A fructose probe, and a preparation method and application thereof

Abstract

The application discloses a fructose probe and a preparation method and application thereof. The fructose probe has a structural formula as shown in formula (I). The fructose probe can be used for high-efficiency affinity enrichment and screening and identification of a fructose direct binding protein, and can provide a new molecular mechanism for glomerular podocyte injury caused by fructose and a new target for treatment of high-fructose related kidney diseases.

Description

Technical Field

[0001] This invention relates to the combination of biotechnology, specifically to a fructose probe, its preparation method, and its application. Background Technology

[0002] Fructose is commonly added to foods and beverages as a sweetener due to its high sweetness, good solubility, and low price. However, excessive fructose intake also leads to many public health problems and disrupts people's lives. Epidemiological studies have found a significant positive correlation between the intake of high-sugar beverages and the incidence of chronic kidney disease. Previous research in our laboratory has confirmed that a long-term high-fructose diet can induce kidney damage in rats, manifested as proteinuria and fusion and shedding of glomerular podocyte foot processes. Chronic kidney disease can alter the structure and function of podocytes, causing them to lose specific differentiation markers and reducing glomerular filtration capacity. Studies have found that high fructose intake accelerates podocyte damage, reducing the structural support provided by podocytes to glomerular capillaries, impairing glomerular filtration and reabsorption functions, and promoting the production of uric acid, creatinine, urea nitrogen, and proteinuria. However, it remains unclear whether fructose directly binds to proteins in glomerular podocytes and regulates podocyte function through these proteins, leading to kidney damage.

[0003] Activity-based protein profiling (ABPP) is a highly valuable proteomics tool used for identifying active small-molecule target proteins, elucidating the function of unknown proteins, and screening small-molecule inhibitors. ABPP utilizes reactive groups on chemical probes to covalently label active sites in the proteome. The labeled proteins are then enriched using reporter groups on the probes, and finally, mass spectrometry analysis is used to identify protein components and site information. In recent years, chemical proteomics has made significant progress. ABPP enables objective and comprehensive analysis of small molecules and their binding proteins, providing a theoretical basis for identifying specific physiological or pathological processes associated with small-molecule proteins. However, a fructose probe based on ABPP technology for screening and identifying fructose-directly binding proteins is currently lacking. Based on previous research, this invention designs and successfully obtains a fructose probe that can be used for screening and identifying fructose-directly binding proteins using ABPP technology. Preliminary results demonstrate that high fructose levels can induce glomerular podocyte dysfunction and kidney damage through these target proteins. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a fructose probe that can be used for screening and identifying fructose-directly binding proteins using ABPP technology, as well as its preparation method and application.

[0005] Therefore, the present invention adopts the following technical solution: A fructose probe of the present invention, wherein the structural formula of the fructose probe is shown in formula (I):

[0006]

[0007] The method for preparing the fructose probe according to the present invention includes the following steps:

[0008]

[0009] (1) Weigh compound 1, p-hydroxybenzoic acid, tert-butyldimethylchlorosilane, and imidazole into a round-bottom flask. After adding N,N-dimethylformamide solution, stir at room temperature for 24 hours using a magnetic stirrer. Transfer the reaction product from the round-bottom flask to a separatory funnel, add diethyl ether and water for extraction, collect the upper organic phase solution, add water to the collected organic phase solution, extract again, collect the upper organic phase solution, transfer it to a beaker, and dehydrate it with anhydrous MgSO4. After dehydration, filter the solution and transfer it to a round-bottom flask. Remove diethyl ether using a rotary evaporator to obtain the product. Then add dichloromethane to dissolve the product and transfer it to a silica gel column. Elute with dichloromethane for purification. Transfer the eluent containing the purified product to a round-bottom flask and remove dichloromethane using a rotary evaporator. After drying, add... A tetrahydrofuran solution, water, and glacial acetic acid were added in a volume ratio of 5:1:4. The mixture was stirred at room temperature for 21 hours using a magnetic stirrer. The reaction solution was then transferred to a separatory funnel, and ether and water were added for extraction in a volume ratio of 2:1. The organic phase solution was collected and transferred to a beaker for dehydration with anhydrous MgSO4. The dehydrated solution was transferred to a round-bottom flask and the ether was removed by rotary evaporation. After drying, a transparent liquid product was obtained. The product was loaded onto a silica gel column and rapidly eluted with dichloromethane for purification. The eluent containing the purified product was transferred to a round-bottom flask, and the dichloromethane was removed again by rotary evaporation. The product was then dried and evacuated for 15 minutes. A white crystalline product was observed in the round-bottom flask, which is compound 2, in which tert-butyldimethylchlorosilane protects the hydroxyl group of p-hydroxybenzoic acid.

