N-benzylated sialylglycan derivatives at position c-9 and their preparation and use

Abstract

A C-9 position N-benzylated sialic acid glycan derivative and a preparation method thereof, a structural formula of which is as follows: wherein R1 is phenyl or substituted phenyl; R2 is acetyl (Ac), glycolyl (Gc) or fluoroacetyl (F-Ac or FAc); and R3 is a glycosyl group. The compound prepared by the method has extremely strong specific binding activity to Siglec protein and can be used for preparing a medicine for treating autoimmune diseases, tumors or organ transplantation.

Description

Technical Field

[0001] This invention relates to a technology in the field of bioengineering, specifically a C-9 N-benzylated sialic acid polysaccharide derivative and its preparation and application. Background Technology

[0002] Sialic acid (Sia, SA) is a class of nonacarbonic monosaccharide molecules, typically located at the terminal ends of glycans on glycoproteins or glycolipids. N-acetylneuraminic acid (Neu5Ac) and N-hydroxyacetylneuraminic acid (Neu5Gc) are common sialic acid structures. In the cell nucleus, sialic acid monosaccharides are first converted to nucleoside sugars (CMP-Sia) by cytidine monophosphate-sialic acid synthase (CMP-Sia Synthetase, CSS), and then transported to the Golgi apparatus. Subsequently, under the action of nearly 20 sialotransferases, α-2,3 / 6 / 8-linked sialosides are formed at the glycan terminals of glycoproteins or glycolipids. Sialosides participate extensively in cell biological processes through specific interactions with sia-binding proteins, playing a crucial role in fields such as epidemiology, immunology, and tumor biology. Summary of the Invention

[0003] This invention addresses the shortcomings of existing technologies, such as the inability to effectively synthesize sialic acid-polysaccharide derivatives, the low water solubility of inhibitors, and the inability to resolve the specific glycosidic bond configuration between sialic acid and polysaccharide, which leads to low specificity and off-target problems. It proposes a C-9 N-benzylated sialic acid-polysaccharide derivative and its preparation method. The resulting compound exhibits extremely strong specific binding activity to Siglec protein and can be used to prepare drugs for the treatment of autoimmune diseases, tumors, or organ transplantation.

[0004] This invention is achieved through the following technical solution:

[0005] This invention relates to a C-9 N-benzylated sialic acid polysaccharide derivative, the structural formula of which is: Wherein: R1 is phenyl or substituted phenyl; R2 is acetyl (Ac), hydroxyacetyl (Gc) or fluoroacetyl (F-Ac or FAc); R3 is glycosyl.

[0006] The structural formula of R1 is as follows: Among them, R4, R5, R6, R7 and R8 are one of hydrogen, chlorine, fluorine, bromine, methyl, methoxy, ethyl, propyl, isopropyl, nitro, hydroxyl, amino, acetamide, benzamide, sulfonamide, carboxyl, phenyl or phenoxy.

[0007] R1 is preferably 2,6-difluoro-4-carboxyphenyl, with the following structural formula:

[0008] The structural formula of R3 is any one of the following:

[0009] Where: R9 is OH (lactose) or NHAc (N-acetyllactosamine); R 10 It is hydrogen, glycosyl group or aglycone.

[0010] The R mentioned 10 Preferably, it contains lactose, N-acetyllactosamine, N-linked glycan, O-linked glycan, or a structural formula thereof. -(CH2CHCO2H) m -R 11 (polyacrylic acid), aglycones of amino acids or polypeptide chains, wherein: m is 0-100, n is 0-20, and the polypeptide chain is a natural protein or a synthetic polypeptide.

[0011] The R mentioned 11 It is one of methyl, methoxy, substituted methoxy, benzyloxy, substituted benzyloxy, hydroxy, amino, acetamino, substituted acetamino, benzoylamino, substituted benzoylamino, N-benzyloxycarbonylamino, N-tert-butoxycarbonylamino, mercapto, azide, allyloxy, propynylated, or maleimide (MAL) group.

[0012] The R3 is preferably a disaccharide Galb1-4GlcNAcb1-ProNH2 (LacNAcbProNH2).

[0013] The C-9 N-benzylated sialic acid polysaccharide derivative is preferably any one of the following structural formulas:

[0014]

[0015] This invention relates to a method for preparing the above-mentioned C-9 N-benzylated sialic acid polysaccharide derivative. The method involves first synthesizing a C-9 NH2-modified sialic acid monosaccharide using a chemical method, then further synthesizing a C-9 NH2-modified sialic acid polysaccharide using an enzymatic method, followed by a reductive amination reaction to synthesize the C-9 N-benzylated sialic acid polysaccharide derivative, and finally removing the protecting group to obtain the final product.

[0016] The aforementioned chemical synthesis of C-9 NH2-modified sialic acid monosaccharides refers to the following: using sialic acid Neu5Ac as a starting material, its carboxyl group is protected with a methyl group; under anhydrous, weakly alkaline, and low-temperature conditions, the 9-position hydroxyl group of sialic acid is selectively protected with Ts (p-toluenesulfonyl group); under heating conditions, the Ts group is substituted with an azide to introduce an azide at the 9-position of sialic acid; and the azide is reduced by palladium-catalyzed hydrogenation on carbon or the Staudinger reaction to obtain C-9 NH2-modified sialic acid monosaccharides, the structural formula of which is as follows.

[0017] The enzymatic synthesis of C-9 NH2-modified sialic acid polysaccharides refers to the following steps: C-9 NH2-modified sialic acid monosaccharides are converted into CMP-activated sialic acid derivatives using CMP-sialic acid synthase (CSS). Then, α2,6-sialic acid transferases (such as Pd2,6ST) transfer the C-9 NH2-modified sialic acid to the 6-hydroxyl group on the non-reducing end of the galactose of the polysaccharide (such as LacNAc), yielding C-9 NH2-modified sialic acid polysaccharides. The structural formula is as follows:

[0018] The reductive amination reaction for synthesizing N-benzylated sialic acid polysaccharide derivatives refers to: in the presence of a reducing agent at room temperature, a sialic acid polysaccharide modified with NH₂ at the C-9 position undergoes a reductive amination reaction with a benzaldehyde derivative, introducing a benzyl group at the 9-position amino group to obtain a C-9 N-benzylated sialic acid polysaccharide derivative, the structural formula of which is...

[0019] The removal of protecting groups includes: removing the Boc protecting group on the amino group by TFA, removing the Fmoc (fluorene methoxycarbonyl) protecting group on the amino group by piperidine, removing the Cbz or Bn protecting group by Pd-C catalytic hydrogenation, and reducing the azide group to an amino group by trimethylphosphine or palladium on carbon catalytic hydrogenation, to obtain the C-9 N-benzylated sialic acid polysaccharide derivative.

