A coumarin-quinoline hybrid near-infrared fluorescent probe compound and a synthesis method and application thereof

Abstract

The application discloses a coumarin-quinoline hybrid near-infrared fluorescent probe compound for reversibly detecting sulfur dioxide and hydrogen peroxide, a synthesis method and application thereof. The probe takes a coumarin derivative as a fluorescent group and is connected with a quinoline salt through a conjugated chain. The probe itself has weak fluorescence, and after a specific Michael addition reaction occurs between the C-4 position of the quinoline salt unit of the probe and HSO3-, strong near-infrared fluorescence emission is generated at 712 nm, the response time is less than 5 seconds, and the detection limit is as low as 15.5 nM. The addition product can be oxidized by H2O2, the original probe structure is restored, and the fluorescence is quenched, so that reversible detection is realized. The probe has high selectivity and strong anti-interference ability. The probe can be used for quantitatively detecting the content of sulfur dioxide in food and water samples, and is successfully applied to imaging of endogenous and exogenous sulfur dioxide in living cells, monitoring of sulfur dioxide in lipid droplets and reversible cycle imaging of sulfur dioxide and hydrogen peroxide, and has important application value in the fields of food safety monitoring and biomedical research.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes and biosensing technology, specifically relating to a coumarin-quinoline hybrid near-infrared fluorescent probe compound, its preparation method, and its application in the detection of sulfur dioxide and hydrogen peroxide, particularly in food detection and bioimaging. Background Technology

[0003] Hydrogen peroxide (H2O2), as one of the most important reactive oxygen species in living organisms, is a crucial signaling molecule and biomarker, playing a vital role in maintaining cellular redox homeostasis and signal transduction. However, when produced in excess, it can damage biological macromolecules such as DNA, thereby inducing a series of diseases such as inflammation, Parkinson's disease, and cancer. J. Hazard. Mater. 2022, 432 , 128605; Sens. Actuators. B. Chem. 2024, 418 ,136311; Food Chem 2023, 410 (135381). Therefore, SO2 and H2O2 are not only key signaling molecules regulating cellular redox homeostasis, but also jointly maintain the dynamic stability of intracellular redox balance. Given their crucial roles in physiological and pathological processes, developing analytical tools capable of real-time monitoring of their dynamic interconversion is of great significance. Near-infrared fluorescent probes have shown broad prospects in the field of bioimaging due to their advantages such as low background interference, minimal photodamage, and strong tissue penetration. However, near-infrared fluorescent probes capable of reversibly imaging the SO2 / H2O2 redox cycle at the living cell level are still rarely reported. Therefore, constructing near-infrared probes for SO2 and H2O2 with both high selectivity and reversible response is of great importance.

[0004] Based on this, this invention designs a fluorescent probe CQB for real-time quantitative reversible detection of sulfur dioxide and hydrogen peroxide. This probe is a hybrid of a coumarin derivative and a quinoline salt. The quinoline salt has high electrophilicity at its C-4 position, allowing it to undergo a Michael addition reaction with SO2, achieving an ultrafast response to SO2. Due to the oxidizing properties of H2O2, the addition product can be reversibly oxidized and regenerated by H2O2 to regenerate the probe CQB, thus achieving reversible detection of sulfur dioxide and hydrogen peroxide. At an excitation wavelength of 530 nm, the probe itself is luminous; however, upon the addition of HSO3... - Subsequently, a strong emission peak appeared at 712 nm in the near-infrared region. Upon further addition of H2O2, the addition product was reversibly oxidized to restore the probe's original structure, and the fluorescence signal weakened accordingly, thus achieving reversible control of the process. Summary of the Invention

[0005] One of the objectives of this invention is to provide a novel coumarin-quinoline hybrid near-infrared fluorescent probe compound.

[0006] A second objective of this invention is to provide a method for synthesizing the aforementioned fluorescent probe compound.

[0007] A third objective of this invention is to provide the application of the above-mentioned fluorescent probe compound in the detection of sulfur dioxide and / or hydrogen peroxide.

[0008] A fourth objective of this invention is to provide the application of the above-mentioned fluorescent probe compound in the preparation of reagents for fluorescence imaging of sulfur dioxide and / or hydrogen peroxide.

