A rapid enzymatic separation method for PEG-modified oligosaccharides

Abstract

The present invention belongs to the technical field of carbohydrate preparation, and relates to a separation and purification method of carbohydrates, and in particular to a method for rapid enzymatic separation of PEG-modified oligosaccharides. Lactose is used as a raw material to prepare a PEG-modified glycosyl receptor by a chemical method, and the PEG-modified glycosyl receptor is coupled with a monosaccharide raw material by an enzymatic method to form a PEG-modified oligosaccharide by a glycosidic bond; a first alcohol solvent is added to the material after the enzymatic reaction and mixed evenly, and then a first solid-liquid separation is performed to obtain purified PEG-modified oligosaccharides; the purified PEG-modified oligosaccharides are redissolved with water, the chemical bond between the oligosaccharide and the PEG is disconnected, and then a second alcohol solvent is added, mixed evenly and a second solid-liquid separation is performed to obtain non-PEG-modified oligosaccharides. The present invention only requires solid-liquid separation operations such as centrifugation on the enzyme reaction solution, and the process is simple to operate, shortens the time of sugar chain synthesis, and improves the efficiency of oligosaccharide synthesis.

Description

Technical Field

[0001] The invention belongs to the technical field of carbohydrate preparation, relates to a method for separating and purifying carbohydrates, and in particular to a method for rapid separation of oligosaccharides modified by PEG (polyethylene glycol). 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] Sugars have complex structures (with multiple hydroxyl groups and glycosidic bonds with stereo configurations) and microscopic heterogeneity of sugar chain structures in glycoconjugates (different glycosidic bond connection methods, glycosylation numbers, etc.). These characteristics make it extremely difficult to obtain oligosaccharides and glycoconjugates with defined structures by separation and extraction. Currently, there are a variety of synthetic methods that can improve the efficiency of oligosaccharide synthesis. However, the separation of oligosaccharides mainly uses ion exchange chromatography, gel permeation chromatography, and a combination of the two. This method has a long separation time and high preparation cost, making it difficult to achieve large-scale separation and preparation of oligosaccharides. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for rapid enzymatic separation of PEG-modified oligosaccharides, which can achieve efficient and high-yield synthesis and separation of target oligosaccharides.

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

[0006] On the one hand, a method for rapid separation of PEG-modified oligosaccharides by enzymatic method, using lactose as a raw material to prepare a PEG-modified glycosyl acceptor by chemical method, and using an enzymatic method to couple the PEG-modified glycosyl acceptor with the monosaccharide raw material by glycosidic bond to form PEG-modified oligosaccharides;

[0007] Adding a first alcohol solvent to the material after the enzymatic reaction, mixing evenly, and performing a first solid-liquid separation to obtain purified PEG-modified oligosaccharides; re-dissolving the purified PEG-modified oligosaccharides with water, breaking the chemical bond between the oligosaccharides and PEG, and then adding a second alcohol solvent, mixing evenly and performing a second solid-liquid separation to obtain non-PEG-modified oligosaccharides;

[0008] The chemical structure of the PEG-modified glycosyl acceptor is shown in Formula I.

[0009]

[0010] R is a polyethylene glycol group having a molecular weight of at least 2,000.

[0011] Enzymatic preparation of oligosaccharides can rapidly synthesize oligosaccharides with a defined structure by controlling the type of enzyme. However, the enzymes used in the enzymatic method are all water-soluble, and the synthesized oligosaccharides are also water-soluble. The separation time is too long when using ion exchange chromatography, gel permeation chromatography or a method of combining the two. In order to reduce the separation time, the present invention first uses a chemical method to prepare a PEG-modified glycosyl receptor, and then uses the glycosyl receptor to prepare PEG-modified oligosaccharides by an enzymatic method. At this time, after the oligosaccharides are modified by PEG, their solubility in alcohol solvents decreases. By adding an alcohol solvent to the aqueous solution after the reaction, the solubility of the PEG-modified oligosaccharides is reduced, so that the PEG-modified oligosaccharides are precipitated from the solution system, and then by solid-liquid separation, the separation of the PEG-modified oligosaccharides and the enzymatic reaction system is achieved. In order to further obtain non-PEG-modified oligosaccharides, the PEG in the PEG-modified oligosaccharides is cut off, and then the principle of the reduced solubility of PEG in alcohol solvents is used to add an alcohol solvent to the reaction system after the cut-off to achieve the separation of oligosaccharides and polyethylene glycol.

[0012] On the other hand, an application of the above-mentioned method for rapid enzymatic separation of PEG-modified oligosaccharides in the preparation of oligosaccharides.

