Peroxynitrite near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a peroxynitrite near-infrared fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of peroxynitrite near-infrared fluorescent probes. According to the technical scheme, the peroxynitrite near-infrared fluorescent probe is characterized in that the structural formula of the peroxynitrite near-infrared fluorescent probe is shown in the specification. The invention further specifically discloses a preparation method of the peroxynitrite near-infrared fluorescent probe and application of the peroxynitrite near-infrared fluorescent probe to selective detection of peroxynitrite in an aqueous solution, a biological cell system and animal liver tissue. The near-infrared fluorescent probe disclosed by the invention has the advantages of fluorescence emission in a near-infrared region, good selectivity, low detection limit and the like.

Description

Technical Field

[0001] This invention belongs to the field of peroxynitrite fluorescent probe technology, specifically relating to a peroxynitrite near-infrared fluorescent probe, its preparation method, and its application. Background Technology

[0002] The liver plays a central role in drug metabolism, and drug poisoning is a leading cause of acute liver injury worldwide. Drug-induced liver injury (DILI) is a disease caused by the death of a large number of hepatocytes, accompanied by inflammation and oxidative stress. Its long-term progression may lead to hepatocellular carcinoma. Studies have shown that the abnormal accumulation of reactive oxygen species (ROS) within hepatocytes is a major cause of hepatocyte damage. OPS (overoxygenated nitrite) is a contributing factor. - ONOO possesses strong oxidizing and nitrifying capabilities, playing a crucial role in cell signal transduction and homeostasis regulation. When ONOO in the body... - When levels of these substances exceed normal ranges, they can damage many important biomolecules (such as nucleic acids, proteins, and lipids), and are a major contributing factor to many diseases, including liver damage, cardiovascular disease, neurodegenerative diseases, and cancer. In the progression of drug-induced liver injury, ONOO within hepatocytes... - The levels often exhibit characteristic abnormal changes, indicating ONOO - It can serve as an important molecular marker for assessing drug-induced liver injury. Therefore, accurate detection of ONOO is crucial. - This is crucial for exploring the physiological and pathological functions of liver injury.

[0003] In recent years, fluorescence imaging technology, with its unique advantages such as high sensitivity, excellent spatiotemporal resolution, and non-destructive testing of biological samples, has been used to detect, monitor, and track biologically important species, including ions, small molecules, reactive species, and biomacromolecules such as DNA / RNAs, proteins, carbohydrates, and lipids. This has promoted interdisciplinary research at the interfaces of physics, chemistry, biology, pharmaceuticals, and medical sciences. Currently, peroxynitrite (ONOO) is specifically detected in hepatocytes. -Research on fluorescent probes is still in its developmental stage, and key performance indicators such as detection sensitivity, selectivity, and biocompatibility require further optimization and improvement. Compared to short-wavelength emission fluorescent probes, near-infrared fluorescent probes have lower phototoxicity, deeper penetration depth, and less background fluorescence, thus offering greater imaging advantages and providing chemists, biologists, and clinical diagnostics with more comprehensive and accurate imaging information at the in vivo level. Anthocyanins are natural pigments with 2-phenylbenzopyran cations as their main backbone, widely found in various flowers and fruits. Anthocyanin molecules have good water solubility, high molar extinction coefficients, large conjugated systems, and are easily structurally modified, facilitating further extension of their conjugated systems to generate near-infrared emission fluorescent signals, making them an ideal platform for constructing peroxynitrite near-infrared fluorescent probes. Summary of the Invention

[0004] Based on the current research status of near-infrared fluorescent probes for peroxynitrite, this invention innovatively designs a near-infrared fluorescent probe for peroxynitrite. This probe uses anthocyanin derivatives as the fluorescent group and a spironolactam structure formed by phenylhydrazine as the recognition group for peroxynitrite, enabling selective and imaging detection of peroxynitrite in living systems. This fluorescent probe features near-infrared fluorescence emission, good selectivity, and a low detection limit.

