Hydrogen peroxide probe as well as preparation method and application thereof

Abstract

The invention relates to a hydrogen peroxide probe and a preparation method and application thereof, the hydrogen peroxide probe connects pyridinium to a difluoro boron indole electron acceptor, can induce fluorescence quenching by distorting intramolecular charge transfer, generates weak fluorescence when the concentration of hydrogen peroxide in mitochondria is low, and can detect the mitochondria. According to the present invention, the hydrogen peroxide probe is used for detecting the hydrogen peroxide concentration in the mitochondria, the continuous fluorescence response is generated when the hydrogen peroxide concentration in the mitochondria is increased, the qualitative and quantitative analysis of the hydrogen peroxide level in the mitochondria is achieved, and the hydrogen peroxide probe can target the increased lipid droplet migration after the oxidative stress is caused by the increase of the hydrogen peroxide concentration so as to well detect the oxidative stress process; the method has important significance for exploring related damage of cell oxidative stress.

Description

Technical Field

[0001] The invention relates to the field of fluorescent probes, and in particular to a hydrogen peroxide probe and a preparation method and application thereof. Background Art

[0002] Hydrogen peroxide, also known as hydrogen peroxide, is an important oxidant that plays a key role in biomedicine and other fields. It not only plays an important role in cell metabolism, but also participates in many pathological processes, such as inflammation and oxidative stress. Therefore, it is particularly important to develop fluorescent probes that can monitor changes in intracellular hydrogen peroxide concentration in real time.

[0003] At the same time, oxidative stress caused by excessive production of reactive oxygen species (ROS) in mitochondria, such as hydrogen peroxide, will increase the number of lipid droplets to store excess lipids, which will further affect cell metabolism and function.

[0004] Therefore, it is necessary to design a hydrogen peroxide probe that can continuously respond to intracellular mitochondrial ROS and lipid droplets to detect the concentration of hydrogen peroxide in cells and the progression of cellular oxidative stress. Summary of the invention

[0005] One object of the present invention is to provide a hydrogen peroxide probe that can continuously respond to changes in hydrogen peroxide concentration in mitochondria of cells and can target the migration of lipid droplets produced after oxidative stress, thereby achieving the purpose of detecting hydrogen peroxide concentration and oxidative stress process in cells.

[0006] The present invention is achieved through the following technical solutions:

[0007] A hydrogen peroxide probe, characterized in that it has a structural formula shown in Formula I:

[0008]

[0009] In Formula I, R1 is selected from C1 to C 16 Chain alkyl, polyethylene glycol group, C1~C 16 Alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols; R2 is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, substituted or unsubstituted phenyl, hydroxyl, amino, carboxyl, formate, sulfonic acid and salts thereof, methoxy; n=0-2.

[0010] In the present technical solution, a hydrogen peroxide probe is constructed by connecting pyridinium to a hemicyanine intermediate, and pyridinium twisted intramolecular charge transfer (TICT) can be used to induce fluorescence quenching, so that the hydrogen peroxide probe produces very weak fluorescence when labeling mitochondria in cells.

[0011] In this technical solution, when hydrogen peroxide is produced in the cell, the hydrogen peroxide probe will react with the hydrogen peroxide, and the borate of the hydrogen peroxide probe will be oxidized and cleaved by the hydrogen peroxide, specifically releasing a compound that can be detected as fluorescent, and the fluorescence intensity increases with the increase of the concentration of hydrogen peroxide, and within a certain range of hydrogen peroxide concentration, the fluorescence intensity is substantially proportional to the hydrogen peroxide, thereby achieving quantitative analysis of hydrogen peroxide in cell mitochondria. Taking the hydrogen peroxide probe HPBCy5-1 in Example 1 as an example, LDBCy5-3 is obtained after reacting with hydrogen peroxide, and its reaction path is:

[0012]

[0013] In this technical solution, the hydrogen peroxide probe can not only label mitochondria and fluorescently respond to hydrogen peroxide produced by mitochondria, but also the product of the probe and hydrogen peroxide can specifically stain lipid droplets and target the migration of lipid droplets, thereby being able to detect the production of lipid droplets caused by oxidative stress caused by excessive hydrogen peroxide concentration in mitochondria, thereby combining the concentration change of hydrogen peroxide to more intuitively and accurately detect the progress of oxidative stress.

[0014] In this technical solution, the group R1 can affect the hydrophilicity and hydrophobicity of the probe. In order to better label mitochondria, the group R1 is selected from C1 to C 16 Chain alkyl, polyethylene glycol group, C1~C 16 Alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols.

[0015] In some preferred embodiments, R1 is selected from C1-C4 chain alkyl, polyethylene glycol group, C1-C4 alkyl carboxylic acid and active ester, C1-C3 alkyl sulfonic acid and salt thereof, C1-C4 alcohol. More preferably, R1 is selected from C1-C4 chain alkyl, polyethylene glycol group, C1-C4 alcohol.

