CN115611834A

CN115611834A - Carbofuran hapten and synthesis process thereof

Info

Publication number: CN115611834A
Application number: CN202211636027.0A
Authority: CN
Inventors: 吕俊海
Original assignee: EXPERIMENTAL RESEARCH CENTER CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
Current assignee: EXPERIMENTAL RESEARCH CENTER CHINA ACADEMY OF CHINESE MEDICAL SCIENCES
Priority date: 2022-12-20
Filing date: 2022-12-20
Publication date: 2023-01-17

Abstract

The invention relates to carbofuran hapten and a synthesis process thereof. The synthesis process comprises the following steps: mixing carbofuran and dianhydride in a solvent, adding a catalyst for reaction, and obtaining a mixed reaction solution after the reaction is finished; drying the mixed reaction liquid, and extracting with ultrapure water to obtain a first water phase; and extracting the first water phase by using dichloromethane to obtain a second water phase, and drying to obtain the carbofuran hapten. Compared with the prior art, the synthesis process only takes carbofuran and dianhydride as raw materials, and has the advantages of less reaction reagent types and dosage, simple synthesis process (only one-step reaction), low reaction condition requirements (which can be carried out in a common EP tube, the reaction can be carried out within the range of 4 to 80 ℃, backflow and the like are not needed), simple product separation and purification (only 2 simple extraction steps are needed), and the synthesized hapten furthest retains the structure (such as free methyl) of carbofuran molecules, so that the accuracy of subsequent application detection is improved.

Description

Carbofuran hapten and synthesis process thereof

Technical Field

The invention belongs to the field of pesticide residue detection, and particularly relates to carbofuran hapten and a synthesis process thereof.

Background

With the rapid development of economy in China, the demand of people on health is higher and higher, and the rapid development of the traditional Chinese medicine industry is promoted, so that the demand of traditional Chinese medicine resources is increased day by day. The artificial planting of the traditional Chinese medicinal materials is an effective way for realizing the regeneration and continuous utilization of traditional Chinese medicine resources, but on the other hand, in the global plant medicine trade, the traditional Chinese medicinal material export proportion in China is very small, the medicine quality is the primary factor influencing the traditional Chinese medicine export in China, the pesticide residue is an important factor influencing the medicine quality, and the traditional Chinese medicine pesticide residue problem has become the bottleneck of the traditional Chinese medicine export, so the pesticide residue pollution becomes an important problem to be solved urgently in the traditional Chinese medicinal material production. Therefore, related regulations for detecting pesticide residues of the traditional Chinese medicines are also made in 2020 edition of Chinese pharmacopoeia; the pesticide residue needs to be inspected for 33 pesticide residues, and carbofuran is one of the 33 pesticide residues.

Carbofuran (CAR) belongs to carbamate pesticides, has a chemical name of 2,3-dihydro-2,2-dimethyl-7-benzofuranyl-N-methyl carbamate, has a trade name of Carbofuran, carbofuran and the like, and belongs to a high-toxicity pesticide. Was developed and produced by FMC corporation and Mobay chemical corporation in 1969. It is used as broad-spectrum pesticide and nematocide in agricultural production, and has the functions of shortening the growth period of crop and promoting the growth and development of crop to raise the yield of crop effectively. But the action mechanism of carbofuran is irreversibly combined with cholinesterase, so that acetylcholine cannot be decomposed and accumulated in the body, nerve center conduction is influenced, and toxicity to human and wild animals is high. Meanwhile, the fertilizer is difficult to degrade in acid soil, is extremely easy to pollute soil and underground water sources, and brings potential threats to the safety and environment of agricultural products including planting traditional Chinese medicinal materials. Therefore, besides enhancing the use management of carbofuran, the detection and supervision of carbofuran should be enhanced while the carbofuran is controlled from the source.

