Activation-triggered in situ anchoring mitochondria-targeted probe for peroxynitrite anion and its application

Abstract

This invention relates to a class of activation-triggered in-situ anchored mitochondrial-targeted fluorescent probes for peroxynitrite anions and their applications. Specifically, the probes of this invention exhibit excellent detection performance for peroxynitrite anions in a pure water system. This is mainly manifested in: achieving precise quantitative detection of ONOOˉ in mitochondria, with significant changes in the fluorescence spectrum generated by the reaction between the probe and ONOOˉ; possessing high selectivity, enabling highly selective detection of ONOOˉ in complex biological environments, with response intensity and speed exceeding those of other analytes; rapid reaction and high sensitivity, with a low detection limit for ONOOˉ; simultaneously, it can respond in a pure water system, making it environmentally friendly; most importantly, this probe can detect peroxynitrite anions in mitochondria and achieve long-term in-situ tracer imaging through covalent binding.

Description

Technical Field

[0001] This invention relates to coumarin compounds as fluorescent probes for peroxynitrite anions. Specifically, the probes of this invention can be used to measure peroxynitrite anions; they can enable the detection and long-term in-situ tracing imaging of peroxynitrite anions in mitochondria. Background Technology

[0002] peroxynitrite (ONOO) ˉ ONOO is a highly reactive reactive oxygen species (ROS) primarily generated within cellular mitochondria. ˉ The formation of superoxide anions (O5) is usually caused by superoxide anions (O5). ˉ It is formed by the reaction of nitric oxide (NO) with nitric oxide and is one of the key signaling molecules in cells. ˉ It plays a crucial role in cell signal transduction, particularly by regulating cellular function through the hydration of nitrated tyrosine residues. It not only participates in cellular immune responses but also regulates fundamental physiological processes such as cell proliferation, apoptosis, and metabolism, playing a vital role in immune surveillance, especially in responding to pathogen invasion. Furthermore, 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 Its accumulation in cells is closely related to cellular dysfunction, especially under certain pathological conditions, where excessive ONOO... ˉ It can cause fatal damage to cells. Mitochondria, as the center of cellular energy production, are also one of the main sources of reactive oxygen species. When ONOO in the mitochondria... ˉ When its concentration is abnormally high, it not only damages mitochondrial function and restricts ATP synthesis, but may also cause cell membrane damage and even promote the occurrence and development of various diseases such as cancer and neurodegenerative diseases (such as Alzheimer's and Parkinson's). Therefore, accurate monitoring of ONOO in mitochondria is crucial. ˉ The concentration and dynamic changes of [the substance] are crucial for revealing its role in cellular physiological and pathological processes.

[0004] More specifically, measuring peroxynitrite (ONOO) in the cell nucleus or cells. ˉ The concentration of ONOO has wide-ranging applications in the biomedical field. ˉ It plays an important role in intracellular signal transduction and is closely related to various pathological processes. Measurement of ONOO... ˉ The concentration of [a specific substance] can help reveal the state of cells under different physiological and pathological conditions. Here are some anticipated applications:

[0005] 1. Cancer detection and treatment

[0006] Early cancer detection: peroxynitrite (ONOO) ˉ ONOO plays a crucial role in the proliferation, metastasis, and drug resistance of cancer cells. In cancer cells, ONOO... ˉ Concentrations are typically high. This is achieved by monitoring ONOO. ˉ Changes in concentration can be used for the early diagnosis of cancer, especially for cancer types caused by oxidative stress.

[0007] Cancer treatment assessment: ONOO ˉ It plays a crucial role in the drug resistance mechanisms of cancer cells, influencing their response to chemotherapy drugs. Monitoring ONOO ˉ Changes in concentration can help assess the effectiveness of cancer treatments, especially redox therapy or targeted therapy.

[0008] Cancer cell-targeted drug development: through the regulation of ONOO ˉ At certain concentrations, it may be possible to develop new anticancer drugs or chemotherapy adjuvants to enhance their 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 these substances in neurodegenerative diseases is closely related to nerve cell damage, apoptosis, and oxidative stress in the brain. ˉ Changes in concentration can be used as biomarkers for these diseases, aiding in early diagnosis and monitoring disease progression.

