Temperature-sensitive material and glycoconjugate, and synthesis method therefor and use thereof in solid-phase glycosynthesis

Abstract

Disclosed in the present invention are a temperature-sensitive material and a glycoconjugate, and a synthesis method therefor and the use thereof in solid-phase glycosynthesis, wherein the temperature-sensitive material is an alkynylated 2-ethyl-2-oxazoline polymer, the azido sugar has a structure of Suger-N-C3N3, and the temperature-sensitive material and the azido sugar are conjugated to form the temperature-sensitive glycoconjugate, thereby realizing the use in solid-phase glycosynthesis. In the present invention, in an actual application stage, the precipitation of glycoconjugate in an aqueous phase can be realized by means of increasing the temperature, the dissolution of a glycoconjugate can be realized by means of reducing the temperature, and the separation of the sugar from the material can also be realized by means of trifluoroacetic acid (TFA). On the basis of the above characteristics, the temperature-sensitive material and the glycoconjugate thereof of the present invention can efficiently and conveniently solve the defects of a sugar intermediate being difficult to separate and purify during polymerization and the steps being uncontrollable, thereby realizing controllable synthesis and purification of sugar, and is of great significance to the research of sugar science.

Description

A thermosensitive material and a glycoconjugate, their synthesis method and application in solid-phase glycosynthesis. Technical Field

[0001] This invention belongs to the field of sugar materials and sugar catalysis, specifically relating to a thermosensitive material and a sugar conjugate, their synthesis method, and their application in solid-phase sugar synthesis. Background Technology

[0002] The diversity and complexity of carbohydrate compounds determine the diversity of their functions in biological processes. Therefore, the stable acquisition of oligosaccharides and glycoconjugates with defined structures is a significant limitation restricting related research. Naturally extracted sugar products, due to differences in processing methods and raw material types, result in heterogeneous structures and numerous impurities, limiting research on the biological functions of sugars. Thus, the synthesis, purification, and development of universal methods for carbohydrate compounds are major bottlenecks hindering the development of carbohydrate science. Chemical synthesis of carbohydrate compounds, through the formation of glycosidic bonds via chemical reactions and the assembly of sugar chains, offers great flexibility. However, the acquisition of specific groups or configurations during the reaction often involves multiple steps of group protection and deprotection, which are cumbersome and result in low yields. Furthermore, various glycosylation methods, protecting group manipulation, and synthetic route design in chemical methods require a high level of organic chemistry expertise from experimenters, making the experiments challenging to master.

[0003] Enzymatic synthesis of carbohydrates involves constructing glycosidic bonds through enzymatic glycosylation reactions, mimicking the natural in vivo synthetic pathway and utilizing enzymes for the in vitro assembly of oligosaccharides. Compared to traditional chemical glycosylation methods, enzymatic glycosylation exhibits unique advantages. It possesses high regio and stereoselectivity, avoids cumbersome group protection and deprotection operations, and offers mild reaction conditions and simple steps. Combining high technical efficiency with environmental friendliness, it aligns with national green biomanufacturing strategies and has become a crucial pathway for obtaining complex oligosaccharides and glycoconjugates. Despite these advantages, the enzymatic synthesis of carbohydrates suffers from drawbacks such as low product separation efficiency and high difficulty, severely limiting the synthetic efficiency of carbohydrate intermediates. Summary of the Invention

[0004] Objective: To address the problems in the separation and purification of sugar intermediates, this invention provides a thermosensitive material and a glycoconjugate based on this material, while also realizing the excellent application of this thermosensitive glycoconjugate in solid-phase sugar synthesis. This invention synthesizes a thermosensitive material with an alkyne group whose precipitation temperature is controlled by the degree of polymerization, and also synthesizes a sugar acceptor with an azido group adapted to the thermosensitive material. The alkyne group on the thermosensitive material undergoes a Click reaction with the azido group on the azido sugar, thereby achieving the conjugation of the sugar with the thermosensitive material (called a glycoconjugate). The temperature-sensitive precipitation property of the thermosensitive material enables the precipitation of the glycoconjugate under high-temperature conditions and the dissolution of the glycoconjugate at low temperatures. This invention discloses a method for synthesizing thermosensitive materials and their sugar conjugates, and their application in solid-phase sugar synthesis. Compared with traditional sugar separation methods, this method is faster and simpler, making solid-phase sugar synthesis controllable and promising for the synthesis of homogeneous sugars. It lays a good foundation for studying the structure-activity relationship of carbohydrate compounds and is of great significance to the development of sugar science. It effectively solves the limitations of existing technologies, such as the difficulty in separating sugars and the non-uniformity of molecular weight, and simplifies purification steps and improves synthesis efficiency.

[0005] Technical solution: To achieve the above objectives, the thermosensitive material of this invention is a type of alkynylated modified 2-ethyl-2-azolino polymer, the structural formula of which is shown below, where n represents the degree of polymerization of 100-2000:

[0006] The method for preparing the thermosensitive material of the present invention includes the following steps:

[0007] 2-Ethyl-2-azolinite as a monomer and initiator are added to the solvent. Under an inert atmosphere, the reaction system is continuously reacted at 100-200℃ for 5-12 hours. After the reaction is completed, the reaction is quenched, the solvent is evaporated, recrystallized, filtered and dried to obtain a white powder solid of alkynylated poly-2-ethyl-2-azolinite, which is the thermosensitive material.

[0008] Preferably, anhydrous acetonitrile is used as the solvent, and the reaction is carried out under the protection of inert gases such as nitrogen and argon. The degree of polymerization is controlled by the ratio of monomer 2-ethyl-2-azolinone to initiator p-toluenesulfonate propyne ester; the preferred reaction formula is:

[0009] The thermosensitive glycoconjugate based on the thermosensitive material described in this invention is formed by combining the thermosensitive material with an azide sugar. The thermosensitive material is an alkynylated modified 2-ethyl-2-azolino polymer, and the azide sugar has a Sugar-N-C3N3 structure.

[0010] The thermosensitive sugar conjugate is formed by combining the alkynyl group on the thermosensitive material with the azido group on the azido sugar, thereby achieving the conjugation of sugar and thermosensitive material.

[0011] The raw materials for the azide sugar are monosaccharides or oligosaccharides, including but not limited to galactose, glucose, acetylglucosamine, acetylglucosamine, or lactose.

[0012] The azidosaccharide is either a galactose-based monosaccharide or a lactose-based disaccharide, and its structure is shown below:

[0013] The method for preparing the thermosensitive glycoconjugate of the present invention includes the following steps:

[0014] A thermosensitive material, azide sugar, ascorbic acid or hydrazine or tris(2-carbonylethyl)phosphohydrochloride, and copper raw material are added to the reaction solvent. The mixture is stirred at room temperature, and after the reaction, it is extracted, concentrated, recrystallized, filtered, and the solid is dried to obtain the thermosensitive glycoconjugate.

