Synthesis of oligosaccharide fragments corresponding to the exopolysaccharide released by Streptococcus macedonicus Sc 136

Abstract

Hexa-, penta- and tetrasaccharide fragments related to the repeating unit of the exopolysaccharide secreted by Streptococcus macedonicus Sc 136 were synthesized in a very efficient manner involving minimum number of steps. A general glycosylation condition has been applied for all glycosylation steps and yields were excellent.

1 Introduction

Lactic acid bacteria (LAB) possess a large number of metabolic properties, for instance, they ferment lactose to lactic acid, which in turn inhibits the growth of pathogenic bacteria. LAB are the organisms that beneficially affect the host animal by improving the intestinal microbial balance [1, 2] by producing an abundant variety of exopolysaccharides (EPS), which provide an important contribution to human health by acting as prebiotic substrates [3]. In general EPS have excellent water-binding properties and so protect the bacterial cells in low moisture environment [4]. EPSs secreted by LAB play a significant role in the protection of microbial cells against phagocytosis, phage attacks, antibiotics, toxic compounds (e.g. toxic metal ions, sulfur dioxide, and ethanol), osmotic stress and bacteriocins such as nisin [5]. Due to their excellent adhesion properties EPS play key role in biofilm formation and dental caries pathogenesis [6, 7]. In the dairy industry, and particularly, yogurt industry, in which the addition of stabilizers is prohibited, LAB have been studied for their ability to influence both the texture and viscosity of fermented milk products [3]. Most of the LAB are nonpathogenic and a number of EPSs secreted by LAB has antigenic properties, which cause modulation of the immune system of the host and/or host-pathogen interactions [8]. In general, yoghurts are used to develop immunogenicity and so the strains used as starter cultures may indirectly be immunostimulators. Various studies directing to the immunomodulatory effects of EPSs have established that EPS produced by Lactobacillus and Streptococcus strains have considerable immunomodulatory effects in the host. Besides acting as immunomodulators [9], EPSs secreted by LAB have several other medicinal activities, which include their use as antihyperlipidemic [10, 11], antitumor [12], antimutagenic [13], antiulcer [14] and antibacterial agents [15].

Streptococcus macedonicus is a Gram-positive lactic acid bacteria, present in the starter flora of the Greek cheese and goat cheese. Several Streptococcus macedonicus strains are used as cultures and natural food preservatives and are found to have several antimicrobial activities especially in cheese [16]. Recently, the structure of a hexasaccharide repeating unit of the exopolysaccharide secreted by Streptococcus macedonicus Sc136, isolated from cheese has been demonstrated, containing a unique galactofuranose moiety at the non-reducing terminus [17] (Fig. 1). In order to study the rheological, pharmaceutical and physicochemical properties of EPS produced by Streptococcus macedonicus, it is essential to have a larger quantity of the hexasaccharide and their close analogs. Whilst oligosaccharides can sometimes be isolated from natural sources, efficient chemical synthetic strategies offer the advantage of having access to large quantities of oligosaccharides as well as analogues of the natural oligosaccharides. We report herein concise chemical syntheses of hexa-, penta- and tetrasaccharide fragments corresponding to the exopolysaccharide secreted by Streptococcus macedonicus Sc136.

Fig. 1

Structure of the extracellular hexasaccharide produced by Streptococcus macedonicus Sc136

2 Results and discussion

The synthesis of compounds 1, 2 and 3 (Fig. 2) from suitably functionalized mono- and disaccharide intermediates are presented in Schemes 1, 2, 3, 4 and 5. Compounds 4–10 [18–24] were synthesized following earlier literature reports (Fig. 3).

Fig. 2

Structures of synthesized hexa- and penta- and tetrasaccharides (1, 2 and 3) corresponding to the exopolysaccharide produced by Streptococcus macedonicus Sc136

Scheme 1

Reagents: a NIS, TMSOTf, CH₂Cl₂, −10°C, 2 h, 78% for 11 and 82% for 15; b CH₃ONa, CH₃OH, r t, 5 h; c 2,2-dimethoxypropane, p-TsOH, DMF, r t, 5 h; d BnBr, solid NaOH, n-Bu₄NBr, THF, r t, 5 h, 60% in three steps; e 80% aq. AcOH, 80°C, 1 h, 90%; f triethyl orthoacetate, p-TsOH, r t, 4 h; g 80% aq. AcOH, r t, 1 h, 85% in two steps; h HClO₄–SiO₂, CH₃CN, r t, 20 min, 90%

Scheme 2

Reagents: a NIS–TMSOTf, −30°C, 2 h, 78% for 17 and 81% for 19; b acetic anhydride, pyridine, r t, 1 h; c HClO₄–SiO₂, CH₃CN, r t, 20 min, 95% in two steps; d (1) NH₂NH₂.H₂O, C₂H₅OH, 80°C, 6 h; (2) Ac₂O, Pyridine, r t, 2 h; e CH₃ONa, CH₃OH, r t, 5 h; f H₂, 20% Pd(OH)₂–C, CH₃OH, r t, 12 h, 69% in three steps

Scheme 3

Reagents: a CH₃ONa, CH₃OH, r t, 5 h; b 2,2-dimethoxypropane, p-TsOH, DMF, r t, 5 h; c BnBr, NaOH, n-Bu₄NBr, THF, r t, 5 h, 78% in three steps; d 80% aq. AcOH, 80°C, 1 h, 87% e triethyl orthoacetate, p-TsOH, r t, 4 h; f 80% aq. AcOH, r t, 1 h, 86% after two steps; g NIS–TMSOTf, −30°C, 1 h, 84%; h HClO₄–SiO₂, CH₃CN, r t, 20 min, 92%

Scheme 4

Reagents: a NIS–TMSOTf, −30°C, 1 h, (82% for 26 and 79% for 28); b HClO₄–SiO₂, CH₃CN, r t, 20 min, 89%; c (1) NH₂NH₂·H₂O, C₂H₅OH, 80°C, 6 h; (2) Ac₂O, Pyridine, rt, 2 h; d CH₃ONa, CH₃OH, r t, 5 h; e H₂, 20% Pd(OH)₂-C, CH₃OH, r t, 12 h, 66% in three steps

Scheme 5

Reagents: a NIS–TMSOTf, CH₂Cl₂, −30°C, 2 h, (47% for 29 and 35% for 30); b (1) NH₂NH₂·H₂O, C₂H₅OH, 80°C, 6 h; (2) Ac₂O, Pyridine, r t, 2 h; c CH₃ONa, CH₃OH, r t, 5 h; d H₂, 20% Pd(OH)₂-C, CH₃OH, r t, 12 h, 70% in three steps

Fig. 3

Mono- and disaccharide intermediates for the synthesis of compounds 1, 2 and 3

The synthesis of compound 1 started with the glycosylation of the glycosyl acceptor 10 [24], prepared from d-glucose in three steps, with ethyl thioglycoside donor 4 [18], prepared from d-lactose octaacetate, in the presence of N-iodosuccinimide-methyltrifluoromethanesulfonate (NIS-TMSOTf) [25] to furnish trisaccharide derivative 11 in 78% yield. The compound 11 was converted to the trisaccharide derivative 12 in 60% over all yield following a sequence of reactions involving deacetylation, 3,4-O-isopropylidenation [26] and benzylation [27]. Removal of isopropylidene group [28] from compound 12 using 80% aq. acetic acid afforded trisaccharide diol 13 in 90% yield, which was selectively acetylated [29] using triethyl orthoacetate to furnish trisaccharide acceptor 14 in 85% yield. Glycosylation of compound 14 with ethyl thioglycoside donor 6 [20], prepared from d-glucosamine hydrochloride in six steps, in the presence of NIS-TMSOTf [25] furnished tetrasaccharide derivative 15 in 82% yield. Removal of the benzylidene acetal [30] from the compound 15 using HClO₄–SiO₂ afforded tetrasaccharide diol 16 in 90% yield (Scheme 1). Selective glycosylation of tetrasaccharide diol acceptor 16 with ethyl thioglycoside donor 8 [22], prepared from d-glucose in five steps, in the presence of NIS-TMSOTf [25] furnished pentasaccharide derivative 17 in 78% yield. Conventional acetylation followed by removal of benzylidene acetal [30] from the compound 17 using HClO₄–SiO₂ afforded pentasaccharide acceptor 18 in 95% yield, which was selectively glycosylated with ethyl thioglycoside donor 9 [23], prepared from d-galactose, in the presence of NIS–TMSOTf [25] to give hexasaccharide derivative 19 in 81% yield. Conversion of N-phthalimido group to N-acetyl group of compound 19 was achieved in two steps involving the treatment with hydrazine hydrate, followed by N-acetylation [31]. Global deprotection of the resulting acetylated hexasaccharide derivative involving deacetylation and hydrogenolysis [32] furnished target hexasaccharide 1 as its methyl glycoside in 69% yield (Scheme 2). The structure of the hexasaccharide 1 was confirmed from its 1 D, 2 D NMR and mass spectral data. Presence of six anomeric signals in the ¹H NMR spectrum [δ 5.02 (d, J = 1.2 Hz, H-1_F), 4.82 (d, J = 8.4 Hz, H-1_D), 4.77 (d, J = 4.0 Hz, H-1_A), 4.50 (d, J = 8.0 Hz, H-1_C), 4.48 (d, J = 8.0 Hz, H-1_B), 4.42 (d, J = 7.6 Hz, 1 H, H-1_E)] and ¹³C NMR spectrum [δ 107.5 (C-1_F), 102.7 (C-1_B), 102.3 (C-1_D), 101.8 (C-1_C), 101.7 (C-1_E) and 98.4 (C-1_A)] supported the formation of the hexasaccharide 1.