[0020] When R2 in the structure is a hydroxyacetyl (Gc) or fluoroacetyl (FAc), Neu5Gc9NH2 or Neu5FAc9NH2 is synthesized first, with the following structural formula: Then, the corresponding C-9 N-benzylated sialic acid polysaccharide derivative is synthesized using the method described above.

[0021] The synthesis of Neu5Gc9NH2 and Neu5FAc9NH2 is achieved using, but not limited to, the techniques described in [Peng, W.; Paulson, J.; J. Am. Chem. Soc. 2017, 139: 12450-12458.].

[0022] When R3 in the structure is a polymer of lactose, N-acetylglucosamine, or a product thereof derived from an aglycone, it is preferable to prepare the polysaccharide aglycone derivative by chemical and / or enzymatic methods and then synthesize its N-benzylated sialic acid polysaccharide derivative by the above method.

[0023] When the sialic acid derivative in the structure is linked in an alpha configuration to the 3-hydroxyl group of the non-reducing terminal galactose as R3, an α2,3-sialyltransferase (e.g., PmST1) is used to enzymatically synthesize sialyl polysaccharide modified with NH2 at the C-9 position.

[0024] This invention relates to the application of the above-mentioned C-9 N-benzylated sialic acid polysaccharide derivative for the preparation of drugs for autoimmune diseases, targeting tumor cells or organ transplantation.

[0025] The autoimmune diseases mentioned refer to: hypersensitivity reactions, rheumatoid arthritis, and systemic lupus erythematosus.

[0026] The targeted tumor cells mentioned refer to B-cell lymphoma and acute myeloid leukemia (AML). Technical effect

[0027] This invention synthesizes a C-9 N-benzyl sialic acid polysaccharide derivative with nearly 10-fold increased affinity and inhibitory activity for mouse-derived Siglec-2 (mCD22) via a reductive amination reaction. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the synthesis route in Example 1;

[0029] Figure 2 This is a schematic diagram of the synthesis route in Example 2.

[0030] Figure 3 and Figure 4 This is a data graph for Example 3. Detailed Implementation Example 1

[0031] like Figure 1 As shown, this embodiment relates to the synthesis of C-9 N-benzylated Neu5Ac sialyl polysaccharide, whose structural formula is as follows: The specific steps include:

[0032] Step a: Prepare Methyl 9-Tosyl Neu5Ac ester (9-p-Toluenesulfonyl-Sialate Methyl ester, 6), with the following structural formula: Specifically, it includes:

[0033] a.1 Sialic acid Neu5Ac (10.0 g, 32.6 mmol) and Dowex acidic resin (10.0 g) were dissolved in 200 mL of methanol and stirred at room temperature for 4 days until the solution was clear. After filtering the resin, the filtrate was concentrated under reduced pressure to obtain product 5.

[0034] a.2 Product 5 was dissolved in anhydrous pyridine (300 mL), and p-toluenesulfonyl chloride (18.6 g, 97.7 mmol, 4.0 equiv.) was added in portions with stirring in an ice bath. The mixture was then allowed to warm to room temperature and stirred overnight. After the reaction was complete, the reaction was quenched with methanol in an ice bath. After filtration, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (EA:MeOH = 30:1) to give product 6 (12.9 g, 27.06 mmol, 83%). 1 HNMR(400MHz,D2O):d 7.20(d,J=8.2Hz,2H),7.08(d,J=8.2Hz,2H),4.23(t,J=9.7Hz,1H),4.10–4.00(m,1H),3.78–3.68(m,1H),3.57–3. 47(m,2H),3.48–3.38(m,2H),3.03(d,J=5.9Hz,1H),2.69(d,J=26.5Hz,1H),2.62–2.50(m,1H),2.32–2.16(s,6H,2x CH3),1.90(s,3H).

[0035] Step b: Prepare 9-Azido Neu5Ac (9-azido-sialic acid, 7), whose structural formula is as follows: Specifically, the process involved dissolving product 6 (9.30 g, 20.0 mmol) and sodium azide (1.95 g, 1.5 equiv.) in a mixture of acetone (80 mL) and water (20 mL). Under nitrogen protection, the mixture was stirred overnight at 65 °C, and the solution was analyzed by TLC (iPrOH:NH). 3. The reaction was monitored using H2O:H2O = 7:2:1. After the reaction was complete, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (EA:MeOH = 1:1) to obtain product 7 (5.67 g, 85%). 1 HNMR(400MHz,D2O):d 4.08–3.88(m,4H),3.62(dd,J=13.1,2.8Hz,1H),3.56–3.45(m,2H),2.23(dd ,J=13.0,4.9Hz,1H),2.07(s,3H),1.84(t,J=12.0Hz,1H).ESI-MS:m / zcalcd forC11H18N4O8[M+H] + 335.1; found 335.3.

[0036] Step c: Prepare 9-Amino Neu5Ac (9-aminosialic acid, 8) using any of the following methods, with the following structural formula: Specifically, it includes:

[0037] Option 1: Dissolve product 7 (2.54 g, 7.6 mmol) in a methanol (25 mL)-water (25 mL) mixture, add a catalytic amount of 10% Pd-C, and insert a hydrogen balloon (1 atm). Stir overnight at room temperature. TLC (iPrOH:NH) 3. The reaction was monitored using H2O:H2O = 5:2:1. After the reaction was complete, the product was filtered through diatomaceous earth, concentrated under reduced pressure, purified by Sephadex G15 gel column (mobile phase: 100 mM NH4HCO3), and freeze-dried to obtain product 8 (2.22 g, 95%).

[0038] Option 2: Dissolve product 7 (1.27 g, 3.8 mmol) in 20 mL of water, and slowly add PMe3 (1.0 M in THF, 1.5 equiv., 5.7 mL). Stir at room temperature for 2 h, and perform TLC (iPrOH:NH) 3. The reaction was monitored using H2O:H2O = 5:2:1. After the reaction was completed, the mixture was concentrated under reduced pressure, purified by Sephadex G15 gel column chromatography (mobile phase: 100 mM NH4HCO3), and freeze-dried to obtain product 8 (1.05 g, 90%).

[0039] 1 H NMR (400MHz, D2O): d 4.09–3.81 (m, 4H), 3.49 (d, J=8.5Hz, 1H), 3.37 (dd, J= 13.1,3.3Hz,1H),2.93(dd,J=13.2,9.5Hz,1H),2.20(dd,J=12.8,4.8Hz,1H),2.03(s,3H),1.81(t,J=12.0Hz,1H); ESI-MS:m / z calcd for C11H20N2O8[M+H] + 308.1; found 308.3.

