Activated and triggered in-situ anchored mitochondrial targeting peroxynitrite anion fluorescent probe and application thereof

Abstract

The invention relates to a peroxynitrite anion-targeting fluorescent probe based on activation-triggering in-situ anchored mitochondria and application of the peroxynitrite anion-targeting fluorescent probe. Specifically, the probe disclosed by the invention can show excellent detection performance on peroxynitrite anions in a pure water system. The method is mainly characterized in that accurate and quantitative detection of ONOO <-> in mitochondria is realized, and the fluorescence spectrum generated by the reaction of the probe and ONOO <-> changes remarkably; high selectivity is achieved, high-selectivity detection on ONOO <-> can be achieved in a complex biological environment, and the response strength and speed are both higher than those of other analytes; the reaction is rapid, the sensitivity is high, and the detection limit on ONOO <-> is low; meanwhile, response can be carried out in a pure water system, and the method is environment-friendly; most importantly, the probe can realize detection of peroxynitrite anions in mitochondria, and can realize long-time in-situ tracing imaging through covalent binding.

Description

Technical Field

[0001] The present invention relates to coumarin compounds as a peroxynitrite anion fluorescent probe. Specifically, the probe of the present invention can be used for the measurement of peroxynitrite anions, and can realize the detection of peroxynitrite anions in mitochondria and long-term in-situ tracing imaging. Background Art

[0002] Peroxynitrite (ONOO ˉ ) is a highly reactive reactive oxygen species (ROS) that is mainly generated in cell mitochondria. ˉ The generation of superoxide anions (O5 ˉ ) reacts with nitric oxide (NO) and is one of the key signaling molecules in cells. ˉ It plays an important role in cell signal transduction, especially regulating cell function by nitrating tyrosine residues. It not only participates in the cell's immune response, but also regulates basic physiological processes such as cell proliferation, apoptosis, and metabolism, especially playing an immune monitoring role in response to pathogen invasion. In addition, ONOO ˉ It also plays an important role in the drug resistance of tumor cells, pathological changes in neurodegenerative diseases, and the pathogenesis of cardiovascular diseases.

[0003] However, ONOO ˉ The accumulation of ONOO in cells is closely related to the disorder of cell function, especially under certain pathological conditions. ˉ It will cause fatal damage to cells. Mitochondria are the production center of cell energy and one of the main sources of reactive oxygen. ˉ When the concentration is abnormally high, it will not only destroy the function of mitochondria and limit ATP synthesis, but also may cause damage to cell membranes and even promote the occurrence and development of various diseases such as cancer and neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease). Therefore, it is necessary to accurately monitor ONOO in mitochondria. ˉ The concentration and dynamic changes of proteins are crucial to revealing their roles in cellular physiological and pathological processes.

[0004] More specifically, the peroxynitrite (ONOO) in the nucleus or cells is measured. ˉ ) concentration, it has a wide range of application value in the biomedical field. ˉ It plays an important role in signal transduction in cells and is closely related to a variety of pathological processes. ˉ The concentration of can help reveal the state of cells under different physiological and pathological conditions. The following are some of the expected applications:

[0005] 1. Cancer detection and treatment

[0006] Early detection of cancer: peroxynitrite (ONOO ˉ ) plays a key role in the proliferation, metastasis and drug resistance of cancer cells. In cancer cells, ONOO ˉ The concentration is usually high. By monitoring ONOO ˉ Changes in the concentration of oxidative stress can be used for early diagnosis of cancer, especially for those types of cancer that are triggered by oxidative stress.

[0007] Cancer Therapy Evaluation: ONOO ˉ It plays an important role in the drug resistance mechanism of cancer cells and affects the response of cancer cells to chemotherapy drugs. ˉ Changes in concentrations can help evaluate the effectiveness of cancer treatment, especially redox therapy or targeted therapy.

[0008] Cancer cell targeted drug development: by regulating ONOO ˉ By increasing the concentration, it is possible to develop new anti-cancer drugs or chemotherapy adjuvant drugs to enhance the killing effect on cancer cells.

[0009] 2. Diagnosis and prevention of neurodegenerative diseases

[0010] Alzheimer's disease (AD) and Parkinson's disease (PD): ONOO ˉ The accumulation of ONOO in neurodegenerative diseases is closely related to nerve cell damage, apoptosis and oxidative stress in the brain. ˉ Changes in concentrations can be used as biomarkers for these diseases, aiding in early diagnosis and monitoring disease progression.

