Fluorescent compound based on aza IR780 skeleton, preparation method and application thereof, and glutathione fluorescent probe reagent

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a fluorescent compound based on an aza IR780 skeleton, a preparation method and application of the fluorescent compound and a glutathione fluorescent probe reagent. The compound has a structure as shown in a formula (I) or a formula (II). The fluorescent compound based on the aza IR780 skeleton, which is provided by the invention, can be used as a probe to show a fluorescent response signal for opening two channels specific to glutathione, and can be successfully applied to visual imaging of the concentration of glutathione in a plant body. Formula (I) and formula (II).

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to fluorescent compounds based on the azaIR780 backbone, their preparation methods and applications, and glutathione fluorescent probe reagents. Background Technology

[0002] Redox homeostasis is crucial for the survival and growth of plants.

[0003] Redox homeostasis refers to the balance between oxidants and antioxidants within cells. In this balance, plants can cope with various biotic and abiotic stresses while maintaining normal physiological functions such as cell protection, enzyme activity regulation, signal transduction, detoxification, and disease resistance.

[0004] However, an imbalance in redox homeostasis can lead to oxidative stress, which can damage plants. For example, abiotic stresses such as high temperature, low temperature, drought, waterlogging, ultraviolet radiation, and pollution can all disrupt plant oxidative balance. Biotic stresses such as pathogen infection, insect predation, and competing plants can activate the plant's oxidative defense system, leading to the production of more reactive oxygen species and oxidative stress. Therefore, assessing redox homeostasis in plants is crucial for timely understanding of plant growth status and guiding agricultural production and management, and is of great significance for improving plant stress resistance and yield.

[0005] Glutathione, as a non-enzymatic antioxidant, is an excellent biomarker for evaluating redox homeostasis in plants under stress. Glutathione-based fluorescent probes have been extensively studied in animals, but fluorescent probes for glutathione in plants are still rarely reported.

[0006] In summary, the development of a highly sensitive glutathione fluorescent probe is of great significance as a new strategy for assessing redox homeostasis in plants under stress, and its application in monitoring redox balance in plants under various biotic and abiotic stresses is also of great significance. Summary of the Invention

[0007] The purpose of this invention is to provide a class of dual-channel glutathione fluorescent probes based on the azide IR780 backbone.

[0008] To achieve the above objectives, a first aspect of the present invention provides a fluorescent compound based on an aza-IR780 framework, the compound having the structure shown in formula (I) or formula (II); Formula (I); Equation (II).

[0009] A second aspect of the present invention provides a method for preparing a fluorescent compound based on an aza-IR780 framework, the fluorescent compound having the structure shown in formula (I), the method comprising: In the presence of a solvent and an alkaline substance, compound Aza-IR780 was reacted with compound NP-SO2Cl in a first contact reaction to obtain the fluorescent compound; Aza-IR780: NP-SO2Cl: ; Formula (I): .

[0010] A third aspect of the present invention provides another method for preparing a fluorescent compound based on an aza-IR780 framework, the fluorescent compound having the structure shown in formula (II), the method comprising: In the presence of a solvent and an alkaline substance, compound Cy-NMe-Pz and compound NP-SO2Cl undergo a second contact reaction to obtain the fluorescent compound; Cy-NMe-Pz: NP-SO2Cl: ; Formula (II): .

[0011] The fourth aspect of the present invention provides the application of the aza-IR780-based fluorescent compound described in the first aspect as a dual-channel glutathione fluorescent probe.

[0012] The fifth aspect of the present invention provides a glutathione fluorescent probe reagent containing a fluorescent compound based on the aza-IR780 backbone as described in the first aspect for monitoring the effective amount of glutathione content.

[0013] This invention provides a class of dual-channel glutathione fluorescent probes through rational molecular design. These fluorescent probes exhibit the characteristics of an aza-IR780 fluorophore and a naphthalimide sulfonamide recognition group.

[0014] The fluorescent compounds based on the azaIR780 backbone provided by this invention can be applied to the dynamic monitoring of glutathione in plants.

[0015] Through the above technical solution, the present invention has at least the following beneficial effects: The dual-channel glutathione fluorescent probe based on the aza-IR780 backbone provided by this invention has good spectral response performance.

[0016] The fluorescent probe provided by this invention exhibits excellent selectivity and sensitivity when detecting glutathione.

[0017] As will be seen from the following text of this invention, the two probe molecules with the structures shown in formulas (I) and (II) provided by this invention have a specific response to GSH.

