Novel disaccharide linker, preparation method therefor and use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

Novel disaccharide linker and its preparation method and use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefits and priority of patent application No. 202410038405.8 filed with the State Intellectual Property Office of China on January 10, 2024 and patent application No. 202510013650.8 filed with the State Intellectual Property Office of China on January 3, 2025, the entire contents of which are hereby incorporated by reference into this document in their entirety. Technical Field

[0003] The present disclosure relates to the fields of medicinal chemistry and biotechnology drugs, and specifically to a novel disaccharide linker, non-natural glycoengineered antibodies obtained by using the disaccharide linker and a novel enzyme, and site-specific and quantitative antibody-drug conjugates based on glycosylation modification, as well as their preparation methods and uses. Background Art

[0004] Antibody-based drug conjugates are a type of biotechnology drug that uses antibodies as targeting carriers to carry effector payloads, thereby achieving enrichment of the effector payload in the targeted area. These payloads can include cytotoxins, radioisotopes, oligonucleotides, immunomodulators, peptides, or protein fragments, forming antibody-toxin conjugates (such as Trastuzumab deruxtecan), antibody-radioisotope conjugates (such as BAY 2315497), antibody-nucleic acid conjugates (such as AOC 1001), antibody-immunomodulator conjugates (such as XMT-2056), and antibody-peptide conjugates (such as AMG 133).

[0005] Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals composed of antibodies, cytotoxins, and linkers. ADCs combine the targeting capabilities of antibodies with the cytotoxicity of cytotoxins, achieving targeted tumor cell destruction through controlled cytotoxin release via the linker. Compared to traditional chemotherapy drugs, ADCs have lower systemic toxicity and a wider therapeutic window. Compared to traditional antibody drugs, ADCs possess more direct, immune-independent tumor-killing capabilities. Currently marketed ADCs primarily rely on naturally occurring lysine (e.g., trastuzumab emtansine) or cysteine ​​(e.g., enfortumab vedotin) in antibodies. These ADCs, produced using random conjugation with natural amino acids, exhibit poor homogeneity and poor conjugation site stability, resulting in poor safety and a narrow therapeutic window. Currently, a variety of methods are available for the preparation of site-directed ADCs, including exogenous cysteine ​​insertion, non-natural amino acid insertion, enzyme-catalyzed conjugation, and glycosyl site-directed conjugation.

[0006] The Fc domain of natural antibodies contains a conserved N-glycosylation site. This site has been exploited to develop a variety of site-specific conjugation techniques. Kathrin Zuberbühler et al. used sodium periodate to oxidize fucose, introducing an aldehyde group for payload loading. Qun Zhou et al. used the glycosyltransferase beta-1,4-Gal-T1 and Sialyltransferase (Sia T) to process antibodies into antibodies with uniform sialic acid termini. These antibodies were then oxidized with sodium periodate to introduce an aldehyde group for conjugation. Wild-type endoglycosidases can be used to convert antibody N-glycosylation into a truncated, uniform form. Building on this, Laixi Wang et al. used mutant endoglycosidases (e.g., Endo SD233Q, Endo S2 D184M) to transfer oxazoline substrates bearing bioorthogonal reactive groups to the N-glycan termini, allowing subsequent payload conjugation via bioorthogonal reactions. Based on glycosylated truncated antibodies, Pradman K. Qasba used the beta-1,4-galactosyltransferase1-Y289L mutant to achieve the transfer of galactose and N-acetylgalactosamine with bioorthogonal reactive groups, thereby achieving payload coupling. Huang Wei's team and Laixi Wang's team reported the use of wild-type endoglycosidases Endo S and Endo S2, respectively, to prepare glycoengineered antibodies and ADCs using disaccharide linkers as substrates. Currently, the disaccharide structures that can be used as disaccharide linkers for glycosylation transfer and the transfer specificity of different enzymes for these disaccharide substrates remain to be explored.

[0007] Detailed Description of the Invention

[0008] The present disclosure aims to provide a novel disaccharide linker that can site-specifically introduce small molecule drugs into antibodies.

[0009] In a first aspect, the present disclosure provides a disaccharide linker represented by formula (I)

[0010] in,

[0011] X is selected from an oxygen atom or a sulfur atom;

[0012] Y is selected from an oxygen atom or a sulfur atom;

[0013] R1 is selected from the following groups:

[0014] wherein n is selected from an integer of 0-30, or an integer of 0-20, or an integer of 0-10;

[0015] R2 is selected from the following groups: hydrogen, -N3, C1-C6 alkyl-N3, -C≡CH, C1-C6 alkyl-C≡CH, -SH, C1-C6 alkyl-SH, C1-C6 alkyl;

[0016] The G ring represents a structure derived from a monosaccharide molecule, which is connected to the monosaccharide through a thioether bond or a glycosidic bond; the monosaccharide molecule is selected from galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine, mannose, N-acetylmannosamine, fucose, and sialic acid sugar; when Y is a sulfur atom, the G ring is connected to the monosaccharide through a thioether bond or a glycosidic bond. The thioether bond is a 1,4-thioether bond, a 2,4-thioether bond or a 3,4-thioether bond; when Y is an oxygen atom, the G ring is connected to the The glycosidic bond is a 1,4-glycosidic bond, a 2,4-glycosidic bond or a 3,4-glycosidic bond; and

[0017] Z is selected from the group consisting of hydrogen, a reactive group containing an azide residue, a reactive group containing a cycloalkyne residue, a reactive group containing a tetrazine residue, a reactive group containing an alkyne residue, a reactive group containing a cycloalkene residue, or a reactive group containing a maleimide residue.

[0018] In some embodiments of the present disclosure, R2 is selected from the following groups: hydrogen, -N3, -CH2-N3, -CH2-CH2-N3, -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -SH, -CH2-SH, -CH2-CH2-SH, C1-C6 alkyl.

[0019] In some embodiments of the present disclosure, the expression "the G ring represents a structure derived from a monosaccharide molecule, which is linked to a monosaccharide through a thioether bond or a glycosidic bond" means that the G ring represents a structure derived from a monosaccharide molecule, which can be linked to another monosaccharide through a thioether bond or a glycosidic bond.

[0020] Z is selected from a reactive group containing an azide residue, a reactive group containing a cycloalkyne residue, a reactive group containing a tetrazine residue, a reactive group containing an alkyne residue, or a reactive group containing a cycloalkene residue.

[0021] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), Y is selected from an oxygen atom, and the G ring is connected to The glycosidic bond is a 1,4-glycosidic bond, a 2,4-glycosidic bond or a 3,4-glycosidic bond, preferably a 1,4-glycosidic bond.

[0022] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), Y is selected from a sulfur atom, and the G ring is connected to The thioether bond is a 1,4-thioether bond, a 2,4-thioether bond or a 3,4-thioether bond, preferably a 1,4-thioether bond.

[0023] In some embodiments of the present disclosure, "G ring represents a structure derived from a monosaccharide molecule" means that the G ring can be selected from galactosyl, N-acetylgalactosamine, glucosyl, N-acetylglucosamine, mannosyl, N-acetylmannosamine, fucosyl or sialic acid glycosyl. In some embodiments of the present disclosure, "G ring can be linked to another monosaccharide through a thioether bond or a glycosidic bond" means that the G ring can be linked to another monosaccharide through a thioether bond or a glycosidic bond. of additional monosaccharide linkages.

[0024] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), the monosaccharide molecule from which the G ring is derived is galactose, glucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, or N-acetylmannosamine.

[0025] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), R1 is selected from the following structures:

[0026] wherein n is selected from an integer of 0-30; or an integer of 0-20, or an integer of 0-10.

[0027] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), R2 is selected from hydrogen, -N3, -CH2-N3, -CH2-CH2-N3, -C≡CH, -SH, -CH3, -CH2-CH3, and -CH2-CH2-CH3.

[0028] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing an azide residue, it is selected from the following groups:

[0029] wherein n, m, and I are independently selected from an integer of 0-30, or an integer of 0-20, or an integer of 0-10;

[0030] Rb is hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl.

[0031] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing an azide residue, it is selected from the following groups:

[0032] wherein n, m, and I are independently selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0033] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a cycloalkyne residue, it is selected from the following groups:

[0034] wherein n, m, and I are independently selected from an integer of 0-30; or an integer of 0-20, or an integer of 0-10;

[0035] Rb is hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl.

[0036] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a cycloalkyne residue, it is selected from the following groups:

[0037] wherein n, m, and I are independently selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0038] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing an alkyne residue, it is selected from the following groups:

[0039] wherein n, m, and I are independently selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0040] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing an alkyne residue, it is selected from the following groups:

[0041] wherein n, m, and I are independently selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0042] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a tetrazine residue, it is selected from the following groups:

[0043] wherein n, m, I, and q are independently selected from an integer of 0-30, or an integer of 0-20, or an integer of 0-10;

[0044] Ra is hydrogen or C1-C6 alkyl; Rb is hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl.

[0045] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a tetrazine residue, it is selected from the following groups:

[0046] wherein n, m, I, and q are independently selected from integers of 0-30, or integers of 0-20, or integers of 0-10; and Ra is hydrogen or a C1-C6 alkyl group.

[0047] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a cycloalkene residue, it is selected from the following groups:

[0048] wherein n, m, and I are independently selected from an integer of 0-30, or an integer of 0-20, or an integer of 0-10;

[0049] Rb is hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl.

[0050] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a cycloalkene residue, it is selected from the following structures:

[0051] wherein n, m, and I are independently selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0052] In some embodiments of the present disclosure, in the disaccharide linker represented by formula (I), when Z is a reactive group containing a maleimide residue, it is selected from the following groups:

[0053] In some embodiments of the present disclosure, the disaccharide linker represented by formula (I) is selected from the following specific compounds:

[0054] wherein n is selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0055] In a second aspect, the present disclosure provides a disaccharide conjugate, the structure of which is represented by formula (II):

[0056] in,

[0057] X, Y, R1, R2, and G ring are as defined above;

[0058] PL-Z'- represents a substituent on the G ring, and the substitution position of PL-Z'- is any position except the first position of the G ring derived from the monosaccharide molecule;

[0059] L is a divalent linker or a multivalent linker connecting Z' and P, which comprises a cleavable linker, a non-cleavable linker or a combination thereof;

[0060] P is selected from cytotoxins, small molecule drugs, near-infrared or fluorescent probes, polypeptides, RNA and related drugs, radioisotope labels, contrast agents and magnetic resonance imaging agents; the small molecule drug is preferably selected from maytansine, DM-1, DM-4, ​​MMAE, MMAF, Auristatin 0101, SN-38, Dxd, exitecan, duocarmycin, amanitin, PBDs, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines, and derivatives of the above compounds, or the small molecule drug is a radioactive therapeutic;

[0061] Z' is a linker fragment connecting the L and G rings, which is independently absent or selected from -(CH2) p -、-(CH2-CH2-O) p -, -(phenyl) p - and any combination of one or two or more of the following groups:

[0062] wherein Ra, Rc, and Rd are selected from hydrogen, C1-C6 alkyl, C6-C 10Aryl, C5-C 10 heteroaryl; and

[0063] Each p is an integer from 1 to 5, for example, 1, 2, 3, 4, and 5.

[0064] In some embodiments of the present disclosure, in the disaccharide conjugate represented by formula (II), Z' is a linker fragment connecting the L and G rings, which is independently absent or -(CH2) p -、-(CH2-CH2-O) p -, wherein p is an integer from 1 to 5, or includes the specific groups listed above.

[0065] In some embodiments of the present disclosure, in the disaccharide conjugate represented by formula (II), L is selected from or comprises one or more of the following combinations: Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly.

[0066] In some embodiments of the present disclosure, in the disaccharide conjugate represented by formula (II), L is selected from or comprises one or more of the following combinations: Val-Cit, Val-Ala, Ala-Ala, and Gly-Gly-Phe-Gly.

[0067] In some embodiments of the present disclosure, in the disaccharide conjugate represented by formula (II), L is selected from or comprises a combination of one or more of the following cleavable linkers:

[0068] In some embodiments of the present disclosure, in the disaccharide conjugate represented by formula (II), L is selected from or comprises a combination of one or more non-cleavable linkers: -(CH2) m -、-(CH2-CH2-O) m -、-(PO4) n -, wherein m and n are independently selected from an integer of 0-30, or an integer of 0-20, or an integer of 0-10.

[0069] In some embodiments of the present disclosure, in the disaccharide conjugate represented by formula (II), when P is a toxin, it is selected from the following groups:

[0070] In some embodiments of the present disclosure, the disaccharide conjugate represented by formula (II) is selected from the following specific compounds:

[0071] wherein n is selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0072] In a third aspect, the present disclosure provides an antibody-based conjugate drug, as shown in the following formula (III):

[0073] wherein, rings X, Y, and G are as defined above;

[0074] Z', L, and P are as defined above, or PL-Z'- is hydrogen;

[0075] t is selected from 0 or 1;

[0076] R1' is selected from R1 or a group generated by the reaction of R1 and a drug linker ELP, and R2' is selected from R2 or a group generated by the reaction of R2 and a drug linker ELP; wherein R1 and R2 are as defined above; in the drug linker ELP, L and P are as defined above, and E comprises the following group:

[0077] wherein Ra, Rc, and Rd are independently selected from hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl;

[0078] Ab is an antibody or antigen-binding fragment selected from monoclonal antibody, bispecific antibody or polyspecific antibody.

[0079] In some embodiments of the present disclosure, in the antibody-based conjugate drug represented by formula (III), the antibody is selected from: Pertuzumab, Trastuzumab, Rituximab, Cetuximab, Margetuximab, Abciximab, Daclizumab, Adalimumab, Palivizumab, Basiliximab, Bevacizumab, Panitumumab, Nimotuzumab, Denosumab, Zolbetuximab b), Ramucirumab, Necitumumab, Ipilimumab, Daratumumab, Alemtuzumab, Elotuzumab, Blinatumomab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Toripalimab, Catumaxomab, Emicizumab, Amivantamab.

[0080] In some embodiments of the present disclosure, in the antibody-based conjugate drug represented by formula (III), the drug linker ELP is selected from the following specific compounds:

[0081] wherein n is selected from an integer of 0-30, an integer of 0-20, or an integer of 0-10.

[0082] In some embodiments of the present disclosure, when the antibody-based drug conjugate of formula (III) has a single payload, it is selected from the following specific structures:

[0083] Wherein, n is selected from an integer of 0-30, or an integer of 0-20, or an integer of 0-10; t is selected from 0 or 1.

[0084] In some embodiments of the present disclosure, when the antibody-based conjugate drug represented by formula (III) has a dual payload, it is selected from the following specific compounds:

[0085] wherein m, n, and I are independently selected from integers of 0-30, or 0-20, or 0-10; and t is selected from 0 or 1.

[0086] In some embodiments of the present disclosure, each n, each m, each I, and each q are independently selected from an integer of 0-30, for example, selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0087] In some embodiments of the present disclosure, each C1-C6 alkyl group is, for example, a C1 alkyl group, a C2 alkyl group, a C3 alkyl group, a C4 alkyl group, a C5 alkyl group, and a C6 alkyl group; each C6-C 10 Aryl is exemplified by phenyl and naphthyl; each C5-C 10 Examples of heteroaryl groups include 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, and 14-membered heteroaryl groups containing one or more (e.g., 2, 3, 4, and 5) heteroatoms selected from N, O, and S.

[0088] In a fourth aspect, the present disclosure provides a method for preparing an antibody-based conjugate drug, the method comprising the following three methods:

[0089] Method 1:

[0090] a) under the action of an endoglycosidase having beta-N-acetylglucosamine hydrolysis activity or an endoglycosidase and a fucosylation hydrolase, the wild-type antibody is hydrolyzed to form a deglycosylated antibody containing one N-acetylglucosamine or a core fucosylated N-acetylglucosamine;

[0091] b) coupling the deglycosylated antibody obtained in step a) with the disaccharide linker described above under the catalysis of an endoglycosidase having transglycosyl activity or a mutant thereof to obtain a glycoengineered antibody;

[0092] c) coupling the glycoengineered antibody containing orthogonal reactive groups obtained in step b) with a drug linker ELP modified with a corresponding group capable of undergoing a specific coupling reaction with the orthogonal reactive group to prepare an antibody-based conjugated drug;

[0093] Method 2:

[0094] a) coupling the wild-type antibody with the disaccharide linker described above under the catalysis of an endoglycosidase having transglycosylation activity or a mutant thereof to obtain a glycoengineered antibody;

[0095] b) coupling the glycoengineered antibody containing orthogonal reactive groups obtained in step a) with a drug linker ELP modified with a corresponding group capable of undergoing a specific coupling reaction with the orthogonal reactive group to prepare an antibody-based conjugated drug;

[0096] Method 3:

[0097] Under the catalysis of an endoglycosidase with glycosyl transfer activity or a mutant thereof, the wild-type antibody and the disaccharide-small molecule drug conjugate described above are co-incubated, or the deglycosylated antibody and the disaccharide-small molecule drug conjugate are co-incubated to prepare an antibody-based conjugate drug.

[0098] In some embodiments of the present disclosure, in the first and second modes, the orthogonal reactive group and the corresponding group capable of undergoing a specific coupling reaction with the orthogonal reactive group are selected from any of the following combinations: an azide group and an alkynyl group, a tetrazine group and a trans-cyclooctene (TCO), a thiol group and a maleimide, a thiol group and a thiol group or an activated form of a thiol group, an aldehyde group and an amino group, an aldehyde group and an aminooxy group or a hydrazine group.

[0099] In some embodiments of the present disclosure, the endoglycosidase having endo-beta-N-acetylglucosaminidase hydrolysis activity is selected from Endo Se2, Endo Si, Endo S, Endo S2, Endo F3, Endo A, Endo D, Endo CC, and mutants thereof.

[0100] In some embodiments of the present disclosure, the endoglycosidase having transglycosylation activity is selected from the group consisting of Endo Se2, Endo Si, Endo S, Endo S2, Endo F3, and mutants thereof.

[0101] In some embodiments of the present disclosure, when preparing deglycosylated antibodies, glycoengineered antibodies, and antibody-based drug conjugates without core fucosylation, an endoglycosidase having endo-beta-N-acetylglucosaminylase hydrolysis activity is used in combination with a fucose hydrolase.

[0102] In a fifth aspect, the present disclosure provides use of the disaccharide linker provided in the first aspect of the present disclosure or the disaccharide conjugate provided in the second aspect of the present disclosure in preparing the antibody-based conjugate drug of the present disclosure.

[0103] In some embodiments of the present disclosure, the antibody-based drug conjugate is the antibody-based drug conjugate provided in the third aspect of the present disclosure.

[0104] In a sixth aspect, the present disclosure provides use of the antibody-based conjugate drug provided in the third aspect of the present disclosure in the preparation of a drug for treating and / or preventing tumors, inflammation, viruses, infectious diseases or other immune diseases.

[0105] In a seventh aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody-based drug conjugate provided in the third aspect of the present disclosure.

[0106] In some embodiments of the present disclosure, the pharmaceutical composition is used to treat and / or prevent diseases, including tumors, inflammation, viruses, infectious diseases or other immune diseases.

[0107] In an eighth aspect, the present disclosure provides a method for treating and / or preventing a disease, the method comprising administering to a patient a therapeutically and / or preventively effective amount of the antibody-based conjugate drug provided by the third aspect of the present disclosure.

[0108] In some embodiments of the present disclosure, the disease comprises a tumor, inflammation, virus, infectious disease or other immune disease.

[0109] The present disclosure describes the preparation of homogenized sugar-chain-modified antibodies using glycoengineering methods. This disclosure relates to the Fc region of an antibody molecule, wherein the Fc region is modified with specific sugar chains that enhance the function of the Fc region. In some embodiments, the specifically sugar-chain-modified Fc region comprises a monoclonal antibody, preferably a humanized monoclonal antibody. Disclosed herein are methods for producing such antibodies or antibody fragments comprising specific sugar chain modifications using glycoengineering methods.

[0110] Unless otherwise defined, all academic terms used in this disclosure have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. All publications and patent documents mentioned in this disclosure can be regarded as suggestions of those with ordinary knowledge in the art to which this disclosure belongs.

[0111] In this disclosure, the term "sugar" refers to a polysaccharide or a molecule containing carbohydrates, whether oxidized or not, including but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. Sugar is also used herein to refer to the carbohydrate portion of a sugar-containing conjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide, or proteoglycan. The term "sugar chain" is used interchangeably with "sugar" herein.

[0112] As used in this disclosure, a "sugar chain linker" as an activated donor molecule for glycosyl coupling can be a synthetic oxazoline- or thiazoline-containing sugar, such as an oligosaccharide with an activated reducing end, preferably an oligosaccharide molecule with an oxazoline structure; or it can be a natural N-glycan oxazoline. The sugar chain linker can also be chemically modified, for example, by introducing functional groups through azidation, alkynylation, aldehyde formation, sulfhydration, etc. The term "disaccharide linker" is a sugar chain linker containing at least two monosaccharide units, preferably two monosaccharide units connected by a glycosidic bond or a thioether bond.

[0113] In the present disclosure, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity. The term "immunoglobulin" is used interchangeably with "antibody" herein. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population.

[0114] Core-fucosylated and non-fucosylated glycoproteins are important classes of molecules that play key roles in many biological events, such as tumor metastasis, cell adhesion, pathogen infection, and immune responses. Natural and recombinant fucosylated and non-fucosylated glycoproteins are typically produced as mixtures of glycoforms that differ only in the structure of the side-chain oligosaccharides.

[0115] The term "antibody-based drug conjugate" disclosed herein refers to a conjugate formed by covalently linking all polypeptides / proteins targeting specific cells to a payload. The polypeptide / protein targeting specific cells can be an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody, a bispecific antibody, or a polyclonal antibody; the payload can be a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a peptide, RNA and related drugs, a radioisotope label, a contrast agent, and a magnetic resonance imaging agent; the covalent conjugate formed can be used for treatment or detection. The term "antibody-drug conjugate" disclosed herein refers to a conjugate formed by covalently linking all polypeptides / proteins targeting specific cells to a cytotoxin.

[0116] The term "endoglycosidase" disclosed herein refers to a glycoside hydrolase having beta-N-acetylglucosaminyl endoglycosidase hydrolysis activity, typically belonging to glycoside hydrolase family 18 or 85. Some endoglycosidases known in the art include Endo S, Endo S2, Endo F3, Endo A, Endo D, Endo CC, and mutants thereof, for example, see those described in WO2022 / 226420. Such enzymes may also have transglycosyltransferase activity, and enzymes known in the art having such activity include Endo S, Endo S2, Endo F3, and mutants thereof, for example, see those described in WO2022 / 226420.

[0117] As used herein, "wild-type antibodies" typically refer to antibodies that are naturally occurring or recombinantly expressed and have N-glycosylation sites (N-glycans). For example, all antibodies with an N297 site in the Fc region are considered "wild-type antibodies," and the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. The term "deglycosylated antibody" herein refers to an antibody containing one N-acetylglucosamine or core-fucosylated N-acetylglucosamine after the wild-type antibody has been treated with glycoside hydrolases.

[0118] The term "drug linker ELP" as used herein refers to a covalent linker comprising an orthogonal reactive group, a linker, and a payload. In a typical ELP, E comprises any of the following structures:

[0119] The following reaction schemes generally illustrate the preparation routes of the glycoengineered antibodies and sugar chain-directed ADCs disclosed in the present disclosure.

[0120] General methods for glycoengineering antibodies:

[0121] General Operation 1: Preparation of Glycoengineered Antibodies

[0122] The concentrations of the wild-type antibody, the prepared disaccharide linker, and endoglycosidase (Endo Si or Endo Se2) were adjusted to 10 mg / mL, 2 mM (30 times the equivalent of the antibody concentration), and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the reaction was incubated at 25°C for 12 hours. The desired non-natural glycoengineered antibody was then purified with protein A.

[0123] General Operation 2: Preparation of Site-Specific ADCs Based on Azide-Modified Glycoengineered Antibodies

[0124] The prepared azide-modified sugar-engineered antibody and cycloalkyne linker-load were adjusted to 5 mg / mL and 0.33 mM concentrations, respectively. The pH of the reaction system was adjusted to 7.4 and incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically coupled ADC was obtained by protein A purification or ultrafiltration.

[0125] General Operation 3: Preparation of Site-Specific ADCs Based on Cycloalkyne-Modified Glycoengineered Antibodies

[0126] The prepared cycloalkynyl-modified sugar-engineered antibody and azide linker-load were adjusted to 5 mg / mL and 0.33 mM concentrations, respectively. The pH of the reaction system was adjusted to 7.4 and incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-site-coupled ADC was obtained by protein A purification or ultrafiltration.

[0127] General Operation 4: Preparation of Site-Specific ADCs Based on Tetrazine-Modified Glycoengineered Antibodies

[0128] The prepared tetrazine-modified sugar-engineered antibody and trans-cyclooctene (TCO)-type linker-load were adjusted to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4 and incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically coupled ADC was obtained by protein A purification or ultrafiltration.

[0129] General Operation 5: One-step preparation of sugar-specific site-conjugated ADC

[0130] The prepared loaded disaccharide linker, wild-type antibody, and endoglycosidase (Endo Si or Endo Se2) were adjusted to 0.4 mM, 5 mg / mL, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the reaction was incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically coupled ADC was obtained by protein A purification or ultrafiltration.

[0131] General Operation 6: Preparation of Dual-Loaded Site-Directed ADCs Based on Azide- and Tetrazine-Modified Glycoengineered Antibodies

[0132] The prepared azide- and tetrazine-modified sugar-engineered antibodies, cycloalkyne linker-payload, and trans-cyclooctene (TCO) linker-payload were adjusted to 5 mg / mL, 0.33 mM, and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the reaction was incubated at 25°C overnight. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was obtained by protein A purification or ultrafiltration.

[0133] General Operation 7: Preparation of Site-Specific ADCs Based on Linear Alkyne-Modified Glycoengineered Antibodies

[0134] Copper sulfate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and sodium ascorbate were premixed to concentrations of 20 mM, 100 mM, and 150 mM, respectively, for use as a catalyst. The prepared linear alkyne-modified glycoengineered antibody, the azide linker-payload, and the catalyst were mixed to concentrations of 5 mg / mL, 0.5 mM, 0.67 mM (copper sulfate), 3.33 mM (THPTA), and 5 mM (sodium ascorbate), respectively. The reaction system was adjusted to pH 7.4 and incubated overnight at 25°C. After LC-MS confirmation of conversion to product, the desired sugar-specifically conjugated ADC was purified by protein A or ultrafiltration.

[0135] Technical Effects

[0136] The present disclosure achieves at least one of the following beneficial technical effects:

[0137] Based on the site-specific structural modification of sugars, multiple new disaccharide linker modification sites have been enriched and developed, providing a variety of new disaccharide linker structures.

[0138] Based on the development of endoglycosidases, the present invention discovered and tested two wild-type endoglycosidases with broader substrate specificity and better transfer efficiency.

[0139] The present invention utilizes an enzyme-catalyzed reaction to achieve site-specific introduction of orthogonal reactive groups onto antibodies, thereby enabling site-specific conjugation with small molecule drugs. This approach is simple to operate and readily adaptable to industrial production. Furthermore, the site-specifically conjugated antibody-drug conjugates possess excellent drugability and can be used to treat tumors, inflammation, viruses, infectious diseases, or other immune disorders.

