A method for synthesizing a controllable polysaccharide of a human sequence

Abstract

The application designs and synthesizes polysaccharide construction monomers (mannose, galactose or glucose) containing azide group and terminal alkyne group at the same time, realizes monosaccharide molecular coupling through copper catalyzed azide-alkyne click chemistry reaction (CuAAC), the rate of copper catalyzed azide-alkyne click chemistry is fast, the yield is high, the product is stable, there is no by-product, the synthesis of polysaccharide molecules composed of single kind of polysaccharide construction monomers and sequence controllable polysaccharide molecules composed of different kinds of polysaccharide construction monomers can be realized. Similarly, the azide-alkyne click chemistry is used for modifying fluorescent molecules at the end of the polysaccharide molecule, and the synthesis of sequence controllable polysaccharide molecules with fluorescent properties is realized.

Description

Technical Field

[0001] This invention relates to the synthesis of non-natural polysaccharide molecules, and more specifically to a method for artificially synthesizing polysaccharides with controllable sequences. Background Technology

[0002] Studies have shown that various cells, including cancer cells, have multiple sugar-specific receptors on their surfaces, such as lectins. For example, galactolectin-3 can bind to β-galactoside and is highly expressed in several cell types, including epithelial cells and immune cells. Glucose and mannose receptors are overexpressed in various tumor cells. Therefore, introducing carbohydrate molecules into drug targeting systems can effectively achieve specific treatment for tumor cells. Furthermore, due to the abnormal glucose metabolism mechanisms of tumor cells, many tumor cells have a large number of sugar-binding receptors on their surface, with expression levels far exceeding those of other types of receptor proteins. Therefore, using carbohydrate molecules as targeting groups can achieve efficient and active drug uptake by tumor cells, further improving tumor targeting capabilities. However, because the binding affinity between monosaccharide molecules and receptors such as lectins is relatively weak and somewhat reversible, directly using monosaccharides as targeting groups is not very effective. If multiple monosaccharide molecules are covalently combined to form a polysaccharide sequence, their binding affinity to receptors can be significantly improved. For example, in 2017, the research group of Chen Guosong and Jiang Ming at Fudan University reported using nanoparticles synthesized with glycosylated polymers to simulate the glycocalyx on the cell surface. They found that nanoparticles with a mixture of galactosylated and mannosylated polymers were more easily absorbed by macrophages. Further optimization of polysaccharide sequences using high-throughput screening methods can further improve the targeting ability, biocompatibility, and selectivity of target sugar molecules. It can be inferred that sugar molecules play important roles in many biological processes; however, current methods for artificially synthesizing sequence-controllable polysaccharides are extremely limited. This is mainly due to the lack of effective and efficient artificial synthesis methods for glycosidic bond structures similar to acetals. Therefore, developing an efficient and precise method for preparing sequence-controllable polysaccharide molecules is crucial for studying the role of polysaccharide molecules in biological processes.

[0003] In polysaccharide chemistry, the coupling reaction between sugars, i.e., glycosylation, has always been a challenge in the artificial synthesis of polysaccharides. Currently, commonly used methods mainly include monosaccharide coupling in solution using halogenated sugars, glycosyltrichloroacetilimes, and thioglycosides; oligosaccharide coupling assembly; and solid-phase polysaccharide synthesis. However, solution-phase synthesis currently requires relatively complex reaction, separation, and purification steps. Furthermore, the similarity in physical and chemical properties among different monosaccharides (natural monosaccharides are mostly optical isomers) poses a significant challenge to the solution-phase synthesis of polysaccharides with controllable sequences. Therefore, the coupling reaction used needs to be highly efficient, have high conversion rates, and be free of byproducts. Among these, copper-catalyzed azide-alkyne cycloaddition (CuAAC), widely used in the biopharmaceutical field, has been applied to the synthesis of peptides and nucleic acids due to its fast reaction rate, high yield, stable products, and lack of byproducts. Therefore, azide-alkyne click chemistry is used in combination with various synthetic techniques to replace the traditional polysaccharide synthesis strategy. The triazole ring structure is used to replace the glycosidic bond structure in natural polysaccharides, which not only achieves efficient polysaccharide synthesis, but also increases the stability of polysaccharides, thereby achieving the precise construction of polysaccharide molecular sequences. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the artificial synthesis of sequence-controllable polysaccharides. This invention, for the first time, utilizes copper-catalyzed azido-yne click chemistry to efficiently and precisely prepare various sequence-controllable polysaccharide molecules. Similarly, azido-yne click chemistry is used to modify the ends of polysaccharide molecules with fluorescent molecules, achieving the synthesis of sequence-controllable polysaccharide molecules with fluorescent properties.

[0005] The sequence-controllable polysaccharide building monomers include: glucose building monomer, galactose building monomer, and mannose building monomer.

[0006] The sequence-controllable polysaccharide monomer structure consists of three parts: a monosaccharide molecule, a terminal alkyne group, and an azide group; the monosaccharide molecule is selected from glucose, galactose, and / or mannose.

[0007] The copper catalyst is a monovalent copper complex formed by the reaction of copper sulfate and sodium ascorbate in a molar ratio of 1:1 to 4.

[0008] The fluorescent molecule is fluorescein isothiocyanate (FITC) containing an azide group.

[0009] The method for artificially synthesizing sequence-controllable polysaccharides involves using controllable polysaccharide building monomers in a copper catalytic system to generate sequence-controllable polysaccharide molecules composed of the same type of polysaccharide building monomers, sequence-controllable polysaccharide molecules composed of different types of polysaccharide building monomers, and sequence-controllable polysaccharide molecules modified with fluorescent molecules by controlling the types of polysaccharide building monomers added.

[0010] The sequence-controllable polysaccharide monomer structure described in this invention consists of three parts: a monosaccharide molecule (glucose, galactose, mannose), a terminal alkyne group, and an azide group, and has the following structural formula:

[0011]

[0012] The sequence-controllable polysaccharide building monomer of the present invention is prepared by the following steps:

[0013] (1) Dissolve glycidyl propargyl ether in dry tetrahydrofuran, slowly add n-butyllithium in n-hexane solution at -49℃ to -100℃ under inert gas protection, react at -49℃ to -100℃ for 0.1h to 1h, then slowly add trimethylsilyl chloride and react for 2h to 4h, then add saturated brine, extract with ethyl acetate, and further purify by column chromatography to obtain trimethylsilyl propargyl glycidyl ether;

[0014] (2) Dissolve trimethylsilylpropynyl glycidyl ether in dry N,N-dimethylformamide, add ammonium chloride and sodium azide in sequence and stir overnight, then add saturated brine, extract with ethyl acetate, and further purify by column chromatography to obtain 1-trimethylsilylpropynyloxy-3-azido-2-propanol.

