Sugar coupling method and sugar conjugate obtained by sugar coupling method

Abstract

The invention discloses a sugar coupling method and a sugar conjugate obtained by the same, a reaction formula of the sugar coupling method is shown in the specification, and the method comprises the following steps: a substrate A containing aldehyde sugar and a substrate B are subjected to Pickering-Schengler reaction to generate a sugar conjugate C. Based on the unique reactivity of aldehyde sugar and protein, polypeptide and the like containing a beta-(hetero) arylethylamine structure in a water phase, selective sugar modification of polypeptide or protein is achieved, a series of glycopeptides and glycoproteins can be conveniently constructed, and the method has the advantages of being mild in reaction condition, easy to operate, free of catalysts or additives, good in biocompatibility, high in repeatability and the like and suitable for industrial production. The method has a series of advantages of high yield and the like, natural amino acid can be used as a substrate, the use of genetic engineering and other means is avoided, and the technical threshold is greatly reduced. Furthermore, the method can be used for carrying out structural modification on sugar in glycopeptide, glycoprotein and glycolipid, and sugar fixed-point coupling of complex glycopeptide and glycoprotein can be realized. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of carbohydrate chemistry, and particularly relates to a method for sugar conjugation and a sugar conjugate obtained thereby. Background Art

[0002] Carbohydrate compounds play important roles in basic life activities such as cell communication, proliferation and differentiation, and immune response. At the same time, they are inevitably associated with the occurrence of various diseases. Through the artificial assembly and splicing of sugar substrate fragments by chemical synthesis, sugar substrates with clear structures can be obtained. At the same time, different sugar fragments and connection methods can greatly expand the diversity and complexity of carbohydrate compounds, which will greatly expand the chemical space of carbohydrate research and promote related glycobiology research fields. Biomacromolecules such as polypeptides and proteins play irreplaceable roles in the basic activities of living organisms. By modifying biomacromolecules, it not only provides a tool for their functional research, but also can endow biomolecules with different characteristics. Further, site-selective modification of proteins is the key to their precise functional research and improving the therapeutic index of protein drugs.

[0003] Compared with the most common reactions of aldehyde groups to form oximes or hydrazones, the Pictet–Spengler reaction generates tetrahydroisoquinoline products through the reaction of tryptamine with aldehyde compounds. The products formed by this reaction have obvious structural stability advantages and have good potential for bioconjugation. However, the traditional Pictet–Spengler reaction has low efficiency under biocompatible conditions (aqueous phase, neutral) and is difficult to be used for the conjugation of biomacromolecules.

[0004]

[0005] To address the problem of low efficiency of the conjugation reaction of biomacromolecules, in 2013, the Bertozzi research group [1] replaced the amino group of tryptamine with a hydroxylamine or hydrazine structure and moved this group from the 3-position of indole to the 2-position. Through such a structural design, on the one hand, the efficiency of imine formation was enhanced, and on the other hand, the activity of the imine intermediate being nucleophilically attacked by the 3-position of indole was also enhanced, thereby greatly improving the reaction efficiency by a large margin. Using this substrate, the Bertozzi research group achieved site-selective modification of aldehyde-labeled proteins and conducted related biological application research. The reaction formula is as follows:

[0006]

[0007] Based on the same idea, Pomplun et al. [2] reported in 2019 an example of substrate design through pyrrole to improve the efficiency of the Pictet–Spengler reaction, and also achieved site-selective modification of aldehyde-labeled proteins. The reaction formula is as follows:

[0008]

[0009] However, the aforementioned strategies for modifying the Pictet–Spengler reaction all start from tryptamine or substrates, and the proteins with aldehyde tags need to introduce aldehyde groups through genetic engineering techniques, which have relatively high technical requirements. Moreover, introducing aldehyde groups by chemical methods has the problem of uneven reactions.

[0010] In 2000, Tam et al. [3] reported the coupling of N-terminal tryptophan-containing polypeptides with aldehyde-containing polypeptides to obtain structurally stable polypeptide–polypeptide conjugates. However, this method needs to be carried out under pure acetic acid conditions and cannot react in the aqueous phase, which greatly limits the further application of this method.

[0011] [1] P. Agarwal, J. van der Weijden, E. M. Sletten, D. Rabuka, C. R. Bertozzi, Proceedings of the National Academy of Sciences 2013, 110, 46 - 51.

[0012] [2] S. Pomplun, M. Y. H. Mohamed, T. Oelschlaegel, C. Wellner, F. Bergmann, Angew. Chem. Int. Ed. 2019, 58, 3542 - 3547.

[0013] [3] X. Li, L. Zhang, S. E. Hall, J. P. Tam, Tetrahedron Lett. 2000, 41, 4069 - 4073. SUMMARY OF THE INVENTION

[0014] Based on the problems existing in the prior art, the present invention utilizes the unique reaction characteristics of aldehyde sugars to achieve the coupling of aldehyde sugars with biomolecules, such as substrates containing β-arylethylamine or β-heteroarylethylamine structures (such as tryptophan, N-aminoethylpyrrole or its derivatives, N-terminal tryptophan-containing polypeptides or proteins, etc.), to obtain stable and homogeneous sugar conjugates, and solves the problem of unstable oxime or hydrazone structures formed by aldehyde compounds. Moreover, the method for synthesizing sugar conjugates provided by the present invention avoids the addition and removal operations of protecting groups in conventional carbohydrate chemistry. In particular, the method of the present invention can be carried out under aqueous phase conditions and has significant advantages such as high efficiency, high biocompatibility, simplicity and high efficiency, and green and low cost. Further, the method of the present invention can be used for site-selective modification of proteins.

[0015] In view of this, an object of the present invention is to provide a method for sugar coupling and a sugar conjugate obtained by this method.

[0016] Another object of the present invention is to provide an antibody-drug conjugate prepared from a sugar conjugate, and its preparation method and application.

[0017] To achieve the above object, the present invention adopts the following technical solutions:

[0018] In the first aspect, the present invention provides a sugar conjugation method, and its reaction formula is as follows:

[0019]

[0020] Wherein, represents a sugar, a glycopeptide, a glycoprotein, a glycolipid or a derivative thereof substituted by a derivatizing group, wherein the derivative refers to a derivative that does not affect the reaction of the glyoxal group in the aldehyde group-containing sugar substrate A with substrate B in the Pictet-Spengler reaction, and the derivatizing group includes: a reactive group, a group derived from a small molecule toxin, a biotin tag, a fluorescent group, a radionuclide, etc.; specifically, the derivatizing group is: 4-nitrophenol group, benzyl, methyl or benzoyl;

[0021] The Ar ring is a C6-C12 aromatic ring or a 5-12 membered heteroaromatic ring, and X is C, CH or N; the heteroaromatic ring refers to an aromatic heterocyclic ring containing 1-3 (for example, 1, 2, 3) heteroatoms selected from N, O, S in the ring atoms, and the rest are carbon atoms, including monocyclic and fused rings;

[0022] R 1 is n electron-donating substituents, n is 0 to 5, for example 0, 1, 2, 3, 4, 5; the electron-donating substituents include: hydroxyl (-OH), C1-C20 alkoxy, amino (-NH2), -NH(C1-C20 alkyl), -N(C1-C20 alkyl)(C1-C20 alkyl), -NHCO-R 3 wherein the R 3 has a structure of -(L) i -(W) j wherein, i and j are each independently an integer between 0 and 10, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. When i and j are both 0, R 3 is H, each L is independently a linking group, for example C1-C20 alkylene, -COO-, -CONH-, or a derivative group of PEG, PAB (p-aminobenzyl), VC (valine-citrulline), BCN (bicyclo[6.1.0]non-4-yne); each W is independently selected from: a reactive group, a group derived from a small molecule toxin, a biotin tag, a fluorescent group, a radionuclide;

[0023] R 2is hydrogen, a carboxyl group (-COOH), -C(=O)NH2, a peptide segment or other derivative groups;

[0024] represents a single bond or a double bond. When represents a single bond, X is CH or N. When represents a double bond, X is C;

[0025] The method includes the following steps:

[0026] Substrate A containing an aldehyde group sugar reacts with substrate B in a Pictet-Spengler reaction to generate a sugar conjugate C.

