A continuous chemical enzymatic synthesis strategy for the synthesis of a structurally comprehensive library of sulfated N-glycans

Abstract

The present application relates to a continuous chemical enzyme synthesis strategy for synthesizing a sulfated complex N-glycan compound library. Specifically, the present application comprehensively uses the branch selectivity of specific glycosyltransferase on N-glycan core pentasaccharide and the site selectivity of sulfotransferase CHST1 and CHST6, designs and uses a continuous chemical enzyme synthesis strategy, and successfully synthesizes a sulfated complex N-glycan molecule with a clear structure.

Description

Technical Field

[0001] This invention relates to the field of oligosaccharide synthesis technology, specifically to a continuous chemoenzymatic synthesis strategy for synthesizing a comprehensive library of sulfonated N-saccharides. Background Technology

[0002] In recent years, tumor immunotherapy, represented by immune checkpoint blockade, has achieved groundbreaking therapeutic effects. However, a significant proportion of cancer patients lack sufficient immune response or show no response to existing immunotherapies. Therefore, there is an urgent need to identify new immune checkpoints and develop corresponding immunotherapies and drugs to participate in anti-tumor immune responses, thereby expanding the patient population that can benefit. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a class of tumor immune checkpoint molecules with great application potential. Tumor cells typically highly express sialic acid oligosaccharides on their surface, while tumor-associated immune cells highly express Siglec proteins. The sialic acid-Siglec axis formed by these two molecules can promote the establishment of a tumor immunosuppressive microenvironment, thereby facilitating the immune evasion of tumor cells. To date, more than a dozen members of the Siglec protein family have been clearly demonstrated to directly participate in tumor immune responses. For example, the CD24 protein, which is highly expressed on tumor cells, can specifically bind to the Siglec-10 protein, which is highly expressed on the surface of macrophages, sending a "don't eat me" signal to macrophages and thus inhibiting their phagocytic activity. In cancer bone metastases, elevated Siglec-15 expression levels may promote tumor-induced osteoblastogenesis and inhibit T-cell responses to specific antigens. Therefore, finding high-affinity sialic acid oligosaccharide ligands for Siglec protein is crucial for developing novel anti-tumor immunotherapies and drugs.

[0003] N-glycans are among the most abundant and abundant sialic acid oligosaccharides in the human body and are important endogenous glycosyl ligands for Siglec proteins. Recent studies have found that changes in the sulfonation level of N-glycans on the cell surface can significantly affect the binding of cells to Siglec proteins. Therefore, sulfonated complex N-glycans may be a class of extremely important Siglec protein ligand molecules. However, due to their complex structure, there is currently no effective method to synthesize these sugar molecules, which hinders research on the interaction between sulfonated complex N-glycans and Siglec proteins.

[0004] Therefore, there is an urgent need in this field to develop a strategy for synthesizing sulfonated complex N-glycan molecules in order to explore the interaction between sulfonated complex N-glycans and Siglec proteins. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synthesizing sulfonated complex N-sugar molecules.

[0006] In a first aspect, the invention provides the use of sulfotransferase CHST6 for sulfonation modification of the 6-position of the terminal N-acetylglucosamine group of the compound shown in formula (I).

[0007]

[0008] In the formula, the cube represents N-acetylglucosamine;

[0009] The light-colored round shape represents galactosyl groups;

[0010] R1 is a monosaccharide residue or a disaccharide residue;

[0011] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0012] R is absent, or a C1-C10 straight-chain or branched alkyl azide group.

[0013] In another preferred embodiment, the square and the light-colored circle are linked by an oxyglycosidic bond at position 1 of the N-acetylglucosamine group and position 3 of the galactose group.

[0014] In another preferred embodiment, the light-colored circle and R1 are linked by an oxyglycosidic bond at the 1-position of the galactose group and the hydroxyl group in R1 excluding the 1-position.

[0015] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R is a C5 linear alkyl azide group.

[0016] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R1 is an N-acetylglucosyl residue or a disaccharide residue composed of N-acetylglucosyl and fucose (GlcNAc-Fuc residue).

[0017] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R2 is not galactosyl.

[0018] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), the light-colored circles in R1-R2 are connected to disaccharide residues that are not N-acetylglucosamine-galactose residues (GlcNAc-Gal residues).

[0019] In another preferred embodiment, in the N-glycosidic material with the structure shown in formula (I), R2 is selected from the group consisting of: none,

[0020]

[0021] In the formula, the cube represents N-acetylglucosamine;

[0022] The light-colored round shape represents galactosyl groups;

[0023] The dark-colored circles represent mannosyl groups;

[0024] The rhombus shape represents an N-acetylneuraminic acid group;

[0025] The triangle represents fucose.

[0026] In another preferred embodiment, the compound represented by formula (I) is selected from the group consisting of:

[0027]

[0028] In the formula, the cube represents N-acetylglucosamine;

[0029] The light-colored round shape represents galactosyl groups;

[0030] The dark-colored circles represent mannosyl groups;

[0031] The rhombus shape represents an N-acetylneuraminic acid group;

[0032] The triangle represents fucose.

[0033] In a second aspect, the present invention provides a method for the continuous chemical enzymatic synthesis of sulfonated N-sugars, comprising the steps of:

[0034] (s1) A premix is ​​provided, the premix comprising:

[0035] (a) N-glycosidic substrate;

[0036] (b) PAPS; and

[0037] (c) Sulfonase, wherein the sulfonase is CHST6;

[0038] The N-glycosidic substrate has the structure shown in formula (I):

[0039]

[0040] In the formula, the cube represents N-acetylglucosamine;

[0041] The light-colored round shape represents galactosyl groups;

[0042] R1 is a monosaccharide residue or a disaccharide residue;

[0043] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0044] R is absent, or a C1-C10 straight-chain or branched alkyl azide group;

[0045] (s2) Under enzyme-catalyzed conditions, the N-sugar substrate undergoes a sulfonation reaction with PAPS to modify the 6-position of the terminal N-acetylglucosamine group in the N-sugar substrate by sulfonation, thereby obtaining a sulfonated N-sugar (compound E6).

[0046] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R is a C5 linear alkyl azide group.

[0047] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R1 is an N-acetylglucosyl residue or a disaccharide residue composed of N-acetylglucosyl and fucose.

[0048] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R2 is not galactosyl.

[0049] In another preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), the light-colored circles in R1-R2 are connected to disaccharide residues that are not N-acetylglucosamine-galactose residues (GlcNAc-Gal residues).

[0050] In another preferred embodiment, in the N-glycosidic material with the structure shown in formula (I), R2 is selected from the group consisting of: none,

[0051]

[0052] In the formula, the cube represents N-acetylglucosamine;

[0053] The light-colored round shape represents galactosyl groups;

[0054] The dark-colored circles represent mannosyl groups;

[0055] The rhombus shape represents an N-acetylneuraminic acid group;

[0056] The triangle represents fucose.

[0057] In another preferred embodiment, the method further includes step (s3) glycosylation of the sulfonated N-sugar using a glycosyltransferase, thereby modifying the end of the sulfonated N-sugar with a glycosyl group to obtain a glycosyl-modified sulfonated N-sugar (compound E7). Preferably, step (s3a) involves galactosylation of the sulfonated N-sugar using B4Gal4 catalyzes the process to obtain a galactosylated sulfonated N-sugar (compound E7).

[0058] In another preferred embodiment, the method further includes step (s4) sialylating compound E7 with a glycosyltransferase, such that the Gal of compound E7 is modified with sialic acid, thereby obtaining a sialylated compound.

[0059] In another preferred embodiment, the sialylated compound is used as a substrate, and the sialylation reaction is performed again using a glycosyltransferase to modify the Gal or Neu5Ac of the sialylated compound with sialic acid.

[0060] In another preferred embodiment, the sialic acid reaction can be performed by further modifying the sialic acid-modified Gal with sialic acid.

[0061] In another preferred embodiment, in step (s4), Gal and / or Neu5Ac at or near the end (preferably at or near the N end) are subjected to a sialylation reaction.

[0062] In another preferred embodiment, step (s4) is repeated 1 to 10 times, more preferably 1 to 8 times, and even more preferably 1 to 5 times.

[0063] In another preferred embodiment, step (s4) is selected from the group consisting of:

[0064] (s4a) The α2,6-sialylation of Gal in compound E7 was catalyzed by ST6Gal1, resulting in the terminal modification of compound E7 with Neu5Ac, thus yielding compound E8.

[0065] (s4b) Using BtST to catalyze the α2,3-sialylation of Gal in compound E7, thereby modifying the end of compound E7 with Neu5Ac to obtain compound E10; and / or

[0066] (s4c) The α2,8-sialylation of Neu5Ac in compound E8 or E10 is catalyzed by CSTII, which further modifies the end of compound E8 or E10 with Neu5Ac to obtain compound E9 or E11.

[0067] (s4d) Using Pd2,6ST to catalyze the α2,6 sialylation of Gal (at or near the end, preferably at or near the N end) in compound E10, the Gal in compound E10 is further sialylated to obtain compound E12.

[0068] In another preferred embodiment, the method further includes step (s5) sulfonating the sialylated compound with a sulfonase to obtain a disulfonated N-sugar, preferably using CHST1 to catalyze the sulfonation of the 6-hydroxyl group of Gal in E10 to obtain compound E13.

[0069] In another preferred embodiment, the method further includes step (s6) of hydrolyzing the sulfonated N-sugar obtained in step (s2) with a glycoside hydrolase, such that the terminal glycosyl groups of the non-sulfonated branches of the sulfonated N-sugar are hydrolyzed.

[0070] In another preferred embodiment, the compound obtained by steps (s3), (s4) and / or (s5) is used as an N-sugar substrate for sulfonation.

[0071] In another preferred embodiment, the glycosyltransferase is selected from the group consisting of Alg1, Alg2, MGAT1, MGAT2, MGAT4, MGAT5, B4GalT1, B4GalT4, B3GNT2, BtST, Pd2,6ST, ST6Gal1, M2,6ST, FucT, CST II, ​​or combinations thereof.

[0072] In another preferred embodiment, the sulfotransferase is selected from the group consisting of CHST1, CHST6, Gal3ST2, or combinations thereof.

[0073] In another preferred embodiment, the glycoside hydrolase is selected from the group consisting of NanA, B3GalH, or combinations thereof.

[0074] In another preferred embodiment, the Alg1 is derived from Saccharomyces cerevisiae.

[0075] In another preferred embodiment, the Alg2 is derived from Saccharomyces cerevisiae.

[0076] In another preferred embodiment, the MGAT1 is sourced from Homo sapiens.

[0077] In another preferred embodiment, the MGAT2 is sourced from Homo sapiens.

[0078] In another preferred embodiment, the MGAT4 is sourced from Homo sapiens.

[0079] In another preferred embodiment, the MGAT5 is sourced from Homo sapiens.

[0080] In another preferred embodiment, the B4GalT1 is derived from Homo sapiens.

[0081] In another preferred embodiment, the B4GalT4 is derived from Homo sapiens.

[0082] In another preferred embodiment, the B3GNT2 is sourced from Homo sapiens.

[0083] In another preferred embodiment, the BtST is derived from Bibersteinia trehalosi.

[0084] In another preferred embodiment, the Pd2,6ST is derived from Photobacterium damsela.

[0085] In another preferred embodiment, the ST6Gal1 is sourced from Homo sapiens.

