Preparation method and application of sulfydryl probe based on alpha, beta-unsaturated acrylate

Abstract

The invention belongs to the technical field of medicines, and discloses a near-infrared fluorescent probe taking an alpha, beta-unsaturated acryloyl ester unit as a response site of sulfydryl. The fluorescent probe has great advantages in the aspects of biological small molecule identification, protein marking and disease diagnosis, and is one of the most convenient and most popular tools for exploring a complex biological process. The fluorescent probe molecule capable of releasing red fluorescence after being activated by sulfydryl is obtained for the first time by connecting a fluorophore with an alpha, beta-unsaturated acryloyl ester unit. The fluorescent probe molecule disclosed by the invention can be used for specifically detecting sulfydryl in an organism, so that the aim of accurately monitoring the sulfydryl in a cell is fulfilled.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry and medicine, and more particularly to a method for preparing a novel fluorescent probe using α,β-unsaturated acrylate as a biological thiol receptor and its use. Background Art

[0002] Biological thiols have strong redox and nucleophilic properties and play a crucial role in regulating various physiological processes. These small molecule thiols are essential for maintaining redox homeostasis, protein function regulation, cell signaling, cell growth, and detoxification. In addition, dysregulation of cellular thiol levels is associated with a range of pathological conditions, including cardiovascular diseases, neurodegenerative diseases, cancer, stroke, and osteoporosis. This association of biological thiols with various health complications emphasizes their importance as potential indicators for the diagnosis and treatment of related diseases. Therefore, the precise and real-time detection of biological thiols has attracted great attention in clinical diagnosis. Due to the excellent temporal and spatial resolution, non-invasiveness, rapid response, and real-time imaging capabilities of fluorescence imaging technology, it has become a powerful tool in the medical field. Recently, many fluorescent probes for detecting biological thiols have been developed, and these probes generally aim to utilize the unique nucleophilicity of the thiol group to initiate specific chemical reactions that lead to fluorescence modulation. In these fluorescence activation reactions, the Michael addition reaction of thiol with α,β-unsaturated carbonyl compounds has been widely used to develop biological thiol-responsive fluorescent probes. The electrophilic nature of these moieties enables them to selectively undergo nucleophilic addition reactions with thiols to trigger fluorescence activation. Based on this, the present invention rationally constructs an activatable red fluorescent probe by modulating the reactivity of the α,β-unsaturated carbonyl unit of the probe. The probe uses the α,β-unsaturated acrylate unit as the response site for thiols and is linked to a red fluorophore as a signal reporter. It shows great potential for tracking its target with high selectivity and sensitivity in living cells and in vivo. Summary of the Invention

[0003] Based on the current status of the above prior art research, the present invention provides a method for preparing a biological thiol-activated red fluorescent probe based on α,β-unsaturated acrylate as an identification unit and its use. The fluorescent probe shown in the present invention can be used for specific recognition of biological thiols and dynamic monitoring of thiol levels in vivo:

[0004] Specifically, the present invention provides a fluorescent probe having the following structure shown in formula (I):

[0005]

[0006] The present invention has excellent specificity, sensitivity, and low cytotoxicity, and thus can be successfully applied to the dynamic monitoring of thiol levels under biological conditions. It realizes the specific recognition of biological thiols and the dynamic monitoring of thiol levels in vivo. The present invention provides a new tool for the diagnosis and treatment evaluation of thiol-related diseases, opens a new window for promoting the development of thiol-activated fluorescent probes, and reveals the potential of α,β-unsaturated acrylate units as novel and specific thiol receptor moieties. The specific preparation method of the fluorescent molecular probe shown in the present invention is as follows:

[0007]

[0008] Synthesis of compound 5a: At 0 °C, n-decanol (15 mmol, 1.0 eq.) was dissolved in anhydrous DCM (30 mL), and then TEA (18 mmol, 1.2 eq.) and acryloyl chloride (16 mmol, 1.1 eq.) were slowly added to the solution. The temperature of the reaction mixture was raised to room temperature and reacted for 30 minutes. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate, and the aqueous phase was extracted three times with DCM, and dried under reduced pressure to collect the product.

[0009] Synthesis of compound 5b: Compound 5a (6 mmol, 1.0 eq) and DABCO (3 mmol, 0.5 eq) were dissolved in acetonitrile (30 mL), and then an aqueous solution of formaldehyde (18 mmol, 3.0 eq) was added, and the reaction was carried out at 50 °C for 30 minutes. After the reaction was completed, the product was extracted with ethyl acetate, washed with saturated sodium bicarbonate, and dried in vacuo. The product was obtained by purification by column chromatography (DCM / MeOH = 50 / 1).

[0010] Synthesis of compound FR-NH2: 4-(Diethylamino)salicylaldehyde (1.03 mmol, 1.0 eq.) and 6-amino-3,4-dihydro-1(2H)-naphthalenone (1.13 mmol, 1.1 eq.) were dissolved in methanesulfonic acid and stirred at 90 °C for 5 hours. The reaction mixture was poured into an ice-water mixture (200 mL). Then perchloric acid (70%; 7 mL) was slowly added to the reaction mixture. After filtration, a black solid product FR-NH2 was obtained (yield 76%).

