An onoo - Near-infrared fluorescent probes, methods of making and using same

Abstract

This invention relates to ONOO ‑ In the field of detection technology, specifically involving an ONOO ‑ Near-infrared fluorescent probe, its preparation method, and its application. This invention constructs a novel fluorescent probe specifically for detecting ONOO. ‑ The ONOO ‑ Near-infrared fluorescent probe for ONOO ‑ It exhibits good reactivity and is capable of specifically imaging endogenous ONOO in living cells or in vivo systems. ‑ Furthermore, it exhibits a large Stokes shift and an emission wavelength of 760 nm, significantly higher than other near-infrared fluorescent probes based on hemicyanine dye structures. In addition, this ONOO... ‑ The near-infrared emission characteristics of near-infrared fluorescent probes enable them to penetrate deeper into tissues and reduce background interference in bioimaging, which is significant for improving imaging sensitivity and accuracy. Based on these characteristics, ONOO... ‑ Near-infrared fluorescent probes can be used for the diagnosis and research of diseases such as atherosclerosis, neurodegenerative diseases, inflammation, and cancer.

Description

Technical Field

[0001] This invention relates to ONOO - In the field of detection technology, specifically involving an ONOO - Near-infrared fluorescent probes, their preparation methods, and applications. Background Technology

[0002] Peroxynitrite (ONOO) - It is composed of nitric oxide (NO) and superoxide anion (O2). - Highly reactive nitrogen species (RNS) are rapidly generated under physiological conditions. - It has strong oxidizing and nitrifying capabilities, enabling it to oxidize lipids, proteins, and DNA, and plays a key role in various pathological processes, including atherosclerosis, neurodegenerative diseases, inflammation, and cancer.

[0003] However, ONOO - With extremely short lifetimes (approximately 10-20 milliseconds), low concentrations, and high reactivity, traditional detection methods such as electron spin resonance (ESR), high-performance liquid chromatography (HPLC), and immunohistochemistry suffer from drawbacks including complex operation, low spatiotemporal resolution, and inability to provide real-time monitoring. Small molecule fluorescent probes, due to their high sensitivity, high selectivity, non-invasiveness, and real-time imaging capabilities, have become the preferred method for detecting ONOO in biological systems. - The ideal tool.

[0004] Near-infrared (NIR, 650–900 nm) fluorescent probes offer advantages such as deep penetration into biological tissues, minimal background fluorescence interference, and low photodamage to biological samples, making them particularly suitable for in vivo imaging. Hememicyanine dyes, a typical D-π-A structure fluorescent dye, possess advantages such as high molar extinction coefficient, moderate fluorescence quantum yield, and easy structural modification, making them excellent frameworks for constructing near-infrared fluorescent probes.

[0005] Currently reported ONOO - Fluorescent probes are mostly based on recognition groups such as borate esters, ketones, or selenide ethers, but they suffer from slow response speed, poor selectivity, or insufficient emission wavelength. Therefore, it is necessary to develop an ONOO probe based on a novel recognition mechanism that exhibits fast response, high selectivity, and near-infrared emission characteristics. - Fluorescent probes have significant scientific value and promising applications in the diagnosis and research of diseases such as atherosclerosis, neurodegenerative diseases, inflammation, and cancer. Summary of the Invention

[0006] This invention aims to overcome the limitations of existing technologies. -The shortcomings of fluorescent probes, such as slow response speed, poor selectivity, and insufficient emission wavelength, necessitate the development of an ONOO... - Near-infrared fluorescent probes, their preparation methods, and applications are proposed to overcome the aforementioned shortcomings.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an ONOO - The near-infrared fluorescent probe has the following structural formula (1): (1).