[0040] Step d: Prepare 9-NH2-Neu5Aca2-6Galb1-4GlcNAcb1-ProN3 (9-aminoNeu5Ac sialoglycoside, 10), the structural formula of which is Specifically, the reaction involved dissolving substrate N-acetyllactosamine 9 (100 mg, 0.22 mmol), product 8 (132.6 mg, 2.0 equiv.), and CTP (231.9 mg, 2.0 equiv.) in 10.7 mL of Tris buffer (100 mM, pH 8.5, containing 20 mM MgCl2). Then, appropriate amounts of recombinant CSS protein (NmCSS) derived from Neisseria meningitidis and sialidyltransferase Pd2,6ST were added, and the mixture was shaken overnight at 37°C. After the reaction, the product was purified using a Sephadex G15 gel column (mobile phase: 100 mM NH4HCO3) and lyophilized to obtain product 10 (147.6 mg, 91%). 1 H NMR (700MHz, D2O):d 4.57(d,J=7.8Hz,1H),4.46(d,J=7.9Hz,1H),4.08(td,J=9.3,2.8Hz,1H),4.02–3.92(m,4H),3.88–3.73(m,5H),3.70–3.6 0(m,5H),3.57–3.50(m,3H),3.47–3.36(m,3H),3.04(dd,J=13.1,9.4Hz,1H),2.67(dd,J=12.6,4.5Hz,1H),2.06(s,6H,2x CH3),1.86(m,2H),1.72(t,J=12.0Hz,1H); 13 C NMR (176MHz, D2O):d 175.0,174.6,103.5,100.9,80.7,74.4,73.6,72.4,72.3,70.7,70.3,68.4,68.0, 67.9,67.2,63.3,60.3,54.9,51.8,47.8,42.3,40.1,28.1,22.3,22.1; ESI-MS:m / z calcd for C 28 H 49 N6O 18 [M+H] + :757.3,found:757.4.

[0041] Step e: Prepare 9-N-benzyl Neu5Ac-Sialoside Derivative (9-N-benzylated Neu5Ac sialoside, 12), with the following structural formula: Specifically, the reaction involved dissolving product 10 (90 mg, 0.12 mmol) and 3,5-difluoro-4-aldehyde benzoic acid 11 (45 mg, 2.0 equiv.) in 1.0 mL of H₂O, slowly adding sodium cyanoborohydride (30 mg, 4.0 equiv.), and stirring overnight at room temperature. After the reaction was complete, the reaction was quenched, and product 12 (96.7 mg, 87%) was obtained by RP-HPLC purification. 1 H NMR (700MHz, D2O):d 7.73(d,J=7.9Hz,2H),4.55(d,J=8.3Hz,1H),4.52(s,2H),4.45(d,J=7.9Hz,1H),4.19( ddd,J=9.8,8.3,3.0Hz,1H),4.02–3.94(m,3H),3.93(d,J=3.5Hz,1H),3.86–3.80(m,4H ),3.74–3.65(m,5H),3.64–3.57(m,3H),3.56–3.49(m,3H),3.23(dd,J=13.0,9.9Hz,1H ),2.67(dd,J=12.6,4.6Hz,1H),2.04(s,6H),1.85–1.84(m,2H),1.76(t,J=12.2Hz,1H); 13 C NMR (176MHz, D2O):d 175.0,174.5,172.4,167.6,135.1,117.2,115.5,113.2,113.1,113.0,111.7,111.6,103.5,100.9,99.7,80.6,74.4,73.5,72. 5,72.4,72.4,70.7,70.5,68.3,67.6,67.1,66.7,63.2,60.3,54.9,51.7,50.1,47.8,39.6,38.4,28.1,22.2,22.0; ESI-MS: m / z calcd forC36H56F2N7O20[M+NH4] + :944.4,found:944.5.

[0042] Step f: Prepare the 9-N-benzyl Neu5Ac-Sialoside Derivative final product (3), with the following structural formula: Specifically, product 12 (50 mg, 54.1 mmol) was dissolved in 200 mL of H2O, and PMe3 (1 M in THF, 108 mL, 2.0 equiv.) was slowly added dropwise while stirring at room temperature for 2 h. After the reaction was completed, product 3 (43.3 mg, 89%) was obtained by RP-HPLC purification. 1 H NMR (700MHz, D2O):d 7.74(d,J=7.9Hz,2H),4.55–4.52(m,3H),4.45(d,J=8.0Hz,1H),4.21–4.17(m,1H),4.05–3. 97(m,3H),3.93(d,J=3.4Hz,1H),3.86–3.77(m,4H),3.76–3.66(m,5H),3.63–3.60(m,2H),3. 58(dd,J=10.1,3.5Hz,1H),3.56–3.49(m,3H),3.23(dd,J=13.0,10.0Hz,1H),3.09(t,J=7.0H z,2H),2.66(dd,J=12.5,4.6Hz,1H),2.07(s,6H),1.98–1.93(m,2H),1.74(t,J=12.2Hz,1H); 13 C NMR(176MHz,D2O):d 174.7,172.7,160.5,135.4,117.2,113.1,113.0,103.6,101.0,99.9,80.8,74.4,73.6,72.4,72.2,70.7, 70.5,68.3,67.9,67.7,66.7,63.3,60.2,54.8,51.7,50.1,39.8,38.4,37.6,26.6,22.2,22.0; ESI-MS:m / z calcd for C36H55F2N4O20[M+H] + :901.3,found:901.5.

