A dual-response fluorescent probe and its preparation method and application

Abstract

The present invention discloses a dual-response fluorescent probe, a preparation method and an application thereof, and relates to the technical field of small molecule probes. The chemical structure, preparation method and application of the dual-response fluorescent probe are provided. The background fluorescence of the fluorescent probe P2 prepared by the present invention itself is negligible. If the probe is in a high-viscosity medium, it can emit near-infrared fluorescence at 819nm, thereby realizing viscosity detection; when peroxynitrite is present in the system, the conjugated structure of the probe is destroyed, and the probe emits green fluorescence at 511nm, thereby achieving the recognition of peroxynitrite. Cell experiments found that the probe can be used for fluorescent imaging of intracellular viscosity and peroxynitrite. The probe can also be used for viscosity imaging analysis of inflammatory and diabetic mice. In addition, we can simultaneously monitor changes in viscosity and peroxynitrite levels during ferroptosis.

Description

Technical Field

[0001] The present invention relates to the technical field of small molecule probes, and more particularly to a dual-response fluorescent probe and a preparation method and application thereof. Background Art

[0002] Mitochondria are the "powerhouses" of many eukaryotic cells, playing a vital role in biological processes such as cell division, autophagy, apoptosis, and cell differentiation. Viscosity, a key parameter of the mitochondrial microenvironment, can influence a variety of physiological processes, such as intracellular transport, signal transduction, and intermolecular interactions. Furthermore, abnormal intracellular viscosity is associated with certain diseases, including diabetes, atherosclerosis, Parkinson's disease, and Alzheimer's disease. Therefore, real-time monitoring of mitochondrial viscosity is of great significance.

[0003] Peroxynitrite is an important reactive oxygen species characterized by strong oxidizing properties, strong nucleophilicity, and a short half-life. Peroxynitrite plays an important role in maintaining redox homeostasis, resisting bacterial invasion, signal transduction, and tumorigenesis. However, excessive peroxynitrite can irreversibly react with biomolecules (such as proteins, nucleic acids, and lipids), leading to functional loss of key cellular components and inducing disease. Therefore, the detection of peroxynitrite in biological samples is essential.

[0004] Fluorescent probes have attracted significant attention for in vivo analyte detection due to their advantages, including simple synthesis, high sensitivity, and good specificity. To this end, researchers have developed a number of fluorescent probes capable of detecting viscosity and peroxynitrite. While these probes exhibit excellent performance, they are only suitable for measuring viscosity or peroxynitrite alone, and are unable to simultaneously detect both. Furthermore, studies have demonstrated that intracellular reactive oxygen species levels may be correlated with viscosity. Therefore, developing a dual-responsive fluorescent probe capable of simultaneously detecting viscosity and peroxynitrite is a pressing challenge for those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a dual-response fluorescent probe for simultaneously detecting viscosity and peroxynitrite, as well as a preparation method and application thereof.

[0006] Dual-responsive fluorescent probes offer advantages for simultaneous detection of viscosity and peroxynitrite. The two fluorescent channels are separated by a large distance, offering the advantage of being immune to fluorescence crosstalk. This dual-function assay is virtually unaffected by fluorescence crosstalk, significantly reducing the probability of false-positive results and improving analytical accuracy.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A dual-response fluorescent probe for simultaneous detection of viscosity and peroxynitrite, with the following chemical formula:

[0009] .

[0010] The present invention also provides a method for preparing the aforementioned dual-response fluorescent probe for simultaneous detection of viscosity and peroxynitrite, comprising the following steps:

[0011] (1) 8-Hydroxyjulolidine and phthalic anhydride were dissolved in toluene, heated under reflux, the solvent was removed, and the compound 3 was obtained by chromatography;

[0012] (2) Compound 3 was reacted with cyclohexanone in concentrated sulfuric acid, poured into crushed ice and stirred, perchloric acid was added and stirred, and the mixture was allowed to stand and then filtered to obtain compound 4;

[0013] (3) Compound 4 and 9-formyljulolidine were dissolved in acetic anhydride, heated under reflux, the solvent was removed, and the fluorescent probe P2 was obtained by chromatography;

[0014] .

[0015] Preferably, in step (1), the molar ratio of 8-hydroxyjulolidine to phthalic anhydride is 1:1-2; the reaction temperature is controlled to be 80-100° C., and the reaction is carried out for 8-24 hours; and chromatography is performed using a silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20-50 as the eluent.

