A fluorescent probe for detecting thiols, its preparation method and application

Abstract

This invention belongs to the fields of synthetic chemistry and medical detection technology, and provides a fluorescent probe for detecting thiols, its preparation method, and its applications. The fluorescent probe can detect dynamic changes in endogenous and exogenous biothiols levels in living cells using fluorescence imaging technology. Furthermore, it can also detect dynamic changes in biothiols levels in the endoplasmic reticulum of cells with drug-induced liver injury. This invention has potential application value in studying the mechanism of action of biothiols in the endoplasmic reticulum of cells with drug-induced liver injury.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic chemistry and medical detection technology, specifically relating to a fluorescent probe for detecting thiols and its application. Background Technology

[0002] Biothiols typically include cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), all of which contain a sulfhydryl group in their molecular structure. These sulfhydryl groups, with their nucleophilic and reducing properties, play a crucial role in maintaining cellular redox homeostasis. Under normal physiological conditions, the intracellular concentration of Cys is typically 30-200 μmol / L, Hcy is 5-12 μmol / L, while GSH concentrations are much higher than Cys and Hcy, reaching 1-10 mmol / L. Imbalances in cellular thiol levels can lead to cardiovascular disease, Alzheimer's disease, age-related cognitive impairment, cancer, and drug-induced liver injury. Therefore, detecting dynamic changes in thiol levels in living systems is of great significance for the early diagnosis of diseases such as drug-induced liver injury.

[0003] The endoplasmic reticulum is a large organelle in eukaryotic cells, involved in processes including protein processing, lipid metabolism, and calcium metabolism. 2+ Thiols play a crucial role in cellular physiological processes such as storage. During endoplasmic reticulum (ER) stress, Cys can capture excess reactive oxygen species (ROS) and free radicals. Hcy, a homologue of Cys, also plays an important role in maintaining cellular homeostasis. Elevated Hcy levels lead to the excessive accumulation of misfolded proteins in the ER, triggering the unfolded protein response (UPR) and generating excessive ROS. GSH is one of the most abundant biogenic thiols, participating in disulfide bond formation in the ER and acting as an important antioxidant to protect cells from oxidative stress damage. Furthermore, these three biogenic thiols are closely related. Cys and Hcy can generate endogenous GSH under the action of enzymes such as cysteine ​​β-synthase (CBS) and cysteine ​​γ-lyase (CSE). Therefore, detecting dynamic changes in thiol levels in the ER is of practical significance for the early diagnosis of drug-induced liver injury.

[0004] Currently, clinical methods for diagnosing liver diseases mainly include medical imaging techniques, routine blood tests, and biopsies. These methods typically have drawbacks such as radiation risks, lack of specificity in biomarkers, and invasive procedures. In contrast, fluorescent probe technology, due to its low cost, speed, real-time nature, non-invasiveness, and high resolution, has become a potential alternative tool for diagnosing the occurrence and development of liver diseases. Therefore, there is an urgent need to develop a fluorescent probe for detecting the dynamic changes in biothiols in the endoplasmic reticulum of cells affected by drug-induced liver injury, in order to elucidate the mechanism of action of biothiols in the physiological and pathological processes of drug-induced liver injury. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a fluorescent probe for detecting biothiols, which has a fast response speed, good photostability, and specific response to thiols. Moreover, this probe can locate the endoplasmic reticulum.

[0006] Another object of the present invention is to provide an application of the above-mentioned fluorescent probe in the detection of biothiols in biological cells.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A fluorescent probe for detecting biothiols, 4-thiocarbamate-N-(2-aminoethyl)-4-methylphenyl-1-sulfonamide naphthalimide, abbreviated as ER-pGSH, has the chemical structural formula shown in formula (I): Formula (I).