[0140] BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1. Detection of transglycosylation activity of compound G0 by different endoglycosidases;

[0142] Figure 2. Detection of transglycosylation activity of compound G1 by different endoglycosidases;

[0143] Figure 3. Detection of transglycosylation activity of compound G2 by different endoglycosidases;

[0144] Figure 4. Efficacy of low-dose sugar-specifically conjugated ADC in the NCI-N87 xenograft tumor model;

[0145] Figure 5. Efficacy of high-dose sugar-site-conjugated ADC in the NCI-N87 xenograft tumor model. Example

[0146] The following describes the specific process of preparing disaccharide linkers, glycoengineered antibodies, and sugar-specifically coupled ADCs using the preparation method disclosed herein through specific examples.

[0147] In this disclosure, sugar linkers were synthesized by Wuhan Tangzhi Pharmaceutical Co., Ltd. DBCO (dibenzocyclooctyne) compounds and tetrazine compounds were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. D-glucosamine hydrochloride and azido acid compounds were purchased from Anaiji Chemical Technology Co., Ltd. Acetonitrile was purchased from Wuhan Shenshi Co., Ltd. Galactose oxidase, catalase, and horseradish peroxidase were purchased from Shanghai Yingxin Laboratory Co., Ltd. All other compounds and reagents, unless otherwise specified, were purchased from Sinopharm Chemical Reagent Co., Ltd. The instruments and chromatographic columns used include: LC-MS Agilent 1290+6135B, analytical high performance liquid chromatograph (Thermo ultimate 3000), Tongwei ELSD-UM500, preparative high performance liquid chromatograph (Wuhan Ruihe Chromatography Technology, LC 2100); Agilent SB C18 (5μm, 4.6×150mm), Waters XBridge BEH Amide (250mm×4.6mm, 5.0μm), Welch HILIC Amide (10μm, 21.2×250mm).

[0148] The antibody used in the embodiments of the present disclosure is a wild-type antibody containing an Fc segment, and the Fc segment sequence is, for example, shown in SEQ ID NO: 5, such as a typical example of Pertuzumab. In the present disclosure, Trastuzumab was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. In the present disclosure, the fillers, chromatographic columns, and instruments used for purification and property determination of antibodies, sugar chain-modified antibodies, ADCs include: 5ml cOmplete TM His-Tag Purification Column (Roche), HiLoad™ 26 / 600 Superdex™ 200 prep grade column (Cytive), AmMag TM Protein A Magnetic Beads (GenScript), SDA030 protein purification system (Sepure), Acquity I-Class / RDa (Waters) liquid chromatography-mass spectrometry, Arc Premier high-performance liquid chromatograph, ACCQUITY UPLC BEH PROTEIN C4 (Waters, 1.7 μm, 2.1 mm × 50 mm) column, TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm) SEC column, TSKgel Butyl-NPR (4.6 mm × 10 cm, 2.5 μm) HIC column.

[0149] In the present disclosure, drug linkers DBCO-GGFG-Dxd and TCO-PEG4-GGFG-Dxd were purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.; drug linkers DBCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and N3-PEG4-VC-PAB-MMAE were purchased from MedChemExpress LLC.

[0150] 1. Preparation of disaccharide linkers

[0151] Example 1: Synthesis of Compound G0

[0152] Step 1: Preparation of 50mM pH 7.0 phosphate buffer: Dissolve 0.68g of potassium dihydrogen phosphate in 100mL of deionized water, and then adjust the pH of the phosphate buffer to 7.0±0.2 with 1N sodium hydroxide solution. At room temperature, add 40mL of 50mM pH 7.0 phosphate buffer and compound 1 (800mg, 2.09mmol) to a 100mL single-necked reaction flask, stir, and continuously introduce oxygen (O2) using a syringe needle for about 0.5-1 hour. Then, add 1kU galactose oxidase, 10kU horseradish peroxidase (HRP), and 200kU catalase in sequence. React at room temperature for 6 hours. After the reaction is complete, stop introducing oxygen. The prepared compound 2 is directly used for the next reaction without purification.

[0153] Step 2: To the reaction mixture of the previous step was added 250 μL of compound 3 (O-(2-azidoethyl)hydroxylamine, CAS: 1397706-70-5, (250 μL, 2.45 mmol), and the reaction was allowed to proceed at 15-25°C overnight. The reaction was monitored by TLC (isopropanol: concentrated aqueous ammonia: deionized water = 10:3:2). After the reaction was complete, most of the water (>95%) was removed by rotary evaporation, and 40 mL of ethanol was added. The insoluble matter was removed by filtration, and the filtrate was dried by rotary evaporation and purified by silica gel column chromatography (dichloromethane:methanol = 10:2-10:3, containing 1% concentrated aqueous ammonia). The collected fractions were concentrated and then lyophilized to obtain compound 4 (350 mg, two-step yield 36%).

[0154] ESI-MS calc.for C 16 H 27 N5O 11 [M+H] + m / z=466.2, found m / z=466.2. 1HNMR(400MHz,deuterium oxide)δ7.47(d,J=4.1Hz,0.7H),6.81(d,J=4.6Hz,0.3H),5.16(q,J=1.5Hz,0.7H),4.80(ddd, J=4.7,3.5,1.2Hz,0.3H),4.67–4.65(m,0.3H),4.49(d,J=7.9Hz,0.65H),4.46(dd,J=7.9,1.6 Hz,0.35H),4.42(ddd,J=4.1,2.8,1.2Hz,0.7H),4.29–4.19(m,2H),4.04(dt,J=2.8,1.3Hz,1H ),3.97–3.89(m,1H),3.88–3.78(m,3H),3.73–3.60(m,3H),3.54(m,3H),2.00(d,J=1.8Hz,3H).

[0155] Step 3: To a 50 mL centrifuge tube, add 5 mL of deionized water, compound 4 (100 mg, 0.21 mmol), and potassium phosphate (684 mg, 3.22 mmol). Cool to 0°C in an ice-water bath with stirring. Slowly add 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 232 mg, 1.07 mmol). After reacting at 0°C for half an hour, warm to room temperature and stir overnight. The reaction endpoint was determined by TLC (dichloromethane:methanol = 10:2, containing 1% concentrated aqueous ammonia). After completion of the reaction, the mixture was purified by C18 column chromatography (deionized water / methanol = 20:1-10:1). The fractions were collected, concentrated, and then lyophilized to obtain compound G0 (63 mg, 66% yield).

[0156] ESI-MS calc.for C 16 H 26 N5O 10 [M+H] + m / z=448.2, found m / z=448.2. 1HNMR (400MHz, deuterium oxide)δ7.49(d,J=4.8Hz,0.7H),6.81(d,J=4.6Hz,0.3H),6.09(dd,J=7.3,2.3Hz ,1H),4.43(d,J=7.8Hz,0.7H),4.38(d,J=7.9Hz,0.3H),4.34(m,0.3H),4.33–4.3( m,1H),4.25–4.15(m,3H),4.11(m,1H),3.98(dd,J=3.4,1.1Hz,0.7H),3.74(m,1H ),3.67–3.57(m,3H),3.53–3.44(m,3H),3.40–3.35(m,1H),1.99(t,J=1.9Hz,3H).

[0157] Example 2: Synthesis of Compound G1

[0158] Step 1: To a 100 mL single-necked reaction flask, add 5 mL of 50 mM pH 7.0 phosphate buffer and compound 5 (230 mg, 0.58 mmol), respectively. Stir in a 30°C water bath and continuously introduce oxygen (O2) using a syringe needle for approximately 0.5 hours. In a separate 5 mL centrifuge tube, add 1 mL of 50 mM pH 7.0 phosphate buffer, 280 U galactose oxidase, 10 kU horseradish peroxidase (HRP), 200 kU catalase, and 0.16 mL of 1 mM copper sulfate aqueous solution. Activate in a 30°C water bath for 0.5 hours. The activated enzyme mixture is then added to a 100 mL single-necked reaction flask and reacted in a 30°C water bath for 6 hours. After the reaction is complete, discontinue oxygen infusion, and the prepared compound 6 proceeds directly to the next step without purification.

[0159] Step 2: Compound 3 (130 μL, 1.27 mmol) was added to the reaction mixture from the previous step. The mixture was allowed to react overnight at 25°C and monitored by TLC (isopropanol: concentrated aqueous ammonia: deionized water = 10:3:2). After the reaction was complete, most of the water (>90%) was removed by rotary evaporation. 20 mL of methanol was then added and the insoluble material was filtered off. The precipitate was washed twice with 10 mL of methanol each. The filtrate was then dried and purified by silica gel column chromatography (dichloromethane:methanol = 10:1-5:1). The collected fractions were concentrated to yield compound 7 (150 mg, 54% yield over two steps).

[0160] Step 3: To a 100 mL single-necked reaction flask, add 10 mL of deionized water and compound 7 (150 mg, 0.31 mmol), respectively. Cool to 0°C with stirring, add potassium phosphate (1020 mg, 4.81 mmol), and cool the reaction solution to -3-0°C. Add CDMBI (330 mg, 1.52 mmol) and react at 0°C overnight. The reaction endpoint was detected by TLC (isopropanol: concentrated aqueous ammonia: deionized water = 50:5:4). After the reaction was complete, the insoluble matter was filtered off and washed with 3 mL of water. The filtrate was purified by C18 column chromatography (5%-15% methanol-water solution) using a preparative high-performance liquid chromatograph (Wuhan Ruihe Chromatography Technology, LC 2100). The collected fractions were concentrated and lyophilized to obtain compound G1 (86 mg, 60% yield).

[0161] ESI-MS calc.for C 16 H 26 N5O9S[M+H] + m / z=464.1, found m / z=464.1. 1 HNMR(400MHz,deuterium oxide)δ7.48(d,J=5.4Hz,0.7H),6.91(d,J=5.6Hz,0.3H),6.01(d,J=7.56,2.3Hz,1H),4.51 (d,J=8.6Hz,1H),4.46(d,J=8.4Hz,0.3H),4.41-4.44(m,1H),4.25(d,J=5.2Hz,0.7H),4.17( d,J=5.6Hz,0.3H),4.17–4.15(m,2H),4.10–4.04(m,1H),3.98(d,J=5.4Hz,0.7H),3.76-3.7 4(m,1H),3.64-3.53(m,2H),3.49-3.34(m,4H),2.91(t,J=8.6Hz,1H).1.95(d,J=1.6Hz,3H).

[0162] Example 3: Synthesis of Compound G2

[0163] Step 1: To a 100 mL three-necked reaction flask, add 10 mL of pyridine and compound 4 (200 mg, 0.43 mmol), respectively. Nitrogen was then introduced and the system was cooled to -15°C. p-Toluenesulfonyl chloride (p-TsCl, 410 mg, 2.15 mmol) was dissolved in 5 mL of pyridine and added dropwise to the 100 mL three-necked reaction flask via syringe. The reaction was allowed to react overnight at approximately -15°C. The reaction endpoint was determined by TLC (dichloromethane:methanol = 10:1, containing 0.1% concentrated aqueous ammonia). Upon completion of the reaction, 15 mL of dichloromethane was added for dilution. The product was then purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1). Fractions were collected and concentrated to yield crude compound 8 (140 mg, 53% yield).

[0164] Step 2: To a 100 mL three-necked reaction flask, 8 mL of deionized water, crude compound 8 (140 mg, 0.23 mmol), and sodium azide (235 mg, 3.61 mmol) were added, respectively. The mixture was purged with nitrogen and stirred at approximately 60°C for 40 hours. After completion of the reaction, the reaction mixture was filtered and the filtrate was purified by C18 column chromatography (deionized water:methanol = 10:1 to 5:1). The collected fractions were concentrated to afford compound 9 (90 mg, 81% yield).

[0165] Step 3: Add 10 mL of deionized water and compound 9 (90 mg, 0.18 mmol) to a 100 mL single-necked reaction flask, and cool the reaction solution to about 0°C. After adding potassium phosphate (584 mg, 2.75 mmol) to the single-necked reaction flask, cool the reaction solution to -3-0°C, add CDMBI (200 mg, 0.92 mmol), and stir the reaction at about 0°C overnight. The reaction endpoint was detected by TLC (isopropanol: concentrated ammonia water: deionized water = 50:5:4). After the reaction was completed, the reaction solution was filtered to remove insoluble matter and washed with 3 mL of water. The filtrate was washed three times with 15 mL of toluene to remove impurities. The aqueous phase was purified by C18 column chromatography (deionized water: methanol = 5:1-3:1, containing 0.1% concentrated NH 3· H2O), the collected fractions were concentrated and then lyophilized to obtain compound G2 (46 mg, yield 53%)

[0166] ESI-MS calc.for C 16 H 25 N8O9[M+H] + m / z=473.1, found m / z=473.1. 1HNMR(400MHz,deuterium oxide)δ7.54(d,J=5.2Hz,0.7H),6.87(d,J=5.8Hz,0.3H),6.01(d,J=7.6Hz,1H),4.38 (d,J=8.4Hz,0.7H),4.34(d,J=8.2Hz,0.3H),4.31(d,J=5.8Hz,0.3H),4.29-4.26(m,1 H),4.21-4.14(m,3H),4.08(d,J=4.4Hz,1H),3.94(d,J=4.2Hz,0.7H),3.68(t,J=8.2H z,1H),3.61-3.47(m,3H),3.47-3.42(m,3H),3.39-3.35(m,1H),1.96(d,J=1.4Hz,3H).

[0167] Example 4: Synthesis of Compound G3

[0168] Step 1: Compound 5 (2.83 g, 7.1 mmol) and 115 mL of 50 mM pH 7.0 phosphate buffer were added to a 1000 mL single-necked reaction flask. Stirring was performed in a 30°C water bath. Oxygen (O2) was continuously introduced using a syringe needle for approximately 0.5 hours. Separately, 10 mL of 50 mM pH 7.0 phosphate buffer, 3 kU galactose oxidase, 30 kU horseradish peroxidase (HRP), 600 kU catalase, and 1.8 mL of 1 mM copper sulfate aqueous solution were added to the reaction mixture in a 30°C water bath for 0.5 hours. The activated enzyme mixture was then added to the 1000 mL single-necked reaction flask and reacted in a 30°C water bath for 6 hours. After the reaction was complete, oxygen was discontinued, and the prepared compound 6 was directly used in the next reaction without purification.

[0169] Step 2: Sodium carbonate (Na2CO3, 376 mg, 3.5 mmol) and hydroxylamine hydrochloride (NH2OH·HCl, 542 mg, 6.4 mmol) were added to the reaction system in the previous step, and the mixture was allowed to react overnight at 25°C. The reaction was monitored by TLC (isopropanol:concentrated aqueous ammonia:deionized water = 10:2:1). After the reaction was completed, 100 mL of methanol was added to the system and the insoluble matter was removed by filtration. The filtrate was dried and purified by C18 column (5% methanol-water solution). The collected fractions were concentrated to obtain compound 10 (2.68 g, 92% yield).

[0170] Step 3: Compound 10 (2.68 g, 6.5 mmol), 50 mL of 50 mM pH 7.0 phosphate buffer, and 100 mL of methanol were added to a 500 mL single-necked reaction flask, stirred, and cooled to 0°C. Nickel chloride hexahydrate (NiCl2·6H2O, 3.2 g, 13.5 mmol) and sodium borohydride (NaBH4, 1.2 g, 31.7 mmol) were added, and the mixture was reacted at 0°C overnight. After the reaction was complete, the reaction solution was filtered, and the filtrate was purified by amide column chromatography (Welch HILIC Amide, 10 μm, 21.2 x 250 mm, 90%-50% acetonitrile-water solution). The collected fractions were concentrated to obtain compound 11 (1.85 g, 71% yield).

[0171] Step 4: To a 100 mL single-necked reaction flask, 5 mL of N,N-dimethylformamide (DMF), compound 11 (113 mg, 0.28 mmol), N,N-diisopropylethylamine (DIPEA, 181 μL, 0.742 g / mL at 25°C, 1.04 mmol), and compound 20 (DBCO-COOSu, 167 mg, 0.42 mmol) were added and reacted at room temperature overnight. The reaction endpoint was determined by TLC (isopropanol:concentrated ammonia:deionized water = 25:5:3). After completion of the reaction, the solvent was evaporated and the product was purified by silica gel column chromatography (methanol:dichloromethane = 1:9-1:4). Fractions were collected and concentrated to yield compound 12 (163 mg, 91% yield).

[0172] Step 5: To a 100 mL single-necked reaction flask, 16 mL of deionized water and compound 12 (163 mg, 0.24 mmol) were added. The reaction system was cooled to approximately 0°C, and potassium phosphate (756 mg, 3.56 mmol) was added. The reaction temperature was maintained at 0°C, and CDMBI (285 mg, 1.31 mmol) was added. The reaction was stirred at approximately 0°C overnight. The reaction endpoint was determined by TLC (isopropanol:concentrated ammonia:deionized water = 25:5:3). After the reaction was complete, the reaction mixture was filtered, and the precipitate was washed with 3 mL of water. The filtrate was collected and washed four times with 30 mL of toluene to remove impurities. The aqueous phase was purified by C18 column chromatography (20%-50% acetonitrile-water solution). The collected fractions were concentrated and lyophilized to obtain compound G3 (white solid product, 70 mg, 44% yield).

[0173] ESI-MS calc.for C 33 H 38 N3O 10 S[M+H] + m / z=668.2, found m / z=668.2. 1HNMR(400MHz,deuterium oxide)δ8.21(d,J=7.6Hz,2H),7.42(d,J=7.6Hz,1H),7.30–7.16(m,5H),7.09(d,J=7.2Hz ,1H),5.91(d,J=7.8Hz,1H),4.87(d,J=7.2Hz,1H),4.32–4.10(m,2H),3.91(d,J=8.2Hz,1 H),3.71–3.62(m,2H),3.52(d,J=6.8Hz,2H),3.46–3.28(m,3H),3.26–3.02(m,3H),3.01– 2.91(m,1H),2.74(d,J=6.4Hz,1H),2.41–2.29(m,1H),2.06(s,2H),1.86(d,J=1.6Hz,3H).

[0174] Example 5: Synthesis of Compound G7

[0175] Step 1: 2-[(Azidoacetyl)amino]-2-deoxy-D-glucose (26.2 g, 100 mmol) and 64.1 g of UDP-Gal (uridine 5′-diphosphate galactose disodium salt, 64.1 g, 105 mmol) were weighed and added to a 1000 mL single-necked reaction flask. 600 mL of deionized water was added and stirred to dissolve. MgCl2 (4 g, 42 mmol) was added, and 0.3 g of β-1,4-galactosyltransferase (NmLgtB-4) was added to catalyze the reaction. The system pH was 8.0, the reaction temperature was 37°C, and the stirring speed was 200 rpm. The reaction was allowed to proceed overnight. After completion of the reaction, an equal volume of ethanol was added to remove the enzyme. After centrifugation, the supernatant was concentrated. The concentrate was purified by electrodialysis, membrane concentration, and purification by silica gel column chromatography (dichloromethane:methanol = 10:1). Fractions were collected and concentrated to obtain compound 13 (8.2 g, 19% yield).

[0176] Step 2: To a 100 mL single-necked reaction flask, add 10 mL of deionized water and compound 13 (100 mg, 0.24 mmol), respectively. The reaction system was cooled to approximately 0°C, potassium phosphate (610 mg, 2.87 mmol) was added, and the temperature was further lowered to -3-0°C. CDMBI (220 mg, 1.01 mmol) was added, and the reaction was stirred at approximately 0°C overnight. TLC was used to determine the reaction endpoint. Upon completion of the reaction, the reaction solution was filtered to remove insoluble matter and washed with 3 mL of water. The filtrate was washed three times with 15 mL of toluene. The aqueous phase was purified by C18 column chromatography (deionized water:methanol = 5:1-3:1, containing 0.1% concentrated aqueous ammonia). The collected fractions were concentrated and then lyophilized to obtain compound G7 (51 mg, 52% yield).

[0177] HRMS calc.for C 14 H 23 N4O 10 [M+H] + m / z=407.14153, found m / z=407.14003. 1 HNMR(400MHz,deuterium oxide)δ5.99(d,J=7.2Hz,1H),4.22–4.17(m,2H),4.07–4.05(m,1H),3.96(dd, J=11.2,3.4Hz,1H),3.7(d,J=3.4Hz,1H),3.61–3.38(m,8H),3.27–3.21(m,2H).

[0178] Example 6: Synthesis of Compound G9

[0179] Step 1: To a 1000 mL three-necked reaction flask, 500 mL of dichloromethane, 2-aminoethanol (10 g, 164 mmol), and triethylamine (34 mL, 245 mmol) were added, respectively. The mixture was purged with nitrogen and di-tert-butyl dicarbonate (Boc2O, 35.7 g, 164 mmol) was added at 0°C. The mixture was stirred at room temperature for 4-5 hours. After completion of the reaction, the dichloromethane was dried and dissolved in 300 mL of ethyl acetate. The mixture was washed with 200 mL of saturated ammonium chloride solution and 100 mL of water, and dried over anhydrous sodium sulfate. The solution was concentrated to obtain compound 14 (20 g, 76% yield).

[0180] Step 2: To a 2L three-necked reaction flask, 1L of tetrahydrofuran (THF), compound 14 (20g, 124mmol), N-hydroxyphthalimide (PHTOH, 21.2g, 130mmol), and triphenylphosphine (PPh3, 35.8g, 136mmol) were added, respectively. The mixture was purged with nitrogen and stirred. The temperature of the reaction system was lowered to approximately 0°C. Diisopropyl azodicarboxylate (DIAD, 26.3mL, 130mmol) was gradually added dropwise. The reaction was allowed to proceed overnight at 0°C. TLC confirmed the reaction was complete (petroleum ether:ethyl acetate = 2:1). After the reaction was complete, 500mL of ethyl acetate was added to the reaction solution. The organic phase was washed with 200mL of saturated sodium chloride solution and 200mL of deionized water, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1-1:1) to obtain 41g of crude compound 15, which was used directly in the next step.

[0181] Step 3: To a 100 mL single-necked reaction flask, add 6 mL of dichloromethane, crude compound 15 (1.25 g, 4.08 mmol), and trifluoroacetic acid (CF3COOH, 3.0 mL, 39.18 mmol) and react at room temperature for 2 hours. The reaction endpoint was monitored by TLC (petroleum ether:ethyl acetate = 10:1). After completion, the reaction was concentrated and pumped dry to obtain 1.3 g of crude compound 16, which was used directly in the next step without purification.

[0182] Step 4: To a 100 mL three-necked reaction flask were added 6 mL of dichloromethane, DBCO-acid (CAS: 1353016-70-2, 0.5 g, 1.64 mmol), N-hydroxysuccinimide (HNS, CAS: 6066-82-6, 0.28 g, 2.43 mmol), and EDCI﹒HCl (CAS: 7084-11-9, 0.47 g, 2.45 mmol), respectively. The mixture was stirred at room temperature for 3-4 hours. Then, the crude product of compound 16 (1.3 g, 4.06 mmol) and N,N-diisopropylethylamine (DIPEA, 3 mL, 17.22 mmol) were added and stirred at room temperature for 2-3 hours. The reaction endpoint was detected by TLC (petroleum ether:ethyl acetate = 10:1). After the reaction was complete, the insoluble matter was removed by filtration, and the insoluble matter was rinsed twice with 10 mL of dichloromethane. The filtrate was washed twice with 20 mL of saturated sodium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate and concentrated through a silica gel column (petroleum ether:ethyl acetate = 1:1-1:2) to obtain compound 17 (0.23 g, yield 29%).

[0183] Step 5: To a 100 mL single-necked reaction flask, 4 mL of dichloromethane, 2 mL of methanol, and compound 17 (0.42 g, 0.85 mmol) were added, followed by hydrazine hydrate (N2H4﹒H2O, 36 μL, 0.94 mmol). The mixture was reacted at room temperature for 2 hours, and the reaction endpoint was monitored by TLC (dichloromethane:methanol = 5:1). After the reaction was complete, 40 mL of deionized water was added, and the aqueous phase was extracted three times with 20 mL of dichloromethane. The organic phases were combined, washed twice with 20 mL of saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated on a silica gel column (methanol:dichloromethane = 1:19-1:9) to obtain compound 18 (0.2 g, 65% yield).

[0184] Step 6: To a 100 mL single-necked reaction flask, add 5 mL of 50 mM pH 7.0 phosphate buffer and compound 5 (120 mg, 0.30 mmol), respectively. Stir in a 30°C water bath and continuously introduce oxygen (O2) using a syringe needle for approximately 0.5 hours. In a separate 5 mL centrifuge tube, add 1 mL of 50 mM pH 7.0 phosphate buffer, 280 U galactose oxidase, 10 kU horseradish peroxidase (HRP), 200 kU catalase, and 0.16 mL of 1 mM copper sulfate aqueous solution. Activate in a 30°C water bath for 0.5 hours. The activated enzyme mixture is then added to a 100 mL single-necked reaction flask and reacted in a 30°C water bath for 6 hours. After the reaction is complete, discontinue oxygen introduction, and the prepared compound 6 proceeds directly to the next step without purification.

[0185] Step 7: Compound 18 (120 mg, 0.33 mmol) was added to a 100 mL single-necked reaction flask and allowed to react overnight at 25°C. The reaction was monitored by TLC (isopropanol: concentrated aqueous ammonia: deionized water = 10:2:1). After the reaction was complete, most of the water (>90%) was removed by rotary evaporation. 20 mL of methanol was added to fully dissolve the mixture. Insoluble matter was removed by filtration and washed twice with 10 mL of methanol. The filtrate was concentrated and purified by silica gel column chromatography (dichloromethane:methanol = 15:1-6:1). The fractions were collected, concentrated again, and purified by C18 column chromatography (30%-70% acetonitrile-water solution). The collected fractions gave compound 19 (120 mg, 54% yield).

[0186] Step 8: To a 100 mL single-necked reaction flask, add 15 mL of deionized water and compound 19 (120 mg, 0.16 mmol), respectively. The reaction system was cooled to approximately 0°C, potassium phosphate (514 mg, 2.42 mmol) was added, and the temperature was further lowered to 0°C. CDMBI (175 mg, 0.81 mmol) was added, and the reaction was stirred at approximately 0°C overnight. The reaction endpoint was determined by TLC (isopropanol:concentrated aqueous ammonia:deionized water = 50:5:3). After completion of the reaction, the insoluble material was removed by filtration and washed with 3 mL of water. The filtrate was purified by C18 column chromatography (30%-70% acetonitrile-water solution). The collected fractions were concentrated and lyophilized to obtain compound G9 (white solid product, 45 mg, 39% yield).

[0187] ESI-MS calc.for C 35 H 43 N4O 12 S[M+H2O+H] + m / z=743.3, found m / z=743.3.1 HNMR(400MHz,deuterium oxide)δ7.68(d,J=5.2Hz,0.7H),7.56-7.35(m,8H),6.84(d,J=5.2Hz,0.3H),6.18-6.13(m,1H),5.1 0(d,J=16.8Hz,1H),4.61(d,J=7.4Hz,0.7H),4.54-4.52(m,1H),4.33(t,J=5.2Hz,1H),4.25(d,J=8. 2Hz,0.3H),4.18-4.16(m,1H),4.07(s,1H),4.03-3.80(m,4H),3.76-3.58(m,3H),3.54-3.46(m,1H) ,3.34-3.19(m,2H),3.04(d,J=9.8Hz,1H),2.57-2.49(m,1H),2.29-2.15(m,3H),2.08-2.01(m,3H).

[0188] Example 7: Synthesis of Compound G10

[0189] Step 1: To a 100 mL single-necked flask, 10 mL of tetrahydrofuran, 11,12-didehydro-γ-oxydibenzo[b,f]azocane-5(6H)-butyric acid (DBCO-acid, 0.5 g, 1.64 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 0.35 g, 1.70 mmol), and N-hydroxysuccinimide (NHS, 0.2 g, 1.74 mmol) were added, respectively. The mixture was purged with nitrogen and allowed to react at room temperature overnight. After the reaction was complete, the solid was filtered off, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1 to 1:1). The fractions were collected and concentrated to obtain compound 20 (0.4 g, 61% yield).

