A fluorescent probe for monitoring glutathione inside and outside cell membranes, a synthesis method and application thereof

Abstract

The application provides a kind of fluorescence probe for monitoring glutathione in and out of cell membrane, synthesis method and application thereof, the synthesis method of the fluorescence probe is that compound 1 and 2-hydroxybenzothiazole are dissolved in DMF, sodium tert-butoxide is added, under nitrogen environment, reaction is carried out at 78-82 DEG C for 11-13 hours, after cooling, filtering, purification, the probe is obtained.The Nap-Mem-GSH probe disclosed in the application has good specificity, good reaction to glutathione, and less reaction to other biological thiols.The Nap-Mem-GSH probe disclosed in the application has strong light stability, strong chemical interference resistance, low cytotoxicity, good cell membrane targeting and GSH responsiveness.

Description

Technical Field

[0001] This invention relates to a fluorescent probe for monitoring glutathione inside and outside the cell membrane, its synthesis method, and its application; it belongs to the field of biomedical technology. Background Technology

[0002] Reduced glutathione (GSH) is a biomarker of oxidative stress and the most abundant non-protein thiol species in cells. Glutathione not only controls and maintains cellular redox balance, participates in the antioxidant protection of peptides and proteins, thiol disulfide exchange, redox-dependent cell signaling, and gene expression regulation, but also detoxifies and synthesizes eicosanoids. Abnormal glutathione levels are associated with various diseases, such as AIDS, cancer, liver damage, and neurodegenerative diseases. Furthermore, high levels of glutathione can lead to enhanced immunity. Therefore, real-time detection of GSH is of great interest.

[0003] The cell membrane is the boundary of the cell, separating various substances and organelles within the cell from the external environment. The cell membrane is responsible for signal transduction, substance transport, and maintaining cellular homeostasis. It is also involved in oxidative stress, which can lead to the breakdown of peroxides and changes in the properties of membrane lipids.

[0004] Glutathione production and metabolism involve intracellular and extracellular circulation, a cycle comprised of six enzymatic reactions involving γ-glutamate. First, the produced glutathione is transported from the cell to the extracellular space via transport proteins on the cytoplasmic membrane. γ-glutamyltransferase (γ-GT), distributed on the extracellular membrane of specific cell types, is a crucial enzyme in the glutathione cycle. It hydrolyzes glutathione, promoting the transfer of γ-glutamine residues to neutral amino acids for intracellular transport. Extracellular glutathione then re-enters the cell under the catalysis of γ-GT, completing the glutathione cycle. Utilizing the cellular glutathione cycle, many modern intelligent drug delivery systems can be developed based on changes in intracellular and extracellular glutathione concentrations. Furthermore, the cell membrane and glutathione interact and influence each other in some cellular activities. Simultaneously visualizing the intracellular and extracellular glutathione levels provides new research strategies for studying their interactions and their circulation within and outside the cell.

[0005] 1,8-Naphthylamine derivatives are fluorophores widely used in cell imaging. Their modifiability, synthetic diversity, and easily tunable photophysical properties allow for the synthesis of a range of fluorescent probes. However, some probes not only have complex synthetic routes but also exhibit weak specificity, leading to low signal-to-noise ratios and fluorescence quenching during imaging. These drawbacks limit the development of responsive probes for imaging.

[0006] According to current reports, fluorescent probes for detecting glutathione have some drawbacks, such as complex synthesis routes and poor photostability. Furthermore, there are currently no commercially available fluorescent probes that can target glutathione on the cell membrane. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a fluorescent probe for monitoring glutathione across cell membranes, its synthesis method, and its applications, achieving the following objectives:

[0008] With good specificity, good photostability, low biotoxicity, and chemical resistance, it can be used for fluorescence imaging of GSH in cells.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A fluorescent probe for monitoring glutathione levels across the cell membrane, the structure of which is as follows:

[0011] .

[0012] The probe is referred to as Nap-Mem-GSH;

[0013] The fluorescent probe is synthesized by dissolving compound 1 and 2-hydroxybenzothiazole in DMF, adding sodium tert-butoxide, reacting at 78-82℃ for 11-13 hours under nitrogen atmosphere, and then cooling, filtering, and purifying to obtain the probe.

