Preparation method and application of fluorescent probe for monitoring redox process

Abstract

The present invention discloses a preparation method and application of a fluorescent probe for monitoring redox processes, and belongs to the technical field of organic small molecule fluorescent probes. The fluorescent probe provided by the present invention is shown in formula (I), which is based on a Rhodol group as a parent, introduces a strong electron-withdrawing group F atom on the bottom ring, and introduces a phenyl selenium group by utilizing the high activity of the fluorine atom on the 4th position of the bottom ring. The redox potential of the selenium atom is used to design and synthesize a fluorescent probe that can detect ClO ‑ / GSH redox process probe. The redox fluorescent probe of the present invention can realize the exogenous ClO ‑ / GSH redox process detection; and ClO ‑ / GSH has specific response, high sensitivity and good optical stability; at the same time, the preparation yield of the probe provided by the present invention is high. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the technical field of molecular fluorescent probes, and in particular relates to a preparation method and application of a fluorescent probe for monitoring redox processes. Background Art

[0002] The dynamic balance of redox is essential for aerobic life to maintain normal life activities. Aerobic respiration produces highly oxidative reactive oxygen species (ROS) while providing energy to the body. As an important member of the reactive oxygen family, HClO plays an important role in the immune response of organisms. Abnormal levels of HClO can cause many immune-related diseases, such as diabetes and cancer. The redox balance of reactive oxidative species and reducing substances can maintain and regulate the normal physiological activities of cells. When cells are in an abnormal state, reducing substances cannot effectively consume excessive ROS, resulting in redox imbalance, further leading to abnormal cell function and ultimately causing various diseases. The chemical fluorescent probe for detecting the redox state has a recognition group that can bind to the signal molecule and a group that emits a fluorescent signal. The redox state is reflected by the intensity of the emitted fluorescence. Therefore, real-time monitoring of the dynamic balance between intracellular ROS and reducing substances can help us understand and regulate the intracellular redox state.

[0003] In recent years, fluorescence imaging and fluorescent probe technology have been widely used in the development of bioactive species detection due to its high sensitivity, high temporal and spatial resolution, real-time in situ and non-invasive detection. There are many fluorescent probes that can detect oxidative species or reductive species, but there are not many probes that can dynamically detect redox processes. Therefore, the development of a simple and effective fluorescent probe that can be used to dynamically monitor the redox process in cells has important research significance and practical value. Summary of the invention

[0004] The purpose of the present invention is to solve the problem of real-time monitoring of the dynamic balance between intracellular reactive oxygen species (ROS) and reducing substances, and to provide a preparation method and application of a fluorescent probe for monitoring the redox process.

[0005] Technical solution of the present invention

[0006] To achieve the above object, the present invention first provides a fluorescent probe for monitoring the redox process. The chemical name of the probe is 2-(6-(diethylamino-3-oxo-3H-xanthene-9-yl)-3,5,6-trifluoro-4-((3-phenylselenoyl)propylamino)benzoic acid, referred to as Rhodol-Se, and the structure is shown in formula (I):

[0007]

[0008] The probe can monitor ClO - / GSH redox process of the redox pair.

[0009] The present invention also provides a method for preparing the fluorescent probe for monitoring the redox process. The raw materials used in the method are easily available, the synthesis steps are simple and the yield is high. The specific synthesis route is as follows:

[0010]

[0011] The synthesis steps are as follows:

[0012] S1, reacting 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid and resorcinol in methanesulfonic acid to obtain Rhodol-F;

[0013] S2, diphenyl diselenide and sodium hydroxide are placed in an organic solvent for reaction, sodium borohydride NaBH4 is then added for reaction, N-(3-bromopropyl)phthalimide is added to the reaction mixture for further reaction to finally obtain the compound shown in product Z1;

[0014] S3, hydrolyzing compound Z1 and hydrazine hydrate in an organic solvent to obtain a compound represented by product Z2;

[0015] S4, subjecting the Rhodol-F obtained in step S1 to a substitution reaction with Z2 in an organic solvent to obtain a compound Rhodol-Se as shown in formula I.

[0016] As a partial embodiment, in S1, the molar ratio of the 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid and resorcinol is 1:1.1-1.5, preferably 1:1.3; if the addition ratio of resorcinol is small, the reaction time will be long and by-products will be easily generated. If the addition ratio of resorcinol is large, the reaction cost will increase. The weight ratio of the total weight of the 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid and resorcinol to the methanesulfonic acid solvent is 1:20-30. The reaction temperature is preferably 60°C to 95°C.

[0017] As a partial implementation mode, in S2, the molar ratio of diphenyl diselenide, sodium hydroxide, sodium borohydride and N-(3-bromopropyl)phthalimide is 1:1.5~3:1~2.5:1.5~2, preferably 1:2.5:2:2; the weight ratio of the total weight of diphenyl diselenide, sodium hydroxide, sodium borohydride and N-(3-bromopropyl)phthalimide to the organic solvent is 1:9~15; the organic solvent is an alcohol organic solvent, preferably ethanol; the reaction temperature is preferably 25°C~50°C.

