Sialyl (α-(2→6))-D-aminogalactopyranose derivative or its salt, sugar conjugate and preparation method thereof

Abstract

The present invention belongs to the technical field of oligosaccharides and their glycoconjugates, and specifically relates to sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof, glycoconjugates and preparation methods thereof. The present invention provides nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof, wherein the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives have a structure shown in Formula 1. Mouse experiments show that the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof provided by the present invention can be coupled with carrier proteins or polypeptides through different linkers to obtain glycoprotein (glycopeptide) conjugates, produce more effective immune responses, and can specifically identify tumor cells expressing STn, thereby achieving an anti-tumor effect; a new skeleton structure is provided for the research and development of anti-tumor sugar vaccines, which is expected to promote the development of anti-tumor sugar vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of oligosaccharides and sugar conjugates thereof, and specifically relates to sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof, sugar conjugates and preparation methods thereof. Background Art

[0002] The research on anti-tumor vaccines based on carbohydrate antigens has become a promising research direction in the current field of tumor immunotherapy. Among them, the carbohydrate antigen STn is a disaccharide structure containing sialic acid, which is often expressed in human breast cancer, colorectal cancer, ovarian cancer, and prostate cancer, but rarely expressed in normal tissues (Holmberg, L. Expert Rev. Vaccines 2004, 3, 655-663.), thus becoming an important target for tumor immunotherapy. Based on this, Canadian Biomira Company developed STn-KLH (keyhole limpet hemocyanin) conjugates— Vaccines are used to prevent and treat colorectal cancer and breast cancer metastasis. However, in Phase III clinical trials, it was found that single use It cannot improve the time to disease progression and overall survival rate, and only when it is used in combination with hormones can it show a certain effect, increasing the survival period from 5.8 months when hormones are used alone to 8.3 months. The anti-tumor activity of steroids depends on the presence of hormones, which affects their anti-tumor activity (Holmberg, L. Expert Rev. Vaccines 2004, 3, 655-663).

[0003] same The difficulties encountered are similar. The main problem encountered by anti-tumor sugar vaccines today is that the vaccine cannot produce an effective immune response in the body. Summary of the invention

[0004] The object of the present invention is to provide a sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof, a sugar conjugate and a preparation method thereof. The sugar conjugate (sugar antigen) obtained by coupling the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof provided by the present invention to a protein through different linkers can produce a more effective immune response, produce more antibodies, and show good activity in anti-tumor vaccines; thereby, the survival period can be prolonged, thereby achieving an anti-tumor effect.

[0005] The present invention provides a nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative has a structure shown in Formula 1:

[0006]

[0007] In Formula 1, R1 is an amide group or -NH2; the amide group 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 ; wherein p or q is independently 0, 1, 2 or 3, and p+q=3; a is any integer from 1 to 20; b is any integer from 2 to 20;

[0008] R2 is a substituent having a double bond, an acetylenic bond, an azido group, an aldehyde group, a protected acetal group, a maleimide group, an N-hydroxysuccinimide group, a thiol group, a protected thiol group, a seleno group, a protected seleno group, -NH2 or -ONH2.

[0009] Preferably, the R1 is -NHC(O)CH p F q or -NHC(O)C a H 2a+1 ; The R2 is allyloxy.

[0010] Preferably, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative has any one of the structures shown in Formula 1-1 to Formula 1-5:

[0011]

[0012] Preferably, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative salt is a salt formed by the reaction of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative having the structure shown in Formula 1 with a base.

[0013] The present invention provides a method for preparing the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative described in the above technical solution.

[0014] When R1 is -NHC(O)CH3, the process comprises the following steps:

[0015] The glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1;

[0016]

[0017] The coupling product of the structure shown in Formula 4-1, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-1;

[0018]

[0019] The debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative;

[0020] When the R1 is -NH2, the method comprises the following steps:

[0021] The glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-2;

[0022]

[0023] The coupling product of the structure shown in Formula 4-2, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-2;

[0024] The debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6;

[0025]

[0026] In a protective gas atmosphere, the selective deacetylation coupling product of the structure described in Formula 6, a polar solvent and an organic base are mixed to remove the trifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative;

[0027] When the R1 is an amide group other than -NHC(O)CH3, the method comprises the following steps:

[0028] The selective deacetylation coupling product of the structure described in Formula 6, a polar solvent, an organic base and an acylating agent are mixed to carry out detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative; the acylating agent is an anhydride, carboxylic acid or carboxylic acid ester corresponding to R1.

[0029] The present invention provides a sugar conjugate, which is obtained by coupling the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme with a polypeptide or a carrier protein through different linkers.

[0030] The present invention provides a method for preparing the glycoconjugate described in the above technical solution, comprising the following steps:

[0031] The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof described in the above technical solution is dissolved in a polar solvent, and an oxidizing gas is introduced to perform an oxidation reaction or an N-hydroxysuccinimide group is introduced by extending the carbon chain to obtain a disaccharide containing an aldehyde group or a N-hydroxysuccinimide group;

[0032] The disaccharide containing an aldehyde group or an N-hydroxysuccinimide group, a protein or a polypeptide, a reducing agent and a buffer solution are mixed and subjected to a coupling reaction to obtain the sugar conjugate.

[0033] The present invention provides the use of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative described in the above technical scheme or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme in the preparation of anti-tumor drugs.

[0034] The present invention provides the use of the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0035] The present invention provides a vaccine for treating tumors, comprising the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution and a pharmaceutically acceptable carrier or excipient.

[0036] The present invention provides a nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative has a structure shown in Formula 1. The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof provided by the present invention has a structure shown in Formula 1, wherein the nitrogen bridge (N(OMe)) connection replaces the oxygen bridge (O) connection in the disaccharide antigen structure, and the structure is novel, and good activity is shown in anti-tumor vaccines. Mouse experiments show that the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt linked to the structure shown in Formula 1 provided by the present invention can be coupled with a carrier protein or polypeptide to obtain a glycoprotein (glycopeptide) conjugate. The vaccine using the glycoprotein (glycopeptide) conjugate as a sugar antigen can produce a more effective immune response, produce more specific antibodies, and can specifically recognize tumor cells expressing STn, thereby achieving an anti-tumor effect; further, compared with the structure linked by oxygen bridge (O), the vaccine obtained by using the sugar antigen prepared by the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt linked to the structure shown in Formula 1 provided by the present invention has a significantly improved antibody titer for recognizing STn, and the third Thirteen days after immunization, the vaccine obtained from the sugar antigen prepared from the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt with nitrogen-linked structure shown in Formula 1 of the present invention had an antibody titer of 4812 for recognizing STn, and the vaccine obtained from the sugar antigen prepared from the hapten with oxygen bridge (O)-linked structure had an antibody titer of 1458 for recognizing STn; 13 days after the fourth immunization, the vaccine obtained from the sugar antigen prepared from the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt with nitrogen-linked structure shown in Formula 1 of the present invention had an antibody titer of 89288 for recognizing STn, and the vaccine obtained from the hapten with oxygen bridge (O)-linked structure had an antibody titer of 5716 for recognizing STn. Moreover, the vaccine obtained from the sugar antigen prepared from the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt with nitrogen-linked structure shown in Formula 1 of the present invention can significantly prolong the survival of mice in a tumor-bearing mouse model test. Figure 6 The results show that the vaccine obtained from the sugar antigen prepared from the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative or its salt provided by the present invention has a survival period of 115 days in a tumor-bearing mouse model test. It is suitable for preparing anticancer drugs such as breast cancer, colorectal cancer, ovarian cancer, and prostate cancer. On the other hand, this is a brand-new compound, which provides a new skeleton structure for the research and development of anti-tumor sugar vaccines, and is expected to promote the development of anti-tumor sugar vaccines.

[0037] The present invention provides a sugar conjugate, which is obtained by coupling the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative or its salt described in the above technical solution, or the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative or its salt prepared by the preparation method described in the above technical solution with a polypeptide or protein. Compared with the oxygen bridge (O)-linked structural galactose derivative, the sugar conjugate provided by the present invention is used as an anti-tumor vaccine to produce a strong immune response in mice. In comparison, the titer of antibodies produced by the sugar conjugates of the present invention increased by 3 to 15 times, and the survival of tumor-bearing mice was significantly prolonged after vaccination. Both the antibody titer and the survival of mice were significantly increased, which is expected to promote the development of anti-tumor sugar vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A synthesis flow chart of a sialic acid (α-(2→6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-1 is provided for Example 1 of the present invention;

[0039] Figure 2 Provides a synthesis flow chart of sialic acid (α-(2→6))-D-aminogalactopyranose derivatives of structures shown in Formula 1-2 to Formula 1-5 for Examples 2 to 5 of the present invention;

[0040] Figure 3 The titer of the serum of each mouse in the STn-KLH and 1-KLH groups after the fourth immunization with 1-KLH prepared in Example 6 of the present invention;

[0041] Figure 4 This is the survival curve of mice after administration of 1-KLH prepared in Example 6 of the present invention;

[0042] Figure 5 This is the tumor growth curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention;

[0043] Figure 6 This is the survival curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention;

[0044] Figure 7 This is the tumor growth curve of mice after administration of NSTn-NHS-CRM197 prepared in Example 8 of the present invention;

[0045] Figure 8 The synthetic flow chart of the glycosyl acceptor of the structure shown in Formula 7 in Example 1 of the present invention;

[0046] Fig. 9 This is a synthetic flow chart of the glycosyl donor with the structure shown in Formula 3 in the embodiment of the present invention;

[0047] Fig.10 is a synthetic flow chart of the glycoprotein conjugate in the embodiment of the present invention;

[0048] Fig.11 1-NHS-CRM197 is a synthetic flow chart of the glycoprotein conjugate 1-NHS-CRM197 in an embodiment of the present invention. DETAILED DESCRIPTION

[0049] The present invention provides a nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative has a structure shown in Formula 1:

[0050]

[0051] In Formula 1, R1 is an amide group or -NH2; the amide group 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 ; wherein p or q is independently 0, 1, 2 or 3, and p+q=3; a is any integer from 1 to 20; b is any integer from 2 to 20;

[0052] R2 is a substituent having a double bond, an acetylenic bond, an azido group, an aldehyde group, a protected acetal group, a maleimide group, an N-hydroxysuccinimide group, a thiol group, a protected thiol group, a seleno group, a protected seleno group, -NH2 or -ONH2.

