Chemical enzymatic total synthesis method of N-glycan

Abstract

The invention provides a chemical enzymatic total synthesis method of N-glycan. Specifically, N-sugar core tetrasaccharide shown in a formula (I) and core hexasaccharide shown in a formula (II) are prepared from monosaccharide through a chemical enzymatic synthesis strategy, and various symmetric or asymmetric N-glycans 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.

Description

Technical Field

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

[0002] Protein N-glycosylation, the attachment of N-acetylglucosamine (GlcNAc) to the nitrogen atom of the asparagine (Asn) side chain via a β-N-linkage, is one of the most common post-translational modifications of proteins. N-glycosylation occurs in most organisms, and the structure of N-glycans is highly conserved throughout evolution. All naturally occurring N-glycans contain a conserved structure, known as the core pentasaccharide, to which varying numbers of monosaccharides can be added or removed, resulting in a vastly diverse array of N-glycan structures within living cells.

[0003] Glycosylation is a frequent and complex modification of proteins that is essential for both single-cell and multicellular life. It regulates a wide range of cellular functions, from protein folding, trafficking, sorting, localization, half-life, and signaling to proliferation, migration, and adhesion to the surrounding environment. For therapeutic antibodies, different glycosylation modifications can have a significant impact on antibody function. For example, knocking out core fucose can greatly enhance antibody-dependent cell-mediated cytotoxicity (ADCC) activity, while high galactose levels can enhance the ability of antibodies to bind to FcγRs.

[0004] Glycoproteins and glycopeptides, which are involved in carbohydrate-carbohydrate, carbohydrate-protein, and carbohydrate-lipid interactions commonly involved in biological processes, have attracted significant research interest due to their potential therapeutic applications. Currently, natural glycoproteins are an important source of templates for the design and development of molecules with therapeutic applications. However, because these molecules exist in heterogeneous environments and at extremely low concentrations, the isolation of large quantities of glycoproteins with the desired purity from biological sources is extremely complex. As an alternative approach to address this problem, the chemical synthesis of glycoproteins has been developed. Within this context, several methods for the synthesis of glycopeptides in solution and / or solid phase have been reported. In most of these methods, glycosylated amino acid derivatives are used as building blocks for solution and solid phase synthesis. The synthetic activity of glycoproteins is a key parameter that allows their use as pharmaceuticals to mitigate the effects of microbial drug resistance and / or cancer. However, the chemical synthesis of glycoproteins is extremely challenging because these molecules possess multiple reactive sites and highly specific stereochemistry. Consequently, the design and implementation of synthetic routes require multiple protection schemes.

[0005] The core tetrasaccharide, formed by removing one reducing end N-acetylglucosamine from the core pentasaccharide, is another important precursor for the synthesis of truncated N-glycans. Although these truncated glycans are not complete N-glycan structures, they can be chemically converted to oxazoline substrates, which are then transferred to the corresponding structures by mutant Endo enzymes. Currently, oxazoline substrates are widely used to synthesize homogeneous glycopeptides, glycoproteins, or homogenized cells using mutant endonases. Summary of the Invention

[0006] 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 efficiently assembles from common starting materials and only requires a few simple chemical reactions and enzymatic reaction steps to synthesize N-sugar core tetrasaccharides and core hexasaccharides (multi-gram levels); next, the core tetrasaccharides and core hexasaccharides are extended by glycosyltransferases to produce a variety of symmetrical and asymmetrical 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 prepared 14 complex asymmetric N-glycans. The method of the present invention can prepare N-glycans on a large scale and efficiently, and does not require tedious purification operations, and has the characteristics of low cost and strong adaptability.

[0007] In view of this, in a first aspect, the present invention provides a method for synthesizing a core tetrasaccharide represented by formula (I), comprising the following steps: S9: under the action of β-N-acetylglucosaminyl endoside enzyme (Endo enzyme) or a protein derived from Endo enzyme that has Endo enzyme activity and has one or more amino acids substituted, deleted or added in the amino acid sequence of Endo enzyme, compound 9 is desorbed from compound 10 to convert it into a core tetrasaccharide represented by formula (I), the reaction formula of which is as follows:

[0008]

[0009] Where,

[0010] R is a C6-C95 linear or branched alkyl, preferably a C6-C50 linear or branched alkyl, more preferably a C6-C20 linear or branched alkyl, and further preferably, R is selected from: More preferably, R is

[0011] represents D-mannopyranose,

[0012] stands for N-acetylglucosamine,

[0013] 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.

[0014] 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.

[0015] α6 indicates 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.

[0016] In some embodiments, the β-N-acetylglucosamine endonuclease (Endo enzyme) can be selected from: β-N-acetylglucosamine endonuclease S (Endo S), β-N-acetylglucosamine endonuclease CC (Endo CC), β-N-acetylglucosamine endonuclease M (Endo M), β-N-acetylglucosamine endonuclease D (Endo D), etc.

[0017] In some embodiments, the β-N-acetylglucosaminidase endo S (Endo S) can be derived from Streptococcus pyogenes.

[0018] In some embodiments, the endo-β-N-acetylglucosaminidase CC (Endo CC) can be derived from Coprinopsis cinerea.

[0019] In some embodiments, the endo-β-N-acetylglucosaminidase M (Endo M) can be derived from Mucor hiemalis.

[0020] In some embodiments, the endo-β-N-acetylglucosaminidase D (Endo D) is derived from Streptococcus pneumonia.

[0021] In some embodiments, the reaction can be carried out in Mg 2+ In the presence of.

[0022] In some embodiments, the reaction can be carried out at a pH value of 6-9, preferably a pH value of 7-8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably 7.0.

[0023] In some embodiments, compound 9 can be prepared by a method comprising the following steps:

[0024] S8: Compound 9 was synthesized from Compound 8 under the action of α-1,3 / 1,6-mannosyltransferase (ALG2) or a protein derived from ALG2 that has ALG2 activity and has one or more amino acids substituted, deleted, or added in its amino acid sequence:

[0025]

[0026] In the formula, the definitions of various symbols are the same as above.

[0027] In some embodiments, in S8, the α-1,3 / 1,6-mannosyltransferase (ALG2) may be of human origin; preferably, its amino acid sequence is as shown in SEQ ID NO: 2.

[0028] In some embodiments, in S8, the reaction can be carried out in Mg 2+ In the presence of.

[0029] In some embodiments, in S8, the reaction can be carried out at a pH value of 7-8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., preferably 7.4-7.5.

[0030] In some embodiments, compound 8 can be prepared by a method comprising the following steps:

[0031] S7: Compound 8 was synthesized from Compound 7 under the action of chitobiosyl 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 its amino acid sequence:

[0032]

[0033] In the formula, the definitions of various symbols are the same as above.

[0034] In some embodiments, in S7, the chitobiosyl diphosphate polyphenol β-mannosyltransferase (ALG1) may be derived from Saccharomyces cerevisiae; in particular, its amino acid sequence is shown in SEQ ID NO: 1.

