A fluorescence probe for detecting superoxide anion and peroxynitrite anion in golgi body and a preparation method and application thereof

Abstract

The present invention discloses a fluorescent probe for dual detection of superoxide anions and peroxynitrosyl ions by targeting the Golgi apparatus, and its preparation method and application, belonging to the technical field of active oxygen probes. The fluorescent probe of the present invention has the molecular formula: C 42 H 54 F3N2O5S + The structural formula is: After the probe reacts with superoxide anions, the fluorescence intensity at 720 nm increases. Subsequently, after reacting with peroxynitrosyl ions, the fluorescence at 720 nm decreases and the fluorescence at 460 nm increases. The probe can also be used to detect superoxide anions and peroxynitrosyl ions in the Golgi apparatus of living cells, generating fluorescence images using confocal fluorescence microscopy. This suggests that the probe has potential practical value.

Description

Technical Field

[0001] The present invention belongs to the technical field of active oxygen probes, and in particular relates to a fluorescent probe for dual detection of superoxide anions and peroxynitrosyl ions by targeting the Golgi apparatus, and a preparation method and application thereof. Background Art

[0002] Superoxide anion is the simplest reactive oxygen species in the body and is the first reactive oxygen species produced. It is the precursor of hydrogen peroxide, peroxynitrite, etc. Therefore, changes in superoxide anion concentration can determine changes in other ROS levels to a certain extent. Peroxynitrosyl ion is the product of superoxide anion and nitric oxide, and has stronger oxidizing and nucleophilic properties than superoxide anion. In addition, it can be converted into more active secondary free radicals, such as hydroxyl radical (·OH), nitro radical (·NO2) and carbonate radical (CO3 ·- ) It causes greater damage to the cardiovascular system or other metabolically active organs. Therefore, the activity level of peroxynitrosyl ions and superoxide anions represents the progress of oxidative stress in important life systems such as the cardiovascular system. The Golgi apparatus is a central organelle bound to the intracellular membrane. It plays a key role in the transport, processing and sorting of newly synthesized membranes, secretory proteins and lipids. Changes in the morphological structure of the Golgi apparatus are considered to be important targets for cardiovascular diseases. Therefore, the invention of a probe that can accurately target the Golgi apparatus and detect superoxide anions and peroxynitrosyl ions is of great significance for the study of diseases caused by Golgi damage.

[0003] Currently, methods for detecting superoxide anions include high performance liquid chromatography (HPLC), electrochemical method, SOD enzyme activity assay, chemiluminescence method, and fluorescence analysis method. However, these methods require a large amount of samples, have a long detection time, and are highly toxic, and are therefore not suitable for biological environmental detection. Therefore, it is very important to explore a convenient and inexpensive method that can qualitatively and quantitatively detect superoxide anions and peroxynitrosyl ions, and can perform in situ visual monitoring of biological samples. When the organic fluorescent probe changes with a specific target, its fluorescence signal will change to achieve the purpose of detection. Fluorescence analysis has the characteristics of low output limit, high sensitivity, good selectivity, small sampling volume, simple and fast method, and can perform non-invasive imaging detection of target molecules in cells, and can observe signal changes in real time online, vividly and specifically. Therefore, it is very necessary to invent a superoxide anion and peroxynitrosyl ion fluorescent probe that can quickly detect and easily observe signals. In addition, molecular fluorescent probes with Golgi-targeting capabilities and comprehensive detection capabilities of superoxide anions and peroxynitrosyl ions have not yet been developed. Considering the unique application of the Golgi apparatus in cardiovascular diseases, the development of a fluorescent probe that can simultaneously distinguish and detect superoxide anions and peroxynitrosyl ions is of great significance for explaining the relationship between the Golgi apparatus and cardiovascular toxic damage. SUMMARY

[0004] The purpose of the present application is to provide a luminescent fluorescent probe, referred to as probe 6, capable of rapidly detecting superoxide anion and peroxynitrite ion, and further to provide a preparation method and application of the probe 6.