[0043] Step g: Prepare Biotinylated 9-N-benzyl Neu5Ac-Sialoside Derivative (biotinylated 9-N-benzylated Neu5Ac sialoside final product, 13), with the following structural formula: Specifically, the reaction involved dissolving product 3 (12 mg, 13.3 mmol) and NaHCO3 (11.2 mg, 10 equiv.) in dimethyl sulfoxide (200 mL) - water (200 mL), adding NHS-LC-LC-Biotin (15.1 mg, 2.0 equiv.) in portions, and stirring overnight at room temperature. After the reaction was complete, methanol was added to quench the reaction, and the product was purified by RP-HPLC after evaporation to obtain product 13 (17.1 mg, 95%). 1 HNMR (700MHz, D2O):d 7.74(d,J=8.0Hz,2H),4.61(dd,J=8.0,4.8Hz,1H),4.53(d,J=8.5Hz,3H),4.4 6–4.41(m,2H),4.19(td,J=9.8,9.2,3.0Hz,1H),4.03–3.96(m,2H),3.95–3.8 9(m,2H),3.85–3.84(m,4H),3.69(ddd,J=13.6,11.0,5.7Hz,4H),3.60(ddt,J =18.6,10.0,5.0Hz,4H),3.53(td,J=10.2,4.6Hz,3H),3.34(dd,J=9.6,4.9Hz, 1H),3.30–3.22(m,2H),3.17(td,J=6.8,3.4Hz,5H),3.00(dt,J=13.1,4.8Hz, 1H), 2.78 (d, J=13.0Hz, 1H), 2.67 (dd, J=12.5, 4.7Hz, 1H), 2.24 (td, J=7.1, 2. 9Hz, 6H), 2.04 (s, 6H), 1.79–1.71 (m, 5H), 1.65–1.58 (m, 7H), 1.51 (dd, J=10.7 ,5.0Hz,5H),1.41(dq,J=13.8,5.1,4.4Hz,2H),1.33–1.28(m,4H); ESI-MS:m / z calcd for C58H91F2N8O24S[M+H] + :1353.6,found:1353.5. Example 2

[0044] like Figure 2 As shown, this embodiment relates to the preparation of C9-N-benzylated Neu5Gc sialyl polysaccharide, whose structural formula is as follows: The specific steps include:

[0045] Step a: Prepare Methyl 2,4,7,8,9-penta-O-Ac Neu5Ac ester (2,4,7,8,9-penta-oxo-acetyl-sialic acid methyl ester, 14), with the following structural formula: Specifically, the reaction proceeded as follows: Product 5 (20.1 g, 62.2 mmol) and a catalytic amount of 4-N,N-dimethylaminopyridine (DMAP) were dissolved in anhydrous pyridine (500.0 mL), and acetic anhydride (58.3 mL, 10.0 equiv.) was added dropwise at 0 °C. The reaction mixture was stirred overnight at room temperature, and the reaction was monitored by TLC (ethyl acetate: petroleum ether = 3:1). After the reaction was complete, the pyridine was removed by concentration under reduced pressure. The residue was diluted with dichloromethane, and then washed successively with water, 1.0 N HCl, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and dried to obtain product 14 (33.2 g, 96%). ESI-MS: m / z calcd for C22H31NO14Na[M+Na] + 556.2; found 556.3.

[0046] Step b: Prepare Methyl p-tolyl-2,4,7,8,9-penta-O-Ac-2-thio Neu5Ac ester (2,4,7,8,9-penta-oxo-acetyl-2-p-toluenethiophenol-sialic acid methyl ester, 15), with the following structural formula: Specifically, the reaction mixture consisted of: dissolving product 14 (16.2 g, 29.1 mmol) and p-methylthiophenol (11.4 g, 3.0 equiv.) in anhydrous dichloromethane (500.0 mL), and adding boron trifluoride diethyl ether solution (24.1 mL, 3.0 equiv.) dropwise to the reaction system at 0 °C. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC (ethyl acetate: petroleum ether = 3:1). After the reaction was complete, the reaction was quenched by adding saturated sodium bicarbonate solution at 0 °C, followed by washing with water, saturated sodium bicarbonate solution, and saturated saline solution, respectively. The organic phase was dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:2) to obtain product 15 (17.0 g, 94%, a / b = 3:1).

[0047] αisomer: 1H NMR (400MHz, CDCl3):d 7.32(d,J=7.9Hz,2H),7.07(d,J=7.8Hz,2H),5.25–5.16(m,2H),5.09(d,J=9.9Hz,1H),4 .77(td,J=11.0,4.6Hz,1H),4.34(dd,J=12.4,2.5Hz,1H),4.14(dd,J=12.4,5.4Hz,1H), 3.94–3.85(m,1H),3.81(dd,J=10.7,1.8Hz,1H),3.54(s,3H),2.71(dd,J=12.9,4.7Hz,1 H),2.30(s,3H),2.07(s,3H),1.99(s,3H),1.98(s,3H),1.95(s,3H),1.93–1.88(m,1H).

[0048] β isomer: 1 H NMR (400MHz, CDCl3):d 7.32(d,J=7.9Hz,2H),7.13(d,J=7.9Hz,2H),5.56(d,J=10.3Hz,1H),5.46(t,J=2.5Hz,1H),5.38(t d,J=11.1,4.8Hz,1H),4.96(dt,J=8.7,2.3Hz,1H),4.61(dd,J=10.5,2.5Hz,1H),4.49(dd,J=12.2,2 .2Hz,1H),4.17–4.07(m,1H),4.02(dd,J=12.3,8.5Hz,1H),3.60(s,3H),2.64(dd,J=13.9,4.8Hz,1H ),2.33(s,3H),2.12–2.10(m,1H),2.10(s,3H),2.08(s,3H),2.03(s,3H),1.96(s,3H),1.90(s,3H).

[0049] ESI-MS:m / z calcd for C27H35NO12SNa[M+Na] + 620.2; found 620.3.

[0050] Step c: Prepare Methyl p-tolyl-2-thioNeu5Ac ester (p-toluenethiophenol-sialoside methyl ester, 16), with the following structural formula: Specifically, the reaction proceeded as follows: Compound 15 (5.0 g, 8.06 mmol) was dissolved in anhydrous methanol (50.0 mL), and a catalytic amount of sodium methoxide was added to the reaction system. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by TLC (DCM:MeOH = 8:1). After the reaction was complete, the reaction was neutralized with Amberlite IR-120 acidic ion exchange resin. The mixture was filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 10:1) to obtain product 16 (3.5 g, 96%). 1 H NMR (400MHz, CD3OD):d 7.48(d,J=7.9Hz,2H),7.18(d,J=7.8Hz,2H),4.52(d,J=10.1Hz,1H),4.12(td,J=10.6,4.5Hz,1H),3.96–3.76(m,3H),3.68(dd,J=10.8,5.0Hz,1 H),3.57(d,J=8.5Hz,1H),3.54(s,3H),2.69(dd,J=13.6,4.6Hz,1H),2.35(s,3H),2.05(s,3H),1.96(dd,J=13.6,11.6Hz,1H).ESI-MS:m / zcalcd for C19H27NO8SNa[M+Na] + 452.2; found 452.2.

