Near-infrared fluorescent probe for detecting H2S in biological system as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for detecting H2S in a biological system as well as a preparation method and application of the near-infrared fluorescent probe. The structural formula of the fluorescent probe is shown in the specification, and the probe has excellent mitochondrial targeting ability and can be successfully applied to H2S detection and fluorescence imaging in a biological system. By virtue of the unique dynamic response characteristic, the probe can quickly distinguish H2S from other sulfur-containing biological small molecules and amino acids. And obvious color change is accompanied in the reaction process, so that naked eye observation is facilitated. The probe can be used for effectively monitoring changes of endogenous and exogenous H2S in onion endoepidermis cells, soybean slice cells, soybean root cells, tobacco root cells and wheat root cells under stress conditions, and is suitable for whole-plant fluorescence imaging analysis of mung bean plants and peanut plants. On the other hand, the prepared fluorescent probe is successfully applied to co-localization imaging of wheat root mitochondria, and real-time monitoring of H2S in juvenile zebrafish bodies is achieved.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a near-infrared fluorescent probe for detecting H2S in biological systems, its preparation method, and its application. Background Technology

[0002] Hydrogen sulfide (H2S) is a key gaseous signaling molecule in plants, playing a central role in regulating stomatal movement, promoting root development, enhancing antioxidant defense, and improving tolerance to abiotic stresses. With in-depth research into the physiological functions of H2S, its central role in plant stress response mechanisms has become increasingly prominent. Therefore, developing technologies for rapid monitoring of H2S in plants is crucial for elucidating its physiological functions. Current H2S detection mainly relies on traditional methods such as gas chromatography, colorimetry, ion chromatography, ultraviolet absorption chromatography, and high-performance liquid chromatography. However, these methods typically require complex experimental equipment, are costly, and, importantly, can damage biological samples. Therefore, they are unsuitable for in-situ real-time detection of plant samples, significantly limiting the ability to monitor H2S in living plants.

[0003] To overcome the technical bottlenecks of traditional detection technologies in in vivo plant applications, the development of a novel H2S detection tool suitable for plant systems and possessing in-situ real-time monitoring capabilities is urgently needed. Molecular probe technology, with its advantages of high selectivity, high sensitivity, low biotoxicity, and good biocompatibility, provides an ideal approach to achieving this goal. These probes can quantitatively detect H2S in plants under non-invasive conditions without affecting normal physiological processes and metabolic activities, providing key technical support for revealing the dynamic behavior of H2S in plant signal transduction and stress responses. Among numerous molecular imaging techniques, near-infrared fluorescence imaging exhibits outstanding technical advantages. Near-infrared light has stronger tissue penetration capabilities, effectively penetrating thicker plant tissues (such as leaves, stems, and roots), significantly reducing signal attenuation caused by light scattering and absorption. Simultaneously, biological tissues exhibit extremely low background autofluorescence in the near-infrared band, which can significantly improve the imaging signal-to-noise ratio, thereby achieving high-resolution, real-time visualization monitoring of the distribution and dynamic changes of H2S in deep plant tissues. Therefore, developing molecular probes based on near-infrared fluorescence imaging technology that can specifically respond to H2S has become the key to advancing research on plant gas signaling molecules.

[0004] The target product of this application has been characterized. Studies have shown that in acetonitrile and HEPES buffer solutions, 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 method for preparing a near-infrared fluorescent probe for detecting H2S in biological systems, as well as its preparation method and application. The fluorescent probe is prepared by condensation using hemicyanine as the parent nucleus, introducing 4-methyl-1-(triethylene glycol monomethyl ether)quinolineonium salt as a mitochondrial localizing group, and 2,4-dinitrofluorobenzene as a recognition site for H2S detection. This fluorescent probe exhibits a relatively stable response to H2S, good linearity and selectivity, and has been successfully used for fluorescence imaging of plants (sections, roots, whole plants, co-localization) and zebrafish.

[0006] The present invention also provides a method for preparing the above-mentioned fluorescent probe and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A near-infrared fluorescent probe for detecting H2S in biological systems, the molecular formula of the fluorescent probe being C 37 H 36 N3O9 + Br - The structural formula is as follows:

[0009] .

[0010] The preparation method and synthetic route of the above-mentioned near-infrared fluorescent probe for detecting H2S in biological systems are as follows:

[0011]

[0012] Specifically, the following steps are included:

[0013] (1) Compound 1 and compound 2 react in ethanol to give CTH-OH;

[0014] (2) The fluorescent probe CTH-OH-NO2 was obtained by reacting CTH-OH and 2,4-dinitrofluorobenzene in DMF.

[0015] Preferably, compound 1 and compound 2 are dissolved in anhydrous ethanol, refluxed at 80-90°C until the reaction is complete, cooled to room temperature, concentrated, and purified by thin-layer chromatography to obtain CTH-OH.

[0016] Preferably, the preparation process of the fluorescent probe CTH-OH-NO2 is as follows: CTH-OH and potassium carbonate are dissolved in ultra-dry N,N-dimethylformamide, 2,4-dinitrofluorobenzene is added and stirred at 70~80 °C until the reaction is complete, the reaction mixture is cooled to room temperature, extracted with dichloromethane, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain CTH-OH-NO2.

[0017] Preferably, in step (1), the molar ratio of compound 1 to compound 2 is (1~2): (1~2).

