Saccharides against sialidases, methods of manufacture and uses thereof

Abstract

Disclosed are methods of preparing 3-fluorosialic acid-containing sugars, and methods of conjugating the same to homogeneous antibodies. Also within the scope of the invention are 3-fluorosialic acid-containing compounds, monoclonal antibodies conjugated to N-glycans terminating in 3-fluorosialic acid glycosides linked in an alpha 2,6 configuration, and methods of using such monoclonal antibodies to treat cancer.

Description

Background Technology

[0001] Sialic acid is a negatively charged monosaccharide, typically located at the terminal ends of glycans on glycolipids and glycoproteins. It participates in many physiological intracellular and intercellular processes, including interactions with other biomolecules and receptors on cells, viruses, and bacteria. 1 Furthermore, sialylation plays a crucial role in regulating the function and fate of secreted glycoproteins and membrane-bound receptors. For example, sialylation of epidermal growth factor receptor (EGFR) has been shown to inhibit EGFR dimerization, thereby interfering with EGF binding and phosphorylation, which is associated with tumorigenesis. 2 Furthermore, sialylation regulates the half-life of glycoproteins in the bloodstream because the desialylation of N-glycans exposes the underlying galactose, which is recognized by hepatic desialylated glycoprotein receptors, leading to the rapid removal of glycoproteins from circulation. 3 Therefore, increasing the degree of sialylation can improve the half-life and adverse effects of glycoprotein therapeutics.

[0002] In recent years, the role of glycosylation in protein structure and function has been extensively studied, inspiring the development of new methods for the synthesis and glycoengineering of proteins, particularly therapeutic monoclonal antibodies (mAbs). 5 For example, afucosylated biantennary N-glycans with terminal α-2,6-linked sialic acid have been shown to have the optimal glycan structure, enhancing antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activity. 6 .

[0003] Sialic acid derivatives with fluoride at the C-3 position are known to inhibit sialyltransferase and sialidase (or neuraminidase), and are more stable. 7 However, glycosylation using fluorinated sugars as donors often presents significant challenges due to the strong electron-withdrawing effect of the fluorine group, which inactivates the glycosylation reaction. Although a sialyltransferase exists capable of transferring 3-fluorosialic acid residues from the corresponding cytidine monophosphate-sialic acid to form α-2,3-linked sialyl glycosides, a corresponding α-2,6-sialyltransferase does not exist. 11 .

[0004] Therefore, there is a need to provide a reliable method for preparing sugars containing α-2,6-linked 3-fluorosialic acid glycosides for the development of glycoprotein therapeutic agents. Summary of the Invention

[0005] One aspect of the present invention is a method for preparing a sugar containing 3-fluorosialic acid. The method includes reacting 3-hydroxysialic acid with a sugar to perform a glycosylation reaction to form an α2,6-linked 3-hydroxysialic acid glycoside, and reacting the α2,6-linked 3-hydroxysialic acid glycoside with a fluorinating agent to perform a fluorination reaction to form a sugar containing 3-fluorosialic acid.

[0006] Another aspect of the present invention is a method for preparing homogeneous antibodies that bind to a sugar containing 3-fluorosialic acid. This method involves glycosylation of a monoclonal antibody using the sugar.

[0007] Another aspect of the present invention relates to compounds of formula (VI):

[0008]

[0009] Where R 1 Is it Ac or H, R? 2 Is it Bz or H, R? 3 It is methyl or H, R 4 It is Bn or H, and X is OH, leaving group or sugar. Note: "Ac" represents acetyl, "Bz" represents benzyl, "Bn" represents benzyl, and the sugar can be monosaccharide, disaccharide or oligosaccharide.

[0010] The scope of this invention also includes a monoclonal antibody that binds to an N-glycan terminated with an α2,6-linked 3-fluorosialic acid glycoside.

[0011] This invention further encompasses a method for treating cancer. The method includes administering to an individual in need an effective amount of a monoclonal antibody bound to an N-glycan terminated at α2,6-linked 3-fluorosialic acid glycoside.

[0012] The following description of the invention will set forth the details of the invention. Other features, objects, and advantages of the invention will become more apparent from the detailed description of the following specific examples and the appended claims. Attached Figure Description

[0013] The following description refers to the accompanying drawings, in which:

[0014] Figure 1 A shows two graphs depicting the enzymatic hydrolysis of three sialic acid glycosides (i.e., 1, 2, and S27) as a function of enzyme concentration. Figure 1 B shows two graphs depicting sialidase inhibition as a function of the concentration of 2,S27 or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) (a sialidase inhibitor);

[0015] Figure 2 These are four N-glycan staining electrophoresis gels: (1) rituxan-G, (2) rituxan-Fax -SCT, (3) rituxan-SCT, and (4) commercially available rituxan; and

[0016] Figure 3 Showing the effects on three N-glycans, namely rituxan-F ax Mass spectra obtained from -SCT, rituxan-SCT, and rituxan. Detailed Implementation

[0017] This article first details a method for preparing sugars containing 3-fluorosialic acid.

[0018] The method includes the following steps: reacting 3-hydroxysialic acid with a sugar to perform a glycosylation reaction, forming an α2,6-linked 3-hydroxysialic acid glycoside; and reacting the α2,6-linked 3-hydroxysialic acid glycoside with a fluorinating agent to perform a fluorination reaction, forming a sugar containing 3-fluorosialic acid. The sugar can be a monosaccharide, disaccharide, or oligosaccharide. In the exemplary method, the sugar is a monosaccharide.

[0019] In certain specific instances of this method, the 3-hydroxysialic acid is 2-bromo-3-hydroxysialic acid, and the glycosylation reaction is carried out in the presence of silver trifluoromethanesulfonate (AgOTf) and disodium hydrogen phosphate (Na2HPO4). Preferably, the glycosylation reaction is carried out in toluene.

[0020] In other specific examples of this method, the fluorinating agent is perfluoro-1-butanesulfonyl fluoride (NfF), and the fluorination reaction is carried out in the presence of a catalyst. For example, the catalyst is 1,8-diacylbicyclo[5,4,0]-undecyl-7-ene (DBU). Preferably, the fluorination reaction is carried out in toluene. Notably, the reaction can be further carried out in the presence of tri(dimethylamino)silicate (TASF).

[0021] In the exemplary method, 3-hydroxysialic acid has formula (I):

[0022]

[0023] Sugars are compounds of formula (II):

[0024]

[0025] α2,6-linked 3-hydroxysialic acid glycoside is a compound of formula (III):

[0026] as well as

[0027] Sugars containing 3-fluorosialic acid are compounds of formula (IV):

[0028]

[0029] In another specific example, the method further includes reacting a sugar containing 3-fluorosialic acid with a second sugar to carry out another glycosylation reaction.

[0030] Also within the scope of this invention is a method for preparing homogeneous antibodies that bind to sugars containing 3-fluorosialic acid. Similarly, this method includes glycosylation of the monoclonal antibody with a sugar containing 3-fluorosialic acid.

[0031] In certain specific instances of this method, the sugar containing 3-fluorosialic acid is obtained by the above-described method and its specific instances, which includes the following steps: reacting 3-hydroxysialic acid with the sugar to perform a glycosylation reaction to form an α2,6-linked 3-hydroxysialic acid glycoside, and reacting the α2,6-linked 3-hydroxysialic acid glycoside with a fluorinating agent to perform a fluorination reaction to form a sugar containing 3-fluorosialic acid.

[0032] In other specific examples, the 3-fluorosialic acid-containing sugar is an N-glycan terminated with an α2,6-linked 3-fluoro-sialic acid glycoside. It may have the structure of formula (V):

[0033]

[0034] Where Z is

[0035] Another aspect of the present invention relates to compounds of formula (VI):

[0036]

[0037] R 1 R 2 R 3 R 4 X is as defined in the above-mentioned invention description section.

[0038] In one specific instance, the compound has the structure of formula (IV):

[0039]

[0040] In another specific example, each of the compounds of formula (VI) comprises X as an N-glycan. The exemplary compound of this specific example has the structure of formula (V):

[0041]

[0042] Where Z is

[0043] The present invention also includes monoclonal antibodies that bind to a sugar containing 3-fluorosialic acid, wherein the sugar is an N-glycan terminated with a 3-fluorosialic acid glycoside in an α2,6 manner. Preferably, the N-glycan terminated with a 3-fluorosialic acid glycoside in an α2,6 manner has the formula (V) shown above.

[0044] The present invention further includes a method for treating cancer or autoimmune diseases, comprising administering an effective amount of the above-mentioned monoclonal antibody to an individual in need of such treatment. For example, the cancer could be leukemia or lymphoma, while the autoimmune disease could be rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura leukemia, lymphoma, Evans syndrome, vasculitis, bullous skin disorders, type 1 diabetes, Sjogren's syndrome, anti-NMDA receptor encephalitis, Devic's disease, Graves' ophthalmopathy, or autoimmune pancreatitis. Pancreatitis, opsoclonus myoclonus syndrome, or IgG4-related diseases.

[0045] The term "treatment" as used herein refers to administering the above-described pharmaceutical composition to an individual suffering from, exhibiting symptoms of, or being susceptible to, the aforementioned disease (i.e., cancer), with the aim of imparting a therapeutic or preventative effect. The term "effective amount" refers to the amount of active pharmaceutical ingredient required to impart this effect. As will be appreciated by those skilled in the art, the effective amount will vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of co-administration with other therapeutic treatments.

[0046] The following provides a more detailed description of certain specific aspects of the present invention.

[0047] Compared to unfluorinated sialic acid derivatives, 3-fluorosubstituted sialic acid derivatives are more stable to sialidase. 7 2,3-Difluorosialic acid (DFSA) can be developed by introducing a fluorine atom into the mutarotation isomer and the C-3 position (procedure 1).8 As a biochemical probe 9 and activity-based proteomic analysis probes to study sialidase 10 With the aid of DFSA, fluorine atoms are positioned along the C-3 axis (3F). ax ) and 3F eq Compared to derivatives, it slows down the deactivation of enzymes (k i ) and reactivation (k hydr The impact is greater. 8b Inspired by this observation, and considering the potential applications of 3-fluorosialic acid-containing sialic acid glycosides in glycoprotein therapy, we wanted to investigate their stability. Specifically, we intend to investigate the effects of incorporating a 3F-terminus into the N-glycan ends of mAbs. ax Can the Neu5Ac motif increase its stability against sialidase and maintain its effector function?

[0048] To achieve our goal, we developed a preparation method for synthesizing α2,6-linked 3F ax -Neu5Ac oligosaccharides, including biantennary N-glycans. We also demonstrated the synthesis of 3-F in the presence of sialidase. ax Neu5Ac-α2,6-Gal binding is more stable, and antibodies with the corresponding biantennary glycans have the same binding affinity as their non-fluorinated counterparts.

[0049] Several methods for synthesizing 3F-Neu5Ac have been reported, including: (a) protected polysaccharide and XeF2-BF3×OEt2 12 Molecular fluorine 13 and fluorine reagent (Selectfluor) 7c (a) Fluorination; (b) Reversal of the equatorial hydroxyl group at the C3 position in sialic acid derivatives 7f ; and (c) the aldolase-catalyzed conversion of ManNAc and 3-fluoro-pyruvate to 3F eq Neu5Ac and 3F ax Enzymatic conversion of Neu5Ac 7a,11a,14 However, to our knowledge, there is currently no description of the synthesis of 3F. ax A method using -Neu5Ac as the terminal N-glycan. The only disclosed method using 3F... ax The preparation of disaccharides from Neu5Ac is limited to the enzymatic synthesis of 3-FaxNeu5Ac-α2,3-Lac, rather than the desired α2,6-linking. 11a Therefore, we focused our efforts on the chemical synthesis of this bond. 15 .

[0050]

[0051] Process 1

[0052] Using 3F ax After screening various glycosylation conditions for the Neu5Ac-based donor (procedure 2a), we investigated alternative strategies, including the use of 3OH in 3OH-Neu5Ac-α2,6-Gal-STol. eq Become 3F ax S N 2. Reaction (Procedure 2b). First, we investigated sialylation at the O-6 position using the conditions reported by Goto et al. (Table 1, Table Values ​​1). 16 The reaction was carried out in toluene at -10°C using 1 equivalent of AgOTf as a promoter and Na2HPO4 as a base (procedure 3). These conditions yielded disaccharide 6 in low yield (15%) with a 3:1 (α:β) mutarotation isomer ratio. Changing the solvent to dichloromethane did not improve the non-mirror image selectivity (Table 2). Increasing the temperature and reaction time improved the yield, but significantly reduced the selectivity (Tables 3–5). Surprisingly, the higher the acceptor equivalent used, the better the stereoselectivity observed (Tables 3 vs. 6). The optimized conditions (Table 7) yielded 6 in 35% (99% brsm) yield with excellent α-selectivity (α:β = 13:1).

[0053]

[0054] Process 2

[0055]

[0056] Process 3

[0057] Table 1. Optimization of glycosylation steps.

[0058]

[0059]

[0060] a by 1 H NMR determination. b Yield of the isolated product. c Based on the yield of recovered starting materials (brsm), the receptor. d The donor and recipient were not completely consumed. e Use with MS's CH2Cl2. f have MS. g Gram weight

[0061] OH eq Reverse to F ax Verification proved to be a challenging task. Using TASF to replace OTf and OMs with fluorine resulted in the decomposition of the starting materials. To optimize this conversion, we screened various fluorinating agents, but none were successful. However, treatment with perfluoro-1-butanesulfonyl fluoride (NfF) in anhydrous toluene at 90 °C for 6 days (process 3) in the presence of 1,8-diacabicocyclo[5,4,0]-undecyl-7-ene (DBU) yielded 4 (Table 2, Table 1) in 6% yield (Process 3) for 2 days. 17 Further optimization of reaction conditions, such as decreasing the reaction temperature (Tables 1, 5, and 6) and increasing the reaction time (Table 7), improved the overall yield of 4. Due to the steric hindrance around C-3, the conversion of 7 to 4 appears to be the rate-determining step. In many cases, we were able to isolate 7 formed in the presence of NfF and DBU within 1 day at room temperature. However, the conversion of 7 to 4 (77% brsm yield) required a long reaction time. Attempts to improve the conversion by increasing the reaction temperature resulted in the decomposition of 7. Therefore, by increasing the reagent amount and reaction time to 15 days, we obtained the best results (Table 8). Finally, we found that the addition of TASF helped improve reaction efficiency, reducing the reaction time to only 2 days (Table 9). Fully protected 3F ax The stereochemistry of the -Neu5Ac-α2,6-Gal-STol disaccharide (4) was confirmed by X-ray diffraction analysis.

[0062]

[0063] Process 4

[0064] Table 2. Optimization of the fluorination process.

[0065]

[0066]

[0067] a Reagents and conditions: (a) NfF, DBU, toluene, 40°C, 15d, 49% (77% brsm). b Yield of the isolated product. c No product was observed. d Add NfF (4 equivalents / day) and DBU (4 equivalents / day) in portions, then stir for 14 days. e Add NfF (4 equivalents / day), DBU (4 equivalents / day) and tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) (2 equivalents / day) in portions.

[0068] Refer to process 5, and use NIS / TMSOTf to enable 3F. ax -Neu5Ac-disaccharide donor 4 was coupled with acceptor (8) to obtain 9. Next, the O-allyl group at the mutarotation isomer position was removed by PdCl2 isomerization in AcOH / NaOAc, and the mutarotation isomer hydroxyl group (10) was further converted to a fluoride (11) and an imide ester (12). Using Cp2HfCl2 / AgOTf conditions, with 3F ax The fluoride donor at the end of -Neu5Ac (11) glycosylated the core disaccharide (13) at the O-3 position to give the hexasaccharide 14 in 85% yield. After removing the benzylidene group, 15 was glycosylated at the O-6 position to give the desired decasaccharide (16) in 70% yield with excellent positional and α-stereoselectivity. We also tested TfOH-promoted glycosylation with the N-phenyltrifluoroacetylimide ester donor (12), but the product yield was low. Then, the fully deprotected polysaccharide (17) was obtained in 40% overall yield after the following series of steps: (a) saponification with LiOH to remove the ester and NHTroc groups; (b) acetylation of the free amine with the alcohol; (c) removal of the OAc group with sodium methoxide; and (d) hydrogenolysis of the O-benzyl group with Pd / C in a mixture of MeOH / water / HCO2H. 4b .

[0069]

[0070] Process 5

[0071] Reagents and conditions in procedure 5: (a) 8, TfOH, NIS, MS, CH2Cl2, -40℃, 2h, 64%. (b) PdCl2, CH3COONa, AcOH / H2O, 82%. (c) DAST, CH2Cl2, -20℃, 73%. (d) ClC(=NPh)CF3, Cs2CO3, CH2Cl2, 0℃ to rt, 3h, 56%. (e) 11, AgOTf, Cp2HfCl2, toluene. MS, 0℃, 3h, 85%; (f) pTSA·H2O, CH3CN, rt, 6h, 75%; (g) 11, AgOTf, Cp2HfCl2, toluene, MS, -15℃, 3h, 70% (80% brsm); (h) 12, TfOH, CH2Cl2, MS, -60 to -20 °C, 3 h, 33% (55% brsm); (i) LiOH, dioxane / H2O (4:1), 90 °C, 16 hrs; (j) Ac2O, Py, 16 h; (k) NaOMe, MeOH, 16 h; (l) Pd(OH)2, MeOH / H2O / HCOOH (6:3:1), H2, 16 h, 40% (4 steps).

[0072] After establishing a stepwise synthesis experimental scheme, we simplified the glycan assembly process (process 6) by developing a programmable [2+2+2] one-pot synthesis of hexasaccharides (14)(17 precursors). The designed one-pot experimental scheme includes using 3F ax The -Neu5Ac- disaccharide donor (4) (RRV = 2053) was initially coupled to the less reactive receptor (18) (RRV = 537) at -40 °C, followed by the injection of the reduced-terminal receptor 13 at -20 °C. After 1 hour at -10 °C and following a standard purification protocol, the hexasaccharide 14 was isolated in 26% yield.

