CN112898343A - Hapten of organophosphorus triazophos pesticide and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明涉及免疫化学分析及农药残留检测技术领域，具体而言，涉及一种有机磷类三唑磷农药的半抗原及其制备方法。本发明的有机磷类三唑磷农药半抗原的制备方法，以1‑苯基‑3‑羟基‑1,2,4‑三唑为原料，通过硝化反应、取代反应、还原反应和引入羧基活性基团反应合成有机磷类三唑磷农药的半抗原，以利于制备出对有机磷类农药特异性强亲和力高的抗体。

The invention relates to the technical field of immunochemical analysis and pesticide residue detection, in particular to a hapten of an organophosphorus triazophos pesticide and a preparation method thereof. The preparation method of the organophosphorus triazophos pesticide hapten of the present invention uses 1-phenyl-3-hydroxy-1,2,4-triazole as a raw material, through nitration reaction, substitution reaction, reduction reaction and introduction of carboxyl activity The haptens of organophosphorus triazophos pesticides are synthesized by group reaction, so as to facilitate the preparation of antibodies with high specificity and high affinity for organophosphorus pesticides.

Description

Hapten of organophosphorus triazophos pesticide and preparation method thereof

Technical Field

The invention relates to the technical field of immunochemical analysis and pesticide residue detection, in particular to a hapten of an organophosphorus triazophos pesticide and a preparation method thereof.

Background

The quality and safety of agricultural products affect the physical health of people and are more and more concerned by people. The pesticide residue in the agricultural products can not only cause environmental pollution, but also cause damage to the health of human bodies. Especially, as the organic phosphorus pesticide is used in an irregular way and is abused, the organic phosphorus pesticide is remained on crops in a large amount, and the health and the ecological environment of human beings are seriously threatened. Therefore, the development of a high-sensitivity and low-detection-limit rapid immunoassay method for agricultural product pesticide residue is an urgent research problem to be solved, and the enhancement of the triazophos detection methodology research has important significance for guaranteeing food safety and agricultural product trade in China.

To establish an immunoassay method, it is first necessary to prepare an antibody having a high affinity for the specificity of a pesticide. The molecular weight of the general pesticide small molecular compound is less than 1000 daltons, and the general pesticide small molecular compound is a small molecular hapten. Pesticide small molecule compounds are generally not immunogenic and do not stimulate an immune response in an animal to produce antibodies. Therefore, the antibody must be coupled with a macromolecular carrier to be immunogenic, namely, the antibody can perform specific binding reaction with a corresponding antibody. The pesticide hapten molecule must have some active groups (-NH2, -COOH, -OH, -SH, etc.) that can be covalently bound to the carrier. The hapten has chemical characteristics similar to those of the substance to be detected so as to reduce cross contamination and ensure that the characteristic structure of the macromolecular compound of the pesticide hapten and the carrier can be easily identified by immunocompetent cells. According to the biotechnological means such as organic synthesis, biochemistry and immunology, an ideal hapten with an active group is designed and synthesized.

If an antibody with high specificity and affinity in immune response is obtained, it is first provided with an artificial antigen with high immunogenicity and specificity. Firstly, hapten which retains the molecular structure of pesticide and has active groups is synthesized, and secondly, the hapten reacts with carrier protein (such as albumin, egg protein) and the like through the active groups (-NH2, -COOH, -OH, -SH and the like) to prepare the antigen. After the pesticide artificial antigen is successfully synthesized, the pesticide artificial antigen usually needs dialysis, column chromatography and other methods for purifying the pesticide artificial antigen.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method of a hapten of an organophosphorus triazophos pesticide, which takes 1-phenyl-3-hydroxy-1, 2, 4-triazole as a raw material and synthesizes the hapten of the organophosphorus triazophos pesticide through nitration, substitution, reduction and reaction by introducing carboxyl active groups, so as to be beneficial to preparing an antibody with high specificity and affinity to the organophosphorus pesticide.

