Near-infrared fluorescent probe DCM-MBS for detecting ONOO <-> as well as preparation method and application of near-infrared fluorescent probe DCM-MBS

Abstract

The invention discloses a near-infrared fluorescent probe DCM-MBS for detecting ONOO <-> as well as a preparation method and application of the near-infrared fluorescent probe DCM-MBS, and belongs to the technical field of peroxynitroso ion detection. The probe DCM-MBS is a water-soluble probe, the detection method is simple, ONOO <-> response is rapid, selectivity is good, specificity is high, detection sensitivity is high, interference of other biological molecules, active substances and the background of the probe can be effectively avoided, repeatability, accuracy and reliability of a detection result are ensured, and the detection method is suitable for large-scale popularization and application. Further, the requirement of real-time monitoring of the rapidly-changed ONOO <-> level in a living body is met; the color change is obvious and easy to observe, and qualitative in-situ real-time detection of ONOO <-> can be realized by naked eyes; the fluorescent probe has very excellent biocompatibility, can realize excellent fluorescence imaging in various cells and living mouse bodies, and does not have obvious toxic or side effects; the synthesis method is simple and efficient, raw materials are low in cost and easy to obtain, large-scale industrial production is facilitated, and wide market application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of peroxynitrite ion detection, in particular to a method for detecting ONOO - Near-infrared fluorescent probe DCM-MBS and its preparation method and application. Background Art

[0002] Drug-induced liver injury (DILI) is a common liver disease in clinical practice and a serious threat to human health. According to incomplete statistics, the number of patients hospitalized due to drug-induced liver injury is increasing year by year worldwide, and some patients may even develop liver failure, which is life-threatening. The pathogenesis of DILI is complex, among which the excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) plays a key role in its pathological occurrence and development. Peroxynitrite (ONOO - ) is a highly oxidizing active nitrogen species, which is composed of nitric oxide (NO) and superoxide anion (O2 - ) Rapid reaction generation. In drug-induced liver injury, due to the disorder of drug metabolism, NO and O2 - The imbalance of ONOO - Excessive ONOO - It will attack biological macromolecules such as proteins, lipids and nucleic acids in cells, triggering oxidative stress and nitrative stress, leading to liver cell damage, apoptosis and even necrosis, further aggravating the liver's inflammatory response and dysfunction.

[0003] Therefore, accurate detection of ONOO in drug-induced liver injury is necessary. - The level of ONOO is of great significance for the in-depth understanding of the pathogenesis of DILI, early diagnosis and the development of effective treatment strategies. Traditional detection methods such as chemiluminescence and electron spin resonance can achieve ONOO to a certain extent. - However, it has the disadvantages of complex operation, low sensitivity, poor selectivity and inability to achieve in situ real-time detection. In contrast, fluorescent probe technology has become a popular method for detecting ONOO due to its unique advantages such as high sensitivity, high selectivity, good biocompatibility and ability to perform in situ, real-time and visual detection of targets. - Therefore, a method to specifically detect ONOO - The water-soluble fluorescent probe has important practical significance. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting ONOO - The near-infrared fluorescent probe DCM-MBS and its preparation method and application are used to solve the problem of ONOO in the prior art. -The detection has problems such as complex operation, low sensitivity, poor selectivity and inability to achieve in-situ real-time detection.

[0005] To achieve the above object, the present invention provides a method for detecting ONOO - The near-infrared fluorescent probe DCM-MBS has the structural formula:

[0006]

[0007] A method for detecting ONOO as described above - The preparation method of the near-infrared fluorescent probe DCM-MBS comprises the following steps:

[0008] S1. Synthesizing Compound 1: Dissolve isophorone and malononitrile in anhydrous ethanol, add piperidine and reflux for reaction. After the reaction, evaporate to dryness to obtain a crude product. Purify the crude product to obtain Compound 1;

[0009] S2, synthesizing compound 2, dissolving compound 1 prepared in S1 and p-acetaminobenzaldehyde in anhydrous acetonitrile, adding piperidine under a protective atmosphere and reflux reacting, and washing the precipitate after the reaction to obtain compound 2;

[0010] S3, synthesizing compound 3, adding compound 2 prepared in S2 to a mixture of concentrated hydrochloric acid and anhydrous ethanol, stirring, neutralizing, extracting, removing water and solvent, and purifying to obtain compound 3;

[0011] S4, synthesizing the probe DCM-MBS, dissolving the compound 3 obtained in S3 in dichloromethane, adding triethanolamine dropwise, stirring, and then adding p-toluenesulfonyl chloride dropwise. Stirring under a protective atmosphere overnight, purifying the organic layer to obtain the probe DCM-MBS;

[0012] The reaction formula is as follows:

[0013]

[0014] Preferably, the equivalent ratio of isophorone:malononitrile:anhydrous ethanol:piperidine in S1 is 1:2:10:0.01; and the reflux reaction conditions are 80-85° C. for 10-15 hours.

