A method for the synthesis of acinetobacter baumannii lipopolysaccharide o-antigen

Abstract

This invention belongs to the field of organic synthesis technology, specifically relating to a method for synthesizing the O-antigen of Acinetobacter baumannii lipopolysaccharide. The O-antigen described in this invention is from Acinetobacter baumannii. A.baumannii. This invention first utilizes retrosynthetic analysis to determine the synthetic strategy of a 2-1 convergent [2+3(1+2)] pentasaccharide repeating unit derivative of Acinetobacter baumannii ATCC 17961 lipopolysaccharide O-antigen, and synthesized a series of monosaccharide and disaccharide building blocks. Then, through optimization of the synthetic conditions, a fully protected pentasaccharide fragment was constructed using stereoselective glycosylation via organocatalysis, followed by the removal of the protecting group. This provides a raw material basis for further immunological research on glycoantigens and vaccine development. The method of this invention features good selectivity, high yield, and mild reaction conditions, making it easy to achieve large-scale mass production and possessing significant application prospects.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a method for synthesizing Acinetobacter baumannii lipopolysaccharide O-antigen. Background Technology

[0002] Bacterial surface polysaccharides mainly include extracellular capsular polysaccharides (CPS), membrane lipopolysaccharides (LPS), and other non-bound polysaccharides. They protect bacteria from external environmental and host immune defenses and play a crucial role in cell adhesion to other biological and abiotic surfaces. Studies have found that the polysaccharide structures on the surface of most pathogenic bacteria are similar in nature to membrane proteins and are important virulence factors that can induce immune responses in host cells.

[0003] Vaccines developed based on bacterial polysaccharide structures aim to generate specific and durable immune responses. Meanwhile, the development of glycoconjugated vaccines requires focusing on identifying a specific immunoprotective oligosaccharide and then combining the polysaccharide antigen with a carrier protein to form a glycoconjugated vaccine. This overcomes the T-cell independence of polysaccharide antigens while preserving the uniqueness of the polysaccharide structure.

[0004] LPS is a potent virulence factor and can also be recognized by the host's immune system as a pathogen-associated antigen. Therefore, vaccine development using LPS as a candidate can provide essential information for the development of vaccines and diagnostic reagents.

[0005] Acinetobacter baumannii is an aerobic Gram-negative bacillus listed by the World Health Organization as a key target for urgently needed novel antimicrobial drugs. It is a common cause of soft tissue and urinary tract infections, sepsis, pneumonia, and meningitis. As a Gram-negative pathogen, the surface polysaccharide LPS of Acinetobacter baumannii is closely related to its pathogenicity and virulence. In 2009, Evgeny et al. reported the chemical structure of the repeating unit of the lipopolysaccharide O-antigen in Acinetobacter baumannii ATCC 17961 strain; however, there are currently no reports on the synthesis method of the lipopolysaccharide O-antigen in Acinetobacter baumannii ATCC 17961.

[0006] Therefore, based on the unique structure and potential immunogenicity of the lipopolysaccharide O-antigen of Acinetobacter baumannii ATCC 17961, this invention introduces an azide group at the C-3 position using the Ferrier rearrangement reaction, starting from 2-nitroglycoene, and efficiently synthesizes diaminoglycan. Through ingenious orthogonal design of the protecting group on the core galactose, the invention achieves the sequential construction of glycosidic bonds at positions 4, 6, and 3. The stereoselectivity of the β-configuration of glycosylation is ensured by the participation effect of the acyl neighboring group at position 2, and the stereoselectivity of the α-configuration of glycosylation is ensured by the use of additives. This invention achieves the synthesis of five sugars through the construction of all glycosidic bonds, laying the foundation for the screening of Acinetobacter baumannii-related glycoantigens. Summary of the Invention

[0007] The purpose of this invention is to provide a method for synthesizing the lipopolysaccharide O-antigen of Acinetobacter baumannii ATCC 17961, which can efficiently synthesize the O-antigen pentasaccharide repeating unit, providing a raw material basis for the development of glycoconjugated vaccines.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0009] A pentasaccharide compound comprising the compound shown in Formula I and the compound shown in Formula II, with the following specific structures:

[0010]

[0011] Specifically, R1 is either Bz or AZMB.

[0012] Specifically, R2 can be TIPS, Bn, or H.

[0013] Specifically, Ac represents acetyl, Bn represents benzyl, AZMB represents 2-azidomethylbenzoyl, TIPS represents triisopropylsilyl, Bz represents benzoyl, TCA represents trichloroacetic acid, and Cbz represents benzyloxycarbonyl.

[0014] Specifically, the compounds represented by Formula II are 2-2, 2-91, or 2-92, and their specific structural formulas are as follows:

[0015]

[0016]

[0017] Furthermore, the present invention also provides a method for synthesizing the pentasaccharide compounds 2-2 and 2-91 of Formula II, comprising the following steps:

[0018] The disaccharide donor and the trisaccharide acceptor are mixed and dissolved in organic solvent I. The mixture is pre-reacted at 0°C for 10-15 min (preferably 10 min) under an inert atmosphere. Catalyst I is added, and the mixture is reacted at room temperature (20-25°C) for 2-3 h (preferably 3 h). The mixture is then quenched, filtered, concentrated, and purified by column chromatography to obtain the final product.

[0019] Specifically, the disaccharide donors are 2-5 and 2-83, with the following specific structural formulas:

[0020]

[0021] Specifically, there are 2-3 trisaccharide receptors, and their specific structural formulas are as follows:

[0022]

[0023] Specifically, the inert atmosphere is formed using nitrogen or argon.

[0024] Specifically, the organic solvent I is dichloromethane, toluene, or chloroform, preferably dichloromethane (DCM).

[0025] Specifically, catalyst I is TMSOTf (trimethylsilyl trifluoromethanesulfonate).

[0026] Specifically, the molar ratio of disaccharide donor to trisaccharide acceptor is (1-2):1; preferably 2:1.

[0027] Specifically, the molar ratio of disaccharide donor to catalyst I is (4-7):1; preferably 6.8:1 or 4.8:1.

[0028] Furthermore, the present invention also provides a method for synthesizing the pentasaccharide compound 2-92 of Formula II, comprising the following steps:

[0029] Compound 2-91 was dissolved in organic solvent II, HF-Py was added at room temperature, and the reaction was carried out in an oil bath at 25-35°C (preferably 30°C) for 72 hours. The mixture was diluted with dichloromethane and washed successively with 1M hydrochloric acid solution, saturated NaHCO3 solution and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain 2-92.

[0030] Specifically, the organic solvent II is a mixture of tetrahydrofuran and pyridine in a volume ratio of 1:1.

[0031] Furthermore, the present invention also provides a method for synthesizing the pentasaccharide compound of Formula I, comprising the following steps:

[0032] (1) Dissolve the compound shown in formula II in organic solvent III, add catalyst II, and react in an ice bath and hydrogen atmosphere to achieve hydrogenolysis of benzyl and trichloroacetyl to obtain crude product;

[0033] (2) The crude product was dissolved in tetrahydrofuran, and LiOH aqueous solution was added. After the reaction was completed at room temperature, the final product was obtained through post-treatment.

[0034] Specifically, the organic solvent III is a mixed solvent of tert-butanol, water, tetrahydrofuran, and glacial acetic acid, with a volume ratio of 5:2:(0.5-1):(0.05-0.1), preferably 5:2:1:0.05.

[0035] Specifically, the catalyst II is Pd(OH)2 / C, and the mass ratio of the intermediate product to the catalyst Pd(OH)2 / C is 1:(2-4), preferably 1:4.

[0036] Specifically, in step (1), the reaction is to exchange H2 in an ice bath for 15 minutes, followed by hydrogenation at room temperature and atmospheric pressure for 6 days to obtain the crude product.

[0037] Specifically, in step (2), the concentration of the LiOH aqueous solution is 1M, and the post-treatment includes quenching with acidic resin, filtration, concentration, and column chromatography.

[0038] Compared with the prior art, the advantages of the present invention are:

[0039] This invention first utilizes retrosynthetic analysis to determine the synthetic strategy of a 2-1 convergent [2+3(1+2)] pentasaccharide repeating unit derivative of the Acinetobacter baumannii ATCC 17961 lipopolysaccharide O-antigen, and synthesizes a series of monosaccharide and disaccharide building blocks. Then, through optimization of synthetic conditions, a fully protected pentasaccharide fragment is constructed using organocatalytic stereoselective glycosylation, followed by the removal of the protecting group. This provides a raw material basis for further research on glycoantigen immunology and vaccine development.

[0040] The method of the present invention has the characteristics of good selectivity, high yield and mild reaction conditions, and is easy to realize large-scale mass production, and has important application prospects. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0042] In the following examples, the abbreviations are: Ac for acetyl, Ac2O for acetic anhydride, AcOH for glacial acetic acid, AZMB for 2-azidomethylbenzoyl, All for allyl, Bn for benzyl, Bu2SnO for di-n-butyltin oxide, Bz for benzoyl, CSA for camphorsulfonic acid, Cbz for benzyloxycarbonyl, DCM for dichloromethane, DBU for 1,8-diazobispyrocyclo[5.4.0 undec-7-ene, DDQ for... 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone, DIPEA is diisopropylethylamine, DMAP is 4,4-dimethylaminopyridine, DMDO is dimethyldioxypropane, DMF is dimethylformamide, DMP is dimethyl phthalate, DTBMP is 2,6-di-tert-butyl-4-methylpyridine, EDCI is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, Et is ethyl, Im is imidazole. i- Pr stands for isopropyl, Me for methyl, MS for molecular sieve, Nap for 2-(bromomethyl)naphthalene, NBS for N-bromosuccinimide, NIS for N-iodosuccinimide, PE for petroleum ether, Ph for phenyl, PiVOH for trimethylacetic acid, PMB for methoxybenzyl, PMBCl for 4-methoxybenzyl chloride, PTFAI for N-phenyltrifluoroacetyl, PPY for 4-pyrrolidinylpyridine, and Py for pyridine. t Bu is tert-butyl, TBAB is tetrabutylammonium bromide, TBAI is tetrabutylammonium iodide, TBAN is tetrabutylammonium nitrate, TBAF is tetrabutylammonium fluoride, TBDPS is tert-butyldiphenylsilyl, TBDPSCl is diphenyltert-butylchlorosilane, TEMPO is 2,2,6,6-tetramethylpiperidine oxide, BAIB is bis(acetoxy)iodobenzene, TBS is tert-butyldimethylsilyl, TBSCl is dimethyltert-butylchlorosilane, TCA is trichloroacetic acid, Tf is trifluoromethanesulfonate, TIPS is triisopropylsilyl, TEA is triethylamine, TFA is trifluoroacetic acid, THF is tetrahydrofuran, TMS is trimethylsilyl, TMSOTf is trimethylsilyl trifluoromethanesulfonate, TBSOTf is tert-butyldimethylsilyl trifluoromethanesulfonate, Tol is toluene, Ts is p-toluenesulfonyl, TsOH is p-toluenesulfonic acid, and TCCA is trichloroisocyanuric acid.

