Method for total synthesis of N-glycan

Abstract

The invention provides a method for total synthesis of N-glycan. Specifically, N-sugar core pentasaccharide as shown in a formula (I) and core heptasaccharide as shown in a formula (II) are prepared from monosaccharide through a chemical enzymatic synthesis strategy, and various symmetric or asymmetric N-glycans as shown in a formula (III) are obtained by further utilizing chemical enzymatic reaction synthesis and taking reversible galactosylation as a selective protection strategy. The method disclosed by the invention can be used for efficiently preparing the N-glycan on a large scale without complicated purification operation, so that the method has the characteristics of low cost and strong adaptability. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of N-glycan synthesis, and particularly relates to a method for the total synthesis of N-glycans. Background Art

[0002] Protein N-glycosylation refers to the attachment of N-acetylglucosamine (GlcNAc) to the nitrogen atom of the asparagine (Asn) side chain via a βN-linkage, which is one of the most common post-translational modifications of proteins. N-glycosylation exists in most organisms, and the N-glycan structure is highly conserved during evolution. All naturally occurring N-glycans contain a conserved structure called the core pentasaccharide, on which different numbers of monosaccharides can be added or removed, thus generating an extremely diverse range of N-glycan structures in living cells.

[0003] The attachment of N-glycans to proteins affects many properties of glycoproteins, thereby regulating many physiological processes, such as protein folding, cell signaling, intracellular trafficking, subcellular localization, and ligand-receptor interactions. It is well known that abnormal N-glycosylation, including abnormal site occupancy and glycan structure, is strongly associated with cancer and many other diseases. Therefore, structurally defined N-glycans have great application potential in basic research and medicinal chemistry. For example, core fucosylated N-glycans are biomarkers approved by the FDA for the early detection of hepatocellular carcinoma (HCC).

[0004] Extraction from natural sources has always been the main method for obtaining structurally defined N-glycans. However, N-glycans exist in nature as a difficult-to-separate mixture at low abundance. In recent years, chemoenzymatic methods have become a popular strategy for obtaining structurally defined N-glycans. This strategy relies on the chemical synthesis of short N-glycan precursors and subsequent enzymatic extension to obtain complex N-glycans. Due to the complexity of the structure, dozens to over a hundred steps are required to prepare advanced intermediates, such as core pentasaccharides and heptasaccharides, to enable enzymatic diversification. Such high-demand syntheses can only be carried out in highly specialized laboratories. Alternatively, enzymatic degradation of sialylglycopeptides (SGP) extracted from egg yolk can also produce N-glycan core precursors for enzymatic extension. However, SGP exists as a mixture in egg yolk at low abundance (only about 0.3% (w / w)), the SGP extraction process requires a large amount of organic solvents, and the operation is quite time-consuming and labor-intensive. Therefore, it has been an obstacle in glycochemistry to obtain sufficient amounts of building blocks or N-glycan core precursors to construct many N-glycan structures in large quantities.

[0005] In living cells, de novo biosynthesis of N-glycans starts from dolichol phosphate on the cytoplasmic side of the endoplasmic reticulum membrane, and the asparagine-linked glycosyltransferase (ALG) family is responsible for the first few steps. ALG7 and ALG13 / 14 catalyze the addition of two GlcNAc residues to dolichol phosphate, resulting in GlcNAc β1,4GlcNAc-PP-dolichol (Gn 2 -PP-Dol). Subsequently, ALG1 and ALG2 add three mannose residues, using GDP-D-Man as the donor to generate Man 3 Gn 2 -PP-Dol. ALG2 is a bifunctional enzyme responsible for adding α-1,3- and α-1,6-linked mannoses to the product of ALG1 (Man 1 Gn 2 -PP-Dol) to form Man 3 Gn 2 -PP-Dol. Then, eight ALG enzymes catalyze the formation of Glc 3 Man 9 Gn 2 -PP-Dol, which is a common precursor before the protein glycosylation step. After assembly, Glc 3 Man 9 Gn 2 -oligosaccharide is transferred to the Asn residue of the protein by β-N-linkage of oligosaccharyltransferase (OST). Further removal or addition of several sugar residues on this structure forms extremely complex N-glycan structures in living cells.

[0006] Although the biosynthetic pathway of N-glycans has been elucidated for many years, it is generally considered impractical to enzymatically prepare N-glycans by fully following the natural biosynthetic pathway. There are three main challenges: First, the enzymes involved in N-glycan biosynthesis are difficult to prepare; second, due to their complex structures, natural dolichols are difficult to synthesize, and the main dolichol in humans is Dol-19 (C95 lipid), which is highly hydrophobic and not suitable for enzymatic synthesis; third, glycosylation donors (sugar nucleotides and dolichol phosphate mannose) are difficult to prepare on a large scale. Summary of the Invention

[0007] Based on the problems existing in the above-mentioned prior art, the inventors have developed a method for the chemoenzymatic total synthesis of N-glycans. This method is efficiently assembled from common starting materials and requires only a few simple chemical reactions and enzymatic reaction steps to synthesize N-glycan core pentasaccharide and core heptasaccharide (multi-gram scale); Next, the core pentasaccharide and core heptasaccharide are extended by glycosyltransferases to produce a variety of symmetric and asymmetric N-glycans. In particular, the present invention provides a reversible enzymatic galactosylation as a protection strategy for the synthesis of asymmetric N-glycans, and successfully prepares 49 complex asymmetric N-glycans. The method described in the present invention can prepare N-glycans on a large scale and efficiently, and does not require cumbersome purification operations, and has the characteristics of low cost and strong adaptability.

[0008] In view of this, in the first aspect, the present invention provides a method for synthesizing the core pentasaccharide represented by formula (I) or the core heptasaccharide represented by formula (II),

[0009]

[0010] In formula (I) or formula (II),

[0011] represents D-mannopyranose,

[0012] represents N-acetylglucosamine,

[0013] β4 means that a glycosidic hydroxyl group of one sugar is connected to a hydroxyl group on the 4-position carbon of another sugar through a β-configured glycosidic bond,

[0014] β2 means that a glycosidic hydroxyl group of one sugar is connected to a hydroxyl group on the 2-position carbon of another sugar through a β-configured glycosidic bond,

[0015] α3 means that a glycosidic hydroxyl group of one sugar is connected to a hydroxyl group on the 3-position carbon of another sugar through an α-configured glycosidic bond,

[0016] α6 means that a glycosidic hydroxyl group of one sugar is connected to a hydroxyl group on the 6-position carbon of another sugar through an α-configured glycosidic bond;

[0017] The synthesis method includes the following steps:

[0018] S5: Compound 5 reacts with compound c to obtain compound 6, and its reaction formula is as follows:

[0019]

[0020] In the formula, R is selected from C6-C95 straight-chain or branched-chain alkyl groups, preferably C6-C50 straight-chain or branched-chain alkyl groups, more preferably C6-C20 straight-chain or branched-chain alkyl groups, and further preferably, R is selected from: Further preferably, R is

[0021] S6: Compound 6 is deacetylated to obtain compound 7, and the reaction formula is as follows:

[0022]

[0023] In the formula, R is defined as in step S5;

[0024] S7: Compound 7 and guanosine diphosphate mannose (GDP-Man) are converted into compound 8 under the action of chitobiosyl diphosphate polyphenol β-mannosyltransferase (ALG1) or a protein derived from ALG1 with one or several amino acids substituted, deleted or added in the amino acid sequence of ALG1 and having ALG1 activity, and the reaction formula is as follows:

[0025]

[0026] In the formula, R is defined as in step S5;

[0027] S8: Under the action of α-1,3 / 1,6-mannosyltransferase (ALG2) or a protein derived from ALG2 with one or several amino acids substituted, deleted or added in the amino acid sequence of ALG2 and having ALG2 activity, compound 8 and guanosine diphosphate mannose (GDP-Man) are converted into compound 9, and the reaction formula is as follows:

[0028]

[0029] In the formula, R is defined as in step S5;

[0030] S9: Compound 9 is de-lipidated at the tail to obtain the core pentasaccharide shown in formula (I), and the reaction formula is as follows:

[0031]

[0032] S10: Under the action of α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) or a protein derived from MGAT1 with one or several amino acids substituted, deleted or added in the amino acid sequence of MGAT1 and having MGAT1 activity, the core pentasaccharide shown in formula (I) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) are converted into compound 28, and the reaction formula is as follows:

[0033]

[0034] S11: Under the action of α-1,6-mannosylglycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT2) or a protein derived from MGAT2 with substitution, deletion or addition of one or more amino acids in the amino acid sequence of MGAT2 and having MGAT2 activity, compound 28 and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) are converted into the core heptasaccharide shown in formula (II), and the reaction formula is as follows:

[0035]

[0036] In a specific embodiment, compound 5 can be prepared by the following method:

[0037] S1: Compound 1 reacts with acetic anhydride to form compound 2, and the reaction formula is as follows:

[0038]

[0039] S2: Compound 2 selectively removes the acetyl group at the 1-position to obtain compound 3, and the reaction formula is as follows:

[0040]

[0041] S3: Compound 3 first reacts with 4,5-dicyanoimidazole and dibenzyl N,N'-diisopropylphosphoramidite, and then reacts with tert-butyl hydroperoxide to obtain compound 4, and the reaction formula is as follows:

[0042]

[0043] S4: Compound 4 is reduced to obtain compound 5, and the reaction formula is as follows:

[0044]

[0045] Step S1

[0046] In some embodiments, the reaction is carried out in solvent 1. For example, the solvent 1 can be pyridine;

[0047] In some embodiments, the molar ratio of compound 1 to acetic anhydride can be 14.5-15.5:1;

[0048] In some embodiments, the reaction is carried out at 20-30 °C under stirring conditions; preferably, the reaction end point can be monitored by thin layer chromatography;

[0049] In some embodiments, after the reaction is completed, it further includes a separation and purification step: removing the solvent in the reaction solution, adding dichloromethane for dissolution, washing, collecting the dichloromethane phase, drying and concentrating to obtain Compound 2; preferably, the washing can be sequentially carried out with 1M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution; preferably, the drying is carried out using anhydrous sodium sulfate.

[0050] Step S2

[0051] In some embodiments, the reaction is carried out in Solvent 2. For example, the Solvent 2 can be tetrahydrofuran;

[0052] In some embodiments, the reaction is carried out under the action of methanol ammonia. For example, in methanol ammonia, the concentration of ammonia is 6 - 8 mol / L. For example, the volume ratio of methanol ammonia to Solvent 2 is 1:2 - 4;

[0053] In some embodiments, the reaction is carried out at 20 - 30 °C under stirring conditions; preferably, the end point of the reaction can be monitored by thin layer chromatography.

[0054] In some embodiments, after the reaction is completed, it further includes a separation and purification step: diluting the reaction solution with dichloromethane, washing and collecting the organic phase, and obtaining Compound 2 after drying, concentrating and column chromatography purification; preferably, the washing can be sequentially carried out with 1M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution; preferably, the drying is carried out using anhydrous sodium sulfate; preferably, the eluent for column chromatography is dichloromethane / methanol = 95:5, v / v.

