A method for synthesizing patulin hapten

Abstract

The present invention belongs to the technical field of fine chemical synthesis, and discloses a method for synthesizing patulin hapten, comprising the following steps: S1, synthesis of compound 01→S2, synthesis of compound 02→S3, synthesis of compound 03→S4, synthesis of compound 04→S5, synthesis of compound 05→S6, synthesis of compound a→S7, synthesis of compound 06. In this scheme, an artificial patulin hapten molecule with immunogenicity is designed and synthesized by a chemical synthesis method, thereby inducing the production of antibodies with strong specificity, high immunity and high sensitivity, providing more options for the synthesis method of patulin hapten, and the present invention can obtain a hapten with a higher degree of overlap with patulin in structure, thereby improving the coupling effect of the hapten and the protein, and the obtained patulin hapten can be used as a precursor of a complete antigen in an enzyme-linked immunosorbent assay method, and has a high use value.

Description

Technical Field

[0001] The invention relates to the technical field of fine chemical synthesis, and more specifically to a method for synthesizing patulin hapten. Background Art

[0002] Patulin (PAT), also known as patulin, patulin, and coral penicillin, is a secondary metabolite produced by fungi such as Aspergillus and Penicillium. It is widely present in various moldy fruits and silage. Its molecular formula is C7H6O4. It is a toxic secondary metabolite of fungi. It has high heat resistance and acid resistance. It can exist stably under acidic and heated conditions, so it is difficult to be destroyed during food processing. Studies have confirmed that patulin is not only toxic to fungi and bacteria, but also to animals and higher plants. Because patulin is very stable and widely present in agricultural products such as fruits and vegetables, it will cause quality problems that cannot be ignored in the production of agricultural products.

[0003] The specific binding of special antigens and antibodies, followed by labeling technology to achieve quantitative and qualitative analysis, is called an immunological detection method. Immunological detection methods have many advantages. Microbiologists have also tried to establish an immunological detection method for patulin. However, due to the special molecular structure of patulin, it is not easy to modify. Therefore, the patulin antibodies currently obtained can only slightly block the patulin, and the serum titer is not high.

[0004] There are only a few reports on patulin immunoassay abroad. Champdoré et al. reported the construction of two structural analogs of patulin by chemical synthesis. The synthesis route is shown in the attached figure of the specification. Figure 2 As shown,

[0005] This method obtains the patulin hapten by protecting the sugar end and then performing ketal reaction, oxidation reaction, ylide reaction, reduction reaction, and reaction of anhydride and alcohol. However, experiments have found that there are problems with this synthesis scheme: the unsaturated lactone in the molecular structure is unstable, and the hydroxyl group at the 7-position is easily eliminated spontaneously or under alkaline conditions (intramolecular elimination, 90%) after esterification, resulting in the failure to obtain the patulin hapten in the final reaction. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for synthesizing patulin hapten.

[0007] To solve the above problems, the present invention adopts the following technical solutions.

[0008] A method for synthesizing patulin hapten comprises the following steps:

[0009] S1. Synthesis of Compound 01: L-arabinose was suspended in freshly distilled benzyl alcohol and cooled in an ice-salt bath, and then dry hydrogen chloride gas was introduced to saturation, and the reaction was purified to obtain Compound 01;

[0010] S2. Synthesis of compound 02: Compound 01 and anhydrous copper sulfate were suspended in acetone and stirred under the condition of nitrogen as protective gas, concentrated sulfuric acid was added, and the pH was neutralized to 8.0 with dry ammonia gas. After filtering through a sand plate, the organic solvent was removed by low-pressure distillation, and the resultant was dissolved with ether, and the insoluble matter was removed by filtration, and the acetone compound 02 was obtained by purification;

[0011] S3. Synthesis of compound 03: Compound 02 was dissolved in ethanol and added to carbon tetrachloride, and hydrated ruthenium dioxide and water were added under stirring.

[0012] Then, the sodium periodate solution was added dropwise to the above-mentioned mixed solution under stirring. The mixed solution was vigorously stirred during the addition process and the pH value was adjusted to 6-7 with saturated sodium bicarbonate to purify and obtain ketone compound 03;

[0013] S4. Synthesis of compound 04: Compound 03 and ylide were dissolved in dry toluene, stirred under nitrogen atmosphere, and then vacuum evaporated to remove the organic solvent, and the obtained compound 04 was purified;

[0014] S5. Synthesis of compound 05: Compound 04 was dissolved in methanol, hydrochloric acid was added, the mixture was heated under reflux, methanol was removed by vacuum evaporation, the hydrochloric acid was neutralized with saturated sodium bicarbonate, and compound 05 was obtained by extraction and purification with dichloromethane;

[0015] S6. Synthesis of compound a: benzyl alcohol was dissolved in ether, and then DMAP and succinic anhydride were added. After stirring at room temperature for 8 hours, succinic anhydride was added continuously, and the reaction was continued for 15 hours to obtain compound a.

