Method for synthesizing Core M3 type O-mannan and extending sugar chain of Core M3 type O-mannan

Abstract

The invention discloses a method for synthesizing Core M3 type O-mannan, and belongs to the technical field of medicines. A convergent chemical synthesis strategy and a modular assembly method are adopted, and the efficient synthesis of the core M3 type O-mannose chain and the related oligosaccharide fragments thereof is successfully realized by accurately controlling the spatial configuration of glucosidic bonds and the connection sequence of sugar units. Corresponding monosaccharide, disaccharide and trisaccharide are used as initial raw materials, orthogonal protecting groups such as Nap, Alloc and TBDPS are adopted, multi-step glycosylation and protecting group operation are carried out to obtain matrix glycan hexasaccharide and tetrasaccharide building blocks, the matrix glycan hexasaccharide and tetrasaccharide building blocks are connected through a phosphate bond to obtain a fully-protected product, then core M3 type O-mannose chains are obtained only through two-step deprotection, and the core M3 type O-mannose chains are obtained. The synthesis strategy is simple and convenient, and large-scale preparation of a target product can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a method for synthesizing Core M3 type O-mannans and extending their sugar chains. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Dystroglycan (DG) is a core component of the dystrophin-glycoprotein complex (DGC), isolated from the skeletal muscle membrane. It is a transmembrane glycoprotein composed of two subunits: α-DG located outside the cell and β-DG located on the cell membrane. Mutations in genes involved in O-mannosylation disrupt the connection between α-DG and its ligands on the ECM, leading to muscle dysfunction and α-dystrophinopathy.

[0004] The O-mannans on the surface of α-DG can be divided into three core types based on their structure: Core M1 (GlcNAcβ1-2Manα), Core M2 (GlcNAcβ1-2(GlcNAcβ1-6)Manα), and Core M3 (GalNAcβ1-3GlcNAcβ1-4-(phospho-6)Manα). Core M3 sugar chains play an important role in maintaining the normal structure and function of muscle and brain. Core M3 O-mannans are only found in α-DG. Structural abnormalities in these glycans can lead to the inactivation of α-DG, disrupting its interaction with the extracellular matrix and ultimately causing α-muscular dystrophy. This disease is a general term for a class of inherited neuromuscular diseases caused by glycosylation defects. Its pathogenic mechanism is the abnormal biosynthesis of Core M3 O-mannans in α-DG, resulting in a loss of extracellular matrix anchoring function. Recent studies have found that the Core M3 oligosaccharide structure of α-DG plays an important role in regulating its physiological functions. Therefore, the synthesis of Core M3 oligosaccharides is of great value for the diagnosis and treatment of this disease.

[0005] Due to the complex biosynthesis pathway of Core M3 O-mannans and the numerous enzymes involved, enzymatic synthesis of complete Core M3 glycan chains is difficult to achieve in large quantities. Chemical and chemoenzymatic methods are alternative approaches for synthesizing these glycans, but because the complete structure of the Core M3 glycan was not elucidated until 2016, reports on its synthesis are limited. Therefore, the development of efficient chemical synthesis methods to obtain structurally defined Core M3-related oligosaccharides is urgently needed to lay the foundation for the development of potential treatments for muscular dystrophy. Summary of the Invention

[0006] In view of this, the present invention provides a method for synthesizing Core M3 type O-mannans and their extended sugar chains. The present invention develops an efficient convergent synthesis strategy to efficiently prepare Core M3 related oligosaccharides using chemical methods, specifically including:

[0007] Compound 1 is a pseudo-decasaccharide Xylα1-3GlcAβ1-3Xylα1-3GlcAβ1-4Xylβ1-4Rbo5P-1Rbo5P

[0008] -3GalNAcβ1-3GlcNAcβ1-4Man6Pα1-O(CH2)5NH2;

[0009] Compound 2 is a pseudo-octose Xylα1-3GlcAβ1-4Xylβ1-4Rbo5P-1Rbo5P-3GalNAcβ1

[0010] -3GlcNAcβ1-4Man6Pα1-O(CH2)5NH2;

[0011] Compound 3 is a pseudohexasaccharide Xylβ1-4Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4Man6Pα1

[0012] -O(CH2)5NH2;

[0013] Compound 4 is a pseudotetrasaccharide Rbo5P-3GalNAcβ1-3GlcNAcβ1-4Man6Pα1-O(CH2)5NH2;

[0014] Compound 5 is a pseudopentasaccharide Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4Man6Pα1

[0015] -O(CH2)5NH2;

[0016] Compound 6 is the phosphorylated trisaccharide GalNAcβ1-3GlcNAcβ1-4Man6Pα1-O(CH2)5NH2;

[0017] Compound 7 trisaccharide GalNAcβ1-3GlcNAcβ1-4Man1-O(CH2)5NH2;

[0018] Its structure is as follows:

[0019]

[0020] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0021] The present invention provides a method for synthesizing Core M3 O-mannans and their extended sugar chains. Using monosaccharides, disaccharides, and trisaccharides as starting materials, orthogonal protecting groups such as Nap, Alloc, and TBDPS are employed. Oligosaccharide fragments are constructed through glycosylation and protective group manipulation. The oligosaccharide fragments are then coupled via phosphorylation. Finally, the protecting groups are removed to obtain the target product, Core M3 O-mannans and their extended sugar chains (matrigosaccharides). This synthetic strategy is simple and can achieve large-scale production of the target product.

[0022] The specific operation of the glycosylation is: using N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) or silver trifluoromethanesulfonate (AgOTf) as glycoside glycosyl donor activators, trimethylsilyl trifluoromethanesulfonate (TMSOTf) or trifluoromethanesulfonate (TfOH) or boron trifluoride ethyl ether (BF3OEt2) as glycosyl trichloroacetimidate donor activators, or trimethylsilyl iodide (TMSI) and triphenylphosphine oxide (Ph3PO) as the activation system, in an organic solvent and an inert gas environment, at a low temperature of -4°C to 0°C, with molecular sieves as a desiccant.

[0023] Preferably, the molecular sieve is Any one or more combinations of molecular sieves.

[0024] Preferably, the organic solvent is a combination of one or more of dichloromethane, chloroform, and toluene, more preferably dichloromethane or toluene.

[0025] Preferably, the inert gas is one or more combinations of nitrogen and argon.

[0026] The phosphorylation comprises the following steps: generating a trivalent phosphorus intermediate under the action of N,N-diisopropylammonium tetrazolium salt and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, and then obtaining a phosphorylated product under the action of tert-butyl peroxide.

[0027] Furthermore, when the matrix polysaccharide is a pseudo-decasaccharide (1), the oligosaccharide fragments are compounds 1-1 and 1-2;

[0028]

[0029] The pseudo-decasaccharide synthesis method is as follows: Compound 1-1 is first reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate 46, which is then reacted with compound 1-2 through a phosphorylation coupling reaction to obtain a fully protected pseudo-decasaccharide 47; finally, deprotection is performed to obtain the pseudo-decasaccharide. The deprotection operation is as follows: Compound 47 first removes the acetyl (Ac), benzoyl (Bz), and methyl (Me) protecting groups, and then removes the cyanoethyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), and benzyl (Bn) protecting groups.

[0030] The specific synthetic route is as follows:

[0031]

[0032] The compound 1-1 is obtained from a monosaccharide donor 1-3, a monosaccharide donor 1-4, a monosaccharide acceptor 1-5 and a pseudo-disaccharide acceptor 1-6 through multi-step glycosidation and protecting group operations.

[0033]

[0034] The specific preparation method of compound 1-1 is as follows: monosaccharide donor 1-4 and monosaccharide acceptor 1-5 are subjected to a pre-activated 1,2-cis xylose glycosylation reaction to obtain disaccharide 10. Monosaccharide donor 1-3 and monosaccharide acceptor 1-5 are subjected to a pre-activated 1,2-cis xylose glycosylation reaction to obtain disaccharide 11. Disaccharide 10 is further glycosylated with pseudodisaccharide acceptor 1-6, and the Nap protecting group is removed to obtain compound 12. Disaccharide 11 is used as a glycosyl donor and compound 12 is used as a glycosyl acceptor to undergo a glycosylation reaction to obtain compound 1-1. The structural formulas of compounds 10, 11, and 12 are:

[0035]

[0036] The compound 1-2 is obtained from trisaccharide 1-7, ribitol 1-8 and five-carbon linker 1-9 through multi-step glycosylation and protecting group operation.