[0016] Preferably, in step (2), the molar ratio of cyclohexanone, compound 3 and concentrated sulfuric acid is 1:1~2:30~100; the reaction temperature of compound 3 and cyclohexanone is 60~90°C, and the reaction time is 1~3 h; the mass ratio of concentrated sulfuric acid to crushed ice is 1:10~30, the temperature is controlled at 0°C, and the stirring time is 2~10 min; the mass fraction of perchloric acid is 50%~70%, the molar ratio of perchloric acid to compound 3 is 1:5~10, and the stirring time is 10~20 min; and the standing time is 1~3 h.

[0017] Preferably, the molar ratio of compound 4 to 9-formyljulolidine in step (3) is 1:1-2; the reaction temperature is 80°C-100°C, and the reaction time is 2-8 h; the chromatography is performed using a silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20-80 as the eluent.

[0018] The present invention also provides the above-mentioned dual-response fluorescent probe for qualitative or quantitative detection of ONOO - It is used as a detection reagent for ions and / or viscosity.

[0019] The present invention also provides the use of the dual-response fluorescent probe in preparing a peroxynitrite and / or viscosity fluorescent imaging agent in living cells.

[0020] The present invention also provides the use of the dual-response fluorescent probe in preparing a fluorescent imaging agent in the process of cell ferroptosis.

[0021] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a dual-response fluorescent probe and application for simultaneously detecting viscosity and peroxynitrite. The technical effect obtained is that the fluorescent probe P2 prepared by the present invention, in a low-viscosity phosphate buffer, because the carbon-carbon single bond can rotate freely, causes the probe itself to not fluoresce; When in a high-viscosity environment (such as glycerol), the single bond rotation is restricted, and the probe can emit near-infrared fluorescence at 819 nm, thereby realizing viscosity detection. On the other hand, in the absence of peroxynitrite, the background fluorescence of the probe itself can be ignored; and after reacting with peroxynitrite, the conjugated structure of the probe is destroyed, and green fluorescence will be emitted at 511 nm, achieving the identification of peroxynitrite. The probe can be used for fluorescence imaging of viscosity and peroxynitrite in biological samples. In addition, the probe can also be used to monitor changes in viscosity and peroxynitrite levels during ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The probe P2 provided by the present invention responds to viscosity, wherein: Figure 1 A in the middle is the fluorescence emission spectrum of the probe in glycerol-phosphate buffer with different ratios; Figure 1 In the figure (B), the linear relationship between the logarithm of the fluorescence intensity of the probe at 819 nm and the logarithm of the viscosity.

[0023] Figure 2 The response graph of probe P2 to peroxynitrite provided by the present invention, wherein: Figure 2 A in the middle is the fluorescence emission spectrum of the probe in peroxynitrite solutions with different concentrations; Figure 2 B in the figure is the linear relationship between the fluorescence intensity of the probe at 511 nm and the concentration of peroxynitrite.

