A signal amplification type glutathione s-transferase fluorescent probe and a preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and relates to a signal amplification type glutathione S-transferase fluorescent probe and a preparation method and application thereof. The compound has a structure shown in formula (I). The glutathione S-transferase fluorescent probe based on a cyclohexenone recognition unit provided by the application shows sensitive detection on glutathione S-transferase, can realize self-amplification signal triggered by mercapto protein in a low-concentration glutathione environment, and can be successfully applied to visual imaging of glutathione S-transferase in various insects and weeds. Formula (I).

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a signal amplification glutathione S-transferase fluorescent probe, its preparation method, and its application. Background Technology

[0002] In modern biological detoxification and metabolic systems, glutathione S-transferases (GSTs) are a key phase II metabolic enzyme, widely involved in the biotransformation of exogenous compounds. GSTs accelerate the water solubility and excretion of toxic substances by catalyzing conjugated reactions between glutathione (GSH) and various endogenous or exogenous electrophilic substrates, playing a crucial detoxification and protective role in the body's resistance to chemical stresses such as pesticides and environmental pollutants. Numerous studies have shown that the GST superfamily is not only widely distributed in mammals and higher plants, but also highly conserved in agricultural pests and field weeds. Changes in their catalytic activity and expression levels are closely related to the formation of resistance to pesticides, herbicides, and other agents in individual organisms.

[0003] In the formation of pesticide resistance in agricultural insect pests and weeds, the GST-mediated metabolic detoxification pathway has been recognized as another core resistance mechanism besides target site mutations. GST can conjugate GSH in various insecticides, herbicides, or their intermediate products after primary metabolism by enzymes such as P450 and carboxylesterases, thereby significantly reducing their toxic burden in vivo. This mechanism was first systematically revealed in the study of triazine herbicide resistance and has since been widely validated in various pesticides and biological populations. Especially in small agricultural pests and weeds, the increased activity level of GST, as an important detoxification enzyme, is often closely related to cross-resistance to multiple pesticides, thus having important indicative significance in resistance monitoring and mechanism analysis. Taking the diamondback moth, a global key pest of cruciferous crops, and *Hemiberlesia lataniae*, a major annual grass weed in the Yangtze River Basin and areas south of it, as typical examples, both types of insect pests and weeds have developed significant resistance to multiple chemical control agents, in which the GST superfamily-mediated metabolic detoxification plays a core role in their resistance evolution.

[0004] Traditionally, research and monitoring of GST activity widely employs GST fluorescent probes that rely on GSH as a reaction substrate. These probes typically react specifically with GSH under GST catalysis, resulting in the generation or enhancement of fluorescence signals, thus enabling visual detection of GST activity. However, in agricultural weeds and insects, which are significantly smaller than large animals, the background content of GSH is often extremely low. This makes it difficult for traditional GST fluorescent probes to obtain fluorescence signals with sufficient intensity and contrast, severely limiting their application in small-sized insect pests and failing to meet the requirements for highly sensitive real-time imaging and quantitative analysis of GST-mediated resistance metabolism.

[0005] Therefore, addressing the technical bottlenecks of low GSH content in agricultural insect pests and the limited signal of traditional probes, it is necessary to develop a GST fluorescent probe system capable of signal self-amplification under ultra-low GSH conditions. By introducing triggerable cascade reactions or amplification processes into the molecular design, the probe can generate continuously enhanced fluorescence output after initial catalysis involving a small amount of GSH, potentially significantly improving the response sensitivity and imaging contrast to GST activity. Constructing such novel GST fluorescent probes with self-amplification characteristics will not only enable highly sensitive detection and detailed visualization of GST activity in small agricultural insect pests such as diamondback moth and sedge, but will also provide a new analytical tool for further elucidating their GST-mediated resistance metabolic mechanisms, thus providing crucial technical support for improving resistance theory and formulating scientific and efficient integrated control strategies. Summary of the Invention

[0006] The purpose of this invention is to provide a class of self-expanding glutathione S-transferase fluorescent probes based on cyclohexenone.

[0007] To achieve the above objectives, a first aspect of the present invention provides a signal-amplifying glutathione S-transferase fluorescent probe having the structure shown in formula (I); Formula (I) Wherein, R is selected from one of the following groups: .

[0008] A second aspect of the present invention provides a method for preparing the signal-amplified glutathione S-transferase fluorescent probe, the method comprising: dissolving a fluorescent compound RH containing an amino or piperazine unit in an organic solvent with a tri-phosphorus gas, and then adding an alkaline substance and compound 2 to react and obtain the fluorescent probe; Compound 2.

[0009] A third aspect of the present invention provides the application of the above-mentioned signal-amplified glutathione S-transferase fluorescent probe, wherein the fluorescent probe is used for the dynamic detection of glutathione S-transferase content at the in vivo level in insects and plants.

[0010] A fourth aspect of the present invention provides another application of the above-mentioned signal amplification glutathione S-transferase fluorescent probe, wherein the fluorescent probe is used to monitor dynamic fluctuations in glutathione S-transferase content caused by an insect resistance model.

[0011] Through the above technical solution, the present invention has at least the following beneficial effects: (1) The self-expanding glutathione S-transferase fluorescent probe based on cyclohexenone provided by the present invention has good spectral response performance.