[0011] Monitoring of oxidative stress and neuroinflammation: ONOO ˉ It participates in the pathogenesis of these diseases by interacting with signal transduction and neuroinflammation between neurons. This is achieved through real-time monitoring of ONOO. ˉ The concentration of [specific substance] can provide new insights for the treatment of neurodegenerative diseases, especially in antioxidant therapy and reducing neuroinflammation.

[0012] 3. Cardiovascular diseases

[0013] Heart disease and high blood pressure: ONOO ˉ It plays a crucial role in oxidative stress in the cardiovascular system and is closely related to arteriosclerosis, impaired vascular endothelial function, and the development of heart disease. Monitoring ONOO ˉ Concentration helps 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 through mechanisms such as promoting vascular endothelial cell damage and lipid peroxidation. By detecting ONOO... ˉ Changes in concentration can be used for the early diagnosis and monitoring of atherosclerosis.

[0015] 4. Metabolic diseases

[0016] Diabetes: ONOO ˉ The production of [a specific substance] is associated with oxidative stress in diabetes, particularly in terms of impaired pancreatic β-cell function and insulin resistance. Monitoring ONOO [a specific substance]... ˉ The concentration of [a specific substance] can be used to assess the pathogenesis of diabetes and its response to drug treatment.

[0017] Obesity and related metabolic disorders: ONOO ˉ Its role in obesity, inflammation of adipose tissue, and metabolic syndrome is receiving increasing attention. Monitoring ONOO ˉ Concentration can help understand the level of oxidative stress in obesity and other metabolic diseases, as well as the response to interventions such as diet and exercise.

[0018] 5. Monitoring of inflammatory responses

[0019] Chronic inflammation and autoimmune diseases: ONOO ˉ Excessive production of ONOO is often accompanied by chronic inflammatory responses and plays an important role in autoimmune diseases such as rheumatoid arthritis and lupus. ˉ Changes in concentration can serve as biomarkers for these diseases, helping to assess disease activity and monitor treatment efficacy.

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

[0021] 6. Monitoring of immune system function

[0022] Immune cell activity assessment: ONOO ˉ As an important mediator of the immune system response, it can participate in the immune response by regulating the function of T cells, B cells, and macrophages. By detecting ONOO... ˉ Concentration can be used to monitor the functional state of the immune system, assess the immune system's response to external pathogens, and the response to immunotherapy.

[0023] Detection of immunosuppressive diseases: In some immunosuppressive diseases (such as HIV infection, post-organ transplant immunosuppression), ONOO ˉChanges in concentration can serve as an indicator of disease, helping to assess immune status and the effectiveness of treatment regimens.

[0024] 7. Toxicological Research and Drug Development

[0025] Drug screening and toxicity assessment: During the drug development process, ONOO ˉ Concentration changes are an important indicator for assessing drug toxicity, especially oxidative stress-related toxicity. By detecting ONOO... ˉ Changes in drug concentration can effectively screen out drugs that are toxic to cells, thereby avoiding harm to the body.

[0026] Research on antioxidant drugs: Many antioxidant drugs work by regulating ONOO ˉ To reduce oxidative damage to cells by adjusting the concentration of ONOO. ˉ Changes in concentration can help assess the effectiveness of antioxidant drugs and optimize treatment plans.

[0027] It is evident that measuring ONOO in the cell nucleus or cells ˉ Concentration has broad application potential, especially in areas such as cancer, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, inflammatory responses, and immune system function. With increasing research into ONOO... ˉ A deeper understanding of its mechanism of action and monitoring its concentration can not only help in the early diagnosis of the disease, but also provide important clinical references for the treatment of the disease.

[0028] Currently, although some positively charged, targetable fluorescent probes have been applied to live cell mitochondria (ONOO) ˉ While these probes have been used in imaging studies, they still have some significant limitations. First, their selectivity is relatively poor, potentially limiting their effectiveness against other oxidizing substances (such as H₂O₂, O₅). ˉ These probes can cause cross-reactions, affecting the accuracy and specificity of the fluorescence signal. Secondly, their sensitivity is often insufficient, making it difficult to accurately detect ONOO at low concentrations. ˉ The changes are significant. More importantly, these probes have long reaction times, making it difficult to capture intracellular ONOO in real time. ˉ Rapid changes in concentration are a significant challenge. Furthermore, the intracellular targeting ability of many targeted probes faces certain challenges. In particular, changes or loss of mitochondrial membrane potential (MMP) often affect the targeting ability of these probes, as 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 prevent probes from effectively targeting mitochondria, thus affecting the accuracy and reliability of experiments.