[0010] (2) Weigh N,N'-dicyclohexylcarbonimide into a beaker and dissolve it in dichloromethane; weigh compound 2, fructose diacetone, and 4-dimethylaminopyridine into a 25 mL round-bottom flask, add dichloromethane, and place on a magnetic stirrer; slowly add the N,N'-dicyclohexylcarbonimide solution to the round-bottom flask under ice bath conditions, remove the ice box after the addition is complete, and stir the reaction solution at room temperature for 12 h to allow fructose diacetone to attach to compound 2; then stir the round-bottom flask under ice bath conditions for 10 min to observe the precipitation of dicyclohexylurea, and then remove the reaction mixture from the round-bottom flask. The product was filtered, and the filtered solution was transferred to a separatory funnel. It was then extracted twice, each time, with 10% Na₂CO₃ solution, 1M HCl solution, and saturated NaCl solution. The organic phase solution was collected and transferred to a beaker, and anhydrous MgSO₄ was added for dehydration. The dehydrated solution was filtered and transferred to a round-bottom flask, and evaporated to dryness using a rotary evaporator. The obtained product was dissolved in 5 mL of petroleum ether / ethyl acetate and then purified by silica gel column chromatography. The eluent was petroleum ether / ethyl acetate. The eluent containing the purified product was collected and evaporated to dryness to obtain compound 3.

[0011] (3) Weigh compound 3 into a round-bottom flask, add tetrahydrofuran to dissolve it, then place it on an ice bath and add tetra-n-butylammonium fluoride dropwise. Stir for 15 min to remove the TBS protecting group from compound 3 using tetra-n-butylammonium fluoride. Then, under ice bath conditions, slowly add 30% H2O2 dropwise to the round-bottom flask until no white precipitate is formed to terminate the reaction. Filter the solution in the round-bottom flask and transfer the filtered solution to a separatory funnel. Add ethyl acetate for extraction and collect the upper organic phase solution. Add saturated NaHCO3 aqueous solution to the organic phase solution in the separatory funnel for extraction. Collect the upper organic phase solution again and transfer it to a flask. Add anhydrous Na2SO4 for dehydration and filter. Transfer the filtered solution to a round-bottom flask and remove organic solvents such as ethyl acetate using a rotary evaporator. Elute and purify the obtained product using silica gel column elution. The eluent is petroleum ether / ethyl acetate. Collect the eluent containing the purified product and evaporate to dryness to obtain compound 4.

[0012] (4) Weigh compound 4 into a round-bottom flask, add N,N-dimethylformamide to dissolve it, then add 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine and K2CO3, place on a magnetic stirrer, stir at 50°C in the dark for 12 h to connect compound 4 to a universal biological probe containing a hetero-nitrogen ring and an alkyne group; then, transfer the round-bottom flask to an ice bath and slowly add water to quench the reaction, then transfer it to a separatory funnel, add ethyl acetate for extraction, and collect the upper organic phase solution; transfer the collected organic phase solution to a beaker, add anhydrous Na2SO4 for dehydration and filter; use a rotary evaporator to remove organic solvents such as ethyl acetate, and elute and purify the obtained product by silica gel column elution, the eluent being petroleum ether / ethyl acetate; collect the eluent containing the purified product and evaporate to dryness to obtain the fructose probe of compound 5.

