WO2024078578A1 - 唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法 - Google Patents

唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法 Download PDF

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WO2024078578A1
WO2024078578A1 PCT/CN2023/124243 CN2023124243W WO2024078578A1 WO 2024078578 A1 WO2024078578 A1 WO 2024078578A1 CN 2023124243 W CN2023124243 W CN 2023124243W WO 2024078578 A1 WO2024078578 A1 WO 2024078578A1
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formula
structure shown
sialic acid
reaction
nitrogen
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French (fr)
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叶新山
霍常鑫
郑秀静
许成豪
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • the present invention belongs to the technical field of oligosaccharides and sugar conjugates thereof, and specifically relates to sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivatives or salts thereof, sugar conjugates and preparation methods thereof.
  • the carbohydrate antigen STn is a disaccharide structure containing sialic acid, which is often expressed in human breast cancer, colorectal cancer, ovarian cancer, and prostate cancer, but rarely expressed in normal tissues (Holmberg, L. Expert Rev. Vaccines 2004, 3, 655-663.), thus becoming an important target for tumor immunotherapy.
  • Canadian Biomira Company developed STn-KLH (keyhole limpet hemocyanin) conjugates— Vaccines are used to prevent and treat colorectal cancer and breast cancer metastasis.
  • the object of the present invention is to provide a sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or a salt thereof, a sugar conjugate and a preparation method thereof.
  • the sugar conjugate (sugar antigen) obtained by coupling the sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or a salt thereof provided by the present invention to a protein through different linkers can produce a more effective immune response, produce more antibodies, and show good activity in anti-tumor vaccines; thereby, the survival period can be prolonged, thereby achieving an anti-tumor effect.
  • the present invention provides a nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative has a structure shown in Formula 1:
  • R2 is a substituent having a double bond, an acetylenic bond, an azido group, an aldehyde group, a protected acetal group, a maleimide group, an N-hydroxysuccinimide group, a thiol group, a protected thiol group, a seleno group, a protected seleno group, -NH2 or -ONH2 .
  • the R 1 is -NHC(O)CH p F q or -NHC(O)C a H 2a+1 ; and the R 2 is allyloxy.
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative has any one of the structures shown in Formula 1-1 to Formula 1-5:
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative salt is a salt formed by the reaction of the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative having the structure shown in Formula 1 with a base.
  • the present invention provides a method for preparing the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative described in the above technical solution.
  • the steps include:
  • glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1;
  • the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative;
  • glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-2;
  • debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6;
  • the selective deacetylation coupling product of the structure of Formula 6 a polar solvent and an organic base are mixed to remove the trifluoroacetyl protection to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative;
  • the 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 to carry out detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative;
  • the acylating agent is an anhydride, carboxylic acid or carboxylic acid ester corresponding to R 1 .
  • the present invention provides a sugar conjugate, which is obtained by coupling the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme with a polypeptide or a carrier protein through different linkers.
  • the present invention provides a method for preparing the glycoconjugate described in the above technical solution, comprising the following steps:
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt described in the above technical solution is dissolved in a polar solvent, and an oxidizing gas is introduced to perform an oxidation reaction or an N-hydroxysuccinimide group is introduced by extending the carbon chain to obtain a disaccharide containing an aldehyde group or a N-hydroxysuccinimide group;
  • the disaccharide containing an aldehyde group or an N-hydroxysuccinimide group, a protein or a polypeptide, a reducing agent and a buffer solution are mixed and subjected to a coupling reaction to obtain the sugar conjugate.
  • the present invention provides the use of the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative described in the above technical scheme or the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme in the preparation of anti-tumor drugs.
  • the present invention provides the use of the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.
  • the present invention provides a vaccine for treating tumors, comprising the sugar conjugate described in the above technical solution or prepared by the preparation method described in the above technical solution.
  • the invention also comprises a sugar conjugate and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative has a structure shown in Formula 1.
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or a salt thereof provided by the present invention has a structure shown in Formula 1, wherein the nitrogen bridge (N(OMe)) connection replaces the oxygen bridge (O) connection in the disaccharide antigen structure, and the structure is novel, and good activity is shown in anti-tumor vaccines.
  • sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt linked to the structure shown in Formula 1 provided by the present invention can be coupled with a carrier protein or polypeptide to obtain a glycoprotein (glycopeptide) conjugate.
  • the vaccine using the glycoprotein (glycopeptide) conjugate as a sugar antigen can produce a more effective immune response, produce more specific antibodies, and can specifically identify tumor cells expressing STn, thereby achieving an anti-tumor effect; further, compared with the structure linked by oxygen bridge (O), the vaccine obtained by using the sugar antigen prepared by the sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt linked to the structure shown in Formula 1 provided by the present invention has a significantly improved antibody titer for recognizing STn, and the third Thirteen days after immunization, the antibody titer of STn recognition obtained by the vaccine obtained from the sugar antigen prepared from the sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose derivative or its salt with a nitrogen-linked structure as shown in Formula 1 of the present invention was 4812, and the antibody titer of STn recognition obtained by the vaccine obtained from the sugar antigen prepared from the hapten with an oxygen bridge (O)
  • the vaccine obtained from the sugar antigen prepared from the sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative or its salt connected with the structure shown in Formula 1 provided by the present invention can significantly prolong the survival of mice in the tumor-bearing mouse model test. From the results of Figure 6, it can be concluded that the vaccine obtained from the sugar antigen prepared from the sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative or its salt connected with the structure shown in Formula 1 provided by the present invention has a survival period of 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 skeleton structure for the research and development of anti-tumor sugar vaccines, and is expected to promote the development of anti-tumor sugar vaccines.
  • the present invention provides a sugar conjugate, which is obtained by coupling the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative or its salt described in the above technical solution, or the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative or its salt prepared by the preparation method described in the above technical solution with a polypeptide or protein.
  • the sugar conjugate provided by the present invention is used as an anti-tumor vaccine to produce a strong immune response in mice.
  • the titer of antibodies produced by the sugar conjugates of the present invention increased by 3 to 15 times, and the survival of tumor-bearing mice was significantly prolonged after vaccination. Both the antibody titer and the survival of mice were significantly increased, which is expected to promote the development of anti-tumor sugar vaccines.
  • FIG1 is a flow chart of the synthesis of a sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-1 provided in Example 1 of the present invention
  • FIG2 is a synthetic flow chart of sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivatives having structures shown in Formulas 1-2 to 1-5 provided in Examples 2 to 5 of the present invention;
  • FIG3 shows the titer of serum of each mouse in the STn-KLH and 1-KLH groups after the fourth immunization with 1-KLH prepared in Example 6 of the present invention
  • FIG4 is a survival curve of mice after administration of 1-KLH prepared in Example 6 of the present invention.
  • FIG5 is a tumor growth curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention.
  • FIG6 is a survival curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention.
  • FIG7 is a tumor growth curve of mice after administration of NSTn-NHS-CRM197 prepared in Example 8 of the present invention.
  • FIG8 is a flow chart of the synthesis of the glycosyl acceptor of the structure shown in Formula 7 in Example 1 of the present invention.
  • FIG9 is a flow chart of the synthesis of the glycosyl donor of the structure shown in Formula 3 in an embodiment of the present invention.
  • FIG10 is a flow chart of the synthesis of glycoprotein conjugates according to an embodiment of the present invention.
  • FIG. 11 is a synthetic flow chart of the glycoprotein conjugate 1-NHS-CRM197 in an embodiment of the present invention.
  • the present invention provides a nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative or a salt thereof, wherein the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative has a structure shown in Formula 1:
  • R2 is a substituent having a double bond, an acetylenic bond, an azido group, an aldehyde group, a protected acetal group, a maleimide group, an N-hydroxysuccinimide group, a thiol group, a protected thiol group, a seleno group, a protected seleno group, -NH2 or -ONH2 .
  • the protected acetal group is a substituent having a protective group on the acetal group
  • the protected thiol group is a substituent having a protective group on the thiol group
  • the protected selenoyl group is a substituent having a protective group on selenium.
  • the present invention has no special requirements on the type of the protective group.
  • the R 1 is preferably -NHC(O)CH p F q or -NHC(O)C a H 2a+1 ; and the R 2 is preferably allyloxy.
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative has any one of the structures shown in Formula 1-1 to Formula 1-5:
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative salt is a salt formed by the reaction of the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative of the structure shown in Formula 1 with a base.
  • the present invention provides a method for preparing the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative described in the above technical solution.
  • the steps include:
  • glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1;
  • the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative;
  • glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-2;
  • debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6;
  • the selective deacetylation coupling product of the structure of Formula 6 a polar solvent and an organic base are mixed to remove trifluoroacetyl protection to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative;
  • the 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 to carry out detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative;
  • the acylating agent is an anhydride, carboxylic acid or carboxylic acid ester corresponding to R 1 .
  • the method for preparing the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative comprises the following steps:
  • glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product of the structure shown in Formula 4-1;
  • the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and an alkaline catalytic agent are mixed and selectively deacetylated to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative.
  • the present invention mixes a glycosyl acceptor with a structure shown in Formula 2-1, a glycosyl donor with a structure shown in Formula 3, a coupling reagent (hereinafter referred to as the first coupling reagent) and a polar solvent to carry out a glycosylation coupling reaction (hereinafter referred to as the glycosylation coupling reaction) to obtain a coupling product with a structure shown in Formula 4-1.
  • the glycosyl acceptor with the structure shown in Formula 2-1 is preferably the glycosyl acceptor with the structure shown in Formula 2-1-1;
  • the method for preparing the glycosyl acceptor of the structure shown in Formula 2-1-1 preferably comprises the following steps:
  • camphorsulfonic acid and polar solvent are mixed to carry out benzyl protection at 3,4 positions to obtain the compound of structure shown in formula 8; the compound of structure shown in formula 8, tetramethylpiperidinyl oxide (TEMPO), iodobenzene diacetic acid (BAIB) and polar solvent are mixed to carry out selective oxidation at 6 position to obtain the compound of structure shown in formula 9; in a protective gas atmosphere, the compound of structure shown in formula 9, NaCNBH3 and polar solvent are mixed to carry out double bond reduction reaction to obtain the glycosyl acceptor of structure shown in formula 2-1-1.
  • TEMPO tetramethylpiperidinyl oxide
  • BAIB iodobenzene diacetic acid
  • polar solvent are mixed to carry out selective oxidation at 6 position to obtain the compound of structure shown in formula 9; in a protective gas atmosphere, the compound of structure shown in formula 9, NaCNBH3 and polar solvent are mixed to carry out double bond reduction reaction to obtain the glycosyl accept
  • the present invention mixes the compound of structure shown in formula 7, camphorsulfonic acid and polar solvent to carry out benzyl protection at positions 3 and 4, thereby obtaining the compound of structure shown in formula 8.
  • the method for preparing the compound of structure shown in formula 7 preferably comprises the following steps: dissolving the compound of structure shown in formula 10 (galactosamine hydrochloride), acetic anhydride and carbonate-type strong alkaline resin in a mixed solvent of methanol and water, and reacting under ice-water bath conditions to obtain the compound of structure shown in formula 11; mixing the compound of structure shown in formula 11, allyl alcohol and ethanol solution of boron trifluoride, then adding ethanol solution of HCl, and reacting under reflux conditions to obtain the compound of structure shown in formula 7;
  • the method for preparing the compound of the structure shown in Formula 11 is preferably as follows: commercially available aminogalactose hydrochloride (2.5 g, 11.6 mmol) shown in Formula 10 and 5.0 g carbonate-type strong alkaline resin, 58 mL water, and 6 mL methanol are mixed, stirred under an ice bath, and 1.5 mL acetic anhydride is added dropwise. After 2 hours, the mixture is filtered and the resin is washed. After the mother liquor is concentrated, it is passed through a strong acid resin column and evaporated to dryness to obtain the compound shown in Formula 11, which is directly used for the next step reaction without purification.
  • the molar ratio of the compound of the structure shown in Formula 7 to camphorsulfonic acid is preferably 3.8:0.23.
  • the polar solvent is preferably dimethyl phthalate (DMP, ⁇ , ⁇ -dimethoxypropane).
  • the present invention has no special requirements for the amount of the polar solvent, as long as the 3,4-benzylidene protection is carried out smoothly.
  • the reaction temperature of the 3,4-benzylidene protection is preferably room temperature, and the reaction insulation time is preferably 22h. After the 3,4-benzylidene protection reaction, a 3,4-benzylidene protection reaction liquid is obtained.
  • the present invention preferably performs post-treatment on the 3,4-benzylidene protection reaction liquid to obtain a compound of the structure shown in Formula 8.
  • the post-treatment preferably includes: mixing the 3,4-benzylidene protection reaction liquid and a saturated sodium bicarbonate aqueous solution to obtain a mixed solution; mixing and extracting the mixed solution with an organic solvent to obtain an extracted organic phase; drying and concentrating the combined extracted organic phase to obtain a concentrated solution; and performing column chromatography separation on the concentrated solution to obtain a compound of the structure shown in Formula 8.
  • the organic solvent is preferably dichloromethane.
  • the drying reagent is preferably anhydrous sodium sulfate.
  • the drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration.
  • the concentration is preferably performed by concentration under reduced pressure.
  • the eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of the petroleum ether to acetone is preferably 2:1.
  • the present invention mixes the structural compound shown in Formula 8, tetramethyl piperidine oxide (TEMPO), iodobenzene diacetic acid (BAIB) and a polar solvent for 6-position selective oxidation to obtain a structural compound shown in Formula 9.
  • TEMPO tetramethyl piperidine oxide
  • BAIB iodobenzene diacetic acid
  • a polar solvent for 6-position selective oxidation to obtain a structural compound shown in Formula 9.
  • the molar ratio of the structural compound shown in Formula 8, TEMPO and BAIB is preferably 0.61:0.06:0.55.
  • the polar solvent is preferably dichloromethane.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the 6-position selective oxidation reaction is carried out smoothly.
  • the temperature of the 6-position selective oxidation reaction is preferably room temperature, and the insulation time of the 6-position selective oxidation reaction is preferably 3h.
  • the 6-position selective oxidation reaction obtains a 6-position selective oxidation reaction liquid.
  • the present invention preferably post-treats the 6-position selective oxidation reaction liquid to obtain a structural compound shown in Formula 9.
  • the post-treatment preferably includes: mixing the 6-position selective oxidation reaction liquid and a saturated sodium bicarbonate aqueous solution to obtain a mixed solution; extracting the mixed solution and an organic solvent to obtain an extracted organic phase; drying the combined extracted organic phase and removing the solvent to obtain a residue; subjecting the residue to column chromatography separation to obtain a structural compound shown in Formula 9.
  • the organic solvent is preferably dichloromethane.
  • the drying reagent is preferably anhydrous sodium sulfate.
  • the drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration.
  • the solvent removal is preferably performed by evaporation.
  • the eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is preferably 1:2.
  • the present invention mixes the compound of structure shown in formula 9, NaCNBH 3 and polar solvent in a protective gas atmosphere to carry out double bond reduction reaction to obtain a glycosyl acceptor of structure shown in formula 2-1-1.
  • the molar ratio of the compound of structure shown in formula 9 and 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 acetic acid and methanol is preferably 1:1.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the double bond reduction reaction is carried out smoothly.
  • the temperature of the double bond reduction reaction is preferably 0°C
  • the insulation time of the double bond reduction reaction is preferably 4h
  • the protective gas is preferably argon.
  • the double bond reduction reaction obtains a double bond reduction reaction liquid
  • the present invention preferably post-treats the double bond reduction reaction liquid to obtain a glycosyl acceptor of structure shown in formula 2-1-1.
  • the post-treatment preferably comprises: mixing the double bond reduction reaction solution and an organic solvent to obtain a mixed solution; removing the solvent from the mixed solution to obtain a residue; and subjecting the residue to column chromatography separation to obtain a glycosyl receptor with the structure shown in 2-1-1.
  • the organic solvent is preferably toluene.
  • the solvent removal is preferably evaporation.