[0037]

[0038] Among them, the specific preparation method of compound 1-2 is as follows: Compound 1-7 and compound 1-9 are subjected to a glycosylation reaction, and the resulting glycosylated product is acetylated after removing the 2,2,2-trichloroethoxycarbonyl (Troc) protecting group to obtain compound 31; then compound 31 is deprotected by tert-butyldiphenylsilane (TBDPS) and phosphorylation is introduced to obtain compound 33, and finally, compound 33 is deprotected by removing the allyloxycarbonyl (Alloc) group to obtain compound 34; compound 1-8 is reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate 37, and compound 37 is added to a system obtained by mixing compound 34 and tetrazolium, and then fully protected pseudo-tetrasaccharide 38 is obtained under the action of tert-butyl peroxide, and the allyloxycarbonyl (Alloc) group is removed to obtain compound 1-2. The structural formulas of the compounds 31, 33, 34, 37, and 38 are:

[0039]

[0040]

[0041] Furthermore, the synthesis method of the pseudo-octasaccharide (2) is as follows: Compound 43 is first reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate 44, which is then reacted with compound 1-2 through a phosphorylation coupling reaction to obtain a fully protected pseudo-octasaccharide 45; finally, a deprotection operation is performed to obtain pseudo-octasaccharide 2. The specific synthesis route is as follows:

[0042]

[0043] The deprotection operation is as follows: compound 45 first removes the acetyl (Ac), benzoyl (Bz), and methyl (Me) protecting groups, and then removes the cyanoethyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), and benzyl (Bn) protecting groups to obtain the pseudo-octasaccharide.

[0044] Furthermore, the synthesis method of the pseudo-hexasaccharide (3) is as follows: compound 40 is reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate 41, which is then reacted with compound 1-2 to obtain a protected pseudo-hexasaccharide 42; finally, a deprotection operation is performed to obtain pseudo-hexasaccharide 3. The specific synthesis route is as follows:

[0045]

[0046] The deprotection operation is as follows: compound 40 first removes the acetyl (Ac) and benzoyl (Bz) protecting groups, and then removes the cyanoethyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz) and benzyl (Bn) protecting groups to obtain a pseudohexasaccharide.

[0047] Furthermore, the synthesis method of the pseudo-tetrasaccharide (4) is as follows: Compound 29 and Compound 1-9 are subjected to a glycosylation reaction to obtain a glycosylated product, the obtained glycosylated product is deprotected by 2,2,2-trichloroethoxycarbonyl and then acetylated to obtain Compound 31; Compound 31 is then deprotected by p-tert-butyldiphenylsilane and phosphorylated to obtain Compound 33; finally, Compound 33 is deprotected by Alloc to obtain Compound 34;

[0048] Compound 35 and benzyl trichloroacetimidate donor were catalyzed by TfOH to obtain a Bn-protected product, and then TIPS was removed to obtain ribitol compound 36; the hydroxyl group of compound 36 was introduced into a protective group phosphite to obtain compound 37; compound 37 was slowly added dropwise after mixing compound 34 and tetrazole, and then fully protected tetrasaccharide 38 was obtained under the action of tert-butyl peroxide; finally, after deprotection operation, pseudo-tetrasaccharide (4) was obtained.

[0049] The deprotection operation is as follows: the acetyl (Ac) protecting group of compound 38 is first removed, and then the allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz) and benzyl (Bn) protecting groups are removed to obtain the pseudo-tetrasaccharide of Core M3 type O-mannan.

[0050] The specific synthetic route is as follows:

[0051]

[0052] The molar ratio of compound 29, compound 1-9, and TMSOTf is 1:0.7-1.0:0.05-0.1; and the volume ratio of the molecular sieve to the solvent is 0.05-0.15 g / mL.

[0053] Furthermore, the synthesis method of the pseudo-pentasaccharide (5) is as follows: Compound 37 is added to a mixture of Compound 1-2 and tetrazole to obtain Compound 39; and finally, after deprotection, the pseudo-pentasaccharide 5 is obtained. The specific synthesis route is as follows:

[0054]

[0055] The deprotection operation is as follows: the acetyl (Ac) protecting group of compound 40 is first removed, and then the allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz) and benzyl (Bn) protecting groups are removed to obtain the pseudo-pentasaccharide.

[0056] Furthermore, the synthesis method of the trisaccharide (7) is as follows: when the glycosyl donor is compound 24 and the glycosyl acceptor is compound 1-9, TMSOTf is used as an activator to obtain compound 25 through glycosidation reaction, compound 25 is first removed from the 2,2,2-trichloroethoxycarbonyl protecting group and then acetyl group is added to obtain compound 26, then the p-tert-butyldiphenylsilane protecting group is removed to obtain compound 27, and finally the acetyl group, benzyloxycarbonyl and benzyl protecting groups are removed to obtain trisaccharide (7). The specific synthesis route is as follows:

[0057]

[0058] The molar ratio of compound 24, compound 1-9, and TMSOTf is 1:1.1-1.2:0.05-0.1; the mass ratio of the molecular sieve to the volume ratio of the solvent is 0.05-0.15 g / mL.

[0059] The deprotection operation is as follows: compound 25 is firstly deprotected from the 2,2,2-trichloroethoxycarbonyl (Troc) protecting group and then acetyl (Ac) is added, then the p-tert-butyldiphenylsilane (TBDPS) protecting group is removed, and finally the acetyl (Ac), benzyloxycarbonyl (Cbz) and benzyl (Bn) protecting groups are removed to obtain a trisaccharide.

[0060] The synthesis method of the phosphorylated trisaccharide is as follows: Compound 27 is phosphorylated to obtain Compound 28, and then deprotected to obtain the phosphorylated trisaccharide (6). The specific synthesis route is as follows:

[0061]

[0062] Compared with the existing technology, the present invention has achieved the following beneficial effects:

[0063] (1) In the synthesis method of the Core M3 O-mannose and its extended matrix polysaccharide of the present invention, the relevant xylose, glucose, ribitol and five-carbon linker glycosyl modules, namely Compound 1-3, Compound 1-4, Compound 1-5, Compound 1-6, Compound 1-7, Compound 1-8 and Compound 1-9, were prepared respectively through reasonable protective group design. Core M3 O-mannose and its extended matrix polysaccharide were chemically synthesized through glycosidation reaction, phosphorylation coupling and deprotection operation.

[0064] The method for synthesizing Core M3 type O-mannans and their extended sugar chains (matrix polysaccharides) of the present invention is simple to operate, has readily available raw materials, is highly practical, has a high yield, and is low in cost, and is of great significance for the industrial production and application of such substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0066] Figure 1 .NMR of compound 1 1 H spectrum;

[0067] Figure 2 .NMR of compound 2 1 H spectrum;

[0068] Figure 3 .NMR of compound 3 1 H spectrum;

[0069] Figure 4 .NMR of compound 5 1 H spectrum;

[0070] Figure 5 .NMR of compound 4 1 H spectrum;

[0071] Figure 6 .NMR of compound 6 1 H spectrum;

[0072] Figure 7 .NMR of compound 7 1 H spectrum. DETAILED DESCRIPTION

[0073] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0074] The technical solution of the present invention is further described below with reference to specific embodiments.

[0075] Example 1. Chemical synthesis of a trisaccharide compound of Core M3 type O-mannan (7)

[0076] 1.1 Synthesis route:

[0077]

[0078] 1.2 Specific experimental steps

[0079] Add the reaction flask Molecular sieves were added to the flask under vacuum, cooled to room temperature, and then purged with argon. Compound 24 (1.0 g, 0.7 mmol) and compound 1-9 (263.7 mg, 0.8 mmol) were added in sequence and dissolved in 7.0 mL of dry dichloromethane. The mixture was stirred at room temperature for 5 min. The reaction solution was cooled to 0°C and TMSOTf (15 μL, 70.0 μmol) was added. The reaction was allowed to react at this temperature for 30 min. The plate was developed with petroleum ether: ethyl acetate: dichloromethane (v / v / v) = 1:1:1. TLC was used to detect the completion of the reaction. The reaction was quenched with triethylamine, the molecular sieves were filtered off through a celite pad, and the mixture was diluted with dichloromethane. The organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 25 as a white foamy solid in a yield of 79%.