[0009] In a first aspect, the present invention provides a coumarin-quinoline hybrid near-infrared fluorescent probe compound for the reversible detection of sulfur dioxide and hydrogen peroxide, with the English name 3-((1E,3E)-4-(8-(diethylamino)-2,2-difluoro-5-oxo-2H,5H-2l4,3l3-[1,3,2]dioxaborinino[5,4-c]chromen-4-yl)buta-1,3-dien-1-yl)-1-methylquinolin-1-ium, named CQB, and with the structural formula as shown in formula (1): Equation (1) The near-infrared fluorescent probe CQB of this invention uses a coumarin derivative as the parent compound and introduces a highly electrophilic quinoline salt cation as a specific recognition group through a conjugated olefin bond. The probe molecule itself is in a fluorescence quenching state. When bisulfite (the main water-soluble form of sulfur dioxide) is present in the system, a Michael addition reaction rapidly occurs at the C-4 position of the quinoline salt, reconstructing the conjugated molecular system and releasing strong characteristic fluorescence in the 712 nm near-infrared band. After introducing hydrogen peroxide into the system, the addition product undergoes an oxidation reaction to restore the original molecular structure of the probe, and the fluorescence signal weakens and fades simultaneously, thus completing the reversible cyclic recognition between the two.

[0010] Secondly, the present invention provides a method for synthesizing the above-mentioned fluorescent probe compound CQB, comprising the following steps: (1) At 0~5℃, phosphorus oxychloride was slowly added to a mixture of malonic acid and phenol. The reaction mixture was heated at 110~120℃ for 1~2 h and then quenched by cooling. The mixture was extracted with ethyl acetate, the organic layer was dried with anhydrous sodium sulfate and concentrated under reduced pressure, and the obtained sample was separated by column chromatography to obtain a white solid compound 1; the molar ratio of malonic acid, phenol and phosphorus oxychloride was 1: (1.5~2.5): (1.2~1.8), preferably 1:2:1.5; The structural formula of compound 1 is (2) Compound 1 and m-hydroxy-N,N-diethylaniline were dissolved in toluene. After refluxing the reaction system for 10-12 h, the mixture was cooled to room temperature, and a solid precipitated out. Subsequently, the solid obtained by vacuum filtration was separated by silica gel column chromatography to obtain yellow solid compound 2; the molar ratio of compound 1 to m-hydroxy-N,N-diethylaniline was 1:(0.8-1.2), preferably 1:1; The structural formula of compound 2 is (3) Under conditions of 0~5℃, N,N-dimethylformamide was slowly added to phosphorus oxychloride, and the reaction was stirred for 30 min. Then, the DMF solution of compound 2 was added to the above reaction system, and the reaction was continued at 80~90℃ for 5~6 h. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into ice water and stirred gently. The precipitate was filtered and washed with isopropanol. The crude product was separated by column chromatography to obtain compound 3. The molar ratio of N,N-dimethylformamide, phosphorus oxychloride and compound 2 was (5~7):(3~5):1, preferably 6:4:1. The structural formula of compound 3 is (4) Dissolve compound 3 in toluene, heat to 110-120°C, slowly add boron trifluoride diethyl ether to the above reaction mixture, and continue heating for 0.5-1 h. After the reaction is complete, cool to room temperature, add petroleum ether, precipitate a yellow solid, and separate by column chromatography to obtain compound 4; the molar ratio of compound 3 to boron trifluoride diethyl ether is 1:(1.2-1.8), preferably 1:1.5; The structural formula of compound 4 is (5) Dissolve (triphenylphosphonic acid) acetaldehyde and 3-quinoline formaldehyde in anhydrous toluene. Stir the reaction system at 60-70℃ for 10-12 h, cool it to room temperature, concentrate it under reduced pressure, and separate the crude product by column chromatography to obtain compound 5; the molar ratio of (triphenylphosphonic acid) acetaldehyde and 3-quinoline formaldehyde is 1: (0.8-1.2), preferably 1:1; The structural formula of compound 5 is (6) Under an argon atmosphere, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 5. The reaction system was stirred at room temperature for 24-26 h, and then concentrated and purified under reduced pressure to obtain compound 6. The molar ratio of compound 5 to methyl trifluoromethanesulfonate was 1: (8-12), preferably 1:10. The structural formula of compound 6 is (7) Add methanesulfonic acid to the acetonitrile solution of compound 4 and compound 6, and heat at 70°C. o After heating and stirring at C for 5-6 h, the organic solvent was removed under reduced pressure. The crude product was purified by column chromatography to obtain the target product CQB. The molar ratio of compound 4 to compound 6 was 1:(0.8-1.2), preferably 1:1.

[0011] Thirdly, the present invention provides the fluorescent probe compound CQB described above for the detection of sulfur dioxide (in the form of HSO3⁻ / SO3). 2 Applications in (in its ⁻ form) and / or hydrogen peroxide (H2O2). The detection includes, but is not limited to, quantitative fluorescence detection, qualitative identification, and dynamic reversible monitoring. In particular, this probe can be used to detect sulfur dioxide content in food (such as white sugar, rock sugar, dried fruit, etc.) or water samples.

