A fluorescence probe for detecting sulfur dioxide based on FRET principle and a preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and relates to a fluorescent probe for detecting sulfur dioxide based on the FRET principle and a preparation method and application thereof. The fluorescent probe developed by the application for sulfur dioxide changes the corresponding fluorescence color and intensity in the presence of sulfur dioxide, and can be used for detecting sulfur dioxide, greatly reducing the interference of external conditions and improving the detection accuracy. Experimental results show that the detection limit of the probe provided by the application for Na2SO3 is about 22nM, indicating that the probe has high sensitivity for Na2SO3. The fluorescent probe provided by the application can be used for intracellular detection, which is of great significance for in-depth study on the kinetic mechanism of the generation, delivery and deposition of sulfur dioxide in organisms, and further understanding of the physiological and toxicological effects of sulfur dioxide. The structural formula of the fluorescent probe for detecting sulfur dioxide is shown as formula III.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and relates to a fluorescent probe for detecting sulfur dioxide based on the FRET principle, its preparation method and application. Background Technology

[0002] Sulfur dioxide (SO2) is a colorless gas with a pungent odor and is one of the most common air pollutants. It is the simplest and most common sulfur oxide, possessing diverse chemical properties and a wide range of applications. SO2 is not only present in the natural environment, released in large quantities during volcanic eruptions, but also produced in many industrial processes, particularly in the combustion of coal and oil. Sulfur dioxide and its derivatives are widely used in chemistry, medicine, agriculture, and many other fields. For example, in winemaking, sulfur dioxide is often used as a preservative to extend the shelf life of wine. Sulfur dioxide reacts with polyphenols in wine, causing changes in its flavor and color. The U.S. Food and Drug Administration (FDA) requires that foods with nitrite content exceeding 10 mg / kg must be labeled; Japan limits the sulfur dioxide derivative content in salted vegetables, starches, and other foods to 30 mg / kg; and Germany limits the sulfur dioxide derivative content in garlic products to 50 mg / kg. Sulfur dioxide can produce corrosive sulfites on the moist mucous membranes of the upper respiratory tract, which can damage the trachea and lungs, potentially causing various respiratory inflammations. Furthermore, sulfur dioxide can be absorbed into the bloodstream and affects the metabolism of carbohydrates and proteins, damaging organs such as the gastrointestinal tract, liver, kidneys, and heart. Long-term exposure to or ingestion of SO2 may also lead to cancer in humans. Therefore, in recent years, research into reliable, rapid, and convenient methods for detecting SO2 has been continuously increasing.

[0003] Fluorescent probes, with their advantages of high sensitivity, strong specificity, fast response speed, strong anti-interference ability, and long lifetime, provide a real-time detection platform that well meets the needs of various testing scenarios. In particular, the emergence of fluorescent probes has provided a new platform for monitoring SO2 concentration in vivo. Compared with traditional "switch" fluorescent probes, dual-fluorophore fluorescent probes based on the principles of intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) have higher research value. Targeting this mechanism, researchers have recently developed various fluorescent probes for SO2 detection using two probes with different emission wavelengths as detection signals. However, most still suffer from drawbacks such as low solubility, poor stability, and small Stokes shift. Therefore, developing a practical fluorescent probe with a large Stokes shift and efficient and stable SO2 detection is particularly important. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a fluorescent probe for detecting sulfur dioxide based on the FRET principle, its preparation method, and its application. The fluorescent probe provided by this invention uses a coumarin derivative as the fluorescent donor, a hemicyanine dye as the acceptor, linked by a piperazine, and with a carbon-carbon double bond as the reaction site.

