A fucose polymer, its preparation method and application

Abstract

This invention discloses a fucose polymer, its preparation method, and its applications. Using natural fucoidan sulfate ester as a template, a fucose polymer with well-defined and homogeneous structures is prepared via an olefinic metathesis ring-opening polymerization reaction. The structure involves different glycosidic bond linkages, degrees of polymerization, sugar chain lengths, and sulfate sites. The amphiphilic fucose polymer self-assembles into spherical nanoparticles, exhibiting inhibitory activity against type I herpes simplex virus infection. The sulfated fucoidan polymer with a degree of polymerization of 20 shows the best antiviral effect. This invention achieves efficient and controllable synthesis of fucoidan oligosaccharide polymers and has the potential value as a novel anti-herpes simplex virus drug, which can be applied in the preparation of anti-herpes simplex virus drugs.

Description

Technical Field

[0001] This invention belongs to the field of polymer material synthesis and preparation technology, specifically relating to a fucose polymer and its preparation method and application. Background Technology

[0002] Fucoidan, primarily derived from brown algae and marine mollusks, is a highly sulfated polysaccharide composed of L-fucose linked by α-glycosidic bonds. The glycosidic bonds are predominantly of type I structures with α(1→3) linkages and type II structures with alternating α(1→3) and α(1→4) linkages. Sulfate content can reach up to 30%, and molecular weight varies widely across species, ranging from tens of thousands to millions. Marine-derived fucoidans possess antiviral, antitumor, anti-inflammatory, anticoagulant, and antithrombotic activities, making them a hot topic in marine glycopharmaceutical research. However, their structure and physiological activities are significantly influenced by factors such as species origin, marine environment, harvesting season, and extraction process, resulting in substantial structural differences and diverse biological activities. This poses challenges to detailed structure-activity relationship studies.

[0003] Research on Fucoidan has primarily focused on the chemical synthesis of oligosaccharide fragments, with limited studies on its bioactivity. The largest oligosaccharide fragment to date is fully sulfated fucoidan. However, due to the small size of chemically synthesized Fucoidan oligosaccharide fragments, total oligosaccharide synthesis is challenging, resulting in low yields and activities, and thus failing to reflect the structural and activity characteristics of natural polysaccharides. Glycopolymers, on the other hand, are a class of polymeric compounds with sugar units introduced into the polymer backbone. Due to their multivalent effects similar to natural sugar chains, they can enhance the affinity and specificity of sugars for proteins, mimicking the structure and bioactivity of natural polysaccharides. Furthermore, their synthesis is simple and yields are high, making them an important method for studying the structure-activity relationship of Fucoidan.

[0004] Currently, the publicly disclosed structures of fucose polymers are mainly fucoidan monosaccharide polymers. A Chinese patent application (publication number: CN108530570B) discloses fucoidan sulfate ester mimics with monosaccharides as structural units. The main structural feature is the difference in sulfation sites, and the structural types are relatively simple. At present, there are no research reports or invention patents on fucose polymers with different structural features and their biological functions. Summary of the Invention

[0005] The purpose of this invention is to develop an efficient method for synthesizing fucoidan oligosaccharide polymers with different structural features, and to study their higher-order structural features and anti-herpes simplex virus activity using a series of fucoidan polymers, summarizing the relationship between their structure and antiviral activity, and providing a theoretical basis for the development of marine sulfated sugar drugs.

[0006] A fucose polymer, comprising unsulfated or fully sulfated fucoidan polymers p-F1-0S and p-F1-234S, α-1,3-linked unsulfated or fully sulfated fucoidan polymers p-F2-13-0S and p-F2-13-234S, and α-1,4-linked unsulfated or fully sulfated fucoidan polymers p-F2-14-0S and p-F2-14-234S; its molecular formula is shown below ( Figure 7 ):

[0007]

[0008]

[0009] In the formula, n = 5 - 180.

[0010] Preferably, the degree of polymerization of the fucose polymer is 10-160, and more preferably, the degree of polymerization of the fucose polymer is 20.