[0027] In some embodiments, the substrate A containing an aldehyde group sugar is selected from sugars containing an aldehyde group, glycopeptides of sugars containing an aldehyde group, glycoproteins of sugars containing an aldehyde group, glycolipids of sugars containing an aldehyde group or derivatives obtained by substituting them with derivative groups; the glycoaldehyde groups of the sugars containing an aldehyde group, glycopeptides of sugars containing an aldehyde group, glycoproteins of sugars containing an aldehyde group, glycolipids of sugars containing an aldehyde group or their derivatives can be those contained in the sugar structure itself or obtained by treatment (such as by chemical (enzymatic) methods or genetic engineering means).

[0028] In some embodiments, the introduction of a glycoaldehyde group by a chemical method can be achieved by oxidizing a substrate containing a sugar structure with 2,2,6,6-tetramethylpiperidine oxide (TEMPO) or sodium periodate.

[0029] In some embodiments, 2,2,6,6-tetramethylpiperidine oxide (TEMPO) is used to oxidize the primary hydroxyl group in the sugar structure to an aldehyde; specifically, a substrate containing a sugar structure with a primary hydroxyl group is reacted with 2,2,6,6-tetramethylpiperidine oxide in the presence of trichloroisocyanuric acid and sodium bicarbonate; the reaction is carried out in a solvent (such as N,N-dimethylformamide); the equivalent ratio of the substrate containing a sugar structure with a primary hydroxyl group to 2,2,6,6-tetramethylpiperidine oxide is 100:1 to 1:1, preferably 40:1; the reaction temperature is -40 to 20 °C, such as 0 °C; the reaction time is 1 to 6 h, such as 4 h.

[0030] In some embodiments, sodium periodate is used to oxidize the primary hydroxyl group in the sugar structure to an aldehyde; specifically, a substrate containing a sugar structure with a primary hydroxyl group is reacted with sodium periodate in an aqueous phase (such as water, phosphate buffer); the equivalent ratio of the substrate containing a sugar structure with a primary hydroxyl group to sodium periodate is 1:20 to 50, such as 1:30; the reaction pH value is 6.0 - 8.0, such as 7.0; the reaction temperature is 0 to 4 °C, such as 0 °C; the reaction time is 15 to 30 min, such as 15 min.

[0031] In some embodiments, the method for introducing uronic groups enzymatically is as follows: using galactose oxidase to selectively oxidize the 6-hydroxy group of the galactose moiety in galactose or a substrate containing a galactose structure (including its derivatives) to an aldehyde group; specifically, reacting galactose or a substrate containing a galactose structure under the action of galactose oxidase, horseradish peroxidase, and catalase, and oxidizing the 6-hydroxy group of the galactose moiety to an aldehyde group in an oxygen atmosphere; the reaction can be carried out in a phosphate buffer solution, with the reaction pH value being 4.0 - 10.0, such as 7.0; the reaction temperature is 10 - 40 °C, such as 20 - 30 °C; the reaction time is 1 - 8 h, such as 2 h.

[0032] In some embodiments, the aldehyde group-containing sugar moiety in the substrate A of the aldehyde group-containing sugar is selected from: monosaccharides: glucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, mannose, N-acetylmannosamine, fructose, ribose, arabinose, xylose, etc.; disaccharides: melibiose, lactose, N-acetyl lactose, etc.; oligosaccharides: sialylated oligosaccharides (SCT), desialylated oligosaccharides (CT), etc.; or oligosaccharides and polysaccharides containing one or more of the aforementioned sugars, as well as derivatives obtained by substituting them with derivatized groups.

[0033] In some embodiments, the substrate A of the aldehyde group-containing sugar is selected from the following:

[0034]

[0035] Wherein, AcNH- represents acetamido; pNP represents 4-nitrophenol group; Ph represents phenyl; Bn represents benzyl; Me represents methyl; Bz represents benzoyl.

[0036] In some embodiments, the substrate A of the aldehyde group-containing sugar is an aldehyde group-containing glycopeptide, which is selected from sialyl glycopeptides containing uronic groups and desialylated glycopeptides containing uronic groups; the sialyl glycopeptides containing uronic groups are obtained by oxidizing the corresponding sialyl glycopeptides with sodium periodate according to the aforementioned method; the desialylated glycopeptides containing uronic groups are obtained by oxidizing the corresponding desialylated glycopeptides with galactose oxidase according to the aforementioned method; in particular, the sialyl glycopeptides containing uronic groups and the desialylated glycopeptides containing uronic groups respectively have structures selected from the following:

[0037]

[0038] In some embodiments, the substrate A of the aldehyde group-containing sugar is an aldehyde group-containing glycoprotein, preferably, the aldehyde group-containing glycoprotein is an antibody.

[0039] Specifically, the antibody is IgG with a conserved N-glycosylation site at N297 in the Fc region;

[0040] Specifically, the antibody is IgG1, IgG2 or IgG4;

[0041] Specifically, the antibody is a monoclonal antibody, polyclonal antibody, bispecific antibody, trispecific antibody, nanobody fused with an Fc domain, therapeutic antibody or functional antibody from different species sources (such as mouse, rat, sheep, rabbit, human, etc.);

[0042] Specifically, the targets of the antibody include HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276 (B7H3), HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPa, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG (5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R;

[0043] Preferably, the antibody is selected from trastuzumab.

[0044] Particularly, the glycoprotein containing aldehyde group sugar is aldehyde group-containing sialic acid complex glycoform-trastuzumab, which is obtained by oxidizing sialic acid complex glycoform-trastuzumab with sodium periodate and has the following structure:

[0045]

[0046] Among them, trastuzumab is linked to the aldehyde group-containing sialic acid complex glycoform as shown above through the Asn297 position of the Fc domain.

[0047] In some embodiments, in substrate B, the Ar ring is a C6-C10 aromatic ring or a 5-10 membered heteroaromatic ring, such as phenyl, indolyl, pyrrolyl.

[0048] In some embodiments, the substrate B has a structure selected from the following:

[0049]

[0050] Among them, R 1 is one or more optionally substituted substituents selected from the following: hydroxyl (-OH), p is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, especially 4 - 7, such as 5.

[0051] R 2 is selected from H, carboxyl (-COOH), -C(=O)NH2, and a peptide segment of 3 - 20 amino acids.

[0052] In some embodiments, the substrate B is a substance selected from the following group:

[0053] tryptophan, dopa, dopamine, 3 - hydroxyphenethylamine, N - aminoethylpyrrole;

[0054] a polypeptide or protein with tryptophan, dopamine or N - aminoethylpyrrole at the N - terminus, and

[0055] a derivative obtained by substituting the aforementioned substance with a derivatizing group.

