CN111349071A

CN111349071A - For detecting ONOO-Xanthene derivative, and synthetic method and application thereof

Info

Publication number: CN111349071A
Application number: CN202010089405.2A
Authority: CN
Inventors: 尤进茂; 陆姣; 李赞; 谭江坤; 孙志伟; 常芮
Original assignee: Qufu Normal University
Current assignee: Qufu Normal University
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2020-06-30

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a method for detecting ONOO^‑The xanthene derivative and a synthetic method and application thereof. Using 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene as a fluorescent group, and using an activated C ═ C double bond as a reaction active group; the xanthene derivative is 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-benzylidene) -1,2,3, 4-tetrahydroxanthene. The invention is to ONOO^‑Has excellent selectivity, obvious fluorescent signal readout, sensitive response and good sensitivity to ONOO^‑The response time of the cell is within seconds, the detection limit is low and is 3.2nm/L which is far lower than that of the two substances in the cellFrequently present in amounts such that excess ONOO is found in a timely manner^‑And can be endogenous and exogenous to the living cells, ONOO^‑And (5) carrying out in-situ detection on the concentration.

Description

For detecting ONOO-Xanthene derivative, and synthetic method and application thereof

Technical Field

The invention relates to the technical field of fluorescent probes, in particular to a method for detecting ONOO^-The xanthene derivative and a synthetic method and application thereof.

Background

Peroxynitrous acid (ONOO)^-) Is a strong oxidant and nucleophile with short half-life, belonging to the reactive oxygen species (HROS). ONOO^-Is formed by nitric oxide free radical (NO) and superoxide radical (O)₂ ^-) Generated in the form of free diffusion, with a diffusion rate of (-1 × 1010M-1s-1) ONOO^-Mainly produced in macrophages, endothelial cells, platelets, leukocytes and neurons and have an important role in the field of biochemistry.

ONOO^-Can directly oxidize/nitrify proteins, esters or nucleic acids in cells, destroy cell structures and functions, and finally cause cell death. Alternatively, the cells may utilize the produced ONOO^-To defend against microbial invasion. Due to direct detection of ONOO^-Has great difficulty in concentration, and thus related to ONOO in the biological field^-The many beneficial functions involved remain controversial or are not clarified. Thus the development of a device capable of monitoring the ONOO specifically^-Horizontal fluorescent probes have become a challenge for a wide range of researchers. Over the past few years, researchers have designed and developed a series of fluorescence-enhanced based ONOO^-Probes, some also have been used in comparative biological studies. However, these fluorescent probes are useful in the quantitative detection of ONOO^-The process is susceptible to interference from various factors, such as different background probe concentrations, different cellular microenvironments, and the like. Therefore, the design and development can detect the ONOO in situ^-Concentration of fluorescent probes remains a challenge.

Disclosure of Invention

Aiming at the defects in the prior art, the methodThe technical problem to be solved by the invention is to provide a new cell capable of carrying out endogenous and exogenous ONOO on living cells^-The concentration is detected in situ, the detection sensitivity is high, the detection limit is low, the harm to cells is small, the influence of external conditions in the biological cell sample imaging is small, the result is accurate, the method also has the advantages of high fluorescence quantum yield, obvious phenomenon, easiness in observation and high accuracy, the equipment is convenient to operate, the implementability is high, the method is suitable for biological cell imaging, and the method is used for detecting the ONOO^-The xanthene derivative and a synthetic method and application thereof.

The technical scheme adopted by the invention for realizing the purpose is as follows: for detecting ONOO^-The xanthene derivative of (1) has 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene as a fluorescent group and activated C ═ C double bond as a reactive group; the xanthene derivative is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-benzylidene) -1,2,3, 4-tetrahydroxanthene, and the chemical structural formula of the xanthene derivative is as follows:

the invention also includes a method for detecting ONOO^-The method for synthesizing the xanthene derivative comprises the following steps:

(1) putting concentrated sulfuric acid into a container, and then adding cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid reacts at the temperature of 70-90 ℃, after the reaction is finished, the solution is cooled to the room temperature, poured into ice water and stirred evenly, and then perchloric acid is dripped into the mixed liquid to separate out solid, thus obtaining an intermediate product F₃₇₆，F₃₇₆Is 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene;

(2) intermediate product F obtained in step (1)₃₇₆Dissolving in anhydrous ethanol, adding 4-methoxybenzaldehyde, stirring and refluxing at 60-90 deg.C, cooling to room temperature, spin drying the filtrate, and purifying with chromatographic column to obtain final product F₄₉₄，F₄₉₄Is 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-benzylidene) -1,2,3, 4-tetrahydroxanthene.