The invention also aims to provide the organophosphorus triazophos pesticide hapten.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a method for preparing hapten of organophosphorus triazophos pesticide comprises the following steps:

(a) 1-phenyl-3-hydroxy-1, 2, 4-triazole is used as a raw material, and the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole is obtained through nitration reaction;

(b) carrying out substitution reaction on the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole and diethyl thiophosphoryl chloride obtained in the step (a) to obtain diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate;

(c) performing nitro reduction reaction on the diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate obtained in the step (b) to obtain diethyl (1-p-aminobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate;

(d) and (c) mixing diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate obtained in the step (c) with succinic anhydride for reaction to obtain triazophos 4- (4- (3-diethyl thiophosphate-1, 2, 4-triazole) -anilino) -4-oxobutyric acid.

Preferably, the nitration reaction comprises the steps of:

dropwise adding fuming nitric acid into a mixture of the 1-phenyl-3-hydroxy-1, 2, 4-triazole and concentrated sulfuric acid, stirring for 4.5-5.5 h at 0-5 ℃ after dropwise adding, and cooling.

Preferably, the dosage ratio of the 1-phenyl-3-hydroxy-1, 2, 4-triazole, the concentrated sulfuric acid and the fuming nitric acid is (5.5-6.5) g, (45-55) mL and (2.2-2.8) g.

Preferably, the substitution reaction comprises the steps of:

stirring and reacting the mixture of the diethyl thiophosphoryl chloride, the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole, the acid-binding agent, the 4-dimethylamino pyridine and the organic solvent at 65-75 ℃ for 1.8-2.2 h;

preferably, the acid-binding agent is triethylamine;

preferably, the organic solvent is dichloroethane;

preferably, the mixture after the stirring reaction is subjected to a post-treatment, which comprises the following steps: and cooling the mixture subjected to the stirring reaction, mixing the mixture with water for liquid separation, extracting the water phase with ethyl acetate for 1-2 times, washing the organic phase with saturated saline solution, concentrating, and purifying by column chromatography.

Preferably, the dosage ratio of the triethylamine, the 4-dimethylaminopyridine, the diethylthiophosphoryl chloride, the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole and the dichloroethane is (3.5-4), (0.28-0.32), (5.8-6.2), (4.8-5.2) and (48-55) mL.

Preferably, the nitro reduction reaction comprises the following steps:

stirring and reacting the mixture of diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate, ammonium chloride, Fe powder, water and ethanol at 78-82 ℃ for 55-65 min;

preferably, the mixture after the stirring reaction is subjected to a post-treatment, which comprises the following steps: and cooling and filtering the mixture after the stirring reaction, concentrating the filtrate to remove ethanol, mixing the filtrate with water, extracting the water phase with ethyl acetate for 2-3 times, washing the organic phase with saturated saline solution, concentrating, and purifying by column chromatography.

Preferably, the using amount ratio of diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate to ammonium chloride to Fe powder to water is (5.8-6.2) g, (0.28-0.32) g, (3.5-4) g, (8-12) mL.

Preferably, in step (d), the mixing reaction comprises the steps of:

reacting the diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate and the succinic anhydride in a solvent at 55-65 ℃ for 55-65 min;

preferably, the solvent is pyridine;

preferably, in step (d), the method further comprises post-treating the reacted mixture, wherein the post-treatment comprises the following steps:

and cooling and concentrating the reacted mixture, mixing the concentrated mixture with water, extracting the water phase with ethyl acetate for 2-3 times, washing the organic phase with saturated saline solution, concentrating, and purifying by column chromatography.

Preferably, the diethyl (1-p-aminobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate, the succinic anhydride and the pyridine are used in a ratio of (0.8-1.2) g to (0.3-0.4) g to (18-22) mL.

The organophosphorus triazophos pesticide hapten has the following structure:

compared with the prior art, the invention has the beneficial effects that:

the hapten of the organophosphorus triazophos pesticide is synthesized by taking 1-phenyl-3-hydroxy-1, 2, 4-triazole as a raw material through nitration, substitution, reduction and reaction of introducing carboxyl active groups, so that the preparation of the antibody with high specificity and affinity to the organophosphorus pesticide is facilitated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a liquid chromatography-mass spectrometry diagram of a 1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole compound in example 1 of the present invention;

FIG. 2 is a liquid mass spectrum of a diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) phosphorothioate compound in example 1 of the present invention;

FIG. 3 is a liquid mass spectrum of a diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate compound in example 1 of the present invention;

FIG. 4 is a liquid mass spectrum of triazophos hapten in example 1 of the present invention;