[0018] In the UV absorption spectrum, the probe of formula (I) exhibited two absorption peaks at 694 nm and 835 nm in the buffer solution. However, after the addition of glutathione, the absorption peaks significantly weakened and transformed into a single broad peak, and the solution color changed from light green to light blue. In contrast, other amino acids had little effect on its absorption peaks; only Hcy showed a slight change in absorption while still maintaining the characteristic of having two absorption peaks.

[0019] In the ultraviolet absorption spectrum, the probe of formula (II) exhibits absorption peaks at 436 nm and 777 nm. After the addition of glutathione, the near-infrared absorption peaks are significantly enhanced, and the solution turns pale green. The addition of other amino acids does not significantly affect the absorption spectrum of the probe, indicating that the probe has good selectivity for glutathione.

[0020] This invention further investigated the fluorescence response of this type of probe in the visible and near-infrared channels.

[0021] Under 390 nm light excitation, the fluorescence intensity of the probe of formula (I) was significantly enhanced in the visible light region. The addition of glutathione increased its fluorescence intensity by nearly 300 times, while homocysteine ​​only caused an increase of one-sixth of the intensity. In the near-infrared region, the addition of glutathione increased the fluorescence intensity by more than 10 times, while the effects of other amino acids were negligible.

[0022] Under 390 nm light excitation, the probe of formula (II) also showed a strong fluorescence enhancement response to glutathione in the visible and near-infrared channels, with enhancements of 122 times and 66 times, respectively.

[0023] Furthermore, in the presence of different amino acids, the fluorescence response of the probes of formula (I) and formula (II) still maintains high selectivity for glutathione, indicating that they can effectively identify glutathione even in complex samples.

[0024] Then, the sensitivity of the probe was studied through titration experiments. The results showed that, under different concentrations of glutathione, this type of probe exhibited a linear fluorescence response and low detection limits for glutathione in both the visible and near-infrared channels.

[0025] In summary, the fluorescent compounds based on the aza-IR780 backbone provided by this invention, as dual-channel fluorescent probes, can specifically respond to glutathione and exhibit excellent sensitivity in both visible and near-infrared channels, showing broad application potential. Attached Figure Description

[0026] Figure 1 This is a synthetic route diagram of the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1; Figure 2 These are the UV absorption spectra of the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1 for different amino acid molecules; Figure 3 The images show the dual-channel fluorescence spectra of the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1 for different amino acid molecules. Figure 4 This is a dual-channel fluorescence spectrum of the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1, used to study their anti-interference performance against GSH. Figure 5 These are the UV absorption spectrum and dual-channel fluorescence emission spectrum of the probe Cy-NMe-NP prepared in Example 1 against GSH. Figure 6 These are the UV absorption spectrum and dual-channel fluorescence emission spectrum of the probe Cy-NH-NP prepared in Example 1 for the titration of GSH. Figure 7 The results are the response time measurements of the Cy-NMe-NP probe prepared in Example 1 interacting with GSH. Figure 8 These are the results of the response time measurement of the probe Cy-NH-NP prepared in Example 1 interacting with GSH; Figure 9 This is a dual-channel fluorescence imaging image of GSH in Arabidopsis root tips using the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1. Figure 10 This is a dual-channel fluorescence imaging image of GSH in living Arabidopsis thaliana using the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1. Figure 11 This is a dual-channel fluorescence imaging image of GSH in Arabidopsis thaliana of the two probe molecules (Cy-NMe-NP and Cy-NH-NP) prepared in Example 1 under salt stress. Figure 12 This is a fluorescence imaging result of co-incubating the probe Cy-NMe-NP with fungal-infected rice leaves. Detailed Implementation

[0027] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] As previously described, a first aspect of the present invention provides a fluorescent compound based on an aza-IR780 framework, the compound having the structure shown in formula (I) or formula (II); Formula (I); Equation (II).

[0029] As previously stated, a second aspect of the present invention provides a method for preparing a fluorescent compound based on an aza-IR780 framework, the fluorescent compound having the structure shown in formula (I), the method comprising: In the presence of a solvent and an alkaline substance, compound Aza-IR780 was reacted with compound NP-SO2Cl in a first contact reaction to obtain the fluorescent compound; Aza-IR780: NP-SO2Cl: ; Formula (I): .

