A block-type sugar polymer and a preparation method and application thereof

Abstract

The present invention discloses a segmented glycopolymer, its preparation method, and application. The segmented glycopolymer uses commercially available fucose and lactose as raw materials. Through chemical enzymatic synthesis, it can controllably prepare fucose-sialyllactose segmented glycopolymers with varying degrees of polymerization, sugar densities, and linker arm lengths. This segmented glycopolymer can simultaneously act on the important viral proteins hemagglutinin and neuraminidase on the surface of influenza A virus, inhibiting influenza A virus infection through a dual-targeting mechanism. This has significant development and application value in the prevention and treatment of influenza A virus and can be used to develop drug-resistant anti-influenza drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high polymer material synthesis and preparation, and particularly relates to a block type sugar polymer and a preparation method and application thereof. BACKGROUND

[0002] Influenza A virus (IAV) belongs to Orthomyxoviridae, is spherical, and has a diameter of 70-130 nm. Two important viral proteins, Hemagglutinin (HA) and Neuraminidase (NA), are distributed on the surface of the virus, mediate recognition and dissociation with host cells, and are important targets for the development of antiviral drugs. Natural fucoidan sulfate KW can specifically bind to the surface NA protein of influenza A virus, thereby inhibiting the hydrolysis of host cell surface sialic acid and preventing the escape of the virus. Sialic acid oligosaccharide is an active sugar unit with high specificity for binding to HA, and is the best choice for developing HA inhibitors. However, the binding specificity and affinity of sialic acid to HA are not only affected by the structure of sialic acid itself, but also depend on the adjacent sugar units, the connection mode, and the sugar density and other factors. Drug-resistant virus strains of currently marketed anti-influenza A virus drugs such as oseltamivir and zanamivir have frequently appeared. Therefore, it is of great significance to design and develop new anti-IAV drugs targeting the conserved surface of the virus.

[0003] Compared with natural polysaccharides, the preparation of sugar polymers with clear structural characteristics by regulating the conditions of the polymerization process realizes the controllability of the sugar unit density, sugar substitution degree, length of the polymer backbone, and higher structure of the sugar polymer, and opens up a new way for the synthesis of structure-controllable functional sugar polymers. According to the structural characteristics of the polymer, linear sugar polymers are divided into homopolymers and block type sugar polymers. The methods for synthesizing sugar polymers are generally divided into two categories: direct monomer polymerization and pre-synthesis of polymer glycosylation after modification of the polymer, which are generally referred to as "monomer polymerization" and "post-modification polymerization".

[0004] The current disclosed influenza A virus drug targeting type is single target inhibition, and the sugar polymer as the influenza A virus drug is mainly homopolymer, dendritic polymer, etc. Chinese application patent (publication number: CN108530570B) discloses that the fucoidan mimetic with C2 sulfate has good inhibitory effect on H1N1 type IAV virus. Chinese application patent (publication number: CN103880975B) discloses that the natural fucoidan sulfate with α-1, 3 connection has strong inhibitory effect on the neuraminidase activity of influenza A H1N1, H5N1 and H3N2 viruses. Chinese application patent (publication number: CN110312529A) discloses that the sialyllactose dendritic polymer inhibits the infection of influenza virus by combining with hemagglutinin. Through the above analysis, it is proved that fucose as a sugar unit targets neuraminidase to inhibit influenza A virus infection, and sialyllactose as a sugar unit targets hemagglutinin to inhibit influenza A virus infection. And the currently disclosed invention patents are all IAV virus drugs with single target of NA protein or HA protein. At present, there is no related patent of block type sugar polymer as IAV drug, and there is no double-targeted IAV inhibitor that can target NA protein and HA at the same time. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of fucose-sialyllactose block type sugar polymer and its application in double-targeted anti-influenza A virus drugs.

[0006] A block type sugar polymer, its general structure is as follows:

[0007] The block type sugar polymer is composed of a norbornene macromolecular skeleton to form the main chain of the polymer, n=10, 25, 50, 75 or 100; Linker is the connecting arm part between the polymer skeleton and the sugar unit, which is divided into three structures of Linker 1, 2 and 3, and m=4 or 24 in the Linker 2; Sugar is the sugar-containing part, which is fucose or sialyllactose, and constitutes the side chain of the polymer.

[0008] The block type sugar polymer includes two structural types: one is a single block sugar polymer, that is, two units of the Sugar part, one of which is only one of fucose or sialyllactose unit, and the other is not modified by a free carboxyl group; the other structural type is a double block sugar polymer, that is, the two units of the Sugar part are both sugar units, which are composed of fucose and sialyllactose.

[0009] The Linker 1 (shorter connecting arm) connects the mono- or bi-blocked sugar polymer; Linker 2 (longer connecting arm) is a connecting arm composed of a triazole heterocycle and polyethylene glycol with different polymerization degrees, wherein m = 4 or 24, connecting mono- or bi-blocked sugar polymer, when the selected sugar unit is sialylated lactose, m = 4, when the selected sugar unit is fucose, m = 24; when the type of sugar polymer is only a mono-blocked sugar polymer, the Sugar part only has one sugar unit connected, and the part without sugar unit modification is a free carboxyl group, that is, Linker 3.