[0005] The present invention also provides a method for preparing the above-mentioned peroxynitrite near-infrared fluorescent probe and its application in selectively detecting peroxynitrite in aqueous solutions, biological cell systems, and animal tissues.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a near-infrared fluorescent probe for peroxynitrite, the structural formula of which is:

[0007]

[0008] The fluorescent probe exhibits near-infrared fluorescence emission and the ability to selectively detect peroxynitrite.

[0009] The preparation method of the peroxynitrite near-infrared fluorescent probe of the present invention comprises the following specific preparation steps:

[0010] Step S1: Add 0.63 g of 8-hydroxyjuloridin, 0.53 g of phthalic anhydride, and 35 mL of toluene to the reaction vessel. Reflux at 120 °C for 5 h. Cool the reaction solution to room temperature and evaporate the solvent to obtain the crude product. Purify the crude product using silica gel column chromatography to obtain a brown solid compound 1 with a yield of 70%. The eluent used in the silica gel column chromatography was a 50:1 (v / v) mixture of dichloromethane and methanol. The corresponding synthetic route is as follows:

[0011]

[0012] Step S2: At 0℃, 15 mL of H2SO4 solution, 1.012 g of compound 1, and 567.8 mg of 6-(dimethylamino)-3,4-dihydronaphth-1(2H)-one were added to the reaction vessel and stirred until homogeneous. The mixture was then heated to 90℃ and reacted for 3 h. After the reaction solution cooled to room temperature, it was slowly poured into ice water, followed by the addition of 1 mL of perchloric acid. After stirring for 2 min, a precipitate was formed. The mixture was filtered through a Buchner funnel, and the precipitate was washed with cold water to obtain the crude product. The crude product was dried and purified by silica gel column chromatography to obtain a dark blue solid compound 2 with a yield of 52%. The eluent used in the silica gel column chromatography was a 15:1 volume ratio of dichloromethane / methanol mixture. The corresponding synthetic route is as follows:

[0013]

[0014] Step S3: Add 30 mL of dichloromethane, 59.0 mg of compound 2, 19.2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 13.5 mg of 1-hydroxybenzotriazole (HOBt) to the reaction vessel and stir until homogeneous at room temperature. Then add 10.8 mg of phenylhydrazine and continue stirring at room temperature for 4 h. The reaction solution is then washed three times with water. The resulting dichloromethane phase is dried over anhydrous Na2SO4 and evaporated to dryness to obtain the crude product. The crude product is purified by silica gel column chromatography to obtain the target fluorescent probe compound NIR-ONOO in 53% yield. The eluent used for silica gel column chromatography is a 50:1 (v / v) mixture of dichloromethane and methanol. The corresponding synthetic route is as follows:

[0015]

[0016] The application of the peroxynitrite near-infrared fluorescent probe described in this invention in the selective detection of peroxynitrite, wherein the fluorescent probe can detect K+ in K+-containing near-infrared fluorescent probes. + Mg 2+ Cu 2+ Fe 3+ Na + Zn 2+ Ca 2+ NO2 - S2 - SO3 2- NO 1 O2, HClO, ·OH, H2O2, O2 ·- Selective detection of peroxynitrite in PBS buffer solutions containing Cys, Hcy, and GSH.

[0017] The present invention relates to the application of the peroxynitrite near-infrared fluorescent probe in the selective detection of peroxynitrite in aqueous solutions, biological cell systems, or animal liver tissue.

[0018] The application of the peroxynitrite near-infrared fluorescent probe described in this invention in the preparation of peroxynitrite fluorescent detection reagents or ultraviolet-visible light detection reagents in aqueous solutions.

[0019] The application of the peroxynitrite near-infrared fluorescent probe described in this invention in the preparation of intracellular peroxynitrite fluorescence imaging detection reagents.

[0020] The application of the peroxynitrite near-infrared fluorescent probe described in this invention in the preparation of a peroxynitrite fluorescence imaging detection reagent in animal liver tissue.