In separate experiments, target pentasaccharide 2 was synthesized following Schemes 3 and 4. The disaccharide derivative 23 was prepared from per-O-acetylated methyl d-lactoside (20) [33] following a three-step reaction sequence. Conventional saponification of compound 20, isopropylidenation [26] in the presence of 2,2-dimethoxypropane and p-TsOH followed by benzylation [27] afforded disaccharide derivative 21 in 78% overall yield. Removal of isopropylidene group [28] from compound 21 using 80% aq. AcOH at elevated temperature resulted in the formation of disaccharide diol 22 in 87% yield. Compound 22 was selectively 4-O-acetylated [29] via the orthoester formation followed by its hydrolysis under acidic condition to give the disaccharide acceptor 23 in 86% overall yield. Glycosylation of 23 with the ethyl thioglycoside donor 6, in the presence of NIS-TMSOTf [25] furnished trisaccharide derivative 24 in 84% yield. Removal of the benzylidene acetal [30] from compound 24 using HClO₄-SiO₂ in acetonitrile furnished trisaccharide diol 25 in 92% yield. Selective glycosylation of diol acceptor 25 with ethyl thioglycoside donor 8 in the presence of NIS-TMSOTf [25] furnished tetrasaccharide derivative 26 in 82% yield. Removal of the benzylidene acetal [30] from compound 26 using HClO₄-SiO₂ in acetonitrile afforded the tetrasaccharide diol 27 in 89% yield. Selective 6-O-glycosylation of compound 27 with ethyl thioglycoside donor 9 in the presence of NIS-TMSOTf [25] furnished the pentasaccharide derivative 28 in 79% yield. Conversion of N-phthalimido group to N-acetyl group of compound 28 was achieved in two steps involving the treatment with hydrazine hydrate, followed by N-acetylation [31]. Global deprotection of the resulting pentasaccharide derivative involving deacetylation and hydrogenolysis [32] furnished target pentasaccharide 2 as its methyl glycoside in 66% yield. Presence of five anomeric signals in the ¹H NMR spectrum [δ 4.98 (d, J = 1.2 Hz, H-1_E), 4.77 (d, J = 8.4 Hz, H-1_C), 4.46 (d, J = 8.0 Hz, H-1_D), 4.44 (d, J = 8.0 Hz, H-1_B) and 4.38 (d, J = 7.6 Hz, H-1_A)] and ¹³C NMR spectrum [δ 107.0 (C-1_E), 102.2 (C-1_A and C-1_B), 101.8 (C-1_C), 100.3 (C-1_D)] confirmed the formation of the required pentasaccharide 2.

In another set of experiments presented in Scheme 5, the preparation of tetrasaccharide 3 started with the selective glycosylation of disaccharide diol 22 with ethyl thioglycoside donor 5 [19] in the presence of NIS-TMSOTf [25] to furnish trisaccharide derivative 29 in 47% yield together with some undesired di-glycosylated product (∼25%). The compound 29 was separated and used for the next step glycosylation reaction using ethyl thioglycoside donor 7 [21]. Condensation of compound 7 with compound 29 in the presence of NIS–TMSOTf [25] furnished tetrasaccharide derivative 30 in 35% yield. Conversion of N-phthalimido group to N-acetyl group of compound 30 was achieved in two steps involving the treatment with hydrazine hydrate, followed by N-acetylation [31]. Global deprotection of the resulting tetrasaccharide derivative involving deacetylation and hydrogenolysis [32] furnished target tetrasaccharide 3 as its methyl glycoside in 70% yield. Presence of four anomeric signals in the ¹H NMR spectrum [δ 4.80 (d, J = 8.1 Hz, H-1_C), 4.73 (d, J = 4.0 Hz, H-1_D), 4.62 (d, J = 8.2 Hz, H-1_B), 4.32 (d, J = 7.5 Hz, H-1_A)] and ¹³C NMR spectrum [δ 101.9 (C-1_B), 101.7 (C-1_A), 100.8 (C-1_C), 100.0 (C-1_D)] confirmed the formation of the required tetrasaccharide 3.

3 Conclusion

In summary, concise synthesis of hexa-, penta- and a tetrasaccharide fragments corresponding to the exopolysaccharide secreted by Streptococcus macedonicus Sc 136 as their methyl glycosides were achieved using thioglycosides as glycosyl donors and a generalized glycosylation conditions. In the course of synthesis of target molecules (1, 2 and 3), a number of novel methodologies developed from our laboratory have been applied. These synthesized oligosaccharide fragments could be evaluated for their use as prebiotic substances to control metabolic disorders [34].

4 Experimental section

General methods

All reactions were monitored by thin layer chromatography over silica gel coated TLC plates. The spots on TLC were visualized by warming ceric sulphate (2% Ce(SO₄)₂ in 2N H₂SO₄) sprayed plates on a hot plate. Silica gel 230–400 mesh was used for column chromatography. ¹H and ¹³C NMR, 2DCOSY, HMQC spectra were recorded on Brucker Advance DPX 300 MHz using CDCl₃ and D₂O as solvents and TMS as internal reference unless stated otherwise. Chemical shift value is expressed in δ ppm. ESI-MS were recorded on a MICROMASS QUTTRO II triple quadrupole mass spectrometer. Elementary analysis was carried out on Carlo ERBA-1108 analyzer. Optical rotations were measured at 25°C on a Rudolf Autopol III polarimeter. Commercially available grades of organic solvents of adequate purity are used in many reactions.

Methyl (2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (11)

To a solution of compound 10 (1.0 g, 2.1 mmol) and ethyl thioglycoside donor 4 (1.8 g, 2.6 mmol) in anhydrous CH₂Cl₂ (20 ml) was added powdered MS 4 Å (1.0 g) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-iodosuccinimide (880 mg, 3.9 mmol) was added to the reaction mixture and it was cooled to −10°C. To the cold reaction mixture was added TMSOTf (15 μl) and the reaction mixture was allowed to stir at −10°C for 2 h. After completion (TLC; hexane-EtOAc 3:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃ and diluted with CH₂Cl₂ (100 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (6:1) as eluant to afford pure trisaccharide derivative 11 (1.7 g, 78%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 15.6{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat) 2365, 1752, 1712, 1370, 1221, 1052, 771 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.62–7.59 (m, 1 H, aromatic protons), 7.36–7.25 (m, 12 H, aromatic protons), 7.10–7.07 (m, 2 H, aromatic protons), 5.33 (br s, 1 H), 5.12–5.09 (m, 1 H), 4.99–4.92 (m, 3 H), 4.79–4.72 (m, 3 H), 4.67–4.65 (m, 1 H), 4.58–4.54 (m, 2 H), 4.48–4,34 (m, 3 H), 4.25–4.18 (m, 1 H), 4.13–4.08 (m, 2 H), 4.00–3.92 (m, 1 H), 3.84–3.73 (m, 4 H), 3.68–3.58 (m, 3 H), 3.48–3.39 (m, 1 H), 3.36 (s, 3 H, OCH ₃), 3.24–3.18 (m, 1 H), 2.15, 2.06, 2.04, 2.01, 1.96, 1.95 (6 s, 21 H, 7 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.5, 170.4, 170.2, 170.1, 169.9, 169.5, 169.1, 139.1–127.0 (aromatic carbons), 101.1 (C-1_B), 99.7 (C-1_C), 98.2 (C-1_A), 79.8, 78.7, 77.0, 75.9, 74.7, 73.6, 73.3, 73.2, 72.2 (2 C), 70.9, 70.5, 69.6, 69.0, 67.5, 66.7, 62.1, 60.8, 55.3 (OCH₃), 21.0, 20.8 (2 C), 20.7, 20.6, 20.5 (2 C); ESI–MS: m/z = 1,105.4 [M+Na]⁺; Anal. Calcd. for C₅₄H₆₆O₂₃ (1,082.4): C, 59.88; H, 6.14; found: C, 59.65; H, 6.40.