[0014] The structural formula of compound 1 is as follows:

[0015] .

[0016] The molar ratio of compound 1 to 2-hydroxybenzothiazole is 1:1.9-2.1; the molar ratio of 2-hydroxybenzothiazole to sodium tert-butoxide is 1:0.24-0.26; and the mass-to-volume ratio of 2-hydroxybenzothiazole to DMF is 58.0-58.5 mg:1 mL.

[0017] The preparation method of compound 1 is as follows: 4-bromo-1,8-naphthalenedicarboxylic anhydride and 1-dodecylamine are dissolved in ethanol, refluxed at 88-92°C for 11-13 hours, and then cooled, filtered and purified to obtain compound 1.

[0018] The molar ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to 1-dodecylamine is 1:1.9-2.1; the mass-volume ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to ethanol is 1g:14.5-15.5mL.

[0019] The fluorescent probe is used for sensing and detecting glutathione inside and outside the cell membrane in biological systems.

[0020] The sensing detection is either fluorescence detection or cell imaging.

[0021] Compared with the prior art, the present invention achieves the following beneficial effects:

[0022] The Nap-Mem-GSH probe described in this invention has good specificity, reacts well to glutathione, and reacts less to other biothiols.

[0023] The Nap-Mem-GSH probe described in this invention exhibits strong photostability. Without the addition of GSH, the fluorescence intensity of Nap-Mem-GSH does not decrease significantly within 30 minutes, and after the addition of GSH, the fluorescence intensity remains stable within 70 minutes, demonstrating that Nap-Mem-GSH has excellent time stability.

[0024] The Nap-Mem-GSH probe described in this invention has strong resistance to chemical interference and high selectivity for GSH.

[0025] The Nap-Mem-GSH probe described in this invention has low cytotoxicity; when the concentration of Nap-Mem-GSH is 20 μM, the activity of HeLa cells is greater than 80%.

[0026] The present invention, Nap-Mem-GSH, exhibits excellent cell membrane targeting and GSH responsiveness, while also providing excellent imaging performance. Attached Figure Description

[0027] Figure 1 It is a probe called Nap-Mem-GSH. 1 H NMR spectrum;

[0028] Figure 2 It is a probe called Nap-Mem-GSH. 13 C NMR spectrum;

[0029] Figure 3 This is a bar chart showing the fluorescence emission intensity of the probe Nap-Mem-GSH in different biothiols;

[0030] Figure 4 It is the ultraviolet absorption spectrum of the probe Nap-Mem-GSH with and without GSH;

[0031] Figure 5 It is the fluorescence emission spectrum of the probe Nap-Mem-GSH in the presence and absence of GSH;

[0032] Figure 6The fluorescence emission spectra of the probe Nap-Mem-GSH vary with GSH concentration. The concentrations of the curves from bottom to top are: 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, and 130 μM.

[0033] Figure 7 This is a standard curve of the fluorescence emission of the probe Nap-Mem-GSH as a function of GSH.

[0034] Figure 8 This is a time stability plot of Nap-Mem-GSH with and without GSH.

[0035] Figure 9 This is a line graph showing the fluorescence emission intensity of the probe Nap-Mem-GSH under different pH conditions;

[0036] Figure 10 This is a bar chart showing the selectivity of the probe Nap-Mem-GSH for GSH and other chemical analytes;

[0037] The chemical analytes corresponding to 1-13 are as follows: 1 is GSH, 2 is NaSH, 3 is Cys, 4 is Hcy, 5 is H2O2, and 6 is Cl. - 7 is Cu 2+ 8 is HClO, 9 is Glu, and 10 is NO. 2- 11 is TBHP, 12 is Fe 2+ 13 is Ag + ;

[0038] Figure 11 This is a bar chart showing the competitive selectivity of the probe Nap-Mem-GSH;

[0039] 1 is blank, 2 is NaSH, 3 is Cys, 4 is Hcy, 5 is H2O2, and 6 is Cl. - 7 is Cu 2+ 8 is HClO, 9 is Glu, and 10 is NO. 2- 11 is TBHP, 12 is Fe 2+ 13 is Ag + ;