[0021] Compared with existing technologies, this invention has the following advantages and beneficial effects: (1) The near-infrared fluorescent probe in this invention has a simple synthesis route and convenient post-processing operation; (2) The peroxynitrite in this invention has high selectivity and high sensitivity (detection limit reaches 20 nM), and can effectively resist interference from other substances in the body; (3) The peroxynitrite in this invention has near-infrared fluorescence emission characteristics (emission wavelength 655 nm), which is suitable for specific imaging detection of peroxynitrite in liver tissue. This probe significantly reduces biological autofluorescence interference through its near-infrared emission and effectively reduces photodamage to biological samples through its long-wavelength excitation, thereby obtaining more accurate and stable optical signals and imaging effects. Therefore, the near-infrared fluorescent probe in this invention provides an efficient tool for the detection of peroxynitrite and provides a new technical means for in-depth research on the mechanism of action of peroxynitrite in physiological and pathological processes. Attached Figure Description

[0022] Figure 1 The fluorescence spectra of the fluorescent probe compound NIR-ONOO prepared in Example 1 after the addition of different concentrations of peroxynitrite are shown.

[0023] Figure 2 This is the UV-Vis absorption spectrum of the fluorescent probe compound NIR-ONOO prepared in Example 1 after the addition of different concentrations of peroxynitrite.

[0024] Figure 3 The graph shows the relationship between the fluorescence intensity of the fluorescent probe compound NIR-ONOO at an emission wavelength of 658 nm and the concentration of peroxynitrite (0-30 μM). The inset is a linear graph showing the relationship between the fluorescence intensity of the fluorescent probe compound NIR-ONOO at an emission wavelength of 658 nm and the concentration of peroxynitrite (1-10 μM).

[0025] Figure 4The fluorescence response of the fluorescent probe compound NIR-ONOO prepared in Example 1 to different species is as follows: (1) Blank; (2) K + (1mM); (3)Mg 2+ (1mM); (4)Cu 2+ (500μM); (5)Fe 3+ (500μM); (6)Na + (1mM); (7)Zn 2+ (1mM); (8)Ca 2+ (1mM); (9)NO2 - (50μM); (10)S2 - (50μM); (11)SO3 2- (50μM); (12)NO (50μM); (13) 1 O2(50μM); (14)HClO(50μM); (15)·OH(50μM); (50)H2O2(100μM); (17)O2 ·- (100μM); (18) Cys (500μM); (19) Hcy (500μM); (20) GSH (5mM); (21) ONOO - (30μM).

[0026] Figure 5 This describes the response of the fluorescent probe compound NIR-ONOO prepared in Example 1 to peroxynitrite under different pH conditions.

[0027] Figure 6 This is a fluorescence imaging image of the fluorescent probe compound NIR-ONOO prepared in Example 1 detecting exogenous peroxynitrite in HeLa cells.

[0028] Figure 7 This is a fluorescence imaging image of the fluorescent probe compound NIR-ONOO prepared in Example 1 detecting endogenous peroxynitrite in RAW 264.7 cells.

[0029] Figure 8 This is a fluorescence imaging image of the fluorescent probe compound NIR-ONOO prepared in Example 1 detecting exogenous peroxynitrite in tissue. Detailed Implementation

[0030] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0031] Example 1

[0032] Synthesis of the fluorescent probe compound NIR-ONOO

[0033] (1) Synthesis of Compound 1

[0034] 0.63 g of 8-hydroxyjuloridin, 0.53 g of phthalic anhydride, and 35 mL of toluene were added to a 100 mL round-bottom flask, and the mixture was refluxed at 120 °C for 5 h. The reaction solution was cooled to room temperature and the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography to give 1780 mg of a brown solid compound, with a yield of 70%. The eluent volume ratio used was dichloromethane / methanol = 50:1. The synthetic route is as follows:

[0035]

