Trimethoprim hapten as well as synthesis method and application thereof

Abstract

The invention discloses a trimethoprim hapten as well as synthesis and application thereof. The structure of the trimethoprim hapten is as shown in a formula I. The trimethoprim hapten provided by the invention can fully expose the antigenic determinant of trimethoprim and maintain the spatial conformation of trimethoprim, has good immunogenicity, and is mild in synthesis conditions, few in reaction steps, high in yield and capable of reducing the cost of raw materials. The trimethoprim colloidal gold detection product provided by the invention has the characteristics of strong specificity and high sensitivity, can realize accurate and rapid detection of trimethoprim in a sample, and has no cross reaction with other drugs. And the formula I of the # imgabs0 # is shown in the specification.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a trimethoprim hapten and a synthesis method and application thereof. Background Art

[0002] As the material basis for the development of modern breeding industry, veterinary drugs have played an important role in reducing animal morbidity and mortality, promoting animal growth, and improving the quality of animal products. However, the misuse and abuse of veterinary drugs have caused some animal-derived foods to exceed the veterinary drug standards, affecting food quality and thus the health of consumers. Regulatory authorities in various parts of my country have continuously strengthened the monitoring of veterinary drug residues and carried out risk detection.

[0003] As a commonly used antibacterial enhancer, trimethoprim is often used in veterinary clinics to treat diseases such as pullorum, fowl cholera, and secondary respiratory bacterial infections. However, due to the widespread use of trimethoprim and irrational use of the drug, the trimethoprim residues in some animal-derived foods do not meet the national food safety standards. Long-term consumption of foods with excessive trimethoprim residues may cause nausea, vomiting, and other reactions. It may also cause allergic reactions and toxic reactions in the human body, produce drug resistance, and lead to the occurrence of "three hazards" (teratogenicity, carcinogenicity, and mutagenicity). For this reason, China and international food committees in Europe and the United States have formulated maximum residue limit standards for trimethoprim in animal-derived foods.

[0004] At present, the main detection methods of trimethoprim include gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and surface enhanced Raman spectroscopy. These methods mainly rely on accurate data acquisition and analysis systems, and can perform quantitative analysis, but they require expensive instruments and well-trained professionals, and are not suitable for on-site and rapid detection of large quantities of samples. In recent years, there are more and more methods for detecting residues using bioanalytical techniques. Enzyme-linked immunosorbent assay is one of the most commonly used immunoassay methods, which has the characteristics of high specificity and rapid detection, but its operation steps are cumbersome, time-consuming and requires a spectrophotometer to read the results, making it unsuitable for field detection, on-site detection, import and export port detection and emergency detection such as exceeding the standard. In comparison, colloidal gold immunochromatography can make up for the above shortcomings. The test strips are easy to carry, easy to operate, have low professional requirements for detection personnel, and have a short reaction time. It is suitable for on-site rapid detection of large quantities of samples and has been widely used in the detection of food, biology, environment, pharmaceuticals, clinical medicine and other fields. Therefore, the present invention provides a trimethoprim hapten with a stable structure, and couples the hapten with a carrier protein to prepare a trimethoprim complete antigen. The colloidal gold test strip prepared by the antigen can realize accurate and rapid detection of trimethoprim.

[0005] The patent with application number CN 202110180167.0 discloses a trimethoprim hapten TMPO, artificial antigen, antibody and their preparation methods and applications. This method retains the molecular skeleton structure of trimethoprim, which is more conducive to immune induction. However, a large amount of hydrochloric acid gas is generated during the quenching process with phosphorus oxychloride, which affects the stability of the Boc protecting group, exposes the amino group, and leads to the generation of other uncontrollable impurities during the formation of the hapten, greatly reducing the yield of the trimethoprim hapten. In contrast, the method for preparing the trimethoprim hapten provided by the present invention also retains the molecular skeleton structure of trimethoprim, and the Cbz group has better stability in protecting the amino group and is not easily deprotected during the post-treatment process. When the Cbz group is deprotected by the palladium carbon / hydrogen system, the reaction is cleaner, resulting in a higher yield of the trimethoprim hapten. Summary of the Invention

[0006] The object of the present invention is to provide a trimethoprim hapten and its synthesis method and application, to achieve accurate and rapid detection of trimethoprim and no cross-reaction with other drugs.

[0007] To achieve the above object, the present invention is realized through the following technical solutions: One of the objects of the present invention is to provide a trimethoprim hapten, and the structure of the trimethoprim hapten is shown in Formula I: Formula I.