At present, the methods for detecting carbofuran residues mainly comprise High Performance Liquid Chromatography (HPLC), gas Chromatography (GC), liquid-mass spectrometry (HPLC-MS) and the like. Although the detection methods have good stability and repeatability, high accuracy and high sensitivity; but the method needs complex, precise and expensive instruments and equipment, has high use cost, multiple detection steps, long time, high technical requirement on operators, high requirement on sample pretreatment, environmental friendliness, unsuitability for field batch detection and the like; thereby limiting the application of the methods to large-scale detection of medicinal plants or field detection of various links from production to application. Therefore, the method has important significance in establishing a technical method which is simple, rapid, sensitive, cheap and suitable for large-scale detection in fields and laboratories.

Immunoassay based on antigen-antibody specific binding reaction has these advantages, and common methods include ELISA, radioimmunoassay, and lateral chromatography rapid detection test paper. Although immunoassay is used for pesticide residue analysis in a short time, immunoassay has the advantages of high detection speed, low cost, low sample requirement and the like, and can be used for on-site rapid detection in various links such as planting, harvesting, production, processing, sale and the like of traditional Chinese medicinal materials, so that immunoassay has great promoting significance for improving the quality of the traditional Chinese medicinal materials, ensuring the health of patients and breaking through the bottleneck of traditional Chinese medicine export.

Immunoassays are premised on the preparation of appropriate antigens and specific antibodies. The carbofuran has a molecular weight of 221, is a typical small molecular compound, is a hapten, does not contain a functional group for coupling, must be subjected to molecular modification or hapten synthesis to obtain a carbofuran molecular modification product or an analogue hapten with a coupling group (usually carboxyl or amino, and more usually carboxyl), and then is coupled with a carrier protein by a covalent bond to form a complete antigen for antibody preparation and subsequent immunoassay.

The existing methods for modifying carbofuran molecules (namely synthesizing carbofuran haptens) mainly comprise the following steps:

1. the carbofuran structural analog with hexanoic acid group or butyric acid group, such as 6- [ [ (2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl ] amino ] hexanoic acid, 4- [ [ (2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl ] amino ] butyric acid, is prepared by one-step synthesis from basic raw materials by adopting a process similar to chemical synthesis of carbofuran.

The synthesis process of the patent with the publication number of CN105399711.B is as follows:

s1, dissolving 1.64g of furan phenol in 50mL of dichloromethane, adding 1.11mg of triethylamine under ice bath, stirring for 10 minutes, dissolving 3.04g of bis (p-nitrophenyl) carbonate in 15mL of dichloromethane, slowly and dropwise adding the solution into the reaction system, gradually heating to 30 ℃, continuing stirring for reaction for 1 hour, and monitoring the completion of the reaction by a TLC plate, wherein a developing agent used is petroleum ether, namely ethyl acetate = 10;

s2, weighing 4-aminobutyl methyl ester hydrochloride which is in equimolar amount with the furan phenol and is alkalized by triethylamine, dissolving the 4-aminobutyl methyl ester hydrochloride in 10mL dichloromethane, adding the dichloromethane into the reaction solution under the ice bath condition, heating the temperature to room temperature, and continuously stirring the mixture overnight; washing with 50mL of saturated saline solution for 2 times; drying the reaction product by using anhydrous sodium sulfate, filtering to remove a drying agent, drying the filtrate in vacuum by spinning, and purifying by using 200-300-mesh silica gel column chromatography to obtain 1.8g of a hapten intermediate, wherein an eluant is petroleum ether, ethyl acetate =10 and the yield is 58%.

S3, dissolving the hapten intermediate obtained in the step S2 into 15mL1, 4-dioxane, adding 18mL of trifluoroacetic acid and 15mL of water, heating at 60 ℃ for reaction for 4 hours, and cooling to room temperature; extracting with 30mL ethyl acetate for 2 times, mixing organic phases, and sequentially washing with 30mL water, saturated sodium bicarbonate, water, and saturated saline solution for 2 times; drying with anhydrous sodium sulfate, filtering to remove desiccant, vacuum drying the filtrate, and purifying with 200-300 mesh silica gel column chromatography to obtain hapten 0.73g, eluent is petroleum ether, ethyl acetate =5:1, and yield is 42%.