[0036] (2) Synthesis of compound 2

[0037] At 0°C, 15 mL of 98 wt% H₂SO₄ solution, 1.012 g of compound 1, and 567.8 mg of 6-(dimethylamino)-3,4-dihydronaphthyl-1(2H)-one were added to a 100 mL round-bottom flask and stirred for 5 min. The mixture was then heated to 90°C and reacted for 3 h. The reaction solution was cooled to room temperature and slowly poured into ice water. 1 mL of perchloric acid was then added, and the mixture was stirred for 2 min, after which a precipitate formed. The mixture was then filtered through a Buchner funnel, and the precipitate was filtered off and washed with cold water to obtain the crude product. The crude product was dried and purified by silica gel column chromatography to obtain 2920.5 mg of a dark blue solid compound, with a yield of 52%. The eluent volume ratio used was dichloromethane / methanol = 15:1. The synthetic route is as follows:

[0038]

[0039] (3) Synthesis of fluorescent probe compound NIR-ONOO

[0040] In a 100 mL round-bottom flask, 30 mL of dichloromethane, 59.0 mg of compound 2, 19.2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 13.5 mg of 1-hydroxybenzotriazole were added and stirred at room temperature for 30 min. Then, 10.8 mg of phenylhydrazine was added and the reaction was continued at room temperature for 4 h. The reaction solution was then washed three times with water. The resulting dichloromethane phase was dried over anhydrous Na2SO4 and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 30.8 mg of the target fluorescent probe compound NIR-ONOO, with a yield of 53%. The eluent volume ratio used was dichloromethane / methanol = 50:1. The synthetic route is as follows:

[0041]

[0042] Example 2

[0043] Determination of fluorescence spectra of the fluorescent probe compound NIR-ONOO reacting with different concentrations of peroxynitrite.

[0044] The fluorescent probe compound NIR-ONOO prepared in Example 1 was dissolved in N,N-dimethylformamide (DMF) to prepare a 1 mM stock solution. 0.02 mL of this stock solution was added to a 5 mL centrifuge tube, and different equivalents (0–30 μM) of peroxynitrite standard solution were added. The solution was diluted to 4 mL with 50 mM PBS buffer (pH = 7.4) (DMF / PBS volume ratio 1:1). The fluorescence spectrum was measured using 620 nm excitation light and a slit width of 5 nm / 5 nm. The fluorescence spectrum is shown below. Figure 1 As shown in the figure, the fluorescence at 658 nm gradually increases with the addition of peroxynitrite. The relationship between the fluorescence intensity and the concentration of peroxynitrite is as follows: Figure 3 As shown, at 1.0×0 -7 Up to 1.0×10 -5 The fluorescence exhibits a linear relationship within the concentration range of mol / L, and the fluorescence measurement instrument used is a Hitachi F-7100 fluorescence spectrophotometer.

[0045] Example 3

[0046] Determination of UV-Vis absorption spectra of the fluorescent probe compound NIR-ONOO reacting with different concentrations of peroxynitrite.

[0047] Figure 2 Figure 2 shows the UV-Vis absorption spectra of the fluorescent probe compound NIR-ONOO prepared in Example 1 after reaction with different concentrations of peroxynitrite. The amount of peroxynitrite added ranged from 0 to 30 μM. As can be seen from Figure 2, an absorption peak is observed at 620 nm, and the absorbance gradually increases with increasing peroxynitrite concentration. The UV-Vis absorption spectra were measured using a Cary 100 UV-Vis spectrophotometer.

[0048] Example 4

[0049] The fluorescent probe compound NIR-ONOO exhibits selectivity for various common substances in various living organisms.

[0050] 0.02 mL of the fluorescent probe stock solution from Example 2 was added to a 5 mL centrifuge tube. The standard solution of the competing substance to be investigated was added, and the solution was diluted to 4 mL with 50 mM PBS buffer (pH = 7.4) (DMF / PBS volume ratio 1:1). A total of 20 common substances in living organisms were investigated, resulting in 21 samples, the last of which was peroxynitrite. Fluorescence spectra were measured using 620 nm excitation light and a slit width of 5 nm / 5 nm. The results are as follows: Figure 4 As shown, common substances in living organisms have almost no effect on the fluorescence of the fluorescent probe compound NIR-ONOO at 658 nm, while the addition of peroxynitrite solution significantly enhances the fluorescence of the fluorescent probe NIR-ONOO at 658 nm.