[0005] Invention Concept: Coumarin and its derivatives, as typical fluorescent groups, possess advantages such as good luminescence performance, high stability, and ease of synthesis. 1-Ethyl-2,3,3-trimethyl-3H-indole exhibits the characteristic of condensing with 4-dimethylaminobenzaldehyde to form the ICT effect, emitting red fluorescence. Therefore, this invention uses a coumarin derivative as the fluorescent donor and a hemicyanine dye as the acceptor. A "coumarin derivative-piperazine-indole" compound structure was designed based on the Michael addition reaction via piperazine linkage. The initial probe exhibits red fluorescence of the acceptor. Upon addition of Na2SO3, an addition reaction occurs at the C=C portion of the acceptor, blocking the intramolecular charge transfer effect of the acceptor. The red fluorescence disappears, the fluorescence resonance energy transfer process is blocked, and the fluorescence emission transforms into green fluorescence emission from the donor, thereby achieving the detection of sulfur dioxide.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] This invention discloses an intermediate I, the structural formula of which is shown below:

[0008]

[0009] Furthermore, the present invention discloses a method for preparing the above-mentioned intermediate I, wherein coumarin 343 and 4-piperazine-1-benzaldehyde undergo a coupling reaction under the action of a condensing agent and a catalyst to obtain intermediate I.

[0010]

[0011] In some embodiments, the condensing agent is any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; the catalyst is any one of 4-dimethylaminopyridine and N,N-diisopropylethylamine.

[0012] In some embodiments, preferably, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and the catalyst is 4-dimethylaminopyridine.

[0013] In some embodiments, the molar ratio of coumarin 343 to 4-piperazin-1-benzaldehyde, condensing agent, and catalyst is 0.8–1.2:1.2–2.0:1.0–2.0:0.1–0.2; the coupling reaction is carried out at a temperature of -10–10°C.

[0014] In some embodiments, preferably, the molar ratio of coumarin 343 to 4-piperazine-1-benzaldehyde, the condensing agent, and the catalyst is 1.0:1.2:1.5:0.2; the coupling reaction is carried out at a temperature of 0°C.

[0015] The solvent used in the coupling reaction is preferably any one or a combination of two of dichloromethane and N,N-dimethylformamide, with dichloromethane being the preferred solvent. There are no special requirements for the amount of solvent used; it is sufficient to dissolve and disperse or dissolve the raw materials evenly.

[0016] The application of the aforementioned intermediate I in the preparation of a fluorescent probe for detecting sulfur dioxide based on the FRET principle is also within the scope of protection of this invention.

[0017] Furthermore, this invention discloses a fluorescent probe for detecting sulfur dioxide based on the FRET principle, the structural formula of which is shown in Formula III below:

[0018]

[0019] Where X is F, Cl, or I.

[0020] Furthermore, this invention discloses the preparation method of the above-mentioned fluorescent probe for detecting sulfur dioxide based on the FRET principle, wherein intermediate I and compound II undergo a Michael addition reaction under the action of an alkali to obtain the fluorescent probe;

[0021]

[0022] Wherein, X is F, Cl or I, preferably I.

[0023] In some embodiments, the base is piperidine.

[0024] In some embodiments, the molar ratio of intermediate I to compound II is 1.0:1.0 to 1.5.

[0025] In some embodiments, preferably, the molar ratio of intermediate I to compound II is 1.0:1.0.

[0026] In the Michael addition reaction, the amount of base used is trace. Preferably, if 1 mmol of intermediate I is used as the raw material, 0.8 mmol to 1.2 mmol of base is sufficient.

[0027] The reaction temperature of the Michael addition reaction is the reflux temperature of the solvent used.

[0028] The solvent used in the Michael addition reaction is preferably any one or a combination of ethanol and methanol, with ethanol being preferred. There are no special requirements for the amount of solvent used; it is sufficient to dissolve and disperse or dissolve the raw materials evenly.

[0029] The application of the aforementioned fluorescent probe for detecting sulfur dioxide based on the FRET principle in detecting sulfur dioxide in water and / or in vivo and / or in vitro is also within the scope of protection of this invention.

[0030] Beneficial effects:

[0031] (1) The fluorescent probe developed by this invention for sulfur dioxide changes in fluorescence color and intensity in the presence of sulfur dioxide, and can be used for the detection of sulfur dioxide, greatly reducing the interference of external conditions and improving the detection accuracy. Experimental results show that the detection limit of the probe provided by this invention for Na2SO3 is about 22 nM, indicating that the probe has high sensitivity to Na2SO3.