However, the disadvantages of this approach are: firstly, the structure lacks a methyl group of the carbofuran molecule, and the deletion of the methyl group has potential adverse effects on the conditions of antigen space structure, antibody recognition and combination, cross reaction of the prepared detection reagent with other similar pesticides and the like; secondly, the synthesis steps are multiple, the process is complex, the requirements on reaction conditions and devices are relatively high, the purification process of the product is relatively complex, and the like, so that the amount of the chemical reagent used in the reaction is relatively large. It is difficult for non-chemical laboratories or production units to implement.

2. 2-n-butylaminopropionic acid is used as a starting material, and is vulcanized and chlorinated to react with 3-hydroxy carbofuran to obtain the structural analogue with carboxyl and based on 3-hydroxy carbofuran. The molecule structurally retains the original methyl of carbofuran, but hydroxyl is added on a furan ring, and atoms such as S, N and the like and another 4-carbon side chain are introduced in the added carboxyl chain, so that the difference of the molecular structure compared with the carbofuran is larger than that of a product obtained by the former method, the possibility of influencing the structure of an epitope of the carbofuran is greatly increased, and the difficulty of combining an antibody with a main body structure of carbofuran is also increased; meanwhile, the synthesis reaction also has the same disadvantages as the method 1.

Therefore, the technical scheme of the invention is provided.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides carbofuran hapten and a synthesis process thereof. The synthesis process only uses carbofuran and dianhydride as raw materials, and compared with the prior art, the synthesis process has the advantages of less reaction reagent types and dosage, simple synthesis process (only one-step reaction), low reaction condition requirement (the reaction can be carried out in a common EP tube, the reaction can be carried out within the range of 4 to 80 ℃, backflow and the like are not needed), simple product separation and purification (only 2 simple steps of extraction are needed), and the synthesized hapten furthest retains the structure (such as free methyl) of carbofuran molecules, so that the accuracy of subsequent application detection is improved.

The scheme of the invention provides a synthesis process of carbofuran hapten, which comprises the following steps:

(1) Mixing carbofuran and dianhydride in a solvent, adding a catalyst for reaction, and obtaining a mixed reaction solution after the reaction is finished;

(2) Drying the mixed reaction liquid, and extracting with ultrapure water to obtain a first water phase;

(3) And extracting the first water phase by using dichloromethane to obtain a second water phase, and drying to obtain the carbofuran hapten.

Preferably, in step (1), the dianhydride is one of succinic anhydride, glutaric anhydride or adipic anhydride.

Preferably, in step (1), the solvent is pyridine.

Preferably, in step (1), the catalyst is one of 4-Dimethylaminopyridine (DMAP) or 4-pyrrolidinylpyridine (4-PPY).

Preferably, in the step (1), the temperature of the reaction is 4 to 80 ℃.

Preferably, the reaction temperature is 56 ℃ and the corresponding reaction time is 5h. The inventor verifies that when the reaction temperature is too low, the required reaction time is longer, namely the time cost is too high; at higher reaction temperatures, side reactions may increase and the stability of carbofuran may decrease. Therefore, 56 ℃ is most suitable.

Preferably, in the step (2), the mixed reaction solution is dried by nitrogen, and then extracted by precooled ultrapure water with the same volume at 4 ℃ to obtain the first water phase.

Preferably, in the step (3), the first aqueous phase is extracted by using dichloromethane with the same volume to obtain a second aqueous phase, and then the second aqueous phase is dried by using nitrogen to obtain the carbofuran hapten.

Based on the same technical concept, the invention further provides carbofuran hapten obtained by the synthesis process.

For the purpose of understanding the present invention, the reaction process of the present invention will be described:

in the process of preparing carbofuran hapten, carbofuran is taken as a basic raw material, dianhydride is taken as an active group donor (specifically, succinic anhydride, glutaric anhydride or adipic anhydride and the like can be selected), acid anhydride reacts with imino on carbofuran molecules to form amide bond for combination, meanwhile, the acid anhydride is subjected to ring opening, and the other end forms carboxyl. The reaction process is shown in figure 1. In subsequent application, the carboxyl on the carbofuran hapten molecule and the free amino on the carrier protein can react to form an amido bond through active ester reaction or other reactions to be connected into a whole. And can be further used for preparing antibodies of carbofuran and various immunoassays, such as lateral immunochromatography assay, ELISA, radioimmunoassay and the like.