[0043] In the following examples, D-galactose, acetylglucosene, 2-glucosamine hydrochloride, D-galactose, and methyl o-methylbenzoate are conventional commercially available products.

[0044] Example 1: A method for synthesizing the lipopolysaccharide O-antigen of Acinetobacter baumannii ATCC 17961

[0045] The lipopolysaccharide O-antigen of Acinetobacter baumannii ATCC 17961 described in this invention is a pentasaccharide repeating unit derivative 2-1, the specific structure of which is shown below. Through retrosynthetic analysis, it was found that compound 2-1 was synthesized by organic catalysis of compound 2-2, and compound 2-2 was synthesized by organic catalysis of disaccharide acceptor 2-4, disaccharide donor 2-5, and monosaccharide donor 2-6.

[0046]

[0047] This invention utilizes the difference in reactivity between the 3- and 6-position hydroxyl groups of the galactose moiety in disaccharide acceptor building blocks 2-4, as well as the selectivity at position 6, to design a synthesis strategy one [2+3(1+2)]: first glycosylate at position 6 to obtain a trisaccharide, and then perform glycosylation at position 3; and a synthesis strategy two [1+2+2]: a one-pot method to assemble a pentasaccharide using three building blocks.

[0048] The specific compounds were synthesized as follows: 1) Pentasaccharide fragment 2-2 was generated by glycosylation of trifluoroacetylimine ester donor 2-5 with trisaccharide acceptor 2-3 under TMSOTf promotion. 2) Trisaccharide acceptor 2-3 was prepared by selective glycosylation of trifluoroacetylimine ester donor 2-6 with disaccharide acceptor 2-4 at position 6. 3) Disaccharide acceptor 2-4 was prepared by removing two PMB protecting groups from a disaccharide fragment obtained by monosaccharide donor 2-7 and monosaccharide acceptor 2-8 under catalytic TMSOTf promotion. 4) Compound 2-5 was prepared from monosaccharide donor 2-9 and monosaccharide acceptor 2-6. During the synthesis, the construction of the four β-glycosidic bonds was accomplished using a convenient and efficient acyl neighboring group participation effect, and the hydroxyl or amino groups at position 2 of the monosaccharide building blocks 2-6, 2-7, and 2-9 were all protected with acyl groups.

[0049] 1. Synthesis of monosaccharide receptors 2-8

[0050]

[0051] This invention uses commercially available D-galactose as the starting material. A permanent benzyl protecting group is used to protect the galactose at position 2, which does not require glycosylation. Then, a readily introduced and easily removed p-methoxybenzyl (PMB) group is used to protect positions 3 and 6, allowing for efficient subsequent removal to obtain disaccharide acceptors 2-4 with disaccharide glycosylation sites. Finally, an acetyl group orthogonal to the p-methoxybenzyl group protects the galactose at position 4, ensuring preferential independent manipulation of position 4 to complete the glycosylation reaction on the inert 4-position axial bond. Regarding the introduction of the amino-containing linker, this invention completes the construction of the 1,2-cis-α-galactosidic bond, and then only requires the removal of the acetyl group at position 4 with sodium methoxide to obtain monosaccharide acceptors 2-8.

[0052] The proportions of raw materials and specific steps in the synthesis of monosaccharide donors 2-8 are as follows:

[0053] Compound 2-12

[0054]

[0055] Compound 2-11 (10.1 g, 37.1 mmol) was dissolved in 100 mL of 2,2-dimethoxypropane to obtain a suspension. After adding camphor sulfonic acid (1.72 g, 7.42 mmol, 0.2 eq), the system gradually became clear. Under nitrogen protection, the reaction was stirred at room temperature for 48 h. The reaction was quenched with triethylamine, and the solvent was removed by concentration under reduced pressure. The crude product was dissolved in 100 mL of DMF. The reaction system was placed in an ice-water bath, and sodium hydride (2.96 g, 74 mmol, 2 eq) was added in portions. The mixture was stirred for 20 min, and benzyl bromide (6.6 mL, 55.5 mmol, 1.5 eq) was slowly added. The mixture was gradually heated to room temperature and reacted overnight. TLC showed that the starting material was exhausted. The reaction was quenched dropwise with methanol, diluted with ethyl acetate, and washed three times with water, once each with saturated NaHCO3 solution and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a further crude product. The crude product was dissolved in 150 mL of 80% acetic acid aqueous solution and refluxed in an oil bath at 75 °C for 4 h. The solution was concentrated under reduced pressure, evaporated to dryness by azeotropic distillation with toluene, acetic acid and water, and separated by column chromatography (dichloromethane:methanol 20:1-16:1) to give a white solid 2-12 (7.53 g, 20.8 mmol, 56%).

[0056] For the specific method of synthesizing compound 2-11 using D-galactose as a starting material, refer to the following reference (Ohlsson J, Magnusson G. Galabiosyl donors; efficient synthesis from 1,2,3,4,6-penta-O-acetyl-β-D-galactopyranose[J].Carbohydrate Research,2000,329(1):49-55.).

[0057] Compound 2-13

[0058]

[0059] Compound 2-12 (1.81 g, 5 mmol) was dissolved in 40 mL of toluene. In a solution dried by MS (to remove water), dibutyltin oxide (2.49 mg, 10 mmol, 2 eq) was added at room temperature. The mixture was refluxed in an oil bath at 120 °C for 6 hours and allowed to cool naturally for 20 minutes. Tetrabutylammonium bromide (1.61 g, 5 mmol, 1 eq) and 4-methoxybenzyl chloride (1.7 mL, 12.5 mmol, 2.5 eq) were added rapidly, and the mixture was heated in an oil bath at 115 °C, maintaining a gentle boil. The mixture was stirred for 5 hours, and the reaction was quenched with triethylamine. The solvent was removed by concentration under reduced pressure, and the mixture was separated by column chromatography (petroleum ether: ethyl acetate 8:1-1:1) to give a white solid 2-13 (2.03 g, 3.35 mmol, 67%).

[0060] Compound 2-14

[0061]

[0062] Under nitrogen protection, compound 2-13 (1.87 g, 3.1 mmol) was dissolved in 18 mL of pyridine. 4-Dimethylaminopyridine (76 mg, 0.6 mmol, 0.2 eq) was added at room temperature, and the mixture was stirred for 5 minutes. Acetic anhydride (0.43 mL, 4.6 mmol, 1.5 eq) was then added, and the reaction was stirred at room temperature. TLC monitoring showed that the reactants were exhausted. The mixture was diluted with ethyl acetate and transferred out, washed twice with 1N HCl solution, once each with saturated NaHCO3 solution and saturated brine, and then dried over anhydrous Na2SO4. The solution was filtered and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 8:1-2:1) yielded a white solid 2-14 (1.84 g, 2.85 mmol, 92%).

[0063] Compound 2-16

[0064]

[0065] Compound 2-14 (1.94 g, 3 mmol) was dissolved in 22 mL of a mixed solvent of acetone and water (10:1 v / v). NBS (1.6 g, 9 mmol, 3 eq) was added in two portions over an ice-water bath. The mixture was slowly brought to room temperature and stirred for 1 hour. TLC was used to monitor the depletion of the reactants. The reaction was quenched by adding saturated sodium thiosulfate solution. The mixture was diluted with DCM and transferred out. The solution was washed successively with water, saturated NaHCO3 solution, and saturated brine. The solution was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 5:1–1:1) yielded colorless syrup 2-15 (1.52 g, 2.76 mmol, 92%).

[0066] Compound 2-15 (1.05 g, 1.9 mmol) was dissolved in 12 mL of acetone, potassium carbonate (320 mg, 2.3 mmol, 1.2 eq) was added, and the mixture was stirred at room temperature for 10 min. PTFAI (0.46 mL, 2.8 mmol, 1.5 eq) was added, and the mixture was reacted at room temperature for 5 h. Triethylamine was added, the mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 16:1-5:1) to obtain colorless syrup 2-16 (1.24 g, 1.71 mmol, 90%, α:β = 1.1:1).

[0067] Compound 2-17

[0068]

[0069] Trifluoroacetylimide donor 2-16 (100 mg, 0.14 mmol, 1 eq) and receptor 2-21 (63 mg, 0.19 mmol, 1.4 eq) were dissolved in 2 mL of DCM ( In MS drying and dehydration, newly activated [product / material] is added. MS (200 mg), under nitrogen protection, stirred at room temperature for 30 min, the reaction was placed at -60 °C, TMSOTf (4 μL, 0.02 mmol, 0.15 eq) was added, the temperature was gradually raised to room temperature and stirred for 2 hours, the reaction was quenched by adding triethylamine, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 12:1-3:1) to obtain a mixture of colorless syrups 2-17 and 2-17b (117 mg, 98%, α:β = 1:6.5).

[0070] Among them, receptors 2-21 were prepared according to the method described in the literature (Noti C, de Paz JL, Polito L, et al. Preparation and use of microarrays containing synthetic heparin oligosaccharides for therapid analysis of heparin–protein interactions[J]. Chemistry–A European Journal, 2006, 12(34): 8664-8686.).

[0071] Compounds 2-8

[0072]

[0073] Compound 2-17 (270 mg, 0.31 mmol) was dissolved in 3 mL of a mixture of dichloromethane and methanol (2:1), and sodium methoxide (4 mg, 0.08 mmol, 0.25 eq) was added. The mixture was reacted at room temperature for 48 hours, and the pH was adjusted to approximately 7 using acidic resin (continuously monitored with pH paper). The mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 5:1-2:1) to obtain colorless syrup 2-8 (246 mg, 0.3 mmol, 97%). [α] D 25 = +23.0 (c 0.8, CHCl3); 1 H NMR (400MHz, CDCl3) δ7.45–7.20(m,18H),7.16(d,J=6.8Hz,1H),6.86(t,J=8.1Hz,4H),5.17(d,J=1 2.9Hz,2H),4.77(t,J=9.4Hz,2H),4.70(d,J=11.0Hz,1H),4.61(d,J=11.1Hz,2H),4.55–4.41(m,4H ),4.03(s,1H),3.86(s,1H),3.81(d,J=6.1Hz,4H),3.78(s,3H),3.68(dd,J=9.7,5.6Hz,1H),3.59( dd,J=16.6,9.5Hz,2H),3.46–3.10(m,3H),2.62(s,1H),1.64–1.45(m,4H),1.31(d,J=19.7Hz,2H). 13 C NMR (100MHz, CDCl3) δ159.3,159.2,138.6,137.9,130.4,130.1,129.5,1 29.4,128.6,128.5,128.4,127.9,127.7,127.8,127.3,127.2,113.7,113 .8,97.4,77.5,75.8,73.3,72.4,69.3,68.4,68.0,67.2,55.31,55.29,5 0.5,50.2,47.2,46.2,29.8,29.1,28.0,27.6,23.5; HRMS(ESI)m / z:Calcd for C 49 H 57 NO 10 [M+Na] + 842.3880, found 842.3876.

