Sialidase-resistant saccharide and method of preparing and using the same

Abstract

To provide a method of preparing a saccharide that contains 3-fluoro-sialic acid and a method of bonding it to a homogeneous antibody.SOLUTION: Provided is a monoclonal antibody having a saccharide containing 3-fluoro-sialic acid, wherein the saccharide containing 3-fluoro-sialic acid is prepared by the steps of: conducting a glycosylation reaction by reacting 3-hydroxy-sialic acid with a saccharide to form an α2,6-linked 3-hydroxy-sialoside; and conducting a fluorination reaction by reacting the α2,6-linked 3-hydroxy-sialoside with a fluorinating agent to form the saccharide containing 3-fluoro-sialic acid.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] Sialic acids are negatively charged monosaccharides that are often represented at the outermost termini of glycans on glycolipids and glycoproteins and are involved in many physiological intra- and intercellular processes, including interactions with other biomolecules and receptors on cells, viruses and bacteria. 1 Sialylation also plays an important role in regulating the function and fate of secreted glycoproteins and membrane-bound receptors. For example, sialylation of the epidermal growth factor receptor (EGFR) has been shown to inhibit EGFR dimerization, thus interfering with EGF binding and phosphorylation, which has been linked to tumorigenesis. 2 Sialylation also regulates the half-life of glycoproteins in the blood circulation, as desialylation of N-glycans exposes the underlying galactose, which is recognized by the hepatic asialoglycoprotein receptor, resulting in rapid clearance of glycoproteins from the circulation. 3 Therefore, increasing the degree of sialylation may increase the half-life and undesirable effects of glycoprotein therapeutics.

[0002] Recently, the role of glycosylation on protein structure and function has been intensively investigated, encouraging the development of new methods for glycan synthesiscampillary and glycoengineering of proteins, especially therapeutic monoclonal antibodies (mAbs). 5 For example, defucosylated biantennary N-glycans with terminal α-2,6-linked sialic acids have been shown to have optimal glycan structures for enhanced antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and anti-inflammatory activity. 6 .

[0003] Sialic acid derivatives with a fluorine atom at the C-3 position are known to inhibit sialyltransferase and sialidase (or neuraminidase) and are more stable. 7However, glycosylation with fluorosugar donors often poses significant challenges due to the strong electronic effect of the fluorine group, which inactivates the glycosylation reaction. Although sialyltransferases exist that can transfer 3-fluoro-sialic acid residues from the corresponding cytidine monophosphate-sialic acid to form α-2,3-linked sialosides, there are no corresponding α-2,6-sialyltransferases available. 11 .

[0004] Thus, there is a need to provide methods for reliably preparing saccharide-containing α-2,6-linked 3-fluoro-sialosides that can be used to develop glycoprotein therapeutics. Summary of the Invention

[0005] An embodiment of the invention is a method for preparing a saccharide comprising 3-fluoro-sialic acid, the method comprising the steps of performing a glycosylation reaction by reacting 3-hydroxy-sialic acid with a saccharide to form an α2,6-linked 3-hydroxy-sialoside, and performing a fluorination reaction by reacting the α2,6-linked 3-hydroxy-sialoside with a fluorinating agent to form a saccharide comprising 3-fluoro-sialic acid.

[0006] Another aspect of the invention is a method for preparing a homogeneous antibody conjugated to a sugar that contains 3-fluoro-sialic acid by glycosylating a monoclonal antibody with the sugar.

[0007] A further aspect of the present invention is a compound of formula (VI): [ka] (In the formula, R 1 is Ac or H, and R 2 is Bz or H, and R 3 is methyl or H, R 4is Bn or H, and X is OH, a leaving group, or a sugar, where "Ac" stands for acetyl, "Bz" stands for benzoyl, "Bn" stands for benzyl, and the sugar may be a monosaccharide, disaccharide, or oligosaccharide.

[0008] Monoclonal antibodies bound to α2,6-linked 3-fluoro-sialoside terminated N-glycans are also within the scope of the present invention.

[0009] Further encompassed by the present invention is a method of treating cancer comprising administering to a subject in need thereof an effective amount of a monoclonal antibody conjugated to an α2,6-linked 3-fluoro-sialoside terminal N-glycan.

[0010] The details of the invention are described in the following detailed description of the invention. Other features, objects and advantages of the invention will become apparent from the following detailed description of several embodiments and the appended claims.

[0011] The following description refers to the accompanying drawings. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1A shows two graphs depicting the enzyme-catalyzed hydrolysis of three sialosides, namely 1, 2 and S27, as a function of enzyme concentration, and FIG. 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. [Diagram 2] Stained electrophoresis gels of four N-glycans: (1) Rituxan-G, (2) Rituxan-Fax-SCT, (3) Rituxan-SCT, and (4) commercial Rituxan. [Diagram 3] Mass spectra obtained for three types of N-glycans, Rituxan-Fax-SCT, Rituxan-SCT and Rituxan, are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Disclosed in detail herein for the first time is a method for preparing saccharides containing 3-fluoro-sialic acid.

[0014] Again, the method includes the steps of performing a glycosylation reaction by reacting 3-hydroxy-sialic acid with a sugar to form an α2,6-linked 3-hydroxy-sialoside, and performing a fluorination reaction by reacting the α2,6-linked 3-hydroxy-sialoside with a fluorinating agent to form a sugar comprising 3-fluoro-sialic acid. The sugar may be a monosaccharide, a disaccharide, or an oligosaccharide. In an exemplary method, the sugar is a monosaccharide.

[0015] In a particular embodiment of the method, the 3-hydroxy-sialic acid is 2-bromo-3-hydroxy-sialic 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.

[0016] In another embodiment of the 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-diazabicyclo[5,4,0]-undec-7-ene (DBU). Preferably, the fluorination reaction is carried out in toluene. However, the reaction can be further carried out in the presence of tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF).

[0017] In an exemplary method, 3-hydroxy-sialic acid is reacted with a compound of formula (I): [ka] and The sugar is a compound of formula (II): [ka] and The α2,6-linked 3-hydroxy-sialoside is a compound of formula (III): [ka] and The sugar containing 3-fluoro-sialic acid is a compound of formula (IV): [ka] It is.

[0018] In a further embodiment, the method further comprises performing another glycosylation reaction by reacting the sugar comprising 3-fluoro-sialic acid with a second sugar.

[0019] Also within the scope of the invention is a method for preparing a homogeneous antibody conjugated to a sugar that contains 3-fluoro-sialic acid, again comprising the step of glycosylating a monoclonal antibody with a sugar that contains 3-fluoro-sialic acid.

[0020] In certain embodiments of the method, the saccharide comprising 3-fluoro-sialic acid is obtained by the above method and its embodiments, which include the steps of performing a glycosylation reaction by reacting 3-hydroxy-sialic acid with a saccharide to form an α2,6-linked 3-hydroxy-sialoside, and performing a fluorination reaction by reacting the α2,6-linked 3-hydroxy-sialoside with a fluorinating agent to form a saccharide comprising 3-fluoro-sialic acid.

[0021] In other embodiments, the saccharide comprising 3-fluoro-sialic acid is an α2,6-linked 3-fluoro-sialoside terminal N-glycan, which is represented by the compound of formula (V): [ka] may be In the formula, Z is [ka] It is.

[0022] Another aspect of the present invention is a compound of formula (VI): [ka] Regarding R 1 , R 2 , R 3 , R 4 and X are defined in the Summary section above.

[0023] In one embodiment, the compound is a compound of formula (IV): [ka] It is.

[0024] In yet another embodiment, the compounds of formula (VI) each comprise an N-glycan, X. Exemplary compounds of this embodiment are compounds of formula (V): [ka] and In the formula, Z is [ka] It is.

[0025] The present invention also encompasses monoclonal antibodies conjugated to a saccharide comprising 3-fluoro-sialic acid, the saccharide comprising 3-fluoro-sialic acid being an α2,6-linked 3-fluoro-sialoside terminal N-glycan. Preferably, the α2,6-linked 3-fluoro-sialoside terminal N-glycan is a compound of formula (V) shown above.

[0026] The present invention further encompasses a method for treating cancer or autoimmune disease, which comprises administering an effective amount of the monoclonal antibody to a subject in need thereof.For example, the cancer may be leukemia or lymphoma, and the autoimmune disease may be rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura leukemia, lymphoma, Evans syndrome, vasculitis, bullous dermatosis, type 1 diabetes, Sjogren syndrome, anti-NMDA receptor encephalitis, Devic's disease, Graves' ophthalmopathy, autoimmune pancreatitis, opsoclonus-myoclonus syndrome, or IgG4-related disease.

[0027] The term "treat" or "treatment" as used herein refers to administering the pharmaceutical composition to a subject having the disease, i.e., cancer, symptoms of such disease, or predisposition to such disease, for the purpose of providing a therapeutic or prophylactic effect. The term "effective amount" refers to the amount of active drug required to provide such an effect. Effective doses will vary depending on the type of disease being treated, route of administration, excipient use, and the possibility of co-use with other therapeutic treatments, as recognized by those skilled in the art.

[0028] A more detailed description of certain specific embodiments of the invention is provided below.

[0029] 3-Fluoro-substituted sialic acid derivatives are known to be more stable to sialidases than non-fluorinated sialic acid derivatives. 7 By introducing a fluorine atom at the C-3 position of the anomeric form, 2,3-difluorosialic acid (DFSA) was developed (Scheme 1). 8 , biochemical probes 9 and activity-based protein profiling probes for studying sialidases 10 With the help of DFSA, the fluorine atom (3F ax ) but on the 3rd floor eq Deactivation of the enzyme (k i ) and reactivation (k hydr) had a larger effect on the deceleration of both 8b Inspired by this observation, we wished to investigate the stability of sialosides using 3-fluorosialic acid for their potential use in glycoprotein therapeutics. Specifically, we attached 3F to the termini of N-glycans on mAbs. ax It is our intention to investigate whether the incorporation of a -Neu5Ac motif can increase its stability towards sialidases while still maintaining its effector function.

[0030] Towards our goal, we have isolated an α2,6-linked 3F-glycan complex that contains biantennary N-glycans. ax We have developed a preparative method for the synthesis of 3-F-Neu5Ac oligosaccharides. ax It was also demonstrated that the Neu5Ac-α2,6-Gal bond was more stable in the presence of sialidase, and antibodies bearing the corresponding biantennary glycans had the same binding affinity as their non-fluorinated counterparts.

[0031] (a)XeF2-BF3×OEt2 12 , molecular fluorine 13 and fluorination of protected glycans with Selectfluor 7c , (b) inversion of the equatorial hydroxyl group at C3 in sialic acid derivatives 7f , and (c) 3F of ManNAc and 3-fluoro-pyruvate. eq Neu5Ac and 3F ax Aldolase-catalyzed enzymatic conversion to Neu5Ac 7a、11a、14 Several methods for the synthesis of 3F-Neu5Ac have been reported, including ax No method has been described so far for the synthesis of -Neu5Ac-terminated N-glycans. ax The only description disclosing the preparation of a disaccharide containing -Neu5Ac is 3-F ax Limited to enzymatic synthesis of Neu5Ac-α2,3-Lac, not the required α2,6-linked form 11aTherefore, we focused our efforts on the chemical synthesis of this bond. 15 . [ka]

[0032] 3F ax After screening various glycosylation conditions using the -Neu5Ac-based donor (Scheme 2a), we found that the 3OH-Neu5Ac-α2,6-Gal-STol eq 3F ax to S N Other strategies involving the two reactions were investigated (Scheme 2b). First, we investigated sialylation at O-6 using the conditions reported by Goto et al. (Table 1, entry 1). 16 The reaction was carried out in toluene at -10 °C with 1 equivalent of AgOTf as promoter and Na2HPO4 as base (Scheme 3). These conditions afforded the disaccharide 6 in low yield (15%) with an anomeric ratio of 3:1 (α:β). Changing the solvent to CH2Cl2 did not improve the diastereoselectivity (entry 2). By increasing the temperature and reaction time, the yield increased, but the selectivity decreased dramatically (entries 3-5). Surprisingly, better stereoselectivity was observed when more equivalents of acceptor were used (entry 3 vs. 6). Optimized conditions (entry 7) afforded 6 in 35% (99% brsm) yield with excellent α-selectivity (α:β=13:1). [ka] [Table 1]