[0016] In some embodiments, the group R1 is a chain alkyl group, which can be a straight chain alkyl group or a branched chain alkyl group. The number of carbon atoms in the chain alkyl group is preferably C1 to C 16 , more preferably C1~C 12 , and more preferably C1 to C4. In one or more embodiments, the chain alkyl group is methyl, ethyl, propyl, isopropyl, butyl or tert-butyl.

[0017] In some embodiments, the group R1 is a polyethylene glycol group, and the degree of polymerization of the polyethylene glycol group is preferably 1-10, and more preferably 1-5.

[0018] In some embodiments, the group R1 is an alkyl carboxylic acid and an active ester, and the alkyl carboxylic acid and the active ester can be acetic acid, propionic acid or butyric acid and their corresponding methyl ester and ethyl ester.

[0019] In some embodiments, the group R1 is an alkyl sulfonic acid and a salt thereof, and the alkyl sulfonic acid and a salt thereof may be ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid and their corresponding ammonium salts, sodium salts and potassium salts.

[0020] In some embodiments, the group R1 is an alcohol, preferably, the group R1 is methanol, ethanol or propanol.

[0021] In the present technical solution, the R2 group is used to adjust the photophysical properties of the hydrogen peroxide probe. Preferably, the R2 group is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, substituted or unsubstituted phenyl, hydroxyl, amino, carboxyl, formate, sulfonic acid and its salt, methoxy.

[0022] In some preferred embodiments, R2 is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, methoxy. Further preferably, R2 is selected from hydrogen, halogen, methyl. In one or more preferred embodiments, R2 is selected from hydrogen, chlorine, bromine or iodine.

[0023] In the present technical scheme, the hydrogen peroxide probe can label mitochondria, and utilize pyridinium to distort the intramolecular charge transfer to induce fluorescence quenching, thereby generating weak fluorescence when the hydrogen peroxide concentration in the mitochondria is low, and generating a continuous fluorescence response when the hydrogen peroxide concentration in the mitochondria increases, thereby achieving qualitative and quantitative analysis of the hydrogen peroxide level in the mitochondria; not only that, when the hydrogen peroxide concentration increases and causes oxidative stress, the hydrogen peroxide probe can also target the increased migration of lipid droplets, thereby better detecting the progress of oxidative stress, which is of great significance for exploring the related damage of cellular oxidative stress.

[0024] In some preferred embodiments, the hydrogen peroxide probe is selected from the following compounds:

[0025]

[0026] The present invention also provides a method for preparing a hydrogen peroxide probe, which is used to prepare any of the above-mentioned hydrogen peroxide probes. The preparation method specifically comprises the following steps:

[0027] The compound represented by formula II is mixed with the compound represented by formula III to obtain an intermediate represented by formula IV;

[0028] The intermediate represented by formula IV is reacted with 4-bromomethylphenylboronic acid pinacol ester to generate the hydrogen peroxide probe represented by formula I;

[0029] Formula II: Formula III:

[0030] Formula IV:

[0031] In formula II and formula IV, R2 is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, substituted or unsubstituted phenyl, hydroxyl, amino, carboxyl, formate, sulfonic acid and its salts, methoxy; in formula III and formula IV, R1 is selected from C1 to C 16 Chain alkyl, polyethylene glycol group, C1~C 16 Alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols; n=0-2.

[0032] In the technical scheme, the compound shown in formula II is a difluoroborindolenine electron acceptor connected to pyridinium, and the compound shown in formula III is a fluoroborindole merocyanine covering far infrared to near infrared. Among them, the fluoroborinole merocyanine can be synthesized in an existing manner, for example, by the process disclosed in patent CN116284089A, or purchased commercially.

[0033] In some preferred embodiments, after being dissolved in the first solvent, the first base is added for condensation, and the reaction system is reacted at 40 to 70°C to obtain the intermediate. In one or more preferred embodiments, the first solvent can be anhydrous dichloromethane, toluene, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, benzene, n-butanol, isopropanol, ethanol, methanol, chlorobenzene, xylene, mesitylene. The first base can be N,N-diisopropylethylamine, piperidine, triethylamine, pyridine, acetate, carbonate, bicarbonate, potassium tert-butoxide. The reaction mixture is stirred at 40 to 70°C for a certain period of time to obtain the intermediate, preferably, the reaction temperature is 50 to 60°C.

[0034] In the technical scheme, the intermediate is further reacted with 4-bromomethylphenylboronic acid pinacol ester to obtain a hydrogen peroxide probe. In some preferred embodiments, the intermediate and 4-bromomethylphenylboronic acid pinacol ester are dissolved in a second solvent and reacted at 100 to 120° C. for 1 to 3 hours to obtain the hydrogen peroxide probe. In one or more embodiments, the second solvent can be toluene, xylene, dimethyl sulfoxide, N,N-dimethylformamide. In one or more embodiments, the reaction temperature is 110° C.

[0035] Furthermore, the preparation method of the compound represented by formula II comprises the following steps:

[0036] After dissolving 2,3,3-trimethyl-5-bromo-3H-indole, pyridine-4-boric acid, a second base and a catalyst in a third solvent, the mixture is reacted at 90 to 120° C. under an inert atmosphere to obtain a pyridinium derivative, and a compound represented by formula II is prepared based on the pyridinium derivative.

