A novel ratio near-infrared fluorescent probe for detecting sulfur dioxide, a preparation method and application thereof

Abstract

The application provides a novel ratio near-infrared fluorescent probe for detecting sulfur dioxide, a preparation method and application of the fluorescent probe, the fluorescent probe is Cou-Oxo, the preparation method comprises the following steps: dissolving a compound Cou-CN and a compound 3 in toluene, adding piperidine to catalyze a reaction, refluxing at 114-116 DEG C under a nitrogen atmosphere for 4.8-5.2 hours, and after the reaction is completed, the fluorescent probe Cou-Oxo is obtained through purification.The ratio fluorescent probe has good light emitting characteristics, can effectively detect sulfur dioxide derivatives, has the potential of quantitative detection, and the lower limit of detection is 0.942 muM.The ratio fluorescent probe has a fast response to the detection of sulfur dioxide derivatives, has good light emitting characteristics and stability, and can detect HSO3 ‑ in a physiological pH range of 7-9; the ratio fluorescent probe has lower biological toxicity and is suitable for cell imaging.

Description

Technical Field

[0001] This invention relates to a novel ratiometric near-infrared fluorescent probe for detecting sulfur dioxide, its preparation method, and its application, belonging to the field of fluorescent probe technology. Background Technology

[0002] Sulfur dioxide (SO2) mainly originates from the mining of sulfur-containing minerals, chemical production activities, and volcanic eruptions. It typically exists in organisms in the form of its metabolic derivatives (sulfites and bisulfites). Studies have shown that sulfur dioxide derivatives can enter the human body through the respiratory tract or food. Excessive inhalation of sulfur dioxide may cause respiratory damage, and even inflammation, nerve damage, and cardiovascular disease. Furthermore, sulfur dioxide derivatives can be endogenously produced through enzymatic catalysis and intracellular oxidative stress responses. Certain specific enzymes endogenously produce SO2 when participating in the oxidative decomposition of sulfur-containing molecules such as hydrogen sulfide, cysteine, and homocysteine. SO2 not only participates in the synthesis and metabolism of plant cells as a signaling molecule in animal cells, but excessive SO2 can also impair plant growth at the physiological and biochemical levels. Therefore, rapid and accurate detection of SO2 and its derivatives (SO3) is crucial. 2- / HSO3 - It is of great significance for physiological and pathological research.

[0003] Traditional detection techniques for sulfur dioxide (SO2) have been widely reported, including infrared spectroscopy, electrochemical potentiometrics, gas chromatography, and capillary chromatography. However, these methods all have certain limitations, such as long processing times, complex operation, high instrument costs, and the inability to perform in-situ real-time detection. Near-infrared fluorescent probes, due to their advantages of ease of operation, in-situ real-time imaging, and non-destructive testing, are highly favored in the biomedical, environmental, and food testing fields. Near-infrared fluorescent probes are also widely used in the biomedical field due to their long emission wavelength, strong tissue penetration, and high signal-to-background ratio.

[0004] There is no existing technology that can simultaneously image SO2 and its derivatives in cells, animal and plant tissues using a ratiometric fluorescent probe of coumarin derivatives. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a novel ratiometric near-infrared fluorescent probe for detecting sulfur dioxide, its preparation method, and its applications, achieving the following objectives:

[0006] We provide ratiometric fluorescent probes for the detection of sulfur dioxide derivatives. These probes offer high detection sensitivity, rapid response to sulfur dioxide derivatives, low biotoxicity, and excellent luminescent properties and stability for detecting sulfur dioxide derivatives. They can be used for imaging detection of SO2 derivatives in cells and animal / plant tissues.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A novel ratiometric near-infrared fluorescent probe for detecting sulfur dioxide, the chemical structure of which is shown below:

[0009] .

[0010] The fluorescent probe is prepared by dissolving compound Cou-CN and compound 3 in toluene, adding piperidine to catalyze the reaction, refluxing at 114-116℃ for 4.8-5.2 hours under a nitrogen atmosphere, and purifying to obtain the fluorescent probe Cou-Oxo.

