CN115521348A

CN115521348A - Sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or salt thereof, glycoconjugate and preparation method thereof

Info

Publication number: CN115521348A
Application number: CN202211248083.7A
Authority: CN
Inventors: 叶新山; 霍常鑫; 郑秀静; 许成豪
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2022-12-27
Also published as: WO2024078578A1

Abstract

The invention belongs to the technical field of oligosaccharide and glycoconjugate thereof, and particularly relates to sialic acid (alpha- (2 → 6)) A) -D-galactopyranose derivative or a salt thereof, a glycoconjugate and a process for the preparation thereof. The invention provides a nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative or a salt thereof, wherein the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative has a structure shown in a formula 1. Mouse experiments show that the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-galactosamine derivative or the salt thereof can be coupled with carrier protein or polypeptide through different linkers to obtain glycoprotein (glycopeptide) conjugate, so that more effective immunoreaction is generated, and the tumor cells expressing STn can be specifically identified, thereby achieving the effect of resisting tumors; provides a new framework structure for the research and development of the anti-tumor saccharide vaccine and is expected to promote the development of the anti-tumor saccharide vaccine.

Description

Sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or salt thereof, glycoconjugate and preparation method thereof

Technical Field

The invention belongs to the technical field of oligosaccharides and glycoconjugates thereof, and particularly relates to sialic acid (alpha- (2 → 6)) -D-galactopyranose derivatives or salts thereof, glycoconjugates and preparation methods thereof.

Background

The study of saccharide antigen-based anti-tumor vaccines is a promising direction for current tumor immunotherapy. The carbohydrate antigen STn is a disaccharide structure containing sialic acid, is mostly expressed in human breast cancer, colorectal cancer, ovarian cancer and prostate cancer, and is rarely expressed in normal tissues (Holmberg, L.Expertrev.vaccines 2004,3, 655-663.), thereby becoming an important target for tumor immunotherapy. The company Biomira, canada, developed an STn-KLH (keyhole limpet hemocyanin) conjugate on the basis of this

Vaccine for the prevention and treatment of colorectal cancer and breast cancer metastasis. But found alone when subjected to phase III clinical trials

It does not improve the time to disease progression and overall survival, but shows some effect only when combined with hormones, increasing survival from 5.8 months to 8.3 months with hormones applied alone. Due to the fact that

The antitumor activity of (a) is dependent on the presence of hormones, so that the antitumor activity thereof is affected (Holmberg, l.expertrev. Vaccines 2004,3, 655-663.).

Is composed of

Similar difficulties are encountered with current anti-tumor saccharide vaccines, where the main problem is that the vaccine is unable to generate an effective immune response in vivo.

Disclosure of Invention

The invention aims to provide a sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or a salt thereof, a glycoconjugate and a preparation method thereof, wherein the glycoconjugate (glycoantigen) obtained by coupling the sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or the salt thereof with protein through different linkers can generate more effective immune response, generate more antibodies and show good activity in the aspect of anti-tumor vaccines; thereby prolonging the life cycle and achieving the anti-tumor effect.

The present invention provides a nitrogen-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative or a salt thereof, having a structure represented by formula 1:

in the formula 1, R ₁ Is an amide group or-NH ₂ (ii) a The amido is-NHC (O) CH _p Cl _q 、-NHC(O)CH _p F _q 、-NHC(O)CH _p Br _q 、-NHC(O)H、-NHC(O)C _a H _2a+1 、-NHC(O)C _a H _2a OH、-NHC(O)C _b H _2b-1 or-NHC (O) C _b H _2b-3 (ii) a Wherein p or q are independently 0, 1, 2or 3, and p + q =3; a is any integer of 1 to 20; b is any integer of 2 to 20;

R ₂ is a compound with double bonds, acetylene bonds, azido groups, aldehyde groups, protective acetal groups, maleimide groups and N-hydroxysuccinimidyl groupsAmino, mercapto, protected mercapto, seleno, protected seleno, -NH ₂ or-ONH ₂ A substituent of (3).

Preferably, said R ₁ is-NHC (O) CH _p F _q or-NHC (O) C _a H _2a+1 (ii) a Said R is ₂ Is allyloxy.

Preferably, the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative has any one of the structures represented by formulas 1-1 to 1-5:

preferably, the salt of the N-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative is a salt formed by reacting the N-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative having the structure represented by formula 1 with a base.

The invention provides a preparation method of the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative in the technical scheme,

the R is ₁ is-NHC (O) CH ₃ The method comprises the following steps:

mixing a glycosyl acceptor with a structure shown in a formula 2-1, a glycosyl donor with a structure shown in a formula 3, a coupling reagent and a polar solvent to carry out glycosylation coupling reaction to obtain a coupling product with a structure shown in a formula 4-1;

mixing the coupling product with the structure shown in the formula 4-1, a polar solvent and an acidic catalytic reagent, and performing debenzylation protection to obtain a debenzylation coupling product with the structure shown in the formula 5-1;

mixing the product of debenzylation fork coupling with the structure shown in the formula 5-1, a polar solvent and an alkaline catalytic reagent, and carrying out selective deacetylation to obtain the derivative of N-linked sialic acid (alpha- (2 → 6)) -D-galactosamine pyran;

the R is ₁ is-NH ₂ The method comprises the following steps:

mixing a glycosyl acceptor with a structure shown in a formula 2-2, a glycosyl donor with a structure shown in a formula 3, a coupling reagent and a polar solvent to carry out glycosylation coupling reaction to obtain a coupling product with a structure shown in a formula 4-2;

mixing the coupling product with the structure shown in the formula 4-2, a polar solvent and an acidic catalytic reagent, and performing debenzylation protection to obtain a debenzylation coupling product with the structure shown in the formula 5-2;

mixing the debenzylation fork coupling product with the structure shown in the formula 5-2, a polar solvent and an alkaline catalytic reagent, and performing selective deacetylation to obtain a selective deacetylation coupling product with the structure shown in the formula 6;

mixing the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent and an organic base in a protective gas atmosphere to perform the protection of the deacetylation, so as to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranopyranosyl galactose derivative;

the R is ₁ Is except-NHC (O) CH ₃ When other amide groups are present, the following steps are included:

mixing the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent, an organic base and an acylation reagent to perform a deacetylation protection and acylation reaction to obtain the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative; the acylating agent is R ₁ The corresponding anhydride, carboxylic acid or carboxylic acid ester.

The invention provides a glycoconjugate, which is obtained by coupling the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative or the salt thereof in the technical scheme or the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative or the salt thereof prepared by the preparation method in the technical scheme with polypeptide or carrier protein through different linkers.

The invention provides a preparation method of glycoconjugate in the technical scheme, which comprises the following steps:

dissolving the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or the salt thereof in a polar solvent, introducing oxidizing gas for oxidation reaction or introducing N-hydroxysuccinimide group by extending carbon chain to obtain disaccharide containing aldehyde group or N-hydroxysuccinimide group;

and mixing the disaccharide containing aldehyde groups or the N-hydroxysuccinimide groups, protein or polypeptide, a reducing agent and a buffer solution, and carrying out coupling reaction to obtain the glycoconjugate.

The invention provides application of the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative or the salt thereof prepared by the preparation method in the technical scheme in preparation of antitumor drugs.

The invention provides application of the glycoconjugate in the technical scheme or the glycoconjugate prepared by the preparation method in the technical scheme in preparation of antitumor drugs.

The invention provides a vaccine for treating tumor, which comprises the glycoconjugate in the technical scheme or the glycoconjugate prepared by the preparation method in the technical scheme and a pharmaceutically acceptable carrier or auxiliary material.

The present invention provides a nitrogen-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative having a structure represented by formula 1 or a salt thereof. The sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative with the structure shown in formula 1 or the salt thereof provided by the invention has the advantages that nitrogen bridge (N (OMe)) connection is used in the structure of the compound to replace oxygen bridge (O) connection in a disaccharide antigen structure, the structure is novel, and the compound has good activity in the aspect of antitumor vaccines. Mouse experiments show that the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative with the structure shown in formula 1 or the salt thereof can be coupled with carrier protein or polypeptide to obtain a glycoprotein (glycopeptide) conjugate, and a vaccine taking the glycoprotein (glycopeptide) conjugate as a glycoantigen can generate more effective immune response, generate more specific antibodies and specifically recognize STn-expressing tumor cells, so that the effect of resisting tumors is achieved; further, compared with the structure linked by an oxygen bridge (O), the antibody titer for recognizing STn obtained by the vaccine prepared by the saccharide antigen prepared by the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyrano galactose derivative with the structure shown in formula 1 or the salt thereof provided by the invention is obviously improved, and the antibody titer for recognizing STn obtained by the vaccine prepared by the saccharide antigen prepared by the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyrano galactose derivative with the structure shown in formula 1 or the salt thereof is 4812 and the antibody titer for recognizing STn obtained by the vaccine prepared by the saccharide antigen prepared by the hapten with the structure linked by the oxygen bridge (O) is 1458, which are measured 13 days after the third immunization; 13 days after the fourth immunization, the STn-recognizing antibody titer of the vaccine obtained from the saccharide antigen prepared from the nitrogen-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative having the structure represented by formula 1 of the present invention or the salt thereof was 89288, and the STn-recognizing antibody titer of the vaccine obtained from the saccharide antigen prepared from the hapten having an oxygen bridge (O) -linked structure was 5716. Furthermore, the vaccine obtained from the saccharide antigen prepared from the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactosamine derivative having the structure represented by formula 1 or the salt thereof provided by the present invention can significantly prolong the survival time of mice in a tumor-bearing mouse model test, and as can be seen from the results of fig. 6, the vaccine obtained from the saccharide antigen prepared from the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactosamine derivative having the structure represented by formula 1 or the salt thereof provided by the present invention has a survival time of 115 days in a tumor-bearing mouse model test. Is suitable for preparing anticancer drugs for breast cancer, colorectal cancer, ovarian cancer, prostate cancer and the like. On the other hand, the compound is a novel compound, provides a novel framework structure for the research and development of the anti-tumor saccharide vaccine, and is expected to promote the development of the anti-tumor saccharide vaccine.

The invention provides a glycoconjugate, which is obtained by coupling the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-galactosamine derivative or the salt thereof in the technical scheme or the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-galactosamine derivative or the salt thereof prepared by the preparation method in the technical scheme with polypeptide or protein. Compared with the structure galactose derivatives connected with an oxygen bridge (O), the glycoconjugate provided by the invention is used as an anti-tumor vaccine to generate strong immune response in mice, and

compared with the prior art, the titer of the antibody generated by the glycoconjugate is increased by 3 to 15 times, and the survival time of the tumor-bearing mice after vaccination is obviously prolonged. The antibody titer and the survival time of the mice are obviously improved, and the development of the anti-tumor sugar vaccine is expected to be promoted.

Drawings

FIG. 1 is a scheme showing the synthesis of a sialic acid (. Alpha. - (2 → 6)) -D-aminopyranosyl galactose derivative having a structure represented by the formula 1-1 in example 1 of the present invention;

FIG. 2 is a scheme showing the synthesis of sialic acid (α - (2 → 6)) -D-galactopyranose derivatives having the structures represented by formulas 1-2 to 1-5 according to examples 2 to 5 of the present invention;

FIG. 3 is a graph showing the serum titers of STn-KLH and 1-KLH group per mouse after the fourth immunization with 1-KLH prepared in example 6 of the present invention;

FIG. 4 is a mouse survival curve following administration of 1-KLH prepared in example 6 of the present invention;

FIG. 5 is a graph of tumor growth in mice after administration of 1-CRM197 prepared in example 7 of the invention;

FIG. 6 is a mouse survival curve following administration of 1-CRM197 prepared in example 7 of the present invention;

FIG. 7 is a mouse tumor growth curve following NSTn-NHS-CRM197 administration, prepared in example 8 of the present invention;

FIG. 8 is a scheme showing a synthesis scheme of a glycosyl acceptor having the structure represented by formula 7 in example 1 of the present invention;

FIG. 9 is a scheme showing the synthesis of a glycosyl donor having the structure shown in formula 3 in the example of the present invention;

FIG. 10 is a scheme showing the synthesis of a glycoprotein conjugate in an example of the present invention;

FIG. 11 is a scheme showing the synthesis of glycoprotein conjugate 1-NHS-CRM197 according to the example of the present invention.