Methyl (2,6-di-O-benzyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (12)

To a solution of trisaccharide derivative 11 (1.5 g, 1.4 mmol) in CH₃OH (20 ml) was added solid CH₃ONa until the pH of the solution became ∼10. The reaction mixture was allowed to stir at room temperature for 5 h. After neutralization with Amberlite IR-120 (H⁺) resin, the reaction mixture was filtered and evaporated to dryness. To a solution of the crude mass in DMF (5.0 ml) was added 2,2-dimethoxypropane (210 μl, 1.7 mmol) and p-toluenesulfonic acid (100 mg) and the reaction mixture was allowed to stir at 60°C for 4 h. The reaction was quenched with triethylamine (0.2 ml) and the solvents were removed under reduced pressure. To a solution of the crude mass in THF (20 ml) were added powdered NaOH (650 mg, 16.2 mmol), benzyl bromide (1.6 ml, 13.8 mmol) and tetrabutylammonium bromide (100 mg) in succession and the reaction mixture was allowed to stir vigorously at room temperature for 8 h. After completion (TLC; hexane-EtOAc 6:1), the reaction mixture was diluted with water (100 ml) and extracted with CH₂Cl₂ (150 ml). The organic layer was washed with water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (9:1) as eluant to furnish pure compound 12 (1.1 g, 60%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} - 23.2{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,927, 1,636, 1,217, 1,054, 770 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.36–7.14 (m, 40 H, aromatic protons), 5.12–5.09 (d, J = 10.5 Hz, 1 H), 4.97–4.93 (d, J = 10.5 Hz, 1 H), 4.80–4.79 (d, J = 2.7 Hz, 1 H), 4.75–4.68 (m, 6 H), 4.63–4.49 (m, 4 H), 4.42–4.38 (d, J = 11.1 Hz, 1 H), 4.38–4.36 (d, J = 7.5 Hz, 1 H, H-1_B), 4.34–4.33 (d, J = 3.9 Hz, 1 H, H-1_A), 4.31–4.27 (m, 3 H), 4.07–4.05 (d, J = 5.4 Hz, 1 H), 3.99–3.94 (m, 2 H), 3.91–3.83 (m, 3 H), 3.70 (br s, 2 H), 3.63–3.58 (m, 3 H), 3.50–3.44 (m, 3 H), 3.39 (s, 3 H, OCH ₃), 3.34–3.26 (m, 3 H), 3.18–3.16 (m, 1 H), 1.40, 1.36 (2 s, 6 H, C(CH ₃)₂); ¹³C NMR (CDCl₃, 75 MHz): δ 139.6–138.0 (aromatic carbons), 128.3–126.9 (aromatic carbons), 109.6, 102.6 (C-1_B), 101.8 (C-1_C), 98.4 (C-1_A), 83.2, 82.3, 80.5 (2 C), 79.4, 78.9, 76.2, 75.4, 75.3, 75.2 (2 C), 73.7, 73.6, 73.3 (2 C), 73.2 (2 C), 72.9, 72.1, 70.1, 68.9, 68.1, 67.9, 55.2 (OCH₃), 28.0, 26.5; ESI-MS: m/z = 1301.6 [M+Na]⁺; Anal. Calcd. for C₇₈H₈₆O₁₆ (1278.6): C, 73.22; H, 6.77; found: C, 73.0; H, 7.00.

Methyl (2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (13)

A solution of compound 12 (1.0 g, 0.8 mmol) in 80% aq. acetic acid (50 ml) was stirred at 80°C for 1 h. After completion (TLC; hexane-EtOAc 3:1), the reaction mixture was concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (4:1) as eluant to furnish pure compound 13 (870 mg; 90%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} - 18.9{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,447, 3,030, 2,922, 1,722, 1,454, 1,363, 1,058, 745, 699 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.43–7.08 (m, 40 H, aromatic protons), 5.10–5.03 (dd, J = 10.5 each Hz, 1 H), 5.04 (d, J = 10.5 Hz, 1 H), 4.86–4.81 (m, 2 H), 4.78–4.69 (m, 4 H), 4.66–4.62 (m, 2 H), 4.60–4.58 (m, 2 H), 4.48–4.36 (m, 5 H), 4.35–4.33 (m, 2 H), 4.12–3.99 (t, J = 9.3 each Hz, 1 H), 3.93–3.79 (m, 3 H), 3.73–3.70 (m, 2 H), 3.61–3.58 (m, 3 H), 3.52–3.46 (m, 3 H), 3.44–3.37 (m, 2 H), 3.36 (s, 3 H, OCH ₃), 3.34–3.30 (m, 3 H), 3.20–3.16 (m, 1 H); ¹³C NMR (CDCl₃, 75 MHz): δ 138.4–135.7 (aromatic carbons), 128.5–125.4 (aromatic carbons), 101.3 (2 C, C-1_B and C-1_C), 97.1 (C-1_A), 81.8, 80.9, 79.1, 78.7, 77.6, 75.1, 74.0, 73.9 (2 C), 73.8, 73.6, 72.3, 72.1 (3 C), 71.9, 71.8, 71.6, 68.8, 67.4 (2 C), 66.9, 66.7, 53.9 (OCH₃); ESI–MS: m/z = 1261.5 [M+Na]⁺; Anal. Calcd. for C₇₅H₈₂O₁₆ (1,238.5): C, 72.68; H, 6.67; found: C, 72.45; H, 6.95.

Methyl (4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (14)

To a solution of compound 13 (850 mg, 0.7 mmol) in DMF (3 ml) was added triethyl orthoacetate (630 μl, 3.4 mmol) followed by p-toluenesulfonic acid (50 mg) and the reaction mixture was allowed to stir at room temperature for 2 h. After completion, the reaction mixture was neutralized with triethylamine (0.1 ml) and the solvents were removed under reduced pressure. A solution of the crude mass in 80% aq. acetic acid (20 ml) was allowed to stir at room temperature for 1 h. After completion (TLC; hexane-EtOAc 3:1), the reaction mixture was concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (5:1) as eluant to furnish pure compound 14 (760 mg, 85%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 23.4{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,018, 2,926, 1,740, 1,455, 1,366, 1,218, 1,067, 767, 699 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.45–7.15 (m, 40 H, aromatic protons), 5.31 (br s, 1 H), 5.11 (d, J = 11.7 Hz, 1 H), 5.02 (d, J = 10.2 Hz, 1 H), 4.84–4.68 (m, 6 H), 4.64–4.60 (m, 3 H), 4.59 (d, J = 4.2 Hz, 1 H, H-1_A), 4.50–4.46 (m, 1 H), 4.45 (d, J = 8.0 Hz, 1 H, H-1_C), 4.39 (d, J = 7.8 Hz, 1 H, H-1_B), 4.38–4.35 (m, 2 H), 4.30–4.26 (m, 2 H), 4.05–4.02 (t, J = 9.9 Hz each, 1 H), 3.92–3.84 (m, 3 H,), 3.70–3.69 (m, 2 H), 3.62–3.57 (m, 2 H), 3.52–3.46 (m, 3 H), 3.40 (s, 3 H, OCH ₃), 3.36–3.31 (m, 5 H), 3.16–3.14 (m, 1 H), 2.04 (s, 3 H, COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.5, 139.5–136.9 (aromatic carbons), 129.6–126.9 (aromatic carbons), 102.5 (C-1_B), 102.2 (C-1_C), 98.3 (C-1_A), 82.9, 82.1, 80.3, 79.5, 78.8, 76.1, 75.2, 75.0 (2 C), 74.9 (2 C), 73.5, 73.2, 73.1, 72.8, 72.2, 71.9, 69.8, 69.5, 68.0, 667.9 (2 C), 67.1, 55.1 (OCH₃), 20.1; ESI–MS: m/z = 1,303.5 [M+Na]⁺; Anal. Calcd. for C₇₇H₈₄O₁₇ (1,280.6): C, 72.17; H, 6.61; found: C, 71.92; H, 6.90.

