阅读说明：本技术 一种α-半乳糖抗原活性前体及其合成方法和应用 (Alpha-galactose antigen active precursor and synthesis method and application thereof ) 是由 曹学峰 李学超 丁冯玲 王鹏 于 2020-05-14 设计创作，主要内容包括：本发明提出了一种α-半乳糖抗原活性前体及其合成方法和应用。本发明的合成方法包括以下步骤：在化合物I中加入尿苷二磷酸葡萄糖、可溶性镁盐和Tris-HCl缓冲液,配制成水溶液；在水溶液中添加UDP-葡萄糖-4-差向异构酶和α-1,3-半乳糖基转移酶,反应,得初产物；在初产物中添加β-半乳糖苷酶,反应,得化合物II；将化合物II加氢反应,得化合物III；化合物III与方型酸二乙酯在室温下搅拌反应,浓缩,纯化,得α-半乳糖抗原活性前体。本发明在糖基转移酶的作用下使糖链延长,通过化学反应转化为活性前体；该合成方法效率高,工艺流程短,操作简单,分离方便,产率高,易于实现产业化,可用于志贺样毒素检测试剂盒。(The invention provides an alpha-galactose antigen active precursor and a synthesis method and application thereof. The synthesis method comprises the following steps: adding uridine diphosphate glucose, soluble magnesium salt and Tris-HCl buffer solution into the compound I to prepare aqueous solution; adding UDP-glucose-4-epimerase and alpha-1, 3-galactosyltransferase into an aqueous solution, and reacting to obtain a primary product; adding beta-galactosidase into the primary product, and reacting to obtain a compound II; performing hydrogenation reaction on the compound II to obtain a compound III; and (3) stirring the compound III and the squaric acid diethyl ester at room temperature for reaction, concentrating and purifying to obtain the alpha-galactose antigen active precursor. The invention prolongs the sugar chain under the action of glycosyltransferase, and converts the sugar chain into an active precursor through chemical reaction; the synthesis method has the advantages of high efficiency, short process flow, simple operation, convenient separation, high yield and easy realization of industrialization, and can be used for a shiga-like toxin detection kit.)

1. A method for synthesizing an alpha-galactose antigen active precursor, which is characterized by comprising the following steps:

1) taking a compound I with a structural formula I, adding uridine diphosphate glucose, soluble magnesium salt and Tris-HCl buffer solution to prepare aqueous solution; the molar ratio of uridine diphosphate glucose to the compound I is 1.3-3.0:1, the molar ratio of soluble magnesium salt to the compound I is 10-30:1, the molar ratio of Tris-HCl buffer solution to the compound I is 20-40:1, and the pH value of the Tris-HCl buffer solution is 5-9;

r is hydroxyl, amino, acetamido, trifluoroacetamido, n is natural number, and the value is 1-10;

2) adding UDP-glucose-4-epimerase and alpha-1, 3-galactosyltransferase into the aqueous solution, wherein the addition amount of the UDP-glucose-4-epimerase is 0.3-3.0%, the addition amount of the alpha-1, 3-galactosyltransferase is 0.2-2.0%, and reacting for 5-100h at 15-40 ℃ to obtain an initial product;

3) adjusting the pH value of the primary product to 6.5-9.0, adding beta-galactosidase, wherein the addition amount of the beta-galactosidase is 0.5-5.0%, reacting at 20-40 ℃ for 1-24h, and purifying to obtain a compound II with a structural formula II;

4) dissolving the compound II in an organic solvent, adding a catalyst, reacting at room temperature for 20-30h under the condition of hydrogen, filtering, and concentrating to obtain a compound III, wherein the structural formula of the compound III is shown as the formula III;

5) dissolving the compound III in an organic solvent to obtain a solution A, adjusting the pH value of the solution A to 7-9 by adopting organic alkali, adding diethyl type acid into the solution A, stirring and reacting for 10-20h at room temperature, concentrating and purifying to obtain an alpha-galactose antigen active precursor, wherein the structural formula of the alpha-galactose antigen active precursor is shown as a formula IV;

2. the method for synthesizing an alpha-galactose antigen active precursor according to claim 1, wherein:

in the step 3), before purification, absolute ethyl alcohol with the same volume as the reaction solution is added into the reaction solution, and incubation is carried out for 20-60min, wherein the temperature of the absolute ethyl alcohol is 2-6 ℃, and the incubation temperature is 2-6 ℃.

