CN115521348A - Sialic acid (α-(2→6))-D-aminogalactopyranose derivative or salt thereof, glycoconjugate and preparation method thereof - Google Patents

Abstract

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

Description

Sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or their salts, sugar conjugates substances and their preparation methods

technical field

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

Background technique

疫苗，用于预防和治疗结直肠癌和乳腺癌转移。但是在进行III期临床试验时发现单独应用

不能改善疾病进展时间和总体存活率，而仅当将其与激素合用时才可以显示一定效果，使生存期由单独应用激素的5.8个月提高到8.3个月。由于

的抗肿瘤活性依赖于激素的存在，使其抗肿瘤活性受到影响(Holmberg,L.ExpertRev.Vaccines 2004,3,655-663.)。Research on anti-tumor vaccines based on carbohydrate antigens has become a promising research direction in the field of tumor immunotherapy. Among them, the sugar antigen STn is a disaccharide structure containing sialic acid, which is mostly 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, Canada Biomira developed the STn-KLH (keyhole limpet hemocyanin) conjugate—

Vaccines for the prevention and treatment of colorectal and breast cancer metastases. However, during the phase III clinical trial, it was found that the single application

It cannot improve the time of disease progression and the overall survival rate, but only when it is combined with hormones can it show a certain effect, so that the survival period can be increased from 5.8 months to 8.3 months when hormones are used alone. because

The anti-tumor activity of Vaccine depends on the presence of hormones, which affects its anti-tumor activity (Holmberg, L. Expert Rev. Vaccines 2004, 3, 655-663.).

遇到的困难类似，现今抗肿瘤糖疫苗遇到的主要问题就是疫苗不能在体内产生有效的免疫应答。same

The difficulties encountered are similar. The main problem encountered by anti-tumor sugar vaccines today is that the vaccines cannot produce effective immune responses in vivo.

Contents of the invention

The object of the present invention is to provide sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof, glycoconjugates and preparation methods thereof, sialic acid (α-( 2→6))-D-aminogalactopyranose derivatives or their salts can produce more effective immune responses and produce more antibodies through the glycoconjugates (sugar antigens) obtained by coupling proteins with different linkers. The anti-tumor vaccine has shown good activity; thus it can prolong the survival period and achieve the anti-tumor effect.

The present invention provides a nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof, the nitrogen-linked sialic acid (α-(2→6)) -D-aminogalactopyranose derivatives have the structure shown in formula 1:

In Formula 1, R ₁ is an amide group or -NH ₂ ; the amide group is -NHC(O)CH _p Cl _q , -NHC(O)CH _p F _q , -NHC(O)CH p Br 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 are 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;

R2 is a group with _double bond, alkyne bond, azido group, aldehyde group, protected acetal group, maleimide group, N-hydroxysuccinimide group, mercapto group, protected mercapto group, selenoyl group, protected selenoyl group , -NH ₂ or -ONH ₂ substituents.

Preferably, the R ₁ is -NHC(O)CH _p F _q or -NHC(O)C _a H _2a+1 ; the R ₂ is allyloxy.

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

Preferably, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative salt is a nitrogen-linked sialic acid (α-(2→6))- The salt formed by the reaction of D-aminogalactopyranose derivatives with base.

The present invention provides a method for preparing nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives described in the above technical scheme,

When said R ₁ is -NHC(O)CH ₃ , it may comprise the following steps:

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

Mix the coupling product of the structure shown in formula 4-1, a polar solvent and an acidic catalytic reagent, and perform debenzylidene protection to obtain the debenzylidene coupling product of the structure shown in formula 5-1;

Mix the debenzylidene coupling product of the structure shown in formula 5-1, a polar solvent and a basic catalytic reagent for selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))- D-aminogalactopyranose derivatives;

When said R ₁ is -NH ₂ , it may comprise the following steps:

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

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

Mixing the debenzylidene coupling product of the structure shown in formula 5-2, a polar solvent and a basic catalytic reagent, and performing selective deacetylation to obtain the selective deacetylation coupling product of the structure of formula 6;

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 for detrifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→ 6))-D-aminogalactopyranose derivatives;

When said R ₁ is an amide group other than -NHC(O)CH ₃ , it may comprise the following steps:

The selective deacetylation coupling product of the structure described in formula 6, a polar solvent, an organic base and an acylating agent are mixed for detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-( 2→6))-D-aminogalactopyranose derivative; the acylating agent is an acid anhydride, carboxylic acid or carboxylic acid ester corresponding to R ₁ .

The present invention provides a glycoconjugate consisting of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the above technical scheme The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method is coupled with a polypeptide or a carrier protein through different linkers.

The present invention provides a method for preparing the glycoconjugate described in the above technical scheme, comprising the following steps:

The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme is dissolved in a polar solvent, and an oxidizing gas is passed through for oxidation reaction or by Extending the carbon chain and introducing N-hydroxysuccinimide groups to obtain disaccharides containing aldehyde groups or N-hydroxysuccinimide groups;

Mix the disaccharide containing aldehyde group or N-hydroxysuccinimide group, protein or polypeptide, reducing agent and buffer solution, and carry out coupling reaction to obtain the sugar conjugate.

The present invention provides the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative described in the above technical scheme or the nitrogen-linked sialic acid ( Application of α-(2→6))-D-aminogalactopyranose derivatives or salts thereof in the preparation of antitumor drugs.

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

The present invention provides a vaccine for treating tumors, comprising the glycoconjugate described in the above technical solution or the glycoconjugate prepared by the preparation method described in the above technical solution and a pharmaceutically acceptable carrier or auxiliary material.

The present invention provides a nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof, the nitrogen-linked sialic acid (α-(2→6)) -D-aminogalactopyranose derivatives have the structure shown in formula 1. The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt of the structure shown in formula 1 provided by the present invention uses a nitrogen bridge (N(OMe)) in the structure of the compound The connection replaces the oxygen bridge (O) connection in the structure of the disaccharide antigen, has a novel structure, and shows good activity in anti-tumor vaccines. Experiments on mice have shown that the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or salt thereof provided by the present invention can be coupled with a carrier protein or polypeptide The resulting glycoprotein (glycopeptide) conjugate, the vaccine using the glycoprotein (glycopeptide) conjugate as a carbohydrate antigen can produce a more effective immune response, produce more specific antibodies, and can specifically recognize and express STn tumor cells, so as to achieve the effect of anti-tumor; further, compared with the structure connected by oxygen bridge (O), the nitrogen-linked sialic acid (α-(2→6) )-D-aminogalactopyranose derivatives or their salts prepared sugar antigen obtained by the vaccine obtained recognition of STn antibody titer significantly improved, measured 13 days after the third immunization, the structure shown in formula 1 of the present invention nitrogen connection The antibody titer of recognizing STn obtained from the sugar antigen prepared by the sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt is 4812, the oxygen bridge (O) linked The antibody titer of recognizing STn obtained by the vaccine obtained from the sugar antigen prepared by the hapten of the structure is 1458; 13 days after the fourth immunization, the nitrogen-linked sialic acid (α-(2→6 ))-D-aminogalactopyranose derivatives or salts thereof prepared sugar antigen obtained by the vaccine to recognize STn antibody titer is 89288, oxygen bridge (O) linked structure hapten prepared sugar antigen obtained The titer of antibody recognizing STn obtained by the vaccine was 5716. Moreover, the vaccine obtained from the sugar antigen prepared by the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt of the structure shown in formula 1 provided by the present invention is effective in tumor-bearing mice In the model test, the survival period of mice can be significantly prolonged. As can be drawn from the results in Figure 6, the nitrogen-linked sialic acid (α-(2→6))-D-aminopyran half of the structure shown in formula 1 provided by the invention The survival period of the vaccine obtained from the sugar antigen prepared by the lactose derivative or its salt was 115 days in the 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 framework structure for the research and development of anti-tumor sugar vaccines, and is expected to promote the development of anti-tumor sugar vaccines.

相比，本发明糖缀合物产生抗体的滴度增加了3倍到15倍，疫苗接种后荷瘤小鼠生存期明显延长。无论是抗体滴度还是小鼠生存期都是明显升高的，有望推动抗肿瘤糖疫苗的发展。The present invention provides a glycoconjugate consisting of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the above technical scheme The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method is coupled with a polypeptide or protein. Compared with the structural galactose derivatives connected by oxygen bridge (O), the vaccine obtained by the glycoconjugate provided by the present invention, as an anti-tumor vaccine, produced a strong anti-tumor vaccine in mice. immune response, and

In comparison, the antibody titer produced by the glycoconjugate of the present invention is increased by 3 to 15 times, and the survival period of tumor-bearing mice after vaccination is obviously prolonged. Both the antibody titer and the survival period of the mice were significantly increased, which is expected to promote the development of anti-tumor sugar vaccines.

Description of drawings

Fig. 1 is a flow chart for the synthesis of sialic acid (α-(2→6))-D-aminogalactopyranose derivatives with the structure shown in formula 1-1 provided in Example 1 of the present invention;

Fig. 2 is a flow chart for the synthesis of sialic acid (α-(2→6))-D-aminogalactopyranose derivatives with structures shown in formulas 1-2 to 1-5 in Examples 2 to 5 of the present invention;

Fig. 3 is the titer of each mouse serum in the STn-KLH and 1-KLH groups after the fourth immunization of 1-KLH prepared in Example 6 of the present invention;

Fig. 4 is the mouse survival curve after administration of 1-KLH prepared in Example 6 of the present invention;

Figure 5 is the mouse tumor growth curve after administration of 1-CRM197 prepared in Example 7 of the present invention;

Figure 6 is the mouse survival curve after administration of 1-CRM197 prepared in Example 7 of the present invention;

Figure 7 is the mouse tumor growth curve after administration of NSTn-NHS-CRM197 prepared in Example 8 of the present invention;

FIG. 8 is a flow chart of the synthesis of the glycosyl acceptor with the structure shown in Formula 7 in Example 1 of the present invention;

Fig. 9 is a synthesis flow diagram of a glycosyl donor with the structure shown in formula 3 in the embodiment of the present invention;

Figure 10 is a flow chart of the synthesis of glycoprotein conjugates in the examples of the present invention;

Fig. 11 is a flow chart of the synthesis of the glycoprotein conjugate 1-NHS-CRM197 in the example of the present invention.

detailed description

The present invention provides a nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or a salt thereof, the nitrogen-linked sialic acid (α-(2→6)) -D-aminogalactopyranose derivatives have the structure shown in formula 1:

In Formula 1, R ₁ is an amide group or -NH ₂ ; the amide group is -NHC(O)CH _p Cl _q , -NHC(O)CH _p F _q , -NHC(O)CH p Br 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 are 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;

R2 is a group with _double bond, alkyne bond, azido group, aldehyde group, protected acetal group, maleimide group, N-hydroxysuccinimide group, mercapto group, protected mercapto group, selenoyl group, protected selenoyl group , -NH ₂ or -ONH ₂ substituents.

