A chemiluminescent probe for detecting hydrogen peroxide and its preparation method and application

Abstract

The present invention relates to the technical field of chemical analysis and detection, and in particular to a chemiluminescent probe for detecting hydrogen peroxide, a preparation method and an application thereof. The probe has a structural formula as shown in formula (I). The compound as shown in formula (I) provided by the present invention has high sensitivity to hydrogen peroxide, has high sensitivity to hydrogen peroxide while targeting the endoplasmic reticulum, has a detection lower limit of 126nM for hydrogen peroxide, can detect hydrogen peroxide with a concentration of 126nM-98μM, and is proportional to its fluorescence intensity within the hydrogen peroxide concentration range of 126nM-98μM. The probe can provide an effective research tool for studying the content of hydrogen peroxide in the endoplasmic reticulum of cells, and can provide early warning for the field of clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a chemiluminescent probe for detecting hydrogen peroxide and a preparation method and application thereof. Background Art

[0002] Hydrogen peroxide is a well-known precursor of reactive oxygen and nitrogen species and an important product of oxygen metabolism. It plays an important role in many biological processes such as immune response and cell signal transduction. However, abnormal production of hydrogen peroxide can damage cellular biomolecules, which is closely related to many diseases. It is known that excessive hydrogen peroxide can cause oxidative stress, which will lead to a large number of plant physiological and chemical processes and various diseases (such as cancer and neurodegenerative diseases). Therefore, the development of highly sensitive and selective fluorescent probes for hydrogen peroxide detection and quantitative determination is very important for monitoring the in vivo production of hydrogen peroxide and elucidating its biological functions. The endoplasmic reticulum is the largest organelle in the cell. It is an important site for protein folding and decomposition in the cell and the main site of lipid synthesis. The endoplasmic reticulum often responds very sensitively to changes in the cellular environment. Therefore, it is very meaningful to develop a probe that can detect hydrogen peroxide on the endoplasmic reticulum.

[0003] At present, the main methods used to detect hydrogen peroxide include electrochemical method, colorimetric method, mass spectrometry, high performance liquid chromatography, etc., which generally have defects such as complex pre-treatment, destruction of cell tissue structure, and high cost. In recent years, fluorescent probe detection method has been favored by scientific researchers due to its advantages such as high sensitivity, low toxicity, and easy operation. At present, the method of using fluorescent small molecules to detect hydrogen peroxide is relatively mature. In 2019, Caixia Yin's team developed a near-infrared detector for detecting hydrogen peroxide. In 2021, Tianyu Liang's team successfully developed a dual-lock and key two-photon fluorescent probe that responds to hydrogen peroxide and viscosity, and applied it to cell imaging and inflammation treatment. However, the fluorescent probes currently used for hydrogen peroxide detection have many limitations, which generally include: (1) The photostability of fluorescent probes is poor and they are easily bleached under continuous light; (2) The selectivity for hydrogen peroxide is not high enough and the detection results are easily interfered by other ROS; (3) Some fluorescent probes cannot effectively avoid the interference of biological background fluorescence, resulting in poor detection accuracy; (4) The fluorescence quantum yield of the probe is low and the imaging effect is poor; (5) The synthesis route is relatively complicated, resulting in high cost and labor-intensive synthesis of the detection reagent.

[0004] The endoplasmic reticulum is a complex organelle in which hydrogen peroxide can be produced in different subregions. The content of hydrogen peroxide in the endoplasmic reticulum is usually low, so its detection requires highly sensitive technology. However, current technologies have certain challenges in detecting trace amounts of hydrogen peroxide in the endoplasmic reticulum. Conventional detection methods may not meet the requirements for sensitive detection of low-concentration hydrogen peroxide, thus affecting the accurate measurement of the content of hydrogen peroxide in the endoplasmic reticulum. Since the detection of hydrogen peroxide content in the endoplasmic reticulum is a challenging task. The existing technologies do have some limitations in this regard, including low positioning accuracy and poor sensitivity. Chemiluminescence, as a type of small molecule fluorescent probe, has also attracted much attention in recent years for its design and application, but there is currently no report on chemiluminescent probes that can target the detection of hydrogen peroxide in the endoplasmic reticulum of cells. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art, provide a chemiluminescent probe for detecting hydrogen peroxide and a preparation method thereof, and apply the chemiluminescent probe to the detection of hydrogen peroxide, so as to overcome the problems of low positioning accuracy and poor sensitivity in the prior art for detecting the content of hydrogen peroxide in the endoplasmic reticulum.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0007] In a first aspect, the present application first provides a chemiluminescent probe for detecting hydrogen peroxide, wherein the probe has a structural formula as shown in formula (I):