  • the eluent used in the column chromatography separation is preferably ethyl acetate.
  • R 2 in the glycosyl acceptor of the structure shown in Formula 2-1 is a substituent of other structures
  • the preparation method is the same as the preparation method when R 2 is a propyleneoxy group, and will not be described in detail here.
  • the method for preparing the glycosyl donor having the structure shown in Formula 3 preferably comprises the following steps:
  • the compound of the structure shown in Formula 12 is dissolved in acetonitrile, DIPEA and (EtO) 2 PCl are added, and the reaction is carried out at 0-25° C. in an Ar atmosphere to obtain a glycosyl donor of the structure shown in Formula 3.
  • the specific preparation method of the glycosyl donor of the structure shown in Formula 3 is preferably: under nitrogen protection, the compound of the structure shown in Formula 12 (1.1 g, 2.20 mmol) is dissolved in 20 mL of acetonitrile solution, 0.94 mL of DIPEA is added, and the mixture is stirred under ice bath cooling, diethylphosphite chloride (0.65 mL, 4.50 mmol) is added, and the ice bath is removed after 5 minutes. After the reaction is completed as monitored by TLC, the reaction system is evaporated to dryness, ethyl acetate is added, and the mixture is filtered with suction.
  • the first coupling reagent is preferably MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).
  • the molar ratio of the glycosyl acceptor of the structure shown in Formula 2-1 to the glycosyl donor of the structure shown in Formula 3 is preferably 0.061:0.091.
  • Structural glycosyl receptors and The mass ratio of MS is preferably 20:100.
  • the polar solvent is preferably dichloromethane.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the first glycosylation coupling reaction is carried out smoothly.
  • the molar ratio of the glycosyl acceptor of the structure shown in Formula 2-1 and TMSOTf is preferably 0.061:0.018.
  • the mixing for the first glycosylation coupling reaction comprises: in a protective gas atmosphere, mixing the glycosyl acceptor of the structure shown in Formula 2-1, the glycosyl donor of the structure shown in Formula 3 and MS is dissolved in a polar solvent, stirred and mixed for 1h to obtain a mixed solution; the mixed solution is cooled to 0°C and mixed with TMSOTf.
  • the present invention preferably uses TLC to detect that the glycosyl donor of the structure shown in Formula 3 is completely reacted, and the first glycosylation coupling reaction is completed.
  • the first glycosylation coupling reaction liquid is obtained after the first glycosylation coupling reaction, and the present invention preferably post-treats the first glycosylation coupling reaction liquid to obtain a coupling product of the structure shown in Formula 4-1.
  • the post-treatment preferably includes: adding triethylamine to the first glycosylation coupling reaction liquid to quench the reaction, and heating the obtained quenched reaction liquid to room temperature; filtering the quenched reaction liquid with diatomaceous earth to obtain a filtrate; removing the solvent from the filtrate to obtain a residue; and separating the residue by column chromatography to obtain a coupling product of the structure shown in Formula 4-1.
  • the solvent removal is preferably evaporation.
  • the column chromatography separation preferably uses a first elution solvent and a second elution solvent in sequence;
  • the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:1;
  • the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1.
  • the present invention mixes the coupling product of the structure shown in Formula 4-1, a polar solvent and an acidic catalyst, and performs debenzylation protection to obtain a debenzylation coupling product of the structure shown in Formula 5-1.
  • the acidic catalyst is preferably pyridine p-toluenesulfonate (PPTS).
  • PPTS pyridine p-toluenesulfonate
  • the mass ratio of the coupling product of the structure shown in Formula 4-1 and the acidic catalyst is preferably 100:47.
  • the polar solvent is preferably methanol. The present invention has no special requirements for the amount of the polar solvent, as long as the debenzylation protection reaction is carried out smoothly.
  • the temperature of the debenzylation protection reaction is preferably 65°C, and the insulation time of the debenzylation protection reaction is preferably 3h.
  • a debenzylation protection reaction liquid is obtained after the debenzylation protection.
  • the present invention preferably performs post-treatment on the debenzylation protection reaction liquid to obtain a debenzylation coupling product of the structure shown in Formula 5-1.
  • the post-treatment preferably includes: removing the solvent from the debenzylation protection reaction liquid to obtain a residue; and separating the residue by column chromatography to obtain a debenzylation coupling product of the structure shown in Formula 5-1.
  • the solvent removal is preferably evaporation.
  • the elution solvents used in the column chromatography separation are preferably ethyl acetate and methanol, and the volume ratio of ethyl acetate to methanol is preferably 15:1.
  • the debenzylidene coupling product of the structure shown in Formula 5-1 is preferably the debenzylidene coupling product of the structure shown in Formula 5-1-1
  • the present invention mixes the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and an alkaline catalyst to perform selective deacetylation to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative.
  • the alkaline catalyst is preferably sodium methoxide and sodium hydroxide aqueous solution, and the molar concentration of the sodium hydroxide aqueous solution is preferably 1 mol/L.
  • the polar solvent is preferably methanol, and the present invention has no special requirements for the amount of the methanol, as long as the selective deacetylation is carried out smoothly.
  • the mixing for selective deacetylation reaction includes the following steps: mixing the debenzylidene coupling product of the structure shown in Formula 5-1, a polar solvent and sodium methoxide to perform a first step reaction; obtaining a reaction solution; mixing the reaction solution and sodium hydroxide aqueous solution to perform a second step reaction to obtain a selective deacetylation reaction solution; the first step reaction is preferably detected by TLC, and the time of the first step reaction is preferably 30 minutes; the second step reaction is preferably detected by TLC, and the time of the second step reaction is preferably 4 hours.
  • a selective deacetylation reaction liquid is obtained.
  • the selective deacetylation reaction liquid is preferably post-treated to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative.
  • the post-treatment preferably includes: introducing carbon dioxide into the selective deacetylation reaction liquid until it becomes neutral, removing the solvent from the obtained neutral reaction liquid to obtain a residue; and subjecting the residue to column chromatography separation to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative.
  • the solvent removal is preferably evaporation.
  • the column chromatography separation is preferably performed by reverse phase column chromatography separation.
  • the elution solvent used in the column chromatography is preferably pure water and methanol, and the volume ratio of the pure water to methanol is preferably 1:4.
  • the preparation method of the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative comprises the following steps:
  • glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3, a coupling reagent (hereinafter referred to as the second coupling reagent) and a polar solvent are mixed to carry out a glycosylation coupling reaction (hereinafter referred to as the second glycosylation coupling reaction) to obtain a coupling product of the structure shown in Formula 4-2;
  • debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and a basic catalyst are mixed to perform selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6;
  • the selective deacetylation coupling product of the structure described in Formula 6 a polar solvent and an organic base are mixed to remove the trifluoroacetyl protection to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative.
  • a glycosyl acceptor having a structure shown in Formula 2-2, a glycosyl donor having a structure shown in Formula 3, a coupling reagent and a polar solvent are mixed to carry out a glycosylation coupling reaction to obtain a coupling product having a structure shown in Formula 4-2.
  • the glycosyl acceptor with the structure shown in Formula 2-2 is preferably the glycosyl acceptor with the structure shown in Formula 2-2-1;
  • the preparation method of the glycosyl acceptor of the structure shown in Formula 2-2-1 preferably comprises the following steps: mixing the compound of the structure shown in Formula 10, a polar solvent, triethylamine and methyl trifluoroacetate to carry out a trifluoroethyl reaction at position 2 to obtain a reaction solution containing a reaction product substituted with trifluoroethyl at position 2; concentrating the reaction solution containing the reaction product substituted with trifluoroethyl at position 2 to obtain a concentrated solution containing a reaction product substituted with trifluoroethyl at position 2; the amount of the compound of the structure shown in Formula 10 and the volume ratio of triethylamine are preferably 11.6mmol:4.1mL; the molar ratio of the compound of the structure shown in Formula 10 and the methyl trifluoroacetate is preferably 11.6:12.6; the polar solvent is preferably methanol, and the present invention has no special requirements for the amount of the polar solvent, as
  • the temperature of the trifluoroethyl reaction at position 2 is preferably room temperature, the insulation time of the trifluoroethyl reaction at position 2 is preferably overnight, and the trifluoroethyl reaction at position 2 is preferably carried out under stirring.
  • the concentration is preferably reduced pressure concentration.
  • the concentrated solution of the reaction product containing trifluoroethyl substitution at position 2, allyl alcohol and ether hydrochloride are mixed to carry out an allyl reaction at position 1 to obtain a reaction solution containing a reaction product containing allyl substitution at position 1; the reaction solution containing the reaction product containing allyl substitution at position 1 is separated into solid and liquid, and the obtained filtrate is concentrated to obtain a concentrated solution containing a reaction product containing allyl substitution at position 1;
  • the molar concentration of the ether hydrochloride is preferably 3 mol/L;
  • the ratio of the amount of the compound of the structure shown in Formula 10 to the volume of the ether hydrochloride is preferably 11.6 mol:18.1 mL;
  • the ratio of the amount of the compound of the structure shown in Formula 10 to the volume of the allyl alcohol is preferably 11.6 mol:28.9 mL;
  • the allyl reaction at position 1 is preferably carried out under reflux conditions, and the reflux time is preferably
  • the concentrated solution of the reaction product containing 1-position allyl substitution, a polar solvent and tert-butyldimethylsilyl chloride (TBDMSCL) are mixed to carry out a 6-position TBDM protection reaction to obtain a compound of the structure shown in Formula 13; the molar ratio of the compound of the structure shown in Formula 10 and the TBDMSCL is preferably 11.6:12.6.
  • the polar solvent is preferably pyridine.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the 6-position TBDM protection reaction is carried out smoothly.
  • the temperature of the 6-position TBDM protection reaction is preferably room temperature
  • the insulation time of the 6-position TBDM protection reaction is preferably 16h
  • the 6-position TBDM protection reaction is preferably carried out under stirring.
  • a 6-position TBDM protection reaction liquid is obtained.
  • the present invention preferably post-treats the 6-position TBDM protection reaction liquid to obtain a compound of the structure shown in Formula 13.
  • the post-treatment preferably includes: concentrating the 6-position TBDM protection reaction liquid to obtain a concentrated solution; mixing the concentrated solution and an extractant to extract to obtain an extracted organic phase, drying the extracted organic phase and concentrating it to obtain a concentrated solution; subjecting the concentrated solution to column chromatography separation to obtain a compound with a structure shown in Formula 13.
  • the concentration is preferably reduced pressure concentration.
  • the extractant is preferably dichloromethane and a saturated sodium bicarbonate aqueous solution.
  • the drying reagent is preferably anhydrous sodium sulfate.
  • the drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration.
  • the concentration is preferably reduced pressure concentration.
  • the eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 4:1 to 2:1;
  • the present invention mixes the compound of the structure shown in Formula 13, a polar solvent and camphorsulfonic acid, and performs 3,4-benzylidene protection to obtain the compound of the structure shown in Formula 14.
  • the molar ratio of the compound of the structure shown in Formula 13 and camphorsulfonic acid is preferably 4.43:2.22.
  • the polar solvent is preferably acetonitrile and dimethyl phthalate (DMP, ⁇ , ⁇ -dimethoxypropane).
  • DMP dimethyl phthalate
  • the present invention has no special requirements for the amount of the polar solvent, and it is sufficient to ensure that the 3,4-benzylidene protection is carried out smoothly.
  • the reaction temperature of the 3,4-benzylidene protection is preferably room temperature, and the reaction insulation time is preferably 15min.
  • a 3,4-benzylidene protection reaction liquid is obtained.
  • the present invention preferably performs post-treatment on the 3,4-benzylidene protection reaction liquid to obtain a compound of the structure shown in Formula 14.
  • the post-treatment preferably includes: extracting the 3,4-benzylidene protection reaction solution and the extractant to obtain an extracted organic phase; drying and concentrating the extracted organic phase to obtain a concentrated solution; subjecting the concentrated solution to column chromatography separation to obtain a compound with a structure shown in Formula 14.
  • the extractant is preferably dichloromethane and saturated brine.
  • the drying reagent is preferably anhydrous sodium sulfate.
  • the drying is preferably performed by concentrating the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably performed by filtration.
  • the concentration is preferably concentrated under reduced pressure.
  • the eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 20:1 to 10:1;
  • the present invention mixes the compound of the structure shown in Formula 14, a polar solvent, acetic acid and tetrabutylammonium fluoride trihydrate, and performs a selective TBDMS removal reaction to obtain a compound of the structure shown in Formula 15.
  • the molar ratio of the compound of the structure shown in Formula 14 and acetic acid is preferably 0.12:1.25.
  • the molar ratio of the compound of the structure shown in Formula 14 and tetrabutylammonium fluoride trihydrate is preferably 0.12:0.5.
  • the polar solvent is preferably tetrahydrofuran.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the selective TBDMS removal reaction is carried out smoothly.
  • the mixed selective TBDMS removal reaction preferably includes the following steps: mixing the compound of the structure shown in Formula 14, a polar solvent and acetic acid to obtain a mixed solution; in a protective gas atmosphere, cooling the mixed solution to 0°C and mixing it with tetrabutylammonium fluoride trihydrate to perform a selective TBDMS removal reaction.
  • the protective gas Preferably, it is argon.
  • the reaction temperature of the selective TBDMS removal reaction is preferably 0°C, and the reaction insulation time is preferably 4h. After the selective TBDMS removal reaction, a selective TBDMS removal reaction liquid is obtained.
  • the present invention preferably performs post-treatment on the selective TBDMS removal reaction liquid to obtain a compound with a structure shown in Formula 15.
  • the post-treatment preferably comprises: concentrating the selective TBDMS removal reaction liquid and mixing it with an extractant to obtain an extracted organic phase; drying the extracted organic phase and concentrating it to obtain a concentrated solution; and subjecting the concentrated solution to column chromatography separation to obtain a compound with a structure shown in Formula 15.
  • the concentration is preferably to concentrate the selective TBDMS removal reaction liquid until the volume is reduced by half.
  • the extractant is preferably dichloromethane and saturated brine.
  • the drying reagent is preferably anhydrous sodium sulfate.
  • the drying is preferably to concentrate the organic extract phase of the solid-liquid separation, and the solid-liquid separation is preferably filtration.
  • the concentration is preferably reduced pressure concentration.
  • the eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to
  • the present invention mixes the compound of the structure shown in Formula 15, a polar solvent, TEMPO and BAIB, and performs an N-methoxy reaction at position 6 to obtain a compound of the structure shown in Formula 16.
  • the molar ratio of the compound of the structure shown in Formula 15, TEMPO and BAIB is preferably 1.92:0.19:2.11.
  • the polar solvent is preferably dichloromethane.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the N-methoxy reaction at position 6 is smoothly carried out.
  • the reaction temperature of the N-methoxy at position 6 is preferably 40°C, and the insulation time of the N-methoxy reaction at position 6 is preferably 6h.
  • the present invention preferably performs post-treatment on the N-methoxy reaction liquid at position 6 to obtain a compound of the structure shown in Formula 16.
  • the post-treatment preferably includes: diluting the N-methoxy group at position 6 with an organic solvent to obtain a diluted reaction solution; stirring and mixing the diluted reaction solution, a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate for 10 minutes to obtain a mixed solution; extracting the mixed solution and an extractant to obtain an extracted organic phase; drying the extracted organic phase and removing the solvent to obtain a residue; subjecting the residue to column chromatography separation to obtain a compound with a structure shown in Formula 16.
  • the organic solvent is preferably dichloromethane.
  • the extractant is preferably dichloromethane.
  • the drying reagent is preferably anhydrous sodium sulfate. The drying is preferably to remove the solvent from the organic extract phase of solid-liquid separation, and the removal of the solvent is preferably evaporation.
  • the eluent used in the column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone is preferably 10:1 to 4:1;
  • the present invention mixes the compound of structure shown in formula 16, NaCNBH 3 and polar solvent in a protective gas atmosphere to carry out double bond reduction reaction to obtain a glycosyl acceptor of structure shown in formula 2-2-1.