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

1. the synthesis process disclosed by the invention is simple in reaction process and few in steps, only one step is needed for chemical reaction, and separation, purification, transfer and the like of intermediate products in multi-step reaction are avoided.

2. The reaction product is simple to separate and purify and only needs two steps of extraction.

3. The reaction condition is simple and mild, high temperature and high pressure are not needed, and the reaction can be carried out in a common EP tube.

4. The usage amount of the reaction raw materials is small.

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, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a diagram of the synthetic reaction of carbofuran hapten according to the invention.

FIG. 2 is a chromatogram of carbofuran hapten according to the invention.

FIG. 3 is a mass spectrum of carbofuran hapten according to the invention;

FIG. 4 is an electrophoretogram of denatured protein in which carbofuran is coupled to BSA and OVA to complete antigen.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1

The embodiment provides a synthesis process of carbofuran hapten, which comprises the following steps:

(1) Dissolving 0.5mmoL of carbofuran and 0.5mmoL of succinic anhydride in 500 mu L of pyridine, adding 0.1mmoL of catalyst DMAP, stirring or shaking for reaction for 5 hours at 56 ℃, and obtaining mixed reaction liquid (brown liquid) after the reaction is finished;

(2) Blowing the mixed reaction solution to dryness by using nitrogen, and extracting by using precooled ultrapure water with the same volume at 4 ℃ to obtain a first water phase;

(3) And extracting the first water phase by using dichloromethane with the same volume to obtain a second water phase, and drying by using nitrogen to obtain the carbofuran hapten.

Example 2

(1) Dissolving 0.5mmoL of carbofuran and 0.5mmoL of succinic anhydride in 500 mu L of pyridine, adding 0.1mmoL of catalyst 4-PPY, and stirring or shaking at 4 ℃ for reaction for 7d to obtain a mixed reaction liquid (brown liquid);

(2) Blowing the mixed reaction solution to be dry by adopting nitrogen, and extracting by adopting precooled ultrapure water with the same volume at 4 ℃ to obtain a first water phase;

Example 3

(1) Dissolving 0.5mmoL of carbofuran and 0.5mmoL of succinic anhydride in 500 mu L of pyridine, adding 0.1mmoL of catalyst DMAP, stirring or shaking for reaction for 3h at the temperature of 80 ℃, and obtaining mixed reaction liquid (brown liquid) after the reaction is finished;

Examination example

To verify the accuracy of the synthesis process of the invention, the following tests were performed for identification:

the identification method comprises the following steps: an appropriate amount of carbofuran hapten (i.e. the substance to be detected) obtained in example 1 is taken and detected by liquid chromatography-mass spectrometry.

Sample treatment: the test substance is dissolved in 1mL of methanol, and diluted 10 times with methanol to be tested.

Chromatographic conditions are as follows: ZORBAX SB-C18.1X 50mm,1.8-Micron, mobile phase, A phase 0.1% aqueous formic acid, B phase 0.1% acetonitrile formic acid, 0.3mL/min, gradient elution, 0-30min, 10% B-90% B; the sample size was 5. Mu.L.

Mass spectrum conditions: esl ion source, positive ion mode, atomization pressure 45Psi, dry gas (N) ₂ ) Flow rate of 12L.min ^-1 The temperature of the drying gas is 300 ℃, and the capillary voltage is 4000V; and (3) adopting a full scanning mode, wherein the scanning range m/z is 90-450.

Wherein, the detected chromatogram is shown in FIG. 2, and the mass spectrogram is shown in FIG. 3.

As can be seen from FIGS. 2 and 3, the peak time of the product chromatogram is 12.659 minutes, and the ion peak of the product molecule is 322.3 m/z (positive ion mode, hapten molecule binds to a hydrogen ion, mass spectrum apparent molecular weight is actual molecular weight plus 1). In addition, the expected molecular weight of the carbofuran hapten is 321 according to the reaction formula shown in fig. 1, so that the molecular weight detected by mass spectrometry is consistent with the expected molecular weight, which indicates that the carbofuran hapten is successfully prepared.