[0074] 2. Synthesis of monosaccharide donors 2-7

[0075] This invention designs an efficient method for constructing diamino sugars. Using 2-nitroglycosides as a precursor, an azide group is introduced at C-3 via Ferrier rearrangement and N-heterogeneous Michael addition, and the glycosylation of the anolyl oxyallyl group is completed, thus introducing two amino precursors (azido and nitro). The diamino compound is then obtained by one-step reduction with zinc powder.

[0076] 2.1 Synthesis of Compound 2-27

[0077]

[0078] This invention uses commercially available all-acetylated glucoseene as the starting material. All acetylation groups are removed using potassium carbonate, yielding a crude product 2-22 after filtration. This crude product is then directly silylated at positions 3 and 6. By controlling the stoichiometric amounts of dimethyl tert-butylchlorosilane and imidazole, silylation at both positions is achieved, yielding compound 2-23. Next, the hydroxyl group at position 4 is protected with a benzyl group. Tetrahydrofuran is chosen as the solvent. Through optimization of the conditions, compound 2-24 can be prepared in tens of grams with a stable yield exceeding 80%. Further removal of the two temporarily protected syl groups using TBAF yields a glucoseene intermediate 2-25 with only the 4-position protected by a benzyl group. After complete reaction, the tetrabutylammonium fluoride residue is removed by repeated silica gel column chromatography purification, finally yielding a white solid compound 2-25. To construct the 3-position acetylated precursor compound 2-27, a sterically hindered syl group is again used to protect the 6-position. By controlling the reagent stoichiometry and reaction system concentration, a TBDPS protecting group is selectively introduced, and finally, an acetylation group is introduced at position 3.

[0079] The proportions of raw materials used in the synthesis of compound 2-27, as well as the specific steps, are as follows:

[0080] Compound 2-23

[0081]

[0082] Peracetylglucose 12 g, 44 mmol was dissolved in a mixture of dichloromethane and methanol (20 mL: 40 mL), and potassium carbonate (3 g, 22 mmol, 0.5 eq) was added. The mixture was reacted at room temperature for 4 hours, filtered, and concentrated under reduced pressure to give crude product 2-22. Crude product 2-22 was dissolved in 40 mL of DMF, and imidazole (11.98 g, 176 mmol, 4 eq) was added at room temperature. Dimethyl tert-butylchlorosilane (14.59 g, 96.8 mmol, 2.2 eq) was added under an ice-water bath, and the mixture was transferred to room temperature and reacted overnight. The mixture was diluted with ethyl acetate, washed three times with water, once each with saturated NaHCO3 solution and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 50:1-20:1) to give colorless syrup 2-23 (14.84 g, 39.6 mmol, 90%).

[0083] Compound 2-25

[0084]

[0085] Compound 2-23 (9 g, 24 mmol) was dissolved in 100 mL of tetrahydrofuran. The reaction system was placed in an ice-water bath, and sodium hydride (1.92 g, 48 mmol, 2 eq) was added in portions. The mixture was stirred for 20 min, and benzyl bromide (4 mL, 33.6 mmol, 1.4 eq) was slowly added. The mixture was gradually brought to room temperature and reacted overnight. TLC showed that the starting material was exhausted. Methanol was added dropwise to quench the reaction, and the mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 80:1) to obtain colorless syrup 2-24 (9.15 g, 19.68 mmol, 82%).

[0086] Compound 2-24 (5.4 g, 11.6 mmol) was dissolved in 35 mL of tetrahydrofuran, and TBAF (6.6 g, 23.2 mmol, 2 eq) was added. The mixture was reacted at room temperature for 3 hours. When the starting material was exhausted by TLC, the mixture was concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 5:1-1:1) to give a white solid 2-25 (2.74 g, 10.44 mmol, 90%).

[0087] Compound 2-27

[0088]

[0089] Compound 2-25 (8.3 g, 35.1 mmol) was dissolved in 330 mL of DMF, and imidazole (3.59 g, 52.7 mmol, 1.5 eq) was added. Diphenyl tert-butylchlorosilane (10 mL, 38.6 mmol, 1.1 eq) was added under ice-water bath. The mixture was transferred to room temperature and reacted for 24 hours. TLC showed that the starting material was exhausted. The mixture was diluted with ethyl acetate and transferred out. It was washed three times with water, once each with saturated NaHCO3 solution and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain crude product 2-26. The crude product was dissolved in a mixed solvent of DCM:Py (120 mL: 60 mL), and 4-dimethylaminopyridine (855 mg, 7 mmol, 0.2 eq) and acetic anhydride (5 mL, 52.6 mmol, 1.5 eq) were added. The mixture was stirred at room temperature, and the reaction mixture was monitored by TLC until the reactants were exhausted. The mixture was diluted with dichloromethane and transferred out, washed twice with 1 N HCl solution, once with saturated NaHCO3 solution, and once with saturated brine. The solution was dried over anhydrous Na2SO4. The mixture was filtered and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 30:1-20:1) was used to obtain colorless syrup 2-27 (15.6 g, 30.18 mmol, 86%).

[0090] 2.2 Synthesis of compounds 2-7

[0091]

[0092]

[0093] Starting with compound 2-27, 2-nitro compound 2-34 was first synthesized using the TBAN-Tf₂O-DTBMP system. Then, starting with 2-nitro compound 2-34, an azide group was introduced at the C-3 position using the same synthetic method, yielding compound 2-35 in 68% yield. A one-pot reduction of the nitro and azide groups was performed, and trichloroacetyl protection was successfully achieved, yielding N-acylated diamino compound 2-36. Next, starting with compound 2-27, the silylation group at position 6 was removed using tetrabutylammonium fluoride, followed by TEMPO oxidation to convert the primary hydroxyl group at position 6 to a carboxyl group. Benzyl protection of the carboxyl group yielded benzyl ester 2-33. Finally, compound 2-33 underwent palladium dichloride removal to remove the anolyl allyl group, preparing the trifluoroacetylimine ester donor 2-7.

[0094] The proportions of raw materials and specific steps in the synthesis of monosaccharide donors 2-7 are as follows:

[0095] Compound 2-35

[0096]

[0097] Compound 2-27 (12 g, 23.2 mmol) was dissolved in 250 mL of dichloromethane. Tetrabutylammonium nitrate (14.13 g, 46.4 mmol, 2 eq) and 2,6-di-tert-butyl-4-methylpyridine (9.53 g, 46.4 mmol, 2 eq) were added at room temperature under nitrogen protection. Trifluoromethanesulfonic anhydride (7.8 mL, 46.4 mmol, 2 eq) was added at -70 °C, and the reaction was carried out at -70 °C for 30 min. The dichloromethane was diluted and transferred out, washed with 1 M HCl solution, washed with saturated brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the salt formed in the reaction system was crystallized out with glacial ethyl acetate. The solution was filtered, evaporated to dryness, and separated by column chromatography (petroleum ether: ethyl acetate 20:1-16:1) to obtain colorless syrup 2-34 (9.6 g, 17.17 mmol, 74%).

[0098] Compound 2-34 (3.1 g, 5.5 mmol) was dissolved in 30 mL of dichloromethane, and 4-pyrrolylpyridine (163 mg, 1.1 mmol, 0.2 eq) and trimethylsilane azide (0.87 mL, 6.6 mmol, 1.2 eq) were added. The reaction was carried out at room temperature for 30 min. TLC showed that the starting material was exhausted. Trimethylacetic acid (337 mg, 3.3 mmol, 0.6 eq) and allyl alcohol (1.75 mL, 11 mmol, 2 eq) were added to the reaction system. The system was sealed and reacted in an oil bath at 35 °C for 48 hours. The mixture was concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 30:1-20:1) to obtain colorless syrup 2-35 (2.25 g, 3.74 mmol, 68%).

[0099] Compound 2-37

[0100]

[0101] Compound 2-35 (770 mg, 1.3 mmol) was dissolved in a mixed solvent of tetrahydrofuran:water (15 mL:1.5 mL). Zinc powder (1.7 g, 26 mmol, 20 eq) and copper sulfate (207 mg, 1.3 mmol, 1 eq) were added, followed by slow addition of hydrochloric acid (0.68 mL, 13 mmol, 10 eq). The mixture was stirred at room temperature, and TLC was used to determine when the starting material was exhausted. The hydrochloric acid was neutralized with saturated sodium bicarbonate solution, and the mixture was filtered. The water was evaporated to dryness by azeotropic distillation with toluene to obtain the crude product. The crude product was dissolved in 15 mL of tetrahydrofuran (… (Stanzas dried on MS), add triethylamine (1.4 mL, 10.4 mmol, 8 eq) and trichloroacetyl chloride (0.6 mL, 5.2 mmol, 4 eq), react at room temperature for 4 h, filter, concentrate under reduced pressure, and separate by column chromatography (petroleum ether: ethyl acetate 16:1-10:1) to obtain colorless syrup 2-36 (640 mg, 0.78 mmol, 60%).

[0102] Compound 2-36 (1.43 g, 1.7 mmol) was dissolved in 5 mL of tetrahydrofuran, and tetrabutylammonium fluoride (444 mg, 1.7 mmol, 1 eq) was added. The mixture was reacted at room temperature for 10 hours. TLC showed that the starting material was exhausted. The mixture was then concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 5:1-1:1) to give a white foamy solid 2-37 (0.9 g, 1.5 mmol, 88%).

