A method for synthesizing thiol-modified oligosaccharides

Abstract

The present invention belongs to the field of sugar, relates to the synthesis and separation of oligosaccharides, and specifically to a method for synthesizing thiol-modified oligosaccharides. Lactose is made into a thiol-containing glycosyl acceptor by a chemical method, and the thiol-containing glycosyl acceptor is coupled with a monosaccharide by an enzymatic method to synthesize thiol-modified oligosaccharides; thiol-modified silica is added to the system after the enzymatic reaction, and the thiol-modified silica and the thiol-modified oligosaccharides are formed into a disulfide bond to obtain a silica-oligosaccharide complex, and the silica-oligosaccharide complex is separated and extracted and then washed with double distilled water, and then the disulfide bond of the silica-oligosaccharide complex is opened with a β-mercaptoethanol solution, and the silica is removed to obtain thiol-modified oligosaccharides. The synthetic method of the present invention realizes the efficient separation of thiol-modified oligosaccharides and the enzyme reaction system by utilizing a thiol-modified silica carrier, while ensuring the efficient conduct of the enzyme reaction, thereby realizing the large-scale preparation of oligosaccharides.

Description

Technical Field

[0001] The invention belongs to the field of sugars, relates to the synthesis and separation of oligosaccharides, and specifically relates to a method for synthesizing thiol-modified oligosaccharides. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Carbohydrate compounds have potential biomedical effects. In order to study their biological functions, it is necessary to effectively obtain pure oligosaccharides and glycoconjugates. Although the chemoenzymatic method uses the flexibility and diversity of chemical synthesis and the specificity of enzymes to prepare a large number of oligosaccharides, the complex separation process of the enzyme reaction system is too cumbersome and still cannot meet the demand for oligosaccharides. In 1997, Blixt and Norberg used disulfide bonds as a coupling / separation strategy. They first connected the disaccharide to Thiopropyl 6B resin. After the enzymatic reaction is completed, dithiothreitol DTT is used to remove the polysaccharide with the linker from the solid support. However, the inventors found that the enzymatic reaction efficiency of the oligosaccharide targeted to the resin is low, the reaction is incomplete, and the use of Thiopropyl 6B resin is relatively expensive and difficult to promote on a large scale. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a method for synthesizing thiol-modified oligosaccharides. The synthesis method of the present invention realizes the efficient separation of thiol-modified oligosaccharides from an enzyme reaction system by utilizing a thiol-modified silica carrier, while ensuring the efficient progress of the enzyme reaction, thereby realizing large-scale preparation of oligosaccharides.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] A method for synthesizing thiol-modified oligosaccharides, comprising: preparing lactose into a thiol-containing glycosyl acceptor by a chemical method, and coupling the thiol-containing glycosyl acceptor with a monosaccharide by an enzymatic method to synthesize the thiol-modified oligosaccharides;

[0007] Adding thiol-modified silica to the system after the enzymatic reaction, and forming a disulfide bond between the thiol-modified silica and the thiol-modified oligosaccharide to obtain a silica-oligosaccharide complex, separating and extracting the silica-oligosaccharide complex, washing with double distilled water, and then opening the disulfide bond of the silica-oligosaccharide complex with a β-mercaptoethanol solution, and removing the silica to obtain a thiol-modified oligosaccharide;

[0008] The chemical structural formula of the sulfhydryl-containing glycosyl acceptor is shown in Formula I:

[0009]

[0010] Wherein, R is a mercapto group or an alkyl group substituted with a mercapto group.

[0011] The present invention studies the separation and purification of oligosaccharide enzymatic synthesis and the cost of the separation materials used. First, thiol-modified silica is used as an oligosaccharide separation material, and a thiol-containing glycosyl receptor is prepared by a chemical method, and then the sugar chain is extended by an enzymatic method to obtain thiol-modified oligosaccharides. Then, a disulfide bond is formed between the thiol-modified oligosaccharide and the thiol-modified silica, and the oligosaccharide and the silica are compounded, so that the silica fixes and separates the oligosaccharide in the solution, and then the disulfide bond is reduced by β-mercaptoethanol, so that the oligosaccharide and the silica are separated, thereby obtaining thiol-modified oligosaccharides.

[0012] Since disulfide bonds are formed by oxidation of two thiol groups, in the process of forming disulfide bonds, disulfide bonds are also formed between thiol-modified oligosaccharides. The oligosaccharides are separated and recovered. However, it is found through experiments that the present invention uses thiol-modified silica as an oligosaccharide separation material with a yield of more than 95%, indicating that the selectivity of the disulfide bonds formed between thiol-modified silica and thiol-modified oligosaccharides is better, and therefore it is beneficial to the separation of thiol-modified oligosaccharides, thereby ensuring the yield of thiol-modified oligosaccharides.

[0013] The beneficial effects of the present invention are:

[0014] The present invention prepares sulfhydryl-containing glycosyl acceptors by chemical methods, adopts an enzymatic method to efficiently extend sugar chains, and uses sulfhydryl-modified silica as an oligosaccharide separation material to quickly and completely separate the efficiently prepared sulfhydryl-modified oligosaccharides from the enzymatic reaction system, which not only simplifies the reaction steps, shortens the separation and purification time, improves the synthesis efficiency of oligosaccharides, but also ensures the overall yield of oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 It is a schematic diagram of the enzymatic modular assembly 1 of the present invention;

[0017] Figure 2 It is a schematic diagram of the enzymatic modular assembly 2 of the present invention;

[0018] Figure 3 It is a schematic diagram of the enzymatic modular assembly 3 of the present invention;

[0019] Figure 4It is a schematic diagram of the enzymatic modular assembly 4 of the present invention;

[0020] Figure 5 It is a schematic diagram of the enzymatic modular assembly 5 of the present invention;

[0021] Figure 6 It is a schematic diagram of the enzymatic modular assembly 6 of the present invention;

[0022] Figure 7 It is a schematic diagram of the enzymatic modular assembly 7 of the present invention;

[0023] Figure 8 It is a schematic diagram of the enzymatic modular assembly 8 of the present invention;

[0024] Fig. 9 The schematic diagram of the chemical method for preparing the thiol-modified silica carrier of the present invention;

[0025] Fig.10 This is a schematic diagram of the reaction process of synthesizing β-configuration lactose compound 1 by chemical method in Example 1 of the present invention;

[0026] Fig.11 This is a schematic diagram of the reaction process of enzymatic modular synthesis of heptasaccharide compound 2 in Example 1 of the present invention;