[0033] OH eq F axThe inversion to proved to be a difficult task. Replacement of OTf and OMs with fluorine using TASF resulted in the decomposition of the starting material. To optimize this transformation, we screened various fluorination reagents without any success. However, treatment of 6 with perfluoro-1-butanesulfonyl fluoride (NfF) in the presence of 1,8-diazabicyclo[5,4,0]-undec-7-ene (DBU) in anhydrous toluene at 90 °C for 2 days (Scheme 3) afforded 4 in 6% yield (Table 2, entry 1). 17 Further optimization of reaction conditions, such as decreasing the reaction temperature (entries 1, 5, and 6) and increasing the reaction time (entry 7), improved the overall yield of 4. The conversion of 7 to 4 appears to be the rate-limiting step due to steric hindrance around C-3. In many cases, we were able to isolate 7, formed at room temperature and in the presence of NfF and DBU, within a day. However, the conversion of 7 to 4 (77% brsm yield) requires a long reaction time. Attempts to improve the conversion by increasing the reaction temperature resulted in the decomposition of 7. Thus, our best results were obtained by increasing the amount of reagents and extending the reaction time to 15 days (entry 8). Finally, we found that the addition of TASF helped to increase the reaction efficiency, thereby shortening the reaction time to only 2 days (entry 9). The fully protected 3F ax The stereochemistry of the -Neu5Ac-α2,6-Gal-STol disaccharide (4) was confirmed by X-ray diffraction analysis. [ka] [Table 2]

[0034] Referring to Scheme 5, NIS / TMSOTf was used to synthesize 3F axThe -Neu5Ac-disaccharide donor 4 was coupled to the acceptor (8) to give 9. The O-allyl group at the anomeric position was then removed by isomerization with PdCl2 in AcOH / NaOAc, and the anomeric hydroxyl group (10) was further converted to the fluoride (11) and imidate (12). ax Glycosylation of the core disaccharide (13) at O-3 with -Neu5Ac terminal fluoride donor (11) gave the hexasaccharide 14 in 85% yield. After removal of the benzylidene group, 15 was glycosylated at the O-6 position to give the desired decasaccharide (16) in 70% yield with excellent regio- and α-stereoselectivity. We also tested TfOH-promoted glycosylation with N-phenyltrifluoroacetimidate donor (12), but this gave poor yields of product. The following sequence of steps was then carried out: (a) saponification with LiOH to remove the ester and NHTroc groups, (b) acetylation of the free amines and alcohols, (c) removal of the OAc groups with sodium methoxide, and (d) hydrogenolysis of the O-benzyl groups with Pd / C in a MeOH / water / HCOH mixture. 4b The fully deprotected glycan (17) was obtained in 40% overall yield according to the procedure described above. [ka]

[0035] Reagents and conditions in Scheme 5: (a) 8, TfOH, NIS, MS 4 Å, CH2Cl2, -40 °C, 2 h, 64%. (b) PdCl2, CH3COONa, AcOH / H2O, 82%. (c) DAST, CH2Cl2, -20 °C, 73%. (d) ClC(=NPh)CF3, Cs2CO3, CH2Cl2, 0 °C to room temperature, 3 h, 56%. (e) 11, AgOTf, Cp2HfCl2, toluene, MS 4 Å, 0 °C, 3 h, 85%. (f) pTSA·H2O, CH3CN, room temperature, 6 h, 75%. (g) 11, AgOTf, Cp2HfCl2, toluene, MS 4 Å, -15 °C, 3 h, 70% (80% brsm). (h) 12, TfOH, CH2Cl2, 4 Å MS, -60 to -20 °C, 3 h, 33% (55% brsm). (i) LiOH, dioxane / H2O (4:1), 90 °C, 16 h. (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).

[0036] After establishing the protocol for the stepwise synthesis, we streamlined the assembly of glycans by developing a programmable [2+2+2] one-pot synthesis of the precursor hexasugar (14) of 17 (Scheme 6). The designed one-pot protocol involves the 3F ax It consisted of the initial coupling of the -Neu5Ac-disaccharide donor (4) (RRV = 2053) with the less reactive acceptor (18) (RRV = 537), followed by injection of the reducing end acceptor 13 at -20 °C. After 1 h at -10 °C and a standard purification protocol, the hexasaccharide 14 was isolated in 26% yield. [ka]

[0037] Reagents and conditions in Scheme 6: (a) TfOH, NIS, 4 Å MS, CH2Cl2, -40°C to -10°C, 3 h, 26%.

[0038] 3F in the presence of sialidase axTo gather preliminary data on the stability of the -Neu5Ac-α2,6-Gal motif, we performed in vitro assays using commercially available sialidases from Clostridium perfringens and Vibrio cholerae. 19 For this purpose, (1) Neu5Ac-α2,6-Gal-pNP and (2) 3F ax The -Neu5Ac analog was prepared as a substrate (Scheme 1). Both enzymes showed the expected hydrolytic activity towards the native substrate 1, but 3F ax -analog 2 (see FIG. 1A). We also observed that 2 did not significantly inhibit the hydrolysis of 1 like DANA did (see FIG. 1B).

[0039] Standard Protocol to Prepare Homogeneous Glycoforms of mAbs 6 Compound 17 was converted to an oxazoline donor according to and coupled to a GlcNAc-modified IgG (containing no core fucose) in the presence of Endo S2(D184Q). ax The binding affinity of the -Neu5Ac-glycoform to FcγRIIIa was analyzed by surface plasmon resonance analysis together with the parent non-fluorinated glycoform (G2S2) and a commercial sample of rituximab (major glycoforms: G1F1, G0F1, G2F1). 6 When compared to a commercial sample of rituximab, the homogenous glycoform of IgG with biantennary N-glycans containing α2,6-sialylation and no core fucose exhibited a 39.9-fold (Neu5Ac-G2S2) and 37.4-fold (3F ax -Neu5Ac-G2S2) (see Table 4). ax The fact that the binding affinities of the -Neu5Ac modified glycoforms were found to be in the same range as the parent glycans supports the conclusion that 3F ax This provides a basis for in vivo studies of -Neu5Ac-glycosylated mAbs, and these results will be reported in due course.

[0040] In conclusion, we have demonstrated that sialidase-resistant oligosaccharides and 3F ax3F as a building block for the synthesis of a representative homogenous antibody containing -Neu5Ac-terminal biantennary N-glycan ax We developed a chemical synthesis of -Neu5Ac-α2,6-Gal-STol. Compared with the commercially available rituximab sample, 3F ax The homogeneous glycoform modified with -Neu5Ac-glycans showed a 37.4-fold improvement in binding to FcγRIIIa. ax -Neu5Ac modified antibody glycoforms displayed similar binding affinity to Fc receptors. Overall, our results reveal a new general strategy for the half-life enhancement of therapeutic glycoproteins.

[0041] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. Thus, the following specific examples are merely illustrative, and should not be construed as limiting in any way to the remainder of the disclosure. All documents cited herein are incorporated by reference in their entirety.

[0042] Example 1: Synthesis of 3-fluoro-sialic acid-containing sugars and precursors All reactions were carried out under an inert atmosphere unless otherwise stated, followed by standard syringe-septum techniques. Solvents were purchased from commercial sources and used without further purification. Fine molecular sieves 4 Å (EMD Millipore) were ground to a powder and activated before use. The progress of all reactions was monitored by TLC glass plates precoated with silica gel 60 F254 (Merck KGaA). TLC was visualized by ultraviolet light (254 nm), p-anisaldehyde and / or ammonium cerium molybdate staining. Column chromatography was performed on Across silica gel (particle size 0.035-0.070 mm, 60 Å). 1 H, 13 C and 19F NMR spectra were recorded on Bruker AVIII-600, DRX-500, AV-400, DPX-400, AVANCE 500 AV and AVANCE 600 spectrometers at 25 °C, and chemical shifts were measured in δ (ppm) using the residual solvent peak as internal standard (δ, 1 H NMR ppm: 7.24 (CHCl3), 4.80 (H2O), 13 77(CDCl3) for C NMR. Coupling constants (J) are measured in Hz. Data are reported as "chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad)". High resolution mass spectra (HRMS) were recorded on an Agilent LC / MSD TOF mass spectrometer for electrospray ionization time-of-flight (ESI-TOF) reflectron experiments. Cu K α Bruker D8 Platinum- with radiation (λ=1.5478) 135 Single crystal X-ray diffraction studies were performed on a CCD diffractometer. HPLC measurements were performed on a Hitachi HPLC D-7000 system. RRV measurements were recorded using normal phase ZORBAX RX-SIL, 5 μm, 4.6 × 250 mm (colloidal silica, Agilent Technologies) with the solvent system EtOAc / Hexane at a flow rate of 1 mL / min and visualized at 254 nm.

[0043] [ka] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-D-erythro-α-L-gluco-non-2-ulopyranosonic acid methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 6α. A mixture of acceptor 3 (1.05 g, 1.80 mmol, 1 equiv.) and donor 5 (1.02 mg, 1.80 mmol, 1 equiv.) in anhydrous toluene (60 mL) was stirred under argon atmosphere for 10 min. The reaction mixture was then cooled to −50° C. and dry Na2HPO4 (1.07 g, 7.54 mmol, 4.2 equiv.) was added followed by AgOTf (692 mg, 2.69 mmol, 1.5 equiv. in 18 mL toluene) with stirring. Upon completion of the reaction (TLC showed disappearance of starting material after 24 hours), the reaction mixture was diluted with EtOAc (80 mL) and washed with 20% aqueous Na2S2O3 (10 mL), saturated aqueous NaHCO3 (5 mL) and brine (5 mL), then the organic layer was dried over MgSO4, filtered and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (3:7) as eluent to give compound 6 (α:β=13:1) as a white powder (680 mg, 35%) along with recovered acceptor 3 (670 mg, 99% brsm). f = 0.36 (silica gel, CHCl3:MeOH = 20:1); 1 H NMR (600MHz, CDCl3): δ7.96-7.94(m,2H,Ar-H),7.91-7.90(m,2H,Ar-H),7.50-7.47(m,2H,Ar-H ),7.40-7.39(m,2H,Ar-H),7.37-7.32(m,5H,Ar-H),7.25-7.18(m,4H,Ar-H),7.06-7.04(m,2H, Ar-H),5.81(dd,J=10.2,9.6Hz,1H),5.59(d,J=10.2Hz,1H),5.38(dd,J=9.0,3.0Hz,1H),5.35- 5.32(m,1H),5.25(dd,J=10.2,9.6Hz,1H),5.24(d,J=7.8,2.4Hz,1H),4.92(d,J=9.6Hz,1H,C1-H β),4.71(d,J=12.0Hz,1H),4.60(d,J=12.0Hz,1H),4.49(dd,J=10.8,1.8Hz,1H),4.30(dd,J=12.6,3.0Hz,1H) ,4.26(ddd,J=10.2,10.2,10.2Hz,1H),4.20(d,J=3.0Hz,1H),4.10(dd,J=10.2,7.2Hz,1H),4.04(dd,J=12.6 ,6.6Hz,1H),4.00(dd,J=6.6,6.6Hz,1H),3.86(d,J=9.6Hz,1H),3.82-3.73(m,2H),3.75(s,3H,-CH3),2.30( s,3H,-CH3),2.09(s,3H,-CH3),2.07(s,3H,-CH3),2.06(s,3H,-CH3),1.96(s,3H,-CH3),1.89(s,3H,-CH3); 13 C NMR(150MHz,CDCl3):δ171.6,170.6,170.3,170.1,169.5,168.5,165.8,165.1, 138.0,137.9,133.4,133.1,129.8,129.7,129.6,128.9,128.6,128.4,128.3,12 8.2,127.7,127.6,100.3,86.6,75.9,74.5,73.7,73.2,73.1,72.5,68.8,68.3, 63.1,62.5,52.8,48.4,23.1,21.2,20.9,20.8,20.7,20.7;HRMS(ESI-TOF)m / e:C 54 H 59 NO 20 SNa[M+Na] + Calculated value: 1096.3243 / Measured value: 1096.3241.

[0044] TIFF2025063181000021.tif22 [ka] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-D-erythro-β-L-gluco-non-2-ulopyranosonic acid methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 6β. β-anomer 6β:Rf =0.42(シリカゲル、CHCl3:MeOH=20:1); 1 H NMR(600MHz,CDCl3):δ7.94-7.92(m,4H,Ar-H),7.50-7.47(m,2H,Ar-H),7.43-7.40(m,6H,Ar-H),7.37-7.31(m,5H,Ar-H),7.13-7.12(m,2H,Ar-H),5.81(dd,J=10.2,10.2Hz,1H),5.51(dd,J=10.2,3.0Hz,1H),5.22(ddd,J=6.0,6.0,3.0Hz,1H),5.14(d,J=13.2Hz,1H),5.07(dd,J=6.0,2.4Hz,1H),4.90(d,J=10.2Hz,1H,C1-H β ),4.66-4.62(m,2H),4.55(dd,J=12.6,2.4Hz,1H),4.29(d,J=1.8Hz,1H),4.14(dd,J=7.8,7.2Hz,1H),4.08(ddd,J=10.8,10.8,10.8Hz,1H),3.98(dd,J=12.6,6.6Hz,1H),3.92-3.88(m,2H),3.85-3.77(m,3H),3.83(s,3H,-CH3),3.53(dd,J=10.8,2.4Hz,1H),2.32(s,3H,-CH3),2.26(s,3H,-CH3),2.07(s,3H,-CH3),2.04(s,3H,-CH3),1.97(s,3H,-CH3),1.68(s,3H,-CH3); 13 C NMR(150MHz,CDCl3):δ171.4,170.6,170.3,169.8,169.8,166.6,165.8,165.2,139.2,138.4,133.5,133.4,133.1,129.9,129.8,129.5,128.9,128.5,128.3,127.2,126.0,99.6,86.9,76.0,75.5,74.3,73.1,72.5,71.7,70.3,68.4,67.2,62.1,62.0,53.3,47.5,22.9,21.5,21.2,20.8,20.7,20.7;HRMS(ESI-TOF)m / e:C 54 H 59 NO 20 S[M+H] +Calculated value: 1074.3424 / Measured value: 1074.3458.