[0016] S7. Synthesis of compound 06: Compound 05 was dissolved in THF, DCC was added, and then compound a dissolved in CH2Cl2 was added, and acetic anhydride was added to continue the reaction. The organic solvent was evaporated in vacuo, and the residue was dissolved in ethyl acetate and then purified by silica gel chromatography to obtain compound 06, i.e., patulin hapten.

[0017] Compared with the prior art, the advantages of the present invention are:

[0018] (1) The synthesis method of the present invention can obtain a hapten that has a higher degree of structural overlap with patulin, thereby improving the coupling effect of the hapten and the protein.

[0019] (2) The patulin hapten obtained by the synthesis method of the present invention can be used as a precursor of a complete antigen in an enzyme-linked immunosorbent assay method and has a high use value.

[0020] (3) The present invention optimizes the reaction of converting carbonyl groups into olefins via phosphine ylides and uses toluene, which can be metabolized by humans, as a solvent, which is more environmentally friendly and easy to operate.

[0021] (4) The synthetic method of the present invention has a high yield and the obtained product has a high chemical purity.

[0022] (5) The present invention overcomes the problem of unstable hydroxyl structure prone to intramolecular elimination and adopts a new synthesis strategy to synthesize the key intermediate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The general route for the synthesis steps of patulin hapten is shown;

[0024] Figure 2 The invention discloses a synthetic route of patulin hapten disclosed in the prior art. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments, and all other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative work are within the scope of protection of the present invention.

[0026] See also Figure 1 , a method for synthesizing patulin hapten, comprising the following steps:

[0027] S1. Synthesis of compound 01:

[0028] L-arabinose (0.313 mol, 47 g) was suspended in 250 mL of freshly distilled benzyl alcohol and cooled in an ice-salt bath, then dry hydrogen chloride gas was passed through until saturated;

[0029] The reaction mixture was then stirred at room temperature for 18 h to obtain white syrupy crystals, which were then purified to obtain crystals of compound 01 with a yield of 70%.

[0030] S2. Synthesis of compound 02:

[0031] Compound 01 (0.069 mol, 16.6 g) and anhydrous copper sulfate (0.41 mol, 65 g) were suspended in 800 mL of analytical grade acetone and stirred in the presence of nitrogen, and then 1 mL of concentrated sulfuric acid was added to obtain a mixture;

[0032] The mixture was stirred at room temperature for 18 h and then neutralized with dry ammonia gas to pH 8.0, and the solution turned bright blue;

[0033] After filtering through a sand plate, the organic solvent was removed by low-pressure distillation to form a syrupy substance, which was dissolved in 150 mL of ether, and the insoluble, unchanged 01 was removed by filtration, and the ether was removed by low-pressure distillation to form crystals;

[0034] Then, it was recrystallized from ether-light petroleum at -10°C to obtain acetone compound 02 in a yield of 72%.

[0035] S3. Synthesis of compound 03:

[0036] Compound 02 (24.4 mmol, 6 g) was dissolved in ethanol and added to 107 mL of carbon tetrachloride, and then 0.11 g of hydrated ruthenium dioxide and 10 mL of water were added under stirring;

[0037] Dissolve 6 g of 0.25 mol / L sodium periodate in 114 mL of water to obtain a mixed solution, and add it drop by drop to the above solution under stirring over 1.5 h;

[0038] The mixture was stirred vigorously during the addition and the pH was adjusted to 6-7 with saturated sodium bicarbonate, but after all was added, the aqueous phase showed a yellow-green color;

[0039] The organic phase was extracted three times with 50 mL of water, then dried and evaporated under low pressure to obtain a grayish yellow-brown syrup (4.5 g, 75%), which was purified to obtain ketone compound 03 (3.3 g, 66%) as a syrup.

[0040] S4. Synthesis of compound 04:

[0041] Take 10g (35.9mmol) of compound 03 and 13.7g (40.9mmol) of ylide and dissolve them in 150mL of dry toluene, stir for 5 hours under the protection of nitrogen, and then evaporate the organic solvent in vacuo. Add 120mL of light petroleum and let it stand overnight, then filter out the precipitate;

[0042] The organic solvent in the filtrate was removed under vacuum evaporation conditions, and the residue was purified by silica gel chromatography (chromatographic liquid: light petroleum ethyl acetate = 5:1) to obtain compound 04 as an oily product with a mass of 8.8 g and a yield of 75%.

