A near-infrared fluorescent probe for detecting peroxynitrite, a preparation method and application thereof

Abstract

The application provides a near-infrared fluorescent probe for detecting peroxynitrite, a preparation method and application thereof, and a structure formula is as follows: The probe half-florin derivative is used as a fluorophore, and a boronic acid group is introduced as a recognition group of peroxynitrite (ONOO ‑ ). After the probe contacts ONOO ‑ , the hydroxyl group is exposed, fluorescence is enhanced, the probe can effectively monitor the dynamic changes of endogenous and exogenous ONOO ‑ in onion inner epidermis cells, mung bean slice cells and wheat root cells under stress environment, and can realize whole-plant fluorescence imaging detection of mung bean plants; meanwhile, the synthesized fluorescent probe can also realize fluorescence co-localization imaging analysis of wheat root Golgi bodies, and has good selectivity and high sensitivity. The probe is successfully applied to the detection and imaging application of ONOO ‑ in a biological system.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a near-infrared fluorescent probe for detecting nitrite peroxide, its preparation method, and its application. Background Technology

[0002] peroxynitrite (ONOO) - ) is nitric oxide (NO) and superoxide anion (•O2). - Highly reactive oxygen species (ROS) generated by coupling reactions within the body. - It is a reactive nitrogen species with a nitrification capacity far exceeding that of its precursors. It can induce nitrification stress in plants and participate in physiological processes such as plant stress resistance, signal transduction, nutrient uptake, and environmental adaptation. Under normal physiological conditions, intracellular ONOO... - The content of it is extremely low. When it is subjected to external stress, such as salt stress or heavy metal stress, ONOO - The concentration of these substances far exceeds normal levels, triggering a nitrogenation reaction that begins to attack cellular DNA and proteins, leading to apoptosis. Therefore, the development of ONOO... - Detection methods for nitrite peroxide are crucial for a deeper understanding of its function in complex biological systems. To date, reported methods for detecting nitrite peroxide primarily rely on traditional techniques: electrochemistry, electron spin resonance (ESR) UV-Vis absorption spectroscopy, liquid chromatography, and electromagnetic resonance spectroscopy. These methods not only require complex sample processing but also damage cell structures. They can only provide static, final data and cannot capture the initial stress state. - The dynamic changes are not suitable for detection of living systems.

[0003] To overcome the technical bottlenecks of traditional detection technologies in the application of in vivo plant samples, a novel ONOO detection method suitable for plant systems with in-situ real-time monitoring capabilities was developed. - Detection tools are urgently needed. Small molecule fluorescent probes offer a feasible path to achieving this goal due to their advantages such as rapid response, non-destructive in-situ detection, real-time monitoring, and high sensitivity. These probes can detect ONOO in plants without damaging cell structure. - Quantitative real-time monitoring is possible. Furthermore, near-infrared imaging, in molecular imaging techniques, reduces signal attenuation caused by light scattering and absorption due to its superior tissue penetration. The extremely low background autofluorescence in the near-infrared band significantly improves the imaging signal-to-noise ratio, thereby enabling the detection of ONOO in deep plant tissues. - High-resolution and real-time visualization monitoring of distribution and dynamic changes. Therefore, development of a near-infrared fluorescence imaging technology with specific ONOO response is needed. - Furthermore, fluorescent probes suitable for real-time imaging of living plant cells are essential.

[0004] The target product of this application has been characterized. Studies have shown that in PBS buffer solution, the compound can selectively recognize hydrogen sulfide, while other ions have almost no interference with the recognition process. Summary of the Invention

[0005] To overcome the problems in the prior art, this invention provides a near-infrared fluorescent probe for detecting nitrite peroxide, its preparation method, and its applications. This fluorescent probe responds rapidly to nitrite peroxide, exhibits good selectivity and linearity, and has been successfully used for plant (section, root, whole plant, co-localization) imaging.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A near-infrared fluorescent probe for detecting nitrite peroxide has the following structural formula:

[0008] .

[0009] The preparation method of the above-mentioned near-infrared fluorescent probe for detecting nitrite peroxide, and the synthetic route are as follows:

[0010]

[0011] Includes the following steps:

[0012] (1) Compound I reacts with pinacol 4-bromomethylphenylboronic acid in DMF to give compound II;

[0013] (2) Compound II reacts with compound III in ethanol to give CY-ONOO.

[0014] Preferably, the preparation process of compound II in step (1) is as follows: compound I and cesium carbonate are dissolved in DMF and stirred in an ice-water bath for 3 to 10 minutes; then 4-bromomethylphenylboronic acid pinacol ester is added under stirring conditions, and stirring is continued for 3 to 10 minutes. The mixture is taken out of the ice bath and stirred at room temperature until the reaction is complete. After the reaction is completed, the solvent is removed to obtain compound II.

[0015] Preferably, the preparation process of CY-ONOO in step (2) is as follows: Compound II and Compound III are dissolved in ultra-dry ethanol, refluxed at 80~85℃ for 10~15 hours, cooled to room temperature, filtered, and the solid is separated and purified by column chromatography to obtain CY-ONOO.