[0008] (Ⅰ)

[0009] The chemiluminescent probe of 1,2-dioxetane is one of the most popular fluorescent probes at present. It has the characteristics of not requiring exogenous light excitation, which makes it have the advantages of low background light, long fluorescence duration, and more convenient detection of substrates compared with traditional fluorescent probes. p-Toluenesulfonamide is a common endoplasmic reticulum targeting group, which is used in the construction of traditional fluorescent probe structures. This targeting group has been applied in many literatures, proving that it has high efficiency and specificity in endoplasmic reticulum targeting. Chemiluminescent probes and fluorescent probes are two different types of probes. So far, chemiluminescent probes targeting the endoplasmic reticulum have not been reported. Considering the necessity of real-time imaging of the endoplasmic reticulum in vivo without exogenous light excitation, the applicant applied the endoplasmic reticulum targeting group to the chemiluminescent probe for the first time and successfully constructed a compound probe as shown in formula (I). The probe is composed of an endoplasmic reticulum targeting group - p-toluenesulfonamide and a chemiluminescent shield - pinacol borate alcohol ester, which can respond to hydrogen peroxide and successfully detect the fluorescence of the probe in the endoplasmic reticulum of the cell.

[0010] The mechanism of action of the compound shown in formula (I) is as follows: after the borate structure is oxidized by hydrogen peroxide, 1,6 racemization occurs, the hydroxyl group is deprotonated, and the compound shown in formula (I) enters an unstable high-energy state, and then the molecule dissociates and chemiluminescence is turned on.

[0011] In a second aspect, the present application provides a method for preparing the above-mentioned chemiluminescent probe for detecting hydrogen peroxide, comprising the following steps:

[0012] (1) dissolving the compound represented by formula (II) in a solvent, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxybenzotriazolone, N-toluenesulfonylethylenediamine, and N,N-diisopropylethylamine to react, and after the reaction is completed, separating and purifying to obtain the compound represented by formula (III);

[0013] (2) dissolving the compound represented by formula (III) in a solvent, adding a base and 4-bromomethylphenylboronic acid pinacol ester, reacting in an inert gas atmosphere, and after the reaction is completed, separating and purifying to obtain the compound represented by formula (IV);

[0014] (3) dissolving the compound represented by formula (IV) in a solvent, adding methylene blue and introducing oxygen, and after the photoreaction is completed, separating and purifying to obtain the compound represented by formula (I);

[0015] (Ⅰ) (Ⅱ) (III) (IV)

[0016] The compound represented by formula (II) in the present invention is a disclosed compound, and the synthesis method thereof can be referred to in the literature: Green, O., Eilon, T., Hananya, N., Gutkin, S., Bauer, CR, & Shabat, D. (2017). Opening a Gateway for Chemiluminescence Cell Imaging: Distinctive Methodology for Design of Bright Chemiluminescent Dioxetane Probes. ACS Cent Sci, 3(4), 349-358.

[0017] In order to obtain more effective ER-targeted compounds, the applicant screened a series of reported ER-targeted group compounds. Two compounds synthesized during the screening process are listed below;

[0018] Compound (a):

[0019]

[0020] Compound (b):

[0021]

[0022] After a series of screening, the endoplasmic reticulum targeting group carried by the compound shown in formula (I) designed by the applicant, namely p-toluenesulfonamide, has higher endoplasmic reticulum targeting, higher sensitivity and better response performance compared with other targeting groups. Therefore, the compound shown in formula (I) was determined as the final compound.

[0023] Preferably, the solvent is one or more of dichloromethane, acetonitrile, toluene, tetrahydrofuran, and dimethylformamide.

[0024] Preferably, the molar ratio of the compound represented by formula (II), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxybenzotriazolone, N-toluenesulfonylethylenediamine and N,N-diisopropylethylamine in step (1) is 1:1.5-2.5:2.5-3.5:1:2.5-3.5.

[0025] It is further preferred that the molar ratio of the compound represented by formula (II), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxybenzotriazolone, N-toluenesulfonylethylenediamine and N,N-diisopropylethylamine in step (1) is 1:2:3:1:3.