In the present invention, the protected acetal group is a substituent with a protecting group on the acetal group, and the protected thiol is a substituent with a protecting group on the mercapto; the protected selenoyl is a substituent with a protecting group on the selenium. Substituents. The present invention has no special requirements on the type of the protecting group.

In the present invention, the R ₁ is preferably -NHC(O)CH _p F _q or -NHC(O)C _a H _2a+1 ; the R ₂ is preferably allyloxy.

In a specific embodiment of the present invention, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative has the structure shown in formula 1-1 to formula 1-5 Either:

In the present invention, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative salt is a nitrogen-linked sialic acid (α-(2→6) )-D-aminogalactopyranose derivatives react with bases to generate salts.

The present invention provides a method for preparing nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives described in the above technical scheme,

When said R ₁ is -NHC(O)CH ₃ , it may comprise the following steps:

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

Mix the coupling product of the structure shown in formula 4-1, a polar solvent and an acidic catalytic reagent, and perform debenzylidene protection to obtain the debenzylidene coupling product of the structure shown in formula 5-1;

Mix the debenzylidene coupling product of the structure shown in formula 5-1, a polar solvent and a basic catalytic reagent for selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))- D-aminogalactopyranose derivatives;

When said R ₁ is -NH ₂ , it may comprise the following steps:

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

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

Mixing the debenzylidene coupling product of the structure shown in formula 5-2, a polar solvent and a basic catalytic reagent, and performing selective deacetylation to obtain the selective deacetylation coupling product of the structure of formula 6;

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 for detrifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→ 6))-D-aminogalactopyranose derivatives;

When said R ₁ is an amide group other than -NHC(O)CH ₃ , it may comprise the following steps:

The selective deacetylation coupling product of the structure described in formula 6, a polar solvent, an organic base and an acylating agent are mixed for detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-( 2→6))-D-aminogalactopyranose derivative; the acylating agent is an acid anhydride, carboxylic acid or carboxylic acid ester corresponding to R ₁ .

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

In the present invention, when the R ₁ is -NHC(O)CH ₃ , the preparation method of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative comprises The following steps:

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

Mix the coupling product of the structure shown in formula 4-1, a polar solvent and an acidic catalytic reagent, and perform debenzylidene protection to obtain the debenzylidene coupling product of the structure shown in formula 5-1;

Mix the debenzylidene coupling product of the structure shown in formula 5-1, a polar solvent and a basic catalytic reagent for selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))- D-aminogalactopyranose derivatives.

In the present invention, 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 are mixed for glycosylation coupling. Linkage reaction (hereinafter referred to as glycosylation coupling reaction) to obtain the coupling product with the structure shown in formula 4-1.

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

In the present invention, the preparation method of the glycosyl acceptor with the structure shown in formula 2-1-1 preferably includes the following steps:

Mix the compound of structure shown in formula 7, camphorsulfonic acid and polar solvent to carry out 3,4 position benzylidene protection, obtain the compound of structure shown in formula 8; Compound of structure shown in formula 8, tetramethyl piperidine oxide (TEMPO ), iodobenzenediacetic acid (BAIB) and a polar solvent are mixed and carried out 6-position selective oxidation to obtain a structural compound shown in formula 9; in a protective gas atmosphere, the structural compound shown in formula 9, NaCNBH ₃ and a polar solvent are mixed The double bond reduction reaction is carried out to obtain the sugar acceptor with the structure shown in formula 2-1-1.

In the present invention, the compound with the structure shown in formula 7, camphorsulfonic acid and polar solvent are mixed for 3 and 4 benzylidene protection to obtain the compound with the structure shown in formula 8. In the present invention, the preparation method of the structure compound shown in formula 7 preferably comprises the following steps: the compound (galactosamine hydrochloride) of structure shown in formula 10, acetic anhydride and carbonate radical type strong basic resin are dissolved in methanol and In a mixed solvent of water, react under ice-water bath conditions to obtain a compound of the structure shown in formula 11; mix the compound of the structure shown in formula 11, allyl alcohol and the ethanol solution of boron trifluoride, and then add the ethanol solution of HCl , react under the condition of reflux to obtain the compound of the structure shown in formula 7;

In a specific embodiment of the present invention, the preparation method of the structural compound shown in formula 11 is specifically preferably: the commercially available galactosamine hydrochloride (2.5g, 11.6mmol) shown in formula 10 and 5.0g carbonate-type strong Basic resin, 58mL water, and 6mL methanol were mixed, stirred in an ice bath, and 1.5mL acetic anhydride was added dropwise. After 2h, filter with suction and wash the resin. The mother liquor was concentrated and passed through a strong acid resin column, and evaporated to dryness to obtain the compound shown in formula 11, which was directly used in the next reaction without purification. Add the prepared compound of formula 11, 0.32 mL of BF ₃ , and Et ₂ O into 28 mL of allyl alcohol, and stir at reflux for 2 h. Then add 0.5 mL of HCl in Et ₂ O solution, and continue to reflux for 1 h. After cooling, diethyl ether was added until cloudiness appeared, and it was left overnight at 4°C. After filtering and washing with ether, 0.9 g of a white solid was obtained, which was the compound of formula 7, and the yield of the two-step reaction was 30%.

In the present invention, the molar ratio of the compound represented by 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 on the amount of the polar solvent to ensure that the 3 and 4-position benzylidene protection is carried out smoothly That's it. The reaction temperature for the protection of the 3 and 4-position benzylidene is preferably room temperature, and the reaction holding time is preferably 22 hours. After the 3,4-position benzylidene protection reaction, the 3,4-position benzylidene protection reaction solution is obtained. In the present invention, the 3,4-position benzylidene protection reaction solution is preferably post-treated to obtain the compound with the structure shown in formula 8. The post-treatment preferably includes: mixing the 3,4-position benzylidene protection reaction solution and saturated aqueous sodium bicarbonate solution to obtain a mixed solution; mixing and extracting the mixed solution and an organic solvent to obtain an extracted organic phase; combining the obtained extracted organic phase The phase was dried and then concentrated to obtain a concentrated solution; the concentrated solution was subjected to column chromatography to obtain a compound of the structure shown in Formula 8. The organic solvent is specifically preferably dichloromethane. The drying agent is preferably anhydrous sodium sulfate. The drying preferably concentrates the organic extract phase of the solid-liquid separation, and the solid-liquid separation is specifically preferably filtering. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 2:1.

After obtaining the structural compound shown in formula 8, the present invention mixes the structural compound shown in formula 8, tetramethylpiperidine oxide (TEMPO), iodobenzenediacetic acid (BAIB) and a polar solvent for selective oxidation at the 6-position to obtain Structural compound shown in formula 9. In the present invention, the molar ratio of the compound of formula 8, TEMPO and BAIB is preferably 0.61:0.06:0.55. The polar solvent is preferably dichloromethane, and the present invention has no special requirements on the amount of the polar solvent, as long as the selective oxidation reaction at the 6-position is ensured to proceed smoothly. The temperature of the 6-position selective oxidation reaction is preferably room temperature, and the holding time of the 6-position selective oxidation reaction is preferably 3 hours. In the present invention, the 6-position selective oxidation reaction is used to obtain a 6-position selective oxidation reaction liquid, and in the present invention, the 6-position selective oxidation reaction liquid is preferably post-treated to obtain the structural compound shown in formula 9. The post-treatment preferably includes: mixing the 6-position selective oxidation reaction liquid and saturated aqueous sodium bicarbonate solution to obtain a mixed solution; mixing and extracting the mixed solution and an organic solvent to obtain an extracted organic phase; drying the combined extracted organic phase Finally, the solvent was removed to obtain a residue; the residue was subjected to column chromatography to obtain a compound of the structure shown in formula 9. The organic solvent is specifically preferably dichloromethane. The drying agent is preferably anhydrous sodium sulfate. The drying preferably concentrates the organic extract phase of the solid-liquid separation, and the solid-liquid separation is specifically preferably filtering. The solvent removal is preferably 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 and ethyl acetate is preferably 1:2.

After obtaining the structural compound shown in Formula 9, the present invention mixes the structural compound shown in Formula 9, NaCNBH ₃ and a polar solvent in a protective gas atmosphere to perform a double bond reduction reaction to obtain the sugar with the structure shown in Formula 2-1-1 Base receptors. In the present invention, the molar ratio of the compound represented by formula 9 to NaCNBH ₃ is preferably 2.14:3.19. The polar solvent is preferably a mixed solvent of acetic acid and methanol, and the volume ratio of the acetic acid and methanol is preferably 1:1. The present invention has no special requirements on the amount of the polar solvent to ensure that the double bond reduction reaction goes smoothly Just proceed. The temperature of the double bond reduction reaction is preferably 0° C., the holding time of the double bond reduction reaction is preferably 4 hours, and the protective gas is preferably argon. In the present invention, the double bond reduction reaction is used to obtain a double bond reduction reaction solution. In the present invention, the double bond reduction reaction solution is preferably post-treated to obtain the glycosyl acceptor with the structure shown in formula 2-1-1. The post-treatment preferably includes: mixing the double bond reduction reaction solution with an organic solvent to obtain a mixed solution; removing the solvent from the mixed solution to obtain a residue; performing column chromatography on the residue to obtain 2-1-1 Structural glycosyl acceptor shown. The organic solvent is specifically preferably toluene. The solvent removal is preferably evaporation. The eluent used in the column chromatography separation is preferably ethyl acetate.

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 for R2 being propenyloxy, and will not be repeated here.

In the present invention, the preparation method of the glycosyl donor with the structure shown in formula 3 preferably includes the following steps:

The compound with the structure shown in Formula 12 was dissolved in acetonitrile, DIPEA and (EtO) ₂ PCl were added, and the reaction was carried out in an Ar atmosphere at 0-25°C to obtain the glycosyl donor with the structure shown in Formula 3.

In a specific embodiment of the present invention, the specific preparation method of the glycosyl donor with the structure shown in formula 3 is preferably: under nitrogen protection, dissolve the compound (1.1 g, 2.20 mmol) with the structure shown in formula 12 in 20 mL of acetonitrile solution Add 0.94mL DIPEA, stir under ice-cooling, add diethylphosphorous oxychloride (0.65mL, 4.50mmol), remove the ice bath after 5min, TLC monitors that the reaction is complete, evaporate the reaction system to dryness, add ethyl acetate, Suction filtration, the filtrate was evaporated to dryness, washed twice with ethyl acetate, separated by column chromatography, eluent (V/V) petroleum ether: ethyl acetate = 1:2, to obtain the glycosyl donor with the structure shown in formula 3 Body 1.1g, yield 89%.