[0040] Figure 12 This is a bar chart showing the cytotoxicity of the Nap-Mem-GSH probe;

[0041] Figure 13 Fluorescence imaging of the probe Nap-Mem-GSH in live HeLa cells;

[0042] Figure 14 Images and cell membrane colocalization curves of HeLa cells under the action of Nap-Mem-GSH and DiD perchlorate;

[0043] Where A is the yellow channel of the probe Nap-Mem-GSH; B is the red channel of the commercial probe DiD; C is the bright field of HeLa cells; D is the mixed channel; E is the cell membrane colocalization curve before adding GSH; F is the cell membrane colocalization curve after adding GSH.

[0044] Figure 15 This is a fluorescence imaging image of the probe Nap-Mem-GSH in zebrafish;

[0045] Figure 16 A bar chart showing the fluorescence intensity of the probe Nap-Mem-GSH in zebrafish. Detailed Implementation

[0046] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. The compound numbers in the embodiments are the same as the compound numbers in the above scheme.

[0047] Example 1: Probe Synthesis:

[0048] The synthesis route is as follows:

[0049] .

[0050] The specific synthesis method is as follows:

[0051] (1) Preparation of compound 1

[0052] 4-Bromo-1,8-naphthalenedicarboxylic anhydride (2 g, 8.6 mmol) and 1-dodecylamine (3.18 g, 17.2 mmol) were dissolved in ethanol (30 ml), refluxed at 90 °C for 12 hours, cooled to room temperature, and the crude product was purified by filtration and column chromatography to obtain a yellow solid, which is compound 1, with a yield of 98%.

[0053] The above yields are calculated based on 4-bromo-1,8-naphthalenedicarboxylic anhydride, by dividing the actual mass of compound 1 obtained by the theoretical mass of compound 1.

[0054] (2) Preparation of probe Nap-Mem-GSH

[0055] Compound 1 (443 mg, 1 mmol) and 2-hydroxybenzothiazole (291 mg, 2 mmol) were dissolved in DMF (5 ml), and sodium tert-butoxide (48 mg, 0.5 mmol) was added. The mixture was reacted at 80 °C for 12 hours under nitrogen atmosphere, cooled to room temperature, filtered to obtain the residue, and purified by silica gel column chromatography to obtain a yellow solid, which was the probe Nap-Mem-GSH, with a yield of 45%.

[0056] 1 H NMR (400 MHz, DMSO) δ 8.57 – 8.43 (m, 2H), 8.37 (dd, J = 7.7, 4.4Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.77 (t, J = 7.3 Hz, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.50 (t, J = 7.0 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.17 (t, J = 6.5 Hz, 2H), 6.50 (d, J = 9.4 Hz, 1H), 4.00 (d, J = 8.5 Hz, 2H), 1.60 (s, 2H), 1.30 (s, 4H), 1.21 (s, 14H), 0.84 (t, J = 6.4 Hz, 3H). 13 C NMR (CDCl3, 101 MHz): δ = 164.30,159.43, 133.70, 131.91, 129.82, 129.04, 127.72, 125.58,123.62, 122.65,122.31, 114.30, 110.05, 40.50, 31.92, 29.63, 28.22, 27.22, 22.70, 14.21. HRMS(ESI + ): m / z calcd for C 24 H 13 N2O3[M + H + ] + :509.2804, found: 509.2808

[0057] The above yields are calculated based on compound 1, by dividing the actual mass of Nap-Mem-GSH obtained by the theoretical mass of Nap-Mem-GSH.

[0058] Example 2: Screening of common biothiols by the probe Nap-Mem-GSH

[0059] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL probe stock solution. Common biothiols were dissolved in DMSO to prepare a 10 mmol / mL biothiol stock solution. Reaction tests were performed using various common biothiols, such as Cys, Hcy, GSH, and H2S. The probe stock solution, biothiol stock solution, and a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) were diluted to prepare a working solution with a probe concentration of 10 μg / mL and a biothiol concentration of 100 μM. The fluorescence intensity of the probe at 545 nm was measured at room temperature with an excitation wavelength of 390 nm. The experimental results are attached. Figure 3 As shown, the results indicate that Nap-Mem-GSH responds well to GSH, while other bio-thiols have a relatively small effect on Nap-Mem-GSH.

