A bifunctional, reversible, quantitative, and Golgi-targeted fluorescent probe for real-time monitoring of intracellular redox homeostasis kinetics

Abstract

The present invention relates to a bifunctional, reversible, quantitative and Golgi-targeted fluorescent probe for real-time monitoring of the dynamics of intracellular redox homeostasis, belonging to the field of fluorescent probes. Its molecular structure is as follows: The probe molecule exhibits red fluorescence. After reacting with ClO-, selenium (Se) is oxidized to selenium oxide (Se=O) and emits green fluorescence. After reacting with GSH, selenium oxide (Se=O) is reduced to selenium (Se) and the red fluorescence is restored, with Golgi targeting. The probe molecule of the present invention can realize the real-time monitoring of the dynamic changes of the ClO- / GSH-mediated redox state in Golgi oxidative stress, and has important application value in the field of biochemical molecular analysis.

Description

[0001] The present invention relates to a bifunctional, reversible, quantitative and Golgi-targeted fluorescent probe PSeZ-Cou-Golgi for real-time monitoring of the kinetics of intracellular redox homeostasis, including synthesis steps, fluorescence spectrum testing and applications in the field of bioimaging, belonging to the field of fluorescent probes. Background Art

[0002] Intracellular redox homeostasis is of great significance for regulating the physiological functions of organisms (such as protein transport, cell proliferation and cell signaling). More specifically, intracellular redox reactions mainly occur in major organelles such as mitochondria, endoplasmic reticulum and Golgi apparatus. So far, intracellular redox in mitochondria has been well studied. However, little attention has been paid to the intracellular redox homeostasis in the Golgi apparatus (a key organelle for cell metabolism). It has been reported that the kinetics of the hypochlorite / glutathione (ClO - / GSH) redox cycle is closely related to intracellular redox homeostasis. Excessive ClO - will disrupt the redox balance, generate cellular oxidative stress, and lead to various diseases such as acute lung injury, acute kidney injury, acute liver injury, Alzheimer's disease and cancer. At the same time, the most abundant biothiol GSH in cells is the main scavenger of ClO - and can maintain the intracellular redox balance. Therefore, real-time monitoring of the dynamics of the ClO - / GSH redox cycle in the Golgi apparatus is very important for understanding the pathological and physiological processes of organisms.

[0003] As a versatile molecular tool, organic fluorescent probes have increasingly attracted the attention of biomedical research scientists due to their good selectivity, high sensitivity, design flexibility and real-time monitoring ability. Many fluorescent probes have been established for the separate detection of ClO - or GSH. To study the intracellular ClO- / GSH redox homeostasis, it is necessary to dynamically detect these two substances simultaneously. The physical combination of two separate single-functional ClO - and GSH probes usually cannot detect ClO - and GSH simultaneously because the two probes exhibit different cellular uptake and have different distributions in cells. At the same time, the ClO - / GSH redox homeostasis is dynamic and its concentration is always fluctuating. Therefore, the fluorescent probe for monitoring the ClO - / GSH redox cycle should be reversible. Recently, several "on-off" reversible probes have been used to detect ClO - / GSH redox cycle. Although these "on-on" probes are useful, they are vulnerable to probe concentration and instrument factors, which is disadvantageous for reliable bioimaging in a variable redox environment. Quantitative fluorescence probes are more advantageous than "on-off" types because the former improves the accuracy of quantitative analysis and simplifies the operation procedure through self-calibration of two independent signals. Therefore, the fluorescence probe for monitoring the ClO - / GSH redox cycle dynamics in cells should be bifunctional, reversible, and ratio-measuring.