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

[0014] (1) The present invention combines the rapid and efficient characteristics of enzymatic synthesis with the advantages of PEG in oligosaccharide separation and purification, and can quickly prepare oligosaccharide chains of different sugar forms in large quantities. The glycosyltransferase, sugar nucleoside generating enzyme and sugar kinase used in the enzymatic method of the present invention are all from prokaryotes, have the advantages of high protein expression, wide substrate adaptability, high catalytic efficiency, etc., and are suitable for large-scale preparation of oligosaccharides; the use of sugar nucleotide generating enzyme can efficiently convert cheap and readily available monosaccharides into expensive nucleoside-activated sugar donors, greatly reducing production costs; compared with chemical synthesis methods with complicated steps and low yields, enzymatic synthesis has obvious advantages in terms of spatial and stereochemical specificity, greatly simplifies the reaction steps, and improves the overall yield of the reaction.

[0015] (2) The present invention avoids the problem of difficult separation and purification commonly encountered in enzymatic synthesis, which requires solid-liquid separation operations such as centrifugation of the enzyme reaction solution. The process is simple to operate and shortens the time for sugar chain synthesis and improves the efficiency of oligosaccharide synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 It is a schematic diagram of the enzymatic modular assembly 1 of the present invention;

[0018] Figure 2It is a schematic diagram of the enzymatic modular assembly 2 of the present invention;

[0019] Figure 3 It is a schematic diagram of the enzymatic modular assembly 3 of the present invention;

[0020] Figure 4 It is a schematic diagram of the enzymatic modular assembly 4 of the present invention;

[0021] Figure 5 It is a schematic diagram of the enzymatic modular assembly 5 of the present invention;

[0022] Figure 6 The synthetic route of the chemically synthesized β-configuration lactose compound 1 in Example 1 of the present invention is shown in FIG.

[0023] Figure 7 The synthetic route of the enzymatic modular synthesis of trisaccharide compound 2 in Example 1 of the present invention;

[0024] Figure 8 The synthetic route of the enzymatic modular synthesis of tetrasaccharide compound 3 in Example 2 of the present invention;

[0025] Fig. 9 The synthetic route of the modular enzymatic synthesis of pentasaccharide compound 4 in Example 3 of the present invention;

[0026] Fig.10 This is a synthetic route for the modular enzymatic synthesis of hexasaccharide compound 5 in Example 4 of the present invention;

[0027] Fig.11 The synthetic route of the modular enzymatic synthesis of pentasaccharide compound 6 in Example 5 of the present invention;

[0028] Fig.12 This is a synthetic route for the modular enzymatic synthesis of hexasaccharide compound 7 in Example 6 of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] In order to solve the problems of long separation time and high cost after the prior oligosaccharide synthesis, the present invention proposes a method for rapid enzymatic separation of PEG-modified oligosaccharides.

[0032] A typical embodiment of the present invention provides a method for rapid enzymatic separation of PEG-modified oligosaccharides, wherein lactose is used as a raw material to prepare a PEG-modified glycosyl acceptor by a chemical method, and the PEG-modified glycosyl acceptor is coupled to a monosaccharide raw material by a glycosidic bond by an enzymatic method to form a PEG-modified oligosaccharide;

[0033] Adding a first alcohol solvent to the material after the enzymatic reaction, mixing evenly, and performing a first solid-liquid separation to obtain purified PEG-modified oligosaccharides; re-dissolving the purified PEG-modified oligosaccharides with water, breaking the chemical bond between the oligosaccharides and PEG, and then adding a second alcohol solvent, mixing evenly and performing a second solid-liquid separation to obtain non-PEG-modified oligosaccharides;

[0034] The chemical structure of the PEG-modified glycosyl acceptor is shown in Formula I.

[0035]

[0036] R is a polyethylene glycol group having a molecular weight of at least 2,000.

[0037] The present invention studies the problem of separation and purification in oligosaccharide enzymatic synthesis. First, lactoside containing PEG is prepared, the sugar chain is extended by an enzymatic modular assembly strategy, and the product in the enzyme reaction solution is purified by centrifugation. This method can quickly and efficiently obtain oligosaccharide chains with different structures, and can provide a large amount of oligosaccharides with determined structures for subsequent functional studies. At the same time, the PEG collected by the present invention can be reused, which is conducive to further reducing costs.

[0038] The first alcohol solvent and the second alcohol solvent described in the present invention are both alcohol solvents, such as methanol, ethanol, propanol, etc. Among them, ethanol has lower toxicity, so using ethanol is more beneficial to environmental protection.

[0039] In some embodiments, the molecular weight of the polyethylene glycol group is 2,000 to 30,000, preferably 5,000 to 10,000.

[0040] In some embodiments, the chemical formula of the PEG-modified glycosyl acceptor is That is, R is Wherein, * is the connection position of the linking group.

[0041] In one or more embodiments, hydrazine is used to break the chemical bond between the oligosaccharide and the PEG.