[0015] Preferably, the molar ratio of alkynylated poly-2-ethyl-2-azolin to azidosaccharide is 1:1.5, and the amounts of ascorbic acid and copper sulfate pentahydrate are 0.5 and 0.3 times the molar amount of alkynylated poly-2-ethyl-2-azolin, respectively. The reaction solvent is a mixed solution of water and ethanol or tert-butanol in a 1 / 1 (v / v) ratio. The reaction formula is shown below:

[0016] The preparation of the azidosaccharide is as follows:

[0017] (a) Compound 1, N-methylhydroxylamine hydrochloride, reacts with ditert-butyl dicarbonate (Boc2) to give compound 2 by replacing the hydrogen atom on the nitrogen of N-methylhydroxylamine hydrochloride with tert-butyloxycarbonyl (Boc).

[0018] (b) Compound 2 reacts with 1-chloro-3-iodopropane, which replaces the hydrogen atom on the hydroxyl group of compound 2, thereby extending the chain and yielding compound 3.

[0019] (c) Compound 3 reacts with sodium azide, and the chlorine on the propyl group of compound 3 is replaced by the azide group to obtain compound 4;

[0020] (d) The tert-butoxycarbonyl (Boc) protecting group on compound 4 was removed by trifluoroacetic acid (TFA), and the hydrogen on the nitrogen of compound 4 was released to obtain compound 5;

[0021] (e) Compound 5 reacts with sugars, using monosaccharides or oligosaccharides as glycosyl donors and compound 5 as a raw material, to obtain azide sugars;

[0022] The reaction formula is shown below:

[0023] The application of the thermosensitive glycoconjugate described in this invention in solid-phase glycosynthesis.

[0024] The application process is as follows:

[0025] (1) Mix the glycosyl donor and the glycoconjugate;

[0026] (2) Add glycosides and shake to react;

[0027] (3) Heat the reaction solution after shaking in step (2), take the precipitated product and wash it with hot water to obtain disaccharide conjugate or polysaccharide conjugate. Repeating steps (1)(2)(3) can increase the sugar units.

[0028] (4) Add trifluoroacetic acid to the disaccharide conjugate or polysaccharide conjugate obtained in step (3), then heat and take the clear liquid to obtain the corresponding disaccharide or polysaccharide.

[0029] The enzymes synthesized in the solid phase 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).

[0030] This invention first synthesizes a thermosensitive material with a specific degree of polymerization, then synthesizes azide sugars with azido groups. The azide sugars are mixed with the thermosensitive material, and the sugars are combined with the thermosensitive material via a Click reaction to achieve the synthesis of glycoconjugates. This invention combines the thermosensitive material with the azido groups on the azide sugar through the alkyne groups on the material, achieving the conjugation of the sugar with the thermosensitive material (hereinafter collectively referred to as glycoconjugates). Solid-phase synthesis of the sugar is then achieved using a corresponding glycoenzyme. In practical applications, the glycoconjugates can be precipitated in the aqueous phase by increasing the temperature, and dissolved by decreasing the temperature (the critical temperature is controlled by the degree of polymerization of the material). The sugars can also be separated from the material using trifluoroacetic acid (TFA) to obtain structurally defined sugar compounds (Figure 1). Based on these characteristics, this invention's method for synthesizing glycoconjugates efficiently and conveniently solves the shortcomings of difficult separation and purification of sugar intermediates during polymerization and the uncontrollable steps, thus achieving controllable synthesis and purification of sugars, which is of great significance to the research of sugar science.

[0031] Specifically, the preparation of the present invention preferably includes three parts:

[0032] Part 1: Preparation of thermosensitive materials with terminal alkyne groups

[0033] Using propargyl p-toluenesulfonate as an initiator and 2-ethyl-2-azoline as a monomer, an inert gas such as nitrogen or argon was introduced into the reaction flask. Anhydrous acetonitrile was added, followed by 2-ethyl-2-azoline, to a concentration of 1–5 M. Subsequently, propargyl p-toluenesulfonate was added dropwise. After the addition was complete, the reaction system was maintained at 100–200 °C for 5–12 h. After the reaction was completed, 1:1 water or saturated sodium carbonate was added to quench the reaction. The quenching process required 50–80 °C for 6–12 h. After quenching, the solvent was evaporated, and the product was redissolved with DCM. Then, anhydrous diethyl ether was added for recrystallization, followed by filtration and drying to obtain a white powdery solid of alkynylated poly-2-ethyl-2-azoline.

[0034] Part Two: Preparation of Azide Sugars

[0035] (1) Compound 1 N-methylhydroxylamine hydrochloride reacts with ditert-butyl dicarbonate (Boc2) to give compound 2 by replacing the hydrogen atom on the nitrogen of N-methylhydroxylamine hydrochloride with tert-butyloxycarbonyl (Boc).

[0036] (2) Compound 2 reacts with 1-chloro-3-iodopropane, and 1-chloro-3-iodopropane replaces the hydrogen on the hydroxyl group of compound 2, thereby extending the chain and obtaining compound 3.

[0037] (3) Compound 3 reacts with sodium azide, and the chlorine on the propyl group of compound 3 is replaced by the azide group to obtain compound 4;

[0038] (4) The tert-butoxycarbonyl (Boc) protecting group on compound 4 is removed by trifluoroacetic acid (TFA), and the hydrogen on the nitrogen of compound 4 is released to obtain compound 5.

[0039] (5) Compound 5 reacts with sugar, using monosaccharides or oligosaccharides as glycosyl donors and compound 5 as raw material to achieve coupling between sugar and azide side chain.

[0040] In step (1), the solvent for compound 1 is a mixture of tetrahydrofuran (THF) and water in a volume ratio of 1:1. Solid K2CO3 is added to the mixture of compound 1, and the amount of ditert-butyl dicarbonate is 0.5 to 1 times the molar number of compound 1. The reaction is carried out at 0°C and can be restored to room temperature after the addition is completed.

[0041] In step (2), the reaction solvent used is anhydrous tetrahydrofuran (THF), the amount of 1-chloro-3-iodopropane is 1 to 2 times the number of moles of compound 2, the amount of sodium hydride is 1 to 2 times the number of moles of compound 2, the reaction requires the sodium hydride to react with compound 1 at 0°C for more than 30 minutes, and then 1-chloro-3-iodopropane is added to restore room temperature; the inert protective gas is nitrogen, helium, argon, etc.

[0042] In step (3), the reaction solvent used is N,N-dimethylformamide (DMF), and the amount of sodium azide is 1 to 2 times the number of moles of compound 3; the reaction is carried out at 90℃ to 100℃.

[0043] In step (4), compound 4 is hydrolyzed and protected with an acid, including but not limited to trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, etc., and dichloromethane (DCM) is used as the solvent. The amount of acidic reagent used is 0.5 to 5 times that of compound 4.

[0044] In step (5), the sugar used includes, but is not limited to, monosaccharides or oligosaccharides such as galactose, glucose, acetylglucosamine, acetylglucosamine, and lactose. The reaction solution is a NaOAc buffer solution (0.1-1M, pH 4.5-5.3). The amount of compound 5 is 1-5 times the number of moles of sugar. The reaction is carried out at 15℃-35℃.