[0073]

[0074] Process 6

[0075] Reagents and conditions in process 6: (a) TfOH, NIS, MS, CH2Cl2, -40℃ to -10℃, 3h, 26%

[0076] In order to collect information about 3F ax Preliminary stability data of the Neu5Ac-α2,6-Gal motif in the presence of sialidase were obtained. We prepared Neu5Ac-α2,6-Gal-pNP(1) and 3F... ax -Neu5Ac analogue (2) was used as a matrix (procedure 1) for in vitro assay with commercially available sialidases from Clostridium perfringens and Vibrio cholerae. 19 Both enzymes showed the expected hydrolytic activity against native matrix 1, but not against 3F. ax - Analog 2 is inactive (see...) Figure 1 A). We also observed that 2 did not inhibit the hydrolysis of 1 as significantly as DANA (see Figure 1 B).

[0077] To prepare homogeneous mAb glycoforms, compound 17 was converted into an oxazoline donor and ligated to a GlcNAc-initiated IgG (without core fucose) in the presence of Endo S2 (D184Q) according to standard experimental protocols. 6 Homogeneous 3F axThe binding affinity of the -Neu5Ac- glycoform to FcγRIIIa was analyzed using surface plasma resonance. 6 The samples were measured together with the parent non-fluorinated glycoform (G2S2) and commercially available rituximab samples (main glycoforms: G1F1, G0F1, G2F1). Compared to commercially available rituximab samples, the homogeneous IgG glycoform with α2,6-sialylated and core-fucose-free biantennary N-glycans showed a 39.9-fold (Neu5Ac-G2S2) and 37.4-fold (3F1) increase in binding affinity. ax -Neu5Ac-G2S2)(see Table 4). 3F was found. ax The fact that the affinity of the Neu5Ac-modified glycoform is in the same range as that of the parent glycan is due to the fact that 3F... ax In vivo studies of -Neu5Ac-glycosylated mAbs provide a basis for this research. These results will be reported in due course.

[0078] In summary, we have developed a 3F ax The chemical synthesis of -Neu5Ac-α2,6-Gal-STol can be used as a synthetic oligosaccharide for sialidase resistance and is representative of 3F ax -Neu5Ac is the building block of homogeneous antibodies with terminal biantennary N-glycans. When compared with commercially available rituximab samples, after 3F... ax -Neu5Ac-polysaccharide-modified homogeneous glycoforms showed a 37.4-fold increase in binding to FcγRIIIa. Furthermore, the parent non-fluorinated and 3F-modified... ax The -Neu5Ac-modified antibody glycoforms exhibited binding affinity similar to the Fc receptor. Overall, our results reveal a new and universal strategy for improving the half-life of therapeutic glycoproteins.

[0079] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the fullest extent based on the above description. Therefore, the following specific embodiments should be interpreted as merely illustrative and not as limiting the remainder of this disclosure in any way. All publications cited herein are incorporated herein by reference in their entirety.

[0080] Example 1: Synthesis of sugars and precursors containing 3-fluorosialic acid

[0081] Unless otherwise specified, all reactions were carried out under an inert atmosphere and following standard syringe-septum techniques. Solvents were commercially available and used without further purification. The pulverized molecular sieves... (EMD Millipore) was ground into powder and activated before use. The progress of all reactions was monitored via TLC glass plates pre-coated with silica gel 60F254 (Merck KGaA). TLC imaging was performed using UV light (254 nm) and staining with anisaldehyde and / or cerium ammonium molybdate. Across silica gel (particle size 0.035–0.070 mm) was used. ) was used for column chromatography. 1 H, 13 C and 19 F NMR spectra were recorded at 25°C using Bruker AVIII-600, DRX-500, AV-400, DPX-400, AVANCE 500AV, and AVANCE 600 spectrometers. Chemical shifts were measured in δ (ppm) using the residual solvent peak as an internal standard (δ, ppm: in 1 The ¹H NMR values ​​are 7.24 (CHCl₃) and 4.80 (H₂O); 13 The value in C NMR is 77 (CDCl3). Coupling constants (J) are measured in Hz. Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad peak). High-resolution mass spectrometry (HRMS) was performed on an Agilent LC / MSD TOF mass spectrometer using electrospray ionization time-of-flight (ESI-TOF) reflectance experiments. Single-crystal X-ray diffraction studies were conducted on a Cu K... α The radiation (=1.5478) of Bruker D8 Platinum- 135 The measurements were performed using a CCD diffractometer. HPLC measurements were performed on a Hitachi HPLC D-7000 system. RRV measurements were recorded using a normal-phase ZORBAX RX-SIL, 5 μm, 4.6 x 250 mm (Colloidal Silica, Agilent Technologies) solvent system of EtOAc / hexane at a flow rate of 1 mL / min and imaged at 254 nm.

[0082]

[0083] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-D-erythrose-α-L-glucose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-4-O-benzyl-1-thio-β-D-galactopyranoside 6α. A mixture of acceptor 3 (1.05 g, 1.80 mmol, 1 equivalence) and donor 5 (1.02 mg, 1.80 mmol, 1 equivalence) in anhydrous toluene (60 mL) was stirred for 10 min under an argon atmosphere. The reaction mixture was then cooled to -50 °C, and anhydrous Na2HPO4 (1.07 g, 7.54 mmol, 4.2 equivalence) was added with stirring, followed by AgOTf (692 mg, 2.69 mmol, 1.5 equivalence, in 18 mL of toluene). After the reaction was complete (TLC showed the starter disappeared after 24 hours), the reaction mixture was diluted with EtOAc (80 mL), washed with 20% Na₂S₂O₃ aqueous solution (10 mL), saturated NaHCO₃ aqueous solution (5 mL), and brine (5 mL), and then the organic layer was dehydrated with MgSO₄, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (3:7) as the precipitate to give compound 6 (α:β = 13:1) as a white powder (680 mg, 35%), along with the recovered acceptor 3 (670 mg, 99% brsm). α-Mutarotation isomer 6α:R f =0.36 (silica gel, CHCl3:MeOH = 20:1); 1 H NMR (600MHz, CDCl3): δ7.96-7.94(m,2H,Ar-H),7.91-7.90(m,2H,Ar-H),7.50-7.47(m,2H,Ar-H ),7.40-7.39(m,2H,Ar-H),7.37-7.32(m,5H,Ar-H),7.25-7.18(m,4H,Ar-H),7.06-7.04(m,2H, Ar-H),5.81(dd,J=10.2.9.6Hz,1H),5.59(d,J=10.2Hz,1H),5.38(dd,J=9.0,3.0Hz,1H),5.35- 5.32(m,1H),5.25(dd,J=10.2,9.6Hz,1H),5.24(d,J=7.8,2.4Hz,1H),4.92(d,J=9.6Hz,1H,C1-H β),4.71(d,J=12.0Hz,1H),4.60(d,J=12.0Hz,1H),4.49(dd,J=10.8,1.8Hz,1H),4.30(dd,J=12.6,3.0Hz,1H) ,4.26(ddd,J=10.2,10.2,10.2Hz,1H),4.20(d,J=3.0Hz,1H),4.10(dd,J=10.2,7.2Hz,1H),4.04(dd,J=12.6 ,6.6Hz,1H),4.00(dd,J=6.6,6.6Hz,1H),3.86(d,J=9.6Hz,1H),3.82-3.73(m,2H),3.75(s,3H,-CH3),2.30( s,3H,-CH3),2.09(s,3H,-CH3),2.07(s,3H,-CH3),2.06(s,3H,-CH3),1.96(s,3H,-CH3),1.89(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.6,170.6,170.3,170.1,169.5,168.5,165.8,165.1, 138.0,137.9,133.4,133.1,129.8,129.7,129.6,128.9,128.6,128.4,128.3,12 8.2,127.7,127.6,100.3,86.6,75.9,74.5,73.7,73.2,73.1,72.5,68.8,68.3, 63.1,62.5,52.8,48.4,23.1,21.2,20.9,20.8,20.7,20.7; HRMS(ESI-TOF)m / e:C 54 H 59 NO 20 SNa[M+Na] + Calculated value: 1096.3243, measured value: 1096.3241.

[0084]

[0085] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-D-erythrose-β-L-glucose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-4-O-benzyl-1-thio-β-D-galactopyranoside 6β.β-mutantisomer 6βR f =0.42 (silica gel, CHCl3:MeOH = 20:1); 1H NMR (600MHz, CDCl3): δ7.94-7.92(m,4H,Ar-H),7.50-7.47(m,2H,Ar-H),7.43-7 .40(m,6H,Ar-H),7.37-7.31(m,5H,Ar-H),7.13-7.12(m,2H,Ar-H),5.81(dd,J=1 0.2.10.2Hz,1H),5.51(dd,J=10.2,3.0Hz,1H),5.22(ddd,J=6.0,6.0,3.0Hz,1H) ,5.14(d,J=13.2Hz,1H),5.07(dd,J=6.0,2.4Hz,1H),4.90(d,J=10.2Hz,1H,C1-H β ),4.66-4.62(m,2H),4.55(dd,J=12.6,2.4Hz,1H),4.29(d,J=1.8Hz,1H),4.14(dd,J=7.8,7. 2Hz,1H),4.08(ddd,J=10.8,10.8,10.8Hz,1H),3.98(dd,J=12.6,6.6Hz,1H),3.92-3.88(m,2 H),3.85-3.77(m,3H),3.83(s,3H,-CH3),3.53(dd,J=10.8,2.4Hz,1H),2.32(s,3H,-CH3),2. 26(s,3H,-CH3),2.07(s,3H,-CH3),2.04(s,3H,-CH3),1.97(s,3H,-CH3),1.68(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.4,170.6,170.3,169.8,169.8,166.6,165.8,165.2, 139.2,138.4,133.5,133.4,133.1,129.9,129.8,129.5,128.9,128.5,128.3,1 27.2,126.0,99.6,86.9,76.0,75.5,74.3,73.1,72.5,71.7,70.3,68.4,67.2,6 2.1,62.0,53.3,47.5,22.9,21.5,21.2,20.8,20.7,20.7; HRMS(ESI-TOF)m / e:C 54 H 59 NO 20 S[M+H] + Calculated value: 1074.3424, measured value: 1074.3458.

[0086]

[0087] 4. To a solution of compound 6α (500 mg, 0.47 mmol, 1 equivalent) in toluene (5 mL) in a screw-capped vial with a stir bar, add DBU (0.28 mL, 1.86 mmol, 4 equivalents), perfluoro-1-butanesulfonyl fluoride (0.33 mL, 1.86 mmol, 4 equivalents), and TASF (256 mg, 0.93 mmol, 2 equivalents). Seal the vial and stir at 40 °C. After 24 hours, the reaction mixture was treated with additional amounts of DBU (0.28 mL, 1.86 mmol, 4 equivalents), perfluoro-1-butanesulfonyl fluoride (0.33 mL, 1.86 mmol, 4 equivalents), and TASF (256 mg, 0.93 mmol, 2 equivalents), and then stirred at 40 °C for another 24 hours. The reaction mixture was directly loaded onto a silica gel column and dissolved in acetone / toluene (7:3). Disaccharide 4 (300 mg, 60%) was obtained as a pale yellow powder, accompanied by perfluoro-1-butanesulfonyl compound 7 (50 mg, 8%) as a pale yellow powder.

[0088] Synthesis of disaccharide 4 from compound 7: DBU (0.64 mL, 4.31 mmol, 8 equivalents) and perfluoro-1-butanesulfonyl fluoride (0.77 mL, 4.31 mmol, 8 equivalents) were added to a solution of compound 7 (730 mg, 0.54 mmol, 1 equivalent) in toluene (7 mL) placed in a screw-capped vial with a stir bar. The container was sealed and stirred at 40 °C for 15 days. The reaction mixture was loaded directly onto a silica gel column and dissolved in acetone / toluene (7:3) as the precipitate; disaccharide 4 (282 mg, 49%) was obtained as a pale yellow powder, accompanied by perfluoro-1-butanesulfonyl compound 7 (267 mg, 77% BRSM) as a pale yellow powder. f =0.43 (silicone, acetone / toluene = 2:3); 11H NMR (600MHz,CDCl3): δ7.95-7.93(m,2H,Ar-H), 7.87-7.85(m,2H,Ar-H), 7.48-7.44(m,2H,Ar-H), 7.41-7.39(m,2H,Ar-H), 7.35-7.33(m,2H,Ar-H), 7.31-7.29(m,2H,Ar-H), 7.25-7.24(m,2H,Ar-H), 7.21-7.19(m,2H,Ar-H), 7.16-7.14(m,1H,Ar-H) 7.04-7.03(m,2H,Ar-H),5.79(dd,J=10.2.9.6Hz,1H),5.49(ddd,J=9.0,5.4,2.4Hz,1H),5.42(dd,J=10.2,3.0Hz,1H),5.31 -5.28(m,2H),5.20(dd,J=27.0,11.4Hz,1H,sia-C4-H),5.01(dd,J=51.6,1.8Hz,1H,sia-C3-H),4.97(d,J=10.2Hz,1H,C1-H β ),4.66(d,J=11.4Hz,1H),4.58(d,J=11.4Hz,1H),4.37(dd,J=12.6,2.4Hz,1H),4.28 -4.26(m,2H),4.17(dd,J=12.6,5.4Hz,1H),4.09-4.05(m,2H),3.98(dd,J=10.2,6.0H z,1H),3.74(dd,J=10.2,8.4Hz,1H),3.72(s,3H,-CH3),2.29(s,3H,-CH3),2.17(s,3 H,-CH3),2.16(s,3H,-CH3),2.09(s,3H,-CH3),1.98(s,3H,-CH3),1.91(s,3H,-CH3); 13C NMR (150MHz, CDCl3): δ170.9,170.7,170.4,170.2,169.8,165.6,165.3,138.3,137 .6,133.2,133.0,132.6,129.8,129.8,129.7,129.5,129.3,129.1,128.4,128.3,12 8.1,127.5,127.3,98.3,98.2,88.1,86.8,86.4,76.2,75.6,74.6,74.0,71.4,69.2,69.0,68.5,68.0,67.3,63.4,62.5,53.2,45.5,23.4,21.2,21.2,20.8,20.7,20.7; 19 F NMR (376MHz, CDCl3): δ-215.9; HRMS (ESI-TOF) m / e: C 54 H 58 FNO 19 SNa[M+Na] + Calculated value: 1098.3200, measured value: 1098.3212.

[0089]

[0090] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-3-O-(perfluoro-1-butane)sulfonyl-D-erythrose-α-L-glucose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-4-O-benzyl-1-thio-β-D-galactopyranoside 7.R f =0.59 (silicone, acetone: toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ7.95-7.94(m,2H,Ar-H),7.90-7.89(m,2H,Ar-H),7.49- 7.45(m,2H,Ar-H),7.40-7.39(m,2H,Ar-H),7.36-7.30(m,4H,Ar-H),7.27-7.26 (m,2H,Ar-H),7.25-7.18(m,3H,Ar-H).7.04-7.03(m,2H,Ar-H),5.82(dd,J=10 .2.9.6Hz,1H),5.62-5.38(m,4H),5.26(d,J=9.0Hz,1H),4.96-4.94(m,2H,C1-H β),4.70-4.64(m,3H),4.35-4.30(m,1H),4.22-4.20(m,2H),4.09(dd,J=9.6,6 .0Hz,1H),4.02(dd,J=12.6,6.0Hz,1H),3.98(dd,J=6.6,6.0Hz,1H),3.93(dd ,J=10.2,7.2Hz,1H),3.76(s,3H,-CH3),2.29(s,3H,-CH3),2.17(s,3H,-CH3) ,2.09(s,3H,-CH3),2.06(s,3H,-CH3),1.94(s,3H,-CH3),1.90(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.6,170.2,170.1,169.3,167.0,165.7,165.2,138.1, 137.7,133.2,133.0,132.5,129.8,129.7,129.7,129.5,129.1,129.0,128.4,1 28.3,128.1,128.0,127.5,127.4,97.8,86.5,83.4,75.7,74.6,74.2,72.6,70.0,68.4,67.9,66.5,64.5,62.4,53.1,49.0,23.0,21.1,20.9,20.7,20.6,20.3; 19 F NMR(376MHz, CDCl3): δ-126.2,-126.2,-121.5,-121.5,-110.4,-110.4,-81.0,-80.9,-80.9; HRMS(ESI-TOF)m / e:C 58 H 58 F9NO 22 S2Na[M+Na] + Calculated value: 1378.2640, measured value: 1378.2644.

[0091]

[0092] Allyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranoside methyl ester]-(2→6)-[2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside 9. The acceptor 8 (1.04 g, 1.03 mmol, 1 equivalent) and donor 4 (1.39 g, 1.29 mmol, 1.25 equivalent) were reacted with activated pulverized... The mixture of MS (0.70 g) in anhydrous CH₂Cl₂ (7 mL) was stirred under argon for 1 hour. The reaction mixture was then cooled to -40 °C, and NIS (465 mg, 2.06 mmol, 2 equivalences) was added with stirring, followed by TfOH (0.5 M in Et₂O, 0.62 mL, 0.31 mmol, 0.3 equivalences). After the reaction was complete (TLC showed the starter disappearing after ~2 hours), the reaction mixture was quenched with Et₃N (0.4 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH₂Cl₂ (30 mL) and washed with 20% Na₂S₂O₃ aqueous solution (10 mL), saturated NaHCO₃ aqueous solution (15 mL), and brine (8 mL). The separated organic layer was dehydrated with MgSO₄ and concentrated. The residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the precipitate to give compound 9 as a white powder (1.30 g, 64%). f =0.51 (silicone, acetone: toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ7.90-7.88(m,4H,Ar-H),7.48-7.45(m,2H,Ar-H),7.34-7.22(m,24H,Ar-H),7.18-7.13(m,10H,Ar-H),5 .85-5.78(m,2H),5.47-5.44(m,1H),5.35-5.32(m,4H),5.24-5.16(m,2H),5.11(dd,J=10.2,1.2Hz,1H),5.06-4.97(m,3H,C1-H β ), 4.85-4.73(m,4H,Cl-H) α C1-H β),4.70-4.54(m,6H),4.53-4.42(m,4H),4.33-4.26(m,2H),4.23-4.18( m,3H),4.14-4.04(m,3H),3.97-3.93(m,1H),3.89-3.81(m,5H),3.73-3. 58(m,10H),3.45-3.43(m,1H),3.32-3.30(m,1H),2.16(s,3H,-CH3),2.1 3(s,3H,-CH3),2.10(s,3H,-CH3),2.01(s,3H,-CH3),1.92(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.7,170.6,170.4,170.2,169.8,165.6,165.4, 165.4,165.1,153.8,138.8,138.6,138.5,138.2,138.2,138.1,133.7,13 3.2,133.1,133.1,130.8,130.0,129.8,129.6,129.4,129.0,128.4,128. 3,128.3,128.2,128.2,128.2,128.1,128.1,128.0,128.0,127.9,127.6, 127.6,127.5,127.4,127.3,127.2,125.2,117.1,100.0,98.3,98.2,96.9,95.6,88.1,86.8,77.9,75.0,74.7,74.6,74.5,74.2,74.1,73.9,73.4,7 3.3,73.1,73.0,72.4,71.9,71.4,70.9,70.9,69.5,69.2,69.1,69.0,68.0,67.9,67.2,62.9,62.3,57.2,53.2,45.5,23.3,21.0,20.7,20.7,20.6; 19 F NMR (376MHz, CDCl3): δ-215.6; HRMS (ESI-TOF) m / e: C 100 H 108 Cl3FN2O 31 Na[M+Na] + Calculated value: 1979.5878, measured value: 1979.5889.

