Disaccharide linkers, disaccharide-small molecule drug conjugates, and glycoside-specific antibody-drug conjugates, methods for producing the same, and uses

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 drug chemistry and biotechnology, and more specifically to disaccharide linkers, non-natural glycotechnology antibodies produced using these disaccharide linkers, and specific and quantitative antibody-drug conjugates based on glycans, as well as methods for producing and using these. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are composed of antibodies, cytotoxins, and linkers. They exert antitumor activity by delivering cytotoxins to tumor tissue through the antibody, achieving targeted toxin delivery. Compared to conventional chemotherapy drugs, ADCs have lower biological toxicity and superior therapeutic index. In early ADCs, cytotoxins were mainly bound to lysine (Lys) and cysteine ​​(Cys), which are abundant in the antibody, by random coupling. ADCs formed in this way have uneven toxin binding sites and amounts, resulting in poor in vivo stability, efficacy, pharmacokinetic properties, and a narrow therapeutic range. Glycoside-specific ADCs can solve these problems. The main specific coupling technologies used in the production of glycoside-specific ADCs include the THIOMAB method, non-natural amino acid insertion methods, enzyme-catalyzed methods, and glycoside-specific methods, each of which has its own 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's Fc domain. Currently, in vitro antibody glycosylation site modification methods mainly include glycosyltransferase technology and glycoside endonuclease technology.

[0004] Glycosyltransferase technology uses galactose transferase or sialic acid transferase to transfer galactose or sialic acid with a reactive functional group to the antibody glycan site, and then couples it with a cytotoxin to produce glycoside-specific ADC compounds. For example, Zhu et al. first hydrolyzed the terminal galactose of the N-glycan of the antibody glycosylation site using β1,4-galactosidase, then used galactose transferase to transfer ketocarbonyl-containing GalNAc to the N-glycan end, and then coupled it with a toxin containing hydroxyamine to obtain a glycoside-specific ADC compound. Qun Zhou et al. used galactose transferase and sialic acid transferase to sequentially transfer galactose and sialic acid to the antibody glycosylation site, then oxidized the terminal sialic acid with sodium periodate to introduce an aldehyde group to the glycosylation site, providing a reaction site for toxin coupling. Floris L. van Delft et al. hydrolyzed the heterogeneous N-glycan chain of the antibody using the glycoside endonuclease Endo-S, then transferred azide-containing GalNAz to the glycosylation site of the antibody using galactose transferase, and finally modified the glycosylation site with a toxin via a click chemistry reaction.

[0005] In glycoside endonuclease technology, glycoside endonucleases and bioorthogonal reactions are used to achieve the production of glycoside-specific ADCs. Our team and Davis's team obtained azide-modified oligosaccharide oxazoline substrates using semi-synthetic modifications, and then sequentially used two glycoside endonucleases, Endo-S and its mutant enzyme Endo-S D233Q, to transfer the bioorthogonal-modified oligosaccharide to the glycosylation site of the antibody, and further obtained glycoside-specific ADC compounds using bioorthogonal reactions.

[0006] While existing glycosyltransferase and glycoside endonuclease technologies can produce more homogeneous ADC compounds compared to random coupling, each has its own drawbacks. Glycosyltransferase technology requires the synthesis of glycoside substrates in the form of activated CMP or UDP, and since glycosyltransferases tend to have weak catalytic activity, reaction times are long, making it difficult to control manufacturing 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 or semi-synthetic modification, and total synthesis is even more difficult. Both glycosyltransferase and glycoside endonuclease technologies involve multiple enzymes and multi-step reactions, limiting their effectiveness and productivity, and not contributing to antibody stability. In addition, both technologies heavily rely on orthogonal reactions to achieve toxic modification of the glycosylation site of the antibody, which limits the drug development of glycoside-specific ADCs.

[0007] This patent invents a series of disaccharide linkers that can efficiently transfer to the glycosylation site of an antibody under the action of the wild-type glycoside endonuclease Endo-S2. When the disaccharide structure has bioorthogonal groups, glycosylated antibodies with bioorthogonal groups can be obtained using enzyme catalysis, and glycoside-specific ADC compounds based on the disaccharide structure can be produced in "two steps" by utilizing bioorthogonal reactions. 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, and as a result, novel ADC molecules with good activity in vivo and in vitro can be obtained. [Overview of the project] [Means for solving the problem]

[0008] A technical object of the present invention is to provide a disaccharide linker capable of specifically and quantitatively introducing a small molecule drug into an antibody. Another technical object of the present invention is to provide the 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 molecule drug conjugate. Another technical object of the present invention is to provide a small molecule drug-antibody conjugate linked by the disaccharide linker. Another technical object of the present invention is to provide the use of the disaccharide linker-small molecule drug conjugate or the small molecule drug-antibody conjugate linked by the disaccharide linker in the production of pharmaceutical or diagnostic reagents.

[0009] In one embodiment, the present invention provides a disaccharide linker represented by the following formula I. [ka] I

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

[0011] ZYX- indicates a substituent on the G ring, and the substitution position of ZYX- is any position on the G ring other than position 1 derived from the monosaccharide molecule. Here, in the structure ZYX-, ZY- may or may not be present, and if ZY- is absent, X is an aldehyde group, a phosphate group, -NH2, -CH2-NH2, -COOH, or -CH2SR. p -CH2SeR p , -N3, -CH2-N3, 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 that connects X and Z, and preferably Y is a group consisting of -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, and -(PO4)n-. Or a combination of a severable fragment and the above-mentioned connected fragment. The following is selected: Here, m and n are each independently selected from the group of integers between 0 and 30, and w is a hydrogen atom, and the polyethylene glycol structure is of various lengths.

[0013] Z is selected from the group consisting of the following cases i) to iv). i) Fragments having bioorthogonal reactive groups or functional molecules Preferably, Z is selected from the group consisting of the following reactive groups: azide residues, aldehyde residues, thiol residues, alkyne residues, alkene residues, halogen residues, tetoazine residues, nitrone residues, hydroxyamine residues, nitrile residues, hydrazine residues, ketone residues, boric acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isocyanide residues, cydonone residues, selenium residues, conjugated diene residues, phosphate residues, cycloalkyne residues, and cycloalkene residues. Alternatively, Z is selected from the group consisting of the following groups. [ka] Here, n is an integer from 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, nanoantibodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotopes, and contrast agents and magnetic resonance imaging agents.

[0014] ii) [ka] Here, L1 is a trivalent linker having three reactive groups. Preferably, L1 is a branched-chain amino acid having reactive functional groups derived from lysine, aspartic acid, glutamic acid, propargylglycine, cysteine, and the following structure. [ka] Here, n is an integer from 1 to 30. L2 and L3 are divalent or multivalent linkers that connect L1 to Z2 and Z3. Preferably, L2 and L3 are independently -(CH2)m-(CH-w)n-, -(CH2-CH2-O)m-(CH-w)n-, and -(PO4)n- structures, respectively. Or a combination of a severable fragment and the above-mentioned connecting fragment. The group is selected from the following, and 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, for example, polyethylene glycol of various lengths. Z' is a linking fragment that couples L1 and the sugar linker, and is independently a non-existent -(CH2)p- or a group reactable with the Z group in case i), and p is an integer between 1 and 5. For example, Z' is selected from the group consisting of the following groups: [ka] Here, R1 and R2 are independently selected from the group consisting of H, -CH3, -CH2CH3, cyclopropyl, or cyclobutyl. The definitions of Z2 and Z3 are the same as the definition of Z in case i).

[0015] iii) [ka] Here, L6 is a tetravalent linker having four reactive groups. Preferably, L6 is selected from the group consisting of dilysine, diglutamic acid, diaspartic acid, aspartic acid-glutamic acid dipeptide structure, aspartic acid-lysine dipeptide structure, and glutamic acid-lysine structure, or 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] Selected from the group consisting of the following: Here, R1 is a hydroxy-OH or azide group -N3, R2 is any group, R3 is either a hydroxy-OH or -NH- containing group R4 is any group, and the dashed line indicates the linkage site.

[0017] In a specific embodiment, the disaccharide linker of formula I is represented by the following formula II. [ka] II In Equation II, X, Y, and X are as defined above.

[0018] In specific embodiments, the disaccharide linker is selected from the group consisting of the following specific compounds. [ka] JPEG0007848224000013.jpg55144 Here, R is a fragment or combination relating to Y and Z above, and l, m, and n are each independent integers between 0 and 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 represented by the following reaction formula. [ka]

[0021] In the above reaction equation, the G ring is as defined above. The modification sites of the monosaccharide are any other modifiable sites other than position 1. U is the introduced active group, selected from the group consisting of aldehyde groups, amino groups, azide groups, and alkynyl groups. The definitions of X, Y, and Z are as defined above.

[0022] The aforementioned method, 1) A disaccharide structure having an acetylglucosamine structure at its terminus is modified by the action of an enzyme or other small molecule compound to obtain a disaccharide structure having an active group U, and after this disaccharide structure having an active group U is derivatized, ZYX- containing orthogonal reactivity or a functional molecular fragment is introduced. 2) A step to obtain a disaccharide linker represented by formula I by introducing ZYX- which has orthogonal reactivity or contains a functional molecular fragment, through a cyclization step, 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 specific embodiments, the derivatization reaction in step 1) is an oxime reaction, a reductive amination, an amino group-involved reaction, or an azide group-involved reaction.

[0025] In a specific embodiment, in step 2), a 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 embodiment, the present invention provides a disaccharide-small molecule drug conjugate having a structure represented by the following formulas III, IV, or V. [ka] III JPEG0007848224000019.jpg3165 IV JPEG0007848224000020.jpg3670 V

[0027] In formulas III, IV, and V above, the G ring, X, Y, Z3, Z', L1, L2, and L3 are 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 a bioorthoth 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 divalent linker that links D, D1, or D2 to the rest of formulas III-V, and preferably L is selected from the group consisting of -(CH2)a-(CH2CH2)b-(NHCO)n-(CH2)c- or from the group consisting of the following groups. [ka] JPEG0007848224000022.jpg5698 JPEG0007848224000023.jpg74115 [ka] JPEG0007848224000025.jpg30121 JPEG0007848224000026.jpg25131 JPEG0007848224000027.jpg26134 Here, V and W are dual-function linkers, and the structure includes lysine and propargylglycine as dual-function linkers. For example, L is [ka] JPEG0007848224000029.jpg44139 JPEG0007848224000030.jpg35145 R3 and R4 are 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 azidable monosaccharides, disaccharides, oligosaccharides, or PEG structures of various lengths having an azide group, or combinations of PEG with chain-like or cyclic monosaccharides, disaccharides, or oligosaccharides. Here, the oligosaccharides include branched oligosaccharide chains. The dashed 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, wherein the small molecule drug is preferably selected from the group consisting of mytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, docamycin, amanitin, PBD derivatives, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, colchicine, estramustine, semadolin, eloiterobin, fluorescent reagents, monosaccharides, disaccharides, oligosaccharides, and derivatives of the above compounds. Alternatively, the small molecule drug is a radiotherapeutic agent. Preferably, D, D1, and D2 each independently represent the group consisting of the following groups. [ka] JPEG0007848224000032.jpg23129 JPEG0007848224000033.jpg26129 JPEG0007848224000034.jpg31112 JPEG0007848224000035.jpg24118 JPEG0007848224000036.jpg24117

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[0028] In specific embodiments, the disaccharide-small molecule drug conjugate is represented by the following formulas VI, VII, and VIII: [ka] VI JPEG0007848224000059.jpg4260 VII JPEG0007848224000060.jpg4762 VIII The definitions of each substituent in formulas VI, VII, and VIII are as defined above.

[0029] In specific embodiments, the disaccharide-small molecule drug conjugate is selected from the group consisting of the following compounds. [ka] Compound DG-1 JPEG0007848224000062.jpg50142 Compound DG-2 JPEG0007848224000063.jpg54133 Compound DG-3 [ka] Compound DG-4 JPEG0007848224000065.jpg62124 Compound DG-5 JPEG0007848224000066.jpg50121 Compound DG-6 [ka] Compound DG-7 JPEG0007848224000068.jpg56142 Compound dDG-1 JPEG0007848224000069.jpg52136 Compound dDG-2 [ka] Compound dDG-3

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

[0031] In yet another embodiment, the present invention provides a glycosylation antibody based on the specific linkage of an antibody Fc domain N-glycosylation site represented by the following formula IX. [ka] IX Here, in formula IX above, the definitions of the G ring 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 derived from a different species. Preferably, Ab is selected from the group consisting of the following options: trastuzumab, pertuzumab, rituximab, cetuximab, morozumab, gemtuzumab, absiximab, darizumab, adalimumab, palizumab, valiximab, bevacizumab, panitumumab, nitrotuzumab, denituzumab, decitumab, lemonibisumab, nexituzumab, eprilimus, daremab, ventocibizumab, alemtuzumab, erlotuzumab, bonatumab, nivolumab, pembrolizumab, atezolizumab, avilumab, dalvalumab, tremelimumab, catumab, verintumomab, emicizumab, and evantozumab (Rybrevant).

[0032] In a specific embodiment, the glycosylation-engineered antibody is represented by 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 glycosylated antibody, wherein the method is carried out by the following Method 1 or Method 2. [ka] Here, in the above reaction equation, m is selected from the group consisting of 0 or 1, and the definitions of the G ring, X, Y, and Z are as defined above.

[0034] Method 1: Wild-type antibodies are hydrolyzed with glycoside endonuclease or a combination of glycoside endonuclease and glucosidase to remove heterogeneous sugar chains from the conservative glycosylation site of the natural antibody, thereby obtaining a deglycosylated antibody. Subsequently, the disaccharide linker and the wild-type antibody are co-incubated, and the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain by catalytic action of wild-type glycoside endonuclease, thereby producing a 1,6-acetylglucosamine disaccharide modified antibody represented by formula IX, which is either fucose-containing or fucose-free, modified with a disaccharide linker represented by formula I containing an orthogonal reactive group. Method 2: The disaccharide linker and wild-type antibody described above are co-incubated, and the N-oligosaccharide structure of the wild-type antibody Fc domain is hydrolyzed by the catalytic action of wild-type glycoside endonuclease. Simultaneously, the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain, thereby producing a 1,6-acetylglucosamine disaccharide modified antibody represented by formula IX, which may or may not contain fucose, modified with a disaccharide linker represented by formula I containing an orthogonal reactive group.

