Disaccharide linkers, disaccharide-small molecule drug conjugates and glycoside-specific antibody-drug conjugates, methods for their preparation and use

Abstract

The present application relates to a disaccharide linker, a disaccharide-small molecule drug conjugate and a glycoside-specific antibody-drug conjugate, and the preparation and use thereof. The structure of the disaccharide linker is shown in the following formula I. The present invention provides a novel specific and quantitative antibody-drug conjugate format, and improves the stability and cytotoxicity of the antibody-drug conjugate. [Formula 1] TIFF2024506979000148.tif40162

Description

[Technical Field]

[0001] The present invention relates to the fields of medicinal chemistry and biotechnology drugs, and more particularly to a disaccharide linker, a non-natural glycoengineered antibody produced using the disaccharide linker, and a specific and quantitative antibody-drug conjugate based on the sugar chain, as well as methods for producing and using the same. [Background technology]

[0002] Antibody-drug conjugates (ADCs) consist of an antibody, a cytotoxin, and a linker. They deliver the cytotoxin to tumor tissue via the antibody, achieving targeted delivery of the toxin and exerting antitumor activity. Compared to conventional chemotherapeutic drugs, ADCs have lower biological toxicity and a superior therapeutic index. In early ADCs, cytotoxins were primarily conjugated to lysine (Lys) or cysteine ​​(Cys) residues, which are abundant in antibodies, via random coupling. ADCs formed in this manner exhibited nonuniform toxin binding sites and amounts, resulting in poor in vivo stability, efficacy, and pharmacokinetic properties, as well as a narrow therapeutic window. Glycoside-specific ADCs can address these issues. The main coupling techniques used to manufacture glycoside-specific ADCs include the THIOMAB method, unnatural amino acid insertion, enzyme catalysis, and glycoside-specific coupling, each with its own unique characteristics.

[0003] Glycoside-specific ADC compounds are produced using glycoside-specific coupling technology, and cytotoxins are site-specifically modified at the N297 glycosylation site of the antibody Fc domain. Currently, in vitro antibody glycosylation site modification methods mainly involve glycosyltransferase technology and glycoside endonuclease technology.

[0004] Glycosyltransferase technology uses galactosyltransferase or sialyltransferase to transfer reactive galactose or sialic acid to antibody glycosylation sites, followed by the coupling of a cytotoxin to produce glycoside-specific ADC compounds. For example, Zhu et al. first hydrolyzed the terminal galactose of the N-glycosylation site of an antibody using β1,4-galactosidase, then used galactosyltransferase to transfer a ketocarbonyl-containing GalNAc to the N-glycosylation site, followed by the coupling of a hydroxylamine-containing toxin to obtain a glycoside-specific ADC compound. Qun Zhou et al. used galactosyltransferase and sialyltransferase to sequentially transfer galactose and sialic acid to the glycosylation site of an antibody, then oxidized the terminal sialic acid with sodium periodate, introducing an aldehyde group at the glycosylation site to provide a reactive site for toxin coupling. Floris L. van Delft et al. used glycoside endonuclease Endo-S to hydrolyze the heterogeneous N-glycans of an antibody, then used galactosyltransferase to transfer an azide group-containing GalNAz to the glycosylation site of the antibody, and finally modified the glycosylation site with a toxin using a click chemistry reaction.

[0005] Glycoside endonuclease technology utilizes glycoside endonucleases and bioorthogonal reactions to achieve glycoside-specific ADC production. Our team and the Davis team used semisynthetic modifications to obtain azide-modified oligosaccharide oxazoline substrates, and then sequentially used two glycoside endonucleases, Endo-S and its mutant enzyme Endo-S D233Q, to transfer the bioorthogonal-modified oligosaccharides to the glycosylation sites of antibodies, followed by bioorthogonal reactions to obtain glycoside-specific ADC compounds.

[0006] Existing glycosyltransferase and glycoside endonuclease technologies both produce more homogeneous ADC compounds than random coupling, but each has its own drawbacks. Glycosyltransferase technology requires the synthesis of activated forms of sugar substrates, such as CMP or UDP. Because glycosyltransferases tend to have weak catalytic activity, the reaction times are long, making it difficult to control production efficiency and costs. Furthermore, the oligosaccharide substrates used in glycoside endonuclease technology are difficult to obtain, requiring complex purification processes, such as extraction from egg yolk and semisynthetic modification, and total synthesis is even more difficult. Both glycosyltransferase and glycoside endonuclease technologies involve multiple enzymes and multistep reactions, limiting their effectiveness and productivity and not contributing to antibody stability. Furthermore, both technologies rely heavily on bioorthogonal reactions to achieve toxin modification at the glycosylation site of antibodies, limiting the drug development of glycoside-specific ADCs.

[0007] This patent invents a series of disaccharide linkers that can be efficiently transferred to the glycosylation site of an antibody under the action of wild-type glycoside endonuclease Endo-S2. When the disaccharide structure contains a bioorthogonal group, glycoengineered antibodies bearing the bioorthogonal group can be obtained by enzyme catalysis, and glycoside-specific ADC compounds based on the disaccharide structure can be produced in two steps using a bioorthogonal reaction. Furthermore, when functional groups such as drugs are directly attached to the disaccharide structure, novel glycoside-specific ADCs can be produced in one step using enzyme catalysis. This invention provides a simple and efficient method for producing glycoside-specific ADC compounds, resulting in novel ADC molecules with good in vivo and in vitro activity. Summary of the Invention [Means for solving the problem]

[0008] A technical object of the present invention is to provide a disaccharide linker that can specifically and quantitatively introduce small molecular weight drugs into antibodies. Another technical object of the present invention is to provide a use of the disaccharide linker in the production of antibody-drug conjugates. Another technical object of the present invention is to provide a disaccharide linker-small molecular weight drug conjugate. Another technical object of the present invention is to provide a small molecular weight drug-antibody conjugate linked by the disaccharide linker. Another technical object of the present invention is to provide a use of the disaccharide linker-small molecular weight drug conjugate or the small molecular weight drug-antibody conjugate linked by the disaccharide linker in the production of a pharmaceutical or diagnostic reagent.

[0009] In one aspect, the present invention provides a disaccharide linker as shown in Formula I below: [ka] I

[0010] In Formula I, The G ring represents a structure derived from a monosaccharide molecule, and is bound to the 4-position of N-acetyl-D-glucosamine, which is cyclized at the 1,2-positions by a glycosidic bond, and the monosaccharide molecule is selected from the group consisting of galactose, N-acetyl-galactose, glucose, mannose, fucose, and sialosugar; and the glycosidic bond is a 1,4-glycosidic bond, a 2,4-glycosidic bond, or a 3,4-glycosidic bond.

[0011] ZYX- represents a substituent on the G ring, and the substitution position of ZYX- is any position other than position 1 of the G ring derived from a monosaccharide molecule. Here, in the structure ZYX-, ZY- may or may not be present, and when ZY- is absent, X represents an aldehyde group, a phosphate group, -NH, -CH-NH, -COOH, or -CHSR. p , -CH2SeR p , -N3, -CH2-N3, and R pis a protecting group. When ZY- is present, X is selected from the group consisting of -CH2-, -CH2-O-, -CH2-S-, -CH2-Se-, -CO-NH-, -ON=CH-, -CONH-N=CH-, -NHCH2-, -CH=CH-, and the following structures: [ka]

[0012] Y is a divalent or polyvalent linker connecting X and Z, and preferably, Y is a group consisting of -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, -(PO4)n-. or a combination of the cleavable fragment and the linked fragment. Here, m and n are each independently selected from the group consisting of integers between 0 and 30, and w is a hydrogen atom or a polyethylene glycol structure having various lengths.

[0013] Z is selected from the group consisting of the following cases i) to iv). i) fragments bearing reactive groups or functional molecules for bioorthogonal reactivity Preferably, Z is selected from the group consisting of the following reactive groups: azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetraazine residue, nitrone residue, hydroxyamine residue, nitrile residue, hydrazine residue, ketone residue, boric acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halocarbonyl residue, isocyanide residue, sydnone residue, selenium residue, conjugated diene residue, phosphate residue, cycloalkyne residue, and cycloalkene residue. Alternatively, Z is selected from the group consisting of the following groups: [ka] wherein n is an integer of 1 to 30, and R1 and R2 are each independently selected from the group consisting of H, -CH3, -CH2CH3, cyclopropyl, or cyclobutyl. Preferably, the functional molecule is selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycol, lipids, polypeptides, nanobodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, and contrast agents and magnetic resonance imaging agents.

[0014] ii) [ka] wherein L1 is a trivalent linker having three reactive groups, and preferably L1 is a branched-chain amino acid having a reactive functional group derived from lysine, aspartic acid, glutamic acid, propargylglycine, cysteine, or the following structure: [ka] Here, n is an integer of 1 to 30. L2 and L3 are divalent or polyvalent linkers connecting L1 to Z2 and Z3. Preferably, L2 and L3 each independently have the structure -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, or -(PO4)n-. or a combination of the cleavable fragment and the above linked fragment where m and n are each independently selected from the group consisting of integers between 0 and 30. w is a hydrogen atom or other side chain structure, such as polyethylene glycol of various lengths. Z' is a linking fragment coupling L1 and the sugar linker and is independently absent, -(CH2)p-, or a group capable of reacting with the Z group in case i), where p is an integer between 1 and 5. For example, Z' is selected from the group consisting of the following groups: [ka] wherein R1 and R2 are each independently selected from the group consisting of H, -CH3, -CH2CH3, cyclopropyl, or cyclobutyl. The definitions of Z2 and Z3 are the same as those of Z in case i).

[0015] iii) [ka] wherein L6 is a tetravalent linker having four reactive groups, and is preferably selected from the group consisting of dilysine, diglutamic acid, diaspartic acid, an aspartic acid-glutamic acid dipeptide structure, an aspartic acid-lysine dipeptide structure, and a glutamic acid-lysine structure, or is selected from the group consisting of the following structures: [ka] Here, n is an integer from 1 to 30, the definitions of L2, L3, and L4 are the same as the definitions of L2 and L3 in case ii), the definition of Z' is the same as the definition of Z' in case ii), and the definitions of Z2, Z3, and Z4 are the same as the definitions of Z2 and Z3 in case ii).

[0016] iv) [ka] Here, the definition of L1 is the same as the definition of L1 in case ii), the definitions of L2, L3, L4, and L5 are the same as the definitions of L2 and L3 in case ii), the definition of Z' is the same as the definition of Z' in case ii), and the definitions of Z2, Z3, Z4, and Z5 are the same as the definitions of Z2 and Z3 in case ii). Alternatively, if Y and Z do not exist, X is [ka] where R1 is hydroxy-OH or an azide group -N3, R2 is any group, R3 is either hydroxy-OH or -NH-containing, R4 is any group, and the wavy line indicates the linkage site.

[0017] In a specific embodiment, the disaccharide linker of formula I is shown in formula II below: [ka] II In Formula II, X, Y, and X are each as defined above.

[0018] In a specific embodiment, the disaccharide linker is selected from the group consisting of the following specific compounds: [ka] JPEG2022174834000013.jpg55144 Here, R is a fragment or combination of Y and Z above, and l, m, and n are each independently an integer of 0-30.

[0019] Preferably, the disaccharide linker is selected from the group consisting of the following structures: [ka] [ka] [ka]

[0020] In another aspect, the present invention provides a method for producing the above-mentioned disaccharide linker, for example, a method shown in the following reaction scheme: [ka]

[0021] In the above reaction scheme, the G ring is as defined above. The monosaccharide modification position is a modifiable site other than position 1. U is the introduced active group, selected from the group consisting of an aldehyde group, an amino group, an azide group, and an alkynyl group. X, Y, and Z are each as defined above.

[0022] The method comprises: 1) modifying a disaccharide structure having a terminal acetylglucosamine structure by the action of an enzyme or other small molecule compound to obtain a disaccharide structure having an active group U, and then introducing ZYX- having orthogonal reactivity or containing a functional molecular fragment after the disaccharide structure having the active group U has been derivatized; 2) a cyclization step of the ZYX-introduced disaccharide structure having orthogonal reactivity or containing functional molecular fragments to obtain the disaccharide linker of formula I; Includes.

[0023] In a specific embodiment, the modification reaction in step 1) is an oxidation reaction, the enzyme is galactose oxidase, and U is an aldehyde group.

[0024] In a specific embodiment, the derivatization reaction in step 1) is an oximation reaction, a reductive amination, a reaction involving an amino group, or a reaction involving an azide group.

[0025] In a specific embodiment, in step 2), the cyclization reaction is carried out using 2-chloro-1,3-dimethylimidazolinium chloride or 2-chloro-1,3-dimethyl-1H-benzimidazolium-3-chloride.

[0026] In another aspect, the present invention provides disaccharide-small molecule drug conjugates having the structure shown in Formula III, IV, or V below. [ka] III JPEG2022174834000019.jpg3165 IV JPEG2022174834000020.jpg3670 V

[0027] In the above formulas III, IV, and V, ring G, X, Y, Z3, Z', L1, L2, and L3 are each as defined above, and in the structure of formula IV or V, L may be the same or different from each other. Z2' and Z3' are linker structures formed from the bioorthogonal group and Z2 and Z3, and Z' may be the same or different from each other, and may exist simultaneously or independently. L is a bivalent linker connecting D, D1, or D2 to the remaining moieties in formulas III-V, and preferably L is selected from the group consisting of -(CH2)a-(CH2CH2)b-(NHCO)n-(CH2)c-, or the group consisting of the following groups: [ka] JPEG2022174834000022.jpg5698 JPEG2022174834000023.jpg74115 [ka] JPEG2022174834000025.jpg30121 JPEG2022174834000026.jpg25131 JPEG2022174834000027.jpg26134 Here, V and W are bifunctional linkers, and include structures in which lysine and propargylglycine are bifunctional linkers. For example, L is [ka] JPEG2022174834000029.jpg44139 JPEG2022174834000030.jpg35145 wherein a, b, c, d, and e are each independently selected from the group consisting of integers between 0 and 30, and m and n are 0 or 1. R3 and R4 are each independently selected from the group consisting of CH3-, (CH3)2CH-, PhCH2, NH2(CH2)4-, and NH2CONH(CH2)3-, and R is selected from the group consisting of azidizable monosaccharides, disaccharides, oligosaccharides, or PEG structures of various lengths having an azide group, or combinations of PEG with linear or cyclic monosaccharides, disaccharides, or oligosaccharides. The oligosaccharides include branched oligosaccharide chains. The wavy lines indicate linkage sites. D, D1, and D2 each independently represent a cytotoxic compound, a group derived from a small molecule drug, or a group derived from a fluorescent probe, and the small molecule drug is preferably selected from the group consisting of maytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, dokamycin, amanitin, PBDs, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estramustine, cemadotin, eleutherobin, a fluorescent reagent, a monosaccharide, a disaccharide, an oligosaccharide, and derivatives of the above compounds. Alternatively, the small molecule drug is a radiotherapeutic drug. Preferably, D, D1, and D2 each independently represent a group consisting of the following groups: [ka] JPEG2022174834000032.jpg23129 JPEG2022174834000033.jpg26129 JPEG2022174834000034.jpg31112 JPEG2022174834000035.jpg24118 JPEG2022174834000036.jpg24117

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[0028] In specific embodiments, the disaccharide-small molecule drug conjugates are represented by the following Formulas VI, VII, and VIII: [ka] VI JPEG2022174834000059.jpg4260 VII JPEG2022174834000060.jpg4762 VIII The definition of each substituent in formulae VI, VII and VIII is as defined above.

[0029] In a specific embodiment, the disaccharide-small molecule drug conjugate is any one selected from the group consisting of the following compounds: [ka] Compound DG-1 JPEG2022174834000062.jpg50142 Compound DG-2 JPEG2022174834000063.jpg54133 Compound DG-3 [ka] Compound DG-4 JPEG2022174834000065.jpg62124 Compound DG-5 JPEG2022174834000066.jpg50121 Compound DG-6 [ka] Compound DG-7 JPEG2022174834000068.jpg56142 Compound dDG-1 JPEG2022174834000069.jpg52136 Compound dDG-2 [ka] Compound dDG-3

[0030] In each of the above structures, the structure of the MMAE portion is: [ka] is.

[0031] In yet another aspect, the present invention provides a glycoengineered antibody based on the specific linkage of an antibody Fc domain N-glycosylation site as shown in Formula IX below. [ka] IX wherein in the above formula IX, the definitions of ring G and X, Y, and Z are as defined above, m is selected from the group consisting of 0 or 1, and n is selected from the group consisting of 1 or 2. Ab is a monoclonal antibody, a bifunctional antibody, or a polyclonal antibody, and is a therapeutic antibody or a functional antibody from a different species. Preferably, the Ab is selected from the group consisting of: trastuzumab, pertuzumab, rituximab, cetuximab, morozumab, gemtuzumab, abciximab, darizumab, adalimumab, palizumab, baliximab, bevacizumab, panitumumab, nitrotuzumab, denitumab, decitumab, lemonivumab, nexituzumab, eprilimus, daremab, bentocivizumab, alemtuzumab, erlotuzumab, bonatumab, nivolumab, pembrolizumab, atezolizumab, avilumab, darvalumab, tremelimumab, catumab, belintumomab, emicizumab, evantozumab (Rybrevant).

[0032] In a specific embodiment, said glycoengineered antibody has the following formula X: [ka] X In the above formula X, the definitions of X, Y, and Z are as defined above, m is selected from the group consisting of 0 and 1, n is selected from the group consisting of 1 or 2, and Ab is an antibody.

[0033] In yet another aspect, the present invention provides a method for producing the above-mentioned glycoengineered antibody, wherein the method is carried out by the following method 1 or the following method 2: [ka] In the above reaction formula, m is selected from the group consisting of 0 and 1, and the definitions of ring G, X, Y, and Z are as defined above.

[0034] Method 1: A wild-type antibody is hydrolyzed with a glycosidic endonuclease or a combination of a glycosidic endonuclease and a glucosidase to remove heterogeneous sugar chains at the conservative glycosylation sites of the native antibody, yielding a deglycosylated antibody. The wild-type antibody is then co-incubated with the disaccharide linker described above, and the disaccharide linker is ligated to the conservative glycosylation sites of the antibody Fc domain by the catalytic action of the wild-type glycosidic endonuclease to produce a fucose-containing or non-fucose-containing 1,6-acetylglucosamine disaccharide-modified antibody represented by Formula IX, which is modified with the disaccharide linker represented by Formula I containing an orthogonal reactive group. Method 2: The above-described disaccharide linker and wild-type antibody are co-incubated, and the N-oligosaccharide structure of the wild-type antibody Fc domain is hydrolyzed by the catalytic action of the wild-type glycosidic endonuclease. At the same time, the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain to produce a fucose-containing or non-fucose-containing 1,6-acetylglucosamine disaccharide-modified antibody represented by Formula IX, which is modified with the disaccharide linker represented by Formula I containing an orthogonal reactive group.

[0035] Preferably, the wild-type glycosidic endonuclease is N-acetylglucosamine endohydrolase, and more preferably, the N-acetylglucosamine endohydrolase is Endo-S2 (Endoglycosidase-S2), for example, Endo-S2 endoglycosidase derived from Streptococcus pyogenes. When producing a compound with coreless fucosylation, a glycosidic endonuclease and a fucose hydrolase must be used in combination.

[0036] In a specific embodiment, the glycoengineered antibody is produced by Method 1 below or Method 2 below. [ka] In the above reaction scheme, m is selected from the group consisting of 0 or 1, and X, Y, and Z are each defined as above.