[0051] Example 5

[0052] Effect of solution pH on the fluorescence response of the fluorescent probe compound NIR-ONOO in the determination of peroxynitrite.

[0053] The effect of pH on the fluorescence intensity of the blank fluorescent probe compound NIR-ONOO and NIR-ONOO + peroxynitrite (15 μM) was investigated. The results are as follows: Figure 5 Within the pH range of 5–9, pH had virtually no effect on the fluorescence intensity of the blank fluorescent probe compound NIR-ONOO. However, in the presence of peroxynitrite (15 μM), the fluorescence intensity of the detection system was highest at pH 7.4, indicating that the fluorescent probe compound NIR-ONOO showed the best response to peroxynitrite at this pH. Figure 5 As can be seen, within the pH range of 5 to 9, the fluorescent probe compound NIR-ONOO showed a good response to peroxynitrite.

[0054] Example 6

[0055] Detection of exogenous peroxynitrite in HeLa cells by the fluorescent probe compound NIR-ONOO

[0056] First, different concentrations of SIN-1 (3-morpholinopyridineimine hydrochloride, peroxynitrite donor) were added to HeLa cells. After incubation for 30 min, the fluorescent probe compound NIR-ONOO (5 μM) was added to the HeLa cells and incubated for another 30 min. The cells were then washed three times with Duchenne phosphate-buffered saline (DPBS) buffer. The HeLa cells were then imaged under a laser confocal microscope. The fluorescence imaging conditions were set as follows: λ ex =638nm, emission wavelength set to 640–750nm. Fluorescence imaging results are as follows: Figure 6 As shown in the figure, the intracellular fluorescence intensity gradually increased with increasing SIN-1 concentrations (0 μM, 500 μM, 1000 μM). However, the experimental group that was pre-incubated with uric acid (200 μM, a peroxynitrite consumable) and SIN-1 (1 mM) for half an hour before adding the probe showed a significant decrease in fluorescence intensity. These results indicate that the fluorescent probe compound NIR-ONOO can effectively detect exogenous peroxynitrite in cells.

[0057] Example 7

[0058] Detection of endogenous peroxynitrite in RAW 264.7 cells using the fluorescent probe compound NIR-ONOO.

[0059] First, RAW 264.7 cells were incubated with different concentrations of LPS / IFN-γ for 12 h. Then, the fluorescent probe compound NIR-ONOO (5 μM) was added for a further 30 min of incubation. The cells were then washed three times with Duchenne phosphate-buffered saline (DPBS) before cell imaging. Figure 7 It can be seen that the fluorescence intensity of RAW 264.7 cells gradually increases with the increase of LPS / IFN-γ concentration. These results indicate that the fluorescent probe compound NIR-ONOO can effectively detect endogenous peroxynitrite in cells.

[0060] Example 8

[0061] Detection of exogenous peroxynitrite in liver tissue sections by the fluorescent probe compound NIR-ONOO

[0062] Considering the excellent two-photon excitation fluorescence properties of compound 2, generated by the reaction of fluorescent probe compound NIR-ONOO with peroxynitrite, this study further applied NIR-ONOO to the imaging detection of exogenous peroxynitrite in liver tissue sections under two-photon imaging mode. In this experiment, mouse liver tissue was used as the imaging target. After preparing sections of mouse liver tissue, the sections were incubated with 50 μM fluorescent probe solution for half an hour, followed by the addition of SIN-1 (5 mM, 3-morpholinopyridineimine hydrochloride, peroxynitrite donor), and incubated for another 30 min. The prepared tissue sections were washed three times with phosphate buffer and then subjected to depth scanning imaging under a confocal microscope. The testing conditions were set as follows: two-photon excitation wavelength of 820 nm and emission wavelength band of 640–750 nm. The results are as follows: Figure 8 As shown, the liver tissue emitted a significant fluorescent signal, with a penetration depth of up to 168 μm. This demonstrates that the fluorescent probe NIR-ONOO can effectively image exogenous peroxynitrite in mouse liver tissue with good imaging depth.