[0004] In this patent, a ratio fluorescence probe, the bifunctional, reversible, and ratio-type fluorescence probe PSeZ-Cou-Golgi, was constructed to detect ClO - / GSH related to redox homeostasis in the Golgi apparatus for the diagnosis of acute lung injury in mice. In the presence of ClO - , the selenium (Se) atom in the PSeZ moiety is oxidized to selenium oxide (Se=O), and the probe PSeZ-Cou-Golgi is converted to PSeZ-Cou-Golgi oxide. Since the oxide of the PSeZ moiety is a weaker electron donor compared to PSeZ, PSeZ-Cou-Golgi oxide exhibits green fluorescence. In the presence of GSH, Se=O is reduced to Se, and PSeZ-Cou-Golgi oxide can be converted back to PSeZ-Cou-Golgi. When the probe PSeZ-Cou-Golgi is continuously treated with ClO - and GSH, a red-green-red fluorescence change will be observed. The probe PSeZ-Cou-Golgi is a useful tool for monitoring the ClO - / GSH redox cycle. Summary of the Invention

[0005] The object of the present invention is to develop a bifunctional, reversible, quantitative, and Golgi-targeted fluorescence probe for real-time monitoring of the dynamics of intracellular redox homeostasis in view of the deficiencies of existing research, and to solve the technical problem of simultaneously and dynamically detecting the ClO - / GSH redox homeostasis in cells with existing probes. The fluorescence probe PSeZ Cou-Golg has the following structural formula:

[0006]

[0007] The synthesis route is as follows:

[0008]

[0009] (a) Dissolve o-bromoaniline and 1-bromo-2-iodo-4-methoxybenzene in toluene, add tris(dibenzylideneacetone)dipalladium(0), 1,1'-bis(diphenylphosphino)ferrocene, and sodium tert-butoxide, and reflux and stir at 120 °C for 18 h. After cooling to room temperature, extract with ethyl acetate. Wash with saturated brine and dry over anhydrous sodium sulfate. Rotate dry under reduced pressure to obtain the crude product, and separate by column chromatography to obtain the colorless oily compound 1;

[0010] (b) Dissolve compound 1, copper(I) iodide, sodium iodide, and N,N'-dimethylethylenediamine in anhydrous dioxane and stir at 110 °C for 24 h. After cooling to room temperature, add aqueous ammonia solution, extract with DCM, and dry over anhydrous sodium sulfate. Rotate dry under reduced pressure to obtain the crude product, and separate by column chromatography to obtain the brown oily compound 2;

[0011] (c) Dissolve compound 2, Se powder, and KOH in anhydrous DMSO and react at 110 °C for 24 h. After cooling to room temperature, extract with DCM, wash with saturated NH4Cl solution, and dry over anhydrous sodium sulfate. Rotate dry under reduced pressure to obtain the crude product, and separate by column chromatography to obtain the yellow solid compound 3;

[0012] (d) Dissolve the compound 3 prepared in step (c) in anhydrous DMF, slowly add NaH, react at 0 °C for 30 min, then add 2-dimethylethanol, stir at room temperature for 5 h, cool to room temperature, pour the reaction solution into water to quench, extract with dichloromethane three times, dry over anhydrous sodium sulfate, rotate dry under reduced pressure to obtain the crude product, and then separate by column chromatography to obtain compound 4;

[0013] (e) Dissolve compound 4, DMAP, and triethylamine in anhydrous DCM and add dropwise CH3COCl. React at room temperature for 12 h, cool to room temperature, pour the reaction solution into H2O, extract with DCM three times, dry over anhydrous sodium sulfate, rotate dry under reduced pressure to obtain the crude product, and separate by column chromatography to obtain compound 5;

[0014] (f) Drop POCl3 into anhydrous DMF and react at 0 °C for 30 min. Then dissolve compound 5 in anhydrous DMF and add it to the above mixture, react at 80 °C for 6 h, cool to room temperature, pour the reaction solution into water to quench. Neutralize with saturated aqueous NaHCO3 solution, extract with DCM three times, and dry over anhydrous Na2SO4. Rotate dry under reduced pressure to obtain the crude product, and separate by column chromatography to obtain compound 6;

[0015] (g) At -78 °C under argon protection, dissolve compound 6 in anhydrous DCM, add BBr3, and react at room temperature for 12 h. Pour the reaction solution into water to quench. Neutralize with saturated aqueous NaHCO3 solution, extract with DCM three times, and dry over anhydrous Na2SO4. Rotate dry under reduced pressure to obtain the crude product, and separate by column chromatography to obtain compound 7;

[0016] (h) Dissolve compound 7, diethyl malonate, and piperidine in EtOH and reflux for 6 hours. Remove the solvent by distillation under reduced pressure to obtain a crude product, and separate compound 8 by column chromatography;

[0017] (i) Dissolve compound 8 in anhydrous THF, add 4-sulfonylbenzoyl chloride and Et3N, and react at room temperature for 6 hours. Remove the solvent by distillation under reduced pressure to obtain a crude product, and separate the probe PSeZ-Cou-Golgi by column chromatography.

