Coumarin fluorescent probe with large Stokes displacement, preparation method and application of coumarin fluorescent probe in HClO detection

Abstract

The invention provides a coumarin fluorescent probe with large Stokes displacement, a preparation method and application of the coumarin fluorescent probe in biological detection, and belongs to the field of coumarin fluorescent probes. The coumarin fluorescent probe with the large Stokes displacement has a molecular structural formula as shown in the specification, in the formula, R is one of dimethylamino, nitryl, methoxyl and hydroxyl. According to the coumarin fluorescent probe with the large Stokes displacement, provided by the invention, on the premise that the large Stokes displacement and near-infrared emission are combined, high-selectivity and quick-response effective detection on endogenous or exogenous HClO of living cells can be realized; meanwhile, the preparation raw materials of the coumarin fluorescent probe are easy to obtain, the preparation is simple, and the preparation efficiency is high.

Description

Technical Field

[0001] This invention relates to the field of coumarin fluorescent probes, and in particular to a coumarin fluorescent probe with a large Stokes shift, its preparation method, and its application in HClO detection. Background Technology

[0002] As a widely used basic fluorescent structure, coumarin uses benzo-α-pyranone as its core, and its native form exhibits almost no visible fluorescence. In existing technologies, introducing electron-donating groups at positions 6 and 7 or electron-withdrawing groups at positions 3 and 4 can extend the molecular conjugation system, resulting in a redshift of the emission wavelength. Coumarin-based fluorescent probes developed based on this strategy possess advantages such as high quantum yield, good photostability, ease of modification, and tunable wavelength, and have been widely used in biosensing, analytical detection, functional materials, and medical diagnostics.

[0003] Hypochlorous acid (HClO) is one of the endogenous reactive oxygen species, produced by the reaction of H2O2 and Cl- catalyzed by myeloperoxidase (MPO). - HClO is generated. Under physiological conditions, HClO exerts its antibacterial function through strong oxidation; however, its abnormal excess is closely related to various pathological processes such as cardiovascular disease, arthritis, and neurodegenerative diseases, and has been identified as a potential biomarker for the early diagnosis of osteoarthritis (OA). Meanwhile, HClO is also present in everyday environments such as drinking water, household bleach, and swimming pools, making the detection of HClO in the environment a widespread need.

[0004] While existing methods such as chemiluminescence, colorimetry, and electrochemistry are sensitive for the detection of HClO in live cells, they cannot meet the requirements for non-invasive real-time imaging of live cells. Reported fluorescent probes suffer from drawbacks such as small Stokes shifts (typically <50 nm), susceptibility to interference from other reactive oxygen species (ROS), poor stability, short emission wavelengths (typically <600 nm), and the inability to detect the spatiotemporal distribution of HClO in live cells in situ. These limitations prevent the effective detection of endogenous or exogenous HClO in live cells with high selectivity and rapid response, while also achieving large Stokes shifts and near-infrared emission. This has become a technical bottleneck for fluorescent probes used in the detection of HClO in live cells.

[0005] Furthermore, existing methods for preparing fluorescent probes for HClO detection in live cells suffer from problems such as difficulty in obtaining raw materials, high preparation difficulty, and low preparation efficiency.

[0006] Based on this, a coumarin-based fluorescent probe with a large Stokes shift is provided, which can achieve highly selective and rapid detection of endogenous or exogenous HClO in living cells under the premise of both large Stokes shift and near-infrared emission. At the same time, the raw materials for the preparation of this coumarin-based fluorescent probe are easy to obtain, the preparation is simple and efficient, and it has important technical significance and research value. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention provides a coumarin-based fluorescent probe with a large Stokes shift, which can achieve highly selective and rapid detection of endogenous or exogenous HClO in living cells while possessing both a large Stokes shift and near-infrared emission. At the same time, the raw materials for preparing this coumarin-based fluorescent probe are easy to obtain, the preparation is simple, and the preparation efficiency is high.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A coumarin-based fluorescent probe with a large Stokes shift has the following molecular structure: ; In the formula, R is one of the following: dimethylamino, nitro, methoxy, or hydroxy.

