A process for the preparation of (4-methylumbelliferyl)-N-acetyl-alpha-D-neuraminic acid sodium salt

Abstract

The application relates to the technical field of organic synthesis, and particularly discloses a method for preparing (4-methyl umbelliferone)-N-acetyl-alpha-D-neuraminic acid sodium salt, which comprises the following steps: taking N-acetylneuraminic acid as raw material, sequentially performing the following steps of methyl esterification under an acid condition, full acetylation in a pyridine and acetic anhydride system, end group chlorination by using a chlorinating reagent, glycosylation with an umbelliferone salt, deacetylation and saponification, and conveniently preparing (4-methyl umbelliferone)-N-acetyl-alpha-D-neuraminic acid sodium salt. Compared with the prior art, the method is simple in operation, high in yield, low in waste, simple in purification mode, high in product purity and easy to realize low-cost, large-scale and rapid preparation.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid. Background Technology

[0002] Neuraminidase, widely distributed on the surface of the influenza virus, can hydrolyze the sialic acid glycosidic bonds at the ends of respiratory sugar chains, further releasing viral particles bound to sialic acid molecules. This allows other host cells to be infected, thus exacerbating influenza symptoms. Influenza virus neuraminidase inhibitors can competitively bind to the influenza virus and, by inhibiting the active site of neuraminidase, slow down or even eliminate the release of viral particles, thereby achieving the goal of treating influenza.

[0003] In influenza virus neuraminidase inhibition experiments, (4-methylumbelliferone)-N-acetyl-α-D-neuraminidase sodium salt (also known as 4-MUNANA, CAS: 76204-02-9) is typically used as a substrate to assess the activity of neuraminidase inhibitors. When neuraminidase on the viral surface recognizes 4-MUNANA, it specifically binds to it and hydrolyzes its α-oxyglycosidic bond, releasing the fluorescent umbelliferone. The neuraminidase inhibitor competitively binds to the active site of neuraminidase, reducing the amount of 4-MUNANA hydrolyzed per unit time, leading to a decrease in the fluorescence value of the umbelliferone. This further results in a lower slope of the fluorescence kinetic curve, and the degree of decrease in the slope of the fluorescence kinetic curve is used to assess the activity of the neuraminidase inhibitor.

[0004] Given the crucial role of (4-methylumbelliferone)-N-acetyl-α-D-neuraminidide sodium salt in investigating the inhibitory activity of influenza virus neuraminidase, it is necessary to conduct in-depth research on its preparation method.

[0005] A search revealed published literature (including but not limited to the following) related to the preparation of sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid, which is relevant to this invention's patent application: 1. Chinese Patent CN107446004B: A method for synthesizing sodium 2-(4'-methylumbelliferone)-alpha-N-acetylneuraminic acid. This patent describes a method for obtaining benzyl ester by esterification of N-acetylneuraminic acid as the starting material. The remaining sites are protected with acetyl groups by glacial acetic acid / acetyl chloride, while a leaving group -Cl is introduced at the 2-position. Subsequently, the 2-position is coupled with 4-methylumbelliferone, and finally the acetyl and benzyl groups are removed to obtain the sodium salt product. However, this synthesis method has the following defects: (1) The preparation of N-acetylneuraminic acid benzyl ester using highly irritating benzyl bromo is inconvenient; (2) After the chlorination reaction, toluene with a high boiling point is used as an azeotropic solvent to remove excess acetyl chloride, which is complicated and the highly active chlorosaccharide is easily degraded at high temperatures; (3) In the glycosylation step, diisopropylethylamine is used as a base, and umbelliferone is used to react with chlorosaccharide to obtain the glycosylated product.