Methyl (3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (15)

To a solution of compound 14 (750 mg, 0.6 mmol) and ethyl thioglycoside donor 6 (340 mg, 0.7 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (1 g) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-iodosuccinimide (240 mg, 1.0 mmol) was added to the reaction mixture and it was cooled to −10°C. To the cold reaction mixture was added TMSOTf (10 μl) and allowed to stir at −10°C for 2 h. After completion (TLC; hexane-EtOAc 3:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃ and diluted with CH₂Cl₂ (50 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (4:1) as eluant to afford pure tetrasaccharide derivative 15 (840 mg, 82%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 11.5{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,928, 2,870, 1,495, 1,454, 1,366, 1,217, 1,090, 758, 699 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.57–6.92 (m, 49 H, aromatic protons), 5.97–5.91 (t, J = 9.6 each, 1 H), 5.65–5.59 (t, J = 9.6 each, 1 H), 5.59 (br s, 1 H, PhCH), 5.44 (br s, 1 H), 5.10 (d, J = 11.4 Hz, 1 H), 4.95 (d, J = 10.5 Hz, 1 H), 4.80–4.73 (m, 4 H), 4.68–4.55 (m, 5 H), 4.50–4.42 (m, 3 H), 4.37–4.29 (m, 5 H), 4.26–4.15 (m, 3 H), 3.94–3.77 (m, 8 H), 3.63–3.56 (m, 3 H), 3.47–3.41 (m, 5 H), 3.38 (s, 3 H, OCH ₃), 3.24–3.22 (m, 2 H), 2.93–2.90 (m, 1 H), 2.12, 1.89 (2 s, 6 H, 2 COCH ₃), ¹³C NMR (CDCl₃, 75 MHz) : δ 170.1, 169.9, 167.6 (2 C), 139.6–134.1 (aromatic carbons), 131.0–123.3 (aromatic carbons), 102.6 (C-1_B), 101.8 (PhCH), 101.6 (C-1_D), 98.8 (C-1_C), 98.4 (C-1_A), 82.8, 82.1, 80.5, 79.7, 78.8 (2 C), 75.5, 75.3, 75.1 (2 C), 74.7, 74.3, 73.6 (2 C), 73.5 (2 C), 73.3, 72.9, 72.4, 69.9 (2 C), 69.6, 68.6, 68.1, 67.8, 67.7, 66.0, 55.7 (C-2_D), 55.3 (OCH₃), 20.9, 20.6; ESI-MS: m/z = 1,724.6 [M+Na]⁺; Anal. Calcd. for C₁₀₀H₁₀₃NO₂₄ (1701.7): C, 70.53; H, 6.10; found: C, 70.27; H, 6.30.

Methyl (3-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galacto-pyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (16)

To a solution of compound 15 (800 mg, 0.5 mmol) in CH₃CN (15 ml) was added HClO₄–SiO₂ (90 mg) and the reaction mixture was allowed to stir at room temperature for 20 min. After completion (TLC; hexane-EtOAc 2:1), the reaction mixture was filtered through a Celite® bed and evaporated to dryness under reduced pressure. The crude compound was passed through a short pad of SiO₂ using hexane-EtOAc (1:1) as eluant to furnish pure compound 16 (685 mg, 90%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 21.4{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,443, 2,926, 1,718, 1,638, 1,219, 1,080, 770 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.46–7.44 (m, 4 H, aromatic protons), 7.33–7.27 (m, 34 H, aromatic protons), 7.11–7.03 (m, 4 H, aromatic protons), 6.75–6.73 (m, 2 H, aromatic protons), 5.69–5.64 (t, J = 9.6 Hz each, 1 H), 5.58 (d, J = 9.4, 1 H, H-1_D), 5.57 (br s, 1 H), 5.06 (d, J = 11.4 Hz, 1 H), 4.94 (d, J = 10.5 Hz, 1 H), 4.76–4.71 (m, 3 H), 4.65–4.58 (m, 2 H), 4.56 (d, J = 3.9 Hz, 1 H, H-1_A), 4.54–4.51 (m, 2 H), 4.41 (d, J = 8.7 Hz, 1 H), 4.35 (d, J = 8.1 Hz, 1 H, H-1_C), 4.34 (d, J = 7.9 Hz, 1 H, H-1_B), 4.31–4.27 (m, 3 H), 4.19–4.10 (m, 4 H), 4.05–3.96 (m, 1 H), 3.95–3.87 (m, 1 H), 3.85–3.74 (m, 5 H), 3.68–3.60 (m, 1 H) 3.58–3.48 (m, 3 H), 3.45–3.37 (m, 4 H), 3.35 (s, 3 H, OCH ₃), 3.35–3.33 (m, 3 H) 3.21–3.17 (m, 1 H), 2.93–2.90 (m, 1 H), 2.95–2.90 (m, 1 H), 2.10, 1.88 (2 s, 6 H, 2 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz) : δ 171.1, 171.0, 168.0 (2 C), 139.6–136.9 (aromatic carbons), 128.4–126.8 (aromatic carbons), 102.5 (C-1_B), 101.7 (C-1_D), 99.1 (C-1_C), 98.4 (C-1_A), 82.8, 82.0, 81.1, 80.5, 78.7, 78.2, 77.0, 75.9, 75.6, 75.3, 75.0, 74.6, 74.2, 73.6 (3 C), 73.5, 73.4, 72.8, 71.9, 70.4, 69.9, 68.6, 67.9, 67.7, 67.6, 61.3, 55.2 (C-2_D), 54.9 (OCH₃), 21.1, 20.6; ESI-MS: m/z = 1,636.6 [M+Na]⁺; Anal. Calcd. for C₉₃H₉₉NO₂₄ (1613.6): C, 69.17; H, 6.18; found: C, 69.0; H, 6.40.

Methyl (2,3-di-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranosyl)-(1→6)-(3-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (17)

To a solution of compound 16 (650 mg, 0.4 mmol) and ethyl thioglycoside donor 8 (250 mg, 0.5 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (500 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-iodosuccinimide (180 mg, 0.8 mmol) was added to the reaction mixture and it was cooled to −30°C. TMSOTf (5 μl) was added to the cold reaction mixture and it was allowed to stir at −30°C for 1 h. After completion (TLC; hexane-EtOAc 5:2), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃, diluted with CH₂Cl₂ (50 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (2:1) as eluant to afford pure pentasaccharide derivative 17 (650 mg, 78%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} - 28.5{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,930, 2,880, 1,510, 1,457, 1,369, 1,090, 710 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.05–8.00 (m, 5 H, aromatic proton), 7.32–7.15 (m, 52 H, aromatic protons), 6.95–6.93 (m, 2 H, aromatic protons), 5.96–5.90 (t, J = 9.9 Hz each, 1 H), 5.60 (br s, 1 H, PhCH), 5.57–5.48 (m, 3 H), 5.42 (br s, 1 H), 5.09 (d, J = 11.2 Hz, 1 H), 5.02–4.96 (m, 2 H), 4.81–4.71 (m, 4 H), 4.66–4.50 (m, 7 H), 4.43–4.31 (m, 5 H), 4.30–4.14 (m, 4 H), 4.04–3.80 (m, 9 H), 3.64–3.59 (m, 3 H), 3.54–3.40 (m, 8 H), 3.38 (s, 3 H, OCH ₃), 3.24–3.23 (m, 2 H), 2.95–2.90 (m, 1 H), 2.09, 1.86 (2 s, 6 H, 2COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 171.0, 170.2, 167.9 (2 C), 165.7, 165.5, 139.6–132.8 (aromatic carbons), 129.9–126.1 (aromatic carbons), 102.5 (C-1_B), 101.9 (PhCH), 101.8 (C-1_D), 101.5 (C-1_C), 98.4 (C-1_E), 98.3 (C-1_A), 82.8, 82.2, 80.4, 79.1, 78.8 (2 C), 76.7, 75.6, 75.3, 75.1 (2 C), 74.9, 74.3, 73.6, 73.5 (2 C), 73.3 (2 C), 73.2, 72.9 (2 C), 72.8, 72.6, 72.0, 70.0, 69.9, 69.6, 69.4, 68.7, 68.4, 67.8, 66.6, 55.3 (C-2_D), 54.8 (OCH₃), 20.9, 20.6; ESI–MS: m/z = 2,094.8 [M+Na]⁺; Anal. Calcd. for C₁₂₀H₁₂₁NO₃₁ (2,071.8): C, 69.52; H, 5.88; found: C, 69.25; H, 6.0.

Methyl (2,3-di-O-benzoyl-β-d-glucopyranosyl)-(1→6)-(3,4-di-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (18)

