CN113372328A - Synthetic method of imidacloprid artificial hapten - Google Patents
Synthetic method of imidacloprid artificial hapten Download PDFInfo
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- CN113372328A CN113372328A CN202110773955.0A CN202110773955A CN113372328A CN 113372328 A CN113372328 A CN 113372328A CN 202110773955 A CN202110773955 A CN 202110773955A CN 113372328 A CN113372328 A CN 113372328A
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- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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Abstract
The invention discloses a synthetic method of imidacloprid artificial hapten, which comprises the following steps: and (3) preparing the liquid A and the liquid B, mixing the liquid A and the liquid B, stirring and preserving heat under a heating condition, dissolving by using DDW after cooling, extracting for multiple times after adjusting the pH value, and drying to obtain the IMI hapten. The hapten preparation method provided by the invention has the effects of reducing the preparation risk and improving the concentration and titer of the hapten.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a synthetic method of an imidacloprid artificial hapten.
Background
IMI is a small molecule substance. It is only reactogenic but not immunogenic, and is only immunogenic after coupling to a carrier protein. Because IMI does not have an active group that is directly coupled to a carrier protein, the IMI molecule needs to be derivatized first, which depends mainly on 3 active sites in its molecular structure, respectively: α -Cl on the pyridine ring; imino H atom on imidazole ring and-NO2Reduction to-NH2. Among them, the hapten synthesis method of replacing alpha-Cl on pyridine ring by affinity substitution reaction method was used for synthesizing IMI artificial hapten at first and has the most extensive application. The substitution of the imino H atom on the imidazole ring is theoretically possible, but the practical feasibility is not high, since pyridine N is bound to acetylcholine receptorThe substitution of the necessary groups by other groups reduces the ability of the compound to bind to nachrs. To NO2Reduction to-NH2In this method, the antibody produced by the hapten has poor specificity, and the imidacloprid can hardly be recognized. The synthesis method of the hapten replacing alpha-Cl on a pyridine ring is the most mature method published by the Zhu national idea. Although this method is feasible and results in a final product, it also has disadvantages such as: high-risk chemical reagents are used in the test process, so that fire is easily caused, the purity of the hapten is not high, the titer of the serum antibody of the immunized mouse is unstable and not high, and the like.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or other problems occurring in the prior art imidacloprid artificial hapten products.
Therefore, one of the purposes of the invention is to provide a synthetic method of imidacloprid artificial hapten, which overcomes the defects of the existing production method of imidacloprid artificial hapten.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a synthetic method of imidacloprid artificial hapten comprises the following steps: and (3) preparing the liquid A and the liquid B, mixing the liquid A and the liquid B, stirring and preserving heat under a heating condition, dissolving by using DDW after cooling, extracting for multiple times after adjusting the pH value, and drying to obtain the IMI hapten.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: the solution A is prepared by mixing IMI and DMSO.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: the solution B is prepared by dissolving 3-mercaptopropionic acid and DMSO in an alkaline environment.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: the preparation method of the solution B comprises the following steps: KOH, 3-mercaptopropionic acid and DMSO are mixed uniformly.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: the liquid A and the liquid B are mixed in a manner that the liquid A is dropwise added into the liquid B.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: in the solution B, the ratio of KOH: 3-mercaptopropionic acid: 0.4-0.5 g of DMSO, 0.35-0.53 g:5mL.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: in the solution B, the ratio of KOH: 3-mercaptopropionic acid: DMSO 0.45g:0.42g:5mL.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: the temperature of the solution A is raised to 100 ℃ before the solution A and the solution B are mixed.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: mixing the solution A and the solution B, stirring and keeping the temperature under the heating condition until the solution A and the solution B are kept in the electric jacket for 2 hours.
As a preferred scheme of the synthetic method of the imidacloprid artificial hapten, the method comprises the following steps: multiple extraction is dichloromethane extraction, and the lower layer is extracted with NaHCO3Extracting, collecting the upper layer, extracting with ethyl acetate, collecting the lower layer, and extracting with NaHCO3Extracting, collecting the upper layer, extracting with ethyl acetate, collecting the upper layer, and extracting with DDW.