[0035] Preferably, the wild-type glycoside endonuclease is N-acetylglucosamine endohlase, and more preferably, the N-acetylglucosamine endohlase is Endo-S2 (Endoglycosidase-S2), for example, Endo-S2 endoglycosidase derived from Streptococcus pyogenes. When producing coreless fucose compounds, the combined use of glycoside endonuclease and fucose hydrolase is required.

[0036] In a specific embodiment, the glycosylated antibody is produced by the following method 1 or method 2. [ka] Here, in the above reaction equation, m is selected from the group consisting of 0 or 1, and the definitions of X, Y, and Z are as defined above, respectively.

[0037] Method 1: Wild-type antibodies are hydrolyzed with glycoside endonuclease or a combination of glycoside endonuclease and glucosidase to remove heterogeneous sugar chains from the conservative glycosylation site of the natural antibody, thereby obtaining a deglycosylated antibody. Subsequently, the disaccharide linker and the wild-type antibody are co-incubated, and the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain by catalytic action of wild-type glycoside endonuclease, thereby producing a 1,6-acetylglucosamine disaccharide modified antibody represented by formula X, which is either fucose-containing or fucose-free, modified with a disaccharide linker represented by formula II containing an orthogonal reactive group. Method 2: The disaccharide linker and wild-type antibody described above are co-incubated, and the N-oligosaccharide structure of the wild-type antibody Fc domain is hydrolyzed by the catalytic action of wild-type glycoside endonuclease. Simultaneously, the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain, thereby producing a 1,6-acetylglucosamine disaccharide modified antibody represented by formula X, which is either fucose-containing or fucose-free, modified with a disaccharide linker represented by formula II containing an orthogonal reactive group.

[0038] Preferably, the wild-type glycoside endonuclease is N-acetylglucosamine endohlase, and more preferably, the N-acetylglucosamine endohlase is Endo-S2 (Endoglycosidase-S2), for example, Endo-S2 endoglycosidase derived from Streptococcus pyogenes. When producing coreless fucose compounds, the combined use of glycoside endonuclease and fucose hydrolase is required.

[0039] In yet another embodiment, the present invention provides an antibody-drug conjugate represented by the following formula XI. [ka] XI In formula XI, the definitions of the G ring and X, Y, Z', L, and D 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 an antibody, and the sugar linkage site is a conservative N-glycosylation site on antibody Fc.

[0040] In a specific embodiment, the antibody-drug conjugate is represented by the following formula XII. [ka] XII In formula XII, the definitions of X, Y, Z', L, and D 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 an antibody, and the sugar structure linkage site is a conservative N-glycosylation site on antibody Fc.

[0041] Furthermore, the present invention further states that in the structures of formulas XI and XII, the -Z'-LD structure is [ka] JPEG0007848224000079.jpg28143 The present invention provides an antibody-drug conjugate substituted with Z', L, L1-L6, and D1 and D2, respectively, as defined above. Z2', Z3', Z4', and Z5' are linked fragments formed by the reaction of the bioorthoth group of the functional molecule with Z2, Z3, Z4, and Z5, respectively, and may exist simultaneously or independently. The definitions of D3 and D4 are the same as those of D1 and D2. If the structures of D1-D4 are the same, the antibody-drug conjugate of formula XI or XII represents a high drug load (drug-to-antibody ratio, DAR value) antibody-drug conjugate carrying the same drug structure. If D1-D4 are different, the antibody-drug conjugate of formula XI or XII represents a multi-drug form antibody-drug conjugate carrying different drug structural compositions.

[0042] In another embodiment, the present invention provides a method for producing the antibody-drug conjugate described above, comprising the following two methods. Method 1: a) The above disaccharide linker and wild-type antibody are co-incubated to hydrolyze the wild-type antibody Fc domain Asn297 N-oligosaccharide structure by catalytic action of wild-type glycoside endonuclease, and simultaneously link the disaccharide linker to the antibody Fc domain Asn297 site, or the above disaccharide linker, deglycosylated antibody, and glycoside endonuclease are co-incubated to produce a 1,6-acetylglucosamine disaccharide modified antibody represented by formula IX, which is fucose-containing or non-containing, modified with a disaccharide linker represented by formula I containing an orthogonal reactive group. Here, the deglycosylated antibody may be obtained by pre-treating the wild-type antibody with glycoside endonuclease, or by simultaneously removing fucose using fucose hydrolase. b) The antibody, which is a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide modified with a disaccharide linker represented by formula I containing an orthogonal reactive group obtained in step a), is coupled with a modified small molecule drug having a corresponding group that can specifically couple with the orthogonal reactive group to produce an antibody-drug conjugate of formula XI or XII. Method 2: The above disaccharide-small molecule drug conjugate and wild-type antibody are co-incubated, and the wild-type antibody Fc domain Asn297 N-oligosaccharide structure is hydrolyzed by the catalytic action of wild-type glycoside endonuclease, simultaneously linking the disaccharide-small molecule drug conjugate to the antibody Fc domain Asn297 site, or the above disaccharide-small molecule drug conjugate, deglycosylated antibody, and glycoside endonuclease are co-incubated to produce an antibody-drug conjugate of formula XI or XII. Here, the deglycosylated antibody may be obtained by pre-treating the wild-type antibody with glycoside endonuclease, or by simultaneously removing fucose using fucose hydrolase.

[0043] In a specific embodiment, the wild-type glycoside 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 core fucosylated compound, it is necessary to use a combination of a glycoside endonuclease and a fucosidase.

[0044] In a specific embodiment, in the method 1, the orthogonal reactive group and the corresponding group capable of specifically coupling with this 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 the method 1, the drug linker has the following groups so as to couple with a low-molecular-weight drug modified with the corresponding group.

Chemical formula

[0046] In a specific embodiment, the low-molecular-weight drug modified with the corresponding group is selected from the group consisting of the following compounds.

Chemical formula

[0047] In a specific embodiment, the method 1 is represented by the following reaction formula. [Chemical] Here, in the above reaction formula, m is selected from the group consisting of 0 or 1, and the definitions of X, Y, Z, Z’, L, and D are as defined above. E is an orthogonal reactive group capable of reacting with Z. Here, the glycoengineered antibody in the reaction formula can be obtained by the method described above. [[ID=#64]]

[0048] In a specific embodiment, the method 1 is represented by the following reaction formula. [ka] Here, in the above reaction equation, m is selected from the group consisting of 0 or 1, and the definitions of X, Y, Z, Z', L, and D are as defined above. E is an orthogonal reactive group that can react with Z, and the glycosylation antibody in the reaction equation is obtained by the method described above.

[0049] In a specific embodiment, the manufacturing method is represented by the following reaction equation. [ka] Here, the definitions of L and D are as defined above. E3 is a corresponding group that reacts orthogonally with the aldehyde group and is selected from the group consisting of cyclosulfidopyrazole, orthoaminobenzamidoxime, and hydroxyamine structures, for example, [ka] X2 is a structure formed by the reaction of an aldehyde group with E3, and E5 is a corresponding group that reacts orthogonally with an azide group, selected from the group consisting of a linear alkynyl group, a DBCO system structure, and a BCN system structure. X4 is a structure formed by the reaction of an azide group with E5.

[0050] In specific embodiments, Method II involves co-incubating a glycoside endonuclease, an antibody, and the disaccharide-small molecule drug conjugate as shown in the following two reaction equations, thereby hydrolyzing the N-oligosaccharide at the Asn297 position of the antibody Fc domain's conservative glycosylation site and simultaneously transferring the disaccharide-small molecule drug conjugate to the Asn297 position (Method 1), or co-incubating the disaccharide-small molecule drug conjugate, a deglycosylating antibody, and a glycoside endonuclease (Method 2) to achieve glycoside-specific and quantitative introduction of a small molecule drug and obtain a corresponding antibody-drug conjugate. Here, the deglycosylating antibody may be obtained by pre-treating a wild-type antibody with a glycoside endonuclease, or by simultaneously removing fucose using fucose hydrolase. [ka] JPEG0007848224000099.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 glycosylation modification or in the production of antibody-drug conjugates.

[0052] In one embodiment, the present invention provides a use of the antibody-drug conjugate in the manufacture of a drug, pharmaceutical composition or diagnostic reagent, wherein the drug in the conjugate is selected from the group consisting of antitumor, anti-inflammatory, antiviral, anti-infective disease or other immunotherapeutic drugs. [Effects of the Invention]

[0053] This invention enhances and develops new linking methods and linking structures based on glycoside-specific structural modification. By introducing other reactions such as amide reactions in addition to orthogonal reactions in the disaccharide structure and drug-linker, it constructs a new form of specific quantitative antibody-drug conjugate, improving the stability and cytotoxicity of the antibody-drug conjugate and achieving superior drug formation characteristics.

[0054] This invention utilizes the fact that wild-type glycoside endonucleases possess both hydrolytic activity towards N-oligosaccharides in the conservative glycosylation site of antibodies and transglycosylation activity towards specific novel disaccharide linker structures. It designs and manufactures disaccharide linkers with orthogonal reactive groups, drug-linkers, or dual drug-linkers, and achieves specific insertion of the orthogonal reactive groups into antibodies via enzyme-catalyzed reactions. Furthermore, it enables specific coupling with low-molecular-weight drugs, or direct, one-step specific quantitative coupling of low-molecular-weight drugs via enzyme-catalyzed reactions. This process eliminates the need for prior hydrolysis of heterogeneous N-oligosaccharides in the conservative glycosylation site of the wild-type antibody Fc domain, resulting in fewer purification steps, simplicity, and ease of industrial production.

[0055] The specifically linked antibody-drug conjugates represented by formulas IX and X, produced from the disaccharide linkers represented by formulas I and II, have a homogeneous chemical structure that allows for specific quantification. Compared to approved antibody-drug conjugates with heterogeneous structures, they offer the advantages of a clearly defined single structure and controllable quality. Furthermore, this manufacturing method has advantages over other specific coupling methods, such as ease of operation and fewer purification steps, good drug formation properties, and ease of industrialization. In addition, the specifically linked antibody-drug conjugates exhibit good antitumor activity. [Brief explanation of the drawing]

[0056] [Figure 1] The results of the initial screening of sugar substrates and glycoside endonucleases in this application are shown. A: Screening of the antibody transglycosylation activity of various glycoside endonucleases against G14; B: Screening of the ability of glycoside endonuclease Endo-S2 to identify various sugar substrates and transfer them to the antibody glycosylation site. [Figure 2]The following are the results of in vitro activity experiments for ADCs linked with some of the disaccharide linkers of this invention. A: Inhibition rate of some gsADCs (gsADC-5, gsADC-21, gsADC-30, gsADC-36) against SK-Br-3 cells, B: Inhibition rate of some gsADCs (gsADC-35, gsADC-34, gsADC-38) against SK-Br-3 cells, C: Inhibition rate of some gsADCs (gsADC-5, gsADC-21, gsADC-30, gsADC-36) against NCI-N87 cells, D: E: Inhibition rate of some gsADCs (gsADC-35, gsADC-34, gsADC-38) on 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] The results of in vivo activity experiments for ADCs linked with some of the disaccharide linkers of this invention are shown. The inhibition status of gsADC-21, gsADC-30, gsADC-35, and gsADC-36 on in vivo tumor volume and their effects on nude mouse body weight are shown, respectively. [Modes for carrying out the invention]

[0057] Terminology: In this application, "multivalent linker" refers to a linker with a valency exceeding two.

[0058] The glycosidases used in this invention were expressed in an E. coli strain and were expressed in the laboratory. The small molecule cytotoxic drugs DM1 and MMAE used in this invention were purchased from Shanghai Zeishan Pharmaceutical Technology Co., Ltd. The DBCO and BCN compounds were purchased from Chengdu Baierkang 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 Biochemistry Co., Ltd. BTTAA was purchased from Taizhou Gelinmeike Co., Ltd. Galactose oxidase, catalase, and horseradish peroxidase were purchased from Biotechnology (Shanghai) Co., Ltd. The amino acid compounds were purchased from Shanghai Jier Biochemistry Co., Ltd. 4-pentic acid was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. Unless otherwise specified, all other compounds and reagents were purchased from Sinopharmaceutical Group Chemical Reagents Co., Ltd.

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

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

[0061] The following outlines the production routes for the disaccharide linker, glycosylation-engineered antibody, and glycoside-specific antibody-drug conjugate (ADC) described in this application, using a schematic reaction diagram.

[0062] General manufacturing example The disaccharide linker synthesis route 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. eO-(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. hO-(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 active ester, pH 7.4. m. CMP-sialic acid (as shown in compound 70 below), α-2,6-sialic acid transferase, 100 mM Tris buffer, pH 8.0. n. DBCO-CONHS 80, pH 7.4 / DMF. [ka] Note: g. Endo-S2, pH 7.0 buffer containing (or not containing) a certain amount of solubilizer such as DMSO, DMA, or DMF, 30°C. h. pH 7.0 buffer containing (or not containing) a certain amount of solubilizer such as DMSO, DMA, or DMF, general procedure 3 to general procedure 10.

[0063] General operations 1 Method for producing non-natural glycosylated 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 incubated at 30°C for 0.5 to 12 hours at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL respectively, with the pH of the reaction system adjusted to 7.0. The mixture was then incubated at 30°C for 0.5 to 12 hours, and subsequently purified with protein A to obtain the desired non-natural glycosylation antibodies Ab-1 to Ab-14. [ka] Ab-1 JPEG0007848224000103.jpg5389 Ab-2 JPEG0007848224000104.jpg5085 Ab-3 [ka] Ab-4 JPEG0007848224000106.jpg4679 Ab-5 JPEG0007848224000107.jpg5086 Ab-6 [ka] Ab-7 JPEG0007848224000109.jpg4782 Ab-8 JPEG0007848224000110.jpg4779 Ab-9 [ka] Ab-10 JPEG0007848224000112.jpg5290 Ab-11 JPEG0007848224000113.jpg46115 Ab-12 [ka] Ab-13 JPEG0007848224000115.jpg62101 Ab-14

[0064] General operations 2 Method for producing non-natural glycosylated antibodies 2 For example, as shown in Examples 51-55 below, the prepared derivatized disaccharide oxazolines (i.e., compounds G3, G8, G10, G13) and sialic acid-derived disaccharide oxazolines (i.e., compound G12), non-fucose antibodies, and wild-type glycoside endonuclease Endo-S2 were incubated at 30°C for 0.5 to 12 hours at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL respectively, with the pH of the reaction system adjusted to 7.0. The mixture was then incubated at 30°C for 0.5 to 12 hours, and subsequently purified with protein A to obtain the desired non-natural glycosylation antibodies Ab-15 to Ab-19. [ka] Ab-15 JPEG0007848224000117.jpg4881 Ab-16 JPEG0007848224000118.jpg42130 Ab-17 [ka] Ab-18 JPEG0007848224000120.jpg5594 Ab-19

[0065] General operations 3 One-step method for producing glycoside-specific antibody-drug conjugates (ADCs) For example, as shown in Examples 89-96 below, the prepared drug-linker-containing 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 incubated at 30°C for 0.5 to 12 hours at concentrations of 0.5 mM, 5 mg / mL, and 0.4 mg / mL respectively, with the reaction system pH adjusted to 7.0. After incubation at 30°C for 0.5 to 12 hours, and confirmation of complete conversion to the product by LC-MS, the mixture was purified with protein A to obtain 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 disaccharide antibodies 1 For example, as shown in Example 56 below, the prepared aldehyde disaccharide antibody (i.e., non-natural glycosylation antibody Ab-2) and a drug-linker containing a 2-aminoaniline oxime group (i.e., compound D2) were incubated at 37°C with the pH adjusted to 7.0-7.4 so that their respective concentrations were sequentially 5 mg / mL and 0.3 mM. After confirming complete conversion to the product by LC-MS, the mixture was purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugate gsADC-1.

