Cell membrane dual-targeting glutathione activated fluorescent probe, preparation and application

Abstract

The invention belongs to the technical field of optical imaging and biosensing, and particularly relates to a cell membrane dual-targeting glutathione activated fluorescent probe and preparation and application thereof.The cell membrane dual-targeting glutathione activated fluorescent probe is characterized in that 2, 4-dinitrobenzenesulfonyl is adopted as a GSH recognition group in the fluorescent probe, in the presence of GSH, the fluorescent probe and GSH are subjected to an aromatic nucleophilic reaction to induce splitting decomposition of the 2, 4-dinitrobenzenesulfonyl, and the cell membrane dual-targeting glutathione activated fluorescent probe is obtained; a fluorophore PM-Red is released, so that the detection of GSH is realized; when the probe is designed, the propanesulfonic acid anion is used as a targeting part of a cell membrane, and decane with a hydrophobic effect is added, so that the fluorescent probe is difficult to dissolve in cell sap of cells and can be deposited on the cell membrane, and finally, accurate targeting detection of endogenous and exogenous GSH of the cell membrane of the living cells is realized.

Description

Technical Field

[0001] The invention belongs to the field of optical imaging and biosensor technology, and specifically relates to a cell membrane dual-targeted glutathione activated fluorescent probe, and its preparation and application. Background Art

[0002] Glutathione (GSH) is a key signaling molecule that regulates cellular physiological activities in organisms. Abnormal expression of GSH in living organisms is often associated with a variety of diseases. It has been found that cardiovascular disease, Alzheimer's disease, cancer and other diseases that seriously threaten human life and health are closely related to abnormal expression of GSH. Therefore, it is of great significance to monitor the fluctuation of GSH in organisms to determine the extent of disease occurrence and the diagnosis and treatment of the disease.

[0003] Organic small molecule fluorescent probes are widely used in the detection and imaging of active molecules in vivo due to their high sensitivity, good selectivity and non-destructive detection. Although a series of fluorescent probes for recognizing GSH have been constructed based on nucleophilic substitution reactions, addition cyclization and Michael addition reactions, these probes have problems such as non-targeting and poor selectivity, and are prone to "false signals" during the detection process, resulting in inaccurate detection. Summary of the invention

[0004] The purpose of the present invention is to provide a cell membrane dual-targeted glutathione-activated fluorescent probe, preparation and application thereof, which can realize accurate targeted detection of endogenous and exogenous glutathione in the cell membrane of living cells, thereby improving the accuracy of glutathione detection, in view of the above-mentioned problems existing in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a cell membrane dual-targeted glutathione-activated fluorescent probe having the following structural formula:

[0007]

[0008] In a second aspect, the present invention provides a method for preparing a cell membrane dual-targeted glutathione-activated fluorescent probe, the preparation method comprising:

[0009] S1. Dissolve 5-methyl salicylaldehyde and compound 1 in DMSO and react at 130-135° C. to obtain compound 2; the structural formula of compound 1 is:

[0010]

[0011] The structural formula of the compound 2 is:

[0012]

[0013] S2. Dissolve compound 2 in HBr solution and react at 110-120° C. to obtain compound 3; the structural formula of compound 3 is:

[0014]

[0015] S3, compound 3 is mixed with potassium carbonate and DMF, and then bromodecane is added to react at 80° C. to obtain compound 4; the structural formula of compound 4 is:

[0016]

[0017] S4, dissolving compound 4 and hexamethylenetetramine in trifluoroacetic acid and reacting at 70-80° C. to obtain compound 5; the structural formula of compound 5 is:

[0018]

[0019] S5. Dissolve compound 5 in toluene, add (formylmethylene)triphenylphosphine, and react at 60-65° C. to obtain compound 6; the structural formula of compound 6 is:

[0020]

[0021] S6. Compound 6 and propanesulfonic acid pyridinium salt are dissolved in ethanol, and piperidine is added to react at 60-70° C. to obtain the fluorophore PM-Red; the structural formula of the fluorophore PM-Red is:

[0022]

[0023] S7. Mix the fluorophore PM-Red and triethylamine in dichloromethane to obtain a mixed solution; dissolve 2,4-dinitrosulfonyl chloride in dichloromethane and slowly add it dropwise to the mixed solution, and react at room temperature to obtain a fluorescent probe with the structural formula as described in claim 1.