[0060] Example 3: Ultraviolet absorption spectrum of probe Nap-Mem-GSH

[0061] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL probe stock solution. GSH was dissolved in DMSO to prepare a 1 mM GSH stock solution. The probe stock solution was diluted with a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) to a 10 μg / mL working solution for UV absorption testing. Then, the GSH stock solution was added to obtain the test solution, with a final GSH concentration of 10 μM. After incubation for 10 minutes, the test solution was subjected to UV testing, as shown in the attached figure. Figure 4 As shown, the maximum absorption wavelengths of the Nap-Mem-GSH solution without the addition of GSH are 390 nm and 475 nm. However, after the addition of GSH, the UV absorption intensity at 390 nm increases, while the UV absorption intensity at 475 nm decreases.

[0062] Example 4: Fluorescence emission spectrum of probe Nap-Mem-GSH

[0063] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL probe stock solution. The probe stock solution was diluted with a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) to prepare a 10 μg / mL working solution. The fluorescence emission intensity of the probe was measured at an excitation wavelength of 390 nm. GSH was dissolved in DMSO to prepare a 1 mM GSH stock solution. The GSH stock solution was added to the above working solution to obtain the test solution. The final concentration of GSH in the test solution was 100 μM. After incubation at room temperature for 10 minutes, the fluorescence emission intensity of the probe in the presence of GSH was measured at an excitation wavelength of 390 nm.

[0064] As attached Figure 5 As shown, the fluorescence emission of Nap-Mem-GSH was tested in the presence or absence of GSH. The results showed that Nap-Mem-GSH emitted weakly in the absence of GSH, while the fluorescence intensity increased significantly to 40 times the original value after the addition of GSH.

[0065] Example 5: Fluorescence spectroscopy test of probe Nap-Mem-GSH at different GSH concentrations.

[0066] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL probe stock solution. GSH was dissolved in DMSO to prepare a 1 mM GSH stock solution. The probe stock solution and GSH stock solution were mixed with DMSO / PBS buffer (v / v = 1:1, 0.1 mol / L, pH = 7.4) to prepare test solutions with different GSH concentrations. In each test solution, the GSH concentration increased from 1 μM to 130 μM. The probe concentration in each test solution was 10 μg / mL. After incubation at room temperature for 2 minutes, the emission intensity of the probe at 545 nm was measured using a 390 nm excitation wavelength, and the fluorescence spectrum was recorded (see Appendix). Figure 6 , attached Figure 6 In the figure, the curves from bottom to top correspond to GSH concentrations of 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, and 130 μM in the test solution, respectively.

[0067] The fluorescence intensity versus concentration at 545 nm wavelength is plotted in the appendix. Figure 7 Within the range of 30 μM to 130 μM, the fluorescence intensity is linearly proportional to the GSH concentration.

[0068] Example 6: Fluorescence performance test of probe Nap-Mem-GSH under different pH conditions.

[0069] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL probe stock solution. GSH was dissolved in DMSO to prepare a 1 mM GSH stock solution. Mixed solutions of DMSO and PBS at different pH values ​​(v / v = 1:1, 0.1 mol / L, pH = 2, 3, 4, 5, 6, 7, 8, 9) were prepared and calibrated using a pH meter. The probe stock solution was diluted with the mixed solutions of DMSO and PBS at different pH values ​​to a 10 μg / mL working solution for fluorescence testing. Then, the GSH stock solution was added to obtain the test solution, with a final GSH concentration of 10 μM. After incubation for 10 minutes, the fluorescence of the test solution was measured. The fluorescence emission of Nap-Mem-GSH was detected at an excitation wavelength of 390 nm, as shown in the attached figure. Figure 9 As shown in the figure. Experimental results indicate that Nap-Mem-GSH exhibits the strongest and most stable fluorescence intensity within a pH range of 7.0-8.0. Meanwhile, since the reactivity of glutathione increases with increasing pH, Nap-Mem-GSH is affected under acidic conditions, leading to a decrease in the reactivity of Nap-Mem-GSH with glutathione, thus resulting in weakened fluorescence intensity. The optimal reactivity of Nap-Mem-GSH with glutathione at pH 7.0-8.0 demonstrates that Nap-Mem-GSH can adapt to the cellular environment and achieve cell imaging.

