A peroxynitrite-responsive near-infrared fluorescent probe based on fluorine atom regulation, and a preparation method and application thereof

Abstract

The application belongs to the field of organic molecule fluorescent probes, and specifically discloses a peroxynitrite (ONOO-) near-infrared fluorescent probe and a preparation method and application thereof. The application takes rhodamine as a near-infrared dye main body and allyl carbonate as an ONOO- response group, proposes a molecular probe design method based on fluorine atom electronic effect regulation, and prepares a molecular fluorescent probe MF-ONOO for sensitive, rapid and high-selectivity response of ONOO-. Specifically, the electron-withdrawing effect of fluorine atom adjusts the electrophilicity of allyl carbonate, so that the allyl carbonate can be specifically oxidized by ONOO- and undergo ester bond cleavage, release the dye, and thus produce a significant fluorescence "turn-on" response. The probe has absorption and emission in the near-infrared region, the fluorescence enhancement factor reaches 41.6 times, the detection limit is as low as 3.03 nM, and the probe exhibits excellent selectivity and sensitivity to ONOO-. The MF-ONOO can be used to monitor the dynamic changes of exogenous and endogenous ONOO- of HepG2 cells in real time. In an acute inflammation model, specific near-infrared fluorescence imaging of inflammation sites in liver and lung tissues can be realized. The probe has simple synthesis route, high specificity and sensitivity, can provide a reliable tool for quantitative testing of various environment and disease related ONOO-, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probe technology, specifically involving a type of rhodamine-like near-infrared fluorescent probe regulated by the electronic effect of fluorine substituents and its preparation method, as well as the application of this probe in the qualitative and quantitative detection of peroxynitrite (ONOO⁻) and the fluorescence imaging diagnosis of inflammatory diseases in solution, live cells, and in vivo inflammatory models. Background Technology

[0002] Overoxygenated nitrite (ONOO⁻) is an important reactive nitrogen / oxygen species in organisms, generated in situ through a diffusion-controlled reaction of superoxide anion and nitric oxide. Under normal physiological conditions, ONOO⁻ participates in key physiological processes such as vascular tone regulation, immune defense, and cell signal transduction. However, when the production and clearance of ONOO⁻ are imbalanced, its excessive accumulation can cause widespread damage to biomolecules such as proteins, lipids, and DNA through oxidative and nitrifying stress, leading to cellular dysfunction and even apoptosis. Numerous studies have shown that abnormally elevated ONOO⁻ levels are closely related to a variety of major diseases, including inflammation, cardiovascular disease, neurodegenerative diseases, and tumors. Therefore, developing detection tools that can track the dynamic changes of ONOO⁻ in biological systems in real time with high sensitivity and selectivity has significant clinical and research value for elucidating the pathogenesis of related diseases and screening and evaluating the efficacy of anti-inflammatory drugs. Among numerous detection methods, detection techniques based on small molecule fluorescent probes have attracted widespread attention due to their advantages such as well-defined structure, high sensitivity, ease of operation, and ability to achieve real-time in-situ imaging. In recent years, researchers have developed a large number of ONOO⁻-responsive fluorescent probes based on various fluorescent precursors (such as rhodamine, hemicyanine, bodily oil, and coumarin). These probes typically utilize response mechanisms such as photoinduced electron transfer (PET), intramolecular charge transfer (ICT), and excited-state intramolecular proton transfer (ESIPT) to achieve specific recognition of ONOO⁻. However, existing ONOO⁻ fluorescent probes still have many shortcomings in performance. On the one hand, the selectivity and sensitivity of existing probes are insufficient. Due to the short half-life and extremely high oxidative activity of ONOO⁻, and the coexistence of a large number of reactive oxygen / nitrogen species, metal ions, and bio-thiol molecules with similar physicochemical properties in organisms, existing probes struggle to specifically distinguish ONOO⁻, easily generating non-specific fluorescence interference. Moreover, most probes have detection limits at the micromolar level, failing to capture nanomolar levels of trace ONOO⁻ signals in organisms. On the other hand, optical performance defects limit imaging of deep tissues. Most probes are excited and emitted in the visible light region, which causes strong interference from the autofluorescence of biological tissues and shallow tissue penetration. In addition, some probe systems rely on external metal catalysts, and metal ions can cause cytotoxicity and disrupt the redox balance in vivo, making them unsuitable for long-term live cell incubation and live animal imaging, and they have poor biocompatibility. Summary of the Invention

[0003] To address the current limitations of existing ONOO⁻ fluorescent probes in achieving a balance between sensitivity, selectivity, biocompatibility, and penetration into living tissue, this invention proposes a molecular probe design method based on the electronic effects of fluorine atoms. This method utilizes a rhodamine-like NIR dye as the main NIR dye and allyl carbonate as the ONOO⁻ responsive group, resulting in a near-infrared molecular fluorescent probe, MF-ONOO, with significantly improved overall performance.