[0010] The application relates to a preparation method of a block type sugar polymer, which is prepared according to the following steps: (1) dissolving non-protecting group or full-sulfated azidoethyl fucose in a methanol solvent, taking carbon-loaded palladium hydroxide as a catalyst, reducing an azido group into an amino group under a hydrogen atmosphere, and performing reaction at room temperature for 0.5-1.5 h; after the reaction is completed, diatomite is used for filtration, filtration is performed, and steam drying is performed, LH-20 gel column purification is performed, and non-sulfated or full-sulfated amino fucose monomers with a short connecting arm are prepared; dissolving non-protecting group or full-sulfated azidoethyl fucose and alkynyl-amino modified 24-polymerization-degree polyethylene glycol chains in a THF / H2O reaction system, taking copper sulfate pentahydrate and sodium ascorbate as catalysts, adding a copper ion stabilizer TBTA, performing a copper-catalyzed azido-alkynyl Click reaction under a nitrogen protection atmosphere, and performing reaction at 40-60 DEG C for 3-5 h; after the reaction is completed, methanol is used for washing and filtration, vacuum concentration is performed, and LH-20 gel column purification is performed, and non-sulfated or full-sulfated amino fucose monomers with a long connecting arm are prepared; (2) full-acetyl azidoethyl lactose is dissolved in a methanol / sodium methoxide reaction system, acetyl groups are removed by adjusting pH to 9-10, non-protecting group azidoethyl lactose intermediates are obtained; the intermediates are dissolved in a water reaction solvent, carbon-loaded palladium hydroxide is used as a catalyst, an azido group is reduced into an amino group under a hydrogen atmosphere, reaction is performed at room temperature for 0.5-1.5 h, diatomite is used for filtration, filtration is performed, and steam drying is performed, and LH-20 gel column purification is performed, and amino lactose monomers with a short connecting arm are prepared; full-acetyl azidoethyl lactose and alkynyl-amino modified 4-polymerization-degree polyethylene glycol chains are dissolved in a THF / H2O reaction system, copper sulfate pentahydrate and sodium ascorbate are used as catalysts, a copper ion stabilizer TBTA is added, a copper-catalyzed azido-alkynyl Click reaction is performed under a nitrogen protection atmosphere, reaction is performed at 40-60 DEG C for 3-5 h; after the reaction is completed, methanol is used for washing and filtration, vacuum concentration is performed, and LH-20 gel column purification is performed, and full-acetyl amino lactose intermediates with a long connecting arm are prepared, acetyl groups of the intermediates are removed in a methanol / sodium methoxide reaction system, and amino lactose monomers with a long connecting arm are prepared; (3) a monomer containing an NHS ester norbornene is dissolved in dichloromethane as a reaction solvent, an olefin hydrogen metathesis ring-opening polymerization reaction is performed, a third-generation Grubbs catalyst (G3 rd) solution, and the reaction was continued at room temperature. After the reaction was completed, the reaction was terminated by adding vinyl ethyl ether, and the reaction mixture was concentrated under reduced pressure. The NHS ester modified norbornene macromolecular polymer backbone was prepared by alcohol precipitation with five times the amount of diethyl ether and centrifugation. (4) The shorter / longer arm non-sulfated or fully sulfated amino fucose monomer of step (1) was acylated with the NHS ester modified norbornene macromolecular polymer backbone of step (3) in 5 / 1 DMF / H2O as the reaction solvent, under the catalysis of weak base triethylamine, at room temperature, for 4-6 h. After the reaction was completed, the reaction mixture was dialyzed with 16% sodium chloride solution and lyophilized to obtain the shorter arm non-sulfated or fully sulfated fucose polymer and the longer arm non-sulfated or fully sulfated fucose polymer. (5) The amino lactose monomer of step (2) was acylated with the NHS ester modified norbornene macromolecular polymer backbone of step (3) by the same method as step (4) to obtain the single-block lactose polymer, i.e., the shorter arm lactose polymer and the longer arm lactose polymer. (6) The amino fucose monomer of step (1) and the amino lactose monomer of step (2) were acylated with the NHS ester modified norbornene macromolecular polymer backbone of step (3) by the same method as step (4) to obtain the double-block fucose-lactose polymer, i.e., the shorter arm non-sulfated or fully sulfated fucose-lactose polymer and the longer arm non-sulfated or fully sulfated fucose-lactose polymer. (7) The single-block lactose polymer of step (5) and the double-block fucose-lactose polymer of step (6) were reacted with CMP-Neu5Ac as the sugar donor, Tris-HCl and MgCl2, 1M NaOH to adjust the pH to 8.0-8.5, H2O, NmCSS, at 37°C, and then the pH was adjusted to weak alkalinity. Pd2,6ST was added as the sialic acid transferase to perform the directional modification of sialic acid at the 6 position of galactose. After the reaction was completed, the enzyme was inactivated by adding an equal volume of ice ethanol, and the mixture was left to stand at -20°C for 20-40 min. The supernatant was obtained by centrifugation, concentrated, and purified by G10 column to obtain the fucose-sialic acid lactose block polymer, i.e., the shorter arm sialic acid lactose polymer and the longer arm sialic acid lactose polymer.

[0011] The polymerization degree ([M] / [C]) of the NHS ester modified norbornene macromolecular polymer backbone of step (3) was 10, 25, 50, 75, or 100.

[0012] The post-modification polymerization method is to construct a sugar monomer containing a specific functional group, and then connect the sugar monomer with a macromolecular polymerization backbone prepared by an olefin hydrogen metathesis ring-opening polymerization reaction through a predetermined reaction, thereby preparing a sugar polymer. By adjusting the ratio of the sugar monomer to the polymerization backbone, the size of the sugar density (DS) is controlled, i.e. a block type sugar polymer with different sugar densities is prepared.

[0013] The reaction conditions of the post-modification polymerization, i.e. the solvent, the reaction temperature and the amount of triethylamine, all affect the effect of the polymerization reaction. The optimal preparation conditions are: the solvent is DMF:H2O at 5:1, the reaction temperature is room temperature, and the amount of triethylamine is 6 equivalents (relative to the polymerization backbone).

[0014] In the fucose-sialyllactose block type polymer, the molar ratio of fucose to sialyllactose is 1:6 or 1:1.

[0015] The single-block fucose polymer, the single-block sialyllactose polymer and the fucose-sialyllactose block type sugar polymer are all amphiphilic structures, including a hydrophilic sugar part and a hydrophobic polynorbornene part. The amphiphilic polymer self-assembles in an aqueous solution to form spherical nanoparticles.