[0008] This invention constructs a novel fluorescent probe specifically for detecting ONOO. - The ONOO - The specific mechanism of action of near-infrared fluorescent probes is as follows: Figure 1 As shown, ONOO - It possesses strong nucleophilicity and oxidizing properties, enabling it to specifically attack the sulfur atoms of trifluoromethanesulfonate, leading to the breaking of the OS bond and the release of free phenolic hydroxyl groups. Upon release of the phenolic hydroxyl groups, the PET process is blocked, the conjugated system of the hemicyanine dye is restored, and the fluorescence intensity is significantly enhanced. Simultaneously, the emission wavelength may undergo a blue or red shift, enabling ratiometric or enhanced detection.

[0009] Meanwhile, the inventors proved through experimental results that ONOO - Near-infrared fluorescent probe for ONOO - It exhibits excellent reactivity and is capable of specifically imaging endogenous ONOO in living cells or in vivo systems. - Furthermore, it exhibits a large Stokes shift and an emission wavelength of 760 nm, significantly higher than other near-infrared fluorescent probes based on hemicyanine dye structures. In addition, this ONOO... - The near-infrared emission characteristics of near-infrared fluorescent probes enable them to penetrate deeper into tissues and reduce background interference in bioimaging, which is significant for improving imaging sensitivity and accuracy. Based on these characteristics, ONOO... - Near-infrared fluorescent probes can be used for the diagnosis and research of diseases such as atherosclerosis, neurodegenerative diseases, inflammation, and cancer.

[0010] This invention provides an ONOO - Methods for preparing near-infrared fluorescent probes include: S1. IR-780 Iodide and 3-hydroxybenzylthiophenol undergo a nucleophilic substitution reaction with triethylamine to give precursor solution A. Precursor solution A is concentrated and purified to give a blue intermediate. S2. The blue intermediate, triethylamine, trifluoromethanesulfonic anhydride, and dichloromethane were reacted with stirring to obtain precursor solution B. Precursor solution B was washed, concentrated, and purified to obtain ONOO. - Near-infrared fluorescent probe.

[0011] The method provided by this invention produces ONOO - A near-infrared fluorescent probe was first synthesized using IR-780 iodide and 3-hydroxybenzylthiophenol with the assistance of triethylamine to obtain a blue intermediate. Subsequently, the blue intermediate, triethylamine, and trifluoromethanesulfonic anhydride were reacted in dichloromethane to yield ONOO, a blue solid. - Near-infrared fluorescent probe. Its reaction formula is as follows: Figure 2 As shown.

[0012] Preferably, in S1, the molar ratio of IR-780 iodide, 3-hydroxythiophenol, and triethylamine is 1:(2~4):(2~4). And / or, the nucleophilic substitution reaction is performed at 50-60°C for 4-10 h; And / or, after the nucleophilic substitution reaction is completed, the solution is concentrated and purified to obtain a blue intermediate.

[0013] Preferably, in S2, the molar ratio of the blue intermediate, triethylamine, and trifluoromethanesulfonic anhydride is 1:(3~4):(2~3). And / or, the reaction conditions are 30-40°C with stirring for at least 12 hours; And / or, trifluoromethanesulfonic anhydride is added under ice bath conditions to react with the blue intermediate and triethylamine.

[0014] Preferably, in S1 and S2, the concentration is carried out by rotary evaporator concentration; and / or the purification is carried out by column chromatography purification of the crude product obtained after concentration; and / or the eluent for column chromatography purification is dichloromethane and methanol, with dichloromethane and methanol eluting at a volume ratio gradient of 100:1 to 10:1.

[0015] This invention provides ONOO - Near-infrared fluorescent probes in ONOO - Applications in in vivo detection and / or imaging.

[0016] Preferably, the in vivo includes: cells, tissues and organs; and / or, the detection is a quantitative detection; and / or, the detection limit is ≤29 nM.

[0017] The ONOO - Near-infrared fluorescent probes possess advantages such as fast response speed, high specificity and selectivity, low susceptibility to background interference, and high sensitivity (detection limit ≤29 nM). With the development of ONOO... - With the increase of concentration, ONOO- The response intensity of the near-infrared fluorescent probe also increases accordingly, making it suitable for ONOO applications. - The detection, quantitative detection, and imaging of ONOO. Furthermore, experiments have proven that this ONOO... - Near-infrared fluorescent probes can be visualized and traced in living cells. - Dynamic changes in levels can affect exogenous ONOO in live mice. - Imaging based on horizontal changes; proving the ONOO - Near-infrared fluorescent probes can be used for detection in cells, tissues, and organs.