[0018] As a partial implementation, in S3, the molar ratio of the compound Z1 to hydrazine hydrate is 1:2-4.5, preferably 1:4.1; the weight ratio of the total weight of the compound Z1 and hydrazine hydrate to the ethanol solvent is 1:1.5-10; the organic solvent for the hydrolysis reaction is an alcohol organic solvent, preferably ethanol; the reaction temperature is preferably 25°C-50°C.

[0019] Since -NH2 and -Se exist in the compound, both of which are easily oxidized, the temperature should not be too high.

[0020] As some embodiments, in the S4 substitution reaction, the molar ratio of Rhodol-F to Z2 is 1:1 to 1.5, preferably 1:1.2; the weight ratio of the total weight of Rhodol-F and Z2 to the DMF solvent is 1:5 to 9. Since DMF has a high boiling point and is not easy to remove, it is not suitable to use a large amount; the reaction temperature is preferably 20°C to 50°C, and the reaction time is 18 to 30 hours.

[0021] As some implementation methods, in S1-S4, silica gel column chromatography is preferably used for purification.

[0022] The present invention also provides application of the fluorescent probe in detecting the cellular redox process.

[0023] Advantages and beneficial effects of the present invention:

[0024] The fluorescent probe provided by the present invention has a high yield and can dynamically monitor ClO - / GSH redox process can be achieved in cells with endogenous and exogenous ClO - / GSH detection, ClO - The redox pair of γ-GSH has a specific response, high sensitivity, low signal-to-noise ratio, and good optical stability. The maximum absorption wavelength of the probe of the present invention is about 530nm, and the maximum emission wavelength is 575nm ( Figure 2 ), the recognition mechanism of the probe for ClO- / GSH is intramolecular induced electron transfer (PET), which has high sensitivity (detection limit less than 10 -8 M / L). Moreover, the probe of the present invention has good biomembrane permeability and low cytotoxicity, and can achieve ClO - / Detection of GSH redox process( Figure 3 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The probe is at different concentrations of ClO - Fluorescence emission spectrum (the horizontal axis is the emission wavelength and the vertical axis is the fluorescence intensity).

[0026] Figure 2The probe adds ClO - The fluorescence spectra of the probe after oxidation to different concentrations of antioxidant GSH (the horizontal axis is the emission wavelength, and the vertical axis is the fluorescence intensity).

[0027] Figure 3 The probe Rhodol-Se is a probe for exogenous ClO - / GSH redox process cell imaging application. DETAILED DESCRIPTION

[0028] The following detailed descriptions are all illustrative and are intended to provide further explanation of the application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs.

[0029] It should be noted that the terms used herein are only for describing specific implementation methods and are not intended to limit the example implementations according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The structural feature of the fluorescent probe for monitoring the redox process provided by the present invention is that Rhodol is used as the parent structure, a halogen atom F with strong electron withdrawal is introduced on the bottom ring, and the F atom at the 4th position on the bottom ring has high reactivity, so a phenyl selenium group is introduced to design and synthesize the probe Rhodol-Se.

[0032] The present invention also relates to the application of the probe for monitoring ClO in HeLa cells. - / GSH redox process; the living biological sample includes living cells or living tissues, preferably living HeLa cells.

[0033] The preparation method of a fluorescent probe for detecting redox processes and the detection mechanism of redox processes in applications described in the present invention are as follows: Se atoms have redox potentials and are easily oxidized by active oxygen. This feature is utilized to introduce them into a fluorophore receptor, and the "off-on" of fluorescence is controlled by a PET mechanism. The electron-donating group on Se can be transferred to the fluorescent receptor, and a PET process occurs, thereby quenching fluorescence. When Se is oxidized to Se=O, the PET mechanism is blocked, and fluorescence is turned on.

[0034] In order to better understand the technical solution of the present invention, it is further described in detail below through specific embodiments.