[0012] The fluorescent probe responds to bisulfite in less than 5 seconds, with a detection limit as low as 15.5 nM for bisulfite and 119.7 nM for hydrogen peroxide. It can complete four or more reversible fluorescence cycles of sulfur dioxide and hydrogen peroxide, exhibiting excellent selectivity and is unaffected by biothiols, reactive oxygen species, amino acids, or common anion and cation matrices.

[0013] Fourthly, this invention provides the application of the aforementioned fluorescent probe compound CQB in the preparation of a fluorescent imaging reagent for detecting sulfur dioxide and / or hydrogen peroxide. This reagent is particularly suitable for real-time, reversible fluorescent imaging monitoring of sulfur dioxide and / or hydrogen peroxide levels in living cells or biological tissues.

[0014] The fluorescent probe CQB can target the lipid droplet structure in HepG2 cells for fluorescence visualization imaging detection of exogenous and endogenous sulfur dioxide in living cells. Utilizing the reversible properties of the probe's addition reaction with bisulfite to enhance fluorescence and its reduction reaction with hydrogen peroxide to quench fluorescence, real-time fluorescence monitoring of the dynamic redox cycle of these two substances within living cells is achieved.

[0015] Compared with the prior art, the present invention has the following advantages: 1. It has reversible response characteristics: Based on the coumarin-quinoline salt hybrid skeleton, it utilizes the high electrophilicity of the C-4 position of the quinoline salt to undergo a specific Michael addition with SO2. The addition product can be reversibly recovered by H2O2 oxidation, thereby realizing reversible and dynamic monitoring of the SO2 / H2O2 redox cycle; 2. Excellent response performance: The probe of this invention can complete a rapid response to bisulfite within 5 seconds, with high detection sensitivity. The detection limit of bisulfite is as low as 15.5 nM, and the detection limit of hydrogen peroxide can reach 119.7 nM, which far exceeds the detection accuracy of conventional fluorescent probes of the same type. 3. Strong anti-interference and selectivity: It shows no obvious fluorescence response to common reactive oxygen species, biothiols, amino acids, and conventional anions and cations in water and food. The matrix has strong anti-interference ability, and the detection results are accurate and reliable. 4. Outstanding advantages of near-infrared emission: The CQB probe emits wavelengths in the near-infrared region (712 nm), which has the advantages of low background interference and strong tissue penetration, making it suitable for imaging complex biological systems; 5. Wide range of application scenarios: It can meet the needs of food sulfur dioxide safety screening and environmental water pollutant detection in the field of people's livelihood, and also meet the scientific research needs of cell physiological mechanism research and redox pathological mechanism exploration in the biomedical field, with high practical value. Attached Figure Description

[0016] Figure 1 The proton nuclear magnetic resonance spectrum of the fluorescent probe CQB prepared in Example 1 of this invention (… 1 H NMR spectrum; Figure 2 The carbon nuclear magnetic resonance spectrum of the fluorescent probe CQB prepared in Example 1 of this invention ( 13 C NMR spectrum; Figure 3 This is a high-resolution mass spectrometry (HRMS) image of the fluorescent probe CQB prepared in Example 1 of this invention; Figure 4 The kinetics of the reaction between the fluorescent probe CQB and (A) sulfur dioxide and (B) the reversible cycle of sulfur dioxide and hydrogen peroxide are shown. Figure 5 The fluorescent probe CQB is used to detect different concentrations of HSO3. - (A) Fluorescence spectrum response diagrams of H2O2 (C), and corresponding linear relationship diagrams of fluorescence intensity versus concentration (B and D); Figure 6 CQB fluorescent probe for HSO3 - (A) and H2O2 (B) selectivity and anti-interference test bar chart; Figure 7 Confocal fluorescence imaging of fluorescent probe targeting CQB lipid droplets; Figure 8 The fluorescent probe CQB was used to monitor exogenous HSO3 in HepG2 cells. - Confocal fluorescence imaging with horizontal variation; Figure 9Confocal fluorescence imaging of the fluorescent probe CQB used to monitor changes in lipid droplets in HepG2 cells; Figure 10 The fluorescent probe CQB was used to monitor endogenous HSO3 in HepG2 cells. - Confocal fluorescence imaging with horizontal variation; Figure 11 The fluorescent probe CQB is used for reversible monitoring of HSO3 in HepG2 cells. - Confocal fluorescence imaging of changes in H2O2. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the reagents and materials used in this invention are commercially available.