[0037] In the technical scheme, 2,3,3-trimethyl-5-bromo-3H-indole and pyridine-4-boric acid are reacted in a third solvent to obtain a pyridine derivative. Among them, the second base can be K2CO3, CsCO3, Na2CO3, TiOH, NaOCH2CH3; the catalyst can be Pd(PPh3)4, Pd(dppf)Cl2. The third solvent can be toluene, a mixed solution of isopropanol and water, a mixed solution of ethanol and water, a mixed solution of isopropanol and water, a mixed solution of ethylene glycol dimethyl ether and water, tetrahydrofuran, toluene. In some preferred embodiments, the third solvent is a mixed solvent composed of toluene, isopropanol and water, so that the solvents are mutually soluble, thereby dissolving the second base, such as potassium carbonate, and converting it into a uniform phase, further improving the reaction yield and speed.

[0038] The present invention also provides an application of a hydrogen peroxide probe. Specifically, the hydrogen peroxide probe is used to mark mitochondria, generates a continuous fluorescence response to hydrogen peroxide in the mitochondria, and the fluorescence intensity is positively correlated with the concentration of hydrogen peroxide.

[0039] Furthermore, the hydrogen peroxide probe can target the migration of lipid droplets in cells after reacting with hydrogen peroxide.

[0040] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0041] 1. The hydrogen peroxide probe provided by the present invention can label mitochondria, and uses pyridinium to distort the intramolecular charge transfer to induce fluorescence quenching, producing weak fluorescence when the hydrogen peroxide concentration in the mitochondria is low, and producing continuous fluorescence response when the hydrogen peroxide concentration in the mitochondria increases, thereby achieving qualitative and quantitative analysis of the hydrogen peroxide level in the mitochondria;

[0042] 2. The hydrogen peroxide probe provided by the present invention can target the increased migration of lipid droplets after the increase in hydrogen peroxide concentration leads to oxidative stress, thereby better detecting the progress of oxidative stress, which is of great significance for exploring the related damage of cell oxidative stress; 3. The preparation process of the present invention is simple, the reaction conditions are mild, and the synthesis path is short, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0044] Figure 1 The HPLC spectrum of the hydrogen peroxide probes HPBCy5-1, LDBCy5-3, and the reaction product of the hydrogen peroxide probe HPBCy5-1 and hydrogen peroxide in the specific embodiment of the present invention is shown;

[0045] Figure 2The following are shown in a specific embodiment of the present invention: (a) a mass spectrum of the reaction product of hydrogen peroxide probe HPBCy5-1 and hydrogen peroxide, (b) a mass spectrum of hydrogen peroxide probe HPBCy5-1;

[0046] Figure 3 The figures show the fluorescence response time of the hydrogen peroxide probes HPBCy5-1 (a) and HPBCy5-2 (b) in the reaction with hydrogen peroxide at different concentrations in a specific embodiment of the present invention;

[0047] Figure 4 The graph shows the fluorescence intensity changes of hydrogen peroxide probes HPBCy5-1 (a) and HPBCy5-2 (b) after incubation with hydrogen peroxide of different concentrations for 1 hour in a specific embodiment of the present invention;

[0048] Figure 5 The selectivity of HPBCy5-1 (a) and HPBCy5-2 (b) for hydrogen peroxide in a specific embodiment of the present invention is shown;

[0049] Figure 6 The fluorescence imaging of mitochondria labeled with the probe HPBCy5-1 in a specific embodiment of the present invention is shown, wherein Hoechst is a commercial cell nucleus stain, Mito-Tracker Green is a commercial mitochondrial stain, the first row is group A, and the second row is group B;

[0050] Figure 7 The fluorescence imaging of the probe HPBCy5-1 targeting lipid droplet migration after adding H2O2 in a specific embodiment of the present invention is shown, wherein Bodipy 493 / 503 It is a commercially available stain for lipid droplets;

[0051] Figure 8 It shows the fluorescence imaging of lipid droplets labeled by the probe HPBCy5-1 after stimulating cells with different concentrations of PMA in a specific embodiment of the present invention;

[0052] Fig. 9 The fluorescence imaging of lipid droplets labeled with the probe HPBCy5-1 after stimulating cells with PMA, oleic acid, and PMA in a specific embodiment of the present invention is shown;

[0053] Fig.10 The experimental results of using the probe HPBCy5-2 in an APAP-induced liver injury animal model in a specific embodiment of the present invention are shown, wherein (a) is in vivo and tissue imaging, HE staining and Oil Red O staining of tissues, (b) is a bar graph of the control group and each experimental group corresponding to (a), and (c) is a bar graph of transaminase ALT and AST in the control group and each experimental group. DETAILED DESCRIPTION

[0054] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0055] The term "connection" used in the present invention, unless otherwise specified, may be directly connected or indirectly connected via other groups. All raw materials of the present invention are not particularly limited in their sources, and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art. All raw materials of the present invention are not particularly limited in their purity, and the present invention preferably adopts analytical grade or conventional purity requirements in the field of fluorescent probes. All raw materials of the present invention, their brands and abbreviations are conventional brands and abbreviations in the field, and each brand and abbreviation is clear and definite in the field of its related use. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the brands, abbreviations and corresponding uses.