[0009] A method for preparing a coumarin-based fluorescent probe with a large Stokes shift, as described above, includes the following steps: a primary reaction, a secondary reaction, and a tertiary reaction; The method of the first reaction is as follows: in a solvent environment, 7-diethylamino-4-methylcoumarin and Lawson's reagent are contacted and reacted by heating to obtain compound 2; Compound 2 has the following molecular structural formula: ; The secondary reaction method is as follows: in a solvent environment, under inert gas protection, in the presence of Lewis acid and organic base, benzothiazole-2-acetonitrile is contacted with compound 2 and reacted at room temperature to obtain compound 3; Compound 3 has the following molecular structural formula: ; The method of the three reactions is as follows: in a solvent environment, under the protection of an inert gas, in the presence of piperidine, compound 3 and compound 4 are contacted and reacted by heating to obtain a coumarin fluorescent probe with a large Stokes shift. Compound 4 has the following molecular structural formula: ; In the molecular structural formula of compound 4, R is one of the following: dimethylamino, nitro, methoxy, or hydroxyl.

[0010] Preferably, in the primary reaction, the molar ratio of 7-diethylamino-4-methylcoumarin to Lawson's reagent is 1:1.2-1.8.

[0011] Preferably, in the primary reaction, the temperature of the heating reaction is 105-115℃, and the holding time of the reaction is 5.5-6.5h.

[0012] Preferably, in the secondary reaction, the molar ratio of compound 2 to benzothiazole-2-acetonitrile is 1:1.05-1.25.

[0013] Preferably, in the secondary reaction, the reaction time at room temperature is 10-12 hours.

[0014] Preferably, in the secondary reaction, the Lewis acid is silver nitrate and the organic base is triethylamine.

[0015] Preferably, in the three reactions, the molar ratio of compound 3 to compound 4 is 1:1.1-1.5.

[0016] Preferably, in the three reactions, the temperature of the heating reaction is 88-92℃, and the holding time is 10-12h.

[0017] Application of the aforementioned coumarin fluorescent probe with a large Stokes shift, or a coumarin fluorescent probe with a large Stokes shift prepared by the aforementioned method, in detecting endogenous or exogenous HClO at the live cell level.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The coumarin-based fluorescent probe of the present invention with a large Stokes shift is a coumarin-benzothiazole "double D-π-A" type near-infrared fluorescent probe with a large Stokes shift (>120 nm). This fluorescent probe achieves an "OFF-ON" type near-infrared-visible blue-shifted fluorescence signal through the epoxy ring-opening response mechanism of HClO. Specifically, the present invention uses benzaldehyde compounds with different substituents and coumarin as the parent nucleus to synthesize a hypochlorous acid (HClO) near-infrared (NIR) probe with a large Stokes shift, providing a double (D–π–a) structure to further expand the Stokes shift. When HClO induces vinyl bridge epoxidation, π-conjugation is interrupted, ICT is suppressed, and an "OFF-ON" type fluorescence signal is generated, with a significant blue shift in emission, which makes the intrinsic HClO NI The R-detection becomes highly sensitive, with a detection limit as low as 14.4 nM and a response time of <5 min, while maintaining high selectivity in pH 4-10 and the presence of various physiological ions / ROS. Simultaneously, cell experiments demonstrate that the coumarin-based fluorescent probe with a large Stokes shift exhibits excellent biocompatibility, enabling real-time imaging of endogenous hypochlorous acid generated by exogenous addition and LPS / PMA stimulation in living cells. This effectively solves the technical challenge of in-situ monitoring of the spatiotemporal distribution of HClO in existing technologies, providing a reliable tool for studying the spatiotemporal distribution, generation, and degradation mechanisms of HClO. Based on this, the coumarin-based fluorescent probe with a large Stokes shift can achieve highly selective and rapid detection of endogenous or exogenous HClO in living cells, while possessing both a large Stokes shift and near-infrared emission.

[0019] (2) The preparation method of the coumarin fluorescent probe with large Stokes shift of the present invention has readily available raw materials, simple preparation method, high preparation efficiency, and is conducive to large-scale production. Attached Figure Description

[0020] Figure 1 The image shows the HR-MS spectrum of the coumarin-based fluorescent probe CouSt-BT (R=N(CH3)2) prepared in Example 1.

