Sialidase-resistant sugars and their preparation and use

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 acid is a negatively charged monosaccharide often found at the outermost end of glycans on glycolipids and glycoproteins, and is involved in many physiological intracellular and intercellular processes, including interactions with other biomolecules and receptors on cells, viruses, and bacteria. 1 Furthermore, sialylation plays a crucial role in regulating the function and fate of secreted glycoproteins and membrane-bound receptors. For example, sialylation of epidermal growth factor receptor (EGFR) is known to inhibit EGFR dimerization, thereby interfering with EGF binding and phosphorylation, which has been linked to tumorigenesis. 2 Furthermore, desialylation of N-glycans exposes the underlying galactose, which is recognized by hepatic asialoglycoprotein receptors, leading to rapid removal of glycoproteins from circulation. Thus, sialylation regulates the half-life of glycoproteins in the bloodstream. 3 Therefore, an increase in the degree of sialylation may enhance the half-life and undesirable effects of glycoprotein therapeutics.

[0002] Recently, the role of glycosylation in protein structure and function has been studied intensively, encouraging the development of new methods for glycan synthesis campillary and glycosylation engineering of proteins, particularly therapeutic monoclonal antibodies (mAbs). 5 For example, defucosylated bibranched N-glycans with terminal α-2,6-linked sialic acid have been found to possess an optimal glycan structure for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and anti-inflammatory activity. 6 .

[0003] Sialic acid derivatives with fluorine at the C-3 position are known to inhibit sialyltransferase and sialidase (or neuraminidase) and be more stable. 7However, glycosylation with fluorinated sugars as donors often poses significant challenges due to the strong electronic effect of the fluorine group that inactivates the glycosylation reaction. There exists a sialyltransferase capable of transferring a 3-fluoro-sialic acid residue from the corresponding cytidine monophosphate-sialic acid to form an α-2,3-linked sialoside, but there is no available corresponding α-2,6-sialyltransferase. 11 .

[0004] Therefore, there is a need to provide a method for reliably preparing a sugar containing an α-2,6-linked 3-fluoro-sialoside that can be used for developing glycoprotein therapeutics.

Summary of the Invention

[0005] An aspect of the present invention is a method for preparing a sugar containing 3-fluoro-sialic acid. This method includes a step of performing a glycosylation reaction by reacting 3-hydroxy-sialic acid with a sugar to form an α2,6-linked 3-hydroxy-sialoside, and a step of performing a fluorination reaction by reacting the α2,6-linked 3-hydroxy-sialoside with a fluorinating agent to form a sugar containing 3-fluoro-sialic acid.

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

[0007] A further aspect of the present invention is a compound of formula (VI):

Chemical Formula

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

[0009] Furthermore, the present invention encompasses methods for treating cancer. This method includes administering an effective amount of a monoclonal antibody conjugated to an α2,6-linked 3-fluoro-sialoside-terminal N-glycan to a subject in need.

[0010] Details of the present invention are described below in embodiments for carrying out the invention. Other features, purposes, and advantages of the present invention will also become apparent from the following detailed descriptions of some embodiments and the appended claims.

[0011] Please refer to the attached diagram for the following explanation. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1A shows two graphs illustrating the enzyme-catalyzed hydrolysis of three sialosides, namely 1, 2, and S27, as a function of enzyme concentration, while Figure 1B shows two graphs illustrating sialidase inhibition as a function of concentration of 2, S27, or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) (a sialidase inhibitor). [Figure 2] The four N-glycans used are (1) lituxan-G, (2) lituxan-Fax-SCT, (3) lituxan-SCT, and (4) commercially available lituxan-stained electrophoresis gels. [Figure 3] The mass spectra obtained for three types of N-glycans, namely lituxan-Fax-SCT, lituxan-SCT, and lituxan, are shown. [Modes for carrying out the invention]

[0013] Disclosed in detail for the first time in this specification is a method for preparing sugars containing 3-fluorosialic acid.

[0014] To reiterate, this method includes the steps of: reacting 3-hydroxysialic acid with a sugar to perform a glycosylation reaction to form an α2,6-linked 3-hydroxysialoside; and reacting the α2,6-linked 3-hydroxysialoside with a fluorinating agent to perform a fluorination reaction to form a sugar containing 3-fluorosialic acid. The sugar may be a monosaccharide, disaccharide, or oligosaccharide. In the exemplary method, the sugar is a monosaccharide.

[0015] In certain embodiments of this method, 3-hydroxysialic acid is 2-bromo-3-hydroxysialic acid, and the glycosylation reaction is carried out in the presence of silver trifluoromethanesulfonate (AgOTf) and disodium hydrogen phosphate (Na2HPO4). Preferably, the glycosylation reaction is carried out in toluene.

[0016] In other embodiments of this method, the fluorinating agent is perfluoro-1-butanesulfonyl fluoride (NfF), and the fluorination reaction is carried out in the presence of a catalyst. For example, the catalyst is 1,8-diazabicyclo[5,4,0]-undeca-7-ene (DBU). Preferably, the fluorination reaction is carried out in toluene. This reaction can also be carried out in the presence of tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF).

[0017] In an exemplary manner, 3-hydroxysialic acid is a compound of formula (I): [ka] And, Sugars are compounds of formula (II): [ka] And, α2,6-linked 3-hydroxy-sialosides are compounds of formula (III): [ka] and Sugars containing 3-fluorosialic acid are compounds of formula (IV): [ka] That is the case.

[0018] In further embodiments, the method further includes a step of carrying out another glycosylation reaction by reacting a sugar containing 3-fluorosialic acid with a second sugar.

[0019] A method for preparing homogeneous antibodies bound to a sugar containing 3-fluorosialic acid is also within the scope of the present invention. In this case as well, the method includes a step of glycosylation of a monoclonal antibody with a sugar containing 3-fluorosialic acid.

[0020] In a particular embodiment of this method, a sugar containing 3-fluorosialic acid is obtained by the above method and its embodiments, which includes the steps of: reacting 3-hydroxysialic acid with a sugar to perform a glycosylation reaction to form an α2,6-linked 3-hydroxysialoside; and reacting the α2,6-linked 3-hydroxysialoside with a fluorinating agent to perform a fluorination reaction to form a sugar containing 3-fluorosialic acid.

[0021] In other embodiments, the sugar containing 3-fluorosialic acid is an α2,6-linked 3-fluorosialoside-terminated N-glycan, which is a compound of formula (V): [ka] Even if that is the case, In the formula, Z is [ka] That is the case.

[0022] Another aspect of the present invention relates to a compound of formula (VI): [ka] Regarding R 1 , R 2 , R 3 , R 4 And X is defined in the section of the summary of the invention described above.

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

[0024] In yet another embodiment, the compound of formula (VI) contains X, which is an N-glycan. An exemplary compound of this embodiment is the compound of formula (V): [ka] And, In the formula, Z is [ka] That is the case.

[0025] The present invention also includes monoclonal antibodies bound to a sugar containing 3-fluorosialic acid, wherein the sugar containing 3-fluorosialic acid is an α2,6-linked 3-fluorosialoside-terminated N-glycan. Preferably, the α2,6-linked 3-fluorosialoside-terminated N-glycan is the compound of formula (V) shown above.

[0026] The present invention further encompasses methods for treating cancer or autoimmune diseases, which include administering an effective amount of the above monoclonal antibody to a subject in need. For example, cancer may be leukemia or lymphoma, and autoimmune diseases 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 mellitus, Sjögren's syndrome, anti-NMDA receptor encephalitis, Devic's disease, Graves' ophthalmopathy, autoimmune pancreatitis, opsoclonus-myoclonus syndrome, or IgG4-related disease.

[0027] The terms “to treat” or “to treat” as used herein refer to administering the pharmaceutical composition to a subject having the disease, namely cancer, symptoms of such disease, or a predisposition to such disease, for the purpose of providing a therapeutic or preventive effect. The term “effective dose” refers to the amount of the active drug required to produce such an effect. The effective dose will vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of concomitant use with other therapeutic procedures, as will be recognized by those skilled in the art.

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

[0029] 3-fluorosubstituted sialic acid derivatives are known to be more stable with respect to sialidase compared to non-fluorinated sialic acid derivatives. 7 By introducing a fluorine atom to the C-3 position of the anomer, we developed 2,3-difluorosialic acid (DFSA) (Scheme 1). 8 , biochemical probes 9 and active protein profiling probes for studying sialidases 10 It was used as follows: With the help of DFSA, the fluorine atom in the axial position at C-3 (3F ax ) is on the 3rd floor eq Deactivation of enzymes is more effective than derivatives (k i ) and reactivation (k hydrIt was found to have a greater effect on deceleration for both of the following: 8b Inspired by this observation, the inventors wished to investigate the stability of sialosides using 3-fluorosialic acid for their potential use in glycoprotein therapeutics. Specifically, the inventors added 3F to the terminus of the N-glycan on the mAb. ax The intention is to study whether incorporating the -Neu5Ac motif can enhance its stability against sialidase while maintaining its effector function.

[0030] Towards our objective, we have developed an α2,6-linked 3F containing a branched N-glycan. ax We have developed a preparative method for the synthesis of -Neu5Ac oligosaccharide. ax We also demonstrated that the Neu5Ac-α2,6-Gal bond is more stable in the presence of sialidase, and that antibodies possessing the corresponding branched glycan have the same binding affinity as their non-fluorinated counterparts.