[0043] S5. Synthesis of compound 05:

[0044] Dissolve 2 g (5.982 mmol) of the mixture of compound 04 in 60 mL of methanol, add 2 mL of 1.2 mol / L hydrochloric acid, and then reflux and heat for 2 h;

[0045] After cooling, the excess hydrochloric acid was purified and neutralized with 20 mL of saturated sodium bicarbonate, and the liquid phase was extracted three times with 60 mL of dichloromethane and filtered;

[0046] The organic solvent was then evaporated in vacuo to obtain 1.2 g of compound 05 in the form of powder with a yield of 76%.

[0047] S6. Synthesis of compound a:

[0048] Benzyl alcohol (1.35 mmol, 145 mg) was dissolved in ether, and then DMAP (0.137 mmol, 16.7 mg) and succinic anhydride (1.37 mmol, 1.37 g) were added. After stirring at room temperature for 8 h, succinic anhydride (6.85 mmol, 685 mg) was added. The reaction was continued for 15 h to obtain compound a with a yield of 80%.

[0049] S7. Synthesis of compound 06:

[0050] Dissolve compound 05 (1.37 mmol, 350 mg) in dry THF, add DCC, then add compound a dissolved in CH2Cl2, and stir at room temperature in the dark for 24 h;

[0051] The organic solvent was evaporated in vacuo, and the residue was dissolved in 25 mL of ethyl acetate, washed with water three times, and then purified by silica gel column chromatography to obtain 330 mg of compound 06.

[0052] In summary: the present invention modifies and improves the prior art, synthesizes compound a, and then uses the DCC condensation method to obtain the patulin hapten intermediate;

[0053] An artificial patulin hapten molecule with immunogenicity is designed and synthesized by chemical synthesis, thereby inducing the production of antibodies with strong specificity, high immunity and high sensitivity, providing more options for the synthesis method of patulin hapten.

[0054] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for synthesizing patulin hapten, characterized in that: The following steps are involved: S1. Synthesis of Compound 01: L-arabinose was suspended in freshly distilled benzyl alcohol and cooled in an ice-salt bath, and then dry hydrogen chloride gas was introduced to saturation, and the reaction was purified to obtain Compound 01; S2. Synthesis of compound 02: Compound 01 and anhydrous copper sulfate were suspended in acetone and stirred under the condition of nitrogen as protective gas, concentrated sulfuric acid was added, and the pH was neutralized to 8.0 with dry ammonia gas. After filtering through a sand plate, the organic solvent was removed by low-pressure distillation, and the resultant was dissolved with ether, and the insoluble matter was removed by filtration, and the acetone compound 02 was obtained by purification; S3. Synthesis of compound 03: Compound 02 was dissolved in ethanol and added to carbon tetrachloride, and hydrated ruthenium dioxide and water were added under stirring. Then, the sodium periodate solution was added dropwise to the above-mentioned mixed solution under stirring. The mixed solution was vigorously stirred during the addition process and the pH value was adjusted to 6-7 with saturated sodium bicarbonate to purify and obtain ketone compound 03; S4. Synthesis of compound 04: Compound 03 and ylide were dissolved in dry toluene, stirred under nitrogen atmosphere, and then vacuum evaporated to remove the organic solvent, and the obtained compound 04 was purified; S5. Synthesis of compound 05: Compound 04 was dissolved in methanol, hydrochloric acid was added, the mixture was heated under reflux, methanol was removed by vacuum evaporation, the hydrochloric acid was neutralized with saturated sodium bicarbonate, and compound 05 was obtained by extraction and purification with dichloromethane; S6. Synthesis of compound a: benzyl alcohol was dissolved in ether, and then DMAP and succinic anhydride were added. After stirring at room temperature for 8 hours, succinic anhydride was added continuously, and the reaction was continued for 15 hours to obtain compound a. S7. Synthesis of compound 06: Compound 05 was dissolved in THF, DCC was added, and then compound a dissolved in CH2Cl2 was added, and acetic anhydride was added to continue the reaction. The organic solvent was evaporated in vacuo, and the residue was dissolved in ethyl acetate, and then purified by silica gel column chromatography to obtain compound 06; The structural formula of the compound 01 is The structural formula of the compound 02 is The structural formula of the compound 03 is The structural formula of the compound 04 is The structural formula of the compound 05 is The structural formula of the compound 06 is The structural formula of the compound a is

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