[0018] This invention provides ONOO - Application of near-infrared fluorescent probes in the preparation of kits for detecting atherosclerosis and / or bioimaging tracers.

[0019] In the development of atherosclerosis (AS), ONOO - Excessive production of ONOO- is an important pathological marker. Endothelial cell dysfunction, macrophage infiltration, and foam cell formation are all accompanied by the production of large amounts of ONOO-, leading to low-density lipoprotein (LDL) oxidation, apoptosis, and plaque instability. Therefore, the ONOO- provided by this invention can be utilized... - Near-infrared fluorescent probes are used to prepare kits for the detection of atherosclerosis and / or bioimaging tracers to monitor ONOO in AS lesions. - The level.

[0020] This invention provides a method for detecting ONOO - A kit comprising ONOO - Near-infrared fluorescent probe.

[0021] This invention provides a method for dynamically monitoring ONOO. - Bioimaging tracers, the bioimaging tracers comprising ONOO - Near-infrared fluorescent probe.

[0022] Therefore, the present invention has the following beneficial effects: (1) This invention constructs a novel ONOO - Near-infrared fluorescent probe, specifically designed for detecting ONOO - It can be applied to the diagnosis and research of diseases such as atherosclerosis, neurodegenerative diseases, inflammation, and cancer.

[0023] (2) The ONOO developed in this invention -Near-infrared fluorescent probes have a large Stokes shift and an emission wavelength of 760 nm, which is significantly higher than other near-infrared fluorescent probes based on hemicyanine dye structures, making them suitable for bioimaging.

[0024] (3) The ONOO developed in this invention - Near-infrared fluorescent probes are stable under physiological conditions, exhibit excellent optical stability, and are also resistant to ONOO. - It has high sensitivity and high selectivity, and can identify ONOO in complex environments. - At the same time, ONOO - It has a fast response capability, thus enabling real-time monitoring of ONOO. - Changes in level.

[0025] (4) The ONOO developed in this invention - Near-infrared fluorescent probes were successfully used to monitor ONOO in atherosclerosis models, cells, and live mice. - The horizontal change successfully achieved ONOO - Visual observation in a mouse model of atherosclerosis, with real-time tracking of ONOO - It has great potential in related pathological conditions and provides an effective tool for the diagnosis of atherosclerosis and the research of related drugs. Attached Figure Description