[0006] 2. Journal: Structural Variations of N-Acetylneuraminic Acid, 11,Synthesis of the 4-Methylumbelliferyl-2-α-Glycosides of 7-Epi-, 8-Epi-, and7,8-Bis(epi)-N-acetylneuraminic Acids, as well as of 7-Deoxy-, 8-Deoxy-, 9-Deoxy-, and 4,7-Dideoxy-N-acetylneuraminic Acids and Their Behavior TowardsSialidase from Vibrio cholerae[J]. Liebigs Annalen der Chemie, 1989, p. 519-526. Erich Zbiral et al. reported a method for obtaining the target molecule from N-neuraminic acid via a process involving trifluoroacetic acid-catalyzed methyl esterification, full acetylation in a pyridine-acetic anhydride system, terminal chlorination at -30°C, glycosylation with umbelliferone quaternary ammonium salt, deoxyacetyl group removal, and alkaline hydrolysis. This method has the following drawbacks: (1) it uses the highly volatile acid trifluoroacetic acid as a catalyst to prepare N-acetylneuraminic acid methyl ester; (2) the chlorination reaction is carried out at -30°C, requiring continuous bubbling of hydrochloric acid gas into the reaction system until saturation, resulting in harsh conditions and complex operation; (3) in the glycosylation step, silver carbonate is used as a base, and umbelliferone tetrabutylammonium salt is used to react with chloroglycosylated sugars to obtain the glycosylated product. However, silver carbonate and umbelliferone tetrabutylammonium salt are expensive, leading to high reaction costs.

[0007] By comparison, this invention differs fundamentally from the aforementioned published literature. This invention successfully prepared sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid through a multi-step reaction and fewer purification steps. Therefore, the motivation for this invention lies in using a novel method to prepare sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid.

[0009] To achieve this technical objective, the present invention adopts the following solution: This invention provides a method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid, comprising the following steps: using N-acetylneuraminic acid as a raw material, the mixture undergoes sequential methylation under acidic conditions, full acetylation in a pyridine and acetic anhydride system, terminal chlorination with a chlorinating agent, glycosylation with umbelliferone sodium salt, removal of all oxyacetyl groups, and saponification under alkaline conditions to prepare sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid. Specifically, the method includes the following steps: S1, Methyl esterification: N-acetylneuraminic acid (compound 1) is methyl esterified with methanol in the presence of a cation exchange resin to obtain N-acetylneuraminic acid methyl ester (compound 2). S2, Fully acetylated: N-acetylneuraminic acid methyl ester (compound 2) undergoes a fully acetylated reaction with acetic anhydride to obtain fully acetylated N-acetylneuraminic acid methyl ester (compound 3). S3, Terminal chlorination: The fully acetylated N-acetylneuraminic acid methyl ester (compound 3) reacts with an organic solution of hydrogen chloride to undergo terminal chlorination, yielding the C-2-chlorinated product (compound 4). S4. Glycosylation: The C-2-chlorinated product (compound 4) and the sodium umbelliferone salt (compound 5) undergo a glycosylation reaction under the action of an inexpensive inorganic base promoter to obtain the glycosylated product (compound 6). S5. Deacetylation reaction: The glycosylated product (compound 6) is deacetylated by sodium methoxide in an amount of catalytic solution to remove all O-acetyl groups, yielding the deacetylated product (compound 7). S6. Saponification: The deacetylated product (compound 7) undergoes a saponification reaction in an aqueous sodium hydroxide solution to give sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid (4-MUNANA).

[0010] The reaction pathway is as follows:

[0011] Furthermore, a preferred embodiment of the present invention is as follows: The specific operation of step S1 is as follows: N-acetylneuraminic acid (compound 1) is suspended in 5 to 10 times the volume of methanol, a cation exchange resin is added, the reaction is carried out at room temperature for 8 to 12 h, the cation exchange resin is removed by filtration, and the filtrate is concentrated to obtain N-acetylneuraminic acid methyl ester (compound 2). The specific operation of step S2 is as follows: N-acetylneuraminic acid methyl ester (compound 2) is dissolved in pyridine, 6-10 equivalents of acetic anhydride are added and reacted for 8-12 hours. After quenching the reaction system with methanol, it is diluted with an appropriate amount of dichloromethane, and then washed with dilute acid to remove pyridine. After concentrating the organic phase, fully acetylated N-acetylneuraminic acid methyl ester (compound 3) is obtained. The specific operation of step S3 is as follows: after reacting fully acetylated N-acetylneuraminic acid methyl ester (compound 3) with 2-5 equivalents of hydrogen chloride organic solution at room temperature for 4-8 hours, the reaction system is slowly poured into an ice-cold sodium bicarbonate solution, separated, the organic phase is dried with sodium sulfate, and concentrated to dryness at room temperature to obtain the C-2-chlorinated product (compound 4). The specific operation of step S4 is as follows: the C-2-chloro product (compound 4) and the umbelliferous ketone sodium salt (compound 5) are simultaneously dissolved in an appropriate amount of DMF. At room temperature, an inorganic base is added, and the reaction is continued to be stirred for 8-12 hours. After the reaction is completed, the mixture is filtered, the filtrate is concentrated to dryness, and the glycosylated product (compound 6) is obtained by rapid column chromatography. The specific operation of step S5 is as follows: the glycosylated product (compound 6) is dissolved in an appropriate amount of anhydrous methanol, a catalytic amount of sodium methoxide solution is added, and the reaction is stirred at room temperature for 2-6 hours. After the reaction is completed, the pH value of the system is adjusted to neutral using a cation exchange resin, the cation exchange resin is removed by filtration, and the filtrate is concentrated to obtain the deacetylated product (compound 7), which can be used for the next step of the reaction without purification. The specific operation of step S6 is as follows: the deacetylated product (compound 7) is dissolved in an aqueous solution of sodium hydroxide, and the reaction is stirred at room temperature for 2-6 hours. After the reaction is complete, the filtrate is concentrated to dryness and separated by gel column to obtain sodium salt of (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid (4-MUNANA).

[0012] Furthermore, in step S2, the dilute acid is dilute sulfuric acid, dilute hydrochloric acid, dilute nitric acid, or dilute phosphoric acid, preferably dilute phosphoric acid.

[0013] Further, in step S3, the organic solution of hydrogen chloride is a hydrogen chloride methanol solution, a hydrogen chloride ethanol solution, a hydrogen chloride dioxane solution, or a hydrogen chloride ethyl acetate solution, preferably a hydrogen chloride ethyl acetate solution.

[0014] Further, in step S4, the preparation method of umbelliferone sodium salt (compound 5) is as follows: umbelliferone is dissolved in an appropriate amount of anhydrous methanol, and reacted with an equivalent amount of sodium methoxide at room temperature for 2-6 hours. After concentration, umbelliferone sodium salt (compound 5) is obtained. The preparation of umbelliferone sodium salt (compound 5) is carried out simultaneously with step S3.

[0015] Further, in step S4, the inorganic base is lithium carbonate, sodium carbonate, silver carbonate, potassium carbonate, or cesium carbonate, preferably potassium carbonate.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses ethyl hydrogen chloride solution to react at room temperature for terminal chlorination, which solves the problems of low reaction temperature, low yield and difficult purification reported in relevant literature.

[0017] 2. This invention uses inorganic potassium carbonate as a glycosylation promoter, which conveniently completes the glycosylation reaction and solves the problems of low yield, high cost and difficult purification reported in related literature when using other promoters for glycosylation reactions.

[0018] 3. This invention solves the problems of low yield and inability to scale up the process in current synthesis processes. Attached Figure Description

[0019] Figure 1 The flowchart shows the synthesis process of sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid. Figure 2 The 1H NMR spectrum of 2-O-4-methylumbelliferone-4,7,8,9-tetra-O-acetyl-N-acetyl-α-D-neuraminic acid methyl ester; Figure 3 The carbon NMR spectrum of 2-O-4-methylumbelliferone-4,7,8,9-tetra-O-acetyl-N-acetyl-α-D-neuraminic acid methyl ester; Figure 4 The 1H NMR spectrum of sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid. Figure 5 The image shows the carbon NMR spectrum of sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0021] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available.

