A fluorescent probe based on aggregation-induced emission of ROS and its preparation method

Abstract

The present invention relates to a ROS-responsive fluorescent probe based on aggregation-induced emission, a compound represented by the following chemical formula: #imgabs0# wherein: R1 and R2 are each independently a hydrogen atom. At the same time, a preparation method and uses of the compound are also disclosed. The fluorescent probe is a fluorescent probe with aggregation-induced emission characteristics, has strong fluorescence efficiency, can achieve highly sensitive detection of ROS, has more accurate detection results, and can be co-cultured with cells, making the application more convenient.

Description

Technical Field

[0001] The invention relates to a fluorescent probe, in particular to a ROS-responsive fluorescent probe based on aggregation-induced luminescence, and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are some of the redox products produced by organisms during mitochondrial oxidative metabolism, including superoxide, peroxide, hydroxyl radical, singlet oxygen, peroxyl radical, alkoxyl, lipid peroxide, peroxynitrite, hypochlorous acid and ozone. Under physiological conditions, low concentrations of reactive oxygen species can regulate physiological functions such as cell signal transduction, cell proliferation and differentiation, energy conversion, and immune response that maintain cell homeostasis. However, excessive ROS produced by pathological stimuli such as bacterial infection will damage nucleic acids, proteins and enzymes, leading to diseases such as sepsis, acute hepatitis, Alzheimer's disease, and tumors.

[0003] Traditional detection methods for ROS include electrochemical methods, chemiluminescence, spectrophotometry, chromatography, and fluorescence spectroscopy. Among them, fluorescence spectroscopy has attracted the attention of scientists due to its advantages of easy operation, low cost, high sensitivity, high selectivity, and non-invasiveness, and has been widely used in the fields of biosensing and biodetection. In recent years, dihydroethylamine (DHE) and mitochondria-targeted mitoSOX have been developed for the detection of ROS in cells and mitochondria. However, since DHE can form ethidium through nonspecific redox reactions and form 2-hydroxyethidium through superoxidation, the fluorescence spectra overlap, and simple fluorescence quantitative detection cannot be performed using confocal microscopy or other fluorescence-based microanalysis methods. Dichlorodihydrofluorescein diacetate (DCFH-DA) is a cell-permeable ester that can be hydrolyzed into dihydroxy-DCFH in cells and is also a fluorescent probe commonly used for intracellular ROS detection. However, since DCFH does not react directly with ROS, it is oxidized to DCF in cells. DCF free radicals can be oxidized by transition metals, cytochrome C, heme, etc. to generate peroxides and superoxides, which can easily lead to false positive results. Summary of the invention

[0004] In view of the existing deficiencies, the present invention provides a ROS-responsive fluorescent probe based on aggregation-induced emission, and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention to solve the technical problem is: a fluorescent probe based on ROS response of aggregation-induced emission, a compound shown in the following chemical formula:

[0006] Wherein: R1 and R2 are each independently a hydrogen atom.

[0007] A method for preparing a fluorescent probe based on aggregation-induced emission of ROS, the steps are as follows:

[0008] S1, dispersing 4-bromo-o-phenylenediamine and ethyl pyruvate in solvent 1 and stirring, and purifying to obtain compound 1 after the reaction is completed;

[0009] S2, dispersing compound 1 in solvent 2, adding allyl bromide and potassium carbonate, and purifying to obtain compound 2 after the reaction is completed;

[0010] S3, compound 2 is dispersed in glacial acetic acid, compound 3 and a catalytic amount of concentrated sulfuric acid are added, and after the reaction is completed, purification is performed to obtain intermediate compound 4;

[0011] S4, dispersing compound 4, compound 5 and bipyraclostrobin in solvent 3, adding potassium carbonate and catalyst (Ph3P)4Pd, and purifying to obtain compound 6 after the reaction is completed;

[0012] S5, dispersing compound 6 in solvent 4, adding iodoethane, and purifying to obtain the final product after the reaction is completed;

[0013] in,

[0014] Compound 1 is a compound of the following structural formula: ;

[0015] Compound 2 is a compound of the following structural formula: ;

[0016] Compound 3 is a compound of the following structural formula: ;

[0017] Compound 4 is a compound of the following structural formula: ;

[0018] Compound 5 is a compound of the following structural formula: ;

[0019] Compound 6 is a compound of the following structural formula: ;

[0020] Wherein: R1 and R2 are each independently a hydrogen atom;

[0021] Solvent 1 is ethanol, solvent 2 is acetone, solvent 3 is deionized water and 1,4-dioxane, and solvent 4 is tetrahydrofuran.