[0008] Another object of the present invention is to provide a method for preparing the above trimethoprim hapten, and the steps are as follows: S1. Accurately weigh the raw material of (2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanol, dissolve it in 1,4-dioxane, protect it with nitrogen, then cool it to 0 °C, add benzyloxycarbonyl succinimide, slowly add triethylamine, keep the temperature at 45 °C, and stir and react for 6 hours to obtain a mixed solution A; S2. Monitor the mixed solution A by TLC, confirm that the reaction is complete, cool it to 0 °C, dropwise add purified water for quenching, extract twice with ethyl acetate, combine the organic phases, wash twice with saturated sodium chloride aqueous solution, and dry with anhydrous sodium sulfate to obtain the trimethoprim hapten intermediate B; S3. Dissolve the intermediate B in dichloromethane, cool it to 0 °C, add bromoacetic acid, slowly add sodium carbonate, and stir and react at room temperature for 12 hours to obtain a mixed solution C; S4. Monitor by TLC that there is no raw material left in step S3, cool it to 0 °C, add purified water for quenching, retain the aqueous phase, free it with 3M hydrochloric acid until the pH is equal to 4, extract with ethyl acetate, wash once with purified water, wash once with saturated sodium chloride aqueous solution, and spin-dry the organic phase to obtain the trimethoprim hapten intermediate D; S5. Dissolve the intermediate D in methanol, add palladium carbon at room temperature, stir and react for 8 hours under a hydrogen atmosphere. Detect by TLC, confirm the completion of the reaction, filter off the palladium carbon, and rotary evaporate the organic phase to obtain the trimethoprim hapten intermediate E; S6. Purification: Purify the intermediate E with a normal-phase FLASH column to obtain the purified trimethoprim hapten.

[0009] Preferably, the detailed steps are as follows: S1. Accurately weigh 200 mg of the raw material (2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanol, dissolve it in 20 mL of 1,4-dioxane, protect it under nitrogen, then cool it to 0 °C, add 334 mg of benzyloxycarbonyl succinimide, slowly add 130 mg of triethylamine, keep the temperature at 45 °C, and stir and react for 6 hours to obtain the mixture A; S2. Monitor the mixture A by TLC, confirm the completion of the reaction, cool it to 0 °C, dropwise add 20 mL of purified water for quenching, extract twice with 15 mL of ethyl acetate, combine the organic phases, wash twice with 10 mL of saturated sodium chloride aqueous solution, and dry with anhydrous sodium sulfate to obtain 460 mg of the trimethoprim hapten intermediate B; S3. Dissolve 460 mg of the intermediate B in 20 mL of dichloromethane, cool it to 0 °C, add 114 mg of bromoacetic acid, slowly add 80 mg of sodium carbonate, and stir and react at room temperature for 12 hours to obtain the mixture C; S4. Monitor by TLC that there is no raw material remaining in step S3, cool it to 0 °C, add 20 mL of purified water for quenching, retain the aqueous phase, free it with 3M hydrochloric acid until the pH is equal to 4, extract with 20 mL of ethyl acetate, retain the organic phase, wash once with 10 mL of purified water and once with 10 mL of saturated sodium chloride aqueous solution, and rotary evaporate the organic phase to obtain 520 mg of the trimethoprim hapten intermediate D; S5. Dissolve 520 mg of the intermediate D in methanol, add 10% w / w palladium carbon at room temperature, stir and react for 8 hours under a hydrogen atmosphere. Detect by TLC, confirm the completion of the reaction, filter off the palladium carbon, and rotary evaporate the organic phase to obtain 130 mg of the trimethoprim hapten intermediate E; S6. Purification: Purify the intermediate E with a normal-phase FLASH column to obtain 89 mg of the purified trimethoprim hapten.

[0010] The third object of the present invention is to provide a trimethoprim complete antigen, which is obtained by conjugating the above trimethoprim hapten with a carrier protein, and the structure of the trimethoprim complete antigen is shown in Formula II: Formula II.

[0011] Among them, Protein represents the carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0012] The fourth object of the present invention is to provide a method for preparing the above-mentioned trimethoprim complete antigen.

[0013] Specifically, the method is the EDC / NHS coupling technology. When the carrier protein is bovine serum albumin (BSA) or chicken ovalbumin (OVA), the preparation method of the trimethoprim complete antigen is as follows: Dissolve the above-mentioned trimethoprim hapten in DMF, slowly add NHS and EDC, and stir at room temperature for 6 h to obtain the trimethoprim hapten activation solution; weigh BSA or OVA and dissolve it in PBS buffer (0.01 mol / L, pH 7.4), stir evenly, and slowly add it dropwise to the activated hapten solution, and stir at room temperature for 16 h. Dialyze the obtained reaction solution with PBS buffer at 0.01 mol / L and pH 7.4, change the buffer once every 12 h during dialysis, and dialyze for 72 h to obtain the trimethoprim complete antigen with BSA or OVA as the carrier protein.