[0037] Method 1: A wild-type antibody is hydrolyzed with a glycosidic endonuclease or a combination of a glycosidic endonuclease and a glucosidase to remove heterogeneous sugar chains at the conservative glycosylation sites of the native antibody, yielding a deglycosylated antibody. The wild-type antibody is then co-incubated with the disaccharide linker described above, and the disaccharide linker is ligated to the conservative glycosylation sites of the antibody Fc domain by the catalytic action of the wild-type glycosidic endonuclease to produce a fucose-containing or non-fucose-containing 1,6-acetylglucosamine disaccharide-modified antibody of Formula X modified with the disaccharide linker of Formula II containing an orthogonal reactive group. Method 2: The above-described disaccharide linker and wild-type antibody are co-incubated, and the N-oligosaccharide structure of the wild-type antibody Fc domain is hydrolyzed by the catalytic action of the wild-type glycosidic endonuclease. At the same time, the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain to produce a fucose-containing or non-fucose-containing 1,6-acetylglucosamine disaccharide-modified antibody represented by Formula X that has been modified with the disaccharide linker represented by Formula II containing an orthogonal reactive group.

[0038] Preferably, the wild-type glycosidic endonuclease is N-acetylglucosamine endohydrolase, and more preferably, the N-acetylglucosamine endohydrolase is Endo-S2 (Endoglycosidase-S2), for example, Endo-S2 endoglycosidase derived from Streptococcus pyogenes. When producing a compound with coreless fucosylation, a combination of a glycosidic endonuclease and a fucose hydrolase is required.

[0039] In yet another aspect, the present invention provides an antibody-drug conjugate having the following formula XI: [ka] XI In Formula XI, the definitions of ring G and X, Y, Z', L, and D are each as defined above, m is selected from the group consisting of 0 or 1, and n is selected from the group consisting of 1 or 2. Ab is an antibody, and the carbohydrate attachment site is a conservative N-glycosylation site on the antibody Fc.

[0040] In a specific embodiment, the antibody-drug conjugate is shown in Formula XII: [ka] XII In Formula XII, X, Y, Z', L, and D are each as defined above, m is selected from the group consisting of 0 or 1, and n is selected from the group consisting of 1 or 2. Ab is an antibody, and the carbohydrate linkage site is a conservative N-glycosylation site on the antibody Fc.

[0041] The present invention further provides a compound of formula XI and formula XII, wherein the structure of -Z'-LD is [ka] JPEG2022174834000079.jpg28143 The antibody-drug conjugate of formula XI or XII is substituted with Z', L, L1-L6, and D1 and D2, respectively. Z2', Z3', Z4', and Z5' are linking fragments formed by reacting Z2, Z3, Z4, and Z5 with a bioorthogonal group of a functional molecule, respectively, and may not exist simultaneously or independently. The definitions of D3 and D4 are the same as those of D1 and D2. When the structures of D1-D4 are the same, the antibody-drug conjugate of formula XI or XII represents an antibody-drug conjugate with a high drug loading (drug-to-antibody ratio, DAR value) and the same drug structure. When D1-D4 are different, the antibody-drug conjugate of formula XI or XII represents a multi-drug antibody-drug conjugate with different drug structural compositions.

[0042] In another aspect, the present invention provides a method for producing the above-mentioned antibody-drug conjugate, which comprises the following two methods: Method 1: a) Co-incubating the disaccharide linker with a wild-type antibody to hydrolyze the N-oligosaccharide structure at Asn297 in the Fc domain of the wild-type antibody by the catalytic action of a wild-type glycosidic endonuclease and simultaneously ligating the disaccharide linker to the Asn297 site in the Fc domain of the antibody, or co-incubating the disaccharide linker with a deglycosylated antibody and a glycosidic endonuclease to produce a fucose-containing or non-fucose-containing 1,6-acetylglucosamine disaccharide-modified antibody of Formula IX modified with the orthogonally reactive group-containing disaccharide linker of Formula I. The deglycosylated antibody can be obtained by previously treating the wild-type antibody with a glycosidic endonuclease, or by simultaneously removing fucose using a fucose hydrolase. b) The fucose-containing or fucose-free 1,6-acetylglucosamine disaccharide-modified antibody of Formula IX modified with the orthogonally reactive group-containing disaccharide linker of Formula I obtained in step a) is coupled to a modified small molecule drug having a corresponding group capable of specific coupling reaction with the orthogonally reactive group to produce an antibody-drug conjugate of Formula XI or XII. Method 2: The disaccharide-small molecule drug conjugate and wild-type antibody are co-incubated to hydrolyze the N-oligosaccharide structure at Asn297 in the Fc domain of the wild-type antibody by the catalytic action of the wild-type glycosidic endonuclease, and simultaneously link the disaccharide-small molecule drug conjugate to the Asn297 site in the Fc domain of the antibody, or the disaccharide-small molecule drug conjugate, a deglycosylated antibody, and a glycosidic endonuclease are co-incubated to produce an antibody-drug conjugate of Formula XI or XII, where the deglycosylated antibody can be obtained by previously treating the wild-type antibody with a glycosidic endonuclease, or by simultaneously removing fucose using a fucose hydrolase.

[0043] In a specific embodiment, the wild-type glycosidic endonuclease is N-acetylglucosamine endohydrolase, and more preferably, the N-acetylglucosamine endohydrolase is Endo-S2 (Endoglycosidase-S2, endoglycosidase Endo-S2 derived from Streptococcus pyogenes). When producing a compound with coreless fucosylation, a glycosidic endonuclease and a fucose hydrolase must be used in combination.

[0044] In a specific embodiment, in the method 1, the orthogonal reactive group and the corresponding group capable of specific coupling reaction with the orthogonal reactive group are any combination selected from the group consisting of an azide group and an alkynyl group, a mercapto group and a maleimide, a mercapto group and a mercapto group or an activated form of a mercapto group, an aldehyde group and an amino group, an aldehyde group and an aminooxy group, or a hydrazine group.

[0045] In a specific embodiment, in step b) of Method 1, the drug linker has the following group for coupling to a small molecule drug modified with the corresponding group: [ka]

[0046] In a specific embodiment, the correspondingly group-modified small molecule drug is selected from the group consisting of the following compounds: [ka] D1 JPEG2022174834000082.jpg33143 D2 JPEG2022174834000083.jpg38151 D3 JPEG2022174834000084.jpg33141 D4 [ka] D5 JPEG2022174834000086.jpg52129 D6 JPEG2022174834000087.jpg43147 D7 JPEG2022174834000088.jpg33156 D8 [ka] D9 JPEG2022174834000090.jpg40139 D10 JPEG2022174834000091.jpg30156 D11 [ka] D12 JPEG2022174834000093.jpg22111 D13

[0047] In a specific embodiment, the method 1 is shown in the following reaction scheme: [ka] In the above reaction scheme, m is selected from the group consisting of 0 and 1, and X, Y, Z, Z', L, and D are each defined as above. E is an orthogonal reactive group capable of reacting with Z, and the glycoengineered antibody in the reaction scheme can be obtained by the method described above.

[0048] In a specific embodiment, the method 1 is shown in the following reaction scheme: [ka] In the above reaction scheme, m is selected from the group consisting of 0 or 1, and X, Y, Z, Z', L, and D are each defined as above. E is an orthogonally reactive group capable of reacting with Z, and the glycoengineered antibody in the reaction scheme is obtained by the method described above.

[0049] In a specific embodiment, the preparation method is shown in the following reaction scheme: [ka] Here, L and D are defined as above, and E3 is a group that reacts orthogonally with the aldehyde group and is selected from the group consisting of cyclosulfidopyrazole, orthoaminobenzamidoxime, and hydroxylamine structures, such as [ka] X2 is a structure formed by reacting an aldehyde group with E3, and E5 is a corresponding group that reacts orthogonally with an azide group and is selected from the group consisting of a linear alkynyl group, a DBCO-based structure, and a BCN-based structure. X4 is a structure formed by reacting an azide group with E5.

[0050] In a specific embodiment, the second method involves co-incubating the disaccharide-small molecule drug conjugate with a glycosidic endonuclease, an antibody, and the disaccharide-small molecule drug conjugate to hydrolyze the N-oligosaccharide at Asn297, a conservative glycosylation site in the antibody Fc domain, and simultaneously transfer the disaccharide-small molecule drug conjugate to Asn297 (Method 1), or co-incubating the disaccharide-small molecule drug conjugate with a deglycosylated antibody and a glycosidic endonuclease (Method 2), thereby achieving glycoside-specific and quantitative introduction of the small molecule drug and obtaining the corresponding antibody-drug conjugate, as shown in the following two reaction schemes: The deglycosylated antibody can be obtained by pretreating a wild-type antibody with a glycosidic endonuclease, or by simultaneously removing fucose using a fucose hydrolase. [ka] JPEG2022174834000099.jpg58144

[0051] In yet another aspect, the present invention provides the use of the disaccharide linker or the disaccharide-small molecule drug conjugate in antibody glycoengineering or in the production of antibody-drug conjugates.

[0052] In one aspect, the present invention provides the use of the antibody-drug conjugate described above in the manufacture of a drug, pharmaceutical composition, or diagnostic reagent, wherein the drug in the conjugate is selected from the group consisting of anti-tumor, anti-inflammatory, anti-viral, anti-infectious disease, or other immunotherapeutic drugs. [Effects of the Invention]

[0053] Based on glycoside-specific structural modifications, the present invention develops and refines new linking methods and linking structures. In addition to orthogonal reactions, other reactions such as amide reactions are introduced in the disaccharide structure and drug-linker to construct a new type of specific and quantitative antibody-drug conjugate format, which improves the stability and cytotoxicity of the antibody-drug conjugate and achieves better drug formation properties.

[0054] The present invention utilizes the fact that wild-type glycosidic endonucleases have both hydrolytic activity for N-oligosaccharides at conservative glycosylation sites on antibodies and transglycosylation activity for specific novel disaccharide linker structures. This allows the design and preparation of disaccharide linkers with orthogonal reactive groups, or disaccharide linkers with drug-linkers or dual drug-linkers, which can be used to specifically insert orthogonal reactive groups into antibodies through enzyme-catalyzed reactions. This allows specific coupling with small molecule drugs, or the specific and quantitative coupling of small molecule drugs directly through enzyme-catalyzed reactions in a single step. This process does not require prior hydrolysis of heterogeneous N-oligosaccharides at conservative glycosylation sites on wild-type antibody Fc domains, is simple, requires fewer purification steps, and is easy to produce industrially.

[0055] The specifically linked antibody-drug conjugates of Formulae IX and X, prepared from the disaccharide linkers of Formulae I and II, have a specific and uniform chemical structure. Compared with the approved antibody-drug conjugates with heterogeneous structures, they have the advantages of a clearly defined single structure and controllable quality. Furthermore, compared with other specific coupling methods, this production method has the advantages of simple operation and fewer purification steps, resulting in good drug formation and ease of industrialization. Furthermore, the specifically linked antibody-drug conjugates exhibit good antitumor activity. [Brief explanation of the drawings]

[0056] [Figure 1] Screening results of the initial sugar substrates and glycosidic endonucleases of this application are shown. A: Screening of antibody transglycosylation activity of various glycosidic endonucleases against G14; B: Screening of the ability of glycosidic endonuclease Endo-S2 to recognize and transfer various sugar substrates to antibody glycosylation sites. [Figure 2]

[0039] Figure 1 shows the results of in vitro activity experiments for some disaccharide linker-linked ADCs of the present application. A: Inhibitory rates of some gsADCs (gsADC-5, gsADC-21, gsADC-30, gsADC-36) against SK-Br-3 cells, B: Inhibitory rates of some gsADCs (gsADC-35, gsADC-34, gsADC-38) against SK-Br-3 cells, C: Inhibitory rates of some gsADCs (gsADC-5, gsADC-21, gsADC-30, gsADC-36) against NCI-N87 cells, D: Inhibition rate of some gsADCs (gsADC-35, gsADC-34, gsADC-38) against NCI-N87 cells. E: Effect of some gsADCs (gsADC-5, gsADC-21, gsADC-30, gsADC-36) on the viability of MDA-MB-231 cells. F: Effect of some gsADCs (gsADC-35, gsADC-34, gsADC-38) on the viability of MDA-MB-231 cells. [Figure 3] Figure 1 shows experimental data on the in vivo activity of some disaccharide-linked ADCs of the present application, showing the effects of gsADC-21, gsADC-30, gsADC-35, and gsADC-36 on tumor volume in vivo and on the body weight of nude mice, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0057] The term "multivalent linker" as used herein means a linker that is more than bivalent.

[0058] The glycosidases used in this invention were expressed in Escherichia coli and were laboratory-produced. The small molecule cytotoxic drugs DM1 and MMAE used in this invention were purchased from Shanghai Zeishan Pharmaceutical Technology Co., Ltd. DBCO-based compounds and BCN-based compounds were purchased from Chengdu Baerkang Biotechnology Co., Ltd. 3-Azidopropylamine was purchased from Shanghai Bailinwei Chemical Technology Co., Ltd. N-Acetyl-D-lactosamine and acetonitrile were purchased from Shanghai Jizhi Biochemical Co., Ltd. BTTAA was purchased from Taizhou Gelinmei Company. Galactose oxidase, catalase, and horseradish peroxidase were purchased from Bioengineering Bioengineering (Shanghai) Co., Ltd. Amino acid compounds were purchased from Shanghai Jier Biochemical Co., Ltd. 4-Pentynoic acid was purchased from Shanghai Bitde Pharmaceutical Technology Co., Ltd. All other compounds and reagents were purchased from Sinopharm Group Chemical Reagents Co., Ltd. unless otherwise specified.

[0059] The devices and chromatography columns used in the present invention include a Waters Xevo G2-XS QTOF, an analytical high-performance liquid chromatography system (Thermo Ultimate 3000), an analytical high-performance liquid chromatography system (Beijing Innovation Tongheng LC3000), a preparative high-performance liquid chromatography system (Beijing Innovation Tongheng LC3000); a Thermo C18 (Acclaim™ 120.5 μm, 4.6 × 250 mm), an Agilent SB-C18 (5 μm, 4.6 × 150 mm), and a Waters C18 column (ACQUITY UPLC BEH C18, 1.7 μm, 2.1 × 50 mm).

[0060] Antibody molecular weight measurement equipment: Liquid chromatography mass spectrometer (LC-MS), Waters Xevo G2-XS QTOF, Waters C4 (ACQUITY UPLC Protein BEH C4, 1.7 μm, 2.1 mm x 50 mm).

[0061] Below, we use reaction schemes to outline the synthesis routes for the disaccharide linkers, glycoengineered antibodies, and glycoside-specific antibody-drug conjugates (ADCs) described in this application.

[0062] General manufacturing example The synthesis route of the disaccharide linker is as follows. [ka] Notes: a. Galactose oxidase (GOase), catalase, horseradish peroxidase (HRP), O2, pH 7.0, 30°C. b. Hydroxyamine hydrochloride, sodium carbonate, rt, sodium borohydride, nickel chloride hexahydrate, 4°C. c. 1H-imidazole-1-sulfonyl azide hydrochloride, potassium carbonate, copper sulfate, 37°C. d. 3-azidopropylamine (NaCNBH3), pH 6.0, 0°C. e. O-(2-azidoethyl)hydroxyamine hydrochloride, pH 7.4, 37°C. f. DMC, triethylamine, 0°C, or CDMBI, potassium phosphate, 0°C. g. Propargylamine (NaCNBH3), pH 6.0, 0°C. h. O-(2-propargyl)hydroxyamine hydrochloride, pH 7.4, 37°C. i. Biotin-ONH2, pH 7.4, 37°C. j. FITC-NCS, pH 7.4, 37°C. k. Azidoacetic acid activated ester, pH 7.4. m. CMP-sialic acid (as shown below in Compound 70), α-2,6-sialic acid transferase, 100 mM Tris buffer, pH 8.0. n. DBCO-CONHS 80, pH 7.4 / DMF. [ka] Notes: g. Endo-S2, pH 7.0 buffer solution containing (or not containing) a certain amount of a solubilizing agent such as DMSO or DMA or DMF, 30°C. h. pH 7.0 buffer solution containing (or not containing) a certain amount of a solubilizing agent such as DMSO or DMA or DMF, General Procedure 3 to General Procedure 10.

[0063] General operations 1 Method for producing non-natural glycoengineered antibodies 1 For example, as shown in Examples 37-50 below, the prepared derivatized disaccharide oxazolines (i.e., compounds G1-G11, G13-G14) and sialic acid-derived disaccharide oxazolines (i.e., compound G12), wild-type antibodies, and wild-type glycoside endonuclease Endo-S2 were reacted at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively, in a reaction system adjusted to pH 7.0. The reaction system was incubated at 30°C for 0.5 to 12 hours, and then purified with protein A to obtain the desired non-natural glycoengineered antibodies Ab-1 to Ab-14. [ka] Ab-1 JPEG2022174834000103.jpg5389 Ab-2 JPEG2022174834000104.jpg5085 Ab-3 [ka] Ab-4 JPEG2022174834000106.jpg4679 Ab-5 JPEG2022174834000107.jpg5086 Ab-6 [ka] Ab-7 JPEG2022174834000109.jpg4782 Ab-8 JPEG2022174834000110.jpg4779 Ab-9 [ka] Ab-10 JPEG2022174834000112.jpg5290 Ab-11 JPEG2022174834000113.jpg46115 Ab-12 [ka] Ab-13 JPEG2022174834000115.jpg62101 Ab-14

[0064] General operations 2 Method for producing non-natural glycoengineered antibodies 2 For example, as shown in Examples 51-55 below, the prepared derivatized disaccharide oxazolines (i.e., compounds G3, G8, G10, and G13) and sialic acid-derived disaccharide oxazolines (i.e., compound G12), non-fucose antibodies, and wild-type glycoside endonuclease Endo-S2 were reacted at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively, in a reaction system adjusted to pH 7.0. The reaction system was incubated at 30°C for 0.5 to 12 hours, and then purified with protein A to obtain the desired non-natural glycoengineered antibodies Ab-15 to Ab-19. [ka] Ab-15 JPEG2022174834000117.jpg4881 Ab-16 JPEG2022174834000118.jpg42130 Ab-17 [ka] Ab-18 JPEG2022174834000120.jpg5594 Ab-19

[0065] General operations 3 A method for producing a one-step glycoside-specific antibody-drug conjugate (ADC) For example, as shown in Examples 89-96 below, the prepared drug-linker-bearing disaccharide oxazolines (i.e., compounds DG-1 to DG-7, dDG-1 to dDG-3), wild-type antibodies, and wild-type glycoside endonuclease Endo-S2 were reacted at pH 7.0 to give concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively, and the mixture was incubated at 30°C for 0.5 to 12 hours. Complete conversion to the product was confirmed by LC-MS, and then purified with protein A to yield the desired glycoside-specific quantitative antibody-drug conjugates gsADC-30 to gsADC-37.

[0066] General operations 4 Method for producing specific ADCs based on aldehyde-group disaccharide antibodies 1 For example, as shown in Example 56 below, the prepared aldehyde-group disaccharide antibody (i.e., non-natural glycoengineered antibody Ab-2) and the 2-aminoaniline oxime group-containing drug-linker (i.e., compound D2) were adjusted to 5 mg / mL and 0.3 mM, respectively, at a pH of 7.0 to 7.4, and incubated at 37°C. Complete conversion to the product was confirmed by LC-MS, and then purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugate gsADC-1.