[0035] Example 1: Preparation of fluorescent probe for monitoring redox processes

[0036] S1. 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid (3 g, 7.8 mmol) and resorcinol (1.1 g, 10 mmol) were placed in a 100 mL round-bottom flask, 50 mL of methanesulfonic acid was added, and the mixture was stirred at 90°C for 24 hours. The reaction was stopped, the reaction solution was cooled to room temperature, poured into 100 mL of ice water, and the pH value of the reaction solution was adjusted to neutral with NaHCO3, extracted with dichloromethane three times (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and then spin-dried to collect the crude product. The crude product was recrystallized with ethyl acetate to obtain 3.10 g of pink solid Rhodol-F with a yield of 85%. Mp179-181°C. ESI: m / z[M+H] + =460.1184; calculated value: 460.1094. 1 HNMR(CDCl3,400MHz,ppm):7.34(d,J=12Hz,1H),7.28(d,J=8.8Hz,1H),7.22(s,1H),7.13(d, J=9.6Hz,1H),7.07(d,J=8.8Hz,1H),6.99(s,1H),3.50(q,J=6.8Hz,4H)1.38(t,J=6.8Hz,6H); 13 C NMR (CDCl3, 100MHz, ppm): δ185.71,164.45,159.69,157.42,149.51,147.91,145.22,144.41, 141.32,135.46,134.38,128.51,128.32,116.54,113.43,105.67,95.34,92.76,47.11,12.78.

[0037] S2, Synthesis and Characterization of Intermediate N-(3-phenylseleno)propyl)phthalimide Z1

[0038] Diphenyl diselenide (3.139 g, 10 mmol) and sodium hydroxide (1.0 g, 25 mmol) were placed in a 250 mL round-bottom flask, 100 mL of ethanol was added, and NaBH4 (756 mg, 20 mmol) was slowly added to the round-bottom flask. The suspension was stirred for one hour under argon atmosphere at room temperature, and the next step was carried out without any treatment. N-(3-bromopropyl)phthalimide (5.34 g, 20 mmol) was added to the reaction mixture, and stirred overnight at 50 ° C. After the reaction was completed, the reaction mixture was poured into 250 mL of ice water, neutralized to neutrality with NaHCO3, and extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried with anhydrous Na2SO4 and then spin-dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 3.60 g of white solid powder Z1 with a yield of 52%. Mp98-100℃. ESI: m / z[M+O+2H] + =364.0452; calculated value: 364.0268. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.96 (dd, J=7.6Hz, 0.8Hz, 1H), 7.55-7.46 (m, 4H), 7.39 (d, J=7.6Hz,1H),7.28-7.22(m,3H),3.50(t,J=6.4Hz,2H),3.00(t,J=7.2Hz,2H),2.03-1.98(m,2H); 13 C NMR(100MHz,CDCl3-CD3OD,5:1,v / v):171.01,168.82,137.74,132.63,131 .80,130.4,129.85,129.77,129.15,127.53,126.96,39.71,29.46,24.90.

[0039] S3, Synthesis and Characterization of Intermediate 3-Phenylseleno-1-propylamine Z2

[0040] Compound Z1 (3.5 g, 10 mmol) and hydrazine hydrate (2 mL, 41 mmol) were placed in a 100 mL round-bottom flask, 10 mL of anhydrous ethanol was added, and the mixture was reacted overnight under an argon atmosphere. After the reaction, the reaction mixture was cooled to room temperature, a white solid was precipitated, and the filtrate was dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 1.45 g of white solid powder Z2, with a yield of 67%, Mp110-112°C. ESI: m / z[M+H] + =216.0300; calculated value: 216.0213. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.49 (d, J = 7.2Hz, 2H), 7.29-7.22 (m, 3H), 2.95 (t, J = 7.2Hz, 2H), 2.86 (t, J = 6.8Hz, 2H), 1.92-1.86 (m, 2H); 13 C NMR (100MHz, CDCl3-CD3OD, 5:1, v / v): 132.67, 129.14, 129.10, 127.07, 39.88, 29.28, 24.19.

[0041] S4. Synthesis and characterization of probe Rhodol-Se

[0042] Rhodol-F (150 mg, 0.33 mmol) and Z2 (85 mg, 0.4 mmol) were placed in a 50 mL round-bottom flask, added to 2 mL DMF, stirred at 40 ° C for 24 h, and after the reaction was completed, poured into 100 mL ice water, extracted with dichloromethane (30 mL × 3), combined organic phases were dried over anhydrous Na2SO4 and then spin-dried, and the crude product was separated by silica gel column chromatography (developing solvent: dichloromethane / ethyl acetate, v / v, 100 / 0-100 / 30) to obtain 141 mg pink solid powder Rhodol-Se, with a yield of 65.5%. Mp193-195 ° C. ESI: m / z[M+H] + =655.1360; calculated value: 655.1245. 1H NMR(CDCl3,400MHz,ppm)7.50(s,2H),7.33-7.38(m,3H),7.27(s,2H),7.22(s,1H),7.04(t,J=11.2Hz,2H),6.92(s ,1H),3.69(d,J=6.8Hz,4H),3.63(t,J=6.2Hz,2H),2.98(t,J=6.8Hz,2H),2.06-2.03(m,2H),1.39(t,J=6.2Hz,6H); 13 C NMR(CDCl3,100MHz,ppm)168.68,159.82,159.42,158.66,158.22,157.8 0,157.38,157.15,156.81,132.60,131.52,130.41,129.48,129.13,127. 11,118.68,118.54,115.84,113.01,110.71,102.77,96.42,77.30,77.0 7,76.75,54.03,53.81,53.58,53.35,53.13,46.53,30.80,24.35,12.38.