[0027] Fig.12 This is a schematic diagram of the reaction process of modular enzymatic synthesis of heptasaccharide compound 3 according to Example 2 of the present invention;

[0028] Fig.13 This is a schematic diagram of the reaction process of enzymatic modular synthesis of heptasaccharide compound 4 in Example 3 of the present invention;

[0029] Fig.14 This is a schematic diagram of the reaction process of enzymatic modular synthesis of heptasaccharide compound 5 according to Example 4 of the present invention;

[0030] Fig.15 This is a schematic diagram of the reaction process of enzymatic modular synthesis of pentasaccharide compound 6 in Example 5 of the present invention;

[0031] Fig.16 This is a schematic diagram of the reaction process of enzymatic modular synthesis of hexasaccharide compound 7 according to Example 6 of the present invention;

[0032] Fig.17 This is a schematic diagram of the reaction process of enzymatic modular synthesis of hexasaccharide compound 8 in Example 7 of the present invention. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] In view of the fact that it is difficult to balance the enzymatic reaction efficiency and the separation efficiency in the existing methods, the present invention provides a method for synthesizing thiol-modified oligosaccharides.

[0036] A typical embodiment of the present invention provides a method for synthesizing thiol-modified oligosaccharides, wherein lactose is prepared into a thiol-containing glycosyl acceptor by a chemical method, and the thiol-containing glycosyl acceptor is coupled with a monosaccharide by an enzymatic method to synthesize the thiol-modified oligosaccharide;

[0037] Adding thiol-modified silica to the system after the enzymatic reaction, and forming a disulfide bond between the thiol-modified silica and the thiol-modified oligosaccharide to obtain a silica-oligosaccharide complex, separating and extracting the silica-oligosaccharide complex, washing with double distilled water, and then opening the disulfide bond of the silica-oligosaccharide complex with a β-mercaptoethanol solution, and removing the silica to obtain a thiol-modified oligosaccharide;

[0038] The chemical structural formula of the sulfhydryl-containing glycosyl acceptor is shown in Formula I:

[0039]

[0040] Wherein, R is a mercapto group or an alkyl group substituted with a mercapto group.

[0041] In some embodiments, the β-mercaptoethanol solution is subjected to ultrasonic degassing treatment before use to avoid oxidation of thiol groups as much as possible.

[0042] In some embodiments, after the enzymatic reaction, the reaction is terminated in a boiling water bath, and the mixture is centrifuged at 1 to 10° C. The supernatant is concentrated, the concentrate is mixed with thiol-modified silica for 20 to 60 minutes, and the precipitate is collected by centrifugation. The precipitate is washed with double distilled water, and then a β-mercaptoethanol solution is added and mixed for 20 to 60 minutes, and the supernatant containing thiol-modified oligosaccharides is collected by centrifugation.

[0043] In some embodiments, R is a thiol group. Studies have shown that when R is a thiol group, the separation effect is better.

[0044] The thiol-modified silica can be obtained from the market or synthesized by itself. In some embodiments, the preparation process of the thiol-modified silica is as follows: The method uses tetraethoxysilane (TEOS) to prepare silicon dioxide in the presence of ammonia water, and uses mercaptosilane to modify the silicon dioxide to obtain mercapto-modified silicon dioxide, such as Fig. 9 As shown. The silica prepared by the method is a nano-scale particle material, which is further modified by mercaptosilane, which is more conducive to the capture of thiol-modified oligosaccharides. The mercaptosilane used can be 3-mercaptopropyltriethoxysilane, 2-mercaptoethyltriethoxysilane, mercaptomethyltriethoxysilane, etc.

[0045] In some embodiments, lactose is subjected to a full acetylation reaction, a brominated reaction, a sulfhydrylation reaction, and a deprotection reaction in sequence to obtain a sulfhydryl-containing glycosyl acceptor.

[0046] Specifically, after lactose is reacted with acetic anhydride, all exposed hydroxyl groups are protected with acetyl groups; hydrogen bromide (acetic acid solution) is then used to prepare α-configuration glycoside, and potassium thioacetate is added to deprotect the glycoside to obtain the glycoside.

[0047] In some embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method comprises: using enzymatic modular assembly 1 (such as Figure 1 As shown in FIG. 2 ), N-acetylglucosamine is coupled to a sulfhydryl-containing glycosyl acceptor via a β1-3 glycosidic bond to synthesize a trisaccharide; 2 (as shown in FIG. 2 ) is assembled modularly using an enzymatic method. Figure 2 As shown in the figure, galactose is coupled to a trisaccharide with a β1-4 glycosidic bond to synthesize a tetrasaccharide; N-acetylglucosamine is coupled to a tetrasaccharide with a β1-3 glycosidic bond by enzymatic modular assembly 1 to synthesize a poly-LacNAc backbone pentasaccharide; galactose is coupled to a poly-LacNAc backbone pentasaccharide with a β1-4 glycosidic bond by enzymatic modular assembly 2 to synthesize a hexasaccharide 1; enzymatic modular assembly 3 (as shown in the figure) is coupled to a trisaccharide with a β1-4 glycosidic bond to synthesize a tetrasaccharide; N-acetylglucosamine is coupled to a tetrasaccharide with a β1-3 glycosidic bond by enzymatic modular assembly 1 to synthesize a poly-LacNAc backbone pentasaccharide; Figure 3 ) coupling fucose to the hexasaccharide 1 via an α1-2 glycosidic bond to synthesize the heptasaccharide shown in formula (II);

[0048]

[0049] Specifically, the enzymes used in the enzymatic modular assembly 1 are a fusion enzyme (NahK / GlmU) of N-acetylhexosaminyl kinase (NahK) and glyconucleoside generating enzyme (GlmU) and β1-3N-acetylglucosamine transferase (HpLgtA).

[0050] Specifically, the enzymes used in the enzymatic modular assembly 2 are galactokinase (GalK), sugar nucleoside generator (BLUSP) and β1-4 galactosyltransferase (NmLgtB).

[0051] Specifically, the enzymes used in the enzymatic modular assembly 3 are glyconucleoside generator (FKP) and α1-2 fucosyltransferase (Hmα1,2FucT).

