CN117924335A - ONOO with endoplasmic reticulum targeting-Fluorescent probe and preparation method and application thereof - Google Patents
ONOO with endoplasmic reticulum targeting-Fluorescent probe and preparation method and application thereof Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an ONOO-fluorescent probe with endoplasmic reticulum targeting, a preparation method and application thereof, and relates to the technical field of in-situ imaging detection in organisms. The endoplasmic reticulum fluorescent probe provided by the invention has the remarkable advantages of low cytotoxicity, high light stability, good endoplasmic reticulum positioning specificity and the like, can specifically detect ONOO-level fluctuation under endoplasmic reticulum oxidative stress in vivo, and is not interfered by other biomolecules, enzymes and the like. After the target probe reacts with ONOO-in the invention, the obtained aggregation-induced emission endoplasmic reticulum probe has good light stability, low cytotoxicity and high endoplasmic reticulum positioning specificity, can be used for live cell real-time dynamic high-resolution fluorescence imaging, has good imaging effect, and can be used for real-time monitoring of liver injury and evaluation of injury degree.
Description
Technical Field
The invention relates to the technical field of in-situ imaging detection in organisms, in particular to an ONOO - fluorescent probe with endoplasmic reticulum targeting, a preparation method and application thereof.
Background
The liver is the largest digestive organ of the human body and is located in the abdomen of the human body. It plays an important role in various physiological processes, including balance of glycogen collection and metabolism of various substances. With the acceleration of the current pace of life, the liver is exposed to the threat of alcohol, drugs and chemicals. Acute liver injury can worsen to liver failure, severe cases can lead to death, severely compromising human health. Acute liver injury occurs in a complex manner, and evaluation is laborious, and its clinical behavior includes all liver-related pathological processes. Drug therapy is a great clinical challenge, mainly due to the lack of effective and accurate diagnosis. At present, for diagnosis of acute liver injury, the traditional clinical method is based on monitoring serum index levels, wherein the indexes comprise alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP), but the biomarkers are repeated with other diseases (such as acute lung injury, skeletal muscle injury and the like) without specificity, so that great uncertainty can be brought to clinical diagnosis, and therefore, how to realize rapid and accurate diagnosis of acute liver injury by using an accurate detection method has epoch-making significance for accelerating urgent and timely treatment of acute liver injury.
Endoplasmic reticulum is an important class of organelles involved in vital activities, involved in synthesis and secretion of many protein, glycogen, lipid and cholesterol substances, and is generally classified into two basic types, i.e., rough endoplasmic reticulum (rough endoplasmic reticulum, RER) and smooth endoplasmic reticulum (smooth endoplasmic reticulum, SER) depending on whether or not there is ribosome attachment on the outer surface of the endoplasmic reticulum, and has detoxification functions, i.e., some enzymes are contained in the smooth endoplasmic reticulum in hepatocytes to remove fat-soluble wastes and metabolically produced harmful substances, so stress detection on the endoplasmic reticulum may be more specific to acute liver injury. Peroxynitrate (ONOO -) is a highly oxidative and nucleophilic reagent, and endoplasmic reticulum oxidative stress is usually accompanied by abnormal fluctuations of ONOO -, whereas under physiological conditions ONOO - has a short lifetime (20 ms), high reactivity and low concentration (nanomolar levels), so detection of endogenous ONOO - is always a problem. To date, electrochemical, uv-vis absorption spectroscopy and immunohistochemical methods have been used to analyze ONOO - concentrations, but none of these methods have been used for in vivo in situ imaging of ONOO -. Recently, due to its high temporal and spatial resolution, high sensitivity and non-invasiveness. Fluorescent probe-based fluorescence imaging techniques have become an ideal tool for exploring the physiological function of ONOO -. Numerous ONOO - specific fluorescent probes have been reported depending on the type of reactive groups including double bonds, phenylboronates, α -ketoamides, indoline-2, 3-diones, trifluoromethyl ketones and N-aminophenols. However, there are few peroxynitrite fluorescent probes with endoplasmic reticulum targeting, and at the same time, few fluorescent probes can evaluate the fluctuation of levels of ONOO - under endoplasmic reticulum oxidative stress.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an ONO-fluorescent probe with endoplasmic reticulum targeting and a preparation method and application thereof, which are used for solving the technical problem that the horizontal fluctuation of ONO-under endoplasmic reticulum oxidative stress is difficult to evaluate in the prior art.