[0011] Synthesis of compound FR-NCO: FR-NH2 (0.28 mmol, 1.0 eq.) was dissolved in 10 mL of dry DCM. Then triphosgene (0.14 mmol, 0.5 eq.) and triethanolamine (0.34 mmol, 1.2 eq.) were added. The reaction mixture was stirred at 45 °C for 4 hours, and the organic phase was concentrated to obtain the crude product FR-NCO.

[0012] Synthesis of Example 1: Dissolve compound FR-NCO (0.28 mmol, 1 eq.) in anhydrous DCM solution, then add 5b (0.28 mmol, 1 eq.) and triethylamine (0.34 mmol, 1.2 eq.) to the solution, react at 0 °C for 1 hour, and purify by column chromatography to obtain a black solid product (yield 62%). Brief Description of the Drawings

[0013] Figure 1 It is a schematic diagram of the UV-visible absorption spectrum of the reaction between Example 1 and GSH.

[0014] Figure 2 It is a schematic diagram of the time-dependent fluorescence spectrum of the reaction between Example 1 and GSH.

[0015] Figure 3 It is a schematic diagram of the concentration-dependent fluorescence spectrum of the reaction between Example 1 and GSH.

[0016] Figure 4 It is a schematic diagram of the fluorescence spectrum of Example 1 for GSH at different pH values.

[0017] Figure 5 It is a schematic diagram of the selective fluorescence spectrum of Example 1 for biothiols.

[0018] Figure 6 It is a schematic diagram of the high-performance liquid chromatography of the reaction between Example 1 and GSH.

[0019] Figure 7 It is a schematic diagram of the orbital of Example 1 for density functional theory calculation.

[0020] Figure 8 It is a schematic diagram of the cytotoxic activity results of Example 1 against HepG2 and HeLa cells.

[0021] Figure 9 It is a schematic diagram of the cell imaging of Example 1 for biothiols (A) and the cell imaging after treatment with NEM (B).

[0022] Figure 10 It is a schematic diagram of the zebrafish imaging of Example 1 for biothiols.

[0023] Test Example 1: UV-visible absorption spectrum experiment of the reaction between Example 1 and GSH Experimental method: Measure the absorbance of Example 1 by incubating Example 1 (2 μM) with GSH in PBS (10 mM, pH = 7.4) at 37 °C for 30 minutes.

[0024] Experimental results: Example 1 showed an obvious absorption peak at 525 nm. After incubation with GSH, a red shift appeared at 580 nm, indicating the reactive release of the fluorophore to GSH.

[0025] Experimental Example 2: Time-dependent Fluorescence Response Experiment of the Reaction between Example 1 and GSH Experimental Method: The time-dependent fluorescence response of Example 1 was measured by incubating Example 1 (2 μM) with GSH in PBS (10 mM, pH = 7.4) at 37 °C and measuring every two minutes for a total of 30 minutes.

[0026] Experimental Results: As Figure 2 shown, an attenuated fluorescence signal centered at λ em = 625 nm was initially observed for Example 1, and this signal gradually increased with time and reached a peak within about 30 minutes. These results confirmed the release of the unmasked fluorophore triggered by GSH, resulting in enhanced fluorescence.

[0027] Experimental Example 3: Concentration-dependent Fluorescence Response Experiment of the Reaction between Example 1 and GSH Experimental Method: The concentration-dependent fluorescence response of Example 1 was measured by incubating Example 1 with different concentrations of GSH in PBS (10 mM, pH 7.4) at 37 °C for 30 minutes.

[0028] Experimental Results: The fluorescence response of Example 1 to different concentrations of GSH (0 μM to 2 mM) was observed. As Figure 3 shown, the fluorescence signal centered at λ em = 625 nm increased significantly with the increase in GSH concentration. These results further supported the excellent and quantitative response characteristics of Example 1 to biothiols.

[0029] Experimental Example 4: Fluorescence Spectrum Response of Example 1 to GSH at Different pH Values Experimental Method: Example 1 and GSH (1 mM) were added to buffer solutions of different pH values to prepare solutions of appropriate concentrations and incubated for a certain time, and then their fluorescence spectra were measured.

[0030] Experimental Results: Example 1 showed a weak fluorescence signal in the pH range of 5.0 - 9.0. When Example 1 was incubated with GSH (1 mM), the fluorescence intensity increased sharply in the pH range of 7.0 - 9.0. In the pH range of 7.0 - 9.0, the relative fluorescence intensity remained stable, indicating that the probe is suitable for physiological conditions.