[0045] [ka] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 4. To a solution of compound 6α (500 mg, 0.47 mmol, 1 equiv.) in toluene (5 mL) in a screw-capped vial containing a stir bar, DBU (0.28 mL, 1.86 mmol, 4 equiv.), perfluoro-1-butanesulfonyl fluoride (0.33 mL, 1.86 mmol, 4 equiv.) and TASF (256 mg, 0.93 mmol, 2 equiv.) were added. The reaction vial was sealed and stirred at 40 °C. After 24 h, the reaction mixture was treated with additional amounts of DBU (0.28 mL, 1.86 mmol, 4 equiv.), perfluoro-1-butanesulfonyl fluoride (0.33 mL, 1.86 mmol, 4 equiv.) and TASF (256 mg, 0.93 mmol, 2 equiv.) and then stirred at 40° C. for another 24 h. The reaction mixture was directly loaded onto a silica gel column and eluted with acetone / toluene (7:3). Disaccharide 4 was obtained as a pale yellow powder (300 mg, 60%) along with perfluoro-1-butanesulfonyl compound 7 (50 mg, 8%) as a pale yellow powder.

[0046] Synthesis of disaccharide 4 from 7: To a solution of compound 7 (730 mg, 0.54 mmol, 1 equiv.) in toluene (7 mL) in a screw-cap vial containing a stir bar, DBU (0.64 mL, 4.31 mmol, 8 equiv.) and perfluoro-1-butanesulfonyl fluoride (0.77 mL, 4.31 mmol, 8 equiv.) were added. The vessel was sealed and stirred at 40° C. for 15 days. The reaction mixture was directly loaded onto a silica gel column and eluted with acetone / toluene (7:3) as the eluent. Disaccharide 4 was isolated as a pale yellow powder (282 mg, 49%) along with perfluoro-1-butanesulfonyl compound 7 (267 mg, 77% brsm) as a pale yellow powder. R f = 0.43 (silica gel, acetone / toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ7.95-7.93(m,2H,Ar-H),7.87-7.85(m,2H,Ar-H),7.48-7.44(m,2H,Ar-H),7.41-7.39(m,2H,Ar-H),7 .35-7.33(m,2H,Ar-H),7.31-7.29(m,2H,Ar-H),7.25-7.24(m,2H,Ar-H),7.21-7.19(m,2H,Ar-H),7.16-7.14(m,1H,Ar-H), 7.04-7.03(m,2H,Ar-H),5.79(dd,J=10.2,9.6Hz,1H),5.49(ddd,J=9.0,5.4,2.4Hz,1H),5.42(dd,J=10.2,3.0Hz,1H),5.31 -5.28(m,2H),5.20(dd,J=27.0,11.4Hz,1H,sia-C4-H),5.01(dd,J=51.6,1.8Hz,1H,sia-C3-H),4.97(d,J=10.2Hz,1H,C1-H β),4.66(d,J=11.4Hz,1H),4.58(d,J=11.4Hz,1H),4.37(dd,J=12.6,2.4Hz,1H),4.28 -4.26(m,2H),4.17(dd,J=12.6,5.4Hz,1H),4.09-4.05(m,2H),3.98(dd,J=10.2,6.0H z,1H),3.74(dd,J=10.2,8.4Hz,1H),3.72(s,3H,-CH3),2.29(s,3H,-CH3),2.17(s,3 H,-CH3),2.16(s,3H,-CH3),2.09(s,3H,-CH3),1.98(s,3H,-CH3),1.91(s,3H,-CH3); 13 C NMR(150MHz, CDCl3):δ170.9,170.7,170.4,170.2,169.8,165.6,165.3,138.3,137 .6,133.2,133.0,132.6,129.8,129.8,129.7,129.5,129.3,129.1,128.4,128.3,12 8.1,127.5,127.3,98.3,98.2,88.1,86.8,86.4,76.2,75.6,74.6,74.0,71.4,69.2,69.0,68.5,68.0,67.3,63.4,62.5,53.2,45.5,23.4,21.2,21.2,20.8,20.7,20.7; 19 F NMR(376MHz,CDCl3):δ-215.9;HRMS(ESI-TOF)m / e:C 54 H 58 FNO 19 SNa[M+Na] + Calculated value: 1098.3200 / Measured value: 1098.3212.

[0047] [ka] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-3-O-(perfluoro-1-butane)sulfonyl-D-erythro-α-L-gluco-non-2-ulopyranosonic acid methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 7. R f<h2 style=";text-align:left;direction:ltr">=0.59(シリカゲル、アセトン:トルエン=2:3);<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> H NMR (600MHz, CDCl3): δ7.95-7.94(m, 2H, Ar-H), 7.90-7.89(m, 2H, Ar-H), 7.49-7.45(m, 2H, Ar-H), 7.40-7.39(m, 2H, Ar-H), 7.36-7.30(m, 4H, Ar-H), 7.27-7.26(m, 2H, Ar-H), 7.25-7.18(m, 3H, Ar-H), 7.04-7.03(m, 2H, Ar-H), 5.82(dd, J=10.2, 9.6Hz, 1H), 5.62-5.38(m, 4H), 5.26(d, J=9.0Hz, 1H), 4.96-4.94(m, 2H, C1-H<h2 style=";text-align:left;direction:ltr"> β <h2 style=";text-align:left;direction:ltr"> ),4.70-4.64(m,3H),4.35-4.30(m,1H),4.22-4.20(m,2H),4.09(dd,J=9.6,6.0Hz,1H),4.02(dd,J=12.6,6.0Hz,1H),3.98(dd,J=6.6,6.0Hz,1H),3.93(dd,J=10.2,7.2Hz,1H),3.76(s,3H,-CH3),2.29(s,3H,-CH3),2.17(s,3H,-CH3),2.09(s,3H,-CH3),2.06(s,3H,-CH3),1.94(s,3H,-CH3),1.90(s,3H,-CH3);<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR(150MHz,CDCl3):δ170.6,170.2,170.1,169.3,167.0,165.7,165.2,138.1, 137.7,133.2,133.0,132.5,129.8,129.7,129.7,129.5,129.1,129.0,128.4,1 28.3,128.1,128.0,127.5,127.4,97.8,86.5,83.4,75.7,74.6,74.2,72.6,70.0,68.4,67.9,66.5,64.5,62.4,53.1,49.0,23.0,21.1,20.9,20.7,20.6,20.3;<h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> F NMR(376MHz,CDCl3):δ-126.2,-126.2,-121.5,-121.5,-110.4,-110.4,-81.0,-80.9,-80.9;HRMS(ESI-TOF)m / e:C<h2 style=";text-align:left;direction:ltr"> 58 <h2 style=";text-align:left;direction:ltr"> H58 F9NO 22 S2Na[M+Na] + Calculated value: 1378.2640 / Measured value: 1378.2644.

[0048] TIFF2025063181000025.tif22 [ka] Allyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl]-(2→6)-[2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside9. A mixture of acceptor 8 (1.04 g, 1.03 mmol, 1 equiv.), donor 4 (1.39 g, 1.29 mmol, 1.25 equiv.) and activated powdered 4 Å MS (0.70 g) in anhydrous CHCl (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 equiv.) was added followed by TfOH (0.5 M in EtO, 0.62 mL, 0.31 mmol, 0.3 equiv.) with stirring. Once the reaction was complete (TLC showed disappearance of starting material after about 2 h), the reaction mixture was quenched with EtN (0.4 mL) and filtered through a pad of Celite. The filtrate was diluted with CH2Cl2 (30 mL) and washed with 20% aqueous Na2S2O3 (10 mL), saturated aqueous NaHCO3 (15 mL) and brine (8 mL). The separated organic layer was dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as eluent to give compound 9 as a white powder (1.30 g, 64%). R f = 0.51 (silica gel, acetone:toluene = 2:3); 1H NMR(600MHz,CDCl3):δ7.90-7.88(m,4H,Ar-H),7.48-7.45(m,2H,Ar-H),7.34-7.22(m,24H,Ar-H),7.18-7.13(m,10H,Ar-H),5.85-5.78(m,2H),5.47-5.44(m,1H),5.35-5.32(m,4H),5.24-5.16(m,2H),5.11(dd,J=10.2,1.2Hz,1H),5.06-4.97(m,3H,C1-H β ),4.85-4.73(m,4H,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); 13C NMR(150MHz,CDCl3):δ170.7,170.6,170.4,170.2,169.8,165.6,165.4, 165.4,165.1,153.8,138.8,138.6,138.5,138.2,138.2,138.1,133.7,13 3.2,133.1,133.1,130.8,130.0,129.8,129.6,129.4,129.0,128.4,128. 3,128.3,128.2,128.2,128.2,128.1,128.1,128.0,128.0,127.9,127.6, 127.6, 127.5, 127.4, 127.3, 127.2, 125.2, 117.1, 100.0, 98.3, 98.2, 96.9, 95.6, 88.1, 86.8, 77.9, 75.0, 74.7, 74.6, 74.5, 74.2, 74.1, 73.9, 73.4, 7 3.3,73.1,73.0,72.4,71.9,71.4,70.9,70.9,69.5,69.2,69.1,69.0,68.0,67.9,67.2,62.9,62.3,57.2,53.2,45.5,23.3,21.0,20.7,20.7,20.6; 19 F NMR(376MHz,CDCl3):δ-215.6;HRMS(ESI-TOF)m / e:C 100 H 108 Cl3FN2O 31 Na[M+Na] + Calculated value: 1979.5878 / Measured value: 1979.5889.

[0049] [ka] [5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl ester]-(2→6)-[2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside 10. To a stirred solution of tetrasaccharide 9 (578 mg, 0.30 mmol, 1 equiv.) in acetic acid (6.0 mL, acetic acid / water, 10:1=v / v), CH3COONa (121 mg, 1.48 mmol, 5 equiv.) was added followed by PdCl2 (105 mg, 0.59 mmol, 2 equiv.) at room temperature. After 20 h, when TLC showed disappearance of starting material, the reaction mixture was diluted with ethyl acetate (20 mL) and poured into saturated aqueous NaHCO3 (20 mL). The aqueous layer was extracted with ethyl acetate (2×10 mL) and the combined organic phase was dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / hexane (2:3) as eluent to give compound 10 as a white powder (465 mg, 82%). R f = 0.23 (silica gel, acetone:hexane = 2:3); 1 H NMR (600MHz, CDCl3): δ7.90-7.87(m,4H,Ar-H),7.48-7.45(m,2H,Ar-H),7.34-7.20(m,24H,Ar-H),7.19- 7.12(m,10H,Ar-H),5.80-5.75(m,1H),5.45-5.43(m,1H),5.33-5.28(m,3H),5.21-4.89(m,5H,man-C1-H α ,man-C1-H β ,C1-H β ), 4.84-4.30(m,15H,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(150MHz,CDCl3):δ170.8,170.7,170.7,170.7,170.6,170.5,170.4,170.3,170.3,170 .2,169.9,169.8,165.6,165.6,165.4,165.2,165.2,154.0,138.6,138.5,138.3,138.2,1 38.1,133.2,133.1,129.8,129.6,129.5,129.0,128.4,128.4,128.3,128.3,128.2,128.2,128.2,128.1,128.0,127.9,127.9,127.7,127.7,127.6,127.5,127.5,127.4,127.4,127 .3,100.0,99.5,98.3,98.2,98.1,95.8,95.7,92.3,88.2,88.1,86.8,76.1,74.8,74.7,74.6,74.6,74.3,74.2,74.2,73.9,73.8,73.3,73.2,73.1,73.1,72.7,72.6,72.4,72.3,71. 6, 71.4, 71.2, 70.9, 70.8, 69.9, 69.5, 69.3, 69.2, 69.1, 69.0, 68.1, 68.0, 67.2, 67.1, 63.0, 62.3, 62.2, 62.1, 53.3, 53.3, 45.5, 45.4, 26.3, 23.3, 21.1, 20.8, 20.7, 20.7, 20.6, 20.6; 19 F NMR(376MHz,CDCl3):δ-215.2,-215.7;HRMS(ESI-TOF)m / e:C 97 H 104 Cl3FN2O 31 Na[M+Na] + Calculated value: 1939.5565 / Measured value: 1939.5619.