[0029] Therefore, developing mitochondrial-targeting fluorescent probes with self-immobilizing properties is particularly important. Probes with self-immobilizing properties can bind stably to target sites (such as mitochondria) without being affected 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, thus 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 of ONOO and its dynamic changes in living cells reveal ˉ Its role in various physiological and pathological processes.

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

[0031] Therefore, constructing mitochondrial-targeting fluorescent probes with high selectivity, high sensitivity, and self-immobilization properties will be crucial for in-depth research on ONOO. ˉ This provides more precise tools to understand the biological functions of organisms and their role in diseases. Summary of the Invention

[0032] Therefore, this invention aims to provide a novel self-immobilizing targeted fluorescent probe and its preparation method for use in mitochondria at physiological levels. ˉ The probe is simple to synthesize, highly sensitive, selective, and can respond rapidly to ONOO. ˉ These characteristics, along with the ability to anchor within mitochondria, enable ONOO ˉ Long-term in-situ tracer imaging.

[0033] Specifically, this invention provides a novel ONOO based on activation-trigger-anchoring. ˉ A fluorescent probe, the chemical structural formula of which is shown in (Ⅰ):

[0034]

[0035] In the formula, 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 The group consisting of hydrogen atoms, straight-chain or branched alkyl groups, straight-chain or branched alkoxy groups, sulfonic acid groups, ester groups, and hydroxyl groups is independently selected; and 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 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 It is a hydrogen atom, R3 is -SO5H and R 18 =-OCH3; 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 R1 is a hydrogen atom, R6 is an ethyl group, and R1 is -SO3H.

[0036] In some specific embodiments of the present invention, the novel ONOO is described. ˉ The fluorescent probes are R1, R2, R3, R4, R5, R6, R7, R8, R9, and R1. 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), consisting entirely of hydrogen atoms, has the following structural formula:

[0037]

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

[0039]

[0040] In the formula, 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 group consisting of hydrogen atoms, straight-chain or branched alkyl groups, straight-chain or branched alkoxy groups, sulfonic acid groups, ester groups, and hydroxyl groups is independently selected; and 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 can be the same or different.

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

[0042] The present invention also provides a method for measuring, screening or detecting ONOO. ˉ A fluorescent probe composition comprising 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] This invention also provides a fluorescent probe of formula (I) or formula (II) for the preparation, measurement, screening or detection of ONOO. ˉ Applications in 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 for determining the content includes the following steps:

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

[0048] b) Determine the fluorescence properties of the fluorescent compound.

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

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

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

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

[0053] This invention also provides the application of (I) or (II) formula fluorescent probe for intracellular colocalization of mitochondria, and for the detection of ONOO within mitochondria. ˉ Capable of long-term tracing imaging.

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

[0055] This invention also provides the application of (I) or (II) formula fluorescent probes in zebrafish fluorescence imaging.

[0056] This invention provides a method for detecting intracellular peroxynitrite (ONOO). ˉ Fluorescent probes at concentrations of 0.05 can target the cell nucleus or mitochondria and reflect changes in fluorescence signals. ˉThe concentration of this probe is [specific value missing]. This probe has wide applications in multiple fields, including early diagnosis and treatment evaluation of cancer, detection and disease progression monitoring of neurodegenerative diseases (such as Alzheimer's and Parkinson's), risk assessment and treatment efficacy monitoring of cardiovascular diseases (such as atherosclerosis, myocardial infarction, and hypertension), detection and intervention efficacy evaluation of oxidative stress in metabolic diseases (such as diabetes and obesity), activity monitoring of chronic inflammatory and autoimmune diseases (such as rheumatoid arthritis and lupus), and monitoring of immune system function. This probe is highly selective and can [specific value missing]. ˉ It generates a significant fluorescence signal response to concentration changes, and ONOO can be monitored in real time. ˉ Dynamic changes within cells. Furthermore, the probe can be used to assess the effectiveness of antioxidant drugs and monitor intracellular ONOO. ˉ The concentration regulation effect helps in screening antioxidant drugs and studying their therapeutic effects.