[0021] Figure 2 The spectral response of the fluorescent probe CouSt-BT (R=N(CH3)2) prepared in Example 1 to five different solvents.

[0022] Figure 3 The spectral response of the fluorescent probe CouSt-BT (R=NO2) prepared in Example 1 to five different solvents.

[0023] Figure 4 The spectral response of the fluorescent probe CouSt-BT (R=OCH3) prepared in Example 1 to five different solvents.

[0024] Figure 5 The spectral response of the fluorescent probe CouSt-BT (R=OH) prepared in Example 1 to five different solvents.

[0025] Figure 6 The image shows the HR-MS spectrum of Cout-BT-ClO after the fluorescent probe Cout-BT (R=N(CH3)2) prepared in Example 1 responds.

[0026] Figure 7 This is a schematic diagram of the response mechanism of the fluorescent probe CouSt-BT (R=N(CH3)2) prepared in Example 1.

[0027] Figure 8 The image shows the spectral response characteristics of the fluorescent probe CouSt-BT to HClO.

[0028] Figure 9 These are the interference immunity test diagram and pH test diagram for the fluorescent probe CouSt-BT in the detection of HClO.

[0029] Figure 10 The fluorescence spectrum shows the kinetic stability of the fluorescent probe CouSt-BT in the HClO system.

[0030] Figure 11 This is a time-dependent fluorescence image of the fluorescent probe CouSt-BT in HepG2 cells.

[0031] Figure 12 This is a fluorescence imaging image of the fluorescent probe CouSt-BT in cells, showing the exogenous HClO.

[0032] Figure 13 This is a fluorescence imaging image of endogenous HClO in cells using the fluorescent probe CouSt-BT. Detailed Implementation

[0033] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, "first," "second," etc., are used to distinguish similar objects and are not used to describe a particular order or sequence. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0035] This invention provides a coumarin-based fluorescent probe, CouSt-BT, with a large Stokes shift, having the following molecular structure: ; In the formula, R is one of the following: dimethylamino, nitro, methoxy, or hydroxy.

[0036] Specifically, the molecular structure of the coumarin-based fluorescent probe CouSt-BT with a large Stokes shift is one of the following: .

[0037] The present invention also provides a method for preparing the coumarin-based fluorescent probe CouSt-BT with a large Stokes shift, comprising the following steps: a primary reaction, a secondary reaction, and a tertiary reaction; The method of the first reaction is as follows: in a solvent environment, 7-diethylamino-4-methylcoumarin (compound 1) is contacted with Lawson's reagent and the reaction is carried out at a higher temperature to obtain compound 2; Compound 2 has the following molecular structural formula: .

[0038] Furthermore, the method of the first reaction is as follows: in a solvent environment, 7-diethylamino-4-methylcoumarin (compound 1) is contacted with Lawson's reagent, heated to 105-115°C, and reacted at this temperature for 5.5-6.5 h. After cooling to room temperature, the product is diluted, washed, dehydrated, concentrated under reduced pressure, and then purified by column chromatography to obtain compound 2.

[0039] Specifically, the method for the first reaction is as follows: 7-diethylamino-4-methylcoumarin (compound 1) and Lawson's reagent are added to anhydrous toluene, heated to 105-115°C, and reacted at this temperature for 5.5-6.5 hours. After cooling to room temperature, the mixture is diluted with ethyl acetate (EtOAc), and the organic layer is washed with deionized water. The organic layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a concentrate. The concentrate is purified by column chromatography to obtain compound 2.

[0040] In the aforementioned primary reaction, the molar ratio of 7-diethylamino-4-methylcoumarin to Lawson's reagent is 1:1.2-1.8.

[0041] The secondary reaction method is as follows: in a solvent environment, under inert gas protection, in the presence of Lewis acid and organic base, benzothiazole-2-acetonitrile is contacted with compound 2 and reacted at room temperature to obtain compound 3; Compound 3 has the following molecular structural formula: .