[0013] Further, in step (1), the molar ratio of compound 1 p-hydroxybenzoic acid, tert-butyldimethylchlorosilane and imidazole is 1:3:3.

[0014] Further, in step (2), the molar ratio of compound 2, fructose diacetone, 4-dimethylaminopyridine and N,N'-dicyclohexylcarboimide is 1:1.2:1:1.5.

[0015] Furthermore, in step (3), the molar ratio of compound 3 to tetra-n-butylammonium fluoride is 10:1, and the volume ratio of tetrahydrofuran to tetra-n-butylammonium fluoride is 10:1.5.

[0016] Furthermore, in step (4), the molar ratio of compound 4, K2CO3 and 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine is 1:2:1.1.

[0017] A kit of the present invention comprises biotin with azide, streptavidin magnetic beads and the fructose probe.

[0018] The application of the fructose probe described in this invention in screening and identifying fructose-directly binding proteins in human glomerular podocytes cultured in vitro.

[0019] The fructose probe described in this invention is used to screen and identify fructose-directly binding proteins in various other cell lines and tissues.

[0020] Beneficial effects: This invention designs and synthesizes a fructose probe capable of click chemistry and ultraviolet cross-linking, which can be used for efficient affinity enrichment, screening, and identification of fructose-bound proteins, providing a new molecular mechanism for fructose-induced glomerular podocyte damage and a new target for the treatment of high-fructose-related kidney diseases.

[0021] Compared with the prior art, the present invention has at least the following advantages:

[0022] (1) This invention is the first to synthesize a fructose probe based on ABPP technology that can perform click chemical reactions and ultraviolet cross-linking, and the fructose probe retains the biological effects and functions of natural fructose molecules.

[0023] (2) The fructose probe of the present invention can be used for efficient affinity enrichment, screening and identification of fructose-bound proteins to discover new therapeutic targets for high fructose-related diseases. Attached Figure Description

[0024] Figure 1 This diagram illustrates how the fructose probe of this invention leads to a decrease in the expression of a signature protein in cultured podocytes.

[0025] Figure 2 This diagram illustrates the decrease in mitochondrial membrane potential in cultured podocytes caused by the fructose probe of this invention.

[0026] Figure 3 This figure shows the increase in mitochondrial ROS levels in in vitro cultured podocytes caused by the fructose probe of this invention.

[0027] Figure 4 This is a diagram of the protein bound by the UV cross-linking covalent labeling and enrichment of fructose probes of the present invention.

[0028] Figure 5 This image shows the identification and screening of fructose probe-binding proteins using the label-free quantitative mass spectrometry technique of this invention. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are only descriptive and not limiting. The scope of protection of the present invention shall be determined by the claims.

[0030] Example 1

[0031] The present invention provides a fructose probe, the structural formula of which is shown in formula (I):

[0032]

[0033] The method for preparing the fructose probe according to the present invention includes the following steps:

[0034]

