A fluorescent probe for distinguishing detection of Hcy, Cys and GSH, and a preparation method and a detection method thereof

Abstract

The application discloses a fluorescent probe for differentiating and detecting Hcy, Cys and GSH and a preparation method and a detection method thereof, and belongs to the technical field of fluorescent probes. The fluorescent probe for differentiating and detecting Hcy, Cys and GSH is C-Jul, and the fluorescent probe is used for differentiating and detecting Hcy, Cys and GSH, effectively interferes with the recognition effect of complex biological factors, and significantly improves the detection sensitivity and the signal-to-noise ratio. The fluorescent probe has the characteristics of differentiating and detecting effects and high detection efficiency, and also has stable pH. The probe has a relatively fast response time in recognizing Hcy, Cys and GSH, has the ability to monitor the dynamic changes of Hcy, Cys and GSH, has strong anti-interference, and can accurately recognize Hcy under the existence of various physiological interference factors in real seminal plasma samples.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a fluorescent probe for distinguishing and detecting Hcy, Cys and GSH, and its preparation and detection methods. Background Technology

[0002] Hcy (Hydroxylcysteine) is a sulfur-containing amino acid, and recent studies have found it to be closely related to male reproductive function. Clinical data show that Hcy levels in the seminal plasma of patients with oligospermia, asthenospermia, and teratospermia are significantly higher than in normal men, and Hcy concentration is negatively correlated with sperm quality. Mechanistic studies have shown that Hcy can induce oxidative stress, leading to the accumulation of reactive oxygen species, which in turn affects sperm motility and damages sperm DNA. Based on the crucial role of Hcy in spermatogenesis, seminal plasma Hcy levels are expected to become a molecular marker reflecting early abnormalities in the male reproductive system. Compared to traditional semen parameters, Hcy detection can indicate potential disease risks before significant changes in sperm morphology and quantity occur, buying valuable time for early clinical intervention and treatment.

[0003] Currently, the detection of hemolytic cytokines (Hcy) in seminal plasma mainly relies on high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). While these methods are accurate, they typically require cumbersome sample pretreatment, expensive equipment, or lengthy incubation processes, limiting their application in clinical testing. Fluorescent probe technology, due to its high sensitivity, ease of operation, and real-time visualization, offers a highly promising alternative for rapid Hcy detection. In contrast, fluorescent probe detection technology, with its high sensitivity, rapid response, ease of operation, strong tissue penetration, and low background interference, has become the ideal choice for Hcy detection. In recent years, fluorescent probes based on mechanisms such as Michael addition, cyclization, and nucleophilic substitution have emerged, achieving selective recognition of bio-thiols. However, due to the high similarity in structure and reactivity of Hcy, cysteine ​​(Cys), and glutathione (GSH), simultaneously distinguishing these three thiols using a single probe remains a major challenge in the field. While the few sensors reported so far have demonstrated discriminative capabilities, they still have shortcomings. More importantly, despite the increasingly clear key role of Hcy in male infertility, research on applying high-performance fluorescent probes to thiol imaging and seminal plasma detection in the reproductive system remains lacking.

[0004] Therefore, developing a fluorescent probe that can simultaneously distinguish Hcy, Cys, and GSH under physiological conditions through multiple non-interfering fluorescent channels is not only of methodological innovation significance, but also fills the gap in its application in the early diagnosis of male infertility. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a fluorescent probe for distinguishing and detecting Hcy, Cys and GSH, as well as a method for preparing and detecting the probe, in order to solve the above problems.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH. The fluorescent probe is characterized by being C-Jul, which is (9-[(4-fluorophenyl)thio]-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxaldehyde). This fluorescent probe is used to distinguish and detect Hcy, Cys, and GSH, effectively mitigating the interference of complex biological factors on the recognition effect and significantly improving detection sensitivity and signal-to-noise ratio.

[0007] Furthermore, the fluorescent probe C-Jul is able to distinguish and detect Hcy, Cys and GSH and generate changes in fluorescence signals, and its product fluorescence has significant color differentiation.