[0004] To achieve the above objectives, the present invention discloses the following technical solution:

[0005] Firstly, this invention employs precise molecular structure design, using a rhodamine-like substance as the near-infrared fluorescent core and allyl carbonate as the ONOO⁻ specific recognition and response group. Electron-withdrawing fluorine substituents are introduced into the side chain of the fluorescent core, and the electrophilicity of the allyl carbonate group is precisely controlled through the electronic effect of the fluorine atom. This results in a specific oxidative cleavage reaction with ONOO⁻, deprotection, and release of a conjugated, extended near-infrared strongly fluorescent dye, achieving a "turn-on" type specific fluorescence response without the need for metal ion catalysis throughout the process. The probe structure is shown in Formula 1: Formula 1.

[0006] Compared with existing technologies, this invention eliminates the dependence on external metal catalysts, fundamentally avoiding the biocompatibility problems that metal catalysts may cause. At the same time, it achieves high selectivity and high sensitivity recognition of ONOO⁻ by regulating the electronic effect of allyl carbonate groups, providing a new approach for the design of ONOO⁻ fluorescent probes.

[0007] Secondly, the preparation method of the molecular fluorescent probe MF-ONOO includes the following steps:

[0008] Step 1: Cyclohexanone was slowly added dropwise to pre-cooled concentrated sulfuric acid under ice bath conditions. 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid was added in portions with stirring. The temperature was then raised to 90°C and the reaction was maintained at this temperature with stirring for 2 hours. After the reaction was complete, the reaction solution was cooled to room temperature and slowly poured into crushed ice. Perchloric acid was added, resulting in the precipitation of a red precipitate. The precipitate was filtered, washed with sufficient cold water, and dried to obtain a red solid intermediate compound 1.

[0009] Step 2: Intermediate compound 1 and 3-fluoro-4-hydroxybenzaldehyde were dissolved in a mixed solvent of acetic acid and ethanol in a volume ratio of 1:1, and the mixture was refluxed at 90°C overnight under nitrogen protection. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to obtain the purple solid intermediate MF-OH.

[0010] Step 3: Under nitrogen protection, the intermediate MF-OH was dissolved in anhydrous dichloromethane, triethylamine was added, and allyl chloroformate was slowly added dropwise while cooling in an ice bath. The mixture was then heated to room temperature and stirred overnight. After the reaction was complete, the solution was diluted with water, extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain a purple-red solid target probe MF-ONOO.

[0011] Thirdly, an application of the MF-ONOO fluorescent molecular probe is provided.

[0012] Specifically, MF-ONOO can be used for qualitative or quantitative detection of ONOO⁻ concentration in solution systems and biological samples. After reacting with ONOO⁻, the probe exhibits a significant change in its UV-Vis absorption spectrum, with the maximum absorption wavelength red-shifted to 635 nm. When this wavelength is used as the excitation source, strong near-infrared fluorescence emission is generated at 735 nm, with a fluorescence enhancement factor of 41.6 times and a detection limit as low as 3.03 nM, achieving highly sensitive detection of ONOO⁻.

[0013] Specifically, MF-ONOO can be used as a real-time fluorescence imaging tool for ONOO⁻ concentration in live cells. After incubating HepG2 cells with the probe, ONOO⁻ levels were upregulated by exogenous administration of SIN-1 and endogenous stimulation (LPS combined with IFN-γ), resulting in a significant increase in fluorescence intensity. However, the fluorescence signal was significantly weakened after adding the superoxide anion scavenger TEMPO, the nitric oxide synthase inhibitor L-NAME, or the ONOO⁻ direct scavenger uric acid (UA), indicating that MF-ONOO can dynamically and sensitively reflect changes in ONOO⁻ concentration in live cells.