[0005] To achieve the above technical purpose, the technical scheme adopted by the present application is as follows:

[0006] A Golgi-targeted fluorescent probe for double detection of superoxide anion and peroxynitrite ion, namely probe 6, has a molecular formula of C 42 H 54 F3N2O5S + , and a structural formula as follows:

[0007] The probe 6 of the present application can resist the interference of hydrogen peroxide, hypochlorous acid, nitric oxide, hydroxyl radical, hydrogen sulfide, cysteine, sulfite and glutathione.

[0008] The synthetic route of the probe 6 of the present application is as follows:

[0009]

[0010] The preparation method of the fluorescent probe 6 comprises the following preparation steps:

[0011] (1) Under ice bath, a mixed solution of 20 mL anhydrous DMF and 20 mL anhydrous DCM is added to a container, and then a mixed solution of 15 mL POCl3 and 20 mL DCM is slowly dropped into the container under inert gas protection using a constant pressure dropping funnel, and finally 5.0 g of 4-cyclohexanone carboxylic acid is dissolved in 5 mL anhydrous DMF and then added dropwise into the reaction, and the temperature is raised to 50℃, and refluxed for 5 h; after the reaction is completed, the reaction liquid is poured into ice water, and placed in a-20℃ refrigerator overnight, and after standing at room temperature, yellow solid is precipitated, and after suction filtration and drying, 6.31 g of compound 2 is obtained without further purification (yield 83%), and the structural formula of the compound 2 is as follows:

[0012] (2) 400 mg of compound 2 is added into a 100 mL round-bottom flask, 150 mg of sodium acetate and 340 mg of 2-methylbenz

[0013] zolium iodide are added as solvents, and the temperature is raised to 90℃ under nitrogen protection, and the reaction is carried out for 2 h, and the solvent is removed by rotary evaporation, and column chromatography is used for separation and purification, and finally 644 mg of green solid compound 3 is obtained (yield 66%), and the structural formula of the solid compound 3 is as follows:

[0014] ​(3) 500 mg of compound 3, 220 mg of tetradecylamine, and 500 mg of HATU were dissolved in 10 mL of DCM, and 80 μL of triethylamine was added. The mixture was reacted at room temperature overnight under nitrogen protection. The solvent was removed by vacuum rotary evaporation, and the mixture was separated and purified by column chromatography and eluted with DCM to obtain 540 mg of green solid compound 4 (yield 79%). The structural formula of compound 4 is:

[0015]

[0016] (4) 75 mg of resorcinol and 83 mg of potassium carbonate were dissolved in 5 mL of acetonitrile and stirred at room temperature for 10 min. Then, 350 mg of compound 4 was added and the temperature was raised to 50°C for 4 h. After the reaction was completed, the mixture was dried under reduced pressure and separated by column chromatography and eluted with DCM to obtain 124 mg of blue-green solid compound 5 (yield 41%). The structural formula of compound 5 is:

[0017]

[0018] (5) 100 mg of compound 5 was dissolved in 5 mL of DCM. 100 μL of trifluoromethylsulfonate and 30 μL of triethylamine were added under ice bath, and the mixture was stirred at 0°C for 10 min and then at room temperature for 30 min. The mixture was dried under reduced pressure, separated by column chromatography, and eluted with DCM to obtain 80 mg of purple solid compound 6 (yield 66%), which was the target probe with the structural formula:

[0019]

[0020] Preferably, in step (2), the column chromatography separation and purification is performed with an eluent composition of DCM:C2H5OH=(10:1).

[0021] Preferably, compounds 3, 4, and 5 are further purified using a silica gel chromatography column before use.

[0022] Preferably, the triethylamine in step (5) is added dropwise; after obtaining the target probe compound 6, it is further purified using a silica gel chromatography column.

[0023] Preferably, during the entire preparation process, the organic solvent, such as dichloromethane, ethanol or acetonitrile, in the reaction solution is removed by reduced pressure distillation using a rotary evaporator.

[0024] The fluorescent probe 6 of the present invention is used to detect superoxide anions and peroxynitrosyl ions in aqueous solutions and in vitro biological samples.