[0011] The fucose polymer has an amphiphilic structure, comprising a hydrophilic fucose moiety and a hydrophobic polynorbornene backbone moiety. The amphiphilic fucose polymer undergoes self-assembly in aqueous solution, i.e., polymerization-induced self-assembly to form advanced nanostructures, distinct from the unassembled aggregated state of natural polysaccharides. The fucose polymer self-assembles into spherical nanoparticles in aqueous solution, and the nanoparticle size increases with increasing degree of polymerization.

[0012] A method for preparing a fucose polymer, comprising the following steps:

[0013] (1) Azide-modified fucose and thioglycoside-modified fucose were protected with benzoyl (Bz), acetyl (Ac), and benzyl (Bn) groups to construct azide ethyl fucosylation acceptor and thioglycoside fucosylation donor.

[0014] (2) The fucoidan acceptor and donor described in step (1) were subjected to a glycosylation coupling reaction under the conditions of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and N-iodosuccinimide (NIS) as glycosylation coupling reagents and anhydrous diethyl ether as solvent. The reaction temperature was -5 to 5℃ and the reaction time was 10 to 20 min. After the reaction was completed, triethylamine was added to neutralize and the mixture was restored to room temperature. After filtration and evaporation, silica gel column chromatography was used to obtain the fully protected azidoethylfucobiose intermediate.

[0015] (3) After the fully protected azidoethylfucobiose intermediate described in step (2) undergoes a deprotection reaction, the unprotected azidoethylfucobiose intermediate and alkynylated norbornene molecules undergo an azide-alkynyl Click reaction under the catalysis of copper sulfate pentahydrate and sodium ascorbate. The reaction temperature is 40-60℃ and the reaction time is 2-4h. After the reaction is completed and cooled to room temperature, the unprotected fucose monomer that can be used for polymerization is purified by silica gel column chromatography.

[0016] (4) The unprotected fucose polymerization monomer described in step (3) is polymerized using the Hoveyda-Grubbs second-generation catalyst (HG 2). nd Under the catalysis of ) and in the emulsion reaction solvent conditions of buffer and organic solvent 1,2-dichloroethane (DCE), the phase transfer catalyst DTAB was added, the reaction temperature was 70-80℃, the reaction time was 10-20min, and the olefinic metathesis ring-opening polymerization (ROMP) was carried out. After the reaction was completed, the mixture was cooled to room temperature, and the polymerization reaction was terminated by adding vinyl ethyl ether as a terminator. After removing the organic solvent by rotary evaporation, the mixture was purified by dialysis to prepare unprotected fucose polymer.

[0017] (5) The unprotected fucose polymer described in step (4) was reacted overnight with formamide as the reaction solvent under the catalysis of sulfur trioxide·triethylamine (SO3·Et3N) as a sulfation reagent and at a reaction temperature of 60-80℃. After the reaction was completed, the reaction was neutralized with saturated sodium bicarbonate at low temperature and purified by dialysis to prepare the fully sulfated fucose polymer.

[0018] After fucose is modified with an azide group, two configurations, α and β, appear at the anodic position. The α configuration, which is the same as that of natural fucose, is selected as the intermediate for the subsequent preparation of fucose polymers.

[0019] Fucosyl donors with fully protected ethoxylated groups were glycosylated and coupled with fucosyl acceptors with exposed hydroxyl groups at the 3- or 4-positions modified with azidoethyl to construct α-1,3-linked or α-1,4-linked fucoidan units, respectively.

[0020] In the olefin-hydrogen metathesis ring-opening polymerization reaction, the degree of polymerization is controlled by adjusting the ratio of monomer to catalyst, i.e., the value of [M] / [C]. The degrees of polymerization of the fucose polymer are 10, 20, 40, 80 and 160, respectively.

[0021] The fucose polymer is prepared by a direct monomer polymerization method, which involves first constructing a fucose polymer monomer containing a bridged ring double bond that can be used for polymerization, and then preparing the fucose polymer by an olefin-hydrogen metathesis ring-opening polymerization reaction of this polymer monomer.

