A hydrogen peroxide probe and its preparation method and application

Abstract

A hydrogen peroxide probe, and its preparation method and application. The hydrogen peroxide probe connects pyridinium to a difluoroborindolenine electron acceptor and can induce fluorescence quenching by distorting intramolecular charge transfer. It produces weak fluorescence when the hydrogen peroxide concentration in the mitochondria is low, and produces 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. At the same time, the hydrogen peroxide probe can also target the increased migration of lipid droplets after oxidative stress caused by increased hydrogen peroxide concentration, thereby better detecting the progression of oxidative stress. This is of great significance for exploring cellular damage related to oxidative stress.

Description

Technical Field

[0001] The present 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 cellular metabolism but is also involved in many pathological processes, such as inflammation and oxidative stress. Therefore, the development of fluorescent probes that can monitor changes in intracellular hydrogen peroxide concentration in real time is particularly important.

[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 progress 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 by having the 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, hydroxy, amino, carboxyl, formate, sulfonic acid and salts thereof, methoxy; n = 0-2.

[0010] In this technical solution, a hydrogen peroxide probe is constructed by linking 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 within cells, the hydrogen peroxide probe reacts with the hydrogen peroxide. The boronate ester in the hydrogen peroxide probe is oxidatively cleaved by the hydrogen peroxide, specifically releasing a compound that can be detected as fluorescent. The fluorescence intensity increases with increasing hydrogen peroxide concentration. Within a certain range of hydrogen peroxide concentrations, the fluorescence intensity is substantially proportional to the hydrogen peroxide, thereby enabling 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 reaction with hydrogen peroxide. Its reaction pathway is:

[0012]

[0013] In this technical solution, the hydrogen peroxide probe not only labels mitochondria and fluorescently responds to hydrogen peroxide produced by mitochondria, but also the product of the probe and hydrogen peroxide can specifically stain lipid droplets and target their migration, thereby detecting the production of lipid droplets caused by oxidative stress in mitochondria due to excessive hydrogen peroxide concentration. Combined with changes in hydrogen peroxide concentration, the progression of oxidative stress can be more intuitively and accurately detected.

[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 their salts, C1-C4 alcohols.

[0015] In some preferred embodiments, R1 is selected from C1-C4 chain alkyl groups, polyethylene glycol groups, C1-C4 alkyl carboxylic acids and active esters, C1-C3 alkyl sulfonic acids and salts thereof, and C1-C4 alcohols. Further preferably, R1 is selected from C1-C4 chain alkyl groups, polyethylene glycol groups, and C1-C4 alcohols.

[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, 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 alkylsulfonic acid and a salt thereof, and the alkylsulfonic acid and a salt thereof can 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 this 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 salts, and 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 this technical solution, the hydrogen peroxide probe can label mitochondria, using pyridinium to distort intramolecular charge transfer to induce fluorescence quenching, producing weak fluorescence when the hydrogen peroxide concentration in the mitochondria is low, and producing 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 increase in hydrogen peroxide concentration leads to 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 cells caused by 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 aforementioned 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 react to obtain the 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 salt, 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 this technical solution, the compound represented by Formula II is a difluoroborindolenine electron acceptor connected to a pyridinium, and the compound represented by Formula III is a fluoroborindole merocyanine covering the far-infrared to near-infrared range. The fluoroborinole merocyanine can be synthesized using existing methods, such as the process disclosed in patent CN116284089A, or purchased commercially.

[0033] In some preferred embodiments, after dissolving 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. 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, or mesitylene. The first base can be N,N-diisopropylethylamine, piperidine, triethylamine, pyridine, acetate, carbonate, bicarbonate, or potassium tert-butoxide. The reaction mixture is stirred at 40-70°C for a certain period of time to obtain the intermediate. Preferably, the reaction temperature is 50-60°C.

[0034] In this technical solution, the intermediate is further reacted with 4-bromomethylphenylboronic acid pinacol ester to produce 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 100-120°C for 1-3 hours to produce the hydrogen peroxide probe. In one or more embodiments, the second solvent can be toluene, xylene, dimethyl sulfoxide, or 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 90-120° 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 this technical solution, 2,3,3-trimethyl-5-bromo-3H-indole and pyridine-4-boronic acid are reacted in a third solvent to produce a pyridine derivative. The second base can be K2CO3, CsCO3, Na2CO3, TiOH, or NaOCH2CH3; the catalyst can be Pd(PPh3)4 or Pd(dppf)Cl2. The third solvent can be toluene, a mixed solution of isopropyl alcohol and water, a mixture of ethanol and water, a mixture of isopropyl alcohol and water, a mixture of ethylene glycol dimethyl ether and water, tetrahydrofuran, or toluene. In some preferred embodiments, the third solvent is a mixed solvent of toluene, isopropyl alcohol, and water. This allows the solvents to dissolve each other, thereby dissolving the second base, such as potassium carbonate, and converting it into a homogeneous 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 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.

