CN116814720A - Synthesis method of sulfhydryl modified oligosaccharide - Google Patents

Synthesis method of sulfhydryl modified oligosaccharide Download PDF

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CN116814720A
CN116814720A CN202310582956.6A CN202310582956A CN116814720A CN 116814720 A CN116814720 A CN 116814720A CN 202310582956 A CN202310582956 A CN 202310582956A CN 116814720 A CN116814720 A CN 116814720A
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enzymatic
oligosaccharide
enzyme
sulfhydryl
modified
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方文元
刘静
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JINING MEDICAL UNIVERSITY
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JINING MEDICAL UNIVERSITY
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/08—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium
    • C07H5/10—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium to sulfur
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Abstract

The invention belongs to the field of sugar, relates to synthesis and separation of oligosaccharide, and in particular relates to a synthesis method of sulfhydryl modified oligosaccharide. Preparing lactose into a glycosyl acceptor containing sulfhydryl by adopting a chemical method, and coupling the glycosyl acceptor containing sulfhydryl with monosaccharide by adopting an enzymatic method to synthesize the sulfhydryl modified oligosaccharide; adding sulfhydryl modified silica into a system after enzymatic reaction, forming disulfide bonds between the sulfhydryl modified silica and the sulfhydryl modified oligosaccharide to obtain a silica-oligosaccharide compound, separating and extracting the silica-oligosaccharide compound, washing the silica-oligosaccharide compound with double distilled water, then opening disulfide bonds of the silica-oligosaccharide compound with beta-mercaptoethanol solution, and removing the silica to obtain the sulfhydryl modified oligosaccharide. According to the synthesis method, the high-efficiency separation of the sulfhydryl modified oligosaccharide and the enzyme reaction system is realized by utilizing the sulfhydryl modified silica carrier, and meanwhile, the high-efficiency performance of the enzyme reaction is ensured, so that the large-scale preparation of the oligosaccharide is realized.

Description

Synthesis method of sulfhydryl modified oligosaccharide
Technical Field
The invention belongs to the field of sugar, relates to synthesis and separation of oligosaccharide, and in particular relates to a synthesis method of sulfhydryl modified oligosaccharide.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Saccharide compounds have potential biomedical roles, and in order to study their biological functions, efficient acquisition of pure oligosaccharides and glycoconjugates is required. Although the chemoenzymatic method utilizes the flexible diversity of chemical synthesis and the specificity of enzymes to prepare a large amount of oligosaccharides, the complex separation process of an enzyme reaction system is too complicated to meet the demands of the oligosaccharides. Blixt and Norberg used disulfide bond as a coupling/separation strategy in 1997, where disaccharides were first linked to Thiopropyl as disulfide bonds6B resin. After the enzymatic reaction has ended, the linker-carrying glycans are removed from the solid support with dithiothreitol DTT. However, the inventors have found that oligosaccharides targeted to the resin undergo an enzymatic reaction with low efficiency, do not react completely and that Thiopropyl +.>The 6B resin is relatively expensive and difficult to popularize and use on a large scale.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a synthesis method of sulfhydryl modified oligosaccharide, which realizes the efficient separation of the sulfhydryl modified oligosaccharide and an enzyme reaction system by utilizing a sulfhydryl modified silica carrier, and ensures the efficient performance of the enzyme reaction at the same time, thereby realizing the large-scale preparation of the oligosaccharide.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for synthesizing sulfhydryl modified oligosaccharide, which comprises preparing lactose into sulfhydryl-containing glycosyl acceptor by chemical method, and coupling sulfhydryl-containing glycosyl acceptor with monosaccharide by enzymatic method to synthesize sulfhydryl modified oligosaccharide;
adding sulfhydryl modified silica into a system after enzymatic reaction, forming disulfide bonds between the sulfhydryl modified silica and the sulfhydryl modified oligosaccharide to obtain a silica-oligosaccharide compound, separating and extracting the silica-oligosaccharide compound, washing the silica-oligosaccharide compound with double distilled water, then opening the disulfide bonds of the silica-oligosaccharide compound with beta-mercaptoethanol solution, and removing the silica to obtain the sulfhydryl modified oligosaccharide;
the chemical structural formula of the sulfhydryl-containing glycosyl acceptor is shown as formula I:
wherein R is mercapto or mercapto-substituted alkyl.
The invention researches the cost problem of separation and purification and the separation materials used in the synthesis of the oligosaccharidases. Firstly, using sulfhydryl modified silicon dioxide as an oligosaccharide separation material, preparing a sulfhydryl-containing glycosyl acceptor by a chemical method, and then extending a sugar chain by an enzymatic method to obtain the sulfhydryl modified oligosaccharide. Then disulfide bonds are formed between the sulfhydryl-modified oligosaccharide and the sulfhydryl-modified silicon dioxide, and the oligosaccharide and the silicon dioxide are compounded, so that the silicon dioxide is used for fixing and separating the oligosaccharide in the solution, and then the disulfide bonds are reduced by beta-mercaptoethanol, so that the oligosaccharide and the silicon dioxide are separated, and the sulfhydryl-modified oligosaccharide is obtained.
Because disulfide bonds are formed by oxidizing two mercapto groups, in the process of forming the disulfide bonds, the disulfide bonds are formed between the mercapto-modified oligosaccharides, and the oligosaccharides are separated and recovered, however, experiments show that the yield is over 95 percent by adopting the mercapto-modified silicon dioxide as an oligosaccharide separation material, which shows that the selectivity of the disulfide bonds formed between the mercapto-modified silicon dioxide and the mercapto-modified oligosaccharides is better, thus being beneficial to the separation of the mercapto-modified oligosaccharides, and further ensuring the yield of the mercapto-modified oligosaccharides.