[0026] Figure 1 For the detection of ONOO by the fluorescent probe of this invention - A schematic diagram of the mechanism; Figure 2 This is a synthesis route diagram of the fluorescent probe in Example 1 of the present invention; Figure 3 The fluorescent probe in Example 1 of this invention 1 H NMR spectrum; Figure 4 The fluorescent probe in Example 1 of this invention 13 C NMR spectrum; Figure 5 The HRMS spectrum of the fluorescent probe in Example 1 of this invention; Figure 6 The fluorescent probe of this invention and ONOO - The relevant optical experimental results are shown in the figure, where a represents the probe and ONOO. - The ultraviolet absorption spectrum, b is the probe and ONOO - The fluorescence emission spectrum, where c is the fluorescence emission spectrum of the probe and ONOO. - Linear fitting at 760 nm, where d is the probe and ONOO -The results of the anti-interference experiment in the presence of other substances, e is the result of the selectivity experiment of the probe with other substances, and f is the result of the pH stability test of the probe; Figure 7 The fluorescent probe of this invention and ONOO - The relevant time results are shown in the graph, where a represents the time between the probe and ONOO. - The time-kinetic results after the reaction are shown in Figure b, where b represents the time-kinetic results of the probe and ONOO. - Time stability monitoring results graph; Figure 8 This is a graph showing the cytotoxicity results of the fluorescent probe of this invention in RAW 264.7 cells; Figure 9 The fluorescent probe of this invention targets ONOO in living cells. - The results of the detection capability are shown in the figure, where a represents the ONOO levels in RAW 264.7 cells after SIN-1 pretreatment. - Imaging, b shows the probe's effect on ONOO in RAW 264.7 cells pretreated with 2-ME. - Imaging; Figure 10 Nile red staining image of RAW 264.7 cells after ox-LDL treatment; Figure 11 The fluorescent probe of this invention targets ONOO in foam cells. - The detection capability results show that ox-LDL induces different levels of ONOO in foam cells at different times. - Imaging and UA-preprocessed RAW 264.7 cells ONOO - Imaging; Figure 12 ONOO in RAW 264.7 cells after pretreatment with the fluorescent probe NAC of this invention - Image; Figure 13 For HKYellow-AM to ONOO - Test result image; Figure 14 HE staining images of the heart, liver, spleen, lungs, and kidneys of mice that were injected with and not injected with the probe; Figure 15 The fluorescent probe of this invention is for the in vitro ONOO in live mice. - The image; Figure 16 To verify the construction results of different degrees of atherosclerosis models, the diagram shows: a) H&E staining, b) Oil Red O staining, c) Nile Red staining, and d) four lipid parameters. Figure 17 The probes used in this embodiment of the invention were tested on WT mice and 12W APOE mice. - / -Mice, 18W APOE - / - ONOO in the model - Live imaging image. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0028] In this section, all chemicals and solvents used are of analytical grade. HPLC-MS analysis was performed on a Waters LCMS system, and NMR spectroscopy was also performed. 1 H and 13 C) Data were recorded on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as an internal control; UV-Vis absorption spectra were obtained using a PerkinElmer Lambda (UV / VIS) spectrophotometer, and fluorescence emission spectra were collected using a PerkinElmer Fluorescence Spectrometer (FL 6500). Absorption and fluorescence test results were recorded at room temperature in PBS containing 30% anhydrous ethanol; cell fluorescence imaging was performed using a Leica confocal microscope, blue channel: λex = 405 nm, λem = 425~504 nm; red channel: λex = 680 nm, λem = 740~790 nm, scale bar: 20 µm; data were analyzed using one-way ANOVA, and the means of the experimental groups were compared with the means of the control group (n=3; , P <0.001).

[0029] The raw materials were sourced as follows: IR-780 iodide was purchased from Sigma, CAS 207399-07-3; 3-hydroxythiophenol was purchased from Maclean, CAS 40248-84-8; dichloromethane was purchased from Shanghai Testing & Certification Center; methanol was purchased from Shanghai Testing & Certification Center; triethylamine was purchased from Aladdin, NEt3, CAS 121-44-8; trifluoromethanesulfonic anhydride was purchased from Anaiji, CAS 358-23-6; and NN-dimethylformamide was purchased from Aladdin, DMF, CAS D111999.

[0030] Unless otherwise specified, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0031] The 8-week-old C57BL / 6J mice and APOE used - / - Mice were purchased from Wuhan Shubaili Biotechnology Co., Ltd., and all animal experiments were conducted in accordance with the institutional animal use and care regulations approved by the Ethics Committee of Jianghan University.

[0032] Example 1 A 10 mL solution of DMF containing IR-780 (333.5 mg, 0.5 mmol) was added to a 10 mL solution of DMF containing 3-hydroxythiophenol (126 mg, 1.0 mmol) and triethylamine (200 μL, 1.44 mmol). The reaction mixture was heated to 60 °C for 8 hours. The resulting mixture was concentrated using a rotary evaporator, and the crude product was purified by column chromatography (eluent: dichloromethane:methanol = 100:1–10:1, v / v) to give a blue solid intermediate. The intermediate (110 mg, 0.25 mmol) and triethylamine (130 μL, 1 mmol) were slowly added to dichloromethane, and trifluoromethanesulfonic anhydride (140 mg, 0.5 mmol) was slowly added in an ice bath with stirring at 38 °C overnight. The resulting mixture was concentrated using a rotary evaporator. The crude product was purified by column chromatography (eluent: dichloromethane:methanol = 100:1~10:1, v / v), and after vacuum drying, a blue solid target probe (44.21 mg, yield 31.57%) was obtained, namely ONOO. - Near-infrared fluorescent probe. The above reaction formula is as follows: Figure 2 As shown.