[0067] General operations 5 Method for producing specific ADCs based on aldehyde-group disaccharide antibodies 2 For example, as shown in Example 57 below, the prepared aldehyde-group disaccharide antibody (i.e., non-natural glycoengineered antibody Ab-2) and aminooxy group-containing drug-linker (i.e., compound D1) were adjusted to 5 mg / mL and 0.3 mM, respectively, at a pH of 7.0 to 7.5, and incubated at 37°C. Complete conversion to the product was confirmed by LC-MS, and then purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugate gsADC-2.

[0068] General operations 6 Method for producing specific ADCs based on aldehyde-group disaccharide antibodies 3 For example, as shown in Examples 58-59 below, the prepared aldehyde-group disaccharide antibody (i.e., non-natural glycoengineered antibody Ab-2) and thioPz group-containing drug-linker (i.e., compounds D3-D4) were sequentially adjusted to 5 mg / mL and 0.3 mM, respectively, and EDTA and 10% Triton X-100 were added to final concentrations of 0.5 mM and 1%. The pH was adjusted to 5.5, and the mixture was incubated at 37°C. Complete conversion to the product was confirmed by LC-MS, and then purified with protein A to obtain the desired glycoside-specific antibody-drug conjugates gsADC-3 to gsADC-4.

[0069] General operations 7 Method for producing specific ADCs based on azidodisaccharide antibodies 1 For example, as shown in Examples 60-76 below, the prepared azidodisaccharide antibodies (i.e., non-natural glycoengineered antibodies Ab-3, Ab-4, Ab-6, Ab-9, and Ab-15) and DBCO-based drug-linkers (i.e., compounds D6-D9 and D13) were adjusted to 5 mg / mL and 0.3 mM, respectively, at pH 7.4, incubated at 37°C, and after confirming complete conversion to the products by LC-MS, purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugates gsADC-5 to gsADC-20 and gsADC-29.

[0070] General operations 8 Method for producing specific ADCs based on azidodisaccharide antibodies 2 As shown in Examples 77-84 below, the prepared azidodisaccharide antibodies (i.e., non-natural glycoengineered antibodies Ab-3, Ab-4, Ab-6, Ab-9, Ab-14, and Ab-15) and BCN-based drug-linkers (i.e., compounds D5, D11, and D12) were incubated at 37°C with concentrations of 5 mg / mL and 0.3 mM, respectively, at a pH of 7.4, and after complete conversion to the products was confirmed by LC-MS, the conjugates were purified with protein A to yield the desired glycoside-specific quantitative antibody-drug conjugates gsADC-21 to gsADC-24, gsADC-39, and gsADC-41 to gsADC-43.

[0071] General operations 9 Method for producing specific ADCs based on azidodisaccharide antibodies3 For example, as shown in Examples 85-89, the prepared azidodisaccharide antibodies (i.e., non-natural glycoengineered antibodies Ab-3, Ab-4, Ab-6, and Ab-9) and linear alkynyl group-based drug-linker (i.e., compound D10) were diluted to 5 mg / mL and 0.3 mM, respectively, with the pH adjusted to 7.4. A 6 mM Cu(I)-BTTAA solution (prepared by sequentially adding 21 μL of ddH2O, 3 μL of 60 mM CuSO4 solution, 3 μL of 300 mM BTTAA solution, and 3 μL of 900 mM sodium ascorbate solution) was added to a final concentration of 0.5 mM. The mixture was incubated at 37°C, and complete conversion to the product was confirmed by LC-MS. Purification with protein A afforded the desired glycoside-specific antibody-drug conjugates gsADC-25 to gsADC-28.

[0072] General operations 10 Methods for producing defucosylated disaccharide-specific antibody-drug conjugates (ADCs) For example, as shown in Example 97 below, the prepared drug-linker-bearing disaccharide oxazoline (i.e., compound DG-6), a non-fucosylated antibody, and wild-type glycoside endonuclease Endo-S2 were mixed at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL, respectively, and the reaction system pH was adjusted to 7.0. The mixture was incubated at 30°C for 1 hour. Complete conversion to the product was confirmed by LC-MS, and the mixture was then purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugate gsADC-38.

[0073] Hereinafter, specific steps for producing each disaccharide linker, glycoengineered antibody, and glycoside-specific ADC by the above-mentioned general production method will be described with reference to specific examples.

[0074] I: Preparation of disaccharide linkers Example 1: Synthesis of Compounds G1-G2 The structures and synthesis methods of compounds G1 and G2 are as follows. [ka] JPEG2022174834000122.jpg2651 Compound G1 Compound G2 JPEG2022174834000123.jpg17142

[0075] Step 1: Compound 1 (20 mg, 52.2 μmol) was dissolved in 800 μL of 50 mM PB, pH 7.0 buffer. 2-Chloro-1,3-dimethyl-1H-benzimidazolium-3-chloride (CDMBI, 56.4 mg, 261 μmol) was added to the reaction mixture, mixed uniformly, and then cooled to 0°C. Potassium phosphate (166 mg, 0.783 mmol) was added, and ddH2O was added to a total volume of 1044 μL. The reaction was allowed to proceed at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain unsalted compound G1 (dissolved in water to make a 50 mM stock). This was then aliquoted and stored at -80°C. HRMS calculated value C 14 H 23 NO10 [M+H] + 366.14, actual value 366.1322.

[0076] Step 2: Compound G1 (5 mg, 274 μL of 50 mM stock) obtained in step 1 was bubbled with O2 for 10 minutes, followed by the addition of 11.9 U of galactose oxidase (GOase), 120 U of horseradish peroxidase (HRP), and 2.38 kU of catalase. The reaction mixture was then filled to 300 μL. After 4 hours at 30 °C and 888 rpm, the mixture was purified using a P2 column. One equivalent of NaOH was added and the mixture was lyophilized to obtain compound G2 (dissolved 50 mM stock, solvent: 50 mM PB, pH 7.0). The mixture was then aliquoted and stored at -80 °C. HRMS calculated value C 14 H 21 NO 10 [M+H] + 364.1243, actual value 364.1201.

[0077] Example 2: Synthesis of Compound G3 The structure and synthesis method of compound G3 are as follows. [ka] Compound G3 JPEG2022174834000125.jpg14141

[0078] Step 1: Compound 1 (20 mg, 52.2 μmol) was dissolved in 1 mL of 50 mM PB, pH 7.0 buffer, and O2 was bubbled in for 10 minutes. After that, galactose oxidase GOase 47.6 U, horseradish peroxidase HRP 480 U, and catalase 9.52 kU were added to the reaction system, and the volume was adjusted to 1.19 mL. After 4 hours at 30 °C and 888 rpm, the mixture was purified using a P2 column and then lyophilized to obtain compound 2 (18 mg, 90.5% yield). HRMS calculated value C 14 H 23 NO 11 [M+H] +382.1349, actual value 382.1331.

[0079] Step 2: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer, and compound 12 (5.3 mg, 51.9 μmol) was added to the reaction mixture and reacted at 37°C for 2 hours. LC-MS confirmed the completion of the reaction, and compound 3 (20 mg, 91% yield) was obtained after isolation and purification using a semi-preparative C18 column. HRMS calculated value C 14 H 21 NO 10 [M+H] + 466.1785, actual value 466.1732.

[0080] Step 3: Compound 3 (20 mg, 43 μmol) was dissolved in 700 μL of 50 mM PB, pH 7.0 buffer, and CDMBI (46.44 mg, 215 μmol) was added to the reaction mixture. After uniform mixing, the mixture was cooled to 0°C, potassium phosphate (137 mg, 0.645 mmol) was added, and ddH2O was added to bring the total volume to 860 μL. The mixture was reacted at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction mixture containing compound G3. HRMS calculated value C 16 H 25 N5O 10 [M+H] + 448.1679, actual value 448.1616. 1 HNMR (600MHz, heavy water) δ7.57(d,J=4.8Hz,0.65H),6.91(d,J=4.6Hz,0.35H),6.01(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),4.3 3-4.3(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).

[0081] Example 3: Synthesis of Compound G4 The structure and synthesis method of compound G4 are as follows. [ka] Compound G4 JPEG2022174834000127.jpg20140

[0082] Step 1: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 0.2 M PB, pH 6.0 buffer solution. Compound 13 (23.6 mg, 236 μmol) and sodium cyanoborohydride (NaCNBH) (59.5 mg, 944 μmol) were added to the reaction mixture and reacted at 0°C for 4 hours. LC-MS confirmed the completion of the reaction, and after purification using a P2 column, compound 4 (16 mg, 72.8% yield) was obtained. HRMS calculated value C 17 H 31 N5O 10 [M+H] + 466.2149, actual value 466.2132.

[0083] Step 2: Compound 4 (16 mg, 34.4 μmol) was dissolved in 500 μL of 50 mM PB, pH 7.0 buffer. CDMBI (37.2 mg, 172 μmol) was added to the reaction mixture and mixed uniformly. The mixture was then cooled to 0°C. Potassium phosphate (109.6 mg, 0.516 mmol) was added, and ddH2O was added to bring the total volume to 688 μL. The mixture was then reacted at 0°C for 2 hours. A large amount of precipitate was observed, which was removed by centrifugation to obtain a supernatant containing compound G4. HRMS calculated value C 17 H 29 N5O9[M+H] + 448.2043, actual value 448.2102. 1HNMR (600MHz, heavy water) δ5.97(d,J=7.3Hz,1H),4.29(d,J=7.9Hz,1H),4.27(dd,J=3.0,1.6 Hz,1H),4.07(m,1H),3.72(d,J=3.5Hz,1H),3.69(dd,J=12.3,2.5Hz,1H),3.63-3.58( m,1H),3.56-3.49(m,2H),3.40-3.35(m,1H),3.31(m,1H),3.29(t,J=6.7Hz,3H),2.93 (q,J=7.4Hz,2H),2.68(q,J=7.3Hz,2H),1.95(d,J=1.7Hz,3H),1.67(p,J=7.1Hz,2H).

[0084] Example 4: Synthesis of Compound G5 The structure and synthesis method of compound G5 are as follows. [ka] Compound G5 JPEG2022174834000129.jpg17143

[0085] Step 1: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.0 buffer. Hydroxyamine hydrochloride (3.6 mg, 52 μmol) and 180 μL of methanol were added to the reaction mixture, mixed uniformly, and then sodium carbonate (2.6 mg, 23.6 μmol) was slowly added. After 3 hours of reaction at room temperature, nickel chloride hexahydrate (28 mg, 118 μmol) and sodium borohydride (26.8 mg, 0.7 mmol) were added to the reaction mixture. The reaction mixture was left overnight at 4°C, centrifuged, and the supernatant was removed. The precipitate was washed twice with water. The combined water washes were purified using a P2 column and lyophilized to give compound 5 (15 mg, 83% yield). HRMS calculated value C 14 H 26 N2O 10 [M+H] + 383.1665, actual value 383.1661.

[0086] Step 2: Compound 5 (15 mg, 39.25 μmol) was dissolved in 600 μL of 50 mM PB, pH 7.0 buffer. CDMBI (42.4 mg, 196.3 μmol) was added to the reaction mixture and mixed uniformly. The mixture was then cooled to 0°C. Potassium phosphate (125 mg, 0.589 mmol) was added, and ddH2O was added to bring the total volume to 785 μL. The mixture was then reacted at 0°C for 2 hours. A large amount of precipitate was observed, which was removed by centrifugation to obtain a supernatant containing compound G5. HRMS calculated value C 14 H 24 N2O9[M+H] + 365.156, actual value 365.1521.

[0087] Example 5: Synthesis of Compound G6 The structure and synthesis method of compound G6 are as follows. [ka] Compound G6 JPEG2022174834000131.jpg17144

[0088] Step 1: Compound 5 (15 mg, 39.35 μmol) was dissolved in 500 μL of CH OH / HO = 1:4 system, and 1H-imidazole-sulfonyl azide hydrochloride (12.3 mg, 58.9 μmol), potassium carbonate (16.3 mg, 117.75 μmol), and copper sulfate (6.3 mg, 39.25 μmol) were added to the reaction system in this order, and the reaction was carried out at 37 °C for 4 hours. LC-MS confirmed that the reaction was almost complete, and the product was isolated and purified using a P2 column, followed by lyophilization to obtain compound 6 (13 mg, 81.3% yield). HRMS calculated value C 14 H 24 N4O 10 [M+H] + 409.157, actual value 409.1526.

[0089] Step 2: Compound 6 (13 mg, 31.85 μmol) was dissolved in 500 μL of 50 mM PB, pH 7.0 buffer. CDMBI (34.4 mg, 159.3 μmol) was added to the reaction mixture and mixed uniformly. The mixture was then cooled to 0°C. Potassium phosphate (101.5 mg, 0.478 μmol) was added, and ddH2O was added to bring the total volume to 637 μL. The mixture was then reacted at 0°C for 2 hours. A large amount of precipitate was observed, which was removed by centrifugation to obtain a supernatant containing compound G6. HRMS calculated value C 14 H 22 N4O9[M+H] + 391.1465, actual value 391.1432.

[0090] Example 6: Synthesis of Compound G7 [ka] Compound G7 JPEG2022174834000133.jpg21143

[0091] Step 1: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 0.2 M PB, pH 6.0 buffer solution. Compound 15 (13 mg, 236 μmol) and sodium cyanoborohydride (59.5 mg, 944 μmol) were added to the reaction mixture and reacted at 0°C for 4 hours. LC-MS confirmed the completion of the reaction, and the product was isolated and purified using a P2 column to obtain compound 7 (14 mg, 70.5% yield). HRMS calculated value: C 17 H 28 N2O 10 [M+H] + 421.1822, actual value 421.1876.

[0092] Step 2: Compound 7 (16 mg, 38 μmol) was dissolved in 500 μL of 50 mM PB, pH 7.0 buffer. CDMBI (41 mg, 190 μmol) was added to the reaction mixture, mixed uniformly, and then cooled to 0°C. Potassium phosphate (121 mg, 0.57 mmol) was added, and ddH2O was added to bring the total volume to 688 μL. The reaction was carried out at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction mixture containing compound G7. HRMS calculated value C 17 H 26 N2O9[M+H] + 403.1716, measured value 403.1733, 1 HNMR (600MHz, heavy water) δ5.97(d,J=7.3Hz,1H),4.33-4.23(m,2H),4.11-4.03(m,1H),3.74(d,J=3.6Hz,1H),3.69(dd,J=12.3,2.5H z,1H),3.63-3.58(m,1H),3.56-3.48(m,2H),3.40-3.35(m,1H),2.90-2.86(m,3H),2.67-2.62(m,2H),1.95(d,J=1.8Hz,3H).

[0093] Example 7: Synthesis of Compound G8 [ka] Compound G8 JPEG2022174834000135.jpg18143

[0094] Step 1: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer, and compound 16 (5.6 mg, 51.9 μmol) was added to the reaction mixture and reacted at 37°C for 2 hours. Completion of the reaction was confirmed by LC-MS, and compound 8 (18.1 mg, 88% yield) was obtained after isolation and purification using a semi-preparative C18 column. HRMS calculated value C 17 H 26 N2O 11 [M+H] + 435.1615, actual value 435.1610.

[0095] Step 2: Compound 8 (18.1 mg, 41.6 μmol) was dissolved in 700 μL of 50 mM PB, pH 7.0 buffer. CDMBI (45 mg, 208 μmol) was added to the reaction mixture, mixed uniformly, and then cooled to 0°C. Potassium phosphate (132.5 mg, 0.624 mmol) was added, and ddH2O was added to bring the total volume to 860 μL. The reaction was carried out at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a supernatant containing compound G8. HRMS calculated value C 17 H 24 N2O 10 [M+H] + 417.1509, actual value 417.1505.

[0096] 1 HNMR (600MHz, heavy water) δ7.53(d,J=4.9Hz,0.65H),6.95(d,J=4.8Hz,0.35H),5.98(d,J=7.3Hz,1H),4.61(d,J=9.9Hz,2H),4.43-4.25(m,3H),4.15-4 .05(m,1.3H),3.95(d,J=3.4Hz,0.7H),3.76-3.67(m,1H),3.65-3.53(m ,3H),3.46(dd,J=10.1,7.8Hz,1H),3.36(m,1H),1.96(d,J=1.6Hz,3H).

[0097] Example 8: Synthesis of Compound G9 [ka] Compound G9 JPEG2022174834000137.jpg21145

[0098] Step 1: Compound 5 (15 mg, 39.3 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer, and compound 17 (23.3 mg, 117.9 μmol) was added to the reaction mixture. The reaction was allowed to proceed at room temperature for 4 hours. LC-MS confirmed the completion of the reaction, and compound 9 (14 mg, 76.7% yield) was obtained after isolation and purification using a P2 gel column. HRMS calculated value C 16 H 27 N5O 11 [M+H] + 466.1785, actual value 466.1725.

[0099] Step 2: Compound 9 (14 mg, 30.1 μmol) was dissolved in 500 μL of DO. CDMBI (32.5 mg, 150.5 μmol) was added to the reaction mixture, mixed uniformly, and then cooled to 0°C. Potassium phosphate (96 mg, 0.45 mmol) was added, and DO was added to a total volume of 602 μL. The reaction was continued at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a supernatant containing compound G9. HRM calculated value C 16 H 25 N5O 10 [M+H] + 448.1679, actual value 448.1666.

[0100] Example 9: Synthesis of Compound G10 [ka] Compound G10 JPEG2022174834000139.jpg21144

[0101] Step 1: Compound 2 (20 mg, 52.48 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer, and compound 18 (22.6 mg, 63 μmol) was added to the reaction system. After standing at room temperature for 8 hours, the reaction was confirmed to be complete by LC-MS. After isolation and purification using a semi-preparative C18 column, compound 10 (32.6 mg, 86% yield) was obtained. HRMS calculated value C 28 H 46 NO14 S[M+H] + 723.2871, actual value 723.2877.

[0102] Step 2: Compound 10 (10 mg, 13.85 μmol) was dissolved in DO, and CDMBI (15 mg, 69.3 μmol) was added to the reaction mixture. After uniform mixing, the mixture was cooled to 0°C, potassium phosphate (44 mg, 208 μmol) was added, and DO was added to a total volume of 277 μL. The mixture was reacted at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a supernatant containing compound G10. HRMS calculated value C 28 H 44 NO 13 S[M+H] + 705.2765, actual value 705.2771.

[0103] Example 10: Synthesis of Compound G11 [ka] Compound G11 JPEG2022174834000141.jpg20143

[0104] Step 1: Compound 2 (20 mg, 52.48 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer, and compound 90 Benzyl-NCS (9.4 mg, 63 μmol) was added to the reaction system. After standing at room temperature for 8 hours, the reaction was confirmed to be complete by LC-MS. After isolation and purification using a semi-preparative C18 column, compound 11 (17 mg, 62%) was obtained. HRMS calculated value C 22 H 33 N3O 10 S[M+H] + 532.1965, actual value 532.1911.

[0105] Step 2: Compound 11 (17 mg, 32.4 μmol) was dissolved in DO, and CDMBI (35 mg, 162 μmol) was added to the reaction mixture. After uniform mixing, the mixture was cooled to 0°C, potassium phosphate (103 mg, 0.486 mmol) was added, and DO was added to a total volume of 650 μL. The mixture was reacted at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a supernatant containing compound G11. HRMS calculated value C 22 H 31 N3O9S[M+H] + 514.1859, actual value 514.1880. 1 HNMR (600MHz, heavy water) δ7.32(t,J=7.6Hz,2H),7.25(dd,J=7.9,5.7Hz,3H),5.97(d,J=7.3Hz,1H),4.7(m,2H),4.39-4.15(m,2H), 4.04(m,1H),3.70(dd,J=12.3,2.5Hz,4H),3.56(dd,J=12.3,6.4Hz,3H),3.51-3.43(m,1H),3.43-3.31(m,2H),1.97(m,3H).