[0070] Example 7: Time stability test of probe Nap-Mem-GSH.

[0071] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL probe stock solution. GSH was dissolved in DMSO to prepare a 10 mmol / mL GSH stock solution. The probe stock solution was diluted with a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) to prepare a 10 μg / mL working solution for fluorescence testing. At room temperature, the fluorescence emission of Nap-Mem-GSH was detected at an excitation wavelength of 390 nm, and the fluorescence intensity was measured every 2 minutes. Then, GSH stock solution was added to obtain the test solution, with a final GSH concentration of 10 μM. After addition, the fluorescence intensity was measured every 2 minutes, and after 20 minutes, it was measured every 10 minutes. The results are attached. Figure 8 As shown, the fluorescence intensity of Nap-Mem-GSH did not decrease significantly within 30 minutes without the addition of GSH, and remained stable within 70 minutes after the addition of GSH, demonstrating that Nap-Mem-GSH has excellent time stability and is of practical significance.

[0072] Example 8: Chemical stability data of probe Nap-Mem-GSH

[0073] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution of 1 mg / mL. Different chemical interfering substances were dissolved in DMSO to prepare an interfering substance stock solution of 10 mmol / mL. GSH was dissolved in DMSO to prepare a GSH stock solution of 10 mmol / mL.

[0074] The probe stock solution, interferon stock solution, and a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) were diluted to prepare a working solution with a probe concentration of 10 μg / mL and a chemical interferon concentration of 100 μM. The probe stock solution, GSH stock solution, and a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) were also diluted to prepare a working solution with a probe concentration of 10 μg / mL and a GSH concentration of 100 μM. All working solutions were reacted at room temperature for 10 minutes before detection. The fluorescence intensity of Nap-Mem-GSH at 545 nm was detected using an excitation wavelength of 390 nm. The selectivity of Nap-Mem-GSH for other analytes, including other amino acids and common biogenic metal ions, was tested. (See attached...) Figure 10 As shown, only the addition of GSH to the Nap-Mem-GSH solution produces selectivity. Only the addition of GSH to the Nap-Mem-GSH solution significantly enhances fluorescence intensity. Other substances include (2) NaSH, (3) Cys, (4) Hcy, (5) H2O2; (6) Cl. - (7)Cu 2+ (8) HClO, (9) Glu, (10) NO2 - (11) TBHP, (12) Fe 2+ (13) Ag + It does not induce fluorescence enhancement. Therefore, Nap-Mem-GSH exhibits high selectivity for GSH.

[0075] To further demonstrate Nap-Mem-GSH's selective recognition ability of GSH, we further tested the competition between GSH and other substances. The probe prepared in Example 1 was dissolved in DMSO to prepare a 1 mg / mL probe stock solution. Different chemical interfering substances were dissolved in DMSO to prepare a 10 mM interfering substance stock solution. The probe stock solution, interfering substance stock solution, and a mixture of DMSO and PBS (v / v = 1:1, 0.1 mol / L, pH = 7.4) were diluted to prepare a working solution with a probe concentration of 10 μg / mL and a chemical interfering substance concentration of 100 μM. After reacting at room temperature for 10 minutes, detection was performed, using 390 nm as the excitation wavelength to detect the fluorescence emission of Nap-Mem-GSH. Then, GSH stock solution was added to obtain the test solution, with a final GSH concentration of 10 μM. After reacting at room temperature for 10 minutes, detection was performed. (See attached...) Figure 11 As shown, the striped bars represent the results before the addition of GSH, and the black bars represent the results after the addition of GSH. Other analytes do not affect the detection of GSH by Nap-Mem-GSH. GSH is also easily detected in the presence of other analytes, and these substances do not cause fluorescence enhancement. Therefore, Nap-Mem-GSH exhibits high selectivity for GSH and strong resistance to interference.

[0076] Example 9: Nap-Mem-GSH probe cytotoxicity assay

[0077] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution of 1 mg / mL.