[0011] Monitoring oxidative stress and neuroinflammation: ONOO ˉ Through the relationship between signal transduction and neuroinflammation between neurons, it participates in the pathogenesis of these diseases. ˉ The concentration of 247 N·g·g·e·s·s may provide new ideas for the treatment of neurodegenerative diseases, especially in antioxidant therapy and reducing neuroinflammation.

[0012] 3. Cardiovascular disease

[0013] Heart disease and high blood pressure: ONOO ˉ ONOO plays an important role in oxidative stress in the cardiovascular system and is closely related to arteriosclerosis, impaired endothelial function, and the occurrence of heart disease. ˉ Concentrations help assess the risk of cardiovascular disease, especially in the early detection and monitoring of diseases such as hypertension and myocardial infarction.

[0014] Atherosclerosis: ONOO ˉIt may participate in the formation of atherosclerosis by promoting damage to vascular endothelial cells and lipid peroxidation. ˉ Concentration changes can be used for early diagnosis and monitoring of atherosclerosis.

[0015] 4. Metabolic diseases

[0016] Diabetes: ONOO ˉ The generation of ONOO is associated with oxidative stress in diabetes, especially in the functional damage of pancreatic β cells and insulin resistance. ˉ The concentration of β-catenin can be used to evaluate the pathogenesis of diabetes and the response to drug treatment.

[0017] Obesity and related metabolic disorders: ONOO ˉ There is increasing interest in the role of ONOO in obesity, adipose tissue inflammation, and metabolic syndrome. ˉ Concentrations can help understand the level of oxidative stress in obesity and other metabolic diseases, as well as the response to intervention measures (such as diet, exercise, etc.).

[0018] 5. Inflammatory response monitoring

[0019] Chronic Inflammation and Autoimmune Diseases: ONOO ˉ Excessive production of ONOO is often associated with chronic inflammatory responses and plays an important role in autoimmune diseases such as rheumatoid arthritis and lupus erythematosus. ˉ Changes in concentrations can serve as biomarkers for these diseases, helping to assess disease activity and monitor therapeutic efficacy.

[0020] Acute inflammatory response monitoring: In acute inflammatory response, ONOO ˉ Rapid generation of ONOO is closely related to tissue damage. ˉ Changes in concentration help assess the intensity of the inflammatory response and its effects on tissues, especially in cases of infection, trauma, etc.

[0021] 6. Immune system function monitoring

[0022] Immune cell activity assessment: ONOO ˉ As an important mediator of immune system response, it can participate in immune response by regulating the functions of T cells, B cells and macrophages. ˉ Concentration can monitor the functional status of the immune system, evaluate the immune system's response to external pathogens, and its response to immunotherapy.

[0023] Detection of immunosuppressive diseases: In some immunosuppressive diseases (such as HIV infection, immunosuppression after organ transplantation), ONOO ˉChanges in concentrations can serve as disease markers and help assess immune status and the effectiveness of treatment options.

[0024] 7.Toxicology research and drug development

[0025] Drug screening and toxicity assessment: In the drug development process, ONOO ˉ Concentration changes are an important indicator for evaluating drug toxicity, especially oxidative stress-related toxicity. ˉ By monitoring the concentration changes, drugs that are toxic to cells can be effectively screened out, thereby avoiding damage to the body.

[0026] Research on antioxidant drugs: Many antioxidant drugs can regulate ONOO ˉ The concentration of ONOO ˉ Changes in concentrations can help evaluate the effects of antioxidant drugs and optimize treatment plans.

[0027] It can be seen that the determination of ONOO in the nucleus or cells ˉ The concentration has a wide range of application potential, especially in cancer, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, inflammatory response, immune system function, etc. ˉ A deep understanding of the mechanism of action and monitoring of its concentration can not only help in the early diagnosis of the disease, but also provide important clinical reference for the treatment of the disease.

[0028] Currently, although some positively charged targetable fluorescent probes have been applied to mitochondrial ONOO in living cells, ˉ However, these probes still have some significant limitations. First, they have relatively poor selectivity and may be sensitive to other oxidizing species (such as H2O2, O5 ˉ Second, the sensitivity of these probes is usually not high enough to accurately detect ONOO at low concentrations. ˉ More importantly, these probes have a long reaction time and are difficult to capture ONOO in real time. ˉ Rapid changes in concentration. In addition, the targeting ability of many targeted probes in cells also faces certain challenges. In particular, changes or disappearance of mitochondrial membrane potential (MMP) often affect the targeting ability of these probes, because many positively charged probes rely on mitochondrial membrane potential to locate to mitochondria. However, under oxidative stress or pathological conditions, the loss of mitochondrial membrane potential may result in the inability of probes to effectively target mitochondria, thus affecting the accuracy and reliability of the experiment.