[0011] The molar ratio of compound Cou-CN to compound 3 is 1:0.8-1.2; the molar ratio of compound Cou-CN to piperidine is 1:0.18-0.22; and the mass-volume ratio of compound Cou-CN to toluene is 4.5-4.7 mg:1 mL.

[0012] The chemical structural formula of the compound Cou-CN is shown below:

[0013] ;

[0014] The chemical structural formula of compound 3 is shown below:

[0015] .

[0016] The compound Cou-CN is prepared by stirring compound 1 with malononitrile, piperidine and pure glacial acetic acid in anhydrous ethanol under nitrogen protection, reacting at 88-92℃ for 5.8-6.2 hours, and then purifying to obtain compound Cou-CN.

[0017] In the preparation steps of compound Cou-CN, the molar ratio of compound 1 to malononitrile is 1:1.4-1.6; the molar ratio of compound 1 to piperidine is 1:0.18-0.22; the molar ratio of piperidine to glacial acetic acid is 1:0.9-1.1; and the mass-volume ratio of glacial acetic acid to anhydrous ethanol is 2.3-2.5 mg:1 mL.

[0018] The preparation method of compound 1 is as follows: ethyl acetoacetate, 4-(diethylamino)-2-hydroxybenzaldehyde and piperidine are dissolved in anhydrous ethanol, and the mixture is refluxed at 88-92°C for 5.8-6.2 hours under nitrogen atmosphere. After the reaction is combined, the mixture is purified to obtain compound 1. The molar ratio of ethyl acetoacetate to 4-(diethylamino)-2-hydroxybenzaldehyde is 1:0.8-1.2; the molar ratio of ethyl acetoacetate to piperidine is 1:0.28-0.32; and the mass-volume ratio of ethyl acetoacetate to anhydrous ethanol is 1g:10-12mL.

[0019] The preparation method of compound 3 is as follows: 4-(diethylamino)-2-hydroxybenzaldehyde and cesium carbonate are dissolved in DMF, and 2-bromo-1-cyclohexene-1-carboxaldehyde is slowly added. After reacting at room temperature for 47-49 hours, compound 3 is obtained by purification. The molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to cesium carbonate is 1:1.8-2.2; the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to 2-bromo-1-cyclohexene-1-carboxaldehyde is 1:2.8-3.2; and the mass-volume ratio of cesium carbonate to DMF is 1g:15-17mL.

[0020] The application of the fluorescent probe in the preparation of reagents for detecting sulfur dioxide.

[0021] The synthetic route of the fluorescent probe described in this invention is as follows:

[0022]

[0023] This invention constructs a fluorescent molecule with a DAD structure, using coumarin and xanthracene as structural units, malononitrile as an electron-withdrawing group, and dimethylamine on both sides as electron-donating groups. This probe possesses a large conjugated backbone and excellent ICT processes, enabling it to emit light in the near-infrared band (approximately 800 nm). The detection of sulfur dioxide is achieved through nucleophilic addition of unsaturated double bonds. Furthermore, due to its high precision (ratio-response), pH stability, and good selectivity, this probe has been successfully used to monitor sulfur dioxide and its derivatives in HeLa cells and zebrafish. This invention also explores an inflammation model in the roots of tobacco seedlings and uses the Cou-Oxo probe to detect the flux of sulfur dioxide derivatives in situ.

[0024] The Cou-Oxo probe of this invention can dynamically sense and detect sulfur dioxide derivatives in the microenvironment; the sensing and detection includes fluorescence detection, cell imaging, etc.

[0025] Compared with the prior art, the present invention achieves the following beneficial effects:

[0026] (1) The ratiometric fluorescent probe of the present invention has good luminescence properties, can effectively detect sulfur dioxide derivatives, has the potential for quantitative detection, and has a detection limit of 0.942 μM.

[0027] (2) The ratiometric fluorescent probe of the present invention has a fast response to the detection of sulfur dioxide derivatives, good luminescence characteristics and stability, and can detect HSO3 in the physiological pH range of 7-9. - .