[0015] Preferably, the evaporation to dryness in S1 is performed by evaporating to dryness under reduced pressure using a rotary evaporator or a freeze dryer; and the crude product is purified by silica gel column chromatography using petroleum ether as the eluent.

[0016] Preferably, in S2, the equivalent ratio of compound 1: p-acetaminobenzaldehyde: anhydrous acetonitrile: piperidine is 1:1:10:0.3; and washing is performed with acetonitrile.

[0017] Preferably, in S3, the equivalent ratio of compound 2: concentrated HCl: absolute ethanol is 0.1:1:2; sodium hydroxide solution is used for neutralization; extraction is carried out using a mixed solution of saturated brine and ethyl acetate, and the volume ratio of saturated brine: ethyl acetate is 1:1.

[0018] Preferably, anhydrous sodium sulfate is used for drying in S3; solvent removal is carried out by vacuum solvent removal method, and the vacuum condition is 0.01 MPa; purification is carried out by silica gel column purification, and the eluent is a mixture of petroleum ether and dichloromethane, and the volume ratio of petroleum ether: dichloromethane = 1:9.

[0019] Preferably, in S4, the equivalent ratio of compound 3: dichloromethane: triethanolamine: tosyl chloride is 1:5:1.2:2; after adding triethanolamine dropwise, stir for 15 - 30 minutes; the organic layer is extracted with ethyl acetate; purification is carried out by silica gel column purification, and the eluent is a mixture of petroleum ether and dichloromethane, and the volume ratio of petroleum ether: dichloromethane = 3:7.

[0020] A method for detecting ONOO - using the above near-infrared fluorescent probe DCM-MBS. After mixing and incubating the probe DCM-MBS, the test solution, and PBS, the change in fluorescence signal is detected using a fluorescence detection instrument, and qualitative and quantitative analysis of ONOO - can be achieved.

[0021] An application of the above-described near-infrared fluorescent probe DCM-MBS for detecting ONOO - in the preparation of an ONOO - detection reagent.

[0022] Therefore, the present invention provides a near-infrared fluorescent probe DCM-MBS for detecting ONOO - and its preparation method and application, and its specific technical effects are as follows:

[0023] (1) The probe DCM-MBS provided by the present invention exhibits fluorescence characteristics at 562 nm in the initial state; when reacting with ONOO - , its fluorescence spectrum redshifts to 648 nm and strong red fluorescence is generated. The color change of the reaction system can be observed with the naked eye, and qualitative in-situ real-time detection of ONOO - can be achieved with the naked eye;

[0024] (2) The probe DCM-MBS provided by the present invention responds to ONOO - rapidly and has high detection sensitivity. The highest fluorescence intensity can be reached in 180 seconds, and the lowest detection limit can reach 197 nM, which can meet the requirement of real-time monitoring of the rapidly changing ONOO - level in vivo;

[0025] (3) The probe DCM-MBS provided by the present invention has good selectivity and strong specificity, and can accurately recognize ONOO in a complex biological system - , which can effectively avoid the interference of other biomolecules, active substances and its own background, and ensure the repeatability, accuracy and reliability of the detection results;

[0026] (4) The probe DCM-MBS provided by the present invention is a water-soluble probe, and the detection method is simple. The reaction can be carried out in a system without adding any organic reagents. It has excellent biocompatibility, can achieve excellent fluorescence imaging in a variety of cells and live mice, and no obvious toxic and side effects are observed;

[0027] (5) The probe DCM-MBS can be successfully synthesized by using the preparation method provided by the present invention. The synthesis method is simple, efficient, the raw material cost is low and easy to obtain, which is conducive to large-scale industrial production and has broad market application prospects.