[0086] In another preferred embodiment, the FucT is derived from Helicobacter pylori.

[0087] In another preferred embodiment, the CST II is derived from Campylobacter jejuni.

[0088] In another preferred embodiment, CHST1 is derived from Homo sapiens.

[0089] In another preferred embodiment, the CHST6 is derived from Homo sapiens.

[0090] In another preferred embodiment, the Gal3ST2 is derived from Homo sapiens.

[0091] In another preferred embodiment, the NanA is derived from Streptococcus penumoniae.

[0092] In another preferred embodiment, the B3GalH is derived from Xanthomonas manihotis.

[0093] In another preferred embodiment, CHST6 is derived from Homo sapiens.

[0094] In another preferred embodiment, in step (s1), the PAPS in the premixed solution is 1 to 5 equivalents of N-sugar substrate, more preferably 1 to 4 equivalents, more preferably 1 to 3 equivalents, for example, about 2 equivalents.

[0095] In another preferred embodiment, in step (s1), the premixed solution also contains Mg. 2+ Mg 2+ The concentration of the substance in the premixed solution is 1–200 mM, preferably 5–100 mM, more preferably 5–50 mM, for example, about 10 mM.

[0096] In another preferred embodiment, in step (s1), the pH of the premixed solution is 6-9, preferably 7-9, for example, about 8.5.

[0097] In another preferred embodiment, in step (s2), the temperature of the sulfonation reaction is 25–40°C, more preferably 30–40°C, even more preferably 35–40°C, for example, about 37°C.

[0098] In another preferred embodiment, in step (s2), the sulfonation reaction takes 0.1 to 24 hours, more preferably 1 to 24 hours, and even more preferably 5 to 12 hours, for example, about 8 hours.

[0099] In another preferred embodiment, the N-glycoside substrate is selected from the group consisting of:

[0100]

[0101] In the formula, R is an empty, or C1-C10 straight-chain or branched alkyl azide group;

[0102] The cube contains N-acetylglucosamine;

[0103] The light-colored round shape represents galactosyl groups;

[0104] The dark-colored circles represent mannosyl groups;

[0105] The rhombus shape represents an N-acetylneuraminic acid group;

[0106] The triangle represents fucose.

[0107] In a third aspect, the present invention provides a sulfonated N-sugar prepared by the method described in the second aspect of the present invention.

[0108] In another preferred embodiment, the sulfonated N-sugar is selected from the group consisting of:

[0109]

[0110]

[0111] In the formula, R is an empty, or C1-C10 straight-chain or branched alkyl azide group; the cube is N-acetylglucosyl;

[0112] The light-colored round shape represents galactosyl groups;

[0113] The dark-colored circles represent mannosyl groups;

[0114] The rhombus shape represents an N-acetylneuraminic acid group;

[0115] The triangle represents fucose.

[0116] In a fourth aspect, the present invention provides an N-sugar molecule conjugate, comprising:

[0117] (a) sulfonated N-sugars as described in the third aspect of the invention; and

[0118] (b) A coupling portion coupled to the sulfonated N-sugar, wherein the coupling portion is selected from the group consisting of: a solid support, a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0119] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0120] In a fifth aspect, the present invention provides a molecular library composed of sulfonated N-sugars prepared by the method described in the second aspect of the present invention.

[0121] In another preferred embodiment, the sulfonated N-sugars in the molecular library are selected from the group consisting of:

[0122]

[0123]

[0124] In the formula, R is an empty, or C1-C10 straight-chain or branched alkyl azide group; the cube is N-acetylglucosyl;

[0125] The light-colored round shape represents galactosyl groups;

[0126] The dark-colored circles represent mannosyl groups;

[0127] The rhombus shape represents an N-acetylneuraminic acid group;

[0128] The triangle represents fucose.

[0129] In a sixth aspect, the present invention provides a method for sulfonating a compound of formula (II), comprising the steps of:

[0130] (z1) A premix is ​​provided, the premix comprising:

[0131] (a) The compound shown in formula (II);

[0132]

[0133] In the formula, the cube represents N-acetylglucosinolates;

[0134] The light-colored round shape represents galactosyl groups;

[0135] R1 is absent or a monosaccharide residue;

[0136] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0137] R is absent, or a C1-C10 straight-chain or branched alkyl azide group;

[0138] R3 is absent or has an N-acetylneuraminic acid group;

[0139] (b) PAPS; and

[0140] (c) A sulfonyltransferase, wherein the sulfonyltransferase is selected from the group consisting of CHST1, Gal3ST2, or a combination thereof;

[0141] (z2) Under enzyme-catalyzed conditions, the compound shown in formula (II) undergoes a sulfonation reaction with PAPS to modify the 3- or 6-position of the galactose group in the compound shown in formula (II) by sulfonation, thereby obtaining the sulfonated compound shown in formula (II).

[0142] In another preferred embodiment, CHST1 is derived from Homo sapiens.

[0143] In another preferred embodiment, the Gal3ST2 is derived from Homo sapiens.

[0144] In step (z1), in the premix, PAPS is 1 to 10 equivalents of N-sugar substrate, preferably 1 to 8 equivalents, more preferably 1 to 5 equivalents, for example, about 4 equivalents or 2 equivalents.

[0145] In another preferred embodiment, in the compound shown in formula (II), the cube and the light-colored circle are linked by a β1,4-oxyglycosidic bond.

[0146] In another preferred embodiment, in the compound shown in formula (II), the cube and R1 are linked by a β1,X (X is any hydroxyl group in the structure of R1 except for position 1) oxyglycosidic bond.

[0147] In another preferred embodiment, in the compound shown in formula (II), the light-colored circle and R3 are linked by an α1,3-oxyglycosidic bond.

[0148] In another preferred embodiment, in the compound shown in formula (II), R is a C5 straight-chain alkyl azide group.

[0149] In another preferred embodiment, in the compound shown in formula (II), R1 is absent or mannose-based.

[0150] In another preferred embodiment, in the compound shown in formula (II), R2 is selected from the group consisting of: none,

[0151]

[0152] The cube contains N-acetylglucosamine;

[0153] The light-colored round shape represents galactosyl groups;

[0154] The dark-colored circles represent mannosyl groups;

[0155] The rhombus shape represents the N-acetylneuraminic acid group.

[0156] In another preferred embodiment, in step (z1), the pH of the premixed solution is 6-9, preferably 7-9, for example, about 8.5.

[0157] In another preferred embodiment, in step (z2), the temperature of the sulfonation reaction is 25–40°C, more preferably 30–40°C, even more preferably 35–40°C, for example, about 37°C.

[0158] In another preferred embodiment, in step (z1), the premixed solution also contains Mg. 2+ Mg 2+ The concentration of the substance in the premixed solution is 1–200 mM, preferably 5–100 mM, more preferably 5–50 mM, for example, about 10 mM.

[0159] In another preferred embodiment, the method further includes step (z3) sialylating the sulfonated compound of formula (II) obtained in step (z2) with a glycosyltransferase, such that the Gal of the sulfonated compound of formula (II) is modified with sialic acid, thereby obtaining a sialylated and sulfonated compound.

[0160] In another preferred embodiment, in step (z3), the glycosyltransferase comprises Pd2,6ST.

[0161] In another preferred embodiment, the compound represented by formula (II) is selected from the group consisting of:

[0162]

[0163] In the formula, R is an empty, or C1-C10 straight-chain or branched alkyl azide group;

[0164] The cube contains N-acetylglucosamine;

[0165] The light-colored round shape represents galactosyl groups;

[0166] The dark-colored circles represent mannosyl groups;

[0167] The rhombus shape represents the N-acetylneuraminic acid group.

[0168] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0169] Figure 1 A schematic diagram of the synthesis method of N-glycoskeletons with different branching patterns is shown. a) Starting from an activated monosaccharide donor, a continuous chemical enzymatic synthesis strategy was used to achieve the large-scale synthesis of the core pentasaccharide (5). Subsequently, compound 5 was treated with glycosyltransferases MGAT4 and MGAT5 to obtain multi-branched skeleton intermediates 6-8. b) Compound 5 was first converted to compound 9 by glycosyltransferase B3GalT, and then converted to compound 10 by glycosyltransferase B4GalT1 and glycoside hydrolase B3GalH. Compounds 9 and 10 were modified with skeleton branching to obtain compounds 11 and 12, respectively. Compound 11 was further modified with enzymes to obtain compounds 13 and 14. The anomeric position of the reducing end GlcNAc in the compound is connected to a C1-C10 straight-chain or branched alkyl azide via an amide bond, that is, R is a C1-C10 straight-chain or branched alkyl azide group.

[0170] Figure 2 A schematic diagram of the synthesis of asymmetric N-sugar molecular skeletons using the substrate specificity of CHST6 is shown. Starting from compound 5, compounds 15 and 16 were synthesized under the catalysis of CHST6. CHST6 exhibited extremely low catalytic activity for compound 5. Using compound 17 as a substrate, compound 18 was synthesized under the catalysis of CHST6, ultimately yielding compound 18 in 93% yield. Based on the above results, compound 19 was used as a substrate. Utilizing the catalytic properties of CHST6, compound 20 was synthesized, and compounds A12 and A13 were further synthesized. In these compounds, the anomeric position of the reducing end GlcNAc is connected via an amide bond to a C1-C10 straight-chain or branched alkyl azide group, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0171] Figure 3Schematic diagrams of the synthesis of different N-sugar terminal fragments are shown. Galactosylation of GlcNAc catalyzed by B4GalT1; N-acetylglucosylation of Gal catalyzed by B3GNT2; 6-hydroxysulfonation of GlcNAc catalyzed by CHST6; galactosylation of (6S)GlcNAc catalyzed by B4GalT4; α2,6-sialylation of Gal catalyzed by ST6Gal1; α2,3-sialylation of Gal catalyzed by BtST; α2,8-sialylation of Neu5Ac catalyzed by CSTII; 6-hydroxysulfonation of Gal catalyzed by CHST1; 3-sulfonation of Gal catalyzed by Gal3ST2; and fucosylation of GlcNAc catalyzed by FucT. R' represents the N-sugar core backbone structure with or without functional linker arms.