[0029] Therefore, it is particularly important to develop mitochondrial-targeted fluorescent probes with self-fixing properties. Probes with self-fixing properties can stably bind to target locations (such as mitochondria) without being disturbed by changes in mitochondrial membrane potential. In this way, these probes can not only effectively target mitochondria, but also maintain their targeting ability under pathological conditions such as oxidative stress, thereby avoiding the problems that traditional probes may encounter under these conditions. In this way, researchers can monitor ONOO more efficiently and accurately. ˉ The concentration and its dynamic changes in living cells reveal that ONOO ˉ Plays a role in various physiological and pathological processes.

[0030] In addition, the design of probes with fast response and high sensitivity is very important for real-time monitoring of ONOO ˉ The dynamic changes of ONOO under different physiological or pathological conditions are of great significance. These probes can help researchers better understand ˉ It plays a role in major diseases such as cancer, neurodegenerative diseases, cardiovascular diseases, and provides new ideas for the diagnosis and treatment of related diseases.

[0031] Therefore, constructing mitochondrial-targeted fluorescent probes with high selectivity, high sensitivity and self-fixation properties will provide a basis for in-depth study of ONOO ˉ It provides more precise tools to understand the biological functions of proteins and their roles in diseases. Summary of the invention

[0032] In view of this, the present invention aims to provide a novel targeted fluorescent probe with self-fixing properties and its preparation for the field, which is used to detect ONOO in mitochondria at physiological levels. ˉ The probe has the advantages of simple synthesis, high sensitivity, good selectivity and rapid response to ONOO ˉ And other characteristics, and can be anchored in the mitochondria to achieve ONOO ˉ Long-term in situ tracing imaging.

[0033] Specifically, the present invention provides a novel ONOO based on activation-trigger-anchoring ˉ Fluorescent probe, the chemical structure of the fluorescent probe is shown in (I):

[0034]

[0035] Wherein, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R16 , R 17 , R 18 , and R 19 is independently selected from the group consisting of a hydrogen atom, a straight or branched alkyl group, a straight or branched alkoxy group, a sulfonic acid group, an ester group and a hydroxyl group; and wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 can be the same or different. Preferably, R1, R2, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is a hydrogen atom, R3 is -SO5H and R 18 is -OCH3; or wherein R1, R2, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is a hydrogen atom, R1 is an ethyl group and R6 is -SO3H.

[0036] In some specific embodiments of the present invention, the novel ONOO ˉ The fluorescent probes are R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 The compound of formula (II) wherein all of them are hydrogen atoms has the following structural formula:

[0037]

[0038] The present invention also provides a method for preparing the fluorescent probe of formula (I) or formula (II), including the following synthetic route and method:

[0039]

[0040] Wherein, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is independently selected from the group consisting of a hydrogen atom, a straight or branched alkyl group, a straight or branched alkoxy group, a sulfonic acid group, an ester group and a hydroxyl group; and wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 Can be the same or different.

[0041] In the present invention, ONOO ˉ In the preparation method of the fluorescent probe, illustratively, the first step is carried out at a reaction temperature of 90°C in an oil bath, the reaction time is 8 hours, and the molar ratio of 7-hydroxycoumarin to thiomorpholine and polyformaldehyde is approximately 1:1:1.2; the second step is carried out at room temperature, the reaction time is 24 hours, and the molar ratio of compound (III) to 4-bromomethylphenylboronic acid pinacol ester is 1:1.2; the third step is also carried out at room temperature, the reaction time is 24 hours, and the molar ratio of compound (IV) to methyl iodide is 1:10.

[0042] The present invention also provides a method for measuring, screening or detecting ONOO ˉ The fluorescent probe composition comprises a fluorescent probe of formula (I) or formula (II).

[0043] In some specific embodiments of the present invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution or a combination thereof.

[0044] The present invention also provides a fluorescent probe of formula (I) or a fluorescent probe of formula (II) for preparing, measuring, screening or detecting ONOO ˉ Application of reagents.

[0045] The present invention also provides a method for detecting ONOO in a sample. ˉ The presence or determination of ONOO in a sample ˉ A kit containing the fluorescent probe of formula (I) or formula (II).