[0015] (3) 1-Trimethylsilylpropoxy-3-azido-2-propanol, succinic anhydride, and N,N-dimethylaminopyridine were dissolved in dichloromethane, stirred overnight at room temperature, and the solvent was removed under vacuum. The succinic acid-1-trimethylsilylpropoxy-3-azido-2-propanol monoester was obtained by column chromatography.

[0016] (4) Dissolve the monosaccharide molecules in concentrated ammonia water, add aminoformamide powder in multiple portions while stirring, stir at room temperature for 2 to 4 days, and remove the solvent under vacuum to obtain α-amino-monosaccharide molecules.

[0017] (5) Succinic acid-1-trimethylsilylpropoxy-3-azido-2-propanol monoester was dissolved in oxalyl chloride and stirred overnight. The solvent was removed under vacuum and redissolved in dry N,N-dimethylformamide. Separately, α-amino-monosaccharide molecules were dissolved in dry N,N-dimethylformamide and dry triethylamine was added. The mixture was heated to 45℃~80℃ to dissolve. Then, the N,N-dimethylformamide solution of succinic acid-1-trimethylsilylpropoxy-3-azido-2-propanol monoester was slowly added to the α-amino-monosaccharide molecule solution. The mixture was slowly cooled to room temperature and stirred overnight. The solvent was removed under vacuum and the monomer was further purified by column chromatography to obtain the sequence-controllable polysaccharide building block.

[0018] The monosaccharide molecules include glucose, galactose, and / or mannose, preferably, the glucose is D-glucose.

[0019] The sequence-controllable polysaccharide building monomers include: glucose building monomers containing terminal alkyne groups and azido groups, galactose building monomers containing terminal alkyne groups and azido groups, and / or mannose building monomers containing terminal alkyne groups and azido groups.

[0020] The sequence-controllable polysaccharide molecule of the present invention, composed of monomers of a single type of sugar, has the following structural formula:

[0021]

[0022] The sequence-controllable polysaccharide composed of monomers of a single type of polysaccharide described in this invention is prepared by the following steps:

[0023] (1) To facilitate the detection of reaction progress and reaction products by liquid chromatography, benzene ring molecules were first modified on the polysaccharide building monomer at the beginning. The polysaccharide building monomer and the starting molecule containing benzene ring were dissolved in a water / methanol mixed solvent at a molar ratio of 1:1 to 3. Then, 0.05 to 1 equivalent of copper catalyst as polysaccharide building monomer was added. The reaction was stirred at room temperature for 0.5 to 2.5 h. Azid-alkyne click chemical coupling reaction occurred between molecules. The monosaccharide molecule containing benzene ring modification synthesized from the polysaccharide building monomer was obtained by preparative high performance liquid chromatography.

[0024] (2) The monosaccharide molecules obtained in step (1) above are dissolved in a water / methanol mixed solvent, and a THF solution of TBAF is added. This reaction system is reacted at a temperature not higher than 10°C for 0.2 h to 2 h, and then reacted at room temperature for 0.5 h to 3 h. After that, lithium nitrate is added to quench the remaining TBAF. The product is purified by high performance liquid chromatography to obtain monosaccharide molecules with terminal alkyne groups after the removal of silane protecting groups.

[0025] (3) The monosaccharide molecules with terminal alkyne groups obtained in step (2) above are mixed with polysaccharide building monomers of the same kind at a molar ratio of 1:1 to 3, dissolved in a water / methanol mixed solvent, and then 0.05 to 1 equivalent of copper catalyst as polysaccharide building monomer is added. The mixture is stirred at room temperature for 0.5 to 2.5 h. The terminal alkyne groups of the monosaccharide molecules continue to undergo azide-alkyne click chemical coupling reaction with the azide groups of the same kind of polysaccharide building monomers. The disaccharide molecules are obtained by purification by high performance liquid chromatography.

[0026] (4) The disaccharide molecule obtained in step (3) above is deprotected by the silane protecting group on the terminal alkyne under the reaction conditions in step (2) to obtain a reactive disaccharide molecule.

[0027] (5) The reactive disaccharide molecule in step (4) above is mixed with the same polysaccharide building monomer at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the disaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of the same polysaccharide building monomer. The trisaccharide molecule is obtained by purification by high performance liquid chromatography.

[0028] (6) The trisaccharide molecules obtained in step (5) above undergo chain growth through steps (2) and (3) above to obtain tetrasaccharide molecules composed of monomers of the same type of sugar.

[0029] (7) The tetrasaccharide molecules obtained in step (6) above are subjected to chain growth through steps (2) and (3) above, respectively, to obtain pentasaccharide molecules composed of monomers of the same type of sugar.

[0030] The sequence-controllable polysaccharide building monomers include: glucose building monomers, galactose building monomers and / or mannose building monomers.

[0031] The copper catalyst is a monovalent copper complex formed by the reaction of copper sulfate and sodium ascorbate in a molar ratio of 1:1 to 4.

[0032] Furthermore, to ensure the efficiency and yield of the azide-acetylene click chemical coupling reaction, the concentration of the polysaccharide building block monomer is controlled at 5–80 mmol / L, the amount of copper catalyst is 0.05–1 equivalent of the polysaccharide building block monomer, and the reaction solvent is a mixture of water and methanol with a volume ratio of 1:20–20:1; preferably, the concentration of the polysaccharide building block monomer is controlled at 30–40 mmol / L.

[0033] Furthermore, in order to ensure the efficiency and yield of the removal of silane protecting groups on the terminal alkynes, TBAF was added dropwise to the reaction system at a temperature not exceeding 10°C, and the reaction was stirred at a temperature not exceeding 10°C for 0.2 h to 2 h, and then stirred at room temperature for 0.5 h to 3 h, with the concentration of TBAF controlled at 0.25 mol / L.

[0034] The polysaccharide molecules with AABB and ABAB sequences composed of different types of polysaccharide building blocks described in this invention have the following structural formulas:

[0035]

[0036] The polysaccharide molecule of the present invention, composed of AABB sequences of different types of polysaccharide building blocks, is prepared by the following steps:

[0037] (1) To facilitate the detection of reaction progress and reaction products by liquid chromatography, benzene ring molecules were first modified on the polysaccharide building monomer at the beginning. The polysaccharide building monomer A obtained in the previous part and the starting molecule containing benzene ring were dissolved in a water / methanol mixed solvent at a molar ratio of 1:1 to 3. Then, 0.05 to 1 equivalent of copper catalyst as polysaccharide building monomer was added. The reaction was stirred at room temperature for 0.5 to 1.5 h, and an azide-alkyne click chemical coupling reaction occurred between molecules. The monosaccharide molecule containing benzene ring modification synthesized from polysaccharide building monomer A was obtained by preparative high performance liquid chromatography.