[0056] The present invention has no particular limitation on the expression of the substituents, and all expressions familiar to those skilled in the art are adopted. Based on common sense, those skilled in the art can correctly understand the meanings according to the expressions.

[0057] 1. Preparation of Hydrogen Peroxide Probe

[0058] [Example 1]

[0059]

[0060] 1) Preparation of pyridinium derivatives

[0061] 2,3,3-Trimethyl-5-bromo-3H-indole (2.4 g, 10 mmol), pyridine-4-boronic acid (1.55 g, 13 mmol), K2CO3 (6.3 g, 36 mmol) and Pd(PPh3)4 (578 mg, 0.5 mmol) were dissolved in a mixed solvent (24 mL of toluene: 12 mL of isopropanol: 6 mL of water) and degassed under argon for 5 minutes. The reaction mixture was heated at 100°C in argon overnight. After cooling, the reaction mixture was partitioned between CH2Cl2 (100 mL) and water (50 mL). The aqueous layer was extracted with CH2Cl2 (2×40 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography to give the pyridinium derivative 10 (1.93 g, 85%).

[0062] 1H NMR (400MHz, CDCl3) δ8.69–8.63(m,2H),7.67–7.58(m,2H),7.56–7.51(m,3H),2.32(s,3H),1.37(s,6H).

[0063] 13 C NMR (101MHz, CDCl3) δ189.47,154.70,150.19,148.52,146.69,135.18,126.85,121.65,120.38,120.01,53.90,23.12,15.60.

[0064] HRMS[M+H]+m / z calcd.for[C16H17N2]+:237.1386found:237.1389.

[0065] 2) Preparation of difluoroborondolenine electron acceptor

[0066]

[0067] Under argon atmosphere, BF3·Et2O (0.8 mL, 6.5 mmol) was added to a solution of pyridinium derivative 10 (1.18 g, 5 mmol) in 8 mL of acetic anhydride. After stirring at 120°C for 4 hours, the solvent was removed using a rotary evaporator, and the residue was purified by silica gel column chromatography to obtain difluoroborondolenine electron acceptor 11 (277 mg, 17%).

[0068] HRMS[M+H]+m / z calcd.for[C18H18BF2N2O]+:327.1475found:327.1479.

[0069] 3) Preparation of hydrogen peroxide probe HPBCy5-1

[0070]

[0071] Dissolve difluoroborindolenine electron acceptor 11 (75.2 mg, 0.2 mmol) and semicyanine intermediate 12 (156.3 mg, 0.3 mmol) in 3 mL toluene solution, and then add acetic acid (25 μL) and piperidine (50 μL). The reaction mixture is stirred at 100°C for 3 hours. The obtained residue is purified by silica gel column chromatography to obtain intermediate 13 (29.8 mg, 21%). Among them, semicyanine intermediate 12 can be prepared by the method disclosed in patent CN116284089A.

[0072] 1H NMR(400MHz, CDCl3)δ8.66(d,J=5.7Hz,2H),7.80–7.67(m,2H),7.63(d,J=8.1Hz,1H),7.57–7.49(m,3H) ,7.35(t,J=13.1Hz,1H),7.20(t,J=7.7Hz,2H),6.95(t,J=7.4Hz,1H),6.80(d,J=7.9Hz,1H),6.19(dd,J= 13.7,11.9Hz,1H),5.99(d,J=14.4Hz,1H),5.70–5.65(m,1H),5.59(d,J=12.5Hz,1H),3.88(t,J=6.3Hz,2 H),3.71(t,J=6.2Hz,2H),3.68–3.56(m,10H),3.58–3.50(m,3H),3.37(s,3H),1.61(s,6H),1.51(s,6H).

[0073] 13 C NMR (101MHz, CDCl3) δ179.46,172.85,161.89,150.25,148.05,146.15,14 4.78,144.04,141.69,139.15,134.65,127.86,127.73,122.76,121.71,1 21.48,121.16,120.37,117.68,115.81,107.53,97.34,91.54,71.93,71. 11,70.68,70.62,70.53,67.33,59.05,49.07,46.61,42.82,28.35,25.43.

[0074] HRMS[M+H]+m / z calcd.for[C41H49BF2N3O5]+:712.3728found:712.3728.

[0075]

[0076] Intermediate 13 (21.3 mg, 0.03 mmol) and 4-bromomethylphenylboronic acid pinacol ester (17.8 mg, 0.06 mmol) were dissolved in toluene (0.3 mL). The reaction mixture was stirred at 110 ° C for 1 hour. After cooling to room temperature, the crude product was precipitated in 10 mL of methyl tert-butyl ether / n-hexane (1:1, v / v) and kept at 4 ° C overnight. The precipitate was then centrifuged and dried in vacuo to obtain the hydrogen peroxide probe HPBCy5-1 (27.5 mg, 91%).