[0012] (2) The fluorescent probe provided by the present invention can achieve sensitive detection of glutathione S-transferase in low glutathione concentration environments (such as below 1 μM).

[0013] (3) The fluorescent probe provided by the present invention exhibits excellent selectivity and sensitivity when detecting glutathione S-transferase.

[0014] (4) The fluorescent compound Fu-Ca-Cy provided by the present invention for detecting glutathione S-transferase in low glutathione concentration environments can realize the visual monitoring of glutathione S-transferase in diamondback moth and sedge.

[0015] (5) The fluorescent compound Fu-Ca-Cy provided by the present invention for detecting glutathione S-transferase in a low glutathione concentration environment has the advantages of low background interference and strong tissue penetration ability.

[0016] (6) The fluorescent compound Fu-Ca-Cy provided by the present invention for detecting glutathione S-transferase in low glutathione concentration environments has broad application prospects in monitoring changes in glutathione S-transferase in living pests, diseases and weeds and in the study of the enzyme’s involvement in biological resistance.

[0017] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0018] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0019] Figure 1 This is the synthetic route diagram for the probe R-Ca-Cy.

[0020] Figure 2This is a synthetic route diagram of the GST fluorescent probe Fu-Ca-Cy prepared in Example 2.

[0021] Figure 3 The image shows the UV absorption and fluorescence spectra of the fluorescent probe BDP-Ca-Cy prepared in Example 1 in response to glutathione S-transferase under a high concentration of glutathione environment.

[0022] Figure 4 The image shows the UV absorption and fluorescence spectra of the fluorescent probe Np-Ca-Cy prepared in Example 1 in response to glutathione S-transferase under a high concentration of glutathione environment.

[0023] Figure 5 The image shows the UV absorption and fluorescence spectra of the fluorescent probe Rh-Ca-Cy prepared in Example 1 in response to glutathione S-transferase under a high concentration of glutathione environment.

[0024] Figure 6 The image shows the UV absorption and fluorescence spectra of the fluorescent probe Fu-Ca-Cy prepared in Example 2 in response to glutathione S-transferase under a high concentration of glutathione environment.

[0025] Figure 7 The UV absorption and fluorescence spectra of the comparative probe DNS-Ca-Cy prepared in Comparative Example 1 in response to glutathione S-transferase under high concentration of glutathione are shown.

[0026] Figure 8 The UV absorption and fluorescence spectra of the comparative probe Cy-Ca-Cy prepared in Comparative Example 1 in response to glutathione S-transferase under high concentration of glutathione are shown.

[0027] Figure 9 The fluorescence spectrum of the fluorescent probe Fu-Ca-Cy prepared in Example 2 in response to glutathione S-transferase under low concentration of glutathione is shown.

[0028] Figure 10 The image shows the selective fluorescence spectrum of the fluorescent probe Fu-Ca-Cy prepared in Example 2 for GST.

[0029] Figure 11 The image shows the fluorescence titration spectrum of GSH using the fluorescent probe Fu-Ca-Cy prepared in Example 2.

[0030] Figure 12 The images show the fluorescence titration diagram and fitting curve of GST using the fluorescent probe Fu-Ca-Cy prepared in Example 2.

[0031] Figure 13 The diagram shows the kinetics of the fluorescent probe Fu-Ca-Cy prepared in Example 2 on GST.

[0032] Figure 14 The fluorescence spectrum of the fluorescent probe Fu-Ca-Cy prepared in Example 2 under the action of thiol protein is shown.

[0033] Figure 15 This is a fluorescence image of GST by the fluorescent probe Fu-Ca-Cy prepared in Example 2 in live diamondback moth.

[0034] Figure 16 This is a fluorescence imaging image of GST in live *Gnaphalium affine* prepared in Example 2 using the fluorescent probe Fu-Ca-Cy. Detailed Implementation

[0035] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0036] This invention provides a signal-amplifying fluorescent probe for glutathione S-transferase, the probe having the structure shown in formula (I); Formula (I) R is selected from one of the following groups (the wavy line indicates the bonding position): .

[0037] This invention provides a class of self-extruding large glutathione S-transferase fluorescent probes through rational molecular design. These fluorescent probes exhibit fluorophore characteristics of cyclohexenone recognition units and conjugated furan rings.

[0038] According to a preferred embodiment of the present invention, the probe has the structure shown in formula (II). Equation (II).

[0039] The present invention also provides a method for preparing the signal-amplifying glutathione S-transferase fluorescent probe, the method comprising: dissolving a fluorescent compound RH containing an amino or piperazine unit with tri-light gas in an organic solvent, and then adding an alkaline substance and compound 2 to react to obtain the corresponding target compound.

[0040]

[0041] RH represents a compound in which the R group is bonded to a hydrogen atom.

[0042] For the fluorescent probe shown in formula (II), the method includes: (1) Under the protection of an inert gas, compound 1, p-aminobenzaldehyde and a first organic solvent were subjected to a first contact reaction to obtain the fluorescent compound Fu-NH2; (2) Compound Fu-NH2 and triphosphorus gas were dissolved in a second organic solvent, and then an alkaline substance and compound 2 were added to carry out a second contact reaction to obtain the fluorescent probe Fu-Ca-Cy; .

[0043] According to a preferred embodiment of the present invention, the alkaline substance is selected from at least one of pyridine, triethylamine, 4-dimethylaminopyridine, and sodium ethoxide.