[0009] The preparation method of the above-mentioned fluorescent probe includes the following steps: (1) 4-Bromo-1,8-naphthalenedicarboxylic anhydride and N-(2-aminoethyl)-4-methylbenzenesulfonamide react under reflux to produce ; (2) In the presence of potassium carbonate and N-hydroxysuccinimide, compound 1 reacts under reflux in dimethyl sulfoxide to produce ; (3) Under nitrogen protection, catalyzed by N,N'-diisopropylethylamine base, compound 2 and phenyl thiochloroformate reacted in anhydrous dichloromethane at room temperature to generate compound ER-pGSH, i.e., a fluorescent probe.

[0010] Application of the above-mentioned fluorescent probe in the detection of biothiols in solutions and cells.

[0011] A reagent for preparing the above-mentioned fluorescent probe.

[0012] The mechanism of this invention is as follows: The fluorescent probe ER-pGSH for detecting biothiols described in this invention uses a phenyl thiocarbamate group as a weak electron-donating group and an amide group as a strong electron-withdrawing group, connected by a π bond. The molecule exhibits a "D-π-A" molecular structure, resulting in a weak intramolecular electron push-pull effect and weak fluorescence. When the probe ER-pGSH interacts with a thiol, the phenyl thiocarbamate group leaves, exposing a hydroxyl group. The hydroxyl group is a strong electron-donating group, enhancing the intramolecular electron push-pull effect and emitting strong fluorescence.

[0013] The present invention has the following advantages: The biothiol fluorescent probe provided in this invention belongs to the small molecule fluorescent probe category. Currently, there are few reported small molecule fluorescent probes for recognizing thiols in the endoplasmic reticulum (ER), especially those recognizing thiols in the ER of cells with drug-induced liver injury. The optical properties of the probe were demonstrated using a UV-Vis spectrophotometer and fluorescence spectroscopy. The biothiol fluorescent probe provided in this invention exhibits advantages such as high fluorescence amplification (49-fold), fast response speed (7 min), specific thiol response, and good photostability. These advantages lay a theoretical foundation for biological imaging applications. Building upon optical testing, the application is further extended to the cellular level. The probe of this invention can detect dynamic changes in endogenous and exogenous biothiol levels in living cells using fluorescence imaging technology. Furthermore, it can also detect dynamic changes in biothiol levels in the ER of cells with drug-induced liver injury. This invention has potential application value in studying the mechanism of action of biothiols in the ER of cells with drug-induced liver injury. Attached Figure Description

[0014] Figure 1 It is a probe ER-pGSH 1 H NMR spectrum; Figure 2 It is a probe ER-pGSH 13 C NMR spectrum; Figure 3 The fluorescence titration spectroscopy test was performed on the interaction between the probe ER-pGSH and GSH; the excitation wavelength was 455 nm, the slit width was 6 nm; the probe concentration was 10 µmol / L, and the GSH concentration was 0-109 µmol / L. Figure 4 This is a standard curve of fluorescence intensity versus concentration of the probe ER-pGSH at 566 nm. Figure 5 The fluorescence kinetics spectrometry of the interaction between the probe ER-pGSH and GSH was measured; the excitation wavelength was 455 nm, the slit width was 6 nm, the probe concentration was 10 µmol / L, the GSH concentration was 100 µmol / L, and the test time was 30 min. Figure 6 The photostability test of the probe ER-pGSH was conducted; the excitation wavelength was 455 nm, the slit was 6 nm, the probe concentration was 10 µmol / L, and the GSH concentration was 100 µmol / L. Figure 7 This is a selective assay for the probe ER-pGSH; the excitation wavelength is 455 nm, the slit is 6 nm; the probe concentration is 10 µmol / L, and the analyte concentration is 100 µmol / L. Figure 8This is an application of endoplasmic reticulum co-localization cell imaging using the probe ER-pGSH and commercial probes; Red channel: excitation wavelength: 561 nm, acquisition band: 570-620 nm; Green channel: excitation wavelength: 405 nm, emission band: 500-570 nm; Figure 9 It is an application of the probe ER-pGSH for exogenous GSH cell imaging; excitation wavelength: 405 nm, acquisition band: 500-570 nm; Figure 10 It is an application of the probe ER-pGSH for endogenous GSH cell imaging; excitation wavelength: 405 nm, acquisition band: 500-570 nm; Figure 11 This is an application of the probe ER-pGSH in GSH imaging of drug-induced liver injury cells; excitation wavelength: 405 nm, acquisition band: 500-570 nm. Detailed Implementation

[0015] 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.