[0031] (a)XeF2-BF3×OEt2 12 , molecular fluorine 13 and fluorination of glycans protected by Selectfluor 7c (b) Inversion of the equatorial hydroxyl group at C3 in sialic acid derivatives 7f , and (c) ManNAc and 3F 3-fluoropyruvic acid eq Neu5Ac and 3F ax Aldolase-catalyzed enzyme conversion to Neu5Ac 7a、11a、14 Several methods for the synthesis of 3F-Neu5Ac, including [specific compound], have been reported. However, to the best of our knowledge, 3F ax There are currently no described methods for the synthesis of Neu5Ac-terminal N-glycans. 3F ax -The only description disclosing the preparation of disaccharides containing Neu5Ac is 3-F ax This enzyme is limited to the synthesis of Neu5Ac-α2,3-Lac and does not produce the required α2,6-linked form. 11aTherefore, the inventors focused their efforts on the chemical synthesis of this bond. 15 . [ka]

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

[0033] OH eq F axThe inversion to was found to be a difficult task. Fluorine substitution of OTf and OMs using TASF resulted in the decomposition of the starting materials. To optimize this conversion, the inventors screened various fluorinating reagents, but without success. However, by treating 6 with perfluoro-1-butanesulfonyl fluoride (NfF) in the presence of 1,8-diazabicyclo[5,4,0]-undeca-7-ene (DBU) in anhydrous toluene for 2 days at 90°C (Scheme 3), 4 was obtained in 6% yield (Table 2, Item 1). 17 Further optimization of reaction conditions, such as lowering the reaction temperature (items 1, 5, and 6) and increasing the reaction time (item 7), improved the overall yield of 4. The conversion from 7 to 4 appears to be the rate-limiting step due to steric hindrance around C-3. In many cases, the inventors were able to isolate 7 formed within 1 day at room temperature and in the presence of NfF and DBU. However, the conversion from 7 to 4 (yield of 77% brsm) requires a long reaction time. Attempts to improve the conversion by increasing the reaction temperature resulted in the decomposition of 7. Thus, the inventors' best results were obtained by increasing the amount of reagent and extending the reaction time to 15 days (item 8). Finally, the inventors found that the addition of TASF increased the reaction efficiency, thereby helping to shorten the reaction time to just 2 days (item 9). 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, 3F using NIS / TMSOTf ax-Neu5Ac- disaccharide donor 4 was attached to acceptor (8) to obtain 9. Next, the O-allyl group at the anomeric position was removed by isomerization with PdCl2 in AcOH / NaOAc, and the hydroxyl group (10) of the anomeric was further converted to fluoride (11) and imidate (12). 3F was performed using Cp2HfCl2 / AgOTf conditions. ax -Hexa sugar 14 was obtained in 85% yield by glycosylation of the core disaccharide (13) at O-3 with the Neu5Ac-terminal fluoride donor (11). After removal of the benzylidene group, 15 was glycosylated at the O-6 position to obtain the desired decasaccharide (16) in 70% yield with excellent regioselectivity and α-stereoselectivity. The inventors also tested TfOH-enhanced glycosylation using the N-phenyltrifluoroacetoimidate donor (12), but this yielded a product in insufficient yield. Next, the following series of steps were performed: (a) saponification with LiOH to remove the ester and NHTroc group, (b) acetylation of the free amine and alcohol, (c) removal of the OAc group with sodium methoxide, and (d) hydrogenation of the O-benzyl group with Pd / C in a MeOH / water / HCO2H mixture. 4b According to this, a completely deprotected glycan (17) was obtained in a total yield of 40%. [ka]

[0035] Reagents and conditions in Scheme 5: (a) 8, TfOH, NIS, MS at 4 Å, CH2Cl2, -40°C, 2 hours, 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 hours, 56%. (e) 11, AgOTf, Cp2HfCl2, toluene, MS at 4 Å, 0°C, 3 hours, 85%. (f) pTSA·H2O, CH3CN, room temperature, 6 hours, 75%. (g) 11, AgOTf, Cp2HfCl2, toluene, MS at 4 Å, -15°C, 3 hours, 70% (80% brsm). (h) 12, TfOH, CH2Cl2, 4 Å MS, -60 to -20℃, 3 hours, 33% (55% brsm). (i) LiOH, dioxane / H2O (4:1), 90℃, 16 hours. (j) Ac2O, Py, 16 hours. (k) NaOMe, MeOH, 16 hours. (l) Pd(OH)2, MeOH / H2O / HCOOH (6:3:1), H2, 16 hours, 40% (4 stages).

[0036] After establishing a protocol for stepwise synthesis, the inventors streamlined the assembly of glycans by developing a programmable [2+2+2] one-pot synthesis of 17 precursor hexasaccharides (14) (Scheme 6). The designed one-pot protocol is 3F at -40°C. ax The process consisted of initial binding of the -Neu5Ac- disaccharide donor (4) (RRV=2053) to the less reactive acceptor (18) (RRV=537), followed by injection of the reducing end acceptor 13 at -20°C. After 1 hour at -10°C and using a standard purification protocol, the hexasaccharide 14 was isolated in 26% yield. [ka]

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

[0038] 3F in the presence of sialidase axTo collect preliminary data on the stability of the -Neu5Ac-α2,6-Gal motif, the inventors conducted an in vitro assay using commercially available sialidases derived from Clostridium perfringens and Vibrio cholerae. 19 For this purpose, (1) Neu5Ac-α2,6-Gal-pNP and (2) 3F ax -Neu5Ac analogs were prepared as substrates (Scheme 1). Both enzymes showed the expected hydrolytic activity against undenatured substrate 1, but 3F ax -It was inactive with respect to analog 2 (see Figure 1A). The inventors also observed that 2 did not significantly inhibit the hydrolysis of 1, as DANA does (see Figure 1B).

[0039] A standard protocol is used to prepare homogeneous glycoforms of mAbs. 6 Compound 17 was converted to an oxazoline donor according to the instructions and linked to GlcNAc-modified IgG (without core fucose) in the presence of Endo S2 (D184Q). Homogeneous 3F ax The binding affinity of the -Neu5Ac- glycoform to FcγRIIIa was analyzed using surface plasmon resonance (SEM) along with its parent non-fluorinated glycoform (G2S2) and commercially available rituximab samples (major glycoforms: G1F1, G0F1, G2F1). 6 The measurements were taken using the following method. Compared to commercially available rituximab samples, homogeneous glycoforms of IgG containing α2,6-sialylation and a branched N-glycan without core fucose showed 39.9 times (Neu5Ac-G2S2) and 37.4 times (3F) binding affinity. ax -Neu5Ac-G2S2) showed improvement (see Table 4). 3F ax The fact that the binding affinity of the -Neu5Ac modified glycoform was found to be within the same range as that of the parent glycan is related to 3F ax This provides a premise for in vivo studies of -Neu5Ac-glycosylated mAbs. These results will be reported in due course.

[0040] In conclusion, the inventors have found that sialidase-resistant oligosaccharides and 3F ax-3F is used as a building block for the synthesis of representative homogeneous antibodies containing Neu5Ac-terminal bibranched N-glycans. ax We developed the chemical synthesis of -Neu5Ac-α2,6-Gal-STol. Compared to commercially available rituximab samples, it showed 3F ax Homogeneous glycoforms modified with -Neu5Ac-glycan showed a 37.4-fold improvement in binding to FcγRIIIa. Furthermore, defluorination of the parent and 3F ax -Neu5Ac-modified antibody glycoforms showed similar binding affinity to the Fc receptor. Overall, our results reveal a novel general strategy for improving the half-life of therapeutic glycoproteins.

[0041] It is expected that those skilled in the art can make the most of the present invention based on the above description, even without further details. Therefore, the following specific examples are merely illustrative and should not be construed as limiting the remainder of this disclosure. All references herein are incorporated by reference in their entirety.

[0042] Example 1: Synthesis of sugars and precursors containing 3-fluorosialic acid Unless otherwise specified, all reactions were carried out under an inert atmosphere, followed by standard syringe-septum filtration. Solvents were purchased from commercial sources and used without further purification. Fine molecular sieves (4 Å, EMD Millipore) were pulverized and activated before use. The progress of all reactions was monitored using TLC glass plates pre-coated with silica gel 60 F254 (Merck KGaA). TLC was visualized using ultraviolet light (254 nm), p-anisaldehyde, and / or ammonium cerium molybdate staining. Column chromatography was performed using Across silica gel (particle size 0.035-0.070 mm, 60 Å). 1 H, 13 C and 191F NMR spectra were recorded at 25°C using Bruker AVIII-600, DRX-500, AV-400, DPX-400, AVANCE 500 AV, and AVANCE 600 spectrometers, and the chemical shift was measured in δ (ppm) using the residual solvent peak as an internal standard (δ, 1 In 1H NMR, ppm values ​​were 7.24 (CHCl3) and 4.80 (H2O). 13 The result was 77 (CDCl3) in ¹³C NMR. The coupling constant (J) was measured in Hz. Data are expressed as "chemical shift, multiplicity (s=single line, d=double line, t=triple line, q=quadruple line, m=multiple line, br=broad)". In electrospray ionization time-of-flight (ESI-TOF) reflectron experiments, high-resolution mass spectra (HRMS) were recorded using an Agilent LC / MSD TOF mass spectrometer. Cu K α Bruker D8 Platinum- equipped with radiation (λ=1.5478) 135 Single-crystal X-ray diffraction studies were performed using a CCD diffractometer. HPLC measurements were performed using a Hitachi HPLC D-7000 system. RRV measurements were recorded using a normal-phase ZORBAX RX-SIL, 5 μm, 4.6 × 250 mm (colloidal silica, Agilent Technologies) with a solvent system of siRNA / hexane at a flow rate of 1 mL / min, and visualized at 254 nm.