[0050] [ka] [5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl ester]-(2→6)-[2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl fluoride 11. To a well-stirred solution of hemiacetal 10 (395 mg, 0.21 mmol, 1 equiv.) in anhydrous CHCl (12 mL) was added DAST (81.6 μL, 0.62 mmol, 3 equiv.) at −20° C. The reaction mixture was stirred vigorously until TLC showed disappearance of the starting material (3 h). The reaction mixture was diluted with CHCl (10 mL) and washed with saturated aqueous NaHCO (5 mL) and brine (4 mL). The organic phase was dried over MgSO, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / hexane (3:4) as eluent to give compound 11 as a white powder (289 mg, 73%). R f = 0.32 (silica gel, acetone:hexane = 3:4); 1 H NMR (600MHz, CDCl3): δ7.89-7.88(m,4H,Ar-H),7.47-7.45(m,2H,Ar-H),7.33-7.22(m,22H,Ar-H) ,7.17-7.12(m,12H,Ar-H),5.78(dd,J=10.2,7.8Hz,1H),5.53-5.40(m,2H),5.33-5.29(m,3H,C1-H α ),5.21(dd,J=27.6,11.4Hz,1H,sia-C4-H),5.05-4.96(m,2H,C1-H β ),4.94(d,J=11.4Hz,1H),4.82-4.79(m,4H,C1-H β), 4.75 - 4.72 (m, 1H), 4.68 - 4.59 (m, 3H), 4.58 - 4.54 (m, 2H), 4.50 - 4.48 (m, 2H), 4.30 (dd, J = 12.6, 3.0 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, 8.4 Hz, 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); 13 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 (2C), 20.6; 19 19F 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.

[0051]

Chemical Structure

[0052] [ka] Benzyl [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl]-(2→6)-[2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy) 14. A mixture of AgOTf (194 mg, 0.75 mmol, 6 equiv. relative to acceptor), bisCp2HfCl2 (167 mg, 0.44 mmol, 3.5 equiv. relative to acceptor) and freshly activated 4 Å MS (1.50 g) in anhydrous toluene (15 mL) was stirred at room temperature under argon for 1 h. The reaction mixture was then cooled to -20°C and treated with a solution of donor 11 (290 mg, 0.15 mmol, 1.2 equiv) and acceptor 13 (115 mg, 0.13 mmol, 1 equiv) in anhydrous toluene (8 mL). The reaction mixture was stirred at 0°C until TLC showed disappearance of starting material (3 h). Upon completion of the reaction, the reaction mixture was quenched with Et3N (0.25 mL), diluted with EtOAc (25 mL), and filtered through a Celite pad. The filtrate was washed twice with saturated aqueous NaHCO3 (10 mL) and brine (6 mL), and the organic phase was dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as eluent to give compound 14 as a white powder (299 mg, 85%).

[0053] One-pot synthesis: A mixture of acceptor 18 (40.0 mg, 0.037 mmol, 1 equiv.), donor 4 (60.1 mg, 0.056 mmol, 1.5 equiv.) and activated powdered 4 Å MS (0.25 g) in anhydrous CHCl (1.25 mL) was stirred under argon for 1 h. The reaction mixture was then cooled to −40° C. and NIS (12.6 mg, 0.056 mmol, 1.5 equiv.) was added followed by TfOH (0.5 M in EtO, 22.3 μL, 0.011 mmol, 0.3 equiv.), and the mixture was then stirred to −20° C. for 2 h. Then, once the starting material had disappeared (monitored by TLC), the reaction mixture was treated successively with acceptor 13 (34.2 mg, 0.037 mmol, 1 equiv.), NIS (16.8 mg, 0.075 mmol, 2 equiv.) and TfOH (0.5 M in Et2O, 22.3 μL, 0.011 mmol, 0.3 equiv.) in anhydrous CHCl2 (1.25 mL) while stirring under argon for 10 min. Once all reagents were added, the mixture was warmed to -10 °C and continued to stir until TLC showed the disappearance of the starting material (1 h). Upon completion, the reaction was quenched with Et3N (0.4 mL) and filtered through a pad of Celite. The filtrate was diluted with CHCl2 (10 mL) and washed with 20% aqueous Na2S2O3 (3 mL), saturated aqueous NaHCO3 (2 mL) and brine (2 mL). The separated organic layer was dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as eluent, followed by a second column chromatography using acetone / hexane (3:4) as eluent to give compound 14 as a white powder (27 mg, 26%). f = 0.43 (silica gel, acetone:toluene = 3:5); 1H NMR(600MHz,CDCl3):δ7.96-7.95(m,2H,Ar-H),7.91-7.90(m,2H,Ar-H),7.51-7.47(m,2H,Ar-H),7.36-7.26(m,35H,Ar-H),7.23-7.10(m,19H,Ar-H),5.84(dd,J=10.2,7.8Hz,1H),5.45-5.42(m,1H),5.34-5.27(m,4H),5.21-5.15(m,2H),5.04-4.94(m,4H,C1-H α ,C1-H β ),4.87-4.78(m,4H),4.71-4.50(m,14H,2C1-H β ),4.46-4.34(m,5H),4.31-4.21(m,4H),4.16(dd,J=12.6,5.4Hz,1H),4.13-4.06(m,2H),3.98-3.95(m,4H,C1-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(150MHz,CDCl3):δ170.8,170.7,170.4,170.2,169.7,169.4,165.6,165.4 ,165.2,153.7,153.6,138.9,138.7,138.4,138.3,137.9,137.1,133.2,129.8, 129.7,129.5,129.1,128.9,128.6,128.5,128.4,128.4,128.3,128.3,128.2,128.1,128.1,128.0,128.0,127.9,127.9,127.9,127.8,127.8,127.6,127.6,12 7.6,127.5,127.4,127.4,127.3,127.2,126.3,102.1,100.1,99.0,98.7,98.6,95.8,95.5,88.2,86.9,78.8,78.4,74.8,74.5,74.3,74.2,73.9,73.8,73.3,7 2.9, 72.3, 71.9, 71.4, 71.1, 70.7, 70.3, 69.2, 69.1, 69.0, 68.4, 68.3, 67.9, 67.2, 66.2, 63.2, 62.3, 57.4, 56.8, 53.3, 45.5, 23.3, 21.0, 20.8, 20.7, 20.6, 20.6; 19 F NMR(376MHz,CDCl3):δ-215.6;HRMS(ESI-TOF)m / e:C 142 H 150 Cl6FN3O 43 Na2[M+2Na] 2+ Calculated value: 1429.8771 / Measured value: 1429.8864.

[0054] [ka] Benzyl [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl]-(2→6)-[2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl]-(1→4)-[3,6-di-O-benzyl-2-deoxy-2-(2,2,2-tetrahydrofuran) Trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-(1→2)-[3,4,6-tri-O-benzyl-α-D-mannopyranosyl]-(1→3)-[2-O-acetyl-β-D-mannopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranoside 15. To a stirred solution of starting material 14 (318 mg, 0.11 mmol, 1 equiv.) in acetonitrile (12 mL) was added p-TsOH·HO (21.5 mg, 0.11 mmol, 1 equiv.). The reaction mixture was stirred vigorously until TLC showed disappearance of starting material (6 h). Upon 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 eluent to give compound 15 as a white powder (231 mg, 75%). f = 0.25 (silica gel, acetone:toluene = 1:2); 1 H NMR (600MHz, CDCl3): δ7.88-7.87(m,4H,Ar-H),7.46(dd,J=7.2,7.2Hz,1H,Ar-H),7.43(dd,J=7.2,7.2Hz,1H,Ar-H),7.33-7.13(m,47H,Ar-H),7.09 -7.08(m,2H,Ar-H),6.01(br,1H),5.76(dd,J=10.2,7.8Hz,1H),5.46-5.4 3(m,1H),5.38(d,J=8.4Hz,1H),5.31-5.29(m,3H),5.19-5.13(m,2H,C1-H α ), 5.05-4.96(m,3H,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=12.0Hz,1H),4.40-4.36(m,2H),4.31(dd,J=12.6,2.4Hz,1H),4.24-4.16(m,4H). ),4.12-4.09(m,2H),4.02(br,1H),3.98-3.93(m,2H),3.91-3.86(m,2H),3.80(dd,J=7.8.6 .0Hz,1H),3.76-3.69(m,6H),3.66-3.37(m,18H),2.96-2.93(m,1H),2.13(s,3H,-CH3),2 1(s,3H,-CH3),2.09(s,3H,-CH3),1.98(s,3H,-CH3),1.93(s,3H,-CH3),1.90(s,3H,-CH3); 13 C NMR(150MHz,CDCl3):δ170.8,170.7,170.4,170.3,170.2,169.8,165.6,165.5,165.3,154.3,153.8,138.6,138.4,138.3,138.1,138.0 ,137.8,137.2,133.3,133.1,129.8,129.6,129.4,129.0,128.5,128.4,128.3,128.2,128.2,127.9,127.9,127.8,127.7,127.6,127.5, . 127.5,127.4,127.4,127.3,100.1,99.1,98.3,98.2,98.1,95.7,95.5,88.2,86.9,78.6,78.2,75.4,75.1,74.8,74.7,74.4,74.2,74.1 ,73.8,73.3,73.2,72.6,71.9,71.8,71.5,71.4,70.9,70.6,69.5,62.3,57.2,56.8,53.8,53.3,45.4,23.3,21.1,21.0,20.7(2C),20.7; 19 F NMR(376MHz,CDCl3):δ-215.6;HRMS(ESI-TOF)m / e:C 135 H 146 Cl6FN3O 43 Na[M+Na] + Origination:2748.7337 / Current:2748.7373.

[0055] [ka] Benzyl [5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl ester -(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)]-[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonic acid methyl ester 16. Methyl-D-erythro-α-L-manno-non-2-ulopyranosone (2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)]-[2-O-acetyl-β-D-mannopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranoside.

[0056] From the fluoride donor: A mixture of AgOTf (142 mg, 0.55 mmol, 8 equiv. relative to acceptor), Cp2HfCl2 (105 mg, 0.28 mmol, 4 equiv. relative to acceptor) and freshly activated 4 Å MS (1 g) in anhydrous toluene (10 mL) was stirred at room temperature for 1 h under argon atmosphere. The reaction mixture was then cooled to -40 °C and a solution of donor 11 (232 mg, 0.12 mmol, 1.75 equiv.) and acceptor 15 (188 mg, 0.069 mmol, 1 equiv.) in anhydrous toluene (1 mL) was added. Stirring was continued for 3 h at -15 °C. The reaction mixture was quenched with Et3N (0.20 mL), diluted with EtOAc (20 mL) and filtered through a pad of Celite. The filtrate was washed twice with saturated aqueous NaHCO3 (8 mL) and brine (4 mL). The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using acetone / toluene (2:3) as eluent to give compound 16 as a white powder (223 mg, 70%) along with recovered 15 (23.0 mg, 80% brsm).