[0056] Specifically, the substrate B is selected from: tryptophan, N - aminoethylpyrrole, N - aminoethylpyrrole derivative, and a polypeptide with tryptophan at the N - terminus;

[0057] Specifically, the N - aminoethylpyrrole derivative has a structure selected from the following:

[0058]

[0059] Specifically, the polypeptide with tryptophan at the N - terminus is selected from polypeptides having the following sequences:

[0060] WPNF (SEQ ID NO: 1, 2a); WDRVYIHPFHL (SEQ ID NO: 2, 2b);

[0061] WDRVYIHPF (SEQ ID NO: 3, 2c); WRVYIHPF (SEQ ID NO: 4, 2d);

[0062] WVYIHPF (SEQ ID NO: 5, 2e); WYGGFM (SEQ ID NO: 6, 2f);

[0063] WYAFHLMD (SEQ ID NO: 7, 2g); WYAFEVVG (SEQ ID NO: 8, 2h);

[0064] WYaFDVVG (SEQ ID NO: 9, 2i); WKLRLEWNR (SEQ ID NO: 10, 2j);

[0065] WLTVSPWY (SEQ ID NO: 11, 2k); WKKLKKLFSKLWNWK (SEQ ID NO: 12, 21).

[0066] In some embodiments, the aldehyde group-containing sugar substrate A reacts with substrate B in a solvent selected from the group consisting of: an aqueous phase system, formic acid, acetic acid, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, methanol, or a combination thereof.

[0067] In some embodiments, the aqueous phase system includes: water, an aqueous buffer, and the aqueous buffer can be: phosphate buffer, 2-(N-morpholino)ethanesulfonic acid buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) buffer, sodium acetate buffer, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane buffer, preferably phosphate buffer.

[0068] In some embodiments, the pH of the reaction system is 4.0 - 10.0, preferably 6.0 - 8.0, such as 7.0; the reaction temperature is 4 - 100 °C, preferably 30 - 40 °C, such as 37 °C; the reaction time is 0.1 - 72 h, preferably 1 - 24 h, such as 1 - 15 h, 1 - 12 h, or 1 - 5 h.

[0069] In some embodiments, the molar ratio of substrate A to substrate B is 1:100 - 100:1, preferably 1:20 - 20:1, such as 10 - 20:1 or 1:10 - 20.

[0070] In some embodiments, the sugar coupling method is selected from one of the following methods:

[0071] Method 1: The aldehyde group-containing sugar substrate A undergoes a Pictet-Spengler reaction with tryptophan as substrate B-1 or a polypeptide or protein containing an N-terminal tryptophan in an aqueous phase system to form a sugar conjugate C-1;

[0072] The reaction formula is as follows:

[0073]

[0074] Method 2: The aldehyde group-containing sugar substrate A undergoes a Pictet-Spengler reaction with N-aminoethylpyrrole or its derivative as substrate B-2 in an aqueous phase system to form a sugar conjugate C-2;

[0075] The reaction formula is as follows:

[0076]

[0077] Among them, R1, R2, the aqueous phase system, the reaction temperature, the reaction pH, and the molar ratio of substrates A and B are the same as those defined above.

[0078] In a second aspect, the present invention provides the sugar conjugate obtained by the above method.

[0079] Specifically, the sugar conjugate product is selected from compounds having the following structures:

[0080]

[0081]

[0082]

[0083]

[0084] In particular, the sugar conjugate is an antibacterial peptide (3r1) having the following structure:

[0085]

[0086] The antibacterial peptide 3rl can be prepared from compound 1r and polypeptide WKKLKKLFSKLWNWK by the method described in the first aspect of the present invention.

[0087] In particular, the sugar conjugate is azide-tagged sialic acid complex glycoform-trastuzumab, which is obtained by azide-tagging the aldehyde group-containing sialic acid-trastuzumab with compound 4a by the method described in the first aspect of the present invention, and has the following structure:

[0088]

[0089] Among them, the antibody is linked to the aldehyde group-containing sialic acid complex glycoform through the Asn297 position of the Fc domain.

[0090] The present invention also provides the use of the following compounds in the preparation of antibacterial drugs:

[0091]

[0092] In particular, provided is the use of the compound in the preparation of a medicament against the following Gram-positive bacteria: MRSA-USA300, Efm-HS-0649, Van (drug-resistant), 06188, and the following Gram-negative bacteria: AB1157 (E. coli), Aba., P. aeruginosa PAO1, Klebsiella pneumoniae. This antimicrobial peptide has a stronger bactericidal effect than antimicrobial peptide 21 and stronger stability against human serum.

[0093] The present invention also provides the use of the method according to the first aspect in sugar or glycopeptide modification and the preparation of antibody-drug conjugates.

[0094] The present invention also provides a method for preparing a trastuzumab-MMAE conjugate, and the reaction formula is as follows:

[0095]

[0096] The reaction includes the following steps:

[0097] (1) The aldehyde group-containing sialic acid complex glycoform-trastuzumab undergoes a Pictet-Spengler reaction with 4a to obtain sialic acid complex glycoform-trastuzumab modified with an azide tag;

[0098] (2) The sialic acid complex glycoform-trastuzumab modified with an azide tag undergoes a click chemical reaction with BCN-PEG4-VC-PAB-MMAE to obtain a sugar-site specific antibody-drug conjugate.

[0099] The structure of the BCN-PEG4-VC-PAB-MMAE is:

[0100]

[0101] In a specific embodiment, the Pictet-Spengler reaction is carried out in a phosphate buffer, the pH of the reaction system is 6.0 - 8.0, such as 7.0; the reaction temperature is 0 - 40 °C, such as 37 °C; the reaction time is 0.5 - 24 h, such as 1 - 5 h, such as 2 h; the dosage of 4a is 10 - 20 equivalents of the aldehyde group-containing antibody.

[0102] In a specific embodiment, the click chemistry reaction is carried out in a phosphate buffer, the pH of the reaction system is 6.0 - 8.0, such as 7.0; the reaction temperature is 0 - 40 °C, such as 37 °C; the reaction time is 0.5 - 24 h, such as 1 - 5 h, such as 2 h; the dosage of BCN-PEG4-VC-PAB-MMAE is 10 - 20 equivalents of the sugar-modified antibody.

[0103] The present invention also provides a trastuzumab-MMAE conjugate prepared by the above method, and its structure is as follows:

[0104]

[0105] The present invention also provides the application of the above trastuzumab-MMAE conjugate in the preparation of anti-tumor drugs. In particular, the tumor is HER2-positive breast cancer.

[0106] Beneficial effects

[0107] 1. Based on the unique reactivity of aldehyde sugars with proteins, polypeptides, etc. containing β-(hetero)aryl ethylamine structures in the aqueous phase, the present invention realizes the selective sugar modification of polypeptides or proteins, and can conveniently construct a series of glycopeptides and glycoproteins, with a series of advantages such as mild reaction conditions, simple operation, no need for catalysts or additives, good biocompatibility, and high yields.

[0108] 2. The method of the present invention can use natural amino acids as substrates, and its characteristics can avoid the use of means such as genetic engineering, greatly reducing the technical threshold.

[0109] 3. The method of the present invention can modify the sugars in glycopeptides, glycoproteins, and glycolipids, and can achieve site-specific coupling of sugars in complex glycopeptides and glycoproteins, such as the preparation of antibody-drug conjugates and cell glycan labeling. Description of the drawings

[0110] Figure 1 It is a stability trend diagram of unglycosylated antimicrobial peptide 21 in human serum in Example 28 of the present invention.