[0045] Part Three: Preparation of Thermosensitive Glycoconjugates

[0046] The ratio of alkynylated poly-2-ethyl-2-azolin to azidosaccharide was 1:2. The amounts of ascorbic acid and copper sulfate pentahydrate were 0.5 and 0.3 times the molar amount of alkynylated poly-2-ethyl-2-azolin, respectively. The reaction solvent was a 1 / 1 (v / v) mixture of water and ethanol. After stirring overnight at room temperature, an aqueous solution was added, and the mixture was extracted with chloroform, concentrated, and then recrystallized with cold ether. After filtration, the white powdery solid was dried in a vacuum drying oven to obtain immobilized lactose.

[0047] Using the above-described process, a thermosensitive glycoconjugate was prepared by conjugating a thermosensitive material with an azide sugar. This imbues the sugar with temperature sensitivity, allowing for convenient separation of the glycoconjugate by controlling the ambient temperature to regulate its dissolution and precipitation. Furthermore, the glycoconjugate can be used in enzymatic reactions to achieve efficient and continuous catalysis of carbohydrates. Upon completion of the reaction, the sugar is released from the glycoconjugate via bond breaking with trifluoroacetic acid, enabling precise synthesis of carbohydrates.

[0048] Design Principle: The diversity and complexity of carbohydrate structures determine the diversity of their functions in biological processes. Therefore, studying the structure-activity relationship (SOR) of structurally defined carbohydrates is of great significance. Enzymatic synthesis of carbohydrates has advantages such as stereospecificity, high efficiency and selectivity, and ease of operation, avoiding cumbersome group protection and deprotection operations, making it a very important method in carbohydrate synthesis. However, enzymatic synthesis also has disadvantages, such as uncontrollable degree of glycan polymerization and difficulties in separation and purification. Therefore, by introducing the sugar substrate into a temperature-sensitive material, convenient purification of carbohydrate compounds can be achieved. At the same time, trifluoroacetic acid can be used to release the sugars in the sugar conjugates, which is beneficial for synthesizing structurally defined carbohydrates and studying their corresponding SOR.

[0049] This invention synthesizes an alkynylated poly(2-ethyl-2-azolinium) thermosensitive material, achieving good control over the dissolution temperature by controlling the degree of polymerization. A strategy for glycoazidation modification was developed, synthesizing a series of azidosaccharides with azido groups, broadening the range of sugar substrates. This invention ingeniously links the thermosensitive material to azidosaccharides via a Glick reaction. This linkage method exhibits high selectivity, mild reaction conditions, and good linkage stability, allowing the synthesized glycoconjugates to remain stable while also possessing good temperature sensitivity.

[0050] The thermosensitive material prepared in this invention controls the dissolution temperature by adjusting the degree of polymerization (a higher degree of polymerization results in a lower precipitation temperature), thereby influencing the dissolution and precipitation of the material and enabling its separable application. Furthermore, the material contains alkynyl groups. The prepared azidosaccharides are produced by azidating natural sugars (monosaccharides, disaccharides, trisaccharides, etc.) using organic chemical methods, resulting in sugars with specific azido groups. The NC bonds connecting the sugar and the azido side chains can be broken using trifluoroacetic acid (TFA), thus restoring the azidosaccharide structure to its original natural form. The prepared glycoconjugates are formed by linking the alkynyl groups on the thermosensitive material with the azido groups of the azidosaccharides via the Glick reaction. This results in glycoconjugates that possess both the temperature sensitivity of the thermosensitive material (allowing for temperature-controlled dissolution and precipitation) and the cleavable structure of the azidosaccharides.

[0051] The thermosensitive material prepared by this invention, through a glycoconjugate formed by linking the thermosensitive material with azidosaccharides, realizes the application of the thermosensitive material in solid-phase sugar synthesis. Specifically, the novel thermosensitive material prepared by this invention can be recognized by enzymes to carry out enzymatic reactions. Normal enzymatic reactions require the addition of sugar units to achieve a continuous reaction from monosaccharide to disaccharide, and from disaccharide to trisaccharide to polysaccharide. After each sugar reaction, the product needs to be separated and purified. Normal steps require a series of complex and time-consuming operations such as column chromatography (silica gel column chromatography, hydrogel column chromatography) and lyophilization. This invention involves sugars participating in enzymatic reactions as glycoconjugates. After the reaction, the product is precipitated directly by controlling the temperature, achieving rapid separation (unlike traditional silica gel column chromatography and gel column chromatography, which are time-consuming and labor-intensive). Based on this, controllable sugar synthesis (solid-phase sugar synthesis) can be achieved. Furthermore, based on the breakable structure of azide sugars, the glycoconjugates can be further separated into sugars and materials, with the sugars being natural sugars (traditional solid-phase synthesized sugars generally require additional groups, resulting in non-natural sugars). This facilitates scientists' research on the structure-activity relationship of sugars. The temperature-sensitive material prepared by this invention enables convenient separation and controllable synthesis of sugars, which is of great significance for the study of sugar structure-activity relationships.

[0052] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0053] 1. This invention achieves temperature sensitivity of the sugar conjugate by synthesizing a temperature-sensitive material and conjugating it with azide sugar. The high-temperature precipitation and low-temperature dissolution of the sugar conjugate can be achieved by controlling the temperature, which is beneficial for the purification and separation of products during the enzymatic reaction and helps to accelerate the enzymatic reaction process.

[0054] 2. Based on the temperature precipitation characteristics of sugar conjugates, these conjugates can be applied to the solid-phase synthesis of sugars, enabling the controllable synthesis of sugars. This is of great significance for the synthesis of sugar compounds with well-defined structures and uniform molecular weights, and also makes a significant contribution to sugar science research.

[0055] 3. Due to the special nature of the linking groups of sugar conjugates, trifluoroacetic acid can be used to cleave the sugar conjugates to obtain natural sugar compounds. This separation method is milder and more efficient. Attached Figure Description

[0056] Figure 1. A real-life photograph of the high-temperature precipitation and low-temperature dissolution of temperature-sensitive materials;

[0057] Figure 2 shows the 1H NMR spectrum of the alkynylated modified poly-2-ethyl-2-azolinite thermosensitive material;

[0058] Figure 3 shows the relationship between the initiator to monomer ratio (M / I) and the critical temperature (LCST).

[0059] Figure 4 shows the relationship between the concentration of the thermosensitive material and the critical temperature (LCST).