[0055] Step S3

[0056] In some embodiments, the reaction is carried out in Solvent 3. For example, the Solvent 3 can be acetonitrile;

[0057] In some embodiments, the molar ratio of Compound 3 to 4,5-dicyanoimidazole is 1:2.5 - 3.5; the molar ratio of Compound 3 to dibenzyl N,N'-diisopropylphosphoramidite is 1:1.5 - 2.5; the molar ratio of Compound 3 to tert-butyl hydroperoxide is 1:3.5 - 4.5;

[0058] In some embodiments, the reactions of Compound 3 with 4,5-dicyanoimidazole, dibenzyl N,N'-diisopropylphosphoramidite, and with tert-butyl hydroperoxide are all carried out at 20 - 30 °C under stirring conditions; preferably, tert-butyl hydroperoxide is added to the reaction system in the form of a decane solution; preferably, the end point of the reaction is monitored by thin layer chromatography.

[0059] In some embodiments, after the reaction is completed, a separation and purification step is further included: the reaction solution is filtered through diatomaceous earth and then concentrated, diluted with dichloromethane, the organic phase is collected after washing, and compound 4 is obtained after drying, concentration, and purification by column chromatography; preferably, the washing can be sequentially carried out with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution; preferably, the drying is carried out using anhydrous sodium sulfate; preferably, the eluent for column chromatography is dichloromethane / methanol = 95:5, v / v.

[0060] Step S4

[0061] In some embodiments, the reaction is carried out in solvent 4. For example, the solvent 4 can be methanol;

[0062] In some embodiments, the reaction is reduced with hydrogen in the presence of palladium on carbon. For example, the mass ratio of compound 4 to palladium on carbon is 1:0.05 - 0.15;

[0063] In some embodiments, the reaction is carried out at 20 - 30 °C under stirring conditions; preferably, the end point of the reaction is monitored by thin layer chromatography;

[0064] In some embodiments, after the reaction is completed, a separation and purification step is further included: the reaction solution is filtered through diatomaceous earth and then concentrated to obtain compound 5.

[0065] Step S5

[0066] In some embodiments, compound 5 is first dissolved in solvent 5. For example, the solvent 5 is methanol, and then triethylamine is added to convert it into a triethylammonium salt; after the triethylammonium salt is activated with N,N - carbonyldiimidazole for the phosphate group in solvent 6, it is then reacted with compound c. For example, the solvent 6 can be N,N - dimethylformamide.

[0067] In some embodiments, the dosage ratio of compound 5 to triethylamine is 1.0 g:0.5 - 1.5 mL; the molar ratio of compound 5 to N,N - carbonyldiimidazole is 1:5 - 6; the dosage ratio of compound 5 to compound c is 1:1.0 - 1.5;

[0068] In some embodiments, it is converted into a triethylammonium salt by concentration, and then co - evaporated with toluene to remove the excess triethylamine. Preferably, it is co - evaporated with toluene 3 times;

[0069] In some embodiments, the reaction conditions for activating the phosphate group with N,N - carbonyldiimidazole are to activate at room temperature for 3 - 6 h; preferably, after the activation reaction is completed, methanol is added to quench the unreacted N,N - carbonyldiimidazole;

[0070] In some embodiments, compound c is dissolved in solvent 7 and added to the aforementioned reaction system in the form of a solution; preferably, solvent 7 is dichloromethane; in some embodiments, the conditions for reacting with compound c are 20-30 °C with stirring, and the reaction time is 2-4 days;

[0071] In some embodiments, after the reaction is completed, it further includes a separation and purification step: concentrating the reaction solution and purifying it by column chromatography to obtain compound 6; preferably, the eluent for column chromatography is ethyl acetate∶methanol∶water = 70∶30∶0.3.

[0072] In some embodiments, compound c can be prepared according to the following method:

[0073] (a) Compound a first reacts with 4,5-dicyanoimidazole and dibenzyl N,N′-diisopropylphosphoramidite, and then reacts with tert-butyl hydroperoxide to obtain phosphorylated compound b;

[0074] (b) Compound b is reduced by hydrogen to obtain compound c;

[0075] The reaction formula is as follows:

[0076]

[0077] wherein, R is defined as in step S5;

[0078] In step (a)

[0079] In some embodiments, compound a reacts in solvent 3. For example, solvent 3 is acetonitrile;

[0080] In some embodiments, the molar ratio of compound a to 4,5-dicyanoimidazole is 1∶2.5-3.5; the molar ratio of compound a to dibenzyl N,N′-diisopropylphosphoramidite is 1∶1.5-2.5; the molar ratio of compound a to tert-butyl hydroperoxide is 1∶3.5-4.5;

[0081] In some embodiments, the reactions of compound a with 4,5-dicyanoimidazole, dibenzyl N,N′-diisopropylphosphoramidite, and with tert-butyl hydroperoxide are all carried out at 20-30 °C under stirring conditions; preferably, tert-butyl hydroperoxide is added to the reaction system in the form of a decane solution; preferably, the reaction end point is monitored by thin layer chromatography;

[0082] In some embodiments, after the reaction is completed, a separation and purification step is further included: the reaction solution is filtered through diatomaceous earth and then concentrated, diluted with dichloromethane, the organic phase is collected after washing, and compound b is obtained after drying, concentration, and purification by column chromatography; preferably, the washing can be sequentially carried out with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution; preferably, the drying is carried out using anhydrous sodium sulfate; preferably, the eluent for column chromatography is dichloromethane / methanol = 85:5, v / v;

[0083] In step (b)

[0084] In some embodiments, compound b is reduced in methanol. In some embodiments, compound b is reduced by hydrogen in the presence of a hydrogenation catalyst. For example, the hydrogenation catalyst is palladium on carbon;

[0085] In some embodiments, after the reaction is completed, a separation and purification step is further included: the reaction solution is filtered through diatomaceous earth and then concentrated to obtain compound c.

[0086] Step S6

[0087] In some embodiments, the reaction is carried out in solvent 8. For example, solvent 8 is a mixed solvent of dichloromethane and methanol. Preferably, the ratio of dichloromethane to methanol is 1:1, v / v;

[0088] In some embodiments, compound 6 undergoes a deacetylation reaction in the presence of sodium methoxide. The molar ratio of compound 6 to sodium methoxide is 1:1.5 - 2.5; preferably, sodium methoxide is added to the reaction system in the form of a solution, and the solvent is preferably methanol;

[0089] In some embodiments, the reaction is carried out at 20 - 30 °C under stirring conditions, and the reaction time is 8 - 14 h;

[0090] In some embodiments, after the reaction is completed, a separation and purification step is further included: the reaction solution is neutralized with a hydrogen-type ion exchange resin and then filtered, and the filtrate is concentrated to obtain compound 7.

[0091] Step S7

[0092] In some embodiments, the chitobiosyl diphosphate polyphenol β-mannosyltransferase (ALG1) is derived from Saccharomyces cerevisiae; in particular, its amino acid sequence is as shown in SEQ ID: 1.

[0093] In some embodiments, the reaction is carried out in the presence of Mg 2+ present;

[0094] In some embodiments, the reaction is carried out under the conditions of a pH value of 6 - 9, such as 6.5, 7.0, 7.2, 7.5, 7.8, 8.0, 8.5, etc., preferably 7.4.

[0095] Step S8

[0096] In some embodiments, the α-1,3 / 1,6-mannosyltransferase (ALG2) is of human origin; in particular, its amino acid sequence is as shown in SEQ ID: 2.

[0097] In some embodiments, the reaction is carried out in the presence of Mg 2+ ;

[0098] In some embodiments, the reaction is carried out under the conditions of a pH value of 7 - 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., preferably 7.4.

[0099] Step S9

[0100] In some embodiments, the reaction is carried out in an HCl solution. For example, the concentration of HCl in the HCl solution is 0.5 - 1.5 M.

[0101] In some embodiments, the reaction temperature is 60 - 80 °C, preferably 70 °C, and the reaction time is 1.5 - 2.5 h.

[0102] Step S10

[0103] In some embodiments, the α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) is of human origin, in particular, its amino acid sequence is as shown in SEQ ID: 3.

[0104] In some embodiments, the reaction is carried out in the presence of Mn 2+ , Mg 2+ ;

[0105] In some embodiments, the reaction is carried out under the conditions of a pH value of 7 - 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., preferably 7.4.

[0106] Step S11

[0107] In some embodiments, the α-1,6-mannosylglycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT2) is of human origin; in particular, its amino acid sequence is as shown in SEQ ID: 4.

[0108] In some embodiments, the reaction is carried out in the presence of Mn 2+, Mg 2+ in the presence of;

[0109] In some embodiments, the reaction is carried out under the conditions of a pH value of 7 - 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., preferably 7.4.

[0110] In some embodiments, steps S7 and S8 can be carried out separately or in the same reaction system according to the one - pot method.

[0111] In a specific embodiment, steps S7 and S8 are carried out in the same reaction system. At this time, compound 7 and guanosine diphosphate mannose (GDP - Man) can be used as substrates, in the presence of metal Mg 2+ in the presence of, under the action of the chitobiosyl diphosphate polyphenol β - mannosyltransferase (ALG1) described in step S7 or a protein derived from ALG1 with one or several amino acids substituted, deleted or added in the amino acid sequence of ALG1 and having ALG1 activity, and the α - 1,3 / 1,6 - mannosyltransferase (ALG2) described in step S8 or a protein derived from ALG2 with one or several amino acids substituted, deleted or added in the amino acid sequence of ALG2 and having ALG2 activity, catalyze the synthesis of Man 3 - Gn 2 - PP - lipid (compound 9), and its reaction formula is as follows:

[0112]

[0113] In a specific embodiment, steps S10 and S11 can be carried out separately or in the same reaction system according to the one - pot method.

[0114] In some embodiments, steps S10 and S11 are carried out in the same reaction system. At this time, the core pentasaccharide shown in formula (I) and uridine diphosphate N - acetylglucosamine (UDP - GlcNAc) can be used as substrates, in the presence of metal Mg 2+ , Mn 2+ in the presence of, under the action of the α - 1,3 - mannosylglycoprotein 2 - β - N - acetylglucosaminyltransferase (MGAT1) described in step S10 or a protein derived from MGAT1 with one or several amino acids substituted, deleted or added in the amino acid sequence of MGAT1 and having MGAT1 activity, and the α - 1,6 - mannosylglycoprotein 2 - β - N - acetylglucosaminyltransferase (MGAT2) described in step S11 or a protein derived from MGAT2 with one or several amino acids substituted, deleted or added in the amino acid sequence of MGAT2 and having MGAT2 activity, catalyze the synthesis of the core heptasaccharide GlcNAc shown in formula (II)2 -Man 3 -GlcNAc 2 , and the reaction formula is as follows:

[0115]

[0116] In a second aspect, the present invention provides a method for preparing an N-glycan represented by formula (III),

[0117]

[0118] In formula (III),

[0119] represents D-mannopyranose,

[0120] represents N-acetylglucosamine,

[0121] represents N-acetylneuraminic acid,

[0122] represents D-galactopyranose,

[0123] represents L-fucopyranose,

[0124] β4 means that a glycosidic hydroxyl group of one sugar and a hydroxyl group on the 4-position carbon of another sugar are connected by a β-configured glycosidic bond,

[0125] β2 means that a glycosidic hydroxyl group of one sugar and a hydroxyl group on the 2-position carbon of another sugar are connected by a β-configured glycosidic bond,

[0126] α3 means that a glycosidic hydroxyl group of one sugar and a hydroxyl group on the 3-position carbon of another sugar are connected by an α-configured glycosidic bond,