[0024] In a third aspect, the present invention provides an application of a cell membrane dual-targeted glutathione-activated fluorescent probe, wherein the fluorescent probe is used for sensing and detecting endogenous and exogenous glutathione in cell membranes and living cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The cell membrane dual-targeted glutathione-activated fluorescent probe of the present invention, on the one hand, uses 2,4-dinitrobenzenesulfonyl as the recognition group of GSH. Since it contains nitro, it has a strong electron-withdrawing effect to form a highly electron-deficient state on the benzene ring. When it is connected to the fluorophore PM-Red as an electron acceptor, it will cause fluorescence quenching. Therefore, when GSH is present, the fluorescent probe and GSH undergo an aromatic nucleophilic reaction, inducing the cleavage of 2,4-dinitrobenzenesulfonyl, thereby releasing the fluorophore PM-Red, and realizing the detection of GSH; on the other hand, when designing the probe, not only the propanesulfonic acid anion is used as the targeting site of the cell membrane, but also decane with a hydrophobic effect is added, so that the fluorescent probe is not easy to dissolve in the cytoplasmic fluid, but is easy to adhere and deposit on the cell membrane, thereby enhancing the cell membrane targeting of the probe. Therefore, the present invention adopts the cell membrane dual-targeting strategy and the specific recognition of GSH strategy to construct a new glutathione-activated fluorescent probe, which can realize the precise targeted detection of endogenous and exogenous GSH in the cell membrane of living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the synthesis route of the fluorescent probe PM-Red-GSH described in Example 1.

[0028] Figure 2 is the compound 2 in Example 1 1 H NRM spectrum.

[0029] Figure 3 is the compound 3 in Example 1 1 H NRM spectrum.

[0030] Figure 4 is the compound 4 in Example 1 1 H NRM spectrum.

[0031] Figure 5 is the compound 5 in Example 1 1 H NRM spectrum.

[0032] Figure 6 is the compound 6 in Example 1 1 H NRM spectrum.

[0033] Figure 7 is the fluorophore PM-Red in Example 1 1 H NRM spectrum.

[0034] Figure 8 is the fluorescent probe PM-Red-GSH in Example 1 1 H NRM spectrum.

[0035] Fig. 9This is the 13C NRM spectrum of the fluorescent probe PM-Red-GSH in Example 1.

[0036] Fig.10 This is the HRMS graph of the fluorescent probe PM-Red-GSH in Example 1.

[0037] Fig.11 This is the UV absorption diagram of the fluorescent probe PM-Red-GSH before and after responding to GSH.

[0038] Fig.12 This is the fluorescence emission diagram of the fluorescent probe PM-Red-GSH before and after responding to GSH.

[0039] Fig.13 The fluorescence emission diagram of the fluorescent probe PM-Red-GSH in response to different concentrations of GSH.

[0040] Fig.14 This is the fluorescence emission intensity diagram of the fluorescent probe PMM-Red-GSH in response to different concentrations of GSH at 713nm.

[0041] Fig.15 This is the ultraviolet absorption graph of the fluorescent probe PM-Red-GSH in response to different concentrations of GSH at 452nm.

[0042] Fig.16 This is the fluorescence emission intensity diagram of the fluorescent probe PM-Red-GSH in response to different pH at 713nm.

[0043] Fig.17 The fluorescence emission intensity of the fluorescent probe PM-Red-GSH in response to 0 and 100 μM GSH at 713 nm in 80 min.

[0044] Fig.18 This is the fluorescence emission intensity diagram of the fluorescent probe PM-Red-GSH in response to different targets at 713nm.

[0045] Fig.19 This is a diagram of the cytotoxicity test of the fluorescent probe PM-Red-GSH.

[0046] Fig. 20 This is a co-localization experiment of the fluorescent probe PM-Red-GSH and the cell membrane green fluorescent probe Dio.

[0047] Fig.21 This is the fluorescence intensity diagram of the fluorescent probe PM-Red-GSH detecting exogenous GSH in cells.

[0048] Fig. 22 This is the fluorescence intensity diagram of the fluorescent probe PM-Red-GSH detecting endogenous GSH in cells. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific implementations and drawings.