[0025] The application of the probe of the present invention specifically includes the following steps:

[0026] (1) Dissolve probe 6 in DMSO to prepare probe stock solution;

[0027] (2) Add the probe stock solution to the test solution and conduct the test.

[0028] First, superoxide anions and peroxynitrosyl ions in aqueous solution can cause changes in the fluorescence spectrum of the fluorescent probe. Therefore, the content of superoxide anions and peroxynitrosyl ions in the solution can be judged by observing the degree of spectral change in the fluorescence spectrometer, thereby achieving quantitative detection. Second, fluorescence imaging of living cells incubated with fluorescent probe 6 and superoxide anions and peroxynitrosyl ions is performed using a confocal microscope, and changes in the blue channel fluorescence signal are observed to achieve the purpose of ratiometric detection of superoxide anions and peroxynitrosyl ions in the biological environment.

[0029] The change in the fluorescence spectrum refers to: the change in the fluorescence peak at 720nm in the fluorescence spectrum; if the peak at 720nm becomes larger, it means that superoxide anions are present; the change in the fluorescence peak at 460nm; if the peak at 460nm becomes larger, it means that peroxynitrosyl ions are present.

[0030] The change in fluorescence imaging refers to the enhancement of red and blue channel fluorescence in live cell imaging under 630nm and 405nm light sources. Preferably, a confocal laser scanning microscope is used.

[0031] The addition of superoxide anions or peroxynitrosyl ions to the probe solution can produce visible changes. These changes are: Upon addition of superoxide anions, the probe solution changes color from blue to green; upon addition of peroxynitrosyl ions, the probe solution changes color from blue to colorless. Purple probe compounds appear blue when dissolved in the solvent DMSO.

[0032] Beneficial effects: (1) The probe is simple to synthesize and has a high yield; (2) The present invention achieves specific and rapid detection of superoxide anions in aqueous solutions; (3) The present invention achieves detection of superoxide anions and peroxynitrosyl ions in the Golgi apparatus of living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is compound 3 in Example 1 1 H NMR spectrum;

[0034] Figure 2 is compound 3 in Example 1 13 C NMR spectrum;

[0035] Figure 3 is the HRMS spectrum of compound 3 in Example 1;

[0036] Figure 4 is the compound 4 in Example 1 1 H NMR spectrum;

[0037] Figure 5 is the H NMR spectrum of compound 4 in Example 1; 13 C NMR spectrum;

[0038] Figure 6 is the HRMS spectrum of compound 4 in Example 1;

[0039] Figure 7 is the H NMR spectrum of compound 5 in Example 1; 1 H NMR spectrum;

[0040] Figure 8 is the C NMR spectrum of compound 5 in Example 1; 13 C NMR spectrum;

[0041] Figure 9 is the HRMS spectrum of compound 5 in Example 1;

[0042] Figure 10 is the H NMR spectrum of probe 6 in Example 1; 1 H NMR spectrum;

[0043] Figure 11 is the C NMR spectrum of probe 6 in Example 1; 13 C NMR spectrum;

[0044] Figure 12 is the HRMS spectrum of probe 6 in Example 1;

[0045] Figure 13 is the change of fluorescence spectrum of probe 6 (10 μmol / L) in PBS buffer solution (concentration 10 mmol / L, pH 7.4, containing 20% DMSO) with the addition of different amounts of superoxide anion in Example 2; in the figure, from bottom to top, the superoxide anion concentration is 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 100 μmol / L of fluorescence spectrum;

[0046] Figure 14 is the change of fluorescence spectrum of probe 6 (10 μmol / L) in PBS buffer solution (concentration 10 mmol / L, pH 7.4, containing 20% DMSO) with the addition of different amounts of peroxynitrite ion after the addition of 5 eq of superoxide anion in Example 2; in the figure, from bottom to top, the peroxynitrite ion concentration is 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 and 20 μmol / L of fluorescence spectrum;

[0047] Figure 15This is a line graph showing the fluorescence intensity changes of probe 6 (10 μM) at 720 nm in PBS buffer (10 mmol / L, pH 7.4, containing 20% ​​DMSO) and 100 μM superoxide anion over 30 minutes. The excitation wavelength is 630 nm.

