Method for identifying chiral triazole bactericide by using P38 protein

Abstract

The invention discloses a method for identifying chiral triazole bactericides by using P38 protein, the affinity constant of unknown triazole bactericide monomers and P38 protein is measured by a surface plasmon resonance technology, and the affinity constant is compared with the affinity constant of a standard substance, so that the configuration of the unknown monomers is judged. The detection method is simple.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticides, and specifically relates to a method for identifying chiral triazole fungicides by using P38 protein. Background Art

[0002] Triazole fungicides are a class of organic heterocyclic compounds, and are widely used in crops such as fruits and vegetables due to their broad-spectrum, high-efficiency, ability to prevent and treat diseases, and regulate plant growth. As the largest category with the most varieties among global fungicides, triazole fungicides are one of the most commonly used pesticides in recent years. Since they entered the pesticide market in the 1970s, many triazole fungicides such as tebuconazole, diniconazole, myclobutanil, difenoconazole, and propiconazole have been successively applied to agricultural production. Triazole fungicides have the characteristics of high efficiency, low toxicity, long-lasting efficacy, and strong systemicity, and can be used to control powdery mildew, rust, and leaf spot diseases, showing great potential in disease prevention and treatment and plant growth regulation. In addition, triazole fungicides have the property of anti-spores and can be used for prevention or as the main agent for the treatment of early fungal infections.

[0003] Due to the high chemical stability, long half-life, low biodegradability, and easy transportation in the environment of triazole fungicides, they have been widely detected in various environmental media. Triazole fungicides such as tebuconazole, difenoconazole, propiconazole, and triadimefon have been widely detected in fruits such as grapes, apples, and pomegranates. Among them, the longest half-life of tebuconazole in grapes can reach 9.8 - 12.2 d, and the residue amount is 0.002 - 0.298 mg / kg. Compared with fruits, the half-life of triazole fungicides in crops is shorter, but the time in paddy soil can be as long as 23 d. It can be seen that triazole fungicides can remain in the environment for a long time in various environmental media and may pose great hazards to animals and humans.

[0004] Existing studies have confirmed that triazole fungicides can cause embryonic developmental toxicity and endanger various metabolic organs such as the liver, kidney, and intestine. Different concentrations of Cyproconazole (1 mg / L and 10 mg / L) can induce weight loss, slow metamorphosis, and abnormal behavioral endpoints in tadpoles of Rana nigromaculata. Propiconazole can significantly inhibit the development of zebrafish embryos and larvae, resulting in a significant decrease in the activities of lipoprotein lipase and fatty acid synthase and a significant up-regulation of the expression levels of lipid metabolism-related genes, posing a potential health risk to the aquatic ecosystem. In addition, in the study on the enantioselective toxicokinetics and toxicity accumulation of myclobutanil, myclobutanil enrichment was observed in tissues and organs such as the liver, fat, skin, intestine, lung, and kidney, and histopathological changes such as hepatocyte hypertrophy, karyopyknosis, vacuolization, and non-zonal large vacuole lipid accumulation were observed in the liver. In the study on the toxic effects of epoxiconazole on the liver and kidney of Wistar rats, severe damage to the overall tissues of the liver and kidney and significant disorders of liver and kidney functions were also found. It can be seen that the potential health risks posed by triazole fungicides to various organisms cannot be ignored, and the development of more advanced, effective, and sensitive pesticide residue detection technologies is particularly important.

[0005] Triazole fungicides are widely used fungicides in the world at present, and the vast majority of triazole fungicides belong to chiral pesticides, having 1 or 2 chiral centers and existing 2 or 4 enantiomers. At present, 14 chiral triazole fungicides are on sale and in use. Although enantiomers have similar physical and chemical properties, they have different activities, degradation behaviors, and toxicities (such as endocrine disruption toxicity, reproductive toxicity, and neurotoxicity) in the environment and organisms.

[0006] So far, there have been extensive studies on the separation of triazole fungicides with one chiral center and the differences in enantiomeric toxicity and environmental behavior. However, due to the 4 enantiomers of triazole fungicides with two chiral centers, the detection and discrimination methods of enantiomers are limited to a certain extent. Therefore, the development of detection technologies for different chiral monomers of triazole fungicides will provide a good technical basis for the above-mentioned related research at the enantiomer level, and has important scientific significance for the future better development of a class of chiral triazole fungicides with high efficiency, ultra-low dosage, high selectivity, and safety for human health. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a method for identifying chiral triazole fungicides using P38 protein, which does not require running standard samples multiple times and has a simple detection method.

[0008] A method for identifying chiral triazole fungicides using P38 protein, comprising the following steps:

[0009] (1) Prepare a protein-coupled running buffer;

[0010] (2) Prepare a gradient solution of the triazole fungicide monomer standard using the running buffer in step (1).