[0111] Figure 2 It is a stability trend diagram of glycosylated antimicrobial peptide 3rl in human serum in Example 28 of the present invention.

[0112] Figure 3 It is the result of the influence of MMAE and antibody-drug conjugate on the survival rate of different tumor cells in Example 29 of the present invention. Detailed implementation manners

[0113] The following examples are used to further illustrate the present invention. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection required by the present invention.

[0114] Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are all conventional raw materials, reagents, and methods in the art.

[0115] The sugar aldehyde group-containing substrates 1a-1r in the following examples are prepared by one of the following methods:

[0116] Method 1: 1a - 1l, 1r, desialylated glycoprotein containing aldehyde group (CHO - ASGP) is prepared by galactose oxidase oxidation method, and its specific preparation method is as follows:

[0117] Dissolve the substrate (1 mmol), galactose oxidase (100 U / mmol), horseradish peroxidase (0.02 mol%), and catalase (0.001 mol%) in phosphate buffer (50 mM, pH 7.0, 20 mL), evacuate and replace with oxygen, then stir and react at room temperature in an oxygen atmosphere for 2 hours. After monitoring the reaction by HPLC and completion of the reaction, purify the product by semi - preparative HPLC.

[0118] Method 2: Substrate 1m - 1p is prepared by 2,2,6,6 - tetramethylpiperidine oxide (TEMPO) oxidation, and its specific preparation method is as follows:

[0119] Dissolve the substrate (0.25 mmol), 2,2,6,6 - tetramethylpiperidine oxide (2.5 mol%), trichloroisocyanuric acid (75 mol%), and sodium bicarbonate (30 equivalents) in dry DMF (50 mL), react at - 0 °C for 4 hours. After monitoring the reaction by HPLC and completion of the reaction, purify the product by semi - preparative HPLC.

[0120] Method 3: Substrate 1q, sialylated glycoprotein containing aldehyde group (CHO - SGP) is prepared by sodium periodate oxidation, and its specific preparation method is as follows:

[0121] Dissolve the substrate (1 mmol) in pure water (20 mL), add sodium periodate (30 equivalents) at 0 °C, after reacting at 0 °C for 15 min, quench the reaction with ethanol, and then purify the product through a Sephadex column.

[0122] The structures of the substrates 1a’ - 1q’ corresponding to compounds 1a - 1q are as follows:

[0123]

[0124] The substrate structures of sialylated glycoprotein containing aldehyde group (CHO - SGP) and desialylated glycoprotein containing aldehyde group (CHO - ASGP) are respectively:

[0125]

[0126] The characterization data of compounds 1a - 1r, CHO - SGP, and CHO - ASGP are shown in Table 1.

[0127] Table 1

[0128]

[0129]

[0130] Peptides 2a-2l were purchased from GenScript Biotechnology Co., Ltd. with a purity of more than 95%.

[0131] Compounds 4a and 4b were purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd. with a purity of more than 95%.

[0132] Antibodies Tras-SCT and BCN-PEG4-VC-PAB-MMAE were purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.

[0133] Example 1:

[0134]

[0135] The peptide 2a (1 mM) with the sequence WPNF (SEQ ID NO: 1) was incubated with the aldehyde-containing acetyllactosamine 1a (10 mM) in a phosphate buffer solution at pH 7.0 at 37°C for 12 hours. After completion of the reaction, the target product 3aa was obtained by HPLC semi-preparative purification and lyophilization with a yield of 99%. ESI-MS: calcd. for C 43 H 55 N7O 16 m / z=925.3705,found926.3681[M+H] + .

[0136] Example 2:

[0137] Replace 1a in Example 1 with 1b, and perform the remaining operations in the same manner.

[0138]

[0139] The yield of the target product 3ba was 99%. ESI-MS: calculated for C 5o H 59 N7O 16 S m / z=1045.3739, found 1046.38[M+H] + .

[0140] Example 3:

[0141] Replace 1a in Example 1 with 1c, and perform the remaining operations in the same manner.

[0142]

[0143] The yield of the target product 3ca was 99%. ESI-MS: calculated for C 49 H 58 N8O 18m / z=1046.3869, found 1047.3922[M+H] + .

[0144] Example 4:

[0145] Replace 1a in Example 1 with 1d, and perform the remaining operations in the same manner.

[0146]

[0147] The yield of the target product 3da was 99%. ESI-MS: calculated for C 51 H 60 N 10 O 15 m / z=1052.4240, found 1053.4294[M+H] + .

[0148] Example 5:

[0149] Replace 1a in Example 1 with 1e, and perform the remaining operations in the same manner.

[0150]

[0151] The yield of the target product 3ea was 92%. ESI-MS: calculated for C 45 H 50 N 10 O 10 m / z=890.3711,found891.4526[M+H] + .

[0152] Example 6:

[0153] Replace 1a in Example 1 with 1f, and perform the remaining operations in the same manner.

[0154]

[0155] The yield of the target product 3fa was 88%. ESI-MS: calculated for C 43 H 47 N9O 10 m / z=849.3446,found850.3892[M+H] + .

[0156] Example 7:

[0157] Replace 1a in Example 1 with 1g, and perform the remaining operations in the same manner.

[0158]

[0159] The yield of the target product 3ga of the reaction was 87%. ESI-MS: calcd. for C 49 H 57 N9O 15 m / z = 1011.3974, found 1012.4[M+H] + .

[0160] Example 8:

[0161] Replace 1a in Example 1 with 1h, and the remaining operations are the same.

[0162]

[0163] The yield of the target product 3ha of the reaction was 88%. ESI-MS: calcd. for C 49 H 57 N9O 15 m / z = 1011.3974, found 1012.4047[M+H] + .

[0164] Example 9:

[0165] Replace 1a in Example 1 with 1i, and the remaining operations are the same.

[0166]

[0167] The yield of the target product 3ia of the reaction was 99%. ESI-MS: calcd. for C 48 H 60 N8O 16 S m / z = 1036.3848, found 1037.3887[M+H] + .

[0168] Example 10:

[0169] Replace 1a in Example 1 with 1j, and the remaining operations are the same.

[0170]

[0171] The yield of the target product 3ja of the reaction was 99%. ESI-MS: calcd. for C 47 H 61 N 11 O 16 S m / z = 1067.4018, found 1068.3971[M+H] + .

[0172] Example 11:

[0173] Replace 1a in Example 1 with 1k, and keep the rest of the operations the same.

[0174]

[0175] The yield of the target product 3ka of the reaction is 99%. ESI-MS: calcd. for C 49 H 64 N8O 16 S m / z = 1052.4161, found 1053.4221 [M+H] + .

[0176] Example 12:

[0177] Replace 1a in Example 1 with 1l, and keep the rest of the operations the same.

[0178]

[0179] The yield of the target product 31a of the reaction is 99%. ESI-MS: calcd. for C 53 H 68 N8O 16 S m / z = 1104.4474, found 1105.4563 [M+H] + .

[0180] Example 13:

[0181] Replace 1a in Example 1 with 1m, and keep the rest of the operations the same.

[0182]

[0183] The yield of the target product 3ma of the reaction is 83%. ESI-MS: calcd. for C 44 H 51 N7O 11 m / z = 853.3647, found 854.3799 [M+H] + .