  • the molar ratio of the compound of structure shown in formula 16 and 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 acetic acid and methanol is preferably 1:1.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the double bond reduction reaction is carried out smoothly.
  • the temperature of the double bond reduction reaction is preferably 0°C
  • the insulation time of the double bond reduction reaction is preferably 4h
  • the protective gas is preferably argon.
  • the double bond reduction reaction obtains a double bond reduction reaction liquid
  • the present invention preferably post-treats the double bond reduction reaction liquid to obtain a glycosyl acceptor of structure shown in formula 2-2-1.
  • the post-treatment preferably includes: mixing the double bond reduction reaction solution and an organic solvent to obtain a mixed solution; removing the solvent from the mixed solution to obtain a residue; and subjecting the residue to column chromatography separation to obtain a glycosyl receptor with the structure shown in 2-2-1.
  • the organic solvent is preferably toluene.
  • the solvent removal is preferably evaporation.
  • the eluent used for the column chromatography separation is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 2:1.
  • R 2 in the glycosyl acceptor of the structure shown in Formula 2-2 is a substituent of other structures
  • the preparation method is the same as the preparation method when R 2 is a propyleneoxy group, and will not be described in detail here.
  • the second coupling reagent is preferably MS and trimethylsilyl trifluoromethanesulfonate (TMSOTf).
  • the molar ratio of the glycosyl acceptor of the structure shown in Formula 2-2 to the glycosyl donor of the structure shown in Formula 3 is preferably 0.98:1.98.
  • the mass ratio of MS is preferably 378:1.6.
  • the polar solvent is preferably dichloromethane.
  • the present invention has no special requirements for the amount of the polar solvent, as long as the second glycosylation coupling reaction is carried out smoothly.
  • the molar ratio of the glycosyl acceptor of the structure shown in Formula 2-2 and TMSOTf is preferably 0.98:0.15.
  • the mixing for the second glycosylation coupling reaction comprises: in a protective gas atmosphere, mixing the glycosyl acceptor of the structure shown in Formula 2-2, the glycosyl donor of the structure shown in Formula 3 and MS is dissolved in a polar solvent, stirred and mixed for 1h to obtain a mixed solution; the mixed solution is cooled to 0°C and mixed with TMSOTf.
  • the present invention preferably uses TLC to detect that the second glycosyl donor of the structure shown in Formula 3 is completely reacted, and the second glycosylation coupling reaction is completed.
  • the second glycosylation coupling reaction is obtained after the second glycosylation coupling reaction.
  • the present invention preferably performs post-treatment on the glycosylation coupling reaction liquid to obtain a coupling product of the structure shown in Formula 4-2.
  • the post-treatment preferably includes: adding triethylamine to the second glycosylation coupling reaction liquid to quench the reaction, and heating the obtained quenched reaction liquid to room temperature; filtering the quenched reaction liquid with diatomaceous earth to obtain a filtrate; removing the solvent from the filtrate to obtain a residue; and separating the residue by column chromatography to obtain a coupling product of the structure shown in Formula 4-2.
  • the solvent removal is preferably evaporation.
  • the eluent for column chromatography separation is preferably a mixed solvent of petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:1.
  • elution is carried out with a first elution solvent and elution is carried out with a second elution solvent in sequence;
  • the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of the petroleum ether and acetone is preferably 1:2;
  • the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1 to 5:1.
  • the coupling product of the structure shown in Formula 4-2, a polar solvent and an acidic catalyst are mixed and debenzylidene protection is performed to obtain a debenzylidene coupling product of the structure shown in Formula 5-2.
  • the acidic catalyst is preferably pyridine p-toluenesulfonate (PPTS).
  • the polar solvent is preferably methanol. There is no special requirement for the amount of the polar solvent, as long as the debenzylidene protection reaction is ensured to proceed smoothly.
  • the temperature of the debenzylidene protection reaction is preferably 65°C, and the insulation time of the debenzylidene protection reaction is preferably 3h.
  • a debenzylidene protection reaction liquid is obtained after the debenzylidene protection, and the present invention preferably performs post-treatment on the debenzylidene protection reaction liquid to obtain a debenzylidene coupling product of the structure shown in Formula 5-2.
  • the post-treatment preferably includes: removing the solvent from the debenzylidene protection reaction liquid to obtain a residue; and performing column chromatography separation on the residue to obtain a debenzylidene coupling product of the structure shown in Formula 5-2.
  • 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 is preferably eluted with a first elution solvent and eluted with a second elution solvent in sequence;
  • the first elution solvent is preferably petroleum ether and acetone, and the volume ratio of petroleum ether and acetone is preferably 1:2;
  • the second elution solvent is preferably toluene and methanol, and the volume ratio of toluene and methanol is preferably 10:1 to 5:1.
  • the debenzylidene coupling product of the structure shown in Formula 5-2 is preferably the debenzylidene coupling product of the structure shown in Formula 5-2-1:
  • the present invention mixes the debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and an alkaline catalyst, and performs selective deacetylation to obtain a selective deacetylation coupling product of the structure shown in Formula 6.
  • the alkaline catalyst is preferably sodium methoxide and sodium hydroxide aqueous solution
  • the sodium methoxide is preferably added in the form of sodium methoxide solution, and the mass percentage of the sodium methoxide solution is preferably 30%
  • the molar concentration of the sodium hydroxide aqueous solution is preferably 2 mol/L.
  • the polar solvent is preferably methanol, and the present invention has no special requirements for the amount of the methanol, as long as the selective deacetylation is smoothly carried out.
  • the mixed selective deacetylation reaction comprises the following steps: mixing the debenzylidene coupling product of the structure shown in Formula 5-2, a polar solvent and sodium methoxide for the first reaction; obtaining a reaction solution; the ratio of the amount of the debenzylidene coupling product of the structure shown in Formula 5-2 to the volume of the sodium methoxide solution is preferably 0.024mmol:0.02mL; mixing the reaction solution with an aqueous sodium hydroxide solution for the second reaction to obtain a selective deacetylation reaction solution; the first reaction is preferably detected by TLC, and the time of the first reaction is preferably 1h; the second reaction is preferably detected by TLC, and the time of the second reaction is preferably 0.5h.
  • a selective deacetylation reaction solution is obtained after the selective deacetylation reaction, and the present invention preferably performs post-treatment on the selective deacetylation reaction solution to obtain the selective deacetylation coupling product of the structure shown in Formula 6.
  • the post-treatment preferably comprises: introducing carbon dioxide into the selective deacetylation reaction solution until it is neutral, removing the solvent from the obtained neutral reaction solution, and obtaining a selective deacetylation coupling product of the structure shown in Formula 6.
  • the solvent removal is preferably evaporation.
  • the present invention After obtaining the selective deacetylation coupling product of the structure described in Formula 6, the present invention, in a protective gas atmosphere, mixes the selective deacetylation coupling product of the structure described in Formula 6, a polar solvent and an organic base for detrifluoroacetyl protection to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative.
  • the organic base is preferably triethylamine.
  • the polar solvent is preferably methanol, and the protective gas is preferably argon.
  • the detrifluoroacetyl protection reaction is preferably carried out under reflux conditions, and the insulation time of the detrifluoroacetyl protection reaction is preferably overnight.
  • the present invention preferably performs post-treatment on the detrifluoroacetyl protection reaction solution obtained after the detrifluoroacetyl protection reaction to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative.
  • the post-treatment preferably includes: removing the solvent from the detrifluoroacetyl protection reaction solution to obtain a residue; subjecting the residue to reverse phase column chromatography to obtain an eluent; subjecting the eluent to ion exchange to remove organic salts to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative.
  • the solvent removal is preferably evaporation.
  • the eluent used in the reverse phase column chromatography is preferably pure water to methanol, and the volume ratio of pure water to methanol is preferably 1:4.
  • the ion exchange is preferably performed using an ion exchange resin column.
  • the method for preparing the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative 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 to carry out detrifluoroacetyl protection and acylation reaction to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative;
  • the acylating agent is an anhydride, carboxylic acid or carboxylic acid ester corresponding to R 1 .
  • the acid anhydride preferably includes acetic anhydride, propionic anhydride, n-butyric anhydride, isobutyric anhydride or n-hexanoic anhydride;
  • the carboxylic acid preferably comprises monofluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, monochloroacetic acid or dichloroacetic acid;
  • the carboxylic acid ester preferably includes methyl monofluoroacetate, methyl difluoroacetate, methyl trifluoroacetate or methyl dichloroacetate.
  • the acylating agent is preferably methyl monofluoroacetate, methyl difluoroacetate or methyl trifluoroacetate.
  • the detrifluoroacetyl protection and acylation reaction are preferably carried out under reflux conditions, and the insulation time of the detrifluoroacetyl protection and acylation reaction is preferably overnight.
  • the acylation reaction solution obtained by the detrifluoroacetyl protection and acylation reaction is preferably post-treated to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative.
  • the post-treatment preferably includes: removing the solvent from the acylation reaction solution to obtain a residue; subjecting the residue to reverse phase column chromatography to obtain an eluent; subjecting the eluent to ion exchange to remove organic salts to obtain the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-amino galactopyranose derivative.
  • the desolventizing is preferably evaporation.
  • the eluent used in the reverse phase column chromatography is preferably pure water to methanol, and the volume ratio of the pure water to methanol is preferably 1:4.
  • the ion exchange is preferably carried out using an ion exchange resin column.
  • the present invention provides a sugar conjugate, which is obtained by coupling the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt described in the above technical scheme, or the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme with a polypeptide or a carrier protein through different linkers.
  • 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 solution, comprising the following steps:
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt described in the above technical solution is dissolved in a polar solvent, and an oxidizing
  • the aldehyde-containing disaccharide or the N-hydroxysuccinimide-containing disaccharide is oxidized by a volatile gas or introduced by extending the carbon chain.
  • the disaccharide containing an aldehyde group or an N-hydroxysuccinimide group, a protein or a polypeptide, a reducing agent and a buffer solution are mixed and subjected to a coupling reaction to obtain the sugar conjugate.
  • the present invention dissolves the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminopyranose galactose derivative or its salt described in the above technical solution in a polar solvent, introduces an oxidizing gas to carry out an oxidation reaction, and obtains a disaccharide containing an aldehyde group.
  • the polar solvent is preferably anhydrous methanol.
  • the oxidizing gas is preferably air containing ozone.
  • the temperature of the oxidation reaction is preferably -72°C; the oxidation reaction liquid obtained after the oxidation reaction is carried out for 30 minutes is a blue solution; and the introduction of the oxidizing gas is stopped.
  • an oxidation reaction liquid is obtained after the oxidation reaction, and the present invention preferably performs post-treatment on the oxidation reaction liquid to obtain a disaccharide containing an aldehyde group.
  • the post-treatment preferably includes: introducing nitrogen into the oxidation reaction liquid to remove unreacted oxidizing gas and then heating it to room temperature; desolventizing the oxidation reaction liquid to obtain a disaccharide containing an aldehyde group.
  • the desolventizing is preferably vacuum desolventizing.
  • the present invention mixes the disaccharide containing an aldehyde group, a protein or a polypeptide, a reducing agent and a buffer solution, and performs a coupling reaction to obtain the glycoconjugate.
  • the reducing agent is preferably sodium cyanoborohydride.
  • the pH value of the buffer solution is preferably 7.6.
  • the coupling reaction temperature is preferably room temperature
  • the coupling reaction temperature holding time is preferably 24 hours
  • the coupling reaction is preferably carried out in a light-proof condition.
  • a coupling reaction solution is obtained after the coupling reaction.
  • the coupling reaction solution is preferably post-treated to obtain the glycoconjugate.
  • the present invention provides the use of the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative described in the above technical scheme or the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative or its salt prepared by the preparation method described in the above technical scheme in the preparation of anti-tumor drugs.
  • the present invention provides the use of the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.
  • the anti-tumor drug preferably includes a therapeutic vaccine or a preventive vaccine.
  • the present invention provides a vaccine for treating tumors, comprising the sugar conjugate described in the above technical solution or the sugar conjugate prepared by the preparation method described in the above technical solution and a pharmaceutically acceptable carrier or excipient.
  • the compound of formula 8 (183 mg, 0.61 mmol) was dissolved in 1 mL of dichloromethane, and TEMPO (9.1 mg, 0.06 mmol) and BAIB were added. (215mg, 0.55mmol), stirred at room temperature for 3 hours. Diluted with 4mL of dichloromethane, added 5.2mL of a co-saturated aqueous solution of sodium thiosulfate and sodium bicarbonate, and stirred vigorously for 10 minutes. Extracted with dichloromethane, dried with sodium sulfate, and evaporated to dryness.
  • the compound of formula 9 (701 mg, 2.14 mmol) was dissolved in a mixed solution of acetic acid/methanol (7 mL/7 mL), and NaCNBH 3 (201.9 mg, 3.19 mmol) was added at 0 ° C under argon protection, and the reaction was completed after 4 hours of stirring. A small amount of toluene was added, and the mixture was concentrated and evaporated to dryness. The residue was separated by column chromatography (ethyl acetate) to obtain an oily substance, which was a glycosyl acceptor of formula 2-1-1 (compound 9 in Figure 1) of 637 mg, with a yield of 90%.
  • the glycosyl donor (55.5 mg, 0.091 mmol) of the structure shown in Formula 3 and the glycosyl acceptor (20.0 mg, 0.061 mmol) of the structure shown in Formula 2-1-1 were added.
  • MS 100 mg was dissolved in dry dichloromethane (1 mL) under nitrogen protection and stirred at room temperature for 1 hour.
  • the reaction system was cooled to 0 ° C and TMSOTf (3.2 ⁇ L, 0.018 mmol) was added. After TLC showed that the glycosyl donor of the structure shown in Formula 3 was basically reacted, a drop of triethylamine was added to quench the reaction. After the reaction system was warmed to room temperature, the reaction system was filtered with diatomaceous earth.
  • Example 2 a synthesis flow chart of a sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-2 is provided:
  • the glycosyl donor of formula 3 (compound 10 in FIG. 2 ) (1209 mg, 1.98 mmol), the glycosyl acceptor of formula 2-2-1 (378 mg, 0.98 mmol) and 1.6 g
  • the molecular sieve was dissolved in 16.1 mL of dichloromethane under nitrogen protection and stirred at room temperature for 1 hour.
  • the reaction system was cooled to 0°C and TMSOTf (26 ⁇ L, 0.15 mmol) was added. After TLC showed that the glycosyl acceptor (compound 17) of the structure shown in formula 2-2-1 was basically reacted, a drop of triethylamine was added to quench the reaction.
  • reaction system was warmed to room temperature, the reaction system was filtered with diatomaceous earth. The filtrate was evaporated to dryness. The residue was separated by column chromatography (petroleum ether: acetone, 1:1) to obtain an oily substance.
  • Salt was obtained as a white solid, which was a nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative of the structure shown in Formula 1-2, 11 mg, with a yield of 75%.
  • Example 2 a synthesis flow chart of a sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-2 is provided:
  • Example 3 a synthesis flow chart of a sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-3 is provided:
  • Example 5 a synthesis flow chart of a sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivative having a structure shown in Formula 1-5 is provided:
  • the nitrogen-linked sialic acid ( ⁇ -(2 ⁇ 6))-D-aminogalactopyranose derivatives of formula 1-1 to 1-5 prepared in Examples 1 to 5 and STn of formula 17 were dissolved in 5 mL of anhydrous methanol, respectively. Air containing ozone was introduced at -72°C. When the system turned blue (about 10-30 minutes), the ozone was stopped. After 10 minutes, the system was still blue. Nitrogen was introduced into the reaction system for about 10 minutes to remove excess ozone. 0.2 mL of dimethyl sulfide was added dropwise. The temperature of the reaction system was then allowed to naturally rise to room temperature. After 2 hours, the solvent was removed from the reaction system under vacuum to obtain the hapten containing an aldehyde group.
  • the hapten containing an aldehyde group was dissolved with KLH in a buffer solution with a pH value of 7.6, sodium cyanoborohydride was added, and the mixture was reacted on a shaker at room temperature in the dark for 24 hours. After dialysis, the glycoprotein conjugate N(OMe)-STn-KLH was obtained, which was recorded as 1-KLH, 2-KLH, 3-KLH, 4-KLH, and 5-KLH.