[0044] According to a preferred embodiment of the present invention, the first organic solvent and the second organic solvent are each independently selected from at least one of tetrahydrofuran, N,N-dimethylformamide, dichloromethane and anhydrous ethanol.

[0045] In the preparation of the fluorescent compound Fu-Ca-Cy, 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 known knowledge in the field.

[0046] This invention does not impose any particular requirements on the preparation method of compound Fu-NH2. According to one specific embodiment, the preparation method of compound Fu-NH2 includes the following steps: under a nitrogen atmosphere, ethanol is added to compound 1 and p-aminobenzaldehyde, and the reaction solution is refluxed. After the reaction is completed, the mixture is cooled to room temperature, extracted with dichloromethane, and the organic layer is dried under reduced pressure with sodium sulfate to obtain a crude product, which is a black powder compound Fu-NH2.

[0047] According to a preferred embodiment of the present invention, the molar ratio of compound 1 to p-aminobenzaldehyde in step (1) is 2:(1.5-1.8). According to a preferred embodiment of the present invention, the temperature of the first contact reaction is 30-100°C.

[0048] This invention does not specify a particular method for preparing compound Fu-Ca-Cy. According to one specific embodiment, the preparation method of compound Fu-Ca-Cy includes the following steps: Under a nitrogen atmosphere, compound Fu-NH2 and triphosgene are added to dichloromethane, and triethylamine is added dropwise. After stirring for 5 h, pyridine and compound 2 are added. After reacting at room temperature, the mixture is extracted with dichloromethane. The organic layer is dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain a crude product. This crude product is then separated by column chromatography to obtain a brown solid compound Fu-Ca-Cy.

[0049] According to a preferred embodiment of the present invention, in step (2), the molar ratio of compound Fu-NH2 to triphosgene is 1:(1-2.5); the molar ratio of the alkaline substance to compound Fu-NH2 is 1:(5-10); and the molar ratio of compound 2 to compound Fu-NH2 is 1:(1.2-2). According to a preferred embodiment of the present invention, the temperature of the second contact reaction is 20-40°C.

[0050] The fluorescent probe of this invention can be applied to the dynamic detection of glutathione S-transferase content at the in vivo level in insects and plants. This includes, but is not limited to, its application in visualization imaging at the in vivo level in insects and weeds.

[0051] The fluorescent probe of the present invention can also be used to monitor the dynamic fluctuations in glutathione S-transferase content caused by insect resistance models.

[0052] The probe of this invention uses cyclohexenone as a recognition group and is modified with various fluorophores. This type of probe can exhibit a fluorescence response signal specific to glutathione S-transferase and has been successfully applied to the visualization imaging of glutathione S-transferase in insects and plants. In the imaging experiments of diamondback moth and sedge, the blank control group without probe treatment showed only extremely low background fluorescence; when incubated with the probe alone, both diamondback moth and sedge root tips produced strong red fluorescence signals. The superimposed channels showed that the fluorescence signal highly matched the outline of the biological tissue, and the imaging contrast was excellent. After further introducing the GSH-specific inhibitor N-ethylmaleimide (NEM) and the GST active site inhibitor ethacrylic acid (EA) for control experiments, the fluorescence intensity in both organisms was significantly reduced. The above results confirm that the enhanced fluorescence signal of the probe Fu-Ca-Cy in agricultural pests and weeds mainly depends on the catalytic activity of GST. It can not only achieve specific imaging of GST, but also provide a powerful in vivo detection tool for the study of GST-related physiological mechanisms in agricultural organisms.

[0053] 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.

[0054] Example 1

[0055] This embodiment illustrates the synthesis of a novel fluorescent probe, commonly named R-Ca-Cy, and its synthetic route is as follows: Figure 1 As shown.

[0056] Example 1A

[0057] Step 1, Synthesis of intermediate BDP-NH2: Under a nitrogen atmosphere, 2,4-dimethylpyrrole (630 mg, 6.6 mmol) and p-nitrobenzaldehyde (453 mg, 3.0 mmol) were dissolved in dry tetrahydrofuran (90 mL), and 10 drops of trifluoroacetic acid were added and stirred at room temperature in the dark for 24 h. Then, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (680 mg, 3.0 mmol) dissolved in dry tetrahydrofuran (100 mL) was added dropwise, and the mixture was stirred for 4–5 h. Triethylamine (18 mL) and boron trifluoride diethyl ether (18 mL) were added in an ice bath, and the mixture was stirred for another 12 h before filtration. The crude product was purified by dichloromethane column chromatography to give an orange-red solid compound. This orange-red intermediate (100 mg, 0.27 mmol) was dissolved in dry ethanol (10 mL), and 10% palladium on carbon (100 mg) and hydrazine hydrate (0.194 mL) were added. The mixture was refluxed for 30 min and then filtered. The filtrate was concentrated and separated by column chromatography to obtain the target product as an orange solid, with a yield of 63%.