[0024] Figure 3 Schematic diagram of the selectivity of probe P2 provided by the present invention, wherein, Figure 3 The vertical axis A is the fluorescence intensity of the probe at 819 nm, and the order of the substances on the horizontal axis is 1. CO3 2- (100 μM); 2. SO4 2- (100 μM); 3. S 2- (100μM); 4. S2O3 2- (100 μM); 5. SO32- (100 μM); 6. Cl - (100 μM); 7. HS - (100 μM); 8.HSO3 - (100 μM); 9. NO3 - (100 μM); 10. Ag + (100 μM); 11. Al 3+ (100 μM); 12. Ca 2+ (100 μM); 13. Cu 2+ (100 μM); 14. Fe 2+ (100 μM); 15. K + (100 μM); 16. Na + (100 μM); 17. Mg 2+ (100 μM); 18. Zn 2+ (100 μM); 19. Alanine (1 mM); 20. Arginine (1 mM); 21. Cysteine ​​(1 mM); 22. Glutamic acid (1 mM); 23. Homocysteine ​​(1 mM); 24. Histidine (1 mM); 25. Serine (1 mM); 26. H2O2 (100 μM); 27. ClO - (100 μM); 28. t-BuOOH (100 μM); 29. ​​•OH (100 μM); 30. t-BuO• (100 μM); 31. 1 O2(100 μM); 32.NO (100 μM); 33.NO2 - (100 μM); 34. C2H5SH (100 μM); 35. Bovine serum albumin (10 μg / mL); 36. Human serum albumin (10 μg / mL); 37. Glycerol. Figure 3 The vertical axis in B is the fluorescence intensity of the probe at 511 nm, and the order of the substances in the horizontal axis is 1. CO3 2- (100 μM); 2. SO4 2- (100 μM); 3. S 2- (100 μM); 4.S2O3 2- (100 μM); 5. SO3 2- (100 μM); 6. Cl -(100 μM); 7. HS - (100 μM); 8. NO3 - (100 μM); 9. Na + (100 μM); 10. K + (100 μM); 11. Mg 2+ (100 μM); 12. Ag + (100 μM); 13. Al 3+ (100 μM); 14. Ca 2+ (100 μM); 15. Cu 2+ (100 μM); 16. Fe 2+ (100 μM);17. Zn 2+ (100 μM); 18. Alanine (1 mM); 19. Arginine (1 mM); 20. Cysteine ​​(1 mM); 21. Glutamic acid (1 mM); 22. Homocysteine ​​(1 mM); 23. Histidine (1 mM); 24. Serine (1 mM); 25. C2H5SH (100 μM); 26. H2O2 (100 μM); 27. ClO - (100 μM); 28. t-BuOOH(100 μM); 29. ​​•OH (100 μM); 30. t-BuO• (100 μM); 31. Blank; 32. 1 O2(100 μM); 33. NO (100 μM); 34. NO2 - (100 μM); 35. Peroxynitrite (30 μM).

[0025] Figure 4 This is a confocal imaging diagram of the probe P2 provided by the present invention in SK-OV-3 cells under different viscosity conditions, wherein: Figure 4 In Figure A, the red channel row is the fluorescence image of the probe in the cell, and the bright field row is the bright field photo of the cell; Figure 4 Middle B is a statistical graph of cell fluorescence intensity.

[0026] Figure 5 This is a confocal imaging diagram of the probe P2 provided by the present invention in HeLa cells after stimulation with exogenous and endogenous drugs, wherein: Figure 5 The green row in A is the peroxynitrite fluorescence image of the probe in the cell, and the bright field row is the bright field photo of the cell; Figure 5 Middle B is a statistical graph of cell fluorescence intensity.

[0027] Figure 6 The probe P2 provided by the present invention is used for imaging of cell ferroptosis, wherein: Figure 6 In center A, the control row shows images of the probe in cells without any treatment (control group), and the erastin row shows images of cells treated with erastin; Figure 6 Middle B is a statistical graph of cell fluorescence intensity. DETAILED DESCRIPTION

[0028] The following describes embodiments of the present invention, examples of which are shown in the accompanying drawings. The embodiments described with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but are not to be construed as limiting the present invention.

[0029] Example 1

[0030] A specific synthesis method of a dual-functional fluorescent probe for simultaneous detection of viscosity and peroxynitrite

[0031] 1.89 g of 8-hydroxyjulolidine (10 mmol) and 1.48 g of phthalic anhydride (10 mmol) were dissolved in 35 mL of toluene. The mixture was heated to 100°C and refluxed for 16 h. After completion of the reaction, the solvent was removed under reduced pressure. The product was then purified by silica gel column chromatography using a 1:20 volume ratio of methanol to dichloromethane as the eluent to afford compound 3 (yellow powder, 2.36 g, 70% yield).

[0032] .

[0033] 10 mL of concentrated sulfuric acid was cooled to 0°C, and then 1.04 mL of cyclohexanone (10 mmol) and 1.69 g of compound 3 (5 mmol) were added sequentially to the concentrated sulfuric acid. The reaction solution was heated to 90°C and stirred for 1.5 hours. The reaction mixture was then poured into 100 g of ice and stirred for 2 minutes. Then, 1 mL of 70% perchloric acid was added dropwise and stirred continuously for 20 minutes. After standing for 1 hour, the precipitate was collected by filtration to obtain compound 4 (a red solid, 1 g, 40% yield).

[0034] .