[0051] Step d: Preparation of Methyl 9-tosyl-p-tolyl-2-thioNeu5Ac ester (9-p-toluenesulfonyl-2-p-toluenethiophenol-sialoside methyl ester, 17), with the following structural formula: Specifically, the reaction proceeded as follows: Compound 16 (4.5 g, 9.96 mmol) was dissolved in anhydrous pyridine (50 mL), and p-toluenesulfonyl chloride (9.0 g, 3.0 equiv.) was added in portions to the reaction mixture at 0 °C. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC (DCM:MeOH = 10:1). After the reaction was complete, methanol was added to quench the reaction. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM:MeOH = 30:1) to give product 17 (4.89 g, 81%). 1HNMR(400MHz,CD3OD):d 7.82(d,J=8.2Hz,2H),7.45(d,J=8.1Hz,2H),7.40(d,J=7.9Hz,2H),7.18(d,J=7.9Hz,2H) ,4.59(s,1H),4.43–4.37(m,1H),4.33(dd,J=9.9,2.3Hz,1H),4.16–4.03(m,2H),3.94–3. 87(m,1H),3.81(t,J=10.2Hz,1H),3.59–3.54(m,1H),3.53(s,3H),2.66(dd,J=13.7,4.7H z,1H),2.47(s,3H),2.35(s,3H),2.04(s,3H),1.91(dd,J=13.7,11.6Hz,1H).ESI-MS:m / z calcd for C26H33NO10S2Na[M+Na] + 606.2; found 606.3.

[0052] Step e: Prepare Methyl 9-azido-p-tolyl-2-thio Neu5Ac ester (9-azido-2-p-toluenethiophenol-sialic acid methyl ester, 18), with the following structural formula: Specifically, the reaction proceeded as follows: Product 17 (3.7 g, 6.1 mmol) and sodium azide (793 mg, 2.0 equiv.) were dissolved in anhydrous N,N-dimethylformamide (100 mL), stirred overnight at 80 °C, and the reaction was monitored by TLC (DCM:MeOH = 15:1). After the reaction was complete, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM:MeOH = 20:1) to obtain product 18 (2.59 g, 89%). 1 H NMR(400MHz,CD3OD):d 7.47–7.41(m,2H),7.19(d,J=7.9Hz,2H),4.49–4.44(m,1H),4.17–4.08(m,1H),3.93–3.83(m,2H),3.64–3.57(m,2H),3.56(s,3H) ,3.42(dd,J=12.8,6.4Hz,1H),2.68(dd,J=13.7,4.7Hz,1H),2.36(s,3H),2.06(s,3H),1.95(dd,J=13.7,11.6Hz,1H).ESI-MS:m / z calcd for C19H26N4O7SNa[M+Na] + 477.2; found 477.2.

[0053] Step f: Prepare Methyl 9-azido-p-tolyl-2-thio Neu5NH2 ester (5-amino-9-azido-2-p-tolyl-thiophenol-sialic acid methyl ester, 19), with the following structural formula: Specifically, product 18 (2.0 g, 4.19 mmol) was dissolved in a methanol-methanesulfonic acid (40 mL) mixture and stirred overnight at 60 °C. The reaction was monitored by TLC (DCM:MeOH = 8:1). After the reaction was complete, saturated sodium bicarbonate solution was added to neutralize the reaction. After concentration under reduced pressure, the product was purified by silica gel column chromatography (DCM:MeOH = 10:1) to obtain product 19 (1.54 g, 89%). 1 H NMR(400MHz,CD3OD):d 7.43(d,J=7.9Hz,2H),7.19(d,J=7.8Hz,2H),4.56(d,J=9.7Hz,1H),4.06–3.9 5(m,1H),3.95–3.88(m,1H),3.80(d,J=8.3Hz,1H),3.63(dd,J=12.9,2.7Hz,1 H),3.59(s,3H),3.45(dd,J=12.9,5.9Hz,1H),2.97(t,J=10.0Hz,1H),2.65(d d,J=13.9,4.0Hz,1H),2.35(s,3H),1.94(t,J=12.4Hz,1H).ESI-MS:m / zcalcd for C17H25N4O6S[M+H] + 413.1; found 413.3.

[0054] Step g: Prepare Methyl 9-azido-p-tolyl-2-thio Neu5Gc ester (5-N-hydroxyacetyl-9-azido-2-p-toluenethiophenol-sialic acid methyl ester, 20), the structural formula of which is Specifically, it includes:

[0055] g.1 Acetoxyacetic acid (571 mg, 2.0 equiv.), HATU (1.84 g, 2.0 equiv.), and DIEA (1.26 mL, 3.0 equiv.) were dissolved in 5.0 mL of anhydrous N,N-dimethylformamide. After stirring at room temperature for 5 min, anhydrous N,N-dimethylformamide (20 mL) containing product 19 (1.0 g, 2.42 mmol) was added. The mixture was stirred at room temperature for 5 h, and the reaction was monitored by TLC (DCM: MeOH = 20:1). After the reaction was completed, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM: EtOAc = 1:3) to obtain the crude product. ESI-MS: m / z calcd for C21H29N4O9S[M+H] + 513.2; found 513.3.

[0056] g.2 The crude product was dissolved in 20.0 mL of anhydrous methanol, and a catalytic amount of sodium methoxide was added to the reaction system at 0 °C. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC (DCM:MeOH = 10:1). After the reaction was completed, Amberlite IR-120 acidic ion exchange resin was added to quench the reaction, and the mixture was filtered and concentrated under reduced pressure to obtain product 20 (0.98 g, 82% in two steps). 1 H NMR(400MHz,CD3OD):d 7.45(d,J=7.9Hz,2H),7.19(d,J=7.9Hz,2H),4.57(d,J=10.5Hz,1H),4.29–4.20(m ,1H),4.09(s,2H),3.97–3.85(m,2H),3.63–3.58(m,2H),3.57(s,3H),3.41(dd,J= 12.8,6.4Hz,1H),2.69(dd,J=13.7,4.7Hz,1H),2.36(s,3H),1.96(dd,J=13.7,11.6Hz,1H).ESIMS:m / z calcd for C19H26N4O8SNa[M+Na] + 493.2; found 493.2.

[0057] Step h: Prepare 9-Azido-p-tolyl-2-thio Neu5Gc (5-N-hydroxyacetyl-9-azido-2-p-toluenethiophenol-sialic acid glycoside, 21), the structural formula of which is Specifically, this includes: 20 (523.4 mg, 1.06 mg) of product... Lithium hydroxide (127.0 mg, 5.0 equiv.) was dissolved in a mixture of THF and water (5.0 mL) at 0 °C. The mixture was stirred at room temperature for 1 h, and the solution was analyzed by TLC (iPrOH:NH4+).3. The reaction was monitored using H2O:H2O = 14:2:1. After the reaction was complete, Amberlite IR-120 acidic ion exchange resin was added to neutralize the reaction, followed by filtration, concentration under reduced pressure, purification using a Sephadex G15 gel column (mobile phase: 100 mM NH4HCO3), and freeze-drying to obtain product 21 (440 mg, 91%). ESI-MS: m / z calcd for C18H25N4O8S[M+H] + 457.1; found 457.1.