[0053] In the present invention, the protected acetal group is a substituent having a protective group on the acetal group, the protected thiol group is a substituent having a protective group on the thiol group, and the protected selenoyl group is a substituent having a protective group on selenium. The present invention has no special requirements on the type of the protective group.

[0054] In the present invention, the R1 is preferably -NHC(O)CH p F q or -NHC(O)C a H 2a+1 ; The R2 is preferably allyloxy.

[0055] In a specific embodiment of the present invention, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative has any one of the structures shown in Formula 1-1 to Formula 1-5:

[0056]

[0057] In the present invention, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative salt is a salt formed by the reaction of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in Formula 1 with a base.

[0058] The present invention provides a method for preparing the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative described in the above technical solution.

[0059] When R1 is -NHC(O)CH3, the process comprises the following steps:

[0060] The glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1;

[0061]

[0062] The coupling product of the structure shown in Formula 4-1, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-1;

[0063]

[0064] The debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative;

[0065] When the R1 is -NH2, the method comprises the following steps:

[0066] The glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-2;

[0067]

[0068] The coupling product of the structure shown in Formula 4-2, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-2;

[0069] The debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6;

[0070]

[0071] In a protective gas atmosphere, the selective deacetylation coupling product of the structure described in Formula 6, a polar solvent and an organic base are mixed to remove the trifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative;

[0072] When the R1 is an amide group other than -NHC(O)CH3, the method comprises the following steps:

[0073] The selective deacetylation coupling product of the structure described in Formula 6, a polar solvent, an organic base and an acylating agent are mixed to carry out detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative; the acylating agent is an anhydride, carboxylic acid or carboxylic acid ester corresponding to R1.

[0074] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0075] In the present invention, when the R1 is -NHC(O)CH3, the method for preparing the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative comprises the following steps:

[0076] The glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1;

[0077]

[0078] The coupling product of the structure shown in Formula 4-1, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-1;

[0079]

[0080] The debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and an alkaline catalytic agent are mixed and selectively deacetylated to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative.

[0081] The present invention mixes a glycosyl acceptor with a structure shown in Formula 2-1, a glycosyl donor with a structure shown in Formula 3, a coupling reagent (hereinafter referred to as the first coupling reagent) and a polar solvent to carry out a glycosylation coupling reaction (hereinafter referred to as the glycosylation coupling reaction) to obtain a coupling product with a structure shown in Formula 4-1.

[0082] In the present invention, the glycosyl acceptor with the structure shown in Formula 2-1 is preferably a glycosyl acceptor with the structure shown in Formula 2-1-1;

[0083]

[0084] In the present invention, the method for preparing the glycosyl acceptor of the structure shown in Formula 2-1-1 preferably comprises the following steps:

[0085] The compound of the structure shown in Formula 7, camphorsulfonic acid and a polar solvent are mixed to carry out benzyl protection at the 3,4 positions to obtain the compound of the structure shown in Formula 8; the compound of the structure shown in Formula 8, tetramethylpiperidinyl oxide (TEMPO), iodobenzene diacetic acid (BAIB) and a polar solvent are mixed to carry out selective oxidation at the 6 position to obtain the compound of the structure shown in Formula 9; in a protective gas atmosphere, the compound of the structure shown in Formula 9, NaCNBH3 and a polar solvent are mixed to carry out a double bond reduction reaction to obtain a glycosyl receptor with the structure shown in Formula 2-1-1.

[0086]

[0087] The present invention mixes the compound of structure shown in formula 7, camphorsulfonic acid and polar solvent to carry out benzyl protection at positions 3 and 4, thereby obtaining the compound of structure shown in formula 8. In the present invention, the preparation method of the compound of structure shown in formula 7 preferably comprises the following steps: dissolving the compound of structure shown in formula 10 (galactosamine hydrochloride), acetic anhydride and carbonate-type strong alkaline resin in a mixed solvent of methanol and water, and reacting under ice-water bath conditions to obtain the compound of structure shown in formula 11; mixing the compound of structure shown in formula 11, allyl alcohol and ethanol solution of boron trifluoride, then adding ethanol solution of HCl, and reacting under reflux conditions to obtain the compound of structure shown in formula 7;

[0088]

[0089] In a specific embodiment of the present invention, the method for preparing the compound of the structure shown in Formula 11 is preferably as follows: commercially available aminogalactose hydrochloride (2.5 g, 11.6 mmol) shown in Formula 10 and 5.0 g carbonate-type strong alkaline resin, 58 mL water, and 6 mL methanol are mixed, stirred under an ice bath, and 1.5 mL acetic anhydride is added dropwise. After 2 hours, filter and wash the resin. After the mother liquor is concentrated, it passes through a strong acid resin column and evaporates to dryness to obtain the compound shown in Formula 11, which is directly added to the next step without purification. The prepared compound of Formula 11 and 0.32 mL BF3 and Et2O are added to 28 mL allyl alcohol and refluxed and stirred for 2 hours. Then add 0.5 mL HCl in Et2O solution and continue to reflux for 1 hour. Cool, add ether until turbidity appears, and place at 4°C overnight. Filter and wash with ether to obtain 0.9 g of white solid, which is the compound of the structure shown in Formula 7, and the two-step reaction yield is 30%.

[0090] In the present invention, the molar ratio of the compound of the structure shown in Formula 7 to camphorsulfonic acid is preferably 3.8:0.23. The polar solvent is preferably dimethyl phthalate (DMP, α, α-dimethoxypropane). The present invention has no special requirements for the amount of the polar solvent, as long as the 3,4-benzylidene protection is carried out smoothly. The reaction temperature of the 3,4-benzylidene protection is preferably room temperature, and the reaction insulation time is preferably 22h. After the 3,4-benzylidene protection reaction, a 3,4-benzylidene protection reaction liquid is obtained. The present invention preferably performs post-treatment on the 3,4-benzylidene protection reaction liquid to obtain a compound of the structure shown in Formula 8. The post-treatment preferably includes: mixing the 3,4-benzylidene protection reaction liquid and a saturated sodium bicarbonate aqueous solution to obtain a mixed solution; mixing and extracting the mixed solution with an organic solvent to obtain an extracted organic phase; drying and concentrating the combined extracted organic phase to obtain a concentrated solution; and performing column chromatography separation on the concentrated solution to obtain a compound of the structure shown in Formula 8. The organic solvent is preferably dichloromethane. The drying reagent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration. The concentration is preferably performed by concentration under reduced pressure. The eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to acetone is preferably 2:1.

[0091] After obtaining the structural compound shown in Formula 8, the present invention mixes the structural compound shown in Formula 8, tetramethyl piperidine oxide (TEMPO), iodobenzene diacetic acid (BAIB) and a polar solvent for 6-position selective oxidation to obtain a structural compound shown in Formula 9. In the present invention, the molar ratio of the structural compound shown in Formula 8, TEMPO and BAIB is preferably 0.61:0.06:0.55. The polar solvent is preferably dichloromethane. The present invention has no special requirements for the amount of the polar solvent, as long as the 6-position selective oxidation reaction is carried out smoothly. The temperature of the 6-position selective oxidation reaction is preferably room temperature, and the insulation time of the 6-position selective oxidation reaction is preferably 3h. In the present invention, the 6-position selective oxidation reaction obtains a 6-position selective oxidation reaction liquid. The present invention preferably post-treats the 6-position selective oxidation reaction liquid to obtain a structural compound shown in Formula 9. The post-treatment preferably includes: mixing the 6-position selective oxidation reaction liquid and a saturated sodium bicarbonate aqueous solution to obtain a mixed solution; extracting the mixed solution and an organic solvent to obtain an extracted organic phase; drying the combined extracted organic phase and removing the solvent to obtain a residue; and subjecting the residue to column chromatography separation to obtain a structural compound shown in Formula 9. The organic solvent is preferably dichloromethane. The drying reagent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration. The solvent removal is preferably performed by evaporation. The eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is preferably 1:2.

[0092] After obtaining the compound of structure shown in formula 9, the present invention mixes the compound of structure shown in formula 9, NaCNBH3 and polar solvent in a protective gas atmosphere to carry out double bond reduction reaction to obtain a glycosyl receptor of structure shown in formula 2-1-1. In the present invention, the molar ratio of the compound of structure shown in formula 9 and NaCNBH3 is preferably 2.14:3.19. The polar solvent is preferably a mixed solvent of acetic acid and methanol, and the volume ratio of acetic acid and methanol is preferably 1:1. The present invention has no special requirements for the amount of the polar solvent, as long as the double bond reduction reaction is carried out smoothly. The temperature of the double bond reduction reaction is preferably 0°C, the insulation time of the double bond reduction reaction is preferably 4h, and the protective gas is preferably argon. In the present invention, the double bond reduction reaction obtains a double bond reduction reaction liquid, and the present invention preferably post-treats the double bond reduction reaction liquid to obtain a glycosyl receptor of structure shown in 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; and subjecting the residue to column chromatography separation to obtain a glycosyl receptor with the structure shown in 2-1-1. The organic solvent is preferably toluene. The solvent removal is preferably evaporation. The eluent used in the column chromatography separation is preferably ethyl acetate.