[0050] Example 1 Preparation of fluorescent probe

[0051] See also Figure 1 The preparation method of the cell membrane dual-targeted glutathione-activated fluorescent probe is carried out in the following steps:

[0052] S1. Compound 1 (0.74 g, 4.8 mmol) was dissolved in 5 mL DMSO and placed in a round-bottom flask. The mixture was reacted at 130° C. overnight. The reaction was monitored by TLC until the reaction was complete. The reaction was stopped, washed with water, extracted three times with ethyl acetate, the organic phases were combined, the solvent was dried under reduced pressure, and purified by column chromatography (EA:PE=1:8) to obtain 0.71 g of a light yellow solid, i.e., compound 2, with a yield of 65.99%;

[0053] Compound 2 1 H NRM spectrum Figure 2 As shown: 1 H NMR (500MHz, DMSO-d6) δ7.82 (d, J=8.5Hz1H), 7.40–7.36 (m, 2H), 7.10-7.06 (m, 2H), 6.85 (d, J=8.3Hz, 1H), 3.82 (s, 3H), 2.40 (s, 3H);

[0054] S2. Compound 2 (0.54 g, 2 mmol) was dissolved in 40 mL of 40% HBr solution (40% HBr solution means 40 mL of HBr and the remaining 60 mL of water in 100 mL of HBr solution), and the mixture was stirred at 110°C for overnight. The reaction was monitored by TLC until the reaction was complete, and the reaction was stopped. 1 mol / L NaOH solution was added to adjust the pH to about 6.0, and then the aqueous phase was adjusted to pH=7.0 with saturated NaHCO3 solution. After a light yellow solid precipitated, the mixture was filtered off under reduced pressure, the initial product was collected, and recrystallized through ether to obtain 0.36 g of a white solid, i.e., compound 3, with a yield of 69.23%.

[0055] Compound 3 1 H NRM spectrum Figure 3 As shown: 1 H NMR (500MHz, DMSO-d6) δ8.64 (s, 1H), 7.79 (d, J = 8.7Hz, 1H), 7.45 (d, J = 1.9Hz, 1 H), 7.39–7.36(m, 1H), 7.10–7.05(m, 1H), 6.91–6.84(m, 2H), 2.42-2.38(m, 3H);

[0056] S3, compound 3 (0.10 g, 0.4 mmol) and potassium carbonate (0.17 g, 1.2 mmol) were placed in a round-bottom flask containing 10.0 mL of DMF, the temperature was raised to 80°C, 0.8 mmol of bromodecane was added and the reaction was allowed to proceed overnight, TLC was monitored until the reaction was complete, water was added to quench the reaction, and the mixture was extracted three times with ethyl acetate, the organic phases were combined, the solvent was dried under reduced pressure, and the mixture was separated by column chromatography (EA:PA=1:3) to obtain 0.11 g of a white solid, i.e., compound 4, with a yield of 73.33%;

[0057] Compound 4 1 H NRM spectrum Figure 4 As shown: 1 H NMR (500MHz, DMSO-d6) δ7.85 (d, J=8.3Hz, 1H), 7.37 (d, J=1.8Hz, 1H), 7.33 (d, J=2.1Hz, 1H), 7.10–7.06 (m, 1H), 7.02 (dd, J=8.3, 1.9Hz, 1H), 6.85 (d , J=8.3Hz, 1H), 4.01 (t, J=6.3Hz, 2H), 2.40 (s, 3H), 1.75 (tt, J=7.5, 6.3Hz , 2H), 1.44 (dq, J=8.0, 7.0Hz, 2H), 1.32–1.25 (m, 12H), 0.92–0.86 (m, 3H);

[0058] S4, compound 4 (0.71 g, 1.8 mmol) and hexamethylenetetramine (0.546 g, 3.9 mmol) were dissolved in 15 mL of trifluoroacetic acid, stirred and refluxed overnight, monitored by TLC until the reaction was complete and cooled to room temperature, and then the pH value was adjusted with potassium hydroxide until the solid was completely precipitated, filtered, washed with water, and vacuum dried, and purified by column chromatography (EA:PA=1:5) to obtain 0.52 g of yellow solid, i.e., compound 5, with a yield of 68.42%;