[0106] Example 11: Synthesis of Compound G12 [ka] Compound G12 JPEG2022174834000143.jpg19142

[0107] Step 1: Compound 1 (10 mg, 26.1 μmol) and compound 70 (16 mg, 26.1 μmol) were dissolved in 1.5 mL of 100 mM pH 8.0 Tris buffer, and 30 μg of α2,6-sialyltransferase (Pd2,6ST) was added to the reaction mixture. After confirming the completion of the reaction by thin-layer chromatography, the mixture was purified by P2 column chromatography to give compound 71 (12.6 mg, 72% yield). HRMS calculated value C 25 H 42 N2O 19 [M+H] +675.246, actual value 675.2433.

[0108] Step 2: Compound 71 (2 mg, 2.97 μmol) was dissolved in DO, and CDMBI (3.2 mg, 14.83 μmol) was added to the reaction mixture. After uniform mixing, the mixture was cooled to 0°C, potassium phosphate (9.5 mg, 44.55 μmol) was added, and DO was added to bring the total volume to 148 μL. The mixture was reacted at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a supernatant containing compound G12. HRMS calculated value C 25 H 40 N2O 18 [M+H] + 657.2354, actual value 657.2301. 1 HnMR (600MHz, heavy water) δ5.71(d,J=7.3Hz,1H),4.07-3.99(m,2H),3.83-3.77(m,1H),3.60-3.47(m,4H),3.46-3.35(m,3 H),3.35-3.18(m,7H),3.15-3.05(m,3H),2.63(m,1H),1.69(d,J=1.9Hz,3H),1.66(s,3H),1.30(t,J=12.1Hz,1H).

[0109] Example 12: Synthesis of Compound G13 [ka] Compound G13 JPEG2022174834000145.jpg29140

[0110] Step 1: Compound 5 (15 mg, 39.3 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer, and compound 80 (23.7 mg, 59 μmol) was added to the reaction mixture. The mixture was allowed to react at room temperature for 4 hours. LC-MS confirmed the completion of the reaction, and compound 81 (22 mg, 83.7% yield) was obtained after isolation and purification using a semi-preparative column. HRMS calculated value C 33 H 39 N3O 12 [M+H] +670.2612, actual value 670.2661.

[0111] Step 2: Compound 81 (10 mg, 14.9 μmol) was dissolved in 500 μL of DO. CDMBI (16.2 mg, 74.7 μmol) was added to the reaction mixture, mixed uniformly, and then cooled to 0°C. Potassium phosphate (31.6 mg, 149 μmol) was added, and DO was added to bring the total volume to 600 μL. The reaction was continued at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a supernatant containing compound G13. HRMS calculated value C 33 H 37 N3O 11 [M+H] + 652.2506, actual value 652.2501.

[0112] Example 13: Synthesis of Compound G14 [ka] Compound G14 JPEG2022174834000147.jpg99145

[0113] Step 1: Compound 82 (369 mg, 1 mmol) was dissolved in 2 mL of DMF, and HATU (1.52 g, 4 mmol), 3-azidopropylamine (500 μL, 50 mmol), and N,N-diisopropylethylamine (DIPEA) (1 mL, 5.9 mmol) were added in sequence. After uniform mixing, the mixture was reacted at room temperature for 1 hour. The reaction system was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to obtain compound 83 (yield 92%) as a white powder. HRMS calculated value C 26 H 31 NO4[M+H] + 534.2499, actual value 534.6498.

[0114] Step 2: Compound 83 (54 mg, 0.1 mMol) was dissolved in 500 μL of methanol, 250 μL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 2 hours. The mixture was then lyophilized to give Compound 84 (yield 94%). HRMS calculated value C 11 H 21 NO2[M+H] + 312.1818, actual value 312.8763.

[0115] Step 3: Compound 84 (9 mg, 0.028 mmol) was dissolved in 500 μL of DMF, and then compound 2 (16.8 mg, 0.044 mmol) and NaCNBH3 (18.4 mg, 0.29 mmol) were added in sequence. After uniform mixing, the mixture was reacted at 37 ° C for 6 hours. The reaction system was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to obtain compound 85 (yield 62%) as a white powder. HRMS calculated value C 25 H 43 N 10 O 11 [M+H] + 659.3113, actual value 659.7651.

[0116] Step 4: Compound 85 (18 mg, 0.027 mmol) was dissolved in 500 μL of 50 mM PB, pH 7.4 buffer, and DMC (65.91 mg, 0.39 mmol) and triethylamine (18 μL, 0.13 mmol) were added. The mixture was allowed to react at 0°C for 2 hours. LC-MS confirmed the completion of the reaction. The mixture was then purified using a semi-preparative column and lyophilized to give compound G14 (79% yield). HRMS calculated value C 25 H 42 N 10 O 10 [M+H] + 643.3119, measured value 643.3786.

[0117] II: Preparation of small molecule drug-linkers Example 14: Synthesis of Compound D1 The structure and synthesis method of compound D1 are as follows. [ka] NH2O-VC-PAB-MMAE (compound D1) JPEG2022174834000149.jpg62141

[0118] Step 1: Compound 20 (5.6 mg, 17.8 μmol) was dissolved in 100 μL of DMF, and HATU (13.5 mg, 35.6 μmol), compound 19 (NH2-VC-PAB-MMAE, 20 mg, 17.8 μmol), and N,N-diisopropylethylamine DIPEA (9.3 μL, 53.4 μmol) were added to the above system, and the reaction was carried out at 37 °C for 2 hours. The reaction system was monitored by LC-MS, and the completion of the reaction was confirmed. After isolation and purification using a semi-preparative C18 column, the mixture was lyophilized to obtain compound 21 (20 mg, 87% yield). HRMS calculated value C 75 H 107 N 11 O 16 [M+H] + 1418.7975, [M+2H] 2+ 709.9026, actual measurements 1418.7913, 709.9021.

[0119] Step 2: Compound 21 (20 mg, 14.1 μmol) was dissolved in 100 μL of DMF, and 100 μL of triethylamine was added to the above system. After uniform mixing, the reaction was allowed to proceed at room temperature for 1 hour. The reaction system was monitored by LC-MS. After completion of the reaction was confirmed, the product was purified using a semi-preparative C18 column and then lyophilized to obtain compound D1 (16.5 mg, 89% yield). HRMS calculated value C 60 H 97 N 11 O 14 [M+H] + 1196.7294, [M+2H] 2+ 598.8685, actual measurements 1196.7263, 598.8622.

[0120] Example 15: Synthesis of Compound D2 The structure and synthesis method of compound D2 are as follows. [ka] Compound D2 JPEG2022174834000151.jpg56141

[0121] Step 1: Compound 23 (352 mg, 2.2 mmol) was dissolved in 10 mL of tetrahydrofuran. NaH (60% in oil, 88 mg, 2.2 mmol) was added to the above system at 0 °C. The reaction mixture was stirred at 0 °C for 15 minutes, and then compound 22 (332 mg, 2 mmol) was slowly added. The mixture was stirred at room temperature for 1 hour. The reaction was quenched with methanol, concentrated, and purified on a silica gel column (petroleum ether:ethyl acetate = 4:2) to give compound 24 (199 mg, 65%).

[0122] Step 2: Compound 24 (153 mg, 0.5 mmol) was dissolved in 10 mL of methanol, and iron (300 mg) and concentrated hydrochloric acid (0.5 mL) were added to the above system. The reaction system was diluted with 10 mL of water and vigorously stirred at 80 °C for 1 hour. The reaction system was filtered, neutralized with sodium bicarbonate, filtered, and concentrated. The mixture was purified by silica gel column (petroleum ether: ethyl acetate 2:1) to give compound 25 (55 mg, 40%).

[0123] Step 3: Compound 25 (55 mg, 0.2 mmol) was dissolved in 5 mL of methanol / water (1:1). LiOH (10 mg, 0.42 mmol) was added to the reaction mixture and stirred at room temperature for 4 hours. The mixture was concentrated and purified on a silica gel column (petroleum ether:ethyl acetate (1:1)) to give compound 26 (44.6 mg, 90%).

[0124] Step 4: Compound 26 (5.6 mg, 0.0225 mmol) was dissolved in 100 μL of DMF, and HATU (8.5 mg, 0.0225 mmol), compound 19 (8.5 mg, 0.0225 mmol), and DIPEA (7.83 μL, 0.045 mmol) were added to the reaction mixture. The reaction was allowed to proceed at 37°C for 1 hour. The reaction was monitored by LC-MS. After completion of the reaction, the product was purified using a semi-preparative C18 column and lyophilized to give compound 27 (24 mg, 80%). HRMS calculated value C 71 H 108 N 12 O 14 [M+H] + 1353.8186, [M+2H] 2+ 677.413, actual measurements 1353.8172, 677.4121.

[0125] Step 5: Compound 27 (13.2 mg, 0.01 mmol) was dissolved in 1 mL of methanol, and 5 equivalents of hydroxylamine hydrochloride and sodium bicarbonate (1:1, dissolved in 0.5 mL of water) were added to the reaction mixture, followed by stirring at 65°C for 24 hours. LC-MS showed the reaction was complete. Compound D2 (13 mg, 94%) was isolated and purified using a semi-preparative C18 column and lyophilized to give HRMS calculated value C. 71 H 111 N 13 O 15 [M+H] + 1386.8401, [M+2H] 2+ 693.9235, Actual value 1386.8395, 693.9264.

[0126] Example 16: Synthesis of Compound D3 The structure and synthesis method of compound D3 are as follows. [ka] ThioPz-Lys(PEG 24 )-VC-PAB-MMAE (compound D3) JPEG2022174834000153.jpg140157

[0127] Step 1: Compound 28 (574 mg, 1.65 mmol), compound 29 (237.4 mg, 1.65 mmol), and DMAP (201.4 mg, 1.65 mmol) were dissolved in 10 mL of anhydrous dichloromethane and the reaction mixture was placed on ice and cooled to 0 °C. DCC (337.4 mg, 1.638 mmol) was added to the above solution and stirred at 0 °C for 30 min, then allowed to warm to room temperature and stirred for 6 h. The mixture was diluted with dichloromethane, filtered, washed with 1N HCl and saturated brine, and the organic layer was dried over MgSO and evaporated. The resulting oil was redissolved in 30 mL of anhydrous ethanol and refluxed for 4 h. Purification using a silica gel column (hexane:ethyl ether = 10:1) gave compound 30 (560 mg, 81% yield).

[0128] Step 2: Compound 30 (499 mg, 1.24 mmol) and ethyl hydrazine chloride (191 mg, 1.23 mmol) were dissolved in 10 mL of ethanol, and triethylamine (17.3 μL, 0.124 mmol) was added. The mixture was reacted at 50° C. for 2 hours. The reaction mixture was concentrated and purified using a silica gel column (hexane:ethyl ether=1:1) to give compound 31 (402 mg, 71% yield).

[0129] Step 3: Compound 31 (236 mg, 0.5 mmol) was dissolved in 10 mL of THF:MeOH:Water (2:3:1). LiOH (25 mg, 1.04 mmol) was added to the above system and stirred for 4 hours. 40 mL of water and 40 mL of ethyl ether were added to the above system, and the aqueous layer was adjusted to pH 2 and washed with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate, concentrated, and purified using a silica gel column (dichloromethane:methanol (4:1)) to obtain compound 32 (160 mg, 72% yield).

[0130] Step 4: Compound 32 (50 mg, 112.6 μmol) was dissolved in 200 μL of DMF, and DCC (34.7 mg, 168.7 μmol) and NHS (19.4 mg, 168.7 μmol) were added to the above system, followed by reaction at 37° C. for 4 hours. The reaction system was monitored by LC-MS, and after completion of the reaction was confirmed, the product was isolated and purified using a semi-preparative C18 column, followed by lyophilization to give compound 33 (55 mg, 90.3% yield).

[0131] Step 5: Compound 35 (CH3O-PEG 24 A solution of 50 mg of -COOH (43 μmol) in 2 mL of anhydrous dichloromethane was added to a 25 mL round-bottom flask, and the solution was refluxed at 50°C for approximately 2 hours under nitrogen. After analysis by thin-layer chromatography using a 1:8 ratio of methanol to dichloromethane showed that the reaction was complete, the reaction mixture was evaporated and vacuumed with an oil pump for 30 minutes to remove thionyl chloride from the reaction mixture.

[0132] Step 6: Compound 34 (Fmoc-Lys-OH, 16 mg, 43 μmol) and NaHCO (18 mg, 215 μmol) were dissolved in 900 μL tetrahydrofuran and 300 μL pure water in a round-bottom flask and clarified.

[0133] Step 7: The system obtained by evaporation in Step 1 was dissolved in 400 μL of anhydrous tetrahydrofuran and slowly added dropwise to the reaction system from Step 2 while stirring in an ice bath, and the reaction was allowed to proceed at room temperature for 30 minutes. The reaction system was monitored by LC-MS, and after the completion of the reaction was confirmed, the product was isolated and purified by semi-preparative separation, and then lyophilized to obtain Compound 36 (42 mg, yield 63.6%). HRMS calculated value C 73 H 126 N2O 30 [M+H] + 1511.8473, [M+2H] 2+ 756.4275, actual values ​​1511.8401, 756.4233.

[0134] Step 8: Compound 36 (10 mg, 6.62 μmol) and HATU (5 mg, 13.24 μmol) were dissolved in 100 L of anhydrous DMF. Compound 19 (NH2-VC-PAB-MMAE, 8.2 mg, 7.28 μmol) was added to the reaction mixture, and DIPEA (3.44 μL, 20 μmol) was added dropwise with stirring. The reaction was allowed to proceed at room temperature for 1 hour. The reaction was monitored by LC-MS. After completion of the reaction was confirmed, the product was purified by semi-preparative separation and lyophilized to give compound 37 (15 mg, 87% yield). HRMS calculated value C 131 H 218 N 12 O 41 [M+2H] 2+ 1308.7745, [M+3H] 3+ 872.852, actual measurements 1308.7761, 872.8542.

[0135] Step 9: Compound 37 (15 mg, 3.8 μmol) was dissolved in 160 μL of DMF, and 40 μL of piperidine was added to the reaction mixture. The mixture was stirred at room temperature for 20 minutes. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was purified by semi-preparative separation and lyophilized to give compound 38 (12.8 mg, 92% yield). HRMS calculated value C 116 H 208 N 12 O 39 [M+2H] 2+ 1197.7405, [M+3H] 3+ 798.8293, actual measurements 1197.7375, 798.8234.

[0136] Step 10: Compound 33 (2.9 mg, 5.37 μmol) was dissolved in 100 μL of DMF, and compound 38 (12.8 mg, 5.37 μmol) and triethylamine (1.5 μL, 10.74 μmol) were added to the reaction mixture. The mixture was then reacted at 37°C for 2 hours. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound 39 (12 mg, 80% yield). HRMS calculated value C 142 H 230 N 14 O 41S[M+2H] 2+ 1410.8105, [M+3H] 3+ 940.876, actual values ​​1410.8123, 940.8771.

[0137] Step 11: Compound 39 (39 mg, 4.28 μmol) was dissolved in 130 μL of dichloromethane, and the reaction mixture was cooled to 0°C. 10 μL of water, 10 μL of triisopropylsilane, and 80 μL of trifluoroacetic acid were added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified by a semi-preparative C18 column and lyophilized to give compound D3 (6 mg, 54.7% yield). HRMS calculated value C 123 H 216 N 14 O 41 S[M+2H] 2+ 1289.756, [M+3H] 3+ 860.173, actual measurements 1289.7552, 860.1741.

[0138] Example 17: Synthesis of Compound D4 The structure and synthesis method of compound D4 are as follows. [ka] ThioPz- VC-PAB-MMAE (Compound D4) JPEG2022174834000155.jpg57163

[0139] Step 1: Compound 33 (11.6 mg, 21.4 μmol) was dissolved in 100 μL of DMF, and compound 19 (20 mg, 17.8 μmol) and triethylamine (3 μL, 21.4 μmol) were added to the reaction mixture, followed by reaction at 37°C for 2 hours. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound 40 (25 mg, 90% yield). HRMS calculated value C 84 H 116 N 12 O14 S[M+2H] 2+ 775.4305, actual value 775.4331.

[0140] Step 2: Compound 40 (20 mg, 12.9 μmol) was dissolved in 130 μL of dichloromethane, and the reaction mixture was cooled to 0°C. 10 μL of water, 10 μL of triisopropylsilane, and 80 μL of trifluoroacetic acid were added to the reaction mixture and stirred at room temperature for 30 minutes. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified by a semi-preparative C18 column and lyophilized to give compound D4 (12.8 mg, 76% yield). HRMS calculated value C 65 H 102 N 12 O 14 S[M+H] + 1307.7437, actual value 1307.7437.

[0141] Example 18: Synthesis of Compound D5 The structure and synthesis method of compound D5 are as follows. [ka] BCN-Lys(PEG 24 )-VC-PAB-MMAE (compound D5) JPEG2022174834000157.jpg46139

[0142] Compound 38 (12.8 mg, 5.38 μmol) was dissolved in 200 μL of DMF, and compound 41 (BCN-O-PNP, 3.38 mg, 10.76 μmol) and triethylamine (1.5 μL, 10.76 μmol) were added. The mixture was then left to stand at 37°C for 3 hours. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound D5 (10 mg, 72.7% yield). HRMS calculated value C 127 H 220 N 12 O 41 [M+2H] 2+1285.7825, [M+3H] 3+ 857.524, actual measurements 1285.7848, 857.5232.

[0143] Example 19: Synthesis of Compound D6 The structure and synthesis method of compound D6 are as follows. [ka] DBCO-Lys(PEG 24 )-MMAE (compound D6) JPEG2022174834000159.jpg80140

[0144] Step 1: Compound 36 (21 mg, 13.93 μmol) and HATU (10.6 mg, 27.86 μmol) were dissolved in 100 L of anhydrous DMF. Compound 42 (MMAE, 10 mg, 13.93 μmol) and DIPEA (7.26 mL, 41.79 μmol) were added to the reaction mixture and allowed to react at room temperature for 1 hour. The reaction mixture was monitored by LC-MS. After completion of the reaction was confirmed, the product was purified by semi-preparative separation and lyophilized to give compound 43 (26 mg, 84.7% yield). HRMS calculated value C 112 H 191 N7O 36 [M+2H] 2+ 1106.174, [M+3H] 3+ 737.785, actual values ​​1106.1722, 737.7832.

[0145] Step 2: Compound 43 (26 mg, 11.76 μmol) was dissolved in 160 μL of DMF, and 40 μL of piperidine was added to the reaction mixture. The mixture was stirred at room temperature for 20 minutes. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was purified by semi-preparative separation and lyophilized to give compound 44 (21 mg, 90% yield). HRMS calculated value C 97 H 181 N7O 34 [M+2H] 2+ 995.14, actual value 995.1442.

[0146] Step 3: Compound 45 (DBCO-COOH, 3.5 mg, 10.56 μmol) and HATU (8 mg, 21.12 μmol) were dissolved in 100 μL of anhydrous DMF. Compound 44 (21 mg, 10.56 μmol) and DIPEA (5.5 μL, 31.68 μmol) were added to the reaction mixture and allowed to react at room temperature for 1 hour. The reaction was monitored by LC-MS. After completion of the reaction, the product was purified by semi-preparative separation and lyophilized to give compound D6 (20.2 mg, 84% yield). HRMS calculated value C 118 H 198 N8O 36 [M+2H] 2+ 1152.7033, [M+3H] 3+ 768.8048, actual measurements 1152.7022, 768.8031.