MS和三氟甲磺酸三甲基硅酯(TMSOTf)。In the present invention, the first coupling reagent is specifically preferably

MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

MS的质量比优选为20:100。所述极性溶剂优选为二氯甲烷，本发明对所述极性溶剂的用量没有特殊要求，确保所述第一糖基化偶联反应顺利进行即可。式2-1所示结构的糖基受体和TMSOTf的摩尔比优选为0.061:0.018。所述混合进行第一糖基化偶联反应包括：在保护气体气氛中，将式2-1所示结构的糖基受体、式3所示结构的糖基供体和

MS溶解于极性溶剂中，搅拌混合反应1h，得到混合混合溶液；将所述混合溶液降温至0℃与TMSOTf混合。本发明优选采用TLC检测式3所示结构的糖基供体完全反应后，所述第一糖基化偶联反应完毕。在本发明中，所述第一糖基化偶联反应后得到第一糖基化偶联反应液，本发明优选对所述第一糖基化偶联反应液进行后处理，得到式4-1所示结构的偶联产物。在本发明中，所述后处理优选包括：将第一糖基化偶联反应液加入三乙胺萃灭反应，将得到的猝灭反应液升温至室温；将淬灭反应液用硅藻土抽滤后得到滤液；将所述滤液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到式4-1所示结构的偶联产物。所述除溶剂优选为蒸发。所述柱层析分离优选依次采用第一洗脱溶剂洗脱和第二洗脱溶剂洗脱；所述第一洗脱溶剂优选为石油醚和丙酮，所述石油醚和丙酮的体积比优选为1:1；所述第二洗脱溶剂优选为甲苯和甲醇，所述甲苯和甲醇的体积比优选为10:1。In the present invention, the molar ratio of the glycosyl acceptor with the structure shown in formula 2-1 to the glycosyl donor with the structure shown in formula 3 is preferably 0.061:0.091. The glycosyl acceptor of the structure shown in formula 2-1 and

The mass ratio of MS is preferably 20:100. The polar solvent is preferably dichloromethane, and the present invention has no special requirements on the amount of the polar solvent, as long as the first glycosylation coupling reaction proceeds smoothly. The molar ratio of the glycosyl acceptor with the structure shown in formula 2-1 to TMSOTf is preferably 0.061:0.018. The mixing to carry out the first glycosylation coupling reaction includes: in a protective gas atmosphere, the glycosyl acceptor with the structure shown in formula 2-1, the glycosyl donor with the structure shown in formula 3 and

MS was dissolved in a polar solvent, stirred and mixed for 1 h to obtain a mixed solution; the mixed solution was cooled to 0° C. and mixed with TMSOTf. In the present invention, TLC is preferably used to detect that the first glycosylation coupling reaction is completed after the glycosyl donor with the structure shown in formula 3 is completely reacted. In the present invention, the first glycosylation coupling reaction solution is obtained after the first glycosylation coupling reaction, and the present invention preferably performs post-treatment on the first glycosylation coupling reaction solution to obtain formula 4- The coupling product of the structure shown in 1. In the present invention, the post-treatment preferably includes: adding triethylamine to the first glycosylation coupling reaction liquid to extract the reaction, and warming the obtained quenching reaction liquid to room temperature; A filtrate was obtained after suction filtration; the filtrate was desolvated to obtain a residue; the residue was separated by column chromatography to obtain a coupling product with the structure shown in formula 4-1. The solvent removal is preferably evaporation. The column chromatography separation preferably adopts the first elution solvent elution and the second elution solvent elution successively; the first elution solvent is preferably sherwood oil and acetone, and the volume ratio of the sherwood oil and acetone is preferably 1:1; the second eluting solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1.

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 catalytic reagent, and performs debenzylidene protection to obtain the coupling product of formula 5-1. The debenzylidene coupling product with the structure shown. In the present invention, the acidic catalytic reagent is preferably pyridine p-toluenesulfonate (PPTS). The mass ratio of the coupling product of the structure shown in formula 4-1 to the acidic catalytic reagent is preferably 100:47. The polar solvent is preferably methanol, and the present invention has no special requirements on the amount of the polar solvent, as long as the debenzylidene protection reaction proceeds smoothly. The temperature of the debenzylidene protection reaction is preferably 65° C., and the holding time of the debenzylidene protection reaction is preferably 3 h. In the present invention, after the debenzylidene protection, the debenzylidene protection reaction solution is obtained. In the present invention, the debenzylidene protection reaction solution is preferably post-treated to obtain the debenzylidene coupling of the structure shown in formula 5-1. product. In the present invention, the post-treatment preferably includes: removing the solvent from the debenzylidene protection reaction solution to obtain a residue; separating the residue by column chromatography to obtain the debenzylidene coupling with the structure shown in Formula 5-1 joint product. The solvent removal is preferably evaporation. The elution solvent used in the column chromatography separation is preferably ethyl acetate and methanol, and the volume ratio of ethyl acetate and methanol is preferably 15:1.

In the present invention, the debenzylidene coupling product of the structure shown in formula 5-1 is specifically preferably the debenzylidene coupling product of the structure shown in formula 5-1-1

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 a basic catalytic reagent to perform selective deacetylation The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative is obtained. In the present invention, the basic catalytic reagent 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 on the amount of methanol, as long as the selective deacetylation is ensured to proceed smoothly. In the present invention, said mixing for selective deacetylation reaction comprises the following steps: mixing the debenzylidene coupling product of the structure shown in formula 5-1, a polar solvent and sodium methoxide for the first step reaction; obtaining the reaction liquid; the reaction solution and aqueous sodium hydroxide solution are mixed for the second step reaction to obtain a selective deacetylation reaction solution; the first step reaction is preferably detected by TLC, and the first step reaction time is preferably 30min ; The second step reaction is preferably detected by TLC, and the time of the second step reaction is preferably 4h. In the present invention, the selective deacetylation reaction liquid is obtained after the selective deacetylation reaction, and the present invention preferably performs post-treatment on the selective deacetylation reaction liquid to obtain the nitrogen-linked sialic acid (α- (2→6))-D-aminogalactopyranose derivatives. In the present invention, the post-treatment preferably includes: introducing carbon dioxide into the selective deacetylation reaction solution to neutrality, removing the solvent from the obtained neutral reaction solution to obtain a residue; Separation by column chromatography to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative. The solvent removal is preferably evaporation. The column chromatography separation preferably adopts 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.

In the present invention, when the R ₁ is -NH ₂ , the preparation method of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative comprises the following steps:

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

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

Mixing the debenzylidene coupling product of the structure shown in formula 5-2, a polar solvent and a basic catalytic reagent, and performing selective deacetylation to obtain the selective deacetylation coupling product of the structure of formula 6;

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 for detrifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→ 6))-D-aminogalactopyranose derivatives.

In the present invention, the glycosyl acceptor with the structure shown in formula 2-2, the glycosyl donor with the structure shown in formula 3, a coupling reagent and a polar solvent are mixed for glycosylation coupling reaction to obtain the glycosyl group shown in formula 4-2. Structured coupling products.

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

In the present invention, the preparation method of the glycosyl acceptor with the structure shown in formula 2-2-1 preferably includes the following steps: mixing the compound with the structure shown in formula 10, a polar solvent, triethylamine and methyl trifluoroacetate Carrying out the reaction of trifluoroethyl on the 2-position to obtain a reaction solution containing a reaction product substituted by a 2-position trifluoroethyl; concentrating the reaction solution containing a reaction product substituted by a 2-position trifluoroethyl to obtain a reaction solution containing a 2-position trifluoroethyl The concentrated solution of the reaction product of replacement; The amount of substance of the compound of structure shown in formula 10 and the volume ratio of triethylamine are preferably 11.6mmol:4.1mL; The compound of structure shown in formula 10 and described methyl trifluoroacetate The molar ratio is preferably 11.6:12.6; the polar solvent is preferably methanol, and the present invention has no special requirements on the amount of the polar solvent, as long as the reaction of the trifluoroethyl group at the 2-position proceeds smoothly. The temperature of the reaction of the trifluoroethyl group at the 2-position is preferably room temperature, the holding time of the reaction of the trifluoroethyl group at the 2-position is preferably overnight, and the reaction of the trifluoroethyl group at the 2-position is preferably carried out under stirring conditions. The concentration is preferably concentration under reduced pressure.

Mix the concentrated solution containing the reaction product substituted by the 2-position trifluoroethyl, allyl alcohol and ether hydrochloride to carry out the reaction of the allylic group on the 1-position to obtain the reaction solution containing the reaction product substituted by the 1-position allyl group; The reaction liquid of the allyl-substituted reaction product is separated from the solid-liquid, and the obtained filtrate is concentrated to obtain a concentrated solution containing the 1-position allyl-substituted reaction product; the molar concentration of the diethyl ether hydrochloride is preferably 3mol/L; Formula 10 The ratio of the amount of substance of the compound of the shown structure and the volume of said diethyl ether hydrochloride is preferably 11.6mol:18.1mL; the ratio of the amount of substance of the compound of the structure shown in formula 10 and the volume of the allyl alcohol is preferably 11.6 mol: 28.9mL; the allyl reaction on the 1-position is preferably carried out under reflux conditions, and the reflux time is preferably 0.5h. The solid-liquid separation is preferably filtration; the concentration is preferably concentration under reduced pressure.

