CN115746056A - Preparation of novel peroxynitrite fluorescent probe and accurate detection in drug-induced liver injury formation and inhibition process - Google Patents

Abstract

The invention relates to a novel peroxynitrite (ONOO) ^‑ ) The preparation of the fluorescent probe and the accurate detection of the fluorescent probe in the process of drug-induced liver injury formation and inhibition are carried out, wherein the name of the compound probe is 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridine-2-diphenyl phosphate (6,8-dichoro-9,9-dimethyl-7-oxo-7,9-diazeparidin-2-yldiphenylphosphinate). The compound molecular sensor of the invention can efficiently, sensitively and selectively detect the ONOO in organisms ^‑ . The probe is not interfered by other ROS and RNS analogues, and can be efficiently and sensitively used in complex environmentQuantitative detection of difficult-to-capture ONOO in biological systems ^‑ The content of (a). And is used for detecting living organisms such as cells or animals with excellent biocompatibility. The probe molecules were applied to APAP-induced model mice and the induced lesions were treated with inhibitors (GSH, glu, NAC). The molecule can realize accurate imaging in the formation and inhibition process of drug-induced liver injury. The method is beneficial to early diagnosis of patients with liver injury and screening of treatment schemes, and has important practical significance.

Description

Preparation of novel peroxynitrite fluorescent probe and accurate detection in drug-induced liver injury formation and inhibition process

Technical Field

The invention relates to preparation of a novel peroxynitrite fluorescent probe and accurate detection in a drug-induced liver injury forming and inhibiting process.

Background

In the human body, the liver is the major organ for the metabolism of various substances, including alcohol, drugs, and chemicals. Given that the liver plays a vital role in many physiological processes, liver damage can lead to loss of normal function, leading to serious dysfunction and even death. The causes of liver injury are complex, with alcohol and drugs being common causes of acute and chronic liver injury. Particularly, drug-induced liver damage has become a serious threat to human health because many drugs, including those for treating liver damage, are metabolized by the liver. Therefore, diagnosis and evaluation of drug-induced liver injury are difficult, resulting in inefficiency of precise treatment. Current diagnosis of liver damage depends on the levels of serum markers including alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), total Bilirubin (TBIL) and dehydrogenase (LDH). However, serum indicators cannot be monitored in real time and in situ. Therefore, developing more accurate tools to describe the formation and inhibition processes of drug-induced liver injury remains an urgent necessity for diagnosis and treatment.

In the search for probe molecules, reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are reported as potential indicators for monitoring liver-related metabolic states. They are important by-products of reactions within the liver and are therefore closely related to the redox state in biological events. Wherein peroxynitrite (ONOO) ^- ) Reported as one of the most important biomarkers in liver injury. In the model of liver damage caused by various factors, ONOO ^- Can be used as a practical index for reflecting the damage degree. Endogenous ONOO ^- The increased concentration is caused by liver dysfunction, which manifests itself as hepatocyte death. Due to ONOO ^- Has a half-life of about 10 ms and is therefore difficult to track for physiological and pathological events. This factor also meets the necessity for real-time and on-site monitoring.

In the last decade, fluorescent probes have attracted extensive attention from researchers due to their advantages of high selectivity, high sensitivity, good biocompatibility and non-invasiveness. By optimizing the fluorescent probe, the dynamic imaging is gradually adapted to the requirements of real time and in situ. We prepared an ONOO ^- The fluorescent probe can be activated and used for accurate imaging of drug-induced liver injury formation and inhibition processes. The probe is used for monitoring ONOO ^- Has good stability, wider pH adaptability and higher specificitySex and sensitivity, can be used to establish a link between ONOO-levels and liver damage. The drug-induced liver injury is taken as an important point, and a specific model is researched to reflect the pathogenesis and the treatment process of the drug-induced liver injury. This information may be helpful in achieving early diagnosis of drug-induced liver injury and screening for therapeutically effective treatment regimens. A series of experiments show that the fluorescent molecule has good performance and high potential practical application value.

Disclosure of Invention

The invention aims to provide preparation of a novel peroxynitrite fluorescent probe and accurate detection of the peroxynitrite fluorescent probe in a drug-induced liver injury forming and inhibiting process.