Detailed Description

R ₂ is a compound with double bonds, acetylene bonds, azido groups, aldehyde groups, protected acetal groups, maleimide groups, N-hydroxysuccinimide groups, mercapto groups, protected mercapto groups, seleno groups, protected seleno groups, -NH ₂ or-ONH ₂ A substituent of (1).

In the present invention, the protective acetal group is a substituent having a protective group on the acetal group, and the protective thiol group is a substituent having a protective group on a thiol group; the selenium-protecting group is a substituent group with a protecting group on selenium. The present invention has no particular requirement on the kind of the protecting group.

In the present invention, said R ₁ preferably-NHC (O) CH _p F _q or-NHC (O) C _a H _2a+1 (ii) a The R is ₂ Allyloxy is preferred.

In a specific embodiment of the present invention, the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative has any one of the structures represented by formulae 1-1 to 1-5:

in the present invention, the salt of the N-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative is a salt produced by reacting the N-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative having a structure represented by formula 1 with a base.

The invention provides a preparation method of the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-galactopyranose derivative in the technical scheme,

said R is ₁ is-NHC (O) CH ₃ The method comprises the following steps:

mixing a debenzylation fork coupling product with a structure shown as a formula 5-1, a polar solvent and an alkaline catalytic reagent, and performing selective deacetylation to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyrano galactose derivative;

the R is ₁ is-NH ₂ The method comprises the following steps:

mixing the coupling product with the structure shown in the formula 4-2, a polar solvent and an acidic catalytic reagent, and carrying out debenzylation protection to obtain a debenzylation coupling product with the structure shown in the formula 5-2;

mixing the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent and an organic base in a protective gas atmosphere to perform the deacetylation protection to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative;

mixing the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent, an organic base and an acylation reagent to carry out the protection of the deacetylation and the acylation reaction to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative; the acylating agent is R ₁ Corresponding acid anhydrides, carboxylic acids or carboxylic acid esters.

In the present invention, all the preparation starting materials/components are commercially available products well known to those skilled in the art, unless otherwise specified.

In the present invention, said R ₁ is-NHC (O) CH ₃ The method for preparing the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-galactopyranose derivative comprises the following steps:

mixing the coupling product with the structure shown in the formula 4-1, a polar solvent and an acidic catalytic reagent, and carrying out debenzylation protection to obtain a debenzylation coupling product with the structure shown in the formula 5-1;

mixing the product of debenzylation fork coupling shown in the formula 5-1, a polar solvent and an alkaline catalytic reagent, and carrying out selective deacetylation to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-galactosamine pyranose derivative.

The present invention provides a coupled product having a structure represented by formula 4-1, which is obtained by mixing a glycosyl acceptor having a structure represented by formula 2-1, a glycosyl donor having a structure represented by formula 3, a coupling reagent (hereinafter referred to as a first coupling reagent) and a polar solvent to perform a glycosylation coupling reaction (hereinafter referred to as a glycosylation coupling reaction).

In the present invention, the glycosyl acceptor with the structure shown in the formula 2-1 is particularly preferably the glycosyl acceptor with the structure shown in the formula 2-1-1;

in the present invention, the method for preparing the glycosyl acceptor having the structure represented by formula 2-1-1 preferably comprises the steps of:

mixing the structural compound shown as the formula 7, camphorsulfonic acid and a polar solvent for benzylidene protection at the 3,4 position to obtain the structural compound shown as the formula 8(ii) a Mixing a structural compound shown as a formula 8, tetramethylpiperidine oxide (TEMPO), iodobenzene diacetic acid (BAIB) and a polar solvent for 6-site selective oxidation to obtain a structural compound shown as a formula 9; in a protective gas atmosphere, a compound with a structure shown in a formula 9 and NaCNBH ₃ Mixing with polar solvent to perform double bond reduction reaction to obtain glycosyl acceptor with the structure shown in formula 2-1-1.

According to the invention, a structural compound shown in a formula 7, camphorsulfonic acid and a polar solvent are mixed for 3,4-position benzylidene protection to obtain a structural compound shown in a formula 8. In the present invention, the method for preparing the compound having the structure represented by formula 7 preferably comprises the steps of: dissolving a compound (galactosamine hydrochloride) with a structure shown in a formula 10, acetic anhydride and carbonate type strong-base resin in a mixed solvent of methanol and water, and reacting under the ice-water bath condition to obtain a compound with the structure shown in a formula 11; mixing a compound with a structure shown in a formula 11, allyl alcohol and an ethanol solution of boron trifluoride, adding an ethanol solution of HCl, and reacting under a reflux condition to obtain a compound with a structure shown in a formula 7;

in a specific embodiment of the present invention, the preparation method of the compound having the structure represented by formula 11 is specifically preferably: commercially available galactosamine hydrochloride represented by formula 10 (2.5 g,11.6 mmol) and 5.0g of carbonate type strongly basic resin, 58mL of water, 6mL of methanol were mixed, stirred in ice bath, and 1.5mL of acetic anhydride was added dropwise. After 2h, the resin was filtered off with suction and washed. Concentrating the mother liquor, passing through a strong acid resin column, evaporating to dryness to obtain the compound shown in formula 11, and directly performing the next reaction without purification. The resulting compound of formula 11 and 0.32mL BF ₃ 、Et ₂ O was added to 28mL of allyl alcohol and stirred at reflux for 2h. Then add 0.5mL HCl in Et ₂ And continuously refluxing in the O solution for 1h. Cooling, adding diethyl ether untilTurbidity appeared and was left overnight at 4 ℃. Filtering, washing with ether to obtain white solid 0.9g, which is the compound of formula 7, and the yield of the two steps is 30%.

In the present invention, the molar ratio of the structural compound represented by the formula 7 to camphorsulfonic acid is preferably 3.8. The polar solvent is preferably dimethyl phthalate (DMP, alpha-dimethylpropane), and the dosage of the polar solvent is not particularly required, so that the 3, 4-benzylidene protection can be successfully carried out. The reaction temperature of the 3,4 benzylidene protection is preferably room temperature, and the reaction incubation time is preferably 22h. After the 3,4 benzylidene protection reaction, a 3,4 benzylidene protection reaction solution is obtained, and in the invention, the 3,4 benzylidene protection reaction solution is preferably subjected to post-treatment to obtain the compound with the structure shown in the formula 8. The post-treatment preferably comprises: mixing the 3,4-position benzylidene protection reaction solution and a saturated sodium bicarbonate water solution to obtain a mixed solution; mixing and extracting the mixed solution and an organic solvent to obtain an extracted organic phase; drying the combined extracted organic phases and concentrating to obtain a concentrated solution; and carrying out column chromatography separation on the concentrated solution to obtain the compound shown in the formula 8. The organic solvent is particularly preferably dichloromethane. The drying agent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating an organic extract phase obtained by solid-liquid separation, and the solid-liquid separation is particularly preferably performed by filtration. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 2.

After the compound with the structure shown in the formula 8 is obtained, the compound with the structure shown in the formula 8, tetramethyl piperidine oxide (TEMPO), iodobenzene diacetic acid (BAIB) and a polar solvent are mixed for 6-site selective oxidation, and the compound with the structure shown in the formula 9 is obtained. In the present invention, the molar ratio of the structural compound represented by formula 8, TEMPO and BAIB is preferably 0.61. The polar solvent is preferably dichloromethane, and the dosage of the polar solvent has no special requirement, so that the 6-site selective oxidation reaction is ensured to be smoothly carried out. The temperature of the 6-site selective oxidation reaction is preferably room temperature, and the holding time of the 6-site selective oxidation reaction is preferably 3 hours. In the present invention, the 6-position selective oxidation reaction produces a 6-position selective oxidation reaction solution, and in the present invention, the 6-position selective oxidation reaction solution is preferably subjected to post-treatment to produce a compound having a structure represented by formula 9. The post-treatment preferably comprises: mixing the 6-site selective oxidation reaction solution with a saturated sodium bicarbonate water solution to obtain a mixed solution; mixing and extracting the mixed solution and an organic solvent to obtain an extracted organic phase; drying the combined extracted organic phases, and removing the solvent to obtain a residue; and carrying out column chromatography separation on the residue to obtain the compound shown in the formula 9. The organic solvent is particularly preferably dichloromethane. The drying agent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating an organic extract phase obtained by solid-liquid separation, and the solid-liquid separation is particularly preferably performed by filtration. The desolventizing agent is preferably evaporated. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is preferably 1.

After obtaining the compound with the structure shown in the formula 9, the invention uses the compound with the structure shown in the formula 9 and NaCNBH in a protective gas atmosphere ₃ Mixing with polar solvent to carry out double bond reduction reaction to obtain glycosyl acceptor with the structure shown in formula 2-1-1. In the present invention, a compound having a structure represented by formula 9 and NaCNBH ₃ Preferably 2.14. The polar solvent is preferably a mixed solvent of acetic acid and methanol, and the volume ratio of the acetic acid to the methanol is preferably 1:1, the invention has no special requirements on the dosage of the polar solvent, and the smooth progress of the double bond reduction reaction is ensured. The temperature of the double bond reduction reaction is preferably 0 ℃, the heat preservation time of the double bond reduction reaction is preferably 4 hours, and the protective gas is preferably argon. In the invention, the double bond reduction reaction is carried out to obtain a double bond reduction reaction solution, and the double bond reduction reaction solution is preferably subjected to post-treatment to obtain the glycosyl acceptor with the structure shown in the formula 2-1-1. The post-treatment preferably comprises: mixing the double bond reduction reaction solution and an organic solvent to obtain a mixed solution; removing the solvent from the mixed solution to obtain a residue; performing column chromatography separation on the residue to obtain glycosyl acceptor with the structure shown in 2-1-1And (3) a body. The organic solvent is particularly preferably toluene. The desolvation is preferably carried out by evaporation. The eluent used for the column chromatography separation is preferably ethyl acetate.

In the present invention, when R2 is a substituent of another structure in the glycosyl acceptor having a structure represented by formula 2-1, the preparation method is the same as the preparation method when R2 is an allyloxy group, and details are not repeated herein.

In the present invention, the method for preparing the glycosyl donor represented by formula 3 preferably comprises the steps of:

dissolving a compound having a structure represented by formula 12 in acetonitrile, adding DIPEA and (EtO) ₂ And (3) reacting PCl at 0-25 ℃ in Ar atmosphere to obtain the glycosyl donor with the structure shown in the formula 3.

In a specific embodiment of the present invention, the specific method for preparing the glycosyl donor having the structure represented by formula 3 is preferably: under the protection of nitrogen, dissolving a compound (1.1g, 2.20mmol) with a structure shown in formula 12 in 20mL of acetonitrile solution, adding 0.94mL of DIPEA, stirring under ice-bath cooling, adding diethyl phosphorosol chloride (0.65mL, 4.50mmol), removing the ice bath after 5min, monitoring the reaction by TLC, evaporating the reaction system to dryness, adding ethyl acetate, performing suction filtration, evaporating the filtrate, washing the filtrate with the evaporated filtrate with the ethyl acetate twice, performing column chromatography separation, and eluting with petroleum ether (V/V): ethyl acetate =1:2, 1.1g of glycosyl donor with the structure shown in the formula 3 is obtained, and the yield is 89%.