[0028] (3) The ratio fluorescent probe of the present invention has low biotoxicity and is suitable for cell imaging.

[0029] (4) The ratiometric fluorescent probe of the present invention is used to detect endogenous / exogenous sulfur dioxide at the cellular level and monitor changes in intracellular sulfur dioxide concentration.

[0030] (5) The ratiometric fluorescent probe of the present invention can be applied to imaging changes of SO2 derivatives in plant tissues and zebrafish, and has broad application prospects in the field of chemical analysis and detection. Attached Figure Description

[0031] Figure 1 It is the probe Cou-Oxo prepared in Example 1. 1 H NMR spectrum;

[0032] Figure 2 It is the probe Cou-Oxo prepared in Example 1. 13 C NMR spectrum;

[0033] Figure 3 This is the HR-MS spectrum of the probe Cou-Oxo prepared in Example 1;

[0034] Figure 4 It is the fluorescence spectrum of the probe Cou-Oxo in response to SO2 derivatives;

[0035] Where (A) is the response spectrum of probe Cou-Oxo to SO2 derivative at an emission wavelength of 550 nm; (B) is the response spectrum of probe Cou-Oxo to SO2 derivative at an emission wavelength of 781 nm; (C) is the fluorescence intensity ratio ( I 550 nm / I 781 nm) and HSO3 - Linear relationship of concentration (0-100µM); (D) is the fluorescence intensity ratio ( I 550 nm / I 781 nm) and HSO3 - Linear relationship of concentration (0-40µM);

[0036] Figure 5 It is probe Cou-Oxo and HSO3 - The kinetic stability fluorescence spectrum of the system;

[0037] Figure 6 This is a bar chart showing the fluorescence intensity of the probe Cou-Oxo in different ions;

[0038] Figure 7 This is a bar chart showing the pH test results for the Cou-Oxo probe.

[0039] Figure 8 This is a bar chart showing the cytotoxicity of the Cou-Oxo probe on HeLa cells;

[0040] Figure 9 The probe Cou-Oxo is used for fluorescence imaging of exogenous SO2 derivatives in cells;

[0041] Figure 10 The probe Cou-Oxo is used for fluorescence imaging of endogenous SO2 derivatives in cells;

[0042] Figure 11 The probe Cou-Oxo is used for SO2 imaging of cross sections of tobacco seedling roots;

[0043] Figure 12 This is a confocal image of the Cou-Oxo probe targeting exogenous SO2 derivatives in zebrafish;

[0044] Figure 13 This is a confocal image of the Cou-Oxo probe targeting endogenous SO2 derivatives in zebrafish. Detailed Implementation

[0045] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0046] Example 1 Synthesis of probe compound Cou-Oxo

[0047] The synthesis route is as follows:

[0048]

[0049] The specific synthesis method is as follows:

[0050] (1) Ethyl acetoacetate (1.30 g, 10.0 mmol), 4-(diethylamino)-2-hydroxybenzaldehyde (1.93 g, 10.0 mmol) and piperidine (0.25 g, 3.0 mmol) were dissolved in anhydrous ethanol (15.0 mL) and heated under nitrogen atmosphere at 90 °C for 6 hours. After the reaction was completed, the remaining solvent was removed by vacuum evaporation. The filter cake was washed with ethanol 2-3 times and dried under vacuum to obtain a pale yellow solid. The crude product was recrystallized from ethanol to obtain the desired compound 1 (3-acetyl-7-(diethylamino)-2H-pyran-2-one, 1.3 g).

[0051] (2) Under nitrogen protection, compound 1 (518.62 mg, 2.0 mmol) was reacted with malononitrile (198.18 mg, 3.0 mmol), piperidine (34.06 mg, 0.4 mmol), and pure glacial acetic acid (24.02 mg, 0.4 mmol) in anhydrous ethanol (10.0 mL) at 90 °C for 6 hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure, and the product was purified by silica gel column chromatography using PE / EtOAc (v / v=20 / 1) as the eluent to obtain compound Cou-CN (399.56 mg).