Further, the specific process of the synthesis method is as follows:

in the step (1), the first step of the method,

the molar ratio of 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to cyclohexanone is preferably 1:2,

the reaction time is preferably 2 hours,

the loading sequence is preferably: adding concentrated sulfuric acid, stirring in ice-water bath for 5 min, adding cyclohexanone, stirring at 0 deg.C for 5 min, adding 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid, and heating to 70-90 deg.C.

Further, the specific process of the synthesis method is as follows:

in the step (2), the step (c),

intermediate product F₃₇₆The molar ratio to 4-methoxybenzaldehyde is preferably 1:2,

the stirring reflux time at (60-90) deg.C is preferably 2 h.

The invention also includes a method for detecting ONOO^-The xanthene derivatives can be used as molecular probes for detecting ONOO^-Total content of, detecting exogenous ONOO in cells^-Concentration or cellular endogenous ONOO^-Concentration and cell ONOO^-And (4) fluorescence imaging.

Further, the cells have endogenous ONOO^-The concentration is detected by adding LPS + PMA drug to induce and make cells self-generate ONOO^-(ii) a Exogenous ONOO to said cells^-The concentration is detected by externally adding ONOO^-And is finally taken up by the cells; the fluorescence imaging is to the ONOO in the cell^-And (6) imaging.

Further, F is established₄₉₄Molecular probes and ONOO^-The steps of the linear working curve are as follows:

(1) preparing a PBS buffer salt solution with the pH value of 7.40 and the concentration of 10 mM; the ONOO with the concentration of 1mM is prepared^-H of (A) to (B)₂O standard solution and F at a concentration of 1mM₄₉₄DMSO solutions of molecular probes;

(2) providing 0 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L,80 μ L, 90 μ L and 100 μ L, respectively, and providing a concentration of 1mM ONOO^-In a total of 11 parts, 10uL of 1mM F was added to each of the standard solutions₄₉₄The molecular probe solution was added with 1mL of 10mM PBS buffer solution, andthen adding 1mL of ethanol, and uniformly mixing;

(3) measuring fluorescence intensity with a fluorescence spectrophotometer to obtain fluorescence intensity ratio, preferably detecting ONOO^-The excitation wavelength of the fluorescence intensity is 425nm, and the reaction time is 30 min;

(4) by ONOO^-The concentration of (A) is the abscissa and the fluorescence intensity ratio is the ordinate, obtaining the probe molecule pair ONOO^-Linear equation of concentration and fluorescence intensity ratio.

Further, F is established₄₉₄Molecular probes and ONOO in the cellular environment^-The steps of the linear working curve are as follows:

(1) preparing a PBS buffer salt solution with the pH of 7.40 and the concentration of 10 mM; HSO was prepared at a concentration of 1mM₃ ^-And ClO^-Standard solution, F at a concentration of 1mM₄₉₄Standard solutions of molecular probes in DMSO;

(2) respectively collecting 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L and 10 μ L of ONOO with concentration of 1mM^-The standard solution is co-cultured for 2h at 37 ℃, then the cells are placed in a buffer solution of PBS (phosphate buffer solution), the cells are placed in a confocal environment for observation and imaging, the fluorescence intensities of different color light channels are collected, and then the fluorescence intensity ratio is carried out, so that the exogenous ONOO related to the cells is obtained^-A linear equation of the ratio of the concentration of (a) to the fluorescence intensity; obtaining exogenously added ONOO^-Cell map and intracellular fluorescence intensity ratio to ONOO^-Linear equation of concentration.