[0059] Compound 5 1 H NRM spectrum Figure 5 As shown: 1H NMR (500MHz, DMSO-d6) δ7.86 (d, J=8.2Hz, 1H), 7.57 (d, J=2.5Hz, 1H), 7.48 (d, J=2.1Hz, 1H), 7.33 (d, J=2.2Hz, 1H), 7.02 (dd, J=8.3, 1.9Hz , 1H), 4.01 (t, J=6.3Hz, 2H), 2.39 (s, 3H), 1.75 (tt, J=7.5, 6.3Hz, 2H), 1.44 (dq, J=8.0, 7.0Hz, 2H), 1.33–1.25 (m, 12H), 0.92–0.86 (m, 3H);

[0060] S5. Compound 5 (2.55 g, 6 mmol) was dissolved in 20 mL of toluene, (formylmethylene)triphenylphosphine (1.86 g, 6.1 mmol) was added, and the mixture was stirred at 60°C for 6 h. The reaction was monitored by TLC until the reaction was complete. Toluene was removed by distillation under reduced pressure, and the mixture was purified by column chromatography (EA:PA=1:4) to obtain 1.32 g of a yellow solid, i.e., compound 6, with a yield of 48.71%.

[0061] Compound 6 1 H NRM spectrum Figure 6 As shown: 1 HNMR (500MHz, DMSO-d6) δ9.66 (dd, J=7.0, 1.3Hz, 1H), 7.86 (d, J=8.2Hz, 1H), 7.52 (dd, J=16.1, 1.3Hz, 1H), 7.37–7.31 (m, 3H), 7.02 (dd, J=8.3, 1.9Hz, 1H) , 6.72–6.67(m, 1H), 4.01(t, J=6.3Hz, 2H), 2.39(s, 3H), 1.75(tt, J=7.5, 6.3 Hz, 2H), 1.44 (dq, J=8.0, 7.0Hz, 2H), 1.32–1.26 (m, 12H), 0.92–0.86 (m, 3H);

[0062] S6. Compound 6 (0.45 g, 1 mmol) and propanesulfonic acid pyridinium salt (0.20 g, 1 mmol) were dissolved in 15 mL of ethanol, 2 drops of piperidine were added, and the mixture was reacted at 60° C. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, the mixture was purified by column chromatography (DCM:MeOH=30:1) to obtain 0.22 g of a red solid, i.e., the fluorophore PM-Red, with a yield of 33.85%.

[0063] Fluorophore PM-Red 1 H NRM spectrum Figure 7 As shown: 1HNMR (500MHz, DMSO-d6) δ8.99–8.95 (m, 2H), 8.19–8.14 (m, 2H), 7.86 (d, J =8.2Hz, 1H), 7.46–7.20 (m, 4H), 7.04–6.95 (m, 2H), 6.85–6.74 (m, 2H), 4.7 4(t, J=7.1Hz, 2H), 4.01(t, J=6.3Hz, 2H), 2.42–2.20(m, 5H), 1.75(tt, J=7 .5, 6.3Hz, 2H), 1.51–1.40 (m, 4H), 1.34–1.24 (m, 13H), 0.92–0.86 (m, 3H);

[0064] S7. Mix the fluorophore PM-Red (648 mg, 1 mmol) and triethylamine (101 mg, 1 mmol) in 10 mL of dichloromethane to obtain a mixed solution; dissolve 2,4-dinitrosulfonyl chloride (319 mg, 1.2 mmol) in 6 mL of dichloromethane, and slowly dropwise add the mixture to the mixture, react overnight at room temperature, monitor the reaction by TLC until the reaction is complete, cool to room temperature, and purify by column chromatography (DCM: MeOH = 30:1) to obtain 252.6 mg of a red solid, i.e., a fluorescent probe named PM-Red-GSH, with a yield of 30%;

