Temperature-sensitive materials, sugar conjugates, methods for synthesizing the same, and their use in sugar solid-phase synthesis.

Abstract

This invention discloses a thermosensitive material, a sugar conjugate, a method for synthesizing the same, and its use in solid-phase sugar synthesis. The thermosensitive material is an alkynylation-modified 2-ethyl-2-oxazoline polymer, and the azide sugar has a Sugar-N-C3N3 structure. By conjugating the thermosensitive material with the azide sugar, a thermosensitive sugar conjugate is formed, enabling its use in solid-phase sugar synthesis. In practical applications, the present invention allows for the precipitation of the sugar conjugate in the aqueous phase by increasing the temperature, the dissolution of the sugar conjugate by decreasing the temperature, and the separation of the sugar from the material using trifluoroacetic acid (TFA). Based on the above properties, the thermosensitive material and its sugar conjugate of the present invention can efficiently and simply solve the drawbacks of difficulty in separating and purifying sugar intermediates during the polymerization process and the inability to control the steps. This enables controllable synthesis and purification of sugars, which has great significance for the study of sugar science.

Description

Technical Field

[0001] The present invention belongs to the fields of sugar materials and sugar catalysts, and specifically relates to thermosensitive materials, sugar conjugates, and their synthesis methods, as well as their use in solid-phase sugar synthesis.

Background Art

[0002] Due to the diversity and complexity of their structures, saccharide compounds determine the diversity of functions involved in biological processes. Therefore, the stable availability of oligosaccharides and sugar conjugates with clear structures is a major constraint for related research. Natural extract sugar products have non-uniform structures and many impurities due to differences in process methods and raw material types, which limits research on the biological functions of sugars. Therefore, the synthesis, purification, and development of general methods for saccharide compounds have become important bottlenecks restricting the development of sugar science. The chemical synthesis method of saccharide compounds is very flexible in that it assembles sugar chains by forming glycosidic bonds through chemical reactions. However, in many cases, to obtain specific groups and arrangements during the reaction, it is necessary to go through multiple steps of group protection and deprotection. The steps are cumbersome and the yield is low. In addition, in chemical methods, a high level of proficiency in organic chemistry is required for various glycosylation methods, manipulation of protecting groups, and design of synthetic routes, and it is difficult to acquire the experiments.

[0003] The enzymatic synthesis method of sugar compounds involves constructing glycosidic bonds through enzymatic glycosylation reactions, mimicking natural biosynthetic pathways in vivo, and assembling oligosaccharides in vitro using biological enzymes. Compared to conventional chemical glycosylation methods, enzymatic glycosylation has unique advantages, including high regioselectivity and stereoselectivity, avoidance of cumbersome steps of group protection and deprotection, mild reaction conditions, simple steps, high technical efficiency, and environmental considerations. Therefore, it aligns with China's green bio-preparation strategy and has become an important approach for obtaining complex oligosaccharides and sugar conjugates. Although the enzymatic synthesis method of sugar compounds has many advantages as described above, it suffers from drawbacks such as low separation efficiency and difficulty of product separation, which severely limits the efficiency of sugar intermediate synthesis. [Overview of the project] [Problems that the invention aims to solve]

[0004] Objective of the Invention: To address the challenges in the separation and purification of sugar intermediates, the present invention provides a thermosensitive material and a sugar conjugate based on the thermosensitive material, and enables the effective use of the thermosensitive sugar conjugate in solid-phase sugar synthesis. The present invention synthesizes a thermosensitive material having an alkynyl group, whose precipitation temperature is controlled by the degree of polymerization, and also synthesizes a sugar acceptor having an azide group compatible with the thermosensitive material. A click reaction occurs between the alkynyl group of the thermosensitive material and the azide group of the azide sugar to realize a conjugate between sugar and the thermosensitive material (sugar conjugate). By utilizing the temperature precipitation property of the thermosensitive material, it is possible to precipitate the sugar conjugate under high temperature conditions and dissolve the sugar conjugate at low temperatures. The present invention discloses a thermosensitive material, a method for synthesizing its sugar conjugate, and its use in solid-phase sugar synthesis. Compared to conventional sugar separation methods, this method is faster and simpler for specific applications, allows for controllable solid-phase sugar synthesis, and offers promising future potential for synthesizing homogeneous sugars. It lays a good foundation for studying the structure-activity relationships of sugar compounds and is of great importance to the development of sugar science. This method effectively solves the limitations of conventional sugar synthesis, such as difficulty in separation and heterogeneity of synthesized molecular weight, simplifies the purification step, and improves synthesis efficiency. [Means for solving the problem]

[0005] Technical solution: To achieve the above objective, the temperature-sensitive material described in the present invention is an alkynylation-modified 2-ethyl-2-oxazoline polymer, the structural formula of which is shown below. [ka] (Here, n represents the degree of polymerization from 100 to 2000.)

[0006] The method for preparing a temperature-sensitive material according to the present invention is: The process includes the steps of adding 2-ethyl-2-oxazoline as a monomer and an initiator to a solvent, reacting the reaction system continuously for 5 to 12 hours at 100 to 200°C under an inert atmosphere, quenching the reaction after completion, dry-evaporating the solvent, recrystallizing, drying by suction filtration, and obtaining a temperature-sensitive material in the form of a white powdery solid of alkynylated poly2-ethyl-2-oxazoline.

[0007] Preferably, the reaction is carried out using anhydrous acetonitrile as the solvent, under the protection of an inert gas such as nitrogen or argon, and the degree of polymerization is controlled by the ratio of the monomer 2-ethyl-2-oxazoline to the initiator p-toluenesulfonic acid propargyl. The reaction equation is preferably as follows. [ka]

[0008] A thermosensitive sugar conjugate based on the thermosensitive material described in the present invention, wherein the thermosensitive sugar conjugate is formed by conjugating the thermosensitive material with an azido sugar, the thermosensitive material is an alkynylation-modified 2-ethyl-2-oxazoline polymer, and the structure of the azido sugar is a Sugar-N-C3N3 structure.

[0009] Here, the thermosensitive sugar conjugate achieves the conjugation of a sugar and a thermosensitive material by bonding the alkynyl group of the thermosensitive material with the azide group of the azide sugar.

[0010] Here, the raw material for the azido sugar is a monosaccharide or oligosaccharide, and includes, but is not limited to, galactose, glucose, acetylglucosamine, acetylglucosamine, or lactose.

[0011] Here, the azide sugar is either an azide sugar based on galactose (Gal) monosaccharide or an azide sugar based on lactose (Lac) disaccharide, and their structures are shown below. [ka]

[0012] The method for preparing a thermosensitive sugar conjugate described in the present invention is: The process includes adding a thermosensitive material, azido sugar, ascorbic acid, hydrazine or tris(2-carbonylethyl)phosphine hydrochloride, and a copper starting material to a reaction solvent, stirring and reacting at room temperature, extracting and concentrating the mixture after the reaction, recrystallizing it, filtering it, and then drying the solid to obtain a thermosensitive sugar conjugate.