The technical scheme of the invention is as follows:

a novel peroxynitrite fluorescent probe is characterized by having the following structure:

a method for preparing the fluorescent probe, which comprises the following steps:

step 1, weighing 1g m-hydroxyacetophenone, adding into a three-neck bottle, and filling nitrogen. Adding about 20 ml anhydrous tetrahydrofuran to dissolve; in ice bath, 5.4 ml methyl magnesium chloride was slowly injected into the center of the reaction system by syringe suction, and 2h was stirred at room temperature. Reflux 10 h at 70 degrees celsius. After the reaction liquid is cooled, 5ml saturated ammonium chloride solution and 16 ml 1M hydrochloric acid are added for acidification, ethyl acetate is added for extraction for three times, the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, decompressed and concentrated, and purified by a silica gel column, the polarity is PE: EA = 40: 1, and pure white solid 672.3 mg is obtained and is the first-step product.

Step 2. 690 mg of 2, 6-dichoro-4- (chloroimino) cyclohexa-2,5-dien-1-one and 500 mg first step product were weighed into a flask, dissolved in 5ml tetrahydrofuran and 5ml water was added. 3.5ml of 2.0M aqueous NaOH solution was slowly added dropwise in an ice bath, and stirred at 0 ℃ for 2 hours. 160ml of saturated ammonium chloride solution and 100ml of ethyl acetate are added, mixed with vigorous stirring, the phases are separated and the aqueous layer is extracted once with ethyl acetate and all ethyl acetate is washed with ammonium chloride solution. Weighing 8.2g sodium hydrosulfite, dissolving in 82ml water, washing ethyl acetate phase twice until the color of blue-black solution becomes light, extracting water layer with ethyl acetate, finally washing ethyl acetate solution with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain brown tar-like product. The product was dissolved with an appropriate amount of methanol, 2M hydrochloric acid solution (16.4 ml) slowly stirred under nitrogen atmosphere was added at room temperature, reacted at 100 ℃ for 1.5h, cooled to room temperature, the solution was extracted twice with an appropriate amount of ethyl acetate, and the resulting ethyl acetate solution was washed with saturated brine. Weighing 1g of sodium periodate, dissolving in a proper amount of water, adding into the ethyl acetate solution, stirring at room temperature for 15min, separating phases, washing an organic phase with brine, drying with sodium sulfate, and concentrating under reduced pressure. At 80 ℃, dropwise adding absolute ethyl alcohol into the product until the absolute ethyl alcohol is completely dissolved, slowly cooling, separating out a solid, filtering and drying. The filtrate was recrystallized twice repeatedly. A total of 370mg of a red-black solid were obtained as the second-step product.

Step 3. Weighing about 200mg of the second step product, dissolving in 5ml of dichloromethane, and dropwise adding triethylamine 80 at room temperatureμL, 0.1eq DMAP is weighed and finally the diphenylphosphinic acid chloride 149 is added dropwiseμL, stirred at room temperature overnight. 2mL saturated NH ₄ Aqueous Cl solution was added to the reaction mixture. The layers were separated and the aqueous layer was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by column chromatography (dichloromethane: petroleum ether =20, 1,v = 20) gave 82 mg as an orange solid as the final probe molecule.

The invention has the advantages that: the compound of the invention has good molecular biocompatibility and low biological toxicity, can be used as a molecular sensor, efficiently, sensitively and selectively detect peroxynitrite in organisms, and starts a fluorescence reaction. The detection mechanism is not interfered by other similar ROS and RNS analogs, can efficiently and sensitively detect peroxynitrite in a complex environment, and can quantitatively detect the content of peroxynitrite difficult to capture in a biological system according to a detection target curve. More particularly, the molecule has excellent biocompatibility, can be used for detection of living organisms such as cells or animals, and has little toxicity. The probe molecules were applied to APAP-induced model mice and the induced lesions were treated with inhibitors (GSH, glu, NAC). The red channel fluorescence enhancement is time-dependent and dose-dependent during the formation of a drug-induced liver injury. In the inhibition assay, inhibition of liver damage is manifested as a decrease in fluorescence intensity. The molecule can realize accurate imaging in the formation and inhibition process of drug-induced liver injury. The information can help the early diagnosis of the liver injury patients and the screening of effective treatment schemes, and has very important practical significance.