[0033] The structure of the aforementioned blue powder probe was characterized by the following tests: 1 H NMR, 13 C NMR and HRMS spectra as follows Figure 3-5 As shown.

[0034] from Figure 3-5 As can be seen from this, the compound has the following properties. 1H NMR (400 MHz, DMSO) δ 8.28-8.24 (d, 1H), 8.04-8.03 (d, 1H), 7.89-7.87 (m, 2H), 7.74-7.72 (m, 1H), 7.62-7.58 (m, 2H), 7.53-7.51(m, 1H), 7.27 (s, 1H), 7.00-6.97 (d, 1H), 4.56-4.52(t, 2H), 2.78-2.75 (t, 2H), 2.73-2.70 (t, 2H), 1.87-1.84 (t, 2H), 1.79 (s,6H), 1.23 (s, 2H), 1.01-0.97 (t, 3H). 13 C10 NMR (101 MHz, DMSO) δ 179.90, 148.88, 147.27, 145.75, 143.46, 141.63, 136.13, 134.19, 132.53, 131.48, 129.96, 129.64, 129.03, 128.91, 123.54, 121.18, 118.81, 115.09, 111.09, 110.55, 51.79, 47.57, 32.24, 29.16, 27.65, 26.90, 21.91, 20.23, 11.41. Chemical formula: C10 29 H 29 F3NO3S2[M+H] + Precision quality: 560.67, Discovery quality: 560.1533.

[0035] The results proved that the synthesis yielded the desired result. Figure 2 The image shows a near-infrared fluorescent probe based on a semicyanine dye.

[0036] Application Example 1 This example demonstrates the probe's interaction with ONOO through an optical experiment. - The detection capability was demonstrated, and all the following experiments were performed in PBS containing 30% anhydrous ethanol, with an excitation wavelength of 680 nm and a slit size of 10 nm / 10 nm.

[0037] (1) Absorption spectroscopy detection.

[0038] Probe (10 μM) and ONOO - The ultraviolet absorption spectrum is as follows Figure 6 As shown in figure a, the probe characteristic peak is at 580 nm; however, after adding ONOO -Subsequently, the initially blue solution turned colorless, and a new absorption peak appeared at 680 nm.

[0039] (2) Fluorescence spectroscopy detection.

[0040] Probe (10 μM) and ONOO - The fluorescence emission spectrum is as follows Figure 6 As shown in b, ONOO is added to the probe solution. - Subsequently, the fluorescence significantly increased, indicating that the probe was effective against ONOO. - The detector exhibits excellent fluorescence response.

[0041] (3) Quantitative feasibility analysis.

[0042] This example demonstrates the use of a detection probe with different concentrations of ONOO. - The fluorescence intensity after the reaction was used to evaluate the probe quantization of ONOO. - The ability to determine concentration. Results are as follows: Figure 6 As shown in c, with ONOO - With increasing concentration, the emission intensity at 750 nm also increases, and the fluorescence intensity is similar to that of ONOO. - A strong linear relationship exists between concentrations. The probe pair ONOO was calculated using the standard formula LOD = 3σ / k (σ refers to the standard deviation of the blank sample signal, and k refers to the slope of the calibration curve). - The detection limit is 29 nM.

[0043] (4) Specific detection.

