CN111303076A - Preparation method of methylene blue hapten and methylene blue immunogen - Google Patents
Preparation method of methylene blue hapten and methylene blue immunogen Download PDFInfo
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- CN111303076A CN111303076A CN202010138477.1A CN202010138477A CN111303076A CN 111303076 A CN111303076 A CN 111303076A CN 202010138477 A CN202010138477 A CN 202010138477A CN 111303076 A CN111303076 A CN 111303076A
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- C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
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Abstract
The invention relates to the technical field of medicines, and provides a preparation method of methylene blue hapten and immunogen, which comprises the steps of firstly carrying out chemical reaction on N, N-dimethyl-p-phenylenediamine, hydrated aluminum sulfate, sodium thiosulfate, zinc chloride and potassium dichromate to obtain an intermediate 1, secondly carrying out chemical reaction on N-methylaniline, lutidine, 5-bromovaleric acid ethyl ester and sodium hydroxide to obtain an intermediate 2, and thirdly carrying out chemical reaction on the intermediate 1 and the intermediate 2 under the action of a catalyst of silver carbonate to obtain the methylene blue hapten. And finally, reacting the methylene blue hapten with Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), and then adding Bovine Serum Albumin (BSA) for reaction to prepare the methylene blue immunogen. The methylene blue immunogen prepared by the method has a coupling ratio of about 28:1, can stimulate mice to produce antibodies, has an antibody titer of about 64000, has a methylene blue concentration of 39.0ng/mL when the inhibition rate is 50%, and has an excellent competitive inhibition effect on the methylene blue.
Description
Technical Field
The invention belongs to the technical field of antigen preparation, and particularly relates to a preparation method of methylene blue hapten and methylene blue immunogen.
Background
Methylene blue (chemical formula is C)16H18ClN3S, molecular weight: 319.86) is a phenothiazine salt, has a dark green bronze luster crystal (trihydrate) appearance, is soluble in water/ethanol, is insoluble in ethers, is relatively stable in air, and has an alkaline and toxic aqueous solution. Methylene blue is widely used in the aspects of chemical indicators, dyes, biological coloring agents, medicines and the like, has certain treatment effect on bacillary dysentery, cancers, bacterial and viral infections and central nervous system diseases, and is also used in aquaculture for treating certain fish diseases or used as a disinfectant.
However, methylene blue has some toxicity, and high concentrations of methylene blue can poison animals and lead to death. The U.S. food administration (FDA), the european union, and japan also set strict detection standards for methylene blue residues in aquatic products.
One effective way to detect methylene blue residues is immunological detection methods based on specific antibodies, which require the preparation of methylene blue immunogens. The preparation of methylene blue immunogen usually needs to implement the process of synthesizing methylene blue immunogen, in the process of synthesizing the methylene blue immunogen, methylene blue metabolite azure A or azure C can be directly used as hapten for synthesis, however, when the methylene blue immunogen synthesized by azure A or azure C is used for immunizing mice, the problem of short exposed group exists, so that polyclonal antibody having competitive inhibition effect on methylene blue is not easy to generate.
Disclosure of Invention
The invention aims to solve the problems and provides a novel methylene blue hapten and a preparation method of a methylene blue immunogen.
In order to achieve the above object, the technical solution of the present invention is summarized as follows:
firstly, carrying out chemical reaction on N, N-dimethyl-p-phenylenediamine, hydrated aluminum sulfate, sodium thiosulfate, zinc chloride and potassium dichromate to obtain an intermediate 1, secondly, carrying out chemical reaction on N-methylaniline, lutidine, 5-bromoethyl valerate and sodium hydroxide to obtain an intermediate 2, and thirdly, carrying out chemical reaction on the intermediate 1 and the intermediate 2 under the action of a catalyst of silver carbonate to obtain the methylene blue hapten. And finally, reacting the methylene blue hapten with Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), and then adding Bovine Serum Albumin (BSA) for reaction to prepare the methylene blue immunogen.
