A bis-antenna type sialoglycan and a synthesis method and application thereof

Abstract

The application discloses a kind of double antenna type sialylglycan and its synthetic method and application, and the application belongs to the field of sugar chemistry and glycoprotein.The double antenna type sialylglycan of the application, the sample is prepared by chemical synthesis, the method is first synthesized a compound with two amino groups and one thiol group as the basic skeleton of double sialic acid oligosaccharide chain, the second step is connected with sialic acid lactose and two amino groups on the skeleton respectively, the third step is covalently connected with the thiol group of basic skeleton and polyethylene glycol maleimide.This bionic double sialic acid oligosaccharide chain structure is similar to natural double antenna oligosaccharide chain, and the oligosaccharide chain is replaced by hydrophilic PEG and retains terminal sialic acid and galactose.It can be applied to the field of protein modification to improve the physiological and biological activity of protein.

Description

Technical Field

[0001] This invention relates to a dual-antenna type sialic acid polysaccharide, its synthesis method and application, belonging to the fields of glycoengineering and biomedicine. Background Technology

[0002] Glycosylation is a crucial post-translational modification of proteins, significantly impacting their stability, spatial folding, and biological function. Many pharmaceutical proteins, such as the monoclonal antibody IgG and erythropoietin (EPO), rely on structurally complex N-glycosylation modifications, particularly the sialic acid residues at the glycan terminals, for their biological activity. Currently, these proteins are primarily expressed in mammalian cells (such as CHO cells), but high culture medium costs, complex processes, and long production cycles limit their widespread application.

[0003] Pichia pastoris and Saccharomyces cerevisiae are commonly used heterologous protein expression systems, offering advantages in high efficiency and low cost for large-scale recombinant protein production. However, the N-glycans synthesized by these yeast systems are typically high-mannose, lacking mammalian-specific glycosylation modifications, leading to reduced activity or insufficient stability of the target protein. Although some studies have focused on glycosylation engineering of Pichia pastoris to achieve humanized glycosylation synthesis, technical bottlenecks remain, such as low efficiency and incomplete structure, in introducing terminal sialic acid. Furthermore, extracting complex N-glycans containing sialic acid from natural products (such as egg yolks) and then linking them to target proteins via chemical or enzymatic methods is another feasible glycosylation modification strategy. However, naturally derived glycans have heterogeneous structures, low sialylation levels, complex extraction processes, and limited yields, making it difficult to meet the needs of large-scale pharmaceutical protein production.

[0004] To address the aforementioned issues, this invention proposes a novel biomimetic dual-antenna sialylated N-glycan synthesis strategy. This method uses readily scalable sialylated lactose as a raw material, constructs a trifunctional linker through lysine activation, and achieves efficient conjugation with drug proteins using bifunctional PEG. This synthetic route is simple to operate, operates under mild reaction conditions, and is cost-effective. Experimental results show that IgG and lactoferrin modified with this biomimetic sialylated N-glycan exhibit significantly enhanced bioactivity and function, demonstrating promising application potential. Summary of the Invention

[0005] This invention provides a technology for the synthesis and application of dual-antenna sialic acid polysaccharides. The method involves generating dual-antenna sialic acid polysaccharides by reductive amination of 3'-SL with synthesized compound c, and generating compound e by Michael addition reaction with MAL-PEG-CHO. Compound e is then reductively aminationed with the ε-amino group of lysine in a protein, thereby giving the protein dual-antenna sialic acid polysaccharides.

[0006] The first objective of this invention is to provide a method for synthesizing dual-antenna type sialic acid polysaccharides, comprising the following steps: S1. Mercaptoethylamine hydrochloride and triphenylchloromethane are dissolved in an organic solvent and stirred under a protective gas atmosphere to obtain compound a; S2. Dissolve (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid in an organic solvent, add a coupling catalyst, a basic catalyst, and a condensing agent to obtain a reaction system. Dissolve compound a obtained in step S1 in an organic solvent and add it dropwise to the reaction system to carry out the reaction. The reaction product is diluted, washed, and subjected to column chromatography to obtain compound b. S3. Dissolve compound b obtained in step S2 in an organic solvent, add triisopropylsilane ether (TIS), stir the reaction, remove the solvent and wash to remove impurities, and freeze dry to obtain compound c. S4. The compound c obtained in step S3, sialyl lactose (3'-SL), and sodium cyanoborohydride (NaBH3CN) are dissolved in an organic solvent and reacted at 50-60°C for 24-48 hours under a protective gas atmosphere. The mixture is then filtered, washed, and dried to obtain compound d. S5. The compound d obtained in step S4 is dissolved in a buffer solution with maleimide-polyethylene glycol-acetaldehyde and reacted under protective gas conditions. After the reaction, the salt is removed to obtain compound e, which is the dual-antenna type sialic acid polysaccharide.