[0046] The present invention also provides a method for detecting ONOO in a sample. ˉ The presence or determination of ONOO in a sample ˉ The method comprises the following steps:

[0047] a) contacting the fluorescent probe of formula (I) or formula (II) with a sample to form a fluorescent compound;

[0048] b) determining the fluorescence property of the fluorescent compound.

[0049] In some specific embodiments of the invention, the sample is a chemical sample.

[0050] In some embodiments of the invention, the sample is a biological sample.

[0051] In some specific embodiments of the invention, the sample is an aqueous environment sample.

[0052] In some specific embodiments of the present invention, the sample is a food sample.

[0053] The present invention also provides the use of the fluorescent probe of formula (I) or (II) for co-localization with mitochondria in cells, and for detecting ONOO in mitochondria. ˉ Capability to perform long-term tracking imaging.

[0054] The present invention also provides the use of the fluorescent probe of formula (I) or (II) in cell fluorescence imaging.

[0055] The present invention also provides the use of the fluorescent probe of formula (I) or (II) in zebrafish fluorescence imaging.

[0056] The present invention provides a method for detecting intracellular peroxynitrite (ONOO ˉ ) concentration of fluorescent probes, which can target the cell nucleus or mitochondria and reflect ONOO by changes in fluorescence signals. ˉThe probe can be widely used in many fields, including early diagnosis and treatment evaluation of cancer, detection and disease progression monitoring of neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), risk assessment and treatment effect monitoring of cardiovascular diseases (such as atherosclerosis, myocardial infarction, and hypertension), oxidative stress detection and intervention effect evaluation in metabolic diseases (such as diabetes and obesity), activity monitoring of chronic inflammatory and autoimmune diseases (such as rheumatoid arthritis and lupus erythematosus), and monitoring of immune system function. The probe is highly selective and can detect ONOO ˉ The concentration of ONOO can be monitored in real time by generating a significant fluorescence signal response. ˉ In addition, the probe can also be used to evaluate the effects of antioxidant drugs and monitor ONOO in cells. ˉ The regulation of concentration can help screen antioxidant drugs and study their therapeutic effects.

[0057] In conclusion, the fluorescent probe of the present invention not only helps to reveal the ˉ The mechanisms of action in various physiological and pathological processes can also provide new biomarkers and tools for early diagnosis of diseases, drug development, and treatment evaluation.

[0058] The present invention has one or more of the following significant advantages and effects compared to the prior art:

[0059] 1. Quantitative detection of ONOO ˉ :ONOO provided by the present invention ˉ Fluorescent probes can interact with ONOO ˉ The fluorescence spectrum changes significantly, thus achieving the effect of ONOO ˉ Accurate quantitative detection.

[0060] 2. Highly selective identification: ONOO provided by the present invention ˉ Fluorescent probes can not only ˉ reaction, and can specifically recognize ONOO in a variety of ions and chemical substances (such as potassium ions, calcium ions, sodium ions, etc.) ˉ , ensuring the accuracy and reliability of detection.

[0061] 3. High sensitivity and rapid response: ONOO provided by the present invention ˉ Fluorescent probes and ONOO ˉ Fast response speed, high sensitivity, able to quickly respond and capture ONOO ˉ The existence of is conducive to timely detection and analysis.

[0062] 4. Targeting mitochondria: ONOO provided by the present invention ˉFluorescent probes can be used to detect ONOO in mitochondria and other organelles. ˉ Due to the different contents, different fluorescence signals are shown in mitochondria and other organelles, thus achieving mitochondrial targeting.

[0063] 5. Pure water system detection: ONOO provided by the present invention ˉ The fluorescent probe can be detected in pure water system, bringing beneficial technical effects such as high sensitivity and accuracy, simplified operation process, rapid response, high selectivity and specificity, environmental friendliness, and easy promotion and application. These effects make the fluorescent probe of the present invention have broad application prospects in the fields of water quality monitoring, biomedical research, etc.

[0064] 6. Long-term tracer imaging

[0065] The fluorescent probe of the present invention utilizes its activation-triggering-anchoring characteristics to covalently bind to proteins in mitochondria to achieve ONOO ˉ Detection and long-term tracking imaging.

[0066] 7. Can be used under physiological conditions

[0067] The fluorescent probe of the present invention can be used under physiological level conditions and can be applied to fluorescent imaging of living cells and zebrafish.