[0127] α6 means that a glycosidic hydroxyl group of one sugar and a hydroxyl group on the 6-position carbon of another sugar are connected by an α-configured glycosidic bond;

[0128] --- means containing or not containing the above monosaccharide structures;

[0129] The method includes: on the basis of the synthesis method of the core pentasaccharide represented by formula (I) or the core heptasaccharide represented by formula (II) in the foregoing N-glycan, extending the core pentasaccharide or core heptasaccharide through an enzymatic reaction or a chemical reaction to prepare the N-glycan represented by formula (III);

[0130] Among them, the enzymatic reaction or chemical reaction is selected from one or more of the following:

[0131] (1) Under the action of β1,3-galactosyltransferase (B3GalT) or a protein derived from B3GalT with substitution, deletion or addition of one or several amino acids in the amino acid sequence of B3GalT and having B3GalT activity, N-glycan reacts with uridine diphosphogalactose (UDP-Gal), and galactose C linked by β1,3-glycosidic bond is added to N-acetylglucosamine A at the antenna end of N-glycan;

[0132] Preferably, the B3GalT is derived from Chromobacterium violaceum; in particular, its amino acid sequence is as shown in SEQ ID: 5;

[0133] Preferably, the above enzymatic reaction is carried out in the presence of Mg 2+ ; the pH value of the reaction system is 6 to 9, preferably about 7.5;

[0134] (2) Under the action of β1,4-galactosyltransferase (B4GalT) or a protein derived from B4GalT with substitution, deletion or addition of one or several amino acids in the amino acid sequence of B4GalT and having B4GalT activity, N-glycan reacts with uridine diphosphogalactose (UDP-Gal), and galactose C and / or D linked by β1,4-glycosidic bond are added to N-acetylglucosaminyl A and / or B at the antenna end of N-glycan;

[0135] Preferably, the B4GalT is of human origin; in particular, its amino acid sequence is as shown in SEQ ID: 6;

[0136] Preferably, the above enzymatic reaction is carried out in the presence of Mn 2+ ; the pH value of the reaction system is 6 to 9, preferably about 7.5;

[0137] (3) Under the action of α2,6-sialyltransferase (Pd2,6ST) or a protein derived from Pd2,6ST with substitution, deletion or addition of one or several amino acids in the amino acid sequence of Pd2,6ST and having Pd2,6ST activity, or under the action of α2,6-sialyltransferase (ST6Gal1) or a protein derived from ST6Gal1 with substitution, deletion or addition of one or several amino acids in the amino acid sequence of ST6Gal1 and having ST6Gal1 activity, N-glycan reacts with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F linked by α2,6-glycosidic bond are added to galactosyl C and / or D at the antenna end of N-glycan;

[0138] Preferably, the Pd2,6ST is derived from Photobacterium damsela; in particular, its amino acid sequence is as shown in SEQ ID: 7; the ST6Ga11 is derived from human, in particular, its amino acid sequence is as shown in SEQ ID: 8;

[0139] Preferably, the above enzymatic reaction is carried out in the presence of Mg 2+ The pH value of the reaction system is 6 to 9, preferably about 7.5;

[0140] (4) Under the action of α2,3-sialyltransferase (BtST) or a protein derived from BtST with one or several amino acids substituted, deleted or added in the amino acid sequence of BtST and having BtST activity, or under the action of α2,3-sialyltransferase (PPST) or a protein derived from PPST with one or several amino acids substituted, deleted or added in the amino acid sequence of PPST and having PPST activity, N-glycan reacts with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F linked by α2,3-glycosidic bond are added to galactosyl C and / or D at the antenna end of N-glycan;

[0141] Preferably, the BtST is derived from Bibersteinia trehalosi; in particular, its amino acid sequence is as shown in SEQ ID: 9; the PPST is derived from Photobacterium phosphoreum; in particular, its amino acid sequence is as shown in SEQ ID: 10;

[0142] Preferably, the above enzymatic reaction is carried out in the presence of Mg 2+ The pH value of the reaction system is 6 to 9, preferably about 7.5.

[0143] (5) Under the action of α1,3-fucosyltransferase (FucT) or a protein derived from FucT with one or several amino acids substituted, deleted or added in the amino acid sequence of FucT and having FucT activity, N-glycan reacts with guanosine diphosphate fucose (GDP-Fucose), and fucose G and / or H linked by α1,3-glycosidic bond are added to N-acetylglucosaminyl A and / or B on the antenna of N-glycan;

[0144] Preferably, the FucT is derived from Helicobacter pylori; in particular, its amino acid sequence is as shown in SEQ ID: 11;

[0145] Preferably, the above enzymatic reaction is carried out in the presence of Mg 2+It is carried out in the presence of; the pH value of the reaction system is 6 to 9, preferably about 7.5.

[0146] (6) Under the action of β1,3-galactosidase hydrolase (B3GalH) or a protein derived from B3GalH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of B3GalH and having B3GalH activity, remove galactose C linked by β1,3-glycosidic bond to N-acetylglucosamine A at the antenna end of N-glycan;

[0147] Preferably, the pH value of the reaction system for the above enzymatic reaction is 6 to 9, preferably about 7.0;

[0148] (7) Under the action of β1,4-galactosidase hydrolase (B4GalH) or a protein derived from B4GalH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of B4GalH and having B4GalH activity, remove galactose C and / or D linked by β1,4-glycosidic bond to N-acetylglucosamine A and / or B at the antenna end of N-glycan;

[0149] Preferably, the pH value of the reaction system for the above enzymatic reaction is 6 to 9, preferably about 7.0;

[0150] (8) Under the action of β1,2-N-acetylglucosaminidase hydrolase (GlcNAcH) or a protein derived from GlcNAcH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of GlcNAcH and having GlcNAcH activity, remove N-acetylglucosamine A and / or B linked by β1,2-glycosidic bond to galactose at the antenna end of N-glycan;

[0151] Preferably, the pH value of the reaction system for the above enzymatic reaction is 6 to 9, preferably about 7.0.

[0152] (9) Under the action of α2,3-sialidase (NanC) derived from Streptococcus penumoniae or a protein derived from NanC with substitution, deletion or addition of one or several amino acids in the amino acid sequence of NanC and having NanC activity, remove N-acetylneuraminic acid E and / or F linked by α2,3-glycosidic bond to galactose C and / or D at the antenna end of N-glycan;

[0153] Preferably, the NanC is derived from Streptococcus penumoniae;

[0154] Preferably, the pH value of the reaction system for the above enzymatic reaction is 6 to 9, preferably about 7.0.

[0155] (10) Under the action of α1,3-fucosidase (FucH) or a protein derived from FucH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of FucH and having FucH activity, fucose G and / or H on N-acetylglucosamine A and / or B linked by α1,3-glycosidic bond on the antenna of N-glycan is removed;

[0156] Preferably, the pH value of the reaction system for the above enzymatic reaction is 6 to 9, preferably about 7.0;

[0157] (11) Under the action of acetic acid, N-acetylneuraminic acid E and / or F linked by α2,6-glycosidic bond to galactose C or D at the end of the N-glycan antenna is removed;

[0158] Preferably, acetic acid is in the form of an acetic acid aqueous solution with a concentration of 1.5 - 2.5 M; the reaction temperature is 70 - 90 °C;

[0159] Preferably, after the reaction is completed, the reaction is neutralized by adding sodium bicarbonate, and concentrated and purified by a P2 column to obtain the target product.

[0160] In the above steps (1)-(11), a separation and purification step may be further included. Specifically, the purification step includes: after the reaction is completed, ethanol is added, and solid-liquid separation is carried out (for example, centrifugation at 10000 g for 10 minutes), and the supernatant is concentrated and purified by a P2 column to obtain the target product.

[0161] In some embodiments, the N-glycan represented by formula (III) is selected from the compounds represented by the following structures:

[0162]

[0163]

[0164]

[0165] In some embodiments, the method for preparing the N-glycan represented by (III) includes the following steps:

[0166]

[0167] (a) Using the core heptasaccharide shown in formula (II) as a substrate, in the presence of β1,3-galactosyltransferase (B3GalT) or a protein derived from B3GalT with substitution, deletion, or addition of one or more amino acids in the amino acid sequence of B3GalT and having B3GalT activity, a galactose C linked by a β1,3-glycosidic bond is added to N-acetylglucosamine A at the antenna end of the core heptasaccharide shown in formula (II) to obtain compound 40;

[0168] (b) Then, in the presence of β1,4-galactosyltransferase (B4GalT) or a protein derived from B4GalT with substitution, deletion, or addition of one or more amino acids in the amino acid sequence of B4GalT and having B4GalT activity, a galactose D linked by a β1,4-glycosidic bond is added to N-acetylglucosamine B at the antenna end of compound 40 to obtain compound 41;

[0169] (c) Subsequently, in the presence of β1,3-galactosylhydrolase (B3GalH) or a protein derived from B4GalH with substitution, deletion, or addition of one or more amino acids in the amino acid sequence of B4GalH and having B4GalH activity, the terminal galactose C linked by a β1,3-glycosidic bond is removed to obtain the asymmetric bi-antennary N-glycan 42.

[0170] In some embodiments, the method for preparing the bi-antennary N-glycan shown in (III) further includes the method shown in the following reaction formula:

[0171]

[0172]

[0173]

[0174] The specific steps include:

[0175] (d) In the presence of β1,4-galactosyltransferase (B4GalT) or a protein derived from B4GalT with substitution, deletion, or addition of one or more amino acids in the amino acid sequence of B4GalT and having B4GalT activity, the compound of formula (II), compound 28, compound 43, compound 49, compound 53, compound 56, compound 61, compound 64, and uridine diphosphogalactose (UDP-Gal) react to add galactose linked by a β1,4-glycosidic bond to the N-acetylglucosaminyl group at the antenna end of the N-glycan to obtain compound 30, compound 35, compound 44, compound 50, compound 54, compound 57, compound 62, and compound 65, respectively;

[0176] (e) Under the action of α2,6-sialyltransferase (Pd2,6ST) or a protein derived from Pd2,6ST with substitution, deletion or addition of one or more amino acids in the amino acid sequence of Pd2,6ST and having Pd2,6ST activity, or under the action of α2,6-sialyltransferase (ST6Gal1) or a protein derived from ST6Gal1 with substitution, deletion or addition of one or more amino acids in the amino acid sequence of ST6Gal1 and having ST6Gal1 activity, compound 30, compound 35, compound 42, compound 44, compound 57, and compound 72 react with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) respectively, and N-acetylneuraminic acid linked by α2,6-glycosidic bond is added to the galactosyl group at the antenna end of N-glycan, obtaining compound 33, compound 38, compound 61, compound 48, compound 68, and compound 77 respectively;

[0177] (f) Under the action of α2,3-sialyltransferase (BtST) or a protein derived from BtST with substitution, deletion or addition of one or more amino acids in the amino acid sequence of BtST and having BtST activity, or under the action of α2,3-sialyltransferase (PPST) or a protein derived from PPST with substitution, deletion or addition of one or more amino acids in the amino acid sequence of PPST and having PPST activity, compound 30, compound 31, compound 35, compound 36, compound 42, compound 45, compound 52, compound 57, compound 65, compound 68, compound 70, compound 72, and compound 74 react with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) respectively, and N-acetylneuraminic acid linked by α2,3-glycosidic bond is added to the galactosyl group at the antenna end of N-glycan, obtaining compound 32, compound 34, compound 37, compound 39, compound 43, compound 46, compound 53, compound 58, compound 66, compound 69, compound 71, compound 75, and compound 76 respectively;