[0035] (1) Weigh 1.38 g of compound 1-hydroxybenzoic acid, 4.52 g of tert-butyldimethylchlorosilane and 2.04 g of imidazole into a 25 mL round-bottom flask, and add 20 mL of the mixture. After reacting with N,N-dimethylformamide solution, the mixture was stirred with a magnetic stirrer for 24 hours. The reaction product was then transferred from the round-bottom flask to a separatory funnel, and 100 mL of diethyl ether and 50 mL of water were added for extraction. The upper organic phase was collected, and 25 mL of water was added to the collected organic phase. Extraction was repeated, and the upper organic phase was collected and transferred to a beaker for dehydration with anhydrous MgSO4. The dehydrated solution was filtered and transferred to a round-bottom flask. Diethyl ether was removed by rotary evaporation to obtain the product. 5 mL of dichloromethane was added to dissolve the product, and the solution was transferred to a silica gel column for elution with dichloromethane. The eluent containing the purified product was transferred to a round-bottom flask, and dichloromethane was removed by rotary evaporation. After drying, 15 mL of tetrahydrofuran solution, 3 mL of water, and 12 mL of MgSO4 solution were added. L of glacial acetic acid was stirred at room temperature on a magnetic stirrer for 21 h. The reaction solution was then transferred to a separatory funnel, and 100 mL of diethyl ether and 50 mL of water were added for extraction. The organic phase solution was collected and transferred to a beaker for dehydration with anhydrous MgSO4. The dehydrated solution was transferred to a round-bottom flask and the diethyl ether was removed by rotary evaporation. After evaporation to dryness, a transparent liquid product was obtained. The product was loaded onto a silica gel column and eluted with dichloromethane to purify it. The eluent containing the purified product was transferred to a round-bottom flask, and the dichloromethane was removed again by rotary evaporation. The product was then evaporated to dryness and evacuated for 15 min. A white crystalline product was observed in the round-bottom flask, which is compound 2 (1.84 g, yield: 73%) in which tert-butyldimethylchlorosilane protects the hydroxyl group of p-hydroxybenzoic acid.

[0036] (2) Weigh N,N'-dicyclohexylcarbonimide (1.3 g) into a beaker and dissolve it in 5 mL of dichloromethane; weigh compound 2 (1.26 g), fructose diacetone (1.3 g), and 4-dimethylaminopyridine (0.61 g) into a 25 mL round-bottom flask, add 20 mL of dichloromethane, and place the flask on a magnetic stirrer; slowly add the N,N'-dicyclohexylcarbonimide solution dropwise into the round-bottom flask under ice bath conditions. After the addition is complete, remove the ice box and stir the reaction solution at room temperature for 12 h to allow the fructose diacetone to attach to compound 2; then stir the round-bottom flask under ice bath conditions for 10 min to observe the precipitation of dicyclohexylurea. The reaction product in the round-bottom flask was then filtered, and the filtered solution was transferred to a separatory funnel. It was then extracted twice, each time, with 10% Na2CO3 solution, HCl solution (1M), and saturated NaCl solution. The organic phase solution was collected and transferred to a beaker, and anhydrous MgSO4 was added for dehydration. The dehydrated solution was filtered and transferred to a round-bottom flask, and evaporated to dryness using a rotary evaporator. The obtained product was dissolved in 5 mL of petroleum ether / ethyl acetate and then purified by silica gel column chromatography. The eluent was petroleum ether / ethyl acetate. The eluent containing the purified product was collected and evaporated to dryness to obtain compound 3 (742 mg, yield: 30%).

[0037] (3) Weigh compound 3 (742 mg) into a round-bottom flask, add tetrahydrofuran to dissolve it, then place it in an ice bath, add tetra-n-butylammonium fluoride dropwise, and stir for 15 min to remove the TBS protecting group from compound 3 using tetrabutylammonium fluoride; then, under ice bath conditions, slowly add 30% H2O2 dropwise to the round-bottom flask until no white precipitate is formed to terminate the reaction; filter the solution in the round-bottom flask, transfer the obtained solution to a separatory funnel, extract with ethyl acetate, and collect the upper organic phase. The organic phase solution in the separatory funnel was extracted with saturated NaHCO3 aqueous solution. The upper organic phase solution was collected again and transferred to a flask. Anhydrous Na2SO4 was added for dehydration and filtration. The filtered solution was transferred to a round-bottom flask and the organic solvents such as ethyl acetate were removed by rotary evaporation. The obtained product was eluted and purified by silica gel column chromatography with petroleum ether / ethyl acetate as the eluent. The eluent containing the purified product was collected and evaporated to dryness to obtain compound 4 (558 mg, yield: 98%).