[0026] Preferably, the molar ratio of the compound represented by formula (III), the base and 4-bromomethylphenylboronic acid pinacol ester in step (2) is 1:3-4:1-1.5.

[0027] More preferably, the molar ratio of the compound represented by formula (III), the base and 4-bromomethylphenylboronic acid pinacol ester in step (2) is 1:3:1.2.

[0028] Preferably, the base in step (2) is one or more of piperidine, triethylamine, potassium carbonate and sodium carbonate.

[0029] Preferably, the inert gas in step (3) is nitrogen.

[0030] Preferably, the molar ratio of the compound represented by formula (IV) to methylene blue in step (3) is 1:0.2-0.3.

[0031] More preferably, the molar ratio of the compound represented by formula (IV) to methylene blue in step (3) is 1:0.3.

[0032] More preferably, the reaction conditions in step (1) are room temperature reaction for 1-3 hours, the reaction in step (2) is for 3-6 hours, and the light irradiation reaction in step (3) is for 30-60 minutes.

[0033] Further preferably, the separation and purification steps in steps (1) and (2) are specifically as follows: extracting the reaction solution obtained after the reaction with ethyl acetate, concentrating the organic phase under reduced pressure, performing column chromatography purification using a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:1 as an eluent, collecting the eluate containing the target product, and concentrating under reduced pressure to obtain a purified reaction product; the separation and purification step in step (3) is specifically as follows: extracting the reaction solution obtained after the reaction with dichloromethane, concentrating the organic phase under reduced pressure, performing column chromatography purification using a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:1 as an eluent, collecting the eluate containing the target product, and concentrating under reduced pressure to obtain a purified reaction product.

[0034] In a third aspect, the present application provides the use of the above-mentioned chemiluminescent probe for detecting hydrogen peroxide or the chemiluminescent probe prepared by the preparation method in the detection of hydrogen peroxide.

[0035] Further preferably, the chemiluminescent probe is used in targeted detection of the content of hydrogen peroxide in the endoplasmic reticulum of cells.

[0036] The present invention has the following beneficial effects:

[0037] (1) The present invention applies the endoplasmic reticulum targeting group to the chemiluminescent probe for the first time, which not only gives the chemiluminescent probe the endoplasmic reticulum targeting property, but also can respond to hydrogen peroxide and stimulate 550nm fluorescence emission while targeting the endoplasmic reticulum;

[0038] (2) Since the chemiluminescent probe shown in formula (I) does not require exogenous light excitation, it has the advantages of low background interference and little light damage to biological samples;

[0039] (3) The compound represented by formula (I) is highly sensitive to hydrogen peroxide, and has high sensitivity to hydrogen peroxide while targeting the endoplasmic reticulum. The detection limit of hydrogen peroxide is 126 nM, and it can detect hydrogen peroxide at a concentration of 126 nM-98 μM. Its fluorescence intensity is proportional to the concentration of hydrogen peroxide in the range of 126 nM-98 μM.

[0040] (4) The compound represented by formula (I) proposed in the present invention can provide an effective research tool for studying the content of hydrogen peroxide in the endoplasmic reticulum of cells, and can provide early warning in the field of clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is the H NMR spectrum of the compound of formula (I) prepared in Example 1 of the present invention.

[0042] Figure 2 This is the NMR carbon spectrum of the compound shown in formula (I) prepared in Example 1 of the present invention.

[0043] Figure 3 This is a fluorescence emission spectrum of the compound of formula (I) prepared in Example 4 of the present invention, added to a PBS buffer solution containing hydrogen peroxide.

[0044] Figure 4 This is a fluorescence diagram of the selectivity results of the compound of formula (I) prepared in Example 5 of the present invention under PBS buffer (pH=7.4).

[0045] Figure 5 This is a fluorescence graph of the compound shown in formula (I) prepared in Example 6 of the present invention under different illumination times and pH conditions.

[0046] Figure 6 This is the MTT test result of the compound represented by formula (I) prepared in Example 7 of the present invention.

[0047] Figure 7 This is the co-localization fluorescence imaging result of the compound represented by formula (I) prepared in Example 8 of the present invention in MCF-7 cells.