A solution of compound 17 (600 mg, 0.3 mmol) in pyridine and acetic anhydride (10 ml; 1:1 v/v) was allowed to stir at room temperature for 5 h. The solvents were removed and co-evaporated with toluene (3 × 10 ml) under reduce pressure. To a solution of the acetylated product in CH₃CN (15 ml) was added HClO₄–SiO₂ (50 mg) and the reaction mixture was allowed to stir at room temperature for 20 min. After completion (TLC; hexane-EtOAc 2:1), the reaction mixture was filtered through a Celite® bed and evaporated to dryness under reduced pressure. The crude product was passed through a short pad of SiO₂ using hexane-EtOAc (1:1) as eluant to furnish pure compound 18 (580 mg, 95%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 26.8{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,460, 2,941, 2,890, 1,506, 1,461, 1,375, 1,092, 715 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.0–7.90 (m, 6 H, aromatic protons), 7.48–7.38 (m, 7 H, aromatic protons), 7.35–7.11 (m, 39 H, aromatic protons), 6.85–6.83 (m, 2 H, aromatic protons), 5.75–5.72 (t, J = 9.9 Hz each, 1 H), 5.58 (d, J = 8.9 Hz, 1 H, H-1_D), 5.50–5.30 (m, 3 H), 5.20–5.08 (m, 1 H), 5.03 (d, J = 11.5 Hz, 1 H), 4.90 (d, J = 11.2 Hz, 1 H), 4.84–4.72 (m, 1 H), 4.70–4.65 (m, 1 H), 4.63 (d, J = 4.1 Hz, 1 H, H-1_A), 4.61–4.59 (m, 1 H), 4.56 (d, J = 8.3 Hz, 1 H, H-1_E), 4.54–4.51 (m, 1 H), 4.49 (d, J = 8.1 Hz, 1 H, H-1_C), 4.46–4.37 (m, 2 H), 4.34 (d, J = 7.8 Hz, 1 H, H-1_B) 4.32–4.19 (m, 4 H), 4.18–4.06 (m, 4 H), 4.01–3.91 (m, 3 H), 3.88–3.70 (m, 7 H), 3.65–3.50 (m, 5 H), 3.48–3.33 (m, 7 H), 3.32 (s, 3 H, OCH ₃), 3.20–3.12 (m, 2 H), 2.88–2.80 (m, 1 H), 2.05, 1.92, 1.76 (3 s, 9 H, 3 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.5, 170.1, 169.9, 167.4 (2 C), 165.9, 165.2, 139.5-137.8 (aromatic carbons), 133.9–123.2 (aromatic carbons), 102.3 (C-1_B), 101.7 (C-1_D), 99.8 (C-1_C), 98.7 (C-1_E), 98.2 (C-1_A), 82.6, 81.9, 80.3, 79.4, 78.6, 78.5, 76.6, 76.0, 75.4, 75.2, 74.9, 74.7, 74.1, 73.6, 73.4 (2 C), 73.1, 72.7, 72.4, 72.2, 72.0, 71.5, 71.4, 70.3, 70.1, 70.0, 69.8, 69.1, 68.1, 67.6, 62.0, 61.5, 55.2 (C-2_D), 54.7 (OCH₃), 20.7, 20.6, 20.3; ESI–MS: m/z = 2,048.7 [M+Na]⁺; Anal. Calcd. for C₁₁₅H₁₁₉NO₃₂ (2025.7): C, 68.14; H, 5.92; found: C, 68.02; H, 6.11.

Methyl (2,3,5,6-tetra-O-benzoyl-β-d-galactofuranosyl)-(1→6)-(2,3-di-O-benzoyl-β-d-glucopyranosyl)-(1→6)-(3,4-di-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (19)

To a solution of compound 18 (550 mg, 0.3 mmol) and ethyl thioglycoside donor 9 (210 mg, 0.3 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (300 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-Iodosuccinimide (100 mg, 0.4 mmol) was added to the reaction mixture and it was cooled to −30°C. To the cold reaction mixture was added TMSOTf (3 μl) and the reaction mixture was allowed to stir at −30°C for 1 h. After completion (TLC; hexane-EtOAc 1:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃ and diluted with CH₂Cl₂ (50 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (1:1) as eluant to afford pure hexasaccharide derivative 19 (635 mg, 81%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 32.4{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,929, 2,876, 1,498, 1,453, 1,365, 1,217, 1,088, 699 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.20–8.07 (m, 4 H, aromatic protons), 8.00–7.88 (m, 9 H, aromatic protons), 7.50–7.42 (m, 13 H, aromatic protons), 7.38–7.05 (m, 48 H, aromatic protons), 6.10–6.09 (m, 1 H), 5.76–5.64 (m, 2 H), 5.69–5.57 (m, 2 H), 5.52 (br s, 1 H), 5.41–5.38 (m, 3 H), 5.05–4.85 (m, 6 H), 4.80–4.69 (m, 4 H), 4.59–4.47 (m, 5 H), 4.41–4.30 (m, 4 H), 4.26–4.05 (m, 8 H), 4.02–3.82 (m, 7 H), 3.80–3.71 (m, 2 H), 3.68–3.57 (m, 3 H), 3.48–3.37 (m, 7 H), 3.33 (s, 3 H, OCH₃), 3.20–3.18 (m, 2 H), 2.98–2.80 (m, 1 H), 2.06, 1.95, 1.72 (3 s, 9 H, 3COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 169.9 (2 C), 169.5, 167.2 (2 C), 166.6, 166.0, 165.7, 165.6 (2 C), 165.3, 139.4–132.8 (aromatic carbons), 129.8–123.2 (aromatic carbons), 106.2 (C-1_F), 102.2 (C-1_B), 101.7 (C-1_D), 100.9 (C-1_C), 98.2 (2 C, C-1_A and C-1_E), 82.6, 82.0, 81.9, 81.1, 80.1, 78.7, 78.6 (2 C), 77.0 (2 C), 76.6, 75.6, 75.1, 74.9 (3 C), 74.1, 73.3 (2 C), 73.1 (3 C), 72.7, 72.3, 71.8, 70.3, 70.1, 69.9, 69.7, 69.6 (2 C), 68.2, 68.0, 67.6 (2 C), 66.5, 63.7, 55.1 (C-2_D), 54.8 (OCH₃), 20.6, 20.5, 20.2; ESI–MS: m/z = 2,626.9 [M+Na]⁺; Anal. Calcd. for C₁₄₉H₁₄₅NO₄₁ (2603.9): C, 68.68; H, 5.61; found: C, 68.45; H, 5.90.

Methyl (3,4-O-isopropylidene-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (21)

To a solution of disaccharide derivative 20 (1.0 g, 1.5 mmol) in CH₃OH (20 ml) was added solid CH₃ONa until the pH of the solution became ∼10. The reaction mixture was allowed to stir at room temperature for 5 h. After neutralization with Amberlite IR-120 (H⁺) resin, the reaction mixture was filtered and evaporated to dryness. To a solution of the crude mass in DMF (5.0 ml) were added 2,2-dimethoxypropane (220 μl, 1.8 mmol) and p-toluenesulfonic acid (100 mg) and the reaction mixture was allowed to stir at 60°C for 4 h. The reaction was quenched with triethylamine (0.5 ml) and the solvents were removed under reduced pressure. To a solution of the crude mass in THF (30 ml) were added powdered NaOH (640 mg, 15.9 mmol), benzyl bromide (1.6 ml, 13.8 mmol) and tetrabutylammonium bromide (50 mg) in succession and the reaction mixture was allowed to stir vigorously at room temperature for 8 h. After completion (TLC; hexane-EtOAc 6:1), the reaction mixture was diluted with water (100 ml) and extracted with CH₂Cl₂ (150 ml). The organic layer was washed with water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (9:1) as eluant to furnish pure compound 21 (990 mg, 78%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 4.8{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,018, 2,926, 1,740, 1,366, 1,218, 1,067, 769 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.36–7.27 (m, 25 H, aromatic protons), 5.07–5.05 (m, 1 H), 5.04–5.02 (dd, J = 5.9 Hz, 1 H), 4.99 (d, J = 5.9 Hz, 1 H), 4.91–4.86 (dd, J = 9.6 and 6.9 Hz, 2 H), 4.78–4.73 (m, 2 H), 4.59–4.53 (m, 2 H), 4.50–4.10 (m, 2 H), 4.21 (d, J = 12.0 Hz, 1 H), 4.22–4.07 (m, 3 H), 3.84 (d, J = 2.4 Hz, 1 H), 3.80–3.69 (m, 3 H), 3.63–3.54 (m, 2 H), 3.54–3.37 (t, J = 7.2 Hz each, 1 H), 3.38 (s, 3 H, OCH ₃), 3.01–2.99 (m, 1 H), 2.88–2.81 (m, 1 H), 1.44 (s, 3 H, (CH ₃)₂C), 1.39 (s, 3 H, (CH ₃)₂C); ¹³C NMR (CDCl₃, 75 MHz): δ 138.9–138.2 (aromatic carbons), 128.7–126.7 (aromatic carbons), 109.8 ((CH₃)₂ C), 102.8 (C-1_B), 102.0 (C-1_A), 82.9, 81.6, 80.6, 79.3, 76.4, 75.5, 75.2 (2 C), 73.6, 73.4 (2 C), 73.2, 73.1, 68.9, 68.2, 55.6 (OCH₃), 28.0, 26.4 (CH₃)₂C); ESI-MS: m/z = 869.4 [M+Na]⁺; Anal. Calcd. for C₅₁H₅₈O₁₁ (846.4): C, 72.32; H, 6.90; found: C, 72.14; H, 7.11.