In the present invention, the first coupling reagent is particularly preferably

MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

In the present invention, the molar ratio of the glycosyl acceptor having the structure represented by formula 2-1 to the glycosyl donor having the structure represented by formula 3 is preferably 0.061. A glycosyl acceptor having a structure represented by formula 2-1 and

the mass ratio of MS is preferably 20. The polar solvent is preferably dichloromethane, and the dosage of the polar solvent is not specially required, so that the first glycosylation coupling reaction is ensured to be smoothly carried out. The molar ratio of glycosyl acceptor of the structure represented by formula 2-1 to TMSOTf is preferably 0.061. The mixing for performing the first glycosylation coupling reaction comprises: subjecting a glycosyl acceptor having a structure represented by formula 2-1, a glycosyl donor having a structure represented by formula 3, and

dissolving MS in a polar solvent, stirring, mixing and reacting for 1h to obtain a mixed solution; and cooling the mixed solution to 0 ℃ and mixing with TMSOTf. According to the invention, after the glycosyl donor with the structure shown in the formula 3 is detected to be completely reacted by adopting TLC, the first glycosylation coupling reaction is finished. In the present invention, a first glycosylation coupling reaction solution is obtained after the first glycosylation coupling reaction, and the present invention preferably performs post-treatment on the first glycosylation coupling reaction solution to obtain a coupled product with a structure shown in formula 4-1. In the present invention, the post-treatment preferably includes: adding triethylamine into the first glycosylation coupling reaction liquid for extraction and quenching reaction, and heating the obtained quenching reaction liquid to room temperature; leaching the quenched reaction liquid with kieselguhr to obtain a filtrate; removing the solvent from the filtrate to obtain a residue; and carrying out column chromatography separation on the residue to obtain the coupling product with the structure shown in the formula 4-1. The desolventizing agent is preferably evaporated. The column chromatography separation preferably sequentially adopts a first elution solvent and a second elution solvent for elution; the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 1; the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene to methanol is preferably 10.

After the coupling product with the structure shown in the formula 4-1 is obtained, the coupling product with the structure shown in the formula 4-1, a polar solvent and an acidic catalytic reagent are mixed for debenzylation protection, and the debenzylation coupling product with the structure shown in the formula 5-1 is obtained. In the present invention, the acidic catalytic agent is preferably pyridine p-toluenesulfonate (PPTS). The mass ratio of the coupling product having the structure represented by formula 4-1 to the acidic catalytic agent is preferably 100. The polar solvent is preferably methanol, and the dosage of the polar solvent has no special requirement, so that the benzylidene protection reaction can be smoothly carried out. The temperature of the debenzylation fork protection reaction is preferably 65 ℃, and the heat preservation time of the debenzylation fork protection reaction is preferably 3 hours. In the invention, the debenzylation fork protection reaction liquid is obtained after the debenzylation fork protection, and the debenzylation fork protection reaction liquid is preferably subjected to post-treatment to obtain the debenzylation fork coupling product with the structure shown in the formula 5-1. In the present invention, the post-treatment preferably comprises: removing the solvent from the debenzylation fork protection reaction liquid to obtain a residue; and carrying out column chromatography separation on the residue to obtain the debenzylation fork coupling product with the structure shown in the formula 5-1. The desolventizing agent is preferably evaporated. The elution solvent used for the column chromatography separation is preferably ethyl acetate and methanol, and the volume ratio of ethyl acetate to methanol is preferably 15.

In the present invention, the benzylidene coupling product having the structure represented by the formula 5-1 is particularly preferably a benzylidene coupling product having the structure represented by the formula 5-1-1

After obtaining the debenzylation fork coupling product with the structure shown in the formula 5-1, mixing the debenzylation fork coupling product with the structure shown in the formula 5-1, a polar solvent and an alkaline catalytic reagent, and carrying out selective deacetylation to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyralactopyranose derivative. In the present invention, the basic catalytic agent is preferably sodium methoxide and an aqueous sodium hydroxide solution, and the molar concentration of the aqueous sodium hydroxide solution is preferably 1mol/L. The polar solvent is preferably methanol, and the dosage of the methanol is not particularly required, so that the selective deacetylation can be smoothly carried out. In the present invention, the mixing for the selective deacetylation reaction comprises the following steps: mixing a benzylidene coupling product with a structure shown as a formula 5-1, a polar solvent and sodium methoxide to carry out a first-step reaction; obtaining a reaction solution; mixing the reaction solution and an aqueous solution of sodium hydroxide for a second step reaction to obtain a selective deacetylation reaction solution; the first-step reaction is preferably detected by TLC, and the time of the first-step reaction is preferably 30min; the second reaction is preferably detected by TLC, and the time of the second reaction is preferably 4h. In the present invention, the selective deacetylation reaction is followed by obtaining a selective deacetylation reaction solution, and in the present invention, it is preferable to carry out a post-treatment of the selective deacetylation reaction solution to obtain the N-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative. In the present invention, the post-treatment preferably comprises: introducing carbon dioxide into the selective deacetylation reaction liquid to neutrality, and removing the solvent from the obtained neutral reaction liquid to obtain a residue; subjecting the residue to column chromatography to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-galactopyranose derivative. The desolventizing agent is preferably evaporated. The column chromatography separation preferably adopts reversed phase column chromatography separation. The elution solvent adopted by the column chromatography is preferably pure water and methanol, and the volume ratio of the pure water to the methanol is preferably 1.

In the present invention, said R ₁ is-NH ₂ The method for preparing the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-galactopyranose derivative comprises the following steps:

mixing a glycosyl acceptor with a structure shown in a formula 2-2, a glycosyl donor with a structure shown in a formula 3, a coupling reagent (hereinafter referred to as a second coupling reagent) and a polar solvent to carry out glycosylation coupling reaction (hereinafter referred to as a second glycosylation coupling reaction) to obtain a coupling product with a structure shown in a formula 4-2;

mixing the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent and an organic base in a protective gas atmosphere to perform the deacetylation protection to obtain the N-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative.

The invention mixes the glycosyl acceptor with the structure shown in the formula 2-2, the glycosyl donor with the structure shown in the formula 3, a coupling reagent and a polar solvent for glycosylation coupling reaction to obtain a coupling product with the structure shown in the formula 4-2.

In the present invention, the glycosyl acceptor with the structure shown in the formula 2-2 is particularly preferably the glycosyl acceptor with the structure shown in the formula 2-2-1;

in the present invention, the method for preparing the glycosyl acceptor having the structure represented by formula 2-2-1 preferably comprises the steps of: mixing a compound with a structure shown in a formula 10, a polar solvent, triethylamine and methyl trifluoroacetate to perform 2-position trifluoroethyl reaction to obtain a reaction solution containing a 2-position trifluoroethyl substituted reaction product; concentrating the reaction liquid containing the reaction product substituted by the 2-position trifluoroethyl to obtain a concentrated liquid containing the reaction product substituted by the 2-position trifluoroethyl; the mass of the compound having the structure represented by formula 10 and the volume ratio of triethylamine are preferably 11.6 mmol; the molar ratio of the compound having the structure represented by formula 10 to the methyl trifluoroacetate is preferably 11.6; the polar solvent is preferably methanol, and the invention has no special requirement on the dosage of the polar solvent and ensures that the trifluoroethyl reaction on the 2-position is smoothly carried out. The temperature of the trifluoroethyl group in the 2-position is preferably room temperature, the incubation time of the trifluoroethyl group in the 2-position is preferably overnight, and the trifluoroethyl group in the 2-position is preferably carried out under stirring. The concentration is preferably concentration under reduced pressure.

Mixing the concentrated solution containing the 2-position trifluoroethyl substituted reaction product, allyl alcohol and ethyl ether hydrochloride to perform 1-position allyl reaction to obtain a reaction solution containing the 1-position allyl substituted reaction product; carrying out solid-liquid separation on the reaction liquid containing the reaction product substituted by the 1-position allyl, and concentrating the obtained filtrate to obtain a concentrated solution containing the reaction product substituted by the 1-position allyl; the molar concentration of the ethyl ether hydrochloride is preferably 3mol/L; the ratio of the amount of substance of the compound having the structure represented by formula 10 to the volume of the diethyl ether hydrochloride is preferably 11.6 mol; the ratio of the amount of substance of the compound having the structure represented by formula 10 to the volume of the allyl alcohol is preferably 11.6 mol; the allyl group at the 1-position is preferably carried out under reflux, and the time of reflux is preferably 0.5h. The solid-liquid separation is preferably filtration; the concentration is preferably concentration under reduced pressure.

Mixing concentrated solution containing 1-site allyl substituted reaction product, polar solvent and tert-butyldimethylsilyl chloride (TBDMSCL) to perform 6-site TBDM protection reaction to obtain a compound with a structure shown in a formula 13; the molar ratio of the compound having the structure represented by formula 10 to the TBDMSCL is preferably 11.6. The polar solvent is preferably pyridine, and the dosage of the polar solvent has no special requirement, so that the 6-bit TBDM protection reaction can be smoothly carried out. The temperature of the 6-bit TBDM protection reaction is preferably room temperature, the heat preservation time of the 6-bit TBDM protection reaction is preferably 16h, and the 6-bit TBDM protection reaction is preferably carried out under the condition of stirring. And (3) obtaining 6-bit TBDM protective reaction liquid after the 6-bit TBDM protective reaction, preferably carrying out post-treatment on the 6-bit TBDM protective reaction liquid to obtain the compound with the structure shown in the formula 13. In the present invention, the post-treatment preferably comprises: concentrating the 6-bit TBDM protective reaction solution to obtain a concentrated solution; mixing the concentrated solution with an extracting agent for extraction to obtain an extracted organic phase, drying the extracted organic phase and concentrating to obtain a concentrated solution; and carrying out column chromatography separation on the concentrated solution to obtain the compound with the structure shown in the formula 13. The concentration is preferably concentration under reduced pressure. The extractant is particularly preferably dichloromethane and saturated aqueous sodium bicarbonate solution. The drying agent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating an organic extract phase obtained by solid-liquid separation, and the solid-liquid separation is particularly preferably performed by filtration. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 4;

after obtaining the compound with the structure shown in the formula 13, a polar solvent and camphorsulfonic acid are mixed, and the benzylidene protection at the 3,4 position is carried out to obtain the compound with the structure shown in the formula 14. In the present invention, the molar ratio of the structural compound represented by formula 13 to camphorsulfonic acid is preferably 4.43. The polar solvent is preferably acetonitrile and dimethyl phthalate (DMP, alpha-dimethyl phthalate), and the dosage of the polar solvent is not particularly required, so that the 3, 4-position benzylidene protection can be successfully carried out. The reaction temperature of the 3,4 benzylidene protection is preferably room temperature, and the reaction incubation time is preferably 15min. After the 3,4 benzylidene protection reaction, a 3,4 benzylidene protection reaction solution is obtained, and in the invention, the 3,4 benzylidene protection reaction solution is preferably subjected to post-treatment to obtain the compound with the structure shown in formula 14. The post-treatment preferably comprises: mixing and extracting 3,4-benzylidene protection reaction liquid and an extracting agent to obtain an extracted organic phase; drying the extracted organic phase and concentrating to obtain a concentrated solution; and carrying out column chromatography separation on the concentrated solution to obtain the compound with the structure shown in the formula 14. The extracting agent is particularly preferably dichloromethane and saturated brine. The drying agent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating an organic extract phase obtained by solid-liquid separation, and the solid-liquid separation is particularly preferably performed by filtration. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 20 to 10;