The invention provides a synthetic method of imidacloprid artificial hapten, which reduces the generation of by-products of 3-mercaptopropionic acid by adjusting and optimizing reaction sequence and reagents.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 shows the antibody titers obtained from the haptens obtained in examples 1 and 2;
the hapten prepared in example 2 is on the left side of the figure, and the hapten prepared in example 1 is on the right side of the figure;
FIG. 2 is a mass spectrum of the hapten prepared in example 1;
FIG. 3 is a mass spectrum of the hapten prepared in example 2;
FIG. 4 is a mass spectrum of the hapten prepared in example 3.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
Weighing 1.3g of IMI, and dissolving the IMI in 15mL of DMSO to obtain solution A; weighing 0.45g of KOH and 0.42g of 3-mercaptopropionic acid, and dissolving the KOH and the 3-mercaptopropionic acid in 5mL of DMSO to obtain solution B; opening the electric heating jacket, and slowly heating the solution A to 100 ℃; introducing nitrogen; slowly dripping the solution B into the solution A, and stirring to fully mix the solution A and the solution B; keeping the temperature at 100 ℃ for 2 h; removing the electric heating jacket, and cooling the reaction system to roomAnd (4) warming. Dissolving 5mL of reaction system by 8mL of LDDW, and adjusting the pH to 3.0 by using 6mol/L of dilute hydrochloric acid; extracting with dichloromethane in a separating funnel, and collecting the lower layer; NaHCO for lower layer3Extracting and collecting the upper layer; extracting the upper layer with ethyl acetate, and collecting the lower layer; the lower layer was adjusted to pH 3.0 with 6mol/L dilute hydrochloric acid, extracted with ethyl acetate, and the upper layer was collected and the product was in the upper layer. The product was extracted with DDW, dried by addition of a suitable amount of anhydrous sodium sulfate, and then evaporated using an evaporator to obtain IMI hapten.
Example 2
5.15g imidacloprid was dissolved in 20mL DMSO and 2.25g KOH was added. 2.10g of 3-mercaptopropionic acid was weighed out separately, dissolved in 10mL of DMSO and transferred to a 50mL constant pressure dropping funnel. Slowly adding the 3-mercaptopropionic acid solution under stirring, slowly heating to 100 ℃ on an oil bath, preserving the temperature for 2 hours, and removing the oil bath. After the product had cooled naturally to room temperature, 50mL of water were added to
The HCl solution was adjusted to pH 3. The mixture was transferred to a 250mL separatory funnel, extracted 3 times with 90mL dichloromethane, and the organic phase was collected. With 75mL NaHCO3The dichloromethane extract was washed with the aqueous solution 3 times and the aqueous phase was collected. The aqueous phase was washed with a small amount of ether and the ether layer was discarded. Reuse of
The pH of the aqueous phase was adjusted to 3 with HCl solution, then the aqueous phase was extracted 3 times with 90mL ethyl acetate and the ethyl acetate extracts were combined. The ethyl acetate was washed with a small amount of distilled water, and the aqueous layer was discarded. The extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a small amount of yellow viscous liquid. Adding a small amount of acetone solution to dissolve, standing overnight, and precipitating yellow crystal. Filtering and taking out crystals.
Example 3
Weighing 1.3g of IMI, and dissolving the IMI in 15mL of DMSO to obtain solution A; weighing 0.45g of KOH and 0.35g of 3-mercaptopropionic acid, and dissolving the KOH and the 3-mercaptopropionic acid in 5mL of DMSO to obtain solution B; opening the electric heating jacket, and slowly heating the solution A to 100 ℃; introducing nitrogen; slowly dripping the solution B into the solution A, and stirring to fully mix the solution A and the solution B; keeping the temperature at 100 ℃ for 2 h; remove electricityAnd (4) heating, and cooling the reaction system to room temperature. Dissolving 5mL of reaction system by 8mL of LDDW, and adjusting the pH to 3.0 by using 6mol/L of dilute hydrochloric acid; extracting with dichloromethane in a separating funnel, and collecting the lower layer; NaHCO for lower layer3Extracting and collecting the upper layer; extracting the upper layer with ethyl acetate, and collecting the lower layer; the lower layer was adjusted to pH 3.0 with 6mol/L dilute hydrochloric acid, extracted with ethyl acetate, and the upper layer was collected and the product was in the upper layer. The product was extracted with DDW, dried by addition of an appropriate amount of anhydrous sodium sulfate, and then evaporated with a rotary evaporator to give IMI hapten.
Example 4
The IMI haptens prepared in examples 1-3 were coupled to the same carrier protein (BSA) to obtain complete antigens. And (3) immunizing 7-week-old female SPF-grade BALB/c mice by using the coupled complete antigen according to a preset immunization program, and performing tail breaking blood collection 7 days after the three-way immunization and the five-way immunization to determine the serum antibody titer of the mice.
Example 5
Mice were immunized with the two complete antigens prepared in example 4, blood was collected after immunization, and serum antibody titers were measured by an indirect ELISA method (ELISA method) as follows:
the data obtained are shown in FIG. 1:
from fig. 1, it can be seen that the serum antibody titer of the mice immunized with the complete antigen obtained by hapten coupling can be stabilized at 1:10000 or more, and it is clear that the index obtained in example 1 is significantly higher than the index obtained by the subsequent treatment of the hapten in example 2, and the serum antibody titer of the mice immunized with the complete antigen obtained by hapten coupling obtained in example 1 should be higher regardless of the dilution. The invention of our invention, in addition to the improvement of the purity of the final product, the embodiment 1 can realize the stable titer of the prepared serum antibody over 1: 10000.