[0190] Step 2: To a 500 mL single-necked reaction flask, 260 mL of N,N-dimethylformamide, compound 1 (5 g, 13 mmol), and 2,2-dimethoxypropane (18.8 g, 181 mmol) were added, stirred, and p-toluenesulfonic acid (TsOH, 0.2 g, 1.2 mmol) was added at room temperature. The mixture was stirred at 25°C for 30 minutes. After completion of the reaction, solid sodium bicarbonate was added to quench the reaction under ice-bath conditions, and the mixture was brought to room temperature and stirred for 1 hour. The solid was filtered off, and the filtrate was concentrated and slurried with ethyl acetate. The mixture was ultrasonically shaken for 30 minutes, filtered, rinsed, and the filter cake was collected and dried using an oil pump to obtain compound 21 (3.5 g, 64% yield).

[0191] Step 3: To a 250 mL three-necked reaction flask, add 100 mL of pyridine and compound 21 (3.5 g, 8.27 mmol), respectively. The nitrogen atmosphere was replaced, the reaction system was cooled to -20°C, stirred, and p-toluenesulfonyl chloride (5.5 g, 28.85 mmol) was added portionwise. The reaction was continued at -20°C overnight. TLC analysis showed the product as the main reaction point (dichloromethane:methanol = 20:1). After completion of the reaction, 100 mL of saturated sodium bicarbonate solution was added to quench the reaction. The reaction was concentrated and purified by silica gel column chromatography (methanol:dichloromethane = 1:9-2:8). After fraction collection and concentration, compound 22 (2.6 g, 54% yield) was obtained.

[0192] Step 4: To a 100 mL three-necked reaction flask, 30 mL of deionized water, compound 22 (1.5 g, 2.60 mmol), and sodium azide (2.5 g, 38.46 mmol) were added, respectively, and the mixture was reacted at 60°C for 36 hours. The reaction endpoint was monitored by TLC (dichloromethane:methanol = 20:1). After the reaction was complete, the product was purified by C18 column chromatography (5%-15% methanol-water solution). The collected fraction was dried to 20 mL and then lyophilized to obtain compound 23 (0.8 g, 68% yield).

[0193] Step 5: To a 100 mL three-necked reaction flask, 10 mL of methanol, compound 23 (0.8 g, 1.78 mmol), and di-tert-butyl dicarbonate (Boc2O, 0.6 g, 2.75 mmol) were added, the nitrogen atmosphere was replaced three times, Pd / C (80 mg, 0.18 mmol), the hydrogen atmosphere was replaced three times, and the mixture was stirred at room temperature for 2 hours under a hydrogen balloon. The end point was detected by TLC (dichloromethane:methanol = 10:1). After completion of the reaction, the product was filtered through celite, concentrated, and purified by C18 column chromatography (5%-20% methanol-water solution). The collected fractions were spin-dried to obtain compound 24 (0.4 g, 43% yield).

[0194] Step 6: To a 50 mL two-necked reaction flask, 2 mL of methanol, 0.2 mL of a 10 mM hydrogen chloride solution in ethanol, and compound 24 (0.1 g, 0.19 mmol) were added, respectively, and stirred at room temperature for 2 hours. The end point was detected by TLC (isopropanol: water: concentrated ammonia solution = 20:4:5). After the reaction was complete, the solvent was dried and purified by C18 column chromatography (5% methanol in water). The collected fractions were dried to 10 mL and then lyophilized to obtain compound 25 (30 mg, 38% yield).

[0195] Step 7: To a 100 mL three-necked reaction flask, 2 mL of N,N-dimethylformamide, compound 25 (40 mg, 0.10 mmol), compound 20 (64 mg, 0.16 mmol), and triethylamine (TEA, 80 mg, 0.50 mmol) were added. The reaction was stirred at room temperature for 2 hours. After completion of the reaction, the DMF was spin-dried and purified by C18 column chromatography (20%-50% methanol in water). The eluate was concentrated to approximately 5 mL to obtain compound 26 (30 mg, 45% yield).

[0196] Step 8: To a 100 mL single-necked reaction flask, add 5 mL of water and compound 26 (30 mg, 0.04 mmol), respectively. The reaction system was cooled to approximately 0°C, potassium phosphate (142 mg, 0.67 mmol) was added, and the temperature was further lowered to -3-0°C. CDMBI (48 mg, 0.22 mmol) was added, and the reaction was stirred at approximately 0°C overnight. The reaction endpoint was determined by TLC (isopropanol: concentrated aqueous ammonia: deionized water = 50:5:4). After completion of the reaction, the reaction mixture was filtered to remove insoluble matter, and the filter cake was washed with 3 mL of deionized water. The filtrate was washed three times with 15 mL of toluene to remove impurities. The product was purified by C18 column chromatography (20%-50% methanol in water). The eluate was evaporated to remove methanol, and then lyophilized to obtain compound G10 (26 mg, 99% yield).

[0197] ESI-MS calc.for C 33 H 38 N3O 11 [M+H] + m / z=652.3, found m / z=652.3. 1 HNMR(400MHz,deuterium oxide)δ7.68-7.34(m,8H),6.00(d,J=8.6Hz,1H),5.12(d,J=14.8Hz,1H),4.44-4.36(m,2H),3.92(d,J=8.4Hz,1H),3.86(d,J= 14.8Hz,1H),3.81-3.74(m,2H),3.68-3.49(m,5H),3.46-3.32(m,3H),2.64-2.53(m,1H),2.34-2.13(m,3H),2.00-1.95(m,3H).

[0198] Example 8: Synthesis of Compound G13

[0199] Step 1: To a 100 mL three-necked reaction flask, 10 mL of N,N-dimethylformamide, D-glucamine hydrochloride (1.1 g, 5.10 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 1.16 g, 5.62 mmol), 1-hydroxybenzotriazole (HOBt, 0.76 g, 5.62 mmol) and triethylamine (TEA, 0.57 g, 5.62 mmol) were added, stirred evenly, and 3-azidopropanoic acid (3-Azidopropanoic acid, 0.65 g, 5.62 mmol) was added under ice-water bath. After the addition was complete, the reaction was stirred at room temperature overnight, concentrated directly, and purified by silica gel column chromatography (dichloromethane: methanol = 10:1). The mixture was concentrated under reduced pressure to give compound 27 (380 mg, yield 27%).

[0200] Step 2: To a 100 mL three-necked reaction flask, 20 mL of deionized water, compound 27 (380 mg, 1.38 mmol), and UDP-Gal (790 mg, 1.29 mmol) were added, stirred, and an appropriate amount of metal salt MgCl2·6H2O (0.1 g) was added. The reaction was then allowed to proceed in the presence of β-1,4-galactosyltransferase (NmLgtB-4, 0.06 g). The reaction solution was maintained at a pH of 8.0, a temperature of 37°C, and a stirring speed of 200 rpm overnight. After completion of the reaction, an equal volume of ethanol was added to remove the enzyme, and the mixture was concentrated. The product was then purified by electrodialysis, membrane concentration, and silica gel column chromatography (dichloromethane:methanol = 10:1). The product was then concentrated under reduced pressure to afford compound 28 (120 mg, 21% yield).

[0201] Step 3: To a 100 mL single-necked reaction flask, add 10 mL of deionized water and compound 28 (100 mg, 0.23 mmol). The reaction system was cooled to approximately 0°C, and potassium phosphate (580 mg, 2.73 mmol) was added. The temperature was then lowered to -3-0°C, and CDMBI (210 mg, 0.97 mmol) was added. The reaction temperature was maintained at approximately 0°C and stirred overnight. The reaction endpoint was determined by TLC. Upon completion of the reaction, insoluble matter was removed by filtration, and the filter cake was washed with 3 mL of water. The filtrate was washed with 15 mL of toluene to remove impurities. The aqueous phase was purified by C18 column chromatography (deionized water:methanol = 5:1-3:1, containing 0.1% concentrated ammonia). The collected fractions were concentrated and lyophilized to obtain compound G13 (58 mg, 60% yield).

[0202] ESI-MS calc.for C 15 H 24 KN4O 10 [M+K] + m / z=459.1, found m / z=459.1. 1HNMR(400MHz,deuterium oxide)δ6.01(d,J=7.8Hz,1H),4.33–4.28(m,2H),4.12(d,J=7.2Hz,1H),3.78( d,J=5.6Hz,1H),3.71–3.46(m,9H),3.42–3.435(m,2H),2.58(t,J=7.4Hz,2H).

[0203] Example 9: Synthesis of Compound G14

[0204] Step 1: To a 100 mL three-necked reaction flask, 10 mL of N,N-dimethylformamide, D-glucamine hydrochloride (1.1 g, 5.10 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 1.16 g, 5.62 mmol), 1-hydroxybenzotriazole (HOBt, 0.76 g, 5.62 mmol) and triethylamine (TEA, 0.57 g, 5.62 mmol) were added, stirred evenly, and 4-azidobutyric acid (4-Azidobutyric acid, 0.73 g, 5.62 mmol) was added under an ice-water bath. After the addition was complete, the reaction was stirred at room temperature overnight, concentrated directly, and purified by silica gel column chromatography (dichloromethane: methanol = 10:1). The mixture was concentrated under reduced pressure to give compound 29 (320 mg, yield 22%).

[0205] Step 2: To a 100 mL three-necked reaction flask, 20 mL of deionized water, compound 29 (320 mg, 1.10 mmol), and UDP-Gal (730 mg, 1.20 mmol) were added, stirred, and an appropriate amount of metal salt MgCl2·6H2O (0.1 g) was added. The reaction was then allowed to proceed in the presence of β-1,4-galactosyltransferase (NmLgtB-4, 0.06 g). The reaction solution was maintained at a pH of 8.0, a temperature of 37°C, and a stirring speed of 200 rpm overnight. After completion of the reaction, an equal volume of ethanol was added to remove the enzyme, and the mixture was concentrated. The product was then purified by electrodialysis, membrane concentration, and silica gel column chromatography (dichloromethane:methanol = 10:1). The product was then concentrated under reduced pressure to afford compound 30 (130 mg, 26% yield).

[0206] Step 3: To a 100 mL single-necked reaction flask, add 10 mL of deionized water and compound 30 (100 mg, 0.22 mmol). The reaction system was cooled to approximately 0°C, and potassium phosphate (580 mg, 2.73 mmol) was added. The temperature was then lowered to -3-0°C, and CDMBI (210 mg, 0.97 mmol) was added. The reaction temperature was maintained at approximately 0°C and stirred overnight. The reaction endpoint was determined by TLC. Upon completion of the reaction, insoluble matter was removed by filtration, and the filter cake was washed with 3 mL of water. The filtrate was washed with 15 mL of toluene to remove impurities. The aqueous phase was purified by C18 column chromatography (deionized water:methanol = 5:1-3:1, containing 0.1% concentrated ammonia). The collected fractions were concentrated and lyophilized to obtain compound G14 (61 mg, 64% yield).

[0207] ESI-MS calc.for C 16 H 26 N4O 10 [M+K] + m / z=473.1, found m / z=473.1. 1 HNMR(400MHz,deuterium oxide)δ5.099(d,J=7.6Hz,1H),4.33–4.28(m,2H),4.11(d,J=7.2Hz,1H),3.77(d,J=5.8Hz,1H),3. 71–3.46(m,7H),3.40–3.34(m,1H),3.31(t,J=7.4Hz,3H),2.37(t,J=6.8Hz,2H),1.77-1.86(m,2H).

[0208] Example 10: Synthesis of Compound G27

[0209] Step 1: To a 1000 mL single-necked reaction flask, add 115 mL of 50 mM pH 7.0 phosphate buffer and compound 5 (2.83 g, 7.09 mmol), respectively. Stir in a 30°C water bath and continuously introduce oxygen (O2) using a syringe needle for approximately 0.5 hours. In a separate 50 mL centrifuge tube, add 10 mL of 50 mM pH 7.0 phosphate buffer, 3 kU galactose oxidase, 30 kU horseradish peroxidase (HRP), 600 kU catalase, and 1.8 mL of 1 mM copper sulfate aqueous solution. Activate in a 30°C water bath for 0.5 hours. The activated enzyme mixture is then added to a 1000 mL single-necked reaction flask and reacted in a 30°C water bath for 6 hours. After the reaction is complete, discontinue oxygen infusion, and the prepared compound 6 proceeds directly to the next step without purification.

[0210] Step 2: Sodium carbonate (Na2CO3, 306 mg, 2.89 mmol) and hydroxylamine hydrochloride (NH2OH·HCl, 442 mg, 6.36 mmol) were added to the reaction mixture from the previous step, and the mixture was allowed to react overnight at 25°C. The reaction was monitored by TLC (isopropanol:concentrated ammonia:deionized water = 10:2:1). After the reaction was complete, 100 mL of methanol was added to the reaction system, and the insoluble matter was removed by filtration. The filtrate was then dried and purified by C18 column chromatography (5% methanol in water) to obtain compound 10 (2.68 g, 92% yield).

[0211] Step 3: Compound 10 (2.68 g, 6.5 mmol), 50 mL of 50 mM pH 7.0 phosphate buffer, and 100 mL of methanol were added to a 500 mL single-necked reaction flask, stirred, and cooled to 0°C. Nickel chloride hexahydrate (NiCl2·6H2O, 3.2 g, 13.5 mmol) and sodium borohydride (NaBH4, 1.2 g, 31.7 mmol) were added, and the mixture was reacted at 0°C overnight. After the reaction was complete, the reaction solution was filtered, and the filtrate was purified by amide column chromatography (Welch HILIC Amide, 10 μm, 21.2 x 250 mm, 90%-50% acetonitrile-water solution). The collected fractions were concentrated to obtain compound 11 (1.85 g, 71% yield).

[0212] Step 4: To a 100 mL single-necked reaction vial were added 5 mL of N,N-dimethylformamide (DMF), compound 11 (250 mg, 0.63 mmol), N,N-diisopropylethylamine (DIPEA, 330 μL, 0.742 g / mL at 25°C, 1.89 mmol), and 2,5-dioxopyrrolidin-1-yl 2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)acetate (MTAB-COOSu, 310 mg, 0.95 mmol). The mixture was reacted overnight at room temperature. The reaction endpoint was determined by TLC (isopropanol:concentrated aqueous ammonia:deionized water = 25:5:3). Upon completion of the reaction, the solvent was evaporated and the product was purified by silica gel column chromatography (methanol:dichloromethane = 3:17-3:7). Fractions were collected and concentrated to yield compound 31 (300 mg, 78% yield).

[0213] Step 5: To a 100 mL single-necked reaction flask, add 30 mL of deionized water and compound 31 (300 mg, 0.49 mmol), respectively. The reaction system was cooled to approximately 0°C, and potassium phosphate (1.56 g, 7.35 mmol) was added. The temperature was then lowered to 0°C, and CDMBI (533 mg, 2.46 mmol) was added. The reaction was stirred at approximately 0°C overnight. The reaction endpoint was determined by TLC (isopropanol:concentrated ammonia:deionized water = 25:5:3). Upon completion of the reaction, insoluble matter was removed by filtration and washed with 3 mL of deionized water. The aqueous phase of the filtrate was washed three times with 30 mL of toluene to remove impurities. The aqueous phase was purified by C18 column chromatography (20%-50% acetonitrile-water solution). The collected fractions were concentrated and lyophilized to yield compound G27 (46 mg, 16% yield).

[0214] ESI-MS calc.for C 25 H 33 N6O9S[M+H] + m / z=593.2, found m / z=593.2. 1 HNMR (400MHz, deuterium oxide)δ7.44(d,J=7.8Hz,2H),7.32(d,J=7.8Hz,2H),5.92(d,J=7.2Hz,1H),4. 32(d,J=7.2Hz,2H),3.99(d,J=6.8Hz,1H),3.77(d,J=6.4Hz,1H),3.67(d,J=7. 8Hz,1H),3.69(s,2H),3.54–3.49(m,2H),3.47–3.43(m,1H),3.38–3.32(m,2H) ,3.28–3.20(m,2H),2.89(s,3H),2.82(d,J=6.8Hz,1H),1.86(d,J=1.6Hz,3H).

[0215] Example 11: Synthesis of Compound G4

[0216] Step 1: Add 1L of methanol and 50g of N-acetyl-D-glucosamine (GlcNAc, 0.23mol) to a 2L single-mouth reaction bottle, stir and dissolve at room temperature. Then add 100g of cationic resin Dowex (H + ), the system temperature was heated to 70°C and stirred for 24 hours. The mixture was cooled to room temperature, the residue was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 38.4 g of white solid compound 32, which was directly used in the next step without further purification.

[0217] Step 2: 1L DMF and compound 32 (20g, 85.02mmol) were added to a 2L three-necked reaction flask, stirred at room temperature to dissolve, and then sodium bicarbonate (214.5g, 2.55mol) and tetramethylpiperidinium oxide (0.4g, 2.56mmol) were added in sequence. The system was replaced with nitrogen protection, cooled to about 0°C, and trichloroisocyanuric acid (19.8g, 85.02mmol) was added. The reaction solution was stirred at 0°C for 4 hours, and then filtered to remove the solid. The filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol = 20:1-10:1) to obtain 9.5g of off-white solid compound 33 with a yield of 48%.

[0218] Step 3: To a 500 mL three-necked reaction flask at 0°C under nitrogen, 75 mL of THF, (methoxymethyl)triphenylphosphine bromide (Ph3PCH2OMeBr, 15.0 g, 38.86 mmol), and a solution of potassium tert-butoxide (t-BuOK, 4.33 g, 38.59 mmol) in THF (70 mL) were added, followed by stirring at 0°C for 2 hours. A solution of compound 33 (5.0 g, 21.44 mmol) in THF (37 mL) was added, followed by warming to room temperature and stirring for another 3 hours. The reaction solution was concentrated under reduced pressure to approximately 40% of its original volume, followed by the addition of 30% sulfuric acid (30 mL) and stirring at 20°C for 3 hours. The reaction solution was adjusted to pH 7 at low temperature using a 2M aqueous sodium hydroxide solution, then concentrated under reduced pressure and redissolved with methanol (150 mL). The solid was filtered off and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 30:1-10:1) to obtain 1.37 g of compound 34 as a yellow solid in a yield of 25%.

[0219] Step 4: To a 50 mL three-necked reaction flask under nitrogen at 20° C., 14 mL of methanol, compound 34 (1.37 g, 5.54 mmol), potassium carbonate (1.53 g, 11.1 mmol), and Bestmann's reagent (1.49 g, 7.76 mmol) were added, respectively. The reaction solution was stirred at 20° C. for 12 hours, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure and purified by C18 column chromatography (acetonitrile:water = 1:20-1:2) to obtain 439 mg of compound 35 as a yellow solid in a yield of 32%.

[0220] Step 5: To a 100 mL two-necked reaction flask, 12 mL of 6 M hydrochloric acid solution and compound 35 (1.37 g, 5.63 mmol) were added, respectively, and the mixture was stirred at 60°C for 3 hours. After the reaction solution cooled to room temperature, the pH was adjusted to approximately 7 with 1 M sodium hydroxide solution. The reaction solution was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 1:20) to obtain 222 mg of off-white solid compound 36, with a yield of 53%.

[0221] Step 6: To a 50 mL two-necked reaction flask at room temperature were added compound 36 (222 mg, 0.97 mmol), UDP-Gal (590 mg, 1.06 mmol), a solution of MgCl2·6H2O (100 mg) in deionized water (20 mL), and β1,4-galactosyltransferase NmLgtB-4 (60 mg), respectively. The reaction was stirred at 37°C overnight. An equal volume of ethanol was added, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to afford 214 mg of solid compound 37 in a 56% yield.

[0222] Step 7: Under 0°C, 10 mL of water, compound 37 (214 mg, 0.55 mmol), potassium phosphate (1.74 g, 8.20 mmol) and CDMBI (0.6 g, 2.76 mmol) were added to a 100 mL three-necked reaction flask in sequence. The reaction solution was stirred at 0°C overnight, the solid was removed by filtration, and the filtrate was extracted with toluene (10 mL × 3). After the aqueous phase was combined, the aqueous phase was purified by C18 preparative purification (acetonitrile: water = 1:10-1:3, preparative high performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100) to give 78 mg of white solid compound G4, with a yield of 38%.

[0223] ESI-MS calc.for C 16 H 24 NO9[M+H] + m / z=374.1, found m / z=374.1. 1 H NMR(400MHz,D2O)δ5.93(d,J=7.4Hz,1H),4.33(d,J=7.8Hz,1H),4.23(s,1H),4.07–4.03(m,1H),3.78( s,1H),3.74–3.53(m,5H),3.51(s,1H),3.42–3.35(m,2H),2.56(s,1H),2.52–2.46(m,1H),1.92(s,3H).

[0224] Example 12: Synthesis of Compound G4b

[0225] Step 1: Add 1L of methanol and 50g of N-acetyl-D-glucosamine (GlcNAc, 0.23mol) to a 2L single-mouth reaction bottle, stir and dissolve at room temperature. Then add 100g of cationic resin Dowex (H +), the system temperature was heated to 70°C and stirred for 24 hours. The mixture was cooled to room temperature, the residue was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 38.4 g of white solid compound 32, which was directly used in the next step without further purification.

[0226] Step 2: 1L DMF and compound 32 (20g, 85.02mmol) were added to a 2L three-necked reaction flask, stirred at room temperature to dissolve, and then sodium bicarbonate (214.5g, 2.55mol) and tetramethylpiperidinium oxide (0.4g, 2.56mmol) were added in sequence. The system was replaced with nitrogen protection, cooled to about 0°C, and trichloroisocyanuric acid (19.8g, 85.02mmol) was added. The reaction solution was stirred at 0°C for 4 hours, and then filtered to remove the solid. The filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography (dichloromethane: methanol = 20:1-10:1) to obtain 9.5g of off-white solid compound 33 with a yield of 48%.

[0227] Step 3: To a 250 mL three-necked reaction flask under nitrogen at 20° C., 95 mL of methanol, compound 33 (9.5 g, 40.73 mmol), potassium carbonate (11.3 g, 81.76 mmol), and Bestmann's reagent (10.2 g, 53.09 mmol) were added. The reaction solution was stirred at 20° C. for 14 hours, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 20:1-15:1) to obtain 5.95 g of compound 38 as a yellow solid in a yield of 63%.

[0228] Step 4: To a 100 mL single-necked reaction flask, 29 mL of 6 M hydrochloric acid solution and compound 38 (1.0 g, 4.36 mmol) were added, respectively, and the mixture was stirred at 60°C for 5 hours. After the reaction solution cooled to room temperature, the pH was adjusted to approximately 7 with 1 M sodium hydroxide solution. The reaction solution was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 1:20) to obtain 460 mg of compound 39 as an off-white solid, with a yield of 49%.

[0229] Step 5: To a 100 mL single-necked reaction flask at room temperature were added compound 39 (460 mg, 2.14 mmol), UDP-Gal (1.31 g, 2.30 mmol), a solution of MgCl2·6H2O (100 mg) in deionized water (20 mL), and β1,4-galactosyltransferase NmLgtB-4 (60 mg). The reaction was stirred at 37°C overnight. An equal volume of ethanol was added, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to afford 320 mg of solid compound 40 in a 39% yield.

[0230] Step 6: At 0°C, 15 mL of water, compound 40 (0.32 g, 0.85 mmol), potassium phosphate (2.7 g, 12.72 mmol), and CDMBI (0.92 g, 4.24 mmol) were added to a 100 mL three-necked reaction flask in sequence. The reaction solution was stirred at 0°C overnight. TLC (isopropanol: concentrated aqueous ammonia: water = 25:5:3) showed that the reaction was complete. The solid was filtered off, and the filtrate was extracted with toluene (10 mL × 3). After the aqueous phase was combined, the aqueous phase was purified by C18 preparative chromatography (acetonitrile: water = 1:10-1:3, preparative high performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100) to obtain 116 mg of white solid compound G4b, with a yield of 38%.

[0231] ESI-MS calc.for C 15 H 22 NO9[M+H] + m / z=360.1, found m / z=360.1. 1 H NMR(400MHz,D2O)δ5.95(d,J=7.2Hz,1H),4.47(d,J=7.8Hz,1H),4.17–4.08(m,2H),4.05(d,J=8.7H z,1H),3.98–3.88(m,1H),3.78(d,J=3.1Hz,1H),3.73–3.43(m,5H),3.44–3.27(m,1H),1.94(s,3H).

[0232] Example 13: Synthesis of Compound G20

[0233] Step 1: To a 500 mL single-necked reaction flask, add 150 mL of dichloromethane, pentaacetylmannose (Ac5Man, 50 g, 0.128 mol), and 4-toluenethiophenol (TolSH, 25 g, 0.201 mol) and stir at room temperature to dissolve. Add boron trifluoride etherate (BF3·Et2O, 40 mL, 0.32 mol) and stir at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 1:1) indicates the reaction is complete. The reaction solution is diluted with dichloromethane (450 mL) and washed with saturated sodium bicarbonate (1000 mL). The organic phase is concentrated under reduced pressure to obtain crude compound 41, which is used in the next step with a theoretical yield of 58 g.

[0234] Step 2: To a 2.0 L single-necked reaction flask, add 600 mL of methanol and compound 41 (58 g, 0.175 mol) separately, stirring at room temperature to dissolve. Then, add 40 mL of a 5 M MeONa solution in methanol, stirring at room temperature for 2 hours. TLC (dichloromethane:methanol = 20:1) indicated the reaction was complete. 1 M dilute hydrochloric acid was added to adjust the pH to 7. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain 30.8 g of an off-white solid, compound 42, in an 82% yield.

[0235] Step 3: 300 mL of DMF, compound 42 (30 g, 0.105 mol) and benzaldehyde dimethyl acetal (40 mL) were added to a 1.0 L single-necked reaction flask and stirred at room temperature to dissolve. TsOH (0.54 g, 0.003 mol) was then added and the reaction was stirred at room temperature overnight. Saturated sodium bicarbonate (40 mL) was added to quench the reaction. After concentration under reduced pressure, the concentrate was slurried with 300 mL of a mixed solution of petroleum ether and ethyl acetate (1:1) to precipitate a solid. The filtered solid was slurried with 250 mL of a mixed solution of water and ethanol (4:1) and filtered to obtain compound 43 as a white solid, 17.8 g, with a yield of 45%.

[0236] Step 4: Under nitrogen, add 350 mL of toluene and compound 43 (17.8 g, 0.048 mol) to a 1.0 L single-necked reaction flask, stir at room temperature to dissolve. Then add dibutyltin oxide (Bu2SnO, 12.1 g, 0.049 mol) and react at 125°C for 3 hours. After cooling to room temperature, add tetrabutylammonium bromide (Bu4NBr, 16.2 g, 0.050 mol), cesium fluoride (CsF, 7.4 g, 0.049 mol), and p-methoxybenzyl chloride (PMBCl, 7.8 g, 0.050 mol) to the reaction system and react at 125°C for 3 hours under nitrogen. After cooling, ethyl acetate (800 mL) was added and the mixture was washed with saturated sodium bicarbonate (600 mL). The organic phase was concentrated under reduced pressure and the concentrate was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to give compound 44 as a colorless oil, 18 g, in a yield of 76%.