The beneficial effects of the invention are as follows:
according to the invention, the sulfhydryl-containing glycosyl acceptor is prepared by a chemical method, the enzymatic method is adopted to carry out efficient extension on the sugar chain, and the efficiently prepared sulfhydryl-modified oligosaccharide is rapidly and completely separated from an enzymatic reaction system by taking the sulfhydryl-modified silicon dioxide as an oligosaccharide separation material, so that the reaction steps are simplified, the separation and purification time is shortened, the synthesis efficiency of the oligosaccharide is provided, and the overall yield of the oligosaccharide is ensured.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic view of an enzymatic modular assembly 1 of the present invention;
FIG. 2 is a schematic view of an enzymatic modular assembly 2 of the present invention;
FIG. 3 is a schematic view of an enzymatic modular assembly 3 of the present invention;
FIG. 4 is a schematic view of an enzymatic modular assembly 4 of the present invention;
FIG. 5 is a schematic view of an enzymatic modular assembly 5 of the present invention;
FIG. 6 is a schematic view of an enzymatic modular assembly 6 of the present invention;
FIG. 7 is a schematic view of an enzymatic modular assembly 7 of the present invention;
FIG. 8 is a schematic view of an enzymatic modular assembly 8 of the present invention;
FIG. 9 is a schematic representation of the chemical preparation of mercapto-modified silica supports of the present invention;
FIG. 10 is a schematic illustration of the chemical synthesis of lactose compound 1 in beta-configuration according to example 1 of the present invention;
FIG. 11 is a schematic diagram showing the enzymatic modular synthesis of heptasaccharide compound 2 according to example 1 of the present invention;
FIG. 12 is a schematic illustration of the enzymatic modular synthesis of heptasaccharide compound 3 according to example 2 of the present invention;
FIG. 13 is a schematic illustration of the enzymatic modular synthesis of heptasaccharide compound 4 according to example 3 of the present invention;
FIG. 14 is a schematic illustration of the enzymatic modular synthesis of heptasaccharide compound 5 according to example 4 of the present invention;
FIG. 15 is a schematic illustration of the enzymatic modular synthesis of pentasaccharide compound 6 according to example 5 of the present invention;
FIG. 16 is a schematic illustration of the enzymatic modular synthesis of hexasaccharide compound 7 according to example 6 of the present invention;
FIG. 17 is a schematic diagram showing the enzymatic modular synthesis of hexasaccharide compound 8 according to example 7 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the difficulty in considering the enzyme reaction efficiency and the separation efficiency of the existing method, the invention provides a synthesis method of sulfhydryl modified oligosaccharide.
In an exemplary embodiment of the invention, a synthesis method of a sulfhydryl modified oligosaccharide is provided, lactose is prepared into a sulfhydryl-containing glycosyl acceptor by a chemical method, and the sulfhydryl-containing glycosyl acceptor is coupled with monosaccharide by an enzymatic method to synthesize the sulfhydryl modified oligosaccharide;
adding sulfhydryl modified silica into a system after enzymatic reaction, forming disulfide bonds between the sulfhydryl modified silica and the sulfhydryl modified oligosaccharide to obtain a silica-oligosaccharide compound, separating and extracting the silica-oligosaccharide compound, washing the silica-oligosaccharide compound with double distilled water, then opening the disulfide bonds of the silica-oligosaccharide compound with beta-mercaptoethanol solution, and removing the silica to obtain the sulfhydryl modified oligosaccharide;
the chemical structural formula of the sulfhydryl-containing glycosyl acceptor is shown as formula I:
wherein R is mercapto or mercapto-substituted alkyl.
In some embodiments, the beta-mercaptoethanol solution is subjected to ultrasonic degasification prior to use, to minimize oxidation of the mercapto groups.
In some embodiments, after the enzymatic reaction, the reaction is stopped in a boiling water bath, the reaction is centrifuged at 1-10 ℃, the supernatant is concentrated, the concentrate is mixed with the mercapto-modified silica for 20-60 min, the precipitate is collected by centrifugation, the precipitate is washed with double distilled water, then the beta-mercaptoethanol solution is added and mixed for 20-60 min, and the supernatant containing the mercapto-modified oligosaccharides is collected by centrifugation.
In some embodiments, R is mercapto. Studies show that when R is mercapto, the separation effect is better.
Mercapto-modified silica may be obtained commercially or by self-synthesis, and in some embodiments, the mercapto-modified silica is prepared by: by passing throughThe method uses Tetraethoxysilane (TEOS) to prepare silicon dioxide under the condition of ammonia water, and uses mercaptosilane to carry out modification reaction on the silicon dioxide to obtain mercapto modified silicon dioxide, as shown in figure 9. By->The silicon dioxide prepared by the method is a nano-scale particle material, and is modified by adopting mercaptosilane, so that the capture of the mercapto-modified oligosaccharide is facilitated. The mercaptosilane used may be 3-mercaptopropyltriethoxysilane, 2-mercaptoethyltriethoxysilane, mercaptomethyltriethoxysilane, or the like.
In some embodiments, the lactose is subjected to a total acetylation reaction, a bromoglycosylation reaction, a sulfation reaction, and a deprotection reaction in sequence to obtain the glycosyl acceptor containing sulfhydryl.
Specifically, lactose and acetic anhydride are reacted, and then all exposed hydroxyl groups are protected by acetyl groups; then preparing alpha-configuration glycoside by using hydrogen bromide (acetic acid solution), then adding potassium thioacetate, and removing protection.
In some embodiments, the process of preparing the sulfhydryl-modified oligosaccharide using an enzymatic method comprises: coupling N-acetamido glucose to a sulfhydryl-containing glycosyl acceptor in beta 1-3 glycosidic bond by using an enzymatic modular assembly 1 (shown in figure 1) to synthesize trisaccharide; coupling galactose to trisaccharide by beta 1-4 glycosidic bond to synthesize tetrasaccharide by utilizing an enzymatic modular assembly 2 (shown in figure 2); coupling N-acetylglucosamine to tetraose through beta 1-3 glycosidic bond by utilizing an enzymatic modularized assembly 1 to synthesize poly-LacNAc skeleton pentasaccharide; coupling galactose to poly-LacNAc skeleton pentasaccharide by beta 1-4 glycosidic bond to synthesize hexasaccharide 1 by using enzyme method modularized assembly 2; coupling fucose to hexasaccharide 1 with alpha 1-2 glycosidic bond by using enzyme method modularized assembly 3 (shown in figure 3) to synthesize heptasaccharide shown in formula (II);
specifically, the enzymes used in the enzymatic modular assembly 1 are fusion enzymes (NahK/GlmU) of N-acetylhexokinase (NahK) and a sugar nucleoside-producing enzyme (GlmU) and beta 1-3N-acetylglucosyl transferase (HpLgtA).