[0172] Figure 4 The diagram illustrates the synthetic methods of compounds A1-A22. a) Compound 10 is extended to compound 23 via a sequential enzymatic process. Compound 23 is then converted to compound A1 by sulfonyltransferase CHST6 and sulfonyl donor PAPS. Finally, compound A1 is extended and modified by a sequential enzymatic process to obtain compounds A2-A11. b) Compounds A13 and A12 are converted to compounds A15-A20 by glycosyltransferases BtST, ST6Gal1, B4GalT4, and B4GalT1, respectively. Compound 20 is converted to A14 by the combined action of glycosyltransferases B4GalT1 and B4GalT4. A14 is further modified by sialylation to obtain compounds A21 and A22. The anomeric position of the reducing end GlcNAc in each compound is connected via an amide bond to a C1-C10 straight-chain or branched alkyl azide group, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0173] Figure 5A schematic diagram of the synthesis methods for compounds A23-A35 is shown. a) Compound D1 is converted to compound 18 by glycosyltransferase B3GNT2 and sulfonyltransferase CHST6. Subsequently, compound 18 is converted to compound A23 by glycosyltransferase B4GalT4. Finally, A23 is treated with sialylglycosyltransferases BtST, Pd2,6ST, and CST II to obtain compounds A24-A28. b) Compound 17 is converted to compound 25 by fucosyltransferase FucT. Compound 25 is treated with sulfonyltransferase CHST6 to obtain compound 26. Compound 26 is converted to compound A29 by glycosyltransferase B4GalT4. Subsequently, compound A29 is treated with sialylglycosyltransferase BtST to obtain compound A30. Finally, compound A30 is treated with sulfonyltransferase CHST1 to obtain compound A31. c) Compounds D1 and D5 are treated with sulfonyltransferase Gal3ST2 to obtain compounds A32 and A34, respectively. A32 was further sialylated to give compound A33. D17 was treated with sulfotransferase CHST1 to give compound A35. The anomeric position of the reducing end GlcNAc in the compound is connected by an amide bond to a C1-C10 straight-chain or branched alkyl azide group, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0174] Figure 6 The diagram shows the synthetic methods of compounds B1-B11. a) Compound 12 was extended by a series of enzymatic modifications to obtain compound 30. Compound 30 was converted to compound B1 by sulfonyltransferase CHST6. Finally, compound B1 was further modified to obtain compounds B2-B4. b) Compound 14 was extended by a series of enzymatic modifications to obtain compound 33. Compound 33 was converted to compounds B5-B11 by a series of glycosyltransferases and sulfonyltransferase CHST1. In these compounds, the anomeric position of the reducing end GlcNAc is connected to a C1-C10 straight-chain or branched alkyl azide via an amide bond, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0175] Figure 7 A schematic diagram of the synthesis methods for compounds B12-B18 is shown. a) Compound 28 is converted to compound B12 by glycosyltransferase B4GalT1 and sulfonyltransferase CHST6. Compound B12 is further modified to obtain compounds B13-B15. b) Compound 13 is converted to compound 36 by glycosyltransferase B3GNT2 and sulfonyltransferase CHST6. Compound 36 is further modified to obtain compounds B16-B18. In each compound, the anomeric position of the reducing end GlcNAc is connected to a C1-C10 straight-chain or branched alkyl azide via an amide bond, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0176] Figure 8 A schematic diagram of the synthesis of compounds B19-B31 is shown. Compounds 6 and 7 in a) and b) are converted to compounds 38 and 40, respectively, by glycosyltransferases B4GalT1 and B3GNT2, and by sulfonyltransferase CHST6. Compounds 38 and 40 are further modified to obtain compounds B19-B25. Compounds D2 and D3 are converted to compounds B26 and B28, respectively, by sulfonyltransferase Gal3ST2. Compounds B26 and B28 are converted to compounds B27 and B29, respectively, by sialylglycosyltransferase. Compounds D18 and D19 are converted to compounds B30 and B31, respectively, by sulfonyltransferase CHST1. In each compound, the anomeric position of the reducing end GlcNAc is connected via an amide bond to a C1-C10 straight-chain or branched alkyl azide group, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0177] Figure 9 A schematic diagram of the synthesis methods for compounds C1-C6 is shown. a) Compound 8 was treated with glycosyltransferases B4GalT1 and B3GNT2, and sulfonyltransferase CHST6 to obtain compound 42. Compound 42 was converted to C1 by glycosyltransferase B4GalT4. Subsequently, compound C1 was modified by sialylation to obtain compounds C2 and C3. b) Compound D4 was converted to compound C4 by sulfonyltransferase Gal3ST2, and compound C4 was sialylated to obtain compound C5. Compound D20 was treated with sulfonyltransferase CHST1 to obtain compound C6. In each compound, the anomeric position of the reducing end GlcNAc is connected by an amide bond to a C1-C10 straight-chain or branched alkyl azide group, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0178] Figure 10 Members of the sulfonated N-sugar characteristic molecular library of the present invention are shown. In the compounds, the anomeric position of the reducing end GlcNAc is connected via an amide bond to a C1-C10 straight-chain or branched alkyl azide group, i.e., R is a C1-C10 straight-chain or branched alkyl azide group.

[0179] Figure 11 The activity comparison between CHST2 and CHST6 is shown. a) SDS-PAGE electrophoresis analysis of CHST2 and CHST6. b) Comparison of the activities of CHST2 and CHST6 to substrate 17. The model reaction conditions were consistent with the general method for enzymatic reactions described in Example 13, with an overnight reaction time. The developing solvent for thin-layer chromatography was isopropanol / ammonia / water (7:3:2, v / v / v). c) Comparison of the activities of CHST2 and CHST6 to substrate E1. The reaction conversion was calculated by HPLC peak area (n=3). Detailed Implementation

[0180] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors have unexpectedly discovered a continuous chemoenzymatic synthesis strategy for synthesizing a library of sulfonated complex N-saccharides. Specifically, this invention comprehensively utilizes the branch selectivity of specific glycosyltransferases on the N-saccharide core pentasaccharide, and the site selectivity of sulfonyltransferases CHST1 and CHST6, to design and successfully synthesize 72 structurally well-defined sulfonated complex N-saccharide molecules using a continuous chemoenzymatic synthesis strategy. The key feature of the designed continuous chemoenzymatic synthesis strategy is the controllable modification of branching by utilizing the branch selectivity of specific glycosyltransferases on the N-saccharide core pentasaccharide, followed by the site selectivity of sulfonyltransferases CHST1 and CHST6 to complete the sulfonation of specific branches. This invention is based on this foundation.

[0181] the term

[0182] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0183] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0184] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0185] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0186] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0187] As used in this article, square It represents N-acetylglucosinolate (GlcNAc).

[0188] As used in this article, light-colored or yellow circles It represents galactosyl (Gal).

[0189] As used in this article, dark or green circles It represents mannosyl (Man).

[0190] As used in this article, rhombus It represents N-acetylneuraminic acid (Neu5Ac).

[0191] As used in this article, triangle It represents fucose.

[0192] As used in this article, the term “PAPS” refers to 3'-adenosine 5'-phosphosulfate, a sulfate donor.

[0193] As used in this article, in the structural formula, "6S" indicates that the 6th position of the residue is sulfonated; "3S" indicates that the 3rd position of the residue is sulfonated.

[0194] sulfonation reaction

[0195] Sulfation refers to the process by which sulfate groups (-OSO3) are converted into sulfuric acid groups in a living organism through an enzymatic reaction. - The process by which 3'-phosphoadenosine-5'-phosphosulfate is transferred from an active sulfuric acid donor (PAPS) to a specific acceptor molecule (such as a protein, glycolipid, polysaccharide, or small molecule metabolite).

[0196] Glycosyltransferase

[0197] Glycosyltransferases are a class of enzymes that catalyze the transfer of sugar groups (such as glucose, galactose, mannose, etc.) from active donors (usually nucleotide sugars, such as UDP-glucose, GDP-mannose) to acceptor molecules (such as proteins, lipids, other sugar chains or small molecules) to form glycosidic bonds.

[0198] In this invention, glycosyltransferases include, but are not limited to, Alg1, Alg2, MGAT1, MGAT2, MGAT4, MGAT5, B4GalT1, B4GalT4, B3GNT2, BtST, Pd2,6ST, ST6Gal1, M2,6ST, FucT, and CSTII. Those skilled in the art can select appropriate glycosyltransferases according to their needs. For example, appropriate glycosyltransferases can be selected based on the glycosyltransferases and their functions shown in Table 1.

[0199] Table 1. Glycosyltransferases and their functional characteristics

[0200]

[0201]

[0202] sulfotransferase

[0203] Sulfonotransferases (SULTs) are a class of enzymes that catalyze the transfer of sulfate groups from active donors (such as 3'-adenosine-5'-phosphate sulfate, PAPS) to various acceptor molecules.

[0204] In this invention, the sulfonyltransferase is selected from the group consisting of CHST1, CHST6, Gal3ST2, or combinations thereof. In this invention, the sulfonyltransferase is capable of sulfonating specific sites of compounds with specific structures.

[0205] The sulfonyltransferase CHST6 can sulfonate the 6-position of the N-acetylglucosamine group at the end of the compound shown in formula (I).

[0206]

[0207] In the formula, the cube represents N-acetylglucosamine;

[0208] The light-colored round shape represents galactosyl groups;

[0209] R1 is a monosaccharide residue;

[0210] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0211] R is absent, or a C1-C10 straight-chain or branched alkyl azide group.

[0212] In a preferred embodiment, in the N-sugar substrate with the structure shown in formula (I), R2 is not galactosyl.

[0213] In a preferred embodiment, in the N-sugar substrate with the structure shown in Formula (I), the light-colored circles in R1-R2 are connected to disaccharide residues that are not N-acetylglucosamine-galactose residues (GlcNAc-Gal residues).

[0214] The sulfonyltransferase CHST1 can sulfonate the 6-position of the galactose group in the compound shown in formula (II);

[0215]

[0216] In the formula, the cube represents N-acetylglucosamine;

[0217] The light-colored round shape represents galactosyl groups;

[0218] R1 is absent or a monosaccharide residue;

[0219] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0220] R is absent, or a C1-C10 straight-chain or branched alkyl azide group;

[0221] R3 is absent or has an N-acetylneuraminic acid group.

[0222] The sulfonyltransferase Gal3ST2 can sulfonate the 3-position of the galactose group in the compound shown in formula (II);

[0223]

[0224] In the formula, the cube represents N-acetylglucosamine;

[0225] The light-colored round shape represents galactosyl groups;

[0226] R1 is absent or a monosaccharide residue;

[0227] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0228] R is absent, or a C1-C10 straight-chain or branched alkyl azide group;

[0229] R3 is absent or has an N-acetylneuraminic acid group.

[0230] The method of the present invention

[0231] The method of this invention refers to a continuous chemical enzymatic synthesis method for sulfonated N-sugars, comprising the following steps:

[0232] (s1) A premix is ​​provided, the premix comprising:

[0233] (a) N-glycosidic substrate;

[0234] (b) PAPS; and

[0235] (c) Sulfonase, wherein the sulfonase is CHST6;

[0236] The N-glycosidic substrate has the structure shown in formula (I):

[0237]

[0238] In the formula, the cube represents N-acetylglucosamine;

[0239] The round shape represents galactosyl groups;

[0240] R1 is a monosaccharide residue or a disaccharide residue;

[0241] R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups;

[0242] R is absent, or a C1-C10 straight-chain or branched alkyl azide group;

[0243] (s2) Under enzyme-catalyzed conditions, the N-sugar substrate undergoes a sulfonation reaction with PAPS to modify the 6-position of the terminal N-acetylglucosamine group in the N-sugar substrate by sulfonation, thereby obtaining a sulfonated N-sugar (compound E6).

[0244] In a preferred embodiment, in the N-sugar substrate with the structure shown in Formula (I), R is a C5 linear alkyl azide group. In a preferred embodiment, in the N-sugar substrate with the structure shown in Formula (I), R1 is N-acetylglucosyl or a disaccharide residue composed of N-acetylglucosyl and fucose. In a preferred embodiment, in the N-sugar substrate with the structure shown in Formula (I), R2 is not galactosyl. In a preferred embodiment, in the N-sugar substrate with the structure shown in Formula (I), the disaccharide residues connected to the light-colored circles in R1-R2 are not N-acetylglucosyl-galactose residues (GlcNAc-Gal residues).