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

[0044] TIFF0007877514000020.tif22 [ka] p-Tolyl[5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-D-erythro-β-L-gluconona-2-uropyranosonate methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 6β. β-anomeric 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-Acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-mannonona-2-uropyranosonic acid methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside 4. Compound 6α (500 mg, 0.47 mmol, 1 equivalent) in a screw-cap vial containing a stirring bar was dissolved in toluene (5 mL), to which DBU (0.28 mL, 1.86 mmol, 4 equivalents), perfluoro-1-butanesulfonyl fluoride (0.33 mL, 1.86 mmol, 4 equivalents) and TASF (256 mg, 0.93 mmol, 2 equivalents) were added. The reaction vial was sealed and stirred at 40°C. After 24 hours, the reaction mixture was treated with additional amounts of DBU (0.28 mL, 1.86 mmol, 4 equivalents), perfluoro-1-butanesulfonyl fluoride (0.33 mL, 1.86 mmol, 4 equivalents), and TASF (256 mg, 0.93 mmol, 2 equivalents), and then stirred at 40°C for a further 24 hours. The reaction mixture was directly packed into 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 eq) in toluene (7 mL) in a screw-cap vial containing a stir bar was added DBU (0.64 mL, 4.31 mmol, 8 eq) and perfluoro-1-butanesulfonyl fluoride (0.77 mL, 4.31 mmol, 8 eq). 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 as a pale yellow powder (267 mg, 77% brsm). R f = 0.43 (silica gel, acetone / toluene = 2:3); 1 1H NMR (600 MHz, CDCl3): δ 7.95 - 7.93 (m, 2H, Ar-H), 7.87 - 7.85 (m, 2H, Ar-H), 7.48 - 7.44 (m, 2H, Ar-H), 7.41 - 7.39 (m, 2H, Ar-H), 7.35 - 7.33 (m, 2H, Ar-H), 7.31 - 7.29 (m, 2H, Ar-H), 7.25 - 7.24 (m, 2H, Ar-H), 7.21 - 7.19 (m, 2H, Ar-H), 7.16 - 7.14 (m, 1H, Ar-H), 7.04 - 7.03 (m, 2H, Ar-H), 5.79 (dd, J = 10.2, 9.6 Hz, 1H), 5.49 (ddd, J = 9.0, 5.4, 2.4 Hz, 1H), 5.42 (dd, J = 10.2, 3.0 Hz, 1H), 5.31 - 5.28 (m, 2H), 5.20 (dd, J = 27.0, 11.4 Hz, 1H, sia-C4-H), 5.01 (dd, J = 51.6, 1.8 Hz, 1H, sia-C3-H), 4.97 (d, J = 10.2 Hz, 1H, C1-H β), 4.66 (d, J = 11.4 Hz, 1H), 4.58 (d, J = 11.4 Hz, 1H), 4.37 (dd, J = 12.6, 2.4 Hz, 1H), 4.28 - 4.26 (m, 2H), 4.17 (dd, J = 12.6, 5.4 Hz, 1H), 4.09 - 4.05 (m, 2H), 3.98 (dd, J = 10.2, 6.0 Hz, 1H), 3.74 (dd, J = 10.2, 8.4 Hz, 1H), 3.72 (s, 3H, -CH3), 2.29 (s, 3H, -CH3), 2.17 (s, 3H, -CH3), 2.16 (s, 3H, -CH3), 2.09 (s, 3H, -CH3), 1.98 (s, 3H, -CH3), 1.91 (s, 3H, -CH3); 13 13C NMR (150 MHz, CDCl3): δ 170.9, 170.7, 170.4, 170.2, 169.8, 165.6, 165.3, 138.3, 137.6, 133.2, 133.0, 132.6, 129.8, 129.8, 129.7, 129.5, 129.3, 129.1, 128.4, 128.3, 128.1, 127.5, 127.3, 98.3, 98.2, 88.1, 86.8, 86.4, 76.2, 75.6, 74.6, 74.0, 71.4, 69.2, 69.0, 68.5, 68.0, 67.3, 63.4, 62.5, 53.2, 45.5, 23.4, 21.2, 21.2, 20.8, 20.7, 20.7; 19 19F NMR (376 MHz, CDCl3): δ -215.9; HRMS (ESI-TOF) m / e: C 54 H 58 FNO 19 SNa [M + Na] + Calculated value: 1098.3200 / Measured value: 1098.3212.

[0047]

Chemical Structure

[0048] TIFF0007877514000024.tif22 [ka] Allyl[5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-mannonona-2-uropyranosonic 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-mannopyranoside 9. A mixture of acceptor 8 (1.04 g, 1.03 mmol, 1 equivalent), donor 4 (1.39 g, 1.29 mmol, 1.25 equivalents), and activated powdered 4 Å MS (0.70 g) in anhydrous CH2Cl2 (7 mL) was stirred under argon for 1 hour. The reaction mixture was then cooled to -40°C, and NIS (465 mg, 2.06 mmol, 2 equivalents) was added, followed by TfOH (0.5 M in Et2O, 0.62 mL, 0.31 mmol, 0.3 equivalents) while stirring. Once the reaction was complete (TLC showed disappearance of the starting materials in about 2 hours), the reaction mixture was inactivated with Et3N (0.4 mL) and filtered through a Celite pad. The filtrate was diluted with CH2Cl2 (30 mL) and washed with 20% Na2S2O3 aqueous solution (10 mL), saturated NaHCO3 aqueous solution (15 mL), and brine (8 mL). The separated organic layer was dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the eluent to obtain compound 9 as a white powder (1.30 g, 64%). 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-Acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-mannonona-2-uropyranosonic 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-mannopyranoside 10. To a stirred solution of tetrasaccharide 9 (578 mg, 0.30 mmol, 1 equivalent) in acetic acid (6.0 mL, acetic acid / water, 10:1 = v / v), CH3COONa (121 mg, 1.48 mmol, 5 equivalents) was added, followed by the addition of PdCl2 (105 mg, 0.59 mmol, 2 equivalents) at room temperature. After 20 hours, when the TLC showed the disappearance of the starting material, the reaction mixture was diluted with ethyl acetate (20 mL) and poured into a saturated NaHCO3 aqueous solution (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 the eluent to obtain compound 10 as a white powder (465 mg, 82%). 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-Acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-mannonona-2-uropyranosonic 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-mannopyranosylfluoride 11. To a well-stirred solution of hemiacetal 10 (395 mg, 0.21 mmol, 1 equivalent) in anhydrous CH2Cl2 (12 mL), DAST (81.6 μL, 0.62 mmol, 3 equivalents) was added at -20°C. The reaction mixture was vigorously stirred until TLC showed the disappearance of the starting material (3 hours). The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with saturated NaHCO3 aqueous solution (5 mL) and brine (4 mL). The organic phase was dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / hexane (3:4) as the eluent to obtain compound 11 as a white powder (289 mg, 73%). 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-mannonona-2-uropyranosonate 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] [C)carbonylamino-β-D-glucopyranosyl]-(1→2)-[3,4,6-tri-O-benzyl-α-D-mannopyranosyl]-(1→3)-[2-O-acetyl-4,6-O-benzylidine-β-D-mannopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl 14. A mixture of AgOTf (194 mg, 0.75 mmol, 6 equivalents relative to the acceptor), bisCp2HfCl2 (167 mg, 0.44 mmol, 3.5 equivalents relative to the acceptor) and newly activated 4 Å MS (1.50 g) in anhydrous toluene (15 mL) was stirred at room temperature under argon for 1 hour. The reaction mixture was then cooled to -20°C and treated with a solution of donor 11 (290 mg, 0.15 mmol, 1.2 equivalents) and acceptor 13 (115 mg, 0.13 mmol, 1 equivalent) in anhydrous toluene (8 mL). The reaction mixture was stirred at 0°C until TLC showed the disappearance of the starting materials (3 hours). After the reaction was complete, the reaction mixture was deactivated with Et3N (0.25 mL), diluted with ELISA (25 mL), and filtered through a Celite pad. The filtrate was washed twice with saturated NaHCO3 aqueous solution (10 mL) and brine (6 mL), 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 the eluent to obtain 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 equivalent), donor 4 (60.1 mg, 0.056 mmol, 1.5 equivalents), and activated powdered 4 Å MS (0.25 g) in anhydrous CH2Cl2 (1.25 mL) was stirred under argon for 1 hour. The reaction mixture was then cooled to -40°C, and NIS (12.6 mg, 0.056 mmol, 1.5 equivalents) was added, followed by TfOH (0.5 M in Et2O, 22.3 μL, 0.011 mmol, 0.3 equivalents). The mixture was then stirred to -20°C for 2 hours. Next, once the starting material had disappeared (monitored by TLC), the reaction mixture was continuously treated for 10 minutes with acceptor 13 (34.2 mg, 0.037 mmol, 1 equivalent), NIS (16.8 mg, 0.075 mmol, 2 equivalents), and TfOH (0.5 M in Et2O, 22.3 μL, 0.011 mmol, 0.3 equivalents) in anhydrous CH2Cl2 (1.25 mL) while stirring under argon. After adding all reagents, the mixture was warmed to -10°C and stirred continuously until the TLC indicated the disappearance of the starting material (1 hour). Once complete, the reaction was inactivated with Et3N (0.4 mL) and filtered through a Celite pad. The filtrate was diluted with CH2Cl2 (10 mL) and washed with 20% Na2S2O3 aqueous solution (3 mL), saturated NaHCO3 aqueous solution (2 mL), and brine (2 mL). The separated organic layer was dried over MgSO4, filtered, and vacuum concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the eluent, followed by a second column chromatography using acetone / hexane (3:4) as the eluent, to obtain 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 Cl6 FN3O 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-mannonona-2-uropyranosonic 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-to [Lichloroethoxy)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 12 mL stirred solution of starting material 14 (318 mg, 0.11 mmol, 1 equivalent) in acetonitrile, p-TsOH·H2O (21.5 mg, 0.11 mmol, 1 equivalent) was added. The reaction mixture was vigorously stirred until TLC showed the disappearance of the starting material (6 hours). Once completed, the reaction mixture was inactivated 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 obtain 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-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro-α-L-mannonona-2-uropyranosonic 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-mannopyranosyl-(1→3)]-[5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro Ro-D-erythro-α-L-mannonona-2-uropyranosonic 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-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 16.