[0057] In summary, the fluorescent probe of this invention not only helps to reveal ONOO ˉ Its mechanisms of action in various physiological and pathological processes can also provide new biomarkers and tools for early disease diagnosis, 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 The ONOO provided by this invention ˉ Fluorescent probes can interact with ONOO ˉ This effect produces a significant linear change in the fluorescence spectrum, thereby achieving the control of ONOO. ˉ Precise quantitative detection.

[0060] 2. High selectivity recognition: The ONOO provided by this invention ˉ Fluorescent probes can not only interact with ONOO ˉ The reaction can also specifically recognize ONOO in a variety of ions and chemical substances (such as potassium ions, calcium ions, sodium ions, etc.). ˉ This ensures the accuracy and reliability of the testing.

[0061] 3. High sensitivity and rapid response: The ONOO provided by this invention ˉ Fluorescent probes and ONOO ˉ It has a fast response speed and high sensitivity, enabling it to quickly respond to and capture ONOO. ˉ The existence of [the substance] facilitates timely detection and analysis.

[0062] 4. Targets mitochondria: The ONOO provided by this invention ˉFluorescent probes can be detected in mitochondria and other organelles via ONOO. ˉ Different concentrations result in different fluorescent signals in mitochondria and other organelles, enabling targeting of mitochondria.

[0063] 5. Pure water system testing: The ONOO system provided by this invention... ˉ Fluorescent probes can be used for detection in pure water systems, offering advantages such as high sensitivity and accuracy, simplified operation procedures, rapid response, high selectivity and specificity, environmental friendliness, and ease of promotion and application. These advantages make the fluorescent probes of this invention promising for broad applications in water quality monitoring, biomedical research, and other fields.

[0064] 6. Long-term tracer imaging

[0065] The fluorescent probe of this invention utilizes its activation-trigger-anchoring properties to covalently bind to proteins within mitochondria, achieving ONOO. ˉ Detection and long-term tracer imaging.

[0066] 7. Can be used under physiological conditions

[0067] The fluorescent probe of this invention can be used under physiological conditions and can be applied to fluorescence imaging of live cells and zebrafish.

[0068] In summary, the ONOO provided by this invention ˉ Fluorescent probes offer multiple beneficial technical advantages, including quantitative detection, highly selective recognition, rapid response, mitochondrial targeting, detection in pure water systems, and long-term tracer imaging, making them ideal for ONOO. ˉ It provides effective tools and methods for detection and analysis. Attached Figure Description

[0069] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

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

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

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

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

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

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

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

[0078] Figure 9 This study investigates the influence of common substances in the human body on the fluorescence intensity of probe (II) (5 μM). The analytes include: calcium ions, copper ions, ferrous ions, ferric ions, potassium ions, magnesium ions, sodium ions, chloride ions, iodide ions, nitrate ions, sulfate ions, sulfide ions, cysteine ​​and homocysteine ​​(500 μM), glutathione (5 mM), nitric oxide, superoxide ions, hydroxyl ions, tert-butyl hydroperoxide, tert-butyl, hydrogen peroxide, and ONOO. ˉ (Unless otherwise specified, all analyte concentrations are 10 μM). The bar chart represents the fluorescence intensity values ​​of the probe at 450 nm in the presence of different analytes;

[0079] Figure 10 It is a test of the ability of fluorescent probes to locate mitochondria within cells;

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

[0081] Figure 12 The fluorescent probe is for the exogenous and endogenous ONOO in HeLa cells. ˉ Fluorescence imaging;

[0082] Figure 13 It is a fluorescent probe for zebrafish endogenous and exogenous ONOO ˉ Fluorescence imaging;

[0083] Figure 14 The toxicity test was performed on HeLa cells treated with the formula (II) probe using CCK-8;

[0084] Figure 15 The toxicity test was performed on HeLa cells treated with the CCK-8 intermediate probe of formula (Ⅳ). Detailed Implementation