[0048] Figure 8 This is the hydrogen peroxide detection result at the cellular level of the compound of formula (I) prepared in Example 9 of the present invention. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0050] The compounds represented by formula (I), (II), (III) and (IV) in the following examples are respectively as follows:

[0051] (Ⅰ) (Ⅱ) (III) (IV)

[0052] The compound represented by formula (II) is a disclosed compound. The synthesis and preparation method thereof can be found in the literature: Green, O., Eilon, T., Hananya, N., Gutkin, S., Bauer, CR, & Shabat, D. (2017). Opening a Gateway for Chemiluminescence Cell Imaging: Distinctive Methodology for Design of Bright Chemiluminescent Dioxetane Probes. ACS Cent Sci, 3(4), 349-358.

[0053] Example 1: Preparation of chemiluminescent probe for detecting hydrogen peroxide

[0054] (1) Preparation of the compound represented by formula (III)

[0055] The compound shown in formula (II) (90 mg, 0.264 mmol) was dissolved in 5 mL of dimethylformamide (DMF), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (101 mg, 0.528 mmol), N-hydroxybenzotriazolone (Hobt) (107 mg, 0.794 mmol), N-toluenesulfonylethylenediamine (56 mg, 0.264 mmol), and N,N-diisopropylethylamine (102 mg, 0.794 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 1 h. After the reaction was completed, the mixture was diluted with 20 mL of ethyl acetate, and washed with 20 mL of 1M HCl, 20 mL of saturated sodium bicarbonate, and 20 mL of saturated brine. The organic phase was concentrated, and the compound shown in formula (III) (43 mg, yield 30.38%) was obtained by column separation and purification under the condition of petroleum ether / ethyl acetate 1:1 as eluent, as a white solid.

[0056] 1H NMR (500MHz, DMSO) δ = 10.14 (s, 1H), 8.11 (s, 1H), 7.69 (s, 2H), 7.63 (s, 1H), 7.57 (s, 1H), 7.40 (s, 3H), 6.83 (s, 1H), 6.75 (s, 1H), 6. 59(s,1H),3.22(s,3H),3.20(s,2H),3.16(s,1H),2.81(s,2H),2.64(s,1H),2.36(s,3H),1.81(ddd,J=45.2,27.4,15.1,12H).13C NMR (126MHz, DMSO) δ = 166.26 (s), 143.33–143.17 (m), 137.83 (s), 134.85 (s), 134.72–134.56 (m), 131.59 (s),126.94–126.78(m),121.92(s),121.50(s),116.49(s),57.86(s),55.25(s),37.34(s),32.24(s).

[0057] (2) Preparation of the compound represented by formula (IV)

[0058] The compound shown in formula (III) (43 mg, 0.080 mmol) was dissolved in 3 mL of DMF, and K2CO3 (33 mg, 0.24 mmol) and 4-bromomethylphenylboronic acid pinacol ester (29 mg, 0.096 mmol) were added to the reaction solution. The reaction was protected by nitrogen during the entire reaction process and the reaction was carried out for 4 hours. The reaction solution was diluted with 15 mL of ethyl acetate and extracted with 10 mL of saturated sodium chloride. After the organic phase was concentrated, petroleum ether / ethyl acetate 1:1 was used as the eluent, and the compound shown in formula (IV) (40 mg, yield 66.48%) was obtained after column separation and purification as a white solid.

[0059] 1H NMR (500MHz, DMSO) δ = 8.16 (s, 1H), 7.76–7.63 (m, 6H), 7.55 (s, 1H), 7.40 (s, 4H), 6.88 (s, 1H), 6.83 (s, 1H), 6.62 (s, 1H) ),5.31(s,2H),3.22(s,2H),3.16(s,3H),3.14(s,1H),2.83(s,2H),2.36(s,3H),1.93–1.55(m,12H),1.29(s,11H).

[0060] (3) Preparation of the compound represented by formula (I)

[0061] The compound (IV) (40 mg, 0.053 mmol) was dissolved in 5 mL of dichloromethane (DCM), and methylene blue (5 mg, 0.015 mmol) was added and oxygen was introduced. The reaction was irradiated with light for 30 min. After the reaction was completed, it was diluted with 10 mL of DCM and washed three times with 10 mL of saturated sodium chloride. The organic phase was concentrated and separated and purified by column chromatography on silica gel with an eluent ratio of petroleum ether / ethyl acetate of 1:1. Compound (1) (30 mg, yield 72.12%) was obtained as a white solid. H NMR spectrum: Figure 1 , NMR carbon spectrum see Figure 2 .