[0178] (g) Under the action of α1,3-fucosyltransferase (FucT) or a protein derived from FucT with substitution, deletion or addition of one or several amino acids in the amino acid sequence of FucT and having FucT activity, compound 31, compound 35, compound 51, compound 54, compound 42, compound 62, compound 65, and compound 72 react with guanosine diphosphate fucose (GDP-Fucose) respectively, and fucose linked by an α1,3-glycosidic bond is added to N-acetylglucosamine on the antenna of N-glycan to obtain compound 32, compound 36, compound 52, compound 55, compound 56, compound 70, compound 67, and compound 74;

[0179] (h) Under the action of β1,3-galactosyl hydrolase (B3GalH) or a protein derived from B3GalH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of B3GalH and having B3GalH activity, the galactose linked by a β1,3-glycosidic bond to N-acetylglucosamine at the antenna end of compound 41 is removed to obtain compound 42;

[0180] (i) Under the action of β1,4-galactosyl hydrolase (B4GalH) or a protein derived from B4GalH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of B4GalH and having B4GalH activity, the galactose linked by a β1,4-glycosidic bond to N-acetylglucosamine at the antenna end of compound 49, compound 59 or compound 72 is removed respectively to obtain compound 50, compound 60, and compound 73;

[0181] (j) Under the action of β1,2-N-acetylglucosaminyl hydrolase (GlcNAcH) or a protein derived from GlcNAcH with substitution, deletion or addition of one or several amino acids in the amino acid sequence of GlcNAcH and having GlcNAcH activity, the N-acetylglucosamine linked by a β1,2-glycosidic bond to galactose at the antenna end of compound 42 is removed to obtain compound 72;

[0182] (k) Under the action of α2,3-sialic acid hydrolase (NanC) derived from Streptococcus penumoniae or a protein derived from NanC with substitution, deletion or addition of one or several amino acids in the amino acid sequence of NanC and having NanC activity, the N-acetylneuraminic acid linked by an α2,3-glycosidic bond to galactosyl at the antenna end of compound 45 or compound 48 is removed respectively to obtain compound 47 and compound 49;

[0183] (1) Under the action of α1,3-fucosidase (FucH) or a protein derived from FucH with substitution, deletion or addition of one or more amino acids in the amino acid sequence of FucH and having FucH activity, the fucose on the N-acetylglucosamine group linked by an α1,3-glycosidic bond to the antenna of the N-glycan of compound 58 is removed to obtain compound 59:

[0184] (m) Under the action of acetic acid, the N-acetylneuraminic acid linked by an α2,6-glycosidic bond to the galactose group at the end of the antenna of compound 50 is removed to obtain compound 51. Detailed implementation mode

[0185] The present invention has been described in detail above, but the above embodiments are essentially only illustrative and are not intended to limit the present invention. In addition, the present application is not limited by any theory described in the foregoing prior art or the summary of the invention or the following examples.

[0186] Unless otherwise clearly stated, the numerical ranges in the entire application document include any sub-ranges therein and any numerical values incremented by the smallest sub-unit of the given value therein. Unless otherwise clearly stated, the numerical values in the entire application document represent an approximate measure or limitation of the scope of embodiments including minor deviations from the given values and having approximately the mentioned values and having the exact values mentioned. Except for the working examples provided at the end of the detailed description, all numerical values of the parameters (such as quantities or conditions) in this application document (including the appended claims) should be understood to be modified by the term "about" in all cases, whether or not "about" actually appears before the numerical value. "About" means that the stated numerical value allows for some imprecision (somewhat close to the exact value; approximately or reasonably close to the value; approximate). If the imprecision provided by "about" is not understood in this ordinary meaning in the art, then "about" as used herein at least represents the variations that can be produced by the ordinary methods of measuring and using these parameters. For example, "about" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1% or less than or equal to 0.5%.

[0187] The present invention has successfully developed a general method for efficiently preparing various symmetric and asymmetric N-glycans. The method includes: by a chemoenzymatic synthesis strategy, preparing N-glycan core pentasaccharide and core heptasaccharide from monosaccharides, and obtaining various symmetric and asymmetric N-glycans by using enzymatic synthesis and reversible galactosylation as a selective protection strategy. By chemoenzymatic method, starting from the disaccharide GlcNAcβ1,4GlcNAc (Compound 1), phosphorylated disaccharides GlcNAc 2 -PP-lipid (Compound 5) of several substrates (C6, C7, C8, C9, C10, C11, C20) with different lipid tails were synthesized through several simple chemical steps, and the relative activities of ALG1 of these substrates were compared. Enzymatic reactions were carried out with yeast-derived ALG1 and ALG2 to generate Man 3 -GlcNAc 2 -PP-lipid (Compound 9), and the core pentasaccharide Man 3 -GlcNAc 2 (the compound shown in Formula (I)) was obtained by heating under acidic conditions. Enzymatic reactions were carried out in the presence of MGAT1 and MGAT2 to generate the core heptasaccharide GlcNAc 2 -Man 3 -GlcNAc 2 (the compound shown in Formula (II)). By using sugar nucleotide synthetic related enzymes from different clone sources to extend the core pentasaccharide and core heptasaccharide, and taking advantage of the ready availability of the core pentasaccharide and core heptasaccharide, 49 different symmetric and asymmetric N-glycans were synthesized. In particular, the present invention has developed a reversible galactosylation catalyzed by β1,3-galactosyltransferase (from the compound shown in Formula (II) to Compound 41), which specifically recognizes a single arm of the N-glycan as a protection strategy for generating asymmetric N-glycans. Compared with most reported methods using HPLC to purify products, all the structures obtained in the present invention are purified by conventional chromatography methods, including size exclusion columns or ion exchange columns. Therefore, the reaction scale can be easily expanded. The method can prepare N-glycans on a large scale and efficiently, and without cumbersome purification operations, so it has the characteristics of low cost and strong adaptability.

[0188] Examples

[0189] (1) Preparation of enzymes using an Escherichia coli expression system

[0190] Artificially synthesize the catalytic domain of glycosyltransferase using an Escherichia coli expression system, including:

[0191] Chitobiosyl diphosphate polyphenol β-mannosyltransferase (ALG1) from Saccharomyces cerevisiae, whose amino acid sequence is:

[0192]

[0193] Human α-1,3 / 1,6-mannosyltransferase (ALG2), the amino acid sequence of which is:

[0194]

[0195] Human α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1, (29 to 445 aa)), the amino acid sequence of which is:

[0196]

[0197] Chromobacterium violaceum β1,3-galactosyltransferase (B3GalT), the amino acid sequence of which is:

[0198]

[0199] α2,6-sialyltransferase (Pd2,6ST) from Photobacterium damselae, the amino acid sequence of which is:

[0200]

[0201] α2,3-sialyltransferase (BtST) from Thalassiosira pseudonana, the amino acid sequence of which is:

[0202]

[0203] α2,3-sialyltransferase (PPST) from Photobacterium phosphoreum, the amino acid sequence of which is:

[0204]

[0205] α1,3-fucosyltransferase (FucT) from Helicobacter pylori, the amino acid sequence of which is:

[0206]

[0207] The above-mentioned related enzymes were prepared by an Escherichia coli expression system and purified using Ni-NTA. These genes were all synthesized by GenScript (Nanjing, China) or Sangon Biotech (Shanghai, China).

[0208] All genes were cloned into the pET-28a vector, with six histidine (His) tags at the N-terminus. The correctly identified plasmid was transformed into Escherichia coli BL21(DE3) for protein expression. Escherichia coli BL21(DE3) cells containing the recombinant plasmid were cultured in two liters of Luria-Bertani (LB) medium containing 50 μg / ml kanamycin at 37 °C with a rotary shaker at 200 rpm. When the OD value reached 0.8, 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and protein expression was induced overnight at 16 °C. Cells were collected by centrifugation at 7000 rpm for 10 min. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl buffer, 300 mM NaCl, 10 mM imidazole; pH 7.5). Cells were disrupted using a microfluidizer and the lysate was centrifuged at 12,000 g for 30 min to remove cell debris. His-tagged proteins were purified using a Ni-NTA agarose column. Before purification, the column was equilibrated with lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole; pH 7.5). The column was washed with 2 column volumes of lysis buffer and the target protein was eluted with elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole; pH 7.5). The eluted target proteins are the enzymes used in the present invention, and these enzymes were desalted by filtration through an ultrafiltration centrifugal tube (Amicon Ultra-5, 10 Kd) for further use. The protein concentration was determined using a BCA protein assay kit.

[0209] Although the sources of the enzymes in the present application are described above, their source pathways are not limited thereto, as long as they can perform the functions intended in the present application.

[0210] (2) Preparation of enzymes using the HEK293 expression system

[0211] The catalytic domains of human glycosyltransferases were artificially synthesized, including:

[0212] α-1,3 / 1,6-mannosyltransferase (HsALG2) from human (full length, amino acid sequence shown in SEQ ID: 2);

[0213] α-1,6-mannosylglycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT2) from human (45 to 447 aa), and its amino acid sequence is:

[0214]

[0215] β1,4-galactosyltransferase (B4GalT) from human (45 to 398 aa), and its amino acid sequence is:

[0216]

[0217] Human-derived α2,6-sialyltransferase (ST6Gal1) (75 to 406 aa), the amino acid sequence of which is:

[0218]

[0219]

[0220] All genes were cloned into a reconstructed pCDNA3.1 vector. This vector is designed to produce a recombinant protein containing a 25-amino acid NH 2 terminal signal sequence of lysosomal α-mannosidase from Trypanosoma cruzi, an 8xHis tag, and a GFP tag (green fluorescent protein). According to the manufacturer's instructions, large-scale protein preparation was achieved by transient transfection of HEK293 suspension cultures. HEK293 suspension cells (Freestyle 293-F cells) were transfected, and the culture supernatant containing the secreted fusion protein was placed on a Ni-NTA agarose column. Before purification, the column was equilibrated with lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole; pH 7.5). The column was washed with 2 column volumes of lysis buffer and eluted with elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole; pH 7.5). The enzyme was desalted with a 10 kDa cut-off molecular weight by filtration (Amicon Ultra-5, 10Kd). Protein concentration was determined using a BCA protein assay kit.

[0221] (3) Enzymes from commercial sources

[0222] The B3GalH (#P0726S), B4GalH (#P0730S), GlcNAcH (#P0744S), NanC (#P0743S), FucH (#P0748S) were purchased from NEB.

[0223] Example 1: Large-scale synthesis and purification of Compound 2:

[0224]

[0225] Compound 1 (4.5 g, 10.6 mmol) was dissolved in anhydrous pyridine (40 mL), and acetic anhydride (15 mL, 159 mmol) was slowly added under nitrogen protection. After stirring at room temperature until the raw materials reacted completely, the solvent was removed by rotary evaporation. Then, dichloromethane (DCM, 200 mL) was added for dissolution, and it was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic layer was collected, the aqueous layer was back-extracted with DCM, and the organic layers were combined after collection and dried over anhydrous sodium sulfate. After vacuum concentration, 6.67 g of crude compound 2 was obtained as a white powder, and the product yield was 93%.