[0016] Preferably, in step (1), the molar ratio of compound I, 4-bromomethylphenylboronic acid pinacol ester and cesium carbonate is 1:1:2.

[0017] Preferably, in step (2), the molar ratio of compound II to compound III is (1.0~1.2):(1.0~1.2).

[0018] The above-mentioned fluorescent probe is used for the fluorescence detection of peroxynitrite in plant systems.

[0019] Furthermore, for fluorescence imaging of plant cells and tissues, including onion inner epidermal cells, mung bean stem slices, and wheat root cells, the samples need to be pre-treated with ONOO before imaging. - Treat with a solution, salt solution, or heavy metal solution, and then stain in 1-10 μM probe solution for 5-30 minutes.

[0020] Specifically, onion inner epidermis, mung bean stem slices, and wheat roots need to be treated in different solutions for 10–30 minutes; then stained in 1–10 μM probe solution for 5–30 minutes, rinsed with water, and then imaged (excitation wavelength 638 nm, detection range 670–730 nm). - The solution concentration is 5 μM to 25 μM, the salt solution concentration is 100 mM to 300 mM, and the heavy metal solution concentration is 50 μM to 150 μM. Preferably, the salt solution is a sodium chloride solution and the heavy metal solution is a chromium chloride solution.

[0021] Furthermore, when the probe is used for fluorescence imaging of the whole plant, it needs to be treated with a salt solution or a heavy metal solution before imaging. Preferably, the salt solution is a sodium chloride solution and the heavy metal solution is a chromium chloride solution.

[0022] Specifically, germinated plant seeds are treated in a salt solution or heavy metal solution for 12 hours to 5 days, then soaked in a 1-10 μM probe solution for 0.5-1 hour, rinsed with water, and then imaged. The seeds are mung bean seeds, the salt solution concentration is 100 mM-200 mM, and the heavy metal solution concentration is 50 μM-150 μM. Preferably, the salt solution is a sodium chloride solution, and the heavy metal solution is a chromium chloride solution.

[0023] Furthermore, the probe is used for colocalization fluorescence imaging of the Golgi apparatus in plant cells. Before imaging, it needs to be treated with 20-100 nM Golgi-Tracker Green solution for 20-40 minutes, and then soaked in 5-15 μM probe solution for 0.5-1 hour.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The probe of this invention has high selectivity and sensitivity, and can specifically identify ONOO. -Molecules, unaffected by common ions and amino acids, can achieve the targeting of ONOO molecules in plant systems. - Reliable testing.

[0026] 2. The probe synthesized in this invention has good biocompatibility, low toxicity, and can perform fluorescence imaging in living plants and can accurately locate the Golgi apparatus.

[0027] 3. The probe synthesized in this invention has a maximum emission wavelength of 705 nm, strong penetration ability, and is more suitable for imaging inside plants. Attached Figure Description

[0028] Figure 1 The near-infrared fluorescent probe CY-ONOO prepared in Example 1 has a hydrogen nuclear magnetic spectrum.

[0029] Figure 2 The near-infrared fluorescent probe CY-ONOO carbon NMR spectrum prepared in Example 1;

[0030] Figure 3 High-resolution mass spectrometry of the near-infrared fluorescent probe CY-ONOO prepared in Example 1;

[0031] Figure 4 The near-infrared fluorescent probe prepared in Example 1 and the probe with ONOO - The high-resolution mass spectrum afterward;

[0032] Figure 5 The near-infrared fluorescent probe prepared in Example 1 and the probe with ONOO - Comparison of changes in ultraviolet absorption spectra after the event;

[0033] Figure 6 The near-infrared fluorescent probe prepared in Example 1 and the probe with ONOO - Comparison of fluorescence spectrum changes after the event;

[0034] Figure 7 Different concentrations of ONOO were added to the near-infrared fluorescent probe prepared in Example 1. - Fluorescence spectrum after solution processing;

[0035] Figure 8 Different concentrations of ONOO were added to the near-infrared fluorescent probe prepared in Example 1. - Linear relationship of fluorescence intensity at 705 nm after solution treatment;

[0036] Figure 9 The near-infrared fluorescent probe prepared in Example 1 and the addition of ONOO - Fluorescence spectra of the solution in different pH systems;

[0037] Figure 10ONOO was added to the near-infrared fluorescent probe prepared in Example 1. - Fluorescence spectra as a function of time;

[0038] Figure 11 The fluorescent probe prepared in Example 1 is used to recognize ONOO. - The selection ability test; from left to right, the numbers are 0.blank, 1.Na. 2+ , 2. Ba 2+ 3. Ca 2+ 4. Zn 2+ 5. Mg 2+ 6. F - 7. I - 8. Br - 9. S 2- 10. HSO 3- 11. SO4 2- , 12. Cys, 13. Hcy, 14. GSH, 15. •OH, 16. 1 O2, 17. ROO•,18. NO, 19. H2O2, 20. ClO - 21. ONOO - A bar chart comparing the fluorescence intensity at 705 nm of the fluorescent probe prepared in Example 1 with that of the fluorescent probe prepared in Example 1;