[0245] In a preferred embodiment, the method further includes step (s3) glycosylation of the sulfonated N-sugar using a glycosyltransferase, thereby modifying the terminal end of the sulfonated N-sugar with a glycosyl group to obtain a glycosyl-modified sulfonated N-sugar (compound E7). Preferably, step (s3a) involves galactosylation of the sulfonated N-sugar using B4Gal4 catalyzes the process to obtain a galactosylated sulfonated N-sugar (compound E7).

[0246] In a preferred embodiment, the method further includes step (s4) sializing compound E7 with a glycosyltransferase to modify the Gal of compound E7 with sialic acid, thereby obtaining a sialylated compound. In a preferred embodiment, step (s4) is selected from the group consisting of: (s4a) using ST6Gal1 to catalyze the α2,6 sialylation of Gal in compound E7, thereby modifying the end of compound E7 with Neu5Ac to obtain compound E8; (s4b) using BtST to catalyze the α2,3 sialylation of Gal in compound E7, thereby modifying the end of compound E7 with Neu5Ac to obtain compound E10; and / or (s4c) using CSTII to catalyze the α2,8 sialylation of Neu5Ac in compound E8 or E10, thereby further modifying the end of compound E8 or E10 with Neu5Ac to obtain compound E9 or E11; (s4d) using Pd2,6ST to catalyze the α2,6 sialylation of Gal (at or near the end) in compound E10, thereby further sialylating Gal in compound E10 to obtain compound E12.

[0247] In a preferred embodiment, the method further includes step (s5) sulfonating the sialylated compound with a sulfonase to obtain a disulfonated N-sugar, preferably using CHST1 to catalyze the sulfonation of the 6-hydroxyl group of Gal in E10 to obtain compound E13.

[0248] In a preferred embodiment, the method further includes step (s6) of hydrolyzing the sulfonated N-sugar obtained in step (s2) with a glycoside hydrolase, such that the terminal glycosyl groups of the non-sulfonated branches of the sulfonated N-sugar are hydrolyzed.

[0249] In one exemplary embodiment, compound 10 is converted to compound 23 via a sequential enzymatic elongation using a glycosyltransferase, which is then catalyzed by the sulfonyltransferase CHST6 to generate compound A1; using compound A1 as a substrate, different glycosyltransferases are used to catalyze the generation of compounds A2-A11. Figure 4 (a)

[0250] In one exemplary embodiment, compounds A13 and A12 are reacted with glycosyltransferases BtST, ST6Gal1, B4GalT4, and B4GalT1 to generate compounds A15-A20, respectively; compound A20 is reacted with glycosyltransferases B4GalT1 and B4GalT4 to generate compound A14, and compound A14 is further sialylated to give compounds A21 and A22. Figure 4 (b)

[0251] In one exemplary embodiment, compound D1 is reacted with a glycosyltransferase to generate compound 17, which is then sulfonated with a sulfonyltransferase CHST6 to generate compound 18; compound 18 is reacted with a glycosyltransferase B4GalT4 to generate compound A23; compound A23 is then sialylated by one or more glycosyltransferases to generate compounds A24-A28. Figure 5 (a) Compound 17 is converted to compound 25 by fucose glycosyltransferase FucT. Compound 25 is sulfonated by sulfonase CHST6 to generate compound 26; compound 26 is converted to compound A29 by glycosyltransferase B4GalT4, and then compound A29 is sialylated to generate compound A30; finally, compound A30 is sulfonated again (by sulfonase CHST1) to generate compound A31. Figure 5 (b) Compounds D1 and D5 are reacted with sulfonyltransferase Gal3ST2 to form compounds A32 and A34; compound A32 is further sialylated to form compound A33; compound D17 is reacted with sulfonyltransferase CHST1 to form compound A35. Figure 5 (c)

[0252] In one exemplary embodiment, compound 12 is continuously enzymatically modified and extended to yield compound 30. Compound 30 is then reacted with sulfonyltransferase CHST6 to generate compound B1. Compound B1 is further reacted with glycosyltransferase to generate compound B2. Compound B2 is then sialylated to generate compounds B3 and B4. Figure 6 (a) Compound 14 was continuously extended by enzymatic modification to obtain compound 32. Compound 32 was reacted with sulfonyltransferase CHST6 to generate compound 33. Compound 33 was reacted with glycosyltransferase to generate compound B5 or B8. Compound B5 was sialylated to generate compounds B6 and B7. Compound B8 was sialylated to generate compound B9. Compound B9 was reacted with sulfonyltransferase CHST1 to generate compound B10, which was subsequently sialylated to generate compound B11. Figure 6 (b)

[0253] In one exemplary embodiment, compound 28 is reacted with glycosyltransferase to generate compound 34, which is then reacted with sulfonyltransferase CHST6 to generate compound B12. Compound B12 is reacted with glycosyltransferase to generate compound B13, which is then sialylated to generate compound B14 or B15. Figure 7 (a) Compound 13 is converted to compound 35 by glycosyltransferase, then to compound 36 by sulfonyltransferase CHST6, and compound 36 is converted to compound B16 by glycosyltransferase, which is then sialylated to generate compound B17 or B18. Figure 7 (b)

[0254] In one exemplary embodiment, compound 37 is reacted with sulfonyltransferase CHST6 to generate compound 38, compound 38 is reacted with glycosyltransferase to generate compound B19, which is then sialylated to generate compound B20 or B21. Figure 8 (a) Compound 39 is converted to compound 40 by sulfonyltransferase CHST6, and compound 40 is converted to compound B22 by glycosyltransferase, which then undergoes one or more sialylation reactions to generate compounds B23, B24, or B25. Figure 8 (b) Compounds D2 and D3, under the action of sulfonyltransferase Gal3ST2, generate compounds B26 and B28, which then undergo sialylation to generate compounds B27 and B29. Compounds D18 and D19, under the action of sulfonyltransferase CHST1, generate compounds B30 and B31. Figure 8 (c)

[0255] In one exemplary embodiment, compound 41 is reacted with sulfonyltransferase CHST6 to generate compound 42, compound 42 is reacted with glycosyltransferase to generate compound C1, which is then sialylated to generate compound C2 or C3. Figure 9 (a) Compound D4 is converted to compound C4 by sulfonyltransferase Gal3ST2, which then undergoes sialylation to generate compound C5. Compound D20 is converted to compound C6 by sulfonyltransferase CHST1. Figure 9 (b)

[0256] The molecular library of the present invention

[0257] As used herein, the terms "molecular library of the present invention", "N-sugar library of the present invention", and "molecular library of the present invention" are used interchangeably and all refer to a molecular library composed of sulfonated N-sugars prepared by the method of the present invention.

[0258] In a preferred embodiment, the shown molecular library consists of sulfonated N-sugars selected from the group consisting of:

[0259]

[0260]

[0261] In the formula, R is an empty, or C1-C10 straight-chain or branched alkyl azide group; the cube is N-acetylglucosyl;

[0262] The light-colored round shape represents galactosyl groups;

[0263] The dark-colored circles represent mannosyl groups;

[0264] The rhombus shape represents an N-acetylneuraminic acid group;

[0265] The triangle represents fucose.

[0266] The main advantages of this invention include:

[0267] 1. A continuous chemoenzymatic method was used to synthesize sulfonated N-sugar compounds. Specific sulfonate transferases were used to sulfonate specific sites in N-sugar compounds, achieving controlled modification. Sulfonate transferases CHST1 and CHST6 were used to sulfonate specific branches of N-sugar compounds, ultimately yielding 72 target compounds.

[0268] 2. A variety of structurally complex sulfonated N-sugars were synthesized through sulfonate transferase, glycosyltransferase and glycoside hydrolase.

[0269] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0270] Example 1: Synthesis of Compound 4

[0271]

[0272] Substrate 3 (14 g, 11 mmol, 1.0 equiv) was dissolved in 150 mL of deionized water. After stirring at 0 °C for 15 min, 2-chloro-1,3-dimethylimidazoline chloromonium (37 g, 220 mmol, 20.0 equiv), sodium azide (43 g, 660 mmol, 60.0 equiv), and 2,6-dimethylpyridine (5 mL, 44 mmol, 40.0 equiv) were added sequentially. The reaction was carried out at 0 °C for 72 h, and the reaction progress was monitored by thin-layer chromatography (TLC) (isopropanol / ammonia / water, 7:3:2, v / v). Once the reaction was complete, the supernatant was centrifuged and directly purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 4 (14.3 g, quantum).

[0273] 1 H NMR(800MHz,D2O)δ5.12(d,J=1.8Hz,1H),4.92(d,J=1.7Hz,1H),4.78–4.74(m,2H),4.61(d,J=8.1Hz,1H),4.56(d,J=8.4Hz,2H),4.25(d, J=2.9Hz,1H),4.19(dd,J=3.5,1.6Hz,1H),4.11(dd,J=3.5,1.7Hz,1H),3.99–3.85(m,9H),3.83–3.40(m,30H),2.09(s,3H),2.06(s,6H). 13C NMR(201MHz,D2O)δ174.77,174.74,174.64,101.32,100.42,99.60,99.57,97.00,88. 50,80.43,79.50,78.68,76.45,76.42,76.29,75.80,75.77,74.36,74.29,73.54,73.3 6,73.25,72.82,72.22,71.96,70.17,69.88,69.44,69.37,67.32,67.27,65.85,65.6 8,61.59,60.60,59.89,55.31,54.88,54.47,22.30,22.19,22.08.ESI-Q-TOF-HRMSm / z calculated for C 50 H 84 N7O 35 [M+H] + :1342.5003,found1342.5003.

[0274] Example 2 Synthesis of Compound 5

[0275]

[0276] Compound 4 (14.3 g, 11 mmol, 1.0 equiv) was dissolved in 500 mL of anhydrous methanol, and 1,3-propanediol and DIPEA were added sequentially. The mixture was stirred at room temperature for 2 h under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography (TLC) (isopropanol / ammonia / water, 7:3:2, v / v). Once the reaction was complete, 300 mL of ice-cold diethyl ether was immediately added to the reaction mixture. After thorough shaking, the mixture was centrifuged for 5 min, and the supernatant was discarded to obtain a white precipitate as the crude product. The crude product was not purified and proceeded directly to the next step.

[0277] 5-Azide-valeric acid (2.8 mL, 22 mmol, 2.0 equiv) was dissolved in 300 mL of anhydrous DMF, followed by the addition of HATU (12.5 g, 33 mmol, 3.0 equiv) and DIPEA (11.5 mL, 66 mmol, 6.0 equiv). After stirring at room temperature for 15 min, the crude product from the previous step was added, and the reaction was allowed to proceed at room temperature for 2 h. The reaction progress was monitored by thin-layer chromatography (TLC) (isopropanol / ammonia / water, 7:3:2, v / v). Once the reaction was complete, 300 mL of deionized water was added to the reaction mixture, and the mixture was extracted once with ethyl acetate. The aqueous phase was concentrated to obtain the crude compound 5. The crude compound was purified by reversed-phase silica gel column chromatography (C18) to obtain compound 5 (11.8 g, 77% for 2 steps).