[0013] Preferably, the molar ratio of alkynylated poly2-ethyl-2-oxazoline to azido sugar is 1:1.5, the amounts of ascorbic acid and copper sulfate pentahydrate used are 0.5 and 0.3 times the number of moles of alkynylated poly2-ethyl-2-oxazoline, respectively, the reaction solvent is a 1 / 1 (v / v) mixed solution of water and ethanol or tert-butanol, and the reaction equation is shown below. [ka]

[0014] Here, the preparation of the azido sugar is as follows: (a) The N-methylhydroxylamine hydrochloride of compound 1 is reacted with di-tert-butyl dicarbonate (Boc2) to replace the hydrogen atom on the nitrogen of the N-methylhydroxylamine hydrochloride with a tert-butyloxycarbonyl group (Boc) to obtain compound 2. (b) Compound 2 is reacted with 1-chloro-3-iodopropane to replace the hydrogen on the hydroxyl group of compound 2 with 1-chloro-3-iodopropane, thereby extending the chain and obtaining compound 3. (c) Compound 3 is reacted with sodium azide to replace the chlorine on the propyl group of compound 3 with an azide group to obtain compound 4. (d) The tert-butyloxycarbonyl (Boc) protecting group of compound 4 is removed by the action of trifluoroacetic acid (TFA), and the hydrogen atom on the nitrogen of compound 4 is released to obtain compound 5. (e) Reacting compound 5 with a sugar, where the sugar used is a monosaccharide or oligosaccharide as a glycosyl donor, and reacting compound 5 as a raw material to obtain an azide sugar. The reaction formula is shown below.

Chemical formula

[0015] Use in the solid-phase synthesis of the thermosensitive sugar conjugate described in the present invention.

[0016] Here, the process of the above use is as follows: (1) Mix a glycosyl donor and a sugar conjugate. (2) Add a carbohydrate enzyme and perform a shaking reaction. (3) Heat-treat the reaction solution shaken in step (2), wash the precipitated product with hot water to obtain a disaccharide conjugate or a polysaccharide conjugate, and repeat steps (1), (2), and (3) to increase the sugar units. (4) Add the disaccharide conjugate or polysaccharide conjugate obtained in step (3) to trifluoroacetic acid, heat it, collect the supernatant to obtain the corresponding disaccharide or polysaccharide.

[0017] Here, the enzymes used in solid-phase synthesis include, but are not limited to, NmLgtA (β1-3 N-acetylglucosaminyltransferase from Neisseria meningitides), Hpα1,2FT (α1,2 fucosyltransferase from Helicobacter pylori), Pd2,6ST (α2-6 sialyltransferase from P. damselaedamsela), NmLgtB (β-4 galactosyltransferase from N. meningitides), PmST1 (α2-3 sialyltransferase from P. multocida), PmHS2 (P. multocida Heparosan Synthase 2), KfoC (E. coli K4 chondroitin polymerase), and PmHAS (Pasteurella multocida Hyaluronan Synthase).

[0018] In this invention, first, a thermosensitive material having a specific degree of polymerization is synthesized, then an azide sugar having an azide group is synthesized, and this azide sugar and the thermosensitive material are mixed and bonded by a click reaction to synthesize a sugar conjugate. In this invention, a conjugate of sugar and thermosensitive material is realized by bonding the thermosensitive material to the azide group of the azide sugar via the alkynyl group of the material (hereinafter collectively referred to as sugar conjugate). Next, solid-phase synthesis of the sugar is performed using the corresponding carbohydrate enzyme. At the practical stage, the sugar conjugate can be precipitated in the aqueous phase by raising the temperature, and the sugar conjugate can be dissolved by lowering the temperature (the critical temperature is controlled by the degree of polymerization of the material). Furthermore, by separating the sugar from the material using trifluoroacetic acid (TFA), a sugar compound with a clearly defined structure can be obtained (Figure 1). Based on the above characteristics, the present invention's method for synthesizing sugar conjugates efficiently and simply solves the drawbacks of difficulty in separating and purifying sugar intermediates during the polymerization process, as well as the inability to control the steps. This enables controllable synthesis and purification of sugars, which has great significance for research in sugar science.

[0019] Specifically, the preparation of the present invention preferably includes the following three parts.

[0020] Part 1: Preparation of temperature-sensitive materials having terminal alkynyl groups Using propargyl p-toluenesulfonic acid as the initiator and 2-ethyl-2-oxazoline as the monomer, an inert gas such as nitrogen or argon is introduced into the reaction flask, anhydrous acetonitrile is added, then 2-ethyl-2-oxazoline is added to a concentration of 1-5 M, and then propargyl p-toluenesulfonic acid is added dropwise. After the dropwise addition is complete, the reaction system is carried out continuously at 100-200°C for 5-12 hours. After the reaction is complete, the reaction is quenched by adding 1:1 water or saturated sodium carbonate, and quenching should be carried out at 50-80°C for 6-12 hours. After quenching is complete, the solvent is dry-evaporated, the product is redissolved in DCM, anhydrous ethyl ether is added and recrystallized, and the product is dried by suction filtration to obtain a white powdery solid of alkynyl-modified poly2-ethyl-2-oxazoline.

[0021] Part 2: Preparation of Azido Sugars (1) The N-methylhydroxylamine hydrochloride of compound 1 is reacted with di-tert-butyl dicarbonate (Boc2) to replace the hydrogen atom on the nitrogen of the N-methylhydroxylamine hydrochloride with a tert-butyloxycarbonyl group (Boc) to obtain compound 2. (2) Compound 2 is reacted with 1-chloro-3-iodopropane to replace the hydrogen on the hydroxyl group of compound 2 with 1-chloro-3-iodopropane, thereby extending the chain and obtaining compound 3. (3) Compound 3 is reacted with sodium azide to replace the chlorine on the propyl group of compound 3 with an azide group to obtain compound 4. (4) The tert-butyloxycarbonyl (Boc) protecting group of compound 4 is removed by the action of trifluoroacetic acid (TFA), and the hydrogen atom on the nitrogen of compound 4 is released to obtain compound 5. (5) Compound 5 is reacted with a sugar, and in the sugar used, a monosaccharide or oligosaccharide is used as the glycosyl donor and compound 5 is used as the raw material to bond the sugar and the azide side chain.

[0022] Here, in step (1), the solvent for compound 1 is a mixed liquid of tetrahydrofuran (THF) and water in a 1:1 volume ratio. The K2CO3 solid is added to the mixed liquid of compound 1, and the amount of di-tert-butyl dicarbonate used is 0.5 to 1 times the number of moles of compound 1. The reaction is carried out at 0°C for the addition, and after the addition is complete, the temperature can return to room temperature.

[0023] Here, the reaction solvent used in step (2) is anhydrous tetrahydrofuran (THF), the amount of 1-chloro-3-iodopropane used is 1 to 2 times the number of moles of compound 2, and the amount of sodium hydride used is 1 to 2 times the number of moles of compound 2. In the reaction, first, sodium hydride is reacted with compound 1 at 0°C for 30 minutes or more, and then 1-chloro-3-iodopropane is added to bring it back to room temperature. The inert protective gas is nitrogen gas, helium gas, argon gas, etc.

[0024] Here, the reaction solvent used in step (3) is N,N-dimethylformamide (DMF), the amount of sodium azide used is 1 to 2 times the number of moles of compound 3, and the reaction is carried out at 90°C to 100°C.

[0025] In step (4), an acid is used for hydrolysis deprotection of compound 4. The acids used include, but are not limited to, trifluoroacetic acid, p-toluenesulfonic acid, and hydrochloric acid. The solvent used is dichloromethane (DCM). The amount of acidic reagent used is 0.5 to 5 times the amount of compound 4.

[0026] Here, the sugars used in step (5) include, but are not limited to, monosaccharides or oligosaccharides such as galactose, glucose, acetylglucosamine, lactose, the reaction solution is NaOAc buffer solution (0.1-1 M, pH 4.5-5.3), the amount of compound 5 used is 1-5 times the number of moles of sugar, and the reaction is carried out at 15°C-35°C.