[0093] In the present invention, when R2 in the glycosyl acceptor of the structure shown in Formula 2-1 is a substituent of other structures, the preparation method is the same as the preparation method when R2 is a propenyloxy group, and will not be described in detail here.

[0094] In the present invention, the method for preparing the glycosyl donor of the structure shown in Formula 3 preferably comprises the following steps:

[0095] The compound of the structure shown in Formula 12 is dissolved in acetonitrile, DIPEA and (EtO)2PCl are added, and the reaction is carried out at 0-25°C in an Ar atmosphere to obtain a glycosyl donor of the structure shown in Formula 3.

[0096]

[0097] In a specific embodiment of the present invention, the specific preparation method of the glycosyl donor of the structure shown in Formula 3 is preferably: under nitrogen protection, the compound of the structure shown in Formula 12 (1.1 g, 2.20 mmol) is dissolved in 20 mL of acetonitrile solution, 0.94 mL of DIPEA is added, and the mixture is stirred under ice bath cooling, diethylphosphite chloride (0.65 mL, 4.50 mmol) is added, and the ice bath is removed after 5 minutes. After the reaction is complete as monitored by TLC, the reaction system is evaporated to dryness, ethyl acetate is added, and suction is filtered. The filtrate is evaporated to dryness, washed twice with ethyl acetate, and separated by column chromatography, the eluent (V / V) is petroleum ether: ethyl acetate = 1:2, and 1.1 g of the glycosyl donor of the structure shown in Formula 3 is obtained, with a yield of 89%.

[0098] In the present invention, the first coupling reagent is preferably MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

[0099] In the present invention, the molar ratio of the glycosyl acceptor of the structure shown in Formula 2-1 to the glycosyl donor of the structure shown in Formula 3 is preferably 0.061:0.091. The mass ratio of MS is preferably 20:100. The polar solvent is preferably dichloromethane. The present invention has no special requirements for the amount of the polar solvent, as long as the first glycosylation coupling reaction is carried out smoothly. The molar ratio of the glycosyl acceptor of the structure shown in Formula 2-1 and TMSOTf is preferably 0.061:0.018. The mixing for the first glycosylation coupling reaction comprises: in a protective gas atmosphere, mixing the glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3 and MS is dissolved in a polar solvent, stirred and mixed for 1h to obtain a mixed solution; the mixed solution is cooled to 0°C and mixed with TMSOTf. The present invention preferably uses TLC to detect that the glycosyl donor of the structure shown in Formula 3 is completely reacted, and the first glycosylation coupling reaction is completed. In the present invention, the first glycosylation coupling reaction liquid is obtained after the first glycosylation coupling reaction, and the present invention preferably post-treats the first glycosylation coupling reaction liquid to obtain a coupling product of the structure shown in Formula 4-1. In the present invention, the post-treatment preferably includes: adding triethylamine to the first glycosylation coupling reaction liquid to quench the reaction, and heating the obtained quenched reaction liquid to room temperature; filtering the quenched reaction liquid with diatomaceous earth to obtain a filtrate; removing the solvent from the filtrate to obtain a residue; and separating the residue by column chromatography to obtain a coupling product of the structure shown in Formula 4-1. The solvent removal is preferably evaporation. The column chromatography separation preferably uses a first elution solvent and a second elution solvent in sequence; the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:1; the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1.

[0100] After obtaining the coupling product of the structure shown in Formula 4-1, the present invention mixes the coupling product of the structure shown in Formula 4-1, a polar solvent and an acidic catalyst, and performs debenzylation protection to obtain a debenzylation coupling product of the structure shown in Formula 5-1. In the present invention, the acidic catalyst is preferably pyridine p-toluenesulfonate (PPTS). The mass ratio of the coupling product of the structure shown in Formula 4-1 and the acidic catalyst is preferably 100:47. The polar solvent is preferably methanol. The present invention has no special requirements for the amount of the polar solvent, as long as the debenzylation protection reaction is carried out smoothly. The temperature of the debenzylation protection reaction is preferably 65°C, and the insulation time of the debenzylation protection reaction is preferably 3h. In the present invention, a debenzylation protection reaction liquid is obtained after the debenzylation protection. The present invention preferably performs post-treatment on the debenzylation protection reaction liquid to obtain a debenzylation coupling product of the structure shown in Formula 5-1. In the present invention, the post-treatment preferably includes: removing the solvent from the debenzylidene protection reaction solution to obtain a residue; and subjecting the residue to column chromatography separation to obtain a debenzylidene coupling product of the structure shown in Formula 5-1. The solvent removal is preferably evaporation. The elution solvents used in the column chromatography separation are preferably ethyl acetate and methanol, and the volume ratio of ethyl acetate to methanol is preferably 15:1.

[0101] In the present invention, the debenzylidene coupling product of the structure shown in Formula 5-1 is preferably the debenzylidene coupling product of the structure shown in Formula 5-1-1

[0102]

[0103] After obtaining the debenzylidene coupling product of the structure shown in Formula 5-1, the present invention mixes the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and an alkaline catalyst to perform selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative. In the present invention, the alkaline catalyst is preferably sodium methoxide and sodium hydroxide aqueous solution, and the molar concentration of the sodium hydroxide aqueous solution is preferably 1 mol / L. The polar solvent is preferably methanol, and the present invention has no special requirements for the amount of the methanol, as long as the selective deacetylation is carried out smoothly. In the present invention, the mixing for selective deacetylation reaction includes the following steps: mixing the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and sodium methoxide to perform a first step reaction; obtaining a reaction solution; mixing the reaction solution and 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 30 minutes; the second step reaction is preferably detected by TLC, and the time of the second step reaction is preferably 4 hours. In the present invention, after the selective deacetylation reaction, a selective deacetylation reaction liquid is obtained. In the present invention, the selective deacetylation reaction liquid is preferably post-treated to obtain the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative. In the present invention, the post-treatment preferably includes: introducing carbon dioxide into the selective deacetylation reaction liquid until it becomes neutral, removing the solvent from the obtained neutral reaction liquid to obtain a residue; and subjecting the residue to column chromatography separation to obtain the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative. The solvent removal is preferably evaporation. The column chromatography separation is preferably performed by reverse phase column chromatography separation. The elution solvent used in the column chromatography is preferably pure water and methanol, and the volume ratio of the pure water to methanol is preferably 1:4.

[0104] In the present invention, when the R1 is -NH2, the preparation method of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative comprises the following steps:

[0105] The glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3, a coupling reagent (hereinafter referred to as the second coupling reagent) and a polar solvent are mixed to carry out a glycosylation coupling reaction (hereinafter referred to as the second glycosylation coupling reaction) to obtain a coupling product of the structure shown in Formula 4-2;

[0106] The coupling product of the structure shown in Formula 4-2, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-2;

[0107] The debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6;

[0108] In a protective gas atmosphere, the selective deacetylation coupling product of the structure described in Formula 6, a polar solvent and an organic base are mixed to remove the trifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative.

[0109] In the present invention, a glycosyl acceptor having a structure shown in Formula 2-2, a glycosyl donor having a structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product having a structure shown in Formula 4-2.

[0110] In the present invention, the glycosyl acceptor with the structure shown in Formula 2-2 is preferably the glycosyl acceptor with the structure shown in Formula 2-2-1;

[0111]

[0112] In the present invention, the preparation method of the glycosyl acceptor of the structure shown in Formula 2-2-1 preferably comprises the following steps: mixing the compound of the structure shown in Formula 10, a polar solvent, triethylamine and methyl trifluoroacetate to carry out a trifluoroethyl reaction at position 2 to obtain a reaction solution containing a reaction product substituted with trifluoroethyl at position 2; concentrating the reaction solution containing the reaction product substituted with trifluoroethyl at position 2 to obtain a concentrated solution containing a reaction product substituted with trifluoroethyl at position 2; the amount of the compound of the structure shown in Formula 10 and the volume ratio of triethylamine are preferably 11.6mmol:4.1mL; the molar ratio of the compound of the structure shown in Formula 10 and the methyl trifluoroacetate is preferably 11.6:12.6; the polar solvent is preferably methanol, and the present invention has no special requirements for the amount of the polar solvent, as long as the trifluoroethyl reaction at position 2 is ensured to proceed smoothly. The temperature of the trifluoroethyl reaction at position 2 is preferably room temperature, the insulation time of the trifluoroethyl reaction at position 2 is preferably overnight, and the trifluoroethyl reaction at position 2 is preferably carried out under stirring. The concentration is preferably reduced pressure concentration.

[0113] The concentrated solution of the reaction product containing trifluoroethyl substitution at position 2, allyl alcohol and ether hydrochloride are mixed to carry out an allyl reaction at position 1 to obtain a reaction solution containing a reaction product containing allyl substitution at position 1; the reaction solution containing the reaction product containing allyl substitution at position 1 is solid-liquid separated, and the obtained filtrate is concentrated to obtain a concentrated solution containing a reaction product containing allyl substitution at position 1; the molar concentration of the ether hydrochloride is preferably 3 mol / L; the ratio of the amount of the compound of the structure shown in Formula 10 to the volume of the ether hydrochloride is preferably 11.6 mol:18.1 mL; the ratio of the amount of the compound of the structure shown in Formula 10 to the volume of the allyl alcohol is preferably 11.6 mol:28.9 mL; the allyl reaction at position 1 is preferably carried out under reflux conditions, and the reflux time is preferably 0.5 h. The solid-liquid separation is preferably filtration; and the concentration is preferably reduced pressure concentration.