[0030] As previously stated, a third aspect of the present invention provides another method for preparing a fluorescent compound based on an aza-IR780 framework, the fluorescent compound having the structure shown in formula (II), the method comprising: In the presence of a solvent and an alkaline substance, compound Cy-NMe-Pz and compound NP-SO2Cl undergo a second contact reaction to obtain the fluorescent compound; Cy-NMe-Pz: NP-SO2Cl: ; Formula (II): .

[0031] Preferably, in the aforementioned second and third aspects, the alkaline substance is pyridine.

[0032] Preferably, in the second and third aspects mentioned above, the solvent is selected from at least one of tetrahydrofuran and N,N-dimethylformamide.

[0033] Preferably, in the aforementioned second and third aspects, the temperature of the first contact reaction and / or the second contact reaction is 30-100°C.

[0034] According to a particularly preferred embodiment, a method for preparing a fluorescent compound based on an aza-IR780 framework with the structure shown in formula (I) includes: S1: Add THF solution and pyridine to compound Aza-IR780, and stir the reaction solution at room temperature (e.g., for 5 min). S2: Cool the reaction solution obtained in S1 (e.g., cool to 0 °C), and then slowly add a tetrahydrofuran solution of NP-SO2Cl. After the addition is complete, reflux the reaction solution overnight. S3: After the reaction is complete, the reaction system is cooled to room temperature, and the THF solution is removed by vacuum distillation. The residue is separated by column chromatography to obtain a dark green solid, which is the fluorescent compound based on the aza-IR780 framework with the structure shown in formula (I). Figure 1 (The compound shown in Cy-NH-NP).

[0035] Preferably, the amount of pyridine used is 6-7 mL relative to 1 mol of the compound Aza-IR780.

[0036] According to another particularly preferred embodiment, a method for preparing a fluorescent compound based on an aza-IR780 framework with the structure shown in formula (II) includes: SS1: Add THF solution and pyridine to compound Cy-NMe-Pz, and stir the reaction solution at room temperature (e.g., for 5 min). SS2: Cool the reaction solution obtained in SS1 (e.g., cool to 0 °C), and then slowly add a tetrahydrofuran solution of NP-SO2Cl. After the addition is complete, reflux the reaction solution overnight. SS3: After the reaction was completed, the reaction system was cooled to room temperature, and the THF solution was removed by vacuum distillation. The residue was separated by column chromatography to obtain a dark green solid, which is the fluorescent compound based on the aza-IR780 framework with the structure shown in formula (I). Figure 1 (The compound shown in Cy-NMe-NP).

[0037] Preferably, the amount of pyridine used is 6-7 mL relative to 1 mol of the compound Cy-NMe-Pz.

[0038] In the preparation of fluorescent compounds based on the aza-IR780 skeleton with the structure shown in formula (I) or (II), there are no special requirements for the amount of solvent used. Those skilled in the art can select an appropriate amount of solvent based on the known knowledge in the art.

[0039] In the process of preparing fluorescent compounds based on the azaIR780 skeleton with the structure shown in formula (I) or (II), there are no special requirements for the type of eluent used for the column chromatography separation. For example, dichloromethane / methanol with a volume ratio of 100:1 can be used as the eluent.

[0040] This invention does not impose any particular requirements on the preparation method of NP-SO2Cl; by way of example, it can be used... Figure 1 The method shown is used for preparation, including: Ethanol was added to OP-SO3K and n-butylamine under a nitrogen atmosphere, and the reaction solution was refluxed. After the reaction was completed, the reaction solution was cooled to room temperature and filtered to obtain a solid. The solid product was washed several times with anhydrous ethanol to obtain a white crude product NP-SO3K. Then, thionyl chloride was added to NP-SO3K under a nitrogen atmosphere. Subsequently, the reaction catalyst DMF was added, and the reaction solution was refluxed overnight. After the reaction was completed, the reaction solution was cooled to room temperature, and the excess thionyl chloride solution was distilled under reduced pressure to obtain a white crude product NP-SO2Cl.

[0041] This invention does not impose any particular requirements on the preparation method of the compound Cy-NMe-Pz. Exemplarily, it can be prepared by using... Figure 1 The method shown is used for preparation, including: Under nitrogen atmosphere, phosphorus oxychloride (POCl3) was added dropwise to dichloromethane and N,N-dimethylformamide (DMF). After stirring for 30 minutes, N-methylpiperidinone (R, for example, H or methyl) was added. After reflux, the mixture was washed several times with dichloromethane / diethyl ether to obtain an oily product. The oily product was refluxed with an indole salt for 3 hours, and the reaction solution was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography to obtain the compound Cy-NMe. Under nitrogen atmosphere, N,N-dimethylformamide was added to Cy-NMe and piperazine, and the reaction system was stirred overnight at 60 °C. After the reaction was completed, most of the solvent was removed by reduced pressure distillation, and the mixture was then separated by column chromatography to obtain the dark blue solid product Cy-NMe-Pz.