[0022] Preferably, the molar ratio of 4-bromo-o-phenylenediamine to ethyl pyruvate is 1:1.1-1:1.5, the reaction temperature of step S1 is room temperature, and the reaction time is 6-12 hours.

[0023] Preferably, the molar ratio of the compound 1 to allyl bromide is 1:1-1:1.5, the reaction temperature of step S2 is 50° C., and the reaction time is 8 to 12 hours.

[0024] Preferably, the molar ratio of compound 2 to compound 3 is 1:1.1-1:1.5, the reaction temperature of step S3 is 80° C., and the reaction time is 12-24 hours.

[0025] Preferably, the molar ratio of compound 4, compound 5 and biboric acid pinacol ester is 1:1:1.2-1:1:1.5, the reaction temperature of step S4 is 80° C., and the reaction time is 48-72 hours.

[0026] Preferably, the molar ratio of compound 6 to ethyl iodide is 1:1.5-1:2.0, the reaction temperature of step S5 is room temperature, and the reaction time is 12-24 hours.

[0027] A use of a fluorescent probe based on aggregation-induced luminescence (AIE) responsive to ROS, using the fluorescent probe as described above to detect the intracellular ROS content.

[0028] The beneficial effects of the present invention are as follows: the fluorescent probe of the invention is a ROS-responsive fluorescent probe based on aggregation-induced emission of a quinoxalinone skeleton. The fluorescent probe based on aggregation-induced emission (AIE) has the advantages of large Stokes shift, high luminescence intensity, strong resistance to photobleaching, no random flickering, good biocompatibility, etc., which well solves the problem that traditional fluorescent probes are prone to photoquenching and photobleaching. At the same time, the fluorescent probe can oxidize the thioether in the molecule into sulfone and sulfoxide under the action of ROS, resulting in a blue shift in the fluorescence emission wavelength, thereby realizing high-sensitivity detection of ROS. After the formation of aggregates, the fluorescence efficiency is enhanced, the detection result is more accurate, and the problems of fluorescence spectrum overlap and false positives generated in traditional ROS detection are solved. In addition, the fluorescent probe can be co-cultured with cells, and is more convenient to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 2 is the fluorescence spectrum of the fluorescent probe (QuinoNS) of the embodiment of the present invention before and after ROS response (2 is before ROS response, 1 is after response);

[0030] Figure 2 This is a study on the uptake of QuinoNS by RAW264.7 cells in the present invention;

[0031] Figure 3 This is a study on the toxicity of QuinoNS to RAW264.7 cells in an embodiment of the present invention;

[0032] Figure 4This is the nuclear magnetic resonance hydrogen spectrum characterization of Example Compound 6 of the present invention; DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the purpose, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments for a clear and complete description. Obviously, the embodiments described are partial embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0034] A fluorescent probe based on aggregation-induced emission of ROS response and its preparation method and application, the fluorescent probe has a compound shown in the following chemical formula:

[0035] Wherein: R1 and R2 are each independently a hydrogen atom.