[0038] (2) The monosaccharide molecules obtained in step (1) above are dissolved in a water / methanol mixed solvent, and a THF solution of TBAF is added. This reaction system is reacted at a temperature not higher than 10°C for 0.2 h to 2 h, and then reacted at room temperature for 0.5 h to 3 h. After that, lithium nitrate is added to quench the remaining TBAF. The product is purified by high performance liquid chromatography to obtain a reactive monosaccharide molecule composed of polysaccharide building monomer A after the removal of the silane protecting group.

[0039] (3) The monosaccharide molecule with terminal alkyne group obtained in step (2) above is mixed with polysaccharide building monomer A of the same kind at a molar ratio of 1:1-3 and dissolved in a water / methanol mixed solvent. Then, 0.05-1 equivalent of copper catalyst as polysaccharide building monomer is added and the reaction is stirred at room temperature for 0.5-1.5 h. The terminal alkyne group of the monosaccharide molecule continues to undergo azide-alkyne click chemical coupling reaction with the azide group of the same kind of polysaccharide building monomer. The AA sequence disaccharide molecule is obtained by purification by high performance liquid chromatography.

[0040] (4) The AA-sequence disaccharide molecule obtained in step (3) above is de-protected by the silane protecting group on the terminal alkyne under the reaction conditions in step (2) to obtain a reactive AA-sequence disaccharide molecule.

[0041] (5) The reactive AA-sequence disaccharide molecule in step (4) above is mixed with polysaccharide building monomer B at a molar ratio of 1:1-3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the AA disaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of polysaccharide building monomer B. The AAB-sequence trisaccharide molecule is obtained by purification by high performance liquid chromatography.

[0042] (6) The AAB sequence trisaccharide molecule obtained in step (5) above is deprotected by the silane protecting group on the terminal alkyne under the reaction conditions in step (2) to obtain a reactive AAB sequence trisaccharide molecule.

[0043] (7) The reactive AAB sequence trisaccharide molecule in step (6) above is mixed with polysaccharide building monomer B at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the AAB trisaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of polysaccharide building monomer B. The tetrasaccharide molecule with the AABB sequence is obtained by purification by high performance liquid chromatography.

[0044] The polysaccharide molecule of the ABAB sequence composed of different types of polysaccharide building blocks described in this invention is prepared by the following steps:

[0045] (1) To facilitate the detection of reaction progress and reaction products by liquid chromatography, a starting molecule containing a benzene ring is first modified on the polysaccharide building monomer at the beginning. The polysaccharide building monomer A obtained in the previous part and the starting molecule containing a benzene ring are dissolved in a water / methanol mixed solvent at a molar ratio of 1:1 to 3. Then, 0.05 to 1 equivalent of copper catalyst as polysaccharide building monomer is added. The reaction is stirred at room temperature for 0.5 to 2 hours. Azid-alkyne click chemical coupling reaction occurs between molecules. The monosaccharide molecule containing benzene ring modification synthesized from polysaccharide building monomer A is obtained by preparative high performance liquid chromatography purification.

[0046] (2) The monosaccharide molecules obtained in step (1) above are dissolved in a water / methanol mixed solvent, and a THF solution of TBAF is added. This reaction system is reacted at a temperature not higher than 10°C for 0.2 h to 2 h, and then reacted at room temperature for 0.5 h to 3 h. After that, lithium nitrate is added to quench the remaining TBAF. The product is purified by high performance liquid chromatography to obtain a reactive monosaccharide molecule composed of polysaccharide building monomer A after the removal of the silane protecting group.

[0047] (3) The monosaccharide molecules with terminal alkyne groups obtained in step (2) above are mixed with polysaccharide building monomer B at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Then, 0.05 to 1 equivalent of copper catalyst as polysaccharide building monomer is added and the mixture is stirred at room temperature for 0.5 to 2 hours. The terminal alkyne groups of the monosaccharide molecules continue to undergo azide-alkyne click chemical coupling reaction with the azide groups of the same type of polysaccharide building monomer. The AB sequence disaccharide molecules are obtained by purification by high performance liquid chromatography.

[0048] (4) The AB sequence disaccharide molecule obtained in step (3) above is de-protected by the silane protecting group on the terminal alkyne under the reaction conditions in step (2) to obtain a reactive AB disaccharide molecule.

[0049] (5) The reactive AB sequence disaccharide molecule in step (4) above is mixed with polysaccharide building monomer A at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the AB sequence disaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of polysaccharide building monomer A. The ABA sequence trisaccharide molecule is obtained by purification by high performance liquid chromatography.

[0050] (6) The ABA sequence trisaccharide molecule obtained in step (5) above is deprotected by the silane protecting group on the terminal alkyne under the reaction conditions in step (2) to obtain a reactive ABA sequence trisaccharide molecule.

[0051] (7) The reactive ABA sequence trisaccharide molecule in step (6) above is mixed with polysaccharide building monomer B at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the ABA sequence trisaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of polysaccharide building monomer B. The tetrasaccharide molecule of the ABAB sequence is obtained by purification by high performance liquid chromatography.

[0052] The sequence-controllable polysaccharide building monomers include: glucose building monomer, galactose building monomer, and mannose building monomer.

[0053] The sequence-controllable polysaccharide building monomers A and B are two different polysaccharide building monomers.

[0054] The copper catalyst is a monovalent copper complex formed by the reaction of copper sulfate and sodium ascorbate in a molar ratio of 1:1 to 4.

[0055] Furthermore, to ensure the efficiency and yield of the azide-acetylene click chemical coupling reaction, the concentration of the polysaccharide building block monomer is controlled at 50–80 mmol / L, the amount of copper catalyst is 0.05–1 equivalent of the polysaccharide building block monomer, and the reaction solvent is a mixture of water and methanol with a volume ratio of 1:20–20:1; preferably, the concentration of the polysaccharide building block monomer is controlled at 30–40 mmol / L.

[0056] Furthermore, in order to ensure the efficiency and yield of the removal of silane protecting groups on the terminal alkynes, TBAF was added dropwise to the reaction system at a temperature not exceeding 10°C, and the reaction was stirred at a temperature not exceeding 10°C for 0.2 h to 2 h, and then stirred at room temperature for 0.5 h to 3 h, with the concentration of TBAF controlled at 0.1 to 2 mol / L.