[0062] 1H NMR (500MHz, DMSO) δ = 8.22 (s, 137H), 7.68 (dt, J = 22.2, 11.2, 1006H), 7.59 (s,119H),7.49(s,110H),7.39(d,J=7.9,592H),6.68(s,176H),5.33(s,26 0H),4.88–4.76(m,1H),3.22(s,337H),3.17(s,381H),3.10(s,178H),2.82 (s,324H),2.37(d,J=10.7,609H),2.11–1.40(m,1817H),1.27(s,2127H).

[0063] Example 2: Preparation of chemiluminescent probe for detecting hydrogen peroxide

[0064] The difference between this embodiment and embodiment 1 is that:

[0065] (1) Preparation of the compound represented by formula (III)

[0066] The compound represented by formula (II) (90 mg, 0.264 mmol) was dissolved in 5 mL of dimethylformamide (DMF), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (76 mg, 0.396 mmol), N-hydroxybenzotriazolone (Hobt) (89 mg, 0.66 mmol), N-toluenesulfonylethylenediamine (56 mg, 0.264 mmol), and N,N-diisopropylethylamine (85 mg, 0.66 mmol) were added to the reaction solution successively, and the compound represented by formula (III) (39 mg, yield 27.56%) was obtained by separation and purification as a white solid.

[0067] (2) Preparation of the compound represented by formula (IV)

[0068] The compound represented by formula (III) (43 mg, 0.080 mmol) was dissolved in 3 mL of DMF, K2CO3 (38.5 mg, 0.28 mmol) and 4-bromomethylphenylboronic acid pinacol ester (29 mg, 0.096 mmol) were added to the reaction solution, and after separation and purification, the compound represented by formula (IV) (37 mg, yield 61.49%) was obtained as a white solid.

[0069] (3) Preparation of the compound represented by formula (I)

[0070] The compound represented by formula (III) (0.10 g, 0.42 mmol), the compound represented by formula (IV) (0.13 g, 0.42 mmol) and piperidine (7.1 mg, 0.084 mmol) were added to 4 ml of ethanol solution to obtain 28 mg of the pure compound represented by formula (I) with a product yield of 67.32% as a white solid. The rest was the same as Example 1.

[0071] Example 3: Preparation of chemiluminescent probe for detecting hydrogen peroxide

[0072] The difference between this embodiment and embodiment 1 is that:

[0073] (1) Preparation of the compound represented by formula (III)

[0074] The compound represented by formula (II) (90 mg, 0.264 mmol) was dissolved in 5 mL of dimethylformamide (DMF), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (126 mg, 0.66 mmol), N-hydroxybenzotriazolone (Hobt) (124 mg, 0.924 mmol), N-toluenesulfonylethylenediamine (56 mg, 0.264 mmol), and N,N-diisopropylethylamine (118 mg, 0.924 mmol) were added to the reaction solution successively, and the compound represented by formula (III) (42 mg, yield was 29.68%) was obtained by separation and purification as a white solid.

[0075] (2) Preparation of the compound represented by formula (IV)

[0076] The compound represented by formula (III) (43 mg, 0.080 mmol) was dissolved in 3 mL of DMF, K2CO3 (44 mg, 0.32 mmol) and 4-bromomethylphenylboronic acid pinacol ester (29 mg, 0.096 mmol) were added to the reaction solution, and after separation and purification, the compound represented by formula (IV) (39 mg, yield 64.81%) was obtained as a white solid.

[0077] (3) Preparation of the compound represented by formula (I)

[0078] The compound represented by formula (III) (0.10 g, 0.42 mmol), the compound represented by formula (IV) (0.13 g, 0.42 mmol) and piperidine (7.1 mg, 0.084 mmol) were added to 4 ml of ethanol solution to obtain 30 mg of the pure compound represented by formula (I) with a product yield of 72.12% as a white solid. The rest was the same as Example 1.

[0079] Example 4: Emission spectrum of the compound represented by formula (I) (5 μM) in a PBS detection system (emission wavelength 550 nm.)