[0016] The block type sugar polymer is used in the preparation of a medicine for inhibiting influenza A virus infection.

[0017] The block type sugar polymer can simultaneously target NA and HA. Fucose sulfate and sialyllactose are respectively targeted at the important target proteins, neuraminidase (NA) and hemagglutinin (HA), on the surface of influenza A virus. Through hemagglutination inhibition (HI) tests and neuraminidase inhibition experiments, it is confirmed that the single-block fucose sulfate polymer and the single-block sialyllactose polymer have targeting specificity and act on the NA and HA target proteins, respectively; the double-block fucose-sialyllactose polymer can simultaneously target NA and HA and exert a double-targeting mechanism. The protein distance between the target proteins, neuraminidase and hemagglutinin, on the surface of the virus is about 14 nm, so the polynorbornene backbone polymerization degree (DP) is designed to be 10, 25, 50, 75 and 100, and the optimal theoretical degree is 25, so as to bridge more target proteins.

[0018] The length of the neuraminidase and hemagglutinin is 6.0 nm and 13.5 nm respectively, and the length difference of the target protein is 7.5 nm, which is matched with the length of the NA and HA protein for better chelation. Therefore, the connecting arm connected with the sulfated fucose is a connecting arm (Linker 2, m = 24) constructed by connecting triazole heterocycle with 24 polymerization degrees of polyethylene glycol, and a shorter connecting arm of Linker 1; the connecting arm connected with sialyl lactose is a connecting arm (Linker 2, m = 4) constructed by connecting triazole with 4 polymerization degrees of polyethylene glycol, and a shorter connecting arm of Linker 1.

[0019] Preferably, the polymerization degree of the block type sugar polymer is 25, the sugar density is 70%, the connecting arm is Linker 1, the fucose has a sulfate group modification, and the molar ratio of sulfated fucose to sialyl lactose is 1:1, which can exert the best effect of inhibiting IAV virus infection.

[0020] The present application has the following beneficial effects: The present application adopts the "post-modification polymerization" strategy, can regulate the polymerization degree, the length of the connecting arm, the sugar density and other structural characteristics of the block type polymer, and increases the diversity of the structure of the block type sugar polymer. The reaction operation is relatively simple, and the yield is high. The fucose-sialyl lactose block type sugar polymer designed in the present application for anti-influenza A virus drug can simultaneously act on the neuraminidase and hemagglutinin two viral target proteins, realizes the double-targeting inhibition effect, simultaneously plays an inhibitory effect in the process of blocking the virus entry and release, and greatly increases the efficacy of preventing and treating influenza A virus. The fucose-sialyl lactose block type sugar polymer described in the present application is a drug-resistant double-targeting influenza A virus inhibitor. Avoids the drug-resistant problem of the marketed zanamivir and sialic acid to the virus strain. The structure of the fucose-sialyl lactose block type sugar polymer designed in the present application is bionic to the structure of the influenza A virus itself and the important target proteins hemagglutinin and neuraminidase, which is a "virus-like" nano inhibitor. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a preparation route map of the fucose-sialyl lactose block type sugar polymer. Figure 2 It is a nuclear magnetic hydrogen spectrum diagram of a representative block type sugar polymer in the present application; in the diagram, the horizontal coordinate f1 represents the chemical shift (ppm). Figure 3 It is a high-level structure characterization diagram of the fucose-sialyl lactose block type sugar polymer. Figure 4 It is a graph of the fucose-sialyl lactose block type sugar polymer acting on the hemagglutination inhibition of influenza A virus. Figure 5 It is a graph of the fucose-sialyl lactose block type sugar polymer acting on the neuraminidase inhibition of influenza A virus. Figure 6A graph of the CPE inhibition rate of fucose-sialylated lactose block type sugar polymers against influenza A virus. DETAILED DESCRIPTION

[0022] For the purpose of promoting the understanding of the present application, a more comprehensive description will be given below. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided for the purpose of making the disclosure of the present application more thorough and comprehensive.

[0023] Example 1 Preparation of fucose monomer

[0024] The fucose monomer is in the α configuration and is divided into two modes of sulfation modification, i.e. non-sulfated and fully sulfated at the 2, 3 and 4 positions, and two lengths of the connecting arm, i.e. shorter connecting arm and longer connecting arm modified by PEG chain. That is, the fucose monomer is divided into four types: fucose monomer with shorter connecting arm and non-sulfated or fully sulfated, and fucose monomer with longer connecting arm and non-sulfated or fully sulfated. The synthetic reaction route is shown in Figure 1 a, and the preparation method comprises the following steps:

[0025] (1) Weigh 10.2 g (62.1 mmol) of L-fucose in a 250 mL round-bottom flask, add 46.6 mL (0.621 mol) of 2-azidoethanol and 9.0 g of hydrogen-type cationic dendron, and reflux at 80°C for 24 h under stirring. The reaction is monitored by TLC. After the reaction is completed, the reaction solution is returned to room temperature, filtered, and evaporated to dryness to obtain an α / β-configuration unprotected azidoethylfucose derivative. This derivative is placed in a 500 mL round-bottom flask, dissolved in 100 mL of pyridine, and 50 mL of acetic anhydride is added to react at room temperature. The reaction is monitored by TLC. After the reaction is completed, the reaction solution is concentrated under reduced pressure, extracted twice with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous sulfuric acid, filtered, evaporated, and then chromatographed on a silica gel column to obtain an α-configuration fully acetylazidoethylfucose derivative. 6.3 g of α-configuration peracetylated fucose was weighed into a 250 mL round-bottom reaction flask and dissolved in 100 mL of methanol. The pH was adjusted to 9.5 with freshly prepared saturated sodium methoxide and allowed to react at room temperature for 2 h. After completion of the reaction, the mixture was neutralized with a hydrogen-type cationic resin, filtered, and evaporated to dryness to obtain the α-configuration unprotected azidoethylfucose. 3.2 g (13.7 mmol) of this unprotected azidoethylfucose was weighed into a round-bottom flask and dissolved in 6 mL of DMF. 111.9 g (617.4 mmol) of the sulfation reagent sulfur trioxide × triethylamine was added and the mixture was heated at 70°C with stirring overnight. After completion of the reaction, the mixture was returned to room temperature, cooled to 0°C, and neutralized with saturated sodium bicarbonate solution for 30 min. The mixture was concentrated under reduced pressure and purified using an LH-20 gel column to obtain the fully sulfated azidoethylfucose derivative. (2) Weigh 1.8 g of the unprotected or fully sulfated azidoethylfucose derivative in step (1) of Example 1 into a round-bottom flask, add methanol to dissolve it, add 10.8 g of carbon-supported palladium hydroxide (6 times the volume of the sugar derivative), and react at room temperature under a hydrogen atmosphere. After the reaction is completed by TLC detection, filter with diatomaceous earth as the auxiliary filter, concentrate under reduced pressure, and purify with an LH-20 gel column to obtain an aminofucose monomer with a shorter linker arm without sulfate or fully sulfated.

[0026] Take 1.0 g (4.3 mmol) of the no-protecting group or full-sulfated azidoethyl fucose derivative in step (1) of Example 1 and 4.8 g (4.3 mmol) of the 24-mer PEG chain modified with an alkynyl-amino group in a round-bottom flask, dissolve in 20 mL of DMF, prepare a CuSO4x5H2O-TBTA complex solution by adding 214.1 mg (0.9 mmol, 26.7 mg / mL) of CuSO4x5H2O and 455.1 mg (0.9 mmol, 88.7 mg / mL) of TBTA in distilled water, add the complex solution to the reaction solution after 10 min of stabilization, then add a 302.1 mg (1.7 mmol, 66.7 mL) aqueous solution of sodium ascorbate, adjust the volume ratio of the DMF:H2O reaction system to 1:1 with deionized water, protect under nitrogen, and react at room temperature. After the reaction is completed as detected by TLC, concentrate under reduced pressure, and purify by LH-20 gel column to obtain the longer-armed no-sulfated or full-sulfated amino fucose monomer.

[0027] Preparation of lactose monomer in Example 2

[0028] The lactose polymer of the present application is in the β configuration and has two lengths of connecting arms, i.e., a shorter connecting arm and a longer connecting arm modified with a PEG chain. That is, it is divided into two types, i.e., a shorter connecting arm lactose monomer and a longer connecting arm lactose monomer. The synthetic reaction route is shown in FIG. b, and the preparation method includes the following steps: Figure 1

[0029] ​(1) Take D-lactose 6.5 g (18.0 mmol) in a 500 mL round-bottom flask, add 200 mL of dry pyridine, dissolve, add 100 mL of acetic anhydride, 4-dimethylaminopyridine 440.8 mg (3.6 mmol), stir at 45°C in an oil bath for 5 h, after TLC detection is completed, restore to room temperature, and the reaction solution is concentrated under reduced pressure, sequentially extracted with 1 M hydrochloric acid, saturated sodium bicarbonate solution, saturated sodium chloride solution, each 2 times, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness, to obtain a full acetyl lactose derivative mainly in β configuration. Take 5.8 g (8.6 mmol) of the full acetyl lactose derivative in a 250 mL round-bottom reaction flask, add 58 mL of tetrahydrofuran, dissolve, add 2.0 mL (17.1 mmol) of benzylamine, and react at room temperature for 2 h. After TLC detection shows that the reaction is completed, sequentially extract with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution, each 2 times, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain a lactose intermediate with selective removal of the anomeric acetyl group in β configuration. Take 4.2 g (6.6 mmol) of the lactose intermediate in a 100 mL round-bottom reaction flask, dissolve in 12 mL of dry dichloromethane, exchange with nitrogen, and add DBU 455.8 µL (3.3 mmol) and trichloroacetonitrile 8.4 mL (46.2 mmol) under nitrogen protection. Stir at room temperature for 5 h. After TLC detection shows that the reaction is completed, concentrate under reduced pressure, and purify by silica gel column to obtain an α configuration anomeric trichloroacetyl imine modified lactose derivative. Take 3.3 g (4.2 mmol) of the lactose derivative in a 50 mL round-bottom reaction flask, dissolve in 8.0 mL of dry dichloromethane, add dry molecular sieves, exchange with nitrogen, add 2-azidoethanol 482.3 µL (6.3 mmol), stir at room temperature for 30 min, cool the reaction solution to -30°C, add TMSOTf 1.1 mL (6.3 mmol), and stir at -30°C under nitrogen protection for 1 h. After TLC detection shows that the reaction is completed, restore to room temperature, neutralize by adding triethylamine, filter, concentrate under reduced pressure, and purify by silica gel column to obtain a β configuration full acetyl azidoethyl lactose derivative. Take 1.5 g of the lactose derivative in a 25 mL round-bottom reaction flask, add 5 mL of methanol, adjust the pH to 9-10 with freshly prepared saturated sodium methoxide, stir at room temperature for 1 h, after TLC detection shows that the reaction is completed, neutralize by adding a hydrogen cation resin, filter, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain an azidoethyl modified lactose derivative.The lactose derivative 780.4 mg was weighed into a 25 mL reaction flask, 2.0 mL of deionized water was added, 4.72 g of carbon-supported palladium hydroxide (6 times the amount of lactose derivative by weight) was added, the reaction solution was bubbled with hydrogen, and the reaction was stirred at room temperature for 5 h. The reaction solution was filtered with diatomite as a filter aid, and purified by LH-20 gel column to obtain the amino lactose monomer, i.e. the lactose monomer with a shorter connecting arm.