[0114] The concentrated solution of the reaction product containing 1-position allyl substitution, a polar solvent and tert-butyldimethylsilyl chloride (TBDMSCL) are mixed to carry out a 6-position TBDM protection reaction to obtain a compound of the structure shown in Formula 13; the molar ratio of the compound of the structure shown in Formula 10 to the TBDMSCL is preferably 11.6:12.6. The polar solvent is preferably pyridine. The present invention has no special requirements for the amount of the polar solvent, as long as the 6-position TBDM protection reaction is carried out smoothly. The temperature of the 6-position TBDM protection reaction is preferably room temperature, the insulation time of the 6-position TBDM protection reaction is preferably 16h, and the 6-position TBDM protection reaction is preferably carried out under stirring. After the 6-position TBDM protection reaction, a 6-position TBDM protection reaction liquid is obtained. The present invention preferably post-treats the 6-position TBDM protection reaction liquid to obtain a compound of the structure shown in Formula 13. In the present invention, the post-treatment preferably includes: concentrating the 6-position TBDM protection reaction liquid to obtain a concentrated solution; mixing the concentrated solution and an extractant to extract to obtain an extracted organic phase, drying the extracted organic phase and concentrating it to obtain a concentrated solution; subjecting the concentrated solution to column chromatography separation to obtain a compound with a structure shown in Formula 13. The concentration is preferably reduced pressure concentration. The extractant is preferably dichloromethane and a saturated sodium bicarbonate aqueous solution. The drying reagent is preferably anhydrous sodium sulfate. The drying is preferably to concentrate the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably filtration. The concentration is preferably reduced pressure concentration. The eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 4:1 to 2:1;

[0115]

[0116] After obtaining the compound of the structure shown in Formula 13, the present invention mixes the compound of the structure shown in Formula 13, a polar solvent and camphorsulfonic acid, and performs 3,4-benzylidene protection to obtain the compound of the structure shown in Formula 14. In the present invention, the molar ratio of the compound of the structure shown in Formula 13 and camphorsulfonic acid is preferably 4.43:2.22. The polar solvent is preferably acetonitrile and dimethyl phthalate (DMP, α, α-dimethoxypropane). The present invention has no special requirements for the amount of the polar solvent, and it is sufficient to ensure that the 3,4-benzylidene protection is carried out smoothly. The reaction temperature of the 3,4-benzylidene protection is preferably room temperature, and the reaction insulation time is preferably 15min. After the 3,4-benzylidene protection reaction, a 3,4-benzylidene protection reaction liquid is obtained. The present invention preferably performs post-treatment on the 3,4-benzylidene protection reaction liquid to obtain a compound of the structure shown in Formula 14. The post-treatment preferably includes: extracting the 3,4-benzylidene protection reaction solution and the extractant to obtain an extracted organic phase; drying and concentrating the extracted organic phase to obtain a concentrated solution; subjecting the concentrated solution to column chromatography separation to obtain a compound with a structure shown in Formula 14. The extractant is preferably dichloromethane and saturated brine. The drying reagent is preferably anhydrous sodium sulfate. The drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration. The concentration is preferably concentrated under reduced pressure. The eluent used for column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 20:1 to 10:1;

[0117]

[0118] After obtaining the compound of the structure shown in Formula 14, the present invention mixes the compound of the structure shown in Formula 14, a polar solvent, acetic acid and tetrabutylammonium fluoride trihydrate, and performs a selective TBDMS removal reaction to obtain a compound of the structure shown in Formula 15. In the present invention, the molar ratio of the compound of the structure shown in Formula 14 to acetic acid is preferably 0.12:1.25. The molar ratio of the compound of the structure shown in Formula 14 to tetrabutylammonium fluoride trihydrate is preferably 0.12:0.5. The polar solvent is preferably tetrahydrofuran. The present invention has no special requirements for the amount of the polar solvent, as long as the selective TBDMS removal reaction is carried out smoothly. The mixed selective TBDMS removal reaction preferably includes the following steps: mixing the compound of the structure shown in Formula 14, a polar solvent and acetic acid to obtain a mixed solution; in a protective gas atmosphere, cooling the mixed solution to 0°C and mixing it with tetrabutylammonium fluoride trihydrate to perform a selective TBDMS removal reaction. The protective gas is preferably argon. The reaction temperature of the selective TBDMS removal reaction is preferably 0°C, and the reaction insulation time is preferably 4h. After the selective TBDMS removal reaction, a selective TBDMS removal reaction liquid is obtained. The present invention preferably performs post-treatment on the selective TBDMS removal reaction liquid to obtain a compound with a structure shown in Formula 15. The post-treatment preferably includes: concentrating the selective TBDMS removal reaction liquid and mixing it with an extractant to obtain an extracted organic phase; drying the extracted organic phase and concentrating it to obtain a concentrated solution; subjecting the concentrated solution to column chromatography separation to obtain a compound with a structure shown in Formula 15. The concentration is preferably to concentrate the selective TBDMS removal reaction liquid until the volume is halved. The extractant is preferably dichloromethane and saturated brine. The drying reagent is preferably anhydrous sodium sulfate. The drying is preferably to concentrate the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably filtration. The concentration is preferably reduced pressure concentration. The eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 2:1 to 1:1;

[0119]

[0120] After obtaining the compound of the structure shown in Formula 15, the present invention mixes the compound of the structure shown in Formula 15, a polar solvent, TEMPO and BAIB, and performs an N-methoxy reaction at position 6 to obtain a compound of the structure shown in Formula 16. In the present invention, the molar ratio of the compound of the structure shown in Formula 15, TEMPO and BAIB is preferably 1.92:0.19:2.11. The polar solvent is preferably dichloromethane. The present invention has no special requirements for the amount of the polar solvent, as long as the N-methoxy reaction at position 6 is smoothly carried out. The reaction temperature of the N-methoxy at position 6 is preferably 40°C, and the insulation time of the N-methoxy reaction at position 6 is preferably 6h. After the N-methoxy reaction at position 6, an N-methoxy reaction liquid at position 6 is obtained. The present invention preferably performs post-treatment on the N-methoxy reaction liquid at position 6 to obtain a compound of the structure shown in Formula 16. The post-treatment preferably includes: diluting the N-methoxy group at position 6 with an organic solvent to obtain a diluted reaction solution; stirring and mixing the diluted reaction solution, a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate for 10 minutes to obtain a mixed solution; extracting the mixed solution and an extractant to obtain an extracted organic phase; drying the extracted organic phase and removing the solvent to obtain a residue; subjecting the residue to column chromatography separation to obtain a compound with a structure shown in Formula 16. The organic solvent is preferably dichloromethane. The extractant is preferably dichloromethane. The drying reagent is preferably anhydrous sodium sulfate. The drying is preferably to remove the solvent from the organic extract phase of solid-liquid separation, and the removal of the solvent is preferably evaporation. The eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 10:1 to 4:1;

[0121]

[0122] After obtaining the compound of structure shown in formula 16, the present invention mixes the compound of structure shown in formula 16, NaCNBH3 and polar solvent in a protective gas atmosphere to carry out double bond reduction reaction to obtain a glycosyl receptor of structure shown in formula 2-2-1. In the present invention, the molar ratio of the compound of structure shown in formula 16 and NaCNBH3 is preferably 0.73:1.09. The polar solvent is preferably a mixed solvent of acetic acid and methanol, and the volume ratio of acetic acid and methanol is preferably 1:1. The present invention has no special requirements for the amount of the polar solvent, as long as the double bond reduction reaction is carried out smoothly. The temperature of the double bond reduction reaction is preferably 0°C, the insulation time of the double bond reduction reaction is preferably 4h, and the protective gas is preferably argon. In the present invention, the double bond reduction reaction obtains a double bond reduction reaction liquid, and the present invention preferably post-treats the double bond reduction reaction liquid to obtain a glycosyl receptor of structure shown in formula 2-2-1. The post-treatment preferably includes: 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 subjecting the residue to column chromatography separation to obtain a glycosyl receptor with the structure shown in 2-2-1. The organic solvent is preferably toluene. The solvent removal is preferably evaporation. The eluent used for the column chromatography separation is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 2:1.

[0123] In the present invention, when R2 in the glycosyl acceptor of the structure shown in Formula 2-2 is a substituent of other structures, the preparation method is the same as the preparation method when R2 is a propenyloxy group, and will not be described in detail here.

[0124] In the present invention, the second coupling reagent is preferably MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

[0125] In the present invention, the molar ratio of the glycosyl acceptor of the structure shown in Formula 2-2 to the glycosyl donor of the structure shown in Formula 3 is preferably 0.98:1.98. The mass ratio of MS is preferably 378:1.6. The polar solvent is preferably dichloromethane. The present invention has no special requirements for the amount of the polar solvent, as long as the second glycosylation coupling reaction is carried out smoothly. The molar ratio of the glycosyl acceptor of the structure shown in Formula 2-2 and TMSOTf is preferably 0.98:0.15. The mixing for the second glycosylation coupling reaction comprises: in a protective gas atmosphere, mixing the glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3 and MS is dissolved in a polar solvent, stirred and mixed for 1h to obtain a mixed solution; the mixed solution is cooled to 0°C and mixed with TMSOTf. The present invention preferably uses TLC to detect that the second glycosyl donor of the structure shown in Formula 3 is completely reacted, and the second glycosylation coupling reaction is completed. In the present invention, the second glycosylation coupling reaction is obtained after the second glycosylation coupling reaction. The present invention preferably performs post-treatment on the glycosylation coupling reaction liquid to obtain a coupling product of the structure shown in Formula 4-2. In the present invention, the post-treatment preferably includes: adding triethylamine to the second glycosylation coupling reaction liquid to quench the reaction, and heating the obtained quenched reaction liquid to room temperature; filtering the quenched reaction liquid with diatomaceous earth to obtain a filtrate; removing the solvent from the filtrate to obtain a residue; and separating the residue by column chromatography to obtain a coupling product of the structure shown in Formula 4-2. The solvent removal is preferably evaporation. The eluent for column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:1.