[0028] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the probe DCM-MBS prepared in Example 1 of the present invention;

[0031] Figure 2 is the nuclear magnetic resonance carbon spectrum of the probe DCM-MBS prepared in Example 1 of the present invention;

[0032] Figure 3 is the high-resolution mass spectrum of the probe DCM-MBS prepared in Example 1 of the present invention;

[0033] Figure 4 is the fluorescence spectrum of the probe DCM-MBS in Example 2 of the present invention when ONOO - is added in different co-solvents and different buffers; among which (A) is the fluorescence spectrum in different organic solvents, and (B) is the fluorescence spectrum in different buffers;

[0034] Figure 5 is the fluorescence emission spectrum, fluorescence intensity and fluorescence intensity ratio (I - of the probe DCM-MBS with the change of ONOO concentration in Example 2 of the present invention 655 / I 572 ) and ONOO - concentration linear relationship; where (A) is the color change of the probe DCM-MBS with increasing ONOO - concentration under naked-eye observation; (B) is the titration fluorescence emission spectrum and fluorescence intensity of the probe DCM-MBS with different concentrations of ONOO - ; (C) is the linear relationship diagram of the fluorescence intensity ratio (I 655 / I 572 ) and ONOO - concentration;

[0035] Figure 6 are the color photos of the probe DCM-MBS after reacting with different ions in Example 2 of the present invention; where (A) is the color change photo under ultraviolet light; (B) is the color change photo under daylight; 1 in the figure is Blank; 2 is ONOO - ; 3 is SO3 2- ; 4 is Cl - ; 5 is I - ; 6 is SO4 2- ; 7 is IO4 - ; 8 is Fe 2+ ; 9 is ClO - ; 10 is Cu 2+ ; 11 is Co 2+ ; 12 is HSO4 - ; 13 is S2O5 2- ; 14 is CN - ; 15 is Fe 3+ ; 16 is HSO3 - ; 17 is ClO4 - ; 18 is S 2- ; 19 is AcO - ; 20 is K + ; 21 is S2O3 2- ; 22 is O2 1 ; 23 is Ca 2+ ; 24 is Mg 2+ ; 25 is Al 3+ ; 26 is Br - ; 27 is Pb 2+ ; 28 is Sn 2+ ; 29 is Cu + ; 30 is Ba 2+ ;

[0036] Figure 7 is the statistical result of the selectivity and competitiveness of the probe DCM-MBS for detecting ONOO - in Example 2 of the present invention; where 1 is ONOO -; 2 is Black; 3 is SO3 2- ; 4 is Cl - ; 5 is I - ; 6 is SO4 2- ; 7 is IO4 - ; 8 is Fe 2+ ; 9 is ClO - ; 10 is Cu 2 + ; 11 is Co 2+ ; 12 is HSO4 - ; 13 is S2O5 2- ; 14 is CN - ; 15 is Fe 3+ ; 16 is HSO3 - ; 17 is ClO4 - ; 18 is S 2- ; 19 is AcO - ; 20 is K + ; 21 is S2O3 2- ; 22 is O2 1 ; 23 is Ca 2+ ; 24 is Mg 2+ ; 25 is Al 3+ ; 26 is Br - ; 27 is Pb 2+ ; 28 is Sn 2 + ; 29 is Cu + ; 30 is Ba 2+ ;

[0037] Figure 8 are the investigation results of the influence of time on the probe DCM-MBS in Example 2 of the present invention; where (A) is the fluorescence emission spectrum of the probe DCM-MBS before and after reacting with ONOO - ; (B) is the ultraviolet absorption spectrum of the probe DCM-MBS before and after reacting with ONOO - ; (C) is the curve of the fluorescence intensity changing with time;

[0038] Figure 9 are the investigation results of the influence of pH on the probe DCM-MBS in Example 2 of the present invention;

[0039] Figure 10 are the experimental results of the cytotoxicity of different concentrations of the probe DCM-MBS on HCT116 cells in Example 2 of the present invention;

[0040] Figure 11 are the confocal imaging diagrams of different concentrations of ONOO - in A549 cells in Example 3 of the present invention;

[0041] Figure 12 It is the confocal imaging diagram of different concentrations of ONOO in Example 3 of the present invention - in HCT116 cells;

[0042] Figure 13 It is the confocal imaging diagram of different concentrations of ONOO in Example 3 of the invention - in Hk2 cells;

[0043] Figure 14 It is the in vivo imaging diagram of the probe DCM-MBS in drug-induced liver injury mice and normal mice and the liver tissue section diagram after dissection in Example 4 of the present invention; wherein (A) is the in vivo fluorescence imaging diagram of normal mice and liver injury model mice after injecting the probe DCM-MBS over time; (B) is the liver tissue section diagram of normal mice and liver injury model mice 1 h after injecting the probe DCM-MBS; (C) is the in vivo fluorescence imaging diagram of the kidneys, lungs, pancreas, liver, and heart of normal mice and liver injury model mice after injecting the probe DCM-MBS; (D) is the photo of the kidneys, lungs, pancreas, liver, and heart of normal mice and liver injury model mice under sunlight after injecting the probe DCM-MBS. Specific embodiments

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and examples.