[0042] In one or more embodiments, all hydroxyl groups of lactose are protected with acetyl groups to obtain fully acetylated lactose, the acetyl group at the anomeric position of the fully acetylated lactose is removed with ammonium acetate, and then a trichloroacetimidation reaction is carried out with trichloroacetonitrile to obtain a glycosyl donor of trichloroacetimidate ester, the glycosyl donor of trichloroacetimidate ester is subjected to a β-configuration glycosidation reaction with an acceptor 2-(2-chloroethoxy)ethanol, and then an azidation reaction and a reduction reaction are carried out to obtain an amino group, the sugar and PEG are connected by diethyl squarate, and finally the acetyl group is removed to obtain a PEG-modified glycosyl acceptor.

[0043] In some embodiments, the method for preparing PEG-modified oligosaccharides by enzymatic method comprises: using enzymatic modular assembly 1 (such as Figure 1 ) coupling N-acetylglucosamine to a PEG-modified glycosyl acceptor via a β1-3 glycosidic bond to synthesize a trisaccharide of formula (II);

[0044]

[0045] 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).

[0046] Specifically, a PEG-modified glycosyl acceptor, N-acetylglucosamine (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 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 to 70 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal in volume to the reaction solution is added and placed at 2 to 6°C for 5 to 15 minutes, and finally the trisaccharide represented by formula (II) is obtained by centrifugation.

[0047] In one or more embodiments, the method further comprises: utilizing enzymatic modular assembly 2 (such as Figure 2 ) coupling galactose to the trisaccharide of formula (II) via a β1-4 glycosidic bond to synthesize the tetrasaccharide of formula (III);

[0048]

[0049] Specifically, the enzymes used in the enzymatic modular assembly 2 are galactokinase (GalK), sugar nucleoside generator (BLUSP) and β1-4 galactosyltransferase (NmLgtB).

[0050] Specifically, the trisaccharide shown in formula (II), 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 β1-4 galactosyltransferase (NmLgtB) are added, and the reaction time is 3 to 70 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal in volume to the reaction solution is added and placed at 2 to 6°C for 5 to 15 minutes, and finally the tetrasaccharide shown in formula (III) is obtained by centrifugation.

[0051] In one or more embodiments, the method further comprises: using enzymatic modular assembly 1 to couple N-acetylglucosamine to the tetrasaccharide of formula (III) via a β1-3 glycosidic bond to synthesize the pentasaccharide of formula (IV);

[0052]

[0053] Specifically, the tetrasaccharide shown in formula (III), N-acetylglucosamine (1.2 to 4.0 equivalents), adenine nucleoside triphosphate (ATP) (1.2 to 4.0 equivalents), uridine nucleoside 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 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 to 70 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal in volume to the reaction solution is added and placed at 2 to 6°C for 5 to 15 minutes, and finally the pentasaccharide shown in formula (IV) is obtained by centrifugation.

[0054] In one or more embodiments, the method further comprises: coupling galactose to the pentasaccharide of formula (IV) by a β1-4 glycosidic bond using enzymatic modular assembly 2 to form a hexasaccharide of formula (V);

[0055]

[0056] Specifically, the pentasaccharide represented by formula (IV), 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 β1-4 galactosyltransferase (NmLgtB) are added, and the reaction time is 3 to 70 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal to the volume of the reaction solution is added and placed at 2 to 6°C for 5 to 15 minutes, and finally the hexasaccharide represented by formula (V) is obtained by centrifugation.

[0057] In one or more embodiments, the method further comprises: utilizing enzymatic modular assembly 5 (such as Figure 5 ) coupling galactose to the trisaccharide of formula (II) via a β1-3 glycosidic bond to synthesize the tetrasaccharide of formula (III');

[0058]

[0059] Specifically, the enzymes used in the enzymatic modular assembly 5 are galactokinase (GalK), sugar nucleoside generating enzyme (BLUSP), and β1-3 galactosyltransferase (Cvβ3GalT).

[0060] Specifically, the trisaccharide represented by formula (II), 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 β1-3 galactosyltransferase (Cvβ3GalT) are added, and the reaction time is 3 to 70 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal in volume to the reaction solution is added and placed at 2 to 6°C for 5 to 15 minutes, and finally centrifuged to obtain the tetrasaccharide represented by formula (III').

[0061] In one or more embodiments, the method further comprises: using enzymatic modular assembly 3 (such as Figure 3 ) coupling fucose to the tetrasaccharide of formula (III') via an α1-4 glycosidic bond to synthesize the pentasaccharide of formula (VI);

[0062]

[0063] Specifically, the enzymes used in the enzymatic modular assembly 3 are glyconucleoside generator (FKP) and α1-4 fucosyltransferase (Hpα1,3 / 4FucT).