[0058] Step i: Prepare 9-Amino-Neu5Gc (9-amino-5-N-hydroxyacetyl-sialic acid, 22), whose structural formula is as follows: Specifically, it includes:

[0059] i.1 Product 21 (440.0 mg, 0.96 mmol) was dissolved in an acetone-water (5.0 mL) mixture, and N-bromosuccinimide (515 mg, 3.0 equiv.) was added at 0 °C. The mixture was stirred at room temperature for 6 h, and TLC (iPrOH:NH) was performed. 3. The reaction was monitored using H2O:H2O = 7:2:1. After the reaction was complete, a saturated sodium bicarbonate solution was added to neutralize the reaction. The solution was concentrated under reduced pressure, purified using a Sephadex G15 gel column (mobile phase: 100 mM NH4HCO3), and then freeze-dried to obtain the crude product. 1 H NMR (600MHz, D2O): d 4.16 (s, 2H), 4.16–4.12 (m, 1H), 4.11 (d, J = 10.6Hz, 1H), 4.01 (t, J = 10.2Hz, 1H), 3.94–3.90 (m, 1H), 3.62(dd,J=13.1,2.8,1H), 3.53(d,J=9.2Hz,1H), 3.49(dd,J=13.1,6.1Hz,1H), 2.24(dd,J=13.0,5.0Hz,1H), 1.86(t,J=12.3Hz,1H); ESI-MS: m / z calcd forC11H18N4O9Na[M+Na] + 373.1; found 373.2.

[0060] i.2 Dissolve the above crude product in 2.0 mL of water, and add PMe3 (1.0 M THF, 2.0 mL, 2.0 equiv.). Stir at room temperature for 2 h, and perform TLC (iPrOH:NH4+). 3.The reaction was monitored using H2O:H2O = 5:2:1. After the reaction was complete, the mixture was concentrated under reduced pressure, purified by Sephadex G15 gel column chromatography (mobile phase: 100 mM NH4HCO3), and lyophilized to obtain product 22 (262 mg). 84%). ESI-MS:m / z calcd for C11H21N2O9[M+H] + 325.1; found 325.2.

[0061] Step j: Prepare 9-Amino-Neu5Gcα-2-6Galβ-1-4GlcNAcβ-1-ProN3 (9-aminoNeu5Gc sialic acid polysaccharide, 24), the structural formula of which is Specifically, the reaction involved dissolving substrate N-acetyllactosamine 9 (50 mg, 0.107 mmol), product 22 (69.7 mg, 2.0 equiv), and CTP (112.8 mg, 2.0 equiv) in 5.35 mL of Tris buffer (100 mM, pH 8.5, containing 20 mM MgCl2), adding appropriate amounts of NmCSS and Pd2,6ST, and shaking overnight at 37°C. After the reaction was complete, the product was purified using a Sephadex G15 gel column (mobile phase: 100 mM NH4HCO3), and then lyophilized to obtain product 24 (73.5 mg, 89%). 1 H NMR(700MHz,D2O):d 4.58(d,J=7.9Hz,1H),4.47(d,J=7.9Hz,1H),4.18–4.07(m,3H),4.02(s,5H),3.92–3.81(m,5H),3.77(m,3H),3.71–3.60(m,5H),3.58–3.5 2(m,3H),3.45–3.36(m,3H),3.05(dd,J=13.1,9.5Hz,1H),2.70(dd,J=12.4,4.5Hz,1H),2.08(s,3H),1.86(m,2H),1.74(t,J=12.1Hz,1H); 13 C NMR(176MHz,D2O):d 175.8,174.6,103.5,100.9,80.6,74.5,73.6,72.4,72.1,70.7,70.2,68.4,68.0, 67.8,67.2,63.3,61.0,60.3,55.0,51.5,47.8,42.2,40.1,28.1,22.3; ESI-MS:m / z calcd forC28H48N6O19Na[M+Na] + :795.3,found:795.7.

[0062] Step k: Prepare 9-N-benzyl Neu5Gc-Sialoside Derivative (9-N-benzylated Neu5Gc sialic acid polysaccharide, 25), with the following structural formula: Specifically, the reaction involved dissolving product 24 (50 mg, 62.9 mmol) and 3,5-difluoro-4-aldehyde benzoic acid 11 (23.4 mg, 2.0 equiv.) in 0.5 mL of H₂O, slowly adding sodium cyanoborohydride (15.8 mg, 4.0 equiv.), and stirring overnight at room temperature. After the reaction was complete, the reaction was quenched, and product 25 (54.0 mg, 91%) was obtained by RP-HPLC purification. 1 H NMR(700MHz,D2O): δ7.64(d,J=8.0Hz,2H),4.50–4.43(m,3H),4.36(d,J=7.9Hz,1H),4.1 1(td,J=9.8,2.8Hz,1H),4.04(d,J=3.5Hz,2H),3.96–3.84(m,4H),3.83–3.78(m,2H),3.7 8–3.70(m,3H),3.66–3.58(m,4H),3.54–3.40(m,6H),3.30(m,2H),3.13(dd,J=13.0,10. 0Hz,1H),2.60(dd,J=12.5,4.7Hz,1H),1.99(s,3H),1.76(m,2H),1.66(t,J=12.2Hz,1H); 13 C NMR (176MHz, D2O): δ175.8,174.5,174.1,173.0,135.4,124.6,122.5,113.6,112.9,112.9,103.5,100.9,100.1,80.5,74.4,73.5, 72.4,72.3,72.1,70.7,70.4,68.3,67.6,67.1,66.9,63.2,61.0,60.3,54.9,51.4,50.1,47.8,39.9,38.4,28.1,22.2; ESI-MS:m / z calcd for C36H53F2N6O21[M+H] + :943.3,found:943.5.

