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

Abstract

The present invention belongs to the field of optical imaging and biosensor technology, and specifically relates to a cell membrane dual-targeted glutathione-activated fluorescent probe, preparation and application. In the fluorescent probe, 2,4-dinitrobenzenesulfonyl is used as a recognition group for GSH. In the presence of GSH, the fluorescent probe undergoes an aromatic nucleophilic reaction with GSH to induce the cleavage of the 2,4-dinitrobenzenesulfonyl group, releasing the fluorophore PM-Red, thereby detecting GSH. When designing the probe, not only is a propanesulfonic acid anion used as the targeting site on the cell membrane, but decane with a hydrophobic effect is also added, so that the fluorescent probe is difficult to dissolve in the cell fluid of the cell and is deposited on the cell membrane, ultimately achieving precise targeted detection of endogenous and exogenous GSH in the cell membrane of living cells.

Description

Technical Field

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

[0002] Glutathione (GSH) is a key signaling molecule that regulates cellular physiological activities in organisms. Abnormal GSH expression in organisms is often associated with a variety of diseases. Currently, 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 GSH expression. Therefore, monitoring the fluctuation of GSH in organisms to determine the severity of disease and the diagnosis and treatment of the disease is of great significance.

[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 GSH recognition have been constructed based on reactions such as nucleophilic substitution, addition cyclization, and Michael addition, these probes suffer from non-targeting and poor selectivity, making them prone to "false signals" during detection and resulting in inaccurate detection. Summary of the Invention

[0004] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and to provide a cell membrane dual-targeted glutathione-activated fluorescent probe that can achieve precise targeted detection of endogenous and exogenous glutathione in the cell membrane of living cells, thereby improving the accuracy of glutathione detection, as well as its preparation and application.

[0005] To achieve the above objectives, the technical solutions of the present invention are 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-methylsalicylaldehyde 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 was mixed with potassium carbonate and DMF, and then bromodecane was added to react at 80° C. to obtain compound 4; the structural formula of compound 4 is:

[0016]

[0017] S4. Dissolve compound 4 and hexamethylenetetramine in trifluoroacetic acid and react 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. Dissolve compound 6 and propanesulfonic acid pyridinium salt in ethanol, add piperidine, and 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 the fluorescent probe with the structural formula as claimed in claim 1.

[0024] In a third aspect, the present invention provides an application of a cell membrane dual-targeted glutathione-activated fluorescent probe, which 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 uses 2,4-dinitrobenzenesulfonyl as the recognition group of GSH. Because it contains a nitro group, it has a strong electron-withdrawing effect, which forms 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, in the presence of GSH, the fluorescent probe and GSH undergo an aromatic nucleophilic reaction, inducing the cleavage of the 2,4-dinitrobenzenesulfonyl group, 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 easily dissolved in the cytoplasmic fluid, but is easily attached and deposited on the cell membrane, thereby enhancing the cell membrane targeting of the probe. Therefore, the present invention adopts a cell membrane dual-targeting strategy and a specific recognition strategy of GSH 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] Figure 9This is the 13C NRM spectrum of the fluorescent probe PM-Red-GSH in Example 1.

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

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

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

[0039] Figure 13 Figure 2 shows the fluorescence emission graph of the fluorescent probe PM-Red-GSH in response to different concentrations of GSH.

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

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

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

[0043] Figure 17 Figure 2 shows 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] Figure 18 This is the fluorescence emission intensity diagram of the fluorescent probe PM-Red-GSH in response to different targets at 713nm.

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

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

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

[0048] Figure 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 will be further described in detail below with reference to specific embodiments and the accompanying 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 of DMSO in a round-bottom flask and reacted overnight at 130° C. The reaction was monitored by TLC until completion. The reaction was stopped, washed with water, and extracted three times with ethyl acetate. The organic phases were combined, the solvent was evaporated 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, i.e., 40 mL of HBr and the remaining 60 mL of water in 100 mL of HBr solution), stirred at 110° C. for overnight, and monitored by TLC until the reaction was complete. The reaction was stopped, and 1 mol / L NaOH solution was added to adjust the pH to about 6.0. The aqueous phase was then adjusted to pH=7.0 with saturated NaHCO 3 solution. After a light yellow solid precipitated, the product was filtered off under reduced pressure and collected. The product was 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 evaporated 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, and the reaction was monitored by TLC until completion and cooled to room temperature. The pH value was adjusted with potassium hydroxide until the solid was completely precipitated. The mixture was filtered, washed with water, and dried in vacuo. The mixture was purified by column chromatography (EA:PA=1:5) to obtain 0.52 g of a 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, and (formylmethylene)triphenylphosphine (1.86 g, 6.1 mmol) was added. The mixture was stirred at 60° C. for 6 h. The reaction was monitored by TLC until completion. Toluene was removed by distillation under reduced pressure, and the product 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 pyridinium propanesulfonate (0.20 g, 1 mmol) were dissolved in 15 mL of ethanol, and 2 drops of piperidine were added. The mixture was reacted at 60° C. and 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. The fluorophore PM-Red (648 mg, 1 mmol) and triethylamine (101 mg, 1 mmol) were mixed in 10 mL of dichloromethane to obtain a mixture; 2,4-dinitrosulfonyl chloride (319 mg, 1.2 mmol) was dissolved in 6 mL of dichloromethane and slowly added dropwise to the mixture. The mixture was reacted at room temperature overnight and 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 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 such as Figure 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 images of PM-Red-GSH are shown in Figure 5. Figure 10 Shown: HRMS [M+H + ]:843.2729.Found:843.290.