[0077] 1 H NMR(400MHz, CDCl3) δ9.36(d,J=6.4Hz,2H),8.21(d,J=6.4Hz,2H),7.92–7.71(m,5H),7.65(d,J=8.2Hz, 1H),7.59(d,J=7.7Hz,2H),7.40(t,J=13.1Hz,1H),7.20(d,J=7.9Hz,2H),6.97(t,J=7.4Hz,1H),6.83(d, J=7.9Hz,1H),6.27–6.12(m,3H),6.00(d,J=14.2Hz,1H),5.71(s,1H),5.63(d,J=12.5Hz,1H),3.94–3.83 (m,2H),3.73(t,J=6.2Hz,2H),3.64–3.51(m,12H),3.37(s,3H),1.61(s,6H),1.52(s,6H),1.31(s,12H).

[0078] 13 C NMR (101MHz, CDCl3) δ179.48,173.93,163.07,155.93,148.05,147.66,144.48,143.86,1 43.29,142.67,139.25,135.98,135.66,129.08,128.97,128.61,127.93,124.10,122.77 ,121.99,121.72,121.53,117.13,115.86,107.87,97.77,92.26,84.11,71.92,71.87,71.11,70.66,70.60,70.51,67.37,63.45,59.04,48.96,46.85,42.95,28.35,25.58,24.84.

[0079] HRMS[M]+m / z calcd.for[C54H66B2F2N3O7]+:928.5049found:928.5067.

[0080] [Example 2]

[0081] 1) Preparation of difluoroborondolenine electron acceptor

[0082]

[0083] The difluoroboranoindole electron acceptor 11 (163 mg, 0.5 mmol) prepared in Example 1 and N-chlorosuccinimide (133 mg, 1 mmol) were dissolved in a mixed solvent (3 mL of chloroform: 0.5 mL of N-methylpyrrolidone), and dimethyl sulfoxide (10 mL, 0.2 mmol) was added. The mixture was stirred at 50°C for 3 hours, and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to obtain difluoroboranoindole electron acceptor 14 (115 mg, 64%).

[0084] 1 H NMR (400MHz, CDCl3) δ8.70(s,2H),7.76(d,J=8.1Hz,1H),7.66(dd,J=8.3,1.7Hz,1H),7.58–7.49(m,3H),2.46(s,3H),1.78(s,6H).

[0085] 13 C NMR (101MHz, CDCl3) δ177.49,176.94,150.06,147.87,142.97,142.72,136.84,128.05,121.74,120.28,117.09,101.93,52.69,22.21,21.89

[0086] HRMS[M+H]+m / z calcd.for[C18H17BClF2N2O]+:361.1085found:361.1084.

[0087] 2) Preparation of hydrogen peroxide probe HPBCy5-2

[0088]

[0089] Dissolve difluoroborindolenine electron acceptor 14 (36.0 mg, 0.1 mmol) and semicyanine intermediate 12 (93.8 mg, 0.18 mmol) in anhydrous dichloromethane, and add N,N-diisopropylethylamine (52 μL, 0.3 mmol). The reaction mixture is stirred at 50°C for 60 minutes. After cooling to room temperature, the reaction solution is purified by silica gel column chromatography to obtain intermediate 15 (35 mg, 47%). 1H NMR(400MHz, CDCl3) δ8.66(d,J=5.1Hz,2H),7.87(dd,J=14.1,12.0Hz,1H),7.68(d,J=8.2Hz,1H), 7.61(dd,J=8.3,1.8Hz,1H),7.53–7.43(m,4H),7.21(d,J=7.4Hz,2H),6.99(s,1H),6.85(d,J=7.7H z,1H),6.55(d,J=14.1Hz,1H),6.36–6.26(m,1H),5.67(d,J=12.7Hz,1H),3.92(t,J=6.1Hz,2H),3. 74(t,J=6.2Hz,2H),3.65–3.59(m,10H),3.56–3.52(m,2H),3.37(s,3H),1.78(s,6H),1.63(s,6H).

[0090] 13 C NMR (101MHz, CDCl3) δ173.83,168.69,163.72,150.19,149.41,148.03,144. 26,144.05,143.77,142.48,139.38,134.99,127.95,127.76,123.43,121.7 7,121.74,121.49,120.01,115.87,112.93,108.01,100.55,98.09,71.93,71.13,70.69,70.63,70.53,67.40,59.05,51.93,47.02,43.02,28.36,22.84.

[0091] HRMS[M]+m / z calcd.for[C41H48BClF2N3O5]+:746.3338found:746.3328.

[0092]

[0093] Intermediate 15 (18.6 mg, 0.025 mmol) and 4-bromomethylphenylboronic acid pinacol ester (22.4 mg, 0.03 mmol) were dissolved in 0.3 mL of toluene. The reaction mixture was stirred at 110 ° C for 2 hours. After cooling to room temperature, the crude product was precipitated in 10 mL of methyl tert-butyl ether / n-hexane (3:2, v / v) and kept at 4 ° C overnight. The precipitate was then centrifuged and dried in vacuo to obtain the hydrogen peroxide probe HPBCy5-2 (28.1 mg, 90%).