[0035] In some embodiments, in S7, the reaction can be carried out in Mg 2+ In the presence of.

[0036] In some embodiments, in S7, the reaction can be carried out at a pH value of 6-9, such as 6.5, 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, 8.5, etc., preferably 7.4-7.5.

[0037] In some embodiments, the present invention provides a one-pot method for synthesizing the core tetrasaccharide of formula (I), that is, the above steps S7, S8 and S9 are carried out in the same reaction system according to the one-pot method, comprising the following steps: compound 7 and guanosine diphosphate mannose (GDP-Man) are used as substrates, and in the presence of metal Mg 2+ In the presence of chitobiose diphosphate polyphenol β-mannosyltransferase (ALG1) or a protein derived from ALG1 having ALG1 activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of ALG1, and α-1,3 / 1,6-mannosyltransferase (ALG2) or a protein derived from ALG2 having ALG2 activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of ALG2, catalyzes the synthesis of compound 8 to obtain compound 9. Compound 10 is then removed under the action of β-N-acetylglucosaminyl endo-enzyme (Endo-enzyme) or a protein derived from Endo-enzyme having Endo-enzyme activity by substitution, deletion or addition of one or several amino acids in the amino acid sequence of Endo-enzyme, to catalyze the synthesis of the core tetrasaccharide represented by formula (I). The reaction formula is as follows:

[0038]

[0039] In the formula, the definitions of various symbols are the same as above.

[0040] In some embodiments, compound 7 can be prepared by a method comprising the following steps:

[0041] S6: Compound 7 is synthesized from compound 6, and the reaction formula is as follows:

[0042]

[0043] In the formula, the definitions of various symbols are the same as above.

[0044] In some embodiments, in S6, the reaction is carried out in solvent 8, preferably, solvent 8 is a mixed solvent of dichloromethane and methanol, preferably, the volume ratio of dichloromethane to methanol is 1:1;

[0045] Preferably, compound 6 undergoes deacetylation in the presence of sodium methoxide; preferably, the molar ratio of compound 6 to sodium methoxide is 1:1.5 to 2.5; preferably, sodium methoxide is added to the reaction system in the form of a solution, and the solvent is preferably methanol;

[0046] Preferably, the reaction is carried out at 20-30° C. with stirring, and the reaction time is 8-14 h.

[0047] In some embodiments, after the reaction is completed, a separation and purification step is further included. Preferably, the separation and purification step includes neutralizing the reaction solution with a hydrogen-type ion exchange resin and then filtering, and concentrating the filtrate to obtain compound 7.

[0048] In some embodiments, compound 6 can be prepared by a method comprising the following steps:

[0049] S5: Compound 6 is synthesized from compound 5 and compound c, and the reaction formula is shown below:

[0050]

[0051] Wherein, the definition of R is the same as above.

[0052] In some embodiments, in S5, compound 5 is dissolved in solvent 5, then triethylamine is added to convert it into a triethylammonium salt. The triethylammonium salt is activated with N,N-carbonyldiimidazole in solvent 6 for phosphate group activation, and then reacted with compound c to obtain compound 7. Preferably, solvent 5 can be methanol; solvent 6 can be N,N-dimethylformamide. Preferably, the ratio of compound 5 to triethylamine can be 1.0 g:0.5-1.5 mL; the molar ratio of compound 5 to N,N-carbonyldiimidazole can be 1:5-6; and the equivalent ratio of compound 5 to compound c can be 1:1.0-1.5. Preferably, the compound is converted into a triethylammonium salt by concentration, and then co-evaporated with toluene to remove excess triethylamine, preferably three times. Preferably, the reaction conditions for N,N-carbonyldiimidazole activation of the phosphate group are room temperature for 3-6 hours. Preferably, after the activation reaction is completed, methanol is added to quench unreacted N,N-carbonyldiimidazole. Preferably, compound c is dissolved in solvent 7 and added to the aforementioned reaction system as a solution. Preferably, solvent 7 can be dichloromethane. The reaction conditions with compound c can be 20-30°C, stirring, and the reaction time can be 2-4 days. Preferably, after the reaction is completed, a separation and purification step is further included; preferably, the separation and purification step includes concentrating the reaction solution and purifying it by column chromatography to obtain compound 6; preferably, the column chromatography solvent is ethyl acetate:methanol:water = 70:30:0.3.

[0053] Preferably, compound c can be prepared according to the following method:

[0054] (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;

[0055] (b) Compound b is reduced by hydrogen to obtain compound c;

[0056] The reaction formula is as follows:

[0057]

[0058] Wherein, R is as defined in step S5;

[0059] Preferably, in step (a):

[0060] In some embodiments, compound a is reacted in solvent 3; preferably, the solvent 3 is acetonitrile.

[0061] 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; and the molar ratio of compound a to tert-butyl peroxide is 1:3.5-4.5.

[0062] In some embodiments, the reaction of compound a 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; preferably, the reaction endpoint is monitored by thin layer chromatography.

[0063] In some embodiments, after the reaction is completed, a separation and purification step is further included. Preferably, the separation and purification step comprises filtering the reaction solution through diatomaceous earth, concentrating it, diluting it with dichloromethane, collecting the organic phase after washing, drying, concentrating it, and purifying it by column chromatography to obtain compound b; preferably, washing can be washing with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence; preferably, drying is drying with anhydrous sodium sulfate; preferably, the column chromatography solvent is dichloromethane / methanol = 85:5, v / v.

[0064] Preferably, in step (b):

[0065] In some embodiments, 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 on carbon.

[0066] In some embodiments, after the reaction is completed, a separation and purification step is further included. Preferably, the separation and purification step includes filtering the reaction solution through diatomaceous earth and then concentrating it to obtain compound c.

[0067] On the other hand, the present invention provides a method for synthesizing a core hexasaccharide of formula (II) using a core tetrasaccharide of formula (I), the method comprising the following steps:

[0068] 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 more amino acids in the amino acid sequence of MGAT1, the core tetrasaccharide represented by formula (I) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) are converted into compound 28, and the reaction formula is shown below:

[0069]

[0070] S11: Compound 28 and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) are converted into the core hexasaccharide represented by formula (II) under the action of α-1,6-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT2) or a protein derived from MGAT2 with MGAT2 activity and substitution, deletion, or addition of one or more amino acids in the amino acid sequence of MGAT2. The reaction formula is as follows:

[0071]

[0072] in,

[0073] 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.

[0074] The definitions of other symbols are the same as above.

[0075] In some embodiments, in S10, the α-1,3-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) may be of human origin, and in particular, its amino acid sequence is shown in SEQ ID NO: 3.

[0076] In some embodiments, in S10, the reaction can be carried out under Mn 2+ Mg 2+ In the presence of.

[0077] In some embodiments, in S10, the reaction is carried out at a pH value of 7-8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., preferably 7.4-7.5.

[0078] In some embodiments, in S11, the α-1,6-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT2) may be of human origin; in particular, its amino acid sequence is shown in SEQ ID NO: 4.

