A method for detecting ONOO - Near-infrared fluorescent probe and its preparation method and application

Abstract

The present invention discloses a method for detecting ONOO ‑ The near-infrared fluorescent probe and its preparation method and application belong to the field of peroxynitrite ion detection technology. ‑ The near-infrared fluorescent probe has a fast detection speed and can reach the highest fluorescence intensity within 60 seconds; the reaction is obvious and easy to observe. ‑ After the reaction occurs, strong red fluorescence is generated, and the color change of the solution can be observed with the naked eye; it has strong selectivity and high sensitivity, which can effectively avoid interference from other biological molecules and active substances, ensuring the accuracy and reliability of the detection results, and the minimum detection limit can reach 107nM; it has good biocompatibility and low toxicity in the body, and will not adversely affect the normal physiological functions of biological samples; the preparation method provided is simple and easy to operate, the raw materials are low in cost and readily available, the synthesis efficiency is high, it can be used for large-scale industrial production, and has broad application prospects.

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 and its preparation method and application. Background Art

[0002] Peroxynitrite ion (ONOO - As an important reactive oxygen and nitrogen species, peroxynitrite (PN) plays a key role in physiological and pathological processes in vivo. Abnormal levels of PN are closely associated with a variety of diseases, such as inflammation, neurodegenerative diseases, and cardiovascular disease. Therefore, developing a method for rapid, sensitive, and highly selective detection of peroxynitrite in vivo with the naked eye is of great scientific significance and clinical application value.

[0003] Near-infrared fluorescent probes have attracted widespread attention in the fields of bioimaging and biodetection due to their advantages, such as deep tissue penetration, low background interference, and minimal damage to biological samples. However, existing peroxynitrite ion fluorescent probes still have limitations in detection speed, sensitivity, selectivity, and biocompatibility, making them difficult to meet the needs of practical applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting ONOO - The invention provides a near-infrared fluorescent probe and its preparation method and application, so as to provide an ONOO with fast detection speed, strong specificity, high sensitivity, good biocompatibility and the result can be observed by naked eyes. - Detection probe.

[0005] To achieve the above object, the present invention provides a method for detecting ONOO - A near-infrared fluorescent probe, the structural formula of the near-infrared fluorescent probe is:

[0006]

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

[0008] S1. Synthesizing Compound 1: Dissolving isophorone and malononitrile in anhydrous ethanol, then adding piperidine, and heating under reflux under a protective atmosphere. After completion of the reaction, spin-drying to obtain a crude product of Compound 1, and purifying the crude product of Compound 1 to obtain Compound 1;

[0009] S2, synthesizing compound 2, dissolving compound 1 and 4-hydroxybenzaldehyde obtained in S1 in anhydrous ethanol, adding piperidine and conducting reflux reaction under a protective atmosphere, and after completion of the reaction, spin-drying to obtain a crude product of compound 2, and purifying the crude product of compound 2 to obtain compound 2;

[0010] S3. Synthesis for detection of ONOO - To prepare a near-infrared fluorescent probe, compound 2 was dissolved in DCM, triethylamine was added dropwise in an ice bath, and 4-(trifluoromethyl)benzoyl chloride was added dropwise after stirring for 20 min. The mixture was stirred at 25°C under a protective gas atmosphere overnight, and the organic layer was extracted and purified.

[0011] The reaction formula is as follows:

[0012]

[0013] Preferably, the equivalent ratio of isophorone:malononitrile:anhydrous ethanol:piperidine in S1 is 1:2:10:0.01; and the heating reflux is carried out at 85° C. and 0.1 MPa for 12 h.

[0014] Preferably, the spin drying in S1 is performed by a rotary evaporator under reduced pressure, and the reduced pressure condition is 0.01 MPa; the crude product of compound 1 is purified by silica gel column chromatography, and the eluent is petroleum ether.