3. The method for synthesizing an alpha-galactose antigen active precursor according to claim 1, wherein:

in the step 4), the organic solvent is methanol, the catalyst is palladium carbon, the mass percentage of palladium in the palladium carbon is 10%, and the mass ratio of the palladium carbon to the compound II is 0.01-0.1: 1.

4. The method for synthesizing an alpha-galactose antigen active precursor according to claim 1, wherein:

in the step 5), the organic solvent used in the solution A is a mixed solution of water and ethanol, and the volume ratio of the water to the ethanol is 2: 1; the organic base is any one of diethylamine, triethylamine, quaternary amine, aniline and benzylamine.

5. The method for synthesizing the alpha-galactose antigen active precursor according to any one of claims 1 to 4, wherein the compound I is prepared by:

s1: taking a compound V with a structural formula shown as a formula V as a raw material, and carrying out reflux reaction for 4-8h at the temperature of 135-140 ℃ under the action of carboxylic anhydride and sodium carboxylate to protect hydroxyl in the compound V so as to obtain a compound VI with a structural formula shown as a formula VI;

s2: dissolving the compound VI obtained in the step S1 in an organic solvent, adding p-toluene thiophenol, and reacting at room temperature for 2-4h under the action of a catalyst to obtain a compound VII with the structural formula shown in the formula VII;

s3: dissolving the compound VII obtained in the step S2 in an organic solvent, adding polyethylene glycol azide, and performing coupling reaction for 0.5-1.5h at room temperature under the action of a catalyst to obtain a compound VIII with a structural formula shown in the formula VIII;

s4: and (4) dissolving the compound VIII obtained in the step S3 in an organic solvent, adding sodium alkyl alkoxide, adjusting the pH value to 9-10, and carrying out substitution reaction at room temperature for 10-20h to obtain a compound I.

6. The method for synthesizing an alpha-galactose antigen active precursor according to claim 5, wherein:

in step S1, the carboxylic anhydride is acetic anhydride and the sodium carboxylate is sodium acetate.

7. The method for synthesizing an alpha-galactose antigen active precursor according to claim 5, wherein:

in step S2, the organic solvent used is dichloromethane, and the catalyst used is boron trifluoride diethyl etherate.

8. The method for synthesizing an alpha-galactose antigen active precursor according to claim 5, wherein:

in the step S3, the organic solvent is dichloromethane, the catalyst is silver trifluoromethanesulfonate and tetrabutylammonium iodide, and the molar ratio of the silver trifluoromethanesulfonate to the tetrabutylammonium iodide is 1: 20-30;

preferably, in step S4, the organic solvent used is methanol, and the sodium alkyl alkoxide used is sodium methoxide.

9. An alpha-galactose antigen active precursor characterized by:

the precursor of alpha-galactose antigen is prepared by the method for synthesizing the precursor of alpha-galactose antigen according to any one of claims 1 to 8.

10. Use of an active precursor of an alpha-galactose antigen, characterized in that:

the alpha-galactose antigen active precursor prepared by the method for synthesizing the alpha-galactose antigen active precursor according to any one of claims 1 to 8 is used in a shiga-like toxin detection kit.

Technical Field

The invention relates to the technical field of carbohydrate synthesis methods, in particular to an alpha-galactose antigen active precursor and a synthesis method and application thereof.