Drawings

FIG. 1 is a graph showing the UV absorption spectrum of the fluorescent molecular probe in 10 mM PBS buffer solution.

FIG. 2 is a fluorescence emission spectrum of the fluorescent molecular probe in 10 mM PBS buffer solution.

FIG. 3 shows the fluorescent molecular probes in 10 mM PBS buffer solution with ONOO ^- Fluorescence spectra and fluorescence intensity fit plots of concentration changes.

FIG. 4 shows fluorescence intensity of the fluorescent molecular probe at 656nm as ONOO in 10 mM PBS buffer solution ^- Linear dependence of concentration change.

FIG. 5 is a graph showing the selectivity and interference of the fluorescent molecular probe in 10 mM PBS buffer solution.

FIG. 6 is a graph of the fluorescence spectra of the fluorescent molecular probe in 10 mM PBS buffer solution as a function of pH.

FIG. 7 is a graph of the fluorescence spectra of the fluorescent molecular probes in 10 mM PBS buffer solution over time.

FIG. 8 is a graph showing the toxicity of the fluorescent molecular probe to HepG2 cells.

FIG. 9 is a time-dependent experimental fluorescence confocal map of the concentration of the fluorescent molecular probe entering a cell in a living cell.

FIG. 10 is a confocal graph of fluorescence measured at different APAP-induced concentrations and at different times for the fluorescent molecular probe in living cells.

FIG. 11 is a fluorescence confocal image of the fluorescent molecular probe for detecting APAP-induced liver damage in living animals (nude mice).

Detailed Description

The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples at all.

The first embodiment is as follows: preparation of peroxynitrite fluorescent probe

Step 1, weighing 1g m-hydroxyacetophenone, adding into a three-necked bottle, and filling nitrogen. Adding about 20 ml anhydrous tetrahydrofuran to dissolve; in ice bath, 5.4 ml methyl magnesium chloride was slowly injected into the center of the reaction system by syringe suction, and 2h was stirred at room temperature. Reflux 10 h at 70 degrees celsius. After the reaction liquid is cooled, 5ml saturated ammonium chloride solution and 16 ml 1M hydrochloric acid are added for acidification, ethyl acetate is added for extraction for three times, the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, decompressed and concentrated, and purified by a silica gel column, the polarity is PE: EA = 40: 1, and pure white solid 672.3 mg is obtained and is the first-step product.

Step 2. 690 mg of 2, 6-dichoro-4- (chloroimino) cyclohexa-2,5-dien-1-one and 500 mg first step product were weighed into a flask, dissolved in 5ml tetrahydrofuran and 5ml water was added. 3.5ml of 2.0M aqueous NaOH solution was slowly added dropwise in an ice bath, and stirred at 0 ℃ for 2 hours. 160ml of saturated ammonium chloride solution and 100ml of ethyl acetate are added, mixed with vigorous stirring, the phases are separated and the aqueous layer is extracted once with ethyl acetate and all ethyl acetate is washed with ammonium chloride solution. Weighing 8.2g sodium hydrosulfite, dissolving in 82ml water, washing ethyl acetate phase twice until the color of blue-black solution becomes light, extracting water layer with ethyl acetate, finally washing ethyl acetate solution with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain brown tar-like product. The product was dissolved with an appropriate amount of methanol, 2M hydrochloric acid solution (16.4 ml) slowly stirred under nitrogen atmosphere was added at room temperature, reacted at 100 ℃ for 1.5h, cooled to room temperature, the solution was extracted twice with an appropriate amount of ethyl acetate, and the resulting ethyl acetate solution was washed with saturated brine. Weighing 1g of sodium periodate, dissolving in a proper amount of water, adding into the ethyl acetate solution, stirring at room temperature for 15min, separating phases, washing an organic phase with brine, drying with sodium sulfate, and concentrating under reduced pressure. At 80 ℃, dropwise adding absolute ethyl alcohol into the product until the absolute ethyl alcohol is completely dissolved, slowly cooling, separating out a solid, filtering and drying. The filtrate was recrystallized twice repeatedly. A total of 370mg of a red-black solid were obtained as the second-step product.