[0015] Preferably, the equivalent ratio of compound 1: 4-hydroxybenzaldehyde: anhydrous ethanol: piperidine in S2 is 1:1.5:10:0.01; and the reaction mixture is refluxed at 90° C. and 0.1 MPa for 4 h.

[0016] Preferably, the spin drying in S2 is performed under reduced pressure using a rotary evaporator, and the reduced pressure condition is 0.01 MPa; the crude product of compound 2 is purified by silica gel column chromatography, and the eluent is a mixed solvent of petroleum ether: ethyl acetate in a volume ratio of 6:1.

[0017] Preferably, the equivalent ratio of compound 2:DCM:triethylamine:4-(trifluoromethyl)benzoyl chloride in S3 is 1:5:1.2:2.

[0018] Preferably, the stirring in S3 is carried out at 0.1 MPa; the extraction is carried out using ethyl acetate; the purification is carried out using a silica gel column, and the eluent is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0019] A method for detecting ONOO using the above method - The method of detecting peroxynitrite ion with near-infrared fluorescent probe will be used to detect ONOO - The near-infrared fluorescent probe is added to the biological sample to be tested, and after incubation, the change of the fluorescence signal is detected by a fluorescence detection instrument to achieve qualitative and quantitative analysis of peroxynitrite ions.

[0020] A method for detecting ONOO - Application of near-infrared fluorescent probe in the detection of peroxynitrite ions.

[0021] Therefore, the present invention provides a method for detecting ONOO - The near-infrared fluorescent probe, preparation method and application thereof, and its specific technical effects are as follows:

[0022] (1) The method for detecting ONOO provided by the present invention - Near-infrared fluorescent probe with fast detection speed and response to ONOO - Fast, reaching the highest fluorescence intensity within 60 seconds, which can meet the real-time monitoring needs of rapidly changing peroxynitrite ion levels in organisms;

[0023] (2) The method for detecting ONOO provided by the present invention - The near-infrared fluorescent probe has a significant and easy-to-observe reaction. - After the reaction occurs, strong red fluorescence is produced, and the change in solution color can be observed with the naked eye;

[0024] (3) The method for detecting ONOO provided by the present invention - The near-infrared fluorescent probe has strong selectivity and high sensitivity, which can effectively avoid interference from other biological molecules and active substances, ensuring the accuracy and reliability of the test results. The minimum detection limit can reach 107nM.

[0025] (4) The method for detecting ONOO provided by the present invention - The near-infrared fluorescent probe has good biocompatibility and low toxicity in vivo, and will not adversely affect the normal physiological functions of biological samples, laying the foundation for its wide application in the biomedical field.

[0026] (5) The method for detecting ONOO provided by the present invention - The method for preparing the near-infrared fluorescent probe is simple and easy to operate, the raw materials are low-cost and readily available, the synthesis efficiency is high, it can be used for large-scale industrial production, and has broad application prospects.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is a high-resolution mass spectrum of the probe DCV-TFB prepared in Example 1 of the present invention;

[0030] Figure 2 is a hydrogen nuclear magnetic resonance spectrum of the probe DCV-TFB prepared in Example 1 of the present invention;

[0031] Figure 3 is the carbon NMR spectrum of the probe DCV-TFB prepared in Example 1 of the present invention;

[0032] Figure 4 The fluorescence spectra of the probe DCV-TFB in Example 2 of the present invention in different organic solvents and different buffer solutions are shown in Figure 1 ; wherein (A) is the fluorescence spectra in different organic solvents, and (B) is the fluorescence spectra in different buffer solutions;

[0033] Figure 5 The probe DCV-TFB with ONOO in Example 2 of the present invention - Fluorescence emission spectra and fluorescence intensity of the concentration changes, as well as the fluorescence intensity ratio (I 655 / I 572 ) and ONOO - The linear relationship between the concentrations; (A) is the fluorescence emission spectrum and fluorescence intensity results; (B) is the fluorescence intensity ratio (I 655 / I 572 ) and ONOO - Linear relationship graph of concentration;

