Fluorescent probe for detecting sulfur dioxide based on FRET (Fluorescence Resonance Energy Transfer) principle and preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent probes, and relates to a fluorescent probe for detecting sulfur dioxide based on an FRET principle, and a preparation method and application thereof. According to the fluorescent probe developed for sulfur dioxide, the corresponding fluorescence color and intensity change in the presence of sulfur dioxide, the fluorescent probe can be used for detecting sulfur dioxide, the interference of external conditions is greatly reduced, the detection accuracy is improved, and experimental results show that the detection limit of the probe for Na2SO3 is about 22nM, and the detection sensitivity is high. The result shows that the probe has high sensitivity to Na2SO3. The fluorescent probe provided by the invention can be used for intracellular detection, and is of great significance for deeply researching the dynamic mechanism of the process of generation, delivery, deposition and the like of sulfur dioxide in a living body and further understanding the physiological and toxicological effects of sulfur dioxide. The structural formula of the fluorescent probe for detecting sulfur dioxide is shown as a formula III. # imgabs0 #

Description

Technical Field

[0001] The invention 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. Background Art

[0002] Sulfur dioxide (SO2) is a colorless gas with a pungent odor and is one of the common pollutants in the atmosphere. It is the simplest and most common sulfur oxide with a variety of chemical properties and a wide range of applications. SO2 not only exists in the natural environment and is released in large quantities during volcanic eruptions, but is also produced in many industrial processes, especially in the combustion of coal and oil. Sulfur dioxide and its derivatives are widely used in many fields such as chemistry, medicine, and agriculture. For example, in the winemaking process, 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 the flavor and color of the wine. The U.S. Food and Drug Administration (FDA) requires that foods with a nitrite content of more than 10 mg / kg must be labeled; Japan limits the content of sulfur dioxide derivatives in salty vegetables, starch and other foods to 30 mg / kg; Germany limits the content of sulfur dioxide derivatives 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, and may cause various respiratory inflammations. In addition, sulfur dioxide can be absorbed by the blood, and it can also affect the metabolism of carbohydrates and proteins, causing damage to the gastrointestinal tract, liver, kidneys, heart and other organs. Long-term exposure or ingestion of SO2 may also cause human cancer. Therefore, in recent years, the research on reliable, fast and convenient methods to detect SO2 has continued to grow.

[0003] Fluorescent probes are real-time detection platforms with the advantages of high sensitivity, strong specificity, fast response speed, strong anti-interference ability, and long life, which well meet the test scenarios. In particular, the emergence of fluorescent probes provides 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. In response to this mechanism, researchers have developed a variety of fluorescent probes for detecting SO2 in recent years using probes with two different emission wavelengths as detection signals. However, most of them still have disadvantages such as low solubility, poor stability, and small Stokes shift. Therefore, it is particularly important to develop a practical fluorescent probe with large Stokes shift and efficient and stable detection of SO2. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a fluorescent probe for detecting sulfur dioxide based on the FRET principle and its preparation method and application in view of the shortcomings of the prior art. The fluorescent probe provided by the present invention uses a coumarin structural derivative as a fluorescent donor, a hemicyanine dye as an acceptor, is connected through piperazine, and uses a carbon-carbon double bond as a reaction site.

[0005] Invention idea: Coumarin and its derivatives, as a typical type of fluorescent group, have the advantages of good luminescence performance, high stability, and easy synthesis; 1-ethyl-2,3,3-trimethyl-3H-indole has the characteristics of being able to condense with 4-dimethylaminobenzaldehyde to form ICT effect and emit red fluorescence. Therefore, the present invention uses coumarin structural derivatives as fluorescence donors and semi-cyanine dyes as receptors, connected by piperazine, and designs the compound structure of "coumarin derivative-piperazine-indole" based on Michael addition reaction. The initial probe shows the red fluorescence of the receptor. After adding Na2SO3, an addition reaction occurs in the C=C part of the receptor, blocking the intramolecular charge transfer effect of the receptor, the red fluorescence disappears, and the fluorescence resonance energy transfer process is blocked, which is converted into the green fluorescence emission of the donor, thereby realizing the detection of sulfur dioxide.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] The present invention discloses an intermediate I, the structural formula of the intermediate I is as follows:

[0008]

[0009] Furthermore, the present invention discloses a method for preparing the intermediate I, wherein coumarin 343 and 4-piperazine-1-benzaldehyde are subjected to a coupling reaction under the action of a condensing agent and a catalyst to obtain the 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'-tetramethyluronium hexafluorophosphate; and 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-piperazine-1-benzaldehyde, condensation agent, and catalyst is 0.8-1.2: 1.2-2.0: 1.0-2.0: 0.1-0.2; and the reaction temperature of the coupling reaction is -10 to 10°C.