Application example

In order to test the application effect of the carbofuran hapten obtained by the synthesis process, the carbofuran complete antigen is further prepared by selecting the carbofuran hapten obtained in example 1.

The method comprises the following steps:

(S1) dissolving 0.1mmoL carbofuran hapten in 300 mu L DMF, adding equal molar DCC and NHS while stirring, continuously stirring for 6h in the dark, standing overnight at 4 ℃, centrifuging at 4 ℃ and 10000r/min, and taking supernatant to obtain active ester;

(S2) dissolving 36mg of Bovine Serum Albumin (BSA) carrier protein in 5mL of carbonate buffer solution with the pH value of 9.0, dropwise adding active ester under the condition of magnetic stirring, continuously stirring for 12h after completion, centrifuging to obtain supernatant, dialyzing with ultrapure water for 5 times, freeze-drying, and storing in a freezer at-80 ℃ to obtain the carbofuran complete antigen (marked as 'g-BSA').

The second method comprises the following steps:

(SS 1) dissolving 0.1mmoL carbofuran hapten in 300 mu L DMF, adding equal molar DCC and NHS while stirring, continuously stirring for 6h in the dark, standing overnight at 4 ℃, centrifuging at 4 ℃ and 10000r/min, and taking supernatant to obtain active ester;

(SS 2) dissolving 30mg of chicken Ovalbumin (OVA) carrier protein in 5mL of carbonate buffer solution with the pH value of 9.0, dropwise adding active ester under the condition of magnetic stirring, continuously stirring for 12h after the completion, centrifuging to obtain supernatant, dialyzing with ultrapure water for 5 times, freeze-drying, and storing in a freezer at-80 ℃ to obtain the carbofuran complete antigen. (denoted as "g-OVA").

Identification of carbofuran complete antigen:

the identification method comprises the following steps: 10. Mu.g of "g-BSA" and "g-OVA" were taken, respectively, and the corresponding carrier proteins BSA and OVA, and the protein molecular weight standard (marked as Marker) were taken. Denatured protein electrophoresis (SDS-PAGE) was performed using 12.5% separation gel, and after completion of the electrophoresis, the gel was stained with Coomassie Brilliant blue for 1 hour, decolorized with a decolorizing solution, and scanned with a gel imager for imaging, as shown in FIG. 4. From the gel image of fig. 4 it can be seen that: the migration speed of the complete antigen band is obviously lower than that of the corresponding carrier protein, and the molecular weight of the complete antigen formed after the coupling of the carrier protein and the hapten is proved to be larger than that of the carrier protein, so that the complete antigen is successfully prepared.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and shall cover the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A synthesis process of carbofuran hapten, which is characterized by comprising the following steps:

2. The process for synthesizing carbofuran hapten according to claim 1, wherein in the step (1), the dianhydride is one of succinic anhydride, glutaric anhydride or adipic anhydride.

3. The process for synthesizing carbofuran hapten according to claim 1, wherein in the step (1), the solvent is pyridine.

4. The process for synthesizing carbofuran hapten according to claim 1, wherein in the step (1), the catalyst is one of 4-dimethylamino pyridine and 4-pyrrolidinyl pyridine.

5. The process for synthesizing carbofuran hapten according to claim 1, wherein the reaction temperature in the step (1) is 4-80 ℃.

6. The process for synthesizing carbofuran hapten as claimed in claim 5, wherein the reaction temperature is 56 ℃, and the reaction time is 5h.

7. The process for synthesizing carbofuran hapten as claimed in claim 1, wherein in the step (2), the mixed reaction solution is dried by nitrogen, and then extracted by precooled ultrapure water with the same volume at 4 ℃ to obtain the first water phase.

8. The process for synthesizing carbofuran hapten as claimed in claim 1, wherein in the step (3), the first aqueous phase is extracted by dichloromethane with the same volume to obtain a second aqueous phase, and then the second aqueous phase is dried by nitrogen to obtain the carbofuran hapten.

9. Carbofuran hapten obtained by the synthesis process of claim 1~8.