[0066] Example 2 GSH detection

[0067] (1) UV absorption of the fluorescent probe PM-Red-GSH before and after responding to 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 Figure 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 measured under 425 nm excitation. The test results are shown in Figure 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 the fluorescence emission intensity was measured under 425 nm excitation. The test results are shown in Figure 13 , it can be seen that with the increase of GSH concentration, the fluorescence intensity of PM-Red-GSH gradually increases; see Figure 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 in 70% PBS-30% CH3CN at 37°C for 40 min and then its UV absorption was tested; the test results are shown in Figure 15 , it can be seen that the UV 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 at 37°C and different pH conditions (pH = 5, 5.5, 6, 6.5, 7, 7.34, 8, 8.5) for 40 min, and the fluorescence emission intensity was measured under 425 nm excitation. The test results are shown in Figure 16 , PM-Red-GSH exhibited essentially no fluorescence and no fluorescence change when reacting with 0 μM GSH under different pH conditions, while exhibited strong fluorescence emission at 713 nm with no fluorescence change when reacting with 100 μM GSH under different pH conditions. This indicates that PM-Red-GSH exhibits good stability under different pH conditions and responds well to GSH under different pH conditions, regardless of pH.

[0077] (6) Kinetic study of different GSHs by 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. The fluorescence emission intensity was measured under 425 nm excitation within 0-80 min. The test results are shown in Figure 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 that it has 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 Figure 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 the 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) and cultured for another 24 hours (three parallel experiments were performed for each concentration). Then, the cells were washed three times with PBS, 0.5mg / L MTT was added, and the cells were incubated for another 4 hours. The culture medium was aspirated, and 100μL DMSO was used to dissolve the formazan for about 15 minutes. The ultraviolet absorption at 490nm was measured using a microplate reader, and the cytotoxicity was calculated according to the following formula: Cytotoxicity = Average absorbance of experimental group cells / Average absorbance of control group cells. The test results are shown in Table 1. Figure 19 It can be seen that BV-2 cells maintained good activity after treatment with probe PM-Red-GSH. When the concentration of probe PM-Red-GH reached 30 μM, the cell survival rate was greater than 85%, indicating that probe PM-Red-GSH had less toxicity to cells.

[0083] (9) Targeting 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 culturing BV-2 cells, they were incubated with the fluorophore PM-Red for 30 minutes, 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 determined using a confocal microscope. Cell co-localization imaging was performed, and it was observed that the probe mainly targeted the cell membrane and had a good co-localization effect with the DIO probe. The results are shown in Figure 20 , Figure 20 Panel A shows a confocal image of the cell membrane using PM-Red, Panel B shows a confocal image of the cell membrane using DIO, Panel C shows a merge of Panels A and B, Panel D shows the calculated Pearson coefficient, and Panel E shows a waveform of the colocalized green and red channel fluorescence intensity values. The Pearson coefficient was 0.95, indicating 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. BV-2 cells were cultured and treated with 0.5 mM NEM (N-ethylmaleimide, a commercial GSH scavenger) for 40 minutes. 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 minutes. Fluorescence detection was performed. Figure 21 , Figure 21 Figure A is a confocal fluorescence imaging 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 minutes. The fluorescence imaging results were shown in Figure 22 In the control group in Figure A, the cells showed strong red fluorescence and good cell imaging ability after 4 hours. RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4), which can inhibit the production of GSH in cells. 10μM probe PM-Red-GSH and 10μM RSL3 were co-incubated with cells for 30 minutes. The fluorescence imaging results are shown in Figure 4. Figure 22 In the RSL3 group in Figure A, the fluorescence of the cells gradually weakened over time, and after 2 hours, the cells were essentially devoid of fluorescence, indicating that RSL3 inhibited GSH production in the cells, leading to decreased fluorescence. Lip-1 is a ferroptosis inhibitor that promotes GSH production in cells. Cells were co-incubated with 10 μM RSL3, 10 μM Lip-1, and 10 μM fluorescent probe PM-Red-GSH for 30 minutes. The fluorescence imaging results are shown in Figure 3. Figure 22 In Figure A, RSL3+Lip-1 group, obvious red fluorescence can be seen in the cell body and good fluorescence imaging capability is maintained after 4 hours. Figure 22 Panel A shows confocal fluorescence images of the control, RSL3, and RSL3+Lip-1 groups, and Panel B shows the average relative fluorescence intensity of Panel A. These results demonstrate that the PM-Red-GSH probe not only exhibits excellent fluorescence imaging capabilities for endogenous GSH but also maintains this capability for a prolonged period of time.

Claims

1. A cell membrane dual-targeted glutathione-activated fluorescent probe, characterized by: 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, wherein: The preparation method comprises: S1. Dissolve 5-methylsalicylaldehyde 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 was mixed with potassium carbonate and DMF, and then bromodecane was added to react at 80° C. to obtain compound 4; the structural formula of compound 4 is: ； S4. Dissolve compound 4 and hexamethylenetetramine in trifluoroacetic acid and react 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. Dissolve compound 6 and propanesulfonic acid pyridinium salt in ethanol, add piperidine, and 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 the fluorescent probe with the structural formula as claimed in claim 1.

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