The technical scheme adopted by the invention is as follows:
An ONOO-fluorescent probe with endoplasmic reticulum targeting, which consists of an AIE skeleton of quinoline, a response group of borate and a targeting group of sulfonamide, and is named QM-ONOO, and has a structural formula shown in formula 1:
the preparation method of the ONOO-fluorescent probe with endoplasmic reticulum targeting comprises the following steps:
(1) Dissolving 2-methylquinoline in toluene solution, adding ethyl bromoacetate under nitrogen condition to react, heating to 100-120 deg.C, refluxing overnight, cooling to room temperature after reaction, precipitating solid, suction filtering, washing the solid with diethyl ether to obtain the final product
Substance 1 represented by formula 2;
(2) Dissolving the substance 1 in ethanol, adding malononitrile and sodium ethoxide under nitrogen, stirring at room temperature for 6-12 h, removing solvent by rotary evaporation after the reaction is completed, extracting with dichloromethane as an organic phase and saturated saline as a water phase, combining and retaining an organic layer, removing the solvent by rotary evaporation, and separating and purifying by a silica gel column to obtain a substance 2 shown in a formula 3;
(3) Dissolving the substance 2 in a THF/H 2 O mixed solution, adding NaOH under the condition of nitrogen, heating to 60-80 ℃, reacting overnight, concentrating the reaction solution, adding water until the components are completely dissolved, dropwise adding dilute hydrochloric acid until the solid is no longer separated out, and carrying out suction filtration to obtain a substance 3 shown in a formula 4;
(4) Dissolving the substance 3 in DMF, slowly adding EDCI and HOBT under the condition of 0 ℃ and stirring for half an hour under the condition of nitrogen, ice-bathing at 0 ℃, slowly adding N- (2-amino ethyl) -4-methylbenzenesulfonamide, reacting at room temperature overnight, extracting with ethyl acetate as an organic phase and saturated saline water as a water phase after the reaction is completed, merging the organic layers, steaming to remove the solvent, and separating and purifying by a silica gel column to obtain a substance 4 shown as a formula 5;
(5) Dissolving substances 4 and 4-formylphenylboronic acid pinacol ester in acetonitrile, dropwise adding piperidine under the condition of nitrogen, reacting to 70-100 ℃ for reflux reaction overnight, extracting with dichloromethane as an organic phase and saturated saline as a water phase after the reaction is finished, merging the organic layers, steaming to remove the solvent, and separating and purifying by a silica gel column to obtain QM-ONOO shown in formula 1.
Further, in the step (1), the molar ratio of the 2-methylquinoline to the ethyl bromoacetate is 1:1-1.5.
Further, in the step (2), the molar ratio of the substance 1, malononitrile and sodium ethoxide is 1:2:2.
Further, in the step (3), the mol ratio of the substance 2 to NaOH is 1:1, and the volume ratio of THF/H 2 O in the THF/H 2 O mixed solution is 5:1.
Still further, in step (4), the molar ratio of the substance 3 to EDCI, HOBT, N- (2-aminoethyl) -4-methylbenzenesulfonamide is 1:1.3:1.3:1.3.
Further, in the step (5), the molar ratio of the substance 4 to 4-formylphenyl boronic acid pinacol ester is 1:2-3.
Use of an ONOO-fluorescent probe with endoplasmic reticulum targeting as a fluorescent probe for detecting ONOO-level fluctuations under endoplasmic reticulum oxidative stress in vivo.