[0126] Preferably, the first elution solvent and the second elution solvent are used for elution in sequence; the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:2; the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1 to 5:1.

[0127] The coupling product of the structure shown in Formula 4-2, a polar solvent and an acidic catalyst are mixed, and debenzylidene protection is performed to obtain a debenzylidene coupling product of the structure shown in Formula 5-2. In the present invention, the acidic catalyst is preferably pyridine p-toluenesulfonate (PPTS). The polar solvent is preferably methanol. The present invention has no special requirements for the amount of the polar solvent, as long as the debenzylidene protection reaction is carried out smoothly. The temperature of the debenzylidene protection reaction is preferably 65°C, and the insulation time of the debenzylidene protection reaction is preferably 3h. In the present invention, a debenzylidene protection reaction liquid is obtained after the debenzylidene protection. The present invention preferably performs post-treatment on the debenzylidene protection reaction liquid to obtain a debenzylidene coupling product of the structure shown in Formula 5-2. In the present invention, the post-treatment preferably includes: removing the solvent from the debenzylidene protection reaction liquid to obtain a residue; and separating the residue by column chromatography to obtain a debenzylidene coupling product of the structure shown in Formula 5-2. The desolventizing is preferably evaporation. The eluting solvents used in the column chromatography separation are preferably ethyl acetate and methanol, and the volume ratio of ethyl acetate to methanol is preferably 15:1. The column chromatography separation preferably uses a first elution solvent and a second elution solvent in sequence; the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:2; the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1 to 5:1.

[0128] In the present invention, the debenzylidene coupling product of the structure shown in Formula 5-2 is preferably the debenzylidene coupling product of the structure shown in Formula 5-2-1:

[0129]

[0130] After obtaining the debenzylidene coupling product of the structure shown in Formula 5-2, the present invention mixes the debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and an alkaline catalyst, and performs selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6. In the present invention, the alkaline catalyst 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 of the sodium methoxide solution is preferably 30%; the molar concentration of the sodium hydroxide aqueous solution is preferably 2 mol / L. The polar solvent is preferably methanol, and the present invention has no special requirements for the amount of the methanol, as long as the selective deacetylation is smoothly carried out. In the present invention, the mixed selective deacetylation reaction comprises the following steps: mixing the debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and sodium methoxide for the first reaction; obtaining a reaction solution; the ratio of the amount of the debenzylidene coupling product of the structure shown in Formula 5-2 to the volume of the sodium methoxide solution is preferably 0.024mmol:0.02mL; mixing the reaction solution with an aqueous sodium hydroxide solution for the second reaction to obtain a selective deacetylation reaction solution; the first reaction is preferably detected by TLC, and the time of the first reaction is preferably 1h; the second reaction is preferably detected by TLC, and the time of the second reaction is preferably 0.5h. In the present invention, a selective deacetylation reaction solution is obtained after the selective deacetylation reaction, and the present invention preferably performs post-treatment on the selective deacetylation reaction solution to obtain the selective deacetylation coupling product of the structure shown in Formula 6. In the present invention, the post-treatment preferably comprises: introducing carbon dioxide into the selective deacetylation reaction solution until it is neutral, removing the solvent from the obtained neutral reaction solution, and obtaining a selective deacetylation coupling product of the structure shown in Formula 6. The solvent removal is preferably evaporation.

[0131] After obtaining the selective deacetylation coupling product of the structure described in Formula 6, the present invention, in a protective gas atmosphere, mixes the selective deacetylation coupling product of the structure described in Formula 6, a polar solvent and an organic base for detrifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative. In the present invention, the organic base is preferably triethylamine. The polar solvent is preferably methanol, and the protective gas is preferably argon. In the present invention, the detrifluoroacetyl protection reaction is preferably carried out under reflux conditions, and the insulation time of the detrifluoroacetyl protection reaction is preferably overnight. The present invention preferably performs post-treatment on the detrifluoroacetyl protection reaction solution obtained after the detrifluoroacetyl protection reaction to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative. The post-treatment preferably includes: removing the solvent from the detrifluoroacetyl protection reaction solution to obtain a residue; subjecting the residue to reverse phase column chromatography to obtain an eluent; subjecting the eluent to ion exchange to remove organic salts to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative. In the present invention, the solvent removal is preferably evaporation. The eluent used in the reverse phase column chromatography is preferably pure water to methanol, and the volume ratio of pure water to methanol is preferably 1:4. The ion exchange is preferably performed using an ion exchange resin column.

[0132] In the present invention, when the R1 is an amide group other than -NHC(O)CH3, the method for preparing the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative comprises the following steps:

[0133] The selective deacetylation coupling product of the structure described in Formula 6, a polar solvent, an organic base and an acylating agent are mixed to carry out detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative; the acylating agent is an anhydride, carboxylic acid or carboxylic acid ester corresponding to R1.

[0134] In the present invention, the acid anhydride preferably includes acetic anhydride, propionic anhydride, n-butyric anhydride, isobutyric anhydride or n-hexanoic anhydride;

[0135] The carboxylic acid preferably comprises monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, monochloroacetic acid or dichloroacetic acid;

[0136] The carboxylic acid ester preferably includes methyl monofluoroacetate, methyl difluoroacetate, methyl trifluoroacetate or methyl dichloroacetate.

[0137] In the present invention, the acylating agent is preferably methyl fluoroacetate, methyl difluoroacetate or methyl trifluoroacetate.

[0138] In the present invention, the detrifluoroacetyl protection and acylation reaction are preferably carried out under reflux conditions, and the insulation time of the detrifluoroacetyl protection and acylation reaction is preferably overnight. In the present invention, the acylation reaction solution obtained by the detrifluoroacetyl protection and acylation reaction is preferably post-treated to obtain the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative. The post-treatment preferably includes: removing the solvent from the acylation reaction solution to obtain a residue; subjecting the residue to reverse phase column chromatography to obtain an eluent; subjecting the eluent to ion exchange to remove organic salts to obtain the nitrogen-linked sialic acid (α-(2→6))-D-amino galactopyranose derivative. In the present invention, the desolventizing is preferably evaporation. The eluent used in the reverse phase column chromatography is preferably pure water to methanol, and the volume ratio of the pure water to methanol is preferably 1:4. The ion exchange is preferably carried out using an ion exchange resin column.

[0139] The present invention provides a sugar conjugate, which is obtained by coupling the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme with a polypeptide or a carrier protein through different linkers.

[0140] In the present invention, the carrier protein is preferably bovine serum albumin (BSA), hemocyanin (KLH) or CRM197.

[0141] The present invention provides a method for preparing the glycoconjugate described in the above technical solution, comprising the following steps:

[0142] The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof described in the above technical solution is dissolved in a polar solvent, and an oxidizing gas is introduced to perform an oxidation reaction or an N-hydroxysuccinimide group is introduced by extending the carbon chain to obtain a disaccharide containing an aldehyde group or a disaccharide containing an N-hydroxysuccinimide group;

[0143] The disaccharide containing an aldehyde group or an N-hydroxysuccinimide group, a protein or a polypeptide, a reducing agent and a buffer solution are mixed and subjected to a coupling reaction to obtain the sugar conjugate.

[0144] The present invention dissolves the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative or its salt described in the above technical solution in a polar solvent, introduces an oxidizing gas to carry out an oxidation reaction, and obtains a disaccharide containing an 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°C; the oxidation reaction liquid obtained after the oxidation reaction is carried out for 30 minutes is a blue solution; and the introduction of the oxidizing gas is stopped. In this invention, an oxidation reaction liquid is obtained after the oxidation reaction, and the present invention preferably performs post-treatment on the oxidation reaction liquid to obtain a disaccharide containing an aldehyde group. The post-treatment preferably includes: introducing nitrogen into the oxidation reaction liquid to remove unreacted oxidizing gas and then heating it to room temperature; desolventizing the oxidation reaction liquid to obtain a disaccharide containing an aldehyde group. The desolventizing is preferably vacuum desolventizing.

[0145] After obtaining the disaccharide containing an aldehyde group, the present invention mixes the disaccharide containing an aldehyde group, a protein or a polypeptide, a reducing agent and a buffer solution to perform a coupling reaction to obtain the glycoconjugate.

[0146] In the present invention, the reducing agent is preferably sodium cyanoborohydride. The pH value of the buffer solution is preferably 7.6.

[0147] In the present invention, the coupling reaction temperature is preferably room temperature, the coupling reaction temperature holding time is preferably 24 hours, and the coupling reaction is preferably carried out in a light-proof condition.

[0148] In the present invention, a coupling reaction solution is obtained after the coupling reaction. In the present invention, the coupling reaction solution is preferably post-treated to obtain the glycoconjugate.

[0149] The present invention provides the use of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative described in the above technical scheme or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme in the preparation of anti-tumor drugs.

[0150] The present invention provides the use of the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0151] In the present invention, the anti-tumor drug preferably includes a therapeutic vaccine or a preventive vaccine.

[0152] The present invention provides a vaccine for treating tumors, comprising the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution and a pharmaceutically acceptable carrier or excipient.