[0039] Figure 12 The near-infrared fluorescent probe prepared in Example 1 is used to identify ONOO. - The interference capability test, from left to right, shows 0. blank, 1. Na. 2+ , 2. Ba 2+ 3. Ca 2+ 4. Zn 2+ 5. Mg 2+ 6. F - 7. I - 8. Br - , 9.S 2- 10. HSO 3- 11. SO4 2- , 12. Cys, 13. Hcy, 14. GSH, 15. •OH, 16. 1 O2, 17.ROO•, 18. NO, 19. H2O2, 20. ClO - 21. ONOO - A bar chart comparing the fluorescence intensity at 705 nm of the fluorescent probe prepared in Example 1 with that of the fluorescent probe prepared in Example 1;

[0040] Figure 13 This is a diagram showing the biocompatibility of near-infrared fluorescent probes with plants.

[0041] Figure 14 The images are bioimaging images of near-infrared fluorescent probes. (a), (b), and (c) are bioimaging images of onion inner epidermis after pretreatment with different solutions. (a1), (b1), and (c1) are fluorescence quantification images of onion inner epidermis in sample A.

[0042] Figure 15 The images are bioimaging images of near-infrared fluorescent probes. (a), (b), and (c) are bioimaging images of mung bean stem slices after pretreatment with different solutions. (a1), (b1), and (c1) are fluorescence quantification images of mung bean stem slice A.

[0043] Figure 16 The images are bioimaging images of near-infrared fluorescent probes. (a) and (b) are bioimaging images of wheat roots after pretreatment with different solutions. (a1) and (b1) are fluorescence quantification images of wheat roots in A.

[0044] Figure 17 The images are bioimaging images of near-infrared fluorescent probes. (a) and (b) are bioimaging images of wheat roots after pretreatment with different solutions for different times. (a1) and (b1) are fluorescence quantification images of wheat roots in A.

[0045] Figure 18 The images show whole-plant imaging of near-infrared fluorescent probes, where (a) is the experimental treatment process of mung bean seeds under salt stress, (b) and (c) are whole-plant fluorescence imaging results after 12 hours and 5 days of treatment with different concentrations of NaCl solution (0, 100, 150, 200 mM), respectively, and (d) and (e) are whole-plant fluorescence quantification images.

[0046] Figure 19 This is a whole-plant imaging image of a near-infrared fluorescent probe, where (a) shows mung bean seeds exposed to cadmium ions (Cd). 2+ (a) Experimental treatment procedure for stress, (b) and (c) are whole-plant fluorescence imaging results after 12 hours and 5 days of treatment with different concentrations of cadmium ions (0, 50, 100, 150 μM), respectively, and (d) and (e) whole-plant imaging fluorescence quantification diagram.

[0047] Figure 20 The images show bioimaging of the near-infrared fluorescent probe, where (a) is a colocalization image of Golgi-Tracker Green and wheat roots treated with the probe. Detailed Implementation

[0048] The present invention will be further described below with reference to embodiments and accompanying drawings, but this is not intended to limit the invention.

[0049] Example 1

[0050] The preparation process of the fluorescent probe in this embodiment specifically includes the following steps:

[0051] (1) Preparation of compound I

[0052] The synthesis route is as follows:

[0053]

[0054] Under ice bath conditions, PBr3 (9 mL, 100 mmol) was slowly added dropwise to a mixture of DMF (7 mL, 90 mmol) and CH2Cl2 (40 mL), and the mixture was stirred for 40 minutes. Cyclohexanone (4 mL, 37 mmol) was then slowly added, the mixture was removed from the ice bath, and stirred at room temperature for 30 hours. After the reaction was complete, the reaction mixture was slowly added to a 100 mL ice-water bath. The pH was adjusted to neutral with anhydrous sodium carbonate. The mixture was extracted with CH2Cl2 (3 × 40 mL), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure to give a reddish-brown oily liquid compound. A total of 17.8 g of the compound was obtained, with a yield of 78.5%. This compound was used directly in subsequent steps without purification; 2,4-dihydroxybenzaldehyde (4.14 g, 30 mmol) and PPTS (pyridine 4-methylbenzenesulfonic acid, 0.38 g) were added to a 250 mL round-bottom flask containing 40 mL of CH2Cl2. Under nitrogen protection, 6.23 mL of 3,4-dihydro-2H-pyran was added to a reaction flask and the mixture was refluxed at 40 °C. After the reaction was complete, the mixture was concentrated and purified by eluent petroleum ether:ethyl acetate (15:1 v / v) to give compound 2 (approximately 7.80 g, 85% yield) as a white solid. Under nitrogen atmosphere, anhydrous potassium carbonate (2.07 g, 15 mmol), compound 1 (0.94 g, 5 mmol), and compound 2 (1.1 g, 5 mmol) were added to a round-bottom flask containing 8 mL of DMF solution and stirred at 40 °C until complete. The reaction mixture was filtered under reduced pressure to remove the potassium carbonate solid. The filtrate was concentrated to give compound 3, which was used directly in subsequent steps without further purification. 50 mL of CH2Cl2 and compound 3 were transferred to a 50 mL round-bottom flask. The pH was adjusted to 3-4 with trifluoroacetic acid, and the mixture was heated and stirred in a water bath at 30 °C. After the reaction was complete, the mixture was filtered under reduced pressure and washed with ice-cold ethanol at 4°C to give a yellow solid product, compound I (approximately 1.30 g), in 63.2% yield. The NMR spectra of compound I are as follows: 1HNMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.92 (s,1H), 6.65 -6.55 (m, 2H), 2.52 (d, J = 7.1 Hz, 2H), 2.28 (t, J = 6.0 Hz, 2H),1.61 (p, J = 5.9 Hz, 2H); 13 C NMR (101 MHz, DMSO-d6) δ 186.59, 160.79, 160.19,153.22, 128.61, 128.09, 125.18, 113.52, 112.48, 111.77, 102.34, 29.42, 21.70,20.48.