[0011] (3) Screen the optimal coupling conditions through ligand pre-enrichment experiments.

[0012] (4) Couple the P38 protein using the optimal coupling conditions in step (3) to prepare a P38 protein chip.

[0013] (5) Use the multi-cycle detection method to detect the affinity constant between the gradient solution of the triazole fungicide monomer standard obtained in step (2) and the P38 protein chip.

[0014] (6) Determine the affinity constant between the unknown triazole fungicide monomer and the P38 protein under the conditions of step (5), and compare it with the detection value of the standard product to judge the configuration of the unknown triazole fungicide monomer.

[0015] In the present invention, the affinity constant between the unknown triazole fungicide monomer and the P38 protein is determined by surface plasmon resonance (SPR) technology and compared with the affinity constant of the standard product, so as to judge the configuration of the unknown monomer. This method does not require running the standard product multiple times, and the detection method is simple.

[0016] Preferably, in step (1), the running buffer for protein coupling is PBS-P buffer with 5% DMSO.

[0017] More preferably, the running buffer for protein coupling is also corrected for solvent using 4.5% DMSO buffer and 5.8% DMSO buffer.

[0018] In the present invention, to avoid channel blockage after injection, when preparing the gradient solution of the triazole fungicide monomer, it is necessary to first dissolve it with 100% DMSO solvent to form a mother liquor. However, since the introduction of high-concentration DMSO will have a great impact on signal detection (1% DMSO can cause a change fluctuation of 1200 RU), the change in the response value caused by a slight change in the DMSO concentration during the preparation of the detection sample will reach the same order of magnitude as the change in the response value caused by the binding of the triazole fungicide monomer sample itself. Therefore, solvent correction is required to deduct the influence of the bulk effect of the organic solvent.

[0019] Preferably, in step (2), the triazole fungicide is metconazole, triadimenol, paclobutrazol or tebuconazole.

[0020] In the present invention, the triazole fungicides may be metaconazole, triadimenol, paclobutrazol or tebuconazole, and their structures are shown as follows:

[0021]

[0022] Among them, metaconazole, triadimenol and paclobutrazol all contain two chiral centers, that is, there are four corresponding isomers; tebuconazole contains only one chiral center, that is, there are two enantiomers.

[0023] Preferably, step (2) is to dilute the mother liquor of the triazole fungicide monomer standard with PBS-P buffer, and then dilute it with 5% DMSO running buffer to the highest concentration of the gradient, and continue to dilute step by step downward to obtain the gradient solution of the triazole fungicide monomer standard.

[0024] Further preferably, the gradient solution of the triazole fungicide monomer standard is 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.562 μM, 0.781 μM, 0.391 μM, 0.195 μM, 0.098 μM, 0.049 μM, 0.024 μM.

[0025] Preferably, in step (3), the steps of the ligand pre-enrichment experiment are to dilute the P38 protein solution to 10 μg / mL with sodium acetate buffer at pH 5.5, 5.0, 4.5, and 4.0 respectively, with a volume of 100 μL, and compare the actual coupling amounts of P38 protein and the chip under each pH condition, and select the pH value of the sodium acetate buffer corresponding to the closest theoretical coupling amount as the optimal coupling condition.

[0026] Further preferably, the ligand is P38 protein.

[0027] Further preferably, the chip is a CM5 chip.

[0028] Further preferably, the theoretical coupling amount = 1.5 × R L , where R L is the ligand coupling level, that is, the coupling amount, and R L is calculated by the following formula:

[0029]

[0030] Among them, R max is the maximum binding capacity on the protein chip surface, and R max = 100 RU; MW Analyte and MW LigandThey are the molecular weights of the triazole fungicide and the protein, respectively; S m is the stoichiometric ratio, S m = 1.

[0031] In the present invention, the molecular weights of the triazole fungicides are all around 300. During the ligand pre-enrichment experiment, 300 can be first used as the molecular weight of the triazole fungicide for calculation to obtain the theoretical coupling amount of the P38 protein ligand and the chip.

[0032] Further preferably, the optimal coupling condition is the sodium acetate buffer solution with pH 4.0.

[0033] Further preferably, in step (4), the coupling method is amino coupling, the coupling time is 900 s, and the flow rate is 10 μL / min.