  • the preparation method is basically the same as that of Example 6, except that CRM197 is used to replace KLH in Example 6 to obtain glycoprotein conjugate N(OMe)-STn-KLH, which are respectively recorded as 1-CRM197, 2-CRM197, 3-CRM197, 4-CRM197, and 5-CRM197.
  • glycoprotein conjugate 1-NHS-CRM197 was prepared according to the synthetic process described in FIG11 :
  • Test compound glycoprotein (polypeptide) conjugate prepared in Examples 6, 7 and 8 of the present invention.
  • mice Six Balb/c female mice, 6-8 weeks old (Number: SCXKjing2007-0001, SPF/VAF) were purchased from the Department of Animal Science, Peking University Health Science Center and raised in the animal department. Mice were immunized with STn-KLH, STn-CRM197, and conjugates of STn derivatives linked to nitrogen and KLH or CRM197 (1-KLH, 2-KLH, 3-KLH, 4-KLH, 5-KLH and 1-CRM197, 2-CRM197, 3-CRM197, 4-CRM197, 5-CRM197), and the glycoprotein (peptide) contained 1-3 ⁇ g of sugar (dissolved in PBS) in each immunization.
  • STn-KLH STn-CRM197
  • conjugates of STn derivatives linked to nitrogen and KLH or CRM197 (1-KLH, 2-KLH, 3-KLH, 4-KLH, 5-KLH and 1-CRM197, 2-CRM197, 3-CRM197, 4-CRM197,
  • Immunization was once every 2 weeks, and the immunization route was intraperitoneal injection, for a total of 4 immunizations. Blood was collected before immunization, 13 days after the second immunization, 13 days after the third immunization, and 14 days after the fourth immunization. Serum was separated and stored in a -80 o C refrigerator for testing.
  • Antigen coating Coat the ELISA plate with 100 ⁇ L of STn-BSA (containing 0.02 ⁇ g of STn) at 4 ° C overnight.
  • washing and blocking Add 200 ⁇ L of washing buffer PBS-Tween20 (0.05%) to each well to wash the plate, wash 3 times, then add 200 ⁇ L of blocking solution (3% BSA-PBS) to each well, incubate at 37°C for 1 hour.
  • Color development Wash three times, add 100 ⁇ L of the prepared color development substrate o-phenylenediamine (OPD) to each well, develop the color for 15 minutes at room temperature in the dark, and add 2M H 2 SO 4 to each well to stop the color development.
  • OPD o-phenylenediamine
  • Result determination Read the OD value at a wavelength of 490 nm using an ELISA reader.
  • the serum dilution factor when the OD value after subtracting the blank serum well reading is 0.1 is used as the antibody titer.
  • mice Eight Balb/c female mice, 6-8 weeks old (Number: SCXKjing2007-0001, SPF/VAF), were purchased from the Department of Animal Science, Peking University Health Science Center and raised in the animal department. On day 0, 5 ⁇ 10 ⁇ 5 CT26 cells were inoculated under the armpit of each mouse, and PBS solutions containing 1-NHS-CRM197, 1-CRM197 and 1-KLH were subcutaneously injected on days 2, 6, 10 and 17, respectively.
  • Figure 3 shows the titer of serum STn-KLH and serum of each mouse in the 1-KLH group after the fourth immunization of 1-KLH prepared in Example 6 of the present invention
  • Figure 4 shows the survival curve of mice after administration of 1-KLH prepared in Example 6 of the present invention
  • Figure 5 shows the tumor growth curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention
  • Figure 6 shows the survival curve of mice after administration of 1-CRM197 prepared in Example 7 of the present invention
  • Figure 7 shows the tumor growth curve of mice after administration of 1-NHS-CRM197 prepared in Example 8 of the present invention.
  • Table 1 shows the antibody titers recognizing STn in mouse serum measured 13 days after the third and fourth immunizations of the sugar conjugates prepared in Example 6
  • Table 2 is a comparison of the sugar loading amounts of 1-CRM197 prepared in Example 7 and 1-NHS-CRM197 prepared in Example 8.
  • a mouse tumor model was constructed using CT-26 colon cancer cells expressing STn glycogen antigens; then the tumor-bearing mice were immunized with the glycoconjugates (1-KLH, 1-CRM197 or 1-NHS-CRM197) in the present invention, and the survival period, tumor volume and antibody titer of the mice were observed.
  • the experimental results showed that compared with the control group, the glycoconjugates 1-KLH, 1-CRM197 or 1-NHS-CRM197 significantly prolonged the survival period of the mice, inhibited tumor growth, and increased antibody titer after inoculation of mice, indicating that they had a good anti-tumor effect.

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Abstract

属于寡糖及其糖缀合物技术领域，具体涉及唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法。提供了氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，其具有式1所示结构。小鼠实验表明，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐能够与载体蛋白质或多肽通过不同连接子偶联得到糖蛋白(糖肽)缀合物，产生更有效的免疫反应，能够特异地识别表达STn的肿瘤细胞，从而达到对抗肿瘤的作用；为抗肿瘤糖疫苗的研发提供新的骨架结构，有望推动抗肿瘤糖疫苗的发展。

Description

唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法
本申请要求于2022年10月12日提交中国专利局、申请号为CN202211248083.7、发明名称为“唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于寡糖及其糖缀合物技术领域，具体涉及唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法。
背景技术
基于糖抗原的抗肿瘤疫苗研究成为当前肿瘤免疫治疗领域的一个有前景的研究方向。其中糖抗原STn是一个含有唾液酸的二糖结构，在人类乳腺癌、结直肠癌、卵巢癌、前列腺癌多有表达，而在正常组织中表达极少(Holmberg,L.ExpertRev.Vaccines 2004,3,655-663.)，因而成为肿瘤免疫治疗的重要靶点。加拿大Biomira公司以此为基础研发了STn-KLH(钥孔虫戚血蓝蛋白)偶联物—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.)。
同遇到的困难类似，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。
发明内容
本发明的目的在于提供唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法，本发明提供的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐通过不同连接子偶联蛋白得到的糖缀合物(糖抗原)能够产生更有效的免疫反应，产生更多的抗体，在抗肿瘤疫苗方面表现出了很好的活性；从而能够延长生存期，从而达到抗肿瘤的作用。
本发明提供了一种氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1所示结构：
式1中，R1为酰胺基或-NH2；所述酰胺基为-NHC(O)CHpClq、-NHC(O)CHpFq、-NHC(O)CHpBrq、-NHC(O)H、-NHC(O)CaH2a+1、-NHC(O)CaH2aOH、-NHC(O)CbH2b-1或-NHC(O)CbH2b-3；其中，p或q独立地为0、1、2或3，且p+q＝3；a为1～20中的任一整数；b为2～20中的任一整数；
R2为带有双键、炔键、叠氮基、醛基、保护缩醛基、马来酰亚胺基、N-羟基琥珀酰亚胺基、巯基、保护巯基、硒基、保护硒基、-NH2或-ONH2的取代基。
优选的，所述R1为-NHC(O)CHpFq或-NHC(O)CaH2a+1；所述R2为烯丙氧基。
优选的，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1-1～式1-5所示结构中的任一结构：
优选的，所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物盐为式1所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物与碱反应生成的盐。
本发明提供了上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的制备方法，
所述R1为-NHC(O)CH3时，包括以下步骤：
将式2-1所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-1所示结构的偶联产物；
将式4-1所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-1所示结构的脱苄叉偶联产物；
将式5-1所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；
所述R1为-NH2时，包括以下步骤：
将式2-2所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-2所示结构的偶联产物；
将式4-2所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-2所示结构的脱苄叉偶联产物；
将式5-2所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到式6所述结构的选择性脱乙酰基偶联产物；
在保护气体气氛中，将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂和有机碱混合进行脱三氟乙酰基保护，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；
所述R1为除-NHC(O)CH3以外的酰胺基时，包括以下步骤：
将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂、有机碱和酰化试剂混合进行脱三氟乙酰基保护和酰化反应，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；所述酰化试剂为R1对应的酸酐、羧酸或羧酸酯。
本发明提供了一种糖缀合物，由上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，或上述技术方案所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐与多肽或载体蛋白质通过不同连接子偶联得到。
本发明提供了上述技术方案所述的糖缀合物的制备方法，包括以下步骤：
将上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐溶解于极性溶剂中，通入氧化性气体进行氧化反应或通过延长碳链引入N-羟基琥珀酰亚胺基，得到含有醛基或含有N-羟基琥珀酰亚胺基的二糖；
将所述含有醛基或含有N-羟基琥珀酰亚胺基的二糖、蛋白质或多肽、还原剂和缓冲溶液混合，进行偶联反应，得到所述糖缀合物。
本发明提供了上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或上述技术方案所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐在制备抗肿瘤药物中的应用。
本发明提供了上述技术方案所述糖缀合物或上述技术方案所述的制备方法制备得到的糖缀合物在制备抗肿瘤药物中的应用。
本发明提供了一种治疗肿瘤的疫苗，包括上述技术方案所述糖缀合物或上述技术方案所述的制备方法制备得到 的糖缀合物和药学上可接受的载体或辅料。
本发明提供了一种氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1所示结构。本发明提供的式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，化合物的结构中用氮桥(N(OMe))连接代替了二糖抗原结构中的氧桥(O)连接，结构新颖，在抗肿瘤疫苗方面表现出了很好的活性。小鼠实验表明，本发明提供的式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐能与够载体蛋白质或多肽进行偶联得到的糖蛋白(糖肽)缀合物，以糖蛋白(糖肽)缀合物作为糖抗原的疫苗能够产生更有效的免疫反应，产生更多的特异性抗体，能够特异地识别表达STn的肿瘤细胞，从而达到对抗肿瘤的作用；进一步，相较于氧桥(O)连接的结构，采用本发明本发明提供的式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐制备的糖抗原得到的疫苗获得的识别STn的抗体滴度明显提高，第三次免疫后13天测定，本发明式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐制备的糖抗原得到的疫苗获得的识别STn的抗体滴度为4812，氧桥(O)连接的结构的半抗原制备的糖抗原得到的疫苗获得的识别STn的抗体滴度为1458；第四次免疫后13天测定，本发明式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐制备的糖抗原得到的疫苗获得的识别STn的抗体滴度为89288，氧桥(O)连接的结构的半抗原制备的糖抗原得到的疫苗获得的识别STn的抗体滴度为5716。而且，本发明提供的式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐制备的糖抗原得到的疫苗在荷瘤小鼠模型试验中能明显延长小鼠生存期，由图6的结果可以得出，本发明提供的式1所示结构氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐制备的糖抗原得到的疫苗在荷瘤小鼠模型试验中生存期为115天。适用于制备乳腺癌、结直肠癌、卵巢癌、前列腺癌等抗癌药物。另一方面，这是一个全新化合物，为抗肿瘤糖疫苗的研发提供新的骨架结构，有望推动抗肿瘤糖疫苗的发展。