after the compound with the structure shown in the formula 14 is obtained, the compound with the structure shown in the formula 14, a polar solvent, acetic acid and tetrabutylammonium fluoride trihydrate are mixed, and a selective TBDMS removal reaction is carried out to obtain the compound with the structure shown in the formula 15. In the present invention, the molar ratio of the structural compound represented by formula 14 to acetic acid is preferably 0.12. The molar ratio of the structural compound represented by the formula 14 to tetrabutylammonium fluoride trihydrate is preferably 0.12. The polar solvent is preferably tetrahydrofuran, and the invention has no special requirement on the dosage of the polar solvent and ensures that the selective TBDMS removal reaction is smoothly carried out. The mixed selective TBDMS removal reaction preferably comprises the steps of: mixing a compound with a structure shown in a formula 14, a polar solvent and acetic acid to obtain a mixed solution; and in the protective gas atmosphere, cooling the mixed solution to 0 ℃, mixing with tetrabutylammonium fluoride trihydrate, and carrying out the selective TBDMS removal reaction. The protective gas is preferably argon. The reaction temperature of the selective TBDMS removal reaction is preferably 0 ℃, and the reaction heat preservation time is preferably 4h. The selective TBDMS removal reaction liquid is obtained after the selective TBDMS removal reaction, and the selective TBDMS removal reaction liquid is preferably subjected to post-treatment to obtain the compound with the structure shown in the formula 15. The post-treatment preferably comprises: concentrating the reaction solution subjected to selective TBDMS removal, and then mixing and extracting the reaction solution with an extracting agent to obtain an extracted organic phase; drying the extracted organic phase and concentrating to obtain a concentrated solution; and carrying out column chromatography separation on the concentrated solution to obtain the compound with the structure shown in the formula 15. The concentration is preferably to concentrate the reaction solution for selectively removing TBDMS until the volume is reduced by half. The extracting agent is particularly preferably dichloromethane and saturated saline. The drying agent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating an organic extract phase obtained by solid-liquid separation, and the solid-liquid separation is particularly preferably performed by filtration. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 2;

after the compound with the structure shown in the formula 15 is obtained, the compound with the structure shown in the formula 15, a polar solvent, TEMPO and BAIB are mixed, and N-methoxyl reaction is carried out on the 6-position, so that the compound with the structure shown in the formula 16 is obtained. In the present invention, the molar ratio of the structural compound represented by formula 15, TEMPO and BAIB is preferably 1.92. The polar solvent is preferably dichloromethane, and the invention has no special requirement on the dosage of the polar solvent and ensures that the N-methoxyl reaction on the 6-position is smoothly carried out. The reaction temperature of the N-methoxyl on the 6 position is preferably 40 ℃, and the heat preservation time of the reaction of the N-methoxyl on the 6 position is preferably 6h. The 6-position N-methoxyl reaction liquid is obtained after the 6-position N-methoxyl reaction, and the invention preferably carries out post treatment on the 6-position N-methoxyl reaction liquid to obtain the compound with the structure shown in the formula 16. The post-treatment preferably comprises: diluting the 6-position N-methoxyl group with an organic solvent to obtain a diluted reaction solution; stirring and mixing the diluted reaction solution and a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate for 10min to obtain a mixed solution; mixing and extracting the mixed solution and an extracting agent to obtain an extracted organic phase; drying the extracted organic phase, and removing the solvent to obtain a residue; and carrying out column chromatography separation on the residue to obtain the compound with the structure shown in the formula 16. The organic solvent is preferably dichloromethane. The extractant is particularly preferably dichloromethane. The drying agent is preferably anhydrous sodium sulfate. The drying preferably removes the solvent from the organic extract phase of the solid-liquid separation, which is preferably evaporated. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 10;

after the compound with the structure shown in the formula 16 is obtained, the compound with the structure shown in the formula 16 and NaCNBH are added in a protective gas atmosphere ₃ Mixing with polar solvent to carry out double bond reduction reaction to obtain glycosyl acceptor with the structure shown in formula 2-2-1. In the present invention, a compound having a structure represented by formula 16 and NaCNBH ₃ The molar ratio of (c) is preferably 0.73. The polar solvent is preferably a mixed solvent of acetic acid and methanol, and the volume ratio of the acetic acid to the methanol is preferably 1:1, the invention has no special requirements on the dosage of the polar solvent, and ensures the reduction reaction of the double bondsThe process is carried out smoothly. The temperature of the double bond reduction reaction is preferably 0 ℃, the heat preservation time of the double bond reduction reaction is preferably 4h, and the protective gas is preferably argon. In the invention, the double bond reduction reaction is carried out to obtain a double bond reduction reaction solution, and the double bond reduction reaction solution is preferably subjected to post-treatment to obtain the glycosyl acceptor with the structure shown in the formula 2-2-1. The post-treatment preferably comprises: mixing the double bond reduction reaction solution and an organic solvent to obtain a mixed solution; removing the solvent from the mixed solution to obtain a residue; and carrying out column chromatography separation on the residue to obtain the glycosyl acceptor with the structure shown in 2-2-1. The organic solvent is particularly preferably toluene. The desolvation is preferably carried out by evaporation. The eluent used for the column chromatography separation is preferably petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is preferably 2.

In the present invention, when R2 is a substituent of another structure in the glycosyl acceptor having a structure represented by formula 2-2, the preparation method is the same as the preparation method when R2 is an allyloxy group, and details are not repeated here.

In the present invention, the second coupling reagent is particularly preferably

MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

In the present invention, the molar ratio of the glycosyl acceptor having a structure represented by formula 2-2 to the glycosyl donor having a structure represented by formula 3 is preferably 0.98. A glycosyl acceptor having a structure represented by formula 2-2 and

the mass ratio of MS is preferably 378. The polar solvent is preferably dichloromethane, and the dosage of the polar solvent is not particularly required, so that the second glycosylation coupling reaction is ensured to be smoothly carried out. The molar ratio of glycosyl acceptor having the structure represented by formula 2-2 to TMSOTf is preferably 0.98. The mixing for performing a second glycosylation coupling reaction comprises: in a protective gas atmosphere, a glycosyl acceptor with a structure shown in formula 2-2, a glycosyl donor with a structure shown in formula 3 and

dissolving MS in a polar solvent, stirring, mixing and reacting for 1h to obtain a mixed solution; and cooling the mixed solution to 0 ℃ and mixing with TMSOTf. According to the invention, after the second glycosyl donor with the structure shown in the formula 3 is completely reacted, the second glycosylation coupling reaction is finished by adopting TLC detection. In the invention, a second glycosylation coupling reaction solution is obtained after the second glycosylation coupling reaction, and the glycosylation coupling reaction solution is preferably subjected to post-treatment to obtain a coupling product with a structure shown in a formula 4-2. In the present invention, the post-treatment preferably comprises: adding triethylamine into the second glycosylation coupling reaction liquid for extraction and quenching reaction, and heating the obtained quenching reaction liquid to room temperature; leaching the quenched reaction liquid with kieselguhr to obtain a filtrate; removing the solvent from the filtrate to obtain a residue; and carrying out column chromatography separation on the residue to obtain the coupling product with the structure shown in the formula 4-2. The desolvation is preferably carried out by evaporation. The eluent for the column chromatographic separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 1.

Preferably, the first elution solvent and the second elution solvent are adopted for elution in sequence; the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 1; the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene to methanol is preferably 10.

And mixing the coupling product with the structure shown in the formula 4-2, a polar solvent and an acidic catalytic reagent for debenzylation protection to obtain the debenzylation coupling product with the structure shown in the formula 5-2. In the present invention, the acidic catalytic agent is preferably pyridine p-toluenesulfonate (PPTS). The polar solvent is preferably methanol, and the dosage of the polar solvent has no special requirement, so that the benzylidene protection reaction can be smoothly carried out. The temperature of the debenzylation fork protection reaction is preferably 65 ℃, and the heat preservation time of the debenzylation fork protection reaction is preferably 3 hours. In the invention, the debenzylation fork protection reaction liquid is obtained after the debenzylation fork protection, and the debenzylation fork protection reaction liquid is preferably subjected to post-treatment to obtain the debenzylation fork coupling product with the structure shown in the formula 5-2. In the present invention, the post-treatment preferably comprises: removing the solvent from the debenzylation fork protection reaction liquid to obtain a residue; and carrying out column chromatography separation on the residue to obtain the debenzylation fork coupling product with the structure shown in the formula 5-2. The desolvation is preferably carried out by evaporation. The elution solvent adopted by the column chromatography separation is preferably ethyl acetate and methanol, and the volume ratio of the ethyl acetate to the methanol is preferably 15. The column chromatography separation preferably sequentially adopts a first elution solvent and a second elution solvent for elution; the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of the petroleum ether to the acetone is preferably 1; the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene to methanol is preferably 10.

In the present invention, the debenzylation coupling product of the structure represented by formula 5-2 is particularly preferably a debenzylation coupling product of the structure represented by formula 5-2-1:

after obtaining the debenzylation fork coupling product with the structure shown in the formula 5-2, mixing the debenzylation fork coupling product with the structure shown in the formula 5-2, a polar solvent and an alkaline catalytic reagent, and performing selective deacetylation to obtain the selective deacetylation coupling product with the structure shown in the formula 6. In the present invention, the alkaline catalytic agent is preferably sodium methoxide and sodium hydroxide aqueous solution, the sodium methoxide is preferably added in the form of sodium methoxide solution, and the mass percentage content of the sodium methoxide solution is preferably 30%; the molar concentration of the aqueous sodium hydroxide solution is preferably 2mol/L. The polar solvent is preferably methanol, and the invention has no special requirement on the using amount of the methanol and ensures that the selective deacetylation is smoothly carried out. In the present invention, the mixing for the selective deacetylation reaction comprises the following steps: mixing a benzylidene coupling product with a structure shown in a formula 5-2, a polar solvent and sodium methoxide to carry out a first-step reaction; obtaining a reaction solution; the ratio of the amount of substance of the benzylidene coupling product having the structure represented by formula 5-2 to the volume of the sodium methoxide solution is preferably 0.024mmol:0.02mL; mixing the reaction solution and a sodium hydroxide aqueous solution to perform a second-step reaction to obtain a selective deacetylation reaction solution; the first-step reaction is preferably detected by TLC, and the time of the first-step reaction is preferably 1h; the second-step reaction is preferably detected by TLC, and the time of the second-step reaction is preferably 0.5h. In the present invention, after the selective deacetylation reaction, a selective deacetylation reaction solution is obtained, and in the present invention, it is preferable to perform post-treatment on the selective deacetylation reaction solution to obtain the selective deacetylation coupling product having the structure represented by formula 6. In the present invention, the post-treatment preferably comprises: and (3) introducing carbon dioxide into the selective deacetylation reaction liquid until the reaction liquid is neutral, and removing the solvent from the obtained neutral reaction liquid to obtain the selective deacetylation coupling product with the structure shown in the formula 6. The desolventizing agent is preferably evaporated.

After the selective deacetylation coupling product with the structure shown in the formula 6 is obtained, the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent and an organic base are mixed in a protective gas atmosphere to carry out the protection of the deacetylation, and the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyralactose derivative is obtained. In the present invention, the organic base is particularly preferably triethylamine. The polar solvent is preferably methanol and the shielding gas is preferably argon. In the present invention, the protection by the dehydrotrifluoroacetyl group is preferably carried out under reflux, and the incubation time for the protection by the dehydrotrifluoroacetyl group is preferably overnight. In the present invention, it is preferable that the N-linked sialic acid (. Alpha. - (2 → 6)) -D-aminopyranosyl galactose derivative is obtained by subjecting the protection reaction solution after the protection reaction to a post-treatment. The post-treatment preferably comprises: removing the solvent from the protection reaction solution to obtain a residue; carrying out reverse phase column chromatography on the residue to obtain an eluent; subjecting the eluate to ion exchange to remove organic salts, thereby obtaining the N-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative. In the present invention, the desolvation is preferably performed by evaporation. The eluent adopted by the reverse phase column chromatography is preferably pure water to methanol, and the volume ratio of the pure water to the methanol is preferably 1. The ion exchange is preferably carried out using an ion exchange resin column.