[0093]

[0094] [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranose methyl ester]-(2→6)-[2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside] [1→2]-3,4,6-tri-O-benzyl-α-D-mannopyranoside 10. At room temperature, CH3COONa (121 mg, 1.48 mmol, 5 equivalents) was added to a stirred solution of tetrasaccharide 9 (578 mg, 0.30 mmol, 1 equivalent) in acetic acid (6.0 mL, acetic acid / water, 10:1 = v / v), followed by the addition of PdCl2 (105 mg, 0.59 mmol, 2 equivalents). After 20 hours, when TLC showed the disappearance of the starting material, the reaction mixture was diluted with ethyl acetate (20 mL) and poured into a saturated aqueous solution of NaHCO3 (20 mL). The aqueous layer was extracted with ethyl acetate (2 × 10 mL), and the combined organic phases were then dehydrated and concentrated with MgSO4. The residue was purified by silica gel column chromatography using acetone / hexane (2:3) as the precipitate to give compound 10 as a white powder (465 mg, 82%). f =0.23 (silicone, acetone:hexane = 2:3); 1 H NMR (600MHz, CDCl3): δ7.90-7.87(m,4H,Ar-H),7.48-7.45(m,2H,Ar-H),7.34-7.20(m,24H,Ar-H),7.19- 7.12(m,10H,Ar-H),5.80-5.75(m,1H),5.45-5.43(m,1H),5.33-5.28(m,3H),5.21-4.89(m,5H,man-C1-H α ,man-C1-H β C1-H β ), 4.84-4.30(m, 15H, Cl-H β ),4.27-3.79(m,12H),3.78-3.43(m,12H),2.13-2.12(m,6H,-2CH3),2.09-2.08(m,3H,-CH3),2.02-2.00(m,3H,-CH3),1.91(br,3H,-CH3); 13C NMR (150MHz, CDCl3): δ170.8,170.7,170.7,170.7,170.6,170.5,170.4,170.3,170.3,170 .2,169.9,169.8,165.6,165.6,165.4,165.2,165.2,154.0,138.6,138.5,138.3,138.2,1 38.1,133.2,133.1,129.8,129.6,129.5,129.0,128.4,128.4,128.3,128.3,128.2,128.2,128.2,128.1,128.0,127.9,127.9,127.7,127.7,127.6,127.5,127.5,127.4,127.4,127 .3,100.0,99.5,98.3,98.2,98.1,95.8,95.7,92.3,88.2,88.1,86.8,76.1,74.8,74.7,74.6,74.6,74.3,74.2,74.2,73.9,73.8,73.3,73.2,73.1,73.1,72.7,72.6,72.4,72.3,71. 6,71.4,71.2,70.9,70.8,69.9,69.5,69.3,69.2,69.1,69.0,68.1,68.0,67.2,67.1,63.0,62.3,62.2,62.1,53.3,53.3,45.5,45.4,26.3,23.3,21.1,20.8,20.7,20.7,20.6,20.6; 19 F NMR(376MHz, CDCl3): δ-215.2,-215.7; HRMS(ESI-TOF)m / e:C 97 H 104 Cl3FN2O 31 Na[M+Na] + Calculated value: 1939.5565, measured value: 1939.5619.

[0095]

[0096] [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranoside methyl ester]-(2→6)-[2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloro] [Ethoxy)carbamate-β-D-glucopyranoside]-(1→2)-3,4,6-tris-O-benzyl-α-D-mannopyranoside fluoride 11. At -20 °C, DAST (81.6 μL, 0.62 mmol, 3 equivalents) was added to a well-stirred solution of hemiacetal 10 (395 mg, 0.21 mmol, 1 equivalent) in anhydrous CH2Cl2 (12 mL). The reaction mixture was stirred vigorously until TLC showed the disappearance of the starting material (3 h). The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with saturated NaHCO3 aqueous solution (5 mL) and brine (4 mL). The organic phase was dehydrated by MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / hexane (3:4) as the precipitate to give compound 11 as a white powder (289 mg, 73%). f =0.32 (silicone, acetone:hexane = 3:4); 1 HNMR (600MHz, CDCl3): δ7.89-7.88(m,4H,Ar-H),7.47-7.45(m,2H,Ar-H),7.33-7.22(m,22H,Ar-H) ,7.17-7.12(m,12H,Ar-H),5.78(dd,J=10.2,7.8Hz,1H),5.53-5.40(m,2H),5.33-5.29(m,3H,C1-H α ),5.21(dd,J=27.6,11.4Hz,1H,sia-C4-H),5.05-4.96(m,2H,C1-H β ),4.94(d,J=11.4Hz,1H),4.82-4.79(m,4H,C1-H β),4.75-4.72(m,1H),4.68-4.59(m,3H),4.58-4.54(m,2H),4.50-4.48(m,2H),4.30(dd,J=12 .6,3.0Hz,1H),4.26(d,J=10.2Hz,1H),4.23-4.15(m,4H),4.08-4.04(m,2H),3.95(dd,J=9.0 ,8.4Hz,1H),3.89-3.78(m,5H),3.72-3.57(m,9H),3.50-3.48(m,1H),3.35(d,J=0.6Hz,1H)2 .14(s,3H,-CH3),2.12(s,3H,-CH3),2.09(s,3H,-CH3),2.00(s,3H,-CH3),1.91(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.8,170.7,170.4,170.2,169.9,165.6,165.4,165.2,153.9,138.2,138.1,137.8,133. 2,133.1,129.8,129.6,129.4,129.0,128.4,128.3,128.2,128.1,128.0,127.9,127.7,127.6,127.6,127.4,127 .3,127.3,106.9,105.4,99.8,99.2,98.3,98.2,95.6,88.1,86.8,74.9,74.8,74.2,74.0,73.9,73.8,73.3,73.2 ,72.5,71.4,71.3,70.9,69.1,69.1,68.9,68.0,67.3,62.8,62.3,56.9,53.3,45.6,23.4,21.1,20.8(2C),20.6; 19 F NMR(376MHz, CDCl3): δ-138.6,-215.7; HRMS(ESI-TOF)m / e:C 97 H 103 Cl3F2N2O 30 Na[M+Na] + Calculated value: 1941.5521, measured value: 1941.5521.

[0097]

[0098] (N-Phenyl)-2,2,2-trifluoroacetylimide ester [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketosepyranose methyl ester]-(2→6)-[2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside] [Glycoside]-(1→2)-3,4,6-tri-O-benzyl-D-mannopyranoside 12. A solution of hemiacetal 10 (395 mg, 0.21 mmol, 1 equivalent) in anhydrous CH2Cl2 (12 mL) was stirred, and Cs2CO3 (83.2 mg, 0.26 mmol, 2 equivalent) and 2,2,2-trifluoro-N-phenyl-iminoacetyl chloride (41 μL, 0.26 mmol, 2 equivalent) were added at 0 °C with stirring. The mixture was then heated to room temperature and stirred for 3 hours. The reaction mixture was diluted with CH2Cl2 (10 mL), filtered through a diatomaceous earth mat, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / hexane (1:2) as the precipitate to give compound 12 as a white foam (150 mg, 56%); a mixture of mutarotatory isomers (α:β = 1:1). R f =0.55 (silicone, acetone:hexane = 1:1); 1 H NMR (600MHz, CDCl3): δ7.93-7.88(m,4H,Ar-H),7.49-7.45(m,2H,Ar-H),7.40-7.12(m,38H,Ar-H),6.75-6.71(m,1H ,Ar-H),5.82-5.78(m,1H),5.46-5.42(m,1H),5.36-5.30(m,3H),5.24-5.16(m,1H),5.08-4.81(m,5H,0.5man-C1-H α ,2C1-H β ),4.78-4.74(m,1H),4.69-4.41(m,9H,0.5man-C1-H β ),4.33-4.06(m,7H),3.95-3.82(m,5H),3.69-3.36(m,10H),2.16-2.10(m,9H,-3CH3),2.01-1.99(m,3H,-CH3),1.92(s,3H,-CH3); 13C NMR (150MHz, CDCl3): δ170.7,170.7,170.7,170.7,170.4,170.2,169.9,169.9,165.6,165.6 ,165.4,165.2,165.1,154.1,153.9,143.3,143.1,138.7,138.5,138.3,138.2,138.2,138.1 ,138.0,138.0,137.8,137.7,133.2,133.1,133.1,129.8,129.6,129.4,129.3,129.0,128.7,128.4,128.4,128.3,128.3,128.3,128.2,128.2,128.1,128.1,128.1,128.0,127.9,127.7 ,127.7,127.6,127.5,127.5,127.5,127.4,127.3,119.4,119.2,102.3,100.2,100.0,98.3,98.2,95.6,95.5,88.0,86.7,80.0,79.3,75.9,75.0,74.7,74.7,74.5,74.3,74.2,73.8,73. 8,73.5,73.3,73.2,73.1,72.8,72.4,72.3,71.5,71.4,71.1,71.0,70.9,70.1,69.8,69.3,69.1,68.2,67.2,67.2,62.8,62.3,62.2,53.3,53.2,45.5,23.3,21.0,21.0,20.7,20.7,20.6; 19 F NMR(376MHz, CDCl3): δ-65.2,-65.4,-65.5,-65.6,-65.6,-65.7,-215.6,-215.7; HRMS(ESI-TOF)m / e:C 105 H 108 Cl3F4N3O 31 Na[M+Na] + Calculated value: 2110.5860, measured value: 2110.5865.

[0099]

[0100] Benzyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranoside methyl ester]-(2→6)-[2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-[3,4,6-tri-O-benzyl] [2-O-acetyl-4,6-O-benzylidene-β-D-mannopyranoside]-(1→3)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside 14. AgOTf (194 mg, 0.75 mmol, 6 equivalents relative to the receptor) and bis(Cp2HfCl2) (167 mg, 0.44 mmol, 3.5 equivalents relative to the receptor) were mixed with freshly activated... The mixture of MS (1.50 g) in anhydrous toluene (15 mL) was stirred at room temperature under argon for 1 h. The reaction mixture was then cooled to -20 °C and treated with a solution of donor 11 (290 mg, 0.15 mmol, 1.2 equivalence) and acceptor 13 (115 mg, 0.13 mmol, 1 equivalence) in anhydrous toluene (8 mL). The reaction mixture was stirred at 0 °C until TLC showed the disappearance of the initiator (3 h). After the reaction was complete, the reaction mixture was quenched with Et3N (0.25 mL), diluted with EtOAc (25 mL), and filtered through a diatomaceous earth mat. The filtrate was washed twice with saturated NaHCO3 aqueous solution (10 mL) and brine (6 mL), the organic phase was dehydrated with MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the precipitate to give compound 14 as a white powder (299 mg, 85%).

[0101] One-pot synthesis: Acceptor 18 (40.0 mg, 0.037 mmol, 1 equivalent), donor 4 (60.1 mg, 0.056 mmol, 1.5 equivalent), and activated pulverized... The mixture of MS (0.25 g) in anhydrous CH2Cl2 (1.25 mL) was stirred under argon for 1 hour. Then, the reaction mixture was cooled to -40 °C and NIS (12.6 mg, 0.056 mmol, 1.5 equivalence) was added, followed by TfOH (0.5 M in Et2O, 22.3 μL, 0.011 mmol, 0.3 equivalence), and the mixture was stirred at -20 °C for 2 hours. Next, after the initiator disappeared (monitored by TLC), the reaction mixture was sequentially treated with acceptor 13 (34.2 mg, 0.037 mmol, 1 equivalence) in anhydrous CH2Cl2 (1.25 mL), NIS (16.8 mg, 0.075 mmol, 2 equivalences), and TfOH (0.5 M in Et2O, 22.3 μL, 0.011 mmol, 0.3 equivalences) with stirring under argon for 10 min. After adding all reagents, the mixture was heated to -10 °C and stirred continuously until TLC showed the initiator had disappeared (1 h). After completion, the reaction mixture was quenched with Et3N (0.4 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH2Cl2 (10 mL) and washed with 20% Na2S2O3 aqueous solution (3 mL), saturated NaHCO3 aqueous solution (2 mL), and brine (2 mL). The separated organic layer was dehydrated with MgSO4, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the solvent, followed by a second silica gel column chromatography using acetone / hexane (3:4) as the solvent, to give compound 14 as a white powder (27 mg, 26%). f =0.43 (silicone, acetone: toluene = 3:5); 1 H NMR (600MHz, CDCl3): δ7.96-7.95(m,2H,Ar-H),7.91-7.90(m,2H,Ar-H),7.51-7.47(m,2H,Ar-H),7.36-7.26(m,35H,Ar-H),7.23- 7.10(m,19H,Ar-H),5.84(dd,J=10.2,7.8Hz,1H),5.45-5.42(m,1H),5.34-5.27(m,4H),5.21-5.15(m,2H),5.04-4.94(m,4H,C1-H α C1-H β ),4.87-4.78(m,4H),4.71-4.50(m,14H,2C1-H β ),4.46-4.34(m,5H),4.31-4.21(m,4H),4.16(dd,J=12.6,5.4Hz,1H),4.13-4.06(m,2H),3.98-3.95(m,4H,C1-Hβ ),3.84-3.57(m,17H),3.51-3.41(m,4H),3.36-3.35(m,2H),3.15(br,1H),2.93(br,1H),2.68-2.67(m,1H),2. 12(s,3H,-CH3),2.11(s,3H,-CH3),2.09(s,3H,-CH3),1.99(s,3H,-CH3),1.91(s,3H,-CH3),1.85(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.8,170.7,170.4,170.2,169.7,169.4,165.6,165.4 ,165.2,153.7,153.6,138.9,138.7,138.4,138.3,137.9,137.1,133.2,129.8, 129.7,129.5,129.1,128.9,128.6,128.5,128.4,128.4,128.3,128.3,128.2,128.1,128.1,128.0,128.0,127.9,127.9,127.9,127.8,127.8,127.6,127.6,12 7.6,127.5,127.4,127.4,127.3,127.2,126.3,102.1,100.1,99.0,98.7,98.6,95.8,95.5,88.2,86.9,78.8,78.4,74.8,74.5,74.3,74.2,73.9,73.8,73.3,7 2.9,72.3,71.9,71.4,71.1,70.7,70.3,69.2,69.1,69.0,68.4,68.3,67.9,67.2,66.2,63.2,62.3,57.4,56.8,53.3,45.5,23.3,21.0,20.8,20.7,20.6,20.6; 19 F NMR (376MHz, CDCl3): δ-215.6; HRMS (ESI-TOF) m / e: C 142 H 150 Cl6FN3O 43 Na₂[M+2Na] 2+ Calculated value: 1429.8771, measured value: 1429.8864.

[0102]

[0103] Benzyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranose methyl ester]-(2→6)-[2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-[3,4,6-tri- [-O-benzyl-α-D-mannopyranoside]-(1→3)-[2-O-acetyl-β-D-mannopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside 15. Add p-TsOH·H2O (21.5 mg, 0.11 mmol, 1 equivalent) to a solution of stirred starting material 14 (318 mg, 0.11 mmol, 1 equivalent) in acetonitrile (12 mL). Stir the reaction mixture vigorously until TLC shows the disappearance of the starting material (6 h). After completion, quench the reaction mixture with Et3N (0.3 mL) and concentrate under high vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (3:5) as the precipitate to give compound 15 as a white powder (231 mg, 75%). f =0.25 (silicone, acetone: toluene = 1:2); 1 H NMR (600MHz, CDCl3): δ7.88-7.87(m,4H,Ar-H),7.46(dd,J=7.2,7.2Hz,1H,Ar-H),7.43(dd,J=7.2,7.2Hz,1H,Ar-H),7.33-7.13(m,47H,Ar-H),7.09 -7.08(m,2H,Ar-H),6.01(br,1H),5.76(dd,J=10.2,7.8Hz,1H),5.46-5.4 3(m,1H),5.38(d,J=8.4Hz,1H),5.31-5.29(m,3H),5.19-5.13(m,2H,C1-H α ), 5.05-4.96(m,3H,Cl-H) β ), 4.89-4.79(m,4H,Cl-H) β ), 4.69-4.60(m,8H,Cl-H) β ), 4.57-4.49(m,8H,Cl-H) β),4.45(d,J=12.0Hz,1H),4.40-4.36(m,2H),4.31(dd,J=12.6,2.4Hz,1H),4.24-4.16(m,4H ),4.12-4.09(m,2H),4.02(br,1H),3.98-3.93(m,2H),3.91-3.86(m,2H),3.80(dd,J=7.8,6 .0Hz,1H),3.76-3.69(m,6H),3.66-3.37(m,18H),2.96-2.93(m,1H),2.13(s,3H,-CH3),2.1 1(s,3H,-CH3),2.09(s,3H,-CH3),1.98(s,3H,-CH3),1.93(s,3H,-CH3),1.90(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.8,170.7,170.4,170.3,170.2,169.8,165.6,165.5,165.3,154.3,153.8,138.6,138.4,138.3,138.1,138.0 ,137.8,137.2,133.3,133.1,129.8,129.6,129.4,129.0,128.5,128.4,128.3,128.2,128.2,127.9,127.9,127.8,127.7,127.6,127.5, 127.5,127.4,127.4,127.3,100.1,99.1,98.3,98.2,98.1,95.7,95.5,88.2,86.9,78.6,78.2,75.4,75.1,74.8,74.7,74.4,74.2,74.1 ,73.8,73.3,73.2,72.6,71.9,71.8,71.5,71.4,70.9,70.6,69.5,62.3,57.2,56.8,53.8,53.3,45.4,23.3,21.1,21.0,20.7(2C),20.7; 19 F NMR (376MHz, CDCl3): δ-215.6; HRMS (ESI-TOF) m / e: C 135 H 146 Cl6FN3O 43 Na[M+Na] + Calculated value: 2748.7337, measured value: 2748.7373.