[0043] Example 2

[0044] S1. 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid (3 g, 7.8 mmol) and resorcinol (990 mg, 9 mmol) were placed in a 100 mL round-bottom flask, 50 mL of methanesulfonic acid was added, and the mixture was stirred at 90°C for 24 hours. The reaction was stopped, the reaction solution was cooled to room temperature, poured into 100 mL of ice water, and the pH value of the reaction solution was adjusted to neutral with NaHCO3, extracted with dichloromethane three times (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and then spin-dried to collect the crude product. The crude product was recrystallized with ethyl acetate to obtain 2.74 g of pink solid Rhodol-F with a yield of 75%. Mp179-181°C. ESI: m / z[M+H] + =460.1184; calculated value: 460.1094. 1 H NMR (CDCl3, 400MHz, ppm): 7.34 (d, J = 12Hz, 1H), 7.28 (d, J = 8.8Hz, 1H), 7.22 (s, 1H), 7.13 (d, J =9.6Hz,1H),7.07(d,J=8.8Hz,1H),6.99(s,1H),3.50(q,J=6.8Hz,4H)1.38(t,J=6.8Hz,6H); 13C NMR (CDCl3, 100MHz, ppm): δ185.71,164.45,159.69,157.42,149.51,147.91,145.22,144.41, 141.32,135.46,134.38,128.51,128.32,116.54,113.43,105.67,95.34,92.76,47.11,12.78.

[0045] S2, Synthesis and Characterization of Intermediate N-(3-phenylseleno)propyl)phthalimide Z1

[0046] Diphenyl diselenide (3.139 g, 10 mmol) and sodium hydroxide (600 mg, 15 mmol) were placed in a 250 mL round-bottom flask, 100 mL of ethanol was added, and NaBH4 (567 mg, 15 mmol) was slowly added to the round-bottom flask. The suspension was stirred for one hour under argon atmosphere at room temperature, and the next step was carried out without any treatment. N-(3-bromopropyl)phthalimide (4.00 g, 15 mmol) was added to the reaction mixture, and stirred overnight at 50 ° C. After the reaction was completed, the reaction mixture was poured into 250 mL of ice water, neutralized to neutrality with NaHCO3, and extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried with anhydrous Na2SO4 and then spin-dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 3.12 g of white solid powder Z1 with a yield of 45%. Mp98-100℃. ESI: m / z[M+O+2H] + =364.0452; calculated value: 364.0268. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.96 (dd, J=7.6Hz, 0.8Hz, 1H), 7.55-7.46 (m, 4H), 7.39 (d, J=7.6Hz,1H),7.28-7.22(m,3H),3.50(t,J=6.4Hz,2H),3.00(t,J=7.2Hz,2H),2.03-1.98(m,2H); 13 C NMR(100MHz,CDCl3-CD3OD,5:1,v / v):171.01,168.82,137.74,132.63,131 .80,130.4,129.85,129.77,129.15,127.53,126.96,39.71,29.46,24.90.

[0047] S3, Synthesis and Characterization of Intermediate 3-Phenylseleno-1-propylamine Z2

[0048] Compound Z1 (3.5 g, 10 mmol) and hydrazine hydrate (1.5 mL, 30 mmol) were placed in a 100 mL round-bottom flask, 10 mL of anhydrous ethanol was added, and the reaction was allowed to proceed overnight under an argon atmosphere. After the reaction was completed, the reaction mixture was cooled to room temperature, a white solid was precipitated, and the filtrate was dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 1.30 g of white solid powder Z2, with a yield of 60%, Mp110-112°C. ESI: m / z[M+H] + =216.0300; calculated value: 216.0213. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.49 (d, J = 7.2Hz, 2H), 7.29-7.22 (m, 3H), 2.95 (t, J = 7.2Hz, 2H), 2.86 (t, J = 6.8Hz, 2H), 1.92-1.86 (m, 2H); 13 C NMR (100MHz, CDCl3-CD3OD, 5:1, v / v): 132.67, 129.14, 129.10, 127.07, 39.88, 29.28, 24.19.