[0080] Compound 25 (780.0 mg, 0.5 mmol) was dissolved in 4.6 mL of acetic acid, and activated zinc powder (1.5 g, 23.0 mmol) was added at 45°C. The reaction was allowed to proceed for 7 h at this temperature. The product was developed using a 30:1 ratio of dichloromethane to methanol (v / v) and a TLC plate. The reaction was complete. The zinc powder was filtered through a celite pad, concentrated, and toluene was added and dried using a diaphragm pump (to completely remove the acetic acid). The crude product was dissolved in 5.0 mL of pyridine solution, and the reaction solution was cooled to 0°C. Ac2O (0.6 mL, 5.6 mmol) and DMAP (13.0 mg, 0.1 mmol) were added in sequence. The temperature was naturally raised and the reaction was allowed to proceed overnight. The product was developed using a 1:1 ratio of petroleum ether to ethyl acetate (v / v) and a TLC plate. The reaction was complete. The reaction mixture was quenched by adding methanol and diluted with dichloromethane. The organic phase was washed sequentially with 1 M hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 1:5) to give 26 as a white foamy solid in a yield of 83%.

[0081] Compound 26 (620.0 mg, 0.4 mmol) was dissolved in 4.4 mL of a 10:9 tetrahydrofuran:pyridine (v / v) mixture. The reaction solution was cooled to 0°C and a 70% hydrogen fluoride pyridine solution (1.7 mL, 13.0 mmol) was added dropwise. The mixture was allowed to warm to room temperature for 4 h. The reaction was then developed using a 30:1 ethyl acetate:methanol (v / v) plate. TLC confirmed the complete reaction. Saturated sodium bicarbonate was slowly added at 0°C to quench the reaction. The mixture was diluted with dichloromethane, and the organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was separated by silica gel column chromatography (30:1 ethyl acetate:methanol) to afford 27 as a white foamy solid in a 96% yield.

[0082] Compound 27 (50.0 mg, 42.0 μmol) was dissolved in 1.5 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 4 h. The developing solvent was ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 2:1:1:0.5. TLC was used to detect the completion of the reaction of the raw material. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The deacylated compound was separated on a Sephadex™ LH20 column. The above product was dissolved in 2 mL of a mixed solution containing methanol:tetrahydrofuran:water:acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (59.0 mg, 84.0 μmol) was added. The reaction flask was placed in a hydrogenation reactor and allowed to react overnight at room temperature. The developer was ethyl acetate:methanol:water:acetic acid (v / v) = 2:2:2:0.8. TLC confirmed the completion of the reaction. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified on a C18 column (eluent: methanol and water) and lyophilized to obtain 7 as a white foamy solid in a 62% yield. 1 H NMR(600MHz,D2O)δ4.87–4.86(m,1H,1a-H),4.54(d,J=8.2Hz,1H,1b-H),4.53(d,J=8.3Hz,1H,1c-H),4.00–3 .98(m,1H,2a-H),3.96(dd,J=12.4,2.4Hz,2H),3.90(dd,J=9.1,3.4Hz,1H,3a-H),3.88–3.82(m,2H,2b-H,2c -H),3.82–3.76(m,4H),3.76–3.70(m,2H),3.70–3.62(m,2H),3.59–3.53(m,3H),3.01(t,J=7.6Hz,2H,linke r-NCH2),2.11(s,3H,COCH3),2.03(s,3H,COCH3),1.75–1.61(m,4H,linker-CH2),1.46(m,2H,linker-CH2). 13 C NMR(150MHz,D2O)δ174.6,173.7,101.4,99.3,80.6,77.6,75.3,75.1,71.0,70.3,69.5,69.4,68 .6,67.7,67.5,61.1,60.7,60.5,54.6,52.7,39.4,27.9,26.5,22.5,22.4,22.3.HRMS(ESI)Calcd for C 27 H 50 N3O 16 + [M+H] +:672.3186,found 672.3195.

[0083] Example 2. Chemical synthesis of phosphorylated trisaccharide compound (6) of Core M3 type O-mannan

[0084] 2.1 Synthesis route:

[0085]

[0086] 2.2 Specific experimental steps

[0087] Add the reaction bottle The mixture was dried over 400 μg of 5-molecular sieves in a vacuum oven, cooled to room temperature, and then purged with argon. Compound 27 (200.0 mg, 0.2 mmol) was added and dissolved in 3.4 mL of dry dichloromethane. Then, 1.0 mL of a tetrazole solution in acetonitrile (tetrazole concentration: 0.45 M) was added. The mixture was stirred at room temperature for 5 min. N,N-diisopropyldibenzylphosphoramidite (226.0 μL, 0.7 mmol) was added and allowed to react at room temperature for 40 min. The reaction was then developed using ethyl acetate:methanol = 30:1. TLC was used to detect the completion of the reaction. The reaction solution was cooled to 0°C, tert-butyl peroxide (92 μL, 0.5 mmol) was added, and the temperature was naturally raised for 2 h. The reaction was then developed using petroleum ether:ethyl acetate (v / v) = 1:3. TLC was used to detect the completion of the reaction. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 1:5) to obtain 28 as a white foamy solid in a yield of 92%.

[0088] Compound 28 (88.0 mg, 60.0 μmol) was dissolved in 2.5 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 4 h. The developing solvent was ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 2:1:1:0.5. TLC was used to detect the completion of the reaction of the raw material. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The deacylated compound was separated on a Sephadex™ LH20 column. The above product was dissolved in 2 mL of a mixed solution containing methanol:tetrahydrofuran:water:acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (85.2 mg, 0.1 mmol) was added. The reaction flask was placed in a hydrogenation reactor and allowed to react overnight at room temperature. The developer was ethyl acetate:methanol:water:acetic acid (v / v) = 2:2:2:0.7. TLC confirmed the completion of the reaction. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified on a C18 column (eluent: methanol and water) and lyophilized to obtain 6 as a white foamy solid in a 58% yield. 1H NMR(600MHz,D2O)δ4.86(d,J=1.7Hz,1H,1a-H),4.61(d,J=8.4Hz,1H,1b-H),4.53(d,J=8.3Hz,1H,1c-H),4.07(dd d,J=11.4,4.5,2.0Hz,1H),4.01(t,J=4.9Hz,1H),3.99(dd,J=3.0,1.7Hz,2H,2a-H),3.98–3.93(m,2H),3.91–3.84 (m,4H,2b-H,2c-H),3.84–3.69(m,8H,linker-OCHa),3.61–3.53(m,3H,linker-OCHb),3.01(t,J=7.6Hz,2H,linke r-NCH2),2.14(s,3H,COCH3),2.03(s,3H,COCH3),1.75–1.60(m,4H,linker-CH2),1.50–1.41(m,2H,linker-CH2). 13 C NMR(150MHz,D2O)δ174.6,173.9,101.5,101.3,99.5,80.7,76.6,75.3,75.0,70.4,70.1,70.0,69.5,69 .3,68.6,67.7,67.5,63.32,63.29,61.0,60.6,54.4,52.6,39.4,27.8,26.4,22.43,22.38,22.37,22.3. 31 P NMR(162MHz,D2O)δ1.95.HRMS(ESI)Calcd for C 27 H 51 N3O 19 P + [M+H] + :752.2849,found 752.2877.

[0089] Example 3. Chemical synthesis of pseudo-tetrasaccharide compound (4) of Core M3 type O-mannan

[0090] 3.1 Synthesis route:

[0091]

[0092] 3.2 Specific experimental steps (1) Preparation of compound 34

[0093] Add the reaction flask Molecular sieves were added to the flask under vacuum, cooled to room temperature, and then purged with argon. Compound 29 (7.2 g, 4.6 mmol) and compound 1-9 (1.6 g, 4.8 mmol) were added in sequence and dissolved in 46.0 mL of dry dichloromethane. The mixture was stirred at room temperature for 5 min. The reaction solution was cooled to 0°C and TMSOTf (83 μL, 43.6 μmol) was added. The reaction was allowed to react at this temperature for 30 min. The plate was developed with petroleum ether: ethyl acetate: dichloromethane (v / v / v) = 2:1:2. TLC was used to detect the completion of the reaction of the raw materials. The reaction was quenched with triethylamine, the molecular sieves were filtered off through a celite pad, and the mixture was diluted with dichloromethane. The organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, filtered and concentrated, and separated by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 30 as a white foamy solid in an 81% yield.