[0007] In one embodiment of the present invention, the organic solvent is one or a mixture of dimethylformamide (DMF), dichloromethane (DCM), trifluoroacetic acid (TFA), and methanol.

[0008] In one embodiment of the present invention, the organic solvent used in step S1 is a mixed solvent of dimethylformamide and dichloromethane, the organic solvent used in step S2 is dimethylformamide, the organic solvent used in step S3 is a mixed solvent of dichloromethane and trifluoroacetic acid, and the organic solvent used in step S4 is methanol.

[0009] In one embodiment of the present invention, in step S1, the molar ratio of mercaptoethylamine hydrochloride and triphenylchloromethane is 1 to 3:1.

[0010] In one embodiment of the present invention, the reaction time in step S1 is 20 to 30 hours.

[0011] In one embodiment of the present invention, in step S1, the product obtained from the reaction is treated as follows: the product is added to a 2-3M sodium hydroxide solution and extracted with dichloromethane. After drying the extracted organic phase, the solvent is removed by rotary evaporation to obtain compound a.

[0012] In one embodiment of the present invention, the coupling catalyst is 1-hydroxybenzotriazole (HObt) or 1-hydroxy-7-azabenzotriazole (HOAT).

[0013] In one embodiment of the present invention, the alkaline catalyst is N-methylmorpholine (NMM), N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), or dimethylaminopyridine (DMAP).

[0014] In one embodiment of the present invention, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl), tetramethylchloroformamidine hexafluorophosphate (TCFH), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), hexafluorophosphate (benzotriazol-1-yloxy)tripyrrolylphosphonate (PyBOP), N,N'-carbonyldiimidazole (CDI), hexafluorophosphate O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU), or hexafluorophosphate O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HBTU).

[0015] In one embodiment of the present invention, in step S2, the molar ratio of (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid to compound a, coupling catalyst, basic catalyst and condensing agent is 1:1.5~2.5:1~1.5:2~4:1.5~2.5.

[0016] In one embodiment of the present invention, in step S2, the washing is performed sequentially using 0.5~2M hydrochloric acid, saturated sodium carbonate solution, and saturated sodium chloride solution.

[0017] In one embodiment of the present invention, in step S2, column chromatography is performed using silica gel column chromatography with an elution gradient of DCM / methanol (CH3OH) 0 / 100~10 / 90.

[0018] In one embodiment of the present invention, in step S3, the amount of triisopropylsilane ether added is greater than 10 times the molar equivalent of compound b.

[0019] In one embodiment of the present invention, the reaction time in step S3 is 0.5 to 2 hours.

[0020] In one embodiment of the present invention, in step S3, washing is performed using anhydrous diethyl ether, and after washing, 0.08~0.15M hydrochloric acid is added and the mixture is repeatedly freeze-dried to obtain compound c.

[0021] In one embodiment of the present invention, in step S4, the molar ratio of compound c to sialyl lactose and sodium cyanoborohydride is 1:2~3:4~6.

[0022] In one embodiment of the present invention, in step S4, the washing is performed using methanol.

[0023] In one embodiment of the present invention, in step S5, the molecular weight of polyethylene glycol in maleimide-polyethylene glycol-acetaldehyde is 500~2000.

[0024] In one embodiment of the present invention, the reaction time in step S5 is 0.5 to 2 hours.

[0025] In one embodiment of the present invention, in step S5, the buffer solution is a phosphate buffer solution with a pH of 6.5 to 7.5.

[0026] In one embodiment of the present invention, desalination is performed by dialysis.

[0027] A second objective of this invention is to provide a dual-antenna type sialic acid polysaccharide prepared by the method, the molecular formula of which is: .

[0028] A third objective of this invention is to provide the application of the aforementioned dual-antenna type sialyl polysaccharide and its derivatives in protein resialylation.