[0042] As previously stated, the fourth aspect of the present invention provides the application of the aza-IR780-based fluorescent compound described in the first aspect as a dual-channel glutathione fluorescent probe.

[0043] Preferably, the fluorescent probe is used for the dynamic detection of glutathione content in plant root tips and in vivo.

[0044] In a preferred embodiment, the fluorescent probe is used to monitor the dynamic fluctuations in glutathione content caused by abiotic stress and biotic stress models in plants.

[0045] As previously described, the fifth aspect of the present invention provides a glutathione fluorescent probe reagent containing a fluorescent compound based on the aza-IR780 backbone as described in the first aspect for monitoring the effective amount of glutathione content.

[0046] The fluorescent compounds based on the nitrogen-containing IR780 framework provided by this invention can be applied to imaging at the plant root tip and in vivo levels.

[0047] In this invention, plants without probe staining showed almost no background fluorescence signal; after co-incubation with the probe of this invention, plants emitted bright fluorescence signals in the green and red fluorescence channels; subsequently, after treating the plants with N-ethylmaleimide (NEM, an intracellular GSH inhibitor), and then staining with the probe for imaging, it was found that the fluorescence intensity of both the green and red fluorescence channels was significantly reduced; these results indicate that the two probes provided by this invention can detect GSH in plants with high imaging contrast, providing a powerful analytical tool for monitoring the dynamic fluctuations of GSH in plants under stress.

[0048] In this invention, fluorescent compounds based on the azaIR780 backbone can be used to image GSH content under salt stress using a dual-channel glutathione fluorescent probe. In this invention, unstained plants show almost no background fluorescence signal; plants incubated with the probe alone emit fluorescence in both the green and red fluorescence channels; after pretreatment under salt stress, the fluorescence signals of probe-stained plants are significantly enhanced in both channels, confirming the probe's ability to respond to dynamic fluctuations in GSH content under salt stress.

[0049] Therefore, the fluorescent compounds based on the azaIR780 skeleton of the present invention can be used in the application of plant stress tracing.

[0050] In summary, this invention relates to the construction and application of a class of dual-channel glutathione fluorescent probes based on the azaIR780 backbone. These probes use azaIR780 as the fluorophore and naphthalimide sulfonamide as the recognition unit, and their structural formulas are shown in formula (I) or (II). This invention provides a method for preparing these glutathione probes and their applications. These probes exhibit a glutathione-specific dual-channel activated fluorescence response signal and have been successfully applied to the visualization of glutathione concentration in plants.

[0051] The present invention will be described in detail below through embodiments. Unless otherwise specified, the raw materials used in the following embodiments are all common commercially available products.

[0052] Example 1 A novel fluorescent probe, named Cy-NMe-NP, was synthesized, and its chemical structure is shown below. Figure 1 As shown in the image.