[0034] Preferably, in step (5), the steps of the multi-cycle detection method are as follows: the gradient solutions of the triazole fungicide monomer standard obtained in step (2) are injected in sequence according to the concentration from low to high, the binding time is 120 s, the flow rate is 30 μL / min, and the dissociation time is 240 s.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The present invention determines the affinity constant between the unknown triazole fungicide monomer and the P38 protein by surface plasmon resonance (SPR) technology and compares it with the affinity constant of the standard product, so as to judge the configuration of the unknown monomer. This method does not require running the standard product multiple times, and the detection method is simple. Description of the Drawings

[0037] Figures 1 to 12 They are the affinity curves of 1R5R-metconazole, 1S5S-metconazole, 1R5S-metconazole, 1S5R-metconazole, 1R2R-triadimenol, 1S2S-triadimenol, 1R2S-triadimenol, 1S2R-triadimenol, 2R3R-paclobutrazol, 2S3S-paclobutrazol, R-tebuconazole, and S-tebuconazole, respectively. Specific Embodiments

[0038] The following combines examples to further describe the present invention in detail, but the embodiments of the present invention are not limited to the following examples.

[0039] All the raw materials used in the present invention are commercially available.

[0040] Example 1

[0041] (1) Prepare the running buffer for protein coupling and the solvent calibration mother liquor

[0042] The running buffer used is PBS-P+ containing 5% DMSO (the DMSO content can be adjusted according to the solubility of the sample, with a maximum tolerance of 10%). Take 52.5 mL of 10x PBS-P and dilute it to 500 mL with deionized water to prepare 1.05x PBS-P. According to the ratio in Table 1, add DMSO to prepare the 5% DMSO running buffer and the solvent calibration mother liquor.

[0043] Table 1: Preparation of the running buffer for protein conjugation and the solvent calibration mother liquor

[0044] Sample 4.5% DMSO 5.8% DMSO 5% DMSO Running Buffer 1.05x PBS-P 9.5 mL 9.5 mL 950 mL 100% DMSO 0.45 mL 0.58 mL 50 mL Final Volume ~10 mL ~10 mL 1000 mL

[0045] Mix the 4.5% and 5.8% mother liquors according to the following table to prepare the 5% DMSO concentration calibration curve.

[0046] Table 2: Concentrations of the 5% DMSO concentration calibration curve

[0047] Sample (μL) 1 2 3 4 5 6 7 8 4.5% DMSO 0 200 400 600 800 1000 1200 1400 5.8% DMSO 1400 1200 1000 800 600 400 200 0

[0048] (2) Prepare the gradient solutions of the triazole fungicide monomer standards

[0049] In the present invention, since the commercially available paclobutrazol currently only consists of the S-enantiomer and the R-enantiomer, therefore, we only selected 2R3R-paclobutrazol and 2S3S-paclobutrazol of paclobutrazol for testing.

[0050] The triazole fungicide monomer standards are respectively metconazole (1R5R-metconazole, 1S5S-metconazole, 1R5S-metconazole, 1S5R-metconazole), triadimenol (1R2R-triadimenol, 1S2S-triadimenol, 1R2S-triadimenol, 1S2R-triadimenol), paclobutrazol (2R3R-paclobutrazol, 2S3S-paclobutrazol) or tebuconazole (R-tebuconazole, S-tebuconazole); the triazole fungicide monomer standards were separated by Daicel Corporation.

[0051] Dilute the 10 mM triazole fungicide monomer standard mother liquor and the P38 protein inhibitor 20-fold with 1.05x PBS-P to obtain the 500 μM triazole fungicide monomer standard mother liquor and the P38 protein inhibitor. Then, dilute the 500 μM triazole fungicide monomer standard mother liquor and the P38 protein inhibitor to 50 μM respectively with the prepared 5% DMSO running buffer as the highest injection concentration, and dilute them serially in half, with 12 concentration gradients, setting one repeated concentration at intervals, and adding a 0 concentration. The specific concentration gradients are shown in Table 3.

[0052] Table 3: Preparation of the gradient solutions of the triazole fungicide monomer standards and the P38 protein inhibitor

[0053] Sample Serial Number Concentration (μM) 1 0 2 0.024 3 0.049 4 0.098 5 0.195 6 0.391 7 0.781 8 1.562 9 3.125 10 6.25 11 12.5 12 25 13 50

[0054] (3) Screen the optimal coupling conditions through ligand pre-enrichment experiments

[0055] Theoretical coupling amount = 1.5 × R L , where R L is the ligand coupling level, i.e., the coupling amount, and R L is calculated by the following formula:

[0056]

[0057] where R max is the maximum binding capacity on the surface of the protein chip, and R max = 100 RU; MW Angalyte and MW Ligand are the molecular weights of the triazole fungicide and the P38 protein, respectively; S m is the stoichiometric ratio, and S m = 1.

[0058] Dilute the P38 protein solution to 10 μg / mL with sodium acetate buffer solutions with pH values of 5.5, 5.0, 4.5, and 4.0, respectively, with a volume of 100 μL, and compare the actual coupling amounts of the P38 protein with the CM5 chip under each pH condition.