[0184] Example 14:

[0185] Replace 1a in Example 1 with 1n, and keep the rest of the operations the same.

[0186]

[0187] The yield of the target product 3na of the reaction is >95%. ESI-MS: calcd. for C 36 H 44 N6O11 m / z = 736.3068, found 737.3275 [M+H] + .

[0188] Example 15:

[0189] Replace 1a in Example 1 with 1o, and keep the rest of the operations the same.

[0190]

[0191] The yield of the target product 3oa of the reaction is 87%. ESI-MS: calcd. for C 36 H 44 N6O 11 m / z = 736.3068, found 737.3203 [M+H] + .

[0192] Example 16:

[0193] Replace 1a in Example 1 with 1p, and keep the rest of the operations the same.

[0194]

[0195] The yield of the target product 3pa of the reaction is 83%. ESI-MS: calcd. for C 36 H 44 N6O 11 m / z = 736.3068, found 737.3256 [M+H] + .

[0196] Example 17:

[0197] Replace 1a in Example 1 with 1q, and keep the rest of the operations the same.

[0198]

[0199] The yield of the target product 3qa of the reaction is >90%. ESI-MS: calcd. for C 101 H 145 N 11 O 58 m / z = 2439.8735, found 2440.8622 [M+H] + .

[0200] Example 18:

[0201] Replace 1a in Example 1 with 1i, and 2a with 2b (SEQ ID NO: 2), and keep the rest of the operations the same.

[0202]

[0203] The yield of the target product 3ib of the reaction was 99%. ESI-MS: calcd. for C 92 H 127 N 21 O 25 S m / z = 1955.8876, found 979.4677 [M+2H] 2+ , 653.3129 [M+3H] 3+ .

[0204] Example 19:

[0205] Replace 1a in Example 1 with 1i, and 2a with 2c (SEQ ID NO: 3), and the remaining operations are the same.

[0206]

[0207] The yield of the target product 3ic of the reaction was 99%. ESI-MS: calcd. for C 80 H 107 N 17 O 23 S m / z = 1705.7446, found 1706.7509 [M+H] + , 853.8915 [M+2H] 2+ .

[0208] Example 20:

[0209] Replace 1a in Example 1 with 1i, and 2a with 2d (SEQ ID NO: 4), and the remaining operations are the same.

[0210]

[0211] The yield of the target product 3id of the reaction was 99%. ESI-MS: calcd. for C 76 H 102 N 16 O 20 S m / z = 1590.7177, found 796.3618 [M+2H] 2+ .

[0212] Example 21:

[0213] Replace 1a in Example 1 with 1i, and 2a with 2e (SEQ ID NO: 5), and the remaining operations are the same.

[0214]

[0215] The yield of the target product 3ie was 99%. ESI-MS: calculated for C 70 H 90 N 12 O 19 S m / zm=1434.6166,found 1435.6204[M+H] + ,718.3197[M+2H] 2+ .

[0216] Example 22:

[0217] In Example 1, 1a was replaced by 1i, and 2a was replaced by 2f (SEQ ID NO: 6). The remaining operations were the same.

[0218]

[0219] The yield of the target product 3if was 99%. ESI-MS: calculated for C 57 H 71 N9O 18 S2 m / z=1233.4358, found 1234.5563[M+H] + .

[0220] Example 23:

[0221] In Example 1, 1a was replaced by 1i, and 2a was replaced by 2g (SEQ ID NO: 7). The remaining operations were the same.

[0222]

[0223] The yield of the target product 3g was 99%. ESI-MS: calculated for C 74 H 98 N 14 O 21 S3 m / z=1614.6193, found 1615.6091[M+H] + , 808.3128[M+2H] 2+ .

[0224] Example 24:

[0225] In Example 1, 1a was replaced by 1i, and 2a was replaced by 2h (SEQ ID NO: 8). The remaining operations were the same.

[0226]

[0227] The yield of the target product 3ih of the reaction is 99%. ESI-MS: calcd. for C 67 H 88 N 12 O 21 S m / z = 1442.6064, found 1443.6149 [M+H] + , 722.3122 [M+2H] 2+ .

[0228] Example 25:

[0229] Replace 1a in Example 1 with 1i, and 2a with 2i (SEQ ID NO: 9), and the remaining operations are the same.

[0230]

[0231] The yield of the target product 3ii of the reaction is 99%. ESI-MS: calcd. for C 67 H 88 N 12 O 21 S m / z = 1428.5908, found 1429.5912 [M+H] + , 715.3003 [M+2H] 2+ .

[0232] Example 26:

[0233] Replace 1a in Example 1 with 1j, and 2a with 2j (SEQ ID NO: 10), and the remaining operations are the same.

[0234]

[0235] The yield of the target product 3jj of the reaction is 99%. ESI-MS: calcd. for C 79 H 120 N 24 O 23 S m / z = 1804.8679, found 903.9427 [M+2H] 2+ .

[0236] Example 27:

[0237] Replace 1a in Example 1 with 1j, and 2a with 2k (SEQ ID NO: 11), and the remaining operations are the same.

[0238]

[0239] The yield of the target product 3jk of the reaction is 99%. ESI-MS: calcd. for C 72 H 97 N 15 O 22 S m / z = 1555.6653, found 1556.6748 [M+H] + .

[0240] Example 28:

[0241] Replace 1a in Example 1 with 1i and 2a with 2l (SEQ ID NO: 12), and the remaining operations are the same.

[0242]

[0243] The yield of the target product 3il of the reaction is 99%. ESI-MS: calcd. for C 122 H 183 N 27 O 28 S m / z = 2506.3447, found 1254.1783 [M+2H] 2+ , 836.4604 [M+3H] 3+ .

[0244] Example 29:

[0245] Replace 1a in Example 1 with 1i and 2a with 2m, and the remaining operations are the same.

[0246]

[0247] The yield of the target product 3im of the reaction is 92%, 10:1 dr. ESI-MS: calcd. for C 30 H 40 N5O 11 S m / z = 677.2367, found 678.2584 [M+H] + .

[0248] 11H NMR (600 MHz, D2O) δ 7.50 (dd, J = 11.5, 8.1 Hz, 2H), 7.20 - 7.15 (m, 1H), 7.08 (t, J = 7.5 Hz, 1H), 4.99 (d, J = 7.8 Hz, 1H), 4.57 (d, J = 10.5 Hz, 1H), 4.47 (d, J = 7.6 Hz, 1H), 4.17 (dd, J = 11.9, 4.6 Hz, 1H), 4.05 (d, J = 7.9 Hz, 1H), 3.96 (d, J = 3.2 Hz, 1H), 3.91 - 3.88 (m, 1H), 3.87 (dd, J = 5.3, 2.6 Hz, 2H), 3.73 (dd, J = 12.5, 4.4 Hz, 1H), 3.72 - 3.67 (m, 2H), 3.63 - 3.56 (m, 2H), 3.51 - 3.46 (m, 1H), 3.42 (d, J = 15.2 Hz, 1H), 3.38 - 3.33 (m, 1H), 3.24 (d, J = 15.2 Hz, 1H), 3.08 - 3.00 (m, 1H), 1.98 (s, 3H). 13 13C NMR (151 MHz, D2O) δ 174.44, 172.20, 171.87, 136.75, 125.03, 123.06, 119.97, 118.34, 112.06, 106.34, 103.92, 84.22, 79.27, 78.95, 78.04, 73.51, 73.23, 72.05, 70.23, 67.77, 59.63, 56.14, 54.67, 52.28, 33.60, 29.09, 23.28, 22.17.