[0057] From the trifluoroacetimidate donor: A mixture of acceptor 15 (32.0 mg, 0.012 mmol, 1 equiv.), donor 12 (42.9 mg, 0.021 mmol, 1.75 equiv.) and activated powdered 4 Å MS (200 mg) in anhydrous CHCl (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 EtO, 5.86 μL, 2.93 μmol, 0.25 equiv. relative to acceptor). After stirring at -20 °C for 3 h, the reaction mixture was quenched with EtN (0.10 mL), diluted with CHCl (10 mL) and filtered through a Celite pad. The filtrate was washed twice with saturated aqueous NaHCO (4 mL) and brine (3 mL). The organic phase was dried over MgSO, filtered and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (2:3) as the eluent to give compound 16 as a white powder (18.0 mg, 33%) along with recovered acceptor (12.6 mg, 55% brsm). f=0.17(shell:shell=1:2); 1 H NMR(600MHz,CDCl3):δ7.91-7.86(m,8H,Ar-H),7.48-7.42(m,4H,Ar-H),7.33-7. 11(m,83H,Ar-H),5.81-5.75(m,2H),5.43-5.38(m,3H),5.31-5.07(m,11H,2C1-H). α ),5.03-4.78(m,9H,2C1-H β ),4.75-4.08(m,48H,4C1-H β ),4.05-3.89(m,7H),3.87-2.99(m,40H),2.13-2.12(m,6H,-2CH3),2.10-2.07(m,15H,-5CH3),1.99-1.97(s,6H,-2CH3),1.90-1.88(m,6H,-2CH3); 13C NMR(150MHz,CDCl3):δ170.7,170.6,170.6,170.4,170.2,169.8,169.7,169.6,165.6,165.5, 165.4,165.4,165.1,154.0,153.6,139.1,138.8,138.4,138.3,138.1,138.0,137.7,133.2,13 3.1,129.8,129.8,129.7,129.6,129.6,129.5,129.5,129.1,129.1,128.4,128.4,128.3,128.2,128.2,128.0,128.0,127.8,127.8,127.8,127.7,127.7,127.5,127.4,127.4,127.3,127.2 ,127.1,100.1,99.8,98.8,98.4,98.2,95.7,88.1,86.8,78.3,77.8,75.3,74.7,74.5,74.4,74.3,74.2,74.0,73.9,73.4,73.3,73.3,73.2,73.1,73.0,72.9,72.5,72.4,72.0,71.9,71.5,7 1.0,71.0,70.9,70.9,70.7,69.5,69.4,69.2,69.1,69.0,69.0,68.5,68.1,68.1,68.0,67.2,62.9,62.2,57.5,57.2,53.8,53.2,53.2,45.5,45.3,23.3,21.0,21.0,20.8,20.7,20.7,20.6; 19 F NMR(376MHz,CDCl3):δ-215.6(2F);HRMS(ESI-TOF)m / e:C 232 H 255 Cl9F2N6O 74 [M+H3O+NH4] 2+ Calculated value: 2330.6770 / Measured value: 2330.6724.

[0058] [ka] [5-acetamido-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonate-(2→6)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-[5-acetamido-3,5-dideoxy-3-fluoro Rho-D-erythro-α-L-manno-non-2-ulopyranosonate-(2→6)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)]-[β-D-mannopyranosyl]-(1→4)-2-acetamido-2-deoxy-D-glucopyranoside 17. A solution of the protected glycan 16 (103 mg, 0.022 mmol, 1 equiv.) and LiOH (51.5 mg, 50% in SMwt) in a 1,4-dioxane / HO mixture (3 mL, 4:1=v / v) was stirred at 90 °C for 16 h. The reaction mixture was concentrated under high vacuum and subjected to acetylation conditions (pyridine (2.5 mL), Ac2O (1.5 mL), room temperature, 16 h). After removal of the solvent, the crude residue was purified by LiChroprep® RP-18 reversed phase column chromatography using HO / MeOH (1:5) as eluent. The product was deacetylated by stirring with NaOMe in MeOH (3 mL, 0.5 M) for 16 h. The reaction mixture was neutralized with IR-120, filtered and concentrated in vacuo. The residue was purified by LiChroprep® RP-18 reversed phase column chromatography using HO / MeOH (1:4) as eluent. The deacetylated crude product was dissolved in a mixture of MeOH / HO / HCOOH (3 mL, 6:3:1=v / v / v) and treated with Pd(OH)2 (51.5 mg, 50% by SMwt) for 20 h. The reaction mixture was filtered through a Celite pad and concentrated in vacuum. The residue was purified by (BIO-RAD) Biogel P-2 column chromatography (eluted with water), followed by LiChroprep® RP-18 reversed-phase column chromatography (eluted with water) to obtain compound 17 (18.3 mg, 40%) as a white powder of anomer mixture (α:β=0.65:0.35). 11H NMR (600 MHz, D2O): δ 5.23 (d, J = 3.0 Hz, 0.65H, a-C1-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, a-C1-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 13C 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; 19 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.

[0059]

Chemical Structure

[0060] [ka] Reagents and conditions in Scheme S1: (a) TFA, MeOH, 60 °C, 16 h, then AcCl, rt, 2 days, then Na2HPO4, CH3CN, reflux, 20 h, 92%. (b) Reference 3, 80% (3 steps). (c) TiBr4, dichloroethane, rt, 10 min, 92%. (d) BH3, Cu(OTf)2, THF, rt, 8 h, 96%.

[0061] [ka] 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2,6-anhydro-D-glycero-D-galacto-non-2-enoic acid methyl ester S3 19To a stirred solution of compound S2 (20 g, 64.7 mmol, 1 equiv.) in MeOH (600 mL) was added TfOH (4.95 mL, 64.7 mmol, 1 equiv.). The reaction mixture was stirred at 60 °C for 16 h. The solvent was removed by rotary evaporation under reduced pressure and coevaporated twice with toluene to remove traces of water. The resulting residue was dissolved in AcCl (200 mL) in a round-bottom flask at 0 °C, sealed, and stirred to room temperature for 2 days. Once the solvent was removed, the resulting residue was diluted with anhydrous acetonitrile (200 mL) and then Na2HPO4 (19.6 g, 155 mmol, 2.4 equiv.) was added under argon. The reaction mixture was stirred vigorously at 90 °C until TLC showed disappearance of starting material (16 h). The solution was filtered through a pad of Celite, dried over MgSO4, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using EtOAc / hexane (4:1) as the eluent to give compound S3 as a brown foam (28.2 g, 92%). Spectroscopic data were consistent with those previously reported in the literature. 19 .R f = 0.30 (silica gel, EtOAc); 1 H NMR (600MHz, CDCl3): δ5.95(d,J=3.0Hz,1H),5.81(d,J=9.0Hz,1H),5.47-5.44(m,2H),5.32-5.30(m,1H),4.59(dd,J=12.0,3.0Hz,1H),4.38-4.33(m ,2H),4.15(d,J=12.0,7.2Hz,1H),3.76(s,3H,-CH3),2.08(s,3H,-CH3),2 .03(s,3H,-CH3),2.02(s,3H,-CH3),2.01(s,3H,-CH3),1.88(s,3H,-CH3); 13 C NMR(150MHz,CDCl3):δ170.7,170.5,170.1,170.1,170.0,161.6,145.0,107.9, 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:C 20 H 27 NO 12 Na[M+Na] +Calculated value: 496.1425 / Measured value: 496.1435.

[0062] [ka] 5-Acetamido-4,7,8,9-tetra-O-acetyl-2-bromo-2,5-dideoxy-D-erythro-α-L-gluconone-2-ulopyranosonic acid methyl ester 3 20 Epoxide S4 was synthesized from sialic acid using a reported procedure. 21 To a stirred solution of epoxide compound S4 (1.10 g, 2.25 mmol, 1 equiv.) in anhydrous 1,2-dichloroethane (18 mL) was added TiBr4 (0.91 g, 2.47 mmol, 1.1 equiv.) under argon for 10 min. 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 (10 mL), 5% aqueous NaHCO3 (10 mL), and then brine (5 mL). The separated organic layer was dried over MgSO4, concentrated in vacuo, and the resulting residue was purified by silica gel column chromatography using acetone / hexane (2:1) as eluent to give compound 3 as a white powder (1.18 g, 92%). The spectroscopic data and protocol were identical to those previously reported. 20 .R f = 0.23 (silica gel, acetone:toluene = 1:1); 1 H NMR (600MHz, CDCl3): δ5.99(d,J=7.2Hz,1H),5.42(dd,J=7.2,1.2Hz,1H),5.21-5.18(m,1H) ),5.10(ddd,J=6.0,6.0,2.4Hz,1H),4.38(dd,J=12.6,2.4Hz,1H),4.32-4.30(m,2H),3.99 (dd,J=12.6,6.0Hz,1H),3.86(s,3H,-CH3),3.78(d,J=9.0Hz,1H),3.66(br,1H,-OH),2.07 (s,3H,-CH3),2.06(s,3H,-CH3),2.05(s,3H,-CH3),2.01(s,3H,-CH3),1.84(s,3H,-CH3); 13C NMR(150MHz, CDCl3):δ171.4,170.6,170.3,169.8,169.7,167.0,98.0,75.0,72.8 ,72.2,69.9,66.5,61.9,54.0,47.2,22.9,21.0,20.7,20.6;HRMS(ESI-TOF)m / e:C 20 H 29 BrNO 13 [M+H] + Calculated value: 570.0817 / Measured value: 570.0822.

[0063] [ka] p-Tolyl 2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 5 22 Starting material S5 23 To a stirred solution of BH3-THF complex (1M in THF, 34.3 mL, 34.3 mmol, 5 equiv.) of (4.00 g, 6.86 mmol, 1 equiv.), Cu(OTf)2 (124 mg, 0.34 mmol, 0.05 equiv.) was added and the reaction mixture was stirred at room temperature for 8 h. Upon completion, the reaction was carefully neutralized with TEA (0.96 mL, 6.86 mmol, 1 equiv.) at 0 °C and then diluted with MeOH (15 mL). After removing the solvent in vacuum, the resulting residue was purified by silica gel column chromatography using EtOAc / Hexane (1:2) as eluent to give compound 5 as a white powder (3.85 g, 96%). Spectroscopic data were consistent with those previously reported in the literature. 22 .R f = 0.45 (silica gel, EtOAc:hexane = 1:1); 1 H NMR (600MHz, CDCl3): δ7.97-7.94(m,4H,Ar-H),7.51-7.48(m,2H,Ar-H),7.38-7.34(m,6H,Ar-H),7.27- 7.21(m,5H,Ar-H),5.85(dd,J=10.2,10.2Hz,1H),5.36(dd,J=9.6,3.0Hz,1H),4.87(d,J=9.6Hz,1H,C1-H β),4.74(d,J=12.0Hz,1H),4.47(d,J=12.0Hz,1H),4.16(d,J=2.4Hz,1H),3.91(d,J=11.4,7.2Hz, 1H),3.76(dd,J=6.0,6.0Hz,1H),3.61(dd,J=11.4,5.4Hz,1H),2.30(s,3H,-CH3),1.83(br,1H); 13 C NMR (150MHz, CDCl3): δ165.9,165.2,138.1,137.4,133.4,133.1,133.0,129.8,129.7,129.6,129.5,128.9,1 28.7,128.5,128.4,128.3,128.1,127.9,86.8,79.0,76.0,74.6,73.7,68.5,61.9,21.1;HRMS(ESI-TOF)m / e:C 34 H 32 O7SNa[M+Na] + Calculated value: 607.1761 / Measured value: 607.1770.

[0064] [ka] Reagents and conditions in Scheme S2: (a) TESOTf, BnCHO, TESH, toluene, THF, 2 h, -20 °C, 90%, (b) NIS, TfOH, 4 Å MS, -40 °C, 1.5 h, CH2Cl2, 75%, (c) NaBH3CN, HCl / ether, AW-300, THF, 0 °C to room temperature, 16 h, 85%.

[0065] [ka] p-Tolyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-β-D-glucopyranoside S7. Starting material S6 24To a stirred solution of (5.17 g, 9.42 mmol, 1 equiv) in a mixture of THF / toluene (45 mL, 1:2=v / v) was added TESOTf (4.26 mL, 18.8 mmol, 2 equiv) under argon at -20 °C. After stirring at -20 °C for 45 min, benzaldehyde (4.79 mL, 47.1 mmol, 5 equiv) and triethylsilane (2.26 mL, 14.1 mmol, 1.5 equiv) were added dropwise to the mixture with stirring. After 2 h at -20 °C, TLC showed disappearance of starting material and the reaction was quenched with saturated aqueous Na2CO3, diluted with EtOAc (100 mL), washed with saturated aqueous Na2CO3 (30 mL) and brine. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using CH2Cl2 / toluene (1:4) as the eluent followed by EtOAc / toluene (1:10) to give compound S7 as a white solid (5.40 g, 90%). f = 0.50 (silica gel, EtOAc:toluene = 1:25); 1 H NMR (600MHz, acetone-d6): δ7.52-7.50(m,2H,Ar-H),7.42-7.36(m,4H,Ar-H),7.32-7.31(m,2H,Ar-H) ,7.29-7.22(m,4H,Ar-H),7.18-7.15(m,2H,Ar-H),5.72(s,1H,Ph-CH),5.03(d,J=10.8Hz,1H,C1-H β ),4.90-4.82(m,3H),4.74(d,J=12.0Hz,1H),4.30(dd,J=10.2,5.4Hz,1H),3.95(dd,J=9.6,9. 0Hz,1H),3.84-3.71(m,3H),3.56(ddd,J=9.6,9.6,4.8Hz,1H),2.83(s,2H),2.32(s,3H,-CH3); 13C 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: C 30 H 30 Cl3NO6SNa[M+Na] + Calculated value: 660.0752 / Actual value: 660.0749.