[0094] Compound 30 (6.3 g, 3.6 mmol) was dissolved in 35.0 mL of acetic acid. Activated zinc powder (12.0 g, 0.2 mol) was added at 45°C and allowed to react for 7 h. The reaction was then developed using a 30:1 ratio of dichloromethane to methanol (v / v) and TLC to detect the complete reaction. The zinc powder was filtered through a celite pad, concentrated, and toluene was added and dried using a diaphragm pump (to completely remove the acetic acid). The crude product was dissolved in 35.0 mL of pyridine solution, cooled to 0°C, and Ac2O (6.6 mL, 70.0 mmol) and DMAP (44.0 mg, 0.4 mmol) were added in sequence. The reaction mixture was allowed to warm naturally and allowed to react overnight. The reaction was then developed using a 1:1 ratio of petroleum ether to ethyl acetate (v / v) and TLC to detect the complete reaction. The reaction mixture was quenched by adding methanol and diluted with dichloromethane. The organic phase was washed sequentially with 1 M hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to afford 31 as a white foamy solid in a yield of 91%.

[0095] Compound 31 (5.2 g, 3.5 mmol) was dissolved in 35.0 mL of a 10:9 tetrahydrofuran:pyridine (v / v) mixture. The reaction solution was cooled to 0°C and a 70% hydrogen fluoride pyridine solution (13.8 mL, 0.1 mol) was added dropwise. The temperature was naturally raised and the reaction was allowed to react for 4 h. The reaction was then developed using a 30:1 ethyl acetate:methanol (v / v) plate. TLC confirmed the reaction was complete. Saturated sodium bicarbonate solution was slowly added at 0°C to quench the reaction. The mixture was diluted with dichloromethane, and the organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. 32 was separated by silica gel column chromatography (30:1 ethyl acetate:methanol) to afford 32 as a white foamy solid in a 91% yield.

[0096] Add the reaction bottle The mixture was dried over a vacuum oven over 1500 nmol molecular sieves, cooled to room temperature, and then purged with argon. Compound 32 (975.0 mg, 0.8 mmol) was added and dissolved in 16.0 mL of dry dichloromethane. Then, 5.0 mL of a tetrazole solution in acetonitrile (tetrazole concentration: 0.45 M) was added. The mixture was stirred at room temperature for 5 min. N,N-diisopropyldibenzylphosphoramidite (1.0 mL, 3.2 mmol) was added and allowed to react at room temperature for 40 min. The reaction was then developed using ethyl acetate:methanol = 30:1. TLC was used to detect the completion of the reaction. The reaction solution was cooled to 0°C, tert-butyl peroxide (440 μL, 2.4 mmol) was added, and the temperature was naturally raised. The reaction was allowed to react for 2 h. The reaction was then developed using pure ethyl acetate. TLC was used to detect the completion of the reaction. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to obtain 33 as a white foamy solid in 89% yield.

[0097] Compound 33 (3.2 g, 2.1 mmol) was dissolved in 21.4 mL of dry tetrahydrofuran, and 1,3-dimethylbarbituric acid (670.0 mg, 4.3 mmol) and tetrakis(triphenylphosphine)palladium (3.2 mg, 21.0 μmol) were added sequentially. The mixture was allowed to react at room temperature for 30 min. The reaction was completed by TLC using pure ethyl acetate as the developing solvent. The mixture was diluted with ethyl acetate, and the organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was separated by silica gel column chromatography (ethyl acetate:methanol = 50:1) to afford 34 as a white foamy solid in 82% yield. 1H NMR(600MHz,CDCl3)δ8.28–8.17(m,1H,NH),7.42–7.32(m,11H,ArH),7.33–7.21(m,8H,ArH),7.15(dd,J=16.3,8.4Hz,2H,ArH,NH),5.32(dd,J=9.6,3.5Hz,1H,3a-H),5.28–5.24(m,1H,4b-H),5.19–5.12(m,3H,2a-H,PhCH2),5.12–5.01(m,4H,PhCH2,PhCH2),4.90(t,J=9.4Hz,1H,4c-H),4.75–4.69(m,1H,1a-H),4.55–4.46(m,3H,6a-H,PhCH2),4.45(d,J=8.6Hz,1H,1c-H),4.29(dd,J=12.4,4.0Hz,1H,6c-H),4.28–4.25(m,1H,1b-H),4.17–4.04(m,3H,6b-H,2c-H,6b’-H),4.01(d,J=11.0Hz,1H,6a’-H),3.96(t,J=9.7Hz,1H,4a-H),3.88(dd,J=12.4,2.4Hz,1H,6c’-H),3.84–3.76(m,2H,5a-H,5b-H),3.68(t,J=9.1Hz,1H,3c-H),3.62–3.47(m,3H,2b-H,3b-H,linker-OCHa),3.38–3.14(m,4H,linker-OCHb,5c-H,linker-NCH2),2.10(d,J=1.1Hz,6H,COCH3(×2)),2.04–1.96(m,18H,COCH3(×6)),1.60–1.41(m,4H,linker-CH2),1.38–1.15(m,2H,linker-CH2). 13C NMR (150MHz, CDCl3) δ175.5,171.7,170.6,170.3,169.7,169.6,169.4,156.8,156.2,137.9,136.9,136.8,135.01,134.9 6,134.89,134.85,129.25,129.23,128.9,128.6,128.50,128.46,128.1,128.0,127.90,127.86,127.33,127.28,101.6,1 00.1,97.7,79.7,74.2,73.9,71.2,71.1,70.7,70.3,70.25,70.21,69.83,69.78,68.7,68.4,68.3,68.1,67.20,67.16,65.2,62.0,61.7,55.6,55.4,50.6,50.3,47.1,46.1,28.9,27.9,27.4,23.2,22.6,20.9,20.8,20.73,20.72,20.68,20.61. 31 P NMR(162MHz,CDCl3)δ-0.29.HRMS(ESI)Calcd for C 68 H 87 N3O 27 P + [M+H] + :1408.5260,found 1408.5255.

[0098] (2) Preparation of Compound 38

[0099] Add the reaction flask The mixture was dried over a vacuum oven with 1% zeolite (3.2 g, 5.6 mmol) and cooled to room temperature, followed by argon gas. Compound 35 (3.2 g, 5.6 mmol) and benzyl trichloroacetimidate (3.2 mL, 16.8 mmol) were added sequentially and dissolved in 18.6 mL of dry 1,4-dioxane. The mixture was stirred at room temperature for 5 min, and TfOH (150 μL, 1.7 mmol) was added. The reaction was allowed to react at room temperature for 20 min. The reaction was developed using a 9:1 ratio of petroleum ether to ethyl acetate (v / v) and the completion of the reaction was monitored by TLC. The reaction was quenched with triethylamine, and the zeolite was filtered through a celite pad. The mixture was diluted with dichloromethane, and the organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in 10.0 mL of TBAF (adjusted to pH 6-7 with acetic acid) and reacted at room temperature for 1 h. The product was separated by spot chromatography on a silica gel column using a developing solvent of petroleum ether:ethyl acetate (v / v) = 6:1 (petroleum ether:ethyl acetate = 4:1) to obtain 36 as a colorless oil in an 80% yield.

[0100] Add the reaction flask Molecular sieves, vacuum-baked the bottle, cooled to room temperature and then purged with argon, compound 36 (144.0 mg, 0.3 mmol) was added and dissolved in 3.0 mL of dry dichloromethane. N, N-diisopropylammonium tetrazolium salt (98.0 mg, 0.6 mmol) and 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (181 μL, 0.6 mmol) were added in sequence, and the reaction was allowed to react at room temperature for 2 h. The plate was developed with petroleum ether: ethyl acetate (v / v) = 2:1, and the reaction was completed by TLC detection. The molecular sieves were filtered out through a diatomaceous earth pad, diluted with dichloromethane, and the organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, filtered and concentrated, toluene was added and dried with a diaphragm pump (to remove moisture) to obtain an oily crude product 37. Add to the reaction flask Molecular sieves were added to the flask under vacuum, cooled to room temperature, and then purged with argon. Compound 34 (300.0 mg, 0.2 mmol) was added and dissolved in 2.0 mL of dry dichloromethane. 1.6 mL of tetrazole in acetonitrile (tetrazole concentration was 0.45 M) was added and stirred at room temperature for 15 min. The crude product from the previous step was dissolved in 1.0 mL of dry dichloromethane and slowly added dropwise to the reaction system. The reaction was allowed to react at room temperature for 40 min. The flask was developed with ethyl acetate:methanol (v / v) = 40:1 and TLC was performed until the starting material disappeared. The reaction solution was cooled to 0°C, tert-butyl peroxide (130 μL, 0.5 mmol) was added, the temperature was naturally raised, and the reaction was allowed to react for 2 h. The flask was developed with pure ethyl acetate and TLC was used to detect the completion of the reaction. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (ethyl acetate:methanol = 50:1) to obtain 38 as a white foamy solid in 86% yield.