The first aspect of the invention provides a preparation method of methylene blue hapten, which comprises the following steps:
A. preparation of intermediate 1 (2-amino-5-dimethylaminophenylthiosulfuric acid)
Under the condition of low temperature, adding reaction raw materials of N, N-dimethyl-p-phenylenediamine, hydrated aluminum sulfate, sodium thiosulfate and zinc chloride into water according to the molar ratio of 1:2:1:1, stirring, dropwise adding a potassium dichromate solution, and continuously stirring for reacting for a certain time; then, the temperature is raised to room temperature, and the product is subjected to post-treatment to obtain an intermediate 1, wherein the reaction formula is as follows:
B. preparation of intermediate 2(5- (methyl (phenyl) amino) pentanoic acid)
Sequentially adding 50-100 mmol/LN-methylaniline, 80-120 mmol/L2, 6-lutidine and 80-120 mmol/L5-ethyl bromovalerate solution into a reaction vessel according to the volume ratio of 8-12: 10-15: 13-17, and carrying out reflux reaction in acetonitrile; after removing the solvent, extracting, washing and drying to obtain oily substances; adding NaOH solution into the oily matter, carrying out reflux reaction, adjusting the pH value with volatile acid, extracting with an organic solvent, separating and purifying to obtain the intermediate 2, wherein the reaction formula is as follows:
C. preparation of methylene blue hapten (3- ((4-carboxybutyl) (methyl) amino) -7- (dimethylamino) -phenothiazine-5-ammonium chloride)
Dissolving the intermediate 1 and the intermediate 2 in a methanol-water solution according to a molar ratio of 1:1, stirring and heating under the protection of gas, adding silver carbonate with a molar number 11-3 times that of the intermediate, performing reflux reaction, separating and purifying to obtain the methylene blue hapten, wherein the reaction formula is as follows:
preferably, in the step A, the molar ratio of the reaction raw materials to water is 1: 3-4, and after the raw materials are added, the water and the reaction raw materials are stirred for 5-10 min at the temperature of 0 ℃; and dropwise adding a potassium dichromate solution with the concentration of 0.5-1.0 mol/L, and after the dropwise adding is finished, stirring and reacting for 2 hours at the temperature of 0 ℃ to obtain a product. And sequentially carrying out suction filtration, washing, acetone leaching, oil pump drying, pulping by using methyl tert-butyl ether, and oil pump drying again to obtain the intermediate 1.
Preferably, in the step B, after the reflux reaction in acetonitrile for 30 hours, the solvent is removed by reduced pressure distillation, and the oily substance is obtained by ethyl acetate extraction, water washing twice, anhydrous sodium sulfate drying and spin drying at 60 ℃.
And then adding 1.5-3.0 mol/L NaOH solution into the oily matter, carrying out reflux reaction for 2 hours, adjusting the pH value to 5 by using HCl, extracting for three times by using ethyl acetate, combining organic phases, drying by using anhydrous sodium sulfate, carrying out spin drying, and carrying out column chromatography separation and purification to obtain the intermediate 2. In the process of column chromatography separation, the adopted eluent is a mixed solution of n-hexane and ethyl acetate with the volume ratio of 1: 1.
Preferably, in the step C, the volume ratio of methanol to water in the methanol-water solution is 5:2, after stirring and heating to 50 ℃ under the protection of nitrogen, adding silver carbonate, completely replacing nitrogen, heating to 50 ℃ for reflux reaction for 2h, and tracing the reaction end by a TLC (thin layer chromatography) plate to obtain a primary product.
And (3) carrying out suction filtration on the primary product through diatomite, spin-drying and column chromatography separation and purification to obtain the methylene blue hapten, wherein in the column chromatography separation process, the adopted eluent is dichloromethane-methanol mixed liquor with the volume ratio of 9: 1.
The second aspect of the present invention is to prepare methylene blue immunogen by using the above methylene blue hapten as raw material, wherein the preparation method comprises the following steps:
step 1: completely dissolving methylene blue hapten, Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) with the same mole number in a Dimethylformamide (DMF) solution, reacting for 8-10 h at room temperature under the stirring condition, and standing overnight at 4 ℃;
step 2: centrifuging at 4 ℃ and 4000rpm for 10min, taking supernatant, adding the supernatant into 10-20 mg/mL bovine serum albumin solution, and reacting for 6-8 h in ice-water bath;
and step 3: and transferring the solution into a treated dialysis bag after reaction, dialyzing for 3-4 days at 4 ℃ by using a phosphate buffer solution as a dialysate, replacing the dialysate for 3-4 times every day, centrifuging for 10min at 4 ℃ and 4000rpm, and taking supernatant, namely the methylene blue immunogen solution.