[0094] 11H NMR (400 MHz, CDCl3) δ 9.38 (d, J = 6.6 Hz, 2H), 8.18 (d, J = 6.5 Hz, 2H), 7.93 (t, J = 13.0 Hz, 1H), 7.82 (d, J = 7.8 Hz, 3H), 7.71 (dd, J = 8.5, 1.8 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 7.8 Hz, 2H), 7.54 (t, J = 13.2 Hz, 1H), 7.23 (d, J = 7.6 Hz, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.90 (d, J = 7.9 Hz, 1H), 6.54 (d, J = 13.9 Hz, 1H), 6.34 (t, J = 12.8 Hz, 1H), 6.20 (s, 2H), 5.74 (d, J = 12.8 Hz, 1H), 3.96 (t, J = 6.0 Hz, 2H), 3.76 (t, J = 6.1 Hz, 2H), 3.61 (s, 10H), 3.53 (t, J = 3.3 Hz, 2H), 3.37 (s, 3H), 1.79 (s, 6H), 1.63 (s, 6H), 1.32 (s, 12H).

[0095] 13 13C NMR (101 MHz, CDCl3) δ 173.15, 169.70, 165.38, 155.84, 151.03, 147.43, 146.31, 144.52, 143.51, 143.27, 139.56, 135.98, 135.65, 129.15, 129.08, 128.63, 128.05, 124.04, 123.56, 122.30, 121.81, 121.40, 115.86, 112.20, 108.49, 101.11, 98.82, 84.12, 71.92, 71.14, 70.67, 70.62, 70.60, 70.52, 70.45, 67.46, 63.43, 59.04, 51.69, 47.37, 43.23, 28.34, 24.85, 22.87.

[0096] HRMS [M]+ m / z calcd. for [C54H65B2ClF2N3O7]+: 962.4660 found: 962.4678.

[0097] 2. Optical property test of hydrogen peroxide probe

[0098]

Example 3

[0099] This example tests the photophysical properties of hydrogen peroxide probes HPBCy5-1 and HPBCy5-2, wherein the quantum yield is measured using rhodamine 800 in ethanol as a reference.

[0100] Tables 1 and 2 show the maximum absorption wavelengths (λ) of HPBCy5-1 and HPBCy5-2 in DMSO and PBS, respectively. Abs ), molar absorption coefficient (ε), maximum emission wavelength (λ em ), Stokes shift (Stokes Shift), quantum yield (QY) and fluorescence brightness (Brightness).

[0101] Table 1: Photophysical properties of HPBCy5-1.

[0102]

[0103]

[0104] Table 2: Photophysical properties of HPBCy5-2.

[0105]

[0106] As can be seen from Tables 1 and 2, both probes have lower fluorescence quantum yields in DMSO and PBS, and in the physiological environment simulated by PBS, the quantum yield of HPBCy5-1 is only 0.0008. This indicates that the fluorescence quenching induced by pyridinium through twisted intramolecular charge transfer (TICT) is very effective for both probes, and is more effective for HPBCy5-1 with weaker receptors. The two probes show almost no fluorescence when not cleaved.

[0107] [Example 4]

[0108] In this example, the hydrogen peroxide probe HPBCy5-1 (20 μM) was reacted with H2O2 (500 μM) in PBS (30% DMF), and the HPLC spectra of the reaction products, HPBCy5-1 and LDBCy5-3 at a wavelength of 660 nm, and the mass spectra of related compounds within a specified minute were detected.

[0109] like Figure 1 and Figure 2 As shown, after reacting with H2O2, the boronate ester of the hydrogen peroxide probe HPBCy5-1 is oxidatively cleaved, specifically releasing LDBCy5-3 (compound 16), which in turn produces fluorescence that can be detected. The reaction is:

[0110]

[0111] [Example 5]

[0112] In this example, fluorescence response experiments were performed on hydrogen peroxide probes HPBCy5-1 and HPBCy5-2.

[0113] Specifically, the hydrogen peroxide probe was incubated with 50 μM, 200 μM, and 500 μM H2O2 at 37°C, and the change of fluorescence intensity over time was measured. Figure 3 As shown, the probe can produce fluorescence response in a short time of contact with H2O2. Within a certain period of time, the fluorescence intensity continues to increase with time. In the same period of time, the higher the concentration of H2O2, the stronger the fluorescence intensity.

[0114] The hydrogen peroxide probe was incubated with different concentrations of H2O2 at 37°C for 1 hour. Figure 4 As shown, the fluorescence intensity of the two hydrogen peroxide probes shows a good linear relationship with the concentration of H2O2. Therefore, the concentration of H2O2 in the physiological environment can be determined based on the fluorescence intensity detected within a certain period of time. In addition, the detection limit (LOD) of the probe HPBCy5-1 is 0.2μM, and the detection limit of HPBCy5-2 is 0.3μM.