[0045] In order to make the purpose, technical solution and advantages of the present application clearer, more thorough and complete, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0046] The instruments, equipment, reagents and materials used in the examples are all obtained through commercial channels; the method steps not described in detail are all conventional technical means in the art.

[0047] Example 1

[0048] Prepare the fluorescent probe DCM-MBS, and the synthesis route is as follows:

[0049]

[0050] The specific steps are as follows:

[0051] (1) Synthesize compound 1.

[0052] Accurately weigh isophorone (5.0 g, 36.2 mmol, 1.0 equiv.) and malononitrile (5.0 g, 75.7 mmol, 2.0 equiv.) in a 250.0 mL round-bottom flask, add 150.0 mL of anhydrous ethanol (EtOH, 10 equiv.) to fully dissolve, then add piperidine (112.0 mg, 0.362 mmol, 0.01 equiv.) and reflux at 82°C for 12 h. After the reaction is complete, evaporate to dryness under reduced pressure on a rotary evaporator (reduced pressure condition is 0.01 MPa), and the resulting product is purified by silica gel column chromatography (eluent is petroleum ether, PE) to obtain a white solid, which is compound 1, totaling 4.5 g (yield is 66.0%, R f =0.39, and the developing solvent was petroleum ether:ethyl acetate (PE:EtOAc) with a volume ratio of 10:1).

[0053] (2) Synthesis of compound 2.

[0054] In a 25.0 mL round-bottom flask, p-acetaminobenzaldehyde (163.2 mg, 1.0 mmol, 1.0 equiv.) and the prepared compound 1 (186.3 mg, 1.0 mmol, 1.0 equiv.) were added and dissolved with 5.0 mL of anhydrous acetonitrile (10.0 equiv.). 60 μL of piperidine (0.6 mmol, 0.3 equiv.) was added at room temperature under nitrogen protection, and the mixture was refluxed at 82°C for 1 hour. After the reaction was completed, the precipitate was washed with acetonitrile to obtain an orange solid, which was compound 2, totaling 246.2 mg (yield 74.3%, R f =0.55, developing solvent was PE:EtOAc volume ratio =6:1).

[0055] (3) Synthesis of compound 3.

[0056] The prepared compound 2 (125.0 mg, 0.38 mmol, 0.1 equiv.) was added to a mixture of concentrated HCl (10 mL, 1.0 equiv.) and EtOH (20 mL, 2.0 equiv.), and the mixture was stirred at 80°C for 5.5 h. After the reaction was completed, the pH was adjusted to 7.0 with 0.1 M sodium hydroxide solution. Then, the aqueous solution was extracted with a mixture of saturated brine and ethyl acetate (volume ratio of 1:1), and the obtained product was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure (reduced pressure condition of 0.01 MPa) to obtain a crude compound 3. The crude compound 3 was purified by silica gel column (eluent: PE:DCM volume ratio = 1:9), and the red solid compound obtained was compound 3, a total of 90.2 mg (yield 82.0%, R f =0.65, developing solvent: PE:EtOAc=6:1).

[0057] (4) Synthesize the probe DCM-MBS.

[0058] Accurately weigh the prepared compound 3 (28.0 mg, 0.1 mmol, 1.0 equiv.) and place it in a 25 mL reaction flask. Add 5 mL of dichloromethane (DCM, 5.0 equiv.) to dissolve it completely. Then, under ice bath conditions, add triethylamine (TEA, 18 μL, 0.12 mmol, 1.2 equiv.) dropwise and stir for 20 min. Next, add p-toluenesulfonyl chloride (38.6 mg, 0.2 mmol, 2.0 equiv.) dropwise into the flask and stir overnight under nitrogen. The obtained product is extracted with ethyl acetate, the organic layer is collected, and purified by silica gel column (the eluent is PE:DCM volume ratio = 3:7). Finally, the obtained yellow solid compound is the probe DCM-MBS, 20 mg in total (yield 42%, R f = 0.55, the developing agent is PE:EtOAc volume ratio = 4:1).