FIG. 5 is a nuclear magnetic spectrum of triazophos hapten in example 1 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

According to one aspect of the invention, the invention relates to a preparation method of a hapten of an organophosphorus triazophos pesticide, which comprises the following steps:

a method for preparing hapten of organophosphorus triazophos pesticide comprises the following steps:

(a) 1-phenyl-3-hydroxy-1, 2, 4-triazole is used as a raw material, and the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole is obtained through nitration reaction;

(b) carrying out substitution reaction on the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole and diethyl thiophosphoryl chloride obtained in the step (a) to obtain diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate;

(c) performing nitro reduction reaction on the diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate obtained in the step (b) to obtain diethyl (1-p-aminobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate;

(d) and (c) mixing diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate obtained in the step (c) with succinic anhydride for reaction to obtain triazophos 4- (4- (3-diethyl thiophosphate-1, 2, 4-triazole) -anilino) -4-oxobutyric acid.

To enhance the immunological recognition of an antibody to a hapten during an immune reaction, it is often necessary to design a spacer (or linker molecule) of 4-6 carbons between the hapten and the carrier protein to reduce steric hindrance of the interaction between the antibody and the hapten. Spacer walls should be avoided as much as possible around the functional groups attached to the target molecule, optimally located distal to the important characteristic functional groups, otherwise the recognition and binding sites of the antibody and antigen will be reduced. In addition, some active groups, such as-NH, need to be artificially and chemically modified for some haptens without reactive groups₂-COOH, -OH, -SH, etc. In the process of designing and synthesizing the pesticide hapten, the molecular structure of the pesticide is retained to the greatest extent for enhancing the specificity and affinity of the antibody and the antigen, so that the chemical structure and the electron distribution of the pesticide molecule are hardly influenced, and the guarantee is provided for obtaining the antibody with high specificity to the pesticide.

The invention relates to a hapten method for synthesizing organophosphorus triazophos pesticide by taking 1-phenyl-3-hydroxy-1, 2, 4-triazole as a raw material and reacting through nitration, substitution, reduction, introduction of carboxyl active groups and the like, which is favorable for preparing an antibody with high specificity and affinity to organophosphorus pesticide.

Preferably, the nitration reaction comprises the steps of:

dropwise adding fuming nitric acid into a mixture of the 1-phenyl-3-hydroxy-1, 2, 4-triazole and concentrated sulfuric acid, stirring for 4.5-5.5 h at 0-5 ℃ after dropwise adding, and cooling.

In one embodiment, the mixture is stirred at 0-5 ℃ for 4.5-5.5 h after the dropwise addition, and optionally 4.5h, 4.6h, 4.7h, 4.8h, 4.9h, 5h, 5.1h, 5.2h, 5.3h, 5.4h or 5.5 h.

Preferably, the dosage ratio of the 1-phenyl-3-hydroxy-1, 2, 4-triazole, the concentrated sulfuric acid and the fuming nitric acid is (5.5-6.5) g, (45-55) mL and (2.2-2.8) g.

Preferably, the 1-phenyl-3-hydroxy-1, 2, 4-triazole, concentrated sulfuric acid and fuming nitric acid are used in a ratio of 6g to 50mL to 2.6 g.

Preferably, the substitution reaction comprises the steps of:

and stirring the mixture of the diethyl thiophosphoryl chloride, the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole, the acid-binding agent, the 4-dimethylamino pyridine and the organic solvent at 65-75 ℃ for reaction for 1.8-2.2 h.

In one embodiment, the temperature of the stirring reaction of the mixture of the diethylthiophosphoryl chloride, the 1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole, the acid-binding agent, the 4-dimethylaminopyridine and the organic solvent is 65 to 75 ℃, and may be 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃.

In one embodiment, the mixture of the diethylthiophosphoryl chloride, the 1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole, the acid-binding agent, the 4-dimethylaminopyridine and the organic solvent is stirred for a reaction time of 1.8h, 1.9h, 2h, 2.1h or 2.2 h.

Preferably, the acid scavenger is triethylamine.

Preferably, the organic solvent is dichloroethane.