[0038] (4) Weigh compound 4 (380 mg) into a round-bottom flask, add 5 mL of N,N-dimethylformamide to dissolve it, then add 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine (273 mg) and K2CO3 (277 mg), place on a magnetic stirrer, and stir at 50 °C in the dark for 12 h to allow compound 4 to be linked to a universal biological probe containing a heteroazo ring and an alkyne group; then, transfer the round-bottom flask to an ice bath and slowly add 20 mL of water dropwise to... The reaction was quenched, and the mixture was then transferred to a separatory funnel. Ethyl acetate was added for extraction, and the upper organic phase solution was collected. The collected organic phase solution was transferred to a beaker, and anhydrous Na2SO4 was added for dehydration and filtration. Organic solvents such as ethyl acetate were removed using a rotary evaporator. The obtained product was eluted and purified by silica gel column chromatography with petroleum ether / ethyl acetate as the eluent. The eluent containing the purified product was collected and evaporated to dryness to obtain the fructose probe of compound 5 (210 mg, yield: 42%).

[0039] A kit of the present invention comprises biotin with azide, streptavidin magnetic beads and the fructose probe.

[0040] The application of the fructose probe described in this invention in screening and identifying fructose-directly binding proteins in human glomerular podocytes cultured in vitro.

[0041] The fructose probe described in this invention is used to screen and identify fructose-directly binding proteins in various other cell lines and tissues.

[0042] Example 2

[0043] The method for preparing the fructose probe of the present invention includes the following steps:

[0044]

[0045] (1) Weigh compound 1, p-hydroxybenzoic acid, tert-butyldimethylchlorosilane, and imidazole into a 25 mL round-bottom flask. Add 20 mL of N,N-dimethylformamide solution and stir with a magnetic stirrer for 24 h. Transfer the reaction product from the round-bottom flask to a separatory funnel and add 100 mL of diethyl ether and 50 mL of water for extraction. Collect the upper organic phase solution. Add 25 mL of water to the collected organic phase solution and extract again. Collect the upper organic phase solution and transfer it to a beaker for dehydration with anhydrous MgSO4. After dehydration, filter the solution and transfer it to a round-bottom flask. Remove the diethyl ether using a rotary evaporator to obtain the product. Then add 5 mL of dichloromethane to dissolve the product and transfer it to a silica gel column. Elute with dichloromethane for purification. Transfer the eluent containing the purified product to a round-bottom flask and remove the dichloromethane using a rotary evaporator. After drying, add 15 mL of tetrahydrofuran solution, 3 mL of water, and 12 mL of glacial acetic acid. Place the column on a magnetic stirrer. After stirring at room temperature for 21 hours, the reaction solution was transferred to a separatory funnel, and 100 mL of diethyl ether and 50 mL of water were added for extraction. The organic phase solution was collected and transferred to a beaker for dehydration with anhydrous MgSO4. The dehydrated solution was transferred to a round-bottom flask and the diethyl ether was removed by rotary evaporation. After drying, a transparent liquid product was obtained, which was loaded onto a silica gel column and rapidly eluted with dichloromethane for purification. The eluent containing the purified product was transferred to a round-bottom flask, and the dichloromethane was removed again by rotary evaporation. The solution was dried and vacuumed for 15 minutes. A white crystalline product was visible in the round-bottom flask, which is compound 2, in which tert-butyldimethylchlorosilane protects the hydroxyl group of p-hydroxybenzoic acid. The molar ratio of p-hydroxybenzoic acid, tert-butyldimethylchlorosilane, and imidazole in compound 1 was 1:3:3.