[0014] Specifically, MF-ONOO can be used for early diagnosis and disease assessment of ONOO⁻-related inflammatory diseases at the in vivo level. In an LPS / D-GalN-induced acute inflammation mouse model, after tail vein injection of MF-ONOO, the near-infrared fluorescence signals of the liver and lungs of inflamed mice were significantly enhanced compared with those of normal mice; histopathological (H&E staining) analysis further confirmed that the intensity of the fluorescence signal was positively correlated with the degree of tissue damage and the severity of inflammation, indicating that MF-ONOO can serve as an in vivo imaging tool for assessing the progression of inflammation and has the potential to guide the evaluation of the efficacy of anti-inflammatory drugs.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention provides a molecular probe MF-ONOO designed using Rhodamine (an NIR dye) as the main component and allyl carbonate as the ONOO⁻ responsive group. The probe employs a fluorine atom electronic effect-based control strategy, and its synthesis method is simple, efficient, and uses readily available raw materials. The probe itself exhibits good biocompatibility and photostability. This probe achieves a specific ONOO⁻-activated response without the need for a metal catalyst, overcoming key problems in existing technologies such as low sensitivity, poor selectivity, and dependence on external catalysts.

[0017] The specific advantages include the following:

[0018] The synthesis process is simple and suitable for large-scale preparation: The probe MF-ONOO described in this invention involves only three organic synthesis reactions with mild reaction temperatures and no harsh anhydrous or oxygen-free extreme conditions; the column chromatography elution system is conventional and readily available, and the yields of the three steps are 42.51%, 65%, and 58%, respectively. The overall synthesis cost is low, the operation threshold is low, and it has the potential for large-scale production.

[0019] Excellent near-infrared fluorescence response performance: The probe excitation wavelength is 635 nm and the emission wavelength is 735 nm, which is within the biological near-infrared window, significantly reducing the background of autofluorescence in cells and tissues; the probe's own fluorescence is weak when there is no ONOO⁻, but the fluorescence is enhanced by 41.6 times after recognition, with a detection limit as low as 3.03 nM, and excellent signal-to-noise ratio of the on-response; the fluorescence intensity reaches the plateau phase within 10 min, with a fast response speed, which is suitable for instantaneous ONOO⁻ dynamic capture.

[0020] Ultra-high selectivity and physiological stability: MF-ONOO shows no obvious fluorescence response to 41 coexisting interfering substances in vivo (various reactive oxygen species / nitrogen, metal cations, anions, amino acids, reduced biological sulfhydryl groups, and antioxidant vitamins), and only specifically recognizes ONOO⁻; the probe's background fluorescence is stable in a wide pH range of 4.0~12.0, and the response intensity does not fluctuate in the physiological pH range of 5.5~7.4, making it adaptable to the acid-base microenvironment in cells and tissues.

[0021] Excellent biocompatibility and no metal catalyst required: The recognition process of this probe does not rely on exogenous metal ion catalysis at all, eliminating the cytotoxicity and in vivo oxidative stress interference caused by metal ions from the root; CCK-8 cytotoxicity experiments confirmed that the cell survival rate was >90% under high concentration of probe, and there were no obvious toxic side effects in animal in vivo experiments, making it suitable for long-term cell incubation and long-term in vivo imaging.

[0022] It has the potential for in vivo inflammation imaging: the probe can achieve in vitro quantitative detection, single-cell level ONOO⁻ tracing, and in vivo inflammatory lesion imaging layer by layer; the fluorescence signal is highly correlated with pathological damage and liver function biochemical indicators, which can be used as a scientific research tool to analyze the pathogenesis of inflammation, and can also be developed into an early visualization diagnostic reagent for inflammation. At the same time, it can establish an in vivo efficacy evaluation platform for anti-inflammatory drugs, with a wide range of application scenarios. Attached Figure Description