[0067] General operations 5 Method 2 for producing specific ADCs based on aldehyde disaccharide antibodies For example, as shown in Example 57 below, the prepared aldehyde disaccharide antibody (i.e., non-natural glycosylation antibody Ab-2) and aminooxy group-containing drug-linker (i.e., compound D1) were incubated at 37°C at a pH of 7.0-7.5 so that their respective concentrations were sequentially 5 mg / mL and 0.3 mM. After confirming complete conversion to the product by LC-MS, the mixture was 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 disaccharide antibodies 3 For example, as shown in Examples 58-59 below, EDTA and 10% TritonX-100 were added to the prepared aldehyde disaccharide antibody (i.e., non-natural glycosylation antibody Ab-2) and thioPz group-containing drug-linker (i.e., compounds D3-D4) so ​​that their concentrations were sequentially 5 mg / mL and 0.3 mM, with final concentrations of 0.5 mM and 1%. The pH was adjusted to 5.5, incubated at 37°C, and confirmed by LC-MS to be completely converted to the product. The mixture was then purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugates gsADC-3 to gsADC-4.

[0069] General operations 7 Method 1 for producing specific ADCs based on azidonissaccharide antibodies For example, as shown in Examples 60-76 below, the prepared azid disaccharide antibodies (i.e., non-natural glycosylation antibodies Ab-3, Ab-4, Ab-6, Ab-9, Ab-15) and DBCO-based drug-linkers (i.e., compounds D6-D9, D13) were incubated at 37°C with the pH adjusted to 7.4 so that their respective concentrations were sequentially 5 mg / mL and 0.3 mM. After confirming complete conversion to the product by LC-MS, the mixture was 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 2 for producing specific ADCs based on azidonissaccharide antibodies As shown in Examples 77-84 below, the prepared azid disaccharide antibodies (i.e., non-natural glycosylation antibodies Ab-3, Ab-4, Ab-6, Ab-9, Ab-14, Ab-15) and BCN-based drug-linkers (i.e., compounds D5, D11, D12) were incubated at 37°C with the pH adjusted to 7.4 so that their respective concentrations were sequentially 5 mg / mL and 0.3 mM. After confirming complete conversion to the product by LC-MS, the mixture was purified with protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugates gsADC-21~gsADC-24, gsADC-39, gsADC-41~gsADC-43.

[0071] General operations 9 Method 3 for producing specific ADCs based on azidonissaccharide antibodies For example, as shown in Examples 85-89, the prepared azido disaccharide antibodies (i.e., non-natural glycotechnology antibodies Ab-3, Ab-4, Ab-6, Ab-9) and linear alkynyl group drug-linker (i.e., compound D10) were sequentially added to a pH of 7.4 so that their respective concentrations were 5 mg / mL and 0.3 mM. 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 then added to achieve a final concentration of 0.5 mM. The mixture was incubated at 37°C, and after confirming complete conversion to the product by LC-MS, it was purified using protein A to obtain the desired glycoside-specific quantitative antibody-drug conjugates gsADC-25 to gsADC-28.

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

[0073] The following describes the specific steps for producing each disaccharide linker, glycosylation-engineered antibody, and glycoside-specific ADC using the general manufacturing method described above, with reference to specific examples.

[0074] I: Manufacturing of disaccharide linker Example 1: Synthesis of compounds G1-G2 The structures and synthesis methods of compounds G1-G2 are as follows. [ka] JPEG0007848224000122.jpg2651 Compound G1 Compound G2 JPEG0007848224000123.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 system and mixed uniformly. The mixture was then cooled to 0°C, potassium phosphate (166 mg, 0.783 mmol) was added, and ddH2O was added to bring the total volume to 1044 μL. The reaction was carried out at 0°C for 2 hours. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain compound G1 (dissolved in water to make a 50 mM stock), which was then divided into smaller portions and stored at -80°C. HRMS calculation value C 14 H 23 NO10 [M+H] + 366.14, measured value 366.1322.

[0076] Step 2: After blowing O2 into the compound G1 (5 mg, 274 μL of 50 mM stock) obtained in Step 1 for 10 minutes, 11.9 U of galactose oxidase GOase, 120 U of horseradish peroxidase HRP, and 2.38 kU of catalase Catalase were added, the reaction system was supplemented to 300 μL, and after 4 hours at 30 °C and 888 rpm, it was separated and purified by a P2 column, and after adding one equivalent of NaOH and freeze-drying, compound G2 (dissolved 50 mM stock, solvent is 50 mM PB, pH 7.0) was obtained. After aliquoting, it was stored at -80 °C. HRMS calculated value C 14 H 21 NO 10 [M+H] + 364.1243, measured value 364.1201.

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

Chemical formula

[0078] Step 1: Dissolve compound 1 (20 mg, 52.2 μmol) in 1 mL of 50 mM PB, dissolve it in a buffer solution with pH 7.0, blow O2 for 10 minutes, then add 47.6 U of galactose oxidase GOase, 480 U of horseradish peroxidase HRP, and 9.52 kU of catalase Catalase to the above reaction system, supplement the volume to 1.19 mL, and after 4 hours at 30 °C and 888 rpm, purify it using a P2 column and then freeze-dry to obtain compound 2 (18 mg, yield 90.5%). HRMS calculated value C 14 H 23 NO 11 [M+H] +382.1349, measured 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. Compound 12 (5.3 mg, 51.9 μmol) was added to the reaction system and reacted at 37°C for 2 hours. After confirming the completion of the reaction by LC-MS, compound 3 (20 mg, 91% yield) was obtained by isolation and purification using a semi-preparative C18 column. HRMS calculated value C 14 H 21 NO 10 [M+H] + 466.1785, measured value 466.1732.

[0080] Step 3: Compound 3 (20 mg, 43 μmol) was dissolved in 700 μL of 50 mM PB and then dissolved in pH 7.0 buffer. CDMBI (46.44 mg, 215 μmol) was added to the reaction system and mixed uniformly. After cooling 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 reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed. The precipitate was removed by centrifugation, and a reaction system containing compound G3 was obtained. HRMS calculation value C 16 H 25 N5O 10 [M+H] + 448.1679, measured 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 JPEG0007848224000127.jpg20140

[0082] Step 1: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 0.2 MPa, pH 6.0 buffer. Compound 13 (23.6 mg, 236 μmol) and sodium borohydride cyanohydride (NaCNBH3) (59.5 mg, 944 μmol) were added to the reaction system, and the mixture was reacted at 0°C for 4 hours. After confirming the completion of the reaction by LC-MS, the compound 4 (16 mg, yield 72.8%) was obtained after purification using a P2 column. HRMS calculated value C 17 H 31 N5O 10 [M+H] + 466.2149, measured 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 system and mixed uniformly. After cooling 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 reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain the supernatant containing compound G4. HRMS calculation value C 17 H 29 N5O9[M+H] + 448.2043, measured 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 JPEG0007848224000129.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 system and mixed homogeneously. Then sodium carbonate (2.6 mg, 23.6 μmol) was slowly added. After reacting at room temperature for 3 hours, nickel chloride hexahydrate (28 mg, 118 μmol) and sodium borohydride (26.8 mg, 0.7 mmol) were added to the system. The reaction system was left overnight at 4°C, centrifuged to remove the supernatant, washed twice with water, and the water washes were combined. After purification using a P2 column, the mixture was freeze-dried to obtain Compound 5 (15 mg, yield 83%). HRMS calculation value C 14 H 26 N2O 10 [M+H] + 383.1665, measured 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 system and mixed uniformly. After cooling 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 reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain the supernatant containing compound G5. HRMS calculated value C 14 H 24 N2O9[M+H] + 365.156, measured value 365.1521.

[0087] Example 5: Synthesis of Compound G6 The structure and synthesis method of compound G6 are as follows: [ka] Compound G6 JPEG0007848224000131.jpg17144

[0088] Step 1: Compound 5 (15 mg, 39.35 μmol) was dissolved in 500 μL of CH3OH / H2O=1:4 system. 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 sequentially added to the reaction system, and the mixture was reacted at 37°C for 4 hours. The reaction was confirmed to be nearly complete by LC-MS, and after isolation and purification using a P2 column, the compound was freeze-dried to obtain compound 6 (13 mg, yield 81.3%). HRMS calculated value C 14 H 24 N4O 10 [M+H] + 409.157, measured 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 system and mixed uniformly. After cooling 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 reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain the supernatant containing compound G6. HRMS calculated value C 14 H 22 N4O9[M+H] + 391.1465, measured value 391.1432.

[0090] Example 6: Synthesis of Compound G7 [ka] Compound G7 JPEG0007848224000133.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. Compound 15 (13 mg, 236 μmol) and sodium borohydride cyanohydride (59.5 mg, 944 μmol) were added to the reaction system, and the mixture was reacted at 0°C for 4 hours. The reaction was confirmed to be complete by LC-MS, and after isolation and purification using a P2 column, compound 7 (14 mg, yield 70.5%) was obtained. HRMS calculated value C 17 H 28 N2O 10 [M+H] + 421.1822, measured 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 system and mixed uniformly. After cooling 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. The formation of a large precipitate was observed, and the precipitate was removed by centrifugation to obtain the reaction system containing compound G7. HRMS calculation 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 JPEG0007848224000135.jpg18143

[0094] Step 1: Compound 2 (18 mg, 47.2 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer. Compound 16 (5.6 mg, 51.9 μmol) was added to the reaction system, and the reaction was carried out at 37°C for 2 hours. After confirming the completion of the reaction by LC-MS, compound 8 (18.1 mg, yield 88%) was obtained by isolation and purification using a semi-preparative C18 column. HRMS calculated value C 17 H 26 N2O 11 [M+H] + 435.1615, measured 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 system and mixed uniformly. After cooling 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. The formation of a large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain the supernatant containing compound G8. HRMS calculation value C 17 H 24 N2O 10 [M+H] + 417.1509, measured 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 JPEG0007848224000137.jpg21145

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

[0099] Step 2: Compound 9 (14 mg, 30.1 μmol) was dissolved in 500 μL of D2O. CDMBI (32.5 mg, 150.5 μmol) was added to the reaction system and mixed uniformly. After cooling to 0°C, potassium phosphate (96 mg, 0.45 mmol) was added, and D2O was added to bring the total volume to 602 μL. The reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain the supernatant containing compound G9. HRM calculation value C 16 H 25 N5O 10 [M+H] + 448.1679, measured value 448.1666.

[0100] Example 9: Synthesis of compound G10 [ka] Compound G10 JPEG0007848224000139.jpg21144

[0101] Step 1: Compound 2 (20 mg, 52.48 μmol) was dissolved in 200 μL of 50 mM PB, pH 7.4 buffer. Compound 18 (22.6 mg, 63 μmol) was added to the reaction system, and after standing at room temperature for 8 hours, the reaction was confirmed to be complete by LC-MS. Compound 10 (32.6 mg, yield 86%) was obtained by isolation and purification using a semi-preparative C18 column. HRMS calculated value C 28 H 46 N6O14 S[M+H] + 723.2871, measured value 723.2877.

[0102] Step 2: Compound 10 (10 mg, 13.85 μmol) was dissolved in D2O, and CDMBI (15 mg, 69.3 μmol) was added to the reaction system. After homogeneous mixing, the mixture was cooled to 0°C, potassium phosphate (44 mg, 208 μmol) was added, and D2O was added to bring the total volume to 277 μL. The reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain the supernatant containing compound G10. HRMS calculated value C 28 H 44 N6O 13 S[M+H] + 705.2765, measured value 705.2771.

[0103] Example 10: Synthesis of compound G11 [ka] Compound G11 JPEG0007848224000141.jpg20143

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

[0105] Step 2: Dissolve Compound 11 (17 mg, 32.4 μmol) in D2O, add CDMBI (35 mg, 162 μmol) to the above reaction system, mix uniformly, then cool to 0 °C, add potassium phosphate (103 mg, 0.486 mmol), add D2O to make the total volume 650 μL, and react at 0 °C for 2 hours. The formation of a large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain the supernatant containing Compound G11.HRMS calculated value C 22 H 31 N3O9S[M+H] + 514.1859, measured value 514.1880. 1 HNMR (600 MHz, heavy water) δ 7.32 (t, J = 7.6 Hz, 2H), 7.25 (dd, J = 7.9, 5.7 Hz, 3H),5.97 (d, J = 7.3 Hz, 1H),4.7 (m, 2H),4.39 - 4.15 (m, 2H),4.04 (m, 1H),3.70 (dd, J = 12.3, 2.5 Hz, 4H),3.56 (dd, J = 12.3, 6.4 Hz, 3H),3.51 - 3.43 (m, 1H),3.43 - 3.31 (m, 2H),1.97 (m, 3H).

[0106] Example 11: Synthesis of Compound G12

Chemical Structure

[0107] Step 1: Dissolve Compound 1 (10 mg, 26.1 μmol) and Compound 70 (16 mg, 26.1 μmol) in 1.5 mL of 100 mM pH 8.0 Tris buffer, add 30 μg of α2,6-sialyltransferase (α2,6-ST) to the above reaction system. After confirming the completion of the reaction by thin-layer chromatography, isolate and purify by P2 column to obtain Compound 71 (12.6 mg, yield 72%).HRMS calculated value C 25 H 42 N2O 19 [M+H] +675.246, measured value 675.2433.