[0068] In summary, the ONOO provided by the present invention ˉ Fluorescent probes have multiple beneficial technical effects such as quantitative detection, highly selective recognition, sensitive response, mitochondrial targeting, pure water system detection, and long-term tracer imaging. ˉ It provides effective tools and methods for detection and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0070] Figure 1 It is the ONOO prepared in Example 1 ˉ H NMR spectrum of probe (II);

[0071] Figure 2 The probe of formula (II) (5 μM) was added to ONOO ˉ Response time spectra before and after;

[0072] Figure 3The intermediate probe (5 μM) of formula (IV) was added to ONOO ˉ Response time spectra before and after;

[0073] Figure 4 The probe of formula (II) (5 μM) was added with ONOO ˉ (10μM), HOCl (10μM), and response time spectra before and after H2O2 (10μM);

[0074] Figure 5 The probe of formula (II) (5 μM) was added to ONOO ˉ Fluorescence spectra before and after (0-10μM).

[0075] Figure 6 is the fluorescence intensity of the probe (II) (5 μM) at 450 nm and ONOO ˉ Linear relationship diagram of (0-1μM);

[0076] Figure 7 The intermediate probe (5 μM) of formula (IV) was added to ONOO ˉ Fluorescence spectra before and after (0-10 μM);

[0077] Figure 8 is the fluorescence intensity of the intermediate probe (IV) (5 μM) at 450 nm and ONOO ˉ Linear relationship graph of (0-10 μM);

[0078] Fig. 9 The effects of common substances in the human body on the fluorescence intensity of the probe of formula (II) (5μM). The analytes include: calcium ion, copper ion, ferrous ion, ferric ion, potassium ion, magnesium ion, sodium ion, chloride ion, iodine ion, nitrate ion, sulfate ion, sulfide ion, cysteine ​​and homocysteine ​​(500μM), glutathione (5mM), nitric oxide, superoxide ion, hydroxyl ion, tert-butyl hydroperoxide, tert-butyl, hydrogen peroxide, ONOO ˉ (Except where otherwise noted, the concentrations of other analytes were 10 μM.) The bar graphs represent the fluorescence intensity values ​​of the probe at 450 nm in the presence of different analytes;

[0079] Fig.10 It is a test of the fluorescent probe's ability to locate mitochondria in cells;

[0080] Fig.11 It is a fluorescent probe for ONOO in mitochondria ˉ Long-term tracking imaging capability;

[0081] Fig.12 It is a fluorescent probe for endogenous ONOO in Hela cells. ˉ Fluorescence imaging of

[0082] Fig.13 It is a fluorescent probe for endogenous ONOO in zebrafish ˉ Fluorescence imaging of

[0083] Fig.14 The toxicity test was conducted on Hela cells treated with the probe of formula (II) using CCK-8;

[0084] Fig.15 This is a toxicity test conducted using CCK-8 on Hela cells treated with the intermediate probe of formula (IV). DETAILED DESCRIPTION

[0085] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present invention.

[0086] Example 1 Synthesis of the fluorescent probe of formula (II), the synthetic design route is as follows:

[0087]

[0088] Embodiment 1: The first step is to dissolve 7-hydroxycoumarin, thiomorpholine and paraformaldehyde in 25 mL of anhydrous ethanol, and heat to reflux at 90°C for 16 h under nitrogen protection; the second step is to dissolve 500 mg of the first step product, 537 mg of 4-bromomethylphenylboronic acid pinacol ester and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h; the third step is to dissolve 346 mg of the second step product and 437 μL of iodomethane in 10 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h. The crude product is passed through a liquid chromatography column with a mixed system of dichloromethane and methanol (v / v, 35:1) to obtain 95 mg of a pure compound of formula (II) with a yield of 21%.

[0089] Embodiment 2: The first step is to dissolve 7-hydroxycoumarin, thiomorpholine and polyformaldehyde in 25 mL of anhydrous ethanol, and heat to reflux at 90°C for 16 h under nitrogen protection; the second step is to dissolve 500 mg of the first step product, 537 mg of 4-bromomethylphenylboronic acid pinacol ester and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h; the third step is to dissolve 346 mg of the second step product and 437 μL of iodomethane in 10 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h. The crude product is passed through a liquid chromatography column with a mixed system of dichloromethane and methanol (v / v, 35:1) to obtain 129 mg of a pure compound of formula (II) with a yield of 29%.

[0090] Embodiment 3: The first step is to dissolve 7-hydroxycoumarin, thiomorpholine and paraformaldehyde in 25 mL of anhydrous ethanol, and heat to reflux at 90°C for 16 h under nitrogen protection; the second step is to dissolve 500 mg of the first step product, 537 mg of 4-bromomethylphenylboronic acid pinacol ester and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h; the third step is to dissolve 346 mg of the second step product and 437 μL of iodomethane in 10 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h. The crude product is passed through a liquid chromatography column with a mixed system of dichloromethane and methanol (v / v, 35:1) to obtain 106 mg of a pure compound of formula (II) with a yield of 24%.