[0079] In some embodiments, in S11, the reaction is carried out under Mn 2+Mg 2+ In the presence of.

[0080] In some embodiments, in S11, the reaction is carried out at a pH value of 7-8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., preferably 7.4-7.5.

[0081] In some embodiments, steps S10 and S11 can be performed separately or in the same reaction system according to a one-pot method.

[0082] In some embodiments, steps S10 and S11 are performed in the same reaction system. In this case, the core tetrasaccharide represented by formula (I), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), can be 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-acetylglucosaminyltransferase (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, and α-1,6-mannose glycoprotein 2-β-N-acetylglucosaminyltransferase (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, catalyzes the synthesis of the core hexasaccharide GlcNAc2-Man3-GlcNAc represented by formula (II), and the reaction formula is as follows:

[0083]

[0084] In the formula, the definitions of various symbols are the same as above.

[0085] In another aspect, the present invention provides a method for preparing N-glycans represented by formula (III),

[0086]

[0087] In formula (III),

[0088] represents D-mannopyranose,

[0089] stands for N-acetylglucosamine,

[0090] stands for N-acetylneuraminic acid,

[0091] represents D-galactopyranose,

[0092] represents L-fucopyranose,

[0093] 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.

[0094] 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.

[0095] 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.

[0096] α6 means that the hemiacetal hydroxyl group of one sugar is connected to the hydroxyl group on the 6-carbon of another sugar through an α-configuration glycosidic bond;

[0097] ---Indicates whether it contains or does not contain the above monosaccharide structure;

[0098] The method comprises: extending the core tetrasaccharide represented by formula (I) or the core hexasaccharide represented by formula (II) by an enzymatic reaction or a chemical reaction to prepare an N-glycan represented by formula (III);

[0099] Wherein, the enzymatic reaction or chemical reaction is selected from one or more of the following:

[0100] (1) N-glycans react with uridine diphosphogalactose (UDP-Gal) under the action of β1,3-galactosyltransferase (B3GalT) or a protein derived from B3GalT that has B3GalT activity and has one or more amino acids substituted, deleted, or added in its amino acid sequence, thereby adding galactose C linked to N-acetylglucosamine A at the antenna end of the N-glycan via a β1,3-glycosidic bond;

[0101] Preferably, the B3GalT is derived from Chromobacterium violaceum; in particular, its amino acid sequence is shown in SEQ ID NO: 5;

[0102] Preferably, the above 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 the pH value is 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.5;

[0103] (2) N-glycans react with uridine diphosphogalactose (UDP-Gal) under the action of β1,4-galactosyltransferase (B4GalT) or a protein derived from B4GalT that has one or more amino acids substituted, deleted, or added in the amino acid sequence of B4GalT and has B4GalT activity, thereby adding galactose C and / or D linked by a β1,4-glycosidic bond to the N-acetylglucosamine group A and / or B at the antenna end of the N-glycan;

[0104] Preferably, the B4GalT is of human origin; in particular, its amino acid sequence is shown in SEQ ID NO: 6;

[0105] Preferably, the above 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 the pH value is 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.5;

[0106] (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), thereby adding N-acetylneuraminic acid E and / or F linked by an α2,6-glycosidic bond to the galactosyl C and / or D at the antennal end of the N-glycan;

[0107] Preferably, the Pd2,6ST is derived from Photobacterium damselae; in particular, its amino acid sequence is shown in SEQ ID NO: 7; the ST6Gal1 is derived from human, in particular, its amino acid sequence is shown in SEQ ID NO: 8;

[0108] Preferably, the above enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of conditions, the pH value of the reaction system is 6 to 9, preferably the pH value is 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.5;

[0109] (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), thereby adding N-acetylneuraminic acid E and / or F linked by an α2,3-glycosidic bond to the galactosyl C and / or D at the antenna end of the N-glycan;

[0110] Preferably, the BtST is derived from Bibersteinia trehalosi, and in particular, its amino acid sequence is shown in SEQ ID NO: 9; the PPST is derived from Photobacterium phosphoreum, and in particular, its amino acid sequence is shown in SEQ ID NO: 10;

[0111] Preferably, the above enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of the conditions; the pH value of the reaction system is 6 to 9, preferably the pH value is 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.5;

[0112] (5) Under the action of α1,3-fucosyltransferase (FucT) or a FucT-derived protein having FucT activity and having one or more amino acids substituted, deleted, or added in the amino acid sequence of FucT, 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;

[0113] Preferably, the FucT is derived from Helicobacter pylori; in particular, its amino acid sequence is shown in SEQ ID NO: 11;

[0114] Preferably, the above enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of the conditions; the pH value of the reaction system is 6 to 9, preferably the pH value is 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.5;

[0115] (6) β1,3-galactosylation hydrolase (B3GalH) or a protein derived from B3GalH that has one or more amino acids substituted, deleted, or added in the amino acid sequence of B3GalH and has B3GalH activity, removes galactose C from the antenna end of the N-glycan that is linked to N-acetylglucosamine A by a β1,3-glycosidic bond;

[0116] Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably pH 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.0;

[0117] (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 that has one or more amino acids substituted, deleted or added in the amino acid sequence of B4GalH and has B4GalH activity;

[0118] Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably pH 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.0;

[0119] (8) 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 one or more amino acids substituted, deleted or added in the amino acid sequence of NanC and having NanC activity;

[0120] Preferably, the NanC is derived from Streptococcus penumoniae;

[0121] Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably pH 7 to 8, such as 7.0, 7.2, 7.4, 7.5, 7.8, 8.0, etc., more preferably about 7.0.

[0122] The above steps (1) to (8) may further include a separation and purification step. Specifically, the separation and purification step includes: after the reaction is completed, adding ethanol, solid-liquid separation (for example, 10000g, centrifugation for 10 minutes), and concentrating and purifying the supernatant using a P2 column to obtain the target product.

[0123] In some embodiments, the N-glycan represented by formula (III) is selected from the compounds represented by the following structures:

[0124]

[0125] In some embodiments, the above method for preparing the N-glycan represented by (III) comprises the following steps:

[0126]

[0127] (a) using the core hexasaccharide represented by formula (II) as a substrate, a galactose C linked by a β1,3-glycosidic bond is added to the N-acetylglucosamine A at the antenna end of the core hexasaccharide represented by formula (II) under the action of β1,3-galactosyltransferase (B3GalT) or a protein derived from B3GalT that has B3GalT activity and has one or more amino acids substituted, deleted, or added in the amino acid sequence of B3GalT, to obtain compound 35;

[0128] (b) adding a galactose D linked by a β1,4-glycosidic bond to the N-acetylglucosamine B at the antennal end of compound 35 under the action of β1,4-galactosyltransferase (B4GalT) or a protein derived from B4GalT that has B4GalT activity and has one or more amino acids substituted, deleted, or added in the amino acid sequence of B4GalT to obtain compound 36;

[0129] (c) Under the action of β1,3-galactosylation hydrolase (B3GalH) or a protein derived from B3GalH with B3GalH activity and substitution, deletion, or addition of one or more amino acids in the amino acid sequence of B3GalH, the terminal β1,3-glycosidic-linked galactose C is removed to obtain asymmetric biantennary N-glycan 37.