Further, using F₄₉₄Molecular probe for detecting endogenous ONOO of living cells^-The concentration steps of (a) are preferably:

(1) selecting RAW cells, culturing the recovered cells in a medium containing 10% bovine embryo serum, 1% double antibody, 89% DMEM, 5% CO at 37 deg.C₂Culturing for 24h in the environment to obtain cells with good growth vigor for later use;

(2) culturing RAW cells in culture medium, and culturing 4 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 10⁷～ 9×10⁷Culturing for 24 hours per mL;

(3) taking 2 groups of culture medium, adding LPS and PMA in the first group, and adding only10 μ L of F at a concentration of 0.5mM₄₉₄DMSO solutions of molecular probes as controls;

(4) to the second group, 100. mu.L of LPS solution at a concentration of 1. mu.g/ml, 100. mu.L of PMA solution at a concentration of 1. mu.g/ml were added to stimulate cells to produce endogenous ONOO^-Then, 10. mu.L of F with a concentration of 0.5mM was added₄₉₄Culturing the DMSO solution, observing and imaging under confocal condition, collecting fluorescence intensity of different color light channels, and performing fluorescence intensity ratio according to intracellular ONOO^-Linear equation of the ratio of concentration to fluorescence intensity of endogenous ONOO^-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the endogenous ONOO of the cells^-The concentration of (c).

Further, using F₄₉₄Molecular probe for detecting exogenous ONOO of living cell^-The concentration steps of (a) are preferably:

(2) culturing RAW cells in culture medium, and culturing 5 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 10⁷～ 9×10⁷Culturing for 24 hours per mL;

(3) collecting 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L and 10 μ L of ONOO with concentration of 1mM ^-10. mu.L of 1mM F was added to each of the standard solutions₄₉₄Co-culturing the probe in DMSO solution at 37 ℃ for 2h, placing the cells in PBS buffer solution, observing and imaging under confocal conditions, collecting fluorescence intensities of different color light channels, and performing fluorescence intensity ratio according to exogenous ONOO of the cells^-The linear equation of the ratio of the concentration to the fluorescence intensity of the exogenous ONOO^-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the exogenous ONOO of the cells^-The concentration of (c).

The invention is used for detecting ONOO^-The xanthene derivative and the synthesis method and the application thereof have the beneficial effects that:

(1) f of the invention₄₉₄The ratiometric fluorescent labeling reagent uses 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene as a fluorescent group and activated C ═ C as a reaction site to enable a probe pair to be ONOO^-Has excellent selectivity and has obvious fluorescent signal read-out.

(2) F of the invention₄₉₄Ratiometric fluorescent labeling reagents are sensitive to ONOO^-Within seconds.

(3) F of the invention₄₉₄The detection limit of the ratiometric fluorescent labeling reagents is low, compared to commercial fluorescent labeling reagents, the pair ONOO proposed by us^-The detection limit of the two substances is respectively 3.2nm/L which is far lower than the normal existing content of the two substances in the cells, so that excessive ONOO can be found in time^-。

(4) F of the invention₄₉₄The ratio-type fluorescent labeling reagent uses the ratio of the fluorescence intensities as a reference rather than the direct fluorescence intensity, and thus greatly reduces the influence from the external environment and instruments, compared with the switch-type fluorescent labeling reagent.

(5) The invention is applied to the detection of living cells, and further promotes the exploration of the function of the biological micromolecules in the biological field.

(6) The change of the ultraviolet color after the reaction is obvious, so that the ultraviolet color can be detected by naked eyes, and the method is convenient and quick.

(7) Due to its long wavelength in the infrared part, ONOO^-And is less harmful to biological cells without excessive damage.

Drawings

FIG. 1 is F in example 1₄₉₄Synthetic schemes for molecular probes;

FIG. 2 shows F in example 1₃₇₆H-NMR of the intermediate product;

FIG. 3 shows F in example 1₃₇₆C-NMR of the intermediate product;

FIG. 4 shows F in example 1₄₉₄Mass spectra of the molecular probe;

FIG. 5 shows F in example 1₄₉₄H-NMR of the molecular probe;

FIG. 6 shows F in example 1₄₉₄C-NMR of the molecular probe;

FIG. 7 shows F in example 2₄₉₄Molecular probe pair ONOO^-The fluorescence titration spectrum of (a);

FIG. 8 is a linear equation for the peroxynitrite titration for the probe of example 2;

FIG. 9 is F₄₉₄Histograms of the selectivity of molecular probes for various interfering examples;

FIG. 10 exogenous addition of ONOO in example 6^-Subsequent confocal images of the cells;

FIG. 11 is the ratio of intracellular fluorescence intensity to ONOO in example 6^-A linear equation of concentration;

FIG. 12 is the endogenous assay ONOO in example 7^-(ii) a map of the relevant cells;

FIG. 13 is the endogenous ONOO of example 7^-A histogram of the confocal fluorescence intensity ratio of (a);

FIG. 14 is a bar graph of cell viability.