Specifically, enzymes used in the enzymatic modular assembly 2 are galactokinase (GalK), sugar nucleoside producing enzyme (BLUSP) and beta 1-4 galactosyltransferase (NmLgtB).
Specifically, enzymes used in the enzymatic modular assembly 3 are a sugar nucleoside producing enzyme (FKP), and an α1-2 fucosyltransferase (hmα1,2 fuct).
Specifically, a sulfhydryl group-containing glycosyl acceptor, N-acetamido glucose (1.2-4.0 equivalents), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalents), uracil nucleoside triphosphate (UTP) (1.2-4.0 equivalents), mgCl 2 (5-100 mM), tris-HCl buffer (10-500 mM, pH 5.0-10.0), then adjusting the pH value of the reaction system to 4.5-8.5, then adding N-acetamido hexokinase (NahK) and sugar nucleoside to generateThe fusion enzyme (NahK/GlmU) of the enzyme (GlmU) and beta 1-3N-acetylglucosaminyl transferase (HpLgtA) react for 3-70 hours, after the reaction is finished, target oligosaccharide is captured by a sulfhydryl modified silica carrier, double distilled water is used for eluting impurities, and finally, the trisaccharide is obtained by eluting and releasing 5-40 mM beta-mercaptoethanol. As a receptor for the next enzyme reaction, galactose (1.2-4.0 equivalents), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalents), uracil nucleoside triphosphate (UTP) (1.2-4.0 equivalents), mgCl 2 (5-100 mM), tris-HCl buffer solution (10-500 mM, pH 5.0-10.0), regulating the pH value of the reaction system to 4.5-8.5, adding galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP) and beta 1-4 galactosyltransferase (NmLgtB), reacting for 3-70 hours, capturing target oligosaccharide through a sulfhydryl modified silica carrier after the reaction is finished, eluting impurities through double distilled water, and finally eluting and releasing through 5-40 mM beta-mercaptoethanol to obtain the tetraose. The repeated use of the above enzyme module can prepare poly-LacNAc hexasaccharide 1 which can be used as a 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), mgCl 2 (5-100 mM), tris-HCl buffer solution (10-500 mM, pH 5.0-10.0), regulating the pH value of the reaction system to 4.5-8.5, adding sugar nucleoside generating enzyme (FKP), alpha 1-2 fucosyltransferase (Hmalpha 1,2 FucT) for 3-70 hours, capturing target oligosaccharides through a sulfhydryl modified silica carrier after the reaction is completed, eluting impurities through double distilled water, and finally eluting and releasing through 5-30 mM beta-mercaptoethanol to obtain the heptasaccharide shown in the formula (II) respectively.
In one or more embodiments, the process of enzymatically preparing the thiol-modified oligosaccharide further comprises: coupling fucose to poly-LacNAc skeleton pentasaccharide with alpha 1-3 glycosidic bond to synthesize hexasaccharide 2 by using enzyme method modularized assembly 7 (shown in figure 7); coupling galactose to hexasaccharide 2 with beta 1-4 glycosidic bond by using enzyme method modularized assembly 2 (shown in figure 2), and synthesizing heptasaccharide shown in formula (III);
specifically, enzymes used in the enzymatic modular assembly 7 are a sugar nucleoside producing enzyme (FKP), and an α1-3 fucosyltransferase (Hpα1,3 FucT).
Specifically, enzymes used in the enzymatic modular assembly 2 are galactokinase (GalK), sugar nucleoside producing enzyme (BLUSP) and beta 1-4 galactosyltransferase (NmLgtB).
Specifically, poly-LacNAc backbone pentasaccharide, fucose (1.2-4.0 equivalents), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalents), guanosine Triphosphate (GTP) (1.2-4.0 equivalents), mgCl 2 (5-100 mM), tris-HCl buffer (10-500 mmol, pH 5.0-10.0), regulating pH of the reaction system to 4.5-8.5, adding sugar nucleoside generating enzyme (FKP), alpha 1-3 fucosyltransferase (Hpalpha 1,3 FucT), reacting for 3-70 hours, capturing target oligosaccharide by using sulfhydryl modified silica carrier after the reaction is completed, eluting impurities by double distilled water, eluting by 5-30 mM beta-mercaptoethanol, and releasing to obtain hexasaccharide 2 as a receptor for the next enzyme reaction, galactose (1.2-4.0 equivalent), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalent), uracil nucleoside triphosphate (UTP) (1.2-4.0 equivalent), mgCl 2 (5-100 mM), tris-HCl buffer solution (10-500 mM, pH 5.0-10.0), regulating the pH value of a reaction system to 4.5-8.5, adding galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP) and beta 1-4 galactosyltransferase (NmLgtB), reacting for 3-70 hours, capturing target oligosaccharide through a sulfhydryl modified silica carrier after the reaction is finished, eluting impurities through double distilled water, and finally eluting and releasing through 5-40 mM beta-mercaptoethanol to obtain the heptasaccharide shown in the formula (III).
In one or more embodiments, the process of enzymatically preparing the thiol-modified oligosaccharide further comprises: coupling sialic acid to hexasaccharide 1 with alpha 2-3 glycosidic bond by using enzyme method modularized assembly 6 (shown in figure 6) to synthesize heptasaccharide shown in formula (IV);
specifically, enzymes used in the enzymatic modular assembly 6 are a sugar nucleoside producing enzyme (NmCSS) and an α2-3 sialyltransferase (PmST 1M 144D).
