Hapten of halofuginone as well as synthesis method and application of hapten

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a halofuginone hapten, a synthesis method thereof and an application thereof. Background Art

[0002] Halofuginone, also known as halofuginone, is a new type of broad-spectrum and highly effective anti-coccidial drug. It is effective against Eimeria tenella, E. necatrix, E. acervulina, E. mivati, E. maxima and E. brunetti in chickens, and has obvious inhibitory and killing effects on the sporozoites, first-generation and second-generation schizonts of coccidia. It is also effective against most Gram-positive bacteria and has no cross-resistance. Since the insecticidal effect of halofuginone is irreversible, there is no recurrence after drug withdrawal. Moreover, it can promote the growth of broilers, improve feed efficiency and reduce the mortality rate. It is suitable for the shuttle combination of various different drugs, and its weight gain and feed-to-meat ratio are better than those of other anti-coccidial drug shuttle combinations.

[0003] At present, halofuginone has been widely used in livestock and poultry breeding. Therefore, it is particularly important to establish a reliable detection method for the residue of halofuginone in animal-derived foods, scientifically estimate its withdrawal period, and ensure food safety. Therefore, many countries have established detection methods for halofuginone residues and stipulated their residue limits. The Ministry of Agriculture and Rural Affairs of China has also stipulated in the document "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Foods" (GB 31650-2019) that halofuginone drugs are prohibited during the lactation period of cows; the maximum residue limits in bovine muscle, fat, liver and kidney tissues are 10, 25, 30 and 30 μg / kg respectively; the maximum residue limits in chicken / turkey muscle, skin / fat and liver tissues are 100, 200 and 130 μg / kg respectively.

[0004] The main methods for detecting halofuginone residues are high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas-liquid chromatography (GLC), capillary electrophoresis (CE), etc. These methods rely on precise data acquisition and analysis systems and can perform quantitative analysis, but they have high requirements for instrument equipment and personnel's detection technology, the sample processing process is relatively complex, and the detection time is relatively long, making it difficult to process a large number of samples simultaneously. In contrast, the colloidal gold immunochromatography method can make up for the above deficiencies. The test strip is convenient to carry, easy to operate, has low professional requirements for the detector, and has a short reaction time. It is suitable for rapid on-site detection of a large number of samples and has now been widely used in the detection of food, biology, environment, drugs, clinical medicine and other fields. Therefore, the present invention provides a halofuginone hapten with stable structure, conjugates it with carrier protein to prepare a halofuginone complete antigen, and the colloidal gold test strip prepared by this antigen has high specificity and good sensitivity, and can achieve accurate and rapid detection of halofuginone. Summary of the Invention

[0005] The object of the present invention is to provide a halofuginone hapten, its synthesis method and application, so as to achieve accurate and rapid detection of halofuginone and no cross - reaction with other drugs.

[0006] 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 halofuginone hapten, and the structure of the halofuginone hapten is shown in Formula I: Formula I.

[0007] Another object of the present invention is to provide a method for preparing the above - mentioned halofuginone hapten, and the steps are as follows: S1. Accurately weigh the halofuginone raw material, dissolve it in dichloromethane, add N,N - diisopropylethylamine and di - tert - butyl dicarbonate, and stir - react at room temperature; S2. After 3 hours, monitor the reaction solution by TLC, confirm that the reaction is completed, add purified water for quenching, extract three times with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and thus obtain the halofuginone hapten intermediate A; S3. Dissolve the intermediate A in dichloromethane, cool down to 0 °C, add triethylamine, protect with nitrogen, slowly add p - nitrophenyl chloroformate, and stir - react at room temperature for 2 hours after restoring to room temperature; S4. Monitor by TLC that there is no raw material left in step S3, add purified water for quenching, extract twice with ethyl acetate, wash once with saturated sodium chloride aqueous solution, spin - dry the organic phase to obtain the crude product of intermediate B, and purify it with a normal - phase FLASH column to obtain the halofuginone intermediate B; S5. Dissolve the intermediate B in dichloromethane, add N,N - diisopropylethylamine and 2 - hydroxypyridine - N - oxide at room temperature, stir - react at room temperature for 2 hours, monitor by TLC, after confirming that the reaction is completed, add purified water to the reaction solution for quenching, extract three times with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, spin - dry the organic phase to obtain the crude product of intermediate C, and purify it with a normal - phase FLASH column to obtain the halofuginone hapten intermediate C; S6. Dissolve the intermediate C in dichloromethane, slowly add trifluoroacetic acid at room temperature, stir - react at room temperature for 2 hours, after monitoring by TLC that there is no raw material left in step S6, spin - dry the organic phase to obtain intermediate D; S7. Purification: Purify the intermediate D with a normal - phase FLASH column to obtain the purified halofuginone hapten.