[0101] (3) Preparation of Compound 4

[0102] Compound 38 (164.0 mg, 80.8 μmol) was dissolved in 3.0 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 6 h and spotted using ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 6:1:1:0.1 as the developing solvent. TLC detected the completion of the reaction of the raw materials. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The compound (acyl, cyanoethyl, and Alloc were removed) was separated on a Sephadex™ LH20 column. The above product was dissolved in 5.0 mL of a mixed solution containing methanol:tetrahydrofuran:water:acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (180.0 mg, 0.3 mmol) was added. The reaction flask was placed in a hydrogenation reactor and allowed to react at room temperature for 15 h. The developer was ethyl acetate:methanol:water:acetic acid (v / v) = 2:2:2:0.7. TLC confirmed the completion of the reaction. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified by a C18 column (eluent: methanol and water) and lyophilized to obtain 4 as a white foamy solid in a 69% yield. 1 HNMR(600MHz,D2O)δ4.86(d,J=1.8Hz,1H,1a-H),4.65(d,J=8.0Hz,1H,1b-H),4.60(d,J=8.3Hz,1H,1c-H),4.23(t ,J=10.0Hz,1H),4.18(d,J=3.1Hz,1H),4.09–4.04(m,2H),4.02(d,J=12.6Hz,2H),4.00–3.90(m,6H),3.89–3.69(m ,11H),3.67(dd,J=11.9,7.2Hz,1H),3.63–3.59(m,1H),3.59–3.54(m,2H),3.00(ddd,J=8.9,6.8,2.3Hz,2H,linke r-NCH2),2.15(s,3H,COCH3),2.05(s,3H,COCH3),1.79–1.60(m,4H,linker-CH2),1.51–1.42(m,2H,linker-CH2). 13 C NMR(150MHz,D2O)δ174.6,173.9,101.3,101.2,99.5,80.9,76.5,75.3,74.72,74.69,74.64,72.1,70.9,70.8,70.04,69 .98,69.5,69.3,68.6,67.5,66.8,66.7,63.45,63.42,62.3,61.0,60.6,54.4,51.4,39.4,27.9,26.4,22.5,22.4,22.3.31 P NMR(162MHz,D2O)δ1.77,1.02,-0.20.HRMS(ESI)Calcd for C 32 H 62 N3O 26 P2 + [M+H] + :966.3092,found 966.3096.

[0103] (4) Preparation of Compound 1-2

[0104] Compound 38 (608 mg, 0.3 mmol) was dissolved in 3.0 mL of dry tetrahydrofuran, and 1,3-dimethylbarbituric acid (94.0 mg, 0.6 mmol) and tetrakis(triphenylphosphine)palladium (3.5 mg, 3.0 μmol) were added sequentially. The mixture was allowed to react at room temperature for 30 min. The reaction was completed by TLC using ethyl acetate:methanol = 30:1 as the developing solvent. The mixture was diluted with ethyl acetate, and the organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (ethyl acetate:methanol = 30:1) to afford 1-2 as a white foamy solid in a 91% yield. 1 H NMR (400MHz, CDCl3) δ7.55(dd,J=23.0,8.2Hz,2H,ArH),7.44–7.11(m,61H,ArH),6.25(t,J=7.1Hz,1H),6.03(d,J=7.1H z,1H),5.43(dd,J=8.1,3.6Hz,1H),5.36–5.26(m,2H),5.21–5.13(m,6H),5.11–4.99(m,7H),4.97–4.81(m,3H),4.76–4 .55(m,12H),4.55–4.37(m,7H),4.35–4.17(m,3H),4.12–3.86(m,13H),3.85–3.69(m,7H),3.65–3.38(m,4H),3.37–3.1 5(m,6H),2.62–2.42(m,4H),2.09–1.90(m,42H,COCH3),1.63–1.42(m,8H,linker-CH2),1.36–1.16(m,4H,linker-CH2). 13CNMR(150MHz,CDCl3)δ171.92,171.89,171.87,170.91,170.88,170.7,170.5,170.4,170.0,169.9,169.7,169.6,169.54,169.48,156.8,156.3,138.24,138.21,138.1,138.0,137.94,137.87,137.8,136.9,136.8,135.30,135.28,135.23,135.19,129.1,129.0,128.9,128.6,128.53,128.51,128.2,128.19,128.12,128.01,127.96,127.90,127.86,127.83,127.3,116.8,116.7,101.4,101.3,99.1,98.7,97.7,97.6,79.1,79.0,78.0,77.9,77.81,77.76,77.72,73.93,73.90,73.86,73.83,73.82,73.65,72.57,72.4,72.2,71.8,70.6,70.5,70.2,70.03,69.99,69.96,69.0,68.9,68.7,68.6,68.3,67.8,67.7,67.65,67.58,67.4,67.3,67.2,65.6,65.5,65.4,62.6,62.38,62.37,62.35,62.31,62.10,62.06,61.31,61.27,61.24,61.18,55.8,54.2,53.8,50.6,50.3,47.1,46.2,28.9,28.0,27.5,23.7,23.6,23.4,23.3,20.91,20.88,20.85,20.73,20.70,20.64,20.58,19.4,19.31,19.26.31PNMR(162MHz,CDCl3)δ2.36,-0.52,-1.90.HRMS(ESI)Calcd for C 97 H 119 N4O 34 P 2+ [M+H] + :1946.7210,found1946.7209;HRMS(ESI)Calcd for C 97 H 118 N4NaO 34 P 2+ [M+Na]+ :1968.7029,found 1968.7028.

[0105] Example 4. Chemical synthesis of pseudo-pentasaccharide compound (5) of Core M3 type O-mannan

[0106] 4.1 Synthesis route:

[0107]

[0108] 4.2 Specific experimental steps (1) Preparation of compound 1-2

[0109] Compound 38 (608 mg, 0.3 mmol) was dissolved in 3.0 mL of dry tetrahydrofuran, and 1,3-dimethylbarbituric acid (94.0 mg, 0.6 mmol) and tetrakis(triphenylphosphine)palladium (3.5 mg, 3.0 μmol) were added sequentially. The mixture was allowed to react at room temperature for 30 min. The reaction was completed by TLC using ethyl acetate:methanol = 30:1 as the developing solvent. The mixture was diluted with ethyl acetate, and the organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (ethyl acetate:methanol = 30:1) to afford 1-2 as a white foamy solid in a 91% yield.

[0110] 1 H NMR (400MHz, CDCl3) δ7.55(dd,J=23.0,8.2Hz,2H,ArH),7.44–7.11(m,61H,ArH),6.25(t,J=7.1Hz,1H),6.03(d,J=7.1H z,1H),5.43(dd,J=8.1,3.6Hz,1H),5.36–5.26(m,2H),5.21–5.13(m,6H),5.11–4.99(m,7H),4.97–4.81(m,3H),4.76–4 .55(m,12H),4.55–4.37(m,7H),4.35–4.17(m,3H),4.12–3.86(m,13H),3.85–3.69(m,7H),3.65–3.38(m,4H),3.37–3.1 5(m,6H),2.62–2.42(m,4H),2.09–1.90(m,42H,COCH3),1.63–1.42(m,8H,linker-CH2),1.36–1.16(m,4H,linker-CH2).