[0042] Furthermore, the secondary reaction method is as follows: in a solvent environment, under inert gas protection, in the presence of silver nitrate and triethylamine, benzothiazole-2-acetonitrile is contacted with compound 2, reacted at room temperature, diluted, washed, dehydrated, concentrated under reduced pressure, and then purified by column chromatography to obtain compound 3.

[0043] Specifically, the secondary reaction method is as follows: benzothiazol-2-acetonitrile and silver nitrate are dissolved in anhydrous ethanol, then an anhydrous ethanol solution of compound 2 is added, and triethylamine is added dropwise. Under inert gas protection, the reaction is carried out at room temperature for 10-12 hours. The mixture is then diluted with ethyl acetate (EtOAc), the organic layer is washed with deionized water, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a concentrate. The concentrate is purified by column chromatography to obtain compound 3.

[0044] In the secondary reaction, the molar ratio of compound 2 to benzothiazole-2-acetonitrile is 1:1.05-1.25.

[0045] The method of the three reactions is as follows: in a solvent environment, under the protection of an inert gas, in the presence of piperidine, compound 3 and compound 4 are contacted and reacted by heating to obtain a coumarin fluorescent probe CouSt-BT with a large Stokes shift. Compound 4 has the following molecular structural formula: ; In the formula, R is one of the following: dimethylamino, nitro, methoxy, or hydroxy.

[0046] The compound 4 is one of the following: p-hydroxybenzaldehyde, p-nitrobenzaldehyde, p-methoxybenzaldehyde, or p-dimethylaminobenzaldehyde.

[0047] Furthermore, the method for the three-stage reaction is as follows: in a solvent environment, under the protection of an inert gas, in the presence of piperidine, compound 3 and compound 4 are contacted, heated to 88-92°C, and reacted at this temperature for 10-12 hours. After cooling to room temperature, the mixture is diluted, washed, dehydrated, concentrated under reduced pressure, and then purified by column chromatography to obtain the coumarin-based fluorescent probe CouSt-BT with a large Stokes shift.

[0048] Specifically, the three-reaction method is as follows: Compound 3, Compound 4, and piperidine are added to acetonitrile, heated to 88-92°C under inert gas protection, and reacted at this temperature for 10-12 hours. After cooling to room temperature, the mixture is diluted with ethyl acetate (EtOAc), and the organic layer is washed with deionized water. The organic layer is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain a concentrate. The concentrate is purified by column chromatography to obtain the coumarin-based fluorescent probe CouSt-BT with a large Stokes shift.

[0049] In the three reactions, the molar ratio of compound 3 to compound 4 was 1:1.1-1.5.

[0050] The preparation method of the coumarin-based fluorescent probe CouSt-BT with a large Stokes shift, and its synthetic route are as follows: .

[0051] This invention also provides the application of the coumarin-based fluorescent probe CouSt-BT with a large Stokes shift in detecting endogenous or exogenous HClO at the live cell level.

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with some specific embodiments.

[0053] Example 1 1. This embodiment provides a method for preparing a coumarin-based fluorescent probe CouSt-BT (R=N(CH3)2) with a large Stokes shift. The specific steps are as follows: (1) 7-Diethylamino-4-methylcoumarin (466.0 mg, 2.0 mmol) and Lawson's reagent (1.202 g, 3.0 mmol) were dissolved in anhydrous toluene (5.0 mL). The mixture was stirred at 110 °C for 6 h, cooled to room temperature and diluted with ethyl acetate (EtOAc) (100 mL). The organic layer was washed with H2O (3 × 100 mL), dried with anhydrous Na2SO4, filtered to remove solids, and concentrated under reduced pressure to obtain a concentrate. The concentrate was subjected to silica gel column chromatography (petroleum ether PE / ethyl acetate EtOAc = 20:1) to give a viscous yellow oil compound 2 (461.4 mg, yield 93.4%).

