CN115703845A - Oligosaccharide linker and side chain hydrophilic fragment combined sugar chain site-directed antibody-drug conjugate, preparation method and application thereof - Google Patents

Abstract

The invention provides a novel oligosaccharide linker and a connection mode, and the invention uses antibody conservative glycosylation sites as coupling sites, double-antenna oligosaccharides as a linker structure, and aldehyde groups modified by terminal sialic acid and terminal galactose and other structures as connection residues to carry out coupling of a series of small molecular compounds. In addition, the invention also provides a synthesis mode of the oligosaccharide linker, a preparation method of the small molecule drug and a preparation method of the antibody drug conjugate. In addition, the invention also provides a test result of the candidate compound on the activity level of cells, and provides the application of the antibody drug conjugate or the drug combination in antitumor, anti-inflammatory, antiviral, anti-infection or other immunotherapy drugs.

Description

A sugar chain-directed antibody-drug conjugate combined with an oligosaccharide linker and a side chain hydrophilic segment Combined matter, its preparation method and use

technical field

The present invention relates to a new type of oligosaccharide linker, using the oligosaccharide linker to prepare novel sugar chain-based site-directed antibody-drug conjugates and their preparation method and application.

Background technique

Antibody-drug conjugates are complexes formed by covalently linking small molecule compounds to antibodies. Among them, the site-specific small molecule coupling technology realizes the introduction of small molecule drugs at specific sites of antibodies through the selective modification of antibodies, showing a good application prospect.

IgG antibodies comprise variable Fab regions as well as constant (crystallizable) Fc domains. In the Fc domain, there is a conserved glycosylation site (Asn297), carrying two structurally heterogeneous N-glycans dominated by biantennary. Utilizing the conservative type of IgG glycosylation sites, the introduction of non-natural oligosaccharide linkers can be realized through the selective hydrolysis and modification of its natural sugar chains, so as to realize the insertion of small molecule coupling sites. The main feature of the present invention is that the insertion of a single-structure N-sugar chain with a reactive group at the glycosylation site of an antibody is realized through sugar chain modification technology, and the reactive group carried by the sugar chain is used to realize the integration of small molecule drugs and The site-directed coupling of antibodies obtains site-directed antibody-drug conjugates, and provides a new method of oligosaccharide connection and small molecule drug-linker design and synthesis.

Contents of the invention

The purpose of the present invention is to provide a sugar chain site-directed antibody-drug conjugate composed of an oligosaccharide linker and a side chain hydrophilic segment, its preparation method and application.

In the first aspect of the present invention, a kind of oligosaccharide linker shown in formula (I) is provided:

in:

结构；X are each independently selected from the structures formed by stable covalent bonds formed by highly reactive functional groups (such as hydroxylamine, amino, and mercapto structures) and aldehyde structures, preferably the following structures: -NHCH ₂ -, -ONH=CH-, -CONHCH ₂ -, -NHCO-CH=CH-,

structure;

Each Y is independently selected from the following groups: -(CH ₂ )m-(CH-w)n1-, -(CH ₂ -CH ₂ -O)m-(CH-w)n1-, wherein m and n1 are An integer between 0-30; w is a hydrogen atom or other side chain structures (such as PEG of different lengths);

Z are each independently selected from the following groups:

Wherein, R ₁ and R ₂ are independently selected from H or CH ₃ , D is a small molecule drug, L is a drug linker structure, and Z' is a connection structure between a drug linker and a sugar structure.

In a second aspect of the present invention, an oligosaccharide linker shown in formula (II) is provided:

in:

结构，或独立地为经高碘酸钠氧化得到的醛基结构(此时无Y/Z结构)；X are each independently selected from a structure formed by a stable covalent bond formed by a highly reactive functional group (such as hydroxylamine, amino, mercapto structure) and an aldehyde structure, preferably the following structure:

structure, or independently the aldehyde structure obtained by oxidation of sodium periodate (no Y/Z structure at this time);

Each Y is independently selected from the following groups: -(CH ₂ )m-(CH-w)n1-, -(CH ₂ -CH ₂ -O)m-(CH-w)n1-, wherein m and n1 are An integer between 0-30; w is a hydrogen atom or other side chain structure (such as PEG with different lengths, such as PEG with different molecular weights from PEG200 to PEG50000);

Z are each independently selected from the following groups:

Wherein, R ₁ and R ₂ are independently selected from H or CH ₃ , D is a small molecule drug, L is a drug linker structure, and Z' is a connection structure between a drug linker and a sugar structure.

In another preferred example, in the oligosaccharide linker described in the first or second aspect of the present invention, Z' is selected from the following groups:

Wherein, R ₁ and R ₂ are independently selected from H or CH ₃ ;

L is selected from the following groups:

In the formula, a, b, and c are each independently an integer ≥ 0 (such as 0-20, preferably 1-10).

In another preferred embodiment, a and c are each independently an integer between 2-6; b is an integer selected from 3-30.

In another preferred example, in the oligosaccharide linker, the side chain of L derives a hydrophilic branch structure R:

Among them, R is polyethylene glycol of different lengths or sugar structures with different lengths and configurations composed of different sugar structures, such as glucose Glu, galactose Gal, mannose Man, N-acetylglucose GlcNAc, N-acetylglucose Oligosaccharide structures composed of lactose GalNAc, sialosaccharide SA, xylose Xyl, fucose Fuc, fructose, deoxyribose monosaccharide and different monosaccharides;

V is a bifunctional linker, including but not limited to a structure with lysine, cysteine, and propargylglycine as a bifunctional linker:

Among them, R1 is a side chain polyethylene glycol PEG structure or a sugar structure, R2 is a linker structure that can be covalently linked to an oligosaccharide structure, or R1 can be divided into a Polyethylene glycol PEG chains or sugar structures of different lengths of the functional groups of the base reaction, R1 and R2 positions can be interchanged, each independently selected from the following structures:

Wherein, a, c and d are respectively independently an integer between 2-6; b is an integer selected from between 3-30,

R ₃ and R ₄ are independently selected from CH ₃ -, CH ₃ CH(CH ₃ )-, PhCH ₂ -, NH ₂ (CH ₂ ) ₄ -, NH ₂ CONH(CH ₂ ) ₃ -;

n is an integer of 0-10;

代表连接位点。

Represents the junction site.

In another preferred example, in the oligosaccharide linker, D is a small molecule drug or a small molecule active compound or a radiotherapy, selected from maytansine, DM-1, MMAE, MMAF, MMAP, auristatin E, Changchun Neosine, vinblastine, vinorelbine, VP-16, camptothecin, SN-38, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estrogen Mustin, cemadotin or argentine; or preferably selected from the following groups:

代表连接位点。in,

Represents the junction site.

In another preferred embodiment, the oligosaccharide linker is selected from the following compounds:

In the third aspect of the present invention, there is provided an intermediate that can be used to prepare the above-mentioned oligosaccharide linker of the present invention, said

The intermediate is asparagine and its polypeptide compound containing sugar structures shown in (III) and (IV):

CHO-ASG-Asn/Peptide(III)

CHO-SG-Asn/Peptide (IV).

In the third aspect of the present invention, a method for preparing the compound represented by formula (I) or (ii) by using the intermediate of the present invention is provided.

A preferred preparation method is as follows:

The method comprises the following steps: the glycopeptide structure of the terminal galactose is obtained by acid hydrolysis or neuraminidase hydrolysis of the sialic acid glycopeptide obtained through egg yolk extraction, and the terminal glycopeptide having an aldehyde active group is obtained by the action of galactose oxidase. Galactose glycopeptide, the aldolylated glycopeptide undergoes oxime formation reaction or reductive amination reaction or other reactions with aldehyde groups, introduces other functional groups with bioorthogonal reaction value, and releases them under the action of endoglycosidase An oligosaccharide chain with a special marker group, and the oligosaccharide linker is obtained through a ring closure process;

Alternatively, the method includes the following steps: the sialic acid glycopeptide obtained by extracting egg yolk is hydrolyzed with sialic acid and oxidized with galactose to obtain a glycopeptide with an aldehyde active group, and after the hydrolyzed glycopeptide is hydrolyzed by a glycosidase, After aldehyde group derivatization reaction, other functional groups with bioorthogonal reaction value are introduced, and oligosaccharide linkers are obtained through the cyclization process.

Alternatively, the method comprises the following steps: the sialic acid glycopeptide obtained by extracting egg yolk is hydrolyzed by acid or neuraminidase to obtain the glycopeptide structure of terminal galactose, and then hydrolyzed by glycosidase to obtain the terminal galactose oligosaccharide chain , the oligosaccharide linker is prepared through galactose oxidation, aldehyde group derivatization reaction, and cyclization process;

Alternatively, the method includes the following steps: the terminal sialooligosaccharide chain of the sialoglycopeptide obtained through glycosidase hydrolysis, hydrolysis of sialic acid or neuraminidase, oxidation of galactose, derivatization of aldehyde groups The oligosaccharide linker was prepared by reaction and ring closure.

In the fifth aspect of the present invention, a method for glycosyl modification of an antibody is provided, comprising the step of: transferring the sugar structure in the structures (III) and (IV) to the The conserved glycosylation site of the antibody, so as to obtain the structure of the glycoengineered antibody with the following structure:

Wherein, the X/Y/Z structure in the structure (VII) is as defined above;

m, p and q are independently selected from 0 or 1;

Ab is an antibody.

In another preferred example, the method is carried out using the above-mentioned intermediates of the present invention.

In the sixth aspect of the present invention, there is provided a glycoengineered antibody or antibody-drug based on site-specific linkage of the N-glycosylation site of the antibody Fc region as shown in the following formula (V), (VI) or (VII) Conjugates:

Wherein, the X/Y/Z structure in the structure (VII) is as defined above;

m, p and q are independently selected from 0 or 1;

Ab is an antibody.

In the seventh aspect of the present invention, there is provided a structural formula (VIII) of a sialic acid aldehyde-based glycoengineered antibody or antibody-drug conjugate:

Wherein, the X/Y/Z structure in the structure (VIII) is as defined above;

m, p and q are independently selected from 0 or 1;

Ab is an antibody;

Preferably, the connection mode at X is a ring thioether pyrazole, anthraniloyl oxime, thiazolidine structure

In another preferred example, the antibody-drug conjugates represented by the formulas (V), (VI), (VII) and (VIII) based on the site-specific linkage of the N-glycosylation site of the Fc region of the antibody , wherein, Ab is selected from the following antibodies: Trastuzumab, Pertuzumab, Rituximab, Cetuximab, Moromonab, Gemtuzumab, Abciximab, Dart Lentizumab, adalimumab, palivizumab, basiliximab, bevacizumab, panitumumab, nimotuzumab, or denosumab.

In another preferred example, in the above-mentioned antibody-drug conjugate of the present invention, D is selected from but not limited to maytansine, DM-1, MMAE, MMAF, MMAP, auristatin E, vincristine, vinblastine, Vinorelbine, VP-16, camptothecin, SN-38, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estramustine, simma Dodin or arucillosu; or preferably selected from the following groups:

代表连接位点。

Represents the junction site.

In the eighth aspect of the present invention, there is provided a method for preparing glycoengineered antibodies and antibody-drug conjugates as shown in formulas (V), (VI), (VII), and (VIII), comprising the following steps:

a) cutting off the N-sugar chain of the antibody by glycosidase to obtain an antibody whose N-glycosylation site in the Fc region is modified with acetylglucosamine monosaccharide or fucose α1,6 acetylglucosamine disaccharide;

b) link the oligosaccharide structure represented by formula (III) or (IV) to the acetylglucosamine monosaccharide or fucose α1,6 acetylglucose obtained in step a) through the combined catalysis of glycosidase or its mutants On the amine-disaccharide-modified antibody, prepare an antibody modified by the oligosaccharide linker shown in formula (V) and (VI) containing a special reactive group;

c) Optionally, the antibody modified with the oligosaccharide linker shown in formula (V) and (VI) containing a special reactive group obtained in step b) is combined with Antibody-drug conjugates represented by formulas (VII) and (VIII) are prepared by coupling the small molecule drugs modified by the corresponding groups with specific coupling reactions.

In the ninth aspect of the present invention, there is provided a method for preparing glycoengineered antibodies and antibody-drug conjugates as shown in (VII) and (VIII), comprising the following steps:

a) cutting off the N-sugar chain of the antibody by glycosidase to obtain an antibody whose N-glycosylation site in the Fc region is modified with acetylglucosamine monosaccharide or fucose α1,6 acetylglucosamine disaccharide;

b) Connect the oligosaccharide linker represented by formula (I) or (II) to the acetylglucosamine monosaccharide or fucose α1,6 acetylglucosamine obtained in step a) through the catalysis of glycosidase or its mutant On the disaccharide-modified antibody, an antibody modified with an oligosaccharide linker shown in formula (I) and (II) containing a special reactive group is prepared;

c) The antibody modified by the oligosaccharide linker shown in formula (I) and (II) containing the special reactive group obtained in step b) is combined with the special reactive group capable of interacting with the sugar chain of the antibody The small molecule drug modified by the corresponding group for specific coupling reaction is coupled to prepare antibody-drug conjugates represented by formulas (VII) and (VIII).

In the tenth aspect of the present invention, there is provided a method for preparing antibody-drug conjugates represented by formulas (IV) and (V), wherein, in steps a) and b), the glycosidase or its mutation The body is fucose hydrolase, N-acetylglucosamine endohydrolase or their mutants, more preferably, said N-acetylglucosamine endohydrolase comprises Endo-S (Streptococcus pyogenes endohydrolase Endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, S. At least one of Streptococcus pyogenes endoglycosidase-Sd) and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); And/or, preferably, steps b) and c) The special reactive groups mentioned above and the corresponding groups that can carry out specific coupling reaction with this group are respectively azide and alkynyl, sulfhydryl and maleimide, sulfhydryl and sulfhydryl or activated form of sulfhydryl, aldehyde and Amino groups, aldehyde groups and hydroxylamine or hydrazine groups correspond to combinations in pairs.

In the eleventh aspect of the present invention, there is provided a method for preparing antibody-drug conjugates represented by formulas (V), (VI), (VII) and (VIII), wherein, in step c), the The above-mentioned small-molecule drug modified with a corresponding group capable of carrying out a specific coupling reaction with the special reactive group (aldehyde group) on the antibody sugar chain is selected from but not limited to the following structures:

ThiolPZ-VC-PAB-MMAE(D7)

ABAO-VC-PAB-MMAE (D8).

In another preferred example, in the antibody-drug conjugates of the formulas (VII) and (VIII), the small molecule drug coupled to the sugar linker is preferably a biological drug containing a side chain PEG structure and a sugar structure. Small molecule compounds with orthogonal groups, but not limited to compounds with the following structures:

DBCO-PEG ₄ -Lys(εPEG ₁₂ )-VC-PAB-MMAE (n=12, D1)

DBCO-PEG ₄ -Lys(εPEG ₂₄ )-VC-PAB-MMAE (n=24, D2)

DBCO-PEG ₄ -Lys(αPEG ₁₂ )-VC-PAB-MMAE (n=12, D3)

DBCO-PEG ₄ -Lys(αPEG ₂₄ )-VC-PAB-MMAE(D4)

Alkyne-PEG ₄ -VC-PAB-MMAE (n=4, D5)

Alkyne-PEG ₂₄ -VC-PAB-MMAE (n=24, D6)

DBCO-Lys(εPEG ₂₄ )-VC-PAB-MMAE(D9)

BCN-Lys(εPEG ₂₄ )-VC-PAB-MMAE(D10)

DBCO-(Gly-Triazol-Glc)-VC-PAB-MMAE(D11)

DBCO-(Gly-Triazol-Glc-Gal)-VC-PAB-MMAE(D12)

DBCO-(Gly-Triazol-Glc3)-VC-PAB-MMAE(D13)

DBCO-(Gly-Triazol-Glc4)-VC-PAB-MMAE(D14).

In the twelfth aspect of the present invention, a pharmaceutical composition is provided, which contains a pharmaceutically acceptable carrier and the above-mentioned antibody-drug conjugate of the present invention.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated here.

Description of drawings

Figure 1 The mass-to-charge ratio (m/z) and deconvoluted mass spectrum of the non-natural sugar chain antibody Ab-1.

Figure 2 The mass-to-charge ratio (m/z) and deconvoluted mass spectrum of the non-natural sugar chain antibody Ab-2.

Figure 3 The mass-to-charge ratio (m/z) and deconvoluted mass spectrum of the non-natural sugar chain antibody Ab-3.

Figure 4 The mass-to-charge ratio (m/z) and deconvoluted mass spectrum of the non-natural sugar chain antibody Ab-4.

Figure 5 is the mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the non-natural sugar chain antibody Ab-5.

Figure 6 The mass-to-charge ratio (m/z) and deconvoluted mass spectrum of the non-natural sugar chain antibody Ab-6.

Figure 7 The deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-1.

Figure 8 The deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-2.

Figure 9 is the deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-3.

Figure 10 The deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-4.

Figure 11 The deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-5.

Figure 12 The deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-6.

Figure 13 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-7.

Figure 14 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-8.

Fig. 15 The mass-to-charge ratio (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-9.

Figure 16 The mass-to-charge ratio map (m/z) and the deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-10.

Figure 17 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-11.

Fig. 18 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-12.

Figure 19 The mass-to-charge ratio map (m/z) and the deconvoluted mass spectrum of the sugar chain site-fixed ADC compound gsADC-13.

Figure 20 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-14.

Figure 21 The mass-to-charge ratio map (m/z) and the deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-15.

Figure 22 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-16.