In the present invention, said R ₁ Is except-NHC (O) CH ₃ When other amide group is present, the method for producing the N-linked sialic acid (. Alpha. - (2 → 6)) -D-aminopyranosyl galactose derivative comprises the steps of:

In the present invention, the acid anhydride preferably includes acetic anhydride, propionic anhydride, n-butyric anhydride, isobutyric anhydride or n-caproic anhydride;

the carboxylic acid preferably comprises monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, monochloroacetic acid or dichloroacetic acid;

the carboxylic acid ester preferably comprises methyl monofluoroacetate, methyl difluoroacetate, methyl trifluoroacetate or methyl dichloroacetate.

In the present invention, the acylating agent is particularly preferably methyl fluoroacetate, methyl difluoroacetate or mL methyl trifluoroacetate.

In the present invention, the dehydrotrifluoroacetyl protection and acylation reaction is preferably carried out under reflux conditions, and the incubation time for the dehydrotrifluoroacetyl protection and acylation reaction is preferably overnight. In the present invention, it is preferable that the acylation reaction solution obtained by the above-mentioned dehydrotrifluoroacetyl protection and acylation reaction is subjected to a post-treatment to obtain the N-linked sialic acid (. Alpha. - (2 → 6)) -D-aminopyranosyl galactose derivative. The post-treatment preferably comprises: removing the solvent from the acylation reaction liquid to obtain a residue; carrying out reversed-phase column chromatography on the residue to obtain an eluent; and (3) carrying out ion exchange on the eluent to remove organic salts to obtain the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative. In the present invention, the desolvation is preferably performed by evaporation. The eluent adopted by the reverse phase column chromatography is preferably pure water to methanol, the volume ratio of the pure water to the methanol is preferably 1.

In the present invention, the carrier protein is preferably Bovine Serum Albumin (BSA), hemocyanin (KLH) or CRM197.

dissolving the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyralactose derivative or the salt thereof in a polar solvent, introducing oxidizing gas to perform oxidation reaction or introducing N-hydroxysuccinimide groups by extending carbon chains to obtain disaccharide containing aldehyde groups or disaccharide containing N-hydroxysuccinimide groups;

and mixing the disaccharide containing aldehyde groups or N-hydroxysuccinimide groups, protein or polypeptide, a reducing agent and a buffer solution, and carrying out coupling reaction to obtain the glycoconjugate.

The invention dissolves the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or the salt thereof in a polar solvent, and introduces oxidizing gas for oxidation reaction to obtain disaccharide containing aldehyde group. In the present invention, the polar solvent is preferably anhydrous methanol. The oxidizing gas is preferably air containing ozone. The temperature of the oxidation reaction is preferably-72 ℃; carrying out the oxidation reaction for 30min to obtain an oxidation reaction solution which is a blue solution; the introduction of the oxidizing gas is stopped. In the present invention, the oxidation reaction solution is obtained after the oxidation reaction, and the oxidation reaction solution is preferably post-treated to obtain the disaccharide containing the aldehyde group. The post-treatment preferably comprises: introducing nitrogen into the oxidation reaction liquid to remove unreacted oxidizing gas, and then heating to room temperature; and removing the solvent from the oxidation reaction solution to obtain the disaccharide containing aldehyde group. The desolventizing is preferably vacuum desolventizing.

After the disaccharide containing aldehyde groups is obtained, the disaccharide containing aldehyde groups, protein or polypeptide, a reducing agent and a buffer solution are mixed for coupling reaction to obtain the glycoconjugate.

In the present invention, the reducing agent is preferably sodium cyanoborohydride. The pH of the buffer solution is preferably 7.6.

In the present invention, the temperature of the coupling reaction is preferably room temperature, and the incubation time of the coupling reaction is preferably 24 hours. The coupling reaction is preferably carried out under exclusion of light.

In the present invention, a coupling reaction solution is obtained after the coupling reaction, and the present invention preferably performs post-treatment on the coupling reaction solution to obtain the glycoconjugate.

In the present invention, the antitumor drug preferably includes a therapeutic vaccine or a prophylactic vaccine.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

FIG. 8 is a scheme for synthesizing a compound of formula 7:

commercially available galactosamine hydrochloride represented by formula 10 (2.5 g,11.6 mmol) and 5.0g of a carbonate type strongly basic resin, 58mL of water, 6mL of methanol were mixed, stirred in ice bath, and 1.5mL of acetic anhydride was added dropwise. After 2h, the resin was filtered off with suction and washed. Concentrating the mother liquor, passing through a strong acid resin column, evaporating to dryness to obtain the compound shown in formula 11, and directly performing the next reaction without purification. The resulting compound of formula 11 and 0.32mL BF ₃ 、Et ₂ O was added to 28mL of allyl alcohol and stirred under reflux for 2h. Then add 0.5mL HCl in Et ₂ And continuously refluxing in the O solution for 1h. After cooling, diethyl ether was added until turbidity appeared and left overnight at 4 ℃. Filtering, washing with ether to obtain white solid 0.9g, which is the compound with the structure shown in formula 7, and the yield of the two-step reaction is 30%.

FIG. 9 shows a scheme for synthesizing glycosyl donors having the structure shown in formula 3:

under the protection of nitrogen, dissolving a compound (1.1g, 2.20mmol) with a structure shown in a formula 12 in 20mL acetonitrile solution, adding 0.94mL DIPEA, stirring under the cooling of an ice bath, adding diethylphosphorous oxychloride (0.65mL, 4.50mmol), removing the ice bath after 5min, monitoring the reaction by TLC, evaporating the reaction system to dryness after the reaction is completely monitored, adding ethyl acetate, performing suction filtration, carrying the filtrate, evaporating to dryness, carrying out ethyl acetate twice, performing column chromatography separation, and eluting an eluent (V/V) petroleum ether: ethyl acetate =1:2, 1.1g of glycosyl donor with the structure shown in the formula 3 is obtained, and the yield is 89%.

A synthetic scheme of a sialic acid (. Alpha. - (2 → 6)) -D-galactopyranose derivative having a structure represented by formula 1-1 shown in FIG. 1:

a compound having a structure represented by formula 7 (compound 6,1.0g,3.8mmol in FIG. 1) was dissolved in DMP (. Alpha.,. Alpha. -dimethoxypropane,24.3mL,198.5 mmol), camphorsulfonic acid (54.5 mg, 0.23mmol) was added, and the mixture was stirred at room temperature for 22 hours. The reaction mixture was poured into saturated aqueous sodium bicarbonate solution and then extracted with dichloromethane; the extracts were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether: acetone, 2: 1) to give 940mg of a compound having the structure represented by formula 8 (compound 7 in fig. 1) as a white solid in a yield of 82%.

¹ H NMR(300MHz,CDCl ₃ ):δ5.89-5.83(1H,m),5.54(1H,d,J＝9.3Hz),5.31-5.21(2H,m),4.85(1H,d,J＝3.6Hz),4.31(1H,dt,J＝3.3Hz,9.3Hz),4.20-4.18(1H,m),4.15(1H,td,J＝5.1Hz),4.12-3.95(4H,m),3.89-3.82(1H,m),2.18(1H,dd,J＝3.3Hz,9.3Hz),2.04(3H,s),1.59(3H,s),1.35(3H,s)。

The compound of the structure described in formula 8 (183mg, 0.61mmol) was dissolved in 1mL of dichloromethane, TEMPO (9.1mg, 0.06mmol) and BAIB (215mg, 0.55mmol) were added, and stirring was carried out at room temperature for 3 hours. Diluted with 4mL of dichloromethane, 5.2mL of a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate was added and stirred vigorously for 10 minutes. Extracted with dichloromethane, dried over sodium sulfate and evaporated to dryness. The residue was dissolved in 2mL pyridine and MeONH was added ₂ HCl (76.1mg, 0.91mmol), stirred at room temperature for 1 hour, evaporated to dryness, and the residue was separated by column chromatography (petroleum ether: ethyl acetate, 1.

¹ H NMR(300MHz,CDCl ₃ )δ7.52(d,1H,J＝7.8Hz),6.87(d,1H,J＝4.8Hz),5.94-5.81(m,2H),5.57(d,2H,J＝9.3Hz),5.32-5.31(m,4H),5.09(dd,1H,J＝2.7Hz,4.8Hz),4.84(dd,2H,J＝3.3Hz,5.1Hz),4.55(dd,1H,J＝2.7Hz,7.2Hz),4.45(dd,1H,J＝3.0Hz,5.1Hz),4.33-4.30(m,2H),4.21-4.07(m,4H),4.00-3.89(m,7H),2.03(s,6H),1.59(s,3H),1.58(s,3H),1.33(s,6H)；HRMS(ESI)Anal.Calcd for C ₁₅ H ₂₅ N ₂ O ₆ [M+H] ⁺ :329.1707,found 329.1701。

Dissolving a compound (701mg, 2.14mmol) with a structure shown in formula 9 in an acetic acid/methanol (7 mL/7 mL) mixed solution, and adding NaCNBH under the protection of argon at 0 DEG C ₃ (201.9mg, 3.19mmol), stirring, after 4 hours the reaction is complete. Adding small amount of toluene, concentrating, evaporating to dryness, and collecting residueThe extract was separated by column chromatography (ethyl acetate) to give 637mg of a glycosyl acceptor (compound 9 in FIG. 1) having a structure represented by formula 2-1-1 as an oily substance with a yield of 90%.

¹ H NMR(300MHz,CDCl ₃ )δ5.96-5.83(m,2H),5.59(d,1H,J＝9.3Hz),3.28(dd,1H,J＝1.5Hz,17.1Hz),5.22(dd,1H,J＝1.2Hz,10.5Hz),4.82(d,1H,J＝3.3Hz),4.31-4.24(m,2H),4.19(dd,1H,J＝5.4Hz,12.9Hz),4.11-4.05(m,2H),3.99-3.93(dd,1H,J＝6.0Hz,12.6Hz),3.54(s,3H),3.28-3.15(m,2H),2.03(s,3H),1.57(s,3H),1.34(s,3H)； ¹³ CNMR(75MHz,CDCl ₃ )δ169.97,133.51,117.84,109.67,96.75,74.72,73.75,68.26,63.09,61.37,52.09,50.50,27.95,26.55,23.46；HRMS(ESI)Anal.Calcd for C ₁₅ H ₂₇ N ₂ O ₆ [M+H] ⁺ :331.1864,found 331.1865。

The glycosyl donor (55.5mg, 0.091mmol) with the structure shown in the formula 3, the glycosyl acceptor (20.0 mg, 0.061mmol) with the structure shown in the formula 2-1-1,

MS (100 mg) was dissolved in dry dichloromethane (1 mL) under nitrogen and stirred at room temperature for 1 hour. The reaction was cooled to 0 ℃ and TMSOTf (3.2. Mu.L, 0.018 mmol) was added. TLC shows that glycosyl donor with the structure shown in the formula 3 is basically completely reacted, and then a drop of triethylamine is added for extraction and quenching reaction. After the reaction system is heated to room temperature, the reaction system is filtered by suction through diatomite. The filtrate was evaporated to dryness. The residue was separated by column chromatography (petroleum ether: acetone, 1, followed by toluene: methanol, 10, 1) to give a compound having a structure represented by formula 4-1-4 (compound 11 in fig. 1) as an oil, 31.0mg, yield 63%.