[0091] Embodiment 4: The first step is to dissolve 7-hydroxycoumarin, thiomorpholine and paraformaldehyde in 25 mL of anhydrous ethanol, and heat to reflux at 90°C for 16 h under nitrogen protection; the second step is to dissolve 500 mg of the first step product, 537 mg of 4-bromomethylphenylboronic acid pinacol ester and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h; the third step is to dissolve 346 mg of the second step product and 437 μL of iodomethane in 10 mL of anhydrous acetonitrile, and stir to react at room temperature for 24 h. The crude product is passed through a liquid chromatography column with a mixed system of dichloromethane and methanol (v / v, 35:1) to obtain 102 mg of a pure compound of formula (II) with a yield of 23%.

[0092] Embodiment 5: According to the synthetic route similar to that of Embodiment 4, a fluorescent probe V having a structure of formula I is synthesized, wherein R1, R2, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19is a hydrogen atom, R3 is -SO3H and R 18 It is -OCH3.

[0093] Embodiment 6: According to the synthetic route similar to that of Embodiment 4, a fluorescent probe VI having a structure of formula I is synthesized, wherein R1, R2, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is a hydrogen atom, R1 is an ethyl group and R6 is -SO3H.

[0094] For example, the product structure characterization data of the fluorescent probe of formula (II) prepared in embodiments 1-4 are as follows (the hydrogen spectrum is shown in the attached Figure 1 shown):

[0095] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 8.06 (d, J = 12.0Hz, 1H), 7.94 (d, J = 6.0Hz, 1H), 7.74 (d, J = 6.0Hz, 2H), 7.55 (d, J = 6.0Hz, 2H), 7.32 (d, J = 6.0Hz, 1H), 6.42 (d, J = 12.0Hz, 1H), 5.40 (s, 2H), 4.73 (s, 2H), 3.66 (m, J = 12.0Hz, 4H), 3.13 (t, J = 12.0Hz, 3H), 2.99 (d, J = 18.0Hz, 4H), 1.30 (s, 12H).

[0096] Example 2: Testing the temporal dynamics of fluorescent probes

[0097] Prepare a 10 mL test system with a concentration of 5 μM of the fluorescent probe of formula (II), and then add 10 μM ONOO ˉ Add to the test system, shake evenly and immediately test the fluorescence intensity change with a fluorescence spectrometer. Perform the same test on the intermediate probe of formula (Ⅳ). In addition, prepare three 10mL test systems with a concentration of 5μM of the fluorescent probe of formula (Ⅱ), and then add 10μM ONOO ˉ, H2O2, and HOCl were added to the test system respectively, and the fluorescence intensity change was immediately tested by fluorescence spectrometer after being shaken evenly. The above measurement was carried out in a pure water (10mM PBS, pH 7.4) system, the probe used was the probe prepared in Example 1, and the fluorescence spectrum was measured at 25°C. Unless otherwise specified, all spectral data are λ ex =350nm, slit: W ex =W em =5nm.

[0098] Test results such as Figure 2 (Formula II), Figure 3 As shown in (Formula IV), it can be clearly seen that when ONOO ˉ After addition, the fluorescence intensity at 450nm reached the maximum value and remained basically unchanged after about 50s. Compared with other active oxygen species ( Figure 4 ), formula (II) fluorescent probe for ONOO ˉ The response of the probe to ONOO ˉ The reaction is faster than that of the intermediate probe (Ⅳ) and can be used for ONOO ˉ Provides a rapid analytical method for determination.

[0099] Example 3: Testing fluorescent probes for ONOO ˉ Fluorescence spectrum of the concentration gradient

[0100] Prepare multiple parallel samples with a probe concentration of 5 μM in 10 mL colorimetric tubes, and then add different concentrations of ONOO ˉ (0-10 μM) was added to the test system, shaken evenly and left to stand for 5 minutes, and the fluorescence intensity change was tested by fluorescence spectrometer. The above measurement was carried out in pure water (10 mM PBS, pH 7.4), and the probes used were the probe of formula (II) and the intermediate probe of formula (IV) prepared in Example 1, and all spectral tests were measured at 25°C. The test results are shown in Figure 5 , 6 , 7, and 8.