[0078] HeLa cells were cultured in 10% fetal bovine serum medium supplemented with penicillin (100 μg / mL), streptomycin (100 U / mL) and 10% heat-inactivated FBS, and cultured in a humid atmosphere of 37 ℃ and 5% CO2 to obtain HeLa cell culture medium.

[0079] 100 μL of HeLa cell culture medium was seeded into 96-well plates, with a cell density of 1 × 10⁶ cells / well. 5 / mL; the probe stock solution was diluted to different final concentrations using a mixture of DMSO and PBS (composition as above), added to the wells, and cultured for 24 hours. The cytotoxicity was then detected using the MTT assay.

[0080] The results are attached. Figure 12 The results showed that even at a Nap-Mem-GSH concentration of 20 μM, the viability of HeLa cells was greater than 80%. Therefore, Nap-Mem-GSH is suitable for cell imaging experiments.

[0081] Example 10: Application of the probe Nap-Mem-GSH to fluorescence imaging of GSH in cells

[0082] The probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL stock solution.

[0083] HeLa cells were cultured in 10% fetal bovine serum medium supplemented with penicillin (100 μg / mL), streptomycin (100 U / mL), and 10% heat-inactivated FBS. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. HeLa cells were then cultured in the above medium to obtain HeLa cell culture medium with a cell density of 1 × 10⁻⁶ cells / mL. 5 / mL, and divided into 4 groups.

[0084] Group A (control): Added 10 μg / mL Nap-Mem-GSH;

[0085] Group B (NEM treatment): 10 μM Nap-Mem-GSH and 5 mM n-ethylmaleimide (NEM, a well-known biothiol scavenger) were added.

[0086] Group C (H2O2 treatment): 10 μg / mL Nap-Mem-GSH and 10 μM H2O2 were added;

[0087] Group D (GSH treatment): 10 μg / mL Nap-Mem-GSH and 10 μM GSH were added; the four groups were incubated at 37℃ for 20 minutes and then fluorescence was detected.

[0088] The fluorescence of Nap-Mem-GSH was collected at 560 nm ± 20 nm. (See attached image) Figure 13 The results showed that after adding Nap-Mem-GSH (10 μg / mL) and incubating for 20 minutes:

[0089] Group A used Nap-Mem-GSH to stain cells individually. The probes were mainly concentrated on the cell membrane, and HeLa cells showed brighter yellow fluorescence.

[0090] After NEM was added to group B, the fluorescence intensity of the probe decreased due to the response of NEM and GSH, but the probe still targeted the cell membrane.

[0091] In group C, H2O2 was added. Because H2O2 disrupted the normal structure of the cell membrane, the probe could no longer target the cell membrane and entered the cell. At the same time, H2O2 could stimulate the cell to produce oxidative stress and generate GSH, so a brighter yellow fluorescence appeared inside the cell.

[0092] Group D was given exogenous GSH. GSH responded to the probe on the cell membrane, producing a brighter yellow fluorescence that accumulated on the cell membrane.

[0093] The phenomena described above demonstrate that Nap-Mem-GSH exhibits excellent cell membrane targeting and GSH responsiveness, along with superior imaging performance, and possesses significant potential for practical applications.

[0094] To further investigate the cell membrane targeting of Nap-Mem-GSH, we used DiD perchlorate, a widely used commercial cell membrane targeting dye, for co-localization studies.

[0095] HeLa cells were cultured in 10% fetal bovine serum medium supplemented with penicillin (100 μg / mL), streptomycin (100 U / mL), and 10% heat-inactivated FBS. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. HeLa cells were then cultured in the above medium to obtain HeLa cell culture medium with a cell density of 1 × 10⁻⁶ cells / mL. 5 / mL, 10 μg / mL Nap-Mem-GSH and 0.5 μM DiD perchlorate were added to the above HeLa cell culture medium, and after co-incubation for 20 minutes, the cells were observed under a fluorescence confocal microscope to obtain Figure 14 The AD images are as follows: Image A shows the yellow channel of the Nap-Mem-GSH probe; Image B shows the red channel of the commercial probe DiD; Image C shows the bright field of HeLa cells; and Image D shows the mixed channels.