[0278] 1H NMR(800MHz,D2O)δ5.13(s,1H),5.07(d,J=9.7Hz,1H),4.93(s,1H),4.78(s,1H),4.63( d,J=8.1Hz,1H),4.56(d,J=8.7Hz,2H),4.26(d,J=2.9Hz,1H),4.20(d,J=3.9Hz,1H),4. 12(d,J=3.7Hz,1H),4.00–3.41(m,39H),3.34(t,J=6.7Hz,2H),2.31(t,J=7.2Hz,2H),2 .09(s,3H),2.06(s,6H),2.02(s,3H),1.65(p,J=7.5Hz,2H),1.57(q,J=7.3Hz,2H).13C NMR(201MHz,D2O)δ177.32,174.77,174.74,174.64,174.59,101.26,100.42,99.60,99.58,97.01,80.43, 79.53,78.57,78.18,76.42,76.29,76.22,75.80,75.78,74.37,74.29,73.54,73.36,73.25,72.83,72.72 ,71.96,70.17,69.89,69.87,69.44,69.38,67.32,67.27,65.84,65.68,61.68,61.60,60.60,59.92,59.8 3,55.31,54.90,53.78,50.64,35.21,27.33,22.38,22.30,22.18,21.96.ESI-Q-TOF-HRMSm / zcalculated for C55H93N8O36,[M+H]+:1441.5687,found 1441.5679.

[0279] Example 3: Reaction of β1,3-Gal using B3GalT

[0280] The substrate molecule (1.0 eq) and UDP-Gal (1.5 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase B3GalT was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and then purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0281] Taking compound 9 as an example, other compounds can be synthesized using the same or similar methods.

[0282] Compound 5 (300 mg, 0.21 mmol) and UDP-Gal (178 mg, 0.32 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). An appropriate amount of glycosyltransferase B3GalT was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 9 (275 mg, 82%).

[0283] 1H NMR(600MHz,D2O)δ5.13(d,J=1.7Hz,1H),5.07(d,J=9.7Hz,1H),4.93(d,J=1.7Hz,1H),4.7 8(s,1H),4.64–4.58(m,2H),4.56(d,J=8.4Hz,1H),4.45(d,J=7.8Hz,1H),4.26(d,J=2.2Hz ,1H),4.21(d,J=3.3Hz,1H),4.11(d,J=3.4Hz,1H),4.00–3.41(m,45H),3.34(t,J=6.8Hz,2 H),2.31(t,J=7.2Hz,2H),2.12–1.98(m,12H),1.65(p,J=7.1Hz,2H),1.61–1.52(m,2H).13C NMR(201MHz,D2O)δ177.32,174.78,174.74,174.64,174.59,103.40,101.26,100.42,99.58,99.53,99.33,97.00,81.72 ,80.40,79.53,78.59,78.18,76.43,76.30,76.22,75.78,75.33,75.25,74.38,74.30,73.54,73.37,72.83,72.72,72.42 ,71.96,70.65,70.18,69.88,69.45,69.36,68.62,68.51,67.33,67.27,65.85,65.70,61.68,61.61,61.02,60.62,60.60 ,59.93,59.84,55.32,54.91,54.42,53.79,50.64,35.22,27.34,22.39,22.36,22.32,22.19,21.97.ESI-Q-TOF-HRMSm / z calculatedfor C61H103N8O41,[M+H]+:1603.6215,found 1603.6211.

[0284] Example 4: Reaction of β1,4-Gal using B4GalT1

[0285] The substrate molecule (1.0 eq) and UDP-Gal (1.5 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase B4GalT1 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0286] Taking compound A13 as an example, other compounds can be synthesized using the same or similar methods.

[0287] Compound 20 (30 mg, 0.016 mmol) and UDP-Gal (14 mg, 0.024 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). An appropriate amount of glycosyltransferase B4GalT1 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound A13 (23 mg, 70%).

[0288] 1H NMR(600MHz,D2O)δ5.12(s,1H),5.07(d,J=9.7Hz,1H),4.93(s,1H),4.77(s,1H),4.69(d,J=8. 5Hz, 1H), 4.62 (d, J=8.0Hz, 1H), 4.58 (d, J=7.8Hz, 2H), 4.47 (dd, J=7.8, 2.3Hz, 2H), 4.33 (dd, J= 11.2,2.2Hz,1H),4.27–4.18(m,4H),4.11(d,J=1.8Hz,1H),4.01–3.43(m,54H),3.34(t,J=6.7H z,2H),2.30(t,J=7.2Hz,2H),2.10–1.98(m,15H),1.65(p,J=7.5Hz,2H),1.59–1.53(m,2H).13C NMR(151MHz,D2O)δ177.30,174.90,174.68,174.62,174.57,102.94,102.89,102.85,101.24,100.41,99.52,99.43,97.03,82.30,80.41 ,79.45,78.63,78.55,78.44,78.16,76.36,76.28,76.20,75.32,75. 02,74.70,74.66,74.38,73.52,73.47,72.81,72.70,72.47,72.06,7 1.98,71.91,70.93,70.16,69.85,69.56,69.42,69.36,68.51,68.24 ,67.31,67.26,67.17,65.71,65.62,61.68,61.62,61.09,60.99,59. 97,59.94,59.90,59.81,55.54,54.93,54.83,54.75,53.78,50.62,35.20,27.32,22.37,22.31,22.19,22.15,21.96.ESI-Q-TOF-HRMSm / z calculatedfor C75H127N9O54S,[M+2H]2+:1024.8589,found m / z1024.8588.

[0289] Using a method similar to that used to synthesize compound A13, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0290] Table 2

[0291]

[0292]

[0293]

[0294]

[0295] Note: Compounds 6, 7, 8, 20, 33, 36, A6, A13 and B10 require the use of B4GalT1 and BtST, B4GalT4 or B3GNT2 in combination to synthesize the corresponding target compounds.

[0296] Example 5: Reaction of β1,4-Gal using B4GalT4

[0297] The substrate molecule (1.0 eq) and UDP-Gal (1.5 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase B4GalT4 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0298] Taking compound B19 as an example, other compounds can be synthesized using the same or similar methods.

[0299] Compound 38 (264 mg, 0.09 mmol) and UDP-Gal (229 mg, 0.41 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase B4GalT4 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound B19 (248 mg, 81%).

[0300] 1H NMR(600MHz,D2O)δ5.12(s,1H),5.08(d,J=9.6Hz,1H),4.93(s,1H),4.77(s,1H),4.74–4.72(m,3H), 4.63(d,J=8.0Hz,1H),4.61–4.56(m,2H),4.56–4.51(m,4H),4.49–4.44(m,3H),4.41(d,J=10.7Hz,3H ),4.33(dd,J=11.0,3.8Hz,3H),4.25–4.17(m,5H),4.12(d,J=3.6Hz,1H),4.07–3.47(m,90H),3.35( t,J=6.7Hz,2H),2.31(t,J=7.2Hz,2H),2.12–1.96(m,24H),1.69–1.62(m,2H),1.61–1.53(m,2H).13C NMR(151MHz,D2O)δ177.31,174.87,174.68,174.63,174.58,174.55,102. 95,102.90,102.78,102.57,101.58,101.26,100.37,99.52,99.45,99.09 ,97.05,82.31,82.24,80.30,79.42,78.59,78.21,78.17,77.50,76.31,7 6.22,75.98,75.33,75.02,74.79,74.72,74.66,74.38,74.34,72.81,72. 70,72.47,72.38,72.11,72.06,71.99,71.94,70.97,70.12,69.86,69.42 ,68.60,68.30,68.26,68.09,67.33,66.48,65.66,61.62,61.10,61.03,6 0.06,59.97,59.92,59.83,55.15,55.09,54.92,54.76,53.80,50.64,35. 22,27.33,22.39,22.33,22.21,22.19,22.16,21.97.ESI-Q-TOF-HRMSm / z calculated for C123H206N12O95S3,[M+2H]2+:1733.5404, found m / z 1733.5404.

[0301] Using a method similar to that used to synthesize compound B19, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0302] Table 3

[0303]

[0304]

[0305]

[0306]

[0307] Note: Compounds E20, A5, A12, 33, 36 and 40 require the use of B4GalT4 and B4GalT1, BtST or ST6Gal1 in combination to synthesize the corresponding target compounds.

[0308] Example 6: Reaction of β1,4-GlcNAc using MGAT4

[0309] The substrate molecule (1.0 eq) and UDP-GlcNAc (1.5 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 5 mM MnCl2, pH 7.5). Glycosyltransferase MGAT4 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0310] Taking compound 6 as an example, other compounds can be synthesized using the same or similar methods.

[0311] Compound 5 (240 mg, 0.17 mmol) and UDP-GlcNAc (166 mg, 0.26 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 5 mM MnCl2, pH 7.5). An appropriate amount of glycosyltransferase MGAT4 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 6 (250 mg, 91%).

[0312] 1H NMR(600MHz,D2O)δ5.12(s,1H),5.07(d,J=9.7Hz,1H),4.92(s,1H),4.77(s,1H),4.62 (d,J=7.9Hz,1H),4.58–4.50(m,3H),4.22(d,J=1.7Hz,2H),4.13–4.10(m,1H),4.05(dd ,J=9.3,3.3Hz,1H),4.00–3.39(m,44H),3.34(t,J=6.7Hz,2H),2.31(t,J=7.2Hz,2H), 2.10–2.08(m,6H),2.06(s,6H),2.01(s,3H),1.68–1.62(m,2H),1.60–1.53(m,2H).13C NMR(151MHz,D2O)δ177.31,174.76,174.73,174.64,174.58,101.64,101.26,100.39,99.66,99.57,99.14,97.00,80.34,7 9.51,78.56,78.31,78.17,76.28,76.22,75.93,75.88,75.81,75.77,74.36,74.28,73.48,73.36,73.25,72.82,72.71,71 .95,71.88,70.12,69.90,69.87,69.66,69.43,68.10,67.32,65.83,65.70,62.46,61.59,61.27,60.71,60.59,60.55,59. 92,59.83,55.59,55.31,55.26,54.88,53.78,50.63,35.21,27.33,22.37,22.30,22.18,22.13,21.96.ESI-Q-TOF-HRMSm / z calculated forC63H107N9O41,[M+2H]2+:822.8277,found m / z 822.8278.

[0313] Using a method similar to that used to synthesize compound 6, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0314] Table 4

[0315]

[0316] Example 7: Reaction of β1,6-GlcNAc using MGAT5

[0317] The substrate molecule (1.0 eq) and UDP-GlcNAc (1.5 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase MGAT5 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used.

[0318] Taking compound 7 as an example, other compounds can be synthesized using the same or similar methods.

[0319] Compound 5 (300 mg, 0.21 mmol) and UDP-GlcNAc (205 mg, 0.32 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 5 mM MnCl2, pH 7.5). An appropriate amount of glycosyltransferase MGAT5 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 7 (311 mg, 91%).

[0320] 1H NMR(600MHz,D2O)δ5.13(s,1H),5.07(d,J=9.7Hz,1H),4.87(s,1H),4.78(s,1H),4.62(d,J=7.9Hz ,1H),4.58(d,J=3.6Hz,1H),4.56(d,J=3.6Hz,1H),4.54(d,J=8.4Hz,1H),4.26(d,J=3.1Hz,1H),4. 23–4.16(m,2H),4.10(dd,J=3.6,1.6Hz,1H),3.97–3.39(m,44H),3.34(t,J=6.7Hz,2H),2.31(t,J= 7.2Hz,2H),2.08(s,3H),2.07–2.04(m,9H),2.02(s,3H),1.69–1.61(m,2H),1.61–1.53(m,2H).13C NMR(151MHz,D2O)δ177.32,174.75,174.63,174.58,174.23,101.65,101.28,100.37,99.74,99.63,99.57,97.18,80 .36,79.39,78.58,78.17,76.53,76.44,76.22,75.79,74.45,74.38,73.65,73.54,73.28,73.26,72.71,71.91,71.5 6,70.19,69.94,69.92,69.88,69.40,69.36,67.50,67.28,65.62,65.50,61.70,60.76,60.59,59.86,59.83,55.56, 55.37,55.30,54.88,53.77,50.63,35.21,27.33,22.47,22.38,22.31,22.20,21.95.ESI-Q-TOF-HRMSm / zcalculated for C63H107N9O41,[M+2H]2+:822.8277, found m / z 822.8280.