[0103] Compound 2-33

[0104]

[0105] Compound 2-37 (1.04 g, 1.7 mmol) was dissolved in a mixed solvent of dichloromethane:water:tert-butanol (2:1:1, 20 mL). TEMPO oxidant (53 mg, 0.34 mmol, 0.2 eq) and BAIB (1.1 g, 3.4 mmol, 2 eq) were added under ice-water bath conditions. The mixture was then reacted overnight at room temperature. TLC analysis showed that the starting material was exhausted. The reaction was quenched with saturated sodium thiosulfate solution, diluted with dichloromethane, washed with water, and back-extracted from the aqueous phase with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain crude product 2-38.

[0106] The crude product 2-38 was dissolved in 10 mL of DMF, and potassium bicarbonate (1.02 g, 10.2 mmol, 6 eq) and benzyl bromide (0.6 mL, 5.1 mmol, 3 eq) were added. The mixture was stirred at room temperature for 18 h. The product was diluted with ethyl acetate and transferred out. It was washed three times with water, once each with saturated NaHCO3 solution and saturated brine, and dried over anhydrous Na2SO4. The mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 12:1-8:1) to give a white solid 2-33 (0.84 g, 1.19 mmol, 70%).

[0107] Compounds 2-7

[0108]

[0109] Compound 2-33 (0.4 g, 0.57 mmol) was dissolved in 18 mL of a mixed solvent of methanol and dichloromethane in a volume ratio of 2:1. Palladium dichloride (25 mg, 0.14 mmol, 0.25 eq) was added, and the mixture was reacted at 35 °C for 3 h. The mixture was filtered through silica gel, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 5:1-1:1) to give a white waxy solid 2-39 (300 mg, 0.45 mmol, 80%).

[0110] Compound 2-39 (300 mg, 0.45 mmol) was dissolved in 4 mL of acetone, and cesium carbonate (176 mg, 0.54 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 10 min, and then PTFAI (0.11 mL, 0.68 mmol, 1.5 eq) was added. The mixture was reacted at room temperature for 5 h, and then triethylamine was added. The mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 12:1-8:1) to give colorless syrup 2-7 (305 mg, 0.36 mmol, 81%). α-Configuration: 1 H NMR(400MHz,d6-Acetone)δ8.67(d,J=8.1Hz,1H),8.38(s,1H),7.49–7.24(m,10H),7.20(dd,J=7.3,2.0Hz,2H),7.13(t,J=7.5Hz,1H),6.85 (d,J=6.9Hz,2H),6.60(s,1H),5.34–5.22(m,2H),4.76(d,J=10.5Hz,1H),4.71(t,J=7.0Hz,2H),4.55(d,J=10.6Hz,1H),4.53–4.34(m,2H). 13 C NMR(100MHz,d6-Acetone)δ167.6,162.5,162.4,143.2,137.6,135.4,128.9,128.6,128.5 ,128.2,127.9,127.7,124.6,119.2,93.0,92.6,92.2,76.6,74.6,73.3,67.3,53.0,52.8.

[0111] 3. Synthesis of monosaccharide donors 2-6

[0112]

[0113] In the synthesis of monosaccharide building blocks 2-6, this invention starts from commercially available 2-glucosamine hydrochloride, first completes the trichloroacetylation of the C-2 amino group to prepare a thioglycoside, synthesizes compound 2-40, then uses NBS to hydrolyze the anolyte thioglycoside to obtain compound 2-41 with exposed anolyte hydroxyl groups, and then uses cesium carbonate as a base to prepare trifluoroacetic acid imine ester donor 2-6.

[0114] The proportions of raw materials used in the synthesis of monosaccharide donors 2-6, as well as the specific steps, are as follows:

[0115] Compounds 2-6

[0116]

[0117] Compound 2-40 (1.74 g, 2.53 mmol) was dissolved in 22 mL of a mixture of acetone and water (10:1 v / v). NBS (1.35 g, 7.59 mmol, 3 eq) was added in two portions over an ice-water bath. The mixture was slowly brought to room temperature and stirred for 4 hours. TLC was used to monitor the depletion of the reactants. The reaction was quenched by adding saturated sodium thiosulfate solution. The mixture was diluted with ethyl acetate and washed successively with water, saturated NaHCO3 solution, and saturated brine. The solution was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 8:1-2:1) yielded colorless syrup 2-41 (1.36 g, 2.23 mmol, 90%).

[0118] The specific method for synthesizing compound 2-40 using 2-glucosamine hydrochloride as a starting material can be found in the literature (Blatter G, Beau JM, Jacquinet J C. The use of 2-deoxy-2-trichloroacetamido-D-glucopyranose derivatives in syntheses of oligosaccharides[J].Carbohydrateresearch,1994,260(2):189-202. and Zhang Y, Knapp S.Simplified beta-glycosylationof peptides[J].Tetrahedron,2018,74(23):2891-2903.).

[0119] Compound 2-41 (770 mg, 1.1 mmol) was dissolved in 8 mL of acetone, and cesium carbonate (424 mg, 1.3 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 10 min, and then PTFAI (0.3 mL, 1.7 mmol, 1.5 eq) was added. The mixture was reacted at room temperature for 5 h, and then triethylamine was added. The mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 16:1-8:1) to give a white waxy solid 2-6 (784 mg, 0.95 mmol, 86%). α-configuration: 1H NMR (400MHz, CDCl3) δ7.53–7.24(m,15H),7.20(dd,J=7.0,2.2Hz,2H),7.10(t,J=7. 4Hz,1H),6.76(d,J=7.7Hz,2H),6.52(d,J=8.3Hz,1H),6.45(s,1H),4.88(d,J=11.2 Hz,1H),4.79(dd,J=18.8,10.9Hz,2H),4.63(dd,J=20.9,11.3Hz,2H),4.53(d,J=12 .0Hz,1H),4.32(s,1H),3.93(s,3H),3.83(d,J=10.1Hz,1H),3.72(d,J=10.8Hz,1H). 13 C NMR (100MHz, CDCl3) δ161.9,143.0,137.7,137.5,137.3,128.8,128.6,128.5,128.23,128.20,128.15,128.1, 128.0,127.9,124.6,119.3,93.7,92.1,78.4,77.6,75.25,75.16,73.7,73.6,67.7,54.0; HRMS(ESI)m / z:Calcd forC 37 H 34 Cl3F3N2O6[M+Na] + 787.1332, found 787.1326.

[0120] 4. Synthesis of disaccharide donors 2-5

[0121] Disaccharide donor 2-5 is composed of a glucose and an aminogalactose linked by a β-glycosidic bond at positions 1 and 3. Due to the relatively poor reactivity of the hydroxyl group at position 3 of the aminogalactose, glycosylation is difficult to achieve in existing synthetic methods. This invention overcomes the problem of glycosidic bond construction at this position by achieving orthogonal removal of the anodic temporary protecting group. The synthesis of disaccharide building block 2-5 begins with the preparation of monosaccharide donors and acceptors. This invention designs and synthesizes the monosaccharide trifluoroacetylimine ester donor 2-9 and monosaccharide acceptors 2-62, 2-63, 2-66, and 2-68.

[0122] 4.1 Synthesis Method 1 for Donors 2-9

[0123]

[0124] This invention starts with compound 2-49, which has a naked hydroxyl group at position 2, introduces an AZMB protecting group at position 2 to obtain compound 2-50, and then synthesizes compound 2-9.

[0125] The proportions of raw materials used in the synthesis of compounds 2-9, as well as the specific steps, are as follows:

[0126] Compound 2-50

[0127]

[0128] Compound 2-49 (1.1 g, 1.98 mmol) was dissolved in 10 mL of dichloromethane. Compound 2-48 (0.7 g, 3.96 mmol, 2 eq), EDCI (0.91 g, 4.75 mmol, 2.4 eq), and DMAP (0.46 g, 3.76 mmol, 1.9 eq) were added. Under nitrogen protection, DIPEA (1.3 mL, 7.92 mmol, 4 eq) was added at room temperature. The mixture was stirred for 5 h, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 10:1-4:1) to give a white solid 2-50 (1.16 g, 1.62 mmol, 82%).

[0129] Compounds 2-49 were prepared according to the method described in the literature (Chayajarus K, Chambers DJ, Chughtai MJ, et al. Stereospecific Synthesis of 1,2-cis Glycosides by Vinyl-Mediated IAD[J]. Organic Letters, 2004, 6(21): 3797-3800.).

[0130] Compound 2-48 (i.e., AZMBOH)

[0131]

[0132] Methyl o-methylbenzoate (3 mL, 20 mmol) was dissolved in 80 mL of carbon tetrachloride. NBS (4.2 g, 22 mmol, 1.1 eq) and benzoyl peroxide (105 mg, 0.4 mmol, 0.02 eq) were added at room temperature. The mixture was then refluxed in an oil bath at 80 °C for 24 h. After filtration and concentration under reduced pressure, crude product 2-46 was obtained. The crude product was dissolved in 60 mL of ethanol, and sodium azide (1.43 g, 22 mmol, 1.1 eq) was slowly added under an ice-water bath. The reaction was carried out at room temperature for 48 h. The reaction was quenched by slow addition of saturated brine. The mixture was extracted twice with ethyl acetate, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 8:1-3:1) yielded a colorless oil 2-47 (3.56 g, 18.6 mmol, 93%).

[0133] Compound 2-47 (3.4 g, 17.8 mmol) was dissolved in 55 mL of a mixture of tetrahydrofuran and water (10:1), and lithium hydroxide (3.73 g, 89 mmol, 5 eq) was added. The mixture was stirred at room temperature for 48 h, diluted with 100 mL of water, and extracted twice with dichloromethane. The aqueous phase was collected and the pH was adjusted to approximately 2 with 2N HCl solution. A white solid precipitated in the reaction system. The mixture was then extracted three more times with dichloromethane, and the organic phase was collected, washed with saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give white solid 2-48 (3.1 g, 17.44 mmol, 98%).

[0134] Compounds 2-9

[0135] Compound 2-50 (600 mg, 1 mmol) was dissolved in 11 mL of a mixed solvent of acetone and water (10:1 v / v). NBS (534 mg, 3 mmol, 3 eq) was added in two portions in an ice-water bath. The mixture was slowly heated to room temperature and stirred for 3 hours. The reaction was quenched with saturated sodium thiosulfate solution, diluted with ethyl acetate, and washed successively with water, saturated NaHCO3 solution, and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The product was purified by column chromatography. The product was dissolved in 3 mL of acetone, and cesium carbonate (391 mg, 1.2 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 10 min, and PTFAI (0.56 mL, 1.5 mmol, 1.5 eq) was added. The mixture was reacted at room temperature for 5 hours, and triethylamine was added. The mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether:ethyl acetate 20:1-15:1) to give compound 2-9 (670 mg, 0.86 mmol, 86%).