[0014] The fifth object of the present invention is to provide a trimethoprim colloidal gold detection product, which is characterized in that the above-mentioned trimethoprim complete antigen is coated on the nitrocellulose membrane.

[0015] The present invention has the following beneficial effects: 1. The present invention provides a linker arm with a specific length and containing the active group -COOH derived from the trimethoprim structure. After the hapten is connected to the carrier protein, the obtained complete antigen can fully expose the antigenic determinant of trimethoprim and maintain the spatial conformation of trimethoprim, and has good immunogenicity.

[0016] 2. The trimethoprim colloidal gold detection product provided by the present invention has the characteristics of high sensitivity, good specificity and strong stability, can realize the accurate and rapid detection of trimethoprim, and has no cross-reaction with other drugs. Description of the Drawings

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0018] The structures, ratios, sizes, etc. shown in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the implementation conditions of the present invention. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the ratio relationship, or adjustment of the size, without affecting the efficacy that the present invention can produce and the purpose that can be achieved, should still fall within the scope covered by the technical content disclosed in the present invention.

[0019] Figure 1 MS spectrum of trimethoprim hapten; Figure 2 Synthesis structure diagram of trimethoprim hapten; Figure 3 Sensitivity detection results of trimethoprim in fresh milk; Figure 4 Specificity detection results of trimethoprim colloidal gold test strip. Detailed implementation manners

[0020] The following specific embodiments illustrate the implementation manners of the present invention. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0021] Example 1. Preparation of trimethoprim hapten The structure of the trimethoprim hapten is shown in Formula I: Formula I.

[0022] The preparation method of the trimethoprim hapten described in this example is as follows in specific operation process: S1. Accurately weigh 200 mg of (2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanol raw material, dissolve it in 20 mL of 1,4-dioxane, protect with nitrogen, then cool down to 0 °C, add 334 mg of benzyloxycarbonyl succinimide, slowly add 130 mg of triethylamine, keep the temperature at 45 °C, and stir and react for 6 hours to obtain a mixed solution A; S2. Monitor the mixed solution A by TLC, confirm that the reaction is complete, cool down to 0 °C, dropwise add 20 mL of purified water for quenching, extract twice with 15 mL of ethyl acetate, combine the organic phases, wash twice with 10 mL of saturated sodium chloride aqueous solution, and dry with anhydrous sodium sulfate to obtain 460 mg of the trimethoprim hapten intermediate product B; S3. Dissolve 460 mg of intermediate product B in 20 mL of dichloromethane, cool down to 0 °C, add 114 mg of bromoacetic acid, slowly add 80 mg of sodium carbonate, and stir the reaction at room temperature for 12 hours to obtain mixture C. S4. Monitor by TLC that there is no raw material remaining in step S3, cool down to 0 °C, add 20 mL of purified water for quenching, retain the aqueous phase, free it with 3M hydrochloric acid until the pH equals 4, extract with 20 mL of ethyl acetate, retain the organic phase, wash once with 10 mL of purified water and once with 10 mL of saturated sodium chloride aqueous solution, spin-dry the organic phase to obtain 520 mg of trimethoprim hapten intermediate product D. S5. Dissolve 520 mg of intermediate product D in methanol, add 10% w / w of palladium carbon at room temperature, stir the reaction under a hydrogen atmosphere for 8 hours, detect by TLC, confirm that the reaction is complete, filter off the palladium carbon, and spin-dry the organic phase to obtain 130 mg of trimethoprim hapten intermediate product E. S6. Purification: Purify intermediate product E with a normal-phase FLASH column to obtain 89 mg of purified trimethoprim hapten.

[0023] After column purification, a trimethoprim carboxyl derivative with a higher purity is obtained. Detect the trimethoprim carboxyl derivative by LC-MS. From Figure 1 it can be seen that the signal of the trimethoprim hapten is 365.20 and the molecular weight of the trimethoprim hapten is 364, thus determining the hapten structure. The synthesis route of the trimethoprim hapten is as Figure 2 shown.