[0029] In one embodiment of the present invention, the application specifically includes reacting a dual-antenna type sialyl polysaccharide, sodium cyanoborohydride, and the protein to be modified in a sodium borate buffer solution, followed by dialysis to remove salts and freeze-drying to obtain a re-sialylated protein.

[0030] In one embodiment of the present invention, the protein is lactoferrin, human immunoglobulin, or erythropoietin.

[0031] In one embodiment of the present invention, the reaction is carried out at a temperature of 0-10 °C. o C reaction takes 24-48 hours.

[0032] In one embodiment of the present invention, the pH of the sodium borate buffer solution is 8-10. Attached Figure Description

[0033] Figure 1 This is a synthetic route diagram of the dual-antenna sialyte in this invention; Figure 2 This is the MALDI-TOF mass spectrum of compound d in this invention; Figure 3 This refers to the determination of the molecular weight of compound e in this invention; Figure 4 This is the 1H NMR spectrum of compound e in this invention; Figure 5 This invention relates to the determination of sialic acid content in resialylated lactoferrin. Figure 6 The present invention describes the determination of the antioxidant activity of resialylated lactoferrin, including (a) reducing power determination; (b) DPPH free radical scavenging rate; (c) hydroxyl free radical scavenging rate; and (d) ABTS free radical scavenging rate. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] The mercaptoethylamine hydrochloride and (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid involved in this invention were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; triphenylchloromethane was obtained from Shanghai Titan Technology Co., Ltd.; maleimide-polyethylene glycol-acetaldehyde was purchased from Shaanxi Xinyan Bomei Biotechnology Co., Ltd.; sialic acid lactose was purchased from Chr. Hansen, Denmark; and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0036] Detection method: (1) Detection method for sialic acid content Preparation of Neu5Ac standard solutions: Accurately weigh 5 mg of Neu5Ac standard, dissolve it in ultrapure water, and dilute to 500 mL in volumetric flasks to prepare standard stock solutions with a mass concentration of 10 μg / mL. After serial dilution, prepare Neu5Ac standard solutions with mass concentration gradients of 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, and 10 μg / mL.

[0037] Protein-binding sialic acid hydrolysis: Weigh lactoferrin, add 4 M acetic acid at a ratio of 1:100, and hydrolyze in a water bath at 80 °C for 180 min. Cool to room temperature on ice, add 10% trichloroacetic acid to precipitate the protein, centrifuge at 8000 rpm for 10 min, and collect the supernatant for derivatization.

[0038] Pre-column derivatization: Take the supernatant above and add a 6:1 volume ratio of DMB derivatization solution (8 mmol / L DMB, 14 mmol / L sodium dithionite, 0.8 mol / L β-mercaptoethanol, 1.5 mol / L glacial acetic acid). React at 50 °C in the dark for 2.5 h. Filter the product through a 0.45 μm filter membrane for HPLC analysis.

[0039] HPLC analysis: Sialic acid in lactoferrin was separated and detected using a Taurus C18-HP 250×4.6mm, 5μm column at 30 ℃. The fluorescence detector used an excitation wavelength of 373 nm and an emission wavelength of 448 nm. The mobile phase was V1 (methanol):V2 (acetonitrile):V3 (ultrapure water) = 7:8:85, the flow rate was 0.9 mL / min, and the injection volume was 20 μL. A standard curve was plotted with the mass concentration of Neu5Ac standard solution on the x-axis and the corresponding peak area on the y-axis. The Neu5Ac content was calculated based on the standard curve equation, and the results were expressed as per milligram of lactoferrin.

[0040] (2) Determination of DPPH free radical scavenging ability Sample solutions of different concentration gradients and equal volumes of 0.2 mmol·L⁻¹ -1 The DPPH solution (dissolved in anhydrous ethanol) was thoroughly mixed and reacted in the dark for 30 min. The absorbance at 517 nm was then measured using a multi-mode microplate reader. Ultrapure water was used as a blank control, and VC aqueous solution was used as a positive control. The DPPH free radical scavenging rate was calculated using the following formula.