[0057] The sequence-controllable polysaccharide molecule of the present invention utilizes an azide-alkyne click chemical coupling reaction to modify the end of the polysaccharide molecule with fluorescent molecules, thereby achieving the synthesis of a sequence-controllable polysaccharide molecule with fluorescent properties.

[0058] The fluorescent molecule described in this invention is a fluorescein isothiocyanate (FITC) containing an azide group, which has the following structural formula:

[0059]

[0060] The fluorescein molecule containing azide group modification described in this invention is prepared by the following steps:

[0061] 2-[2-(2-azidoethoxy)ethoxy]ethylamine was dissolved in dry tetrahydrofuran, and N,N-diisopropylethylamine and fluorescein isothiocyanate were added sequentially. After stirring overnight at room temperature, the solvent was removed under vacuum, and the azido-modified fluorescein was further purified by column chromatography.

[0062] The fluorescently modified sequence-controllable polysaccharide molecule of the present invention has the following structural formula:

[0063]

[0064] The fluorescently modified sequence-controllable polysaccharide molecule of the present invention is prepared by the following steps:

[0065] (1) The sequence-controllable polysaccharide molecule obtained above was dissolved in a water / methanol mixed solvent, and a THF solution of TBAF was added. This reaction system was reacted at a temperature not higher than 10℃ for 0.2h to 2h, and then reacted at room temperature for 0.5h to 3h. Then, lithium nitrate was added to quench the excess TBAF. The product was purified by high performance liquid chromatography, thereby obtaining a reactive sequence-controllable polysaccharide molecule with desilane protecting groups.

[0066] (2) The sequence-controllable polysaccharide molecule with terminal alkyne group obtained in step (1) above is mixed with fluorescein containing azido group at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Then, 0.5 to 4 equivalents of copper catalyst for polysaccharide building monomer are added and the reaction is stirred at room temperature for 0.5 to 2 hours. The terminal alkyne group of the polysaccharide molecule and the azido group of the fluorescent molecule undergo an azido-alkyne click chemical coupling reaction. The sequence-controllable polysaccharide molecule modified with fluorescent molecule is obtained by purification by high performance liquid chromatography.

[0067] The sequence-controllable polysaccharide molecules include: sequence-controllable polysaccharide molecules composed of a single type of polysaccharide building monomer and sequence-controllable polysaccharide molecules composed of different types of polysaccharide building monomers.

[0068] The copper catalyst is a monovalent copper complex formed by the reaction of copper sulfate and sodium ascorbate in a molar ratio of 1:1-4.

[0069] Furthermore, in order to ensure the efficiency and yield of the removal of silane protecting groups on the terminal alkynes, TBAF was added dropwise to the reaction system at a temperature not exceeding 10°C, and the reaction was stirred at a temperature not exceeding 10°C for 0.2 h to 2 h, and then stirred at room temperature for 0.5 h to 3 h, with the concentration of TBAF controlled at 0.1 to 2 mol / L.

[0070] Furthermore, to ensure the efficiency and yield of the azide-acetylene click chemical coupling reaction, the concentrations of polysaccharide molecules and fluorescent molecules are controlled at 5–80 mmol / L, the amount of copper catalyst is 1–4 equivalents of polysaccharide molecules, and the reaction solvent is a mixture of water and methanol with a volume ratio of 1:20–20:1; preferably, the concentrations of polysaccharide molecules and fluorescent molecules are controlled at 30–40 mmol / L.

[0071] Therefore, the present invention provides a synthetically produced sequence-controllable polysaccharide building monomer, characterized in that the sequence-controllable polysaccharide building monomer comprises a monosaccharide molecule, a terminal alkyne group, and an azide group, and its structural formula is shown below:

[0072]

[0073] Wherein, R is a monosaccharide molecule, and R is selected from any one or more of glucose, galactose, and mannose.

[0074] The present invention also provides a synthetically produced sequence-controllable polysaccharide, characterized in that the synthetically produced sequence-controllable polysaccharide is selected from any one or more of the following: a sequence-controllable polysaccharide molecule composed of monomers of the same type of polysaccharide, a sequence-controllable polysaccharide molecule composed of monomers of different types of polysaccharides, or a sequence-controllable polysaccharide molecule composed of monomers of the same or different types of polysaccharides modified by fluorescent molecules.

[0075] The polysaccharide building monomer comprises a monosaccharide molecule, a terminal alkyne group, and an azide group, and its structural formula is shown below:

[0076]

[0077] Wherein, R is a monosaccharide molecule, and R is selected from any one or more of glucose, galactose, and mannose;

[0078] The artificially synthesized sequence-controllable polysaccharide is generated in a copper-catalyzed system by controlling the type and amount of monomers added to the polysaccharide.

[0079] This invention also provides a synthetically produced sequence-controlled polysaccharide, selected from one or more of the following:

[0080] A. A sequence-controllable polysaccharide molecule composed of mannose, with an example structural formula as follows:

[0081]

[0082] B. A sequence-controlled polysaccharide molecule composed of galactose, with an example structural formula as follows:

[0083]

[0084] C. A sequence-controllable polysaccharide molecule composed of glucose, with an example structural formula as follows:

[0085]

[0086] The n is a natural number that is not zero, preferably a natural number from 1 to 10, and more preferably 1, 2, 3, 4, or 5.

[0087] This invention also provides another artificially synthesized sequence-controlled polysaccharide, selected from any one or more of the following:

[0088] (1) Contains [mannose (A) + mannose (A) + galactose (B) + galactose (B)] n1 Sequence-controllable polysaccharide molecules, specifically AABB sequence-controllable polysaccharide molecules, have the following structural examples:

[0089]

[0090] (2) Contains [mannose (A) + galactose (B)] n2 Sequence-controllable polysaccharide molecules, specifically polysaccharide molecules with controllable AB sequences, are illustrated in the following structural example:

[0091]

[0092] The n1 and n2 are natural numbers that are not zero; preferably, n1 or n2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; more preferably, n1 or n2 is 1, 2, 3, 4; even more preferably, n2 is 2. n And n2≥2, that is, a polysaccharide molecule with a controllable ABAB sequence.