[0034] Figure 6 The DCV-TFB pair in the second embodiment of the present invention detects ONOO - The selectivity and competitiveness of ONOO - ; 2 is Black; 3 is SO3 2- ; 4 is Cl - 5 is 1 - ; 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 S2O52- ; 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+ ;

[0035] Figure 7 This is the result of investigating the effect of time on the probe DCV-TFB in Example 2 of the present invention;

[0036] Figure 8 This is the result of investigating the effect of pH on the probe DCV-TFB in Example 2 of the present invention;

[0037] Figure 9 This is the result of investigating the cytotoxicity of the probe DCV-TFB at different concentrations on HepG2 cells in Example 2 of the present invention;

[0038] Figure 10 The three different concentrations of ONOO in the embodiment of the present invention are - Confocal microscopy images of HCT-116 cells;

[0039] Figure 11 The three different concentrations of ONOO in the embodiment of the present invention are - Confocal microscopy images of HepG2 cells;

[0040] Figure 12 The three different concentrations of ONOO in the embodiment of the present invention are - Confocal microscopy images of A549 cells;

[0041] Figure 13 The three different concentrations of ONOO in the embodiment of the present invention are - Confocal microscopy images of HK2 cells. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0043] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0044] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources; the method steps not described in detail are conventional techniques in the art.

[0045] Example 1

[0046] Preparation of a method for detecting ONOO - The near-infrared fluorescent probe (DCV-TFB) was synthesized as follows:

[0047]

[0048] The specific steps are as follows:

[0049] (1) Synthesis of compound 1.

[0050] Accurately weigh isophorone (5.0 g, 36.2 mmol, 1.0 equiv.) and malononitrile (5.0 g, 75.7 mmol, 2.0 equiv.) and place them in a 250.0 mL round-bottom flask. Then add 150.0 mL (10 equiv.) of anhydrous ethanol to fully dissolve them. Then add piperidine (112.0 mg, 0.362 mmol, 0.01 equiv.) and reflux for 12 h at 85 ° C, 0.1 MPa, and nitrogen atmosphere. After the reaction is completed, the product is evaporated to dryness under reduced pressure on a rotary evaporator (reduced pressure condition is 0.01 MPa). 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 66.0%, R f =0.39, and the developing solvent was petroleum ether:ethyl acetate (PE:EtOAc) with a volume ratio of 10:1).

[0051] (2) Synthesis of compound 2.

[0052] 4-Hydroxybenzaldehyde (366.4 mg, 3.0 mmol, 1.5 equiv.) and prepared compound 1 (372.5 mg, 2.0 mmol, 1.0 equiv.) were added to a 25.0 mL round-bottom flask, dissolved in 10.0 mL of anhydrous ethanol (10 equiv.), and then 60 μL of piperidine (0.6 mmol, 0.3 equiv.) was added. The mixture was refluxed at 90° C., 0.1 MPa, and nitrogen atmosphere for 4 h. After the reaction was completed, the mixture was dried under reduced pressure on a rotary evaporator to obtain a crude compound 2. The crude compound 2 was purified by silica gel column chromatography (eluent: PE: EtOAc volume ratio = 6: 1). The obtained orange solid was the pure compound 2, a total of 411.9 mg (yield: 70.9%, R f =0.62, developing solvent: PE:EtOAc=3:1).

[0053] (3) Synthesis of DCV-TFB.