[0041] DPPH free radical scavenging rate (%) =

[0042] (3) Determination of hydroxyl radical scavenging ability Different concentration gradient sample solutions were sequentially mixed with an equal volume of 9 mmol·L⁻¹ -1 Ferrous sulfate solution and 9 mmol·L -1 Mix the hydrogen peroxide solution (prepared fresh before use) thoroughly, react at 37 °C for 10 min, and after the reaction is complete, add an equal volume of 9 mmol·L⁻¹. -1 The salicylic acid solution (dissolved in anhydrous ethanol) was reacted again at 37 °C for 30 min. The absorbance at 510 nm was then measured using a multi-mode microplate reader. Ultrapure water was used as a blank control, and VC aqueous solution was used as a positive control. The hydroxyl radical scavenging rate was calculated using the following formula.

[0043] Hydroxyl radical scavenging rate (%) =

[0044] (4) Determination of ABTS free radical scavenging ability Use 2.45 mmol·L -1 Prepare 7 mmol·L⁻¹ solution using potassium persulfate as solvent. -1 ABTS solution, incubated in the dark at room temperature for 12–16 h. Use 0.01 mol·L⁻¹ -1The ABTS solution was diluted with phosphate buffered saline (PBS) at pH 7.2 until the absorbance at 734 nm was approximately 0.7. 20 μL of samples of different concentrations were mixed thoroughly with 2 mL of the aforementioned solution and allowed to stand for 10 min. The absorbance at 734 nm was then measured using a multi-mode microplate reader. An ABTS solution without sample was used as a blank control, and a vitamin C aqueous solution was used as a positive control. The ABTS free radical scavenging rate was calculated using the following formula.

[0045] ABTS free radical scavenging rate (%) =

[0046] (5) Reducing power test Take 100 μL of sample solutions of different concentrations and 250 μL of 0.2 mol·L⁻¹ -1 Mix PBS solution (pH 6.6) and 250 μL potassium ferricyanide solution (1% by volume) thoroughly. Incubate at 50°C with shaking for 20 min. After the reaction, add 2.5 mL trichloroacetic acid solution (10% by volume) and mix well. Centrifuge at 8000 rpm for 5 min. Mix 250 μL of supernatant, 250 μL of ultrapure water, and 500 μL of ferric chloride solution (0.1% by volume). Then, measure the absorbance of the mixture at 700 nm using a multi-mode microplate reader. Use ultrapure water as a blank control and VC aqueous solution as a positive control. The absorbance value obtained is the reducing power value.

[0047] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0048] Example 1 0.25 g of mercaptoethylamine hydrochloride and 0.618 g of triphenylchloromethane were weighed and dissolved in a DMF:DCM mixture of 1:1. The mixture was stirred at room temperature for 24 hours under nitrogen protection. The solvent was removed by rotary evaporation. The yellow oily liquid was added to a 3M sodium hydroxide aqueous solution and extracted with DCM (3 × 10 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain a dark orange-yellow oily compound a. Figure 1 Compound 3) 0.56 g, yield 80.12%.

[0049] ESI-MS: m / z 273 [(MeOTr)] + ], 350 [(M + H) + ]. 1H NMR (400 MHz, CDCl3) δ7.46 – 7.35 (m, 5H), 7.35 – 7.22 (m, 5H), 7.26 – 7.13 (m, 2H), 6.84 – 6.76(m, 2H), 3.77 (s, 3H), 2.92 (s, 3H), 2.86 (s, 3H), 2.58 (t, J = 6.5 Hz, 2H), 2.33 (t, J = 6.6 Hz, 2H), 1.77 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 162.52,158.08, 145.20, 136.94, 130.76, 130.70, 129.48, 129.41, 127.95, 127.84,126.74, 126.59, 113.23, 113.12, 66.09, 55.20, 40.97, 36.43, 36.06, 31.40.

[0050] Example 2 Weigh 0.346 g of (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid and dissolve it in 15 ml of anhydrous DMF. Add 0.15 g of HObt, 0.33 mL of NMM, and 0.383 g of EDCI·HCl. After reacting at room temperature for 1 hour, dissolve compound a from Example 1 in anhydrous DMF and add it dropwise to the reaction system. After the reaction is complete, dilute the reaction system with 20 mL of deionized water and 20 mL of ethyl acetate. Wash the organic phase with 1 M hydrochloric acid (3 × 20 mL), saturated sodium carbonate solution (3 × 20 mL), and saturated brine (3 × 20 mL). Purify the crude product by silica gel column chromatography with a column chromatography elution gradient of DCM / methanol (CH3OH) 0 / 100~10 / 90. Remove the solvent by rotary evaporation to obtain a white powder compound b. Figure 1 Compound 5) 0.156 g, yield 23%.