[0018] Preferably, in step (2), the molar ratio of CTH-OH, 2,4-dinitrofluorobenzene and potassium carbonate is 1:(2~4):(4~6).

[0019] Furthermore, for fluorescence imaging of plant cells and tissues, the plant cells and tissues include onion inner epidermal sections, soybean stem sections, soybean roots, tobacco roots, and wheat roots. Before imaging, the samples need to be treated with NaHS solution or D-cys solution, and then stained in 1~10μM CTH-OH-NO2 probe solution for 2~10 minutes. The concentration of NaHS solution is 50μM~200μM, and the concentration of D-cys solution is 0.1~1 mM.

[0020] Specifically, onion epidermal cells, soybean stem slices, soybean roots, tobacco roots, and wheat roots were first treated in different solutions for 3-10 minutes, then stained in 1-10 μM probe solution for 3-8 minutes, rinsed with water, and then imaged.

[0021] Furthermore, the probe is used for imaging endogenous and exogenous H2S in wheat root cells. Before imaging, the sample needs to be treated with NaHS solution for 20-40 minutes, D-cys solution for 1-6 hours, NaCl solution for 12-36 hours, AlCl3 solution for 3-12 hours, or exposed to air for 3-9 hours. Then, it is stained in 1-10 μM CTH-OH-NO2 probe solution for 20-40 minutes, rinsed with water, and then imaged. The concentration of NaHS solution is 60-180 μM, the concentration of D-cys solution is 0.1-1 mM, the concentration of NaCl solution is 50-150 mM, and the concentration of AlCl3 solution is 20-100 μM.

[0022] Furthermore, when the probe is used for fluorescence imaging of the whole plant, it needs to be treated with a salt solution or a metal solution before imaging processing. The concentration of the metal solution is 20-100 μM, and the concentration of the salt solution is 50-200 mM. Preferably, the salt is sodium chloride, and the metal solution is an AlCl3 solution.

[0023] Specifically, the germinated plant seeds are treated with a salt solution or a metal solution for 10 hours to 5 days, then soaked in a 1-10 μM fluorescent probe CTH-OH-NO2 solution for 0.5 to 2 hours, rinsed with water, and then imaged. The plant seeds refer to mung bean or peanut seeds.

[0024] Furthermore, the probe is used for colocalization fluorescence imaging of mitochondria in plant cells. Before imaging, it needs to be treated with 20-100 nM Mito Tracker Green solution for 20-40 minutes, and then soaked in 5-15 μM probe solution for 0.5-2 hours.

[0025] Furthermore, the probe is used for imaging zebrafish, and it needs to be treated with NaHS solution with a concentration of 50~200 μM before imaging processing.

[0026] Specifically, zebrafish were pretreated in a culture medium containing NaHS solution (final NaHS solution concentration of 50-200 μM) for 20-40 minutes, then incubated in a culture medium containing 5-15 μM fluorescent probe solution for 20-40 minutes before imaging.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention has high selectivity and sensitivity, can specifically identify H2S molecules, is not affected by common ions and amino acids, and can reliably detect H2S in biological samples.

[0029] 2. This probe has good biocompatibility and applicability, good water solubility and low toxicity. It has been successfully used for real-time, in-situ imaging at the plant cell, tissue and even whole plant level, and can realize mitochondrial colocalization monitoring.

[0030] 3. This probe combines ease of synthesis with practical potential. The preparation route is mature, the yield is high, and the cost is controllable, providing a stable and reliable analytical tool for plant physiological research and agricultural environmental monitoring. Attached Figure Description

[0031] Figure 1 The 1H NMR spectrum of the near-infrared fluorescent probe prepared in Example 1;

[0032] Figure 2 The carbon NMR spectrum of the near-infrared fluorescent probe prepared in Example 1;

[0033] Figure 3 The images show the near-infrared fluorescent probe prepared in Example 1 and its high-resolution mass spectrum after adding NaHS.

[0034] Figure 4 Comparison of the UV absorption spectra of the near-infrared fluorescent probe, the parent core, and the probe after adding NaHS to the probe prepared in Example 1;

[0035] Figure 5 The image shows a comparison of the fluorescence spectra of the near-infrared fluorescent probe, the parent core, and the probe after adding NaHS to the probe prepared in Example 1.

[0036] Figure 6 The fluorescence spectra of the near-infrared fluorescent probe prepared in Example 1 after adding different concentrations of NaHS solution are shown.

[0037] Figure 7 Linear relationship of fluorescence intensity at 730 nm after adding different concentrations of NaHS solution to the near-infrared fluorescent probe prepared in Example 1;

[0038] Figure 8 Here are bar charts showing the near-infrared fluorescent probe prepared in Example 1 and its fluorescence spectra in different pH systems after the addition of NaHS solution;

[0039] Figure 9 The fluorescence spectrum of the near-infrared fluorescent probe prepared in Example 1 after the addition of NaHS is shown as a change over time.

[0040] Figure 10 Different ions (in the figure: from left to right: 1, Zn) 2+ , 2. Ni 2+ 3. K + 4. Co 2+ , 5.Ba 2+ 6. Mn 2+ 7. Fe 3+ 8. Cu 2+ 9. Cd 2+ 10. Na + , 11. Ca 2+ 12. N2H4, 13. S 2- 14. F - , 15. Cl - , 16. Br - 17. ClO - 18. HSO3 - A bar chart comparing the fluorescence intensity of the fluorescent probe prepared in Example 1 with that of the following: 19. Glutamic acid, 20. Lysine, 21. Arginine, 22. Tryptophan, 23. Phenylalanine, 24. Cysteine, 25. Glutathione, 26. Homocysteine, 27. NaHS) at 730 nm.