[0054] (2) Add AgNO3 (952 mg, 5.6 mmol) and benzothiazole-2-acetonitrile (390 mg, 2.24 mmol) to a 100 mL round-bottom flask, and add anhydrous ethanol (10 mL) to completely dissolve benzothiazole-2-acetonitrile; then add a solution of compound 2 (460 mg, 1.87 mmol) in anhydrous ethanol (5 mL), and add three drops of triethylamine. Under nitrogen atmosphere, react overnight at room temperature, and dilute with ethyl acetate EtOAc (100 mL); wash the organic layer with H2O (3 × 100 mL), dry with anhydrous Na2SO4, filter to remove solids, concentrate under reduced pressure to obtain concentrate; pass the concentrate by column chromatography (petroleum ether PE / ethyl acetate EtOAc = 5:1) to obtain orange-red solid compound 3 (302.5 mg, yield 41.8%).

[0055] (3) Compound 3 (193.5 mg, 0.50 mmol) and p-dimethylaminobenzaldehyde (R=N(CH3)2, 90 mg, 0.6 mmol) were added to a pressure-resistant tube. Two drops of piperidine were added in acetonitrile (5 mL) as solvent. The reaction was stirred at 90 °C for 12 h under nitrogen atmosphere protection, cooled to room temperature and diluted with ethyl acetate (EtOAc) (100 mL). The organic layer was washed with H2O (3 × 100 mL), dried with anhydrous Na2SO4, filtered to remove solids, and concentrated under reduced pressure to obtain a concentrate. The concentrate was subjected to silica gel column chromatography (petroleum ether PE / ethyl acetate EtOAc = 1:1) to obtain the final product, a red solid, namely the coumarin fluorescent probe CouSt-BT (R=N(CH3)2, 138.4 mg, yield 53.3%) with a large Stokes shift. The HR-MS spectrum is shown below. Figure 1 As shown.

[0056] 2. This embodiment also provides a method for preparing a coumarin-based fluorescent probe CouSt-BT (R=NO2) with a large Stokes shift. The specific method is the same as the preparation method of the aforementioned coumarin-based fluorescent probe CouSt-BT (R=N(CH3)2), except that in step 3, p-nitrobenzaldehyde (R=NO2, 91mg, 0.6mmol) is used instead of p-dimethylaminobenzaldehyde; the final coumarin-based fluorescent probe CouSt-BT with a large Stokes shift is obtained (R=NO2, 113.7mg, yield 43.6%).

[0057] 3. This embodiment also provides a method for preparing a coumarin-based fluorescent probe CouSt-BT (R=OCH3) with a large Stokes shift. The specific method is the same as the preparation method of the aforementioned coumarin-based fluorescent probe CouSt-BT (R=N(CH3)2), except that in step 3, p-methoxybenzaldehyde (R=OCH3, 81.6 mg, 0.6 mmol) is used instead of p-dimethylaminobenzaldehyde; the final coumarin-based fluorescent probe CouSt-BT with a large Stokes shift is obtained (R=OCH3, 128.4 mg, yield 50.9%).

[0058] 4. This embodiment also provides a method for preparing a coumarin-based fluorescent probe CouSt-BT (R=OH) with a large Stokes shift. The specific method is the same as the preparation method of the aforementioned coumarin-based fluorescent probe CouSt-BT (R=N(CH3)2), except that in step 3, p-hydroxybenzaldehyde (R=OCH3, 73mg, 0.6mmol) is used instead of p-dimethylaminobenzaldehyde; the final coumarin-based fluorescent probe CouSt-BT with a large Stokes shift is obtained (R=OH, 132.7mg, yield 54.1%).

[0059] Example 2 Spectroscopic response characteristics of the fluorescent probe CouSt-BT to HClO; The fluorescent probes CouSt-BT (R1=N(CH3)2, 5.18 mg), CouSt-BT (R2=NO2, 5.20 mg), CouSt-BT (R3=OCH3, 5.05 mg), and CouSt-BT (R4=OH, 4.91 mg) prepared in Example 1 were dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain four different 1 mM fluorescent probe test solutions.