Action and Effect of the invention
According to the preparation method of the methylene blue derivative hapten, traditional reactions such as Friedel-crafts acylation and Huang Minlon reduction are not adopted, long carbon chain carboxyl is introduced into an aromatic ring structure, N-dimethyl-p-phenylenediamine and N-methylaniline are respectively used as raw materials to respectively synthesize two intermediates, then through a closed-loop reaction, five carbon chain carboxyl is directionally introduced into a nitrogen methyl side chain on one side, and the methylene blue derivative hapten is coupled with protein through carboxyl to obtain the methylene blue antigen. The reaction operation is simple, and the raw materials are easy to obtain.
The methylene blue immunogen prepared according to the method of the invention has a coupling ratio of about: 28:1, and capable of stimulating the production of antibodies in mice, having an antibody titer of about 64000, a methylene blue concentration of 39.0ng/mL at an inhibition rate of 50% (IC50), and an excellent competitive inhibition effect on methylene blue.
Drawings
FIG. 1 is a methylene blue semi-antigen hydrogen spectrum (1HNMR) representation;
FIG. 2 is a methylene blue immunogen UV-visible scanning spectrum;
FIG. 3 is the results of methylene blue polyclonal antibody titers;
figure 4 is a competition inhibition curve for methylene blue antibody.
Detailed Description
The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.
The first embodiment is as follows: preparation of methylene blue hapten
1. Synthesis of 2-amino-5-dimethylaminophenylthiosulfuric acid (intermediate 1)
Adding 18 hydrated aluminum sulfate, sodium thiosulfate, zinc chloride and N, N-dimethyl-p-phenylenediamine into water with the mole number 3-4 times of the total mole number of the reaction raw materials according to the molar ratio of 1:2:1:1, wherein the reaction temperature is 0 ℃, and the stirring time is 5-10 min.
Then, according to the molar ratio of potassium dichromate to 18 hydrated aluminum sulfate of 1: 3.5-4.0, 0.5-1.0 mol/L potassium dichromate solution is dripped into the mixture to react for 2 hours at the temperature of 0 ℃ to obtain a product; slowly heating to room temperature, carrying out suction filtration, sequentially washing a filter cake with the reaction solution with the same volume of water and washing with acetone with the half volume, carrying out oil pump drying, pulping with the reaction solution with the same volume of methyl tert-butyl ether, and carrying out oil pump drying to obtain an intermediate 1.
2. Synthesis of intermediate 2(5- (methyl (phenyl) amino) pentanoic acid)
Adding 10mL of 50-100 mmol/L (preferably 90mmol/L) N-methylaniline, 12mL of 80-120 mmol/L (preferably 90mmol/L)2, 6-lutidine and 15mL of 80-120 mmol/L (preferably 100mmol/L) 5-bromoethyl valerate into a 250mL single-neck flask in sequence, carrying out reflux reaction in 100mL acetonitrile for 30h, evaporating under reduced pressure to remove the solvent, adding 50mL of ethyl acetate, washing with 20mL of water twice, drying with anhydrous sodium sulfate, and carrying out spin-drying at 60 ℃ to obtain an oily substance.
Adding 30mL of 1.5-3.0 mol/L sodium hydroxide solution into the oily substance, refluxing for 2h, cooling to room temperature, adjusting the pH value to 5 by using HCl, extracting for 3 times by using 30mL of ethyl acetate, combining organic phases, drying by using anhydrous sodium sulfate, filtering, evaporating to remove an organic solvent to obtain an initial product, and performing column chromatography separation and purification (eluent: n-hexane/ethyl acetate 1:1) to obtain an intermediate 2.
3. Synthesis of methylene blue hapten
Dissolving the intermediate 1 and the intermediate 2 in a methanol-water solution (volume ratio of 5:2) according to a molar ratio of 1:1, stirring and heating to 50 ℃ under the protection of nitrogen, slowly adding silver carbonate with a molar multiple of 11-3 times (preferably 2 times) of the intermediate, completely adding the displaced nitrogen, heating and refluxing for 2 hours, and finishing the TLC point plate tracking reaction. Filtering with diatomite, spin drying to obtain the initial product, and separating and purifying by column chromatography (eluent: dichloromethane/methanol 9:1) to obtain methylene blue hapten.