[0108] Step 2: Compound 71 (2 mg, 2.97 μmol) was dissolved in D2O, CDMBI (3.2 mg, 14.83 μmol) was added to the reaction system, and after uniformly mixing, it was cooled to 0 °C, potassium phosphate was added (9.5 mg, 44.55 μmol), D2O was added to make the total volume 148 μL, and the reaction was carried out at 0 °C for 2 hours. The formation of a large amount of precipitate was observed, the precipitate was removed by centrifugation, and the supernatant containing compound G12 was obtained. HRMS calculated value C 25 H 40 N2O 18 [M+H] + 657.2354, measured value 657.2301. 1 1HnMR (600 MHz, heavy water) δ 5.71 (d, J = 7.3 Hz, 1H), 4.07 - 3.99 (m, 2H), 3.83 - 3.77 (m, 1H), 3.60 - 3.47 (m, 4H), 3.46 - 3.35 (m, 3H), 3.35 - 3.18 (m, 7H), 3.15 - 3.05 (m, 3H), 2.63 (m, 1H), 1.69 (d, J = 1.9 Hz, 3H), 1.66 (s, 3H), 1.30 (t, J = 12.1 Hz, 1H).

[0109] Example 12: Synthesis of Compound G13

Chemical formula

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

[0111] Step 2: Compound 81 (10 mg, 14.9 μmol) was dissolved in 500 μL of D2O. CDMBI (16.2 mg, 74.7 μmol) was added to the reaction system and mixed uniformly. After cooling to 0°C, potassium phosphate (31.6 mg, 149 μmol) was added, and D2O was added to bring the total volume to 600 μL. The reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain the supernatant containing compound G13. HRMS calculated value C 33 H 37 N3O 11 [M+H] + 652.2506, measured value 652.2501.

[0112] Example 13: Synthesis of Compound G14 [ka] Compound G14 JPEG0007848224000147.jpg99145

[0113] Step 1: Compound 82 (369 mg, 1 mmol) was dissolved in 2 mL of DMF, and HATU (1.52 g, 4 mmol), compound 3-azidopropylamine (500 μL, 50 mmol), and N,N-diisopropylethylamine DIPEA (1 mL, 5.9 mmol) were sequentially added and mixed homogeneously. The mixture was then reacted at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction. After separation and purification by semi-preparative C18 column, the compound was lyophilized to obtain compound 83 (yield 92%) as a white powder. HRMS calculated value C 26 H 31 N9O4[M+H] + 534.2499, actual measured 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. After lyophilization, compound 84 (yield 94%) was obtained. HRMS calculation value C 11 H 21 N9O2[M+H] + 312.1818, measured value 312.8763.

[0115] Step 3: Compound 84 (9 mg, 0.028 mmol) was dissolved in 500 μL of DMF, and Compound 2 (16.8 mg, 0.044 mmol) and NaCNBH3 (18.4 mg, 0.29 mmol) were sequentially added and mixed uniformly. The mixture was then reacted at 37°C for 6 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After separation and purification by semi-preparative C18 column, the compounds were freeze-dried to obtain Compound 85 (yield 62%), a white powder. HRMS calculated value C 25 H 43 N 10 O 11 [M+H] + 659.3113, measured 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 reacted at 0°C for 2 hours, and the reaction was confirmed to be complete by LC-MS. The compound was separated and purified by semi-preparative column chromatography, lyophilized, and compound G14 (yield 79%) was obtained. HRMS calculated value C 25 H 42 N 10 O 10 [M+H] + 643.3119, measured value 643.3786.

[0117] II: Manufacturing of Low-Molecular-Weight Drugs - 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) JPEG0007848224000149.jpg62141

[0118] Step 1: Compound 20 (5.6 mg, 17.8 μmol) was dissolved in 100 μL of DMF. 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 sequentially added to the system, and the reaction was carried out at 37°C for 2 hours. The reaction system was monitored by LC-MS to confirm completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound 21 (20 mg, yield 87%). HRMS calculated value C 75 H 107 N 11 O 16 [M+H] + 1418.7975, [M+2H] 2+ 709.9026, measured value 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 system. After homogeneous mixing, the mixture was reacted at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound D1 (16.5 mg, yield 89%). HRMS calculated value C 60 H 97 N 11 O 14 [M+H] + 1196.7294, [M+2H] 2+ 598.8685, measured value 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 JPEG0007848224000151.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 system at 0°C, and the reaction system was stirred at 0°C for 15 minutes. Then, compound 22 (332 mg, 2 mmol) was slowly added. The mixture was stirred at room temperature for 1 hour. The reaction was stopped with methanol, the reaction system was concentrated, and purified by silica gel column (petroleum ether:ethyl acetate = 4:2) to obtain 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 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 again, and concentrated. It was purified by silica gel column (petroleum ether:ethyl acetate 2:1) to obtain compound 25 (55 mg, 40%).

[0123] Step 3: Compound 25 (55 mg, 0.2 mmol) was dissolved in 5 mL of methanol / water in a 1:1 ratio. LiOH (10 mg, 0.42 mmol) was added to the reaction system, and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 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 sequentially added to the reaction system. The reaction was carried out at 37°C for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction, after which the compound was isolated and purified using a semi-preparative C18 column, and then lyophilized to obtain 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, measured value 1353.8172, 677.4121.

[0125] Step 5: Compound 27 (13.2 mg, 0.01 mmol) was dissolved in 1 mL of methanol. Five equivalents of hydroxylamine hydrochloride and sodium bicarbonate (dissolved in 0.5 mL of water) were added to the reaction system, and the mixture was stirred at 65°C for 24 hours. The reaction system was detected by LC-MS, indicating completion of the reaction. After isolation and purification using a half-size C18 column, the compound was freeze-dried to obtain compound D2 (13 mg, 94%). HRMS calculated value: C 71 H 111 N 13 O 15 [M+H] + 1386.8401, [M+2H] 2+ 693.9235, measured 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) JPEG0007848224000153.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 system was cooled to 0°C on ice. DCC (337.4 mg, 1.638 mmol) was added to the above solution, and the mixture was stirred at 0°C for 30 minutes, then returned to room temperature and stirred for 6 hours. The mixture was diluted with dichloromethane, filtered, washed with 1 N HCl and saturated brine, and the organic layer was dried over MgSO4 and evaporated. The resulting oily substance was again dissolved in 30 mL of anhydrous ethanol and refluxed for 4 hours. The mixture was purified by silica gel column (hexane:ethyl ether = 10:1) to obtain Compound 30 (560 mg, yield 81%).

[0128] Step 2: Compound 30 (499 mg, 1.24 mmol) and ethyl hydrazine hydrochloride (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 system was concentrated and purified by silica gel column chromatography (hexane:ethyl ether = 1:1) to obtain compound 31 (402 mg, yield 71%).

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

[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 system and reacted at 37°C for 4 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction, after which the compound was isolated and purified using a semi-preparative C18 column, and then lyophilized to obtain compound 33 (55 mg, yield 90.3%).

[0131] Step 5: Compound 35 (CH3O-PEG 24 50 mg (43 μmol) of thionyl chloride (-COOH) was dissolved in 2 mL of anhydrous dichloromethane in a 25 mL round-bottom flask. Two drops of thionyl chloride were added dropwise, and the mixture was refluxed under nitrogen at 50°C for approximately 2 hours. Thin-layer chromatography was performed using a methanol:dichloromethane ratio of 1:8 to confirm that the reaction was complete. The reaction system was then evaporated, and the system was evaporated using an oil pump for 30 minutes to remove any remaining thionyl chloride.

[0132] Step 6: Compound 34 (Fmoc-Lys-OH, 16 mg, 43 μmol) and NaHCO3 (18 mg, 215 μmol) were dissolved in 900 μL of tetrahydrofuran and 300 μL of pure water in a round-bottom flask until clear.

[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. The reaction was carried out at room temperature for 30 minutes. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification by semi-preparative fractionation, compound 36 (42 mg, yield 63.6%) was obtained by lyophilization. HRMS calculated value C 73 H 126 N2O 30 [M+H] + 1511.8473, [M+2H] 2+ 756.4275, measured value 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 system, and DIPEA (3.44 μL, 20 μmol) was added dropwise while stirring, and the reaction was carried out at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction, after which the compound was isolated and purified by semi-preparative fractionation, and then freeze-dried to obtain compound 37 (15 mg, yield 87%). HRMS calculated value C 131 H 218 N 12 O 41 [M+2H] 2+ 1308.7745, [M+3H] 3+ 872.852, measured value 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 system. The mixture was stirred at room temperature for 20 minutes. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification by semi-preparative fractionation, compound 38 (12.8 mg, 92% yield) was obtained by lyophilization. HRMS calculated value C 116 H 208 N 12 O 39 [M+2H] 2+ 1197.7405, [M+3H] 3+ 798.8293, measured value 1197.7375, 798.8234.

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

[0137] Step 11: Compound 39 (39 mg, 4.28 μmol) was dissolved in 130 μL of dichloromethane, and the reaction system was cooled to 0°C. 10 μL of water, 10 μL of triisopropylsilane, and 80 μL of trifluoroacetic acid were added to the reaction system, and the mixture was stirred at room temperature for 30 minutes. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, compound D3 (6 mg, yield 54.7%) was obtained after lyophilization. HRMS calculated value C 123 H 216 N 14 O 41 S[M+2H] 2+ 1289.756, [M+3H] 3+ 860.173, measured value 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) JPEG0007848224000155.jpg57163

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

[0140] Step 2: Compound 40 (20 mg, 12.9 μmol) was dissolved in 130 μL of dichloromethane, and the reaction system was cooled to 0°C. 10 μL of water, 10 μL of triisopropylsilane, and 80 μL of trifluoroacetic acid were added to the reaction system, and the mixture was stirred at room temperature for 30 minutes. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, compound D4 (12.8 mg, 76% yield) was obtained after lyophilization. HRMS calculated value C 65 H 102 N 12 O 14 S[M+H] + 1307.7437, measured 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) JPEG0007848224000157.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 allowed to stand at 37°C for 3 hours. The reaction system was monitored by LC-MS to confirm completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound D5 (10 mg, yield 72.7%). HRMS calculated value C 127 H 220 N 12 O 41 [M+2H] 2+1285.7825, [M+3H] 3+ 857.524, measured value 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) JPEG0007848224000159.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 system and reacted at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification by semi-preparative fractionation, compound 43 (26 mg, yield 84.7%) was obtained by lyophilization. HRMS calculated value C 112 H 191 N7O 36 [M+2H] 2+ 1106.174, [M+3H] 3+ 737.785, measured value 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 system. The mixture was stirred at room temperature for 20 minutes. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification by semi-preparative fractionation, compound 44 (21 mg, 90% yield) was obtained by lyophilization. HRMS calculated value C 97 H 181 N7O 34 [M+2H] 2+ 995.14, actual measured 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 system, and the mixture was reacted at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification by semi-preparative fractionation, compound D6 (20.2 mg, yield 84%) was obtained by lyophilization. HRMS calculated value C 118 H 198 N8O 36 [M+2H] 2+ 1152.7033, [M+3H] 3+ 768.8048, measured values ​​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) JPEG0007848224000161.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 system and reacted at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification by semi-preparative fractionation, compound D7 (12 mg, 84%) was obtained by lyophilization. HRMS calculated value C 137 H 225 N 13 O 41 [M+2H] 2+ 1355.3039, [M+3H]3+ 903.8718, measured value 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. Sodium azidide (96 mg, 1.5 mmol) and CDMBI (32.4 mg, 150 μmol) were added to the reaction system. The reaction system was cooled to 0°C on ice, potassium phosphate (96 mg, 450 μmol) was added, and the reaction was carried out at 0°C for 4 hours to produce compound 50. The reaction system was not treated further, but was divided into smaller portions and stored at -80°C.

[0151] Step 2: Compound 46 (6 mg, 17.9 μmol) was dissolved in 100 μL of DMF. 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 sequentially added to the reaction system, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound 47 (21 mg, yield 82%). HRMS calculated value C 78 H 109 N 11 O 15 [M+2H] 2+ 720.913, measured 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 system. The reaction was carried out at room temperature for 20 minutes. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a C18 semi-preparative column, compound 48 (16 mg, 90% yield) was obtained. HRMS calculated value C 63 H 99 N 11 O 13 [M+H] + 1218.7502, [M+2H] 2+ 609.879, measured value 1218.7552, 609.8776.

[0153] Step 4: Preparation of Cu(I)-BTTAA solution: 2.5 μL of 60 mM CuSO4, 39 μL of 300 mM BTTAA, and 286 μL of 0.9 M sodium ascorbate were sequentially mixed uniformly and then stored.

[0154] Step 5: Compound 48 (16 mg, 13.1 μL) was added to the reaction system in Step 1 (calculated based on 100% yield, compound 50 (17.9 mg, 25.9 μL)), and after homogeneous mixing, the total volume of Cu(I)-BTTAA solution from Step 4 was added, and the reaction was carried out at 37°C for 4 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction, and after isolation and purification using a semi-preparative C18 column, compound 51 (20 mg, 80% yield) was obtained by lyophilization. HRMS calculated value C 87 H 140 N 14 O 33 [M+2H] 2+ 955.493, actual measured value 955.4887.

[0155] Step 6: Compound 45 (DBCO-COOH, 3.5 mg, 10.47 μmol) was dissolved in 100 μL of DMF. HATU (8 mg, 20.94 μmol), compound 51 (20 mg, 10.47 μmol), and DIPEA (5.46 mL, 31.41 μmol) were sequentially added to the reaction system, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain 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, measured 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) JPEG0007848224000165.jpg43140

[0157] Step 1: Compound 52 (SH-DM1, 20 mg, 27.13 μmol) was dissolved in 100 μL of DMF. Compound 53 (SMCC, 9 mg, 27.13 μmol) was added to the reaction system, and the reaction was carried out at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, compound 54 (24 mg, yield 82.7%) was obtained by lyophilization. HRMS calculated value C 51 H 66 ClN5O 16 S[M+H] + 1072.3992, measured value 1072.3966.