[0237] Step 5: Under nitrogen protection, 350 mL of DMF and compound 44 (18 g, 0.036 mol) were added to a 1.0 L single-necked reaction flask, stirred at room temperature to dissolve. Sodium hydride (NaH, 2.2 g, 60 wt%, 0.055 mol) was then added and stirred at room temperature for 10 minutes. Benzyl bromide (BnBr, 6.5 mL, 0.055 mol) was then added and reacted at room temperature for 6 hours. Saturated ammonium chloride (20 mL) was added to quench the reaction. After the solution was concentrated under reduced pressure, ethyl acetate (800 mL) was added to the concentrate and washed with water (300 mL × 2). After the organic phase was concentrated under reduced pressure, it was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1-20:1) to obtain colorless gum compound 45, 19.2 g, with a yield of 90%.

[0238] Step 6: Under nitrogen protection, add 150 mL of dichloromethane and compound 45 (5.6 g, 0.010 mol) to a 500 mL three-necked reaction flask, stir at room temperature to dissolve. Then add dry Molecular sieves (28 g) were added and stirred at room temperature for 2 hours. The system was cooled to -60°C, and 1-(phenylsulfonyl)pyrrole (BSP, 2.0 g, 0.010 mol) and tri-tert-butylphosphine (TTBP, 5.0 g, 0.025 mol) were added and stirred for 45 minutes. Trifluoromethanesulfonic anhydride (Tf2O, 2.0 mL, 0.012 mol) was then slowly added dropwise, and the reaction was maintained at -60°C for 30 minutes. A solution of compound 46 (3.2 g, 0.007 mol) in dichloromethane (10 mL) was then added, and the reaction was continued at -60°C for 3 hours. TLC (petroleum ether:ethyl acetate = 5:1) showed that the reaction was complete. The reaction solution was filtered, and the filter residue was rinsed with dichloromethane (300 mL). The filtrate was washed once with water (200 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate=8:1) to obtain colorless gum compound 47, 14.1 g, in a yield of 65%.

[0239] Step 7: To a 250 mL single-necked reaction flask, add 56 mL of dichloromethane, 3.2 mL of water, and compound 47 (1.6 g, 1.71 mmol) respectively and stir at room temperature until completely dissolved. The system was cooled to 0°C and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 0.9 g, 3.96 mmol) was added. The reaction was carried out at 0°C for 0.5 hours, then the temperature was raised to room temperature and stirring was continued for 1 hour. The reaction solution was diluted with dichloromethane (50 mL) and washed with saturated sodium bicarbonate (30 mL x 2). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-7:1) to obtain yellow gum compound 48, 1.39 g, with a yield of 99%.

[0240] Step 8: To a 250 mL single-necked reaction flask, 75 mL of tetrahydrofuran, 20 mL of water, and compound 48 (1.39 g, 1.73 mmol) were added, respectively, and stirred at room temperature until completely dissolved. The system was replaced with nitrogen protection, and Pd / C (2 g, 10 wt%, 1.88 mmol) and HCl (5 mL, 1 M) aqueous solution were added to the system. The system was filled with hydrogen (15 psi). Stirring was carried out at room temperature overnight. TLC (isopropanol: concentrated aqueous ammonia: water = 4:2:1) showed that the reaction was complete. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain compound 49 as a yellow solid, which was used directly in the next step without purification (estimated yield 0.6 g, yield 93%).

[0241] Step 9: Under nitrogen, a 100 mL single-necked flask was charged with a solution of compound 49 (600 mg, 1.59 mmol), azidoacetic acid (N3CH2COOH, 241 mg, 2.38 mmol), N-hydroxysuccinimide (HOSu, 329 mg, 2.86 mmol), triethylamine (Et3N, 0.66 mL, 4.77 mmol) in DMF (10 mL), and N,N'-dicyclohexylcarbodiimide (DCC, 721 mg, 3.49 mmol). The mixture was stirred at room temperature overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered, concentrated under reduced pressure, and purified by amide column chromatography to afford compound 50 as a white solid, 0.5 g, in a 74% yield.

[0242] Step 10: To a 100 mL three-necked reaction flask at 0°C were added 10 mL of water, compound 50 (160 mg, 0.38 mmol), potassium phosphate (K3PO4, 1200 mg, 5.65 mmol), and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 409 mg, 1.88 mmol), respectively. The reaction was stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated completion of the reaction. The precipitate was removed by filtration, and the filtrate was purified by amide column chromatography to afford 120 mg of compound G20 as a white solid in a 78% yield.

[0243] ESI-MS calc.for C 14 H 23 N4O 10 [M+H] + m / z=407.1, found m / z=407.1. 1H NMR(400MHz,D2O)δ6.02(d,J=7.1Hz,1H),4.54(s,1H),4.24(s,1H),4.14–4.07(m,1H),4.02(s,1H ),3.82–3.67(m,3H),3.65–3.53(m,3H),3.52–3.34(m,3H),3.32–3.23(m,1H),3.24–3.12(m,1H).

[0244] Example 14: Synthesis of Compound G21

[0245] Step 1: To a 500 mL single-necked reaction flask, 150 mL of dichloromethane, pentaacetyl glucose (Ac5Glc, 50 g, 0.128 mol) and 4-toluene thiophenol (TolSH, 27 g, 0.217 mol) were added, stirred at room temperature to dissolve. Boron trifluoride etherate (BF3·Et2O, 40 mL, 0.32 mol) was then added and stirred at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 3:1) showed that the reaction was complete. The reaction solution was diluted with dichloromethane (800 mL) and washed with saturated sodium bicarbonate (1000 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 7:1-3:1) to obtain 48 g of white solid compound 51, with a yield of 82%.

[0246] Step 2: Under nitrogen protection, 200 mL of dichloromethane, compound 51 (12.5 g, 0.028 mol) and compound 46 (10.1 g, 0.021 mol) were added to a 500 mL three-necked reaction flask, and stirred at room temperature to dissolve. Molecular sieves (35 g) were added and stirred at room temperature for 2 hours. The temperature was lowered to -10°C, and N-iodosuccinimide (NIS, 8.8 g, 0.039 mol) and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 2.9 mL, 0.016 mol) were added to the reaction solution. After the addition was complete, the temperature was raised to room temperature and stirred at room temperature overnight. TLC (petroleum ether: ethyl acetate = 3:1) showed that the reaction was complete. The reaction solution was filtered and the filter residue was rinsed with dichloromethane (300 mL). Water (300 mL) was added to the filtrate. Sodium thiosulfate was slowly added under vigorous stirring until the organic phase faded. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 7:1-4:1) to obtain 17 g of white solid compound 52 in a yield of 99%.

[0247] Step 3: Add 200 mL of methanol and compound 52 (17 g, 21.09 mmol) to a 1.0 L single-necked reaction flask, stir at room temperature to dissolve. Then add 5 M MeONa in methanol (1 mL) and stir at room temperature for 2 hours. TLC (petroleum ether:ethyl acetate = 10:1) showed that the reaction was complete. Add cationic resin (H + ) and adjusted to pH 6-7. Filter and concentrate the filtrate under reduced pressure to obtain 12.6 g of light yellow solid compound 53 in a yield of 93%. The compound was used directly in the next step without further purification.

[0248] Step 4: To a 250 mL single-necked reaction flask, add 60 mL of tetrahydrofuran, 7.5 mL of water, and compound 53 (2 g, 3.14 mmol) respectively, stirring at room temperature until completely dissolved. The system was replaced with nitrogen, and a Pd / C (2 g, 10 wt%, 1.88 mmol) and HCl (12.5 mL, 1 M) aqueous solution were added. H₂ (15 psi) was introduced, and the mixture was stirred at room temperature overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 4:2:1) indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 54 as a yellow solid, which was used directly in the next step without purification (theoretical yield: 1.18 g).

[0249] Step 5: Under nitrogen, compound 54 (1.18 g, 3.12 mmol), azidoacetic acid (N3CH2COOH, 473 mg, 4.68 mmol), N-hydroxysuccinimide (HOSu, 647 mg, 5.62 mmol), triethylamine (Et3N, 1.3 mL, 9.40 mmol) in DMF (15 mL), and N,N'-dicyclohexylcarbodiimide (DCC, 1.42 g, 6.88 mmol) were added to a 100 mL single-necked flask and stirred overnight at room temperature. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered, concentrated under reduced pressure, and purified by amide column chromatography to afford 370 mg of compound 55 as a white solid in a 28% yield.

[0250] Step 6: To a 100 mL three-necked reaction flask at 0°C were added 10 mL of water, compound 55 (120 mg, 0.28 mmol), potassium phosphate (K3PO4, 900 mg, 4.24 mmol), and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 307 mg, 1.41 mmol), respectively. The reaction was stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated completion of the reaction. The precipitate was removed by filtration, and the filtrate was purified by amide column chromatography to afford 60 mg of compound G21 as a white solid in a 52% yield.

[0251] ESI-MS calc.for C 14 H 23 N4O 10 [M+H] + m / z=407.1, found m / z=407.2. 1 H NMR(400MHz,D2O)δ6.04(dd,J=7.3,2.9Hz,1H),4.38–4.22(m,2H),4.17–4.07(m,1H),4.02(d,J=22.6H z,2H),3.80–3.61(m,3H),3.61–3.44(m,2H),3.35–3.25(m,3H),3.25–3.16(m,1H),3.15–3.03(m,1H).

[0252] Example 15: Synthesis of Compound G22

[0253] Step 1: To a 500 mL single-necked reaction flask, add 125 mL of DMF, compound 13 (5 g, 0.012 mol), and 4-methoxybenzaldehyde dimethyl acetal (6.4 g, 0.035 mol) and stir at room temperature to dissolve. Then, add p-toluenesulfonic acid (TsOH, 0.2 g, 0.001 mol) and stir at room temperature for 2 hours. Then, add saturated sodium bicarbonate (100 mL) to quench the reaction, filter out the solid, and concentrate the filtrate under reduced pressure to obtain the crude product of compound 56, which was directly used in the next step based on a 6.39 g amount.

[0254] Step 2: To a 500 mL single-necked reaction flask, 100 mL of pyridine, compound 56 (6.39 g, 0.012 mol), and 4-dimethylaminopyridine (DMAP, 0.14 g, 0.001 mol) were added, stirred at room temperature, and dissolved. Acetic anhydride (Ac2O, 20 mL, 0.216 mol) was then added to the system and stirred at room temperature overnight. TLC (petroleum ether:ethyl acetate = 1:1) showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1-0:1) to obtain 7.8 g of off-white solid compound 57, with a yield of 88%.

[0255] Step 3: To a 250 mL single-necked reaction flask, 80 mL of acetic acid (AcOH), 20 mL of water, and compound 57 (7.8 g, 0.010 mol) were added, respectively, and stirred at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 1:4) showed the reaction was complete. After concentration under reduced pressure, water (150 mL) was added to the concentrate, and then extracted with ethyl acetate (150 mL x 2). The organic phases were combined and concentrated under reduced pressure to obtain 5.9 g of off-white solid compound 58, which was directly used in the next step without further purification.

[0256] Step 4: To a 250 mL single-necked reaction flask, add 60 mL of pyridine and compound 58 (5.9 g, 9.30 mmol), stir at room temperature to dissolve, and cool to 20°C to -15°C. Add p-toluenesulfonyl chloride (TsCl, 6.8 g, 35.67 mmol) at -20°C to -15°C, and stir overnight. Add 20 mL of methanol to quench the reaction, and then concentrate the reaction solution under reduced pressure. After concentration, add ethyl acetate (400 mL) and 1 M HCl (1000 mL), and then separate the organic layer. The aqueous phase is extracted with ethyl acetate (400 mL), and the organic phases are combined and washed with saturated sodium bicarbonate aqueous solution (200 mL). The organic phase is concentrated under reduced pressure to obtain 10 g of white solid compound 59, which is used directly in the next step without purification (estimated yield 5 g, yield 68%).

[0257] Step 5: Under nitrogen protection and room temperature, add 120 mL of DMF and compound 59 (5 g, 7.55 mmol) to a 250 mL three-necked reaction flask. Then add sodium azide (NaN3, 6.18 g, 95.06 mmol), then heat to 60 ° C and stir for 60 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Add water (200 mL) to the residue and extract with ethyl acetate (100 mL × 2). The organic phases are combined and concentrated under reduced pressure to obtain 2.4 g of white solid compound 60, which is directly used in the next step without further purification.

[0258] Step 6: Add 100 mL of methanol and compound 60 (2.4 g, 3.64 mmol) to a 250 mL single-necked reaction flask, stir at room temperature and dissolve completely. Then add a methanol solution of sodium methoxide (MeONa, 0.7 mL) and stir at room temperature overnight. + ) was adjusted to pH 6-7 and filtered. The filtrate was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water=1:20) to obtain 500 mg of off-white solid compound 61 with a yield of 30%.

[0259] Step 7: At 0°C, 10 mL of water, compound 61 (125 mg, 0.28 mmol), potassium phosphate (K3PO4, 905 mg, 4.26 mmol), and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 295 mg, 1.36 mmol) were added to a 50 mL three-necked reaction flask, followed by stirring at 0°C overnight. TLC (isopropanol: ammonia: water = 25:5:3) showed that the reaction was complete. The reaction solution was warmed to room temperature and then extracted with toluene (20 mL x 4) to remove impurities. The aqueous layer was directly purified by C18 column chromatography (acetonitrile: water = 1:10-1:3, preparative high performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100)) to obtain 60 mg of white solid compound G22, with a yield of 50%.

[0260] ESI-MS calc.for C 14 H 22 N7O9[M+H] + m / z=432.1, found m / z=432.1. 1 H NMR(400MHz,D2O)δ6.04(d,J=7.2Hz,1H),4.38–4.24(m,2H),4.11(d,J=6.8Hz,1H),4.02(d,J=14.8Hz,1H),3. 99–3.79(m,1H),3.70(s,1H),3.67–3.53(m,3H),3.54–3.38(m,3H),3.36–3.27(m,2H),3.24(d,J=13.1Hz,1H).

[0261] Example 16: Synthesis of Compound G23

[0262] Step 1: To a 500 mL single-necked reaction flask, 200 mL of DMF, compound 13 (5 g, 0.012 mol) and benzaldehyde dimethyl acetal (2.15 g, 0.014 mol) were added, stirred at room temperature to dissolve. p-Toluenesulfonic acid (TsOH, 0.2 g, 0.001 mol) was then added and stirred at room temperature overnight. Saturated sodium bicarbonate (100 mL) was then added to quench the reaction, the solid was filtered off, and the filtrate was concentrated under reduced pressure and purified by C18 column chromatography (methanol: water = 2:3-1:1) to give 4.0 g of compound 62 as a white solid in a yield of 66%.

[0263] Step 2: To a 250 mL single-necked reaction flask at -20°C to -15°C, add 100 mL of pyridine and compound 62 (4.0 g, 0.008 mol) respectively and stir to dissolve. Then, add p-toluenesulfonyl chloride (TsCl, 2.98 g, 0.016 mol) and stir overnight. The reaction was quenched by adding 20 mL of methanol and then concentrated under reduced pressure. The residue was used directly in the next step without purification (calculated based on a theoretical yield of 5.2 g).

[0264] Step 3: To a 250 mL single-necked reaction flask, 100 mL of 1 M HCl aqueous solution and compound 63 (5.2 g, 0.007 mol) were added, respectively, and stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 1:4 to 1:1) to afford 1 g of compound 64 as a yellow solid in a 22% yield.

[0265] Step 4: Under nitrogen, 20 mL of DMF and compound 64 (1 g, 0.0016 mol) were added to a 100 mL three-necked reaction flask, stirred at room temperature, and dissolved. Sodium azide (NaN3, 1.05 g, 0.016 mol) was then added, and the mixture was heated to 60°C and stirred for 48 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by C18 column chromatography (acetonitrile:water = 1:20 to 1:2) to obtain 0.22 g of compound 65, a 30% yield.

[0266] Step 5: To a 50 mL three-necked reaction flask, add 10 mL of water and compound 65 (150 mg, 0.334 mmol) respectively, and stir at room temperature to dissolve. Cool to 0°C, then add potassium phosphate (K3PO4, 1.06 g, 5.0 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 362 mg, 1.67 mmol), and stir at 0°C overnight. TLC (isopropanol: ammonia: water = 25:5:3) showed that the reaction was complete. The reaction solution was warmed to room temperature, and then the impurities were extracted with toluene (20 mL x 4). The aqueous layer was directly purified by C18 column chromatography (acetonitrile: water = 1:20-1:2, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to obtain 100 mg of white solid compound G23, with a yield of 69%.

[0267] ESI-MS calc.for C 14 H 22 N7O9[M+H] + m / z=432.1, found m / z=432.1. 1H NMR(400MHz,D2O)δ6.30-6.21(m,1H),4.50-4.41(m,2H),4.36-4.29(m,1H), 4.30-4.14(m,1H),3.95-3.90(m,1H),3.87-3.59(m,8H),3.59-3.48(m,2H).

[0268] Example 17: Synthesis of Compound G24

[0269] Step 1: To phosphate buffer (40 mL) were added galactose oxidase (18 mL), copper sulfate (CuSO₄, 2 mM, 10 mL), horseradish peroxidase (HRP, 340 mg), and catalase (10 mL), followed by activation in a 30°C water bath for 30 minutes. To a 2.0 L three-necked reaction flask at 30°C, phosphate buffer (200 mL) and compound 66 (13 g, 29.45 mmol) were added, respectively. O₂ was introduced for 15-30 minutes, and the enzyme mixture was then added to the reaction system. The reaction was allowed to react in a 30°C water bath for 6 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. O₂ introduction was stopped, and the reaction solution was used directly in the next step without further processing.

[0270] Step 2: To the reaction solution, add NaCO (1.56 g, 14.72 mmol) and hydroxylamine hydrochloride (NHOH·HCl, 2.25 g, 32.38 mmol). The mixture was allowed to react at room temperature for 2 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. 300 mL of methanol was added, and the precipitate was removed by filtration through celite. The precipitate was rinsed with 300 mL of methanol, and the filtrate was used directly in the next reaction (compound 68, 13.0 g, 97% yield).

[0271] Step 3: To a 2.0 L single-necked reaction flask at 0°C, 600 mL of methanol, 280 mL of phosphate buffer, compound 68 (13 g, 0.029 mol), and nickel chloride hexahydrate (NiCl2·6H2O, 17.0 g, 0.072 mol) were added and stirred. Sodium borohydride (NaBH4, 5.4 g, 0.143 mol) and 9-fluorenylmethyl N-succinimidyl carbonate (FmocOSu, 19.3 g, 0.057 mol) were then added and stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 50:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 1:3-1:2) to afford 16 g of compound 69 as a white solid in an 84% yield.

[0272] Step 4: To a 250 mL single-necked reaction flask, add 45 mL of methanol, compound 69 (3.3 g, 0.005 mol), and aqueous HCl (20 mL, 5 M) at room temperature for 4 hours. TLC (isopropanol:concentrated ammonia:water = 50:5:3) indicated the reaction was complete. The mixture was concentrated under reduced pressure to afford crude compound 70, which was used directly in the next step based on a theoretical yield of 2.98 g.

[0273] Step 5: To a 250 mL reaction flask at room temperature, 100 mL of methanol and compound 70 (2.98 g, 0.005 mol) were added, followed by sodium methoxide to adjust the pH to 7. Under nitrogen, Boc-glycine-N-hydroxysuccinimide ester (BocNHCH2COOSu, 6.1 g, 0.022 mol) was added and stirred overnight. The reaction solution was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 1:3-2:3) to obtain 3.1 g of compound 71 as a white solid, in an 86% yield.

[0274] Step 6: To a 250 mL single-necked reaction flask, add 40 mL of methanol, compound 71 (3.3 g, 0.005 mol), and aqueous HCl (17 mL, 5 M) at room temperature for 4 hours. TLC (isopropanol:concentrated ammonia:water = 50:5:3) indicated the reaction was complete. The mixture was concentrated under reduced pressure to afford crude compound 72, which was used directly in the next step (2.8 g).

[0275] Step 7: Preparation of trifluoromethanesulfonyl azide (TfN3): To a mixture of NaN3 (3.5 g, 53.2 mmol, 3 eq) in H2O (25 mL) and DCM (40 mL) in a 250 mL three-necked reaction flask at -5-0°C was added Tf2O (3 mL, 17.7 mmol, 1 eq), followed by stirring for 2.5 hours. The mixture was separated, and the aqueous phase was extracted with DCM (10 mL x 2). The combined DCM extracts were washed with saturated sodium bicarbonate (40 mL) to obtain a TfN3 solution in DCM. 3.0 g (17.1 mmol, 97%) of TfN3 was used.

[0276] The TfN3 solution in dichloromethane obtained above was added to a solution of compound 72 (2.8 g, 0.004 mol), K2CO3 (0.65 g, 0.005 mol), and CuSO4 (6.8 mg, 0.043 mmol) in water (60 mL) and methanol (120 mL) in a 1.0 L single-necked flask at room temperature. The mixture was allowed to react overnight at room temperature. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered, and the methanol and dichloromethane were evaporated under reduced pressure. The product precipitated from the water. 3 g of crude compound 73 was filtered and used directly in the next step (estimated actual content 80%, yield 87%).

[0277] Step 8: To a 100 mL single-necked flask at room temperature, add 20 mL of DMF and compound 73 (1.5 g, 80 wt%, 0.002 mol) and stir to dissolve. Add piperidine (2 mL, 0.020 mol) and allow to react for 2 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. Concentrate under reduced pressure to obtain crude compound 74, which (0.78 g) was used directly in the next step.

[0278] Step 9: To a 100 mL single-necked flask under nitrogen at room temperature were added 15 mL of DMF, compound 74 (0.78 g, 1.8 mmol), N-hydroxysuccinimide (HOSu, 0.32 g, 2.8 mmol), N3-PEG3-COOH (0.68 g, 2.8 mmol), and N,N'-dicyclohexylcarbodiimide (DCC, 0.76 g, 3.7 mmol), respectively. The mixture was allowed to react overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The product was filtered, and the filtrate was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 1:5-1:2) to afford 0.63 g of compound 75 as a white solid in a 52% yield.

[0279] Step 10: Potassium phosphate (K3PO4, 634 mg, 2.99 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 216 mg, 0.99 mmol) were added to a solution of compound 75 (130 mg, 0.20 mmol) in water (8 mL) (100 mL three-necked reaction flask) at 0°C, and then stirred at 0°C overnight. TLC (isopropanol: concentrated aqueous ammonia: water = 25:5:3) showed that the reaction was complete. The reaction solution was filtered and the filtrate was directly purified by C18 column chromatography (acetonitrile: water = 1:5-1:2, preparative high performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100) to give 82 mg of white solid compound G24, with a yield of 65%.

[0280] ESI-MS calc.for C 23 H 39 N8O 13 [M+H] + m / z=635.2, found m / z=635.2. 1 H NMR(400MHz,D2O)δ6.19(d,J=7.3Hz,1H),4.44(dd,J=3.1,1.6Hz,1H),4.37(d,J=7.8Hz,1H),4.27(ddd,J=8.6,3.1,1.5Hz,1H),4.24-4.11(m, 2H),3.85(dd,J=3.4,0.9Hz,1H),3.81-3.74(m,3H),3.72-3.63(m,13H),3.58(dd,J=9.9,3.4Hz,1H),3.52-3.40(m,6H),2.54(t,J=6.1Hz,2H).

[0281] Example 18: Synthesis of Compound G25

[0282] Step 1: At room temperature, add D-(+)-galactosamine hydrochloride (GalNH 2· To a suspension of HCl (50 g, 0.23 mol) in MeOH (400 mL) (1.0 L single-necked flask), add MeONa (13.2 g, 0.24 mol). Stir at room temperature for 20 minutes. Then, add phthalic anhydride (PhthO, 37.8 g, 0.26 mol), and stir overnight. Concentrate under reduced pressure to obtain crude compound 76, which is used directly in the next step (calculated as 71 g).

[0283] Step 2: To a 2.0 L single-necked reaction flask, 700 mL of pyridine, compound 76 (71 g, 0.23 mol), and 4-dimethylaminopyridine (DMAP, 1.4 g, 0.01 mol) were added, stirred, and dissolved. Acetic anhydride (Ac2O, 195 mL, 2.08 mol) was then added to the system and stirred at room temperature overnight. TLC (petroleum ether:ethyl acetate = 1:1) showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1-2:1) to obtain 59 g of solid compound 77 in a yield of 54%.

[0284] Step 3: To a 500 mL single-necked reaction flask, 180 mL of dichloromethane, compound 77 (59 g, 0.12 mol), 4-toluene thiophenol (TolSH, 24.5 g, 0.20 mol), and boron trifluoride etherate (BF3·Et2O, 50 mL, 0.40 mol) were added and stirred at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 3:1) showed that the reaction was complete. The mixture was diluted with dichloromethane (900 mL) and washed with saturated sodium bicarbonate aqueous solution (1200 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 7:1-3:1) to give 43 g of compound 78 as a white solid in a 64% yield.

[0285] Step 4: Under nitrogen protection, dry Molecular sieves (30 g) were added and stirred at room temperature for 2 hours. The temperature was lowered to about -10°C, and N-iodosuccinimide (NIS, 7.0 g, 31.11 mmol) and silver trifluoromethanesulfonate (AgOTf, 0.66 g, 2.57 mmol) were added, and then the mixture was warmed to room temperature and stirred overnight. TLC (petroleum ether:ethyl acetate = 3:1) showed that the reaction was complete. The reaction solution was filtered and the filter residue was rinsed with dichloromethane (300 mL). Water (300 mL) was added to the filtrate. Sodium thiosulfate was slowly added under vigorous stirring until the organic phase faded. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1-1:1) to obtain 15 g of colorless gum compound 80 in an 87% yield.

[0286] Step 5: Under nitrogen protection and 0°C, sodium methoxide (1.5 mL, 5 M methanol solution) was added to a solution of compound 80 (15 g, 15.04 mmol) in methanol (300 mL) (1.0 L single-necked reaction bottle), and the mixture was stirred at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 1:3) showed that the reaction was complete, and cationic resin (H +) and adjusted to pH 6-7. Filter and concentrate the filtrate under reduced pressure to obtain 13 g of crude pale yellow solid compound 81, which was used directly in the next step without further purification.

[0287] Step 6: Under nitrogen, hydrazine hydrate (N2H4·H2O, 14.5 mL, 298.91 mmol) was added to a solution of compound 81 (13 g, 14.93 mmol) in ethanol (400 mL) in a 1.0 L single-necked reaction flask. The mixture was stirred at 80°C overnight. TLC (dichloromethane:methanol = 10:1) indicated the reaction was complete. The mixture was cooled and filtered. The filtrate was concentrated under reduced pressure and purified by C18 column chromatography (methanol:water = 2:3 to 2:1) to afford 7.8 g of compound 82 as a white solid in an 85% yield.

[0288] Step 7: To a mixture of compound 82 (2 g, 3.27 mmol) in tetrahydrofuran (240 mL) and water (70 mL) in a 500 mL single-necked flask, palladium on carbon (2 g, 10 wt %, 1.88 mmol) and 1 M dilute HCl (13 mL) were added. H₂ (15 psi) was introduced and stirred at room temperature overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 4:2:1) indicated the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to afford compound 83 as a white solid, which was used directly in the next step without purification (1.1 g was used in the next step).

[0289] Step 8: Under nitrogen, to a solution of compound 83 (1.1 g, 2.66 mmol), azidoacetic acid (N3CH2COOH, 807 mg, 7.99 mmol), N-hydroxysuccinimide (HOSu, 1.1 g, 9.56 mmol), and triethylamine (3 mL) in DMF (15 mL) (50 mL single-necked flask) was added N,N'-dicyclohexylcarbodiimide (DCC, 2.2 g, 10.66 mmol). The mixture was allowed to stand at room temperature overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered, concentrated under reduced pressure, and the residue was purified by amide column chromatography to afford 0.8 g of compound 84 as a white solid in a 59% yield.