[0032] (2) The fluorescent probe provided by the present invention can be used for intracellular detection, which is of great significance for in-depth study of the dynamic mechanism of sulfur dioxide generation, delivery and deposition in organisms, and further understanding of the physiological and toxicological effects of sulfur dioxide.

[0033] (3) The probe preparation process provided by the present invention is simple, easy to operate, and has a novel structure, providing a new approach for the detection of sulfur dioxide. Attached Figure Description

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0035] Figure 1 The image shows the 1H NMR spectrum of intermediate I.

[0036] Figure 2 This is a mass spectrometry data graph of intermediate I.

[0037] Figure 3 This is the 1H NMR spectrum of the probe NPB.

[0038] Figure 4 This is a mass spectrometry data graph of the NPB probe.

[0039] Figure 5 This is a schematic diagram illustrating the principle of NPB probe detection of sulfur dioxide.

[0040] Figure 6 This is a mass spectrometry data graph of compound IV.

[0041] Figure 7 The fluorescence intensity of NPB probe against sulfur dioxide at different pH values ​​(I) 487 / I 591 Ratio change curve.

[0042] Figure 8 Fluorescence spectra and fluorescence intensity I of different analytes were determined using the NPB probe. 487 / I 591 Ratio chart.

[0043] Figure 9 The graph shows the relationship between the fluorescence intensity of the probe NPB and the sulfur dioxide concentration, as well as the fluorescence intensity I. 487 / I 591 Relationship between sulfur dioxide concentration and sulfur dioxide concentration.

[0044] Figure 10 Confocal microscopy images of the NPB probe for detecting exogenous sulfur dioxide in cells and fluorescence intensity of the NPB probe.

[0045] Figure 11 Confocal microscopy images of the NPB probe for detecting endogenous sulfur dioxide in cells and fluorescence intensity of the NPB probe. Detailed Implementation

[0046] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0047] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0048] All compound structural formulas in this invention were drawn using KingDraw software.

[0049] The following ingredients were used in this invention: Coumarin 343, CAS No. 55804-65-4, with a molecular weight of 285.29; EDCI, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, with a molecular weight of 191.7; DMAP, 4-dimethylaminopyridine, with a molecular weight of 122.168; and 4-piperazine-1-benzaldehyde, with a molecular weight of 190.24.

[0050] Example 1: Preparation of Intermediate I

[0051]

[0052] Coumarin 343 (285 mg, 1 mmol), EDCI (287 mg, 1.5 mmol), and DMAP (25 mg, 0.2 mmol) were dissolved in 20 mL of dichloromethane and stirred at 0 °C for 2 h. Subsequently, 4-piperazin-1-benzaldehyde (228 mg, 1.2 mmol) was added to the reaction system, and stirring was continued at 0 °C overnight. After the reaction was completed, the reaction solution was washed three times with water, the organic phase was collected, the solvent was recovered by vacuum concentration, and the solution was eluted by column chromatography on silica gel (DCM / EA = 10 / 1, V / V) to give a pale yellow solid intermediate I, 411 mg, with a yield of 90%.

[0053] The 1H NMR spectrum of intermediate I is shown below. Figure 1 Mass spectrum (see) Figure 2 The specific NMR and mass spectrometry data are as follows:

[0054] 1 H NMR(400MHz,Chloroform-d)δ9.79(s,1H),7.84(s,1H),7.79(m,2H),6.91(d,J=8.7Hz,3H),3.96(m,2H),3.59(s,2H),3.51 (d,J=10.5Hz,4H),3.31(q,J=5.0Hz,4H),2.88(t,J=6.4Hz,2H),2.76(t,J=6.3Hz,2H),1.97(ddt,J=11.6,9.4,4.6Hz,4H). 13 C NMR(101MHz,Chloroform-d)δ190.44,165.66,159.54,154.70,152.43,147.39,146.17,131.87,127.51,12 6.00,119.19,114.28,113.84,107.66,106.02,50.13,49.73,27.46,21.23,20.29,20.12.HRMS(ESI+):m / z calculated for[M+H] + :458.2002.found458.1998.