[0153] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0154] Example 1

[0155] according to Figure 8 The synthesis flow chart of the compound of the structure shown in Formula 7 is shown:

[0156] The commercially available aminogalactose hydrochloride (2.5 g, 11.6 mmol) shown in formula 10, 5.0 g carbonate-type strong alkaline resin, 58 mL water, and 6 mL methanol were mixed, stirred under ice bath, and 1.5 mL acetic anhydride was added dropwise. After 2 hours, the mixture was filtered and the resin was washed. After the mother liquor was concentrated, it was passed through a strong acid resin column and evaporated to dryness to obtain the compound shown in formula 11, which was directly used for the next step reaction without purification. The prepared compound of formula 11 and 0.32 mL BF3 and Et2O were added to 28 mL allyl alcohol and refluxed and stirred for 2 hours. Then 0.5 mL HCl in Et2O solution was added and refluxed for 1 hour. Cool, add ether until turbidity appears, and place at 4°C overnight. Filter and wash with ether to obtain 0.9 g of white solid, which is the compound of the structure shown in formula 7, and the yield of the two-step reaction is 30%.

[0157]

[0158] according to Fig. 9 The synthesis flow chart of the glycosyl donor of the structure shown in Formula 3 is shown:

[0159] Under nitrogen protection, the compound of the structure shown in formula 12 (1.1 g, 2.20 mmol) was dissolved in 20 mL of acetonitrile solution, 0.94 mL of DIPEA was added, and the mixture was stirred under ice bath cooling. Diethylphosphite chloride (0.65 mL, 4.50 mmol) was added, and the ice bath was removed after 5 minutes. After the reaction was completed as monitored by TLC, the reaction system was evaporated to dryness, ethyl acetate was added, and suction filtered. The filtrate was evaporated to dryness, and ethyl acetate was taken twice for column chromatography separation. The eluent (V / V) was petroleum ether: ethyl acetate = 1:2, and 1.1 g of the glycosyl donor of the structure shown in formula 3 was obtained, with a yield of 89%.

[0160]

[0161] according to Figure 1 The synthesis flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in Formula 1-1 is shown as follows:

[0162] The compound represented by the structure of formula 7 ( Figure 1Compound 6 (1.0 g, 3.8 mmol) was dissolved in DMP (α, α-dimethoxypropane, 24.3 mL, 198.5 mmol), camphorsulfonic acid (54.5 mg, 0.23 mmol) was added, and stirred at room temperature for 22 hours. The reaction mixture was poured into a saturated sodium bicarbonate aqueous solution, and then extracted with dichloromethane; the extracts were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by column chromatography (petroleum ether: acetone, 2:1) to obtain a white solid compound having the structure of formula 8 ( Figure 1 Compound 7) 940 mg, yield 82%.

[0163] 1 H NMR (300MHz, CDCl3): 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).

[0164] The compound of formula 8 (183 mg, 0.61 mmol) was dissolved in 1 mL of dichloromethane, TEMPO (9.1 mg, 0.06 mmol) and BAIB (215 mg, 0.55 mmol) were added, and the mixture was stirred at room temperature for 3 hours. Diluted with 4 mL of dichloromethane, 5.2 mL of a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate were added, and the mixture was stirred vigorously for 10 minutes. Extracted with dichloromethane, dried with sodium sulfate, and evaporated to dryness. The residue was dissolved in 2 mL of pyridine, MeONH2·HCl (76.1 mg, 0.91 mmol) was added, stirred at room temperature for 1 hour, and evaporated to dryness. The residue was separated by column chromatography (petroleum ether: ethyl acetate, 1:2) to obtain a white solid compound of formula 9 ( Figure 1 The product of compound 8) was 161 mg, with a yield of 81% (Z / E=1 / 1).

[0165] 1H NMR (300MHz, CDCl3) δ7.52(d,1H,J=7.8Hz),6.87(d,1H,J=4.8Hz),5.94-5.81(m,2H),5.57(d,2 H, 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 N2O6[M+H] + :329.1707,found329.1701.

[0166] The compound of formula 9 (701 mg, 2.14 mmol) was dissolved in a mixed solution of acetic acid / methanol (7 mL / 7 mL), and NaCNBH3 (201.9 mg, 3.19 mmol) was added at 0°C under argon protection. The mixture was stirred and the reaction was completed after 4 hours. A small amount of toluene was added, and the mixture was concentrated and evaporated to dryness. The residue was separated by column chromatography (ethyl acetate) to obtain an oily substance, which was a glycosyl receptor of formula 2-1-1 ( Figure 1 Compound 9) 637 mg, yield 90%.

[0167] 1 H NMR (300MHz, CDCl3) δ5.96-5.83 (m, 2H), 5.59 (d, 1H, J = 9.3Hz), 3.28 (dd, 1H, J = 1.5 Hz,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=6.0H z,12.6Hz),3.54(s,3H),3.28-3.15(m,2H),2.03(s,3H),1.57(s,3H),1.34(s,3H); 13CNMR (75MHz, CDCl3) δ169.97,133.51,117.84,109.67,96.75,74.72,73.75,6 8.26,63.09,61.37,52.09,50.50,27.95,26.55,23.46; HRMS(ESI)Anal.Calcd for C 15 H 27 N2O6[M+H] + :331.1864, found 331.1865.

[0168] The glycosyl donor (55.5 mg, 0.091 mmol) of the structure shown in Formula 3 and the glycosyl acceptor (20.0 mg, 0.061 mmol) of the structure shown in Formula 2-1-1 were added. MS (100 mg) was dissolved in dry dichloromethane (1 mL) under nitrogen protection and stirred at room temperature for 1 hour. The reaction system was cooled to 0°C and TMSOTf (3.2 μL, 0.018 mmol) was added. After TLC showed that the glycosyl donor of the structure shown in Formula 3 was basically reacted, a drop of triethylamine was added to quench the reaction. After the reaction system was warmed to room temperature, the reaction system was filtered with diatomaceous earth. The filtrate was evaporated to dryness. The residue was separated by column chromatography (petroleum ether: acetone, 1:1, followed by toluene: methanol, 10:1) to obtain an oily substance as a compound of the structure shown in Formula 4-1-4 ( Figure 1 Compound 11), 31.0 mg, yield 63%.

[0169] 1H NMR (500MHz, CDCl3) 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),5.2 7(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 CNMR(125MHz,CDCl3)δ170.90,170.69,170.24,170.07,170.04,170.00,167 .64,133.52,117.55,109.37,96.92,94.89,74.44,73.00,69.82,69.66,68.2 9,67.93,65.11,63.96,62.39,53.12,52.70,50.37,49.33,35.11,29.63,28 .06,26.55,23.42,23.13,21.00,20.82,20.75,20.68; HRMS(ESI)Anal.Calcd for C 35 H 54 N3O 18 [M+H] + :804.3397,found804.3425.

[0170] The compound of the structure shown in formula 4-1-4 ( Figure 1 Compound 11) (100 mg, 0.124 mmol) was dissolved in dry methanol (1 mL), PPTS (47 mg) was added to the reaction system, stirred at 65 ° C for 3 h, evaporated to dryness, and the residue was separated by column chromatography (ethyl acetate: methanol, 15: 1) to obtain a white solid compound of the structure shown in formula 5-1-1 ( Figure 1Compound 12), 92 mg, yield 97%.

[0171] 1 H NMR (500MHz, CDCl3) 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,12.5 Hz),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, CDCl3) 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 forC 32 H 50 N3O 18 [M+H] + :764.3084,found 764.3088.

[0172] The compound represented by the structure of formula 5-1-1 ( Figure 1Compound 12) (30 mg, 0.039 mmol) was dissolved in methanol (2 mL), stirred at room temperature, and a drop of 30% sodium methoxide was added. After 30 minutes, TLC showed that the reaction was complete. After concentrating and draining the solvent, 1 N NaOH aqueous solution (1.3 mL) was added. After 4 hours, TLC showed that the reaction was complete. Carbon dioxide gas was introduced until neutral. After evaporation, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1:4) to obtain a white solid with a nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose derivative of the structure shown in formula 1-1, 19 mg, and a yield of 83%.

[0173] 1 HNMR(400MHz,D2O)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,D2O)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 N3O 14 [M+H] + :582.2505, found 582.2517.

[0174] Example 2

[0175] according to Figure 2Example 2 shows a synthetic flow chart of a sialic acid (α-(2→6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-2:

[0176] The compound of the structure shown in Formula 10 (D-galactosamine hydrochloride, 2.5 g, 11.6 mmol) was dissolved in 34.7 mL of methanol, 4.1 mL of triethylamine and methyl trifluoroacetate (1.5 mL, 12.6 mmol) were added, and the mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was dissolved in 28.9 mL of allyl alcohol, 18.1 mL of 3M hydrochloric acid ether was added, and refluxed for 0.5 h. Filtered, and the filtrate was concentrated. The residue was dissolved in 18.6 mL of pyridine, TBDMSCl (1.9 g, 12.6 mmol) was added, and stirred at room temperature for 16 h. After concentrated under reduced pressure, it was extracted with dichloromethane and saturated sodium bicarbonate, the organic phase was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was separated by column chromatography (petroleum ether: ethyl acetate, 4:1 to 2:1) to obtain an oily compound of the structure shown in Formula 13 ( Figure 2 Compound 13), 2.3 g, yield 46%.

[0177] 1 H NMR (400MHz, CDCl3) 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=5.3H z,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, CDCl3) 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.CalcdforC 17 H 34 N2O6F3Si[M+NH4] + :447.2133,found447.2122.

[0178] Compound 13 (1.9 g, 4.43 mmol) was dissolved in 53.2 mL of acetonitrile, and DMP (10.8 mL, 88.2 mmol) and camphorsulfonic acid (506 mg, 2.22 mmol) were added, and stirred at room temperature for 15 minutes. The mixture was extracted with dichloromethane and saturated brine, and the organic phase was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was separated by column chromatography (petroleum ether: ethyl acetate, 20:1 to 10:1) to obtain an oily compound having the structure shown in Formula 14 ( Figure 2 Compound 14), 1.1 g, yield 51%.