Preferably, the mixture after the stirring reaction is subjected to a post-treatment, which comprises the following steps: and cooling the mixture subjected to the stirring reaction, mixing the mixture with water for liquid separation, extracting the water phase with ethyl acetate for 1-2 times, washing the organic phase with saturated saline solution, concentrating, and purifying by column chromatography.

Preferably, the dosage ratio of the triethylamine, the 4-dimethylaminopyridine, the diethylthiophosphoryl chloride, the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole and the dichloroethane is (3.5-4), (0.28-0.32), (5.8-6.2), (4.8-5.2) and (48-55) mL.

Preferably, the triethylamine, 4-dimethylaminopyridine, diethylthiophosphoryl chloride, 1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole and dichloroethane are used in a ratio of 3.7g:0.3g:6g:5g:50 mL.

Preferably, the nitro reduction reaction comprises the following steps:

and (2) stirring and reacting the mixture of diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate, ammonium chloride, Fe powder, water and ethanol at 78-82 ℃ for 55-65 min.

In one embodiment, the temperature for stirring and reacting the mixture of diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate, ammonium chloride, Fe powder, water and ethanol is 78-82 ℃, and may be 78 ℃, 79 ℃, 80 ℃, 81 ℃ or 82 ℃.

In one embodiment, the stirring reaction time of the mixture of diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate, ammonium chloride, Fe powder, water and ethanol is 55-65 min, and can be 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min or 65 min.

Preferably, the mixture after the stirring reaction is subjected to a post-treatment, which comprises the following steps: and cooling and filtering the mixture after the stirring reaction, concentrating the filtrate to remove ethanol, mixing the filtrate with water, extracting the water phase with ethyl acetate for 2-3 times, washing the organic phase with saturated saline solution, concentrating, and purifying by column chromatography.

Preferably, the using amount ratio of diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate to ammonium chloride to Fe powder to water is (5.8-6.2) g, (0.28-0.32) g, (3.5-4) g, (8-12) mL.

Preferably, the diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate, the ammonium chloride, the Fe powder and the water are used in a ratio of 6g:0.3g:3.7g:10 mL.

Preferably, in step (d), the mixing reaction comprises the steps of:

reacting the diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate and the succinic anhydride in a solvent at 55-65 ℃ for 55-65 min.

Preferably, the solvent is pyridine.

In one embodiment, the reaction temperature is 55-65 ℃, and 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃ or 65 ℃ can be selected.

In one embodiment, the reaction time is 55-65 min, and can be 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min or 65 min.

Preferably, in step (d), the method further comprises post-treating the reacted mixture, wherein the post-treatment comprises the following steps:

and cooling and concentrating the reacted mixture, mixing the concentrated mixture with water, extracting the water phase with ethyl acetate for 2-3 times, washing the organic phase with saturated saline solution, concentrating, and purifying by column chromatography.

Preferably, the diethyl (1-p-aminobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate, the succinic anhydride and the pyridine are used in a ratio of (0.8-1.2) g to (0.3-0.4) g to (18-22) mL.

Preferably, the diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate, succinic anhydride and pyridine are used in a ratio of 1g:0.36g:20 mL.

The synthesis route of the organophosphorus triazophos pesticide hapten is as follows:

according to another aspect of the invention, the invention relates to an organophosphorus triazophos pesticide hapten which has the following structure:

the invention will be further explained with reference to specific examples.

Example 1

A preparation method of organophosphorus triazophos pesticide hapten comprises the following steps:

(a) adding 6.0g of 1-phenyl-3-hydroxy-1, 2, 4-triazole into 50mL of concentrated sulfuric acid, dropwise adding 2.6g of fuming nitric acid into the reaction solution at 0 ℃, and stirring the reaction solution for 5 hours at 0 ℃ after dropwise adding; LCMS detection of figure 1 shows complete consumption of starting material with peak product formation; pouring the 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole reaction solution into ice water, firstly filtering precipitated solid, pulping with ethyl acetate, and then filtering to obtain a light yellow solid 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole compound; 5.0g of 1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole compound, yield 65%;