[0016] Example 1 Synthesis of the fluorescent probe ER-pGSH The reaction proceeds according to the following route: .

[0017] (1) Synthesis of intermediate 1 4-Bromo-1,8-naphthalenedicarboxylic anhydride (3.0 mmol) and N-(2-aminoethyl)-4-methylbenzenesulfonamide (3.0 mmol) were heated to reflux at 85 °C for 7.5 h in anhydrous ethanol. After the reaction was monitored for completeness by thin-layer chromatography, the reaction system was cooled to room temperature, filtered under reduced pressure, and the filter cake was washed repeatedly with small amounts of anhydrous ethanol. The mixture was then dried under vacuum to give compound 1, a brown solid, in 91% yield. 1 H NMR (400 MHz, DMSO- d 6) δ 8.54 (d, J = 5.6 Hz, 1H), 8.53 (d, J = 4.0 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.0 (t, J = 8.0 Hz, 1H), 7.79(s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 4.11 (t,J = 6.4 Hz, 2H), 3.05-3.16 (m, 2H), 2.25 (s, 3H).

[0018] (2) Synthesis of intermediate 2 Intermediate 1 (2.7 mmol) was dissolved in 10 mL of dimethyl sulfoxide, followed by the addition of K₂CO₃ (9.0 mmol) and N-hydroxysuccinimide (3.0 mmol). The mixture was heated under nitrogen protection at 100 °C for 6 h under reflux. After monitoring the reaction for completeness by thin-layer chromatography, the mixture was cooled to room temperature, and the reaction solution was poured into ice water to adjust the pH to 6. The mixture was then filtered under reduced pressure, and the filter cake was washed three times with small amounts of water. The solid was then dried under vacuum. The dried solid was purified by column chromatography (using [eluent]). V DCM : V MeOH = 50 : 1) yielded yellow solid intermediate 2, yield: 11%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.89 (br s, 1H), 8.56 (d, J = 8.4Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.34 (d, J = 8.2 Hz, 1H), 7.76-7.80 (m, 1H),7.72-7.75 (m, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.17 (d, J =8.2 Hz, 1H), 4.10 (t, J = 6.4 Hz, 2H), 3.08 (q, J = 6.4 Hz, 2H), 2.27 (s, 1H).

[0019] (3) Synthesis of compound ER-pGSH Intermediate 2 (0.3 mmol) was dissolved in 3 mL of anhydrous dichloromethane. Then, 3 drops of N,N'-diisopropylethylamine were added to the reaction system. Under nitrogen protection, the reaction system was placed in a cryogenic reactor and stirred at 0°C for 30 min. Next, an anhydrous dichloromethane solution containing phenyl thiocarbamate (0.6 mmol) was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and the reaction continued for 24 h. After the reaction was monitored for completeness by thin-layer chromatography, the reaction solution was poured into three layers of water and adjusted to neutral with a 2.7% sodium bicarbonate solution. The reaction solution was then extracted with dichloromethane solution (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate, then distilled under reduced pressure and dried under vacuum. The crude product was purified by column chromatography (elution). V DCM : V EA =50 : 1) yielded a white solid compound ER-pGSH, yield: 74%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.51-8.59 (m, 3H), 8.00 (t, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 6.4 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 6.4 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 4.14 (t, J = 6.4 Hz, 2H), 3.11(q, J = 6.4 Hz, 2H), 2.29 (s, 1H). 13 C NMR (101 MHz, DMSO- d 6) δ193.99, 163.76, 163.22, 153.75, 153.43, 142.99, 138.07, 131.81, 131.76, 130.51, 130.01, 129.32, 128.80, 128.17, 127.77, 126.86, 124.83, 123.28, 122.27, 121.80, 121.21, 21.41; 1 H NMR spectrum as follows Figure 1 ; 13 C NMR spectra as follows Figure 2 .