Methyl (2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (22)

A solution of compound 21 (950 mg, 1.1 mmol) in 80% aq. acetic acid (30 ml) was stirred at 80°C for 1 h. After completion (TLC; hexane-EtOAc 3:1), the reaction mixture was concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (4:1) as eluant to furnish pure compound 22 (785 mg; 87%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 3.2{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,018, 2,926, 1,740, 1,366, 1,218, 1,067, 769 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.40–7.38 (m, 2 H, aromatic protons), 7.32–7.23 (m, 23 H, aromatic protons), 5.03–4.97 (m, 2 H), 4.87 (d, J = 8.0 Hz, 1 H, H-1_B), 4.83–4.78 (m, 3 H), 4.69 (d, J = 12.0 Hz, 1 H), 4.55 (d, J = 12.0 Hz, 1 H), 4.47–4.41 (m, 4 H, H-1_A and 2 PhCH ₂), 4.06–4.02 (t, J = 8.0 Hz each, 1 H), 3.94 (br s, 1 H), 3.81 (br s, 1 H), 3.76 (br s, 1 H), 3.68–3.61 (m, 3 H), 3.53–3.49 (m, 2 H), 3.46–3.45 (m, 2 H), 3.44 (s, 3 H, OCH ₃) 3.40–3.37 (m, 1 H); ¹³C NMR (CDCl₃, 75 MHz): δ 139.1–138.1 (aromatic carbons), 128.5–127.4 (aromatic carbons), x102.8 (2 C, C-1_A and C-1_B), 82.8, 81.6, 80.1, 76.7, 75.3 (3 C), 75.1, 75.0, 73.5 (2 C), 73.2, 73.0, 68.8 (2 C), 55.6 (OCH₃); ESI-MS: m/z = 829.4 [M+Na]⁺; Anal. Calcd. for C₄₈H₅₄O₁₁ (806.4): C, 71.44; H, 6.75; found: C, 70.98; H, 6.90.

Methyl (4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (23)

To a solution of compound 22 (750 mg, 0.9 mmol) in DMF (5 ml) were added triethyl orthoacetate (920 μl, 5.0 mmol) and p-toluenesulfonic acid (50 mg) and the reaction mixture was allowed to stir at room temperature for 2 h. After completion (TLC; hexane-EtOAc 5:1), the reaction mixture was neutralized with triethylamine (0.1 ml) and evaporated to dryness. A solution of the crude mass in 80% aq. acetic acid (15 ml) was allowed to stir at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (7:1) as eluant to furnish pure compound 23 (660 mg, 86%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 11.2{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,018, 2,926, 1,740, 1,366, 1,218, 1,067, 769 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.43–7.21 (m, 25 H, aromatic protons), 5.33–5.32 (m, 1 H), 5.07–5.01 (m, 2 H), 4.88–4.73 (m, 5 H), 4.58 (d, J = 12.0 Hz, 1 H), 4.53–4.43 (m, 3 H), 4.28 (d, J = 12.0 Hz, 1 H), 4.08–4.02 (t, J = 9.0 Hz each, 1 H), 3.77–3.75 (m, 2 H), 3.70–3.65 (m, 3 H), 3.57–3.41 (m, 3 H), 3.37 (br s, 3 H, OCH ₃), 3.36–3.34 (m, 2 H), 2.06 (s, 3 H, COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.7, 139.1–127.3 (aromatic carbons), 102.9 (C-1_B), 102.5 (C-1_A), 82.6, 81.5, 80.2, 76.5, 75.3 (2 C), 75.1 (2 C), 73.4, 73.2, 72.5, 72.1, 69.8, 68.2, 67.3, 55.4; ESI-MS: m/z = 871.3 [M+Na]⁺; Anal. Calcd. for C₅₀H₅₆O₁₂ (848.4): C, 70.74; H, 6.65; found: C, 70.56; H, 6.90.

Methyl (3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (24)

To a solution of compound 23 (650 mg, 0.8 mmol) and ethyl thioglycoside donor 6 (435 mg, 0.9 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (300 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-iodosuccinimide (300 mg, 1.3 mmol) was added to the reaction mixture and it was cooled to −10°C. TMSOTf (5 μl) was added to the cold reaction mixture and it was allowed to stir at −10°C for 1 h. After completion (TLC; hexane-EtOAc 3:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃, diluted with CH₂Cl₂ (50 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (5:1) as eluant to afford pure tetrasaccharide derivative 24 (855 mg, 84%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 14.3{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,012, 2,870, 1,459, 1,370, 1,220, 1,090, 758 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.55–6.95 (m, 34 H, aromatic protons), 5.95–5.89 (t, J = 9.9 and 9.9 Hz, 1 H, H-3_C), 5.60 (d, J = 8.1 Hz, 1 H, H-1_C), 5.57 (br s, 1 H, PhCH), 5.45 (br s, 1 H), 4.95 (d, J = 11.1 Hz, 1 H), 4.81–4.72 (m, 3 H), 4.48–4.43 (m, 3 H), 4.38–4.20 (m, 6 H), 3.95–3.92 (m, 1 H), 3.90–3.72 (m, 4 H), 3.69–3.64 (m, 2 H), 3.52–3.46 (m, 5 H), 3.45–3.38 (m, 2 H), 3.36 (s, 3 H, OCH ₃), 3.18–3.12 (m, 1 H), 2.06, 1.87 (2 s, 6 H, 2 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.1, 169.9, 167.6 (2 C), 139.0–123.4 (aromatic carbons), 102.8 (PhCH), 102.0 (C-1_B), 101.6 (C-1_C), 98.9 (C-1_A), 82.7, 81.5, 78.9, 78.7, 75.8, 75.4, 75.2 (2 C), 74.9, 74.5, 73.6, 73.1, 72.4, 69.8, 69.6, 68.7, 68.1, 67.8, 66.1, 55.6 (2 C, OCH₃ and C-2_C), 20.8, 20.5 (2 COCH₃); ESI–MS: m/z = 1,292.5 [M+Na]⁺; Anal. Calcd. for C₇₃H₇₅NO₁₉ (1,269.5): C, 69.02; H, 5.95; found: C, 68.79; H, 6.20.

Methyl (3-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (25)

To a solution of compound 24 (800 mg, 0.6 mmol) in CH₃CN (10 ml) was added HClO₄–SiO₂ (50 mg) and the reaction mixture was allowed to stir at room temperature for 20 min. After completion (TLC; hexane-EtOAc 2:1), the reaction mixture was filtered through a Celite® bed and evaporated to dryness under reduced pressure. The crude product was passed through a short pad of SiO₂ using hexane-EtOAc (1:1) as eluant to furnish pure compound 25 (690 mg, 92%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 6.5{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,449, 2,930, 1,725, 1,646, 1,225, 1,090, 779 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.45–7.05 (m, 29 H, aromatic protons), 5.66–5.56 (m, 3 H), 4.94–4.90 (m, 2 H), 4.77–4.66 (m, 3 H), 4.41–4.33 (m, 2 H), 4.29 (br s, 1 H), 4.27–4.26 (m, 1 H), 4.22–4.18 (m, 2 H), 4.15–4.08 (m, 2 H), 4.03–4.01 (m, 1 H), 3.86–3.73 (m, 4 H), 3.63–3.59 (m, 1 H), 3.56–3.51 (m, 2 H), 3.44 (s, 3 H, OCH ₃), 3.41–3.32 (m, 6 H), 3.09–3.06 (m, 1 H), 2.10, 1.87 (2 s, 6 H, 2 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 169.5, 169.4, 167.5 (2 C), 137.8–136.7 (aromatic carbons), 132.2–124.8 (aromatic carbons), 101.5 (C-1_B), 100.6 (C-1_C), 97.8 (C-1_A), 81.3, 80.1, 79.9, 77.1, 75.0, 74.5, 73.7, 73.6, 73.0, 72.3, 72.2, 71.7, 70.8, 69.2, 67.5, 66.7, 66.4, 60.1, 58.8, 54.0 (C-2_C), 53.8 (OCH₃), 19.7, 19.2 (2 COCH₃); ESI–MS: m/z = 1,204.4 [M+Na]⁺; Anal. Calcd. for C₆₆H₇₁NO₁₉ (1181.5): C, 67.05; H, 6.05; found: C, 66.82; H, 6.30.