[0023] To make the above and / or other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described below: Figure 1 This is a schematic diagram of the synthetic route and response mechanism of the molecular fluorescent probe MF-ONOO; Figure 2 These are (a) proton NMR spectrum, (b) fluorine spectrum, and (c) carbon NMR data for MF-ONOO. Figure 3 The UV-Vis absorption spectrum of the ONOO⁻ response of the probe MF-ONOO; Figure 4 The ONOO⁻ response fluorescence emission spectrum of the probe MF-ONOO; Figure 5 The time-response curve of the fluorescence kinetics of the probe MF-ONOO for identifying ONOO⁻; Figure 6 The bar chart shows the selective fluorescence test of the probe MF-ONOO against 41 coexisting biological interfering substances. Figure 7 The curves show the fluorescence intensity comparison of the probe MF-ONOO in response to ONOO⁻ under different pH conditions; Figure 8 The fluorescence intensity change spectrum of the probe MF-ONOO in response to the ONOO⁻ concentration gradient; Figure 9 The test curve for the lowest detection limit of the ONOO⁻ response of the probe MF-ONOO; Figure 10 Bar graph showing cell viability of HepG2 cells after treatment with different concentrations of probe MF-ONOO for 24 h; Figure 11 The fluorescence imaging test of the probe MF-ONOO in HepG2 cells is shown. In Figure (a), the fluorescence imaging image is shown after incubating the probe with different concentrations of ONOO⁻ in the cells, and the bar graph of the relative fluorescence intensity of the probe in the red channel in Figure (a) is shown. Figure 12This image shows in vivo fluorescence imaging of the probe MF-ONOO in a mouse model of acute inflammation induced by LPS / D-GalN. Figure a shows fluorescence images of the liver and lungs of mice in the normal and inflammatory groups after intraperitoneal injection of MF-ONOO; figure b shows fluorescence images of ex vivo organs of mice in the normal and inflammatory groups; figure c shows the relative fluorescence intensity of the organs in figure (b); figure d shows H&E-stained pathological sections of lung tissue; figure e shows H&E-stained pathological sections of liver tissue; and figure f shows the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in the normal and inflammatory groups. Detailed Implementation

[0024] The following embodiments are used to fully illustrate the technical solution of the present invention and are only preferred embodiments of the present invention. They do not constitute a limitation on the scope of protection of the present invention. Any conventional parameter adjustments made by those skilled in the art based on the ideas of the present invention to the reaction feed amount, solvent ratio, and reaction temperature fall within the scope of protection of the present invention.

[0025] Example 1: Preparation of probe molecule MF-ONOO The synthetic route is attached. Figure 1 As shown, the details are as follows:

[0026] Step 1: Cyclohexanone (0.66 mL, 6.38 mmol) was slowly added dropwise to pre-cooled concentrated sulfuric acid (7 mL, 130 mmol) under ice bath conditions. 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid (1.00 g, 3.19 mmol) was added in portions with stirring. The mixture was then heated to 90°C and stirred for 2 hours. After the reaction was complete, the reaction solution was cooled to room temperature and slowly poured into crushed ice. Perchloric acid (0.70 mL, 18.60 mmol) was added, resulting in a red precipitate. The precipitate was filtered, washed with sufficient cold water, and dried to give a red solid intermediate compound 1, with a yield of 42.51%.

[0027] Step 2: Intermediate compound 1 (2.00 g, 4.20 mmol) and 3-fluoro-4-hydroxybenzaldehyde (0.59 g, 4.20 mmol) were dissolved in a mixed solvent of acetic acid and ethanol (volume ratio 1:1) (20 mL), and the mixture was refluxed at 90°C overnight under nitrogen protection. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, volume ratio) to give a purple solid intermediate MF-OH in 65% yield.

[0028] Step 3: Under nitrogen protection, intermediate MF-OH (0.24 g, 0.40 mmol) was dissolved in anhydrous dichloromethane (7 mL), and triethylamine (0.20 mL, 1.40 mmol) was added. Allyl chloroformate (560 μL, 5.28 mmol) was slowly added dropwise under ice bath cooling, followed by stirring at room temperature overnight. After the reaction was complete, the mixture was diluted with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30:1, v / v) to give the target product MF-ONOO as a purple-red solid, in 58% yield.

[0029] Example 2: Structural characterization of the probe molecule MF-ONOO The proton, fluorine, and carbon spectra were performed using a 400 MHz nuclear magnetic resonance spectrometer, with deuterated DMSO as the solvent. The corresponding spectra are attached. Figure 2 The characteristic chemical shift data are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 7.90 (d, J = 7.5 Hz, 1H), 7.77 (s, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.55 – 7.39 (m, 2H), 7.39 – 7.24 (m, 3H), 6.48 (s, 1H), 6.47 – 6.31 (m, 2H), 5.39 (d, J = 19.0 Hz, 1H), 5.30 (d, J = 10.4 Hz,1H), 4.74 (d, J = 7.1 Hz, 2H), 3.32 (d, J = 6.9 Hz, 4H), 3.26 (s, 1H), 2.70(d, J = 9.3 Hz, 1H), 2.59 (s, 1H), 1.54 (s, 3H), 1.09 (s, 6H); 19 F NMR (376 MHz, DMSO- d6 ) δ -130.18; 13 C NMR (101 MHz, DMSO- d6) δ 169.55, 152.50, 152.34, 152.12, 137.17,137.10, 135.88, 132.00, 130.49, 128.79, 127.17, 126.67, 124.10, 119.73,117.81, 97.24, 69.93, 44.24, 26.98, 23.09, 22.34, 12.91.