[0059] The 1H NMR spectrum of the probe DCM-MBS is as Figure 1 shown, and the 13C NMR spectrum is as Figure 2 shown, and the high-resolution mass spectrum is as Figure 3 shown.

[0060] The 1H NMR data of the probe DCM-MBS are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.2 Hz, 2H), 7.23 (s, 1H), 7.18 (s, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.81 (s, 1H), 2.60 (s, 3H), 2.32 (s, 4H), 0.99 (s, 6H).

[0061] The 13C NMR data of the probe DCM-MBS are as follows: 13 C NMR (101 MHz, DMSO-d6) δ 170.39, 156.07, 143.60, 139.14, 137.08, 136.60, 131.50, 129.87, 129.05, 128.58, 126.81, 122.47, 119.39, 76.02, 42.36, 38.20, 31.76, 27.49, 21.05.

[0062] Example 2

[0063] Perform performance tests on the fluorescent probe DCM-MBS prepared in Example 1 as follows:

[0064] (1) Fluorescence spectrum test.

[0065] 1) Determine the optimal co-solvent. Add 100.0 μL of probe DCM-MBS (0.1 mmol / L) and 100.0 μL of ONOO - ion solution (0.1 mmol / L) into a colorimetric tube, then add 1.0 mL of different organic reagents (DMSO, ACN, DMF, MeOH, THF, DXA, with deionized water as the control) and 1.0 mL of phosphate buffer solution (PBS) respectively, and then fix the reaction system to 5.0 mL with deionized water, and scan the fluorescence spectra and ultraviolet absorption spectra of each reaction system. The excitation and emission slit widths of the fluorescence spectrum are set to 10 nm and 10 nm respectively, and the voltage is set to 700 V. The results are as Figure 4 shown in (A). The experiment shows that in the system of deionized water and PBS buffer solution, the fluorescence intensity of the fluorescence probe is the best and the Stokes shift is the largest. Therefore, no organic co-solvent is added in the subsequent experiments.

[0066] 2) Determine the optimal buffer solution. Add 100.0 μL of probe DCM-MBS (0.1 mmol / L) and 100.0 μL of ONOO - ion solution (0.1 mmol / L) into a colorimetric tube, then add 1.0 mL of deionized water and 1.0 mL of different buffer solutions (PBS, HEPES, Tris) respectively, and then fix the reaction system to 5.0 mL with deionized water, and scan the fluorescence spectra and ultraviolet absorption spectra of each reaction system. The excitation and emission slit widths of the fluorescence spectrum are set to 10 nm and 10 nm respectively, and the voltage is set to 700 V. The results are as Figure 4 shown in (B). The probe has the best response performance in PBS, and in the system of deionized water and PBS, that is, in the system without adding any organic reagents, the probe has the best response effect.

[0067] 3) Determine the lowest detection limit.

[0068] Add 100.0 μL of probe DCM-MBS (0.1 mmol / L) and 100.0 μL of ONOO with different concentrations - (4.0 - 16.0 μM) ion solution into a colorimetric tube, then add 1.0 mL of deionized water and 1.0 mL of PBS buffer solution respectively, and then fix the reaction system to 5.0 mL with deionized water, and scan the fluorescence spectra and ultraviolet absorption spectra of each reaction system. The excitation and emission slit widths of the fluorescence spectrum are set to 10 nm and 10 nm respectively, and the voltage is set to 700 V. The results are as Figure 5 shown. The probe DCM-MBS shows a good linear response to ONOO - (4.0 - 10.0 μM), and the correlation coefficient R2 Reached 0.9901, and the lowest detection limit was 197.0 nM, indicating that the probe DCM-MBS can be used to monitor the concentration change of ONOO - .

[0069] 4) Investigate the selectivity and anti-interference ability (competitiveness) of the probe DCM-MBS. Add 100.0 μL of the probe DCM-MBS (0.1 mmol / L), 100.0 μL of 0.1 M ONOO - ion solution or some common interfering ions (such as Fe 3+ , K + , Ca 2+ , Mg 2+ , etc.) into a colorimetric tube, then add 1.0 mL of deionized water and 1.0 mL of PBS buffer respectively, and then fix the reaction system to 5.0 mL with deionized water. Scan the fluorescence spectra and ultraviolet absorption spectra of each reaction system. The excitation and emission slit widths of the fluorescence spectra are set to 10 nm and 10 nm respectively, and the voltage is set to 700 V. The photos of the probe DCM-MBS after reacting with different ions are as shown in Figure 6 , and the statistical results are as shown in Figure 7 . After adding ONOO - , the fluorescence signal of the probe DCM-MBS at 655 nm increased significantly, while adding other analytes hardly caused a fluorescence shift.