[0226] Example 2: Large-scale synthesis and purification of compound 3:

[0227]

[0228] Compound 2 (6.50 g, 9.61 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 60 mL). Under an ice bath, 7.0 M ammonia in methanol solution (20 mL) was slowly added, and the reaction was carried out at room temperature until the raw materials reacted completely, and the reaction was monitored by TLC (EA∶MeOH = 8∶1). After the reaction was completed, the reaction mixture was diluted with DCM (300 mL) and washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic layer was collected, the aqueous layer was back-extracted with DCM, and the organic layers were combined after collection and dried over anhydrous sodium sulfate. After concentration under vacuum, it was loaded onto silica gel by dry loading and purified by column chromatography (DCM∶MeOH = 95∶5) to obtain 5.06 g of compound 3 as a white powder, and the yield was 83%.

[0229] Example 3: Large-scale synthesis and purification of compound 4:

[0230]

[0231] Compound 3 (4.80 g, 7.57 mmol), 4,5-dicyanoimidazole (2.68 g, 22.70 mmol) and 4A molecular sieve were suspended in 30 mL of ultradry acetonitrile under nitrogen protection. Then, dibenzyl N,N-diisopropylphosphoramidite (3.8 mL, 11.35 mmol) was added dropwise under an ice bath. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature until the raw materials reacted completely and then placed in an ice bath to cool the temperature to 0 °C. Then, a 5.0 M - 6.0 M solution of tert-butyl hydroperoxide in decane (6 mL, 30.3 mmol) was added dropwise. After the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature until the reaction was complete. The insoluble solids were removed by filtration through diatomaceous earth, and the solution was concentrated under reduced pressure. The concentrated solution was diluted with DCM (200 mL) and washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic layer was collected, the aqueous layer was back-extracted with DCM, the organic layers were collected and combined, and then dried over anhydrous sodium sulfate. After concentration under vacuum, silica gel was added for dry loading, and purification was carried out by column chromatography (DCM∶MeOH = 95∶5) to obtain 4.87 g of compound 4 as a white powder with a yield of 72%.

[0232] Example 4: Large-scale synthesis and purification of compound 5:

[0233]

[0234] Compound 4 (4.87 g, 5.44 mmol) was dissolved in anhydrous methanol (MeOH, 20 mL), and 10% Pd-C (500 mg) was added. The reaction mixture was vigorously stirred at room temperature under a hydrogen atmosphere until the raw materials reacted completely. After filtration through diatomaceous earth, it was concentrated under vacuum to obtain 3.5 g of compound 5 as a white powder with a yield of 90%.

[0235] Example 5: Preparation of compounds c1 - c7

[0236]

[0237] (1) Compounds a1 - a7 (2 g) were separately suspended in 30 mL of anhydrous acetonitrile with 4,5 - dicyanoimidazole (3 equivalents) and 4A molecular sieve under nitrogen protection. Then, dibenzyl N,N - diisopropylphosphoramidite (1.5 equivalents) was added dropwise under an ice bath. After the addition, the ice bath was removed, and the reactants were stirred at room temperature until the raw materials reacted completely. Then, the reaction was placed in an ice bath to cool the temperature to 0 °C. Subsequently, a 5.0 M - 6.0 M solution of tert - butyl hydroperoxide in decane (4 equivalents) was added dropwise. After the addition, the ice bath was removed, and the reactants were stirred at room temperature until the product of the previous step reacted completely. The insoluble solids were removed by filtration through diatomaceous earth, and the solution was concentrated under reduced pressure. The mixture was diluted with DCM (200 mL) and washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic layer was collected, the aqueous layer was back - extracted with dichloromethane, and the organic layers were combined after collection and dried over anhydrous sodium sulfate. After concentration under vacuum, the residue was loaded onto silica gel by dry loading and purified by column chromatography (PE∶EA = 85∶15) to obtain the target products b1 - b7 respectively.

[0238] (2) Compounds b1 - b7 (3 g) were dissolved in anhydrous MeOH (15 mL), and 10% Pd - C (300 mg) was added. The reactants were vigorously stirred under a hydrogen atmosphere at room temperature until the raw materials reacted completely. After filtration through diatomaceous earth, it was concentrated under vacuum to obtain the target products c1 - c7.

[0239] Example 6: Preparation of Compounds 6a - 6g

[0240]

[0241] Compound 5 (1.0 g, 1.4 mmol) was dissolved in anhydrous MeOH (10 mL), and 1 mL of triethylamine was added. The mixture was concentrated under vacuum to obtain bis(triethylammonium) phosphate. The crude material was co - evaporated with toluene (3×10 mL) three times to remove the excess triethylamine. The resulting triethylammonium salt form of compound 5 was dissolved in anhydrous DMF (20 mL), and then a solution of N,N′ - carbonyldiimidazole (1.2 g, 7.7 mmol) in anhydrous DMF (20 mL) was slowly added under nitrogen protection. The reactants were stirred at room temperature for 4.5 h. Then, methanol (0.6 mL, 14.0 mmol) was added, and the reaction was stirred for 30 minutes to quench the unreacted CDI. A solution of compounds c1 - c7 (1.2 equivalents) in DCM (10 mL) was added to the reaction mixture, and the reaction was stirred at room temperature for 3 days. After the raw materials reacted completely, the reactants were concentrated under vacuum and the residue was purified by column chromatography (EA∶MeOH∶H 2 O = 70∶30∶0.3) to obtain the target products 6a - 6g.

[0242] Example 7: Preparation of Compounds 7a - 7g

[0243]

[0244] Dissolve Compounds 6a - 6g in anhydrous MeOH / DCM (1:1, 20 mL). Under nitrogen protection, slowly add a MeOH solution of 5.4 M MeONa (0.5 equivalent). React at room temperature for 12 hours. After completion, neutralize the reaction mixture with a hydrogen - type ion - exchange resin. After filtration, concentrate the solvent to obtain the target products 7a - 7g.

[0245] Example 8: Large - scale Synthesis and Purification of Compound d

[0246]

[0247] Dissolve Compound 5 (208 mg, 0.23 mmol) in 1.5 mL of 25% aqueous ammonium hydroxide solution. Stir the reaction at room temperature until the reaction is complete. After completion, purify through a P - 2 column. After lyophilization, 95 mg of Compound d, a white powder, is obtained with a yield of 81%.

[0248] Example 9: Large - scale Synthesis and Purification of Compound e

[0249]

[0250] Dissolve Compound 1 (500 mg, 1.18 mmol) in 10 mM of MgCl 2 and BtMP in 10 mM Tris buffer (pH 7.5) and incubate. The reaction is carried out at 37 °C and monitored by TLC. Once no starting material is observed on TLC, quench the reaction by adding cold ethanol. Remove the insoluble precipitate by centrifugation. Concentrate and purify the supernatant through a P2 column to obtain Compound e.

[0251] Example 10: Preparation of Compounds 8a - 8g

[0252]

[0253] Incubate a 50 μL mixture (50 mM Tris buffer, pH 7.4) containing 10 mM substrate (d, 7a, 7b, 7c, 7d, 7e, 7f, or 7g), 10 mM GDP - Man, 5 mM Mg 2+ and 375 μg of cell lysate (ALG1) at 37 °C for 20 minutes; terminate the reaction by diluting the reaction 10 - fold with a buffer of cooled acetonitrile / 100 mM aqueous ammonium acetate solution (pH 4.5 (60% acetonitrile)). Use a ZIC - cHILIC chromatographic column through a The diluted solution was analyzed at 254 nm. The chromatographic column was eluted at a flow rate of 0.6 mL / min with acetonitrile / 100 mM ammonium acetate aqueous solution pH 4.5 (65% acetonitrile) at 40 °C. The enzyme activity was evaluated by quantifying the newly formed GDP, and the results are shown in Table 1.

[0254] Table 1

[0255] Substrate Relative enzyme activity (ALG1) d ND 7a 100％ 7b 92％ 7c 79％ 7d 47％ 7e 19％ 7f 4.7％ 7g 3.8％

[0256] Compound 7a - 7g (200 mg) was incubated with 10 mM MgCl 2 and ALG1 lysate in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37 °C and monitored by TLC. Once no starting material was observed on TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified using a P2 column to obtain compounds 8a - 8g for the test in Example 11.

[0257] Example 11: Preparation of Compounds g, 9a - 9g

[0258]

[0259] A 50 μL mixture (50 mM Tris buffer, pH 7.4) containing 10 mM substrate (e or 8a - 8g), 20 mM GDP-Man, 5 mM Mg 2+ and 625 μg cell lysate (ALG2 or HsALG2) was incubated at 37 °C for 20 minutes; the reaction was terminated by diluting the reaction 10 - fold with a buffer of cooled acetonitrile / 100 mM ammonium acetate aqueous solution (pH 4.5 (60% acetonitrile)) to prepare compounds g, 9a - 9g. The diluted solution was analyzed at 254 nm using a ZIC - cHILIC chromatographic column equipped with a UV detector. The chromatographic column was eluted at a flow rate of 0.6 mL / min with acetonitrile / 100 mM ammonium acetate aqueous solution pH 4.5 (65% acetonitrile) at 40 °C. The enzyme activity was evaluated by quantifying the newly formed GDP, and the results are shown in Table 2. The diluted solution was analyzed at 254 nm. The chromatographic column was eluted at a flow rate of 0.6 mL / min with acetonitrile / 100 mM ammonium acetate aqueous solution pH 4.5 (65% acetonitrile) at 40 °C. The enzyme activity was evaluated by quantifying the newly formed GDP, and the results are shown in Table 2.

[0260] Table 2

[0261] Substrate Relative enzyme activity (ALG2) Relative enzyme activity (HsALG2) e ND ND 8a 100％ 100％ 8b 134％ 143％ 8c 201％ 308％ 8d 140％ 207％ 8e 151％ 192％ 8f 210％ 330％ 8g 21％ 12％

[0262] Combining the results in Table 1 and Table 2, it shows that phosphorylated disaccharide Gn with different lipid tails (C6, C7, C8, C9, C10, C11, C20) 2The ALG1 activity of the -PP-lipid substrate is related to the length of the lipid tail. When R is C10 or C11, its ALG1 activity is better. Considering the activities of both ALG1 and ALG2, compound 7b is used for the large-scale preparation of core pentasaccharide (I) and core heptasaccharide (II).

[0263] Example 12: Large-scale preparation of core pentasaccharide (I) and core heptasaccharide (II)

[0264]

[0265] (1) 15 g of compound 1 was prepared according to the steps of Examples 1-7 to obtain compound 7b. After purification by a P2 column, compound 7b was incubated with ALG1 and an excess of GDP-Man. After the reaction was completed, the reaction was quenched by adding cold ethanol. The supernatant was roughly concentrated and purified by a P2 column to obtain compound 8b. Then, compound 8b was incubated with ALG2 and an excess of GDP-Man to generate compound 9b. After the reaction was completed, the reaction was quenched by adding cold ethanol. The supernatant was concentrated, 1 M hydrochloric acid was added, and the reactant was incubated at 70 °C for 2 hours to release the core pentasaccharide. After completion, the product was purified by a P2 column to obtain 4.84 g of core pentasaccharide (I) with an overall yield of 14.5%.