[0136] 4.2 Method 2 for the synthesis of donors 2-9

[0137]

[0138] This invention also proposes another synthetic route for compound 2-9, using glycoene as a raw material to synthesize compound 2-52 with a hydroxyl group exposed at position 2, then introducing an AZMB protecting group under the same condensation conditions, and subsequently removing the anolyl allyl group with palladium dichloride to obtain product 2-54, which is the same as the product after hydrolysis of the thioglycoside of compound 2-50, and then preparing donor 2-9.

[0139] The proportions of raw materials used in the synthesis of compounds 2-9, as well as the specific steps, are as follows:

[0140] Compound 2-53

[0141] Compound 2-52 (2.1 g, 4.3 mmol) was dissolved in 20 mL of dichloromethane, and compound 2-48 (1.52 g, 5.94 mmol, 2 eq), EDCI (1.97 g, 10.3 mmol, 2.4 eq), and DMAP (1.0 g, 8.2 mmol, 1.9 eq) were added. Under nitrogen protection, DIPEA (2.85 mL, 17.2 mmol, 4 eq) was added at room temperature, and the mixture was stirred for 5 h. The mixture was concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 10:1-5:1) to obtain colorless syrup 2-53 (2.65 g, 4.08 mmol, 95%).

[0142] Compounds 2-52 were prepared according to the method described in the literature (Somasundaram D, Balasubramanain KK, Shanmugasundaram B. Simple and mild stereoselective O-glycosidation using 1,2-anhydrosugars under neutral conditions[J].Tetrahedron Letters,2019,60(11):764-767.).

[0143] Compounds 2-9

[0144]

[0145] Compound 2-53 (2.63 g, 4 mmol) was dissolved in 150 mL of methanol, and palladium dichloride (177 mg, 1 mmol, 0.25 eq) was added. The mixture was reacted at 35 °C for 3 h, filtered through silica gel, concentrated under reduced pressure, and separated by rapid column chromatography (petroleum ether: ethyl acetate 6:1-3:1) to obtain colorless syrup 2-54 (2.1 g, 3.44 mmol, 86%).

[0146] Compound 2-54 (900 mg, 1.5 mmol) was dissolved in 10 mL of acetone, and cesium carbonate (586 mg, 1.8 mmol, 1.2 eq) was added. Under nitrogen protection, PTFAI (0.36 mL, 2.2 mmol, 1.5 eq) was added, and the mixture was reacted at room temperature for 5 hours. Triethylamine was added, the mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 20:1-10:1) to give colorless syrup 2-9 (1.06 g, 0.96 mmol, 91%). α-Configuration: 11H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.8 Hz, 1H), 7.64–7.47 (m, 3H), 7.43–7.27 (m, 10H), 7.26–7.16 (m, 7H), 7.12 (t, J = 7.8 Hz, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.64 (s, 1H), 6.45 (d, J = 6.6 Hz, 2H), 5.35 (dd, J = 9.4, 2.2 Hz, 1H), 4.87 (t, J = 9.9 Hz, 2H), 4.82–4.70 (m, 2H), 4.68–4.50 (m, 4H), 4.23 (t, J = 9.5 Hz, 1H), 4.07 (d, J = 9.6 Hz, 1H), 3.93 (t, J = 9.6 Hz, 1H), 3.84 (dd, J = 11.0, 3.4 Hz, 1H), 3.75 (d, J = 10.9 Hz, 1H). 13 13C NMR (100 MHz, CDCl3) δ 165.2, 143.1, 138.4, 138.0, 137.80, 137.75, 133.3, 131.0, 129.6, 129.4, 128.64, 128.56, 128.5, 128.4, 128.2, 128.1, 128.04, 127.96, 127.9, 127.82, 127.80, 127.5, 126.4, 124.3, 120.5, 119.1, 93.0, 79.7, 77.3, 75.6, 75.5, 73.60, 73.56, 72.4, 68.0, 52.9.

[0147] β-configuration: 1 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 7.7 Hz, 1H), 7.69–7.50 (m, 2H), 7.46–

[0148] 7.12 (m, 19H), 7.08 (t, J = 7.4 Hz, 1H), 6.73 (s, 2H), 5.91 (s, 1H), 5.50 (s, 1H), 4.81 (d, J = 12.0 Hz, 2H), 4.78–4.63 (m, 4H), 4.58 (dd, J = 11.3, 8.2 Hz, 2H), 4.02–3.51 (m, 5H). 13CNMR (100MHz, CDCl3) δ164.7,143.2,138.0,137.8,137.7,137.6,133.2,131.0,129.6,128.8,128.52,128.47,128.4, 128.05,128.02,127.93,127.88,127.84,127.81,124.4,119.3,95.0,82.2,76.0,75.1,75.0,73.5,72.5,68.0,52.9.

[0149] Compounds 2-9: HRMS(ESI) m / z: Calcd for C 43 H 39 F3N4O7[M+Na] + Synthesis of compound 2-66 (803.2668, found 803.2672.4.3)

[0150]

[0151] In synthesizing different 2-aminogalactosyl receptors 2-66 and 2-68, this invention first synthesizes compound 2-59, then selects 1-bromomethylnaphthalene to protect the 3-position, and adjusts the protecting groups at the 4,6-positions to benzyl groups to obtain the key intermediate building block 2-62. Then, the Nap protecting group at the 3-position is removed by DDQ oxidation to obtain the monosaccharide receptor 2-63. Starting from compound 2-62, this invention uses TBS or All to temporarily protect the anomeric position of the aminogalactosyl receptor.

[0152] Specifically, starting with compound 2-62, the anothioglycoside was hydrolyzed with NBS to obtain a naked hydroxyl group at position 1, followed by silylation with TBS. The two steps yielded a β-configuration compound 2-65 in an overall yield of 89%. Next, starting with compound 2-62, a simple thioglycoside pre-activation was performed, followed by glycosylation with an added allyl alcohol acceptor, yielding compound 2-67 in a good yield and a single β-stereoconfiguration. Then, using the same synthetic method as compound 2-63, the Nap protecting group at position 3 was removed, and compounds 2-65 and 2-67 were used to obtain the corresponding 2-aminogalactose acceptors 2-66 and 2-68.

[0153] The proportions of raw materials used in the synthesis of compound 2-66, as well as the specific steps, are as follows:

[0154] Compound 2-60

[0155]

[0156] Compound 2-59 (4.26 g, 7.71 mmol) was dissolved in 22.5 mL of a mixed solvent of DMF and THF (1:8). Under an ice-water bath, sodium hydride (0.62 g, 15.42 mmol, 2 eq) and TBAI (0.57 g, 1.54 mmol, 0.2 eq) were slowly added and stirred for 10 min. Then, 1-bromomethylnaphthalene (2.56 g, 11.56 mmol, 1.5 eq) was added, and the mixture was gradually brought to room temperature and reacted overnight. The THF was evaporated to dryness, diluted with ethyl acetate, and washed three times with water, once each with saturated NaHCO3 solution and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 8:1-2:1) to obtain a foamy solid 2-60 (6.28 g, 7.32 mmol, 95%).

[0157] Compounds 2-59 were prepared by the method described in the literature (Ramadan S, Yang W, Zhang Z, et al. Synthesis of chondroitin sulfate A bearing syndecan-1 glycopeptide[J]. Organic letters, 2017, 19(18): 4838-4841.).

[0158] Compound 2-62

[0159]

[0160] Compound 2-60 (4.81 g, 7.3 mmol) was dissolved in 50 mL of a mixed solvent of methanol and dichloromethane (3:2), p-toluenesulfonic acid (2.51 g, 14.61 mmol, 2 eq) was added, and the mixture was stirred in an oil bath at 50 °C for 6 h. The mixture was then concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 3:1-1:1) to give a waxy solid 2-61 (3.75 g, 6.57 mmol, 90%).

[0161] Compound 2-61 (2.66 g, 4.66 mmol) was dissolved in 27 mL of a mixed solvent of DMF and THF (1:8). Under an ice-water bath, sodium hydride (0.78 g, 19.55 mmol, 4.2 eq) and TBAI (0.34 g, 0.93 mmol, 0.2 eq) were slowly added, and the mixture was stirred for 10 min. Benzyl bromide (2.2 mL, 18.62 mmol, 4 eq) was then added, and the mixture was gradually brought to room temperature and reacted for 3 h. The THF was evaporated to dryness, and the mixture was diluted with ethyl acetate and transferred out. The solution was washed three times with water, once each with saturated NaHCO3 solution and saturated brine, and dried over anhydrous Na2SO4. The solution was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether:ethyl acetate 16:1-10:1) to give a white solid 2-62 (2.7 g, 3.59 mmol, 77%).

[0162] Compound 2-63

[0163]

[0164] Compound 2-62 (320 mg, 0.43 mmol) was dissolved in 5 mL of DCM, and 0.5 mL of buffer solution was added. DDQ (0.12 g, 0.52 mmol, 1.2 eq) was added under ice-water bath conditions. The reaction was carried out at room temperature for 4 h, quenched with saturated sodium thiosulfate solution, diluted with ethyl acetate, filtered, and washed successively with water, saturated NaHCO3 solution, and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 8:1-3:1) to obtain colorless syrup 2-63 (226 mg, 0.37 mmol, 87%).

[0165] Compound 2-66

[0166]

[0167] Compound 2-62 (2 g, 2.71 mmol) was dissolved in 22 mL of a mixture of acetone and water in a volume ratio of 10:1. NBS (1.45 g, 8.14 mmol, 3 eq) was added in two portions over an ice-water bath. The mixture was slowly brought to room temperature and stirred for 1 hour. TLC was used to monitor the depletion of the reactants. The reaction was quenched by adding saturated sodium thiosulfate solution. The mixture was diluted with DCM and washed successively with water, saturated NaHCO3 solution, and saturated brine. The solution was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 5:1-2:1) yielded colorless syrup 2-64 (1.64 g, 2.55 mmol, 94%).

[0168] Compound 2-64 (3.09 g, 4.8 mmol) was dissolved in 15 mL of DMF, and imidazole (1.08 g, 7.18 mmol, 2 eq) and dimethyl tert-butylchlorosilane (652 mg, 9.58 mmol, 1.5 eq) were added sequentially. Under nitrogen protection, the mixture was stirred overnight at room temperature. The solution was diluted with ethyl acetate and transferred out. The solution was washed three times with water, once each with saturated NaHCO3 solution and saturated brine, and dried over anhydrous Na2SO4. The solution was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 30:1-20:1) to obtain colorless syrup 2-65 (3.05 g, 4 mmol, 84%, β configuration).