Mix the concentrated solution containing the reaction product substituted by 1-position allyl group, polar solvent and tert-butyldimethylsilyl chloride (TBDMSCL) to carry out the 6-position TBDM protection reaction to obtain the compound with the structure shown in formula 13; The molar ratio of the compound with the shown structure to the TBDMSCL is preferably 11.6:12.6. The polar solvent is preferably pyridine, and the present invention has no special requirements on the amount of the polar solvent, as long as the 6-position TBDM protection reaction proceeds smoothly. The temperature of the 6-position TBDM protection reaction is preferably room temperature, the holding time of the 6-position TBDM protection reaction is preferably 16 hours, and the 6-position TBDM protection reaction is preferably carried out under stirring conditions. After the 6-position TBDM protection reaction, the 6-position TBDM protection reaction solution is obtained. In the present invention, the 6-position TBDM protection reaction solution is preferably post-treated to obtain the compound with the structure shown in formula 13. In the present invention, the post-processing preferably includes: concentrating the 6-position TBDM protection reaction solution to obtain a concentrated solution; mixing the concentrated solution and an extractant for extraction to obtain an extracted organic phase, and drying the extracted organic phase Concentrate afterward to obtain a concentrated solution; the concentrated solution is subjected to column chromatography to obtain a compound with the structure shown in Formula 13. The concentration is preferably concentration under reduced pressure. The extractant is specifically preferably dichloromethane and saturated aqueous sodium bicarbonate solution. The drying agent is preferably anhydrous sodium sulfate. The drying preferably concentrates the organic extract phase of the solid-liquid separation, and the solid-liquid separation is specifically preferably filtering. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 4:1 to 2:1;

After obtaining the compound with the structure shown in Formula 13, the present invention mixes the compound with the structure shown in Formula 13, a polar solvent and camphorsulfonic acid, and performs 3 and 4 benzylidene protection to obtain the compound with the structure shown in Formula 14. In the present invention, the molar ratio of the compound represented by formula 13 to 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 on the amount of the polar solvent to ensure that the 3,4-position benzylidene protection It goes without a hitch. The reaction temperature for the protection of the 3 and 4-position benzylidene is preferably room temperature, and the reaction holding time is preferably 15 minutes. After the 3,4-position benzylidene protection reaction, the 3,4-position benzylidene protection reaction solution is obtained. In the present invention, the 3,4-position benzylidene protection reaction solution is preferably post-treated to obtain the compound with the structure shown in formula 14. The post-treatment preferably includes: mixing and extracting the 3 and 4-position 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; performing column chromatography on the concentrated solution Analysis and separation, the compound of the structure shown in formula 14 was obtained. The extractant is specifically preferably dichloromethane and saturated brine. The drying agent is preferably anhydrous sodium sulfate. The drying preferably concentrates the organic extract phase of the solid-liquid separation, and the solid-liquid separation is specifically preferably filtering. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 20:1 to 10:1;

After obtaining the compound with the structure shown in Formula 14, the present invention mixes the compound with the structure shown in Formula 14, a polar solvent, acetic acid and tetrabutylammonium fluoride trihydrate, and performs a selective de-TBDMS reaction to obtain the structure shown in Formula 15 compound. In the present invention, the molar ratio of the compound represented by formula 14 to acetic acid is preferably 0.12:1.25. The molar ratio of the compound represented by formula 14 to tetrabutylammonium fluoride trihydrate is preferably 0.12:0.5. The polar solvent is preferably tetrahydrofuran, and the present invention has no special requirements on the amount of the polar solvent, as long as the selective TBDMS removal reaction proceeds smoothly. The mixed selective de-TBDMS 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 Butylammonium fluoride mixed for selective removal of TBDMS reaction. The protective gas is preferably argon. The reaction temperature of the selective TBDMS removal reaction is preferably 0° C., and the reaction holding time is preferably 4 hours. After the selective removal of TBDMS reaction, a selective removal of TBDMS reaction solution is obtained. In the present invention, the selective removal of TBDMS reaction solution is preferably post-treated to obtain a compound with the structure shown in formula 15. The post-treatment preferably includes: concentrating the selective TBDMS removal reaction solution and mixing it with an extractant to obtain an extracted organic phase; drying and concentrating the extracted organic phase to obtain a concentrated solution; performing column chromatography on the concentrated solution Separated to obtain the compound of the structure shown in formula 15. The concentration is preferably to concentrate the selective removal of TBDMS reaction liquid until the volume is reduced by half. The extractant is specifically preferably dichloromethane and saturated brine. The drying agent is preferably anhydrous sodium sulfate. The drying preferably concentrates the organic extract phase of the solid-liquid separation, and the solid-liquid separation is specifically preferably filtering. The concentration is preferably concentration under reduced pressure. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 2:1 to 1:1;

After obtaining the compound with the structure shown in Formula 15, the present invention mixes the compound with the structure shown in Formula 15, a polar solvent, TEMPO and BAIB, and performs N-methoxyl reaction at the 6-position to obtain the compound with the structure shown in Formula 16. In the present invention, the molar ratio of the compound of formula 15, TEMPO and BAIB is preferably 1.92:0.19:2.11. The polar solvent is preferably dichloromethane, and the present invention has no special requirements on the amount of the polar solvent, as long as the N-methoxyl reaction at the 6-position proceeds smoothly. The reaction temperature of the N-methoxy group at the 6-position is preferably 40° C., and the holding time for the N-methoxy group at the 6-position reaction is preferably 6 hours. After the N-methoxyl reaction at the 6th position is selected, the N-methoxyl reaction solution at the 6th position is obtained. In the present invention, the N-methoxyl reaction solution at the 6th position is preferably post-treated to obtain the formula 16 Structured compounds. The post-treatment preferably includes: diluting the N-methoxy group at the 6-position with an organic solvent to obtain a diluted reaction solution; stirring and mixing the diluted reaction solution, sodium thiosulfate and sodium bicarbonate co-saturated aqueous solution for 10 minutes to obtain A mixed solution; mixing and 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; performing column chromatography on the residue to obtain the formula 16 Structured compounds. The organic solvent is preferably dichloromethane. The extractant is specifically preferably dichloromethane. The drying agent is preferably anhydrous sodium sulfate. The drying preferably performs solvent removal on the solid-liquid separated organic extract phase, and the solvent removal is preferably evaporation. The eluent used for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 10:1 to 4:1;

After obtaining the structural compound shown in Formula 16, the present invention mixes the structural compound shown in Formula 16, NaCNBH ₃ and a polar solvent in a protective gas atmosphere to perform a double bond reduction reaction to obtain the sugar with the structure shown in Formula 2-2-1 Base receptors. In the present invention, the molar ratio of the compound represented by formula 16 to NaCNBH ₃ is preferably 0.73:1.09. The polar solvent is preferably a mixed solvent of acetic acid and methanol, and the volume ratio of the acetic acid and methanol is preferably 1:1. The present invention has no special requirements on the amount of the polar solvent to ensure that the double bond reduction reaction goes smoothly Just proceed. The temperature of the double bond reduction reaction is preferably 0° C., the holding time of the double bond reduction reaction is preferably 4 hours, and the protective gas is preferably argon. In the present invention, the double bond reduction reaction is used to obtain a double bond reduction reaction solution. In the present invention, the double bond reduction reaction solution is preferably post-treated to obtain a glycosyl acceptor having the structure shown in formula 2-2-1. The post-treatment preferably includes: mixing the double bond reduction reaction solution with an organic solvent to obtain a mixed solution; removing the solvent from the mixed solution to obtain a residue; performing column chromatography on the residue to obtain 2-2-1 Structural glycosyl acceptor shown. The organic solvent is specifically 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 the petroleum ether and ethyl acetate is preferably 2:1.

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 that in which R2 is propenyloxy, and details will not be repeated here.

MS和三氟甲磺酸三甲基硅酯(TMSOTf)。In the present invention, the second coupling reagent is specifically preferably

MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

MS的质量比优选为378:1.6。所述极性溶剂优选为二氯甲烷，本发明对所述极性溶剂的用量没有特殊要求，确保所述第二糖基化偶联反应顺利进行即可。式2-2所示结构的糖基受体和TMSOTf的摩尔比优选为0.98:0.15。所述混合进行第二糖基化偶联反应包括：在保护气体气氛中，将式2-2所示结构的糖基受体、式3所示结构的糖基供体和

MS溶解于极性溶剂中，搅拌混合反应1h，得到混合混合溶液；将所述混合溶液降温至0℃与TMSOTf混合。本发明优选采用TLC检测式3所示结构的第二糖基供体完全反应后，所述第二糖基化偶联反应完毕。在本发明中，所述第二糖基化偶联反应后得到第二糖基化偶联反应液，本发明优选对所述糖基化偶联反应液进行后处理，得到式4-2所示结构的偶联产物。在本发明中，所述后处理优选包括：将第二糖基化偶联反应液加入三乙胺萃灭反应，将得到的猝灭反应液升温至室温；将淬灭反应液用硅藻土抽滤后得到滤液；将所述滤液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到式4-2所示结构的偶联产物。所述除溶剂优选为蒸发。所述柱层析分离的洗脱剂优选为石油醚和丙酮的混合溶剂，所述石油醚和丙酮的体积比优选为1:1。In the present invention, the molar ratio of the glycosyl acceptor with the structure shown in formula 2-2 to the glycosyl donor with the structure shown in formula 3 is preferably 0.98:1.98. The glycosyl acceptor of structure shown in formula 2-2 and

The mass ratio of MS is preferably 378:1.6. The polar solvent is preferably dichloromethane, and the present invention has no special requirements on the amount of the polar solvent, as long as the second glycosylation coupling reaction proceeds smoothly. The molar ratio of the glycosyl acceptor with the structure shown in formula 2-2 to TMSOTf is preferably 0.98:0.15. The mixing for the second glycosylation coupling reaction includes: in a protective gas atmosphere, the glycosyl acceptor with the structure shown in formula 2-2, the glycosyl donor with the structure shown in formula 3 and

MS was dissolved in a polar solvent, stirred and mixed for 1 h to obtain a mixed solution; the mixed solution was cooled to 0° C. and mixed with TMSOTf. In the present invention, TLC is preferably used to detect the complete reaction of the second glycosyl donor with the structure shown in formula 3, and the second glycosylation coupling reaction is completed. In the present invention, the second glycosylation coupling reaction solution is obtained after the second glycosylation coupling reaction. In the present invention, the glycosylation coupling reaction solution is preferably post-treated to obtain the formula 4-2 The coupling product with the structure shown. In the present invention, the post-treatment preferably includes: adding the second glycosylation coupling reaction liquid to triethylamine to extract the reaction, and warming the quenched reaction liquid to room temperature; A filtrate was obtained after suction filtration; the filtrate was desolvated to obtain a residue; the residue was subjected to column chromatography separation to obtain a coupled product with the structure shown in formula 4-2. The solvent removal is preferably evaporation. The eluent for the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 1:1.

Preferably adopt first elution solvent elution and second elution solvent elution successively; Described first elution solvent is preferably sherwood oil and acetone, and the volume ratio of described sherwood oil and acetone is preferably 1:2; The described The second elution solvent is preferably toluene and methanol, and the volume ratio of the toluene and methanol is preferably 10:1 to 5:1.

Mix the coupling product with the structure shown in Formula 4-2, a polar solvent and an acidic catalytic reagent, and perform debenzylidene protection to obtain the debenzylidene coupled product with the structure shown in Formula 5-2. In the present invention, the acidic catalytic reagent is preferably pyridine p-toluenesulfonate (PPTS). The polar solvent is preferably methanol, and the present invention has no special requirements on the amount of the polar solvent, as long as the debenzylidene protection reaction proceeds smoothly. The temperature of the debenzylidene protection reaction is preferably 65° C., and the holding time of the debenzylidene protection reaction is preferably 3 h. In the present invention, after the debenzylidene protection, the debenzylidene protection reaction solution is obtained. In the present invention, the debenzylidene protection reaction solution is preferably post-treated to obtain the debenzylidene coupling with the structure shown in formula 5-2. product. In the present invention, the post-treatment preferably includes: removing the solvent from the debenzylidene protection reaction solution to obtain a residue; separating the residue by column chromatography to obtain the debenzylidene coupling with the structure shown in formula 5-2. joint product. The solvent removal is preferably evaporation. The elution solvent used in the column chromatography separation is preferably ethyl acetate and methanol, and the volume ratio of ethyl acetate and methanol is preferably 15:1. The column chromatography separation preferably adopts the first elution solvent elution and the second elution solvent elution successively; the first elution solvent is preferably sherwood oil and acetone, and the volume ratio of the sherwood oil and acetone is preferably 1:2; the second elution solvent is preferably toluene and methanol, and the volume ratio of the toluene and methanol is preferably 10:1 to 5:1.