[0321] Using a method similar to that used to synthesize compound 7, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0322] Table 5

[0323]

[0324] Example 8: Reaction of β1,3-GlcNAc using B3GNT2

[0325] The substrate molecule (1.0 eq) and UDP-GlcNAc (1.5 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase B3GNT2 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0326] Taking compound 30 as an example, other compounds can be synthesized using the same or similar methods.

[0327] Compound 29 (200 mg, 0.07 mmol) and UDP-GlcNAc (68 mg, 0.11 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). An appropriate amount of glycosyltransferase B3GNT2 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 30 (161 mg, 75%).

[0328] 1H NMR(600MHz,D2O)δ5.14(s,1H),5.07(d,J=9.6Hz,1H),4.95(s,1H),4.77(s,1H),4.69(d,J=8.4Hz,1H),4.65–4.58(m,3H),4.55(d,J=8.3Hz,1H),4.48–4.41(m,3H),4.25–4.21(m,2H),4.15(d,J=3.3Hz,1H),4.12(d,J=2.8Hz,1H),4.07–3.44(m,82H),3.34(t,J=6.7Hz,2H),2.71–2.65(m,2H),2.31(t,J=7.2Hz,2H),2.11–2.00(m,4H),1.73(t,J=12.2Hz,2H),1.69–1.61(m,2H),1.61–1.53(m,2H).13C NMR(126MHz,D2O)δ177.31,174.96,174.90,174.88,174.72,174.64,174.58,174.55,173.52,103.54,102.88,102.85,101.59,101.27,100.43,100.15,100.12,99.35,99.24,99.10,96.95,81.81,80.70,80.60,80.35,79.61,78.61,78.19,77.97,76.21,75.96,75.63,74.86,74.77,74.40,74.35,74.30,73.67,73.54,72.81,72.70,72.52,72.39,72.13,72.05,71.95,71.70,70.72,70.13,70.01,69.67,69.44,68.37,68.34,68.20,68.11,67.31,65.88,65.71,63.31,62.63,61.60,61.25,60.95,60.45,60.30,60.20,59.95,59.83,55.63,55.11,54.89,54.63,54.55,53.77,51.85,50.63,40.05,35.21,27.32,22.47,22.38,22.22,22.14,22.02,21.95.ESI-Q-TOF-HRMSm / z calculated for C111H180N12O77,[M-2H]2-:1456.5275,found m / z 1456.5273.

[0329] Using a method similar to that used to synthesize compound 30, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0330] Table 6

[0331]

[0332]

[0333] Note: Compounds 5, 6 and 7 require the use of B3GNT2 and B4GalT1, B3GalH, B3GalT or B4GalT1 in combination to synthesize the corresponding target compounds.

[0334] Example 9: Reaction of α2,3-Neu5Ac using BtST

[0335] The substrate molecule (1.0 eq) and CMP-Neu5Ac (2 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 8.5). Glycosyltransferase BtST was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, ion exchange column purification was performed.

[0336] Taking compound B23 as an example, other compounds can be synthesized using the same or similar methods.

[0337] For example, the synthesis of compound B23: Compound B22 (80 mg, 0.02 mmol) and CMP-Neu5Ac (74 mg, 0.12 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 8.5). An appropriate amount of glycosyltransferase BtST was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction. The supernatant was collected by centrifugation, concentrated, and then purified by Bio-P2 size exclusion chromatography with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound B23 (57 mg, 57%).

[0338] 1H NMR(800MHz,D2O)δ5.12(s,1H),5.06(d,J=9.7Hz,1H),4.87(s,1H),4.76(s,1H),4.72–4.69(m,3H),4.63–4.56(m,6H),4.54(d,J=8.0Hz,1H),4.48–4.44(m,3H),4.39(d,J=10.7Hz,3H),4.32(d,J=10.6Hz,3H),4.25(s,1H),4.23–4.16(m,5H),4.12(dd,J=9.9,3.1Hz,3H),4.08(d,J=3.8Hz,1H),4.02–3.94(m,6H),3.93–3.53(m,99H),3.50(t,J=9.7Hz,1H),3.39(t,J=9.7Hz,1H),3.33(t,J=6.6Hz,2H),2.74(dd,J=12.5,4.6Hz,3H),2.30(t,J=7.2Hz,2H),2.09–1.98(m,33H),1.80(t,J=12.1Hz,3H),1.67–1.61(m,2H),1.59–1.53(m,2H).13C NMR(126MHz,D2O)δ177.31,174.91,174.86,174.68,174.62,174.58,174.49,174.09,173.85,102.95,102.86,102.81,102.17,101.60,101.32,100.28,99.68,99.63,99.52,97.27,82.35,80.24,79.13,78.68,78.60,78.55,78.17,77.29,77.26,76.58,76.49,76.21,75.32,75.06,75.02,74.67,74.39,73.54,72.84,72.69,72.37,72.20,72.06,71.92,71.88,71.48,70.38,70.28,70.15,69.85,69.42,69.36,68.39,68.25,68.05,67.54,67.46,67.27,66.49,65.61,62.52,61.71,61.10,61.03,60.10,59.95,59.82,55.13,54.97,54.90,54.79,53.78,51.68,50.63,39.54,35.21,27.33,22.53,22.38,22.33,22.24,22.17,22.04,21.97.ESI-Q-TOF-HRMSm / z calculated for C156H252N15O119S3,[M-3H]3-:1445.1102, found m / z 1445.1104.

[0339] Using a method similar to that used to synthesize compound B23, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0340] Table 7

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] Note: Compounds A6, A5, A13, A12 and B10 require the combined use of BtST and B4GalT1 to synthesize the corresponding target compounds.

[0347] Example 10 uses Pd2,6ST, ST6Gal1 or M2,6ST to mount the reaction of α2,6-Neu5Ac.

[0348] The substrate molecule (1.0 eq) and CMP-Neu5Ac (2 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 8.5). Glycosyltransferases Pd2,6ST, ST6Gal1, or M2,6ST were added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, ion exchange column purification was performed.

[0349] Taking compound C3 as an example, other compounds can be synthesized using the same or similar methods.

[0350] Compound C1 (30 mg, 0.007 mmol) and CMP-Neu5Ac (34 mg, 0.056 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 8.5). An appropriate amount of glycosyltransferase ST6Gal1 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound C3 (19 mg, 51%).

[0351] 1H NMR(800MHz,D2O)δ5.13(s,1H),5.06(d,J=9.7Hz,1H),4.86(s,1H),4.76–4.72(m,5H),4.61(d,J=7.8Hz,2H),4.57(d,J=8.0Hz,1H),4.54(d,J=6.9Hz,2H),4.50–4.44(m,8H),4.41(d,J=10.7Hz,4H),4.32–4.25(m,4H),4.25–4.17(m,7H),4.10–4.08(m,1H),4.05–4.03(m,1H),4.02–3.95(m,8H),3.95–3.48(m,128H),3.40(t,J=9.7Hz,1H),3.34(t,J=6.8Hz,2H),2.67(dd,J=12.5,4.7Hz,4H),2.30(t,J=7.2Hz,2H),2.11–1.95(m,42H),1.72(t,J=12.2Hz,4H),1.67–1.61(m,2H),1.59–1.53(m,2H).13C NMR(126MHz,D2O)δ177.30,174.88,174.67,174.62,174.57,174.53,174.48,174.03,173.54,103.44,102.98,102.88,102.85,102.60,101.66,101.54,101.36,100.12,99.66,99.63,99.01,97.35,82.32,80.33,80.31,80.26,79.97,78.95,78.68,78.60,78.25,78.17,76.68,76.22,76.10,75.02,74.79,74.69,74.64,74.40,73.63,72.68,72.52,72.36,72.27,72.13,72.05,71.94,71.84,71.68,71.45,70.74,70.12,69.85,69.37,68.39,68.36,68.25,68.20,67.51,66.88,65.60,63.28,62.63,61.13,60.03,59.95,59.90,59.81,55.09,54.93,54.83,53.78,51.88,50.63,40.05,35.21,27.33,22.57,22.40,22.37,22.28,22.24,22.16,22.02,21.96.ESI-Q-TOF-HRMSm / z calculated forC195H318N18O150S4,[M+3H]3+:1814.2303, found m / z 1814.2308.

[0352] Using a method similar to that used to synthesize compound C1, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0353] Table 8

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] Note: Compounds A13 and A12 require the combined use of ST6Gal1, B4GalT1, and B4GalT4 to synthesize the corresponding target compounds.

[0360] Example 11: Reaction of α2,8-Neu5Ac using CST II

[0361] The substrate molecule (1.0 eq) and CMP-Neu5Ac (2 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 8.5). Glycosyltransferase CST II was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, ion exchange column purification was performed.

[0362] Taking compound A27 as an example, other compounds can be synthesized using the same or similar methods.

[0363] Compound A25 (13 mg, 0.004 mmol) and CMP-Neu5Ac (9.8 mg, 0.016 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 8.5). An appropriate amount of glycosyltransferase CST II was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound A27 (8 mg, 53%).

[0364] 1H NMR(800MHz,D2O)δ5.12(s,1H),5.07(d,J=9.8Hz,1H),4.93(s,1H),4.77(s,1H),4.74–4.72(m,2H),4.62(d,J=8.3Hz,1H),4.58(d,J=8.1Hz,2H),4.50–4.44(m,4H),4.42(d,J=10.5Hz,2H),4.29(dd,J=11.2,5.7Hz,2H),4.25(d,J=3.0Hz,1H),4.22–4.16(m,5H),4.15–4.09(m,3H),4.02–3.47(m,93H),3.34(t,J=6.8Hz,2H),2.78(dd,J=12.4,4.7Hz,2H),2.58(dd,J=12.3,4.4Hz,2H),2.30(t,J=7.3Hz,2H),2.11–1.97(m,30H),1.75(t,J=12.2Hz,2H),1.68–1.61(m,4H),1.60–1.53(m,2H).13C NMR(126MHz,D2O)δ177.31,175.01,174.97,174.86,174.69,174.62,174.58,173.41,173.36,103.56,102.97,102.95,102.62,101.26,100.79,100.48,99.55,99.52,97.06,82.31,82.29,80.63,80.42,79.35,78.58,78.48,78.17,76.53,76.38,76.21,75.03,74.68,74.65,74.42,74.36,74.09,73.76,73.54,72.82,72.70,72.63,72.34,72.27,72.12,72.08,71.98,71.92,71.75,70.70,70.18,69.84,69.78,69.43,69.38,68.49,68.44,68.26,68.14,67.74,67.33,67.28,66.95,65.48,63.77,62.56,61.68,61.13,59.93,59.82,54.97,54.80,54.75,54.68,53.80,52.34,51.74,50.63,40.50,40.09,35.21,27.33,22.38,22.32,22.29,22.21,22.03,21.97.ESI-Q-TOF-HRMSm / z calculated for C139H223N14O104S2,[M-3H]3-:1272.0683, found m / z 1272.0671.