In summary, compared with the prior art, the invention has the following advantages and beneficial effects:
1. The invention designs and synthesizes an aggregation-induced emission endoplasmic reticulum fluorescent probe with a quinoline skeleton, and the probe molecule consists of the quinoline skeleton, a targeting group and a reaction group. The aggregation-induced emission endoplasmic reticulum fluorescent probe QM-ONOO provided by the invention has the remarkable advantages of low cytotoxicity, high light stability, good endoplasmic reticulum positioning specificity and the like, can specifically detect ONOO-level fluctuation under endoplasmic reticulum oxidative stress in vivo, and is not interfered by other biomolecules, enzymes and the like.
2. After the target probe reacts with ONOO-, the method has obvious aggregation-induced emission behavior, and the obtained aggregation-induced emission endoplasmic reticulum probe has good light stability, low cytotoxicity and high endoplasmic reticulum positioning specificity, can be used for live cell real-time dynamic high-resolution fluorescence imaging, has good imaging effect, and can be used for real-time monitoring of liver injury and evaluation of injury degree.
3. The ONO-responsive probe obtained by the preparation method can qualitatively display the ONO-spatial distribution of the endoplasmic reticulum in HepG2 cells and the ONO-horizontal fluctuation of the endoplasmic reticulum when the mice are damaged by liver, and has important scientific research and economic value for researching ONO-related physiological and pathological processes.
4. The invention has simple synthetic route, mild reaction condition and higher yield; the raw materials are simple and easy to obtain, and the synthesis cost is low.
Drawings
FIG. 1 is a synthetic scheme employed by fluorescent probe QM-ONOO.
FIG. 2 shows a nuclear magnetic resonance hydrogen spectrum of the fluorescent probe material 2 in CDCL 3.
FIG. 3 is a nuclear magnetic resonance carbon spectrum of the fluorescent probe material 2 in CDCL 3.
FIG. 4 shows a nuclear magnetic resonance hydrogen spectrum of fluorescent probe material 3 in DMSO.
FIG. 5 is a nuclear magnetic resonance carbon spectrum of fluorescent probe material 3 in DMSO.
FIG. 6 shows a nuclear magnetic resonance hydrogen spectrum of fluorescent probe material 4 in DMSO.
FIG. 7 is a nuclear magnetic resonance carbon spectrum of fluorescent probe material 4 in DMSO.
FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of fluorescent probe QM-ONOO in DMSO.
FIG. 9 is a nuclear magnetic resonance carbon spectrum of fluorescent probe QM-ONOO in DMSO.
FIG. 10 is a diagram showing the photophysical properties of fluorescent probe QM-ONOO: (A) Fluorescence emission spectra of fluorescent probe QM-ONOO in mixed solvent of water/DMSO with different proportions; (B) The maximum fluorescence intensity ratio I/I 0 of the fluorescent probe QM-ONOO in mixed solvents of tetrahydrofuran/water with different proportions is shown in a graph; (C) Is the absorption of fluorescent probe QM-ONOO before and after incubation with ONOO; (D) For the fluorescence probe QM-ONOO, the fluorescence spectrum changes along with the increase of ONOO; (E) FIG. 10 (D) shows a linear relationship between fluorescence intensity and ONOO concentration; (F) Increasing fluorescence spectrum variation with time for fluorescent probe QM-ONOO; (G) The fluorescence spectrum of the fluorescent probe QM-ONOO is changed with different pH values; (H) A selectivity experiment of fluorescent probe QM-ONOO.
FIG. 11 is a confocal fluorescence image of fluorescent probe QM-ONOO under different concentrations of exogenous stimulus.
FIG. 12 is a confocal fluorescence image of fluorescent probe QM-ONOO under different endogenous stimuli.