¹ H NMR(500MHz,CDCl ₃ )δ5.91-5.83(m,1H),5.57(d,1H,J＝9.0Hz),5.38(dd,1H,J＝2.5Hz,7.0Hz),5.34(dt,1H,J＝1.5Hz,10.0Hz),5.27(dq,1H,J＝1.5Hz,17.0Hz),5.22-5.18(m,2H),4.84-4.79(m,2H),4.40(dd,1H,J＝2.5Hz,12.5Hz),4.30-4.25(m,2H),4.20-4.13(m,2H),4.12-4.01(m,3H),3.97(ddt,1H,J＝1.0Hz,6.5Hz,13.0Hz),3.81(s,3H),3.61(s,3H),3.27(dd,1H,J＝7.0Hz,14.5Hz),3.17(dd,1H,J＝6.5Hz,15.0Hz),2.60(dd,1H,J＝4.5Hz,10.0Hz),2.04(m,1H),2.14,2.12,2.04,2.04,2.03,1.89,1.57,1.36(s,8*3H)； ¹³ C NMR(125MHz,CDCl ₃ )δ170.90,170.69,170.24,170.07,170.04,170.00,167.64,133.52,117.55,109.37,96.92,94.89,74.44,73.00,69.82,69.66,68.29,67.93,65.11,63.96,62.39,53.12,52.70,50.37,49.33,35.11,29.63,28.06,26.55,23.42,23.13,21.00,20.82,20.75,20.68；HRMS(ESI)Anal.Calcd for C ₃₅ H ₅₄ N ₃ O ₁₈ [M+H] ⁺ :804.3397,found 804.3425。

The compound represented by the structure of formula 4-1-4 (compound 11 in fig. 1) (100mg, 0.124mmol) was dissolved in dried methanol (1 mL), PPTS (47 mg) was added to the reaction system, stirred at 65 ℃ for 3h, evaporated to dryness, and the residue was separated by column chromatography (ethyl acetate: methanol, 15, 1) to give a white solid as the compound represented by the structure of formula 5-1-1 (compound 12 in fig. 1), 92mg, yield 97%.

¹ H NMR(500MHz,CDCl ₃ )δ5.95(d,1H,J＝8.5Hz),5.95-5.86(m,1H),5.40-5.37(m,2H),5.35(dd,1H,J＝2.5Hz,7.5Hz),5.30(dd,1H,J＝1.5Hz,17Hz),5.23(dd,1H,J＝1.0Hz,15.5Hz),4.87-4.80(m,2H),4.38(dd,1H,J＝1.5Hz,12.5Hz),4.35-4.30(m,1H),4.23(dd,1H,J＝5.0Hz,13.0Hz),4.14-4.02(m,5H),4.00(dd,1H,J＝6.0Hz,13.0Hz),3.90(t,1H,J＝6.0Hz),3.80(s,3H),3.80-3.77(m,1H),3.59(s,3H),3.21(dd,1H,J＝7.5Hz,14.0Hz),3.12(dd,1H,J＝5.5Hz,14.5Hz),3.09(d,1H,J＝3.5Hz),2.59(dd,1H,J＝4.5Hz,12.5Hz),2.21(t,1H,J＝12.5Hz),2.14,2.14,2.07,2.04,2.04,1.88(s,6*3H)； ¹³ C NMR(100MHz,CDCl ₃ )δ172.39,170.94,170.79,170.30,170.26,170.21,167.94,133.54,117.75,96.51,94.76,72.94,71.21,69.73,69.48,68.51,68.16,67.90,67.80,63.92,62.52,52.89,52.83,50.68,49.39,34.95,23.32,23.15,21.08,20.84,20.81,20.77；HRMS(ESI)Anal.Calcd for C ₃₂ H ₅₀ N ₃ O ₁₈ [M+H] ⁺ :764.3084,found 764.3088。

A compound having a structure represented by formula 5-1-1 (Compound 12 in FIG. 1) (30mg, 0.039mmol) was dissolved in methanol (2 mL), stirred at room temperature, 30% sodium methoxide was added in one drop, and the reaction was complete after 30 minutes by TLC. After concentrating and draining the solvent, 1N NaOH aqueous solution (1.3 mL) was added. TLC showed completion of the reaction after 4 hours, passed through carbon dioxide gas to neutrality, and after evaporation to dryness the residue was chromatographed on a reverse phase column (pure water to methanol: water, 1.

¹ HNMR(400MHz,D ₂ O)δ5.88-5.80(m,1H),5.24(dq,1H,J＝1.6Hz,17.6Hz),5.15(dd,1H,J＝1.6Hz,10.8Hz),4.83(d,1H,J＝3.6Hz),4.13(ddt,1H,J＝1.2Hz,5.2Hz,13.2Hz),4.05(dd,1H,J＝4Hz,11.2Hz),4.02-3.94(m,3H),3.84(dd,1H,J＝3.2Hz,11.2Hz),3.78-3.72(m,2H),3.70-3.64(m,2H),3.57-3.45(m,6H),3.10(dd,1H,J＝6.8Hz,14.4Hz),2.98(dd,1H,J＝6.4Hz,14.4Hz),2.61(dd,1H,J＝4.4Hz,12.4Hz),1.93(s,3H),1.92(s,3H),1.80(t,1H,J＝12.4Hz)； ¹³ C NMR(100MHz,D ₂ O)δ174.94,174.60,173.11,133.63,118.03,96.56,95.66,72.88,71.92,69.14,68.79,68.70,68.63,68.38,67.76,64.01,62.62,52.94,51.83,49.87,37.70,22.03,21.92；HRMS(ESI)Anal.Calcd for C ₂₃ H ₄₀ N ₃ O ₁₄ [M+H] ⁺ :582.2505,found 582.2517。

Example 2

A synthetic scheme for sialic acid (. Alpha. - (2 → 6)) -D-galactopyranose derivatives of the structure shown in formula 1-2 is provided according to example 2 shown in FIG. 2:

the compound having the structure represented by formula 10 (D-galactosamine hydrochloride, 2.5g,11.6 mmol) was dissolved in 34.7mL of methanol, 4.1mL of triethylamine and methyl trifluoroacetate (1.5 mL,12.6 mmol) were added, and the mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was dissolved in 28.9mL of allyl alcohol, 18.1mL of 3M ethyl hydrochloride was added, and the mixture was refluxed for 0.5h. Filtering, and concentrating the filtrate. The residue was dissolved in 18.6mL of pyridine, TBDMSCl (1.9g, 12.6 mmol) was added, and the mixture was stirred at room temperature for 16h. After concentration under reduced pressure, extraction with dichloromethane and saturated sodium bicarbonate, drying of the organic phase with sodium sulfate, suction filtration, concentration of the filtrate, and separation of the residue by column chromatography (petroleum ether: ethyl acetate, 4.

¹ H NMR(400MHz,CDCl ₃ )δ6.53(d,1H,J＝8.9Hz),5.91-5.84(m,1H),5.30-5.24(m,2H),4.97(d,1H,J＝3.7Hz),4.41(td,1H,J＝3.6Hz,9.9Hz),4.19(dd,1H,J＝5.3Hz,12.9Hz),4.14(s,1H),4.01(dd,1H,J＝6.3Hz,12.9Hz),3.94(d,2H,J＝4.4Hz),3.82-3.71(m,2H),3.63(s,1H),2.72-2.70(m,1H),0.92(s,9H),0.12(s,6H)； ¹³ C NMR(100MHz,CDCl ₃ )δ158.01(q,J＝37.0Hz),133.10,118.28,115.79(q,J＝286.0Hz),96.11,69.84,69.84,69.69,68.43,63.81,51.08,25.76,18.20,-5.54,-5.57；HRMS(ESI)Anal.Calcd forC ₁₇ H ₃₄ N ₂ O ₆ F ₃ Si[M+NH ₄ ] ⁺ :447.2133,found447.2122。

Compound 13 (1.9g, 4.43mmol) was dissolved in 53.2mL of acetonitrile, and DMP (10.8mL, 88.2mmol) and camphorsulfonic acid (506mg, 2.22mmol) were added, and the mixture was stirred at room temperature for 15 minutes. Extraction was performed with dichloromethane and saturated brine, the organic phase was dried over sodium sulfate, suction filtration was performed, the filtrate was concentrated, and the residue was separated by column chromatography (petroleum ether: ethyl acetate, 20.

¹ H NMR(400MHz,CDCl ₃ )δ6.40(d,1H,J＝9.3Hz),5.90-5.80(m,1H),5.28-5.22(m,2H),4.83(d,1H,J＝3.3Hz),4.27-4.14(m,3H),4.11(dd,1H,J＝4.9Hz,8.8Hz),4.03(td,1H,J＝2.1Hz,6.5Hz),3.97(dd,1H,J＝6.4Hz,12.8Hz),3.89(dd,1H,J＝6.7Hz,10.0Hz),3.82(dd,1H,J＝6.6Hz,10.0Hz),1.55(s,3H),1.33(s,3H),0.90(s,9H),0.08(s,6H)； ¹³ C NMR(100MHz,CDCl ₃ )δ157.17(q,J＝37.0Hz),132.98,118.41,115.78(q,J＝286.2Hz),109.84,95.89,74.02,72.26,68.39,68.34,62.19,51.52,27.93,26.39,25.76,18.20,-5.42,-5.55；HRMS(ESI)Anal.Calcd for C ₂₀ H ₃₄ NO ₆ F ₃ SiK[M+K] ⁺ :508.1734,found508.1734。

Compound 14 (58mg, 0.12mmol) was dissolved in 3.2mL tetrahydrofuran, acetic acid (65.8. Mu.L, 1.25 mmol) was added, and tetrabutylammonium fluoride trihydrate (157.7 mg, 0.50mmol) was added at 0 ℃ under argon. After stirring at 50 ℃ for 4 hours, the system was concentrated to half the original volume, extracted with dichloromethane and saturated brine, the organic phase was dried over sodium sulfate, filtered with suction, the filtrate was concentrated, and the residue was separated by column chromatography (petroleum ether: ethyl acetate, 2.

¹ H NMR(400MHz,CDCl ₃ )δ6.50(d,1H,J＝8.9Hz),5.90-5.81(m,1H),5.30-5.23(m,2H),4.89(d,1H,J＝3.1Hz),4.29-4.14(m,4H),4.12-4.05(m,1H),4.03-3.94(m,2H),3.91-3.81(m,1H),2.32(dd,1H,J＝2.8Hz,8.8Hz),1.56(s,3H),1.34(s,3H)； ¹³ C NMR(100MHz,CDCl ₃ )δ157.28(q,J＝38.0Hz),132.83,118.59,115.76(q,J＝286.0Hz),110.29,96.10,74.03,73.31,68.66,67.87,62.52,51.38,27.89,26.47；HRMS(ESI)Anal.Calcd forC ₁₄ H ₂₀ NO ₆ F ₃ Na[M+Na] ⁺ :378.1135,found 378.1134。

Compound 15 (681mg, 1.92mmol) was dissolved in 3.2mL of dichloromethane, TEMPO (29mg, 0.19mmol) and BAIB (679mg, 2.11mmol) were added, and stirring was carried out at 40 ℃ for 6 hours. Diluted with 15mL of dichloromethane, 20mL of a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate was added and stirred vigorously for 10min. Extracted with dichloromethane, dried over sodium sulfate and evaporated to dryness. The residue was dissolved in 6.3mL pyridine and MeONH was added ₂ HCl (240mg, 2.85mmol), stirred at room temperature for 1 hour, evaporated to dryness, and the residue was separated by column chromatography (petroleum ether: ethyl acetate, 10.

¹ H NMR(400MHz,CDCl ₃ )δ7.52(d,2H,J＝7.3Hz),6.87(d,1H,J＝4.9Hz),6.32(d,3H,J＝8.9Hz),5.91-5.81(m,3H),5.36-5.22(m,6H),5.13(dd,1H,J＝2.7Hz,4.8Hz),4.90-4.88(m,3H),4.59(dd,2H,J＝2.4Hz,7.3Hz),4.48(dd,1H,J＝2.7Hz,4.9Hz),4.33-4.12(m,11H),4.05-3.97(m,3H),3.94(s,3H),3.91(s,6H),1.59(s,6H),1.58(s,3H),1.35(s,9H)；HRMS(ESI)Anal.Calcd for C ₁₅ H ₂₁ N ₂ O ₆ F ₃ K[M+K] ⁺ :421.0978,found421.0982。

Dissolving compound 16 (279mg, 0.73mmol) in glacial acetic acid/methanol (2.4 mL/2.4 mL) mixed solution, adding NaCNBH under argon protection at 0 DEG C ₃ (69mg, 1.09mmol) with stirringAfter stirring, the reaction was completed for 4 hours, a small amount of toluene was added, the reaction solution was evaporated to dryness, and the residue was separated by column chromatography (petroleum ether: ethyl acetate, 2: 1) to obtain 268mg of a glycosyl acceptor having the structure represented by formula 2-2-1 (compound 17 in fig. 2) as a white solid in a yield of 96%.

