Synthesis of Modular Building Blocks using Glycosyl Phosphate Donors for the Construction of Asymmetric N-Glycans

Abstract

Glycosyl phosphates are known as versatile donors for the synthesis of complex oligosaccharides both chemically and enzymatically. Herein, we report the stereoselective construction of modular building blocks for the synthesis of N-glycan using glycosyl phosphates as donors. We have synthesized four trisaccharide building blocks with orthogonal protecting groups, namely, Manβ2GlcNAc(OAc)₃β6GlcNAc (9), Manβ2GlcNAc-β6GlcNAc(OAc)₃ (15), Manβ2GlcNAc(OAc)₃β4GlcNAc (18) and Manβ2GlcNAcβ4GlcNAc(OAc) (22) for further selective elongation using glycosyltransferases. The glycosylation reaction using glycosyl phosphate was found to be high yielding with shorter reaction time. Initially, The phthalimide protected glucosamine donor was exploited to ensure the formation of β-glycosidic linkage and later converted to the N-acetyl group before the enzymatic synthesis. The selective deprotection of O-benzyl group was performed prior to enzymatic synthesis to avoid its negative interference.

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1. Introduction

Glycans are composed of a number of monosaccharides linked together via glycosidic bond(s) and constitute the carbohydrate portion of glycoproteins, glycolipids or other glycoconjugates. In glycoproteins, glycans are broadly divided into O-glycans and Nglycans. They affect several biological processes in the cell such as protein folding, cell signaling, embryogenesis etc.¹ N-Glycans are assembled during protein biosynthesis through attachment to the amide nitrogen of asparagine in a defined peptide chain. The synthesis and processing of N-glycans generally takes place in the endoplasmic reticulum and Golgi complex involving different stages of glycosylation and deglycosylation.² The glycan precursor Glc₃Man₉GlcNAc₂ is first transferred from a lipid pyrophosphate derivative to a growing polypeptide in the endoplasmic reticulum (ER) and further trimmed by ER glucosidases before travelling to the Golgi complex for further processing³ down to Man₅GlcNAc₂, which is further glycosylated under the catalysis of N-acetylglucosamine transferase (GnT) to convert it to a complex or hybrid-type glycan. Next, a series of enzymes of the GnT family modify the glycoform with the addition of GlcNAc moieties to the D1 arm of the glycan.⁴ Then, the Golgi-generated glycoforms is converted to highly diverse glycan species through a series of sequential galactosylation, sialylation and fucosylation. The naturally occurring glycans can only be isolated in minor quantities and as complex mixtures, difficult to separate, and cannot provide a reliable source for detailed biological studies. More than twenty thousand structures of N-glycans exist depending on the linkages of monosaccharides, and many of which are difficult to sequence due to their isomeric nature. In order, to study the effect of individual N-glycans on the function of a particular glycoprotein would ideally require a structurally diverse collection of synthetic N-glycans with systematic variations in compositions and branching, terminal sugars, and core modifications. The chemical synthesis to access diverse and complex N-glycan structures is challenging due to step-wise synthesis and multiple purification processes. However, over the last two decades, chemical and chemo-enzymatic synthesis methods have been reported for the synthesis of complex and hybrid type N-glycans.^5,6 In 2013, we reported a convergent synthesis of multi-antennary N-glycans and their application to the study of HIV-antigenicity.^7a Recently, we also developed a modular chemo-enzymatic approach for the synthesis of high-mannose, hybrid- and complex-type N-glycan structures for the development of glycan arrays for the rapid screening and identification of epitopes on HIV-1 recognized by the broadly neutralizing antibodies isolated from HIV-1 patients.^7b

In the last 100 years, synthetic chemists have been developing efficient glycosyl donors for the synthesis of complex oligosaccharides. Numerous versatile glycosyl donors have been developed, among which glycosyl trichloroacetimidates⁸ and thioglycosides⁹ are more commonly utilized. However, the application of glycosyl donors, such as glycosyl sulfoxides,¹⁰ n-pentenyl glycosides,¹¹ glycosyl phosphites,¹² glycosyl halides,¹³ anhydrosugars¹⁴, glucals¹⁵, has been also widely documented for the synthesis of complex oligosaccharide. Since mid-1990’s, glycosyl phosphates have become an attractive class of glycosylating donors due to their stability, ease of activation and high reactivity at low temperature. The use of glycosyl phosphate donors for enzymatic syntheses is also well known. It is known that glycosyltransferases use nucleotide 5′-phosphosugars (NDPs) for the biosynthesis of complex oligosaccharides.¹⁶ Seeberger and co-workers reported the synthesis of glycosyl phosphates for the synthesis of Oglycosides,¹⁷ C-glycosides¹⁸, and complex oligosaccharides.¹⁹ Previously, we have reported the synthesis of glycosyl phosphates and their application in the chemical and chemoenzymatic synthesis of oligosaccharides.¹² In the current context, we extend our chemo-enzymatic modular approach using glycosyl phosphates and glycosyltransferases to construct several modular building blocks which are useful for the assembly of symmetric and asymmetric N-glycans.

2. Result and Discussion

The synthesis of the targeted trisaccharide 9 was initiated from orthogonally protected glycosyl donor 2 and acceptor 1, which were synthesized from commercially available D-mannose and Dglucosamine, respectively, using known procedures.^7b Initially, glucosamine donor 2 and acceptor 1 were coupled using NIS/TMSOTf to furnish disaccharide 3 with 1,2-β-glycosidic linkage in 86% yield. Selective opening of the 4,6-O-benzylidene ring in the presence of BH₃-THF/n-Bu₂BOTf led to 6-hydroxy mannosyl acceptor 4 in 82%. On the other hand, the glycosyl phosphate donor 6 was prepared from thioglycoside donor 2, which upon deacetylation under Zemplén condition followed by benzyl protection using BnBr/NaH provided O-benzyl protected thioglycoside 5. The anomeric phosphate group was installed using dibutyl phosphate in the presence of NIS/TfOH to generate 6 with excellent yield. Next, disaccharide 4 and phosphate donor 6 were coupled using TMSOTf at −50 ^oC to obtain trisaccharide 7 in 80% yield. Later, removal of phthalimide protecting groups using ethylene diamine/n-BuOH followed by N-acetylation using Ac₂O/Py generated trisaccharide 8. In order to avoid any negative interference of the bulky -OBn groups during the enzymatic reaction, we planned to remove the benzyl groups using Pd(OH)₂/C-H₂ to furnish 9 (Scheme 1). The C4 hydroxyl group at the GlcNAc (β6 arm) of trisaccharide 9 can be elongated by enzymatic glycosylation to form the desired N-glycan structure.

Scheme 1:

Synthesis of Manβ2GlcNAc(OAc)₃β6GlcNAc

To further investigate the application of the phosphate donor towards the synthesis of diverse branching units, we installed triO-acetyl glucosamine entity on the β6 arm of the trisaccharide motif. Glycosyl phosphate 6 was coupled with mannosyl acceptor 1 using TMSOTf to furnish disaccharide 10 in good yield. The presence of doublet at 5.27 ppm with a coupling constant (J = 8.4 Hz) in ¹H NMR spectrum along with the signal at 96.9 ppm in ¹³C NMR indicated the formation of 1,2-β-linked disaccharide 10. Selective opening of the 4,6-O-benzylidene ring in the presence of BH₃-THF/n-Bu₂BOTf led to the 6-hydroxy disaccharide acceptor 11 in 78% yield. The absence of signal at 5.42 ppm and 101.5 ppm in ¹H and ¹³C NMR spectrum, respectively, indicated the cleavage of 4,6-O-benzylidene ring. Next, the disaccharide acceptor 11 was coupled with phosphate donor 12 using TMSOTf to furnish the β2-β6 linked trisaccharide 13. The presence of two doublet signals at 5.20 (d, J = 8.5 Hz, 1H) and 5.10 (d, J = 8.4 Hz, 1H) in ¹H NMR confirmed that the two glucosamine units are β2-β6 linked with mannose in 13. Removal of the phthalimide protecting group using ethylene diamine at 90 °C, followed by N-acetylation with Ac₂O/Py generated 14. Finally, Pd(OH)₂/C-H₂ mediated hydrogenation furnished the desired trisaccharide 15 in 87% (Scheme 2).