In FIG. 13, (A) and (C) are co-localized fluorescence images of fluorescent probe QM-ONOO and mitochondrial commercial stain ER-Tracker-Green and endoplasmic reticulum commercial stain Lyso-Tracker-Green, (B) co-localized effect of target probe corresponding to (A) and endoplasmic reticulum, and (D) co-localized effect of target probe corresponding to (A) and endoplasmic reticulum.
In FIG. 14, (A) and (D) are images of fluorescence of the fluorescent probe QM-ONOO and living and in vitro fluorescence of mice with different degrees of liver injury, and (B) and (C) correspond to the quantitative fluorescence intensity of (A).
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The present application will be described in detail with reference to examples and experimental data.
Example 1
Fig. 1 is a synthesis route diagram adopted by the fluorescent probe QM-ONOO according to the present embodiment, and the specific synthesis process is as follows:
1. Synthesis of substance 1
The compound 2-methylquinoline (2.86 g,20 mmol) was taken up in 50ml of toluene and ethyl bromoacetate (5 g,30 mmol) was added under nitrogen and the reaction was warmed to 120℃and refluxed overnight. After the reaction was completed, the solid was cooled to room temperature and precipitated, and the solid after suction filtration was washed with diethyl ether to finally obtain 5.2g of an orange-yellow solid in 84% yield without further purification.
2. Synthesis of substance 2
Substance 1 (5 g,25 mmol) was dissolved in 40 ml of ethanol, malononitrile (3.4 g,50 mmol) and sodium ethoxide (3.4 g,50 mmol) were added under nitrogen, stirred overnight at room temperature, monitored by TLC, the solvent was removed by rotary evaporation after completion of the reaction, dichloromethane extraction, the organic layer was washed with water and saturated brine, the organic layers were combined and dried over sodium sulfate. The solvent is removed by rotary evaporation, and the product with the yield of 22% is obtained by separating and purifying with a silica gel column, wherein the nuclear magnetic resonance hydrogen spectrum and the carbon spectrum are respectively shown in the figure 2/3. The related map data are:
1H NMR(400MHz,Chloroform-d)δ8.91(d,J＝8.4Hz,1H),7.70(t,J＝7.9Hz,1H),7.36(t,J＝8.6Hz,2H),6.71(s,1H),4.98(s,2H),4.28(q,J＝7.0Hz,2H),2.51(s,3H),1.30(t,J＝7.1Hz,3H).
13C NMR(101MHz,Chloroform-d)δ14.011,21.721,49.234,62.748,109.443,115.420,118.329,119.610,120.804,124.706,126.494,133.416,138.736,148.038,153.651,166.617.
HRMS:m/z,cal:293.1164,found:316.1056[M+Na+].
3. Synthesis of substance 3
Material 2 (800 mg,2.73 mmol) was dissolved in a mixed solution of THF/H2O (35 mL/7 mL), naOH (1.09 g,27.3 mmol) was added under nitrogen, warmed to 70℃and reacted overnight. The reaction was concentrated by TLC, water was added until the components were completely dissolved, and dilute hydrochloric acid (1M) was added dropwise until no more solids precipitated. Suction filtration gave 720 mg of substance 3 in 99% yield. The nuclear magnetic resonance hydrogen spectrum and the carbon spectrum are respectively shown in figures 4/5. The related map data are:
1H NMR(400MHz,DMSO-d6)δ8.89(d,J＝8.4Hz,1H),7.88(t,J＝7.8Hz,1H),7.80(m,1H),7.59(m,1H),6.83(s,1H),5.28(s,2H),2.58(s,3H).
13C NMR(101MHz,DMSO-d6)δ21.335,47.793,49.542,108.588,117.525,118.726,120.146,125.069,133.642,138.858,151.055,153.012,168.810.
HRMS:m/z,cal:265.0851,found:266.0935[M+H+].