[0048] Figure 16 This is a line graph showing the change in fluorescence intensity at 460 nm of probe 6 after adding 80 μM superoxide anion and then 20 μM peroxynitrosyl ion in PBS buffer solution (concentration 10 mmol / L, pH 7.4, containing 20% ​​DMSO) within 30 minutes, with an excitation wavelength of 360 nm.

[0049] Figure 17 This is the selectivity column fluorescence data of probe 6 for different interfering analytes; in the figure, 1. Blank; 2. H2O2; 3. . OH; 4.HClO; 5.NO; 6.H2S; 7.Cys; 8.SO3 2- ;9.GSH;10.ONOO - ;11.O2 ·- The probe (10 μM) reacted with different interfering analytes in PBS buffer (10 mmol / L, pH 7.4, containing 30% DMSO); the ordinate represents the fluorescence intensity at 720 nm, with an excitation wavelength of 630 nm;

[0050] Figure 18 This is a columnar fluorescence data diagram of the selectivity of probe 6 for different interfering analytes after adding 80 μM superoxide anion; in the figure, 1. Blank; 2. H2O2; 3. . OH; 4.HClO; 5.NO; 6.H2S; 7.Cys; 8.SO3 2- ;9.GSH;10.ONOO - ;11.O2 ·- The probe (10 μM) reacted with different interfering analytes in PBS buffer (10 mmol / L, pH 7.4, containing 20% ​​DMSO); the ordinate represents the fluorescence intensity at 460 nm, with an excitation wavelength of 360 nm;

[0051] Figure 19 is the pH-dependent response of probe 6 (10 μM) to superoxide anion (100 μM);

[0052] Figure 20 is the pH-dependent response of probe 6 (10 μM) after the addition of 80 μM superoxide anion followed by the addition of 20 μM peroxynitrosyl ion;

[0053] Figure 21is the confocal fluorescence imaging map of probe 6 (10 μM) detecting superoxide anion and peroxynitrosyl ion in H9c2 cells; excitation wavelength: blue channel: 405 nm, red channel: 630 nm; Scale bar = 10 μm.

[0054] Figure 22 is the confocal fluorescence imaging map of probe 6 (10 μM) detecting Golgi and other subcellular organelle colocalization in H9c2 cells; excitation wavelength: blue channel: 405 nm, red channel: 630 nm; Scale bar = 10 μm. DETAILED DESCRIPTION

[0055] The technical solutions of the present application are further described below in combination with specific embodiments, but are not limited thereto.

[0056] Example 1

[0057] Synthesis of compound 2-6

[0058] A mixture solution of 20 mL anhydrous DMF and 20 mL anhydrous DCM was added to a container under ice bath, and then a mixture of 15 mL POCl3 and 20 mL DCM was slowly dropped into the container using a constant pressure dropping funnel under inert gas protection, and finally 5.0 g of 4-cyclohexanone carboxylic acid was dissolved in 5 mL anhydrous DMF and then added dropwise into the reaction, and the temperature was increased to 50 °C and refluxed for 5 h. After the reaction was completed, the reaction liquid was poured into ice water, and then placed in a-20 °C refrigerator overnight, and then a yellow solid was precipitated after standing at room temperature, and then the solid was extracted by suction filtration and dried, and 6.31 g of compound 2 was obtained without further purification (yield 83%).