In the present invention, the debenzylidene coupling product of the structure shown in formula 5-2 is specifically preferably the debenzylidene coupling product of the structure shown in formula 5-2-1:

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 a basic catalytic reagent to perform selective deacetylation group, to obtain the selective deacetylation coupling product of the structure described in formula 6. In the present invention, the basic catalytic reagent 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 aqueous sodium hydroxide solution is preferably 2mol/L. The polar solvent is preferably methanol, and the present invention has no special requirements on the amount of methanol, as long as the selective deacetylation is ensured to proceed smoothly. In the present invention, said mixing for 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 step reaction; obtaining the reaction liquid; 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; the reaction solution is mixed with aqueous sodium hydroxide solution for the second step reaction to obtain selective deacetylation reaction solution; the first step reaction is preferably detected by TLC, and the time of the first step reaction is preferably 1h; the second step reaction is preferably detected by TLC, and the second step reaction is preferably detected by TLC. The time of step reaction is preferably 0.5h. In the present invention, the selective deacetylation reaction liquid is obtained after the selective deacetylation reaction, and the present invention preferably carries out post-treatment on the selective deacetylation reaction liquid to obtain the selective deacetylation reaction liquid described in the formula 6. Sexual deacetylation coupling product. In the present invention, the post-treatment preferably includes: introducing carbon dioxide into the selective deacetylation reaction solution to neutrality, and removing the solvent from the neutral reaction solution obtained to obtain the selective deacetylation reaction solution of formula 6. Acetyl coupling product. The solvent removal is preferably evaporation.

After obtaining the selective deacetylation coupling product of the structure described in formula 6, the present invention mixes the selective deacetylation coupling product of the structure described in formula 6, a polar solvent and an organic base in a protective gas atmosphere for deacetylation. Trifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative. In the present invention, the organic base is specifically preferably triethylamine. The polar solvent is preferably methanol, and the protective gas is preferably argon. In the present invention, the de-trifluoroacetyl protection reaction is preferably carried out under the condition of reflux, and the incubation time of the de-trifluoroacetyl protection reaction is preferably overnight. In the present invention, it is preferred that you post-treat the detrifluoroacetyl protection reaction solution obtained after the detrifluoroacetyl protection reaction to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyridine Galactanose derivatives. The post-treatment preferably includes: removing the solvent from the trifluoroacetyl protection reaction solution to obtain a residue; performing reverse-phase column chromatography on the residue to obtain an eluate; performing ionization on the eluate The organic salt is removed by exchange to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose 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 the pure water to methanol is preferably 1:4. The ion exchange is preferably performed using an ion exchange resin column.

In the present invention, when the R ₁ is an amide group other than -NHC(O)CH ₃ , the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative The preparation method comprises the following steps:

The selective deacetylation coupling product of the structure described in formula 6, a polar solvent, an organic base and an acylating agent are mixed for detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-( 2→6))-D-aminogalactopyranose derivative; the acylating agent is an acid anhydride, carboxylic acid or carboxylic acid ester corresponding to R ₁ .

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

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

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

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

In the present invention, the de-trifluoroacetyl protection and acylation reactions are preferably carried out under reflux conditions, and the incubation time for the de-trifluoroacetyl protection and acylation reactions is preferably overnight. In the present invention, you preferably post-treat the acylation reaction solution obtained from the detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminopyran semi Lactose derivatives. The post-treatment preferably includes: removing the solvent from the acylation reaction solution to obtain a residue; performing reverse-phase column chromatography on the residue to obtain an eluate; performing ion exchange on the eluate to remove organic salts , to obtain the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose 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 the pure water to methanol is preferably 1:4. The ion exchange is preferably performed using an ion exchange resin column.

The present invention provides a glycoconjugate consisting of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the above technical scheme The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method is coupled with a polypeptide or a carrier protein through different linkers.

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

The present invention provides a method for preparing the glycoconjugate described in the above technical scheme, comprising the following steps:

The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme is dissolved in a polar solvent, and an oxidizing gas is passed through for oxidation reaction or by Extending the carbon chain and introducing N-hydroxysuccinimide groups to obtain disaccharides containing aldehyde groups or disaccharides containing N-hydroxysuccinimide groups;

Mix the disaccharide containing aldehyde group or N-hydroxysuccinimide group, protein or polypeptide, reducing agent and buffer solution, and carry out coupling reaction to obtain the sugar conjugate.

In the present invention, the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme is dissolved in a polar solvent, and an oxidizing gas is passed through to carry out the oxidation reaction , to obtain disaccharides containing aldehyde groups. 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 solution obtained after the oxidation reaction is carried out for 30 minutes is a blue solution; the feeding of the oxidizing gas is stopped. In the present invention, the oxidation reaction solution is obtained after the oxidation reaction, and in the present invention, the oxidation reaction solution is preferably post-treated to obtain disaccharides containing aldehyde groups. The post-treatment preferably includes: blowing the oxidation reaction liquid into nitrogen gas to remove unreacted oxidizing gas and then raising the temperature to room temperature; removing the solvent from the oxidation reaction liquid to obtain disaccharides containing aldehyde groups. The solvent removal is preferably vacuum solvent removal.

After the disaccharide containing aldehyde group is obtained, the present invention mixes the disaccharide containing aldehyde group, protein or polypeptide, reducing agent and buffer solution, and performs a coupling reaction to obtain the glycoconjugate.

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

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

In the present invention, the coupling reaction liquid is obtained after the coupling reaction, and in the present invention, the coupling reaction liquid is preferably post-treated to obtain the glycoconjugate.

The present invention provides the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative described in the above technical scheme or the nitrogen-linked sialic acid ( Application of α-(2→6))-D-aminogalactopyranose derivatives or salts thereof in the preparation of antitumor drugs.

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

In the present invention, the anti-tumor drugs preferably include therapeutic vaccines or preventive vaccines.

The present invention provides a vaccine for treating tumors, comprising the glycoconjugate described in the above technical solution or the glycoconjugate prepared by the preparation method described in the above technical solution and a pharmaceutically acceptable carrier or auxiliary material.

In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

According to the synthetic flow chart of the compound of structure shown in Figure 8 shown in formula 7:

Commercially available galactosamine hydrochloride (2.5g, 11.6mmol) shown in formula 10 and 5.0g carbonate type strong basic resin, 58mL water and 6mL methanol were mixed, stirred under ice bath, and 1.5mL ethyl alcohol was added dropwise. anhydride. After 2h, filter with suction and wash the resin. The mother liquor was concentrated and passed through a strong acid resin column, and evaporated to dryness to obtain the compound shown in formula 11, which was directly used in the next reaction without purification. Add the prepared compound of formula 11, 0.32 mL of BF ₃ , and Et ₂ O into 28 mL of allyl alcohol, and stir at reflux for 2 h. Then add 0.5mL HCl in Et ₂ O solution, continue to reflux for 1h. After cooling, diethyl ether was added until cloudiness appeared, and it was left overnight at 4°C. After filtering and washing with ether, 0.9 g of a white solid was obtained, which was the compound of formula 7, and the yield of the two-step reaction was 30%.

According to the synthesis flow diagram of the sugar group donor with the structure shown in formula 3 shown in Figure 9:

Under the protection of nitrogen, the compound (1.1g, 2.20mmol) of the structure shown in formula 12 was dissolved in 20mL acetonitrile solution, 0.94mL DIPEA was added, stirred under ice bath cooling, diethylphosphorous oxychloride (0.65mL, 4.50mmol ), after 5min, the ice bath was withdrawn, and after the TLC monitoring reaction was complete, the reaction system was evaporated to dryness, ethyl acetate was added, suction filtered, the filtrate was evaporated to dryness, ethyl acetate was taken twice, column chromatography was separated, and the eluent ( V/V) Petroleum ether: ethyl acetate = 1:2, to obtain 1.1 g of the sugar group donor with the structure shown in formula 3, with a yield of 89%.

According to the synthetic flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in the formula 1-1 shown in Figure 1:

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

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

The compound of 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 stirred at room temperature for 3 hours. Dilute with 4 mL of dichloromethane, add 5.2 mL of a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate, and stir vigorously for 10 minutes. Extract with dichloromethane, dry over sodium sulfate and evaporate to dryness. Dissolve the residue in 2 mL of pyridine, add MeONH ₂ ·HCl (76.1 mg, 0.91 mmol), stir at room temperature for 1 hour, evaporate to dryness, and separate the residue by column chromatography (petroleum ether: ethyl acetate, 1:2), to obtain The white solid was the compound of formula 9 (compound 8 in Figure 1), 161 mg, and the yield was 81% (Z/E=1/1).

¹ H NMR (300MHz, CDCl ₃ ) δ7.52 (d, 1H, J = 7.8Hz), 6.87 (d, 1H, J = 4.8Hz), 5.94-5.81 (m, 2H), 5.57 (d, 2H, J＝9.3Hz), 5.32-5.31(m, 4H), 5.09(dd, 1H, J＝2.7Hz, 4.8Hz), 4.84(dd, 2H, J＝3.3Hz, 5.1Hz), 4.55(dd, 1H ,J=2.7Hz,7.2Hz),4.45(dd,1H,J=3.0Hz,5.1Hz),4.33-4.30(m,2H),4.21-4.07(m,4H),4.00-3.89(m,7H ),2.03(s,6H),1.59(s,3H),1.58(s,3H),1.33(s,6H); HRMS(ESI)Anal.Calcd for C ₁₅ H ₂₅ N ₂ O ₆ [M+H ] ⁺ :329.1707,found329.1701.

The compound (701mg, 2.14mmol) of the structure shown in formula 9 was dissolved in acetic acid/methanol (7mL/7mL) mixed solution, and NaCNBH ₃ (201.9mg, 3.19mmol) was added under the protection of argon at 0°C, stirred, and after 4 hours The response is complete. A small amount of toluene was added, concentrated and evaporated to dryness, and the residue was separated by column chromatography (ethyl acetate) to obtain 637 mg of the oily substance as a sugar acceptor (compound 9 in Figure 1) with the structure shown in formula 2-1-1. The yield was 90%.