[0169] The synthesis method of compound 2-66 is the same as that of compound 2-62 to compound 2-63, except that 3.3 g of compound 2-65 was added to obtain colorless syrup compound 2-66 (2.34 g, 3.78 mmol, 87%).

[0170] 4.4 Synthesis of compounds 2-5

[0171]

[0172]

[0173] In the synthesis of disaccharide 2-72, the synthetic conditions were optimized, and glycosylation was performed using 2-9 as the donor, 2-66 as the acceptor, and TBS as the anodic temporary protecting group to obtain disaccharide 2-72, with the yield increased to 87%. The migration yield was calculated with the acceptor as 1 eq.

[0174]

[0175] In the synthesis of compound 2-5, the disaccharide 2-72 needs to be further attached with a leaving group to prepare it as a donor. First, the temporary protecting group TBS at the anomeric position of compound 2-72 is removed using TBAF. An equimolar amount of acetic acid is added during the reaction, yielding compound 2-73 in 90% yield. The leaving group PTFAI is attached using a conventional method for preparing trifluoroacetylimine ester donors. The starting material is purified, and then an equimolar amount of organic base (triethylamine) is added to the reaction system to ensure a basic reaction environment. The order of addition is then changed: cesium carbonate, triethylamine, and PTFAI are first dissolved in acetone under nitrogen protection and stirred for 10 min before the reaction substrate is added, yielding the target product 2-5 in 70% yield.

[0176] The proportions of raw materials and specific steps for the synthesis of disaccharide donors 2-5 are as follows:

[0177] Compound 2-72

[0178]

[0179] Donor 2-9 (353 mg, 0.45 mmol, 2 eq) and acceptor 2-66 (140 mg, 0.23 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and dissolved in 2 mL of DCM. Add to MS drying and dehydration (x2) MS (200 mg), under nitrogen protection, stirred at -40 °C for 10 min, added TBSOTf (10 μL, 0.045 mmol, 0.2 eq), reacted at -40 °C for 3 h, quenched with triethylamine, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 16:1-8:1) to obtain colorless syrup 2-72 (238 mg, 0.2 mmol, 87%).

[0180] Compound 2-73

[0181]

[0182] Compound 2-72 (0.64 g, 0.53 mmol) was dissolved in 10 mL of tetrahydrofuran, and tetrabutylammonium fluoride (166 mg, 0.63 mmol, 1.2 eq) and glacial acetic acid (36 μL, 0.63 mmol, 1.2 eq) were added. The mixture was reacted at room temperature for 5 hours. TLC showed that the starting material was exhausted, and the mixture was directly concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 8:1-2:1) to obtain colorless syrup 2-73 (0.52 g, 0.48 mmol, 90%).

[0183] Compounds 2-5

[0184]

[0185] Cesium carbonate (182 mg, 0.56 mmol, 1.2 eq) and PTFAI (0.11 mL, 0.7 mmol, 1.5 eq) were dissolved in 5 mL of acetone and stirred at room temperature for 20 min. Compound 2-73 (510 mg, 0.46 mmol) was dissolved in 1 mL of acetone and added to the reaction system. The reaction was carried out at room temperature for 3 h. Triethylamine was added, the mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 15:1-8:1) to obtain colorless syrup 2-5 (410 mg, 0.32 mmol, 70%) and colorless syrup 2-74 (105 mg, 0.1 mmol, 21%). 2-5: α-configuration 1H NMR(400MHz,d6-Acetone)δ8.29(d,J=8.2Hz,1H),7.99(dd,J=7.8,0.8Hz,1H),7.61(ddd,J=21.3,10.8,3.9Hz,2H ),7.50–7.40(m,3H),7.39–7.21(m,20H),7.21–7.11(m,5H),7.07(t,J=7.5Hz,1H),6.74(d,J=6.7Hz,2H),6.36(s ,1H),5.44–5.31(m,1H),5.25(d,J=7.9Hz,1H),5.21(d,J=10.8Hz,1H),4.94–4.78(m,4H),4.75(d,J=11.2Hz,1H) ,4.72–4.52(m,7H),4.49(d,J=11.9Hz,1H),4.43(s,1H),4.19(s,1H),4.04–3.80(m,4H),3.79–3.51(m,3H).β-configuration 1 H NMR(400MHz,d6-Acetone)δ8.37(d,J=8.5Hz,1H),8.01(d,J=7.8Hz,1H),7.60(dd,J=12.1,7.2Hz,2 H),7.51–7.13(m,28H),7.10(t,J=7.4Hz,1H),6.77(d,J=7.5Hz,2H),6.00(s,1H),5.34(t,J=8.6Hz ,1H),5.20–5.14(m,1H),5.03(d,J=8.1Hz,1H),4.92(d,J=14.4Hz,1H),4.85(dd,J=10.8,4.9Hz,2H ),4.80–4.36(m,10H),4.20(s,1H),4.07–3.75(m,5H),3.71(d,J=7.8Hz,2H),3.63(d,J=6.1Hz,1H).

[0186] 5. Synthesis of disaccharide receptors 2-4

[0187]

[0188] Disaccharide 2-75 was prepared by direct glycosylation of the naked hydroxyl group on the axial bond of the galactose acceptor 2-8 with the trifluoroacetylimine diaminouronic acid donor 2-7. TBSOTf was used as a promoter and dichloromethane as a solvent. By adjusting the reaction conditions and controlling the reaction temperature at -40℃, the reaction yield was preferably increased to 93% by increasing the equivalence of donor 2-7, and the result was a single β-configuration.

[0189]

[0190] Then, at 0°C, the obtained disaccharide 2-75 was dissolved in a dichloro solution containing 5% trifluoroacetic acid. After acid treatment, the target disaccharide 2-4 was finally obtained in 94% yield.

[0191] The proportions of raw materials and specific steps involved in the synthesis of disaccharide receptor 2-4 are as follows:

[0192] Compound 2-75

[0193]

[0194] Donor 2-7 (104 mg, 0.12 mmol, 1.5 eq) and acceptor 2-8 (68 mg, 0.083 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and dissolved in 2 mL of DCM. Add to MS drying and dehydration (x2) MS (200 mg), under nitrogen protection, stirred at -40 °C for 10 min, added TBSOTf (4 μL, 0.017 mmol, 0.2 eq), reacted at -40 °C for 2 h, quenched with triethylamine, filtered, concentrated under reduced pressure, and separated by rapid column chromatography (petroleum ether: ethyl acetate 8:1-2:1) to obtain colorless transparent syrup 2-75 (106 mg, 0.072 mmol, 87%).

[0195] Compounds 2-4

[0196]

[0197] Compound 2-75 (180 mg, 0.12 mmol, 1 eq) was dissolved in 5 mL of dichloromethane. TFA (0.25 mL) was added under ice-water bath conditions, and the reaction was gradually brought to room temperature. TLC monitoring showed that the starting material was exhausted. The reaction mixture was poured into an ice-cold saturated sodium bicarbonate solution, extracted twice with dichloromethane, and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether:ethyl acetate 5:1-1:1) to give colorless syrup 2-4 (138 mg, 0.11 mmol, 94%). [α] D 25 = +9.5 (c 2.0, CHCl3); 1H NMR(400MHz,d6-Acetone)δ8.45(d,J=9.4Hz,1H),8.23(d,J=8.7Hz,1H),7.53–7.06(m,25H) ,5.33(d,J=8.4Hz,1H),5.23(s,2H),5.16(s,2H),4.94(s,1H),4.75(d,J=10.6Hz,1H),4.67 –4.45(m,6H),4.34(d,J=3.9Hz,1H),4.32–4.25(m,2H),4.25–4.12(m,2H),4.06(s,1H),3.8 9–3.68(m,3H),3.67–3.51(m,2H),3.37(s,1H),3.24(s,3H),1.54(s,4H),1.42–1.30(m,2H). 13 C NMR(100MHz,d6-Acetone)δ168.2,162.1,161.9,156.3,155.7,139.2,138.6,137. 9,137.4,135.5,128.5,128.44,128.36,128.09,128.07,127.85,127.79,127.7,12 7.53,127.49,127.3,127.1,102.1,96.4,93.0,92.8,77.7,77.1,76.4,75.0,74.2,71.3,70.0,69.1,67.4,67.1,66.6,60.8,57.2,55.4,50.1,47.0,46.1,29.1,23.4.

[0198] 6. Synthesis of pentasaccharides 2-2 and 2-1

[0199] 6.1 Synthesis of compound 2-2:

[0200] For the synthesis of the pentasaccharide repeating unit 2-2, this invention first employs a [2+3(1+2)] convergent glycosylation strategy. By controlling the donor equivalence and reaction temperature, the regioselectivity of the trifluoroacetylimine ester donor 2-6 with the naked hydroxyl group at position 6 of the disaccharide acceptor 2-4 is ensured. Through optimization of the reaction conditions—using 1.2 equivalence donors, reacting at -60°C, and using TMSOTf as a promoter—the reaction time was shortened to 1 hour, the yield increased to 90%, and no tetrasaccharide byproducts were observed. This invention also attempts to synthesize the pentasaccharide 2-2 using a [2+3] glycosylation strategy with the disaccharide donor 2-5 and the trisaccharide acceptor 2-3 under TMSOTf as a promoter.

[0201] This invention also utilizes a one-pot strategy to synthesize the pentasaccharide 2-2 by sequentially adding trifluoroacetylimine ester donors 2-6 and 2-5 to the disaccharide acceptor 2-4 under the condition of TMSOTf as a promoter. Both glycosylation strategies resulted in a residual amount of trisaccharide acceptor 2-3. The reaction systems were purified by gel column chromatography, yielding the pentasaccharide products in yields of 60% and 56%, respectively.