Detailed Description

The invention is further explained in detail with reference to the drawings and the specific embodiments;

example 1:

for detecting ONOO^-The xanthene derivative of (1) is characterized in that 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene is used as a fluorescent group, and an activated C ═ C double bond is used as a reactive group; the xanthene derivative is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-benzylidene) -1,2,3, 4-tetrahydroxanthene, and the chemical structural formula of the xanthene derivative is as follows:

(1) adding concentrated sulfuric acid into flask, stirring for 5 min in ice water bath, adding cyclohexanone, stirring for 5 min at 0 deg.C, and adding 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid, of which 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid,Reacting cyclohexanone at the molar ratio of 1:2 (70-90) DEG C for 2h, after the reaction is finished, cooling the solution to room temperature, pouring the solution into ice water, stirring the solution uniformly, then dropwise adding perchloric acid into the mixed liquid, and separating out a solid to obtain an intermediate product 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene, which is named as F₃₇₆Intermediate product F₃₇₆The H-NMR is shown in FIG. 2, and the C-NMR is shown in FIG. 3.

(2) Dissolving the intermediate product 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene obtained in the step (1) by using absolute ethanol, adding 4-methoxybenzaldehyde, wherein the molar ratio of the intermediate product to the 4-methoxybenzaldehyde is 1:2, stirring and refluxing at the temperature of (60-90) DEG for 2 hours, cooling to room temperature, spin-drying filtrate, and obtaining a final product 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-benzylidene) -1,2,3, 4-tetrahydroxanthene through a chromatographic column method, wherein the final product is named as F₄₉₄The synthetic route is shown in figure 1, F₄₉₄The mass spectrum is shown in FIG. 4, the H-NMR is shown in FIG. 5, and the C-NMR is shown in FIG. 6.

The invention also includes a method for detecting ONOO^-The xanthene derivatives can be used as molecular probes for detecting ONOO^-Total content of, detecting exogenous ONOO in cells^-Concentration or cellular endogenous ONOO^-Concentration and cell ONOO^-And (4) fluorescence imaging. Endogenous ONOO of cells^-The concentration is detected by adding LPS + PMA drug to induce and make cells self-generate ONOO^-(ii) a Exogenous to cells ONOO^-The concentration is detected by externally adding ONOO^-And is finally taken up by the cells; fluorescence imaging of the ONOO in cells^-And (6) imaging.

Example 2:

F₄₉₄molecular probe pair ONOO^-Responsive fluorescence spectral property of

(1) Preparing a PBS buffer salt solution with the pH value of 7.40 and the concentration of 10 mM; the ONOO with the concentration of 1mM is prepared^-H of (A) to (B)₂A standard solution of O, a DMSO solution of a probe at a concentration of 1 mM;

(2) 10uL of the probe solution was added to a 5mL Ep tube, 1mL of 10mM PBS buffer solution was added, and then 1mL of ethanol was added and mixed well. Is divided intoRespectively collecting 0 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L,80 μ L, 90 μ L and 100 μ L of ONOO with concentration of 1mM^-A total of 11 parts of the solution (2) were added to each Ep tube.

(3) After reacting for 30min, testing fluorescence intensity by a fluorescence spectrophotometer to obtain fluorescence intensity ratio, and detecting ONOO^-The excitation wavelength of the fluorescence intensity is 425 nm; to obtain F₄₉₄The fluorescence titration spectra of the molecular probes are shown in FIG. 7.

(4) By ONOO^-The concentration of (A) is the abscissa and the fluorescence intensity ratio is the ordinate, obtaining the molecular probe pair ONOO^-The linear equation of the ratio of concentration to fluorescence intensity is shown in FIG. 8.