[0041] Figure 11 The near-infrared fluorescent probe prepared in Example 1 was used to test its ability to recognize H2S interference. (In the figure: from left to right, 1. Zn) 2+ , 2. Ni2+ 3. K + 4. Co 2+ , 5. Ba 2+ 6. Mn 2+ 7. Fe 3+ 8. Cu 2+ 9. Cd 2 + 10. Na + , 11. Ca 2+ 12. N2H4, 13. S 2- 14. F - , 15. Cl - , 16. Br - 17. ClO - , 18.HSO3 - , 19. Glutamic acid, 20. Lysine, 21. Arginine, 22. Tryptophan, 23. Phenylalanine, 24. Cysteine, 25. Glutathione, 26. Homocysteine, 27. NaHS);

[0042] Figure 12 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of the inner epidermis of onions after pretreatment with different solutions, and (a) is a fluorescence quantification image of the inner epidermis of onions in A.

[0043] Figure 13 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of soybean stem slice cells after pretreatment with different solutions, and (a) is a fluorescence quantification image of soybean stem slice cells in A.

[0044] Figure 14 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of soybean root cells after pretreatment with different solutions, and (a) is a fluorescence quantification image of soybean root cells in A.

[0045] Figure 15 These are bioimaging images of near-infrared fluorescent probes, where (B) is a bioimaging image of tobacco root cells after pretreatment with different solutions, and (b) is a fluorescence quantification image of tobacco root cells in A.

[0046] Figure 16These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of wheat root cells pretreated with different concentrations of NaHS solution, and (a) is a fluorescence quantification image of wheat root cells in A.

[0047] Figure 17 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of wheat root cells pretreated in D-cys solution for different times, and (a) is a fluorescence quantification image of wheat root cells in A.

[0048] Figure 18 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of wheat root cells after pretreatment in NaCl solution for different times, and (a) is a fluorescence quantification image of wheat root cells in A.

[0049] Figure 19 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of wheat root cells exposed to air for different times, and (a) is a fluorescence quantification image of wheat root cells in A.

[0050] Figure 20 This is a bioimaging image of a near-infrared fluorescent probe, where (A) shows the effect of the probe on Al. 3+ Bioimaging images of wheat root cells after pretreatment in solution for different times, (a) is the fluorescence quantification image of wheat root cells in A;

[0051] Figure 21 This is a whole-plant imaging image of a near-infrared fluorescent probe, where (A) shows mung bean seeds exposed to aluminum ions (Al). 3+ (A) Experimental treatment procedure for stress, (B) and (C) are whole-plant fluorescence imaging results after 15 hours and 3 days of treatment with different concentrations of aluminum ions (0, 20, 50, 80, 100 μM), respectively, and (D) and (E) whole-plant imaging fluorescence quantification diagram.

[0052] Figure 22 This is a whole-plant imaging image of a near-infrared fluorescent probe, where (A) shows peanut seeds exposed to aluminum ions (Al). 3+ (A) Experimental treatment procedure for stress, (B) and (C) are whole-plant fluorescence imaging results after 12 hours and 4 days of treatment with different concentrations of aluminum ions (0, 20, 50, 80, 100 μM), respectively, and (D) and (E) whole-plant imaging fluorescence quantification diagram.

[0053] Figure 23The images show whole-plant imaging of near-infrared fluorescent probes. (A) shows the experimental treatment process of mung bean seeds under salt stress. (B) and (C) are whole-plant fluorescence imaging results after 15 hours and 4 days of treatment with different concentrations of NaCl solution (0, 50, 100, 150, 200 mM), respectively. (D) and (E) are whole-plant fluorescence quantification images.

[0054] Figure 24 The images show whole-plant imaging of near-infrared fluorescent probes. (A) shows the experimental treatment process of peanut 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, 50, 100, 150, 200 mM), respectively. (D) and (E) are whole-plant fluorescence quantification images.

[0055] Figure 25 The images show bioimaging of the near-infrared fluorescent probe, where (A) is a colocalization image of MitoTracker Green and wheat roots treated with the probe.

[0056] Figure 26 These are bioimaging images of near-infrared fluorescent probes, where (A) is a bioimaging image of zebrafish treated with NaHS, and (a) is a fluorescence quantification image of the zebrafish in A. Detailed Implementation

[0057] The present invention will be further described below with reference to embodiments and accompanying drawings, but this is not intended to limit the invention.

[0058] Example 1

[0059] The preparation process of the fluorescent probe in this embodiment specifically includes the following steps:

[0060] (1) Preparation of compound 1

[0061] The synthesis route is as follows:

[0062]

[0063] 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 7.8 g of compound I 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 (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. After the reaction was complete, the mixture was concentrated and purified by eluent petroleum ether:ethyl acetate (60:1 v / v) to give a white solid, compound II (approximately 7.80 g, 85% yield). Under nitrogen atmosphere, anhydrous potassium carbonate (2.07 g, 15 mmol), compound I (0.94 g, 5 mmol), and compound II (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 solid potassium carbonate. The filtrate was concentrated to give compound III, which was used directly in subsequent steps without further purification. 50 mL of CH2Cl2 and compound III 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 30°C water bath. 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 1 (approximately 1.30 g), with a yield of 63.2%. The NMR spectra of compound 1 are as follows:

[0064] 1 H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H), 6.92 (s, 1H), 6.66-6.56 (m, 2H), 2.54 (d, J = 6.8 Hz, 2H), 2.28 (t, J = 6.0Hz, 2H), 1.61 (p, J = 6.1 Hz, 2H).