[0093] This invention also provides another artificially synthesized sequence-controlled polysaccharide, selected from one or more of the following:

[0094]

[0095] The aforementioned artificially synthesized sequence-controllable polysaccharide is a sequence-controllable polysaccharide modified with a fluorescent molecule, wherein the fluorescent molecule contains an azide group; preferably, the fluorescent molecule has the following structural formula:

[0096]

[0097] This invention also provides another artificially synthesized sequence-controlled polysaccharide, selected from one or more of the following:

[0098] (1) A polysaccharide molecule with sequence controllable mannose modified by fluorescent molecules, the structural formula of which is shown below:

[0099]

[0100] (2) A polysaccharide molecule with sequence controllable galactose modified by fluorescent molecules, with the following structural example:

[0101]

[0102] (3) A sequence-controllable polysaccharide molecule containing glucose modified with fluorescent molecules, with the following structural example:

[0103]

[0104] (4) Polysaccharide molecules with controllable AABB sequences modified by fluorescent molecules, with the following structural examples:

[0105]

[0106] (5) Polysaccharide molecules with controllable AB sequences modified by fluorescent molecules, with the following structural example:

[0107]

[0108] The n is a natural number that is not zero, preferably a natural number from 1 to 10, and more preferably 1, 2, 3, 4, or 5;

[0109] The n1 and n2 are natural numbers that are not zero; preferably, n1 or n2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; more preferably, n1 or n2 is 1, 2, 3, 4; even more preferably, n2 is 2. n And n2≥2, that is, polysaccharide molecules with controllable ABAB sequences modified by fluorescent molecules.

[0110] This invention also provides another artificially synthesized sequence-controlled polysaccharide, selected from one or more of the following:

[0111]

[0112]

[0113] This invention also provides applications of the above-mentioned polysaccharide building monomers, sequence-controllable polysaccharides, and methods, wherein the applications are selected from any one or more of the following:

[0114] (1) Application in the preparation of pharmaceutical compositions;

[0115] (2) Application in the preparation of reagents for cell detection;

[0116] (3) Application in the preparation of reagents for the diagnosis or treatment of diseases;

[0117] (4) Application in in vitro drug screening;

[0118] (5) Application in in vitro testing not for the purpose of disease diagnosis.

[0119] The present invention also provides compositions containing the above-described sequence-controlled polysaccharides.

[0120] The present invention also provides a drug or detection reagent containing the above-mentioned sequence-controllable polysaccharide, wherein the drug or detection reagent further contains a pharmaceutically or medically acceptable carrier or excipient.

[0121] Furthermore, the present invention also provides applications of the said composition, the said drug or diagnostic reagent, wherein the application is selected from one or more of the following:

[0122] (1) Application in in vitro drug screening;

[0123] (2) Application in in vitro testing not for disease diagnosis

[0124] Furthermore, the aforementioned cells include normal cells or tumor cells.

[0125] Furthermore, the aforementioned diseases include tumors.

[0126] Furthermore, the tumor includes carcinoma, including basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, nasopharyngeal carcinoma, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma, head cancer, tongue cancer, oral cancer, etc.

[0127] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0128] 1. For the first time, precise and controllable synthesis of polysaccharide sequences has been achieved, which greatly improves the efficiency of artificial polysaccharide synthesis while increasing the stability of polysaccharides.

[0129] 2. For the first time, copper-catalyzed azide-alkyne click chemistry was used to synthesize polysaccharide molecules, replacing the natural glycosidic bond structure and providing a new design idea for the artificial synthesis of polysaccharides.

[0130] 3. For the first time, the construction of a sequence-controllable polysaccharide molecule modified with fluorescent molecules was achieved. Attached Figure Description

[0131] Figure 11H NMR spectrum of monosaccharide molecules synthesized from mannose monomers in deuterated DMSO;

[0132] Figure 2 Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF-MS) of monosaccharide molecules synthesized from mannose monomers;

[0133] Figure 3 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of disaccharide molecules synthesized from mannose monomers;

[0134] Figure 4 1H NMR spectrum of the trisaccharide molecule synthesized from mannose monomers in deuterated DMSO;

[0135] Figure 5 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of the trisaccharide molecules synthesized from mannose monomers;

[0136] Figure 6 1H NMR spectrum of the tetrasaccharide molecule synthesized from mannose monomers in deuterated DMSO;

[0137] Figure 7 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of the tetrasaccharide molecules synthesized from mannose monomers;

[0138] Figure 8 1H NMR spectrum of the pentasaccharide molecule synthesized from mannose monomers in deuterated DMSO;

[0139] Figure 9 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of the pentasaccharide molecules synthesized from mannose monomers;

[0140] Figure 10 High-resolution mass spectra (HRMS) of disaccharide molecules synthesized from galactose monomers;

[0141] Figure 11 High-resolution mass spectra (HPMS) of monosaccharide molecules synthesized from mannose monomers modified with fluorescein isothiocyanate;

[0142] Figure 12 High-resolution mass spectra (HPMS) of trisaccharide molecules synthesized from fluorescein isothiocyanate-modified mannose monomers;

[0143] Figure 13Matrix-assisted laser desorption / ion-time mass spectrometry (MALDI-TOF-MS) of a tetrasaccharide molecule with a fluorescein isothiocyanate-modified mannose-galactose-mannose-galactose (ABAB) sequence;

[0144] Figure 14 Matrix-assisted laser desorption / ion-time mass spectrometry (MALDI-TOF-MS) of a tetrasaccharide molecule with a fluorescein isothiocyanate-modified mannose-mannose-galactose-galactose (AABB) sequence. Detailed Implementation

[0145] The present invention will be further described in detail below through specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0146] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0147] Example 1: Synthesis of mannose building blocks

[0148] (1) Dissolve glycidyl propargyl ether in dry tetrahydrofuran, slowly add n-butyllithium in n-hexane solution at -78℃ under Ar protection, react at -78℃ for 0.5 h, then slowly add trimethylsilyl chloride and react for 3 h, then add saturated brine, extract with ethyl acetate, and further purify by column chromatography to obtain trimethylsilyl propargyl glycidyl ether;

[0149] (2) Dissolve trimethylsilylpropynyl glycidyl ether in dry N,N-dimethylformamide, add ammonium chloride and sodium azide in sequence and stir overnight, then add saturated brine, extract with ethyl acetate, and further purify by column chromatography to obtain 1-trimethylsilylpropynyloxy-3-azido-2-propanol.

[0150] (3) 1-Trimethylsilylpropoxy-3-azido-2-propanol, succinic anhydride, and N,N-dimethylaminopyridine were dissolved in dichloromethane, stirred overnight at room temperature, and the solvent was removed under vacuum. The succinic acid-1-trimethylsilylpropoxy-3-azido-2-propanol monoester was obtained by column chromatography.