Example 3:

probe pair ONOO^-Response time of

(2) 10uL of the probe solution was added to a 5mL Ep tube, 1mL of 10mM PBS buffer solution was added, and then 1mL of ethanol was added and mixed well. Respectively collecting 0 μ L, 50 μ L and 100 μ L of ONOO with concentration of 1mM^-A total of 3 parts of the solution of (2) were added to each Ep tube.

(3) After reacting for 30min, testing fluorescence intensity by a fluorescence spectrophotometer to obtain fluorescence intensity ratio, and detecting ONOO^-The excitation wavelength of the fluorescence intensity was 425 nm.

Example 4:

establishing F₄₉₄Molecular probes and ONOO^-Linear working curve of (d):

(1) preparing a PBS buffer salt solution with the pH value of 7.40 and the concentration of 10 mM; the ONOO with the concentration of 1mM is prepared^-H of (A) to (B)₂O standard solution, F at a concentration of 1mM₄₉₄DMSO solutions of molecular probes;

(2) providing 0 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L,80 μ L, 90 μ L and 100 μ L, respectively, and providing a concentration of 1mM ONOO^-In a total of 11 parts, 10uL of each of the standard solutions of (1) were added to the mixture1mM of F₄₉₄Adding 1mL of PBS buffer salt solution with the concentration of 10mM into the probe solution, then adding 1mL of ethanol, and uniformly mixing;

(4) by ONOO^-The concentration of (A) is the abscissa and the fluorescence intensity ratio is the ordinate, obtaining the molecular probe pair ONOO^-Linear equation of concentration and fluorescence intensity ratio.

Example 5:

establishing F₄₉₄Molecular probes and ONOO in the cellular environment^-Linear working curve of (d):

Example 6:

detection of exogenous ONOO in living cells^-The concentration of (a):

(1) the ONOO with the concentration of 1mM is prepared^-A standard solution of water, a DMSO standard solution of a probe at a concentration of 1mM, an LPS solution at a concentration of 1. mu.g/ml, and a PMA solution at a concentration of 1. mu.g/ml.

(2) Cell culture: selecting RAW cells, culturing the recovered cells in a medium containing 10% bovine embryo serum, 1% double antibody, 89% DMEM, 5% CO at 37 deg.C₂In an environment of 24h, obtaining cells with good growth vigor for later use.

(3) Culturing live RAW cells in culture medium, and culturing 5 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 10⁷～ 9×10⁷Incubating for 24h at a concentration of 1mM probe in DMSO of 10. mu.L, respectively 0. mu.L, 2.5. mu.L, 5. mu.L, 7.5. mu.L and 10. mu.L of ONOO at a concentration of 1mM^-The standard solution is co-cultured for 2h at 37 ℃, then the cells are placed in a buffer solution of PBS (phosphate buffer solution), the cells are placed in a confocal environment for observation and imaging, the fluorescence intensities of different color light channels are collected, and then the fluorescence intensity ratio is carried out, so that the exogenous ONOO related to the cells is obtained^-A linear equation of the ratio of the concentration of (a) to the fluorescence intensity; FIG. 10 is the exogenous addition of ONOO^-FIG. 11 is a graph of the ratio of the fluorescence intensity in cells to the ONOO^-Linear equation of concentration.

Example 7:

detection of endogenous ONOO in living cells^-Concentration of (2)

(1) Culturing live RAW cells in culture medium, and culturing 4 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 10⁷～ 9×10⁷Culturing for 24 hours per mL;

(2) 2 groups of medium were used, the first group was supplemented with LPS and PMA, and 10. mu.L of F at a concentration of 0.5mM₄₉₄DMSO solution as control. To the second group, 100. mu.L of LPS solution at a concentration of 1. mu.g/ml, 100. mu.L of PMA solution at a concentration of 1. mu.g/ml were added to stimulate cells to produce endogenous ONOO^-Then, 10. mu.L of F with a concentration of 0.5mM was added₄₉₄Culturing the DMSO solution, observing and imaging in confocal condition, collecting fluorescence intensity of different color light channels, and performing fluorescence intensity ratio, FIG. 12 shows endogenous detection ONOO^-FIG. 13 is a cellular map of intracellular ONOO^-The concentration of LPS and PMA in the graph corresponds to 0 on the abscissa of FIG. 13, the concentration of the endogenous ONOO in the cell is obtained by a linear equation of the control in the graph in which the test group without LPS and PMA corresponds to 1 on the abscissa of FIG. 13^-The concentration of (c).