Figure 23 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-17.

Figure 24 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-18.

Figure 25 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-19.

Figure 26 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-20.

Figure 27 The mass-to-charge ratio map (m/z) and deconvoluted mass spectrum of the sugar chain-fixed ADC compound gsADC-21.

Figure 28 is the deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-22.

Figure 29 The deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-23.

Figure 30 is the deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-24.

Figure 31 is the deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-25.

Figure 32 is the deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-26.

Figure 33 is the deconvoluted mass spectrum of the sugar chain-directed ADC compound gsADC-27.

Figure 34 Compound 8-H NMR.

Figure 35 Compound 8-13C NMR.

Figure 36 Compound 13c-H NMR.

Figure 37 Compound 13c-13C NMR.

Figure 38 Compound D2-H NMR.

Figure 39 Compound D3-H NMR.

Figure 40 Compound D4-H NMR.

Figure 41 Compound D5-H NMR.

Figure 42 Compound D9-H NMR.

Figure 43 Compound D10-H NMR.

Figure 44 Compound D11-H NMR.

Figure 45 Compound D15-H NMR.

Figure 46 Cell activity results of sugar chain site-directed ADC compounds.

Figure 47 is a curve graph of tumor size over time and a curve graph of body weight over time.

Detailed ways

After extensive and in-depth research, the present inventors unexpectedly discovered for the first time a new type of oligosaccharide linker and its connection method. Specifically, the present invention provides a new type of oligosaccharide linker and its connection method. The conservative glycosylation site of the antibody is used as the coupling site, the biantennary oligosaccharide is used as the linker structure, and the terminal sialic acid and terminal galactose are modified. Aldehyde and other structures are the coupling of a series of small molecular compounds for linking residues. The site-specific linked antibody-drug conjugate of the present invention has good antitumor activity. The present invention has been accomplished on this basis.

The reagents used in the present invention are commercially available or prepared by conventional methods. Among them, the glycoside hydrolase and its mutant enzymes are all expressed in the Escherichia coli system. 3-Azidopropylamine, propargylamine and acetonitrile were purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.; small molecule cytotoxic drugs DM1 and MMAE were purchased from Nanjing Lianning Biopharmaceutical Company; click chemical linker DBCO-PEG ₄ -acid was purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd.; amino acid compounds were purchased from Jill Biochemical (Shanghai) Co., Ltd.; 4-pentynoic acid was purchased from Shanghai Beide Pharmaceutical Technology Co., Ltd.; PEG compounds were purchased from Wuhan Fusheng Kanghua Technology Co., Ltd. ; Lysine was purchased from Bi De Pharmaceutical Technology Co., Ltd. Other compounds, reagents and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. unless further explained.

Instruments and chromatographic columns used in the present invention include: analytical high performance liquid chromatograph (Beijing Innovation Tongheng LC3000), preparative high performance liquid chromatography (Beijing Innovation Tongheng LC3000); Agilent SB-C18 (5 μ m, 4.6 x150mm), Waters OBD-C18 (5μm, 19x250mm);

Antibody molecular weight determination instrument: liquid chromatography-mass spectrometry (LC-MS), Agilent 6230LC-TOF-MS, equipped with Agilent C-18column (3.5μm, 50x2.1mm) at 55°C.

The main advantages of the present invention include

The antibody-drug conjugates of site-specific connection shown in formulas (IV) and (V) prepared by the oligosaccharide linker shown in formula (I) (II) (III), its chemical structure is a uniform structure of site-specific quantification , compared with the marketed antibody-drug conjugates with heterogeneous structures, it has the advantages of clear and single structure and controllable quality. Meanwhile, the preferred site-specific linked antibody-drug conjugate exhibits good antitumor activity, and the IC ₅₀ can reach 0.01nM (1.578ng/mL).

Below in conjunction with specific embodiment, further state the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method that does not indicate detailed condition in the following examples, generally according to conventional conditions such as people such as Sambrook, molecular cloning: the condition described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example

The synthesis route of gsADC is as follows:

Legend: a. 0.1% TFA, 80°C or asialic acid enzyme means; b. Galactose oxidase, O ₂ , horseradish peroxidase, catalase; c. 3-azidopropylamine, NaCNBH ₃ , pH 6.0, 0°C; dO-(2-azidoethyl) hydroxylamine hydrochloride, pH 7.4; e. NaIO ₄ , protected from light, 15 min; f. Endo-M, pH 6.2-6.5;

g. DMC, triethylamine or CDMBI, K ₃ PO ₄ ,

General operation 1: Preparation method 1 of non-natural glycoengineered antibody

The prepared derivatized oligosaccharide oxazoline (i.e. compound 13a-13d), deglycosylated Herceptin Fucα1, 6GlcNAc-Herceptin, mutant endoglycosidase Endo-S D233Q, so that the concentrations of each item are 1.5mM, 5 mg/mL, 0.15 mg/mL, and incubate at 30°C for about 2 hours to obtain the antibodies Ab-1 to Ab-4 of Example 20-23 through protein A.

General operation 2: Preparation method 2 of non-natural glycoengineered antibody

The prepared aldehyde-modified glycopeptide structures (2 and 8), deglycosylated Herceptin Fucα1, 6GlcNAc-Herceptin (or wild-type Herceptin), mutant endoglycosidase Endo-S D233Q, Endo-M For N175Q, the respective concentrations were 3.3 mM, 5 mg/mL, 0.6 mg/mL and 1 mg/mL in turn, and incubated at 30°C for 6-12 hours, and the antibodies Ab-5 to Ab-6 of Example 24-25 were obtained through protein A .

General operation 3: Method 1 for the preparation of sugar chain-directed ADC molecules based on aldose engineering antibodies

The prepared aldehyde-modified glycoengineering antibodies (Ab-5~Ab-6) were dissolved in 50mM PB, pH 7.5 buffer system, and drug-linker D9 containing hydroxylamine was added to the above system to make the final concentrations of each 5mg/mL and 0.55mM, adjust the pH of the reaction system to 7.5, incubate at 37°C for 4h, SDS-PAGE and MS analysis show that about 4 drug-linkers are coupled, purified by protein A, concentrated by ultrafiltration tubes and exchanged liquid Afterwards, the corresponding sugar chain-directed ADC molecules gsADC-1-gsADC-2 of Examples 26-27 were obtained.

General operation 4: Method 2 for the preparation of sugar chain-directed ADC molecules based on aldose engineering antibodies

The prepared aldehyde-modified glycoengineering antibodies (Ab-5~Ab-6) were dissolved in 50mM PB, pH 7.5 buffer system, and the drug-linker D8 containing ABAO functional group was added to the above system to make the final concentration of each 5mg/mL and 1.3mM in turn, adjust the pH of the reaction system to 5.0, incubate overnight at 37°C, SDS-PAGE and MS analysis show that about 4 drug-linkers are coupled, purified by protein A, concentrated by ultrafiltration tubes and replaced with liquid After the operation, the corresponding sugar chain site-directed ADC molecules gsADC-3-gsADC-4 of Examples 28-29 were obtained.

General operation five: sugar chain-directed ADC molecule preparation method three based on aldose engineering antibody

The prepared aldehyde-modified glycoengineered antibodies (Ab-5~Ab-6) were dissolved in 50mM PB, pH 7.5 buffer system, and the drug-linker D7, EDTA and 10% TritonX-containing thioPz were added to the above-mentioned system. 100, so that the final concentration of each item is 5mg/mL, 1.3mM, 1mM and 1%, adjust the pH of the reaction system to 5.5, and incubate overnight at 37°C. SDS-PAGE and MS analysis show that about 4 drug-linkers are coupled, After protein A purification, ultrafiltration tube concentration, and liquid exchange operations, the corresponding sugar chain-directed ADC molecules gsADC-5-gsADC-6 of Examples 30-31 were obtained.

General operation 6: Method 1 for the preparation of sugar chain-directed ADC molecules based on azido-glycoengineering antibodies

The prepared azido-modified glycoengineered antibodies (Ab-1~Ab-4) were dissolved in 50mM PB, pH 7.5 buffer system, and drug-linkers D1-D4 containing ring strained alkynyl structures were added to the above system , D10-D15, so that the final concentration of each item is 5mg/mL and 0.55mM in turn, adjust the pH of the reaction system to 7.5, incubate at 37°C for 4h, SDS-PAGE and MS analysis show that about 4 drug-linkers are coupled, and the protein After A purification, ultrafiltration tube concentration and liquid exchange operations, the corresponding sugar chain-directed ADC molecules gsADC-7~gsADC-21 and gsADC-28~gsADC-29 of Examples 32-46 and 53-54 were obtained.

General operation 7: Preparation method of sugar chain site-directed ADC molecules based on azido-glycoengineering antibodies II

The prepared azido-modified glycoengineering antibodies (Ab-1~Ab-4) were dissolved in 50mM PB, pH 7.5 buffer system, and drug-linkers D5-D6 and Cu(I)-BTTAA solution (preparation method: sequentially add 21 μL ddH ₂ O, 3 μL 60 mM CuSO ₄ solution, 3 μL 300 mM BTTAA solution, 3 μL 900 mM Vc-Na solution, mix well and set aside), so that the final concentration of each item is 5 mg/ mL, 0.55mM and 1mM, adjust the pH of the reaction system to 7.5, incubate at 37°C for 4h, SDS-PAGE and MS analysis show that about 4 drug-linkers are coupled, after protein A purification, ultrafiltration tube concentration and medium exchange operation The corresponding sugar chain-directed ADC molecules gsADC-22-gsADC-27 of Examples 47-52 were obtained.

Example 1: Preparation of N ₃ -ON=CH-SCT-ox 13a

Dissolve 50mg of sialoglycopeptide SGP 1 in 2.5mL of 0.2M PB, pH 7.1 buffer system, add 2.5mL of prepared 30mM sodium periodate solution to the above reaction system, mix well and place it on ice in the dark for 15 minutes. The CHO-SGP fraction was collected and lyophilized to obtain CHO-SGP 2 (42 mg, yield 88%). HRMS, calcd for C ₁₀₈ H ₁₇₇ N ₁₅ O ₆₆ [M+3H] ³⁺ 914.3730, [M+H ₂ O+3H] ³⁺ 920.3765, found 914.3739, 920.3778. ¹ H NMR (500MHz, D ₂ O) δ5.06 (1H, s, H1c), 4.98 (1H, d, J=9.7Hz, H1a'), 4.9 (3H, m, H1c', CHO–C(H) -(OH) ₂ ),4.72(1H,s,H1b),4.62(1H,t,J=6.7Hz,Hαof Asn),4.55 (3H,d,J=7.4Hz,H1a,H1d,H1d'), 4.41(1H,d,J=3.5Hz,Hαof Thr),4.39(1H,d,J=2.5Hz,H1e),4.37-4.27(3H,m,H1e',Hβof Thr,Hαof Lys),4.25(1H ,q,J＝7.1Hz,Hαof Ala), 4.18(1H,s,H2b),4.14(1H,d,J＝3.4Hz,H2c),4.09(1H,d,J＝7.5Hz,Hαof Val), 4.04(1H, d, H2c'), 4.00(3H, t, J＝6.4Hz, Hαof Lys), 2.94(m, 4H, Hεof Lys), 2.83-2.6(2H, m, Hβof Asn), 2.53(2H ,dd,H3feq,H3f'eq),2.11-1.89(19H,6x3Ac,Hβof Val),1.83(6H,Hβof Lys, H3fax,H3f'ax),1.76-1.58(4H,m,J＝7.5Hz,Hδof Lys),1.38(4H,m,Hγof Lys),1.31(3H,d, J=7.5Hz,Hβof Ala),1.13(3H,J=6.5Hz,Hγof Thr),0.89(6H,d,7.0Hz, Hγof Val).

Dissolve 30mg CHO-SGP 2 in 300μL 0.2M PB, pH 7.4 buffer system, add 60μL 100mg/mL O-(2-azidoethyl) hydroxylamine hydrochloride (dissolved in 0.2M PB, pH 7.4 buffer system), incubated at 37°C, and after 4 hours, the reaction system was monitored by analytical HPLC and analyzed by mass spectrometry, which showed that the reaction was almost complete. Separation and purification were carried out on a semi-preparative C18 column, and the target components were collected and freeze-dried to obtain N ₃ -ON=CH-SGP 3 (27 mg, yield 85%). HRMS ^{, calcd for C112H185N23O66 [M+3H]3+} _{970.402 , found 970.4034} . ¹ H NMR (500MHz, D ₂ O) δ7.43 (1.6H, dd, J＝7.2, 3.3Hz, H1 of oxime), 6.86 (0.4H, dd, J＝6.7, 2.6Hz, H1 of oxime), 5.06(1H,s,H1c),4.97(1.4H,H1a',H6f,H6f'),4.89(1H,s,H1c'),4.78(1H,s,H1b),4.6(1H,t,HαofAsn) ,4.55(3H,d,J=6.8Hz,H1a,H1d,H1d'),4.41(1H,d,J=3.5Hz,Hαof Thr),4.38 (1H,d,J=2.5Hz,H1e),4.34 (1H,d,J=3Hz,H1e'),4.35-4.29(2H,Hβof Thr,Hαof Lys), 4.25(1H,q,J=7Hz,Hαof Ala),4.18(6.6H,m,H2b,H2 of oxime, H6f, H6f'), 4.13 (1H, d, J = 2.1Hz, H2c), 4.09 (1H, d, J = 8.1Hz, Hαof Val), 4.05 (1H, d, J = 2Hz, H2c' ),4.00(1H,t,J＝6.4Hz,Hαof Lys),2.94(4H,q,J＝7.5Hz,Hεof Lys),2.82-2.61(2H,m,Hβof Asn), 2.63-2.52(2H, H3feq, H3f'eq), 2.05-1.88 (19H, 6x3Ac, Hβof Val), 1.88-1.68 (6H, Hβof Lys, H3fax, H3f'ax), 1.67 (4H, m, J＝7.5Hz, Hδof Lys), 1.45-1.33 (4H, m, Hγof Lys), 1.32 (3H, d, J＝7.5Hz, Hβof Ala), 1.15 (3H, J＝6.5Hz, Hγof Thr), 0.89 (6H, d, J＝7.0Hz ,HγofVal).

N ₃ -ON=CH-SGP 3 (10μmoL, 1eq) was dissolved in 100μL 50mM PB, pH 6.5 buffer solution, 1M hydrochloric acid and 1M sodium hydroxide to adjust the pH to 6.5, and 1.7μL 30mg/mL Endo -M, incubated at 30°C, after 4 hours, use analytical HPLC to monitor the reaction system and perform mass spectrometry, which shows that the reaction is complete. Use C18 semi-preparative column and Sephadex G25 to purify to obtain N ₃ -ON=CH-SCT-OH 4 (Yield 83%). HRMS, calcd for _{C76H121N13O53} [M+2H] ²⁺ _1032.8665 , [M+3H _] ³⁺ 688.9136, found 1032.8622, _688.9119 . ¹ H NMR (500MHz, deuterium oxide) δ7.41 (dd, J=7.2, 4.9Hz, 1.3H, H1 of oxime), 6.83 (dd, J=6.6, 4.1Hz, 0.7H, H1 of oxime), 5.12 (d, J=3.1Hz, 0.8H, H1a), 5.07(s, 1H), 4.96(dd, J=9.0, 6.6 Hz, 0.7H), 4.88(s, 1H), 4.52(d, J=7.3 Hz,2H),4.35(d,J=7.9Hz,2H),4.24-4.13(m,6.3H),4.11(s,1H),4.03(s,1H),3.93-3.77(m,14H), 3.69(m,20H),3.59-3.38(m,21H),1.99-1.87(m,15H),2.57(d,J＝11.8Hz,2H,),2.02-1.87(m,15H),1.77(t ,J=11.4Hz,2H).

Weigh 10 mg of N ₃ -ON=CH-SCT-OH and dissolve in 480 μL of distilled water, add 12.5 mg of DMC and 30.3 μL of triethylamine to the above system, mix well and put it on ice to react for 2 hours, use dextran G25 gel Column purification afforded N ₃ -ON=CH-SCT-ox 13a (yield 78%). HRMS, calcd for _{C76H119N13O52} [M+2H] ²⁺ _1023.8612 , [M+ _3H ] ³⁺ 682.91, found 1023.8616, _682.9202 . ¹ H NMR (500MHz, deuterium oxide) δ7.39(d, J=7.2Hz, 1.6H), 6.82(d, J=6.6Hz, 0.4H,), 6.03(d, J=7.2Hz, 1H, H1 of-ox), 5.13-4.98(m, 1.4H), 4.9(s, 1H), 4.68(s, 1H), 4.55(dd, J=5.7Hz, 2H), 4.37(d, J=6.6Hz, 2H), 4.32(s, 1H), 4.28-4.2(dd, 1.6H), 4.17(t, J=4.6Hz, 4H), 4.13(m, 1H), 4.11(m, 1H), 2.57(d, J＝13.2Hz, 2H), 2.15–1.84(m, 15H), 1.68(t, 2H).