[0018] Example 1 Synthesis of fluorescent probe compounds (1) Synthesis of compound 1 Phosphorus oxychloride (45 mmol, 4.2 mL) was slowly added dropwise to a mixture of malonic acid (30 mmol, 3.12 g) and phenol (60 mmol, 5.65 g) in an ice bath at 0 °C. After the addition was complete, the reaction mixture was heated to 115 °C and reacted for 1.5 hours. After the reaction was completed, the mixture was cooled to room temperature, the upper layer was poured into 400 mL of water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting sample was separated by column chromatography (petroleum ether / ethyl acetate = 200:1, v / v) to give compound 1 as a white solid in 82% yield. 1 H NMR (400 MHz, CDCl3) δ 7.41 – 7.35 (m, 4H), 7.27 – 7.22 (m, 2H), 7.17 – 7.13 (m, 4H), 3.83 (s, 2H). 13 C NMR (151 MHz, CDCl3)δ 164.8, 150.4, 129.6, 126.4, 121.4, 41.7. (2) Synthesis of compound 2 Compound 1 (25 mmol, 6.402 g) and m-hydroxy-N,N-diethylaniline (25 mmol, 4.131 g) were dissolved in toluene. The reaction mixture was refluxed at 118 °C for 12 h, and then cooled to room temperature, resulting in the precipitation of a solid. The solid obtained by vacuum filtration was then separated by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1) to give compound 2 as a yellow solid (yield 91%). 1 H NMR (400 MHz, DMSO -d6 ) δ 11.88 (s, 1H), 7.52 (d, J = 9.0 Hz, 1H), 6.63 –6.60 (m, 1H), 6.42 (d, J = 2.5 Hz, 1H), 5.24 (s, 1H), 3.40 – 3.34 (m, 5H), 1.08(t, J = 7.0 Hz, 6H). 13 C NMR (151 MHz, DMSO -d6 ) δ 166.9, 163.2, 156.6, 151.3,124.5, 108.6, 103.9, 96.8, 86.6, 44.4, 12.7. (3) Synthesis of compound 3 At 0 °C, N,N-dimethylformamide (60 mmol) was slowly added to phosphorus oxychloride (24 mmol, 2.24 mL), and the mixture was stirred for 30 min. Then, a DMF solution (10 mL) of compound 2 (6 mmol, 1.398 g) was added to the reaction system, and the reaction was continued at 80 °C for 5 h. After the reaction was completed, the mixture was cooled to room temperature, poured into 200 mL of ice water, and gently stirred. The precipitate was filtered and washed with isopropanol. The crude product was separated by column chromatography (petroleum ether / ethyl acetate = 30:1) to give compound 3 in 83% yield. 1 H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 9.2 Hz, 1H), 6.60 –6.57 (m, 1H), 6.36 (d, J = 2.5 Hz, 1H), 3.46 – 3.41 (m, 4H), 2.70 (s, 3H), 1.23(t, J = 7.2 Hz, 6H). 13C NMR (151 MHz, CDCl3) δ 204.5, 177.8, 161.3, 157.4, 153.8, 127.0, 109.1, 102.8, 98.45, 96.4, 45.0, 29.8, 12.4. (4) Synthesis of compound 4 Compound 3 (5 mmol, 1.38 g) was dissolved in toluene (20 mL), heated to 118 °C, and boron trifluoride diethyl ether (7.5 mmol, 1.06 g) was slowly added to the reaction mixture. The reaction was continued for 0.5 h. After the reaction was completed, the mixture was cooled to room temperature, and petroleum ether was added. A yellow solid precipitated, which was separated by column chromatography (petroleum ether / ethyl acetate = 6:1) to give compound 4 in 56% yield. 1 H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 9.3 Hz, 1H), 6.67 – 6.63 m,1H), 6.36 (d, J = 2.4 Hz, 1H), 3.53 – 3.48 (m, 4H), 2.83 (s, 4H), 1.28 (t, J = 7.2Hz, 6H). 13 C NMR (151 MHz, CDCl3) δ 196.3, 173.2, 159.2, 159.1, 156.2, 129.3,110.5, 101.9, 96.6, 45.6, 26.3, 12.4. (5) Synthesis of compound 5 (Triphenylphosphonic acid)acetaldehyde (6 mmol, 1.826 g) and 3-quinoline carbaldehyde (6 mmol, 0.942 g) were dissolved in toluene (30 mL). The reaction mixture was stirred at 60 °C for 10 h, cooled to room temperature, concentrated under reduced pressure, and the crude product was separated by column chromatography (petroleum ether / ethyl acetate = 25:1) to give compound 5 in 92% yield. 1 H NMR (400 MHz, CDCl3)δ 9.79 (d, J = 7.5 Hz, 1H), 9.11 (d, J = 2.2 Hz, 1H), 8.31 (d, J= 2.5 Hz, 1H),8.15 – 8.11 (m, 1H), 7.90 – 7.87 (m, 1H), 7.82 – 7.77(m, 1H), 7.66 – 7.59 (m,2H), 6.96 – 6.90 (m, 1H). 13 C NMR (151 MHz, CDCl3) δ 193.0, 149.2, 149.0,148.7, 136.0,131.2, 129.9, 129.5, 128.5, 127.7, 127.5, 127.0. (6) Synthesis of compound 6 Under an argon atmosphere, methyl trifluoromethanesulfonate (20 mmol, 2.06 mL) was added to a chloroform solution of compound 5 (2 mmol, 0.366 g). The reaction mixture was stirred at room temperature for 24 h, and then concentrated under reduced pressure to remove the solvent. The crude product was washed with chloroform to give compound 6 in 90% yield. 1 H NMR (400 MHz, DMSO) -d6 ) δ 10.02 (s, 1H), 9.85 (d, J = 7.5 Hz, 1H), 9.58 (s, 1H), 8.55 (d, J = 9.0 Hz, 1H), 8.48 (d, J = 8.3 Hz,1H), 8.35 – 8.31 (m, 1H), 8.10 (t, J = 7.6 Hz, 1H), 7.99 (d, J = 16.1 Hz, 1H), 7.29 – 7.23 (m, 1H), 4.67 (s, 3H). 13 C NMR (151 MHz, DMSO -d6 ) δ 194.7, 150.4,145.9, 145.8, 138.5, 136.8, 132.8, 131.5, 131.1, 129.2, 128.6, 119.8, 46.1. (7) Synthesis of compound CQB Methanesulfonic acid (5 μL) was added to a solution of compound 4 (1 mmol, 0.323 g) and compound 6 (1 mmol, 0.347 g) in acetonitrile (10 mL). The reaction mixture was heated and stirred at 70 °C for 5 h. The organic solvent was removed by concentration under reduced pressure. The crude product was purified by column chromatography (dichloromethane / ethanol = 20:1) to obtain the target product CQB in 41% yield.