[0018] In step (a), the molar ratio of 1-bromo-2-iodo-4-methoxybenzene, o-bromoaniline, tris(dibenzylideneacetone)dipalladium(0), 1,1'-bis(diphenylphosphino)ferrocene, and sodium tert-butoxide is 1:1.2:0.05:0.1:1.4.

[0019] In step (b), the molar ratio of compound 1, copper(I) iodide, sodium iodide, and N,N'-dimethylethylenediamine is 1:0.1:0.2:4.

[0020] In step (c), the molar ratio of compound 2, selenium powder, and KOH is 1:2:4.

[0021] In step (d), the molar ratio of compound 3, NaH, and 2-dimethylethanol is 1:6.8:1.1.

[0022] In step (e), the molar ratio of compound 4, DMAP, triethylamine, and CH3COCl is 1:0.1:1.5:1.5.

[0023] In step (f), the molar ratio of compound 5 and POCl3 is 1:3.6.

[0024] In step (g), the molar ratio of compound 6 and BBr3 is 1:5.3.

[0025] In step (h), the molar ratio of compound 7 and diethyl malonate is 1:1.2.

[0026] In step (i), the molar ratio of compound 8 and 4-sulfonylbenzoyl chloride is 1:4.

[0027] In the steps (a), (b), (c), (d), (e), (f), (g), (h), and (i), the eluents used in column chromatography are (pure hexane), (pure hexane), (Vhexane:Vdichloromethane = 3:7), (Vpetroleum ether:Vdichloromethane = 1:1), (Vpetroleum ether:Vdichloromethane = 2:1), (Vpetroleum ether:Vdichloromethane = 3:1), (Vpetroleum ether:Vdichloromethane = 2:1), (Vethyl acetate:Vdichloromethane = 1:15), (Vpetroleum ether:Vdichloromethane = 2:1).

[0028] The fluorescence probe test method of the present invention is as follows: The probe molecule is dissolved in acetonitrile / PBS (3:7, v / v, 10 mM, pH = 7.40) and tested at room temperature. It can qualitatively and quantitatively detect ClO - / GSH, and the specific implementation method is introduced in detail in the implementation examples.

[0029] The response mechanism of the fluorescence probe of the present invention is as follows: For the fluorescence probe PSeZ-Cou-Golgi, in the presence of ClO-, the selenium (Se) atom in the PSeZ part is oxidized to selenium oxide (Se=O), and the probe PSeZ-Cou-Golgi is transformed into PSeZ-Cou-Golgi oxide. Since the oxide of the PSeZ part is a weaker electron donor compared to PSeZ, PSeZ-Cou-Golgi oxide shows green fluorescence. In the presence of GSH, Se=O is reduced to Se, and PSeZ-Cou-Golgi oxide can be transformed back to PSeZ-Cu-Golgi. When the probe PSeZ-Cou-Golgi is continuously treated with ClO- and GSH, a red-green-red fluorescence change will be observed, and the detection of the ClO - / GSH redox cycle can be achieved.

[0030] The fluorescence probe parent body of the present invention exhibits red fluorescence. After complete response with ClO - , the red fluorescence at 644 nm weakens, and the fluorescence at 514 nm enhances, showing green fluorescence; after complete response with GSH, the green fluorescence at 514 nm weakens, and the fluorescence at 644 nm enhances. It shows that the fluorescence intensity ratio (I 514nm / I 644nm ) of the fluorescence probe PSeZ-Cu-Golgi has a good linear relationship with the ClO - / GSH concentration, achieving the effect of simultaneous response.