[0365] Using a method similar to that used to synthesize compound A27, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0366] Table 9

[0367]

[0368] Example 12: Reaction of α1,4-Fuc using FucT

[0369] The substrate molecule (1.0 eq) and GDP-Fuc (2 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase FucT was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and then purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0370] Taking compound 25 as an example, other compounds can be synthesized using the same or similar methods.

[0371] Compound 17 (166 mg, 0.08 mmol) and GDP-Fuc (101 mg, 0.16 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Glycosyltransferase FucT was added to the reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 25 (172 mg, 91%).

[0372] 1H NMR(600MHz,D2O)δ5.15–5.09(m,3H),5.06(d,J=10.1Hz,1H),4.91(s,1H),4.84–4.81(m,2H),4.7 7(s,1H),4.71–4.66(m,2H),4.64–4.55(m,3H),4.46–4.40(m,2H),4.25(s,1H),4.18(d,J=3.5Hz, 1H),4.12–4.08(m,3H),4.02–3.42(m,67H),3.34(t,J=6.7Hz,2H),2.30(t,J=7.2Hz,2H),2.08(s, 3H),2.06–2.02(m,12H),2.01(s,3H),1.69–1.61(m,2H),1.60–1.52(m,2H),1.17–1.13(m,6H).13C NMR(151MHz,D2O)δ177.31,174.88,174.63,174.57,174.47,174.45,10 2.77,101.75,101.24,100.48,99.50,99.16,98.65,96.84,81.54,81.5 1,80.36,79.65,78.57,78.16,76.28,76.21,76.16,75.59,75.22,75.1 9,74.61,74.56,74.42,74.33,74.27,73.52,73.47,72.99,72.81,72.7 1,71.97,71.83,70.53,70.16,69.66,69.42,69.35,69.13,68.22,67.6 3,67.32,67.27,66.68,66.66,65.80,65.70,61.69,61.59,61.41,60.4 3,59.91,59.82,59.70,55.68,55.60,54.87,53.77,50.62,35.20,27.3 2,22.39,22.38,22.18,22.12,21.95,15.27,15.24.ESI-Q-TOF-HRMSm / z calculated for C95H156N10O64,[M-2H]2-:1230.4635,found m / z1230.4599.

[0373] Using a method similar to that used to synthesize compound 25, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0374] Table 10

[0375] Substrate target compound Mass spectrometry of the target compound Compound E5 Compound E19

[0376] Example 13 uses CHST6 to install the 6-O-sulfonation reaction

[0377] The substrate molecule (1.0 eq) and PAPS (2 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Sulfonyltransferase CHST6 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, ion exchange column purification was performed.

[0378] Taking compound 18 as an example, other compounds can be synthesized using the same or similar methods.

[0379] Compound 17 (100 mg, 0.046 mmol) and PAPS (93 mg, 0.18 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). An appropriate amount of sulfonyltransferase CHST6 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and then purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 18 (100 mg, 93%).

[0380] 1H NMR(800MHz,D2O)δ5.12(s,1H),5.07(d,J=9.7Hz,1H),4.94(s,1H),4.77(s,1H),4.71–4.68(m,2H),4 .62(d,J=8.3Hz,1H),4.60–4.57(m,2H),4.49–4.45(m,2H),4.36–4.32(m,2H),4.26(d,J=3.0Hz,1H), 4.23(dd,J=11.3,5.8Hz,2H),4.21–4.18(m,2H),4.11(d,J=3.6Hz,1H),4.02–3.46(m,57H),3.34(t,J =6.7Hz,2H),2.31(t,J=7.3Hz,2H),2.11–1.98(m,18H),1.65(p,J=7.4Hz,2H),1.59–1.54(m,2H).13C NMR(201MHz,D2O)δ177.32,174.91,174.69,174.68,174.63,174.59,102.95,102.92,102.86,101.25,100.41,99.54,99.45,99 .44,97.02,82.25,80.44,79.43,78.57,78.18,76.39,76.28,76.21,75.04,74.70,74.66,74.39,73.48,73.47,72.82,72.71,7 2.07,71.99,71.92,70.17,69.86,69.57,69.43,69.38,68.27,68.26,67.33,67.26,67.19,65.59,61.69,61.63,61.10,59.97, 59.82,55.55,54.94,54.78,54.76,53.78,50.63,35.21,27.33,22.38,22.31,22.20,22.15,22.13,21.96.ESI-Q-TOF-HRMSm / z calculated forC83H140N10O62S2,[M+2H]2+:1166.377, found m / z 1166.3764.

[0381] Using a method similar to that used to synthesize compound 18, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0382] Table 11

[0383]

[0384]

[0385]

[0386] Example 14: 6-O-sulfonation reaction using CHST1

[0387] The substrate molecule (1.0 eq) and PAPS (4 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Sulfonyltransferase CHST1 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, ion exchange column purification was performed.

[0388] Taking compound A17 as an example, other compounds can be synthesized using the same or similar methods.

[0389] Compound A16 (10 mg, 0.004 mmol) and PAPS (4 mg, 0.008 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). An appropriate amount of sulfonyltransferase CHST1 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound A17 (4 mg, 40%).

[0390] 1H NMR(800MHz,D2O)δ5.12(s,1H),5.07(d,J=9.7Hz,1H),4.93(s,1H),4.77(s,1H),4.71(d,J=8.4Hz,1H),4.62(d,J=8.1Hz,2H),4.58(d,J=8.0Hz,2H),4.47(dd,J=7.8,1.7Hz,2H),4.46–4.42(m,1H),4.30(dd,J=11.1,5.7Hz,1H),4.25(d,J=3.0Hz,1H),4.22–4.17(m,4H),4.15(dd,J=9.9,3.1Hz,1H),4.11(d,J=3.0Hz,1H),4.02(d,J=3.2Hz,1H),4.01–3.46(m,63H),3.34(t,J=6.7Hz,2H),2.75(dd,J=12.4,4.7Hz,1H),2.31(t,J=7.3Hz,2H),2.08(s,3H),2.06–2.02(m,12H),2.01(s,3H),1.82(t,J=12.1Hz,1H),1.68–1.62(m,2H),1.61–1.53(m,2H).13C NMR(126MHz,D2O)δ177.31,174.88,174.69,174.63,174.58,102.92,102.90,102.74,102.37,101.25,100.43,99.92,99.54,99.44,97.06,82.42,80.43,79.47,78.81,78.63,78.56,78.45,78.18,76.37,76.28,76.21,75.32,75.07,74.70,74.39,73.53,72.82,72.71,72.58,72.48,72.37,71.93,71.55,70.94,70.17,69.83,69.29,68.50,68.10,67.34,62.51,62.45,61.68,61.13,61.00,59.95,55.13,54.94,54.83,54.73,53.79,51.66,50.63,39.44,35.21,27.33,22.70,22.38,22.31,22.19,22.03,21.96.ESI-Q-TOF-HRMSm / z calculated for C92H154N10O70S2,[M+2H]2+:1291.4114,found m / z 1291.4117.

[0391] Using a method similar to that used to synthesize compound A17, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0392] Table 12

[0393]

[0394]

[0395] Example 15 uses Gal3ST2 to install the 3-O-sulfonation reaction.

[0396] The substrate molecule (1.0 eq) and PAPS (2 eq) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). Sulfonyltransferase Gal3ST2 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, ion exchange column purification was performed.

[0397] Taking compound A32 as an example, other compounds can be synthesized using the same or similar methods.

[0398] Compound D1 (40 mg, 0.027 mmol) and PAPS (55 mg, 0.11 mmol) were dissolved in Tris-HCl aqueous solution (100 mM, 10 mM MgCl2, pH 7.5). An appropriate amount of sulfonyltransferase Gal3ST2 was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and purified by Bio-P2 size exclusion chromatography, with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound A32 (18 mg, 41%).

[0399] 1H NMR(600MHz,D2O)δ5.12(s,1H),5.07(d,J=9.6Hz,1H),4.93(s,1H),4.77(s,1H),4.62(d,J=8. 1Hz,1H),4.61–4.55(m,4H),4.36–4.32(m,2H),4.31–4.28(m,2H),4.25(d,J=2.9Hz,1H),4.19( d,J=3.4Hz,1H),4.12(d,J=3.5Hz,1H),4.01–3.56(m,45H),3.50(q,J=9.5Hz,2H),3.34(t,J=6 .7Hz,2H),2.30(t,J=7.2Hz,2H),2.10–1.98(m,12H),1.68–1.61(m,2H),1.59–1.53(m,2H).13C NMR(151MHz,D2O)δ177.31,174.69,174.68,174.63,174.58,102.50,102.47,101.25,100.39,99.53,99.49,99.44,97.0 3,80.42,79.97,79.42,78.57,78.36,78.33,78.17,76.41,76.25,76.20,74.89,74.68,74.64,74.39,74.34,73.52,72.8 1,72.70,72.05,71.97,71.91,70.16,69.42,69.36,69.09,67.31,67.26,66.79,65.71,65.57,61.69,61.62,60.89,59. 90,59.81,54.93,54.84,54.82,53.77,50.62,35.20,27.32,22.37,22.31,22.19,21.95.ESI-Q-TOF-HRMSm / zcalculated for C67H110N8O52S2,[M-2H]2-:961.2831,found m / z 961.2821.

[0400] Using a method similar to that used to synthesize compound A32, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0401] Table 13

[0402]

[0403]

[0404] Example 16: Hydrolysis of β1,3-Gal using B3GalH

[0405] The substrate molecule (1.0 eq) was dissolved in a Tris-HCl aqueous solution (50 mM, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM. An appropriate amount of B3GalH was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and then purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0406] Taking compound 10 as an example, other compounds can be synthesized using the same or similar methods.

[0407] Compound 9 (250 mg, 0.16 mmol) was dissolved in a Tris-HCl aqueous solution (50 mM, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM. An appropriate amount of B3GalH was added to the reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction. The supernatant was collected by centrifugation, concentrated, and then purified by Bio-P2 size exclusion chromatography with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was collected and concentrated, and then lyophilized to obtain the intermediate compound. The intermediate compound was synthesized into compound 10 (218 mg, two-step yield 87%) according to Example 4.

[0408] 1H NMR(600MHz,D2O)δ5.12(s,1H),5.07(d,J=10.7Hz,1H),4.93(s,1H),4.77(s,1H),4.62(d,J =8.0Hz,1H),4.59(d,J=8.2Hz,1H),4.56(d,J=8.3Hz,1H),4.48(d,J=8.8Hz,1H),4.25(d,J= 2.8Hz,1H),4.19(d,J=3.7Hz,1H),4.12(d,J=3.8Hz,1H),4.01–3.43(m,45H),3.34(t,J=6.6 Hz,2H),2.30(t,J=7.3Hz,2H),2.15–1.98(m,12H),1.68–1.62(m,2H),1.60–1.53(m,2H).13C NMR (151MHz, DMSO) δ174.67,172.10,172.06,171.98,171.92,100.26,98.59,97.75,96.93,96.75,94.34,77.78,76. 79,75.90,75.79,75.51,73.75,73.60,73.56,73.12,72.67,72.00,71.72,71.68,70.87,70.58,70.16,70.05,69.81, 69.40,69.32,68.29,67.51,67.22,66.76,66.71,65.85,64.66,64.61,63.09,62.97,59.03,58.96,58.34,57.93,57 .27,57.25,57.16,52.64,52.28,52.17,51.13,47.97,32.55,24.67,19.73,19.66,19.55,19.31.ESI-Q-TOF-HRMSm / z calculated forC61H103N8O41,[M+H]+:1603.6215,found 1603.6212.