[0030] Example 3: In vitro spectroscopic performance testing of the probe molecule MF-ONOO The reaction system for the in vitro test was as follows: a 384-well microplate was used, with a total test volume of 100 μL per well. The solution contained 99.90% PBS buffer (100 mM, pH = 7.4), 0.10% DMSO, and a final concentration of 10 μM for the probe MF-ONOO. The test temperature was 37°C. Specific experimental results are as follows: In vitro UV-Vis absorption spectroscopy: The probe molecule MF-ONOO was dissolved in PBS buffer (10 mM, pH = 7.4, containing 0.1% DMSO), and ONOO⁻ was added to a final concentration of 10 μM. After incubation at 37°C for 10 minutes, the absorption spectrum in the range of 300–800 nm was recorded using a UV-Vis spectrophotometer (see attached). Figure 3 Experimental results show that after the addition of ONOO⁻, the absorption peak of MF-ONOO at 635 nm is significantly enhanced, and the absorption peak undergoes a significant red shift, proving that the probe reacts with ONOO⁻ to generate a conjugated system with an extended luminescent product.

[0031] In vitro fluorescence response assay: The probe molecule MF-ONOO was dissolved in PBS buffer (10 mM, pH = 7.4, containing 0.1% DMSO), and its fluorescence emission spectrum in the range of 650–850 nm was measured using 635 nm as the excitation wavelength (see attached image). Figure 4 Upon addition of ONOO⁻ (10 μM), MF-ONOO exhibited strong near-infrared fluorescence emission at 735 nm, with a fluorescence enhancement factor of 41.6 times. In the absence of ONOO⁻, the probe's own background fluorescence was extremely weak, indicating that MF-ONOO possesses excellent "on" response characteristics.

[0032] In vitro kinetics assay: After mixing the probe molecules MF-ONOO (10 μM) and ONOO⁻ (10 μM) in PBS buffer (10 mM, pH = 7.4, containing 0.1% DMSO), the change in fluorescence intensity at 735 nm over time was immediately recorded (see attached diagram). Figure 5Experimental results show that the fluorescence intensity reaches a plateau within 10 minutes, and the fluorescence intensity of the probe itself does not change significantly under the same conditions, demonstrating the rapid response capability and good stability of MF-ONOO to ONOO⁻.

[0033] In vitro selectivity assay: The probe molecule MF-ONOO (10 μM) was added to various interfering substances (including H2O2, ·OH, O2•⁻, ... 1 O2, ClO⁻, NO, NO2⁻, Na + K + Mg 2+ Ca 2+ Zn 2+ Fe 3+ Al 3+ Hg 2+ Mn 2+ Pd 2+ , CO, NO, Br⁻, Cl⁻, F⁻, Co 2+ Fe 2+ SO3 2 ⁻、HS⁻、CO3 2 ⁻, HCO3⁻, HSO3⁻, NO2⁻, NO3⁻, SO4 2 ⁻、S 2 ⁻、Cu 2+ Tryptophan, L-lysine, glutathione (GSH), cysteine ​​(Cys), homocysteine ​​(Hcy), Vitamin C, and Vitamin E were added. The final concentration of the interfering substances was 100 μM (10 μM for ONOO⁻). After incubation at 37°C for 10 minutes, the fluorescence intensity at 735 nm was measured (see attached image). Figure 6 Experimental data show that only ONOO⁻ can induce a significant fluorescence enhancement in MF-ONOO, while other interfering substances do not induce a significant fluorescence response, demonstrating that MF-ONOO has excellent selectivity for ONOO⁻.