Example 2: Preparation of N ₃ -NH-SCT-ox 13b

Dissolve 30mg CHO-SGP 2 in 500μL 0.2M PB, pH=6.0 buffer system, add 32μL 3-azidopropylamine to the above system, adjust the pH to 6.5 with acetic acid, add 140μL 100mg/mL to the above reaction system Sodium cyanoborohydride mother liquor, the reaction system was incubated on ice, and the reaction system was monitored overnight by analytical HPLC-MS, showing that the reaction was almost complete. After separation and purification on a semi-preparative C18 column, N ₃ -NH-SGP 5 (21 mg, yield 67%) was obtained by lyophilization. HRMS ^{, calcd for C114H193N23O64 [M+3H]3+} _{970.4263 , found 970.4233} . ¹ H NMR (500 MHz, deuterium oxide) δ5.05(1H, s, H1c), 4.95(1H, d, J=9.6Hz, H1a'), 4.85(1H, s, H1c'), 4.72(1H, s, H1b), 4.57 (1H, t, J = 6.7Hz, Hαof Asn), 4.52 (3H, H1a, H1d, H1d'), 4.38 (1H, d, J = 3.5Hz, Hαof Thr), 4.36 (1H ,d,J=2.5Hz,H1e),4.35(1H,d,J=3Hz,H1e'),4.32(2H,m,Hαof Lys1,Hβof Thr),4.23(1H,q,J=7.2Hz,Hαof Ala),4.16(1H,s,H2b),4.11(1H,d,J=2.7 Hz,H2c),4.07(1H,d,J=7.6Hz,Hαof Val),4.02(1H,d,J=3.5 Hz,H2c'),4.01-3.91(3H,m, Hαof Lys,H6f,H6f'),3.25-3.05(8H,m,2x-CH ₂ -NH-CH ₂ -),2.93(4H,q,J ＝7.5Hz, Hεof Lys), 2.83-2.62(2H, m, Hβof Asn), 2.62-2.51(2H,dd, J＝12.5Hz, 2.5Hz, H3feq, H3f'eq), 2.05-1.91(19H,m ,6x3Ac,Hβof Val),1.92-1.86(4H,m,H3g),1.85-1.66(6H,m,Hβof Lys, H3fax,H3f'ax),1.6(4H,m,J＝7.5Hz,Hδof Lys) ,1.42-1.31(4H,m,Hγof Lys),1.28(3H,d,J＝7.5Hz,Hβof Ala),1.12(3H,J＝6.5Hz,Hγof Thr),0.87(6H,d,J＝7.0 Hz,Hγof Val).

N ₃ -NH-SGP 5 (10μmoL, 1eq) was dissolved in 100μL 50mM PB, pH 6.5 buffer, 1M hydrochloric acid and 1 M sodium hydroxide to adjust the pH to 6.5, and 1.7μL 30mg/mL Endo- M, incubated at 30°C, after 4 h, the reaction system was monitored by analytical HPLC and mass spectrometry showed that the reaction was complete, and purified by C18 semi-preparative column and Sephadex G25 to obtain N ₃ -NH-SCT-OH 6 (produced rate of 76%). HRMS, calcd for _{C78H129N13O51} [M+2H] ²⁺ _1032.9028 , [M+ _3H ] ³⁺ 688.9378, found 1032.9012, _688.9322 .

Weigh 10 mg of N ₃ -NH-SCT-OH 6 and dissolve in 480 μL of distilled water, add 12.5 mg of DMC and 30.3 μL of triethylamine to the above system, mix well and place it on ice for 2 hours to react. Use Sephadex G25 gel column Purification afforded N ₃ -NH-SCT-ox 13b (65% yield). HRMS _, calcd for _{C78H127N13O50} [M+2H] ²⁺ 1023.8976, [M+ _3H ] ³⁺ 682.9343, found _1023.8989 , 682.9331.

Example 3: Preparation of N ₃ -ON=CH-CT-ox 13c

Dissolve 200 mg of sialic acid glycopeptide SGP 1 in 2 mL of 0.1% TFA aqueous solution, heat the above reaction system in a water bath at 80°C for 2 h, monitor the complete hydrolysis of the terminal sialic acid structure by HPLC, use a semi-preparative C18 column to separate and purify, and collect The target fraction was lyophilized to obtain ASGP 7 (150 mg, yield 94%). HRMS ^{, Calcd. for C90H155N13O54 [M+3H]3+} _{761.6672 , found 761.667} . ¹ H NMR (500MHz, D ₂ O) δ5.06(s, 1H, H1c), 4.98(d, J = 9.6Hz, 1H, H1a'), 4.86(s, 1H, H1c'), 4.62(t, J=6.7Hz, 1H), 4.75(s, 1H, H1b), 4.62(dd, J= 14.1, 7.6Hz, 1H, Asn-H), 4.53(m, 3H, H1a/d/d'), 4.43 (d,J=3Hz,1H,Thr-H),4.40(d,J=7.6Hz,2H,H1e/e'),4.34(m,2H,Lys(KV)-H,Thr-H),4.26 (q,J=7.1Hz,1H,Ala-H),4.18(m,1H),4.13(dd,J=3.5,1.6Hz,1H,H2c),4.10(d,J=7.6Hz,1H,Val -H), 4.05(dd, J=3.5, 1.6 Hz, 1H, H2c'), 4.01(t, J=6.7Hz, 1H, Lys(NKT)-H), 2.94(m, 4H, Lys-H) ,2.73(m,2H,Asn-H),2.08-1.91(m,13H,Val-H,Ac-CH ₃ ),1.84(m,4H,Lys-H),1.77-1.58(m,4H,Lys -H), 1.39(m,4H,Lys-H),1.32(d,J=7.2Hz,3H,Ala-H),1.14(d,J=6.4Hz,3H,Thr-H),0.90(d , J＝6.7Hz, 6H, Val-H). ¹³ C NMR (126MHz, D ₂ O) δ174.71, 174.68, 174.62, 174.34, 173.83, 173.47, 172.53, 172.17, 172.04, 169.66, 163.07, 162.78, 115.18, ,102.95-102.93(C1e/e'),101.31(C1b),100.41(AsnC),99.56(C1c),99.42,97.02(C1c'),78.69,78.55(C1a'),78.25,76.45(C2c),76.29 (C2c'), 76.19, 75.35, 74.74, 74.70, 74.40, 73.56, 72.84, 72.80, 72.51, 72.08, 71.95, 70.97, 69.45, 69.39, 68.54, 61.01, 59.99 (Val-Cα), 58.15 (Thr-Cα) ,,52.72 (Lys(NKT)-Cα),49.51(Ala-Cα),39.17(Lys-Cε),38.99(Lys-Cε),36.38(Asn-Cβ),30.47 (Thr-Cβ),30.13(Lys -Cβ), 26.33(Lys-Cδ), 22.35(CH ₃ CO-), 21.90(Lys-Cγ), 18.85(Thr-Cγ), 17.77(Val-Cγ), 16.74(Ala-Cβ).

Dissolve 100mg ASGP 7 in 1.7mL 50mM PB, pH=7.0 buffer system, blow oxygen into the above system for 10 minutes, then add 80U galactose oxidase, 2.4KU horseradish peroxidase and 6KU catalase in sequence , the above reaction system was placed on a metal bath and incubated at 30°C. After 1 hour, the reaction system was monitored by analytical HPLC and identified by mass spectrometry, which showed that the reaction was complete. Separation and purification were performed using a semi-preparative C18 column, and the target fraction was collected and lyophilized to obtain CHO-ASGP 8 (72 mg, yield 72%). HRMS, Calcd. for _{C90H151N13O54} [M+3H] ³⁺ _{760.3235 ,} found _760.3233 . ¹ H NMR (500MHz, deuterium oxide) δ5.08(d, J=1Hz, 1H, H6e), 5.07(d, J=1Hz, 1H, H6e'), 5.06(s, 1H, H1c), 4.98(d ,J=9.6Hz,1H,H1a'),4.86(s,1H,H1c'),4.75(s,1H,H1b),4.53(dd,J=14.1,7.5Hz,1H,Asn-Hα),4.44 -4.38(m,3H,H1a/d/d'),4.43(d,J＝3Hz,1H, Thr-Hα),4.40(d,J＝7.6Hz,2H,H1e/e'),4.34(m , 2H, Lys(KV)-Hα, Thr-Hβ), 4.26(q, J=7.1 Hz, 1H, Ala-Hα), 4.13(dd, J=3.5, 1.6Hz, 1H, H2c), 4.10(d ,J=7.6Hz,1H,Val-Hα),4.05 (dd,J=3.5,1.6Hz,1H,H2c'),4.02(m,3H,H5e/e',Lys(NKT)-Hα),2.94 (m,4H,Lys-Hε), 2.73(m,2H,Asn-Hβ),2.04-1.92(m,13H,Val-Hβ,Ac-CH ₃ ),1.84(m,4H,Lys-Hβ), 1.75-1.59 (m, 4H, Lys-Hδ), 1.44-1.34 (m, 4H, Lys-Hγ), 1.32 (d, J = 7.2Hz, 3H, Ala-Hβ), 1.14 (d, J = 6.4 Hz , 3H, Thr-Hγ), 0.91 (d, J=6.7Hz, 6H, Val-Hγ). ¹³ C NMR (126MHz, deuterium oxide) δ174.74, 174.71, 174.65, 174.35, 173.84, 173.53, 172.55, 172.19 ,172.06,163.09,162.81,117.51,115.19,106.46,103.28&103.26(C1e/e'),99.65(C1c),97.10(C1c'),88.02(C6e/e',HO-C-OH),79.50 , 78.70,78.26(C1a'),76.92,76.44(C2c),76.23(C2c'),74.59,74.57,74.41,72.84,72.81,72.44, 72.22,72.09,71.99,70.70,70.19(C2b),699.45 ,67.99(C5e/e'),67.35,67.31,67.08 (Thr-Cβ),60.09,59.96,59.89,59.46(Val-Cα),58.20(Thr-Cα),54.96,54.78,53.71,53.57 (Lys- Cα), 52.72 (Lys-Cα), 49.95 (Asn-Cα), 49.47 (Ala-Cα), 39.17 & 38.99 (Lys-Cε), 36.38 (Asn-Cβ), 30.47 (Lys-Cβ), 30.13 ( Val-Cβ), 26.33&26.23(Lys-Cδ), 22.35&22.25&22.12& 21.90(CH ₃ CO-), 21.15(Lys-Cγ), 18.85(Thr-Cγ), 18.37(Val-Cγ), 17.77 (Val-Cγ), 16.74 (Ala-Cβ).

Dissolve 30mg CHO-ASGP 8 in 300μL 0.2M PB, pH 7.4 buffer system, add 73μL 100mg/mL O-(2-azidoethyl) hydroxylamine hydrochloride (dissolved in 0.2M PB, pH 7.4 buffer system), incubated at 37°C, and after 4 hours, the reaction system was monitored by analytical HPLC and identified by mass spectrometry, which showed that the reaction was almost complete. The target fraction was collected and lyophilized to obtain N ₃ -ON=CH-ASGP 9 (28 mg, yield 87%). HRMS, calcd for _{C94H159N21O54} [M+3H] ³⁺ _{816.3525 ,} found _816.3533 . ¹ H NMR (500MHz, deuterium oxide) δ7.56 (dd, J=4.1, 1.3Hz, 1.3H, H6e/e'), 6.91 (dd, J=4.6, 1.7Hz, 0.7H, H6e/e') ,5.07 (s,1H,H1c),4.99(d,J=9.6Hz,1H,H1a'),4.88(s,1H,H1c'),4.88(m,0.7H,H5e/e'),4.63( t, J＝6.8Hz,1H,Asn-Hα),4.60-4.51(m,3H,H1a,H1d,H1d'),4.47(m,2H,H1e/e'),4.42(m,1.3H,H5e /e'), 4.4(d, J=3.4Hz, Thr-Hα), 4.35(m, m, 2H, Lys(KV)-Hα, Thr-Hβ), 4.27(m, 2H), 4.22-4.18( m,5H,H7,H2b),4.14(m,1H,1H,H2c),4.11(d,J＝7.6Hz,1H,Val-Hα), 4.04(m,3H,H4e/e',H2c', Lys(NKT)-Hα),3.00-2.91(m,4H,Lys-Hε),2.85-2.62(m,2H,Asn-Hβ),2.11-1.92(m,13H,4x3Ac,Val-Hβ),1.90 -1.60(m,8H,Lys-Hβ,-Hδ),1.45-1.35(m,4H,Lys-Hγ),1.33(d,J=7.2Hz,3H,Ala-Hγ),1.15(d,J= 6.4Hz, 3H, Thr-Hγ), 0.92 (d, J=6.7Hz, 6H, Val-Hγ). ¹³ C NMR (126MHz, deuterium oxide) δ174.74, 174.64, 174.35, 173.80, 173.64, 172.55, 172.20, 172.06 ,169.68,163.11,162.84,149.71(C6e/e'),148.88(C6e/e'),117.51, 115.19,103.06(C1e/e'),102.62,101.33,100.44(C1b),99.58(C1c),99.45 ,99.36,97.03(C1c'), 80.40,79.54,79.17,78.71,78.27(C1a'),76.46(C2c),76.27(C2c'),76.20,74.64,74.41,73.57, 73.02,72.81,72.45(C5e/ ( Val-Cα), 58.29 (Thr-Cα), 54.95, 54.87, 53.71, 53.58 (Lys-Cα), 52.73 (Lys-Cα), 49.95 (Asn-Cα), 49.76, 49.47 (Ala-Cα), 39.18&39 .00(Lys-Cε), 36.48(Asn-Cβ), 30.48(Lys-Cβ), 30.13(Val-Cβ), 26.34& 26.24(Lys-Cδ), 22.34&22.26&22.13&21.90(CH ₃ CO -), 21.16(Lys-Cγ), 18.88(Thr-Cγ), 18.37(Val-Cγ), 17.77(Val-Cγ), 16.75(Ala-Cβ).

N ₃ -ON=CH-ASGP 9 (10μmoL, 1eq) was dissolved in 100μL 50mM PB, pH 6.5 buffer solution, 1M hydrochloric acid and 1M sodium hydroxide to adjust the pH to 6.5, and 1.7μL 30mg/mL Endo -M, incubated at 30°C, after 4 hours, use analytical HPLC to monitor the reaction system and perform mass spectrometry, which shows that the reaction is complete. Use C18 semi-preparative column and Sephadex G25 to purify to obtain N ₃ -ON=CH-CT-OH 10 (Yield 85%). _HRMS , _calcd for _C58H95N11O41 [M+2H] ²⁺ 801.7922, found _801.7929 . ¹ H NMR (500MHz, deuterium oxide) δ7.55(d, J=4.1Hz, 1.4H, H6e/e'), 6.90(d, J=4.6Hz, 0.6H, H6e/e'), 5.15(d ,J=3.1Hz,0.6H,H1a-α), 5.06(s,1H,H1c),4.87(s,1H,H1c'),4.79(dd,J=4.1Hz,1.7Hz,0.6H,H5e/ e'),4.53(m,2H, H1d/d'),4.47(dd,J＝7.9,1.9Hz,1.4H,H1e/e'),4.44(dd,J＝7.9,1.9Hz,0.6H, H1e/e'), 4.41(dd, J=4.1Hz, 1.7Hz, 1.4H, H5e/e'), 4.25(t, J=4.9Hz, 1H, H2b), 4.20(t, J=5.0Hz, 4H, H7e/e'), 4.13(s, 1H, H2c), 4.05(s, 1H, H2c'), 4.03(d, J=3.2Hz, 2H, H4e/e'), 2.02-1.96(m, 9H, H-Ac). ¹³ C NMR (126MHz, deuterium oxide) δ174.60(3 x CH ₃ CO-), 149.73(-N=CH-), 148.86 (-N=CH-), 103.05(C1e/ e'), 102.60(C1e/e'), 100.42(C1b), 99.55(C1c), 99.45, 99.41-99.39 (C1d/d'), 97.04(C1c'), 94.91(C1a-β), 90.50(C1a -α),80.47,80.10,79.72,79.15,78.29,76.42(C2c),76.32(C2c'),74.72,74.69,74.63,74.59,73.58,73.56,73.02(C2b),72.88,72.45(C5e/e' ), 72.10-72.05(C7e/e'),71.90,70.61,70.44,70.25(C5e/e'),69.93,69.41,68.84,67.34,67.30, 65.88,61.71,61.65,60.03,59.94,59.896,54.9 54.86, 49.94, 49.75 (C8e/e'), 22.34 ( _CH3CO- ).

Weigh 10 mg of N ₃ -ON=CH-CT-OH 10 and dissolve it in 625 μL of distilled water, add 16 mg of DMC and 39 μL of triethylamine to the above system, mix well and put it on ice for 2 hours to react. Use Sephadex G25 gel column Purification afforded N ₃ -ON=CH-CT-ox 13c (yield 82%). HRMS, calcd for _C58H93N11O40 [M+2H] ²⁺ _{792.7868 ,} found _792.7871 . ¹ HNMR (500MHz, deuterium oxide) δ7.56(d, J=4.1Hz, 1.4H, H6e/e'), 6.91(d, J=4.6 Hz, 0.6H, H6e/e'), 6.04(d, J=7.3Hz, 1H, H1a of GlcNAc-oxa), 5.07(s, 1H, H1c), 4.88(s, 1H, H1c'), 4.79(d, J=3.9Hz, 0.6H, H5e/e') ,4.53(m,2H,H1d/d'),4.50-4.43(m,2H,H1e/e'),4.42(d,J＝3.9Hz,1.4H,H5e/e'),4.26(t,J =4.8Hz,1H,H2b),4.20(t,J=5.0Hz,4H,H7e/e'),4.14(s,1H,H2c),4.09(t,J=3.7Hz,1H),4.06(s , 1H, H2c'), 4.04 (d, J=3.2Hz, 2H, H4e/e'), 2.00 (m, 9H, H-Ac). ¹³ C NMR (126MHz, deuterium oxide) δ174.64 (3 x CH ₃ CO-),149.75(C6e/e'),148.88(C6e/e'),103.06(C1e/e'),102.60(C1e/e'),100.44(C1b),99.56(C1c,C1a-oxa ), 99.42(C1d/d'), 97.05(C1c'), 80.48, 79.17, 76.43(C2c), 76.33(C2c'), 74.64, 73.56, 73.02(C2b), 72.89, 72.46, 72.11(C7e/e'),71.91,70.45,70.26(C5e/e'),69.94,69.42,68.85,67.31,61.71,59.94,55.00,54.86,49.95,49.76(C8e/e'),22.34(CH ₃ CO-).

