of compounds 1-4 are shown in the following formulas: 、 、 、 。 3. The preparation method according to claim 2, characterized in that, The solvents for the cyclization reaction include aminosulfonic acid, acetonitrile, and water.

4. The preparation method according to claim 3, characterized in that, The molar ratio of compound 1 to aminosulfonic acid is 1:0.

1.

5. The preparation method according to claim 2 or 3, characterized in that, The cyclization reaction took 24 hours.

6. The preparation method according to claim 2, characterized in that, The molar ratio of compound 3 to compound 4 is 1:1.

5.

7. The preparation method according to claim 2, characterized in that, The Williamson synthesis reaction is carried out in an alkaline environment provided by K2CO3.

8. The preparation method according to claim 2, characterized in that, The Williamson synthesis reaction was carried out at a temperature of 90-100°C for 12-24 hours.

9. The application of the dual-site benzothiazole fluorescent probe of claim 1 in the simultaneous detection of H2O2 and hypochlorite.

10. The application of the dual-site benzothiazole fluorescent probe of claim 1 in the preparation of a detection reagent, wherein the detection reagent is used to simultaneously detect H2O2 and hypochlorite.

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