[0064] Specifically, the tetrasaccharide shown in (III'), fucose (1.2 to 4.0 equivalents), adenine 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 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 sugar nucleoside generating enzyme (FKP) and α1-4 fucosyltransferase (Hpα1,3 / 4FucT) are added. The reaction time is 3 to 70 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal to the volume of the reaction solution is added and placed at 2 to 6°C for 5 to 15 minutes. Finally, the pentasaccharide shown in formula (VI) can be obtained by centrifugation.

[0065] In one or more embodiments, the method further comprises: utilizing enzymatic modular assembly 4 (such as Figure 4 ) coupling sialic acid to the pentasaccharide of formula (VI) via an α2-3 glycosidic bond to synthesize the hexasaccharide of formula (VII);

[0066]

[0067] Specifically, the enzymes used in the enzymatic modular assembly 4 are nucleoside synthase (NmCSS) and α2-3 sialyltransferase (PmST1M144D).

[0068] Specifically, the pentasaccharide shown in formula (VI), 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) 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 (NmCSS) and α2-3 sialyltransferase (PmST1M144D) are added. The reaction time is 0.5 to 30 hours. After the reaction is completed, the enzyme is removed in a boiling water bath, and anhydrous ethanol equal to the volume of the reaction solution is added and placed at 2 to 6° C. for 5 to 15 minutes. Finally, the hexasaccharide shown in formula (VII) can be obtained by centrifugation.

[0069] Unless otherwise specified, the "equivalent" in the present invention refers to the ratio of the amounts of substances when the substances interact with each other, such as: N-acetylglucosamine (1.2 to 4.0 equivalents) means that the ratio of the amounts of N-acetylglucosamine to lactoside represented by formula (I) is 1.2 to 4.0.

[0070] The reaction temperature in the enzyme modular assembly 1-5 of the present invention is 0-37°C and the rotation speed is 0-240r / min. The method of stopping and separating the enzyme reaction at the same time is to add 2-6°C anhydrous ethanol of the same volume as the reaction solution to the reaction system and incubate at 2-6°C for 1-15 minutes.

[0071] 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. In the preliminary experiments, 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 pylori β1,3-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 multifunctional 2,3-sialyltransferase 1M144D (PmST1 M144D); Bacteroides fragilis bifunctional L-fucokinase / GDP-fucose pyrophosphorylase (FKP) and Helicobacter pylori α1,3 / 4-fucosyltransferase (Hpα1,3 / 4FucT); Chromobacterium violaceum (Cvβ3GalT) are used as the enzymes used in the present invention, and have the best catalytic effect, high synthesis efficiency, and simple purification. In addition, the above enzymes can be expressed and purified in large quantities in a conventional Escherichia coli expression system.

[0072] Another embodiment of the present invention provides an application of the above-mentioned method for rapid enzymatic separation of PEG-modified oligosaccharides in the preparation of oligosaccharides.

[0073] 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.

[0074] Example 1

[0075] Chemical synthesis of β-lactoside 1 [Galβ(l-4)GlcβOR]:

[0076] Lactose 8 (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 under stirring 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 9 (18.70 g, 94%).

[0077] Compound 9 (15.0 g, 22.12 mmol), ammonium acetate (6.8 g, 88.21 mmol) and methanol / tetrahydrofuran (v / v, 1 / 1) were added to a 250 mL round-bottom flask, and the mixture was reacted at room temperature for 8 hours, concentrated, and purified by rapid silica gel chromatography (petroleum ether / ethyl acetate 1:2, v / v) to obtain a white syrupy compound. The compound was dissolved in anhydrous dichloromethane, trichloroacetonitrile and DBU were added to the reaction solution, reacted at room temperature for 1 hour, concentrated, and purified by rapid silica gel chromatography (petroleum ether / ethyl acetate 1:2, v / v) to obtain a light yellow syrupy compound. The compound (6.0 g, 8.70 mmol), receptor (2-chloroethoxyethanol, 1.4 g, 0.31 mmol), activated The molecular sieve was dissolved in anhydrous dichloromethane, stirred at room temperature for 20 minutes, then stirred in a low-temperature stirrer at -30°C for 10 minutes, trimethylsilyl trifluoromethanesulfonate TMSOTf (38 μL) was added, and the reaction was continued at -30°C for 1 hour. Triethylamine was added to terminate the reaction, filtered, concentrated, and separated and purified by silica gel column to obtain white syrupy compound 10 (6.2 g, 89%).

[0078] Compound 10 (4.80 g, 6.74 mmol), N,N-dimethylformamide (60 mL), sodium azide (4.2 g) and tetrabutylammonium iodide (0.12 g) were added to a 250 mL round-bottom flask and stirred at 110° C. for 12 hours. After the reaction was complete as detected by thin layer chromatography (PE:EA=1:1), the mixture was filtered using diatomaceous earth, concentrated by rotary evaporation, and purified by rapid silica gel column separation to obtain a light yellow solid compound 11 (4.2 g, 90%).