[0290] Step 9: To a solution of compound 84 (130 mg, 0.26 mmol) in water (10 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 817 mg, 3.85 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 278 mg, 1.28 mmol). The mixture was then stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated completion of the reaction. The precipitate was removed by filtration, and the filtrate was purified using preparative C18 (acetonitrile:water = 1:20 to 1:4) to afford 80 mg of compound G25 as a white solid in a 63% yield.

[0291] ESI-MS calc.for C 16 H 25 N8O 10 [M+H] + m / z=489.1, found m / z=489.1. 1 H NMR(400MHz,D2O)δ6.18(d,J=7.3Hz,1H),4.56(d,J=8.4Hz,1H),4.46(dd,J=3.1,1.7Hz,1H),4.26(ddd,J=7.4,3.2,1.6Hz,1H ),4.17(dd,J=4.0,1.8Hz,2H),4.04(d,J=1.2Hz,2H),3.95-3.88(m,2H),3.85-3.55(m,7H),3.33(ddd,J=8.8,6.4,2.4Hz,1H).

[0292] Example 19: Synthesis of Compound G28b

[0293] Step 1: Under nitrogen, sodium azide (54 g, 0.83 mol) was added to 3-chloropropylamine hydrochloride (36 g, 0.28 mol) in water (220 mL) in a 500 mL reaction flask. The mixture was stirred at 80°C overnight. Sodium hydroxide (33 g, 0.83 mol) was added, and the mixture was extracted with ether (200 mL x 5). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to approximately 100 mL of compound 85, which was then used directly in the next step (based on a 22 g yield of 79%).

[0294] Step 2: To a mixed solution of glutamic acid (52 g, 0.19 mol) in DMF (300 mL) and water (200 mL) in a 1.0 L single-necked reaction flask, triethylamine (98 mL) was added and stirred at room temperature for 10 minutes. Di-tert-butyl dicarbonate (Boc2O, 81 g, 0.37 mol) was then added and allowed to react for 2.5 hours. Saturated saline (600 mL) and aqueous HCl (4 M, 160 mL) were added, and the mixture was extracted with ethyl acetate (400 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was slurried with dichloromethane (200 mL) to obtain 85 g of compound 86 as a white powder solid, with a yield of 97%.

[0295] Step 3: Under nitrogen, EDCI (39 g, 0.20 mol) and N,N-diisopropylethylamine (DIPEA, 84 mL, 0.48 mol) were added to a suspension of compound 85 (10 g, 0.10 mol), compound 86 (14 g, 0.06 mol), and 1-hydroxybenzotriazole (HOBt, 27 g, 0.20 mol) in dichloromethane (200 mL) in a 500 mL reaction flask. The mixture was stirred overnight at room temperature. TLC (ethyl acetate:methanol = 1:0) indicated the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was added with saturated sodium bicarbonate (400 mL). The mixture was then extracted with ethyl acetate (200 mL x 3). The combined organic phases were concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate:methanol = 1:0-5:1) to afford 8 g of compound 87 as a light yellow oil in a 48% yield.

[0296] Step 4: To a solution of compound 87 (8 g, 0.02 mol) in dichloromethane (30 mL) in a 100 mL reaction flask was added trifluoroacetic acid (14.5 mL) and allowed to react at room temperature for 2 hours. TLC (ethyl acetate:methanol = 10:1) indicated the reaction was complete. After concentration under reduced pressure, the product was purified by C18 column chromatography (methanol:water = 1:10 to 2:3) to afford 4.5 g of compound 88 as a colorless oil in a 74% yield.

[0297] Step 5: To a solution of N-hydroxyphthalimide (50 g, 0.31 mol) in DMF (30 mL) (2.0 L three-necked reaction flask) was added potassium carbonate (85 g, 0.62 mol) and tert-butyl bromoacetate (47 mL, 0.32 mol) and stirred at 60°C for 2 hours. TLC (ethyl acetate:methanol = 10:1) showed the reaction was complete. The reaction solution was cooled to room temperature and then poured into ice water (2 L). A white solid precipitated with stirring, which was filtered. The filter cake was washed with water (300 mL) and then slurried with petroleum ether (300 mL) to give 60 g of compound 89 as a white solid in a 70% yield.

[0298] Step 6: To a solution of compound 89 (20 g, 0.07 mol) in dichloromethane (80 mL) in a 500 mL reaction flask was added trifluoroacetic acid (43 mL) and stirred at room temperature for 6 hours. TLC (ethyl acetate:methanol = 10:1) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was slurried with ethyl acetate (50 mL) to afford 12 g of compound 90 as a white solid, in a 75% yield.

[0299] Step 7: Under nitrogen, N,N'-dicyclohexylcarbodiimide (DCC, 4.4 g, 21.33 mmol) was added to a mixture of compound 88 (5 g, 16.06 mmol), compound 90 (3.9 g, 17.63 mmol), and 1-hydroxybenzotriazole (HOBt, 2.9 g, 21.46 mmol) in DMF (65 mL) and acetonitrile (65 mL) in a 500 mL reaction flask. The mixture was stirred overnight at room temperature. TLC (isopropanol:water = 20:1) indicated the reaction was complete. The white precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain crude compound 91, which was used directly in the next step without purification (8.26 g was used in the next step).

[0300] Step 8: To a solution of compound 91 (8.26 g, 16.05 mmol) in methanol (200 mL) in a 500 mL reaction flask was added hydrazine hydrate (5.1 mL, 105.14 mmol) and allowed to react at room temperature for 2 hours. TLC (isopropanol:concentrated ammonia:water = 20:1:1) indicated the reaction was complete. The product was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:3-1:2) to afford 6 g of compound 92 as a white solid in a 97% yield.

[0301] Step 9: To phosphate buffer (5 mL), galactose oxidase (3 mL), horseradish peroxidase (HRP, 50 mg), catalase (1.5 mL), and copper sulfate (CuSO4, 1 mM, 0.9 mL) were added sequentially and activated in a 30°C water bath for 30 minutes. Oxygen (O2) was bubbled through a 25 mL phosphate buffer solution containing compound 5 (600 mg, 1.50 mmol) in a 100 mL single-necked reaction vial at 30°C for 30 minutes. The enzyme mixture was then added to the reaction system and allowed to react in a 30°C water bath for 6 hours. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete, and the oxygen flow was stopped. Compound 92 (693 mg, 1.80 mmol) was added and the reaction was allowed to proceed overnight at room temperature. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) showed the reaction was complete. Methanol (300 mL) was added and the precipitate was removed by filtration through celite. The precipitate was rinsed with methanol (300 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:10-1:4) to give 700 mg of compound 93 as a white powdery solid in a 61% yield.

[0302] Step 10: To a solution of compound 93 (170 mg, 0.22 mmol) in water (8 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 709 mg, 3.34 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 242 mg, 1.11 mmol). The mixture was then stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was filtered, and the filtrate was directly purified by C18 column chromatography (acetonitrile:water = 1:4-7:3) to afford 112 mg of compound G28b as a white solid in a 67% yield.

[0303] ESI-MS calc.for C 27 H 44 N 11 O 12 S[M+H] + m / z=746.2, found m / z=746.2. 1 H NMR(400MHz,D2O)δ7.55(d,J=4.6Hz,0.7H),6.85(d,J=4.5Hz,0.3H),5.97(d,J=7.3Hz,1H),4.51(d,J=5.0Hz,2H),4.44 (d,J=10.1Hz,1H),4.38(d,J=7.0Hz,1H),4.25(s,1H),4.22–4.16(m,1H),4.06(s,1H),4.00–3.94(m,1H),3.73(d,J=11. 9Hz,1H),3.61(d,J=17.7Hz,2H),3.47(d,J=9.6Hz,1H),3.37(d,J=7.5Hz,1H),3.24(d,J=9.5Hz,4H),3.17(d,J=6.6Hz,4 H),2.90(s,1H),2.19(d,J=6.1Hz,2H),2.04–1.96(m,1H),1.92(s,3H),1.84(dd,J=14.8,7.6Hz,1H),1.68–1.60(m,4H).

[0304] Example 20: Synthesis of Compound G29

[0305] Step 1: To a solution of compound 1 (10 g, 26.09 mmol) and TsOH (0.45 g, 2.61 mmol) in DMF (250 mL) (500 mL single-necked reaction flask) was added 2,2-dimethoxypropane (51 mL) and stirred at room temperature for 25 minutes. The reaction was then quenched by adding saturated sodium bicarbonate (50 mL), the solid was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 (methanol:water = 1:20) to obtain 4.9 g of compound 21 as a white solid in a 44% yield.

[0306] Step 2: To a solution of compound 21 (4.9 g, 11.57 mmol) in pyridine (200 mL) in a 500 mL single-necked reaction flask at -20°C was added TsCl (4.0 g, 20.98 mmol) and stirred overnight. The reaction was quenched by adding 20 mL of methanol and then concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 2:3 to 1:1) to afford 2.3 g of compound 22 as a yellow solid in a 34% yield.

[0307] Step 3: Compound 22 (2.3 g, 3.98 mmol) was dissolved in 30 mL of 1.5 M aqueous HCl in a 250 mL single-necked reaction flask and stirred at room temperature for 2 hours. TLC (dichloromethane:methanol = 20:1) indicated the reaction was complete, and the mixture was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:2-1:1) to afford 1.8 g of Compound 94 as a yellow solid, in an 86% yield.

[0308] Step 4: To a solution of compound 94 (2.3 g, 4.28 mmol) in water (15 mL) was added dropwise in a 100 mL single-necked reaction flask containing N,O-dimethylhydroxylamine hydrochloride (MeNHOMe·HCl, 1.09 g, 11.17 mmol) and sodium acetate (AcONa, 0.91 g, 11.09 mmol) in water (10 mL). The mixture was allowed to react overnight at room temperature. TLC (dichloromethane:methanol = 20:1) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:2 to 1:1) to afford 1.2 g of compound 95, a 48% yield.

[0309] Step 5: Under nitrogen, NaN (1.1 g, 16.9 mmol) was added to a solution of compound 95 (1.2 g, 2.07 mmol) in DMF (30 mL) in a 100 mL three-necked reaction flask. The mixture was then heated to 60°C and allowed to react overnight. TLC (isopropanol:water:concentrated aqueous ammonia = 25:5:3) indicated the reaction was complete. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:20 to 1:4) to afford 0.9 g of compound 96, in a yield of 96%.

[0310] Step 6: To a solution of compound 96 (0.9 g, 1.99 mmol) in methanol (20 mL) in a 100 mL single-necked reaction flask was added Pd / C (1 g, 5 wt %, 0.47 mmol) and Pd(OH)2 / C (1 g, 20 wt %, 1.42 mmol). Hydrogen was introduced (15 psi) and the mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was used directly in the next step (0.8 g, 94% calculated).

[0311] Step 7: To a solution of compound 97 (0.8 g, 1.88 mmol) in methanol (20 mL) in a 100 mL single-necked reaction flask was added N(CH)COOSu (1.28 g, 5.66 mmol) and EtN (0.4 mL). The mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:20 to 1:4) to afford 0.41 g of compound 98, a 40% yield.

[0312] Step 8: Compound 98 (0.41 g, 0.76 mmol) was dissolved in acetic acid (60 mL) and water (15 mL) and reacted at 60°C for 4 hours. TLC (i-PrOH:NH 3· The reaction was complete (H₂O:H₂O = 25:5:3). The reaction solution was concentrated under reduced pressure, and the residue was purified by C₁₈ column chromatography (methanol:water = 1:4-3:7) to afford 0.34 g of compound 99 as a white solid, in a 90% yield.

[0313] Step 9: To a solution of compound 99 (200 mg, 0.41 mmol) in water (13 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 1.29 g, 6.08 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 440 mg, 2.03 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was filtered, and the filtrate was directly used for preparative HPLC (acetonitrile:water = 1:5-2:3, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to obtain 140 mg of compound G29 as a white solid, with a yield of 72%.

[0314] ESI-MS calc.for C 18 H 30 N5O 10 [M+H] + m / z=476.2, found m / z=476.2. 1H NMR(400MHz,D2O)δ6.09(d,J=7.3Hz,1H),4.48(d,J=7.8Hz,1H),4.43(dd,J=3.0,1.5Hz,1H),4.21(dq,J=6.7,1.7Hz,1H),3 .96-3.90(m,1H),3.87-3.62(m,5H),3.61-3.35(m,6H),2.39(t,J=7.2Hz,2H),2.07(d,J=1.8Hz,3H),1.91(q,J=7.0Hz,2H).

[0315] Example 21: Synthesis of Compound G32

[0316] Step 1: To a solution of compound 1 (3.0 g, 7.83 mmol) in pyridine (30 mL) in a 100 mL single-necked reaction flask was added TsCl (3.7 g, 19.41 mmol) at 25°C and stirred for 3 hours. TLC (acetonitrile:water = 10:1) indicated the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 50:1-10:1) to afford 2.2 g of compound 100 as a yellow solid, in a 40% yield.

[0317] Step 2: Under nitrogen protection, NaN3 (1.03 g, 15.8 mmol) was added to a solution of compound 100 (2.2 g, 3.18 mmol) in DMF (15 mL) (100 mL three-necked reaction flask), and then the temperature was raised to 80°C and stirred for 12 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was prepared using C18 (acetonitrile: water = 1:10-1:2, preparative high performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100)) to obtain 160 mg of white solid compound 101, with a yield of 11%.

[0318] Step 3: Potassium phosphate (K3PO4, 1.18 g, 5.56 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 400 mg, 1.84 mmol) were added to a solution of compound 101 (160 mg, 0.37 mmol) in water (12 mL) in a 100 mL three-necked reaction flask at 0°C, followed by stirring at 0°C overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. Impurities were extracted with toluene (20 mL x 4), and the aqueous layer was directly purified by C18 chromatography (acetonitrile:water = 1:10-1:1, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to afford 120 mg of compound G32 as a white solid, in a yield of 78%.

[0319] ESI-MS calc.for C 14 H 22 N7O8[M+H] + m / z=416.1, found m / z=416.1. 1 H NMR(400MHz,D2O)δ5.96(d,J=7.3Hz,1H),4.40–4.23(m,2H),4.07(d,J=8.1Hz,1H),3.74(s,1H),3 .71–3.61(m,2H),3.57–3.42(m,4H),3.43–3.33(m,2H),3.29(dd,J=13.2,3.7Hz,1H),1.93(s,3H).

[0320] Example 22: Synthesis of Compound G33

[0321] Steps 1 and 2: Galactose oxidase (12.5 mL), horseradish peroxidase (HRP, horseradish peroxidase, 210 mg), catalase (6.25 mL), and an aqueous solution of copper sulfate (CuSO4, 1 mM, 4 mL) were added sequentially to a 25 mL phosphate buffer solution and activated in a 30°C water bath for 30 min. Oxygen (O2) was then bubbled through a 125 mL phosphate buffer solution containing compound 1 (5 g, 13.04 mmol) in a 2.0 L single-necked reaction flask at 30°C. After 30 min, the enzyme mixture was added to the reaction system. The reaction was incubated in a 30°C water bath for 6 h. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete, and the oxygen bubble was discontinued. NaCO (691 mg, 6.52 mmol) and NHOH·HCl (997 mg, 14.35 mmol) were then added to the reaction solution, and the mixture was stirred at room temperature for 3 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. Methanol (300 mL) was added, and the precipitate was removed by filtration through celite. The precipitate was rinsed with methanol (300 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 6:1-3:1) to afford 4.0 g of compound 102 in a 77% yield.

[0322] Step 3: To a mixture of compound 102 (5.5 g, 13.88 mmol) and NiCl2·6H2O (8.25 g, 34.71 mmol) in methanol (160 mL) and phosphate buffer (PB, 80 mL) in a 500 mL single-necked reaction flask were added NaBH4 (3.15 g, 83.27 mmol) and Boc2O (9.09 g, 41.65 mmol) at 0°C. The mixture was stirred overnight at 0°C. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:5-1:2) to afford 3.2 g of compound 103 as a white solid in a yield of 48%.

[0323] Step 4: To a solution of compound 103 (1.6 g, 3.32 mmol) in pyridine (32 mL) in a 100 mL three-necked reaction flask at -20°C was added TsCl (0.7 g, 3.67 mmol) and stirred overnight. The mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:4 to 3:1) to afford 0.6 g of compound 104, in a 28% yield.

[0324] Step 5: Under nitrogen, to a solution of compound 104 (0.6 g, 0.94 mmol) in DMF (20 mL) in a 100 mL three-necked reaction flask was added NaN (0.61 g, 9.38 mmol), and the mixture was heated to 60°C and stirred for 24 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:3-1:2) to afford 0.4 g of compound 105 as a white solid in an 84% yield.

[0325] Step 6: Compound 105 (0.4 g, 0.79 mmol) was dissolved in dilute HCl (5 mL, 1.5 M) in a 25 mL single-necked reaction vial and stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure to afford crude compound 106, which was used directly in the next step (yield calculated to be 300 mg).

[0326] Step 7: To a solution of compound 106 (300 mg, 0.68 mmol), HOSu (140 mg, 1.22 mmol), N3-PEG3-COOH (250 mg, 1.01 mmol), and Et3N (0.28 mL, 2.01 mmol) in DMF (5 mL) (25 mL single-necked flask) was added DCC (314 mg, 1.52 mmol) under nitrogen at room temperature. The mixture was allowed to react overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The product was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:4-3:7) to afford 0.23 g of compound 107 as a white solid, in a 55% yield.

[0327] Step 8: Potassium phosphate (K3PO4, 1150 mg, 5.42 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 392 mg, 1.81 mmol) were added to a solution of compound 107 (230 mg, 0.36 mmol) in water (10 mL) (50 mL three-necked reaction flask) at 0°C, and then stirred at 0°C overnight. TLC (isopropanol: concentrated aqueous ammonia: water = 25:5:3) showed that the reaction was complete. The reaction solution was filtered and the filtrate was directly purified by C18 column chromatography (acetonitrile: water = 1:4-1:1, preparative high performance liquid chromatography (Wuhan Ruihe Chromatography Technology, LC 2100) to give 80 mg of white solid compound G33, with a yield of 36%.

[0328] ESI-MS calc.for C 23 H 39 N8O 12 [M+H] + m / z=619.2, found m / z=619.2. 1 H NMR(400MHz,D2O)δ6.11(d,J=7.3Hz,1H),4.43-4.36(m,2H),4.22(dq,J=6.7,1.6Hz,1H),3.91-3.87(m,1H) ,3.80(t,J=6.2Hz,2H),3.77-3.54(m,15H),3.54-3.40(m,6H),2.57(t,J=6.2Hz,2H),2.07(d,J=1.8Hz,3H).

[0329] Example 23: Synthesis of Compound G35

[0330] Step 1: To a solution of compound 13 (2.0 g, 4.71 mmol) in pyridine (60 mL) in a 100 mL three-necked reaction flask was added TsCl (2.7 g, 14.16 mmol) at -15°C and stirred for 13 hours. TLC (acetonitrile:water = 10:1) indicated the reaction was complete. Ethyl acetate (200 mL) was added for dilution, followed by a saturated aqueous sodium bicarbonate solution (100 mL). The mixture was separated, and the organic phase was washed with saturated brine (150 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford 2.8 g of compound 108 as a yellow solid in an 81% yield.

[0331] Step 2: To a solution of compound 108 (2.8 g, 3.82 mmol) in DMF (50 mL) in a 100 mL three-necked reaction flask was added sodium azide (3.73 g, 57.38 mmol) and stirred at 60°C for 40 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The product was concentrated under reduced pressure and purified by C18 column chromatography (acetonitrile:water = 1:10 to 3:7) to afford 1.8 g of crude compound 109 as a white solid.

[0332] Step 3: Potassium phosphate (K3PO4, 2.35 g, 11.07 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 0.8 g, 3.68 mmol) were added to a solution of compound 109 (0.35 g, 0.74 mmol) in water (15 mL) in a 100 mL three-necked reaction flask at 0°C, followed by stirring at 0°C overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. Impurities were extracted with toluene (20 mL x 4), and the aqueous layer was directly purified by C18 chromatography (acetonitrile:water = 1:10-1:3, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to obtain 166 mg of compound G35 as a white solid, with a yield of 49%.

[0333] ESI-MS calc.for C 14 H 21 N 10 O8[M+H] + m / z=457.1, found m / z=457.1. 1 H NMR(400MHz,D2O)δ6.07(d,J=7.3Hz,1H),4.40–4.25(m,2H),4.20–4.11(m,1H),4 .10–3.92(m,1H),3.72(d,J=2.8Hz,1H),3.65(d,J=8.3Hz,2H),3.57–3.20(m,8H).

[0334] Example 24: Synthesis of Compound G36

[0335] Steps 1 and 2: Galactose oxidase (4 mL), copper sulfate (1 mM, 1.2 mL), horseradish peroxidase (HRP, 66 mg), and catalase (2 mL) were added sequentially to phosphate buffer (8 mL) and activated in a 30°C water bath for 30 min. Oxygen (O2) was bubbled through a 40 mL phosphate buffer solution containing LacNPr (1.6 g, 4.03 mmol) in a 1.0 L single-necked reaction flask at 30°C for 30 min. The enzyme mixture was then added to the reaction system and incubated in a 30°C water bath for 6 h. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. Oxygen bubble was discontinued and the reaction solution was used directly in the next step without further processing. N3C2H4ONH2 (0.62 g, 6.07 mmol) was added and allowed to react at room temperature for 14 hours. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The product was concentrated under reduced pressure, and the residue was redissolved in 40 mL of methanol. The product was filtered, and the filtrate was concentrated under reduced pressure. The residue was used to prepare C18 (acetonitrile:water = 1:20-1:3, using a preparative high-performance liquid chromatograph (Wuhan Ruihe Chromatography Technology, LC 2100) to give 0.84 g of compound 111 as a white solid, in a 43% yield.

[0336] Step 3: To a solution of compound 111 (840 mg, 1.75 mmol) in water (30 mL) in a 100 mL three-necked flask at 0°C were added potassium phosphate (K3PO4, 5.58 g, 26.29 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 1.9 g, 8.75 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was filtered, and the filtrate was directly analyzed by preparative HPLC (methanol:water = 1:20-1:4, Wuhan Ruihe Chromatography Technology, LC 2100) to obtain 417 mg of compound G36 as a white solid, with a yield of 51%.

[0337] ESI-MS calc.for C 17 H 28 N5O 10 [M+H] + m / z=462.2, found m / z=462.2. 1H NMR(400MHz,D2O)δ7.51(d,J=4.8Hz,0.7H),6.85(d,J=5.9Hz,0.3H),5.95(d,J=7.3Hz,1H ),4.40–4.32(m,1H),4.32–4.22(m,2H),4.19–4.17(m,0.3H),4.17–4.11(m,2H),4.04(d, J=4.1Hz,1H),3.91(d,J=3.3Hz,0.7H),3.68(d,J=12.3Hz,1H),3.54(t,J=7.9Hz,3H),3.4 3(dd,J=12.0,6.3Hz,3H),3.32(d,J=6.4Hz,1H),2.31–2.23(m,2H),1.03(t,J=8.2Hz,3H).

[0338] Example 25: Synthesis of Compound G37

[0339] Steps 1 and 2: Galactose oxidase (4 mL), copper sulfate (1 mM, 1.2 mL), horseradish peroxidase (HRP, 66 mg), and catalase (2 mL) were added sequentially to phosphate buffer (8 mL) and activated in a 30°C water bath for 30 min. Oxygen (O2) was bubbled through a 40 mL phosphate buffer (1.6 g, 3.89 mmol) solution of LacNBu (1.6 g, 3.89 mmol) in a 1.0 L single-necked reaction flask at 30°C for 30 min. The enzyme mixture was then added to the reaction system and allowed to react in a 30°C water bath for 5 h. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete, and the oxygen flow was discontinued. N3C2H4ONH2 (0.6 g, 5.88 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 14 h. TLC (isopropanol:concentrated aqueous ammonia:water=25:5:3) showed the reaction was complete, and the product was concentrated under reduced pressure. The residue was redissolved in methanol (40 mL) and filtered. The filtrate was concentrated under reduced pressure, and the residue was used for preparation of C18 (acetonitrile:water=1:20-1:3, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to give 0.6 g of compound 113 as a white solid in a yield of 31%.

[0340] Step 3: Potassium phosphate (K3PO4, 3.2 g, 15.08 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 1.1 g, 5.07 mmol) were added to a solution of compound 113 (0.5 g, 1.01 mmol) in water (40 mL) in a 100 mL three-necked flask at 0°C, and then stirred overnight at 0°C. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) showed the reaction was complete. The reaction solution was filtered, and the filtrate was directly purified by C18 column chromatography (acetonitrile:water = 1:10-1:3, preparative HPLC (methanol:water = 1:20-1:4, Wuhan Ruihe Chromatography Technology, LC 2100) to obtain 400 mg of white solid compound G37, with a yield of 83%.

[0341] ESI-MS calc.for C 18 H 30 N5O 10 [M+H] + m / z=476.2, found m / z=476.2. 1 H NMR(400MHz, Deuterium Oxide)δ7.67(d,J=4.8Hz,0.7H),7.01(d,J=4.6Hz,0.3H),6.11(d,J=7.3Hz,1H),4.85(dd,J=4.6, 1.2Hz,0.3H),4.52(d,J=7.8Hz,0.7H),4.50–4.39(m,2H),4.38–4.26(m,2.3H),4.22(ddd,J=6.6, 3.0,1.5Hz,1H),4.07(d,J=3.2Hz,0.7H),3.87–3.80(m,1H),3.71(td,J=10.5,5.8Hz,3H),3.65–3 .53(m,3H),3.51–3.44(m,1H),2.39(t,J=7.4Hz,2H),1.68(h,J=7.5Hz,2H),0.98(t,J=7.4Hz,3H).

[0342] Example 26: Synthesis of Compound G38

[0343] Steps 1 and 2: Galactose oxidase (12.5 mL), horseradish peroxidase (HRP, 210 mg), catalase (6.25 mL), and copper sulfate (CuSO4, 1 mM, 4 mL) were added sequentially to phosphate buffer (25 mL) and activated in a 30°C water bath for 30 min. Oxygen (O2) was bubbled through a 500 mL three-necked reaction flask containing compound 1 (5 g, 13.04 mmol) in phosphate buffer (125 mL) at 30°C for 30 min. The enzyme mixture was then added to the reaction system and incubated in a 30°C water bath for 6 h. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete, and the oxygen bubble was discontinued. To the reaction solution were added NaCO (691 mg, 6.52 mmol) and NHOH·HCl (997 mg, 14.35 mmol), and the mixture was allowed to react at room temperature for 3 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. Methanol (300 mL) was added, and the precipitate was removed by filtration through celite. The precipitate was rinsed with methanol (300 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 6:1-3:1) to afford 4.0 g of compound 102 in a 77% yield.

[0344] Step 3: To a mixture of compound 102 (5.5 g, 13.88 mmol) and NiCl2·6H2O (8.25 g, 34.71 mmol) in MeOH (160 mL) and phosphate buffer (PB, 80 mL) in a 500 mL single-necked reaction flask at 0°C were added NaBH4 (3.15 g, 83.27 mmol) and Boc2O (9.09 g, 41.65 mmol). The mixture was stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:5-1:2) to afford 3.2 g of compound 103 as a white solid in a yield of 48%.

[0345] Step 4: To a solution of compound 103 (2.5 g, 5.18 mmol) in pyridine (50 mL) in a 250 mL single-necked reaction flask at -20°C was added TsCl (1.83 g, 9.60 mmol) and stirred overnight. The mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:4 to 3:1) to afford 1.83 g of compound 104, in a 55% yield.

