Saccharides against sialidases, methods of manufacture and uses thereof

Abstract

The present application relates to saccharides against sialidases, methods of making and uses thereof. Disclosed herein are methods of making 3-fluoro sialic acid-containing saccharides, and methods of conjugating the same to homogeneous antibodies. Also within the scope of the present application are 3-fluoro sialic acid-containing compounds, monoclonal antibodies conjugated to N-glycans terminated with 3-fluoro sialic acid glycosides linked in an alpha 2,6 configuration, and methods of treating cancer with such monoclonal antibodies.

Description

[0001] This application is a divisional application of the invention patent application filed on March 10, 2020, with application number "202080022413.3" and titled "Sialidase-resistant sugars, their manufacturing method and uses". Technical Field

[0002] This application generally relates to sialidase-resistant sugars, their manufacturing methods, and their uses. Background Technology

[0003] 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.

[0004] 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 .

[0005] 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 .

[0006] 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

[0007] 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.

[0008] 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.

[0009] Another aspect of the present invention relates to compounds of formula (VI):

[0010] ,

[0011] 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 a monosaccharide, disaccharide, or oligosaccharide.

[0012] 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.

[0013] 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 terminally linked to α2,6-linked 3-fluorosialic acid glycoside.

[0014] 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

[0015] The following description refers to the accompanying drawings, in which:

[0016] 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 1B 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);

[0017] Figure 2 The electrophoretic gels are stained with four N-glycans: (1) rituxan-G, (2) rituxan-F. ax -SCT, (3) rituxan-SCT, and (4) commercially available rituxan; and

[0018] Figure 3 Showing three types N- glycans, i.e., rituxan-F ax Mass spectra obtained from -SCT, rituxan-SCT, and rituxan. Detailed Implementation

[0019] This article first details a method for preparing sugars containing 3-fluorosialic acid.

[0020] The method includes the following steps: reacting 3-hydroxysialic acid with a sugar to perform a glycosylation reaction, forming an α,6-linked 3-hydroxysialic acid glycoside; and reacting the α,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.

[0021] 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.

[0022] 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).

[0023] In the exemplary method, 3-hydroxysialic acid has formula (I):

[0024] ,

[0025] Sugars are compounds of formula (II):

[0026] ,

[0027] α-2,6-linked 3-hydroxysialic acid glycoside is a compound of formula (III):

[0028] ,as well as

[0029] Sugars containing 3-fluorosialic acid are compounds of formula (IV):

[0030] .

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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):

[0035] ,

[0036] Where Z is or .

[0037] Another aspect of the present invention relates to compounds of formula (VI):

[0038] ,

[0039] R 1 R 2 R 3 R 4 X is as defined in the above-mentioned invention description section.

[0040] In one specific instance, the compound has the structure of formula (IV):

[0041] .

[0042] 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):

[0043] ,

[0044] Where Z is or .

[0045] 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.

[0046] 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.

[0047] 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 dose" 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 dose 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.

[0048] The following provides a more detailed description of certain specific aspects of the present invention.

[0049] 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?

[0050] To achieve our goal, we developed a preparation method for synthesizing α-2,6-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.

[0051] Several methods for synthesizing 3F-Neu5Ac have been reported, including: (a) protected polysaccharide with 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-linked disaccharide. 11a Therefore, we focused our efforts on the chemical synthesis of this bond. 15 .

[0052]

[0053] Process 1

[0054] 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 use the conditions reported by Goto et al. to investigate... O Sialization at position -6 (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 ratio. α : β Mutarotation isomer ratio. Changing the solvent to dichloromethane did not improve non-mirror selectivity (Table 2). Increasing 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 observed stereoselectivity (Tables 3 vs. 6). Optimized conditions (Table 7) provided 6 with a 35% (99% brsm) yield, exhibiting excellent... α -selectivity( α:β = 13:1).

[0055] Process 2

[0056]

[0057] Process 3

[0058] Table 1. Optimization of glycosylation steps.

[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 CH2Cl2 with 4 Å MS was used. f It has 4 Å 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, in the presence of 1,8-diacabinitro[5,4,0]-undecyl-7-ene (DBU), at 90°C... o At C, anhydrous toluene was treated with perfluoro-1-butanesulfonyl fluoride (NfF) for 6 days (process 3) to obtain 4 in 6% yield (Table 2, Table 1). 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 one 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] Process 4

[0063] Table 2. Optimization of the fluorination process.

[0064]

[0065] a Reagents and conditions: (a) NfF, DBU, toluene, 40 o C, 15 d, 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.

[0066] Refer to process 5, and use NIS / TMSOTf to enable 3F. ax -Neu5Ac- disaccharide donor 4 is coupled with acceptor (8) to obtain 9. Then, the mutarotation isomer position is reduced by PdCl2 isomerization in AcOH / NaOAc. O -Allyl group removal, and further conversion of the mutarotatory isomer hydroxyl group (10) into fluoride (11) and imide ester (12). Using Cp2HfCl2 / AgOTf conditions, with 3F ax -Neu5Ac is the terminal fluoride donor (11) O Glycosylation of the core disaccharide (13) at the -3 position yielded the hexasaccharide 14 in 85% yield. After removal of the benzylidene group, 15 was... O -6 position glycosylation yielded the desired decasaccharide (16) in 70% yield, exhibiting excellent position selectivity and α - Stereoselectivity. We also use N The phenyl trifluoroacetyl imide ester donor (12) was tested for TfOH-promoted glycosylation, but the product yield was low. Subsequently, a fully deprotected polysaccharide (17) was obtained in 40% overall yield following a series of steps: (a) saponification with LiOH to remove ester and NHTroc groups; (b) acetylation of free amines with alcohols; (c) removal of OAc groups with sodium methoxide; and (d) hydrogenolysis with Pd / C in a mixture of MeOH / water / HCO2H. O -Benzyl 4b .

[0067]

[0068] Process 5

[0069] Reagents and conditions in procedure 5: (a) 8, TfOH, NIS, 4Å MS, CH2Cl2, -40 o C, 2 h, 64%. (b) PdCl2, CH3COONa, AcOH / H2O, 82%. (c) DAST, CH2Cl2, -20 o C, 73%. (d) ClC(=NPh)CF3, Cs2CO3, CH2Cl2, 0 o C to rt, 3 h, 56%; (e)11, AgOTf, Cp2HfCl2, toluene, 4 ÅMS, 0 o C, 3 h, 85%; (f) p TSA•H2O, CH3CN, rt, 6 h, 75%; (g)11, AgOTf, Cp2HfCl2, toluene, 4Å MS, -15 o C, 3 h, 70% (80% brsm); (h)12, TfOH, CH2Cl2, 4Å MS, -60 to -20 o C, 3 h, 33% (55% brsm); (i) LiOH, dioxane / H2O (4:1), 90 o 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).

[0070] After establishing the stepwise synthesis experimental protocol, we simplified the glycan assembly process (procedure 6) by developing a programmable [2 + 2 + 2] one-pot synthesis of hexasaccharides (14) (precursors of 17). The designed one-pot experimental protocol includes using 3F ax -Neu5Ac-disaccharide donor (4) (RRV = 2053) with less reactive receptor (18) (RRV = 537) at -40 o Initial coupling was performed at C, followed by -20 o Inject the reduced-terminal receptor 13 at C. At -10 o After 1 hour at C and following the standard purification protocol, hexasaccharide 14 was isolated in 26% yield.

[0071]

[0072] Process 6

[0073] Reagents and conditions in Procedure 6: (a) TfOH, NIS, 4Å MS, CH2Cl2, -40 o C to -10 o C, 3 h, 26%

[0074] In order to collect information about 3F ax -Neu5Ac- α Preliminary stability data of the 2,6-Gal motif in the presence of sialidase were obtained, and Neu5Ac- α 2,6-Gal- p NP(1) and 3F ax -Neu5Ac analogue (2) as a matrix (procedure 1), with Clostridium perfringens ( C. perfingens ) and Vibrio cholerae ( V. cholera In vitro assay of commercially available sialidase. 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 A). Figure 1 B).

[0075] To prepare homogeneous mAb glycoforms, compound 17 was converted into an oxazoline donor and ligated to a GlcNAc-induced IgG (without core fucose) in the presence of Endo S2 (D184Q) according to standard experimental protocols. 6 Homogeneous 3F ax The binding affinity of the -Neu5Ac- glycoform to FcγRIIIa was analyzed using surface plasma resonance. 6 The sample was 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, those with fluorinated glycoforms were measured. α 2,6-Sialotropic and core-fucose-free double tentacles N - The homogeneous IgG glycoform of the polysaccharide showed a 39.9-fold (Neu5Ac-G2S2) and a 37.4-fold (3F) 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.

[0076] In summary, we have developed a 3F ax -Neu5Ac- α The chemical synthesis of 2,6-Gal-STol can be used as a synthetic oligosaccharide for the synthesis of anti-sialidase and representative oligosaccharides with 3F ax -Neu5Ac is the tip of the double antennae. N - Polysaccharides are the building blocks of homogeneous antibodies. 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.

[0077] 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.

[0078] Example 1: Synthesis of sugars and precursors containing 3-fluorosialic acid

[0079] 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. Powdered molecular sieves (EMD Millipore) were ground into powder and activated before use. The progress of all reactions was monitored by TLC using TLC plates pre-coated with silica gel 60 F254 (Merck KGaA). TLC imaging was performed by UV light (254 nm) and staining with anisaldehyde and / or cerium ammonium molybdate. Column chromatography was performed on Across silica gel (particle size 0.035–0.070 mm, 60 Å). 1 H, 13 C and 19 F NMR spectroscopy was performed using Bruker AVIII-600, DRX-500, AV-400, DPX-400, AVANCE 500 AV, and AVANCE 600 spectrometers at 25°C. o Recorded under C, chemical shift is measured using the residual solvent peak as an internal standard in units of δ (ppm) (δ, ppm: in 1 The ¹H NMR values ​​are 7.24 (CHCl₃) and 4.80 (H₂O); 13 The coupling constant is 77 (CDCl3) in C NMR. JMeasurements are taken in Hz. Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiply, br = broad peak). High-resolution mass spectrometry (HRMS) is 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 are conducted on a Cu K... α radiation( = 1.5478) Bruker D8 Platinum- 135 The measurements were performed on 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, 5µm, 4.6 x 250mm (Colloidal Silica, Agilent Technologies) solvent system of EtOAc / hexane at a flow rate of 1 mL / min and imaged at 254 nm.

[0080]

[0081] p-Tolyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-5-deoxy-D-erythrose- α [-L-glucose-non-2-ketopyranoyl 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). The organic layer was then 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%), accompanied by recovered receptor 3 (670 mg, 99% brsm). α-Mutarotation isomer 6α: R f = 0.36 (silica gel, CHCl3:MeOH = 20:1); 1 H NMR (600 MHz, 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. 9.6 Hz, 1H), 5.59 (d, J = 10.2 Hz, 1H), 5.38 (dd, J = 9.0, 3.0 Hz, 1H), 5.35-5.32 (m, 1H), 5.25 (dd, J = 10.2, 9.6Hz, 1H), 5.24 (d, J = 7.8, 2.4 Hz, 1H), 4.92 (d, J = 9.6 Hz, 1H, C1-H β ), 4.71 (d, J = 12.0 Hz, 1H), 4.60 (d, J = 12.0 Hz, 1H), 4.49 (dd, J = 10.8, 1.8 Hz, 1H), 4.30(dd, J = 12.6, 3.0 Hz, 1H), 4.26 (ddd, J = 10.2, 10.2, 10.2 Hz, 1H), 4.20 (d, J =3.0 Hz, 1H), 4.10 (dd, J = 10.2, 7.2 Hz, 1H), 4.04 (dd, J = 12.6, 6.6 Hz, 1H), 4.00 (dd, J = 6.6, 6.6 Hz, 1H), 3.86 (d, J= 9.6 Hz, 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 (150 MHz, CDCl3): δ 171.6, 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, 128.2,127.7, 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.