[0065] 13C NMR (101 MHz, DMSO-d6) δ 186.57, 160.77, 160.17, 153.21, 128.60,128.08, 125.17, 113.51, 112.47, 111.76, 102.33, 29.42, 21.70, 20.48.

[0066] (2) Preparation of compound 2

[0067] The synthesis route is as follows:

[0068]

[0069] Under a nitrogen atmosphere, 4-methylquinoline (0.8 mL, 5.75 mmol) and diethylene glycol 2-bromoethyl methyl ether (4.95 mL, 17.25 mmol) were added to a 100 mL round-bottom flask containing 10 mL of acetonitrile solution. The mixture was stirred at 120 °C for 8 hours. After filtering the reaction mixture under reduced pressure, 8 mL of petroleum ether was added. The solution was concentrated in a refrigerator to give a purple solid. After filtration, compound 2 (1.43 g, yield 67%) was obtained. The NMR information of compound 2 is as follows:

[0070] 1 H NMR (400 MHz, DMSO-d6) δ 9.48 (d, J = 6.1 Hz, 1H), 8.81 (d, J = 9.0Hz, 1H), 8.68 (dd, J = 8.5, 1.4 Hz, 1H), 8.38 (ddd, J = 8.7, 6.9, 1.4 Hz,1H), 8.21 (dd, J = 11.1, 6.9 Hz, 2H), 5.40 (t, J = 4.9 Hz, 2H), 4.09 (t, J =4.9 Hz, 2H), 3.64 (dd, J = 3.9, 2.0 Hz, 2H), 3.50 (s, 2H), 3.44 – 3.42 (m,2H), 3.42 – 3.40 (m, 2H), 3.15 (s, 3H).

[0071] 13C NMR (100 MHz, DMSO-d6) δ 159.34, 149.65, 137.39, 135.44, 130.03,129.30, 127.57, 122.72, 120.02, 71.61, 70.31, 70.05, 69.96, 68.23, 58.51,56.92, 20.26.

[0072] (3) Preparation of CTH-OH

[0073] The synthesis route is as follows:

[0074]

[0075] Compound 1 (86.51 mg, 0.379 mmol) and compound 2 (140.23 mg, 0.379 mmol) were dissolved in anhydrous ethanol (10 mL). The mixture was refluxed at 80 °C for 12 hours. After cooling to room temperature, the crude product was concentrated under reduced pressure and then purified by preparative thin-layer chromatography (TLC) using a dichloromethane / methanol (60:1 v / v) mixed solvent system as eluent. The final product obtained and collected was CTH-OH, a deep blue solid (81.42 mg, yield 37%). Specific NMR data are as follows:

[0076] 1 H NMR (400 MHz, Methanol-d4) δ 8.46 (d, J = 6.9 Hz, 1H), 7.94 (d, J =8.6 Hz, 1H), 7.87 (d, J = 14.8 Hz, 1H), 7.79-7.75 (m, 1H), 7.71 (dd, J =15.9, 8.6 Hz, 2H), 7.46 (t, J = 7.5 Hz, 1H), 6.48 (d, J = 14.9 Hz, 1H), 6.43-6.36 (m, 2H), 6.25 (dd, J = 8.3, 2.3 Hz, 1H), 5.92 (s, 1H), 4.62-4.51 (m,2H), 3.89 (t, J = 4.9 Hz, 2H), 3.67-3.60 (m, 2H), 3.59-3.51 (m, 4H), 3.47(dd, J = 5.9, 3.0 Hz, 2H), 3.31 (s, 3H), 2.36-2.24 (m, 2H), 2.21-2.11 (m,2H), 1.64 (p, J = 7.0, 6.1 Hz, 2H).

[0077] 13 C NMR (100 MHz, Methanol-d4) δ 159.61, 155.26, 153.37, 151.86,145.00, 137.72, 137.37, 133.71, 127.57, 127.50, 127.47, 125.66, 125.31,125.01, 117.69, 113.96, 112.21, 112.12, 112.03, 111.47, 101.66, 71.57, 70.28,70.10, 69.94, 67.82, 57.86, 55.20, 28.68, 24.13, 20.34.

[0078] (4) Preparation of CTH-OH-NO2

[0079] The synthesis route is as follows:

[0080]

[0081] The preparation process of the fluorescent probe CTH-OH-NO2 is as follows: CTH-OH (53 mg, 0.091 mmol) and potassium carbonate (62.9 mg, 0.455 mmol) were dissolved in 10 mL of ultra-dry N,N-dimethylformamide, and 2,4-dinitrofluorobenzene (0.0343 mL, 0.273 mmol) was added and stirred at 80 °C for 9 hours. After the reaction mixture was cooled to room temperature, it was extracted with dichloromethane, and purified by preparative thin-layer chromatography (TLC) using a dichloromethane / methanol (90:1 v / v) mixed solvent system as the eluent. The final product obtained and collected was CTH-OH-NO2, a deep purple solid (37.2 mg, yield 67%). The 1H and 1C NMR spectra of the fluorescent probe prepared in this example are shown below. Figure 1 and Figure 2 As shown, the specific NMR data are as follows:

[0082] 1H NMR (400 MHz, Methanol-d4) δ 8.86 (d, J = 2.7 Hz, 1H), 8.81 (d, J =6.7 Hz, 1H), 8.52 (d, J = 8.6 Hz, 1H), 8.47 (dd, J = 8.9, 2.7 Hz, 1H), 8.28(s, 2H), 8.09 (d, J = 6.7 Hz, 1H), 8.03 (t, J = 7.9 Hz, 1H), 7.83 (t, J = 7.7Hz, 1H), 7.38 (d, J = 9.2 Hz, 1H), 7.18 – 7.12 (m, 2H), 7.08 – 7.02 (m, 1H),6.83 (dd, J = 8.3, 2.2 Hz, 1H), 6.56 (s, 1H), 4.97 (t, J = 4.7 Hz, 2H), 4.01(t, J = 4.7 Hz, 2H), 3.63 – 3.58 (m, 2H), 3.51 (t, J = 4.3 Hz, 2H), 3.44 (dd,J = 6.2, 3.4 Hz, 2H), 3.37 (s, 2H), 3.25 (s, 3H), 2.63 (t, J = 5.9 Hz, 2H),2.54 (t, J = 6.0 Hz, 2H), 1.85 (p, J = 5.9 Hz, 2H).

[0083] 13 C NMR (100 MHz, Methanol-d4) δ 155.53, 154.24, 153.58, 153.45,153.18, 146.40, 142.58, 140.27, 138.21, 137.41, 134.47, 130.03, 129.19,128.43, 128.14, 127.82, 126.22, 125.62, 124.88, 121.64, 120.89, 120.73,119.68, 118.28, 114.91, 114.41, 113.55, 113.49, 106.48, 71.48, 70.30, 70.03,69.85, 67.91, 57.71, 55.86, 35.17, 31.67, 29.15, 24.21, 20.34, 13.89, 13.10.

[0084] The parent nucleus CTH-OH, the fluorescent probe CTH-OH-NO2, and the fluorescent probe CTH-OH-NO2 prepared in this invention were subjected to high-resolution mass spectrometry analysis with NaHS. Figure 3 As shown. The probe data showed good agreement, confirming the correct structure of the target product, and the probe data after the response matched the CTH-OH data. ESI-MS (m / z) for C 37 H 36 N3O9 + [M] + : Calcd 666.2446, found666.2443.

[0085] The application tests of the fluorescent probe prepared in this embodiment are as follows:

[0086] 1) Preparation of stock solution for testing

[0087] a. Fluorescent probe CTH-OH-NO2 for detecting H2S sample solution (1.00×10⁻⁶) -3 Preparation of (mol / L): Accurately weigh 0.0035 g (M=746.7) of fluorescent probe and dissolve it in 4.7 mL of dimethyl sulfoxide to prepare a concentration of 1.00×10⁻⁶ mol / L. -3 A solution of mol / L.

[0088] b. CTH-OH sample solution (1.00 × 10⁻⁶) -3 Preparation of (mol / L): Accurately weigh 0.0046 g (M=580.6) of fluorescent probe and dissolve it in 8 mL of dimethyl sulfoxide to prepare a concentration of 1.00×10⁻⁶ mol / L. -3 A solution of mol / L.

[0089] c. All amino acids and ions were prepared with deionized water to a concentration of 1.0 × 10⁻⁶. -2 A solution of mol / L.

[0090] d. Preparation of HEPES buffer solution (10 mM, pH=7.4):

[0091] Accurately weigh 1.19 g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) using a balance, dissolve it completely in 400 mL of Wahaha purified water, and then prepare a 1.0 × 10⁻⁶ solution. -2 Add the prepared sodium hydroxide aqueous solution (mol / L) to a solution with a concentration of 1.0 × 10⁻⁶ mol / L. -2 Adjust the pH to 7.4 with a sodium hydroxide aqueous solution of mol / L, then bring the volume to 500 mL in a volumetric flask. After use, store in the refrigerator for future use.

[0092] Spectroscopic properties experiment of the probe

[0093] Use a pipette to transfer three 3 mL acetonitrile and HEPES buffer (1:9, v / v) systems, and add 30 μL of probe stock solution (1.00 × 10⁻⁶) to each system. -3 30 μL of nucleus stock solution (1.00 × 10⁻⁶ mol / L) -3 mol / L), and 30 μL of probe stock solution (1.00×10⁻⁶ mol / L). -3 45 μL of NaHS (1.00 × 10⁻⁶ mol / L) and 45 μL of NaHS (1.00 × 10⁻⁶ mol / L) -2 A mol / L solution was prepared. After thorough mixing and ensuring no bubbles were present, the UV absorption spectrum was measured at room temperature (parameters: scan range 350-900 nm, fast scan speed, 1 nm interval). Results are as follows: Figure 4 ,Depend on Figure 4 It can be seen that the probe reverts to the parent nucleus after responding to H2S, and its maximum absorption peak shifts from 560 nm to 620 nm. Further measurement of its fluorescence spectrum (parameters: excitation wavelength 630 nm, excitation slit width 5.0 nm, emission slit width 10.0 nm) yielded the following results: Figure 5 ,Depend on Figure 5 It can be seen that the fluorescence of the probe is enhanced after responding to H2S. Different concentrations of NaHS solution were added to a 3 mL system of acetonitrile and HEPES buffer (1:9, v / v), and the fluorescence spectra were measured. The results are as follows: Figure 6 As shown, with the increase of NaHS concentration, the fluorescence intensity of the solution system at 730 nm gradually increases. The linear equation of CTH-OH-NO2 obtained by linear fitting is: Y = 363.9167 + 4.9723[NaHS], where R 2 =0.999, the calculated detection limit is 0.035 μM. The probe CTH-OH-NO2 has a detection limit within the NaHS concentration range of 0-90 μM. log I 730 There is a good linear relationship between it and [NaHS], as shown in the experimental results. Figure 7 As shown.