[0008] Furthermore, the fluorescent probe C-Jul exhibits low toxicity and good biocompatibility in biological systems.

[0009] Furthermore, a method for preparing a fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH includes the following steps: S1. Weigh 8-hydroxyjulonidine and bis(2,4,6-trichlorophenyl) malonate and dissolve them in toluene. After thorough stirring and dissolution, heat under reflux at 110°C for 10 h. After the reaction is complete, return to room temperature, filter under reduced pressure, and wash with petroleum ether and ethyl acetate to obtain a white product LM-2. The LM-2 is (9-hydroxy-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-11-one). S2. Under nitrogen protection, phosphorus oxychloride was added dropwise to anhydrous N,N-dimethylformamide. After stirring at room temperature for 60 minutes, a light pink solution was obtained. Then, LM-2 dissolved in DMF was added dropwise to the light pink solution. The solution was then heated to 60°C and refluxed until the reaction was complete. The reaction mixture was then poured into ice water, and after the solid precipitated, it was filtered under reduced pressure, washed with pre-cooled ethanol, and dried to obtain an orange solid product LM-3. The LM-3 is (9-chloro-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxaldehyde). S3. Weigh LM-3 and dissolve it in dichloromethane, then add 4-fluorothiophenol and a catalytic amount of triethylamine. React at room temperature for 12 hours. After the reaction is complete, remove the solvent under reduced pressure. The crude product is purified by silica gel column chromatography, which is probe C-Jul.

[0010] Further, in step S1, the molar ratio of 8-hydroxyjulonidine to bis(2,4,6-trichlorophenyl) malonate is 1:1.1.

[0011] Further, in step S2, the molar ratio of LM-2 to phosphorus oxychloride is 1:5.

[0012] Further, in step S3, the molar ratio of LM-3 to 4-fluorothiophenol is 1:1, the eluent phase for column chromatography purification is composed of petroleum ether and ethyl acetate in a volume ratio of 4:1, and the catalytic amount is 60 μL.

[0013] Furthermore, a detection method for distinguishing between Hcy, Cys, and GSH includes the following steps: P1. Introduce probe C-Jul into the biological sample to be tested; P2 induces the generation of fluorescence signals; P3. Detect the generated fluorescence signal to determine the presence or concentration of Hcy, Cys, and GSH.

[0014] Furthermore, the biological sample is selected from live cells and seminal plasma.

[0015] Furthermore, the detection method is used for imaging visualization and tracking of exogenous Hcy in living cells.

[0016] The beneficial effects of this invention are as follows: 1. Coumarin compounds, as a classic class of fluorescent dyes, possess advantages such as high fluorescence quantum yield, good photostability, and ease of structural modification, making them ideal fluorophores for constructing fluorescent probes. By rationally modifying the coumarin matrix and introducing specific recognition groups, selective fluorescence responses to target analytes can be achieved. Based on this, this invention uses coumarin as a backbone, introduces a p-fluorophenylthio group as a photoinduced electron transfer quenching unit, and uses an aldehyde group as an auxiliary recognition site. Through optimized screening, an activated fluorescent probe C-Jul capable of efficiently detecting homocysteine ​​was successfully prepared.

[0017] 2. The fluorescent probe of the present invention has the characteristics of distinguishing detection effects, high detection efficiency, and stable pH properties.

[0018] 3. The probe of the present invention has a fast response time in identifying Hcy, Cys and GSH, and has the ability to monitor the dynamic changes of Hcy, Cys and GSH; it also has strong anti-interference ability and can accurately identify Hcy in real seminal plasma samples with a variety of physiological interference factors.

[0019] 4. The fluorescent probe of the present invention is suitable for live cell imaging and has good biocompatibility; the detection method provided by the present invention is simple to operate, uses inexpensive solvents, and is convenient for post-processing.