Specifically, hexasaccharide 1, N-acetylneuraminic acid (Neu 5 Ac) (1.2-4.0 eq), cytidine Triphosphate (CTP) (1.2-4.0 eq), mgCl 2 (5-100 mM), tris-HCl buffer solution (10-500 mM, pH 5.0-10.0), regulating the pH value of the reaction system to 4.5-8.5, adding sugar nucleoside generating enzyme (NmCSS) and alpha 2-3 sialyltransferase (PmST 1M 144D), reacting for 0.5-36 hours, capturing target oligosaccharide through a sulfhydryl modified silica carrier after the reaction is finished, eluting impurities through double-distilled water, and finally eluting and releasing through 5-40 mM beta-mercaptoethanol to obtain the heptasaccharide shown in the formula (IV).
In one or more embodiments, the process of enzymatically preparing the thiol-modified oligosaccharide further comprises: using enzyme method to make modularized assembly 8 (shown in figure 8), coupling sialic acid to hexasaccharide 1 with alpha 2-6 glycosidic bond to synthesize heptasaccharide shown in formula (V);
specifically, enzymes used in the enzymatic modular assembly 8 are a sugar nucleoside producing enzyme (NmCSS), and an α2-6 sialyltransferase (Pd 2,6 ST).
Specifically, hexasaccharide 1, N-acetylneuraminic acid (Neu 5 Ac) (1.2-4.0 eq), cytidine Triphosphate (CTP) (1.2-4.0 eq), mgCl 2 (5-100 mM), tris-HCl buffer (10-500 mM, pH 5.0-10.0), regulating pH of the reaction system to 4.5-8.5, adding sugar nucleoside generating enzyme (NmCSS) and alpha 2-6 sialyltransferase (Pd 2,6 ST), reacting for 0.5-36 hr, capturing target oligosaccharide with mercapto-modified silica carrier after the reaction, eluting impurities with double distilled water, and finally eluting with 5-40 mM beta-mercaptoEluting with ethanol to release to obtain heptasaccharide shown in formula (V).
In one or more embodiments, the process of enzymatically preparing the thiol-modified oligosaccharide further comprises: coupling fucose to tetrasaccharide by using an enzymatic modular assembly 3 to synthesize pentasaccharide represented by formula (VI) by an alpha 1-2 glycosidic bond;
specifically, enzymes adopted in the enzymatic modular assembly 3 are a sugar nucleoside generating enzyme (FKP) and an alpha 1-2 fucosyltransferase (Hmalpha 1-2 FucT).
Specifically, the tetraose, fucose (1.2-4.0 equivalents), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalents), guanine nucleoside triphosphate (GTP) (1.2-4.0 equivalents), mgCl in the synthesis of formula (II) 2 (5-100 mM), tris-HCl buffer solution (10-500 mmol, pH 5.0-10.0), regulating the pH value of the reaction system to 4.5-8.5, adding sugar nucleoside generating enzyme (FKP), alpha 1-3 fucosyltransferase (Hpalpha 1,3 FucT) for 3-70 hours, capturing target oligosaccharide through a sulfhydryl modified silica carrier after the reaction is completed, eluting impurities through double distilled water, and finally eluting and releasing through 5-40 mM beta-mercaptoethanol to obtain the heptasaccharide shown in the formula (VI).
In one or more embodiments, the process of enzymatically preparing the thiol-modified oligosaccharide further comprises: coupling N-acetamido galactose with alpha 1-3 glycosidic bond to pentasaccharide shown in formula (VI) to synthesize hexasaccharide shown in formula (VII) by using enzyme method modularization assembly 4 (shown in figure 4);
specifically, the enzymes used in the enzymatic modular assembly 4 are a fusion enzyme (NahK/GlmU) of N-acetylhexokinase (NahK) and a sugar nucleoside-producing enzyme (GlmU) and an alpha 1-3N-acetylgalactosyl transferase (BgtA).
Specifically, a compound represented by the formula (VI)Pentasaccharide, N-acetamido galactose (1.2-4.0 equivalent), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalent), uracil nucleoside triphosphate (UTP) (1.2-4.0 equivalent), mgCl 2 (5-100 mM), tris-HCl buffer solution (10-500 mM, pH 5.0-10.0), regulating the pH value of a reaction system to 4.5-8.5, adding fusion enzyme (NahK/GlmU) of N-acetylhexokinase (NahK) and sugar nucleoside generating enzyme (GlmU), alpha 1-3N-acetylgalactosyltransferase (BgtA), reacting for 3-70 hours, capturing target oligosaccharide through a sulfhydryl modified silica carrier after the reaction is finished, eluting impurities through double distilled water, and finally eluting and releasing through 5-40 mM beta-mercaptoethanol to obtain the heptasaccharide shown in the formula (VII).
In one or more embodiments, the process of enzymatically preparing the thiol-modified oligosaccharide further comprises: coupling galactose with alpha 1-3 glycosidic bond to pentasaccharide shown in formula (VI) by using enzyme method modularization assembly 5 (shown in figure 5) to synthesize hexasaccharide shown in formula (VIII);
specifically, enzymes used in the enzymatic modular assembly 5 are galactokinase (GalK), sugar nucleoside producing enzyme (BLUSP), and alpha 1-3 Galactosyltransferase (GTB).
Specifically, pentasaccharide represented by formula (VI), galactose (1.2-4.0 equivalents), adenine nucleoside triphosphate (ATP) (1.2-4.0 equivalents), uracil nucleoside triphosphate (UTP) (1.2-4.0 equivalents), mgCl 2 (5-100 mM), tris-HCl buffer solution (10-500 mM, pH 5.0-10.0) to prepare aqueous solution, then regulating the pH value of a reaction system to 4.5-8.5, then adding galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP) and alpha 1-3 galactose Glycosyltransferase (GTB), reacting for 3-70 hours, capturing target oligosaccharide through a sulfhydryl modified silica carrier after the reaction is finished, eluting impurities through double-steaming water, and finally eluting and releasing through 5-30 mM beta-mercaptoethanol to obtain the heptasaccharide shown in the formula (VIII).
"equivalent weight" in the present invention refers to the ratio of the amounts of substances at the time of substance interactions, such as: n-acetylglucosamine (1.2-4.0 eq.) has the meaning: the ratio of the amount of N-acetylglucosamine to the amount of the thiol-containing glycosyl acceptor substance is 1.2-4.0.