[0034] In vitro pH selectivity and anti-interference test: The probe molecule MF-ONOO (10 μM) was dissolved in PBS buffer at different pH values ​​(pH 4.0–12.0), and ONOO⁻ (10 μM) was added. After incubation at 37°C for 10 minutes, the fluorescence intensity at 735 nm was measured. A control group without ONOO⁻ was also included to investigate the effect of pH on the probe's intrinsic fluorescence background and stability (see attached). Figure 7Experimental results show that, without the addition of ONOO⁻, MF-ONOO exhibits extremely weak and stable autofluorescence over a wide pH range; under physiologically relevant pH conditions (pH 5.5–7.4), the fluorescence response intensity remains essentially consistent after the addition of ONOO⁻, indicating that MF-ONOO possesses good pH adaptability and stability, enabling accurate detection of ONOO⁻ under different acidic and alkaline environments without being affected by pH fluctuations.

[0035] In vitro concentration gradient response and detection limit determination: Different concentrations of ONOO⁻ (0–10 μM) were added to the test system containing MF-ONOO (10 μM), and after incubation for 10 minutes, the fluorescence intensity at 735 nm was recorded (see attached figure). Figure 8 The results showed that the fluorescence intensity of MF-ONOO increased in a concentration-dependent manner with increasing ONOO⁻ concentration, and exhibited a good linear relationship in the range of 0–7 μM (R²). 2 > 0.90), and the calculated limit of detection (LOD) is as low as 3.03 nM, indicating that MF-ONOO has extremely high detection sensitivity for ONOO⁻ (see attached). Figure 9 ).

[0036] Example 4: Cytotoxicity and Cell Imaging Study of Probe Molecule MF-ONOO This section describes the culture of HepG2 cells in DMEM high-glucose medium at 37°C and 5% CO2. The medium contained 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin, 100 U / mL).

[0037] CCK-8 cytotoxicity assay: HepG2 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were placed in 96-well cell culture plates and incubated for 24 hours. Subsequently, different concentrations of probes (0–50 μM, containing 0.5% DMSO) were added to the cells, and incubation continued for another 24 hours under dark conditions. After washing the cells with PBS, 100 μL of fresh culture medium and 10 μL of Cell Counting Kit-8 (CCK-8) were added to each well, and incubation was carried out at 37°C for 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated (see attached image). Figure 10 Experimental results showed that MF-ONOO maintained cell viability >90% in the concentration range of 0–50 μM, indicating that the probe has good biocompatibility.

[0038] Imaging of exogenous ONOO⁻ cells: HepG2 cells were packaged at 1 × 10⁻ cells per well. 5Cells were placed in confocal culture dishes at a ratio of 1:1 and incubated overnight at 37°C and 5% CO2. The experimental group was incubated with SIN-1 (800 μM) for 30 minutes to provide exogenous ONOO⁻, followed by incubation for 1 hour with the probe MF-ONOO (20 μM, 0.2% DMSO). The control group received only the same amount of probe without SIN-1 treatment. After washing the cells with PBS, imaging was performed using a confocal laser scanning microscope (see attached image). Figure 11 Excitation wavelength: 633 nm, emission channel: 680–740 nm, scale bar: 20 μm. Experimental results showed that cells treated with SIN-1 exhibited strong near-infrared fluorescence signals, while the untreated group showed extremely weak fluorescence signals, proving that MF-ONOO can effectively detect the entry of exogenous ONOO⁻.

[0039] Imaging of endogenous ONOO⁻ cells: HepG2 cells were treated as follows: ① Co-stimulation with LPS (20 μg / mL) and IFN-γ (300 ng / mL) for 6 hours to induce endogenous ONOO⁻ production; ② Co-treatment with either the superoxide anion scavenger TEMPO (1 mM) or the nitric oxide synthase inhibitor L-NAME (3 mM) in addition to LPS / IFN-γ stimulation; ③ Addition of uric acid (UA, 600 μM), a direct ONOO⁻ scavenger. After treatment, all cells were incubated with MF (20 μM, 0.2% DMSO) for 1 hour, washed with PBS, and then subjected to confocal imaging (see attached image). Figure 11 Experimental results showed that the fluorescence intensity of cells stimulated by LPS / IFN-γ was significantly enhanced; however, the fluorescence signal was significantly weakened after the addition of TEMPO, L-NAME, or UA, further confirming that MF-ONOO can specifically respond to the endogenous ONOO⁻ produced by living cells, and that the fluorescence intensity is positively correlated with the intracellular ONOO⁻ level.

[0040] Example 5: In vivo imaging study of probe molecule MF-ONOO in an acute inflammatory mouse model All animal experiments involved in this study were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (8th edition, 2011), and all animals were approved by the relevant agency's animal management and use committee.