[0052] (3) A mixture of N,N-dimethylformamide (DMF, 23.5 mL) and chloroform (100 mL) was added to a flask. Phosphorus tribromide (255.0 mmol, 24.2 mL) was added in small amounts several times under nitrogen atmosphere at 0 °C. After reacting for 1.5 hours, cyclohexanone (102.0 mmol, 10.5 mL) was added, and the mixture was reacted at room temperature for 12 hours to obtain a clear orange solution. Sodium bicarbonate was added to the solution to neutralize the pH to 7. The solution was extracted with a mixture of dichloromethane and water. After extraction, anhydrous sodium sulfate was added to remove water from the solution. The product was then purified by column chromatography using petroleum ether as the eluent. The final product was a pale yellow transparent liquid compound 2 (2-bromo-1-cyclohexene-1-carboxaldehyde, 7.7 g).

[0053] (4) In a flask containing DMF (25.0 mL) as solvent, 4-(diethylamino)-2-hydroxybenzaldehyde (4.1 mmol, 792.3 mg) and cesium carbonate (8.2 mmol, 1.5630 g) were added. The mixture was stirred thoroughly to dissolve the solid. Then, compound 2 (12.4 mmol, 2.3442 g) was slowly added. After reacting at room temperature for 48 hours, the solution was extracted with dichloromethane and water. The extract was dehydrated with anhydrous sodium sulfate and then purified by column chromatography using n-hexane and ethyl acetate (20:1, v / v) as eluents. The final product was a yellow solid compound 3 (581 mg).

[0054] (5) Compound Cou-CN (68.85 mg, 0.224 mmol) and compound 3 (63.6 mg, 0.224 mmol) were dissolved in toluene (15 mL), and piperidine (3.83 mg, 0.045 mmol) was added to catalyze the reaction. The mixture was refluxed at 115 °C for 5 hours under a nitrogen atmosphere. After the reaction was completed, the crude product was purified by silica gel column chromatography and eluted with PE / EtOAc (v / v = 15 / 1) to obtain Cou-Oxo (16 mg).

[0055] Example 2: Fluorescence response behavior of probe Cou-Oxo to SO2

[0056] The probe Cou-Oxo (5.15 mg) prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to obtain a 1 mM probe stock solution.

[0057] Add 20 μL of the probe stock solution to a cuvette, then add a mixture of DMSO and PBS (V:V=1:1) to the cuvette to obtain a final volume of 2 mL of probe solution, resulting in a final probe concentration of 10 μM.

[0058] Take an appropriate amount of sodium bisulfite solid and prepare a sodium bisulfite solution with a final concentration of 10 μM using PBS solution. The concentration of the PBS solution is 0.01 M and the pH is 7.4.

[0059] The prepared sodium bisulfite solution was added to cuvettes containing the probe solution to achieve a final concentration of 0-100 μM (0-10 eq). After incubation for 3 hours, fluorescence was measured. The results are shown below. Figure 4 .

[0060] Depend on Figure 4 As can be seen, after adding NaHSO3 solution (the main form of SO2 in aqueous solution) to the probe solution, the fluorescence of the Cou-Oxo probe at the emission wavelength of 781 nm decreased at an excitation wavelength of 460 nm. At the emission wavelength of 550 nm, a new emission peak gradually appeared and increased (Figures 4A and 4B), showing obvious ratiometric characteristics. When 10 equivalents of NaHSO3 were added, the emission intensity increased by 100 times compared to the original (Figure 4C).

[0061] The above results indicate that SO2 derivatives (HSO3) - This disrupted the conjugated structure of Cou-Oxo, resulting in a significant ratiometric fluorescence signal. The fluorescence ratio was correlated with that of the sulfur dioxide derivative (HSO3). - The correlation between concentrations is reliable (Y = 0.12321 + 0.74827X, R0). 2= 0.998), and the detection limit of Cou-Oxo was 0.942 μM (based on the 3σ / k standard method) (Figure 4D). These results indicate that Cou-Oxo can effectively detect sulfur dioxide derivatives and has the potential for quantitative detection.