[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 induce fluorescence quenching by pyridinium-distorted intramolecular charge transfer. It produces weak fluorescence when the hydrogen peroxide concentration in the mitochondria is low, and produces 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.

[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 HPLC spectra of hydrogen peroxide probes HPBCy5-1, LDBCy5-3, and the reaction product of hydrogen peroxide probe HPBCy5-1 and hydrogen peroxide in specific embodiments of the present invention are shown;

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

[0046] Figure 3 The fluorescence response time of hydrogen peroxide probes HPBCy5-1 (a) and HPBCy5-2 (b) in response to different concentrations of hydrogen peroxide in a specific embodiment of the present invention is shown;

[0047] Figure 4 The graph shows the fluorescence intensity changes of hydrogen peroxide probes HPBCy5-1 (a) and HPBCy5-2 (b) after incubation with different concentrations of hydrogen peroxide 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 Figure 2 shows fluorescence imaging of mitochondria labeled with the probe HPBCy5-1 in a specific embodiment of the present invention, wherein Hoechst is a commercial nuclear stain and 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 commercial lipid droplet stain;

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

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

[0053] Figure 10 The experimental results of using the probe HPBCy5-2 in an animal model of APAP-induced liver injury in a specific embodiment of the present invention are shown, wherein (a) shows in vivo and tissue imaging, HE staining, and Oil Red O staining of tissues; (b) shows a bar graph of the in vivo and tissue images of the control group and each experimental group corresponding to (a); and (c) shows a bar graph of the transaminases 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 examples and drawings. The exemplary embodiments of the present invention and their descriptions 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 may be directly connected or indirectly connected via other groups unless otherwise specified. 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. 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 of this art. Each brand and abbreviation is clear and unambiguous in the field of its related use. Those skilled in the art can purchase them from the market or prepare them by conventional methods based on the brand, abbreviation and corresponding use.

[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 meaning of the substituents 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), KCO (6.3 g, 36 mmol), and Pd(PPh) (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 under argon overnight. After cooling, the reaction mixture was partitioned between CHCl (100 mL) and water (50 mL). The aqueous layer was extracted with CHCl (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 yield 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] To a solution of the pyridinium derivative 10 (1.18 g, 5 mmol) in 8 mL of acetic anhydride under argon was added BF3·Et2O (0.8 mL, 6.5 mmol). After stirring at 120°C for 4 hours, the solvent was removed using a rotary evaporator, and the resulting residue was purified by silica gel column chromatography to afford the 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] Difluoroborondolenine electron acceptor 11 (75.2 mg, 0.2 mmol) and hemicyanine intermediate 12 (156.3 mg, 0.3 mmol) were dissolved in 3 mL of toluene, followed by the addition of acetic acid (25 μL) and piperidine (50 μL). The reaction mixture was stirred at 100°C for 3 hours. The resulting residue was purified by silica gel column chromatography to yield intermediate 13 (29.8 mg, 21%). Hemicyanine intermediate 12 can be prepared using the method disclosed in 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 stored at 4°C overnight. The precipitate was then centrifuged and dried in vacuo to yield 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 difluoroborindolenine 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). 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 resulting residue was purified by silica gel column chromatography to yield the difluoroborindolenine 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] Difluoroborondolenine electron acceptor 14 (36.0 mg, 0.1 mmol) and hemicyanine intermediate 12 (93.8 mg, 0.18 mmol) were dissolved in anhydrous dichloromethane, and N,N-diisopropylethylamine (52 μL, 0.3 mmol) was added. The reaction mixture was stirred at 50°C for 60 minutes. After cooling to room temperature, the reaction solution was 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 stored at 4°C overnight. The precipitate was then centrifuged and dried in vacuo to yield 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, 143.5, 143.27, 139.56, 135.98, 135.65, 129.15, 129.08, 128.63, 128.05, 124.04, 123.56, 122.3, 121.81, 121.4, 115.86, 112.2, 108.49, 101.11, 98.82, 84.12, 71.92, 71.14, 70.67, 70.62, 70.6, 70.52, 70.45, 67.46, z 63.43, 59.04, 51.69, y 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

[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, 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 in Tables 1 and 2, both probes exhibited lower fluorescence quantum yields in DMSO and PBS. In the physiological environment simulated by PBS, the quantum yield of HPBCy5-1 was only 0.0008. This indicates that pyridinium-induced fluorescence quenching via twisted intramolecular charge transfer (TICT) is highly effective for both probes, and is particularly effective for HPBCy5-1, which has a weaker receptor. Both probes exhibited virtually no fluorescence when uncleaved.