Probe pair ONOO^-Selective experiment of

(1) OH, O at a concentration of 1mM₂ ^-,H₂O₂,HClO,GSH,Cys,Hcy,Tempo,NO₃ ^-,NO₂ ^-,ONOO^-1And O₂In an aqueous solution of (1), Cu is prepared at a concentration of 30mM²⁺,Cu⁺And Fe³⁺An aqueous solution of (a).

(2) 10uL of 1mM probe solution was added to a 5mL Ep tube, 1mL of 10mM PBS buffer solution was added, and then 1mL of ethanol was added and mixed well. 10uL of the prepared solution 10uL was added to each Ep tube.

(3) After reacting for 30min, testing fluorescence intensity by a fluorescence spectrophotometer to obtain fluorescence intensity ratio, and detecting ONOO^-The excitation wavelength of the fluorescence intensity is 425 nm;

(4) using the species as the abscissa and the fluorescence intensity ratio as the ordinate to obtain the probe molecule pair ONOO^-Selective histogram, see fig. 9.

Cell viability assay

Cell viability experiments primary validation F₄₉₄The effect of toxicity on cell life. Adding F with different concentrations into cell culture solution₄₉₄Probes (0M,2.5M,5M,7.5M,10M and 12.5M) at 37 ℃ 5% CO₂Was cultured in the incubator of (1) for 24 hours, and 25. mu.L of 4-methylthiazolyltetrazole MTT (5mg mL) was added^-1) The cells were cultured for 4 hours in a cell culture medium, and the cell viability was evaluated by the MTT cuvette method. Will not add F₄₉₄Cell survival of (2) was set to 100%, with different concentrations of F₄₉₄The data associated with the added experimental groups are plotted in a relative histogram 14, in which the concentrations 0M,2.5M,5M,7.5M,10M and 12.5M correspond in turn to 1,2,3,4, 5 and 6 on the abscissa of FIG. 14.

The macrophages in the above examples were mouse kidney macrophages, product number CP-M187, available from Wuhan Poussel Life technologies Ltd, HPLC-MS analysis was performed using an Agilent 1100 Mass Spectrometry System (Agilent, USA) equipped with a degasser, a quaternary pump, and an autosampler, and HPLC separation was performed using a Hypersil GOLD C18 column (2.1mm × 50 mm,1.8 μ M i.d., Agilent, USA)The light detection is carried out by using Hitachi F-4600 fluorescence spectrometer for ONOO^-The detection excitation wavelength is 500nm, the excitation and emission slit widths are both 10.0nm, the voltage is 400V, and the scanning speed is 2400 nm/min. Fluorescence imaging observations were performed by Olympus Fluo View FV1000 (Japan) confocal, with 40-fold objective. The separation and purification of the compound are realized by adopting a thin-layer chromatography silica gel column, wherein the filler is 300-400 meshes.

The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. All equivalent changes or modifications made in accordance with the spirit of the present disclosure are intended to be covered by the scope of the present disclosure.

Claims

1. For detecting ONOO^-The xanthene derivative of (1) is characterized in that 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene is used as a fluorescent group, and an activated C ═ C double bond is used as a reactive group; the xanthene derivative is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-benzylidene) -1,2,3, 4-tetrahydroxanthene, and the chemical structural formula of the xanthene derivative is as follows:

2. a method for detecting ONOO according to claim 1^-The method for synthesizing a xanthene derivative is characterized by comprising the following steps:

(1) putting concentrated sulfuric acid into a container, and then adding cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid reacts at the temperature of 70-90 ℃, after the reaction is finished, the solution is cooled to the room temperature, poured into ice water and stirred evenly, and then dropped into the mixed liquidAdding perchloric acid, separating out solid to obtain intermediate product F₃₇₆，F₃₇₆Is 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene;

3. The method for detecting ONOO according to claim 2^-The method for synthesizing a xanthene derivative according to (1), which comprises: the specific process of the synthesis method comprises the following steps:

in the step (1), the first step of the method,

the reaction time is preferably 2 hours,

4. The method for detecting ONOO according to claim 2^-The method for synthesizing a xanthene derivative according to (1), which comprises: the specific process of the synthesis method comprises the following steps:

in the step (2), the step (c),

the stirring reflux time at (60-90) deg.C is preferably 2 h.