[0044] This example selected common metal ions, anions, ROS, RNS, and various other substances for selective experiments to determine the probe's response to ONOO in complex environments. - The specificity of the probe (10 μM) and other substances (50 μM) is shown in the experimental results. Figure 6 As shown in d, where 1-21 represent Na + K + Fe 3+ Fe 2+ AL 3+ Ca 2+ Zn 2+ NH4 + Cl - NO3 - CO3 2- HCO3 - SO4 - GSH, Hcy, Cys, ClO - H2O2, O2 - HS - ONOO- The results showed that only ONOO - It can react with the probe and produce a significant fluorescence change.

[0045] Furthermore, the probe (10 μM) was coupled with ONOO - The anti-interference experiment results in the presence of other substances (100 μM) are as follows: (50 μM) Figure 6 As shown in e, where 1-20 represent Na + K + Fe 3+ Fe 2+ AL 3+ Ca 2+ Zn 2+ NH4 + Cl - NO3 - CO3 2- HCO3 - SO4 - GSH, Hcy, Cys, ClO - H2O2, O2 - HS - The results show that, in the presence of other interfering substances, the probe is effective against ONOO. - The specificity is unaffected.

[0046] The results of selective and anti-interference experiments show that the probe can identify ONOO in complex environments. - This could potentially enable the precise identification of ONOO in the biological environment. - .

[0047] (5) Optical stability test.

[0048] To evaluate the practical applicability of the probe, the effect of pH on its performance was systematically studied. The results are as follows: Figure 6 As shown in f, with or without ONOO - Under these conditions, the probe maintains good stability at pH values ​​ranging from 6.0 to 11.0. These results indicate that the probe retains good reactivity at physiological pH levels, thus enabling it to function reliably in biological applications.

[0049] (6) Response time detection.

[0050] This example demonstrates the use of a detection probe with different concentrations of ONOO. - Fluorescence intensity within 240 min after reaction, high concentration ONOO - It takes a certain amount of time to reach reaction equilibrium. Figure 7 a). Therefore, 1 h was chosen as the reaction node to detect the reaction of the fluorescent probe.

[0051] Further detection of the probe within 24 hours with ONOO - The fluorescence intensity after the reaction indicates the stability of the fluorescence after the reaction. Figure 7 b).

[0052] Application Example 2 This example uses mouse mononuclear macrophage leukemia cells (RAW 264.7) to demonstrate the effect of the probe on ONOO within the cells. - Its imaging capabilities.

[0053] The RAW 264.7 cells used in this example were cultured in complete medium containing 1% penicillin-streptomycin, 10% fetal bovine serum, and 90% DMEM. All cell plates and culture dishes were placed in a humidified incubator at 37°C, 5% CO2, and 95% O2.

[0054] Before conducting imaging experiments, the MTT assay was used to assess the cytotoxicity of the probe in RAW264.7 cells. Figure 8 ).

[0055] Based on the above, probes were used to monitor exogenous ONOO in RAW 264.7 cells. - Specifically, 0-1 mM exogenous ONOO was added to cultured SH-SY5Y cells. - (SIN-1), incubate for 1 hour, then add the probe (5 µM) and incubate for another 30 minutes. Results are as follows: Figure 9 As shown in Figure a, the fluorescence intensity in RAW 264.7 cells increased with increasing SIN-1 concentration, indicating that the probe can effectively detect exogenous ONOO in cells. - ONOO - O2 - The rapid reaction with NO produces 2-methoxyestradiol (2-ME, 0, 5, 10 µg / mL), which is then incubated for 30 minutes, followed by incubation with the probe for another 30 minutes. Results are as follows... Figure 9 b. Fluorescence intensity increases with increasing 2-ME concentration, further verifying that the probe can target intracellular ONOO. - Quantitative analysis. The above results confirm that the probe is suitable for monitoring intracellular ONOO. - Horizontal fluctuations.

[0056] Application Example 3 This example establishes a foam cell model to verify the effectiveness of the probe in dynamically monitoring AS in real time. - The ability to change horizontally.