[0151] (4) Dissolve monosaccharide molecules in concentrated ammonia water, add aminoformamide powder in multiple portions while stirring, stir at room temperature for three days, and remove the solvent under vacuum to obtain α-amino-D-glucose.

[0152] (5) Succinic acid-1-trimethylsilylpropoxy-3-azido-2-propanol monoester was dissolved in oxalyl chloride and stirred overnight. The solvent was removed under vacuum and redissolved in dry N,N-dimethylformamide. α-amino-D-glucose was dissolved in dry N,N-dimethylformamide and dry triethylamine was added. The mixture was heated to 60°C to dissolve. Subsequently, the N,N-dimethylformamide solution of succinic acid-1-trimethylsilylpropoxy-3-azido-2-propanol monoester was slowly added to the α-amino-D-glucose solution. The mixture was slowly cooled to room temperature and stirred overnight. The solvent was removed under vacuum and the mannose monomer was further purified by column chromatography.

[0153] ¹H NMR spectroscopy confirmed the successful synthesis of mannose building blocks (such as...). Figure 1-2 (as shown); using the same reaction conditions and reaction amounts, the reactant α-amino-D-glucose in step (5) above was replaced with D-mannose and D-galactose respectively. The 1H NMR test showed that the galactose building monomer and glucose building monomer were successfully synthesized.

[0154] Example 2: Synthesis of a sequence-controlled polysaccharide composed of monomers from a single type of mannose

[0155] (1) To facilitate the detection of reaction progress and reaction products by liquid chromatography, benzene ring molecules were first modified on the polysaccharide building monomer at the beginning. 120 mg of mannose building monomer and 55 mg of the starting molecule containing benzene ring were dissolved in 6.0 mL of water / methanol mixed solvent with a volume ratio of 1:1. Then, 6.1 mg of copper sulfate pentahydrate and 9.7 mg of sodium ascorbate were weighed and dissolved in 60 μL of water to obtain a monovalent copper complex, which was added to the above reaction system. The reaction was stirred at room temperature for 1 h, and an azide-alkyne click chemical coupling reaction occurred between molecules. The mannose monosaccharide molecule containing benzene ring was obtained by preparative high performance liquid chromatography.

[0156] (2) Weigh 20 mg of mannose monosaccharide molecules and dissolve them in 1.6 mL of a 1:1 water / methanol mixed solvent. Add 0.6 mL of TBAF in THF solution. After reacting at 0 °C for 0.5 h, react at room temperature for another 1.5 h. Then add 81 mg of lithium nitrate to quench the remaining TBAF. The product is purified by high performance liquid chromatography to obtain mannose monosaccharide molecules with desilane protecting groups that are reactive.

[0157] (3) Weigh 33 mg of reactive mannose monosaccharide molecules and 33 mg of mannose building monomers, dissolve them in 1.5 mL of a 1:1 water / methanol mixed solvent, then weigh 2.8 mg of copper sulfate pentahydrate and 4.5 mg of sodium ascorbate, dissolve them in 20 μL of water to obtain a monovalent copper complex, add it to the above reaction system, stir the reaction at room temperature for 1 h, the terminal alkyne group of mannose monosaccharide molecules continues to undergo an azide-alkyne click chemical coupling reaction with the azide group of mannose building monomers, and obtain mannose disaccharide molecules by high performance liquid chromatography.

[0158] (4) Weigh 20 mg of mannose disaccharide molecules and dissolve them in 1.0 mL of a 1:1 water / methanol mixed solvent. Add 90 μL of TBAF in THF solution. After reacting the reaction system at 0 °C for 0.5 h, react it at room temperature for 1.5 h. Then add 13 mg of lithium nitrate to quench the remaining TBAF. The product is purified by high performance liquid chromatography to obtain the mannose disaccharide molecules with reactive properties after the removal of the silane protecting group.

[0159] (5) Weigh 30 mg of reactive mannose disaccharide molecules and 18 mg of mannose building monomers, dissolve them in 0.8 mL of a 1:1 water / methanol mixed solvent, then weigh 1.5 mg of copper sulfate pentahydrate and 2.4 mg of sodium ascorbate, dissolve them in 20 μL of water to obtain a monovalent copper complex, add it to the above reaction system, stir the reaction at room temperature for 1 h, the terminal alkyne group of mannose monosaccharide molecules continues to undergo an azide-alkyne click chemical coupling reaction with the azide group of mannose building monomers, and obtain mannose trisaccharide molecules by high performance liquid chromatography.

[0160] (6) Weigh 50 mg of mannose trisaccharide molecules and dissolve them in 1.7 mL of a 1:1 water / methanol mixed solvent. Add 0.7 mL of TBAF in THF solution. After reacting at 0 °C for 0.5 h, react at room temperature for 1.5 h. Then add 92 mg of lithium nitrate to quench the remaining TBAF. The product is purified by high performance liquid chromatography to obtain the mannose trisaccharide molecules with desilane protecting groups.

[0161] (7) Weigh 20 mg of reactive mannose trisaccharide molecules and 8.2 mg of mannose building monomers, dissolve them in 0.4 mL of a 1:1 water / methanol mixed solvent, then weigh 0.7 mg of copper sulfate pentahydrate and 1.1 mg of sodium ascorbate, dissolve them in 20 μL of water to obtain a monovalent copper complex, add it to the above reaction system, stir the reaction at room temperature for 1 h, the terminal alkyne group of mannose monosaccharide molecules continues to undergo an azide-alkyne click chemical coupling reaction with the azide group of mannose building monomers, and obtain mannose tetrasaccharide molecules by high performance liquid chromatography.

[0162] 1H NMR and matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) tests confirmed the successful synthesis of monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides (e.g., mannose-based monomers). Figure 3-9 (As shown).

[0163] Similarly, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides composed of galactose or glucose monomers were synthesized (e.g., ...). Figure 10 (As shown).

[0164] Example 3: Synthesis of a sequence-controlled polysaccharide composed of monomers from a single type of mannose

[0165] 2-[2-(2-azidoethoxy)ethoxy]ethylamine was dissolved in dry tetrahydrofuran, and N,N-diisopropylethylamine and fluorescein isothiocyanate were added sequentially. The mixture was stirred overnight at room temperature, and the solvent was removed under vacuum. Further purification by column chromatography yielded the azide-modified fluorescein. Figure 11 As shown.

[0166] Example 4 Synthesis of a trisaccharide molecule composed of mannose monomers modified with fluorescein isothiocyanate

[0167] (1) 30 mg of mannose was dissolved in a 1.0 mL water / methanol mixed solvent, and 0.4 mL of TBAF in THF solution was added. The reaction system was reacted at 0 °C for 0.5 h, and then at room temperature for 1.5 h. Then, 55 mg of lithium nitrate was added to quench the excess TBAF. The product was purified by high performance liquid chromatography to obtain a reactive, sequence-controllable polysaccharide molecule with desilane protecting groups.