[0035] 500 mg of compound 4 (1 mmol) and 241 mg of 9-formyljulolidine (1.2 mmol) were dissolved in 15 mL of acetic anhydride, and the mixture was refluxed at 100°C for 4 h. After completion of the reaction, the solvent was removed under reduced pressure. The product was then purified by silica gel column chromatography using a 1:20 volume ratio of methanol to dichloromethane as the eluent to obtain the fluorescent probe P2 (blue powder, 410 mg, 60% yield). 1 H-NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 8.17 (d, J = 7.6 Hz, 1H), 7.83 (dd, J = 7.0 Hz, 1H), 7.78-7.69 (m, 2H), 7.31 (d, J = 7.2 Hz, 1H), 7.20(s, 2H), 6.48 (s, 1H), 3.56-3.44 (m, 4H), 3.31 (s, 4H), 3.06 (d, J = 6.5 Hz,2H), 2.89 (s, 2H), 2.75 (d, J = 5.8 Hz, 4H), 2.68-2.62 (m, 2H), 2.19 (s, 2H), 2.05 (s, 2H), 1.88 (dt, J = 12.0, 5.4 Hz, 6H), 1.78-1.63 (m, 2H). 13 C-NMR (100MHz, CDCl3) δ 161.31, 157.51, 151.65, 149.60, 144.45, 138.01, 134.47, 131.98,130.79, 130.68, 128.81, 128.61, 127.87, 125.28, 123.68, 121.75, 121.17,120.26, 119.84, 114.96, 103.51, 49.73, 49.32, 49.12, 26.74, 26.60, 24.86,20.35, 20.31, 19.41, 18.84, 18.60. HRMS m / z: calcd for C 39 H 39 N2O3 + [M] + : m / z583.2955; found: m / z 583.2963.

[0036] .

[0037] Example 2

[0038] Responsiveness of the probe P2 prepared in Example 1 of the present invention to viscosity

[0039] Probe P2 was prepared into a 1 mM stock solution using dimethyl sulfoxide. Glycerol and pH 7.4 phosphate buffer were mixed in different proportions to adjust the sample viscosity. The probe was dissolved in solutions of different viscosities, and the final concentration of the probe was maintained at 10 μM. Figure 1 As shown in Figure A, as the volume ratio of glycerol in the medium increases (glycerol increases from 0% to 99%), the fluorescence intensity of the probe at 819 nm gradually increases. In addition, the logarithm of the fluorescence intensity of the probe at 819 nm shows a good linear relationship with the logarithm of the viscosity, as shown in Figure 4. Figure 1 As shown in B.

[0040] Example 3

[0041] Responsiveness of probe P2 prepared in Example 1 of the present invention to peroxynitrite

[0042] In the phosphate buffer with a concentration of 10 μM probe P2, the fluorescence intensity of the probe at 511 nm gradually increased with the increase of the added peroxynitrite concentration (from 0 to 30 μM), as shown in Figure 2. Figure 2 In addition, there is a good linear relationship between the fluorescence intensity of the probe at 511 nm and the concentration of peroxynitrite, ONOO - The detection limit is 18nM. Figure 2 As shown in B.

[0043] Example 4

[0044] The selectivity of the probe P2 prepared in Example 1 of the present invention

[0045] In a phosphate buffer solution containing probe P2 at a concentration of 10 μM, common ions, reactive oxygen species, and amino acids were added to investigate the selectivity of the probe for viscosity and peroxynitrite. Figure 3 As shown in Figure A, after adding the above substances, the fluorescence signal of the probe at 819 nm remained almost unchanged, but bright fluorescence was observed in glycerol, indicating that the probe has high selectivity for viscosity. Similarly, these species did not cause any change in the fluorescence of the probe at 511 nm, while the presence of peroxynitrite significantly increased the fluorescence, as shown in Figure 4. Figure 3 As shown in B, it shows that the probe can specifically recognize peroxynitrite.

[0046] Example 5

[0047] Probe P2 prepared in Example 1 of the present invention is used for cell viscosity imaging

[0048] SK-OV-3 cells were divided into three groups: the first group was incubated with 10 μM probe P2 for 30 minutes (control group); the second group was treated with 25 μM nystatin for 30 minutes and then incubated with 10 μM probe for 30 minutes; the third group was cultured with serum-free incomplete culture medium for 30 minutes and then incubated with 10 μM probe for 30 minutes. The cells were then irradiated with 640 nm excitation light and the fluorescence signal at 650-750 nm was collected. Figure 4 As shown in middle A, compared with the control group, the cells in the second and third groups showed stronger fluorescence signals. Figure 4 B is the statistical value of the fluorescence intensity of the cells. The experimental results show that the probe can be used for imaging analysis of cell viscosity.