[0053] The synthesis method of fluorescent probes specifically includes the following steps: Step 1, Synthesis of Cy-NMe-Pz: Under a nitrogen atmosphere, 10 mL of dry DMF was added to Cy-NMe (682 mg, 1.0 mmol) and piperazine (861 mg, 10.0 mmol), and the reaction mixture was stirred overnight at 60 °C. After the reaction was complete, most of the solvent was removed by vacuum distillation, and the mixture was then separated by column chromatography (eluent: dichloromethane / methanol = 150:1) to give a dark blue solid product in 65% yield. 1 H NMR (600 MHz, CDCl3): δ (ppm) = 7.76 (d, J = 12.0 Hz,1H), 7.38-7.28 (m, 2H), 7.17 (t, J = 6.0 Hz, 1H), 7.01 (d, J = 6.0 Hz, 1H), 5.75 (d, J = 12.0 Hz, 1H), 3.99-3.96 (m, 3H), 3.63 (s, 2H), 3.32 (s, 2H), 2.76(s, 2H), 2.73 (s, 2H), 1.90-1.86 (m, 2H), 1.74 (s, 6H), 1.06 (t, J = 6.0 Hz, 3H). Step 2, Synthesis of Cy-NMe-NP: Under a nitrogen atmosphere, 20 mL of dry THF solution and 2 mL of pyridine were added to the intermediate Cy-NMe-Pz (220 mg, 0.3 mmol), and the reaction mixture was stirred at room temperature for 5 minutes. Then, the reaction mixture was cooled to 0 °C, and a tetrahydrofuran solution of NP-SO2Cl (422 mg, 1.2 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was refluxed overnight. After the reaction was complete, the mixture was cooled to room temperature, and the THF solution was removed by vacuum distillation. The residue was separated by column chromatography (eluent: dichloromethane / methanol = 100:1) to give a dark gray solid in 41% yield. 1 H NMR (600 MHz, CDCl3): δ (ppm) = 9.06-8.93(m, 1.5H), 8.76-8.53 (m, 4H), 8.41 (d, J= 6.0 Hz, 1H), 7.97 (s, 1H), 7.89 (s,1H), 7.57 (t, J = 18.0 Hz, 1.5H), 7.25-7.15 (m, 3H), 7.07-7.03 (m, 1.5H), 6.89(s, 1.5H), 6.60 (d, J = 18.0 Hz, 0.5H), 5.96 (d, J = 18.0 Hz, 0.5H), 4.38 (s,2H), 4.17 (s, 3H), 4.10 (m, 4H), 3.83 (s, 4H), 3.69 (s, 2H), 3.59 (s, 4H),2.96 (s, 2H), 1.77 (m, 6H), 1.56 (s, 2H), 1.42 (m, 12H), 1.01 (t, J = 6.0 Hz, 6H), 0.98 (m, 3H). Step 3, Synthesis of Cy-NH-NP: Under a nitrogen atmosphere, 20 mL of dry THF solution and 2 mL of pyridine were added to intermediate Aza-IR780 (200 mg, 0.3 mmol), and the reaction mixture was stirred at room temperature for 5 minutes. The reaction mixture was then cooled to 0 °C, and a tetrahydrofuran solution of NP-SO2Cl (422 mg, 1.2 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was refluxed overnight. After the reaction was complete, the mixture was cooled to room temperature, and the THF solution was removed by vacuum distillation. The residue was separated by column chromatography (eluent: dichloromethane / methanol = 80:1) to give 112 mg of a dark green solid, with a yield of 38%. 1 H NMR (400 MHz, CDCl3): δ(ppm) = 9.13 (d, J = 8.0 Hz, 1H), 8.58-8.52 (m, 3H), 7.86 (t, J = 8.0 Hz, 1H), 7.79 (d, J = 16.0 Hz, 2H), 7.45-7.39 (m, 4H), 7.32 (d, J = 8.0 Hz, 2H), 7.24(d, J = 8.0 Hz, 2H), 6.19 (d, J = 16.0 Hz, 2H), 4.8 (s, 4H), 4.30 (t, J= 8.0Hz, 4H), 4.15 (t, J = 8.0 Hz, 2H), 3.26-2.89 (m, 1H), 2.0-1.95 (m, 4H), 1.84(s, 1H), 1.71-1.67 (m, 2H), 1.49-1.40 (m, 4H), 1.26 (s, 6H), 1.11 (t, J = 8.0Hz, 6H), 0.98 (t, J = 8.0 Hz, 3H), 0.88-0.83 (m, 2H). Example 2 To investigate the selectivity of two probe molecules (Cy-NMe-NP and Cy-NH-NP) for GSH.

[0054] like Figure 2 The image shows the changes in UV absorption spectra when two probe molecules were added to different types of amino acids (Hcy, Cys, Glu, Ser, Tyr, Lys, His, Arg, Met, Gly, Phe, GSH, Ala, and Gln) (all at a concentration of 100 μM). Figure 2 In the image, (A) represents the UV absorption spectra of the probe Cy-NMe-NP in different amino acid solutions and a photograph of the solution under sunlight. Figure 2 (B) in the image represents the UV absorption spectrum of the probe Cy-NH-NP in different amino acid solutions and a photograph of the solution under sunlight.

[0055] Cy-NMe-NP itself exhibits two sharp absorption peaks at 694 nm and 835 nm in HEPES buffer solution. The addition of Hcy reduces the intensity of its absorption peaks, but it still maintains the dual absorption peak characteristic. Only with the addition of GSH does the intensity of its absorption peak decrease significantly and transform into a broad peak covering the 715 nm-816 nm region. The solution color also changes from pale green to pale blue. Under the same conditions, other amino acids do not show significant changes in the intensity of the absorption peaks of the probe Cy-NMe-NP.