[0056] From the fluoride donor: A mixture of AgOTf (142 mg, 0.55 mmol, 8 equivalents relative to the acceptor), Cp2HfCl2 (105 mg, 0.28 mmol, 4 equivalents relative to the acceptor), and newly activated 4 Å MS (1 g) in anhydrous toluene (10 mL) was stirred at room temperature under an argon atmosphere for 1 hour. The reaction mixture was then cooled to -40 °C, and a solution of donor 11 (232 mg, 0.12 mmol, 1.75 equivalents) and acceptor 15 (188 mg, 0.069 mmol, 1 equivalent) in anhydrous toluene (1 mL) was added. Stirring was continued at -15 °C for 3 hours. The reaction mixture was deactivated with Et3N (0.20 mL), diluted with SiO2 (20 mL), and filtered through a Celite pad. The filtrate was washed twice with saturated NaHCO3 aqueous solution (8 mL) and brine (4 mL). The organic phase was dried over MgSO4, filtered, and vacuum concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (2:3) as the eluent, and compound 16 was obtained as a white powder (223 mg, 70%) together with the recovered compound 15 (23.0 mg, 80% brsm).

[0057] From trifluoroacetoimidate donors: A mixture of acceptor 15 (32.0 mg, 0.012 mmol, 1 equivalent), donor 12 (42.9 mg, 0.021 mmol, 1.75 equivalents), and activated powdered 4 Å MS (200 mg) in anhydrous CH2Cl2 (2 mL) was stirred under argon for 30 minutes. The reaction mixture was then cooled to -60°C and treated with TfOH (0.5 M in Et2O, 5.86 μL, 2.93 μmol, 0.25 equivalents relative to the acceptor). After stirring at -20°C for 3 hours, the reaction mixture was deactivated with Et3N (0.10 mL), diluted with CH2Cl2 (10 mL), and filtered through a Celite pad. The filtrate was washed twice with saturated NaHCO3 aqueous solution (4 mL) and brine (3 mL). The organic phase was dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (2:3) as the eluent, and compound 16 was obtained as a white powder (18.0 mg, 33%) together with the 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-Acetamide-3,5-dideoxy-3-fluoro-D-erythro-α-L-mannonona-2-uropyranosonate-(2→6)-β-D-galactopyranosyl-(1→4)-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-[5-Acetamide-3,5-dideoxy-3-fluoro L-D-erythro-α-L-mannonona-2-uropyranosonate-(2→6)-β-D-galactopyranosyl-(1→4)-2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)]-[β-D-mannopyranosyl]-(1→4)-2-acetamide-2-deoxy-D-glucopyranoside 17. Protected glycan 16 (103 mg, 0.022 mmol, 1 equivalent) and LiOH (51.5 mg, 50% SMwt) were mixed in a 1,4-dioxane / H2O mixture (3 mL, 4:1=v / v) and stirred at 90°C for 16 hours. The reaction mixture was concentrated under high vacuum and subjected to acetylation conditions (pyridine (2.5 mL), Ac2O (1.5 mL), room temperature, 16 hours). After removing the solvent, the crude residue was purified by LiChroprep® RP-18 reversed-phase column chromatography using H2O / MeOH (1:5) as the eluent. The product was deacetylated by stirring with NaOMe in MeOH (3 mL, 0.5 M) for 16 hours. The reaction mixture was neutralized with IR-120, filtered, and concentrated under vacuum. The residue was purified by LiChroprep® RP-18 reversed-phase column chromatography using H2O / MeOH (1:4) as the eluent. The deacetylated crude product was dissolved in a mixture of MeOH / H2O / HCOOH (3 mL, 6:3:1 = v / v / v) and treated with Pd(OH)2 (51.5 mg, 50% SMwt) for 20 hours. The reaction mixture was filtered through a Celite pad and concentrated under vacuum. The residue was purified by (BIO-RAD) Biogel P-2 column chromatography (elution with water), and then by LiChroprep® RP-18 reversed-phase column chromatography (elution with water) to obtain compound 17 as a white powder of an anomer mixture (α:β = 0.65:0.35) (18.3 mg, 40%). 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 hours, then AcCl, room temperature, 2 days, then Na2HPO4, CH3CN, reflux, 20 hours, 92%. (b) Reference 3, 80% (3 steps). (c) TiBr4, dichloroethane, room temperature, 10 minutes, 92%. (d) BH3, Cu(OTf)2, THF, room temperature, 8 hours, 96%.

[0061] [ka] 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2,6-anhydro-D-glycero-D-galactonone-2-methyl ethanol S3 19Compound S2 (20 g, 64.7 mmol, 1 equivalent) was mixed with MeOH (600 mL) in a stirred solution, to which TfOH (4.95 mL, 64.7 mmol, 1 equivalent) was added. The reaction mixture was stirred at 60°C for 16 hours. The solvent was removed by rotary evaporation under reduced pressure, and trace amounts of water were removed by simultaneous evaporation twice with toluene. 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. After removing the solvent, the resulting residue was diluted with anhydrous acetonitrile (200 mL), and then Na2HPO4 (19.6 g, 155 mmol, 2.4 equivalents) was added under argon. The reaction mixture was vigorously stirred at 90°C until TLC showed the disappearance of the starting material (16 hours). This solution was filtered through a Celite pad, dried over MgSO4, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using SiO / hexane (4:1) as the eluent to obtain compound S3 as a brown foamy substance (28.2 g, 92%). The spectroscopic data were consistent with those previously reported in the literature. 19 R f =0.30(silica gel, alkyl) 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-uropyranosonate methyl3 20 The synthesis of epoxide S4 was carried out from sialic acid using the reported procedure. 21 To a stirred solution of epoxide compound S4 (1.10 g, 2.25 mmol, 1 equivalent) in anhydrous 1,2-dichloroethane (18 mL), TiBr4 (0.91 g, 2.47 mmol, 1.1 equivalents) was added under argon for 10 minutes. The solvent was removed by rotational evaporation under high vacuum. The resulting residue was diluted with SiO4 (30 mL) and washed with saturated aqueous solution of Na2SO4 (10 mL), 5% aqueous solution of NaHCO3 (10 mL), and then brine (5 mL). The separated organic layer was dried over MgSO4, concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography using acetone / hexane (2:1) as the eluent to obtain 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 (1 M, 34.3 mL, 34.3 mmol, 5 equivalents in THF) (4.00 g, 6.86 mmol, 1 equivalent), Cu(OTf)2 (124 mg, 0.34 mmol, 0.05 equivalents) was added, and the reaction mixture was stirred at room temperature for 8 hours. After completion, the reaction system was carefully neutralized at 0°C with TEA (0.96 mL, 6.86 mmol, 1 equivalent), and then diluted with MeOH (15 mL). After removing the solvent under vacuum, the resulting residue was purified by silica gel column chromatography using siRNA / hexane (1:2) as the eluent to obtain compound 5 as a white powder (3.85 g, 96%). The spectroscopic data were consistent with those previously reported in the literature. 22 R f =0.45 (silica gel, SiO: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 hours, -20°C, 90%. (b) NIS, TfOH, 4 Å MS, -40°C, 1.5 hours, CH2Cl2, 75%. (c) NaBH3CN, HCl / ether, AW-300, THF, 0°C to room temperature, 16 hours, 85%.

[0065] [ka] p-Tolyl3-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 THF / toluene (5.17 g, 9.42 mmol, 1 equivalent) in 45 mL (1:2=v / v), TESOTf (4.26 mL, 18.8 mmol, 2 equivalents) was added under argon at -20°C. After stirring at -20°C for 45 minutes, benzaldehyde (4.79 mL, 47.1 mmol, 5 equivalents) and triethylsilane (2.26 mL, 14.1 mmol, 1.5 equivalents) were added dropwise to the mixture with stirring. After 2 hours at -20°C, TLC showed the disappearance of the starting materials, and the reaction was deactivated with saturated aqueous solution of Na2CO3, diluted with SiO2 (100 mL), and washed with saturated aqueous solution of Na2CO3 (30 mL) and brine. The combined organic layers were dried over MgSO4, filtered, and concentrated under vacuum. The resulting residue was first purified by silica gel column chromatography using CH2Cl2 / toluene (1:4) as the eluent, followed by SiO2 / toluene (1:10), to obtain compound S7 as a white solid (5.40 g, 90%). f =0.50 (silica gel, Â: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); 13¹¹C NMR (150MHz, acetone-d6):δ 155.3, 139.8, 139.4, 138.6, 133.3, 130.7, 130.5, 129.8, 129.6, 129.1, 129.0, 129.0, 128.5, 128.2, 127.1, 101.8, 97.1, 88.6, 82.7, 80.8, 75.0, 75.0, 71.2, 69.1, 57.4, 21.1; HRMS (ESI-TOF) m / e:C 30 H 30 Cl3NO6SNa[M+Na] + Calculated value: 660.0752 / Measured value: 660.0749.