[0046] (2) Weigh N,N'-dicyclohexylcarbonimide into a beaker and dissolve it in 5 mL of dichloromethane. Weigh compound 2, fructose diacetone, and 4-dimethylaminopyridine into a 25 mL round-bottom flask, add 20 mL of dichloromethane, and place the flask on a magnetic stirrer. Slowly add the N,N'-dicyclohexylcarbonimide solution to the round-bottom flask under ice bath conditions. After the addition is complete, remove the ice box and stir the reaction solution at room temperature for 12 h to allow fructose diacetone to attach to compound 2. Then, stir the round-bottom flask under ice bath conditions for 10 min to observe the precipitation of dicyclohexylurea. Subsequently, filter the reaction product in the round-bottom flask and transfer the filtered solution to a separatory funnel. The organic phase was extracted twice, sequentially with 10% Na₂CO₃ solution, 1M HCl solution, and saturated NaCl solution. The organic phase solution was collected and transferred to a beaker, and anhydrous MgSO₄ was added for dehydration. The dehydrated solution was filtered and transferred to a round-bottom flask, and evaporated to dryness using a rotary evaporator. The obtained product was dissolved in 5 mL of petroleum ether / ethyl acetate and then purified by silica gel column chromatography. The eluent was petroleum ether / ethyl acetate. The eluent containing the purified product was collected and evaporated to dryness to obtain compound 3. The molar ratio of compound 2, fructose diacetone, 4-dimethylaminopyridine, and N,N'-dicyclohexylcarboimide was 1:1.2:1:1.5.

[0047] (3) Weigh compound 3 into a round-bottom flask, add 10 mL of tetrahydrofuran to dissolve it, then place it on an ice bath and add 1.5 mL of tetra-n-butylammonium fluoride dropwise. Stir for 15 min to remove the TBS protecting group from compound 3 using tetra-n-butylammonium fluoride. Then, under ice bath conditions, slowly add 30% H2O2 dropwise to the round-bottom flask until no white precipitate is formed to terminate the reaction. Filter the solution in the round-bottom flask, transfer the filtered solution to a separatory funnel, add ethyl acetate for extraction, collect the upper organic phase solution, and add... Extraction was performed using a saturated NaHCO3 aqueous solution. The upper organic phase solution was collected again and transferred to a flask. Anhydrous Na2SO4 was added for dehydration and filtration. The filtered solution was transferred to a round-bottom flask, and organic solvents such as ethyl acetate were removed by rotary evaporation. The obtained product was eluted and purified by silica gel column chromatography with petroleum ether / ethyl acetate as the eluent. The eluent containing the purified product was collected and evaporated to dryness to obtain compound 4. The molar ratio of compound 3 to tetrabutylammonium fluoride was 10:1, and the volume ratio of tetrahydrofuran to tetrabutylammonium fluoride was 10:1.5.

[0048] (4) Weigh compound 4 into a round-bottom flask, add 5 mL of N,N-dimethylformamide to dissolve it, then add 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine and K2CO3, place on a magnetic stirrer, stir at 50°C in the dark for 12 h to connect compound 4 to a universal biological probe containing a hetero-nitrogen ring and an alkyne group; then, transfer the round-bottom flask to an ice bath and slowly add 20 mL of water to quench the reaction, then transfer it to a separatory funnel, add ethyl acetate for extraction, and collect the upper organic phase solution; transfer the collected organic phase solution to a beaker, add anhydrous Na2SO4 for dehydration and filter; use a rotary evaporator to remove organic solvents such as ethyl acetate, and elute and purify the obtained product by silica gel column elution, the eluent being petroleum ether / ethyl acetate; collect the eluent containing the purified product and evaporate to dryness to obtain the fructose probe of compound 5. The molar ratio of compound 4, K2CO3 and 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine is 1:2:1.1.

[0049] Experimental Example 1

[0050] 1. Synthesis of fructose probe based on ABPP technology principle

[0051] This invention designs and synthesizes a fructose probe that can "fish out" fructose-bound proteins directly through ultraviolet cross-linking and click chemistry. Natural fructose was used as a positive control to evaluate the biological function of the fructose probe. This invention found that the fructose probe exhibits similar biological effects to fructose, including a reduction in mitochondrial respiratory function in cultured podocytes. Figure 1 Increased mitochondrial ROS levels Figure 2 ) and decrease in mitochondrial membrane potential ( Figure 3 These results indicate that the fructose probe retains the biological functions of natural fructose and can be used to "fish out" fructose-binding proteins in cultured podocytes in vitro.