[0030] Preparation of the NHS ester modified poly norbornene skeleton

[0031] The synthetic reaction route is as follows Figure 1c, the preparation method specifically comprises the following steps: (1) 1.0 g of norbornene diacid anhydride (6.1 mmol) is weighed into a 100 mL round-bottom reaction bottle, dissolved in 32 mL of toluene, 6-amino hexanoic acid 2.4 g (18.3 mmol) and triethylamine 8.5 mL (60.9 mmol) are added, and the reaction is heated to reflux at 120°C for 5.5 h in an oil bath. After the reaction solution is restored to room temperature, it is concentrated under reduced pressure, and then extracted with 1M hydrochloric acid and saturated sodium chloride solution twice, respectively. After drying with anhydrous sulfuric acid, filtering, evaporating to dryness and separating and purifying by silica gel column chromatography, a norbornene derivative is obtained. 1.6 g of the derivative is weighed into a 50 mL round-bottom reaction bottle, dissolved in 16 mL of dichloromethane, and then EDS 1.6 g (8.1 mmol) and NHS 936.3 mg (8.1 mmol) are added. The reaction is carried out at room temperature under stirring. After the reaction is completed as detected by TLC, the norbornene monomer derivative containing NHS ester is obtained by separation and purification by silica gel column. rd A solution of 14.2 mg x mL of DCM is freshly prepared. -1 The amount of catalyst is calculated according to the ratio of sugar monomer to catalyst, i.e. [M] / [C] is 25, 75, and the catalyst is added at -78°C. After the reaction is carried out at this temperature for 20 min in the dark, the reaction solution is restored to room temperature and reacted for 1 h. After the reaction is completed as detected by TLC, the reaction is terminated by adding vinyl ether and stirring for 30 min. Then, the reaction solution is concentrated under reduced pressure, and the polymerization degree of the NHS ester modified norbornene polymer skeleton is 25, 75.

[0032] Preparation of a monoblock fucose polymer

[0033] The synthesis reaction route is shown in Figure 1 d, the preparation method specifically comprises the following steps: (1) 12.0 mg of the shorter arm amino fucose monomer without sulfuric acid or fully sulfuric acid in step (1) of Example 1 is dissolved in 297.6 µL of deionized water, and 18.6 mg of the NHS ester containing poly-norbornene skeleton in step 2) of Example 3 (0.048 mmol, 12.5 mg x mL -1) was dissolved in DMF 1.49 mL and added to the aqueous solution containing the sugar monomers, and triethylamine 40 μL (0.29 mmol) was added, and the reaction was stirred at room temperature for 5 h. After the reaction was completed, the product was dialyzed against 16% sodium chloride solution and lyophilized to obtain the shorter-armed non-sulfated or fully sulfated fucose polymer.

[0034] Preparation of a single-block lactose polymer

[0035] The synthetic reaction route is shown in Figure 1 d, and the preparation method specifically includes the following steps: (1) 20.5 mg (0.052 mmol) of the shorter-armed lactose monomer in step (1) of Example 2 was dissolved in 328 μL of deionized water, and 20.5 mg (0.052 mmol, 12.5 mg x mL -1 ) was dissolved in DMF 1.64 mL and added to the aqueous solution containing the lactose monomers, and triethylamine 44.3 μL (0.32 mmol) was added to the reaction solution, and the reaction was stirred at room temperature for 5 h. After the reaction was completed, the product was dialyzed against 16% sodium chloride solution and lyophilized to obtain the shorter-armed lactose polymer. (2) The longer-armed lactose monomer in step (2) of Example 2 was used together with the NHS ester-containing polynorbornene skeleton in step (2) of Example 3 to prepare a longer-armed lactose polymer using the same post-modification polymerization method as described above.

[0036] Preparation of a fucose-lactose block type sugar polymer

[0037] The synthetic reaction route is shown in Figure 1 d, and the preparation method specifically includes the following steps: (1) 20.5 mg (0.052 mmol) of the shorter-armed lactose monomer in step (1) of Example 2 was dissolved in 328 μL of deionized water, and 20.5 mg (0.052 mmol, 12.5 mg x mL -1) was dissolved in 800 μL DMF and added to an aqueous solution containing two sugar monomers. 21.8 μL (0.16 mmol) of triethylamine was added to the reaction solution and stirred at room temperature for 5 h. After the reaction was completed, it was dialyzed with 16% sodium chloride solution and freeze-dried to obtain a shorter linker arm non-sulfated or fully sulfated fucose-lactose block type sugar polymer. (2) The longer linker arm non-sulfated or fully sulfated fucose monomer in step (2) of Example 1, the longer linker arm lactose monomer in step (2) of Example 2, and the NHS ester-containing polynorbornene skeleton in step (2) of Example 3 were weighed, and the same post-modification polymerization method as above was used to prepare a longer linker arm non-sulfated or fully sulfated fucose-lactose block type polymer.

[0038] Example 7 Preparation of Fucose-Sialyl Lactose Block Glycopolymer

[0039] The synthetic reaction route is as follows Figure 1 As shown in Figure d, the preparation method specifically includes the following steps: Using a chemoenzymatic synthesis technique, 600 mg of the glycosyl donor CMP-Neu5Ac was weighed into a centrifuge tube, 1.535 g of CTP, 605.7 mg of Tris-HCl, and 203.3 mg of MgCl2 were added, and the mixture was dissolved in deionized water. The pH was adjusted to 8.2 with 1 M NaOH, 5 mL of NmCSS was added, and the mixture was filled to 50 mL with H2O. The reaction was incubated at 37°C for 1 hour. After TLC confirmed the formation of CMP-Sia, the pH was again adjusted to a weak base with 1 M NaOH. The reaction solution containing CMP-Sia was then proportionally added to the shorter linker / longer linker lactose polymer of Example 5 and the shorter linker / longer linker non-sulfated or fully sulfated fucose-lactose block glycopolymer of Example 6. Sialyltransferase Pd2,6ST was then added, and the reaction was incubated at 37°C overnight. After the reaction is complete, the enzyme is inactivated with an equal volume of icy ethanol. After standing at -20°C for 30 min, the supernatant is centrifuged, concentrated, and purified using a G10 column. Sialyl lactose polymers with shorter linkers / longer linkers, and glycopolymers with shorter linkers / longer linkers containing either no or fully sulfated fucose sialyllactose are prepared.