[0096] HeLa cell culture medium was incubated with 10 μg / mL Nap-Mem-GSH and 0.5 μM DiD perchlorate for 20 minutes. Dual-channel imaging was then performed, and a region at the cell membrane was selected for fluorescence intensity measurement. Figure 14 E; then add 10 μM GSH, continue incubation for 20 minutes, and select another area at the cell membrane for fluorescence intensity testing to obtain... Figure 14 F.

[0097] The results showed that the probe Nap-Mem-GSH had excellent imaging performance, and the calculated Pearson colocalization coefficient was 0.95, confirming that Nap-Mem-GSH specifically targets the cell membrane of live HeLa cells.

[0098] Example 11: Application of the Nap-Mem-GSH probe in fluorescence imaging of zebrafish

[0099] In Example 1, Nap-Mem-GSH was dissolved in DMSO to prepare a probe stock solution with a concentration of 1 mg / mL. GSH was dissolved in DMSO to prepare a 10 mmol / mL GSH stock solution. First, 5-day-old zebrafish were pretreated with 1 mmol / mL PTU and then incubated with 10 μg / mL probe working solution at room temperature for 30 minutes. After rinsing three times with PBS, fluorescence imaging was performed using CLSM (see [link to CLSM]). Figure 15 (Ctrl). Nap-Mem-GSH was excited at 405 nm (single photon). The fluorescence of Nap-Mem-GSH was collected at 560 nm ± 20 nm.

[0100] like Figure 15 As shown in the Ctrl key, yellow fluorescence can be seen inside the zebrafish.

[0101] Then, GSH stock solution was added to the zebrafish after co-incubation with the PTU and probe. The final concentration of GSH was 10 μM. After incubation at room temperature for 10 minutes, fluorescence detection was performed, and enhanced yellow fluorescence was found in the zebrafish (see...). Figure 15 This demonstrates that Nap-Mem-GSH can detect exogenous GSH in zebrafish. Then, exogenous NEM (final concentration: 5 mM) was added to the co-incubation solution of GSH and zebrafish, and the mixture was incubated for 10 minutes. Changes in fluorescence intensity were detected at room temperature, revealing a decrease in fluorescence intensity within the zebrafish (see GSH treatment). Figure 15 (NEM treatment), indicating that Nap-Mem-GSH has the ability to detect GSH in zebrafish.

Claims

1. A fluorescent probe for monitoring glutathione levels across the cell membrane, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. The method for synthesizing the fluorescent probe according to claim 1, characterized in that: The fluorescent probe is synthesized by dissolving compound 1 and compound 2 in DMF, adding sodium tert-butoxide, reacting at 78-82℃ for 11-13 hours under nitrogen atmosphere, cooling, filtering, and purifying to obtain the probe. The structural formula of compound 1 is as follows: ; The structural formula of compound 2 is as follows: .

3. The method for synthesizing the fluorescent probe according to claim 2, characterized in that: The molar ratio of compound 1 to compound 2 is 1:1.9-2.1; the molar ratio of compound 2 to sodium tert-butoxide is 1:0.24-0.26; and the mass-volume ratio of compound 2 to DMF is 58.0-58.5 mg:1 mL.

4. The method for synthesizing the fluorescent probe according to any one of claims 2-3, characterized in that: The preparation method of compound 1 is as follows: 4-bromo-1,8-naphthalenedicarboxylic anhydride and 1-dodecylamine are dissolved in ethanol, refluxed at 88-92°C for 11-13 hours, and then cooled, filtered and purified to obtain compound 1.

5. The method for synthesizing the fluorescent probe according to claim 4, characterized in that: The molar ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to 1-dodecylamine is 1:1.9-2.1; the mass-volume ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to ethanol is 1g:14.5-15.5mL.

6. The use of the fluorescent probe of claim 1 in reagents for sensing and detecting glutathione inside and outside the cell membrane in biological systems.

7. The application of the fluorescent probe according to claim 6 in the preparation of reagents for sensing and detecting glutathione across cell membranes in biological systems, characterized in that: The sensing detection is either fluorescence detection or cell imaging.

Images