[0094] Use a 3 mL mixture of acetonitrile and HEPES buffer (1:9, v / v). Adjust the pH of the HEPES buffer to the desired level with 1 mol / L hydrochloric acid or 1 mol / L NaOH solution, then mix it with acetonitrile at the required volume ratio. Add 30 μL (1.00 × 10⁻⁶) of the mixture to each volume. -3 Add 30 μL (1.00 × 10⁻⁶ mol / L) probe stock solution to another sample. -3 mol / L) probe stock solution and 45 μL NaHS (1.00×10⁻⁶ mol / L) -2A mol / L solution was shaken thoroughly until no bubbles were present, and its fluorescence intensity was measured at room temperature (730 nm). The results are as follows: Figure 8 As shown. By Figure 8 It can be seen that the fluorescence intensity of the probe remains relatively stable; the probe exhibits the best response at pH 6-8 after the addition of NaHS solution, therefore, this probe is suitable for plant and animal environments. Using a pipette, 3 mL of acetonitrile and HEPES buffer solution (1:9, v / v) was transferred, and 30 μL of probe stock solution (1.00 × 10⁻⁶) was added. -3 45 μL of NaHS (1.00 × 10⁻⁶ mol / L) and 45 μL of NaHS (1.00 × 10⁻⁶ mol / L) -2 A mol / L solution. Shake well until no bubbles are present, and perform tests every three minutes at room temperature. The results are as follows: Figure 9 As shown, the probe responds completely in about 40 minutes.

[0095] Using a 3 mL transfer of acetonitrile and HEPES buffer solution (1:9, v / v), add 30 μL (1.00 × 10⁻⁶) to the solution. -3 1 mol / L) probe stock solution was added, and then the analyte (45 μL, 1.00 × 10⁻⁶ mol / L) was added sequentially. -2 mol / L)( 1.Zn 2+ ,2.Ni 2+ 3.K + ,4.Co 2+ 5.Ba 2+ 6.Mn 2+ 7.Fe 3+ 8.Cu 2+ 9.Cd 2+ ,10.Na + ,11.Ca 2+ , 12.N2H4, 13.S 2- 14.F - 15.Cl - ,16.Br - 17.ClO - 18.HSO3 - 19. Glutamic acid, 20. Lysine, 21. Arginine, 22. Tryptophan, 23. Phenylalanine, 24. Cysteine, 25. Glutathione, 26. Homocysteine, 27. Sodium hydrosulfide), their fluorescence spectra were detected, and the results are as follows. Figure 10 As shown, the fluorescence emission of the probe is significantly enhanced in the NaHS system, and the fluorescence intensity of the probe does not change significantly after the addition of other different ions.

[0096] Using a 3 mL transfer of acetonitrile and HEPES buffer solution (1:9, v / v), add 30 μL (1.00 × 10⁻⁶) to the solution. -3 50 μL of probe stock solution (mol / L) was added, followed by the addition of 50 μL of 1.00 × 10⁻⁶ mol / L probe stock solution. -2 mol / L) interfering substance (1. Zn 2+ ,2.Ni 2+ 3.K + ,4.Co 2 + 5.Ba 2+ 6.Mn 2+ 7.Fe 3+ 8.Cu 2+ 9.Cd 2+ ,10.Na + ,11.Ca 2+ , 12.N2H4, 13.S 2- 14.F - 15.Cl - ,16.Br - 17.ClO - 18.HSO3 - 19. Glutamic acid, 20. Lysine, 21. Arginine, 22. Tryptophan, 23. Phenylalanine, 24. Cysteine, 25. Glutathione, 26. Homocysteine, 27. Sodium hydrosulfide), mixed thoroughly, and subjected to fluorescence spectroscopy scanning. The fluorescence intensity at 730 nm was plotted as a bar chart. Figure 11 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 responds well to hydrogen sulfide in the presence of common anions, cations, and amino acid interfering substances, and has strong anti-interference ability against a variety of common anions and cations, demonstrating the excellent performance of the fluorescent probe.

[0097] 2) Imaging of the probe

[0098] Onion inner epidermis, soybean stem tissue sections, and soybean and tobacco root tissues were pretreated for 30 minutes in 1 mM HT solution, different concentrations of NaHS solution (50 μM and 200 μM), and 0.5 mM D-cys solution, respectively. Then, they were stained for 5 minutes in 5 μM CTH-OH-NO2 probe solution (probe stock solution diluted to 5 μM in acetonitrile and HEPES buffer (1:9, v / v)). All samples were washed with water after treatment and then subjected to fluorescence imaging. Fluorescence imaging showed ( Figure 12-15Both NaHS treatments showed a concentration-dependent increase in fluorescence intensity, confirming the effective detection of endogenous H2S. Furthermore, D-cysteine ​​pretreatment led to a significant fluorescence enhancement, indicating that the probe can monitor the increase in endogenous H2S. Notably, all samples pretreated with HT showed a decrease in fluorescence, suggesting that HT may effectively block H2S accumulation in plant cells by inhibiting H2S synthase activity or interfering with its metabolic pathway. These results collectively demonstrate that the CTH-OH-NO2 probe possesses good stress response capability and biocompatibility, enabling reliable real-time tracking and visualization of H2S dynamic changes in various plant tissues under different treatment conditions.