[0409] Using a method similar to that used to synthesize compound 10, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0410] Table 14

[0411]

[0412] Example 17: The reaction of hydrolyzing β1,4-Gal using B4GalH

[0413] The substrate molecule (1.0 eq) was dissolved in a Tris-HCl aqueous solution (50 mM, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM. An appropriate amount of B4GalH was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and then purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0414] Taking compound 24 as an example, other compounds can be synthesized using the same or similar methods.

[0415] Compound A5 (60 mg, 0.03 mmol) was dissolved in a Tris-HCl aqueous solution (50 mM, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM. An appropriate amount of B4GalH was added to the reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction. The supernatant was collected by centrifugation, concentrated, and then purified by Bio-P2 size exclusion chromatography with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound 24 (50 mg, 91%).

[0416] 1H NMR(600MHz,D2O)δ5.10(s,1H),5.05(d,J=9.7Hz,1H),4.90(s,1H),4.76(s,1H),4.67(d,J=8.4Hz,1H),4.60(d,J=7.9Hz, 1H),4.56(d,J=7.5Hz,1H),4.54(d,J=8.4Hz,1H),4.44(d,J=7.8Hz,1H),4.32(dd,J=11.2,2.1Hz,1H),4.23(d,J=2.4Hz,1H ),4.20(dd,J=11.2,5.7Hz,1H),4.18–4.16(m,2H),4.09(dd,J=3.5,1.6Hz,1H),4.00–3.38(m,48H),3.32(t,J=6.7Hz,2H), 2.28(t,J=7.2Hz,2H),2.06(s,3H),2.04–2.03(m,6H),2.02(s,3H),1.99(s,3H),1.67–1.59(m,2H),1.58–1.50(m,2H).13C NMR(126MHz,D2O)δ177.32,174.91,174.77,174.68,174.64,174.59,102.91,102.86,101.26,100.41,99.57,99.54,99.46,97.0 0,82.29,80.42,79.53,78.59,78.18,76.38,76.28,76.22,75.77,75.03,74.72,74.37,74.29,73.53,73.48,73.36,72.82,72.7 1,71.95,71.93,70.17,69.87,69.57,69.43,69.37,68.26,67.32,67.26,67.19,65.87,65.69,61.69,61.60,61.11,60.59,59.9 9,59.92,59.84,55.54,55.31,54.90,54.78,53.78,50.63,35.21,27.33,22.38,22.31,22.18,22.14,21.96.ESI-Q-TOF-HRMSm / z calculated forC69H117N9O49S,[M+2H]2+:943.8325, found m / z 943.8325.

[0417] Using a method similar to that used to synthesize compound A5, the corresponding target compounds were synthesized from the substrates shown in the table below.

[0418] Table 15

[0419]

[0420] Example 18: Reaction of Hydrolysis of Neu5Ac with NanA

[0421] The substrate molecule (1.0 eq) was dissolved in a Tris-HCl aqueous solution (50 mM, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM. An appropriate amount of NanA was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was concentrated and then purified by Bio-P2 size exclusion chromatography, using 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain the target compound. For further purification, a Hypercarb SPE column was used for product purification.

[0422] Taking compound A5 as an example, other compounds can be synthesized using the same or similar methods.

[0423] Compound A1 (90 mg, 0.04 mmol) was dissolved in a Tris-HCl aqueous solution (50 mM, 5 mM MgCl2, pH 7.5) to a final concentration of 5 mM. An appropriate amount of NanA was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, an equal volume of ethanol was added to quench the reaction. The supernatant was collected by centrifugation, concentrated, and then purified by Bio-P2 size exclusion chromatography with 50 mM NH4HCO3 solution as the eluent. The fraction containing the product was then collected, concentrated, and lyophilized to obtain compound A5 (75 mg, 95%).

[0424] 1H NMR(800MHz,D2O)δ5.12(s,1H),5.07(d,J=9.7Hz,1H),4.93(s,1H),4.77(s,1H),4.69(d,J=8.5Hz,1H),4.62( d,J=8.1Hz,1H),4.60–4.56(m,2H),4.48(d,J=7.9Hz,1H),4.46(d,J=8.0Hz,1H),4.33(dd,J=11.1,2.1Hz,1H), 4.25(d,J=2.9Hz,1H),4.22(dd,J=11.2,5.7Hz,1H),4.20–4.18(m,2H),4.11(d,J=3.0Hz,1H),4.00–3.45(m,54 H),3.34(t,J=6.0Hz,2H),2.30(t,J=7.2Hz,2H),2.09–1.99(m,15H),1.68–1.62(m,2H),1.60–1.53(m,2H).13C NMR(126MHz,D2O)δ177.31,174.92,174.71,174.68,174.63,174.59,102.92,102.86,101.26,100.40,99.53,99.45,99.41,97.01,82.28,80 .42,79.45,78.60,78.57,78.48,78.17,76.39,76.27,76.22,75.33,7 5.03,74.71,74.67,74.38,74.36,73.52,73.48,73.46,72.82,72.71,7 2.49,72.06,71.98,71.92,70.95,70.19,70.17,69.86,69.57,69.42, 69.38,68.51,68.27,67.32,67.26,67.18,65.77,65.65,61.69,61.62, 61.11,60.99,59.95,59.84,55.54,54.95,54.83,54.78,53.79,50.63,35.21,27.33,22.38,22.32,22.21,22.15,21.97.ESI-Q-TOF-HRMSm / z calculated forC75H127N9O54S,[M+2H]2+:1024.8589, found m / z 1024.859.

[0425] Based on the method described in Examples 1-18, the sulfonated N-sugar of the present invention was synthesized.

[0426] Example 19

[0427] The proteins CHST2 and CHST6 were diluted with SDS-PAGE sample buffer and boiled for 10 minutes. The heat-denatured proteins were then added to the corresponding electrophoresis wells, and protein gel electrophoresis was performed. After gel electrophoresis, the gel was stained with Coomassie staining solution. The gel was then destained with a destaining solution. Finally, the positions of the proteins CHST2 and CHST6 were observed using protein control standards.

[0428] The results are as follows Figure 11 As shown in Figure a, the molecular weight of CHST2 is around 100 kDa, and the molecular weight of CHST6 is around 80 kDa.

[0429] Set up three parallel reaction tubes (A, B, and C), each containing 50 μL of reaction solution (containing 2 mM compound 17, 16 mM PAPS, 10 mM MgCl2, and 100 mM Tris-HCl buffer (pH 7.5)). Additionally, tube B contained an appropriate amount of sulfonyltransferase CHST2, and tube C contained an appropriate amount of sulfonyltransferase CHST6 (the same amount as in tube B). Incubate the three parallel reaction tubes at 37°C overnight. Monitor the reaction by TLC.

[0430] The results are as follows Figure 11 As shown in Figure b, compared with the composition of compounds in tube A, compound 17 in tube B is slightly reduced, compound 17 disappears in tube C, and a new compound 18 appears.

[0431] Six parallel reaction tubes (A, B, C, D, E, and F) were prepared, each containing 400 μL of reaction solution (containing 5 mM compound E1, 20 mM PAPS, 10 mM MgCl2, and 100 mM Tris-HCl buffer (pH 7.5)). Additionally, tubes A, B, and C each contained 80 μg of sulfonyltransferase CHST2, and tubes D, E, and F each contained 80 μg of sulfonyltransferase CHST6. The six parallel reaction tubes were incubated at 37°C. Samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h for HPLC analysis, and the conversion rate was calculated based on the integrated area of ​​the product peaks. The average conversion rates of reaction groups A, B, and C at each time point were calculated, and the average conversion rates of reaction groups D, E, and F at each time point were calculated. Finally, a conversion-reaction-time function graph was plotted.

[0432] The results are as follows Figure 11 As shown in Figure c, the conversion rate of CHST2 after 12 hours of reaction is only 10%, while the conversion rate of CHST6 after 6 hours of reaction is close to 100%.

[0433] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of sulfotransferase CHST6, characterized in that, Used for sulfonation modification of the 6-position of the N-acetylglucosamine group at the end of the compound shown in formula (I); In the formula, the cube represents N-acetylglucosinolates; The light-colored round shape represents galactosyl groups; R1 is a monosaccharide residue or a disaccharide residue; R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups; R is absent, or a C1-C10 straight-chain or branched alkyl azide group.

2. The use as described in claim 1, characterized in that, The squares and light-colored circles are linked by an oxyglycosidic bond at position 1 of the N-acetylglucosamine group and position 3 of the galactose group.

3. The use as described in claim 1, characterized in that, The light-colored circle and R1 are linked by an oxyglycosidic bond at the 1 position on the galactose group and the hydroxyl group in R1 except at the 1 position.

4. A method for the continuous chemical enzymatic synthesis of sulfonated N-sugars, characterized in that, Including the following steps: (s1) A premix is ​​provided, the premix comprising: (a) N-glycosidic substrate; (b) PAPS; and (c) Sulfonase, wherein the sulfonase is CHST6; The N-glycosidic substrate has the structure shown in formula (I): In the formula, the cube represents N-acetylglucosamine; The light-colored round shape represents galactosyl groups; R1 is a monosaccharide residue or a disaccharide residue; R2 is absent, or contains sugar residues, sugar fragments, sugar derivatives, or non-sugar derivatives with functional groups; R is absent, or a C1-C10 straight-chain or branched alkyl azide group; (s2) Under enzyme-catalyzed conditions, the N-sugar substrate undergoes a sulfonation reaction with PAPS to modify the 6-position of the terminal N-acetylglucosamine group in the N-sugar substrate by sulfonation, thereby obtaining a sulfonated N-sugar (compound E6).

5. The method as described in claim 4, characterized in that, The CHST6 mentioned is derived from Homo sapiens.

6. The method as described in claim 4, characterized in that, In step (s1), in the premixed solution, PAPS is 1 to 5 equivalents of N-sugar substrate, preferably 1 to 4 equivalents, more preferably 1 to 3 equivalents.

7. The sulfonated N-sugar prepared by the method of claim 4.

8. The sulfonated N-sugar as described in claim 7, characterized in that, The sulfonated N-sugar is selected from the following group: In the formula, R is an empty, or C1-C10 straight-chain or branched alkyl azide group; the cube is N-acetylglucosyl; The light-colored round shape represents galactosyl groups; The dark-colored circles represent mannosyl groups; The rhombus shape represents an N-acetylneuraminic acid group; The triangle represents fucose.

9. An N-sugar molecule conjugate, characterized in that, include: (a) the sulfonated N-sugar as described in claim 7; and (b) A coupling portion coupled to the sulfonated N-sugar, wherein the coupling portion is selected from the group consisting of: a solid support, a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

10. A molecular library composed of sulfonated N-sugars prepared by the method of claim 4.

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