[0049] S4. Synthesis and characterization of probe Rhodol-Se

[0050] Rhodol-F (150 mg, 0.33 mmol) and Z2 (68 mg, 0.5 mmol) were placed in a 50 mL round-bottom flask, added to 2 mL DMF, stirred at room temperature for 24 h, poured into 100 mL ice water after the reaction, extracted with dichloromethane (30 mL × 3), combined organic phases were dried over anhydrous Na2SO4 and then spin-dried, the crude product was separated by silica gel column chromatography (developing solvent: dichloromethane / ethyl acetate, v / v, 100 / 0-100 / 30), and 129 mg pink solid powder Rhodol-Se was obtained, with a yield of 60%. Mp193-195 ° C. ESI: m / z [M+H] + =655.1360; calculated value: 655.1245. 1H NMR(CDCl3,400MHz,ppm)7.50(s,2H),7.33-7.38(m,3H),7.27(s,2H),7.22(s,1H),7.04(t,J=11.2Hz,2H),6.92(s ,1H),3.69(d,J=6.8Hz,4H),3.63(t,J=6.2Hz,2H),2.98(t,J=6.8Hz,2H),2.06-2.03(m,2H),1.39(t,J=6.2Hz,6H); 13 C NMR(CDCl3,100MHz,ppm)168.68,159.82,159.42,158.66,158.22,157.8 0,157.38,157.15,156.81,132.60,131.52,130.41,129.48,129.13,127. 11,118.68,118.54,115.84,113.01,110.71,102.77,96.42,77.30,77.0 7,76.75,54.03,53.81,53.58,53.35,53.13,46.53,30.80,24.35,12.38.

[0051] Example 3

[0052] S1. 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid (3 g, 7.8 mmol) and resorcinol (1.21 g, 11 mmol) were placed in a 100 mL round-bottom flask, 50 mL of methanesulfonic acid was added, and the mixture was stirred at 70°C for 24 hours. The reaction was stopped, the reaction solution was cooled to room temperature, poured into 100 mL of ice water, and the pH value of the reaction solution was adjusted to neutral with NaHCO3, extracted with dichloromethane three times (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and then spin-dried to collect the crude product. The crude product was recrystallized with ethyl acetate to obtain 2.55 g of pink solid Rhodol-F with a yield of 70%. Mp179-181°C. ESI: m / z[M+H] + =460.1184; calculated value: 460.1094. 1 H NMR (CDCl3, 400MHz, ppm): 7.34 (d, J = 12Hz, 1H), 7.28 (d, J = 8.8Hz, 1H), 7.22 (s, 1H), 7.13 (d, J =9.6Hz,1H),7.07(d,J=8.8Hz,1H),6.99(s,1H),3.50(q,J=6.8Hz,4H)1.38(t,J=6.8Hz,6H); 13C NMR (CDCl3, 100MHz, ppm): δ185.71,164.45,159.69,157.42,149.51,147.91,145.22,144.41, 141.32,135.46,134.38,128.51,128.32,116.54,113.43,105.67,95.34,92.76,47.11,12.78.

[0053] S2, Synthesis and Characterization of Intermediate N-(3-phenylseleno)propyl)phthalimide Z1

[0054] Diphenyl diselenide (3.139 g, 10 mmol) and sodium hydroxide (800 mg, 20 mmol) were placed in a 250 mL round-bottom flask, 100 mL of ethanol was added, and NaBH4 (945 mg, 25 mmol) was slowly added to the round-bottom flask. The suspension was stirred for one hour under argon atmosphere at room temperature, and the next step was carried out without any treatment. N-(3-bromopropyl)phthalimide (4.81 g, 18 mmol) was added to the reaction mixture, and stirred overnight at 50 ° C. After the reaction was completed, the reaction mixture was poured into 250 mL of ice water, neutralized to neutrality with NaHCO3, and extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried with anhydrous Na2SO4 and then spin-dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 3.32 g of white solid powder Z1 with a yield of 48%. Mp98-100℃. ESI: m / z[M+O+2H] + =364.0452; calculated value: 364.0268. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.96 (dd, J=7.6Hz, 0.8Hz, 1H), 7.55-7.46 (m, 4H), 7.39 (d, J=7.6Hz,1H),7.28-7.22(m,3H),3.50(t,J=6.4Hz,2H),3.00(t,J=7.2Hz,2H),2.03-1.98(m,2H); 13 C NMR(100MHz, CDCl3-CD3OD,5:1,v / v):171.01,168.82,137.74,132.63,131 .80,130.4,129.85,129.77,129.15,127.53,126.96,39.71,29.46,24.90.

[0055] S3, Synthesis and Characterization of Intermediate 3-Phenylseleno-1-propylamine Z2

[0056] Compound Z1 (3.5 g, 10 mmol) and hydrazine hydrate (1.7 mL, 35 mmol) were placed in a 100 mL round-bottom flask, 10 mL of anhydrous ethanol was added, and the mixture was reacted overnight under an argon atmosphere. After the reaction, the reaction mixture was cooled to room temperature, a white solid was precipitated, filtered, and the filtrate was spin-dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 1.34 g of white solid powder Z2, with a yield of 62%, Mp110-112°C. ESI: m / z[M+H] + =216.0300; calculated value: 216.0213. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.49 (d, J = 7.2Hz, 2H), 7.29-7.22 (m, 3H), 2.95 (t, J = 7.2Hz, 2H), 2.86 (t, J = 6.8Hz, 2H), 1.92-1.86 (m, 2H); 13 C NMR (100MHz, CDCl3-CD3OD, 5:1, v / v): 132.67, 129.14, 129.10, 127.07, 39.88, 29.28, 24.19.