[0027] Part 3: Preparation of thermosensitive sugar conjugates The molar ratio of alkynylated poly2-ethyl-2-oxazoline to azido sugar is 1:2, and the amounts of ascorbic acid and copper sulfate pentahydrate used are 0.5 and 0.3 times the number of moles of alkynylated poly2-ethyl-2-oxazoline, respectively. The reaction solvent is a 1 / 1 (v / v) mixed solution of water and ethanol. After stirring overnight at room temperature, the aqueous solution is added, extracted with chloroform, concentrated, then recrystallized with cold ethyl ether, filtered, and the resulting white powdery solid is dried in a vacuum oven to obtain immobilized lactose.

[0028] By employing the above process, a thermosensitive material and an azide sugar are conjugated to prepare a thermosensitive sugar conjugate. This imparts thermosensitivity to the sugar. By controlling the ambient temperature, the dissolution and precipitation of the sugar conjugate can be controlled, enabling easy separation of the sugar conjugate. Furthermore, by using the sugar conjugate in enzymatic reactions, the synthesis of sugar compounds can be catalyzed efficiently and continuously. After the reaction is complete, the sugar is released from the sugar conjugate by bond cleavage with trifluoroacetic acid, enabling the precise synthesis of sugar compounds.

[0029] Design Principle: The diversity and complexity of structures in sugar compounds determine the diversity of functions involved in biological processes. Therefore, studying the structure-activity relationships of structurally defined sugar compounds is of great importance. Enzymatic synthesis of sugar compounds has advantages such as stereospecificity, high selectivity, and ease of operation, and avoids the troublesome protection and deprotection operations of the groups, making it a very important method in the synthesis of sugar compounds. However, enzymatic synthesis also has disadvantages, such as the inability to control the degree of polymerization of sugar chains and the difficulty of separation and purification. Therefore, introducing sugar substrates into thermosensitive materials enables the easy purification of sugar compounds. In addition, the use of trifluoroacetic acid can liberate sugars from sugar conjugates, which is useful for the synthesis of structurally defined sugar compounds and the study of the corresponding structure-activity relationships.

[0030] In this invention, an alkynylation-modified poly2-ethyl-2-oxazoline thermosensitive material was synthesized. By controlling the degree of polymerization of the material, the dissolution temperature was successfully controlled. Furthermore, a sugar azidation modification strategy was developed, and by synthesizing a series of azido sugars having azide groups, the variety of sugar substrates was increased. In this invention, the thermosensitive material and azido sugars are cleverly linked by a Glick reaction. This linkage method has high selectivity, mild reaction conditions, and good linkage stability, and as a result, the synthesized sugar conjugate has high stability and good thermosensitivity.

[0031] The thermosensitive material prepared according to the present invention controls the dissolution temperature of the material by controlling the degree of polymerization of the material (the higher the degree of polymerization, the lower the precipitation temperature), thereby influencing the dissolution and precipitation of the material and enabling separable use. Furthermore, the material contains an alkynyl group. The prepared azido sugar is obtained by azidating natural sugars (monosaccharides, disaccharides, trisaccharides, etc.) by organic chemical means to confer a specific azide group to the sugar. Furthermore, the structure of the azido sugar can be returned to the original natural sugar by cleaving the bond at the NC bond site linking the sugar and the azide side chain with trifluoroacetic acid (TFA). Prepared sugar conjugate: Since the alkynyl group of the thermosensitive material and the azide group of the azido sugar are linked by a Glick reaction to form a sugar conjugate, this sugar conjugate possesses both the temperature sensitivity of the thermosensitive material (dissolution and precipitation can be controlled by temperature) and the cleavage properties of the azido sugar structure.

[0032] Regarding the thermosensitive material prepared according to the present invention, the sugar conjugate obtained by linking the thermosensitive material with an azide sugar enables the use of the thermosensitive material in solid-phase sugar synthesis. Specifically, the sugar conjugate synthesized using the novel thermosensitive material prepared according to the present invention can be recognized by enzymes and subjected to enzymatic reactions. In conventional enzymatic reactions, the addition of sugar units is required to sequentially react monosaccharides to disaccharides, disaccharides to trisaccharides, and then polysaccharides. After each step of the sugar reaction, the product needs to be separated and purified. Conventional steps require a series of operations such as column chromatography (silica gel column chromatography, hydrogel column chromatography) and freeze-drying, which are complex and time-consuming. In the present invention, sugar participates in the enzymatic reaction in the form of a sugar conjugate. After the reaction is complete, rapid separation is possible by directly precipitating the product by temperature control (unlike conventional silica gel column chromatography and gel column chromatography, which are time-consuming and labor-intensive). This enables controllable sugar synthesis (solid-phase sugar synthesis). Furthermore, the cleavage properties of the azide sugar structure make it possible to separate sugar conjugates into sugar and material forms. Here, since the sugar is a natural sugar (in conventional solid-phase sugar synthesis, additional groups are usually required, resulting in the production of unnatural sugars), it facilitates structure-activity relationship studies by scientists. By applying the thermosensitive material prepared by this invention to sugar synthesis, simple sugar separation and controllable synthesis become possible, which is of great significance to the study of sugar structure-activity relationships. [Effects of the Invention]

[0033] Beneficial effects: Compared to the prior art, the present invention has the following advantages.

[0034] 1. The present invention achieves temperature sensitivity of a sugar conjugate by synthesizing a temperature-sensitive material and conjugating it with an azido sugar. Temperature control enables high-temperature precipitation and low-temperature dissolution of the sugar conjugate, which is advantageous for the purification and separation of products during enzymatic reactions and also contributes to accelerating the enzymatic reaction process.

[0035] 2. Based on the temperature precipitation characteristics of sugar conjugates, applying these sugar conjugates to solid-phase sugar synthesis enables the controlled synthesis of sugars. This has great significance for the synthesis of sugar compounds with clear structures and uniform molecular weights, and will greatly contribute to research in sugar science.

[0036] 3. Based on the special properties of the linking groups of sugar conjugates, it is possible to cleave sugar conjugates using trifluoroacetic acid to obtain natural sugar compounds. This separation method is milder and more efficient. [Brief explanation of the drawing]

[0037] [Figure 1] This is a photograph of high-temperature precipitation and low-temperature dissolution of a temperature-sensitive material. [Figure 2] This is the NMR hydrogen spectrum of an alkynylation-modified poly2-ethyl-2-oxazoline thermosensitive material. [Figure 3] This figure shows the relationship between the ratio of initiator to monomer (M / I) and the critical temperature (LCST). [Figure 4] This figure shows the relationship between the concentration of a temperature-sensitive material and its critical temperature (LCST). [Figure 5] This is the NMR hydrogen spectrum of the azide group side chain. [Figure 6] This is an ESI image of GlcA-N-C3N3. [Figure 7] This is an ESI image of GalNAc-N-C3N3. [Figure 8] This is an ESI image of Gal-N-C3N3. [Figure 9] This is an ESI image of Lac-N-C3N3. [Figure 10] This is an ESI image of chondroitin disaccharide-N-C3N3. [Figure 11] This is the HR-MS spectrum of Lacto-N-triose. [Figure 12] This is the HR-MS spectrum of (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc. [Figure 13] This is the HR-MS spectrum of GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc. [Figure 14] This is the HR-MS spectrum of Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc. [Figure 15] This is the HR-MS spectrum of (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc. [Figure 16] This is the HR-MS spectrum of Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc. [Figure 17] This is the HR-MS spectrum of the (Fucα1-2Galβ1-4(Fucα1-3)Glc) tetrasaccharide. [Modes for carrying out the invention]

[0038] The present invention can be better understood by the following examples. However, it will be readily apparent to those skilled in the art that the contents described in the examples are merely for illustrative purposes and should not limit, nor should they limit, the present invention as described in detail in the claims.