[0080] In phosphate buffer (PBS, 10mM, pH=7.4), after the compound represented by formula (I) (5μM) came into contact with hydrogen peroxide (40μM), a chemiluminescent signal appeared immediately; compared with the control group without H2O2, the compound represented by formula (I) detected obvious chemiluminescence at the maximum emission of 550nm, and the fluorescence intensity of the compound represented by formula (I) increased by nearly 40 times compared with the control group. The fluorescence spectrum is shown in Figure 3 .

[0081] Example 5: Fluorescence spectrum detection (emission wavelength 550 nm) of the selective results of the compound represented by formula (I) (5 μM) in a PBS detection system (pH 7.4).

[0082] We investigated the anti-interference ability of the compound shown in formula (I) against the fluorescence signals of common interfering substances in different organisms, including hydrogen peroxide, trivalent iron ions, calcium ions, potassium ions, divalent copper ions, magnesium ions, sodium ions, bromide ions, chloride ions, fluoride ions, iodide ions, phosphate ions, cysteine, glutamic acid, glutathione, hydrogen sulfide, potassium superoxide, hypochlorous acid, nitro group, and blank. The concentration of all substrates to be tested was 20 μM. The compound shown in formula (I) will not respond to interfering substances other than H2O2 under physiological conditions, indicating that the compound shown in formula (I) has a high selectivity for H2O2, and the anti-interference ability of the chemiluminescent signal can meet the imaging conditions at the cellular level. The fluorescence spectrum is shown in Figure 4 , where 1-20 are hydrogen peroxide, trivalent iron ion, calcium ion, potassium ion, divalent copper ion, magnesium ion, sodium ion, bromide ion, chloride ion, fluoride ion, iodine ion, phosphate ion, cysteine, glutamic acid, glutathione, hydrogen sulfide, potassium superoxide, hypochlorous acid, nitro group, and blank. The emission wavelength is 550nm.

[0083] Example 6: Stability determination of the compound represented by formula (I) (5 μM)

[0084] In order to explore the stability of the compound shown in formula (I), we conducted the photostability experiment of the probe and the chemiluminescence response of the compound shown in formula (I) under different pH conditions with the addition of H2O2 (20 μM). The compound shown in formula (I) can still maintain structural stability within 12 hours of illumination, and the chemiluminescence signal is enhanced only between pH 7-9, reaching the maximum value at pH 8. The results show that the photostability and optimal pH environment of the compound shown in formula (I) are sufficient to meet the detection requirements of the probe in the organism, and the fluorescence spectrum is shown in Figure 5 .

[0085] Example 7: MTT cell assay of the compound represented by formula (I).

[0086] We use the MTT method to detect the cytotoxicity of the compound shown in formula (I). The principle is that the mitochondria in living cells contain succinate dehydrogenase and cytochrome C. Thiazole blue (MTT) generates water-insoluble blue-purple formazan crystals under the action of these two, which are then deposited in the cells. Subsequently, DMSO is used to dissolve the blue-purple formazan crystals in the cells, and the absorbance value is detected at a wavelength of 550nm using an enzyme reader. At the same time, dead cells do not contain succinate dehydrogenase and cannot form blue-purple formazan with MTT. Therefore, the cell survival rate is proportional to the deposition of blue-purple formazan, and the larger the OD value, the higher the cell survival rate.

[0087] The detection steps are as follows: First, HELa cells are cultured in a 96-well cell culture plate at a cell seeding density of about 2×104 cells / well, and incubated in a 37°C cell thermostat containing 5% CO2 for 24 hours. Fetal bovine serum and appropriate amounts of antibodies (penicillin and streptomycin) are added to the culture medium. Then, different concentrations of the compound represented by formula (Ⅰ) (0-50 μM) are added and incubated for 24 hours. 50 μL MTT (1 mg / mL PBS buffer) is added to each well, and the incubation is continued at 37°C, 5% CO2 for 4 hours. The culture medium is removed, and 150 μL DMSO is added to each well. After shaking for 5 minutes, the absorbance is detected at 590 nm. The results of the MTT experiment are shown in Figure 6 .

[0088] Example 8: Cell co-localization imaging of the compound represented by formula (I).