Background

Monoclonal antibody drugs are taking an important position in the biopharmaceutical industry, and in the global market, antibody drugs are becoming the focus of international pharmaceutical industry competition. Among the antibody drugs, the development and application of therapeutic monoclonal antibody drugs represented by rituximab and trastuzumab become the most successful representatives in the field of tumor molecular targeted therapy. The monoclonal antibody drug takes tumor-targeted antigen as a molecular target, and mediates multiple mechanisms such as complement-dependent cytotoxicity, antibody-dependent cytotoxicity and the like to kill cancer cells by combining with the antigen. In the process of killing cancer cells by monoclonal antibody drugs, the selection of proper target antigen is the prerequisite and key for successfully designing and developing tumor therapeutic monoclonal antibodies.

Alpha-galactose antigen active precursor (alpha-gal for short in English) is a carbohydrate epitope widely present in non-primate mammals such as pigs, cows and mice, however, human alpha-1, 3Galactosyltransferase (alpha-1, 3 galctosyltransferase, alpha-1, 3GT) gene functions are inactivated without expressing alpha-galactosyl (alpha-gal) epitope, but a large amount of anti-alpha-gal antibody naturally exists; the results of xenotransplantation studies suggest that the re-expression of the xenotransplantation antigen α -gal on human tumor cells may induce an anti-tumor effect similar to that of host-versus-graft hyperacute rejection.

Shiga-like toxins are also known as VT Toxins (VT), O157: H7 Escherichia coli produce shiga-like toxins. Since 1982, the incidence of Escherichia coli O157: H7 infection has been increasing in developed countries. In 1996, between 5 and 8 months, the greatest outbreak worldwide occurred in japan, and 10000 people infected, and nearly 10 people died, and received worldwide attention. The alpha-galactose antigen active precursor has an inhibiting effect on Shiga-like toxin, and is expected to be used as a lead molecule to be developed into a high-sensitivity diagnostic reagent for early diagnosis of Shiga toxin-producing bacteria infection.

At present, in the prior art, the alpha-galactose antigen active precursor is mostly prepared by pure chemical synthesis, specifically, oligosaccharide chains are prepared by chemical synthesis, then protected oligosaccharide molecules and linker molecules are coupled by a chemical coupling method, and finally, the ends of the linker molecules are converted into activated groups. The chemical method needs complicated operations of protecting functional groups and protecting de-functional groups in the process of synthesizing the alpha-galactose antigen active precursor, has long process route, difficult separation and low yield, and is not beneficial to realizing industrial production.

Disclosure of Invention

The invention provides an alpha-galactose antigen active precursor, a synthesis method and application thereof, aiming at solving the problems of long process route, difficult separation and low yield caused by fussy functional group protection and functional group removing operation in the process of synthesizing the alpha-galactose antigen active precursor by a chemical method in the prior art.

In order to solve the technical problem, the technical scheme of the invention is realized as follows:

in one aspect, the present invention provides a method for synthesizing an alpha-galactose antigen active precursor, comprising the steps of:

1) taking a compound I with a structural formula I, adding uridine diphosphate glucose, soluble magnesium and a Tris-HCl buffer solution to prepare an aqueous solution; the molar ratio of uridine diphosphate glucose to the compound I is 1.3-3.0:1, the molar ratio of soluble magnesium to the compound I is 10-30:1, the molar ratio of Tris-HCl buffer solution to the compound I is 20-40:1, and the pH value of the Tris-HCl buffer solution is 5-9;

r is hydroxyl, amino, acetamido, trifluoroacetamido, n is natural number, and the value is 1-10;

2) adding UDP-glucose-4-epimerase and alpha-1, 3-galactosyltransferase into the aqueous solution, wherein the addition amount of the UDP-glucose-4-epimerase is 0.3-3.0%, the addition amount of the alpha-1, 3-galactosyltransferase is 0.2-2.0%, and reacting for 5-100h at 15-40 ℃ to obtain an initial product;

3) adjusting the pH value of the primary product to 6.5-9.0, adding beta-galactosidase, wherein the addition amount of the beta-galactosidase is 0.5-5.0%, reacting at 20-40 ℃ for 1-24h, and purifying to obtain a compound II with a structural formula II;