[0070] In the competitive experiment, in 100.0 μL of the probe DCM-MBS (0.1 mmol / L), 100.0 μL of ONOO - (0.1 mmol / L), 1.0 mL of deionized water and 1.0 mL of PBS buffer, then add 100.0 μL of each interfering ion solution (0.1 mmol / L) respectively, and then fix the reaction system to 5.0 mL with deionized water. Scan the fluorescence spectra and ultraviolet absorption spectra of each reaction system. The excitation and emission slit widths of the fluorescence spectra are set to 10 nm and 10 nm respectively, and the voltage is set to 700 V. The photos of the probe DCM-MBS after reacting with different ions are as shown in Figure 6 , and the statistical results are as shown in Figure 7 . It was found that other interfering factors did not affect the detection of ONOO - by the probe DCM-MBS. The above results indicate that the probe DCM-MBS has excellent selectivity and strong anti-interference ability for ONOO - .

[0071] 5) Investigate the stability and pH tolerance of the probe DCM-MBS.

[0072] First, investigate the reaction of the probe DCM-MBS (10.0 μM) with ONOO- (4.0 - 10.0 μM) The kinetic properties of the reaction. As Figure 8 shown in (A) of, in the absence of ONOO - , the probe DCM-MBS has strong fluorescence at 562 nm. When ONOO - is added, the fluorescence signal of the probe undergoes a red shift, and the fluorescence signal at 648 nm is significantly enhanced and reaches its peak within 3 min.

[0073] Then, it was evaluated whether the probe DCM-MBS could detect ONOO under physiological pH conditions. - By changing the pH value (2 - 11) of the buffer solution in the reaction system and performing spectral scanning, the experimental results showed that the probe DCM-MBS had a good fluorescence response to ONOO - in the range of pH = 6 - 10 ( Figure 9 ), indicating that the probe DCM-MBS can detect changes in the concentration of ONOO under physiological conditions. -

[0074] (2) Biocompatibility test.

[0075] The CCK-8 method was used to evaluate the toxicity of the probe DCM-MBS to HCT-116 cells. First, 100.0 μL of cell suspension was added to a 96-well plate, and then 100 μL of DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin was added. The 96-well plate was placed in an incubator at 37 °C and 5% CO2 for 24 hours. Subsequently, 8 different concentrations of the probe DCM-MBS at 0.0 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, 10.0 μM, 15.0 μM, and 20.0 μM were added to the above system, and incubation was continued for 24 hours. Then, 0.1 mL of CCK-8 solution was added, and incubation was continued for 4 hours. Finally, the absorbance at 450 nm of each well (or reaction system) was recorded, and the cell viability was calculated using the following formula:

[0076] Cell viability (%) = (A 加药 - A 空白 ) / (A 不加药 - A 空白 ) × 100%

[0077] where A 加药 refers to the cell group after co-incubation of the probe solution and the CCK-8 solution; A 空白 refers to the blank control, which is the cell group without adding the probe solution or the CCK-8 solution; A 不加药 refers to the negative control group, which is the cell group incubated only with the CCK-8 solution.

[0078] The results are as Figure 10 shown, and even at a concentration of 20 μM ONOO - , the survival rate of the cells can still reach more than 80%.

[0079] Example 3

[0080] The fluorescent probe DCM-MBS prepared in Application Example 1 was used to detect peroxynitrite ions in cells, as follows:

[0081] An appropriate amount of HCT-116, A549, and Hk2 cell suspensions were routinely cultured in a 37 °C, 5% CO2 incubator until the logarithmic growth phase. After the culture was completed, 100 μL of a probe DCM-MBS solution with a concentration of 10.0 μM was added to each culture system, and incubation was continued for 20 minutes to allow the probe to fully interact with the cells. After incubation, washing was performed 3 times with PBS to completely remove the unbound probe. Subsequently, 4 different concentrations (0.0 μM, 5.0 μM, 10.0 μM, 20.0 μM in sequence) of peroxynitrite ion (ONOO - ) solutions were added to the above-mentioned cell systems after washing treatment, and incubation was continued for 15 minutes to allow ONOO - to fully react with the cells and the probe. After the reaction was completed, washing was performed 3 times again with PBS to terminate the reaction and remove the residual ONOO - solution. The treated cell samples were placed under a confocal microscope for fluorescence imaging analysis to obtain relevant information such as the distribution and intensity changes of the fluorescence signal in the cells.