[0130] In some embodiments, the above method for preparing the biantennary N-glycan represented by (III) further comprises the method represented by the following reaction formula:

[0131]

[0132] The specific steps include:

[0133] (d) reacting the compound of formula (II), compound 35, or compound 38 with uridine diphosphogalactose (UDP-Gal) in the presence of β1,4-galactosyltransferase (B4GalT) or a protein derived from B4GalT having B4GalT activity and having one or more amino acids substituted, deleted, or added in its amino acid sequence to add galactose linked by a β1,4-glycosidic bond to the N-acetylglucosamine group at the antennal end of the N-glycan to obtain compound 30, compound 36, or compound 39, respectively;

[0134] (e) Compound 30 or Compound 39 reacts with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac) in the presence 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 more amino acids in the amino acid sequence of Pd2,6ST, or in the presence of α2,6-sialyltransferase (ST6Gal1) or a protein derived from ST6Gal1 having ST6Gal1 activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of ST6Gal1, to add N-acetylneuraminic acid linked to the galactose group at the antennal end of the N-glycan via an α2,6-glycosidic bond to obtain Compound 33 or Compound 40;

[0135] (f) Compound 30, Compound 31 or Compound 37 reacts with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac) in the presence 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 in the presence 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, to add N-acetylneuraminic acid linked by an α2,3-glycosidic bond to the galactose group at the antennal end of the N-glycan to obtain Compound 34, Compound 32 or Compound 38;

[0136] (g) Compound 30 reacts with guanosine diphosphate-fucose (GDP-Fucose) under the action of α1,3-fucosyltransferase (FucT) or a FucT-derived protein having FucT activity and having one or more amino acids substituted, deleted, or added in the amino acid sequence of FucT, thereby adding fucose linked to the N-acetylglucosamine group on the antenna of the N-glycan via an α1,3-glycosidic bond to obtain Compound 31;

[0137] (h) removing galactose linked to N-acetylglucosamine via a β1,3-glycosidic bond at the antenna end of compound 36 under the action of β1,3-galactosylation hydrolase (B3GalH) or a protein derived from B3GalH having B3GalH activity and having substitution, deletion, or addition of one or more amino acids in the amino acid sequence of B3GalH, to obtain compound 37;

[0138] (i) removing galactose linked to N-acetylglucosamine via a β1,4-glycosidic bond at the antenna end of compound 36 or compound 41 under the action of β1,4-galactosylating 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, to obtain compound 37 or compound 42, respectively;

[0139] (j) Under the action of α2,3-sialyl hydrolase (NanC) from Streptococcus penumoniae or a protein derived from NanC with NanC activity and substitution, deletion or addition of one or more amino acids in the amino acid sequence of NanC, N-acetylneuraminic acid linked to the galactosyl group via an α2,3-glycosidic bond at the antennal end of compound 40 was removed to obtain compound 41. DETAILED DESCRIPTION

[0140] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the above prior art or invention summary or the following examples.

[0141] Unless otherwise expressly stated, numerical ranges throughout this application include any subranges therein and any numerical values ​​in increments of the smallest subunit of a given value therein. Unless otherwise expressly stated, numerical values ​​throughout this application represent approximate measures or limits of the range of embodiments that include minor deviations from the given value and have approximately the stated value as well as the stated exact value. Except for the working examples provided at the end of the detailed description, all numerical values ​​for parameters (e.g., quantities or conditions) in this application (including the appended claims) should be understood in all cases as being modified by the term "approximately", regardless of whether "approximately" actually appears before the value. "Approximately" means that the stated value allows for slight imprecision (some approach to exactness in the value; approximately or reasonably close to the value; approximately). If the imprecision provided by "approximately" is not understood in this ordinary sense in the art, "approximately" as used herein at least represents the variation that can be produced by ordinary methods of measuring and using these parameters. For example, "approximately" 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%.

[0142] The present invention successfully developed a universal method for efficiently preparing a variety of symmetrical and asymmetrical N-glycans. The method comprises: preparing N-glycan core tetrasaccharides and core hexasaccharides from monosaccharides through a chemoenzymatic synthesis strategy, and utilizing enzymatic synthesis and reversible galactosylation as a selective protection strategy to obtain a variety of symmetrical and asymmetrical N-glycans. Several phosphorylated disaccharides GlcNAc2-PP-lipid (Compound 5) with substrates having different lipid tails (C6, C7, C8, C9, C10, C11, C20) were synthesized through a few simple chemical steps. Yeast-derived ALG1 and ALG2 were used to generate Man3-GlcNAc2-PP-lipid (Compound 9) through an enzymatic reaction. Compound 10 was then removed by Endo enzyme to obtain the core tetrasaccharide Man3-GlcNAc (compound represented by Formula (I)). In the presence of MGAT1 and MGAT2, an enzymatic reaction was performed to generate the core hexasaccharide GlcNAc2-Man3-GlcNAc2 (compound represented by Formula (II)). By using sugar nucleotide synthesis-related enzymes from different cloned sources to extend core tetrasaccharides and core hexasaccharides, and taking advantage of the ready availability of core tetrasaccharides and core hexasaccharides, 14 different symmetric and asymmetric N-glycans were synthesized. In particular, the present invention developed a reversible galactosylation (from the compound represented by formula (II) to compound 35) catalyzed by a β1,3-galactosyltransferase that specifically recognizes a single arm of the N-glycan as a protection strategy for producing asymmetric N-glycans. Compared with most reported methods that use HPLC to purify products, all structures obtained in the present invention were purified using conventional chromatographic methods, including size exchange 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 without the need for tedious purification operations, thus having the characteristics of low cost and strong adaptability.

[0143] Example

[0144] (1) Enzyme preparation using E. coli expression system

[0145] The catalytic domain of the glycosyltransferase was artificially synthesized using the E. coli expression system, including:

[0146] Chitobiosyl diphosphate polyphenol β-mannosyltransferase (ALG1) from Saccharomyces cerevisiae has the following amino acid sequence:

[0147]

[0148]

[0149] Human α-1,3 / 1,6-mannosyltransferase (ALG2), whose amino acid sequence is:

[0150]

[0151] Human α-1,3-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1, (29 to 445aa)), the amino acid sequence of which is:

[0152]

[0153] Chromobacterium violaceum β1,3-galactosyltransferase (B3GalT), the amino acid sequence of which is:

[0154]

[0155] α2,6-sialyltransferase (Pd2,6ST) from Photobacterium damselae, whose amino acid sequence is:

[0156]

[0157]

[0158] α2,3-sialyltransferase (BtST) from Bibersteinia trehalosi, the amino acid sequence of which is:

[0159]

[0160] α2,3-sialyltransferase (PPST) from Phosphorobacterium phosphogenum, whose amino acid sequence is:

[0161]

[0162] α1,3-fucosyltransferase (FucT) from Helicobacter pylori, its amino acid sequence is:

[0163]

[0164] The above enzymes were expressed in E. coli and purified using Ni-NTA. These genes were synthesized by GenScript (Nanjing, China) or Sangon Biotech (Shanghai, China).