[0147] Example 20: Synthesis of Compound D7 The structure and synthesis method of compound D7 are as follows: [ka] DBCO-Lys(PEG 24 )-VC-PAB-MMAE (compound D7) JPEG2022174834000161.jpg50136

[0148] Compound 45 (DBCO-COOH, 1.8 mg, 5.38 μmol) and HATU (4.1 mg, 10.76 μmol) were dissolved in 100 μL of anhydrous DMF. Compound 38 (12.8 mg, 5.38 μmol) and DIPEA (2.8 μL, 16.14 μmol) were added to the reaction mixture and allowed to react at room temperature for 1 hour. The reaction mixture was monitored by LC-MS. After completion of the reaction, the product was purified by semi-preparative separation and lyophilized to give compound D7 (12 mg, 84%). HRMS calculated value C 137 H 225 N 13 O 41 [M+2H] 2+ 1355.3039, [M+3H]3+ 903.8718, actual values ​​1355.302, 903.8707.

[0149] Example 21: Synthesis of Compound D8 The structure and synthesis method of compound D8 are as follows. [ka] DBCO-Gly(maltotetraose)-VC-PAB-MMAE (Compound D8) [ka]

[0150] Step 1: Compound 49 (maltotetraose, 20 mg, 30 μmol) was dissolved in 600 μL of ddH2O, and sodium azide (96 mg, 1.5 mmol) and CDMBI (32.4 mg, 150 μmol) were added to the reaction mixture. The reaction mixture was cooled to 0°C on ice, and potassium phosphate (96 mg, 450 μmol) was added. The reaction mixture was then allowed to react at 0°C for 4 hours to produce compound 50. The reaction mixture was aliquoted and stored at -80°C without further processing.

[0151] Step 2: Compound 46 (6 mg, 17.9 μmol) was dissolved in 100 μL of DMF, and HATU (13.6 mg, 35.8 μmol), compound 19 (NH2-VC-PAB-MMAE, 20 mg, 17.9 μmol), and DIPEA (0.88 mL, 53.7 μmol) were added to the reaction mixture, and the mixture was allowed to react at 37 °C for 2 hours. The reaction mixture was monitored by LC-MS, and after completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound 47 (21 mg, 82% yield). HRMS calculated value C 78 H 109 N 11 O 15 [M+2H] 2+ 720.913, actual value 720.9121.

[0152] Step 3: Compound 47 (21 mg, 14.58 μmol) was dissolved in 80 μL of DMF, and 20 μL of piperidine was added to the reaction mixture. The mixture was allowed to react at room temperature for 20 minutes. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was purified by a C18 semi-preparative column to give compound 48 (16 mg, 90% yield). HRMS calculated value C 63 H 99 N 11 O 13 [M+H] + 1218.7502, [M+2H] 2+ 609.879, actual values ​​1218.7552, 609.8776.

[0153] Step 4: Preparation of Cu(I)-BTTAA solution: 32.5 μL of 60 mM CuSO4, 39 μL of 300 mM BTTAA, and 286 μL of 0.9 M sodium ascorbate were mixed uniformly in sequence and then stored.

[0154] Step 5: Compound 48 (16 mg, 13.1 μmol) was added to the reaction mixture in Step 1 (calculated based on a 100% yield, compound 50 (17.9 mg, 25.9 μmol)) and mixed uniformly. After that, the entire volume of Cu(I)-BTTAA solution in Step 4 was added and reacted at 37°C for 4 hours. The reaction mixture was monitored by LC-MS. After the reaction was complete, the product was purified using a semi-preparative C18 column and lyophilized to obtain compound 51 (20 mg, 80% yield). HRMS calculated value C 87 H 140 N 14 O 33 [M+2H] 2+ 955.493, actual value 955.4887.

[0155] Step 6: Compound 45 (DBCO-COOH, 3.5 mg, 10.47 μmol) was dissolved in 100 μL of DMF, and HATU (8 mg, 20.94 μmol), compound 51 (20 mg, 10.47 μmol), and DIPEA (5.46 mL, 31.41 μmol) were added to the reaction mixture, and the mixture was allowed to react at 37°C for 2 hours. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound D8 (21 mg, 91% yield). HRMS calculated value C 108 H 157 N 15 O 35 [M+2H] 2+ 1113.0562, [M+3H] 3+ 742.3734, Actual value 1113.0505, 742.3704.

[0156] Example 22: Synthesis of Compound D9 The structure and synthesis method of compound D9 are as follows. [ka] DBCO-SMCC-DM1 (Compound D9) JPEG2022174834000165.jpg43140

[0157] Step 1: Compound 52 (SH-DM1, 20 mg, 27.13 μmol) was dissolved in 100 μL of DMF, and compound 53 (SMCC, 9 mg, 27.13 μmol) was added to the reaction mixture and reacted at 37°C for 2 hours. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound 54 (24 mg, 82.7% yield). HRMS calculated value C 51 H 66 ClNO 16 S[M+H] + 1072.3992, actual value 1072.3966.

[0158] Step 2: Compound 54 (SMCC-DM1, 24 mg, 22.4 μmol) was dissolved in 100 μL of DMF, and compound 55 (DBCO-NH2, 6.2 mg, 22.4 μmol) and triethylamine (6.3 μL, 44.8 μmol) were added to the reaction mixture and allowed to react at 37°C for 1 hour. The reaction mixture was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound D9 (20 mg, 72.6% yield). HRMS calculated value C 65 H 77 ClNO 14 S[M+H] + 1233.4985, [M+2H] 2+ 617.253, actual measurements 1233.4923, 617.2555.

[0159] Example 23: Synthesis of Compound D10 The structure and synthesis method of compound D10 are as follows. [ka] Straight-chain alkynyl group -Lys(PEG 24 )-VC-PAB-MMAE (compound D10) JPEG2022174834000167.jpg48140

[0160] Compound 56 (0.6 mg, 6.12 μmol) was dissolved in 100 μL of DMF, and HATU (4.1 mg, 10.7 μmol) was added to the reaction mixture. After uniform mixing, compound 38 (12.8 mg, 5.35 μmol) and DIPEA (2.8 μL, 16.05 μmol) were added sequentially and reacted at 37 ° C for 2 hours. The reaction was monitored by LC-MS, and after completion of the reaction was confirmed, the product was isolated and purified using a semi-preparative C18 column, and then lyophilized to obtain compound D10 (10 mg, 75.6% yield). HRMS calculated value C 121 H 212 N 12 O 40 [M+2H] 2+ 1237.7535, [M+3H] 3+825.505, actual measurements 1237.7532, 825.5060.

[0161] Example 24: Synthesis of Compound D11 The structure and synthesis method of compound D11 are as follows. [ka] BCN-PEG2-CH2- VC-PAB-MMAE (Compound D11) JPEG2022174834000169.jpg53141

[0162] Step 1: Compound 86 (8.24 mg, 0.0356 mMol) was dissolved in 82.4 μL of DMF. HATU (13.6 mg, 0.0356 mmol), compound 19 (20 mg, 0.0178 mmol), and DIPEA (9.34 μL, 0.0536 mmol) were added to the reaction mixture, and the mixture was allowed to react at 37°C for 2 hours. The reaction mixture was monitored by LC-MS. After the reaction was shown to be nearly complete, 107 μL of triethylamine was added, the mixture was mixed uniformly, and the mixture was allowed to react at room temperature for 15 minutes. The product was separated and purified using a semi-preparative C18 column. The title product was collected and lyophilized to give compound 87 (20.8 mg, 92% yield). HRMS calculated value C 64 H 105 N 11 O 15 [M+H] + 1268.787, measurement 1268.7815.

[0163] Step 2: Compound 87 (20.8 mg, 0.0164 mmol) was dissolved in 208 μL of DMF, and compound 41 (10.35 mg, 0.0328 mmol) and triethylamine (9.13 μL, 0.0657 mmol) were added. The reaction was allowed to proceed at 37°C for 2 hours. The reaction was monitored by LC-MS. After the reaction was shown to be nearly complete, the product was separated and purified using a semi-preparative C18 column. The title product was collected and lyophilized to give compound D11 (14.7 mg, 62% yield). HRMS calculated value C 75 H 117 N11 O 17 [M+H] + 1444.8707, measured value 1444.8662.

[0164] Example 25: Synthesis of Compound D12 The structure and synthesis method of compound D12 are as follows. [ka] BCN-PEG2-CH2-CH2-VC-PAB-MMAE (Compound D12) JPEG2022174834000171.jpg53145

[0165] Step 1: Compound 88 (10.68 mg, 0.0267 mmol) was dissolved in 106.8 μL of DMF. HATU (20.34 mg, 0.0534 mmol), compound 19 (30 mg, 0.0267 mmol), and DIPEA (9.34 μL, 0.0802 mmol) were added to the reaction mixture, and the mixture was allowed to react at 37°C for 2 hours. The reaction mixture was monitored by LC-MS. After the reaction was shown to be nearly complete, 156 μL of triethylamine was added, the mixture was mixed uniformly, and the mixture was allowed to react at room temperature for 15 minutes. The product was separated and purified using a semi-preparative C18 column. The title product was collected and lyophilized to give compound 89 (31.1 mg, 90.7% yield). HRMS calculated value C 65 H 107 N 11 O 15 [M+H] + 1282.8026, measurement 1282.8041.

[0166] Step 2: The above product, compound 89 (31.1 mg, 0.0234 mmol), was dissolved in 208 μL of DMF, and compound 41 (10.35 mg, 0.0351 mmol) and triethylamine (9.13 μL, 0.0936 mmol) were added. The reaction was allowed to proceed at 37°C for 2 hours. The reaction was monitored by LC-MS. After the reaction was shown to be nearly complete, the product was separated and purified using a semi-preparative C18 column. The title product was collected and lyophilized to give compound D12 (28.5 mg, 83.5% yield). HRMS calculated value C 76 H 119 N 11 O 17 [M+H]+1458.8864, measured value 1458.8792.

[0167] Example 26: Synthesis of Compound D13 The structure and synthesis method of compound D12 are as follows. [ka] DBCO-PEG4-mMAE (Compound D13) JPEG2022174834000173.jpg23144

[0168] Compound DBCO-PEG4-COOH (9.8 mg, 17.8 μmol) was dissolved in 100 μL of DMF, and HATU (13.5 mg, 35.6 μmol), MMAE (12.8 mg, 17.8 μmol), and DIPEA (18.6 μL, 106.8 μmol) were added to the above system, and the reaction was carried out at 37 °C for 2 hours. The reaction system was monitored by LC-MS, and after the reaction was completed, the product was separated and purified using a semi-preparative C18 column. The title compound was collected and lyophilized to obtain compound D13 (15.8 mg, 71% yield). HRMS calculated value C 69 H 101 N7O 14 [M+H] + 1252.7485, actual value 1252.7479.

[0169] III: Synthesis of DG5 and dDG-1 from disaccharide-small molecule drug conjugate DG-1 Example 27: Synthesis of Compound DG-1 The structure and synthesis method of compound DG-1 are as follows. [ka] MMAE-PAB-VC-ON=CH-LacNAc-ox (compound DG-1) JPEG2022174834000175.jpg49141

[0170] Step 1: Compound D1 (10 mg, 8.5 μmol) was dissolved in 100 μL of DMF, and compound 2 (3.3 mg, 8.5 μmol) was dissolved in 100 μL of 0.2 M PB buffer, pH 7.5. The two systems were mixed and reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS. After completion of the reaction, the mixture was purified using a semi-preparative C18 column and lyophilized to obtain compound 57 (10 mg, 74% yield). HRMS calculated value C 74 H 118 N 12 O 24 [M+2H] 2+ 780.4265, actual value 780.4221.

[0171] Step 2: Compound 57 (10 mg, 6.4 μmol) was dissolved in 100 mL of DMF / 50 mM, pH 7.5, PB = 1:1. CDMBI (6.9 mg, 32 μmol) was added to the above system, mixed uniformly, and then cooled to 0 °C on ice. Potassium phosphate (20.4 mg, 96 μmol) was added and the reaction was continued at 0 °C for 12 hours. LC-MS confirmed that the reaction essentially produced a cyclized product. After isolation and purification using a basic C18 semi-preparative column, compound DG-1 (7.2 mg, 72% yield) was obtained. HRMS calculated value C 74 H 116 N 12 O 23 [M+2H] 2+ 771.4215, actual value 771.4221.

[0172] Example 28: Synthesis of Compound DG-2 The structure and synthesis method of compound DG-2 are as follows. [ka] Compound DG-2 JPEG2022174834000177.jpg72140

[0173] Step 1: Compound 38 (20 mg, 8.36 μmol) was dissolved in 100 μL of DMF, and compound 58 (13.6 mg, 41.8 μmol) and triethylamine (3.5 L, 25.1 μmol) were added to the above system, followed by reaction at 37°C for 2 hours. The reaction system was monitored by LC-MS, and after completion of the reaction was confirmed, the product was isolated and purified using a semi-preparative C18 column, followed by lyophilization to give compound 59 (18 mg, 83% yield). HRMS calculated value C 125 H 217 N 13 O 44 [M+2H] 2+ 1303.2645, [M+3H] 3+ 869.1792, actual measurements 1303.2667, 869.1799.

[0174] Step 2: Compound 59 (18 mg, 6.9 μmol) was dissolved in 100 L of DMF. Compound 5 (2.64 mg, 6.9 μmol) and triethylamine (2.9 μmol, 20.8 μmol) were added to the reaction mixture, and the mixture was allowed to react at room temperature for 3 hours. Compound 5 (2.64 mg, 6.9 μmol) was added, and the mixture was allowed to react at room temperature for another 3 hours. The reaction was monitored by LC-MS, and after completion of the reaction was confirmed, the mixture was purified using a semi-preparative C18 column and lyophilized to give compound 60 (14.2 mg, 71% yield). HRMS calculated value C 135 H 238 N 14 O 51 [M+3H] 3+ 958.2231, [M+4H] 4+ 718.9193, actual measurements 958.2233, 718.9192.

[0175] Step 3: Compound 60 (14.2 mg, 4.95 μmol) was dissolved in 100 μL of 50 mM PB (pH 7.5). CDMBI (5.4 mg, 24.8 μmol) was added to the reaction mixture, mixed uniformly, and then placed on ice. After cooling to 0°C, potassium phosphate (15.8 mg, 74.25 μmol) was added and the mixture was allowed to react at 0°C for 12 hours. The reaction was monitored for near completion by LC-MS. The product was isolated and purified using a basic C18 semi-preparative column, then lyophilized to obtain compound DG-2 (10 mg, 70.5% yield). HRMS calculated value C 135 H 236 N 14 O 50 [M+3H] 3+ 952.2196, actual value 952.2112.

[0176] Example 29: Synthesis of Compound DG-3 The structure and synthesis method of compound DG-3 are as follows. [ka] Compound DG-3 JPEG2022174834000179.jpg55120

[0177] Step 1: Compound 38 (20 mg, 8.36 μmol) was dissolved in 100 μL of DMF / 0.2M pH 6.0 PB = 1:1. Compound 2 (9.6 mg, 25.1 μmol) was added to the reaction mixture. The reaction mixture was adjusted to pH 6.0 with NaOH / HCl, and then sodium cyanoborohydride (5.3 mg, 83.6 μmol) was added and the reaction was carried out at 37 °C for 3 hours. The reaction was monitored for near completion by LC-MS. The product was isolated and purified using a semi-preparative C18 column, followed by lyophilization to give compound 61 (18 mg, 78.3% yield). HRMS calculated value C 130 H 231 N 13 O 49 [M+3H] 3+ 920.5406, actual value 920.5353.

[0178] Step 2: Compound 61 (18 mg, 6.53 μmol) was dissolved in 100 L of 50 mM PB (pH 7.5) at a 1:1 ratio. CDMBI (7 mg, 32.63 μmol) was added to the reaction mixture, mixed uniformly, and then placed on ice. The mixture was cooled to 0°C, and potassium phosphate (20.8 mg, 98 μmol) was added. The mixture was allowed to react at 0°C for 12 hours. LC-MS showed that the reaction was nearly complete. The mixture was purified using a basic C18 column and then lyophilized to obtain compound DG-3 (11 mg, 61.5% yield). HRMS calculated value C 130 H 229 N 13 O 48 [M+3H] 3+ 914.537, actual value 914.5310.

[0179] Example 30: Synthesis of Compound DG-4 The structure and synthesis method of compound DG-4 are as follows. [ka] Compound DG-4 JPEG2022174834000181.jpg51140

[0180] Step 1: Compound 54 (10 mg, 9.3 μmol) was dissolved in 100 μL of DMF, and compound 5 (3.6 mg, 9.3 μmol) and triethylamine (3.8 μL, 27.9 μmol) were added to the above system, followed by a reaction at 37°C for 2 hours. Compound 5 (3.6 mg, 9.3 μmol) was then added, followed by a reaction at 37°C for 2 hours. The reaction was monitored for near completion by LC-MS, and the product was isolated and purified using a semi-preparative C18 column, followed by lyophilization to give compound 62 (10 mg, 80%). HRMS calculated value C 61 H 87 ClNO 23 S[M+2H] 2+ 670.2694, actual value 670.2669.

[0181] Step 2: Compound 62 (10 mg, 7.47 μmol) was dissolved in 100 μL of DMF / 50 mM PB, pH 7.5. CDMBI (8 mg, 37.35 μmol) was added to the reaction mixture and mixed uniformly. The reaction mixture was then placed on ice and cooled to 0°C. Potassium phosphate (23.8 mg, 112 μmol) was added and the mixture was allowed to react at 0°C for 12 hours. The reaction was monitored for near completion by LC-MS. The product was isolated and purified using a basic C18 column, then lyophilized to give compound DG-4 (7 mg, 71% yield). HRMS calculated value C 61 H 85 ClNO 22 S[M+2H] 2+ 661.2641, actual value 661.2660.

[0182] Example 31: Synthesis of Compound DG-5 The structure and synthesis method of compound DG-5 are as follows. [ka] Compound DG-5 JPEG2022174834000183.jpg70141

[0183] Step 1: Compound 52 (20 mg, 27.12 μmol) was dissolved in 200 μL of DMF, and compound 63 (6.5 mg, 27.12 μmol) and 100 μL of 0.2 M NaHPO were added to the above system and reacted at room temperature for 2 hours. After monitoring the reaction for near completion by LC-MS, 200 μL of 1% NaOH was added to the above system, and the reaction changed from pale yellow to pale red. After 1 hour, the reaction was monitored for completion, and the compound was isolated and purified using a semi-preparative C18 column, followed by lyophilization to obtain compound 64 (19 mg, 78%). HRMS calculated value C 41 H 58 ClNO 13 S[M+H] + 896.3518, actual value 896.3533.

[0184] Step 2: Compound 64 (19 mg, 21.2 μmol) was dissolved in 400 L of DMF / 0.2 M pH 6.0 PB = 1:1. Compound 2 (32.3 mg, 84.8 μmol) was added to the reaction mixture. The reaction mixture was adjusted to pH 6.0 with NaOH / HCl, and then sodium cyanoborohydride (10.7 mg, 169.6 μmol) was added and the reaction was carried out at 37 °C for 3 hours. The reaction was monitored for near completion by LC-MS. The product was isolated and purified using a semi-preparative C18 column, followed by lyophilization to give compound 65 (20 mg, 75% yield). HRMS calculated value C 55 H 81 ClNO 23 S[M+2H] 2+ 631.2459, actual value 631.2424.