[0249] Example 30:

[0250]

[0251] The sialylglycopeptide (CHO - SGP) or desialylated glycopeptide (CHO - ASGP) (0.5 mM) containing aldehyde groups was incubated with the azide - tagged aminoethylpyrrole substrate 4a (2.5 mM) in phosphate buffer (pH 7.0) at 37 °C for 1 hour. After monitoring the completion of the reaction by HPLC, the target product was obtained by semi - preparative purification and freeze - drying.

[0252] The conversion rate of sialylglycopeptide (CHO - SGP) was 99%. Modified SGP: ESI - MS: calcd. for C 146 H 241 N 27 O 76 m / z = 3588.5823, found 1796.4119 [M + 2H] 2+, 1197.9574 [M+3H] 3+ , 898.4801 [M+4H] 4+ .

[0253] The conversion rate of desialylated glycopeptide (CHO-ASGP) is 99%. Modified ASGP: ESI-MS: calcd. for C 128 H 215 N 25 O 64 m / z = 3126.4338, found 1564.6313 [M+2H] 2+ , 1043.4398 [M+3H] 3+ .

[0254] Example 31:

[0255]

[0256] Replace 4a in Example 30 with 4b, and the remaining operations are the same.

[0257] The conversion rate of sialylated glycopeptide (CHO-SGP) is 99%. Modified SGP: ESI-MS: calcd. for C 140 H 223 N 25 O 68 S2 m / z = 3406.4202, found 1705.3282 [M+2H] 2+ , 1137.2345 [M+3H] 3+ , 853.1909 [M+4H] 4+ .

[0258] The conversion rate of desialylated glycopeptide (CHO-ASGP) is 99%. Modified ASGP: ESI-MS: calcd. for C 122 H 197 N 23 O 56 S2 m / z = 2944.2716, found 1473.5547 [M+2H] 2+ , 982.6901 [M+3H] 3+ .

[0259] Example 32:

[0260] Antimicrobial peptides are a class of polypeptides with broad-spectrum antibacterial activity. Due to their unique antibacterial mechanism and broad antibacterial spectrum, they are expected to become a new treatment method to overcome bacterial drug resistance. However, for polypeptide drugs, their poor in vivo stability and metabolic properties greatly limit their application. Using the bioconjugation strategy of the present invention, glycosylated antimicrobial peptides can be rapidly constructed, thereby improving metabolic properties while enhancing antibacterial activity, that is, enhancing drug-likeness.

[0261] In this example, an antimicrobial peptide with the amino acid sequence WKKLKKLFSKLWNWK(21) was used as an example, and the glycosylation process was as follows:

[0262] Compound 1r (10 mM) and antimicrobial peptide WKKLKKLFSKLWNWK (SEQ ID NO: 12) (1 mM) were incubated in phosphate buffer (pH 7.0) at 37 °C for 15 hours. After the reaction was monitored by HPLC and completed, the target product was obtained by semi-preparative purification and then freeze-dried. The conversion rate was >95%; the molecular weight of the glycosylated antimicrobial peptide product: ESI-MS: calcd. for C 116 H 170 N 26 O 23 m / z = 2295.2932, found 1148.7316 [M+2H] 2+ , 766.1025 [M+3H] 3+ .

[0263]

[0264] Bactericidal effect test:

[0265] The minimum inhibitory concentrations (MICs) of the unmodified antimicrobial peptide 21 and the glycosylated antimicrobial peptide 3r1 against MRSA-USA300, Efm-HS-0649, Van (resistant), 06188, AB1157 (E. coli), Aba., P. Aemginosa PAO1, Klebsiella pneumoniate were tested. The results are shown in Table 2 below, indicating that the modified antimicrobial peptide 3rl has enhanced killing effects on different strains such as Efm-HS-0649, Van (resistant), 06188, Klebsiella pneumoniate compared with the unmodified antimicrobial peptide 2l.

[0266] Table 2.

[0267]

[0268] Serum stability test:

[0269] Human serum stability was carried out in 1×PBS containing 25% (v / v) human serum. The test peptide was diluted to a final concentration of 2.5 mg / mL with the serum solution, incubated at 37 °C, and 10 μL of the sample was taken at different time points (0 h, 48 h, 72 h) and added to 10 μL of acetonitrile. After centrifugation at 4 °C for 15 min, the supernatant was taken for HPLC analysis. The results are shown in Figures 1-2 .

[0270] As can be seen from the figure, after incubation for 48 h and 72 h, a large amount of the prototype peptide of unmodified antimicrobial peptide 21 ( Figure 1 ) had been decomposed; in contrast, the glycoconjugated antimicrobial peptide 3rl ( Figure 2 ) showed no obvious decomposition after incubation for the same time, indicating that the stability of the glycoconjugated peptide in human serum was significantly enhanced, which provided a simple and reliable means to improve the drugability for the research of antimicrobial peptides.

[0271] Example 33

[0272] Numerous studies have shown that site-specific antibody-drug conjugates exhibit better in vivo antitumor activity and metabolic properties compared to randomly conjugated ones. Antibody glycosylation occurs at the asparagine site at position 297 of the heavy chain CH segment. Antibody-drug conjugates based on this glycosylation site not only have significant advantages in terms of hydrophilicity, but also the asparagine at position 297 and its neighboring sites are beneficial to improving the stability and in vivo activity of antibody-drug conjugates.

[0273] In this example, using the bioconjugation strategy of the present invention, an antibody-drug conjugate based on the asparagine glycosylation site at position 297 can be rapidly constructed, and the glycosylation process is as follows:

[0274]

[0275]

[0276] The preparation method of the antibody-drug conjugate is as follows:

[0277] (1) The antibody Tras-SCT (10 mg / mL) was incubated with sodium periodate (20 equivalents) in phosphate buffer (pH 7.0) at 0 °C for 30 minutes. After monitoring the completion of the reaction by LCMS, the excess sodium periodate was removed by ultrafiltration to obtain an antibody containing aldehyde groups (selectively oxidizing the 6-position hydroxyl group of galactose to aldehyde), with a conversion rate > 95% and a molecular weight of 149710 (about 4 aldehyde groups).

[0278] (2) Incubate the antibody containing aldehyde groups with 4a (the structure is shown in Example 30 above) in phosphate buffer (pH 7.0) at 37 °C for 2 hours. After monitoring the completion of the reaction by LCMS, ultrafiltration is used to remove the excess 4a to obtain the antibody modified with an azide tag. The conversion rate is >95%, and the molecular weights are 150186, 150553, 150968, 151381 (averagely containing 3 azides).

[0279] (3) Subsequently, incubate the modified antibody (5 mg / mL) with BCN-PEG4-VC-PAB-MMAE (20 equivalents) in phosphate buffer (pH 7.0) at 37 °C for 2 hours. After monitoring the completion of the reaction by LCMS, ultrafiltration is used to remove the excess BCN-PEG4-VC-PAB-MMAE, and freeze-drying gives the antibody-drug conjugate (ADC). The conversion rate is >95%, and the molecular weights are 149783, 151740, 153676, 155646 (averagely containing 3 cytotoxic drugs).