[0082] The results are as Figures 11 - 13 shown, and it can be clearly observed from these figures that as the concentration of ONOO - increases, the red fluorescence in the cells gradually increases.

[0083] Example 4

[0084] Examine the in vivo imaging effect of the fluorescent probe DCM-MBS prepared in Example 1 in mice with drug-induced liver injury, as follows:

[0085] BALB / C mice weighing between 18 and 22 g were selected as the experimental model, and the experimental procedures complied with international ethical guidelines. The mice were randomly divided into two groups: a control group and a drug-induced liver injury model group (referred to as the model group), with 10 mice in each group. The mice in the model group were intraperitoneally injected with acetaminophen (APAP) solution at a dose of 300 mg / kg to construct a drug-induced liver injury mouse model. The mice in the control group were intraperitoneally injected with an equal volume of normal saline. Twelve hours after the injection of APAP or normal saline, a fluorescent probe was injected into the two groups of mice via the tail vein, and the injection dose of the probe was 10 mg / kg for both groups. At 0.5 h, 1 h, 2 h, 4 h, and 6 h after the injection of the fluorescent probe, the mice were placed on the stage of the in vivo imaging system, the position was adjusted, and the distribution and intensity changes of the fluorescent signal in the mice were collected under the condition of an excitation wavelength of 520 nm.

[0086] The results were as Figure 14 shown. The in vivo imaging results showed that only weak fluorescent signals appeared in the liver of the control group mice 0.5 h after the injection of the fluorescent probe, and the fluorescence intensity did not change significantly over time. In contrast, obvious fluorescent signals appeared in the liver of the drug-induced liver injury model group mice 0.5 h after the injection of the fluorescent probe, and although the fluorescence intensity decreased with the extension of time, strong fluorescent signals could still be observed at 4 h ( Figure 14 A). Subsequently, the control group and the model group mice were dissected, and their visceral tissues were removed. Observed under daylight, the fresh liver of the control group mice was darker in color, with clear structure, tight and shiny membranes, and clear outlines of the liver lobes. In contrast, the livers of the liver injury group mice showed signs of collapse and lighter color, unclear lobulation, loose capsules, flat and collapsed areas, pale color, and thin edges ( Figure 14 D). Observed under fluorescence, the fluorescence intensity of the livers of the drug-induced liver injury model group mice was significantly higher than that of the control group mice ( Figure 14 C). This clearly indicated that the fluorescent probe could specifically accumulate in the liver tissues with drug-induced liver injury in mice and react with ONOO - generated due to drug injury in the liver tissue to produce obvious fluorescent signals, thereby achieving the effective detection of ONOO - in drug-induced liver injury.

[0087] In addition, HE staining was performed on the mouse liver tissues ( Figure 14 B). The hepatocytes in the control group were arranged in an orderly manner, and no pathological conditions were observed. In contrast, the liver tissues treated with APAP showed hepatocyte necrosis, hepatocyte edema, and signs of venous congestion, and the results of these liver tissue observations confirmed the successful establishment of the liver injury model. This result further confirmed that the fluorescent probe could accurately detect ONOO in the liver tissues with drug-induced liver injury in vivo- The generation and distribution provide a powerful visualization tool for in-depth study of the pathological mechanism of drug-induced liver injury.