After the reaction was completed, the progress of the reaction was followed by Thin Layer Chromatography (TLC) with the formula (I), (II) and the developing agent EtOAc: meOH: H 2 O: HOAc=4:2:1:0.2 and synthetic expanders of formulae (III) - (VIII) were EtOAc: meOH: H 2 O:HOAc＝2:2:1:0.2。
In the enzymatic method, the reaction temperature is 0-37 ℃ and the rotating speed is 0-240 r/min; the stopping method of the enzyme reaction is that the reaction liquid is boiled in a boiling water bath for 5 to 10 minutes.
The enzyme method module adopted by the invention contains three enzymes of glucokinase, sugar nucleoside generating enzyme and glycosyltransferase, which play a role in high-efficiency catalysis and cooperate with each other to form an organic enzyme reaction system. The enzymes used above were subjected to several optimizations and screens during the assay, which resulted in the finding: nahK/GlmU, bifidobacterium longum N-acetylhexosamine-1-kinase (NahK) and E.coli N-acetylglucosamine uridyltransferase (GlmU), and Helicobacter mustelae. Alpha.1, 3-N-galactosyltransferase (BgtA); helicobacter pylori beta 1,3-N-acetylglucosaminyltransferase (HpLgtA); e.coli galactokinase (GalK), bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP) and Neisseria meningitides β1,4-galactosyltransferase (NmLgtB); neisseria meningitides CMP-sialic acid synthetase (NmCSS), and Pasteurella multocida multifunctional2,3-sialyltransferase 1M144D (PmST 1M 144D); human alpha 1,3-Galactosyltransferase (GTB); bacteroides fragilis bifunctional L-fucokinase/GDP-fucose pyrophosphorylase (FKP) and Helicobacler muslelae. Alpha.l, 2-fucosynltransferase (Hmα1,2 FucT); helicobacter pylori. Alpha.1, 3-fucosynltransferase (Hpα1,3 FucT); photobacterium damselae2,6-sialyltransferase mutant (Pd 2,6 ST) as the enzyme used in the present invention has the advantages of optimal catalytic effect, high synthesis efficiency and simple purification, and the enzyme can be expressed and purified in a large amount in a conventional E.coli expression system.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
Preparation of mercapto-modified silica support:
200mL of absolute ethyl alcohol is added into a 250mL single-neck flask, 15mL of ammonia water is added, the mixture is stirred uniformly, 6mL of tetraethyl silicate is added into the single-neck flask dropwise, and the reaction is carried out for 5h at the temperature of 60 ℃ after the dropwise addition. Centrifugation was carried out at 15000rpm for 30min, and washing was carried out three times with a mixed solvent of absolute ethanol and deionized water in a volume ratio of 1:1. The silica nanoparticles prepared above were uniformly dispersed by adding a suitable amount of absolute ethanol thereto, and 15mL of 3-mercaptopropyl triethoxysilane was added thereto to react for 8 hours under a nitrogen atmosphere. Centrifuging at 15000rpm for 30min, washing with mixed solvent of absolute ethanol and deionized water at volume ratio of 1:1 for three times, and washing with absolute ethanol once.
Synthesis of beta-configured lactobionic glycosides 1[ Gal beta (l-4) GlcβOR ]:
lactose 9 (10 g,29.23 mmol), acetic anhydride (55 mL) and sodium acetate (9.6 g) were added to a 500mL round bottom flask and stirred at 160℃under reflux for 6 hours. After completion of the reaction, which was detected by thin layer chromatography (PE: ea=1:2), the reaction mixture was concentrated by rotary evaporation. The resulting solid was redissolved in 250mL of dichloromethane, extracted twice with half-saturated brine, extracted three times with saturated sodium bicarbonate solution, extracted three times with double distilled aqueous solution, after which the organic phase was separated, dried over anhydrous sodium sulfate and concentrated by rotary evaporation to give compound 10 (18.70 g, 94%) as a pale yellow solid.
To a 250mL round bottom flask was added compound 10 (15.0 g,22.12 mmol) in hydrogen bromide (33% acetic acid solution) in dichloromethane and stirred at room temperature under argon atmosphere for 2h and the progress of the reaction was monitored by TLC. When the reaction was completed, the reaction solution was poured into water, extracted with DCM, washed with aqueous sodium bicarbonate solution, and the organic phase was dehydrated and dried over anhydrous sodium sulfate to obtain compound 11.
To a 250mL round bottom flask was added lactose bromoglycoside 11 (5.0 g,7.12 mmol) dissolved in anhydrous DMF (20 mL), potassium thioacetate (2.6 g,22.01 mmol) was added to the solution, the solution was stirred at room temperature for 5h and the solution turned dark brown, and the progress of the reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was diluted with methylene chloride, eluted with a large amount of double distilled water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, and then concentrated and purified by a silica gel column (PE/ea=3:2, v/v) to give compound 12 (4.2 g, 85%) as a yellow solid.
To a 250mL round bottom flask was added compound 12 (5.2 g,6.94 mmol) dissolved in methanol and sodium methoxide pH 11 was added for 30 minutes at room temperature and the progress of the reaction was monitored by TLC. MeOH/H after completion of the reaction 2 Diluting with O (2/1, v/v), adding acidic ion resin50WX8-200(H + form), and filtration to obtain lactose acceptor compound 1 (2.8 g).
The synthetic route for lactose acceptor compound 1 is shown in figure 10.