Example 4: Preparation of N ₃ -NH-CT-ox 13d

Dissolve 30mg of CHO-ASGP 8 in 500μL of 0.2M PB, pH=6.0 buffer system, add 39μL of 3-azidopropylamine to the above system, adjust the pH to 6.5 with acetic acid, add 166μL of 100mg/mL to the above reaction system Sodium cyanoborohydride mother liquor, and the reaction system were incubated on ice. After overnight, the reaction system was monitored by analytical HPLC and identified by mass spectrometry, showing that the reaction was almost complete. The target fraction was collected and freeze-dried to obtain N ₃ -NH-ASGP 11 (21 mg, yield 65%). HRMS, Calcd. for _{C96H167N21O52} [M+3H] ³⁺ _{816.3768 ,} found _816.3766 .

N ₃ -NH-ASGP 11 (10μmoL, 1eq) was dissolved in 100μL 50mM PB, pH 6.5 buffer solution, 1M hydrochloric acid and 1M sodium hydroxide to adjust the pH to 6.5, and 1.7μL 30mg/mL Endo-M was added to the above mixed system , incubated at 30°C, and after 4 hours, analytical HPLC was used to monitor the reaction system and mass spectrometry showed that the reaction was complete, and purified using C18 semi-preparative column and Sephadex G25 to obtain N ₃ -NH-CT-OH 12 (yield 68 %). HRMS ^{, calcd for C60H103N11O39 [M+2H]2+} _{801.8286 , found 801.8279} .

Weigh 10 mg of N ₃ -ON=CH-CT-OH 12 and dissolve it in 625 μL of distilled water, add 16 mg of DMC and 39 μL of triethylamine to the above system, mix well and place it on ice for 2 hours to react. Use Sephadex G25 gel column Purification afforded N ₃ -NH-CT-ox 13d (68% yield). HRMS, calcd for _{C60H101N11O38} [M+2H] ²⁺ _{792.8233 ,} found _792.8232 .

Example 5: Synthesis of DBCO-PEG ₄ -Lys(εPEG ₁₂ )-VC-PAB-MMAE D1

Weigh 500mg Fmoc-NH-VC-PAB-OH and dissolve in 5mL N,N-dimethylformamide (DMF), add 760mg bis(p-nitrophenyl)carbonate and 286μL DIPEA in sequence, stir overnight at room temperature, HPLC Monitoring of the reaction system and identification by mass spectrometry showed that the reaction was almost complete. Using semi-preparative C18 column separation and purification, the target components were collected and lyophilized to obtain compound S2 (550 mg, yield 86.3%). HRMS _, calcd for _C40H42N6O10 [M+H] ⁺ 767.304 _, found _767.3044 .

Weigh 100mg Fmoc-NH-VC-PAB-PNP S2 and dissolve it in 200μL DMF, add 112mg MMAE, 4mgHOBt, 300μL pyridine in sequence, stir overnight at room temperature, monitor the reaction system by HPLC and identify it by mass spectrometry, showing that the reaction is almost complete. Add 130 μL of piperidine to the above reaction system, vortex and mix, and place at room temperature for 15 minutes. HPLC-MS monitors that Fmoc has been completely removed. Use a semi-preparative C18 column to separate and purify, collect the target components and freeze-dry to obtain compound S3 (120 mg, Yield 82%). HRMS _, calcd. for _C58H94N10O12 [M+H] ⁺ _1123.7131 , found _1123.7179 . ¹ H NMR (500MHz, DMSO-d6) δ10.19(s, 1H), 8.68(d, J=6.9Hz, 1H), 8.21– 8.12(m, 1H), 7.92(d, J=8.6Hz, 1H ),7.72(d,J＝8.6Hz,1H),7.56–7.54(m,2H),7.33–7.22 (m,6H),7.19–7.07(m,1H),5.10–4.98(m,2H), 4.71(s,1H),4.60(s,1H),4.49–4.40(m,2H),4.41(d,J＝6.6Hz,5H),4.20(q,J＝10.6Hz,2H),3.73(d ,J＝9.7Hz,1H),3.64(s,1H), 3.56-3.49(m,2H),3.29(d,J＝10.2Hz,1H),3.26–3.17(m,5H),3.15(s, 2H), 3.09(s, 2H), 3.07–2.92(m, 4H), 2.84(d, J＝18.8Hz, 3H), 2.37(d, J＝15.6Hz, 1H), 2.32–2.17(m, 1H ), 2.17–2.01(m,3H),1.98–190(m,1H),1.85–1.57(m,5H),1.58–1.34(m,5H),1.28(s,1H), 1.02(dd,J =6.3,4.1Hz,3H),0.99(d,J=6.6Hz,3H),0.93(d,J=6.6Hz,6H),0.86(d,J=6.4Hz,2H),0.82(dd,J ＝9.5,6.7Hz,6H),0.79–0.69(m,9H). ¹³ C NMR(125MHz, DMSO-d6)δ173.0,170.5,170.2,169.5,168.2,159.8,156.8,43.8,143.7,138.7,132.4, 128.6, 128.2, 127.2, 126.9, 119.5, 85.7, 82.1, 78.1, 77.3, 75.3, 66.5, 64.0, 61.3, 60.7, 59.1, 58.7, 57.6, 57.6, 55.7, 54.5, 53.6, 50.0, 49.5, 47.7 43.64, 37.47, 35.52, 32.24, 32.00, 30.41, 30.24, 30.03, 29.47, 26.93, 25.67, 24.68, 23.5, 19.2, 19.0, 18.9, 18.7, 18.6, 18.0, 16.4, 16.0, 15.9, 17.2.

Weigh 50mg of CH ₃ O-PEG ₁₂ -COOH S5 into a 25mL round-bottomed flask, dissolve it with 2mL of anhydrous dichloromethane, add two drops of thionyl chloride dropwise, and reflux at 50°C for about 2h under nitrogen protection. Thin-layer chromatographic analysis, the developer ratio of methanol: dichloromethane = 1:8, shows that the reaction is complete, the reaction system is spin-dried, and the oil pump is vacuumed for 30 minutes to ensure that the reaction system is free of thionyl chloride, and re-dissolved with 400 μL of anhydrous tetrahydrofuran , to obtain solution A. Weigh 30 mg of Fmoc-Lys-OH S4 and 33.2 mg of NaHCO ₃ , dissolve in 900 μL of tetrahydrofuran and 300 μL of pure water for clarification, and obtain solution B. Solution A was slowly added dropwise to solution B under stirring in an ice bath. After overnight reaction at room temperature, it was separated and purified using a semi-preparative C18 column and lyophilized to obtain compound S6 (52 mg, yield 66.8%). HRMS _, calcd for _C49H78N2O18 [M+H] ⁺ _{983.5328 ,} found 982.5258.

Weigh 10 mg of compound S6 and 7.8 mg of HATU and dissolve in 100 μL of anhydrous DMF. Weighed 12.6 mg of compound S3 and added it to the above reaction system, and added 5.3 μL DIPEA dropwise under stirring, and reacted at room temperature for 1 hour, and the reaction was almost complete as monitored by LC-MS. Add 20 μL of piperidine to the above reaction system and stir for 20 min. The completion of the reaction was monitored by LC-MS, and compound S7 (7.7 mg, yield 86%) was obtained by separation, purification and lyophilization on a semi-preparative C18 column. HRMS, calcd for _{C92H160N12O27} [M+2H] ²⁺ _{933.5836 ,} found _933.5832 . ¹ H NMR (500MHz, DMSO-d ₆ )δ10.07(s, 1H), 8.41(d, J=8.5Hz, 1H), 8.29(d, J=7.42Hz, 1H), 8.25(s, 0.56H ), 8.11(d, J=5.2Hz, 4H), 8.01(s, 0.44H), 7.90(d, J=8.7Hz, 0.6H), 7.84(t, J=5.6Hz, 1H), 7.65(d ,J＝8.5Hz,0.4H),7.58(dd, J＝8.7,3.6Hz,3H),7.39–7.23(m,8H),7.20–7.07(m,1H),6.12(s,1H),5.18 –4.91(m,2H),4.69(m,1H),4.50(d,J＝5.8Hz,1H),4.42(m,3H),4.34–4.22(m,3H),3.89–3.74(m,1H ),3.59(t,J=6.5Hz,3H),3.51(d,J=1.9Hz,37H),3.48(m,3H),3.45–3.42(m,3H),3.39–3.30(m,1H) ,3.25(d,J=3.8Hz,8H),3.20(d,J=12.4Hz,4H),3.12(s,2H),3.01(m,6H),2.87(dd,J=18.4,5.5Hz, 4H), 2.42(d, J＝16.0Hz, 1H), 2.31(t, J＝6.51Hz, 4H), 2.19–1.89(m, 3H), 1.87–1.66(m, 4H), 1.65–1.43(m ,2H),1.43–1.24(m,6H),1.02(ddd,J＝15.5,14.1,6.6Hz,7H),0.96–0.71(m,18H). ¹³ C NMR(126MHz,DMSO)δ172.88,171.09, 170.98,170.50,170.33,170.27,169.25,169.03,159.58,159.26,158.98,158.70,158.41,144.13,144.08,128.62,128.24,128.19,127.17,127.11,126.93,126.87,119.43,119.28,117.57,115.24, 85.89, 82.12,77.41,75.30,75.27,71.75,70.25,70.13,70.05,69.97,67.28,61.36,60.72,59.14, 58.65,58.50,58.33,57.61,57.57,54.56,53.64,52.46,50.24,49.65,47.67,46.70, 44.22,43.68, 39.02,38.70,37.63,36.58,35.62,31.97,31.44,30.98,30.34,30.14,29.78,29.14,27.21,25.77, 24.79,23.56,22.03,19.60,19.33,19.21,18.99,18.72,16.32, 16.07, 15.87, 15.77, 15.71, 15.41, 10.83, 10.73.

Weigh 1.7 mg of compound S8 and 2.1 mg of HATU and dissolve in 100 μL of anhydrous DMF. 5 mg of compound S7 was added to the above reaction system, added to the above reaction system, 1.4 μL DIPEA was added dropwise under stirring, and after 1 hour of reaction at room temperature, the reaction was complete as monitored by HPLC. Compound D1 (6 mg, yield 86%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS, calcd for _{C124H198N14O34} [M+3H] ³⁺ _810.1476 , _{found 810.1466} . ¹ H NMR (500MHz, DMSO-d6) δ10.01(s, 1H), 8.11(d, J=7.3Hz, 1H), 8.02(d, J=7.9Hz, 2H), 7.89(d, J=8.7 Hz, 1H), 7.79(t, J=5.7Hz, 1H), 7.73–7.66(m, 2H), 7.63(d, J=7.6Hz, 1H), 7.61–7.54(m, 3H), 7.50(m ,1H),7.46(m,2H),7.41–7.23(m,8H),7.20–7.13(m,1H),5.17–4.92(m,3H), 4.69(m,1H),4.57–4.35(m ,3H),4.34–4.17(m,4H),3.63–3.43(m,65H),3.33(q,J＝5.7 Hz,3H),3.29–3.23(m,7H),3.20(d,J＝12.1 Hz,4H),3.11(m,4H),3.10–2.92(m,4H),2.87(m,2H),2.39(m,3H),2.30(t,J＝6.5Hz,3H),2.23–1.90 (m,7H),1.85(t,J＝7.0Hz,2H), 1.83–1.66(m,3H),1.66–1.42(m,2H),1.36(m,3H),1.34–1.10(m,3H ),1.09–0.96(m, 7H),0.94–0.69(m,29H). ¹³ C NMR(126MHz,DMSO)δ172.87,172.36,172.19,172.17, 172.16,171.49,171.38,170.97,170.69,170.323,169 159.46,158.96,152.29,148.93,132.87, 129.91,129.36,128.62,128.47,128.25,128.20,128.14,127.25,126.92,126.87,125.61,122.96,121.88,114.83,108.65,82.12,75.27,71.76,70.25,70.15, 70.06,70.01,697,69.95,69.57, 67.33,67.27,61.37,59.14,58.51,57.85,57.58,55.25,53.00, 36.40,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39.39,39.39,39.9.9. 29.31, 27.15, 25.80, 25.09, 24.94, 24.80, 23.57, 23.24, 19.62, 19.35, 18.50, 15.89, 15.43, 10.86, 10.75.

Example 6: Synthesis of DBCO-PEG ₄ -Lys(εPEG ₂₄ )-VC-PAB-MMAE D2

Weigh 50mg of CH ₃ O-PEG ₂₄ -COOH S9 into a 25mL round bottom flask, dissolve it with 2mL of anhydrous dichloromethane, add two drops of thionyl chloride dropwise, and reflux at 50°C for about 2h under nitrogen protection. Thin-layer chromatographic analysis, the developer ratio of methanol: dichloromethane = 1:8, shows that the reaction is complete, the reaction system is spin-dried, and the oil pump is vacuumed for 30 minutes to ensure that the reaction system is free of thionyl chloride, and re-dissolved with 400 μL of anhydrous tetrahydrofuran , to obtain solution A. Weigh 16 mg of Fmoc-Lys-OH S4 and 18 mg of NaHCO ₃ , dissolve in 900 μL of tetrahydrofuran and 300 μL of pure water for clarification, and obtain solution B. Solution A was slowly added dropwise to solution B under stirring in an ice bath, and reacted at room temperature for 1 h. The reaction system was monitored by LC-MS. The liquid phase yield was about 65%, and there was no improvement after extending the reaction time. Compound S10 (47 mg, yield 71%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS ^{, calcd for C73H126N2O30 [M+2H]2+} _{756.4275 , found 756.4223} .

Weigh 10 mg of compound S10 and 7.8 mg of HATU and dissolve in 100 μL of anhydrous DMF. Weighed 8.2 mg of compound S3 and added it to the above reaction system, and added 3.5 μL DIPEA dropwise under stirring, and reacted at room temperature for 1 hour, and the reaction was almost complete as monitored by LC-MS. Add 20 μL of piperidine to the above reaction system and stir for 20 min. The completion of the reaction was monitored by LC-MS, and compound S11 (13 mg, yield 84%) was obtained by separation, purification and lyophilization on a semi-preparative C18 column. HRMS, calcd for _{C116H208N12O39} [M+3H] ³⁺ _{798.8299 ,} found _798.8272 . ¹ H NMR (500MHz, DMSO-d ₆ )δ10.06 (s, 1H), 8.41(d, J=8.4Hz, 1H), 8.28(d, J=7.41Hz, 1H), 8.24(s, 0.52H ),8.10(s,3H),8.01(s,0.48H),7.90(d,J＝8.7Hz,0.58H),7.84(s,1H),7.65(d,J＝8.7Hz,0.42H), 7.57(m,2H), 7.37–7.22(m,7H),7.23–7.12(m,1.6H),7.07(s,0.4H),6.06(s,1H),5.04(m,2H),4.68( m, 1H), 4.50(d, J＝5.8Hz, 1H), 4.42(q, J＝8.6, 7.8Hz, 2H), 4.33–4.21(m, 2H), 4.10–3.92(m, 2H), 3.86 (s,1H),3.78(dd,J＝9.4,2.3Hz,1H),3.74–3.29(m,104H),3.27–3.22(m,8H), 3.19(d,J＝12.5Hz,4H), 3.12(s,2H),3.02(m,4H),2.87(m,2H),2.41(d,J＝16.0Hz,1H), 2.31(t,J＝6.5Hz,2H),2.18–1.91(m ,4H),1.69(m,3H),1.64–1.42(m,1H),1.41–1.25(m, 6H),1.02(ddd,J＝15.6,13.4,6.7Hz,6H),0.95–0.70(m ,18H). ¹³ C NMR(126MHz,DMSO) δ172.89,171.08,170.99,170.53,170.27,169.26,169.03,159.54,144.11,144.07,128.63,128.25,128.20,127.18,127.13,126.93,126.87,119.42,119.27, 85.87,82.12,75.31,75.27, 71.75,70.25,70.13,70.05,69.96,67.27,61.37,60.72,59.13,58.65,58.51,58.34,57.61,57.57, 54.56,53.63,52.46,50.23,49.64,47.67,46.71, 44.21,43.68,38.70,36.58,31.95,31.44, 3098,29.77,27.23,24.79,23.55,22.03,19.46,19.21,18.72,16.35, 15.87,87,8,8,7,8,7,87,7,87,7,87,7,87,7,87,7,87,87,87,87,87. 10.74.