[0055] (2) Preparation of compound II

[0056] The synthesis route is as follows:

[0057]

[0058] Under nitrogen protection, compound I (228 mg, 0.1 mmol), cesium carbonate (505 mg, 0.2 mmol), and 5 mL of LDM were mixed and stirred in an ice-water bath for 5 minutes. Then, 268 mg, 0.1 mmol of 4-bromomethylphenylboronic acid pinacol ester was added under stirring, and stirring was continued for another 5 minutes. The mixture was then removed from the ice bath and stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed, yielding 253 mg of a yellow solid VII, in 56.9% yield. This solid was not purified and was used directly for subsequent reactions.

[0059] (3) Preparation of compound III

[0060] The synthesis route is as follows:

[0061]

[0062] Under nitrogen protection, benzenesulfonamide (1.72 g, 10 mmol) was suspended in acetone, and chloroacetyl chloride (1.34 g, 12 mmol) was added dropwise at room temperature, with stirring at 70 °C for 1 hour. After cooling the reaction mixture to room temperature, ice water was added and the mixture was stirred for 10 minutes. The resulting solid was filtered under reduced pressure, washed with ice water, and recrystallized from ethanol to give compound 4 (2.36 g), a white solid, in 95.1% yield. Compound 4 (1.29 g, 5 mmol) was dissolved in acetonitrile under nitrogen protection, and 2,3,3-trimethyl-3H-indole (0.8 g, 6 mmol) was added, with stirring under reflux at 85 °C for 12 hours. After the reaction was complete, the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in dichloromethane and shaken thoroughly, then filtered under vacuum to give compound III (1.69 g, 91% yield). The NMR spectra of compound III are as follows: ¹H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 8.04 -7.92 (m, 1H), 7.92-7.85 (m, 1H), 7.80 (s, 5H), 7.68-7.60 (m, 2H), 7.32 (s, 2H), 5.69 (s, 2H), 2.88 (s, 3H), 1.60 (s, 6H); 13 C NMR (101 MHz, DMSO) δ 199.75,162.35, 141.39, 141.08, 139.27, 129.58, 129.14, 126.78, 123.63, 118.96,114.90, 54.45, 50.27, 22.11, 14.47.

[0063] (3) Preparation of CYOH

[0064] The synthesis route is as follows:

[0065]

[0066] Under nitrogen protection, compound III (372 mg, 0.1 mmol), compound I (228 mg, 0.1 mmol), and 10 mL of ethanol were added to a 50 mL round-bottom flask, and the reaction mixture was refluxed at 80 °C overnight. After the reaction was complete, the mixture was cooled to room temperature, and the solid product was collected by vacuum filtration. The product was purified by recrystallization in petroleum ether to give the target product CYOH (355 mg, 61% yield). 1H NMR (500 MHz, DMSO-d6) δ 11.68 (s, 1H), 11.04 (s, 1H), 8.61 (d,J = 14.6 Hz, 1H), 7.88 - 7.77 (m, 4H), 7.76 (d, J = 7.4 Hz, 1H), 7.62 (s,1H), 7.59 -7.46 (m, 3H), 7.40 (t, J = 7.5 Hz, 1H), 7.31 (s, 2H), 7.01 (d, J =2.2 Hz, 1H), 6.92 (dd, J = 8.5, 2.3 Hz, 1H), 6.55 (d, J = 14.6 Hz, 1H), 2.71(t, J = 6.0 Hz, 2H), 2.63 (t, J = 6.2 Hz, 2H), 1.80 (s, 8H); 13 C NMR (126 MHz, DMSO-d6) δ 178.21, 164.58, 162.61, 162.52, 154.79, 145.28, 142.55, 141.72,141.65, 139.45, 136.25, 129.88, 129.25, 127.26, 126.81, 126.37, 123.16,119.34, 115.59, 115.06, 114.98, 112.58, 103.82, 102.47, 50.49, 48.24, 28.70,28.29, 20.40, February 19, 2019.