[0020] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0021] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 This is a synthetic route diagram of the fluorescent probe C-Jul of the present invention; Figure 2 (a) is the proton NMR spectrum of the fluorescent probe C-Jul of this invention; Figure 2 (b) Carbon NMR spectrum of fluorescent probe C-Jul; Figure 3 (a) is a graph showing the experimental results of the selectivity of the fluorescent probe C-Jul of the present invention for common ions in body fluids; Figure 3 (b) is a graph showing the anti-interference experimental results of the fluorescent probe C-Jul of the present invention against common ions in body fluids; Figure 4 (a) The fluorescence spectrum and linear fitting diagram of the fluorescent probe C-Jul of the present invention after reacting with 25-50 μM Hcy; Figure 4 (b) shows the fluorescence spectrum and linear fitting diagram of the fluorescent probe C-Jul of the present invention after reacting with 25-50 μM Cys; Figure 4 (c) shows the fluorescence spectrum and linear fitting pattern of the fluorescent probe C-Jul of the present invention after reacting with 25-50 μM GSH; Figure 5 The fluorescence trend diagram of the fluorescent probe C-Jul of the present invention at different concentrations and at different times with Hcy, Cys and GSH; Figure 6(a) is a graph showing the cytotoxicity assessment of the fluorescent probe C-Jul of the present invention in NTERA-2 cl.D1 cells; Figure 6 (b) is a real-time intracellular fluorescence imaging diagram of exogenous Hcy by the fluorescent probe C-Jul of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the present invention.

[0023] like Figures 1-6 As shown, this invention discloses a fluorescent probe for distinguishing and detecting Hcy, Cys and GSH, as well as its preparation and detection methods.

[0024] Example 1 A fluorescent probe for distinguishing and detecting Hcy, Cys and GSH.

[0025] S1. In a 50 mL round-bottom flask, 8-hydroxyjulonidine (2 g, 10.57 mmol) and bis(2,4,6-trichlorophenyl) malonate (5.38 g, 11.62 mmol) were dissolved in 10 mL of toluene. After thorough stirring, the mixture was heated under reflux at 110 °C for 10 h until the reaction was complete and the solution turned into a dark turbid liquid. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed with pre-cooled petroleum ether and ethyl acetate. The product was dried to give a white product LM-2, wherein LM-2 is (9-hydroxy-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-11-one). S2. Under nitrogen protection, phosphorus oxychloride (1.5 mL) was added dropwise to anhydrous N,N-dimethylformamide (1.5 mL), and stirred at room temperature for about 60 minutes to obtain a light pink solution. Then, LM-2 (1 g, 3.89 mmol) was dissolved in 5 mL of DMF and then slowly added dropwise to the light pink solution. The resulting mixture was heated to 60 °C and refluxed until the reaction was complete. The reaction mixture was then poured into 80 mL of ice water, and a large amount of solid precipitated. The filter cake was collected and washed with pre-cooled ethanol to obtain the yellow product LM-3, which is (9-chloro-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxaldehyde). S3. Dissolve LM-3 (500 mg, 1.65 mmol) in 5 mL of dichloromethane, then add 4-fluorothiophenol (211.5 mg, 1.65 mmol) and a catalytic amount of triethylamine. Stir at room temperature until the reaction is complete, remove the solvent under vacuum, and purify the crude product by silica gel column chromatography to obtain the fluorescent probe C-Jul. C-Jul is (9-[(4-fluorophenyl)thio]-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxaldehyde). The fluorescent probe is used to distinguish and detect Hcy, Cys, and GSH, effectively reducing the interference of complex biological factors on the recognition effect and significantly improving the detection sensitivity and signal-to-noise ratio.