[0052] Specifically, a sulfhydryl-containing glycosyl acceptor, N-acetylglucosamine (1.2-4.0 equivalents), adenosine triphosphate (ATP) (1.2-4.0 equivalents), uridine triphosphate (UTP) (1.2-4.0 equivalents), MgCl2 (5-100 mM), and Tris-HCl buffer (10-500 mM, pH 5.0-10.0) are prepared into an aqueous solution, and then the pH value of the reaction system is adjusted to 4.5-8.5, and then a fusion enzyme (NahK / GlmU) of N-acetylhexosamine kinase (NahK) and glyconucleoside generating enzyme (GlmU) and β1-3N-acetylglucosamine transferase (HpLgtA) are added, and the reaction time is 3-70 hours. After the reaction is completed, the target oligosaccharide is captured by a sulfhydryl-modified silica carrier, impurities are eluted with double distilled water, and finally the trisaccharide is released by elution with 5-40 mM β-mercaptoethanol. It is used as the acceptor of the next enzyme reaction, galactose (1.2-4.0 equivalents), adenosine triphosphate (ATP) (1.2-4.0 equivalents), uridine triphosphate (UTP) (1.2-4.0 equivalents), MgCl2 (5-100mM), Tris-HCl buffer (10-500mM, pH 5.0-10.0) are used to prepare an aqueous solution, and then the pH value of the reaction system is adjusted to 4.5-8.5, and then galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP) and β1-4 galactosyltransferase (NmLgtB) are added, and the reaction time is 3-70 hours. After the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, impurities are eluted with double distilled water, and finally eluted and released by 5-40mM β-mercaptoethanol to obtain tetrasaccharide. The poly-LacNAc hexasaccharide 1 can be prepared by repeatedly using the above enzymatic module, which can be used as the receptor for the next enzyme reaction. Fucose (1.2-4.0 equivalents), adenosine triphosphate (ATP) (1.2-4.0 equivalents), guanosine triphosphate (GTP) (1.2-4.0 equivalents), MgCl2 (5-100mM), and Tris-HCl buffer (10-500mM, pH 5.0-10.0) are used to prepare an aqueous solution, and then the pH value of the reaction system is adjusted to 4.5-8.5, and then sugar nucleoside generating enzyme (FKP) and α1-2 fucosyltransferase (Hmα1,2FucT) are added. The reaction time is 3-70 hours. After the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, and the impurities are eluted with double distilled water. Finally, the heptasaccharide shown in formula (II) can be obtained by eluting and releasing with 5-30mM β-mercaptoethanol.

[0053] In one or more embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method further comprises: using enzymatic modular assembly 7 (such as Figure 7 As shown in FIG. 2 ), fucose is coupled to the poly-LacNAc backbone pentasaccharide via an α1-3 glycosidic bond to synthesize hexasaccharide 2; enzymatic modular assembly 2 (as shown in FIG. Figure 2 ) coupling galactose to the hexasaccharide 2 via a β1-4 glycosidic bond to synthesize the heptasaccharide shown in formula (III);

[0054]

[0055] Specifically, the enzymes used in the enzymatic modular assembly 7 are glyconucleoside generator (FKP) and α1-3 fucosyltransferase (Hpα1,3FucT).

[0056] Specifically, the enzymes used in the enzymatic modular assembly 2 are galactokinase (GalK), sugar nucleoside generator (BLUSP) and β1-4 galactosyltransferase (NmLgtB).

[0057] Specifically, poly-LacNAc backbone pentasaccharide, fucose (1.2 to 4.0 equivalents), adenosine triphosphate (ATP) (1.2 to 4.0 equivalents), guanosine triphosphate (GTP) (1.2 to 4.0 equivalents), MgCl2 (5 to 100 mM), Tris-HCl buffer (10 to 500 mmol, pH 5.0~10.0) to prepare an aqueous solution, then adjust the pH value of the reaction system to 4.5~8.5, then add sugar nucleoside generating enzyme (FKP), α1-3 fucosyltransferase (Hpα1,3FucT), the reaction time is 3~70 hours, after the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, the impurities are eluted with double distilled water, and finally eluted and released by 5~30mM β-mercaptoethanol to obtain hexasaccharide 2, which serves as the next enzyme reaction receptor, galactose (1.2~4.0 equivalents), adenosine triphosphate (ATP) (1.2~4.0 equivalents), uridine triphosphate (UTP) (1.2~4.0 equivalents), MgCl2 (5~100mM), Tris-HCl buffer (10~500mM, pH 5.0~10.0) to prepare an aqueous solution, then adjust the pH value of the reaction system to 4.5~8.5, then add galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP) and β1-4 galactosyltransferase (NmLgtB), the reaction time is 3~70 hours, after the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, the impurities are eluted with double distilled water, and finally eluted and released by 5~40mM β-mercaptoethanol to obtain the heptasaccharide shown in formula (III).

[0058] In one or more embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method further comprises: using enzymatic modular assembly 6 (such as Figure 6 ) coupling sialic acid to the hexasaccharide 1 via an α2-3 glycosidic bond to synthesize the heptasaccharide shown in formula (IV);

[0059]

[0060] Specifically, the enzymes used in the enzymatic modular assembly 6 are nucleoside synthase (NmCSS) and α2-3 sialyltransferase (PmST1M144D).

[0061] Specifically, an aqueous solution of hexasaccharide 1, N-acetylneuraminic acid (Neu5Ac) (1.2 to 4.0 equivalents), cytosine triphosphate (CTP) (1.2 to 4.0 equivalents), MgCl2 (5 to 100 mM), and Tris-HCl buffer (10 to 500 mM, pH 5.0 to 10.0) is prepared, and then the pH value of the reaction system is adjusted to 4.5 to 8.5, and then sugar nucleoside generating enzyme (NmCSS) and α2-3 sialyltransferase (PmST1M144D) are added, and the reaction time is 0.5 to 36 hours. After the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, and impurities are eluted with double distilled water. Finally, the heptasaccharide shown in formula (IV) is obtained by elution and release with 5 to 40 mM β-mercaptoethanol.

[0062] In one or more embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method further comprises: using enzymatic modular assembly 8 (such as Figure 8 ) wherein sialic acid is coupled to the hexasaccharide 1 via an α2-6 glycosidic bond to synthesize the heptasaccharide of formula (V);

[0063]

[0064] Specifically, the enzymes used in the enzymatic modular assembly 8 are nucleoside synthase (NmCSS) and α2-6 sialyltransferase (Pd2,6ST).