[0165] All genes were cloned into the pET-28a vector with a hexa-histidine (His) tag at the N-terminus. Correctly identified plasmids were transformed into Escherichia coli BL21(DE3) for protein expression. E. coli BL21(DE3) cells containing the recombinant plasmids were cultured in two liters of Luria-Bertani (LB) medium supplemented with 50 μg / ml kanamycin at 37°C and 200 rpm on a rotary shaker. When the OD value reached 0.8, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and protein expression was induced overnight at 16°C. Cells were harvested by centrifugation at 7000 rpm for 10 minutes. 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 minutes to remove cell debris. The His-tagged protein was purified using a Ni-NTA agarose column. Prior to 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 protein, the enzyme used in the present invention, was filtered and desalted using an ultrafiltration centrifuge tube (Amicon Ultra-5, 10Kd) for further use. Protein concentration was determined using a BCA protein assay kit.

[0166] Although the sources of the enzymes in this application are described above, their sources are not limited thereto, as long as they can achieve the functions intended in this application.

[0167] (2) Enzyme preparation using HEK293 expression system

[0168] The catalytic domain of human glycosyltransferase was artificially synthesized, including:

[0169] Human α-1,3 / 1,6-mannosyltransferase (ALG2) (full length, amino acid sequence shown in SEQ ID NO: 2);

[0170]

[0171] Human α-1,6-mannose glycoprotein 2-β-N-acetylglucosamine transferase (MGAT2) (45 to 447aa), the amino acid sequence of which is:

[0172]

[0173] Human β1,4-galactosyltransferase (B4GalT) (45 to 398aa), the amino acid sequence of which is:

[0174]

[0175]

[0176] Human α2,6-sialyltransferase (ST6Gal1) (75 to 406aa), the amino acid sequence of which is:

[0177]

[0178] All genes were cloned into the commercially available pCDNA3.1 vector (purchased from Thermo Fisher (Cat. No. V79020)) with BamHI-XhoI restriction sites. The construction method was similar to that reported in the literature (Nature Chemical Biology, volume 14, pages 156–162 (2018)). Large-scale protein production was achieved by transient transfection of HEK293 suspension cultures according to the manufacturer's instructions. HEK293 suspension cells (Freestyle 293-F) were transfected, and the culture supernatant containing the secreted fusion protein was applied to a Ni-NTA agarose column. Prior to 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 by filtration (Amicon Ultra-5, 10Kd) with a 10 kDa cut-off molecular weight. The protein concentration was determined using the BCA protein assay kit.

[0179] (3) Enzymes from commercial sources

[0180] The Endo S (#P0741S), Endo D (#P0742S), B3GalH (#P0726S), B4GalH (#P0730S), and NanC (#P0743S) were purchased from NEB.

[0181] Example 1: Preparation of compounds c1-c7

[0182]

[0183] (1) Compounds a1-a7 (2 g) were suspended in 30 mL of anhydrous acetonitrile with 4,5-dicyanoimidazole (3 equivalents) and 4A molecular sieves, respectively, under nitrogen protection. Dibenzyl N,N-diisopropylphosphoramidite (1.5 equivalents) was then added dropwise under an ice bath. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at room temperature until the reaction of the raw materials was complete. The reaction mixture was then placed in an ice bath and the temperature was lowered to 0°C. A 5.0 M-6.0 M tert-butyl peroxide solution in decane (4 equivalents) was then added dropwise. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at room temperature until the reaction of the product in the previous step was complete. The insoluble solid was removed by filtration through diatomaceous earth and the solution was concentrated under reduced pressure. The mixture was diluted with DCM (200 mL) and washed with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence. The organic layer was collected and the aqueous layer was stripped with dichloromethane. The organic layers were collected and combined and dried over anhydrous sodium sulfate. After concentration under vacuum, silica gel was added for dry loading and the residue was purified by column chromatography (PE:EA=85:15) to obtain the target products b1-b7, respectively.

[0184] (2) Compound b1-b7 (3 g) was dissolved in anhydrous MeOH (15 mL) and 10% Pd-C (300 mg) was added. The reaction was stirred vigorously at room temperature under a hydrogen atmosphere until the reaction of the starting materials was complete. After filtration through celite, the mixture was concentrated under vacuum to obtain the target product c1-c7.

[0185] Example 2: Preparation of Compounds 6a-6g

[0186]

[0187] 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 three times with toluene (3×10 mL) to remove excess triethylamine. The triethylammonium salt of the obtained 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 CDI that had not reacted completely. A solution of compound 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 reaction of the starting material was complete, the reaction was concentrated under vacuum, and the residue was purified by column chromatography (EA:MeOH:H2O=70:30:0.3) to obtain the target product 6a-6g.

[0188] Example 3: Preparation of Compounds 7a-7g

[0189]

[0190] Compounds 6a-6g were dissolved in anhydrous MeOH / DCM (1:1, 20 mL). Under nitrogen, a 5.4 M MeONa solution (0.5 equivalent) in MeOH was slowly added. The reaction was allowed to proceed at room temperature for 12 hours. Upon completion, the reaction mixture was neutralized with hydrogen-form ion exchange resin. After filtration, the solvent was concentrated to yield the desired products 7a-7g.

[0191] Example 4: Preparation of Compounds 8a-8g

[0192]

[0193] Compounds 7a-7g (200 mg) were incubated with 10 mM MgCl2 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, which were used for testing in Example 11.

[0194] Example 5: Preparation of Compounds 9a-9g

[0195]

[0196] Compounds 8a-8g (200 mg) were incubated with 10 mM MgCl2 and ALG2 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 yield compounds 9a-9g.

[0197] Example 6: Large-Scale Preparation of Core Pentasaccharide (I) and Core Heptasaccharide (II)

[0198]

[0199] (1) Compound 7b was prepared from compound 6b according to the steps of Examples 1-3. After purification using a P2 column, compound 7b was incubated with ALG1 and excess GDP-Man. After completion of the reaction, the reaction was quenched by adding cold ethanol, and the supernatant was roughly concentrated and purified by passing it through a P2 column to obtain compound 8b. Compound 8b was then incubated with ALG2 and excess GDP-Man. After completion of the reaction, the reaction was quenched by adding cold ethanol, and the supernatant was roughly concentrated and purified by passing it through a P2 column to obtain compound 9b. Compound 9b was then incubated with Endo S. After completion of the reaction, the reaction was quenched by adding cold ethanol, and the supernatant was purified by passing it through a P2 column to obtain core tetrasaccharide (I).