[0067] (4) Preparation of CY-ONOO

[0068] The synthesis route is as follows:

[0069]

[0070] Under nitrogen protection, compound III (37.2 mg, 0.01 mmol), compound II (53.3 mg, 0.012 mmol), and 5 mL of ethanol were added to a 25 mL round-bottom flask. The mixture was refluxed at 80 °C for 12 hours, then cooled to room temperature. The solid was precipitated by vacuum filtration and purified by silica gel column chromatography using a dichloromethane:methanol (9:1) mixed solvent. A total of 58 mg of probe CY-ONOO was finally obtained in 72.6% yield. The 1H NMR, 1C NMR, and high-resolution mass spectra of probe CY-ONOO are shown below. Figures 1 to 3The NMR data for the CY-ONOO probe are as follows: ¹H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 8.63–8.58 (m, 1H), 8.13 (s, 1H), 7.84 (d, J = 9.1 Hz, 3H), 7.80 (s, 1H), 7.78–7.75 (m, 3H), 7.62–7.57 (m, 3H), 7.55 (s, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.33 (s, 2H), 7.23–7.20 (m, 1H), 7.12–7.09 (m, 1H), 6.62 (d, J = 14.7 Hz, 1H), 5.55 (s, 2H), 5.34 (d, J = 15.9 Hz, 2H), 2.71 (s, 2H), 2.63(s, 2H), 1.80 (s, 8H), 1.29 (s, 12H). 13 C NMR (101 MHz, DMSO-d6) δ 179.17,164.42, 162.01, 161.82, 154.30, 145.78, 142.45, 141.91, 141.69, 140.16,139.50, 135.21, 134.86, 134.67, 129.51, 129.35, 127.65, 127.52, 127.31,127.26, 123.17, 119.35, 116.20, 115.09, 112.95, 104.88, 102.42, 84.21, 70.50,50.78, 48.48, 28.81, 28.24, 25.43, 25.15, 24.18, 20.32. Specific high-resolution mass spectrometry data are as follows: HR-MS (ESI): m / z calcd for C 46 H 49 BN3O7S + : 798.3379, found: 798.3383.

[0071] The application tests of the fluorescent probe prepared in this embodiment are as follows:

[0072] 1) Preparation of stock solution for testing

[0073] a. Fluorescent probe sample solution (1.00 × 10⁻⁶) -3Preparation of (mol / L): Accurately weigh 0.0020 g (M=798) of fluorescent probe CY-ONOO, dissolve it in 2.5 mL of dimethyl sulfoxide, and prepare a probe stock solution with a concentration of 1 mM.

[0074] b. CYOH sample solution of the parent nucleus (1.00 × 10⁻⁶) -3 Preparation of (mol / L): Accurately weigh 0.0020 g (M=582) of fluorescent probe and dissolve it in 3.44 mL of dimethyl sulfoxide to prepare a concentration of 1.00×10⁻⁶ mol / L. -3 A solution of mol / L.

[0075] c. Nitrite peroxide: Add 0.486 g NaNO2 to a 10 ml centrifuge tube, add 0.8 mL H2O2 dropwise under ice bath conditions, then add 0.417 mL of commercially available concentrated hydrochloric acid dropwise. Separately, dissolve 0.24 g of sodium hydroxide solid in 5 mL of deionized water and slowly add it dropwise to the above solution under ice bath conditions. Calculate the concentration using a UV spectrophotometer and measure the absorbance A at 302 nm. The concentration is calculated as c = A / Kb, where b = 1 cm and K = 1670 M. -1 cm -1 The concentration obtained was 3.00 × 10⁻⁶. -3 mol / L ONOO - Mother liquor. All other ions were prepared with deionized water to a concentration of 1.0 × 10⁻⁶. -2 A solution of mol / L was prepared; all amino acids and ions were prepared with deionized water to a concentration of 1.0 × 10⁻⁶ mol / L. -2 A solution of mol / L.

[0076] All buffer solutions used in the following tests were PBS (10 mM, pH=7.4), and all water used in the experiments was deionized water.

[0077] 2) Detection and Analysis

[0078] Two 3 mL PBS buffer solutions were pipetted together, and 30 μL of probe stock solution (1.00 × 10⁻⁶) was added to each. -3 mol / L), and 30 μL of probe stock solution (1.00×10⁻⁶ mol / L). -3 mol / L) and 20 μL of ONOO - (3.00×10 -3 A mol / L solution was prepared. After thorough shaking to ensure no bubbles were present, the high-resolution mass spectrum was measured at room temperature. The near-infrared fluorescent probe and the probe were then treated with ONOO. - The high-resolution mass spectrum after that is as follows Figure 4 As shown, by Figure 4It can be seen that, compared with the HR-MS of CY-ONOO itself, the molecular ion peak of the probe disappears at m / z 798.3383, indicating that CY-ONOO and ONOO... - It is consumed after complete response; simultaneously, a CYOH peak was observed at m / z 582.2065, indicating that CYOH is composed of CY-ONOO and ONOO. - The products formed after the reaction. Similarly, their UV absorption spectra were measured at room temperature (parameters: scan range 400-800 nm, fast scan speed, 1 nm interval). The results are as follows: Figure 5 ,Depend on Figure 5 It can be seen that when CY-ONOO and ONOO - After incubation in buffer, the maximum absorption peak red-shifted from 620 nm to 675 nm. The fluorescence spectrum was then measured (parameters: excitation wavelength 620 nm, excitation slit width 10.0 nm, emission slit width 10.0 nm), and the results are as follows: Figure 6 ,Depend on Figure 6 It can be seen that the probe response is ONOO - Subsequently, fluorescence enhancement occurred. Different concentrations of ONOO were added to the buffer solution system. - The solution was analyzed, and its fluorescence spectrum was measured. The results are as follows: Figure 7 As shown, with the addition of ONOO - With increasing concentration, the fluorescence intensity of the solution system at 705 nm gradually increased. Linear fitting yielded the following linear equation for CY-ONOO: Y = -3590.85304 + 822.42418[ONOO] - ], where R 2 =0.99339, the calculated detection limit is 0.049 μM, and the probe CY-ONOO is in the ONOO range of 8-20 μM. - Within the concentration range, log I 705 with [ONOO - There is a good linear relationship between them, as shown in the experimental results. Figure 8 As shown.