[0065] Specifically, an aqueous solution of hexasaccharide 1, N-acetylneuraminic acid (Neu5Ac) (1.2 to 4.0 equivalents), cytosine triphosphate (CTP) (1.2 to 4.0 equivalents), MgCl2 (5 to 100 mM), and Tris-HCl buffer (10 to 500 mM, pH 5.0 to 10.0) is prepared, and then the pH value of the reaction system is adjusted to 4.5 to 8.5, and then sugar nucleoside generating enzyme (NmCSS) and α2-6 sialyltransferase (Pd2,6ST) are added, and the reaction time is 0.5 to 36 hours. After the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, and impurities are eluted with double distilled water. Finally, the heptasaccharide shown in formula (V) is obtained by elution and release with 5 to 40 mM β-mercaptoethanol.

[0066] In one or more embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method further comprises: coupling fucose to tetrasaccharide by α1-2 glycosidic bond to synthesize pentasaccharide shown in formula (VI) by enzymatic modular assembly 3;

[0067]

[0068] Specifically, the enzymes used in the enzymatic modular assembly 3 are glyconucleoside generator (FKP) and α1-2 fucosyltransferase (Hmα1-2FucT).

[0069] Specifically, the tetrasaccharide synthesized in formula (II), fucose (1.2 to 4.0 equivalents), adenosine triphosphate (ATP) (1.2 to 4.0 equivalents), guanosine triphosphate (GTP) (1.2 to 4.0 equivalents), MgCl2 (5 to 100 mM), and Tris-HCl buffer (10 to 500 mmol, pH 5.0 to 10.0) are prepared into an aqueous solution, and then the pH value of the reaction system is adjusted to 4.5 to 8.5, and then sugar nucleoside generating enzyme (FKP) and α1-3 fucosyltransferase (Hpα1,3FucT) are added, and the reaction time is 3 to 70 hours. After the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, and the impurities are eluted with double distilled water. Finally, the heptasaccharide shown in formula (VI) is obtained by elution and release with 5 to 40 mM β-mercaptoethanol.

[0070] In one or more embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method further comprises: using enzymatic modular assembly 4 (such as Figure 4 ) coupling N-acetylgalactosamine to the pentasaccharide of formula (VI) via an α1-3 glycosidic bond to synthesize the hexasaccharide of formula (VII);

[0071]

[0072] Specifically, the enzymes used in the enzymatic modular assembly 4 are a fusion enzyme (NahK / GlmU) of N-acetylhexosaminyl kinase (NahK) and glyconucleoside generator (GlmU) and α1-3N-acetylgalactosyl transferase (BgtA).

[0073] Specifically, the pentasaccharide represented by formula (VI), N-acetylgalactosamine (1.2 to 4.0 equivalents), adenosine triphosphate (ATP) (1.2 to 4.0 equivalents), uridine triphosphate (UTP) (1.2 to 4.0 equivalents), MgCl2 (5 to 100 mM), Tris-HCl buffer (10 to 500 mM, pH 5.0~10.0) to prepare an aqueous solution, then adjust the pH value of the reaction system to 4.5~8.5, then add a fusion enzyme (NahK / GlmU) of N-acetylhexosaminidokinase (NahK) and glyconucleoside generating enzyme (GlmU) and α1-3N-acetylgalactose glycosyltransferase (BgtA), the reaction time is 3~70 hours, after the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, the impurities are eluted with double distilled water, and finally eluted and released by 5~40mM β-mercaptoethanol to obtain the heptasaccharide shown in formula (VII).

[0074] In one or more embodiments, the process of preparing thiol-modified oligosaccharides by enzymatic method further comprises: using enzymatic modular assembly 5 (such as Figure 5 ) coupling galactose to the pentasaccharide of formula (VI) via an α1-3 glycosidic bond to synthesize the hexasaccharide of formula (VIII);

[0075]

[0076] Specifically, the enzymes used in the enzymatic modular assembly 5 are galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP), and α1-3 galactosyltransferase (GTB).

[0077] Specifically, the pentasaccharide shown in formula (VI), galactose (1.2 to 4.0 equivalents), adenosine triphosphate (ATP) (1.2 to 4.0 equivalents), uridine triphosphate (UTP) (1.2 to 4.0 equivalents), MgCl2 (5 to 100 mM), and Tris-HCl buffer (10 to 500 mM, pH 5.0 to 10.0) are prepared into an aqueous solution, and then the pH value of the reaction system is adjusted to 4.5 to 8.5, and then galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP), and α1-3 galactose glycosyltransferase (GTB) are added, and the reaction time is 3 to 70 hours. After the reaction is completed, the target oligosaccharide is captured by a thiol-modified silica carrier, and the impurities are eluted with double distilled water. Finally, the heptasaccharide shown in formula (VIII) is obtained by eluting and releasing with 5 to 30 mM β-mercaptoethanol.

[0078] In the present invention, "equivalent" refers to the ratio of the amounts of substances when substances interact with each other, such as: N-acetylglucosamine (1.2-4.0 equivalents) means that the ratio of the amounts of N-acetylglucosamine to the sulfhydryl-containing glycosyl acceptor is 1.2-4.0.

[0079] After the reaction is completed, thin layer chromatography (TLC) is used to track the reaction progress. The developing solvent for formula (I) and (II) is EtOAc:MeOH:H2O:HOAc=4:2:1:0.2 and the synthetic developing solvent for formula (III)-(VIII) is EtOAc:MeOH:H2O:HOAc=2:2:1:0.2.

[0080] In the enzyme method of the present invention, the reaction temperature is 0-37°C and the rotation speed is 0-240r / min; the enzyme reaction is stopped by boiling the reaction solution in a boiling water bath for 5-10 minutes.

[0081] The enzymatic module used in the present invention contains three types of enzymes: sugar kinase, sugar nucleoside generating enzyme and sugar transferase, which play a highly efficient catalytic role and cooperate with each other to form an organic enzyme reaction system. During the experiment, the enzymes used above were optimized and screened for many times. The results showed that NahK / GlmU, Bifidobacterium longum N-acetylhexosamine-1-kinase (NahK) and E.coli N-acetylglucosamine uridyltransferase (GlmU), as well as Helicobacter mustelaeα1,3-N-galactosyltransferase (BgtA); Helicobacter pyloriβ1,3-N-acetylglucosaminyltransferase (HpLgtA); E.coligalactokinase (GalK), Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP) and Neisseria meningitidesβ1,4-galactosyltransferase (NmLgtB); Neisseriameningitides CMP-sialic acid synthetase (NmCSS), and Pasteurella multocidamultifunctional2,3-sialyltransferase 1M144D (PmST1 M144D); Humanα1,3-galactosyltransferase (GTB); Bacteroides fragilis bifunctional L-fucokinase / GDP-fucose pyrophosphorylase (FKP) and Helicobacler muslelaeαl,2-fucosyltransferase (Hmα1,2FucT); Helicobacter pyloriα1,3-fucosyltransferase (Hpα1,3FucT); Photobacterium damselae2,6-sialyltransferase mutant (Pd2,6ST) are the enzymes used in the present invention, which have the best catalytic effect, high synthesis efficiency and simple purification, and the above enzymes can be expressed and purified in large quantities in a conventional Escherichia coli expression system.