[0060] Take the test stock solutions of each fluorescent probe and add them to a cuvette containing a mixture of five solvents: ethanol (EtOH), methanol (MeOH), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), acetonitrile (ACN), and PBS buffer (V:V = 1:1). Control the final detection solution volume to 2 mL to achieve a final fluorescent probe concentration of 10 μM. Then, take an appropriate amount of 10 wt% sodium hypochlorite solution and prepare a 10 μM sodium hypochlorite solution using a 0.1 mol / L sodium hydroxide aqueous solution. Add 200 μM sodium hypochlorite solution to the aforementioned test solution and react for 10 min. Perform UV absorption, fluorescence spectra before and after the reaction in the five solvents. The spectral response of each fluorescent probe to the five different solvents is as follows: Figure 2-5As shown in the figures, the comparison reveals that the fluorescent probe CouSt-BT (R=N(CH3)2) exhibits superior response performance compared to the fluorescent probe CouSt-BT (R=NO2, R=OCH3, R=OH). Furthermore, the comparison of solvents demonstrates that ethanol (EtOH) performs better as the test solution. Therefore, subsequent tests were conducted using a mixture of ethanol (EtOH) and PBS buffer (V:V=1:1), and the fluorescent probe CouSt-BT (R=N(CH3)2) was used as the test sample.

[0061] Further, 20 μL of the fluorescent probe CouSt-BT (R=N(CH3)2) test solution was added to a cuvette, followed by a mixture of ethanol (EtOH) and PBS buffer (V:V=1:1) to maintain a final detection volume of 2 mL, resulting in a probe concentration of 10 μM. An appropriate amount of 10 wt% sodium hypochlorite solution was prepared using a 0.1 mol / L sodium hydroxide aqueous solution to achieve a final sodium hypochlorite concentration of 10 μM. Then, 0-450 μM of the prepared sodium hypochlorite solution was added to the aforementioned detection solution to each cuvette, and the mixture was incubated for 10 min before testing.

[0062] Upon detection, after the addition of HClO, at an excitation wavelength of 400 nm, the fluorescent probe CouSt-BT showed a new emission peak at an emission wavelength of 470 nm, which gradually appeared and increased with the concentration of HClO (e.g., ...). Figure 8 As shown in Figure a), it exhibits fluorescence enhancement properties.

[0063] The above results indicate that the strong oxidizing property of HClO disrupts the conjugated structure of CouSt-BT, and the fluorescence intensity shows a linear relationship with HClO, y = 20.68 + 1.04[HClO], R 2 =0.996, detection limit is 14.4 nM (based on the 3σ / k standard method) (e.g. Figure 8 (As shown in b). The aforementioned results indicate that the fluorescent probe CouSt-BT can effectively detect HClO and has the potential for quantitative detection.

[0064] Simultaneously, the response mechanism of the fluorescent probe CouSt-BT (R1=N(CH3)2) was verified. 500 μM hypochlorous acid solution was added to a pure methanol solution of the fluorescent probe CouSt-BT (R1=N(CH3)2), and the response was tested after 10 min. The HR-MS spectrum of the responded CouSt-BT-ClO is shown below. Figure 6 As shown, through with Figure 1The comparison shows that the responded Coutst-BT-ClO exhibits a new peak at 535 m / z, verifying that its response mechanism is consistent with our prediction. The specific response mechanism is as follows: Figure 7 As shown.

[0065] Example 3 Anti-interference study and pH test of fluorescent probe CouSt-BT (R=N(CH3)2) for HClO detection: First, an anti-interference test was conducted. 20 μL of the 1 mM fluorescent probe CouSt-BT (R=N(CH3)2) test stock solution prepared in Example 2 was added to a 2 mL mixture of ethanol (EtOH) and PBS buffer (V:V=1:1). Then, 20 μL of standard solutions of different interfering ions (concentration 10 μM) were added. After shaking, fluorescence detection was performed for 5 min (λex=400nm, λem=470nm). Then, the same amount of HClO as the interfering ion was added, and fluorescence detection was performed again (λex=400nm, λem=470nm). A bar chart of fluorescence intensity versus each interfering ion was established.

[0066] Specific test results are as follows: Figure 9 As shown in the middle left figure, the interfering ions added in the figure, numbered 1-21, are Cu, ... 2+ SO4 2- Na + NO 2- HS - S 2- Fe 3+ Cys, Hcy, CO3 2- K + Ca 2+ Mg 2+ Hg 2+ Zn 2+ The components are N2H4·H2O, Arg, Trp, Ala, H2O2, and Ser. It can be seen that the fluorescent probe CouSt-BT only shows a strong response to the solution after the addition of hypochlorite. The interference of other interfering substances on the probe's fluorescence performance is negligible, and the fluorescence intensity of other interfering substances changes almost nothing. Therefore, the fluorescent probe CouSt-BT has good anti-interference ability.