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

[0015] The solvent used in the coupling reaction is preferably any one of dichloromethane and N,N-dimethylformamide or a combination of the two, preferably dichloromethane; there is no special requirement for the amount of the solvent, as long as the raw materials are dissolved and dispersed or dissolved uniformly.

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

[0017] Furthermore, the present invention discloses a fluorescent probe for detecting sulfur dioxide based on the FRET principle. The structural formula of the fluorescent probe for detecting sulfur dioxide based on the FRET principle is shown in the following formula III:

[0018]

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

[0020] Furthermore, the present invention discloses a method for preparing the above-mentioned fluorescent probe for detecting sulfur dioxide based on the FRET principle, wherein the intermediate I and the compound II undergo Michael addition reaction under the action of a base 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 the intermediate I to the compound II is 1.0:1.0-1.5.

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

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

[0027] Wherein, 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 of ethanol and methanol or a combination of the two, preferably ethanol; there is no special requirement for the amount of the solvent, as long as the raw materials are dissolved and dispersed or dissolved uniformly.

[0029] The application of the above-mentioned fluorescent probe for detecting sulfur dioxide based on the FRET principle in detecting sulfur dioxide in water and / or in vivo and / or outside of organisms is also within the protection scope of the present invention.

[0030] Beneficial effects:

[0031] (1) The fluorescent probe developed by the present invention for sulfur dioxide changes its corresponding 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 the present invention for Na2SO3 is about 22nM, indicating that the probe has a 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 research on the kinetic mechanism of the production, delivery and deposition of sulfur dioxide in organisms and further understanding of the physiological and toxicological effects of sulfur dioxide.

[0033] (3) The probe provided by the present invention has a simple preparation process, easy operation, and novel structure, which provides a new idea for the detection of sulfur dioxide. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0035] Figure 1 This is the H NMR spectrum of intermediate I.

[0036] Figure 2 The mass spectrum data of intermediate I is shown in FIG.

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

[0038] Figure 4 This is the mass spectrum data of probe NPB.

[0039] Figure 5 Schematic diagram of the principle of detecting sulfur dioxide using probe NPB.

[0040] Figure 6 This is the mass spectrum data of compound IV.

[0041] Figure 7 is the fluorescence intensity of probe NPB and its response to sulfur dioxide at different pH values ​​I 487 / I 591 Ratio change curve.

[0042] Figure 8 Fluorescence spectra and fluorescence intensities of different analytes measured by probe NPB 487 / I 591 Ratio graph.

[0043] Fig. 9 The relationship between the fluorescence intensity of the probe NPB and the sulfur dioxide concentration and the fluorescence intensity I 487 / I 591 Relationship diagram with sulfur dioxide concentration.

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

[0045] Fig.11 Confocal microscopy images of the probe NPB detecting endogenous sulfur dioxide in cells and the fluorescence intensity diagram of the probe NPB. DETAILED DESCRIPTION

[0046] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

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

[0048] All the structural formulas of the compounds of the present invention are drawn by KingDraw software.

[0049] The following are used in the examples of the present invention: Coumarin 343, CAS No. 55804-65-4, molecular weight 285.29; EDCI, Chinese name 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, molecular weight 191.7; DMAP, Chinese name 4-dimethylaminopyridine, molecular weight 122.168; 4-piperazine-1-benzaldehyde, molecular weight 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-piperazine-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 with water three times, and the organic phase was collected and concentrated in vacuo to recover the solvent. The solvent was eluted by column chromatography on silica gel (DCM / EA=10 / 1, V / V) to obtain a light yellow solid intermediate I, 411 mg, with a yield of 90%.

[0053] The H NMR spectrum of intermediate I is shown in 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] 1mmol 1-ethyl-2,3,3-trimethylindolium iodide (i.e., intermediate II-1) and 1mmol intermediate I were mixed in 10mL ethanol, and 2 drops of piperidine (about 1mmol) were added to catalyze the Michael addition reaction, and the reaction was refluxed overnight. After the reaction was completed, 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 obtain a purple solid probe compound III-1 (referred to as probe NPB), 0.7mmol, yield 70%.