[0022] The fucose polymer is prepared by a post-polymerization sulfation method to obtain a fully sulfated fucose polymer. The post-polymerization sulfation method involves first using unprotected fucose monomers as reactants to perform an olefin-hydrogen metathesis ring-opening polymerization reaction to prepare a non-sulfated fucose polymer, and then modifying this non-sulfated fucose polymer with sulfation under the catalysis of a sulfating reagent to obtain a fully sulfated fucose polymer.

[0023] The application of the fucose polymer in the preparation of drugs that inhibit herpes simplex virus type I infection.

[0024] Preferably, the degree of polymerization of the fucose polymer is 20 or 40.

[0025] Among sulfated fucoidan polymers with different degrees of polymerization (DP 10, 20, 40, 80, 160), fucoidan polymers with degrees of polymerization of 20 and 40, especially 20, showed the best inhibitory effect on HSV-1 infection. This highlights that fucoidan polymers with medium molecular chain length can exert optimal biological activity, which is related to the optimal chelation mechanism of medium-length polymers with HSV-1 viral proteins.

[0026] At the same degree of polymerization (20), the fucoidan polymer showed a higher ability to inhibit HSV-1 infection than the α-1,3-linked fucoidan polymer, which in turn showed a higher ability than the α-1,4-linked fucoidan polymer. This indicates that longer sugar chains do not necessarily equate to better biological function in polymers.

[0027] Antiviral activity was evaluated using a cytopathic effect (CPE) inhibition assay. The IC50 of sulfated fucoidan polymers with a degree of polymerization of 20 was also measured. 50 1.34 μg·mL -1 Furthermore, its anti-HSV-1 activity was higher than that of natural fucoidan sulfate (2.33 μg·mL⁻¹). -1 ) and the already marketed positive control drug acyclovir (3.96 μg·mL) -1 ).

[0028] The beneficial effects of this invention are as follows: This invention employs a "one-pot" method for selective protection of different sites, selecting appropriate protecting groups for operation, and designing and constructing fucosylated donors and acceptors. Reaction intermediates can be directly added to the next reaction without separation and purification, reducing cumbersome purification processes and simplifying the reaction operation, enabling rapid preparation of donors and acceptors. This invention uses a "polymerization-after-sulfation" method to directly sulfate unprotected fucose polymers, avoiding incomplete sulfation reactions caused by sulfation on sugar monomers and reducing the generation of byproducts. The post-processing operation of direct polymer dialysis purification is simpler and yields higher results compared to the post-processing step of gel column purification after sugar monomer sulfation. This provides a simple and efficient method for the large-scale synthesis of sulfated sugar polymers. This invention uses natural fucoidan sulfate as a template and, based on its structural characteristics, comprehensively considers its configuration, sugar chain length, glycosidic bond linkage, and sulfation mode to design a fucoidan polymer structure. This structure can better serve as a biomimetic structure of natural fucoidan, providing greater accuracy and reliability for subsequent biological function studies and structure-activity relationship evaluations. The sulfated fucoidan polymer of this invention exhibits significant inhibitory activity against type I herpes simplex virus (HSV-1), and its inhibitory effect on HSV-1 infection is higher than that of natural fucoidan sulfate. This indicates that the sulfated fucoidan polymer can not only mimic the structure of natural fucoidan but also better mimic its biological functions. The sulfated fucoidan polymer of this invention shows higher inhibitory activity against HSV-1 than the currently marketed anti-HSV-1 drug acyclovir, demonstrating its potential as a novel glycosyl anti-herpes simplex virus drug. Attached Figure Description

[0029] Figure 1 A route diagram for the preparation of fucosylation donors and acceptors.

[0030] Figure 2 This is a route diagram for the preparation of fucose polymerization monomers.

[0031] Figure 3 This is a route diagram for the preparation of fucose polymers.

[0032] Figure 4 The figures show the 1H NMR spectra of different fucose polymers with different structures in this invention. In the figures, the horizontal axis f1 represents the chemical shift (ppm).