[0266] (2) 4.05 g of core pentasaccharide (I) was incubated with MGAT1 and an excess of UDP-GlcNAc to generate compound 28. The reaction was monitored by TLC. After completion, MGAT2 was added. When no intermediate or starting material was observed on TLC, the reaction was quenched by adding cold ethanol. The supernatant was concentrated and purified by a P2 column. Finally, 5.28 g of core heptasaccharide (II) was obtained with a yield of 92%.

[0267] Example 13: Preparation of compounds 30 - 77

[0268] Based on the core pentasaccharide (I), core heptasaccharide (II) or compound 28 synthesized in Example 12, this example prepares the bis-antennary N-glycan compounds 30 - 77 (shown in the following formula (III)) by enzymatic reaction or chemical reaction extension. The reaction route is as follows:

[0269]

[0270]

[0271]

[0272]

[0273] The general experimental protocol for each enzymatic reaction and chemical reaction is as follows:

[0274] (1) General experimental protocol for β1,3-galactosylation using β1,3-galactosyltransferase (B3GalT)

[0275] Under the action of β1,3-galactosyltransferase (B3GalT), N-glycan reacts with uridine diphosphogalactose (UDP-Gal) to add galactose C linked by β1,3-glycosidic bond to N-acetylglucosamine A at the antenna end of N-glycan. The specific steps include:

[0276] Incubate the starting material N-glycan with 2 equivalents of UDP-Gal, 10 mM of Mg 2+ and B3GalT in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 37 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified by P2 column to obtain the target product.

[0277] The compound transformation of the above enzymatic reaction using B3GalT includes: Compound 29 → Compound 40.

[0278] (2) General experimental protocol for β1,4-galactosylation using β1,4-galactosyltransferase (B4GalT)

[0279] Under the action of β1,4-galactosyltransferase (B4GalT), N-glycan reacts with uridine diphosphogalactose (UDP-Gal) to add galactose C and / or D linked by β1,4-glycosidic bond to N-acetylglucosaminyl A and / or B at the antenna end of N-glycan. The specific steps include:

[0280] Incubate the starting material N-glycan with 2 equivalents of UDP-Gal, 10 mM of MnCl 2 and B4GalT in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 37 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified by P2 column to obtain the target product.

[0281] The compound transformation of the above enzymatic reaction using B4GalT includes: Compound (II) → Compound 30, Compound 28 → Compound 35, Compound 40 → Compound 41, Compound 43 → Compound 44, Compound 49 → Compound 50, Compound 53 → Compound 54, Compound 56 → Compound 57, Compound 61 → Compound 62, Compound 64 → Compound 65.

[0282] (3) General experimental protocol for α2,6-sialylation using α2,6-sialyltransferase (Pd2,6ST or ST6Gal1)

[0283] Under the action of α2,6-sialyltransferase (Pd2,6ST) or α2,6-sialyltransferase (ST6Gall), N-glycan reacts with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac). N-glycan reacts with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F linked by α2,6-glycosidic bond are added to galactosyl C and / or D at the antenna terminal of N-glycan. The specific steps include:

[0284] Incubate the starting material N-glycan with 2 equivalents of CMP-Neu5Ac and 10 mM MgCl 2 and Pd26ST or ST6Gal1 in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 37 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified through P2 column and ion exchange column to obtain the target product.

[0285] The compound conversions in the above enzymatic reactions using Pd2,6ST include: compound 30 → compound 33, compound 35 → compound 38, compound 42 → compound 61, compound 57 → compound 68, compound 72 → compound 77;

[0286] The compound conversions in the above enzymatic reactions using ST6Gal1 include: compound 44 → compound 48.

[0287] (4) General experimental protocol for α2,3-sialylation using α2,3-sialyltransferase (BtST or PPST)

[0288] Under the action of α2,3-sialyltransferase (BtST) or α2,3-sialyltransferase (PPST), N-glycan reacts with cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac). N-glycan and cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) react, and N-acetylneuraminic acid E and / or F linked by α2,3-glycosidic bond are added to galactosyl C and / or D at the antenna terminal of N-glycan. The specific steps include:

[0289] Incubate the starting material N-glycan with 2 equivalents of CMP-Neu5Ac and 10 mM MgCl 2BtST was incubated in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37 °C and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified using a P2 column to obtain the target product.

[0290] The compound conversions in the above enzymatic reactions with BtST included: compound 30 → compound 34, compound 35 → compound 39, compound 42 → compound 43, compound 57 → compound 58, compound 65 → compound 66, compound 72 → compound 76.

[0291] To synthesize structures containing the sialyl Lewis X epitope, the starting material N-glycan was incubated with 10 equivalents of CMP-Neu5Ac, 10 mM MgCl 2 and PPST in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37 °C and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation.

[0292] The compound conversions in the above enzymatic reactions with PPST included: compound 31 → compound 32, compound 36 → compound 37, compound 45 → compound 46, compound 52 → compound 53, compound 68 → compound 69, compound 70 → compound 71, compound 74 → compound 75.

[0293] (5) General experimental protocol for α1,3-fucosylation using α1,3-fucosyltransferase (FucT)

[0294] Under the action of α1,3-fucosyltransferase (FucT), N-glycan reacts with guanosine diphosphate fucose (GDP-Fucose) to add fucose G and / or H linked by an α1,3-glycosidic bond to N-acetylglucosaminyl A and / or B on the antennae of N-glycan. The specific steps include:

[0295] The starting material N-glycan was incubated with 2 equivalents of GDP-L-Fuc, 10 mM MgCl 2 and FucT in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37 °C and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified using a P2 column to obtain the target product.

[0296] The compound conversions in the above enzymatic reactions using FucT include: compound 31 → compound 32, compound 35 → compound 36, compound 51 → compound 52, compound 54 → compound 55, compound 42 → compound 56, compound 62 → compound 70, compound 65 → compound 67, compound 72 → compound 74.

[0297] (6) General experimental protocol for using β1,3-galactosyl hydrolase (B3GalH)

[0298] Under the action of β1,3-galactosyl hydrolase (B3GalH), the galactose C linked by β1,3-glycosidic bond to N-acetylglucosamine A at the antenna terminal of N-glycan is removed. The specific steps include:

[0299] Incubate the starting material N-glycan with 10 mM MgCl 2 and B4GalH in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 30 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified with a P2 column to obtain the target product.

[0300] The compound conversions in the above enzymatic reactions using B3GalH include: compound 41 → compound 42.

[0301] (7) General experimental protocol for using β1,4-galactosyl hydrolase (B4GalH)

[0302] Under the action of β1,4-galactosyl hydrolase (B4GalH), the galactose C and / or D linked by β1,4-glycosidic bond to N-acetylglucosamine A and / or B at the antenna terminal of N-glycan are removed. The specific steps include:

[0303] Incubate the starting material N-glycan with 10 mM MgCl 2 and B4GalH in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 30 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified with a P2 column to obtain the target product.

[0304] The compound conversions in the above enzymatic reactions using B4GalH include: compound 49 → compound 50, compound 59 → compound 60, compound 72 → compound 73.

[0305] (8) General experimental protocol for using β1,2-N-acetylglucosaminyl hydrolase (GlcNAcH)

[0306] Under the action of β1,2-N-acetylglucosaminidase (GlcNAcH), N-acetylglucosamine A linked by β1,2-glycosidic bond to galactose at the end of the N-glycan antenna is removed. The specific steps include:

[0307] The starting material N-glycan is incubated with 10 mM MgCl 2 and GlcNAcH in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 30 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified with a P2 column to obtain the target product.

[0308] The compound transformation of the above enzymatic reaction with GlcNAcH includes: compound 42 → compound 72.

[0309] (9) General experimental protocol using α2,3-sialidase (NanC)

[0310] Under the action of α2,3-sialidase (NanC), N-acetylneuraminic acid E and / or F linked by α2,3-glycosidic bond to galactose C and / or D at the end of the N-glycan antenna is removed. The specific steps include:

[0311] The starting material N-glycan is incubated with 10 mM MgCl 2 and NanC in 10 mM Tris buffer (pH 7.5). The reaction is carried out at 30 °C and monitored by TLC. Once no starting material is observed on TLC, the reaction is quenched by adding cold ethanol. The insoluble precipitate is removed by centrifugation. The supernatant is concentrated and purified with a P2 column to obtain the target product.

[0312] The compound transformation of the above enzymatic reaction with NanC includes: compound 45 → compound 47, compound 48 → compound 49.

[0313] (10) General experimental protocol using α1,3-fucosidase (FucH)

[0314] Under the action of α1,3-fucosidase (FucH), fucose G and / or H linked by α1,3-glycosidic bond to N-acetylglucosamine A and / or B on the N-glycan antenna is removed. The specific steps include:

[0315] The starting material N-glycan is incubated with 10 mM MgCl 2It was incubated with FucH in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 30 °C and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified using a P2 column to obtain the target product.

[0316] The compound conversion in the above enzymatic reaction with FucH includes: compound 58 → compound 59.

[0317] (11) Removal of α2,6-glycosidic bond-linked N-acetylneuraminic acid by acidolysis

[0318] Under the action of acetic acid, N-acetylneuraminic acids E and / or F linked by α2,6-glycosidic bonds to galactose C or D at the end of the N-glycan antenna were removed. The specific steps include:

[0319] The starting material N-glycan was mixed with 2 M AcOH and reacted at 80 °C, and monitored by TLC. Once no starting material was observed on the TLC, the reaction was neutralized by adding sodium bicarbonate. It was concentrated and purified using a P2 column to obtain the target product.

[0320] The compound conversion carried out by the above reaction includes: compound 50 → compound 51.

[0321] Example 13: Large-scale preparation of compound 42 from core heptasaccharide using a "one-pot multi-enzyme" method with a reversible galactosylation strategy

[0322] 3 grams of core heptasaccharide (II) was incubated with 2 equivalents of UDP-Gal, 10 mM Mg 2+ and B3GalT in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37 °C and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by boiling in a 100 °C water bath for 30 min. The insoluble precipitate was removed by centrifugation. 2 equivalents of UDP-Gal and 10 mM Mn 2+ were added to the supernatant and reacted with B4GalT at 37 °C, and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by boiling in a 100 °C water bath for 30 min. The insoluble precipitate was removed by centrifugation. B3GalH was added to the supernatant and reacted at 30 °C, and monitored by TLC. Once no starting material was observed on the TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation. The supernatant was concentrated and purified using a P2 column to obtain 3.03 grams of the target compound 42 with a yield of 90%.

[0323] For the compound prepared above 1 1H NMR and13 The 13C NMR data are listed in Table 3 below.