[0079] Use a pipette to transfer 3 mL of the PBS buffer system. Adjust the pH of the PBS buffer to the appropriate level using 1 mol / L hydrochloric acid or 1 mol / L NaOH solution. Add 30 μL (1.00 × 10⁻⁶) of the solution to each buffer. -3 Add 30 μL (1.00 × 10⁻⁶ mol / L) probe stock solution to another sample. -3 mol / L) probe stock solution and 20 μL of ONOO - (1.00×10 -3To prepare a solution of the corresponding concentration, dissolve an appropriate volume of the stock solution in 3 mL of PBS, shake well until no bubbles are present, and test its fluorescence intensity (at 705 nm) at room temperature. The results are as follows: Figure 9 As shown. By Figure 9 It can be seen that the fluorescence intensity of the probe remains basically stable; the addition of ONOO to the probe... - After solution preparation, the best response was observed at pH 7-10; therefore, this probe is suitable for both plant and animal environments. Using a pipette, transfer 3 mL of PBS buffer solution and add 30 μL of probe stock solution (1.00 × 10⁻⁶). -3 mol / L) and 20 μL of ONOO - (1.00×10 -3 A mol / L solution. Shake well until no bubbles are present, and perform tests every minute at room temperature. The results are as follows: Figure 10 As shown, the probe responds completely in about 7 minutes.

[0080] Use a pipette to transfer 3 mL of PBS buffer solution and add 30 μL (1.00 × 10⁻⁶) to the solution. -3 1 mol / L) probe stock solution, then 20 μL (1.00×10 mol / L) was added sequentially. -2 Other analytes (mol / L) or 20 μL (1.00×10⁻⁶ mol / L) -3 mol / L)ONOO - (0. blank, 1. Na 2+ , 2. Ba 2+ 3. Ca 2+ 4. Zn 2+ 5. Mg 2+ 6. F - 7. I - , 8.Br - 9. S 2- 10. HSO 3- 11. SO4 2- , 12. Cys, 13. Hcy, 14. GSH, 15. •OH, 16. 1 O2,17. ROO• [R=-C(CH3)2C(NH2)=NH], 18. NO, 19. H2O2, 20. ClO - 21. ONOO - The fluorescence spectrum of the sample was detected, and the results are as follows: Figure 11 As shown, the probe is at ONOO -The fluorescence emission was significantly enhanced in the system, and the fluorescence intensity of the probe did not change significantly after the addition of other different ions.

[0081] Use a pipette to transfer 3 mL of PBS buffer solution and add 30 μL (1.00 × 10⁻⁶) to the solution. -3 1 mol / L) probe stock solution, then add 20 μL (1.00×10 mol / L) of the stock solution. -2 A certain interfering substance (mol / L) and 20 μL (1.00×10 mol / L) -3 mol / L)ONOO - (0. blank, 1. Na 2+ , 2. Ba 2+ 3. Ca 2+ 4. Zn 2+ 5. Mg 2+ 6. F - 7. I - , 8.Br - 9. S 2- 10. HSO 3- 11. SO4 2- , 12. Cys, 13. Hcy, 14. GSH, 15. •OH, 16. 1 O2,17. ROO• [R=-C(CH3)2C(NH2)=NH], 18. NO, 19. H2O2, 20. ClO - 21. ONOO - Mix thoroughly, perform fluorescence spectroscopy scanning, and plot a bar chart of the fluorescence intensity at 705 nm. Figure 12 It can be seen that the remaining ions and amino acids have very little interference with the probe, and basically do not react. Therefore, this indicates that the probe does not react with ONOO in the presence of common anions, cations, and amino acids. - The response is good, and it has strong anti-interference ability against a variety of common anions and cations, which demonstrates the excellent performance of the fluorescent probe.