[0082]

[0083] p-Tolyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-5-deoxy-D-erythrose- β [-L-glucose-non-2-ketopyranoyl methyl ester]-(2→6)-2,3-di- O -benzyl-4- O -benzyl-1-thio- β -D-galactopyranoside 6β,β-mutantomer 6βR f = 0.42 (silica gel, CHCl3:MeOH = 20:1); 1H NMR (600 MHz,CDCl3): δ 7.94-7.92 (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 = 10.2.10.2 Hz, 1H), 5.51 (dd, J = 10.2, 3.0 Hz, 1H), 5.22 (ddd, J = 6.0, 6.0, 3.0 Hz,1H), 5.14 (d, J = 13.2 Hz, 1H), 5.07 (dd, J = 6.0, 2.4 Hz, 1H), 4.90 (d, J = 10.2Hz, 1H, C1-H β ), 4.66-4.62 (m, 2H), 4.55 (dd, J = 12.6, 2.4 Hz, 1H), 4.29 (d, J =1.8 Hz, 1H), 4.14 (dd, J = 7.8, 7.2 Hz, 1H), 4.08 (ddd, J = 10.8, 10.8, 10.8 Hz,1H), 3.98 (dd, J = 12.6, 6.6 Hz, 1H), 3.92-3.88 (m, 2H), 3.85-3.77 (m, 3H),3.83 (s, 3H, -CH3), 3.53 (dd, J = 10.8, 2.4 Hz, 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); 13C NMR (150 MHz, CDCl3): δ 171.4, 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, 127.2, 126.0, 99.6, 86.9, 76.0, 75.5, 74.3, 73.1,72.5, 71.7, 70.3, 68.4, 67.2, 62.1, 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.

[0084]

[0085] p-Tolyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl methyl ester]-(2→6)-2,3-di- O -benzyl-4- O -benzyl-1-thio- β 4. 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) to a solution of compound 6α (500 mg, 0.47 mmol, 1 equivalent) in toluene (5 mL) placed in a screw-capped vial with a stir bar. Seal the vial and incubate at 40 °C. o Stirred at 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. oStir for another 24 hours. Load the reaction mixture directly onto a silica gel column and dissolve with acetone / toluene (7:3). Obtain disaccharide 4 (300 mg, 60%) as a pale yellow powder, accompanied by perfluoro-1-butanesulfonyl compound 7 (50 mg, 8%) as a pale yellow powder.

[0086] From 7, synthesize disaccharides 4: Add DBU (0.64 mL, 4.31 mmol, 8 equivalents) and perfluoro-1-butanesulfonyl fluoride (0.77 mL, 4.31 mmol, 8 equivalents) 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. Seal the container and incubate at 40°C. o Stirred at C for 15 days. The reaction mixture was directly loaded onto a silica gel column and dissolved in acetone / toluene (7:3) to obtain disaccharide 4 (282 mg, 49%) 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); 1 H NMR (600 MHz, 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. 9.6 Hz, 1H), 5.49 (ddd, J = 9.0,5.4, 2.4 Hz, 1H), 5.42 (dd, J = 10.2, 3.0 Hz, 1H), 5.31-5.28 (m, 2H), 5.20 (dd, J = 27.0, 11.4 Hz, 1H, sia-C4-H), 5.01 (dd, J = 51.6, 1.8 Hz, 1H, sia-C3-H),4.97 (d, J= 10.2 Hz, 1H, C1-H β ), 4.66 (d, J = 11.4 Hz, 1H), 4.58 (d, J = 11.4 Hz, 1H), 4.37 (dd, J = 12.6, 2.4 Hz, 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, 6.0 Hz, 1H), 3.74 (dd, J = 10.2,8.4 Hz, 1H), 3.72 (s, 3H, -CH3), 2.29 (s, 3H, -CH3), 2.17 (s, 3H, -CH3), 2.16(s, 3H, -CH3), 2.09 (s, 3H, -CH3), 1.98 (s, 3H, -CH3), 1.91 (s, 3H, -CH3); 13 CNMR (150 MHz, CDCl3): δ 170.9, 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, 128.1, 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 (376 MHz, 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.

[0087]

[0088] 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-ketopyranoyl 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): δ 7.95-7.94 (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. 9.6 Hz, 1H), 5.62-5.38 (m, 4H), 5.26 (d, J = 9.0 Hz, 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, 6.0 Hz, 1H), 4.02 (dd, J = 12.6, 6.0 Hz, 1H), 3.98 (dd, J = 6.6, 6.0Hz, 1H), 3.93 (dd, J = 10.2, 7.2 Hz, 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 (150 MHz, CDCl3): δ 170.6, 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, 128.3, 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 (376 MHz, 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.

[0089]

[0090] Allyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl 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. A mixture of acceptor 8 (1.04 g, 1.03 mmol, 1 equivalence), donor 4 (1.39 g, 1.29 mmol, 1.25 equivalence), and activated pulverized 4 Å MS (0.70 g) in anhydrous CH2Cl2 (7 mL) was stirred under argon for 1 h. The reaction mixture was then cooled to -40 °C, and NIS (465 mg, 2.06 mmol, 2 equivalence) was added with stirring, followed by TfOH (0.5 M in Et2O, 0.62 mL, 0.31 mmol, 0.3 equivalence). After the reaction was complete (TLC showed that the starter disappeared 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 resulting 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 (600 MHz, CDCl3): δ 7.90-7.88 (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, 1.2 Hz, 1H), 5.06-4.97 (m, 3H, C1-H β ), 4.85-4.73 (m, 4H, C1-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), 2.13 (s, 3H, -CH3),2.10 (s, 3H, -CH3), 2.01 (s, 3H, -CH3), 1.92 (s, 3H, -CH3); 13 C NMR (150 MHz,CDCl3): δ 170.7, 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, 133.2, 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, 73.3, 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 (376 MHz, CDCl3): δ -215.6; HRMS(ESI-TOF) m / e : C 100 H 108 Cl3FN2O 31Na [M+Na] + Calculated value: 1979.5878, measured value: 1979.5889.

[0091]

[0092] [5-acetamido-4,7,8,9-tetra- O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl 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 10. At room temperature, in a stirred mixture of tetrasaccharide 9 (578 mg, 0.30 mmol, 1 equivalent) and acetic acid (6.0 mL, acetic acid / water, 10:1 = 10). v / v CH3COONa (121 mg, 1.48 mmol, 5 equivalences) was added to the solution, followed by PdCl2 (105 mg, 0.59 mmol, 2 equivalences). 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 resulting 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 (600 MHz, CDCl3): δ 7.90-7.87 (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-Hα , man-C1-H β ,C1-H β ), 4.84-4.30 (m, 15H, C1-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); 13 C NMR (150 MHz, 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, 138.1, 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 ; 19F NMR (376 MHz, 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.

[0093]

[0094] [5-acetamido-4,7,8,9-tetra- O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl 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 fluorine 11. In -20 o At 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 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 H NMR (600 MHz, CDCl3): δ 7.89-7.88 (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, 7.8 Hz, 1H), 5.53-5.40 (m, 2H), 5.33-5.29 (m, 3H, C1-H α ), 5.21(dd, J = 27.6, 11.4 Hz, 1H, sia-C4-H), 5.05-4.96 (m, 2H, C1-H β ), 4.94 (d, J =11.4 Hz, 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, 3.0 Hz, 1H), 4.26 (d, J = 10.2 Hz, 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 =0.6 Hz, 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); 13C NMR (150 MHz, CDCl3): δ 170.8, 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 (2 C), 20.6; 19 F NMR (376 MHz, 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.

[0095]

[0096] ( 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-ketopyranoyl 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-D-mannopyranoside 12. Add a well-stirred solution of hemiacetal 10 (395 mg, 0.21 mmol, 1 equivalent) to anhydrous CH2Cl2 (12 mL), and stir at 0°C. o Cs₂CO₃ (83.2 mg, 0.26 mmol, 2 equivalents) and 2,2,2-trifluoro- were added at C. N -Phenyl-imine acetyl chloride (41 µL, 0.26 mmol, 2 equivalents), then the mixture was heated to room temperature and stirred for 3 hours. The reaction mixture was diluted with CH2Cl2 (10 mL), filtered through a diatomaceous earth pad, 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 (600 MHz, CDCl3): δ 7.93-7.88 (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 (150 MHz, CDCl3): δ 170.7,170.7, 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, 127.4, 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 (376 MHz, 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.

[0097]

[0098] Benzyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl 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-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 14. A mixture of AgOTf (194 mg, 0.75 mmol, 6 equivalents relative to the acceptor), bis(Cp2HfCl2) (167 mg, 0.44 mmol, 3.5 equivalents relative to the acceptor), and freshly activated 4 ÅMS (1.50 g) in anhydrous toluene (15 mL) was stirred at room temperature for 1 hour under argon. The reaction mixture was then cooled to -20 °C and treated with a solution of donor 11 (290 mg, 0.15 mmol, 1.2 equivalents) and acceptor 13 (115 mg, 0.13 mmol, 1 equivalent) in anhydrous toluene (8 mL). The reaction mixture was then subjected to 0... o The mixture was stirred at C until the starting material disappeared on TLC (3 hours). 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 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%).

[0099] One-pot synthesis: A mixture of acceptor 18 (40.0 mg, 0.037 mmol, 1 equivalence), donor 4 (60.1 mg, 0.056 mmol, 1.5 equivalence), and activated and pulverized 4 Å MS (0.25 g) in anhydrous CH2Cl2 (1.25 mL) was stirred under argon atmosphere for 1 hour. The reaction mixture was then 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). The mixture was then stirred to -20 °C. o C was set for 2 hours. Then, after the starter 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 minutes. After adding all reagents, the mixture was heated to -10°C. o C and stirring continued until the starting material disappeared on TLC (1 hour). Afterward, 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 (600 MHz, 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, 7.8 Hz,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, 5.4 Hz, 1H), 4.13-4.06 (m, 2H),3.98-3.95 (m, 4H, C1-H β ), 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); 13C NMR (150 MHz, 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, 127.6, 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, 72.9, 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 (376 MHz, 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.

[0100]

[0101] Benzyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl 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 to a solution of starting material 14 (318 mg, 0.11 mmol, 1 equivalent) in acetonitrile (12 mL) under stirring. p -TsOH . H₂O (21.5 mg, 0.11 mmol, 1 equivalent). The reaction mixture was stirred vigorously until the starting material disappeared on TLC (6 h). After completion, the reaction mixture was quenched with Et₃N (0.3 mL) and concentrated 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 HNMR (600 MHz, CDCl3): δ 7.88-7.87 (m, 4H, Ar-H), 7.46 (dd, J = 7.2, 7.2 Hz, 1H,Ar-H), 7.43 (dd, J = 7.2, 7.2 Hz, 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, 7.8 Hz, 1H), 5.46-5.43(m, 1H), 5.38 (d, J = 8.4 Hz, 1H), 5.31-5.29 (m, 3H), 5.19-5.13 (m, 2H, C1-H α ), 5.05-4.96 (m, 3H, C1-H) β), 4.89-4.79 (m, 4H, C1-H β ), 4.69-4.60 (m, 8H, C1-H β ),4.57-4.49 (m, 8H, C1-H β ), 4.45 (d, J = 12.0 Hz, 1H), 4.40-4.36 (m, 2H), 4.31(dd, J = 12.6, 2.4 Hz, 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, 6.0 Hz, 1H),3.76-3.69 (m, 6H), 3.66-3.37 (m, 18H), 2.96-2.93 (m, 1H), 2.13 (s, 3H, -CH3),2.11 (s, 3H, -CH3), 2.09 (s, 3H, -CH3), 1.98 (s, 3H, -CH3), 1.93 (s, 3H, -CH3), 1.90 (s, 3H, -CH3); 13C NMR (150 MHz, CDCl3): δ 170.8, 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 (376 MHz, 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.