[0324] Table 3

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

Claims

1. A method for synthesizing a core pentasaccharide of formula (I) or a core heptasaccharide of formula (II), characterized in that: In formula (I) or formula (II), represents D-mannopyranose, stands for N-acetylglucosamine, β4 means that a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 4-carbon of another sugar through a β-configuration glycosidic bond. β2 means that a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 2-carbon of another sugar through a β-configuration glycosidic bond. α3 means that a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 3-carbon of another sugar through an α-configuration glycosidic bond. α6 means that the hemiacetal hydroxyl group of a sugar is connected to the hydroxyl group on the 6-carbon of another sugar through an α-configuration glycosidic bond; The synthesis method comprises the following steps: S5: Compound 5 reacts with compound c to obtain compound 6, and the reaction formula is as follows: In the formula, R is selected from C6-C95 straight chain or branched alkyl, preferably C6-C50 straight chain or branched alkyl, more preferably C6-C20 straight chain or branched alkyl, and further preferably, R is selected from: More preferably, R is S6: Compound 6 is deacetylated to obtain compound 7, and the reaction formula is as follows: Wherein, R is as defined in step S5; S7: Compound 7 and guanosine diphosphate mannose (GDP-Man) are converted into compound 8 under the action of chitobiose diphosphate polyphenol β-mannosyltransferase (ALG1) or a protein derived from ALG1 that has ALG1 activity and has one or more amino acids substituted, deleted or added in the amino acid sequence of ALG1. The reaction formula is as follows: Wherein, R is as defined in step S5; S8: Under the action of α-1,3 / 1,6-mannosyltransferase (ALG2) or a protein derived from ALG2 having ALG2 activity and having one or more amino acids substituted, deleted or added in the amino acid sequence of ALG2, compound 8 and guanosine diphosphate mannose (GDP-Man) are converted into compound 9, and the reaction formula is shown below: Wherein, R is as defined in step S5; S9: Compound 9 is freed from the lipid tail to obtain the core pentasaccharide represented by formula (I), and the reaction formula thereof is shown below: S10: Under the action of α-1,3-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) or a protein derived from MGAT1 having MGAT1 activity and having substitution, deletion or addition of one or several amino acids in the amino acid sequence of MGAT1, the core pentasaccharide represented by formula (I) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) are converted into compound 28, and the reaction formula is shown below: S11: Under the action of α-1,6-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT2) or a protein derived from MGAT2 having MGAT2 activity and having substitution, deletion or addition of one or several amino acids in the amino acid sequence of MGAT2, compound 28 and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) are converted into the core heptasaccharide represented by formula (II), and the reaction formula is as follows:

2. The method for synthesizing the core pentasaccharide of formula (I) or the core heptasaccharide of formula (II) according to claim 1, characterized in that: Compound 5 was prepared by the following method: S1: Compound 1 reacts with acetic anhydride to generate compound 2, and the reaction formula is as follows: S2: Compound 2 selectively removes the acetyl group at position 1 to obtain compound 3, and the reaction formula is as follows: S3: Compound 3 first reacts with 4,5-dicyanoimidazole and dibenzyl N,N′-diisopropylphosphoramidite, and then reacts with tert-butyl peroxide to obtain compound 4, the reaction formula of which is shown below: S4: Compound 4 is reduced to obtain compound 5, and the reaction formula is as follows:

3. The method for synthesizing the core pentasaccharide of formula (I) or the core heptasaccharide of formula (II) according to claim 2, characterized in that: In step S1, The reaction is carried out in solvent 1; preferably, the solvent 1 is pyridine; The molar ratio of compound 1 to acetic anhydride is 14.5-15.5:1; The reaction is carried out at 20-30° C. with stirring; and / or In step S2: The reaction is carried out in solvent 2; preferably, the solvent 2 is tetrahydrofuran; The reaction is carried out under the action of methanol ammonia; preferably, the concentration of ammonia in methanol ammonia is 6-8 mol / L; the volume ratio of methanol ammonia to solvent 2 is 1:2-4; The reaction is carried out at 20-30°C with stirring; and / or In step S3: The reaction is carried out in solvent 3; preferably, the solvent 3 is acetonitrile; The molar ratio of compound 3 to 4,5-dicyanoimidazole is 1:2.5-3.5; the molar ratio of compound 3 to dibenzyl N,N'-diisopropylphosphoramidite is 1:1.5-2.5; the molar ratio of compound 3 to tert-butyl peroxide is 1:3.5-4.5; The reaction of compound 3 with 4,5-dicyanoimidazole, dibenzyl N,N′-diisopropylphosphoramidite, and the reaction with tert-butyl peroxide are all carried out at 20-30° C. with stirring; preferably, tert-butyl peroxide is added to the reaction system in the form of a decane solution; and / or In step S4: The reaction is carried out in solvent 4; preferably, the solvent 4 is methanol; The reaction is reduced with hydrogen in the presence of palladium carbon; preferably, the mass ratio of compound 4 to palladium carbon is 1:0.05-0.15; The reaction is carried out at 20-30°C with stirring.

4. The method for synthesizing the core pentasaccharide of formula (I) or the core heptasaccharide of formula (II) according to claim 1, characterized in that: In step S5: First, compound 5 is dissolved in solvent 5, and then triethylamine is added to convert it into triethylammonium salt; after the triethylammonium salt is activated with N, N-carbonyldiimidazole in solvent 6, it is reacted with compound c; Preferably, the solvent 5 is methanol; the solvent 6 is N,N-dimethylformamide; The molar ratio of compound 5 to triethylamine is 1.0 g: 0.5-1.5 mL; the molar ratio of compound 5 to N, N-carbonyldiimidazole is 1: 5-6; the molar ratio of compound 5 to compound c is 1: 1.0-1.5; Preferably, the mixture is converted into triethylammonium salt by concentration, and then co-evaporated with toluene to remove excess triethylamine, preferably, co-evaporated with toluene 3 times; Preferably, the reaction conditions for activating the phosphoric acid group with N,N-carbonyldiimidazole are activation at room temperature for 3 to 6 hours; preferably, after the activation reaction is completed, methanol is added to quench the unreacted N,N-carbonyldiimidazole; Preferably, compound c is dissolved in solvent 7 and added to the aforementioned reaction system in the form of a solution; preferably, solvent 7 is dichloromethane; the reaction conditions with compound c are 20-30° C., stirring reaction, and the reaction time is 2-4 days; Preferably, after the reaction is completed, a separation and purification step is further included: the reaction solution is concentrated and purified by column chromatography to obtain compound 6; preferably, the chromatographic solution of the column chromatography is ethyl acetate: methanol: water = 70: 30: 0.3; Preferably, compound c is prepared according to the following method: (a) Compound a is first reacted with 4,5-dicyanoimidazole and dibenzyl N,N′-diisopropylphosphoramidite, and then reacted with tert-butyl peroxide to obtain phosphorylated compound b; (b) Compound b is reduced by hydrogen to obtain compound c; The reaction formula is as follows: Wherein, R is as defined in step S5; Preferably, in step (a): Compound a reacts in solvent 3; preferably, the solvent 3 is acetonitrile; The molar ratio of compound a to 4,5-dicyanoimidazole is 1:2.5-3.5; the molar ratio of compound a to dibenzyl N,N'-diisopropylphosphoramidite is 1:1.5-2.5; the molar ratio of compound a to tert-butyl peroxide is 1:3.5-4.5; The reaction of compound a with 4,5-dicyanoimidazole, dibenzyl N,N′-diisopropylphosphoramidite, and with tert-butyl peroxide are all carried out at 20-30° C. with stirring; preferably, tert-butyl peroxide is added to the reaction system in the form of a decane solution; Preferably, in step (b): Compound b is reduced in methanol. Preferably, compound b is reduced by hydrogen in the presence of a hydrogenation catalyst, and the hydrogenation catalyst is palladium carbon.

5. The method for synthesizing the core pentasaccharide of formula (I) or the core heptasaccharide of formula (II) according to claim 1, characterized in that: In step S6: The reaction is carried out in solvent 8, which is a mixed solvent of dichloromethane and methanol, preferably, the ratio of dichloromethane to methanol is 1:1, v / v; Compound 6 undergoes a deacetylation reaction in the presence of sodium methoxide; the molar ratio of compound 6 to sodium methoxide is 1:1.5-2.5; preferably, sodium methoxide is added to the reaction system in the form of a solution, and the solvent is preferably methanol; The reaction is carried out at 20-30° C. with stirring for 8-14 hours; and / or In step S7: The chitobiose diphosphate polyphenol β-mannosyltransferase (ALG1) is derived from Saccharomyces cerevisiae; preferably, its amino acid sequence is shown in SEQ ID: 1; Reaction in Mg 2+ to be carried out in the presence of; The reaction is carried out at a pH of 6-9, preferably 7.5; and / or In step S8: α-1,3 / 1,6-mannosyltransferase (ALG2) is of human origin; preferably, its amino acid sequence is shown in SEQ ID: 2; Reaction in Mg 2+ to be carried out in the presence of; The reaction is carried out at a pH of 7-8, preferably 7.5; and / or In step S9: The reaction is carried out in an HCl solution; preferably, the concentration of HCl in the HCl solution is 0.5 to 1.5 M; The reaction temperature is 60-80°C, preferably 70°C, and the reaction time is 1.5-2.5h; and / or In step S10: α-1,3-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) is of human origin, and preferably, its amino acid sequence is shown in SEQ ID: 3; Reaction in Mn 2+ Mg 2+ to be carried out in the presence of; The reaction is carried out at a pH of 7-8, preferably 7.5; and / or In step S11: The α-1,6-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT2) is of human origin; preferably, its amino acid sequence is shown in SEQ ID:

4. Reaction in Mn 2+ Mg 2+ to be carried out in the presence of; The reaction is carried out at a pH of 7-8, preferably 7.5; or Steps S7 and S8 are carried out in the same reaction system according to a one-pot method, comprising the following steps: Compound 7 and guanosine diphosphate mannose (GDP-Man) were used as substrates in the presence of metal Mg 2+ In the presence of β-mannosyltransferase (ALG1) or a protein derived from ALG1 having ALG1 activity and having substitution, deletion or addition of one or more amino acids in the amino acid sequence of ALG1 described in step S7 and α-1,3 / 1,6-mannosyltransferase (ALG2) or a protein derived from ALG2 having ALG2 activity and having substitution, deletion or addition of one or more amino acids in the amino acid sequence of ALG2 described in step S8, compound 9 is catalyzed to synthesize, and the reaction formula is as follows: or Steps S10 and S11 are carried out in the same reaction system, comprising the following steps: The core pentasaccharide represented by formula (I), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was used as a substrate in the presence of metal Mg 2+ , Mn 2+ In the presence of α-1,3-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) described in step S10 or a protein derived from MGAT1 having MGAT1 activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of MGAT1, and α-1,6-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT2) described in step S11 or a protein derived from MGAT2 having MGAT2 activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of MGAT2, catalyzes the synthesis of the core heptasaccharide represented by formula (II), and the reaction formula is as follows:

6. A method for preparing N-glycan represented by formula (III), characterized in that: In formula (III), represents D-mannopyranose, stands for N-acetylglucosamine, stands for N-acetylneuraminic acid, represents D-galactopyranose, represents L-fucopyranose, β4 means that a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 4-carbon of another sugar through a β-configuration glycosidic bond. β2 means that a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 2-carbon of another sugar through a β-configuration glycosidic bond. α3 means that a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 3-carbon of another sugar through an α-configuration glycosidic bond. α6 means that the hemiacetal hydroxyl group of a sugar is connected to the hydroxyl group on the 6-carbon of another sugar through an α-configuration glycosidic bond; ---Indicates whether it contains or does not contain the above monosaccharide structure; The method comprises: on the basis of the synthesis method of the core pentasaccharide of formula (I) or the core heptasaccharide of formula (II) in the N-glycan of any one of claims 1 to 6, extending the core pentasaccharide or the core heptasaccharide by enzymatic reaction or chemical reaction to prepare the biantennary N-glycan of formula (III); Wherein, the enzymatic reaction or chemical reaction is selected from one or more of the following: (1) N-glycan reacts with uridine diphosphogalactose (UDP-Gal) under the action of β1,3-galactosyl transferase (B3GalT) or a protein derived from B3GalT having B3GalT activity and in which one or several amino acids are substituted, deleted or added in the amino acid sequence of B3GalT, thereby adding galactose C linked by a β1,3-glycosidic bond to N-acetylglucosamine A at the antenna end of the N-glycan; Preferably, the B3GalT is derived from Chromobacterium violaceum; preferably, its amino acid sequence is shown in SEQ ID: 5; Preferably, the enzymatic reaction is carried out in Mg 2+ The reaction is carried out in the presence of; the pH value of the reaction system is 6 to 9, preferably 7.5; (2) under the action of β1,4-galactosyl transferase (B4GalT) or a protein derived from B4GalT having B4GalT activity and having one or more amino acids substituted, deleted or added in the amino acid sequence of B4GalT, the N-glycan reacts with uridine diphosphogalactose (UDP-Gal), and galactose C and / or D linked by a β1,4-glycosidic bond are added to the N-acetylglucosamine group A and / or B at the antenna end of the N-glycan; Preferably, the B4GalT is of human origin; preferably, its amino acid sequence is shown in SEQ ID: 6; Preferably, the enzymatic reaction is carried out in the presence of Mn 2+ The reaction is carried out in the presence of; the pH value of the reaction system is 6 to 9, preferably 7.5; (3) under the action of α2,6-sialyltransferase (Pd2,6ST) or a protein derived from Pd2,6ST having Pd2,6ST activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of Pd2,6ST, or under the action of α2,6-sialyltransferase (ST6Gal1) or a protein derived from ST6Gal1 having ST6Gal1 activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of ST6Gal1, N-glycan reacts with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F linked with α2,6-glycosidic bond are added to galactosyl C and / or D at the antenna end of N-glycan; Preferably, the Pd2,6ST is derived from Photobacterium damsela; preferably, its amino acid sequence is shown in SEQ ID: 7; the ST6Gal1 is derived from human, preferably, its amino acid sequence is shown in SEQ ID: 8; Preferably, the enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of a pH value of 6 to 9, preferably 7.5; (4) under the action of α2,3-sialyltransferase (BtST) or a protein derived from BtST having BtST activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of BtST, or under the action of α2,3-sialyltransferase (PPST) or a protein derived from PPST having PPST activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of PPST, N-glycan reacts with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F linked with α2,3-glycosidic bond are added to galactosyl C and / or D at the antenna end of N-glycan; Preferably, the BtST is derived from Bibersteinia trehalosi, and preferably, its amino acid sequence is shown in SEQ ID: 9; the PPST is derived from Photobacterium phosphoreum, and preferably, its amino acid sequence is shown in SEQ ID: 10; Preferably, the enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of a pH value of 6 to 9, preferably 7.5; (5) under the action of α1,3-fucosyltransferase (FucT) or a FucT-derived protein having FucT activity and in which one or more amino acids are substituted, deleted or added in the amino acid sequence of FucT, the N-glycan reacts with guanosine diphosphate-fucose (GDP-Fucose), and fucose G and / or H linked by α1,3-glycosidic bonds are added to the N-acetylglucosamine groups A and / or B on the antenna of the N-glycan; Preferably, the FucT is derived from Helicobacter pylori; preferably, its amino acid sequence is shown in SEQ ID: 11; Preferably, the enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of a pH value of 6 to 9, preferably 7.5; (6) removing galactose C linked to N-acetylglucosamine A by a β1,3-glycosidic bond at the antenna end of the N-glycan under the action of β1,3-galactosylation hydrolase (B3GalH) or a protein derived from B3GalH that has substituted, deleted or added one or more amino acids in the amino acid sequence of B3GalH and has B3GalH activity; Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably 7.0; (7) removing galactose C and / or D linked to N-acetylglucosamine A and / or B by β1,4-glycosidic bond at the antenna end of N-glycan under the action of β1,4-galactosylation hydrolase (B4GalH) or a protein derived from B4GalH having B4GalH activity and having one or more amino acids substituted, deleted or added in the amino acid sequence of B4GalH; Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably 7.0; (8) removing N-acetylglucosamine A and / or B linked to galactose by β1,2-glycosidic bond at the antenna end of N-glycan under the action of β1,2-N-acetylglucosamine hydrolase (GlcNAcH) or a GlcNAcH-derived protein having GlcNAcH activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of GlcNAcH; Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably 7.

0. (9) removing N-acetylneuraminic acid E and / or F linked to galactosyl C and / or D by α2,3-glycosidic bond at the end of N-glycan antenna by the action of α2,3-sialyl hydrolase (NanC) from Streptococcus penumoniae or a protein derived from NanC with NanC activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of NanC; Preferably, the NanC is derived from Streptococcus penumoniae; Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably about 7.0; (10) removing fucose G and / or H from N-acetylglucosamine groups A and / or B on the antenna of N-glycans linked by α1,3-glycosidic bonds under the action of α1,3-fucosyl hydrolase (FucH) or a protein derived from FucH having FucH activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of FucH; Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably about 7.0; (11) under the action of acetic acid, N-acetylneuraminic acid E and / or F connected to galactosyl C or D by α2,6-glycosidic bond at the end of N-glycan antenna is removed; Preferably, the acetic acid is in the form of an aqueous solution of acetic acid with a concentration of 1.5-2.5 M; the reaction temperature is 70-90° C.; Preferably, after the reaction is completed, sodium bicarbonate is added to neutralize the reaction, and the mixture is concentrated and purified using a P2 column to obtain the target product.

7. The method for preparing N-glycan represented by formula (III) according to claim 6, characterized in that: The N-glycan represented by formula (III) is selected from the compounds represented by the following structures:

8. The method for preparing N-glycan represented by formula (III) according to claim 6 or 7, characterized in that: The method for preparing the N-glycan represented by (III) comprises the following steps: (a) using the core heptasaccharide represented by formula (II) as a substrate, adding a galactose C linked by a β1,3-glycosidic bond to the N-acetylglucosamine A at the antenna end of the core heptasaccharide represented by formula (II) under the action of β1,3-galactosyl transferase (B3GalT) or a protein derived from B3GalT having B3GalT activity and having substitution, deletion or addition of one or several amino acids in the amino acid sequence of B3GalT, to obtain compound 40; (b) adding a galactose D linked by a β1,4-glycosidic bond to N-acetylglucosamine B at the antenna end of compound 40 under the action of β1,4-galactosyl transferase (B4GalT) or a protein derived from B4GalT having B4GalT activity and having substitution, deletion or addition of one or several amino acids in the amino acid sequence of B4GalT, to obtain compound 41; (c) Under the action of β1,3-galactosylation hydrolase (B3GalH) or a protein derived from B4GalH having B4GalH activity and in which one or more amino acids are substituted, deleted or added in the amino acid sequence of B4GalH, the terminal galactose C linked by a β1,3-glycosidic bond is removed to obtain asymmetric N-glycan 42.

9. The method for preparing N-glycan represented by formula (III) according to any one of claims 6 to 8, characterized in that: The method for preparing the N-glycan represented by (III) further comprises the following steps: (d) reacting the compound of formula (II), compound 28, compound 43, compound 49, compound 53, compound 56, compound 61, compound 64 with uridine diphosphogalactose (UDP-Gal) under the action of β1,4-galactosyl transferase (B4GalT) or a protein derived from B4GalT having B4GalT activity and in which one or more amino acids are substituted, deleted or added in the amino acid sequence of B4GalT, to add galactose linked by a β1,4-glycosidic bond to the N-acetylglucosamine group at the antenna end of the N-glycan to obtain compound 30, compound 35, compound 44, compound 50, compound 54, compound 57, compound 62, and compound 65, respectively; (e) under the action of α2,6-sialyltransferase (Pd2,6ST) or a protein derived from Pd2,6ST having Pd2,6ST activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of Pd2,6ST, or α2,6-sialyltransferase (ST6Gal1) or a protein derived from ST6Gal1 having ST6Gal1 activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of ST6Gal1, compound 30, compound 35, compound 42, compound 44, compound 57, and compound 72 react with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac) to add N-acetylneuraminic acid linked by an α2,6-glycosidic bond to the galactose group at the antenna end of the N-glycan to obtain compound 33, compound 38, compound 61, compound 48, compound 68, and compound 77, respectively; (f) under the action of α2,3-sialyltransferase (BtST) or a protein derived from BtST having BtST activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of BtST, or under the action of α2,3-sialyltransferase (PPST) or a protein derived from PPST having PPST activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of PPST, Compound 52, compound 57, compound 65, compound 68, compound 70, compound 72, and compound 74 were reacted with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac) to add N-acetylneuraminic acid linked by α2,3-glycosidic bond to the galactose group at the antenna end of N-glycan to obtain compound 32, compound 34, compound 37, compound 39, compound 43, compound 46, compound 53, compound 58, compound 66, compound 69, compound 71, compound 75, and compound 76, respectively; (g) under the action of α1,3-fucosyltransferase (FucT) or a FucT-derived protein having FucT activity and in which one or more amino acids are substituted, deleted or added in the amino acid sequence of FucT, compound 31, compound 35, compound 51, compound 54, compound 42, compound 62, compound 65 and compound 72 react with guanosine diphosphate-fucose (GDP-Fucose) to add fucose linked by an α1,3-glycosidic bond to the N-acetylglucosamine group on the antenna of the N-glycan to obtain compound 32, compound 36, compound 52, compound 55, compound 56, compound 70, compound 67 and compound 74; (h) removing galactose linked to N-acetylglucosamine by a β1,3-glycosidic bond at the antenna end of compound 41 under the action of β1,3-galactosylation hydrolase (B3GalH) or a protein derived from B3GalH having B3GalH activity and in which one or more amino acids are substituted, deleted or added in the amino acid sequence of B3GalH, to obtain compound 42; (i) removing galactose linked to N-acetylglucosamine by β1,4-glycosidic bond at the antenna end of compound 49, compound 59 or compound 72 under the action of β1,4-galactosylating hydrolase (B4GalH) or a protein derived from B4GalH having B4GalH activity and in which one or more amino acids are substituted, deleted or added in the amino acid sequence of B4GalH, to obtain compound 50, compound 60 and compound 73, respectively; (j) removing N-acetylglucosamine linked to galactose by a β1,2-glycosidic bond at the antenna end of compound 42 under the action of β1,2-N-acetylglucosamine hydrolase (GlcNAcH) or a protein derived from GlcNAcH having GlcNAcH activity and having substitution, deletion or addition of one or more amino acids in the amino acid sequence of GlcNAcH, to obtain compound 72; (k) removing N-acetylneuraminic acid linked to the galactosyl group via an α2,3-glycosidic bond at the antenna end of compound 45 or compound 48 under the action of α2,3-sialyl hydrolase (NanC) from Streptococcus penumoniae or a protein derived from NanC having NanC activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of NanC, to obtain compound 47 or compound 49, respectively; (l) under the action of α1,3-fucosyl hydrolase (FucH) or a protein derived from FucH having FucH activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of FucH, the fucose on the N-acetylglucosamine group on the antenna of the N-glycan of compound 58 is removed by α1,3-glycosidic bond to obtain compound 59: (m) Under the action of acetic acid, N-acetylneuraminic acid connected to the galactosyl group via an α2,6-glycosidic bond at the antenna end of compound 50 was removed to obtain compound 51.

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