[0063] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe for peroxynitrite, characterized in that... The structural formula of the fluorescent probe is: The fluorescent probe exhibits near-infrared fluorescence emission and the ability to selectively detect peroxynitrite.

2. A method for preparing the peroxynitrite near-infrared fluorescent probe according to claim 1, characterized in that... The specific preparation steps are as follows: Step S1: Add 0.63 g of 8-hydroxyjuloridin, 0.53 g of phthalic anhydride, and 35 mL of toluene to the reaction vessel. Reflux at 120 °C for 5 h. Cool the reaction solution to room temperature and evaporate the solvent to obtain the crude product. Purify the crude product using silica gel column chromatography to obtain a brown solid compound 1 with a yield of 70%. The eluent used in the silica gel column chromatography was a 50:1 (v / v) mixture of dichloromethane and methanol. The corresponding synthetic route is as follows: Step S2: At 0℃, 15 mL of H2SO4 solution, 1.012 g of compound 1, and 567.8 mg of 6-(dimethylamino)-3,4-dihydronaphthyl-1(2H)-one were added to the reaction vessel and stirred until homogeneous. The mixture was then heated to 90℃ and reacted for 3 h. After the reaction solution cooled to room temperature, it was slowly poured into ice water, followed by the addition of 1 mL of perchloric acid. After stirring for 2 min, a precipitate was formed. The mixture was filtered through a Buchner funnel, and the precipitate was washed with cold water to obtain the crude product. The crude product was dried and purified by silica gel column chromatography to obtain a dark blue solid compound 2 with a yield of 52%. The eluent used in the silica gel column chromatography was a 15:1 volume ratio of dichloromethane / methanol mixture. The corresponding synthetic route is as follows: Step S3: Add 30 mL of dichloromethane, 59.0 mg of compound 2, 19.2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 13.5 mg of 1-hydroxybenzotriazole to the reaction vessel and stir until homogeneous at room temperature. Then add 10.8 mg of phenylhydrazine and continue stirring at room temperature for 4 h. The reaction mixture is then washed three times with water. The resulting dichloromethane phase is dried over anhydrous Na2SO4 and evaporated to dryness to obtain the crude product. The crude product is purified by silica gel column chromatography to obtain the target fluorescent probe compound NIR-ONOO in 53% yield. The eluent used for silica gel column chromatography is a 50:1 (v / v) mixture of dichloromethane and methanol. The corresponding synthetic route is as follows:

3. The application of the near-infrared fluorescent probe for peroxynitrite as described in claim 1 in the selective detection of peroxynitrite, wherein the fluorescent probe can detect peroxynitrite in the presence of K + Mg 2+ Cu 2+ Fe 3+ Na + Zn 2+ Ca 2+ NO2 - S2 - SO3 2- NO 1 O2, HClO, ·OH, H2O2, O2 ·- Selective detection of peroxynitrite in PBS buffer solutions containing Cys, Hcy, and GSH.

4. The application of the peroxynitrite near-infrared fluorescent probe of claim 1 in the selective detection of peroxynitrite in aqueous solution, biological cell system or animal liver tissue.

5. The application of the peroxynitrite near-infrared fluorescent probe according to claim 1 in the preparation of peroxynitrite fluorescent detection reagents or ultraviolet-visible light detection reagents in aqueous solutions.

6. The application of the peroxynitrite near-infrared fluorescent probe according to claim 1 in the preparation of peroxynitrite fluorescence imaging detection reagents in biological cell systems.

7. The application of the peroxynitrite near-infrared fluorescent probe according to claim 1 in the preparation of a peroxynitrite fluorescence imaging detection reagent in animal liver tissue.

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