[0099] To evaluate the ability of the CTH-OH-NO2 probe to detect endogenous and exogenous H2S in plant tissues, this application conducted a systematic stress experiment using wheat roots. In all the following experiments, the roots were stained with the CTH-OH-NO2 probe solution for 30 minutes after stress treatment. Figure 16 As shown, after treatment with different NaHS concentrations (0–180 μM) for 30 minutes, the experimental results showed that the fluorescence intensity of the roots increased with increasing H2S levels, indicating that the probe has a sensitive response to exogenous H2S. To further investigate the dynamic changes of endogenous H2S, this application used 0.5 mM D-cysteine ​​to induce endogenous H2S generation. With prolonged treatment time (0–6 hours), the fluorescence intensity gradually increased. The addition of HT significantly inhibited the fluorescence enhancement effect, indicating that it interfered with the endogenous H2S generation process. Figure 17 In salt stress experiments, wheat roots treated with 100 mM NaCl solution for 0, 12, 24, or 36 hours showed a continuous increase in fluorescence intensity over time. Figure 18 This confirmed that salt stress can induce hydrogen sulfide accumulation in wheat roots. In the air exposure experiment, the fluorescence signal of isolated wheat roots gradually increased after 0, 3, 6, and 9 hours of air exposure (…). Figure 19 This indicates that the oxidative stress condition also promotes H2S formation. In Al... 3+ In the stress experiment, the fluorescence intensity of wheat roots treated with 50 μM AlCl3 solution for 0, 3, 6, and 12 hours significantly increased with increasing treatment time. Figure 20 This indicates that metal stress can also effectively activate the H2S signaling pathway in wheat roots. These results collectively demonstrate that CTH-OH-NO2 can reliably capture H2S signaling pathways in wheat roots caused by salt stress, air exposure, and Al2O3. 3+ The study investigated the dynamic changes in H2S levels induced by various abiotic stresses, including treatment. These findings confirm the reliability and applicability of CTH-OH-NO2 in simultaneously detecting endogenous and exogenous H2S in complex plant systems.

[0100] To evaluate the imaging application of the probe CTH-OH-NO2 in whole plants, this invention uses mung beans and peanuts as plant models for testing. Dynamic H2S changes in plant tissues were simulated by applying exogenous metal stress and salt stress. The experiment was divided into a control group and an experimental group. First, mung bean and peanut seeds were disinfected with 75% alcohol for 30 seconds, rinsed three times with sterile water, and then cultured at room temperature in the dark. The seeds were then germinated in distilled water. The control group consisted of untreated plant seeds. In the experimental group, the germinated seeds were directly placed in different concentrations of AlCl3 (0, 20, 50, 80, 100 μM) and NaCl (0, 50, 100, 150, 200 mM) aqueous solutions for different durations to obtain mung bean sprouts / seedlings and peanut sprouts / seedlings. After culturing, all plant materials were immersed in a 5 μM (stock solution diluted to 5 μM with DMSO) fluorescent probe CTH-OH-NO2 solution for 1 hour, followed by rinsing off any residual probe solution with water. Finally, whole-plant imaging was performed. Figure 21 Al at different concentrations (0, 20, 50, 80, 100 μM) was demonstrated. 3+ Imaging of the entire mung bean plant in solution 15 hours and 3 days after germination, and... Figure 22 This demonstrates that in the same Al 3+ Whole-plant imaging of peanuts 12 hours and 4 days after germination under different concentration gradients. Whole-plant imaging results show that both plants exhibit significant Al2O3 concentrations. 3+ Concentration and time dependence: with Al 3+ With increasing concentration (20→100 μM) and prolonged exposure time (15 hours→3 / 4 days), the fluorescence intensity in plant tissues increased. This result confirms that the probe CTH-OH-NO2 can detect Al. 3+ Stress leads to an increase in H2S in plant tissues, and the increase in fluorescence intensity is positively correlated with the intensity and duration of metal stress. Figure 23 The images show the whole plant images of mung beans after germination for 15 hours and 4 days in NaCl solutions of different concentrations (0, 50, 100, 150, 200 mM). Figure 24 The images show whole-plant imaging of peanuts cultured under the same salt concentration gradient for 12 hours and 5 days. The whole-plant imaging results show that both plants exhibit significant salt concentration and time-dependent responses: with increasing salt concentration (50→200 mM), the fluorescence intensity in plant tissues significantly increased, and with prolonged treatment time, the fluorescence signal intensity also showed a clear increasing trend. This test result confirms that the probe CTH-OH-NO2 can monitor the increase of H2S in plant tissues caused by salt stress, and that the increase in fluorescence intensity is positively correlated with the intensity and duration of salt stress.