Methyl (2,3-di-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranosyl)-(1→6)-(3-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (26)

To a solution of compound 25 (600 mg, 0.5 mmol) and ethyl thioglycoside donor 8 (315 mg, 0.6 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (300 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-Iodosuccinimide (200 mg, 0.9 mmol) was added to the reaction mixture and it was cooled to −30°C. To the cold reaction mixture was added TMSOTf (5 μl) and it was allowed to stir at −30°C for 1 h. After completion (TLC; hexane-EtOAc 2:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃, diluted with CH₂Cl₂ (25 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (2:1) as eluant to afford pure tetrasaccharide derivative 26 (675 mg, 82%) as yellow oil; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 9.7{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,926, 1,730, 1,650, 1,625, 1,219, 1,096, 779 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.02–7.96 (m, 4 H, aromatic protons), 7.55–6.19 (m, 40 H, aromatic protons), 5.60–5.57 (t, J = 8.7 Hz each, 1 H), 5.57–5.54 (m, 2 H), 5.53–5.45 (m, 2 H), 5.41–5.40 (m, 1 H), 4.97 (br s, 1 H), 4.95–4.93 (m, 1 H), 4.92–4.82 (ABq, J = 11.4 Hz, 2 H), 4.80–4.70 (m, 2 H), 4.51–4.47 (m, 2 H), 4.35–4.24 (m, 4 H), 4.23–4.13 (m, 5 H), 3.97–3.89 (m, 5 H), 3.88–3.75 (m, 2 H), 3.61–3.47 (m, 5 H), 3.46 (br s, 3 H, OCH ₃), 3.38–3.34 (m, 3 H), 3.15–3.14 (m, 1 H), 2.06, 1.85 (2 s, 6 H, 2 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 171.1, 170.2, 168.2 (2 C), 165.6, 165.5, 138.8-123.3 (aromatic carbons), 102.5 (PhCH), 102.0 (C-1_B), 101.9 (C-1_A), 101.5 (C-1_C), 98.2 (C-1_D), 85.5, 82.5, 81.4, 79.3, 78.9, 78.7, 77.3, 75.7, 75.2, 74.9, 74.6, 74.2, 73.5, 73.1, 73.0, 72.7, 72.5, 71.8, 70.3, 69.5, 68.5, 68.1, 67.7, 66.6, 56.8 (C-2_C), 54.7 (OCH₃), 20.9, 20.8 (2 COCH₃); ESI–MS: m/z = 1662.6 [M+Na]⁺; Anal. Calcd. for C₉₃H₉₃NO₂₆ (1,639.6): C, 68.08; H, 5.71; found: C, 67.85; H, 6.00.

Methyl (2,3-di-O-benzoyl-β-d-glucopyranosyl)-(1→6)-(3-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (27)

To a solution of compound 26 (600 mg, 0.4 mmol) in CH₃CN (10 ml) was added HClO₄-SiO₂ (50 mg) and the reaction mixture was allowed to stir at room temperature for 20 min. After completion (TLC; hexane-EtOAc 1:1), the reaction mixture was filtered through a Celite® bed and evaporated to dryness under reduced pressure. The crude product was passed through a short pad of SiO₂ to furnish pure compound 27 (555 mg, 89%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} + 18.7{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 3,443, 2,930, 2,875, 1,710, 1,652, 1,454, 1,370, 1,226, 1,100, 775 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.99–7.96 (m, 4 H, aromatic protons), 7.51–7.16 (m, 32 H, aromatic protons), 6.90–6.88 (m, 3 H, aromatic protons), 5.87–5.80 (m, 1 H), 5.61–5.58 (m, 1 H), 5.55–5.48 (m, 2 H), 5.43–5.40 (m, 2 H), 4.95–4.84 (m, 4 H), 4.75 (d, J = 10.8 Hz, 1 H), 4.68–4.63 (m, 2 H), 4.46–4.39 (m, 1 H), 4.35–4.27 (m, 3 H), 4.21–4.12 (m, 4 H), 4.05–4.02 (m, 2 H), 3.92–3.86 (m, 2 H), 3.81–3.78 (m, 2 H), 3.70–3.60 (m, 3 H), 3.56–3.50 (m, 2 H), 3.46–3.41 (m, 3 H), 3.36 (s, 3 H, OCH ₃), 3.35–3.31 (m, 2 H), 3.09–3.06 (m, 1 H), 2.04, 1.82 (2 s, 6 H, 2 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.1, 170.0, 168.0 (2 C), 167.0, 165.8, 139.0–123.2 (aromatic carbons), 102.5 (C-1_B), 101.8 (C-1_A), 100.7 (C-1_C), 98.6 (C-1_D), 82.4, 81.3, 79.2, 78.9, 76.1, 75.6, 75.1 (2 C), 74.9, 74.7, 74.5, 73.5, 73.1, 73.0 (2 C), 72.5, 72.1, 70.1, 69.7, 69.4, 68.5, 68.2, 67.6, 62.0, 56.7 (C-2_C), 54.3 (OCH₃), 20.8, 20.5 (2 COCH₃); ESI–MS: m/z = 1,574.5 [M+Na]⁺; Anal. Calcd. for C₈₆H₈₉NO₂₆ (1,551.5): C, 66.53; H, 5.78; found: C, 66.25; H, 6.00.

Methyl (2,3,5,6-tetra-O-benzoyl-β-d-galactofuranosyl)-(1→6)-(2,3-di-O-benzoyl-β-d-glucopyranosyl)-(1→6)-(3-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(4-O-acetyl-2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (28)

To a solution of tetrasaccharide acceptor 27 (500 mg, 0.3 mmol) and ethyl thioglycoside donor 9 (230 mg, 0.4 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (500 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-Iodosuccinimide (230 mg, 0.4 mmol) was added to the reaction mixture and it was cooled to −30°C. To the cold reaction mixture was added TMSOTf (3 μl) and it was allowed to stir at −30°C for 1 h. After completion (TLC; hexane-EtOAc 1:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃, diluted with CH₂Cl₂ (25 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (1:1) to afford pure pentasaccharide derivative 28 (510 mg, 79%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} + 12.9{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,929, 2,376, 1,765, 1,723, 1,660, 1,370, 1,229, 1,082, 770 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.08–7.72 (m, 12 H, aromatic protons), 7.53–6.90 (m, 47 H, aromatic protons), 5.84–5.78 (m, 1 H), 5.62–5.52 (m, 3 H), 5.48–5.32 (m, 4 H), 4.95–4.82 (m, 4 H), 4.77–4.63 (m, 4 H), 4.55–4.18 (m, 8 H), 4.17–4.11 (m, 4 H), 4.02–3.86 (m, 4 H), 3.79–3.75 (m, 1 H), 3.68–3.47 (m, 7 H), 3.43 (s, 3 H, OCH ₃), 3.37–3.32 (m, 4 H), 3.12–3.09 (m, 1 H), 1.98, 1.83 (2 s, 6 H, 2 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.3, 170.0, 167.8 (2 C), 165.4 (2 C), 165.1 (2 C), 165.0 (2 C), 138.8–122.8 (aromatic carbons), 105.7 (C-1_E), 102.1 (C-1_B), 101.5 (C-1_A), 100.5 (C-1_C), 97.7 (C-1_D), 85.0, 82.2, 82.1, 81.8, 80.9, 78.7, 78.6, 75.6, 75.2, 74.7 (2 C), 74.5, 74.2, 73.8, 73.0, 72.6 (2 C), 72.1, 71.7, 71.0, 70.8, 70.6, 69.7, 69.1, 67.9, 67.8, 67.3, 63.3, 63.1, 56.3 (C-2_C), 54.5 (OCH₃), 20.3, 20.1; ESI-MS: m/z = 2,152.7 [M+Na]⁺; Anal. Calcd. for C₁₂₀H₁₁₅NO₃₅ (2129.7): C, 67.63; H, 5.44; found: C, 67.40; H, 5.75.

Methyl (3,4,6-tri-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)-(2,6-di-O-benzyl-β-d-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (29)

To a solution of compound 22 (700 mg, 0.9 mmol) and ethyl thioglycoside donor 5 (480 mg, 1.0 mmol) in anhydrous CH₂Cl₂ (10 ml) was added powdered MS 4 Å (500 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-iodosuccinimide (270 mg, 1.2 mmol) was added to the reaction mixture and it was cooled to −30°C. To the cold reaction mixture was added TMSOTf (5 μl) and it was allowed to stir at −30°C for 1 h. After completion (TLC; hexane-EtOAc 4:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃ and diluted with CH₂Cl₂ (50 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (5:1) as eluant to afford pure trisaccharide derivative 29 (520 mg, 47%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} + 23.4{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,928, 2,870, 1,705, 1,495, 1,454, 1,366, 1,217, 1,090, 758, 699 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.70–7.68 (m, 29 H, aromatic protons), 5.83–5.76 (t, J = 9.3 and 1.5 Hz, 1 H, H-3_C), 5.63 (d, J = 8.4 Hz, 1 H, H-1_C), 5.34–5.27 (t, J = 9.9 Hz each, 1 H, H-4_C), 5.03 (d, J = 10.8 Hz, 1 H), 4.97 (d, J = 11.1 Hz, 1 H), 4.82 (d, J = 11.1 Hz, 1 H), 4.74–4.69 (m, 2 H), 4.48–4.36 (m, 4 H), 4.34–4.23 (m, 3 H), 4.07 (br s, 1 H), 3.97–3.92 (t, J = 8.4 Hz each, 1 H), 3.84–3.72 (m, 3 H), 3.68–3.63 (m, 1 H), 3.60–3.52 (m, 7 H), 3.48 (s, 3 H, OCH ₃), 3.47–3.44 (m, 2 H), 3.19–3.16 (m, 1 H), 2.06, 1.92, 1.87 (3 s, 9 H, 3 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.2 (2 C), 169.1, 167.8 (2 C), 139.5–123.8 (aromatic carbons), 103.2 (C-1_B), 102.3 (C-1_A), 99.1 (C-1_C), 84.2, 83.2, 81.9, 78.5, 76.2, 75.9, 75.7, 75.4, 75.1, 74.9, 74.6, 74.5, 73.8, 73.4, 71.2, 69.6, 69.1, 64.5, 62.8, 55.7 (C-2″), 55.2 (OCH₃), 20.9, 20.8, 19.6; ESI–MS: m/z = 1,246.5 [M+Na]⁺; Anal. Calcd. for C₆₈H₇₃NO₂₀ (1,223.5): C, 66.71; H, 6.01; found: C, 66.48; H, 6.30.