Synthesis of heptasaccharide compound by enzymatic Module Assembly 1
2[Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]：
Lactose acceptor 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 MgCl 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, nahK/GlmU (2.0 mg) and HpLgtA (2.0 mg) were added, double distilled water was added to a total volume of 20mL, and the reaction system was placed in a shaker and incubated at 37℃for 16 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=8:3:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. Then centrifuging the reaction system at 4℃at 12000rpm for 20 minutes, collecting the supernatant, spin-concentrating, blending the concentrated solution with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier is 1:10) for 30 minutes, centrifuging at 12000rpm for 10 minutes, collecting the precipitate, washing with double distilled water three times to remove non-mercapto impurities, blending 25mM beta-mercaptoethanol with the precipitate for 30 minutes, centrifuging at 12000rpm for 10 minutes, collecting the supernatant, and obtaining a trisaccharide compound (265)0mg, 96%). 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 MgCl 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, galK (2.0 mg), BLUSP (2.0 mg) and NmLgtB (2.0 mg) were added, double distilled water was added to a total volume of 15mL, and the reaction system was placed in a shaker and incubated at 37℃for 110r/min for 12 hours. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. The reaction system was then centrifuged at 4℃at 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier was 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, followed by washing with double distilled water three times to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and the supernatant was collected by centrifugation at 12000rpm for 10 minutes to obtain a tetrasugar compound (174.1 mg, 95%). Tetraose (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 MgCl 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, nahK/GlmU (2.0 mg) and HpLgtA (2.0 mg) were added, double distilled water was added to a total volume of 20mL, and the reaction system was placed in a shaker and incubated at 37℃for 16 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. The reaction system was then centrifuged at 4℃at 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with the mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier was 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, followed by washing with double distilled water three times to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended 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 MgCl 2 (20 mM) (Tris andMgCl 2 the amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, galK (2.0 mg), BLUSP (2.0 mg) and NmLgtB (2.0 mg) were added, double distilled water was added to a total volume of 15mL, and the reaction system was placed in a shaker and incubated at 37℃for 110r/min for 12 hours. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. The reaction system was then centrifuged at 4℃at 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier was 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, followed by washing with double distilled water three times to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended 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%). Subsequently, hexasaccharide (160 mg), fucose (31.2 mg), ATP (104.7 mg), GTP (114.6 mg), tris-HCl buffer (100 mM, pH 7.5) and MgCl were added 2 (20 mM) (Tris and MgCl) 2 The amount of (B) was determined by calculation from the volume of the final reaction mixture) dissolved in a 50mL centrifuge tube, and FKP (2.00 mg) and Hmα1,2FucT (2.00 mg) were added, and after adding double distilled water to a total volume of 10mL, the reaction system was placed in a shaker and incubated at 37℃for 4 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. Then, the reaction system was centrifuged at 4℃for 20 minutes at 12000r/min, the supernatant was collected, concentrated by spin distillation, and the supernatant was collected by blending the concentrated solution with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier is 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, followed by washing with double distilled water three times to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and centrifuged at 12000rpm for 10 minutes to collect the supernatant, to obtain white heptasaccharide compound 2 (179.9 mg, 96%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)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),4.13(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,D 2 O)δ176.2,174.7,174.6,102.8,102.7,102.6,102.4,102.0,101.6,99.5,81.9,81.6,78.2,78.1,75.3,75.0,74.8,74.7,74.4,74.3,74.2,72.7,72.4,72.0,71.8,70.9,70.4,69.9,69.4,69.1,68.7,68.5,68.2,68.2,67.7,66.6,61.4,61.0,60.9,60.0,59.8,38.8,34.3,23.0,22.2,22.1,15.2。
the synthetic route for heptasaccharide compound 2 is shown in figure 11.
Example 2
Synthesis of heptasaccharide compound 3 by enzymatic Module Assembly 2
[Galβ(l-4)Fucα(1-3)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]
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 MgCl prepared in example 1 were mixed 2 (20 mM) (Tris and MgCl) 2 The amount of (B) was determined by calculation from the volume of the final reaction mixture) dissolved in a 50mL centrifuge tube, and then FKP (2.00 mg) and Hpα1,3FucT (2.00 mg) were added, and after adding double distilled water to a total volume of 10mL, the reaction system was placed in a shaker and incubated at 37℃for 4 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. The reaction system was then centrifuged at 4℃at 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier was 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, then washed three times with double distilled water to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended 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%). Hexasaccharide (70 mg), galactose (23.3 mg), ATP (56.7 mg), UTP (57.1 mg), tris-HCl buffer (100 mM, pH 7.5) and MgCl 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, galK (2.0 mg), BLUSP (2.0 mg) and NmLgtB (2.0 mg) were added, double distilled water was added to a total volume of 15mL, and the reaction system was placed in a shaker and incubated at 37℃for 110r/min for 12 hours. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) detection reaction was completed, boilingThe reaction was stopped by boiling in water bath for 5 minutes. Then, the reaction system was centrifuged at 4℃and 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier is 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, then washed three times with double distilled water to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and centrifuged at 12000rpm for 10 minutes to collect the supernatant, to obtain white heptasaccharide compound 3 (87.1 mg, 95%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)δ5.04(d,J＝4.0Hz,1H),4.64(dd,J＝8.5,5.4Hz,2H),4.42(d,J＝8.0Hz,1H),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,D 2 O)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。
the synthetic route for heptasaccharide compound 3 is shown in figure 12.
Example 3
Enzymatic modular 3 synthesis of heptasaccharide compounds
4[Neu5Acα(2-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]：
The hexasaccharide compound (50 mg), neu5Ac (14.4 mg), CTP (23.1 mg), tris-HCl buffer (100 mM, pH 8.0) and MgCl prepared in example 1 2 (20 mM) (Tris and MgCl) 2 The amount of (B) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, nmCSS (2.0 mg) and PmST1M144D (2.0 mg) were added, double distilled water was added to a total volume of 15mL, and the reaction system was placed in a shaker and incubated at 37℃for 1 hour at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=2:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. Then centrifuging the reaction system at 4 ℃ and 12000rpm for 20 minutes, collecting supernatant, concentrating by rotary evaporation, blending the concentrated solution with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier is 1:10) for 30 minutes, and centrifuging at 12000rpm for 10min, collecting the precipitate, washing with double distilled water three times to remove non-sulfhydryl impurities, blending 25mM beta-mercaptoethanol with the precipitate for 30min, centrifuging at 12000rpm for 10min, and collecting the supernatant to obtain white heptasaccharide compound 4 (59.1 mg, 95%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)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,D 2 O)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。
the synthetic route for heptasaccharide compound 4 is shown in figure 13.