Example 6
The hapten prepared in the examples 1, 2 and 3 is subjected to mass spectrometry (maXis ultra-high resolution time-of-flight mass spectrometer, Bruker Dalton), and the measured mass spectra are shown in fig. 2, 3 and 4, so that the peak height in fig. 2 is obviously higher, and the hapten concentration is clearly improved by comparing fig. 2, 3 and 4, and the effect of reducing the oxidation by-products of 3-mercaptopropionic acid is achieved, and the method provided in the example 1 can be achieved through the peak height data, so that the antibody titer is obviously improved.
A complete antigen-immunized mouse was prepared using the haptens obtained in examples 1 to 3, and the serum antibody titer of the immunized mouse was obtained, and the complete antigen further prepared using the hapten synthesized in example 1 produced a higher titer of antibody in the mouse, and the amount of 3-mercaptopropionic acid used in example 1 was 0.42g, which is a preferable amount of 3-mercaptopropionic acid.
The method used in the invention of the inventor reduces the amount of the by-product generated by 3-mercaptopropionic acid, thereby realizing the reduction of production risk and the improvement of hapten purity and the titer of the antibody in the serum of the immunized mouse.
Claims (10)
1. A synthetic method of imidacloprid artificial hapten is characterized by comprising the following steps: the method comprises the following steps: and (3) preparing the liquid A and the liquid B, mixing the liquid A and the liquid B, stirring under a heating condition, then preserving heat, cooling after heat preservation is finished, dissolving by using DDW after cooling, adjusting the pH, extracting for multiple times, and drying to obtain the IMI hapten.
2. The method for synthesizing imidacloprid artificial hapten according to claim 1, which is characterized in that: the solution A is prepared by mixing IMI and DMSO.
3. The method for synthesizing imidacloprid artificial hapten according to claim 1, which is characterized in that: the solution B is prepared by dissolving 3-mercaptopropionic acid and DMSO in an alkaline environment.
4. The method for synthesizing imidacloprid artificial hapten according to claim 3, which is characterized in that: the preparation method of the solution B comprises the following steps: KOH, 3-mercaptopropionic acid and DMSO are mixed uniformly.
5. The method for synthesizing imidacloprid artificial hapten according to claim 1, which is characterized in that: the mixing mode of the solution A and the solution B is to drop the solution A into the solution B.
6. The method for synthesizing imidacloprid artificial hapten according to claim 4, which is characterized in that: in the solution B, the ratio of KOH: 3-mercaptopropionic acid: 0.4-0.5 g of DMSO, 0.35-0.53 g:5mL.
7. The method for synthesizing imidacloprid artificial hapten according to claim 6, which is characterized in that: in the solution B, the ratio of KOH: 3-mercaptopropionic acid: DMSO 0.45g:0.42g:5ml.
8. The method for synthesizing imidacloprid artificial hapten according to claim 1, which is characterized in that: when the solution A and the solution B are mixed, the temperature of the solution A is 100 ℃.
9. The method for synthesizing imidacloprid artificial hapten according to claim 1, which is characterized in that: and mixing the solution A and the solution B, stirring under a heating condition, and then preserving heat for 2 hours in an electric heating sleeve.
10. The method for synthesizing imidacloprid artificial hapten according to claim 1, which is characterized in that: the multiple extraction is dichloromethane extraction, and then the lower layer is taken out and is added with NaHCO3Extracting, extracting the upper layer with ethyl acetate, and extracting the lower layer with NaHCO3Extracting, extracting the upper layer with ethyl acetate, collecting the upper layer, and extracting with DDW.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000191698A (en) * | 1998-12-24 | 2000-07-11 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of imidacloprid, antibody and measurement |
| CN1569840A (en) * | 2004-04-30 | 2005-01-26 | 浙江大学 | Production method and use for imidacloprid artificial hapten, artificial antigen and specific antibody |
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- 2021-07-08 CN CN202110773955.0A patent/CN113372328A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000191698A (en) * | 1998-12-24 | 2000-07-11 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of imidacloprid, antibody and measurement |
| CN1569840A (en) * | 2004-04-30 | 2005-01-26 | 浙江大学 | Production method and use for imidacloprid artificial hapten, artificial antigen and specific antibody |
Non-Patent Citations (2)
| Title |
|---|
| LI KAI等: "Development of an Enzyme-Linked Immunosorbent Assay for the Insecticide Imidacloprid", 《J. AGRIC. FOOD CHEM.》 * |
| 朱国念 等: "吡虫啉人工抗原的合成与鉴定", 《中国农业科学》 * |
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