[0057] S4. Synthesis and characterization of probe Rhodol-Se

[0058] Rhodol-F (150 mg, 0.33 mmol) and Z2 (91 mg, 0.43 mmol) were placed in a 50 mL round-bottom flask, added to 2 mL DMF, stirred at 40 ° C for 24 h, and after the reaction was completed, poured into 100 mL ice water, extracted with dichloromethane (30 mL × 3), combined organic phases were dried over anhydrous Na2SO4 and then spin-dried, and the crude product was separated by silica gel column chromatography (developing solvent: dichloromethane / ethyl acetate, v / v, 100 / 0-100 / 30) to obtain 136 mg pink solid powder Rhodol-Se, with a yield of 63%. Mp193-195 ° C. ESI: m / z[M+H] + =655.1360; calculated value: 655.1245. 1H NMR(CDCl3,400MHz,ppm)7.50(s,2H),7.33-7.38(m,3H),7.27(s,2H),7.22(s,1H),7.04(t,J=11.2Hz,2H),6.92(s ,1H),3.69(d,J=6.8Hz,4H),3.63(t,J=6.2Hz,2H),2.98(t,J=6.8Hz,2H),2.06-2.03(m,2H),1.39(t,J=6.2Hz,6H); 13 C NMR(CDCl3,100MHz,ppm)168.68,159.82,159.42,158.66,158.22,157.8 0,157.38,157.15,156.81,132.60,131.52,130.41,129.48,129.13,127. 11,118.68,118.54,115.84,113.01,110.71,102.77,96.42,77.30,77.0 7,76.75,54.03,53.81,53.58,53.35,53.13,46.53,30.80,24.35,12.38.

[0059] Example 4

[0060] S1. 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid (3 g, 7.8 mmol) and resorcinol (1.32 g, 12 mmol) were placed in a 100 mL round-bottom flask, 50 mL of methanesulfonic acid was added, and the mixture was stirred at 80°C for 24 hours. The reaction was stopped, the reaction solution was cooled to room temperature, poured into 100 mL of ice water, and the pH value of the reaction solution was adjusted to neutral with NaHCO3, extracted with dichloromethane three times (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and then spin-dried to collect the crude product. The crude product was recrystallized with ethyl acetate to obtain 2.92 g of pink solid Rhodol-F with a yield of 80%. Mp179-181°C. ESI: m / z[M+H] + =460.1184; calculated value: 460.1094. 1 H NMR (CDCl3, 400MHz, ppm): 7.34 (d, J = 12Hz, 1H), 7.28 (d, J = 8.8Hz, 1H), 7.22 (s, 1H), 7.13 (d, J =9.6Hz,1H),7.07(d,J=8.8Hz,1H),6.99(s,1H),3.50(q,J=6.8Hz,4H)1.38(t,J=6.8Hz,6H); 13C NMR (CDCl3, 100MHz, ppm): δ185.71,164.45,159.69,157.42,149.51,147.91,145.22,144.41, 141.32,135.46,134.38,128.51,128.32,116.54,113.43,105.67,95.34,92.76,47.11,12.78.

[0061] S2, Synthesis and Characterization of Intermediate N-(3-phenylseleno)propyl)phthalimide Z1

[0062] Diphenyl diselenide (3.139 g, 10 mmol) and sodium hydroxide (720 mg, 18 mmol) were placed in a 250 mL round-bottom flask, 100 mL of ethanol was added, and NaBH4 (756 mg, 20 mmol) was slowly added to the round-bottom flask. The suspension was stirred for one hour under an argon atmosphere at room temperature, and the next step was carried out without any treatment. N-(3-bromopropyl)phthalimide (4.27 g, 16 mmol) was added to the reaction mixture, and stirred overnight at 50 ° C. After the reaction was completed, the reaction mixture was poured into 250 mL of ice water, neutralized to neutrality with NaHCO3, and extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried with anhydrous Na2SO4 and then spin-dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 3.46 g of white solid powder Z1, with a yield of 50%. Mp98-100℃. ESI: m / z[M+O+2H] + =364.0452; calculated value: 364.0268. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.96 (dd, J=7.6Hz, 0.8Hz, 1H), 7.55-7.46 (m, 4H), 7.39 (d, J=7.6Hz,1H),7.28-7.22(m,3H),3.50(t,J=6.4Hz,2H),3.00(t,J=7.2Hz,2H),2.03-1.98(m,2H); 13 C NMR(100MHz,CDCl3-CD3OD,5:1,v / v):171.01,168.82,137.74,132.63,131 .80,130.4,129.85,129.77,129.15,127.53,126.96,39.71,29.46,24.90.