[0051] ESI-MS: m / z 273 [(MeOTr)] + ], 700 [(M + Na) + ]. 1H NMR (400 MHz, CDCl3) δ7.44 – 7.35 (m, 4H), 7.28 (tdd, J = 8.0, 5.9, 2.4 Hz, 6H), 7.26 – 7.14 (m,2H), 6.86 – 6.77 (m, 2H), 3.78 (s, 3H), 3.07 (q, J = 6.1 Hz, 4H), 2.40 (tq, J= 12.8, 6.3 Hz, 2H), 1.90 – 1.72 (m, 1H), 1.42 (d, J = 9.8 Hz, 18H), 1.39 –1.22 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 171.84, 158.18, 156.14, 155.68,144.95, 136.68, 130.73, 129.44, 129.35, 127.94, 127.87, 126.77, 126.73,113.23, 113.20, 80.04, 79.12, 77.25, 66.35, 55.24, 54.37, 39.96, 38.25,32.09, 31.85, 29.71, 28.46, 28.33, 22.62.

[0052] Example 3 0.2 g of compound b was weighed and dissolved in a mixture of DCM (3.75 ml) and TFA (1.25 ml). 0.5 mL of TIS was added, and the mixture was stirred at room temperature for 1 h. The solvent was removed by rotary evaporation, and impurities were washed with anhydrous diethyl ether. The mixture was then repeatedly lyophilized with 0.1 M hydrochloric acid to obtain yellow crystalline compound c. Figure 1 Compound 6) 62.6 mg, yield 75%.

[0053] ESI-MS: m / z 206 [(M + H) + ], 1 H NMR (400 MHz, MeOD) δ 3.84 (dt, J = 9.8,6.5 Hz, 1H), 3.54 – 3.32 (m, 2H), 3.01 – 2.89 (m, 2H), 2.85 – 2.55 (m, 2H),1.99 – 1.78 (m, 2H), 1.77 – 1.64 (m, 2H), 1.49 (p, J = 8.1 Hz, 2H), 1.32 (s,1H). 13C NMR (101 MHz, MeOD) δ 168.73, 161.11, 118.05, 115.15, 52.82, 48.10, 42.54, 39.91, 38.87, 30.69, 30.01, 26.73, 22.94, 21.51.

[0054] Example 4 13.9 mg of compound c and 81.9 mg of sialyl lactose (3'-SL) and 15.7 mg of sodium cyanoborohydride (NaBH3CN) were weighed and dissolved in anhydrous methanol. The mixture was reacted at 50-60°C for 24-48 hours, filtered, washed with methanol, and dried under vacuum to obtain a white powder, compound d. Figure 1 Compound 8) 41.6 mg, yield 58%. MALDI-TOF mass spectrum as shown. Figure 2 As shown.

[0055] MALDI-TOF-MS: m / z 1440.573 [(M + H) + ] , m / z 1462.571[(M + Na) + ],m / z1484.542 [(M + 2Na-H) + ],m / z 1506.554[(M + 3Na-2H) + ].

[0056] Example 5 10 mg of compound d and 10 mg of maleimide-polyethylene glycol-acetaldehyde (MAL-PEG1000-CHO) were weighed and dissolved in 5 ml of phosphate buffer solution (pH 6.5-7.5). The mixture was reacted under nitrogen protection for one hour, and the solution was dialyzed to remove salts. The mixture was then freeze-dried to obtain a yellow compound e. Figure 1 Compound 10 (PEG-disia).

[0057] Molecular weight determination: Mw 3473 ( Figure 3 ), nuclear magnetic resonance spectrum ( Figure 4 The chemical shift of the hydrogen on the carbon atom linking the hydroxyl group in the sugar chain is between 3.4 ppm and 4.7 ppm, and the peak shift of the methylene group in the polyethylene glycol ether bond is 3.69 ppm.

[0058] Example 6 Weigh out 60 mg of compound e, sodium cyanoborohydride (NaBH3CN) to a final concentration of 5 mg / ml and lactoferrin 20 mg, dissolve in 5 ml sodium borate buffer (pH 9.2), react at 0℃ for 24-48 hours, dialyze to remove salt, and freeze dry to obtain the product.