Weigh 2.5 mg of compound S8 and 3.2 mg of HATU and dissolve in 100 μL of anhydrous DMF. 10 mg of compound S11 was added to the above reaction system, added to the above reaction system, 2.3 μL DIPEA was added dropwise under stirring, and after 1 hour of reaction at room temperature, the reaction was complete as monitored by HPLC. Compound D2 (10 mg, yield 81%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS ^{, calcd for C148H246N14O46[M+3H]3+} _{986.2525 , found 986.2515} . ¹ H NMR (500MHz, DMSO-d ₆ )δ10.00(s,1H),8.27(s,0.42H),8.11(d,J=7.4Hz,1H),8.02(d,J=7.8Hz,2H ), 7.89(d,J＝8.7Hz,0.58H),7.79(t,J＝5.5Hz,1H),7.68(q,J＝7.1,5.1Hz,2H),7.63(d,J＝7.3Hz, 1H),7.60–7.54(m,2H),7.53–7.43(m,2H),7.42–7.22(m,7H),7.19(m,1H),6.00(s,1H),5.03(m,3H) ,4.69(m,1H),4.50(d,J＝5.9Hz,1H),4.47–4.35(m,2H),4.31–4.23 (m,2H),4.20(t,J＝7.7Hz,1H), 3.99(m,1H),3.68–3.57(m,7H),3.51(s,96H),3.27–3.16(m,8H),3.15–2.92(m,7H),2.87(d,J＝18.1Hz, 2H),2.51(s,10H),2.39(m,2H),2.30(t,J＝6.5Hz,2H),2.21–1.91(m,4H),1.85(t,J＝6.9Hz,1H), 1.81–1.11(m,25H),1.02(td,J＝15.7,13.9,6.5Hz,7H),0.93–0.69(m,29H). ¹³ C NMR(126MHz,DMSO)δ172.87,172.83, 172.36,172.19, 172.17,172.16,171.49,171.38,171.07,170.97,170.69,170.33,169.24,159.46,158.96,152.29,148.93,132.87,129.91,129.36,128.62,128.58,128.47,128.25,128.20,128.14, 127.25,127.19,127.12, 126.92,126.87,125.61,122.96,121.88,119.46,119.32,114.83,108.65, 82.12,75.27,71.76,70.25,70.17,70.15,70.06,70.01,69.97,69.95,69.57,67.33,67.27,61.37, 58.65,58.51, 57.62,57.58,5.25,53.61,53.00,386,36.61,36.40,39,34.41.96,31.08, 29.80,27.15,25.09,23.5.5.5.5, 15.5, 119.5,19.5,19.5,19.5,8.5,8.5,19.5,8.5,19.5,19.5,8.5,19.5,8.5,19.5,19.5,19.5. 15.43, 10.86, 10.75.

Example 7: Synthesis of DBCO-PEG ₄ -Lys(αPEG ₁₂ )-VC-PAB-MMAE D3

Weigh 50mg of CH ₃ O-PEG ₁₂ -COOH S5 into a 25mL round-bottomed flask, dissolve it with 2mL of anhydrous dichloromethane, add two drops of thionyl chloride dropwise, and reflux at 50°C for about 2h under nitrogen protection. Thin-layer chromatographic analysis, the developer ratio of methanol: dichloromethane = 1:8, shows that the reaction is complete, the reaction system is spin-dried, and the oil pump is vacuumed for 30 minutes to ensure that the reaction system is free of thionyl chloride, and re-dissolved with 400 μL of anhydrous tetrahydrofuran , to obtain solution A. Weigh 30 mg of N'-Fmoc-Lys-OH S12 and 33.2 mg of NaHCO ₃ , dissolve in 900 μL of tetrahydrofuran and 300 μL of pure water for clarification, and obtain solution B. Solution A was slowly added dropwise to solution B under stirring in an ice bath, and reacted at room temperature for 1 h. The reaction system was monitored by LC-MS. The liquid phase yield was about 65%, and there was no improvement after prolonging the reaction time. Compound S13 (42 mg, yield 54%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS _, calcd for _C49H78N2O18 [M+H] ⁺ _{983.5328 ,} found 982.5250.

Weigh 10 mg of compound S13 and 7.74 mg of HATU and dissolve in 100 μL of anhydrous DMF. Weighed 12.6 mg of compound S3 and added it to the above reaction system, and added 5.26 μL DIPEA dropwise under stirring, and reacted at room temperature for 1 hour, and the reaction was almost complete as monitored by LC-MS. Add 20 μL of piperidine to the above reaction system and stir for 20 min. The completion of the reaction was monitored by LC-MS, and compound S14 (8.3 mg, yield 93%) was obtained by separation, purification and freeze-drying using a semi-preparative C18 column. HRMS, Calcd. for _{C92H160N12O27} [M+2H] ²⁺ _933.5836 , _{found 933.5818} . ¹ H NMR (500MHz, DMSO-d ₆ )δ10.02 (s, 1H), 8.26 (s, 0.5H), 8.13 (s, 1H), 8.06 (d, J=7.99Hz, 1H), 8.03 (s ,0.5H),7.89(d,J＝8.7Hz,0.65H),7.72(m,5H),7.63(d,J＝8.5Hz,0.35H),7.59(m,2H),7.38–7.24(m ,7H),7.23–7.12(m,1H),6.10(s,1H),5.17–4.95(m,2H),4.83–4.08(m,6.2H),3.99(m,2.4H),3.79 (dd ,J＝9.3,2.3Hz,0.4H),3.67–3.56(m,3H),3.51(d,J＝1.9Hz,50H),3.46–3.41(m,2H), 3.35(m,1H),3.30 –3.24(m,7H),3.20(d,J＝12.1Hz,3H),3.12(s,2H),3.04(m,2H),3.00– 2.92(m,2H),2.87(m,2H), 2.76(m,1H),2.47–2.34(m,2H),2.28(dd,J＝15.8,9.5Hz,1H), 2.18–2.06(m,2H),2.00(m,2H),1.89–1.64( m,2H),1.54(m,5H),1.41–1.23(m,2H),1.11–0.97(m,7H),0.96–0.71(m,29H). ¹³ C NMR(126MHz,DMSO)δ172.36,172.31 ,171.53, 170.87,170.47,170.20,169.83,169.77,168.72,159.03,158.72,158.43,158.15,157.86,143.64, 143.62,128.14,127.74,127.69,126.67,126.61,126.43,126.37,119.28,119.26,118.95,118.84 ,118.80,116.93,114.61,112.29,85.40,81.62,77.65,74.79,74.76,71.26,69.75,69.65,69.56, 69.44,66.76,60.87,60.23,58.63,58.15,58.01,57.39,57.11,57.08,57.07,54.06 ,53.07,52.16, 49.73,49.14,47.17,46.19,43.72,43.17,38.73,35.91,31.47,31.23,30.56,29.32,26.65,26.61, 25.26,24.30,23.07,22.19,19.12,18.86,17.98,15.83,15.61 ,15.39,15.23,14.94,10.35,10.24.

Weigh 3.4 mg of compound S8 and 4.1 mg of HATU and dissolve in 100 μL of anhydrous DMF. Add 10 mg of compound S14 to the above reaction system, add it to the above reaction system, add 2.76 μL DIPEA dropwise under stirring, react at room temperature for 1 hour, and monitor the completion of the reaction by HPLC. Compound D3 (11.5 mg, yield 88%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS, calcd for _{C124H198N14O34} [M+3H] ³⁺ _810.1476 , _{found 810.1478} . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ10.01(s, 1H), 8.26(s, 0.45H), 8.11(d, J=7.4Hz, 1H), 8.02(d, J=8.0Hz, 1H), 7.89(d, J=8.6Hz, 0.55H), 7.80(t, J=5.6Hz, 1H), 7.69(m, 2H), 7.62(d, J=7.2Hz, 0.65H), 7.60– 7.52(m,3H),7.51–7.42(m,2H),7.41–7.22(m,8.35H),7.21–7.12(m,1H), 5.06(m,3H),4.69(m,1H),4.47 (dd,1H),4.42–3.85(m,11H),3.65–3.55(m,5H),3.55–3.44(m,64H),3.43(dd,J＝5.9,3.6Hz,2H),3.33(q ,J＝6.2Hz,2H),3.24(d,J＝5.2Hz,6H), 3.20(d,J＝12.6Hz,3H),3.12(d,J＝6.2Hz,3H),3.08–2.93(m ,3H),2.86(d,J＝18.4Hz, 3H),2.39(m,3H),2.30(t,J＝6.5Hz,3H),2.06(m,3H),1.85(t,J＝7.0Hz ,1H),1.83–1.11 (m,12H),1.10–0.96(m,7H),0.90–0.70(m,29H). ¹³ C NMR(126MHz,DMSO)δ173.03, 172.88,172.41,172.21,172.17 ,171.39,170.96,170.76,170.38,170.27,169.26,159.58,159.33, 159.03,158.73,158.42,152.27,148.91,144.10,144.09,132.86,129.89,129.35,128.61,128.57,128.46,128.25,128.19,128.13,127.24 ,127.17,127.11,126.92,126.87,125.60,122.95,121.88, 119.46,119.30,119.13,116.83,114.82,114.53,108.64,85.88,82.13,75.28,71.75,70.25, 70.17,70.14,70.05,70.00,69.96,69.54 ,67.31,67.26,61.35,60.72,59.14,58.66,58.49,57.88, 57.61,57.57,55.24,54.56,53.62,53.03,50.23,49.65,47.68,46.71,44.22,43.68,38.86,38.84, 36.59,36.41,35.38 ,34.40,31.93,31.07,30.47,29.76,29.29,27.07,25.78,25.75,25.09,24.93, 24.79,23.55,23.23,19.60,19.31,19.20,18.99,18.48,16.29,16.07,15.86,15.76,15.71,15.40 , 10.83, 10.72.

Example 8: Synthesis of DBCO-PEG ₄ -Lys(αPEG ₂₄ )-VC-PAB-MMAE D4

Weigh 50mg of CH ₃ O-PEG ₂₄ -COOH S9 into a 25mL round bottom flask, dissolve it with 2mL of anhydrous dichloromethane, add two drops of thionyl chloride dropwise, and reflux at 50°C for about 2h under nitrogen protection. Thin-layer chromatographic analysis, the developer ratio of methanol: dichloromethane = 1:8, shows that the reaction is complete, the reaction system is spin-dried, and the oil pump is vacuumed for 30 minutes to ensure that the reaction system is free of thionyl chloride, and re-dissolved with 400 μL of anhydrous tetrahydrofuran , to obtain solution A. Weigh 16 mg of N'-Fmoc-Lys-OH S12 and 18 mg of NaHCO ₃ , dissolve in 900 μL of tetrahydrofuran and 300 μL of pure water for clarification, and obtain solution B. Solution A was slowly added dropwise to solution B under stirring in an ice bath, and reacted at room temperature for 1 h. The reaction system was monitored by LC-MS. The liquid phase yield was about 65%, and there was no improvement after extending the reaction time. Compound S15 (33 mg, yield 50%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS ^{, calcd for C73H126N2O30 [M+2H]2+} _{756.4275 , found 756.4272} .

Weigh 10 mg of compound S15 and 7.74 mg of HATU and dissolve in 100 μL of anhydrous DMF. Weighed 8.2 mg of compound S3, added it to the above reaction system, added 3.44 μL DIPEA dropwise under stirring, and reacted at room temperature for 1 hour, and the reaction was almost complete as monitored by LC-MS. Add 20 μL of piperidine to the above reaction system and stir for 20 min. The completion of the reaction was monitored by LC-MS, and compound S16 (16 mg, yield 91%) was obtained by separation, purification and lyophilization on a semi-preparative C18 column. HRMS _, calcd for _{C116H208N12O39} [M+3H] ³⁺ 798.8299 _, found _798.8298 . ¹ H NMR (500MHz, DMSO-d ₆ )δ10.02 (s, 1H), 8.26 (s, 0.8H), 8.12 (d, J = 7.3Hz, 1H), 8.06 (d, J = 8.0Hz, 1H ),8.02(s,0.2H),7.90(d, J=8.7Hz,1H),7.73(d,J=7.7Hz,6H),7.64(d,J=8.5Hz,1H),7.58(t, J＝5.7Hz, 3H), 7.39-7.24(m, 6H), 7.21-7.12(m, 1H), 6.08(s, 1H), 5.05(m, 3H), 4.69(m, 1H), 4.50(d ,J=5.8 Hz,1H),4.47–4.36(m,2H),4.35–4.30(m,1H),4.27–4.17(m,2H),4.09–3.92(m,1H), 3.68–3.58(m ,3H),3.51(s,89H),3.45–3.34(m,3H),3.25(m,9H),3.20(d,J＝12.2Hz,4H), 3.12(s,2H),3.05(t, J＝10.3Hz, 1H), 2.97(m, 2H), 2.87(d, J＝18.0Hz, 2H), 2.76(m, 2H), 2.46–2.34(m, 3H), 2.32–2.22(m, 1H ),2.19–1.89(m,3H),1.88–1.63(m,1H),1.55(m, 3H),1.41–1.23(m,2H),1.09–0.97(m,8H),0.92–0.69(m ,25H). ¹³ C NMR(126MHz, DMSO)δ172.87,172.05,171.39,170.98,170.74,170.33,170.27,169.24,159.53,159.12, 158.85,158.59,158.33,144.13,144.09,139.04,138.92,138.90,128.63, 128.24,128.19,127.17, 127.11,126.93,126.87,119.46,119.31,118.33,115.97,85.89,82.12,75.29,75.26,71.76, 70.26,70.15,70.06,69.94,67.25,63.81,61.37,60.73,59.14,58.65, 58.51,57.92,57.61,57.57, 54.56,53.57,52.68,50.24,49.64,47.67,46.70,44.21,43.67,39.23,37.63,36.40,35.62,32.29, 31.97,31.71,31.03,29.80,27.17,27.10,25.78, 24.80, 23.56, 22.68, 19.62, 19.34, 19.22, 19.01, 18.76, 18.48, 16.32, 16.08, 15.88, 15.79, 15.71, 15.42, 10.85, 10.74.

Weigh 2.5 mg of compound S8 and 3.2 mg of HATU and dissolve in 100 μL of anhydrous DMF. 10 mg of compound S16 was added to the above reaction system, added to the above reaction system, 2.3 μL DIPEA was added dropwise under stirring, and after 1 hour of reaction at room temperature, the reaction was complete as monitored by HPLC. Compound D4 (11 mg, yield 88%) was obtained by separation, purification and lyophilization using a semi-preparative C18 column. HRMS, calcd for _{C148H246N14O46} [M+3H] ³⁺ _{986.2525 ,} found _986.2522 . ¹ H NMR (500MHz, DMSO-d ₆ )δ10.01(s,1H),8.27(s,1H),8.11(d,J=7.3Hz,1H),8.02(d,J=8.0Hz,2H) ,7.89 (d,J=8.6Hz,1H),7.79(t,J=5.7Hz,1H),7.68(q,J=6.6,5.8Hz,2H),7.63(d,J=7.6Hz, 1H) ,7.61–7.53(m,3H),7.52–7.44(m,3H),7.42–7.23(m,6H),7.21–7.13(m,1H),5.17–4.96(m,3H),4.69(m, 1H), 4.50(d, J＝5.9Hz, 1H), 4.47–4.35(m, 2H), 4.33–4.18(m, 4H), 3.64–3.56(m, 6H), 3.51(d, J＝1.6Hz ,115H),3.43(dd,J＝5.9,3.7Hz,3H),3.39–3.31(m, 3H),3.30–3.17(m,12H),3.12(m,3H),3.09–2.92(m,5H ),2.87(d,J=18.9Hz,2H),2.40(m,2H),2.30(t,J=6.5Hz,2H),2.06(m,5H),1.85(t,J=7.0Hz,2H ),1.84–1.11(m,9H), 1.02(td,J＝15.7,14.2,6.6Hz,7H),0.93–0.72(m,29H). ¹³ C NMR(126MHz,DMSO)δ 172.37,172.34,171.88 ,171.69,171.66,170.88,170.47,170.22,169.84,169.77,168.75,159.00, 158.49,158.19,151.78,148.41,143.61,143.59,132.36,129.40,128.86,128.11,128.07,127.96,127.75,127.69,127.63,126.75 ,126.68,126.61,126.42,126.37,125.10,122.46,121.38,118.96, 118.79,116.37,114.32,114.07,108.15,81.62,74.77,71.26,69.76,69.68,69.65,69.56,69.51, 69.47,69.46,69.06,66.82 ,66.77,60.86,60.22,58.64,58.15,58.01,57.36,57.11,57.07,54.75, 54.06,53.11,52.51,49.74,46.20,43.72,43.18,38.36,36.10,35.91,34.88,33.91,31.45,30.58, 29.28 ,28.80,26.62,26.04,25.30,24.59,24.43,24.30,23.06,22.73,19.11,18.84,18.48,17.99, 15.38,15.28,15.21,14.91,10.24

Example 9: Synthesis of Alkyne-PEG ₄ -VC-PAB-MMAE D5

Weigh compound S17 (4.9 mg, 10 μmoL) and dissolve it in 100 μL DMF, add HATU (7.6 mg, 20 μmoL), compound S3 (NH ₂ -VC-PAB-MMAE, 11 mg, 10 μmoL) and DIPEA (5.3 μL, 30μmoL), react at 37°C for 2h. LC-MS monitoring of the reaction system showed that the reaction was complete. The compound S18 (12 mg, yield 88%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for _{C69H115N11O17} [M+H] _{+ 1370.855} , [M+2H] ²⁺ _685.9314 , found 1370.8552, ^685.9311 .