[0066] [ka] Allyl [3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside S9. Acceptor S8 in anhydrous CHCl (20 mL) 25 A mixture of NIS (523 mg, 0.94 mmol, 1 equiv), donor S7 (900 mg, 1.41 mmol, 1.5 equiv) and activated powdered 4 Å MS (2.00 g) 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 equiv) and TfOH (0.5 M in Et2O, 0.47 mL, 0.23 mmol, 0.25 equiv). After stirring at that temperature for 1.5 h, TLC analysis indicated disappearance of starting material and the reaction mixture was quenched with Et3N (0.2 mL) and filtered through a Celite pad. The filtrate was diluted with CH2Cl2 (20 mL) and washed with 20% aqueous Na2S2O3 (5 mL), saturated aqueous NaHCO3 (10 mL) and brine (5 mL). The separated organic layer was dried over MgSO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using EtOAc / toluene (1:10) as the eluent to give compound S9 as a white foam (710 mg, 75%). f = 0.36 (silica gel, EtOAc:toluene = 1:10); 11H 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 = 7.8 Hz, 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); 13 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.

[0067]

Chemical Structure

[0068] [ka] Reagents and conditions in Scheme S3: (a) NaBH3CN, HCl / ether, AW-300, THF, 0 °C to rt, 16 h, 90%. (b) Ac2O, pyridine, rt, 16 h, 98%. (c) NIS, TfOH, HOPO(OBu)2, 4 Å MS, -30 °C, 2 h, CH2Cl2, 90%. (d) TMSOTf, 4 Å MS, -50 °C, 1 h, CH2Cl2, 87%. (e) NaOMe, MeOH / CH2Cl2, 0 °C to rt, 76%.

[0069] [ka] p-Tolyl 3,6-di-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-β-D-glucopyranoside S10 28 To a stirred solution of starting material S7 (1.00 g, 1.56 mmol, 1 equiv) in anhydrous THF (30 mL) was added activated powdered AW300MS (3.00 g) under argon. The reaction mixture was then cooled to 0 °C and NaCNBH3 (0.98 g, 15.6 mmol, 10 equiv) was added followed by slow addition of HCl·Et2O (2M in Et2O, 7.04 mL, 14.1 mmol, 9 equiv) and the mixture was stirred until TLC showed disappearance of starting material (16 h). Upon completion, the reaction mixture was quenched with saturated aqueous NaHCO3 (0.5 mL) and filtered through a pad of Celite. The filtrate was diluted with CHCl (50 mL) and then washed with saturated aqueous NaHCO3 (15 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography using EtOAc / Hexane / CH2Cl2 (1:3:1) as eluent to give compound S10 as a white foam (902 mg, 90%). 28 The results were consistent with those reported in f = 0.29 (silica gel, EtOAc:hexane = 1:3); 1 H NMR(600MHz,CDCl3):δ7.40-7.38(m,2H,Ar-H),7.36-7.27(m,10H,Ar-H),7.04-7.03(m,2H,Ar-H),5.20(d,J=7.8Hz,1H),4.84(d,J=10.2Hz,1H,C1-H β ),4.77-4.72(m,4H),4.58-4.52(m,2H),3.76(d,J=4.8Hz,2H),3.71-3.64(m,2 H),3.50-3.49(m,1H),3.42-3.38(m,1H),2.90(br,1H,-OH),2.29(s,3H,-CH3); 13C NMR(150MHz,CDCl3):δ153.8,138.1,138.1,137.7,133.0,129.6,128.6,128.5,128.4,128.1,127.9, 127.8,127.7,95.5,86.1,81.9,78.0,74.5,74.4,73.6,72.5,70.4,55.9,21.1;HRMS(ESI-TOF)m / e:C 30 H 32 Cl3NO6SNa[M+Na] + Calculated value: 662.0908 / Measured value: 662.0919.

[0070] [ka] p-Tolyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxy)carbamoylamino-β-D-glucopyranoside S11. To a stirred solution of starting material S10 (0.97 g, 1.51 mmol, 1 eq) in pyridine (12 mL) was added Ac2O (6 mL). The reaction mixture was stirred vigorously at room temperature for 16 h, then concentrated in vacuo and purified by silica gel column chromatography using EtOAc / Hexane / CHCl2 (1:4:1) as eluent to give compound S11 as a white powder (1.01 g, 98%). R f = 0.47 (silica gel, EtOAc:hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ7.41-7.40(m,2H,Ar-H),7.34-7.25(m,8H,Ar-H),7.22-7.21(m ,2H,Ar-H),7.03-7.01(m,2H,Ar-H),5.29(d,J=7.2Hz,1H),5.04(d,J=10.2Hz,1H,C1-H β), 4.98 (dd, J = 9.6, 9.6 Hz, 1H), 4.78 (d, J = 12.0 Hz, 1H), 4.70 (d, J = 12.0 Hz, 1H), 4.62 (d, J = 11.4 Hz, 1H), 4.57 (d, J = 11.4 Hz, 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 13C 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.

[0071]

Chemical formula

[0072] [ka] p-Tolyl[4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl]-(1→2)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside S14. Acceptor S13 in anhydrous CHCl (2 mL) 29 A mixture of S12 (407 mg, 0.73 mmol, 1 equiv.) and donor S12 (900 mg, 1.17 mmol, 1.6 equiv.) and activated powdered 4 Å molecular sieves (200 mg) was stirred under argon for 30 min. It was then cooled to -50 °C, after which TMSOTf (0.21 mL, 1.17 μmol, 1.6 equiv. relative to acceptor) was added with stirring until TLC analysis showed disappearance of starting material (1 h). The reaction mixture was quenched with Et3N (0.30 mL), diluted with CH2Cl2 (20 mL), and filtered through a pad of Celite. The filtrate was washed twice with saturated aqueous NaHCO3 (8 mL) and brine (4 mL). The organic phase was dried over MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using EtOAc / hexane (1:3) as the eluent to give compound S14 as a white powder (710 mg, 87%). f = 0.26 (silica gel, EtOAc:hexane = 1:3); 1 H NMR(600MHz,CDCl3):δ7.37-7.36(m,2H,Ar-H),7.33-7.14(m,25H,Ar-H),7.05-7.03(m,2H,Ar-H),5.33(d,J=1.8Hz,1H,C1-H α ),5.26(d,J=4.8Hz,1H),5.07(d,J=9.0Hz,1H,C1-H β), 4.93 - 4.90 (m, 2H), 4.77 (d, J = 11.4 Hz, 1H), 4.60 - 4.52 (m, 5H), 4.46 - 4.34 (m, 5H), 4.27 (dd, J = 8.4, 8.4 Hz, 1H), 4.19 (dd, J = 9.6, 2.4 Hz, 1H), 4.15 (d, J = 12.0 Hz, 1H), 4.07 (dd, J = 9.6, 9.0 Hz, 1H), 3.84 - 3.81 (m, 2H), 3.64 - 3.63 (m, 2H), 3.57 (dd, J = 10.8, 6.0 Hz, 1H), 3.51 (dd, J = 11.4, 3.0 Hz, 1H), 3.01 (dd, J = 6.6 Hz, 1H), 2.28 (s, 3H, -CH3), 1.82 (s, 3H, -CH3); 13 13C NMR (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.

[0073]

Chemical Structure

[0074] [ka] Reagents and conditions in Scheme S4: (a) NIS, TfOH, 4 Å MS, -40°C, 1 h, CHCl, 93%. (b) Ethylenediamine / BuOH (1:4), 90°C, 2 h, then NaHCO3, TrocCl, CHCl, 0°C, 3 h, 81%. (c) Tf2O, pyridine, CHCl, 0°C, 4 h. (d) Bu4NOAc, toluene, sonication, rt, 8 h, then NaHCO3, TrocCl, CHCl, 0°C, 3 h, 61% (2 steps). (e) DDQ, CHCl, phosphate buffer (pH=7), 0°C-rt, 3 h, 85%. (f) Hydrazine acetate, THF, rt, 16 h, 92%. (g) Tf2O, pyridine, CHCl, 0°C, 2 h. (h) Bu4NOAc, toluene, sonication, room temperature, 8 h, 93% (2 steps). (i) Ethylenediamine / BuOH (1:4), 90 °C, 2 h, then NaHCO3, TrocCl, CH2Cl2, 0 °C, 3 h. (j) Ac2O, pyridine, CH2Cl2, room temperature, 16 h, 83% (2 steps).

[0075] [ka] Benzyl [4,6-O-benzylidine-3-Op-methoxy-benzyl-2-O-levulinoyl-β-D-glucopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside S17. Mixture of acceptors S16 30 A mixture of acceptor S16 in anhydrous CH2Cl2 (50 mL) 30 (1.59 g, 2.74 mmol, 1 equiv.), donor S15 26 (2.11 g, 3.57 mmol, 1.3 equiv) and activated powdered 4 Å MS (5.00 g) were stirred under argon for 1 h. The reaction mixture was then cooled to -40°C and NIS (1.23 g, 0.49 mmol, 2 equiv) was added followed by TfOH (0.5 M in Et2O, 1.37 mL, 0.69 mmol, 0.25 equiv). The reaction was continued until TLC showed disappearance of starting material (2 h). Upon completion, the reaction mixture was quenched with Et3N (0.7 mL) and filtered through a pad of Celite. The filtrate was diluted with CH2Cl2 (50 mL) and washed with 20% aqueous Na2S2O3 (15 mL), saturated aqueous NaHCO3 (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using EtOAc / hexane (2:3) as the eluent to give compound S17 as a white foam (2.68 g, 93%). f = 0.43 (silica gel, acetone:toluene = 1:6); 1 H NMR (600MHz, CDCl3): δ7.75(br,1H,Ar-H),7.63(br,2H,Ar-H),7.50(br,1H,Ar-H ),7.46-7.45(m,2H,Ar-H),7.39-7.32(m,7H,Ar-H),7.29-7.26(m,1H,Ar-H),7.20 -7.18(m,2H,Ar-H),7.07-7.05(m,1H,Ar-H),7.02-7.01(m,4H,Ar-H),6.98-6.97 (m,2H,Ar-H),6.87-6.83(m,5H,Ar-H),5.44(s,1H,Ph-CH),5.10-5.08(m,1H,C1-H β), 4.92 (dd, J = 9.0, 8.4 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.

[0076]

Chemical Structure

[0077] [ka] Benzyl [2-O-acetyl-4,6-O-benzylidine-3-Op-methoxy-benzyl-β-D-mannopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranoside S19. From S18: To a stirred solution of the disaccharide S18 (1.02 g, 1.02 mmol, 1 equiv) in anhydrous CHCl (13 mL) was added anhydrous pyridine (0.58 mL, 7.17 mmol, 7 equiv), followed by the dropwise addition of TfO (0.30 mL, 1.79 mmol, 1.75 equiv) under argon at 0° C. The reaction mixture was stirred at 0° C. until TLC showed disappearance of the starting material (4 h). Upon completion, it was diluted with CHCl (20 mL) and washed with 0.5N HCl (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO and concentrated in vacuo. The crude residue was mixed with BuNOAc (927 mg, 3.07 mmol, 3 equiv) and dissolved in toluene (20 mL). The solvent was removed in vacuo and the residue was coevaporated twice with toluene, then redissolved in anhydrous toluene (13 mL) and the mixture was sonicated for 8 h. During this time, we observed partial deprotection of NHTroc and the reaction mixture was concentrated under high vacuum. The resulting residue was dissolved in CHCl (13 mL) and treated with NaHCO (430 mg, 5.12 mmol, 5 equiv) and 2,2,2-trichloroethyl chloroformate (0.71 mL, 5.12 mmol, 5 equiv) under argon at 0°C. After stirring for 3 h, the reaction mixture was diluted with CH2Cl2 (20 mL) and washed with water (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using EtOAc / toluene (1:5) as eluent to give compound S19 as a white powder (650 mg, 61%).