[0115] [Example 6]

[0116] In order to verify the selectivity of the hydrogen peroxide probe to H2O2, in this example, the probes HPBCy5-1 and HPBCy5-2 were reacted selectively to different substances in PBS (pH 7.4, 30% DMF) at 37°C for 1 h, including 100 μM Na + , K + , Ca 2+ , Cu 2+ , Zn 2+ ,Mg 2+ , Fe 3+ , S 2- , glutathione and cysteine, HOCl at a concentration of 25 μM, NO and ·OH, ONOO at a concentration of 10 μM - , and a concentration of 200 μM H2O2.

[0117] The experimental results are as follows Figure 5 As shown in the results, probes HPBCy5-1 and HPBCy5-2 showed good selectivity for the H2O2 surface and were able to specifically detect H2O2.

[0118] [Example 7]

[0119] Excessive production of reactive oxygen species (ROS) such as H2O2 in mitochondria of cells can lead to oxidative stress. During this process, cells may increase the number of lipid droplets to store excess lipids, which will further affect cell metabolism and function. This example conducted an imaging experiment of H2O2 and lipid droplets (LDs) at the cellular level using a probe.

[0120] Specifically, the confocal microscope A and B were laid the day before. Group A was incubated with 1μM hoechst and 1μM Mito-Tracker Green for 30 minutes, washed once with PBS, and then incubated with 2μM HPBCy5-1 for 30 minutes, washed once with PBS, and imaged. Group B was first treated with 100μM H2O2 for 10 minutes, then incubated with 1μM hoechst and 1μM Mito-Tracker Green for 30 minutes, washed once with PBS, and then incubated with 2μM HPBCy5-1 for 30 minutes, washed once with PBS, and imaged. The experimental results are shown in Figure 2. Figure 6 As shown, the probe HPBCy5-1 can well label mitochondria and has a good overlap effect with the commercial mitochondrial probe Mito-Tracker Green.

[0121] Furthermore, three groups of confocal microscopes were laid the day before. Group 1 was treated with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 min, washed once with PBS, and then incubated with 2 μM HPBCy5-1 for 30 min, washed once with PBS, and imaged. Group 2 was first treated with 100 μM H2O2 for 10 min, then incubated with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 min, washed once with PBS, then incubated with 2 μM HPBCy5-1 for 30 min, washed once with PBS, and imaged. Group 3 was first treated with 200 μM oleic acid, then incubated with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 min, washed once with PBS, then incubated with 0.5 μM LDBCy5-3 for 30 min, washed once with PBS, and imaged.

[0122] like Figure 7 As shown in the figure, in the control group (HPBCy5-1) without H2O2 pretreatment, the dark light mainly came from mitochondria due to the low background fluorescence of the probe HPBCy5-1. Figure 7 As shown in the second row, when A549 cells were pretreated with H2O2 and probe HPBCy5-1 was added (HPBCy5-1+H2O2), the fluorescence signal was significantly enhanced, and part of the product LDBCy5-3 specifically stained lipid droplets. As the reaction proceeded, after the probe HPBCy5-1 reacted with H2O2 for a period of time and produced a large amount of LDBCy5-3, as shown in Figure 7As shown in the third row, the product LDBCy5-3 can be combined with the commercial lipid droplet probe Bodipy 493 / 503 The colocalization indicated that the probe had the ability to target lipid droplet migration after reacting with H2O2.

[0123] [Example 8]

[0124] In this example, the feasibility of the probe for detecting endogenous H2O2 was evaluated.

[0125] Specifically, three groups of confocal microscopes were laid the day before. Group 1 was first treated with PMA (0 μg / ml) for 1 h, and then 1 μM hoechst and 1 μM Bodipy493 / 503 were added for co-incubation for 30 min, washed once with PBS, and then 2 μM HPBCy5-1 was added for co-incubation for 30 min, washed once with PBS, and then imaged; Group 2 was first treated with PMA (3 μg / ml) for 1 h, and then 1 μM hoechst and 1 μM Bodipy493 / 503 were added for co-incubation for 30 min, washed once with PBS, and then 2 μM HPBCy5-1 was added for co-incubation for 30 min, washed once with PBS, and then imaged; Group 3 was first treated with PMA (10 μg / ml) for 1 h, and then 1 μM hoechst and 1 μM Bodipy493 / 503 were added for co-incubation for 30 min, washed once with PBS, and then 2 μM HPBCy5-1 was added for co-incubation for 30 min, washed once with PBS, and then imaged.

[0126] The experimental results are as follows Figure 8 As shown, A549 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 1 hour and then incubated with the probe HPBCy5-1. Compared with A549 cells not treated with PMA, HPBCy5-1 produced a stronger fluorescence signal in lipid droplets, and the higher the concentration of PMA, the more lipid droplets were produced and the stronger the fluorescence signal.

[0127] Two groups of confocal microscopes were laid the day before. Group 1 was treated with PMA (10 μg / mL) for 1 hour, then incubated with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 minutes, washed once with PBS, and incubated with 2 μM HPBCy5-1 for 30 minutes, washed once with PBS, and imaged. Group 3 was treated with 200 μM oleic acid for 6 hours, then treated with PMA (10 μg / mL) for 1 hour, then incubated with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 minutes, washed once with PBS, and incubated with 0.5 μM LDBCy5-3 for 30 minutes, washed once with PBS, and imaged. Fig. 9 As shown, after oleic acid (OA) and PMA were used to further promote the secretion of lipid droplets in vivo, the fluorescence of the cells was further enhanced and was almost completely localized in the lipid droplets.