[0082] 3) Probe biocompatibility

[0083] Mung bean seeds were disinfected with 75% alcohol for 30 seconds and rinsed three times with sterile water. They were then incubated with probe solutions (5 μM, 10 μM, 15 μM, and 20 μM, respectively, prepared by dissolving the probe in PBS buffer. Germination was observed and counted after 24 hours, and the germination rate was calculated. The results showed (…). Figure 13Even seeds pretreated with a probe solution (20 μM) at a concentration far exceeding the working concentration of 5 μM still exhibited a germination rate of over 90%, indicating that the CY-ONOO probe has low biotoxicity and high biocompatibility. It can be used in related applications in plant systems.

[0084] 4) Imaging of the probe

[0085] Onion epidermal cells and mung bean stem slices were first treated with different concentrations of ONOO. - After pretreatment for 30 minutes in a solution (10-25 μM, prepared by dissolving an appropriate volume of stock solution in 3 mL of PBS), the tissue was stained for 5 minutes in a 5 μM CY-ONOO probe solution (the probe was prepared by dissolving it in PBS buffer). In addition, onion inner epidermal cells and mung bean sprout stem sections were pretreated for 30 minutes in different concentrations of NaCl solution (100 mM and 300 mM) and different concentrations of cadmium ion solution (chromium chloride, 50 μM and 150 μM). An experimental group was set up where the pretreated plant tissues were pretreated in 1 mM UA solution for 30 min, and then stained in 5 μM CY-ONOO probe solution (the probe was prepared by dissolving the probe in PBS buffer) for 5 min. In the control group, onion inner epidermal cells and mung bean stem sections were pretreated in 1 mM UA solution for 30 min, and then stained in 5 μM CY-ONOO probe solution (the probe was prepared by dissolving the probe in PBS buffer) for 5 min. All samples were washed with water after treatment and then imaged (excitation wavelength 638 nm, detection range 670-730 nm). Fluorescence imaging showed ( Figure 14 ,15), with ONOO - The increased concentration led to a significant increase in fluorescence intensity, indicating that the probe exhibits exogenous ONOO. - The probe demonstrated its ability to detect endogenous ONOO under stress; and pretreatment with NaCl solution and cadmium ion solution resulted in significant fluorescence enhancement, indicating that the probe could detect endogenous ONOO under stress. - The fluorescence intensity increased. In addition to the above phenomena, all samples pretreated with UA showed a significant decrease in fluorescence intensity, indicating that UA removed some ONOO. - This leads to a decrease in fluorescence intensity, further verifying the probe's effect on both endogenous and exogenous ONOO. - The above results demonstrate that the CY-ONOO probe possesses good stress response capability and adaptability to plant biological systems, and can detect ONOO in various plant tissues under different treatment conditions. - Reliable dynamic and visual analysis of the content changes.

[0086] To further test the probe's applicability to different plant tissues, this application conducted a systematic oxidative stress experiment using wheat roots. For example... Figure 16 As shown, wheat root tissues were pretreated for 30 minutes in different concentrations of NaCl solutions (100 mM and 300 mM) and different concentrations of cadmium ion solutions (chromium chloride, 50 μM and 150 μM). An experimental group was also prepared by pretreating the pretreated plant tissues in a 1 mM UA solution for 30 minutes, followed by staining in a 5 μM CY-ONOO probe solution (the probe was dissolved in PBS buffer) for 20 minutes. All samples were washed with water after treatment and then imaged (excitation wavelength 638 nm, detection range 670-730 nm). The results showed that under both stress conditions, the fluorescence intensity of the root tip tissue increased with increasing stress intensity, indicating that the probe can sensitively capture ONOO within the root tip tissue. - Content changes, and after ONOO - The fluorescence intensity of root tip tissue treated with the scavenger UA decreased significantly, further illustrating the connection between enhanced fluorescence signal and endogenous ONOO. - The formation of [something] is directly related to [something]. Furthermore, with increasing stress time, the fluorescence intensity exhibited by root tip cells gradually increases. Figure 17 The above results demonstrate that this probe can reliably and sensitively detect ONOO in wheat root tip tissue under different stresses. - The change in level reflects its ability to detect ONOO in complex plant systems. - Its good applicability and its potential for use in plants (ONOO) - Dynamic monitoring.

[0087] To test whether the probe's monitoring capabilities could extend beyond the tissue level to the overall plant level, this invention used mung bean (Vigna radiata cv. Zhonglv No. 1) as the experimental subject, applying NaCl salt stress and Cd... 2+ Heavy metal stress was used to test oxidative stress. Mung bean seeds were disinfected with 75% alcohol for 30 seconds, rinsed three times with sterile water, and cultured in the dark at room temperature. First, the mung bean seeds were germinated in distilled water, and then subjected to different concentrations of NaCl solution (100, 150, 200 mM) and Cd... 2+ The plant materials were incubated for different times under stress conditions of 50, 100, and 150 μM solutions. After incubation, all plant materials were uniformly immersed in 5 μM fluorescent probe CY-ONOO solution (dissolved in PBS buffer at pH 7.4) and incubated in the dark for 1 h. The surface was rinsed with water to remove any residual probe solution, and then imaging was performed (excitation wavelength 638 nm, detection range 670-730 nm). Figure 18 The images show the whole plant germination of mung beans 12 hours and 5 days after germination in NaCl solutions of different concentrations (0, 100, 150, 200 mM). Figure 19 This shows mung beans at different concentrations of Cd. 2+ Whole-plant imaging was performed 12 hours and 5 days after germination under stress conditions of 50, 100, and 150 μM solutions. It was clearly observed that the higher the stress concentration, the slower the stem growth with increasing stress duration. Furthermore, the fluorescence signal significantly increased with both the concentration and duration of stress, and the fluorescence began to extend towards the roots. These results confirm that the probe CY-ONOO can monitor ONOO in plant tissues induced by abiotic stress. - The increase in fluorescence intensity was positively correlated with stress intensity and stress duration.