[0060] Figure 5 shows the 1H NMR spectrum of the side chain of the azide group;

[0061] Figure 6 shows the ESI diagram of GlcA-N-C3N3;

[0062] Figure 7 shows the ESI diagram of GalNAc-N-C3N3;

[0063] Figure 8 shows the ESI diagram of Gal-N-C3N3;

[0064] Figure 9 shows the ESI diagram of Lac-N-C3N3;

[0065] Figure 10 shows the ESI diagram of chondroitin disaccharide-N-C3N3;

[0066] Figure 11 shows the HR-MS spectrum of Lacto-N-triose;

[0067] Figure 12 shows the HR-MS spectrum of (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc;

[0068] Figure 13 shows the HR-MS spectrum of GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc;

[0069] Figure 14 shows the HR-MS spectrum of Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc;

[0070] Figure 15 shows the HR-MS spectrum of (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc;

[0071] Figure 16 shows the HR-MS spectrum of Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc;

[0072] Figure 17 shows the HR-MS spectrum of the tetrasaccharide (Fucα1-2Galβ1-4(Fucα1-3)Glc). Detailed Implementation

[0073] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0074] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0075] Among them, NmLgtA (abbreviation: LgtA, catalog number: SE-1001, 1U), Hpα1,2FT (abbreviation: α1,2FucT, catalog number: SE-1019, 1U), Hpα1,3FT (abbreviation: α1,3FucT, catalog number: SE-1018, 1U), Pd2,6ST (abbreviation: Pd26ST, catalog number: SE-1013, 10U), NmLgtB (abbreviation: LgtB, catalog number: SE-1002, 1U), and PmST1 (catalog number: SE-1014, 50U) were all purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd.

[0076] Chondroitin polymerase KfoC (GenBank accession number AB079602) can be purchased or synthesized artificially based on the sequence on NCBI and obtained by fermentation according to literature, such as: Self-assembly immobilization of a universal catalytic microreactor for glycosyltransferases, Process Biochemistry 133(2023)261–269.

[0077] 2-Ethyl-2-azolin: Purchased from Maclean's, catalog number: E830884-25g, CAS: 10431-98-8

[0078] Propylene toluenesulfonate: Purchased from Aladdin, catalog number: P817001-1ml, CAS number: 6165-76-0

[0079] N-Methylhydroxylamine hydrochloride: Purchased from Sigma, item number: M50400, CAS: 4229-44-1

[0080] 3-Iodo-1-chloropropane: Purchased from Sigma, catalog number: 234478, CAS: 6940-76-7

[0081] Gal: Purchased from Sigma, item number: G0750-25g, CAS: 59-23-4;

[0082] GlcA: Purchased from Aladdin, item number: D810404-25g, CAS: 6556-12-3

[0083] GalNAc: Purchased from Sigma, item number: A2795, CAS: 1811-31-0

[0084] UDP-GalNAc: Purchased from Sigma, item number: U5252, CAS: 108320-87-2

[0085] UDP-GlcA: Purchased from Sigma, part number: U5625, CAS: 43195-60-4

[0086] UDP-Gal: Purchased from Sigma, item number: 670111-M, CAS: 137868-52-1

[0087] UDP-GlcNAc: Purchased from Sigma, part number: U4375, CAS: 91183-98-1

[0088] GDP-fucose: Purchased from MCE, item number: HY-134433, CAS: 15839-70-0

[0089] CMP-Sialic acid: Purchased from Sigma, product number: 233264, CAS: 149007-28-3

[0090] The synthesis and application of the glycoconjugates of this invention are as follows:

[0091] Example 1

[0092] Synthesis of temperature-sensitive materials

[0093] 1. Synthesis of thermosensitive materials - alkyne-modified poly-2-ethyl-2-azolinium

[0094] 1.98 g (20 mmol) of monomer 2-ethyl-2-azolin was weighed into a clean pressure-resistant tube. Nitrogen gas was purged three times in a double-row tube to remove all air, ensuring the tube was filled with a nitrogen atmosphere. Then, 5 ml of anhydrous acetonitrile was added to the tube. At room temperature, propyne p-toluenesulfonate initiator (21 mg, 100 μmol) was added. After the system was thoroughly mixed, the reaction was heated at 130 °C for 12 h. After the reaction was complete, the reaction solution was analyzed by TLC. The developing solvent polarity was PE:EA = 1:1. Using alkaline potassium permanganate for color development, it was found that the starting material was almost exhausted at Rf = 0.6. 5 ml of water was added for post-treatment, and the reaction was quenched by stirring at 80 °C for 12 h. The solvent was then evaporated, and the product was redissolved with DCM. Anhydrous diethyl ether was then added for recrystallization. The product was filtered and dried to obtain 1.5 g (75%) of alkynylated poly-2-ethyl-2-azolin as a white powder. The prepared thermosensitive material was subjected to critical temperature solubility test. It was found that the thermosensitive material has the good characteristics of high temperature precipitation and low temperature dissolution. The effect is shown in Figure 1, and the NMR 1H spectrum of the thermosensitive material is shown in Figure 2.

[0095] 2. Relationship between initiator to monomer ratio (M / I) and critical temperature (LCST)

[0096] Weigh 1.98 g (20 mmol) of monomer 2-ethyl-2-azolinoline into four clean, pressure-resistant tubes. Purge the tubes three times with nitrogen to ensure a nitrogen atmosphere. Add 5 ml of anhydrous acetonitrile to each tube. At room temperature, add propargyl p-toluenesulfonate as the initiator. Add different molar ratios of monomer and initiator to the four pressure-resistant tubes: (M / I = 100, 42 mg, 200 μmol); (M / I = 200, 21 mg, 100 μmol); (M / I = 300, 14 mg, 66 μmol); (M / I = ...100, 42 mg, 200 μmol); (M / I = 100, 42 mg, 200 μmol); (M / I = 100, 21 mg, 100 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); (M / I = 14 mg, 66 μmol); After the system was mixed evenly (I = 500, 8.4 mg, 40 μmol), the reaction was heated at 130 °C for 12 h. After the reaction was completed, the reaction solution was analyzed by TLC. The polarity of the developing solvent was PE:EA = 1:1. It was found by developing with alkaline potassium permanganate that the raw material was basically exhausted at Rf = 0.6. After post-treatment, 5 ml of water was added and stirred at 80 °C for 12 h to quench the reaction. Then, the solvent was evaporated and the product was redissolved with DCM. Subsequently, anhydrous diethyl ether was added for recrystallization, and the product was filtered and dried to obtain white powder solids of alkyne-modified poly(2-ethyl-2-azolinium) with M / I = 100, 200, 300 and 500 respectively.

[0097] To investigate the effect of thermosensitive materials synthesized with different M / I ratios on LCST, materials with M / I ratios of 100, 200, 300, and 500 were dissolved in ultrapure water to a concentration of 10 mg / mL. The LCST was then measured by slowly heating the solution in a water bath. The results showed that the larger the M / I ratio, the lower the critical temperature LCST (as shown in Figure 3).

[0098] 3. Relationship between the concentration of thermosensitive materials and the critical temperature (LCST)

[0099] To investigate the effect of different concentrations of thermosensitive materials on LCST, a solid material with M / I = 200 was dissolved in ultrapure water to obtain concentrations of 1, 5, 10, 20, 30, and 50 mg / mL. The LCST was then measured by slowly heating the material in a water bath. The results, shown in Figure 4, indicate that the higher the concentration of the thermosensitive material, the lower its critical temperature (LCST). Furthermore, the LCST decreased most rapidly in the concentration range of 1-20 mg / mL before stabilizing.

[0100] Based on the above experiments, the material with an M / I ratio of 200 was selected from the thermosensitive materials with M / I ratios of 100, 200, 300, and 500 as the solid phase for subsequent oligosaccharide synthesis because its LCST would not be very high, and its loading capacity was higher than that of materials with M / I ratios of 300 and 500. A concentration of 10 mg / mL (i.e., 0.5 mM) was chosen, as this concentration resulted in a relatively lower LCST for the thermosensitive material and avoided increasing the viscosity of the reaction system due to excessively high material concentrations.