[0185] Step 3: Compound 65 (20 mg, 15.86 μmol) was dissolved in 500 μL of 50 mM PB (pH 7.5). CDMBI (17.2 mg, 79.3 μmol) was added to the reaction mixture and mixed uniformly. The reaction mixture was then placed on ice and cooled to 0°C. Potassium phosphate (50.5 mg, 237.9 μmol) was added and the mixture was allowed to react at 0°C for 12 hours. The reaction was monitored for near completion by LC-MS. The product was isolated and purified using a basic C18 column, then lyophilized to give compound DG-5 (12.6 mg, 64% yield). HRMS calculated value C 55 H 79 ClNO 22 S[M+2H] 2+ 622.2406, actual value 622.2409.

[0186] Example 32: Synthesis of Compound DG-6 [ka] Compound DG-6 JPEG2022174834000185.jpg59140

[0187] Step 1: Compound 19 (10 mg, 8.9 μmol) was dissolved in 100 μL of DMF, and compound 2 (13.8 mg, 35.6 μmol) was dissolved in 100 μL of 0.2 M PB buffer (pH 6.0). After confirming that the pH of the reaction system was 6.0, NaCNBH (5.3 mg, 89 μmol) was added, the reaction system was mixed uniformly, and the reaction was allowed to proceed at 37 °C for 2 hours. LC-MS monitoring of the reaction system showed that the majority of the product had been produced. The product was then isolated and purified using a semi-preparative C18 column, followed by lyophilization to obtain compound 70 (10 mg, 75.4% yield). HRMS calculated value C 72 H 117 N 11 O 22 [M+2H] 2+ 744.9187, actual value 744.9110.

[0188] Step 2: Compound 70 (10 mg, 6.7 μmol) was dissolved in 100 μL of DMF / 50 mM, pH 7.5, PB = 1:1. CDMBI (7.2 mg, 33.5 μmol) was added to the above system, mixed uniformly, and then cooled to 0 °C on ice. Potassium phosphate (21.4 mg, 100.5 μmol) was added and the reaction was carried out at 0 °C for 12 hours. LC-MS confirmed that the reaction essentially produced the cyclized product, which was isolated and purified using a basic C18 semi-preparative column to give compound DG-6 (7.2 mg, 73.4% yield). HRMS calculated value C 72 H 115 N 11 O 21 [M+2H] 2+ 735.9134, actual value 735.9133.

[0189] Example 33: Synthesis of Compound DG-7 [ka] Compound DG-7 JPEG2022174834000187.jpg84140

[0190] Step 1: Compound 71 (Fmoc-VA-PAB-OH, 20 mg, 38.8 μmol) was dissolved in 400 μL of DMF, and compound (PNP)2O (23.6 mg, 77.6 μmol) was dissolved in the above mixture. 3.2 L of DIPEA was added, and after homogeneous mixing, the mixture was allowed to react at room temperature overnight. The reaction system was monitored by LC-MS, and after it was confirmed that the majority of the product had been produced, it was isolated and purified using a semi-preparative C18 column, and then lyophilized to obtain compound 72 (23 mg, 87% yield). HRMS calculated value C 37 H 37 N5O8[M+H] + 680.272, actual value 680.2712.

[0191] Step 2: Compound 72 (23 mg, 33.8 μmol) was dissolved in 400 μL of DMF, and MMAE (24.3 mg, 33.8 μmol) was added to the above system. After uniform mixing, HOBt (0.92 mg, 6.76 μmol) and 82 μL of pyridine were added and the reaction was carried out at room temperature for 12 hours. LC-MS confirmed that the reaction essentially produced the cyclized product, which was isolated and purified using a basic C18 semi-preparative column to give compound 73 (35.3 mg, 83% yield). HRMS calculated value C 70 H 98 N8O 13 [M+2H] 2+ 630.3705, actual value 630.3701.

[0192] Step 3: Compound 73 (30 mg, 23.8 μmol) was dissolved in 100 μL of DMF, and 20 μL of piperidine was added to the reaction mixture. After reacting at room temperature for 20 minutes, the reaction was determined to be complete by LC-MS. The product was purified using a semi-preparative C18 column and then lyophilized to give compound 74 (22.9 mg, 93% yield). HRMS calculated value C 55 H 88 N8O 11 [M+H] + 1037.6651, actual value 1037.6559.

[0193] Step 4: Compound 74 (22.9 mg, 22.1 μmol) was dissolved in 100 μL of DMF, and compound 2 (34.2 mg, 88.4 μmol) was dissolved in 100 μL of 0.2 M PB buffer, pH 6.0, and added to the above system. After confirming that the pH of the reaction system was 6.0, NaCNBH3 (131.6 mg, 221 μmol) was added, the reaction system was mixed uniformly, and the reaction was allowed to proceed at 37°C for 6 hours. LC-MS monitoring of the reaction system showed that the majority of the product had been produced. The product was then isolated and purified using a semi-preparative C18 column, followed by lyophilization to obtain compound 75 (23.5 mg, 76% yield). HRMS calculated value C 69 H 111 N9O 21 [M+2H] 2+ 701.9025, actual value 701.9022.

[0194] Step 5: Compound 75 (10 mg, 6.7 μmol) was dissolved in 100 μL of DMF / 50 mM pH 7.5 PB = 1:1, and CDMBI (7.6 mg, 35.5 μmol) was added to the above system. After homogeneous mixing, the mixture was cooled to 0 °C on ice, and potassium phosphate (22.7 mg, 106.5 μmol) was added and the reaction was continued at 0 °C for 12 hours. LC-MS confirmed that the reaction essentially produced the cyclized product, which was isolated and purified using a basic C18 semi-preparative column to give compound DG-7 (6.8 mg, 69% yield). HRMS calculated value C 69 H 109 N9O 20 [M+2H] 2+ 692.8973, actual value 692.8910.

[0195] Example 34: Synthesis of compound dDG-1 The structure and synthesis method of compound dDG-1 are as follows. [ka] Compound dDG-1 [ka]

[0196] Step 1: Compound 5 (10 mg, 26.17 μmol) was dissolved in 500 μL of CH OH / HO = 1:4 system, and 1H-imidazole-sulfonyl azide hydrochloride (8.2 mg, 39.3 μmol), potassium carbonate (10.9 mg, 78.6 μmol), and copper sulfate (6.2 mg, 39.3 μmol) were added to the reaction mixture in sequence, and the mixture was allowed to react at 37 ° C for 4 hours. The reaction was monitored for near completion by LC-MS, and the product was isolated and purified using a P2 column, followed by lyophilization to obtain compound 6 (9 mg, 85% yield).

[0197] Step 2: Preparation of Cu(I)-BTTAA solution: 55 μL of 60 mM CuSO, 66 μL of 300 mM BTTAA, and 490 μL of 0.9 M sodium ascorbate were mixed uniformly in sequence and then stored.

[0198] Step 3: Compound 6 (9 mg, 22 μmol) was dissolved in 50 μL of 50 mM PB buffer, pH 7.5. Compound 46 (7.4 mg, 22 μmol) was added to the reaction mixture and mixed uniformly. After that, the entire volume of Cu(I)-BTTAA solution in Step 7 was added and the mixture was reacted at 37°C for 4 hours. The reaction mixture was monitored by LC-MS. After the completion of the reaction was confirmed, the product was purified using a semi-preparative C18 column and lyophilized to obtain compound 67 (14.2 mg, 88% yield).

[0199] Step 4: Compound 67 (14.2 mg, 19.1 μmol) was dissolved in 100 μL of DMF. HATU (14.6 mg, 38.2 μmol), compound 19 (21.5 mg, 19.1 μmol), and DIPEA (10 μL, 57.3 μmol) were added to the reaction mixture, and the mixture was allowed to react at 37° C. for 2 hours. After the reaction was complete, 20 μL of piperidine was added. After 15 minutes, LC-MS showed that the reaction was complete. The mixture was purified using a semi-preparative C18 column and lyophilized to give compound 68 (26 mg, 83% yield).

[0200] Step 5: Compound 68 (20 mg, 12.3 μmol) was dissolved in 100 μL of DMF, and compound 54 (SMCC-DM1, 13.2 mg, 12.3 μmol) and triethylamine (5 μL, 36.9 μmol) were added to the reaction mixture, followed by reaction at 37° C. for 2 hours. The reaction mixture was monitored by LC-MS. After completion of the reaction, the product was purified using a semi-preparative C18 column and lyophilized to give compound 69 (24 mg, 76% yield).

[0201] Step 6: Compound 69 (24 mg, 9.3 μmol) was dissolved in 100 μL of DMF / 50 mM pH 7 PB = 1:1, and CDMBI (10 mg, 46.5 μmol) was added to the reaction system. After uniform mixing, the mixture was placed on ice and cooled to 0 ° C. Potassium phosphate (29.6 mg, 139.5 μmol) was added and reacted at 0 ° C. for 12 hours. The reaction was monitored for near completion by LC-MS, and the mixture was isolated and purified using a basic C18 column, followed by lyophilization to obtain compound dDG-1 (15.5 mg, 65% yield).

[0202] Example 35: Synthesis of compound dDG-2 [ka] Compound dDG-2 JPEG2022174834000191.jpg84140

[0203] Step 1: Compound 34 (Fmoc-Lys-OH, 20 mg, 54.3 μmol) was dissolved in 200 μL of DMF, and compound 17 (11.82 mg, 59.8 μmol) and 22.6 μL of triethylamine were added to the above system. After uniform mixing, the mixture was allowed to react at room temperature for 2 hours. The completion of the reaction was monitored by LC-MS, and the mixture was isolated and purified using a semi-preparative C18 column, followed by lyophilization to give compound 76 (22.5 mg, 92% yield). HRMS calculated value C 23 H 25 N5O5[M+H] + 452.1934, actual value 452.1991.

[0204] Step 2: Compound 76 (20 mg, 44.3 μmol) and HATU (33.5 mg, 88.6 μmol) were dissolved in 100 μL of anhydrous DMF. Compound 19 (NH2-VC-PAB-MMAE, 49.9 mg, 44.3 μmol) was added to the above reaction system, and DIPEA (22.8 μL, 132.9 μmol) was added dropwise with stirring. The reaction was allowed to proceed at room temperature for 1 hour. The reaction was monitored by LC-MS. After completion of the reaction was confirmed, the product was purified by semi-preparative separation and lyophilized to give compound 77 (60 mg, 88% yield). HRMS calculated value C 81 H 117 N 15 O 16 [M+2H] 2+ 778.9479, actual value 778.9477.

[0205] Step 3: Compound 77 (20 mg, 12.8 μmol) was dissolved in 100 μL of DMF, and 20 μL of piperidine was added to the reaction mixture. After reacting at room temperature for 20 minutes, the reaction was monitored for completion by LC-MS. The mixture was purified using a semi-preparative C18 column and then lyophilized to give compound 78 (15.8 mg, 92% yield). HRMS calculated value C 66 H 107 N 15 O 14 [M+2H] 2+ 667.9139, actual value 667.9140.

[0206] Step 4: Compound 78 (15.8 mg, 11.8 μmol) was dissolved in 100 μL of DMF, and compound 2 (18.3 mg, 47.2 μmol) was dissolved in 100 μL of 0.2 M PB buffer (pH 6.0). This was added to the above system. After confirming that the pH of the reaction system was 6.0, NaCNBH3 (70.3 mg, 118 μmol) was added, the reaction system was mixed uniformly, and the reaction was allowed to proceed at 37 °C for 6 hours. The reaction system was monitored by LC-MS, and after confirming that the majority of the product had been produced, it was isolated and purified using a semi-preparative C18 column, then lyophilized to obtain compound 79 (15 mg, 75% yield). HRMS calculated value C 80 H 130 N 16 O 24[M+2H] 2+ 850.48, actual value 850.4721.

[0207] Step 5: Compound 79 (10 mg, 5.9 μmol) was dissolved in 100 L of DMF / 50 mM, pH 7.5, PB = 1:1, and CDMBI (6.7 mg, 31.2 μmol) was added to the above system. After homogeneous mixing, the mixture was cooled to 0 °C on ice, and potassium phosphate (20 mg, 93.7 μmol) was added. The reaction was allowed to proceed at 0 °C for 12 hours. LC-MS confirmed that the reaction essentially produced the cyclized product, which was isolated and purified using a basic C18 semi-preparative column to give compound dDG-2 (7 mg, 71% yield). HRMS calculated value C 69 H 109 N9O 20 [M+2H] 2+ 842.4747, actual value 842.4721.

[0208] Example 36: Synthesis of compound dDG-3 [ka] Compound dDG-3 JPEG2022174834000193.jpg89157

[0209] Step 1: Compound 82 (6 mg, 0.0162 mmol) was dissolved in 60 μL of DMF. HATU (17.9 mg, 0.0486 mmol), compound 19 (33 mg, 0.0292 mmol), and DIPEA (8.86 μL, 0.0649 mmol) were added to the reaction mixture, and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was monitored by LC-MS. After the reaction was shown to be nearly complete, 128.5 μL of triethylamine was added, the mixture was mixed uniformly, and the mixture was allowed to react at room temperature for 15 minutes. The product was separated and purified using a semi-preparative C18 column. The title product was collected and lyophilized to give compound 91 (21.5 mg, 57.15% yield). HRMS calculated value C 121 H 193 N 21 O 26 [M+2H] 2+1179.2291, [M+3H] 3+ 786.4887, measurements 1179.2211, 786.484.

[0210] Step 2: Compound 91 (21.5 mg, 9.12 μmol) was dissolved in 210 μL of DMF. CHO-LacNAc (10.44 mg, 0.0273 mmol) was added to the reaction mixture. DMF was added to adjust the DMF / 0.2M PB ratio to 1:1. After adjusting the pH of the reaction mixture to 6.0 with NaOH / HCl, sodium cyanoborohydride (5.74 mg, 0.0912 mmol) was added and the reaction was allowed to proceed at room temperature for 3-4 hours. The reaction was monitored for near completion by LC-MS. The product was separated and purified using a semi-preparative C18 column. The title compound was collected and lyophilized to give compound 92 (10.5 mg, 50.5% yield). HRMS calculated value C 135 H 216 N 22 O 36 [M+2H] 2+ 1361.7952, [M+3H] 3+ 908.1994, measurements 1361.7889, 908.1909.

[0211] Step 3: Compound 92 (10.5 mg, 3.86 μmol) was dissolved in 210 μL of DMF / HO = 1:1, DMC (13 mg, 32.63 μmol) was added to the reaction mixture, and triethylamine (32.2 μL, 0.231 mmol) was added. The mixture was then mixed uniformly and placed on ice, cooled to 0 °C, and reacted for 2 hours. The reaction was monitored for near completion by LC-MS, and the product was separated and purified using a basic C18 column. The title product was collected and lyophilized to give compound dDG-3 (7.1 mg, 68% yield). HRMS calculated value C 135 H 214 N 22 O 35 [M+2H] 2+ 1352.7899, [M+3H] 3+ 902.1959, measurements 1352.7881, 902.1952.

[0212] IV: Synthesis of glycoengineered antibodies Ab-1 to Ab-19 Example 37: Synthesis of Ab-1 The non-natural glycoengineered antibody Ab-1 was obtained by applying general procedure 1 to compound G1 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146536.

[0213] Example 38: Synthesis of Ab-2 The non-natural glycoengineered antibody Ab-2 was obtained by applying general procedure 1 to compound G2 and the wild-type antibody Herceptin.

[0214] Example 39: Synthesis of Ab-3 The non-natural glycoengineered antibody Ab-3 was obtained by applying general procedure 1 to compound G3 and wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146702.

[0215] Example 40: Synthesis of Ab-4 The non-natural glycoengineered antibody Ab-4 was obtained by applying general procedure 1 to compound G4 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146699.

[0216] Example 41: Synthesis of Ab-5 The non-natural glycoengineered antibody Ab-5 was obtained by applying general procedure 1 to compound G5 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146170.

[0217] Example 42: Synthesis of Ab-6 The non-natural glycoengineered antibody Ab-6 was obtained by applying general procedure 1 to compound G6 and the wild-type antibody Herceptin.

[0218] Example 43: Synthesis of Ab-7 The non-natural glycoengineered antibody Ab-7 was obtained by applying general procedure 1 to compound G7 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146642.

[0219] Example 44: Synthesis of Ab-8 The non-natural glycoengineered antibody Ab-8 was obtained by applying general procedure 1 to compound G8 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146642.

[0220] Example 45: Synthesis of Ab-9 The non-natural glycoengineered antibody Ab-9 was obtained by applying general procedure 1 to compound G9 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146701.

[0221] Example 46: Synthesis of Ab-10 The non-natural glycoengineered antibody Ab-10 was obtained by applying general procedure 1 to compound G10 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 147218.

[0222] Example 47: Synthesis of Ab-11 The non-natural glycoengineered antibody Ab-11 was obtained by applying general procedure 1 to compound G11 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 146831.

[0223] Example 48: Synthesis of Ab-12 The non-natural glycoengineered antibody Ab-12 was obtained by applying general procedure 1 to compound G12 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 147120.

[0224] Example 49: Synthesis of Ab-13 The non-natural glycoengineered antibody Ab-13 was obtained by applying general procedure 1 to compound G13 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 147110.

[0225] Example 50: Synthesis of Ab-14 The non-natural glycoengineered antibody Ab-14 was obtained by applying general procedure 1 to compound G14 and the wild-type antibody Herceptin. The observed value after HRMS deconvolution was 147152.

[0226] Example 51: Synthesis of Ab-15 The non-natural glycoengineered antibody Ab-15 was obtained by applying general procedure 2 to compound G3 and the non-fucose antibody Herceptin. The observed value after HRMS deconvolution was 146423.

[0227] Example 52: Synthesis of Ab-16 The non-natural glycoengineered antibody Ab-16 was obtained by applying general procedure 2 to compound G8 and the non-fucose antibody Herceptin. The observed value after HRMS deconvolution was 146362.

[0228] Example 53: Synthesis of Ab-17 The non-natural glycoengineered antibody Ab-17 was obtained by applying general procedure 2 to compound G10 and the non-fucose antibody Herceptin. The observed value after HRMS deconvolution was 146939.

[0229] Example 54: Synthesis of Ab-18 The non-natural glycoengineered antibody Ab-18 was obtained by applying general procedure 2 to compound G12 and the non-fucose antibody Herceptin. The observed value after HRMS deconvolution was 146819.

[0230] Example 55: Synthesis of Ab-19 The non-natural glycoengineered antibody Ab-19 was obtained by applying general procedure 2 to compound G13 and the non-fucose antibody Herceptin.

[0231] V: Synthesis of glycoside-specific antibody-drug conjugates gsADC-1 to gsADC-43 Example 56: Synthesis of gsADC-1 (gsADC-displaying sugar site-specific antibody-drug conjugate) The antibody-drug conjugate gsADC-1 was obtained by applying procedure 4 to compound D2 and the non-natural glycoengineered antibody Ab-2.

[0232] Example 57: Synthesis of gsADC-2 The antibody-drug conjugate gsADC-2 was obtained by applying procedure 5 to compound D1 and the non-natural glycoengineered antibody Ab-2. Found value after HRMS deconvolution: 148892.

[0233] Example 58: Synthesis of gsADC-3 The antibody-drug conjugate gsADC-3 was obtained by applying procedure 6 to compound D3 and the non-natural glycoengineered antibody Ab-2.

[0234] Example 59: Synthesis of gsADC-4 The antibody-drug conjugate gsADC-4 was obtained by applying procedure 6 to compound D4 and the non-natural glycoengineered antibody Ab-2.

[0235] Example 60: Synthesis of gsADC-5 Antibody-drug conjugate gsADC-5 was obtained by applying procedure 7 to compound D6 and non-natural glycoengineered antibody Ab-3. Found value after HRMS deconvolution: 151,464.