4) dissolving the compound II in an organic solvent, adding a catalyst, reacting at room temperature for 20-30h under the condition of hydrogen, filtering, and concentrating to obtain a compound III, wherein the structural formula of the compound III is shown as the formula III;

5) dissolving the compound III in an organic solvent to obtain a solution A, adjusting the pH value of the solution A to 7-9 by adopting organic alkali, adding diethyl type acid into the solution A, stirring and reacting for 10-20h at room temperature, concentrating and purifying to obtain an alpha-galactose antigen active precursor, wherein the structural formula of the alpha-galactose antigen active precursor is shown as a formula IV;

in the method for synthesizing the alpha-galactose antigen active precursor of the present invention, first, a sugar chain is extended by the catalytic action of alpha-1, 3-galactosyltransferase; wherein the uridine diphosphate glucose (UDP-glucose) is epimerized into uridine diphosphate galactose (UDP-galactose) by the action of UDP-glucose-4-epimerase (GalE), or uridine diphosphate galactose (UDP-galactose) can be used as it is; then, the precursor is converted into an active precursor by a chemical reaction method and reacted with squaric acid diethyl ester. The method integrates the structural universality of a chemical method and the high-efficiency specificity of the enzyme method through the organic combination of the enzyme method and the chemical method, can efficiently synthesize the alpha-galactose antigen active precursor and the derivatives thereof, has strong universality, and has the advantages of short process flow, simple operation, convenient separation, high yield and easy realization of industrialization; the antigen active precursor has an inhibiting effect on shiga-like toxin, and can be used in a shiga-like toxin detection kit.

In a preferred embodiment, in step 3), before purification, an equal volume of absolute ethanol to the reaction solution is added to the reaction solution, and the reaction solution is incubated for 20-60min, wherein the temperature of the absolute ethanol is 2-6 ℃ and the temperature of the incubation is 2-6 ℃. In the process of extending sugar chain by enzyme method, beta-galactosidase (LacZ) is used to remove unreacted disaccharide by hydrolysis, so as to improve the purity of sugar.

In a preferred embodiment, in step 4), the organic solvent is methanol, the catalyst is palladium on carbon, the mass percentage of palladium in the palladium on carbon is 10%, and the mass ratio of the palladium on carbon to the compound II is 0.01-0.1: 1. The palladium-carbon catalyst is used for hydrogenating the tail end of the trisaccharide compound, and the method is convenient to operate and easy to realize industrialization.

In a preferred embodiment, in the step 5), the organic solvent used for the solution a is a mixed solution of water and ethanol, and the volume ratio of the water to the ethanol is 2: 1; the organic base is any one of diethylamine, triethylamine, quaternary amine, aniline and benzylamine. In the invention, the compound III and the squaric acid diethyl ester are reacted in the solution, the reaction is easy to control, and the reaction speed is also improved.

As a preferred embodiment, the compound I is prepared by a process comprising:

s1: taking a compound V with a structural formula shown as a formula V as a raw material, and carrying out reflux reaction for 4-8h at the temperature of 135-140 ℃ under the action of carboxylic anhydride and sodium carboxylate to protect hydroxyl in the compound V so as to obtain a compound VI with a structural formula shown as a formula VI;

s2: dissolving the compound VI obtained in the step S1 in an organic solvent, adding p-toluene thiophenol, and reacting at room temperature for 2-4h under the action of a catalyst to obtain a compound VII with the structural formula shown in the formula VII;

s3: dissolving the compound VII obtained in the step S2 in an organic solvent, adding polyethylene glycol azide, and performing coupling reaction for 0.5-1.5h at room temperature under the action of a catalyst to obtain a compound VIII with a structural formula shown in the formula VIII;

s4: and (4) dissolving the compound VIII obtained in the step S3 in an organic solvent, adding sodium alkyl alkoxide, adjusting the pH value to 9-10, and carrying out substitution reaction at room temperature for 10-20h to obtain a compound I.

The compound I is synthesized by a chemical method, the chemical structure universality is fully exerted, and the universality of the alpha-galactose antigen active precursor and the derivative thereof is further improved.