[0101] Example 2

[0102] Synthesis of glucuronide conjugates

[0103] 1. Synthesis methods of azidosaccharides

[0104] Synthesis of compound 2

[0105] 1N-methylhydroxylamine hydrochloride (3.0 g, 35.9 mmol) and K₂CO₃ (9.9 g, 71.6 mmol) were weighed into a clean flask. The sample was thoroughly dissolved in 32 mL of a 1:1 H₂O:THF mixture. The flask was then placed in an ice-water bath, and di-tert-butyl dicarbonate (5.78 mL, 25.2 mmol) was added. The mixture was stirred at room temperature for 20.5 h. After the reaction was complete, a TLC analysis was performed. The developing solvent was PE:EA (1:1). Phosphomolybdic acid was used for colorimetric analysis, and a new spot was observed at approximately 0.8 Rf, indicating complete reaction of the starting materials. The reaction mixture was then post-treated by diluting with ethyl acetate, followed by extraction and separation three times. The mixture was washed three times with water, then rinsed with saturated brine, and finally dried with anhydrous sodium sulfate powder. After concentration by rotary evaporation, silica gel column chromatography was performed (PE:EA = 5:1 to 1:1), and compound 2 was finally obtained as a yellow oily liquid (3.6 g, 96%).

[0106] 1 H NMR (400MHz, CDCl3): δ3.15(s,3H),1.47(s,9H).

[0107] Synthesis of Compound 3

[0108] Compound 2 (890.0 mg, 6.0 mmol) was weighed into a clean flask. Nitrogen gas was purged three times using a double-row tube to ensure the flask was filled with a nitrogen atmosphere. Then, 17 mL of ultra-dry tetrahydrofuran was added to dissolve compound 2. NaH (60% dispersed in mineral oil, 290.0 mg, 7.3 mmol) was added under ice bath conditions. The mixture was stirred under ice bath conditions for 30 min, then 1.0 mL of 3-iodo-1-chloropropane (9.3 mmol) was added, and the mixture was further stirred at room temperature for 4.5 h. After the reaction was complete, the reaction solution was analyzed by TLC. The developing solvent polarity was PE:EA = 10:1. Phosphomolybdic acid colorimetric analysis revealed a new spot at Rf = 0.5, indicating that the starting material was largely consumed. Post-treatment involved adding ice water dropwise to quench any unreacted NaH under ice bath conditions. Then, an appropriate amount of ethyl acetate was added for dilution, followed by extraction and separation three times. The reaction solution was washed three times with water, then rinsed with saturated brine. Finally, the reaction solution was dried with anhydrous sodium sulfate powder. After concentration by rotary evaporation, silica gel column chromatography (PE:EA = 10:1) was performed, and compound 3 was finally obtained as a colorless oily liquid (1.16 g, 85%).

[0109] 1 H NMR (400MHz, CDCl3): δ3.98(t,J=5.8Hz,2H),3.68(t,J=6.2Hz,2H),3.10(s,3H),2.06(q,J=6.2Hz,2H),1.49(s,9H)

[0110] Synthesis of compound 4

[0111] Compound 3 (533.5 mg, 2.4 mmol) was weighed into a pressure-resistant tube and dissolved thoroughly in 10 mL of DMF solution. Then, NaN3 (0.3 g, 4.6 mmol) was added. After stirring evenly, the tube was placed in an oil bath and heated to 95 °C with a magnetic stirrer for 18 h. After the reaction was complete, the reaction solution was analyzed by TLC. The developing solvent was PE:EA = 10:1. A new spot was observed at Rf = 0.6 using phosphomolybdic acid. In post-treatment, unreacted NaN3 was quenched dropwise with ice water in an ice bath. Then, an appropriate amount of ethyl acetate was added for dilution, and the mixture was extracted and separated three times. The reaction solution was washed three times with water and then rinsed with saturated brine. Finally, the reaction solution was dried with anhydrous sodium sulfate powder. After concentration by rotary evaporation, silica gel column chromatography (PE:EA = 10:1) was performed to obtain compound 4 as a colorless oily liquid (501.8 mg, 91%).

[0112] 1 H NMR (400MHz, CDCl3): δ3.89 (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);

[0113] Synthesis of Compound 5

[0114] Compound 4 (230.26 mg, 1 mmol) was weighed into a clean gaiwan 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 h. After the reaction was complete, the reaction solution was analyzed by TLC. The developing solvent was PE:EA = 1:1. Phosphomolybdic acid was used for color development, and a new spot was observed at Rf = 0.2, indicating that the starting material was almost completely consumed. The post-treatment involved adjusting the pH to neutral with saturated sodium bicarbonate solution, followed by extraction and separation three times. The reaction solution was washed three times with water, rinsed with saturated brine, and finally dried with anhydrous sodium sulfate powder. After concentration using a rotary evaporator, silica gel column chromatography (PE:EA = 1:1) was performed, yielding compound 5 as a colorless oily liquid (130.15 mg, 91%). The NMR spectrum of compound 5 is shown in Figure 5.

[0115] 1 H NMR (400MHz, CDCl3) δ3.74(t,J=6.1Hz,2H),3.37(t,J=6.8Hz,2H),2.70(s,3H),1.88–1.81(m,2H),1.34–1.19(m,1H).

[0116] Synthesis of GlcA-N-C3N3

[0117] 75 mg (0.382 mmol) of glucuronic acid (GlcA) was placed in a reaction flask and dissolved in 2 mL of NaOAc buffer solution (0.1 M, pH 4.5). Then, compound 5 (149 mg, 1.14 mmol) was added and the mixture was stirred at room temperature for 41 h. After the reaction was completed, the reaction solution was analyzed by TLC. The polarity of the developing solvent was EA:MeOH:water = 5:2:0.5. The ammonium molybdate colorimetric analysis showed a new spot with a smaller polarity than that of the starting glucuronic acid (GlcA) at an Rf value of about 0.5, which is consistent with the polarity change after the hydroxyl group was substituted. After post-reaction processing, the reaction solution was rapidly frozen in liquid nitrogen and then placed in a freeze dryer to remove water. The crude product was mixed with about 0.5g of silica gel powder and dry-loaded for silica gel column chromatography (DCM:MeOH = 10:1). The final product was a colorless syrup (71mg, 61%) of azide glucuronic acid GlcA-N-C3N3. The ESI spectrum of GlcA-N-C3N3 is shown in Figure 6.

[0118] 1 H NMR(400MHz,D2O)δ4.01(t,J=6.1Hz,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=6.3Hz,2H).

[0119] 2. Synthesis of glucuronic acid conjugates

[0120] The thermosensitive material was selected according to Example 1. 5 g (0.25 mmol) of alkynylated modified poly-2-ethyl-2-azolinium (M / I 200) and azidoglucuronic acid (0.114 g, 0.375 mmol) were weighed and added to a 10 mL mixture of 1 / 1 (v / v) water and ethanol. Subsequently, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added. The reaction solution 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 a small amount of cold diethyl ether was added for recrystallization. After filtration, the white powdery solid was dried in a vacuum drying oven to obtain the glucuronic acid conjugate.