¹ H NMR(400MHz,CDCl ₃ )δ6.38(d,1H,J＝9.3Hz),5.99-5.75(m,2H),5.28-5.22(m,2H),4.84(d,1H,J＝3.4Hz),4.33-4.31(m,1H),4.25-4.17(m,2H),4.14-4.10(m,2H),3.97(dd,1H,J＝6.3Hz,12.7Hz),3.52(s,3H),3.25(dd,1H,J＝3.8Hz,14.1Hz),3.18(dd,1H,J＝8.9Hz,14.1Hz),1.55(s,3H),1.33(s,3H)； ¹³ C NMR(100MHz,CDCl ₃ )δ157.19(q,J＝37.1Hz),132.97,118.49,115.77(q,J＝286.3Hz),110.04,95.84,74.19,73.58,68.45,63.32,61.39,51.97,51.47,27.91,26.46；HRMS(ESI)Anal.Calcd for C ₁₅ H ₂₄ N ₂ O ₆ F ₃ [M+H] ⁺ :385.1581,found 385.1570。

A glycosyl donor (Compound 10 in FIG. 2) (1209mg, 1.98mmol) having a structure shown in formula 3, a glycosyl acceptor (378mg, 0.98mmol) having a structure shown in 2-2-1 and 1.6g

The molecular sieve was dissolved in 16.1mL of dichloromethane under nitrogen and stirred at room temperature for 1 hour. The reaction was cooled to 0 ℃ and TMSOTf (26. Mu.L, 0.15 mmol) was added. TLC showed that glycosyl acceptor (compound 17) with the structure shown in formula 2-2-1 was substantially completely reacted, and then one drop of triethylamine was added to kill the reaction. After the reaction system is heated to room temperature, the reaction system is filtered by suction through diatomite. The filtrate was evaporated to dryness. The residue was separated by column chromatography (petroleum ether: acetone, 1. The oil was dissolved in 7.9mL of methanol, PPTS (373mg, 1.49mmol) was added to the reaction system, stirred at 65 ℃ for 3 hours, evaporated to dryness, and the residue was separated by column chromatography (petroleum ether: acetone, 1.

¹ H NMR(400MHz,CDCl ₃ )δ6.74(d,1H,J＝9.0Hz),5.93-5.84(m,1H),5.46-5.21(m,5H),4.96(d,1H,J＝3.7Hz),4.92-4.81(m,1H),4.46-4.33(m,2H),4.25(dd,1H,J＝5.1Hz,13.0Hz),4.13-4.00(m,5H),3.97(t,1H,J＝5.8Hz),3.90-3.79(m,4H),3.61(s,3H),3.35(d,1H,J＝4.4Hz),3.21(d,2H,J＝5.9Hz),3.09(d,1H,J＝9.4Hz),2.64(dd,1H,J＝4.4Hz,12.6Hz),2.20(t,1H,J＝12.4Hz),2.16(s,3H),2.16(s,3H),2.06(s,3H),2.05(s,3H),1.90(s,3H)； ¹³ C NMR(100MHz,CDCl ₃ )δ170.97,170.94,170.47,170.41,170.20,168.07,157.89(q,J＝37.0Hz),133.22,118.13,115.82(q,J＝285.9Hz),95.97,94.53,72.89,69.60,69.39,69.23,68.57,68.48,67.68,63.48,62.62,52.98,52.91,51.13,49.42,34.84,23.09,21.11,20.81,20.79,20.73；HRMS(ESI)Anal.Calcd for C ₃₂ H ₄₇ N ₃ O ₁₈ F ₃ [M+H] ⁺ :818.2801,found 818.2808。

Compound 18 (20mg, 0.024mmol) was dissolved in 1.1mL of methanol, stirred at room temperature, and 0.02mL of 30% sodium methoxide was added, and completion of the reaction was indicated by TLC after 1 hour. After the solvent was concentrated and drained, 0.6mL of 2M aqueous sodium hydroxide solution was added, and after 0.5 hour, TLC showed completion of the reaction, carbon dioxide gas was introduced to the reaction mixture to neutrality, followed by evaporation to dryness. The residue was dissolved in 1.2mL of methanol, 0.5mL of triethylamine and 0.2mL of methyl fluoroacetate were added under argon protection, the mixture was refluxed and stirred overnight, and after evaporation, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1, 4), followed by removal of the residual triethylamine salt through an ion exchange resin column to give a white solid which is a nitrogen-linked sialic acid (α - (2 → 6)) -D-aminopyranopyranose derivative of the structure represented by formula 1-2, 11mg, in a yield of 75%.

¹ H NMR(400MHz,D ₂ O)δ5.93-5.85(m,1H),5.28(dd,1H,J＝1.2Hz,17.3Hz),5.19(d,1H,J＝10.5Hz),4.91(d,1H,J＝3.8Hz),4.87(d,1H,J＝46.4Hz),4.25-4.14(m,2H),4.07(t,1H,J＝6.2Hz),4.04-3.94(m,3H),3.83-3.67(m,4H),3.62-3.48(m,6H),3.16(dd,1H,J＝6.5Hz,14.2Hz),3.03(dd,1H,J＝5.9Hz,14.1Hz),2.66(dd,1H,J＝4.5Hz,12.3Hz),1.97(s,3H),1.85(t,1H,J＝12.0Hz)； ¹³ C NMR(100MHz,D ₂ O)δ174.96,173.12,171.09(d,J＝18.6Hz),133.61,118.14,96.49,95.70,79.77(d,J＝179.6Hz),72.92,71.97,69.18,68.81,68.75,68.67,68.49,67.63,64.05,62.66,52.96,51.87,49.60,37.74,22.06；HRMS(ESI)Anal.Calcd for C ₂₃ H ₃₈ N ₃ O ₁₄ FNa[M+Na] ⁺ :622.2230,found 622.2229。

Example 3

the compound represented by the formula 5-2-1 was obtained by following the preparation method in example 2;

a compound having a structure represented by formula 5-2-1 (compound 18 in FIG. 2) (20mg, 0.024mmol) was dissolved in 1.1mL of methanol. Stirring was carried out at room temperature, 0.02ml of 30% sodium methoxide was added, and the reaction was complete after 1 hour by TLC. After concentrating and draining the solvent, 0.6mL of 2M aqueous sodium hydroxide solution was added. After 0.5 hour TLC indicated complete reaction, carbon dioxide gas was added to neutral and evaporated to dryness. The residue was dissolved in 1.2mL of methanol, 0.5mL of triethylamine and 0.2mL of methyl difluoroacetate were added under an argon atmosphere, and the mixture was stirred under reflux overnight, after which the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1.

¹ H NMR(400MHz,D ₂ O)δ6.10(t,1H,J＝53.6Hz),5.87(ddd,1H,J＝5.8Hz,11.0Hz,22.4Hz),5.26(dd,1H,J＝1.5Hz,17.3Hz),5.18(d,1H,J＝10.5Hz),4.92(d,1H,J＝3.8Hz),4.22-4.13(m,2H),4.06(t,1H,J＝6.3Hz),4.03-3.94(m,3H),3.83-3.76(m,2H),3.76-3.66(m,2H),3.62-3.52(m,5H),3.50(dd,1H,J＝1.4Hz,8.9Hz),3.15(dd,1H,J＝6.4Hz,14.3Hz),3.02(dd,1H,J＝6.0Hz,14.4Hz),2.65(dd,1H,J＝4.5Hz,12.3Hz),1.95(s,3H),1.84(t,1H,J＝12.0Hz)； ¹³ C NMR(100MHz,D ₂ O)δ179.92,178.09,170.29(t,J＝25.7Hz),138.52,123.17,113.14(t,J＝245.6Hz),101.10,100.65,77.88,76.93,74.16,73.74,73.71,73.62,73.45,72.39,69.00,67.61,57.91,56.82,55.13,42.69,27.02；HRMS(ESI)Anal.Calcd for C ₂₃ H ₃₇ N ₃ O ₁₄ F ₂ Na[M+Na] ⁺ :640.2136,found 640.2139。

Example 4

A synthetic scheme for sialic acid (. Alpha. - (2 → 6)) -D-galactopyranose derivatives of the structures shown in formulas 1 to 3 is provided according to example 3 shown in FIG. 2:

a compound having a structure represented by formula 5-2-1 (compound 18 in FIG. 2) (40mg, 0.049mmol) was dissolved in 2.3mL of methanol. Stirring was carried out at room temperature, 0.02ml of 30% sodium methoxide was added, and the reaction was complete after 1 hour by TLC. After concentrating the solvent and draining it, 1.1mL of a 2M aqueous sodium hydroxide solution was added. After 0.5 hour TLC indicated complete reaction, carbon dioxide gas was added to neutral and evaporated to dryness. The residue was dissolved in 2.4mL of methanol, 1.0mL of triethylamine and 0.5mL of methyl trifluoroacetate were added under an argon atmosphere, the mixture was refluxed and stirred overnight, and after evaporation to dryness, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1.

¹ H NMR(400MHz,D ₂ O)δ5.94-5.80(m,1H),5.27(dd,1H,J＝1.5Hz,17.3Hz),5.19(d,1H,J＝10.4Hz),4.94(d,1H,J＝3.8Hz),4.23-4.14(m,2H),4.07(t,1H,J＝6.3Hz),4.04-3.97(m,3H),3.84-3.77(m,2H),3.77-3.67(m,2H),3.62-3.53(m,5H),3.51(dd,1H,J＝1.4Hz,8.9Hz),3.15(dd,1H,J＝6.4Hz,14.3Hz),3.03(dd,1H,J＝6.1Hz,14.3Hz),2.65(dd,1H,J＝4.5Hz,12.3Hz),1.96(s,3H),1.84(t,1H,J＝12.0Hz)； ¹³ C NMR(100MHz,D ₂ O)δ180.00,178.13,164.36(q,J＝37.5Hz),138.58,123.28,120.83(q,J＝284.3Hz),100.95,100.74,77.95,77.00,74.24,73.84,73.76,73.71,73.56,72.21,69.05,67.71,57.98,56.91,55.84,42.76,27.10；HRMS(ESI)Anal.Calcd for C ₂₃ H ₃₇ N ₃ O ₁₄ F ₃ [M+H] ⁺ :636.2222,found 636.2232。

Example 5

A synthetic scheme for sialic acid (. Alpha. - (2 → 6)) -D-galactopyranose derivatives of the structures shown in formulae 1 to 5 is provided according to example 5 shown in FIG. 2:

a compound having a structure represented by formula 5-2-1 (compound 18 in FIG. 2) (40mg, 0.049mmol) was dissolved in 2.3mL of methanol. Stirring was carried out at room temperature, 0.02ml of 30% sodium methoxide was added, and the reaction was complete after 1 hour by TLC. After the solvent was concentrated and drained, 1.1mL of 2M aqueous sodium hydroxide solution was added. After 0.5 hour TLC indicated complete reaction, carbon dioxide gas was added to neutral and evaporated to dryness. The residue was dissolved in 4.9mL of methanol, propionic anhydride (25.2. Mu.L, 0.20 mmol) was added at 0 ℃ and stirred for 0.5h. After evaporation to dryness, the residue was separated by reverse phase column chromatography (purified water to methanol: water, 1.