[0059] 400 mg of the above compound 2 was added into a 100 mL round-bottom flask, 150 mg of sodium acetate and 340 mg of 2-methylbenzoindole iodide were added, 40 mL of acetic anhydride was used as a solvent, and the temperature was increased to 90 °C under nitrogen protection, and the reaction was carried out for 2 h. The solvent was removed by rotary evaporation, and then column chromatography was used for separation and purification, and the eluent was composed of DCM: C2H5OH = (10:1), and finally 644 mg of green solid compound 3 was obtained (yield 66%). 1 H NMR (500 MHz, CDCl3) δ 8.29 (d, J = 14.1 Hz, 2H), 7.33 (dd, J = 20.5, 7.5 Hz, 4H), 7.22-7.17 (m, 3H), 7.13 (d, J = 7.9 Hz, 2H), 6.22 (d, J = 14.1 Hz, 2H), 3.69 (s, 6H), 3.25 (d, J = 13.2 Hz, 2H), 3.03 (s, 1H), 2.80-2.69 (m, 2H), 2.04 (s, 1H), 1.66 (s, 12H). 13C NMR (126MHz, CDCl3) δ173.29,149.79,149.74,144.64,142.74,141.00,128.88,125.50,1 25.16,122.16,110.91,101.97,49.32,37.90,32.54,28.83,28.14,28.08.HRMS(ESI)m / z calcd forC 33 H 36 ClN2O2 + [M] + 527.2460, found 527.2476.

[0060] The synthetic route of compound 3 is:

[0061]

[0062] The synthetic route of compound 4 is:

[0063]

[0064] 500 mg of compound 3, 220 mg of tetradecylamine, and 500 mg of HATU were dissolved in 10 mL of DCM, and 80 μL of triethylamine was added. The mixture was reacted at room temperature overnight under nitrogen protection. The solvent was removed by rotary evaporation under reduced pressure, and the mixture was separated and purified by column chromatography and eluted with DCM to obtain 540 mg of green solid compound 4 (yield 79%). 1H NMR(500MHz,MeOD)δ8.42(d,J=14.2Hz,2H),7.52(d,J=7.4Hz,2H),7.43(t,J=7.7H z,2H),7.35(d,J=8.0Hz,2H),7.29(t,J=7.4Hz,2H),6.28(d,J=14.2Hz,2H),3.69( s,6H),3.28(q,J=6.6Hz,2H),3.23–3.17(m,1H),3.07(d,J=12.6Hz,2H),2.71(d,J =14.2Hz,2H),1.72(s,12H),1.29(s,24H),0.91(d,J=5.9Hz,3H).13CNMR(126MHz, MeOD)δ174.97,173.68,148.22,143.99,142.86,141.17,128.50,125.27,124.49, 122.00,110.80,101.12,49.22,48.16,47.99,47.82,47.65,47.48,47.31,47.14, 44.98,39.33,38.94,31.70,30.52,29.58,29.46,29.41,29.39,29.32,29.24,29. 12,29.10,29.04,28.97,28.75,26.84,26.72,26.41,22.37,13.12.HRMS(ESI)m / z calcd forC 47 H 65 ClN3O + [M] + 722.4816,found722.4821.

[0065] The synthetic route of compound 5 is:

[0066]

[0067] 75 mg of resorcinol and 83 mg of potassium carbonate were dissolved in acetonitrile and stirred at room temperature for 10 min. Then 350 mg of compound 4 was added and the temperature was raised to 50°C for 4 h. After the reaction was completed, the mixture was dried under reduced pressure and separated by column chromatography and eluted with DCM to obtain 124 mg of blue-green solid compound 5 (yield 41%). 1H NMR(500MHz,MeOD)δ8.52(d,J=14.5Hz,1H),7.55(d,J=7.4Hz,1H),7.49–7.41(m,2H), 7.34(td,J=14.4,13.9,7.7Hz,3H),6.75(d,J=8.7Hz,1H),6.65(s,1H),6.24(d,J=14. 5Hz,1H),3.70(s,3H),2.94(d,J=12.0Hz,1H),2.85(d,J=7.3Hz,2H),2.68(d,J=13.3H z,2H),1.75(d,J=4.9Hz,6H),1.58–1.49(m,2H),1.26(s,24H),0.87(t,J=6.8Hz,3H). 13 C NMR(126MHz,MeOD)δ175.36,174.70,168.28,161.03,155.94,142.68,142.54,14 1.25,137.19,129.40,128.57,125.74,122.03,117.69,114.49,112.42,111.20,1 01.94,100.82,49.63,48.13,47.96,47.79,47.62,47.45,47.28,39.19,38.55,31 .70,31.26,30.78,29.44,29.38,29.08,27.10,22.36,13.09.HRMS(ESI)m / zcalcd for C 41 H 55 N2O3 + [M] + 623.4208,found623.4386.