4. Synthesis of substance 4
Material 3 (265 mg,1 mmol) was dissolved in 10 mL of DMF and EDCI (249 mg,1.3 mmol) and HOBT (176 mg,1.3 mmol) were added slowly at 0deg.C and stirred under nitrogen for half an hour. N- (2-aminoethyl) -4-methylbenzenesulfonamide (279 mg,1.3 mmol) was slowly added to the ice bath at 0deg.C and reacted overnight at room temperature, monitored by TLC. After the completion of the reaction, the reaction mixture was extracted with ethyl acetate, the organic layer was washed with water and saturated brine, dried over sodium sulfate, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography on silica gel to give 290 mg of substance 4 in 62% yield. The nuclear magnetic resonance hydrogen spectrum and the carbon spectrum are respectively shown in figure 6/7. The related map data are:
1H NMR(400MHz,DMSO-d6)δ8.89(d,J＝8.4Hz,1H),8.56(t,J＝5.7Hz,1H),7.85(t,J＝8.0Hz,1H),7.70(d,J＝13.8Hz,3H),7.66(d,2H),7.59(q,J＝7.6,6.8Hz,2H),7.40(d,J＝8.1Hz,2H),6.82(s,1H),5.07(s,2H),3.17(d,J＝6.2Hz,2H),2.79(d,J＝6.3Hz,2H),2.55(s,3H),2.38(s,3H).
13C NMR(151MHz,DMSO-d6)δ20.970,21.503,38.852,41.749,47.261,50.527,108.639,117.465,118.903,120.113,120.332,124.824,126.467,126.506,129.718,137.394,139.090,142.759,151.503,152.953,165.848.
HRMS:m/z,cal:461.1522,found:484.1421[M+Na+].
5. Synthesis of QM-ONOO
Substance 4 (100 mg,0.217 mmol) and 4-formylphenylboronic acid pinacol ester (146 mg,0.627 mmol) were dissolved in 10 ml of acetonitrile and 200 μl piperidine were added dropwise under nitrogen and the reaction was brought to reflux at 80 ℃. After completion of the reaction, dichloromethane was used for extraction, and the organic layer was washed with water and saturated brine. The combined organic layers were dried over sodium sulfate, the solvent removed by rotary evaporation and purified by column chromatography on silica gel to give 45 mg of QM-ONOO in 31% yield. The nuclear magnetic resonance hydrogen spectrum and the carbon spectrum are respectively shown in the figure 8/9. The related map data are:
1H NMR(400MHz,DMSO-d6)δ8.92(dd,J＝8.5,1.5Hz,1H),8.60(t,J＝5.8Hz,1H),7.95–7.85(m,1H),7.80–7.68(m,5H),7.70–7.58(m,4H),7.41(dd,J＝18.0,6.9Hz,4H),7.05(s,1H),5.17(s,2H),3.18(d,2H),2.79(d,2H),2.38(s,3H),1.32(s,12H).
13C NMR(101MHz,DMSO-d6)δ20.944,24.680,24.933,41.658,48.615,51.445,73.489,83.842,106.695,117.750,120.213,121.928,124.962,125.085,126.460,127.394,129.654,133.825,134.803,137.377,137.725,138.990,139.436,142.694,149.861,152.776,166.007.
HRMS:m/z,cal:675.2687,found:698.2576[M+Na+]
experimental example 1: photophysical property determination of the resulting QM-ONOO
(1) 1.0MM QM-ONOO concentrate was prepared in DMSO. Fluorescence spectroscopy experiments were performed in HEPES buffer (10 mM, ph=7.4, containing 1mM CTAB). The excitation wavelength of the fluorescence spectrum is 488nm, and the emission range is 510-750 nm. At the same time, the slit width for excitation and emission was set to 5nm.
Aggregation-induced emission properties of target probes in water: in the mixed solvent of DMSO/H 2 O, the fluorescence intensity of QM-OH is remarkably enhanced with the increase of the water content, and when the water content reaches 95%, the fluorescence intensity reaches a peak value, which indicates that the probe QM-OH has obvious aggregation-induced emission properties (as shown in FIGS. 10A and 10B).