The prepared methylene blue hapten is subjected to nuclear magnetic resonance hydrogen spectrum characterization, and the result is shown in figure 1, which shows that the synthesized substance is 3- ((4-carboxybutyl) (methyl) amino) -7- (dimethylamino) -phenothiazine-5-ammonium chloride.
EXAMPLE II preparation of methylene blue immunogen
Putting the methylene blue hapten, Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) prepared in the first example into a beaker according to the molar ratio of 1:1:1, adding a Dimethylformamide (DMF) solution into the beaker, completely dissolving, reacting for 8 hours at room temperature under the condition of magnetic stirring, and standing overnight at 4 ℃; after the reaction, the mixture was centrifuged at 4000 r.p.m. at 4 ℃ for 10min to obtain a supernatant. And adding the supernatant into 5mL of BSA solution with the concentration of 10-20 mg/mL, and reacting for 6h in an ice-water bath. After the reaction, the solution is transferred into a treated dialysis bag, and is dialyzed for 3d at 4 ℃ by taking phosphate buffer solution as dialysate, and the dialysate is replaced 3-4 times per day. After dialysis, the mixture was subjected to dialysis at 4 ℃,
centrifuging for 10min at 4000r.p.m, and taking supernatant, namely the methylene blue immunogen. The coupling result is identified by ultraviolet scanning, and as shown in figure 2, the protein absorption peak after coupling is obviously shifted, thus proving that the coupling is successful.
EXAMPLE III verification of the results of methylene blue immunogenicity
Diluting methylene blue immunogen with phosphate buffer solution to obtain 2mg/mL immunogen solution, adding 100 μ L immunogen solution into 100 μ L Freund's complete adjuvant, mixing, and injecting 3 Balb/c mice of 4 weeks old into abdomen for immunization; after two weeks of immunization, 100 mul of immunogen solution is taken and added with 100 mul of Freund incomplete adjuvant, and after being fully and uniformly mixed, the mixture is injected into the abdomen for immunization; one week later, 100 μ L immunogen solution is taken for tail vein boosting; ten days later, 50. mu.L of the immunogen solution was taken for tail vein injection for booster immunization. On the third day of the last immunization, blood was collected from the orbit, and then subjected to 37 ℃ water bath for 1 hour, and the supernatant was collected, and the serum titer and sensitivity were measured by ELISA.
According to FIG. 3, the immune antibody titers generated by mice No. 2 and 3 were about 64000; the concentration of methylene blue at 50% inhibition (IC50) was 39.0ng/mL by antibody competitive inhibition assay (FIG. 4).
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. A preparation method of methylene blue hapten is characterized by comprising the following steps:
A. preparation of intermediate 1
Under the condition of low temperature, adding reaction raw materials of N, N-dimethyl-p-phenylenediamine, hydrated aluminum sulfate, sodium thiosulfate and zinc chloride into water according to the molar ratio of 1:2:1:1, stirring, dropwise adding a potassium dichromate solution, and continuously stirring for reacting for a certain time; then heating to room temperature, and carrying out post-treatment on the product to obtain an intermediate 1;
B. preparation of intermediate 2
Sequentially adding 50-100 mmol/L N-methylaniline, 80-120 mmol/L2, 6-lutidine and 80-120 mmol/L ethyl 5-bromovalerate solution into a reaction container in a volume ratio of 8-12: 10-15: 13-17, and carrying out reflux reaction in acetonitrile; after removing the solvent, extracting, washing and drying to obtain oily substances;
adding NaOH solution into the oily matter, carrying out reflux reaction, adjusting the pH value with acid, extracting with an organic solvent, separating and purifying to obtain an intermediate 2;
C. preparation of methylene blue hapten
And dissolving the intermediate 1 and the intermediate 2 in a methanol-water solution according to a molar ratio of 1:1, stirring and heating under the protection of gas, adding silver carbonate with the molar number 11-3 times that of the intermediate, performing reflux reaction, separating and purifying to obtain the methylene blue hapten.