[0065] PM-Red-GSH 1 H NRM spectrum Figure 8 As shown: 1 HNMR (500MHz, DMSO-d6) δ9.00–8.95 (m, 2H), 8.89 (d, J=1.9Hz, 1H), 8.53 (dd, J=8.1, 1.9Hz, 1H), 8.33 (d, J =8.0Hz, 1H), 8.19–8.13 (m, 2H), 7.84 (d, J = 8.3Hz, 1H), 7.57–7.53 (m, 2H), 7.40 (d, J = 2.0Hz, 2H), 7.16–7.0 6 (m, 2H), 6.89–6.77 (m, 2H), 4.66 (t, J=7.2Hz, 2H), 4.01 (t, J=6.3Hz, 2H), 2.37–2.22 (m, 5H), 1.75 (tt, J=7 .4, 6.3Hz, 2H), 1.52–1.39(m, 4H), 1.36–1.27(m, 12H), 1.27–1.21(m, 3H), 0.94–0.85(m, 3H); PM-Red-GSH 13 CNMR spectrum Fig. 9 As shown: 13CNMR (125MHz, DMSO-d6) δ162.15, 160.74, 157.66, 151.01, 148.50, 147.57, 146.78, 146.11 ,145.21,136.47,135.52,132.48,132.36,132.20,131.70,131.57,131.48,130.21,130.1 8, 128.56, 127.62, 124.47, 124.14, 121.36, 120.95, 115.96, 102.21, 68.76, 60.44, 59.40, 31.82, 29.57, 29.54, 29.40, 29.37, 26.41, 24.47, 22.71, 21.15, 14.09; HRMS graph of PM-Red-GSH is shown in Fig.10 Shown: HRMS [M+H + ]:843.2729.Found:843.290.

[0066] Example 2 GSH detection

[0067] (1) Fluorescent probe PM-Red-GSH responds to UV absorption before and after GSH

[0068] 10 μM fluorescent probe PM-Red-GSH was incubated with 0 μM and 100 μM GSH in 70% PBS-30% CH3CN at 37°C for 40 min and then its UV absorption was tested. The test results are shown in Fig.11 It can be seen that the fluorescent probe PM-Red-GSH absorbs ultraviolet light at 330nm and 425nm before and after responding to 0μM and 100μM GSH.

[0069] (2) Fluorescence emission of the fluorescent probe PM-Red-GSH before and after responding to GSH

[0070] 10 μM PM-Red-GSH was incubated with 0 μM and 100 μM GSH in 70% PBS-30% CH3CN at 37°C for 40 min, and then the fluorescence emission was tested under 425 nm excitation. The test results are shown in Fig.12 It can be seen that PM-Red-GSH has basically no fluorescence after responding to 0μM GSH, but has strong fluorescence emission at 713nm after responding to 100μM GSH, indicating that PM-Red-GSH can respond to GSH.

[0071] (3) Fluorescence linear relationship between the fluorescent probe PM-Red-GSH and GSH response

[0072] 10 μM fluorescent probe PM-Red-GSH was incubated with 0, 2, 5, 15, 30, 50, 80, 100, 200, and 500 μM GSH in 70% PBS-30% CH3CN at 37°C for 40 min, and then its fluorescence emission intensity was tested under 425 nm excitation. The test results are shown in Fig.13 , it can be seen that with the increase of GSH concentration, the fluorescence intensity of PM-Red-GSH gradually increases; see Fig.14 , showing good linearity in the range of 0-80 μM GSH concentration, y = 406.6X + 7373, and the linear relationship is R 2 =0.9861.

[0073] (4) Linear relationship between the UV absorption of the fluorescent probe PM-Red-GSH and the GSH response

[0074] 10 μM fluorescent probe PM-Red-GSH was incubated with 0, 1, 2, 3, 4, 6, 8, 10, 15, and 20 μM GSH at 70% PBS-30% CH3CN and 37°C for 40 min, and then its UV absorption was tested; the test results are shown in Fig.15 It can be seen that the ultraviolet absorption of PM-Red-GSH gradually increases with the increase of GSH concentration, and at the same time, it shows good linearity in the range of 0-20 μM, y=0.01087X+0.2898, and the linear relationship is R 2 =0.9715.

[0075] (5) pH stability of the fluorescent probe PM-Red-GSH

[0076] 10 μM fluorescent probe PM-Red-GSH was co-incubated with 0 and 100 μM GSH in 70% PBS-30% CH3CN, 37°C and different pH (pH=5, 5.5, 6, 6.5, 7, 7.34, 8, 8.5) for 40 min, and then its fluorescence emission intensity was tested under 425 nm excitation; the test results are shown in Fig.16 It can be seen that PM-Red-GSH has basically no fluorescence and no fluorescence change after responding to 0μM GSH under different pH conditions, and has strong fluorescence emission at 713nm and no fluorescence change after responding to 100μM GSH under different pH conditions. This shows that PM-Red-GSH has good stability under different pH conditions, and PM-Red-GSH has good response to GSH under different pH conditions and is not affected by pH.