[0067] Then, a pH test was performed. 20 μL of the 1 mM probe stock solution prepared in Example 2 and a 10 μM sodium hypochlorite solution were taken and diluted to 2 mL with PBS solutions of different pH (4-10) so that the final concentration of the probe in the test solution was 10 μM and the final concentration of the hypochlorite ion was 200 μM.

[0068] The maximum emission peak of the probe was measured at an excitation wavelength of 400 nm under pH conditions ranging from 4 to 10. Figure 9 As shown in the middle right figure, it can be seen that the fluorescent probe CouSt-BT exhibits strong fluorescence emission in the commonly used pH range of 4-10 when hypochlorite is present, which indicates that the fluorescent probe CouSt-BT is feasible for detecting hypochlorite within the physiological pH range.

[0069] Example 4 Kinetic stability test of fluorescent probe CouSt-BT (R=N(CH3)2) in HClO system: The 1 mM fluorescent probe CouSt-BT (R=N(CH3)2) test stock solution prepared in Example 2 was diluted with a mixture of ethanol (EtOH) and PBS buffer (V:V=1:1). Then, 20 eq of prepared HClO solution was added to control the final probe concentration in the test solution after adding HClO to be 10 μM and the final hypochlorite ion concentration to be 200 μM. After preparing the test solution, direct testing was performed, and fluorescence detection was continuously conducted within 30 min. The concentration of PBS buffer in the test solution was 0.01 M, and the pH was 7.4. The fluorescence intensity in each system was obtained by fluorescence detection, and a standard curve of fluorescence intensity versus time was established. For example... Figure 10 As shown in the figure, after the addition of hypochlorite ions, the fluorescence intensity of the fluorescent probe CouSt-BT at a wavelength of 470 nm continuously increased and then tended to stabilize (5 min). The fluorescent probe CouSt-BT exhibits inherent stability and resistance to ClO₂. - Its responsiveness makes it a suitable choice for long-term, real-time tracking of respondents.

[0070] Example 5 Time-dependent fluorescence assay of the fluorescent probe CouSt-BT (R=N(CH3)2) in HepG2 cells: HepG2 cells were cultured in Dulbecco modified Eagle medium (DMEM, Hyclone) containing 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin, Hyclone) for 3 days at a cell density of 1 × 10⁻⁶ cells / mL. 5 / mL. 1 mL of solution with a density of 1×10⁻⁶ 5 / mL of cell culture medium was seeded into glass-bottomed culture dishes and placed in an incubator to allow the cells to adhere to the walls. When the adhered cells exceeded 70% of the bottom of the culture dish, the following steps were taken: Time-delayed confocal fluorescence microscopy was used to monitor changes in CUT-BT fluorescence intensity in cells over time. Figure 11 As shown, HepG2 cells were co-incubated with the fluorescent probe CouSt-BT (R=N(CH3)2) and HClO solution for 2 min, and a weak fluorescence signal was observed. After co-incubation for 5 min, the fluorescence signal in the cells significantly increased (final probe concentration: 10 μM; final hypochlorite ion concentration: 50 μM). With prolonged culture time, the fluorescence signal tended to stabilize and gradually increased. Therefore, CouSt-BT is an effective tool for real-time detection of HClO.

[0071] Example 6 Fluorescence imaging assay of intracellular / exogenous HClO in HepG2 cells using the fluorescent probe CouSt-BT (R=N(CH3)2): HepG2 cells were cultured in Dulbecco modified Eagle medium (DMEM, Hyclone) containing 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin, Hyclone) for 3 days at a cell density of 1 × 10⁻⁶ cells / mL. 5 / mL. 1 mL of solution with a density of 1×10⁻⁶ 5 / mL of cell culture medium was seeded into glass-bottomed culture dishes and placed in an incubator to allow the cells to adhere to the walls. When the adhered cells exceeded 70% of the bottom of the culture dish, the following steps were taken: Probe control group: Wash cells three times with PBS, and then incubate with the fluorescent probe CouSt-BT (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min.