[0056] The probe Cy-NH-NP exhibits two sets of absorption peaks at 436 nm and 777 nm. The addition of GSH significantly increases the absorption intensity of its near-infrared peak, turning the solution color pale green. However, the addition of other amino acids decreases the near-infrared absorption peak, changing the solution color from yellow-green to yellow. These results indicate that both probe molecules exhibit good specificity to GSH.

[0057] Example 3 Based on the significant changes in ultraviolet absorption, we further investigated the fluorescence changes of the two probe molecules in the visible and near-infrared channels.

[0058] Figure 3 The dual-channel fluorescence spectra of two probe molecules (Cy-NMe-NP and Cy-NH-NP) for different amino acid molecules are shown. Figure 3 In the diagram, (A) represents the fluorescence emission spectrum of the probe Cy-NMe-NP in different amino acid solutions under visible light. Figure 3 In the diagram, (B) represents the fluorescence emission spectrum of the probe Cy-NMe-NP in different amino acid solutions under the near-infrared channel (B); Figure 3 In this diagram, (C) represents the fluorescence emission spectrum of the probe Cy-NH-NP in different amino acid solutions under visible light. Figure 3 In the diagram, (D) represents the fluorescence emission spectrum of the probe Cy-NH-NP in different amino acid solutions in the near-infrared channel (D).

[0059] Furthermore, such as Figure 3 As shown, the probe Cy-NMe-NP exhibits almost no fluorescence in HEPES solution (λ). ex =390 nm), except for Cys, which slightly enhanced the fluorescence intensity at 498 nm (50-fold) and GSH, which significantly enhanced the fluorescence emission at 498 nm (nearly 300-fold), the addition of other amino acids did not cause significant fluorescence changes.

[0060] Similarly, the probe Cy-NH-NP exhibited specific fluorescence responses to GSH in both visible and near-infrared channels. When excited at 390 nm and 740 nm, the probe produced bright fluorescence at 498 nm and 784 nm, respectively, with fluorescence intensities increased by 122-fold and 66-fold, respectively. These results indicate that both probes Cy-NMe-NP and Cy-NH-NP have specific fluorescence responses to GSH, with probe Cy-NH-NP exhibiting better selectivity.

[0061] Furthermore, anti-interference performance is an important indicator for evaluating sensors. Therefore, we further investigated its fluorescence response to GSH in the presence of other amino acids.

[0062] Figure 4 The dual-channel fluorescence spectra of two probe molecules (Cy-NMe-NP and Cy-NH-NP) against GSH are shown. Figure 4 (A) in the diagram represents the fluorescence response of the probe Cy-NMe-NP to GSH in the presence of different amino acids within the visible light channel. Figure 4(B) in the figure represents the fluorescence response of the probe Cy-NMe-NP to GSH in the presence of different amino acids in the near-infrared channel. Figure 4 In the diagram, (C) represents the fluorescence response of the probe Cy-NH-NP to GSH in the presence of different amino acids within the visible light channel. Figure 4 (D) in the figure represents the fluorescence response of the probe Cy-NH-NP to GSH in the presence of different amino acids in the near-infrared channel.

[0063] Furthermore, Figure 4 The results showed that the fluorescence intensity of both probe molecules was significantly enhanced upon the addition of GSH. Furthermore, the fluorescence intensity did not change significantly in the presence of other amino acids, further demonstrating the good anti-interference ability of the two probe molecules against GSH.

[0064] Example 4 Further investigation was conducted into the sensitivity of probes Cy-NMe-NP and Cy-NH-NP for GSH detection in both channels.

[0065] Figure 5 The image shows the UV absorption spectrum and dual-channel fluorescence emission spectrum of the probe Cy-NMe-NP titration of GSH. Figure 5 In the figure, (A) represents the titration UV absorption spectrum of the probe Cy-NMe-NP at different GSH concentrations. Figure 5 (B) in the figure represents the dual-channel fluorescence emission spectrum of probe Cy-NMe-NP titration at different GSH concentrations.

[0066] like Figure 5 As shown, with increasing GSH concentration, the near-infrared absorption of the probe Cy-NMe-NP in the 600-900 nm range gradually weakens, and the absorption peak gradually transforms from two sharp shoulder peaks to a broad absorption peak. Conversely, its fluorescence intensity in both the visible and near-infrared channels gradually increases. The calculated detection limits are 3 × 10⁻⁶. -8 M and 1.9×10 -7 M.