[0202] The proportions of raw materials and specific steps in the synthesis of pentasaccharide 2-2 are as follows:

[0203] Compounds 2-3

[0204]

[0205] Donor 2-6 (168 mg, 0.22 mmol, 1.2 eq) and acceptor 2-4 (220 mg, 0.018 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and dissolved in 10 mL of DCM ( Add to MS drying and dehydration (x2) MS (1 g), under nitrogen protection, stirred at -60°C for 10 min, then TBSOTf (9 μL, 0.04 mmol, 0.2 eq) was added, and the reaction was carried out at -60°C for 2 h. The mixture was quenched with triethylamine, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 8:1-2:1) to give white syrup 2-3 (279 mg, 0.015 mmol, 86%). [α] D 25 = +2.5 (c 1.0, CHCl3); 1 H NMR (400MHz, CDCl3) δ7.51 (d, J = 7.3Hz, 1H), 7.40–6.97 (m, 41H), 5.28–5.05 (m, 4H) ),4.97(d,J=7.1Hz,1H),4.81(d,J=7.2Hz,1H),4.72(dd,J=12.2,7.5Hz,4H),4.6 4–4.38(m,9H),4.32(d,J=8.7Hz,1H),4.16(d,J=8.0Hz,1H),4.13–3.92(m,5H),3 .92–3.43(m,10H),3.22(s,3H),2.59(s,1H),1.51(d,J=5.7Hz,4H),1.27(s,2H). 13C NMR (100MHz, CDCl3) δ167.8,162.8,162.2,161.8,138.4,138.1,138.04,137.95,136.9,134.9,128 .6,128.5,128.41,128.39,128.34,128.32,128.28,127.89,127.86,127.83,127.80,127.74,127. 67,127.62,127.58,127.2,101.5,100.1,96.7,92.8,92.3,92.2,80.0,78.1,75.6,75.1,74.42,74.38,74.3,73.6,71.9,69.5,69.4,68.6,68.4,67.6,67.2,60.1,57.0,56.2,54.5,50.5,27.7,23.6.

[0206] Synthetic Method 1 for Compound 2-2 ([2+3] Glycosylation)

[0207]

[0208] Donor 2-5 (61 mg, 0.048 mmol, 2 eq) and acceptor 2-3 (43 mg, 0.024 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and dissolved in 2 mL of DCM. Add to MS drying and dehydration (x2) MS (200 mg), under nitrogen protection, stirred at 0 °C for 10 min, added TMSOTf (2 μL, 0.01 mmol, 0.3 eq), gradually increased to room temperature for 3 h, quenched with triethylamine, filtered, concentrated under reduced pressure, and separated by gel column chromatography (dichloromethane:methanol 1:1) to give compound 2-2 (41 mg, 0.014 mmol, 60%). 1H NMR(400MHz,CDCl3)δ7.78(t,J=8.9Hz,2H),7.61(d,J=7.9Hz,1H),7.49(t,J=7.4Hz,1H),7.46–6.92(m,68H),6.86(d,J=8.3Hz,1H),5.34(t,1H),5.22–4.98(m,6H),4.91(dd,J=25.4,17.2Hz,2H),4.84–4.55(m,12H),4.54–4.35(m,10H),4.35–4.10(m,5H),4.10–3.63(m,18H),3.63–3.45(m,5H),3.45–2.79(m,6H),1.60–1.36(m,4H),1.23–1.04(m,2H). 13 C NMR(100MHz,CDCl3)δ168.07,165.03,163.57,162.36,161.95,156.25,138.93,138.85,138.12,138.10,137.97,137.95,137.90,137.84,137.76,137.73,137.65,137.29,137.04,132.88,131.13,130.99,130.43,129.94,129.18,128.61,128.51,128.48,128.38,128.33,128.30,128.27,128.22,128.04,127.97,127.93,127.83,127.80,127.76,127.71,127.68,127.57,127.48,127.32,127.20,101.56,101.09,99.87,99.42,97.30,92.44,92.43,91.82,91.79,82.68,80.93,78.49,78.03,77.57,76.23,75.83,75.28,75.16,75.12,75.08,75.02,74.87,74.76,74.69,74.64,74.48,74.47,73.76,73.66,73.64,73.41,72.78,69.20,69.03,68.73,68.60,67.91,67.70,67.61,67.52,67.21,57.60,57.24,57.19,56.87,55.72,54.64,54.60,53.11,52.91,50.11,49.89,47.39,28.02,27.26,23.60,22.75.

[0209] Synthesis Method 2 for Compound 2-2 (One-pot Method)

[0210]

[0211] Donor 2-6 (28 mg, 0.036 mmol, 1.2 eq) and acceptor 2-4 (37 mg, 0.03 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and then mixed with 200 mg of freshly activated [substance / material]. MS was dissolved together in 2 mL of DCM ( Under nitrogen protection, the mixture was stirred at -60°C for 10 min in MS (drying and dehydration x2). TMSOTf (1 μL, 0.006 mmol, 0.2 eq) was added, and the reaction was carried out at -60°C for 2 h. The dried donor 2-5 (76 mg, 0.06 mmol, 2 eq) was dissolved in 1 mL of dry DCM and added to the reaction system. The temperature was then raised to 0°C, and TMSOTf (1 μL, 0.006 mmol, 0.2 eq) was added. The reaction was then carried out at room temperature for 2 h. Triethylamine was added to quench the reaction mixture, which was then filtered, concentrated under reduced pressure, and separated by gel column chromatography (dichloromethane:methanol 1:1) to obtain compound 2-2 (48 mg, 0.017 mmol, 56%).

[0212] 6.2 Synthesis of Compound 2-1:

[0213] The present invention also improves the [2+3] convergent synthesis strategy by adjusting the protecting groups on disaccharide donors 2-5 to achieve the synthesis of the target compound.

[0214] 6.2.1 Synthesis of compound 2-83:

[0215]

[0216] Synthesis of novel donors and acceptors: Starting from the existing building block compound 2-44, this invention introduces a Bz protecting group onto the exposed hydroxyl group at position 2, and then uses borane to reduce and cleave the benzyl acetal protecting group to obtain compound 2-77 with an exposed hydroxyl group at position 6. Next, a TIPS protecting group is used to protect the hydroxyl group at position 6, and finally, donor 2-80 is prepared.

[0217] The acceptor 2-68, with an anolyl-protected allyl group, was reacted with the donor 2-80 to obtain the disaccharide 2-81 using the same synthetic method as that used for the disaccharide 2-72. The anolyl allyl group was further removed using palladium dichloride to give compound 2-82 in a two-step overall yield of 67%. A novel trifluoroacetylimine ester donor 2-83 was prepared using the same method as that used for compound 2-5.

[0218] The proportions of raw materials used in the synthesis of compound 2-83, as well as the specific steps, are as follows:

[0219] Compound 2-77

[0220]

[0221] Compound 2-44 (5.9 g, 13.09 mmol) was dissolved in 60 mL of dichloromethane, and DMAP (0.64 g, 5.24 mmol, 0.4 eq) and triethylamine (7.3 mL, 52.36 mmol, 4 eq) were added. Under nitrogen protection, B2Cl (3.1 mL, 26.18 mmol, 2 eq) was slowly added under ice-water bath and stirred for 4 h. The mixture was concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 10:1-5:1) to give a white solid 2-76 (5.66 g, 10.21 mmol, 78%).

[0222] Compound 2-76 (1.28 g, 2.3 mmol) was placed in a 50 mL round-bottom flask, and borane tetrahydrofuran solution (14 mL, 14 mmol, 6 eq) was added. The mixture was stirred in an ice-water bath for 10 min, and copper trifluoromethanesulfonate (42 mg, 0.12 mmol, 0.05 eq) was added. The mixture was gradually brought to room temperature and stirred overnight. The reaction was quenched dropwise with methanol, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 12:1-5:1) to give a white solid 2-77 (1.2 g, 2.16 mmol, 94%).

[0223] Compounds 2-44 were prepared according to the method described in the literature (Shie CR, Tzeng ZH, Kulkarni SS, et al. Cu(OTf)2 as an Efficient and Dual-Purpose Catalyst in the Regioselective Reductive Ring Opening of Benzylidene Acetals[J]. Angewandte Chemie International Edition, 2005, 44(11): 1665-1668.).

[0224] Compound 2-80

[0225]

[0226] Compound 2-77 (5.35 g, 9.61 mmol) was dissolved in 5.5 mL of DMF, and imidazole (0.98 g, 14.42 mmol, 1.5 eq) and DMAP (0.12 g, 0.96 mmol, 0.1 eq) were added. TIPSCl (2.5 mL, 11.68 mmol, 1.2 eq) was added under ice-water bath, and the mixture was transferred to room temperature and reacted for 3 h. The mixture was diluted with ethyl acetate and transferred out. It was washed three times with water, once each with saturated NaHCO3 solution and saturated brine, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 100:1-50:1) to obtain colorless syrup 2-78 (6.28 g, 8.17 mmol, 85%).

[0227] Compound 2-78 (6.28 g, 8.18 mmol) was dissolved in 80 mL of a mixed solvent of acetone and water (3:1 volume ratio). TCCA (2 g, 8.59 mmol, 1.05 eq) and NaHCO3 (3.43 g, 40.88 mmol, 5 eq) were added in two portions in an ice-water bath. The reaction was carried out at 0 °C for 1 hour, quenched with saturated sodium thiosulfate solution, diluted with ethyl acetate, and washed successively with water, saturated NaHCO3 solution, and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Column chromatography (petroleum ether:ethyl acetate 20:1–10:1) yielded colorless syrup 2-79 (4.26 g, 6.46 mmol, 79%).

[0228] Compound 2-79 (1 g, 1.61 mmol) was dissolved in 10 mL of acetone, and cesium carbonate (630 mg, 1.93 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 10 min, and PTFAI (0.4 mL, 2.41 mmol, 1.5 eq) was added. The mixture was reacted at room temperature for 5 h, and triethylamine was added. The mixture was filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 100:1-60:1) to obtain colorless syrup 2-80 (1 g, 1.26 mmol, 78%).

[0229] Compound 2-68

[0230]

[0231] Under nitrogen protection, compound 2-62 (110 mg, 0.15 mmol, 1 eq) was... MS (500 mg) dissolved in 5 mL DCM ( In MS (drying), after stirring at 0°C for 10 min, allyl alcohol (0.1 mL, 1.46 mmol, 10 eq), NIS (46 mg, 0.2 mmol, 1.4 eq), and trifluoromethanesulfonic acid (8 μL, 0.09 mmol, 0.6 eq) were added. The mixture was stirred at 0°C overnight for 6 h. Triethylamine and saturated sodium thiosulfate solution were added to quench the reaction. The molecular sieve was removed by filtration, the mixture was concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 16:1-10:1) to obtain colorless syrup 2-67 (73 mg, 0.12 mmol, 80%).

[0232] The method for synthesizing compound 2-68 is the same as that for synthesizing compound 2-63 using compound 2-62, except that adding 1.9 g of compound 2-67 yields colorless syrup 2-68 (1.24 g, 2.3 mmol, 82%).