[0088] Therefore, the probe DCM-MBS provided by the present invention is a water-soluble probe, with a simple detection method, responsive to ONOO - rapidly, with good selectivity, strong specificity, and high detection sensitivity. It can effectively avoid the interference of other biomolecules, active substances, and its own background, ensuring the repeatability, accuracy, and reliability of the detection results, and thus meeting the need for real-time monitoring of the rapidly changing ONOO level in vivo; with a significant color change and easy to observe, qualitative in-situ real-time detection of ONOO can be achieved with the naked eye; having excellent biocompatibility, it can achieve excellent fluorescence imaging in various cells and live mice, and no obvious toxic and side effects are observed; the synthesis method is simple and efficient, the raw material cost is low and easy to obtain, which is conducive to large-scale industrial production, and has broad market application prospects. - The color change is significant and easy to observe, and qualitative in-situ real-time detection of ONOO can be achieved with the naked eye. - Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A near-infrared fluorescence probe DCM-MBS for detecting ONOO - , characterized in that The structural formula of the near-infrared fluorescent probe is as follows:

2. A preparation method of a near-infrared fluorescence probe DCM-MBS for detecting ONOO - , characterized in that It includes the following steps: S1. Synthesize compound 1. Dissolve isophorone and malononitrile in absolute ethanol, add piperidine and reflux for reaction. After the reaction is completed, evaporate to dryness to obtain a crude product, and purify the crude product to obtain compound 1. S2. Synthesize compound 2. Dissolve compound 1 prepared in S1 and p-acetamidobenzaldehyde in absolute acetonitrile, add piperidine and reflux for reaction under a protective atmosphere. After the reaction is completed, wash the precipitate to obtain compound 2. S3. Synthesize compound 3. Add compound 2 prepared in S2 to a mixed solution of concentrated hydrochloric acid and absolute ethanol, stir, then neutralize and extract successively. After removing water and solvent, purify to obtain compound 3. S4. Synthesize the probe DCM-MBS. Dissolve compound 3 obtained in S3 in dichloromethane, add triethanolamine dropwise, stir, then add p-toluenesulfonyl chloride dropwise, stir overnight under a protective atmosphere, and purify the organic layer to obtain the probe DCM-MBS. The reaction formula is as follows:

3. A preparation method of a near-infrared fluorescence probe DCM-MBS for detecting ONOO - , characterized in that: In S1, the equivalent ratio of isophorone: malononitrile: absolute ethanol: piperidine is 1:2:10:0.01; the reflux reaction conditions are 80 - 85 °C for 10 - 15 hours.

4. A method for preparing a near-infrared fluorescence probe DCM-MBS for detecting ONOO - , characterized by: In S1, evaporation to dryness is carried out by rotary evaporator under reduced pressure or freeze dryer; the purification of the crude product is carried out by silica gel column chromatography, and the eluent is petroleum ether.

5. A method for preparing a near-infrared fluorescent probe DCM-MBS for detecting ONOO - , characterized in that: In S2, the equivalent ratio of compound 1: p-acetamidobenzaldehyde: absolute acetonitrile: piperidine is 1:1:10:0.3; washing is carried out with acetonitrile.

6. A preparation method of a near-infrared fluorescent probe DCM-MBS for detecting ONOO - , characterized by: In S3, the equivalent ratio of compound 2: concentrated HCl: absolute ethanol is 0.1:1:2; neutralization is carried out with sodium hydroxide solution; extraction is carried out with a mixed solution of saturated brine and ethyl acetate, and the volume ratio of saturated brine: ethyl acetate is 1:

1.

7. A method for preparing a near-infrared fluorescent probe DCM-MBS for detecting ONOO - , characterized in that: In S3, drying is carried out with anhydrous sodium sulfate; removal of solvent is carried out by reduced pressure solvent removal method, and the reduced pressure condition is 0.01 MPa; purification is carried out by silica gel column purification, and the eluent is a mixture of petroleum ether and dichloromethane, and the volume ratio of petroleum ether: dichloromethane = 1:

9.

8. A preparation method of a near-infrared fluorescent probe DCM-MBS for detecting ONOO - , characterized in that: In S4, the equivalent ratio of compound 3: dichloromethane: triethanolamine: p-toluenesulfonyl chloride is 1:5:1.2:2; stir for 15 - 30 minutes after adding triethanolamine dropwise; the organic layer is extracted with ethyl acetate; purification is carried out by silica gel column purification, and the eluent is a mixture of petroleum ether and dichloromethane, and the volume ratio of petroleum ether: dichloromethane = 3:

7.

9. A method for detecting ONOO using the near-infrared fluorescence probe DCM-MBS described in claim 1 - , characterized in that: After incubating the probe DCM-MBS, the test solution, and PBS, the change in fluorescence signal is detected using a fluorescence detection instrument, and qualitative and quantitative analysis of ONOO - can be achieved.

10. An application of the near-infrared fluorescent probe DCM-MBS for detecting ONOO as described in claim 1 - in the preparation of a detection reagent for ONOO - is provided.

Images