Scheme 2:

Synthesis of Manβ2GlcNAcβ6GlcNAc(OAc)₃

With the success towards the β2-β6 linked trisaccharide unit, we were further intrigued to investigate the reactivity of phosphate donor towards the systematic synthesis of β2-β4 linked modules. In this regard, the benzylidene protected disaccharide 3 was treated Et₃SiH-TFA to provide C-4 hydroxy derivative 16, selectively. Next, glycosylation of the phosphate donor 6 with acceptor 16 in the presence of TMSOTf led to the trisaccharide with required β2-β4 linkage. Further, the phthalimide protecting groups of trisaccharide were deprotected followed by Nacetylation using Ac₂O/Py generated 17 in 77%. Finally, removal of the O-benzyl group using Pd(OH)₂/C-H₂ led to the formation of trisaccharide 18 in 90% yield (Scheme 3). To show the diversity of phosphate donors towards various trisaccharide modules, we also focused on the synthesis of the trisaccharide building block containing C-4-OAc N-acetyl glucosamine unit attached with β4 glycosidic linkages to the -OPMP-mannose unit. Selective opening of the 4,6-O-benzylidene ring of 10 in the presence of Et₃SiH/TFA led to 4-hydroxy disaccharide acceptor 20 in 74% yield. Next, the phosphate donor 19 was coupled with disaccharide acceptor 20 using TMSOTf to generate trisaccharide 21. The removal of phthalimide protecting groups, followed by N-acetylation and hydrogenolysis provided trisaccharide 22 with GlcNAc(OAc) at the β4 arm in 86 % yield (Scheme 4).

Scheme 3:

Synthesis of Manβ2GlcNAc(OAc)₃β4GlcNAc

Scheme 4:

Synthesis of Man β2GlcN-β4GlcN(OAc)

The structural patterns of all these trisaccharides 9, 15, 18 and 22 provide a platform for the selective elongation at the GlcNAc unit using enzymes to create libraries of asymmetric branching motifs. We utlized one of the trisaccharide, 18 to demonstrate the applicabilty of enzymatic elongation. Initially, trisaccharide 18 was treated with β−1,4-galactosyltransferase (GalT) using UDPGal in the presence of MnCl₂ to furnish tetrasaccharide 23 with complete stereospecificity in 75% yield. To further extend the chain length and complexity, tetrasaccharide 23 was treated with α−2,3-sialyltransferase (ST) along with ATP (0.05 eq.), CTP (0.05 eq.), phosphoenolpyruvate (2.4 eq.), cytidine monophosphate kinase (CMK, 5 units) and CMP-sialic acid synthetase (CSS, 10U) in Tris buffer (50mM, pH 7.5) to generate pentasaccharide 24 in 83% yield (Scheme 5).

Scheme 5: Stepwise Enzymatic elongation.

Reaction conditions: (i) β-1,4-GalT (15 U), UDP-Gal, Tris buffer (50 mM), MnCl₂ (5mM), 37 °C, 12 h, 75%. (ii) α-2,3-ST (15U), CMK (5 U), CSS (10 U), ATP (0.05 eq.), CTP (0.05 eq.), phosphoenolpyruvate (2.4 eq.), 37 °C, 54 h, 83%.

In conclusion, we have developed a new and highly efficient chemo-enzymatic strategy for the synthesis of modular building blocks using glycosyl phosphate as donors for the synthesis of complex/hybrid N-glycans. The phosphate donors were synthesized from the corresponding thioglycosides. Overall, the glycosylation reactions using phosphate donors were found to be high yielding and highly stereoselective. One of the trisaccharide building block 18 was further exploited for the enzymatic synthesis. With the aid of purified enzymes β−1,4-GalT and α2,3-ST, we were able to elongate the β4 arm of the trisaccharide even in the presence of three O-acetyl moieties at the β2 arm. This result indicates that the presence of multiple OAc protecting groups can be tolerated by the enzymes during the enzymatic reactions. This work emphasizes the use of glycosyl phosphate donors and enzymes to access complex and diverse N-glycan structures.

Experimental Section:

General procedures.

All chemicals were purchased as reagent grade and used without further purification. All anhydrous solvents were purchased from commercial source and further dried in accordance with standard procedures. Molecular sieves (MS 4 Å) were purchased from Aldrich, which were ground into powdered form and activated before use. Reactions were performed under an inert atmosphere and strictly anhydrous conditions. Reactions were monitored with analytical thin-layer chromatography (TLC) on silica gel 60 F254 plates and visualized under UV (254 nm) and/or by spraying with 20% anisaldehyde in ethanol or with a solution of (NH4)₆Mo₇O₂₄·4H₂O 25 g/L. ¹H and ¹³C NMR were recorded on 600/500 MHz and 151 MHz spectrometers, respectively, with the residual solvent signal acting as the internal standard. Chemical shift (in ppm) was determined relative to tetramethylsilane in deuterated chloroform (δ 0 ppm). Coupling constant(s) in hertz (Hz) were measured from one-dimensional spectra. Highresolution mass spectra were obtained from a Q-TOF instrument by the electrospray ionization (ESI) technique.

General procedure for glycosyl phosphate coupling:

To a solution of glycosyl phosphate donor (1.2 equiv.) and acceptor (1.0 eq) in CH₂Cl₂ was mixed with molecular sieves (4 Å). After stirring for 1 h under N₂-atmosphere at room temperature, the mixture was cooled to −50 °C and was added TMSOTf (1.0 equiv.). After the reaction mixture was stirred for additional 1 h at the same temperature, triethylamine (2 equiv.) was added and the mixture was filtered through a pad of Celite and concentrated in vacuo. The crude residue was purified through a silica gel column chromatography to afford the corresponding glycosylated product.

General procedure for the synthesis of glycosyl phosphates:

A solution of thioglycoside donor (1.2 equiv.) and molecular sieves 4 Å (1.0 equiv.) in CH₂Cl₂ was stirred for 1 h under N₂atmosphere at room temperature and then cooled to −40 °C. Dibutyl phosphate (1.0 equiv.), NIS (1.2 equiv.) and TfOH (0.3 equiv.) were added to the mixture, and stirring was continued until the complete conversion of the donor. The reaction mixture was quenched with Et₃N (2 equiv.) and filtered through a Celite pad. The mixture was diluted with CH₂Cl₂ (2 × 30 mL), washed with sat. NaHCO₃ (25 mL), Na₂S₂O₃ (25 mL) and brine (20 mL). The crude product was dried over MgSO₄ and concentrated in vacuo, and the residue was purified through a silica gel column chromatography to afford the corresponding glycosylated product.

General procedure for 4,6-O-benzylidene ring opening using BH₃-THF:

A solution of compound (0.50 mmol) in anhydrous THF (2mL) was mixed with BH₃-THF complex (1M solution in THF), followed by n-Bu₂BOTf (1M solution in CH₂Cl₂) at 0 °C under N₂-atmosphere. After the complete conversion of the starting material to product, Et₃N (2 equiv.) was added. The reaction mixture was further quenched with dropwise addition of MeOH. When no more hydrogen was evolved, the reaction mixture was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel to afford the corresponding 6-hydroxy product.

General procedure for 4,6-O-benzylidene ring opening using Et₃SiH/TFA:

To a solution of 4,6-O-benzylidene protected compound (1.0 mmol) in anhydrous CH₂Cl₂ (20 mL) was added triethyl silane (10 equiv.) followed by trifluroacetic acid (10 equiv.) at 0 ^oC. After 2 h, the reaction mixture was slowly quenched with sodium bicarbonate solution (50 mL). The aqueous layer was further extracted with CH₂Cl₂ (3 × 50 mL), and the combined organic layer was washed with brine solution (50 mL), dried over MgSO₄, filtered and concentrated in vacuo. The crude reaction mixture was purified by flash column chromatography on silica gel to afford the corresponding 4hydroxy product.

General procedure for phthalimide deprotection and Nacetylation:

A protected trisaccharide (1 mmol) was dissolved in a mixture of ethylene diamine: ⁿBuOH (1:4) and stirred at 90 ^oC for 12 h. Solvents were evaporated in vacuo and the reaction mixture was dried for 2 h on high vacuum. The crude product was treated with a mixture of Ac₂O/pyridine (1: 2) at 0 ^oC. Then, the reaction mixture was stirred at room temperature for 12 h. After complete conversion of the starting material, solvents were evaporated in vacuo. The mixture was diluted with EtOAc (2 × 30 mL), washed with sat. NaHCO₃ (25 mL), and brine (20 mL). It was dried over MgSO₄ and concentrated in vacuo. The crude residue was purified through a silica gel column chromatography to afford the corresponding N-acetyl amine derivative.

General procedure for hydrogenolysis:

A protected trisaccharide derivative (0.5 mmol) was dissolved in 10 mL MeOH : HCOOH (9 : 1), and 20%-Pd(OH)₂/C was added. The mixture was placed under an atmosphere of hydrogen gas bubbling through a needle. After 12h, the reaction mixture was filtered through a pad of Celite and washed with a mixture of CH₃OH and H₂O (1/1, v/v, 6 mL). Solvents were evaporated in vacuo and the crude mixture was purified using silica gel column chromatography to furnish the protecting group free trisaccharide.