[0068] The synthetic route of probe 6 is:

[0069]

[0070] Dissolve 100 mg of compound 5 in 5 mL of DCM, add 100 μL of trifluoromethylsulfonate and 30 μL of triethylamine in an ice bath, and stir at 0°C for 10 min, then at room temperature for 30 min. Dried to dryness under reduced pressure, separate by column chromatography, and elute with DCM to obtain 80 mg of purple solid compound 6 (yield 66%). 1H NMR(500MHz,MeOD-d4)δ8.75(d,J=15.4Hz,1H),7.72(d,J=7.3Hz,1H),7.68(d,J=7.9Hz,1H),7.63–7.55(m,4H),7.30(d,J=10.8Hz ,2H),6.73(d,J=15.4Hz,1H),3.99(s,3H),3.01–2.78(m,5H),1.85(s,6H),1.58–1.51(m,2H),1.29(s,24H),0.89(d,J=7.1Hz,3H). 13 C NMR(126MHz,MeOD)δ180.38,174.48,157.27,152.92,150.18,145.92,142.72,142.10,13 0.21,129.28,128.80,128.28,122.38,121.98,121.67,119.14,117.74,113.46,113.02, 109.47,107.32,51.45,48.12,47.95,47.78,47.61,47.44,47.10,46.59,44.97,39.30,3 8.01,32.32,31.67,30.42,29.37,29.04,26.61,26.08,22.33,13.04,7.83.HRMS(ESI)m / z calcd for C 42 H 54 F3N2O5S + ,[M] + 755.3700, found 755.3708.

[0071] Example 2

[0072] Fluorescence spectrum changes of probe 6 in response to different equivalents of potassium superoxide

[0073] The probe 6 prepared in Example 1 was dissolved in DMSO to prepare a 1 mmol / L probe stock solution (the concentration of probe 6 was 1 mmol / L). Potassium superoxide was prepared into solutions of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 100 μmol / L using DMSO. 30 μL of the probe stock solution was taken out and added to a 5 mL centrifuge tube. Different equivalents (0-10 eq) of potassium superoxide stock solution (the equivalent refers to the molar number of potassium superoxide in the potassium superoxide stock solution relative to the molar number of probe in the probe stock solution) were added. The solution was diluted to 3 mL with 0.870 mL of DMSO and different volumes of PBS aqueous solution (concentration 10 mmol / L, pH 7.4) to prepare a test solution with a probe concentration of 10 μmol / L and 20% DMSO. The fluorescence spectrum changes of the probe and the reaction solution with different equivalents of superoxide anions were tested using a fluorescence spectrometer (excitation wavelength is 630nm). The fluorescence spectrum changes are as follows: Figure 13 As shown. Figure 13 It can be seen that as the amount of potassium superoxide added gradually increases, the fluorescence peak of probe 6 solution at 720 nm gradually increases. Figure 14 As shown in the figure, when 5eq potassium superoxide was added, the same concentration of peroxynitrosyl ions was gradually added, and the concentration of peroxynitrosyl ions was 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18 and 20 μmol / L, respectively. The fluorescence at 720 nm gradually decreased (the excitation wavelength was 630 nm), and the fluorescence at 460 nm gradually increased (the excitation wavelength was 360 nm).

[0074] Example 3

[0075] Fluorescence changes of probe 6 and superoxide anion over time

[0076] 30 μL of the fluorescent probe mother solution from Example 2 was taken and added to a 5 mL centrifuge tube. 30 μL of 10 mmol / L potassium superoxide mother solution was added, and then diluted to 3 mL with 0.870 ml of DMSO and 2.100 mL of PBS aqueous solution (concentration 25 mmol / L, pH 7.4). This prepared a test solution with a probe concentration of 10 μmol / L, a potassium superoxide concentration of 10 mmol / L, and 20% DMSO. The fluorescence spectrum was measured over time using an excitation wavelength of 630 nm. Figure 15 It can be seen that the fluorescence at 720nm reaches a peak at 20min and remains unchanged. First, 10eq potassium superoxide is added to react for 30min, and then 2eq peroxynitrosyl ion is added. Figure 16 As shown, the fluorescence at 460 nm reached a peak at 20 min and remained constant.