[0111] 13C NMR(150MHz,CDCl3)δ171.92,171.89,171.87,170.91,170.88,170.7,170.5,170.4,170.0,169.9,169.7,169.6,169.54,169.48,156.8,156.3,138.24,138.21,138.1,138.0,137.94,137.87,137.8,136.9,136.8,135.30,135.28,135.23,135.19,129.1,129.0,128.9,128.6,128.53,128.51,128.2,128.19,128.12,128.01,127.96,127.90,127.86,127.83,127.3,116.8,116.7,101.4,101.3,99.1,98.7,97.7,97.6,79.1,79.0,78.0,77.9,77.81,77.76,77.72,73.93,73.90,73.86,73.83,73.82,73.65,72.57,72.4,72.2,71.8,70.6,70.5,70.2,70.03,69.99,69.96,69.0,68.9,68.7,68.6,68.3,67.8,67.7,67.65,67.58,67.4,67.3,67.2,65.6,65.5,65.4,62.6,62.38,62.37,62.35,62.31,62.10,62.06,61.31,61.27,61.24,61.18,55.8,54.2,53.8,50.6,50.3,47.1,46.2,28.9,28.0,27.5,23.7,23.6,23.4,23.3,20.91,20.88,20.85,20.73,20.70,20.64,20.58,19.4,19.31,19.26.

[0112] 31 P NMR(162MHz,CDCl3)δ2.36,-0.52,-1.90.

[0113] HRMS(ESI)Calcd for C 97 H 119 N4O 34 P2 + [M+H] + :1946.7210,found 1946.7209;HRMS(ESI)Calcd for C97 H 118 N4NaO 34 P2 + [M+Na] + :1968.7029,found 1968.7028.

[0114] (2) Preparation of Compound 5

[0115] Add to the reaction flask Molecular sieves were added to the flask, which was vacuum-baked and cooled to room temperature after purging with argon. Compound 1-2 (150.0 mg, 77.0 μmol) was added and dissolved in 0.5 mL of dry dichloromethane. 579 μL of a tetrazole solution in acetonitrile (tetrazole concentration was 0.45 M) was added and stirred at room temperature for 15 min. Compound 37 was dissolved in 0.5 mL of dry dichloromethane and slowly added dropwise to the reaction system. The reaction was allowed to react at room temperature for 50 min. The flask was developed with ethyl acetate:methanol (v / v) = 40:1 and TLC was performed until the starting material disappeared. The reaction solution was cooled to 0°C and tert-butyl peroxide (47 μL, 0.3 mmol) was added. The temperature was naturally raised and the reaction was allowed to react for 2 h. The flask was developed with pure ethyl acetate and TLC was performed to determine the completion of the reaction. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (ethyl acetate:methanol = 40:1) to obtain 39 as a white foamy solid in 80% yield.

[0116] Compound 39 (115.0 mg, 44.8 μmol) was dissolved in 2.0 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 6 h and spotted using ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 6:1:1:0.1 as the developing solvent. TLC detected the completion of the reaction of the raw materials. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The compound (acyl, cyanoethyl, and Alloc groups had been removed) was separated on a Sephadex™ LH20 column. The above product was dissolved in 2.8 mL of a mixed solution containing methanol:tetrahydrofuran:water:acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (140 mg, 0.2 mmol) was added and the reaction flask was placed in a hydrogenation reactor. The reaction was allowed to proceed at room temperature for 15 h. The developer was ethyl acetate:methanol:water:acetic acid (v / v) = 2:2:2:0.3. TLC was used to determine the completion of the reaction. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified sequentially via a C18 column (eluent: methanol and water) and then a P2 column. The product was lyophilized to obtain 5 as a white foamy solid in a 68% yield.

[0117] 1H NMR(600MHz,D2O)δ4.86(d,J=1.8Hz,1H,1a-H),4.65(d,J=8.0Hz,1H,1b-H),4.60(d,J=8.4Hz,1H,1c-H),4.24(t, J=9.9Hz,1H),4.18(d,J=3.1Hz,1H),4.12–4.02(m,3H),4.02–3.93(m,9H),3.91(d,J=9.5Hz,1H),3.91–3.72(m,13 H),3.71(d,J=9.0Hz,1H),3.67(dd,J=11.9,7.2Hz,1H),3.64–3.53(m,4H),3.00(ddd,J=8.8,7.0,2.8Hz,2H,linke r-NCH2),2.15(s,3H,COCH3),2.05(s,3H,COCH3),1.79–1.59(m,4H,linker-CH2),1.50–1.42(m,2H,linker-CH2).

[0118] 13 C NMR(150MHz,D2O)δ174.6,174.0,101.4,101.1,99.5,80.9,76.2,75.2,74.6,72.0,71.7,71.2,70.92,70.86,70.86,70.77,70.5,6 9.6,69.3,68.6,67.4,66.8,66.6,66.54,66.51,66.47,66.40,66.36,62.28,61.0,60.7,54.4,39.4,27.8,26.3,22.6,22.5,22.2.

[0119] 31 P NMR(243MHz,D2O)δ2.19,1.27,-0.13.

[0120] HRMS(ESI)Calcd for C 37 H 73 N3O 33 P3 + [M+H] + :1180.3334,found 1180.3354.

[0121] Example 5. Chemical synthesis of pseudo-hexasaccharide compound (3) of Core M3 type O-mannan

[0122] 5.1 Synthesis route:

[0123]

[0124] 5.2 Specific experimental steps

[0125] Add the reaction flask Molecular sieves, vacuum-baked the bottle, cooled to room temperature and then purged with argon, compound 40 (95.0 mg, 0.1 mmol) was added and dissolved in 1.1 mL of dry dichloromethane. N, N-diisopropylammonium tetrazolium salt (38.0 mg, 0.2 mmol) and 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (55 μL, 0.2 mmol) were added in sequence, and the reaction was allowed to react at room temperature for 2 h. The plate was developed with petroleum ether: ethyl acetate (v / v) = 3:1. The reaction was detected by TLC. The molecular sieves were filtered out through a diatomaceous earth pad and diluted with dichloromethane. The organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, filtered and concentrated, toluene was added and dried with a diaphragm pump (to remove moisture) to obtain an oily crude product 41. Add to the reaction flask. Molecular sieves, vacuum-baked the flask, cooled to room temperature, and then purged with argon. Compound 1-2 (161.0 mg, 82.7 μmol) was added and dissolved in 0.5 mL of dry dichloromethane. 620 μL of tetrazole in acetonitrile (tetrazole concentration was 0.45 M) was added and stirred at room temperature for 15 min. The crude product from the previous step was dissolved in 0.6 mL of dry dichloromethane and slowly added dropwise to the reaction system. The reaction was allowed to react at room temperature for 50 min. The flask was developed with ethyl acetate:methanol (v / v) = 40:1 and TLC was performed until the starting material disappeared. The reaction solution was cooled to 0°C, tert-butyl peroxide (50 μL, 0.3 mmol) was added, the temperature was naturally raised, and the reaction was allowed to react for 2 h. The flask was developed with pure ethyl acetate and TLC was used to detect the completion of the reaction. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (ethyl acetate:methanol = 40:1) to obtain 42 as a white foamy solid in a yield of 79%.

[0126] Compound 42 (93.0 mg, 32.0 μmol) was dissolved in 2.0 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 6 h and spotted with ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 8:1:1:0.2 as the developing solvent. TLC detected the completion of the reaction of the raw materials. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The compound (acyl, cyanoethyl, and Alloc were removed) was separated on a Sephadex™ LH20 column. The above product was dissolved in 2.4 mL of a mixed solution containing methanol:tetrahydrofuran:water:acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (90.0 mg, 127.0 μmol) was added. The reaction flask was placed in a hydrogenation reactor and allowed to react at room temperature for 24 h. The developer was ethyl acetate:methanol:water:acetic acid (v / v) = 2:2:2:0.5. TLC was used to determine the completion of the reaction. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified sequentially via a C18 column (eluent: methanol and water) and then a P2 column. Freeze-dried, the product afforded 3 as a white foamy solid in a 67% yield.