[0158] Step 2: Compound 54 (SMCC-DM1, 24 mg, 22.4 μmol) was dissolved in 100 μL of DMF. Compound 55 (DBCO-NH2, 6.2 mg, 22.4 μmol) and triethylamine (6.3 μL, 44.8 μmol) were added to the reaction system, and the mixture was reacted at 37°C for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound D9 (20 mg, yield 72.6%). HRMS calculated value C 65 H 77 ClN6O 14 S[M+H] + 1233.4985, [M+2H] 2+ 617.253, measured value 1233.4923, 617.2555.

[0159] Example 23: Synthesis of Compound D10 The structure and synthesis method of compound D10 are as follows. [ka] Linear alkynyl group -Lys(PEG) 24 )-VC-PAB-MMAE (compound D10) JPEG0007848224000167.jpg48140

[0160] Compound 56 (0.6 mg, 6.12 μmol) was dissolved in 100 μL of DMF. HATU (4.1 mg, 10.7 μmol) was added to the reaction system and mixed uniformly. Then, compound 38 (12.8 mg, 5.35 μmol) and DIPEA (2.8 μL, 16.05 μmol) were sequentially added, and the reaction was carried out at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, compound D10 (10 mg, yield 75.6%) was obtained by lyophilization. HRMS calculated value C 121 H 212 N 12 O 40 [M+2H] 2+ 1237.7535, [M+3H] 3+825.505, measured value 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) JPEG0007848224000169.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 sequentially added to the reaction system, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS, and after indicating that the reaction was nearly complete, 107 μL of triethylamine was added, mixed homogeneously, and the mixture was reacted at room temperature for 15 minutes. Separation and purification were performed using a semi-preparative C18 column, and the product described in the title was collected. The product was lyophilized to obtain Compound 87 (20.8 mg, 92% yield). HRMS calculated value C 64 H 105 N 11 O 15 [M+H] + 1268.787, measured value 1268.7815.

[0163] Step 2: Compound 87 (20.8 mg, 0.0164 mmol) was dissolved in 208 μL of DMF, compound 41 (10.35 mg, 0.0328 mmol) and triethylamine (9.13 μL, 0.0657 mmol) were added, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate that the reaction was almost complete. The mixture was then separated and purified using a semi-preparative C18 column, and the product described in the title was collected. The product was lyophilized to obtain compound D11 (14.7 mg, yield 62%). 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) JPEG0007848224000171.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 sequentially added to the reaction system, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS, and after indicating that the reaction was nearly complete, 156 μL of triethylamine was added, mixed homogeneously, and the mixture was reacted at room temperature for 15 minutes. Separation and purification were performed using a semi-preparative C18 column, and the product described in the title was collected. The product was lyophilized to obtain Compound 89 (31.1 mg, yield 90.7%). HRMS calculated value C 65 H 107 N 11 O 15 [M+H] + 1282.8026, measured value 1282.8041.

[0166] Step 2: Compound 89 (31.1 mg, 0.0234 mmol) of the above product was dissolved in 208 μL of DMF, compound 41 (10.35 mg, 0.0351 mmol) and triethylamine (9.13 μL, 0.0936 mmol) were added, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate that the reaction was almost complete. After separation and purification using a semi-preparative C18 column, the product described in the title was collected, lyophilized, and compound D12 (28.5 mg, yield 83.5%) was obtained. 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) JPEG0007848224000173.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 sequentially added to the system and reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction, and then separated and purified using a semi-preparative C18 column to collect the component indicated in the title. The component was lyophilized to obtain compound D13 (15.8 mg, yield 71%). HRMS calculated value C 69 H 101 N7O 14 [M+H] + 1252.7485, measured value 1252.7479.

[0169] III: Synthesis of DG5 and dDG-1 from the 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) JPEG0007848224000175.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 pH 7.5 PB buffer. The two systems were mixed and reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound 57 (10 mg, yield 74%). HRMS calculated value C 74 H 118 N 12 O 24 [M+2H] 2+ 780.4265, measured 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 system and mixed uniformly. The mixture was then cooled to 0°C on ice, potassium phosphate (20.4 mg, 96 μmol) was added, and the mixture was reacted at 0°C for 12 hours. The reaction was monitored by LC-MS to ensure that the product was essentially a cyclization product. After isolation and purification using a basic C18 semi-preparative column, compound DG-1 (7.2 mg, yield 72%) was obtained. HRMS calculated value C 74 H 116 N 12 O 23 [M+2H] 2+ 771.4215, measured 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 JPEG0007848224000177.jpg72140

[0173] Step 1: Compound 38 (20 mg, 8.36 μmol) was dissolved in 100 μL of DMF. Compound 58 (13.6 mg, 41.8 μmol) and triethylamine (3.5 L, 25.1 μmol) were added to the system and reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain compound 59 (18 mg, yield 83%). HRMS calculated value C 125 H 217 N 13 O 44 [M+2H] 2+ 1303.2645, [M+3H] 3+ 869.1792, measured value 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 μL, 20.8 μmol) were added to the reaction system and reacted at room temperature for 3 hours. Compound 5 (2.64 mg, 6.9 μmol) was added and the reaction was continued at room temperature for another 3 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, compound 60 (14.2 mg, yield 71%) was obtained by lyophilization. HRMS calculated value C 135 H 238 N 14 O 51 [M+3H] 3+ 958.2231, [M+4H] 4+ 718.9193, measured value 958.2233, 718.9192.

[0175] Step 3: Compound 60 (14.2 mg, 4.95 μmol) was dissolved in 100 μL of 50 mM pH 7.5 PB. CDMBI (5.4 mg, 24.8 μmol) was added to the reaction system and mixed uniformly. The mixture was then allowed to stand on ice and cooled to 0°C. Potassium phosphate (15.8 mg, 74.25 μmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm completion. The compound was isolated and purified using a basic C18 semi-preparative column, then lyophilized to obtain compound DG-2 (10 mg, yield 70.5%). HRMS calculated value C 135 H 236 N 14 O 50 [M+3H] 3+ 952.2196, measured 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 JPEG0007848224000179.jpg55120

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

[0178] Step 2: Compound 61 (18 mg, 6.53 μmol) was dissolved in 100 L of 50 mM pH 7.5 PB = 1:1. CDMBI (7 mg, 32.63 μmol) was added to the reaction system and mixed uniformly. The mixture was then allowed to stand on ice and cooled to 0°C. Potassium phosphate (20.8 mg, 98 μmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm completion. The compound was isolated and purified using a basic C18 column, then lyophilized to obtain compound DG-3 (11 mg, yield 61.5%). HRMS calculated value C 130 H 229 N 13 O 48 [M+3H] 3+ 914.537, actual measured 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 JPEG0007848224000181.jpg51140

[0180] Step 1: Compound 54 (10 mg, 9.3 μmol) was dissolved in 100 μL of DMF. Compound 5 (3.6 mg, 9.3 μmol) and triethylamine (3.8 μL, 27.9 μmol) were added to the system and reacted at 37°C for 2 hours. After adding compound 5 (3.6 mg, 9.3 μmol), the reaction was continued at 37°C for another 2 hours. The reaction was monitored by LC-MS to confirm completion, and the compound was isolated and purified using a semi-preparative C18 column, then lyophilized to obtain compound 62 (10 mg, 80%). HRMS calculated value C 61 H 87 ClN6O 23 S[M+2H] 2+ 670.2694, measured value 670.2669.

[0181] Step 2: Compound 62 (10 mg, 7.47 μmol) was dissolved in 100 μL of DMF / 50 mM pH 7.5 PB. CDMBI (8 mg, 37.35 μmol) was added to the reaction system and mixed uniformly. The reaction system was then cooled to 0°C on ice, potassium phosphate (23.8 mg, 112 μmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm that it was almost complete. The compound was isolated and purified by a basic C18 column, then lyophilized to obtain compound DG-4 (7 mg, yield 71%). HRMS calculated value C 61 H 85 ClN6O 22 S[M+2H] 2+ 661.2641, measured 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 JPEG0007848224000183.jpg70141

[0183] Step 1: Compound 52 (20 mg, 27.12 μmol) was dissolved in 200 μL of DMF. Compound 63 (6.5 mg, 27.12 μmol) and 100 μL of 0.2 M Na2HPO4 were added to the system, and the mixture was reacted at room temperature for 2 hours. After monitoring that the reaction was almost complete by LC-MS, 200 μL of 1% NaOH was added to the system. The reaction changed from pale yellow to pale red, and after 1 hour, the reaction was monitored to confirm completion. The compound was isolated and purified using a semi-preparative C18 column, then lyophilized to obtain compound 64 (19 mg, 78%). HRMS calculated value C 41 H 58 ClN5O 13 S[M+H] + 896.3518, measured 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 system, and the reaction system was adjusted to pH 6.0 with NaOH / HCl. Then sodium borohydride cyanohydride (10.7 mg, 169.6 μmol) was added, and the reaction was carried out at 37°C for 3 hours. The completion of the reaction was monitored by LC-MS. After isolation and purification using a semi-preparative C18 column, compound 65 (20 mg, yield 75%) was obtained by lyophilization. HRMS calculated value C 55 H 81 ClN6O 23 S[M+2H] 2+ 631.2459, measured value 631.2424.

[0185] Step 3: Compound 65 (20 mg, 15.86 μmol) was dissolved in 500 μL of 50 mM pH 7.5 PB. CDMBI (17.2 mg, 79.3 μmol) was added to the reaction system and mixed uniformly. The reaction system was then cooled to 0°C on ice, potassium phosphate (50.5 mg, 237.9 μmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm that it was almost complete. The compound was isolated and purified by a basic C18 column, then freeze-dried to obtain compound DG-5 (12.6 mg, yield 64%). HRMS calculated value C 55 H 79 ClN6O 22 S[M+2H] 2+ 622.2406, measured value 622.2409.

[0186] Example 32: Synthesis of compound DG-6 [ka] Compound DG-6 JPEG0007848224000185.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 pH 6.0 PB buffer. After confirming that the reaction system pH was 6.0, NaCNBH3 (5.3 mg, 89 μmol) was added, and the reaction system was homogeneously mixed. The reaction was then carried out at 37°C for 2 hours. The reaction system was monitored by LC-MS to show that the majority of the product had been formed. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain Compound 70 (10 mg, yield 75.4%). HRMS calculated value C 72 H 117 N 11 O 22 [M+2H] 2+ 744.9187, measured 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 system and mixed uniformly. The mixture was then cooled to 0°C on ice, potassium phosphate (21.4 mg, 100.5 μmol) was added, and the mixture was reacted at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm that it basically produced a cyclization product. After isolation and purification using a basic C18 semi-preparative column, compound DG-6 (7.2 mg, yield 73.4%) was obtained. HRMS calculated value C 72 H 115 N 11 O 21 [M+2H] 2+ 735.9134, measured value 735.9133.

[0189] Example 33: Synthesis of compound DG-7 [ka] Compound DG-7 JPEG0007848224000187.jpg84140

[0190] Step 1: Compound 71 (Fmoc-VA-PAB-OH, 20 mg, 38.8 μmol) was dissolved in 400 μL of DMF, 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 reacted overnight at room temperature. The reaction system was monitored by LC-MS to show that the majority of the product had been formed. After isolation and purification by semi-preparative C18 column, the compound was lyophilized to obtain compound 72 (23 mg, yield 87%). HRMS calculated value C 37 H 37 N5O8[M+H] + 680.272, measured value 680.2712.

[0191] Step 2: Compound 72 (23 mg, 33.8 μmol) was dissolved in 400 μL of DMF. MMAE (24.3 mg, 33.8 μmol) was added to the system and mixed uniformly. Then HOBt (0.92 mg, 6.76 μmol) and 82 μL of pyridine were added, and the mixture was reacted at room temperature for 12 hours. LC-MS was used to monitor the reaction, confirming that it primarily produced a cyclization product. After isolation and purification using a basic C18 semi-preparative column, compound 73 (35.3 mg, 83% yield) was obtained. HRMS calculated value C 70 H 98 N8O 13 [M+2H] 2+ 630.3705, measured value 630.3701.

[0192] Step 3: Compound 73 (30 mg, 23.8 μmol) was dissolved in 100 μL of DMF, 20 μL of piperidine was added to the reaction system, and the reaction was reacted at room temperature for 20 minutes. After detecting completion of the reaction by LC-MS, the compound was isolated and purified by half-fill C18 column, and then lyophilized to obtain compound 74 (22.9 mg, yield 93%). HRMS calculated value C 55 H 88 N8O 11 [M+H] + 1037.6651, measured 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 pH 6.0 PB buffer 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, and the reaction system was homogenized and reacted at 37°C for 6 hours. The reaction system was monitored by LC-MS to show that most of the product had been formed, and after isolation and purification by semi-preparative C18 column, compound 75 (23.5 mg, yield 76%) was obtained by lyophilization. HRMS calculated value C 69 H 111 N9O 21 [M+2H] 2+ 701.9025, measured 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. CDMBI (7.6 mg, 35.5 μmol) was added to the system and mixed uniformly. The mixture was then cooled to 0°C on ice, potassium phosphate (22.7 mg, 106.5 μmol) was added, and the mixture was reacted at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm that it basically produced a cyclization product. After isolation and purification using a basic C18 semi-preparative column, compound DG-7 (6.8 mg, yield 69%) was obtained. HRMS calculated value C 69 H 109 N9O 20 [M+2H] 2+ 692.8973, actual measured 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 CH3OH / H2O=1:4 system. 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 sequentially added to the reaction system, and the mixture was reacted at 37°C for 4 hours. The reaction was monitored by LC-MS to confirm completion, and after isolation and purification using a P2 column, the compound was freeze-dried to obtain compound 6 (9 mg, 85% yield).

[0197] Step 2: Preparation of Cu(I)-BTTAA solution: 55 μL of 40 mM CuSO4, 66 μL of 300 mM BTTAA, and 490 μL of 0.9 M sodium ascorbate were sequentially mixed uniformly and then stored.

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

[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 sequentially added to the reaction system, and the mixture was reacted at 37°C for 2 hours. After monitoring the completion of the reaction by LC-MS, 20 μL of piperidine was added. After 15 minutes, when LC-MS indicated the completion of the reaction, the compound was isolated and purified by semi-preparative C18 column, then lyophilized to obtain compound 68 (26 mg, 83% yield).

[0200] Step 5: Compound 68 (20 mg, 12.3 μmol) was dissolved in 100 μL of DMF. Compound 54 (SMCC-DM1, 13.2 mg, 12.3 μmol) and triethylamine (5 μL, 36.9 μmol) were added to the reaction system, and the mixture was reacted at 37°C for 2 hours. The reaction system was monitored by LC-MS to indicate completion of the reaction. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain 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. CDMBI (10 mg, 46.5 μmol) was added to the reaction system and mixed homogeneously. The mixture was then allowed to stand on ice and cooled to 0°C. Potassium phosphate (29.6 mg, 139.5 μmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm that it was almost complete. The compound was isolated and purified by a basic C18 column, then lyophilized to obtain compound dDG-1 (15.5 mg, yield 65%).