¹ H NMR (300MHz, CDCl ₃ ) δ5.96-5.83 (m, 2H), 5.59 (d, 1H, J = 9.3Hz), 3.28 (dd, 1H, J = 1.5Hz, 17.1Hz), 5.22 (dd ,1H,J=1.2Hz,10.5Hz),4.82(d,1H,J=3.3Hz),4.31-4.24(m,2H),4.19(dd,1H,J=5.4Hz,12.9Hz),4.11- 4.05(m,2H),3.99-3.93(dd,1H,J＝6.0Hz,12.6Hz),3.54(s,3H),3.28-3.15(m,2H),2.03(s,3H),1.57(s ,3H),1.34(s,3H); ¹³ CNMR(75MHz,CDCl ₃ )δ169.97,133.51,117.84,109.67,96.75,74.72,73.75,68.26,63.09,61.37,52.09,50.50,27.95,26.565,23. (ESI) Anal. Calcd for C ₁₅ H ₂₇ N ₂ O ₆ [M+H] ⁺ : 331.1864, found 331.1865.

MS(100mg)在氮气保护下溶于干燥的二氯甲烷(1mL)中，室温搅拌1小时。将反应体系冷却至0℃，加入TMSOTf(3.2μL,0.018mmol)。TLC显示式3所示结构的糖基供体基本反应完全后，加一滴三乙胺萃灭反应。待反应体系升至室温后，将反应体系用硅藻土抽滤。将滤液蒸干。残留物用柱层析分离(石油醚：丙酮，1：1，随后甲苯：甲醇，10：1)得油状物为式4-1-4所示结构的化合物(图1中的化合物11)，31.0mg，收率63％。The glycosyl donor (55.5 mg, 0.091 mmol) of the structure shown in formula 3, the glycosyl acceptor (20.0 mg, 0.061 mmol) of the structure shown in formula 2-1-1,

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. TLC showed that the glycosyl donor with the structure shown in formula 3 was substantially completely reacted, and then a drop of triethylamine was added to extract the reaction. After the reaction system was raised to room temperature, the reaction system was suction-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 the compound of the structure shown in formula 4-1-4 (compound 11 in Figure 1), 31.0 mg, yield 63%.

¹ H NMR (500MHz, CDCl ₃ ) δ5.91-5.83 (m, 1H), 5.57 (d, 1H, J = 9.0Hz), 5.38 (dd, 1H, J = 2.5Hz, 7.0Hz), 5.34 (dt ,1H,J=1.5Hz,10.0Hz),5.27(dq,1H,J=1.5Hz,17.0Hz),5.22-5.18(m,2H),4.84-4.79(m,2H),4.40(dd,1H ,J=2.5Hz,12.5Hz),4.30-4.25(m,2H),4.20-4.13(m,2H),4.12-4.01(m,3H),3.97(ddt,1H,J=1.0Hz,6.5Hz ,13.0Hz),3.81(s,3H),3.61(s,3H),3.27(dd,1H,J=7.0Hz,14.5Hz),3.17(dd,1H,J=6.5Hz,15.0Hz),2.60 (dd, 1H, J=4.5Hz, 10.0Hz), 2.04(m, 1H), 2.14, 2.12, 2.04, 2.04, 2.03, 1.89, 1.57, 1.36(s, 8*3H); ¹³ CNMR (125MHz, CDCl ₃ )δ170.90,170.69,170.24,170.07,170.04,170.00,167.64,133.52,117.55,109.37,96.92,94.89,74.44,73.00,69.82,69.66,68.29,67.93,65.11,63.96,62.39,53.12,52.70,50.37, _49.33 ^, _35.11 , _29.63 , 28.06, 26.55, 23.42, 23.13, 21.00, 20.82, 20.75, 20.68 _; .

The compound with the structure shown in formula 4-1-4 (compound 11 in Figure 1) (100 mg, 0.124 mmol) was dissolved in dry methanol (1 mL), PPTS (47 mg) was added to the reaction system, and 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 as a compound (compound 12 in Figure 1) of the structure shown in Formula 5-1-1, 92mg, yield 97%.

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

Dissolve the compound shown in formula 5-1-1 (compound 12 in Figure 1) (30 mg, 0.039 mmol) in methanol (2 mL), stir at room temperature, add a drop of 30% sodium methoxide, and TLC shows a reaction after 30 minutes completely. After concentrating and draining the solvent, 1N NaOH aqueous solution (1.3 mL) was added. After 4 hours, TLC showed that the reaction was complete, and carbon dioxide gas was introduced to neutrality, and the residue was evaporated to dryness and separated by reverse phase column chromatography (pure water to methanol: water, 1:4) to obtain a white solid represented by formula 1-1. Nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative with the structure shown, 19 mg, yield 83%.

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

Example 2

According to Example 2 shown in Figure 2, the synthesis flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in the formula 1-2 is provided:

The compound of the structure shown in formula 10 (D-galactosamine hydrochloride, 2.5g, 11.6mmol) was dissolved in 34.7mL of methanol, 4.1mL of triethylamine and methyl trifluoroacetate (1.5mL, 12.6mmol) were added, After stirring overnight at room temperature, it was concentrated under reduced pressure. The residue was dissolved in 28.9 mL of allyl alcohol, 18.1 mL of 3M diethyl ether hydrochloride was added, and refluxed for 0.5 h. Filter and concentrate the filtrate. 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 concentration under reduced pressure, extract with dichloromethane and saturated sodium bicarbonate, dry the organic phase with sodium sulfate, filter with suction, concentrate the filtrate, and separate the residue by column chromatography (petroleum ether: ethyl acetate, 4:1 to 2: 1) The oily substance was obtained as a compound of formula 13 (compound 13 in Fig. 2), 2.3 g, yield 46%.

¹ H NMR (400MHz, CDCl ₃ ) δ6.53 (d, 1H, J = 8.9Hz), 5.91-5.84 (m, 1H), 5.30-5.24 (m, 2H), 4.97 (d, 1H, J = 3.7 Hz), 4.41(td, 1H, J=3.6Hz, 9.9Hz), 4.19(dd, 1H, J=5.3Hz, 12.9Hz), 4.14(s, 1H), 4.01(dd, 1H, J=6.3Hz ,12.9Hz),3.94(d,2H,J＝4.4Hz),3.82-3.71(m,2H),3.63(s,1H),2.72-2.70(m,1H),0.92(s,9H),0.12 (s, 6H); ¹³ C NMR (100MHz, CDCl ₃ ) δ158.01(q, J=37.0Hz), 133.10, 118.28, 115.79(q, J=286.0Hz), 96.11, 69.84, 69.84, 69.69, 68.43 , 63.81, 51.08, 25.76, 18.20, -5.54, -5.57; HRMS (ESI) Anal. Calcd for C ₁₇ H ₃₄ N ₂ O ₆ F ₃ Si[M+NH ₄ ] ⁺ : 447.2133, found 447.2122.

Compound 13 (1.9 g, 4.43 mmol) was dissolved in 53.2 mL of acetonitrile, 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. Extract with dichloromethane and saturated brine, dry the organic phase with sodium sulfate, filter with suction, concentrate the filtrate, and separate the residue by column chromatography (petroleum ether: ethyl acetate, 20:1 to 10:1) to obtain an oily The compound is a compound of the structure shown in formula 14 (compound 14 in Fig. 2), 1.1 g, and the yield is 51%.

¹ H NMR (400MHz, CDCl ₃ ) δ6.40(d, 1H, J=9.3Hz), 5.90-5.80(m, 1H), 5.28-5.22(m, 2H), 4.83(d, 1H, J=3.3 Hz), 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,CDCl ₃ )δ157.17(q,J=37.0Hz),132.98,118.41,115.78(q,J=286.2Hz),109.84 ,95.89,74.02,72.26,68.39,68.34,62.19,51.52,27.93,26.39,25.76,18.20,-5.42,-5.55; HRMS(ESI)Anal.Calcd for C ₂₀ H ₃₄ NO ₆ F ₃ SiK[M+K ] ⁺ :508.1734,found508.1734.

Compound 14 (58 mg, 0.12 mmol) was dissolved in 3.2 mL THF, 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 with suction, the filtrate was concentrated, and the residue was separated by column chromatography (petroleum ether : ethyl acetate, 2:1 to 1:1), to obtain 38 mg of the compound (compound 15 in Figure 2) having the structure shown in formula 15 as an oily product, with a yield of 87%.

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

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 stirred at 40°C for 6 hours. Dilute with 15mL of dichloromethane, add 20mL of co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate, and stir vigorously for 10min. Extract with dichloromethane, dry over sodium sulfate and evaporate to dryness. The residue was dissolved in 6.3 mL of pyridine, MeONH ₂ ·HCl (240 mg, 2.85 mmol) was added, stirred at room temperature for 1 hour, evaporated to dryness, and the residue was separated by column chromatography (petroleum ether: ethyl acetate, 10:1 to 4: 1) to obtain a white solid of the compound represented by formula 16 (compound 16 in Figure 2), 735 mg, and the yield of the two-step reaction was 100% (Z/E=1/2or2/1).

¹ H NMR (400MHz, CDCl ₃ ) δ7.52 (d, 2H, J = 7.3Hz), 6.87 (d, 1H, J = 4.9Hz), 6.32 (d, 3H, J = 8.9Hz), 5.91-5.81 (m,3H),5.36-5.22(m,6H),5.13(dd,1H,J=2.7Hz,4.8Hz),4.90-4.88(m,3H),4.59(dd,2H,J=2.4Hz, 7.3Hz), 4.48(dd, 1H, J=2.7Hz, 4.9Hz), 4.33-4.12(m, 11H), 4.05-3.97(m, 3H), 3.94(s, 3H), 3.91(s, 6H) ,1.59(s,6H),1.58(s,3H),1.35(s,9H);HRMS(ESI)Anal.Calcd for C ₁₅ H ₂₁ N ₂ O ₆ F ₃ K[M+K] ⁺ :421.0978, Found421.0982.

Compound 16 (279mg, 0.73mmol) was dissolved in a mixed solution of glacial acetic acid/methanol (2.4mL/2.4mL), and NaCNBH ₃ (69mg, 1.09mmol) was added at 0°C under the protection of argon, stirred, and the reaction was complete in 4 hours. Add a small amount of toluene, evaporate to dryness, and the residue is separated by column chromatography (petroleum ether: ethyl acetate, 2:1), and the white solid is the glycosyl acceptor of the structure shown in formula 2-2-1 (the Compound 17) 268 mg, yield 96%.