[0036] The preparation process is to use 4-bromo-o-phenylenediamine as the starting material and then cyclize it with ethyl pyruvate, and then construct a fluorescent probe with aggregation-induced emission characteristics through nucleophilic substitution, condensation, Suzuki coupling, and ethylation reaction. The reaction route of the preparation method is as follows: ;

[0037] S1, dispersing 4-bromo-o-phenylenediamine and ethyl pyruvate in solvent 1 and stirring, wherein ethanol is selected as solvent 1, and purifying after the reaction to obtain compound 1, wherein the molar ratio of 4-bromo-o-phenylenediamine and ethyl pyruvate is 1:1.1-1:1.5, the reaction temperature is room temperature, i.e., 25° C. usually referred to in chemical experiments, and the reaction time is 6-12 hours; the obtained compound 1 forms a quinoxalinone skeleton, and its structural formula is: In the example, the molar ratio of 4-bromo-o-phenylenediamine to ethyl pyruvate is 1:1.3, and the reaction time is 10 hours to prepare compound 1;

[0038] S2, dispersing the compound 1 obtained in the above step in solvent 2, which is acetone, and then adding allyl bromide and potassium carbonate, and purifying after the reaction to obtain compound 2; at this time, the molar ratio of compound 1 to allyl bromide is 1:1-1:1.5, the reaction temperature is 50°C, and the reaction time is 8 to 12 hours;

[0039] Compound 2 is a compound of the following structural formula: In the embodiment, the molar ratio of compound 1, allyl bromide and potassium carbonate is 1:1.2:1.5, and the reaction time is 11 hours;

[0040] S3, compound 2 is dispersed in glacial acetic acid, compound 3 and a catalytic amount of concentrated sulfuric acid are added, that is, the added concentrated sulfuric acid acts as a catalyst, and its molar concentration is 18 mol / L at this time, and the amount added is preferably 2-4 drops. After the reaction is completed, purification is performed to obtain an intermediate compound 4; the molar ratio of compound 2 to compound 3 is 1:1.1-1:1.5, the reaction temperature is 80°C, and the reaction time is 12-24 hours;

[0041] Compound 3 is a compound of the following structural formula: ;

[0042] Compound 4 is a compound of the following structural formula: ;

[0043] In the example, 3 drops of concentrated sulfuric acid were added, the molar ratio of compound 2 to compound 3 was 1:1.2, and the reaction time was 23 hours;

[0044] S4, dispersing compound 4, compound 5 and bipyraclostrobin in solvent 3, wherein solvent 3 is deionized water and 1,4-dioxane, adding potassium carbonate and catalyst (Ph3P)4Pd, and purifying after the reaction to obtain compound 6; the molar ratio of compound 4, compound 5 and bipyraclostrobin is 1:1:1.2-1:1:1.5, the reaction temperature is 80°C, and the reaction time is 48-72 hours;

[0045] Compound 5 is a compound of the following structural formula: ;

[0046] Compound 6 is a compound of the following structural formula: ;

[0047] In the embodiment, the molar ratio of compound 4, compound 5, biboric acid pinacol ester and potassium carbonate is 1:1:1.3:1.5, the amount of catalyst (Ph3P)4Pd added is 0.03-0.05g, and a Suzuki reaction system is formed, and the reaction time is 70 hours;

[0048] S5, dispersing compound 6 in solvent 4, which is tetrahydrofuran, adding ethyl iodide, and purifying after the reaction to obtain the final product, i.e. The molar ratio of compound 6 to iodine is 1:1.5-1:2.0, the reaction temperature of step S5 is room temperature, the reaction time is 12-24 hours, and the molar ratio of compound 6 to iodine is 1:1.8 in the embodiment;

[0049] Meanwhile, in the aforementioned structural formula: R1 and R2 are independently hydrogen atoms. In the embodiment, R1 and R2 are both hydrogen atoms. Stirring and dispersion are performed in the reaction of each step. The reaction temperature can be controlled by a water bath. Purification is separated and purified by a silica gel column. The amount of solvent in each step can be selected as 50 ml, 50 ml of ethanol, 50 ml of acetone, 50 ml of glacial acetic acid, 25 ml of deionized water and 1,4-dioxane are selected to form 50 ml of solvent, and 50 ml of tetrahydrofuran is selected. In step S1, 10 g and 7.54 g of 4-bromo-o-phenylenediamine and ethyl pyruvate are weighed respectively. In step S2, all or part of the amount of compound 1 prepared in step S1 is weighed for reaction. The amount of allyl bromide is weighed accordingly according to the amount of compound 1 and the molar ratio between them. In other steps, the amount of each compound is weighed accordingly.