Example 4
Synthesis of heptasaccharide compound by enzyme method modularization 4
5[Neu5Acα(2-6)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]：
The hexasaccharide compound (50 mg), neu5Ac (14.4 mg), CTP (23.1 mg), tris-HCl buffer (100 mM, pH 8.0) and MgCl prepared in example 1 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, nmCSS (2.0 mg), pd2,6ST (2.0 mg) were added, double distilled water was added to a total volume of 15mL, and the reaction system was placed in a shaking table and incubated at 37℃for 1 hour at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=2:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. Then, the reaction system was centrifuged at 4℃and 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier is 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, then washed three times with double distilled water to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and centrifuged at 12000rpm for 10 minutes to collect the supernatant, thereby obtaining white heptasaccharide compound 5 (58.9 mg, 94%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)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)； 13 C NMR(150MHz,D 2 O)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。
the synthetic route for heptasaccharide compound 5 is shown in figure 14.
Example 5
Synthesis of pentasaccharide compound by enzymatic Module Assembly 5
6[Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]：
The tetraose compound (160 mg), fucose (62.2 mg), ATP (204.7 mg), GTP (214.6 mg), tris-HCl buffer (100 mM, pH 7.5) and MgCl prepared in example 1 were mixed 2 (20 mM) (Tris and MgCl) 2 The amount of (B) was determined by calculation from the volume of the final reaction mixture) dissolved in a 50mL centrifuge tube, and then FKP (2.00 mg) and Hmα1,2FucT (2.00 mg) were added, and after adding double distilled water to a total volume of 10mL, the reaction system was placed in a shaker and incubated at 37℃for 10 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. The reaction system was then centrifuged at 4℃at 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier was 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, then washed three times with double distilled water to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and centrifuged at 12000rpm for 10 minutes to collect the supernatant, thereby obtaining a white pentasaccharide compound 6 (218.9 mg, 96%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)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,D 2 O)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。
the synthesis route of pentasaccharide compound 6 is shown in fig. 15.
Example 6
Synthesis of hexasaccharide from enzyme method module assembly 6
7[GalNAcα(1–3)Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]：
Pentasaccharide compound 6 (50 mg), N-acetamido galactose (11.1 mg), ATP (27.6 mg), UTP (24.2 mg), tris-HCl buffer (100 mM, pH 8.0) and MgCl prepared in example 5 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, nahK/GlmU (2.0 mg) and BgtA (2.0 mg) were added, double distilled water was added to a total volume of 10mL, and the reaction system was placed in a shaker and incubated at 37℃for 16 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. The reaction system was then centrifuged at 4℃at 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier was 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, then washed three times with double distilled water to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and centrifuged at 12000rpm for 10 minutes to collect the supernatant, to obtain white hexasaccharide compound 7 (59.8 mg, 96%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)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,D 2 O)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。
the synthetic route for hexasaccharide compound 7 is shown in figure 16.
Example 7
Synthesis of hexasaccharide Compound 8 by enzymatic Module Assembly 7
[Galα(1–3)Fucα(1-2)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]：
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 MgCl prepared in example 5 2 (20 mM) (Tris and MgCl) 2 The amount of (C) was determined by calculation from the volume of the final reaction solution) dissolved in a 50mL centrifuge tube, galK (2.0 mg), BLUSP (2.0 mg) and GTB (2.0 mg) were added, double distilled water was added to a total volume of 10mL, and the reaction system was placed in a shaker and incubated at 37℃for 12 hours at 110 r/min. Thin layer chromatography (EtOAc: meOH: H) 2 O etoh=4:2:1:0.2) the reaction was terminated by boiling the boiling water bath for 5 minutes after completion of the reaction. Then, the reaction system was centrifuged at 4℃and 12000rpm for 20 minutes, the supernatant was collected, concentrated by spin distillation, the concentrated solution was blended with a mercapto-modified silica carrier (the mass ratio of the oligosaccharide to the mercapto-modified silica carrier is 1:10) for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the precipitate was collected, then washed three times with double distilled water to remove non-mercapto impurities, finally 25mM beta-mercaptoethanol was blended with the precipitate for 30 minutes, and centrifuged at 12000rpm for 10 minutes to collect the supernatant, thereby obtaining white hexasaccharide compound 8 (58.3 mg, 95%). The parameters are as follows: 1 H NMR(600MHz,D 2 O)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)； 13 C NMR(150MHz,D 2 O)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。
the synthetic route for hexasaccharide compound 8 is shown in figure 17.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A synthesis method of sulfhydryl modified oligosaccharide is characterized in that lactose is prepared into a sulfhydryl-containing glycosyl acceptor by a chemical method, and the sulfhydryl-containing glycosyl acceptor is coupled with monosaccharide by an enzymatic method to synthesize the sulfhydryl modified oligosaccharide;
adding sulfhydryl modified silica into a system after enzymatic reaction, forming disulfide bonds between the sulfhydryl modified silica and the sulfhydryl modified oligosaccharide to obtain a silica-oligosaccharide compound, separating and extracting the silica-oligosaccharide compound, washing the silica-oligosaccharide compound with double distilled water, then opening the disulfide bonds of the silica-oligosaccharide compound with beta-mercaptoethanol solution, and removing the silica to obtain the sulfhydryl modified oligosaccharide;
the chemical structural formula of the sulfhydryl-containing glycosyl acceptor is shown as formula I:
wherein R is mercapto or mercapto-substituted alkyl.
2. The method for synthesizing a mercapto-modified oligosaccharide according to claim 1, wherein the β -mercaptoethanol solution is subjected to ultrasonic deaeration treatment prior to use, so as to avoid oxidizing mercapto groups as much as possible.
3. The method for synthesizing the mercapto-modified oligosaccharides as set forth in claim 1, wherein after the enzymatic reaction, the reaction is terminated in a boiling water bath, and the reaction is centrifuged at 1 to 10 ℃, the supernatant is concentrated, the concentrate is mixed with the mercapto-modified silica for 20 to 60 minutes, the precipitate is collected by centrifugation, the precipitate is washed with double distilled water, then the beta-mercaptoethanol solution is added and mixed for 20 to 60 minutes, and the supernatant containing the mercapto-modified oligosaccharides is collected by centrifugation.