[0346] Step 5: Under nitrogen, to a solution of compound 104 (1.83 g, 2.87 mmol) in DMF (40 mL) in a 100 mL three-necked reaction flask was added NaN (1.87 g, 28.76 mmol). The mixture was then heated to 60°C and stirred for 48 hours. The reaction mixture was filtered, the filtrate concentrated under reduced pressure, and the residue purified by C18 column chromatography (methanol:water = 1:4 to 1:2) to afford 1.35 g of compound 105 as a white solid, in a 92% yield.

[0347] Step 6: Dissolve compound 105 (0.5 g, 0.99 mmol) in dilute HCl (6.6 mL, 1.5 M) in a 25 mL single-necked reaction vial and stir at room temperature for 2 hours. Then concentrate under reduced pressure to obtain crude compound 106, which was directly used in the next step (350 mg yield, 80% yield).

[0348] Step 7: To a solution of compound 106 (350 mg, 0.79 mmol), HOSu (163 mg, 1.42 mmol), 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzoic acid (239 mg, 1.11 mmol), and EtN (0.4 mL, 2.88 mmol) in DMF (10 mL) (25 mL single-necked flask) was added DCC (407 mg, 1.97 mmol) under nitrogen at room temperature. The mixture was stirred overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The product was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:4 to 1:1) to afford 0.25 g of compound 114 as a red solid in a 52% yield.

[0349] Step 8: To a solution of compound 114 (250 mg, 0.41 mmol) in water (40 mL) and acetonitrile (20 mL) in a 250 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 1310 mg, 6.17 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 448 mg, 2.06 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was extracted with toluene (30 mL x 4), and the aqueous phase was directly purified by C18 column chromatography (acetonitrile:water = 1:4-1:1, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to afford 80 mg of compound G38 as a red solid, in a 33% yield.

[0350] ESI-MS calc.for C 24 H 30 N9O9[M+H] +m / z=588.2, found m / z=588.2. 1 H NMR (400MHz, D2O) δ8.59-8.47(m,2H),8.09-7.98(m,2H),6.05(d,J=7.3Hz,1H),4.44(d,J=7.8Hz,1H),4. 40(dd,J=3.1,1.7Hz,1H), 4.14(ddt,J=7.0,3.3,1.7Hz,1H), 4.00(d,J=3.5Hz,1H), 3.92(t,J=6.6Hz,1H), 3.78(dt,J=8.7,1.6Hz,1H),3.72(d,J=6.5Hz,2H),3.68(dd,J=10.0,3.4Hz,1H),3.62(dd,J=13.2,2.6Hz, 1H), 3.56 (ddd, J=10.0, 6.8, 4.1Hz, 2H), 3.46 (dd, J=13.2, 6.1Hz, 1H), 3.11 (s, 3H), 2.07 (d, J=1.8Hz, 3H).

[0351] Example 27: Synthesis of Compound G39

[0352] Steps 1 and 2: Galactose oxidase (12.5 mL), horseradish peroxidase (HRP, 210 mg), catalase (6.25 mL), and copper sulfate (CuSO4, 1 mM, 4 mL) were added sequentially to phosphate buffer (25 mL) and activated in a 30°C water bath for 30 min. Oxygen (O2) was bubbled through a 500 mL single-necked reaction flask containing compound 1 (5 g, 13.04 mmol) in phosphate buffer (125 mL) at 30°C for 30 min. The enzyme mixture was then added to the reaction system and stirred in a 30°C water bath for 6 h. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete, and the oxygen bubble was discontinued. NaCO (691 mg, 6.52 mmol) and NHOH·HCl (997 mg, 14.35 mmol) were added, and the mixture was allowed to react at room temperature for 3 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. Methanol (300 mL) was added, and the precipitate was removed by filtration through celite. The precipitate was rinsed with methanol (300 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 6:1-3:1) to obtain 4.0 g of compound 102 in a 77% yield.

[0353] Step 3: To a mixture of compound 102 (5.5 g, 13.88 mmol) and NiCl2·6H2O (8.25 g, 34.71 mmol) in MeOH (160 mL) and phosphate buffer (PB, 80 mL) in a 500 mL single-necked reaction flask at 0°C were added NaBH4 (3.15 g, 83.27 mmol) and Boc2O (9.09 g, 41.65 mmol). The mixture was stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:5-1:2) to afford 3.2 g of compound 103 as a white solid in a yield of 48%.

[0354] Step 4: To a solution of compound 103 (2.5 g, 5.18 mmol) in pyridine (50 mL) in a 250 mL single-necked reaction flask at -20°C was added TsCl (1.83 g, 9.60 mmol) and stirred overnight. The mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:4 to 3:1) to afford 1.83 g of compound 104, in a 55% yield.

[0355] Step 5: Under nitrogen, to a solution of compound 104 (1.83 g, 2.87 mmol) in DMF (40 mL) in a 100 mL three-necked reaction flask was added NaN (1.87 g, 28.76 mmol). The mixture was then heated to 60°C and stirred for 48 hours. The reaction mixture was filtered, the filtrate concentrated under reduced pressure, and the residue purified by C18 column chromatography (methanol:water = 1:4 to 1:2) to afford 1.35 g of compound 105 as a white solid, in a 92% yield.

[0356] Step 6: Dissolve compound 105 (0.5 g, 0.99 mmol) in dilute HCl (6.6 mL, 1.5 M) in a 25 mL single-necked reaction vial and stir at room temperature for 2 hours. Then concentrate under reduced pressure to obtain crude compound 106, which was directly used in the next step (350 mg yield, 80% yield).

[0357] Step 7: To a solution of compound 106 (500 mg, 1.13 mmol), HOSu (233 mg, 2.02 mmol), 2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)acetic acid (337 mg, 1.46 mmol), and EtN (0.5 mL, 3.60 mmol) in DMF (10 mL) was added DCC (581 mg, 2.82 mmol) in a 50 mL single-necked flask under nitrogen at room temperature. The mixture was stirred overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The product was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:4 to 1:1) to afford 0.35 g of compound 115 as a red solid in a 50% yield.

[0358] Step 8: To a solution of compound 115 (150 mg, 0.24 mmol) in water (50 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 771 mg, 3.63 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 263 mg, 1.21 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was extracted with toluene (20 mL x 4), and the aqueous phase was directly purified by C18 column chromatography (acetonitrile:water = 1:4-1:1, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to afford 63 mg of compound G39 as a red solid, in a 43% yield.

[0359] ESI-MS calc.for C 25 H 32 N9O9[M+H] + m / z=602.2, found m / z=602.2. 1 H NMR (400MHz, D2O) δ8.44(d,J=8.0Hz,2H),7.62(d,J=8.0Hz,2H),6.06(d,J=7.3Hz,1H),4.34(d,J=7.7Hz,1H),4.27(t,J=2.7Hz,1H),4.14 (d,J=7.3Hz,1H),3.89(d,J=3.3Hz,1H),3.80(s,2H),3.74-3.68(m,1H),3.63-3.40(m,8H),3.09(d,J=1.4Hz,3H),2.06(q,J=1.2Hz,3H).

[0360] Example 28: Synthesis of Compound G40

[0361] Step 1: To a solution of LacNPr (10 g, 25.17 mmol) and TsOH (0.56 g, 3.25 mmol) in DMF (220 mL) (500 mL single-necked reaction flask) was added 4-methoxybenzaldehyde dimethyl acetal (11 mL, 64.59 mmol) and stirred at room temperature for 12 h. The reaction was then quenched by the addition of Et3N (10 mL), concentrated under reduced pressure, and the residue purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 6:1) to afford 6.5 g of Compound 116 as a white solid, in a 50% yield.

[0362] Step 2: To a solution of compound 116 (6.5 g, 12.61 mmol) and DMAP (0.077 g, 0.63 mmol) in pyridine (100 mL) (500 mL single-necked reaction flask) was added Ac2O (12 mL, 127.77 mmol) and stirred at room temperature overnight. TLC (petroleum ether:ethyl acetate = 1:2) showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was added with ethyl acetate (500 mL) and then washed with water (500 mL). The organic phase was concentrated under reduced pressure to give 9.1 g of off-white solid compound 117, which was directly used in the next step without further purification, with a yield of 99%.

[0363] Step 3: A mixture of compound 117 (9.1 g, 12.54 mmol) in acetic acid (80 mL) and water (20 mL) in a 250 mL single-necked reaction flask was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3 to 0:1) to afford 2.8 g of solid compound 118 in a 36% yield.

[0364] Step 4: To a solution of compound 118 (2.8 g, 4.64 mmol) in pyridine (100 mL) in a 250 mL single-necked reaction flask at -20°C was added TsCl (1.4 g, 7.34 mmol), followed by stirring overnight. The reaction was quenched by the addition of 20 mL of methanol, concentrated under reduced pressure, and the residue purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:2-1:3) to afford 1.9 g of solid compound 119 in a 54% yield.

[0365] Step 5: Under nitrogen, NaN3 (1.46 g, 22.46 mmol) was added to a solution of compound 119 (1.9 g, 2.99 mmol) in DMF (40 mL) in a 100 mL three-necked reaction flask. The mixture was then heated to 70°C and stirred for 60 hours. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Water (100 mL) was added to the residue, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined and concentrated under reduced pressure to afford the crude white solid compound 120, which was used directly in the next step without further purification (theoretical yield: 1.58 g).

[0366] Step 6: To a solution of compound 120 (1.58 g, 2.50 mmol) in methanol (30 mL) (100 mL single-necked reaction bottle), add a methanol solution of sodium methoxide (0.5 mL, 5 M) and stir at room temperature for 3 hours. + ) was adjusted to pH 6-7 and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by C18 column chromatography (methanol:water = 1:20-1:10) to obtain 380 mg of solid compound 121 in a yield of 36%.

[0367] Step 7: To a solution of compound 121 (200 mg, 0.47 mmol) in water (15 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 1.51 g, 7.11 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 0.51 g, 2.35 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered and directly subjected to C18 preparative HPLC (acetonitrile:water = 1:20-1:4, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to afford 115 mg of compound G40 as a white solid, in a 60% yield.

[0368] ESI-MS calc.for C 15 H 25 N4O9[M+H] + m / z=405.1, found m / z=405.1. 1H NMR(400MHz,D2O)δ6.12(d,J=7.3Hz,1H),4.50(s,1H),4.48(d,J=1.1Hz,1H),4.22(ddd,J=6.3,3.1,1.6Hz,1H),3.93-3.88 (m,1H),3.88-3.80(m,2H),3.79-3.61(m,4H),3.58-3.41(m,3H),2.43(qq,J=7.5,1.4Hz,2H),1.20(td,J=7.6,1.2Hz,3H).

[0369] Example 29: Synthesis of Compound G42

[0370] Steps 1 and 2: Galactose oxidase (11 mL), horseradish peroxidase (HRP, 200 mg), catalase (6 mL), and a 2 mM copper sulfate (CuSO4, 6 mL) aqueous solution were added sequentially to phosphate buffer (30 mL) and activated in a 30°C water bath for 30 minutes. Oxygen (O2) was bubbled through a 150 mL phosphate buffer solution containing LacNPr (6 g, 15.10 mmol) in a 500 mL single-necked reaction flask at 30°C for 30 minutes. The enzyme mixture was then added to the reaction system and incubated in a 30°C water bath for 6 hours. TLC (isopropanol:concentrated ammonia:water = 4:2:1) indicated the reaction was complete, and the oxygen bubble was discontinued. To the reaction mixture were added NaCO (0.8 g, 7.55 mmol) and NHOH·HCl (1.15 g, 16.55 mmol), and stirred at room temperature for 3 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 4:2:1) indicated the reaction was complete. Methanol (200 mL) was added, and the precipitate was removed by filtration through celite. The filter cake was rinsed with methanol (200 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:20) to afford 5.0 g of compound 122 in 80% yield.

[0371] Step 3: To a mixture of compound 122 (5 g, 12.18 mmol) and NiCl2·6H2O (7.24 g, 30.46 mmol) in methanol (400 mL) and phosphate buffer (PB, 200 mL) in a 500 mL single-necked reaction flask at 0°C were added NaBH4 (2.77 g, 73.22 mmol) and Boc2O (7.98 g, 36.56 mmol). The mixture was stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:4-1:2) to afford 5.1 g of compound 123 as a white solid in 84% yield.

[0372] Step 4: To a solution of compound 123 (5.1 g, 10.27 mmol) in pyridine (100 mL) in a 250 mL single-necked reaction flask at -20°C was added TsCl (3.9 g, 20.46 mmol) and stirred overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to afford 6.6 g of compound 124, a 99% yield.

[0373] Step 5: Under nitrogen, to a solution of compound 124 (6.6 g, 10.14 mmol) in DMF (120 mL) in a 500 mL three-necked reaction flask was added NaN (6.6 g, 101.52 mmol), and the mixture was heated to 60°C and stirred overnight. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:3 to 2:3) to afford 2.0 g of compound 125 as a white solid, in a 38% yield.

[0374] Step 6: Compound 125 (0.6 g, 1.15 mmol) was dissolved in HCl (7.5 mL, 1.5 M in H2O) in a 100 mL single-necked reaction vial and stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure to afford crude compound 126, which was used directly in the next step (450 mg, 85% yield).

[0375] Step 7: To a solution of compound 126 (450 mg, 0.98 mmol), HOSu (164 mg, 1.42 mmol), 2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)acetic acid (294 mg, 1.28 mmol), and EtN (0.5 mL, 3.60 mmol) in DMF (10 mL) (50 mL single-necked flask) was added DCC (324 mg, 1.57 mmol) under nitrogen at room temperature. The mixture was stirred overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:2 to 1:1) to afford 0.42 g of compound 127 as a red solid in a 67% yield.

[0376] Step 8: To a mixture of compound 127 (200 mg, 0.32 mmol) in water (30 mL) and acetonitrile (10 mL) in a 50 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 1010 mg, 4.76 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 343 mg, 1.49 mmol). The mixture was then stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was extracted with toluene (20 mL x 4), and the aqueous phase was directly purified by C18 column chromatography (acetonitrile:water = 1:2-2:1, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to afford 153 mg of compound G42 as a red solid, in a yield of 78%.

[0377] ESI-MS calc.for:C 26 H 34 N9O9[M+H] + m / z=616.2, found m / z=616.2. 1 H NMR (400MHz, D2O) δ8.44(d,J=7.9Hz,2H),7.62(d,J=7.9Hz,2H),6.06(d,J=7.2Hz,1H),4.34(d,J=7.5Hz,1H),4.29(d,J=3.3Hz,1H),4.15(dd,J=7 .2,3.5Hz,1H),3.89(d,J=3.4Hz,1H),3.83-3.66(m,3H),3.63-3.40(m,8 H), 3.09 (d, J = 2.3Hz, 3H), 2.42-2.36 (m, 2H), 1.16 (dd, J = 8.7, 6.6Hz, 3H).

[0378] Example 30: Synthesis of Compound G44

[0379] Steps 1 and 2: Galactose oxidase (9 mL), horseradish peroxidase (HRP, 167 mg), catalase (5 mL), and a 2 mM copper sulfate (CuSO4, 5 mL) aqueous solution were sequentially added to phosphate buffer (20 mL) and activated in a 30°C water bath for 30 minutes. Oxygen (O2) was bubbled through a 500 mL single-necked reaction flask containing LacNPr (5 g, 12.6 mmol) in 100 mL phosphate buffer at 30°C for 30 minutes. The enzyme mixture was then added to the reaction system and stirred in a 30°C water bath for 6 hours. TLC (isopropanol:concentrated ammonia:water = 4:2:1) indicated the reaction was complete, and the oxygen bubble was discontinued. To the reaction solution were added NaCO (0.67 g, 6.32 mmol) and NHOH·HCl (0.96 g, 13.81 mmol), and the mixture was allowed to react at room temperature for 3 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 4:2:1) indicated the reaction was complete. Methanol (200 mL) was added, and the precipitate was removed by filtration through celite. The filter cake was rinsed with methanol (200 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:20) to afford 4.0 g of compound 122 in a 77% yield.

[0380] Step 3: To a mixture of compound 122 (4 g, 9.75 mmol) and NiCl2·6H2O (5.79 g, 24.36 mmol) in methanol (400 mL) and phosphate buffer (PB, 200 mL) in a 1.0 L single-necked reaction flask were added NaBH4 (2.21 g, 58.42 mmol) and Boc2O (6.38 g, 29.23 mmol) at 0°C. The mixture was stirred overnight at 0°C. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:4-1:2) to afford 4.5 g of compound 123 as a white solid in a 92% yield.

[0381] Step 4: A solution of compound 123 (1.1 g, 2.22 mmol) in dilute aqueous hydrochloric acid (13 mL, 1.5 M) was stirred at room temperature for 2 h, then concentrated under reduced pressure to afford compound 128 as a white solid, which was directly used in the next step (yield 94%, based on 900 mg).

[0382] Step 5: To a solution of ethyl bromooctanoate (27 g, 0.11 mol) in DMSO (500 mL) in a 1.0 L single-necked reaction flask, add NaCO (11.4 g, 0.11 mol) and KI (17.9 g, 0.11 mol) and stir at 80°C overnight. The reaction mixture is poured into ice water (3 L) and extracted with petroleum ether:ethyl acetate (400 mL:400 mL x 3). The combined organic phases are concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1-15:1) to obtain 11.0 g of compound L-1 as a white oil in a 55% yield.

[0383] Step 6: To a mixture of compound L-1 (3 g, 16.11 mmol) and N6-Boc-L-lysine (6 g, 24.36 mmol) in methanol (320 mL) and water (80 mL) (1.0 L single-necked reaction flask) was added NaBH3CN (2 g, 31.83 mmol) at -10°C and stirred overnight at -10°C. TLC (dichloromethane:methanol = 7:1) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 5.7 g of compound L-2 as a light yellow liquid, with a yield of 85%.

[0384] Step 7: To a mixed solution of compound L-2 (3 g, 7.20 mmol) in DMF (200 mL) and H2O (40 mL) (1.0 L single-necked reaction flask) was added Boc2O (15.7 g, 71.9 mmol) and Et3N (2 mL, 14.39 mmol) at room temperature and stirred overnight. TLC (dichloromethane: methanol = 10:1) showed that the reaction was complete. The reaction solution was poured into water (2 L) and then extracted with ethyl acetate (400 mL × 3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: methanol = 20:1-15:1) to obtain 1.7 g of light yellow liquid compound L-3, with a yield of 45%.

[0385] Step 8: Under nitrogen, DCC (1.22 g, 5.91 mmol) was added to a DMF solution (40 mL) containing compound L-3 (1.7 g, 3.29 mmol), N3-PEG6-NH2 (1.15 g, 3.28 mmol), and HOSu (0.45 g, 3.91 mmol) in a 250 mL single-necked reaction flask. The mixture was stirred overnight at room temperature. TLC (dichloromethane:methanol = 10:1) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, re-dissolved in DCM (100 mL), and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 100:1-30:1) to obtain 2.3 g of compound L-4 as a light yellow liquid, with a yield of 82%.

[0386] Step 9: At 25°C, LiOH (0.2g, 8.35mmol) was added to a mixed solution of THF (20mL) and H2O (40mL) (250mL single-necked reaction bottle) containing compound L-4 (2.3g, 2.71mmol) and stirred overnight. TLC (dichloromethane:methanol=50:1) showed that the reaction was complete. Water (50mL) was added to the reaction solution, and then extracted with ethyl acetate (150mL×3). The organic phases were combined and concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water=1:1-2:1) to obtain 1.5g of white solid compound L-5 with a yield of 67%.

[0387] Step 10: To a solution of compound 128 (0.9 g, 2.08 mmol), compound L-5 (1.5 g, 1.83 mmol), HOSu (0.32 g, 2.78 mmol), and EtN (1 mL, 7.19 mmol) in DMF (40 mL) (250 mL single-necked flask) was added DCC (0.75 g, 3.63 mmol) under nitrogen at room temperature. The mixture was stirred overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 50:5:3) indicated the reaction was complete. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:1 to 7:3) to afford 0.75 g of compound 129 as a white solid in a 34% yield.

[0388] Step 11: To a solution of compound 129 (250 mg, 0.21 mmol) in methanol (4 mL) in a 100 mL single-necked flask was added concentrated hydrochloric acid (1 mL, 10 M). The mixture was allowed to react at room temperature overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The mixture was concentrated under reduced pressure to afford compound 130 as a white solid, which was used directly in the next step (yield 200 mg, 90%).

[0389] Step 12: Under nitrogen, MTAB-OSu (98 mg, 0.30 mmol) was added to a solution of compound 130 (200 mg, 0.19 mol) and EtN (0.1 mL, 0.72 mmol) in methanol (5 mL) in a 100 mL single-necked flask. The mixture was stirred overnight at room temperature. TLC (isopropanol:concentrated ammonia:water = 50:5:3) indicated the reaction was complete. The product was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (methanol:water = 1:1 to 7:3) to afford 160 mg of compound 131 as a red solid, in a 70% yield.

[0390] Step 13: To a solution of compound 131 (150 mg, 0.12 mmol) in water (60 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 394 mg, 1.86 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 134 mg, 0.62 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 50:5:3) indicated the reaction was complete. The reaction solution was filtered, and the filtrate was purified by C18 column chromatography (acetonitrile:water = 1:2-2:1, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100)) to afford 51 mg of compound G44 as a red solid, in a 34% yield.

[0391] ESI-MS calc.for:C 54 H 89 N 12 O 18 [M+H] + m / z=1193.6, found m / z=1193.6. 1 H NMR(400MHz,D2O)δ8.22(d,J=8.1Hz,2H),7.39(d,J=8.2Hz,2H),5.89(d,J=7.3Hz,1H),4.29–4.12 (m,2H),4.00(d,J=6.1Hz,1H),3.69(d,J=3.1Hz,1H),3.61(d,J=10.4Hz,1H),3.55–3.43(m,24H), 3.42–3.12(m,12H),3.03(t,J=6.6Hz,2H),2.98–2.91(m,1H),2.89(s,3H),2.30–2.15(m,4H),2.0 7(t,J=7.4Hz,2H),1.52–1.30(m,6H),1.28–1.13(m,4H),1.14–1.02(m,8H),0.98(t,J=7.6Hz,3H).

[0392] Example 31: Synthesis of Compound G46

[0393] Step 1: To a suspension of D-glucosamine hydrochloride (GlcNH2·HCl, 21 g, 0.097 mol) in methanol (200 mL) at room temperature (500 mL single-necked flask), sodium methoxide (5.5 g, 0.102 mol) was added. The mixture was stirred at room temperature for 30 minutes. Phthalic anhydride (PhthO, 15.9 g, 0.107 mol) was then added and stirred overnight. The reaction mixture was concentrated under reduced pressure to afford crude compound 132, which was used directly in the next step (based on a 30 g charge).

[0394] Step 2: To a solution of compound 132 (30 g, 0.097 mol) and DMAP (0.6 g, 0.005 mol) in pyridine (500 mL) in a 2.0 L single-necked reaction flask was added AcO (100 mL, 1.065 mol) and stirred overnight at room temperature. TLC (petroleum ether:ethyl acetate = 1:1) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1-2:1) to afford 19.5 g of solid compound 133, a 42% yield.

[0395] Step 3: To a solution of compound 133 (19.5 g, 0.041 mol) and TolSH (7.6 g, 0.061 mol) in dichloromethane (300 mL) (1.0 L single-necked reaction flask), add BF3·Et2O (23 mL, 0.186 mol) and stir at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 3:1) showed the reaction was complete. The mixture was diluted with dichloromethane (500 mL) and washed with saturated sodium bicarbonate (800 mL). The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 7:1-3:1) to afford 16 g of compound 134 as a white solid in a 72% yield.

[0396] Step 4: Under nitrogen protection, dry Molecular sieves (30 g) were added and stirred at room temperature for 2 hours. The temperature was lowered to about -10°C, and NIS (7.0 g, 0.031 mol) and AgOTf (0.53 g, 0.0021 mol) were added. The mixture was then warmed to room temperature and stirred overnight. TLC (petroleum ether:ethyl acetate = 3:1) showed that the reaction was complete. The reaction mixture was filtered and the filter cake was rinsed with dichloromethane (400 mL). Water (500 mL) was added to the filtrate. Sodium thiosulfate was slowly added with vigorous stirring until the organic phase faded. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1-1:1) to obtain 18.2 g of colorless gum compound 135 in an 88% yield.

[0397] Step 5: Under nitrogen protection and 0°C, a solution of compound 135 (18.2 g, 0.018 mol) in methanol (200 mL) was added to a 500 mL single-necked reaction bottle, and stirred at 0°C for 3 hours. TLC (petroleum ether:ethyl acetate = 1:3) showed that the reaction was complete, and a cationic resin (H +) and adjusted to pH 6-7. Filter and concentrate the filtrate under reduced pressure to obtain 15.9 g of crude light yellow solid compound 136, which was used directly in the next step without further purification.

[0398] Step 6: Under nitrogen, N₂H₄·H₂O (10.5 mL, 0.216 mol) was added to a solution of compound 136 (13 g, 0.148 mol) in ethanol (700 mL) in a 2.0 L single-necked reaction flask. The mixture was stirred at 80°C overnight. TLC (dichloromethane:methanol = 10:1) indicated the reaction was complete. After cooling, the mixture was filtered and the filtrate was concentrated under reduced pressure to afford 10.5 g of compound 137 as a white solid in a 94% yield. The product was used directly in the next step without further purification.

[0399] Step 7: To a mixture of compound 137 (2 g, 3.17 mmol) in tetrahydrofuran (120 mL) and water (40 mL) in a 100 mL three-necked flask, palladium / carbon (1.75 g, 10 wt%, 1.64 mmol) and concentrated HCl (1.3 mL, 10 M) were added. H₂ (15 psi) was introduced and the mixture was stirred at room temperature overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 4:2:1) indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford a crude white solid, compound 138, which was used directly in the next step without purification (1.1 g was used in the next step).

[0400] Step 8: Under nitrogen, DCC (2.2 g, 10.66 mmol) was added to a solution of compound 138 (1.1 g, 2.66 mmol), N3CH2COOH (807 mg, 7.98 mmol), HOSu (1.1 g, 9.56 mmol), and Et3N (3 mL) in DMF (15 mL) (100 mL single-necked flask). The mixture was stirred overnight at room temperature. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 5:1-4:1) to afford 0.8 g of crude compound 139 as a white solid. This was then purified by amide column chromatography to afford 0.26 g of compound 139 as a white solid, in a 19% yield.

[0401] Step 9: To a solution of compound 139 (160 mg, 0.32 mmol) in water (12 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 1010 mg, 4.76 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 343 mg, 1.58 mmol). The mixture was then stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was purified using preparative C18 (acetonitrile:water = 1:20 to 1:4) to afford 140 mg of compound G46 as a white solid in a 91% yield.

[0402] ESI-MS calc.for C 16 H 25 N8O 10 [M+H] + m / z=489.1, found m / z=489.1. 1 H NMR(400MHz,D2O)δ6.18(dd,J=7.3,1.2Hz,1H),4.65-4.59(m,1H),4.46(dt,J=2.8,1.3Hz,1H),4.27(dp,J=7.8,1.5Hz,1 H),4.23-4.09(m,2H),4.05(t,J=1.2Hz,2H),3.95-3.86(m,1H),3.78-3.54(m,6H),3.50-3.39(m,2H),3.36-3.29(m,1H).

[0403] Example 32: Synthesis of Compound G47

[0404] Step 1: To a solution of Ac5Glc (45 g, 0.115 mol) and TolSH (21.5 g, 0.173 mol) in DCM (250 mL) (500 mL single-necked reaction flask), BF3·Et2O (43 mL, 0.348 mol) was added and stirred at room temperature for 3 hours. TLC (petroleum ether:ethyl acetate = 3:1) showed the reaction was complete. The mixture was diluted with DCM (600 mL) and washed with saturated sodium bicarbonate (1.0 L). The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 7:1-3:1) to afford 47 g of Compound 51 as a white solid in a 90% yield.