[0040] Example 8 Structural Characterization of Block Glycopolymers

[0041] The structural characterization of the block glycopolymer specifically comprises the following steps:

[0042] (1) Nuclear magnetic resonance analysis. Weigh 5 mg of each block polymer, add 500 μL of heavy water, freeze-dry, repeat the heavy water exchange three times, add 500 μL of heavy water and transfer to an NMR tube, and analyze with a 500 MHz NMR spectrometer. Figure 2As shown, the proton signal of the norbornene cyclic olefin (δ6.31 ppm) was shifted to the low field to δ5.20-5.90 ppm, which was the main chain olefinic proton of the polymer, proving the success of the polymerization reaction. The anomeric signals of the monoblock fucose polymer and the monoblock lactose polymer were also observed in the difucosyl-lactose block polymer, proving the success of the difucosyl-lactose block polymer preparation reaction. In addition, in the fucosyl-sialyl-lactose block glycopolymer, the signal of the dd peak of the protons of the flanking bond appeared at 2.6 ppm, the signal of the characteristic peak of the acetyl protons of the aminoacetyl group of the sialic acid five-position appeared at 1.88 ppm, and the signal of the t peak of the protons of the sialic acid three-position appeared at 1.62 ppm, proving the success of the sialic acid modification.

[0043] (2) High-level structure characterization. The high-level structure of the fucosyl-sialyl-lactose block glycopolymer was characterized by transmission electron microscopy (TEM). Specifically, the block glycopolymer was dispersed in water and configured to 1.0 mg x mL -1 , and dropped on a hydrophilic copper grid loaded with a copper mesh, and negatively stained with 2.0% phosphotungstic acid. After drying, imaging was performed by transmission electron microscopy (TEM). As shown in Figure 3 , the high-level nanostructure of the fucosyl-sialyl-lactose block glycopolymer was spherical nanoparticles.

[0044] Example 9 Inhibition of influenza A virus infection by fucosyl-sialyl-lactose block glycopolymer through dual targeting

[0045] (1) The effect of the block glycopolymer on the inhibition of IAV by targeting HA was studied by hemagglutination inhibition (HI) assay. In the 96-well plate, 50 μL of phosphate buffer solution was added to the first row, and 25 μL of phosphate buffer solution was added to each well of the 96-well plate except for the second row. The second row was injected with each glycopolymer solution (200 μg x mL -1 , 50 μL), and 25 μL of the solution in the first well was transferred to the second well for mixing and two-fold serial dilution. Each well was injected with 25 μL of influenza virus solution [A / Aichi / 2 / 1968 (H3N2), 4 HAU], and the 96-well plate was incubated at 37°C for 1 h. After gently shaking, 4% chicken red blood cells were diluted to 1%, and 100 μL was injected into each well, and the 96-well plate was incubated at 4°C (n = 3) for 1 h. The sedimentation of red blood cells was observed with the naked eye. The red blood cells of the negative group were deposited and formed a red punctate morphology, while the positive wells had an opaque appearance without sedimentation. The hemagglutinin titer was expressed in hemagglutination units / mL (HAU / mL). The inhibitor constant Ki (HAI) reflects the minimum inhibitor concentration necessary to completely inhibit virus-induced hemagglutination. As shown in Figure 4as shown, the mono-blocked fucose polymer had no HA inhibitory effect, while the mono-blocked shorter linker sialyllactose polymer (Ki of 3.125 μg x mL -1 ) and the di-blocked shorter linker fucose-sialyllactose polymer (Ki of 6.25 μg x mL -1 ) had the effect of targeting HA to inhibit IAV.

[0046] (2) The NA activity was studied by using 2'-(4-methylumbelliferyl)-α-D-Neu5Ac (MU-NANA) assay, i.e. neuraminidase inhibition assay, to analyze the effect of the block-type sugar polymer on inhibiting IAV by targeting NA. 30 μL of virus solution (1 x 10 6 PFU / mL) was incubated with 10 μL of the block-type sugar polymer in reaction buffer (150 mM sodium acetate buffer, 1 mM CaCl2, pH 7.0) at a final concentration of 200 μg x mL -1 at 37 °C for 60 min. 10 μL of MU-NANA (160 μM, reaction buffer) was added to the virus / sugar polymer mixture, which was incubated at 37 °C for 45 min in the dark. The reaction was stopped by adding a stop solution (25% ethanol, 0.1 mol L glycine, pH = 10.7). The fluorescence intensity was detected using a microplate reader, and the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 440 nm was read to determine the activity of NA. The experimental settings included a blank control group without the addition of virus, a negative control group with the addition of virus only, and a positive drug zanamivir control group. As shown in Figure 5 , the mono-blocked sialyllactose polymer had no NA inhibitory activity, while the shorter linker mono-blocked sulfated fucose polymer (NA inhibition rate of 44.6%) and the shorter linker di-blocked sulfated fucose-sialyllactose polymer (NA inhibition rate of 47%) had the effect of targeting NA to inhibit IAV.