[0024] Example 2. Preparation of Trimethoprim Complete Antigen 1. Use the EDC / NHS coupling technology to prepare the trimethoprim complete antigen S1. Weigh 40 mg of the pure hapten prepared in Example 1, dissolve it in 1 mL of DMF, slowly add 13 mg of NHS and 23 mg of EDC, and stir at room temperature for 6 h to obtain the trimethoprim hapten activation solution. S2. Weigh 120 mg of BSA or OVA, dissolve it in 10 mL of PBS buffer solution with 0.01 mol / L and pH 7.4, stir evenly, slowly drop it into the activated hapten solution, and stir at room temperature for 16 h. Dialyze with PBS buffer solution with 0.01 mol / L and pH 7.2, change the buffer solution once every 12 h during dialysis, and dialyze for 72 h to obtain the trimethoprim complete antigen with BSA or OVA as the carrier protein.

[0025] Its reaction route is as follows: or

[0026] Example 3: Rapid Detection of Trimethoprim Residue in Milk by Colloidal Gold Immunochromatography I. Preparation of Trimethoprim Colloidal Gold Detection Reagent (1)Preparation of Colloidal Gold Take 1 mL of 1% (mass percentage) chloroauric acid solution, add 99 mL of ultrapure water to prepare a chloroauric acid solution with a final concentration of 0.01% (mass percentage). After heating to boiling, take 1.5 mL of 1% (mass percentage) trisodium citrate and quickly add it to the boiling chloroauric acid solution at one time. Continue heating until the solution changes from light yellow to grayish black and finally to orange red. After the color is stable, continue heating for 5 min, cool to room temperature, and supplement ultrapure water to the original volume.

[0027] (2)Preparation of Colloidal Gold Labeled with Trimethoprim Monoclonal Antibody Adjust the pH value of the colloidal gold solution to 8.0, stir evenly with a constant-speed stirrer, and at the same time add trimethoprim monoclonal antibody drop by drop. The amount of labeled antibody is 1 μg / mL of colloidal gold. After 1 hour, add BSA equivalent to the amount of antibody, fully react for 30 min, then add PEG equivalent to the amount of antibody. After adding, continue stirring for 30 min. Centrifuge at 9000 rpm for 10 min to obtain a homogeneous gold-labeled antibody precipitate, and then resuspend it with PBS for standby to obtain a colloidal gold solution labeled with trimethoprim monoclonal antibody. Dilute this colloidal gold solution by 2 times and spray it on the glass fiber at a spraying amount of 4 μL / cm, dry it at 40 °C and store it for standby. This is the colloidal gold conjugate pad.

[0028] (3)Preparation of Trimethoprim Colloidal Gold Detection Test Strip Spray 1.0 mg / mL trimethoprim-coated antigen solution on the nitrocellulose membrane to form the test line T line, and spray 0.5 mg / mL goat anti-mouse IgG to form the control line C line. The test line and the control line are parallel to each other, and the distance between them is 0.5 cm. Then, on the bottom plate, in the same direction, sequentially lap and adhere the sample pad, the colloidal gold conjugate pad, the nitrocellulose membrane with the test line T line sprayed with trimethoprim-coated antigen and the control line C line sprayed with goat anti-mouse IgG, and the absorbent paper.

[0029] Among them, the test line is close to the colloidal gold conjugate pad and the sample pad, and the control line is close to the absorbent paper. Cut the adhered test strip into test strips of equal width and assemble them into a test card. Seal and dry the test card for storage.

[0030] II. Sample Detection Using competitive immuno-chromatographic technology, qualitatively detect trimethoprim residues in samples. Coat trimethoprim antigen on the test line (T line) of the nitrocellulose membrane, and label the corresponding trimethoprim monoclonal antibody on the colloidal gold. During the detection, when there is no trimethoprim in the sample to be tested, the colloidal gold particles will chromatograph to the test line position and bind to the trimethoprim antigen coated on the membrane, and the T line will show color. When there is trimethoprim in the sample to be tested, it will bind to the gold-labeled specific monoclonal antibody on the conjugate pad to form an immune complex. When the complex chromatographs to the test line position, it will not bind to the trimethoprim-coated antigen on the membrane, and the T line will not show color or the color will become lighter.

[0031] (1) Detection steps: The method is as follows: Take 100 μL of fresh milk and add it to the sample addition hole of the test strip card. After incubating at 20 - 25 °C for 10 minutes, read the result. If both the T line and the C line show purple-red bands and the color of the T line is stronger than that of the C line, the result is negative; if the color of the T line is lighter than that of the C line or the C line shows color while the T line does not show color, the result is positive; if neither the C line nor the T line shows color, the test reagent has expired.

[0032] (2) Sensitivity detection of the trimethoprim colloidal gold test strip card: Perform spiking experiments on negative milk to a final concentration of 5, 10, 15, 20 μg / kg respectively. The results are as Figure 3 shown. The sensitivity of the trimethoprim colloidal gold test strip card in the present invention can reach 10 μg / kg, and the CV value is less than 15%.