[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 CH2Cl2 (20 mL). 25 A mixture of (523 mg, 0.94 mmol, 1 equivalent), donor S7 (900 mg, 1.41 mmol, 1.5 equivalents), and activated powdered 4 Å MS (2.00 g) was stirred under argon for 1 hour. The reaction mixture was then cooled to -40°C and treated with NIS (423 mg, 1.88 mmol, 2 equivalents) and TfOH (0.5 M in Et2O, 0.47 mL, 0.23 mmol, 0.25 equivalents). After stirring at that temperature for 1.5 hours, TLC analysis showed the disappearance of the starting material. The reaction mixture was deactivated with Et3N (0.2 mL) and filtered through a Celite pad. The filtrate was diluted with CH2Cl2 (20 mL) and washed with 20% Na2S2O3 aqueous solution (5 mL), saturated NaHCO3 aqueous solution (10 mL), and brine (5 mL). The separated organic layer was dried over MgSO4 and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using dimethyl / toluene (1:10) as the eluent to obtain compound S9 as a white foamy substance (710 mg, 75%). f =0.36 (silica gel, toluene: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 formula

[0068] [ka] Reagents and conditions in Scheme S3: (a) NaBH3CN, HCl / ether, AW-300, THF, 0°C to room temperature, 16 hours, 90%. (b) Ac2O, pyridine, room temperature, 16 hours, 98%. (c) NIS, TfOH, HOPO(OBu)2, MS at 4 Å, -30°C, 2 hours, CH2Cl2, 90%. (d) TMSOTf, MS at 4 Å, -50°C, 1 hour, CH2Cl2, 87%. (e) NaOMe, MeOH / CH2Cl2, 0°C to room temperature, 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 1.00 g, 1.56 mmol, 1 equivalent of the starting material S7, was added to a stirred solution of anhydrous THF (30 mL) under argon. The reaction mixture was then cooled to 0°C, and NaCNBH3 (0.98 g, 15.6 mmol, 10 equivalents) was added, followed by the slow addition of HCl·Et2O (2 M in Et2O, 7.04 mL, 14.1 mmol, 9 equivalents). The mixture was stirred until TLC showed the disappearance of the starting material (16 hours). Once complete, the reaction mixture was deactivated with saturated NaHCO3 aqueous solution (0.5 mL) and filtered through a Celite pad. The filtrate was diluted with CH2Cl2 (50 mL) and then washed with saturated NaHCO3 aqueous solution (15 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography using SiO / hexane / CH2Cl2 (1:3:1) as the eluent to obtain compound S10 as a white foamy substance (902 mg, 90%). Spectroscopic data can be found in the literature. 28 This was consistent with what had been reported in R. f =0.29 (silica gel, siRNA: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. Ac2O (6 mL) was added to a stirred solution of starting material S10 (0.97 g, 1.51 mmol, 1 equivalent) in pyridine (12 mL). The reaction mixture was vigorously stirred at room temperature for 16 hours, then concentrated under vacuum. The compound S11 was purified by silica gel column chromatography using siRNA / hexane / CH2Cl2 (1:4:1) as the eluent to obtain compound S11 as a white powder (1.01 g, 98%). f =0.47 (silica gel, toluene:hexane = 1:3); 1 H NMR (600MHz, CDCl3): δ7.41-7.40(m,2H,Ar-H),7.34-7.25(m,8H,Ar-H),7.22-7.21(m ,2H,Ar-H),7.03-7.01(m,2H,Ar-H),5.29(d,J=7.2Hz,1H),5.04(d,J=10.2Hz,1H,C1-H β),4.98(dd,J=9.6,9.6Hz,1H),4.78(d,J=12.0Hz,1H),4.70(d,J=12.0Hz,1H),4.62(d,J=11.4Hz,1H),4.57(d,J=11.4Hz,1H),4.50(br,2H) ,4.05(dd,J=9.6,9.0Hz,1H),3.65-3.63(m,1H),3.59-3.55(m,2H),3.33(ddd,J=9.6,9.6,9.6Hz,1H),2.29(s,3H,-CH3),1.87(s,3H,-CH3); 13 C NMR (150MHz, CDCl3): δ169.8,153.7,138.4,138.0,137.7,133.3,129.8,128.6,128.5,128.4,128.2,128.1,128 .1,127.9,127.9,127.7,95.5,85.3,79.4,77.7,74.4,73.6,71.4,69.7,56.5,21.1,20.9;HRMS(ESI-TOF)m / e:C 32 H 34 Cl3NO7SNa[M+Na] + Calculated value: 704.1014 / Measured value: 104.1031.

[0071]

change

[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 CH2Cl2 (2 mL). 29 A mixture of (407 mg, 0.73 mmol, 1 equivalent), donor S12 (900 mg, 1.17 mmol, 1.6 equivalents), and activated powdered 4 Å molecular sieve (200 mg) was stirred under argon for 30 minutes. The mixture was then cooled to -50°C, and TMSOTf (0.21 mL, 1.17 μmol, 1.6 equivalents relative to the acceptor) was added with stirring until TLC analysis showed the disappearance of the starting material (1 hour). The reaction mixture was deactivated with Et3N (0.30 mL), diluted with CH2Cl2 (20 mL), and filtered through a Celite pad. The filtrate was washed twice with saturated NaHCO3 aqueous solution (8 mL) and brine (4 mL). The organic phase was dried over MgSO4, filtered, and vacuum concentrated. The resulting residue was purified by silica gel column chromatography using phenylethylamine (1:3) as the eluent to obtain compound S14 as a white powder (710 mg, 87%). f =0.26 (silica gel, siRNA: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<000031(END]]SNa [M + Na] + Calculated value: 1136.2950 / Measured value: 1136.2978.

[0073]

Chemical formula

[0074]

Chem.

[0075]

Chem.

[0077] [ka] Benzyl[2-O-acetyl-4,6-O-benzyridine-3-Op-methoxybenzyl-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 disaccharide S18 (1.02 g, 1.02 mmol, 1 equivalent) in anhydrous CH2Cl2 (13 mL), anhydrous pyridine (0.58 mL, 7.17 mmol, 7 equivalents) was added, followed by the dropwise addition of Tf2O (0.30 mL, 1.79 mmol, 1.75 equivalents) under argon at 0°C. The reaction mixture was stirred at 0°C until TLC showed the disappearance of the starting material (4 hours). Once complete, it was diluted with CH2Cl2 (20 mL) and washed with 0.5 N HCl (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated under vacuum. The crude residue was mixed with Bu4NOAc (927 mg, 3.07 mmol, 3 equivalents) and dissolved in toluene (20 mL). The solvent was removed under vacuum, the residue was evaporated twice simultaneously with toluene, and then redissolved in anhydrous toluene (13 mL). The mixture was sonicated for 8 hours. During this time, the inventors observed partial deprotection of NHTroc, and the reaction mixture was concentrated under high vacuum. The resulting residue was dissolved in CH2Cl2 (13 mL) and treated with NaHCO3 (430 mg, 5.12 mmol, 5 equivalents) and 2,2,2-trichloroethyl chloroformate (0.71 mL, 5.12 mmol, 5 equivalents) under argon at 0°C. After stirring for 3 hours, the reaction mixture was diluted with CH2Cl2 (20 mL) and washed with water (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using Depositphotos / toluene (1:5) as the eluent to obtain compound S19 as a white powder (650 mg, 61%).

[0078] From S21: A solution of disaccharide S21 (1.20 g, 1.21 mmol, 1 equivalent) in ethylenediamine / n-BuOH (15 mL, 2:8=v / v) was stirred at 90°C for 2 hours. After removal of the solvent, the crude product was evaporated twice simultaneously with toluene. The resulting residue was dissolved in CH2Cl2 (30 mL) and treated with NaHCO3 (508 mg, 6.05 mmol, 5 equivalents) and 2,2,2-trichloroethyl chloroformate (0.83 mL, 6.05 mmol, 5 equivalents) under argon at 0°C. After 3 hours, the reaction mixture was diluted with CH2Cl2 (45 mL) and washed with water (30 mL) and brine (15 mL). The organic layer was dried over MgSO4, concentrated, evaporated twice simultaneously with toluene, and then evaporated under vacuum. The resulting residue was subjected to acetylation conditions by slowly warming it from 0°C to room temperature over 16 hours, and directly to Ac2O (7 mL) in pyridine (14 mL). The reaction mixture was concentrated under high vacuum and purified by silica gel column chromatography using acetone / toluene (1:5) as the eluent to obtain compound S19 as a white powder (1.04 g, 83%). f =0.54 (silica gel, toluene: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-benzyridine-3-Op-methoxybenzyl-β-D-glucopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside S20. To a stirred solution of starting material S17 (2.48 g, 2.37 mmol, 1 equivalent) in anhydrous THF (55 mL), hydrazine acetate (327 mg, 3.55 mmol, 1.5 equivalents) was added under argon at room temperature. The reaction mixture was vigorously stirred until TLC showed the disappearance of the starting material (16 hours). Once complete, the reaction mixture was diluted with siRNA (150 mL) and washed with water (60 mL) and brine (30 mL). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography using SiO2 / hexane (1:2) as the eluent to obtain compound S20 as a white foamy substance (2.07 g, 92%). f =0.54 (silica gel, SiO: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 / Measured value:972.3567.