[0031] The probe molecule described in the present invention has good stability and biocompatibility, and can achieve highly selective and sensitive detection of the dynamic changes of the ClO - / GSH-mediated redox state in Golgi oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 1H NMR spectrum of the fluorescent probe PSeZ-Cu-Golgi of the present invention in deuterated DMSO. The chemical shift is on the abscissa and the intensity is on the ordinate.

[0033] Figure 2 13C NMR spectrum of the fluorescent probe PSeZ-Cu-Golgi of the present invention in deuterated DMSO. The chemical shift is on the abscissa and the intensity is on the ordinate.

[0034] Figure 3 UV absorption spectra of the fluorescent probe PSeZ-Cu-Golgi (10 μM) of the present invention in PBS buffer (0.01 M, pH = 7.4, 30% CH3CN) before and after reacting with 500 μM of ClO - and 600 μM of GSH. The wavelength is on the abscissa and the fluorescence intensity ratio is on the ordinate.

[0035] Figure 4 Fluorescence spectral changes of the fluorescent probe PSeZ-Cu-Golgi (10 μM) of the present invention in PBS buffer (0.01 M, pH = 7.4, 30% CH3CN) before and after reacting with 500 μM of ClO - and 600 μM of GSH. The wavelength is on the abscissa and the absorbance or fluorescence intensity is on the ordinate.

[0036] Figure 5 Fluorescence spectral changes of the fluorescent probe PSeZ-Cu-Golgi (10 μM) of the present invention in PBS buffer (0.01 M, pH = 7.4, 30% CH3CN) after reacting with related substances (ClO - 、ONOO - 、H2O2、KO2、TBHP、ROO·、HO·、 1 O2、Cl - 、NO 3- 、CO3 2- 、K + 、Na + 、Mg 2+ 、Ca 2+ 、S 2- 、Cys, Hcy, GSH. The wavelength is on the abscissa and the fluorescence intensity is on the ordinate.

[0037] Figure 6The MTT experiment of the fluorescent probe PSeZ-Cu-Golgi of the present invention in PBS buffer (0.01 M, pH = 7.4, 30% CH3CN) in HeLa cells. The abscissa is the concentration, and the ordinate is the cell survival rate after incubating with the probe PSeZ-CouGolgi at different concentrations for 24 hours.

[0038] Figure 7 It is the HRMS spectrum of the fluorescent probe PSeZ-Cu-Golgi. The abscissa is the mass-to-charge ratio, and the ordinate is the ion intensity.

[0039] Figure 8 It is the mechanism diagram of the response process of the fluorescent probe of this application.

[0040] Specific implementation examples

[0041] Example 1: Synthesis of Compound 1

[0042] Dissolve o-bromoaniline and 1-bromo-2-iodo-4-methoxybenzene in toluene, add tris(dibenzylideneacetone)dipalladium(0), 1,1'-bis(diphenylphosphino)ferrocene, and sodium tert-butoxide, and reflux and stir at 120 °C for 18 h. After cooling to room temperature, extract with ethyl acetate. Wash with saturated brine and dry with anhydrous sodium sulfate. Rotate and evaporate under reduced pressure to obtain the crude product, and separate it by column chromatography to obtain a colorless oily compound 1 with a yield of 70.5%. The structural characterization of the molecule is as follows: 1 HNMR(400MHz,DMSO-d6)δ(ppm)7.64(dd,J=8.0,1.6Hz,1H),7.50(d,J=8.3Hz,1H),7.31(t,J=7.7Hz,1H),7.12(dd,J=8.1,1.7Hz,1H),6.99(s,1H),6.95(t,J=7.7Hz,1H),6.56-6.53(m,2H),3.68(s,3H). 13 CNMR(100MHz,DMSO-d6)δ(ppm)159.4(s,),141.4(s,),140.2(s,),133.4(s,),133.1(s,),128.6(s,),123.6(s,),120.8(s,),115.1(s,),108.6(s,),105.3(s,),104.8(s,),55.3(s,).HRMS(ESI)m / z calculated C 13 H 12 NOBr2[M+H] + The molecular weight of is 355.9280, and the corresponding value is found at 355.9282.