[0077] Example 4

[0078] Selective study of probe 6 to different interfering analytes

[0079] 30 μL of the probe mother liquor from Example 2 was taken and added into a 5 mL centrifuge tube, and 1.00 mM of the analyte was added: 1. Blank; 2. H202; 3. . OH; 4. HCIO; 5. NO; 6. H2S; 7. Cys; 8. SO3 2- ; 9. GSH; 10. ONOOH - ; 11. O2 ·- was added. 0.970 mL of DMSO and 2.000 mL of PBS aqueous solution (concentration 20 mmol / L, pH 7.4) were added to dilute to 3 mL, and the test solution was prepared with a probe concentration of 10 μmol / L and 20% DMSO. The fluorescence spectrum of the test solution was detected after 30 minutes of reaction. It can be found from Figure 17 and Figure 18 that the fluorescence intensity of the test solution of various analytes did not change significantly relative to the blank test solution. However, the fluorescence intensity of the test solution added with potassium superoxide and peroxynitrite ions at 720 nm and 460 nm emission wavelengths was significantly enhanced. The experimental results show that probe 6 has good selectivity to superoxide anion and peroxynitrite ions. It can be found from Figure 19 and Figure 20 that probe 6 responds best under neutral conditions.

[0080] Example 5

[0081] 10 μL of the probe mother liquor from Example 2 was taken and added into a culture dish of H9c2 cells (containing 1 mL of DMEM medium) to make the probe concentration 10 μmol / L, and incubated for 30 minutes as a control group; the sample of experimental group 1 was incubated with endotoxin and IFN-γ for 12 h in advance to stimulate intracellular superoxide anion and peroxynitrite ions, the sample of experimental group 2 was incubated with endotoxin, IFN-γ and nitric oxide for 12 h in advance, the sample of experimental group 3 was incubated with endotoxin, IFN-γ and Tempo for 12 h in advance, the sample of experimental group 4 was incubated with endotoxin, IFN-γ and Tiron for 12 h in advance, and the sample of experimental group 5 was incubated with endotoxin, IFN-γ and hydrogen sulfide for 12 h in advance. Subsequently, the control group and the experimental groups were subjected to fluorescence imaging using a confocal microscope, and the light sources with excitation wavelengths of 630 nm and 405 nm were used to excite respectively, and the fluorescence of the red and blue channels was collected, and the results are as shown in Figure 21As shown in the fluorescence imaging of the control group, only weak blue and blue fluorescence were observed; however, in experimental group 1, significant red and blue channel fluorescence was observed, experimental group 2 showed weakened red fluorescence and further enhanced blue fluorescence, and experimental groups 3, 4, and 5 showed weak red and blue fluorescence. These experimental results demonstrate that probe 6 can detect superoxide anions and peroxynitrite ions in cellular environments using confocal microscopy and has potential practical applications.

[0082] 10 μL of the fluorescent probe mother solution in Example 2 was taken out and added to a culture dish (containing 1 mL of DMEM culture medium) containing H9c2 cells. The probe concentration was 10 μmol / L and incubated for 30 minutes as a control group. The experimental group 1 sample was incubated with endotoxin and IFN-γ for 12 hours in advance to stimulate intracellular superoxide anions and peroxynitrosyl ions, and the cell organelles were stained using Biyuntian commercial Golgi apparatus, mitochondria, endoplasmic reticulum, and lysosome-targeted fluorescent probes (green). After staining, the cells were incubated with probe 6 working solution for 30 minutes. Subsequently, fluorescence imaging of the control group and the experimental group was performed using a confocal microscope, using a light source with an excitation wavelength of 405 nm to excite, and collecting fluorescence in the green, red, and blue channels. The results are as follows: Figure 22 As shown in the figure, the green, red, and blue Golgi bodies in the experimental group colocalize well, while colocalization with other organelles is poor. These results demonstrate that Probe 6 can be well targeted to the Golgi body and has potential practical applications.