[0081] [ka] Benzyl[2-O-acetyl-4,6-O-benzyridine-3-Op-methoxybenzyl-β-D-mannopyranosyl]-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside S21. Disaccharide S20 (936 mg, 0.985 mmol, 1 equivalent) was stirred in anhydrous CH2Cl2 (12 mL) to which anhydrous pyridine (0.56 mL, 6.90 mmol, 7 equivalents) was added, followed by the dropwise addition of trifluoromethanesulfonic acid anhydride (0.29 mL, 1.72 mmol, 1.75 equivalents) under argon at 0°C. The mixture was stirred at 0°C until TLC showed the disappearance of the starting material (2 hours). Upon completion, the reaction mixture was diluted with CH2Cl2 (20 mL) and washed with 0.5 N HCl (20 mL) and brine (10 mL). The separated organic layer was dried over MgSO4 and concentrated under vacuum. The resulting residue was used directly for further reactions. Bu4NOAc (594 mg, 1.97 mmol, 2 equivalents) was added to the residue and dissolved in toluene (18 mL). The solvent was removed under vacuum, and the residue was evaporated twice simultaneously with toluene to remove trace amounts of water. The residue was redissolved in anhydrous toluene (12 mL), and the mixture was sonicated for 8 hours. After completion, the reaction mixture was diluted with HCl (20 mL) and washed with water (20 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 HCl / hexane (1:2) as the eluent to obtain compound S21 as a white powder (913 mg, 93%). f =0.29 (silica gel, siRNA: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-mannonona-2-uropyranosonic acid 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 equivalent) in anhydrous CH2Cl2 (2 mL), bromine (6.02 μL, 0.12 mmol, 1.1 equivalents) was added at 0°C. After vigorously stirring the reaction mixture for 10 minutes, the solvent was removed under vacuum, and the residue was evaporated twice simultaneously with toluene to remove any trace amounts of water. The resulting residue was dissolved in anhydrous CH3CN (2 mL) and treated with 4-nitrophenol (25.3 mg, 0.18 mmol, 1.7 equivalents) and Ag2O (124 mg, 0.53 mmol, 5 equivalents). The reaction mixture was vigorously stirred in N2 in the dark until TLC showed the disappearance of the starting material (1 hour). Once complete, the reaction mixture was diluted with SiO2 (10 mL) and filtered through a Celite pad. The filtrate was washed twice with saturated NaHCO3 aqueous solution (4 mL) and brine (3 mL). The organic phase was dried over MgSO4, filtered, and vacuum concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the eluent to obtain compound S22 as a white powder (85 mg, 73%). 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: 11,13.3131.

[0084]

Chemical Structure

[0085]

Chemical Structure

[0086] [ka] p-Tolyl[5-Acetamide-3,4,7,8,9-Penta-O-acetyl-5-deoxy-D-erythro-α-L-gluconone-2-uropyranosonate methyl]-(2→6)-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-galactopyranoside S24. Ac2O (1 mL) was added to a stirred solution of starting material 6 (174 mg, 0.16 mmol, 1 equivalent) in pyridine (2 mL). The reaction mixture was vigorously stirred at room temperature for 16 hours, and then concentrated under high vacuum. The resulting residue was purified by silica gel column chromatography using acetone / toluene (1:2) as the eluent to obtain compound S24 as a white powder (162 mg, 90%). 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[5-acetamido-3,4,7,8,9-penta-O-acetyl-5-deoxy-D-erythro-α-L-gluconona-2-uropyranosonic acid methyl]-(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 equivalent) in anhydrous CH2Cl2 (2.5 mL), bromine (6.67 μL, 0.13 mmol, 1.1 equivalents) was added at 0°C. After vigorous stirring for 10 minutes, the solvent was removed under vacuum, and the residue was evaporated twice simultaneously with toluene to remove trace amounts 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 equivalents) and Ag2O (137 mg, 0.59 mmol, 5 equivalents). The reaction mixture was vigorously stirred in N2 in the dark until TLC showed the disappearance of the starting material (1 hour). After the reaction was complete, the reaction mixture was diluted with SiO2 (12 mL) and filtered through a Celite pad. The filtrate was washed twice with saturated NaHCO3 aqueous solution (5 mL) and brine (3 mL). The organic phase was dried over MgSO4, filtered, and vacuum concentrated. The resulting residue was purified by silica gel column chromatography using acetone / toluene (3:5) as the eluent to obtain compound S25 as a white powder (99 mg, 74%). 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-galactonona-2-uropyranosylonate]-(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 (BIO-RAD) Biogel P-2 column chromatography using water as the eluent. The fraction containing Neu5Ac-α2,6-Gal-pNP was recovered and freeze-dried 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: We received β-galactosidase from Aspergillus oryzae (G5160), as well as sialidases derived from Vibrio cholerae (11080725001) and Clostridium perfringens (11585886001) from Sigma Aldrich.

[0092] Enzyme assay for sialidase: This assay was performed in a 96-well plate in a double-row configuration at 37°C with 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: Clostridium perfringens (1 mU), sodium acetate buffer (50 mM) (pH 5.0), and CaCl2 (10 mM); Vibrio cholerae (2 mU), sodium acetate buffer (50 mM) (pH 5.5), CaCl2 (10 mM), and NaCl (150 mM). These reactions were carried out for 40 minutes to 2 hours for Clostridium perfringens and overnight for Vibrio 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 using a microplate reader in the reaction mixture A 405nm This was determined by measuring three compounds (Neu5Ac-α2, 6-GalβpNP, 3F ax -Neu5Ac-α2,6-GalβpNP and 3OH eq -Neu5Ac-α2,6-GalβpNP) was tested as a substrate for the enzyme. All three compounds were incubated at 37°C for 1 hour in the absence of sialidase and then converted to 20 mM. 405nm It has a background absorbance of three compounds, namely 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 for pNP was determined by stepwise 2-fold dilution of 0.35 mM pNP, and then A for the concentration of pNP. 405nm The standard curve for pNP was obtained by graphing the data.

[0093] Inhibition assays for sialidase: The assay was performed in a 96-well plate in double rows at 37°C with a final volume of 50 μL containing the substrate Neu5Ac-α2,6-GalβpNP (0.6 mM), β-galactosidase (100 mU), and sialidase, either in the absence or presence of various inhibitor concentrations (0–20 mM). The reaction was allowed to proceed for 40 minutes to 2 hours for Clostridium perfringens and overnight for Vibrio cholerae. The assay was stopped by adding CAPS buffer (65 μL, 0.5 M, pH 10.5). The amount of p-nitrophenolate formed was measured using a microplate reader in the reaction mixture A 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 Neu5Ac and homogeneous mAbs modified with Neu5Ac Enzyme expression: Endoglycosidases Endo-S, Endo-S2, Endo-S2 mutant (D184Q), and α-L-fucosidase derived from Bacteroides fragilis NCTC9343 were expressed in E. 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 hours. LC-MS and SDS-PAGE analysis showed complete cleavage of N-glycans on the heavy chain. The reaction mixture was subjected to affinity chromatography using a protein A-agarose resin column (1 mL, GE Healthcare) pre-equilibriumated with Tris-HCl buffer (50 mM, pH 7.4). The column was then washed with Tris-HCl buffer (50 mM, pH 7.4, 20 mL). The bound IgG was liberated 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). The fractions containing the antibody were combined and concentrated by centrifugation (Amicon Ultra centrifugal filter, Millipore, Billrica, Massachusetts) to obtain mono-GlcNAc rituximab (2.4 mg). The product was trypsin-treated, and the glycopeptides, namely TKPREEQYNSTYR (m / z=1391.58) and EEQYNSTYR (m / z=1873.88), were analyzed using nanospray LC / MS to confirm the glycosylation pattern of mono-GlcNAc.

[0096] Glycosyl group transfer of mono-GlcNAc rituximab by glycan oxazoline: Glycan oxazoline was added to a mixture of Endo-S2D184Q and mono-GlcNAc rituximab in 50 mM Tris buffer (pH 7.4). This solution was incubated at 37°C for 30 minutes. The reaction mixture was then purified using a protein-A affinity column, followed by an anion exchange column (Capto Q, GE Healthcare) to recover the desired product.

[0097] SDS-PAGE detection of glycotechnology-engineered Herceptin antibodies: All SDS-PAGE analyses were performed using NuPAGE® Novex® 4-12% Bis-Tris gel (Invitrogen) in MOPS buffer, along with 2-mercaptoethanol present in the sample (Figure 2).