[0062] Example 3: Stability test of probe Cou-Oxo

[0063] The 1 mM probe stock solution prepared in Example 2 was diluted with a mixed solution of DMSO and PBS (V:V=1:1), and sodium bisulfite solution (5 eq.) was added. The final concentration of the probe in the detection solution after adding sodium bisulfite was 10 μM, and the final concentration of bisulfite ions was 50 μM. The detection solution was obtained, and fluorescence detection was performed continuously over 90 minutes at different excitation wavelengths to obtain the fluorescence intensity in each system and establish a standard curve of fluorescence intensity versus time.

[0064] The PBS concentration was 0.01M, and the pH was 7.4.

[0065] like Figure 5 As shown, in a solution without added bisulfite ions, Cou-Oxo remained stable for 90 minutes with minimal fluctuations (Probe; λ). em =550nm; pentagram). However, in HSO3 - In the presence of HSO3, the fluorescence emission of Cou-Oxo occurs within 30 seconds (Probe + HSO3). - ;λ em =781nm; the triangle) weakened instantaneously, indicating that the probe was sensitive to HSO3. - The reaction is rapid. Furthermore, the fluorescence intensity at 550 nm wavelength continuously increases and then tends to stabilize (Probe + HSO3). - ;λ em =550nm; spherical). The probe Cou-Oxo exhibits inherent stability and resistance to HSO3. - Its responsiveness makes it a suitable choice for long-term, real-time tracking of respondents.

[0066] Example 4: Study on pH value and anti-interference of probe Cou-Oxo

[0067] Take the 1 mM probe stock solution prepared in Example 2, add 20 μL of the probe stock solution to 2 mL of PBS solution, and then add 20 μL of 10 μM standard solutions of different analytes. After shaking well, perform fluorescence detection (λ) three hours later. ex =460nm,λ em =550nm), construct a bar chart of fluorescence intensity versus each ion, such as Figure 6 As shown.

[0068] The graph shows that only sulfite and bisulfite solutions exhibit strong responses. Interference from other analytes is negligible. Fluorescence intensity changes for other analytes are almost nonexistent.

[0069] like Figure 7 As shown, the 20 μL of 1 mM probe stock solution and the 10 μM sodium bisulfite solution prepared in Example 2 were diluted to 2 mL with PBS solutions of different pH values, so that the final concentration of the probe in the test solution was 10 μM and the final concentration of bisulfite was 50 μM.

[0070] The maximum emission peak of the probe was measured at an excitation wavelength of 460 nm in environments ranging from pH 1 to 10, and a bar graph was plotted with pH value on the x-axis and the emission peak at 550 nm on the y-axis. The Cou-Oxo probe is inert at pH < 6 because the Michael addition reaction requires a base to catalyze the generation of carbanions. In acidic environments, a large amount of H+... + The presence of SO2 derivatives (HSO3) will inhibit this process. - When present, it exhibits strong fluorescence emission at physiological pH 7-9, indicating that HSO3 can be detected within the physiological pH range. - It is feasible.

[0071] Example 5: MTT cytotoxicity assay of probe Cou-Oxo

[0072] 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, and seed cells into 96-well plates (add approximately 100 μL to each well, approximately 10 mL). 4 (1 cell), and then add probe Cou-Oxo (diluted with 99.9% DMEM and 0.1% DMSO) at 0, 5, 10, 15 and 20 μM (final concentration) for incubation.

[0073] Subsequently, cells were cultured at 37 °C for 24 hours in an environment of 5% carbon dioxide and 95% air, and cytotoxicity was detected using the MTT assay. Figure 8As can be seen, using the absorbance of the cell group without Cou-Oxo (0 μg / mL) as a baseline, as the probe concentration increased, the cell viability at 5, 10, 15, and 20 μg / mL was 95.93%, 91.97%, 89.27%, and 87.08%, respectively, all above 85%, indicating that this probe with low biotoxicity is suitable for cell imaging.