[0200] (2) The core tetrasaccharide (I) was incubated with MGAT1 and an excess of UDP-GlcNAc to produce compound 28. The reaction was monitored by TLC. Upon completion, MGAT2 was added. When no intermediates or starting materials were observed on TLC, the reaction was quenched by adding cold ethanol. The supernatant was concentrated and purified using a P2 column. Finally, 1.91 g of the core hexasaccharide (II) was obtained with a total yield of 10.2%.

[0201] Example 7: Preparation of Compounds 30-42

[0202] In this example, based on the core tetrasaccharide (I) and core hexasaccharide (II) synthesized in Example 6, biantennary N-glycan compounds 30-42 (shown in the following formula (III)) were prepared by enzymatic reaction or chemical reaction extension. The reaction scheme is shown below:

[0203]

[0204]

[0205] The general experimental protocols for each enzymatic reaction and chemical reaction are as follows:

[0206] (1) General protocol for β1,3-galactosylation using β1,3-galactosyltransferase (B3GalT)

[0207] Under the action of β1,3-galactosyltransferase (B3GalT), N-glycan reacts with uridine diphosphogalactose (UDP-Gal), adding galactose C linked by a β1,3-glycosidic bond to N-acetylglucosamine A at the antenna end of the N-glycan. The specific steps include:

[0208] The starting material N-glycans were mixed with 2 equivalents of UDP-Gal, 10 mM Mg 2+The reaction was incubated with B3GalT in 10 mM Tris buffer (pH 7.5) at 37°C and monitored by TLC. Once no starting material was observed on TLC, the reaction was quenched by adding cold ethanol. Insoluble precipitates were removed by centrifugation. The supernatant was concentrated and purified using a P2 column to obtain the desired product.

[0209] The compound conversion in the above enzymatic reaction using B3GalT includes: compound (II) → compound 35.

[0210] (2) General experimental protocol for β1,4-galactosylation using β1,4-galactosyltransferase (B4GalT)

[0211] Under the action of β1,4-galactosyltransferase (B4GalT), N-glycan reacts with uridine diphosphogalactose (UDP-Gal), and galactose C and / or D connected 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. The specific steps include:

[0212] The starting material N-glycan was incubated with 2 equivalents of UDP-Gal, 10 mM MnCl2, and B4GalT in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37°C and monitored by TLC. Once the starting material was no longer 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 the desired product.

[0213] The compound conversions of the above enzymatic reaction using B4GalT include: compound of formula (II) → compound 30, compound 35 → compound 36, and compound 38 → compound 39.

[0214] (3) General protocol for α2,6-sialylation using α2,6-sialyltransferase (Pd2,6ST or ST6Gal1)

[0215] Under the action of α2,6-sialyltransferase (Pd2,6ST) or α2,6-sialyltransferase (ST6Gal1), N-glycan reacts with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F connected by an α2,6-glycosidic bond are added to the galactosyl C and / or D at the antenna end of the N-glycan, and the specific steps include:

[0216] The starting material N-glycans were incubated with 2 equivalents of CMP-Neu5Ac, 10 mM MgCl2, and Pd26ST or ST6Gal1 in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37°C and monitored by TLC. Once the starting material was no longer 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 and an ion exchange column to obtain the desired product.

[0217] The compound conversions of the above enzymatic reaction using Pd2,6ST include: compound 30 → compound 33;

[0218] The compound conversion by the above enzymatic reaction using ST6Gal1 includes: Compound 39 → Compound 40.

[0219] (4) General experimental protocol for α2,3-sialylation using α2,3-sialyltransferase (BtST or PPST)

[0220] Under the action of α2,3-sialyltransferase (BtST) or α2,3-sialyltransferase (PPST), N-glycan reacts with cytidine monophosphoryl N-acetylneuraminic acid (CMP-Neu5Ac), and N-acetylneuraminic acid E and / or F connected by an α2,3-glycosidic bond are added to the galactosyl C and / or D at the antenna end of the N-glycan. The specific steps include:

[0221] The starting material N-glycan was incubated with 2 equivalents of CMP-Neu5Ac, 10 mM MgCl2, and BtST 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 the desired product.

[0222] The compound conversions of the above enzymatic reaction using BtST include: compound 30 → compound 34, compound 37 → compound 38.

[0223] To synthesize structures containing the sialyl Lewis X epitope, the starting N-glycan was incubated with 10 equivalents of CMP-Neu5Ac, 10 mM MgCl2, and PPST in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37°C and monitored by TLC. Once the starting material was no longer visible on TLC, the reaction was quenched by adding cold ethanol. The insoluble precipitate was removed by centrifugation.

[0224] The compound conversion using PPST in the above enzymatic reaction includes: compound 31 → compound 32.

[0225] (5) General experimental protocol for α1,3-fucosylation using α1,3-fucosyltransferase (FucT)

[0226] Under the action of α1,3-fucosyltransferase (FucT), N-glycan reacts with guanosine diphosphate-fucose (GDP-Fucose), and fucose G and / or H connected by α1,3-glycosidic bonds are added to N-acetylglucosamine groups A and / or B on the antenna of the N-glycan. The specific steps include:

[0227] The starting material N-glycan was incubated with 2 equivalents of GDP-L-Fuc, 10 mM MgCl2, and FucT in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 37°C and monitored by TLC. Once the starting material was no longer 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 the desired product.

[0228] The compound conversion using FucT in the above enzymatic reaction includes: Compound 30 → Compound 31.

[0229] (6) General experimental protocol using β1,3-galactosylating hydrolase (B3GalH)

[0230] Under the action of β1,3-galactosyl hydrolase (B3GalH), galactose C at the antenna end of the N-glycan, which is connected to N-acetylglucosamine A by a β1,3-glycosidic bond, is removed. The specific steps include:

[0231] The starting material N-glycans were incubated with 10 mM MgCl2 and B4GalH in 10 mM Tris buffer (pH 7.5). The reaction was carried out at 30°C and monitored by TLC. Once the starting material was no longer 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 the desired product.

[0232] The compound conversion by the above enzymatic reaction using B3GalH includes: Compound 36 → Compound 37.

[0233] (7) General experimental protocol using β1,4-galactosylating hydrolase (B4GalH)

[0234] Under the action of β1,4-galactosyl hydrolase (B4GalH), galactose C and / or D at the antenna end of the N-glycan connected to N-acetylglucosamine A and / or B by β1,4-glycosidic bond are removed. The specific steps include:

[0235] The starting material N-glycans were incubated with 10 mM MgCl2 and B4GalH 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 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 desired product.

[0236] The compound conversion by the above enzymatic reaction using B4GalH includes: Compound 41 → Compound 42.

[0237] (8) General experimental protocol using α2,3-sialyl hydrolase (NanC)

[0238] Under the action of α2,3-sialyl hydrolase (NanC), N-acetylneuraminic acid E and / or F connected to galactosyl C and / or D via α2,3-glycosidic bond at the antenna end of N-glycan is removed. The specific steps include:

[0239] The starting material N-glycans were incubated with 10 mM MgCl2 and NanC 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 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 desired product.

[0240] The compound conversion by the above enzymatic reaction using NanC includes: Compound 40 → Compound 41.