[0236] Example 61: Synthesis of gsADC-6 The antibody-drug conjugate gsADC-6 was obtained by applying procedure 7 to compound D7 and the non-natural glycoengineered antibody Ab-3.

[0237] Example 62: Synthesis of gsADC-7 The antibody-drug conjugate gsADC-7 was obtained by applying procedure 7 to compound D8 and the non-natural glycoengineered antibody Ab-3.

[0238] Example 63: Synthesis of gsADC-8 The antibody-drug conjugate gsADC-8 was obtained by applying procedure 7 to compound D9 and the non-natural glycoengineered antibody Ab-3.

[0239] Example 64: Synthesis of gsADC-9 The antibody-drug conjugate gsADC-9 was obtained by applying procedure 7 to compound D6 and the non-natural glycoengineered antibody Ab-4.

[0240] Example 65: Synthesis of gsADC-10 The antibody-drug conjugate gsADC-10 was obtained by applying procedure 7 to compound D7 and the non-natural glycoengineered antibody Ab-4.

[0241] Example 66: Synthesis of gsADC-11 The antibody-drug conjugate gsADC-11 was obtained by applying procedure 7 to compound D8 and the non-natural glycoengineered antibody Ab-4.

[0242] Example 67: Synthesis of gsADC-12 The antibody-drug conjugate gsADC-12 was obtained by applying procedure 7 to compound D9 and the non-natural glycoengineered antibody Ab-4.

[0243] Example 68: Synthesis of gsADC-13 The antibody-drug conjugate gsADC-13 was obtained by applying procedure 7 to compound D6 and the non-natural glycoengineered antibody Ab-6.

[0244] Example 69: Synthesis of gsADC-14 The antibody-drug conjugate gsADC-14 was obtained by applying procedure 7 to compound D7 and the non-natural glycoengineered antibody Ab-6.

[0245] Example 70: Synthesis of gsADC-15 The antibody-drug conjugate gsADC-15 was obtained by applying procedure 7 to compound D8 and the non-natural glycoengineered antibody Ab-6.

[0246] Example 71: Synthesis of gsADC-16 The antibody-drug conjugate gsADC-16 was obtained by applying procedure 7 to compound D9 and the non-natural glycoengineered antibody Ab-6.

[0247] Example 72: Synthesis of gsADC-17 The antibody-drug conjugate gsADC-17 was obtained by applying procedure 7 to compound D6 and the non-natural glycoengineered antibody Ab-9.

[0248] Example 73: Synthesis of gsADC-18 The antibody-drug conjugate gsADC-18 was obtained by applying procedure 7 to compound D7 and the non-natural glycoengineered antibody Ab-9.

[0249] Example 74: Synthesis of gsADC-19 The antibody-drug conjugate gsADC-19 was obtained by applying procedure 7 to compound D8 and the non-natural glycoengineered antibody Ab-9.

[0250] Example 75: Synthesis of gsADC-20 The antibody-drug conjugate gsADC-20 was obtained by applying procedure 7 to compound D9 and the non-natural glycoengineered antibody Ab-9.

[0251] Example 76: Synthesis of gsADC-29 The antibody-drug conjugate gsADC-29 was obtained by applying general procedure 7 to compound D13 and the non-natural glycoengineered antibody Ab-3.

[0252] Example 77: Synthesis of gsADC-21 The antibody-drug conjugate gsADC-21 was obtained by applying procedure 8 to compound D5 and the non-natural glycoengineered antibody Ab-3. Found value after HRMS deconvolution: 151832.

[0253] Example 78: Synthesis of gsADC-39 The antibody-drug conjugate gsADC-39 was obtained by applying procedure 8 to compound D11 and the non-natural glycoengineered antibody Ab-3. Found value after HRMS deconvolution: 149589.

[0254] Example 79: Synthesis of gsADC-41 The antibody-drug conjugate gsADC-41 was obtained by applying procedure 8 to compound D12 and the non-natural glycoengineered antibody Ab-3. Found value after HRMS deconvolution: 149617.

[0255] Example 80: Synthesis of gsADC-22 The antibody-drug conjugate gsADC-22 was obtained by applying procedure 8 to compound D5 and the non-natural glycoengineered antibody Ab-4. Found value after HRMS deconvolution: 151833.

[0256] Example 81: Synthesis of gsADC-23 The antibody-drug conjugate gsADC-23 was obtained by applying procedure 8 to compound D5 and the non-natural glycoengineered antibody Ab-6.

[0257] Example 82: Synthesis of gsADC-24 The antibody-drug conjugate gsADC-24 was obtained by applying procedure 8 to compound D5 and the non-natural glycoengineered antibody Ab-9.

[0258] Example 83: Synthesis of gsADC-42 The antibody-drug conjugate gsADC-42 was obtained by applying procedure 8 to compound D11 and the non-natural glycoengineered antibody Ab-14. Found value after HRMS deconvolution: 152801.

[0259] Example 84: Synthesis of gsADC-43 The antibody-drug conjugate gsADC-43 was obtained by applying procedure 8 to compound D5 and the non-natural glycoengineered antibody Ab-15.

[0260] Example 85: Synthesis of gsADC-25 The antibody-drug conjugate gsADC-25 was obtained by applying procedure 9 to compound D10 and the non-natural glycoengineered antibody Ab-3.

[0261] Example 86: Synthesis of gsADC-26 The antibody-drug conjugate gsADC-26 was obtained by applying procedure 9 to compound D10 and the non-natural glycoengineered antibody Ab-4.

[0262] Example 87: Synthesis of gsADC-27 The antibody-drug conjugate gsADC-27 was obtained by applying procedure 9 to compound D10 and the non-natural glycoengineered antibody Ab-6.

[0263] Example 88: Synthesis of gsADC-28 The antibody-drug conjugate gsADC-28 was obtained by applying procedure 9 to compound D10 and the non-natural glycoengineered antibody Ab-9.

[0264] Example 89: Synthesis of gsADC-30 Antibody-drug conjugate gsADC-30 was obtained by applying procedure 3 to compound DG-1 and wild-type antibody Herceptin. Found value 148892 after HRMS deconvolution.

[0265] Example 90: Synthesis of gsADC-31 The antibody-drug conjugate gsADC-31 was obtained by applying procedure 3 to compound DG-2 and the wild-type antibody Herceptin.

[0266] Example 91: Synthesis of gsADC-32 The antibody-drug conjugate gsADC-32 was obtained by applying procedure 3 to compound DG-3 and the wild-type antibody Herceptin.

[0267] Example 92: Synthesis of gsADC-33 The antibody-drug conjugate gsADC-33 was obtained by applying procedure 3 to compound DG-4 and the wild-type antibody Herceptin.

[0268] Example 93: Synthesis of gsADC-34 The antibody-drug conjugate gsADC-34 was obtained by applying procedure 3 to compound DG-5 and wild-type antibody Herceptin. Found value 148295 after HRMS deconvolution.

[0269] Example 94: Synthesis of gsADC-35 The antibody-drug conjugate gsADC-35 was obtained by applying procedure 3 to compound DG-6 and the wild-type antibody Herceptin. Found value 148751 after HRMS deconvolution.

[0270] Example 95: Synthesis of gsADC-36 The antibody-drug conjugate gsADC-36 was obtained by applying procedure 3 to compound DG-7 and the wild-type antibody Herceptin. Found value 148576 after HRMS deconvolution.

[0271] Example 96: Synthesis of gsADC-37 The antibody-drug conjugate gsADC-37 was obtained by applying procedure 3 to compound dDG-1 and the wild-type antibody Herceptin.

[0272] Example 97: Synthesis of gsADC-38 The antibody-drug conjugate gsADC-38 was obtained by applying procedure 10 to compound DG-6 and the defucosylated antibody Herceptin. Found value 148448 after HRMS deconvolution.

[0273] VI: Screening of sugar substrates and glycosidic endonucleases Production of sugar oxazolines Various sugar substrates (1 equivalent, including mono-, di-, and trisaccharide structures) were dissolved in 50 mM PB buffer, pH 7.0, and CDMBI (5 equivalents) was added to the reaction system. After uniform mixing, the system was cooled to 0°C, and potassium phosphate (15 eq) was added to bring the final sugar substrate concentration to 10 mM. The reaction was carried out at 0°C for 2 hours. A large amount of precipitate was observed, and the precipitate was removed by centrifugation. The supernatant, which contained salt and contained sugar oxazoline substrates G1, G12, G15-G20, was used directly for the next screening step.

[0274] Synthesis of compound G15 [ka] HRMS calculated value C8H 13 NO5[M+H] + 204.0872, actual value 204.0809. 1 HNMR (600MHz, heavy water) δ6.01(d,J=7.3Hz,1H),4.04(ttd,J=6.2,4.4,3.9,2.4Hz,1H),3.90(t,J=3.6Hz,1H),3.74-3. 70(m,1H),3.60(dd,J=12.5,6.3Hz,1H),3.57-3.51(m,1H),3.29(ddd,J=8.9,6.3,2.5Hz,1H),1.97-1.95(m,3H).

[0275] Synthesis of compound G16 [ka] HRMS calculated value C8H 13 NO5[M+H] + 204.0872, actual value 204.0859. 1 HNMR (600MHz, heavy water) δ6.00(d,J=7.2Hz,0.85H),5.14(d,J=3.7Hz,0.15H),4.02-3.97(m,1H),3.86-3.84(m,1H) ),3.81(ddd,J=7.0,4.9,1.8Hz,1H),3.76(td,J=7.1,1.3Hz,1H),3.70-3.59(m,2H),1.94(d,J=1.3Hz,3H).

[0276] Synthesis of compound G17

change

[0277] Synthesis of compound G18

change

[0278] Synthesis of compound G19

change

[0279] Synthesis of compound G20 [ka] HRMS calculated value C 20 H 33 NO 14 [M+H] + 512.1979, actual value 512.1966. 1 HNMR (600MHz, heavy water) δ6.01(d,J=7.3Hz,1H),5.10(d,J=4.0Hz,1H),4.38(dd,J=3.0,1.2Hz,1H),4.34(d,J=7.8Hz,1H),4.2 8-4.24(m,1H),4.11(q,J=6.5Hz,1H),3.96(dt,J=8.7,1.4Hz,1H),3.84-3.32(m,12H),1.98(d,J=1.8Hz,3H),1.12(3H).

[0280] Screening for glycosidic endonucleases G14, deglycosylated antibody (Fucα1,6)GlcNAc-Herceptin, and various glycosidic endonucleases were added sequentially to 50 mM Tris-HCl buffer (pH 7.2) to final concentrations of 1.5 mM, 5 mg / mL, and 0.2 mg / mL, respectively. After 3 hours of incubation at 30°C, the enzyme activity was measured by mass spectrometry. A control group containing no glycosidic endonucleases was also prepared to eliminate the effects of non-enzymatic reactions. Using these screening conditions, ten glycosidic endonucleases were screened: Endo-S, Endo-S D233Q, Endo-S2, Endo-S2 D184M, Endo-F3, Endo-F3 D165A, Endo-D, Endo-D Q431A, Endo-D N322Q, and Endo-A. For example, as shown by A in Figure 1, Endo-S2 was found to have weak transposition activity only for G14, and the transposition yield was approximately 5.6%.

[0281] Sugar substrate screening Sugar oxazoline substrates (G1, G12, G15-G20), deglycosylated antibody (Fucα1,6)GlcNAc-Herceptin, and glycoside endonuclease Endo-S2 were sequentially added to 50 mM Tris-HCl buffer, pH 7.2, to give final concentrations of 1.5 mM, 5 mg / mL, and 0.2 mg / mL, respectively. After incubation at 30°C for 3 hours, the results were measured by mass spectrometry. A control group was also set up without the addition of glycoside endonuclease to eliminate the effects of non-enzymatic reactions. As shown in Figure 1B, the results showed that Endo-S2 had a high recognition efficiency of 68% for the transfer of G1 to the deglycosylated antibody. G12, obtained by introducing sialic acid into the 6-position of the galactose of G1, was also well recognized by Endo-S2. However, the transfer activity of the sugar substrates G19 and G20, modified at the 3-position of galactose or the 3-position of N-acetylglucosamine, was significantly reduced by the action of Endo-S2, and other sugar structures could not be recognized well.

[0282] Pharmacological Example 1 In vitro activity data experiment process and result analysis The activity of some of the disaccharide ADCs described above was evaluated at the cellular level, and a total of three cell lines were selected. SK-Br-3 cells and NCI-N87 cells were Her2-positive cells, and MDA-MB-231 cells were Her2-negative cells. The cellular activity and toxicity of the ADC molecules were measured using the MTT method. Specifically, 100 μL of PBS was added to the outermost well of a 96-well plate, and medium alone was added to three other wells. Approximately 6,000 corresponding cells were added to each of the remaining wells, and the plates were incubated overnight at 37°C in a CO2 incubator. Ten microliters of each ADC molecule (starting with a 5-fold gradient dilution of each ADC molecule from a highest concentration of 100 nM, a total of nine dilutions were performed, with each concentration in triplicate wells) was added to three wells of a 96-well plate. For the other three wells, 10 μL of medium was added to the medium wells as a control and blank. The 96-well plate was then incubated at 37°C in a CO2 incubator for 72 hours. Ten microliters of 5 mg / mL MTT was added to each well and incubated at 37°C for 4 hours. Then, 90 μL of SDS lysate was added to each well and incubated at 37°C for 7 hours to allow the cells to fully divide. Finally, the OD value at 570 nm of each well was measured, and the data were processed using GraphPad Prism 6. The results are shown in Figure 2.

[0283] In Figure 2, gsADC-40 was prepared by ring tension-promoted click chemistry using N3-NH-SCT-Her (see Chinese Patent Application CN107778372A for details; produced from the oligosaccharide structure Az-NH-SCT, e.g., the structure shown in the diagram below) and BCN drug-linker D5. Specifically, the reaction conditions were: N3-NH-SCT-Her was 5 mg / mL, and the pH was adjusted to 7.4 to give compound D5 at 0.55 mM. After confirming complete conversion to the product by LC-MS, protein A purification was used. [ka] N3-NH-SCT-Her

[0284] As can be seen from the results in Figure 2, the disaccharide ADC has in vitro cellular activity comparable to the approved T-DM1 and is non-toxic in negative cells.

[0285] Pharmacological Example 2 In vivo antitumor activity experiment process and result analysis A BALB / c nude mouse tumor model was established using gastric cancer cell line NCI-N87. Using ear markers and the principle of dividing mice into large, medium, and small groups, each containing five mice.

[0286] For the activity evaluation at the animal level, four disaccharide ADC compounds, gsADC-21, gsADC-30, gsADC-35, and gsADC-36, were selected. A Cys random-linked ADC compound (DAR≒4) was used as a positive control, and PBS was used as a negative control. All samples were diluted to 0.2 mg / mL with 1x PBS and decontaminated using a 0.22 mm filter membrane before administration.

[0287] All samples were intraperitoneally administered at a concentration of 3 mg / kg every three days for a total of three doses. Tumor size and mouse weight were measured every three days after the first dose using a Vernier caliper. The experimental procedure was conducted in accordance with animal ethics requirements. Data were graphically analyzed using GraphPad Prism 6 software. As shown in Figure 3, the glycoside-specific ADC compounds produced using this patented technology had good in vivo activity.

Claims

1. A disaccharide linker as shown in formula I below: [Formula 1] In formula I, G ring represents a structure derived from a monosaccharide molecule and is bound to the 4-position of N-acetyl-D-glucosamine cyclized at the 1,2-positions by a glycosidic bond, the monosaccharide molecule being selected from the group consisting of galactose, N-acetyl-galactose, glucose, mannose, fucose, and sialosugar, and the glycosidic bond being a 1,4-glycosidic bond, a 2,4-glycosidic bond, or a 3,4-glycosidic bond; Z-Y-X- represents a substituent on the G ring, and the substitution position of Z-Y-X- is any position other than position 1 of the G ring derived from a monosaccharide molecule; In the structure Z-Y-X-, Z-Y- may be present or absent; When Z-Y- is not present, X is an aldehyde group, a phosphate group, or -NH ２ , -CH ２ -NH ２ , -COOH, -CH ２ S.R. ｐ , -CH ２ SeR ｐ , -N ３ , -CH ２ -N ３ and R ｐ is a protecting group, When Z-Y- is present, X is -CH ２ --, --CH ２ -O-, -CH ２ -S-, -CH ２ -Se-, -CO-NH-, -ON=CH-, -CONH-N=CH-, -NHCH ２ -, -CH=CH-, or the following structure: [Case 2] Y is a divalent or polyvalent linker connecting X and Z; Preferably, Y is -(CH ２ )m-(CH-w)n-,-(CH ２ -CH ２-O)m-(CH-w)n-,-(PO ４ ) n-, where m and n are each independently selected from the group consisting of integers between 0 and 30, and w is a hydrogen atom or a polyethylene glycol structure of various lengths, or a combination of cleavable fragments and the above linked fragments; Z is selected from the group consisting of the following cases i) to iv): i) Fragments having reactive groups or functional molecules for bioorthogonal reactivity Preferably, Z is selected from the group consisting of the following reactive groups: azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetraazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boric acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halocarbonyl residue, isocyanide residue, sydnone residue, selenium residue, conjugated diene residue, phosphoric acid residue, cycloalkyne residue and cycloalkene residue, or selected from the group consisting of the following groups: [C3] n is an integer from 1 to 30; R １ and R ２ are each independently H, -CH ３ , -CH ２ CH ３ , cyclopropyl or cyclobutyl; Preferably, the functional molecule is selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycols, lipids, polypeptides, nanobodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, contrast agents and magnetic resonance imaging agents; ii) [C4] L １ is a trivalent linker having three reactive groups, Preferably, L １is a branched chain amino acid having a reactive functional group derived from lysine, aspartic acid, glutamic acid, propargylglycine, cysteine, and the structure [C5] n is an integer from 1 to 30; L ２ and L ３ L １ and Z ２ and Z ３ and Preferably, L ２ and L ３ are each independently selected from the group consisting of the following structures: -(CH ２ )m-(CH-w)n-,-(CH ２ -CH ２ -O)m-(CH-w)n-,-(PO ４ n-, m and n are each independently selected from the group consisting of integers between 0 and 30, and w is a hydrogen atom or other side chain structure, such as polyethylene glycol of various lengths, or a combination of cleavable fragments and the above linked fragments; Z′ is a coupling L １ and a sugar linker, and does not exist independently; -(CH ２ ) p-, or a group capable of reacting with the Z group in case i), where p is an integer from 1 to 5, For example, Z' is selected from the group consisting of: [C6] R １ and R ２ are each independently H, -CH ３ , -CH ２ CH ３ , cyclopropyl or cyclobutyl; Z ２ and Z ３ The definition of is the same as the definition of Z in case i), iii) [C7] L ６is a tetravalent linker having four reactive groups, Preferably, L ６ is selected from the group consisting of dilysine, diglutamic acid, diaspartic acid, aspartic acid-glutamic acid dipeptide structure, aspartic acid-lysine dipeptide structure, glutamic acid-lysine structure, or is selected from the group consisting of the following structures: [C8] n is an integer from 1 to 30; L ２ , L ３ , L ４ The definition of L in ii) ２ , L ３ The definition of Z' is the same as the definition of Z' in ii), and Z ２ , Z ３ , Z ４ The definition of Z in ii) ２ , Z ３ is the same as the definition of iv) [C9] L １ The definition of L in ii) １ The definition is the same as that of L ２ , L ３ , L ４ , L ５ The definition of L in ii) ２ , L ３ The definition of Z' is the same as the definition of Z' in ii), and Z ２ , Z ３ , Z ４ , Z ５ The definition of Z in ii) ２ , Z ３ is the same as the definition of Or, if Y and Z are not present, X is [C10] Selected from the group consisting of R １ is a hydroxyl group -OH or an azide group -N ３ , R ２ is any group, R ３ is either a hydroxy-OH or -NH-containing group, R ４ is an optional group and the wavy line indicates the site of attachment, a disaccharide linker.