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

分子筛在氮气保护下溶于16.1mL二氯甲烷中，室温搅拌1小时。将反应体系冷却至0℃，加入TMSOTf(26μL,0.15mmol)。TLC显示式2-2-1所示结构的糖基受体(化合物17)基本反应完全后，加一滴三乙胺萃灭反应。待反应体系升至室温后，将反应体系用硅藻土抽滤。将滤液蒸干。残留物用柱层析分离(石油醚：丙酮，1：1)得油状物。将油状物溶解在7.9mL甲醇中，将PPTS(373mg，1.49mmol)加入到反应体系中，65℃搅拌3小时，蒸干，残留物用柱层析分离(石油醚：丙酮，1：2，随后甲苯：甲醇＝10：1至5：1)得油状物为式5-2-1所示结构的化合物(图2中的化合物18)，319mg，两步收率40％。The glycosyl donor (compound 10 in Fig. 2) (1209mg, 1.98mmol) of the structure shown in formula 3, the glycosyl acceptor (378mg, 0.98mmol) of the structure shown in 2-2-1 and 1.6g

Molecular sieves were 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. TLC showed that the glycosyl acceptor (compound 17) with the structure shown in formula 2-2-1 was substantially completely reacted, and then a drop of triethylamine was added to quench the reaction. After the reaction system was raised to room temperature, the reaction system was suction-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 oil. The oil 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 the oily compound (compound 18 in Figure 2) with the structure shown in formula 5-2-1, 319 mg, yield 40% in two steps.

¹ H NMR (400MHz, CDCl ₃ ) δ6.74(d, 1H, J=9.0Hz), 5.93-5.84(m, 1H), 5.46-5.21(m, 5H), 4.96(d, 1H, J=3.7 Hz), 4.92-4.81(m, 1H), 4.46-4.33(m, 2H), 4.25(dd, 1H, J=5.1Hz, 13.0Hz), 4.13-4.00(m, 5H), 3.97(t, 1H ,J=5.8Hz),3.90-3.79(m,4H),3.61(s,3H),3.35(d,1H,J=4.4Hz),3.21(d,2H,J=5.9Hz),3.09(d ,1H,J=9.4Hz),2.64(dd,1H,J=4.4Hz,12.6Hz),2.20(t,1H,J=12.4Hz),2.16(s,3H),2.16(s,3H), 2.06(s,3H),2.05(s,3H),1.90(s,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ170.97,170.94,170.47,170.41,170.20,168.07,157.89(q,J＝37.0Hz ), 133.22, 118.13, 115.82 (q, J=285.9Hz), 95.97, 94.53, 72.89, 69.60, 69.39, 69.23, 68.57, 68.48, 67.68, 63.48, 62.62, 52.98, 52.91, 51.13, 49.49, 2 21.11, 20.81, 20.79, 20.73; HRMS (ESI) Anal. Calcd for C ₃₂ H ₄₇ N ₃ O ₁₈ F ₃ [M+H] ⁺ : 818.2801, found 818.2808.

Compound 18 (20 mg, 0.024 mmol) was dissolved in 1.1 mL of methanol, stirred at room temperature, 0.02 mL of 30% sodium methoxide was added, and TLC showed that the reaction was complete after 1 hour. After concentrating and draining the solvent, 0.6 mL of 2M aqueous sodium hydroxide solution was added. After 0.5 hours, TLC showed that the reaction was complete, and carbon dioxide gas was introduced until neutral, and evaporated to dryness. Dissolve the residue in 1.2mL methanol, add 0.5mL triethylamine and 0.2mL methyl fluoroacetate under the protection of argon, reflux and stir overnight, evaporate to dryness and separate the residue by reverse phase column chromatography (pure water to methanol:water , 1:4), followed by an ion-exchange resin column to remove the residual triethylamine salt to obtain a white solid of nitrogen-linked sialic acid (α-(2→6))-D- Aminogalactopyranose derivatives, 11 mg, yield 75%.

¹ H NMR (400MHz, D ₂ O) δ5.93-5.85 (m, 1H), 5.28 (dd, 1H, J = 1.2Hz, 17.3Hz), 5.19 (d, 1H, J = 10.5Hz), 4.91 ( d,1H,J=3.8Hz),4.87(d,1H,J=46.4Hz),4.25-4.14(m,2H),4.07(t,1H,J=6.2Hz),4.04-3.94(m,3H ),3.83-3.67(m,4H),3.62-3.48(m,6H),3.16(dd,1H,J＝6.5Hz,14.2Hz),3.03(dd,1H,J＝5.9Hz,14.1Hz), 2.66 (dd, 1H, J = 4.5Hz, 12.3Hz), 1.97 (s, 3H), 1.85 (t, 1H, J = 12.0Hz); ¹³ C NMR (100MHz, D ₂ O) δ 174.96, 173.12, 171.09 ( d,J=18.6Hz),133.61,118.14,96.49,95.70,79.77(d,J=179.6Hz),72.92,71.97,69.18,68.81,68.75,68.67,68.49,67.63,64.05,62.66,52.96,51.87, 49.60, 37.74, 22.06; HRMS (ESI) Anal. Calcd for C ₂₃ H ₃₈ N ₃ O ₁₄ FNa[M+Na] ⁺ : 622.2230, found 622.2229.

Example 3

According to Example 2 shown in Figure 2, the synthesis flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in the formula 1-2 is provided:

According to the preparation method in Example 2, the compound with the structure shown in formula 5-2-1 was prepared;

The compound represented by formula 5-2-1 (compound 18 in Fig. 2) (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, and TLC showed that the reaction was complete after 1 hour. After concentrating and draining the solvent, 0.6 mL of 2M aqueous sodium hydroxide solution was added. After 0.5 hours, TLC showed that the reaction was complete, and carbon dioxide gas was introduced until neutral, and evaporated to dryness. Dissolve the residue in 1.2mL of methanol, add 0.5mL of triethylamine and 0.2mL of methyl difluoroacetate under the protection of argon, and stir at reflux overnight. After evaporation to dryness, the residue is separated by reverse phase column chromatography (pure water to methanol: water, 1:4), followed by an ion-exchange resin column to remove residual triethylamine salt to obtain a white solid of nitrogen-linked sialic acid (α-(2→6))-D -Aminogalactopyranose derivative, 13 mg, yield 86%.

¹ H NMR (400MHz, D ₂ O) δ6.10 (t, 1H, J = 53.6Hz), 5.87 (ddd, 1H, J = 5.8Hz, 11.0Hz, 22.4Hz), 5.26 (dd, 1H, J = 1.5Hz, 17.3Hz), 5.18(d, 1H, J=10.5Hz), 4.92(d, 1H, J=3.8Hz), 4.22-4.13(m, 2H), 4.06(t, 1H, J=6.3Hz ),4.03-3.94(m,3H),3.83-3.76(m,2H),3.76-3.66(m,2H),3.62-3.52(m,5H),3.50(dd,1H,J＝1.4Hz,8.9 Hz), 3.15(dd, 1H, J=6.4Hz, 14.3Hz), 3.02(dd, 1H, J=6.0Hz, 14.4Hz), 2.65(dd, 1H, J=4.5Hz, 12.3Hz), 1.95( s,3H),1.84(t,1H,J=12.0Hz); ¹³ C NMR(100MHz,D ₂ O)δ179.92,178.09,170.29(t,J=25.7Hz),138.52,123.17,113.14(t,J ＝245.6Hz), 101.10, 100.65, 77.88, 76.93, 74.16, 73.74, 73.71, 73.62, 73.45, 72.39, 69.00, _67.61 , 57.91, 56.82, 55.13, 42.69, 27.02; ₃₇ N ₃ O ₁₄ F ₂ Na[M+Na] ⁺ : 640.2136, found 640.2139.

Example 4

According to Example 3 shown in Figure 2, the synthesis flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in the formula 1-3 is provided:

According to the preparation method in Example 2, the compound with the structure shown in formula 5-2-1 was prepared;

The compound of the structure represented by formula 5-2-1 (compound 18 in Fig. 2) (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, and TLC showed that the reaction was complete after 1 hour. After concentrating and draining the solvent, 1.1 mL of 2M aqueous sodium hydroxide solution was added. After 0.5 hours, TLC showed that the reaction was complete, and carbon dioxide gas was introduced until neutral, and evaporated to dryness. Dissolve the residue in 2.4 mL of methanol, add 1.0 mL of triethylamine and 0.5 mL of methyl trifluoroacetate under the protection of argon, reflux and stir overnight, evaporate to dryness and separate the residue by reverse phase column chromatography (pure water to methanol: water, 1:4), followed by an ion-exchange resin column to remove residual triethylamine salt to obtain a white solid of nitrogen-linked sialic acid (α-(2→6))-D -Aminogalactopyranose derivative, 18 mg, yield 58%.

¹ H NMR (400MHz, D ₂ O) δ5.94-5.80 (m, 1H), 5.27 (dd, 1H, J = 1.5Hz, 17.3Hz), 5.19 (d, 1H, J = 10.4Hz), 4.94 ( d,1H,J=3.8Hz),4.23-4.14(m,2H),4.07(t,1H,J=6.3Hz),4.04-3.97(m,3H),3.84-3.77(m,2H),3.77 -3.67(m,2H),3.62-3.53(m,5H),3.51(dd,1H,J＝1.4Hz,8.9Hz),3.15(dd,1H,J＝6.4Hz,14.3Hz),3.03(dd , 1H, J=6.1Hz, 14.3Hz), 2.65(dd, 1H, J=4.5Hz, 12.3Hz), 1.96(s, 3H), 1.84(t, 1H, J=12.0Hz); ¹³ C NMR ( 100MHz, _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 N ₃ O ₁₄ F ₃ [M+H] ⁺ : 636.2222, found 636.2232.

Example 5

According to Example 5 shown in Figure 2, the synthesis flow chart of the sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in the formula 1-5 is provided:

According to the preparation method in Example 2, the compound with the structure shown in formula 5-2-1 was prepared;

The compound of the structure represented by formula 5-2-1 (compound 18 in Fig. 2) (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, and TLC showed that the reaction was complete after 1 hour. After concentrating and draining the solvent, 1.1 mL of 2M aqueous sodium hydroxide solution was added. After 0.5 hours, TLC showed that the reaction was complete, and 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 hour. After evaporation to dryness, the residue was separated by reverse phase column chromatography (pure water to methanol: water, 1:4), and the white solid was nitrogen-linked sialic acid (α-(2→6)) with the structure shown in formula 1-5. -D-aminogalactopyranose derivatives, 131 mg, yield 100%.

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

Example 6

Prepare according to the glycoprotein conjugate synthesis process described in Figure 10:

Nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives with structures shown in formulas 1-1 to 1-5 prepared in Examples 1 to 5, and structures shown in formula 17 Dissolve STn in 5mL of anhydrous methanol respectively, and pass air containing ozone at -72°C. When the system turns blue (about 10-30 minutes), stop passing ozone, and the system is still blue after 10 minutes. . Nitrogen was bubbled through the reaction system for about 10 minutes to remove excess ozone. 0.2 mL of dimethyl sulfide was added dropwise, and then the temperature of the reaction system was naturally raised to room temperature. After 2 hours, the solvent was removed from the reaction system under vacuum to obtain a hapten containing an aldehyde group.