[0052] 2. Screening and Identification of Fructose-Binding Proteins Based on ABPP Technology: This invention utilizes ABPP technology to identify fructose-binding proteins in in vitro cultured podocytes using fructose probes, and further identifies these proteins by UV cross-linking and covalently labeling. Figure 4 After enrichment by click chemistry, probe-labeled proteins were identified and screened using label-free quantitative mass spectrometry. Figure 5 Compared with the negative control group, when the screening criteria were set at a fold change of more than 2 times and the number of peptides ≥ 2, a total of 144 potential fructose-directly binding proteins were obtained. Figure 5 A). Bioinformatics analysis indicates that these proteins mainly belong to metabolic pathways and biological processes such as the tricarboxylic acid cycle, pyruvate metabolism, fatty acid degradation, oxidative phosphorylation, and gluconeogenesis / glycolysis. Figure 5B) may be related to podocyte damage and kidney disease caused by high fructose, which can be used for further research on the molecular mechanisms of high fructose-related diseases and provide potential new therapeutic targets.

[0053] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A fructose probe, characterized in that: The fructose probe is shown in formula (I):

2. The method for preparing the fructose probe according to claim 1, characterized in that... Includes the following steps: (1) Weigh compound 1, p-hydroxybenzoic acid, tert-butyldimethylchlorosilane, and imidazole into a round-bottom flask. After adding N,N-dimethylformamide solution, stir at room temperature for 24 hours using a magnetic stirrer. Transfer the reaction product from the round-bottom flask to a separatory funnel, add diethyl ether and water for extraction, collect the upper organic phase solution, add water to the collected organic phase solution, extract again, collect the upper organic phase solution, transfer it to a beaker, and dehydrate it with anhydrous MgSO4. After dehydration, filter the solution and transfer it to a round-bottom flask. Remove diethyl ether using a rotary evaporator to obtain the product. Then add dichloromethane to dissolve the product and transfer it to a silica gel column. Elute with dichloromethane for purification. Transfer the eluent containing the purified product to a round-bottom flask and remove dichloromethane using a rotary evaporator. After drying, add... A tetrahydrofuran solution, water, and glacial acetic acid were added in a volume ratio of 5:1:

4. The mixture was stirred at room temperature for 21 hours using a magnetic stirrer. The reaction solution was then transferred to a separatory funnel, and ether and water were added for extraction in a volume ratio of 2:

1. The organic phase solution was collected and transferred to a beaker for dehydration with anhydrous MgSO4. The dehydrated solution was transferred to a round-bottom flask and the ether was removed by rotary evaporation. After drying, a transparent liquid product was obtained. The product was loaded onto a silica gel column and rapidly eluted with dichloromethane for purification. The eluent containing the purified product was transferred to a round-bottom flask, and the dichloromethane was removed again by rotary evaporation. The product was then dried and evacuated for 15 minutes. A white crystalline product was observed in the round-bottom flask, which is compound 2, in which tert-butyldimethylchlorosilane protects the hydroxyl group of p-hydroxybenzoic acid. (2) Weigh N,N'-dicyclohexylcarbonimide into a beaker and dissolve it in dichloromethane; weigh compound 2, fructose diacetone, and 4-dimethylaminopyridine into a 25 mL round-bottom flask, add dichloromethane, and place on a magnetic stirrer; slowly add the N,N'-dicyclohexylcarbonimide solution to the round-bottom flask under ice bath conditions, remove the ice box after the addition is complete, and stir the reaction solution at room temperature for 12 h to allow fructose diacetone to attach to compound 2; then stir the round-bottom flask under ice bath conditions for 10 min to observe the precipitation of dicyclohexylurea, and then remove the reaction mixture from the round-bottom flask. The product was filtered, and the filtered solution was transferred to a separatory funnel. It was then extracted twice, each time, with 10% Na₂CO₃ solution, 1M HCl solution, and saturated NaCl solution. The organic phase solution was collected and transferred to a beaker, and anhydrous MgSO₄ was added for dehydration. The dehydrated solution was filtered and transferred to a round-bottom flask, and evaporated to dryness using a rotary evaporator. The obtained product was dissolved in 5 mL of petroleum ether / ethyl acetate and then purified by silica gel column chromatography. The eluent was petroleum ether / ethyl acetate. The eluent containing the purified product was collected and evaporated to dryness to obtain compound 3. (3) Weigh compound 3 into a round-bottom flask, add tetrahydrofuran to dissolve it, then place it on an ice bath and add tetra-n-butylammonium fluoride dropwise, stirring for 15 min. This is to remove the TBS protecting group from compound 3 using tetra-n-butylammonium fluoride. Then, under ice bath conditions, slowly add 30% H2O2 dropwise to the round-bottom flask until no white precipitate is formed to terminate the reaction. Filter the solution in the round-bottom flask, transfer the filtered solution to a separatory funnel, add ethyl acetate for extraction, collect the upper organic phase solution, add saturated NaHCO3 aqueous solution to the organic phase solution in the separatory funnel for extraction, collect the upper organic phase solution again, and transfer it to a flask. Add anhydrous Na2SO4 for dehydration and filter. Transfer the filtered solution to a round-bottom flask, remove the ethyl acetate organic solvent by rotary evaporator, and elute and purify the obtained product by silica gel column elution. The eluent is petroleum ether / ethyl acetate. Collect the eluent containing the purified product and evaporate to dryness to obtain compound 4. (4) Weigh compound 4 into a round-bottom flask, add N,N-dimethylformamide to dissolve it, then add 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine and K2CO3, place it on a magnetic stirrer, stir at 50°C in the dark for 12 h to connect compound 4 to a universal biological probe containing a hetero-nitrogen ring and an alkyne group; then, transfer the round-bottom flask to an ice bath and slowly add water to quench the reaction, then transfer it to a separatory funnel, add ethyl acetate for extraction, and collect the upper organic phase solution; transfer the collected organic phase solution to a beaker, add anhydrous Na2SO4 for dehydration and filter; use a rotary evaporator to remove the ethyl acetate organic solvent, and elute and purify the obtained product by silica gel column elution, the eluent being petroleum ether / ethyl acetate; collect the eluent containing the purified product and evaporate to dryness to obtain compound 5 fructose probe.

3. The method for preparing the fructose probe according to claim 2, characterized in that: In step (1), the molar ratio of compound 1 p-hydroxybenzoic acid, tert-butyldimethylchlorosilane and imidazole is 1:3:

3.

4. The method for preparing the fructose probe according to claim 2, characterized in that: In step (2), the molar ratio of compound 2, fructose diacetone, 4-dimethylaminopyridine and N,N'-dicyclohexylcarboimide is 1:1.2:1:1.

5.

5. The method for preparing the fructose probe according to claim 2, characterized in that: In step (3), the molar ratio of compound 3 to tetra-n-butylammonium fluoride is 10:1, and the volume ratio of tetrahydrofuran to tetra-n-butylammonium fluoride is 10:1.

5.

6. The method for preparing the fructose probe according to claim 2, characterized in that: In step (4), the molar ratio of compound 4, K2CO3 and 3-(3-yn-1-butyl)-3-(2-iodoethyl)-3H-bisacrylidine is 1:2:1.

1.

7. A reagent kit, characterized in that: The kit includes biotin with azide, streptavidin magnetic beads, and the fructose probe of claim 1.

8. The use of the fructose probe of claim 1 in the preparation of reagents for screening and identifying fructose-binding proteins in human glomerular podocytes cultured in vitro.

9. The use of the fructose probe of claim 1 in the preparation of reagents for screening and identifying fructose-binding proteins in cell lines and tissues.

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