General procedure for enzyme expression and purifications:

Recombinant enzymes β−1,4 galactosyltransferase (β−1,4-GalT), α−2,3 sialyltransferase, pyruvate kinase (PK), pyrophosphatase (PPA), cytidine monophosphate kinase (CMK) and CMP-sialic acid synthetases (CSS) were over expressed and purified from E. coli BL21(DE3) using Ni-NTA column as our previous protocol.^7b

General procedure for enzymatic β−1, 4-galactosylation:

A trisaccharide (1 equiv.) and UDP-Gal (2 equiv.) were dissolved in Tris buffer (50 mM, pH 7.5) containing MnCl₂ (5 mM) in a small capped glass tube with β−1,4-GalT (15 units). The resulting reaction mixture was incubated at 37 ^oC for 12 h. The protein was removed by heating and centrifugation followed by purification using C18 column chromatography. The pure fractions were pooled and concentrated to give the corresponding product.

General procedure for enzymatic α−2,3-sialylation:

Trisaccharide (1 eq.), Neu5Ac (2 equiv.), ATP (0.05 equiv.), CTP (0.05 equiv.), phosphoenolpyruvate (2.4 equiv.), cytidine monophosphate kinase (CMK, 5U), CMP-sialic acid synthetases (CSS, 10U), pyruvate kinase (PK, 5 units), pyrophosphatase (PPA, 2.5U) and α−2,3 sialyltransferase (15 units) were dissolved in Tris buffer (50 mM, pH 7.5) and transferred to a small glass tube. The reaction was incubated at 37 °C and monitored by TLC until completion. Heating and centrifugation was performed to remove the protein and followed by purification using C18 column chromatography. The pure fractions were pooled and concentrated to give the respective product.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-4,6-O-benzylidene-3-Obenzyl-α-D-mannopyranoside (3)

Glycosyl donor 2 (0.5 g, 0.924 mmol, 1.2 equiv. based on acceptor) and acceptor 1 (0.36 g, 0.775 mmol) in dry CH₂Cl₂ was added 4 Å molecular sieves and the mixture was stirred under N₂atmosphere for 1h, then cooled to −50 ^oC. NIS (0.249 g, 1.10 mmol) and TMSOTf (25 μL, 0.272 mmol) were added, and the reaction mixture was stirred for another 1 h. After complete conversion of the starting materials, the reaction mixture was quenched with Et₃N (2 equiv.) and filtered through a Celite pad. The mixture was diluted with CH₂Cl₂ (2 × 30 mL), washed with sat. NaHCO₃ (25 mL), Na₂S₂O₃ (50 mL) and brine (50 mL). It was then dried over MgSO₄ and concentrated for column chromatography purification on silica gel (PhCH₃: EtOAc, 9:1) to furnish 3 (0.7 g, 86 %) as yellow oil. R_f 0.52 (PhCH₃/EtOAc 9:1); ¹H NMR (600 MHz, CDCl₃) δ 7.89 – 7.85 (m, 2H), 7.78 – 7.73 (m, 2H), 7.41 (td, J = 7.1, 6.5, 3.8 Hz, 4H), 7.34 – 7.31 (m, 4H), 7.31 – 7.28 (m, 2H), 6.76 – 6.72 (m, 2H), 6.67 – 6.63 (m, 2H), 5.89 (dd, J = 10.9, 9.1 Hz, 1H), 5.52 (d, J = 8.5 Hz, 1H), 5.46 (s, 1H), 5.22 (dd, J = 10.1, 9.1 Hz, 1H), 5.13 (d, J = 2.0 Hz, 1H), 4.79 – 4.71 (m, 2H), 4.52 (dd, J = 11.0, 8.5 Hz, 1H), 4.34 – 4.31 (m, 1H), 4.27 (dd, J = 3.2, 1.9 Hz, 1H), 4.23 (dd, J = 12.2, 2.4 Hz, 1H), 4.04 (dd, J = 10.0, 3.1 Hz, 1H), 3.97 (t, J = 9.5 Hz, 1H), 3.90 (ddt, J = 9.4, 4.6, 2.3 Hz, 1H), 3.76 (s, 3H), 3.68 – 3.60 (m, 2H), 3.18 – 3.11 (m, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 1.91 (s, 3H).¹³C NMR (151 MHz, CDCl₃) δ 170.8, 170.3, 169.5, 155.1, 149.5, 138.4, 137.5, 134.4, 133.9, 131.7, 130.1, 128.9, 128.3, 128.2, 127.7, 127.6, 126.1, 123.5, 117.0, 114.6, 101.6, 97.0, 96.24, 78.1, 77.3, 77.1, 76.9, 75.6, 73.7, 72.3, 71.9, 70.4, 69.1, 68.2, 64.4, 62.2, 55.7, 54.6, 20.8, 20.7, 20.6; m/z (HRMS) calcd for C₄₇H₄₇NO₁₆ [M+Na]⁺: 904.2793, found: 904.2789.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-Dmannopyranoside (4)

Disaccharide 4 was prepared from 3 (0.7 g, 0.774 mmol) using the general procedure for 4,6-O-benzylidene ring opening using BH₃-THF, followed by purification using silica gel column chromatography (PhCH₃: EtOAc = 8:2). Yield (0.575 g, 82 %). ¹H NMR (600 MHz, CDCl₃) δ 7.88 (dt, J = 7.2, 3.6 Hz, 2H), 7.79 (dd, J = 5.5, 3.0 Hz, 2H), 7.45 – 7.41 (m, 2H), 7.34 – 7.28 (m, 5H), 7.28 – 7.23 (m, 3H), 6.70 – 6.64 (m, 2H), 6.52 – 6.48 (m, 2H), 5.97 (dd, J = 10.8, 9.0 Hz, 1H), 5.44 (d, J = 8.5 Hz, 1H), 5.22 (dd, J = 10.2, 9.0 Hz, 1H), 5.01 (d, J = 2.2 Hz, 1H), 4.87 (d, J = 10.8 Hz, 1H), 4.81 (d, J = 11.1 Hz, 1H), 4.62 (d, J = 11.2 Hz, 1H), 4.58 – 4.50 (m, 2H), 4.35 (dd, J = 12.2, 5.3 Hz, 1H), 4.26 –4.22 (m, 2H), 4.03 (dd, J = 9.1, 3.1 Hz, 1H), 3.93 (ddd, J = 10.2, 5.3, 2.4 Hz, 1H), 3.81 (t, J = 9.4 Hz, 1H), 3.73 (s, 3H), 3.47 (ddd, J = 10.0, 4.6, 2.6 Hz, 1H), 3.38 (ddd, J = 12.1, 5.1, 2.6 Hz, 1H), 3.29 (ddd, J = 12.3, 8.2, 4.6 Hz, 1H), 2.05 (app d, J = 3.4 Hz, 6H), 1.92 (s, 3H);¹³C NMR (151 MHz, CDCl₃) δ 170.6, 170.2, 169.5, 154.8, 149.5, 138.3, 138.0, 128.4, 128.3, 128.2, 128.0, 127.8, 127.7, 116.8, 114.5, 97.5, 95.6, 77.5, 75.2, 74.5, 73.9, 72.5, 72.1, 71.4, 70.2, 69.0, 62.4, 62.1, 55.6, 54.8, 20.7, 20.6, 20.5; m/z (HRMS) calcd for C₄₇H₄₉NO₁₆ [M+Na]⁺: 906.2649, found: 906.2655.

p-Tolyl-3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-1-thio-βD-glucopyranoside (5)

Compound 2 (0.6 g, 1.09 mmol) was dissolved in anhydrous methanol (8 mL) and sodium methoxide (0.15 g) was added. The reaction mixture was stirred at room temperature for 3 h, and was neutralized using acid resin (IR-120), filtered and concentrated in vacuo, and later co-evaporated with toluene to remove traces of waters. A solution of the tri hydroxy intermediate (0.5 g, 1.20 mmol) in DMF (10 mL) was added tetrabutylammonium iodide (TBAI, 40 mg, 0.12 mmol) and benzyl bromide (0.49 mL, 4.21 mmol) under N₂ atmosphere. The solution was cooled to 0 ^oC and then NaH (60% dispersion in mineral oil, 0.24 g, 6.0 mmol) was added slowly. The reaction mixture was stirred at 0 ^oC for 1 h and then stirred at room temperature for additional 7 h. The reaction mixture was quenched with aqueous NH₄Cl and was diluted with CH₂Cl₂ (2 × 30 mL), washed with sat. NH₄Cl (25 mL), and brine (50 mL). It was dried over MgSO₄ and concentrated in vacuo. The crude mixture was purified using silica gel column chromatography (hexane: EtOAc, 9:1) to furnish 5 (0.57 g, 69 %) as colorless oil.¹H NMR (600 MHz, CDCl₃) δ 7.74 (d, J = 7.3 Hz, 1H), 7.65 – 7.53 (m, 3H), 7.34 –7.32 (m, 4H), 7.32 – 7.29 (m, 6H), 7.25 – 7.21 (m, 5H), 6.96 –6.92 (m, 2H), 6.85 – 6.82 (m, 1H), 6.82 – 6.78 (m, 1H), 5.46 (dd, J = 10.0, 2.7 Hz, 1H), 4.78 (d, J = 10.9 Hz, 1H), 4.73 (d, J = 12.1 Hz, 1H), 4.64 – 4.57 (m, 2H), 4.53 – 4.49 (m, 1H), 4.39 (d, J = 12.1 Hz, 1H), 4.32 (dd, J = 10.4, 8.5 Hz, 1H), 4.21 (td, J = 10.3, 3.4 Hz, 1H), 3.77 (td, J = 12.2, 11.6, 3.2 Hz, 2H), 3.74 –3.67 (m, 1H), 3.62 (ddd, J = 9.9, 4.4, 2.1 Hz, 1H), 2.22 (s, 3H);¹³C NMR (151 MHz, CDCl₃) δ 168.2, 167.5, 141.1, 138.3, 138.2, 138.0, 137.8, 134.0, 133.9, 133. 5, 131.7, 131.5, 129.7, 129.7, 128.7, 128.7 (2), 128.5, 128.5, 128.4, 128.2, 128.1, 128.1 (2), 127.9, 127.8, 127.7, 127.7(2), 127.6, 127.6(2), 127.5, 127.0, 123.5(2), 123.4, 83.5, 80.3, 79.5, 79.4, 75.1, 75.0, 73.5, 68.9, 55.1, 21.2.; m/z (HRMS) calcd for C₄₂H₃₉NSO₆ [M+Na]⁺: 708.2396, found: 708.2399.