[0082] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0083] Example 1

[0084] Preparation of thiol-modified silica support:

[0085] Add 200mL of anhydrous ethanol to a 250mL single-necked flask, add 15mL of ammonia water and stir evenly, add 6mL of tetraethyl silicate dropwise to the single-necked flask and react at 60°C for 5h. Centrifuge at 15000rpm for 30min, and wash three times with a mixed solvent of anhydrous ethanol and deionized water in a volume ratio of 1:1. Add an appropriate amount of anhydrous ethanol to the silica nanoparticles prepared above to make them evenly dispersed, then add 15mL of 3-mercaptopropyltriethoxysilane and react for 8h under a nitrogen atmosphere. Centrifuge at 15000rpm for 30min, wash three times with a mixed solvent of anhydrous ethanol and deionized water in a volume ratio of 1:1, and finally wash once with anhydrous ethanol.

[0086] Chemical synthesis of β-lactoside 1 [Galβ(l-4)GlcβOR]:

[0087] Lactose 9 (10 g, 29.23 mmol), acetic anhydride (55 mL) and sodium acetate (9.6 g) were added to a 500 mL round-bottom flask and refluxed and stirred at 160 ° C for 6 hours. After the reaction was complete by thin layer chromatography (PE: EA = 1: 2), the mixture was concentrated by rotary evaporation. The obtained solid was redissolved in 250 mL of dichloromethane, extracted twice with semi-saturated brine, three times with saturated sodium bicarbonate solution, and three times with double distilled water solution. The organic phase was then separated and dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a light yellow solid compound 10 (18.70 g, 94%).

[0088] Compound 10 (15.0 g, 22.12 mmol) and hydrogen bromide (33% acetic acid solution) in dichloromethane were added to a 250 mL round-bottom flask and stirred at room temperature for 2 h under an argon atmosphere. The reaction progress was monitored by TLC. When the reaction was completed, the reaction solution was poured into water, extracted with DCM, washed with sodium bicarbonate aqueous solution, and the organic phase was dehydrated and dried with anhydrous sodium sulfate to obtain compound 11.

[0089] Add lactobromide 11 (5.0 g, 7.12 mmol) to a 250 mL round-bottom flask and dissolve it in anhydrous DMF (20 mL), add potassium thioacetate (2.6 g, 22.01 mmol) to the solution, stir the solution at room temperature for 5 h, the solution turns dark brown, and the reaction progress is monitored by TLC. After the reaction is completed, dilute with dichloromethane, and elute the reaction solution with a large amount of double distilled water and saturated brine in turn. After drying the organic phase with anhydrous sodium sulfate, it is concentrated and purified by silica gel column (PE / EA=3:2, v / v) to obtain yellow solid compound 12 (4.2 g, 85%).

[0090] Compound 12 (5.2 g, 6.94 mmol) was added to a 250 mL round-bottom flask and dissolved in methanol. Sodium methoxide was added at pH 11 and reacted at room temperature for 30 minutes. The reaction progress was monitored by TLC. After the reaction was completed, it was diluted with MeOH / H2O (2 / 1, v / v) and an acidic ion resin was added. 50WX8-200(H + form) was neutralized and filtered to obtain lactose receptor compound 1 (2.8 g).

[0091] The synthetic route of lactose receptor compound 1 is as follows Fig.10 shown.

[0092] Enzymatic modular assembly 1 for the synthesis of heptasaccharide compounds

[0093] 2[Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0094] Lactose receptor compound 1 (200 mg), N-acetylglucosamine (108.4 mg), ATP (270.1 mg), UTP (237.2 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, NahK / GlmU (2.0 mg) and HpLgtA (2.0 mg) were added, and double distilled water was added to a total volume of 20 mL. The reaction system was placed in a shaker and incubated at 37°C and 110 r / min for 16 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 8: 3: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12000rpm for 20 minutes, the supernatant was collected, and the concentrate was concentrated by rotary evaporation. The concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities. Finally, 25mMβ-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes to obtain a trisaccharide compound (265.0 mg, 96%). The trisaccharide compound (150 mg), galactose (47.3 mg), ATP (144.7 mg), UTP (127.1 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, and GalK (2.0 mg), BLUSP (2.0 mg) and NmLgtB (2.0 mg) were added. After adding double distilled water to a total volume of 15 mL, the reaction system was placed in a shaker and incubated at 37°C and 110 r / min for 12 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12000rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25mMβ-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes to obtain a tetrasaccharide compound (174.1 mg, 95%).Tetrasaccharide compound (150.4 mg), N-acetylglucosamine (50.2 mg), ATP (98.1 mg), UTP (86.2 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, NahK / GlmU (2.0 mg) and HpLgtA (2.0 mg) were added, and double distilled water was added to a total volume of 20 mL. The reaction system was placed in a shaker and incubated at 37°C and 110 r / min for 16 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12000rpm for 20 minutes, the supernatant was collected, and the concentrate was concentrated by rotary evaporation. The concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities. Finally, 25mMβ-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes to obtain a pentasaccharide compound (196.2 mg, 94%). Further, pentasaccharide compound (180 mg), galactose (31.3 mg), ATP (63.7 mg), UTP (72.1 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, GalK (2.0 mg), BLUSP (2.0 mg) and NmLgtB (2.0 mg) were added, and double distilled water was added to a total volume of 15 mL. The reaction system was placed in a shaker and incubated at 37°C and 110 r / min for 12 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12000rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities. Finally, 25mMβ-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes to obtain a hexasaccharide compound (209.3 mg, 95%).Then, hexasaccharide compound (160 mg), fucose (31.2 mg), ATP (104.7 mg), GTP (114.6 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, enzyme FKP (2.00 mg) and Hmα1,2FucT (2.00 mg) were added, and double distilled water was added to a total volume of 10 mL. The reaction system was placed in a shaker and incubated at 37°C and 110 r / min for 4 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 r / min for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white heptasaccharide compound 2 (179.9 mg, 96%). The parameters are as follows: 1 H NMR(600MHz,D2O)δ5.12(d,J=4.2Hz,1H),4.68(t,J=8.3Hz,2H),4.50(d,J=8.1Hz,1H),4.47-4.39(m,3H),4.34(q,J=6.6Hz,1H),4.1 3(d,J=3.5Hz,1H),4.08(d,J=3.2Hz,1H),4.06-3.29(m,45H),2.36(t,J=7.4Hz,2H),2.00(s,3H),2.00(s,3H),1.13(d,J=6.7Hz,3H); 13 C NMR(150MHz,D2O)δ176.2,174.7,174.6,102.8,102.7,102.6,102.4,102.0,1 01.6,99.5,81.9,81.6,78.2,78.1,75.3,75.0,74.8,74.7,74.4,74.3,74.2, 72.7,72.4,72.0,71.8,70.9,70.4,69.9,69.4,69.1,68.7,68.5,68.2,68.2,67.7,66.6,61.4,61.0,60.9,60.0,59.8,38.8,34.3,23.0,22.2,22.1,15.2.