[0058] Step 2: Synthesis of probe BDP-Ca-Cy: Under nitrogen protection, compound BDP-NH2 (0.66 mmol) and triphosgene (293 mg, 0.99 mmol) were dissolved in 15 mL of anhydrous dichloromethane. Triethylamine was added dropwise at 0 °C, and the reaction was carried out at room temperature for 3 h. Then, pyridine (20 drops) and compound 2 (125 mg, 0.99 mmol) were added and reacted for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. The crude product was then separated by column chromatography to obtain a red solid with a yield of 51%. 1 H NMR (400 MHz, CDCl3) δ = 7.46 (d, J = 12 Hz, 2H), 7.12 (d, J = 8 Hz,2H), 7.07 (s, 1H), 6.95 (s, 1H), 5.90 (s, 2H), 4.78 (s, 2H), 2.47 (s, 6H),2.44-2.38 (m, 4H), 1.99-1.95 (m, 2H), 1.18 (s, 6H). 13C NMR (100 MHz, CDCl3) δ200.8, 198.5, 155.4, 153.2, 149.9, 147.1, 143.2, 141.5, 139.0, 138.2, 134.6,131.7, 129.5, 128.7, 121.2, 118.7, 62.2, 61.9, 45.8, 44.5, 38.3, 38.2, 25.9,25.7, 22.8, 22.7, 14.7. Example 1B Step 1, Synthesis of intermediate Rh-NH2: m-Aminophenol (2.18 g, 20.00 mmol) and m-chlorophenol (2.56 g, 20.00 mmol) were added to a 100 mL two-necked reaction flask. Under anhydrous and anaerobic conditions, 20 mL of benzenesulfonic acid and phthalic anhydride (2.96 g, 20.00 mmol) were added sequentially, and the mixture was stirred until the reactants were completely dissolved. The reaction flask was refluxed at 140 °C for 40 h, and the reaction system turned milky yellow. After the reaction was completed, the reaction solution was neutralized with sodium hydroxide solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was separated by column chromatography to obtain a pale yellow solid with a yield of approximately 25%.

[0059] Step 2, Synthesis of the probe Rh-Ca-Cy: Under nitrogen protection, compound Rh-NH2 (0.66 mmol) and triphosgene (293 mg, 0.99 mmol) were dissolved in 15 mL of anhydrous dichloromethane. Triethylamine was added dropwise at 0 °C, and the reaction was carried out at room temperature for 3 h. Then, pyridine (20 drops) and compound 2 (125 mg, 0.99 mmol) were added and reacted for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. The crude product was then separated by column chromatography to obtain a pale yellow solid with a yield of 20%. 1 H NMR (600 MHz, CDCl3) δ 8.02 (d, J = 6 Hz, 1H), 7.67-7.61 (m, 2H), 7.55(s, 1H), 7.30 (s, 1H), 7.12 (d, J = 6.0 Hz, 2H), 7.00 (d, J = 12 Hz, 1H), 6.90 (d, J= 12 Hz, 1H), 6.86 (s, 1H), 6.74-6.70 (m, 2H), 4.83 (s, 2H), 2.48-2.43 (m, 4H), 2.04-2.02 (m, 2H). 13 C NMR (150 MHz, CDCl3) δ 198.2, 169.1,152.8, 151.4, 151.3, 149.8, 140.1, 135.9, 135.1, 134.2, 129.8, 128.9, 128.4,125.9, 125.1, 124.1, 123.6, 117.4, 117.2, 114.3, 113.0, 106.0, 81.7, 62.2,37.8, 29.5, 25.7, 22.4. Example 1C Step 1, Synthesis of intermediate NP-NH2: Under nitrogen protection, 10 mL of N,N-dimethylformamide was added to 4-bromo-N-(2-morpholinoethyl)-1,8-naphthalimide (200 mg, 0.52 mmol) and sodium azide (100 mg, 1.54 mmol), and the reaction was carried out at 100 °C for 4 h. The reaction solution was poured into an ice-water mixture, extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellowish-brown crude product, which could be used directly in the next step without purification. 10 mL of N,N-dimethylformamide was added to this crude product (100 mg, 0.30 mmol), and after stirring to dissolve, 1 mL of an aqueous solution of sodium sulfide nonahydrate (342 mg, 1.50 mmol) was added dropwise, and the reaction was continued for 4 h. The reaction solution was extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a bright yellow crude product with a yield of approximately 70%, which could be used directly in subsequent reactions without purification.

[0060] Step 2, Synthesis of the probe NP-Ca-Cy: Under nitrogen protection, compound NP-NH2 (0.66 mmol) and triphosgene (293 mg, 0.99 mmol) were dissolved in 15 mL of anhydrous dichloromethane. Triethylamine was added dropwise at 0 °C, and the reaction was carried out at room temperature for 3 h. Then, pyridine (20 drops) and compound 2 (125 mg, 0.99 mmol) were added and reacted for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. The crude product was then separated by column chromatography to obtain a yellow solid with a yield of 35%. 1 H NMR (600 MHz, CDCl3) δ 8.60 (d,J = 6.0 Hz, 1H), 8.56 (d, J = 6.0 Hz, 1H), 8.36 (d, J = 6.0 Hz, 1H), 8.19 (d, J = 6.0 Hz, 1H), 7.75 (t, J = 6.0 Hz,1H), 7.63 (s, 1H), 7.20 (s, 1H), 4.92 (s, 2H), 4.33 (t, J = 6.0 Hz, 2H), 3.69(s, 4H), 2.72 (t, J = 6.0 Hz, 2H), 2.62 (s, 4H), 2.54-2.49 (m, 2H), 2.49-2.45(m, 2H), 2.06 (m, J = 6.0 Hz, 2H); 13 C NMR (150 MHz, CDCl3) δ 198.6, 164.1,163.6, 152.7, 148.3, 138.9, 135.3, 132.4, 131.3, 129.0, 126.7, 126.4, 123.3,118.0, 117.4, 90.3, 67.1, 56.2, 53.8, 38.2, 37.2, 29.7, 25.8, 22.8. Example 2 The merging path of the probe Fu-Ca-Cy is as follows Figure 2 As shown, the specific steps include: Step 1, Synthesis of intermediate Fu-NH2: Compound 1 (386 mg, 2 mmol) and p-aminobenzaldehyde (1.32 g, 1.65 mmol) were dissolved in 5 mL of anhydrous ethanol under nitrogen protection and reacted at 78 °C for 12 h. After the reaction was completed, the mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. Subsequently, the crude product was separated by column chromatography to obtain the black powder compound Fu-NH2, with a yield of 50%.