[0085] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0086] Example 1: Synthesis of Formula (II) fluorescent probe. The synthetic design route is as follows:

[0087]

[0088] Implementation Scheme 1: The first step involves dissolving 7-hydroxycoumarin, thiomorpholine, and paraformaldehyde in 25 mL of anhydrous ethanol and refluxing at 90 °C for 16 h under nitrogen protection. The second step involves dissolving 500 mg of the product from the first step, 537 mg of pinacol 4-bromomethylphenylboronic acid, and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The third step involves dissolving 346 mg of the product from the second step, 437 μL of iodomethane, in 10 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The crude product was then passed through a liquid chromatography column using a mixture 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] Implementation Scheme 2: The first step involves dissolving 7-hydroxycoumarin, thiomorpholine, and paraformaldehyde in 25 mL of anhydrous ethanol and refluxing at 90 °C for 16 h under nitrogen protection. The second step involves dissolving 500 mg of the product from the first step, 537 mg of 4-bromomethylphenylboronic acid pinacol ester, and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The third step involves dissolving 346 mg of the product from the second step and 437 μL of iodomethane in 10 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The crude product was then passed through a liquid chromatography column using a mixture 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] Implementation Scheme 3: The first step involves dissolving 7-hydroxycoumarin, thiomorpholine, and paraformaldehyde in 25 mL of anhydrous ethanol and refluxing at 90 °C for 16 h under nitrogen protection. The second step involves dissolving 500 mg of the product from the first step, 537 mg of 4-bromomethylphenylboronic acid pinacol ester, and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The third step involves dissolving 346 mg of the product from the second step and 437 μL of iodomethane in 10 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The crude product was then passed through a liquid chromatography column using a mixture 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] Implementation Scheme 4: The first step involves dissolving 7-hydroxycoumarin, thiomorpholine, and paraformaldehyde in 25 mL of anhydrous ethanol and refluxing at 90 °C for 16 h under nitrogen protection. The second step involves dissolving 500 mg of the product from the first step, 537 mg of pinacol 4-bromomethylphenylboronic acid, and 494.5 mg of potassium carbonate in 25 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The third step involves dissolving 346 mg of the product from the second step, 437 μL of iodomethane, in 10 mL of anhydrous acetonitrile and stirring at room temperature for 24 h. The crude product was then passed through a liquid chromatography column using a mixture 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] Implementation Scheme 5: Following a similar synthetic route to Implementation Scheme 4, synthesize a fluorescent probe V with the structure of Formula I, 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 19It is a hydrogen atom, R3 is -SO3H and R 18 It is -OCH3.

[0093] Implementation Scheme 6: Following a similar synthetic route to Implementation Scheme 4, synthesize a fluorescent probe VI with the structure of Formula I, 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 R1 is a hydrogen atom, R6 is an ethyl group, and R1 is -SO3H.

[0094] For example, the product structure characterization data of the fluorescent probe of formula (II) prepared according to embodiments 1-4 are as follows (the proton spectrum is attached). Figure 1 As 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 time dynamics of a fluorescent probe

[0097] Prepare a 10 mL test system with a concentration of 5 μM for the (II) fluorescent probe, and then add 10 μM ONOO. ˉ Add the sample to the test system, shake well, and immediately measure the change in fluorescence intensity using a fluorescence spectrometer. Perform the same test on the intermediate probe of formula (IV). In addition, prepare three 10 mL test systems of formula (II) fluorescent probes with a concentration of 5 μM, and then add 10 μM ONOO. ˉH₂O₂ and HOCl were added to the test system, and after shaking thoroughly, the fluorescence intensity changes were immediately measured using a fluorescence spectrometer. The above measurements were performed in a pure water (10 mM PBS, pH 7.4) system. The probe used was the probe prepared in Example 1, and the fluorescence spectra were measured at 25°C. Unless otherwise specified, all spectral data are [data missing]. ex Acquired at 350nm, slit size: W ex =W em =5nm.

[0098] Test results are as follows Figure 2 (Formula II) Figure 3 As shown in (Equation IV), it can be clearly seen that when ONOO ˉ After addition, the fluorescence intensity at 450nm reached its maximum value after approximately 50 seconds and remained essentially constant, compared to other reactive oxygen species ( Figure 4 Formula (II) fluorescent probe for ONOO ˉ The response is more rapid, indicating that the probe is more compatible with ONOO. ˉ The reaction is faster than that of intermediate probe (Ⅳ), and can provide ONOO ˉ This provides a rapid analytical method for determination.