[0063] Synthesis and characterization of S3 and intermediate 3-phenylseleno-1-propylamine Z2

[0064] Compound Z1 (3.5 g, 10 mmol) and hydrazine hydrate (0.98 mL, 20 mmol) were placed in a 100 mL round-bottom flask, 10 mL of anhydrous ethanol was added, and the mixture was reacted overnight under an argon atmosphere. After the reaction, the reaction mixture was cooled to room temperature, a white solid was precipitated, filtered, and the filtrate was spin-dried to collect the crude product. The crude product was separated by silica gel column chromatography (developing solvent: ethyl acetate / ethanol, v / v, 100 / 0-100 / 25) to obtain 1.41 g of white solid powder Z2, with a yield of 65%, Mp110-112°C. ESI: m / z[M+H] + =216.0300; calculated value: 216.0213. 1 H NMR (400MHz, CDCl3-CD3OD, 5:1, v / v): 7.49 (d, J = 7.2Hz, 2H), 7.29-7.22 (m, 3H), 2.95 (t, J = 7.2Hz, 2H), 2.86 (t, J = 6.8Hz, 2H), 1.92-1.86 (m, 2H); 13 C NMR (100MHz, CDCl3-CD3OD, 5:1, v / v): 132.67, 129.14, 129.10, 127.07, 39.88, 29.28, 24.19.

[0065] S4. Synthesis and characterization of probe Rhodol-Se

[0066] Rhodol-F (150 mg, 0.33 mmol) and Z2 (98 mg, 0.46 mmol) were placed in a 50 mL round-bottom flask, added to 2 mL DMF, stirred at 30 ° C for 24 h, poured into 100 mL ice water after the reaction, extracted with dichloromethane (30 mL × 3), combined organic phases were dried over anhydrous Na2SO4 and then spin-dried, and the crude product was separated by silica gel column chromatography (developing solvent: dichloromethane / ethyl acetate, v / v, 100 / 0-100 / 30) to obtain 133 mg pink solid powder Rhodol-Se, with a yield of 62%. Mp193-195 ° C. ESI: m / z[M+H] + =655.1360; calculated value: 655.1245. 1H NMR(CDCl3,400MHz,ppm)7.50(s,2H),7.33-7.38(m,3H),7.27(s,2H),7.22(s,1H),7.04(t,J=11.2Hz,2H),6.92(s ,1H),3.69(d,J=6.8Hz,4H),3.63(t,J=6.2Hz,2H),2.98(t,J=6.8Hz,2H),2.06-2.03(m,2H),1.39(t,J=6.2Hz,6H); 13 C NMR(CDCl3,100MHz,ppm)168.68,159.82,159.42,158.66,158.22,157.8 0,157.38,157.15,156.81,132.60,131.52,130.41,129.48,129.13,127. 11,118.68,118.54,115.84,113.01,110.71,102.77,96.42,77.30,77.0 7,76.75,54.03,53.81,53.58,53.35,53.13,46.53,30.80,24.35,12.38.

[0067] Probe Rhodol-Se and addition of different concentrations of ClO - Fluorescence spectrum of

[0068] The Rhodol-Se prepared above was prepared in dimethyl sulfoxide (DMSO) to prepare 5×10 -3 The mother solution of M is ready for use;

[0069] Take 6 μL of 5×10 -3 mol / L standard parent solution was added to 3 mL of the detection system to prepare a main solution with a concentration of 10 μM. Different concentrations of ClO - The guest solution makes the ClO in the sample bottle - The concentration ratios of the probe Rhodol-Se were 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, and the mixture was shaken for fluorescence testing (excitation wavelength 500 nm, slit width 5 nm). The fluorescence intensity in each system was measured. Figure 1 As shown by Figure 1 It can be seen that with the increase of ClO - With the increase of concentration, the fluorescence intensity gradually increased.

[0070] Take 6 μL of 5×10 -3 mol / L standard parent solution was added to 3mL detection system to prepare a main solution with a concentration of 10μM. 3eq ClO- was added to the main solution and the solution was shaken. GSH was then added to make the concentration ratio of GSH to probe Rhodol-Se in the sample bottle to be 0.8, 1.4, 1.8, 2.2, 2.6, 3, 4, 5 respectively. The solution was shaken and the fluorescence test was performed (excitation wavelength 500nm, slit width 5nm). The fluorescence intensity in each system was measured, as shown in Fig. Figure 2 As shown by Figure 2 It can be seen that with the increase of GSH concentration, the fluorescence intensity gradually decreases.