[0008] Preferably, the detailed steps are as follows: S1. Accurately weigh 500 mg of the halofuginone raw material, dissolve it in 10 mL of dichloromethane, add 470 mg of N,N - diisopropylethylamine and 320 mg of di - tert - butyl dicarbonate, and stir - react at room temperature; S2. After 2 to 3 hours, monitor the reaction solution by TLC. After confirming the completion of the reaction, add 15 mL of purified water to quench the reaction, and extract with 15 mL of dichloromethane three times. Combine the organic phases and dry them with anhydrous sodium sulfate to obtain 420 mg of the febrantel hapten intermediate product A. S3. Dissolve 420 mg of intermediate product A in 8 mL of dichloromethane, cool down to 0 °C, add 250 mg of triethylamine, protect with nitrogen, and slowly add 240 mg of p-nitrophenyl chloroformate. After restoring to room temperature, stir and react for 2 hours. S4. After monitoring by TLC that there is no remaining raw material in step S3, add 10 mL of purified water to quench the reaction, then extract twice with 10 mL of ethyl acetate, wash once with 10 mL of saturated sodium chloride aqueous solution, and rotary evaporate the organic phase to obtain 470 mg of the crude product of intermediate product B. Purify it using a normal-phase FLASH column to obtain 380 mg of the febrantel intermediate product B. S5. Dissolve 380 mg of intermediate product B in 5 mL of dichloromethane, add 220 mg of N,N-diisopropylethylamine and 60 mg of 2-hydroxypyridine-N-oxide at room temperature, stir and react at room temperature for 2 hours. After monitoring by TLC and confirming the completion of the reaction, add 10 mL of purified water to the reaction solution to quench the reaction, extract three times with 10 mL of dichloromethane, combine the organic phases, dry them with anhydrous sodium sulfate, rotary evaporate the organic phase to obtain 400 mg of the crude product of intermediate C. Purify it using a normal-phase FLASH column to obtain 310 mg of the febrantel hapten intermediate product C. S6. Dissolve 310 mg of intermediate product C in 5 mL of dichloromethane, slowly add 5 mL of trifluoroacetic acid at room temperature, stir and react at room temperature for 2 hours. After monitoring by TLC that there is no remaining raw material in step S6, rotary evaporate the organic phase to obtain 270 mg of intermediate product D. S7. Purification: Purify intermediate product D using a normal-phase FLASH column to obtain 220 mg of the purified febrantel hapten.

[0009] The third object of the present invention is to provide a febrantel complete antigen, which is obtained by conjugating the above-mentioned febrantel hapten with a carrier protein. The structure of the febrantel complete antigen is shown in Formula II: Formula II.

[0010] Wherein, Protein represents the carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0011] The fourth object of the present invention is to provide a method for preparing the above-mentioned febrantel complete antigen.

[0012] 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 halofuginone complete antigen is as follows: Dissolve the above-mentioned halofuginone hapten in DMF, slowly add NHS and EDC, and stir at room temperature for 5 h to obtain a halofuginone hapten activation solution; weigh bovine serum albumin or chicken ovalbumin and dissolve it in PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.4, stir evenly, slowly add it dropwise to the activated hapten solution, stir at room temperature for 8 h, and dialyze the obtained reaction solution with PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.4. Change the buffer solution every 4 h during dialysis and dialyze for 36 h to obtain a halofuginone complete antigen with bovine serum albumin or chicken ovalbumin as the carrier protein.

[0013] The fifth object of the present invention is to provide a halofuginone colloidal gold detection product, which is characterized in that the above-mentioned halofuginone complete antigen is coated on the nitrocellulose membrane.