[0055] Example 2: Preparation of probe compound III-1

[0056]

[0057] 1 mmol of 1-ethyl-2,3,3-trimethylindolonium iodide (intermediate II-1) and 1 mmol of intermediate I were mixed in 10 mL of ethanol, and a Michael addition reaction was catalyzed by 2 drops of piperidine (approximately 1 mmol). The reaction was refluxed overnight. After the reaction was complete, the solvent was recovered by vacuum concentration, and the crude product was eluted by column chromatography on silica gel (DCM / methanol = 300 / 1, v / v) to give 0.7 mmol of purple solid probe compound III-1 (denoted as probe NPB), in a yield of 70%.

[0058] The 1H NMR spectrum of the probe NPB is shown below. Figure 3 Mass spectrometry data graphs are shown below. Figure 4 The specific NMR and mass spectrometry data are as follows:

[0059] 1 H NMR(500MHz,Chloroform-d)δ8.17–8.09(m,3H),7.87(s,1H),7.57–7.49(m,3H),7.4 9–7.40(m,2H),6.97(d,J=8.8Hz,2H),6.93(s,1H),4.80(q,J=7.3Hz,2H),3.88(s,1H) ,3.69–3.64(m,4H),3.62(s,1H),3.33(q,J=5.7Hz,4H),3.19(t,J=5.7Hz,2H),2.87( t,J=6.4Hz,2H),2.76(t,J=6.2Hz,2H),1.98(q,J=6.4,5.9Hz,4H),1.81–1.57(m,9H). 13 C NMR(126MHz,Chloroform-d)δ179.18,165.90,159.68,154.92,154.73,152.44,14 7.53,146.30,142.61,140.63,135.16,129.45,128.38,126.15,123.77,122.63,11 9.29,113.96,113.71,113.47,107.62,106.17,105.94,51.41,50.15,44.52,42.7 5,29.70,27.60,27.47,22.19,21.87,21.20,20.27,20.12,13.94.HR-MS(ESI):m / z calculated for[M] + :627.3330,found 627.3331.

[0060] Example 3: Determination of sulfur dioxide by probe NPB

[0061] For a detailed schematic diagram of the NPB probe for detecting sulfur dioxide, please refer to [link / reference needed]. Figure 5 The sulfite ion attacks the C=C bond of the acceptor moiety, blocking the ICT process at the acceptor moiety via Michael addition, thus preventing the transfer of energy from the donor to the acceptor. Therefore, the energy manifests as fluorescence in the donor moiety, causing the fluorescence to change from red in the acceptor to green in the donor. This ultimately yields compound IV, whose structural formula is shown below:

[0062]

[0063] Sodium sulfite was dissolved in water (100 μM) to obtain an aqueous sodium sulfite solution. Probe NPB (10 μM, prepared in Example 2) was dissolved in DMSO:H₂O (1:9, V / V) to obtain a probe NPB solution. Using sulfite ions from the sodium sulfite aqueous solution as a substitute for sulfur dioxide, the sodium sulfite aqueous solution and the probe NPB solution were mixed at a volume ratio of 1:1. The detection performance of the probe NPB was evaluated using a fluorescence spectrophotometer (excitation wavelength set to 410 nm).

[0064] Compared to the NPB probe solution with added sodium sulfite aqueous solution, the NPB probe solution alone appears red under fluorescence excitation; the NPB probe solution with added sodium sulfite aqueous solution appears pale green under fluorescence excitation, indicating that the Michael addition reaction between the sulfite ion and the C=C bond on the acceptor portion of the NPB probe successfully occurs, blocking the transfer of energy from the donor. Mass spectrometry analysis of the pale green reaction solution yielded the following results: Figure 6 As shown, the mixture was analyzed by mass spectrometry. The mass spectrometry data of compound IV are as follows: HR-MS (ESI): m / z [M+H] + 709.4710.