[0202] Example 35: Synthesis of compound dDG-2 [ka] Compound dDG-2 JPEG0007848224000191.jpg84140

[0203] Step 1: Compound 34 (Fmoc-Lys-OH, 20 mg, 54.3 μmol) was dissolved in 200 μL of DMF. Compound 17 (11.82 mg, 59.8 μmol) and 22.6 μL of triethylamine were added to the system and mixed uniformly. The mixture was then reacted at room temperature for 2 hours. The reaction was monitored by LC-MS to confirm completion. The compound was isolated and purified using a semi-preparative C18 column, then lyophilized to obtain compound 76 (22.5 mg, 92% yield). HRMS calculated value C 23 H 25 N5O5[M+H] + 452.1934, measured 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 reaction system, and DIPEA (22.8 μL, 132.9 μmol) was added dropwise while stirring, and the reaction was carried out at room temperature for 1 hour. The reaction system was monitored by LC-MS to indicate completion of the reaction, after which the compound was isolated and purified by semi-preparative fractionation, and then lyophilized to obtain compound 77 (60 mg, yield 88%). HRMS calculated value C 81 H 117 N 15 O 16 [M+2H] 2+ 778.9479, measured value 778.9477.

[0205] Step 3: Compound 77 (20 mg, 12.8 μmol) was dissolved in 100 μL of DMF, 20 μL of piperidine was added to the reaction system, and the reaction was reacted at room temperature for 20 minutes. The completion of the reaction was monitored by LC-MS, and after isolation and purification by semi-preparative C18 column, compound 78 (15.8 mg, yield 92%) was obtained by lyophilization. HRMS calculated value C 66 H 107 N 15 O 14 [M+2H] 2+ 667.9139, measured 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 pH 6.0 PB buffer. After adding these to the above system and confirming that the pH of the reaction system was 6.0, NaCNBH3 (70.3 mg, 118 μmol) was added, and the reaction system was homogeneously mixed. The reaction was then carried out at 37°C for 6 hours. The reaction system was monitored by LC-MS to show that the majority of the product had been formed. After isolation and purification using a semi-preparative C18 column, the compound was lyophilized to obtain Compound 79 (15 mg, yield 75%). HRMS calculated value C 80 H 130 N 16 O 24[M+2H] 2+ 850.48, measured 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. CDMBI (6.7 mg, 31.2 μmol) was added to the system and mixed uniformly. The mixture was then cooled to 0°C on ice, potassium phosphate (20 mg, 93.7 μmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction was monitored by LC-MS to confirm that it basically produced a cyclization product. After isolation and purification using a basic C18 semi-preparative column, compound dDG-2 (7 mg, yield 71%) was obtained. HRMS calculated value C 69 H 109 N9O 20 [M+2H] 2+ 842.4747, measured value 842.4721.

[0208] Example 36: Synthesis of compound dDG-3 [ka] Compound dDG-3 JPEG0007848224000193.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 sequentially added to the reaction system, and the mixture was reacted at room temperature for 2 hours. The reaction system was monitored by LC-MS, and after indicating that the reaction was nearly complete, 128.5 μL of triethylamine was added, mixed homogeneously, and the mixture was reacted at room temperature for 15 minutes. Separation and purification were performed using a semi-preparative C18 column, and the product described in the title was collected. Lyophilization was performed to obtain Compound 91 (21.5 mg, yield 57.15%). HRMS calculated value C 121 H 193 N 21 O 26 [M+2H] 2+1179.2291, [M+3H] 3+ 786.4887, measured value 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 system, and DMF was added to make a PB ratio of DMF / 0.2 M (1:1). The pH of the reaction system was adjusted to 6.0 with NaOH / HCl, and then sodium borohydride cyanohydride (5.74 mg, 0.0912 mmol) was added. The reaction was carried out at room temperature for 3-4 hours. The completion of the reaction was monitored by LC-MS. The product was separated and purified using a semi-preparative C18 column, the title component was collected, and lyophilized to obtain compound 92 (10.5 mg, yield 50.5%). HRMS calculated value C 135 H 216 N 22 O 36 [M+2H] 2+ 1361.7952, [M+3H] 3+ 908.1994, measured value 1361.7889, 908.1909.

[0211] Step 3: Compound 92 (10.5 mg, 3.86 μmol) was dissolved in 210 μL of DMF / H2O = 1:1. DMC (13 mg, 32.63 μmol) was added to the reaction system, and triethylamine (32.2 μL, 0.231 mmol) was homogeneously mixed. The mixture was then allowed to stand on ice and cooled to 0°C for 2 hours. The reaction was monitored by LC-MS to confirm completion. The product was separated and purified using a basic C18 column, collected, and lyophilized to obtain compound dDG-3 (7.1 mg, yield 68%). HRMS calculated value C 135 H 214 N 22 O 35 [M+2H] 2+ 1352.7899, [M+3H] 3+ 902.1959, measured value 1352.7881, 902.1952.

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

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

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

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

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

[0217] Example 42: Synthesis of Ab-6 The non-natural glycosylation 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 glycosylation antibody Ab-7 was obtained by applying general procedure 1 to compound G7 and the wild-type antibody Herceptin. The measured value after HRMS deconvolution was 146642.

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

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

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

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

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

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

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

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

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

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

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

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

[0231] V: Synthesis of gsADC-43 from glycoside-specific antibody-drug conjugate gsADC-1 Example 56: Synthesis of gsADC-1 (gsADC-represented sugar site-specific antibody-drug conjugate) The antibody-drug conjugate gsADC-1 was obtained by applying operation 4 to compound D2 and the non-natural glycosylated 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 glycosylated antibody Ab-2. The measured value after HRMS deconvolution was 148892.

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

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

[0235] Example 60: Synthesis of gsADC-5 The antibody-drug conjugate gsADC-5 was obtained by applying procedure 7 to compound D6 and the non-natural glycosylated antibody Ab-3. The measured value after HRMS deconvolution was 151464.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0250] Example 75: Synthesis of gsADC-20 The antibody-drug conjugate gsADC-20 was obtained by applying operation 7 to compound D9 and the non-natural glycosylation-engineered 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 glycosylation-engineered 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 glycosylated antibody Ab-3. The measured value after HRMS deconvolution was 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 glycosylated antibody Ab-3. The measured value after HRMS deconvolution was 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 glycosylated antibody Ab-3. The measured value after HRMS deconvolution was 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 glycosylated antibody Ab-4. The measured value after HRMS deconvolution was 151833.

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

[0257] Example 82: Synthesis of gsADC-24 The antibody-drug conjugate gsADC-24 was obtained by applying operation 8 to compound D5 and the non-natural glycosylation-engineered 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 glycosylated antibody Ab-14. The measured value after HRMS deconvolution was 152801.

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

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

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

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

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

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

[0265] Example 90: Synthesis of gsADC-31 The antibody-drug conjugate gsADC-31 was obtained by applying operation 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 operation 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 operation 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 the wild-type antibody Herceptin. The measured value after HRMS deconvolution was 148295.

[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. The measured value after HRMS deconvolution was 148751.

[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. The measured value after HRMS deconvolution was 148576.

[0271] Example 96: Synthesis of gsADC-37 The antibody-drug conjugate gsADC-37 was obtained by applying procedure 3 to the 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 defucosified antibody Herceptin. The measured value after HRMS deconvolution was 148448.

[0273] VI: Screening of sugar substrates and glycoside endonucleases Manufacturing of sugar oxazolines Various sugar substrates (containing monosaccharide, disaccharide, and trisaccharide structures per equivalent) were dissolved in 50 mM pH 7.0 PB buffer. CDMBI (5 equivalents) was added to the system and mixed uniformly. The mixture was then cooled to 0°C, potassium phosphate (15 eq) was added to bring the final concentration of the sugar substrates in the reaction system to 10 mM, and the reaction was carried out at 0°C for 2 hours. The formation of a large amount of precipitate was observed, and the precipitate was removed by centrifugation. The supernatant consisted of salt-containing sugar oxazoline substrates G1, G12, and G15-G20, which were used directly for screening in the next step.

[0274] Synthesis of compound G15 [ka] HRMS calculated value C8H 13 NO5 [M+H] + 204.0872, measured 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, measured 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, measured 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 of glycoside endonucleases G14, deglycosylating antibody (Fucα1,6)GlcNAc-Herceptin, and various glycoside endonucleases were sequentially added to 50 mM pH 7.2 Tris-HCl buffer. After incubation at 30°C for 3 hours until the final concentrations were 1.5 mM, 5 mg / mL, and 0.2 mg / mL, the concentrations were measured by mass spectrometry. Simultaneously, a control group without glycoside endonucleases was established to eliminate the influence of non-enzymatic reactions. Ten glycoside endonucleases were screened under these screening conditions, and they were identified as 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 in A in Figure 1, it was found that Endo-S2 has weak transfer activity only to G14, and the transfer yield was approximately 5.6%.

[0281] Screening of sugar substrates To a 50 mM pH 7.2 Tris-HCl buffer, sugar oxazoline substrates (G1, G12, G15-G20), deglycosylating antibody (Fucα1,6) GlcNAc-Herceptin, and glycoside endonuclease Endo-S2 were sequentially added. After incubation at 30°C for 3 hours until the final concentrations were 1.5 mM, 5 mg / mL, and 0.2 mg / mL, the concentrations were measured by mass spectrometry. A control group without glycoside endonuclease was simultaneously established to eliminate the influence of non-enzymatic reactions. As shown in Figure 1B, the results indicate that Endo-S2 has a high recognition efficiency of 68% for the transfer of G1 to the deglycosylating antibody. G12, obtained by introducing sialic acid to the 6-position modification of G1 galactose, was also well recognized by Endo-S2. However, the transfer activity of sugar substrates G19 and G20, which are modified at the 3-position of galactose or N-acetylglucoseamine at the 3-position, was significantly reduced by the action of Endo-S2, and other sugar structures were also not well recognized.

[0282] Pharmacological Example 1 In vitro activity data experiment process and results analysis The activity of some of the disaccharide ADCs mentioned above was evaluated at the cellular level, and a total of three cell lines were selected. Here, SK-Br-3 cells and NCI-N87 cells are Her2-positive cells, and MDA-MB-231 is a Her2-negative cell. The cellular activity and toxicity of the ADC molecule were measured by the MTT method. In the specific procedure, 100 μL of PBS was added to the outermost layer of a 96-well plate, and only culture medium was added to three other wells. Approximately 6000 corresponding cells were added to each of the remaining wells, and the plates were incubated overnight at 37°C in a CO2 incubator. 10 μL of each ADC molecule was added (starting with a 5-fold gradient dilution of each ADC molecule from the highest concentration of 100 nM, resulting in a total of 9 dilutions, with each concentration having 3 overlapping wells). Three cells were inoculated into a 96-well plate, and 10 μL of medium was added to the medium wells of the other three wells as a control and blank group. The 96-well plate was incubated in a CO2 incubator at 37°C for 72 hours. 10 μL of 5 mg / mL MTT was added to each well, followed by incubation at 37°C for 4 hours. Then, 90 μL of SDS lysate was added to each well, and the cells were incubated at 37°C for 7 hours to allow sufficient cell division. Finally, the OD value at 570 nm was measured for each well, and the data was processed using GraphPad Prism 6. The results are shown in Figure 2.

[0283] In Figure 2, gsADC-40 was prepared by click chemistry accelerated by ring tension using N3-NH-SCT-Her (for details, see Chinese patent application CN107778372A, produced by the oligosaccharide structure Az-NH-SCT, for example, the structure shown in the following figure) and BCN drug-linker D5. Specifically, the reaction conditions were as follows: pH was adjusted to 7.4 so that N3-NH-SCT-Her was 5 mg / mL and compound D5 was 0.55 mM. After confirming that the reaction had completely transferred to the product by LC-MS, protein A purification was performed. [ka] N3-NH-SCT-Her

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

[0285] Pharmacological Example 2 Experimental process and results analysis of in vivo antitumor activity A BALB / c nude mouse transplant tumor model was constructed using gastric cancer cells NCI-N87, and the principle of grouping mice into large, medium, and small groups was applied using ear canal markers. Each group contained 5 mice.

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

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

Claims

1. A disaccharide linker represented by the following formula II, 【Chemistry 1】 II In Equation II, In Z-Y-X-, Z-Y- may or may not exist. If Z-Y- is not present, X is an aldehyde group, -CH ２ -NH ２ , or -CH ２ -N ３ is, or 【Chemistry 2】 Selected from, The wavy line indicates the connection point. If Z-Y- exists, then X is -CH ２ -, *-CH ２ -O-, -CO-NH-CH ２ -*, -ON=CH-*, -CONH-N=CH-*, -NHCH ２ - Selected from the group consisting of * and the following structures, 【Transformation 3】 * indicates the position where the galactose ring in formula II is bonded. Y is a divalent linker connecting X and Z, or does not exist. Z is selected from the following cases i) to iv): Case i) Fragments having bioorthogonal reactive groups or functional molecules The functional molecules are selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycol, lipids, polypeptides, nanoantibodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, contrast agents, and magnetic resonance imaging agents. Case II) 【Chemistry 4】 L １ is a trivalent linker having three reactive groups, L ２ and L ３ is, L １ and Z ２ and Z ３ It is a divalent linker that connects and Z' is L １ A linking fragment that connects to the remaining part of the disaccharide linker, which does not exist independently, or -(CH ２ ) a linking group formed from p- or the Z group of case i), where p is an integer from 1 to 5, Z ２ and Z ３ The definition of is the same as the definition of Z in case i) above, Case III) 【Transformation 5】 L ６ It is a tetravalent linker having four reactive groups, L ２ , L ３ , L ４ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ The definition of Z in case ii) above is ２ Z ３ This is the same as the definition, Case iv) 【Transformation 6】 L １ The definition of is L in case ii) above. １ This is the same as the definition of L ２ , L ３ , L ４ , L ５ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ Z ５ The definition of Z in case ii) above is ２ Z ３ A disaccharide linker, which is the same as the definition.