[0039] The experimental methods described in the examples are conventional methods unless otherwise specified, and the reagents and materials described are commercially available unless otherwise specified.

[0040] Here, NmLgtA (abbreviation: LgtA, product code: SE-1001, 1U), Hpα1,2FT (abbreviation: α1,2FucT, product code: SE-1019, 1U), Hpα1,3FT (abbreviation: α1,3FucT, product code: SE-1018, 1U), Pd2,6ST (abbreviation: Pd26ST, product code: SE-1013, 10U), NmLgtB (abbreviation: LgtB, product code: SE-1002, 1U), and PmST1 (product code: SE-1014, 50U) were all purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd.

[0041] Chondroitin polymerase KfoC (GenBank accession number AB079602) can be obtained by purchasing or synthesizing based on the NCBI sequence and fermenting according to the reference: Self-assembly immobilization of a universal catalytic microreactor for glycosyltransferases, Process Biochemistry 133 (2023) 261-269.

[0042] 2-Ethyl-2-oxazoline: Purchased from Maclin, Catalog No.: E830884-25g, CAS: 10431-98-8 [ka] p-Propargyl toluenesulfonate: Purchased from Aladdin, Catalog Number: P817001-1ml, CAS Number: 6165-76-0 [ka] N-methylhydroxylamine hydrochloride: Purchased from Sigma, Catalog No.: M50400, CAS No.: 4229-44-1 [ka] 3-Iodo-1-chloropropane: Purchased from Sigma, catalog number: 234478, CAS number: 6940-76-7 [ka] Purchased from Gal:sigma, product code: G0750-25g, CAS number: 59-23-4; [ka] GlcA: Purchased from Aladdin, Part Number: D810404-25g, CAS Number: 6556-12-3 [ka] GalNAc: Purchased from Sigma, part number: A2795, CAS number: 1811-31-0 [ka] UDP-GalNAc: Purchased from Sigma, Part Number: U5252, CAS Number: 108320-87-2 [ka] UDP-GlcA: Purchased from Sigma, part number: U5625, CAS number: 43195-60-4 [ka] UDP-Gal: Purchased from Sigma, part number: 670111-M, CAS number: 137868-52-1 [ka] UDP-GlcNAc: Purchased from Sigma, Part Number: U4375, CAS: 91183-98-1 [ka] GDP-fucose: Purchased from MCE Corporation, Part Number: HY-134433, CAS Number: 15839-70-0 [ka] CMP-Sialic acid: Purchased from Sigma, catalog number: 233264, CAS number: 149007-28-3 [ka] The synthesis and use of the sugar conjugate of the present invention are shown below. [ka]

[0043] Example 1 Synthesis of temperature-sensitive materials 1. Synthesis of temperature-sensitive materials - alkynylation-modified poly2-ethyl-2-oxazoline 1.98 g of the monomer 2-ethyl-2-oxazoline (20 mmol) was weighed into a clean pressure tube, and nitrogen gas was aspirated and replaced three times using a two-row pipe to completely remove the air. After filling the pressure tube with a nitrogen gas atmosphere, 5 ml of anhydrous acetonitrile was added to the pressure tube, and then 21 mg of the initiator p-toluenesulfonic acid propargyl (100 μmol) was added at room temperature. After the system was homogenized, the reaction was heated at 130 °C for 12 hours. After the reaction was complete, the reaction solution was analyzed by TLC, and when the developing agent polarity PE:EA = 1:1 and alkaline potassium permanganate was used to develop the color, an Rf value of 0.6 indicated that the starting material was basically used up. As a post-treatment, 5 ml of water was added and the mixture was stirred at 80°C for 12 hours to quench the reaction. After dry evaporation of the solvent, the product was redissolved in DCM, then anhydrous ethyl ether was added and recrystallized. The mixture was then filtered by suction and dried to obtain a white powdery solid (1.5 g, 75%) of alkynylated poly-2-ethyl-2-oxazoline. When the solubility of the prepared thermosensitive material at critical temperature was tested, it was found that the thermosensitive material had the good properties of precipitating at high temperatures and dissolving at low temperatures. The effect diagram is shown in Figure 1, and the NMR hydrogen spectrum of the thermosensitive material is shown in Figure 2. [ka]

[0044] 2. Relationship between initiator-monomer ratio (M / I) and critical temperature (LCST) Weigh 1.98 g, 20 mmol of the monomer 2-ethyl-2-oxazoline into four clean pressure tubes. Completely remove air by suction displacement with nitrogen gas three times using a two-row pipe, filling the pressure tubes with a nitrogen gas atmosphere. Then, add 5 ml of anhydrous acetonitrile to the pressure tubes, and at room temperature, add the initiator p-toluenesulfonic acid propargyl. In each of the four pressure tubes, different molar ratios of monomer and initiator (M) were added. (M / I=100, 42 mg, 200 μmol) (M / I=200, 21 mg, 100 μmol) (M / I=300, 14 mg, 66 μmol) (M / I=500, 8.4 mg, 40 μmol) were added, and the system was homogeneously mixed. The reaction was then heated at 130°C for 12 hours. After the reaction was complete, the reaction solution was analyzed by TLC. When the developing agent polarity was set to PE:EA=1:1 and alkaline potassium permanganate was used to develop the color, an Rf value of 0.6 indicated that the starting materials were basically used up. As a post-treatment, 5 ml of water was added and the mixture was stirred at 80°C for 12 hours to quench the reaction. After dry evaporation of the solvent, the product was redissolved in DCM, then anhydrous ethyl ether was added and the mixture was recrystallized. The mixture was then filtered by suction and dried to obtain white powdered solids of alkynylated poly2-ethyl-2-oxazoline with M / I ratios of 100, 200, 300, and 500, respectively. To investigate the effect of different M / I ratios on the critical temperature (LCST) of temperature-sensitive materials, materials with M / I ratios of 100, 200, 300, and 500 were dissolved in ultrapure water to a concentration of 10 mg / mL, and then slowly heated in a water bath. The LCSTs were measured, and the results showed that the higher the M / I ratio, the lower the critical temperature (LCST) (see Figure 3).

[0045] 3. Relationship between the concentration of a temperature-sensitive material and its critical temperature (LCST) To investigate the effect of different concentrations of temperature-sensitive materials on the critical temperature (LCST), solid materials with an M / I ratio of 200 were dissolved in ultrapure water to concentrations of 1, 5, 10, 20, 30, and 50 mg / mL. The LCSTs were then measured by slowly raising the temperature in a water bath. As shown in Figure 4, the higher the concentration of the temperature-sensitive material, the lower its critical temperature (LCST). Furthermore, the rate of decrease in LCST was fastest in the concentration range of 1 to 20 mg / mL, and stabilized thereafter. Through the above experiments, the material with an M / I of 200 was selected as the solid phase for subsequent oligosaccharide synthesis from among the temperature-sensitive materials with M / I ratios of 100, 200, 300, and 500 because its LCST was not excessively high, and the load was higher than that for M / I ratios of 300 and 500. The concentration was set to 10 mg / mL (i.e., 0.5 mM), and at this concentration, the LCST of the temperature-sensitive material was relatively low, and the increase in viscosity of the reaction system due to excessively high material concentration was avoided.