4. The method for synthesizing a mercapto-modified oligosaccharide according to claim 1, wherein R is a mercapto group.
5. The method for synthesizing a mercapto-modified oligosaccharide according to claim 1, wherein the mercapto-modified silica is prepared by: by passing throughThe method prepares silicon dioxide by tetraethoxysilane under the condition of ammonia water, and adopts mercaptosilane to carry out modification reaction on the silicon dioxide to obtain mercapto modified silicon dioxide.
6. The method for synthesizing a thiol-modified oligosaccharide as claimed in claim 1, wherein the thiol-group-containing glycosyl acceptor is obtained by sequentially subjecting lactose to a peracetylation reaction, a bromoglycosidation reaction, a sulfation reaction and a deprotection reaction.
7. The method for synthesizing the thiol-modified oligosaccharide according to claim 1, wherein the step of preparing the thiol-modified oligosaccharide by using an enzymatic method comprises: coupling N-acetamido glucose to a sulfhydryl-containing glycosyl receptor through beta 1-3 glycosidic bond by utilizing an enzymatic modular assembly 1 to synthesize trisaccharide; coupling galactose to trisaccharide through beta 1-4 glycosidic bond by utilizing an enzymatic modular assembly 2 to synthesize tetrasaccharide; coupling N-acetylglucosamine to tetraose through beta 1-3 glycosidic bond by utilizing an enzymatic modularized assembly 1 to synthesize poly-LacNAc skeleton pentasaccharide; coupling galactose to poly-LacNAc skeleton pentasaccharide by beta 1-4 glycosidic bond to synthesize hexasaccharide 1 by using enzyme method modularized assembly 2; coupling fucose to hexasaccharide 1 through alpha 1-2 glycosidic bond by using enzyme method modularized assembly 3 to synthesize heptasaccharide shown in formula (II);
preferably, the enzyme adopted in the enzymatic modular assembly 1 is a fusion enzyme of N-acetamidohexokinase and a sugar nucleoside generating enzyme, and beta 1-3N-acetamidoglucosyltransferase;
preferably, the enzyme employed in enzymatic modular assembly 2 is galactokinase, a glyconucleoside-producing enzyme, and a beta 1-4 galactosyltransferase;
preferably, the enzyme employed in enzymatic modular assembly 3 is a sugar nucleoside producing enzyme, alpha 1-2 fucosyltransferase.
8. The method for synthesizing a thiol-modified oligosaccharide as claimed in claim 7, wherein the step of preparing the thiol-modified oligosaccharide by an enzymatic method further comprises: coupling fucose to poly-LacNAc skeleton pentasaccharide by using enzyme method modularized assembly 7 to synthesize hexasaccharide 2; coupling galactose to hexasaccharide 2 through beta 1-4 glycosidic bond by utilizing an enzymatic modularized assembly 2 to synthesize heptasaccharide shown in a formula (III);
preferably, the enzyme used in the enzymatic modular assembly 7 is a sugar nucleoside producing enzyme, alpha 1-3 fucosyltransferase;
preferably, the enzyme employed in enzymatic modular assembly 2 is galactokinase, a glyconucleoside-producing enzyme, and a beta 1-4 galactosyltransferase;
alternatively, the enzymatic preparation of the sulfhydryl-modified oligosaccharide may further comprise: coupling sialic acid to hexasaccharide 1 by alpha 2-3 glycosidic bond by using enzyme method modularized assembly 6 to synthesize heptasaccharide shown in formula (IV);
preferably, the enzyme used in the enzymatic modular assembly 6 is a sugar nucleoside producing enzyme, alpha 2-3 sialyltransferase;
alternatively, the enzymatic preparation of the sulfhydryl-modified oligosaccharide may further comprise: synthesizing a heptasaccharide shown in a formula (V) by coupling sialic acid to hexasaccharide 1 through an alpha 2-6 glycosidic bond by utilizing an enzymatic modular assembly 8;
preferably, the enzyme employed in the enzymatic modular assembly 8 is a sugar nucleoside producing enzyme, alpha 2-6 sialyltransferase.
9. The method for synthesizing a thiol-modified oligosaccharide as claimed in claim 7, wherein the step of preparing the thiol-modified oligosaccharide by an enzymatic method further comprises: coupling fucose to tetrasaccharide by using an enzymatic modular assembly 3 to synthesize pentasaccharide represented by formula (VI) by an alpha 1-2 glycosidic bond;
preferably, the enzyme adopted in the enzymatic modular assembly 3 is a sugar nucleoside generating enzyme or alpha 1-2 fucosyltransferase;
preferably, the enzymatic preparation of the sulfhydryl-modified oligosaccharide further comprises: coupling N-acetamido galactose to pentasaccharide shown in formula (VI) through alpha 1-3 glycosidic bond by utilizing an enzymatic modular assembly 4 to synthesize hexasaccharide shown in formula (VII);
preferably, the enzyme used in the enzymatic modular assembly 4 is a fusion enzyme of N-acetylhexosaminidase and a sugar nucleoside-producing enzyme, and alpha 1-3N-acetylgalactosamine transferase;
preferably, the enzymatic preparation of the sulfhydryl-modified oligosaccharide further comprises: coupling galactose to pentasaccharide shown in formula (VI) by alpha 1-3 glycosidic bond by using an enzymatic modular assembly 5 to synthesize hexasaccharide shown in formula (VIII);
preferably, the enzymes used in the enzymatic modular assembly 5 are galactokinase, a glyconucleoside producing enzyme, an alpha 1-3 galactosyltransferase.
10. The method for synthesizing a mercapto-modified oligosaccharide according to claim 1, wherein in the enzymatic method, the reaction temperature is 0-37 ℃ and the rotation speed is 0-240 r/min; the stopping method of the enzyme reaction is that the reaction liquid is boiled in a boiling water bath for 5 to 10 minutes.
CN202310582956.6A 2023-05-19 2023-05-19 Synthesis method of sulfhydryl modified oligosaccharide Pending CN116814720A (en)

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