[0102]

[0103] Benzyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α -L-mannose-non-2-ketopyranolate 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)]-[5-acetamido-4,7,8,9-tetra- O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α -L-mannose-non-2-ketopyranolate 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→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.

[0104] From fluoride donors: A mixture of AgOTf (142 mg, 0.55 mmol, 8 equivalents relative to the receptor), Cp₂HfCl₂ (105 mg, 0.28 mmol, 4 equivalents relative to the receptor), and freshly activated 4 Å 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 equivalents) and receptor 15 (188 mg, 0.069 mmol, 1 equivalent) in anhydrous toluene (1 mL) was added. o The mixture was stirred continuously at 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 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 acetone / toluene (2:3) as the precipitate to give compound 16 as a white powder (223 mg, 70%), along with the recovered 15 (23.0 mg, 80% brsm).

[0105] From trifluoroacetylimide donor: The mixture of acceptor 15 (32.0 mg, 0.012 mmol, 1 equivalent), donor 12 (42.9 mg, 0.021 mmol, 1.75 equivalent), and activated pulverized 4 Å MS (200 mg) in 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, relative to 0.25 equivalent of acceptor). At -20 °C... o After stirring at C for 3 hours, 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 with 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%), along with the recovered receptor (12.6 mg, 55% brsm). f =0.17 (Silicone, Acetone:Toluene = 1:2); 1 H NMR (600 MHz, CDCl3): δ 7.91-7.86 (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, 4Cl-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): δ 170.7, 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, 129.8, 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, 127.1, 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 (376 MHz, 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.

[0106]

[0107] [5-Acetamido-3,5-Dideoxy-3-fluoro-D-erythrose-] α -L-mannose-non-2-ketopyranolate-(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-ketopyranolate-(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. Protected polysaccharide 16 (103 mg, 0.022 mmol, 1 equivalent) was mixed with LiOH (51.5 mg, 50% by SM wt) in 1,4-dioxane / H2O (3 mL, 4:1 = v / v The solution in the mixture is at 90 o The mixture was stirred at C for 16 hours. 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 solvent removal, the crude residue was subjected to LiChroprep ® 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 IR-120, filtered, and concentrated under vacuum. The residue was purified by LiChroprep chromatography. ® 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 MeOH / H2O / HCOOH (3 mL, 6:3:1). v / v / v The mixture was treated with Pd(OH)₂ (51.5 mg, 50% by SM wt) 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 (precipitation with water), followed by LiChroprep chromatography. ®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 (600 MHz, D2O): δ5.23 (d, J = 3.0 Hz, 0.65H, α-Cl-H α ), 5.14 (d, J = 52.8 Hz, 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, α-Cl-H β ), 4.60 (d, J = 7.8 Hz, 2H, dd'-C1-H β ), 4.48 (d, J = 7.8 Hz, 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 (150 MHz, D2O): δ 174.5, 174.2,174.1, 102.7, 102.6, 100.0, 99.1, 98.9, 98.9, 98.7, 98.6, 96.6, 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, 72.9,72.7, 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; 19F NMR (376 MHz, 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.

[0108]

[0109] [5-Acetamido-3,5-Dideoxy-3-fluoro-D-erythrose-] α -L-mannose-non-2-ketopyranolate-(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-ketopyranolate-(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-glucopyrano)-[2,1- d ]-2-Methyloxazoline S1. Gather Representative synthesis of glycoxazolin. A solution of polysaccharide 17 (14.5 mg, 7.05 µmol, 1 equivalent), 2-chloro-1,3-dimethyl-1H-benzimidazole-3-onium chloride (CDMBI) (12.2 mg, 0.056 mmol, 8 equivalents) and Et3N (19.9 μL) in water (121.1 μL) was prepared at 4... o Stir at C for 1 hour. Allow the reaction mixture to undergo gel filtration chromatography on a Sephadex G-25 column, followed by precipitation with 0.05% Et3N aqueous solution. Combine the fractions containing the polysaccharide oxazoline product and lyophilize to obtain the desired product S1 as a white powder (12.6 mg, 87% yield). 1 H NMR (600 MHz, D2O + 0.05%Et3N): δ 6.09 (d, J= 7.2 Hz, 1H, oxa-C1-H α ), 5.12 (d, J = 52.2 Hz, 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); 13 C NMR (150 MHz, D2O + 0.05% Et3N): δ 174.9, 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 (470 MHz, D2O + 0.05% Et3N): δ -217.3 (d, J FH = 51.3 Hz), -217.3 (d, J FH =51.3 Hz); HRMS (ESI-TOF) m / e : C 76 H 120 F2N5O 56 [M-H] -Calculated value: 2036.6658, measured value: 2036.6672.

[0110]

[0111] Process S1.

[0112] Reagents and conditions in process S1: (a) TFA, MeOH, 60 o C, 16 h, then AcCl, rt, 2d, then Na2HPO4, CH3CN, reflux, 20 h, 92%. (b) ref. 3, 80% (three steps). (c) TiBr4, dichloroethane, rt, 10 min, 92%. (d) BH3, Cu(OTf)2, THF, rt, 8 h, 96%.

[0113]

[0114] 5-Acetamido-4,7,8,9-Tetra- O -Acetyl-3,5-dideoxy-2,6-dehydro-D-glycero-D-galactose–non-2-enoate methyl ester S3. 19 Add TfOH (4.95 mL, 64.7 mmol, 1 equivalent) to a stirred solution of compound S2 (20 g, 64.7 mmol, 1 equivalent) in MeOH (600 mL). Let the reaction mixture reach 60 °C. o Stirred at C for 16 hours. The solvent was removed by rotary evaporation under reduced pressure and co-evaporated twice with toluene to remove water traces. At 0... o The resulting residue was dissolved in AcCl (200 mL) in a round-bottom flask at 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 equivalences) was added under argon atmosphere. The reaction mixture was heated to 90 °C. o The solution was stirred vigorously at C until the starting material disappeared under TLC (16 hours). The solution was filtered through a diatomaceous earth mat, dehydrated with MgSO4, and concentrated under vacuum. The resulting 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); 1H NMR (600 MHz, CDCl3): δ5.95 (d, J = 3.0 Hz, 1H), 5.81 (d, J = 9.0 Hz, 1H), 5.47-5.44 (m, 2H), 5.32-5.30(m, 1H), 4.59 (dd, J = 12.0, 3.0 Hz, 1H), 4.38-4.33 (m, 2H), 4.15 (d, J = 12.0,7.2 Hz, 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 (150 MHz, 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 value of C 20 H 27 NO 12 Na [M+Na] + : 496.1425, measured value: 496.1435.

[0115]

[0116] 5-Acetamido-4,7,8,9-Tetra- O -Acetyl-2-bromo-2,5-dideoxy-D-erythrose- α -L-glucose-non-2-ketopyranoside methyl ester 3. 20 The epoxide S4 was synthesized from sialic acid using a reported procedure. 21A 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 (600 MHz, CDCl3): δ 5.99 (d, J = 7.2 Hz, 1H), 5.42(dd, J = 7.2, 1.2 Hz, 1H), 5.21-5.18 (m, 1H), 5.10 (ddd, J = 6.0, 6.0, 2.4 Hz,1H), 4.38 (dd, J = 12.6, 2.4 Hz, 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 = 9.0 Hz, 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 (150 MHz, CDCl3): δ 171.4, 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 H29 BrNO 13 [M+H] + Calculated value: 570.0817, measured value: 570.0822.

[0117]

[0118] p-Tolyl 2,3-di- O -benzyl-4- O -benzyl-1-thio- β -D-galactopyranoside 5. 22 To the stirring starting material S5 23 Cu(OTf)₂ (124 mg, 0.34 mmol, 0.05 equivalence) was added to a solution of BH₃-THF complex (1 M in THF, 34.3 mL, 34.3 mmol, 5 equivalence), and the reaction mixture was stirred at room temperature for 8 hours. After completion, the solution was... o The reaction was carefully neutralized with TEA (0.96 mL, 6.86 mmol, 1 equivalence) at C, and then diluted with MeOH (15 mL). After removing the solvent under vacuum, the 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 spectroscopic data were consistent with those reported in previous literature. 22 R f = 0.45 (Silicone, EtOAc:Hexane = 1:1); 1 H NMR (600 MHz, CDCl3): δ 7.97-7.94 (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, 10.2 Hz, 1H), 5.36 (dd, J = 9.6, 3.0 Hz, 1H), 4.87 (d, J = 9.6 Hz, 1H, C1-H β ), 4.74 (d, J = 12.0 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.16 (d, J = 2.4 Hz, 1H), 3.91 (d,J = 11.4, 7.2 Hz, 1H), 3.76 (dd, J = 6.0, 6.0 Hz, 1H), 3.61 (dd, J =11.4, 5.4 Hz, 1H), 2.30 (s, 3H, -CH3), 1.83 (br, 1H); 13 C NMR (150 MHz, CDCl3): δ 165.9, 165.2, 138.1, 137.4, 133.4, 133.1, 133.0, 129.8, 129.7, 129.6,129.5, 128.9, 128.7, 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.

[0119]

[0120] Process S2

[0121] Reagents and conditions in process S2: (a) TESOTf, BnCHO, TESH, toluene, THF, 2 h, -20 °C o C,90%. (b) NIS, TfOH, 4Å MS, -40 o C, 1.5 h, CH2Cl2, 75%. (c) NaBH3CN, HCl / ether, AW-300, THF, 0 o C to rt, 16 h, 85%.

[0122]

[0123] p-Tolyl 3- O -benzyl-4,6- O -benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino- β -D-glucopyranoside S7. At -20°C under argon atmosphere. o C, to the starting material S6 24(5.17 g, 9.42 mmol, 1 equivalent) in THF / toluene (45 mL, 1:2 = v / v Add TESOTf (4.26 mL, 18.8 mmol, 2 equivalents) to the solution in the mixture. At -20°C... o After stirring at C for 45 minutes, benzaldehyde (4.79 mL, 47.1 mmol, 5 equivalents) and triethylsilane (2.26 mL, 14.1 mmol, 1.5 equivalents) were added dropwise to the mixture with stirring. At -20°C... o After 2 hours at C, TLC showed the disappearance of the starting material. The reaction was quenched with saturated Na₂CO₃ aqueous solution, diluted with EtOAc (100 mL), and washed with saturated Na₂CO₃ aqueous solution (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 (600 MHz, acetone-d6): δ 7.52-7.50 (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 = 10.8 Hz, 1H, C1-H β ), 4.90-4.82 (m, 3H), 4.74 (d, J = 12.0 Hz, 1H), 4.30 (dd, J = 10.2, 5.4Hz, 1H), 3.95 (dd, J = 9.6, 9.0 Hz, 1H), 3.84-3.71 (m, 3H), 3.56 (ddd, J = 9.6,9.6, 4.8 Hz, 1H), 2.83 (s, 2H), 2.32 (s, 3H, -CH3); 13C10 NMR (150 MHz, 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: C10 30 H 30 Cl3NO6SNa [M+Na] + Calculated value: 660.0752, measured value: 660.0749.