Weigh pentynoic acid (0.8 mg, 7.5 μmoL) and dissolve it in 100 μL DMF, add HATU (5.7 mg, 15 μmoL), compound S18 (10.2 mg, 7.5 μmoL) and DIPEA (4 μL, 22.5 μmoL) to the above system in sequence, Reaction at 37°C for 2h. LC-MS monitoring of the reaction system showed that the reaction was complete. Compound D5 (17 mg, yield 79%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS _, calcd for _{C74H119N11O18} [M+H] ⁺ _1450.8813 , [M+2H] ²⁺ _725.9446 , found 1450.8801, 725.9434. ¹ H NMR (500MHz, DMSO-d ₆ ) δ9.96(d, J=8.5Hz, 1H), 8.26(s, 0.6H), 8.11(d, J=7.5Hz, 1H), 8.01(d, J ＝7.9Hz,0.4H),7.94(t,J＝5.7Hz,1H), 7.87(t,J＝7.8Hz,1H),7.66–7.53(m,2H),7.40–7.23(m,6H), 7.22–7.12(m,1H),5.04(m,2H),4.50(d,J＝5.9Hz,0.65H),4.47–4.36(m,2H),4.32–4.18(m,2H),4.08–3.91 (m,2H), 3.78(dd,J=9.4,2.3Hz,0.35H),3.68–3.54(m,3H),3.49(dd,J=6.7,2.8Hz,17H),3.40(t,J= 5.9Hz, 2H), 3.32(d, J＝10.0Hz, 0.5H), 3.28–3.12(m, 10.5H), 3.09–3.00(m, 1H), 2.97(d, J＝5.4Hz, 2H), 2.86(m,3H),2.72(t,J=2.6Hz,1H),2.47(t,J=7.0Hz,1H),2.39(d,J=6.6Hz,1H),2.35(m,2H), 2.27(dd,J=7.7,5.8Hz,3H),2.17–1.91(m,3H),1.75(m,3H),1.65–1.26(m,4H),1.01(td,J=15.8,14.7,6.7 Hz,7H),0.91–0.72(m,29H). ¹³ C NMR(126MHz,DMSO)δ172.86,172.82,171.63,171.00,170.84,170.34,170.28,169.23,159.55,159.33,159.03,154.713,154 144.11,128.61,128.25,128.19,127.18,127.11,126.92,126.87, 119.47,119.30,116.80,114.50,85.90,84.20,82.12,75.28,71.66,70.26,70.19,70.07,69.95, 69.58,67.39,66.55,61.36, 60.72,59.14,58.65,58.00,57.61,57.57,55.48,54.56,53.60, 50.23,49.64,47.70,44.22,40.40.29,39.79.79.79.79.49.49,39,9.79,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,39,9.7. 35.61, 34.56, 31.96, 31.04, 29.74, 27.11, 25.80, 25.76, 24.80, 23.56, 19.62, 19.35, 19.22, 19.00, 18.55, 16.30, 16.08, 15.88, 15.77, 15.71, 145.84, 145.72

Example 10: Synthesis of Alkyne-PEG ₂₄ -VC-PAB-MMAE D6

Compound S19 (13.7 mg, 10 μmoL) was weighed and dissolved in 100 μL DMF, and HATU (7.6 mg, 20 μmoL), compound S3 (NH ₂ -VC-PAB-MMAE, 10.2 mg, 10 μmoL) and DIPEA ( 5.3mL, 30μmoL), reacted at 37°C for 2h. LC-MS monitoring of the reaction system showed that the reaction was complete, and the semi-preparative C18 column was used for separation and purification, and the target components were collected and freeze-dried to obtain compound S20 (18 mg, yield 81%). HRMS ^{, calcd for C109H195N11O37 [M+2H]2+} _{1126.1936 , found 1126.1918} .

Weigh pentynoic acid (0.5 mg, 5 μmoL) and dissolve it in 100 μL DMF, add HATU (3.8 mg, 10 μmoL), compound S20 (11.2 mg, 5 μmoL) and DIPEA (2.6 μL, 15 μmoL) to the above system in sequence, Reaction 2h. LC-MS monitoring of the reaction system showed that the reaction was complete. Compound D6 (8.4 mg, yield 72%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS ^{, calcd for C114H199N11O38 [M+2H]2+} _{1165.7028 , found 1165.7044} . ¹ H NMR (500MHz, DMSO-d ₆ ) δ9.97(s, 1H), 8.26(m, 0.65H), 8.11(d, J=7.5Hz, 1H), 8.01(m, 0.35H), 7.94( t,J＝5.6Hz,1H),7.88(t,J＝9.8Hz,2H),7.68–7.56(m,3H), 7.42–7.24(m,6H),7.24–7.14(m,1H),5.04 (m,3H),4.50(d,J=6.0Hz,1H),4.47–4.36(m,2H),4.33–4.21(m,2H),3.63(dq,J=17.8,6.0,5.4Hz,3H ),3.52(s,98H),3.41(t,J＝5.9Hz, 2H),3.39–3.31(m,1H),3.25(d,J＝7.1Hz,4H),3.20(td,J＝8.0, 4.0Hz, 6H), 3.12(s, 2H), 3.09–2.83(m, 3H), 2.73(t, J＝2.6Hz, 1H), 2.51(p, J＝1.9Hz, 4H), 2.47(t, J=7.0Hz,1H), 2.41(m,1H),2.35(tt,J=7.2,1.5Hz,3H),2.28(m,3H),2.19–2.05(m,1H),1.99(m,1H ), 1.88–1.66(m,2H), 1.65–1.23(m,3H), 1.02(td,J＝15.8,14.7,6.6Hz,7H),0.92–0.72(m, 29H). ¹³ C NMR(126MHz ,DMSO)δ172.87,171.62,171.01,170.85,170.28,169.25,159.47, 158.98,158.68,144.12,128.61,128.25,128.19,127.18,127.12,126.92,126.87,119.46,119.31,116.93,114.63,85.89,84.21,82.12 ,75.28,71.66,70.26,70.09,69.96,69.58,67.40,66.54, 61.37,60.73,59.14,58.66,58.01,57.62,57.58,54.67,54.56,53.60,50.23,49.65,47.68,46.71, 44.22,43.68,40.45 ,40.28,40.12,39.95,39.78,39.62,39.45,39.07,36.41,34.56,31.02,29.74, 27.18,25.79,24.80,23.57,19.62,19.34,19.21,18.54,16.30,15.88,15.77,15.71,15.41,14.66 , 10.84, 10.74.

Example 11: Synthesis of ThioPz-VC-PAB-MMAE D7

Weigh compound S21 (574mg, 1.65mmoL), compound S22 (237.4mg, 1.65mmoL) and DMAP (201.4mg, 1.65mmoL) were dissolved in 10mL of anhydrous dichloromethane, the reaction system was placed on ice and cooled to 0 °C . Add DCC (337.4mg, 1.638mmoL) to the above solution, stir at 0°C for 30min, return to room temperature, and stir for 6h. Dilute and filter with dichloromethane, wash with 1N HCl and saturated brine, dry the organic layer with MgSO ₄ , and spin dry. The resulting oil was redissolved in 30 mL of absolute ethanol, and refluxed for 4 h. Purification on a silica gel column (hexane:ether=10:1) gave 3-oxo-5-(triphenylthio)pentanoate S23 (560 mg, yield 81%). ¹ H NMR (400MHz, CDCl ₃ ) δ7.45–7.39(m, 6H), 7.29(t, J=7.5Hz, 6H), 7.21(t, J=7.2Hz, 3H), 4.15(q, J= 7.1Hz, 2H), 3.29(s, 2H), 2.43(dd, J＝9.2, 5.2Hz, 4H), 1.25(t, J＝7.1Hz, 3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ201. 0, 166.9, 144.7, 129.7, 128.1, 126.8, 67.0, 61.5, 49.3, 42.1, 25.6, 14.2.

Compound S23 (499mg, 1.24mmoL), ethyl hydrazinoacetate hydrochloride (191mg, 1.23mmoL) was weighed and dissolved in 10mL of ethanol, triethylamine (17.3L, 0.124mmoL) was added, and reacted at 50°C for 2h. The reaction system was concentrated and purified by silica gel column (hexane:ether=1:1) to obtain compound S24 (402 mg, yield 71%). ¹ H NMR (400MHz, CDCl ₃ )δ7.46–7.38(m,6H),7.29(t,J=7.5Hz,6H),7.22(dd,J=8.3,6.1Hz,3H),4.38(s, 2H), 4.19(q, J=7.1Hz, 2H), 3.01(s, 2H), 2.46–2.31(m, 4H), 1.25(t, J=7.1Hz, 3H). ¹³ C NMR (100MHz, CDCl ₃ ) δ172.6, 168.0, 157.7, 144.6, 129.7, 128.1, 126.9, 67.2, 61.7, 45.6, 39.8, 30.5, 28.7, 14.2.

Compound S24 (236mg, 0.5mmoL) was weighed and dissolved in 10mL THF:MeOH:Water=2:3:1, LiOH (25mg, 1.04mmoL) was added to the above system, and stirred for 4h. Pour 40 mL of water and 40 mL of ether into the above system, adjust the pH of the water layer to 2, and wash 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 S25 (160 mg, yield 72%). ¹ H NMR (400MHz, DMSO-d6) δ7.37–7.29 (m,12H),7.27–7.22(m,3H),5.14(s,1H),4.54(s,2H),2.41(t,J= 7.2Hz, 2H), 2.31 (t, J = 7.2Hz, 2H). ¹³ C NMR (100MHz, DMSO-d6) δ169.2, 154.1, 148.6, 144.5, 129.1, 128.1, 126.7, 85.9, 66.2, 47.1, 30.7, 27.3.

Compound S25 (50 mg, 112.6 μmoL) was weighed and dissolved in 200 μL DMF, DCC (34.7 mg, 168.7 μmoL) and NHS (19.4 mg, 168.7 μmoL) were added to the above system, and reacted at 37 °C for 4 h. The reaction system was monitored by HPLC and identified by mass spectrometry, showing that the reaction was almost complete. Take 40 μL of the above reaction system and add it to compound S3 (20 mg, dissolved in 200 μD MF), and add 3 μL triethylamine, incubate at 37 ° C for 2 h, LC-MS monitors that the reaction is almost complete, use a semi-preparative C18 column to separate and purify, and collect the target The fractions were lyophilized to give Trt-thioPz-VC-PAB-MMAE S27 (25 mg, 90% yield). HRMS _, calcd for _{C84H116N12O14S} [M+2H] ²⁺ _775.4305 , found _775.4331 .

Compound S27 (20 mg, 12.9 μmoL) was weighed and 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 above reaction system, and stirred at room temperature for 30 min. LC-MS monitoring of the reaction system showed that the reaction was complete. ThioPz-VC-PAB-MMAE D7 (11 mg, yield 65%) was obtained after separation, purification and lyophilization by a semi-preparative C18 column. HRMS, calcd. for _{C65H102N12O14S} [M+H] ⁺ _1307.7437 , _{found 1307.7329} .

Example 12: Synthesis of ABAO-VC-PAB-MMAE D8

Weigh compound S28 (5.6mg, 0.0225mmol) and dissolve it in 100μL DMF, add HATU (8.5mg, 0.0225mmol), compound S3 (8.5mg, 0.0225mmol) and DIPEA (7.83μL, 0.045mmol) to the above reaction system in sequence ), react at 37°C for 1h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. The target product was collected and lyophilized to obtain compound S29 (24 mg, yield 80%) using a semi-preparative C18 column for separation and purification. HRMS, calcd for _{C71H108N12O14} [M+ _H ] ⁺ _1353.8186 , [M+2H] ²⁺ _677.413 , found 1353.8184, 677.4121. ¹ H NMR (500MHz, CD ₃ OD)δ7.58-7.57(m,2H),7.39-7.29(m,6H),7.22-7.19(m,1H),7.06-6.92(m,3H),5.19- 5.13(m,2H),5.06(dd,J＝13.0,3.4Hz,1H),4.68-4.52(m,4H),4.24-4.18(m,3H), 4.07(br s,1H),3.89(t ,J=6.3Hz,2H),3.86(dd,J=9.1,2.0Hz,1H),3.74-3.66(m,2H), 3.56-3.52(m,1),3.43-3.37(m,2H), 3.35-3.34(m,4H),3.29-3.26(m,3H),3.22-3.15(m,2H), 3.13-3.06(m,2H),2.99-2.88(m,3H),2.53-2.46(m ,2H),2.32-2.29(m,2H),2.25-2.19(m,2H),2.14-2.11(m,1H),2.10-2.03(m,2H),1.99-1.83(m,4H),1.80 -1.71(m,4H),1.69-1.66(m,2H),1.62-1.54(m,3H),1.51-1.45(m,2H),143-140(m,2H),1.31-1.23(m, 1H), 1.77(dd, J=6.6, 4.4Hz, 2H), 1.13(dd, J=10.2, 6.8Hz, 2H), 1.08-1.04(m, 1H), 0.99(d, J=6.5Hz, 2H ),0.97-0.91(m,8H) and 0.90-0.68(m,11H); ¹³ C NMR(125MHz,CD ₃ OD)δ176.4,175.7,175.4,175.1,174.0,172.2,171.7,162.3,158.8,144.0, 143.8,139.5,133.9,130.1,129.6,129.5,129.2,128.6,128.4,128.0,127.9,124.1,121.1,120.1,118.1,117.3,115.6,86.7,83.5,667.5,737.3,268.9 60.8,60.6,60.5,58.6,58.4,56.0,54.9,51.4,50.9,48.1,45.9,45.5,40.4,36.6,31.7,30.4,30.0,27.8,27.0,26.7,26.7,26.6,25.8,25.7,24.5, 19.8, 19.7, 18.9, 15.9, 15.8, 15.0 and 11.0.

Compound S29 (13.2mg, 0.01mmol) was weighed and dissolved in 1mL of methanol, and 5 times the equivalent of hydroxylamine hydrochloride and sodium bicarbonate (dissolved in 0.5mL of water) were added to the above reaction system, and stirred at 65°C for 24h. LC-MS monitoring of the reaction system showed that the reaction was complete, and the compound D8 (13 mg, 94%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for _{C71H111N13O15} [M+H] ⁺ _1386.8401 , [M+2H] ²⁺ _693.9235 , found _1386.8385 , 693.9264. ¹ H NMR (500MHz, CD ₃ OD) δ7.58-7.57(m,2H),7.39-7.28(m,6H),7.22-7.19(m,1H), 7.02-6.90(m,3H),5.19- 5.05(m,2H),4.68-4.49(m,4H),4.26-4.18(m,4H),4.06(t,J=6.2Hz,1H),3.93(t,J=6.2Hz,2H),3.86 (dd,J＝9.1,1.9Hz,1H),3.72-3.66(m,1H),3.57-3.52(m,1H),3.43-3.37(m,1H),3.35-3.34(m,4H),3.28 (d,J＝14.1Hz,3H),3.23-3.15(m,2H), 3.10-3.06(m,2H),2.96-2.92(m,3H),2.54-2.46(m,2H),2.34-2.30 (m,2H),2.24-2.19(m,2H),2.15-2.03(m,2H),2.00-1.86(m,4H),1.84-1.66(m,7H),1.62-1.42(m,5H) ,1.40-1.37(m,2H), 1.33-1.29(m,1H),1.18(t,J＝6.1Hz,3H),1.13(dd,J＝11.5,6.8Hz,3H),1.00-0.96(m ,10H), 0.94-0.92(m,3H),0.90-0.83(m,9H)and 0.80-0.76(m,2H); ¹³ C NMR(125MHz,CD ₃ OD)δ 176.4,175.7,175.4,175.1, 174.0, 172.2, 171.7, 162.3, 144.1, 143.8, 139.5, 134.0, 129.6, 129.5, 129.2, 128.6, 128.4, 128.0, 127.9, 121.7, 121.5, 121.1, 116.1, 115.4, 36, 3.6, 9, 7 66.0,62.0,61.5,60.8,60.5,58.6,58.4,56.1,56.0,54.9,51.4,50.8,49.5,48.1,45.9,45.5,36.6,31.9,31.7,30.5,30.3,30.0,29.9,27.8,27.0, 26.7, 26.7, 26.6, 25.8, 25.6, 24.5, 19.8, 19.8, 18.9, 16.0, 15.8, 15.0, 10.9.

Example 13: Synthesis of NH ₂ O-VC-PAB-MMAE D9

Compound S30 (5.6 mg, 17.8 μmoL) was weighed and dissolved in 100 μL DMF, and HATU (13.5 mg, 35.6 μmoL), compound S3 (NH ₂ -VC-PAB-MMAE, 20 mg, 17.8 μmoL) and DIPEA (9.3mL, 53.4μmol) was reacted at 37°C for 2h. LC-MS monitoring of the reaction system showed that the reaction was complete, and the semi-preparative C18 column was used for separation and purification, and the target components were collected and freeze-dried to obtain compound S31 (22 mg, yield 87%). HRMS, calcd for _{C75H107N11O16} [M+H] ⁺ _1418.7975 , [M+ ^2H]2+ _709.9027 , found 1418.7975, _709.9027 .