[0047] (3) The anti-IAV viral activity of the block-type sugar polymer was evaluated by cytopathic effect (CPE) inhibition assay. MDCK cells were uniformly plated in a 96-well plate, and after they formed a monolayer, 200 μg x mL -1The sugar polymer inhibitors at different concentrations were used to treat the four processes of virus adsorption into the cells (pretreatment of cells, pretreatment of viruses, administration during adsorption, and administration after adsorption). Pretreatment of cells: the sugar polymer inhibitor was first allowed to act on the cells for 1 h, and then the virus was adsorbed for 1 h before being replaced with a maintenance solution; pretreatment of viruses: the sugar polymer inhibitor was incubated with the virus for 1 h, and then the treated virus was adsorbed for 1 h before being replaced with a maintenance solution; administration during adsorption: the sugar polymer inhibitor was added at the same time as the virus was adsorbed, and then the solution was replaced with a maintenance solution after 1 h; administration after adsorption: the virus was adsorbed for 1 h, and then the solution was replaced with a maintenance solution containing the sugar polymer inhibitor. When the virus group developed lesions to 80%-90%, the supernatant was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 20 min. After the formaldehyde was removed, the cells were stained with 0.1% (w / v) crystal violet at 37°C for 30 min. After being washed with water and dried, the OD value was measured at a wavelength of 570 nm using an enzyme-labeled instrument, and the IC50 was calculated according to the cell survival rate. Cell viability value (%) = (OD value of the experimental group-OD value of the virus group / OD value of the blank group-OD value of the virus group) x 100%. As shown in FIG. 1, the sugar polymer inhibitor with a degree of polymerization of 25, a short connecting arm, a sugar density of 70%, and fucose modified with a sulfate group and a sulfate fucose: sialyllactose ratio of 1:1 can exert the best inhibitory effect on IAV virus infection. Figure 6

[0048] According to the analysis of the HA inhibition, NA inhibition, and virus inhibition rate of the block-type sugar polymer described in Example 9, it can be found that the single block sialyllactose has a single targeting effect on the HA protein, the single block sulfated fucose polymer has a single targeting effect on the NA protein, and the sulfate fucose-sialyllactose polymer can simultaneously target the HA and NA proteins, exert a double-targeting mechanism, and inhibit the infection of the H3N2 type influenza A virus, thereby having the potential to develop a new type of influenza A virus drug for prevention and treatment.

[0049] In summary, the block-type sugar polymers with different structural characteristics, such as different lengths of connecting arms, sulfation sites, sugar densities, and degrees of polymerization, are successfully prepared. Taking fucose and sialyllactose as examples, a total of 4 single block fucose polymers, 2 single block sialyllactose polymers, and 4 double block fucose-sialyllactose polymers are prepared. The preparation method of the block-type sugar polymer described in the present application is not limited to fucose and sialyllactose, but can also be applied to the preparation of other oligosaccharide block-type sugar polymers, and enriches the structure of the block-type sugar polymer. The fucose-sialyllactose block-type sugar polymer described in the present application can simultaneously act on the important target proteins hemagglutinin and neuraminidase on the surface of the influenza A virus, exert a double-targeting mechanism to inhibit the infection of the influenza A virus, and avoid the drug resistance problem of some virus strains to zanamivir and oseltamivir. The fucose-sialyllactose block-type sugar polymer has the potential and value to develop a new type of double-targeting drug-resistant influenza A virus drug. ​

[0050] The above embodiments only express several implementation manners of the present application, which are described in a more specific and detailed manner, but cannot be understood as a limitation on the patent scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A block sugar polymer, characterized in that Its general structural formula is shown below: ， The block sugar polymer consists of a norbornene macromolecular skeleton forming the main chain of the polymer, with n = 10, 25, 50, 75 or 100; the linker is the connecting arm part between the polymer backbone and the sugar unit, which is divided into three structures: Linker 1, 2 or 3, and m = 4 or 24 in the Linker 2; the sugar is the sugar-containing part, which is fucose or sialyllactose, and constitutes the side chain of the polymer.

2. The block sugar polymer according to claim 1, characterized in that The block glycopolymers include two structural types: one is a monoblock glycopolymer, that is, the two units constituting the Sugar portion, one of which is a fucose or sialyllactose unit, and the other is not modified by the sugar unit and is a free carboxyl group; the other structural type is a diblock glycopolymer, that is, the two units constituting the Sugar portion are both sugar units, consisting of fucose and sialyllactose.

3. The block sugar polymer according to claim 1, characterized in that The Linker 1 connects monoblock or diblock sugar polymers; Linker 2 is a linker arm composed of a triazole heterocycle connected to polyethylene glycol with different degrees of polymerization, where m=4 or 24, connecting monoblock or diblock sugar polymers. When the selected sugar unit is sialyllactose, m=4; when the selected sugar unit is fucose, m=24; when the sugar polymer type is only a monoblock sugar polymer, the sugar part has only one sugar unit connected, and the part not modified by the sugar unit is a free carboxyl group, i.e., Linker 3.