[0280] Selective tumor cell killing test:

[0281] MTT method: Seed Sk-Br-3 cells and MDA-MB-231 cells in a 96-well plate, with 5000 cells per well and 90 μL of culture medium, and incubate overnight in a cell culture incubator. Then, serially dilute ADC and MMAE from 100 nmol / L by 5-fold with McCOY’5A medium, and finally prepare 9 concentrations. For each concentration, add 10 μL of ADC or MMAE to three replicate wells. Incubate all plates in the cell culture incubator for another three days. Next, add 10 μL of MTT solution (5 mg / mL) to each well and incubate at 37 °C for 4 h. Then, supplement with 10% SDS solution to dissolve formazan, and measure the final optical density (OD) value of the whole plate at 570 nm using a BioTek Epoch microplate reader. All data are analyzed using GraphPad Prism software, and the IC 50 value is calculated. The results are shown in Figure 3 and Table 3.

[0282] Table 3 IC of antibody-drug conjugate (ADC) and MMAE against breast cancer cells 50

[0283]

[0284] From Figure 3 and the results in Table 3, it can be seen that compared with MMAE, the IC 50 of the antibody-drug conjugate prepared in this example against the HER2-positive SK-Br-3 cell line is much lower than the IC 50, indicating that it has a significant effect of selectively killing HER2.

[0285] As can be seen from the above examples, the method of the present application can be well used for the modification of glycopeptides, various antimicrobial peptides, and the synthesis of ADC drugs, and thus has good application prospects in the modification and synthesis of sugars, glycopeptides, and glycoproteins.

Claims

1. A sugar coupling method, and its reaction formula is as follows: Among them, represents a sugar, glycopeptide, glycoprotein, glycolipid or a derivative thereof substituted with a derivatizing group, wherein the derivative refers to a derivative that does not affect the reaction of the glycoaldehyde group in the aldehyde group-containing sugar substrate A with substrate B in the Pictet-Spengler reaction, and the derivatizing group includes: a reactive group, a group derived from a small molecule toxin, a biotin tag, a fluorescent group, a radionuclide; for example, the derivatizing group is: 4-nitrophenol group, benzyl, methyl or benzoyl; The Ar ring is a C6-C12 aromatic ring or a 5-12 membered heteroaromatic ring, and X is C, CH or N; the heteroaromatic ring refers to an aromatic heterocycle containing 1-3 (e.g., 1, 2, 3) heteroatoms selected from N, O, S in the ring atoms, and the rest are carbon atoms, including monocyclic and fused rings; R 1 are n electron-donating substituents, where n is an integer from 0 to 5, such as 0, 1, 2, 3, 4, 5; the electron-donating substituents include: hydroxyl, C1-C20 alkoxy, amino, -NH(C1-C20 alkyl), -N(C1-C20 alkyl)(C1-C20 alkyl), -NHCO-R 3 , where the R 3 has the structure -(L) i -(W) j , where i and j are each independently integers between 0 and 10, and when i and j are both 0, R 3 is H, each L is independently a linking group, such as C1-C20 alkylene, -COO-, -CONH-, or a derivative group of PEG, p-aminobenzyl, valine-citrulline, bicyclo[6.1.0]non-4-yne; each W is independently selected from: a reactive group, a group derived from a small molecule toxin, a biotin tag, a fluorescent group, a radionuclide R 2 is hydrogen, carboxyl (-COOH), -C(=O)NH2, peptide segment or other derivative group; represents a single bond or a double bond. When it represents a single bond, X is CH or N. When it represents a double bond, X is C; The method includes the following steps: The substrate A containing an aldehyde group sugar reacts with the substrate B in a Pictet-Spengler reaction to generate a sugar conjugate C.

2. The sugar conjugation method according to claim 1, wherein The substrate A containing an aldehyde group sugar is selected from an aldehyde group-containing sugar, a glycopeptide containing an aldehyde group sugar, a glycoprotein containing an aldehyde group sugar, a glycolipid containing an aldehyde group sugar, or a derivative obtained by substituting them with a derivatizing group; the aldehyde group of the aldehyde group-containing sugar, the glycopeptide containing an aldehyde group sugar, the glycoprotein containing an aldehyde group sugar, the glycolipid containing an aldehyde group sugar, or their derivatives can be contained in the sugar structure itself, or can be introduced by chemical (enzymatic) methods or genetic engineering means; Preferably, the aldehyde group is introduced chemically by oxidizing a sugar structure-containing substrate with 2,2,6,6-tetramethylpiperidine oxide or sodium periodate; More preferably, the primary hydroxyl group in the sugar structure is oxidized to an aldehyde using 2,2,6,6-tetramethylpiperidine oxide; Specifically, a substrate containing a sugar structure with a primary hydroxyl group is reacted with 2,2,6,6-tetramethylpiperidine oxide in the presence of trichloroisocyanuric acid and sodium bicarbonate; the reaction is carried out in a solvent; preferably, the solvent is N,N-dimethylformamide; the equivalent ratio of the substrate containing a sugar structure with a primary hydroxyl group to 2,2,6,6-tetramethylpiperidine oxide is 100:1 to 1:1, preferably 40:1; the reaction temperature is -40 to 20 °C, e.g., 0 °C; the reaction time is 1 to 6 h, e.g., 4 h; More preferably, the primary hydroxyl group in the sugar structure is oxidized to an aldehyde using sodium periodate; Specifically, a substrate containing a sugar structure with a primary hydroxyl group is reacted with sodium periodate in an aqueous phase; preferably, the aqueous phase is water or a phosphate buffer; the equivalent ratio of the substrate containing a sugar structure with a primary hydroxyl group to sodium periodate is 1:20 to 50, e.g., 1:30; the reaction pH value is 6.0 - 8.0, e.g., 7.0; the reaction temperature is 0 to 4 °C, e.g., 0 °C; the reaction time is 15 to 30 min, e.g., 15 min; Preferably, the method for introducing an aldehyde group by an enzymatic method is: selectively oxidizing the 6-position hydroxyl group of the galactose part in galactose or a substrate containing a galactose structure to an aldehyde using galactose oxidase; Specifically, galactose or a substrate containing a galactose structure is oxidized to an aldehyde at the 6-position hydroxyl group of the galactose part in an oxygen atmosphere under the action of galactose oxidase, horseradish peroxidase, and catalase; preferably, the reaction is carried out in a phosphate buffer; the reaction pH value is 4.0 - 10.0, e.g., 7.0; the reaction temperature is 10 to 40 °C, e.g., 20 to 30 °C; the reaction time is 1 to 8 h, e.g., 2 h.

3. The sugar conjugation method according to claim 1 or 2, wherein The aldose moiety contained in the aldose-containing substrate A is selected from: monosaccharides: glucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, mannose, N-acetylmannosamine, fructose, ribose, arabinose, xylose; disaccharides: melibiose, lactose, N-acetyllactose; oligosaccharides: sialylated oligosaccharides, desialylated oligosaccharides; or oligosaccharides and polysaccharides containing one or more of the aforementioned sugars, and derivatives obtained by substituting them with derivatizing groups; Preferably, the aldose-containing substrate A is selected from the following: wherein, AcNH- represents acetamido; pNP represents 4-nitrophenol group; Ph represents phenyl; Bn represents benzyl; Me represents methyl; Bz represents benzoyl.