[0124]

[0125] 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 will bind to receptor S8. 25 A mixture of 523 mg (0.94 mmol, 1 equivalent), donor S7 (900 mg, 1.41 mmol, 1.5 equivalent), and activated pulverized 4 Å MS (2.00 g) in anhydrous CH2Cl2 (20 mL) was stirred under argon for 1 h. The reaction mixture was then cooled to -40 °C and treated with NIS (423 mg, 1.88 mmol, 2 equivalent) and TfOH (0.5 M in Et2O, 0.47 mL, 0.23 mmol, 0.25 equivalent). After stirring at this temperature for 1.5 hours, TLC analysis showed the disappearance of the starting materials. 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 (600 MHz, CDCl3): δ 7.50-7.48 (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, 5.4 Hz, 1H), 5.55 (s, 1H, Ph-CH), 5.26 (d, J =1.2 Hz, 1H), 5.23-5.20 (m, 1H), 5.16 (dd, J = 10.8, 1.2 Hz, 1H), 5.01 (d, J = 7.8Hz, 1H, C1-H β ), 4.90 (d, J = 10.8 Hz, 1H), 4.83 (d, J = 11.4 Hz, 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, 2.4 Hz, 1H), 4.00-3.97 (m, 1H), 3.94 (dd, J = 9.6, 3.0 Hz, 1H),3.89 (dd, J = 13.2, 6.0 Hz, 1H), 3.82-3.78 (m, 2H), 3.72-3.66 (m, 3H), 3.50-3.46 (m, 1H), 3.12 (d, J = 6.0 Hz, 1H); 13C NMR (150 MHz, CDCl3): δ 153.8, 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, 126.0, 117.2, 101.2, 98.9, 96.9, 95.5, 82.4,78.5, 75.6, 75.2, 74.6, 74.4, 74.2, 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.

[0126]

[0127] 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 300 MS (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 spectroscopic data were consistent with those reported in the literature. 27 R f = 0.17 (Silicone, EtOAc:Toluene = 1:10); 1 H NMR (600 MHz, CDCl3): δ 7.36-7.23 (m, 23H, Ar-H), 7.20-7.19 (m, 2H, Ar-H), 5.84 (ddd, J = 16.2. 10.2, 6.0 Hz, 1H), 5.35 (br, 1H), 5.21(dd, J = 16.2, 1.2 Hz, 1H), 5.14 (dd, J = 10.2, 1.2 Hz, 1H), 4.95 (d, J = 7.2 Hz, 1H, C1-H β ), 4.88 (d, J = 10.8 Hz, 1H), 4.78 (d, J = 1.8 Hz, 1H, C1-H α ), 4.75 (d, J =11.4 Hz, 1H), 4.70-4.63 (m, 5H), 4.60 (d, J = 11.4 Hz, 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, 4.2 Hz, 1H), 3.73-3.70 (m, 3H), 3.66 (dd, J = 10.8, 1.8 Hz, 1H), 3.61 (dd, J = 9.6, 8.4 Hz, 1H),3.54-3.51 (m, 1H), 3.08 (m, 1H), 2.71 (m, 1H, -OH); 13 C NMR (150 MHz, CDCl3): δ154.0, 138.5, 138.5, 138.4, 138.1, 137.6, 133.7, 128.5, 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.

[0128]

[0129] Process S3

[0130] Reagents and conditions in process S3: (a) NaBH3CN, HCl / ether, AW-300, THF, 0 o (a) C to rt, 16 h, 90%. (b) Ac₂O, pyridine, rt, 16 h, 98%. (c) NIS, TfOH, HOPO(OBu)₂, 4 Å MS, -30 o C, 2 h, CH2Cl2, 90%. (d) TMSOTf, 4Å MS, -50 o C, 1 h, CH2Cl2, 87%. (e) NaOMe,MeOH / CH2Cl2, 0 o C to rt, 76%.

[0131]

[0132] p-Tolyl 3,6-di- O -Benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino- β -D-glucopyranoside S10. 28 Under argon atmosphere, activated pulverized AW 300 MS (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 then washed with saturated NaHCO3 aqueous solution (15 mL) and brine (10 mL). The separated organic layer was dehydrated and concentrated using MgSO4. The resulting 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 (Silicone, EtOAc:Hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ 7.40-7.38 (m, 2H, Ar-H), 7.36-7.27 (m, 10H, Ar-H), 7.04-7.03(m, 2H, Ar-H), 5.20 (d, J = 7.8 Hz, 1H), 4.84 (d, J = 10.2 Hz, 1H, C1-H β ), 4.77-4.72 (m, 4H), 4.58-4.52 (m, 2H), 3.76 (d, J = 4.8 Hz, 2H), 3.71-3.64 (m, 2H), 3.50-3.49 (m, 1H), 3.42-3.38 (m, 1H), 2.90 (br, 1H, -OH), 2.29 (s, 3H, -CH3); 13C NMR (150 MHz, CDCl3): δ 153.8, 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.

[0133]

[0134] p-Tolyl 4- O -Acetyl-3,6-bis- 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 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 hours, 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 (Silicone, EtOAc:Hexane = 1:3); 1 H NMR (600 MHz, CDCl3): δ 7.41-7.40 (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 = 7.2 Hz, 1H), 5.04 (d, J = 10.2 Hz, 1H, C1-H β ), 4.98 (dd, J = 9.6, 9.6 Hz, 1H), 4.78 (d, J = 12.0Hz, 1H), 4.70 (d, J= 12.0 Hz, 1H), 4.62 (d, J = 11.4 Hz, 1H), 4.57 (d, J = 11.4Hz, 1H), 4.50 (br, 2H), 4.05 (dd, J = 9.6, 9.0 Hz, 1H), 3.65-3.63 (m, 1H), 3.59-3.55 (m, 2H), 3.33 (ddd, J = 9.6, 9.6, 9.6 Hz, 1H), 2.29 (s, 3H, -CH3), 1.87 (s, 3H, -CH3); 13 C NMR (150 MHz, CDCl3): δ 169.8, 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.

[0135]

[0136] 4- O -Acetyl-3,6-bis- O -Benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino- α or β-D-glucopyranoside dibutyl phosphate S12a, b. A mixture of compound S11 (990 mg, 1.45 mmol, 1 equivalence), dibutyl phosphate (1.15 mL, 5.80 mmol, 3 equivalence), and activated pulverized 4 Å MS (1.80 g) in anhydrous CH2Cl2 (18 mL) was stirred under argon for 1 h. The mixture was then cooled to -30 °C with stirring, and NIS (978 mg, 4.35 mmol, 2 equivalence) was added, followed by TfOH (0.5 M in Et2O, 0.87 mL, 0.43 mmol, 0.3 equivalence). After 2 h, 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 resulting 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 mutarotatory isomers ( α : β= 1:3). β-mutant isomer S12b: R f = 0.32 (Silicone, EtOAc:Toluene = 1:3); 1 H NMR (600 MHz, CDCl3): δ 7.30-7.22 (m, 10H, Ar-H), 5.81 (br, 1H), 5.35 (dd, J = 7.8, 7.8 Hz, 1H, C1-H β ), 5.09 (dd, J = 9.6, 9.0 Hz, 1H), 4.74 (d, J = 12.0 Hz, 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, 9.0 Hz, 1H),3.77-3.74 (m, 1H), 3.69 (ddd, J= 9.6, 4.2, 4.2 Hz, 1H), 3.53-3.52 (m, 2H), 1.83 (s, 3H, -CH3), 1.61-1.54 (m, 4H), 1.36-1.31 (m, 4H), 0.93-0.85 (m, 6H); 13 C NMR (150 MHz, CDCl3): δ 169.5, 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, 73.9,73.6,73.5,70.6,69.2,68.1,68.1,68.1,68.0,57.2,57.2,32.1,32.0,32.0,20.7,18.6,18.6,13.5; : 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 (600 MHz, CDCl3): δ 7.31-7.22 (m, 10H, Ar-H), 5.67 (dd, J = 6.0, 3.6 Hz, 1H, C1-H α ), 5.21-5.16 (m, 2H), 4.75 (d, J = 12.0 Hz, 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, 9.6 Hz, 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); 13C NMR (150 MHz, CDCl3): δ 169.3, 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.

[0137]

[0138] p-Tolyl[4- O -Acetyl-3,6-bis- 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 will bind to receptor S13 29 A mixture of 407 mg (0.73 mmol, 1 equivalent) of donor S12 (900 mg, 1.17 mmol, 1.6 equivalent) and activated pulverized 4 Å molecular sieve (200 mg) in anhydrous CH2Cl2 (2 mL) was stirred under argon atmosphere for 30 min. The mixture was then cooled to -50 °C, followed by the addition of TMSOTf (0.21 mL, 1.17 μmol, 1.6 equivalent 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 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 1H NMR (600 MHz, CDCl3): δ 7.37 - 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 J = 1.8 Hz, 1H, C1 - H α ), 5.26 (d, J J = 4.8 Hz, 1H), 5.07 (d, J J = 9.0 Hz, 1H, C1 - H β ), 4.93 - 4.90 (m, 2H), 4.77 (d, J J = 11.4 Hz, 1H), 4.60 - 4.52 (m, 5H), 4.46 - 4.34 (m, 5H), 4.27 (dd, J J = 8.4, 8.4 Hz, 1H), 4.19 (dd, J J = 9.6, 2.4 Hz, 1H), 4.15 (d, J J = 12.0 Hz, 1H), 4.07 (dd, J J = 9.6, 9.0 Hz, 1H), 3.84 - 3.81 (m, 2H), 3.64 - 3.63 (m, 2H), 3.57 (dd, J J = 10.8, 6.0 Hz, 1H), 3.51 (dd, J J = 11.4, 3.0 Hz, 1H), 3.01 (dd, J J = 6.6 Hz, 1H), 2.28 (s, 3H, -CH3), 1.82 (s, 3H, -CH3); 13CNMR (150 MHz, CDCl3): δ 169.7, 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, 127.7, 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.

[0139]

[0140] 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. In 0 o At temperature C, compound S14 (203 mg, 0.18 mmol, 1 equivalent) was thoroughly stirred and added to a CH2Cl2 / MeOH mixture (10 mL, 1:1 ratio). v / v NaOMe (2.95 mg, 0.055 mmol, 0.3 equivalence) was added to the solution. 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 hours). Afterward, the reaction mixture was neutralized with IR-120, filtered, and concentrated under vacuum. The resulting 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 1H NMR (600 MHz, CDCl3): δ 7.39 - 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 J = 1.8 Hz, 1H, C1-H α ), 5.24 (d, J J = 4.8 Hz, 1H), 5.00 (d, J J = 7.8 Hz, 1H, C1-H β ), 4.93 (d, J J = 10.8 Hz, 1H), 4.78 (d, J J = 11.4 Hz, 1H), 4.69 - 4.64 (m, 2H), 4.61 (d, J J = 11.4 Hz, 2H), 4.57 - 4.51 (m, 4H), 4.43 (d, J J = 11.4 Hz, 1H), 4.34 (dd, J J = 2.4, 2.4 Hz, 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 J = 7.2 Hz, 1H), 2.68 (br, 1H, -OH), 2.30 (s, 3H, -CH3); 13C NMR (150 MHz, CDCl3): δ 154.2,138.5, 138.4, 138.3, 137.9, 137.7, 137.6, 132.5, 130.3, 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.