And 3, weighing about 200mg of the product obtained in the second step, dissolving the product in 5ml of dichloromethane, dropwise adding 80 mu L of triethylamine at room temperature, weighing 0.1eq of DMAP, finally dropwise adding 149 mu L of diphenylphosphinic acid chloride, and stirring at room temperature overnight. 2mL saturated aqueous NH4Cl was added to the reaction mixture. The layers were separated and the aqueous layer was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by column chromatography (dichloromethane: petroleum ether =20, 1,v = 20) gave 82 mg as an orange solid as the final probe molecule. ¹ H NMR (600 MHz, DMSO-d6) δ 7.98 (ddt, J = 12.4, 6.9, 1.4 Hz, 4H), 7.81 (s, 1H), 7.68 (dd, J = 2.6, 1.0 Hz, 1H), 7.67 – 7.62 (m, 2H), 7.58 (ddd, J = 11.2, 9.0, 6.1 Hz, 5H), 7.34 (ddd, J = 8.6, 2.6, 1.0 Hz, 1H), 1.74 (s, 6H). ¹³ C NMR (151 MHz, DMSO) δ 173.04, 153.96, 153.91, 149.90, 140.84, 140.37, 139.86, 137.73, 136.46, 134.61, 133.49, 133.48, 133.35, 132.12, 132.05, 131.11, 130.21, 129.50, 129.41, 120.81, 120.78, 120.16, 120.13, 39.17, 26.38.

The property and activity of the fluorescent molecular compound are tested by applying experiments, the fluorescent molecular probe prepared in the first embodiment is tested in the second to tenth embodiments, and specific data and analysis are as follows:

example two:

FIG. 1: the ultraviolet absorption spectrum of the fluorescent molecular probe in 10 mM PBS buffer solution

Will 10μM fluorescent molecular probe was dissolved in PBS buffer (pH 7.4, 10 mM, 1% DMSO) solution, peroxynitrite (50% nitrite) was addedμM), after incubation at 37 ℃, detection is carried out on an Shimadzu UV-2550 instrument, and the ultraviolet-visible absorption spectrum of the fluorescent molecular probe is shown in figure 1.

Example three:

FIG. 2 is a schematic diagram: the fluorescence emission spectrum of the fluorescent molecular probe in 10 mM PBS buffer solution

Will 10μM fluorescent molecular probe was dissolved in PBS buffer (pH 7.4, 10 mM, 1% DMSO) solution, peroxynitrite (50% nitrite) was addedμM), after incubation at 37 ℃, the fluorescence emission spectrum of the fluorescent molecular probe is shown in FIG. 2 when the fluorescence emission spectrum is detected on Hitachi F-7000 instrument.

Example four:

FIG. 3: the fluorescent molecular probe was conjugated with ONOO in 10 mM PBS buffer solution ^- Fluorescence spectra of concentration changes and fluorescence intensity fit plots.

mu.M fluorescent molecular probes were dissolved in PBS buffer (pH 7.4, 10 mM, 1% DMSO) in different ONOO solutions ^- Detecting the fluorescence spectrum characteristics under the concentration, and incubating at 37 ℃ for 1 h to obtain ONOO ^- The concentration range is 0-60 μ M, and the detection is carried out on Hitachi F-7000 instrument. The results show that the ONOO is treated at 656nm ^- From 0 to 60μM (6 equivalents relative to probe) gave a standard curve; following ONOO ^- Increasing concentration, increasing fluorescence intensity, ONOO ^- When the concentration reaches about 4 equivalent, the fluorescence intensity reaches the maximum and keeps stable

Example five:

FIG. 4 shows fluorescence intensity of the fluorescent molecular probe with ONOO at 656nm in 10 mM PBS buffer solution ^- Linear plot of concentration change

At 656nmFluorescence intensity and ONOO ^- The concentration is 0-10μM shows a strong linear relation, and the correlation coefficient is 0.9985.

Example six:

FIG. 5 is a schematic view of: experimental graph of selectivity and interference of the fluorescent molecular probe in 10 mM PBS buffer solution

Will 10μDissolving M fluorescent molecular probe in PBS buffer solution (pH 7.4, 10 mM, 1% DMSO), adding different analytes for detection, ONOO ^- Has a concentration of 60µM, concentration of other substances tested was 100µM,37 ℃ incubation 1 h, in Hitachi F-7000 instrument for detection.

The fluorescent molecular probe can be used for specifically detecting ONOO ^- Other tested substances are anions and cations, amino acids, ROS, RNS, etc. The fluorescent molecular probe pair ONOO ^- Has good selectivity and interference.