Compound S31 (22 mg, 15.4 μmoL) was weighed and dissolved in 100 μD MF, and 100 μL triethylamine was added to the above system, mixed and reacted at room temperature for 1 h. LC-MS monitoring of the reaction system showed that the reaction was complete, and the compound D9 (16.5 mg, yield 89%) was obtained after separation and purification by a semi-preparative column type C18 column and lyophilization. HRMS, calculated for C ₆₀ H ₉₇ N ₁₁ O ₁₄ [M+H] ⁺ 1196.7294, [M+2H] ²⁺ 598.8685, measured 1196.7263, 598.8622. ¹ H NMR (500MHz, DMSO-d ₆ ) δ10.04( s,1H),8.33(d,J=7.3Hz,1H),8.20(d,J=8.2Hz,1H),8.10(m,0.4H),7.93(d,J=8.8Hz,0.6H), 7.89(m,0.6H),7.73(d,J＝8.5Hz,0.4H),7.54(d,J＝8.0Hz,2H),7.28(m,6H), 7.13(m,1H),5.19–4.91 (m,2H),4.77–4.58(m,1H),4.53(s,2H),4.5–4.25(m,6H),3.72 (d,J＝9.4Hz,1H),3.59–3.41(m,2H ),3.34–2.90(m,15H),2.91–2.71(m,4H),2.45–2.17(m,2H),2.00(m,4H),1.86–1.23(m,9H),1.01(dd,J ＝13.5,6.7Hz,6H),0.79(ddt,J＝43.6,15.6,7.3Hz,29H). ¹³ C NMR(126MHz,DMSO-d ₆ )δ173.32,173.16,171.27,171.03,170.26,169.58,167.78, 159.89,159.61,159.33,159.05,158.78,156.87,156.29,143.84,143.70,138.79,132.42,128.73,128.59,128.28,127.24,126.94,119.64,119.48,117.74,115.41,85.68,82.07, 78.13,77.32,75.46, 71.51,66.60,64.15,61.34,60.71,59.12,58.77,57.83,57.69,57.58,55.77, 54.63,54.53,53.82,50.13,49.60,47.77,46.80,44.21,43.73,39.10,37.43,35.51,32.24,32.02, 31.08, 30.40, 30.03, 29.48, 27.01, 25.67, 24.70, 23.47, 19.44, 19.32, 19.20, 19.10, 18.85, 18.63, 18.31, 16.52, 16.00, 15.69, 15.22, 10.72, 10.67

Example 14: Synthesis of DBCO-Lys(εPEG ₂₄ )-VC-PAB-MMAE D10

Compound S32 (DBCO-COOH, 1.8 mg, 5.38 μmoL) and HATU (4.1 mg, 10.76 μmoL) were weighed and dissolved in 100 μL of anhydrous DMF. Compound S11 (12.8 mg, 5.38 μmoL) and DIPEA (2.8 μL, 16.14 μmoL) were added to the above reaction system, and reacted at room temperature for 1 h. LC-MS monitoring of the reaction system showed that the reaction was complete, and the semi-preparative separation and purification were followed by lyophilization to obtain compound D10 (12 mg, 84%). HRMS, calculated for C ₁₃₇ H ₂₂₅ N ₁₃ O ₄₁ [M+2H] ²⁺ 1355.3039, [M+3H] ³⁺ 903.8718, measured 1355.302, 903.8707. ¹ H NMR (500MHz, DMSO-d ₆ ) δ9.99 (s,1H),8.27(s,1H),8.08(d,J=7.3Hz,1H),8.02(s,1H),7.89(d,J=8.7Hz,1H),7.83(d,J= 7.8Hz, 1H), 7.77(t, J＝5.6Hz, 1H), 7.63(d, J＝7.7Hz, 2H), 7.57(m, 4H), 7.52–7.43 (m, 3H), 7.42–7.24( m,10H),7.22–7.13(m,1H),5.97(s,1H),5.41(s,3H),5.18–4.95(m,3H),4.69(m,1H),4.50(d,J＝ 5.9Hz,1H),4.48–4.33(m,2H),4.26(dd,J＝13.8,10.8Hz,1H),4.22–4.12(m,2H),4.07–3.92(m,3H),3.78(dd ,J＝9.4,2.3Hz,0.5H),3.65(t,J＝4.88Hz,0.5H),3.63–3.56(m,3H),3.51(d,J＝1.6Hz,95H),3.49–3.42( m,4H),3.27–3.23(m,7H),3.20(d,J＝12.1Hz,4H),3.12(s,2H),3.08–2.91(m,3H),2.87(d,J＝18.2Hz ,3H), 2.45–2.36(m,1H),2.29(t,J＝6.6Hz,3H),2.24–1.88(m,5H),1.87–1.12(m,20H),1.08–0.96(m,7H ),0.92–0.71(m,24H). ¹³ C NMR(126MHz,DMSO)δ173.05,172.87,172.83, 172.50,172.27,171.85,171.61,171.35,170.98,170.32,170.28,169.24,159.40,144.12,132.87, 131.36 ,129.91,129.37,128.61,128.47,128.26,128.21,128.14,127.25,127.20,126.92,126.87,125.61,122.96,121.88,108.64,75.27,71.76,70.26,70.14,70.06,69.97,67.33,58.52,55.26, 53.59 ,52.94,50.29,50.23,49.65,44.21,43.67,40.47,40.30,40.14,39.97,39.80,39.64,39.47, 38.80,36.61,35.26,34.36,31.10,30.16,29.78,29.28,27.19,25.80,25.13,24.94 ,24.80,23.57, 23.28,19.58,19.41,19.03,18.44,18.40,16.32,15.90,15.74.

Example 15: Synthesis of DBCO-Gly(glucose)-VC-PAB-MMAE D11

Compound S33 (6 mg, 17.9 μmoL) was weighed and dissolved in 100 μL DMF, and HATU (13.6 mg, 35.8 μmoL), compound S3 (20 mg, 17.9 μmoL) and DIPEA (9.3 μL, 53.7 μmoL) were sequentially added to the above reaction system, Reaction at 37°C for 2h. LC-MS monitoring of the reaction system showed that the Fmoc protecting group was completely removed. Add 20 μL of piperidine to the above reaction system, and react at room temperature for 20 minutes. LC-MS monitoring of the reaction system showed that the reaction was complete, and the compound S34 (16.5 mg, yield 76%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. _HRMS , _calcd for _C63H99N11O13 [M+H] ⁺ _1218.7502 , [M+2H] ²⁺ 609.879, found 1218.7552, 609.8776.

Weigh compound S35 (glucose, 5.4 mg, 30 μmoL) and dissolve it in 600 μL ddH ₂ O, add sodium azide (96 mg, 1.5 mmoL) and CDMBI (32.4 mg, 150 μmoL) to the above reaction system, and place the reaction system in Cool on ice to 0°C, add potassium phosphate (96 mg, 450 μmoL), react overnight at 0°C and add compound S34 (16 mg, 13.1 μmoL). Prepare Cu(I)-BTTAA solution: take 60mM CuSO ₄ 32.5μL, 300mM BTTAA 39μL and 0.9M sodium ascorbate 286μL and mix in turn. Add the whole Cu(I)-BTTAA solution into the above reaction system, mix well and react at 37°C for 4h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound S36 (16 mg, yield 80%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. _HRMS , Calcd. _{for C69H110N14O18} [M+H] ⁺ _1423.8201 , found 1423.8172.

Weigh compound S37 (6 mg, 10.5 μmoL) and dissolve it in 100 μL DMF, add HATU (8 mg, 21 μmoL), compound S36 (15 mg, 10.5 μmoL) and DIPEA (5.5 μL, 31.5 μmoL) to the above reaction system in sequence, 37 ° C Reaction 2h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound D11 (15 mg, yield 84%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS _, calcd for _{C90H127N15O20} [M+2H] ²⁺ 869.9769 _, found _869.9766 . ¹ HNMR (500MHz, DMSO-d6) δ9.75(d, J＝4.2Hz, 1H), 8.34–8.16(m, 1H), 8.03(d, J＝37.1Hz, 4H), 7.88(d, J＝ 8.7Hz,1H),7.66–7.49(m,4H),7.37–7.23(m,7H), 7.22–7.11(m,1H),6.05(s,1H),5.49(d,J＝9.2Hz,1H ),5.18–4.91(m,2H),4.69(d,J＝54.6Hz,1H),4.43(ddd,J＝37.9,26.7,6.4Hz,4H),4.30–4.17(m,2H),4.15– 3.74(m,4H), 3.73–3.63(m,2H),3.62–3.53(m,1H),3.52–3.31(m,5H),3.29–3.10(m,11H),3.09–2.80(m,10H ),2.41(d,J＝16.0Hz,1H),2.28(dd,J＝15.9,9.2Hz,1H),2.22–1.95(m,8H), 1.89–1.27(m,10H),1.25–1.14( m,1H),1.10–0.95(m,7H),0.90–0.65(m,29H). ¹³ C NMR(126MHz,DMSO)δ172.86,172.83,172.56,171.28,170.91,170.34,170.28,169.25, 159.42,158.97 ,158.67,156.93,144.12,144.10,143.33,128.58,128.25,128.20,127.19,127.12, 126.92,126.87,122.55,119.50,119.35,116.98,114.67,99.42,87.83,85.89,82.12,80.39,77.46,75.27,72.66 ,70.12,62.44,61.37,61.23,60.73,59.13,58.65,58.22,57.62,57.58,55.54, 55.01,54.97,54.69,54.57,53.74,50.84,50.22,49.63,46.70,44.22,43.67,30.77,30.40,29.41 28.95,28.92,28.39,27.24,25.75,240,23.57,21.27, 20.00,19.54,19.36,19.36,19.02,18.8.7.8.8.8.8.855.8.8 ,15.42,10.85,10.74.

Example 16: Synthesis of DBCO-Gly(lactose)-VC-PAB-MMAE D12

Weigh compound S38 (lactose, 10.3mg, 30μmoL) and dissolve it in 600μL ddH ₂ O, add sodium azide (96mg, 1.5mmoL) and CDMBI (32.4mg, 150μmoL) to the above reaction system, and place the reaction system in Cool on ice to 0°C, add potassium phosphate (96 mg, 450 μmoL), react overnight at 0°C and add compound S34 (16 mg, 13.1 μmoL). Prepare Cu(I)-BTTAA solution: take 60mM CuSO ₄ 32.5μL, 300mM BTTAA 39μL and 0.9M sodium ascorbate 286μL and mix in turn. Add the whole Cu(I)-BTTAA solution into the above reaction system, mix well and react at 37°C for 4h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound S39 (17 mg, yield 82%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for _{C75H120N14O23} [M+2H] ²⁺ _793.4404 , found _{793.4433 .} ¹ H NMR (500MHz, DMSO-d ₆ )δ10.04(s, 1H), 8.58(d, J=8.7Hz, 1H), 8.34(d, J=7.6Hz, 1H), 8.23(d, J= 5.0Hz, 3H), 8.11(s, 1H), 8.01(s, 1H), 7.89(d, J＝8.6Hz, 0.5H), 7.63(d, J＝8.6Hz, 0.5H), 7.57(dd, J=8.8,3.6Hz,2H),7.39–7.22(m,6H),7.21–7.04(m,2H),6.06(s,1H),5.62(d,J=9.2Hz,1H),5.45(s ,4H),5.20–4.89(m,3H),4.67(m, 1H),4.49(t,J＝6.6Hz,1H),4.41(dt,J＝13.1,7.2Hz,2H),4.36–4.22( m,4H),3.98(m,2H), 3.84–3.73(m,3H),3.69–3.47(m,11H),3.27–3.17(m,5H),3.11(d,J＝2.9Hz,2H) ,3.09– 2.93(m,3H),2.85(dd,J＝18.3,6.5Hz,2H),2.40(d,J＝16.0Hz,1H),2.26(dd,J＝15.4,9.5Hz,1H), 2.19–1.89(m,4H),1.87–1.65(m,3H),1.63–1.25(m,4H),1.01(ddd,J＝15.8,13.1,6.7Hz,7H),0.95–0.68(m,29H ). ¹³ C NMR(126MHz,DMSO)δ172.84,170.92, 170.24,169.22,168.12,159.54,144.09,141.21,128.64,128.35,128.23,128.19,127.17,127.11, 126.92,126.86,123.12,119.45,119.29,104.24, 87.44,85.87,82.11,80.50,80.39,78.13,76.09, 75.56,75.26,73.73,72.48,71.04,68.64,66.56,61.37,60.93,60.73,60.63,57.97,57.58,53.62,52.12,50.22,49.62,47.67, 46.71,44.21,43.67,39.05,31.45,29.78,28,27.32,25.79,24.91, 24.79,23.56,19.53,19.23,19.77,18.43,16.7.9,16.9,16.9,16.9,16.9,16.9,16.9,16.9,16.9,16.9,16.9,16.9,16.9. 10.86, 10.77.

Compound S37 (5.5 mg, 9.5 μmoL) was weighed and dissolved in 100 μL DMF, and HATU (7.2 mg, 19 μmoL), compound S39 (15 mg, 9.5 μmoL) and DIPEA (5 μL, 28.5 μmoL) were sequentially added to the above reaction system, 37 ℃ reaction 2h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound D12 (14.5 mg, yield 81%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for _{C96H137N15O25} [M+2H] ²⁺ _{951.0033 ,} found _951.0078 .

Example 17: Synthesis of DBCO-Gly(maltotriose)-VC-PAB-MMAE D13

Weigh compound S40 (maltotriose, 15 mg, 30 μmoL) and dissolve it in 600 μL ddH ₂ O, add sodium azide (96 mg, 1.5 mmoL) and CDMBI (32.4 mg, 150 μmoL) to the above reaction system, and set the reaction system to Cool on ice to 0°C, add potassium phosphate (96 mg, 450 μmoL), react overnight at 0°C and add compound S34 (16 mg, 13.1 μmoL). Prepare Cu(I)-BTTAA solution: take 60mM CuSO ₄ 32.5μL, 300mM BTTAA 39μL and 0.9M sodium ascorbate 286μL and mix in turn. Add the whole Cu(I)-BTTAA solution into the above reaction system, mix well and react at 37°C for 4h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound S41 (17 mg, yield 74%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for _{C81H130N14O28} [M+2H] ²⁺ _{874.4668 ,} found _874.4661 .

Compound S37 (5.5 mg, 9.5 μmoL) was weighed and dissolved in 100 μL DMF, and HATU (7.2 mg, 19 μmoL), compound S41 (16.5 mg, 9.5 μmoL) and DIPEA (5 μL, 28.5 μmoL) were sequentially added to the above reaction system, React at 37°C for 2h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound D13 (16 mg, yield 82%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. _HRMS , _Calcd . for _{C102H147N15O30} [M+2H] ²⁺ 1032.0297, found _1032.0272 .

Example 18: Synthesis of DBCO-Gly(maltotetraose)-VC-PAB-MMAE D14

Weigh maltotriose (20 mg, 30 μmoL) and dissolve it in 600 μL ddH ₂ O, add sodium azide (96 mg, 1.5 mmoL) and CDMBI (32.4 mg, 150 μmoL) to the above reaction system, and place the reaction system on ice Cool to 0°C, add potassium phosphate (96 mg, 450 μmoL), react overnight at 0°C and add compound S34 (16 mg, 13.1 μmoL). Prepare Cu(I)-BTTAA solution: Take 60mM CuSO ₄ 32.5μL, 300mM BTTAA 39μL and 0.9M sodium ascorbate 286μL and mix in turn. Add the whole Cu(I)-BTTAA solution into the above reaction system, mix and react at 37°C for 4h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound S42 (20 mg, yield 80%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, Calcd. for _{C87H140N14O33} [M+2H] ²⁺ _955.493 , _{found 955.4887} .

Compound S37 (3.5 mg, 10.5 μmoL) was weighed and dissolved in 100 μL DMF, and HATU (8 mg, 21 μmoL), compound S42 (20 mg, 10.5 μmoL) and DIPEA (5.5 μL, 31.5 μmoL) were sequentially added to the above reaction system, 37 ℃ reaction 2h. LC-MS monitoring of the reaction system showed that the reaction was almost complete. Compound D14 (18.5 mg, yield 81%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for C ₁₀₈ H ₁₅₇ N ₁₅ O ₃₅ [M+2H] ²⁺ 1113.0562, [M+3H] ³⁺ 742.3734, found 1113.0505, 742.3704.

Example 19: Synthesis of BCN-Lys(εPEG ₂₄ )-VC-PAB-MMAE D15

Weigh compound S11 (12.8 mg, 5.38 μmoL) and dissolve it in 200 μL DMF, add compound S43 (BCN-O-PNP, 3.38 mg, 10.76 μmoL) and triethylamine (1.5 μL, 10.76 μmoL), and let stand at 37 ° C for 3 h . LC-MS monitoring of the reaction system showed that the reaction was complete. Compound D15 (10 mg, yield 72.7%) was obtained after separation and purification by a semi-preparative C18 column and lyophilization. HRMS, calcd for _{C127H220N12O41} [M+2H] ²⁺ 1285.7825, [M+ _{3H ]} ³⁺ 857.524, found _1285.7848 , 857.5232. ¹ H NMR (500MHz, DMSO-d ₆ )δ9.99(s, 1H), 8.27(s, 0.5H), 8.08(d, J=7.4Hz, 1H), 8.01(d, J=8.5Hz, 0.5 H),7.89(d,J=8.7Hz,1H),7.83(d,J=7.9Hz,1H),7.77(t,J=5.6Hz,1H),7.63(t,J=7.7Hz,1.5H ),7.57(m,4.5H),7.48(m,3H), 7.42–7.24(m,10H),7.24–7.14(m,1H),6.01(s,1H),5.16–4.91(m,3H) ,4.69(m,1H),4.50(d,J=5.9Hz,1H),4.44(d,J=6.6Hz,1H),4.37(q,J=7.5Hz,1H),4.26(dd,J= 13.8, 10.8 Hz, 1H), 4.17(m, 2H), 3.99(m, 2H), 3.78(dd, J＝9.3, 2.4Hz, 1H), 3.64–3.55(m, 4H), 3.55– 3.32(m ,96H),3.28–3.23(m,7H),3.20(d,J＝12.3Hz,4H),3.12(s,2H),3.08–2.92(m,4H), 2.86(dd,J＝18.0,4.2 Hz,3H),2.47–2.36(m,1H),2.29(t,J＝6.6Hz,3H),2.22–1.87(m,5H),1.87–1.62(m,2H),1.63–1.40(m, 3H),1.39–1.12(m,9H),1.08–0.97(m,7H),0.93–0.72(m,29H). ¹³ CNMR(126MHz,DMSO)δ172.87,172.52,172.28,172.16,171.35,170.98,170.34 ,170.27,169.25,168.06,159.43,158.86,152.27,148.91,144.11,144.09,132.86,130.06,129.91,129.36,128.62,128.47,128.26,128.20,127.25,127.13,126.92,126.87,125.61,122.95, 121.87,119.44 ,119.30,114.83,108.63,75.29,75.28,71.75,70.25,70.14,70.05,69.97,67.33, 61.37,58.51,57.77,57.61,57.58,55.26,53.59,36.60,35.26,34.36,31.08,29.76,29.27,25.79 , 25.12, 24.94, 23.56, 23.28, 19.58, 18.43, 18.40, 18.05, 15.89, 15.79, 15.75, 15.42.