[0141] Process S4

[0142] Reagents and conditions in procedure S4: (a) NIS, TfOH, 4 Å MS, -40 o C, 1 h, CH2Cl2, 93%. (b) Ethylenediamine / BuOH (1:4), 90%. o C, 2 h, then NaHCO3, TrocCl, CH2Cl2, 0 o C, 3 h, 81%.(c) Tf2O, pyridine, CH2Cl2, 0 o C, 4h. (d) Bu4NOAc, toluene, ultrasonic treatment, rt, 8h, then NaHCO3, TrocCl, CH2Cl2, 0 o C, 3 h, 61% (2 steps). (e) DDQ, CH2Cl2, phosphate buffer (pH = 7), 0 o C to rt, 3h, 85%. (f) Hydrazine acetate, THF, rt, 16h, 92%. (g) Tf2O, pyridine, CH2Cl2, 0 o C, 2h. (h) Bu4NOAc, toluene, ultrasonic treatment, rt, 8h, 93% (2 steps). (i) Ethylenediamine / BuOH (1:4), 90o C, 2 h, then NaHCO3, TrocCl, CH2Cl2, 0 o C, 3 h. (j)Ac2O, pyridine, CH2Cl2, rt, 16 h, 83% (2 steps).

[0143]

[0144] benzyl[4,6-] O -benzyl-3- O - p -Methoxy-benzyl-2- O -levulinoyl- β [-D-glucopyranoside]-(1→4)-3,6-di- O -Benzyl-2-deoxy-2-phthalimide- β -D-glucopyranoside S17 will bind to receptor S16. 30 (1.59 g, 2.74 mmol, 1 equivalent), donor S15 26 A mixture of 2.11 g (3.57 mmol, 1.3 equivalences) and activated, pulverized 4 Å 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 resulting 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); 1H NMR (600 MHz,CDCl3): δ 7.75 (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, 8.4 Hz, 1H), 4.81-4.71 (m, 4H), 4.58(d, J = 7.8 Hz, 1H, C1-H β ), 4.56 (d, J = 4.2 Hz, 1H), 4.47 (d, J = 6.0 Hz, 1H),4.45 (d, J = 6.6 Hz, 1H), 4.36 (d, J = 12.6 Hz, 1H), 4.23 (dd, J = 10.2, 4.8 Hz,1H), 4.20-4.19 (m, 2H), 4.09-4.06 (m, 1H), 3.87 (dd, J = 11.4, 3.0 Hz, 1H),3.79-3.78 (m, 1H), 3.78 (s, 3H, -CH3), 3.59 (dd, J = 9.6, 9.0 Hz, 1H), 3.55-3.51 (m, 2H), 3.43 (dd, J = 10.2, 10.2 Hz, 1H), 3.17 (ddd, J = 9.6, 9.6, 4.8 Hz,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 (150 MHz, CDCl3): δ 206.2, 171.1, 159.2, 138.6, 138.1, 137.3,133.5, 131.6, 130.4, 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.

[0145]

[0146] benzyl[4,6-] O -benzyl-3- O - p -Methoxy-benzyl- β [-D-glucopyranoside]-(1→4)-3,6-di- O -Benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbamate- β -D-glucopyranoside S18. Disaccharide S17 (625 mg, 0.596 mmol, 1 equivalent) was reacted with ethylenediamine / n- BuOH (7 ml, 2:8 = v / v The solution in ) at 90 o Stirred at C for 2 hours. 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 heated under argon at 0°C. oC was treated with NaHCO3 (250 mg, 2.98 mmol, 5 equivalences) and trichloroethyl 2,2,2-chloroformate (0.41 mL, 2.98 mmol, 5 equivalences) for 3 hours. 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 (600 MHz, CDCl3): δ 7.47-7.46 (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, 3.6 Hz, 1H), 3.87 (br, 1H), 3.80 (dd, J = 11.4, 1.8 Hz, 1H),3.78 (s, 3H, -CH3), 3.56 (dd, J = 9.6, 9.0 Hz, 1H), 3.52-3.45 (m, 5H), 3.15(ddd, J = 9.6, 9.6, 4.8 Hz, 1H), 3.03 (br, 1H, -OH); 13C NMR (150 MHz, CDCl3): δ159.3, 153.8, 138.4, 137.7, 137.3, 137.2, 130.4, 129.6, 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.

[0147]

[0148] benzyl[2- O -acetyl-4,6- O -benzyl-3- O - p -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: under argon at 0 o Anhydrous pyridine (0.58 mL, 7.17 mmol, 7 equivalences) was added dropwise to a solution of disaccharide S18 (1.02 g, 1.02 mmol, 1 equivalence) stirred in anhydrous CH2Cl2 (13 mL), followed by the dropwise addition of Tf2O (0.30 mL, 1.79 mmol, 1.75 equivalences). The reaction mixture was then heated to 0°C. oStirring at C until the starting material disappeared as shown by TLC (4 hours). Afterward, 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 time, 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 concentrated under argon at 0. o C was treated with NaHCO3 (430 mg, 5.12 mmol, 5 equivalences) and 2,2,2-trichloroethyl chloroformate (0.71 mL, 5.12 mmol, 5 equivalences). 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%).

[0149] From S21: Disaccharide S21 (1.20 g, 1.21 mmol, 1 equivalent) was reacted with ethylenediamine / n- BuOH (15 ml, 2:8 = v / v The solution in ) at 90 o Stirred at C for 2 hours. After removing the solvent, the crude product was co-evaporated twice with toluene. The resulting residue was dissolved in CH2Cl2 (30 mL) and heated under argon at 0°C. o C was treated with NaHCO3 (508 mg, 6.05 mmol, 5 equivalences) and 2,2,2-trichloroethyl chloroformate (0.83 mL, 6.05 mmol, 5 equivalences). 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 Ac2O (7 mL) in pyridine (14 mL) at temperatures ranging from 0°C to 0.5°C over 16 hours. oC was slowly increased to room temperature. 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): δ 7.48-7.46 (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 = 3.0 Hz, 1H), 5.09 (br, 1H), 4.92 (d, J = 10.8 Hz, 1H), 4.88 (d, J = 12.0 Hz, 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, 4.8 Hz, 1H), 4.04 (dd, J = 9.0, 8.4 Hz, 1H),3.87-3.84 (m, 2H), 3.78-3.72 (m, 5H, -CH3), 3.59 (dd, J = 10.2, 10.2 Hz, 1H),3.45-3.43 (m, 2H), 3.38 (ddd, J = 8.4, 8.4, 8.4 Hz, 1H), 3.10 (ddd, J = 9.6, 9.6,4.8 Hz, 1H), 2.07 (s, 3H, -CH3); 13C NMR (150 MHz, CDCl3): δ 170.3, 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, 127.8, 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.

[0150]

[0151] 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. In 0 o At temperature C, add stirred S19 (120 mg, 0.12 mmol, 1 equivalent) to a CH2Cl2 / phosphate buffer (pH = 7) mixture (3 mL, 9:1 ratio). v / v 2,3-Dichloro-5,6-dicyanobenzoquinone (52.5 mg, 0.23 mmol, 2.2 equivalences) was added to the solution. The reaction mixture was stirred vigorously until the starting material disappeared on TLC (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 (600 MHz, CDCl3): δ 7.45-7.44 (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 = 3.6 Hz, 1H),5.06 (br, 1H), 4.91 (d, J = 11.4 Hz, 1H), 4.87 (d, J = 12.0 Hz, 1H), 4.74 (d, J =12.0 Hz, 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, 4.8 Hz, 1H), 4.04 (dd, J = 9.0, 9.0 Hz, 1H), 3.83(br, 1H), 3.78 (dd, J = 11.4, 3.0 Hz, 1H), 3.73-3.70 (m, 2H), 3.64 (dd, J = 9.6,3.6 Hz, 1H), 3.56 (dd, J = 10.2, 10.2 Hz, 1H), 3.43-3.36 (m, 2H), 3.11 (ddd, J =9.6, 9.6, 4.8 Hz, 1H), 2.21 (d, J = 3.6 Hz, 1H), 2.11 (s, 3H, -CH3); 13C NMR (150MHz, CDCl3): δ 170.5, 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.

[0152]

[0153] benzyl[4,6-] O -benzyl-3- O - p -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 solution of the stirred 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) and washed with water (60 mL) and brine (30 mL). The organic phase was dehydrated by MgSO4, filtered, and concentrated under vacuum. The resulting 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 (Silicone, EtOAc:Hexane = 1:1); 1H NMR (600 MHz, CDCl3): δ 7.79-7.54 (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 = 8.4 Hz, 1H, C1-H β ), 4.85 (d, J = 11.4 Hz, 1H), 4.77 (d, J = 12.6 Hz, 1H), 4.75 (d, J = 4.2 Hz,1H), 4.73 (d, J = 4.2 Hz, 1H), 4.68 (d, J = 11.4 Hz, 1H), 4.62 (d, J = 7.2 Hz, 1H,C1-H β ), 4.56 (d, J = 12.0 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H), 4.38 (d, J = 12.0 Hz,1H), 4.32 (dd, J = 10.8, 8.4 Hz, 1H), 4.21 (d, J = 10.8, 8.4 Hz, 1H), 4.15-4.10(m, 2H), 4.04 (dd, J = 11.4, 3.0 Hz, 1H), 3.82 (dd, J = 11.4, 1.8 Hz, 1H), 3.78(s, 3H, -CH3), 3.61 (ddd, J = 9.6, 3.0, 3.0 Hz, 1H), 3.56 (dd, J = 3.0, 3.0 Hz,1H), 3.52-3.45 (m, 3H), 3.18 (ddd, J= 9.6, 9.6, 4.8 Hz, 1H), 2.98 (br, 1H, -OH); 13 C NMR (150 MHz, CDCl3): δ 167.9, 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, 74.7, 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.

[0154]

[0155] benzyl[2- O -acetyl-4,6- O -benzyl-3- O - p -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), and then the mixture was heated under argon at 0°C. o Add trifluoromethanesulfonic anhydride (0.29 mL, 1.72 mmol, 1.75 equivalents) dropwise to C, and then mix at 0°C. oStirring at C until the starting material disappears as indicated by TLC (2 hours). Afterward, dilute the reaction mixture with CH₂Cl₂ (20 mL), wash with 0.5 N HCl (20 mL) and brine (10 mL). Dehydrate the separated organic layer with MgSO₄, concentrate under vacuum, and use the residue as is for further reactions. Add Bu₄NOAc (594 mg, 1.97 mmol, 2 equivalences) to the residue and dissolve in toluene (18 mL). Remove the solvent under vacuum, and co-evaporate the residue and toluene twice to remove water. Redissolve the residue in anhydrous toluene (12 mL) and sonicate the mixture for 8 hours. Afterward, dilute the reaction mixture with EtOAc (20 mL), wash with water (20 mL) and brine (10 mL). Dehydrate the separated organic layer with MgSO₄ and concentrate. The residue 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%). f = 0.29 (Silicone, EtOAc:Hexane = 1:2); 1 H NMR (600 MHz, CDCl3): δ 7.76-7.49 (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 = 3.0 Hz, 1H), 5.09 (d, J = 7.8 Hz, 1H, C1-H β ), 4.82-4.76 (m, 3H), 4.68 (s, 1H, C1-H β ), 4.58 (d, J = 12.0 Hz, 1H), 4.49-4.44 (m,3H), 4.38 (d, J= 12.6 Hz, 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, 3.6 Hz, 1H), 3.13 (ddd, J = 9.6, 9.6, 4.8 Hz, 1H), 2.15 (s, 3H, -CH3); 13 C NMR (150 MHz, CDCl3): δ 170.2, 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, 127.5, 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.