[0059] Test example: The sialic acid content and antioxidant activity of resialylated lactoferrin were determined. Results are as follows: Figure 5 and Figure 6 As shown, Figure 5 The results showed that, after the reaction, the sialic acid content measured by covalently linking 20 mg of natural lactoferrin with 60 mg of compound e (LF-PEG-disia) was 27.6 μg / mg (based on per mg of lactoferrin), and the sialic acid content of protolactoferrin was 1.7 μg / mg (based on per mg of lactoferrin). The sialic acid content measured by covalently linking 5 mg of desialized lactoferrin with 60 mg of compound e (desiaLF-PEG-disia) was 177.3 μg / mg, and the sialic acid content of protolactoferrin was 0.8 μg / mg (based on per mg of lactoferrin). The control group consisted of natural lactoferrin without compound e.

[0060] Figure 6 The results showed that at a mass concentration of 4 g / L, the ABTS free radical scavenging rate of resialylated lactoferrin (sia-LF) was 23.9%, significantly higher than that of ( P <0.05) native-LF 14.8%, DPPH free radical scavenging rate 42.4%, significantly higher than ( P <0.05) Prolactin 13.4%, hydroxyl radical scavenging rate was 37.6%, significantly higher than ( P <0.05) Prolactin was 20.58%, while the reducing power assay showed no significant difference. This indicates that resialylated lactoferrin significantly improves the scavenging rate of free radicals, and exhibits a dose-dependent effect.

[0061] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for synthesizing a dual-antenna type sialic acid polysaccharide, characterized in that, Includes the following steps: S1. Mercaptoethylamine hydrochloride and triphenylchloromethane are dissolved in an organic solvent and stirred under a protective gas atmosphere to obtain compound a; S2. Dissolve (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid in an organic solvent, add a coupling catalyst, a basic catalyst, and a condensing agent to obtain a reaction system. Dissolve compound a obtained in step S1 in an organic solvent and add it dropwise to the reaction system to carry out the reaction. The reaction product is diluted, washed, and subjected to column chromatography to obtain compound b. S3. Dissolve compound b obtained in step S2 in an organic solvent, add triisopropylsilane ether, stir the reaction, remove the solvent and wash to remove impurities, and freeze dry to obtain compound c. S4. The compound c obtained in step S3, sialylated lactose, and sodium cyanoborohydride are dissolved in an organic solvent and reacted at 50-60°C for 24-48 hours under a protective gas atmosphere. The mixture is then filtered, washed, and dried to obtain compound d. S5. The compound d obtained in step S4 is dissolved in a buffer solution with maleimide-polyethylene glycol-acetaldehyde and reacted under protective gas conditions. After the reaction, the salt is removed to obtain compound e, which is the dual-antenna type sialic acid polysaccharide. In step S1, the molar ratio of mercaptoethylamine hydrochloride to triphenylchloromethane is 1~3:1; In step S2, the molar ratio of (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid to compound a, coupling catalyst, basic catalyst, and condensing agent is 1:1.5~2.5:1~1.5:2~4:1.5~2.5; The coupling catalyst is 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole; The alkaline catalyst is N-methylmorpholine, N,N-diisopropylethylamine, triethylamine, or dimethylaminopyridine; The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, tetramethylchloroformamidine hexafluorophosphate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, hexafluorophosphate (benzotriazol-1-yloxy)tripyrrolylphosphide, N,N'-carbonyldiimidazole, hexafluorophosphate O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea or hexafluorophosphate O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea.

2. The synthesis method according to claim 1, characterized in that, The organic solvent is one or a mixture of dimethylformamide, dichloromethane, trifluoroacetic acid, and methanol.

3. The synthesis method according to claim 1, characterized in that, In step S2, the washing process involves sequentially washing with 0.5-2M hydrochloric acid, saturated sodium carbonate solution, and saturated sodium chloride solution.

4. The synthesis method according to claim 1, characterized in that, In step S3, the amount of triisopropylsilane ether added is greater than 10 times the molar equivalent of compound b.

5. The synthesis method according to claim 1, characterized in that, In step S4, the molar ratio of compound c to sialyl lactose and sodium cyanoborohydride is 1:2~3:4~6.

6. A dual-antenna type sialic acid polysaccharide prepared by the synthesis method according to any one of claims 1 to 5, having the molecular formula: ， in, n=11~45。 7. The application of the dual-antenna type sialyl polysaccharide of claim 6 in protein resialylation.

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