[0241] Example 8: Large-scale preparation of compound 37 from the core hexasaccharide using a reversible galactosylation strategy using a "one-pot multi-enzyme" approach

[0242] 0.89 g of core hexasaccharide (II) was mixed with 2 equivalents of UDP-Gal, 10 mM Mg 2+ The reaction was incubated with B3GalT in 10 mM Tris buffer (pH 7.5) at 37°C and monitored by TLC. Once no starting material was observed on 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, 10 mM Mn 2+The reaction was carried out with B4GalT at 37°C and monitored by TLC. Once the starting material was no longer observed by TLC, the reaction was quenched by boiling in a 100°C waterbath for 30 minutes. The insoluble precipitate was removed by centrifugation. B3GalH was added to the supernatant and the reaction was continued at 30°C with TLC monitoring. Once the starting material was no longer observed by 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 yield 0.94 g of the target compound 37 in a 92% yield.

[0243] The compound prepared above 1 H NMR and 13 C NMR data are listed in Table 3 below.

[0244] Table 3

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

Claims

1. A method for synthesizing the core tetrasaccharide represented by formula (I), comprising the following steps: S9: Under the action of β-N-acetylglucosaminyl endoside enzyme (Endo enzyme) or a protein derived from Endo enzyme that has Endo enzyme activity and has one or more amino acids substituted, deleted or added in the amino acid sequence of Endo enzyme, Compound 9 removes Compound 10 and is converted into a core tetrasaccharide represented by formula (I). The reaction formula is as follows: Where, R is a C6-C95 linear or branched alkyl, preferably a C6-C50 linear or branched alkyl, more preferably a C6-C20 linear or branched alkyl, and further preferably, R is selected from: More preferably, R is 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. 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 indicates 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.

2. The synthesis method according to claim 1, wherein: The Endo enzyme is selected from the group consisting of Endo S, Endo CC, Endo M, and Endo D. Preferably, the Endo S is derived from Streptococcus pyogenes. Preferably, the Endo CC is derived from Coprinopsis cinerea. Preferably, the Endo M is derived from Mucor hiemalis. Preferably, the Endo D is derived from Streptococcus pneumonia. Preferably, the reaction is carried out in Mg 2+ In the presence of Preferably, the reaction is carried out at a pH of 6-9, more preferably at a pH of 7-8, and even more preferably at 7.

0.

3. The synthesis method according to claim 1, wherein compound 9 is prepared by a method comprising the following steps: S8: Compound 9 was synthesized from Compound 8 under the action of α-1,3 / 1,6-mannosyltransferase (ALG2) or a protein derived from ALG2 that has ALG2 activity and has one or more amino acids substituted, deleted, or added in its amino acid sequence: In the formula, the definitions of the symbols are the same as those in claim 1, Preferably, the ALG2 is of human origin; more preferably, its amino acid sequence is as shown in SEQ ID NO: 2, Preferably, the reaction is carried out in Mg 2+ In the presence of Preferably, the reaction is carried out at a pH of 7-8, more preferably 7.4-7.

5.

4. The synthesis method according to claim 3, wherein compound 8 is prepared by a method comprising the following steps: S7: Compound 8 was synthesized from Compound 7 under the action of chitobiosyl 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 its amino acid sequence: In the formula, the definitions of the symbols are the same as those in claim 3, Preferably, the ALG1 is derived from Saccharomyces cerevisiae; in particular, its amino acid sequence is shown in SEQ ID NO: 1, Preferably, the reaction is carried out in Mg 2+ In the presence of Preferably, the reaction is carried out at a pH of 6-9, more preferably 7.4-7.

5.

5. The synthesis method according to claim 1, wherein The method is carried out in a one-pot process, comprising the following steps: compound 7 and guanosine diphosphate mannose are used as substrates, and the reaction mixture is stirred at room temperature in the presence of metal Mg 2+ In the presence of ALG1 or a protein derived from ALG1 having ALG1 activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of ALG1, and ALG2 or a protein derived from ALG2 having ALG2 activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of ALG2, catalyzes the synthesis of compound 8 to obtain compound 9. Compound 10 is then removed under the action of Endo enzyme or a protein derived from Endo enzyme having Endo enzyme activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence of Endo enzyme, to catalyze the synthesis of the core tetrasaccharide represented by formula (I). The reaction formula is as follows: Where, R is a C6-C95 linear or branched alkyl, preferably a C6-C50 linear or branched alkyl, more preferably a C6-C20 linear or branched alkyl, and further preferably, R is selected from: More preferably, R is 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. 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 indicates 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.

6. The method according to claim 4 or 5, wherein compound 7 is prepared by a method comprising the following steps: S6: Compound 7 is synthesized from compound 6, and the reaction formula is as follows: In the formula, the definitions of the symbols are the same as those in claim 5, Preferably, the reaction is carried out in solvent 8, more preferably, solvent 8 is a mixed solvent of dichloromethane and methanol, and even more preferably, the volume ratio of dichloromethane to methanol is 1:1; Preferably, compound 6 undergoes deacetylation in the presence of sodium methoxide; more preferably, the molar ratio of compound 6 to sodium methoxide is 1:1.5 to 2.5; even more preferably, sodium methoxide is added to the reaction system in the form of a solution, and the solvent is preferably methanol; Preferably, the reaction is carried out at 20-30° C. with stirring, and the reaction time is 8-14 h; Preferably, after the reaction is completed, a separation and purification step is further included. More preferably, the separation and purification step includes neutralizing the reaction solution using a hydrogen-type ion exchange resin and then filtering it, and concentrating the filtrate to obtain compound 7.

7. The method according to claim 6, wherein compound 6 is prepared by a method comprising the following steps: S5: Compound 6 is synthesized from compound 5 and compound c, and the reaction formula is shown below: Wherein, the definition of R is the same as that in claim 6, Preferably, compound 5 is dissolved in solvent 5, and then triethylamine is added to convert it into triethylammonium salt; the triethylammonium salt is activated with N,N-carbonyldiimidazole in solvent 6, and then reacted with compound c to obtain compound 7. Preferably, the solvent 5 is methanol; the solvent 6 is N,N-dimethylformamide, Preferably, the usage 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 equivalent 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, more preferably, co-evaporated with toluene 3 times. Preferably, the reaction conditions for activating the phosphate 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. More preferably, solvent 7 is dichloromethane. Preferably, 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; more preferably, the separation and purification step includes concentrating the reaction solution and purifying it by column chromatography to obtain compound 6. More preferably, the column chromatography solution is ethyl acetate: methanol: water = 70:30:0.3, Preferably, compound c is prepared as follows: (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: in, R is as defined in step S5; Preferably, in step (a): Compound a is reacted in solvent 3; more preferably, the solvent 3 is acetonitrile, Preferably, 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. Preferably, the reaction of compound a with 4,5-dicyanoimidazole and dibenzyl N,N'-diisopropylphosphoramidite, and the reaction with tert-butyl peroxide, are carried out at 20-30° C. with stirring; more preferably, tert-butyl peroxide is added to the reaction system in the form of a decane solution; still more preferably, the reaction endpoint is monitored by thin layer chromatography. Preferably, after the reaction is completed, a separation and purification step is further included. More preferably, the separation and purification step comprises filtering the reaction solution through diatomaceous earth and concentrating it, adding dichloromethane to dilute it, collecting the organic phase after washing, drying, concentrating, and purifying it by column chromatography to obtain compound b; preferably, washing is washing with 1M hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution in sequence; preferably, drying is drying using anhydrous sodium sulfate; preferably, the chromatographic fluid for column chromatography is dichloromethane / methanol = 85:5, v / v; Preferably, in step (b): Compound b is reduced in methanol. Preferably, compound b is reduced by hydrogen in the presence of a hydrogenation catalyst, wherein the hydrogenation catalyst is palladium carbon. After the reaction is completed, a separation and purification step is further included. Preferably, the separation and purification step includes filtering the reaction solution through diatomaceous earth and then concentrating it to obtain compound c.