[0065] Example 4: Determination of sulfur dioxide by NPB probe at different pH levels

[0066] Probe NPB solutions with different pH values ​​were prepared using hydrochloric acid aqueous solution and sodium hydroxide aqueous solution (concentration 10 μM, NPB probe prepared in Example 2). Sodium sulfite was dissolved in water (concentration 100 μM) to obtain sodium sulfite aqueous solution.

[0067] Experimental group: Sodium sulfite aqueous solution and probe NPB solution were mixed at a volume ratio of 1:1. The fluorescence intensity of the mixture was measured using a fluorophotometer (xenon lamp as the light source, 410nm excitation wavelength) to evaluate the detection performance of probe NPB at different pH values.

[0068] Control group: NPB solutions with different pH values ​​(concentration of 10 μM, prepared by probe NPB in Example 2) were used without sodium sulfite aqueous solution. The fluorescence intensity was measured using a fluorophotometer (xenon lamp as the light source, excitation wavelength of 410 nm).

[0069] The curves showing the changes in fluorescence intensity of the NPB probe and its response to sulfur dioxide at different pH values ​​are shown below. Figure 7 As shown in the figure, the fluorescence intensity I of the pure probe NPB is... 487 / I 591 The ratio gradually increases from pH=7, and increases significantly in the pH=7-12 range; the fluorescence intensity I of the probe mixture solution after adding Na2SO3 487 / I 591 The trend of the ratio change was basically consistent with that of the control group, indicating that NPB has the characteristic of pH physiological adaptation.

[0070] Example 5: Selectivity experiment of NPB probe for sulfur dioxide

[0071] Different analytes were prepared into aqueous solutions (analytes were F). - Cl - ,Br - I - Na + K + Ca 2+ Mg 2+ CN - NO3 - HSO4 - ,Cys,GSH,Hcy,Glu,PhSH,NaHS,HSO3 - SO3 2- Different analyte solutions were obtained by mixing DMSO and H2O2 (analyte concentration was 100 μM). Probe NPB (concentration was 10 μM, prepared in Example 2) was dissolved in DMSO:H2O (1:9, V / V) to obtain probe NPB solution.

[0072] Different analyte solutions were mixed with probe NPB solution at a volume ratio of 1:1, and the fluorescence intensity of the mixtures was measured using a fluorophotometer (xenon lamp as the light source, excitation wavelength 410 nm).

[0073] The selectivity test results of the NPB probe for sulfur dioxide are as follows: Figure 8 As shown, Figure a shows the fluorescence spectra of different analytes measured by the NPB probe, and Figure b shows the fluorescence intensity I of different analytes measured by the NPB probe. 487 / I 591 Ratio plot. The results show that fluorescence intensity is only high in H2O2 and HSO3.- SO3 2 Fluctuations occur when the NPB probe is present. This demonstrates that the NPB probe exhibits good selectivity, which is highly advantageous for its application in complex in vivo fluorescence imaging.

[0074] Example 6: Relationship between fluorescence intensity of probe NPB and sulfur dioxide concentration

[0075] Experimental procedure:

[0076] Sodium sulfite aqueous solutions of different concentrations were prepared (concentrations of 0 μM, 4 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM, 40 μM, 44 μM, 48 μM, 52 μM, 56 μM, 60 μM, 64 μM, 68 μM, 72 μM, 76 μM, and 80 μM, respectively). The probe NPB (concentration of 10 μM, prepared in Example 2) was dissolved in DMSO:H2O (1:9, v / v) to obtain the probe NPB solution.

[0077] Sodium sulfite aqueous solutions of different concentrations were mixed with probe NPB solution at a volume ratio of 1:1. The fluorescence intensity of the mixtures was measured using a fluorophotometer (xenon lamp as the light source, excitation wavelength 410 nm). The control group consisted of probe NPB solution mixed with an equal volume of water, without the addition of sodium sulfite aqueous solution, while keeping other experimental conditions unchanged.