[0043] Example 2: Synthesis of Compound 2

[0044] Compound 1, cuprous iodide, sodium iodide, and N,N'-dimethylethylenediamine were dissolved in anhydrous dioxane and stirred at 110 °C for 24 hours. After cooling to room temperature, an aqueous ammonia solution was added, and the mixture was extracted with DCM and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a crude product, which was separated by column chromatography to obtain a brown oily compound 2 with a yield of 86.3%. The structural characterization of the molecule is as follows: 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 7.85 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 6.78 (t, J = 7.7 Hz, 1H), 6.59 (s, 1H), 6.53 (d, J = 2.8 Hz, 1H), 6.42 (dd, J = 8.7, 2.9 Hz, 1H), 3.67 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ (ppm) 160.3 (s), 144.3 (s), 143.1 (s), 139.6 (s), 139.5 (s), 129.3 (s), 124.0 (s), 119.7 (s), 109.5 (s), 104.6 (s), 92.3 (s), 79.8 (s), 55.2 (s). HRMS (ESI) m / z calculated for C 13 H 12 NOI2 [M+H] + is 451.9003, and the corresponding value was found at 451.8993.

[0045] Example 3: Synthesis of Compound 3

[0046] Compound 2, Se powder, and KOH were dissolved in anhydrous DMSO and reacted at 110 °C for 24 hours. After cooling to room temperature, the mixture was extracted with DCM, washed with a saturated NH4Cl solution, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a crude product, which was separated by column chromatography to obtain a yellow solid compound 3 with a yield of 86.3%. The structural characterization of the molecule is as follows: 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 8.58 (s, 1H), 7.09 (d, J = 7.7 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.98 (dd, J = 8.2, 1.5 Hz, 1H), 6.76 (dt, J = 6.9, 3.1 Hz, 2H), 6.44–6.39 (m, 2H), 3.68 (s, 3H) 1313C NMR (100 MHz, DMSO-d6) δ (ppm) 159.5 (s), 143.2 (s), 141.9 (s), 129.3 (s), 128.8 (s), 127.6 (s), 122.1 (s), 115.2 (s), 111.9 (s), 107.9 (s), 101.7 (s), 101.3 (s), 55.0 (s). HRMS (ESI) m / z calculated for C 13 H 12 NOSe ([M+H] + ) is 278.0079, and the corresponding value was found at 278.0082.

[0047] Example 4: Synthesis of Compound 4

[0048] Compound 3 was dissolved in anhydrous DMF, and NaH was slowly added. The reaction was carried out at 0 °C for 30 minutes, then 2-dimethylethanol was added, and the mixture was stirred at room temperature for 5 hours. After cooling to room temperature, the reaction solution was poured into water to quench, and extracted three times with dichloromethane. After drying with anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain a crude product, which was further separated by column chromatography to obtain Compound 4 with a yield of 75%. The structural characterization of the molecule is as follows: 1 1H NMR (400 MHz, CDCl3) δ 7.40 (dd, J = 7.6, 1.5 Hz, 1H), 7.29 (d, J = 3.1 Hz, 1H), 7.25–7.21 (m, 1H), 7.04 (dd, J = 8.1, 0.9 Hz, 1H), 7.00 (td, J = 7.5, 1.2 Hz, 1H), 6.65 (d, J = 2.5 Hz, 1H), 6.60 (dd, J = 8.4, 2.5 Hz, 1H), 4.15 (t, J = 5.2 Hz, 2H), 3.85 (d, J = 5.3 Hz, 2H), 3.81 (s, 3H). 13 13C NMR (100 MHz, CDCl3) δ 160.1, 146.6, 145.1, 130.4, 130.2, 127.6, 124.2, 123.6, 117.6, 114.1, 108.7, 105.1, 58.6, 55.6, 51.0 HRMS (ESI) m / z calculated for C 15 H 16 NO2Se [M+H] + : 322.0341, and the corresponding value was found at 322.0223.