Dibutyl-3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-Dglucopyranoside phosphate (6)

Dibutyl phosphate derivative 6 was prepared from 5 (0.57 g, 0.774 mmol) using the general procedure for the synthesis of glycosyl phosphates, followed by purification using silica gel column chromatography (hexane: EtOAc = 7:3). Yield (0.564 g, 88 %).¹H NMR (600 MHz, CDCl₃) δ 7.70 – 7.64 (m, 4H), 7.37 –7.33 (m, 3H), 7.33 – 7.27 (m, 3H), 7.26 – 7.22 (m, 3H), 6.99 (dt, J = 6.2, 1.4 Hz, 2H), 6.90 – 6.86 (m, 2H), 6.86 – 6.82 (m, 2H), 5.82 (dd, J = 8.5, 7.2 Hz, 1H), 4.84 (d, J = 10.8 Hz, 1H), 4.80 (d, J = 12.2 Hz, 1H), 4.66 (dd, J = 11.5, 9.2 Hz, 2H), 4.54 (d, J = 12.0 Hz, 1H), 4.50 – 4.42 (m, 2H), 4.27 (dd, J = 10.8, 8.5 Hz, 1H), 3.99 – 3.95 (m, 1H), 3.95 – 3.90 (m, 1H), 3.89 – 3.85 (m, 1H), 3.85 – 3.81 (m, 1H), 3.78 (d, J = 1.9 Hz, 1H), 3.77 – 3.75 (m, 1H), 3.75 – 3.72 (m, 1H), 3.72 – 3.69 (m, 1H), 3.69 – 3.66 (m, 1H), 1.54 – 1.48 (m, 2H), 1.27 (ddt, J = 14.5, 11.9, 6.3 Hz, 4H), 1.07 – 1.00 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H), 0.69 (t, J = 7.4 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 167.7, 138.0, 138.0(2), 137.9, 133.8, 131.6, 128.5, 128.4, 128.1, 128.0, 127.9, 127.8, 127.8(2), 127.7, 127.4, 123.3, 94.2, 94.2(2), 79.0, 78.7, 75.5, 75.1, 74.1, 73.6, 68.3, 68.0, 68.0 (2), 67.8, 67.8, 56.3, 56.3, 32.1, 32.1, 31.9, 31.8, 18.6, 18.4, 13.6, 13.5.; m/z (HRMS) calcd for C₄₃H₅₀NPO₁₀ [M+Na]⁺: 794.3070, found: 794.3078.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-2deoxy-2-phthalimido-β-D-glucopyranosyl)-3,4-di-O-benzyl-αD-mannopyranoside (7)

Trisaccharide 7 was prepared from glycosyl phosphate donor 6 (0.104 g, 0.135 mmol) and disaccharide acceptor 4 (0.1 g, 0.113 mmol) using the general procedure for glycosyl phosphate coupling and followed by purification using silica gel column chromatography (PhCH₃: EtOAc = 8:2). Yield (0.130 g, 80 %).¹H NMR (600 MHz, CDCl₃) δ 7.84 – 7.54 (m, 4H), 7.52 (dd, J = 5.5, 3.0 Hz, 4H), 7.33 – 7.21 (m, 16H), 7.10 – 7.07 (m, 3H), 6.98 – 6.95 (m, 2H), 6.91 – 6.81 (m, 4H), 6.65 – 6.58 (m, 4H), 5.83 (dd, J = 10.8, 9.0 Hz, 1H), 5.45 (d, J = 8.5 Hz, 1H), 5.16 (dd, J = 10.2, 9.0 Hz, 1H), 4.96 (dd, J = 5.5, 2.9 Hz, 2H), 4.80 (d, J = 10.9 Hz, 1H), 4.74 (d, J = 12.0 Hz, 1H), 4.62 (dd, J = 11.2, 5.9 Hz, 2H), 4.50 – 4.45 (m, 3H), 4.43 – 4.42 (m, 1H), 4.42 – 4.37 (m, 2H), 4.27 (dd, J = 12.2, 5.0 Hz, 1H), 4.22 (dd, J = 10.7, 8.6 Hz, 1H), 4.19 (d, J = 2.4 Hz, 1H), 4.18 –4.14 (m, 1H), 4.12 (t, J = 2.9 Hz, 1H), 3.99 (dd, J = 10.7, 8.4 Hz, 1H), 3.85 (dd, J = 8.2, 3.2 Hz, 1H), 3.82 (ddd, J = 10.2, 5.1, 2.4 Hz, 1H), 3.74 (s, 3H), 3.73 – 3.71 (m, 1H), 3.65 (dd, J = 11.4, 2.3 Hz, 2H), 3.63 – 3.58 (m, 1H), 3.58 – 3.54 (m, 1H), 3.48 (ddd, J = 9.9, 4.6, 1.9 Hz, 1H), 3.38 (dd, J = 9.5, 8.2 Hz, 1H), 3.10 (dd, J = 11.1, 6.2 Hz, 1H), 2.04 (s, 3H), 2.02 (s, 3H), 1.88 (s, 3H);¹³C NMR (151 MHz, CDCl₃) δ 170.8, 170.3, 169.6, 154.9, 150.3, 138.3, 138.3(2), 138.1, 138.1, 138.0, 134.1, 131.5, 128.5, 128.5(2), 128.4, 128.3, 128.3, 128.2, 128.1, 128.1(2), 128.0, 128.0(2), 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 117.9, 114.4, 98.7, 97.3, 96.6, 79.8, 79.3, 77.9, 75.1, 74.9, 74.9, 73.5, 72.2, 71.7, 71.7, 70.6, 69.4, 69.2, 68.8, 62.4, 55.8, 55.7, 54.6, 20.8, 20.8, 20.6; m/z (HRMS) calcd for C₈₂H₈₀N₂O₂₂ [M+Na]: 1467.5100, found: 1467.5116.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-2acetamido-2-deoxy-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-Dmannopyranoside (8)

Trisaccharide 8 was prepared from trisaccharide 7 (0.130 g, 0.09 mmol) using the general procedure for phthalimide deprotection and N-acetylation. The crude mixture was purified using silica gel column chromatography (PhCH₃: EtOAc = 6:4). Yield (0.09 g, 79 %).¹H NMR (600 MHz, CDCl₃) δ 7.82 – 7.72 (m, 2H), 7.45 – 7.42 (m, 2H), 7.39 – 7.35 (m, 2H), 7.35 – 7.31 (m, 4H), 7.31 – 7.25 (m, 9H), 7.25 – 7.23 (m, 3H), 7.23 – 7.16 (m, 3H), 6.90 – 6.87 (m, 2H), 6.81 – 6.78 (m, 2H), 5.83 (t, J = 10.0 Hz, 1H), 5.50 (d, J = 1.9 Hz, 1H), 5.46 – 5.36 (m, 1H), 5.09 – 5.04 (m, 1H), 4.89 (dd, J = 16.7, 11.5 Hz, 2H), 4.80 (dd, J = 11.3, 7.8 Hz, 2H), 4.67–4.63 (m 2H), 4.59 – 4.55 (m, 1H), 4.55 –4.49 (m, 3H), 4.37 (dd, J = 3.5, 2.0 Hz, 1H), 4.25 (dd, J = 12.2, 5.0 Hz, 1H), 4.14 (dd, J = 9.1, 3.5 Hz, 1H), 4.10 (dd, J = 12.3, 2.3 Hz, 1H),3.92 – 3.86 (m, 2H), 3.84 (t, J = 9.4 Hz, 1H), 3.72 (app s, 4H), 3.67 (d, J = 3.3 Hz, 2H), 3.62 – 3.54 (m, 2H), 3.53 –3.49 (m, 1H), 3.42–3.39 (m, 2H), 3.33 (dt, J = 10.0, 3.3 Hz, 1H), 2.03 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H), 1.92 (s, 3H), 1.82 (s, 3H);¹³C NMR (151 MHz, CDCl₃) δ 173.1, 171.2, 170.7, 170.5, 169.7, 154.8, 149.8, 139.5, 138.5(2), 138.4, 138.1, 137.96, 128.5127.6, 116.8, 114.7, 101.6, 97.8, 95.1, 80.6, 79.1, 78.1, 75.0, 74.9, 74.6, 73.4, 73.4, 72.5, 71.7, 71.6, 71.1, 70.9, 70.1, 69.8, 68.7, 67.3, 62.5, 55.7, 23.9, 23.8, 20.7, 20.7(2), 20.6; m/z (HRMS) calcd for C₇₀H₈₀N₂O₂₀ [M+Na]⁺: 1291.5202, found: 1291.5212.