(b)5.0g of 1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole, 3.7g of triethylamine (Et)₃N), 0.3g of 4-Dimethylaminopyridine (DMAP) and 6.0g of diethylthiophosphoryl chloride were sequentially added to 50mL of Dichloroethane (DCE), and the reaction mixture was stirred at 70 ℃ for 2 hours; LCMS detection of fig. 2 shows complete consumption of starting material with a major product peak; after cooling the diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) phosphorothioate reaction solution, adding 30mL of water for liquid separation, extracting the aqueous phase once with 50mL of ethyl acetate, washing the organic phase with 50mL of saturated saline solution, and concentrating; purifying by column chromatography, and concentrating to obtain light yellow solid diethyl (1-p-nitrobenzene-3-hydroxy-1, 2, 4-triazole) thiophosphate compound; 6.0g of diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) phosphorothioate compound, the yield was 69%;

(c)6.0g diethyl (1-p-nitrophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate, 3.7g Fe powder, 0.3g NH4Cl and 10mL of water were sequentially added to ethanol, and stirred at 80 ℃ for 1 hour; LCMS detection of fig. 3 shows complete consumption of starting material with a major product peak; cooling diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate reaction liquid, filtering, concentrating the filtrate to remove ethanol, adding 20mL of water, extracting the water phase with 50mL of ethyl acetate for three times, washing 50mL of organic phase with saturated saline solution, and concentrating; purifying by column chromatography, and concentrating to obtain yellow solid diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate compound; diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) phosphorothioate compound was 4.0g, and the yield was 73%;

(d)1.0g diethyl (1-p-aminophenyl-3-hydroxy-1, 2, 4-triazole) thiophosphate and 0.36g succinic anhydride were added to 20mL pyridine, and reacted at 60 ℃ for 1 hour; LCMS detection of fig. 4 shows complete consumption of starting material with a major product peak; cooling 4- (4- (3-diethyl thiophosphate-1, 2, 4-triazole) -anilino) -4-oxobutyric acid, concentrating to remove pyridine, adding 20mL of water, extracting the water phase with 50mL of ethyl acetate for three times, washing 50mL of the organic phase with saturated saline solution, and concentrating; after purification by column chromatography and concentration, a pale yellow solid, triazophos 4- (4- (3-diethyl phosphorothioate-1, 2, 4-triazole) -anilino) -4-oxobutanoic acid compound, 0.78g of triazophos 4- (4- (3-diethyl phosphorothioate-1, 2, 4-triazole) -anilino) -4-oxobutanoic acid compound, was obtained in 60% yield.

Test examples

The nuclear magnetic spectrum of the triazophos hapten of the invention is shown in figure 5,¹H NMR(500MHz,DMSO-d₆)δ12.17(s,1H),10.23(s,1H),9.10(s,1H),7.80-7.70(m,4H),4.38-4.26(m,4H),2.61(t,J＝6.0Hz,2H),2.56(d,J＝6.0Hz,2H),1.36(t,J＝7.0Hz,6H)。

finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

Translated fromChinese

1.一种有机磷类三唑磷农药的半抗原的制备方法，其特征在于，包括以下步骤：1. a preparation method of the hapten of an organophosphorus triazophos pesticide, is characterized in that, comprises the following steps:(a)以1-苯基-3-羟基-1,2,4-三唑为原料，通过硝化反应得到1-对硝基苯-3-羟基-1,2,4-三唑；(a) Using 1-phenyl-3-hydroxy-1,2,4-triazole as raw material, 1-p-nitrobenzene-3-hydroxy-1,2,4-triazole is obtained through nitration reaction;(b)将步骤(a)得到的1-对硝基苯-3-羟基-1,2,4-三唑和二乙基硫代磷酰氯进行取代反应，得到二乙基(1-对硝基苯-3-羟基-1,2,4-三唑)硫代磷酸酯；(b) subjecting the 1-p-nitrobenzene-3-hydroxy-1,2,4-triazole obtained in step (a) to a substitution reaction with diethyl phosphorothioate to obtain diethyl (1-p-nitrobenzene) phenyl-3-hydroxy-1,2,4-triazole) phosphorothioate;(c)将步骤(b)得到的二乙基(1-对硝基苯-3-羟基-1,2,4-三唑)硫代磷酸酯进行硝基还原反应，得到二乙基(1-对氨基苯-3-羟基-1,2,4-三唑)硫代磷酸酯；(c) subjecting the diethyl (1-p-nitrobenzene-3-hydroxy-1,2,4-triazole) phosphorothioate obtained in step (b) to a nitro reduction reaction to obtain diethyl (1 - p-aminobenzene-3-hydroxy-1,2,4-triazole) phosphorothioate;(d)将步骤(c)得到的二乙基(1-对氨基苯-3-羟基-1,2,4-三唑)硫代磷酸酯和丁二酸酐混合反应，得到三唑磷4-(4-(3-二乙基硫代磷酸酯-1,2,4-三唑)-苯胺基)-4-氧代丁酸。(d) mixing the diethyl (1-p-aminobenzene-3-hydroxy-1,2,4-triazole) phosphorothioate obtained in step (c) with succinic anhydride to obtain triazophos 4- (4-(3-Diethylphosphorothioate-1,2,4-triazole)-anilino)-4-oxobutanoic acid.2.根据权利要求1所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，所述硝化反应包括以下步骤：2. the preparation method of the hapten of organophosphorus triazophos pesticide according to claim 1, is characterized in that, described nitration reaction comprises the following steps:向1-苯基-3-羟基-1,2,4-三唑和浓硫酸的混合物中滴加发烟硝酸，滴加后于0～5℃搅拌4.5～5.5h，冷却。Fuming nitric acid is added dropwise to the mixture of 1-phenyl-3-hydroxy-1,2,4-triazole and concentrated sulfuric acid, and after the dropwise addition, the mixture is stirred at 0 to 5° C. for 4.5 to 5.5 hours, and cooled.3.根据权利要求2所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，所述1-苯基-3-羟基-1,2,4-三唑、浓硫酸和发烟硝酸的用量比例为(5.5～6.5)g:(45～55)mL:(2.2～2.8)g。3. The preparation method of the hapten of organophosphorus triazophos pesticide according to claim 2, wherein the 1-phenyl-3-hydroxy-1,2,4-triazole, concentrated sulfuric acid and The dosage ratio of fuming nitric acid is (5.5-6.5) g: (45-55) mL: (2.2-2.8) g.4.根据权利要求1所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，所述取代反应包括以下步骤：4. the preparation method of the hapten of organophosphorus triazophos pesticide according to claim 1, is characterized in that, described substitution reaction comprises the following steps:将所述二乙基硫代磷酰氯、所述1-对硝基苯-3-羟基-1,2,4-三唑、缚酸剂、4-二甲氨基吡啶和有机溶剂的混合物于65～75℃搅拌反应1.8～2.2h；The mixture of the diethyl phosphorothioate chloride, the 1-p-nitrobenzene-3-hydroxy-1,2,4-triazole, the acid binding agent, 4-dimethylaminopyridine and the organic solvent was dissolved in 65 ～75℃ stirring reaction for 1.8～2.2h;优选地，所述缚酸剂为三乙胺；Preferably, the acid binding agent is triethylamine;优选地，所述有机溶剂为二氯乙烷；Preferably, the organic solvent is dichloroethane;优选地，对所述搅拌反应后的混合物进行后处理，所述后处理包括以下步骤：将所述搅拌反应的混合物冷却，与水混合进行分液，水相用乙酸乙酯萃取1～2次，有机相用饱和食盐水洗涤、浓缩，再经过柱层析纯化。Preferably, post-processing is performed on the mixture after the stirring reaction, and the post-processing includes the following steps: cooling the stirring reaction mixture, mixing with water for liquid separation, and extracting the aqueous phase with ethyl acetate for 1-2 times , the organic phase was washed with saturated brine, concentrated, and purified by column chromatography.5.