[0022] Please see Figure 1 This invention provides a method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid (4-MUNANA), the specific steps of which are as follows: Step 1, Synthesis of N-acetylneuraminic acid methyl ester (Compound 2): N-acetylneuraminic acid (62 g, 0.2 mol) was suspended in 1000 mL of methanol. 3 g of cation exchange resin was added to the system, and the reaction was carried out at room temperature for 12 h, resulting in a pale yellow, clear solution. The cation exchange resin was removed by filtration, and the filtrate was concentrated to dryness to obtain a grayish-white solid, which is Compound 2. Compound 2 can be used directly in the next step of the reaction without purification.

[0023] Step 2, Synthesis of 2,4,7,8,9-penta-O-acetyl-N-acetyl-α-D-neuraminic acid methyl ester (compound 3): Compound 2 was dissolved in 300 mL of pyridine. Acetic anhydride (153 g, 1.5 mol) was added dropwise under an ice-water bath. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 h. Methanol (19.2 g, 0.6 mol) was then slowly added dropwise to the reaction system to quench excess acetic anhydride, and the mixture was stirred for another 0.5 h. The reaction system was then diluted with 1000 mL of dichloromethane. The reaction system was then slowly poured into a 10% dilute phosphoric acid solution containing ice. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (400 mL * 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the dichloromethane was removed by rotary evaporation, yielding a pale yellow foamy solid (106 g, two-step yield 97.0%), which is compound 3. No purification is required, and it can be used directly in the next reaction.

[0024] Step 3, Synthesis of 2-deoxy-2-chloro-4,7,8,9-penta-O-acetyl-N-acetyl-α-D-neuraminic acid methyl ester (compound 4): Compound 3 (21.2 g, 40 mmol) was dissolved in 300 mL of 2 mol / L ethyl hydrochloride solution and reacted at room temperature for 8 h under nitrogen protection. Thin-layer chromatography (ethyl acetate as the developing solvent) was used to monitor the reaction. After the reactants had completely reacted, the reaction system was carefully poured into 500 mL of ice-cold sodium bicarbonate solution. The separated organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and then rotary evaporated to dryness at a strictly controlled temperature not exceeding 30 °C to obtain a pale yellow foamy solid (17.6 g, crude yield 86.3%), which is compound 4. This product does not require purification and should be used as soon as possible in the next reaction step.

[0025] Step 4, Synthesis of Umbelliferone Sodium Salt (Compound 5): Umbelliferone (14 g, 80 mmol) was dissolved in 140 mL of anhydrous methanol. Under nitrogen protection, an equivalent amount of sodium methoxide in methanol (14.4 g, 80 mmol, 30%) was added dropwise. The reaction was carried out at room temperature for 3 h to fully convert the phenolic hydroxyl groups of the starting material into sodium methoxide. The reaction system was rotary evaporated to dryness to obtain 15.8 g of a pale yellow solid product, which is Compound 5. This product is the sodium salt of umbelliferone and can directly undergo glycosylation reaction with Compound 4. This reaction was carried out simultaneously with Step 3.