2. Y is - (CH ２ )m-(CH-w)n-,-(CH ２ -CH ２ -O) ｍ - (CH-w) ｎ - or - (PO ４ ) ｎ - A linked fragment selected from, or a combination of a cleavable fragment and the linked fragment Y, where m and n are independently selected from integers from 0 to 30, and w is hydrogen or a polyethylene glycol structure having different lengths. Z is selected from the following cases i) to iv): Case i) Z is a bioorthogonal reactive group, and the reactive groups are: amino groups, azide residues, aldehyde residues, thiol residues, alkyne residues, alkene residues, halogen groups, tetrazine residues, nitrone residues, hydroxyamine residues, nitrile residues, hydrazine residues, ketone residues, boric acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isonitrile residues, cydonone residues, conjugated diene residues, phosphate residues, cycloalkyne residues, cycloalkene residues, and 【Transformation 7】 Selected from, Case II) 【Transformation 8】 L １ This is a trivalent linker derived from lysine, aspartic acid, glutamic acid, propargylglycine, cysteine, or selected from the following structures: 【Chemistry 9】 n is an integer between 1 and 30. L ２ and L ３ is, L １ and Z ２ and Z ３ It is a divalent linker that connects and L ２ and L ３ Each is independently - (CH ２ )m-(CH-w)n-,-(CH ２ -CH ２ -O)m-(CH-w)n-, or -(PO ４ ) selected from n- or selected from a combination of a severable fragment and the connecting fragment, where m and n are each independently selected from integers between 0 and 30, and w is a hydrogen atom or polyethylene glycol having different lengths. Z' is L １ A linking fragment that connects to the remaining part of the linking fragment of the disaccharide linker, which does not exist independently, or -(CH ２ ) a group that can react with the Z group in case i) p- or the Z group in case i), where p is an integer from 1 to 5, Z ２ and Z ３ The definition of is the same as the definition of Z in case i) above, Case III) 【Chemistry 10】 L ６ It is a tetravalent linker having four reactive groups, selected from dilysine, diglutamic acid, diaspartic acid, aspartic acid-glutamic acid dipeptide structure, aspartic acid-lysine dipeptide structure, glutamic acid-lysine structure, or selected from the following structures: 【Chemistry 11】 n is an integer between 1 and 30. L ２ , L ３ , L ４ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ The definition of Z in case ii) above is ２ Z ３ This is the same as the definition, Case iv) 【Chemistry 12】 L １ is defined in the same way as L in case ii) above, and the three Ls １ are each independently defined, and the definition of L １ , L ２ , L ３ , L ４ , L ５ is defined in the same way as L in case ii) above, and the definition of L ２ , L ３ is defined in the same way as L in case ii) above, the definition of Z' is defined in the same way as Z' in case ii) above, and Z ２ , Z ３ , Z ４ , Z ５ is defined in the same way as Z ２ , Z ３ in case ii) above. The disaccharide linker according to claim 1.

3. In case i) above, Z is selected from the following groups: 【Chemistry 13】 In the above case ii), Z' is selected from the following elements: 【Chemistry 14】 n is a natural number from 1 to 30, and R １ and R ２ are each independently H, -CH ３ , -CH ２ CH ３ , cyclopropyl, and cyclobutyl, and the disaccharide linker according to claim 1.

4. A disaccharide linker selected from the following compounds: 【Chemistry 15】 R is represented as -Y-Z, Y is either a divalent linker or does not exist. Z is selected from the following cases i) to iv): Case i) Fragments having bioorthogonal reactive groups or functional molecules The functional molecules are selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycol, lipids, polypeptides, nanoantibodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, contrast agents, and magnetic resonance imaging agents. Case II) 【Chemistry 16】 L １ It is a trivalent linker having three reactive groups, L ２ and L ３ is, L １ and Z ２ and Z ３ It is a divalent linker that connects and Z' is L １ A linking fragment that connects to the remaining part of the disaccharide linker, which does not exist independently, or -(CH ２ ) a group that can react with the Z group in case i) p- or the Z group in case i), where p is an integer from 1 to 5, Z ２ and Z ３ The definition of is the same as the definition of Z in case i) above, Case III) 【Chemistry 17】 L ６ It is a tetravalent linker having four reactive groups, L ２ , L ３ , L ４ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ The definition of Z in case ii) above is ２ Z ３ This is the same as the definition, Case iv) [Chemistry 18] L １ The definition of is L in case ii) above. １ This is the same as the definition of L ２ , L ３ , L ４ , L ５ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ Z ５ The definition of Z in case ii) above is ２ Z ３ This is the same as the definition, l, m, and n are independent integers between 0 and 30, forming a disaccharide linker.

5. A disaccharide linker having a structure selected from the following structures. 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】

6. A method for producing a disaccharide linker according to claim 1, the reaction formula being as follows: 【Chemistry 23】 In the above reaction equation, 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. The aforementioned manufacturing method is 1) A step of modifying a disaccharide structure having an acetylglucosamine structure at its terminus to obtain a disaccharide structure having the active group U, and after the disaccharide structure having the active group U is derivatized, introducing Z-Y-X- which has bioorthogonal reactivity or contains a functional molecular fragment, 2) A method for producing disaccharides, comprising the step of obtaining a disaccharide linker represented by formula II through a cyclization step of the disaccharide structure into which the Z-Y-X- having bioorthogonal reactivity or containing the functional molecular fragment is introduced.

7. If the modification in step 1) is an oxidation reaction, and the oxidation reaction is carried out in the presence of an enzyme, then the enzyme is galactose oxidase, and the active group U is an aldehyde group, The derivatization in step 1) is an oxime reaction, a reductive amination, an amino group-involved reaction, or an azide group-involved reaction. The manufacturing method according to claim 6, wherein in step 2), the cyclization step is carried out using 2-chloro-1,3-dimethylimidazolinium chloride or 2-chloro-1,3-dimethyl-1H-benzimidazolium-3-chloride.

8. A disaccharide-small molecule drug conjugate produced from the disaccharide linker described in claim 1, The structure is represented by the following formulas VI, VII, or VIII, 【Chemistry 24】 In the structures of equations VI to VIII, L are the same or different from each other, and Z ２ ', Z ３ ' is a bioorthogonal reactive group and Z ２ Z ３ The linker structure is formed from and, where Z' is the same or different from each other, exists simultaneously or independently. L is D, D １ Or D ２ It is a divalent linker that connects the remaining parts in equations VI to VIII, D, D １ and D ２ Each of the groups independently represents a cytotoxic compound, a group derived from a small molecule drug, or a fluorescent group, wherein the small molecule drug is selected from the group consisting of mytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, docamycin, amanitin, PBD derivatives, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, colchicine, estramustine, semadin, eloiterobin, monosaccharides, oligosaccharides, and derivatives of the above compounds, or is a radiotherapeutic drug, forming a disaccharide-small molecule drug conjugate.

9. L is - (CH ２ ) ａ - (OCH ２ CH ２ ) ｂ - (NHKCO) ｎ - (CH ２ ) ｃ -, or selected from the following bases, 【Chemistry 25】 【Chemistry 26】 V and W are dual-function linkers, and include a structure in which lysine and propargylglycine are dual-function linkers. a, b, c, d, and e are each independently chosen from integers between 0 and 30, m and n are 0 or 1, and R ３ and R ４ Each is independently CH ３ -, (CH ３ ) ２ CH-, PhCH ２ NH ２ (CH ２ ) ４ -, NH ２ CONH(CH ２ ) ３ - Selected from, where R is a polyethylene glycol structure of different lengths comprising an azide-forming monosaccharide, disaccharide, oligosaccharide, or azide group, or a combination of polyethylene glycol with a linear or cyclic monosaccharide and an oligosaccharide, wherein the oligosaccharide includes a branched oligosaccharide chain. The disaccharide-low molecular weight drug conjugate according to claim 8, wherein the wavy line indicates the linking site.

10. L 【Chemistry 27】 Selected from, a, b, c, d, e, m, n, R, R ３ , and R ４ The disaccharide-small molecule drug conjugate according to claim 8, as defined similarly to claim 9.

11. The disaccharide-low molecular weight drug conjugate according to claim 8, wherein D, D1, and D2 are each independently selected from the following groups. 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 【Chemistry 31】 【Chemistry 32】 【Transformation 33】

12. Selected from the following compounds, 【Transformation 34】 【Chemistry 35】 【Transformation 36】 【Chemistry 37】 DM1 in the aforementioned compound dG-1 is represented by the following structural formula: 【change】 In each of the above structures, the structure of the MMAE portion is 【Transformation 38】 This is a disaccharide-small molecule drug conjugate.

13. A glycosylation antibody based on the specific linkage of the antibody Fc domain N-glycosylation site represented by the following formula X, 【Chemistry 39】 In the above equation X, In Z-Y-X-, Z-Y- may or may not exist. If Z-Y- is not present, X is an aldehyde group, -CH ２ -NH ２ , or -CH ２ -N ３ is, or 【Chemistry 40】 Selected from, The wavy line indicates the connection point. If Z-Y- exists, then X is -CH ２ -, *-CH ２ -O-, -CO-NH-CH ２ -*, -ON=CH-*, -CONH-N=CH-*, -NHCH ２ - *, and selected from the following structures, 【Chemistry 41】 * indicates the position where the galactose ring in the above formula X is bonded. Y is a divalent linker connecting X and Z, or does not exist. Z is selected from the group consisting of the following cases i) to iv), Case i) Fragments having bioorthogonal reactive groups or functional molecules The functional molecules are selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycol, lipids, polypeptides, nanoantibodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, contrast agents, and magnetic resonance imaging agents. Case II) 【Chemistry 42】 L １ It is a trivalent linker having three reactive groups, L ２ and L ３ is, L １ and Z ２ and Z ３ It is a divalent linker that connects and Z' is L １ A connecting fragment that joins Y and does not exist independently, -(CH ２ ) p-, or a group that can react with the Z group in case i), where p is an integer from 1 to 5. Z ２ and Z ３ The definition of is the same as the definition of Z in case i) above, Case III) 【Chemistry 43】 L ６ It is a tetravalent linker having four reactive groups, L ２ , L ３ , L ４ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ The definition of Z in case ii) above is ２ Z ３ This is the same as the definition, Case iv) 【Chemistry 44】 L １ The definition of is L in case ii) above. １ This is the same as the definition of L ２ , L ３ , L ４ , L ５ The definition is L in case ii) above. ２ , L ３ The definition is the same as the definition of Z', and the definition of Z' is the same as the definition of Z' in case ii) above, Z ２ Z ３ Z ４ Z ５ The definition of Z in case ii) above is ２ Z ３ This is the same as the definition, 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 derived from a different species, and is a glycotechnology-engineered antibody.

14. Select from the following groups: 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 [Transformation 50] 【Chemistry 51】 The downward-pointing triangle represents fucose, and the square represents N-acetylglucosamine; these are glycosylated antibodies.

15. The glycosylation antibody according to claim 13, wherein Ab is selected from the group consisting of trastuzumab, pertuzumab, rituximab, cetuximab, morozumab, gemtuzumab, absiximab, darizumab, adalimumab, palizumab, valiximab, bevacizumab, panitumumab, nitrotumab, denitumab, decitumab, lemonibisumab, nexituzumab, eprilimus, daremab, ventocibizumab, alemtuzumab, erlotuzumab, bonatumab, nivolumab, pembrolizumab, atezolizumab, avilumab, dalvalumab, tremelimumab, katumab, verintumomab, emicizumab, and evantozumab (Rbrevant).

16. The glycosylation antibody according to claim 15, wherein Ab is trastuzumab.

17. A method for producing a glycosylation-engineered antibody according to claim 13, wherein the method is carried out by the following method 3 or 4: 【Chemistry 52】 In the above reaction equation, m is selected from 0 or 1. In the above method 3, Wild-type antibodies are hydrolyzed with glycoside endonuclease or a combination of glycoside endonuclease and fucosidase to remove heterogeneous sugar chains from the conservative glycosylation site of the natural antibody, thereby obtaining deglycosylated antibodies. Subsequently, the disaccharide linker shown in the above scheme and the deglycosylated antibody are co-incubated, and the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain by catalytic action of glycoside endonuclease, thereby producing 1,6-acetylglucosamine disaccharide-modified antibodies represented by formula X, which are modified with the disaccharide linker containing bioorthogonal reactive groups, with or without fucose. In the aforementioned method 4, The disaccharide linker and the wild-type antibody shown in the above scheme are co-incubated, the N-oligosaccharide structure of the wild-type antibody Fc domain is hydrolyzed by the catalytic action of a glycoside endonuclease, and at the same time the disaccharide linker is linked to the conservative glycosylation site of the antibody Fc domain, thereby producing a 1,6-acetylglucosamine disaccharide modified antibody represented by formula X, which is either fucose-containing or non-containing, modified with the disaccharide linker containing a bioorthogonal reactive group. The method for producing glycosylated antibodies, wherein the glycoside endonuclease is Endo-S2.

18. The method for producing a glycosylation-engineered antibody according to claim 17, wherein the Endo-S2 is Endo-S2 derived from Streptococcus pyogenes.