[0121] Example 3

[0122] Synthesis of acetylgalactosyl conjugates

[0123] Synthesis of GalNAc-N-C3N3

[0124] The synthesis steps of the thermosensitive materials and compounds 2-5 were as described in Examples 1 and 2. 85 mg (0.382 mmol) of acetaminophen GalNAc 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 h. After the reaction, the reaction solution was analyzed by TLC. The developing solvent polarity was DCM:MeOH = 3:1. Ammonium molybdate colorimetric analysis revealed a new spot with a smaller polarity than the starting acetaminophen GalNAc at approximately Rf value of 0.8, consistent with the polarity change after hydroxyl substitution. The reaction solution was rapidly frozen with liquid nitrogen and placed in a freeze dryer to remove water. The crude product was mixed with about 0.5 g of silica gel powder and dry-loaded for silica gel column chromatography (DCM:MeOH = 15:1). The final product was a colorless syrup (94 mg 74%), and the ESI spectrum of GalNAc-N-C3N3 is shown in Figure 7.

[0125] 1 H NMR(400MHz,D2O)δ4.12(t,J=8.7Hz,1H),4.09–3.95(m,1H),3.92–3.81(m,2H),3.76–3.54(m,5H),3.36(d t,J=13.5,6.6Hz,2H),3.28(s,2H),2.69–2.63(m,3H),1.97(d,J=2.4Hz,3H),1.79(dt,J=20.8,6.4Hz,2H).

[0126] Synthesis of acetylgalactosyl conjugates

[0127] The thermosensitive material was selected according to Example 1. 5 g (0.25 mmol) of alkynylated modified poly-2-ethyl-2-azolinium (M / I 200) and 0.124 g (0.375 mmol) of acetaminophen (1 / 1) were weighed and added to a 10 mL mixture of 1 / 1 (v / v) water and ethanol. Subsequently, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added. 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 a small amount of cold ether was added for recrystallization. After filtration, the white powdery solid was dried in a vacuum drying oven to obtain the acetaminophen conjugate.

[0128] Example 4

[0129] Galactose conjugate synthesis

[0130] Synthesis of Gal-N-C3N3

[0131] The synthesis steps of the thermosensitive material and compounds 2-5 were as described in 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 h. After the reaction was completed, the reaction solution was analyzed by TLC. The polarity of the developing solvent was DCM:MeOH = 3:1. The ammonium molybdate colorimetric analysis showed that a new spot with a smaller polarity than that of the starting material Gal was generated at an Rf value of about 0.6, which is consistent with the polarity change after the hydroxyl group was substituted. The reaction solution was rapidly frozen with liquid nitrogen and placed in a freeze dryer to remove water. The crude product was mixed with about 0.5 g of silica gel powder and dry-loaded for silica gel column chromatography (DCM:MeOH = 3:1). The final product was a colorless syrup (76.9 mg, 69%). The ESI spectrum of Gal-N-C3N3 is shown in Figure 8.

[0132] 1 H NMR(400MHz,D2O)δ4.17–4.07(m,1H),3.94–3.81(m,2H),3.73(dd,J=12.3,4.7Hz,1H),3 .54–3.45(m,2H),3.38(dq,J=14.9,7.8,7.3Hz,4H),2.74(s,2H),1.84(t,J=6.4Hz,1H).

[0133] Galactose conjugate synthesis

[0134] The thermosensitive material was selected according to Example 1. 5 g (0.25 mmol) of alkynylated modified poly-2-ethyl-2-azolinite (M / I 200) and galactosyl azidophosphate (0.118 g, 0.375 mmol) were weighed and added to a 10 mL mixture of 1 / 1 (v / v) water and ethanol. Subsequently, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added. 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 a small amount of cold diethyl ether was added for recrystallization. After filtration, the white powdery solid was dried in a vacuum drying oven to obtain the galactosyl conjugate.

[0135] Example 5

[0136] Synthesis of lactose conjugates

[0137] Lactose-N-C3N3 Synthesis

[0138] The synthesis steps of the thermosensitive material and compounds 2-5 were as described in Examples 1 and 2. Lactose (140 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 h. After the reaction was completed, the reaction solution was analyzed by TLC. The polarity of the developing solvent was EA:MeOH:water = 6:3:2. The ammonium molybdate colorimetric analysis showed a new spot with a smaller polarity than that of the raw lactose at an Rf value of about 0.6, which is consistent with the polarity change after the hydroxyl group was substituted. The reaction solution was rapidly frozen with liquid nitrogen and placed in a freeze dryer to remove water. The crude product was mixed with about 0.5 g of silica gel powder and dry-loaded for silica gel column chromatography (DCM:MeOH = 5:1). The final result was a colorless syrup of lactose-N-C3N3 (103.2 mg, 59%). The ESI spectrum of lactose-N-C3N3 is shown in Figure 9.

[0139] 1 H NMR (500MHz, D2O): δ4.48(d,J=7.8Hz,1H), 4.18(d,J=9.1Hz,1H), 4.00(dd,J=2.1,12.3Hz,1H), 3.97-3. 73(m,7H),3.72-3.63(m,3H),3.62-3.52(m,3H),3.45(t,J=6.8Hz,2H),2.78(s,3H),1.92-1.84(m,2H).

[0140] Synthesis of lactose conjugates

[0141] The thermosensitive material was selected according to Example 1. 5 g (0.25 mmol) of alkynylated modified poly-2-ethyl-2-azolin (M / I 200) and 0.17 g (0.375 mmol) of azidolactose were weighed and added to a 10 mL mixture of 1 / 1 (v / v) water and ethanol. Subsequently, ascorbic acid (22 mg, 0.125 mmol) and CuSO4·5H2O (18.7 mg, 0.075 mmol) were added. 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 a small amount of cold diethyl ether was added for recrystallization. After filtration, the white powdery solid was dried in a vacuum drying oven to obtain the lactose conjugate.

[0142] Example 6

[0143] Synthesis of chondroitin disaccharide conjugates

[0144] The synthesis of the glucuronic acid conjugate was performed according to Examples 1 and 2. The concentration of the glucuronic acid conjugate was prepared according to Example 1. The glucuronic acid conjugate was prepared to 10 mg / mL (0.5 mM) (at the final concentration) and added to the enzyme reaction system, which included Tris-HCl (20 mM), UDP-GalNAc (1 mM), MnCl2 (10 mM), and KfoC (0.01 mg / mL). After the reaction was carried out at room temperature with shaking (800 rpm) for 4 h, the system was heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the chondroitin disaccharide conjugate. The solution was dissolved in water to prepare 100 mM, and then 0.12% (v / v) TFA was added to the final concentration. The mixture was heated to 90 °C and filtered while hot to separate the sugar from the temperature-sensitive material. The filtrate was freeze-dried to obtain the chondroitin disaccharide compound with a yield of 74%. The ESI spectrum of chondroitin disaccharide-N-C3N3 is shown in Figure 10.