[0179] 1 H NMR (400MHz, CDCl3) 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 CNMR(100MHz, CDCl3)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 NO6F3SiK[M+K] + :508.1734,found508.1734.

[0180] Compound 14 (58 mg, 0.12 mmol) was dissolved in 3.2 mL of tetrahydrofuran, acetic acid (65.8 μL, 1.25 mmol) was added, and tetrabutylammonium fluoride trihydrate (157.7 mg, 0.50 mmol) was added under argon protection at 0°C. After stirring at 50°C for 4 hours, the system was concentrated to half of the original volume, extracted with dichloromethane and saturated brine, the organic phase was dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was separated by column chromatography (petroleum ether: ethyl acetate, 2:1 to 1:1), and the oily substance was a compound with the structure shown in Formula 15 ( Figure 2 Compound 15) 38 mg, yield 87%.

[0181] 1 H NMR (400MHz, CDCl3) 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, CDCl3)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.CalcdforC 14 H 20 NO6F3Na[M+Na] + :378.1135, found 378.1134.

[0182] Compound 15 (681 mg, 1.92 mmol) was dissolved in 3.2 mL of dichloromethane, TEMPO (29 mg, 0.19 mmol) and BAIB (679 mg, 2.11 mmol) were added, and the mixture was stirred at 40 °C for 6 hours. Diluted with 15 mL of dichloromethane, 20 mL of a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate was added, and the mixture was stirred vigorously for 10 min. Extracted with dichloromethane, dried over sodium sulfate, and evaporated to dryness. The residue was dissolved in 6.3 mL of pyridine, MeONH2·HCl (240 mg, 2.85 mmol) was added, stirred at room temperature for 1 hour, and evaporated to dryness. The residue was separated by column chromatography (petroleum ether: ethyl acetate, 10:1 to 4:1) to obtain a white solid having the structure of Formula 16 ( Figure 2 Compound 16), 735 mg, two-step reaction yield 100% (Z / E=1 / 2 or 2 / 1).

[0183] 1 H NMR (400MHz, CDCl3) 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,3 H),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 N2O6F3K[M+K] + :421.0978,found421.0982.

[0184] Compound 16 (279 mg, 0.73 mmol) was dissolved in a mixed solution of glacial acetic acid / methanol (2.4 mL / 2.4 mL), and NaCNBH3 (69 mg, 1.09 mmol) was added at 0°C under argon protection. The mixture was stirred for 4 hours and the reaction was completed. A small amount of toluene was added and the mixture was evaporated to dryness. The residue was separated by column chromatography (petroleum ether: ethyl acetate, 2:1) to obtain a white solid glycosyl receptor with the structure shown in formula 2-2-1 ( Figure 2 Compound 17) 268 mg, yield 96%.

[0185] 1 H NMR (400MHz, CDCl3) 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); 13C NMR(100MHz, CDCl3)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 N2O6F3[M+H] + :385.1581, found 385.1570.

[0186] The glycosyl donor ( Figure 2 Compound 10) (1209 mg, 1.98 mmol), glycosyl acceptor of the structure shown in 2-2-1 (378 mg, 0.98 mmol) and 1.6 g The molecular sieve was dissolved in 16.1 mL of dichloromethane under nitrogen protection and stirred at room temperature for 1 hour. The reaction system was cooled to 0°C and TMSOTf (26 μL, 0.15 mmol) was added. After TLC showed that the glycosyl acceptor (compound 17) with the structure shown in formula 2-2-1 was basically reacted, a drop of triethylamine was added to quench the reaction. After the reaction system was warmed to room temperature, the reaction system was filtered with diatomaceous earth. The filtrate was evaporated to dryness. The residue was separated by column chromatography (petroleum ether: acetone, 1:1) to obtain an oily substance. The oily substance was dissolved in 7.9 mL of methanol, PPTS (373 mg, 1.49 mmol) was added to the reaction system, stirred at 65°C for 3 hours, evaporated to dryness, and the residue was separated by column chromatography (petroleum ether: acetone, 1:2, then toluene: methanol = 10:1 to 5:1) to obtain an oily substance as a compound with the structure shown in formula 5-2-1 ( Figure 2 Compound 18), 319 mg, two-step yield 40%.

[0187] 1H NMR(400MHz,CDCl3)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,CDCl3)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 N3O 18 F3[M+H] + :818.2801,found 818.2808。

[0188] Compound 18 (20 mg, 0.024 mmol) was dissolved in 1.1 mL methanol, stirred at room temperature, and 0.02 mL 30% sodium methoxide was added. After 1 hour, TLC showed that the reaction was complete. After concentrating and draining the solvent, 0.6 mL 2M sodium hydroxide aqueous solution was added. After 0.5 hours, TLC showed that the reaction was complete. Carbon dioxide gas was introduced until neutral and evaporated to dryness. The residue was dissolved in 1.2 mL methanol, and 0.5 mL triethylamine and 0.2 mL methyl fluoroacetate were added under argon protection. The mixture was refluxed and stirred overnight. After evaporation, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1:4), and then the residual triethylamine salt was removed by an ion exchange resin column to obtain a white solid with a nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in formula 1-2, 11 mg, and a yield of 75%.

[0189] 1 H NMR(400MHz,D2O)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),4.0 4-3.94(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(d d,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,D2O)δ174.96,173.12,171.09(d,J=18.6Hz),133.61,118.14,96.49,95.70,79.77(d,J=179.6Hz),72.92,7 1.97,69.18,68.81,68.75,68.67,68.49,67.63,64.05,62.66,52.96,51.87,49.60,37.74,22.06; HRMS(ESI)Anal.Calcd for C 23 H 38 N3O 14 FNa[M+Na] + :622.2230, found 622.2229.

[0190] Example 3

[0191] according to Figure 2 Example 2 shows a synthetic flow chart of a sialic acid (α-(2→6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-2:

[0192] The compound of the structure shown in Formula 5-2-1 was prepared according to the preparation method in Example 2;

[0193] The compound of the structure shown in Formula 5-2-1 ( Figure 2 Compound 18) (20 mg, 0.024 mmol) was dissolved in 1.1 mL of methanol. After stirring at room temperature, 0.02 mL of 30% sodium methoxide was added. After 1 hour, TLC showed that the reaction was complete. After concentrating and draining the solvent, 0.6 mL of 2M sodium hydroxide aqueous solution was added. After 0.5 hours, TLC showed that the reaction was complete. Carbon dioxide gas was introduced until neutral and evaporated to dryness. The residue was dissolved in 1.2 mL of methanol, and 0.5 mL of triethylamine and 0.2 mL of methyl difluoroacetate were added under argon protection. The mixture was refluxed and stirred overnight. After evaporation, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1:4), and then the residual triethylamine salt was removed by an ion exchange resin column to obtain a white solid with a nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose derivative of the structure shown in formula 1-3, 13 mg, and a yield of 86%.

[0194] 1 H NMR (400MHz, D2O) 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); 13C NMR(100MHz,D2O)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 N3O 14 F2Na[M+Na] + :640.2136,found 640.2139.

[0195] Example 4

[0196] according to Figure 2 Example 3 shows a synthetic flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in Formula 1-3:

[0197] The compound of the structure shown in Formula 5-2-1 was prepared according to the preparation method in Example 2;

[0198] The compound of the structure shown in Formula 5-2-1 ( Figure 2 Compound 18) (40 mg, 0.049 mmol) was dissolved in 2.3 mL of methanol. After stirring at room temperature, 0.02 mL of 30% sodium methoxide was added. After 1 hour, TLC showed that the reaction was complete. After concentrating and draining the solvent, 1.1 mL of 2M sodium hydroxide aqueous solution was added. After 0.5 hours, TLC showed that the reaction was complete. Carbon dioxide gas was introduced until neutral and evaporated to dryness. The residue was dissolved in 2.4 mL of methanol, and 1.0 mL of triethylamine and 0.5 mL of methyl trifluoroacetate were added under argon protection. The mixture was refluxed and stirred overnight. After evaporation, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1:4), and then the residual triethylamine salt was removed by an ion exchange resin column to obtain a white solid with a nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose derivative of the structure shown in formula 1-4, 18 mg, and a yield of 58%.

[0199] 1H NMR(400MHz,D2O)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,D2O)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 N3O 14 F3[M+H] + :636.2222, found 636.2232.

[0200] Example 5

[0201] according to Figure 2 Example 5 shows a synthetic flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in Formula 1-5:

[0202] The compound of the structure shown in Formula 5-2-1 was prepared according to the preparation method in Example 2;

[0203] The compound of the structure shown in Formula 5-2-1 ( Figure 2Compound 18) (40 mg, 0.049 mmol) was dissolved in 2.3 mL of methanol. After stirring at room temperature, 0.02 mL of 30% sodium methoxide was added. After 1 hour, TLC showed that the reaction was complete. After concentrating and draining the solvent, 1.1 mL of 2M sodium hydroxide aqueous solution was added. After 0.5 hours, TLC showed that the reaction was complete. Carbon dioxide gas was introduced until neutral and evaporated to dryness. The residue was dissolved in 4.9 mL of methanol, propionic anhydride (25.2 μL, 0.20 mmol) was added at 0°C, and stirred for 0.5 hours. After evaporation, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1:4) to obtain a white solid with a nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose derivative of the structure shown in formula 1-5, 131 mg, and a yield of 100%.

[0204] 1 H NMR(400MHz,D2O)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,D2O)δ178.60,174.95,173.11,133.60,118.14,96.57,95.67,72.91,71.95,69.20,68.87,68.7 3,68.65,68.42,67.71,64.02,62.65,52.99,51.86,49.80,37.74,29.12,22.05,9.57; HRMS(ESI)Anal.Calcd forC 24 H 41 N3O 14 Na[M+Na] + :618.2481, found 618.2487.