[0014] The present invention has the following beneficial effects: 1. The present invention provides a derivative based on the structure of halofuginone itself without destroying the structure of the raw material, thus ensuring the integrity of the existence of the antigenic determinant. The introduced linker contains an active group -COOH that can efficiently couple with the carrier protein. After it is connected to the carrier protein, the antigenic determinant of halofuginone can be directly and fully exposed, with a stable structure and a good spatial configuration, and has good immunogenicity.

[0015] 2. The halofuginone 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 halofuginone, and has no cross-reaction with other drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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, other implementation drawings can be obtained by extension according to the provided drawings without creative efforts.

[0017] The structures, ratios, sizes, etc. shown in this specification are only used to cooperate with the content disclosed in the specification for those who are familiar with this technology to understand and read, and are not used to limit the limited conditions under which the present invention can be implemented. Therefore, they do not have technical substance significance. Any modification of the structure, change of the proportional relationship, or adjustment of the size should still fall within the scope covered by the technical content disclosed in the present invention without affecting the effects that the present invention can produce and the purposes that can be achieved.

[0018] Figure 1 The MS spectrum of the halofuginone hapten; Figure 2 The synthetic route diagram of the halofuginone hapten; Figure 3 The sensitivity detection results of halofuginone in fresh milk; Figure 4 The specificity detection results of the halofuginone colloidal gold test strip. Specific implementation manners

[0019] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art 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 of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0020] Example 1. Preparation of the halofuginone hapten The structure of the halofuginone hapten is shown in Formula I: Formula I.

[0021] The preparation method of the halofuginone hapten described in this example is as follows in specific operation process: S1. Accurately weigh 500 mg of the halofuginone raw material, dissolve it in 10 mL of dichloromethane, add 470 mg of N,N-diisopropylethylamine and 320 mg of di-tert-butyl dicarbonate, and stir and react at room temperature; S2. After 3 hours, monitor the reaction solution by TLC, confirm that the reaction is completed, add 15 mL of purified water to quench, extract with 15 mL of dichloromethane for three times in total, combine the organic phases, and dry with anhydrous sodium sulfate to obtain 420 mg of the halofuginone hapten intermediate A; S3. Dissolve 420 mg of the intermediate A in 8 mL of dichloromethane, cool down to 0 °C, add 250 mg of triethylamine, protect with nitrogen, slowly add 240 mg of p-nitrophenyl chloroformate, and stir and react for 2 hours after restoring to room temperature; S4. Monitor by TLC that there is no raw material remaining in step S3, add 10 mL of purified water to quench, extract twice with 10 mL of ethyl acetate, wash once with 10 mL of saturated sodium chloride aqueous solution, spin-dry the organic phase to obtain 470 mg of the crude product of the intermediate B, and purify it using a normal-phase FLASH column to obtain 380 mg of the halofuginone intermediate B; S5. Dissolve 380 mg of intermediate B in 5 mL of dichloromethane. Add 220 mg of N,N - diisopropylethylamine and 60 mg of 2 - hydroxypyridine - N - oxide at room temperature. Stir the reaction mixture at room temperature for 2 hours. Monitor the reaction by TLC. After confirming the completion of the reaction, add 10 mL of purified water to the reaction solution to quench it. Extract with 10 mL of dichloromethane three times. Combine the organic phases, dry them using anhydrous sodium sulfate, and rotary - evaporate the organic phase to obtain 400 mg of crude intermediate C. Purify it using a normal - phase FLASH column to obtain 310 mg of the febrantel hapten intermediate C. S6. Dissolve 310 mg of intermediate C in 5 mL of dichloromethane. Slowly add 5 mL of trifluoroacetic acid at room temperature. Stir the reaction mixture at room temperature for 2 hours. After monitoring by TLC and confirming no remaining raw materials in step S6, rotary - evaporate the organic phase to obtain 270 mg of intermediate D. S7. Purification: Purify intermediate D using a normal - phase FLASH column to obtain 220 mg of the purified febrantel hapten.

[0022] After column purification, a febrantel carboxyl derivative with a higher purity is obtained. Detect the febrantel carboxyl derivative by LC - MS. From Figure 1 it can be seen that the signal of the febrantel hapten is 543.10 and the molecular weight of the febrantel hapten is 542.06, thus determining the hapten structure. The synthetic route of the febrantel hapten is as Figure 2 shown.