[0095] The synthetic route of heptasaccharide compound 2 is as follows Fig.11 shown.

[0096] Example 2

[0097] Enzymatic module assembly 2 synthesis of heptasaccharide compound 3

[0098] [Galβ(l-4)Fucα(1-3)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]

[0099] The pentasaccharide compound (150 mg), fucose (31.2 mg), ATP (104.7 mg), GTP (114.6 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) prepared in Example 1 were dissolved in a 50 mL centrifuge tube, enzyme FKP (2.00 mg) and Hpα1,3FucT (2.00 mg) were added, and after adding double distilled water to a total volume of 10 mL, the reaction system was placed in a shaker and incubated at 37° C. and 110 r / min for 4 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12000rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities. Finally, 25mMβ-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes to obtain a hexasaccharide compound (176.9 mg, 96%). The hexasaccharide compound (70 mg), galactose (23.3 mg), ATP (56.7 mg), UTP (57.1 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, and GalK (2.0 mg), BLUSP (2.0 mg) and NmLgtB (2.0 mg) were added. After adding double distilled water to a total volume of 15 mL, the reaction system was placed in a shaker and incubated at 37°C and 110 r / min for 12 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white heptasaccharide compound 3 (87.1 mg, 95%). The parameters are as follows: 1H NMR(600MHz,D2O)δ5.04(d,J=4.0Hz,1H),4.64(dd,J=8.5,5.4Hz,2H),4.42(d,J=8.0Hz,1 H),4.37(dd,J=10.5,7.8Hz,3H),4.11-3.21(m,40H),1.95(s,6H),1.08(d,J=6.5Hz,3H); 13 C NMR(150MHz,D2O)δ174.8,174.5,102.7,102.6,102.4,101.9,101.5,98 .6,91.8,81.9,81.7,81.4,78.1,75.1,74.9,74.9,74.7,74.6,74.3,74. 2,72.6,72.3,71.7,70.5,70.4,69.8,69.0,68.3,68.2,68.1,68.1,67.5 ,66.5,61.3,60.9,60.8,60.3,59.8,59.4,55.8,54.5,22.1,22.1,15.1.

[0100] The synthetic route of heptasaccharide compound 3 is as follows Fig.12 shown.

[0101] Example 3

[0102] Enzymatic modular synthesis of heptasaccharide compounds

[0103] 4[Neu5Acα(2-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0104] The hexasaccharide compound (50 mg) prepared in Example 1, Neu5Ac (14.4 mg), CTP (23.1 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, NmCSS (2.0 mg) and PmST1M144D (2.0 mg) were added, and after adding double distilled water to a total volume of 15 mL, the reaction system was placed in a shaker and incubated at 37° C. and 110 r / min for 1 hour. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 2:2:1:0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white heptasaccharide compound 4 (59.1 mg, 95%). The parameters are as follows: 1 H NMR(600MHz,D2O)δ4.69(d,J=7.7Hz,1H),4.65(d,J=8.3Hz,1H),4.47(d,J=8.0Hz,1H),4.45-4.38( m,3H),4.16-3.26(m,43H),2.63(dd,J=12.4,4.7Hz,1H),2.02-1.99(m,9H),1.69(t,J=12.2Hz,1H); 13 C NMR(150MHz,D2O)δ174.8,174.7,173.4,102.8,102.7,102.6,102.4,101 .9,99.98,91.8,81.9,80.3,78.2,78.0,74.7,74.6,74.4,74.1,74.1,73 .5,72.6,72.4,72.3,72.1,72.0,71.6,70.6,69.8,68.2,68.2,68.1,63. 2,62.5,60.8,59.9,59.8,59.7,55.0,54.8,51.7,39.5,22.1,22.0,21.9.

[0105] The synthetic route of heptasaccharide compound 4 is as follows Fig.13 shown.

[0106] Example 4

[0107] Enzymatic modular synthesis of heptasaccharide compounds

[0108] 5[Neu5Acα(2-6)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0109] The hexasaccharide compound (50 mg) prepared in Example 1, Neu5Ac (14.4 mg), CTP (23.1 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, NmCSS (2.0 mg) and Pd2,6ST (2.0 mg) were added, and double distilled water was added to a total volume of 15 mL. The reaction system was placed in a shaker and incubated at 37° C. and 110 r / min for 1 hour. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 2:2:1:0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white heptasaccharide compound 5 (58.9 mg, 94%). The parameters are as follows: 1 H NMR(600MHz,D2O)δ4.69(d,J=7.7Hz,1H),4.65(d,J=8.3Hz,1H),4.47(d,J=8.0Hz,1H),4.45-4.38( m,3H),4.16-3.26(m,43H),2.45(dd,J=12.4,4.7Hz,1H),2.02-1.99(m,9H),1.69(t,J=12.2Hz,1H); 13C NMR(150MHz,D2O)δ174.8,174.7,173.4,102.8,102.7,102.6,102.4,101 .9,99.98,91.8,81.9,80.3,78.2,78.0,74.7,74.6,74.4,74.1,74.1,73 .5,72.6,72.4,72.3,72.1,72.0,71.6,70.6,69.8,68.2,68.2,68.1,63. 2,62.5,60.8,59.9,59.8,59.7,55.0,54.8,51.7,39.5,22.1,22.0,21.9.

[0110] The synthetic route of heptasaccharide compound 5 is as follows Fig.14 shown.