8. A method for synthesizing a core hexasaccharide of formula (II) using a core tetrasaccharide of formula (I), the method comprising the following steps: 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 more amino acids in the amino acid sequence of MGAT1, the core tetrasaccharide 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 more amino acids in the amino acid sequence of MGAT2, compound 28 and UDP-GlcNAc are converted into the core hexasaccharide represented by formula (II), and the reaction formula is as follows: in, 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. 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 a sugar hemiacetal hydroxyl group is connected to the hydroxyl group on the 6-carbon of another sugar through an α-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. Preferably, in S10, MGAT1 is of human origin, and in particular, its amino acid sequence is as shown in SEQ ID NO: 3, Preferably, in S10, the reaction is carried out in the presence of Mn 2+ Mg 2+ In the presence of Preferably, in S10, the reaction is carried out at a pH of 7-8, more preferably 7.4-7.

5. Preferably, in S11, MGAT2 is of human origin; in particular, its amino acid sequence is as shown in SEQ ID NO: 4, Preferably, in S11, the reaction is carried out under the conditions of Mn 2+ Mg 2+ Carry out in the presence of Preferably, in S11, the reaction is carried out at a pH of 7-8, more preferably 7.4-7.

5. Preferably, steps S10 and S11 are performed separately or in the same reaction system according to a one-pot method. Further preferably, steps S10 and S11 are carried out in the same reaction system, comprising the following steps: using the core tetrasaccharide represented by formula (I), UDP-GlcNAc as a substrate, 2+ 、Mn 2+ In the presence of MGAT1 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 MGAT2 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 hexasaccharide GlcNAc2-Man3-GlcNAc represented by formula (II), and the reaction formula is as follows:

9. A method for preparing N-glycans represented by formula (III), 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 one 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: The core tetrasaccharide represented by formula (I) or the core hexasaccharide represented by formula (II) is extended by an enzymatic reaction or a chemical reaction to prepare an N-glycan represented by formula (III); Wherein, the enzymatic reaction or chemical reaction is selected from one or more of the following: (1) Under the action of β1,3-galactosyltransferase B3GalT or a protein derived from B3GalT that has one or more amino acids substituted, deleted, or added in its amino acid sequence and has B3GalT activity, N-glycan reacts with uridine diphosphogalactose (UDP-Gal), 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; in particular, its amino acid sequence is shown in SEQ ID NO: 5; Preferably, the above 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 the pH value is 7 to 8, more preferably 7.5; (2) Under the action of β1,4-galactosyltransferase B4GalT or a protein derived from B4GalT that has one or more amino acids substituted, deleted, or added in the amino acid sequence of B4GalT and has B4GalT activity, N-glycan reacts with UDP-Gal to add galactose C and / or D linked by a β1,4-glycosidic bond to the N-acetylglucosamine group A and / or B at the antenna end of the N-glycan; Preferably, the B4GalT is of human origin; in particular, its amino acid sequence is shown in SEQ ID NO: 6; Preferably, the above 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 the pH value is 7 to 8, more 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 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), thereby adding N-acetylneuraminic acid E and / or F linked by an α2,6-glycosidic bond to the galactosyl C and / or D at the antennal end of the N-glycan; Preferably, the Pd2,6ST is derived from Photobacterium damselae; in particular, its amino acid sequence is shown in SEQ ID NO: 7; the ST6Gal1 is derived from human, in particular, its amino acid sequence is shown in SEQ ID NO: 8; Preferably, the above 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 a pH value of 7 to 8, more 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 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 CMP-Neu5Ac to add N-acetylneuraminic acid E and / or F linked by an α2,3-glycosidic bond to the galactosyl C and / or D at the antenna end of the N-glycan; Preferably, the BtST is derived from Bibersteinia trehalosi, and in particular, its amino acid sequence is shown in SEQ ID NO: 9; the PPST is derived from Photobacterium phosphoreum, and in particular, its amino acid sequence is shown in SEQ ID NO: 10; Preferably, the above 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 a pH value of 7 to 8, more preferably 7.5; (5) Under the action of α1,3-fucosyltransferase FucT or a FucT-derived protein having FucT activity and having one or more amino acids 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 an α1,3-glycosidic bond is added to the N-acetylglucosamine group A and / or B on the antenna of the N-glycan; Preferably, the FucT is derived from Helicobacter pylori; in particular, its amino acid sequence is shown in SEQ ID NO: 11; Preferably, the above enzymatic reaction is carried out in Mg 2+ The reaction is carried out under the presence of a pH of 6 to 9, preferably a pH of 7 to 8, more preferably 7.5, (6) galactose C linked to N-acetylglucosamine A by a β1,3-glycosidic bond at the antenna end of the N-glycan is removed by the action of β1,3-galactosylation hydrolase B3GalH or a protein derived from B3GalH that has one or more amino acids substituted, deleted, or added 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 to 8, more preferably 7.0; (7) removing galactose C and / or D linked to N-acetylglucosamine A and / or B by β1,4-glycosidic bonds at the antenna ends of N-glycans under the action of β1,4-galactosylation hydrolase B4GalH or a protein derived from B4GalH that has one or more amino acids substituted, deleted or added in the amino acid sequence of B4GalH and has B4GalH activity; Preferably, the pH value of the reaction system of the above enzymatic reaction is 6 to 9, preferably 7 to 8, more preferably 7.0; (8) 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 under the action of α2,3-sialyl hydrolase NanC from Streptococcus penumoniae or a protein derived from NanC with one or more amino acids substituted, deleted or added in the amino acid sequence of NanC and having NanC activity; 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 7 to 8, more preferably 7.

0. Preferably, the above steps (1)-(8) further include a separation and purification step. Preferably, the separation and purification step includes: after the reaction is completed, adding ethanol, solid-liquid separation, and concentrating and purifying the supernatant using a P2 column to obtain the target product.

10. The method according to claim 9, wherein: The N-glycan represented by formula (III) is selected from the compounds represented by the following structures:

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