[0057] Following atherosclerosis, the level of reactive oxygen species (ROS) increases significantly, leading to oxidative stress and increased ONOOS. - Generate. Therefore, real-time ONOO - Imaging techniques are crucial for the timely diagnosis of atherosclerosis. Foam cells are a cell model of atherosclerosis. RAW 264.7 cells were induced to transform into foam cells at different time points (0 h, 24 h, 48 h) using oxidized low-density lipoprotein (ox-LDL, 50 ug / mL). To confirm foam cell formation, Nile red staining was used to observe intracellular lipid accumulation. Figure 10 This dye fluoresces strongly in hydrophobic lipid environments. Figure 11 As shown, with increasing ox-LDL treatment time, foam cells exhibited an increase in the number of bright red fluorescent spots, representing lipid droplets, while control group cells showed only weak fluorescence, confirming that RAW 264.7 cells had been successfully transformed into foam cells of varying degrees.

[0058] Subsequently, the foam cell models at different stages of modeling were imaged using a probe (5 μM), and the fluorescence intensity was found to increase with the deepening of modeling. After foam cells were formed, uric acid (UA, 50 μM, 100 μM) was added. - The scavenging agent caused the red fluorescence intensity to gradually decrease, confirming that the probe could specifically respond to ONOO produced during the pathological process in the cell model. - And it can also monitor ONOO - The dynamic changes.

[0059] At the same time, adding different concentrations of NAC (N-Acetyl-L-Cysteine) antioxidants to foam cells prevents low-density lipoprotein from turning into oxidized low-density lipoprotein, thus reducing ONOO at the source. - The generation of fluorescence; as the NAC concentration increases, the fluorescence intensity decreases, such as... Figure 12 As shown. These results further demonstrate that the probe can monitor ONOO. - The dynamic changes.

[0060] Additionally, HKYellow-AM (6 / 12-mixture) is a yellow fluorescent probe that can detect ONOO in live cells and tissues with high selectivity and sensitivity. - .like Figure 13 This further verified that in ox-LDL-induced foam cells, prolonged incubation with ox-LDL (0 h, 24 h, 48 h) resulted in increased ONOO - Increased content and the addition of UA to ONOO - The content decreased. The above results verify that the probe can monitor ONOO under different conditions.- Its dynamic capabilities demonstrate its strong application potential in AS research.

[0061] Application Example 4 This example validates the ability of probes to monitor ONOO in an animal model. - The ability to fluctuate is as follows: (1) In vivo and exogenous ONOO in live mice - Imaging.

[0062] First, the probe's in vivo toxicity was tested to ensure its safety in live animal experiments. For example... Figure 14 As shown, APOE was injected with 100 μL of 1 mg / kg probe (experimental group) or 100 μL of PBS (control group). - / - H&E staining of the heart, liver, spleen, lungs and kidneys of WT mice showed no obvious pathological changes, confirming that the probe is safe under experimental conditions.

[0063] Subsequently, the probe's ability to detect exogenous ONOO in mice was evaluated. - To assess the effectiveness, three mice were intraperitoneally injected with PBS containing the probe (1 mg / kg, 100 μL); 2.5 μM ONOO. - +Probe, used to perform in vivo imaging of mice at a specific time after injection. For example... Figure 15 As shown, with exogenous ONOO - The fluorescence intensity increased significantly with increasing concentration, proving that the probe can effectively detect exogenous ONOO in live mice. - .

[0064] (2) APOE - / - ONOO in mouse model - Imaging.

[0065] First, the successful establishment of the animal model of atherosclerosis was verified by Nile Red staining, Oil Red O staining, H&E staining, and four blood lipid tests. Figure 16 As shown, Nile Red staining, H&E staining, and Oil Red O staining revealed that ApoE... / Obvious atherosclerotic plaques were visible in the blood vessels of mice, and the plaques became more pronounced with prolonged high-fat feeding. Further validation using four lipid parameters (triglycerides (TG), total cholesterol (T-CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL)) confirmed the successful establishment of in vivo mouse models of atherosclerosis at different stages.