¹ H NMR(400MHz,D ₂ O)δ5.87(dq,1H,J＝5.8Hz,10.7Hz),5.27(d,1H,J＝17.3Hz),5.18(d,1H,J＝10.4Hz),4.87(d,1H,J＝3.6Hz),4.17(dd,1H,J＝5.0Hz,12.9Hz),4.09(dd,1H,J＝3.4Hz,11.1Hz),4.06-3.95(m,3H),3.88(dd,1H,J＝2.5Hz,11.2Hz),3.78-3.68(m,4H),3.62-3.48(m,6H),3.13(dd,1H,J＝6.1Hz,14.2Hz),3.02(dd,1H,J＝6.1Hz,14.3Hz),2.65(dd,1H,J＝4.2Hz,12.2Hz),2.23(q,2H,J＝7.6Hz),1.96(s,3H),1.83(t,1H,J＝11.9Hz),1.04(t,3H,J＝7.6Hz)； ¹³ C NMR(100MHz,D ₂ O)δ178.60,174.95,173.11,133.60,118.14,96.57,95.67,72.91,71.95,69.20,68.87,68.73,68.65,68.42,67.71,64.02,62.65,52.99,51.86,49.80,37.74,29.12,22.05,9.57；HRMS(ESI)Anal.Calcd for C ₂₄ H ₄₁ N ₃ O ₁₄ Na[M+Na] ⁺ :618.2481,found 618.2487。

Example 6

Prepared according to the glycoprotein conjugate synthesis scheme depicted in fig. 10:

nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivatives of the structures represented by the formulae 1-1 to 1-5 and STn of the structure represented by the formula 17 prepared in examples 1 to 5 were dissolved in 5mL of anhydrous methanol, and ozone-containing air was introduced at-72 ℃ until the system became blue (about 10 to 30 minutes), and then the introduction of ozone was stopped, and the system remained blue after 10 minutes. The reaction system was purged with nitrogen for about 10 minutes to remove excess ozone. 0.2mL of dimethyl sulfide is dripped, then the temperature of the reaction system is naturally raised to the room temperature, and after 2 hours, the solvent is removed from the reaction system under vacuum, thus obtaining the hapten containing aldehyde group.

And (3) dissolving the hapten containing aldehyde groups and KLH into a buffer solution with the pH value of 7.6, adding sodium cyanoborohydride, and reacting for 24 hours on a shaking table at room temperature in a dark place. After dialysis, glycoprotein conjugates N (OMe) -STn-KLH were obtained, which were designated as 1-KLH, 2-KLH,3-KLH,4-KLH, and 5-KLH, respectively.

Example 7

The preparation method is basically the same as that of example 6, except that: replacement of KLH in example 6 with CRM197 yielded glycoprotein conjugates N (OMe) -STn-KLH, noted 1-CRM197, 2-CRM197,3-CRM197,4-CRM197,5-CRM197, respectively.

Example 8

Prepared according to the synthetic scheme for glycoprotein conjugate 1-NHS-CRM197 depicted in figure 11:

10mg of the N-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative of the formula 1-5 prepared in example 5 and 2.88mg of mercaptoethylamine hydrochloride were dissolved in 1mL of deoxygenated deionized water, and after the reaction for 10min under ultraviolet irradiation at room temperature, the mixture was concentrated under reduced pressure, purified by using a dextran G10 gel column, and the product obtained by the purification was dissolved in 1mL of super-dry DMF, dropped into 1mL of super-dry DMF containing 57.8mg of bis (2, 5-dioxopyrrolidin-1-yl) adipate, and reacted at room temperature for 2 hours with vigorous stirring. After the reaction is finished, decompressing and concentrating, re-dissolving by methanol, and freeze-drying after HPLC separation to obtain a white solid product hapten N (OMe) -STn-NHS.

Dissolving N (OMe) -STn-NHS with CRM197 in K at pH =8.0 ₂ HPO ₄ In PBS buffer, on a shaker for 12 hours at room temperature, obtaining glycoprotein conjugate 1-NHS-CRM197 after ultrafiltration.

Application example

1. Test materials and sources

1. Test compounds: glycoprotein (polypeptide) conjugates prepared in examples 6, 7 and 8 of the present invention;

2. test method

(I) immunization of mice

6 Balb/c female mice per group, 6-8 weeks old (Number: SCXKjing2007-0001, SPF/VAF), purchased from department of animal sciences of the department of medicine of Beijing university and raised in the animal department. Mice were immunized with STn-KLH, STn-CRM197 and nitrogen-linked STn derivatives conjugated to KLH or CRM197 (1-KLH, 2-KLH,3-KLH,4-KLH,5-KLH and 1-CRM197, 2-CRM197,3-CRM197,4-CRM197,5-CRM 197), respectively, with glycoproteins (polypeptides) immunized each time containing 1-3 μ g of sugar (dissolved in PBS) once every 2 weeks, the route of immunization was intraperitoneal injection, for a total of 4 immunizations. Blood was collected before immunization, 13 days after 2 nd immunization, 13 days after 3 rd immunization and 14 days after 4 th immunization, and serum was separated and frozen in a refrigerator at-80 ℃ to be tested.

(II) determination of antibody titer in serum before and after immunization of mice

The titer of the pooled sera from each group of mice, as well as the titers of sera from each mouse of the 1-KLH, 2-KLH,3-KLH,4-KLH,5-KLH and 1-CRM197, 2-CRM197,3-CRM197,4-CRM197, and 5-CRM197 immunized groups prepared in examples 6 and 7, were determined by ELISA.

1 coating antigen: the plate was coated with 100. Mu.L of STn-BSA (containing 0.02. Mu.g of STn) overnight at 4 ℃.

2, washing and sealing: the plate was washed 3 times by adding 200. Mu.L of washing buffer PBS-Tween20 (0.05%) per well, and then 200. Mu.L of blocking solution (3% BSA-PBS), 37 ℃ for 1 hour per well.

3 plus primary antibody (i.e. immune serum): washing was performed 3 times (the same method as above). Serum was diluted in antibody dilutions (1% BSA-PBS) in a double ratio from one dilution, 100. Mu.L per well, 37 ℃ for 1 hour.

4 adding an enzyme-labeled secondary antibody: after washing 3 times, 100. Mu.L of a secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (. Gamma. -chain-specific)) diluted 5000-fold with an antibody diluent was added to each well at 37 ℃ for 1 hour.

5 color development: washing for 3 times, adding in-situ prepared chromogenic substrate o-phenylenediamine (OPD) 100 μ L per well, developing for 15min at room temperature in dark place, and adding 2M H per well ₂ SO ₄ The color development was terminated.

6, judging the result: OD was read with a microplate reader at 490nm wavelength. The antibody titer was determined as the dilution of the serum at an OD of 0.1 after subtracting the readings of the blank serum wells.

(III) immunotherapy in mice

Each group of 8 Balb/c female mice, 6-8 weeks old (Number: SCXKjing2007-0001, SPF/VAF), purchased from department of animal science of department of medicine of Beijing university and raised in the animal department. On day 0 mice were inoculated with 5X 10^5 CT26 cells in the axilla, and on

days

2, 6, 10, and 17, 1-NHS-CRM197, 1-CRM197, and 1-KLH solutions of PBS containing 1-3. Mu.g of sugar were injected subcutaneously, respectively.

3. Test results

The test results are shown in table 1 and fig. 3 to 6. Wherein, FIG. 3 shows the serum titers of STn-KLH and 1-KLH in each mouse after the fourth immunization with 1-KLH prepared in example 6 of the present invention; FIG. 4 is a mouse survival curve following administration of 1-KLH prepared in example 6 of the present invention; FIG. 5 is a graph of tumor growth in mice after administration of 1-CRM197 prepared in example 7 of the invention; FIG. 6 is the survival curve of mice after administration of 1-CRM197 prepared in example 7 of the invention, and FIG. 7 is the tumor growth curve of mice after administration of 1-NHS-CRM197 prepared in example 8 of the invention.

Table 1 shows the measurement of STn-recognizing antibody titers in mouse sera 13 days after the third and fourth immunizations of the glycoconjugates prepared in example 6

Table 2 compares the sugar loadings of 1-CRM197 prepared in example 7 and 1-NHS-CRM197 prepared in example 8.

TABLE 2 sugar loadings of 1-CRM197 and 1-NHS-CRM197

Constructing a mouse tumor-bearing model by using CT-26 colon cancer cells expressing STn carbohydrate antigen; tumor-bearing mice were then immunized with the glycoconjugates of the present invention (1-KLH, 1-CRM197 or 1-NHS-CRM 197) and observed for survival, tumor volume, and antibody titers. The experimental result shows that compared with a control group, the glycoconjugate 1-KLH, 1-CRM197 or 1-NHS-CRM197 inoculated mice obviously prolongs the survival period of the mice, inhibits the growth of tumors, increases the antibody titer and has good anti-tumor effect.

Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments belong to the protection scope of the present invention.

Claims

1. A nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative or a salt thereof, the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative having a structure represented by formula 1:

R ₂ is a compound with double bonds, acetylene bonds, azido groups, aldehyde groups, protected acetal groups, maleimide groups, N-hydroxysuccinimide groups, mercapto groups, protected mercapto groups, seleno groups, protected seleno groups, -NH ₂ or-ONH ₂ A substituent of (3).

2. The nitrogen-linked sialic acid (a- (2 → 6)) -D-aminopyranosyl galactose derivative or salt thereof according to claim 1 wherein R is ₁ is-NHC (O) CH _p F _q or-NHC (O) C _a H _2a+1 (ii) a Said R is ₂ Is allyloxy.

3. The nitrogen-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative or the salt thereof according to claim 1, wherein the nitrogen-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative has any one of the structures represented by formulae 1-1 to 1-5:

4. the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative or the salt thereof according to any one of claims 1 to 3, wherein the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative salt is a salt formed by reacting the nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative of the formula 1 with a base.

5. A process for producing a nitrogen-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative according to any one of claims 1 to 3,

the R is ₁ is-NHC (O) CH ₃ The method comprises the following steps:

the R is ₁ is-NH ₂ The method comprises the following steps:

the R is ₁ Is except-NHC (O) CH ₃ When other amide groups are present, the method comprises the following steps:

mixing the selective deacetylation coupling product with the structure shown in the formula 6, a polar solvent, an organic base and an acylation reagent to perform a deacetylation protection and acylation reaction to obtain the nitrogen-linked sialic acid (alpha- (2 → 6)) -D-aminopyranosyl galactose derivative; the acylating agent is R ₁ Corresponding acid anhydrides, carboxylic acids or carboxylic acid esters.

6. A glycoconjugate, characterized by: the N-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative or a salt thereof according to any one of claims 1 to 4 or the preparation process according to claim 5, and a polypeptide or a carrier protein to which the N-linked sialic acid (α - (2 → 6)) -D-galactopyranose derivative or a salt thereof is coupled via a different linker.

7. The process for preparing a glycoconjugate according to claim 6, comprising the steps of:

dissolving the N-linked sialic acid (alpha- (2 → 6)) -D-amino galactopyranose derivative or the salt thereof in a polar solvent, and introducing oxidizing gas for oxidation reaction or introducing N-hydroxysuccinimide group by extending a carbon chain to obtain disaccharide containing aldehyde group or N-hydroxysuccinimide group;

8. Use of the N-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative or salt thereof according to any one of claims 1 to 4 or the N-linked sialic acid (α - (2 → 6)) -D-aminopyranosyl galactose derivative or salt thereof produced by the production process according to claim 5 for producing an antitumor agent.

9. Use of the glycoconjugate of claim 6 or the glycoconjugate prepared by the preparation method of claim 7 in the preparation of an antitumor medicament.

10. A vaccine for treating tumor, comprising the glycoconjugate of claim 6 or the glycoconjugate prepared by the preparation method of claim 7 and a pharmaceutically acceptable carrier or adjuvant.