[0026] Figure 2 (a) is the ¹H NMR spectrum of C-Jul: ¹H NMR (400 MHz, Chloroform-d) δ 10.30 (s, 1H), 7.42 (s, 1H), 7.34 (dd, J = 8.9, 5.0 Hz, 2H), 7.00 (t, J = 8.6 Hz, 2H), 3.39 – 3.33 (m, 4H), 2.90 (t, J = 6.4 Hz, 2H), 2.62 (t, J = 6.9 Hz, 2H), 2.03 – 1.92 (m, 4H). Figure 2 (b) is the C-Jul NMR spectrum: ¹³C NMR (101 MHz, CDCl3) δ 188.17, 163.59, 161.12, 160.52, 158.59, 151.67, 148.99, 132.56, 130.77, 126.57, 119.45, 116.90, 116.68, 108.93, 106.03, 50.43, 50.01, 27.61, 21.19, 20.28. Example 2 A detection method using a fluorescent probe to distinguish between Hcy, Cys, and GSH.

[0027] P1. Accurately weigh the fluorescent probe C-Jul prepared in Example 1 and dissolve it in dimethyl sulfoxide (DMSO) solution to prepare a 1 mM solution for later use. P2. Prepare stock solutions of Hcy, Cys, GSH and other amino acids and metal ions using deionized water; add 20 µL of 1 mM probe C-Jul stock solution and DMSO solution to centrifuge tubes to dilute the system to a total volume of 2 mL. P3. Finally, add 20 µL of solutions of different concentrations of Hcy, Cys and GSH, as well as common biomolecules or ions. Shake to mix thoroughly and immediately collect fluorescence spectral data. The fluorescence test parameters are set as follows: excitation slit 5 nm, emission slit 5 nm, excitation wavelength 405 / 488 nm.

[0028] The results are as follows Figure 3 (a) and Figure 3 As shown in (b), when the fluorescent probe C-Jul was interacted with common biomolecules or ions including Gly, Lys, Ser, Trp, Tyr, Phe, Ala, Asn, Met, Ca2+, Fe2+, Fe3+, K+, HClO, H2O2 and ONOO-, as well as Hcy, Cys and GSH, only Hcy, Cys and GSH showed significant fluorescence enhancement at 520 / 565 nm.

[0029] The detection method provided by this invention is simple to operate, uses inexpensive solvents, and has convenient post-processing.

[0030] Example 3 Fluorescent probe C-Jul was tested by fluorescence spectroscopy titration with Hcy, Cys and GSH.

[0031] like Figure 4 (a) Figure 4 (b) and Figure 4 As shown in (c), when excited by 405 / 488 nm light, the fluorescence intensity of Hcy, Cys, and GSH (25-500 µM) was significantly enhanced at 520 and 565 nm, respectively. The three thiols (Hcy, Cys, and GSH) showed good linearity (R2=0.997, 0.994, and 0.997) at concentrations of 10-90 μM, with detection limits of 22 nM, 11 nM, and 28 nM, respectively.

[0032] Example 4 Kinetic analysis of the reaction process between the fluorescent probe C-Jul and Hcy, Cys and GSH.

[0033] like Figure 5 As shown, when the excitation wavelength is 405 / 488 nm, a graph is plotted with the fluorescence intensity at 520 / 560 nm as the ordinate and time as the abscissa. After the addition of Hcy, Cys and GSH, the fluorescence of the fluorescent probe C-Jul at 520 / 560 nm gradually increased, and the fluorescence intensity tended to stabilize at 8, 16 and 4 min.

[0034] The above results indicate that the probe of the present invention has a fast response speed and can quickly detect the content of Hcy, Cys and GSH.

[0035] Example 5 Cell imaging applications.

[0036] The fluorescent probe C-Jul was applied to NTERA-2 cl.D1 cells for the following experiments: Cytotoxicity assessment: The effect of probes at different concentrations on the survival of NTERA-2 cl.D1 cells was evaluated using the MTT assay. After incubation of NTERA-2 cl.D1 cells with 0-20 µM C-Jul for 24 h, cell viability remained above 90%, indicating that C-Jul had no significant toxic effect on NTERA-2 cl.D1 cells. result Figure 6 As shown in (a), it has good biocompatibility and can be applied to live cell imaging.