[0127] 1 H NMR(600MHz,D2O)δ4.87–4.86(bs,1H,1a-H),4.64(d,J=7.8Hz,2H,1b-H,1f-H),4.60(d,J=8.4Hz,1H,1c-H),4.27 –4.14(m,4H),4.13–4.01(m,5H),4.01–3.94(m,9H),3.94–3.86(m,4H),3.86–3.79(m,6H),3.79–3.74(m,2H),3.75 –3.69(m,1H),3.67–3.55(m,5H),3.48(t,J=9.2Hz,1H),3.38–3.28(m,3H),3.00(ddd,J=8.9,7.0,2.2Hz,2H,linke r-NCH2),2.15(s,3H,COCH3),2.05(s,3H,COCH3),1.78–1.60(m,4H,linker-CH2),1.52–1.41(m,2H,linker-CH2).

[0128] 13C NMR(150MHz,D2O)δ174.6,174.0,102.9,101.4,101.1,99.5,80.9,78.93,78. 88,76.2,75.6,75.2,74.8,74.7,74.6,73.2,71.6,71.5,71.2,70.9,70.83,70 .81,70.80,70.5,70.4,69.6,69.3,68.6,67.4,66.8,66.6,66.5,66.4,65.1,64.41,64.38,62.6,61.0,60.7,54.4,51.4,39.4,27.8,26.3,22.6,22.5,22.2.

[0129] 31 P NMR(243MHz,D2O)δ2.00,1.14,-0.13.

[0130] HRMS(ESI)Calcd for C 42 H 81 N3O 37 P3 + [M+H] + :1312.3757, found 1312.3772; HRMS(ESI)Calcd for C 42 H 80 N3NaO 37 P3 + [M+Na] + :1334.3576,found 1334.3581.

[0131] Example 6. Chemical synthesis of pseudo-octasaccharide compound of Core M3 type O-mannan (2)

[0132] 6.1 Synthesis route:

[0133]

[0134] 6.2 Specific experimental steps

[0135] Add the reaction flask Molecular sieves, vacuum-baked the bottle, cooled to room temperature and then purged with argon, compound 43 (181.0 mg, 0.1 mmol) was added and dissolved in 1.0 mL of dry dichloromethane. N, N-diisopropylammonium tetrazolium salt (34.0 mg, 0.2 mmol) and 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (64 μL, 0.2 mmol) were added in sequence, and the reaction was allowed to react at room temperature for 2 h. The plate was developed with petroleum ether: ethyl acetate (v / v) = 2:1, and the reaction was completed by TLC detection. The molecular sieves were filtered out through a diatomaceous earth pad, diluted with dichloromethane, and the organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, filtered and concentrated, toluene was added and dried with a diaphragm pump (to remove moisture) to obtain an oily crude product 44. Add to the reaction flask Molecular sieves, vacuum-baked the flask, cooled to room temperature, and then purged with argon. Compound 1-2 (155.0 mg, 80.0 μmol) was added and dissolved in 0.5 mL of dry dichloromethane. 550 μL of tetrazole in acetonitrile (tetrazole concentration was 0.45 M) was added and stirred at room temperature for 15 min. The crude product from the previous step was dissolved in 0.6 mL of dry dichloromethane and slowly added dropwise to the reaction system. The reaction was allowed to react at room temperature for 50 min. The flask was developed with ethyl acetate:methanol (v / v) = 40:1 and TLC was performed until the starting material disappeared. The reaction solution was cooled to 0°C, tert-butyl peroxide (55 μL, 0.3 mmol) was added, the temperature was naturally raised, and the reaction was allowed to react for 2 h. The flask was developed with pure ethyl acetate and TLC was performed to determine the completion of the reaction. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (ethyl acetate:methanol = 50:1) to obtain 45 as a white foamy solid in 80% yield.

[0136] Compound 45 (100.0 mg, 28.0 μmol) was dissolved in 5.0 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 10 h. The solution was spotted with ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 4:1:1:0.1 as the developing solvent. TLC was performed until the starting material disappeared. 200 μL of water was added and stirred at room temperature for 12 h. TLC was performed until the product spot before adding water disappeared. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The compound (acyl, cyanoethyl, and Alloc groups had been removed) was separated on a Sephadex™ LH20 column. The above product was dissolved in 4.0 mL of a mixed solution containing methanol: tetrahydrofuran: water: acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (140.0 mg, 0.2 mmol) was added and the reaction flask was placed in a hydrogenation reactor and allowed to react at room temperature for 60 h. Mass spectrometry was performed to detect complete removal of the protecting group. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified sequentially through a C18 column (eluent: methanol and water) and a P2 column, and lyophilized to obtain a white foamy solid 2 in a yield of 67%.

[0137] 1 H NMR(600MHz,D2O)δ5.34(d,J=3.8Hz,1H,1h-H),4.86(d,J=1.7Hz,1H,1a-H),4.66(d,J=7.7Hz,1H), 4.63(d,J=8.3Hz,1H),4.60(d,J=8.3Hz,1H),4.56(d,J=8.0Hz,1H),4.28–4.14(m,4H),4.15–4.01(m ,4H),4.01–3.92(m,9H),3.92–3.71(m,18H),3.71–3.56(m,8H),3.54(dd,J=9.8,3.9Hz,1H),3.45– 3.34(m,4H),3.00(t,J=7.6Hz,2H),2.15(s,3H),2.05(s,4H),1.80–1.57(m,4H),1.52–1.41(m,2H).

[0138] 13C NMR(150MHz,D2O)δ174.7,173.9,102.7,101.4,101.2,99.5,98.8,81.1,80.9,78.9,78. 8,76.7,76.4,75.7,75.6,75.3,74.7,73.8,73.1,73.0,72.3,71.7,71.6,71.5,71.3,71. 1,70.85,70.80,70.14,70.08,69.5,69.4,69.3,68.6,67.5,66.8,66.6,66.54,66.47,64.41,64.37,62.9,62.6,61.4,61.0,60.7,54.5,51.4,39.4,27.9,26.4,22.6,22.4,22.3.

[0139] 31 P NMR(243MHz,D2O)δ1.13,0.31,-0.13.

[0140] HRMS(ESI)Calcd for C 53 H 97 N3NaO 47 P3 2+ [M+H+Na] 2+ :821.7196, found 821.7257; HRMS(ESI)Calcd for C 53 H 97 N3KO 47 P3 2+ [M+H+K] 2+ :829.7066,found 829.7135.

[0141] Example 7. Chemical synthesis of pseudo-decasaccharide compound of Core M3 type O-mannan (1)

[0142] 7.1 Synthesis route:

[0143]

[0144] 7.2 Specific experimental steps

[0145] Add the reaction flask Molecular sieves, vacuum-baked the bottle, cooled to room temperature and then purged with argon, compound 1-1 (150.0 mg, 70.0 μmol) was added and dissolved in 1.0 mL of dry dichloromethane. N, N-diisopropylammonium tetrazolium salt (24.0 mg, 140.0 μmol) and 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (46 μL, 140.0 μmol) were added in sequence, and the reaction was allowed to react at room temperature for 2 hours. The plate was developed with petroleum ether: ethyl acetate (v / v) = 2:1, and the reaction was completed by TLC detection. The molecular sieves were filtered out through a celite pad and diluted with dichloromethane. The organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, filtered and concentrated, toluene was added and dried with a diaphragm pump (to remove moisture) to obtain an oily crude product 46. Add to the reaction flask. Molecular sieves, vacuum-baked the flask, cooled to room temperature and then purged with argon, added compound 1-2 (109.0 mg, 60.0 μmol), dissolved in 0.5 mL of dry dichloromethane, added 400 μL of tetrazole in acetonitrile (tetrazole concentration is 0.45 M), stirred at room temperature for 15 minutes, dissolved the crude product from the previous step in 0.5 mL of dry dichloromethane and slowly added dropwise to the reaction system, reacted at room temperature for 50 minutes, and spotted with ethyl acetate: methanol (v / v) = 40:1 as the developing solvent. TLC detection was performed until the starting material disappeared. The reaction solution was cooled to 0°C, tert-butyl peroxide (32 μL, 0.3 mmol) was added, and the temperature was naturally raised to room temperature. The reaction was allowed to react for 2 hours, spotted with pure ethyl acetate as the developing solvent, and the reaction was completed by TLC detection. The molecular sieves were filtered off through a celite pad and diluted with dichloromethane. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated by silica gel column chromatography (ethyl acetate:methanol = 50:1) to obtain 47 as a white foamy solid in an 85% yield.