[0077] (6) Kinetic study of different GSH using the fluorescent probe PM-Red-GSH

[0078] 10 μM PM-Red-GSH was co-incubated with 0 and 100 μM GSH in 70% PBS-30% CH3CN at 37°C for different time periods, and the fluorescence emission intensity was tested under 425 nm excitation within 0-80 min. The test results are shown in Fig.17 It can be seen that when the GSH concentration is 0 μM, PM-Red-GSH has basically no fluorescence at 713 nm and does not change with time, indicating good stability. When the GSH concentration is 100 μM, PM-Red-GSH fluoresces at 713 nm and the fluorescence increases with time. The fluorescence reaches stability at 30 minutes and the fluorescence intensity does not change with time after 30 minutes.

[0079] (7) Study on the selectivity of the fluorescent probe PM-Red-GSH for different targets

[0080] 10 μM fluorescent probe PM-Red-GSH was co-incubated with different targets in 70% PBS-30% CH3CN at 37°C, and the fluorescence emission intensity was tested under 425 nm excitation from an external light source. The test results are shown in Fig.18 , 10 μM PM-Red-GSH was mixed with 200 μM Sec (selenocysteine), Hcy (homocysteine), Cys (cysteine) and 200 μM metal ions (Fe 2+ , Fe 3+ , K + , Na + , Ca 2+ , Zn 2+ ), sulfide (sulfide, the main component is S2O3 2- After incubation with 200 μM ROS / RNS for 40 min, there was basically no fluorescence at 713 nm. Only after incubation with 100 μM GSH did it cause obvious fluorescence changes and the fluorescence was enhanced by 5 times.

[0081] (8) Study on the cytotoxicity of fluorescent probe PM-Red-GSH

[0082] The cytotoxicity of the fluorescent probe PM-Red-GSH was evaluated by the MTT method. BV-2 cells were cultured in DMEM in a 96-cell microplate in an incubator for 24 hours, and then the DMEM medium was replaced with the fluorescent probe PM-Red-GSH containing different concentrations (0-30μM) for another 24 hours (three parallel experiments were performed for each concentration), and then washed three times with PBS, 0.5mg / L MTT was added and incubated for another 4 hours, the culture medium was aspirated, 100μL DMSO was used to dissolve the formazan for about 15 minutes, and the ultraviolet absorption at 490nm was measured using an enzyme reader. The cytotoxicity was calculated according to the following formula: Cytotoxicity = Average absorbance of cells in the experimental group / Average absorbance of cells in the control group. The test results are shown in Fig.19 It can be seen that the BV-2 cells treated with the probe PM-Red-GSH maintained good activity. When the concentration of the probe PM-Red-GH reached 30 μM, the cell survival rate was greater than 85%, indicating that the probe PM-Red-GSH had less toxicity to the cells.

[0083] (9) Targeted study of fluorescent probe PM-Red-GSH on cell membrane

[0084] The co-localization ability of the fluorescent probe PM-Red-GSH and the cell membrane targeting probe DIO was investigated. After the BV-2 cells were cultured, they were incubated with the fluorescent group PM-Red for 30 minutes, and then the cell culture medium was aspirated and incubated with the cell membrane targeting probe DIO for 40 minutes. After washing three times, PBS buffer was added, and then the cell morphology was found with a confocal microscope, and cell co-localization imaging was performed. It was observed that the probe mainly targeted the cell membrane and had a good co-localization effect with the DIO probe. The observation results are shown in Fig. 20 , Fig. 20 Figure A is a cell membrane confocal imaging image of PM-Red, Figure B is a cell membrane confocal imaging image of DIO, Figure C is a merge of Figure A and Figure B, Figure D is a calculated Pearson coefficient graph, and Figure E is a waveform graph of the co-localized green channel and red channel fluorescence intensity values. The Pearson coefficient is 0.95, which shows that the probe PM-Red-GSH prepared by the present invention has good cell membrane targeting ability.