[0089] For the endoplasmic reticulum co-localization imaging of the compound as shown in formula (I), we used MCF-7 cells. After incubating the MCF-7 cells for 24 hours, hydrogen peroxide was added to the cells, and the excess culture medium was rinsed with PBS buffer after incubation for 1 hour. The compound as shown in formula (I) was added to the cells and incubated for 1 hour. After the cells were washed three times with PBS, the commercial red dye for the endoplasmic reticulum, ER-Tracker Red, was then added to the cells and incubated for 15 minutes. The cells were washed again three times with PBS buffer, and the images were taken using a confocal microscope. The co-localization results were calculated by LAX S. The results show that the green fluorescence of the compound as shown in formula (I) is well matched with the red fluorescence of the commercial probe for the endoplasmic reticulum, and the co-localization coefficient is 0.93. This shows that the compound as shown in formula (I) can well target the endoplasmic reticulum and complete fluorescence imaging in living cells. The fluorescence spectrum is shown in Figure 7 .

[0090] Example 9: Detection of hydrogen peroxide at the cellular level by the compound represented by formula (I) (5 μM).

[0091] For hydrogen peroxide imaging at the cellular level of the compound as shown in formula (I), we used HeLa cells. First, the cells to be tested were washed three times with PBS buffer to wash away the excess culture fluid, and then DMEM culture medium was added thereto. In order to achieve a more obvious imaging effect, the cells were divided into two groups. After the two groups of cells were cultured with hydrogen peroxide (40 μM) and without hydrogen peroxide for 0.5 h, they were washed twice with PBS buffer, and the compound as shown in formula (I) (10 μM) was immediately added to the two groups of cells and incubated for 20 min. The cells were observed under the green channel using a confocal microscope. There was no obvious fluorescence display in the cells without the addition of exogenous hydrogen peroxide, while the cells with the addition of exogenous hydrogen peroxide showed strong green fluorescence. This shows that hydrogen peroxide has a decisive role in the chemiluminescence release of the compound as shown in formula (I), and the compound as shown in formula (I) can detect changes in hydrogen peroxide at the cellular level. The experimental results show that the compound can be used for hydrogen peroxide detection at the cellular level, and the fluorescence spectrum is shown in Figure 8 .

Claims

1. A chemiluminescent probe for detecting hydrogen peroxide, characterized in that: The probe has a structural formula as shown in formula (I), (Ⅰ) 2. A method for preparing the chemiluminescent probe for detecting hydrogen peroxide as claimed in claim 1, characterized in that: The following steps are involved: (1) dissolving the compound represented by formula (II) in a solvent, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxybenzotriazolone, N-toluenesulfonylethylenediamine, and N,N-diisopropylethylamine to react, and after the reaction is completed, separating and purifying to obtain the compound represented by formula (III); (2) dissolving the compound represented by formula (III) in a solvent, adding a base and 4-bromomethylphenylboronic acid pinacol ester, reacting in an inert gas atmosphere, and after the reaction is completed, separating and purifying to obtain the compound represented by formula (IV); (3) dissolving the compound represented by formula (IV) in a solvent, adding methylene blue and introducing oxygen, and after the photoreaction is completed, separating and purifying to obtain the compound represented by formula (I); (Ⅰ) (Ⅱ) (Ⅲ) 3. The preparation method according to claim 2, characterized in that: The solvent is one or more of dichloromethane, acetonitrile, toluene, tetrahydrofuran and dimethylformamide.

4. The preparation method according to claim 2 or 3, characterized in that: The molar ratio of the compound represented by formula (II), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxybenzotriazolone, N-toluenesulfonylethylenediamine and N,N-diisopropylethylamine in step (1) is 1:1.5-2.5:2.5-3.5:1:2.5-3.

5.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the compound represented by formula (III), the base and 4-bromomethylphenylboronic acid pinacol ester in the step (2) is 1:3-4:1-1.

5.

6. The preparation method according to claim 2 or 5, characterized in that: The base in step (2) is one or more of piperidine, triethylamine, potassium carbonate and sodium carbonate.

7. The preparation method according to claim 2, characterized in that: The inert gas in step (3) is nitrogen.

8. The preparation method according to claim 2 or 7, characterized in that: The molar ratio of the compound represented by formula (IV) and methylene blue in the step (3) is 1:0.2-0.

3.

9. Use of the chemiluminescent probe for detecting hydrogen peroxide according to claim 1 or the chemiluminescent probe prepared by the preparation method according to any one of claims 2 to 8 in the detection of hydrogen peroxide for non-diagnostic purposes.

10. The use according to claim 9, characterized in that The chemiluminescent probe is used in targeted detection of the content of hydrogen peroxide in the endoplasmic reticulum of cells.

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