[0079] Compound 11 (3.40 g, 3.1 mmol), methanol (40 mL), sodium borohydride (0.5 g) and copper sulfate (0.1 g) were added to a 250 mL round-bottom flask and stirred at room temperature for 6 hours. After the reaction was complete as detected by thin layer chromatography (PE:EA=1:1), the mixture was filtered using diatomaceous earth, concentrated by rotary evaporation, and purified by rapid silica gel column separation to obtain a light yellow solid compound 12 (2.9 g, 90%).

[0080] Compound 12 (2.8 g), methanol (20 mL) and sodium methoxide were added to a 50 mL round-bottom flask until the pH value of the solution reached 9. The mixture was stirred at room temperature for 2 hours. After the reaction was completed by thin layer chromatography (EtOAc:MeOH:H2O:HOAc=4:2:1:0.2), the mixture was filtered and concentrated by rotary evaporation to obtain compound 13 (1.90 g, 98%) as a white solid.

[0081] Compound 13 (1.6 g) and diethyl squarate (0.6 g) were added to a 50 mL round-bottom flask. The pH value of the solution was adjusted to 8 and the mixture was stirred at room temperature for 2 hours. The mixture was filtered and concentrated by rotary evaporation to obtain compound 14 (2.1 g, 98%) as a white solid.

[0082] Compound 14 (1.0 g) and PEG (weight average molecular weight 5000, 8.0 g) were added to a 100 mL round-bottom flask until the pH value of the solution reached about 9-10. The mixture was stirred at room temperature for 2 hours, ethanol was added, and centrifuged to obtain a white solid lactose receptor compound 1 (8.90 g, 98%).

[0083] The synthetic route of lactose receptor compound 1 is as follows Figure 6 shown.

[0084] Enzymatic module assembly 1 to synthesize trisaccharide compound 2 [GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0085] Lactose receptor compound 1 (200 mg), N-acetylglucosamine (10.3 mg), ATP (25.4 mg), UTP (22.0 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 (1.0 mg) and HpLgtA (1.0 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. Boil water for five minutes to remove the enzyme. Anhydrous ethanol was added in an equal volume to the reaction system, and the reaction system was placed in a refrigerator at 4°C for 10 minutes. The precipitate was collected by centrifugation to obtain a white trisaccharide compound 2 (201.2 mg, 96%). Finally, PEG was removed with 5% hydrazine, and PEG was precipitated by adding ethanol. The oligosaccharides in the supernatant were obtained by vacuum evaporation. The parameters are as follows: 1 H NMR(600MHz,D2O)δ4.70(d,J=8.4Hz,1H),4.50(d,J=7.8Hz,1H),4.45(d,J=7.8Hz,1H),4.16(d ,J=3.0Hz,1H),4.03–3.46(m,20H),3.32(t,J=8.4Hz,1H),2.05(s,3H),1.93(p,J=6.6Hz,2H); 13 C NMR(151MHz,D2O)δ174.95,102.92,102.84,102.10,81.96,78.34,75.64,74.88,74.76,74.35 ,73.55,72.79,70.00,69.68,68.33,67.36,60.96,60.47,60.06,55.65,47.87,28.23,22.19.

[0086] The synthetic route of trisaccharide compound 2 is as follows Figure 7 shown.

[0087] Example 2

[0088] Enzymatic module assembly 2 to synthesize tetrasaccharide compound 3 [Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0089] The trisaccharide compound 2 (150 mg), galactose (8.7 mg), ATP (21.1 mg), UTP (20.2 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) prepared in Example 1 were dissolved in a 50 mL centrifuge tube, and GalK (1.0 mg), BLUSP (1.0 mg) and NmLgtB (1.0 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. Boil in boiling water for five minutes to remove the enzyme. Anhydrous ethanol was added in an equal volume to the reaction system, and the reaction system was placed in a refrigerator at 4°C for 10 minutes and centrifuged to collect the precipitate to obtain a white tetrasaccharide compound 3 (151.8 mg, 95%). Finally, PEG was removed with 5% hydrazine, and PEG was precipitated by adding ethanol. The oligosaccharides in the supernatant were obtained by vacuum evaporation. The parameters are as follows: 1 HNMR(600MHz,D2O)δ4.73(d,J=8.4Hz,1H),4.50(d,J=7.8Hz,1H),4.49(d,J= 7.8Hz,1H),4.46(d,J=8.4Hz,1H),4.17(d,J=3.6Hz,1H),4.03–3.96(m,3H), 3.94(d,J=3.0Hz,1H),3.87(dd,J=4.8,12.6Hz,1H),3.84–3.54(m,19H),3.4 8(t,J=6.6Hz,2H),3.33(t,J=8.4Hz,1H),2.05(s,3H),1.93(p,J=6.6Hz,2H); 13 CNMR(151MHz,D2O)δ174.89,102.92,102.84,102.73,102.09,82.05,78.35,78.14,75.33,74.87,74.75,74.53,74.3 4,72.79,72.49,72.16,70.96,69.95,68.55,68.31,67.36,61.04,60.96,60.06,59.85,55.18,47.87,28.24,22.22.