[0156]

[0157] Process S5

[0158] Reagents and conditions in process S5: (a) Br2, CH2Cl2, 0 o C, 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, 0o C, 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%.

[0159]

[0160] p-Nitrophenyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl methyl ester]-(2→6)-2,3-di- O -benzyl-4- O -Benzyl- β -D-galactopyranoside S22. In 0 o A solution of glucosinolate 4 (115 mg, 0.11 mmol, 1 equivalence) in anhydrous CH2Cl2 (2 mL) was stirred at C and bromine (6.02 µL, 0.12 mmol, 1.1 equivalence) was added. After vigorous stirring of 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 starter (1 h). After completion, the reaction mixture was diluted with EtOAc (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 with 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); 11H NMR (600 MHz, CDCl3): δ 8.13–8.11 (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, 7.8 Hz, 1H), 5.65-5.59 (dd, J = 10.2, 3.0 Hz, 1H, C1-H β ), 5.35 (d, J = 9.6 Hz, 1H), 5.28 (dd, J = 9.6, 1.8 Hz, 1H), 5.19 (dd, J =27.0, 10.8 Hz, 1H, sia-C4-H), 5.04 (dd, J = 51.0, 1.8 Hz, 1H, sia-C3-H), 4.72-4.67 (m, 2H), 4.44 (dd, J = 12.6, 3.6 Hz, 1H), 4.40 (d, J = 3.0 Hz, 1H), 4.37(dd, J = 8.4, 6.6 Hz, 1H), 4.31 (d, J = 10.8 Hz, 1H), 4.18 (ddd, J = 4.2, 4.2, 4.2Hz, 1H), 4.08 (dd, J = 12.6, 6.6 Hz, 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); 13C NMR (150MHz, CDCl3): δ 171.1, 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, 128.1, 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 (376 MHz, 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.

[0161]

[0162] p-Nitrophenyl[5-acetamido-4,7,8,9-tetra-] O -Acetyl-3,5-dideoxy-3-fluoro-D-erythrose- α [-L-mannose-non-2-ketopyranoyl 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 initiator (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 (600 MHz, CDCl3): δ 8.13-8.12 (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, 9.8 Hz, 1H), 5.53 (ddd, J =9.0, 6.0, 3.0 Hz, 1H), 5.49-5.45 (m, 2H, C1-H β ), 5.36 (d, J = 9.0 Hz, 1H), 5.27(dd, J = 9.0, 1.8 Hz, 1H), 5.19 (dd, J = 27.0, 11.4 Hz, 1H, sia-C4-H), 5.05 (dd, J = 51.6, 1.8 Hz, 1H, sia-C3-H), 4.47 (d, J = 3.0 Hz, 1H), 4.37 (dd, J = 12.6, 3.0Hz, 1H), 4.26 (dd, J= 10.8, 1.2 Hz, 1H), 4.18 (dd, J = 6.6, 6.6 Hz, 1H), 4.13-4.07 (m, 1H), 4.05 (dd, J = 12.6, 6.6 Hz, 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 (150 MHz, 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 (376 MHz, 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.

[0163]

[0164] p-Nitrophenyl[5-acetamido-3,5-dideoxy-3-fluoro-D-erythrose-] α [-L-mannose-non-2-ketopyranoyl methyl ester]-(2→6)- β-D-Galactopyranoside 2. LiOH·H₂O (22.2 mg, 0.53 mmol, 1 equivalence) dissolved in water (1 mL) was added to a solution of compound S23 (53 mg, 0.053 mmol, 1 equivalence) in methanol (5 mL). After stirring at room temperature for 16 hours, the reaction mixture was neutralized with IR-120, 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 H NMR (600 MHz, D2O): δ 8.29-8.28 (m, 2H, Ar-H), 7.30-7.29 (m, 2H, Ar-H), 5.21 (dd, J = 51.6, 2.4 Hz, 1H, sia-C3-H), 5.18 (d, J = 7.8 Hz, C1-H β ),4.17 (dd, J = 9.0, 9.0 Hz, 1H), 4.07-4.00 (m, 3H), 3.91-3.77 (m, 7H), 3.63 (dd, J = 12.0, 6.6 Hz, 1H), 3.57 (dd, J = 9.0, 1.8 Hz, 1H), 2.04 (s, 3H, -CH3); 13 C NMR (150 MHz, D2O): δ 175.0, 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 (376 MHz, 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.

[0165]

[0166] p-Tolyl[5-acetamido-3,4,7,8,9-penta-] O -Acetyl-5-deoxy-D-erythrose- α [-L-glucose-non-2-ketopyranoyl 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 equivalence) in pyridine (2 mL). The reaction mixture was vigorously stirred at room temperature for 16 hours, 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 (600 MHz, CDCl3): δ 7.94-7.92 (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 = 10.2.9.6 Hz, 1H), 5.44-5.42 (m, 2H), 5.35-5.29 (m, 3H), 5.25 (dd, J = 8.4, 1.8 Hz, 1H), 4.95 (d, J = 10.2 Hz, 1H, C1-H β ), 4.67-4.61 (m, 3H), 4.33 (ddd, J= 10.2,10.2, 10.2 Hz, 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); 13 C NMR (150 MHz, CDCl3): δ 171.0, 170.5, 170.3, 170.0, 169.3, 168.5,168.0, 165.7, 165.2, 138.4, 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.

[0167]

[0168] p-Nitrophenyl[5-acetamido-3,4,7,8,9-penta-] O -Acetyl-5-deoxy-D-erythrose- α [-L-glucose-non-2-ketopyranoyl methyl ester]-(2→6)-2,3-di- O -benzyl-4- O -Benzyl- β -D-galactopyranoside S25. Towards 0 oA solution of thioglycoside S24 (132 mg, 0.12 mmol, 1 equivalence) stirred at C in anhydrous CH2Cl2 (2.5 mL) was supplemented with bromine (6.67 µL, 0.13 mmol, 1.1 equivalence). 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 with MgSO4, filtered, and concentrated under vacuum. The 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); 1 H NMR (600 MHz, CDCl3): δ 8.15-8.13 (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, 7.8 Hz, 1H), 5.54 (dd, J = 10.2, 3.0 Hz, 1H), 5.52 (d, J = 7.8 Hz, 1H, C1-H β ), 5.48 (ddd, J = 10.2, 7.2, 3.0 Hz, 1H), 5.39(d, J = 10.2 Hz, 1H), 5.31-5.29 (m, 2H), 5.23 (dd, J = 9.6, 1.8 Hz, 1H), 4.73(dd, J= 10.8, 2.4 Hz, 1H), 4.71-4.65 (m, 2H), 4.32-4.30 (m, 3H), 4.19 (dd, J =7.2, 7.2 Hz, 1H), 3.97 (dd, J = 12.6, 7.2 Hz, 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 (150 MHz, CDCl3): δ 171.0, 170.7 (2C), 170,0, 169.6, 168.5, 168.3, 165.7, 165.3, 161.7, 142.7,137.9, 133.3, 133.2, 129.9, 129.7, 129.3, 128.9, 128.4, 128.3, 128.2, 128.0,127.6, 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.

[0169]

[0170] p-Nitrophenyl[5-acetamido-3,4,7,8,9-penta-] O -Acetyl-5-deoxy-D-erythrose- α [-L-glucose-non-2-ketopyranoyl 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 equivalence) in EtOAc (1.2 mL) was mixed with NaBrO3 (54.6 mg, 0.36 mmol, 4.5 equivalence) dissolved in H2O (0.9 mL), followed by the slow addition of Na2S2O4 (85%, 56.0 mg, 0.32 mmol, 4 equivalence) dissolved in H2O (1.8 mL). The reaction mixture was stirred until TLC showed the disappearance of the starting material (1.5 h). After this, the reaction mixture was diluted with EtOAc (15 mL), 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 (600 MHz, CDCl3): δ 8.18-8.16 (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, 7.8 Hz, 1H), 5.46-5.34 (m, 5H, C1-H β ),5.30 (d, J = 10.2 Hz, 1H), 5.22 (dd, J = 7.8, 1.8 Hz, 1H), 4.60 (dd, J = 10.8, 1.8Hz, 1H), 4.43 (d, J = 3.0 Hz, 1H), 4.33 (dd, J = 12.6, 2.4 Hz, 1H), 4.26 (ddd, J =10.2, 10.2, 10.2 Hz, 1H), 4.12 (dd, J = 6.6, 6.6 Hz, 1H), 4.05 (dd, J= 10.2, 6.6Hz, 1H), 3.99 (dd, J = 10.2, 6.6 Hz, 1H), 3.93 (dd, J = 12.6, 7.2 Hz, 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 (150 MHz, CDCl3): δ 171.1, 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.

[0171]

[0172] p-Nitrophenyl[5-acetamido-5-deoxy-D-erythrose-] α [-L-glucose-non-2-ketopyranolate]-(2→6)- β-D-galactopyranoside S27. A solution of compound S26 (75 mg, 0.072 mmol, 1 equivalence) in methanol (5 mL) was supplemented with LiOH·H₂O (30 mg, 0.72 mmol, 10 equivalence) 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 (600 MHz, D2O): δ 8.29-8.28 (m, 2H, Ar-H), 7.28-7.27 (m, 2H, Ar-H), 5.20 (d, J = 7.2 Hz, C1-H β ), 4.09-4.06 (m, 2H), 4.01 (dd, J = 10.8, 7.8 Hz,1H), 3.92-3.79 (m, 6H), 3.71 (d, J = 10.8 Hz, 1H), 3.64-3.60 (m, 2H), 3.53 (d, J = 9.6 Hz, 1H), 3.49 (d, J = 9.6 Hz, 1H), 2.02 (s, 3H, -CH3); 13 C NMR (150 MHz, D2O): δ 174.8, 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.

[0173]

[0174] p-Nitrophenyl[5-acetamido-5-deoxy-D-glycero-] α [-D-galactose-non-2-ketopyranose ester]-(2→6)- β-D-galactopyranoside 1. Neu5Ac-α2,6-Gal- p NP: Neu5Ac-α2,6-Gal- 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-sialyltransferase (SiaT, 15 U) in 15 mL of Tris buffer (pH 7.0) with 5 mM mgCl2 and 5 mM MnCl2. p NP. After removing proteins by heating and centrifugation, the product was purified by (BIO-RAD) Biogel P-2 column chromatography using water as the chromatography solvent. The product containing Neu5Ac-α2,6-Gal- p The fractions of NP were collected and freeze-dried to give compound 1 (50%). 1 H NMR (600 MHz, D2O): δ 8.33-8.32 (m, 2H, Ar-H), 7.31-7.29 (m, 2H, Ar-H), 5.23 (d, J = 7.8 Hz, C1-H β ), 4.06-4.04 (m, 2H), 3.99 (dd, J = 10.2, 8.4 Hz, 1H), 3.90-3.86 (m, 3H), 3.83 (dd, J = 10.2, 3.6 Hz, 1H), 3.80-3.76 (m, 1H), 3.73-3.69(m, 3H), 3.65-3.62(m, 2H), 3.57 (d, J = 8.4 Hz, 1H), 2.79 (dd, J = 12.6, 4.8 Hz, 1H), 2.04 (s, 3H,-CH3), 1.68 (dd, J = 12.6, 12.0 Hz, 1H); 13 C NMR (150 MHz, D2O): δ 174.6, 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: C23 H 33 N2O 16 [M+H] + Calculated value: 593.1825, measured value: 593.1825.