[0019] Its proton spectrum is as follows Figure 1 : 1 H NMR (400 MHz, DMSO -d6 ) δ 9.88 (s, 1H), 9.35 (s, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.34 (d, J = 8.2 Hz, 1H), 8.20 (t, J = 7.9 Hz, 1H), 8.01 – 7.93 (m, 3H), 7.79 – 7.69 (m, 2H), 7.57 (d, J = 15.4 Hz, 1H), 6.78 (d, J =9.5 Hz, 1H), 6.51 (s, 1H), 4.64 (s, 3H), 3.55 – 3.49 (m, 4H), 1.16 (t, J = 7.1Hz, 6H). Its carbon spectrum is as follows Figure 2 : 13 C NMR (151 MHz, DMSO -d6 ) δ 179.2, 172.1, 158.9,158.8, 156.5, 150.0, 148.4, 144.1, 138.3, 137.9, 136.2, 133.1, 131.1,130.9,130.2, 129.4, 128.7, 126.7, 122.2, 120.1, 119.6, 111.9, 102.1, 99.1, 96.9,46.0, 45.6, 12.9. Its high-resolution mass spectrum is as follows Figure 3 HRMS (ESI) m / z calcd for C 28 H 28 BF2N2O4(M): 503.1948. Found: 503.1947, error: 0.2 ppm. Example 2: Kinetics and Reversibility Testing of Fluorescent Probe CQB like Figure 4 As shown in Figure A, different concentrations of HSO3 were used. - Fluorescence spectral changes at different time gradients were measured by mixing HSO3 (10, 20, and 40 μM) with probe CQB (10 μM). The results showed that the addition of different concentrations of HSO3... - Subsequently, the fluorescence intensity at 712 nm rapidly increased and then plateaued within 5 seconds. Following this, with the continued addition of H₂O₂ to the reaction mixture, the fluorescence signal at 712 nm gradually weakened with increasing reaction time, and after 20 minutes, the fluorescence signal essentially returned to its initial level. Figure 4 B). Furthermore, under the same conditions, after more than four cycles, there were no significant changes in response time and fluorescence intensity. This indicates that the CQB probe has good reversible response performance and can dynamically monitor HSO3. - And changes in H2O2 concentration.