本发明提供了一种糖缀合物，由上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，或上述技术方案所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐与多肽或蛋白质偶联得到。本发明提供的糖缀合物得到的疫苗相较于氧桥(O)连接的结构半乳糖衍生物，将本发明提供的糖缀合物作为抗肿瘤疫苗，在小鼠体内产生了很强的免疫应答，与相比，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。
附图说明
图1为本发明实施例1提供式1-1所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图；
图2为本发明实施例2～5提供式1-2～式1-5所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图；
图3为本发明实施例6制备的1-KLH第四次免疫后血清STn-KLH和1-KLH组每只小鼠血清的滴度；
图4为本发明实施例6制备的1-KLH施用后小鼠生存曲线；
图5为本发明实施例7制备的1-CRM197施用后小鼠肿瘤生长曲线；
图6为本发明实施例7制备的1-CRM197施用后小鼠生存曲线；
图7为本发明实施例8制备的NSTn-NHS-CRM197施用后小鼠肿瘤生长曲线；
图8为本发明实施例1中式7所示结构的糖基受体的合成流程图；
图9为本发明实施例中式3所示结构的糖基供体的合成流程图；
图10为本发明实施例中糖蛋白缀合物的合成流程图；
图11为本发明实施例中糖蛋白缀合物1-NHS-CRM197的合成流程图。
具体实施方式
本发明提供了一种氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1所示结构：
式1中，R1为酰胺基或-NH2；所述酰胺基为-NHC(O)CHpClq、-NHC(O)CHpFq、-NHC(O)CHpBrq、-NHC(O)H、-NHC(O)CaH2a+1、-NHC(O)CaH2aOH、-NHC(O)CbH2b-1或-NHC(O)CbH2b-3；其中，p或q独立地为0、1、2或3，且p+q＝3；a为1～20中的任一整数；b为2～20中的任一整数；
R2为带有双键、炔键、叠氮基、醛基、保护缩醛基、马来酰亚胺基、N-羟基琥珀酰亚胺基、巯基、保护巯基、硒基、保护硒基、-NH2或-ONH2的取代基。
在本发明中，所述保护缩醛基为缩醛基上具有保护基团的取代基，保护巯基为巯基上具有保护基团的取代基；所述保护硒基为硒上具有保护基团的取代基。本发明对所述保护基团的种类没有特殊要求。
在本发明中，所述R1优选为-NHC(O)CHpFq或-NHC(O)CaH2a+1；所述R2优选为烯丙氧基。
在本发明的具体实施例中，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1-1～式1-5所示结构中的任意一种：
在本发明中，所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物盐为式1所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物与碱反应生成的盐。
本发明提供了上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的制备方法，
所述R1为-NHC(O)CH3时，包括以下步骤：
将式2-1所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-1所示结构的偶联产物；
将式4-1所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-1所示结构的脱苄叉偶联产物；
将式5-1所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；
所述R1为-NH2时，包括以下步骤：
将式2-2所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-2所示结构的偶联产物；
将式4-2所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-2所示结构的脱苄叉偶联产物；
将式5-2所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到式6所述结构的选择性脱乙酰基偶联产物；
在保护气体气氛中，将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂和有机碱混合进行脱三氟乙酰基保护，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；
所述R1为除-NHC(O)CH3以外的酰胺基时，包括以下步骤：
将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂、有机碱和酰化试剂混合进行脱三氟乙酰基保护和酰化反应，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；所述酰化试剂为R1对应的酸酐、羧酸或羧酸酯。
在本发明中，若无特殊说明，所有制备原料/组分均为本领域技术人员熟知的市售产品。
在本发明中，所述R1为-NHC(O)CH3时，所述所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的制备方法包括以下步骤：
将式2-1所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-1所示结构的偶联产物；
将式4-1所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-1所示结构的脱苄叉偶联产物；
将式5-1所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。
本发明将式2-1所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂(以下称为第一偶联试剂)和极性溶剂混合进行糖基化偶联反应(以下称为糖基化偶联反应)，得到式4-1所示结构的偶联产物。
在本发明中，式2-1所示结构的糖基受体具体优选为式2-1-1所示结构的糖基受体；
在本发明中，式2-1-1所示结构的糖基受体的制备方法优选包括以下步骤：
将式7所示结构化合物、樟脑磺酸和极性溶剂混合进行3,4位苄叉保护，得到式8所示结构化合物；将式8所示结构化合物、四甲基哌啶氧化物(TEMPO)、碘苯二乙酸(BAIB)和极性溶剂混合进行6位选择性氧化，得到式9所示结构化合物；在保护气体气氛中，将式9所示结构化合物、NaCNBH3和极性溶剂混合进行双键还原反应，得到式2-1-1所示结构的糖基受体。
本发明将式7所示结构化合物、樟脑磺酸和极性溶剂混合进行3,4位苄叉保护，得到式8所示结构化合物。在本发明中，式7所示结构化合物的制备方法优选包括以下步骤：将式10所示结构的化合物(氨基半乳糖盐酸盐)、乙酸酐和碳酸根型强碱性树脂溶解于甲醇和水的混合溶剂中，在冰水浴条件下进行反应，得到式11所述结构的化合物；将式11所示结构的化合物、烯丙醇和三氟化硼的乙醇溶液混合，然后加入HCl的乙醇溶液，在回流的条件下进行反应，得到式7所示结构的化合物；
在本发明的具体实施例中，式11所示结构化合物的制备方法具体优选为：将式10所示市售的氨基半乳糖盐酸盐(2.5g,11.6mmol)和5.0g碳酸根型强碱性树脂，58mL水，6mL甲醇混合，冰浴下搅拌，逐滴加入1.5mL乙酸酐。2h后，抽滤，洗涤树脂。母液浓缩后通过一根强酸性树脂柱，蒸干,得到式11所示化合物，无需纯化直接投下一步反应。将制得的式11化合物和0.32mL BF3、Et2O加入到28mL烯丙醇中，回流搅拌2h。然后加入0.5mL HCl的Et2O溶液中，继续回流1h。冷却，加入乙醚直到出现混浊，4℃下放置过夜。过滤，用乙醚洗，得到白色固体0.9g，即为式7所示结构的化合物，两步反应收率为30％。
在本发明中，所述式7所示结构化合物和樟脑磺酸的摩尔比优选为3.8:0.23。所述极性溶剂优选为邻苯二甲酸二甲酯(DMP，α,α-dimethoxypropane)，本发明对所述极性溶剂的用量没有特殊要求，确保所述3,4位苄叉保护顺利进行即可。所述3,4位苄叉保护的反应温度优选为室温，反应保温时间优选为22h。所述3,4位苄叉保护反应后得到3,4位苄叉保护反应液，本发明优选对所述3,4位苄叉保护反应液进行后处理，得到式8所示结构的化合物。所述后处理优选包括：将3,4位苄叉保护反应液和饱和碳酸氢钠水溶液混合得到混合溶液；将所述混合溶液和有机溶剂混合萃取，得到萃取有机相；将合并得到的萃取有机相干燥后浓缩，得到浓缩液；将所述浓缩液进行柱层析分离，得到式8所示结构化合物。所述有机溶剂具体优选为二氯甲烷。所述干燥用试剂优选为无水硫酸钠。所述干燥优选对固液分离的有机萃取相进行浓缩，所述固液分离具体优选为过滤。所述浓缩优选为减压浓缩。所述柱层析分离使用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2:1。
得到式8所示结构化合物后，本发明将式8所示结构化合物、四甲基哌啶氧化物(TEMPO)、碘苯二乙酸(BAIB)和极性溶剂混合进行6位选择性氧化，得到式9所示结构化合物。在本发明中，式8所示结构化合物、TEMPO和BAIB的摩尔比优选为0.61:0.06:0.55。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6位选择性氧化反应顺利进行即可。所述6位选择性氧化反应的温度优选为室温，所述6位选择性氧化反应的保温时间优选为3h。在本发明中，所述6位选择性氧化反应得到6位选择性氧化反应液，本发明优选对所述6位选择性氧化反应液进行后处理，得到式9所示结构化合物。所述后处理优选包括：将6位选择性氧化反应液和饱和碳酸氢钠水溶液混合得到混合溶液；将所述混合溶液和有机溶剂混合萃取，得到萃取有机相；将合并得到的萃取有机相干燥后除溶剂，得到残留物；将所述残留物进行柱层析分离，得到式9所示结构化合物。所述有机溶剂具体优选为二氯甲烷。所述干燥用试剂优选为无水硫酸钠。所述干燥优选对固液分离的有机萃取相进行浓缩，所述固液分离具体优选为过滤。所述除溶剂优选为蒸发。所述柱层析分离使用的洗脱剂优选为石油醚和乙酸乙酯的混合溶剂，所述石油醚和乙酸乙酯的体积比优选为1:2。
得到式9所示结构化合物后，本发明在保护气体气氛中，将式9所示结构化合物、NaCNBH3和极性溶剂混合进行双键还原反应，得到式2-1-1所示结构的糖基受体。在本发明中，式9所示结构化合物和NaCNBH3的摩尔比优选为2.14:3.19。所述极性溶剂优选为乙酸和甲醇的混合溶剂，所述乙酸和甲醇的体积比优选为1：1，本发明对所述极性溶剂的用量没有特殊要求，确保所述双键还原反应顺利进行即可。所述双键还原反应的温度优选为0℃，所述双键还原反应的保温时间优选为4h，所述保护气体优选为氩气。在本发明中，所述双键还原反应得到双键还原反应液，本发明优选对所述双键还原反应液进行后处理，得到式2-1-1所示结构糖基受体。所述后处理优选包括：将双键还原反应液和有机溶剂混合得到混合溶液；将所述混合溶液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到2-1-1所示结构糖基受体。所述有机溶剂具体优选为甲苯。所述除溶剂优选为蒸发。所述柱层析分离使用的洗脱剂优选为乙酸乙酯。
在本发明中，式2-1所示结构的糖基受体中R2为其他结构的取代基时，制备方法与R2为丙烯氧基的制备方法相同，在此不再一一赘述。
在本发明中，式3所示结构的糖基供体的制备方法优选包括以下步骤：
将式12所示结构的化合物溶解于乙腈中，加入DIPEA和(EtO)2PCl，在Ar气氛中，于0～25℃进行反应，得到式3所示结构的糖基供体。
在本发明的具体实施例中，式3所示结构的糖基供体的具体制备方法优选为：氮气保护下，将式12所示结构的化合物(1.1g,2.20mmol)溶于20mL乙腈溶液中，加入0.94mL DIPEA，冰浴冷却下搅拌，加入二乙基亚磷酰氯(0.65mL，4.50mmol)，5min后撤冰浴，TLC监测反应完全后，将反应体系蒸干，加入乙酸乙酯，抽滤，将滤液、蒸干，乙酸乙酯洗涤两次，柱层析分离，洗脱剂(V/V)石油醚：乙酸乙酯＝1：2，得式3所示结构的糖基供体1.1g，收率89％。
在本发明中，第一偶联试剂具体优选为MS和三氟甲磺酸三甲基硅酯(TMSOTf)。
在本发明中，式2-1所示结构的糖基受体和式3所示结构的糖基供体的摩尔比优选为0.061:0.091。式2-1所示 结构的糖基受体和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。
得到式4-1所示结构的偶联产物后，本发明将式4-1所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-1所示结构的脱苄叉偶联产物。在本发明中，所述酸性催化试剂优选为吡啶对甲苯磺酸盐(PPTS)。式4-1所示结构的偶联产物和酸性催化试剂的质量比优选为100:47。所述极性溶剂优选为甲醇，本发明对所述极性溶剂的用量没有特殊要求，确保所述脱苄叉保护反应顺利进行即可。所述脱苄叉保护反应的温度优选为65℃，所述脱苄叉保护反应的保温时间优选为3h。在本发明中，所述脱苄叉保护后得到脱苄叉保护反应液，本发明优选对所述脱苄叉保护反应液进行后处理，得到式5-1所示结构的脱苄叉偶联产物。在本发明中，所述后处理优选包括：将脱苄叉保护反应液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到式5-1所示结构的脱苄叉偶联产物。所述除溶剂优选为蒸发。所述柱层析分离采用的洗脱溶剂优选为乙酸乙酯和甲醇，所述乙酸乙酯和甲醇的体积比优选为15:1。
在本发明中，式5-1所示结构的脱苄叉偶联产物具体优选为式5-1-1所示结构的脱苄叉偶联产物
得到式5-1所示结构的脱苄叉偶联产物后，本发明将式5-1所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。在本发明中，所述碱性催化试剂优选为甲醇钠和氢氧化钠水溶液，所述氢氧化钠水溶液的摩尔浓度优选为1mol/L。所述极性溶剂优选为甲醇，本发明对所述甲醇的用量没有特殊要求，确保所述选择性脱乙酰基顺利进行即可。在本发明中，所述混合进行选择性脱乙酰基反应包括以下步骤：将式5-1所示结构的脱苄叉偶联产物、极性溶剂和甲醇钠混合进行第一步反应；得到反应液；将所述反应液和氢氧化钠水溶液混合进行第二步反应，得到选择性脱乙酰基反应液；所述第一步反应优选采用TLC检测，所述第一步反应的时间优选为30min；所述第二步反应优选采用TLC检测，所述第二步反应的时间优选为4h。在本发明中，所述选择性脱乙酰基反应后得到选择性脱乙酰基反应液，本发明优选对所述选择性脱乙酰基反应液进行后处理，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。在本发明中，所述后处理优选包括：向所述选择性脱乙酰基反应液中通入二氧化碳至中性，将得到的中性反应液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。所述除溶剂优选为蒸发。所述柱层析分离优选采用反相柱层析分离。所述柱层析采用的洗脱溶剂优选为纯水和甲醇，所述纯水和甲醇的体积比优选为1:4。
在本发明中，所述R1为-NH2时，所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的制备方法包括以下步骤：
将式2-2所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂(以下称为第二偶联试剂)和极性溶剂混合进行糖基化偶联反应(以下称为第二糖基化偶联反应)，得到式4-2所示结构的偶联产物；
将式4-2所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-2所示结构的脱苄叉偶联产物；
将式5-2所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到式6所述结构的选择性脱乙酰基偶联产物；
在保护气体气氛中，将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂和有机碱混合进行脱三氟乙酰基保护，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。
本发明将式2-2所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-2所示结构的偶联产物。
在本发明中，式2-2所示结构的糖基受体具体优选为式2-2-1所示结构的糖基受体；
在本发明中，式2-2-1所示结构的糖基受体的制备方法优选包括以下步骤：将式10所示结构的化合物、极性溶剂、三乙胺和三氟乙酸甲酯混合进行2位上三氟乙基反应，得到含有2位三氟乙基取代的反应产物的反应液；将含有2位三氟乙基取代的反应产物的反应液浓缩得到含有2位三氟乙基取代的反应产物的浓缩液；式10所示结构的化合物的物质的量和三乙胺的体积比优选为11.6mmol:4.1mL；式10所示结构的化合物和所述三氟乙酸甲酯的摩尔比优选为11.6:12.6；所述极性溶剂优选为甲醇，本发明对所述极性溶剂的用量没有特殊要求，确保所述2位上三氟乙基反应顺利进行即可。所述2位上三氟乙基反应的温度优选为室温，所述2位上三氟乙基反应的保温时间优选过夜，所述2位上三氟乙基反应优选在搅拌的条件下进行。所述浓缩优选为减压浓缩。
将含有2位三氟乙基取代的反应产物的浓缩液、烯丙醇和盐酸乙醚混合进行1位上烯丙基反应，得到含有1位烯丙基取代的反应产物的反应液；将含有1位烯丙基取代的反应产物的反应液固液分离，将得到的滤液浓缩，得到含有1位烯丙基取代的反应产物的浓缩液；所述盐酸乙醚的摩尔浓度优选为3mol/L；式10所示结构的化合物的物质的量和所述盐酸乙醚的体积之比优选为11.6mol:18.1mL；式10所示结构的化合物的物质的量和所述烯丙醇的体积之比优选为11.6mol:28.9mL；所述1位上烯丙基反应优选在回流的条件下进行，所述回流的时间优选为0.5h。所述固液分离优选为过滤；所述浓缩优选为减压浓缩。