[0078] From S21: A solution of the disaccharide S21 (1.20 g, 1.21 mmol, 1 equiv) in ethylenediamine / n-BuOH (15 mL, 2:8=v / v) was stirred at 90 °C for 2 h. After removal of the solvent, the crude product was coevaporated twice with toluene. The resulting residue was dissolved in CHCl (30 mL) and treated with NaHCO (508 mg, 6.05 mmol, 5 equiv) and 2,2,2-trichloroethyl chloroformate (0.83 mL, 6.05 mmol, 5 equiv) under argon at 0 °C. After 3 h, the reaction mixture was diluted with CHCl (45 mL) and washed with water (30 mL) and brine (15 mL). The organic layer was dried over MgSO, concentrated, coevaporated twice with toluene and evaporated in vacuum. The resulting residue was directly subjected to acetylation conditions Ac2O (7 mL) in pyridine (14 mL) with the temperature slowly warming from 0° C. to room temperature over 16 h. The reaction mixture was concentrated under high vacuum and purified by silica gel column chromatography using acetone / toluene (1:5) as eluent to give compound S19 as a white powder (1.04 g, 83%). f = 0.54 (silica gel, EtOAc:toluene = 1:3); 1 H NMR (600MHz, CDCl3): δ7.48-7.46(m,2H,Ar-H),7.39-7.35(m,3H,Ar-H),7.33-7.25(m,14H,Ar-H),7.24-7.21(m,3H,Ar-H),6.84-6.82(m, 2H,Ar-H),5.50(s,1H,Ph-CH),5.41(d,J=3.0Hz,1H),5.09(br,1H),4.92(d,J=10.8Hz,1H),4.88(d,J=12.0Hz,1H),4.72-4.62(m,5H,2C1-H β),4.58-4.56(m,3H),4.47-4.44(m,2H),4.08(dd,J=10.2,4.8Hz,1H),4.04(dd,J=9.0,8.4Hz,1H),3.87-3.84(m,2H),3.78-3.72(m,5H,-CH3 ),3.59(dd,J=10.2,10.2Hz,1H),3.45-3.43(m,2H),3.38(ddd,J=8.4,8.4,8.4Hz,1H),3.10(ddd,J=9.6,9.6,4.8Hz,1H),2.07(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ170.3,159.3,153.8,138.4,137.8,137.4,137.1, 129.7,129.2,128.9,128.5,128.4,128.3,128.2,128.0,128.0,127.9,1 27.8,127.7,126.1,113.8,101.4,99.1,99.1,77.8,75.3,74.4,73.5,71 .4,70.8,69.1,68.5,68.4,67.0,57.5,55.2,21.0;HRMS(ESI-TOF)m / e:C 53 H 56 Cl3NO 14 Na[M+Na] + Calculated value: 1058.2658 / Measured value: 1058.2657.

[0079]

change

[0080] [ka] Benzyl[4,6-O-benzylidine-3-Op-methoxy-benzyl-β-D-glucopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside S20. To a stirred solution of starting material S17 (2.48 g, 2.37 mmol, 1 equiv.) in anhydrous THF (55 mL) was added hydrazine acetate (327 mg, 3.55 mmol, 1.5 equiv.) at room temperature under argon. The reaction mixture was stirred vigorously until TLC showed disappearance of starting material (16 h). Upon completion, the reaction mixture was diluted with EtOAc (150 mL) and washed with water (60 mL) and brine (30 mL). The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography using EtOAc / hexane (1:2) as the eluent to give compound S20 as a white foam (2.07 g, 92%). f = 0.54 (silica gel, EtOAc:hexane = 1:1); 1H NMR(600MHz,CDCl3):δ7.79-7.54(m,4H,Ar-H),7.51-7.44(m,2H,Ar-H),7.38-7.33(m,7H,Ar-H),7.30-7.27(m,3H,Ar-H),7.08-7.04(m,1H,Ar-H),7.02-7.01(m,4H,Ar-H),6.98-6.97(m,2H,Ar-H),6.89-6.81(m,5H,Ar-H),5.46(s,1H,Ph-CH),5.09(d,J=8.4Hz,1H,C1-H β ),4.85(d,J=11.4Hz,1H),4.77(d,J=12.6Hz,1H),4.75(d,J=4.2Hz,1H),4.73(d,J=4.2Hz,1H),4.68(d,J=11.4Hz,1H),4.62(d,J=7.2Hz,1H,C1-H β ),4.56(d,J=12.0Hz,1H),4.46(d,J=12.0Hz,1H),4.38(d,J=12.0Hz,1H),4.32(dd,J=10.8,8.4Hz,1H),4.21(d,J=10.8,8.4Hz,1H),4.15-4.10(m,2H),4.04(dd,J=11.4,3.0Hz,1H),3.82(dd,J=11.4,1.8Hz,1H),3.78(s,3H,-CH3),3.61(ddd,J=9.6,3.0,3.0Hz,1H),3.56(dd,J=3.0,3.0Hz,1H),3.52-3.45(m,3H),3.18(ddd,J=9.6,9.6,4.8Hz,1H),2.98(br,1H,-OH); 13 C NMR(150MHz,CDCl3):δ167.9,159.3,138.4,137.8,137.3,137.2,133.6,131.6,130.5,129.7,128.9,128.4,128.2,128.1,128.0,127.9,127.8,127.5,127.4,127.1,126.0,123.2,113.8,103.4,101.1,97.5,81.3,79.5,78.8,77.8,75.0,74.7,74.5,74.2,73.6,70.8,68.6,68.2,66.2,55.8,55.3;HRMS(ESI-TOF)m / e:C 56 H 55 NO13 Calculated value of Na [M+Na] + :972.3565 / Actual value:972.3567.

[0081] [ka] Benzyl [2-O-acetyl-4,6-O-benzylidine-3-Op-methoxy-benzyl-β-D-mannopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside S21. To a stirred solution of disaccharide S20 (936 mg, 0.985 mmol, 1 equiv) in anhydrous CHCl (12 mL) was added anhydrous pyridine (0.56 mL, 6.90 mmol, 7 equiv), followed by dropwise addition of trifluoromethanesulfonic anhydride (0.29 mL, 1.72 mmol, 1.75 equiv) under argon at 0° C. and the mixture was stirred at 0° C. until TLC showed disappearance of starting material (2 h). Upon completion, the reaction mixture was diluted with CHCl (20 mL) and washed with 0.5 N HCl (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4, concentrated in vacuum, and the obtained residue was used as such for further reaction. To the above obtained residue was added Bu4NOAc (594 mg, 1.97 mmol, 2 equiv.) and dissolved in toluene (18 mL). The solvent was removed in vacuum, and the residue was coevaporated twice with toluene to remove traces of water. The residue was redissolved in anhydrous toluene (12 mL) and the mixture was sonicated for 8 h. Upon completion, the reaction mixture was diluted with EtOAc (20 mL) and washed with water (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated. The obtained residue was purified by silica gel column chromatography using EtOAc / Hexane (1:2) as eluent to obtain compound S21 as a white powder (913 mg, 93%). R f = 0.29 (silica gel, EtOAc:hexane = 1:2); 11H 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 13C 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.

[0082]

Chemical Structure

[0083] [ka] p-Nitrophenyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-manno-non-2-ulopyranosonate methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-β-D-galactopyranoside S22. To a stirred solution of thioglycoside 4 (115 mg, 0.11 mmol, 1 equiv.) in anhydrous CHCl (2 mL) was added bromine (6.02 μL, 0.12 mmol, 1.1 equiv.) at 0° C. After vigorously stirring the reaction mixture for 10 min, the solvent was removed in vacuo and the residue was coevaporated twice with toluene to remove traces of 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 equiv) and Ag2O (124 mg, 0.53 mmol, 5 equiv). The reaction mixture was vigorously stirred in the dark under N2 until TLC showed disappearance of the starting material (1 h). Upon completion, the reaction mixture was diluted with EtOAc (10 mL) and filtered through a Celite pad. The filtrate was washed twice with saturated aqueous NaHCO3 (4 mL) and brine (3 mL). The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The residue obtained was purified by silica gel column chromatography using acetone / toluene (1:2) as eluent to give compound S22 as a white powder (85 mg, 73%). R f = 0.46 (silica gel, acetone:toluene = 2:3); 1 H NMR(600MHz,CDCl3):δ8.13-8.11(m,2H,Ar-H),7.96-7.94(m,2H,Ar-H),7.91-7.89(m,2H,Ar-H),7.49-7.44(m,2H,Ar-H),7.35-7.30(m,6H,Ar-H ),7.24-7.22(m,2H,Ar-H),7.20-7.18(m,2H,Ar-H),7.13-7.11(m,1H,Ar-H),6.09(dd,J=10.2,7.8Hz,1H),5.65-5.59(dd,J=10.2,3.0Hz,1H,C1-H β), 5.35 (d, J = 9.6 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, 4.2 Hz, 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); 13 13C NMR (150 MHz, 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 19F 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.

[0084]

Chemical Structure

[0085]

Chemical Structure

[0086] [ka] p-Tolyl[5-acetamido-3,4,7,8,9-penta-O-acetyl-5-deoxy-D-erythro-α-L-gluconone-2-ulopyranosonic acid methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside S24. To a stirred solution of starting material 6 (174 mg, 0.16 mmol, 1 equiv.) in pyridine (2 mL) was added Ac2O (1 mL). The reaction mixture was stirred vigorously at room temperature for 16 h and then concentrated under high vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as eluent to give compound S24 as a white powder (162 mg, 90%). R f = 0.49 (silica gel, acetone:toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ7.94-7.92(m,2H,Ar-H),7.88-7.87(m,2H,Ar-H),7.48-7.44(m ,2H,Ar-H),7.40-7.38(m,2H,Ar-H),7.35-7.27(m,6H,Ar-H),7.23-7.20(m,2H,Ar-H), 7.18-7.16(m,1H,Ar-H),7.05-7.03(m,2H,Ar-H),5.80(dd,J=10.2.9.6Hz,1H),5.44-5 .42(m,2H),5.35-5.29(m,3H),5.25(dd,J=8.4,1.8Hz,1H),4.95(d,J=10.2Hz,1H,C1-H β ),4.67-4.61(m,3H),4.33(ddd,J=10.2,10.2,10.2Hz,1H),4.25-4.22(m,2H),4.03-3.97(m,3H),3.92-3.89(m,1H),3.75(s,3H,-CH3) ,2.29(s,3H,-CH3),2.15(s,3H,-CH3),2.07(s,3H,-CH3),2.00(s,3H,-CH3),1.95(s,3H,-CH3),1.94(s,3H,-CH3),1.89(s,3H,-CH3); 13C NMR(150MHz,CDCl3):δ171.0,170.5,170.3,170.0,169.3,168.5,168.0,165.7,165.2,13 8.4,137.6,133.1,132.9,132.5,129.8,129.7,129.7,129.5,129.2,129.1,128.3,128.2, 128.0,127.4,127.2,98.9,86.5,76.9,75.5,74.6,74.3,72.7,71.6,71.5,68.5,68.3,66 .9,62.6,62.5,52.7,48.4,23.0,21.1,20.8,20.7,20.6,20.6,20.5;HRMS(ESI-TOF)m / e:C 56 H 61 NO 21 SNa[M+Na] + Calculated value: 1138.3349 / Measured value: 1138.3358.

[0087] [ka] p-Nitrophenyl[methyl 5-acetamido-3,4,7,8,9-penta-O-acetyl-5-deoxy-D-erythro-α-L-gluco-non-2-ulopyranosonate]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-β-D-galactopyranoside S25. To a stirred solution of thioglycoside S24 (132 mg, 0.12 mmol, 1 equiv.) in anhydrous CHCl (2.5 mL) was added bromine (6.67 μL, 0.13 mmol, 1.1 equiv.) at 0° C. After vigorous stirring for 10 min, the solvent was removed in vacuo and the residue was coevaporated twice with toluene to remove traces of 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 equiv) and Ag2O (137 mg, 0.59 mmol, 5 equiv). The reaction mixture was vigorously stirred in the dark under N2 until TLC showed disappearance of the starting material (1 h). Upon completion of the reaction, the reaction mixture was diluted with EtOAc (12 mL) and filtered through a Celite pad. The filtrate was washed twice with saturated aqueous NaHCO3 (5 mL) and brine (3 mL). The organic phase was dried over MgSO4, filtered, concentrated in vacuo, and the resulting residue was purified by silica gel column chromatography using acetone / toluene (3:5) as eluent to give compound S25 as a white powder (99 mg, 74%). R f = 0.51 (silica gel, acetone:toluene = 2:3); 1 H NMR (600MHz, CDCl3): δ8.15-8.13(m,2H,Ar-H),7.95-7.92(m,4H,Ar-H),7.50-7.44(m,2H,Ar-H),7.36-7.31(m,6H,Ar-H),7.24- 7.19(m,2H,Ar-H),7.17-7.15(m,3H,Ar-H),6.10(dd,J=10.2,7.8Hz,1H),5.54(dd,J=10.2,3.0Hz,1H),5.52(d,J=7.8Hz,1H,C1-H β),5.48(ddd,J=10.2,7.2,3.0Hz,1H),5.39(d,J=10.2Hz,1H),5.31-5.29(m,2H),5.23(dd,J=9.6 ,1.8Hz,1H),4.73(dd,J=10.8,2.4Hz,1H),4.71-4.65(m,2H),4.32-4.30(m,3H),4.19(dd,J=7.2, 7.2Hz,1H),3.97(dd,J=12.6,7.2Hz,1H),3.88-3.86(m,2H),3.83(s,3H,-CH3),2.24(s,3H,-CH3 ),2.12(s,3H,-CH3),2.01(s,3H,-CH3),1.98(s,3H,-CH3),1.90(s,3H,-CH3),1.88(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ171.0,170.7(2C),170,0,169.6,168.5,168.3,165.7,165.3,1 61.7,142.7,137.9,133.3,133.2,129.9,129.7,129.3,128.9,128.4,128.3,128.2,12 8.0,127.6,125.7,116.8,99.7,98.4,75.1,74.1,74.0,73.9,72.8,71.6,71.1,69.7, 67.6,67.0,63.8,63.3,52.9,48.4,23.0,20.9,20.8,20.7,20.6;HRMS(ESI-TOF)m / e:C 55 H 59 N2O 24 [M+H] + Calculated value: 1131.3452 / Measured value: 1131.3440.