[0111] Example 5

[0112] Enzymatic modular assembly 5 for the synthesis of pentasaccharide compounds

[0113] 6[Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0114] The tetrasaccharide compound (160 mg), fucose (62.2 mg), ATP (204.7 mg), GTP (214.6 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) prepared in Example 1 were dissolved in a 50 mL centrifuge tube, enzyme FKP (2.00 mg) and Hmα1,2FucT (2.00 mg) were added, and after adding double distilled water to a total volume of 10 mL, the reaction system was placed in a shaker and incubated at 37° C. and 110 r / min for 10 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white pentasaccharide compound 6 (218.9 mg, 96%). The parameters are as follows: 1H NMR(600MHz,D2O)δ5.26(d,J=3.2Hz,1H),4.67(d,J=8.4Hz,1H),4.49(d,J=8.0Hz,1H),4.45(d ,J=7.8Hz,1H),4.40(d,J=7.9Hz,1H),4.20-3.29(m,28H),2.00(s,3H),1.14(d,J=6.6Hz,3H); 13 C NMR(150MHz,D2O)δ103.3,102.8,102.4,101.9,99.2,81.8,81.5,78.7,75.6,75.1,75.0,74.5,74.2,73.6,73.2,72.6,72.5,72 .4,72.2,71.1,70.6,70.5,70.3,69.7,69.6,68.5,68.3,68.2,64.2,62.5,61.8,61.5,60.8,60.4,60.2,35.4,33.9,22.3,15.2.

[0115] The synthetic route of pentasaccharide compound 6 is as follows Fig.15 shown.

[0116] Example 6

[0117] Enzymatic modular assembly 6 for the synthesis of hexasaccharide compounds

[0118] 7[GalNAcα(1–3)Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0119] The pentasaccharide compound 6 (50 mg) prepared in Example 5, N-acetylgalactosamine (11.1 mg), ATP (27.6 mg), UTP (24.2 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) were dissolved in a 50 mL centrifuge tube, NahK / GlmU (2.0 mg) and BgtA (2.0 mg) were added, and after adding double distilled water to a total volume of 10 mL, the reaction system was placed in a shaker and incubated at 37° C. and 110 r / min for 16 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white hexasaccharide compound 7 (59.8 mg, 96%). The parameters are as follows: 1 H NMR(600MHz,D2O)δ5.26(d,J=3.2Hz,1H),5.11(d,J=3.6Hz,1H),4.68(d,J=8.4Hz,1H),4.49(d,J=8.0Hz,1H),4. 45(d,J=7.8Hz,1H),4.41(d,J=7.9Hz,1H),4.21-3.29(m,35H),2.02(s,3H),2.01(s,3H),1.14(d,J=6.6Hz,3H); 13 C NMR (150MHz, D2O) δ174.8,103.3,102.8,102.4,101.9,99.2,91.1,81.9,81.5,78.7,75.6,75.1,74.9,74.5,74.1,73.6,73.3,72 .6,72.5,72.4,72.2,71.1,70.6,70.5,70.3,69.7,69.6,68.5,68.3,68.2,64.2,62.5,61.5,60.8,60.4,60.2,,22.3,22.1,15.2.

[0120] The synthetic route of hexasaccharide compound 7 is as follows Fig.16 shown.

[0121] Example 7

[0122] Enzymatic modular assembly 7 to synthesize hexasaccharide compound 8

[0123] [Galα(1–3)Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0124] The pentasaccharide compound 6 (50 mg), galactose (9.0 mg), ATP (27.6 mg), UTP (24.2 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) (the amount of Tris and MgCl2 is calculated and determined by the volume of the final reaction solution) prepared in Example 5 were dissolved in a 50 mL centrifuge tube, and GalK (2.0 mg), BLUSP (2.0 mg) and GTB (2.0 mg) were added. After adding double distilled water to a total volume of 10 mL, the reaction system was placed in a shaker and incubated at 37° C. and 110 r / min for 12 hours. After the reaction was completed by thin layer chromatography (EtOAc: MeOH: H2O: EtOH = 4: 2: 1: 0.2), the reaction was terminated by boiling in a boiling water bath for 5 minutes. The reaction system was then centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was collected, and the concentrate was mixed with a thiol-modified silica carrier (the mass ratio of the oligosaccharide to the thiol-modified silica carrier was 1:10) for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and then washed three times with double distilled water to remove non-thiol impurities, and finally 25 mM β-mercaptoethanol was mixed with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes to obtain a white hexasaccharide compound 8 (58.3 mg, 95%). The parameters are as follows: 1 H NMR(600MHz,D2O)δ5.27(d,J=3.2Hz,1H),5.10(d,J=3.1Hz,1H),4.67(d,J=8.4Hz,1H),4.49(d,J=8.0Hz, 1H),4.44(d,J=7.8Hz,1H),4.41(d,J=7.9Hz,1H),4.21-3.30(m,33H),2.03(s,3H),1.13(d,J=6.6Hz,3H); 13C NMR(150MHz,D2O)δ,103.3,102.8,102.3,101.8,99.3,93.2,81.8,81.5,78.3,75.6,75.1,74.9,74.5,74.2,73.7,73.2,72 .6,72.5,72.4,72.2,71.1,70.6,70.4,70.3,69.7,69.6,68.5,68.3,68.2,64.2,62.5,61.5,60.8,60.4,60.2,22.1,15.2.

[0125] The synthetic route of hexasaccharide compound 8 is as follows Fig.17 shown.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for synthesizing thiol-modified oligosaccharides, characterized in that: Lactose is prepared into a sulfhydryl-containing glycosyl acceptor by chemical method, and the sulfhydryl-containing glycosyl acceptor is coupled with monosaccharide by enzymatic method to synthesize sulfhydryl-modified oligosaccharide; Adding thiol-modified silica to the system after the enzymatic reaction, and forming a disulfide bond between the thiol-modified silica and the thiol-modified oligosaccharide to obtain a silica-oligosaccharide complex, separating and extracting the silica-oligosaccharide complex, washing with double distilled water, and then opening the disulfide bond of the silica-oligosaccharide complex with a β-mercaptoethanol solution, and removing the silica to obtain a thiol-modified oligosaccharide; The chemical structural formula of the sulfhydryl-containing glycosyl acceptor is shown in Formula I: Wherein, R is a thiol group; The preparation process of thiol-modified silica is as follows: The method uses tetraethoxysilane to prepare silicon dioxide in the presence of ammonia water, and uses mercaptosilane to modify the silicon dioxide to obtain mercapto-modified silicon dioxide; The preparation process of the sulfhydryl-containing glycosyl acceptor is as follows: lactose is subjected to full acetylation reaction, brominated reaction, glucosidation reaction and deprotection reaction in sequence to obtain the sulfhydryl-containing glycosyl acceptor.