[0058] The H NMR spectrum of probe NPB is shown in Figure 3 , mass spectrum data are shown in 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] See the schematic diagram of the principle of probe NPB detection of sulfur dioxide for details. Figure 5 , sulfite attacks the C=C bond of the acceptor part, and blocks the ICT process of the acceptor part through the Michael addition reaction, resulting in the inability of the donor energy to be transferred to the interior of the acceptor. Therefore, the energy is manifested as the fluorescent property of the donor part, resulting in the fluorescence changing from the red of the acceptor to the green of the donor. Finally, compound IV is obtained, and the structural formula of compound IV is shown below:

[0062]

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

[0064] Compared with the probe NPB solution with sodium sulfite aqueous solution added, the probe NPB solution alone was red under fluorescence excitation; the probe NPB solution with sodium sulfite aqueous solution was light green under fluorescence excitation, proving that the sulfite and the C=C bond of the acceptor part on the probe NPB successfully underwent Michael addition reaction, blocking the transfer of donor energy. The light green reaction solution was subjected to mass spectrometry detection, and the detection results were as follows: Figure 6 As shown, the mixed solution was subjected to mass spectrometry detection, and the mass spectrum data of compound IV was: HR-MS (ESI): m / z [M+H] + :709.4710.

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

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

[0067] Experimental group: Sodium sulfite aqueous solution and probe NPB solution were mixed in a volume ratio of 1:1, and the fluorescence intensity of the mixture was tested using a fluorescence photometer (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 in Example 2 of probe NPB) were prepared without adding sodium sulfite aqueous solution, and the fluorescence intensity was tested using a fluorescence photometer (xenon lamp as light source, 410 nm excitation wavelength) for fluorescence measurement.

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

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

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

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

[0073] The selectivity test results of probe NPB for sulfur dioxide are as follows: Figure 8 As shown, Figure a is the fluorescence spectra of different analytes measured by probe NPB, and Figure b is the fluorescence intensity I of different analytes measured by probe NPB 487 / I 591 Ratio diagram. The results show that the fluorescence intensity is only- 、SO3 2 It can be seen that the probe NPB has good selectivity, which is very beneficial for its application in complex in vivo fluorescence imaging.

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

[0075] Experimental operation:

[0076] Sodium sulfite aqueous solutions of different concentrations were prepared (the concentrations were 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). Probe NPB (concentration of 10 μM, prepared in Example 2 of probe NPB) was dissolved in DMSO:H2O (1:9, V / V) to obtain a probe NPB solution.

[0077] Sodium sulfite aqueous solutions of different concentrations were mixed with the probe NPB solution at a volume ratio of 1:1, and the fluorescence intensity of the mixed solution was tested using a fluorescence photometer (xenon lamp as the light source, 410nm excitation wavelength). The control group was a mixture of the probe NPB solution and an equal volume of water, without the addition of sodium sulfite aqueous solution, and other experimental conditions remained 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 sulfur dioxide concentration is as follows: Fig. 9 As shown, a is the relationship between the fluorescence intensity of the probe NPB and the sulfur dioxide concentration, and b is the fluorescence intensity I of the probe NPB. 487 / I 591 The relationship between the concentration of sulfur dioxide and the concentration of sulfur dioxide is shown in the figure. As can be seen from the figure, under 410nm excitation, NPB (10μM) has two channels centered at 487nm and 591nm, with an initial ratio of 0.99 (I 487 / I 591 ), the wider emission band gap (104nm) effectively ensures that the two channels do not interfere with each other.

[0080] (2) As the concentration of sodium sulfite increases, the fluorescence emission focused strongly at 591 nm gradually weakens and eventually disappears, while the fluorescence intensity at 487 nm rapidly increases, and the red fluorescence turns to green fluorescence (in the EP tube in the upper right corner of Figure a, red is the probe NPB solution alone, and green is the 40 μM sodium sulfite aqueous solution + 10 μM probe NPB solution). Until the Na2SO3 concentration increases to 40 μM, the fluorescence intensity changes gradually and shows a linear relationship in the range of 0 μM to 30 μM (R 2 =0.9904), indicating that the probe NPB can accurately measure the concentration of Na2SO3.