[0046] Example 2 Synthesis of glucuronide sugar conjugates

[0047] 1. Method for synthesizing azido sugars Synthesis of Compound 2 N-methylhydroxylamine hydrochloride (3.0 g, 35.9 mmol) and K2CO3 (9.9 g, 71.6 mmol) of compound 1 were weighed into a clean round-bottom flask. After thoroughly dissolving the sample in the flask with 32 mL of H2O:THF=1:1 mixed solution, the round-bottom flask was placed in an ice bath, di-tert-butyl dicarbonate (5.78 mL, 25.2 mmol) was added, and the mixture was stirred at room temperature for 20.5 hours. After the reaction was complete, the reaction solution was analyzed by TLC. When the developing agent was polar PE:EA=1:1 and phosphomolybdic acid was used for color development, a new spot was formed at an Rf value of approximately 0.8, indicating that the starting materials had reacted completely. Next, the reaction was post-processed. First, an appropriate amount of ethyl acetate was added to dilute the mixture, then it was extracted and separated into three liquid-liquid solutions. The reaction solution was washed three times with water, then wetted with saturated brine, and finally dried with anhydrous sodium sulfate powder. After concentration treatment using a rotary evaporator, the compound 2 was finally obtained as a yellow oily liquid (3.6 g, 96%) by silica gel column chromatography (PE:EA = 5:1 to 1:1). 1 H NMR (400 MHz, CDCl3): δ 3.15 (s, 3H), 1.47 (s, 9H).

[0048] Synthesis of Compound 3 Compound 2 (890.0 mg, 6.0 mmol) was weighed into a clean pear-shaped flask, and nitrogen gas was aspirated and replaced three times using a double-row pipe to completely remove the air. After filling the flask with nitrogen gas, 17 mL of ultra-dried tetrahydrofuran was added to dissolve Compound 2, and NaH (60% dispersed in mineral oil, 290.0 mg, 7.3 mmol) was added under ice bath conditions. The mixture was stirred for 30 minutes under ice bath conditions, then 3-iodo-1-chloropropane (1.0 mL, 9.3 mmol) was added, and the mixture was allowed to return to room temperature and stirred for 4.5 hours. After the reaction was complete, the reaction solution was analyzed by TLC, and when color development was performed with phosphomolybdic acid using a polarity PE:EA = 10:1, a new spot was formed at an Rf value of 0.5, indicating that the starting material had been essentially used up. As a post-treatment, ice water was added dropwise in an ice bath environment to quench the unreacted NaH, then an appropriate amount of ethyl acetate was added for dilution, followed by extraction and three liquid-liquid divisions. The reaction solution was washed three times with water, then wetted with saturated saline solution, and finally dried with anhydrous sodium sulfate powder. After concentration treatment using a rotary evaporator, the solution was subjected to silica gel column chromatography (PE:EA = 10:1) to finally obtain compound 3 as a colorless oily liquid (1.16 g, 85%). 1 H NMR (400 MHz, CDCl3): δ 3.98 (t, J = 5.8 Hz, 2H), 3.68 (t, J = 6.2 Hz, 2H), 3.10 (s, 3H), 2.06 (q, J = 6.2 Hz, 2H), 1.49 (s, 9H)

[0049] Synthesis of Compound 4 Compound 3 (533.5 mg, 2.4 mmol) was weighed into a pressure tube and thoroughly dissolved in DMF (10 mL) solution, after which NaN3 (0.3 g, 4.6 mmol) was added. After homogeneous stirring, the magnetic stirrer was heated to 95°C, the pressure tube was placed in an oil bath pot and heated, and the reaction was stirred for 18 hours. After the reaction was complete, the reaction solution was analyzed by TLC, and when the developing agent polarity was set to PE:EA = 10:1 and phosphomolybdic acid was used for color development, a new spot was formed with an Rf value of 0.6. As a work-up, ice water was added dropwise in an ice bath environment to quench the NaN3 that had not been completely reacted, then an appropriate amount of ethyl acetate was added to dilute it, and after extraction, the solution was divided into three liquid-liquid steps, the reaction solution was washed three times with water, wetted with saturated saline solution, and finally the reaction solution was dried with anhydrous sodium sulfate powder. After concentration treatment using a rotary evaporator, the compound 4 was subjected to silica gel column chromatography (PE:EA = 10:1) to obtain a colorless oily liquid (501.8 mg, 91%). 1 H NMR (400 MHz, CDCl3): δ 3.89 (t, J = 5.9 Hz, 2H), 3.42 (t, J = 6.9 Hz, 2H), 3.07 (s, 3H), 1.85 (qui, J = 6.4 Hz, 2H), 1.47 (s, 9H);

[0050] Synthesis of Compound 5 Compound 4 (230.26 mg, 1 mmol) was weighed into a clean round-bottom flask and dissolved in 5 mL of dichloromethane. The reaction system was pre-cooled to 0°C, and trifluoroacetic acid (228 mg, 2 mmol) was added dropwise. The reaction was allowed to proceed for 1 hour. After the reaction was complete, the reaction solution was analyzed by TLC. Using a polarity PE:EA = 1:1 development agent, phosphomolybdic acid was used to develop a new spot with an Rf value of 0.2, indicating that the starting material was essentially used up. As a work-up, the pH was adjusted to neutral with saturated sodium bicarbonate solution. Then, the solution was extracted and separated three times, the reaction solution was washed three times with water, wetted with saturated brine, and finally dried with anhydrous sodium sulfate powder. After concentration treatment with a rotary evaporator, the mixture was subjected to silica gel column chromatography (PE:EA = 1:1) to obtain compound 5 as a colorless oily liquid (130.15 mg, 91%). The NMR hydrogen spectrum of compound 5 is shown in Figure 5. 1 H NMR (400 MHz,CDCl3) δ 3.74 (t, J = 6.1 Hz, 2H), 3.37 (t, J = 6.8 Hz, 2H), 2.70 (s, 3H), 1.88 - 1.81 (m, 2H), 1.34 - 1.19 (m, 1H).

[0051] Synthesis of GlcA-N-C3N3 Glucuronic acid GlcA (75 mg, 0.382 mmol) was placed in a reaction flask, and 2 mL of NaOAc buffer solution (0.1 M, pH 4.5) was added to dissolve it. Then compound 5 (149 mg, 1.14 mmol) was added, and the reaction was stirred at room temperature for 41 hours. After the reaction was complete, the reaction solution was analyzed by TLC, and when the developing agent polarity was set to EA:MeOH:water = 5:2:0.5 and ammonium molybdate was used for color development, a new spot with an Rf value of approximately 0.5 and lower polarity than the starting material GlcA glucuronic acid was formed, which was consistent with the change in polarity due to the substitution of the hydroxyl group. As a post-reaction treatment, the reaction solution was rapidly frozen with liquid nitrogen, then placed in a freeze-dryer to remove water from the reaction solution. The crude product was mixed with approximately 0.5 g of silica gel powder and dry-loaded, followed by silica gel column chromatography (DCM:MeOH = 10:1) to obtain the azidoglucuronic acid compound GlcA-N-C3N3 as a colored syrup (71 mg, 61%). The ESI spectrum of GlcA-N-C3N3 is shown in Figure 6. 1 H NMR (400 MHz,D2O) δ 4.01 (t, J = 6.1 Hz, 2H), 3.84 - 3.78 (m, 1H), 3.69 - 3.61 (m, 2H), 3.44 - 3.35 (m, 5H), 2.83 (s, 3H), 2.68 (s, 1H), 1.86 (p, J = 6.3 Hz, 2H).