[0104]

[0105] Benzyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose methyl pyranoside-(2→6)-2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside-(1→3)]-[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D- Erythrose-α-L-mannose-non-2-ketose methyl pyranoside-(2→6)-2,3-di-O-benzyl-4-O-benzyl-1-β-D-galactopyranoside-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside-(1→6)]-[2-O-acetyl-β-D-mannopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside 16.

[0106] From fluoride donors: AgOTf (142 mg, 0.55 mmol, 8 equivalents relative to receptor) and Cp2HfCl2 (105 mg, 0.28 mmol, 4 equivalents relative to receptor) were compared with freshly activated... The mixture of MS (1 g) in anhydrous toluene (10 mL) was stirred at room temperature for 1 hour under an argon atmosphere. The reaction mixture was then cooled to -40 °C, and a solution of donor 11 (232 mg, 0.12 mmol, 1.75 equivalence) and acceptor 15 (188 mg, 0.069 mmol, 1 equivalence) in anhydrous toluene (1 mL) was added. Stirring continued at -15 °C for 3 hours. The reaction mixture was quenched with Et3N (0.20 mL), diluted with EtOAc (20 mL), and filtered through a diatomaceous earth mat. The filtrate was washed twice with a saturated aqueous solution of NaHCO3 (8 mL) and brine (4 mL). The organic phase was dehydrated with MgSO4, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (2:3) as the precipitate to give compound 16 as a white powder (223 mg, 70%), along with recovered compound 15 (23.0 mg, 80% BRSM).

[0107] From trifluoroacetylimide donor:Receptor 15 (32.0 mg, 0.012 mmol, 1 equivalent) and donor 12 (42.9 mg, 0.021 mmol, 1.75 equivalent) were combined with activated pulverized... The mixture of MS (200 mg) and anhydrous CH2Cl2 (2 mL) was stirred under argon for 30 min. The reaction mixture was then cooled to -60 °C and treated with TfOH (0.5 M in Et2O, 5.86 μL, 2.93 μmol, 0.25 equivalents relative to the acceptor). After stirring at -20 °C for 3 h, the reaction mixture was quenched with Et3N (0.10 mL), diluted with CH2Cl2 (10 mL), and filtered through a diatomaceous earth mat. The filtrate was washed twice with saturated NaHCO3 aqueous solution (4 mL) and brine (3 mL). The organic phase was dehydrated by MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (2:3) as the precipitate to give compound 16 as a white powder (18.0 mg, 33%), accompanied by the recovered acceptor (12.6 mg, 55% brsm). f =0.17 (silicone, acetone: toluene = 1:2); 1 H NMR (600MHz, CDCl3): δ7.91-7.86(m,8H,Ar-H),7.48-7.42(m,4H,Ar-H),7.33-7. 11(m,83H,Ar-H),5.81-5.75(m,2H),5.43-5.38(m,3H),5.31-5.07(m,11H,2C1-H α ), 5.03-4.78(m,9H,2C1-H β ), 4.75-4.08 (m, 48H, 4C1-H) β ),4.05-3.89(m,7H),3.87-2.99(m,40H),2.13-2.12(m,6H,-2CH3),2.10- 2.07(m,15H,-5CH3),1.99-1.97(s,6H,-2CH3),1.90-1.88(m,6H,-2CH3); 13C NMR(150 MHz, CDCl3): δ170.7,170.6,170.6,170.4,170.2,169.8,169.7,169.6,165.6,165.5,165.4, 165.4,165.1,154.0,153.6,139.1,138.8,138.4,138.3,138.1,138.0,137.7,133.2,133.1,1 29.8,129.8,129.7,129.6,129.6,129.5,129.5,129.1,129.1,128.4,128.4,128.3,128.2,128.2,128.0,128.0,127.8,127.8,127.8,127.7,127.7,127.5,127.4,127.4,127.3,127.2,12 7.1,100.1,99.8,98.8,98.4,98.2,95.7,88.1,86.8,78.3,77.8,75.3,74.7,74.5,74.4,74.3,74.2,74.0,73.9,73.4,73.3,73.3,73.2,73.1,73.0,72.9,72.5,72.4,72.0,71.9,71.5,71 .0,71.0,70.9,70.9,70.7,69.5,69.4,69.2,69.1,69.0,69.0,68.5,68.1,68.1,68.0,67.2,62.9,62.2,57.5,57.2,53.8,53.2,53.2,45.5,45.3,23.3,21.0,21.0,20.8,20.7,20.7,20.6; 19 F NMR (376MHz, CDCl3): δ-215.6 (2F); HRMS (ESI-TOF) m / e: C 232 H 255 Cl9F2N6O 74 [M+H3O+NH4] 2+ Calculated value: 2330.6770, measured value: 2330.6724.

[0108]

[0109] [5-Acetamido-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranoside ester-(2→6)-β-D-galactopyranoside-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside-(1→2)-α-D-mannose pyranoside-(1→3)]-[5-acetamido-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranoside ester] -(2→6)-β-D-galactopyranoside-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside-(1→2)-α-D-mannopyranoside-(1→6)]-[β-D-mannopyranoside]-(1→4)-2-acetamido-2-deoxy-D-glucopyranoside 17. A solution of protected polysaccharide 16 (103 mg, 0.022 mmol, 1 equivalent) with LiOH (51.5 mg, 50% by SMwt) in a mixture of 1,4-dioxane / H2O (3 mL, 4:1 = v / v) was stirred at 90 °C for 16 h. The reaction mixture was concentrated under high vacuum and subjected to acetylation conditions (pyridine (2.5 mL), Ac2O (1.5 mL), rt, 16 h). After removal of the solvent, the crude residue was subjected to Purification was performed by RP-18 reverse-phase column chromatography using H₂O / MeOH (1:5) as the solvent. The product was deacetylated by stirring with a NaOMe solution in MeOH (3 mL, 0.5 M) for 16 hours. The reaction mixture was neutralized with an IR-120 filter, filtered, and concentrated under vacuum. The residue was then purified by... Purification was performed by RP-18 reverse-phase column chromatography using H₂O / MeOH (1:4) as the solvent. The deacetylated crude product was dissolved in a MeOH / H₂O / HCOOH mixture (3 mL, 6:3:1 = v / v / v) and treated with Pd(OH)₂ (51.5 mg, 50% by SMwt) for 20 hours. The reaction mixture was filtered through a diatomaceous earth mat and concentrated under vacuum. The residue was purified by (BIO-RAD) Biogel P-2 column chromatography (with water precipitation), followed by... Compound 17 was purified by RP-18 reverse-phase column chromatography (water precipitation) to give a white powder (18.3 mg, 40%) of a mixture of mutarotation isomers (α:β = 0.65:0.35). 1 H NMR (600MHz, D2O): δ5.23 (d, J=3.0Hz, 0.65H, a-C1-H α ),5.14(d,J=52.8Hz,2H,sia-C3-H),5.14(br,1H,c-C1-H α),4.96(br,1H,c'-C1-H α ),4.79(br,1H,b-C1-H β ),4.73(br,0.35H,a-Cl-H β ),4.60(d,J=7.8Hz,2H,dd'-C1-H β ),4.48(d,J=7.8Hz,2H,ee'-C1-H β ),4.28-4.21(m,4H),4.13(br,1H),4.00-3.51(m,57H),2.09-2.08(m,9H,-3CH3),2.05(br,6H,-2CH3); 13 C NMR (150MHz, D2O): δ174.5,174.2,174.1,102.7,102.6,100.0,99.1,98.9,98.9,98.7,98.6,9 6.6,94.4,91.0,90.0,89.8,80.0,79.7,79.3,78.9,76.0,75.8,74.1,74.0,73.9,73.1,72.9,7 2.7,72.4,71.9,71.9,71.6,71.5,71.1,70.3,69.8,69.4,69.2,69.1,69.0,68.7,68.0,67.8,66.9,66.8,65.4,65.2,63.6,62.2,61.2,61.2,59.7,59.6,55.6,54.4,53.1,46.3,21.9,21.6; 19 F NMR(376MHz,D2O):δ-217.4,-217.4; HRMS(ESI-TOF)m / e:C 76 H 121 F2N5O 57 [M-2H] -2 Calculated value: 1026.8351, measured value: 1026.8299.

[0110]

[0111] [5-Acetamido-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketose pyranoside ester-(2→6)-β-D-galactopyranoside-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside-(1→2)-α-D-mannopyranoside-(1→3)]-[5-acetamido-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2- Representative synthesis of ketose pyranoside-(2→6)-β-D-galactopyranoside-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside-(1→2)-α-D-mannopyranoside-(1→6)]-[β-D-mannopyranoside]-(1→4)-(2-acetamido-1,2-dideoxy-D-glucopyranoside)-[2,1-d]-2-methyloxazoline S1. Polysaccharide oxazoline. A solution of polysaccharide 17 (14.5 mg, 7.05 μmol, 1 equivalent), 2-chloro-1,3-dimethyl-1H-benzimidazol-3-onium chloride (CDMBI) (12.2 mg, 0.056 mmol, 8 equivalent), and Et3N (19.9 μL) in water (121.1 μL) was stirred at 4 °C for 1 hour. The reaction mixture was subjected to gel filtration chromatography on a Sephadex G-25 column, and precipitated with 0.05% Et3N aqueous solution. The fractions containing the polysaccharide-oxazoline product were combined and lyophilized to give the desired product S1 as a white powder (12.6 mg, 87% yield). 1 H NMR (600MHz, D2O+0.05%Et3N): δ6.09 (d, J=7.2Hz, 1H, oxa-C1-H α ),5.12(d,J=52.2Hz,2H,sia-C3-H),5.11(br,1H,c-C1-H α ),4.95(br,1H,c'-C1-H α ),4.74(br,1H,b-C1-H β ),4.60-4.57(m,2H,dd'-C1-H β ),4.46-4.45(m,2H,ee'-C1-H β ),4.39(br,1H),4.22-4.15(m,5H),3.98-3.49(m,55H),3.43-3.40(m,1H),2.07-2.02(m,15H,-5CH3); 13C NMR (150MHz, D2O+0.05%Et3N): δ174.9,174.7,174.7,170.7,170.7,168.5,103.1,103.1,101. 4,99.9,99.5,99.4,99.2,99.1,99.0,96.5,91.4,90.2,80.4,79.4,77.9,76.4,76.0,74.6,74 .2,73.4,73.4,72.8,72.4,72.3,72.0,71.9,71.5,70.8,70.7,70.2,69.6,69.5,69.4,69.1,68.5,68.2,67.2,65.8,65.7,65.1,64.1,62.6,61.6,61.6,60.1,54.9,46.7,22.4,22.0,12.9; 19 F NMR (470MHz, D2O+0.05%Et3N): δ-217.3(d,J FH =51.3Hz), -217.3(d,J FH =51.3Hz); HRMS(ESI-TOF)m / e:C 76 H 120 F2N5O 56 [MH] - Calculated value: 2036.6658, measured value: 2036.6672.

[0112]

[0113] Process S1.

[0114] Reagents and conditions in process S1: (a) TFA, MeOH, 60℃, 16h, then AcCl, rt, 2d, then Na2HPO4, CH3CN, reflux, 20h, 92%. (b) ref. 3, 80% (three steps). (c) TiBr4, dichloroethane, rt, 10min, 92%. (d) BH3, Cu(OTf)2, THF, rt, 8h, 96%.

[0115]

[0116] 5-Acetamido-4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-2,6-dehydr-D-glycero-D-galactose-non-2-enoate methyl ester S3. 19TfOH (4.95 mL, 64.7 mmol, 1 equivalence) was added to a solution of compound S2 (20 g, 64.7 mmol, 1 equivalence) in MeOH (600 mL). The reaction mixture was stirred at 60 °C for 16 h. The solvent was removed by rotary evaporation under reduced pressure and co-evaporated twice with toluene to remove water. The resulting residue was dissolved in AcCl (200 mL) in a round-bottom flask at 0 °C, sealed, and stirred at room temperature for 2 days. After removing the solvent, the resulting residue was diluted with anhydrous acetonitrile (200 mL), and then Na2HPO4 (19.6 g, 155 mmol, 2.4 equivalence) was added under argon. The reaction mixture was stirred vigorously at 90 °C until TLC showed the disappearance of the starting material (16 h). The solution was filtered through a diatomaceous earth mat, dehydrated with MgSO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography using EtOAc / hexane (4:1) as the precipitate to give compound S3 as a brown foam (28.2 g, 92%). The spectroscopic data were consistent with those reported in previous literature. 19 R f =0.30 (silicone, EtOAc); 1 H NMR (600MHz, CDCl3): δ5.95(d,J=3.0Hz,1H),5.81(d,J=9.0Hz,1H),5.47-5.44(m,2H),5.32-5.30(m,1H),4.59(dd,J=12.0,3.0Hz,1H),4.38-4.33(m ,2H),4.15(d,J=12.0,7.2Hz,1H),3.76(s,3H,-CH3),2.08(s,3H,-CH3),2 .03(s,3H,-CH3),2.02(s,3H,-CH3),2.01(s,3H,-CH3),1.88(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ 170.7, 170.5, 170.1, 170.1, 170.0, 161.6, 145.0, 107.9, 70.6, 67.8, 67.6, 61.9, 52.5, 46.5, 23.1, 20.8, 20.7, 20.7; HRMS (ESI-TOF) m / e: calculated values ​​of C 20 H 27 NO 12 Na[M+Na] + :496.1425, measured value:496.1435.

[0117]

[0118] 5-Acetamido-4,7,8,9-Tetra-O-acetyl-2-bromo-2,5-dideoxy-D-erythrose-α-L-glucose-non-2-ketosepyranose methyl ester 3. 20 The epoxide S4 was synthesized from sialic acid using a reported procedure. 21 A solution of epoxide S4 (1.10 g, 2.25 mmol, 1 equivalent) in anhydrous 1,2-dichloroethane (18 mL) was stirred and TiBr4 (0.91 g, 2.47 mmol, 1.1 equivalent) was added over 10 min under argon atmosphere. The solvent was removed by rotary evaporation under high vacuum. The resulting residue was diluted with EtOAc (30 mL) and washed with saturated aqueous Na2SO4 solution (10 mL), 5% NaHCO3 aqueous solution (10 mL), and brine (5 mL). The separated organic layer was dehydrated with MgSO4 and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using acetone / hexane (2:1) as the precipitate to give compound 3 as a white powder (1.18 g, 92%). The spectroscopic data and experimental protocol were the same as previously reported. 20 R f =0.23 (silicone, acetone: toluene = 1:1); 1 H NMR (600MHz, CDCl3): δ5.99 (d, J=7.2Hz, 1H), 5.42 (dd, J=7.2, 1.2Hz, 1H), 5.21-5.18 (m, 1H ),5.10(ddd,J=6.0,6.0,2.4Hz,1H),4.38(dd,J=12.6,2.4Hz,1H),4.32-4.30(m,2H),3.99 (dd,J=12.6,6.0Hz,1H),3.86(s,3H,-CH3),3.78(d,J=9.0Hz,1H),3.66(br,1H,-OH),2.07 (s,3H,-CH3),2.06(s,3H,-CH3),2.05(s,3H,-CH3),2.01(s,3H,-CH3),1.84(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.4,170.6,170.3,169.8,169.7,167.0,98.0,75.0,72.8 ,72.2,69.9,66.5,61.9,54.0,47.2,22.9,21.0,20.7,20.6; HRMS(ESI-TOF)m / e:C 20 H 29 BrNO 13 [M+H] + Calculated value: 570.0817, measured value: 570.0822.

[0119]

[0120] p-Tolyl 2,3-di-O-benzyl-4-O-benzyl-1-thio-β-D-galactopyranoside 5. 22 To the stirring starting material S5 23 A solution of (4.00 g, 6.86 mmol, 1 equivalence) of the BH3-THF complex (1 M in THF, 34.3 mL, 34.3 mmol, 5 equivalence) was added to Cu(OTf)2 (124 mg, 0.34 mmol, 0.05 equivalence), and the reaction mixture was stirred at room temperature for 8 hours. After completion, the reaction was carefully neutralized at 0 °C with TEA (0.96 mL, 6.86 mmol, 1 equivalence) and then diluted with MeOH (15 mL). After removing the solvent under vacuum, the resulting residue was purified by silica gel column chromatography using EtOAc / hexane (1:2) as the precipitate to give compound 5 as a white powder (3.85 g, 96%). The spectral data were consistent with those reported in previous literature. 22 R f =0.45 (silica gel, EtOAc:hexane = 1:1); 1 H NMR (600MHz, CDCl3): δ7.97-7.94(m,4H,Ar-H),7.51-7.48(m,2H,Ar-H),7.38-7.34(m,6H,Ar-H),7.27- 7.21(m,5H,Ar-H),5.85(dd,J=10.2,10.2Hz,1H),5.36(dd,J=9.6,3.0Hz,1H),4.87(d,J=9.6Hz,1H,C1-H β ),4.74(d,J=12.0Hz,1H),4.47(d,J=12.0Hz,1H),4.16(d,J=2.4Hz,1H),3.91(d,J=11.4,7.2Hz, 1H),3.76(dd,J=6.0,6.0Hz,1H),3.61(dd,J=11.4,5.4Hz,1H),2.30(s,3H,-CH3),1.83(br,1H); 13C NMR (150MHz, CDCl3): δ165.9,165.2,138.1,137.4,133.4,133.1,133.0,129.8,129.7,129.6,129.5,128.9,1 28.7,128.5,128.4,128.3,128.1,127.9,86.8,79.0,76.0,74.6,73.7,68.5,61.9,21.1; HRMS(ESI-TOF)m / e:C 34 H 32 O7SNa[M+Na] + Calculated value: 607.1761, measured value: 607.1770.

[0121]

[0122] Process S2

[0123] Reagents and conditions in process S2: (a) TESOTf, BnCHO, TESH, toluene, THF, 2h, -20℃, 90%. (b) NIS, TfOH, MS, -40℃, 1.5h, CH2Cl2, 75%. (c) NaBH3CN, HCl / ether, AW-300, THF, 0℃ tor.t., 16h, 85%.