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

[0100] Prepare multiple parallel samples with a probe concentration of 5 μM in 10 mL colorimetric tubes, and then use different concentrations of ONOO. ˉ (0-10 μM) was added to the test system, shaken well, and allowed to stand for 5 minutes. The change in fluorescence intensity was then measured using a fluorescence spectrometer. The above measurements were performed in pure water (10 mM PBS, pH 7.4). The probes used were the formula (II) probe and the formula (IV) intermediate probe prepared in Example 1. All spectral measurements were taken at 25°C. The test results are as follows: Figure 5 , 6 As shown in Figures 7 and 8.

[0101] from Figure 5 , 7 It can be clearly seen from the image that, with ONOO ˉ With increasing concentration, the fluorescence intensity at 450 nm gradually increases. Furthermore, due to... Figure 6 , 8 It can be seen from the addition of ONOO to the probe (5μM) ˉ After (0-1μM), its fluorescence intensity at 450nm is compared with ONOO. ˉA good linear relationship was observed between the concentrations, demonstrating that this fluorescent probe can effectively target ONOO. ˉ It enables quantitative analysis and has a more sensitive detection effect compared to intermediate probes.

[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, and ONOO. ˉ (Unless otherwise specified, all analyte concentrations were 10 μM). The bar chart represents the fluorescence intensity of the probe at 450 nm in the presence of different analytes. The above measurements were performed in a pure water (10 mM PBS, pH 7.4) system. The probe used was the formula (II) probe prepared in Example 1, and all spectral measurements were taken at 25°C. Specifically, multiple parallel samples with a probe concentration of 5 μM were prepared in 10 mL colorimetric tubes, a certain amount of analyte was added, the mixture was shaken, and the fluorescence intensity was measured after standing for 10 minutes. The results are as follows: Figure 9 As shown.

[0104] from Figure 9 It can be seen that the probe of the present invention is effective against ONOO. ˉ It has high selectivity and can selectively interact with ONOO. ˉ The reaction will proceed.

[0105] Example 5: Detection limit test and calculation of the probe

[0106] The limit of detection (LOD) was calculated using fluorescence titration. The formula for calculating the LOD 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. Therefore, the probe-to-ONOO ratio is calculated using equation (II). ˉ The detection limit is 9.0 nM; the formula (V) probe for ONOO ˉ The detection limit is approximately 6.0 nM; Formula (VI) probe for ONOO ˉ The detection limit is approximately 7.0 nM.

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

[0111] ONOO was mediated using probes and Mito-Tracker Green (MTG, a commercially available mitochondrial-selective fluorescent labeling dye). ˉ The treated HeLa cells were stained and imaged using a confocal microscope.

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

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

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

[0115] Example 8: Fluorescent probes targeting endogenous and exogenous ONOO in HeLa cells ˉ Fluorescence microscopy

[0116] HeLa cells were divided into seven groups: Group A consisted of HeLa cells under normal conditions; Group B was incubated with a probe (10 μM) for 10 min; Group C was first incubated with a probe (10 μM) for 10 min, and then incubated with ONOO. ˉ (5μM) incubation for 10 min; Group D was first incubated with the probe (10μM) for 10 min, then with ONOO ˉ (50μM) incubation for 10 min; Group E was first incubated with the probe (10μM) for 10 min, then with ONOO ˉ Group F was pretreated with LPS (1 μg / mL) for 1 h, followed by incubation with the probe (10 μM) for 10 min. Group G was pretreated with SIN-1 (100 μM) for 1 h, followed by incubation with the probe (10 μM) for 10 min. Finally, confocal microscopy was performed on the cells in each of the seven groups, and the results are shown below. Figure 12As shown.

[0117] from Figure 12 It can be seen that this probe can detect endogenous and exogenous ONOO in HeLa cells. ˉ Experiments have shown that this probe can be applied to ONOO in biological samples. ˉ Testing.