[0052] 2. Synthesis of glucuronide conjugates Referring to Example 1, a thermosensitive material was selected. Alkynylated poly-2-ethyl-2-oxazoline (5 g, 0.25 mmol) with an M / I of 200 and azidoglucuronic acid (0.114 g, 0.375 mmol) were weighed out and added to a total of 10 mL of a 1 / 1 (v / v) mixed solution of water and ethanol. Then, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added, and the reaction mixture was stirred overnight at room temperature. After adding 10 mL of aqueous solution, an equal volume of chloroform was added for extraction. The chloroform phase was concentrated, and then recrystallized by adding a small amount of cold ethyl ether. After filtration, the white powdery solid was dried in a vacuum oven to obtain glucuronic acid conjugate.

[0053] Example 3 Synthesis of acetylglucosamine conjugates Synthesis of GalNAc-N-C3N3 The steps for the synthesis of the temperature-sensitive material and compounds 2-5 refer to Examples 1 and 2. Acetylglucosamine GalNAc (85 mg, 0.382 mmol) was placed in a reaction flask, dissolved in 2 mL of NaOAc buffer solution (0.1 M, pH 4.5), and then compound 5 (149 mg, 1.14 mmol) was added. The mixture was stirred at room temperature for 41 hours to allow the reaction to complete. After the reaction was complete, the reaction solution was analyzed by TLC. When the developing agent polarity was set to DCM:MeOH = 3:1 and ammonium molybdate was used for color development, a new spot with an Rf value of approximately 0.8 and lower polarity than the starting material acetylglucosamine GalNAc was formed, which was consistent with the change in polarity due to the substitution of the hydroxyl group. As a post-treatment, the reaction solution was rapidly frozen with liquid nitrogen, then placed in a freeze-dryer to remove water from the reaction solution. The crude product was mixed with approximately 0.5 g of silica gel powder and dry-loaded, followed by silica gel column chromatography (DCM:MeOH = 15:1) to obtain the GalNAc-N-C3N3 compound as a colorless syrup (94 mg 74%). The ESI spectrum of GalNAc-N-C3N3 is shown in Figure 7. 1H NMR (400 MHz,D2O) δ 4.12 (t, J = 8.7 Hz, 1H), 4.09 - 3.95 (m, 1H), 3.92 - 3.81 (m, 2H), 3.76 - 3.54 (m, 5H), 3.36 (dt, J = 13.5, 6.6 Hz, 2H), 3.28 (s, 2H), 2.69 - 2.63 (m, 3H), 1.97 (d, J = 2.4 Hz, 3H), 1.79 (dt, J = 20.8, 6.4 Hz, 2H).

[0054] Synthesis of acetylglucosamine conjugates Referring to Example 1, a temperature-sensitive material was selected. Alkynylated poly-2-ethyl-2-oxazoline (5 g, 0.25 mmol) with an M / I of 200 and azidoacetylglucosamine (0.124 g, 0.375 mmol) were weighed out and added to a total of 10 mL of a 1 / 1 (v / v) mixed solution of water and ethanol. Then, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added, and the reaction mixture was stirred overnight at room temperature. After adding 10 mL of aqueous solution, an equal volume of chloroform was added for extraction. The chloroform phase was concentrated, and then recrystallized by adding a small amount of cold ethyl ether. After filtration, the white powdery solid was dried in a vacuum oven to obtain acetylglucosamine conjugate.

[0055] Example 4 Synthesis of galactose conjugate Synthesis of Gal-N-C3N3 The steps for the synthesis of the temperature-sensitive material and compounds 2-5 refer to Examples 1 and 2. Galactose (68 mg, 0.382 mmol) was placed in a reaction flask, and 2 mL of NaOAc buffer solution (0.1 M, pH 4.5) was added to dissolve it. Then compound 4 (149 mg, 1.14 mmol) was added, and the mixture was stirred at room temperature for 41 hours to allow the reaction to complete. After the reaction was complete, the reaction solution was analyzed by TLC, and when the developing agent polarity was set to DCM:MeOH = 3:1 and ammonium molybdate was used for color development, a new spot with an Rf value of approximately 0.6 and lower polarity than the starting material Gal was formed, which was consistent with the change in polarity due to the placement of the hydroxyl group. As a post-treatment, the reaction solution was rapidly frozen with liquid nitrogen, then placed in a freeze-dryer to remove water from the reaction solution. The crude product was mixed with approximately 0.5 g of silica gel powder and dry-loaded, followed by silica gel column chromatography (DCM:MeOH=3:1) to finally obtain the Gal-N-C3N3 compound as a colorless syrup (76.9 mg, 69%). The ESI spectrum of Gal-N-C3N3 is shown in Figure 8. 1 H NMR (400 MHz,D2O) δ 4.17 - 4.07 (m, 1H), 3.94 - 3.81 (m, 2H), 3.73 (dd, J = 12.3, 4.7 Hz, 1H), 3.54 - 3.45 (m, 2H), 3.38 (dq, J = 14.9, 7.8, 7.3 Hz, 4H), 2.74 (s, 2H), 1.84 (t, J = 6.4 Hz, 1H).

[0056] Synthesis of galactose conjugate Referring to Example 1, a thermosensitive material was selected. Alkynylated poly2-ethyl-2-oxazoline (5 g, 0.25 mmol) and azidogalactose (0.118 g, 0.375 mmol) with an M / I of 200 were weighed and added to a total of 10 mL of a 1 / 1 (v / v) mixed solution of water and ethanol. Then, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added, and the reaction mixture was stirred overnight at room temperature. After adding 10 mL of aqueous solution, an equal volume of chloroform was added for extraction. The chloroform phase was concentrated, and then recrystallized by adding a small amount of cold ethyl ether. After filtration, the white powdery solid was dried in a vacuum oven to obtain galactose conjugate.

[0057] Example 5 Synthesis of lactose conjugate Synthesis of lactose-N-C3N3 The steps for the synthesis of the temperature-sensitive material and compounds 2-5 refer to Examples 1 and 2. Lactose (140 mg, 0.382 mmol) was placed in a reaction flask, and 2 mL of NaOAc buffer solution (0.1 M, pH 4.5) was added to dissolve it. Then compound 5 (149 mg, 1.14 mmol) was added, and the mixture was stirred at room temperature for 41 hours to allow it to react. After the reaction was complete, the reaction solution was analyzed by TLC, and when the developing agent polarity was set to EA:MeOH:water = 6:3:2 and ammonium molybdate was used for color development, a new spot with an Rf value of approximately 0.6 and lower polarity than the starting lactose was formed, which was consistent with the change in polarity due to the placement of the hydroxyl group. As a post-treatment, the reaction solution was rapidly frozen with liquid nitrogen, then placed in a freeze-dryer to remove water from the reaction solution. The crude product was mixed with approximately 0.5 g of silica gel powder and dry-loaded, followed by silica gel column chromatography (DCM:MeOH=5:1) to obtain the lactose-N-C3N3 compound as a colorless syrup (103.2 mg, 59%). The ESI spectrum of lactose-N-C3N3 is shown in Figure 9. 1H NMR (500 MHz, D2O): δ 4.48 (d, J = 7.8 Hz, 1H), 4.18 (d, J = 9.1 Hz, 1H), 4.00 (dd, J = 2.1, 12.3 Hz, 1H), 3.97-3.73 (m, 7H), 3.72-3.63 (m, 3H), 3.62-3.52 (m, 3H), 3.45 (t, J = 6.8 Hz, 2H), 2.78 (s, 3H), 1.92-1.84 (m, 2H).