2. The method for synthesizing thiol-modified oligosaccharides according to claim 1, characterized in that: The β-mercaptoethanol solution was degassed by ultrasonic treatment before use to avoid oxidation of thiol groups.

3. The method for synthesizing thiol-modified oligosaccharides according to claim 1, characterized in that: After the enzymatic reaction, the reaction is terminated in a boiling water bath, and the mixture is centrifuged at 1 to 10° C. The supernatant is concentrated, the concentrate is mixed with thiol-modified silica for 20 to 60 minutes, the precipitate is collected by centrifugation, the precipitate is washed with double distilled water, and then β-mercaptoethanol solution is added and mixed for 20 to 60 minutes, and the supernatant containing thiol-modified oligosaccharides is collected by centrifugation.

4. The method for synthesizing thiol-modified oligosaccharides according to claim 1, characterized in that: The process of preparing thiol-modified oligosaccharides by enzymatic method includes: using enzymatic modular assembly 1 to couple N-acetylglucosamine to a thiol-containing glycosyl receptor with a β1-3 glycosidic bond to synthesize a trisaccharide; using enzymatic modular assembly 2 to couple galactose to a trisaccharide with a β1-4 glycosidic bond to synthesize a tetrasaccharide; using enzymatic modular assembly 1 to couple N-acetylglucosamine to a tetrasaccharide with a β1-3 glycosidic bond to synthesize a poly-LacNAc backbone pentasaccharide; using enzymatic modular assembly 2 to couple galactose to a poly-LacNAc backbone pentasaccharide with a β1-4 glycosidic bond to synthesize a hexasaccharide 1; using enzymatic modular assembly 3 to couple fucose to a hexasaccharide 1 with an α1-2 glycosidic bond to synthesize a heptasaccharide shown in formula (II); 5. The method for synthesizing thiol-modified oligosaccharides according to claim 4, characterized in that: The enzymes used in the enzymatic modular assembly 1 are a fusion enzyme of N-acetylhexokinase and sugar nucleoside generating enzyme and β1-3N-acetylglucosamine transferase.

6. The method for synthesizing thiol-modified oligosaccharides according to claim 4, characterized in that: The enzymes used in enzymatic modular assembly 2 are galactokinase, sugar nucleoside generator and β1-4 galactosyltransferase.

7. The method for synthesizing thiol-modified oligosaccharides according to claim 4, characterized in that: The enzymes used in enzymatic modular assembly 3 are sugar nucleoside generating enzyme and α1-2 fucosyltransferase.

8. The method for synthesizing thiol-modified oligosaccharides according to claim 4, characterized in that: The process of preparing thiol-modified oligosaccharides by enzymatic method also includes: using enzymatic modular assembly 7 to couple fucose to the poly-LacNAc backbone pentasaccharide with an α1-3 glycosidic bond to synthesize hexasaccharide 2; using enzymatic modular assembly 2 to couple galactose to the hexasaccharide 2 with a β1-4 glycosidic bond to synthesize the heptasaccharide shown in formula (III); Alternatively, the process of preparing the thiol-modified oligosaccharide by enzymatic method further comprises: coupling sialic acid to the hexasaccharide 1 by α2-3 glycosidic bond by enzymatic modular assembly 6 to synthesize the heptasaccharide shown in formula (IV); Alternatively, the process of preparing the thiol-modified oligosaccharide by enzymatic method further comprises: using enzymatic modular assembly 8 to couple sialic acid to the hexasaccharide 1 by α2-6 glycosidic bond to synthesize the heptasaccharide shown in formula (V); 9. The method for synthesizing thiol-modified oligosaccharides according to claim 8, characterized in that: The enzymes used in the enzymatic modular assembly 7 are sugar nucleoside generating enzyme and α1-3 fucosyltransferase.

10. The method for synthesizing thiol-modified oligosaccharides according to claim 8, characterized in that: The enzymes used in enzymatic modular assembly 2 are galactokinase, sugar nucleoside generator and β1-4 galactosyltransferase.

11. The method for synthesizing thiol-modified oligosaccharides according to claim 8, characterized in that: The enzymes used in the enzymatic modular assembly 6 are sugar nucleoside generating enzyme and α2-3 sialyltransferase.

12. The method for synthesizing thiol-modified oligosaccharides according to claim 8, characterized in that: The enzymes used in the enzymatic modular assembly 8 are sugar nucleoside generating enzyme and α2-6 sialyltransferase.

13. The method for synthesizing thiol-modified oligosaccharides according to claim 4, characterized in that: The process of preparing thiol-modified oligosaccharides by enzymatic method also includes: coupling fucose to tetrasaccharide by α1-2 glycosidic bond to synthesize pentasaccharide shown in formula (VI) by enzymatic modular assembly 3; 14. The method for synthesizing thiol-modified oligosaccharides according to claim 13, characterized in that: The enzymes used in enzymatic modular assembly 3 are sugar nucleoside generating enzyme and α1-2 fucosyltransferase.

15. The method for synthesizing thiol-modified oligosaccharides according to claim 13, characterized in that: The process of preparing thiol-modified oligosaccharides by enzymatic method also includes: using enzymatic modular assembly 4 to couple N-acetylgalactosamine to the pentasaccharide of formula (VI) by α1-3 glycosidic bond to synthesize the hexasaccharide of formula (VII); 16. The method for synthesizing thiol-modified oligosaccharides according to claim 15, characterized in that: The enzymes used in the enzymatic modular assembly 4 are a fusion enzyme of N-acetylhexosaminyl kinase and sugar nucleoside generating enzyme and α1-3N-acetylgalactosyl transferase.

17. The method for synthesizing thiol-modified oligosaccharides according to claim 13, characterized in that: The process of preparing thiol-modified oligosaccharides by enzymatic method also includes: using enzymatic modular assembly 5 to couple galactose to the pentasaccharide of formula (VI) by α1-3 glycosidic bond to synthesize the hexasaccharide of formula (VIII); 18. The method for synthesizing thiol-modified oligosaccharides according to claim 17, characterized in that: The enzymes used in the enzymatic modular assembly 5 are galactokinase, sugar nucleoside generating enzyme, and α1-3 galactosyltransferase.

19. The method for synthesizing thiol-modified oligosaccharides according to claim 1, characterized in that: In the enzyme method, the reaction temperature is 0-37°C and the rotation speed is 0-240r / min; the method for stopping the enzyme reaction is to boil the reaction solution in a boiling water bath for 5-10 minutes.

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