[0168] (2) 15 mg of a tetrasaccharide molecule with the mannose-galactose-mannose-galactose (ABAB) sequence and 9.7 mg of fluorescein isothiocyanate containing an azide group were dissolved in 0.25 mL of a water / methanol mixed solvent with a volume ratio of 1:3. Then, 8.13 mg of copper sulfate pentahydrate and 12.9 mg of sodium ascorbate were weighed and dissolved in 60 μL of water to obtain a monovalent copper complex, which was added to the above reaction system. The reaction was stirred at room temperature for 1 h. The terminal alkyne group of the polysaccharide molecule and the azide group of the fluorescent molecule underwent an azide-alkyne click chemical coupling reaction. The trisaccharide molecule composed of mannose building monomers modified with fluorescein isothiocyanate was obtained by purification by preparative high performance liquid chromatography.

[0169] High-resolution mass spectra show that the mannose-based monomer modified with fluorescein isothiocyanate was successfully synthesized, resulting in a trisaccharide molecule (such as...). Figure 12 (As shown).

[0170] Example 5: Synthesis of tetrasaccharide molecules with the fluorescein isothiocyanate-modified mannose-galactose-galactose (ABAB) sequence and tetrasaccharide molecules with the fluorescein isothiocyanate-modified mannose-galactose-galactose (AABB) sequence.

[0171] (1) 20 mg of a tetrasaccharide molecule with the mannose-galactose-mannose-galactose (ABAB) sequence was dissolved in 0.6 mL of a water / methanol mixed solvent, and 0.2 mL of a TBAF THF solution was added. This reaction system was reacted at 0 °C for 0.5 h, and then at room temperature for 1.5 h. After that, lithium nitrate was added to quench the excess TBAF. The product was purified by high performance liquid chromatography, thereby obtaining a reactive, sequence-controllable polysaccharide molecule with the silane protecting group removed.

[0172] (2) 15 mg of a tetrasaccharide molecule with the mannose-galactose-mannose-galactose (ABAB) sequence and 9.9 mg of fluorescein isothiocyanate containing an azide group were dissolved in 0.25 mL of a water / methanol mixed solvent with a volume ratio of 1:3. Then, 8.13 mg of copper sulfate pentahydrate and 12.9 mg of sodium ascorbate were weighed and dissolved in 60 μL of water to obtain a monovalent copper complex, which was added to the above reaction system. The reaction was stirred at room temperature for 1 h. The terminal alkyne group of the polysaccharide molecule and the azide group of the fluorescent molecule underwent an azide-alkyne click chemical coupling reaction. The tetrasaccharide molecule with the mannose-galactose-mannose-galactose (ABAB) sequence modified by fluorescein isothiocyanate was obtained by preparative high performance liquid chromatography purification.

[0173] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) confirmed the successful synthesis of a tetrasaccharide molecule with a fluorescein isothiocyanate-modified mannose-galactose-mannose-galactose (ABAB) sequence. Figure 13 As shown.

[0174] Using the same method described above, a tetrasaccharide molecule with a fluorescein isothiocyanate-modified mannose-mannose-galactose-galactose (AABB) sequence was prepared. Its matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALS-TOF-MS) is shown below. Figure 14 As shown.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a sequence-controllable polysaccharide composed of polysaccharide building blocks of the same type, characterized in that, Includes the following steps: (1) A benzene ring molecule is modified at the starting point of the polysaccharide building monomer. The polysaccharide building monomer and the starting molecule containing the benzene ring are dissolved in a water / methanol mixed solvent at a molar ratio of 1:1 to 3. Then, 0.05 to 1 equivalent of copper catalyst of the polysaccharide building monomer is added. The reaction is stirred at room temperature for 0.5 to 2.5 h. An azide-alkyne click chemical coupling reaction occurs between molecules. The monosaccharide molecule containing the benzene ring is purified. (2) Dissolve the monosaccharide molecules obtained in step (1) in a water / methanol mixed solvent and add a THF solution of TBAF; after reacting this reaction system at no higher than 10℃ for 0.2-1h, react it at room temperature for 0.5-3h and then quench the remaining TBAF to obtain a monosaccharide molecule with terminal alkyne group removed by removing silane protecting group. (3) The monosaccharide molecules with terminal alkyne groups obtained in step (2) are mixed with polysaccharide building monomers of the same kind at a molar ratio of 1:1 to 3, dissolved in a water / methanol mixed solvent, and then 0.05 to 1 equivalent of copper catalyst for polysaccharide building monomers are added. The mixture is stirred at room temperature for 0.5 to 3 hours. The terminal alkyne groups of the monosaccharide molecules continue to undergo azide-alkyne click chemical coupling reaction with the azide groups of the same kind of polysaccharide building monomers. The disaccharide molecules are purified to obtain disaccharide molecules. (4) The disaccharide molecule obtained in step (3) is de-protected by the silane protecting group on the terminal alkyne under the reaction conditions in step (2) to obtain a reactive disaccharide molecule. (5) The reactive disaccharide molecule in step (4) is mixed with the same polysaccharide building monomer at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the disaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of the same polysaccharide building monomer, and the trisaccharide molecule is purified. (6) The trisaccharide molecules obtained in step (5) are subjected to chain growth through the above steps (2) and (3) respectively to obtain tetrasaccharide molecules composed of monomers of the same type of sugar; (7) The tetrasaccharide molecules obtained in step (6) are subjected to chain growth through steps (2) and (3) above to obtain pentasaccharide molecules composed of monomers of the same type of sugar; (8) By analogy, n-sugar molecules composed of monomers of the same type of polysaccharide are obtained, that is, the same type of sequence-controllable polysaccharide; The polysaccharide building monomer in step (1) includes a monosaccharide molecule, a terminal alkyne group, and an azide group, and its structural formula is shown below: Wherein, R is a monosaccharide molecule, and R is selected from any one or more of glucose, galactose, and mannose; The polysaccharide building monomer is a glucose building monomer, a galactose building monomer, or a mannose building monomer, and n is 1, 2, 3, 4, or 5.