Photophysical Properties of target Probe QM-ONOO: the target probe QM-ONOO buffer prepared in example 1 was diluted to a concentration of 10. Mu.M, and its UV-visible spectrum and fluorescence spectrum were measured using a UV-visible spectrophotometer and a fluorescence spectrophotometer as shown in FIG. 10C. Fluorescence spectrum change of the target probe QM-ONOO on ONOO -- As shown in FIGS. 10D and 10E, the target probe QM-ONOO prepared in example 1 was diluted to a concentration of 10. Mu.M with HEPES buffer, and ONOO - - (0-50 um) was added at different concentrations to test the fluorescence spectrum change of the system. The result shows that the fluorescence intensity of the simple probe QM-ONOO is lower, and the fluorescence intensity sequentially increases with the increase of ONOO --, and the fluorescence intensity reaches a peak value when the alkaline phosphatase concentration reaches 50 um. FIG. 10F is a response time experiment result of the probe, confirming that the probe can respond at 30s and that the fluorescence intensity is stable within 25 min. FIG. 10G shows the fluorescence intensity of the probe at different pH conditions, confirming that pH in the range of 6-13 works on ONOO --. FIG. 10H shows a test for the selectivity of the probe to ONO -- using a variety of representative oxidative molecules and enzymes, and the change in fluorescence spectrum measured under the same conditions, confirming that the probe has a single selectivity to ONO --. The invention can be seen that the synthesized small molecular fluorescent probe can qualitatively/quantitatively detect the ONOO -- level in and out of the body.
Experimental example 2: confocal imaging of target probes QM-ONOO and HepG2 cells
HepG2 cells were cultured in DMEM medium (10% fetal bovine serum) at 37℃in an incubator with 5% CO 2 humidity. Before the fluorescent imaging experiments, hepG2 cells were washed 3 times with PBS buffer (ph=7.4). The excitation wavelength of the confocal image is 488nm, and the emission wavelength is 600-700 nm. The target probe QM-ONOO (10. Mu.M) prepared in example 1 was co-cultured with HepG2 cells, followed by observation with a confocal microscope, as shown in FIGS. 11 and 12. Experimental results show that the probe has obvious imaging effect on HepG2 cells which generate ONOO -. With the addition of endogenous and exogenous ONOO -, the fluorescence intensity was also increased. Inhibitor experiments show that cells previously treated with UA do not exhibit significant fluorescent signals. Compared with a blank control group, the enhancement of fluorescent signals after the target probe is added can be visually seen; pretreatment with inhibitor UA resulted in fluorescence signal comparable to the blank.
Experimental example 3: co-localization experiments of target Probe QM-ONOO on lysosome/endoplasmic reticulum
The target probe QM-ONOO prepared in example 1 was co-cultured with HepG2 cells and co-localized with lysosomes and endoplasmic reticulum-stained Lyso-Tracker and endoplasmic reticulum-stained ER-Tracker. As shown in FIG. 13, the target probe QM-ONOO (10. Mu.M) prepared in example 1 was co-cultured with HepG2 cells for 30min, followed by addition of Lyso-Tracker or ER-Tracker, respectively, and further co-culture for 30min. Experimental results show that in HepG2 cells, the Pelson coefficients of the co-localization effect of the probe QM-ONOO on the endoplasmic reticulum and the lysosome are 0.95 and 0.40 respectively, indicating that the probe is mainly localized on the endoplasmic reticulum of the cells, indicating that the probe has targeting on the endoplasmic reticulum.