[0041] Inflammation model establishment: BALB / c mice (6-8 weeks old, female) were selected and an acute inflammation mouse model was established by intraperitoneal injection of LPS (4 mg / kg) and D-GalN (200 mg / kg) for 6 hours. Normal control mice were injected with an equal volume of physiological saline.

[0042] In vivo imaging application: The model group and control group were injected via tail vein with MF-ONOO solution (250 μM, 200 μL, physiological saline containing 0.4% DMSO and 1% Tween 80). Thirty minutes after injection, the mice were anesthetized and their abdomens opened. Near-infrared fluorescence images of the liver and lung regions were acquired using an in vivo imaging system (excitation filter: 605 nm, emission filter: 740 nm) (see attached image). Figure 12 a). Experimental results showed that the fluorescence signals in the liver and lung regions of the inflammation model group mice were significantly enhanced compared with the normal control group, indicating that MF-ONOO can effectively image the abnormal increase of ONOO⁻ at the in vivo level.

[0043] Verification of ex vivo organs and pathological indicators: After imaging, mice were euthanized, and liver and lung tissues were removed for near-infrared fluorescence imaging (see attached). Figure 12 (b&c). The results showed that significantly enhanced fluorescence signals were observed in the liver and lungs of mice, verifying the abnormal increase of ONOO⁻ in the liver and lungs of mice with acute inflammation. Organs were fixed in 4% paraformaldehyde, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) (see attached). Figure 12 d&e). Histopathological analysis showed that the lung tissue in the inflammation model group exhibited obvious acute injury pathological features; the liver tissue also showed inflammatory cell infiltration in the portal area and dilation and congestion of the hepatic sinusoids. The results were consistent with in vivo fluorescence imaging, indicating that the fluorescence signal intensity of MF-ONOO can accurately reflect the degree of tissue inflammation, confirming the application potential of MF-ONOO as an in vivo imaging tool for ONOO⁻-related inflammatory diseases. Specifically, we tested the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum (see attached). Figure 12 f) ALT and AST are commonly used indicators for evaluating liver function in clinical practice, and their elevation usually indicates hepatocellular damage or abnormal liver function. Compared with the control group, the ALT and AST levels in the inflammatory mice were significantly enhanced, which was consistent with the results of probe fluorescence imaging.

Claims

1. A near-infrared fluorescent probe MF-ONOO with function for detecting peroxynitrite (ONOO⁻), characterized in that, The probe comprises a core fluorophore composed of a rhodamine-like dye and an allyl carbonate as an ONOO⁻ responsive group. The structural formula of the probe is shown in Formula I: Formula I.

2. The fluorescent probe for detecting peroxynitrite (ONOO⁻) according to claim 1, characterized in that, The detection limit of the fluorescent probe is 3.03 nM.

3. A method for preparing the molecular probe MF-ONOO according to any one of claims 1-2, characterized in that, Includes the following steps: Step 1: In the presence of concentrated sulfuric acid, cyclohexanone was subjected to a cyclization reaction with 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid at a temperature of 0°C to 90°C, followed by salt formation with perchloric acid to obtain intermediate compound 1; Step 2: Under an inert atmosphere, intermediate compound 1 and 3-fluoro-4-hydroxybenzaldehyde are refluxed in a mixed solvent of acetic acid and ethanol, and purified to obtain intermediate MF-OH; wherein the purification is performed by silica gel column chromatography. Step 3: Under an inert atmosphere, the intermediate MF-OH and allyl chloroformate were subjected to an esterification reaction in anhydrous dichloromethane with triethylamine as an acid-binding agent, and the probe molecule MF-ONOO was obtained after purification; wherein the purification was carried out by silica gel column chromatography.

4. Use of the molecular probe MF-ONOO according to any one of claims 1-2 in the preparation of a reagent for detecting the concentration of peroxynitrite (ONOO⁻) in a solution system or biological sample.

5. Use of the molecular probe MF-ONOO according to any one of claims 1-2 in the preparation of a reagent for real-time fluorescence imaging of intracellular peroxynitrite (ONOO⁻).

6. The use of the molecular probe MF-ONOO according to any one of claims 1-2 in the preparation of peroxynitrite (ONOO⁻) for in vivo imaging of inflammatory diseases.

7. The use as described in claim 6, characterized in that, The inflammatory disease is an acute inflammation induced by LPS / D-GalN.

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