[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 reaction products, HPLC spectra of HPBCy5-1 and LDBCy5-3 at a wavelength of 660 nm, and 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 then produces detectable fluorescence. 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 in fluorescence intensity over time was measured. Figure 3 As shown in the figure, the probe can generate fluorescence response in a short time after contact with H2O2. Within a certain period of time, the fluorescence intensity continues to increase with time. Moreover, within 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℃ for 1 hour. Figure 4 As shown, the fluorescence intensity of the two hydrogen peroxide probes exhibits a good linear relationship with the H2O2 concentration. Therefore, the H2O2 concentration in the physiological environment can be determined based on the fluorescence intensity detected over a certain period of time. Furthermore, the limit of detection (LOD) for probe HPBCy5-1 was calculated to be 0.2 μM, and that for HPBCy5-2 was 0.3 μM.

[0115] [Example 6]

[0116] To verify the selectivity of the hydrogen peroxide probe for H2O2, in this example, the probes HPBCy5-1 and HPBCy5-2 were reacted selectively with different substances in PBS (pH 7.4, 30% DMF) at 37°C for 1 h, including 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 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 can lead to oxidative stress. During this process, cells may increase the number of lipid droplets to store excess lipids, which further affects cellular metabolism and function. This example demonstrates the use of probes to image H2O2 and lipid droplets (LDs) at the cellular level.

[0120] Specifically, the confocal microscope A and B were set up the day before. Group A was treated 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 then imaged. Group B was 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 then 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 then 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 then 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 then imaged.

[0122] like Figure 7 As shown in the figure, in the control group (HPBCy5-1) without H2O2 pretreatment, the background fluorescence of the probe HPBCy5-1 was low, and the dark light mainly came from the mitochondria. 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 the figure below. Figure 7As shown in the third row, the product LDBCy5-3 can be combined with the commercial lipid droplet probe Bodipy 493 / 503 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 hour, and then 1 μM hoechst and 1 μM Bodipy493 / 503 were added for co-incubation for 30 minutes, washed once with PBS, and then 2 μM HPBCy5-1 was added for co-incubation for 30 minutes, washed once with PBS, and then the microscope was imaged. Group 2 was first treated with PMA (3 μg / ml) for 1 hour, and then 1 μM hoechst and 1 μM Bodipy493 / 503 were added for co-incubation for 30 minutes, washed once with PBS, and then 2 μM HPBCy5-1 was added for co-incubation for 30 minutes, washed once with PBS, and then the microscope was imaged. Group 3 was first treated with PMA (10 μg / ml) for 1 hour, and then 1 μM hoechst and 1 μM Bodipy493 / 503 were added for co-incubation for 30 minutes, washed once with PBS, and then 2 μM HPBCy5-1 was added for co-incubation for 30 minutes, washed once with PBS, and then the microscope was 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. 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 placed the day before. Group 1 was first treated with PMA (10 μg / mL) for 1 hour, then added with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 minutes, washed once with PBS, added with 2 μM HPBCy5-1 for 30 minutes, washed once with PBS, and imaged. Group 3 was first treated with 200 μM oleic acid for 6 hours, then added with PMA (10 μg / mL) for 1 hour, added with 1 μM hoechst and 1 μM Bodipy493 / 503 for 30 minutes, washed once with PBS, added with 0.5 μM LDBCy5-3 for 30 minutes, washed once with PBS, and imaged. Figure 9 As shown, after oleic acid (OA) and PMA were used to further promote lipid droplet secretion in vivo, the fluorescence of the cells was further enhanced and almost completely localized in the lipid droplets.

[0128] It can be seen that the hydrogen peroxide probe can simultaneously assess 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 this animal model to explore the relationship between hydrogen peroxide and lipid droplet accumulation in mitochondria.

[0131] The experimental results are as follows Figure 10 As shown in (a), compared with the control group (Control), the fluorescence of the 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 the mice in the treatment group using N-acetylcysteine ​​and APAP antidote (NAC+APAP) was significantly reduced. At the same time, Figure 10 As shown in (b), the results of in vitro liver tissue were consistent with those of in vivo liver tissue. Figure 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 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 The hydrogen peroxide probe is selected from the following compounds:

Images