将含有1位烯丙基取代的反应产物的浓缩液、极性溶剂和叔丁基二甲基氯硅烷(TBDMSCL)混合进行6位TBDM保护反应，得到式13所示结构的化合物；式10所示结构的化合物和所述TBDMSCL的摩尔比优选为11.6:12.6。所述极性溶剂优选为吡啶，本发明对所述极性溶剂的用量没有特殊要求，确保所述6位TBDM保护反应顺利进行即可。所述6位TBDM保护反应的温度优选为室温，所述6位TBDM保护反应的保温时间优选为16h，所述6位TBDM保护反应优选在搅拌的条件下进行。所述6位TBDM保护反应后得到6位TBDM保护反应液，本发明优选对所述6位TBDM保护反应液进行后处理，得到式13所示结构的化合物。在本发明中，所述后处理优选包括：将所述6位TBDM保护反应液浓缩得到浓缩液；将所述浓缩液和萃取剂混合进行萃取，得到萃取有机相，将所述萃取有机相干燥后浓缩，得到浓缩液；将所述浓缩液进行柱层析分离，得到式13所示结构的化合物。所述浓缩优选为减压浓缩。所述萃取剂具体优选为二氯甲烷和饱和碳酸氢钠水溶液。所述干燥用试剂优选为无水硫酸钠。所述干燥优选对固液分离的有机萃取相进行浓缩，所述固液分离具体优选为过滤。所述浓缩优选为减压浓缩。所述柱层析分离使用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4:1至2:1；
得到式13所示结构的化合物后，本发明将式13所示结构的化合物、极性溶剂和樟脑磺酸混合，进行3,4位苄叉保护，得到式14所示结构化合物。在本发明中，所述式13所示结构化合物和樟脑磺酸的摩尔比优选为4.43:2.22。所述极性溶剂优选为乙腈和邻苯二甲酸二甲酯(DMP，α,α-dimethoxypropane)，本发明对所述极性溶剂的用量没有特殊要求，确保所述3,4位苄叉保护顺利进行即可。所述3,4位苄叉保护的反应温度优选为室温，反应保温时间优选为15min。所述3,4位苄叉保护反应后得到3,4位苄叉保护反应液，本发明优选对所述3,4位苄叉保护反应液进行后处理，得到式14所示结构的化合物。所述后处理优选包括：将3,4位苄叉保护反应液和萃取剂混合萃取，得到萃取有机相；将所述萃取有机相干燥后浓缩，得到浓缩液；将所述浓缩液进行柱层析分离，得到式14所示结构的化合物。所述萃取剂具体优选为二氯甲烷和饱和食盐水。所述干燥用试剂优选为无水硫酸钠。所述干燥优选对固液分离的有机萃取相进行浓缩，所述固液分离具体优选为过滤。所述浓缩优选为减压浓缩。所述柱层析分离使用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20:1至10:1；
得到式14所示结构的化合物后，本发明将式14所示结构的化合物、极性溶剂、乙酸和三水合四丁基氟化铵混合，进行选择性脱TBDMS反应，得到式15所示结构化合物。在本发明中，所述式14所示结构化合物和乙酸的摩尔比优选为0.12:1.25。所述式14所示结构化合物和三水合四丁基氟化铵的摩尔比优选为0.12:0.5。所述极性溶剂优选为四氢呋喃，本发明对所述极性溶剂的用量没有特殊要求，确保所述选择性脱TBDMS反应顺利进行即可。所述混合选择性脱TBDMS反应优选包括以下步骤：将式14所示结构的化合物、极性溶剂和乙酸混合，得到混合溶液；在保护气体气氛中，将混合溶液降温至0℃和三水合四丁基氟化铵混合进行选择性脱TBDMS反应。所述保护气体 优选为氩气。所述选择性脱TBDMS反应的反应温度优选为0℃，反应保温时间优选为4h。所述选择性脱TBDMS反应后得到选择性脱TBDMS反应液，本发明优选对所述选择性脱TBDMS反应液进行后处理，得到式15所示结构的化合物。所述后处理优选包括：将选择性脱TBDMS反应液浓缩后和萃取剂混合萃取，得到萃取有机相；将所述萃取有机相干燥后浓缩，得到浓缩液；将所述浓缩液进行柱层析分离，得到式15所示结构的化合物。所述浓缩优选为将选择性脱TBDMS反应液浓缩至体积减半。所述萃取剂具体优选为二氯甲烷和饱和食盐水。所述干燥用试剂优选为无水硫酸钠。所述干燥优选对固液分离的有机萃取相进行浓缩，所述固液分离具体优选为过滤。所述浓缩优选为减压浓缩。所述柱层析分离使用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2:1至1:1；
得到式15所示结构的化合物后，本发明将式15所示结构的化合物、极性溶剂、TEMPO和BAIB混合，进行6位上N-甲氧基反应，得到式16所示结构化合物。在本发明中，所述式15所示结构化合物、TEMPO和BAIB的摩尔比优选为1.92:0.19:2.11。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6位上N-甲氧基反应顺利进行即可。所述6位上N-甲氧基的反应温度优选为40℃，6位上N-甲氧基反应的保温时间优选为6h。所述选6位上N-甲氧基反应后得到6位上N-甲氧基反应液，本发明优选对所述6位上N-甲氧基反应液进行后处理，得到式16所示结构的化合物。所述后处理优选包括：将6位上N-甲氧基加有机溶剂稀释，得到稀释反应液；将所述稀释反应液、硫代硫酸钠和碳酸氢钠的共饱和水溶液搅拌混合10min，得到混合溶液；将所述混合溶液和萃取剂混合萃取，得到萃取有机相；将所述萃取有机相干燥后除溶剂，得到残留物；将所述残留物进行柱层析分离，得到式16所示结构的化合物。所述有机溶剂优选为二氯甲烷。所述萃取剂具体优选为二氯甲烷。所述干燥用试剂优选为无水硫酸钠。所述干燥优选对固液分离的有机萃取相进行除溶剂，所述除溶剂优选为蒸发。所述柱层析分离使用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10:1至4:1；
得到式16所示结构化合物后，本发明在保护气体气氛中，将式16所示结构化合物、NaCNBH3和极性溶剂混合进行双键还原反应，得到式2-2-1所示结构的糖基受体。在本发明中，式16所示结构化合物和NaCNBH3的摩尔比优选为0.73:1.09。所述极性溶剂优选为乙酸和甲醇的混合溶剂，所述乙酸和甲醇的体积比优选为1：1，本发明对所述极性溶剂的用量没有特殊要求，确保所述双键还原反应顺利进行即可。所述双键还原反应的温度优选为0℃，所述双键还原反应的保温时间优选为4h，所述保护气体优选为氩气。在本发明中，所述双键还原反应得到双键还原反应液，本发明优选对所述双键还原反应液进行后处理，得到式2-2-1所示结构糖基受体。所述后处理优选包括：将双键还原反应液和有机溶剂混合得到混合溶液；将所述混合溶液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到2-2-1所示结构糖基受体。所述有机溶剂具体优选为甲苯。所述除溶剂优选为蒸发。所述柱层析分离使用的洗脱剂优选为石油醚和乙酸乙酯，所述石油醚和乙酸乙酯的体积比优选为2:1。
在本发明中，式2-2所示结构的糖基受体中R2为其他结构的取代基时，制备方法与R2为丙烯氧基的制备方法相同，在此不再一一赘述。
在本发明中，第二偶联试剂具体优选为MS和三氟甲磺酸三甲基硅酯(TMSOTf)。
在本发明中，式2-2所示结构的糖基受体和式3所示结构的糖基供体的摩尔比优选为0.98:1.98。式2-2所示结构的糖基受体和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。
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1:2；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至5:1。
将式4-2所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-2所示结构的脱苄叉偶联产物。在本发明中，所述酸性催化试剂优选为吡啶对甲苯磺酸盐(PPTS)。所述极性溶剂优选为甲醇，本发明 对所述极性溶剂的用量没有特殊要求，确保所述脱苄叉保护反应顺利进行即可。所述脱苄叉保护反应的温度优选为65℃，所述脱苄叉保护反应的保温时间优选为3h。在本发明中，所述脱苄叉保护后得到脱苄叉保护反应液，本发明优选对所述脱苄叉保护反应液进行后处理，得到式5-2所示结构的脱苄叉偶联产物。在本发明中，所述后处理优选包括：将脱苄叉保护反应液除溶剂，得到残留物；将所述残留物进行柱层析分离，得到式5-2所示结构的脱苄叉偶联产物。所述除溶剂优选为蒸发。所述柱层析分离采用的洗脱溶剂优选为乙酸乙酯和甲醇，所述乙酸乙酯和甲醇的体积比优选为15:1。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1:2；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至5:1。
在本发明中，式5-2所示结构的脱苄叉偶联产物具体优选为式5-2-1所示结构的脱苄叉偶联产物：
得到式5-2所示结构的脱苄叉偶联产物后，本发明将式5-2所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到式6所述结构的选择性脱乙酰基偶联产物。在本发明中，所述碱性催化试剂优选为甲醇钠和氢氧化钠水溶液，所述甲醇钠优选以甲醇钠溶液的形式加入，所述甲醇钠溶液的质量百分含量优选为30％；所述氢氧化钠水溶液的摩尔浓度优选为2mol/L。所述极性溶剂优选为甲醇，本发明对所述甲醇的用量没有特殊要求，确保所述选择性脱乙酰基顺利进行即可。在本发明中，所述混合进行选择性脱乙酰基反应包括以下步骤：将式5-2所示结构的脱苄叉偶联产物、极性溶剂和甲醇钠混合进行第一步反应；得到反应液；式5-2所示结构的脱苄叉偶联产物的物质的量和甲醇钠溶液的体积之比优选为0.024mmol：0.02mL；将所述反应液和氢氧化钠水溶液混合进行第二步反应，得到选择性脱乙酰基反应液；所述第一步反应优选采用TLC检测，所述第一步反应的时间优选为1h；所述第二步反应优选采用TLC检测，所述第二步反应的时间优选为0.5h。在本发明中，所述选择性脱乙酰基反应后得到选择性脱乙酰基反应液，本发明优选对所述选择性脱乙酰基反应液进行后处理，得到所述式6所述结构的选择性脱乙酰基偶联产物。在本发明中，所述后处理优选包括：向所述选择性脱乙酰基反应液中通入二氧化碳至中性，将得到的中性反应液除溶剂，得到式6所述结构的选择性脱乙酰基偶联产物。所述除溶剂优选为蒸发。
得到式6所述结构的选择性脱乙酰基偶联产物后，本发明在保护气体气氛中，将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂和有机碱混合进行脱三氟乙酰基保护，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。在本发明中，所述有机碱具体优选为三乙胺。所述极性溶剂优选为甲醇，所述保护气体优选为氩气。在本发明中，脱三氟乙酰基保护反应优选在回流的条件下进行，所述脱三氟乙酰基保护反应的保温时间优选为过夜。本发明优选你对所述脱三氟乙酰基保护反应后得到的脱三氟乙酰基保护反应液进行后处理，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。所述后处理优选包括：将所述脱三氟乙酰基保护反应液除溶剂，得到残留物；将所述残留物进行反相柱层析，得到洗脱液；将所述洗脱液进行离子交换去除有机盐，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。在本发明中，所述除溶剂优选为蒸发。所述反相柱层析采用的洗脱剂优选为纯水至甲醇，所述纯水和甲醇的体积比优选为1:4。所述离子交换优选采用离子交换树脂柱进行。
在本发明中，所述R1为除-NHC(O)CH3以外的酰胺基时，所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的制备方法包括以下步骤：
将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂、有机碱和酰化试剂混合进行脱三氟乙酰基保护和酰化反应，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；所述酰化试剂为R1对应的酸酐、羧酸或羧酸酯。
在本发明中，所述酸酐优选包括乙酸酐、丙酸酐、正丁酸酐、异丁酸酐或正己酸酐；
所述羧酸优选包括单氟乙酸、二氟乙酸、三氟乙酸、单氯乙酸或二氯乙酸；
所述羧酸酯优选包括单氟乙酸甲酯、二氟乙酸甲酯、三氟乙酸甲酯或二氯乙酸甲酯。
在本发明中，所述酰化试剂具体优选为单氟乙酸甲酯、二氟乙酸甲酯或三氟乙酸甲酯。
在本发明中，脱三氟乙酰基保护和酰化反应优选在回流的条件下进行，所述脱三氟乙酰基保护和酰化反应的保温时间优选为过夜。本发明优选你对所述脱三氟乙酰基保护和酰化反应得到的酰化反应液进行后处理，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。所述后处理优选包括：将所述酰化反应液除溶剂，得到残留物；将所述残留物进行反相柱层析，得到洗脱液；将所述洗脱液进行离子交换去除有机盐，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物。在本发明中，所述除溶剂优选为蒸发。所述反相柱层析采用的洗脱剂优选为纯水至甲醇，所述纯水和甲醇的体积比优选为1:4.所述离子交换优选采用离子交换树脂柱进行。
本发明提供了一种糖缀合物，由上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，或上述技术方案所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐与多肽或载体蛋白质通过不同连接子偶联得到。
在本发明中，所述载体蛋白质优选为牛血清蛋白(BSA)、血蓝蛋白(KLH)或CRM197。
本发明提供了上述技术方案所述的糖缀合物的制备方法，包括以下步骤：
将上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐溶解于极性溶剂中，通入氧化 性气体进行氧化反应或通过延长碳链引入N-羟基琥珀酰亚胺基，得到含有醛基的二糖或含有N-羟基琥珀酰亚胺基的二糖；
将所述含有醛基或含有N-羟基琥珀酰亚胺基的二糖、蛋白质或多肽、还原剂和缓冲溶液混合，进行偶联反应，得到所述糖缀合物。
本发明将上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐溶解于极性溶剂中，通入氧化性气体进行氧化反应，得到含有醛基的二糖。在本发明中，所述极性溶剂优选为无水甲醇。所述氧化性气体优选为含有臭氧的空气。所述氧化反应的温度优选为-72℃；所述氧化反应进行30min得到的氧化反应液为蓝色溶液；停止通入氧化性气体。在额本本发明中，所述氧化反应后得到氧化反应液，本发明优选对所述氧化反应液进行后处理，得到含有醛基的二糖。所述后处理优选包括：将所述氧化反应液通入氮气除去未反应的氧化性气体后升温至室温；将所述氧化反应液除溶剂，得到含有醛基的二糖。所述除溶剂优选为真空除溶剂。
得到含有醛基的二糖后，本发明将所述含有醛基的二糖、蛋白质或多肽、还原剂和缓冲溶液混合，进行偶联反应，得到所述糖缀合物。
在本发明中，所述还原剂优选为氰基硼氢化钠。所述缓冲溶液的pH值优选为7.6。
在本发明中，所述偶联反应的温度优选为室温，所述偶联反应的保温时间优选为24h。所述偶联反应优选在避光条件下进行。
在本发明中，所述偶联反应后得到偶联反应液，本发明优选对所述偶联反应液进行后处理，得到所述糖缀合物。
本发明提供了上述技术方案所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或上述技术方案所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐在制备抗肿瘤药物中的应用。
本发明提供了上述技术方案所述糖缀合物或上述技术方案所述的制备方法制备得到的糖缀合物在制备抗肿瘤药物中的应用。
在本发明中，所述抗肿瘤药物优选包括治疗性疫苗或预防性疫苗。
本发明提供了一种治疗肿瘤的疫苗，包括上述技术方案所述糖缀合物或上述技术方案所述的制备方法制备得到的糖缀合物和药学上可接受的载体或辅料。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的技术方案进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
按照图8所示式7所示结构的化合物的合成流程图：
将式10所示市售的氨基半乳糖盐酸盐(2.5g,11.6mmol)和5.0g碳酸根型强碱性树脂，58mL水，6mL甲醇混合，冰浴下搅拌，逐滴加入1.5mL乙酸酐。2h后，抽滤，洗涤树脂。母液浓缩后通过一根强酸性树脂柱，蒸干,得到式11所示化合物，无需纯化直接投下一步反应。将制得的式11化合物和0.32mL BF3、Et2O加入到28mL烯丙醇中，回流搅拌2h。然后加入0.5mL HCl的Et2O溶液中，继续回流1h。冷却，加入乙醚直到出现混浊，4℃下放置过夜。过滤，用乙醚洗，得到白色固体0.9g，即为式7所示结构的化合物，两步反应收率为30％。
按照图9所示式3所示结构的糖基供体的合成流程图：
氮气保护下，将式12所示结构的化合物(1.1g,2.20mmol)溶于20mL乙腈溶液中，加入0.94mL DIPEA，冰浴冷却下搅拌，加入二乙基亚磷酰氯(0.65mL，4.50mmol)，5min后撤冰浴，TLC监测反应完全后，将反应体系蒸干，加入乙酸乙酯，抽滤，将滤液、蒸干，乙酸乙酯带两次，柱层析分离，洗脱剂(V/V)石油醚：乙酸乙酯＝1：2，得式3所示结构的糖基供体1.1g，收率89％。
按照图1所示式1-1所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图：
将式7所示结构的化合物(图1中的化合物6，1.0g，3.8mmol)溶于DMP(α,α-dimethoxypropane，24.3mL，198.5mmol)，加入樟脑磺酸(54.5mg,0.23mmol)，室温搅拌22小时。将反应混合物倒入饱和碳酸氢钠水溶液，然后用二氯甲烷萃取；萃取液合并后用无水硫酸钠干燥，过滤，减压浓缩，残留物用柱层析分离(石油醚：丙酮，2:1)，得白色固体为式8所述结构的化合物(图1中的化合物7)940mg,收率82％。
1H NMR(300MHz,CDCl3):δ5.89-5.83(1H,m),5.54(1H,d,J＝9.3Hz),5.31-5.21(2H,m),4.85(1H,d,J＝3.6Hz),4.31(1H,dt,J＝3.3Hz,9.3Hz),4.20-4.18(1H,m),4.15(1H,td,J＝5.1Hz),4.12-3.95(4H,m),3.89-3.82(1H,m),2.18(1H,dd,J＝3.3Hz,9.3Hz),2.04(3H,s),1.59(3H,s),1.35(3H,s)。
将式8所述结构的化合物(183mg,0.61mmol)溶于1mL二氯甲烷，加入TEMPO(9.1mg,0.06mmol)和BAIB (215mg,0.55mmol)，室温搅拌3小时。用4mL二氯甲烷稀释，加入5.2mL硫代硫酸钠和碳酸氢钠的共饱和水溶液，剧烈搅拌10分钟。用二氯甲烷萃取，硫酸钠干燥，蒸干。将残留物溶于2mL吡啶，加入MeONH2·HCl(76.1mg,0.91mmol)，室温搅拌1小时，蒸干，残留物用柱层析分离(石油醚：乙酸乙酯，1:2)，得白色固体为式9所示结构的化合物(图1中的化合物8)，为161mg,收率81％(Z/E＝1/1)。