[0067] Figure 6 The image shows the UV absorption spectrum and dual-channel fluorescence emission spectrum of the probe Cy-NH-NP titration of GSH. Figure 6 In the figure, (A) represents the titration UV absorption spectrum of the probe Cy-NH-NP at different GSH concentrations. Figure 6 (B) in the figure represents the titration dual-channel fluorescence emission spectrum of the probe Cy-NH-NP at different GSH concentrations.

[0068] Furthermore, such as Figure 6As shown, the increasing GSH concentration led to a gradual increase in the UV absorption of the probe Cy-NH-NP solution, and correspondingly, a gradual increase in fluorescence emission. These results demonstrate the probe's sensitive response to GSH. The detection limit of the probe in the visible light channel was calculated to be 3.9 × 10⁻⁶. -8 The detection limit for M in the near-infrared channel is 1.9 × 10⁻⁶. -7 M.

[0069] The results showed that both probe molecules had excellent sensitivity to GSH.

[0070] Example 5 The response times of two probe molecules to GSH were studied, and the results are as follows: Figure 7 and Figure 8 As shown in the image.

[0071] Figure 7 In the diagram, (A) represents the time-dependent fluorescence intensity change spectrum of the probe Cy-NMe-NP in the visible light channel for the amino acids GSH, Cys, and Hcy. Figure 7 In the diagram, (B) represents the time-dependent fluorescence intensity change spectrum of the probe Cy-NMe-NP in the near-infrared channel (B) for amino acids GSH, Cys, and Hcy.

[0072] Figure 8 In the image, (A) represents the time-dependent fluorescence intensity change spectrum of the probe Cy-NH-NP in the visible light channel for the amino acids GSH, Cys, and Hcy. Figure 8 (B) in the figure represents the time-dependent fluorescence intensity change spectrum of the probe Cy-NH-NP in the near-infrared channel for amino acids GSH, Cys and Hcy.

[0073] The time response times of probes Cy-NMe-NP and Cy-NH-NP to GSH were 3.5 h and 6 h, respectively, indicating that the two probes can specifically respond to GSH and generate dual-channel enhanced fluorescence signals.

[0074] Example 6 Application of fluorescent probes in GSH imaging of Arabidopsis root tips Arabidopsis seedlings were incubated for 1.5 hours with a mixture of probe Cy-NMe-NP solution (40 μM) and CTAB (100 μM).

[0075] Figure 9 The image shows a dual-channel fluorescence imaging pattern of GSH by two probe molecules (Cy-NMe-NP and Cy-NH-NP) in Arabidopsis root tips, as shown below. Figure 9As shown, Arabidopsis root tips were uniformly stained with the probe and emitted bright fluorescence in both the green and red channels. The merged channel diagram showed that the fluorescence signals in the two channels were almost identical to the shape of the root tips in the bright field, while the blank areas had extremely low background fluorescence signals.

[0076] Similarly, the root tip imaging of Cy-NH-NP stained probe further illustrates that the probe can also achieve visualization imaging of GSH in two channels, indicating that the probe molecule has the potential to serve as a dual-channel GSH imaging probe.

[0077] Example 7 Application of fluorescent probes in GSH imaging in living Arabidopsis thaliana Arabidopsis plants were incubated with probe Cy-NMe-NP and CTAB solution for 1.5 hours. After rinsing off excess dye from the plant surface, fluorescence images of the plants were collected.

[0078] like Figure 10 As shown, the roots, stems, and leaves of Arabidopsis thaliana emit bright fluorescent signals in both the red and green channels, with shapes and outlines almost identical to those in the bright field. The leaf outlines are clearer in the red channel, demonstrating better imaging. Through signal overlay mapping, it can be observed that the green fluorescent signal areas almost completely overlap with the red fluorescent signals, resulting in a perfect yellow superposition of fluorescence.

[0079] After culturing Arabidopsis plants with probe Cy-NH-NP and CTAB solution for 2 hours, the plants emitted bright fluorescence in both channels. Simultaneously, leaf curling was clearly observed in the fluorescence imaging. These results indicate that probes Cy-NMe-NP and Cy-NH-NP can visualize GSH at the in vivo plant level in a dual-channel configuration.