[0037] Real-time imaging of exogenous Hcy: The probe C-Jul was first directly incubated with NTERA-2 cl.D1 cells, and then treated with NEM. The cells were then stimulated with Hcy at a concentration of 10-50 μM for 24 h. The NTERA-2 cl.D1 cells were then incubated with the probe C-Jul and the imaging was monitored by live-cell fluorescence microscopy.

[0038] like Figure 6 As shown in (b), after the probe C-Jul was directly incubated with NTERA-2 cl.D1 cells, the intracellular fluorescence intensity increased with the increase of exogenous Hcy stimulation concentration, showing a good concentration-dependent response characteristic.

[0039] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH, characterized in that: The fluorescent probe is C-Jul, which is (9-[(4-fluorophenyl)thio]-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxaldehyde). The fluorescent probe is used to distinguish and detect Hcy, Cys and GSH, effectively reducing the interference of complex biological factors on the recognition effect, and significantly improving the detection sensitivity and signal-to-noise ratio.

2. The fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH according to claim 1, characterized in that: The fluorescent probe C-Jul can distinguish and detect Hcy, Cys and GSH and generate changes in fluorescence signals, and its product fluorescence has significant color differentiation.

3. The fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH according to claim 2, characterized in that: The fluorescent probe C-Jul exhibits low toxicity and good biocompatibility in biological systems.

4. A fluorescent probe as described in any one of claims 1-3, further comprising a method for preparing a fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH, characterized in that: Includes the following steps: S1. Weigh 8-hydroxyjulonidine and bis(2,4,6-trichlorophenyl) malonate and dissolve them in toluene. After thorough stirring and dissolution, heat under reflux at 110°C for 10 h. After the reaction is complete, return to room temperature, filter under reduced pressure, and wash with petroleum ether and ethyl acetate to obtain a white product LM-2. The LM-2 is (9-hydroxy-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-11-one). S2. Under nitrogen protection, phosphorus oxychloride was added dropwise to anhydrous N,N-dimethylformamide. After stirring at room temperature for 60 minutes, a light pink solution was obtained. Then, LM-2 dissolved in DMF was added dropwise to the light pink solution. The solution was then heated to 60°C and refluxed until the reaction was complete. The reaction mixture was then poured into ice water, and after the solid precipitated, it was filtered under reduced pressure, washed with pre-cooled ethanol, and dried to obtain an orange solid product LM-3. The LM-3 is (9-chloro-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxaldehyde). S3. Weigh LM-3 and dissolve it in dichloromethane, then add 4-fluorothiophenol and a catalytic amount of triethylamine. React at room temperature for 12 h. After the reaction is complete, remove the solvent under reduced pressure. The crude product is purified by silica gel column chromatography, which is probe C-Jul.

5. The method for preparing a fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH according to claim 4, characterized in that: In step S1, the molar ratio of 8-hydroxyjulonidine to bis(2,4,6-trichlorophenyl) malonate is 1:1.

1.

6. The method for preparing a fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH according to claim 5, characterized in that: In step S2, the molar ratio of LM-2 to phosphorus oxychloride is 1:

5.

7. The method for preparing a fluorescent probe for distinguishing and detecting Hcy, Cys, and GSH according to claim 6, characterized in that: In step S3, the molar ratio of LM-3 to 4-fluorothiophenol is 1:1, the eluent phase for column chromatography purification consists of petroleum ether and ethyl acetate in a volume ratio of 4:1, and the catalytic amount is 60 μL.

8. A detection method for distinguishing between Hcy, Cys, and GSH, characterized in that: Includes the following steps: P1. Introduce probe C-Jul into the biological sample to be tested; P2 induces the generation of fluorescence signals; P3. Detect the generated fluorescence signal to determine the presence or concentration of Hcy, Cys, and GSH.

9. The detection method according to claim 8, characterized in that, The biological samples were selected from live cells and seminal plasma.

10. The detection method according to claim 8, characterized in that, The detection method is used for imaging visualization and tracking of exogenous Hcy in living cells.

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