p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-6-O-(2-acetamido-2-deoxy-β-Dglucopyranosyl)-α-D-mannopyranoside (9)

Trisaccharide 9 was prepared from trisaccharide 8 (0.09 g, 0.07 mmol) using the general procedure for hydrogenolysis and followed by purification using silica gel column chromatography (MeOH: CHCl₃ = 1:20). Yield (0.05 g, 91 %).¹H NMR (600 MHz, MeOD) δ 8.10 (s, 2H), 7.06 – 6.96 (m, 2H), 6.90 – 6.84 (m, 2H), 5.55 (td, J = 10.3, 3.7 Hz, 1H), 5.38 (s, 1H), 4.47 – 4.41 (m, 1H), 4.45–4.40 (m, 1H), 4.32 (ddd, J = 12.3, 4.4, 1.8 Hz, 1H), 4.17 – 4.11 (m, 1H), 4.10 (dd, J = 12.4, 2.4 Hz, 1H), 4.06 (d, J = 10.7 Hz, 1H), 3.94 – 3.87 (m, 1H), 3.86–3.84 (m, 1H), 3.76 (s, 3H), 3.73 – 3.68 (m, 2H), 3.68 – 3.59 (m, 2H), 3.66–3.60 (m, 2 H), 3.48 (ddd, J = 10.3, 8.4, 1.7 Hz, 1H), 3.35 – 3.33 (m, 1H), 3.30 – 3.22 (m, 2H), 2.04 (s, 3H), 2.01 (s, 3H), 1.99 (s, 6H), 1.87 (s, 3H); ¹³C NMR (151 MHz, MeOD) δ 174.6, 173.9, 172.3, 171.9, 171.2, 164.7, 156.6, 151.9, 119.1, 119.1, 115.7, 103.1, 99.5, 97.8, 78.5, 77.8, 75.9, 73.9, 72.9, 72.8, 72.1, 71.3, 70.3, 69.7, 68.5, 63.1, 62.7, 57.3, 56.3, 56.1, 23.7, 23.7(2), 23.4, 23.4(2), 20.6, 20.5; m/z (HRMS) calcd for C₃₅H₅₀N₂O₁₀ [M+Na]⁺: 841.2855, found: 841.2860.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-3-O-benzyl-4,6-Obenzylidene-α-D-mannopyranoside (10)

Disaccharide 10 was prepared from glycosyl phosphate donor 6 (0.6 g, 0.775 mmol) and acceptor 1 (0.4 g, 0.646 mmol) using the general procedure for glycosyl phosphates coupling and followed by purification using silica gel column chromatography (PhCH₃: EtOAc = 20:1). Yield (0.530 g, 80 %).¹H NMR (600 MHz, CDCl₃) δ 7.72 – 7.62 (m, 2H), 7.43 – 7.35 (m, 5H), 7.35– 7.28 (m, 9H), 7.27 – 7.20 (m, 5H), 7.07 – 7.02 (m, 4H), 6.93– 6.87 (m, 2H), 6.87 – 6.82 (m, 2H), 6.74 – 6.67 (m, 2H), 6.65– 6.58 (m, 2H), 5.42 (s, 1H), 5.27 (d, J = 8.4 Hz, 1H), 5.06 (d, J = 1.9 Hz, 1H), 4.87 (d, J = 10.8 Hz, 1H), 4.83 (d, J = 12.2 Hz, 1H), 4.78 (d, J = 12.3 Hz, 1H), 4.67 (dd, J = 16.5, 11.6 Hz, 2H), 4.60 (d, J = 12.1 Hz, 1H), 4.55 (d, J = 12.2 Hz, 1H), 4.48 (d, J = 12.2 Hz, 1H), 4.41 (dd, J = 10.8, 8.3 Hz, 1H), 4.35 (dd, J = 10.8, 8.3 Hz, 1H), 4.24 (dd, J = 3.1, 1.9 Hz, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.98 (dd, J = 10.0, 3.1 Hz, 1H), 3.96– 3.91 (m, 1H), 3.82 – 3.76 (m, 2H), 3.74 (s, 3H), 3.72 – 3.68 (m, 1H), 3.59 – 3.53 (m, 2H), 3.21 (s, 1H), 3.06 – 2.99 (m, 1H);¹³C NMR (151 MHz, CDCl₃) δ 176.4, 171.2, 155.0, 149.7, 138.5–137.6, 133.8, 132.0, 128.8– 127.4, 126.1, 123.1, 117.1, 114.6, 101.5, 96.9, 96.3, 79.8, 79.2, 78.1, 75.6, 75.2, 74.9, 74.9(2), 73.7, 73.4, 71.5, 69.3, 68.3, 64.3, 60.5, 59.9, 56.0, 55.7; m/z (HRMS) calcd for C₆₂H₅₉N₂O₁₃ [M+Na]⁺: 1048.3884, found: 1048.3889.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-3,4-di-O-benzyl-α-Dmannopyranoside (11)

Disaccharide 11 was prepared from 10 (0.53 g, 0.687 mmol) using the general procedure for 4,6-O-benzylidene ring opening with BH₃-THF, followed by purification using silica gel column chromatography (PhCH₃: EtOAc = 8:2). Yield (0.413 g, 78 %). ¹H NMR (600 MHz, CDCl₃) δ 7.78 – 7.51 (m, 5H), 7.35 – 7.32 (m, 2H), 7.28 – 7.21 (m, 8H), 7.21 – 7.12 (m, 9H), 7.00 – 6.97 (m, 2H), 6.83 (dd, J = 8.2, 6.8 Hz, 2H), 6.78 – 6.74 (m, 1H), 6.58 – 6.54 (m, 2H), 6.40 – 6.35 (m, 2H), 5.10 (d, J = 8.5 Hz, 1H), 4.83 (d, J = 2.2 Hz, 1H), 4.80 (s, 1H), 4.79 – 4.78 (m, 2H), 4.76 (dd, J = 11.8, 1.9 Hz, 2H), 4.58 (d, J = 10.9 Hz, 1H), 4.53 – 4.48 (m, 2H), 4.48 – 4.43 (m, 2H), 4.43 – 4.38 (m, 2H), 4.27 (dd, J = 10.8, 8.5 Hz, 1H), 4.11 (t, J = 6.0 Hz, 1H), 3.89 (dd, J = 9.2, 3.1 Hz, 1H), 3.72 – 3.68 (m, 1H), 3.68 (d, J = 4.0 Hz, 1H), 3.65 (app s, 4H), 3.36 – 3.31 (m, 2H) 3.24 (dd, J = 12.7, 2.8 Hz, 1H); ¹³C NMR (151 MHz, CDCl₃) δ 168.8, 168.0, 154.8, 149.7, 138.5, 138.3, 138.3(2), 138.2, 138.1, 137.8, 134.2, 133.7, 128.6 −127.4, 116.9, 114.5, 97.6, 95.8, 79.7, 79.1, 75.3, 75.2, 74.8, 74.1, 73.7, 72.4, 70.9, 70.7, 69.5, 62.5, 56.3, 55.7; m/z (HRMS) calcd for C₆₂H₅₉N₂O₁₃ [M+Na]⁺: 1048.3884, found: 1048.3889.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→2)-6-O-(3,4,6-tri-Oacetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→6)3,4-di-O-benzyl-α-D-mannopyranoside (13)