Example seven:

FIG. 6: the fluorescence spectrum of the fluorescent molecular probe responding to pH in 10 mM PBS buffer solution

Will 10μThe M fluorescent molecular probe is dissolved in a PBS buffer solution (pH 7.4, 10 mM, 1% DMSO), and the performance of the M fluorescent molecular probe is detected under different pH values respectively, wherein the pH value ranges are as follows: 3-12. Adding ONOO ^- (60 μM), 1 h was incubated at 37 ℃ and then tested on Hitachi F-7000 apparatus.

The fluorescent molecular probe itself is hardly affected by pH. The fluorescent molecular probe and ONOO ^- (60 μM) response, it can be seen that the fluorescence properties are stable at pH 7-12.

Example eight:

FIG. 7: the fluorescence spectrum of the fluorescent molecular probe responding with time in 10 mM PBS buffer solution

Will 10µDissolving M fluorescent molecular probe in PBS buffer solution (pH 7.4, 10 mM, 1% DMSO), adding ONOO ^- (60 μM) was incubated at 37 ℃ and detected in the Time-scan mode on an F-7000 instrument.

The fluorescent molecular probe shows strong fluorescence increase (basically reaches a peak value) within 1 min, and the fluorescence performance is continuously stable after the time reaches 40 min. By prolonging the response time for more than 48h, the stability of the fluorescent molecular probe detection system is illustrated.

Example nine:

FIG. 8: experimental diagram of the fluorescent molecular probe for HepG2 cytotoxicity

The cytotoxicity of the fluorescent molecular probe is tested by a CCK8 method. The test cell was HepG2. The cell culture medium is complete medium (MEM) containing 20% FBS, 1% penicillin and streptomycin antibiotic solution, and the cells are seeded in 96-well plate and cultured at 37 deg.C and 5% CO ₂ In the incubator, the cell density was 5000 cells/well.

The fluorescent molecular probe has low cytotoxicity, and the survival rate of cells can still reach more than 80% even under high probe concentration, which proves that the fluorescent molecular probe can be further used for clinical tests.

Example ten:

FIG. 9: in living cells, the fluorescent molecular probe is used for testing the concentration and time dependence of entering cells

Will 10µM the fluorescent molecular probe and HepG2 cells at 37 ℃ and 5% CO ₂ Incubating for 15min in incubator, adding ONOO with different concentrations ^- （0, 10, 20, 30, 40, 50, 60 μM) reacting for 30min, and then collecting a confocal image. Incubating the probe and cells for 15min, adding ONOO ^- （60 μM) reaction time, images were collected at 0,5,10, 15, 20, 25, 30min time points, scale bar: 25µm。

The fluorescence intensity of the fluorescent molecular probe is positively correlated with the increase of the peroxynitrite concentration, and the fluorescent molecular probe can be used as an open fluorescent sensor and has better performance in the aspects of cell permeation and living tissue imaging.

Example eleven:

FIG. 10: in living cells, detecting the fluorescence confocal images of the fluorescent molecular probe at different APAP induction concentrations and different times

HepG2 at 37 ℃ 5% CO ₂ After culturing 12h in the incubator, APAP (0, 100, 200, 400, 600, 800, 1000) of different concentrations was added to the cellsμM) incubating for 12h, adding the fluorescent molecular probe, and collecting a fluorescent image after 30 min. Or adding APAP 1 mM into the cells, incubating at different time points (0,2, 4,6,8,10 and 12 h), adding the fluorescent molecular probe, and collecting a fluorescent image after 30 min.

The concentration of endogenous ONOO-of cells induced by APAP is changed, and the fluorescence intensity of fluorescence imaging can be accurately mapped to endogenous ONOO ^- A change in concentration. The result shows that the compound can be used as a molecular sensor and can be sensitive. Selective detection of ONOO in living cells ^- And the fluorescence reaction is started, so that a novel method for detecting the endogenous sulfite of the living cells is provided, and the method has very important practical significance.