[0061] Step 2, Synthesis of Fu-Ca-Cy: Under nitrogen protection, compound Fu-NH2 (200 mg, 0.66 mmol) and triphosgene (293 mg, 0.99 mmol) were dissolved in 15 mL of anhydrous dichloromethane. 1 mL of triethylamine was added dropwise at 0 °C, and the reaction was carried out at room temperature for 5 h. Then, pyridine (20 drops) was added and reacted with compound 2 (125 mg, 0.99 mmol) for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. Subsequently, the crude product was separated by column chromatography to obtain the brown solid compound Fu-Ca-Cy in 35% yield. 1 H NMR (600 MHz, CDCl3) δ = 7.61-7.58 (m, 3H), 7.52(m, 2H), 7.14 (s, 1H), 6.97-6.92 (m, 2H), 4.85 (s, 2H), 2.52-2.48 (m, 2H), 2.46 (s, 2H), 2.05 (m, 2H), 1.79 (s, 6H). 13 C NMR (150 MHz, CDCl3) δ 172.8,151.5, 149.4, 145.7, 141.3, 129.6, 127.6, 117.6, 112.2, 110.7, 109.9, 109.3,96.5, 61.8, 37.1, 28.7, 25.5, 24.9, 21.6. Comparative Example 1 This comparative example is used to illustrate the synthesis of the control probe.

[0062] Comparative Example 1A

[0063] Step 1, Synthesis of intermediate DNS-NH2: Anhydrous piperazine (516.00 mg, 6.00 mmol) was added to a 100 mL two-necked flask and completely dissolved in 35.00 mL of anhydrous dichloromethane. Then, dansyl chloride (540.00 mg, 3.00 mmol) was added in portions to the reaction mixture, and the mixture was stirred at 25 °C for 4 h. The reaction system gradually changed from white to turbid. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude product; this was then purified by column chromatography to finally obtain a pale yellow powder product of 479.00 mg, with a yield of approximately 50%.

[0064] Step 2, compare the synthesis of the probe DNS-Ca-Cy: Under nitrogen protection, compound DNS-NH2 (0.66 mmol) and triphosgene (293 mg, 0.99 mmol) were dissolved in 15 mL of anhydrous dichloromethane. Triethylamine was added dropwise at 0 °C, and the reaction was carried out at room temperature for 3 h. Then, pyridine (20 drops) and compound 2 (125 mg, 0.99 mmol) were added and reacted for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. The crude product was then separated by column chromatography to obtain a pale yellow solid with a yield of 40%. 1 H NMR (400 MHz, CDCl3) δ = 8.56 (d, J = 8 Hz, 1H), 8.46 (d, J = 12 Hz, 1H), 8.18 (d, J = 8 Hz, 1H), 7.53 (t, J = 8 Hz, 2H), 7.18 (d, J = 8 Hz, 1H), 6.84 (s, 1H), 3.19 (s, 4H), 3.08 (s, 2H), 2.89 (s, 6H), 2.46 (s, 4H), 2.37(m, 4H), 1.94 (m, 2H). 13 C NMR (100 MHz, CDCl3) δ 199.1, 151.7, 148.6, 134.9,132.5, 130.7, 130.6, 130.1, 128.0, 123.2, 119.8, 115.2, 55.7, 52.4, 45.7,45.5, 38.4, 26.0, 22.9. Comparative Example 1B Step 1, Synthesis of intermediate Cy-NH2: Under nitrogen protection, indocyanine green IR-780 (667 mg, 1.0 mmol) and piperazine (345 mg, 4.0 mmol) were dissolved in 10 mL of N,N-dimethylformamide. The reaction mixture was then stirred overnight at room temperature. After the reaction was complete, the N,N-dimethylformamide solvent was removed under reduced pressure. The residue was concentrated and recrystallized from the mixture of dichloromethane and anhydrous diethyl ether to give a blue solid product in 76% yield.