[0054] Pure compound 2 (116.0 mg, 0.4 mmol, 1.0 equiv.) was weighed into a 25 mL reaction flask and thoroughly dissolved in 5 mL of DCM (5 equiv.). Triethylamine (TEA, 74.0 μL, 0.5 mmol, 1.2 equiv.) was then added dropwise in an ice bath and stirred for 20 min. 4-(Trifluoromethyl)benzoyl chloride (119.0 μL, 0.8 mmol, 2.0 equiv.) was then added dropwise to the reaction flask. Under a nitrogen atmosphere, the mixture was stirred at 25°C overnight (at 200 rpm for approximately 12 h). The resulting product was extracted with ethyl acetate, and the organic layer was collected and purified on a silica gel column (eluent: PE:EtOAc, volume ratio = 10:1). The resulting yellow solid, 105.4 mg (yield: 57.7%), was DCV-TFB (developing solvent: PE:EtOAc, volume ratio = 4:1, R f =0.60).

[0055] The structure of the DCV-TFB was identified. The high-resolution mass spectrum of DCV-TFB is shown in Figure 2. Figure 1 As shown, the H NMR spectrum is Figure 2 As shown, the carbon NMR spectrum is as follows Figure 3 The H NMR spectrum data is as follows: 1 H NMR(400MHz,DMSO-d6)δ8.33(d,J=8.1Hz,2H),8.00(d,J=8.3Hz,2H),7.85-7.7 8(m,2H),7.49-7.30(m,4H),6.91(s,1H),2.60(d,J=25.0Hz,4H),1.03(s,6H).

[0056] The carbon NMR spectrum data are: 13 C NMR(101MHz,DMSO-d6)δ170.45,163.50,155.76,151.26,136.48,134.26,133.36,132.77,130.8 3,130.20,130.02,129.14,126.09,123.12,122.50,113.10,76.71,42.38,38.25,31.79,27.53.

[0057] Example 2

[0058] The performance of the DCV-TFB prepared in Example 1 was tested as follows:

[0059] (1) Investigating the spectral characteristics of the probe DCV-TFB:

[0060] 100.0 μL probe DCV-TFB (0.1 mmol / L), 100.0 μL ONOO - Ionic solution (0.1mmol / L) was added to a colorimetric tube, and then 1.0mL of different organic reagents (DMSO, ACN, DMF, MeOH, THF, DXA, with H2O as a control) and 1.0mL of different buffer solutions (PBS, HEPES, Tris) were added. The reaction system was fixed to 5.0mL with deionized water. The fluorescence spectrum and UV absorption spectrum of each reaction system were scanned. The excitation and emission slit widths of the fluorescence spectrum were set to 10nm and 10nm respectively, and the voltage was set to 700V. The results are shown in Figure 2. Figure 4 As shown in (A) and (B), the buffer used in (A) is PBS, and the organic reagent used in (B) is DMSO, indicating that the response effect of the probe DCV-TFB is the best in the DMSO and PBS systems.

[0061] The optimal reaction system was determined as follows: 100.0 μL probe DCV-TFB (0.1 mmol / L), 1.0 mL DMSO, 1.0 mL PBS, and then 20 to 400 μL ONOO - Ion solution (0.1mmol / L), fix the reaction system to 5.0mL with deionized water, and make ONOO - The final concentration range of the ion solution was 2.0-40.0 μM. The fluorescence spectrum and ultraviolet absorption spectrum of each reaction system were scanned. The excitation and emission slit widths of the fluorescence spectrum were set to 10 nm and 10 nm, respectively, and the voltage was set to 700 V.

[0062] The results are as follows Figure 5 As shown in (A) and (B), the probe DCV-TFB is sensitive to ONOO- (14.0~32.0μM) showed a good linear response, and the correlation coefficient R 2 The detection limit was 107.0 nM, indicating that the probe DCV-TFB can be used to monitor ONOO - concentration changes.