[0145] Example 7

[0146] Synthesis of chondroitin trisaccharide conjugates

[0147] The synthesis of the chondroitin disaccharide conjugate was performed according to Example 6, and the concentration of the saccharide conjugate was prepared according to Example 1. The chondroitin disaccharide conjugate was prepared to a concentration of 10 mg / mL (0.5 mM) and added to the enzyme reaction system, which included Tris-HCl (20 mM), UDP-GlcA (1 mM), MnCl2 (10 mM), and KfoC (0.01 mg / mL). After the reaction was carried out at room temperature with shaking (800 rpm) for 4 h, the system was heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the chondroitin trisaccharide conjugate. The chondroitin trisaccharide conjugate was dissolved in water to prepare a concentration of 100 mM, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C and filtered while hot to separate the sugar from the temperature-sensitive material. The filtrate was freeze-dried to obtain the chondroitin trisaccharide compound with a yield of 80%.

[0148] Example 8

[0149] Synthesis of Lacto-N-triose II (LNT II)

[0150] The synthesis of the lactose conjugate was performed according to Example 5, and the concentration of the conjugate was prepared according to Example 1. A final concentration of 10 mg / mL (0.5 mM) of the lactose conjugate was added to the enzyme reaction system, which included Tris-HCl (20 mM), UDP-GlcNAc (1 mM), MgCl2 (10 mM), and NmLgtA (β1-3N-acetylglucosaminyltransferase from Neisseria meningitides) (0.01 mg / mL). After reacting at room temperature with shaking (800 rpm) for 2 hours (step a), the system was heated to 90°C, resulting in the precipitation of a white solid. The solid was filtered while hot, washed with 90°C hot water, and dried to obtain the LNT II trisaccharide conjugate. The solution was dissolved in water to prepare 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 (step b). The solution was filtered while hot to separate the sugar chains from the thermosensitive material. The filtrate was lyophilized to obtain the LNT II trisaccharide with a yield of 91%. The HR-MS spectrum of the II trisaccharide is shown in Figure 11.

[0151] Example 9

[0152] Synthesis of 2'-Fucosyllactose (2'-FL)

[0153] The synthesis of the lactose conjugate was as described in Example 5. The concentration of the glycoconjugate was prepared according to Example 1. The lactose conjugate (10 mg / mL, approximately 0.5 mM final concentration) was added to the enzyme reaction system, which included 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 with shaking (800 rpm) for 2 h, the system was heated to 90 °C, resulting in the precipitation of a white solid. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the 2'-FL trisaccharide conjugate. This was dissolved in water to prepare a 100 mM solution, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain the 2'-FL trisaccharide, with a yield of 89%.

[0154] Example 10

[0155] Synthesis of 3-Fucosyllactose (3-FL)

[0156] The synthesis of the lactose conjugate was performed according to Example 5, and the concentration of the sugar conjugate was prepared according to Example 1. The lactose conjugate (10 mg / mL, approximately 0.5 mM final concentration) was added to the enzyme reaction system, 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 the reaction was carried out at room temperature with shaking (800 rpm) for 2 h, the system was heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the 3-FL trisaccharide conjugate. The 3-FL trisaccharide was dissolved in water to prepare 100 mM, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain the 3-FL trisaccharide with a yield of 93%.

[0157] Example 11

[0158] Synthesis of 6'-Sialyllactose (6'-SL)

[0159] The synthesis of the lactose conjugate was performed according to Example 5, and the concentration of the lactose conjugate was prepared according to Example 1. The lactose conjugate (10 mg / mL, approximately 0.5 mM final concentration) was added to the enzyme reaction system, 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 the reaction was carried out at room temperature with shaking (800 rpm) for 2 h, the system was heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the 6'-SL trisaccharide conjugate. The solution was dissolved in water to prepare 100 mM, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain 6′-SL, with a yield of 86%.

[0160] Example 12

[0161] Synthesis of Lacto-N-neotetrose (LNnT)

[0162] The synthesis of the LNT II conjugate was performed according to Example 8, and the concentration of the LNT II conjugate was prepared according to Example 1. The LNT II (10 mg / mL, approximately 0.5 mM final concentration) conjugate was added to the enzyme reaction system, 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 the reaction was carried out at room temperature with shaking (800 rpm) for 2 h, the system was heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the LNnT tetrasaccharide conjugate. The conjugate was dissolved in water to prepare 100 mM, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain the LNnT tetrasaccharide, with a total yield of 84%.

[0163] Example 13

[0164] Synthesis of Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc

[0165] The synthesis of the LNnT tetrasaccharide conjugate was carried out according to Example 12. The precipitate was dissolved again, and the concentration of the conjugate was prepared according to Example 1, with a final concentration of 10 mg / mL (0.5 mM) added to a new reaction system, 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 the reaction was carried out at room temperature with shaking (800 rpm) for 2 h, the system was heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the pentasaccharide conjugate. The pentasaccharide conjugate was dissolved in water to prepare 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. The filtrate was lyophilized to obtain Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc, with an overall yield of 79%.

[0166] Example 14

[0167] Synthesis of (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc

[0168] The synthesis of the LNnT tetrasaccharide conjugate was performed according to Example 12. The precipitate was dissolved again, and the concentration of the conjugate was prepared according to Example 1, adding it to a new reaction system at a final concentration of 10 mg / mL (0.5 mM). This included Tris-HCl (20 mM), CMP-Sialic acid (1 mM), MnCl2 (10 mM), and Pd2,6ST (α2-6sialyltransferase from... P. damselaedamsela (0.01 mg / ml) was reacted at room temperature with shaking (800 rpm) for 2 h. The system was then heated to 90 °C, and a white solid precipitated. The mixture was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the hexasaccharide conjugate. The hexasaccharide was dissolved in water to prepare a 100 mM solution, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain (Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc, with 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.

[0169] Example 15

[0170] Synthesis of GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc

[0171] The synthesis of the LNT II conjugate was performed according to Example 8, and the concentration of the conjugate was prepared according to Example 1. The LNT II conjugate (10 mg / mL, approximately 0.5 mM final concentration) was added to the enzyme reaction system, 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) was reacted at room temperature with shaking (800 rpm) for 2 h. The system was then heated to 90 °C, and a white solid precipitated. The mixture was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide conjugate. The mixture was dissolved in water to prepare a 100 mM solution, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide with a total yield of 85%. The HR-MS spectrum of the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide is shown in Figure 13.

[0172] Example 16

[0173] Synthesis of Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc

[0174] The synthesis of the GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide conjugate was performed according to Example 15. The precipitate was dissolved again, and the concentration of the conjugate was prepared according to Example 1, adding it to a new reaction system at a final concentration of 10 mg / mL (0.5 mM). This included Tris-HCl (20 mM), UDP-Gal (1 mM), MnCl2 (10 mM), and NmLgtB (β-4 galactosyltransferase from... N. meningitides (0.01 mg / ml) was reacted at room temperature with shaking (800 rpm) for 2 h. The system was then heated to 90 °C, and a white solid precipitated. The solid was filtered while hot and washed with hot water at 90 °C to obtain the Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc pentasaccharide conjugate. The conjugate was dissolved in water to prepare a 100 mM solution, and then 0.12% (v / v) TFA was added to a final concentration. The mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc, with a total yield of 76%. The HR-MS spectrum of the Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc pentasaccharide is shown in Figure 14.