Example 20: Synthesis of Ab-1

The non-natural sugar chain antibody Ab-1 is obtained by compound 13a and deglycosylated Herceptin Fucα1,6GlcNAc-Herceptin through general procedure 1.

Example 21: Synthesis of Ab-2

The non-natural sugar chain antibody Ab-2 is obtained from compound 13b and deglycosylated Herceptin Fucα1,6GlcNAc-Herceptin through general procedure 1.

Example 22: Synthesis of Ab-3

The non-natural sugar chain antibody Ab-3 was obtained from compound 13c and deglycosylated Herceptin Fucα1,6GlcNAc-Herceptin through general procedure 1.

Example 23: Synthesis of Ab-4

The non-natural sugar chain antibody Ab-4 was obtained by compound 13d and deglycosylated Herceptin Fucα1,6GlcNAc-Herceptin through general procedure 1.

Example 24: Synthesis of Ab-5

The non-natural sugar chain antibody Ab-5 is obtained by compound 2 and deglycosylated Herceptin Fucα1,6GlcNAc-Herceptin through general procedure 2.

Example 25: Synthesis of Ab-6

The non-natural sugar chain antibody Ab-6 is obtained by compound 8 and deglycosylated Herceptin Fucα1,6GlcNAc-Herceptin through general procedure 2.

Example 26: Synthesis of gsADC-1

Antibody-drug conjugate gsADC-1 is obtained through operation three of compound D9 and non-natural sugar chain antibody Ab-5.

Example 27: Synthesis of gsADC-2

Antibody-drug conjugate gsADC-2 is obtained through operation three of compound D9 and non-natural sugar chain antibody Ab-6.

Example 28: Synthesis of gsADC-3

Antibody-drug conjugate gsADC-3 is obtained through operation 4 of compound D8 and non-natural sugar chain antibody Ab-5.

Example 29: Synthesis of gsADC-4

Antibody-drug conjugate gsADC-4 is obtained through operation 4 of compound D8 and non-natural sugar chain antibody Ab-6.

Example 30: Synthesis of gsADC-5

Antibody-drug conjugate gsADC-5 is obtained through operation five of compound D7 and non-natural sugar chain antibody Ab-5.

Example 31: Synthesis of gsADC-6

Antibody-drug conjugate gsADC-6 is obtained through operation five of compound D7 and non-natural sugar chain antibody Ab-6.

Example 32: Synthesis of gsADC-7

Antibody-drug conjugate gsADC-7 is obtained by compound D1 and non-natural sugar chain antibody Ab-2 through operation six.

Example 33: Synthesis of gsADC-8

Antibody-drug conjugate gsADC-8 is obtained by compound D2 and non-natural sugar chain antibody Ab-2 through operation six.

Example 34: Synthesis of gsADC-9

Antibody-drug conjugate gsADC-9 is obtained by compound D3 and non-natural sugar chain antibody Ab-2 through operation six.

Example 35: Synthesis of gsADC-10

Antibody-drug conjugate gsADC-10 is obtained by compound D4 and non-natural sugar chain antibody Ab-2 through operation six.

Example 36: Synthesis of gsADC-11

Antibody-drug conjugate gsADC-11 is obtained by compound D10 and non-natural sugar chain antibody Ab-2 through operation six.

Example 37: Synthesis of gsADC-12

Antibody-drug conjugate gsADC-12 is obtained by compound D1 and non-natural sugar chain antibody Ab-3 through operation six.

Example 38: Synthesis of gsADC-13

Antibody-drug conjugate gsADC-13 is obtained by compound D2 and non-natural sugar chain antibody Ab-3 through operation six.

Example 39: Synthesis of gsADC-14

Antibody-drug conjugate gsADC-14 is obtained by compound D3 and non-natural sugar chain antibody Ab-3 through operation six.

Example 40: Synthesis of gsADC-15

Antibody-drug conjugate gsADC-15 is obtained by compound D4 and non-natural sugar chain antibody Ab-3 through operation six.

Example 41: Synthesis of gsADC-16

Antibody-drug conjugate gsADC-16 is obtained through operation six of compound D10 and non-natural sugar chain antibody Ab-3.

Example 42: Synthesis of gsADC-17

Antibody-drug conjugate gsADC-17 is obtained through operation six of compound D1 and non-natural sugar chain antibody Ab-4.

Example 43: Synthesis of gsADC-18

Antibody-drug conjugate gsADC-18 is obtained through operation six of compound D2 and non-natural sugar chain antibody Ab-4.

Example 44: Synthesis of gsADC-19

Antibody-drug conjugate gsADC-19 is obtained by compound D3 and non-natural sugar chain antibody Ab-4 through operation six.

Example 45: Synthesis of gsADC-20

Antibody-drug conjugate gsADC-20 is obtained by compound D4 and non-natural sugar chain antibody Ab-4 through operation six.

Example 46: Synthesis of gsADC-21

Antibody-drug conjugate gsADC-21 is obtained by compound D10 and non-natural sugar chain antibody Ab-4 through operation six.

Example 47: Synthesis of gsADC-22

The antibody-drug conjugate gsADC-22 is obtained through operation 7 of compound D5 and non-natural sugar chain antibody Ab-2.

Example 48: Synthesis of gsADC-23

Antibody-drug conjugate gsADC-23 is obtained by compound D6 and non-natural sugar chain antibody Ab-2 through operation 7.

Example 49: Synthesis of gsADC-24

Antibody-drug conjugate gsADC-24 is obtained through operation 7 of compound D5 and non-natural sugar chain antibody Ab-3.

Example 50: Synthesis of gsADC-25

Antibody-drug conjugate gsADC-25 is obtained through operation 7 of compound D6 and non-natural sugar chain antibody Ab-3.

Example 51: Synthesis of gsADC-26

Antibody-drug conjugate gsADC-26 is obtained through operation 7 of compound D5 and non-natural sugar chain antibody Ab-4.

Example 52: Synthesis of gsADC-27

Antibody-drug conjugate gsADC-27 is obtained through operation 7 of compound D6 and non-natural sugar chain antibody Ab-4.

Example 53: Synthesis of gsADC-28

Antibody-drug conjugate gsADC-28 is obtained through operation six of compound D15 and non-natural sugar chain antibody Ab-1.

Example 54: Synthesis of gsADC-29

Antibody-drug conjugate gsADC-29 is obtained through operation six of compound D15 and non-natural sugar chain antibody Ab-3.

Pharmacological Example 1:

Experimental process and result analysis of cell activity data

Cell-level activity evaluation was performed on some sugar chain site-specific ADC compounds above, and three cell lines were selected, among which SK-Br-3 cells and NCI-N87 cells were Her2-positive cells, and MDA-MB-231 was Her2-negative cells. The cell viability and toxicity of ADC molecules were tested by MTT assay. The specific operation is as follows: add 100 μL PBS to the outermost circle of the 96-well plate, select another three wells to add only medium, and add about 6000 corresponding cells to each well, and incubate overnight at 37°C in a CO ₂ incubator. Add 10 μL of each ADC molecule (various ADC molecules are diluted in a 5-fold gradient starting from the highest concentration of 100nM, a total of 9 concentrations are diluted, and each concentration has 3 replicate wells), and the remaining 3 in each 96-well plate are covered with cells and the other 3 Add 10 μL of culture medium to the wells where only the culture medium was added to serve as the control group and the blank group, and place the 96-well plate in a CO ₂ incubator for 72 hours at 37°C. After adding 10 μL of 5 mg/mL MTT to each well, incubate at 37°C for 4 hours, then add 90 μL of SDS lysate to each well, and incubate at 37°C for 7 hours to fully lyse the cells. Finally, the OD value of each well at 570 nm was measured, and GraphPad Prism 6 was used for data processing. Part of the GraphPad Prism 6 analysis results are shown in Figure 46 below, and the IC ₅₀ results of sugar chain-specific ADC compounds are shown in the table below:

Table 1. Cell activity data of sugar chain-directed ADC compounds

Pharmacological embodiment 2:

Two kinds of sugar chain-specific ADC molecules, gsADC-28 and gsADC-29, were selected for in vivo activity evaluation in mice. In addition, a Cys randomly coupled ADC molecule (DAR≈4) was selected as a positive control, and its drug-linker was MC- VC-PAB-MMAE, the negative control is PBS group.

The specific operation is as follows: the nude mice used for animal-level activity evaluation are purchased from the Phase III animal room of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and 5-week-old BALB/c female nude mice with SPF grade are selected. After three days of adaptive culture, tumor cells are inoculated to construct NCI-N87 transplantation. The tumor model was used to evaluate the in vivo activity of sugar chain-directed ADC molecules in mice.

Construction of NCI-N87 Nude Mice Transplanted Tumor Model

Prepare the cell suspension: culture a sufficient amount of NCI-N87 cells, use trypsin to remove the supernatant medium, wash the cells twice with cell-grade 1 x PBS, centrifuge to settle the cells, and remove residual serum, etc. After adding an appropriate amount of 1 x PBS, count using a cytometer. Finally, use a pipette to add an appropriate amount of 1 x PBS and Matrigel gel to make the final concentration of the cell suspension 3.5 x ¹⁰ cells/mL. The Matrigel gel needs to be melted in the refrigerator at 4°C 6 hours in advance, and the pipettes used also need to be placed in advance. Cool in the refrigerator. After the cell suspension was prepared, it was kept on ice until use.

Cell suspension inoculation: After re-mixing the cell suspension in the animal room, use an insulin syringe to inoculate the nude mice with the cell suspension. The inoculation site is the subcutaneous injection of the left upper leg. Rats were injected with 4.3x10 ⁶ NCI-N87 cells. Note that before inoculation, use 75% alcohol to sterilize the inoculation site on the left upper leg of the nude mouse. When the insulin syringe is injected, its mouth should be upward and tilted at 10-20 degrees. After the injection, a small white pimple should appear at the injection site, indicating that the subcutaneous injection was successful.

Animal experiment grouping and ear hole marking: One week after inoculation of the cell suspension, tumors ranging in size from 100 to 250 ^mm3 could be observed, and ear hole marking and weighing were performed on each mouse. The groups were grouped according to the principle of large, medium and small tumors, with 5 mice in each group.

Animal experiment operation

Dosing operation: Firstly, all the samples to be administered were diluted to 0.2 mg/mL with 1 x PBS, and sterilized with a 0.22 μm filter membrane before use. The mice were grouped one day in advance, and the volume of each mouse administered was calculated according to body weight. The administration concentration was 3 mg/kg, administered intraperitoneally once every three days, and administered three times in total. The operation of intraperitoneal administration is to use a disposable syringe to draw an appropriate volume of sample, and vertically insert the syringe into the mouse at the midline of the abdomen and the middle of the right lower leg. You can feel a sense of void after the syringe enters the body, indicating that the intraperitoneal injection is successful. After the first administration, the tumor size and the body weight of the mice were measured every three days, and the mice were killed after a total of one and a half months of measurement, and the tumors were taken to take pictures (according to animal ethics requirements, due to the tumor size of the PBS control group on the 24th day The number has reached 2000, so they were executed ahead of time to collect tumors and placed in a -80°C refrigerator). The measured data were plotted using GraphPad Prism 6 software to obtain a graph of the change in tumor size over time and a graph of the change in body weight over time. The results are shown in Figure 47 below.

The results showed that gsADC-28 and gsADC-29 had a higher inhibitory effect on tumor volume than the positive control.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. An oligosaccharide linker of formula (I):

wherein:

each X is independently selected from the group consisting of a structure formed by a functional group with high reactivity (such as hydroxylamine, amino group, mercapto group structure) and an aldehyde group structure to form a stable covalent bond, and the following structure is preferred: -NHCH ₂ -、-ONH＝CH-、-CONHCH ₂ -、-NHCO-CH＝CH-、

Structure (c);

each Y is independently selected from the following groups: - (CH) ₂ )m-(CH-w)n1-、-(CH ₂ -CH ₂ -O) m- (CH-w) n1-, wherein m and n1 are integers between 0 and 30; w is a hydrogen atom or other side chain structure (e.g., PEG of different lengths);

each Z is independently selected from the following groups:

wherein R is ₁ And R ₂ Are each independently selected from H or CH ₃ D is a small molecule drug, L is a drug linker structure, and Z' is a linking structure between the drug linker and the sugar structure.

2. An oligosaccharide linker of formula (II):

wherein:

each X is independently selected from the group consisting of a functional group having high reactivity (such as hydroxylamine, amino group, mercapto group structure) and an aldehyde group structure forming a stable covalent bond, preferably the following structure:

structure, or independently an aldehyde structure obtained by oxidation with sodium periodate (in this case no Y/Z structure);

each Y is independently selected from the following groups: - (CH) ₂ )m-(CH-w)n1-、-(CH ₂ -CH ₂ -O) m- (CH-w) n1-, wherein m and n1 are integers between 0 and 30; w is a hydrogen atom or other side chain structure (e.g., PEG of different lengths);

each Z is independently selected from the following groups:

wherein R is ₁ And R ₂ Are respectively independentIs selected from H or CH ₃ D is a small molecule drug, L is a drug linker structure, and Z' is a linking structure between the drug linker and the sugar structure.

3. The oligosaccharide linker of claim 1 or 2 wherein Z' is selected from the group consisting of:

wherein R is ₁ And R ₂ Each independently selected from H or CH ₃ ；

L is selected from the following groups:

wherein a, b and c are each independently an integer of 0 or more.

4. The oligosaccharide linker of claim 1 or 2 wherein the side chain of L is derivatized with a hydrophilic branching structure R:

wherein, R is polyethylene glycol with different lengths or sugar structures with different lengths and configurations composed of different sugar structures, such as oligosaccharide structures composed of glucose Glu, galactose Gal, mannose Man, N-acetylglucose GlcNAc, N-acetylgalactosamine GalNAc, sialyl SA, xylose Xyl, fucose Fuc, fructose, deoxyribose monosaccharide and different monosaccharides;

v is a bifunctional linker, including but not limited to structures with lysine, cysteine, propargylglycine as bifunctional linkers:

wherein, R1 is a side chain polyethylene glycol PEG structure or a sugar structure, R2 is a linker structure which can be covalently linked with an oligosaccharide structure, or R1 can be divided into polyethylene glycol PEG chains or sugar structures with different lengths and containing functional groups which can react with amino, carboxyl, sulfydryl, alkynyl and azido, and the positions of R1 and R2 can be interchanged and are respectively and independently selected from the following structures:

wherein a, c and d are each independently an integer between 2 and 6; b is an integer selected from between 3 and 30,

R ₃ and R ₄ Are each independently selected from CH ₃ -、CH ₃ CH(CH ₃ )-、PhCH ₂ -、NH ₂ (CH ₂ ) ₄ -、NH ₂ CONH(CH ₂ ) ₃ -；

n is an integer of 0 to 10;

represents a linking site.

5. The oligosaccharide linker of claim 3 wherein D is a small molecule drug or a small molecule active compound or a radiotherapeutic selected from the group consisting of maytansine, DM-1, MMAE, MMAF, MMAP, auristatin E, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, SN-38, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, estramustine, cimadrol or exelisocycline; or preferably selected from the following groups:

wherein,

represents a linking site.

6. The oligosaccharide linker of claim 3 which is selected from the group consisting of:

7. an intermediate useful in the preparation of the oligosaccharide linker of claim 1 or 2, wherein the intermediate is an asparagine and polypeptide compound thereof containing the sugar structures shown in (III) and (IV):

8. a method of glyco-modifying an antibody, comprising the steps of: transferring the sugar structure in the structures (III) and (IV) to a conserved glycosylation site of the antibody under the action of endoglycosidase, thereby obtaining the glycoengineered antibody structure with the following structure:

wherein the X/Y/Z structure in structure (VII) is as described in any of claims 1-6;

m, p and q are each independently selected from 0 or 1;

ab is an antibody.

9. A site-directed engineered glyco-engineered antibody or antibody-drug conjugate based on N-glycosylation sites of the Fc region of an antibody, as represented by the following formula (V), (VI) or (VII):

wherein the X/Y/Z structure in structure (VII) is as in any of claims 1-6;

m, p and q are each independently selected from 0 or 1;

ab is an antibody.

10. A glycoengineered antibody or antibody drug conjugate based on sialylaldehyde groups of the formula (VIII):

wherein the X/Y/Z structure in structure (VIII) is as described in any one of claims 1-6;

m, p and q are each independently selected from 0 or 1;

ab is an antibody;

preferably, the connection mode at X is a cyclic thioether pyrazole, anthranilic oxime or thiazolidine structure

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