[0033] Figure 5 This is a characterization diagram of the higher-order structure of the fucoidan polymer in this invention.

[0034] Figure 6 This diagram illustrates how the fucose polymer inhibits type I herpes simplex virus infection in this invention.

[0035] Figure 7The diagram shows the molecular structures of the six fucose polymers of this invention. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0037] Example 1: Preparation of fucosylation donors and acceptors

[0038] Fucosylation donors are α-configured fucose derivatives with an anomeric position of the sugar ring modified by an ethioglycoside group, and benzyl, acetyl, and benzoyl groups at positions 2, 3, and 4, respectively. Acceptors are of two types: α-1,3-linked and α-1,4-linked fucosylation acceptors, both being α-configured fucose derivatives with an anomeric position of the sugar ring modified by an azide ethyl group. In the α-1,3-linked fucosylation acceptor, the 3-hydroxyl group of the sugar ring is exposed, while positions 2 and 4 are protected by benzyl and benzoyl groups, respectively. In the α-1,4-linked fucosylation acceptor, the 4-hydroxyl group is exposed, while positions 2 and 3 are protected by benzyl and benzoyl groups, respectively. The preparation route is as follows: Figure 1 As shown, the preparation method specifically includes the following steps:

[0039] Step (1): Preparation of fucosylation donors

[0040] 14.15 g of L-fucose was weighed into a 500 mL round-bottom flask, dissolved in 120 mL of pyridine, and reacted with 60 mL of acetic anhydride at room temperature. After the reaction was detected by TLC, the reaction solution was concentrated and extracted twice each with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The extract was dried under anhydrous sulfuric acid, filtered, and evaporated to dryness to obtain the α / β configuration of peracetylfucose derivative. 12.2 g (36.7 mmol) of this derivative was weighed into a 100 mL round-bottom flask, and 7.0 mL (55.1 mmol) of boron trifluoride·diethyl ether and 13.74 mL (183.6 mmol) of ethanethiol were added under ice bath conditions. After the reaction was detected by TLC, the extract was extracted twice each with saturated sodium bicarbonate solution and saturated sodium chloride solution. The extract was dried under anhydrous sulfuric acid, filtered, and evaporated to dryness, followed by silica gel column chromatography to obtain the α configuration of peracetylethioglycoside fucose derivative.

[0041] 6.2 g of the above α-configuration peracetyl ethioglycoside fucose derivative was weighed into a 100 mL round-bottom flask, dissolved in methanol, and the pH was adjusted to 9-10 with freshly prepared saturated sodium methoxide solution. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC, the fucose was neutralized with hydrogen-form cation exchange resin, filtered, and evaporated to dryness to obtain the α-configuration unprotected ethioglycoside fucose derivative. 3.8 g (18.2 mmol) of this fucose derivative was weighed into a 100 mL round-bottom flask, dissolved in acetonitrile, and then 1.7 g (7.3 mmol) of camphorsulfonic acid and 24.6 mL (109.5 mmol) of triethyl orthobenzoate were added sequentially. The reaction was carried out under reflux and stirring at 80 °C. After the reaction was completed, the mixture was allowed to return to room temperature, neutralized with triethylamine, and the reaction solution was evaporated to dryness to continue the next reaction. Dissolved in N,N-dimethylformamide, 1.46 g (36.5 mmol) of sodium hydride and 4.36 mL (36.5 mmol) of benzyl bromide were added under ice bath conditions. The reaction was carried out at room temperature for 1 h. After the reaction was completed by TLC, methanol was added for neutralization, and ring-opening was performed with 1 M hydrochloric acid. After the reaction was completed, the reaction solution was evaporated to dryness to continue the next step of the reaction. 50 mL of pyridine and 25 mL of acetic anhydride were added for dissolution, and the reaction was carried out at room temperature. After the reaction was completed, the solution was extracted twice each with saturated sodium bicarbonate solution, 1 M hydrochloric acid, and saturated sodium chloride solution, respectively. After drying with anhydrous sulfation, the solution was filtered, evaporated to dryness, and then purified by silica gel column chromatography to obtain α-configuration ethoxylated fucose, which is a fucoidylation donor.