[0124]

[0125] p-Tolyl 3-O-benzyl-4,6-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-β-D-glucopyranoside S7. The mixture was prepared by adding the starting material S6 to the mixture under argon atmosphere at -20°C. 24TESOTf (4.26 mL, 18.8 mmol, 2 equivalences) was added to a solution of THF / toluene (45 mL, 1:2 = v / v) mixture. After stirring at -20 °C for 45 min, benzaldehyde (4.79 mL, 47.1 mmol, 5 equivalences) and triethylsilane (2.26 mL, 14.1 mmol, 1.5 equivalences) were added dropwise to the mixture with stirring. After 2 h at -20 °C, TLC showed the disappearance of the starting material. The reaction was quenched with a saturated aqueous solution of Na₂CO₃, diluted with EtOAc (100 mL), and washed with a saturated aqueous solution of Na₂CO₃ (30 mL) and brine. The combined organic layers were dehydrated with MgSO₄, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using CH2Cl2 / toluene (1:4) followed by EtOAc / toluene (1:10) as the precipitate to give compound S7 as a white solid (5.40 g, 90%). f =0.50 (Silicone, EtOAc:Toluene = 1:25); 1 H NMR (600MHz, acetone-d6): δ7.52-7.50(m,2H,Ar-H),7.42-7.36(m,4H,Ar-H),7.32-7.31(m,2H,Ar-H), 7.29-7.22(m,4H,Ar-H),7.18-7.15(m,2H,Ar-H),5.72(s,1H,Ph-CH),5.03(d,J=10.8Hz,1H,C1-H β ),4.90-4.82(m,3H),4.74(d,J=12.0Hz,1H),4.30(dd,J=10.2,5.4Hz,1H),3.95(dd,J=9.6,9. 0Hz,1H),3.84-3.71(m,3H),3.56(ddd,J=9.6,9.6,4.8Hz,1H),2.83(s,2H),2.32(s,3H,-CH3); 13 C NMR (150MHz, acetone-d6): δ 155.3, 139.8, 139.4, 138.6, 133.3, 130.7, 130.5, 129.8, 129.6, 129.1, 129.0, 129.0, 128.5, 128.2, 127.1, 101.8, 97.1, 88.6, 82.7, 80.8, 75.0, 75.0, 71.2, 69.1, 57.4, 21.1; HRMS (ESI-TOF) m / e: C 30 H 30 Cl3NO6SNa[M+Na] +Calculated value: 660.0752, measured value: 660.0749.

[0126]

[0127] Allyl[3-O-benzyl-4,6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside S9. Receptor S8 25 (523 mg, 0.94 mmol, 1 equivalent), donor S7 (900 mg, 1.41 mmol, 1.5 equivalent) and activated pulverized The mixture of MS (2.00 g) and anhydrous CH2Cl2 (20 mL) was stirred under argon for 1 hour. The reaction mixture was then cooled to -40 °C and treated with NIS (423 mg, 1.88 mmol, 2 equivalences) and TfOH (0.5 M in Et2O, 0.47 mL, 0.23 mmol, 0.25 equivalences). After stirring at this temperature for 1.5 hours, TLC analysis showed the disappearance of the starting material. The reaction mixture was quenched with Et3N (0.2 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH2Cl2 (20 mL) and washed with 20% Na2S2O3 aqueous solution (5 mL), saturated NaHCO3 aqueous solution (10 mL), and brine (5 mL). The separated organic layer was dehydrated with MgSO4 and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using EtOAc / toluene (1:10) as the precipitate to give compound S9 as a white foam (710 mg, 75%). f =0.36 (Silicone, EtOAc:Toluene = 1:10); 1 H NMR (600MHz, CDCl3): δ7.50-7.48(m,2H,Ar-H),7.39-7.36(m,6H,Ar-H),7.33-7.25(m,15H,Ar-H),7.23-7.22(m,2H,Ar-H),5.84(ddd,J=16 .2.10.8,5.4Hz,1H),5.55(s,1H,Ph-CH),5.26(d,J=1.2Hz,1H),5.23-5.20(m,1H),5.16(dd,J=10.8,1.2Hz,1H),5.01(d,J=7.8Hz,1H,C1-H β ),4.90(d,J=10.8Hz,1H),4.83(d,J=11.4Hz,1H),4.75-4.62(m,6H,C1-H α),4.55-4.51(m,3H),4.33-4.29(m,2H),4.13-4.10(m,1H),4.08(dd,J=2.4,2.4Hz,1H),4.00-3.97(m,1H),3.94(dd,J=9.6 ,3.0Hz,1H),3.89(dd,J=13.2,6.0Hz,1H),3.82-3.78(m,2H),3.72-3.66(m,3H),3.50-3.46(m,1H),3.12(d,J=6.0Hz,1H); 13 C NMR (150MHz, CDCl3): δ153.8,138.6,138.5,138.3,138.1,137.3,133.7, 129.0,128.4,128.3,128.2,128.1,128.0,127.8,127.6,127.6,127.4,12 6.0,117.2,101.2,98.9,96.9,95.5,82.4,78.5,75.6,75.2,74.6,74.4,7 4.2,73.3,72.2,72.2,69.2,68.6,68.0,66.1,58.1; HRMS(ESI-TOF)m / e:C 53 H 56 Cl3NO 12 Na[M+Na] + Calculated value: 1026.2760, measured value: 1026.2780.

[0128]

[0129] Allyl[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside 8. 27Under argon atmosphere, activated pulverized AW 300MS (1.20 g) was added to a stirred solution of starting material S9 (500 mg, 0.50 mmol, 1 equivalence) in anhydrous THF (12 mL). The reaction mixture was then cooled to 0 °C and NaCNBH3 (313 mg, 4.97 mmol, 10 equivalence) was added, followed by the slow addition of HCl·Et2O (2 M in Et2O, 2.24 mL, 4.48 mmol, 9 equivalence). The mixture was then stirred until TLC showed the disappearance of the starting material (16 h). After this, the reaction mixture was quenched with saturated NaHCO3 aqueous solution (0.5 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH2Cl2 (20 mL) and then washed with saturated NaHCO3 aqueous solution (8 mL) and brine. The separated organic layer was dehydrated with MgSO4 and concentrated. The residue was purified by silica gel column chromatography using EtOAc / toluene (1:5) as the precipitate to give compound 8 as a white foam (425 mg, 85%). The spectral data were consistent with those reported in the literature. 27 R f =0.17 (Silicone, EtOAc:Toluene = 1:10); 1 H NMR (600MHz, CDCl3): δ7.36-7.23(m,23H,Ar-H),7.20-7.19(m,2H,Ar-H),5.84(ddd,J=16.2.10.2,6.0Hz ,1H),5.35(br,1H),5.21(dd,J=16.2,1.2Hz,1H),5.14(dd,J=10.2,1.2Hz,1H),4.95(d,J=7.2Hz,1H,C1-H β ),4.88(d,J=10.8Hz,1H),4.78(d,J=1.8Hz,1H,C1-H α ),4.75(d,J=11.4Hz,1H),4.70-4.63(m,5H),4.60(d,J=11.4Hz,1H),4.55-4.49(m,4H),4.14-4.07(m,3H),3.96-3.87(m,3H),3.77(dd,J=10. 8,4.2Hz,1H),3.73-3.70(m,3H),3.66(dd,J=10.8,1.8Hz,1H),3.61(dd,J=9.6,8.4Hz,1H),3.54-3.51(m,1H),3.08(m,1H),2.71(m,1H,-OH); 13C NMR (150MHz, CDCl3): δ154.0,138.5,138.5,138.4,138.1,137.6,133.7,1 28.5,128.5,128.3,128.3,128.3,128.2,128.0,128.0,127.8,127.7,127 .7,127.6,127.6,127.5,117.3,79.2,78.2,75.1,74.6,74.3,74.2,73.8, 73.7,73.4,73.3,72.0,71.7,70.9,69.3,68.0,57.5; HRMS(ESI-TOF)m / e:C 53 H 58 Cl3NO 12 Na[M+Na] + Calculated value: 1028.2917, measured value: 1028.2927.

[0130]

[0131] Process S3

[0132] Reagents and conditions in process S3: (a) NaBH3CN, HCl / ether, AW-300, THF, 0℃ to rt, 16h, 90%. (b) Ac2O, pyridine, rt, 16h, 98%. (c) NIS, TfOH, HOPO(OBu)2, MS, -30℃, 2h, CH2Cl2, 90%. (d) TMSOTf, MS, -50℃, 1h, CH2Cl2, 87%. (e) NaOMe, MeOH / CH2Cl2, 0℃ to rt, 76%.

[0133]

[0134] p-Tolyl 3,6-di-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-β-D-glucopyranoside S10. 28Under argon atmosphere, activated pulverized AW 300MS (3.00 g) was added to a stirred solution of starting material S7 (1.00 g, 1.56 mmol, 1 equivalent) in anhydrous THF (30 mL). The reaction mixture was then cooled to 0 °C, and NaCNBH3 (0.98 g, 15.6 mmol, 10 equivalent) was added, followed by the slow addition of HCl·Et2O (2 M in Et2O, 7.04 mL, 14.1 mmol, 9 equivalent). The mixture was stirred until TLC showed the disappearance of the starting material (16 h). After completion, the mixture was quenched with saturated NaHCO3 aqueous solution (0.5 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH2Cl2 (50 mL) and washed with saturated NaHCO3 aqueous solution (15 mL) and brine (10 mL). The separated organic layer was dehydrated with MgSO4 and concentrated. The residue was purified by silica gel column chromatography using EtOAc / hexane / CH2Cl2 (1:3:1) as the precipitate to give compound S10 as a white foam (902 mg, 90%). The spectral data were consistent with those reported in the literature. 28 R f =0.29 (silica gel, EtOAc:hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ7.40-7.38(m,2H,Ar-H),7.36-7.27(m,10H,Ar-H),7.04-7.03(m,2H,Ar-H),5.20(d,J=7.8Hz,1H),4.84(d,J=10.2Hz,1H,C1-H β ),4.77-4.72(m,4H),4.58-4.52(m,2H),3.76(d,J=4.8Hz,2H),3.71-3.64(m,2 H),3.50-3.49(m,1H),3.42-3.38(m,1H),2.90(br,1H,-OH),2.29(s,3H,-CH3); 13 CNMR (150MHz, CDCl3): δ153.8,138.1,138.1,137.7,133.0,129.6,128.6,128.5,128.4,128.1,127.9 ,127.8,127.7,95.5,86.1,81.9,78.0,74.5,74.4,73.6,72.5,70.4,55.9,21.1; HRMS(ESI-TOF)m / e:C 30 H 32 Cl3NO6SNa[M+Na] + Calculated value: 662.0908, measured value: 662.0919.

[0135]

[0136] p-Tolyl-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-β-D-glucopyranoside S11. Ac₂O (6 mL) was added to a solution of the stirred starting material S10 (0.97 g, 1.51 mmol, 1 equivalent) in pyridine (12 mL). The reaction mixture was vigorously stirred at room temperature for 16 h, then concentrated under vacuum and purified by silica gel column chromatography using EtOAc / hexane / CH₂Cl₂ (1:4:1) as the precipitate to give compound S11 as a white powder (1.01 g, 98%). f =0.47 (silica gel, EtOAc:hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ7.41-7.40(m,2H,Ar-H),7.34-7.25(m,8H,Ar-H),7.22-7.21(m ,2H,Ar-H),7.03-7.01(m,2H,Ar-H),5.29(d,J=7.2Hz,1H),5.04(d,J=10.2Hz,1H,C1-H β ),4.98(dd,J=9.6,9.6Hz,1H),4.78(d,J=12.0Hz,1H),4.70(d,J=12.0Hz,1H),4.62(d,J=11.4Hz,1H),4.57(d,J=11.4Hz,1H),4.50(br,2H) ,4.05(dd,J=9.6,9.0Hz,1H),3.65-3.63(m,1H),3.59-3.55(m,2H),3.33(ddd,J=9.6,9.6,9.6Hz,1H),2.29(s,3H,-CH3),1.87(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ169.8,153.7,138.4,138.0,137.7,133.3,129.8,128.6,128.5,128.4,128.2,128.1,128 .1,127.9,127.9,127.7,95.5,85.3,79.4,77.7,74.4,73.6,71.4,69.7,56.5,21.1,20.9; HRMS(ESI-TOF)m / e:C 32 H 34 Cl3NO7SNa[M+Na] + Calculated value: 704.1014, measured value: 104.1031.

[0137]

[0138] 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-α or β-D-glucopyranoside dibutyl phosphate S12a,b. Compound S11 (990 mg, 1.45 mmol, 1 equivalent), dibutyl phosphate (1.15 mL, 5.80 mmol, 3 equivalents) and activated pulverized The mixture of MS (1.80 g) and anhydrous CH2Cl2 (18 mL) was stirred under argon for 1 hour. It was then cooled to -30 °C with stirring, and NIS (978 mg, 4.35 mmol, 2 equivalences) was added, followed by TfOH (0.5 M in Et2O, 0.87 mL, 0.43 mmol, 0.3 equivalences). After 2 hours, TLC analysis showed the disappearance of the starting material. The reaction mixture was quenched with saturated NaHCO3 aqueous solution (0.5 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH2Cl2 (50 mL) and washed with 20% Na2S2O3 aqueous solution (10 mL), saturated NaHCO3 aqueous solution (5 mL), and brine (5 mL). The separated organic layer was dehydrated with MgSO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography using EtOAc / toluene (1:3) as the precipitate to give compound S12 as a white foam (1.00 g, 90%); a mixture of mutarotation isomers (α:β = 1:3). β-Mutarotation isomer S12b:R f =0.32 (Silicone, EtOAc:Toluene = 1:3); 1 H NMR (600MHz, CDCl3): δ7.30-7.22(m,10H,Ar-H),5.81(br,1H),5.35(dd,J=7.8,7.8Hz,1H,C1-H β ),5.09(dd,J=9.6,9.0Hz,1H),4.74(d,J=12.0Hz,1H),4.65-4.58(m,3H),4.49-4.44(m,2H),4.06-3.97(m,4H),3.88(dd,J=9.6,9.0Hz,1H),3.7 7-3.74(m,1H),3.69(ddd,J=9.6,4.2,4.2Hz,1H),3.53-3.52(m,2H),1.8 3(s,3H,-CH3),1.61-1.54(m,4H),1.36-1.31(m,4H),0.93-0.85(m,6H); 13C NMR (150MHz, CDCl3): δ169.5,154.2,137.6,137.6,128.5,128.4,128.3,127.9,127.9,127.7,127.7,96.5,95.3,78.3,74.5,7 3.9,73.6,73.5,70.6,69.2,68.1,68.1,68.1,68.0,57.2,57.2,32.1,32.0,32.0,20.7,18.6,18.6,13.5; HRMS(ESI-TOF)m / e:C 33 H 45 Cl3NO 11 PNa[M+Na] + Calculated value: 790.1688, measured value: 790.1685. α-Mutantisomer S12a:R f =0.44 (Silicone, EtOAc:Toluene = 1:3); 1 H NMR (600MHz, CDCl3): δ7.31-7.22(m,10H,Ar-H),5.67(dd,J=6.0,3.6Hz,1H,C1-H α ),5.21-5.16(m,2H),4.75(d,J=12.0Hz,1H),4.62-4.56(m,3H),4.49-4.44(m,2H),4.11-3.99(m,6H),3.77(dd,J =10.2,9.6Hz,1H),3.50-3.49(m,2H),1.89(s,3H,-CH3),1.60-1.55(m,4H),1.35-1.31(m,4H),0.92-0.86(m,6H); 13 C NMR (150MHz, CDCl3): δ169.3,154.0,137.4,137.4,128.4,128.2,127.8,127.8,127.8,127.6,127.6,127.6,96.4,96.3,95.2,76. 4,74.6,73.6,73.5,71.1,70.1,68.6,68.1,68.0,68.0,67.9,54.4,54.3,32.1,32.0,20.7,18.5,18.5,13.5; HRMS(ESI-TOF)m / e:C 33 H 45 Cl3NO 11 PNa[M+Na] + Calculated value: 790.1688, measured value: 790.1688.

[0139]

[0140] p-Tolyl[4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside S14. The acceptor S13 29 (407 mg, 0.73 mmol, 1 equivalent), donor S12 (900 mg, 1.17 mmol, 1.6 equivalent) and activated pulverized A mixture of molecular sieve (200 mg) and anhydrous CH2Cl2 (2 mL) was stirred under argon for 30 min. It was then cooled to -50 °C, followed by the addition of TMSOTf (0.21 mL, 1.17 μmol, 1.6 equivalents relative to the acceptor) with stirring until TLC analysis showed the disappearance of the initiator (1 h). The reaction mixture was quenched with Et3N (0.30 mL), diluted with CH2Cl2 (20 mL), and filtered through a diatomaceous earth mat. The filtrate was washed twice with saturated NaHCO3 aqueous solution (8 mL) and brine (4 mL). The organic phase was dehydrated with MgSO4, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using EtOAc / hexane (1:3) as the precipitate to give compound S14 as a white powder (710 mg, 87%). f =0.26 (silica gel, EtOAc:hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ7.37-7.36(m,2H,Ar-H),7.33-7.14(m,25H,Ar-H),7.05-7.03(m,2H,Ar-H),5.33(d,J=1.8Hz,1H,C1-H α ),5.26(d,J=4.8Hz,1H),5.07(d,J=9.0Hz,1H,C1-H β),4.93-4.90(m,2H),4.77(d,J=11.4Hz,1H),4.60-4.52(m,5H),4.46-4.34(m,5H),4.27 (dd,J=8.4,8.4Hz,1H),4.19(dd,J=9.6,2.4Hz,1H),4.15(d,J=12.0Hz,1H),4.07(dd,J= 9.6,9.0Hz,1H),3.84-3.81(m,2H),3.64-3.63(m,2H),3.57(dd,J=10.8,6.0Hz,1H),3.5 1(dd,J=11.4,3.0Hz,1H),3.01(dd,J=6.6Hz,1H),2.28(s,3H,-CH3),1.82(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ169.7,154.2,138.5,138.3,138.0,137.8,137.7,137.6,132.5,130.3, 129.8,129.7,129.0,128.4,128.4,128.3,128.2,128.2,128.2,128.2,127.9,127.8,127.8,1 27.7,127.7,127.6,127.6,127.6,127.4,125.2,97.1,95.4,86.2,78.3,75.2,75.1,74.5,74. 1,74.0,73.6,73.4,73.1,72.5,71.7,71.3,70.0,69.2,58.1,21.1,20.8; HRMS(ESI-TOF)m / e:C 59 H 62 Cl3NO 12 SNa[M+Na] + Calculated value: 1136.2950, ​​measured value: 1136.2978.