[0081] (3) According to the formula LOD = 3σ / k (where k is Fig. 9 The slope of the fitting curve in Figure b, σ is the standard deviation of the measured fluorescence intensity data) calculated that the detection limit of the probe NPB for Na2SO3 is about 22nM, indicating that the probe has a high sensitivity to Na2SO3. The above results show that the probe NPB has a good detection ability for Na2SO3.

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

[0083] The probe NPB exogenous SO2 imaging experiment was performed by adding Na2SO3. HepG2 cells were placed in a MEM medium containing 5% CO2 and a stable temperature of 37°C, and then fluorescence was captured. The specific operation is as follows:

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

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

[0086] Test results such as Fig.10 As shown, a is the confocal microscope image of the probe NPB detecting exogenous sulfur dioxide in cells, b is the data diagram of the fluorescence intensity ratio in the Red channel and the Green channel in each group of confocal microscope images, and c is the fluorescence intensity I of each group of probe NPB detecting exogenous sulfur dioxide in cells 487 / I 591Ratio graph. As can be seen from the figure, with the increase of Na2SO3 concentration, the red channel does not change much, but the green fluorescence gradually increases; the final result shows that the fluorescence in the red channel disappears completely, and the fluorescence intensity in the green channel is close to the peak value. Therefore, the probe NPB can detect exogenous SO2 in cells.

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

[0088] HepG2 cells were placed in a MEM medium containing 5% CO2 and incubated at a stable temperature of 37°C, and then the fluorescence was captured. Thiosulfate sulfotransferase (TST) is a common enzyme in HepG2 cells, which can cause cells to produce endogenous SO2 in an environment of Na2S2O3 and GSH. TNBS (2,4,6-trinitrobenzene sulfonate) is considered to be an inhibitor of TST enzyme and can effectively inhibit the cell product SO2. The NPB used in this example was prepared by Example 2.

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

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

[0091] GSH group: HepG2 cells were incubated with MEM medium containing GSH (final concentration of 500 μM) at 5% CO 2 and 37° C. for 1 h, and then probe NPB (final concentration of 10 μM) was added and incubated for another 1 h.

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

[0093] The fluorescence intensity of the mixed solution after incubation was tested using a fluorescence photometer (xenon lamp as light source, 410nm excitation wavelength). Fig.11 As shown, a is the confocal microscope image of the probe NPB detecting endogenous sulfur dioxide in cells, b is the data diagram of the fluorescence intensity ratio in the Red channel and the Green channel in each group of confocal microscope images, and c is the fluorescence intensity I of each group of probe NPB detecting endogenous sulfur dioxide in cells487 / I 591 Ratio graph. As can be seen from the figure, compared with the Control group, the green channel of the GSH+Na2S2O3 group was significantly enhanced, while the red fluorescence was very weak; the experimental results of the GSH group can exclude the reaction of GSH to the fluorescent probe; compared with the Control group, the fluorescence of the TNBS group was consistent.

[0094] The experimental results show that TNBS can be used as an effective TST enzyme inhibitor to inhibit the production of SO2, and the probe NPB can successfully identify endogenous SO2. The sulfur dioxide probe NPB designed in the present invention is a colorimetric fluorescent probe, that is, after the introduction of sulfur dioxide, the acceptor red fluorescence is converted into the donor green fluorescence based on the Michael addition reaction.

[0095] The present invention provides a fluorescent probe for detecting sulfur dioxide based on the FRET principle, and a method for preparing and applying the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. An intermediate I, characterized in that: The structural formula of the intermediate I is shown below:

2. The method for preparing the intermediate I according to claim 1, characterized in that: Coumarin 343 and 4-piperazine-1-benzaldehyde undergo coupling reaction under the action of a condensing agent and a catalyst to obtain intermediate I; 3. The preparation method according to claim 2, characterized in that: The condensing agent is any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the catalyst is any one of 4-dimethylaminopyridine and N,N-diisopropylethylamine.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the coumarin 343 to 4-piperazine-1-benzaldehyde, the condensation agent and the catalyst is 0.8-1.2: 1.2-2.0: 1.0-2.0: 0.1-0.2; the reaction temperature of the coupling reaction is -10-10°C.

5. Use of the intermediate I described in claim 1 in preparing a fluorescent probe for detecting sulfur dioxide based on the FRET principle.

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

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

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

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

5.

10. Use of the fluorescent probe for detecting sulfur dioxide based on the FRET principle as claimed in claim 6 in detecting sulfur dioxide in water and / or in vivo and / or in vitro.

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