[0049] Example 6

[0050] Probe P2 for cellular peroxynitrite imaging

[0051] HeLa cells were divided into four groups: the first group was treated with 5 μM probe P2 for 30 min (control group); the second group was treated with 200 μM lindole for 4 h, and then incubated with 5 μM probe for 30 min; the third group was treated with 1 μg / mL lipopolysaccharide and 100 ng / mL interferon γ for 8 h, and then incubated with 5 μM probe for 30 min; the fourth group was treated with 1 μg / mL lipopolysaccharide and 100 ng / mL interferon γ for 8 h, and then incubated with 10 nM phorbol ester for 30 min, and then incubated with 5 μM probe for 30 min. Finally, the cells were irradiated with 405 nm excitation light, and the fluorescence signal at 500-540 nm was collected. Figure 5 As shown in middle A, cells in groups 2, 3, and 4 showed stronger fluorescence compared with the control group. Figure 5 B is the statistical value of the fluorescence intensity of the cells. The experimental results show that the probe can be used for imaging analysis of intracellular peroxynitrite.

[0052] Example 7

[0053] Probe P2 for imaging viscosity and peroxynitrite during ferroptosis

[0054] SK-OV-3 cells were divided into two groups: the first group was incubated with probe P2 at a concentration of 5 μM for 30 minutes (control group); the second group was treated with 20 μM elastin for 12 hours and then incubated with probe P2 at a concentration of 5 μM for 30 minutes. The cells were then irradiated with 405 nm and 640 nm excitation light, respectively, and fluorescence signals were collected at 500-540 nm and 650-750 nm, respectively. Figure 6 As shown in Figure A, cells undergoing ferroptosis exhibited stronger red and green fluorescence compared to the control group, indicating that the ferroptosis process was accompanied by higher viscosity and higher levels of peroxynitrite. Figure 6 B is the statistical value of the fluorescence intensity of the cells. The experimental results show that the probe can simultaneously monitor the changes in viscosity and peroxynitrite levels during cell ferroptosis.

[0055] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0056] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A dual-response fluorescent probe, characterized in that: The chemical structure is as follows: 。 2. The method for preparing a dual-response fluorescent probe according to claim 1, wherein The following steps are involved: (1) 8-Hydroxyjulolidine and phthalic anhydride were dissolved in toluene, heated under reflux, the solvent was removed, and compound 3 was obtained by chromatography; (2) After compound 3 reacts with cyclohexanone in concentrated sulfuric acid, the mixture is poured into crushed ice and stirred, perchloric acid is added and stirred, and the mixture is allowed to stand and then filtered to obtain compound 4; (3) Compound 4 and 9-aldehyde julolidine were dissolved in acetic anhydride, heated under reflux, the solvent was removed, and the fluorescent probe P2 was obtained by chromatography; 。 3. The preparation method according to claim 2, wherein In step (1), the molar ratio of 8-hydroxyjulolidine to phthalic anhydride is 1:1-2; the reaction temperature is controlled to be 80-100°C, and the reaction is carried out for 8-24 hours; and the chromatography is performed using a silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20-50 as the eluent.

4. The preparation method according to claim 2, wherein In step (2), the molar ratio of cyclohexanone, compound 3 and concentrated sulfuric acid is 1:1~2:30~100; the reaction temperature of compound 3 and cyclohexanone is 60~90°C, and the reaction time is 1~3 h; the mass ratio of concentrated sulfuric acid to crushed ice is 1:10~30, the temperature is controlled at 0°C, and the stirring time is 2~10 min; the mass fraction of perchloric acid is 50%~70%, the molar ratio of perchloric acid to compound 3 is 1:5~10, and the stirring time is 10~20 min; and the standing time is 1~3 h.

5. The preparation method according to claim 2, wherein In step (3), the molar ratio of compound 4 to 9-formyljulolidine is 1:1-2; the reaction temperature is 80°C-100°C, and the reaction time is 2-8 h; the chromatography is performed using a silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20-80 as the eluent.

6. The dual-response fluorescent probe according to claim 1 is used in the preparation of a method for qualitative or quantitative detection of ONOO - Application of ionic and / or viscosity reagents.

7. Use of the dual-response fluorescent probe according to claim 1 in preparing a peroxynitrite and / or viscosity fluorescent imaging agent in living cells.

8. Use of the dual-response fluorescent probe according to claim 1 in preparing a fluorescent imaging agent in the process of cell ferroptosis.

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