In a preferred embodiment, in step S1, the carboxylic anhydride is acetic anhydride and the sodium carboxylate is sodium acetate. In the hydroxyl protection process of disaccharide, acetic anhydride is used as protection, and the reaction is fully carried out under the action of sodium acetate, so that additional impurities are not brought, and the purity and the yield of the product are improved.

In a preferred embodiment, in step S2, the organic solvent used is dichloromethane, and the catalyst used is boron trifluoride diethyl etherate. In the process of substituting the disaccharide active group, dichloromethane is used as an organic solvent, boron trifluoride ethyl ether is used as a catalyst, and the method has the advantages of high reaction speed and high efficiency.

In a preferred embodiment, in step S3, the organic solvent is dichloromethane, the catalyst is silver trifluoromethanesulfonate and tetrabutylammonium iodide, and the molar ratio of the silver trifluoromethanesulfonate to the tetrabutylammonium iodide is 1: 20-30; preferably, in step S4, the organic solvent used is methanol, and the sodium alkyl alkoxide used is sodium methoxide. In the coupling reaction process of the disaccharide and the connecting arm, dichloromethane is used as an organic solvent, silver trifluoromethanesulfonate and tetrabutylammonium iodide are used as catalysts, so that molecules of the connecting arm are accurately and effectively connected to an active substituent group of the disaccharide; meanwhile, in the deprotection process of the compound VIII, under the protection action of sodium methoxide, hydroxyl in methanol and sodium ions on the compound VIII are subjected to substitution reaction, so that the compound I is obtained.

In another aspect, the present invention provides an α -galactose antigen precursor prepared according to the method for synthesizing an α -galactose antigen precursor according to any one of the above aspects. The alpha-galactose antigen active precursor has strong universality, short synthetic process flow, simple operation, convenient separation, high yield and easy realization of industrialization.

In still another aspect, the invention relates to an application of the alpha-galactose antigen active precursor, wherein the alpha-galactose antigen active precursor prepared by the synthesis method of the alpha-galactose antigen active precursor is used in a shiga-like toxin detection kit. The antigen active precursor has an inhibiting effect on shiga-like toxin, and can be used in a shiga-like toxin detection kit.

Compared with the prior art, the invention has the beneficial effects that: in the method for synthesizing the alpha-galactose antigen active precursor of the present invention, first, a sugar chain is extended by the catalytic action of alpha-1, 3-galactosyltransferase; then, the precursor is converted into an active precursor through a chemical reaction method and a square diethyl ester; the method integrates the structural universality of a chemical method and the high-efficiency specificity of the enzyme method through the organic combination of the enzyme method and the chemical method, can efficiently synthesize the alpha-galactose antigen active precursor and the derivatives thereof, has strong universality, and has the advantages of short process flow, simple operation, convenient separation, high yield and easy realization of industrialization; the antigen active precursor has an inhibiting effect on shiga-like toxin, and can be used in a shiga-like toxin detection kit.

Drawings

FIG. 1 shows α -galactose antigen active precursor obtained by the present invention¹H-NMR chart;

FIG. 2 shows α -galactose antigen active precursor obtained by the present invention¹³C-NMR chart;

FIG. 3 is a mass spectrum of the alpha-galactose antigen active precursor obtained by the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention relates to a method for synthesizing an alpha-galactose antigen active precursor, which comprises the following steps:

1) taking a compound I with a structural formula I, adding uridine diphosphate glucose, soluble magnesium and a Tris-HCl buffer solution to prepare an aqueous solution; the molar ratio of uridine diphosphate glucose to the compound I is 1.3-3.0:1, the molar ratio of soluble magnesium to the compound I is 10-30:1, the molar ratio of Tris-HCl buffer solution to the compound I is 20-40:1, and the pH value of the Tris-HCl buffer solution is 5-9;

r is hydroxyl, amino, acetamido, trifluoroacetamido, n is natural number, and the value is 1-10;