Haptens containing aldehyde groups were dissolved together with KLH in a buffer solution with a pH value of 7.6, sodium cyanoborohydride was added, and reacted on a shaker at room temperature in the dark for 24 hours. After dialysis, the glycoprotein conjugates N(OMe)-STn-KLH were obtained, which were respectively denoted as 1-KLH, 2-KLH, 3-KLH, 4-KLH, and 5-KLH.

Example 7

The preparation method is basically the same as in Example 6, except that: KLH in Example 6 is replaced by CRM197 to obtain glycoprotein conjugates N(OMe)-STn-KLH, which are respectively denoted as 1-CRM197 and 2-CRM197, 3-CRM197, 4-CRM197, 5-CRM197.

Example 8

Prepared according to the synthesis process of glycoprotein conjugate 1-NHS-CRM197 described in Figure 11:

Dissolve 10 mg of the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the formula 1-5 prepared in Example 5 and 2.88 mg of mercaptoethylamine hydrochloride in 1 mL In deoxygenated deionized water, after 10 min of ultraviolet irradiation reaction at room temperature, concentrated under reduced pressure, purified using a dextran G10 gel column, dissolved the purified product in 1 mL of ultra-dry DMF, and added dropwise a solution containing 57.8 mg of bis(2,5 -Dioxopyrrolidin-1-yl) adipate in 1 mL of ultra-dry DMF, stirred vigorously at room temperature for 2 hours. After the reaction was completed, it was concentrated under reduced pressure, redissolved in methanol, separated by HPLC, and freeze-dried to obtain the white solid product hapten N(OMe)-STn-NHS.

Dissolve N(OMe)-STn-NHS and CRM197 in K ₂ HPO ₄ -PBS buffer at pH=8.0, react on a shaker at room temperature for 12 hours, and obtain glycoprotein conjugate 1-NHS- CRM197.

Application example

1. Test materials and sources

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

2. Test method

(1) Immunization of mice

Six Balb/c female mice in each group, aged 6-8 weeks (Number: SCXKjing2007-0001, SPF/VAF), were purchased from the Department of Animal Science, Peking University Health Science Center and raised in the Department of Animals. Conjugates of STn derivatives with STn-KLH, STn-CRM197 and nitrogen-linked with KLH or CRM197 (1-KLH, 2-KLH, 3-KLH, 4-KLH, 5-KLH and 1-CRM197, 2- CRM197, 3-CRM197, 4-CRM197, 5-CRM197) were used to immunize mice respectively, and each immunized glycoprotein (polypeptide) contained 1-3 μg of sugar (dissolved in PBS), which was immunized once every 2 weeks, and the immunization route was as follows: Intraperitoneal injection, a total of 4 times of immunization. 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.

(2) Determination of antibody titers in serum of mice before and after immunization

The titer of mixed serum of each group of mice, and 1-KLH, 2-KLH, 3-KLH, 4-KLH, 5-KLH and 1-CRM197, 2-CRM197, 3-KLH prepared in embodiment 6 and embodiment 7 The serum titer of each mouse in the CRM197, 4-CRM197, and 5-CRM197 immunized groups was detected by ELISA method.

1 Coating antigen: Coat 100 μL of STn-BSA (containing 0.02 μg of STn) on the microtiter plate overnight at 4°C.

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, 37° C. for 1 hour.

3. Add primary antibody (ie, immune serum): wash 3 times (the specific method is the same as above). The serum was serially diluted with antibody diluent (1% BSA-PBS) from a certain dilution, and 100 μL was added to each well, at 37° C. for 1 hour.

4 Add enzyme-labeled secondary antibody: wash 3 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, 37 °C, 1 hour.

5 Color development: wash 3 times, add 100 μL of the ready-to-use chromogenic substrate o-phenylenediamine (OPD) to each well, develop color at room temperature in the dark for 15 minutes, add 2M H ₂ SO ₄ to each well to stop color development.

6 Judgment of results: Read the OD value at a wavelength of 490nm with a microplate reader. The serum dilution factor when the OD value after subtracting the blank serum well reading was 0.1 was regarded as the antibody titer.

(3) Mouse immunotherapy

Eight Balb/c female mice in each group, aged 6-8 weeks (Number: SCXKjing2007-0001, SPF/VAF), were purchased from the Animal Science Department of Peking University Health Science Center and raised in the Animal Department. On day 0, each mouse was inoculated with 5×10^5 CT26 cells in the armpit, and on days 2, 6, 10, and 17, 1-NHS-CRM197, 1-CRM197, and 1- KLH solution in PBS.

3. Test results

The test results are shown in Table 1 and Figures 3-6. Wherein, Fig. 3 is the titer of each mouse serum in the serum STn-KLH and 1-KLH groups after the fourth immunization of 1-KLH prepared in Example 6 of the present invention; Fig. 4 is the titer of 1-KLH prepared in Example 6 of the present invention Mouse survival curve after KLH administration; Fig. 5 is the mouse tumor growth curve after 1-CRM197 administration prepared in Example 7 of the present invention; Fig. 6 is the mouse survival curve after 1-CRM197 administration prepared in Example 7 of the present invention, Fig. 7 is the mouse tumor growth curve after administration of 1-NHS-CRM197 prepared in Example 8 of the present invention.

Table 1 is the third and fourth immunization of the glycoconjugates prepared in Example 6 to measure the antibody titer of recognizing STn in the mouse serum 13 days later

Table 2 is the comparison of sugar loading of 1-CRM197 prepared in Example 7 and 1-NHS-CRM197 prepared in Example 8.

Table 2 Sugar loading of 1-CRM197 and 1-NHS-CRM197

A tumor-bearing mouse model was constructed with CT-26 colon cancer cells expressing STn sugar antigen; then the tumor-bearing mice were immunized with the glycoconjugate (1-KLH, 1-CRM197 or 1-NHS-CRM197) in the patent of the present invention Rats were used to observe the survival period, tumor volume and antibody titer of the mice. The experimental results showed that compared with the control group, glycoconjugates 1-KLH, 1-CRM197 or 1-NHS-CRM197 significantly prolonged the survival period of mice, inhibited tumor growth, and increased antibody titers after inoculation of mice, indicating that they have Good antitumor effect.

Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and other embodiments can also be obtained according to the present embodiment without inventive step, and these embodiments are all Belong to the protection scope of the present invention.

Claims (10)

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

In Formula 1, R ₁ is an amide group or -NH ₂ ; the amide group is -NHC(O)CH _p Cl _q , -NHC(O)CH _p F _q , -NHC(O)CH p Br 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 are 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; R2 is a group with _double bond, alkyne bond, azido group, aldehyde group, protected acetal group, maleimide group, N-hydroxysuccinimide group, mercapto group, protected mercapto group, selenoyl group, protected selenoyl group , -NH ₂ or -ONH ₂ substituents. 2. nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof according to claim ₁ , wherein said R is-NHC(O )CH _p F _q or -NHC(O)C _a H _2a+1 ; the R ₂ is allyloxy. 3. The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or salt thereof according to claim 1, wherein the nitrogen-linked sialic acid (α -(2→6))-D-aminogalactopyranose derivatives have any of the structures shown in formula 1-1 to formula 1-5:

4. The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt according to any one of claims 1 to 3, characterized in that the nitrogen-linked sialic acid Acid (α-(2→6))-D-aminogalactopyranose derivative salt is a nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative of the structure shown in formula 1 salts formed by the reaction of substances with bases. 5. The method for preparing the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative according to any one of claims 1 to 3, characterized in that, When said R ₁ is -NHC(O)CH ₃ , it may comprise the following steps: Mix the glycosyl acceptor with the structure shown in formula 2-1, the glycosyl donor with the structure shown in formula 3, coupling reagent and polar solvent to carry out glycosylation coupling reaction to obtain the glycosyl group with the structure shown in formula 4-1 coupling product;

Mix the coupling product of the structure shown in formula 4-1, a polar solvent and an acidic catalytic reagent, and perform debenzylidene protection to obtain the debenzylidene coupling product of the structure shown in formula 5-1;

Mix the debenzylidene coupling product of the structure shown in formula 5-1, a polar solvent and a basic catalytic reagent for selective deacetylation to obtain the nitrogen-linked sialic acid (α-(2→6))- D-aminogalactopyranose derivatives; When said R ₁ is -NH ₂ , it may comprise the following steps: Mix the glycosyl acceptor with the structure shown in formula 2-2, the glycosyl donor with the structure shown in formula 3, coupling reagent and polar solvent to carry out glycosylation coupling reaction to obtain the glycosyl group with the structure shown in formula 4-2 coupling product;

Mixing the coupling product of the structure shown in formula 4-2, a polar solvent and an acidic catalytic reagent, performing debenzylidene protection to obtain the debenzylidene coupling product of the structure shown in formula 5-2; Mixing the debenzylidene coupling product of the structure shown in formula 5-2, a polar solvent and a basic catalytic reagent, and performing selective deacetylation to obtain the selective deacetylation coupling product of the structure of formula 6;

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 for detrifluoroacetyl protection to obtain the nitrogen-linked sialic acid (α-(2→ 6))-D-aminogalactopyranose derivatives; When said R ₁ is an amide group other than -NHC(O)CH ₃ , it may comprise the following steps: The selective deacetylation coupling product of the structure described in formula 6, a polar solvent, an organic base and an acylating agent are mixed for detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid (α-( 2→6))-D-aminogalactopyranose derivative; the acylating agent is an acid anhydride, carboxylic acid or carboxylic acid ester corresponding to R ₁ . 6. A glycoconjugate, characterized in that: the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt, or the nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in claim 5 is coupled with the polypeptide or carrier protein through different linkers Get it. 7. The preparation method of the glycoconjugate according to claim 6, characterized in that, comprising the following steps: The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt is dissolved in a polar solvent, and an oxidizing gas is passed through for oxidation reaction or by extending the carbon chain Introducing N-hydroxysuccinimide groups to obtain disaccharides containing aldehyde groups or N-hydroxysuccinimide groups; Mix the disaccharide containing aldehyde group or N-hydroxysuccinimide group, protein or polypeptide, reducing agent and buffer solution, and carry out coupling reaction to obtain the sugar conjugate. 8. The nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivative or its salt according to any one of claims 1 to 4 or prepared by the preparation method described in claim 5 Application of nitrogen-linked sialic acid (α-(2→6))-D-aminogalactopyranose derivatives or salts thereof in the preparation of antitumor drugs. 9. The application of the glycoconjugate according to claim 6 or the glycoconjugate prepared by the preparation method according to claim 7 in the preparation of antitumor drugs. 10. A vaccine for treating tumors, comprising the glycoconjugate of claim 6 or the glycoconjugate prepared by the preparation method of claim 7 and a pharmaceutically acceptable carrier or adjuvant.

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