[0175] Example 17

[0176] Synthesis of (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc

[0177] The synthesis of the Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc pentasaccharide conjugate was performed according to Example 16. The precipitate was dissolved again, and the concentration of the conjugate was prepared according to Example 1, adding it to a new reaction system at a final concentration of 10 mg / mL (0.5 mM). This 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) was reacted at room temperature with shaking (800 rpm) for 2 h. The system was then heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the hexasaccharide conjugate. The hexasaccharide was dissolved in water to prepare a 100 mM solution. Then, 0.12% (v / v) TFA was added to the final concentration, and the mixture was heated to 90 °C to release the sugar chains. The filtrate was freeze-dried to obtain (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc, with an overall yield of 65%. The HR-MS spectrum of the hexasaccharide (Neu5Acα2-3)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc is shown in Figure 15.

[0178] Example 18

[0179] Synthesis of Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc

[0180] The synthesis of the 6'-Sialyllactose (6'-SL) trisaccharide conjugate was performed according to Example 11. The precipitate was dissolved again, and the concentration of the conjugate was prepared according to Example 1, adding it to a new reaction system at a final concentration of 10 mg / mL (0.5 mM). This system 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) was reacted at room temperature with shaking (800 rpm) for 2 h. The system was then heated to 90 °C, and a white solid precipitated. The solid was filtered while hot, and the precipitate was washed with hot water at 90 °C to obtain the tetrasaccharide conjugate. The conjugate was dissolved in water to prepare 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. The filtrate was lyophilized to obtain Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc, with an overall yield of 82%. The HR-MS spectrum of the Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide is shown in Figure 16.

[0181] Example 19

[0182] Synthesis of (Fucα1-2Galβ1-4(Fucα1-3)Glc)

[0183] The synthesis of the 2'-Fucosyllactose (2'-FL) trisaccharide conjugate was performed according to Example 9. The precipitate was dissolved again, and the concentration of the conjugate was prepared according to Example 1, adding it to a new reaction system at a final concentration of 10 mg / mL (0.5 mM). This included Tris-HCl (20 mM), GDP-fucose (1 mM), MnCl2 (10 mM), and Hpα1,3FT (α1,3fucosyltransferase from Helicobacter pylori). The reaction was carried out at room temperature with shaking (800 rpm) for 2 h. The system was then heated to 90 °C, and a white solid precipitated. The solid was filtered while hot and the precipitate was washed with hot water at 90 °C to obtain the tetrasaccharide conjugate. The conjugate was dissolved in water to prepare a 100 mM solution. Then, 0.12% (v / v) TFA was added to the solution and the mixture was heated to 90 °C to release the sugar chains. The filtrate was lyophilized to obtain (Fucα1-2Galβ1-4(Fucα1-3)Glc), with a total yield of 76%. The HR-MS spectrum of the tetrasaccharide (Fucα1-2Galβ1-4(Fucα1-3)Glc) is shown in Figure 17.

Claims

1. A temperature-sensitive material, characterized in that, The thermosensitive material is a type of alkynylated modified 2-ethyl-2-azolino polymer, with the structural formula shown below, where n represents the degree of polymerization of 100-2000:

2. A method for preparing the thermosensitive material according to claim 1, characterized in that, Includes the following steps: 2-Ethyl-2-azolinite as a monomer and initiator are added to the solvent. Under an inert atmosphere, the reaction system is continuously reacted at 100-200℃ for 5-12 hours. After the reaction is completed, the reaction is quenched, the solvent is evaporated, recrystallized, filtered and dried to obtain a white powder solid of alkynylated poly-2-ethyl-2-azolinite, which is the thermosensitive material.

3. A thermosensitive glycoconjugate based on the thermosensitive material of claim 1, characterized in that, The thermosensitive glycoconjugate is formed by combining a thermosensitive material with an azide sugar. The thermosensitive material is an alkynylated 2-ethyl-2-azolino polymer, and the azide sugar has a Sugar-N-C3N3 structure.

4. The thermosensitive glycoconjugate according to claim 3, characterized in that, The thermosensitive sugar conjugate is formed by combining the alkynyl group on the thermosensitive material with the azido group on the azido sugar, thereby achieving the conjugation of sugar and thermosensitive material.

5. The thermosensitive glycoconjugate according to claim 3, characterized in that, The raw material for the azide sugar is a monosaccharide or oligosaccharide, preferably including but not limited to galactose, glucose, acetylglucosamine, acetylglucosamine, or lactose.

6. The thermosensitive glycoconjugate according to claim 3, characterized in that, The azidosaccharide is either a galactose-based monosaccharide or a lactose-based disaccharide, and its structure is shown below:

7. A method for preparing the thermosensitive glycoconjugate according to claim 3, characterized in that, Includes the following steps: A thermosensitive material, azide sugar, ascorbic acid or hydrazine or tris(2-carbonylethyl)phosphohydrochloride, and copper raw material are added to the reaction solvent. The mixture is stirred at room temperature, and after the reaction, it is extracted, concentrated, recrystallized, filtered, and the solid is dried to obtain the thermosensitive glycoconjugate.

8. The method for preparing the thermosensitive glycoconjugate according to claim 7, characterized in that, The azide sugar is prepared as follows: (a) Compound 1, N-methylhydroxylamine hydrochloride, reacts with ditert-butyl dicarbonate (Boc2) to give compound 2 by replacing the hydrogen atom on the nitrogen of N-methylhydroxylamine hydrochloride with tert-butyloxycarbonyl (Boc). (b) Compound 2 reacts with 1-chloro-3-iodopropane, which replaces the hydrogen atom on the hydroxyl group of compound 2, thereby extending the chain and yielding compound 3. (c) Compound 3 reacts with sodium azide, and the chlorine on the propyl group of compound 3 is replaced by the azide group to obtain compound 4; (d) The tert-butoxycarbonyl (Boc) protecting group on compound 4 was removed by trifluoroacetic acid (TFA), and the hydrogen on the nitrogen of compound 4 was released to obtain compound 5; (e) Compound 5 reacts with sugars, using monosaccharides or oligosaccharides as glycosyl donors and compound 5 as a raw material, to obtain azide sugars; The reaction formula is shown below:

9. The application of the thermosensitive glycoconjugate of claim 3 in solid-phase glycosynthesis.

10. The application according to claim 9, characterized in that, The application process is as follows: (1) Mix the glycosyl donor and the glycoconjugate; (2) Add glycosides and shake to react; (3) Heat the reaction solution after shaking in step (2), take the precipitated product and wash it with hot water to obtain disaccharide conjugate or polysaccharide conjugate. Repeating steps (1)(2)(3) can increase the sugar units. (4) Add trifluoroacetic acid to the disaccharide conjugate or polysaccharide conjugate obtained in step (3), then heat and take the clear liquid to obtain the corresponding disaccharide or polysaccharide.

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