[0020] Example 3: Sensitivity test of fluorescent probe CQB Figure 5 A and 5C are fluorescent probes CQB reacting with different concentrations of HSO3. - The fluorescence spectrum after the reaction, where the concentration of probe CQB is 10 μM, and HSO3... - The concentration varied within the range of 0-50 μM, and the buffer solution used was PBS solution containing 50% 1,4-dioxane (pH = 7.4). The test method was as follows: HSO3 at different pre-prepared concentrations... - The CQB was added sequentially to the PBS buffer system, and after thorough mixing, the fluorescence spectrum changes in the wavelength range of 550-850 nm were immediately measured using a fluorescence spectrophotometer to detect the response to HSO3. - The excitation and emission wavelengths were 561 nm and 712 nm, respectively, and the relationship between the fluorescence spectrum and SO2 concentration was obtained. Figure 5 As can be seen from A, with the sequential increase of SO2 concentration, the fluorescence intensity at 712 nm is related to that of HSO3. - The concentration showed a positive correlation. This indicates that the probe CQB provided by this invention can respond efficiently to HSO3. - It also exhibits good concentration dependence. Figure 5 B is a probe for different concentrations of HSO3. - The fluorescence linear response diagram shows the fluorescence intensity of the probe versus HSO3. - The concentration of HSO3 showed a good linear relationship within a certain range, and calculations yielded the probe's response to HSO3. - The detection limit is 15.5 nM. Figure 5 B and 5D are H2O2 for CQB and HSO3 -The effect of the reaction was that the fluorescence signal at 712 nm gradually decreased with the addition of different concentrations of H2O2 (0 - 400 μM). Figure 5 D represents CQB and HSO3. - The fluorescence linear response diagrams of the reaction system to different concentrations of H2O2 show that CQB and HSO3... - The fluorescence intensity after the reaction showed a good linear relationship with the H₂O₂ concentration (100-300 μM), and the calculated detection limit was 119.7 nM. These results indicate that the probe CQB can effectively detect HSO₃⁻. - Highly sensitive detection of H2O2.

[0021] Example 4: Selectivity and anti-interference test of fluorescent probe CQB Figure 6 A and 6B represent the selectivity and anti-interference properties of the fluorescent probe CQB for SO2. The compatibility of probe CQB (10 μM) with HSO3 was measured. - (30 μM), reactive oxygen species ( t BuOOH, 1 O2, O2 –• , • OH, ONOO – Biothiols (Cys, Hcy, GSH), amino acids (DL, Ser, His, Lys, Pro, Leu, Phe), and anions (NO2) - SO4 2- , PO4 3- OAc - NO3 - ) and cations (K) + Na + Cu 2+ Zn 2+ The fluorescence spectrum changes in response to (200 μM) were analyzed, resulting in a bar chart of fluorescence intensity at 712 nm versus different analytes. Figure 6 As shown in A, only the presence of SO2 can cause the probe to produce strong fluorescence emission at 712 nm. Various other reactive oxygen species, biothiols, amino acids, anions, and cations do not cause changes in the CQB fluorescence signal, nor do they affect HSO3. - The response does not produce any interference. Therefore, the probe CQB is able to achieve HSO3 - High selectivity detection. Simultaneously, in CQB and HSO3... - After adding the various analytes mentioned above to the reaction system, it was found that only the presence of H2O2 could weaken the fluorescence signal at 712 nm. Figure 6B). The above results demonstrate that CQB can achieve highly selective and reversible detection of SO2 and H2O2.

[0022] Example 5: Quantitative Detection of SO2 Content in Water and Food Samples Using the Fluorescent Probe CQB The processing methods for tap water, drinking water, and green tea samples are as follows: Adjust the pH of each sample to 7.4. Then, take 1 mL of each pH-adjusted sample and dilute it to 10 mL to prepare the test solution. After preparing the test samples, add 3 mL of each sample to a cuvette, then add the probe CQB (10 μM) to measure its fluorescence spectrum change. Simultaneously, add different concentrations of HSO3. - The changes in fluorescence signal were measured after (0, 5, 10, 15, and 20 μM). All experiments were repeated three times. The experimental results are shown in Table 1.

[0023] The food sample preparation method is as follows: Weigh 1.5 g of granulated sugar or rock sugar, dissolve it in 10 mL of PBS buffer solution, and adjust the pH to 7.4 to prepare the test sample; cut and weigh 1.5 g of solid food samples such as dried mango, oatmeal, dried lily bulbs, raisins, and kumquat slices appropriately, and extract them with PBS buffer by sonication for 2 hours, then filter to prepare the test solution. Adjust the pH of the test solution to 7.4, then take 1 mL of the test solution and dilute it with buffer solution (10 mM, pH = 7.4, containing 50% 1,4-dioxane) to 10 mL to prepare the test sample. After preparing the test sample, take 3 mL of the sample and add it to a cuvette, then add probe CQB (10 μM) to measure its fluorescence spectrum change, and at the same time add different concentrations of HSO3. - The changes in fluorescence signal were measured after (0, 10, 20, 30, and 40 μM). All experiments were repeated three times. The experimental results are shown in Table 1.