[0090] The synthetic route of tetrasaccharide compound 3 is as follows Figure 8 shown.

[0091] Example 3

[0092] Enzymatic modular synthesis of pentasaccharide 4

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

[0094] The tetrasaccharide compound 3 (100 mg), N-acetylglucosamine (7.1 mg), ATP (15.1 mg), UTP (14.8 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) prepared in Example 2 were dissolved in a 50 mL centrifuge tube, and NahK / GlmU (1.0 mg) and HpLgtA (1.0 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 16 hours. Boil the water for five minutes to remove the enzyme. Anhydrous ethanol was added in an equal volume to the reaction system, and the reaction system was placed in a refrigerator at 4°C for 10 minutes and centrifuged to collect the precipitate to obtain a white pentasaccharide compound 4 (104.5 mg, 96%). Finally, PEG was removed with 5% hydrazine, and PEG was precipitated by adding ethanol. The oligosaccharides in the supernatant were obtained by vacuum evaporation. The parameters are as follows: 1 HNMR(600MHz,D2O)δ4.67(dd,J=11.6,8.4Hz,2H),4.46(dd,J=12.0,7.9Hz,2H),4.42(d,J=7.9Hz,1H),4.13(d,J=3.0Hz,2H),4.00 –3.87(m,4H),3.82(dd,J=4.2,12Hz,1H),3.80–3.40(m,26H),3.29(m,J=8.4Hz,1H),2.02(d,J=3.0Hz,6H),1.90(p,J=6.6Hz,2H); 13 C NMR(151MHz,D2O)δ174.87,174.82,102.89,102.84,102.81,102.69,102.07,81.98,81.94,78.31,78.13,75.61,74.84,74.82,74.72, 74.50,74.31,73.51,72.75,72.12,69.95,69.91,69.64,68.28,67.31,60.92,60.45,60.02,59.82,55.62,55.10,47.84,28.21,22.17.

[0095] The synthetic route of pentasaccharide compound 4 is as follows Fig. 9 shown.

[0096] Example 4

[0097] Enzymatic modular assembly 2 to synthesize hexasaccharide compound 5

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

[0099] The pentasaccharide compound 4 (50 mg), galactose (2.2 mg), ATP (5.9 mg), UTP (4.0 mg), Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (20 mM) prepared in Example 3 were dissolved in a 50 mL centrifuge tube, and GalK (1.0 mg), BLUSP (1.0 mg) and NmLgtB (1.0 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. Boil in boiling water for five minutes to remove the enzyme. Anhydrous ethanol was added in an equal volume to the reaction system, and the reaction system was placed in a refrigerator at 4°C for 10 minutes, and the precipitate was collected by centrifugation to obtain a white hexasaccharide compound 5 (50.9 mg, 95%). Finally, PEG was removed with 5% hydrazine, and PEG was precipitated by adding ethanol. The oligosaccharides in the supernatant were obtained by vacuum evaporation. The parameters are as follows: 1 HNMR(600MHz,D2O)δ4.65(d,J=7.8Hz,2H),4.45(d,J=7.2Hz,1H),4.43(d,J=6.6Hz,1H),4.42(d,J=7.8Hz,1H),4.39(d,J=7.8Hz, 1H),4.11(d,J=3.0Hz,2H),3.98–3.48(m,35H),3.42(t,J=6.6Hz,2H),3.27(t,J=8.4Hz,1H),1.99(s,6H),1.87(p,J=6.6Hz,2H); 13 C NMR (151MHz, D2O) δ174.77,102.81,102.75,102.72,102.65,102.63,101.98,81.94,81.90,78.17,77.97,75.22,74.74,74.64,74. 41,74.22,72.65,72.36,72.04,70.83,69.82,68.41,68.20,67.23,60.91,60.83,59.90,59.70,55.05,55.01,47.73,28.10,22.04.

[0100] The synthetic route of hexasaccharide compound 5 is as follows Fig.10 shown.