Trisaccharide 13 was prepared from glycosyl phosphate donor 12 (0.302 g, 0.483 mmol) and disaccharide acceptor 11 (0.413 g, 0.402 mmol) using the general procedure for glycosyl phosphates coupling and followed by column chromatography on silica gel (PhCH₃: EtOAc = 8:2). Yield (0.471 g, 81 %).¹H NMR (600 MHz, CDCl₃) δ 7.61 (s, 2H), 7.49 (s, 3H), 7.31 – 7.12 (m, 21H), 7.06 – 7.02 (m, 2H), 6.98 – 6.94 (m, 2H), 6.85 – 6.81 (m, 2H), 6.80 – 6.76 (m, 1H), 6.61 – 6.53 (m, 2H), 6.52 – 6.41 (m, 2H), 5.59 (dd, J = 10.7, 9.0 Hz, 1H), 5.20 (d, J = 8.5 Hz, 1H), 5.10 (d, J = 8.4 Hz, 1H), 4.90 – 4.86 (m, 2H), 4.85 (d, J = 10.9 Hz, 1H), 4.79 (d, J = 12.1 Hz, 1H), 4.74 (d, J = 11.7 Hz, 1H), 4.63–4.58 (m, 2H), 4.56 (d, J = 12.1 Hz, 1H), 4.51 (s, 1H), 4.49 (d, J = 3.5 Hz, 1H), 4.47 – 4.44 (m, 2H), 4.40 (dd, J = 10.8, 8.3 Hz, 1H), 4.28 (dd, J = 10.8, 8.5 Hz, 1H), 4.19 (d, J = 11.0 Hz, 1H), 4.14 – 4.12 (m, 1H), 4.05 (dd, J = 10.7, 8.4 Hz, 1H), 3.85 – 3.82 (m, 2H), 3.77 (s, 3H), 3.75 – 3.72 (m, 2H), 3.72 – 3.69 (m, 1H), 3.67 (dq, J = 9.9, 2.3 Hz, 1H), 3.56– 3.51 (m, 2H), 3.30 (dd, J = 9.7, 8.3 Hz, 1H), 2.02 – 1.96 (m, 3H), 1.91 (s, 3H), 1.84 (s, 3H);¹³C NMR (151 MHz, CDCl₃) δ 170.7, 170.2, 169.6, 154.8, 150.4, 138.4, 138.3, 138.2, 138.0, 134.3, 133.6, 128.6–127.4, 123.6, 117.8, 114.3, 98.6, 97.5, 97.0, 79.7, 79.3, 75.2, 75.1, 74.8, 73.6, 71.5, 71.4, 70.9, 70.6, 69.4, 69.2, 62.3, 56.0, 55.7, 54.6, 31.1, 20.7, 20.7, 20.5. m/z (HRMS) calcd for C₈₂H₈₀N₂O₂₂ [M+Na]: 1467.5100, found:1467.5109.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-6-O-(3,4,6-tri-O-acetyl-2acetamido-2-deoxy-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-Dmannopyranoside (14)

Trisaccharide 14 was prepared from trisaccharide 13 (0.471 g, 0.326 mmol) using the general procedure for phthalimide deprotection and N-acetylation and followed by column chromatography on silica gel (PhCH₃: EtOAc = 6:4). Yield (0.302 g, 73 %).¹H NMR (600 MHz, CDCl₃) δ 7.68 (s, 2H), 7.56 (s, 2H), 7.33 – 7.29 (m, 4H), 7.29 – 7.26 (m, 5H), 7.25 – 7.23 (m, 2H), 7.22 (dt, J = 6.4, 1.7 Hz, 3H), 7.13 – 7.10 (m, 2H), 7.04 –7.01 (m, 2H), 6.92 – 6.88 (m, 2H), 6.87 – 6.84 (m, 1H), 6.65 –6.63 (m, 2H), 6.57 – 6.54 (m, 2H), 5.59 (dd, J = 10.7, 9.0 Hz, 1H), 5.20 (d, J = 8.5 Hz, 1H), 5.10 (d, J = 8.4 Hz, 1H), 4.91 –4.86 (m, 2H), 4.85 (d, J = 10.9 Hz, 1H), 4.79 (d, J = 12.1 Hz, 1H), 4.74 (d, J = 11.7 Hz, 1H), 4.63 (d, J = 10.9 Hz, 1H), 4.59 (d, J = 10.9 Hz, 1H), 4.56 (d, J = 12.1 Hz, 1H), 4.51 (s, 1H), 4.49 (d, J = 3.5 Hz, 1H), 4.48 – 4.43 (m, 2H), 4.40 (dd, J = 10.8, 8.3 Hz, 1H), 4.28 (dd, J = 10.8, 8.5 Hz, 1H), 4.19 (d, J = 11.0 Hz, 1H), 4.13 (t, J = 6.0 Hz, 1H), 4.05 (dd, J = 10.7, 8.4 Hz, 1H), 3.85 – 3.82 (m, 3H), 3.77 (s, 3H), 3.75 – 3.71 (m, 2H), 3.71 – 3.67 (m, 2H), 3.56 – 3.52 (m, 2H), 3.30 (dd, J = 9.7, 8.3 Hz, 1H), 3.10 – 3.04 (m, 1H), 1.99 (s, 3H), 1.95 (s, 3H), 1.91 (s, 3H), 1.84 (s, 3H), 1.82 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 170.7, 170.2, 169.6, 154.8, 150.4, 138.4, 138.3, 138.2, 138.0, 134.3, 133.6, 128.6–127.4, 123.6, 117.8, 114.3, 98.6, 97.5, 97.0, 79.7, 79.3, 75.2, 75.1, 74.8, 73.6, 71.5, 71.4, 70.9, 70.6, 69.4, 69.2, 62.3, 56.0, 55.7, 54.6, 31.1, 20.7, 20.7, 20.5; m/z (HRMS) calcd for C₇₀H₈₀N₂O₂₀ [M+Na]⁺: 1291.5202, found: 1291.5208

p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-6-O-(2-acetamido-2-deoxy-β-Dglucopyranosyl)-α-D-mannopyranoside (15)

Trisaccharide 15 was prepared from trisaccharide 14 (0.3 g, 0.236 mmol) using the general procedure for hydrogenolysis and followed by column chromatography on silica gel (MeOH: CHCl₃ = 1:20). Yield (0.168 g, 87 %).¹H NMR (600 MHz, MeOD) δ 7.88 – 7.80 (m, 1H), 7.11 – 7.00 (m, 2H), 6.93 – 6.81 (m, 2H), 5.34 – 5.30 (m, 1H), 5.24 (ddd, J = 10.5, 9.3, 1.1 Hz, 1H), 4.96 – 4.90 (m, 1H), 4.85–4.82 (m, 4H), 4.71 – 4.67 (m, 1H), 4.63 (dd, J = 8.2, 1.3 Hz, 1H), 4.16 (dd, J = 12.2, 4.9 Hz, 1H), 4.11 (dd, J = 3.4, 1.9 Hz, 1H), 4.05 (ddd, J = 12.4, 10.0, 2.2 Hz, 2H), 3.90 – 3.84 (m, 3H), 3.84 – 3.79 (m, 1H), 3.77 (s, 3H), 3.76 – 3.72 (m, 1H), 3.69 (dt, J = 11.8, 5.7 Hz, 1H), 3.61 – 3.56 (m, 1H), 3.55–3.48 (m, 2H), 3.20 (q, J = 7.3 Hz, 1H), 2.02 (app d, 6H), 2.01 (s, 3H), 1.98 (s, 3H), 1.89 (s, 3H);¹³C NMR (151 MHz, MeOD) δ 174.3, 173.4, 172.3, 171.7, 171.2, 156.6, 152.0, 119.5, 115.6, 102.5, 100.9, 98.5, 79.3, 79.1, 78.9, 78.4, 77.9, 74.7, 73.9, 73.8, 72.6, 72.0, 71.4, 70.8, 70.2, 68.7, 63.2, 62.4, 57.7, 56.1, 55.6, 23.5, 23.1, 23.0, 20.7, 20.6; m/z (HRMS) calcd for C₃₅H₅₀N₂O₁₀ [M+Na]⁺: 841.2855, found: 841.2860.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-4,6-di-O-benzyl-α-Dmannopyranoside (16)

Disaccharide 16 was prepared from 3 (0.3 g, 0.34 mmol) using the general procedure for 4,6-O-benzylidene ring opening using Et₃SiH/TFA, followed by purification using silica gel column chromatography (PhCH₃: EtOAc = 8:2). Yield (0.210 g, 70 %).¹H NMR (600 MHz, CDCl₃) δ 7.86 – 7.75 (m, 1H), 7.73 –7.64 (m, 1H), 7.44 – 7.40 (m, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.32 – 7.28 (m, 2H), 7.28 – 7.25 (m, 2H), 7.25 – 7.22 (m, 2H), 7.09 –7.03 (m, 2H), 6.79 – 6.75 (m, 2H), 6.75 – 6.69 (m, 2H), 5.83 (dd, J = 10.8, 9.1 Hz, 1H), 5.54 (d, J = 8.5 Hz, 1H), 5.26 – 5.14 (m, 2H), 4.83 (d, J = 11.0 Hz, 1H), 4.53 (d, J = 11.0 Hz, 1H), 4.47 (dd, J = 10.8, 8.5 Hz, 1H), 4.31 – 4.28 (m, 1H), 4.27 (d, J = 4.9 Hz, 1H), 4.21 (dd, J = 12.2, 2.4 Hz, 1H), 4.07 (s, 2H), 3.89 (ddd, J = 10.3, 5.0, 2.4 Hz, 1H), 3.84 (dd, J = 9.1, 3.1 Hz, 1H), 3.76 –3.72 (m, 4H), 3.70 – 3.65 (m, 1H), 3.41 (dd, J = 10.7, 3.2 Hz, 1H), 3.04 (dd, J = 10.7, 6.4 Hz, 1H), 2.61 (dt, J = 7.3, 2.3 Hz, 1H), 2.03 (s, 3H), 2.03 – 2.00 (s, 3H), 1.87 (s, 3H);¹³C NMR (151 MHz, CDCl₃) δ 170.7, 170.2, 169.5, 155.0, 149.9, 138.2, 137.9, 137.8, 134.2, 129.1–127.3, 125.3, 123.5, 117.5, 114.5, 96.8, 95.9, 77.0, 73.2, 72.9, 72.1, 71.7, 70.9, 70.5, 70.5(2), 69.0, 67.5, 62.2, 55.6, 54.5, 21.5, 20.7, 20.7, 20.5; m/z (HRMS) calcd for C₄₇H₄₉NO₁₆ [M+Na]⁺: 906.2649, found: 906.2653.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2deoxy-β-D-glucopyranosyl)-4-O-(3,4,6-tri-O-benzyl-2acetamido-2-deoxy-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-Dmannopyranoside (17)