[0026] Step 5, Synthesis of 2-O-4-methylumbelliferyl-4,7,8,9-tetra-O-acetyl-N-acetyl-α-D-neuraminic acid methyl ester (compound 6): Compound 5 (7.52 g, 38.0 mmol) was dissolved in 200 mL of DMF, and potassium carbonate (5.72 g, 41.44 mmol) was added. After stirring at room temperature for 0.5 h, 100 mL of DMF solution containing compound 4 (17.6 g, 34.5 mmol) was added dropwise. The system gradually turned pale yellow, and the reaction was stirred at room temperature for 12 h. The solid was removed by filtration, and the solid was washed with an appropriate amount of DMF. The filtrates were combined, and DMF was removed by vacuum pump to obtain a brownish-yellow oil. This oil was then purified by rapid column chromatography to obtain a pale yellow solid (18.9 g, yield 84.4%), which is compound 6. The 1H NMR spectrum of compound 6 is shown in [reference needed]. Figure 2 See the carbon NMR spectrum. Figure 3 The results are as follows: 1 H NMR (400 MHz, CDCl3): δ 7.51 (d, J =8.76 Hz, 1H), 7.07-7.04 (m, 1H), 6.99 (d, J =2.4 Hz, 1H), 6.19 (s, 1H), 5.38-5.36 (m, 3H), 5.01-4.95 (m, 1H), 4.52-4.49 (m, 1H), 4.30-4.27 (m, 1H), 4.14-4.09 (m, 2H), 3.68 (s, 1H), 2.72(dd, J =4.68 Hz, J =13.04 Hz, 1H), 2.41 (s, 3H), 2.25 (t, J=12.56 Hz, 1H), 2.15(s, 3H), 2.13 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.91 (s, 3H); 13 C NMR (100 MHz, CDCl3): δ 170.91, 170.66, 170.28, 170.16, 170.06,168.00, 160.91, 156.61, 154.40, 152.29, 125.76, 116.19, 115.53, 113.47,107.67, 99.85, 73.71, 68.99, 68.41, 67.26, 62.04, 53.27, 49.39, 38.24, 23.24,21.03, 20.86, 20.78, 18.75.

[0027] Step 6, Synthesis of (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid methyl ester (compound 7): Compound 6 (6.0 g, 9.24 mmol) was dissolved in 60 mL of anhydrous methanol, and a catalytic amount of sodium methoxide solution was added. The reaction was carried out at room temperature for 4 h. After the reaction was complete as monitored by thin-layer chromatography (ethyl acetate as the developing solvent), the pH was carefully adjusted to neutral using a cation exchange resin, and the cation exchange resin was removed by filtration. The filtrate was concentrated by rotary evaporation to obtain a pale yellow solid product after the O-acetyl group was removed, which is compound 7. No purification is required, and it can be used for the next step of saponification reaction.

[0028] Step 7, Synthesis of (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid sodium salt (4-MUNANA): Compound 7 obtained in Step 6 was dissolved in 100 mL of 0.1 mol / L sodium hydroxide aqueous solution and reacted at room temperature for 2 h. After the reaction was complete as monitored by thin-layer chromatography (dichloromethane:methanol = 10:1, V / V), the filtrate was concentrated to dryness to obtain the crude product. The crude product was purified by gel column chromatography using deionized water as the eluent, and the eluent containing the product was lyophilized to obtain (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid sodium salt (4-MUNANA) (3.65 g, 84.5%, two-step yield). The 1H NMR spectrum of 4-MUNANA is shown below. Figure 4 See the carbon NMR spectrum. Figure 5 The results are as follows: 1 H NMR (400 MHz, D2O): δ 7.43 (d, J=8.84 Hz, 1H), 7.02-6.96 (m, 2H), 5.95 (s, 1H), 4.01-3.98 (m, 1H), 3.91-3.80 (m, 3H), 3.75-3.68 (m, 1H), 3.61-3.52 (m, 1H), 2.82-2.77 (m, 1H), 2.16 (s, 3H), 1.97 (s, 3H), 1.92 (t, J =13.32Hz, 1H); 13 C NMR (100 MHz, D2O): δ 175.04, 172.59, 164.20, 157.31, 155.92,153.06, 126.01, 117.50, 115.69, 111.38, 107.72, 102.58, 73.56, 71.62, 68.28,67.85, 62.73, 51.75, 40.89, 22.06, 17.88.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; if these modifications and variations fall within the scope of the claims of the present invention and their equivalents, they should all be considered to be within the protection scope of the present invention.