2. The disaccharide linker of formula I is shown in formula II below: [C11] 2. The disaccharide linker of claim 1, wherein in formula II, X, Y and X are as defined in claim 1.

3. The disaccharide linker is selected from the group consisting of the following specific compounds: [C12] 3. The disaccharide linker of claim 1 or 2, wherein R is a fragment or combination according to Y, Z of claim 1, and l, m, and n are each independently an integer from 0 to 30.

4. A method for producing the disaccharide linker according to any one of claims 1 to 3, for example, as shown in the following reaction formula: [C13] In the reaction formula, the definition of the G ring is the same as that in claims 1 to 3, the modification position of the monosaccharide is a modifiable site other than the 1-position, U is an introduced active group selected from the group consisting of an aldehyde group, an amino group, an azide group, and an alkynyl group, and the definitions of X, Y, and Z are as defined in claims 1 to 3, respectively; The method comprises: 1) modifying a disaccharide structure having an acetylglucosamine terminal by the action of an enzyme or other small molecule compound to obtain a disaccharide structure having an active group U, and introducing Z-Y-X- having orthogonal reactivity or containing a functional molecular fragment after the disaccharide structure having the active group U has been derivatized; 2) subjecting the disaccharide structure having Z-Y-X- introduced therein, which has orthogonal reactivity or contains a functional molecular fragment, to a cyclization step to obtain the disaccharide linker represented by formula I.

5. In step 1), the modification reaction is an oxidation reaction, the enzyme is galactose oxidase, and U is an aldehyde group; or the derivatization reaction in step 1) is an oxime-formation reaction, a reductive amination reaction, a reaction involving an amino group, or a reaction involving an azide group; The method according to claim 4, wherein in step 2) the cyclization reaction is carried out using 2-chloro-1,3-dimethylimidazolinium chloride or 2-chloro-1,3-dimethyl-1H-benzimidazolium-3-chloride.

6. A disaccharide-small molecule drug conjugate having the structure shown in Formula III, IV or V: [C14] In the above formulas III, IV and V, the G ring, X, Y, Z ３ , Z', L １ , L ２ , L ３ are as defined in claims 1 to 3, and in the structure of formula IV or V, L may be the same or different from each other, Z ２ ', Z ３ ' is a bioorthogonal group and Z ２ , Z ３ and Z' may be the same or different, and may be present simultaneously or not present independently; L is D, D １ Or D ２ and the remainder of Formula III-V, Preferably, L is -(CH ２ ) a-(CH ２ CH ２ )b-(NHCO)n-(CH ２ ) c- or selected from the group consisting of the following groups: [C15] [C16] V and W are bifunctional linkers, and include structures in which lysine and propargylglycine are bifunctional linkers; For example, L is [C17] Selected from the group consisting of a, b, c, d and e are each independently selected from the group consisting of integers between 0 and 30, m and n are each 0 or 1, R ３ and R ４ are each independently CH ３ -, (CH ３ ) ２ CH-, PhCH ２ , N.H. ２ (CH ２ ) ４ -, NH ２ CONH (CH ２ ) ３ -, R is selected from the group consisting of azidizable monosaccharides, disaccharides, oligosaccharides, PEG structures of various lengths having an azido group, or combinations of PEG with linear or cyclic monosaccharides, disaccharides, or oligosaccharides, said oligosaccharides including branched oligosaccharide chains, The wavy lines indicate the linkage sites. D,D １ and D. ２ each independently represents a cytotoxic compound, a group derived from a small molecule drug, or a fluorescent group, said small molecule drug being preferably selected from the group consisting of maytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, dokamycin, amanitin, PBDs, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estramustine, cemadotin, eleutherobin, fluorescent agents, monosaccharides, disaccharides, oligosaccharides, and derivatives of the above compounds, or a radioactive therapeutic drug; Preferably, D, D １ and D. ２ are each independently selected from the group consisting of the following groups: [C18] [C19] [C20] [C21] [C22] [C23] Disaccharide-small molecule drug conjugates.

7. The disaccharide-small molecule drug conjugates are represented by the following formulas VI, VII and VIII: [C24] The disaccharide-small molecule drug conjugate of claim 6, wherein the definitions of each substituent in formulae VI, VII and VIII are as defined in claim 6, respectively.

8. The disaccharide-low molecular weight drug conjugate is any one selected from the group consisting of the following compounds: [C25] [C26] [C27] [C28] In each of the above structures, the structure of the MMAE portion is [C29] The disaccharide-small molecule drug conjugate according to claim 6 or 7,

9. A glycoengineered antibody based on specific linkage of an antibody Fc domain N-glycosylation site as shown in formula IX below, [C30] In the above formula IX, the definitions of ring G, X, Y and Z are the same as those in claims 1 to 3, m is selected from the group consisting of 0 or 1, n is selected from the group consisting of 1 or 2, Ab is a monoclonal antibody, a bifunctional antibody or a polyclonal antibody, a therapeutic antibody or a functional antibody from a different species; Preferably, the Ab is a glycoengineered antibody selected from the group consisting of trastuzumab, pertuzumab, rituximab, cetuximab, morozumab, gemtuzumab, abciximab, darizumab, adalimumab, palizumab, baliximab, bevacizumab, panitumumab, nitrotuzumab, denitumab, decizumab, lemonivismab, nexituzumab, eprilimus, daremab, bentocivizumab, alemtuzumab, erlotuzumab, bonatumab, nivolumab, pembrolizumab, atezolizumab, avilumab, daruvalumab, tremelimumab, catumab, belintumomab, emicizumab, evantozumab (Rybrevant).

10. The glycoengineered antibody is represented by the following formula X: [C31] 10. The glycoengineered antibody of claim 9, wherein in the above formula X, the definitions of X, Y and Z are the same as those in claims 1 to 3, respectively, m is selected from the group consisting of 0 or 1, n is selected from the group consisting of 1 or 2, and Ab is an antibody.

11. A method for producing a glycoengineered antibody according to claim 9, said method being carried out by the following method 1 or the following method 2, [C32] In the above reaction formula, m is selected from the group consisting of 0 and 1, and the definitions of ring G, X, Y, and Z are the same as those in claims 1 to 3, In method 1, a wild-type antibody is hydrolyzed with a glycosidic endonuclease or a combination of a glycosidic endonuclease and a glucosidase to remove non-uniform glycans at the conservative glycosylation sites of the native antibody to obtain a deglycosylated antibody; and then the disaccharide linker according to any one of claims 1 to 3 and the wild-type antibody are co-incubated to link the disaccharide linker to the conservative glycosylation sites of the antibody Fc domain by catalytic action of the wild-type glycosidic endonuclease to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula IX modified with the disaccharide linker of formula I containing an orthogonal reactive group; In method 2, co-incubating the disaccharide linker according to any one of claims 1 to 3 and a wild-type antibody, hydrolyzing the N-oligosaccharide structure of the wild-type antibody Fc domain by the catalytic action of the wild-type glycosidic endonuclease, and simultaneously linking the disaccharide linker to the conservative glycosylation site of the antibody Fc domain to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula IX modified with the disaccharide linker of formula I containing an orthogonal reactive group; Preferably, the wild-type glycosidic endonuclease is N-acetylglucosamine endohydrolase, more preferably, the N-acetylglucosamine endohydrolase is endoglycosidase Endo-S2, for example, endoglycosidase Endo-S2 derived from Streptococcus pyogenes, and the method for producing a glycoengineered antibody requires the combined use of a glycosidic endonuclease and a fucose hydrolase when producing a coreless fucosylated compound.

12. A method for producing a glycoengineered antibody according to claim 10, said method being carried out by the following method 1 or the following method 2, [C33] In the above reaction scheme, m is selected from the group consisting of 0 or 1, and the definitions of X, Y, and Z are the same as those in claims 1 to 3, respectively; In method 1, a wild-type antibody is hydrolyzed with a glycosidic endonuclease or a combination of a glycosidic endonuclease and a glucosidase to remove non-uniform glycans at the conservative glycosylation sites of the native antibody to obtain a deglycosylated antibody; and then the disaccharide linker according to any one of claims 1 to 3 and the wild-type antibody are co-incubated to link the disaccharide linker to the conservative glycosylation sites of the antibody Fc domain by catalytic action of the wild-type glycosidic endonuclease to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula X modified with a disaccharide linker of formula II containing an orthogonal reactive group; In method 2, co-incubating the disaccharide linker according to any one of claims 1 to 3 and a wild-type antibody, hydrolyzing the N-oligosaccharide structure of the wild-type antibody Fc domain by the catalytic action of the wild-type glycosidic endonuclease, and simultaneously linking the disaccharide linker to the conservative glycosylation site of the antibody Fc domain to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula X modified with the disaccharide linker of formula II containing an orthogonal reactive group; Preferably, the wild-type glycosidic endonuclease is N-acetylglucosamine endohydrolase, more preferably, the N-acetylglucosamine endohydrolase is endoglycosidase Endo-S2, for example, endoglycosidase Endo-S2 from Streptococcus pyogenes; A method for producing glycoengineered antibodies, which requires the combined use of a glycosidic endonuclease and a fucose hydrolase when producing a coreless fucosylated compound.

13. An antibody-drug conjugate as shown in formula XI below: [C34] An antibody-drug conjugate wherein, in formula XI, the definitions of ring G and X, Y, Z', L and D are the same as those in claims 6 to 8, m is selected from the group consisting of 0 or 1, n is selected from the group consisting of 1 or 2, Ab is an antibody, and the glycostructure attachment site is a conservative N-glycosylation site on the antibody Fc.

14. The antibody-drug conjugate is shown in Formula XII: [C35] The antibody-drug conjugate of claim 13, wherein in formula XII, the definitions of X, Y, Z', L and D are the same as those in claims 6 to 8, m is selected from the group consisting of 0 or 1, n is selected from the group consisting of 1 or 2, Ab is an antibody, and the glycostructure linkage site is a conservative N-glycosylation site on the antibody Fc.

15. In the structures of formula XI and formula XII, the structure -Z'-LD is [C36] and wherein the antibody-drug conjugate is substituted with Z', L, L １ -L ６ and D. １ and D. ２ are the same as those in claims 1 to 3 and 6 to 8, respectively; Z ２ ', Z ３ ', Z ４ ', Z ５ ' and Z ' are the bioorthogonal groups of the functional molecules, respectively. ２ , Z ３ , Z ４ , Z ５ and D, which may not exist simultaneously or independently. ３ , D ４ The definition of D １ , D ２ and when D1-D4 have the same structure, the antibody-drug conjugate of formula XI or XII represents an antibody-drug conjugate with high drug loading (drug-to-antibody ratio, DAR value) carrying the same drug structure, and when D1-D4 are different, the antibody-drug conjugate of formula XI or XII represents an antibody-drug conjugate of a multi-drug format carrying different drug structural compositions. The antibody-drug conjugate of claim 13 or 14.

16. A method for producing an antibody-drug conjugate according to claim 13, comprising the following two steps: In method 1, a) co-incubating the disaccharide linker of claim 1 and a wild-type antibody, hydrolyzing the N-oligosaccharide structure of the wild-type antibody Fc domain Asn297 by catalytic action of the wild-type glycosidic endonuclease, and simultaneously linking the disaccharide linker to the Asn297 site of the antibody Fc domain; or co-incubating the disaccharide linker of claim 1, a deglycosylated antibody and a glycosidic endonuclease to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula IX modified with a disaccharide linker of formula I containing an orthogonal reactive group, wherein the deglycosylated antibody can be obtained by treating the wild-type antibody with a glycosidic endonuclease in advance, or by simultaneously removing fucose using a fucose hydrolase; b) coupling the fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula IX, which has been modified with a disaccharide linker of formula I containing an orthogonal reactive group obtained in step a), with a modified low molecular weight drug having a corresponding group capable of specific coupling reaction with the orthogonal reactive group to prepare an antibody-drug conjugate of formula XI; and co-incubating the disaccharide-small molecule drug conjugate according to claim 6 with a wild-type antibody, hydrolyzing the N-oligosaccharide structure at Asn297 in the wild-type antibody Fc domain by catalytic action of the wild-type glycoside endonuclease, and simultaneously linking the disaccharide-small molecule drug conjugate to the Asn297 site in the antibody Fc domain, or co-incubating the disaccharide-small molecule drug conjugate according to claim 6 with a deglycosylated antibody and a glycoside endonuclease to prepare an antibody-drug conjugate represented by formula XI, wherein the deglycosylated antibody may be obtained by treating the wild-type antibody with a glycoside endonuclease in advance, or by removing fucose using a fucose hydrolase at the same time.

17. A method for producing the antibody-drug conjugate according to claim 14 or 15, comprising the following two steps: In method 1, a) co-incubating the disaccharide linker according to claim 2 and a wild-type antibody, hydrolyzing the N-oligosaccharide structure of the wild-type antibody Fc domain Asn297 by catalytic action of the wild-type glycosidic endonuclease, and simultaneously linking the disaccharide linker to the Asn297 site of the antibody Fc domain; or co-incubating the disaccharide linker according to claims 2-3, a deglycosylated antibody and a glycosidic endonuclease to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide-modified antibody of formula X modified with a disaccharide linker of formula II containing an orthogonal reactive group, wherein the deglycosylated antibody may be obtained by treating the wild-type antibody with glycosidic endonuclease in advance, or by simultaneously removing fucose using fucose hydrolase; b) coupling the 1,6-acetylglucosamine disaccharide-modified antibody having or without fucose and having formula X, which has been modified with a disaccharide linker having formula II and containing an orthogonal reactive group obtained in step a), with a modified small molecule drug having a corresponding group capable of specific coupling reaction with the orthogonal reactive group to produce an antibody-drug conjugate having formula XII; In method 2, A method for preparing an antibody-drug conjugate, comprising co-incubating the disaccharide-small molecule drug conjugate according to claim 7 to 8 with a wild-type antibody, hydrolyzing the N-oligosaccharide structure at Asn297 of the wild-type antibody Fc domain by catalytic action of the wild-type glycoside endonuclease, and simultaneously linking the disaccharide-small molecule drug conjugate to the Asn297 site of the antibody Fc domain, or co-incubating the disaccharide-small molecule drug conjugate according to claim 7 to 8 with a deglycosylated antibody and a glycoside endonuclease to prepare an antibody-drug conjugate represented by formula XII, wherein the deglycosylated antibody may be obtained by treating the wild-type antibody with a glycoside endonuclease in advance, or by removing fucose using a fucose hydrolase at the same time.

18. 18. The method of claim 16 or 17, the wild-type glycosidic endonuclease is N-acetylglucosamine endohydrolase, more preferably, the N-acetylglucosamine endohydrolase is endoglycosidase Endo-S2 or Streptococcus pyogenes-derived endoglycosidase Endo-S2, and when producing a compound with coreless fucosylation, a combination of glycosidic endonuclease and fucose hydrolase is required; Preferably, in the method 1, the orthogonal reactive group and the corresponding group capable of specific coupling reaction with the orthogonal reactive group are any combination selected from the group consisting of an azide group and an alkynyl group, a mercapto group and a maleimide, a mercapto group and a mercapto group or an activated form of a mercapto group, an aldehyde group and an amino group, an aldehyde group and an aminooxy group or a hydrazine group; Preferably, in step b) of method 1, the drug linker has the following group for coupling with a small molecule drug modified with the corresponding group: [C37] Preferably, the correspondingly group-modified small molecule drug is selected from the group consisting of the following compounds: [C38] [C39] [C40] [C41] 18. The method of claim 16 or 17.

19. Method 1 is shown in the following reaction scheme: [C42] 17. The method of claim 16, wherein in the above reaction scheme, m is selected from the group consisting of 0 or 1, the definitions of X, Y, Z, Z', L, and D are the same as those in claims 1 to 3 or claims 6 to 8, respectively, and E is an orthogonal reactive group capable of reacting with Z, and the glycoengineered antibody in the reaction scheme is obtained by the method of claim 11.

20. Method 1 is shown in the following reaction scheme: [C43] In the above reaction scheme, m is selected from the group consisting of 0 or 1, the definitions of X, Y, Z, Z', L, and D are the same as those in claims 1 to 3 or claims 6 to 8, respectively, and E is an orthogonally reactive group capable of reacting with Z, and the glycoengineered antibody in the reaction scheme is obtained by the method according to claim 12, Preferably, the method is as shown in the following reaction scheme: [C44] The definitions of L and D are the same as those in claims 6 to 8, and E ３ is a corresponding group which reacts orthogonally with the aldehyde group and is selected from the group consisting of cyclosulfidopyrazole, orthoaminobenzamidoxime, and hydroxylamine structures, e.g. [C45] and X ２ is an aldehyde group and E ３ is the structure formed by the reaction of E ５ is a corresponding group that reacts orthogonally with the azide group, and is selected from the group consisting of a linear alkynyl group, a DBCO-based structure, and a BCN-based structure; X ４ is an azide group and E ５ The method of claim 16 , wherein:

21. The method 2 is As shown in the following reaction formula, the method includes co-incubating a glycoside endonuclease, an antibody, and the disaccharide-small molecule drug conjugate according to claim 6 to hydrolyze the N-oligosaccharide at the conservative glycosylation site Asn297 of the antibody Fc domain and simultaneously transferring the disaccharide-small molecule drug conjugate to Asn297 (method 1); or co-incubating the disaccharide-small molecule drug conjugate according to claim 6, a deglycosylated antibody, and a glycoside endonuclease (method 2), thereby realizing glycoside-specific and quantitative introduction of a small molecule drug, and obtaining a corresponding antibody-drug conjugate, wherein the deglycosylated antibody can be obtained by treating a wild-type antibody with a glycoside endonuclease in advance, or by simultaneously removing fucose using a fucose hydrolase. [C46] 17. The method of claim 16.

22. The method 2 is As shown in the following reaction scheme, the method includes co-incubating a glycoside endonuclease with an antibody and the disaccharide-small molecular drug conjugate according to claims 6 to 8, hydrolyzing the N-oligosaccharide at the conservative glycosylation site Asn297 of the antibody Fc domain and transferring the disaccharide-small molecular drug conjugate to Asn297 (Scheme 1); or co-incubating the disaccharide-small molecular drug conjugate according to claims 6 to 8, a deglycosylated antibody and a glycoside endonuclease (Scheme 2), thereby realizing glycoside-specific and quantitative introduction of a small molecular drug, and obtaining a corresponding antibody-drug conjugate, wherein the deglycosylated antibody can be obtained by treating a wild-type antibody with a glycoside endonuclease in advance, or by simultaneously removing fucose using a fucose hydrolase: [C47] 20. The method of claim 17.

23. Use of a disaccharide linker according to any one of claims 1 to 3 or a disaccharide-small molecule drug conjugate according to any one of claims 6 to 8 in antibody glycoengineering or in the manufacture of antibody-drug conjugates.

24. 16. Use of the antibody-drug conjugate of any one of claims 13 to 15 in the manufacture of a drug, pharmaceutical composition or diagnostic reagent, wherein the drug in the conjugate is selected from the group consisting of anti-tumor, anti-inflammatory, anti-viral, anti-infectious disease or other immunotherapy drugs.

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