[0205] Example 6

[0206] according to Fig.10 The glycoprotein conjugate synthesis process is as follows:

[0207] The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives of formula 1-1 to formula 1-5 prepared in Examples 1 to 5 and STn of formula 17 were dissolved in 5 mL of anhydrous methanol, respectively, and air containing ozone was introduced at -72°C. When the system turned blue (about 10-30 minutes), the ozone was stopped, and the system remained blue after 10 minutes. Nitrogen was introduced into the reaction system for about 10 minutes to remove excess ozone. 0.2 mL of dimethyl sulfide was added dropwise, and the temperature of the reaction system was then allowed to rise naturally to room temperature. After 2 hours, the reaction system was freed of solvent under vacuum to obtain a hapten containing an aldehyde group.

[0208] The hapten containing an aldehyde group was dissolved with KLH in a buffer solution with a pH value of 7.6, sodium cyanoborohydride was added, and the mixture was reacted on a shaker at room temperature in the dark for 24 hours. After dialysis, the glycoprotein conjugate N(OMe)-STn-KLH was obtained, which was recorded as 1-KLH, 2-KLH, 3-KLH, 4-KLH, and 5-KLH.

[0209]

[0210] Example 7

[0211] The preparation method is basically the same as that of Example 6, except that CRM197 is used to replace KLH in Example 6 to obtain glycoprotein conjugate N(OMe)-STn-KLH, which are respectively recorded as 1-CRM197, 2-CRM197, 3-CRM197, 4-CRM197, and 5-CRM197.

[0212] Example 8

[0213] according to Fig.11 The synthetic process of the glycoprotein conjugate 1-NHS-CRM197 is as follows:

[0214] 10 mg of the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative of the structure shown in Formula 1-5 prepared in Example 5 and 2.88 mg of mercaptoethylamine hydrochloride were dissolved in 1 mL of deoxygenated deionized water, and reacted under ultraviolet irradiation at room temperature for 10 minutes, then concentrated under reduced pressure, purified using a dextran G10 gel column, and the purified product was dissolved in 1 mL of ultra-dry DMF, and dropped into 1 mL of ultra-dry DMF containing 57.8 mg of bis(2,5-dioxopyrrolidin-1-yl) adipate, and vigorously stirred for reaction at room temperature for 2 hours. After the reaction was completed, it was concentrated under reduced pressure, re-dissolved in methanol, separated by HPLC, and freeze-dried to obtain a white solid product hapten N(OMe)-STn-NHS.

[0215] N(OMe)-STn-NHS and CRM197 were dissolved together in K2HPO4-PBS buffer at pH=8.0, reacted on a shaker at room temperature for 12 hours, and the glycoprotein conjugate 1-NHS-CRM197 was obtained after ultrafiltration.

[0216] Application Examples

[0217] 1. Test materials and sources

[0218] 1. Test compound: glycoprotein (polypeptide) conjugate prepared in Examples 6, 7 and 8 of the present invention;

[0219] 2. Test methods

[0220] (I) Mouse immunization

[0221] Six Balb / c female mice, 6-8 weeks old (Number: SCXKjing2007-0001, SPF / VAF) were purchased from the Department of Animal Science, Peking University Health Science Center and raised in the animal department. Mice were immunized with STn-KLH, STn-CRM197, and conjugates of STn derivatives linked to nitrogen and KLH or CRM197 (1-KLH, 2-KLH, 3-KLH, 4-KLH, 5-KLH and 1-CRM197, 2-CRM197, 3-CRM197, 4-CRM197, 5-CRM197), and the glycoprotein (peptide) contained 1-3 μg of sugar (dissolved in PBS) in each immunization. Immunization was once every 2 weeks, and the immunization route was intraperitoneal injection, for a total of 4 immunizations. Blood was collected before immunization, 13 days after the second immunization, 13 days after the third immunization, and 14 days after the fourth immunization, and the serum was separated and stored in a -80°C refrigerator for testing.

[0222] (II) Determination of antibody titers in mouse serum before and after immunization

[0223] The titer of the mixed serum of each group of mice, as well as the titer of the serum of each mouse in the 1-KLH, 2-KLH, 3-KLH, 4-KLH, 5-KLH and 1-CRM197, 2-CRM197, 3-CRM197, 4-CRM197, 5-CRM197 immunization groups prepared in Examples 6 and 7 were detected by ELISA.

[0224] 1 Antigen coating: 100 μL STn-BSA (containing 0.02 μg STn) was coated on the ELISA plate at 4°C overnight.

[0225] 2 Washing and blocking: Add 200 μL of washing buffer PBS-Tween20 (0.05%) to each well to wash the plate, wash 3 times, then add 200 μL of blocking solution (3% BSA-PBS) to each well, incubate at 37°C for 1 hour.

[0226] 3. Add primary antibody (i.e. immune serum): Wash 3 times (specific method is the same as above). Serum is diluted with antibody diluent (1% BSA-PBS) starting from a certain dilution, and 100 μL is added to each well, incubated at 37°C for 1 hour.

[0227] 4 Add enzyme-labeled secondary antibody: wash three times, add 100 μL of secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (γ-chain specific)) diluted 5000 times with antibody diluent to each well, and incubate at 37°C for 1 hour.

[0228] 5 Color development: Wash three times, add 100 μL of the prepared color development substrate o-phenylenediamine (OPD) to each well, develop the color for 15 minutes at room temperature in the dark, and add 2M H2SO4 to each well to stop the color development.

[0229] 6. Result determination: Read the OD value at a wavelength of 490 nm using an ELISA reader. The serum dilution factor when the OD value after subtracting the blank serum well reading is 0.1 is used as the antibody titer.

[0230] (III) Immunotherapy in mice

[0231] Eight Balb / c female mice, 6-8 weeks old (Number: SCXKjing2007-0001, SPF / VAF), were purchased from the Department of Animal Science, Peking University Health Science Center and raised in the animal department. On day 0, 5×10^5 CT26 cells were inoculated under the armpit of each mouse, and PBS solutions containing 1-NHS-CRM197, 1-CRM197 and 1-KLH were subcutaneously injected on days 2, 6, 10 and 17, respectively.

[0232] 3. Test results

[0233] The test results are shown in Table 1 and Figures 3 to 6 As shown. Among them, Figure 3The titer of the serum of each mouse in the STn-KLH and 1-KLH groups after the fourth immunization with 1-KLH prepared in Example 6 of the present invention; Figure 4 This is the survival curve of mice after administration of 1-KLH prepared in Example 6 of the present invention; Figure 5 This is the tumor growth curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention; Figure 6 This is the survival curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention. Figure 7 This is the tumor growth curve of mice after administration of 1-NHS-CRM197 prepared in Example 8 of the present invention.

[0234] Table 1 shows the antibody titers recognizing STn in mouse serum measured 13 days after the third and fourth immunizations of the sugar conjugates prepared in Example 6

[0235]

[0236] Table 2 is a comparison of the sugar loading amounts of 1-CRM197 prepared in Example 7 and 1-NHS-CRM197 prepared in Example 8.

[0237] Table 2 Sugar loading of 1-CRM197 and 1-NHS-CRM197

[0238]

[0239] A mouse tumor model was constructed using CT-26 colon cancer cells expressing STn glycogen antigens; then the tumor-bearing mice were immunized with the glycoconjugates (1-KLH, 1-CRM197 or 1-NHS-CRM197) in the present invention, and the survival period, tumor volume and antibody titer of the mice were observed. The experimental results showed that compared with the control group, the glycoconjugates 1-KLH, 1-CRM197 or 1-NHS-CRM197 significantly prolonged the survival period of the mice, inhibited tumor growth, and increased antibody titer after inoculation of mice, indicating that they had a good anti-tumor effect.

[0240] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative has a structure shown in Formula 1-1:

2. The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof according to claim 1, characterized in that The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative salt is a salt formed by the reaction of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative with the structure shown in Formula 1-1 and a base.

3. The method for preparing the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative according to claim 1 or 2, characterized in that: The following steps are involved: The glycosyl acceptor of the structure shown in Formula 2-1-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1; The coupling product of the structure shown in Formula 4-1, a polar solvent and an acidic catalyst are mixed to perform debenzylide protection to obtain a debenzylide coupling product of the structure shown in Formula 5-1; The debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and an alkaline catalytic agent are mixed and selectively deacetylated to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyranose galactose derivative.

4. A sugar conjugate, characterized in that: The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt according to claim 1 or 2, or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method according to claim 3, is coupled with a polypeptide or a carrier protein through different linkers.

5. The method for preparing the sugar conjugate according to claim 4, characterized in that: The following steps are involved: The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof is dissolved in a polar solvent, and an oxidizing gas is introduced to perform an oxidation reaction or an N-hydroxysuccinimide group is introduced by extending the carbon chain to obtain a disaccharide containing an aldehyde group or an N-hydroxysuccinimide group; The disaccharide containing an aldehyde group or an N-hydroxysuccinimide group, a protein or a polypeptide, a reducing agent and a buffer solution are mixed and subjected to a coupling reaction to obtain the sugar conjugate.

6. Use of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt according to claim 1 or 2, or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method according to claim 3 in the preparation of an anti-tumor drug, wherein the anti-tumor drug is a drug for treating tumors that can specifically express STn.

7. Use of the sugar conjugate according to claim 4 or the sugar conjugate prepared by the preparation method according to claim 5 in the preparation of an anti-tumor drug, wherein the anti-tumor drug is a drug for treating tumors that can specifically express STn.

8. A vaccine for treating tumors, comprising the sugar conjugate according to claim 4 or the sugar conjugate prepared by the preparation method according to claim 5 and a pharmaceutically acceptable carrier or excipient.

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