[0020] Example 2: Response of fluorescent probe to different concentrations of GSH Prepare 4 mL of a 10 mmol / L GSH aqueous solution and 4 mL of a 1 mmol / L fluorescent probe stock solution. Using a PBS buffer solution containing 30% dimethyl sulfoxide as the test system, the fluorescence emission spectra of the interaction between the probe ER-pGSH (10 μmol / L) and different concentrations of GSH (0-109 μmol / L) were measured (excitation wavelength 455 nm, slit width 6 nm). A standard curve was established comparing the fluorescence intensity at the maximum emission wavelength (566 nm) with the GSH concentration. Figure 3 , Figure 4 As shown, within the tested concentration range, the fluorescence intensity of the reaction system gradually increased with increasing GSH concentration, exhibiting good linear correlation, with a linear correlation coefficient R0. 2 = 0.966.

[0021] Example 3: Fluorescence kinetics of the fluorescent probe against GSH Prepare 4 mL of 10 mmol / L GSH aqueous solution and 4 mL of 1 mmol / L fluorescent probe stock solution. Using a PBS buffer solution containing 30% dimethyl sulfoxide as the test system, add 100 μmol / L GSH solution to 10 μmol / L probe test solution and measure its fluorescence emission spectrum. Test every 1 min for 30 min. Construct a scatter plot of fluorescence intensity at the maximum emission wavelength (566 nm) versus reaction time. Figure 5 As shown, the fluorescence intensity of the reaction system reaches equilibrium after approximately 7 minutes of reaction.

[0022] Example 4: Photostability of Fluorescent Probes Prepare 4 mL of a 10 mmol / L GSH aqueous solution and 4 mL of a 1 mmol / L fluorescent probe stock solution. Using a PBS buffer solution containing 30% dimethyl sulfoxide as the test system, measure the fluorescence emission spectra of the probe ER-pGSH (10 μmol / L) under both illuminated and non-illuminated conditions, as well as the fluorescence emission spectra of the probe ER-pGSH (10 μmol / L) after interaction with GSH (100 μmol / L) under both illuminated and non-illuminated conditions. Tests were performed every 5 min for a total of 60 min. Figure 6 It can be observed that illumination and non-illumination conditions have almost no effect on the fluorescence emission of the probe ER-pGSH, indicating that the probe has good photostability.

[0023] Example 5: Selectivity of fluorescent probes for different types of analytes Prepare 4 mL of 10 mmol / L aqueous solutions of various analytes and 4 mL of 1 mmol / L fluorescent probe stock solution. Using PBS buffer solution containing 30% dimethyl sulfoxide as the test system, add 100 μmol / L aqueous solutions of various analytes to 10 μmol / L probe test solution. After the probe interacts with the analytes for 20 min, measure their fluorescence emission spectra. Construct a bar chart of fluorescence intensity at the maximum emission wavelength (566 nm) versus various analytes. The results are shown in [Figure number missing]. Figure 7 The ions added in groups 1-24 are: probe, alanine, methionine, lysine, leucine, tryptophan, threonine, valine, isoleucine, copper sulfate, ferric sulfate, ferrous sulfate, potassium chloride, magnesium sulfate, sodium chloride, formaldehyde, acetone, hydrogen peroxide, sodium hypochlorite, sodium hydrosulfide, sodium bisulfite, cysteine, homocysteine, and glutathione. Figure 7 It can be observed that other ions (or amino acids) have almost no effect on the fluorescence of the probe ER-pGSH, while the addition of thiols significantly enhances the fluorescence of the probe ER-pGSH.