[0175] Example 2: Analysis of the stability against sialidase-catalyzed hydrolysis and the inhibitory effect of sialidase. 31

[0176] Material:

[0177] From Aspergillus oryzae ( Aspergillus oryzae β-galactosidase (G5160) and sialidase from Vibrio cholerae (11080725001) and Clostridium perfringens (11585886001) were obtained from Sigma Aldrich.

[0178] Enzyme assay for sialic acid oxidase:

[0179] This measurement was taken at 37. o The assays were repeated in 96-well plates at C, with a final volume of 50 µL containing the matrix (0–20 mM) and β-galactosidase (100 mU). The assay conditions for both 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). *Clostridium perfringens* reactions were carried out for 40 minutes to 2 hours, while *Vibrio cholerae* reactions were carried out overnight. The assays were performed by adding 65 µL of CAPS buffer (…). N The assay was stopped by adding cyclohexyl-3-aminopropanesulfonic acid (0.5 M, pH 10.5). The amount of p-nitrophenolate formed was measured in the reaction mixture using a microdisk reader. 405nm To determine this, three compounds (Neu5Ac-α2,6-Galβ) were used. p NP, 3F ax -Neu5Ac-α2,6-Galβ p NP and 3OH eq -Neu5Ac-α2,6-Galβ p NP) was used as an enzyme substrate for testing. In the absence of sialidase, at 37... o After 1 hour of incubation, all three compounds showed A at 20 mM. 405nm Background absorbance. Three compounds, Neu5Ac-α2,6-Galβ. p NP, 3F ax-Neu5Ac-α2,6-Galβ p NP and 3OH eq -Neu5Ac-α2,6-Galβ p The absorbance values ​​of NP are 0.044, 0.129 and 0.072, respectively. p The standard curve for NP was determined as follows: 0.35 mM NP was continuously diluted twice. p NP, then A 405nm right p Plotting the concentration of NP yields p The standard curve of NP.

[0180] Assay for the inhibition of sialidase:

[0181] This measurement was taken at 37. o C was placed in a 96-well disk with a final volume of 50 µL, containing the matrix Neu5Ac-α2,6-Galβ. p NP (0.6 mM), β-galactosidase (100 mU), and sialidase were tested repeatedly with or without different concentrations (0-20 mM) of inhibitors. The *Clostridium perfringens* reaction was allowed to proceed for 40 minutes to 2 hours, while the *Vibrio cholerae* reaction was 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 ).

[0182] Example 3: Preparation of 3F ax -Neu5Ac and Neu5Ac-modified homogeneous mAbs were used to investigate their effects on FcγRIIIa binding using surface plasma resonance (SPR) analysis.

[0183] Enzyme performance:

[0184] The endosidases Endo-S, Endo-S2, and Endo-S2 mutants (D184Q) in *E. coli* and those from *Bacteroides fragilis* ( Bacteroides fragilis Fucosidase from NCTC 9343 was obtained and purified using Ni-NTA agarose beads.

[0185] Preparation of single GlcNAc-rituximab:

[0186] As previously stated 32Rituximab (3.0 mg; Rituxan® Roche) in Tris-HCl buffer (50 mM, pH 7.4, 1.5 mL) was incubated at 37 °C for 24 h with Endo-S (120 μg), Endo-S2 (240 μg), and BfFucH (4.5 mg). LC-MS and SDS-PAGE analyses showed that the heavy chain... N - The glycan was completely broken down. 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 fractions containing the antibody were combined and concentrated by centrifugation (Amicon Ultra centrifuge filter, Millipore, Billerica, MA) to obtain rituximab monoclonal 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 single GlcNAc.

[0187] Transglycosylation of rituximab using polysaccharide-oxazoline:

[0188] Add the glycan-oxazoline to a mixture of Endo-S2 D184Q and rituximab mono-GlcNAc in 50 mM Tris buffer (pH 7.4). Incubate the solution at 37°C. o Incubate at C for 30 minutes. Then, purify the reaction mixture using a protein-A affinity column followed by a Capto Q anion exchange column (GE Healthcare) to collect the desired product.

[0189] SDS-PAGE assay of glycoengineered Herceptin antibody:

[0190] All SDS-PAGE analyses were performed using NuPAGE® Novex® 4–12% Bis-Tris gel (Invitrogen) in MOPS buffer, with 2-mercaptoethanol present in the samples. Figure 2 ).

[0191] MS spectral analysis of glycoengineered mAbs:

[0192] 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). The digestion solution (6 μL) was injected at a flow rate of 10 μL / min into a self-packed pre-column (150 μm ID x 20 mm, 5 μm, 100 Å). Chromatographic separation was performed on a self-filled reverse-phase C18 nanotube column (75 μm ID x 300 mm, 5 μm, 100 Å) 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. Full-scan MS conditions were investigated: mass range m / z 320–2000, with a resolution of 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. o C ( Figure 3 (and Table 3).

[0193] Surface plasma resonance (SPR) analysis

[0194] 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 a rate of 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.

[0195] Table 3: N - Correction for relative abundance of glycans 33

[0196]

[0197] Table 4: Binding affinity of glycoengineered rituximab IgG1 to FcγRIIIa measured by SPR

[0198]

[0199] Example 4: Relative Reaction Values ​​(RRV) of Compounds 4 and 18

[0200] 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) Perform the measurement.

[0201]

[0202] Other specific examples

[0203] 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.

[0204] 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.

[0205] References:

[0206] (1)Varki, A. Sialic acids in human health and disease. Trends Mol. Med. 2008, 14 , 351.

[0207] (2)(a) Liu, Y.-C.; Yen, H.-Y.; Chen, C.-Y.; Chen, C.-H.; Cheng, P.-F.; Juan, Y.-H.; Chen, C.-H.; Khoo, K.-H.; Yu, C.-J.; epidermal growth factorreceptor suppresses its dimerization and activation in lung cancer cells. Proc. Natl. Acad. Sci. USA 2011, 108 , 11332. (b) Yen, H.-Y.; Liu, Y.-C.; Chen, N.-Y.; Tsai, C.-F.; Wang, Y.-T.; Chen, Y.-J.; Hsu, T.-L.; Proc. Natl. Acad. Sci. USA 2015, 112 , 6955.

[0208] (3)(a) Ashwell, G.; Harford, J. Carbohydrate-specific receptors ofthe liver. Annu. Rev. Biochem. 1982, 51 , 531. (b) Weigel, PH; Yik, JHGlycans as endocytosis signals: the cases of the asialoglycoprotein andhyaluronan / chondroitin sulfate receptors. Biochim. Biophys. Acta 2002, 1572 ,341.

[0209] (4)(a) Wang, Z.; Chinoy , ZS ; Ambre, SG; Peng, W.; McBride , R. ;de Vries , RP ; Glushka , J. ; Paulson , JC ; Boons, GJ A general strategy for the chemoenzymatic synthesis of asymmetrically branched N -glycans. Science 2013, 341 , 379. (b) Shivatare, SS; Chang , SH ; Tsai, TI; Tseng, SY; Shivatare, VS; Lin , YS ; Cheng , YY ; Ren , CT ;Lee , CC ; Pawar , S. ; Tsai, CS; Shih , HW ; Zeng , YF ; Liang , CH ; Kwong , PD ; Burton , DR ; Wu, CY; Wong, CH Modular synthesis of N -glycans and arrays for the hetero-ligand binding analysis of HIVantibodies. Nat. Chem. 2016, 8 , 338. (c) Li, L.; Liu, Y.; Ma, C.; Qu, J.;Calderon, A. D.; Wu, B.; Wei, N.; Wang, X.; Guo, Y.; Xiao, Z.; Song, J.;Sugiarto, G.; Li, Y.; Yu, H.; Chen, X.; Wang, P. G. Efficient chemoenzymaticsynthesis of an N -glycan isomer library. Chem. Sci. 2015, 6 , 5652.

[0210] (5)Li, C.; Wang, L.-X. Chemoenzymatic Methods for the Synthesis ofGlycoproteins. Chem. Rev. 2018, 118 , 8359.

[0211] (6)(a) Lin, C.-W.; Tsai, M.-H.; Li, S.-T.; Tsai, T.-I.; Chu, K.-C.;Liu, Y.-C.; Lai, M.-Y.; Wu, C.-Y.; Tseng, Y.-C.; Shivatare, S. S.; Wang, C.-H.; Chao, P.; Wang, S.-Y.; Shih, H.-W.; Zeng, Y.-F.; You, T.-H.; Liao, J.-Y.;Tu, Y.-C.; Lin, Y.-S.; Chuang, H.-Y.; Chen, C.-L.; Tsai, C.-S.; Huang, C.-C.;Lin, N.-H.; Ma, C.; Wu, C.-Y.; Wong, C.-H. A common glycan structure onimmunoglobulin G for enhancement of effector functions. Proc. Natl. Acad. Sci. USA 2015, 112, 10611. (b) Tsai, T.-I.; Lee, S.-T.; Liu, C.-P.; Chen , KY ; Shivatare , SS ; Lin, C.-W.; Liao, S.-F.; Lin, C.-W.; Hsu , T.-L. ; Wu , Y.-T. ;Tsai , M.-H. ; Lai, M.-Y.; Lin, N.-H.; Wu, C.-Y.; Wong, C.-H. An EffectiveBacterial Fucosidase for Glycoprotein Remodeling. ACS Chem. Biol. 2017, 12 , 63.(c) Liu, C.-P.; Tsai, T.-I.; Cheng, T.; Shivatare, VS; Wu, C.-Y.; Wu, C.-Y.; Wong, C.-H. Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation. Proc. Natl. Acad. Sci. USA 2018, 115 , 720.

[0212] (7)(a) Gantt, R.; Millner , S. ; Binkley, SB Inhibition of N -Acetylneuraminic Acid Aldolase by 3-Fluoroshialic Acid. Biochemistry 1964, 3 ,1952. (b) Hagiwara, T.; Kijima-Suda, I.; Ido, T.; Ohrui, H.; Tomita, K.Inhibition of bacterial and viral sialidases by 3-fluoro- . N -acetylneuraminic acid. Carbohydr. Res. 1994, 263, 167. (c) Burkart, M. D.; Zhang, Z.; Hung, S.-C.; Wong, C.-H. A New Method for the Synthesis of Fluoro-Carbohydrates andGlycosides Using Selectfluor. J. Am. Chem. Soc. 1997, 119 , 11743. (d) D.Burkart, M.; P. Vincent, S.; Wong, C.-H. An efficient synthesis of CMP-3-fluoroneuraminic acid. Chem. Commun. 1999, 1525. (e) Burkart, M. D.; Vincent,S. P.; Duffels, A.; Murray, B. W.; Ley, S. V.; Wong, C.-H. Chemo-enzymaticsynthesis of fluorinated sugar nucleotide: useful mechanistic probes forglycosyltransferases. Bioorg. Med. Chem. 2000, 8 , 1937. (f) Sun, X.‐L.; Kanie,Y.; Guo, C.‐T.; Kanie, O.; Suzuki, Y.; Wong, C.‐H. Syntheses of C‐3‐ModifiedSialylglycosides as Selective Inhibitors of Influenza Hemagglutinin andNeuraminidase. Eur. J. Org. Chem. 2000, 2643. (g) Rillahan, C. D.;Antonopoulos, A.; Lefort, C. T.; Sonon, R.; Azadi, P.; Ley, K.; Dell, A.;Haslam, S. M.; Paulson, J. C. Global metabolic inhibitors of sialyl- andfucosyltransferases remodel the glycome. Nat. Chem. Biol. 2012, 8 , 661.