[0088] The above experimental results demonstrate that the CY-ONOO probe exhibits excellent biocompatibility and superior imaging performance in plant tissues. Because its molecular structure contains p-aminobenzenesulfonamide—a structure previously shown to have Golgi apparatus targeting capabilities in animal cells—this application further investigated its subcellular localization within plant root tip cells. Wheat root tip tissue was first incubated with 50 nM Golgi-Tracker Green for 30 minutes, followed by treatment with 10 μM CY-ONOO probe (prepared by dissolving the probe in PBS buffer) for 1 hour. This experiment was repeated multiple times in parallel. Confocal microscopy imaging (excitation wavelength 638 nm, detection range 670-730 nm) is shown in the attached results. Figure 20 ,Depend on Figure 20 It was observed that the red near-infrared fluorescence signal was clearly distributed within the well-defined root tip cells, and highly overlapped with the green fluorescence signal of Golgi-Tracker Green, with an overlap coefficient of approximately 0.85. These results indicate that CY-ONOO is localized in the Golgi apparatus of plant cells, and its targeting behavior is consistent with that of structural analogs in animal cells.

Claims

1. A near-infrared fluorescent probe for detecting nitrite peroxide, characterized in that, The structure of the fluorescent probe is as follows: The fluorescent probe is designated CY-ONOO.

2. The method for preparing the near-infrared fluorescent probe for detecting nitrite peroxide as described in claim 1, characterized in that, The synthesis route is as follows: ， (1) Compound I reacts with pinacol 4-bromomethylphenylboronic acid in DMF to give compound II; (2) Compound II reacts with compound III in ethanol to give CY-ONOO.

3. The method for preparing the near-infrared fluorescent probe for detecting nitrite peroxide according to claim 2, characterized in that, Specifically, it includes the following steps; (1) Compound I and cesium carbonate were dissolved in DMF and stirred in an ice-water bath for 3 to 10 minutes; then 4-bromomethylphenylboronic acid pinacol ester was added under stirring and stirring was continued for 3 to 10 minutes. The mixture was removed from the ice bath and stirred at room temperature until the reaction was complete. After the reaction was completed, the solvent was removed to obtain compound II. (2) Compound II and Compound III were dissolved in ultra-dry ethanol, refluxed at 80-85°C for 10-15 hours, cooled to room temperature, filtered, and the solid was purified by column chromatography to obtain CY-ONOO.

4. The method for preparing the near-infrared fluorescent probe for detecting nitrite peroxide according to claim 2 or 3, characterized in that, In step (1), the molar ratio of compound I, 4-bromomethylphenylboronic acid pinacol ester and cesium carbonate is 1:1:2; in step (2), the molar ratio of compound II to compound III is (1.0~1.2):(1.0~1.2).

5. The application of the fluorescent probe according to claim 1 in the detection of nitrite peroxide in a plant system, characterized in that, Used for the fluorescent detection of peroxynitrite in plant systems.

6. The application according to claim 5, characterized in that, The probe is used for fluorescence imaging of plant cells and tissues, including onion inner epidermal cells, mung bean stem slices, and wheat root cells. Before imaging, the samples need to be treated with ONOO. - Treatment with solution, salt solution, or heavy metal solution, followed by staining in 1-10 μM CY-ONOO probe solution for 10-30 minutes, ONOO - The solution concentration was 10 μM to 25 μM, the salt solution concentration was 100 mM to 300 mM, and the heavy metal solution concentration was 50 μM to 150 μM.

7. The application according to claim 5, characterized in that, The probe is used for fluorescence imaging of the whole plant, including mung bean plants. Before imaging, the whole plant needs to be subjected to stress treatment with a salt solution or a heavy metal solution, and then soaked in 1~10 μM CY-ONOO probe solution for 0.5~1 hour. The concentration of the salt solution is 100 mM~200 mM, and the concentration of the heavy metal solution is 50 μM~150 μM.

8. The application according to claim 6 or 7, characterized in that, The salt solution is a NaCl solution, and the heavy metal solution is a cadmium ion solution.

9. The application according to claim 5, characterized in that, The probe is used for colocalization fluorescence imaging of the Golgi apparatus in plant cells.

10. The application according to claim 9, characterized in that, Before imaging, the probe needs to be treated with 20-100 nM Golgi-TrackerGreen solution for 20-40 minutes, and then soaked in 5-15 μM probe solution for 0.5-1 hour.

Images