[0098] MS spectroscopy analysis of glycobiologically manipulated mAbs: For the analysis of trypsin-treated glycopeptides, high-resolution and high-mass-accuracy nanoflow LC-MS / MS experiments were performed using an LTQFT Ultra (linear quadrupole ion trap Fourier transform ion cyclotron resonance) mass spectrometer (Thermo Electron, San Jose, California) equipped with a nanoelectrospray ion source (New Objective), an Agilent 1100 Series two-component high-performance liquid chromatography pump (Agilent Technologies, Palo Alto, California), and a Famos autosampler (LC Packings, San Francisco, California). Digestion solution (6 μL) was injected into a self-packed pre-column (150 μm (inner diameter) × 20 mm, 5 μm, 100 Å) at a flow rate of 10 μL / min. Chromatographic separation was performed using a self-packed reversed-phase C18 nanocolumn (75 μm (inner diameter) × 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, operating at a flow rate of 300 nL / min. Full scan MS conditions: 100,000 investigation at a mass range of m / z 320~2000 and a resolution of m / z 400. Ten of the strongest ions were continuously isolated for MS2 by LTQ. The electrospray voltage was maintained at 1.8 kV and the capillary temperature was set to 200 °C (Figure 3 and Table 3).

[0099] Surface plasmon resonance (SPR) analysis All SPR experiments were performed at 25°C using a single-cycle kinetic method with a BIACORE T200, using HBS-EP (10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) as the electrophoresis buffer. FcγRIIIa was transfected into HEK-293 cells, and complex glycosylated recombinant protein was expressed as the analyte. To analyze 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. Rituximab was then captured on the active channel to interact with serial dilutions of the FcγRIIIa analyte (2.5, 5, 10, 20, 40 nM for 2,6-FluoSCT and 2,6-SCT; 8, 24, 72, 216, 648 nM for commercially available rituximab) at a rate of 30 μL / min over a 240-second binding period followed by a 420-second dissociation period. Rituximab data were processed with a dual reference for background removal. The rituximab data were fitted to a 1:1 Langmuir binding model in BiaEvaluation software (GE Healthcare) to obtain reaction rate / affinity constants (Table 4). The antibodies to be analyzed were captured using 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 Previously reported experimental procedure 34 RRV was measured in a triple series according to the following procedure. The RRV (2053) of disaccharide donor 4 was compared to that of competing reference donor S34. 23 The measurement was performed against (RRV=1791). The RRV (537) of disaccharide donor 18 was compared to that of competing reference donor S. 23 Measurements were taken for (RRV=286). [ka]

[0101] Other Embodiments All of the features disclosed herein may be combined into a single feature in any combination. Each feature disclosed herein may be replaced by another feature that serves the same, equivalent, or similar purpose. Thus, unless expressly otherwise specified, each disclosed feature is merely an example of a general set of equivalent or similar features.