[0101] from Figure 5 , 7 It can be clearly seen that with ONOO ˉ With the increase of concentration, the fluorescence intensity at 450nm gradually increases. Figure 6 , 8 It can be seen that the probe (5 μM) was added to ONOO ˉ After 0-1 μM, the fluorescence intensity at 450 nm was similar to that of ONOO ˉThe concentrations of ONOO and ONOO showed a good linear relationship, which proved that the fluorescent probe can be used to detect ONOO ˉ Quantitative analysis can be performed and the probe has a more sensitive detection effect compared with the intermediate probe.

[0102] Example 4: Fluorescent Probe for ONOO ˉ Selective testing

[0103] The analytes were blank, calcium ion, copper ion, ferrous ion, ferric ion, potassium ion, magnesium ion, sodium ion, chloride ion, iodide ion, nitrate ion, sulfate ion, sulfide ion, cysteine ​​and homocysteine ​​(500 μM), glutathione (5 mM), nitric oxide, superoxide ion, hydroxyl ion, tert-butyl hydroperoxide, tert-butyl, hydrogen peroxide, ONOO ˉ (Unless otherwise specified, the concentration of other analytes is 10 μM). The bar graph represents the fluorescence intensity of the probe at 450 nm in the presence of different analytes. The above determination was carried out in a pure water (10 mM PBS, pH 7.4) system. The probe used was the probe of formula (II) prepared in Example 1, and all spectral tests were measured at 25°C. Specifically, multiple parallel samples with a probe concentration of 5 μM were placed in a 10 mL colorimetric tube, and then a certain amount of analyte was added, shaken, and the fluorescence intensity value was measured after standing for 10 minutes. The results are shown in the figure. Fig. 9 shown.

[0104] from Fig. 9 It can be seen that the probe of the present invention is effective for ONOO ˉ It has high selectivity and can selectively react with ONOO ˉ to react.

[0105] Example 5: Probe detection limit test and calculation

[0106] The detection limit was calculated by fluorescence titration. The detection limit calculation formula is as follows:

[0107] Detection limit = 3σ / k

[0108]

[0109] σ is the standard deviation of the fluorescence intensity of the blank probe, and k is the slope of the linear relationship graph. ˉ The detection limit of the probe of formula (V) is 9.0 nM for ONOO ˉ The detection limit of the probe of formula (VI) is about 6.0 nM for ONOO ˉ The detection limit was approximately 7.0 nM.

[0110] Example 6: Targeting ability of fluorescent probes to mitochondria in cells

[0111] ONOO was detected by using probes and Mito-Tracker Green (MTG), a commercial mitochondrial selective fluorescent labeling dye. ˉ Treated HeLa cells were stained and imaged using confocal microscopy.

[0112] like Fig.10 As shown in (A), the blue channel of the probe of formula (II) overlaps well with the red channel of MTG, and the Pearson coefficient reaches 0.9513. Repeat the above operation on the intermediate probe, such as Fig.10 As shown in (B), the Pearson coefficient is only 0.6579, which indicates that the probe of formula (II) has unique mitochondrial targeting ability.

[0113] Example 7: Fluorescent probe for ONOO in mitochondria ˉ Long-term tracking capability

[0114] LPS-pretreated cells were incubated with the probe of formula (II) (10 μM) and the intermediate probe of formula (IV) (10 μM) and imaged within 0-2 hours. Fig.11 Compared with the control cells, the cell fluorescence at 0.5h, 1h, 2h and 3h increased. After the response, the fluorescence intensity of the cells incubated with the probe of formula (II) remained basically unchanged over time, while the fluorescence intensity of the cells incubated with the intermediate probe of formula (IV) gradually decreased over time. This proves that the probe of formula (II) can be fixed on the mitochondria of cells and can be used to monitor mitochondrial ONOO under abnormal conditions. ˉ Changes in levels.

[0115] Example 8: Fluorescent probes for endogenous ONOO in Hela cells ˉ Fluorescence microscopy imaging

[0116] Hela cells were divided into seven groups: Group A was Hela cells in normal state; Group B was incubated with the probe (10 μM) for 10 min; Group C was first incubated with the probe (10 μM) for 10 min, and then with ONOO ˉ (5 μM) for 10 min; Group D was first incubated with the probe (10 μM) for 10 min, and then with ONOO ˉ (50 μM) for 10 min; Group E was first incubated with the probe (10 μM) for 10 min, and then with ONOO ˉ The cells in group F were pretreated with LPS (1 μg / mL) for 1 hour, and then incubated with the probe (10 μM) for 10 minutes. The cells in group G were pretreated with SIN-1 (100 μM) for 1 hour, and then incubated with the probe (10 μM) for 10 minutes. Finally, confocal microscopy imaging was performed on the seven groups of cells. The test results are shown in the figure. Fig.12shown.