Experimental example 4: fluorescence imaging of liver damaged mice with target probe QMP
A liver injury model of mice was established according to the prior art, followed by injection of a target probe QM-ONOO, and live imaging effects were observed in real time, as shown in fig. 14. The experimental results show that the fluorescence signal in normal mice is not obvious, but gradually increases with the increase of the liver injury degree, and is mainly concentrated in the liver part. Thus, it can be shown that liver injury in mice may result in enhanced levels of ONOO-and that the target probe can qualitatively detect ONOO-levels in liver-injured mice.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
Claims (8)
1. An ONOO-fluorescent probe with endoplasmic reticulum targeting, which is characterized by consisting of an AIE framework of quinoline, a response group of boric acid ester and a targeting group of sulfonamide, and is named QM-ONOO, and the structural formula is shown as formula 1:
2. The method for preparing the ONOO - fluorescent probe with endoplasmic reticulum targeting according to claim 1, wherein the ONOO - fluorescent probe is prepared by the following steps:
(1) Dissolving a compound 2-methylquinoline in toluene solution, adding ethyl bromoacetate under the condition of nitrogen to react, heating to 100-120 ℃ and refluxing for 6-12 h, cooling to room temperature after the reaction is finished, separating out solid, and washing the solid after suction filtration by diethyl ether to obtain a substance 1 shown in a formula 2;
(2) Dissolving the substance 1 in ethanol, adding malononitrile and sodium ethoxide under nitrogen, stirring at room temperature for 6-12 h, removing solvent by rotary evaporation after the reaction is completed, extracting with dichloromethane as an organic phase and saturated saline as a water phase, combining and retaining an organic layer, removing the solvent by rotary evaporation, and separating and purifying by a silica gel column to obtain a substance 2 shown in a formula 3;
(3) Dissolving the substance 2 in a THF/H 2 O mixed solution, adding NaOH under the condition of nitrogen, heating to 60-80 ℃, reacting for 6-12H, concentrating the reaction solution, adding water until the components are completely dissolved, dropwise adding dilute hydrochloric acid until the solid is not separated out, and carrying out suction filtration to obtain a substance 3 shown in a formula 4;
(4) Dissolving the substance 3 in DMF, slowly adding EDCI and HOBT under the condition of 0 ℃ and stirring for half an hour under the condition of nitrogen, ice-bathing at 0 ℃, slowly adding N- (2-amino ethyl) -4-methylbenzenesulfonamide, reacting at room temperature for 12 hours, extracting with ethyl acetate as an organic phase and saturated saline as a water phase after the reaction is completed, merging the organic layers, removing the solvent by rotary evaporation, and separating and purifying by a silica gel column to obtain a substance 4 shown in a formula 5;
(5) Dissolving substances 4 and 4-formylphenylboronic acid pinacol ester in acetonitrile, dropwise adding piperidine under the condition of nitrogen, reacting to 70-100 ℃ for reflux reaction overnight, extracting with dichloromethane as an organic phase and saturated saline as a water phase after the reaction is finished, merging the organic layers, steaming to remove the solvent, and separating and purifying by a silica gel column to obtain QM-ONOO shown in formula 1.
3. The method of claim 2, wherein in step (1), the molar ratio of 2-methylquinoline to ethyl bromoacetate is 1:1-1.5.
4. The method of claim 2, wherein in step (2), the molar ratio of the substance 1, malononitrile and sodium ethoxide is 1:2:2.
5. The method of claim 2, wherein in the step (3), the molar ratio of the substance 2 to NaOH is 1:1, and the volume ratio of THF/H 2 O in the mixed solution of THF/H 2 O is 5:1.
6. The method of claim 2, wherein in step (4), the molar ratio of the substance 3 to EDCI, HOBT, N- (2-aminoethyl) -4-methylbenzenesulfonamide is 1:1.3:1.3:1.3.
7. The method of claim 2, wherein in step (5), the molar ratio of the substance 4 to 4-formylphenylboronic acid pinacol ester is 1:2-3.
8. Use of an ONOO-fluorescent probe with endoplasmic reticulum targeting according to claim 1 as a fluorescent probe for detecting ONOO-level fluctuations under endoplasmic reticulum oxidative stress in vivo.
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