[0042] Step (2): Preparation of fucosylation receptor

[0043] 9.92 g (60.4 mmol) of L-fucose was weighed into a 250 mL round-bottom flask, and 45.4 mL (0.604 mol) of 2-azidoethanol and 8.5 g of hydrogen-form cationic dendrites were added. The mixture was heated to reflux at 80 °C with stirring for 24 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was allowed to return to room temperature, filtered, and evaporated to dryness to obtain an α / β configuration unprotected azidoethylfucose derivative. This derivative was placed in a 500 mL round-bottom flask, dissolved in 100 mL of pyridine, and 50 mL of acetic anhydride was added. The reaction was carried out at room temperature and monitored by TLC. After the reaction was completed, the reaction solution was concentrated and extracted twice each with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The extract was dried anhydrous by sulfation, filtered, evaporated to dryness, and then subjected to silica gel column chromatography to obtain an α configuration peracetylazidoethylfucose derivative.

[0044] Weigh 4.6 g (12.8 mmol) of the above-mentioned α-configuration peracetylfucose derivative into a 250 mL round-bottom reaction flask, add 90 mL of methanol to dissolve it, adjust the pH to 9-10 with freshly prepared saturated sodium methoxide solution, react at room temperature for 2 h, and after the reaction is completed by TLC, neutralize with hydrogen-form cation exchange resin, filter, and evaporate to dryness to obtain α-configuration unprotected azidoethylfucose, which can be directly used for the next reaction without purification. First dissolve in acetonitrile, add 593.3 mg (2.6 mmol) of camphorsulfonic acid and 8.61 mL (38.3 mmol) of triethyl orthobenzoate, and react under reflux at 80 °C. After the reaction is completed, restore to room temperature, neutralize with triethylamine, evaporate the reaction solution to dryness, and continue to the next reaction. Dissolved in N,N-dimethylformamide, 1021.7 mg (25.5 mmol) of sodium hydride and 3.06 mL (25.5 mmol) of benzyl bromide were added under ice bath conditions. The reaction was carried out at room temperature for 1 h. After the reaction was completed by TLC, methanol was added for neutralization, and ring-opening was performed with 1 M hydrochloric acid. After the reaction was completed, the sample was extracted twice each with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The sample was dried over anhydrous sulfuric acid, filtered, evaporated to dryness, and then purified by silica gel column chromatography to obtain a 1,3-linked fucosylated acceptor.

[0045] 3.2 g of the 1,3-linked fucosylated receptor was weighed into a 100 mL round-bottom flask, dissolved in methanol, and the pH of the reaction solution was adjusted to 11 with freshly prepared saturated sodium methoxide. The reaction was carried out at room temperature. After the reaction was detected by TLC, the solution was neutralized with hydrogen cation exchange resin, filtered, evaporated to dryness, and purified by silica gel column chromatography to obtain a fucose derivative with anomeric azidoethyl modification and benzyl protection at the C2 position. 1.83 g of this derivative was weighed and dissolved in a 19:1 tetrahydrofuran aqueous solution. Under nitrogen protection, 1.87 mL (11.3 mmol) of N,N-diisopropylethylamine, 63.6 mg (0.29 mmol) of dimethyltin dichloride, and 722.6 μL (6.2 mmol) of benzoyl chloride were added sequentially. The reaction was stirred at room temperature. After the reaction was completed, the solution was extracted twice each with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried anhydrous sulfonated, filtered, evaporated to dryness, and purified by silica gel column chromatography to obtain a 1,4-linked fucosylated receptor.