1H NMR(300MHz,CDCl3)δ7.52(d,1H,J＝7.8Hz),6.87(d,1H,J＝4.8Hz),5.94-5.81(m,2H),5.57(d,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 C15H25N2O6[M+H]+:329.1707,found 329.1701。
将式9所示结构的化合物(701mg,2.14mmol)溶于乙酸/甲醇(7mL/7mL)混合溶液，0oC、氩气保护下加入NaCNBH3(201.9mg,3.19mmol)，搅拌，4小时后反应完全。加入少量甲苯，浓缩蒸干，残留物用柱层析分离(乙酸乙酯)得油状物为式2-1-1所示结构的糖基受体(图1中的化合物9)637mg,收率90％。
1H NMR(300MHz,CDCl3)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)；13C NMR(75MHz,CDCl3)δ169.97,133.51,117.84,109.67,96.75,74.72,73.75,68.26,63.09,61.37,52.09,50.50,27.95,26.55,23.46；HRMS(ESI)Anal.Calcd for C15H27N2O6[M+H]+:331.1864,found 331.1865。
将式3所示结构的糖基供体(55.5mg,0.091mmol)，式2-1-1所示结构的糖基受体(20.0mg,0.061mmol)，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％。
1H NMR(500MHz,CDCl3)δ5.91-5.83(m,1H),5.57(d,1H,J＝9.0Hz),5.38(dd,1H,J＝2.5Hz,7.0Hz),5.34(dt,1H,J＝1.5Hz,10.0Hz),5.27(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)；13C NMR(125MHz,CDCl3)δ170.90,170.69,170.24,170.07,170.04,170.00,167.64,133.52,117.55,109.37,96.92,94.89,74.44,73.00,69.82,69.66,68.29,67.93,65.11,63.96,62.39,53.12,52.70,50.37,49.33,35.11,29.63,28.06,26.55,23.42,23.13,21.00,20.82,20.75,20.68；HRMS(ESI)Anal.Calcd for C35H54N3O18[M+H]+:804.3397,found 804.3425。
式4-1-4所示结构的化合物(图1中的化合物11)(100mg,0.124mmol)溶解在干燥的甲醇(1mL)中，将PPTS(47mg)加入到反应体系中，65℃搅拌3h，蒸干，残留物用柱层析分离(乙酸乙酯：甲醇，15：1)得白色固体为式5-1-1所示结构的化合物(图1中的化合物12)，92mg，收率97％。
1H NMR(500MHz,CDCl3)δ5.95(d,1H,J＝8.5Hz),5.95-5.86(m,1H),5.40-5.37(m,2H),5.35(dd,1H,J＝2.5Hz,7.5Hz),5.30(dd,1H,J＝1.5Hz,17Hz),5.23(dd,1H,J＝1.0Hz,15.5Hz),4.87-4.80(m,2H),4.38(dd,1H,J＝1.5Hz,12.5Hz),4.35-4.30(m,1H),4.23(dd,1H,J＝5.0Hz,13.0Hz),4.14-4.02(m,5H),4.00(dd,1H,J＝6.0Hz,13.0Hz),3.90(t,1H,J＝6.0Hz),3.80(s,3H),3.80-3.77(m,1H),3.59(s,3H),3.21(dd,1H,J＝7.5Hz,14.0Hz),3.12(dd,1H,J＝5.5Hz,14.5Hz),3.09(d,1H,J＝3.5Hz),2.59(dd,1H,J＝4.5Hz,12.5Hz),2.21(t,1H,J＝12.5Hz),2.14,2.14,2.07,2.04,2.04,1.88(s,6*3H)；13C NMR(100MHz,CDCl3)δ172.39,170.94,170.79,170.30,170.26,170.21,167.94,133.54,117.75,96.51,94.76,72.94,71.21,69.73,69.48,68.51,68.16,67.90,67.80,63.92,62.52,52.89,52.83,50.68,49.39,34.95,23.32,23.15,21.08,20.84,20.81,20.77；HRMS(ESI)Anal.Calcd for C32H50N3O18[M+H]+:764.3084,found 764.3088。
将式5-1-1所示结构的化合物(图1中的化合物12)(30mg,0.039mmol)溶于甲醇(2mL)，室温下搅拌，加入30％甲醇钠一滴，30分钟后TLC显示反应完全。浓缩抽干溶剂后，加入1NNaOH水溶液(1.3mL)。4小时后TLC显示反应完全，通入二氧化碳气体至中性，蒸干后残留物用反相柱层析分离(纯水至甲醇：水，1：4)，得到白色固体为式1-1所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物，19mg，收率83％。
1H NMR(400MHz,D2O)δ5.88-5.80(m,1H),5.24(dq,1H,J＝1.6Hz,17.6Hz),5.15(dd,1H,J＝1.6Hz,10.8Hz),4.83(d,1H,J＝3.6Hz),4.13(ddt,1H,J＝1.2Hz,5.2Hz,13.2Hz),4.05(dd,1H,J＝4Hz,11.2Hz),4.02-3.94(m,3H),3.84(dd,1H,J＝3.2Hz,11.2Hz),3.78-3.72(m,2H),3.70-3.64(m,2H),3.57-3.45(m,6H),3.10(dd,1H,J＝6.8Hz,14.4Hz),2.98(dd,1H,J＝6.4Hz,14.4Hz),2.61(dd,1H,J＝4.4Hz,12.4Hz),1.93(s,3H),1.92(s,3H),1.80(t,1H,J＝12.4Hz)；13C NMR(100MHz,D2O)δ174.94,174.60,173.11,133.63,118.03,96.56,95.66,72.88,71.92,69.14,68.79,68.70,68.63,68.38,67.76,64.01,62.62,52.94,51.83,49.87,37.70,22.03,21.92；HRMS(ESI)Anal.Calcd for C23H40N3O14[M+H]+:582.2505,found 582.2517。
实施例2
按照图2所示实施例2提供式1-2所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图：
将式10所示结构的化合物(D-氨基半乳糖盐酸盐，2.5g，11.6mmol)溶于34.7mL甲醇，加入4.1mL三乙胺和三氟乙酸甲酯(1.5mL，12.6mmol)，室温搅拌过夜后减压浓缩。将残留物溶于28.9mL烯丙醇，加入18.1mL3M盐酸乙醚，回流0.5h。过滤，将滤液浓缩。将残留物溶于18.6mL吡啶，加入TBDMSCl(1.9g，12.6mmol)，室温搅拌16h。减压浓缩后用二氯甲烷和饱和碳酸氢钠萃取，有机相用硫酸钠干燥，抽滤，将滤液浓缩，残留物用柱层析分离(石油醚：乙酸乙酯，4：1至2：1)，得到油状物为式13所示结构的化合物(图2中的化合物13)，2.3g，收率46％。
1H NMR(400MHz,CDCl3)δ6.53(d,1H,J＝8.9Hz),5.91-5.84(m,1H),5.30-5.24(m,2H),4.97(d,1H,J＝3.7Hz),4.41(td,1H,J＝3.6Hz,9.9Hz),4.19(dd,1H,J＝5.3Hz,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)；13C NMR(100MHz,CDCl3)δ158.01(q,J＝37.0Hz),133.10,118.28,115.79(q,J＝286.0Hz),96.11,69.84,69.84,69.69,68.43,63.81,51.08,25.76,18.20,-5.54,-5.57；HRMS(ESI)Anal.Calcd for C17H34N2O6F3Si[M+NH4]+:447.2133,found 447.2122。
将化合物13(1.9g，4.43mmol)溶于53.2mL乙腈，加入DMP(10.8mL，88.2mmol)和樟脑磺酸(506mg，2.22mmol)，室温搅拌15分钟。用二氯甲烷和饱和食盐水萃取，有机相用硫酸钠干燥，抽滤，将滤液浓缩，残留物用柱层析分离(石油醚：乙酸乙酯，20：1至10：1)，得到油状物为式14所示结构的化合物(图2中的化合物14)，1.1g，收率51％。
1H NMR(400MHz,CDCl3)δ6.40(d,1H,J＝9.3Hz),5.90-5.80(m,1H),5.28-5.22(m,2H),4.83(d,1H,J＝3.3Hz),4.27-4.14(m,3H),4.11(dd,1H,J＝4.9Hz,8.8Hz),4.03(td,1H,J＝2.1Hz,6.5Hz),3.97(dd,1H,J＝6.4Hz,12.8Hz),3.89(dd,1H,J＝6.7Hz,10.0Hz),3.82(dd,1H,J＝6.6Hz,10.0Hz),1.55(s,3H),1.33(s,3H),0.90(s,9H),0.08(s,6H)；13C NMR(100MHz,CDCl3)δ157.17(q,J＝37.0Hz),132.98,118.41,115.78(q,J＝286.2Hz),109.84,95.89,74.02,72.26,68.39,68.34,62.19,51.52,27.93,26.39,25.76,18.20,-5.42,-5.55；HRMS(ESI)Anal.Calcd for C20H34NO6F3SiK[M+K]+:508.1734,found 508.1734。
将化合物14(58mg，0.12mmol)溶于3.2mL四氢呋喃，加入乙酸(65.8μL，1.25mmol)，0℃氩气保护下加入三水合四丁基氟化铵(157.7mg，0.50mmol)。50℃下搅拌4小时后，将体系浓缩至原体积一半，用二氯甲烷和饱和食盐水萃取，有机相用硫酸钠干燥，抽滤，将滤液浓缩，残留物用柱层析分离(石油醚：乙酸乙酯，2：1至1：1)，得到油状物为式15所示结构的化合物(图2中的化合物15)38mg，收率87％。
1H NMR(400MHz,CDCl3)δ6.50(d,1H,J＝8.9Hz),5.90-5.81(m,1H),5.30-5.23(m,2H),4.89(d,1H,J＝3.1Hz),4.29-4.14(m,4H),4.12-4.05(m,1H),4.03-3.94(m,2H),3.91-3.81(m,1H),2.32(dd,1H,J＝2.8Hz,8.8Hz),1.56(s,3H),1.34(s,3H)；13C NMR(100MHz,CDCl3)δ157.28(q,J＝38.0Hz),132.83,118.59,115.76(q,J＝286.0Hz),110.29,96.10,74.03,73.31,68.66,67.87,62.52,51.38,27.89,26.47；HRMS(ESI)Anal.Calcd for C14H20NO6F3Na[M+Na]+:378.1135,found 378.1134。
化合物15(681mg,1.92mmol)溶于3.2mL二氯甲烷，加入TEMPO(29mg，0.19mmol)和BAIB(679mg，2.11mmol)，40℃搅拌6小时。用15mL二氯甲烷稀释，加入20mL硫代硫酸钠和碳酸氢钠的共饱和水溶液，剧烈搅拌10min。用二氯甲烷萃取，硫酸钠干燥，蒸干。将残留物溶于6.3mL吡啶，加入MeONH2·HCl(240mg，2.85mmol)，室温搅拌1小时，蒸干，残留物用柱层析分离(石油醚：乙酸乙酯，10：1至4：1)，得到白色固体为式16所示结构的化合物(图2中的化合物16)，735mg，两步反应收率100％(Z/E＝1/2or 2/1)。
1H NMR(400MHz,CDCl3)δ7.52(d,2H,J＝7.3Hz),6.87(d,1H,J＝4.9Hz),6.32(d,3H,J＝8.9Hz),5.91-5.81(m,3H),5.36-5.22(m,6H),5.13(dd,1H,J＝2.7Hz,4.8Hz),4.90-4.88(m,3H),4.59(dd,2H,J＝2.4Hz,7.3Hz),4.48(dd,1H,J＝2.7Hz,4.9Hz),4.33-4.12(m,11H),4.05-3.97(m,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 C15H21N2O6F3K[M+K]+:421.0978,found 421.0982。
将化合物16(279mg,0.73mmol)溶于冰醋酸/甲醇(2.4mL/2.4mL)混合溶液，0℃、氩气保护下加入NaCNBH3(69mg，1.09mmol)，搅拌，4小时反应进行完全，加入少量甲苯，蒸干，残留物用柱层析分离(石油醚：乙酸乙酯，2：1)，得到白色固体为式2-2-1所示结构的糖基受体(图2中的化合物17)268mg，收率96％。
1H NMR(400MHz,CDCl3)δ6.38(d,1H,J＝9.3Hz),5.99-5.75(m,2H),5.28-5.22(m,2H),4.84(d,1H,J＝3.4Hz),4.33-4.31(m,1H),4.25-4.17(m,2H),4.14-4.10(m,2H),3.97(dd,1H,J＝6.3Hz,12.7Hz),3.52(s,3H),3.25(dd,1H,J＝3.8Hz,14.1Hz),3.18(dd,1H,J＝8.9Hz,14.1Hz),1.55(s,3H),1.33(s,3H)；13C NMR(100MHz,CDCl3)δ157.19(q,J＝37.1Hz),132.97,118.49,115.77(q,J＝286.3Hz),110.04,95.84,74.19,73.58,68.45,63.32,61.39,51.97,51.47,27.91,26.46；HRMS(ESI)Anal.Calcd for C15H24N2O6F3[M+H]+:385.1581,found 385.1570。
将式3所示结构的糖基供体(图2中的化合物10)(1209mg,1.98mmol)，2-2-1所示结构的糖基受体(378mg,0.98mmol)和1.6g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％。
1H NMR(400MHz,CDCl3)δ6.74(d,1H,J＝9.0Hz),5.93-5.84(m,1H),5.46-5.21(m,5H),4.96(d,1H,J＝3.7Hz),4.92-4.81(m,1H),4.46-4.33(m,2H),4.25(dd,1H,J＝5.1Hz,13.0Hz),4.13-4.00(m,5H),3.97(t,1H,J＝5.8Hz),3.90-3.79(m,4H),3.61(s,3H),3.35(d,1H,J＝4.4Hz),3.21(d,2H,J＝5.9Hz),3.09(d,1H,J＝9.4Hz),2.64(dd,1H,J＝4.4Hz,12.6Hz),2.20(t,1H,J＝12.4Hz),2.16(s,3H),2.16(s,3H),2.06(s,3H),2.05(s,3H),1.90(s,3H)；13C NMR(100MHz,CDCl3)δ170.97,170.94,170.47,170.41,170.20,168.07,157.89(q,J＝37.0Hz),133.22,118.13,115.82(q,J＝285.9Hz),95.97,94.53,72.89,69.60,69.39,69.23,68.57,68.48,67.68,63.48,62.62,52.98,52.91,51.13,49.42,34.84,23.09,21.11,20.81,20.79,20.73；HRMS(ESI)Anal.Calcd for C32H47N3O18F3[M+H]+:818.2801,found 818.2808。
化合物18(20mg,0.024mmol)溶于1.1mL甲醇，室温下搅拌，加入0.02mL 30％甲醇钠，1小时后TLC显示反应完全。浓缩抽干溶剂后，加入0.6mL 2M氢氧化钠水溶液，0.5小时后TLC显示反应完全，通入二氧化碳气体至中性，蒸干。将残留物溶于1.2mL甲醇，氩气保护下加入0.5mL三乙胺和0.2mL氟乙酸甲酯，回流搅拌过夜，蒸干后残留物用反相柱层析分离(纯水至甲醇：水，1：4)，随后通过一根离子交换树脂柱除去残留的三乙胺 盐，得到白色固体为式1-2所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物，11mg，收率75％。
1H NMR(400MHz,D2O)δ5.93-5.85(m,1H),5.28(dd,1H,J＝1.2Hz,17.3Hz),5.19(d,1H,J＝10.5Hz),4.91(d,1H,J＝3.8Hz),4.87(d,1H,J＝46.4Hz),4.25-4.14(m,2H),4.07(t,1H,J＝6.2Hz),4.04-3.94(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)；13C NMR(100MHz,D2O)δ174.96,173.12,171.09(d,J＝18.6Hz),133.61,118.14,96.49,95.70,79.77(d,J＝179.6Hz),72.92,71.97,69.18,68.81,68.75,68.67,68.49,67.63,64.05,62.66,52.96,51.87,49.60,37.74,22.06；HRMS(ESI)Anal.Calcd for C23H38N3O14FNa[M+Na]+:622.2230,found 622.2229。
实施例3
按照图2所示实施例2提供式1-2所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图：
按照实施例2中的制备方法制备得到式5-2-1所示结构的化合物；
式5-2-1所示结构的化合物(图2中的化合物18)(20mg,0.024mmol)溶于1.1mL甲醇。室温下搅拌，加入0.02mL 30％甲醇钠，1小时后TLC显示反应完全。浓缩抽干溶剂后，加入0.6mL 2M氢氧化钠水溶液。0.5小时后TLC显示反应完全，通入二氧化碳气体至中性，蒸干。将残留物溶于1.2mL甲醇，氩气保护下加入0.5mL三乙胺和0.2mL二氟乙酸甲酯，回流搅拌过夜，蒸干后残留物用反相柱层析分离(纯水至甲醇：水，1：4)，随后通过一根离子交换树脂柱除去残留的三乙胺盐，得到白色固体为式1-3所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物，13mg，收率86％。