5. For detecting ONOO^-The use of a xanthene derivative having the structure of claim 1 or prepared by the method of any one of claims 2 to 4 as a molecular probe for the detection of ONOO^-Total content of, detecting exogenous ONOO in cells^-Concentration or cellular endogenous ONOO^-Concentration and cell ONOO^-And (4) fluorescence imaging.

6. The method for detecting ONOO according to claim 5^-The xanthene derivative of (1) is characterized in that: endogenous ONOO of the cell^-The concentration is detected by adding LPS + PMA drug to induce and make cells self-generate ONOO^-(ii) a Exogenous ONOO to said cells^-The concentration is detected by externally adding ONOO^-And is finally taken up by the cells; the fluorescence imaging is to the ONOO in the cell^-And (6) imaging.

7. The method for detecting ONOO according to claim 5^-The xanthene derivative of (1) is characterized in that: establishing F₄₉₄Molecular probes and ONOO^-The steps of the linear working curve are as follows:

(2) providing 0 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L,80 μ L, 90 μ L and 100 μ L, respectively, and providing a concentration of 1mM ONOO^-In a total of 11 parts, 10uL of 1mM F was added to each of the standard solutions₄₉₄Adding 1mL of PBS buffer salt solution with the concentration of 10mM into the probe solution, then adding 1mL of ethanol, and uniformly mixing;

8. The method for detecting ONOO according to claim 5^-Oxygen oxide ofThe application of the anthracene derivative is characterized in that: establishing F₄₉₄Molecular probes and ONOO in the cellular environment^-The steps of the linear working curve are as follows:

9. The method for detecting ONOO according to claim 5^-The xanthene derivative of (1) is characterized in that: using F₄₉₄Molecular probe for detecting endogenous ONOO of living cells^-The concentration steps of (a) are preferably:

(2) culturing RAW cells in culture medium, and culturing 4 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 10⁷～9×10⁷Culturing for 24 hours per mL;

(3) 2 groups of medium were used, the first group was supplemented with LPS and PMA, and 10. mu.L of F at a concentration of 0.5mM₄₉₄DMSO solutions of molecular probes as controls;

(4) to the second group, 100. mu.L of LPS solution at a concentration of 1. mu.g/ml and 100. mu.L of PMA solution at a concentration of 1. mu.g/ml were added to stimulate cells to produce endogenous ONOO^-Then, 10. mu.L of F with a concentration of 0.5mM was added₄₉₄DMSO solubilization of molecular probesCulturing in liquid, observing and imaging in confocal condition, collecting fluorescence intensities of different color light channels, and performing fluorescence intensity ratio according to intracellular ONOO^-Linear equation of the ratio of concentration to fluorescence intensity of endogenous ONOO^-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the endogenous ONOO of the cells^-The concentration of (c).

10. The method for detecting ONOO according to claim 5^-The xanthene derivative of (1) is characterized in that: using F₄₉₄Molecular probe for detecting exogenous ONOO of living cell^-The concentration steps of (a) are preferably:

(2) culturing RAW cells in culture medium, and culturing 5 groups respectively, wherein the inoculum size in each group of culture medium is 2 × 10⁷～9×10⁷Culturing for 24 hours per mL;

(3) collecting 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L and 10 μ L of ONOO with concentration of 1mM^-10. mu.L of 1mM F was added to each of the standard solutions₄₉₄Co-culturing a DMSO solution of a molecular probe at 37 ℃ for 2h, placing the cells in a buffer solution of PBS, observing and imaging under confocal conditions, collecting the fluorescence intensities of different color light channels to perform fluorescence intensity ratio, and performing cell exogenous ONOO^-The linear equation of the ratio of the concentration to the fluorescence intensity of the exogenous ONOO^-To the ratio of the concentration to the fluorescence intensity of the cells, thereby obtaining the exogenous ONOO of the cells^-The concentration of (c).