[0085] (10) Detection of exogenous GSH in cells using the fluorescent probe PM-Red-GSH

[0086] The ability of the fluorescent probe PM-Red-GSH to detect exogenous GSH in cells was investigated. After BV-2 cells were cultured, they were treated with 0.5 mM NEM (N-ethylmaleimide, a commercial GSH scavenger) for 40 min, and then 10 μM fluorescent probe PM-Red-GSH and different concentrations of GSH (0, 5, 10, 20 μM) were added and incubated for another 30 min. The fluorescence detection was shown in Fig. Fig.21 , Fig.21 Figure A is a confocal fluorescence image, and Figure B is the average fluorescence intensity of Figure A. It can be seen that as the GSH concentration increases, the cells show enhanced fluorescence.

[0087] (11) Detection of endogenous GSH in cells using the fluorescent probe PM-Red-GSH

[0088] The ability of the fluorescent probe PM-Red-GSH to detect endogenous GSH in cells was investigated. After culturing BV-2 cells, 10 μM fluorescent probe PM-Red-GSH was co-incubated with the cells for 40 min. The fluorescence imaging results were shown in Fig. 22 In the control group in Figure A, we can see that the cells show strong red fluorescence and can still show good cell imaging ability after 4 hours. RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4), which can inhibit the production of intracellular GSH. 10μM probe PM-Red-GSH and 10μM RSL3 were co-incubated with cells for 30 minutes. The fluorescence imaging results are shown in Fig. 22 In the RSL3 group in Figure A, we can see that the cell fluorescence gradually weakened over time, and after 2 hours, the cells were basically free of fluorescence, indicating that RSL3 inhibited the production of GSH in the cell body, resulting in reduced fluorescence. Lip-1 is a ferroptosis inhibitor that can promote the production of GSH in cells; 10μM RSL3, 10μM Lip-1, and 10μM fluorescent probe PM-Red-GSH were co-incubated with cells for 30 minutes, and the fluorescence imaging results were shown in Fig. 22 In Figure A, in the RSL3+Lip-1 group, obvious red fluorescence can be seen in the cell body and good fluorescence imaging capability is maintained after 4 hours. Fig. 22 Figure A is the confocal fluorescence imaging of the control group, RSL3 group, and RSL3+Lip-1 group, and Figure B is the average relative fluorescence intensity of Figure A. The above results can show that the probe PM-Red-GSH not only has good fluorescence imaging ability for endogenous GSH in cells, but also has a longer fluorescence imaging ability.

Claims

1. A cell membrane dual-targeted glutathione-activated fluorescent probe, characterized in that: The structural formula of the fluorescent probe is:

2. The method for preparing the cell membrane dual-targeted glutathione-activated fluorescent probe according to claim 1, characterized in that: The preparation method comprises: S1. Dissolve 5-methyl salicylaldehyde and compound 1 in DMSO and react at 130-135° C. to obtain compound 2; the structural formula of compound 1 is: The structural formula of the compound 2 is: S2. Dissolve compound 2 in HBr solution and react at 110-120° C. to obtain compound 3; the structural formula of compound 3 is: S3, compound 3 is mixed with potassium carbonate and DMF, and then bromodecane is added to react at 80° C. to obtain compound 4; the structural formula of compound 4 is: S4, dissolving compound 4 and hexamethylenetetramine in trifluoroacetic acid and reacting at 70-80° C. to obtain compound 5; the structural formula of compound 5 is: S5. Dissolve compound 5 in toluene, add (formylmethylene)triphenylphosphine, and react at 60-65° C. to obtain compound 6; the structural formula of compound 6 is: S6. Compound 6 and propanesulfonic acid pyridinium salt are dissolved in ethanol, and piperidine is added to react at 60-70° C. to obtain the fluorophore PM-Red; the structural formula of the fluorophore PM-Red is: S7. Mix the fluorophore PM-Red and triethylamine in dichloromethane to obtain a mixed solution; dissolve 2,4-dinitrosulfonyl chloride in dichloromethane and slowly add it dropwise to the mixed solution, and react at room temperature to obtain a fluorescent probe with the structural formula as described in claim 1.

3. The use of the cell membrane dual-targeted glutathione-activated fluorescent probe according to claim 1, characterized in that: The fluorescent probe is used for sensing and detecting endogenous and exogenous glutathione in cell membrane.

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