[0065] Step 2, compare the synthesis of the probe Cy-Ca-Cy: Under nitrogen protection, compound Cy-NH2 (0.66 mmol) and triphosgene (293 mg, 0.99 mmol) were dissolved in 15 mL of anhydrous dichloromethane. Triethylamine was added dropwise at 0 °C, and the reaction was carried out at room temperature for 3 h. Then, pyridine (20 drops) and compound 2 (125 mg, 0.99 mmol) were added and reacted for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and then evaporated under reduced pressure to obtain the crude product. The crude product was then separated by column chromatography to obtain a green solid with a yield of 60%. 1 H NMR (400 MHz, CDCl3) δ = 7.86-7.73 (m, 2H), 7.45 (s, 6H), 7.18-7.13 (m,2H), 6.04-5.92 (m, 2H), 4.08-3.69 (m, 12H), 3.41 (s, 2H), 2.61 (s, 8H), 2.17(s, 4H), 1.79 (s, 12H), 1.69 (s, 2H), 1.61 (s, 2H), 1.16 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 199.3, 198.3, 171.1, 169.9, 155.1, 149.4, 142.7, 142.6,141.7, 141.6, 141.4, 140.3, 134.4, 128.7, 128.5, 125.9, 124.2, 123.8, 122.1,110.1, 109.8, 109.7, 97.8, 96.4, 85.8, 63.0, 54.9, 54.1, 48.4, 45.6, 45.4,43.6, 38.3, 29.2, 28.9, 26.3, 26.0, 25.3, 25.0, 22.8, 22.7, 21.8, 21.6, 20.6,20.4, 18.8, 17.5, 11.8. Test Example 1 This test case is used to investigate the responsiveness of the probe R-Ca-Cy to GST.

[0066] In the presence of normal concentration GSH (10 μM), the responses of six different GST probes (DNS-Ca-Cy, BDP-Ca-Cy, Cy-Ca-Cy, NP-Ca-Cy, Rh-Ca-Cy, and Fu-Ca-Cy, all 10 μM) to GST (10 μg mL) were compared. - The UV-Vis absorption and fluorescence emission spectra of ¹) were systematically characterized.

[0067] Figure 3 The results are for the detection of the probe BDP-Ca-Cy. Ultraviolet-Vis absorption spectrum (UV-Vis) Figure 3 A) shows that, compared with the single probe BDP-Ca-Cy (black curve) and the condition with only GSH added (red curve), the absorption intensity (blue curve) is significantly enhanced at approximately 520 nm when both GSH and GST are added; the fluorescence emission spectrum ( Figure 3 As shown in B), the fluorescence intensity of the probe with both GSH and GST added (blue curve) decreased by almost half compared to the probe alone (black curve) and the probe with GSH added (red curve).

[0068] Figure 4 The results are for the detection of the probe NP-Ca-Cy. Ultraviolet-Vis absorption spectrum (UV-Vis) Figure 4 A) shows that, compared with the single probe NP-Ca-Cy (black curve), the addition of GSH alone (red curve) and the simultaneous addition of GSH and GST (blue curve) resulted in a subtle new peak at approximately 420-480 nm; fluorescence emission spectra ( Figure 4 B) shows that the probe with both GSH and GST added (blue curve) has about twice the fluorescence intensity compared to the probe alone (black curve) and the probe with only GSH added (red curve).

[0069] Figure 5 The results are for the detection of the probe Rh-Ca-Cy. Ultraviolet-visible fluorescence spectrum (UV-Vis) Figure 5 As shown in Figure A), compared with the probe alone (black curve) and the condition with only GSH added (red curve), the fluorescence intensity (blue curve) of GSH and GST added simultaneously was significantly enhanced at approximately 450 nm; the fluorescence emission spectrum ( Figure 5 B) shows that the probe with both GSH and GST added (blue curve) has significantly enhanced fluorescence intensity in the visible light range of 490-600 nm compared to the probe alone (black curve) and the probe with GSH added (red curve).

[0070] Figure 6 The results are for the detection of the probe Fu-Ca-Cy. Ultraviolet-Vis absorption spectrum (UV-Vis) Figure 6A) shows that the probe Fu-Ca-Cy alone (black curve) exhibits only weak background absorption in the wavelength range of 300 nm to 700 nm; with the addition of GSH alone (red curve), the absorption intensity of the probe slightly increases in the range of 400 nm to 600 nm, with the maximum absorption peak located at around 460 nm; when GSH and GST are added to the system simultaneously (blue curve), the probe shows a significantly enhanced characteristic absorption peak in the range of 450 nm to 600 nm, with a maximum absorption wavelength of approximately 500 nm, demonstrating that GST can significantly alter the ultraviolet absorption characteristics of the probe in the presence of GSH. Fluorescence emission spectrum ( Figure 6 Figure B) shows that the probe Fu-Ca-Cy alone (black curve) and the system with only GSH added (red curve) did not produce detectable fluorescence signals in the wavelength range of 550 nm to 750 nm. When both GSH and GST were added to the system (blue curve), the probe produced strong fluorescence emission in the 600 nm to 700 nm range, with a maximum emission wavelength at 610 nm. Compared with the group with only GSH added, the fluorescence enhancement was nearly 10 times. Furthermore, this probe has a longer fluorescence emission wavelength, exhibiting superior imaging performance in in vivo plant imaging. These results confirm that the probe Fu-Ca-Cy has a good response to GST.