[0141]

[0142] p-Tolyl[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside]-(1→2)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside 18. NaOMe (2.95 mg, 0.055 mmol, 0.3 equivalence) was added to a solution of compound S14 (203 mg, 0.18 mmol, 1 equivalent) in a CH2Cl2 / MeOH mixture (10 mL, 1:1 = v / v) at 0 °C with thorough stirring. After 20 minutes, the ice bath was removed and the reaction mixture was heated to room temperature with stirring until TLC analysis showed the disappearance of the starting material (4 h). Afterward, the reaction mixture was neutralized with an IR-120, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography using EtOAc / hexane (1:2) as the precipitate to give compound 18 as a white foam (149 mg, 76%). f =0.14 (silica gel, EtOAc:hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ7.39-7.37(m,2H,Ar-H),7.34-7.24(m,25H,Ar-H),7.06-7.05(m,2H,Ar-H),5.33(d,J=1.8Hz,1H,C1-H α ),5.24(d,J=4.8Hz,1H),5.00(d,J=7.8Hz,1H,C1-H β ),4.93(d,J=10.8Hz,1H),4.78(d,J=11.4Hz,1H),4.69-4.64(m,2H),4.61(d,J=11.4 Hz,2H),4.57-4.51(m,4H),4.43(d,J=11.4Hz,1H),4.34(dd,J=2.4,2.4Hz,1H),4.22 -4.20(m,2H),4.07-4.04(m,2H),3.84-3.82(m,2H),3.77-3.72(m,2H),3.67-3.65(m ,1H),3.59-3.54(m,2H),3.01(d,J=7.2Hz,1H),2.68(br,1H,-OH),2.30(s,3H,-CH3); 13C NMR (150MHz, CDCl3): δ154.2,138.5,138.4,138.3,137.9,137.7,137.6,132.5,1 30.3,129.8,128.5,128.5,128.4,128.3,128.1,128.0,127.9,127.8,127.8,127. 7,127.6,127.6,127.5,97.5,95.4,86.2,79.1,78.4,75.3,75.2,74.6,74.4,74.2 ,73.8,73.8,73.2,73.1,72.5,71.4,70.9,69.3,57.8,21.1; HRMS(ESI-TOF)m / e:C 57 H 60 Cl3NO 11 SNa[M+Na] + Calculated value: 1094.2845, measured value: 1094.2881.

[0143]

[0144] Process S4

[0145] Reagents and conditions in process S4: (a) NIS, TfOH, MS, -40℃, 1h, CH2Cl2, 93%. (b) Ethylenediamine / BuOH (1:4), 90℃, 2h, then NaHCO3, TrocCl, CH2Cl2, 0℃, 3h, 81%. (c) Tf2O, pyridine, CH2Cl2, 0℃, 4h. (d) Bu4NOAc, toluene, ultrasonic treatment, rt, 8h, then NaHCO3, TrocCl, CH2Cl2, 0℃, 3h, 61% (2 steps). (e) DDQ, CH2Cl2, phosphate buffer. (pH=7), 0℃ to rt, 3h, 85%. (f) Hydrazine acetate, THF, rt, 16h, 92%. (g) Tf2O, pyridine, CH2Cl2, 0℃, 2h. (h) Bu4NOAc, toluene, ultrasonic treatment, rt, 8h, 93% (2 steps). (i) Ethylenediamine / BuOH (1:4), 90℃, 2h, then NaHCO3, TrocCl, CH2Cl2, 0℃, 3h. (j) Ac2O, pyridine, CH2Cl2, rt, 16h, 83% (2 steps).

[0146]

[0147] Benzyl[4,6-O-benzylidene-3-Op-methoxy-benzyl-2-O-levulinoyl-β-D-glucopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside S17. Receptor S16 30 (1.59 g, 2.74 mmol, 1 equivalent), donor S15 26 (2.11 g, 3.57 mmol, 1.3 equivalents) with activated pulverized The mixture of MS (5.00 g) in anhydrous CH₂Cl₂ (50 mL) was stirred under argon for 1 h. The reaction mixture was then cooled to -40 °C and NIS (1.23 g, 0.49 mmol, 2 equivalences) was added, followed by TfOH (0.5 M in Et₂O, 1.37 mL, 0.69 mmol, 0.25 equivalences). The reaction was continued until TLC showed the disappearance of the starting material (2 h). After completion, the mixture was quenched with Et₃N (0.7 mL) and filtered through a diatomaceous earth mat. The filtrate was diluted with CH₂Cl₂ (50 mL) and washed with 20% Na₂S₂O₃ aqueous solution (15 mL), saturated NaHCO₃ aqueous solution (20 mL), and brine (10 mL). The separated organic layer was dehydrated with MgSO₄ and concentrated under vacuum. The residue was purified by silica gel column chromatography using EtOAc / hexane (2:3) as the precipitate to give compound S17 as a white foam (2.68 g, 93%). f =0.43 (silicone, acetone: toluene = 1:6); 1 H NMR (600MHz, CDCl3): δ7.75(br,1H,Ar-H),7.63(br,2H,Ar-H),7.50(br,1H,Ar-H ),7.46-7.45(m,2H,Ar-H),7.39-7.32(m,7H,Ar-H),7.29-7.26(m,1H,Ar-H),7.20 -7.18(m,2H,Ar-H),7.07-7.05(m,1H,Ar-H),7.02-7.01(m,4H,Ar-H),6.98-6.97 (m,2H,Ar-H),6.87-6.83(m,5H,Ar-H),5.44(s,1H,Ph-CH),5.10-5.08(m,1H,C1-H β ),4.92(dd,J=9.0,8.4Hz,1H),4.81-4.71(m,4H),4.58(d,J=7.8Hz,1H,C1-H β),4.56(d,J=4.2Hz,1H),4.47(d,J=6.0Hz,1H),4.45(d,J=6.6Hz,1H),4.36(d,J=12.6Hz,1H),4.23(dd,J =10.2,4.8Hz,1H),4.20-4.19(m,2H),4.09-4.06(m,1H),3.87(dd,J=11.4,3.0Hz,1H),3.79-3.78(m,1H) ,3.78(s,3H,-CH3),3.59(dd,J=9.6,9.0Hz,1H),3.55-3.51(m,2H),3.43(dd,J=10.2,10.2Hz,1H),3.17( ddd,J=9.6,9.6,4.8Hz,1H),2.81-2.76(m,1H),2.70-2.65(m,1H),2.50-2.38(m,2H),2.20(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ206.2,171.1,159.2,138.6,138.1,137.3,133.5,131.6,13 0.4,129.4,129.0,128.5,128.2,128.1,127.9,127.8,127.7,127.5,127.5,127.0 ,126.0,123.1,113.6,101.1,100.6,97.4,81.7,78.0,77.9,76.6,74.8,74.5,73. 7,73.6,70.7,68.6,67.7,65.8,55.7,55.3,37.7,29.9,27.8; HRMS(ESI-TOF)m / e:C 61 H 61 NO 15 Na[M+Na] + Calculated value: 1070.3933, measured value: 1070.3933.

[0148]

[0149] Benzyl[4,6-O-benzylidene-3-Op-methoxy-benzyl-β-D-glucopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside S18. A solution of disaccharide S17 (625 mg, 0.596 mmol, 1 equivalent) in ethylenediamine / n-BuOH (7 mL, 2:8 = v / v) was stirred at 90 °C for 2 h. The solvent was removed by rotary evaporation under high vacuum and co-evaporated twice with toluene to remove water traces. The resulting residue was dissolved in anhydrous CH₂Cl₂ (15 mL) and treated with NaHCO₃ (250 mg, 2.98 mmol, 5 equivalences) and trichloroethyl 2,2,2-chloroformate (0.41 mL, 2.98 mmol, 5 equivalences) at 0 °C under argon for 3 h. Afterward, the reaction mixture was diluted with CH₂Cl₂ (20 mL) and washed with water (20 mL) and brine (10 mL). The organic layer was dehydrated with MgSO₄ and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using EtOAc / toluene (1:4) as the precipitate to give compound S18 as a white powder (481 mg, 81%). f =0.60 (Silicone, EtOAc:Toluene = 1:2); 1 H NMR (600MHz, CDCl3): δ7.47-7.46(m,2H,Ar-H),7.40-7.25(m,20H,Ar-H),6.86-6.85(m, 2H,Ar-H),5.46(s,1H,Ph-CH),5.12(br,1H),4.88-4.85(m,3H),4.71-4.55(m,9H,2C1-H β ),4.06-4.03(m,2H),3.99(dd,J=11.4,3.6Hz,1H),3.87(br,1H),3.80(dd,J=11.4,1.8Hz,1H),3.78(s,3H, -CH3),3.56(dd,J=9.6,9.0Hz,1H),3.52-3.45(m,5H),3.15(ddd,J=9.6,9.6,4.8Hz,1H),3.03(br,1H,-OH); 13C NMR (150MHz, CDCl3): δ159.3,153.8,138.4,137.7,137.3,137.2,130.4,12 9.6,128.9,128.4,128.4,128.3,128.2,128.0,127.8,127.8,127.6,127.4, 126.0,113.8,103.2,101.1,99.2,95.5,81.3,79.9,79.2,77.7,74.9,74.5 ,74.3,74.2,73.5,70.7,68.6,68.2,66.2,57.6,55.2; HRMS(ESI-TOF)m / e:C 51 H 54 Cl3NO 13 Na[M+Na] + Calculated value: 1016.2553, measured value: 1016.2554.

[0150]

[0151] Benzyl[2-O-acetyl-4,6-O-benzylidene-3-Op-methoxy-benzyl-β-D-mannopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside S19. From S18: Anhydrous pyridine (0.58 mL, 7.17 mmol, 7 equivalences) was added dropwise to a stirred solution of disaccharide S18 (1.02 g, 1.02 mmol, 1 equivalence) in anhydrous CH2Cl2 (13 mL) at 0 °C under argon atmosphere, followed by the dropwise addition of Tf2O (0.30 mL, 1.79 mmol, 1.75 equivalences). The reaction mixture was stirred at 0 °C until the starting material disappeared on TLC (4 h). After completion, it was diluted with CH2Cl2 (20 mL) and washed with 0.5 N HCl (20 mL) and brine (10 mL). The separated organic layer was dehydrated with MgSO4 and concentrated under vacuum. The crude residue was mixed with Bu4NOAc (927 mg, 3.07 mmol, 3 equivalences) and dissolved in toluene (20 mL). The solvent was removed under vacuum, and the residue was co-evaporated twice with toluene; then dissolved in anhydrous toluene (13 mL), and the mixture was ultrasonicated for 8 hours. During this period, partial deprotection of NHTroc was observed, and the reaction mixture was concentrated under high vacuum. The resulting residue was dissolved in CH2Cl2 (13 mL) and treated with NaHCO3 (430 mg, 5.12 mmol, 5 equivalences) and 2,2,2-trichloroethyl chloroformate (0.71 mL, 5.12 mmol, 5 equivalences) at 0 °C under argon. After stirring for 3 hours, the reaction mixture was diluted with CH2Cl2 (20 mL) and washed with water (20 mL) and brine (10 mL). The separated organic layer was dehydrated with MgSO4 and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using EtOAc / toluene (1:5) as the precipitate to give compound S19 as a white powder (650 mg, 61%).

[0152] From S21: A solution of disaccharide S21 (1.20 g, 1.21 mmol, 1 equivalent) in ethylenediamine / n-BuOH (15 mL, 2:8 = v / v) was stirred at 90 °C for 2 h. After removing the solvent, the crude product was co-evaporated twice with toluene. The resulting residue was dissolved in CH2Cl2 (30 mL) and treated at 0 °C under argon with NaHCO3 (508 mg, 6.05 mmol, 5 equivalent) and 2,2,2-trichloroethyl chloroformate (0.83 mL, 6.05 mmol, 5 equivalent). After 3 hours, the reaction mixture was diluted with CH2Cl2 (45 mL) and washed with water (30 mL) and brine (15 mL). The organic layer was dehydrated with MgSO4, concentrated, co-evaporated twice with toluene, and then evaporated under vacuum. The resulting residue was subjected directly to acetylation conditions in Ac₂O (7 mL) in pyridine (14 mL) under conditions of slow temperature increase from 0 °C to room temperature over 16 hours. The reaction mixture was concentrated under high vacuum and purified by silica gel column chromatography using acetone / toluene (1:5) as the precipitate to give compound S19 as a white powder (1.04 g, 83%). f =0.54 (Silicone, EtOAc:Toluene = 1:3); 1 H NMR (600MHz, CDCl3): δ7.48-7.46(m,2H,Ar-H),7.39-7.35(m,3H,Ar-H),7.33-7.25(m,14H,Ar-H),7.24-7.21(m,3H,Ar-H),6.84-6.82(m, 2H,Ar-H),5.50(s,1H,Ph-CH),5.41(d,J=3.0Hz,1H),5.09(br,1H),4.92(d,J=10.8Hz,1H),4.88(d,J=12.0Hz,1H),4.72-4.62(m,5H,2C1-H β ),4.58-4.56(m,3H),4.47-4.44(m,2H),4.08(dd,J=10.2,4.8Hz,1H),4.04(dd,J=9.0,8.4Hz,1H),3.87-3.84(m,2H),3.78-3.72(m,5H,-CH3 ),3.59(dd,J=10.2,10.2Hz,1H),3.45-3.43(m,2H),3.38(ddd,J=8.4,8.4,8.4Hz,1H),3.10(ddd,J=9.6,9.6,4.8Hz,1H),2.07(s,3H,-CH3); 13C NMR (150MHz, CDCl3): δ170.3,159.3,153.8,138.4,137.8,137.4,137.1, 129.7,129.2,128.9,128.5,128.4,128.3,128.2,128.0,128.0,127.9,1 27.8,127.7,126.1,113.8,101.4,99.1,99.1,77.8,75.3,74.4,73.5,71 .4,70.8,69.1,68.5,68.4,67.0,57.5,55.2,21.0; HRMS(ESI-TOF)m / e:C 53 H 56 Cl3NO 14 Na[M+Na] + Calculated value: 1058.2658, measured value: 1058.2657.

[0153]

[0154] Benzyl[2-O-acetyl-4,6-O-benzyl-β-D-mannopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate-β-D-glucopyranoside 13. At 0 °C, 2,3-dichloro-5,6-dicyanobenzoquinone (52.5 mg, 0.23 mmol, 2.2 equivalences) was added to a stirred solution of S19 (120 mg, 0.12 mmol, 1 equivalence) in a CH2Cl2 / phosphate buffer (pH=7) mixture (3 mL, 9:1 = v / v). The reaction mixture was stirred vigorously until TLC showed the disappearance of the starting material (3 h). After completion, the reaction mixture was diluted with CH₂Cl₂ (10 mL), washed with saturated NaHCO₃ aqueous solution (8 mL) and brine (4 mL), and then the organic phase was dehydrated with MgSO₄, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using EtOAc / toluene (1:4) as the precipitate to give compound 13 as a white powder (90 mg, 85%). f =0.29 (Silicone, EtOAc:Toluene = 1:3); 1H NMR (600MHz, CDCl3): δ7.45-7.44(m,2H,Ar-H),7.39-7.34(m,7H,Ar-H),7.33-7.26(m,11H,Ar-H),5.47(s,1H,Ph-CH),5.23( d,J=3.6Hz,1H),5.06(br,1H),4.91(d,J=11.4Hz,1H),4.87(d,J=12.0Hz,1H),4.74(d,J=12.0Hz,1H),4.68-4.61(m,4H,2C1-H β ),4.58-4.56(m,2H),4.48(d,J=12.0Hz,1H),4.08(dd,J=10.2,4.8Hz,1H),4.04 (dd,J=9.0,9.0Hz,1H),3.83(br,1H),3.78(dd,J=11.4,3.0Hz,1H),3.73-3.70(m ,2H),3.64(dd,J=9.6,3.6Hz,1H),3.56(dd,J=10.2,10.2Hz,1H),3.43-3.36(m, 2H),3.11(ddd,J=9.6,9.6,4.8Hz,1H),2.21(d,J=3.6Hz,1H),2.11(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.5,153.8,138.5,137.8,137.1,137.0,129.3,128.6,128.6,128.4,128.4,128.3,128.1,128.0,128.0,127. 9,127.8,127.7,126.2,102.1,99.1,99.0,78.5,78.2,74.4,74.4,73.6,71.2,70.8,69.7,68.4,68.4,66.7,57.5; HRMS(ESI-TOF)m / e:C 45 H 49 Cl3NO 13 [M+H] + Calculated value: 916.2264, measured value: 916.2252.

[0155]

[0156] Benzyl[4,6-O-benzylidene-3-Op-methoxy-benzyl-β-D-glucopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside S20. Hydrazine acetate (327 mg, 3.55 mmol, 1.5 equivalence) was added to a stirred solution of starting material S17 (2.48 g, 2.37 mmol, 1 equivalence) in anhydrous THF (55 mL) at room temperature under argon atmosphere. The reaction mixture was vigorously stirred until TLC showed the disappearance of the starting material (16 h). After completion, the reaction mixture was diluted with EtOAc (150 mL), washed with water (60 mL) and brine (30 mL). The organic phase was dehydrated by MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography using EtOAc / hexane (1:2) as the precipitate to give compound S20 as a white foam (2.07 g, 92%). f =0.54 (silica gel, EtOAc:hexane = 1:1); 1 H NMR (600MHz, CDCl3): δ7.79-7.54(m,4H,Ar-H),7.51-7.44(m,2H,Ar-H),7.38-7.33(m,7H,Ar-H),7.30-7.27(m,3H,Ar-H),7.08-7.04( m,1H,Ar-H),7.02-7.01(m,4H,Ar-H),6.98-6.97(m,2H,Ar-H),6.89-6.81(m,5H,Ar-H),5.46(s,1H,Ph-CH),5.09(d,J=8.4Hz,1H,C1-H β ),4.85(d,J=11.4Hz,1H),4.77(d,J=12.6Hz,1H),4.75(d,J=4.2Hz,1H),4.73(d,J=4.2Hz,1H),4.68(d,J=11.4Hz,1H),4.62(d,J=7.2Hz,1H,C1-H β),4.56(d,J=12.0Hz,1H),4.46(d,J=12.0Hz,1H),4.38(d,J=12.0Hz,1H),4.32(dd,J=10.8 ,8.4Hz,1H),4.21(d,J=10.8,8.4Hz,1H),4.15-4.10(m,2H),4.04(dd,J=11.4,3.0Hz,1H),3 .82(dd,J=11.4,1.8Hz,1H),3.78(s,3H,-CH3),3.61(ddd,J=9.6,3.0,3.0Hz,1H),3.56(dd, J=3.0,3.0Hz,1H),3.52-3.45(m,3H),3.18(ddd,J=9.6,9.6,4.8Hz,1H),2.98(br,1H,-OH); 13 C NMR (150MHz, CDCl3): δ167.9,159.3,138.4,137.8,137.3,137.2,133.6,131.6 ,130.5,129.7,128.9,128.4,128.2,128.1,128.0,127.9,127.8,127.5,127.4, 127.1,126.0,123.2,113.8,103.4,101.1,97.5,81.3,79.5,78.8,77.8,75.0,7 4.7,74.5,74.2,73.6,70.8,68.6,68.2,66.2,55.8,55.3; HRMS(ESI-TOF)m / e:C 56 H 55 NO 13 Na[M+Na] + Calculated value: 972.3565, measured value: 972.3567.