[0024] Example 6: Co-localization fluorescence imaging of fluorescent probes and commercial probes Logarithmically growing HepG2 liver cancer cells were seeded into confocal cell culture dishes and cultured in an incubator (37℃, 5% CO2). After cell attachment, confocal cell imaging experiments were performed. 10 μmol / L of the probe ER-pGSH and 1 μmol / L of endoplasmic reticulum red localization dye were added to the cells and incubated for 30 min. Confocal fluorescence imaging was then performed using a confocal fluorescence microscope. Results are as follows: Figure 8As shown, a) is a bright-field plot of the interaction between HepG2 cells and the probe ER-pGSH and the commercial endoplasmic reticulum co-localization dye ER Tracker Red; b) is a red fluorescence field plot of the interaction between HepG2 cells and the probe ER-pGSH and the commercial endoplasmic reticulum co-localization dye ER Tracker Red; c) is a green fluorescence field plot of the interaction between HepG2 cells and the probe ER-pGSH and the commercial endoplasmic reticulum co-localization dye ER Tracker Red; d) is a superimposed plot of a), b), and c); e) is a scatter plot of fluorescence channel intensities; and f) is a correlation plot of the intensities of the probe ER-pGSH and ER Tracker Red. The test results show that the Pearson correlation coefficient is as high as 0.9150, indicating that the endoplasmic reticulum-targeting thiol fluorescent probe ER-pGSH can highly target the endoplasmic reticulum of cells.

[0025] Example 7: Imaging application of fluorescent probes to exogenous GSH in living cells HepG2 liver cancer cells in logarithmic growth phase were seeded into cell culture dishes and cultured in an incubator (37℃, 5% CO2). After cell attachment, HepG2 cells were pretreated with 500 μmol / L N-ethylmaleimide (NEM) for 30 min, and then the cells were divided into four groups for imaging. (1) Add 5 μmol / L probe ER-pGSH, incubate for 30 min and then image; (2) Add 50 μmol / L glutathione ethyl ester (GSH-MEE) and incubate for 30 min. Finally, add 5 μmol / L probe ER-pGSH and incubate for 30 min before imaging. (3) Add 100 μmol / L GSH-MEE and incubate for 30 min, then add 5 μmol / L probe ER-pGSH and incubate for 30 min before imaging; (4) Add 200 μmol / L GSH-MEE and incubate for 30 min. Finally, add 5 μmol / L probe ER-pGSH and incubate for 30 min before imaging.

[0026] See results Figure 9The diagrams are as follows: a) is a bright-field image of HepG2 cells interacting with probes ER-pGSH and NEM; e) is a fluorescence field image of HepG2 cells interacting with probes ER-pGSH and NEM; i) is a superimposed image of a) and e); b)-d) are bright-field images of HepG2 cells interacting with probes ER-pGSH, NEM, and different concentrations of GSH-MEE; f)-h) are fluorescence field images of HepG2 cells interacting with probes ER-pGSH, NEM, and different concentrations of GSH-MEE; j)-l) are superimposed images of b) and f), c) and g), d) and h). As the concentration of added GSH-MEE increases, the fluorescence intensity emitted by HepG2 cells gradually increases, indicating that the probe ER-pGSH can be used for the dynamic detection of GSH levels in live cells.

[0027] Example 8: Imaging application of fluorescent probes to endogenous GSH in living cells HepG2 liver cancer cells in the logarithmic growth phase were seeded into cell culture dishes and then cultured in an incubator (37℃, 5% CO2). After the cells adhered, they were divided into two groups for fluorescence imaging experiments. (1) Imaging of HepG2 liver cancer cells after incubation with 5 μmol / L probe ER-pGSH for 30 min; (2) HepG2 cells were pretreated with 500 μmol / L N-ethylmaleimide for 30 min, and then 5 μmol / L probe ER-pGSH was added. After incubation for 30 min, imaging was performed.

[0028] See results Figure 10 The images show: a) a bright-field image of HepG2 cells interacting with the ER-pGSH probe; b) a fluorescence field image of HepG2 cells interacting with the ER-pGSH probe; c) a superimposed image of a) and b); d) a bright-field image of HepG2 cells interacting with the ER-pGSH probe and NEM; e) a fluorescence field image of HepG2 cells interacting with the ER-pGSH probe and NEM; and f) a superimposed image of d) and e). This demonstrates that the ER-pGSH probe can be used for the dynamic detection of endogenous GSH in cells.