[0071] Figure 7 To compare the detection results of the probe DNS-Ca-Cy, the ultraviolet-visible absorption spectrum was used. Figure 7 A) shows that, compared with the single probe DNS-Ca-Cy (black curve) and the condition with only GSH added (red curve), the absorption intensity of the probe is slightly enhanced when both GSH and GST are added (blue curve); fluorescence emission spectrum ( Figure 7 B) shows that the fluorescence intensity of the probe with added GSH (red curve) and the probe with both GSH and GST added (blue curve) is almost unchanged compared to the probe alone (black curve), indicating that the probe DNS-Ca-Cy has almost no response to GST.

[0072] Figure 8 The results are for the detection of the probe Cy-Ca-Cy. Ultraviolet-Vis absorption spectrum (UV-Vis) Figure 8 A) shows that, compared with the single probe Cy-Ca-Cy (black curve), the absorption intensity is significantly enhanced at approximately 600-800 nm when GSH is added alone (red curve) and when both GSH and GST are added simultaneously (blue curve); fluorescence emission spectra ( Figure 8 B) shows that the fluorescence intensity of the probe with added GSH (red curve) and the probe with both GSH and GST added (blue curve) is almost unchanged compared to the probe alone (black curve).

[0073] Test Example 2

[0074] Given the good response of the probe Fu-Ca-Cy to GST, this test case further investigates the response of the probe to GST at extremely low GSH concentrations.

[0075] Figure 9 The system showed that the single probe Fu-Ca-Cy (1 μM) and the system with only a low concentration of GSH (1 μM) exhibited only extremely low background fluorescence signals in the wavelength range of 550 nm to 750 nm; when low concentrations of GSH and GST (10 μg / mL) were added to the system simultaneously... -1 When GSH was added, the probe emitted strong fluorescence, with a maximum emission wavelength of approximately 610 nm. The fluorescence enhancement factor was more than 10 times that of the group with only GSH added. These results confirm that even in the presence of low concentrations of GSH, the probe Fu-Ca-Cy can still produce a significant fluorescence "on" response to GST, further demonstrating the high sensitivity of the probe to GST.

[0076] Test Example 3

[0077] This test case illustrates the selectivity of the probe Fu-Ca-Cy for GST detection.

[0078] Figure 10 This study presents the fluorescence intensity selectivity test results of the probe Fu-Ca-Cy in the presence of various potential interfering substances. The tested substances included common salts (KBr, NaCl, MgSO4, Cu(NO3)2, NaHS), amino acids (Cys, Hcy, Arg, Tyr, GSH), enzymes and proteins (AchE, BchE, BSA, GGT, CE2, NQO1, NTR, HPPD), and a blank control group (Blank). The results showed that under the same experimental conditions, most tested substances only elicited extremely low fluorescence signals from the probe; only when GSH and GST were co-present (GSH+GST group) did the probe's fluorescence intensity significantly increase, exceeding that of all other tested groups. These results indicate that the probe Fu-Ca-Cy exhibits high selectivity for GST.

[0079] Test Example 4

[0080] This test case further investigates the sensitivity of the probe Fu-Ca-Cy for GST detection.

[0081] like Figure 11As shown, with the increase of GSH concentration, in the concentration range of 0-5 μM, the fluorescence intensity of the probe increases in a concentration-dependent manner with the increase of GSH concentration, and the maximum emission wavelength stabilizes at about 610 nm, which proves that the probe can be adapted to different physiological concentrations of GSH environment, providing a reliable concentration adaptation basis for subsequent GST response. Figure 12 Further titration experiments with GST concentrations showed that, in the range of 0-10 μg / mL... - Within the specified concentration range, the fluorescence intensity of the probe increased significantly in a concentration-dependent manner with increasing GST concentration, and the fluorescence intensity showed a good linear positive correlation with GST concentration (R²≈0.9948). The detection limit was determined to be 0.033 μg / mL. - ¹.

[0082] The above results indicate that the probe molecule has excellent sensitivity to GST.

[0083] Test Example 5

[0084] This test case is used to study the response time of probe molecules to GST.

[0085] Figure 13 The time-dependent fluorescence intensity changes of the probe Fu-Ca-Cy under conditions of GSH-only and GSH-GST co-existence are shown. The curves indicate that in the presence of GSH alone, the probe's fluorescence intensity shows only a slight increasing trend throughout the 80-minute test period; when GSH and GST are co-existing, the probe's fluorescence intensity increases rapidly in the initial stage (0-20 minutes), then the rate of increase slows down and stabilizes after approximately 30 minutes. These results confirm that the probe Fu-Ca-Cy has a specific response to GST.

[0086] Test Example 6

[0087] This test case is used to study the signal amplification effect of the probe Fu-Ca-Cy under the action of thiol proteins.

[0088] according to Figure 14As shown, the blank control group (Blank) and the group containing only GSH (GSH) exhibited only extremely low background fluorescence signals. When GSH and GST coexisted (GSH+GST), the probe produced a significant fluorescence response. Furthermore, the addition of thiol amino acids or proteins such as bovine serum albumin (BSA), papain, cysteine ​​(Cys), homocysteine ​​(Hcy), catalase, or glutathione reductase (GR) resulted in a significant stepwise increase in the probe's fluorescence intensity. These results confirm that the fluorescence response signal of the Fu-Ca-Cy probe can be significantly amplified by the action of thiol amino acids or proteins, indicating that this probe has the potential to further enhance detection sensitivity in complex biological environments. This provides a new signal amplification strategy for high-sensitivity detection of GST under conditions of extremely small individuals and extremely low GSH concentrations.