[0059] (4) Prepare the P38 protein chip.

[0060] Select the optimal coupling condition with the pH value of 4.0 (the actual coupling amount is 7600 RU) of the sodium acetate buffer solution closest to the theoretical coupling amount, run the immobilization module, select the CM5 chip in the chip type, select the amino coupling method, set the coupling time to 900 s, select the flow rate of 10 μL / min, and select the test temperature of 25°C. Place the amino coupling reagent and the target protein solution in the sampling box.

[0061] Fix the p38 protein on channel 2, and use channel 1 as the control blank channel. Click to run the software, save the result file, and the software will automatically generate the coupling result to check whether the actual coupling reaches the target coupling amount.

[0062] (5) Detection of the affinity constant of the triazole fungicide monomer standard

[0063] Adopt the multi-cycle detection method, select the Affinity module, input a series of concentrations of the compound in the setting interface, the binding time is 120 s, the flow rate is 30 μL / min, the dissociation time is 240 s, the solvent correction detection is set once at the beginning and once at the end, and use 50% DMSO to clear the residual compound in the pipeline. The detection temperature is defaulted to 25°C.

[0064] Five concentrations were selected to plot the affinity test curve of the standard product of the triazole fungicide monomer. The curve is as Figures 1 to 12 shown, and the affinity constants are shown in Table 4.

[0065] Table 4: Affinity Constants of the Standard Product of the Triazole Fungicide Monomer

[0066]

[0067] (6) Determine the affinity constant of the unknown triazole fungicide monomer and the P38 protein under the conditions of step (5), and compare it with the detected value of the standard product to judge the configuration of the unknown triazole fungicide monomer.

[0068] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for identifying chiral triazole fungicides using P38 protein, characterized in that: The following steps are involved: (1) Prepare protein coupling running buffer; (2) preparing a gradient solution of a triazole fungicide monomer standard using the running buffer of step (1); (3) Screening the optimal coupling conditions through ligand pre-enrichment experiments; (4) coupling the P38 protein using the optimal coupling conditions of step (3) to prepare a P38 protein chip; (5) using a multi-cycle detection method to detect the affinity constant of the gradient solution of the triazole fungicide monomer standard obtained in step (2) and the P38 protein chip; (6) Determine the affinity constant of the unknown triazole fungicide monomer to the P38 protein under the conditions of step (5), and compare it with the detection value of the standard to determine the configuration of the unknown triazole fungicide monomer.

2. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: In step (1), the protein coupling running buffer is PBS-P buffer containing 5% DMSO.

3. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: The protein coupling running buffer also used 4.5% DMSO buffer and 5.8% DMSO buffer for solvent correction.

4. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: In step (2), the triazole fungicide is metconazole, triadimenol, paclobutrazol or tebuconazole.

5. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: The step (2) is to dilute the mother solution of the triazole fungicide monomer standard with PBS-P buffer, then dilute it to the highest concentration of the gradient with 5% DMSO running buffer, and continue to dilute it step by step to obtain a gradient solution of the triazole fungicide monomer standard.

6. The method for identifying chiral triazole fungicides using P38 protein according to claim 5, characterized in that: The gradient solution of the triazole fungicide monomer standard is 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.562 μM, 0.781 μM, 0.391 μM, 0.195 μM, 0.098 μM, 0.049 μM, and 0.024 μM.

7. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: In step (3), the steps of the ligand pre-enrichment experiment are to dilute the P38 protein solution to 10 μg / mL using sodium acetate buffers with pH values ​​of 5.5, 5.0, 4.5, and 4.0, respectively, with a volume of 100 μL, compare the actual coupling amounts of the P38 protein with the chip under various pH conditions, and select the pH value of the sodium acetate buffer corresponding to the closest theoretical coupling amount as the optimal coupling condition.

8. The method for identifying chiral triazole fungicides using P38 protein according to claim 7, characterized in that: The theoretical coupling amount = 1.5 × R L , where R L is the ligand coupling level, i.e., the coupling amount, R L Calculated by the following formula: Among them, R max is the maximum binding capacity of the protein chip surface, R max =100RU;MW Analyte and MW Ligand are the molecular weights of triazole fungicides and proteins, respectively; S m is the stoichiometric ratio, S m =1.

9. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: In step (4), the optimal coupling condition is sodium acetate buffer at pH 4.

0.

10. The method for identifying chiral triazole fungicides using P38 protein according to claim 1, characterized in that: In step (5), the multi-cycle detection method comprises injecting the gradient solutions of the triazole fungicide monomer standard obtained in step (2) in order from low to high concentration, with a binding time of 120 s, a flow rate of 30 μL / min, and a dissociation time of 240 s.

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