[0071] The probe Rhodol-Se was used to detect exogenous ClO - Cellular imaging of GSH

[0072] HeLa cells of appropriate density were inoculated into a 6-well culture dish and cultured in a 37°C incubator containing 5% CO2. After the cells adhered to the wall, the fluorescent probe Rhodol-Se (concentration of 1 μM) was added and incubated for half an hour for fluorescence imaging (excitation wavelength 488 nm, collection band 520-620 nm). The imaging results are shown in Figure 3 shown.

[0073] Depend on Figure 3 It can be seen that Figure 3 In a1), live Hela cells were first treated with 1mM N-ethylmaleimide (NEM) to remove the biothiol substances contained in the cells, and then treated with the probe Rhodol-Se (1μM). The cells showed weak fluorescence in the green channel. Figure a2) is a cell imaging image under bright field. Figure a3) is an overlay of a1) and a2). In b1), live Hela cells were first treated with 1mM N-ethylmaleimide (NEM) to remove the biothiol substances contained in the cells, and then treated with 1μM Rhodol-Se and ClO - (2μM) was co-cultured for 30 minutes, and the fluorescence signal in the cells was significantly enhanced. Figure b2) is a cell imaging image under bright field, and Figure b3) is an overlay of b1) and b2). In Figure c1), 2μM GSH was added to the cells and co-cultured for 30 minutes on the basis of Figure b1), and the fluorescence signal of the green channel in the cells was significantly reduced. Figure d) is ClO added again - , and found that the green channel fluorescence signal in the cell was enhanced again.

Claims

1. A fluorescent probe for monitoring redox processes, the chemical name of which is 2-(6-(diethylamino-3-oxo-3H-xanthene-9-yl)-3,5,6-trifluoro-4-((3-phenylselenoyl)propylamino)benzoic acid, referred to as Rhodol-Se, which can monitor ClO - The redox process of the redox pair of GSH is shown in the following formula (I):

2. A method for preparing a fluorescent probe for monitoring redox processes according to claim 1, characterized in that: The specific preparation route is as follows: The synthesis steps are as follows: S1, adding 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid and resorcinol into a container containing methanesulfonic acid to react and obtain Rhodol-F; S2, dissolving diphenyl diselenide and sodium hydroxide in an organic solvent, and slowly adding NaBH4 to the mixture; then adding N-(3-bromopropyl)phthalimide to the reaction mixture to continue the reaction, to obtain the compound shown in Z1; S3, dissolving compound Z1 and hydrazine hydrate in an organic solvent for hydrolysis reaction to obtain a compound represented by Z2; S4, subjecting Rhodol-F and Z2 to a substitution reaction in an organic solvent to obtain the target product Rhodol-Se.

3. The preparation method according to claim 2, characterized in that: The molar ratio of 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid to resorcinol in step S1 is 1:1.1-1.

5.

4. The preparation method according to claim 3, characterized in that: The molar ratio of 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid to resorcinol is 1:1.

3.

5. The preparation method according to claim 2, characterized in that The weight ratio of the total weight of 2-(4-(diethylamino-2-hydroxybenzoyl)-tetrafluorobenzoic acid and resorcinol to methanesulfonic acid in step S1 is 1:20-30; and the reaction temperature is 60°C-95°C.

6. The preparation method according to claim 2, characterized in that The molar ratio of diphenyl diselenide, sodium hydroxide, sodium borohydride and N-(3-bromopropyl)phthalimide described in step S2 is 1:1.5-3:1-2.5:1.5-2.

7. The preparation method according to claim 6, characterized in that: The molar ratio of diphenyl diselenide, sodium hydroxide, sodium borohydride and N-(3-bromopropyl)phthalimide is 1:2.5:2:

2.

8. The preparation method according to claim 2, characterized in that The weight ratio of the total weight of diphenyl diselenide, sodium hydroxide, sodium borohydride and N-(3-bromopropyl)phthalimide to the organic solvent in step S2 is 1:9-15; the organic solvent is an alcohol organic solvent; and the reaction temperature is 25°C-50°C.

9. The preparation method according to claim 2, characterized in that The molar ratio of the compound Z1 and hydrazine hydrate in step S3 is 1:2-4.5; the weight ratio of the total weight of the compound Z1 and hydrazine hydrate to the organic solvent is 1:1.5-10.

10. The preparation method according to claim 9, characterized in that The molar ratio of the compound Z1 to hydrazine hydrate is 1:4.

1.

11. The preparation method according to claim 2, characterized in that In step S3, the organic solvent for the hydrolysis reaction is an alcohol organic solvent; the reaction temperature is 25°C to 50°C.

12. The preparation method according to claim 2, characterized in that The molar ratio of the compound Rhodol-F to Z2 in step S4 is 1:1-1.5; the substitution reaction temperature in S4 is 20-50°C and the time is 18-30 hours.

13. The preparation method according to claim 12, characterized in that The molar ratio of the compound Rhodol-F to Z2 is 1:1.2.

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