[0078] Experimental results:

[0079] (1) Fluorescence spectrum and fluorescence intensity I of probe NPB 487 / I 591 The linear fitting curve of the ratio and the change in sulfur dioxide concentration is as follows: Figure 9 As shown, a is the relationship between the fluorescence intensity of the NPB probe and the sulfur dioxide concentration, and b is the fluorescence intensity I of the NPB probe. 487 / I 591 The graph shows the relationship between NPB (10 μM) and sulfur dioxide concentration. It indicates that under 410 nm excitation, NPB has two channels centered at 487 nm and 591 nm, with an initial ratio of 0.99 (I0). 487 / I 591 The relatively wide emission bandgap (104nm) effectively ensures that the two channels do not interfere with each other.

[0080] (2) As the sodium sulfite concentration increased, the fluorescence emission strongly focused at 591 nm gradually weakened and eventually disappeared, while the fluorescence intensity at 487 nm rapidly increased, and the red fluorescence turned into green fluorescence (in the EP tube in the upper right corner of Figure a, the red is the NPB solution alone, and the green is 40 μM sodium sulfite aqueous solution + 10 μM NPB solution). Until the Na2SO3 concentration increased to 40 μM, the fluorescence intensity change tended to level off, showing a linear relationship in the range of 0 μM to 30 μM (R0). 2 =0.9904), indicating that the NPB probe can accurately measure the Na2SO3 concentration.

[0081] (3) According to the formula LOD = 3σ / k (where k is... Figure 9 The slope of the fitted curve in Figure b (where σ is the standard deviation of the measured fluorescence intensity data) calculates the detection limit of the NPB probe for Na2SO3 to be approximately 22 nM, indicating that the probe has high sensitivity to Na2SO3. These results demonstrate that the NPB probe has good detection capability for Na2SO3.

[0082] Example 7: Detection of exogenous sulfur dioxide by probe NPB

[0083] Exogenous SO2 imaging experiments using the NPB probe were performed with the addition of Na2SO3. HepG2 cells were incubated in MEM medium containing 5% CO2 at a stable temperature of 37°C, and then fluorescence was captured. The specific procedures are as follows:

[0084] Experimental group: HepG2 cells were first cultured in MEM medium containing NPB (final concentration 10 μM) at 5% CO2 and 37℃ for 1 h. Then, Na2SO3 (final concentrations of 50 μM, 100 μM, 150 μM, 250 μM, and 500 μM) was added to the medium and cultured for another 1 h. After incubation, the fluorescence intensity of the incubation solution was measured using a fluorometer (xenon lamp as the light source, excitation wavelength 410 nm).

[0085] Control group: HepG2 cells were cultured in MEM medium containing NPB (final concentration of 10 μM) alone, without the addition of Na2SO3, and other experimental conditions were the same.

[0086] Test results as follows Figure 10 As shown, a) is a confocal microscopy image of NPB probe detecting exogenous sulfur dioxide in cells; b) is a graph showing the fluorescence intensity ratio of the Red and Green channels in each group of confocal microscopy images; and c) is the fluorescence intensity I of NPB probe detecting exogenous sulfur dioxide in cells in each group. 487 / I 591Ratio plot. The plot shows that as the Na2SO3 concentration increases, the red channel shows little change, but the green fluorescence gradually increases; ultimately, the fluorescence in the red channel completely disappears, while the fluorescence intensity in the green channel approaches its peak. Therefore, the NPB probe can detect exogenous SO2 within cells.

[0087] Example 8: Detection of endogenous sulfur dioxide in cells using NPB probe

[0088] HepG2 cells were incubated in MEM medium containing 5% CO2 at a stable temperature of 37°C, and then fluorescence was captured. Thiosulfate-transferase (TST) is a common enzyme in HepG2 cells, which can lead to the production of endogenous SO2 in the presence of Na2S2O3 and GSH. TNBS (2,4,6-trinitrobenzenesulfonate) is considered an inhibitor of TST enzyme and can effectively inhibit the cellular product SO2. The NPB used in this example was prepared in Example 2.

[0089] Control group: Incubated with MEM medium containing probe NPB (final concentration 10 μM) at 5% CO2 and 37°C for 2 h.