4. The sugar conjugation method according to any one of claims 1 to 3, characterized in that The aldose-containing substrate A is an aldose-containing glycopeptide, which is selected from sialylated glycopeptides containing aldose groups and desialylated glycopeptides containing aldose groups; In particular, the sialylated glycopeptide containing aldose groups and the desialylated glycopeptide containing aldose groups have structures selected from the following, respectively: or The aldose-containing substrate A is an aldose-containing glycoprotein. Preferably, the aldose-containing glycoprotein is an antibody; Specifically, the antibody is IgG with a conserved N-glycosylation site at N297 in the Fc region; Specifically, the antibody is IgG1, IgG2 or IgG4; Specifically, the antibody is a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a trispecific antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody from different species (such as mice, rats, sheep, rabbits, humans, etc.); Specifically, the targets of the antibody include HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276 (B7H3), HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG (5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R; Preferably, the antibody is selected from trastuzumab; Specifically, the glycoprotein containing aldehyde group sugar is sialic acid complex glycoform containing aldehyde group-trastuzumab, which has the following structure: Among them, trastuzumab is linked to the sialic acid complex glycoform containing aldehyde group as shown above through the Ash297 position of the Fc domain.

5. The sugar conjugation method according to any one of claims 1-4, characterized in that, In substrate B, the Ar ring is a C6-C10 aromatic ring or a 5-10 membered heteroaromatic ring, such as phenyl, indolyl, pyrrolyl; Preferably, the substrate B has a structure selected from the following: wherein R 1 is one or more optionally substituted substituents selected from the following: hydroxyl group, p is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, especially 4 to 7, such as 5; R 2 selected from H, a carboxyl group, -C(=O)NH2, and a peptide segment of 3 to 20 amino acids; More preferably, the substrate B is a substance selected from the following group: Tryptophan, dopa, dopamine, 3-hydroxy phenethylamine, N-aminoethyl pyrrole; Polypeptides or proteins with tryptophan, dopamine or N-aminoethyl pyrrole at the N-terminus, and Derivatives obtained by substituting the aforementioned substances with derivatizing groups; Specifically, the substrate B is selected from: tryptophan, N-aminoethyl pyrrole, N-aminoethyl pyrrole derivatives, polypeptides with tryptophan at the N-terminus; Specifically, the N-aminoethyl pyrrole derivatives have a structure selected from the following: Specifically, the polypeptides with tryptophan at the N-terminus are selected from the polypeptides of the following sequences: WPNF (SEQ ID NO: 1, 2a); WDRVYIHPFHL (SEQ ID NO: 2, 2b); WDRVYIHPF (SEQ ID NO: 3, 2c); WRVYIHPF (SEQ ID NO: 4, 2d); WVYIHPF (SEQ ID NO: 5, 2e); WYGGFM (SEQ ID NO: 6, 2f); WYAFHLMD (SEQ ID NO: 7, 2g); WYAFEVVG (SEQ ID NO: 8, 2h); WYaFDVVG (SEQ ID NO: 9, 2i); WKLRLEWNR (SEQ ID NO: 10, 2j); WLTVSPWY (SEQ ID NO: 11, 2k); WKKLKKLFSKLWNWK (SEQ ID NO: 12, 21).

6. The sugar conjugation method according to any one of claims 1-5, characterized in that, The substrate A containing aldehyde group sugar and the substrate B react in a solvent, and the solvent is selected from the following group: aqueous phase system, formic acid, acetic acid, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, tetrahydrofuran, methanol or a combination thereof; Preferably, the aqueous phase system includes: water, aqueous phase buffer; The aqueous phase buffer is: phosphate buffer, 2-morpholinoethanesulfonic acid buffer, piperazine-N, N'-bis(2-ethanesulfonic acid) buffer, sodium acetate buffer, N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane buffer, preferably phosphate buffer; and / or The pH of the reaction system is 4.0 - 10.0, preferably 6.0 - 8.0, such as 7.0; The reaction temperature is 4 - 100 °C, preferably 30 - 40 °C, such as 37 °C; The reaction time is 0.1 - 72 h, preferably 1 - 24 h, such as 1 - 15 h, 1 - 12 h, or 1 - 5 h; The molar ratio of the substrate A to the substrate B is 1:100 to 100:1, preferably 1:20 to 20:1, such as 10 - 20:1 or 1:10 - 20.

7. The sugar conjugation method according to any one of claims 1-5, characterized in that, The sugar conjugation method is selected from one of the following methods: Method 1: The substrate A containing aldehyde group sugar reacts with tryptophan as the substrate B-1 or a polypeptide or protein containing N-terminal tryptophan in an aqueous phase system to undergo a Pictet-Spengler reaction to generate a sugar conjugate C-1; The reaction formula is as follows: Method 2: The substrate A containing aldehyde group sugar reacts with N-aminoethylpyrrole or its derivative as the substrate B-2 in an aqueous phase system to undergo a Pictet-Spengler reaction to generate a sugar conjugate C-2; The reaction formula is as follows: Among them, R1, R2, the aqueous phase system, the reaction temperature, the reaction pH, and the molar ratio of substrates A and B are defined in the same manner as those defined in claims 1-6.

8. The sugar conjugate obtained by the sugar conjugation method according to any one of claims 1 - 7, Specifically, the sugar conjugate product is selected from compounds having the following structures: Particularly, the sugar conjugate is the antimicrobial peptide 3r1 having the following structure: Particularly, the sugar conjugate is azide-tagged sialic acid complex glycoform-trastuzumab, which has the following structure: Among them, The antibody is linked to the aldehyde group-containing sialic acid complex glycoform through the Asn297 position of the Fc domain.

9. Use of the following compound in the preparation of an antibacterial drug: Particularly, the use of the compound in the preparation of drugs against the following Gram-positive bacteria: MRSA-USA300, Efm-HS-0649, Van (resistant), 06188, and the following Gram-negative bacteria: AB1157 (E. coli), Aba., P. Aeruginosa PAO1, Klebsiella pneumoniate.

10. Use of the sugar conjugation method according to any one of claims 1 - 7 in sugar or glycopeptide modification and the preparation of antibody-drug conjugates.

11. A method for preparing a trastuzumab-MMAE conjugate, the reaction formula is as follows: (1) (2) The reaction includes the following steps: (1) The aldehyde group-containing sialic acid complex glycoform-trastuzumab reacts with 4a to undergo a Pictet-Spengler reaction to obtain azide-tagged sialic acid complex glycoform-trastuzumab; (2) The azide-tagged sialic acid complex glycoform-trastuzumab undergoes a click chemical reaction with BCN-PEG4-VC-PAB-MMAE to obtain a sugar-site specific antibody-drug conjugate, The structure of the BCN-PEG4-VC-PAB-MMAE is: Preferably, the Pictet-Spengler reaction is carried out in a phosphate buffer, the pH of the reaction system is 6.0 - 8.0, such as 7.0; the reaction temperature is 0 - 40 °C, such as 37 °C; the reaction time is 0.5 - 24 h, such as 1 - 5 h, such as 2 h; the dosage of 4a is 10 - 20 equivalents of the aldehyde group-containing antibody; Preferably, the click chemistry reaction is carried out in a phosphate buffer solution, the pH of the reaction system is 6.0 - 8.0, such as 7.0; the reaction temperature is 0 - 40 °C, such as 37 °C; the reaction time is 0.5 - 24 h, such as 1 - 5 h, such as 2 h; the dosage of BCN-PEG4-VC-PAB-MMAE is 10 - 20 equivalents of the sugar-modified antibody.

12. The trastuzumab-MMAE conjugate prepared by the method according to claim 11, having the following structure:

13. Use of the trastuzumab-MMAE conjugate according to claim 12 in the preparation of an anti-tumor drug, in particular, the tumor is HER2-positive breast cancer.

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