[0101] The above experimental results demonstrate that the CTH-OH-NO2 probe exhibits excellent biocompatibility and superior imaging performance in plant tissues. Given that its molecular structure contains a positively charged quinoline ion—a structure previously shown to have mitochondrial 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 MitoTracker Green for 30 minutes, followed by treatment with 10 μM CTH-OH-NO2 probe for 1 hour. This experiment was repeated multiple times in parallel. Confocal microscopy imaging showed (…) Figure 25 The red near-infrared fluorescence signal was clearly distributed within well-defined root tip cells, highly overlapping with the green fluorescence signal of MitoTracker Green, with an overlap coefficient of approximately 0.9. These results indicate that CTH-OH-NO2 is localized in the mitochondria of plant cells, and its targeting behavior is consistent with that of structural analogs in animal cells.

[0102] In the zebrafish model, this invention set up three groups: a blank control group consisting of normally cultured 4-day-old zebrafish larvae; experimental group I was incubated in 10 μM fluorescent probe CTH-OH-NO2 solution for 30 minutes; experimental group II was pretreated with 50 μM HT solution for 30 minutes, and then transferred to 10 μM fluorescent probe CTH-OH-NO2 solution for 30 minutes; experimental group III was pretreated with 50 μM and 200 μM NaHS solutions for 30 minutes, respectively, and then transferred to 10 μM fluorescent probe CTH-OH-NO2 solution for 30 minutes. All operations were performed under culture conditions of 28℃. The experimental results showed that no significant fluorescence signal was observed in the blank control group of zebrafish larvae; experimental group I, treated only with fluorescent probe CTH-OH-NO2, showed weak fluorescence; experimental group II, pretreated with HT and then loaded with the probe, also did not show significant fluorescence enhancement, and the signal intensity was comparable to that of experimental group I; while experimental group III, pretreated with NaHS and then loaded with the probe, showed a significantly enhanced fluorescence signal. The fluorescence intensity exhibited a gradient change (blank group < probe group < probe + NaHS group).

Claims

1. A near-infrared fluorescent probe for detecting H2S in biological systems, characterized in that, The molecular formula of the fluorescent probe is C 37 H 36 N3O9 + Br - The structural formula is as follows: CTH-ON-NO2.

2. The method for preparing the near-infrared fluorescent probe for detecting H2S in biological systems according to claim 1, characterized in that, The synthesis route is as follows: Specifically, the following steps are included: (1) Compound 1 and compound 2 react in ethanol to give CTH-OH; (2) The fluorescent probe CTH-OH-NO2 was obtained by reacting CTH-OH and 2,4-dinitrofluorobenzene in DMF.

3. The method for preparing a near-infrared fluorescent probe for detecting H2S in biological systems according to claim 2, characterized in that, Specifically, the following steps are included: (1) Specific preparation process of CTH-OH: Compound 1 and Compound 2 are dissolved in anhydrous ethanol, refluxed at 80~90℃ until the reaction is complete, cooled to room temperature, the reaction solution is concentrated, purified by thin layer chromatography, and CTH-OH is obtained; (2) The preparation process of the fluorescent probe CTH-OH-NO2 is as follows: CTH-OH and potassium carbonate are dissolved in ultra-dry N,N-dimethylformamide, 2,4-dinitrofluorobenzene is added and stirred at 70~80 °C until the reaction is complete. After the reaction mixture is cooled to room temperature, it is extracted with dichloromethane, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain CTH-OH-NO2.

4. The method for preparing a near-infrared fluorescent probe for detecting H2S in biological systems according to claim 2 or 3, characterized in that, In step (1), the molar ratio of compound 1 to compound 2 is (1~2): (1~2); in step (2), the molar ratio of CTH-OH, 2,4-dinitrofluorobenzene and potassium carbonate is 1:(2~4):(4~6).

5. The application of the near-infrared fluorescent probe of claim 1 for detecting H2S in biological systems, characterized in that, Fluorescent detection of H2S in biological 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, soybean stem sections, soybean roots, tobacco roots, and wheat roots. Before imaging, the samples need to be treated with NaHS solution or D-cys solution, and then stained in 1~10μM CTH-OH-NO2 probe solution for 2~10 minutes. The concentration of NaHS solution is 50μM~200μM, and the concentration of D-cys solution is 0.1~1 mM.

7. The application according to claim 5, characterized in that, The probe is used for imaging endogenous and exogenous H2S in wheat root cells. Before imaging, the sample needs to be treated with NaHS solution for 20-40 minutes, D-cys solution for 1-6 hours, NaCl solution for 12-36 hours, AlCl3 solution for 3-12 hours, or exposed to air for 3-9 hours. Then, it is stained in 1-10 μM TH-OH-NO2 probe solution for 20-40 minutes. The concentration of NaHS solution is 60-180 μM, the concentration of D-cys solution is 0.1-1 mM, the concentration of NaCl solution is 50-150 mM, and the concentration of AlCl3 solution is 20-100 μM.

8. The application according to claim 5, characterized in that, When the probe is used for fluorescence imaging of the whole plant, the plant includes mung bean plants and peanut plants. Before imaging, the plant needs to be subjected to stress treatment with a salt solution with a concentration of 50~200 mM or a metal solution with a concentration of 20~100 μM, and then immersed in a 1~10 μM fluorescent probe CTH-OH-NO2 solution for 0.5~2 hours.

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

10. The application according to claim 5, characterized in that, The probe is used for fluorescence imaging of zebrafish. Before imaging, it needs to be pretreated in a culture medium with a final concentration of 50-200 μM NaHS solution for 20-40 minutes, and then incubated in a culture medium containing 5-15 μM fluorescent probe solution for 20-40 minutes before imaging.

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