4. The method for preparing the block sugar polymer according to claim 1, characterized in that: Follow these steps: (1) Dissolve unprotected or fully sulfated azidoethylfucose in methanol solvent, use carbon-supported palladium hydroxide as a catalyst, and reduce the azide group to amino group in a hydrogen atmosphere. The reaction temperature is room temperature and the reaction time is 0.5-1.5 h. After the reaction, filter with diatomaceous earth, filter and evaporate to dryness, and purify with LH-20 gel column to prepare aminofucose monomers with shorter linker arms without sulfate or fully sulfated. Unprotected or fully sulfated azidoethylfucose and 24-degree-of-polymerization polyethylene glycol chains modified with alkynyl-amino groups were dissolved in a THF / H2O reaction system. Copper sulfate pentahydrate and sodium ascorbate were used as catalysts. TBTA, a copper ion stabilizer, was added to carry out a copper-catalyzed azide-alkyne click reaction under a nitrogen atmosphere. The reaction temperature was 40-60°C and the reaction time was 3-5 hours. After the reaction, the mixture was washed with methanol and filtered, concentrated under reduced pressure, and purified using an LH-20 gel column to prepare aminofucose monomers with longer linker arms without or with full sulfate. (2) The acetyl group of the fully acetylated azidoethyl lactose is removed in a methanol / sodium methoxide reaction system at a pH of 9-10 to obtain an unprotected azidoethyl lactose intermediate. This intermediate is reduced to an amino group in a hydrogen atmosphere in a water-based reaction solvent using carbon-supported palladium hydroxide as a catalyst. The reaction temperature is room temperature for 0.5-1.5 h. After the reaction is completed, the mixture is filtered with diatomaceous earth, evaporated to dryness, and purified on an LH-20 gel column to prepare an amino lactose monomer with a shorter connecting arm. Fully acetylated azidoethyl lactose and a polyethylene glycol chain modified with an alkynyl-amino group and having a degree of polymerization of 4 are dissolved in a THF / H2O reaction system, and a copper sulfate pentahydrate and sodium ascorbate are used as catalysts. A copper ion stabilizer, TBTA, is added to carry out a copper-catalyzed azide-alkyne click reaction under a nitrogen atmosphere at a reaction temperature of 40-60°C for 3-5 hours. After the reaction, the mixture is washed with methanol, filtered, concentrated under reduced pressure, and purified using an LH-20 gel column to prepare a fully acetylated amino lactose intermediate with a longer linker arm. The acetyl group of the intermediate is removed in a methanol / sodium methoxide reaction system to prepare an amino lactose monomer with a longer linker arm. (3) NHS ester norbornene monomers were dissolved in dichloromethane as the reaction solvent and subjected to olefin hydrogen metathesis ring-opening polymerization. The third-generation Grubbs catalyst solution was slowly added at a reaction temperature of -78°C and in the dark. The reaction was continued after returning to room temperature. After the reaction was completed, vinyl ethyl ether was added to terminate the reaction. After vacuum concentration, the mixture was precipitated with five times the amount of ether alcohol and centrifuged to prepare the NHS ester-modified norbornene macromolecular polymer skeleton. (4) adopting a post-modification polymerization method, dissolving the aminofucose monomer with non-sulfated or fully sulfated shorter / longer linker arm described in step (1) and the NHS ester-modified norbornene macromolecular polymer backbone described in step (3) in a 5 / 1 DMF / H2O reaction solvent, and carrying out an amidation reaction under the catalysis of weak alkaline triethylamine, the reaction temperature is room temperature, and the reaction time is 4-6 hours; after the reaction is completed, dialyzing with 16% sodium chloride salt solution and freeze-drying to prepare a fucose polymer with non-sulfated or fully sulfated shorter linker arm and a fucose polymer with non-sulfated or fully sulfated longer linker arm; (5) combining the amino lactose monomer with the shorter / longer connecting arm described in step (2) with the NHS ester-modified norbornene macromolecular polymer backbone described in step (3) by the same method as step (4) to prepare a single-block lactose polymer, i.e., a lactose polymer with a shorter connecting arm and a lactose polymer with a longer connecting arm; (6) The aminofucose monomer with the shorter / longer linker arm not sulfated or fully sulfated in step (1) is combined with the aminolactose monomer with the shorter / longer linker arm in step (2) and the NHS ester-modified norbornene macromolecular polymer backbone in step (3) by the same method as step (4) to prepare a diblock fucose-lactose polymer, i.e., a fucose-lactose polymer with the shorter linker arm not sulfated or fully sulfated and a fucose-lactose polymer with the longer linker arm not sulfated or fully sulfated; (7) The monoblock lactose polymer described in step (5) and the diblock fucose-lactose polymer described in step (6) are reacted at a constant temperature of 37°C with CMP-Neu5Ac as a glycosyl donor, Tris-HCl and MgCl2 are added, the pH is adjusted to 8.0-8.5 with 1M NaOH, H2O and NmCSS are added, and the reaction is continued. After CMP-Sia is generated, the pH is adjusted to a weak alkaline state again, Pd2,6ST is used as a sialyltransferase, and sialic acid is directed modified at the 6-position of galactose. The reaction is continued at a constant temperature of 37°C. After the reaction is completed, the enzyme is inactivated with an equal volume of ice ethanol. After standing at -20°C for 20-40 min, the supernatant is collected and concentrated, and purified with a G10 column to prepare a fucose-sialyllactose block polymer, i.e., a sialyllactose polymer with a shorter connecting arm and a sialyllactose polymer with a longer connecting arm.

5. The method for preparing a block sugar polymer according to claim 4, characterized in that: The degree of polymerization of the NHS ester-modified norbornene macromolecular polymer backbone in step (3) is 10, 25, 50, 75 or 100.

6. The method for preparing a block sugar polymer according to claim 4, characterized in that: The post-modification polymerization method is as follows: first construct a sugar monomer containing a specific functional group, and then connect the sugar monomer with a macromolecular polymer skeleton prepared in advance by olefin hydrogen metathesis ring-opening polymerization through a predetermined reaction, thereby preparing a sugar polymer.

7. The method for preparing a block sugar polymer according to claim 4, characterized in that: In the fucose-sialyllactose block polymer, the molar ratio of fucose:sialyllactose is 1:6 or 1:

1.

8. Use of the block glycopolymer according to claim 1 in the preparation of a medicament for inhibiting influenza A virus infection.

9. Use of the block glycopolymer according to claim 8 in the preparation of a medicament for inhibiting influenza A virus infection, characterized in that: The segmented glycopolymer can target neuraminidase and hemagglutinin simultaneously.

10. Use of the block glycopolymer according to claim 8 in the preparation of a medicament for inhibiting influenza A virus infection, characterized in that: The block glycopolymer has a degree of polymerization of 25, a sugar density of 70%, a linker arm of Linker 1, fucose modified with a sulfate group, and a molar ratio of sulfated fucose to sialyllactose of 1:1.

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