1H NMR(400MHz,D2O)δ6.10(t,1H,J＝53.6Hz),5.87(ddd,1H,J＝5.8Hz,11.0Hz,22.4Hz),5.26(dd,1H,J＝1.5Hz,17.3Hz),5.18(d,1H,J＝10.5Hz),4.92(d,1H,J＝3.8Hz),4.22-4.13(m,2H),4.06(t,1H,J＝6.3Hz),4.03-3.94(m,3H),3.83-3.76(m,2H),3.76-3.66(m,2H),3.62-3.52(m,5H),3.50(dd,1H,J＝1.4Hz,8.9Hz),3.15(dd,1H,J＝6.4Hz,14.3Hz),3.02(dd,1H,J＝6.0Hz,14.4Hz),2.65(dd,1H,J＝4.5Hz,12.3Hz),1.95(s,3H),1.84(t,1H,J＝12.0Hz)；13C NMR(100MHz,D2O)δ179.92,178.09,170.29(t,J＝25.7Hz),138.52,123.17,113.14(t,J＝245.6Hz),101.10,100.65,77.88,76.93,74.16,73.74,73.71,73.62,73.45,72.39,69.00,67.61,57.91,56.82,55.13,42.69,27.02；HRMS(ESI)Anal.Calcd for C23H37N3O14F2Na[M+Na]+:640.2136,found 640.2139。
实施例4
按照图2所示实施例3提供式1-3所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图：
按照实施例2中的制备方法制备得到式5-2-1所示结构的化合物；
式5-2-1所示结构的化合物(图2中的化合物18)(40mg,0.049mmol)溶于2.3mL甲醇。室温下搅拌，加入0.02mL 30％甲醇钠，1小时后TLC显示反应完全。浓缩抽干溶剂后，加入1.1mL 2M氢氧化钠水溶液。0.5小时后TLC显示反应完全，通入二氧化碳气体至中性，蒸干。将残留物溶于2.4mL甲醇，氩气保护下加入1.0mL三乙胺和0.5mL三氟乙酸甲酯，回流搅拌过夜，蒸干后残留物用反相柱层析分离(纯水至甲醇：水，1：4)，随后通过一根离子交换树脂柱除去残留的三乙胺盐，得到白色固体为式1-4所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物，18mg，收率58％。
1H NMR(400MHz,D2O)δ5.94-5.80(m,1H),5.27(dd,1H,J＝1.5Hz,17.3Hz),5.19(d,1H,J＝10.4Hz),4.94(d,1H,J＝3.8Hz),4.23-4.14(m,2H),4.07(t,1H,J＝6.3Hz),4.04-3.97(m,3H),3.84-3.77(m,2H),3.77-3.67(m,2H),3.62-3.53(m,5H),3.51(dd,1H,J＝1.4Hz,8.9Hz),3.15(dd,1H,J＝6.4Hz,14.3Hz),3.03(dd,1H,J＝6.1Hz,14.3Hz),2.65(dd,1H,J＝4.5Hz,12.3Hz),1.96(s,3H),1.84(t,1H,J＝12.0Hz)；13C 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 C23H37N3O14F3[M+H]+:636.2222,found 636.2232。
实施例5
按照图2所示实施例5提供式1-5所示结构的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的合成流程图：
按照实施例2中的制备方法制备得到式5-2-1所示结构的化合物；
式5-2-1所示结构的化合物(图2中的化合物18)(40mg,0.049mmol)溶于2.3mL甲醇。室温下搅拌，加入0.02mL 30％甲醇钠，1小时后TLC显示反应完全。浓缩抽干溶剂后，加入1.1mL 2M氢氧化钠水溶液。0.5小时后TLC显示反应完全，通入二氧化碳气体至中性，蒸干。将残留物溶于4.9mL甲醇，0℃加入丙酸酐(25.2μL，0.20mmol)，搅拌0.5小时。蒸干后残留物用反相柱层析分离(纯水至甲醇：水，1：4)，得到白色固体为式1-5所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物，131mg，收率100％。
1H NMR(400MHz,D2O)δ5.87(dq,1H,J＝5.8Hz,10.7Hz),5.27(d,1H,J＝17.3Hz),5.18(d,1H,J＝10.4Hz),4.87(d,1H,J＝3.6Hz),4.17(dd,1H,J＝5.0Hz,12.9Hz),4.09(dd,1H,J＝3.4Hz,11.1Hz),4.06-3.95(m,3H),3.88(dd,1H,J＝2.5Hz,11.2Hz),3.78-3.68(m,4H),3.62-3.48(m,6H),3.13(dd,1H,J＝6.1Hz,14.2Hz),3.02(dd,1H,J＝6.1Hz,14.3Hz),2.65(dd,1H,J＝4.2Hz,12.2Hz),2.23(q,2H,J＝7.6Hz),1.96(s,3H),1.83(t,1H,J＝11.9Hz),1.04(t,3H,J＝7.6Hz)；13C NMR(100MHz,D2O)δ178.60,174.95,173.11,133.60,118.14,96.57,95.67,72.91,71.95,69.20,68.87,68.73,68.65,68.42,67.71,64.02,62.65,52.99,51.86,49.80,37.74,29.12,22.05,9.57；HRMS(ESI)Anal.Calcd for C24H41N3O14Na[M+Na]+:618.2481,found 618.2487。
实施例6
按照图10所述的糖蛋白缀合物合成流程制备：
将实施例1～5制备的式1-1～式1-5所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物、式17所示结构的STn分别溶解于5mL无水甲醇中，-72℃下通入含有臭氧的空气，当体系变成蓝色(约需10-30分钟)后，停止通臭氧，10分钟后体系仍然为蓝色。向反应体系通入氮气约10分钟，以便除去过量的臭氧。滴加二甲硫醚0.2mL， 之后让反应体系温度自然升至室温，2小时后将反应体系在真空下除去溶剂，即得到了含有醛基的半抗原。
将含有醛基的半抗原分别与KLH共同溶解于pH值为7.6的缓冲液中，加入氰基硼氢化钠，室温下在摇床上避光反应24小时。透析后即得到糖蛋白缀合物N(OMe)-STn-KLH，分别记为1-KLH、2-KLH，3-KLH，4-KLH，5-KLH。
实施例7
与实施例6的制备方法基本相同，不同之处在于：采用CRM197替换实施6中的KLH，得到糖蛋白缀合物N(OMe)-STn-KLH，分别记为1-CRM197、2-CRM197，3-CRM197，4-CRM197，5-CRM197。
实施例8
按照图11所述的糖蛋白缀合物1-NHS-CRM197的合成流程制备：
将实施例5制备的式1-5所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物10mg与巯基乙胺盐酸盐2.88mg溶解于1mL去氧去离子水中，室温下紫外照射反应10min后，减压浓缩，使用葡聚糖G10凝胶柱纯化，将纯化所得产物溶于1mL超干DMF中，滴入含有57.8mg双(2,5-二氧代吡咯烷-1-基)己二酸酯的1mL超干DMF中，剧烈搅拌室温反应2小时。反应结束，减压浓缩，甲醇复溶，HPLC分离后，冷冻干燥，得白色固体产物半抗原N(OMe)-STn-NHS。
将N(OMe)-STn-NHS与CRM197共同溶解在PH＝8.0的K2HPO4-PBS缓冲液中，室温下在摇床上反应12小时，超滤后得到糖蛋白缀合物1-NHS-CRM197。
应用例
一、试验材料及来源
1、供试化合物：本发明实施例6、7、8所制备的糖蛋白(多肽)缀合物；
二、试验方法
(一)小鼠免疫
每组6只Balb/c雌性小鼠，6—8周龄(Number:SCXKjing2007-0001,SPF/VAF)，购自北京大学医学部动物科学部并于动物部饲养。用STn-KLH、STn-CRM197以及氮连接的STn衍生物与KLH或CRM197的缀合物(1-KLH、2-KLH、3-KLH、4-KLH、5-KLH和1-CRM197、2-CRM197、3-CRM197、4-CRM197、5-CRM197)分别免疫小鼠，每次免疫的糖蛋白(多肽)中含有1—3μg的糖(溶解于PBS)，每2周免疫一次，免疫途径为腹腔注射，共免疫4次。分别于免疫前、第2次免疫后13天、第3次免疫后13天及第4次免疫后14天取血，分离血清，-80oC冰箱冻存待测。
(二)小鼠免疫前后血清中抗体滴度的测定
各组小鼠混合血清的滴度，以及实施例6和实施例7制备的1-KLH、2-KLH、3-KLH、4-KLH、5-KLH和1-CRM197、2-CRM197、3-CRM197、4-CRM197、5-CRM197免疫组每只小鼠血清的滴度均采用ELISA方法检测。
1包被抗原：酶标板上包被100μL的STn-BSA(包含0.02μg的STn)4oC过夜。
2洗涤与封闭：每孔加入200μL洗涤缓冲液PBS-Tween20(0.05％)洗板，洗3次，然后每孔加入200μL封闭液(3％BSA-PBS)，37℃，1小时。
3加一抗(即免疫血清)：洗涤3次(具体方法同上)。血清用抗体稀释液(1％BSA-PBS)从某一稀释度开始倍比稀释，每孔加100μL，37℃，1小时。
4加酶标二抗：洗涤3次，每孔加入用抗体稀释液5000倍稀释的100μL的二抗(为辣根过氧化物酶标记的羊抗小鼠IgG(γ-chain特异))，37℃，1小时。
5显色：洗涤3次，每孔加入现配的显色底物邻苯二胺(OPD)100μL，室温避光显色15分钟，每孔加入2M H2SO4终止显色。
6结果判断：用酶标仪于490nm波长读取OD值。把减去空白血清孔读数后的OD值为0.1时的血清稀释倍数作为抗体滴度。
(三)小鼠免疫治疗
每组8只Balb/c雌性小鼠，6—8周龄(Number:SCXKjing2007-0001,SPF/VAF)，购自北京大学医学部动物科学部并于动物部饲养。在第0天每只小鼠腋下接种5×10^5个CT26细胞，第2、6、10、17天分别皮下注射含有1～3μg糖的1-NHS-CRM197、1-CRM197以及1-KLH的PBS溶液。
三、试验结果
测试结果如表1和图3～6所示。其中，图3为本发明实施例6制备的1-KLH第四次免疫后血清STn-KLH和1-KLH组每只小鼠血清的滴度；图4为本发明实施例6制备的1-KLH施用后小鼠生存曲线；图5为本发明实施例7制备的1-CRM197施用后小鼠肿瘤生长曲线；图6为本发明实施例7制备的1-CRM197施用后小鼠生存曲线，图7为本发明实施例8制备的1-NHS-CRM197施用后小鼠肿瘤生长曲线。
表1为实施例6制备的糖缀合物第三次和第四次免疫后13天测定小鼠血清中识别STn的抗体滴度
表2为实施例7制备的1-CRM197和实施例8制备的1-NHS-CRM197的糖负载量的比较。
表2 1-CRM197和1-NHS-CRM197的糖负载量
用表达STn糖抗原的CT-26结肠癌细胞构建了小鼠荷瘤模型；然后用本发明专利中的糖缀合物(1-KLH、1-CRM197或1-NHS-CRM197)免疫荷瘤小鼠，观察小鼠生存期、肿瘤体积以及抗体滴度。实验结果表明与对照组相比，糖缀合物1-KLH、1-CRM197或1-NHS-CRM197接种小鼠后明显延长了小鼠的生存期、抑制肿瘤生长、增加抗体滴度，表明具有良好的抗肿瘤效果。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (15)

  1. 一种氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1所示结构：
    式1中，R1为酰胺基或-NH2；所述酰胺基为-NHC(O)CHpClq、-NHC(O)CHpFq、-NHC(O)CHpBrq、-NHC(O)H、-NHC(O)CaH2a+1、-NHC(O)CaH2aOH、-NHC(O)CbH2b-1或-NHC(O)CbH2b-3；其中，p或q独立地为0、1、2或3，且p+q＝3；a为1～20中的任一整数；b为2～20中的任一整数；
    R2为带有双键、炔键、叠氮基、醛基、保护缩醛基、马来酰亚胺基、N-羟基琥珀酰亚胺基、巯基、保护巯基、硒基、保护硒基、-NH2或-ONH2的取代基。
  2. 根据权利要求1所述的氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，其特征在于，所述R1为-NHC(O)CHpFq或-NHC(O)CaH2a+1；所述R2为烯丙氧基。
  3. 根据权利要求1所述的氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，其特征在于，所述氮连接的唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物具有式1-1～式1-5所示结构中的任一结构：
  4. 根据权利要求1～3任一项所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐，其特征在于，所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物盐为式1所示结构的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物与碱反应生成的盐。
  5. 权利要求1～3任一项所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物的制备方法，其特征在于，
    所述R1为-NHC(O)CH3时，包括以下步骤：
    将式2-1所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-1所示结构的偶联产物；
    将式4-1所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-1所示结构的脱苄叉偶联产物；
    将式5-1所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；
    所述R1为-NH2时，包括以下步骤：
    将式2-2所示结构的糖基受体、式3所示结构的糖基供体、偶联试剂和极性溶剂混合进行糖基化偶联反应，得到式4-2所示结构的偶联产物；
    将式4-2所示结构的偶联产物、极性溶剂和酸性催化试剂混合，进行脱苄叉保护，得到式5-2所示结构的脱苄叉偶联产物；
    将式5-2所示结构的脱苄叉偶联产物、极性溶剂和碱性催化试剂混合，进行选择性脱乙酰基，得到式6所述结构的选择性脱乙酰基偶联产物；
    在保护气体气氛中，将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂和有机碱混合进行脱三氟乙酰基保护，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；
    所述R1为除-NHC(O)CH3以外的酰胺基时，包括以下步骤：
    将式6所述结构的选择性脱乙酰基偶联产物、极性溶剂、有机碱和酰化试剂混合进行脱三氟乙酰基保护和酰化反应，得到所述氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物；所述酰化试剂为R1对应的酸酐、羧酸或羧酸酯。
  6. 根据权利要求5所述的制备方法，其特征在于，所述式2-1所示结构的糖基受体为式2-1-1所示结构的糖基受体；
    式2-1-1所示结构的糖基受体的制备方法包括以下步骤：
    将式7所示结构化合物、樟脑磺酸和极性溶剂混合进行3,4位苄叉保护，得到式8所示结构化合物；将式8所示结构化合物、四甲基哌啶氧化物、碘苯二乙酸和极性溶剂混合进行6位选择性氧化，得到式9所示结构化合物；在保护气体气氛中，将式9所示结构化合物、NaCNBH3和极性溶剂混合进行双键还原反应，得到式2-1-1所示结构的糖基受体。
  7. 根据权利要求6所述的制备方法，其特征在于，式7所示结构化合物的制备方法包括以下步骤：将式10所示结构的化合物、乙酸酐和碳酸根型强碱性树脂溶解于甲醇和水的混合溶剂中，在冰水浴条件下进行反应，得到式11所述结构的化合物；将式11所示结构的化合物、烯丙醇和三氟化硼的乙醇溶液混合，然后加入HCl的乙醇溶液，在回流的条件下进行反应，得到式7所示结构的化合物；
  8. 根据权利要求5所述的制备方法，其特征在于，式3所示结构的糖基供体的制备方法包括以下步骤：
    将式12所示结构的化合物溶解于乙腈中，加入DIPEA和(EtO)2PCl，在Ar气氛中，于0～25℃进行反应，得到式3所示结构的糖基供体。
  9. 一种糖缀合物，其特征在于，由权利要求1～4任一项所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生 物或其盐，或权利要求5～8任一项所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐与多肽或载体蛋白质通过不同连接子偶联得到。
  10. 根据权利要求9所述的糖缀合物，其特征在于，所述载体蛋白质为牛血清蛋白、血蓝蛋白或CRM197。
  11. 权利要求9或10所述的糖缀合物的制备方法，其特征在于，包括以下步骤：
    将所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐溶解于极性溶剂中，通入氧化性气体进行氧化反应或通过延长碳链引入N-羟基琥珀酰亚胺基，得到含有醛基或含有N-羟基琥珀酰亚胺基的二糖；
    将所述含有醛基或含有N-羟基琥珀酰亚胺基的二糖、蛋白质或多肽、还原剂和缓冲溶液混合，进行偶联反应，得到所述糖缀合物。
  12. 权利要求1～4任一项所述的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐或权利要求5～8任一项所述的制备方法制备得到的氮连接唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐在制备抗肿瘤药物中的应用。
  13. 权利要求9或10所述糖缀合物或权利要求11所述的制备方法制备得到的糖缀合物在制备抗肿瘤药物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述抗肿瘤药物包括治疗性疫苗或预防性疫苗。
  15. 一种治疗肿瘤的疫苗，包括权利要求9或10所述糖缀合物或权利要求11所述的制备方法制备得到的糖缀合物和药学上可接受的载体或辅料。
PCT/CN2023/124243 2022-10-12 2023-10-12 唾液酸(α-(2→6))-D-氨基吡喃半乳糖衍生物或其盐、糖缀合物及其制备方法 WO2024078578A1 (zh)

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