[0405] Step 2: To a solution of compound 51 (47 g, 0.103 mol) in methanol (500 mL) in a 1.0 L single-necked reaction flask was added a 5 M solution of sodium methoxide (10 mL) in methanol and stirred at room temperature for 2 hours. TLC (dichloromethane:methanol = 10:1) indicated the reaction was complete. Cationic resin (H+) was added for neutralization, and the pH was adjusted to 6-7. The filtrate was filtered and concentrated under reduced pressure to afford 28 g of compound 140 as a white solid in a 95% yield. The product was used directly in the next step without further purification.

[0406] Step 3: To a solution of compound 140 (8 g, 0.028 mol) and benzaldehyde dimethyl acetal (8.4 mL) in DMF (100 mL) (250 mL single-necked reaction bottle) was added dextrorotatory camphorsulfonic acid (CAS: 3144-16-99, 1.62 g, 0.007 mol) and stirred at room temperature overnight. The reaction was quenched by adding triethylamine (3 mL) and concentrated under reduced pressure. The residue was washed with dichloromethane (150 mL), saturated sodium bicarbonate (50 mL) and water (50 mL). The organic phase was concentrated under reduced pressure and the residue was slurried with dichloromethane:n-hexane = 1:3 (100 mL) to give 9 g of white solid compound 141, with a yield of 86%.

[0407] Step 4: Under nitrogen, Bu2SnO (6.9 g, 0.028 mol) was added to a solution of compound 141 (9 g, 0.024 mol) in toluene (75 mL) in a 250 mL single-necked reaction flask. The mixture was refluxed at 125°C for 4 hours to remove moisture. The mixture was cooled to room temperature and concentrated under reduced pressure. DMF (50 mL), Bu4NBr (8.5 g, 0.026 mol), CsF (4.0 g, 0.026 mol), and PMBCl (4.1 g, 0.015 mol) were added to the residue. The mixture was reacted at room temperature under nitrogen overnight. The mixture was concentrated under reduced pressure, and ethyl acetate (200 mL) was added to the residue. The mixture was washed with saturated sodium bicarbonate (100 mL) and water (100 mL). The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate:dichloromethane = 2:1:1) to obtain 10 g of compound 142 as a white solid in an 84% yield.

[0408] Step 5: To a solution of compound 142 (10 g, 0.02 mol) and DMAP (0.12 g, 0.001 mol) in pyridine (100 mL) in a 500 mL single-necked reaction flask was added benzoyl chloride (BzCl, 3.98 g, 0.028 mol) and stirred overnight at room temperature. TLC (petroleum ether:ethyl acetate = 2:1) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 1:1) to afford 11.5 g of compound 143 as an off-white solid in a 95% yield.

[0409] Step 6: Under nitrogen protection, dry Molecular sieves (30 g) were added and stirred at room temperature for 2 hours. The reaction mixture was cooled to -10°C and NIS (5.4 g, 0.024 mol) and TMSOTf (2.1 g, 0.009 mol) were added to the reaction mixture. After the addition was complete, the mixture was warmed to room temperature and stirred overnight. TLC (petroleum ether:ethyl acetate = 5:1) showed that the reaction was complete. The reaction mixture was filtered and the residue was rinsed with DCM (400 mL). Water (400 mL) was added to the filtrate. Sodium thiosulfate was slowly added with vigorous stirring until the organic phase faded. The organic phase was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1-7:1) to obtain 12 g of colorless gum compound 144 in a yield of 79%.

[0410] Step 7: To a solution of compound 144 (12 g, 0.013 mol) in MeOH (200 mL) (500 mL single-necked reaction flask), add MeONa (1.26 mL, 5 M) in methanol and stir at room temperature for 2 hours. TLC (petroleum ether: ethyl acetate = 3:1) showed that the reaction was complete. Add cationic resin (H + ) and adjusted to pH 6-7. Filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1-3:1) to afford 10 g of compound 145 as a white solid in a yield of 94%. The product was used directly in the next step without further purification.

[0411] Step 8: Under nitrogen protection, trifluoromethanesulfonic anhydride (TfO, 3 mL) was added to a solution of compound 145 (10 g, 0.012 mol) in dichloromethane (100 mL) and pyridine (3 mL) in a 250 mL three-necked reaction flask at 0°C. The reaction was stirred at 0°C for 2 hours. TLC (petroleum ether:ethyl acetate = 3:1) indicated the reaction was complete. The solution was diluted with dichloromethane (100 mL) and washed with dilute hydrochloric acid (1 M, 100 mL) and saturated sodium bicarbonate (100 mL). The organic phase was concentrated under reduced pressure to afford 11 g of compound 146 as a light yellow solid in a 95% yield. The product was used directly in the next step without further purification.

[0412] Step 9: To a solution of compound 146 (11 g, 11.41 mmol) in DMF (150 mL) in a 1.0 L single-necked reaction flask was added NaN (2.9 g, 44.61 mmol) and stirred at 60°C overnight. TLC (petroleum ether:ethyl acetate = 4:1) indicated the reaction was complete. The mixture was diluted with water (1000 mL) and extracted with ethyl acetate (250 mL x 2). The combined organic phases were washed with saturated sodium carbonate (100 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 8:1) to afford 7.2 g of compound 147 as a white solid in a 73% yield.

[0413] Step 10: To a mixture of compound 147 (3.2 g, 3.73 mmol) in DCM (45 mL) and H₂O (2.5 mL) in a 250 mL single-necked reaction flask was added DDQ (1.92 g, 8.46 mmol) at 0°C. The reaction was stirred for 0.5 h, then warmed to room temperature and stirred for 1 h. The reaction solution was diluted with dichloromethane (200 mL) and washed with saturated sodium bicarbonate (200 mL). The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 3:1) to afford 2.1 g of compound 148 as a light yellow gum in 76% yield.

[0414] Step 11: To a mixture of compound 148 (2.1 g, 2.85 mmol) in THF (180 mL) and H₂O (60 mL) in a 500 mL single-necked reaction flask was added Pd / C (2 g, 10 wt%, 1.88 mmol) and an aqueous solution of HCl (1.12 mL, 10 M). The mixture was then purged with H₂ (15 psi) and stirred overnight at room temperature. TLC (isopropanol:concentrated ammonia:water = 4:2:1) indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford compound 149 as a pale yellow solid. This was used directly in the next step without purification (1.1 g (95% yield) was used in the next step).

[0415] Step 12: Under nitrogen, DCC (2.2 g, 10.66 mmol) was added to a solution of compound 149 (1.1 g, 2.66 mmol), N3CH2COOH (807 mg, 7.98 mmol), HOSu (1.1 g, 9.56 mmol), and Et3N (3 mL) in DMF (15 mL) (100 mL single-necked flask). The mixture was allowed to react overnight at room temperature. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 5:1-4:1) to obtain crude compound 150. This was then purified by amide column chromatography to afford 0.3 g of compound 150 as a white solid in a 22% yield.

[0416] Step 13: To a solution of compound 150 (150 mg, 0.30 mmol) in water (8 mL) in a 50 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 943 mg, 4.44 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 321 mg, 1.48 mmol). The mixture was then stirred at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was purified using C18 preparative purification (acetonitrile:water = 1:20 to 1:5) to afford 98 mg of compound G47 as a white solid, in a 67% yield.

[0417] ESI-MS calc.for C 16 H 25 N8O 10 [M+H] + m / z=489.1, found m / z=489.1. 1 H NMR(400MHz,D2O)δ6.17(d,J=7.8Hz,1H),4.85(s,1H),4.47(d,J=4.3Hz,1H),4.43-4.38(m,1H),4.25(d,J=7.4Hz,1H),4 .13(s,2H),4.06(s,2H),3.92-3.66(m,5H),3.64-3.55(m,1H),3.53-3.44(m,1H),3.42-3.34(m,1H),3.34-3.29(m,1H).

[0418] Example 33: Synthesis of Compound G5

[0419] Step 1: Compound 66 (0.8 g, 1.81 mmol) was dissolved in 10 mL of 1.5 M aqueous HCl in a 100 mL single-necked reaction vial and stirred at room temperature for 2 hours. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to afford compound 151 as a white solid, which was used directly in the next step without purification (yield 0.65 g).

[0420] Step 2: Under nitrogen, DCC (710 mg, 3.44 mmol) was added to a solution of compound 151 (650 mg, 1.72 mmol), 3-alkynylbutyric acid (217 mg, 2.58 mmol), HOSu (356 mg, 3.09 mmol), and EtN (0.6 mL) in DMF (10 mL) (100 mL single-necked flask). The mixture was allowed to react overnight at room temperature. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was filtered, concentrated under reduced pressure, and the residue was purified by amide column chromatography to afford 0.12 g of compound 152 as a white solid in a 17% yield.

[0421] Step 3: To a solution of compound 152 (120 mg, 0.29 mmol) in water (16 mL) in a 100 mL three-necked reaction flask at 0°C were added potassium phosphate (K3PO4, 750 mg, 3.53 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 256 mg, 1.18 mmol), followed by stirring at 0°C overnight. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was purified by C18 column chromatography (acetonitrile:water = 1:20-1:5) to afford 82 mg of compound G5 as a white solid, in a 71% yield.

[0422] ESI-MS calc.for C 16 H 24 NO 10 [M+H] + m / z=390.1, found m / z=390.1. 1 H NMR(400MHz,D2O)δ6.14(d,J=7.2Hz,1H),5.86(t,J=6.6Hz,1H),5.37(dd,J=6.7,1.8Hz,2H),4.43(dd,J=3.2,1.8Hz,1H),4.41(d ,J=7.7Hz,1H),4.28(dq,J=7.2,1.6Hz,1H),3.89(dd,J=3.4,0.9Hz,1H),3.85-3.71(m,4H),3.71-3.58(m,3H),3.54-3.41(m,2H).

[0423] Example 34: Synthesis of Compound G45

[0424] Step 1: To phosphate buffer (30 mL) were added galactose oxidase (9.5 mL), copper sulfate (CuSO₄, 2 mM, 5 mL), horseradish peroxidase (HRP, 150 mg), and catalase (4.5 mL), followed by activation in a 30°C water bath for 30 minutes. Phosphate buffer (120 mL) and compound 66 (6 g, 13.59 mmol) were added to a 1.0 L three-necked reaction flask at 30°C. Oxygen (O₂) was introduced for 15-30 minutes, and the enzyme mixture was then added to the reaction system. The reaction was allowed to react in a 30°C water bath for 6 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. O₂ addition was discontinued, and the reaction solution was used directly in the next step without further processing.

[0425] Step 2: To the reaction solution, add NaCO (0.72 g, 6.79 mmol) and hydroxylamine hydrochloride (NHOH·HCl, 1.04 g, 14.97 mmol). The mixture was allowed to react at room temperature for 3 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. 200 mL of methanol was added, and the precipitate was removed by filtration through celite. The precipitate was rinsed with 200 mL of methanol, and the filtrate was used directly in the next reaction (compound 68, 5 g, yield 81%).

[0426] Step 3: To a mixture of compound 68 (5.0 g, 11.00 mmol) and NiCl2·6H2O (8.25 g, 34.71 mmol) in MeOH (400 mL) and phosphate buffer (PB, 170 mL) in a 2000 mL single-necked reaction flask was added NaBH4 (2.08 g, 55.03 mmol) at 0°C. The mixture was reacted overnight at 0°C. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by C18 column chromatography (methanol:water = 1:20, hydrochloric acid system) to afford 2.95 g of compound 153 as a white solid, in a yield of 56%.

[0427] Step 4: CFCOOEt (2 mL) was added to a solution of compound 153 (1.8 g, 3.77 mmol) and EtN (1 mL) in MeOH (40 mL) in a 100 mL single-necked flask. The mixture was allowed to react overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The filtrate was concentrated under reduced pressure, and the residue was purified using C18 preparative purification (methanol:water = 1:3 to 1:1) to afford 1.2 g of compound 154 as a white solid in a 54% yield.

[0428] Step 5: Compound 154 (0.6 g, 1.12 mmol) was dissolved in 10 mL of 1.5 M HCl (10 mL) in a 100 mL single-necked reaction flask and stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure to afford crude compound 155, which was used directly in the next step (yield calculated to be 500 mg).

[0429] Step 6: To a solution of compound 155 (500 mg, 1.06 mmol), EtN (0.3 mL), NCHCOOH (128 mg, 1.27 mmol), and HOSu (182 mg, 1.58 mmol) in DMF (10 mL) (100 mL single-necked flask) was added DCC (393 mg, 1.90 mmol). The mixture was allowed to react at room temperature overnight. TLC (isopropanol:concentrated ammonia:water = 25:5:3) indicated the reaction was complete. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified using C18 preparative purification (methanol:water = 1:20-1:10) to afford 400 mg of compound 156 as a white solid in a 49% yield.

[0430] Step 7: Compound 156 (200 mg, 0.39 mmol) was added to a solution of NaOH (50 mg, 1.25 mmol) in HO (2.5 mL) in a 10 mL single-necked reaction vial. The mixture was stirred at room temperature for 2 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) indicated the reaction was complete. The pH was then adjusted to 3-4 with dilute hydrochloric acid. The mixture was concentrated under reduced pressure to afford crude compound 157, which was used directly in the next step (yield: 150 mg).

[0431] Step 8: To a solution of compound 158 (1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid, 325 mg, 0.53 mmol) and HOSu (73 mg, 0.63 mmol) in DMF (5 mL) (25 mL single-necked bottle) was added DCC (132 mg, 0.64 mmol) and the reaction was carried out at room temperature overnight. A solution of compound 157 (150 mg, 0.35 mmol) in DMF (5 mL) and Et3N (0.1 mL) were added, and the reaction was carried out at room temperature for 5 hours. TLC (isopropanol:concentrated aqueous ammonia:water = 25:5:3) showed that the reaction was complete. The filtrate was concentrated under reduced pressure, and the residue was purified by C18 preparative purification (methanol:water = 1:4-1:1) to give 150 mg of red solid compound 159 in a yield of 41%.

[0432] Step 9: Potassium phosphate (K3PO4, 469 mg, 2.21 mmol) and 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI, 160 mg, 0.74 mmol) were added to a solution of compound 159 (150 mg, 0.15 mmol) in water (45 mL) (250 mL three-necked reaction flask) at 0°C, and then stirred at 0°C overnight. TLC (isopropanol: concentrated aqueous ammonia: water = 25:5:3) showed that the reaction was complete. The reaction solution was filtered, and the filtrate was directly purified by C18 column chromatography (acetonitrile: water = 1:10-3:2, preparative HPLC (Wuhan Ruihe Chromatography Technology, LC 2100) to obtain 108 mg of red solid compound G45, with a yield of 73%.

[0433] ESI-MS calc.for C 42 H 66 N9O 19 [M+H] + m / z=1000.4, found m / z=1000.4; C 42 H 65 N9O 19 K[M+K] + m / z=1038.3, found m / z=1038.3. 1 H NMR(400MHz,D2O)δ8.29(d,J=8.6Hz,2H),7.15(d,J=8.6Hz,2H),6.13(d,J= 7.3Hz,1H),4.38(dd,J=3.1,1.6Hz,1H),4.31(d,J=7.7Hz,1H),4.27(dd,J=5 .7,2.9Hz,2H),4.23-4.05(m,3H),3.94-3.87(m,2H),3.80(d,J=3.3Hz,1H) ,3.77-3.49(m,35H),3.48-3.29(m,4H),2.97(s,3H),2.48(t,J=6.1Hz,2H).

[0434] Example 35: Synthesis of Compound G40-DBCO-PEG4-VC-PAB-MMAE

[0435] G40 (8.1 mg, 0.02 mmol) and DBCO-VC-PEG4-PAB-MMAE (16.58 mg, 0.01 mmol) were dissolved in 1 mL of 7.4% PBS and 1 mL of DMSO, respectively. The mixture was then mixed and allowed to react overnight at 25°C. LC-MS confirmed the reaction was complete. The reaction solution was purified by C18 preparative purification (acetonitrile:water = 1:50 to 5:1) to yield 13 mg of G40-DBCO-PEG4-VC-PAB-MMAE as a white solid in a 63% yield. The NMR sample was first dissolved in deuterated methanol and lyophilized, then dissolved in deuterated DMSO for use as a sample.

[0436] ESI-MS calc.for C 103 H 154 N 16 O 28 [M+2H] 2+ m / z=1031.2, found m / z=1032.0.1H NMR (400MHz, DMSO-d6) δ10.01(s,1H),8.38–8.11(m,1H),8.10–7.76(m,2H),7.63(dq,J=28.4,6.3,4.0Hz,4H),7.5 5–7.21(m,7H),7.21–7.09(m,1H),6.08–5.77(m,2H),5.41(s,1H),5.18–4.85(m,3H),4.81–4.32(m,6H),4.32–3.8 3(m,6H),3.82–3.52(m,4H),3.47(d,J=3.4Hz,9H),3.24(d,J=6.4Hz,3H),3.19(d,J=9.9Hz,2H),3.11(t,3H),3.07 –2.56(m,6H),2.44–1.90(m,8H),1.70(d,J=7.1Hz,3H),1.63–1.25(m,5H),1.13–0.95(m,6H),0.93–0.61(m,16H).

[0437] 2. Recombinant expression and purification of enzymes

[0438] Example 36: Recombinant expression and purification of enzymes

[0439] The following four enzyme coding genes were cloned into the pET22b vector (GenScript). The four enzymes are: Endo Se2 from Streptococcus equi subsp. zooepidemicus Sz105, whose amino acid sequence is 37-1011 of SEQ ID NO: 1; Endo Si from Streptococcus iniae, whose amino acid sequence is 34-928 of SEQ ID NO: 2; Endo S2 from Streptococcus pyogenes NZ131 (serotype M49), whose amino acid sequence is 38-843 of SEQ ID NO: 3; and Endo S from Streptococcus pyogenes, whose amino acid sequence is 37-995 of SEQ ID NO: 4.

[0440] Transform the plasmid containing the target gene into Escherichia coli BL21 (DE3), spread it on a 2×YT solid plate containing 100μg / mL ampicillin, and culture it at 37°C overnight. Pick a single colony and inoculate it into 4ml of 2×YT liquid medium containing 100μg / mL ampicillin and culture it overnight. Pipette 4ml of bacterial liquid into 1L of 2×YT broth medium containing 100μg / mL ampicillin and culture it at 37°C until the OD 600 Then, 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture and cultured at 20°C to induce protein overexpression. After 16 hours, the cells were harvested by centrifugation. TM Cell pellets were lysed using Bacterial Protein Extraction Reagent (Thermo) following the manufacturer's instructions. TM The recombinant proteins of Endo S, Endo S2, Endo Si and Endo Se2 were purified using His-Tag Purification Column (Roche) and SDA030 protein purification system (Sepure). The proteins were concentrated using Amicon centrifugal filters (30 kDa, Millipore) and further purified using HiLoad TM 26 / 600Superdex TMPurification was performed by size exclusion using a 200prep grade column (Cytiva). Fractions containing Endo S, Endo S2, Endo Si, and Endo Se2 fusion proteins were concentrated using Amicon centrifugal filters (30 kDa, Millipore) and stored in storage buffer (20 mM PB, pH 7.5). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel imaging using a Gel Doc EZ Imager (Bio-RAD) and Image Lab scanning software. Protein concentration was quantified using a spectrophotometer (Nano-300).

[0441] 3. Study on the transfer activity of disaccharide linkers

[0442] Example 37: Study on the transfer activity of endoglycosidases such as Endo Se2 and Endo Si on various disaccharide linkers

[0443] The wild-type antibody containing the Fc segment of SEQ ID NO: 5 was dissolved in 20 mM phosphate, 150 mM NaCl, pH 7.4 buffer (10 mg / mL), and hydrolyzed by adding wild-type Endo S2 at a final concentration of 0.4 mg / mL. The antibody was incubated at 37°C overnight and purified using Protein A magnetic beads to obtain a deglycosylated antibody containing one N-acetylglucosamine or core fucosylated N-acetylglucosamine.

[0444] The prepared deglycosylated antibody, disaccharide linker (i.e., compounds G0-G2), and wild-type endoglycosidase (i.e., Endo S or Endo S2 or Endo Si or Endo Se2) were prepared at concentrations of 10 mg / mL, 1.67 mM (25 times the equivalent of the antibody concentration), and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the reaction temperature was 25°C. 4 μL samples were removed every 0.5, 1, 2, 3, and 6 hours, and the reaction was terminated by adding 36 μL of PBS solution containing 0.1% formic acid. The results were analyzed by reduction LC-MS. The results are shown in Figures 1-3 and Table 1.

[0445] For compound G0, at 1 hour, the glycosylation transfer efficiency of Endo Se2 and Endo Si was comparable, and significantly better than Endo S2 and Endo S; at 6 hours, the glycosylation transfer efficiency of Endo Se2, Endo Si and Endo S2 was comparable, and significantly better than Endo S (Figure 1, Table 1).

[0446] For compound G1, at 1 hour, Endo Si showed better transglycosylation efficiency than Endo Se2 and significantly better than Endo S2. Endo S showed weaker transglycosylation efficiency for compound G1. At 6 hours, Endo Se2 and Endo Si showed comparable transglycosylation efficiency, and Endo S2 showed better transglycosylation efficiency (Figure 2, Table 1).

[0447] For compound G2, at 1 hour, the glycosylation efficiency of Endo Si was comparable to that of Endo Se2, and significantly superior to that of Endo S2. Endo S had a weaker transfer efficiency for compound G2. At 6 hours, the glycosylation efficiency of Endo Se2 was comparable to that of Endo Si, and superior to that of Endo S2 (Figure 3, Table 1).

[0448] Table 1 Transglycosidation activity of different endoglycosidases

[0449] Example 38: Study on the substrate specificity of endoglycosidases such as Endo Se2 and Endo Si for various disaccharide linkers

[0450] Wild-type pertuzumab, disaccharide linker, and wild-type endoglycosidase (i.e., Endo S, Endo S2, Endo Si, or Endo Se2) were added at concentrations of 10 mg / mL, 1.67 mM (25 times the antibody concentration), and 0.4 mg / mL, respectively. The reaction pH was adjusted to 7.4 and the reaction temperature was 25°C. After 6 hours, 4 μL of sample was removed and the reaction was terminated by adding 36 μL of PBS solution containing 0.1% formic acid. The sample was then analyzed by LC-MS.

[0451] For disaccharide linker compounds, Endo Se2, Endo Si, Endo S2, and Endo S showed different substrate specificities and transglycosylation efficiencies. Endo Se2 and Endo Si had broader substrate specificity than Endo S2 and Endo S, and the optimal endoglycosidase for each substrate was generally Endo Se2 or Endo Si (Table 2).

[0452] Table 2 Transglycosidation activity of different endoglycosidases

[0453] 4. Glycoengineered Antibodies

[0454] Example 39: Preparation of glycoengineered antibody Ab-G1

[0455] The non-natural glycoengineered antibody Ab-G1 was obtained by combining compound G1 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G1 was 49893.48 Da.

[0456] Example 40: Preparation of glycoengineered antibody Ab-G2

[0457] The non-natural glycoengineered antibody Ab-G2 was obtained by combining compound G2 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G2 was 49902.46 Da.

[0458] Example 41: Preparation of glycoengineered antibody Ab-G7

[0459] The non-natural glycoengineered antibody Ab-G7 was obtained by combining compound G7 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G7 was 49810.12 Da.

[0460] Example 42: Preparation of glycoengineered antibody Ab-G9

[0461] The non-natural glycoengineered antibody Ab-G9 was obtained by combining compound G9 and wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G9 was 50154.80 Da.

[0462] Example 43: Preparation of glycoengineered antibody Ab-G10

[0463] The non-natural glycoengineered antibody Ab-G10 was obtained by combining compound G10 and wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G10 was 50081.15 Da.

[0464] Example 44: Preparation of glycoengineered antibody Ab-G13

[0465] The non-natural glycoengineered antibody Ab-G13 was obtained by combining compound G13 and the wild-type antibody Pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G13 was 49807.70 Da.

[0466] Example 45: Preparation of glycoengineered antibody Ab-G27

[0467] The unnatural glycoengineered antibody Ab-G27 was obtained by reacting compound G27 with the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G27 after HRMS deconvolution was 50024.69 Da.

[0468] Example 46: Preparation of glycoengineered antibody Ab-G4

[0469] The unnatural glycoengineered antibody Ab-G4 was obtained by reacting compound G4 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G4 after HRMS deconvolution was 49802.29 Da.

[0470] Example 47: Preparation of glycoengineered antibody Ab-G4b

[0471] The non-natural glycoengineered antibody Ab-G4b was obtained by reacting compound G4b with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G4b after HRMS deconvolution was 49789.06 Da.

[0472] Example 48: Preparation of glycoengineered antibody Ab-G5

[0473] The non-natural glycoengineered antibody Ab-G5 was obtained by combining compound G5 and the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G5 was 49818.48 Da.

[0474] Example 49: Preparation of glycoengineered antibody Ab-G20

[0475] The non-natural glycoengineered antibody Ab-G20 was obtained by reacting compound G20 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G20 after HRMS deconvolution was 49836.03 Da.

[0476] Example 50: Preparation of glycoengineered antibody Ab-G21

[0477] The unnatural glycoengineered antibody Ab-G21 was obtained by reacting compound G21 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G21 after HRMS deconvolution was 49835.47 Da.

[0478] Example 51: Preparation of glycoengineered antibody Ab-G22

[0479] The non-natural glycoengineered antibody Ab-G22 was obtained by reacting compound G22 and the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G22 was 49835.38 Da.

[0480] Example 52: Preparation of glycoengineered antibody Ab-G23

[0481] The non-natural glycoengineered antibody Ab-G23 was obtained by reacting compound G23 and the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G23 after HRMS deconvolution was 49835.18 Da.

[0482] Example 53: Preparation of glycoengineered antibody Ab-G24

[0483] The non-natural glycoengineered antibody Ab-G24 was obtained by reacting compound G24 and the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G24 after HRMS deconvolution was 50064.00 Da.

[0484] Example 54: Preparation of glycoengineered antibody Ab-G25

[0485] The non-natural glycoengineered antibody Ab-G25 was obtained by reacting compound G25 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G25 after HRMS deconvolution was 49893.33 Da.

[0486] Example 55: Preparation of glycoengineered antibody Ab-G28b

[0487] The non-natural glycoengineered antibody Ab-G28b was obtained by reacting compound G28b with the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G28b after HRMS deconvolution was 50175.80 Da.

[0488] Example 56: Preparation of glycoengineered antibody Ab-G32

[0489] The non-natural glycoengineered antibody Ab-G32 was obtained by reacting compound G32 and the wild-type antibody Pertuzumab via General Procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G32 was 49844.75 Da.

[0490] Example 57: Preparation of glycoengineered antibody Ab-G33

[0491] The non-natural glycoengineered antibody Ab-G33 was obtained by reacting compound G33 with the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G33 after HRMS deconvolution was 50047.05 Da.

[0492] Example 58: Preparation of glycoengineered antibody Ab-G35

[0493] The non-natural glycoengineered antibody Ab-G35 was obtained by reacting compound G35 with the wild-type antibody pertuzumab via general procedure 1. The HRMS deconvolution value of the heavy chain of the glycoengineered antibody Ab-G35 was 49886.09 Da.

[0494] Example 59: Preparation of glycoengineered antibody Ab-G36

[0495] The unnatural glycoengineered antibody Ab-G36 was obtained by reacting compound G36 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G36 after HRMS deconvolution was 49891.14 Da.