[0063] (2) Investigation of the probe DCV-TFB pair for detecting ONOO - Selectivity and anti-interference ability. Using some common interfering ions in the human body, such as common metal ions (such as Na + , K + , Ca 2+ Mg 2+ etc.), common reactive oxygen species (such as ONOO - , ClO - 、O2 1 etc.), common active sulfur (such as SO3 2- 、SO4 2- 、HSO4 - 、S2O5 2- 、S 2- 、S2O3 2- Selectivity and competitiveness studies were conducted. In the selectivity study, 100.0 μL of probe DCV-TFB (0.1 mmol / L), 1.0 mL of DMSO, and 1.0 mL of PBS were added, followed by 100.0 μL of each ion solution (0.1 mmol / L). In the competitive study, 100.0 μL of probe DCV-TFB (0.1 mmol / L), 100.0 μL of ONOO - (0.1mmol / L), 1.0mL DMSO, 1.0mL PBS, and then 100.0μL of each interfering ion solution (0.1mmol / L) were added, and the reaction system was fixed to 5.0mL with deionized water. Figure 6 As shown, add ONOO - After addition, the fluorescence signal of probe DCV-TFB at 655 nm was significantly enhanced, while the addition of other analytes produced almost no fluorescence. In addition, in the competitive experiment, it was found that other interfering ions did not affect the fluorescence of probe DCV-TFB to ONOO - The results showed that the probe DCV-TFB could detect ONOO - It has excellent selectivity and strong anti-interference ability.

[0064] (3) Investigate the stability and pH tolerance of the probe DCV-TFB.

[0065] 1) The effect of time on the probe DCV-TFB was investigated.

[0066] First, the probe DCV-TFB (10.0 μM) and ONOO - The kinetic properties of the reaction. - When ONOO - When the probe reacts rapidly, the fluorescence signal red-shifts, and the fluorescence signal at 655 nm is significantly enhanced and reaches a peak within 1 min ( Figure 7 ).

[0067] 2) Investigate the effect of pH on the probe DCV-TFB.

[0068] We evaluated whether the probe DCV-TFB could detect ONOO at physiological pH. - The probe DCV-TFB was sensitive to ONOO in the pH range of 6 to 10. - Good fluorescence response ( Figure 8 ), indicating that the probe DCV-TFB can detect ONOO under physiological conditions - concentration changes.

[0069] (4) Test the biocompatibility of the probe DCV-TFB.

[0070] The toxicity of the probe DCV-TFB to HepG2 cells was evaluated using the CCK-8 assay. 100.0 μL of HepG2 cell suspension and DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin were added to a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. Six different concentrations of the probe DCV-TFB (0.0 μM, 1.0 μM, 2.5 μM, 5.0 μM, 10.0 μM, and 15.0 μM) were then added and incubated for a further 24 hours. CCK-8 solution was then added and incubated for a further 4 hours. The absorbance of each well (reaction system) at 450 nm was recorded, and cell viability was calculated using the following formula:

[0071] Cell viability (%) = (A 加药 -A 空白 ) / (A 不加药 -A 空白 )×100%.

[0072] The results are as follows Figure 9 As shown, even at 20 μM ONOO - At this concentration, the cell survival rate can still reach over 80%.

[0073] Example 3

[0074] The application of the probe DCV-TFB prepared in Example 1 in the detection of intracellular peroxynitrite ions was investigated as follows:

[0075] HCT-116, HepG2, A549, and Hk2 cells were cultured in a 37°C, 5% CO2 incubator for 24 hours, and then 100 μL of the probe DCV-TFB (10.0 μM) was added and mixed. The cells were incubated for 20 minutes and then washed three times with PBS to remove excess probe. Subsequently, four different concentrations of ONOO (0.0 μM, 5.0 μM, 10.0 μM, and 20.0 μM) were added. - The cells were incubated for 15 min, washed three times with PBS buffer, and fluorescence imaging was performed under a confocal microscope.

[0076] Different concentrations of ONOO - Confocal microscopy images of HCT-116 cells are shown in Figure 2. Figure 10 As shown, confocal microscopy images of HepG2 cells are shown in Figure 11 As shown, confocal microscopy images of A549 cells are shown in Figure 12 As shown, confocal microscopy images of Hk2 cells are shown in Figure 13 As shown in these figures, we can clearly observe that as ONOO - With the increase of concentration, the red fluorescence in the cells gradually increased.