[0101] Example 5

[0102] Enzymatic assembly of modules 3 and 5 to synthesize pentasaccharide 6

[0103] [Galβ(l-3)(Fucα1-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0104] The trisaccharide compound 2 (200 mg) prepared in Example 1, galactose (10.2 mg), ATP (25.9 mg), UTP (24.0 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 Cvβ3GalT (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. Boil the water for five minutes to remove the enzyme. Add anhydrous ethanol of the same volume as the reaction system, place in a refrigerator at 4°C for 10 minutes, centrifuge and collect the precipitate to obtain a white tetrasaccharide compound; tetrasaccharide compound (200 mg), fucose (10.4 mg), ATP (35.1 mg), GTP (38.4 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) are dissolved in a 50 mL centrifuge tube, enzyme FKP (2.00 mg) and Hpα1,3 / 4FucT (2.00 mg) are added, and double distilled water is added to a total volume of 10 mL, and the reaction system is placed in a shaker and incubated at 37°C and 110 r / min for 4 hours. Boil in boiling water for five minutes to remove the enzyme. Add anhydrous ethanol of the same volume as the reaction system, place in a refrigerator at 4°C for 10 minutes, centrifuge and collect the precipitate to obtain white pentasaccharide compound 6 (206.9 mg, 96%), finally remove PEG with 5% hydrazine, add ethanol to precipitate PEG, and obtain the oligosaccharide in the supernatant by vacuum evaporation. The parameters are as follows: 1H NMR(600MHz,D2O)δ5.12(d,J=3.6Hz,1H),4.84(m,1H),4.70(d,J=8.4Hz,1H),4. 48(d,J=7.8Hz,1H),4.45(d,J=7.8Hz,1H),4.42(d,J=8.4Hz,1H),4.15(d,J=3.0 Hz,1H),4.05–3.56(m,26H),3.48(dd,J=7.9,9.6Hz,1H),3.46(t,J=6.6Hz,2H), 3.30(t,J=8.5Hz,1H),2.02(s,3H),1.91(p,J=6.5Hz,2H),1.17(d,J=6.6Hz,3H); 13 C NMR(151MHz,D2O)δ174.58,102.84,102.43,102.01,101.67,98.51,81.97,78.24,75.01,74.80,74.76,74.66,74.26,72.96,72.69,7 2.38,71.81,70.95,69.85,69.10,68.25,68.20,67.61,67.26,66.59,61.40,60.87,59.97,59.54,55.86,47.78,28.15,22.18,15.25.

[0105] The synthetic route of pentasaccharide compound 6 is as follows Fig.11 shown.

[0106] Example 6

[0107] Enzymatic modular assembly 4 to synthesize hexasaccharide compound 7

[0108] [Neu5Acα(2-3)Galβ(l-3)(Fucα1-4)GlcNAcβ(l-3)Galβ(l-4)GlcβOR]:

[0109] The pentasaccharide compound 6 (100 mg), Neu5Ac (9.2 mg), CTP (14.3 mg), Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 (20 mM) prepared in Example 5 (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 NmCSS (2.0 mg) and PmST1M144D (2.0 mg) were added. After adding double distilled water 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 1 hour. Boil in boiling water for five minutes to remove the enzyme. Anhydrous ethanol was added in an equal volume to the reaction system, and the reaction system was placed in a refrigerator at 4°C for 10 minutes. The precipitate was collected by centrifugation to obtain a white hexasaccharide compound 7 (104.4 mg, 96%). Finally, PEG was removed with 5% hydrazine, and PEG was precipitated by adding ethanol. The oligosaccharides in the supernatant were obtained by vacuum evaporation. The parameters are as follows: 1 H NMR(600MHz,D2O)δ5.08(d,J=4.2Hz,1H),4.67(d,J=7.8Hz,1H),4.49(d,J=7.8Hz,1H),4.45(d,J=8.4Hz, 1H),4.40(d,J=7.8Hz,1H),4.13(d,J=3.0Hz,1H),4.05(dd,J=2.6,9.8Hz,1H),4.04(d,J=2.4Hz,1H),4.0 0–3.52(m,33H),3.49(t,J=8.8Hz,1H),3.43(t,J=6.6Hz,2H),3.30(t,J=8.4Hz,1H),2.73(dd,J=4.2,12. 6Hz,1H),2.00(s,3H),1.99(s,3H),1.88(p,J=6.6Hz,2H),1.76(t,J=12.0Hz,1H),1.13(d,J=6.6Hz,3H); 13 C NMR(151MHz,D2O)δ174.88,174.55,173.74,102.81,102.47,101.97,101.42,99.51,98. 46,81.97,78.19,75.52,74.89,74.79,74.76,74.64,74.51,74.23,72.88,72.78,72.65, 71.77,71.73,69.82,69.13,69.04,68.17,67.96,67.57,67.23,67.18,66.53,62.46,61.37,60.85,59.92,59.37,59.18,55.83,51.56,47.73,39.65,28.11,22.12,21.90,15.16.

[0110] The synthetic route of hexasaccharide compound 7 is as follows Fig.12 shown.