Disaccharide acceptor 16 upon glycosylation with glysocyl donor 6 using general procedure for glycosyl phosphates coupling provided trisaccharide. The phthalimide groups of trisaccharide were converted to NHAc using general procedure for phthalimide deprotection and N-acetylation and followed by column chromatography on silica gel (PhCH₃: EtOAc = 6:4) to provide trisaccharide 17. Yield (0.141 g, 77 %).¹H NMR (600 MHz, CDCl₃) δ 7.41 – 7.36 (m, 3H), 7.33 – 7.21 (m, 20H), 7.19 – 7.15 (m, 2H), 6.98 – 6.94 (m, 2H), 6.81 – 6.74 (m, 2H), 5.91 (s, 1H), 5.54 (s, 1H), 5.36 (d, J = 2.8 Hz, 1H), 5.30 (d, J = 8.8 Hz, 1H), 5.11 (d, J = 8.4 Hz, 1H), 5.01 (t, J = 9.7 Hz, 1H), 4.77 (d, J = 6.9 Hz, 1H), 4.76 – 4.74 (m, 2H), 4.74 – 4.67 (m, 3H), 4.60 – 4.55 (m, 2H), 4.54 (d, J = 10.9 Hz, 1H), 4.48 (q, J = 12.2 Hz, 2H), 4.41 (d, J = 11.7 Hz, 1H), 4.26 (d, J = 2.8 Hz, 1H), 4.15 (dd, J = 12.2, 4.6 Hz, 1H), 4.13 – 4.08 (m, 3H), 3.89 – 3.85 (m, 1H), 3.84 – 3.81 (m, 1H), 3.75 (s, 3H), 3.73 – 3.70 (m, 1H), 3.68 – 3.64 (m, 1H), 3.64 – 3.59 (m, 1H), 3.58 (dd, J = 10.5, 4.6 Hz, 1H), 3.52 – 3.44 (m, 1H), 3.37 (ddd, J = 9.3, 4.7, 2.2 Hz, 1H), 1.98 (s, 3H), 1.92 (s, 3H), 1.89 (s, 3H), 1.70 (app s, 6H);¹³C NMR (151 MHz, CDCl₃) δ 171.2, 170.7, 170.4, 170.2, 169.5, 155.0, 150.3, 138.6, 138.4, 138.4(2), 138.2, 138.0, 128.5–127.6, 117.6, 114.7, 100.5, 98.3, 96.7, 81.9, 78.4, 77.3, 74.9, 74.7, 74.6, 73.4, 73.1 72.3, 71.7, 71.5, 69.3, 69.0, 68.9, 62.1, 56.3, 55.7, 55.4, 23.5, 23.2, 20.7, 20.6, 20.6(2); m/z (HRMS) calcd for C₇₀H₈₀N₂O₂₀ [M+Na]⁺: 1291.5202, found: 1291.5208.

p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-4-O-(2-acetamido-2-deoxy-β-Dglucopyranosyl)-α-D-mannopyranoside (18)

Trisaccharide 18 was prepared from trisaccharide 17 (0.14 g, 0.110 mmol) using the general procedure for hydrogenolysis and followed by column chromatography on silica gel (MeOH: CHCl₃ = 1:20). Yield (0.081 g, 90 %).¹H NMR (600 MHz, MeOD) δ 7.02 (d, J = 9.0 Hz, 2H), 6.90 – 6.77 (m, 2H), 5.47 –5.36 (m, 1H), 5.31 – 5.20 (m, 1H), 4.87 – 4.77 (m, 1H),4.67 –4.51 (m, 1H), 4.50 (d, J = 8.4 Hz, 1H), 4.33 (dp, J = 11.9, 6.8 Hz, 2H), 4.23 – 4.10 (m, 2H), 4.07 (tt, J = 6.4, 3.9 Hz, 1H), 3.99 – 3.86 (m, 2H), 3.87 – 3.75 (m, 2H), 3.74 (app s, 3H), 3.73 – 3.67 (m, 2H), 3.68 – 3.55 (m, 2H), 3.55 – 3.44 (m, 1H), 3.42 – 3.32 (m, 2H), 2.04 (app s, 3H), 1.99 (app s, 6H), 1.96 (app s, 6H);¹³C NMR (151 MHz, MeOD) δ 174.2, 173.8, 172.5, 171.9, 171.3, 164.7, 156.7, 151.7(2), 119.1, 119.1, 119.0, 115.6, 103.1, 101.2, 97.9, 78.5, 78.4, 78.0, 75.8, 73.9, 73.5, 72.9, 71.8, 71.8(2), 70.0, 69.7, 63.1, 62.5, 62.1, 62.0, 57.3, 56.1, 55.4, 23.3, 23.0, 20.7, 20.5; m/z (HRMS) calcd for C₃₅H₅₀N₂O₁₀ [M+Na]⁺: 841.2855, found:841.2859.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-3,6-di-O-benzyl-α-Dmannopyranoside (20)

Disaccharide 20 was obtained from the disaccharide 10 (0.4 g, 0.39 mmol) using the general procedure for 4,6-O-benzylidene ring opening using Et₃SiH/TFA and followed by column chromatography on silica gel (PhCH₃: EtOAc = 9:1). Yield (0.294 g, 74 %).¹H NMR (500 MHz, CDCl₃) δ 7.51 (dd, J = 5.5, 3.0 Hz, 2H), 7.42 – 7.37 (m, 2H), 7.33 – 7.21 (m, 18H), 7.05 – 6.98 (m, 4H), 6.90 – 6.85 (m, 2H), 6.85 – 6.81 (m, 1H), 6.77 – 6.72 (m, 2H), 6.71 – 6.67 (m, 2H), 5.29 (d, J = 8.3 Hz, 1H), 5.08 (d, J = 1.9 Hz, 1H), 4.86 (dd, J = 11.0, 8.2 Hz, 2H), 4.80 (d, J = 12.1 Hz, 1H), 4.64 (d, J = 10.9 Hz, 1H), 4.53 (d, J = 11.9 Hz, 1H), 4.48 (s, 1H), 4.47 – 4.43 (m, 2H), 4.36 (dd, J = 10.9, 8.0 Hz, 1H), 4.32 – 4.26 (m, 2H), 3.80 – 3.74 (m, 4H), 3.73 (app s, 4H), 3.71 – 3.59 (m, 3H), 3.38 (dd, J = 10.7, 3.1 Hz, 1H), 2.92 (dd, J = 10.7, 6.4 Hz, 1H); m/z (HRMS) calcd for C₆₂H₆₁NO₁₃ [M+Na]⁺: 1050.4041, found: 1050.4043.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-4-O-(4-O-acetyl-2-deoxy-2phthalimido-β-D-glucopyranosyl)-3,4-di-O-benzyl-α-Dmannopyranoside (21)