[0029] Example 9: Imaging application of fluorescent probes to GSH in drug-induced liver injury cells HepG2 liver cancer cells in the logarithmic growth phase were seeded into cell culture dishes and then cultured in an incubator (37℃, 5% CO2). After the cells adhered, they were divided into six groups for fluorescence imaging experiments. (1) Imaging of HepG2 liver cancer cells after incubation with 5 μmol / L probe ER-pGSH for 30 min; (2) HepG2 liver cancer cells were pretreated with 500 μmol / L acetaminophen (APAP) for 8 h, then the original culture medium was removed, and 5 μmol / L probe ER-pGSH was added and incubated for 30 min before imaging. (3) HepG2 liver cancer cells were pretreated with 500 μmol / L APAP and 15 μmol / L ferroptosis inhibitor (Fer-1) for 8 h, and finally incubated with 5 μmol / L probe ER-pGSH for 30 min before imaging. (4) HepG2 liver cancer cells were pretreated with 500 μmol / L APAP for 8 h, then treated with 100 μmol / L NaHS for 1 h, and finally incubated with 5 μmol / L probe ER-pGSH for 30 min before imaging. (5) HepG2 liver cancer cells were pretreated with 500 μmol / L APAP for 8 h, then treated with 20 μmol / L methylprednisolone (MPDN) for 1 h, and finally incubated with 5 μmol / L probe ER-pGSH for 30 min before imaging. (6) HepG2 liver cancer cells were pretreated with 500 μmol / L APAP for 8 h, then incubated with 20 μmol / L vitamin C (VC) for 1 h, and finally incubated with 5 μmol / L probe ER-pGSH for 30 min before imaging.

[0030] See results Figure 11The diagrams are as follows: a) is a bright field image of HepG2 cells interacting with the probe ER-pGSH; g) is a fluorescence field image of HepG2 cells interacting with the probe ER-pGSH; m) is an overlay of a) and g); b) is a bright field image of HepG2 cells interacting with the probes ER-pGSH and APAP; h) is a fluorescence field image of HepG2 cells interacting with the probes ER-pGSH and APAP; n) is an overlay of b) and h); c)-f) are bright field images of HepG2 cells interacting with the probe ER-pGSH and different therapeutic drugs (Fer-1, NaHS, MPDN, VC); i)-l) are fluorescence field images of HepG2 cells interacting with the probe ER-pGSH and different therapeutic drugs (Fer-1, NaHS, MPDN, VC); o)-r) are overlays of c) and i), d) and j), e) and k), f) and l). The fluorescence intensity of cells with drug-induced liver injury was decreased compared to normal cells, indicating a lower GSH level in these cells. Treatment with ferroptosis inhibitors, sodium hydrosulfide, methylprednisolone, and vitamin C increased the fluorescence intensity of these cells, suggesting that drug treatment can increase GSH levels. These experimental results demonstrate that the probe ER-pGSH can be used for the dynamic detection of GSH levels in cells with drug-induced liver injury.

Claims

1. A fluorescent probe for detecting biothiols, the chemical structure of which is shown in formula (I): Formula (I).

2. A method for preparing the fluorescent probe according to claim 1, characterized by, Includes the following steps: (1) 4-bromo-1,8-naphthalic anhydride and N-(2-aminoethyl)-4-methylbenzenesulfonamide are heated to reflux to produce ; (2) Compound 1 was reacted in dimethyl sulfoxide in the presence of potassium carbonate, N-hydroxy succinimide, and heated to reflux to form ; (3) Under nitrogen protection, compound 2 and phenyl thiochloroformate reacted in anhydrous dichloromethane at room temperature to generate a fluorescent probe, catalyzed by N,N'-diisopropylethylamine base.

3. The application of the fluorescent probe according to claim 1 in the detection of biothiols in solutions and cells.

4. A reagent for preparing the fluorescent probe according to claim 1.

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