2) adding UDP-glucose-4-epimerase and alpha-1, 3-galactosyltransferase into the aqueous solution, wherein the addition amount of the UDP-glucose-4-epimerase is 0.3-3.0%, the addition amount of the alpha-1, 3-galactosyltransferase is 0.2-2.0%, and reacting for 5-100h at 15-40 ℃ to obtain an initial product;

3) adjusting the pH value of the primary product to 6.5-9.0, adding beta-galactosidase, wherein the addition amount of the beta-galactosidase is 0.5-5.0%, reacting at 20-40 ℃ for 1-24h, and purifying to obtain a compound II with a structural formula II;

4) dissolving the compound II in an organic solvent, adding a catalyst, reacting at room temperature for 20-30h under the condition of hydrogen, filtering, and concentrating to obtain a compound III, wherein the structural formula of the compound III is shown as the formula III;

5) dissolving the compound III in an organic solvent to obtain a solution A, adjusting the pH value of the solution A to 7-9 by adopting organic alkali, adding diethyl type acid into the solution A, stirring and reacting for 10-20h at room temperature, concentrating and purifying to obtain an alpha-galactose antigen active precursor, wherein the structural formula of the alpha-galactose antigen active precursor is shown as a formula IV;

preferably, in the step 3), before purification, absolute ethyl alcohol with the same volume as the reaction solution is added into the reaction solution, and the reaction solution is incubated for 20-60min, wherein the temperature of the absolute ethyl alcohol is 2-6 ℃, and the temperature of the incubation is 2-6 ℃.

Further, in the step 4), the organic solvent is methanol, the catalyst is palladium carbon, the mass percentage of palladium in the palladium carbon is 10%, and the mass ratio of the palladium carbon to the compound II is 0.01-0.1: 1.

Specifically, in the step 5), the organic solvent used in the solution A is a mixed solution of water and ethanol, and the volume ratio of the water to the ethanol is 2: 1; the organic base is any one of diethylamine, triethylamine, quaternary amine, aniline and benzylamine.

Again preferably, the preparation method of the compound I is:

s1: taking a compound V with a structural formula shown as a formula V as a raw material, and carrying out reflux reaction for 4-8h at the temperature of 135-140 ℃ under the action of carboxylic anhydride and sodium carboxylate to protect hydroxyl in the compound V so as to obtain a compound VI with a structural formula shown as a formula VI;

s2: dissolving the compound VI obtained in the step S1 in an organic solvent, adding p-toluene thiophenol, and reacting at room temperature for 2-4h under the action of a catalyst to obtain a compound VII with the structural formula shown in the formula VII;

s3: dissolving the compound VII obtained in the step S2 in an organic solvent, adding polyethylene glycol azide, and performing coupling reaction for 0.5-1.5h at room temperature under the action of a catalyst to obtain a compound VIII with a structural formula shown in the formula VIII;

s4: and (4) dissolving the compound VIII obtained in the step S3 in an organic solvent, adding sodium alkyl alkoxide, adjusting the pH value to 9-10, and carrying out substitution reaction at room temperature for 10-20h to obtain a compound I.

Preferably, in step S1, the carboxylic anhydride is acetic anhydride, and the sodium carboxylate is sodium acetate.

Preferably, in step S2, the organic solvent used is dichloromethane, and the catalyst used is boron trifluoride ethyl ether.

Preferably, in the step S3, the organic solvent used is dichloromethane, the catalyst used is silver trifluoromethanesulfonate and tetrabutylammonium iodide, and the molar ratio of the silver trifluoromethanesulfonate to the tetrabutylammonium iodide is 1: 20-30; preferably, in step S4, the organic solvent used is methanol, and the sodium alkyl alkoxide used is sodium methoxide.

An alpha-galactose antigen active precursor of the present invention, which is prepared according to any one of the above methods for synthesizing an alpha-galactose antigen active precursor.

The application of the alpha-galactose antigen active precursor is characterized in that the alpha-galactose antigen active precursor prepared by the synthesis method of any one of the alpha-galactose antigen active precursors is used in a Shiga-like toxin detection kit.