[0128] It can be seen that the hydrogen peroxide probe can simultaneously evaluate the levels of lipid droplets and H2O2 after mitochondrial oxidative stress.

[0129] [Example 9]

[0130] In this example, an acetaminophen (APAP)-induced liver injury animal model was established, and HPBCy5-2 was used in the animal model to explore the relationship between hydrogen peroxide and lipid droplet accumulation in mitochondria.

[0131] The experimental results are as follows Fig.10 As shown in (a), compared with the control group (Control), the fluorescence of mice in the APAP experimental group was significantly enhanced, indicating that a large amount of H2O2 was produced, and the fluorescence intensity increased further with the increase of the treatment experiment. However, the fluorescence intensity of mice in the treatment group using N-acetylcysteine ​​and APAP antidote (NAC+APAP) was significantly reduced. At the same time, Fig.10 As shown in (b), the results of in vitro liver tissue are consistent with those of in vivo liver tissue. Fig.10 (c) shows the changes of two transaminases ALT and AST, demonstrating the effectiveness of the APAP-induced liver injury animal model.

[0132] The terms “first”, “second”, etc. (eg, first solvent, second solvent, etc.) used herein are used only to distinguish corresponding components for the sake of clarity of description and are not intended to limit any order or emphasize importance.

[0133] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A hydrogen peroxide probe, characterized in that It has the structural formula shown in Formula I: In Formula I, R1 is selected from C1 to C 16 Chain alkyl, polyethylene glycol group, C1~C 16 Alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols; R2 is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, substituted or unsubstituted phenyl, hydroxyl, amino, carboxyl, formate, sulfonic acid and salts thereof, methoxy; n=0-2.

2. A hydrogen peroxide probe according to claim 1, characterized in that: R1 is selected from C1-C4 chain alkyl, polyethylene glycol group, C1-C4 alkyl carboxylic acid and active ester, C1-C3 alkyl sulfonic acid and its salt, C1-C4 alcohol; R2 is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, methoxy; n=1-2.

3. A hydrogen peroxide probe according to claim 2, characterized in that: R1 is selected from C1-C4 chain alkyl, polyethylene glycol group, C1-C4 alcohol; R2 is selected from hydrogen and halogen; n=1-2.

4. A hydrogen peroxide probe according to any one of claims 1 to 3, characterized in that: The hydrogen peroxide probe is selected from the following compounds:

5. A method for preparing a hydrogen peroxide probe, characterized in that: For preparing the hydrogen peroxide probe according to any one of claims 1 to 4, the preparation method comprises the following steps: The compound represented by formula II is mixed with the compound represented by formula III to obtain an intermediate represented by formula IV; The intermediate represented by formula IV is reacted with 4-bromomethylphenylboronic acid pinacol ester to generate the hydrogen peroxide probe represented by formula I; Formula II: Formula III: Formula IV: In formula II and formula IV, R2 is selected from hydrogen, halogen, methyl, trifluoromethyl, vinyl, ethynyl, substituted or unsubstituted phenyl, hydroxyl, amino, carboxyl, formate, sulfonic acid and its salts, methoxy; in formula III and formula IV, R1 is selected from C1 to C 16 Chain alkyl, polyethylene glycol group, C1~C 16 Alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols; n=0-2.

6. The method for preparing a hydrogen peroxide probe according to claim 5, characterized in that: After the compound represented by formula II and the compound represented by formula III are dissolved in a first solvent, a first base is added for condensation, and the reaction system is reacted at 40-70° C. to obtain the intermediate.

7. The method for preparing a hydrogen peroxide probe according to claim 5, characterized in that: The intermediate and 4-bromomethylphenylboronic acid pinacol ester are dissolved in a second solvent and reacted at 100 to 120° C. for 1 to 3 hours to obtain the hydrogen peroxide probe.

8. The method for preparing a hydrogen peroxide probe according to claim 5, characterized in that: The preparation method of the compound represented by formula II comprises the following steps: After dissolving 2,3,3-trimethyl-5-bromo-3H-indole, pyridine-4-boric acid, a second base and a catalyst in a third solvent, the mixture is reacted at 90 to 120° C. under an inert atmosphere to obtain a pyridinium derivative, and a compound represented by formula II is prepared based on the pyridinium derivative.

9. An application of a hydrogen peroxide probe, characterized in that: The hydrogen peroxide probe is the hydrogen peroxide probe according to any one of claims 1 to 4. The hydrogen peroxide probe is used to label mitochondria, generates a continuous fluorescence response to hydrogen peroxide in the mitochondria, and the fluorescence intensity is positively correlated with the concentration of hydrogen peroxide.

10. The use according to claim 9, characterized in that: The hydrogen peroxide probe can target the migration of lipid droplets in cells after reacting with hydrogen peroxide.

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