[0049] Example 5: Synthesis of Compound 5

[0050] Compound 4, DMAP, and triethylamine were dissolved in anhydrous DCM, and CH3COCl was added dropwise. The reaction was carried out at room temperature for 12 hours, cooled to room temperature, and the reaction solution was poured into H2O. It was extracted three times with DCM, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography to obtain compound 5 with a yield of 76.1%. The structural characterization of the molecule is as follows: 1H NMR (400 MHz, CDCl3) δ 7.35 (dd, J = 7.6, 1.4 Hz, 1H), 7.24–7.17 (m, 2H), 6.99 (d, J = 8.1 Hz, 1H), 6.95 (td, J = 7.5, 0.9 Hz, 1H), 6.62 (d, J = 2.5 Hz, 1H), 6.55 (dd, J = 8.4, 2.5 Hz, 1H), 4.37 (t, J = 6.4 Hz, 2H), 4.17 (t, J = 6.4 Hz, 2H), 3.79 (s, 3H), 2.04 (s, 4H). 13C NMR (100 MHz, CDCl3) δ 171.0, 160.1, 146.4, 145.1, 130.4, 130.2, 127.6, 123.4, 123.3, 117.0, 113.1, 108.5, 104.5, 61.1, 55.6, 47.1, 20.9. HRMS (ESI) m / z calcd for C 17 H 17 NNaO3Se [M+Na] + : 386.0266, found at 386.0147.

[0051] Example 6: Synthesis of Compound 6

[0052] POCl3 was added dropwise to anhydrous DMF, and the reaction was carried out at 0 °C for 30 minutes. Then compound 5 was dissolved in anhydrous DMF and added to the above mixture. The reaction was carried out at 80 °C for 6 hours, cooled to room temperature, and the reaction solution was quenched by pouring it into water. It was neutralized with saturated aqueous NaHCO3, extracted three times with DCM, and dried over anhydrous Na2SO4. It was concentrated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography to obtain compound 6 with a yield of 68.8%. The structural characterization of the molecule is as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 7.60 (s, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.04 (t, J = 7.3 Hz, 1H), 6.89 (s, 1H), 4.35 (d, J = 5.1 Hz, 2H), 4.30 (d, J = 5.0 Hz, 2H), 3.97 (s, 3H), 1.95 (s, 3H). 1313C NMR (100 MHz, DMSO-d6) δ 187.1, 170.7, 163.0, 152.8, 143.5, 130.5, 129.2, 128.4, 124.7, 121.7, 120.4, 118.7, 112.4, 102.2, 60.7, 56.7, 47.7, 21.1. HRMS (ESI) m / z calcd for C 18 H 18 NO4Se [M+H] + : 392.0396, found 392.0455.

[0053] Example 7: Synthesis of Compound 7

[0054] At -78 °C under argon protection, compound 6 was dissolved in anhydrous DCM, and BBr3 was added. The reaction was carried out at room temperature for 12 h. The reaction mixture was poured into water to quench. It was neutralized with saturated aqueous NaHCO3 solution and extracted three times with DCM. After drying over anhydrous Na2SO4, the solvent was removed under reduced pressure to obtain the crude product, which was separated by column chromatography to obtain compound 7 with a yield of 36%. The structural characterization of the molecule is as follows: 1 1H NMR (400 MHz, CDCl3) δ 11.26 (s, 1H), 9.69 (s, 1H), 7.45 (s, 1H), 7.42–7.36 (m, 2H), 7.05 (d, J = 7.8 Hz, 2H), 6.58 (s, 1H), 4.18 (t, J = 5.3 Hz, 2H), 3.87 (t, J = 5.1 Hz, 2H). 13 13C NMR (100 MHz, CDCl3) δ 192.9, 192.8, 162.0, 152.7, 142.0, 133.4, 129.4, 127.0, 123.7, 121.4, 117.2, 116.3, 111.2, 104.37, 103.87, 57.70, 50.58. HRMS (ESI) m / z calcd for C 15 H 14 NO3Se [M+H] + : 336.0133, found 336.0088.