[0080] Example 9 Visualization of GSH levels in plants under salt stress using fluorescent probes Three-week-old Arabidopsis thaliana plants were treated with 150 mM NaCl for 48 h to establish a salt stress model. Subsequently, the plants were co-incubated with Cy-NMe-NP and CTAB solutions for 1.5 h, rinsed, and then fluorescence imaging was performed.

[0081] The results showed that the roots, stems, and leaves of salt-stressed plants exhibited significantly enhanced fluorescence signals, with the fluorescence intensity in the visible light channel more than doubling (see details). Figure 11 ). Figure 11Image (A) shows the in vivo GSH fluorescence imaging of the probe Cy-NMe-NP in Arabidopsis thaliana under salt stress. As shown in the figure, in both the green and red channels, the blank group shows weak fluorescence, while the fluorescence intensity of the probe group (Cy-NMe-NP) and the salt-stressed probe group (Cy-NMe-NP + NaCl) gradually increases. The merged field shows similar results. Figure 11 (B) in the image represents the in vivo GSH fluorescence imaging of Arabidopsis thaliana using the Cy-NH-NP probe under salt stress. Similarly, the probe group subjected to stress (Cy-NH-NP + NaCl) showed significantly enhanced fluorescence intensity in both channels compared to the probe group (Cy-NH-NP). These results indicate that both Cy-NMe-NP and Cy-NH-NP can effectively visualize the dynamic changes of GSH in in vivo plants under salt stress.

[0082] Example 10 Visualization of GSH levels in plants under pathogen infection using fluorescent probes The probe Cy-NMe-NP was co-incubated with CTAB solution (100 μM) on fungal-infected rice leaves for 12 hours, followed by fluorescence imaging (see results). Figure 12 ).

[0083] Figure 12 In the bright field, the infected leaves turn yellow, with background fluorescence present in the green channel and extremely low background fluorescence in the red channel. After Cy-NMe-NP staining, fluorescence in the green channel significantly increased at both ends and the middle of the leaves, completely overlapping with the fungal infection site, especially the spot-like fluorescence in the middle of the leaves, which highly matched the infection morphology; the corresponding area in the near-infrared channel also showed significant enhancement, confirming elevated GSH levels in this area. In the enlarged view, it can be clearly observed that the merged signal overlay shows a high degree of overlap of fluorescent areas, indicating that fungal infection causes changes in redox levels.

[0084] The results showed that Cy-NMe-NP could sensitively respond to changes in GSH caused by pathogen infection.

[0085] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A fluorescent compound based on an aza-IR780 framework, characterized in that, The compound has the structure shown in formula (I) or formula (II); Formula (I); Equation (II).

2. A method for preparing fluorescent compounds based on the aza-IR780 framework, characterized in that, The fluorescent compound has the structure shown in formula (I), and the method includes: In the presence of a solvent and an alkaline substance, compound Aza-IR780 was reacted with compound NP-SO2Cl in a first contact reaction to obtain the fluorescent compound; Aza-IR780: ;NP-SO2Cl: ; Equation (I): .

3. A method for preparing fluorescent compounds based on the aza-IR780 framework, characterized in that, The fluorescent compound has the structure shown in formula (II), and the method includes: In the presence of a solvent and an alkaline substance, compound Cy-NMe-Pz and compound NP-SO2Cl undergo a second contact reaction to obtain the fluorescent compound; Cy-NMe-Pz: ;NP-SO2Cl: ; Formula (II): .

4. The method according to claim 2 or 3, characterized in that, The alkaline substance is pyridine.

5. The method according to claim 2 or 3, characterized in that, The solvent is selected from at least one of tetrahydrofuran and N,N-dimethylformamide.

6. The method according to claim 2 or 3, characterized in that, The temperature of the first contact reaction and / or the second contact reaction is 30-100℃.

7. The application of the fluorescent compound based on the aza-IR780 backbone as described in claim 1 as a dual-channel glutathione fluorescent probe.

8. The application according to claim 7, characterized in that, The fluorescent probe is used for the dynamic detection of glutathione content in plant root tips and in vivo.

9. The application according to claim 7, characterized in that, The fluorescent probe is used to monitor the dynamic fluctuations in glutathione content caused by abiotic and biotic stress models in plants.

10. A glutathione fluorescent probe reagent, characterized in that, The fluorescent probe reagent contains an effective amount of the fluorescent compound based on the aza-IR780 backbone as described in claim 1 for monitoring glutathione content.

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