[0146] Compound 47 (30.0 mg, 7.0 μmol) was dissolved in 1.0 mL of methanol, and 5 M sodium methoxide-methanol solution was added to adjust the pH to 9-10. The mixture was stirred at room temperature for 12 h. The solution was spotted with ethyl acetate:methanol:water:acetic acid (v / v / v / v) = 4:1:1:0.5 as the developing solvent. TLC was performed until the starting material disappeared. 100 μL of water was added and stirred at room temperature for 12 h. TLC was performed until the product spot before adding water disappeared. A cation exchange resin was added to adjust the pH to 6-7. The resin was removed by filtration, and the mixture was concentrated. The compound (acyl, cyanoethyl, and Alloc groups had been removed) was separated on a Sephadex™ LH20 column. The above product was dissolved in 1.0 mL of a mixed solution containing methanol: tetrahydrofuran: water: acetic acid in a volume ratio of 10:5:4:1. 20% palladium hydroxide on carbon (40.0 mg, 56.0 μmol) was added and the reaction flask was placed in a hydrogenation reactor and allowed to react at room temperature for 72 hours. Mass spectrometry was performed to detect complete removal of the protecting group. The palladium hydroxide on carbon was filtered through a celite pad and concentrated. The product was purified sequentially by a C18 column (eluent: methanol and water) and a P2 column, and lyophilized to obtain 1 as a white foamy solid in a yield of 68%. 1 H NMR(600MHz,D2O)δ5.36(d,J=3.8Hz,2H,1j-H,1h-H),4.86(d,J=1.7Hz,1H,1a-H),4.75(d,J=8.0Hz,1H),4.66(d,J=5.4Hz,1H),4 .64(d,J=6.1Hz,1H),4.60(d,J=8.3Hz,1H),4.56(d,J=8.0Hz,1H),4.24(t,J=9.8Hz,1H),4.22–4.12(m,3H),4.14–4.00(m,4H),4. 01–3.93(m,8H),3.93–3.86(m,5H),3.86–3.79(m,6H),3.79–3.74(m,5H),3.74–3.67(m,3H),3.68–3.52(m,8H),3.51(dd,J=9.2, 8.0Hz,1H),3.46–3.34(m,4H),3.00(ddd,J=8.6,6.8,2.2Hz,2H),2.15(s,3H),2.06(s,3H),1.79–1.59(m,4H),1.53–1.38(m,2H). 13CNMR(150MHz,D2O)δ175.7,175.6,174.6,174.0,102.8,102.6,101.4,101.2,101.1,99.5,98.8,98.7,82.3,81.2 ,81.0,80.9,79.00,78.95,76.7,76.1,75.7,75.6,75.2,74.6,73.8,73.1,73.0,72.3,72.0,71.7,71.6,71.5,71. 2,71.15,71.11,70.9,70.84,70.81,70.78,70.5,70.4,69.6,69.4,69.3,68.6,68.0,67.4,66.8,66.6,66.54,66.47,66.43,64.35,64.31,62.9,62.6,62.5,61.5,61.2,61.0,60.7,54.4,51.4,39.4,27.8,26.3,22.6,22.5,22.3. 31 P NMR(243MHz,D2O)δ1.93,1.14,-0.14.HRMS(ESI)Calcd for C 64 H 113 N3NaO57P3 2+ [M+H+Na] 2+ :975.7568, found 975.7580; HRMS(ESI)Calcd for C 64 H 113 N3KO 57 P3 2+ [M+H+K] 2+ :983.7438,found 983.7459.

[0147] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for synthesizing Core M3 type O-mannans and extending their sugar chains, characterized in that: Using monosaccharides, disaccharides, and trisaccharides as starting materials, and employing Nap, Alloc, and TBDPS protecting groups, oligosaccharide fragments are constructed through glycosylation and protective group manipulation. The oligosaccharide fragments are then coupled via phosphorylation. Finally, the protecting groups are removed to yield the product, Core M3 O-mannans, and their extended sugar chains. The Core M3 type O-mannose chain and its extended sugar chain are: pseudo decaose (1), pseudo octaose (2), pseudo hexaose (3), pseudo pentasaccharide (5), pseudo tetrasaccharide (4), phosphorylated trisaccharide (6), trisaccharide (7); its structural formula is:

2. The synthesis method according to claim 1, wherein The synthesis method of the pseudo-decaose is as follows: Compound 1-1 is first reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate, which is then reacted with Compound 1-2 through a phosphorylation coupling reaction to obtain a fully protected pseudo-decaose; and finally, the pseudo-decaose is obtained through deprotection. The structural formulas of Compounds 1-1 and 1-2 are:

3. The synthesis method according to claim 2, wherein Compound 1-1 is obtained from monosaccharide donor 1-3, monosaccharide donor 1-4, monosaccharide acceptor 1-5 and pseudo-disaccharide acceptor 1-6 through multi-step glycosylation and protecting group operation; the structural formulas of compounds 1-3, 1-4, 1-5 and 1-6 are:

4. The synthesis method according to claim 2, wherein Compound 1-2 is obtained from trisaccharide 1-7, ribitol 1-8, and five-carbon linker 1-9 through multi-step glycosylation and protecting group manipulation; the structural formulas of compounds 1-7, 1-8, and 1-9 are:

5. The synthesis method according to claim 1, wherein The synthesis method of the pseudo-octasaccharide is as follows: Compound 43 is reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate 44, which is then reacted with compound 1-2 to obtain a fully protected pseudo-octasaccharide; and finally, a deprotection operation is performed to obtain the pseudo-octasaccharide. The structural formulas of compounds 43, 44, and 1-2 are:

6. The synthesis method according to claim 1, wherein The synthesis method of the pseudo-hexasaccharide is as follows: Compound 40 is reacted with a phosphoric acid reagent and a tetrazolium salt to generate a trivalent phosphorus intermediate 41, which is then reacted with compound 1-2 to obtain a protected pseudo-hexasaccharide; and finally, a deprotection operation is performed to obtain the pseudo-hexasaccharide. The structural formulas of compounds 40 and 41 are:

7. The synthesis method according to claim 1, wherein The synthesis method of the pseudo-pentasaccharide is as follows: compound 37 is added to a mixture of pseudo-tetrasaccharide 1-2 and tetrazole to obtain compound 39; and finally, a deprotection operation is performed to obtain the pseudo-pentasaccharide. The structures of compounds 37 and 39 are:

8. The synthesis method according to claim 1, wherein The synthesis method of the pseudo-tetrasaccharide is as follows: Compound 29 and Compound 1-9 are subjected to a glycosylation reaction to obtain a glycosylated product, the glycosylated product is deprotected by 2,2,2-trichloroethoxycarbonyl and then acetylated to obtain Compound 31; Compound 31 is then deprotected by p-tert-butyldiphenylsilane and phosphorylated to obtain Compound 33; and finally, Compound 33 is deprotected by Alloc to obtain Compound 34. Compound 35 and benzyl trichloroacetimidate donor were catalyzed by TfOH to obtain a Bn-protected product, and then TIPS was removed to obtain ribitol compound 36; a protecting group phosphite was introduced into the hydroxyl group of compound 36 to obtain compound 37; compound 37 was slowly added dropwise to a mixture of compound 34 and tetrazole, and then fully protected tetrasaccharide 38 was obtained under the action of tert-butyl peroxide; finally, a deprotection operation was performed to obtain a pseudo-tetrasaccharide; the structural formulas of the compounds 29, 1-9, 31, 33, 34, 36, 37, and 38 are:

9. The synthesis method according to claim 1, wherein The synthesis method of the trisaccharide is as follows: the glycosyl donor is compound 24, the glycosyl acceptor is compound 1-9, TMSOTf is used as an activator, and compound 25 is obtained by glycosidation reaction. The 2,2,2-trichloroethoxycarbonyl protecting group of compound 25 is first removed and then an acetyl group is added to obtain compound 26. The p-tert-butyldiphenylsilane protecting group is then removed to obtain compound 27. Finally, the acetyl group, benzyloxycarbonyl group and benzyl protecting groups are removed to obtain the trisaccharide. The structures of compounds 24, 1-9, 25, 26 and 27 are as follows:

10. The synthesis method according to claim 9, wherein The synthesis method of the phosphorylated trisaccharide is as follows: Compound 27 is phosphorylated to obtain Compound 28, followed by deprotection operation to obtain the phosphorylated trisaccharide; the structural formula of Compound 28 is:

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