Trisaccharide 21 was prepared from glycosyl phosphate donor 19 (0.225 g, 0.291 mmol) and disaccharide acceptor 20 (0.250 g, 0.243 mmol) using the general procedure for glycosyl phosphate coupling and followed by column chromatography on silica gel (PhCH₃: EtOAc = 8:2). Yield (0.292 g, 78 %).¹H NMR (600 MHz, CDCl₃) δ 7.63–7.56 (m, 6H), 7.43 – 7.33 (m, 1H), 7.29 (qd, J = 5.9, 2.8 Hz, 12H), 7.25 – 7.14 (m, 10H), 7.08 – 6.99 (m, 5H), 6.99 – 6.95 (m, 2H), 6.95 – 6.81 (m, 7H), 6.63 – 6.58 (m, 2H), 6.58 – 6.51 (m, 2H), 5.21 (d, J = 8.4 Hz, 1H), 4.93 – 4.86 (m, 3H), 4.84 (d, J = 10.9 Hz, 1H), 4.78 (d, J = 12.0 Hz, 1H), 4.70 (d, J = 11.5 Hz, 1H), 4.62 (d, J = 10.9 Hz, 1H), 4.55 (dd, J = 12.0, 9.5 Hz, 2H), 4.50 (d, J = 12.0 Hz, 2H), 4.45 (dd, J = 11.7, 2.9 Hz, 2H), 4.38 (dd, J = 10.8, 8.4 Hz, 1H), 4.33 –4.28 (m, 3H), 4.28 – 4.22 (m, 2H), 4.13 (t, J = 2.8 Hz, 1H), 4.10 (d, J = 11.0 Hz, 1H), 3.92 (dd, J = 10.7, 8.4 Hz, 1H), 3.82 (dd, J = 8.4, 3.2 Hz, 1H), 3.74 (app s, 4H), 3.72 – 3.68 (m, 1H), 3.66 (td, J = 7.2, 3.6 Hz, 2H), 3.55 (dd, J = 11.3, 2.3 Hz, 1H), 3.51 (ddd, J = 9.8, 5.7, 4.0 Hz, 1H), 3.33 – 3.27 (m, 2H), 3.18 (dd, J = 10.6, 5.6 Hz, 1H), 3.06 (dd, J = 11.2, 6.7 Hz, 1H), 2.36 (s, 1H), 1.89 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 169.8, 154.8, 150.5, 138.4, 138.2, 138.2, 138.1, 138.0, 137.7, 133.8, 131.6, 129.1–127.4, 125.4, 117.8, 114.4, 98.7, 97.3, 96.4, 79.7, 79.4, 77.6, 75.2, 75.1, 74.9, 74.8, 73.9, 73.6, 73.5, 73.1, 72.9, 71.3, 71.1, 70.1, 69.9, 69.4, 55.9, 55.7, 55.4, 21.0; m/z (HRMS) calcd for C₉₂H₈₈N₂O₂₂ [M+Na]⁺: 1563.5822, found: 1563.5859.

p-Methoxyphenyl-2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(4-O-acetyl-2-acetamido-2-deoxy-β-Dglucopyranosyl)-α-D-mannopyranoside (22)

Trisaccharide 21 (0.2 g, 0.13 mmol) was converted to protecting group free trisaccharide 22 in a two steps sequence: (i) deprotection of phthalimide groups and converted to NHAc using general procedure for phthalimide deprotection and N-acetylation (ii) catalytic hydrogenation in the presence of Pd(OH)₂/C removed all benzyl ethers. Finally, the crude trisaccharide was purified by column chromatography on silica gel (MeOH: CHCl₃ = 1:9) to furnish 24. Yield (0.059 g, 86 %).¹H NMR (600 MHz, MeOD) δ 7.11 – 7.02 (m, 2H), 6.95 – 6.85 (m, 2H), 5.36 (s, 1H), 4.87 – 4.81 (m, 3H), 4.78 (t, J = 9.2 Hz, 1H), 4.68 (d, J = 8.3 Hz, 1H), 4.54 (d, J = 7.6 Hz, 1H), 4.17 – 4.07 (m, 2H), 3.95 – 3.86 (m, 2H), 3.78 (s, 3H), 3.77 – 3.74 (m, 1H), 3.73 – 3.68 (m, 1H), 3.66 – 3.59 (m, 3H), 3.59 – 3.54 (m, 2H), 3.52 (dd, J = 12.2, 5.8 Hz, 1H), 3.45 (ddd, J = 8.9, 6.0, 2.5 Hz, 1H), 2.11 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H). ¹³C NMR (151 MHz, MeOD) δ 174.5, 172.1, 156.8, 152.0, 119.5, 115.7, 102.9, 98.7, 78.9, 78.0, 75.8, 74.8, 74.0, 73.3, 73.2, 72.1, 71.5, 70.1, 68.8, 62.5, 62.4, 57.8, 57.6, 56.1, 23.6, 23.3, 20.9. m/z (HRMS) calcd for C₃₁H₄₆N₂O₁₈ [M+Na]⁺: 757.2638, found: 757.2647.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2deoxy-β-D-glucopyranosyl)-4-O-(β-D-galactopyranosyl-2acetamido-2-deoxy-β-D-glucopyranosyl)-α-Dmannopyranoside (23)

Glycan 23 was prepared from 18 using the general procedure for enzymatic β−1, 4-galactosylation. The crude compound was purified using C18 column chromatography using MeOH/H₂O as eluent. Fractions containing the product were combined, evaporated, and lyophilized to give the tetrasaccharide 23 (9 mg, 75 %). ¹H NMR (600 MHz, D₂O) δ 7.16 – 7.12 (m, 2H), 7.03 –6.99 (m, 2H), 5.49 (d, J = 2.1 Hz, 1H), 5.28 (dd, J = 10.7, 9.2 Hz, 1H), 5.12 (dd, J = 10.1, 9.3 Hz, 1H), 4.91 (d, J = 8.4 Hz, 1H), 4.58 (d, J = 8.3 Hz, 1H), 4.50 (d, J = 7.9 Hz, 1H), 4.45 (dd, J = 12.6, 3.4 Hz, 1H), 4.35 (dd, J = 3.4, 2.1 Hz, 1H), 4.26 – 4.20 (m, 2H), 4.17 – 4.11 (m, 1H), 4.06 – 4.03 (m, 1H), 4.04 – 3.99 (m, 1H), 3.95 (d, J = 3.5 Hz, 1H), 3.90 – 3.86 (m, 1H), 3.83 (s, 3H), 3.82 – 3.79 (m, 1H), 3.81 – 3.76 (m, 1H), 3.76 (ddd, J = 5.0, 3.6, 2.0 Hz, 2H), 3.75 – 3.71 (m, 1H), 3.71 – 3.68 (m, 1H), 3.62 –3.57 (m, 1H), 3.57 – 3.54 (m, 1H), 2.15 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.06 – 2.04 (m, 3H), 1.99 (s, 3H), ¹³C NMR (151 MHz, D₂O) δ 177.2, 177.0, 176.2, 175.7, 175.4, 157.3, 152.3, 121.2, 117.6, 105.4, 104.0, 102.1, 98.9, 80.6, 79.8, 79.2, 77.9, 77.4, 75.0, 74.9, 74.7, 73.7, 73.5, 71.1, 71.0, 70.7, 65.0, 64.3, 63.5, 63.2, 62.5, 58.3, 57.7, 56.0, 25.8, 24.7, 24.6, 22.6, 22.6(2), 22.5; m/z (HRMS) calcd for C₄₁H₆₀N₂O₂₃ [M+Na]⁺: 1003.3383, found:1003.3384.

p-Methoxyphenyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2deoxy-β-D-glucopyranosyl)-(1→2)-O-(5-Acetamido-3,5dideoxy-D-glycero-α-D-galacto-2-nonulopyrano-sylonate(2→3)-α-D-galactopyranosyl-β-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-α-D-mannopyranoside (24)

Glycan 24 was prepared from 23 using the general procedure for enzymatic α−2, 3-sialylation. The crude compound was purified using C18 column chromatography using MeOH/H₂O as eluent. Fractions containing the product were combined, evaporated, and lyophilized to give the pentasaccharide 24 (11.8 mg, 83 %). ¹H NMR (600 MHz, D₂O) δ 7.06 (d, J = 8.3 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 5.40 (s, 1H), 5.19 (t, J = 10.0 Hz, 1H), 5.04 (t, J = 9.7 Hz, 1H), 4.82 (d, J = 8.3 Hz, 1H), 4.74–4.78 (m, 1H), 4.50 (d, J = 8.3 Hz, 1H), 4.41 (d, J = 7.5 Hz, 1H), 4.37 (d, J = 12.7 Hz, 1H), 4.27 (s, 1H), 4.18 – 4.10 (m, 2H), 3.98 – 3.90 (m, 3H), 3.86 (s, 1H), 3.83 – 3.76 (m, 1H), 3.75 (d, J = 2.1 Hz, 3H), 3.72 – 3.63 (m, 12H), 3.63 – 3.54 (m, 2H), 3.54 – 3.44 (m, 2H), 2.97 – 2.87 (m, 1H), 2.07 (app s, 3H), 2.04 – 1.98 (app m, 9H), 1.97 (s, 3H), 1.93 (s, 3H), 1.29 – 1.18 (m, 1H); ¹³C NMR (151 MHz, D₂O) δ 174.7, 174.5, 173.6, 173.2, 172.8, 154.7, 149.7, 118.6, 115.1, 102.9, 101.5, 99.5, 96.3, 78.1, 77.3, 76.7, 75.3, 74.8, 72.5, 72.4, 72.1, 72.0, 71.1, 70.9, 68.5, 68.4, 68.2, 62.5, 61.8, 61.0, 59.9, 59.5, 55.8, 55.1, 53.5, 30.2, 23.2, 22.1, 22.1, 20.1, 20.1(2), 19.9.