[0024] The CQB probe was applied to the quantitative detection of SO2 residues in drinking water, milk, yogurt, green tea, and various solid foods (Table 1). The spiked recovery rate was good, demonstrating its practical value in the fields of food safety and environmental monitoring.

[0025] Example 6: Subcellular Co-localization Experiment of Fluorescent Probe CQB HepG2 cells were selected for confocal imaging. First, HepG2 cells were incubated with CQB (10 μM) for 30 min, then incubated with the lipid droplet green fluorescent dye LDs-tracker green (2 μM) for 30 min before cell imaging. The experimental results are as follows: Figure 7 As shown. By Figure 7As can be seen, the red fluorescence signal of HepG2 cells stained with CQB has a high degree of overlap with the green fluorescence signal of LDs-tracker green, and the Pearson's correlation coefficient is as high as 0.92. This demonstrates that CQB has excellent lipid droplet targeting ability.

[0026] Example 7: Application of the fluorescent probe CQB in monitoring changes in exogenous SO2 levels HepG2 liver cancer cells were selected for confocal microscopy imaging using different concentrations of HSO3. - HepG2 cells were incubated with (0, 20, 50, 100, 200, 500 μM) for 1 h, then incubated with the probe CQB (10 μM) for another 30 min. After discarding the culture medium, the cells were washed with PBS buffer and imaged using a confocal microscope. The experimental results are shown below. Figure 8 As shown. By Figure 8 It can be seen that when HepG2 cells are incubated with CQB alone, only a weak red fluorescence signal is observed. Figure 8 A) When HSO3 is added - As the concentration increases sequentially, the fluorescence signal in the red channel gradually increases. Figure 8 BF). This indicates that the CQB probe can monitor changes in SO2 content in cells. To further confirm the probe's ability to detect changes in SO2 levels in lipid droplets, HepG2 cells were first incubated with different concentrations of oleic acid (0, 50, 100, 200, 400 μM) for 4 h, followed by the addition of HSO3. - Cells were incubated with (500 μM) for 1 h, followed by incubation with the CQB probe for 30 min. The culture medium was then discarded, and the cells were washed with PBS buffer. The cells were then imaged using a confocal microscope. The experimental results are shown below. Figure 9 As shown. By Figure 9 It can be seen that with the increase of oleic acid content, the number and size of lipid droplets significantly increase. These results indicate that the CQB probe can detect the SO2 content in lipid droplets.

[0027] Example 8: Application of the fluorescent probe CQB in monitoring changes in endogenous SO2 levels HepG2 liver cancer cells were selected for confocal microscopy imaging. HepG2 cells were incubated with different concentrations of cysteine ​​(Cys, 100, 200, and 500 μM) for 2 h, followed by incubation with the CQB probe (10 μM) for 30 min. For the inhibitor group, cells were incubated with Cys (500 μM) for 2 h, then incubated with the reactive oxygen species scavenger N-ethylmaleimide (NEM) for 1 h, followed by incubation with the CQB probe (10 μM) for 30 min. The culture medium was then discarded, and the cells were washed with PBS buffer. Confocal microscopy imaging was performed, and the experimental results are shown below. Figure 10 As shown. By Figure 10 It can be seen that when HepG2 cells are incubated with CQB alone, only a weak red fluorescence signal is observed. Figure 10 A); As the concentration of Cys increased sequentially, the fluorescence signal in the red channel gradually increased ( Figure 10 BD); when the inhibitor NEM was added, the enhancement of fluorescence signal was significantly reversed ( Figure 10 E). The above results demonstrate that the probe provided by this invention can monitor changes in endogenous SO2 levels.

[0028] Example 9: Monitoring HSO3 in HepG2 cells using the fluorescent probe CQB - Applications related to changes in H2O2 concentration levels HepG2 liver cancer cells were selected for confocal microscopy imaging. HepG2 cells were first pre-incubated with oleic acid (400 μM) for 4 hours, then treated with HSO3. - Cells were incubated with 500 μM H2O2 for 1 h, followed by incubation with different concentrations of H2O2 (200, 500, 800, and 1000 μM) for 2 h, and finally incubated with the CQB probe (10 μM) for 30 min. The culture medium was then discarded, cells were washed with PBS buffer, and images were taken using a confocal microscope. The experimental results are shown below. Figure 11 As shown. By Figure 11 It can be seen that, compared with the group incubated only with CQB ( Figure 11 A) Compared to adding HSO3 - The red fluorescence signal was significantly enhanced afterward. Figure 11 B); As the concentration of added H2O2 increases sequentially, the fluorescence signal in the red channel gradually weakens. Figure 11 (CF). The above results demonstrate that the probe provided by this invention can reversibly detect changes in SO2 and H2O2 levels.