[0063] Step 1: Prepare 9-N-benzyl Neu5Gc-Sialoside Derivative final product (9-N-benzylated Neu5Gc sialic acid polysaccharide final product, 4), the structural formula of which is Specifically, the reaction involved dissolving product 25 (50 mg, 53.0 mmol) in 200 mL of H2O, slowly adding PMe3 (1 M in THF, 106 mL, 2.0 equiv.), and stirring at room temperature for 2 h. After the reaction was complete, product 4 (43.8 mg, 90%) was obtained by RP-HPLC purification. 1 H NMR(700MHz,D2O):d 7.73(d,J=8.3Hz,2H),4.57–4.51(m,3H),4.45(d,J=7.8Hz,1H),4.25–4.15(m,1H),4.13(d,J=2.6Hz,2H),4.05–3.97(m,3H ),3.93(d,J=3.5Hz,1H),3.90(d,J=4.9Hz,2H),3.86–3.77(m,3H),3.76–3.66(m,5H),3.65–3.60(m,2H),3.60(s,1H),3.58– 3.48(m,3H),3.23(dd,J=12.9,10.0Hz,1H),3.09(t,J=7.0Hz,2H),2.68(dd,J=12.5,4 .6Hz,1H),2.06(s,3H),1.99–1.92(m,2H),1.75(t,J=12.2Hz,1H),1.65–1.52(m,2H); 13 C NMR(176MHz,D2O):d 175.8,174.7,174.7,172.9,133.1,126.4,117.7,117.2,113.1,112.9,103.6,100.9,100.0,80.7,74.4,73.6,72.4,72. 2,72.1,70.7,70.4,68.3,67.9,67.5,66.9,63.4,61.0,60.2,54.9,51.4,50.1,39.9,37.6,26.6,22.2; ESI-MS:m / zcalcd for C36H55F2N4O21[M+H] + :917.8,found:918.5.

[0064] Step m: Prepare Biotinylated 9-N-benzyl Neu5Gc-Sialoside Derivative (biotinylated 9-N-benzylated Neu5Gc sialoside, 26), with the following structural formula: Specifically, the reaction involved dissolving compound 4 (10 mg, 10.9 mmol) and NaHCO3 (9.2 mg, 10 equiv.) in dimethyl sulfoxide-water (200 mL), adding NHS-LC-LC-Biotin (15.1 mg, 2.0 equiv.) in portions, and stirring overnight at room temperature. After the reaction was complete, methanol was added to quench the reaction, and the product was purified by RP-HPLC after evaporation to obtain product 26 (13.7 mg, 92%). 1 H NMR(700MHz,D2O):d 7.55(s,2H),4.56–4.40(m,4H),4.16(d,J=47.5Hz,2H),4.05–3.80(m,7H),3. 79–3.67(m,3H),3.65–3.48(m,5H),3.37–3.30(m,1H),3.30–3.24(m,1H),3.22 -3.12(m,5H),3.00(dd,J=13.0,5.0Hz,1H),2.79(d,J=12.9Hz,1H),2.68(d,J=11.8Hz,1H),2.25(dd,J=8.7,5.6Hz,5H),2.04( s,3H),1.79–1.71(m,3H),1.67–1.57(m,7H),1.51(h,J=6.1,4.9Hz,4H),1.44–1.38(m,2H),1.31(p,J=7.9Hz,4H); ESI-MS:m / z calcd forC58H91F2N8O25S[M+H] + :1368.6,found:1369.5. Example 3

[0065] This embodiment relates to in vitro activity testing, specifically including:

[0066] Step 1: ELISA method to detect the binding constant (K) between sialic acid polysaccharide derivative and mCD22 protein. d): 50 μL / well of Streptavidin in PBS solution (10 μg / mL) was added to each well of a 384-well high-affinity surface microplate and incubated overnight at 4°C. After washing the 384-well plate three times with 100 μL of PBST (PBS + 0.05% Tween-20), 100 μL of blocking buffer (PBS + 3% BSA) was added to each well, and the plate was blocked for 1 hour at room temperature. After removing the blocking buffer, 50 μL x 10.0 μM of biotinylated sialic acid derivatives (e.g., 13 and 26; unmodified sialyptoglycosides were used as a negative control; and the known compound BPA-Neu5Gc was used as a positive control) was added to each well, and the plate was incubated on a shaker for 2 hours at room temperature. After washing three times with 100 μL of PBST, 50 μL of serially diluted mCD22 protein was added to each well (repeat in triplicate), and the plate was incubated for 1 hour at room temperature. After washing three times with 100 μL of PBST, add 50 μL of premixed antibody solution (Rabbit anti-Human IgG Fc and Goat anti-rabbit HRP) to each well and incubate at room temperature for 90 minutes. After washing three times with 100 μL of PBST, add 50 μL of TMB chromogenic solution to each well and incubate at room temperature for 15 minutes. Then, stop the reaction by adding 50 μL of 2M H2SO4. After 20 minutes, measure the OD value at 450 nm using a microplate reader. Plot the protein binding curve and calculate the binding constant between mCD22 protein and sialic acid derivative using GraphPad Prism software.

[0067] The binding curve of the compound to mCD22 is as follows: Figure 3 As shown, the calculated affinities (Kd values) were 20.63 ± 0.25 ug / mL (compound 13), 0.32 ± 0.04 ug / mL (compound 26), and 2.87 ± 0.05 ug / mL (BPA-Neu5Gc). Compared with the positive compound BPA-Neu5Gc, the affinity of the novel structure 26 (C9 N-benzylated Neu5Gc sialic acid derivative) for mCD22 was increased by nearly 9 times.

[0068] Step 2: ELISA was used to detect the inhibitory activity of sialic acid derivatives on mCD22 protein-binding glycoproteins (IC50). 50Fetuin PBS solution (10 μg / mL, 50 μL / well) was added to 384-well high-affinity surface microplates and incubated overnight at 4°C. After washing the 384-well plates three times with 100 μL of PBST (PBS + 0.05% Tween-20), 100 μL of blocking buffer (PBS + 3% BSA) was added to each well and the plates were blocked at room temperature for 1 hour. After removing the blocking buffer, 25 μL of serially diluted sialic acid derivatives (e.g., compounds 3 and 4, unmodified sialyptoglycoside as a negative control, and known compound BPA-Neu5Gc as a positive control) was added to each well. After three replicates, 25 μL of mCD22 premixed with secondary antibodies (Rabbit anti-Human IgG Fc and Goat anti-rabbit HRP) was added and the plates were incubated at room temperature for 90 minutes. After washing three times with 100 μL of PBST, 50 μL of TMB chromogenic solution was added to each well. After developing the color at room temperature for 15 minutes, 50 μL of 2M H2SO4 was added to terminate the reaction. After 20 minutes, the OD value was measured at 450 nm using a microplate reader. The inhibition curve was plotted and the inhibitory activity of sialic acid derivatives on the binding of mCD22 protein to Fetuin glycoprotein was calculated using GraphPad Prism software.