[0050] The fluorescent probe prepared in this way is a ROS-responsive fluorescent probe based on the aggregation-induced luminescence of the quinoxaline skeleton. Its properties are exactly opposite to those of porphyrin photosensitizers. After forming aggregates in the aqueous phase, the fluorescence efficiency will be significantly enhanced. At the same time, the fluorescent probe can oxidize the thioether in the molecule into sulfone and sulfoxide under the action of ROS, resulting in a blue shift in the fluorescence emission wavelength, thereby achieving high-sensitivity detection of ROS, and more accurate detection results. It solves the problems of fluorescence spectrum overlap and false positives generated in traditional ROS detection, and can be used to detect the intracellular ROS content. During the cell culture process, it can be directly added to the cell culture medium and co-cultured with the cells, which is convenient for detecting the intracellular ROS level and more convenient for application.

[0051] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A fluorescent probe based on ROS response based on aggregation-induced emission, characterized in that: The compound shown in the following chemical formula: Wherein: R1 and R2 are each independently a hydrogen atom.

2. A method for preparing a fluorescent probe based on aggregation-induced emission of ROS, characterized in that: Here are the steps: S1, dispersing 4-bromo-o-phenylenediamine and ethyl pyruvate in solvent 1 and stirring, and purifying to obtain compound 1 after the reaction is completed; S2, dispersing compound 1 in solvent 2, adding allyl bromide and potassium carbonate, and purifying to obtain compound 2 after the reaction is completed; S3, compound 2 is dispersed in glacial acetic acid, compound 3 and a catalytic amount of concentrated sulfuric acid are added, and after the reaction is completed, purification is performed to obtain intermediate compound 4; S4, dispersing compound 4, compound 5 and bipyraclostrobin in solvent 3, adding potassium carbonate and catalyst (Ph3P)4Pd, and purifying to obtain compound 6 after the reaction is completed; S5, dispersing compound 6 in solvent 4, adding iodoethane, and purifying to obtain the final product after the reaction is completed; in, Compound 1 is a compound with the following structural formula: ; Compound 2 is a compound with the following structural formula: ; Compound 3 is a compound with the following structural formula: ; Compound 4 is a compound with the following structural formula: ; Compound 5 is a compound with the following structural formula: ; Compound 6 is a compound with the following structural formula: ; Wherein: R1 and R2 are each independently a hydrogen atom; Solvent 1 is ethanol, solvent 2 is acetone, solvent 3 is deionized water and 1,4-dioxane, and solvent 4 is tetrahydrofuran.

3. The method for preparing a fluorescent probe based on ROS response based on aggregation-induced emission according to claim 2, characterized in that: The molar ratio of 4-bromo-o-phenylenediamine to ethyl pyruvate is 1:1.1-1:1.5, the reaction temperature of step S1 is room temperature, and the reaction time is 6-12 hours.

4. The method for preparing a fluorescent probe based on ROS response based on aggregation-induced emission according to claim 1, characterized in that: The molar ratio of the compound 1 to allyl bromide is 1:1-1:1.5, the reaction temperature of step S2 is 50° C., and the reaction time is 8 to 12 hours.

5. The method for preparing a fluorescent probe based on ROS response based on aggregation-induced emission according to claim 1, characterized in that: The molar ratio of the compound 2 to the compound 3 is 1:1.1-1:1.5, the reaction temperature of step S3 is 80° C., and the reaction time is 12-24 hours.

6. The method for preparing a fluorescent probe based on ROS response based on aggregation-induced emission according to claim 1, characterized in that: The molar ratio of compound 4, compound 5 and biboric acid pinacol ester is 1:1:1.2-1:1:1.5, the reaction temperature of step S4 is 80° C., and the reaction time is 48-72 hours.

7. The method for preparing a fluorescent probe based on ROS response based on aggregation-induced emission according to claim 1, characterized in that: The molar ratio of the compound 6 to ethyl iodide is 1:1.5-1:2.0, the reaction temperature of step S5 is room temperature, and the reaction time is 12-24 hours.

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