[0088]

change

[0089]

Chemical Structure

[0090] [ka] p-Nitrophenyl[5-acetamido-5-deoxy-D-glycero-α-D-galacto-nona-2-ulopyranosilonate]-(2→6)-β-D-galactopyranoside 1. Neu5Ac-α2,6-Gal-pNP:Neu5Ac-α2,6-Gal-pNP was synthesized by mixing pNP-β-Gal (1.0 mmol), sialic acid (1.2 mmol), cytidine triphosphate (1.2 mmol), CMP-sialic acid synthase (CSS, 12 U), pyrophosphatase (PPA, 1 U) and α-2,6-sialyltransferase (SiaT, 15 U) in 15 mL of Tris buffer (pH 7.0) with 5 mM MgCl2 and 5 mM MnCl2. After removing proteins by heating and centrifugation, the product was purified by Biogel P-2 column chromatography using water as the eluent (BIO-RAD). The fractions containing Neu5Ac-α2,6-Gal-pNP were collected and lyophilized to obtain compound 1 (50%). 1 H NMR(600MHz,D2O):δ8.33-8.32(m,2H,Ar-H),7.31-7.29(m,2H,Ar-H),5.23(d,J=7.8Hz,C1-H β ),4.06-4.04(m,2H),3.99(dd,J=10.2,8.4Hz,1H),3.90-3.86(m,3H),3.83(dd,J=10.2,3.6Hz,1H),3.80-3.76(m,1H),3.73-3.69 (m,3H),3.65-3.62(m,2H),3.57(d,J=8.4Hz,1H),2.79(dd,J=12.6,4.8Hz,1H),2.04(s,3H,-CH3),1.68(dd,J=12.6,12.0Hz,1H); 13 C NMR(150MHz,D2O):δ174.6,173.1,161.4,142.1,125.7,116.0,99.8,99.4,73.6,72.1, 71.9,71.3,69.8,68.0,67.7,67.7,62.5,62.2,51.4,39.8,21.6;HRMS(ESI-TOF)m / e:C 23 H 33 N2O 16 [M+H] + Calculated value: 593.1825 / Measured value: 593.1825.

[0091] Example 2: Analysis of stability against sialidase-catalyzed hydrolysis and sialidase inhibition 31 material: β-Galactosidase from Aspergillus oryzae (G5160) and sialidases from Vibrio cholerae (11080725001) and Clostridium perfringens (11585886001) were received from Sigma Aldrich.

[0092] Enzyme assay for sialidase: The assay was performed at 37 °C in duplicate in a 96-well plate in a final volume of 50 μL containing substrate (0-20 mM) and β-galactosidase (100 mU). The assay conditions for the two sialidases were as follows: C. perfringens (1 mU), sodium acetate buffer (50 mM) (pH 5.0), and CaCl2 (10 mM); V. cholerae (2 mU), sodium acetate buffer (50 mM) (pH 5.5), CaCl2 (10 mM), and NaCl (150 mM). The reactions were performed for 40 min to 2 h for C. perfringens and overnight for V. cholerae. The assay was stopped by adding 65 μL of CAPS buffer (N-cyclohexyl-3-aminopropanesulfonic acid, 0.5 M, pH 10.5). The amount of p-nitrophenolate formed was measured by measuring the A of the reaction mixture using a microplate reader. 405nm The results were determined by measuring the activity of three compounds (Neu5Ac-α2, 6-GalβpNP, 3F ax -Neu5Ac-α2,6-GalβpNP and 3OH eq -Neu5Ac-α2,6-GalβpNP) were tested as substrates for the enzyme. All three compounds showed a 1 h A at 20 mM after 1 h incubation at 37 °C in the absence of sialidase. 405nm The three compounds, Neu5Ac-α2,6-GalβpNP, 3F ax -Neu5Ac-α2,6-GalβpNP and 3OH eqThe absorbances of -Neu5Ac-α2,6-GalβpNP were 0.044, 0.129, and 0.072, respectively. The standard curve of pNP was determined by serial two-fold dilutions of 0.35 mM pNP, and then the A 405nm A standard curve for pNP was obtained by graphing the above.

[0093] Inhibition assay for sialidase: The assays were performed in duplicate in 96-well plates at 37°C in a final volume of 50 μL containing the substrate Neu5Ac-α2,6-GalβpNP (0.6 mM), β-galactosidase (100 mU) and sialidase in the absence or presence of various concentrations (0-20 mM) of inhibitors. Reactions were allowed to proceed for 40 min to 2 h for C. perfringens and overnight for V. cholerae. The assays were stopped by adding CAPS buffer (65 uL, 0.5 M, pH 10.5). The amount of p-nitrophenolate formed was monitored using a microplate reader by measuring the A of the reaction mixture. 405nm This was determined by measuring (Figure 1).

[0094] Example 3: 3F to study the effect on binding to FcγRIIIa by surface plasmon resonance (SPR) analysis ax Preparation of homogeneous mAbs modified with -Neu5Ac and Neu5Ac Enzyme expression: Endoglycosidases Endo-S, Endo-S2, Endo-S2 mutant (D184Q), and α-L-fucosidase from Bacteroides fragilis NCTC9343 were expressed in Escherichia coli, and the enzymes were purified using Ni-NTA agarose beads.

[0095] Preparation of mono-GlcNAc-Rituximab: As previously stated 32Rituximab (3.0 mg, Rituxan®, Roche) in Tris-HCl buffer (50 mM, pH 7.4, 1.5 mL) was incubated with Endo-S (120 μg), Endo-S2 (240 μg) and BfFucH (4.5 mg) at 37° C. for 24 h. LC-MS and SDS-PAGE analysis showed complete cleavage of N-glycans on the heavy chain. The reaction mixture was subjected to affinity chromatography on a Protein A-agarose resin column (1 mL, GE Healthcare) pre-equilibrated with Tris-HCl buffer (50 mM, pH 7.4). The column was then washed with Tris-HCl buffer (50 mM, pH 7.4, 20 mL). Bound IgG was released with glycine-HCl (100 mM, pH 3.0, 10 mL) and the eluted fraction was directly neutralized with Tris-HCl buffer (1.0 M, pH 8.3). Fractions containing the antibody were combined and concentrated by centrifugal filtration (Amicon Ultra centrifugal filters, Millipore, Billerica, MA) to yield mono-GlcNAc rituximab (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 mono-GlcNAc.

[0096] Transglycosylation of mono-GlcNAc Rituximab with Glycan Oxazoline: Glycan oxazoline was added to the Endo-S2D184Q and mono-GlcNAc rituximab mixture in 50 mM Tris buffer (pH 7.4). The solution was incubated at 37° C. for 30 min. The reaction mixture was then purified on a Protein-A affinity column followed by a Capto Q (GE Healthcare) anion exchange column to recover the desired product.

[0097] SDS-PAGE detection of glycoengineered Herceptin antibody: All SDS-PAGE analyses were performed using NuPAGE® Novex® 4-12% Bis-Tris gels (Invitrogen) in MOPS buffer with 2-mercaptoethanol present in the samples (Figure 2).

[0098] MS Spectroscopic Analysis of Glycoengineered mAbs: For the analysis of tryptic glycopeptides, high-resolution and high-mass accuracy nanoflow LC-MS / MS experiments were performed on a LTQFT Ultra (linear quadrupole ion trap Fourier transform ion cyclotron resonance) mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source (New Objective), 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 onto a self-packed precolumn (150 μm (id) × 20 mm, 5 μm, 100 Å) at a flow rate of 10 μL / min. Chromatographic separation was performed by a self-packed reversed-phase C18 nanocolumn (75 μm (id) × 300 mm, 5 μm, 100 Å) with 0.1% formic acid / water as mobile phase A and 0.1% formic acid / 80% acetonitrile as mobile phase B operated at a flow rate of 300 nL / min. Full scan MS conditions: mass range m / z 320-2000, 100,000 surveys at resolution m / z 400. The 10 most intense ions were sequentially isolated for MS2 by LTQ. The electrospray voltage was maintained at 1.8 kV and the capillary temperature was set at 200 °C (Figure 3 and Table 3).

[0099] Surface plasmon resonance (SPR) analysis All SPR experiments were performed using a single cycle kinetic method on a BIACORE T200 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 running buffer. FcγRIIIa was transfected into HEK-293 cells to express complex glycosylated recombinant protein as the analyte. For analysis of Rituximab binding to the FcγRIIIa receptor, anti-human Fab antibodies in a human Fab capture kit (GE Healthcare) were immobilized on both the reference and active channels of a CM5 sensor chip, and then Rituximab was captured on the active channel for interaction with serial dilutions of FcγRIIIa analytes (2.5, 5, 10, 20, 40 nM for 2,6-FluoSCT and 2,6-SCT; 8, 24, 72, 216, 648 nM for commercial Rituximab) at 30 μL / min for 240 s of association followed by a dissociation time of 420 s. Rituximab data was processed by double referencing for background subtraction. Rituximab data was fitted to a 1:1 Langmuir binding model in BiaEvaluation software (GE Healthcare) to obtain kinetics / affinity constants (Table 4). The antibodies of interest were captured by a human Fab capture kit and detected by a single cycle kinetic method. [Table 3] [Table 4]

[0100] Example 4: Relative Reactivity Values ​​(RRV) of Compounds 4 and 18 Experimental procedures reported previously 34 The RRV of disaccharide donor 4 (2053) was measured in triplicate according to the competitive reference donor S34 23 The RRV of disaccharide donor 18 (537) was measured against the competitive reference donor S 23 (RRV=286). [ka]

[0101] Other embodiments All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by other features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0102] Furthermore, from the above description, those skilled in the art can easily ascertain the essential characteristics of the present invention without departing from the spirit and scope thereof, and can make various changes and modifications to the present invention to adapt it to various uses and conditions. Accordingly, other embodiments are within the scope of the appended claims.

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Claims

1. A monoclonal antibody having a saccharide that comprises 3-fluoro-sialic acid, the saccharide comprising 3-fluoro-sialic acid comprising reacting 3-hydroxy-sialic acid with a sugar to form a glycosylation reaction to form an α2,6-linked 3-hydroxy-sialoside; reacting the α2,6-linked 3-hydroxy-sialoside with a fluorinating agent to form a sugar containing 3-fluoro-sialic acid; Prepared from The 3-hydroxy-sialic acid is a compound of formula (I): 【Chemistry 1】 and The sugar is a compound of formula (II): 【Chemistry 2】 and The α2,6-linked 3-hydroxy-sialoside is a compound of formula (III): 【Chemistry 3】 and The 3-fluoro-sialic acid containing saccharide is a compound of formula (IV): 【Chemistry 4】 That is, a monoclonal antibody.

2. The glycosylation reaction was carried out using silver trifluoromethanesulfonate (AgOTf) and disodium hydrogen phosphate (Na ２ H.P.O. ４ 2. The monoclonal antibody of claim 1, wherein the antibody is detected in the presence of a .

3. The monoclonal antibody of claim 1, wherein the fluorinating agent is perfluoro-1-butanesulfonyl fluoride (NfF).

4. The monoclonal antibody of claim 1 , wherein the fluorination reaction is carried out in the presence of a catalyst.

5. The monoclonal antibody of claim 4, wherein the catalyst is 1,8-diazabicyclo[5,4,0]-undec-7-ene (DBU).

6. The monoclonal antibody of claim 1 , wherein the glycosylation and fluorination reactions are each carried out in toluene.

7. The monoclonal antibody of claim 1 for treating cancer or an autoimmune disease.

8. A monoclonal antibody having a saccharide that comprises 3-fluoro-sialic acid, said saccharide comprising 3-fluoro-sialic acid being a compound of formula (V): 【Chemistry 5】 and In the formula, Z is 【Chemistry 6】 That is, a monoclonal antibody.

9. The monoclonal antibody of claim 8 for treating cancer or an autoimmune disease.

10. Compound of formula (VI): 【Chemistry 7】 (In the formula, R １ is Ac or H, R ２ is Bz or H, R ３ is methyl or H, R ４ is Bn or H, and X is OH, a leaving group or a sugar).

11. The compound is a compound of formula (IV): 【Chemistry 8】 11. The compound of claim 10,

12. 11. The compound of claim 10, wherein X is an N-glycan.

13. The compound is a compound of formula (V): 【Chemistry 9】 13. The compound of claim 12, wherein

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