2. The method for preparing methylene blue hapten according to claim 1, wherein:
in the step A, the molar ratio of the reaction raw materials to water is 1: 3-4, and after the raw materials are added, the water and the reaction raw materials are stirred for 5-10 min at the temperature of 0 ℃;
the concentration of the potassium dichromate solution is 0.5-1.0 mol/L, and after the dropwise addition is finished, the solution is stirred and reacts for 2 hours at the temperature of 0 ℃.
3. The method for preparing methylene blue hapten according to claim 1, wherein:
and B, in the step A, sequentially carrying out suction filtration, washing, acetone leaching, oil pump drying, pulping by using methyl tert-butyl ether, and oil pump drying again on the product to obtain the intermediate 1.
4. The method for preparing methylene blue hapten according to claim 1, wherein:
and in the step B, after the reflux reaction is carried out in acetonitrile for 30 hours, the solvent is removed through reduced pressure distillation, and the oily matter is obtained through ethyl acetate extraction, water washing twice, anhydrous sodium sulfate drying and spin drying at 60 ℃.
5. The method for preparing methylene blue hapten according to claim 1, wherein:
wherein in the step B, 1.5-3.0 mol/L NaOH solution is added into the oily matter and refluxed for reaction for 2 hours, then HCl is used for adjusting the pH value to 5, ethyl acetate is used for extraction for three times and organic phase is merged, anhydrous sodium sulfate is dried and dried, and the intermediate 2 is obtained after column chromatography separation and purification,
in the process of column chromatography separation, the adopted eluent is a mixed solution of n-hexane and ethyl acetate with the volume ratio of 1: 1.
6. The method for preparing methylene blue hapten according to claim 1, wherein:
wherein, in the step C, the volume ratio of methanol to water in the methanol-water solution is 5:2, the silver carbonate is added after the stirring and the temperature rise to 50 ℃ under the protection of nitrogen, the nitrogen is completely replaced by the silver carbonate, the temperature rise to 50 ℃ is carried out reflux reaction for 2h to obtain a primary product,
the primary product is filtered by diatomite, dried by spinning and separated and purified by column chromatography to obtain the methylene blue hapten,
in the process of column chromatography separation, the adopted eluent is dichloromethane-methanol mixed liquor with the volume ratio of 9: 1.
7. A preparation method of methylene blue immunogen is characterized by comprising the following steps:
step 1: completely dissolving methylene blue hapten, dicyclohexylcarbodiimide and N-hydroxysuccinimide in the same molar number in a dimethylformamide solution, reacting for 8-10 hours at room temperature under the condition of stirring, and standing overnight at low temperature, wherein the preparation method of the methylene blue hapten is as defined in any one of claims 1-6;
step 2: centrifuging at low temperature, taking supernatant, adding the supernatant into 10-20 mg/mL bovine serum albumin solution, and reacting for 6-8 h in ice-water bath;
and step 3: and transferring the solution into a treated dialysis bag after reaction, dialyzing at low temperature for 3-4 days, replacing the dialysate 3-4 times per day, and centrifuging at low temperature to obtain a supernatant, namely the methylene blue immunogen solution.
8. The method of preparing a methylene blue immunogen according to claim 7, wherein:
wherein, in the step 1, after the room temperature reaction, the reaction solution is placed at 4 ℃ and stands overnight;
in the step 2, the centrifugal conditions of the reaction solution are 4 ℃,4000rpm and 10 min;
and 3, in the dialysis process, performing low-temperature dialysis by taking phosphate buffer solution as dialysate, performing dialysis for 3d at 4 ℃, replacing the dialysate for 3-4 times per day, centrifuging for 10min at 4000rpm at 4 ℃ after dialysis, and taking supernatant, namely the methylene blue immunogen.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113372295A (en) * | 2021-07-08 | 2021-09-10 | 河北大学 | Phenothiazine hapten, complete antigen, preparation method and application thereof |
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| CN113372295A (en) * | 2021-07-08 | 2021-09-10 | 河北大学 | Phenothiazine hapten, complete antigen, preparation method and application thereof |
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Application publication date: 20200619 |