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

[0103] References (1) Varki, A. Sialic acids in human health and disease. Trends Mol. Med. 2008, 14, 351. (2) (a) Liu, Y.-C.; Yen, H.-Y.; Chen, C.-Y.; Chen, C.-H.; Cheng, P.-F.; Juan, Y.-H.; Chen, C.-H.; Khoo, K.-H.; Yu, C.-J.; Yang, P.-C.; Hsu, T.-L.; Wong, C.-H. Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 11332. (b) Yen, H.-Y.; Liu, Y.-C.; Chen, N.-Y.; Tsai, C.-F.; Wang, Y.-T.; Chen, Y.-J.; Hsu, T.-L.; Yang, P.-C.; Wong, C.-H. Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 6955. (3) (a) Ashwell, G.; Harford, J. Carbohydrate-specific receptors of the liver. Annu. Rev. Biochem. 1982, 51, 531. (b) Weigel, P. H.; Yik, J. H. Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan / chondroitin sulfate receptors. Biochim. Biophys. Acta 2002, 1572, 341. (4) (a) Wang, Z.; Chinoy, Z. S.; Ambre, S. G.; Peng, W.; McBride, R.; de Vries, R. P.; Glushka, J.; Paulson, J. C.; Boons, G. J. A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 2013, 341, 379. (b) Shivatare, S. S.; Chang, S. H.; Tsai, T. I.; Tseng, S. Y.; Shivatare, V. S.; Lin, Y. S.; Cheng, Y. Y.; Ren, C. T.; Lee, C. C.; Pawar, S.; Tsai, C. S.; Shih, H. W.; Zeng, Y. F.; Liang, C. H.; Kwong, P. D.; Burton, D. R.; Wu, C. Y.; Wong, C. H. Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies. Nat. Chem. 2016, 8, 338. (c) Li, L.; Liu, Y.; Ma, C.; Qu, J.; Calderon, A. D.; Wu, B.; Wei, N.; Wang, X.; Guo, Y.; Xiao, Z.; Song, J.; Sugiarto, G.; Li, Y.; Yu, H.; Chen, X.; Wang, P. G. Efficient chemoenzymatic synthesis of an N-glycan isomer library. Chem. Sci. 2015, 6, 5652. (5) Li, C.; Wang, L.-X. Chemoenzymatic Methods for the Synthesis of Glycoproteins. Chem. Rev. 2018, 118, 8359.(6) (a) Lin, C.-W.; Tsai, M.-H.; Li, S.-T.; Tsai, T.-I.; Chu, K.-C.; Liu, Y.-C.; Lai, M.-Y.; Wu, C.-Y.; Tseng, Y.-C.; Shivatare, S. S.; Wang, C.-H.; Chao, P.; Wang, S.-Y.; Shih, H.-W.; Zeng, Y.-F.; You, T.-H.; Liao, J.-Y.; Tu, Y.-C.; Lin, Y.-S.; Chuang, H.-Y.; Chen, C.-L.; Tsai, C.-S.; Huang, C.-C.; Lin, N.-H.; Ma, C.; Wu, C.-Y.; Wong, C.-H. A common glycan structure on immunoglobulin G for enhancement of effector functions. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 10611. (b) Tsai, T.-I.; Li, S.-T.; Liu, C.-P.; Chen, K. Y.; Shivatare, S. S.; Lin, C.-W.; Liao, S.-F.; Lin, C.-W.; Hsu, T.-L.; Wu, Y.-T.; Tsai, M.-H.; Lai, M.-Y.; Lin, N.-H.; Wu, C.-Y.; Wong, C.-H. An Effective Bacterial Fucosidase for Glycoprotein Remodeling. ACS Chem. Biol. 2017, 12, 63. (c) Liu, C.-P.; Tsai, T.-I.; Cheng, T.; Shivatare, V. S.; Wu, C.-Y.; Wu, C.-Y.; Wong, C.-H. Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation. Proc. Natl. Acad. Sci. U.S.A.2018, 115, 720. (7) (a) Gantt, R.; Millner, S.; Binkley, S. B. Inhibition of N-Acetylneuraminic Acid Aldolase by 3-Fluorosialic Acid. Biochemistry 1964, 3, 1952. (b) Hagiwara, T.; Kijima-Suda, I.; Ido, T.; Ohrui, H.; Tomita, K. Inhibition of bacterial and viral sialidases by 3-fluoro-N-acetylneuraminic acid. Carbohydr. Res. 1994, 263, 167. (c) Burkart, M. D.; Zhang, Z.; Hung, S.-C.; Wong, C.-H. A New Method for the Synthesis of Fluoro-Carbohydrates and Glycosides Using Selectfluor. J. Am. Chem. Soc. 1997, 119, 11743. (d) D. Burkart, M.; P. Vincent, S.; Wong, C.-H. An efficient synthesis of CMP-3-fluoroneuraminic acid. Chem. Commun. 1999, 1525. (e) Burkart, M. D.; Vincent, S. P.; Duffels, A.; Murray, B. W.; Ley, S. V.; Wong, C.-H. Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases. Bioorg. Med. Chem. 2000, 8, 1937. (f) Sun, X.‐L.; Kanie, Y.; Guo, C.‐T.; Kanie, O.; Suzuki, Y.; Wong, C.‐H.Syntheses of C‐3‐Modified Sialylglycosides as Selective Inhibitors of Influenza Hemagglutinin and Neuraminidase. Eur. J. Org. Chem. 2000, 2643. (g) Rillahan, C. D.; Antonopoulos, A.; Lefort, C. T.; Sonon, R.; Azadi, P.; Ley, K.; Dell, A.; Haslam, S. M.; Paulson, J. C. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat. Chem. Biol. 2012, 8, 661. (8) Ishiwata, K.; Ido, T.; Nakajima, T.; Ohrui, H.; Kijima-Suda, I.; Itoh, M. Tumor uptake study of 18F-labeled N-acetylneuraminic acids. International journal of radiation applications and instrumentation. Int. J. Rad. Appl. Instrum. B 1990, 17, 363. (9) (a) Watts, A. G.; Damager, I.; Amaya, M. L.; Buschiazzo, A.; Alzari, P.; Frasch, A. C.; Withers, S. G. Trypanosoma cruzi Trans-sialidase Operates through a Covalent Sialyl-Enzyme Intermediate: Tyrosine Is the Catalytic Nucleophile. J. Am. Chem. Soc. 2003, 125, 7532. (b) Buchini, S.; Gallat, F. X.; Greig, I. R.; Kim, J. H.; Wakatsuki, S.; Chavas, L. M.; Withers, S. G. Tuning Mechanism-Based Inactivators of Neuraminidases: Mechanisticand Structural Insights. Angew. Chem. Int. Ed. Engl. 2014, 53, 3382. (c) Kim, J. H.; Resende, R.; Wennekes, T.; Chen, H. M.; Bance, N.; Buchini, S.; Watts, A. G.; Pilling, P.; Streltsov, V. A.; Petric, M.; Liggins, R.; Barrett, S.; McKimm-Breschkin, J. L.; Niikura, M.; Withers, S. G. Mechanism-Based Covalent Neuraminidase Inhibitors with Broad-Spectrum Influenza Antiviral Activity. Science 2013, 340, 71. (10) Tsai, C. S.; Yen, H. Y.; Lin, M. I.; Tsai, T. I.; Wang, S. Y.; Huang, W. I.; Hsu, T. L.; Cheng, Y. S.; Fang, J. M.; Wong, C.-H. Cell-permeable probe for identification and imaging of sialidases. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 2466. (11) (a) Chokhawala, H. A.; Cao, H.; Yu, H.; Chen, X. Enzymatic Synthesis of Fluorinated Mechanistic Probes for Sialidases and Sialyltransferases. J. Am. Chem. Soc. 2007, 129, 10630. (b) McArthur, J. B.; Yu, H.; Zeng, J.; Chen, X. Converting Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) to a regioselective α2,6-sialyltransferase by saturation mutagenesis and regioselective screening. Org. Biomol. Chem., 2017, 15, 1700. (12) Petrie, C. R.; Sharma, M.; Simmons, O. D.; Korytnyk, W. Synthesis of analogs of N-acetylneuraminic acid and their effect on CMP-sialate synthase. Carbohydr. Res. 1989, 186, 326. (13) Nakajima, T.; Hori, H.; Ohrui, H.; Meguro, H.; Ido, T. Synthesis of N-Acetyl-3-fluoro-neuraminic Acids Agric. Biol. Chem. 1988, 52, 1209. (14) Watts, A. G.; Withers, S. G. The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli. Can. J. Chem 2004, 82, 1581. (15) Hayashi, T.; Kehr, G.; Gilmour R. Stereospecific α‐sialylation by site‐selective fluorination. Angew. Chem. Int. Ed. Engl. 10.1002 / anie.201812963. (16) Okamoto, K.; Kondo, T.; Goto, T. An effective synthesis of α-glycosides of N-acetylneuraminic acid derivatives by use of 2-deoxy-2β-halo-3β-hydroxy-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester. Tetrahedron 1987, 43, 5919. (17) Bennua-Skalmowski, B.; Vorbruggen, H. A facile conversion of primary or secondary alcohols with n-perfluorobutane-sulfonyl fluoride / 1,8-diazabicyclo[5.4.0]undec-7-ene into their corresponding fluorides. Tetrahedron Lett. 1995, 36, 2611. (18) Cao, H.; Li, Y.; Lau, K.; Muthana, S.; Yu, H.; Cheng, J.; Chokhawala, H. A.; Sugiarto, G.; Zhang, L.; Chen, X. Sialidase substrate specificity studies using chemoenzymatically synthesized sialosides containing C5-modified sialic acids. Org. Biomol. Chem., 2009, 7, 5137. (19) Orlova, A. V.; Shpirt, A. M.; Kulikova, N. Y.; Kononov, L. O. N,N-Diacetylsialyl chloride-a novel readily accessible sialyl donor in reactions with neutral and charged nucleophiles in the absence of a promoter. Carbohydr. Res. 2010, 345, 721. (20) Okamoto, K.; Kondo, T.; Goto. T. Functionalization of 2-Deoxy-2,3-dehydro-N-acetylneuraminic Acid Methyl Ester. Bull. Chem. Soc. Jpn. 1987, 60, 631. (21) Pascolutti, M.; Madge, P. D.; Thomson, R. J.; von Itzstein, M. Access to 3-O-Functionalized N-Acetylneuraminic Acid Scaffolds. J. Org. Chem. 2015, 80, 7746. (22) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Kulkarni, S. S.; Huang, Y.-W.; Lee, C.-C.; Chang, K.-L.; Hung, S.-C. Regioselective one-pot protection of carbohydrates. Nature 2007, 446, 896. (23) Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong, C.-H. Programmable One-Pot Oligosaccharide Synthesis. J. Am. Chem. Soc. 1999, 121, 734. (24) Huang, L.; Huang, X. Highly Efficient Syntheses of Hyaluronic Acid Oligosaccharides. Chem. Eur. J. 2007, 13, 529. (25) Shivatare, S. S.; Chang, S.-H.; Tsai, T.-I; Ren, C.-T.; Chuang, H.-Y.; Hsu, L.; Lin, C.-W.; Li, S.-T.; Wu, C.-Yi; Wong, C.-H. Efficient Convergent Synthesis of Bi-, Tri-, and Tetra-antennary (26) Complex Type N-Glycans and Their HIV-1 Antigenicity. J. Am. Chem. Soc. 2013, 135, 15382. (27) Shivatare, SS; Chang, S.-H.; Tsai, T.-I; Tseng, SY; Shivatare, VS; Lin , Y.-S. ; Cheng, Y.-Y.; Ren, C.-T.; Lee, C.-CD; Pawar , S. ; Tsai, C.-S.; Shih, H.-W.; Zeng, Y.-F.; Liang, C.-H.; Kwong, PD; Burton , DR ; Wu, C.-Y.; Wong, C.-H. Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies. Nat. Chem. Rev. 2016, 8, (28) Owner, TK-K.; Huang, C.-Y.; Wong, C.-H. A New Reactivity-Based One-Pot Synthesis of N-Acetyllactosamine Oligomers. J. Org. Chem. Rev. 2003 , 68 , (29) Dinkelaar, J.; Duivenvoorden, BA; Wennekes , T. ; Supercleft, HS; Booth , RG ; Aerts , JMFG ; JDC code; van der Marel, GA A Preparative Synthesis of Human Chitinase Fluorogenic Substrate (4′-Deoxychitobiosyl)-4-methylumbelliferone. Eur. J. Org. Chem. 2010 , 2565 . (30) Chayajarus, K.; Chambers, D. J.; Chughtai, M. J.; Fairbanks, A. J. Stereospecific Synthesis of 1,2-cis Glycosides by Vinyl-Mediated IAD. Org. lett. 2004, 6, 3797-3800. (31) Cao, H.; Li, Y.; Lau, K.; Muthana, S.; Yu, H.; Cheng, J.; Chokhawala, H. A.; Sugiarto, G.; Zhang, L.; Chen, X. Sialidase substrate specificity studies using chemoenzymatically synthesized sialosides containing C5-modified sialic acids. Org. Biomol. Chem., 2009, 7, 5137. (32) Lin, C.-W.; Tsai, M.-H.; Li, S.-T.; Tsai, T.-I.; Chu, K.-C.; Liu, Y.-C.; Lai, M.-Y.; Wu, C.-Y.; Tseng, Y.-C.; Shivatare, S. S.; Wang, C.-H.; Chao, P.; Wang, S.-Y.; Shih, H.-W.; Zeng, Y.-F.; You, T.-H.; Liao, J.-Y.; Tu, Y.-C.; Lin, Y.-S.; Chuang, H.-Y.; Chen, C.-L.; Tsai, C.-S.; Huang, C.-C.; Lin, N.-H.; Ma, C.; Wu, C.-Y.; Wong, C.-H. A common glycan structure on immunoglobulin G for enhancement of effector functions. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 10611. (33) Stavenhagen, K.; Hinneburg, H.; Thaysen-Andersen, M.; Hartmann, L.; Silva, D. V.; Fuchser, J.; Kaspar, S.; Rapp, E.; Seebergera, P. H.; Kolaricha D. Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J. Mass Spectrom. 2013, 48, 627. (34) Lee, J.-C.; Greenberg, W. A; Wong, C.-H. Programmable reactivity-based one-pot oligosaccharide synthesis. Nat. Protoc. 2006, 1, 3143.

Claims

1. A monoclonal antibody having a sugar containing 3-fluorosialic acid, wherein the sugar containing 3-fluorosialic acid is A step of forming an α2,6-linked 3-hydroxysialoside by reacting 3-hydroxysialic acid with a sugar to carry out a glycosylation reaction, The process involves reacting the α2,6-linked 3-hydroxysialoside with a fluorinating agent to carry out a fluorination reaction and form a sugar containing 3-fluorosialic acid, Prepared from, The aforementioned 3-hydroxysialic acid is a compound of formula (I): 【Chemistry 1】 And, The aforementioned sugar is a compound of formula (II): 【Chemistry 2】 And, The α2,6-linked 3-hydroxysialoside is a compound of formula (III): 【Transformation 3】 And, The sugar containing 3-fluorosialic acid is a compound of formula (IV): 【Chemistry 4】 This is a monoclonal antibody.

2. The glycosylation reaction is carried out by silver trifluoromethanesulfonate (AgOTf) and disodium hydrogen phosphate (Na ２ HPO ４ The monoclonal antibody according to claim 1, performed in the presence of ).

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

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

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

6. The monoclonal antibody according to claim 1, wherein the glycosylation and fluorination reactions are carried out in toluene, respectively.

7. A monoclonal antibody according to claim 1, for treating cancer or an autoimmune disease.

8. A monoclonal antibody having a sugar containing 3-fluorosialic acid, wherein the sugar containing 3-fluorosialic acid is a compound of formula (V): 【Transformation 5】 And, In the formula, Z is 【Transformation 6】 This is a monoclonal antibody.

9. A monoclonal antibody according to claim 8, for treating cancer or an autoimmune disease.

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

11. The aforementioned compound is a compound of formula (IV): 【Transformation 8】 The compound according to claim 10.

12. The compound according to claim 10, wherein X is an N-glycan.

13. The aforementioned compound is a compound of formula (V): 【Chemistry 9】 The compound according to claim 12.

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