[0233] Compound 2-83

[0234]

[0235] Donor 2-80 (120 mg, 0.15 mmol, 1.5 eq) and acceptor 2-68 (55 mg, 0.1 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and dissolved in 2 mL of DCM. Add to MS drying and dehydration (x2) MS (200 mg), under nitrogen protection, stirred at -20 °C for 10 min, added TBSOTf (5 μL, 0.02 mmol, 0.2 eq), reacted at -20 °C for 4 h, quenched with triethylamine, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether: ethyl acetate 20:1-10:1) to obtain colorless transparent syrup 2-81 (92 mg, 0.081 mmol, 81%).

[0236] Compound 2-81 (561 mg, 0.49 mmol) was dissolved in 28 mL of a mixed solvent of methanol and dichloromethane (3:1). Palladium dichloride (22 mg, 0.12 mmol, 0.25 eq) was added at room temperature, and the mixture was transferred to an oil bath at 35 °C and reacted for 3 h. The mixture was filtered through silica gel, concentrated under reduced pressure, and separated by rapid column chromatography (petroleum ether: ethyl acetate 6:1-2:1) to give colorless foamy 2-82 (450 mg, 83%).

[0237] Under nitrogen protection, cesium carbonate (108 mg, 0.33 mmol, 1.5 eq), PTFAI (0.1 mL, 0.55 mmol, 2.5 eq), and TEA (46 μL, 0.33 mmol, 1.5 eq) were dissolved in 5 mL of acetone and stirred at room temperature for 10 min. Compound 2-82 (245 mg, 0.22 mmol) was dissolved in 1 mL of acetone and added to the reaction system. The reaction was carried out at room temperature for 2 hours. Triethylamine was added, the mixture was filtered, concentrated under reduced pressure, and separated by rapid column chromatography (petroleum ether: ethyl acetate 20:1-10:1) to obtain colorless syrup 2-83 (197 mg, 0.15 mmol, 70%) and a small amount of oxazoline product.

[0238] 6.2.2 Synthesis of compound 2-91:

[0239] Using a protected disaccharide donor 2-83 and acceptor 2-3, with dichloromethane as solvent and TMSOTf as promoter, a fully protected pentasaccharide repeating unit 2-91 was synthesized at room temperature with a yield of 78%. The specific steps are as follows:

[0240] Compound 2-91

[0241]

[0242] Donor 2-83 (87 mg, 0.068 mmol, 2 eq) and acceptor 2-3 (61 mg, 0.034 mmol, 1 eq) were mixed, azeotropically twice with toluene, evaporated to dryness, and dissolved in 3 mL of DCM. In MS drying and dehydration (x2), newly activated [product / method] is added. MS (300 mg), under nitrogen protection, stirred at 0 °C for 10 min, added TMSOTf (2 μL, 0.01 mmol, 0.3 eq), gradually increased to room temperature for 3 hours, quenched with triethylamine, filtered, concentrated under reduced pressure, purified by gel column chromatography (dichloromethane:methanol 1:1), and then purified by silica gel column chromatography (petroleum ether:ethyl acetate 10:1-3:1) to obtain colorless syrup 2-91 (89 mg, 0.026 mmol, 78%, α:β = 1:1.7). 1H NMR (400MHz, CDCl3) δ8.01(t,J=7.8Hz,2H),7.75(d,J=6.7Hz,1H),7.54(t,J=7.4Hz,1 H),7.46–6.98(m,64H),6.98–6.77(m,2H),5.40–5.25(m,1H),5.25–4.96(m,6H),4.96 –4.86(m,2H),4.86–4.54(m,12H),4.54–4.07(m,13H),4.08–3.66(m,18H),3.66–3.28 (m,7H),3.28–2.83(m,3H),1.65–1.35(m,4H),1.24–1.10(m,2H),1.10–0.90(m,21H).

[0243] 6.2.3 Synthesis of compound 2-92:

[0244] Finally, the protecting groups of the pentasaccharide mixture were partially removed to facilitate purification. After reacting at 30°C for 3 days using HF-Py conditions, a product with desiliconized groups and a single stereoconfiguration was obtained in 67% yield. When using HF-Py conditions to remove silicon groups, the β-configuration product of the isomer pair 2-91 showed good desiliconization. The specific steps are as follows:

[0245] Compound 2-92

[0246]

[0247] Compound 2-91 (132 mg, 0.046 mmol) was dissolved in 2 mL of a mixed solvent of tetrahydrofuran and pyridine (1:1). HF-Py (0.1 mL) was added at room temperature, and the mixture was reacted in an oil bath at 30 °C for 72 h. The solution was diluted with dichloromethane and washed successively with 1 M hydrochloric acid solution, saturated NaHCO3 solution, and saturated brine. The solution was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography (petroleum ether:ethyl acetate 5:1-1:1) to obtain colorless syrup 2-92 (84 mg, 0.031 mmol, 67%, single GalpN-β configuration). GalpN-β pentasaccharide: 1H NMR (400MHz, CDCl3) δ8.00(d,J=7.4Hz,2H),7.77(s,1H),7.63(d,J=5.8Hz,1H),7.54(t,J=7.4Hz,1H),7.48–6.95(m, 64H),6.90(d,J=5.8Hz,2H),5.29(t,J=8.3Hz,1H),5.24–5.02(m,5H),4.98(d,J=7.8Hz,1H),4.93–4.54(m,15H),4.48 (s,4H),4.41(d,J=6.2Hz,1H),4.39–4.20(m,4H),4.17(d,J=11.4Hz,1H),3.98(d,J=9.1Hz,2H),3.95–3.77(m,10H),3 .77–3.64(m,6H),3.64–3.47(m,4H),3.39(d,J=16.4Hz,4H),3.31–2.82(m,3H),1.59–1.37(m,4H),1.22–1.04(m,2H). 13 C NMR (101MHz, CDCl3) δ167.97,165.18,163.27,162.45,161.89,161.60,156.73,1 56.20,139.10,138.64,138.06,137.92,137.82,137.73,137.69,137.46,137.18 ,136.53,135.01,133.23,130.06,129.61,129.15,128.55,128.48,128.42,128.30,128.27,128.18,128.06,127.96,127.88,127.77,127.74,127.66,127.56,12 7.29, 127.15, 101.64, 100.96, 99.38, 99.04, 97.10, 92.76, 92.36, 91.72, 82.44, 80.80, 78.18, 77.95, 76.16, 75.60, 75.38, 75.28, 75.18, 74.95, 74.66, 74.56, 74 .33,73.82,73.71,72.81,69.05,68.88,68.71,67.79,67.54,67.18,61.53,57.92,57.23,55.70,54.48,50.52,50.09,47.35,46.22,28.01,27.27,23.56,22.69.

[0248] 6.2.4 Synthesis of Compound 2-1:

[0249]

[0250] Further removal of protecting groups: Compound 2-92 currently contains only benzoyl, benzyl, and acyl groups on the amino group. The target molecule 2-1 was obtained by removing the benzoyl group with a base and then hydrogenating the remaining protecting groups using Pd(OH)₂ / C. Specifically, the benzyl and trichloroacetyl groups were hydrogenated using Pd(OH)₂ / C in a mixed solvent of tert-butanol, water, tetrahydrofuran, and glacial acetic acid. After simple purification of the crude product, it was treated with 1M lithium hydroxide solution, followed by neutralization with acidic resin and purification by gel column chromatography. After 6 days of atmospheric pressure hydrogenation and 8 hours of treatment with 1M lithium hydroxide solution, the reaction system of compound 2-92, when developed with ethanol:ammonia:water (5:2:1), showed two obvious byproducts with low polarity, making it difficult to purify completely using a Sephadex LH-20 column (methanol:water). NMR data analysis showed that the aromatic protecting groups had been completely removed. The specific steps are as follows:

[0251] Compound 2-92 (28 mg, 0.01 mmol) was dissolved in 1 mL of tetrahydrofuran, and 5 mL of tert-butanol, 2 mL of H2O, and 0.05 mL of AcOH were added. 120 mg of Pd(OH)2 / C was added, and the mixture was ventilated with H2 in an ice bath for 15 min. The reaction was carried out at room temperature for 6 days under 1 atm H2 conditions. The mixture was filtered, concentrated, and the crude product was dissolved in 3 mL of tetrahydrofuran. 3 mL of 1 M LiOH aqueous solution was added, and the reaction was carried out at room temperature for 8 h. The reaction was quenched with acidic resin, filtered, concentrated, and purified by Sephadex LH-20 column chromatography (methanol:water) to obtain the final product.

Claims

1. A method for synthesizing a pentasaccharide compound, characterized in that, The structure of the pentasaccharide compound is as follows: Equation I; The synthesis method includes the following steps: 28 mg of compound 2-92 was dissolved in 1 mL of tetrahydrofuran, and 5 mL of tert-butanol, 2 mL of H2O, and 0.05 mL of AcOH were added. 120 mg of Pd(OH)2 / C was added, and the mixture was ventilated with H2 in an ice bath for 15 min. The reaction was carried out at room temperature for 6 days under 1 atm H2 conditions. The mixture was filtered, concentrated, and the crude product was dissolved in 3 mL of tetrahydrofuran. 3 mL of 1M LiOH aqueous solution was added, and the reaction was carried out at room temperature for 8 h. The reaction was quenched with acidic resin, filtered, concentrated, and purified by Sephadex LH-20 column chromatography to obtain the compound described in Formula I. Compound 2-92 was prepared by the following steps: Compound 2-91 was dissolved in organic solvent II, HF-Py was added at room temperature, and the reaction was carried out in an oil bath at 25~35℃ until complete. The mixture was diluted with dichloromethane and washed successively with 1M hydrochloric acid solution, saturated NaHCO3 solution and saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain 2-92. Compound 2-92 has the following structural formula: ; The organic solvent II is a mixture of tetrahydrofuran and pyridine in a volume ratio of 1:1; Compound 2-91 was prepared by the following steps: The disaccharide donor and the trisaccharide acceptor were mixed and dissolved in organic solvent I. The mixture was pre-reacted at 0°C for 10-15 min under an inert atmosphere. Catalyst I was added, and the mixture was reacted at room temperature for 2-3 h. The reaction was quenched, filtered, concentrated, and purified by column chromatography to obtain the final product. Compound 2-91 has the following structural formula: ; The disaccharide donor is 2-83, and its structural formula is: ； The trisaccharide receptors are 2-3, and their structural formulas are as follows: ； The inert atmosphere is formed using nitrogen or argon. The organic solvent I is dichloromethane, toluene, or chloroform; The catalyst I is TMSOTf; The molar ratio of disaccharide donor to trisaccharide acceptor is (1-2):1; The molar ratio of disaccharide donor to catalyst I is (4-7):1.

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