[0055] Example 8: Synthesis of Compound 8

[0056] Compound 7, diethyl malonate and piperidine were dissolved in EtOH and refluxed for 6 h. The solvent was removed by distillation under reduced pressure to obtain the crude product, which was separated by column chromatography to obtain compound 8 with a yield of 68.3%. The structural characterization of the molecule is as follows: 11H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.84 (s, 1H), 7.39 (dd, J = 7.6, 1.3 Hz, 1H), 7.32–7.25 (m, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H), 7.05 (t, J = 7.1 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 4.09 (t, J = 5.9 Hz, 2H), 3.70 (t, J = 5.7 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H). 13 13C NMR (100 MHz, DMSO-d6) δ 163.3, 156.7, 156.5, 151.4, 148.4, 143.3, 130.5, 130.4, 128.7, 124.8, 120.0, 118.6, 116.8, 113.9, 113.8, 104.0, 61.4, 58.4, 52.2, 14.6. HRMS (ESI) m / z calcd for C 20 H 17 NNaO5Se [M+Na] + : 454.0164, found 454.0036.

[0057] Example 9: Synthesis of Probe Molecule PSeZ-Cou-Golgi

[0058] Compound 8 was dissolved in anhydrous THF, 4-sulfonylbenzoyl chloride and Et3N were added, and the reaction was carried out at room temperature for 6 hours. The solvent was removed by distillation under reduced pressure to obtain a crude product, which was separated by column chromatography to obtain probe PSeZ-Cou-Golgi with a yield of 38.5%. The structural characterization of the molecule is as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 5.0 Hz, 2H), 7.52 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.34 (s, 3H), 7.10–7.05 (m, 1H), 4.56 (d, J = 18.5 Hz, 3H), 4.27 (d, J = 7.0 Hz, 2H), 1.30 (t, J = 6.8 Hz, 3H). 1313C NMR (100 MHz, DMSO-d6) δ 165.0, 163.2, 156.7, 156.5, 151.3, 148.5, 148.5, 143.4, 132.7, 130.8, 130.7, 130.3, 128.8, 126.4, 125.0, 121.2, 119.2, 117.9, 114.3, 114.2, 104.8, 62.0, 61.5, 14.6. HRMS (ESI) m / z calculated for C 27 H 21 N2O8SSe [M-H] - : 613.0189, found at 613.0168.

[0059] Example 10: Study on the Detection Response of Probe PSeZ-Cou-Golgi

[0060] The probe was dissolved in the test solution CH3CN / PBS (3:7, v / v, 10 mM, pH = 7.40) to prepare a 1.0×10 - 3 mol / L solution, and the corresponding test solution was added to test the changes in its UV absorption spectrum and fluorescence spectrum over time. For the fluorescence spectrum, after adding ClO - molecules, a rapid increase in fluorescence intensity at 514 nm and a decrease in fluorescence intensity at 644 nm could be observed. After adding GSH, the fluorescence at 514 nm decreased, and red fluorescence appeared at 644 nm with an increase in intensity. Its detection time was short, and qualitative, quantitative, and reversible detection of ClO - and GSH could be achieved.

[0061] Example 11: Selectivity Experiment of Probe PSeZ-Cou-Golgi

[0062] The probe was dissolved in the test solution CH3CN / PBS (3:7, v / v, 10 mM, pH = 7.40) to prepare a 1.0×10 - 3 mol / L solution, and 500 μM ONOO - , H2O2, KO2, TBHP, ROO·, HO·, 1 O2, Cl - , NO 3- , CO3 2- , K + , Na + , Mg 2+ , Ca 2+ , S 2- , Cys, Hcy did not cause any change in fluorescence, while the addition of ClO -, GSH produced a good linear relationship with the ratio fluorescence signal, Figure 5 indicating that this single-wavelength excitation dual-response probe has high selectivity for both ClO - and GSH. It can be seen that the fluorescence probe of the present invention has good detection performance, which confirms the applicability of the probe.

Claims

1. A bifunctional, reversible, quantitative, and Golgi-targeted fluorescent probe for real-time monitoring of intracellular redox homeostasis kinetics, with the structure as follows:

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