[0072] Exogenous HClO stimulation group: cells were washed three times with PBS, then incubated with the fluorescent probe CouSt-BT (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min, and then incubated with HClO (40, 80 or 120 μM) under the same conditions for 30 min.

[0073] Endogenous HClO stimulation group: cells were pretreated with LPs and PMA (10 μM) for 8 h, washed with PBS 3 times, and then incubated with the fluorescent probe CouSt-BT (10 μM) at 37 °C, 5% carbon dioxide and 95% air for 30 min.

[0074] In the fluorescence imaging experiment of this embodiment, the green channel has the following parameters: excitation wavelength = 400 nm, acquisition wavelength = 440-500 nm, and scale bar = 25 μm.

[0075] like Figure 12As shown, the green channel signal is weak without the addition of hypochlorite. However, the fluorescence intensity of the green channel gradually increases with increasing exogenous hypochlorite concentration. This experiment demonstrates that the fluorescent probe CouSt-BT has the ability to detect changes in external hypochlorite levels at the cellular level.

[0076] Then, the response of the fluorescent probe CouSt-BT to endogenous HClO was evaluated in HepG2 cells. According to the literature, LPs scavenge intracellular hypochlorite, while PMA indirectly leads to an increase in intracellular hypochlorite levels. Figure 13 As shown, the green channel signal was weak in the group without added LPs and PMA. The fluorescence intensity of the green channel was significantly enhanced in the group with added LPs and PMA (10 μM). These results indicate that the fluorescent probe CouSt-BT can detect endogenous / exogenous hypochlorous acid at the cellular level and monitor changes in intracellular hypochlorous acid concentration.

[0077] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0078] 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 coumarin-based fluorescent probe with a large Stokes shift, characterized in that, It has the following molecular structural formula: ； In the formula, R is one of the following: dimethylamino, nitro, methoxy, or hydroxy.

2. A method for preparing a coumarin-based fluorescent probe with a large Stokes shift as described in claim 1, characterized in that, It includes the following steps: primary reaction, secondary reaction, and tertiary reaction; The method of the first reaction is as follows: in a solvent environment, 7-diethylamino-4-methylcoumarin and Lawson's reagent are contacted and reacted by heating to obtain compound 2; Compound 2 has the following molecular structural formula: ； The secondary reaction method is as follows: in a solvent environment, under inert gas protection, in the presence of Lewis acid and organic base, benzothiazole-2-acetonitrile is contacted with compound 2 and reacted at room temperature to obtain compound 3; Compound 3 has the following molecular structural formula: ； The method of the three reactions is as follows: in a solvent environment, under the protection of an inert gas, in the presence of piperidine, compound 3 and compound 4 are contacted and reacted by heating to obtain a coumarin fluorescent probe with a large Stokes shift. Compound 4 has the following molecular structural formula: ； In the molecular structural formula of compound 4, R is one of the following: dimethylamino, nitro, methoxy, or hydroxyl.

3. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the aforementioned primary reaction, the molar ratio of 7-diethylamino-4-methylcoumarin to Lawson's reagent is 1:1.2-1.

8.

4. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the first reaction, the temperature for heating the reaction is 105-115℃, and the holding time for the reaction is 5.5-6.5h.

5. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the secondary reaction, the molar ratio of compound 2 to benzothiazole-2-acetonitrile is 1:1.05-1.

25.

6. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the secondary reaction, the reaction time at room temperature is 10-12 hours.

7. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the secondary reaction, the Lewis acid is silver nitrate; the organic base is triethylamine.

8. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the three reactions, the molar ratio of compound 3 to compound 4 was 1:1.1-1.

5.

9. The method for preparing a coumarin-based fluorescent probe with a large Stokes shift according to claim 2, characterized in that, In the three reactions, the temperature of the heating reaction is 88-92℃, and the holding time is 10-12h.

10. The application of a coumarin fluorescent probe with a large Stokes shift as described in claim 1, or a coumarin fluorescent probe with a large Stokes shift prepared by any one of claims 2-9, in detecting endogenous or exogenous HClO at the live cell level.

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