[0023] Example 2. Preparation of the febrantel complete antigen 1. Use the EDC / NHS coupling technology to prepare the febrantel complete antigen S1. Weigh 80 mg of the pure hapten prepared in Example 1 and dissolve it in 2 mL of DMF. Slowly add 26 mg of NHS and 46 mg of EDC, and stir at room temperature for 5 h to obtain the activated febrantel hapten solution. S2. Weigh 240 mg of BSA or OVA and dissolve it in 10 mL of 0.01 mol / L PBS buffer with pH 7.4. Stir evenly and slowly add it dropwise to the activated hapten solution. Stir at room temperature for 8 h. Dialyze with 0.01 mol / L PBS buffer with pH 7.2, changing the buffer every 4 h for 36 h to obtain the febrantel complete antigen with the carrier protein being BSA or OVA.

[0024] The reaction route is as follows:

[0025] Among them, Protein represents the carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0026] Example 3: Rapid Detection of Halofuginone Residue in Milk by Colloidal Gold Immunochromatography I. Preparation of Colloidal Gold Detection Reagent for Halofuginone (1)Preparation of Colloidal Gold Take 8 mL of 1% (mass percentage) chloroauric acid solution, add 792 mL of ultrapure water to prepare a chloroauric acid solution with a final concentration of 0.01% (mass percentage). After heating to boiling, take 1.0 mL of 1.2% (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 wine red. After the color is stable, continue heating for 6 min, cool to room temperature, and supplement ultrapure water to a total volume of 800 mL.

[0027] (2)Preparation of Colloidal Gold Labeled with Halofuginone Monoclonal Antibody Take the colloidal gold solution, adjust the pH value to 8.5 with 0.1 M potassium carbonate solution, stir evenly with a constant speed stirrer, and at the same time dropwise add the halofuginone monoclonal antibody (purchased from Power Biotech (Shenzhen) Co., Ltd.) to make the final labeling concentration 2.5 μg / mL of colloidal gold. React at room temperature for 1 hour, add 10% bovine serum albumin solution to a final concentration of 1%; after fully reacting for 30 min, add 10% polyethylene glycol - 20000 to a final concentration of 1%; continue stirring for 30 min. Centrifuge at 10500 rpm for 7 min to obtain a homogeneous gold-labeled antibody precipitate, and then resuspend it with 0.01 mol / L PBS buffer solution with a pH of 7.2 to make its volume 1 / 10 of the original volume, that is, obtain the colloidal gold solution labeled with halofuginone monoclonal antibody. Dilute this colloidal gold solution by 3 times and spray it on the glass fiber at a spraying amount of 4 μL / cm, dry it at 37℃ and store it for later use. This is the colloidal gold conjugate pad.

[0028] (3)Preparation of Colloidal Gold Detection Test Strip for Halofuginone Spray 0.8 mg / mL halofuginone-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 the two is 0.65 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 halofuginone-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 with an equal width of 4.05 mm and assemble them into a test card. Seal and dry the test card for storage.

[0030] II. Sample Detection Using competitive immuno-chromatographic technology to qualitatively detect halofuginone residues in samples. The test line (T line) on the nitrocellulose membrane is coated with halofuginone antigen, and the corresponding halofuginone monoclonal antibody is labeled on the colloidal gold. During the test, when there is no halofuginone in the test sample, the colloidal gold particles will combine with the halofuginone antigen coated on the membrane when chromatographing to the test line position, and the T line will show color. When there is halofuginone in the test sample, it will combine with 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 combine with the halofuginone-coated antigen on the membrane, and the T line will not show color or the color will become lighter.

[0031] (1) Detection steps: Add 120 μL of fresh milk to the sample addition hole of the test strip card, and read the result after incubating at 20°C - 25°C for 10 minutes.

[0032] Result reading criteria: Negative (-): Both the C line and the T line show color and the T line shows a stronger color than the C line, indicating that the halofuginone drug concentration in the sample is lower than the detection limit; Positive (+): The C line shows color, the T line shows color, the T line shows the same color as the C line, the T line shows a weaker color than the C line or the T line does not show color, indicating that the halofuginone drug concentration in the sample is equal to or higher than the detection limit; Invalid: The C line does not appear, indicating an incorrect operation process or the test strip card has deteriorated and become ineffective.