[0090] GSH+Na2S2O3 group: HepG2 cells were incubated for 1 h in MEM medium containing GSH (final concentration 500 μM) and Na2S2O3 (final concentration 250 μM) at 5% CO2 and 37℃, and then the probe NPB (final concentration 10 μM) was added and incubated for another 1 h.

[0091] GSH group: HepG2 cells were incubated for 1 h in MEM medium containing GSH (final concentration 500 μM) at 5% CO2 and 37°C, and then NPB probe (final concentration 10 μM) was added for another 1 h of incubation.

[0092] TNBS group: HepG2 cells were incubated for 1 h in MEM medium containing GSH (final concentration 500 μM) and Na2S2O3 (final concentration 250 μM) at 5% CO2 and 37℃, then incubated for another 1 h with the addition of probe NPB (final concentration 10 μM); then incubated for another 20 min with the addition of TNBS (final concentration 10 mM).

[0093] The fluorescence intensity of the incubated mixture was measured using a fluorophotometer (xenon lamp as the light source, excitation wavelength 410 nm). The results are as follows: Figure 11 As shown, a) is a confocal microscopy image of NPB probe detecting endogenous sulfur dioxide in cells; b) is a graph showing the fluorescence intensity ratio of the Red and Green channels in the confocal microscopy images of each group; and c) is the fluorescence intensity I of NPB probe detecting endogenous sulfur dioxide in cells of each group.487 / I 591 Ratio plot. The plot shows that, compared to the Control group, the green channel in the GSH+Na2S2O3 group is significantly enhanced, while the red fluorescence is very faint; the experimental results for the GSH group rule out any reaction of GSH to the fluorescent probe; the fluorescence in the TNBS group is consistent with that of the Control group.

[0094] Experimental results show that TNBS can serve as an effective TST enzyme inhibitor to suppress SO2 production, and the NPB probe can successfully recognize endogenous SO2. The sulfur dioxide probe NPB designed in this invention is a colorimetric fluorescent probe; that is, after the introduction of sulfur dioxide, it changes from red fluorescence of the acceptor to green fluorescence of the donor based on the Michael addition reaction.

[0095] This invention provides a fluorescent probe for detecting sulfur dioxide based on the FRET principle, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. An intermediate I characterized by, The structural formula of intermediate I is shown below: 。 2. Process for the preparation of intermediate I according to claim 1, characterized in that, Coumarin 343 and 4-piperazine-1-benzaldehyde undergo a coupling reaction in the presence of a condensing agent and a catalyst to obtain intermediate I. 。 3. The production method according to claim 2, characterized by, The condensing agent is any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; the catalyst is any one of 4-dimethylaminopyridine and N,N-diisopropylethylamine.

4. The production method according to claim 2, characterized by, The molar ratio of coumarin 343 to 4-piperazine-1-benzaldehyde, condensing agent, and catalyst is 0.8~1.2:1.2~2.0:1.0~2.0:0.1~0.2; the coupling reaction is carried out at a temperature of -10~10°C.

5. The application of intermediate I according to claim 1 in the preparation of a fluorescent probe for detecting sulfur dioxide based on the FRET principle.

6. A fluorescent probe for detecting sulfur dioxide based on the principle of FRET, characterized in that, The structure of the fluorescent probe for detecting sulfur dioxide based on the FRET principle is shown in Formula III below: ； Where X is F, Cl, or I.

7. The method for preparing the fluorescent probe for detecting sulfur dioxide based on the principle of FRET according to claim 6, characterized in that, Intermediate I and compound II undergo a Michael addition reaction under the action of a base to obtain the fluorescent probe; ； Where X is F, Cl, or I.

8. The preparation method according to claim 7, characterized in that, X is I.

9. The preparation method according to claim 7, characterized in that, The base is piperidine.

10. The preparation method according to claim 7, characterized in that, The molar ratio of intermediate I to compound II is 1:1.0~1.

5.

11. The application of the fluorescent probe for detecting sulfur dioxide based on the FRET principle as described in claim 6 in the detection of sulfur dioxide in water and / or in vivo and / or in vitro biological samples for the diagnosis and treatment of non-disease conditions.

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