[0046] Example 2: Preparation of fucose polymerization monomers

[0047] Fucose monomers are composed of norbornene containing bridged double bonds, linked to sugar units of different structures via triazole heterocycles. These monomers can be used for polymerization. The preparation route is as follows: Figure 2 As shown, the preparation method specifically includes the following steps:

[0048] Step (1): Preparation of key intermediates of α-1,3-linked / α-1,4-linked fucoidan by glycosylation coupling reaction

[0049] Take 2.4 g (5.4 mmol) of the fucoidylation donor from Example 1 and 2.8 g (6.5 mmol) of the α-1,3-linked or α-1,4-linked fucoidylation acceptor in a 50 mL round-bottom flask. Add 2.4 g (10.8 mmol) of N-iodosuccinimide and 97.7 μL (0.54 mmol) of trimethylsilyl trifluoromethanesulfonate under ice bath conditions. Add dry molecular sieves, and under nitrogen protection, react for 30 min under ice bath conditions. After the reaction is complete, neutralize with triethylamine, filter, concentrate the reaction solution, and purify by silica gel column chromatography to obtain the key intermediate of α-1,3-linked or α-1,4-linked fully protected fucoidan.

[0050] Step (2): Copper-catalyzed azide-alkynyl Click reaction to prepare fucoidan or disaccharide monomers.

[0051] Weigh 1.5 g (1.85 mmol) of the key intermediate of α-1,3-linked or α-1,4-linked fully protected fucoidan from step (1) of Example 2 into a 100 mL round-bottom flask, and add 37.3 mL (11.1 mmol, 45 mg·mL) of sodium bromate aqueous solution. -1 77.4 mL of sodium dithionite aqueous solution (11.1 mmol, 25 mg / mL) -1 The reaction mixture was stirred vigorously at room temperature. After the reaction was completed by TLC, it was neutralized with sodium thiosulfate aqueous solution, extracted twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by silica gel column chromatography to obtain α-1,3-linked or α-1,4-linked fucoidan derivatives protected by an acetyl group at the 3-position and a benzoyl group at the 4-position. 943 mg of the above fucoidan derivative was weighed into a round-bottom flask, dissolved in 3.0 mL of methanol, and the pH of the reaction solution was adjusted to 11 with freshly prepared saturated sodium methoxide. The reaction was heated under reflux in an oil bath at 75°C. After the reaction was completed by TLC, hydrogen-form cationic dendrites were added for neutralization, filtered, evaporated to dryness, and purified by silica gel column chromatography to obtain α-1,3-linked or α-1,4-linked unprotected azidoethyl fucoidan derivatives.

[0052] Weigh 218.0 mg (0.6 mmol) of the unprotected azidoethylfucoidan derivative from step (2) of Example 1 and the α-1,3-linked or α-1,4-linked unprotected azidoethylfucoidan derivative from step (2) of Example 2, and 173.3 mg (0.9 mmol) of the alkynyl-modified norbornene monomer. Dissolve in 6.25 mL of tetrahydrofuran. Purge the reaction solution with nitrogen for 10 min. Under nitrogen protection, add 28.7 mg (0.1 mmol, 25 mg·mL) of freshly prepared copper sulfate pentahydrate aqueous solution. -1 ) and sodium ascorbate aqueous solution 136.6 mg (0.7 mmol, 100 mg·mL) -1Add deionized water to adjust the volume ratio of tetrahydrofuran to water in the reaction system to 1:1. Heat and stir at 50°C for 3 hours, and the reaction is considered complete by TLC. After returning to room temperature, filter, evaporate to dryness, and purify by silica gel column chromatography to obtain fucoidan, 1,3-linked fucoidan, and 1,4-linked fucoidan monomers.

[0053] Example 3: Preparation of fucose polymer

[0054] Synthetic reaction route as follows Figure 3 As shown, the preparation method specifically includes the following steps:

[0055] Step (1): Weigh 20.0 mg (0.034 mmol) of fucoidan, 1,3-linked fucoidan, and 1,4-linked fucoidan monomers from Step (2) of Example 2 into a round-bottom flask, dissolve in 500 μL of bis-Tris buffer (pH 5.9, 100 mM), add 10.5 mg (0.055 mmol) of DTAB, and catalyst HG 2. nd Freshly prepared with DCE to 4.0 mg / mL -1 The amount of catalyst to be added was calculated based on the ratio of sugar monomer to catalyst, i.e., [M] / [C] of 10, 20, 40, 80, and 160. The catalyst was added, and DCE was added to adjust the volume ratio of bis-Tris buffer to DCE in the reaction solution to 2:1. The reaction solution was heated at 75°C and stirred for 30 min. The reaction was stopped by TLC. After cooling to room temperature, excess vinyl ether was added and stirred at room temperature for 30 min to terminate the reaction. The reaction solution was concentrated under reduced pressure to remove the organic phase DCE. The remaining aqueous phase was dialyzed against 16% sodium chloride (using a 3500 Da dialysis bag) and lyophilized to obtain pure fucoidan monomers, 1,3-linked fucoidan, and 1,4-linked fucoidan polymers.

[0056] Step (2): Weigh 10 mg (0.017 mmol) of the fucoidan monomers, 1,3-linked fucoidan, and 1,4-linked fucoidan polymers from Step (1) of Example 3 into a round-bottom flask, add 500 μL of formamide to dissolve, add 312.2 mg (1.7 mmol) of sulfur trioxide-triethylamine, and react overnight at 70°C with stirring. After the reaction is complete, return to room temperature, cool to 0°C, add saturated sodium bicarbonate solution to neutralize, dialyze with 16% sodium chloride solution (using a 3500 Da dialysis bag), and freeze-dry to obtain pure fully sulfated fucoidan polymer, 1,3-linked fully sulfated fucoidan polymer, and 1,4-linked fully sulfated fucoidan polymer.

[0057] Example 4: Structural characterization of fucose polymers

[0058] The structural characterization of fucose polymers specifically includes the following steps:

[0059] Step (1): Nuclear magnetic resonance analysis

[0060] Weigh 5 mg of each of the series of fucose polymers from Example 3, add 500 μL of heavy water, freeze-dry, repeat the heavy water exchange three times, add 500 μL of heavy water each time, and transfer to an NMR tube for analysis using a 500 MHz NMR spectrometer. Figure 4 As shown, the proton signal of the norbornene cyclic olefin (δ 6.31 ppm) shifts to a lower field to δ 5.20-5.90 ppm, which corresponds to the protons of the polymer's main chain olefin. This confirms the successful polymerization reaction. Furthermore, the proton signal of the sulfated fucose polymer's sugar ring shifts to an even lower field compared to the unsulfated fucose polymer's sugar ring. Particularly noteworthy is the C1 proton signal: the C1 proton signal of sulfated fucose is δ 5.25 ppm, while the C1 proton signal of the unsulfated fucose polymer is δ 5.0 ppm, further demonstrating the success of the sulfation reaction.

[0061] Step (2): High-level structural characterization

[0062] The higher-order structure of the fucose polymer of this invention was characterized using a combination of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy. Figure 5 As shown, the specific methods include the following:

[0063] Method (1): A series of fucose polymers were dispersed in water and prepared into a 1.0 mg·mL solution. -1 The sample was dropped onto a hydrophilic stencil film loaded with a copper mesh, negatively stained with 2.0% phosphotungstic acid, and imaged using a transmission electron microscope (TEM) after drying. Figure 5 As shown in ab, the advanced nanostructure of fucose polymer consists of spherical nanoparticles, which differs from the unassembled aggregated state of natural fucose. Furthermore, the nanoparticle size increases with the degree of polymerization.

[0064] Method (2): The powdered or flocculent fucose polymer sample was evenly sprinkled onto a sample stage coated with conductive adhesive. Excess sample was then blown away with a bulb syringe. After coating to make the sample conductive, it was imaged using a scanning electron microscope (SEM). Figure 5 As shown in c, it was also observed that the advanced nanostructure of the fucose polymer consists of spherical nanoparticles.

[0065] Method (3): A series of fucose polymers were dispersed in water and prepared into a solution of 0.25 mg·mL⁻¹. -1 The sample was dropped onto a mica sheet and, after drying, imaged using an atomic force microscope (AFM). Figure 5As shown in d, the advanced nanostructure of the fucose polymer is consistent with that of methods (1) and (2), and consists of spherical nanoparticles.