[0496] Example 60: Preparation of glycoengineered antibody Ab-G37

[0497] The unnatural glycoengineered antibody Ab-G37 was obtained by reacting compound G37 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G37 after HRMS deconvolution was 49904.60 Da.

[0498] Example 61: Preparation of glycoengineered antibody Ab-G38

[0499] The non-natural glycoengineered antibody Ab-G38 was obtained by reacting compound G38 and wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G38 after HRMS deconvolution was 50018.20 Da.

[0500] Example 62: Preparation of glycoengineered antibody Ab-G39

[0501] The non-natural glycoengineered antibody Ab-G39 was obtained by reacting compound G39 with the wild-type antibody Pertuzumab via General Procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G39 after HRMS deconvolution was 50032.24 Da.

[0502] Example 63: Preparation of glycoengineered antibody Ab-G40

[0503] The non-natural glycoengineered antibody Ab-G40 was obtained by reacting compound G40 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G40 after HRMS deconvolution was 49833.19 Da.

[0504] Example 64: Preparation of glycoengineered antibody Ab-G42

[0505] The non-natural glycoengineered antibody Ab-G42 was obtained by reacting compound G42 and the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G42 after HRMS deconvolution was 50046.26 Da.

[0506] Example 65: Preparation of glycoengineered antibody Ab-G44

[0507] The non-natural glycoengineered antibody Ab-G44 was obtained by reacting compound G44 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G44 after HRMS deconvolution was 50624.49 Da.

[0508] Example 66: Preparation of glycoengineered antibody Ab-G45

[0509] The non-natural glycoengineered antibody Ab-G45 was obtained by reacting compound G45 with the wild-type antibody Pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G45 after HRMS deconvolution was 50405.15 Da.

[0510] Example 67: Preparation of glycoengineered antibody Ab-G46

[0511] The unnatural glycoengineered antibody Ab-G46 was obtained by reacting compound G46 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G46 after HRMS deconvolution was 49865.77 Da.

[0512] Example 68: Preparation of glycoengineered antibody Ab-G47

[0513] The unnatural glycoengineered antibody Ab-G47 was obtained by reacting compound G47 with the wild-type antibody pertuzumab via general procedure 1. The measured value of the heavy chain of the glycoengineered antibody Ab-G47 after HRMS deconvolution was 49866.18 Da.

[0514] 5. Preparation of Sugar-Directed Antibody-Drug Conjugates

[0515] Example 69: Preparation of sugar-directed ADC Ab-G1-DXd

[0516] Ab-G1-DXd is obtained by combining compound DBCO-GGFG-Dxd and non-natural glycoengineered antibody Ab-G1 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 51021.77 Da.

[0517] Example 70: Preparation of sugar-directed ADC Ab-G2-DXd

[0518] Ab-G2-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G2 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52158.93 Da.

[0519] Example 71: Preparation of sugar-directed ADC Ab-G7-DXd

[0520] Ab-G7-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G7 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50964.74 Da.

[0521] Example 72: Preparation of sugar-directed ADC Ab-G9-MMAE

[0522] Ab-G9-MMAE is obtained by combining compound N3-PEG4-VC-PAB-MMAE and non-natural glycoengineered antibody Ab-G9 through general operation three. The measured value of the heavy chain after HRMS deconvolution is 51551.58 Da.

[0523] Example 73: Preparation of sugar-directed ADC Ab-G10-MMAE

[0524] Ab-G10-MMAE is obtained by combining compound N3-PEG4-VC-PAB-MMAE and non-natural glycoengineered antibody Ab-G10 through general operation three. The measured value of the heavy chain after HRMS deconvolution is 51478.22 Da.

[0525] Example 74: Preparation of sugar-directed ADC Ab-G13-DXd

[0526] Ab-G13-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G13 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50978.76 Da.

[0527] Example 75: Preparation of sugar-directed ADC Ab-G27-DXd

[0528] Ab-G27-DXd is obtained by combining the compound TCO-PEG4-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G27 through general operation 4. The measured value of the heavy chain after HRMS deconvolution is 51234.41 Da.

[0529] Example 76: Preparation of sugar-directed ADC Ab-G4b-MMAE

[0530] Ab-G4b-MMAE is obtained by combining compound N3-PEG4-VC-PAB-MMAE and non-natural glycoengineered antibody Ab-G4b through general operation seven. The measured value of the heavy chain after HRMS deconvolution is 51185.38 Da.

[0531] Example 77: Preparation of sugar-directed ADC Ab-G20-DXd

[0532] Ab-G20-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G20 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50963.63 Da.

[0533] Example 78: Preparation of sugar-directed ADC Ab-G21-DXd

[0534] Ab-G21-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G21 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50963.46 Da.

[0535] Example 79: Preparation of sugar-directed ADC Ab-G22-DXd

[0536] Ab-G22-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G22 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52117.98 Da.

[0537] Example 80: Preparation of sugar-directed ADC Ab-G23-DXd

[0538] Ab-G23-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G23 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52116.81 Da.

[0539] Example 81: Preparation of sugar-directed ADC Ab-G24-DXd

[0540] Ab-G24-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G24 through general procedure 2. The measured value of the heavy chain after HRMS deconvolution is 52320.37 Da.

[0541] Example 82: Preparation of sugar-directed ADC Ab-G25-DXd

[0542] Ab-G25-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G25 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52174.43 Da.

[0543] Example 83: Preparation of sugar-directed ADC Ab-G28b-DXd

[0544] Ab-G28b-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G28b through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52432.16 Da.

[0545] Example 84: Preparation of sugar-directed ADC Ab-G32-DXd

[0546] Ab-G32-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G32 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52101.64 Da.

[0547] Example 85: Preparation of sugar-directed ADC Ab-G33-DXd

[0548] Ab-G33-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G33 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 52304.05 Da.

[0549] Example 86: Preparation of sugar-directed ADC Ab-G35-DXd

[0550] Ab-G35-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G35 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 53271.19 Da.

[0551] Example 87: Preparation of sugar-directed ADC Ab-G36-DXd

[0552] Ab-G36-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G36 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 51018.64 Da.

[0553] Example 88: Preparation of sugar-directed ADC Ab-G37-DXd

[0554] Ab-G37-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G37 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 51032.43 Da.

[0555] Example 89: Preparation of sugar-directed ADC Ab-G38-DXd-MMAE:

[0556] Ab-G38-DXd-MMAE was obtained by combining the compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G38 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 52887.29 Da.

[0557] Example 90: Preparation of sugar-directed ADC Ab-G39-DXd-MMAE

[0558] Ab-G39-DXd-MMAE was obtained by combining the compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G39 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 52900.70 Da.

[0559] Example 91: Preparation of sugar-directed ADC Ab-G40-DXd

[0560] Ab-G40-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G40 through general operation 2. The measured value of the heavy chain after HRMS deconvolution is 50961.38 Da.

[0561] Example 92: Preparation of sugar-directed ADC Ab-G42-DXd-MMAE

[0562] Ab-G42-DXd-MMAE was obtained by combining compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G42 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 52914.89 Da.

[0563] Example 93: Preparation of sugar-directed ADC Ab-G44-DXd-MMAE

[0564] Ab-G44-DXd-MMAE was obtained by combining compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G44 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 53492.60 Da.

[0565] Example 94: Preparation of sugar-directed ADC Ab-G45-DXd-MMAE

[0566] Ab-G45-DXd-MMAE was obtained by combining the compounds TCO-PEG4-GGFG-Dxd, DBCO-PEG4-VC-PAB-MMAE and the non-natural glycoengineered antibody Ab-G45 through general operation six. The measured value of the heavy chain after HRMS deconvolution was 53300.04 Da.

[0567] Example 95: Preparation of sugar-directed ADC Ab-G46-DXd

[0568] Ab-G46-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G46 through general procedure 2. The measured value of the heavy chain after HRMS deconvolution is 52174.17 Da.

[0569] Example 96: Preparation of sugar-directed ADC Ab-G47-DXd

[0570] Ab-G47-DXd is obtained by combining the compound DBCO-GGFG-Dxd and the non-natural glycoengineered antibody Ab-G47 through general procedure 2. The measured value of the heavy chain after HRMS deconvolution is 52173.90 Da.

[0571] Example 97: Preparation of sugar-directed ADC Ab-G40-DBCO-PEG4-VC-PAB-MMAE

[0572] Ab-G40-DBCO-PEG4-VC-PAB-MMAE is a combination of wild-type antibody pertuzumab and payload-disaccharide complex G40-DBCO-PEG4-VC-PAB-MMAE obtained by general procedure five. The measured value of the heavy chain after HRMS deconvolution is 51491.63 Da.

[0573] 6. Preparation of sugar-targeted ADCs based on core-free fucosylated antibodies

[0574] Example 98: Preparation of sugar-targeted ADC based on core-free fucosylated antibody

[0575] Wild-type pertuzumab was dissolved in 20 mM phosphate, 150 mM NaCl, pH 7.4 buffer to a concentration of 10 mg / mL. Wild-type Alfc (from Lacticaseibacillus paracasei, GENBANK accession number WP_012492118.1) was added to a final concentration of 0.5 mg / mL for hydrolysis. The mixture was incubated overnight at 37°C and purified using Protein A magnetic beads to obtain a defucosylating antibody (Ab-defuct) containing one N-acetylglucosamine.

[0576] The prepared defucosylated antibody, disaccharide linker, and wild-type endoglycosidase Endo Se2 or Endo Si were added at concentrations of 10 mg / mL, 1.67 mM (25 times the antibody concentration), and 0.4 mg / mL, respectively. The reaction system was adjusted to pH 7.4 and the reaction temperature was 25°C. After 6 hours, a 4 μL sample was removed and terminated with 36 μL of PBS containing 0.1% formic acid. The sample was then analyzed by LC-MS. The deconvoluted HRMS values ​​for the heavy chain are shown in Table 3.

[0577] The defucosylating non-natural glycoengineered antibodies Ab-defuc-G7, G20, G21, G23, G33, G35, and G40, and the compound DBCO-GGFG-Dxd were used to obtain the corresponding ADCs through general operation 2. The actual HRMS deconvolution values ​​of the heavy chain are shown in Table 3.

[0578] The defucosylating non-natural glycoengineered antibodies Ab-defuc-G38, G39, G42, G44, and G45 were reacted with the compounds TCO-PEG4-GGFG-Dxd and DBCO-PEG4-VC-PAB-MMAE to obtain the corresponding ADCs, respectively. The measured values ​​of heavy chain HRMS deconvolution are shown in Table 3.

[0579] Table 3 Preparation of sugar-targeted ADCs based on core-free fucosylated antibodies

[0580] Conclusion: The ADC preparation method disclosed in the present invention can be used to prepare ADC without core fucosylation.

[0581] 7. Efficacy of Sugar-Directed Antibody-Drug Conjugates

[0582] Example 99: Preparation of HER2-targeted sugar-directed ADC

[0583] (1) Synthesis of Tmab-G7, G9, G22, G23, G24, and G28b

[0584] Wild-type antibody trastuzumab (Tmab), disaccharide oxazolines (i.e., compounds G7, G9, G22, G23, G24, and G28b), wild-type endoglycosidases Endo Si (for compounds G7, G9, G23, and G28b), and Endo Se2 (for compounds G22 and G24) were added to concentrations of 10 mg / mL, 1.67 mM (25 times the antibody concentration), and 0.6 mg / mL, respectively. The reaction system was adjusted to pH 7.4, and the reaction temperature was 25°C, at 800 rpm, for 3 hours. Protein A purification was performed to remove small molecules and glycoside hydrolases to obtain the corresponding transglycosylating antibodies.

[0585] (2) Preparation of corresponding ADC

[0586] The prepared transglycosylated antibody Tmab-G7 and DBCO-PEG4-VC-PAB-MMAE were adjusted to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. The desired sugar-specifically coupled ADC Tmab-G7-MMAE was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC.

[0587] The prepared transglycosylated antibody Tmab-G9 and N3-PEG4-VC-PAB-MMAE were adjusted to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C overnight. The desired sugar-specifically coupled ADC Tmab-G9-MMAE was obtained by ultrafiltration and analyzed by LC-MS, HIC, and SEC. The prepared transglycosylated antibodies Tmab-G22, G23, G24, and G28b, and DBCO-PEG4-VC-PAB-MMAE were adjusted to 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and 8 equivalents of DBCO-PEG4-VC-PAB-MMAE were added after incubation at 25°C overnight. The total reaction time was 24 hours. The desired sugar-specifically coupled ADC Tmab-G22 / G23 / G24 / G28b-MMAE was obtained by ultrafiltration and analyzed by LC-MS, hydrophobic chromatography (HIC), and molecular group chromatography (SEC).

[0588] (3) Detection method

[0589] SEC: Column: TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm); Mobile phase: 0.2 M phosphate buffer, 5–15% isopropanol, pH 7.0; Flow rate: 0.5–1 mL / min; Detection wavelength: 280 nm, 248 nm, and 360 nm; Column temperature: Room temperature; Sample load: 30 μg; Elution method: Isocratic.

[0590] HIC: Chromatographic column: TSKgel Butyl-NPR (4.6 mm × 10 cm, 2.5 μm) column; mobile phase A: 0.05 M phosphate buffer, 1.2 M ammonium sulfate, pH 7.0; mobile phase B: 0.05 M phosphate buffer, 20% isopropanol, pH 7.0; flow rate: 0.5 mL / min; detection wavelength: 280 nm & 248 nm & 360 nm; column temperature: room temperature; sample load: 30 μg; elution method: mobile phase B increased from 0% to 100% within 20 minutes.

[0591] The quality control data of the prepared HER2-targeted sugar-specific ADC are shown in Table 4.

[0592] Table 4 HER2 targeted sugar fixed-point ADC quality control data

[0593] Example 100: In vitro efficacy of HER2-targeted carbohydrate-directed ADC

[0594] The purpose of this experiment is to detect the in vitro inhibitory activity of the ADC compounds disclosed herein against SK-BR-3 (human breast cancer cells), NCI-N87 (human gastric cancer cells) and MDA-MB-468 (human breast cancer cells).

[0595] Tumor cells SK-BR-3 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), NCI-N87 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), or MDA-MB-468 (source: Cell Bank, Committee for Type Culture Collection, Chinese Academy of Sciences) in the logarithmic growth phase were added to a cell plate at a rate of 2000 cells / well. The cell plate was incubated in a 37°C, 5% CO2 cell culture incubator for 12-16 hours. 100 μL of sample (starting at 50 μg / ml, 5-fold dilution, 9 concentrations) was added to each well, gently shaken, and then placed in the incubator for incubation. After incubation for 144 hours, 70 μL CellTiter-Glo was added. TM (Promega, Cat. No. G7572) working solution was added, gently shaken to lyse the cells, and the plate was read on a microplate reader. The cell proliferation inhibition rate was calculated as follows: Cell proliferation inhibition rate = (1 - sample well / control well) × 100%. GraphPad Prism 8.0 software was used to plot the logarithm of sample concentration against cytotoxicity % on the ordinate. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC value of each test sample. 50 The specific results are shown in Table 5.

[0596] Table 5 In vitro anti-tumor activity test of the ADC compounds disclosed herein

[0597] Conclusion: The ADC drugs disclosed in the present invention have significant proliferation inhibitory activity against HER2-positive cells SK-BR-3 and NCI-N87; at the same time, they have weak proliferation inhibitory activity against HER2-negative cells MDA-MB-468, showing good selectivity.

[0598] Example 101: In vivo efficacy of HER2-targeted carbohydrate-directed ADC

[0599] BALB / c Nude mice (purchased from Beijing Weitonglihua) were used as test animals to evaluate the efficacy of anti-HER2-ADC after tail vein injection on nude mice bearing human gastric cancer cell NCI-N87 xenografts.

[0600] Mice were subcutaneously inoculated with NCI-N87 cells (source: ATCC) (5×10 6The tumors were grown for 7 days to an average tumor volume of 150 mm 3 The animals were randomly divided into groups according to the tumor volume on day 7 (D7), with 7 animals in each group.

[0601] The mice were injected into the tail vein once a day, and the tumor volume and body weight were measured twice a week and the data were recorded. 28 -T7) / (V 28 -V7)]×100,T 28 T7 and T8 are the tumor volumes of the experimental group on the 28th and 7th day after inoculation, respectively. 28 V and V7 are the tumor volumes of the blank control group (Vehicle, PBS) on day 28 and day 7 after inoculation, respectively. Detailed experimental results at the end of the experiment on day 28 after inoculation are shown in Table 6 and Figures 4-5.

[0602] Table 6 In vivo efficacy evaluation of the disclosed ADC compounds

[0603] Conclusion: The ADC drug disclosed in the present invention exhibits excellent anti-tumor activity against NCI-N87 xenograft tumors with high HER2 expression.

Claims

1. A disaccharide linker represented by formula (I): Among them, X is selected from an oxygen atom or a sulfur atom; Y is selected from an oxygen atom or a sulfur atom; R1 is selected from the following groups: Among them, n is selected from an integer of 0 - 30, or an integer of 0 - 20, or an integer of 0 - 10; R2 is selected from the following groups: hydrogen, -N3, C1-C6 alkyl - N3, -C≡CH, C1-C6 alkyl - C≡CH, -SH, C1-C6 alkyl - SH, and C1-C6 alkyl; The G ring represents a structure derived from a monosaccharide molecule, which can be linked to another monosaccharide through a thioether bond or a glycosidic bond, wherein the monosaccharide molecule is selected from galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine, N-acetylmannosamine, mannose, fucose, and sialic acid sugar; when Y is a sulfur atom, the G ring is linked to a connection, the thioether bond being a 1,4-thioether bond, 2,4-thioether bond or 3,4-thioether bond; when Y is an oxygen atom, the G ring is connected to connected, and the glycosidic bond is a 1,4-glycosidic bond, 2,4-glycosidic bond, or 3,4-glycosidic bond; Z is selected from hydrogen, a reactive group containing an azide residue, a reactive group containing a cycloalkyne residue, a reactive group containing a tetrazine residue, a reactive group containing an alkyne residue, a reactive group containing a cycloalkene residue, or a reactive group containing a maleimide residue.

2. The disaccharide linker according to claim 1, wherein the glycosidic bond is a 1,4-glycosidic bond, or the thioether bond is a 1,4-thioether bond.

3. The disaccharide linker according to any one of claims 1 - 2, wherein the monosaccharide molecule is selected from galactose, glucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine.

4. The disaccharide linker according to any one of claims 1 - 3, wherein the R1 is selected from the following structures: Among them, n is selected from an integer of 0 - 30, or an integer of 0 - 20, or an integer of 0 - 10.

5. The disaccharide linker according to any one of claims 1 - 4, wherein R2 is selected from the following groups: hydrogen, -N3, -CH2-N3, -CH2-CH2-N3, -C≡CH, -CH2-C≡CH, -CH2-CH2-C≡CH, -SH, -CH2-SH, -CH2-CH2-SH, C1-C6 alkyl; or, R2 is selected from hydrogen, -N3, -CH2-N3, -CH2-CH2-N3, -C≡CH, -SH, -CH3, -CH2-CH3, and -CH2-CH2-CH3.

6. The disaccharide linker according to any one of claims 1-5, wherein Z is a reactive group containing an azide residue, and is selected from the following groups: or The reactive group containing an azide residue is selected from the following groups: Or Z is a reactive group containing a cycloalkyne residue, and it is selected from the following groups: Or The reactive group containing a cycloalkyne residue is selected from the following groups: Or Z is a reactive group containing an alkynyl residue, and it is selected from the following groups: Or The reactive group containing an alkynyl residue is selected from the following groups: Or Z is a reactive group containing a tetrazine residue, and it is selected from the following groups: Or The reactive group containing a tetrazine residue is selected from the following structures: Or Z is a reactive group containing a norbornene residue, and is selected from the following groups: Or The reactive group containing a cycloolefin residue is selected from the following structures: Or Z is a reactive group containing a maleimide residue, and it is selected from the following groups: Among them, Each n, each m, each I, and each q are independently selected from integers from 0 to 30, or integers from 0 to 20, or integers from 0 to 10; each Ra is hydrogen or a C1-C6 alkyl group; Rb is hydrogen, a C1-C6 alkyl group, a C6-C 10 aryl group, a C5-C 10 heteroaryl group.

7. The disaccharide linker according to claim 1, which is selected from the following specific compounds: Among them, n is selected from an integer of 0 - 30, or an integer of 0 - 20, or an integer of 0 - 10.

8. Disaccharide conjugate represented by formula (II): Among them, X, Y, R1, R2, and G ring are defined as in any one of claims 1 - 6; P-L-Z’- represents a substituent on the G ring, and the substitution position of P-L-Z’- is any position other than the 1st position of the G ring derived from the monosaccharide molecule; L is a divalent linker or multivalent linker connecting Z’ and P, and it is selected from a cleavable linker, a non-cleavable linker, or a combination thereof; P is selected from a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a polypeptide, an RNA and related drugs, a radioisotope label, a contrast agent, and a magnetic resonance imaging agent; the small molecule drug is, for example, maytansine, DM-1, DM-4, MMAE, MMAF, Auristatin 0101, SN-38, Dxd, irinotecan, duocarmycin, amanitin, PBD class, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines or their derivatives, or the small molecule drug is, for example, a radiotherapy agent; Z’ is a linking segment connecting the L and G rings, and it is independently absent or selected from the group consisting of -(CH2) p -, -(CH2-CH2-O) p -, -(phenyl) p -, and any combination of one or two or more selected from the following groups: wherein Ra, Rc and Rd are each independently selected from hydrogen, C1-C6 alkyl, C6-C 10 aryl and C5-C 10 heteroaryl; and Each p is an integer from 1 to 5.

9. The disaccharide conjugate according to claim 8, wherein L is selected from or comprises a combination of one or more of the following: Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly; or, L is selected from or comprises a combination of one or more of the following: Val-Cit, Val-Ala, Ala-Ala, and Gly-Gly-Phe-Gly; or The L is selected from or comprises a combination of one or more of the following cleavable linkers: or L is selected from or comprises a combination of one or more non-cleavable linkers as follows: -(CH2) m -, -(CH2-CH2-O) m -, -(PO4) n -, where each m and each n are independently selected from integers from 0 to 30, or integers from 0 to 20, or integers from 0 to 10.

10. The disaccharide conjugate according to any one of claims 8-9, wherein P is a cytotoxin selected from the group consisting of:

11. The disaccharide conjugate according to claim 8, which is selected from the following specific compounds: Among them, n is an integer selected from 0 to 30, or an integer selected from 0 to 20, or an integer selected from 0 to 10.

12. Antibody-based conjugate drug shown in formula (III): Among them, The X, Y, and G rings are defined as in any one of claims 1-6; Z′, L, and P are defined as in any one of claims 8-10; or P-L-Z′- is hydrogen; t is selected from 0 or 1; R1’ is selected from R1 or a group generated after the reaction of R1 with the drug linker E-L-P, and R2’ is selected from R2 or a group generated after the reaction of R2 with the drug linker E-L-P; wherein R1 and R2 are as defined in any one of claims 1 and 4-5; L and P in the drug linker E-L-P are as defined in any one of claims 8-10 and E comprises the following groups: Among them, Ra, Rc, and Rd are hydrogen, C1-C6 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl; Ab is an antibody or antigen-binding fragment, selected from monoclonal antibody, bispecific antibody, or multivalent antibody.

13. The antibody-based conjugate drug according to claim 12, wherein the drug linker E-L-P is selected from the following specific compounds: Among them, n is an integer selected from 0 to 30, or an integer selected from 0 to 20, or an integer selected from 0 to 10.

14. The antibody-based conjugate drug according to claim 12 or 13, wherein the antibody is selected from: Pertuzumab, Trastuzumab, Rituximab, Cetuximab, Margetuximab, Abciximab, Daclizumab, Adalimumab, Palivizumab, Basiliximab, Bevacizumab, Panitumumab, Nimotuzumab, Denosumab, Zolbetuximab, Ramucirumab, Necitumumab, Ipilimumab, Daratumumab, Alemtuzumab, Elotuzumab, Blinatumomab, Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Toripalimab, Catumaxomab, Emicizumab, Amivantamab.

15. The antibody-based conjugate drug according to claim 12 or 14, which has a single payload and is selected from the following specific structures: Among them, n is an integer selected from 0 to 30, or an integer from 0 to 20, or an integer from 0 to 10; t is selected from 0 or 1.

16. The antibody-based conjugated drug according to claim 12 or 14, which has a dual payload and is selected from the following specific compounds: Among them, n and I are each independently selected from integers from 0 to 30, or integers from 0 to 20, or integers from 0 to 10; t is selected from 0 or 1.

17. A method for preparing an antibody-based conjugate drug, the method comprising the following three methods: Method 1: a) Under the action of an endoglycosidase having beta-N-acetylglucosaminidase hydrolytic activity or an endoglycosidase and a fucosidase, the wild-type antibody is hydrolyzed to form a deglycosylated antibody containing one N-acetylglucosamine or core fucosylated N-acetylglucosamine; b) The deglycosylated antibody obtained in step a) is conjugated with the disaccharide linker according to any one of claims 1-7 under the catalysis of an endoglycosidase having glycosyltransferase activity or a mutant thereof to obtain a glycoengineered antibody; c) Coupling the glycoengineered antibody containing orthogonal reactive groups obtained in step b) with the drug linker E-L-P as claimed in claim 12 or 13 modified with a corresponding group capable of specifically coupling with the orthogonal reactive group to prepare an antibody-based conjugate drug; Method 2: a) Under the catalysis of an endoglycosidase with glycosyltransferase activity or its mutant, coupling a wild-type antibody with the disaccharide linker as claimed in any one of claims 1-7 to obtain a glycoengineered antibody; b) Coupling the glycoengineered antibody containing orthogonal reactive groups obtained in step a) with the drug linker E-L-P as claimed in claim 12 or 13 modified with a corresponding group capable of specifically coupling with the orthogonal reactive group to prepare an antibody-based conjugate drug; Method 3: Under the catalysis of an endoglycosidase with glycosyltransferase activity or its mutant, co-incubating a wild-type antibody and the disaccharide-small molecule drug conjugate as claimed in any one of claims 8-11, or co-incubating a deglycosylated antibody and the disaccharide-small molecule drug conjugate to prepare an antibody-based conjugate drug.

18. The method according to claim 17, wherein, In the said Method 1 and Method 2, the orthogonal reactive group and the corresponding group capable of specifically coupling with the orthogonal reactive group are selected from any one of the following combinations: azide group and alkyne group, tetrazine group and trans-cyclooctene (TCO), thiol group and maleimide, thiol group and thiol group or activated form of thiol group, aldehyde group and amino group, aldehyde group and aminooxy group or hydrazide group.

19. The method according to claim 17 or 18, wherein The endoglycosidase with beta-N-acetylglucosaminidase hydrolytic activity is selected from Endo Se2, Endo Si, Endo S, Endo S2, Endo F3, Endo A, Endo D, Endo CC and their mutants; And / or the endoglycosidase with glycosyltransferase activity is selected from Endo Se2, Endo Si, Endo S, Endo S2, Endo F3 and their mutants.

20. According to the method as claimed in claim 17 or 18, when preparing a deglycosylated antibody without core fucosylation, a glycoengineered antibody, or an antibody-based conjugate drug, it includes co-using an endoglycosidase with beta-N-acetylglucosaminidase hydrolytic activity and a fucosidase.

21. Use of the disaccharide linker as claimed in any one of claims 1-7 or the disaccharide conjugate as claimed in any one of claims 8-11 in the preparation of an antibody-based conjugate drug.

22. Use of the antibody-based conjugate drug as claimed in any one of claims 12-16 in the preparation of a drug for treating and / or preventing tumors, inflammation, viruses, infectious diseases or other immune diseases.