[0074] Example 6: Fluorescence imaging of intracellular / exogenous SO2 by probe Cou-Oxo

[0075] HeLa 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 a glass-bottomed culture dish and placed in an incubator to allow the cells to adhere to the wall. When the adhered cells exceeded 70% of the bottom of the culture dish, the following steps were taken:

[0076] Probe control group: Wash cells three times with PBS, and then incubate with Cou-Oxo (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min.

[0077] Exogenous SO2 stimulation group: cells were washed three times with PBS, then incubated with Cou-Oxo (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min, and then incubated with NaHSO3 (10, 20 or 30 μM) under the same conditions for 30 min.

[0078] Endogenous SO2 stimulation group: cells were washed three times with PBS, then incubated with Cou-Oxo (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min, and then incubated with 2,4-dinitrobenzenesulfonamide (10, 20 or 30 μM) under the same conditions for 30 min.

[0079] Yellow channel: Excitation wavelength = 460 nm, acquisition wavelength = 510~570 nm. Scale bar = 25 μm.

[0080] like Figure 9 As shown, without adding HSO3 - At that time, the yellow channel signal was very weak. However, with exogenous HSO3... -With increasing concentration, the fluorescence intensity of the yellow channel gradually increased. This experiment demonstrates that Cou-Oxo has the ability to detect changes in external SO2 levels at the cellular level.

[0081] Then, the response of Cou-Oxo to endogenous SO2 was evaluated in HeLa cells. According to the literature, 2,4-dinitrobenzenesulfonamide responds to intracellular thiol groups, releasing endogenous sulfur dioxide. As shown in Figure 10, the yellow channel signal was weaker in the group without 2,4-dinitrobenzenesulfonamide. With increasing 2,4-dinitrobenzenesulfonamide concentration, the fluorescence intensity of the yellow channel gradually increased. These results indicate that Cou-Oxo can detect endogenous / exogenous sulfur dioxide at the cellular level and monitor changes in intracellular sulfur dioxide concentration.

[0082] Example 7: Cou-Oxo probe for imaging the root and stem of tobacco seedlings.

[0083] Purchase local common tobacco seedlings and place them separately in HSO3 - Seedlings were planted and cultured in soil contaminated with (0, 5, 10, 15, 20 μM) for 3 days, and then pulled out and immersed in probe solution Cou-Oxo (10 μM) for 8 hours.

[0084] Each group of HSO3 - Transverse sections of roots from tobacco seedlings treated with different concentrations were imaged using confocal microscopy. Before imaging, surface deposits were removed three times with clear distilled water. Finally, cells were imaged using a Leica SP8 inverted fluorescence confocal microscope. Yellow channel: excitation wavelength = 460 nm, acquisition wavelength = 510 - 570 nm. Scale bar = 250 μm.

[0085] Figure 11 shows that cross-sections of tobacco seedling roots cultured in uncontaminated soil exhibit only weak fluorescence in the yellow channels. With HSO3 - With increasing concentration, the fluorescence intensity of the yellow channel gradually increased. The results indicate that the Cou-Oxo probe is effective in detecting HSO3 in plant tissues. - An effective tool that holds promise for detecting HSO3 in tobacco. - The content of.

[0086] Example 8: Probe Cou-Oxo for Zebrafish Imaging

[0087] Exogenous SO2 stimulation group: Zebrafish were incubated with 10 μM Cou-Oxo for 30 min. They were then incubated with NaHSO3 (10, 25, or 50 μM) under the same conditions for 30 min, transferred to another imaging plate containing trace amounts of water, and imaged under a confocal microscope. Yellow light channel: excitation wavelength = 460 nm, acquisition wavelength = 510~570 nm. Scale bar = 500 μm.

[0088] Endogenous SO2 stimulation group: Zebrafish were incubated with 10 μM Cou-Oxo for 30 min, followed by incubation with 2,4-dinitrobenzenesulfonamide (5, 10, or 25 μM) under the same conditions for 30 min. They were then transferred to another imaging plate containing trace amounts of water and imaged under a confocal microscope. Yellow light channel: excitation wavelength = 460 nm, acquisition wavelength = 510~570 nm. Scale bar = 500 μm.