[0069] The inhibition curve of the compound on mCD22 is as follows: Figure 4 As shown, the calculated half-inhibitory concentration (IC50) is... 50 The concentrations were 11.96 ± 0.11 μM (compound 3), 0.27 ± 0.10 μM (compound 4), and 3.45 ± 0.04 μM (BPA-Neu5Gc), respectively. Compared with the positive compound BPA-Neu5Gc, the novel structure 4 (C9 N-benzylated Neu5Gc sialic acid derivative) showed a 12.7-fold increase in inhibitory activity against mCD22.

[0070] The above-described specific implementations can be partially adjusted by those skilled in the art in different ways without departing from the principles and purpose of the present invention. The scope of protection of the present invention is defined by the claims and is not limited to the above-described specific implementations. All implementation schemes within the scope of the claims are bound by the present invention.

Claims

1. A C-9 N-benzylated sialic acid polysaccharide derivative, characterized in that, The structural formula is: Wherein: R1 is phenyl or substituted phenyl; R2 is acetyl, hydroxyacetyl or fluoroacetyl; R3 is glycosyl.

2. The C-9 N-benzylated sialic acid polysaccharide derivative according to claim 1, characterized in that, The R3 mentioned above is galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine, mannose, lactose, N-acetylgalactosamine, or polysaccharides composed of these, as well as glycoproteins. The structural formula of R1 is as follows: Among them, R4, R5, R6, R7 and R8 are one of hydrogen, chlorine, fluorine, bromine, methyl, methoxy, ethyl, propyl, isopropyl, nitro, hydroxyl, amino, acetamide, benzamide, sulfonamide, carboxyl, phenyl or phenoxy.

3. The C-9 N-benzylated sialic acid polysaccharide derivative according to claim 1 or 2, characterized in that, The structural formula of R3 is any one of the following: Wherein: R9 is lactose or N-acetyllactosamine; R 10 It is hydrogen, glycosyl group or aglycone; The R mentioned 10 It is lactose, N-acetyllactosamine, N-linked sugar, O-linked sugar, or has the following structural formula: Aglycones of polyacrylic acid, amino acids, or polypeptide chains, wherein: m is 0-100, n is 0-20, and the polypeptide chain is a natural protein or a synthetic polypeptide; The R mentioned 11 It is one of methyl, methoxy, substituted methoxy, benzyloxy, substituted benzyloxy, hydroxy, amino, acetamino, substituted acetamino, benzoylamino, substituted benzoylamino, N-benzyloxycarbonylamino, N-tert-butoxycarbonylamino, mercapto, azide, allyloxy, propynylated, or maleimide.

4. The C-9 N-benzylated sialic acid polysaccharide derivative according to claim 1 or 2, characterized in that, R1 is 2,6-difluoro-4-carboxyphenyl, and its structural formula is as follows: R3 is a disaccharide Galb1-4GlcNAcb1-ProNH2.

5. The C-9 N-benzylated sialic acid polysaccharide derivative according to any one of claims 1-4, characterized in that, Specifically, it can be any of the following structural formulas:

6. A method for preparing the C-9 N-benzylated sialic acid polysaccharide derivative according to any one of claims 1-5, characterized in that, After synthesizing sialic acid monosaccharides modified with NH2 at the C-9 position using chemical methods, sialic acid polysaccharides modified with NH2 at the C-9 position were further synthesized by enzymatic methods. C-9 N-benzylated sialic acid polysaccharide derivatives were synthesized by reductive amination reaction, and finally the protecting group was removed to obtain the product.

7. The preparation method according to claim 6, characterized in that, The aforementioned chemical synthesis of C-9 NH2-modified sialic acid monosaccharides refers to the following: using sialic acid Neu5Ac as a starting material, its carboxyl group is protected with a methyl group; under anhydrous, weakly alkaline, and low-temperature conditions, the 9-position hydroxyl group of sialic acid is selectively protected with a p-toluenesulfonyl group; under heating conditions, the Ts group is substituted with an azide to introduce an azide at the 9-position of sialic acid; and the azide is reduced by palladium-catalyzed hydrogenation on carbon or the Staudinger reaction to obtain C-9 NH2-modified sialic acid monosaccharides, the structural formula of which is as follows.

8. The preparation method according to claim 6, characterized in that, The enzymatic synthesis of C-9 NH2-modified sialic acid polysaccharide refers to the following steps: C-9 NH2-modified sialic acid monosaccharide is converted into a CMP-activated sialic acid derivative using CMP-sialic acid synthase; then, α2,6-sialic acid transferase is used to transfer the C-9 NH2-modified sialic acid to the 6-hydroxyl group on the non-reducing end of the galactose of the polysaccharide, yielding C-9 NH2-modified sialic acid polysaccharide with the following structural formula:

9. The preparation method according to claim 6, characterized in that, The reductive amination reaction for synthesizing N-benzylated sialic acid polysaccharide derivatives refers to: in the presence of a reducing agent at room temperature, a sialic acid polysaccharide modified with NH₂ at the C-9 position undergoes a reductive amination reaction with a benzaldehyde derivative, introducing a benzyl group at the 9-position amino group to obtain a C-9 N-benzylated sialic acid polysaccharide derivative, the structural formula of which is...

10. The preparation method according to claim 6, characterized in that, The removal of protecting groups includes: removing the Boc protecting group on the amino group by TFA, removing the fluorene methyloxycarbonyl protecting group on the amino group by piperidine, removing the Cbz or Bn protecting group by Pd-C catalytic hydrogenation, and reducing the azide group to an amino group by trimethylphosphine or palladium on carbon catalytic hydrogenation, to obtain the C-9 N-benzylated sialic acid polysaccharide derivative.

11. The preparation method according to claim 6, characterized in that, When R2 is hydroxyacetyl or fluoroacetyl, Neu5Gc9NH2 or Neu5FAc9NH2 is synthesized first, and then the corresponding C-9 N-benzylated sialic acid polysaccharide derivative is synthesized. When R3 is a polymer of lactose, N-acetyl lactose or its composition derived from an aglycone, the polysaccharide aglycone derivative is prepared by chemical and / or enzymatic methods and then its N-benzylated sialic acid polysaccharide derivative is synthesized. When a sialic acid derivative is attached to the 3-position hydroxyl group of the non-reducing terminal galactose (R3) in an alpha configuration, sialic acid polysaccharides modified with NH2 at the C-9 position are synthesized by enzymatic synthesis using α2,3-sialic acid transferase.

12. An application of the C-9 N-benzylated sialic acid polysaccharide derivative according to any one of claims 1-11, characterized in that, Drugs used to prepare for autoimmune diseases, targeting tumor cells, or organ transplantation.

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