[0213] (8)Ishiwata, K.; Ido, T.; Nakajima, T.; Ohrui, H.; Kijima-Suda, I.;Itoh, M. Tumor uptake study of 18F-labeled N -acetylneuraminic acids.International journal of radiation applications and instrumentation. Int. J. Rad. Appl. Instrum. B 1990, 17 , 363.

[0214] (9)(a) Watts, A. G.; Damager, I.; Amaya, M. L.; Buschiazzo, A.;Alzari, P.; Frasch, A. C.; Withers, S. G. Trypanosoma cruzi Trans-sialidaseOperates through a Covalent Sialyl-Enzyme Intermediate:  Tyrosine Is theCatalytic Nucleophile. J. Am. Chem. Soc. 2003, 125 , 7532. (b) Buchini, S.;Gallat, F. X.; Greig, I. R.; Kim, J. H.; Wakatsuki, S.; Chavas, L. M.;Withers, S. G. Tuning Mechanism-Based Inactivators of Neuraminidases:Mechanisticand Structural Insights. Angew. Chem. Int. Ed. Engl. 2014, 53, 3382.(c) Kim, JH; Resende, R.; Wennekes , T. ; Chen , HM ; Bance, N.; Buchini,S.; Watts , AG ; Pilling , P. ; Streltsov, VA; Petric, M.; Liggins , R. ;Barrett , S. ; McKimm-Breschkin , JL ; Niikura, M.; Withers, SG Mechanism-Based Covalent Neuraminidase Inhibitors with Broad-Spectrum InfluenzaAntiviral Activity. Science 2013, 340 , 71.

[0215] (10)Tsai, CS; Yen, HY; Lin, MI; Tsai, TI; Wang , SY ; Huang , WI ; Hsu, TL; Cheng , YS ; Fang , JM ; Wong, C.-H. Cell-permeable probe for identification and imaging of sialidases. Proc. Natl. Acad. Sci. USA 2013, 110 , 2466.

[0216] (11)(a) Chokhawala, HA; Cao, H.; You, H.; Chen, X. EnzymaticSynthesis of Fluorinated Mechanistic Probes for Sialidases andSialyltransferases. J. Am. Chem. Soc. 2007, 129, 10630. (b) McArthur, J. B.; Yu,H.; Zeng, J.; Chen, X. Converting Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) to a regioselective α2,6-sialyltransferase bysaturation mutagenesis and regioselective screening. Org. Biomol. Chem. ,2017, 15 , 1700.

[0217] (12)Petrie, C. R.; Sharma, M.; Simmons, O. D.; Korytnyk, W. Synthesisof analogs of N -acetylneuraminic acid and their effect on CMP-sialatesynthase. Carbohydr. Res. 1989, 186 , 326.

[0218] (13)Nakajima, T.; Hori, H.; Ohrui, H.; Meguro, H.; Ido, T. Synthesisof N -Acetyl-3-fluoro-neuraminic Acids Agric. Biol. Chem. 1988, 52 , 1209.

[0219] (14)Watts, A. G.; Withers, S. G. The synthesis of some mechanisticprobes for sialic acid processing enzymes and the labeling of a sialidasefrom Trypanosoma rangeli. Can. J. Chem 2004, 82 , 1581.

[0220] (15)Hayashi, T.; Kehr, G.; Gilmour R. Stereospecific α‐sialylation bysite‐selective fluorination. Angew. Chem. Int. Ed. Engl. 10.1002 / anie.201812963.

[0221] (16)Okamoto, K.; Kondo, T.; Goto, T. An effective synthesis of α-glycosides of N -acetylneuraminic acid derivatives by use of 2-deoxy-2β-halo-3β-hydroxy-4,7,8,9-tetra- O - acetyl- N -acetylneuraminic acid methylester. Tetrahedron 1987, 43 , 5919.

[0222] (17)Bennua-Skalmowski, B.; Vorbrüggen, H. A facile conversion ofprimary or secondary alcohols with n-perfluorobutane-sulfonyl fluoride / 1,8-diazabicyclo[5.4.0]undec-7-ene into their corresponding fluorides. Tetrahedron Lett. 1995, 36 , 2611.

[0223] (18)Cao, H.; Li, Y.; Lau, K.; Muthana, S.; Yu, H.; Cheng, J.;Chokhawala, H. A.; Sugiarto, G.; Zhang, L.; Chen, X. Sialidase substratespecificity studies using chemoenzymatically synthesized sialosidescontaining C5-modified sialic acids. Org. Biomol. Chem.,2009, 7 , 5137.

[0224] (19)Orlova, A. V.; Shpirt, A. M.; Kulikova, N. Y.; Kononov, L. O. N , N -Diacetylsialyl chloride—a novel readily accessible sialyl donor in reactionswith neutral and charged nucleophiles in the absence of a promoter. Carbohydr. Res. 2010, 345 , 721.

[0225] (20)Okamoto, K.; Kondo, T.; Goto. T. Functionalization of 2-Deoxy-2,3-dehydro- N -acetylneuraminic Acid Methyl Ester. Bull. Chem. Soc. Jpn. 1987, 60 ,631.

[0226] (21)Pascolutti, M.; Madge, P. D.; Thomson, R. J.; von Itzstein, M.Access to 3- O -Functionalized N-Acetylneuraminic Acid Scaffolds. J. Org. Chem. 2015, 80 , 7746.

[0227] (22)Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Kulkarni, S. S.; Huang, Y.-W.; Lee, C.-C.; Chang, K.-L.; Hung, S.-C. Regioselective one-pot protectionof carbohydrates. Nature 2007, 446 , 896.

[0228] (23)Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.;Wong, C.-H. Programmable One-Pot Oligosaccharide Synthesis. J. Am. Chem. Soc. 1999, 121 , 734.

[0229] (24)Huang, L.; Huang, X. Highly Efficient Syntheses of HyaluronicAcid Oligosaccharides. Chem. Eur. J. 2007, 13 , 529.

[0230] (25)Shivatare, S. S.; Chang, S.-H.; Tsai, T.-I; Ren, C.-T.; Chuang,H.-Y.; Hsu, L.; Lin, C.-W.; Li, S.-T.; Wu, C.-Yi; Wong, C.-H. EfficientConvergent Synthesis of Bi-, Tri-, and Tetra-antennary

[0231] (26)Complex Type N-Glycans and Their HIV-1 Antigenicity. J. Am. Chem. Soc. 2013, 135 , 15382.

[0232] (27)Shivatare, S. S.; Chang, S.-H.; Tsai, T.-I; Tseng, S. Y.;Shivatare, V. S.; Lin, Y.-S.; Cheng, Y.-Y.; Ren, C.-T.; Lee, C.-C. D.; Pawar,S.; Tsai, C.-S.; Shih, H.-W.; Zeng, Y.-F.; Liang, C.-H.; Kwong, P. D.;Burton, D. R.; Wu, C.-Y.; Wong, C.-H. Modular synthesis of N-glycans andarrays for the hetero-ligand binding analysis of HIV antibodies. Nat. Chem. 2016, 8 , 338.

[0233] (28)Mong, TK-K.; Huang, C.-Y.; Wong, C.-H. A New Reactivity-BasedOne-Pot Synthesis of N -Acetyllactosamine Oligomers. J. Org. Chem. 2003, 68 , 2135.

[0234] (29)Dinkelaar, J.; Duivenvoorden, BA; Wennekes, T.; Overkleeft, HS; Boot, RG; Aerts, JMFG; Codée, JDC; van der Marel, GAAPreparative Synthesis of Human Chitinase Fluorogenic Substrate (4'-Deoxychitobiosyl)-4-methylumbelliferone. Eur. J. Org. Chem. 2010, 2565.

[0235] (30)Chayajarus, K.; Chambers, DJ; Chughtai, MJ; Fairbanks, AJ Stereospecific Synthesis of 1,2-cis Glycosides by Vinyl-Mediated IAD. Org. lett. 2004, 6 , 3797-3800.

[0236] (31)Cao, H.; Li, Y.; Lau, K.; Muthana, S.; Yu, H.; Cheng, J.;Chokhawala, HA; Sugiarto, G.; Zhang, L.; Chen, X. Sialidase substratespecificity studies using chemoenzymatically synthesized sialosidescontaining C5-modified sialic acids. Org. Biomol. Chem., 2009, 7 , 5137.

[0237] (32) Lin, C.-W.; Tsai, M.-H.; Li, S.-T.; Tsai, T.-I.; Chu, K.-J.;Liu, Y.-J.; Lai, M.-Y.; Wu, J.-Y.; Tseng, Y.-J.; Shivatare, SS; Wang, C.-H.; Chao, P.; Wang, S.-Y.; Shih, H.-W.; Zeng, Y.-F.; You, T.-H.; Liao, J.-Y.;Tu, Y.-J.; Lin, Y.-S.; Chuang, H.-Y.; Chen, C.-L.; Tsai, C.-S.; Huang, C.-C.;Lin, N.-H.; Ma, C.; Wu, J.-Y.; Wong, C.-H. A common glycan structure onimmunoglobulin G for enhancement of effector functions. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 , 10611.

[0238] (33)Stavenhagen, K.; Hinneburg, H.; Thaysen-Andersen, M.; Hartmann,L.; Silva, D. V.; Fuchser, J.; Kaspar, S.; Rapp, E.; Seebergera, P. H.;Kolaricha D. Quantitative mapping of glycoprotein micro-heterogeneity andmacro-heterogeneity: an evaluation of mass spectrometry signal strengthsusing synthetic peptides and glycopeptides. J. Mass Spectrom. 2013, 48 , 627.

[0239] (34) Lee, J.-C.; Greenberg, W. A; Wong, C.-H. Programmablereactivity-based one-pot oligosaccharide synthesis. Nat. Protoc. 2006, 1 , 3143.

Claims

1. A compound of formula (VI): ， 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 N-glycan.

2. The compound of claim 1, wherein the compound has formula (IV): 。 3. The compound of claim 1, wherein X is an N-polysaccharide.

4. A monoclonal antibody that binds to a sugar containing 3-fluorosialic acid, wherein the sugar containing 3-fluorosialic acid has formula (VI): ， 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 an N-glycan.

5. The monoclonal antibody of claim 4, wherein the 3-fluorosialic acid-containing sugar is an N-glycan terminated by an α2,6-linked 3-fluoro-sialic acid glycoside, wherein the 3-fluorosialic acid-containing sugar has the structure of formula (V): ， Where Z is or .

6. The monoclonal antibody as described in claim 4 or 5, wherein the monoclonal antibody is rituximab.

7. Use of the monoclonal antibody as described in claim 6 in the preparation of a medicament for treating leukemia, lymphoma, or autoimmune diseases.

8. The use as described in claim 7, wherein the autoimmune disease is 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, autoimmune pancreatitis, and opsoclonusmyoclonus syndrome. (syndrome) or IgG4-related diseases.

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