[0157]

[0158] Benzyl[2-O-acetyl-4,6-O-benzylidene-3-Op-methoxy-benzyl-β-D-mannopyranoside]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside S21. Anhydrous pyridine (0.56 mL, 6.90 mmol, 7 equivalences) was added to a stirred solution of disaccharide S20 (936 mg, 0.985 mmol, 1 equivalence) in anhydrous CH2Cl2 (12 mL), followed by dropwise addition of trifluoromethanesulfonic anhydride (0.29 mL, 1.72 mmol, 1.75 equivalences) at 0 °C under argon atmosphere, and the mixture was stirred at 0 °C until the starting material disappeared on TLC (2 h). After completion, the reaction mixture was diluted with CH₂Cl₂ (20 mL) and washed with 0.5 N HCl (20 mL) and brine (10 mL). The separated organic layer was dehydrated with MgSO₄ and concentrated under vacuum, with the residue used as is for further reactions. The residue obtained above was added to Bu₄NOAc (594 mg, 1.97 mmol, 2 equivalences) and dissolved in toluene (18 mL). The solvent was removed under vacuum, and the residue was co-evaporated twice with toluene to remove water. The residue was redissolved in anhydrous toluene (12 mL), and the mixture was ultrasonicated for 8 hours. After completion, the reaction mixture was diluted with EtOAc (20 mL) and washed with water (20 mL) and brine (10 mL). The separated organic layer was dehydrated with MgSO₄ and concentrated. The residue obtained was purified by silica gel column chromatography using EtOAc / hexane (1:2) as the precipitate to give compound S21 as a white powder (913 mg, 93%). R f =0.29 (silica gel, EtOAc:hexane = 1:2); 1 H NMR (600MHz, CDCl3): δ7.76-7.49(m,4H,Ar-H),7.48-7.46(m,2H,Ar-H),7.39-7.31(m,7H,Ar-H),7.26-7.22(m,3H,Ar-H),7.10-7.06(m,1H,Ar-H ),7.04-7.03(m,4H,Ar-H),6.99-6.98(m,2H,Ar-H),6.90-6.84(m,5H,Ar-H),5.49(s,1H,Ph-CH),5.44(d,J=3.0Hz,1H),5.09(d,J=7.8Hz,1H,C1-H β ),4.82-4.76(m,3H),4.68(s,1H,C1-H β),4.58(d,J=12.0Hz,1H),4.49-4.44(m,3H),4.38(d,J=12.6Hz,1H),4.26-4.19(m,2H),4.17-4.11(m,2H),3.86-3.82(m,2H),3.78(s ,3H,-CH3),3.78-3.76(m,1H),3.57-3.54(m,2H),3.44(dd,J=10.2,3.6Hz,1H),3.13(ddd,J=9.6,9.6,4.8Hz,1H),2.15(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.2,167.8,159.2,138.5,137.8,137.4,137.1,133.6,131 .5,129.7,129.2,128.9,128.5,128.1,128.1,127.9,127.9,127.8,127.7,127.6,12 7.5,127.1,126.0,123.1,113.8,101.4,99.5,97.3,79.1,77.7,76.9,75.4,74.5,74 .3,73.5,71.3,70.7,69.1,68.4,68.3,66.9,55.6,55.2,21.0; HRMS(ESI-TOF)m / e:C 58 H 57 NO 14 Na[M+Na] + Calculated value: 1014.3671, measured value: 1014.3699.

[0159]

[0160] Process S5

[0161] Reagents and conditions in process S5: (a) Br2, CH2Cl2, 0℃, 10 min, then p-nitrophenol, Ag2O, CH3CN, 1 h, 73%. (b) NaBrO3, Na2S2O4, H2O, EtOAc, rt, 1.5 h, 93%. (c) LiOH·H2O, MeOH, rt, 16 h, 84%. (d) Ac2O, pyridine, rt, 16 h, 90%. (e) Br2, CH2Cl2, 0℃, 10 min, then p-nitrophenol, Ag2O, CH3CN, 1 h, 74%. (f) NaBrO3, Na2S2O4, H2O, EtOAc, rt, 1.5 h, 97%. (g) LiOH·H2O, MeOH, rt, 16 h, 94%.

[0162]

[0163] p-Nitrophenyl [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-4-O-benzyl-β-D-galactopyranoside S22. Bromine (6.02 μL, 0.12 mmol, 1.1 equivalent) was added to a solution of thioglycoside 4 (115 mg, 0.11 mmol, 1 equivalent) in anhydrous CH2Cl2 (2 mL) at 0 °C. After vigorously stirring the reaction mixture for 10 min, the solvent was removed under vacuum, and the residue was co-evaporated twice with toluene to remove water. The residue thus obtained was dissolved in anhydrous CH3CN (2 mL) and treated with 4-nitrophenol (25.3 mg, 0.18 mmol, 1.7 equivalence) and Ag2O (124 mg, 0.53 mmol, 5 equivalence). The reaction mixture was stirred vigorously in the dark under N2 until TLC showed the disappearance of the starting material (1 h). After completion, the reaction mixture was diluted with EtOAc (10 mL) and filtered through a diatomaceous earth pad. The filtrate was washed twice with a saturated aqueous solution of NaHCO3 (4 mL) and brine (3 mL). The organic phase was dehydrated by MgSO4, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the precipitate to give compound S22 as a white powder (85 mg, 73%). f =0.46 (silicone, acetone: toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ8.13-8.11(m,2H,Ar-H),7.96-7.94(m,2H,Ar-H),7.91-7.89(m,2H,Ar-H),7.49-7.44(m,2H,Ar-H),7.35-7.30(m,6H,Ar-H ),7.24-7.22(m,2H,Ar-H),7.20-7.18(m,2H,Ar-H),7.13-7.11(m,1H,Ar-H),6.09(dd,J=10.2,7.8Hz,1H),5.65-5.59(dd,J=10.2,3.0Hz,1H,C1-H β),5.35(d,J=9.6Hz,1H),5.28(dd,J=9.6,1.8Hz,1H),5.19(dd,J=27.0,10.8Hz,1H,sia-C4-H),5.04(dd,J =51.0,1.8Hz,1H,sia-C3-H),4.72-4.67(m,2H),4.44(dd,J=12.6,3.6Hz,1H),4.40(d,J=3.0Hz,1H),4.37( dd,J=8.4,6.6Hz,1H),4.31(d,J=10.8Hz,1H),4.18(ddd,J=4.2,4.2,4.2Hz,1H),4.08(dd,J=12.6,6.6Hz, 1H),3.89-3.80(m,5H,-CH3),2.26(s,6H,-2CH3),2.10(s,3H,-CH3),1.93(s,3H,-CH3),1.92(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.1,170.8,170.4,170.3,170.2,165.5,165.5,165.4,165.3, 161.8,142.6,138.0,133.2,133.1,129.8,129.7,129.4,129.0,128.4,128.3,128.2,12 8.1,128.0,127.5,125.6,116.8,98.8,98.7,98.2,88.1,86.8,75.0,73.9,73.7,73.2,71.6,69.7,69.1,69.0,67.3,67.2,63.4,63.3,53.4,45.1,23.3,21.2,20.8,20.7,20.6; 19 F NMR (376MHz, CDCl3): δ-215.5; HRMS (ESI-TOF) m / e: C 53 H 55 FN2O 22 Na[M+Na] + Calculated value: 1113.3123, measured value: 1113.3131.

[0164]

[0165] p-Nitrophenyl [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythrose-α-L-mannose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-β-D-galactopyranoside S23. To a solution of compound S22 (63.0 mg, 0.058 mmol, 1 equivalence) in EtOAc (0.8 mL), NaBrO3 (85%, 39.2 mg, 0.26 mmol, 4.5 equivalence) dissolved in H2O (0.6 mL) was added, followed by the slow addition of Na2S2O4 (40.2 mg, 0.23 mmol, 4 equivalence) dissolved in H2O (1.2 mL) at room temperature. The reaction mixture was stirred until TLC showed the disappearance of the starting material (1.5 h). After the reaction was complete, the reaction mixture was diluted with EtOAc (10 mL) and washed with 20% Na₂S₂O₃ aqueous solution (5 mL) and brine (3 mL). The organic phase was dehydrated with MgSO₄, filtered, and concentrated under vacuum. The resulting residue was then purified by silica gel column chromatography using acetone / toluene (3:5) as the precipitate to give compound S23 as a white powder (54 mg, 93%). f =0.40 (silicone, acetone: toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ8.13-8.12(m,2H,Ar-H),7.99-7.98(m,2H,Ar-H),7.95-7.94(m,2H,Ar-H),7.50-7.46(m,2H,Ar-H),7.37-7 .32(m,4H,Ar-H),7.19-7.18(m,2H,Ar-H),6.05(dd,J=10.2,9.8Hz,1H),5.53(ddd,J=9.0,6.0,3.0Hz,1H),5.49-5.45(m,2H,C1-H β),5.36(d,J=9.0Hz,1H),5.27(dd,J=9.0,1.8Hz,1H),5.19(dd,J=27.0,11.4Hz,1H,sia-C4-H),5.05(dd,J=51.6 ,1.8Hz,1H,sia-C3-H),4.47(d,J=3.0Hz,1H),4.37(dd,J=12.6,3.0Hz,1H),4.26(dd,J=10.8,1.2Hz,1H),4.18( dd,J=6.6,6.6Hz,1H),4.13-4.07(m,1H),4.05(dd,J=12.6,6.6Hz,1H),3.99-3.98(m,2H),3.85(s,3H,-CH3),2. 99(br,1H,-OH),2.17(s,3H,-CH3),2.08(s,3H,-CH3),2.07(s,3H,-CH3),1.93(s,3H,-CH3),1.88(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.9,170.8,170.5,170.3,165.6,165.6,165.3,161.8 ,142.9,133.4,133.3,129.8,129.7,129.2,129.1,129.0,128.4,128.4,128.2, 125.6,125.3,117.1,99.0,98.1,98.0,88.3,87.0,73.9,73.3,71.6,69.2,69.1,69.0,67.9,67.2,66.5,63.6,62.9,53.4,45.1,23.3,21.1,20.7,20.6,20.6; 19 F NMR (376MHz, CDCl3): δ-216.0; HRMS (ESI-TOF) m / e: C 46 H 50 FN2O 22 [M+H] + Calculated value: 1001.2834, measured value: 1001.2833.

[0166]

[0167] 2. A solution of compound S23 (53 mg, 0.053 mmol, 1 equivalent) in methanol (5 mL) was reacted with LiOH·H2O (22.2 mg, 0.53 mmol, 10 equivalent) dissolved in water (1 mL). After stirring at room temperature for 16 hours, the reaction mixture was neutralized with an IR-120 filter, filtered, and concentrated under vacuum. The resulting residue was purified by (BIO-RAD) Biogel P-2 column chromatography (precipitation with water) to give compound 2 (27 mg, 84%). 1 HNMR (600MHz, D2O): δ8.29-8.28(m,2H,Ar-H),7.30-7.29(m,2H,Ar-H),5.21(dd,J=51.6,2.4Hz,1H,sia-C3-H),5.18(d,J=7.8Hz,C1-H β ),4.17(dd,J=9.0,9.0Hz,1H),4.07-4.00(m,3H),3.91-3.77(m,7H),3.63(dd,J=12.0,6.6Hz,1H),3.57(dd,J=9.0,1.8Hz,1H),2.04(s,3H,-CH3); 13 C NMR (150MHz, D2O): δ175.0,170.8,161.9,142.5,126.1,116.5,100.0,98.9,98.9,91.6, 90.3,73.9,72.4,72.4,71.6,70.3,69.7,69.5,68.4,68.1,63.7,62.6,46.8,46.8,22.0; 19 F NMR (376MHz, CDCl3): δ-217.6; HRMS (ESI-TOF) m / e: C 23 H 32 FN2O 16 [M+H] + Calculated value: 611.1730, measured value: 611.1732.

[0168]

[0169] p-Tolyl[5-acetamido-3,4,7,8,9-penta-O-acetyl-5-deoxy-D-erythrose-α-L-glucose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-4-O-benzyl-1-thio-β-D-galactopyranoside S24. Ac2O (1 mL) was added to a solution of starting material 6 (174 mg, 0.16 mmol, 1 equivalent) in pyridine (2 mL). The reaction mixture was stirred vigorously at room temperature for 16 h, and then concentrated under high vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the precipitate to give compound S24 as a white powder (162 mg, 90%). f =0.49 (silicone, acetone: toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ7.94-7.92(m,2H,Ar-H),7.88-7.87(m,2H,Ar-H),7.48-7.44(m ,2H,Ar-H),7.40-7.38(m,2H,Ar-H),7.35-7.27(m,6H,Ar-H),7.23-7.20(m,2H,Ar-H), 7.18-7.16(m,1H,Ar-H),7.05-7.03(m,2H,Ar-H),5.80(dd,J=10.2.9.6Hz,1H),5.44-5 .42(m,2H),5.35-5.29(m,3H),5.25(dd,J=8.4,1.8Hz,1H),4.95(d,J=10.2Hz,1H,C1-H β ),4.67-4.61(m,3H),4.33(ddd,J=10.2,10.2,10.2Hz,1H),4.25-4.22(m,2H),4.03-3.97(m,3H),3.92-3.89(m,1H),3.75(s,3H,-CH3) ,2.29(s,3H,-CH3),2.15(s,3H,-CH3),2.07(s,3H,-CH3),2.00(s,3H,-CH3),1.95(s,3H,-CH3),1.94(s,3H,-CH3),1.89(s,3H,-CH3); 13C NMR (150MHz, CDCl3): δ171.0,170.5,170.3,170.0,169.3,168.5,168.0,165.7,165.2,13 8.4,137.6,133.1,132.9,132.5,129.8,129.7,129.7,129.5,129.2,129.1,128.3,128.2, 128.0,127.4,127.2,98.9,86.5,76.9,75.5,74.6,74.3,72.7,71.6,71.5,68.5,68.3,66 .9,62.6,62.5,52.7,48.4,23.0,21.1,20.8,20.7,20.6,20.6,20.5; HRMS(ESI-TOF)m / e:C 56 H 61 NO 21 SNa[M+Na] + Calculated value: 1138.3349, measured value: 1138.3358.

[0170]

[0171] p-Nitrophenyl [5-acetamido-3,4,7,8,9-penta-O-acetyl-5-deoxy-D-erythrose-α-L-glucose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-4-O-benzyl-β-D-galactopyranoside S25. Bromine (6.67 μL, 0.13 mmol, 1.1 equivalent) was added to a solution of thioglycoside S24 (132 mg, 0.12 mmol, 1 equivalent) stirred at 0 °C in anhydrous CH2Cl2 (2.5 mL). After vigorous stirring for 10 min, the solvent was removed under vacuum, and the residue was co-evaporated twice with toluene to remove water. The resulting residue was dissolved in anhydrous CH3CN (2.5 mL) and treated with 4-nitrophenol (28.0 mg, 0.20 mmol, 1.7 equivalence) and Ag2O (137 mg, 0.59 mmol, 5 equivalence). The reaction mixture was stirred vigorously in the dark under N2 until TLC showed the disappearance of the starting material (1 h). After the reaction was complete, the reaction mixture was diluted with EtOAc (12 mL) and filtered through a diatomaceous earth mat. The filtrate was washed twice with saturated NaHCO3 aqueous solution (5 mL) and brine (3 mL). The organic phase was dehydrated by MgSO4, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (3:5) as the precipitate to give compound S25 as a white powder (99 mg, 74%). f =0.51 (silicone, acetone: toluene = 2:3); 1H NMR (600MHz, CDCl3): δ8.15-8.13(m,2H,Ar-H),7.95-7.92(m,4H,Ar-H),7.50-7.44(m,2H,Ar-H),7.36-7.31(m,6H,Ar-H),7.24- 7.19(m,2H,Ar-H),7.17-7.15(m,3H,Ar-H),6.10(dd,J=10.2,7.8Hz,1H),5.54(dd,J=10.2,3.0Hz,1H),5.52(d,J=7.8Hz,1H,C1-H β ),5.48(ddd,J=10.2,7.2,3.0Hz,1H),5.39(d,J=10.2Hz,1H),5.31-5.29(m,2H),5.23(dd,J=9.6 ,1.8Hz,1H),4.73(dd,J=10.8,2.4Hz,1H),4.71-4.65(m,2H),4.32-4.30(m,3H),4.19(dd,J=7.2, 7.2Hz,1H),3.97(dd,J=12.6,7.2Hz,1H),3.88-3.86(m,2H),3.83(s,3H,-CH3),2.24(s,3H,-CH3 ),2.12(s,3H,-CH3),2.01(s,3H,-CH3),1.98(s,3H,-CH3),1.90(s,3H,-CH3),1.88(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.0,170.7(2C),170,0,169.6,168.5,168.3,165.7,165.3,1 61.7,142.7,137.9,133.3,133.2,129.9,129.7,129.3,128.9,128.4,128.3,128.2,12 8.0,127.6,125.7,116.8,99.7,98.4,75.1,74.1,74.0,73.9,72.8,71.6,71.1,69.7, 67.6,67.0,63.8,63.3,52.9,48.4,23.0,20.9,20.8,20.7,20.6; HRMS(ESI-TOF)m / e:C 55 H 59 N2O 24 [M+H] + Calculated value: 1131.3452, measured value: 1131.3440.