[0031] Test Example 5: Selective Fluorescence Spectroscopy Experiment of Example 1 on Biological Thiols Experimental Method: Example 1 and various test substances (1. Blank; 2. Ala; 3. Asp; 4. Gly; 5. Ile; 6. Met; 7. Val; 8. Trp; 9. Lys; 10. Thr; 11. His; 12. Pro; 13. Arg; 14. Ser; 15. Phe; 16. Leu; 17. Tyr; 18. Glu; 19. Gln; 20. MgCl2; 21. FeCl3; 22. FeSO4; 23. CuSO4; 24. NiCl2; 25. KCl; 26. HgCl2; 27. CH3COOH; 28. NaHS; 29. H2O2; 30. O 2- ; 31. ClO - ; 32. ONOO - ; 33. Hcy; 34. Cys; 35. GSH) were respectively added to PBS buffer solution to prepare solutions with appropriate concentrations and incubated for a certain time, and then their fluorescence spectra were measured.

[0032] Experimental Results: Even at higher concentrations (100 eq.), the incubation of Example 1 (2 μM) with all these potential interfering species had a negligible effect on the fluorescence signal of the designed probe, except for Cys, Hcy, and GSH, showing a significantly enhanced fluorescence signal. These observations highlight the significant selectivity of Example 1 for biological thiols (including Cys, Hcy, and GSH).

[0033] Test Example 6: High-Performance Liquid Chromatography Experiment to Elucidate the Reaction Mechanism between Example 1 and GSH Experimental Method: Example 1 was added to PBS buffer solution to prepare a solution with an appropriate concentration, and then an appropriate amount of GSH was added, and then its high-performance liquid chromatography was measured.

[0034] Experimental Results: When Example 1 with a retention time of 10.7 minutes was incubated with GSH (1 mM), a product FR-NH2 with a retention time of 5.2 minutes was produced. These results indicate that Example 1 reacts with GSH, resulting in the cleavage of the carbamate bond and the formation of FR-NH2.

[0035] Test Example 7: Density Functional Theory Calculation Experiment of Example 1 Experimental Method: Density functional theory (DFT) calculation was performed on Example 1 using the B3LYP / 6-311G method.

[0036] Experimental results: Calculations show that the HOMO-LUMO energy gap of Example 1 is relatively large, at 2.5838 eV, which may be related to its optimal quenching effect on fluorophores. In addition, the HOMO-LUMO energy gap of the product FR-NH2 is calculated to be 2.5958 eV. The slight difference in the energy gap between Example 1 and FR-NH2 is related to the red shift observed in the absorption spectrum after reaction with GSH.

[0037] Test Example 8: Cytotoxicity experiment of Example 1 on HepG2 and HeLa cells Experimental method: In a 96-well plate, add 1×10 4 HepG2 and HeLa cells to each well, and treat them with the compound in Example 1 for 24 h, and finally determine the cytotoxicity by the CCK-8 method.

[0038] Experimental results: As Figure 8 shown, even when treated with a higher concentration of Example 1 (up to 20 μM), the survival rate of HepG2 and HeLa cells remained above 90%. These observations confirm the low cytotoxicity and high biocompatibility of Example 1 to the living environment.

[0039] Test Example 9: Cellular imaging test of Example 1 on biological thiols Experimental method: Use Experimental Example 1 to image cellular biological thiols in living HepG2 cells, and take fluorescence images at 10-minute intervals for a total of 30 minutes. Next, incubate HepG2 cells with N-ethylmaleimide (NEM) at 37 °C for 30 minutes to block cellular biological thiols. Then incubate the NEM-incubated cells with Experimental Example 1 at 37 °C for 30 minutes.

[0040] Experimental results: Strong fluorescence signals appeared in HepG2 cells treated with Experimental Example 1, as Figure 9 (A) shows, and it was observed that the signal gradually increased with time. After incubation with NEM, due to the blocking of cellular biological thiols by NEM, a weakened fluorescence signal was observed in the test cells, as Figure 9 (B) shows. These results further verify the selective reaction of Experimental Example 1 with small molecule thiols to release fluorescence, and have the ability to act as a highly sensitive probe to detect biological thiols in solutions and living cells.

[0041] Test Example 10: Zebrafish imaging test of Example 1 on biological thiols Experimental method: Use Experimental Example 1 to image zebrafish. Next, incubate the zebrafish with NEM, then treat and image them with Experimental Example 1, and then treat and image them with GSH.

[0042] Experimental results: As Figure 10As shown, there was no fluorescence in the control group, while the zebrafish treated with Experimental Example 1 showed strong fluorescence signals. The zebrafish were pretreated with NEM and then treated with Experimental Example 1, and there was almost no fluorescence, indicating that the biothiols were blocked. After treatment with GSH, the fluorescence release was significantly enhanced, indicating that the blocked biothiols would regenerate after exposure to GSH. These results convincingly verified that Experimental Example 1 could effectively detect and image biothiols in vivo.

Claims

1. The present invention discloses a preparation method and application of a thiol-activated red fluorescent probe based on α,β-unsaturated acrylate as a recognition unit, the structure of which is shown in the following formula (I). The fluorescent probe shown in the present invention can be used for specific recognition of biological thiols and dynamic monitoring of thiol levels in organisms:

2. An α,β-unsaturated acrylate derivative, characterized in that: Its structure is shown in the following formula (I):

3. A new use of the fluorescent probe as claimed in claim 1, characterized in that: It can be used for specific identification of biological thiols and dynamic monitoring of thiol levels in organisms.

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