[0033] (2) Sensitivity detection of the halofuginone colloidal gold test strip card: Perform spiking experiments on negative milk until the final concentration of halofuginone is 0.5, 1, 1.5, 2, 2.5, 3 μg / kg. The results are as Figure 3 shown. The sensitivity of the halofuginone colloidal gold test strip card in the present invention can reach 1 - 1.5 μg / kg.

[0034] (3) Specificity detection of the halofuginone colloidal gold test strip card: Add 500 μg / kg of halofuginone, amprolium, clopidol, diclazuril, monensin, nicarbazin, robenidine, salinomycin, and toltrazuril 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 halofuginone, indicating that the halofuginone colloidal gold test strip card has excellent specificity and no cross-reaction with the above added drugs.

[0035] (4) Product stability experiment The halofuginone colloidal gold product of the present invention was subjected to an accelerated test at 4°C and 37°C respectively. The halofuginone standard at 1.5 μg / kg was detected at 0 d, 7 d, 14 d, and 30 d respectively to evaluate the stability of the product. The results are shown in Table 1. When aging to 30 days, when the halofuginone colloidal gold test strip was used to detect the sample with the added halofuginone drug concentration of 1.5 μg / kg at 4°C and 37°C, it still had good sensitivity, with no significant difference from the detection results on the first day, and the CV was <10% for both, indicating that the test strip provided by the present invention has good stability.

[0036] Table 1 Stability of the halofuginone colloidal gold test strip

[0037] 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 halofuginone in fresh milk.

Claims

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

2. A synthetic method for preparing the halofuginone hapten described in claim 1, characterized in that: S1. Accurately weigh the halofuginone raw material, dissolve it in dichloromethane, add N,N-diisopropylethylamine and di-tert-butyl dicarbonate, and stir and react at room temperature; S2. After 3 hours, monitor the reaction solution by TLC. After confirming that the reaction is complete, add purified water for quenching, extract three times with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and the halofuginone hapten intermediate A can be obtained; S3. Dissolve the intermediate A in dichloromethane, cool down to 0 °C, add triethylamine, protect with nitrogen, slowly add p-nitrophenyl chloroformate, and stir and react at room temperature for 2 hours after returning to room temperature; S4. Monitor by TLC that there is no raw material remaining in step S3, add purified water for quenching, extract twice with ethyl acetate, wash once with saturated sodium chloride aqueous solution, spin-dry the organic phase to obtain the crude product of intermediate B, and purify it with a normal-phase FLASH column to obtain the halofuginone intermediate B; S5. Dissolve the intermediate B in dichloromethane, add N,N-diisopropylethylamine and 2-hydroxypyridine-N-oxide at room temperature, stir and react at room temperature for 2 hours. After monitoring by TLC and confirming that the reaction is complete, add purified water to the reaction solution for quenching, extract three times with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, spin-dry the organic phase to obtain the crude product of intermediate C, and purify it with a normal-phase FLASH column to obtain the halofuginone hapten intermediate C; S6. Dissolve the intermediate C in dichloromethane, slowly add trifluoroacetic acid at room temperature, stir and react at room temperature for 2 hours. After monitoring by TLC that there is no raw material remaining in step S6, spin-dry the organic phase to obtain intermediate D; S7. Purification: Purify the intermediate D with a normal-phase FLASH column to obtain the purified halofuginone hapten.

3. A halofuginone complete antigen, characterized in that, It is prepared by conjugating the halofuginone hapten described in claim 1 with a carrier protein, and the carrier protein is any one of bovine serum albumin or chicken ovalbumin.

4. A method for preparing the halofuginone complete antigen according to claim 3, characterized in that, When the carrier protein is bovine serum albumin or chicken ovalbumin, the method includes: Dissolve the halofuginone hapten described in claim 1 in DMF, slowly add NHS and EDC, and stir at room temperature for 5 h to obtain the activated solution of the halofuginone hapten; weigh bovine serum albumin or chicken ovalbumin and dissolve it in PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.4, stir evenly, slowly drop it into the activated hapten solution, stir at room temperature for 8 h, dialyze the obtained reaction solution with PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.4, change the buffer solution once every 4 h during the period, and dialyze for 36 h to obtain the complete halofuginone antigen with bovine serum albumin or chicken ovalbumin as the carrier protein.

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

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