[0118] Example 9: Fluorescent probes targeting endogenous and exogenous ONOO in zebrafish ˉ Fluorescence microscopy

[0119] Zebrafish were divided into six groups. Group A was incubated with a probe (10 μM) for 10 min; Group B was first incubated with a probe (10 μM) for 10 min, and then with ONOO. ˉ (5μM) Incubate for 10 min; Group C was first incubated with the probe (10μM) for 10 min, then with ONOO ˉ (10μM) incubation for 10 min; Group D was first incubated with the probe (10μM) for 10 min, then with ONOO ˉ Group E was pretreated with LPS (1 μg / mL) for 1 h, followed by incubation with the probe (10 μM) for 10 min. Group F was pretreated with SIN-1 (100 μM) for 1 h, followed by incubation with the probe (10 μM) for 10 min. Finally, confocal microscopy was performed on the cells in all six groups, and the results are shown below. Figure 13 As shown.

[0120] from Figure 13 It can be seen that this probe can detect endogenous and exogenous ONOO in zebrafish. ˉ Experiments have shown that this probe can be applied to ONOO in biological samples. ˉ Testing.

[0121] Example 10: The toxic effect of fluorescent probes on HeLa cells

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

[0123] like Figure 14 , 15 As shown, when the concentration reaches 10 μM, the toxic effects of the fluorescent probe on organisms are negligible.

[0124] Although the present invention has been described with reference to 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 all such modifications and variations are within the protection scope of the present invention.

Claims

1. A novel ONOOˉ fluorescent probe based on activation-trigger-anchoring, characterized in that, The chemical structural formula of the fluorescent probe is shown in (Ⅰ): In the formula, 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 group consisting of hydrogen atoms, straight-chain or branched alkyl groups, straight-chain or branched alkoxy groups, sulfonic acid groups, ester groups, and hydroxyl groups is independently selected; 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 They can be the same or different.

2. The novel ONOOˉ fluorescent probe as described in 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 All of them are 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 It is a hydrogen atom, R3 is -SO3H and R 18 It is -OCH3; Or 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 R1 is a hydrogen atom, R6 is an ethyl group, and R1 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 reaction includes the following steps: The first step is a reaction at 90°C in an oil bath for 16 hours, with a molar ratio of 7-hydroxycoumarin to thiomorpholine and paraformaldehyde of approximately 1:1:1.2; the second step is a reaction at room temperature for 24 hours, with a molar ratio of compound (III) to pinacol 4-bromomethylphenylboronic acid ester of 1:1.2; the third step is also a reaction at room temperature for 24 hours, with a molar ratio of compound (IV) to iodomethane of 1:

10.

5. A fluorescent probe composition for measuring, screening or detecting ONOOˉ, comprising the fluorescent probe of 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. The use of the fluorescent probe of claim 1 or 2 in the preparation of reagents for measuring, screening or detecting ONOOˉ.

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

9. A method for detecting the presence of ONOOˉ in a sample or for determining ONOOˉ in a sample, comprising the step of contacting the fluorescent probe of claim 1 or 2 with the sample, wherein the method is not a diagnostic or therapeutic method for a disease.

10. The method according to claim 9, wherein the sample is a chemical sample or a biological sample.

11. The application of the fluorescent probe according to claim 1 or 2, which is used for fluorescence imaging detection or photoacoustic imaging detection, wherein the application is not a method for diagnosing or treating a disease.

12. The application of the fluorescent probe according to claim 1 or 2 for intracellular mitochondrial targeted detection, wherein the application is not a diagnostic or therapeutic method for a disease.

13. The application of the fluorescent probe according to claim 1 or 2 for fluorescence imaging in cells, wherein the application is not a diagnostic or therapeutic method for a disease.

14. The application of the fluorescent probe according to claim 1 or 2 for fluorescence imaging in zebrafish, wherein the application is not a diagnostic or therapeutic method for a disease.

15. Use of the fluorescent probe of claim 1 or 2 in the preparation of a formulation for in situ fluorescence imaging detection of cancer or tumor, wherein the use is not a method for diagnosing or treating a disease.

16. Use of the fluorescent probe of claim 1 or 2 in the preparation of formulations for in situ photoacoustic imaging detection of cancer or tumors.

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