[0077] Therefore, the present invention provides a method for detecting ONOO - The near-infrared fluorescent probe has a fast detection speed and can reach the highest fluorescence intensity within 60 seconds; the reaction is obvious and easy to observe. - After the reaction occurs, strong red fluorescence is generated, and the color change of the solution can be observed with the naked eye; it has strong selectivity and high sensitivity, which can effectively avoid interference from other biological molecules and active substances, ensuring the accuracy and reliability of the detection results, and the minimum detection limit can reach 107nM; in addition, it has good biocompatibility and low toxicity in the body, and will not adversely affect the normal physiological functions of biological samples; the preparation method provided is simple and easy to operate, the raw materials are low in cost and readily available, the synthesis efficiency is high, it can be used for large-scale industrial production, and has broad application prospects.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for detecting ONOO - A near-infrared fluorescent probe, characterized in that The structural formula of the near-infrared fluorescent probe is:

2. A method for detecting ONOO according to claim 1. - The method for preparing a near-infrared fluorescent probe is characterized in that: The steps include: S1. Synthesizing Compound 1: Dissolving isophorone and malononitrile in anhydrous ethanol, then adding piperidine, and heating under reflux under a protective atmosphere. After completion of the reaction, spin-drying to obtain a crude product of Compound 1, and purifying the crude product of Compound 1 to obtain Compound 1; S2, synthesizing compound 2, dissolving compound 1 and 4-hydroxybenzaldehyde obtained in S1 in anhydrous ethanol, adding piperidine and conducting reflux reaction under a protective atmosphere, and after completion of the reaction, spin-drying to obtain a crude product of compound 2, and purifying the crude product of compound 2 to obtain compound 2; S3. Synthesis for detection of ONOO - To prepare a near-infrared fluorescent probe, compound 2 was dissolved in DCM, triethylamine was added dropwise in an ice bath, and 4-(trifluoromethyl)benzoyl chloride was added dropwise after stirring for 20 min. The mixture was stirred at 25°C under a protective gas atmosphere overnight, and the organic layer was extracted and purified. The reaction formula is as follows:

3. A method for detecting ONOO according to claim 2. - A method for preparing a near-infrared fluorescent probe, characterized in that: The equivalent ratio of isophorone:malononitrile:anhydrous ethanol:piperidine in S1 is 1:2:10:0.01; heating and reflux are carried out at 85° C. and 0.1 MPa for 12 h.

4. A method for detecting ONOO according to claim 2 - A method for preparing a near-infrared fluorescent probe, characterized in that: The dried product in S1 was dried under reduced pressure using a rotary evaporator at a pressure of 0.01 MPa. The crude product of compound 1 was purified by silica gel column chromatography using petroleum ether as eluent.

5. A method for detecting ONOO according to claim 2. - A method for preparing a near-infrared fluorescent probe, characterized in that: The equivalent ratio of compound 1 in S2: 4-hydroxybenzaldehyde: anhydrous ethanol: piperidine is 1:1.5:10:0.3; the mixture is refluxed at 90° C. and 0.1 MPa for 4 h.

6. A method for detecting ONOO according to claim 2. - A method for preparing a near-infrared fluorescent probe, characterized in that: The dried product in S2 was dried under reduced pressure using a rotary evaporator at a pressure of 0.01 MPa. The crude product of compound 2 was purified by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 as the eluent.

7. A method for detecting ONOO according to claim 2. - A method for preparing a near-infrared fluorescent probe, characterized in that: The equivalent ratio of compound 2:DCM:triethylamine:4-(trifluoromethyl)benzoyl chloride in S3 is 1:5:1.2:

2.

8. A method for detecting ONOO according to claim 2. - A method for preparing a near-infrared fluorescent probe, characterized in that: The mixture was stirred in S3 under 0.1 MPa; ethyl acetate was used for extraction; and silica gel column purification was used as the eluent, which was a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1.

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