2. A method for preparing a sequence-controllable polysaccharide composed of different types of polysaccharide building blocks, characterized in that, Includes the following steps: (1) A benzene ring molecule was modified at the beginning of polysaccharide building monomer A. Polysaccharide building monomer A and the starting molecule containing benzene ring were dissolved in a water / methanol mixed solvent at a molar ratio of 1:1 to 3. Then, 0.05 to 1 equivalent of copper catalyst, which is polysaccharide building monomer, was added. The reaction was stirred at room temperature for 0.5 to 2.5 h. Azid-alkyne click chemical coupling reaction occurred between molecules. The monosaccharide molecule containing benzene ring modification was obtained by purification from polysaccharide building monomer A. (2) Dissolve the monosaccharide molecules obtained in step (1) in a water / methanol mixed solvent, add a THF solution of TBAF, and react the reaction system at a temperature not higher than 10°C for 0.2 to 1 h, then react at room temperature for 0.5 to 3 h to quench the remaining TBAF, purify the product, and thus obtain a reactive monosaccharide molecule composed of polysaccharide building monomer A after the removal of silane protecting groups; (3) The monosaccharide molecule with terminal alkyne group obtained in step (2) is mixed with polysaccharide building monomer A of the same type or polysaccharide building monomer B of different types at a molar ratio of 1:1 to 3, dissolved in water / methanol mixed solvent, and then 0.05 to 1 equivalent of copper catalyst for polysaccharide building monomer is added. The mixture is stirred at room temperature for 0.5 to 2.5 h. The terminal alkyne group of the monosaccharide molecule continues to undergo azide-alkyne click chemical coupling reaction with the azide group of the same type of polysaccharide building monomer, and the AA or AB sequence disaccharide molecule is purified. (4) The AA or AB sequence disaccharide molecule obtained in step (3) is desilane protecting group removed from the terminal alkyne under the reaction conditions in step (2) to obtain a reactive AA or AB sequence disaccharide molecule. (5) The reactive AA sequence disaccharide molecule in step (4) is mixed with polysaccharide building monomer A or B at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne group of the AA or AB disaccharide molecule continues to undergo an azide-alkyne click chemical reaction with the azide group of polysaccharide building monomer A or B, and the AAB or ABA sequence trisaccharide molecule is purified to obtain the AA or ABA sequence trisaccharide molecule. (6) The AAB or ABA sequence trisaccharide molecules obtained in step (5) above are de-protected by the silane protecting group on the terminal alkyne using the reaction conditions in step (2) to obtain a reactive AAB or ABA sequence trisaccharide molecule. (7) The reactive AAB or ABA sequence trisaccharide molecules in step (6) above are mixed with polysaccharide building monomer B at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Using the reaction conditions and catalyst dosage in step (3), the terminal alkyne groups of the AAB or ABA trisaccharide molecules continue to undergo an azide-alkyne click chemical reaction with the azide groups of polysaccharide building monomer B, and the tetrasaccharide molecules with the AABB or ABAB sequence are purified to obtain the tetrasaccharide molecules. (8) By analogy, sequence-controllable polysaccharides composed of monomers of different types of polysaccharides are obtained, namely [AB]. n1 Sequence-controlled polysaccharides or [AABB] n2 Sequence-controlled polysaccharides; The polysaccharide building monomer A or B is a different polysaccharide building monomer, and its structure is as described in claim 1.

3. A method for preparing a sequence-controllable polysaccharide molecule modified with fluorescent molecules, characterized in that, Includes the following steps: (1) The sequence-controllable polysaccharide prepared by the method of claim 1 is dissolved in a water / methanol mixed solvent, and a THF solution of TBAF is added; after the reaction system is reacted at no higher than 10°C for 0.2 to 2 hours, the excess TBAF is quenched after reacting at room temperature for 0.5 to 3 hours, and the product is purified to obtain a reactive sequence-controllable polysaccharide molecule with the silane protecting group removed; (2) The sequence-controllable polysaccharide molecule with terminal alkyne group obtained in step (1) is mixed with the fluorescent molecule containing azido group at a molar ratio of 1:1 to 3 and dissolved in a water / methanol mixed solvent. Then, 0.5 to 4 equivalents of copper catalyst for polysaccharide building monomers are added and the reaction is stirred at room temperature for 0.5 to 2.5 h. The terminal alkyne group of the polysaccharide molecule and the azido group of the fluorescent molecule undergo an azido-alkyne click chemical coupling reaction. The sequence-controllable polysaccharide molecule modified with fluorescent molecule is purified to obtain the polysaccharide molecule.

4. The method according to claim 3, wherein the preparation method of the fluorescent molecule containing an azide group comprises the following: Steps: Dissolve 2-[2-(2-azidoethoxy)ethoxy]ethylamine in dry tetrahydrofuran, then add N,N-diisopropylethylamine and fluorescein isothiocyanate in sequence. Stir overnight at room temperature, then remove the solvent under vacuum and purify to obtain a fluorescent molecule containing an azide group.

5. The method according to claim 3, wherein the fluorescent molecule is a fluorescein isothiocyanate containing an azide group, and its structural formula is shown below:

6. The preparation method according to claim 1, wherein the copper catalyst contains a monovalent copper complex formed by the reaction of copper sulfate and sodium ascorbate in a molar ratio of 1:1 to 4.

7. The preparation method according to claim 1, wherein the copper catalyst contains a monovalent copper complex formed by the reaction of copper sulfate and sodium ascorbate in a 1:2 molar ratio.

8. According to the preparation method of claim 1, the concentration of the polysaccharide building monomer in the azide-acetylene click chemical coupling reaction is controlled at 5-80 mmol / L, the amount of copper catalyst is 0.05-1 equivalent of the polysaccharide building monomer, and the reaction solvent is a mixture of water and methanol, with a volume ratio of water to methanol of 1:20-20:

1.

9. In the preparation method according to claim 1, the concentration of the polysaccharide building monomer in the azide-acetylene click chemical coupling reaction is controlled at 25-50 mmol / L.

10. The preparation method according to claim 1, wherein the amount of copper catalyst used is 0.1 to 0.2 equivalents of the polysaccharide building block.

11. The preparation method according to claim 1, wherein the volume ratio of water to methanol in the reaction solvent is 1:10 to 10:

1.

12. According to the preparation method of claim 1, in the reaction of removing the silane protecting group from the terminal alkyne, TBAF is added dropwise to the reaction system at 0°C and stirred at 0°C for 0.5 h, and then stirred at room temperature for 1.5 h, wherein the concentration of TBAF is controlled at 0.1-1 mol / L.

13. The preparation method according to claim 1, wherein the concentration of TBAF is controlled at 0.2–0.5 mol / L.

14. The preparation method according to claim 1, wherein the concentration of TBAF is controlled at 0.25 mol / L.

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