[0117] from Fig.12 It can be seen that the probe can detect endogenous ONOO in Hela cells. ˉ ; Experiments have shown that this probe can be applied to ONOO in biological samples ˉ Detection.

[0118] Example 9: Fluorescent Probes for Endogenous ONOO in Zebrafish ˉ Fluorescence microscopy imaging

[0119] The zebrafish were divided into six groups. Group A was incubated with the probe (10 μM) for 10 min; Group B was first incubated with the probe (10 μM) for 10 min and then with ONOO ˉ (5 μM) for 10 min; Group C was first incubated with the probe (10 μM) for 10 min, and then with ONOO ˉ (10 μM) for 10 min; Group D was first incubated with the probe (10 μM) for 10 min, and then with ONOO ˉ Group E was pretreated with LPS (1 μg / mL) for 1 hour, and then incubated with the probe (10 μM) for 10 minutes. Group F was pretreated with SIN-1 (100 μM) for 1 hour, and then incubated with the probe (10 μM) for 10 minutes. Finally, confocal microscopy imaging was performed on the six groups of cells. The test results are shown in the figure. Fig.13 shown.

[0120] from Fig.13 It can be seen that this probe can detect endogenous ONOO in zebrafish ˉ ; Experiments have shown that this probe can be applied to ONOO in biological samples ˉ Detection.

[0121] Example 10: Toxic effect of fluorescent probe on Hela cells

[0122] The toxicity test was performed on live HeLa cells incubated with various concentrations (0, 5, 10 and 20 μM) of the probe of formula (II) for 12 h using a CCK-8 kit. The toxicity test was also performed on the intermediate probe of formula (IV).

[0123] like Fig.14 , 15 As shown, when the concentration reaches 10 μM, the toxic effect of the fluorescent probe on organisms can be basically ignored.

[0124] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. A novel ONOO-fluorescent probe based on activation-trigger-anchoring, characterized in that: The chemical structural formula of the fluorescent probe is shown in (I): Wherein, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is independently selected from the group consisting of a hydrogen atom, a straight or branched alkyl group, a straight or branched alkoxy group, a sulfonic acid group, an ester group and a hydroxyl group; and wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 Can be the same or different.

2. The novel ONOO-fluorescent probe according to claim 1, characterized in that R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 They are all hydrogen atoms, and their structural formula is as follows: Or, where R1, R2, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is a hydrogen atom, R3 is -SO3H and R 18 is -OCH3; or among which R1, R2, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 is a hydrogen atom, R1 is an ethyl group and R6 is -SO3H.

3. A method for preparing the fluorescent probe according to claim 1 or 2, comprising the following reaction steps:

4. The preparation method according to claim 3, characterized in that: The method comprises the following steps: the first step is to react at a 90°C oil bath temperature, the reaction time is 16 hours, and the molar ratio of 7-hydroxycoumarin to thiomorpholine and polyformaldehyde is about 1:1:1.2; the second step is to react at room temperature, the reaction time is 24 hours, and the molar ratio of compound (III) to 4-bromomethylphenylboronic acid pinacol ester is 1:1.2; the third step is also to react at room temperature, the reaction time is 24 hours, and the molar ratio of compound (IV) to methyl iodide is 1:

10.

5. A fluorescent probe composition for measuring, screening or detecting ONOOˉ, comprising the fluorescent probe according to claim 1 or 2.

6. The fluorescent probe composition according to claim 5, characterized in that The fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution or a combination thereof.

7. Use of the fluorescent probe according to claim 1 or 2 in the preparation of a reagent for measuring, screening or detecting ONOOˉ.

8. A kit for detecting the presence of ONOO- in a sample or determining the content of ONOO- in a sample, comprising the fluorescent probe according to claim 1 or 2.

9. A method for detecting the presence of ONOO" in a sample or measuring ONOO" in a sample (eg, a chemical sample, a biological sample), comprising the step of contacting the fluorescent probe of claim 1 or 2 with the sample.

10. The use of the fluorescent probe according to claim 1 or 2, which is used for fluorescence imaging detection or photoacoustic imaging detection. Preferably, the fluorescent probe is used for in situ fluorescence imaging detection or in situ photoacoustic imaging detection of cancer or tumors, for intracellular mitochondrial targeted detection, for fluorescence imaging in cells, or for use in zebrafish fluorescence imaging.

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