[0058] Synthesis of lactose conjugate Referring to Example 1, a thermosensitive material was selected. Alkynylated poly-2-ethyl-2-oxazoline (5 g, 0.25 mmol) and azidolactose (0.17 g, 0.375 mmol) with an M / I of 200 were weighed out and added to a total of 10 mL of a 1 / 1 (v / v) mixed solution of water and ethanol. Then, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added, and the reaction mixture was stirred overnight at room temperature. After adding 10 mL of aqueous solution, an equal volume of chloroform was added for extraction, and the chloroform phase was concentrated. After recrystallization with the addition of a small amount of cold ethyl ether, the mixture was filtered, and the resulting white powdery solid was dried in a vacuum oven to obtain lactose conjugate.

[0059] Example 6 Synthesis of chondroitin disaccharide conjugates The synthesis of the glucuronide conjugate is described in Examples 1 and 2, and the setting of the sugar conjugate concentration is described in Example 1. The glucuronide conjugate is added to the enzymatic reaction system at a final concentration of 10 mg / mL (0.5 mM), which contains Tris-HCl (20 mM), UDP-GalNAc (1 mM), MnCl2 (10 mM), and KfoC (0.01 mg / ml). After reacting at room temperature for 4 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain a chondroitin disaccharide conjugate. This was dissolved in water to a 100 mM solution, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C, filtered while still hot to separate the sugar from the thermosensitive material, and the filtrate was freeze-dried to obtain the chondroitin disaccharide compound in 74% yield. The ESI spectrum of chondroitin disaccharide-N-C3N3 is shown in Figure 10.

[0060] Example 7 Synthesis of chondroitin trisaccharide conjugates The synthesis of the chondroitin disaccharide conjugate is described in Example 6, and the setting of the sugar conjugate concentration is described in Example 1. The chondroitin disaccharide conjugate was added to the enzymatic reaction system at a final concentration of 10 mg / mL (0.5 mM), which contains Tris-HCl (20 mM), UDP-GlcA (1 mM), MnCl2 (10 mM), and KfoC (0.01 mg / ml). After reacting at room temperature for 4 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid, filtered while hot, and washed with hot water at 90°C to obtain the chondroitin trisaccharide conjugate. This was dissolved in water to a final concentration of 100 mM, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C, filtered while hot to separate the sugar from the thermosensitive material, and the filtrate was freeze-dried to obtain the chondroitin trisaccharide compound in 80% yield.

[0061] Example 8 Synthesis of Lacto-N-triose II (LNT II) Refer to Example 5 for the synthesis of lactose conjugate, and refer to Example 1 for setting the concentration of the sugar conjugate. The lactose conjugate is brought to a final concentration of 10 mg / mL (0.5 mM) and added to the enzyme reaction system, which contains Tris-HCl (20 mM), UDP-GlcNAc (1 mM), MgCl2 (10 mM), and NmLgtA (β1-3 N-acetylglucosaminyltransferase from Neisseria meningitides) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm) (step a), the system was heated to 90°C to precipitate a white solid, filtered while still hot, washed the precipitate with 90°C hot water, and dried to obtain an LNT II trisaccharide conjugate. This was dissolved in water to 100 mM, then 0.12% (v / v) TFA was added, and the mixture was heated to 90°C to release the sugar chains (step b). The mixture was filtered while still hot to separate the sugar chains from the thermosensitive material, and the filtrate was freeze-dried to obtain LNT II trisaccharide in 91% yield. The HR-MS spectrum of LNT II trisaccharide is shown in Figure 11. [ka]

[0062] Example 9 Synthesis of 2'-Fucosyllactose (2'-FL) The synthesis of lactose conjugate is as described in Example 5. Refer to Example 1 for setting the concentration of the sugar conjugate. Lactose conjugate (10 mg / mL, final concentration approximately 0.5 mM) is added to the enzymatic reaction system, which contains Tris-HCl (20 mM), GDP-fucose (1 mM), MnCl2 (10 mM), and Hpα1,2FT (α1,2 fucosyltransferase from Helicobacter pylori) (0.01 mg / mL). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid, filtered while still hot, and washed with 90°C hot water to obtain a 2'-FL trisaccharide conjugate. This was dissolved in water to a 100 mM solution, then 0.12% (v / v) TFA was added, and the mixture was heated to 90°C to release the sugar chains. The filtrate was freeze-dried to obtain 2'-FL trisaccharide in 89% yield.

[0063] Example 10 Synthesis of 3-Fucosyllactose (3-FL) Refer to Example 5 for the synthesis of lactose conjugate, and refer to Example 1 for setting the concentration of the sugar conjugate. Lactose conjugate (10 mg / mL, final concentration approximately 0.5 mM) is added to the enzymatic reaction system, which contains Tris-HCl (20 mM), GDP-fucose (1 mM), MnCl2 (10 mM), and Hpα1,3FT (α1,3 fucosyltransferase from Helicobacter pylori) (0.01 mg / mL). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain a 3-FL trisaccharide conjugate. This was dissolved in water to a 100 mM solution, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain 3-FL trisaccharide in 93% yield.

[0064] Example 11 Synthesis of 6'-Sialyllactose (6'-SL) Refer to Example 5 for the synthesis of lactose conjugate, and refer to Example 1 for setting the concentration of lactose conjugate. Lactose conjugate (10 mg / mL, final concentration approximately 0.5 mM) is added to the enzymatic reaction system, which contains Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and Pd2,6ST (α2-6 sialyltransferase from P. damselaedamsela) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid, filtered while still hot, and washed with 90°C hot water to obtain a 6'-SL trisaccharide conjugate. This was dissolved in water to a 100 mM solution, then 0.12% (v / v) TFA was added, and the mixture was heated to 90°C to release the sugar chains. The filtrate was freeze-dried to obtain 6'-SL in 86% yield.

[0065] Example 12 Synthesis of Lacto-N-neotetrose (LNnT) Refer to Example 8 for the synthesis of the LNT II conjugate, and refer to Example 1 for setting the concentration of the LNT II conjugate. The LNT II (10 mg / mL, approximately 0.5 mM final concentration) conjugate is added to the enzyme reaction system, which contains Tris-HCl (20 mM), UDP-Gal (1 mM), MnCl2 (10 mM), and NmLgtB (β-4 galactosyltransferase from N. meningitides) (0.01 mg / mL). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain an LNnT tetrasaccharide conjugate. This was dissolved in water to a 100 mM solution, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain LNnT tetrasaccharide in an overall yield of 84%.

[0066] Example 13 Synthesis of Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc Refer to Example 12 for the synthesis of the LNnT tetrasaccharide conjugate. The precipitate was redissolved. Refer to Example 1 for setting the conjugate concentration. A new reaction system was added to a final concentration of 10 mg / mL (0.5 mM), which included Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and PmST1 (α2-3 sialyltransferase from P. multocida) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid, filtered while still hot, and washed with 90°C hot water to obtain a pentasaccharide conjugate. This was dissolved in water to a 100 mM concentration, then 0.12% (v / v) TFA was added, and the mixture was heated to 90°C to release the sugar chains. The filtrate was freeze-dried to obtain Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc in a total yield of 79%.

[0067] Example 14 Synthesis of (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc Refer to Example 12 for the synthesis of the LNnT tetrasaccharide conjugate. The precipitate was redissolved. Refer to Example 1 for setting the conjugate concentration. A new reaction system was added to a final concentration of 10 mg / mL (0.5 mM), which included Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and Pd2,6ST (α2-6 sialyltransferase from P. damselaedamsela) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain a hexasaccharide conjugate. This was dissolved in water to a 100 mM concentration, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc in an overall yield of 81%. The HR-MS spectrum of the hexasaccharide (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc is shown in Figure 12.