[0172]

[0173] p-Nitrophenyl [5-acetamido-3,4,7,8,9-penta-O-acetyl-5-deoxy-D-erythrose-α-L-glucose-non-2-ketosepyranoside methyl ester]-(2→6)-2,3-di-O-benzyl-β-D-galactopyranoside S26. At room temperature, a solution of compound S25 (91.0 mg, 0.080 mmol, 1 equivalent) in EtOAc (1.2 mL) was added to a solution of NaBrO3 (54.6 mg, 0.36 mmol, 4.5 equivalents) dissolved in H2O (0.9 mL), followed by the slow addition of Na2S2O4 (85%, 56.0 mg, 0.32 mmol, 4 equivalents) dissolved in H2O (1.8 mL). The reaction mixture was stirred until TLC showed the disappearance of the starting material (1.5 h). After completion, the reaction mixture was diluted with EtOAc (15 mL) and washed with 20% Na₂S₂O₃ aqueous solution (7 mL) and brine (4 mL). The organic phase was dehydrated with MgSO₄, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (3:5) as the precipitate to give compound S26 as a white powder (81 mg, 97%). f =0.23 (silicone, acetone: toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ8.18-8.16(m,2H,Ar-H),8.00-7.99(m,2H,Ar-H),7.94-7.93(m,2H,Ar-H),7.51-7.46(m, 2H,Ar-H),7.38-7.32(m,4H,Ar-H),7.15-7.13(m,2H,Ar-H),6.07(dd,J=10.2,7.8Hz,1H),5.46-5.34(m,5H,C1-H β),5.30(d,J=10.2Hz,1H),5.22(dd,J=7.8,1.8Hz,1H),4.60(dd,J=10.8,1.8Hz,1H),4.43(d,J=3.0Hz,1H),4.3 3(dd,J=12.6,2.4Hz,1H),4.26(ddd,J=10.2,10.2,10.2Hz,1H),4.12(dd,J=6.6,6.6Hz,1H),4.05(dd,J=10.2,6 .6Hz,1H),3.99(dd,J=10.2,6.6Hz,1H),3.93(dd,J=12.6,7.2Hz,1H),3.83(s,3H,-CH3),3.17(br,1H,-OH),2. 14(s,3H,-CH3),2.05(s,3H,-CH3),2.02(s,3H,-CH3),2.00(s,3H,-CH3),1.87(s,3H,-CH3),1.85(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.1,171.0,170.4,170.0,169.9,168.8,167.9,165. 8,165.2,161.7,142.9,133.4,133.3,129.9,129.7,129.3,129.1,128.4,128. 4,125.8,116.9,99.1,98.9,74.0,73.9,73.0,71.1,70.6,69.2,68.7,67.2,66 .5,63.0,62.5,53.0,48.5,23.0,20.8,20.7,20.6,20.6; HRMS(ESI-TOF)m / e:C 48 H 53 N2O 24 [M+H] + Calculated value: 1041.2983, measured value: 1041.2976.

[0174]

[0175] p-Nitrophenyl [5-acetamido-5-deoxy-D-erythrose-α-L-glucose-non-2-ketosepyranose ester]-(2→6)-β-D-galactopyranoside S27. A solution of compound S26 (75 mg, 0.072 mmol, 1 equivalent) in methanol (5 mL) was added to a solution of LiOH·H2O (30 mg, 0.72 mmol, 10 equivalent) dissolved in water (1 mL). After stirring at room temperature for 16 hours, the reaction mixture was neutralized with an IR-120 filter, filtered, and concentrated. The resulting residue was purified by (BIO-RAD) Biogel P-2 column chromatography using water as the precipitate to give compound S27 (41 mg, 94%). 1 H NMR (600MHz, D2O): δ8.29-8.28(m,2H,Ar-H),7.28-7.27(m,2H,Ar-H),5.20(d,J=7.2Hz,C1-H β ),4.09-4.06(m,2H),4.01(dd,J=10.8,7.8Hz,1H),3.92-3.79(m,6H),3.71(d,J=10.8Hz ,1H),3.64-3.60(m,2H),3.53(d,J=9.6Hz,1H),3.49(d,J=9.6Hz,1H),2.02(s,3H,-CH3); 13 C NMR (150MHz, D2O): δ174.8,173.0,161.9,142.5,126.1,116.4,99.9,98.1,76.1,73.9, 73.5,72.4,72.2,71.5,70.3,68.4,68.0,63.0,62.6,50.7,21.9; HRMS(ESI-TOF)m / e:C 23 H 33 N2O 17 [M+H] + Calculated value: 609.1774, measured value: 609.1774.

[0176]

[0177] p-Nitrophenyl [5-acetamido-5-deoxy-D-glycero-α-D-galactosyl-non-2-ketosepyranose ester]-(2→6)-β-D-galactopyranoside 1. Neu5Ac-α2,6-Gal-pNP: Neu5Ac-α2,6-Gal-pNP was synthesized by mixing pNP-β-Gal (1.0 mmol), sialic acid (1.2 mmol), cytidine triphosphate (1.2 mmol), CMP-sialic acid synthase (CSS, 12 U), pyrophosphatase (PPA, 1 U), and α-2,6-sialyl transferase (SiaT, 15 U) in 15 mL Tris buffer (pH 7.0) with 5 mM mgCl2 and 5 mM MnCl2. After removing the protein by heating and centrifugation, the product was purified by (BIO-RAD) Biogel P-2 column chromatography using water as the precipitate. The fraction containing Neu5Ac-α2,6-Gal-pNP was collected and lyophilized to give compound 1 (50%). 1 H NMR (600MHz, D2O): δ8.33-8.32(m,2H,Ar-H),7.31-7.29(m,2H,Ar-H),5.23(d,J=7.8Hz,C1-H β ),4.06-4.04(m,2H),3.99(dd,J=10.2,8.4Hz,1H),3.90-3.86(m,3H),3.83(dd,J=10.2,3.6Hz,1H),3.80-3.76(m,1H),3.73-3.69 (m,3H),3.65-3.62(m,2H),3.57(d,J=8.4Hz,1H),2.79(dd,J=12.6,4.8Hz,1H),2.04(s,3H,-CH3),1.68(dd,J=12.6,12.0Hz,1H); 13 C NMR (150MHz, D2O): δ174.6,173.1,161.4,142.1,125.7,116.0,99.8,99.4,73.6,72.1, 71.9,71.3,69.8,68.0,67.7,67.7,62.5,62.2,51.4,39.8,21.6; HRMS(ESI-TOF)m / e:C 23 H 33 N2O 16 [M+H] + Calculated value: 593.1825, measured value: 593.1825.

[0178] Example 2: Analysis of the stability against sialidase-catalyzed hydrolysis and the inhibitory effect of sialidase. 31

[0179] Material:

[0180] β-galactosidase from Aspergillus oryzae (G5160) and sialidase from Vibrio cholerae (11080725001) and Clostridium perfringens (11585886001) were obtained from Sigma Aldrich.

[0181] Enzyme assay for sialidase:

[0182] This assay was performed repeatedly in a 96-well plate at 37°C, with a final volume of 50 μL containing the matrix (0-20 mM) and β-galactosidase (100 mU). The assay conditions for the two sialidases were as follows: *Clostridium perfringens* (1 mU), sodium acetate buffer (50 mM) pH 5.0, and CaCl2 (10 mM); *Vibrio cholerae* (2 mU), sodium acetate buffer (50 mM) pH 5.5, CaCl2 (10 mM), and NaCl (150 mM). The *Clostridium perfringens* reaction was carried out for 40 minutes to 2 hours, while the *Vibrio cholerae* reaction was carried out overnight. The assay was stopped by adding 65 μL of CAPS buffer (N-cyclohexyl-3-aminopropanesulfonic acid (0.5 M, pH 10.5)). The amount of p-nitrophenolate formed was measured using a microdisk reader to determine the amount of A in the reaction mixture. 405nm To determine this, three compounds (Neu5Ac-α2,6-GalβpNP, 3F) were used. ax -Neu5Ac-α2,6-GalβpNP and 3OH eq -Neu5Ac-α2,6-GalβpNP) was used as the enzyme substrate for testing. In the absence of sialidase, after incubation at 37°C for 1 hour, all three compounds showed A at 20 mM. 405nm Background absorbance. Three compounds, Neu5Ac-α2,6-GalβpNP, 3F... ax -Neu5Ac-α2,6-GalβpNP and 3OH eq The absorbances of -Neu5Ac-α2,6-GalβpNP were 0.044, 0.129, and 0.072, respectively. The standard curve for pNP was determined as follows: 0.35 mM pNP was continuously diluted twice, and then A... 405nm A standard curve for pNP is obtained by plotting the concentration of pNP.

[0183] Assay for the inhibition of sialidase:

[0184] This assay was performed at 37°C in 96-well plates with a final volume of 50 μL, containing the matrix Neu5Ac-α2,6-GalβpNP (0.6 mM), β-galactosidase (100 mU), and sialidase, repeated with or without different concentrations (0-20 mM) of inhibitors. *Clostridium perfringens* reactions were allowed to proceed for 40 minutes to 2 hours, while *Vibrio cholerae* reactions were allowed to proceed overnight. The assay was stopped by adding CAPS buffer (65 μL, 0.5 M, pH 10.5). The amount of p-nitrophenolate formed was measured using a microdisk reader to determine the amount of A in the reaction mixture. 405nm To determine ( Figure 1 ).

[0185] Example 3: Preparation of 3F ax The effects of Neu5Ac and Neu5Ac-modified homogeneous mAbs on FcγRIIIa binding were investigated using surface plasma resonance (SPR) analysis.

[0186] Enzyme performance:

[0187] Endo-S, Endo-S2, and the Endo-S2 mutant (D184Q) were expressed in Escherichia coli, as well as fucosidase from Bacteroides fragilis NCTC 9343, and the enzymes were purified using Ni-NTA agarose beads.

[0188] Preparation of single GlcNAc-rituximab:

[0189] As previously stated 32 Rituximab (3.0 mg) was placed in Tris-HCl buffer (50 mM, pH 7.4, 1.5 mL). Roche incubated the mixture at 37°C for 24 hours with Endo-S (120 μg), Endo-S2 (240 μg), and BfFucH (4.5 mg). LC-MS and SDS-PAGE analysis showed complete cleavage of the N-glycans on the heavy chain. The reaction mixture was subjected to affinity chromatography on a protein A-agarose resin column (1 mL; GE Healthcare) pre-equilibrated with Tris-HCl buffer (50 mM, pH 7.4). The column was then washed with Tris-HCl buffer (50 mM, pH 7.4, 20 mL). The bound IgG was released with glycine-HCl (100 mM, pH 3.0, 10 mL), and the precipitate was immediately neutralized with Tris-HCl buffer (1.0 M, pH 8.3). The antibody-containing fractions were combined and concentrated by centrifugation (Amicon Ultra centrifuge filter, Millipore, Billerica, MA) to obtain rituximab mono-GlcNAc (2.4 mg). The product was trypsinized, and the glycopeptides, TKPREEQYNSTYR (m / z = 1391.58), and EEQYNSTYR (m / z = 1873.88) were analyzed by nanospray LC / MS to confirm the glycosylation pattern of the mono-GlcNAc.

[0190] Transglycosylation of rituximab using polysaccharide-oxazoline:

[0191] Glycan oxazoline was added to a mixture of Endo-S2 D184Q and rituximab mono-GlcNAc in 50 mM Tris buffer (pH 7.4). The solution was incubated at 37°C for 30 minutes. The reaction mixture was then purified using a protein-A affinity column followed by a Capto Q anion exchange column (GE Healthcare) to collect the desired product.

[0192] SDS-PAGE assay of glycoengineered Herceptin antibody:

[0193] All SDS-PAGE analyses were performed using... 4–12% Bis-Tris gel electrophoresis (Invitrogen) was performed in MOPS buffer, with 2-mercaptoethanol present in the sample. Figure 2 ).

[0194] MS spectral analysis of glycoengineered mAbs:

[0195] To analyze trypsinized glycopeptides, high-resolution and high-quality nanofluidic LC-MS / MS experiments were performed on an LTQFT Ultra (linear quadrupole ion trap Fourier transform ion cyclotron resonance) mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanofluidic spray ion source (New Objective, Inc.), an Agilent 1100 series binary high-performance liquid chromatography pump (Agilent Technologies, Palo Alto, CA), and a Famos autosampler (LC Packings, San Francisco, CA). Digestion solution (6 μL) was injected at a flow rate of 10 μL / min into a self-packed pre-column (150 μm IDx20 mm, 5 μm, ...). ). In a self-filled reverse-phase C18 nanotube column (75μm IDx 300mm, 5μm, Chromatographic separation was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid in 80% acetonitrile as mobile phase B, at a rate of 300 nL / min. The full scan MS conditions were investigated: mass range m / z 320-2000, resolution 100,000 at m / z 400. The ten strongest ions for MS2 were sequentially separated using LTQ. The electrospray voltage was maintained at 1.8 kV, and the capillary temperature was set at 200 °C. Figure 3 (and Table 3).

[0196] Surface plasma resonance (SPR) analysis

[0197] All SPR experiments were performed using a BIACORE T200 electrophoresis apparatus at 25°C with HBS-EP (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) as the electrophoresis buffer, employing a single-cycle kinetic method. FcγRIIIa was transfected into HEK-293 cells to express the complex-type glycosylated recombinant protein as the analyte. To analyze the binding of rituximab to the FcγRIIIa receptor, anti-human Fab antibody from the Human Fab Capture Kit (GE Healthcare) was immobilized on the control and active channels of the CM5 sensor chip. Rituximab was then captured on the active channel for interaction with serially diluted FcγRIIIa analytes (2.5, 5, 10, 20, 40 nM for 2,6-FluoSCT and 2,6-SCT; 8, 24, 72, 216, 648 nM for commercially available rituximab) at 30 μl / min for 240 seconds, followed by dissociation for 420 seconds. Rituximab data were processed with dual references for background subtraction. The rituximab data were fitted to a 1:1 Langmuir binding model in BiaEvaluation software (GE Healthcare) to obtain the kinetic / affinity constants (Table 4). The analyzed antibody was captured using the Human Fab Capture Kit and detected using a single-cycle kinetic method.

[0198] Table 3: Correction for relative abundance of N-glycans 33

[0199]

[0200] Table 4: Binding affinity of glycoengineered rituximab IgG1 to FcγRIIIa measured by SPR

[0201] sample ka(1 / Ms) kd(1 / s) KD(M) Rmax(RU) multiple Rituximab 2.31E+05 0.07054 3.06E-07 32.33 1-time <![CDATA[2,6-F ax SCT]]> 2.44E+05 0.001996 8.18E-09 71.28 37.4-times 2,6-SCT 2.68E+05 0.002059 7.67E-09 60.64 39.9-times

[0202] Example 4: Relative Reaction Values ​​(RRV) of Compounds 4 and 18

[0203] RRV was measured three times according to the previously reported experimental procedure. 34 The RRV(2053) of disaccharide donor 4 is a comparison with the competing control donor S34. 23 (RRV = 1791) was measured. The RRV (537) of disaccharide donor 18 was compared with that of the competing control donor S. 23 (RRV=286) Take measurements.

[0204]

[0205] Other specific examples

[0206] All features disclosed in this specification can be combined in any way. Each feature disclosed in this specification can be replaced by an alternative feature having the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0207] Furthermore, based on the above description, those skilled in the art can readily determine the basic features of the present invention, and various changes and modifications can be made to adapt the present invention to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other specific examples are also within the scope of the patent application.

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Claims

1. A method for preparing a sugar containing 3-fluorosialic acid, wherein the method comprises: 3-hydroxysialic acid is glycosylated by reacting it with a sugar to form α,6-linked 3-hydroxysialic acid glycoside, wherein... The 3-hydroxysialic acid has the structure of formula (I): , The sugar is a compound of formula (II): , The α,2,6-linked 3-hydroxysialic acid glycoside is a compound of formula (III): ,as well as The glycosylation reaction was carried out in the presence of silver trifluoromethanesulfonate (AgOTf), disodium hydrogen phosphate (Na2HPO4), and toluene. The α,2,6-linked 3-hydroxysialic acid glycoside is fluorinated with perfluoro-1-butanesulfonyl fluoride (NfF) to form a sugar containing 3-fluorosialic acid. The fluorination reaction is carried out in the presence of 1,8-diaceridine-bicyclo[5,4,0]-undecyl-7-ene (DBU), toluene, and tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF). The sugar containing 3-fluorosialic acid is a compound of formula (IV): 。 2. The method of claim 1, wherein the temperature of the fluorination reaction is 25 to 90 degrees Celsius. o C.

3. The method of claim 2, wherein the fluorination reaction is carried out at a temperature of 40°C. o C or 50 o C.

4. The method according to any one of claims 1 to 3, wherein the fluorination reaction is carried out for 2 to 17 days.

5. The method of claim 4, wherein the equivalent of NfF / DBU is 4 / 4, 6 / 6, or 8 / 8.

6. A method for preparing glycosylated monoclonal antibodies, wherein the method comprises glycosylating the monoclonal antibody with a sugar containing 3-fluorosialic acid, wherein the sugar containing 3-fluorosialic acid is prepared by the method of any one of claims 1 to 5.

7. A method for preparing homogeneous antibodies that bind to sugars containing 3-fluorosialic acid, wherein the method comprises glycosylation of the monoclonal antibody with a sugar containing 3-fluorosialic acid. The 3-fluorosialic acid-containing sugar is an N-glycan terminated at α2,6-linked 3-fluoro-sialic acid glycosides, and the N-glycan terminated at α2,6-linked 3-fluorosialic acid glycosides has the structure of formula (V): ， Where Z is or .

8. A dual-antennae N-glycan compound having formula (V): ， Where Z is or .

9. Use of a glycosylated monoclonal antibody in the preparation of a reagent for enhancing binding to FcγRIIIa, wherein the glycosylated monoclonal antibody is prepared by the method of claim 7.

10. The use as described in claim 9, wherein the monoclonal antibody is rituximab.

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