Example twelve:

FIG. 11: in living animals (nude mice), the fluorescence confocal picture of the fluorescent molecular probe for detecting the APAP-induced liver injury

Nude mice were injected with 100 injections via the abdominal cavityμAfter M probe molecules, 100 is injected into the abdominal cavity at the same site in the abdominal cavityμM ONOO ^- And collecting fluorescent images of the small animals at different time points (0,5,10, 15, 20, 30, 45 and 60 min). The nude mice are injected with APAP to induce 0,2,4,6,8,10,12 hours in the abdominal cavity and then injected with 100 caudal veinμM probe molecules, and collecting fluorescence images within 1 hour.

According to the picture display, the fluorescent probe can efficiently and accurately detect the liver injury degree induced by the APAP drug, and the information can help the early diagnosis of the liver injury patient and the screening of an effective treatment scheme, so that the fluorescent probe has very important practical significance.

Claims (3)

1. A novel peroxynitrite fluorescent probe is characterized by consisting of the following structural formula:

。

2. the peroxynitrite probe of claim 1, comprising the steps of:

step 1, weighing 1g m-hydroxyacetophenone, adding the m-hydroxyacetophenone into a three-necked bottle, and filling nitrogen; adding about 20 ml anhydrous tetrahydrofuran to dissolve; in ice bath, 5.4 ml methyl magnesium chloride is sucked by a syringe and slowly injected into the center of the reaction system, and 2h is stirred at room temperature; refluxing at 70 deg.C for 10 h; after the reaction liquid is cooled, adding 5ml saturated ammonium chloride solution and 16 ml 1M hydrochloric acid for acidification, adding ethyl acetate for extraction for three times, washing an organic phase with saturated saline solution, drying the organic phase with anhydrous sodium sulfate, concentrating the organic phase under reduced pressure, purifying the organic phase by a silica gel column, selecting the polarity of PE: EA = 40: 1, and obtaining a pure white solid 672.3 mg which is a first-step product;

step 2, weighing 690 mg of 2, 6-dichoro-4- (chloroimino) cyclohexa-2,5-dien-1-one and 500 mg first step product, adding the first step product into a flask, dissolving in 5ml of tetrahydrofuran, and adding 5ml of water; slowly dropwise adding 3.5ml of 2.0M NaOH aqueous solution in ice bath, and stirring for 2h at 0 ℃; adding 160ml of saturated ammonium chloride solution and 100ml of ethyl acetate, mixing under vigorous stirring, separating the phases, extracting the water layer once with ethyl acetate, and washing all the ethyl acetate with ammonium chloride solution; weighing 8.2g of sodium hydrosulfite, dissolving in 82ml of water, washing an ethyl acetate phase twice until the color of a blue-black solution becomes light, extracting a water layer by using ethyl acetate, finally washing the ethyl acetate solution by using saturated saline solution, drying by using anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a brown tar-like product; dissolving the product with appropriate amount of methanol, adding 2M hydrochloric acid solution (16.4 ml) under nitrogen protection and slowly stirring at room temperature, reacting at 100 deg.C for 1.5h, cooling to room temperature, extracting the solution with appropriate amount of ethyl acetate twice, and washing the obtained ethyl acetate solution with saturated saline; weighing 1g of sodium periodate, dissolving in a proper amount of water, adding into the ethyl acetate solution, stirring at room temperature for 15min, separating phases, washing an organic phase with brine, drying with sodium sulfate, and concentrating under reduced pressure; at 80 ℃, dropwise adding absolute ethyl alcohol into the product until the absolute ethyl alcohol is completely dissolved, slowly cooling, separating out solids, filtering and drying; repeatedly recrystallizing the filtrate for two times; 370mg of red-black solid is obtained as a second step product;

step 3. Weighing about 200mg of the second step product, dissolving in 5ml of dichloromethane, and dropwise adding triethylamine 80 at room temperatureμL, 0.1eq DMAP is weighed, and finally, the diphenyl phosphinic acid chloride 149 is drippedμL, stirring at room temperature overnight; 2mL saturated NH ₄ Adding an aqueous solution of Cl into the reaction mixture; extracting the aqueous layer with dichloromethane after layering; drying the organic phase with anhydrous sodium sulfate, and concentrating under reduced pressure; purification by column chromatography (dichloromethane: petroleum ether =20, 1,v = 20) gave 82 mg as an orange solid as the final probe molecule.

3. The peroxynitrite fluorescent molecule of claims 1 and 2, for spectroscopic characterization, in vivo imaging and practical applications.

Images