Claims

1. A method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid, characterized in that, Specifically, the following steps are included: S1. Methyl esterification: N-acetylneuraminic acid and methanol are methyl esterified in the presence of a cation exchange resin to obtain N-acetylneuraminic acid methyl ester. S2, Fully acetylated: N-acetylneuraminic acid methyl ester is subjected to a fully acetylated reaction with acetic anhydride to obtain fully acetylated N-acetylneuraminic acid methyl ester; S3, Terminal chlorination: Peracetylated N-acetylneuraminic acid methyl ester reacts with an organic solution of hydrogen chloride to undergo terminal chlorination, yielding the C-2-chlorinated product; S4. Glycosylation: The C-2-chloro product undergoes a glycosylation reaction with the sodium umbelliferone salt under the action of an inorganic base promoter to obtain the glycosylated product. S5. Deacetylation reaction: Under the action of sodium methoxide as a catalyst, all O-acetyl groups of the glycosylation product are removed to obtain the deacetylated product; S6. Saponification: The deacetylated product undergoes a saponification reaction in sodium hydroxide solution to obtain (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid sodium salt.

2. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 1, characterized in that, The specific operation of step S1 is as follows: N-acetylneuraminic acid is suspended in 5 to 10 times its volume of methanol, cation exchange resin is added, the reaction is carried out at room temperature for 8 to 12 hours, the cation exchange resin is removed by filtration, and the filtrate is concentrated to obtain N-acetylneuraminic acid methyl ester.

3. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 1, characterized in that, The specific operation of step S2 is as follows: N-acetylneuraminic acid methyl ester is dissolved in pyridine, 6-10 equivalents of acetic anhydride are added and reacted for 8-12 hours. After quenching the reaction system with methanol, it is diluted with an appropriate amount of dichloromethane, and then washed with dilute acid to remove pyridine. After concentrating the organic phase, fully acetylated N-acetylneuraminic acid methyl ester is obtained. The dilute acid is dilute sulfuric acid, dilute hydrochloric acid, dilute nitric acid or dilute phosphoric acid.

4. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 1, characterized in that, The specific operation of step S3 is as follows: after reacting fully acetylated N-acetylneuraminic acid methyl ester with 2-5 equivalents of hydrogen chloride organic solution at room temperature for 4-8 hours, the reaction system is slowly poured into an ice-cold sodium bicarbonate solution, separated, the organic phase is dried with sodium sulfate, and concentrated to dryness at room temperature to obtain the C-2-chlorinated product.

5. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 4, characterized in that, In step S3, the organic solution of hydrogen chloride is a hydrogen chloride methanol solution, a hydrogen chloride ethanol solution, a hydrogen chloride dioxane solution, or a hydrogen chloride ethyl acetate solution.

6. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 1, characterized in that, The specific operation of step S4 is as follows: the C-2-chloro product and the umbelliferone sodium salt are dissolved in an appropriate amount of DMF, an inorganic base is added at room temperature, and the reaction is continued to be stirred for 8-12 hours. After the reaction is completed, the mixture is filtered, the filtrate is concentrated to dryness, and the glycosylated product is obtained by rapid column chromatography.

7. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 6, characterized in that, In step S4, the preparation method of umbelliferone sodium salt is as follows: dissolve umbelliferone in an appropriate amount of methanol, react with an equivalent amount of sodium methoxide at room temperature for 2-6 hours, and then concentrate to obtain umbelliferone sodium salt.

8. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 6, characterized in that, In step S4, the inorganic base is lithium carbonate, sodium carbonate, silver carbonate, potassium carbonate, or cesium carbonate.

9. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 1, characterized in that, The specific operation of step S5 is as follows: the glycosylated product is dissolved in an appropriate amount of anhydrous methanol, a catalytic amount of sodium methoxide solution is added, and the reaction is stirred at room temperature for 2-6 hours. After the reaction is completed, the pH of the system is adjusted to neutral using a cation exchange resin, the cation exchange resin is removed by filtration, and the filtrate is concentrated to obtain the deacetylated product.

10. The method for preparing sodium (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid according to claim 1, characterized in that, The specific operation of step S6 is as follows: the deacetylated product is dissolved in an aqueous solution of sodium hydroxide, and the reaction is stirred at room temperature for 2-6 hours. After the reaction is complete, the filtrate is concentrated to dryness and separated by gel column chromatography to obtain sodium salt of (4-methylumbelliferone)-N-acetyl-α-D-neuraminic acid.

Images