根据权利要求4所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，所述三乙胺、4-二甲氨基吡啶、二乙基硫代磷酰氯、1-对硝基苯-3-羟基-1,2,4-三唑与二氯乙烷的用量比例为(3.5～4)g:(0.28～0.32)g:(5.8～6.2)g:(4.8～5.2)g:(48～55)mL。5. the preparation method of the hapten of organophosphorus triazophos pesticide according to claim 4, is characterized in that, described triethylamine, 4-dimethylaminopyridine, diethyl phosphorothioate chloride, 1- The dosage ratio of p-nitrobenzene-3-hydroxy-1,2,4-triazole to dichloroethane is (3.5～4)g:(0.28～0.32)g:(5.8～6.2)g:(4.8～ 5.2) g: (48-55) mL.6.根据权利要求1所述的有机磷类三唑磷农药半抗原的制备方法，其特征在于，所述硝基还原反应包括以下步骤：6. the preparation method of organophosphorus triazophos pesticide hapten according to claim 1, is characterized in that, described nitro group reduction reaction comprises the following steps:将所述二乙基(1-对硝基苯-3-羟基-1,2,4-三唑)硫代磷酸酯、氯化铵、Fe粉、水和乙醇的混合物于78～82℃搅拌反应55～65min；The mixture of diethyl (1-p-nitrobenzene-3-hydroxy-1,2,4-triazole) phosphorothioate, ammonium chloride, Fe powder, water and ethanol was stirred at 78～82°C Reaction 55～65min;优选地，对所述搅拌反应后的混合物进行后处理，所述后处理包括以下步骤：将所述搅拌反应后的混合物冷却、过滤，滤液浓缩去除乙醇后与水混合，水相用乙酸乙酯萃取2～3次，有机相用饱和食盐水洗涤、浓缩，再经过柱层析纯化。Preferably, post-processing is performed on the mixture after the stirring reaction, and the post-processing includes the following steps: cooling and filtering the mixture after the stirring reaction, concentrating the filtrate to remove ethanol and mixing with water, and using ethyl acetate for the water phase After 2-3 extractions, the organic phase was washed with saturated brine, concentrated, and purified by column chromatography.7.根据权利要求6所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，所述二乙基(1-对硝基苯-3-羟基-1,2,4-三唑)硫代磷酸酯、氯化铵、Fe粉和水的用量比例为(5.8～6.2)g:(0.28～0.32)g:(3.5～4)g:(8～12)mL。7. The preparation method of the hapten of organophosphorus triazophos pesticide according to claim 6, wherein the diethyl (1-p-nitrobenzene-3-hydroxy-1,2,4- The dosage ratio of triazole) phosphorothioate, ammonium chloride, Fe powder and water is (5.8-6.2) g: (0.28-0.32) g: (3.5-4) g: (8-12) mL.8.根据权利要求1所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，在步骤(d)中，所述混合反应包括以下步骤：8. the preparation method of the hapten of organophosphorus triazophos pesticide according to claim 1, is characterized in that, in step (d), described mixing reaction comprises the following steps:将所述二乙基(1-对氨基苯-3-羟基-1,2,4-三唑)硫代磷酸酯、所述丁二酸酐在溶剂中于55～65℃反应55～65min；The diethyl (1-p-aminobenzene-3-hydroxy-1,2,4-triazole) phosphorothioate and the succinic anhydride are reacted in a solvent at 55-65° C. for 55-65 minutes;优选地，所述溶剂为吡啶；Preferably, the solvent is pyridine;优选地，在步骤(d)中，还包括对反应后的混合物进行后处理，所述后处理包括以下步骤：Preferably, in step (d), it also includes post-processing the reacted mixture, and the post-processing comprises the following steps:将所述反应后的混合物经过冷却后浓缩，再与水混合，水相用乙酸乙酯萃取2～3次，有机相用饱和食盐水洗涤、浓缩，再经过柱层析纯化。The reacted mixture was cooled, concentrated, and mixed with water. The aqueous phase was extracted with ethyl acetate for 2 to 3 times. The organic phase was washed with saturated brine, concentrated, and purified by column chromatography.9.根据权利要求8所述的有机磷类三唑磷农药的半抗原的制备方法，其特征在于，所述二乙基(1-对氨基苯-3-羟基-1,2,4-三唑)硫代磷酸酯、丁二酸酐和吡啶的用量比例为(0.8～1.2)g:(0.3～0.4)g:(18～22)mL。9 . The preparation method of the hapten of organophosphorus triazophos pesticides according to claim 8 , wherein the diethyl (1-p-aminobenzene-3-hydroxy-1,2,4-triethyl) azole) phosphorothioate, succinic anhydride and pyridine in a ratio of (0.8-1.2) g: (0.3-0.4) g: (18-22) mL.10.有机磷类三唑磷农药半抗原，其特征在于，具有如下结构：10. Organophosphorus triazophos pesticide hapten, characterized in that it has the following structure:

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