19. A method for producing an antibody-drug conjugate, wherein the method is the following method 5 and method 6. In the aforementioned method 5, a) Co-incubate a disaccharide linker and a wild-type antibody to hydrolyze the N-oligosaccharide structure of the wild-type antibody Fc domain Asn297 by catalytic action of a glycoside endonuclease, and simultaneously link the disaccharide linker to the Asn297 site of the antibody Fc domain, or co-incubate the disaccharide linker, a deglycosylated antibody, and a glycoside endonuclease to produce a fucose-containing or non-containing 1,6-acetylglucosamine disaccharide modified antibody modified with the disaccharide linker containing a bioorthogonal reactive group, wherein the deglycosylated antibody is obtained by pre-treating the wild-type antibody with a glycoside endonuclease, or by simultaneously removing fucose using fucosidase. b) The antibody-drug conjugate is produced by coupling the fucose-containing or non-containing 1,6-acetylglucosamine disaccharide modified antibody with the bioorthogonal reactive group-containing disaccharide linker obtained in step a) with a modified low-molecular-weight drug having a corresponding group that can specifically coupling with the bioorthogonal reactive group. The aforementioned disaccharide linker is represented by the following formula II: 【Chemistry 53】 II In Equation II, In Z-Y-X-, Z-Y- may or may not exist. If Z-Y- is not present, X is an aldehyde group, -CH2-NH2, or -CH2-N3, or 【Chemistry 54】 Selected from, The wavy line indicates the connection point. If Z-Y- exists, X is selected from the group consisting of -CH2-, *-CH2-O-, -CO-NH-CH2-*, -ON=CH-*, -CONH-N=CH-*, -NHCH2-*, and the following structures: 【Transformation 55】 * indicates the position where the galactose ring in formula II is bonded. Y is a divalent linker connecting X and Z, or does not exist. Z is selected from the following cases i) to iv): Case i) Fragments having bioorthogonal reactive groups or functional molecules The functional molecules are selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycol, lipids, polypeptides, nanoantibodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, contrast agents, and magnetic resonance imaging agents. Case II) 【Transformation 56】 L1 is a trivalent linker having three reactive groups. L2 and L3 are divalent linkers that connect L1 to Z2 and Z3. Z' is a linking fragment that connects L1 to the rest of the disaccharide linker, does not exist independently, or is a group that can react with -(CH2)p- or the Z group in case i), where p is an integer from 1 to 5. The definitions of Z2 and Z3 are the same as the definition of Z in case i) above. Case III) 【Chemistry 57】 L6 is a tetravalent linker having four reactive groups. 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. Case iv) 【Transformation 58】 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. In Method 6, a deglycosylated antibody is obtained by treating a wild-type antibody with a glycoside endonuclease according to the following reaction formula, and removing fucose using fucosidase as needed. The antibody-drug conjugate is then prepared by co-incubating a disaccharide-small molecule drug conjugate represented by the following formulas VI, VII, or VIII with the deglycosylated antibody and Endo-S2 (Method I). The disaccharide-small molecule drug complex is co-incubated with a wild-type antibody and Endo-S2, and the Fc domain Asn297 N-oligosaccharide structure of the wild-type antibody is hydrolyzed by the catalytic action of Endo-S2, and at the same time the disaccharide-small molecule drug complex is linked to the antibody Fc domain Asn297 site to prepare the antibody-drug complex (Method II). 【Chemistry 59】 【Transformation 60】 In the structures of formulas VI to VIII, L are the same or different from each other, Z2' and Z3' are linker structures formed from bioorthogonal reactive groups and Z2 and Z3, and Z' are the same or different from each other, existing simultaneously or independently. L is a divalent linker that connects D, D1 or D2 with the rest of formulas VI to VIII. L1 is a trivalent linker, and L2 and L3 are independently divalent linkers. Z3 is a fragment having a bioorthogonal reactive group or a functional molecule. D, D1, and D2 each independently represent a cytotoxic compound, a group derived from a small molecule drug, or a fluorescent group, and the small molecule drug is selected from the group consisting of mytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, docamycin, amanitin, PBD derivatives, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, colchicine, estramustine, semadin, eloiterobin, monosaccharides, oligosaccharides, and derivatives of the above compounds, or is a radiotherapeutic agent. X and Y are defined in the same way as in method 5 when Z-Y- exists, Z' is a linking segment that links L or L1 to the rest of the disaccharide linker, does not exist independently or is -(CH2)p-, where p is an integer from 1 to 5. m is 0 or 1, The glycoside endonuclease is Endo-S2, and when producing a coreless fucose compound, the glycoside endonuclease is required to be used in combination with a fucosidase, in a method for producing antibody-drug conjugates.

20. In method 5 described above, the bioorthogonal reactive group and the corresponding group capable of specific coupling reaction with the bioorthogonal reactive group are any combination selected from the group consisting of an azide group and an alkynyl group, a mercapto group and a maleimide group, 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. In step b) of method 5 described above, the disaccharide linker has the following groups so as to couple with the low molecular weight drug modified with the corresponding group: 【Chemistry 61】 n is a natural number from 1 to 30, and R １ and R ２ These are independently H, -CH ３ ,―CH ２ CH ３ Selected from cyclopropyl group and cyclobutyl group, The method for producing the corresponding low-molecular-weight drug according to claim 19, wherein the low-molecular-weight drug with the corresponding group modification is selected from the group consisting of the following compounds. 【Transformation 62】 【Transformation 63】 【Chemistry 64】 【Transformation 65】

21. The method 5 includes step b) shown in the following reaction equation: 【Chemical Formula 66】 The antibody-drug conjugate is obtained by binding the glycosylated antibody obtained from step a) above to E-L-D. In the above reaction equation, m is selected from the group consisting of 0 or 1, and E is a bioorthogonal reactive group that can react with Z. L is a divalent linker that connects D and Z', Z' is a group formed by the orthogonal reaction between E and Z. The manufacturing method according to claim 19, wherein D is as defined in claim 19.

22. The above manufacturing method is represented by the following reaction equation: 【Transformation 67】 E ３ This is a corresponding group that undergoes a bioorthogonal reaction with an aldehyde group, and is selected from the following groups: 【Transformation 68】 X ２ is an aldehyde group and E ３ It is a structure formed by a reaction, E ５ X is a corresponding group that undergoes a bioorthogonal reaction with the azide group, and is selected from the group consisting of a linear alkynyl group, a DBCO system structure, and a BCN system structure. ４ is an azide group and E ５ It is a structure formed by a reaction, L is a divalent linker that connects X2 or X4 to D. The method according to claim 21, wherein D is as defined in claim 19.

23. An antibody-drug conjugate produced by the manufacturing method described in any one of claims 19 to 22, and represented by the following formula XII, 【Transformation 69】 X is selected from the group consisting of -CH2-, *-CH2-O-, -CO-NH-CH2-*, -ON=CH-*, -CONH-N=CH-*, -NHCH2-*, and the following structures: 【Transformation 70】 * indicates the position where the galactose ring in formula XII is bonded. Y is a divalent linker that connects X and Z', Z' is a linked fragment formed through the reaction between the bioorthogonal reactive groups, L is a divalent linker that connects D and Z', D represents a group derived from a cytotoxic compound, a small molecule drug, or a fluorescent group, wherein the small molecule drug is selected from the group consisting of mytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, docamycin, amanitin, PBD derivatives, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, colchicine, estramustine, semadin, eloiterobin, monosaccharides, oligosaccharides, and derivatives of the above compounds, or is a radiotherapeutic agent. m is 0 or 1, An antibody-drug conjugate where n is 1 or 2.

24. In equation XII above, -Z'-L-D can be replaced with one of the following: 【Chemistry 71】 Z ２ ', Z ３ ', Z ４ ', Z ５ ' represents the bioorthogonal group of the functional molecule and Z respectively. ２ Z ３ Z ４ Z ５ A linked fragment formed by the reaction of and, existing simultaneously or independently, or not existing, D ３ , D ４ The definition is D １ , D ２ This is the same as the definition of D １ -D ４ If the structures are the same, the antibody-drug complex of formula XII represents an antibody-drug complex with a high drug load carrying the same drug structure, D １ -D ４ If the values ​​are different, the antibody-drug conjugate of formula XII represents a multi-drug antibody-drug conjugate carrying different drug structural compositions. Z2, Z3, Z4, and Z5 are each independently fragments having bioorthogonal reactive groups or functional molecules. The functional molecules are selected from the group consisting of toxins, drugs, fluorescent probes, polyethylene glycol, lipids, polypeptides, nanoantibodies, DNA and related drugs, RNA and related drugs, cholesterol, antibiotics or radioisotope labels, contrast agents, and magnetic resonance imaging agents. D1, D2, D3, and D4 each independently represent a cytotoxic compound, a group derived from a small molecule drug, or a fluorescent group, and the small molecule drug is selected from the group consisting of mytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, docamycin, amanitin, PBD derivatives, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, colchicine, estramustine, semadin, eloiterobin, monosaccharides, oligosaccharides, and derivatives of the above compounds, or is a radiotherapeutic agent. L, L2, L3, L4, and L5 are each independently divalent linkers. L1 is a trivalent linker, The antibody-drug conjugate according to claim 23, wherein L6 is a tetravalent linker.

25. The antibody-drug conjugate gsADC-1 produced by the method described in claim 19, 5, from the compound D2 described in claim 20 and the glycosylation-engineered antibody Ab-2 described in claim 14, The antibody-drug conjugate gsADC-2 produced by the method described in claim 19, which is produced from the compound D1 described in claim 20 and the glycosylation-engineered antibody Ab-2 described in claim 14. The antibody-drug conjugate gsADC-3 produced by the method described in claim 19, 5, from compound D3 described in claim 20 and the glycosylation-engineered antibody Ab-2 described in claim 14, The antibody-drug conjugate gsADC-4 produced by the method described in claim 19, 5, from compound D4 described in claim 20 and the glycosylation-engineered antibody Ab-2 described in claim 14, The antibody-drug conjugate gsADC-5 produced by the method described in claim 19, which is made from compound D6 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14. The antibody-drug conjugate gsADC-6 produced by method 5 of claim 19 from compound D7 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14, The antibody-drug conjugate gsADC-7 produced by method 5 of claim 19 from compound D8 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14, The antibody-drug conjugate gsADC-8 produced by method 5 of claim 19 from compound D9 according to claim 20 and the glycosylation-engineered antibody Ab-3 according to claim 14, The antibody-drug conjugate gsADC-9 produced by method 5 of claim 19 from compound D6 described in claim 20 and the glycosylation-engineered antibody Ab-4 described in claim 14, The antibody-drug conjugate gsADC-10 produced by method 5 of claim 19 from compound D7 described in claim 20 and the glycosylation-engineered antibody Ab-4 described in claim 14, The antibody-drug conjugate gsADC-11 produced by the method described in claim 19, 5, from compound D8 described in claim 20 and the glycosylation-engineered antibody Ab-4 described in claim 14, The antibody-drug conjugate gsADC-12 produced by the method of claim 19, which is produced from compound D9 according to claim 20 and the glycosylation-engineered antibody Ab-4 according to claim 14. The antibody-drug conjugate gsADC-13 produced by the method described in claim 19, 5, from compound D6 described in claim 20 and the glycosylation-engineered antibody Ab-6 described in claim 14, The antibody-drug conjugate gsADC-14 produced by the method described in claim 19, 5, from compound D7 described in claim 20 and the glycosylation-engineered antibody Ab-6 described in claim 14, The antibody-drug conjugate gsADC-15 produced by the method described in claim 19, which is made from compound D8 described in claim 20 and the glycosylation-engineered antibody Ab-6 described in claim 14. The antibody-drug conjugate gsADC-16 produced by the method described in claim 19, 5, from compound D9 described in claim 20 and the glycosylation-engineered antibody Ab-6 described in claim 14, The antibody-drug conjugate gsADC-17 produced by the method described in claim 19, 5, from compound D6 described in claim 20 and the glycosylation-engineered antibody Ab-9 described in claim 14, The antibody-drug conjugate gsADC-18 produced by the method described in claim 19, 5, from compound D7 described in claim 20 and the glycosylation-engineered antibody Ab-9 described in claim 14, The antibody-drug conjugate gsADC-19 produced by the method of claim 19, which is produced from compound D8 described in claim 20 and the glycosylation-engineered antibody Ab-9 described in claim 14, The antibody-drug conjugate gsADC-20 produced by method 5 of claim 19 from compound D9 described in claim 20 and the glycosylation-engineered antibody Ab-9 described in claim 14, The antibody-drug conjugate gsADC-29 produced by the method described in claim 19, which is produced from compound D13 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14. The antibody-drug conjugate gsADC-21 produced by the method described in claim 19, 5, from compound D5 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14, The antibody-drug conjugate gsADC-39 produced by the method of claim 19, which is produced from compound D11 according to claim 20 and the glycosylation-engineered antibody Ab-3 according to claim 14, The antibody-drug conjugate gsADC-41 produced by the method described in claim 19, 5, from the compound D12 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14, The antibody-drug conjugate gsADC-22 produced by the method described in claim 19, which is made from compound D5 described in claim 20 and the glycosylation-engineered antibody Ab-4 described in claim 14. The antibody-drug conjugate gsADC-23 produced by the method described in claim 19, which is made from compound D5 described in claim 20 and the glycosylation-engineered antibody Ab-6 described in claim 14. The antibody-drug conjugate gsADC-24 produced by the method described in claim 19, which is made from compound D5 described in claim 20 and the glycosylation-engineered antibody Ab-9 described in claim 14. The antibody-drug conjugate gsADC-42 produced by the method described in claim 19, 5, from the compound D11 described in claim 20 and the glycosylation-engineered antibody Ab-14 described in claim 14, The antibody-drug conjugate gsADC-43 produced by the method described in claim 19, 5, from compound D5 described in claim 20 and the glycosylation-engineered antibody Ab-15 described in claim 14, The antibody-drug conjugate gsADC-25 produced by the method described in claim 19, which is produced from compound D10 described in claim 20 and the glycosylation-engineered antibody Ab-3 described in claim 14. The antibody-drug conjugate gsADC-26 produced by the method described in claim 19, 5, from the compound D10 described in claim 20 and the glycosylation-engineered antibody Ab-4 described in claim 14, The antibody-drug conjugate gsADC-27 produced by the method described in claim 19, 5, from the compound D10 described in claim 20 and the glycosylation-engineered antibody Ab-6 described in claim 14, The antibody-drug conjugate gsADC-28 produced by the method described in claim 19, 5, from the compound D10 described in claim 20 and the glycosylation-engineered antibody Ab-9 described in claim 14, The antibody-drug conjugate gsADC-30 produced by the method described in claim 19, from the compound DG-1 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-31 produced by the method described in claim 19, from the compound DG-2 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-32 produced by the method described in claim 19, from the compound DG-3 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-33 produced by the method described in claim 19, from the compound DG-4 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-34 produced by the method described in claim 19, from the compound DG-5 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-35 produced by the method described in claim 19, from the compound DG-6 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-36 produced by the method described in claim 19, from the compound DG-7 described in claim 12 and the wild-type antibody Herceptin, The antibody-drug conjugate gsADC-37 produced by the method described in claim 19, 6, from the compound dDG-1 described in claim 12 and the wild-type antibody Herceptin, and The antibody-drug conjugate gsADC-38 produced from the compound DG-6 described in claim 12 and the defucosified antibody Herceptin by the method described in claim 19, method 6. An antibody-drug conjugate according to claim 23, selected from the group consisting of the following.

26. Use of the disaccharide linker as defined in claim 1 or the disaccharide-low molecular weight drug conjugate as defined in claim 8 in antibody glycosylation modification or the manufacture of antibody-drug conjugates.

27. Use of the antibody-drug conjugate according to claim 23 in the manufacture of a drug, pharmaceutical composition or diagnostic reagent, wherein the drug in the antibody-drug conjugate is selected from the group consisting of antitumor, anti-inflammatory, antiviral, anti-infective disease or other immunotherapeutic drugs.

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