[0068] Example 15 Synthesis of GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc For the synthesis of the LNT II conjugate, refer to Example 8, and for setting the conjugate concentration, refer to Example 1. The LNT II conjugate (10 mg / mL, final concentration approximately 0.5 mM) is added to the enzyme reaction system, which contains Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and Pd2,6ST (α2-6 sialyltransferase from P. damselaedamsela) (0.01 mg / mL). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide conjugate. This was dissolved in water to a 100 mM concentration, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide in an overall yield of 85%. The HR-MS spectrum of the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide is shown in Figure 13.

[0069] Example 16 Synthesis of Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc For the synthesis of the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide conjugate, refer to Example 15. The precipitate was redissolved. For setting the concentration of the conjugate, refer to Example 1. A new reaction system was added to a final concentration of 10 mg / mL (0.5 mM), which included Tris-HCl (20 mM), UDP-Gal (1 mM), MnCl2 (10 mM), and NmLgtB (β-4 galactosyltransferase from N. meningitides) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with hot water at 90°C to obtain the Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc pentasaccharide conjugate. This was dissolved in water to 100 mM, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc in total yield of 76%. The HR-MS spectrum of the Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc pentasaccharide is shown in Figure 14.

[0070] Example 17 Synthesis of (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc For the synthesis of the Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc pentasaccharide conjugate, refer to Example 16. The precipitate was redissolved. For setting the concentration of the conjugate, refer to Example 1. A new reaction system was added to achieve a final concentration of 10 mg / mL (0.5 mM), which included Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and PmST1 (α2-3 sialyltransferase from P. multocida) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain a hexasaccharide conjugate. This was dissolved in water to a 100 mM concentration, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc in total yield of 65%. The HR-MS spectrum of the hexasaccharide (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc is shown in Figure 15.

[0071] Example 18 Synthesis of Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc Refer to Example 11 for the synthesis of the 6'-Sialyllactose (6'-SL) trisaccharide conjugate. The precipitate was redissolved. Refer to Example 1 for setting the conjugate concentration. A new reaction system was added to a final concentration of 10 mg / mL (0.5 mM), which included Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and PmST1 (α2-3 sialyltransferase from P. multocida) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain a tetrasaccharide conjugate. This was dissolved in water to a 100 mM concentration, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc in an overall yield of 82%. The HR-MS spectrum of the Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide is shown in Figure 16.

[0072] Example 19 Synthesis of (Fucα1-2Galβ1-4(Fucα1-3)Glc) Refer to Example 9 for the synthesis of the 2'-Fucosyllactose (2'-FL) trisaccharide conjugate. The precipitate was redissolved. Refer to Example 1 for setting the conjugate concentration. A new reaction system was added to a final concentration of 10 mg / mL (0.5 mM), which included Tris-HCl (20 mM), GDP-fucose (1 mM), MnCl2 (10 mM), and Hpα1,3FT (α1,3 fucosyltransferase from Helicobacter pylori) (0.01 mg / ml). After reacting at room temperature for 2 hours with shaking (800 rpm), the system was heated to 90°C to precipitate a white solid. The precipitate was filtered while still hot, and washed with 90°C hot water to obtain a tetrasaccharide conjugate. This was dissolved in water to a 100 mM concentration, and then 0.12% (v / v) TFA was added. The mixture was heated to 90°C to release the sugar chains, and the filtrate was freeze-dried to obtain (Fucα1-2Galβ1-4(Fucα1-3)Glc) in an overall yield of 76%. The HR-MS spectrum of the (Fucα1-2Galβ1-4(Fucα1-3)Glc) tetrasaccharide is shown in Figure 17.

Claims

1. A thermosensitive sugar conjugate of a thermosensitive material, wherein the thermosensitive sugar conjugate is formed by conjugating a thermosensitive material with an azido sugar. The aforementioned temperature-sensitive material is an alkynylation-modified 2-ethyl-2-oxazoline polymer, the structural formula of which is shown below. The aforementioned azide sugar is an azide sugar based on a galactose (Gal) monosaccharide or an azide sugar based on a lactose (Lac) disaccharide, and the structures of these are as shown below, characterized in that they are thermosensitive sugar conjugates for thermosensitive materials. 【Chemistry 22】 (Here, n represents the degree of polymerization from 100 to 2000.) 【Chemistry 23】

2. The thermosensitive sugar conjugate for a thermosensitive material according to claim 1, characterized by comprising the steps of adding 2-ethyl-2-oxazoline as a monomer and an initiator to a solvent, reacting the reaction system continuously for 5 to 12 hours under conditions of 100 to 200°C in an inert atmosphere, quenching the reaction after the reaction is complete, dry evaporating the solvent, recrystallizing, drying by suction filtration, and obtaining a thermosensitive material which is a white powdery solid of alkynylation-modified poly2-ethyl-2-oxazoline.

3. The thermosensitive sugar conjugate according to claim 1, characterized in that it achieves conjugation of a sugar and a thermosensitive material by bonding the alkynyl group of the thermosensitive material to the azide group of the azide sugar.

4. The thermosensitive sugar conjugate according to claim 1, characterized in that the raw material for the azido sugar is a monosaccharide or oligosaccharide, preferably containing but not limited to galactose, glucose, acetylglucosamine, acetylglucosamine, or lactose.

5. A method for preparing a thermosensitive sugar conjugate according to claim 1, characterized by comprising the steps of adding a thermosensitive material, azido sugar, ascorbic acid, hydrazine or tris(2-carbonylethyl)phosphine hydrochloride, and a copper raw material to a reaction solvent, stirring and reacting at room temperature, extracting and concentrating the mixture after the reaction, further recrystallizing it, filtering it, and then drying the solid to obtain a thermosensitive sugar conjugate.

6. The preparation of the aforementioned azido sugar is as follows: (a) The N-methylhydroxylamine hydrochloride of compound 1 is converted to di-tert-butyl dicarbonate (Boc ２ By reacting it with ), the hydrogen atom on the nitrogen of N-methylhydroxylamine hydrochloride is replaced with a tert-butyloxycarbonyl group (Boc) to obtain compound 2. (b) Compound 2 is reacted with 1-chloro-3-iodopropane to replace the hydrogen on the hydroxyl group of compound 2 with 1-chloro-3-iodopropane, thereby extending the chain and obtaining compound 3. (c) Compound 3 is reacted with sodium azide to replace the chlorine on the propyl group of compound 3 with an azide group to obtain compound 4. (d) The tert-butyloxycarbonyl (Boc) protecting group of compound 4 is removed by the action of trifluoroacetic acid (TFA), and the hydrogen atom on the nitrogen of compound 4 is released to obtain compound 5. (e) Reacting compound 5 with a sugar, where the sugar used is a monosaccharide or oligosaccharide as the glycosyl donor and compound 5 as the raw material, to obtain an azido sugar. The method for preparing a thermosensitive sugar conjugate according to claim 5, characterized in that the reaction equation is shown below. 【Chemistry 24】

7. Use of the thermosensitive sugar conjugate according to claim 1 in sugar solid-phase synthesis.

8. The aforementioned usage process is (1) Mix the glycosyl donor and the sugar conjugate, (2) Add the sugar compound enzyme and shake to carry out the reaction. (3) Heat the reaction solution shaken in step (2), wash the precipitated product with hot water to obtain a disaccharide conjugate or polysaccharide conjugate, and repeat steps (1), (2), and (3) to increase the sugar units. (4) The use according to claim 7, characterized in that the disaccharide conjugate or polysaccharide conjugate obtained in step (3) is added to trifluoroacetic acid, and then heated and the supernatant is collected to obtain the corresponding disaccharide or polysaccharide.

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