[0029] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. 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 coumarin-quinoline hybrid near-infrared fluorescent probe compound, characterized in that, The structural formula of the fluorescent probe compound is: 。 2. A method for synthesizing the coumarin-quinoline hybrid near-infrared fluorescent probe compound as described in claim 1, characterized in that, Includes the following steps: (1) Under low temperature conditions, phosphorus oxychloride was slowly added to a mixture of malonic acid and phenol. The reaction mixture was heated at 110~120℃ for 1~2 h, then quenched, extracted, dried and concentrated. Compound 1 was obtained by column chromatography. The structural formula of compound 1 is ； (2) Compound 1 and m-hydroxy-N,N-diethylaniline were dissolved in toluene, refluxed at 110~120℃ for 10~12 h, cooled and filtered, and compound 2 was obtained by column chromatography. The structural formula of compound 2 is ； (3) Under low temperature conditions, N,N-dimethylformamide was added to phosphorus oxychloride and stirred for 25-35 min. Then, DMF solution of compound 2 was added and the reaction was continued at 80-90℃ for 5-6 h. After the reaction was completed, the mixture was quenched, filtered, washed, and separated by column chromatography to obtain compound 3. The structural formula of compound 3 is ； (4) Dissolve compound 3 in toluene, heat to 110~120℃, add boron trifluoride diethyl ether, heat to react for 0.5~1h, cool and precipitate after the reaction is completed, and separate by column chromatography to obtain compound 4; The structural formula of compound 4 is ； (5) Dissolve (triphenylphosphonic acid) acetaldehyde and 3-quinoline formaldehyde in anhydrous toluene, stir and react at 60~70℃ for 10~12h, concentrate, and separate by column chromatography to obtain compound 5; The structural formula of compound 5 is ； (6) Under an inert atmosphere, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 5, and the mixture was stirred at room temperature for 24-26 h. After concentration and purification, compound 6 was obtained. The structural formula of compound 6 is ； (7) Add methanesulfonic acid to the acetonitrile solution of compound 4 and compound 6, heat and stir at 60~80℃ for 5~6 h, concentrate, and purify by column chromatography to obtain the target fluorescent probe compound CQB.

3. The synthesis method according to claim 2, characterized in that, In step (1), the molar ratio of malonic acid, phenol and phosphorus oxychloride is 1:(1.5~2.5):(1.2~1.8); in step (2), the molar ratio of compound 1 to m-hydroxy-N,N-diethylaniline is 1:(0.8~1.2); in step (3), the molar ratio of N,N-dimethylformamide, phosphorus oxychloride and compound 2 is (5~7):(3~5):1; in step (4), the molar ratio of compound 3 to boron trifluoride ether is 1:(1.2~1.8); in step (5), the molar ratio of (triphenylphosphonic acid)acetaldehyde to 3-quinoline carbaldehyde is 1:(0.8~1.2); in step (6), the molar ratio of compound 5 to methyl trifluoromethanesulfonate is 1:(8~12); in step (7), the molar ratio of compound 4 to compound 6 is 1:(0.8~1.2).

4. The synthesis method according to claim 2 or 3, characterized in that, In steps (1) and (3), the low temperature condition is 0~5℃.

5. The application of the coumarin-quinoline hybrid near-infrared fluorescent probe compound as described in claim 1 in the detection of sulfur dioxide and / or hydrogen peroxide.

6. The application according to claim 5, characterized in that, The detection method is any one of quantitative fluorescence detection, qualitative fluorescence identification, or dynamic reversible fluorescence monitoring.

7. The application according to claim 5, characterized in that, The objects of the test are food samples or water samples.

8. The use of the coumarin-quinoline hybrid near-infrared fluorescent probe compound as described in claim 1 in the preparation of a fluorescent imaging reagent for detecting sulfur dioxide and / or hydrogen peroxide.

9. The application according to claim 8, characterized in that, The fluorescence imaging reagent is used for fluorescence imaging of sulfur dioxide and / or hydrogen peroxide in live cells or biological samples.

10. The application according to claim 9, characterized in that, The fluorescence imaging is a reversible, real-time fluorescence imaging of sulfur dioxide and hydrogen peroxide.

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