[0089] Test Example 7

[0090] This test case illustrates the application of the probe Fu-Ca-Cy in GST imaging of live diamondback moths.

[0091] Based on the excellent performance of the probe Fu-Ca-Cy in in vitro experiments, we further explored its specific imaging application for GST in diamondback moths. After feeding the moths with artificial feed containing the probe, we performed in vivo imaging using a fluorescence imaging system. Figure 15 Imaging results showed that the blank control group exhibited only extremely low background fluorescence, while the experimental group treated with only the probe (Probe) produced a strong red fluorescence signal. However, in the control groups (Probe + NEM, Probe + EA) simultaneously treated with the probe and the specific inhibitors N-ethylmaleimide (NEM) for GSH and ethacrylic acid (EA) for GST, the fluorescence intensity within the diamondback moth was significantly reduced. Quantitative fluorescence intensity analysis further confirmed that, compared with the probe control group without inhibitors, the fluorescence intensity of the NEM and EA treatment groups was reduced by approximately 40% and 30%, respectively. These results indicate that the enhanced fluorescence signal of the probe Fu-Ca-Cy within the diamondback moth mainly depends on the catalytic activity of GST, thus verifying the probe's specific imaging ability for GST in agricultural pests and providing an effective tool for physiological detection and related research of agricultural pests.

[0092] Test Example 8

[0093] This test case further expands the application of the probe Fu-Ca-Cy in the specific imaging of GST in agricultural weeds.

[0094] *Gnaphalium affine* was selected as the model weed for this study. Imaging was performed after co-incubating *Gnaphalium affine* with a probe solution of Fu-Ca-Cy. The imaging results are as follows: Figure 16As shown, under bright field conditions, the root tip morphology of each group of *Gnaphalium affine* remained consistent. Under fluorescent field conditions, the experimental group treated with only the probe (Probe) exhibited a strong red fluorescent signal. The merged channel showed that the fluorescent signal highly matched the outline of the root tip in the bright field, while the blank control group showed only extremely low background fluorescence. When the probe was simultaneously treated with the GSH-specific inhibitor NEM or the GST activity inhibitor EA (Probe + NEM, Probe + EA), the fluorescence intensity of the *Gnaphalium affine* root tip was significantly reduced. Quantitative fluorescence intensity analysis further confirmed that the fluorescence intensity of both the NEM and EA treatment groups was significantly reduced compared to the probe control group without inhibitors.

[0095] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A signal-amplifying fluorescent probe for glutathione S-transferase, characterized in that, The probe has the structure shown in formula (I); Equation (I) Wherein, R is selected from one of the following groups: 。 2. The signal-amplifying glutathione S-transferase fluorescent probe according to claim 1, characterized in that, The probe has the structure shown in formula (II). Equation (II).

3. The method for preparing the signal-amplified glutathione S-transferase fluorescent probe according to claim 1 or 2, characterized in that, The method includes: dissolving a fluorescent compound RH containing an amino or piperazine unit with tri-phosphorus gas in an organic solvent, then adding an alkaline substance and compound 2 to react and obtain a fluorescent probe; Compound 2.

4. The method for preparing the signal-amplifying glutathione S-transferase fluorescent probe according to claim 3, characterized in that, The method includes: (1) Under the protection of an inert gas, compound 1, p-aminobenzaldehyde and a first organic solvent were subjected to a first contact reaction to obtain the fluorescent compound Fu-NH2; (2) Compound Fu-NH2 and triphosphorus gas were dissolved in a second organic solvent, and then an alkaline substance and compound 2 were added to carry out a second contact reaction to obtain the fluorescent probe Fu-Ca-Cy; 。 5. The method for preparing the signal-amplifying glutathione S-transferase fluorescent probe according to claim 3 or 4, characterized in that, The alkaline substance is selected from at least one of pyridine, triethylamine, 4-dimethylaminopyridine, and sodium ethoxide; the first organic solvent and the second organic solvent are each independently selected from at least one of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, and anhydrous ethanol.

6. The method for preparing the signal-amplifying glutathione S-transferase fluorescent probe according to claim 4, characterized in that, In step (1), the molar ratio of compound 1 to p-aminobenzaldehyde is 2:(1.5-1.8).

7. The method for preparing the signal-amplifying glutathione S-transferase fluorescent probe according to claim 4, characterized in that, In step (2), the molar ratio of compound Fu-NH2 to triphosgene is 1:(1-2.5); the molar ratio of alkaline substance to compound Fu-NH2 is 1:(5-10); and the molar ratio of compound 2 to compound Fu-NH2 is 1:(1.2-2).

8. The method for preparing the signal-amplifying glutathione S-transferase fluorescent probe according to claim 4, characterized in that, The temperature of the first contact reaction is 30-100℃; the temperature of the second contact reaction is 20-40℃.

9. The application of the signal-amplifying glutathione S-transferase fluorescent probe according to claim 1 or 2, characterized in that, The fluorescent probe is used for the dynamic detection of glutathione S-transferase content in insects and plants at the in vivo level.

10. The application of the signal-amplifying glutathione S-transferase fluorescent probe according to claim 1 or 2, characterized in that, The fluorescent probe was used to monitor the dynamic fluctuations in glutathione S-transferase levels induced by an insect resistance model.

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