[0083] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

Claims

1. A Golgi apparatus-targeted fluorescent probe for dual detection of superoxide anions and peroxynitrosyl ions, characterized in that: Its molecular formula is: C 42 H 54 F3N2O5S + , the structural formula is: .

2. A method for preparing the Golgi apparatus-targeted fluorescent probe for dual detection of superoxide anions and peroxynitrosyl ions according to claim 1, characterized in that: The method comprises the following preparation steps: (1) A mixed solution of 20 mL of anhydrous DMF and 20 mL of anhydrous DCM was added to a container under ice bath, and then a mixed solution of 15 mL of POCl3 and 20 mL of DCM was slowly added dropwise using a constant pressure dropping funnel under inert gas protection. Finally, 5.0 g of 4-cyclohexanonecarboxylic acid was dissolved in anhydrous DMF and added dropwise to the reaction, the temperature was raised to 50°C, and refluxed for 5 h. After the reaction was completed, the reaction solution was poured into ice water and placed in a -20°C refrigerator overnight. After standing at room temperature, a yellow solid was precipitated, which was filtered and dried without further purification to obtain 6.31 g of compound 2. The structural formula of compound 2 is: ; (2) 400 mg of compound 2 was added to a 100 mL round-bottom flask, along with 150 mg of sodium acetate and 340 mg of 2-methylbenzidindol iodide, and 40 mL of acetic anhydride as a solvent. The mixture was heated to 90° C. under nitrogen protection and reacted for 2 h. The solvent was removed by rotary evaporation and purified by column chromatography to obtain 644 mg of green solid compound 3. The structural formula of solid compound 3 is: ; (3) 500 mg of compound 3, 220 mg of tetradecylamine, and 500 mg of HATU were dissolved in 10 mL of DCM, and 80 μL of triethylamine was added. The mixture was reacted at room temperature overnight under nitrogen protection. The solvent was removed by vacuum rotary evaporation, and the mixture was separated and purified by column chromatography and eluted with DCM to obtain 540 mg of green solid compound 4. The structural formula of compound 4 is: ; (4) 75 mg of resorcinol and 83 mg of potassium carbonate were dissolved in 5 mL of acetonitrile and stirred at room temperature for 10 min. Then, 350 mg of compound 4 was added and the temperature was raised to 50°C for 4 h. After the reaction was completed, the mixture was dried under reduced pressure and separated by column chromatography and eluted with DCM to obtain 124 mg of blue-green solid compound 5. The structural formula of compound 5 is: ; (5) 100 mg of compound 5 was dissolved in 5 mL of DCM, and 100 μL of trifluoromethanesulfonate and 30 μL of triethylamine were added in an ice bath. The mixture was stirred at 0°C for 10 min and then at room temperature for 30 min. The mixture was dried under reduced pressure and separated by column chromatography. The mixture was eluted with DCM to obtain 80 mg of purple solid compound 6, which was the target probe. The structural formula is: .

3. The method for preparing a fluorescent probe for dual detection of superoxide anion and peroxynitrosyl ion targeting the Golgi apparatus according to claim 2, wherein: In step (2), the product was separated and purified by column chromatography, and the eluent composition was DCM:C2H5OH=(10:1).

4. The method for preparing a fluorescent probe for dual detection of superoxide anion and peroxynitrosyl ion targeting the Golgi apparatus according to claim 2, wherein: Before use, compounds 3, 4, and 5 were further purified by silica gel chromatography.

5. The method for preparing a fluorescent probe for dual detection of superoxide anion and peroxynitrosyl ion by targeting the Golgi apparatus according to claim 2, wherein: In step (5), triethylamine is added dropwise; after obtaining the target probe compound 6, it is further purified using a silica gel chromatography column.

6. Use of the Golgi apparatus-targeted fluorescent probe for dual detection of superoxide anions and peroxynitrosyl ions according to claim 1 in non-disease diagnosis and treatment, for detecting superoxide anions and peroxynitrosyl ions in aqueous solutions and in vitro biological samples.

Images