[0066] Subsequently, to WT, 12W ApoE / Mice, 18W ApoE / Mice were subjected to in vivo imaging at specific time points after a tail vein injection of a probe (1 mg / kg, 100 μL). Figure 17 As shown, in vivo imaging revealed that the fluorescence signal in the atherosclerosis model mice was significantly stronger than that in C57, and that ApoE levels increased with the progression of atherosclerosis. / The fluorescence intensity in mice gradually increased. These results indicate that the probe can accurately detect ONOO in animal models. - level.

[0067] The above results confirm that the probe can track ONOO in vivo. - Horizontal fluctuations.

[0068] In summary, the probe provided by this invention can selectively and sensitively detect ONOO. - , for ONOO - ONOO has a rapid response capability and has been successfully applied in cell and animal models. - Imaging can accurately reflect the regulated ONOO in cell and animal models. - The level demonstrates its ability to monitor ONOO in real time under pathological conditions such as AS. - Its application value.

Claims

1. An ONOO - Near-infrared fluorescent probe, characterized in that, Its structural formula is shown in equation (1) below: （1）。 2. The ONOO as described in claim 1 - A method for preparing a near-infrared fluorescent probe, characterized in that, include: S1. IR-780 Iodide and 3-hydroxybenzylthiophenol undergo a nucleophilic substitution reaction with triethylamine to give precursor solution A. Precursor solution A is concentrated and purified to give a blue intermediate. S2. The blue intermediate, triethylamine, trifluoromethanesulfonic anhydride, and dichloromethane were reacted with stirring to obtain precursor solution B. Precursor solution B was concentrated and purified to obtain ONOO. - Near-infrared fluorescent probe.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the IR-780 iodide, 3-hydroxythiophenol and triethylamine is 1:(2~4):(2~4); And / or, the nucleophilic substitution reaction is performed at 50-60°C for 4-10 h; And / or, after the nucleophilic substitution reaction is completed, the solution is concentrated and purified to obtain a blue intermediate.

4. The preparation method according to claim 2, characterized in that, In S2, the molar ratio of the blue intermediate, triethylamine, and trifluoromethanesulfonic anhydride is 1:(3~4):(2~3). And / or, the reaction conditions are 30-40°C with stirring for at least 12 hours; And / or, trifluoromethanesulfonic anhydride is added under ice bath conditions to react with the blue intermediate and triethylamine.

5. The preparation method according to any one of claims 2 to 4, characterized in that, In S1 and S2, the concentration is performed by rotary evaporator concentration; and / or the purification is performed by column chromatography purification of the crude product obtained after concentration; and / or the column chromatography purification uses dichloromethane and methanol as the eluent, with dichloromethane and methanol eluting at a volume ratio gradient of 100:1 to 10:

1.

6. The ONOO as described in claim 1 - Near-infrared fluorescent probes or ONOO prepared by any of the preparation methods described in claims 2 to 5 - Near-infrared fluorescent probes in ONOO - Applications in in vivo detection and / or imaging.

7. The application as described in claim 6, characterized in that, The in vivo includes: cells, tissues and organs; and / or, the detection is a quantitative detection; and / or, the detection limit of the detection is ≤29 nM.

8. The ONOO as described in claim 1 - Near-infrared fluorescent probes or ONOO prepared by any of the preparation methods described in claims 2 to 5 - Application of near-infrared fluorescent probes in the preparation of kits for detecting atherosclerosis and / or bioimaging tracers.

9. A method for detecting ONOO - The reagent kit is characterized by, The kit comprises the ONOO as described in claim 1. - Near-infrared fluorescent probes or ONOO prepared by any of the preparation methods described in claims 2 to 5 - Near-infrared fluorescent probe.

10. A method for dynamically monitoring ONOO - A bioimaging tracer, characterized in that, The bioimaging tracer includes the ONOO as described in claim 1. - Near-infrared fluorescent probes or ONOO prepared by any of the preparation methods described in claims 2 to 5 - Near-infrared fluorescent probe.

Images