[0066] Example 5: Fucosylate polymers have anti-HSV-1 virus infection effects.

[0067] The anti-HSV-1 activity of fucose polymers was evaluated using a cytopathic effect (CPE) inhibition assay. Vero cells were cultured in 96-well plates. After the cells reached a confluent monolayer, the culture medium was discarded, and the fucose polymer inhibitors were diluted to 20, 10, 5, 2.5, 1.25, and 0.625 μg / mL. -1 Three replicates were set for each concentration to conduct the entire drug delivery process, including pretreatment of cells, pretreatment of viruses, drug delivery during adsorption, and drug delivery after adsorption. In addition, a blank group, a virus group, and a positive control group (acyclovir) were set up. Cell pretreatment: Fucose polymer was co-incubated with cells at 37℃ for 1 h and then aspirated; Virus pretreatment: Fucose polymer was mixed with HSV-1 (MOI = 0.1) and co-incubated at 37℃ for 1 h; Drug administration during adsorption: Pretreated virus was added to a 96-well plate after pretreatment of cells, placed in a 37℃ incubator for 1 h for adsorption, then aspirated, and maintenance medium was added; Drug administration after adsorption: Serially diluted fucose polymer was added to maintenance medium, placed in a 37℃ incubator, and the cytopathic effect of the virus group was observed. After the cells became round and shiny and the cytopathic effect detached, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, aspirated, stained with 0.4% (w / v) crystal violet at room temperature for 15 min, aspirated and rinsed, and the A540 nm value was measured using an ELISA reader. Based on the results, the cytopathic effect of the sample was reduced by 50% (IC50). 50 The required sample concentration at that time. Figure 6 As shown, the structure-activity relationship of fucose polymers against HSV-1 virus is as follows: sulfated fucose polymers can inhibit HSV-1 virus, while non-sulfated fucose polymers do not have this effect; among fucose polymers with different chain lengths of polymerization degrees of 10, 20, 40, 80, and 160, sulfated fucose polymers with polymerization degrees of 20 and 40, especially the 20-degree-of-polymerization fucose polymer, showed the best anti-HSV-1 effect (IC50). 50 = 1.34 μg·mL -1 Furthermore, under the same sulfation modification and degree of polymerization of 20, the anti-HSV-1 activity of fucoidan polymers was higher than that of α-1,3-linked fucoidan polymers, and also higher than that of α-1,4-linked fucoidan polymers. In conclusion, sulfated fucoidan polymers at a degree of polymerization of 20 are highly active anti-HSV-1 glycomolecules with superior antiviral efficacy compared to natural fucoidan (IC50). 50 = 2.33 μg·mL -1 ) and the already marketed anti-HSV-1 drug, acyclovir (IC)50 = 3.96 μg·mL -1 ).

[0068] In summary, this invention successfully prepared a series of fucose polymers with different structural characteristics, including varying sugar chain lengths, glycosidic bond linkages, degrees of polymerization, and sulfation sites. This enriches the structure of fucose oligosaccharides and sugar polymers, enabling the large-scale and efficient preparation of fucose sulfate esters. This invention is applicable to the characterization of the morphology and particle size of higher-order structures resulting from the polymerization-induced self-assembly of amphiphilic polymers, and to the study of the true morphology of fucose polymers in aqueous solutions. The sulfated fucose polymers described in this invention exhibit significant inhibitory effects against type I herpes simplex virus infection, showing potential for development into anti-herpes simplex virus drugs.

[0069] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of a fucoidan in the manufacture of a medicament for inhibiting infection by herpes simplex virus type I, characterized in that, The fucose polymers include non-sulfated or fully sulfated fucosyl monosaccharide polymers p-F1-0S, p-F1-234S, non-sulfated or fully sulfated fucosyl disaccharide polymers with α-1,3 linkage p-F2-13-0S, p-F2-13-234S, non-sulfated or fully sulfated fucosyl disaccharide polymers with α-1,4 linkage p-F2-14-0S, p-F2-14-234S; the molecular formula is as follows: ； In the formula, n = 20.

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