[0089] like Figure 12 and Figure 13 As shown, the Cou-Oxo probe (10 µM) exhibited weak fluorescence in the yellow channel after incubation with zebrafish. However, the fluorescence signal in the yellow channel significantly increased upon the addition of exogenous SO2 (10, 25, 50 μM) (Fig. 12). Fluorescence was also observed when zebrafish were stimulated solely with endogenous SO2 (5, 10, 25 μM) (Fig. 13). These imaging results confirm the ability of the Cou-Oxo probe to detect HSO3 in vivo. - Its application potential.

Claims

1. A novel ratiometric near-infrared fluorescent probe for detecting sulfur dioxide, characterized in that: The chemical structural formula of the fluorescent probe is shown in the following: 。 2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The compound Cou-CN and the compound 3 are dissolved in toluene, piperidine is added to catalyze the reaction, under the nitrogen atmosphere, reflux at 114-116 DEG C for 4.8-5.2 hours, after the reaction is completed, the fluorescent probe Cou-Oxo is obtained after purification; The chemical structural formula of the compound Cou-CN is shown in the following: ； The chemical structural formula of the compound 3 is shown in the following: 。 3. The method of claim 2, wherein: The molar ratio of the compound Cou-CN and the compound 3 is 1:0.8-1.2; the molar ratio of the compound Cou-CN and piperidine is 1:0.18-0.22; the mass-volume ratio of the compound Cou-CN and toluene is 4.5-4.7 mg:1 mL.

4. The method of claim 2, wherein: The preparation method of the compound Cou-CN is that under the nitrogen protection, the compound 1, malononitrile, piperidine and pure glacial acetic acid are uniformly stirred in anhydrous ethanol, reaction is carried out at 88-92 DEG C for 5.8-6.2 hours, after the reaction is completed, the compound Cou-CN is obtained after purification; The chemical structural formula of the compound 1 is shown in the following: 。 5. The method of claim 4, wherein: In the preparation step of the compound Cou-CN, the molar ratio of the compound 1 and malononitrile is 1:1.4-1.6; the molar ratio of the compound 1 and piperidine is 1:0.18-0.22; the molar ratio of piperidine and glacial acetic acid is 1:0.9-1.1; the mass-volume ratio of the glacial acetic acid and anhydrous ethanol is 2.3-2.5 mg:1 mL.

6. The production method according to claim 4 or 5, characterized in that: The preparation method of the compound 1 is that ethyl acetoacetate, 4-(diethylamino)-2-hydroxybenzaldehyde and piperidine are dissolved in anhydrous ethanol, under the nitrogen environment, heating is carried out at 88-92 DEG C for 5.8-6.2 hours, after the reaction is completed, the compound 1 is obtained after purification; the molar ratio of the ethyl acetoacetate and 4-(diethylamino)-2-hydroxybenzaldehyde is 1:0.8-1.2; the molar ratio of the ethyl acetoacetate and piperidine is 1:0.28-0.32; the mass-volume ratio of the ethyl acetoacetate and anhydrous ethanol is 1 g:10-12 mL.

7. The method of claim 2, wherein: The preparation method of the compound 3 is that after 4-(diethylamino)-2-hydroxybenzaldehyde and cesium carbonate are dissolved in DMF, 2-bromo-1-cyclohexene-1-formaldehyde is slowly added, after reaction is carried out at room temperature for 47-49 hours, the compound 3 is obtained after purification; the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde and cesium carbonate is 1:1.8-2.2; the molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde and 2-bromo-1-cyclohexene-1-formaldehyde is 1:2.8-3.2; the mass-volume ratio of cesium carbonate and DMF is 1 g:15-17 mL.

8. The application of the fluorescent probe in claim 1 in the preparation of a reagent for detecting sulfur dioxide.

Images