[0111] 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 rapid enzymatic separation of PEG-modified oligosaccharides, characterized in that: Lactose is used as a raw material to prepare a PEG-modified glycosyl acceptor by a chemical method, and the PEG-modified glycosyl acceptor is coupled with a monosaccharide raw material by a glycosidic bond by an enzymatic method to form a PEG-modified oligosaccharide; Adding a first alcohol solvent to the material after the enzymatic reaction, mixing evenly, and performing a first solid-liquid separation to obtain purified PEG-modified oligosaccharides; re-dissolving the purified PEG-modified oligosaccharides with water, breaking the chemical bond between the oligosaccharides and PEG, and then adding a second alcohol solvent, mixing evenly and performing a second solid-liquid separation to obtain non-PEG-modified oligosaccharides; The first alcohol solvent is anhydrous ethanol; the second alcohol solvent is ethanol; The chemical structure of the PEG-modified glycosyl acceptor is .

2. The method for rapid separation of oligosaccharides modified by PEG as claimed in claim 1, characterized in that: Hydrazine was used to break the chemical bond between the oligosaccharide and PEG.

3. The method for rapid separation of oligosaccharides modified by PEG as claimed in claim 1, characterized in that: All hydroxyl groups of lactose are protected with acetyl groups to obtain fully acetylated lactose, the acetyl group at the anomeric position of the fully acetylated lactose is removed with ammonium acetate, and then a trichloroacetimidation reaction is carried out with trichloroacetonitrile to obtain a glycosyl donor of trichloroacetimidate ester, the glycosyl donor of trichloroacetimidate ester is subjected to a β-configuration glycosidation reaction with the acceptor 2-(2-chloroethoxy)ethanol, and then an azidation reaction and a reduction reaction are carried out to obtain an amino group, and the sugar and PEG are connected by diethyl squarate, and finally the acetyl group is removed to obtain a PEG-modified glycosyl acceptor.

4. The method for rapid separation of oligosaccharides modified by PEG according to claim 1, characterized in that: A method for preparing PEG-modified oligosaccharides by enzymatic method, comprising: using enzymatic modular assembly 1 N -acetylglucosamine is coupled to the PEG-modified glycosyl acceptor via a β1-3 glycosidic bond to synthesize the trisaccharide shown in formula (II); ； The enzyme used in the enzymatic modular assembly 1 is N -Acetylhexosamine kinase and sugar nucleoside synthase fusion enzyme and β1-3 N -acetylglucosyltransferase.

5. The oligosaccharide enzymatic rapid separation method modified by PEG as claimed in claim 4 is characterized in that, include: Using enzymatic modular assembly 2, galactose is coupled to the trisaccharide of formula (II) via a β1-4 glycosidic bond to synthesize the tetrasaccharide of formula (III); ； The enzymes used in the enzymatic modular assembly 2 are galactokinase, sugar nucleoside generator and β1-4 galactosyltransferase.

6. The oligosaccharide enzymatic rapid separation method modified by PEG as claimed in claim 5, characterized in that, include: Enzymatic modular assembly N -acetylglucosamine is coupled to the tetrasaccharide of formula (III) via a β1-3 glycosidic bond to synthesize the pentasaccharide of formula (IV); 。 7. The method for rapid separation of oligosaccharides modified by PEG as claimed in claim 6, characterized in that include: Using enzymatic modular assembly 2, galactose is coupled to the pentasaccharide of formula (IV) via a β1-4 glycosidic bond to form a hexasaccharide of formula (V); 。 8. The oligosaccharide enzymatic rapid separation method modified by PEG as claimed in claim 4 is characterized in that, include: Using enzymatic modular assembly 5, galactose is coupled to the trisaccharide of formula (II) via a β1-3 glycosidic bond to synthesize the tetrasaccharide of formula (III'); ； The enzymes used in the enzymatic modular assembly 5 are galactokinase, sugar nucleoside generating enzyme, and β1-3 galactosyltransferase.

9. The oligosaccharide enzymatic rapid separation method modified by PEG as claimed in claim 8, characterized in that, include: The pentasaccharide of formula (VI) is synthesized by coupling fucose to the tetrasaccharide of formula (III') via an α1-4 glycosidic bond using enzymatic modular assembly 3; ； The enzymes used in the enzymatic modular assembly 3 are sugar nucleoside generating enzyme and α1-4 fucosyltransferase.

10. The method for rapid separation of oligosaccharides modified by PEG as claimed in claim 9, characterized in that include: Using enzymatic modular assembly 4, sialic acid is coupled to the pentasaccharide of formula (VI) via an α2-3 glycosidic bond to synthesize the hexasaccharide of formula (VII); ； The enzymes used in the enzymatic modular assembly 4 are sugar nucleoside generating enzyme and α2-3 sialyltransferase.

11. Use of the method for rapid enzymatic separation of PEG-modified oligosaccharides according to any one of claims 1 to 10 in the preparation of oligosaccharides.

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