[0033] (3) Specificity detection of the trimethoprim colloidal gold test strip card: Add drugs such as 500 μg / kg trimethoprim, sulfadiazine, tetracycline, streptomycin, gentamicin, and amoxicillin to negative fresh milk respectively to verify the specificity of the test strip card. The specific detection results are shown in Figure 4 . The detection results are all negative except for trimethoprim, indicating that trimethoprim has excellent specificity and no cross-reaction with the above added drugs.

[0034] (4) Product stability experiment Accelerate the test of the trimethoprim colloidal gold product in the present invention at 4 °C and 37 °C respectively. Detect the trimethoprim standard product at 10 µg / kg at 0 d, 7 d, 14 d, and 30 d respectively to evaluate the stability performance of the product. The results are shown in Table 1. When aging to 30 days, the trimethoprim colloidal gold detection products at 4 °C and 37 °C still have good sensitivity when detecting the 10 μg / kg standard product, with no obvious difference from the detection results on the first day, and the CVs are all <10%, indicating that the test strip card provided by the present invention has good stability.

[0035] Table 1 Stability of the trimethoprim colloidal gold test strip card

[0036] In summary, the colloidal gold test strip of the present invention has the characteristics of high sensitivity, strong specificity and good stability, and can be applied to the rapid detection of trimethoprim in fresh milk.

Claims

1. A trimethoprim hapten, characterized in that: The structure of the trimethoprim hapten is shown in Formula I, Formula I.

2. A synthetic method for preparing the trimethoprim hapten according to claim 1, characterized in that: S1. Accurately weigh (2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl)methanol raw material, dissolve it in 1,4-dioxane, protect with nitrogen, then cool to 0°C, add benzyloxycarbonyl succinimide, slowly add triethylamine, maintain the temperature at 45°C, stir and react for 6 hours to obtain mixed solution A; S2. Monitor the mixed solution A by TLC to confirm that the reaction is complete, cool to 0°C, add purified water dropwise for quenching, extract twice with ethyl acetate, combine the organic phases, wash twice with saturated sodium chloride aqueous solution, and dry over anhydrous sodium sulfate to obtain trimethoprim hapten intermediate B; S3, dissolving the intermediate product B in dichloromethane, cooling to 0°C, adding bromoacetic acid, slowly adding sodium carbonate, maintaining room temperature for stirring and reacting for 12 hours, to obtain a mixed solution C; S4, monitoring step S3 by TLC to see if there is no raw material remaining, cooling to 0°C, adding purified water for quenching, retaining the aqueous phase, freeing with 3M hydrochloric acid until the pH is equal to 4, extracting with ethyl acetate, washing once with purified water, washing once with saturated sodium chloride aqueous solution, and spinning the organic phase to obtain trimethoprim hapten intermediate D; S5, dissolving the intermediate product D in methanol, adding palladium carbon at room temperature, stirring and reacting for 8 hours under hydrogen environment, and performing TLC detection to confirm that the reaction is complete, filtering off the palladium carbon, and drying the organic phase to obtain the trimethoprim hapten intermediate product E; S6. Purification: Use normal phase FLASH column to purify intermediate product E to obtain the purified trimethoprim hapten.

3. A trimethoprim complete antigen, characterized in that: The method is prepared by coupling the trimethoprim hapten described in claim 1 with a carrier protein, wherein the carrier protein is any one of bovine serum albumin and chicken ovalbumin.

4. A method for preparing the trimethoprim complete antigen according to claim 3, characterized in that: When the carrier protein is bovine serum albumin or chicken ovalbumin, the method comprises: The trimethoprim hapten described in claim 1 is dissolved in DMF, NHS and EDC are slowly added, and the mixture is stirred at room temperature for 6 hours to obtain a trimethoprim hapten activation solution; bovine serum albumin or chicken ovalbumin is weighed and dissolved in a 0.01 mol / L PBS buffer solution with a pH of 7.4, stirred evenly, and slowly added dropwise to the activated hapten solution, stirred at room temperature for 16 hours, and the obtained reaction solution is dialyzed with a 0.01 mol / L PBS buffer solution with a pH of 7.4, during which the buffer solution is changed every 12 hours, and the dialyzation is performed for 72 hours to obtain a trimethoprim complete antigen with a carrier protein of bovine serum albumin or chicken ovalbumin.

5. Provide a trimethoprim colloidal gold detection product, characterized in that The nitrocellulose membrane is coated with the trimethoprim complete antigen according to claim 3 or claim 4.

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