Methyl [(3,4,6-tri-O-acetyl-2-deoxy-2-N-phthalimido-β-d-glucopyranosyl)-(1→3)]-(2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl)-(1→4)-(2,6-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (30)

To a solution of compound 29 (500 mg, 0.4 mmol) and ethyl thioglycoside donor 7 (290 mg, 0.5 mmol) in anhydrous CH₂Cl₂ (5 ml) was added powdered MS 4 Å (500 mg) and the reaction mixture was allowed to stir at room temperature under argon for 1 h. N-Iodosuccinimide (170 mg, 0.8 mmol) was added to the reaction mixture and it was cooled to −30°C. To the cold reaction mixture was added TMSOTf (3 μl) and it was allowed to stir at −30°C for 1 h. After completion (TLC; hexane-EtOAc 4:1), the reaction mixture was quenched by adding 5% aq. Na₂S₂O₃, diluted with CH₂Cl₂ (30 ml). The organic layer was washed successively with aq. NaHCO₃ and water, dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified over SiO₂ using hexane-EtOAc (2:1) as eluant to afford pure tetrasaccharide derivative 30 (250 mg, 35%) as yellow oil; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} + 9.3{\left( {c\,1.0,\,{\text{CHCl}}_{3} } \right)}$; IR (neat): 2,928, 2,870, 1,705, 1,495, 1,454, 1,366, 1,217, 1,090, 758, 699 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): 7.44–7.42 (m, 5 H, aromatic protons), 7.35–7.22 (m, 32 H, aromatic protons), 7.09–7.06 (m, 8 H, aromatic protons), 6.92–6.89 (m, 2 H, aromatic protons), 5.65–5.62 (m, 2 H, aromatic protons), 5.69–5.60 (m, 2 H, H-1_C and H-3_C), 5.13 (d, J = 3.0 Hz, 1 H, H-1_D), 4.98–4.95 (d, J = 11.4 Hz, 1 H), 4.88–4.84 (d, J = 11.1 Hz, 1 H), 4.81–4.77 (d, J = 10.5 Hz, 1 H, H-1_B), 4.70 (br s, 1 H), 4.69–4.57 (m, 6 H), 4.54–4.42 (m, 5 H), 4.37–4.24 (m, 6 H), 4.23–4.17 (m, 3 H), 4.15–4.06 (m, 1 H), 3.95–3.79 (m, 6 H), 3.78–3.65 (m, 2 H), 3.60–3.47 (m, 6 H), 3.46 (s, 3 H, OCH ₃), 3.44–3.42 (m, 3 H), 3.10–3.02 (m, 1 H), 1.93, 1.91, 1.81 (3 s, 9 H, 3 COCH ₃); ¹³C NMR (CDCl₃, 75 MHz): δ 170.1 (3 C), 167.2 (2 C), 138.5–122.9 (aromatic carbons), 102.7 (C-1_B), 102.4 (C-1_A), 101.0 (C-1_C), 99.2 (C-1_D), 83.0, 82.3, 82.1, 81.2, 80.6, 78.7, 78.1, 78.0, 77.8, 76.4, 75.3, 75.2, 75.1, 74.8, 74.7, 74.1, 73.7, 73.7, 73.4, 73.1, 73.0, 71.0, 70.0, 68.7, 68.6, 67.6, 66.9, 65.8, 56.7 (OCH₃), 55.4 (C-2_C), 20.5 (3 C); ESI-MS: m/z = 1,768.7 [M+Na]⁺; Anal. Calcd. for C₁₀₂H₁₀₇NO₂₅ (1745.7): C, 70.13; H, 6.17; found: C, 69.91; H, 6.39.

Methyl (β-d-galactofuranosyl)-(1→6)-(β-d-glucopyranosyl)-(1→6)-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-(β-d-galactopyranosyl)-(1→4)-(β-d-glucopyranosyl)-(1→4)-α-d-glucopyranoside (1)

To a solution of the hexasaccharide derivative 19 (600 mg, 0.2 mmol) in EtOH (5.0 ml) was added hydrazine hydrate (1.0 ml) and the reaction mixture was allowed to stir at 80°C for 8 h. The solvents were removed and the crude mass was treated with pyridine (2.0 ml) and acetic anhydride (2.0 ml) for 2 h at room temperature, concentrated and purified over SiO₂ using toluene-EtOAc (1:1) as eluant. To a solution of the N-acetylated hexasaccharide derivative in CH₃OH (10 ml) was added solid CH₃ONa until the pH of the solution reached ∼10. The reaction mixture was allowed to stir at room temperature for 5 h and neutralized with Amberlite IR-120 (H⁺) resin. The reaction mixture was filtered and evaporated to dryness. To a solution of the crude product in CH₃OH (5 ml) was added 20% Pd(OH)₂-C (100 mg) and the reaction mixture was allowed to stir at room temperature under a positive pressure of hydrogen for 12 h. After completion (TLC; CH₃CN-AcOH-H₂O 10:5:1), the reaction mixture was filtered through a Celite® bed and concentrated to a white powder, which was further purified through a Sephadex LH-20 column using CH₃OH-H₂O (4:1) as eluant to furnish pure hexasaccharide 1 (170 mg, 69%) as an amorphous powder; ${\left[ \alpha \right]}^{{{\text{25}}}}_{{\text{D}}} - 35.6{\left( {c\,1.0,\,{\text{H}}_{2} {\text{O}}} \right)}$; IR (KBr): 3,447, 3,062, 3,030, 2,922, 2,365, 1,363, 1,055, 699 cm⁻¹; ¹H NMR (D₂O, 400 MHz): δ 5.02 (d, J = 1.2 Hz, 1 H, H-1_F), 4.82 (d, J = 8.4 Hz, 1 H, H-1_D), 4.77 (d, J = 4.0 Hz, 1 H, H-1_A), 4.50 (d, J = 8.0 Hz, 1 H, H-1_C), 4.48 (d, J = 8.0 Hz, 1 H, H-1_B), 4.42 (d, J = 7.6 Hz, 1 H, H-1_E), 4.22–4.13 (m, 2 H, H-2_F and H-3_D), 4.11–4.00 (m, 3 H, H-3_F, H-4_E and H-6_aF), 3.98–3.95 (m, 1 H, H-4_A), 3.94–3.86 (m, 4 H, H-2_E, H-3_A, H-4_B and H-6_bB), 3.84–3.77 (m, 3 H, H-3_B, H-4_D and H-6_aB), 3.76–3.65 (m, 10 H, H-2_A, H-3_C, H-4_C, H-4_F, H-5_A, H-6_a,bA, H-6_a,bD and H-6_bF), 3.64–3.53 (m, 10 H, H-3_E, H-5_B, H-5_C, H-5_D, H-5_E, H-5_F, H-6_a,bC and H-6_a,bE), 3.38 (s, 3 H, OCH ₃), 3.33–3.28 (m, 3 H, H-2_C, H-2_B and H-2_D), 1.87 (s, 3 H, NHCOCH ₃); ¹³C NMR (D₂O, 100 MHz): δ 172.6 (NHCOCH₃), 107.5 (C-1_F), 102.7 (C-1_B), 102.3 (C-1_D), 101.8 (C-1_C), 101.7 (C-1_E), 98.4 (C-1_A), 82.4 (C-3_F), 81.7 (C-4_B), 80.4 (C-3_D), 78.0 (C-5_A), 77.7 (C-5_C), 76.2 (C-4_E), 75.1 (C-4_F), 74.5 (C-2_A), 74.4 (C-5_B), 74.3 (C-5_D), 74.2 (C-5_E), 73.5 (C-5_F), 72.6 (C-2_C), 72.3 (C-2_B), 70.4 (2 C, C-3_E and C-3_C), 70.3 (C-4_D), 69.7 (C-3_B), 69.3, (C-4_A), 69.2 (C-3_A), 69.0 (C-2_E), 68.0 (C-4_C), 67.8 (C-2_F), 66.5 (C-6_E), 62.2 (2 C, C-6_A and C-6_D), 60.5 (C-6_F), 59.4 (C-6_C), 59.3 (C-6_B), 55.9 (C-2_D), 54.6 (OCH₃), 19.5 (NHCOCH₃); ESI–MS: m/z = 1,068.3 [M+Na]⁺; Anal. Calcd. for C₃₉H₆₇NO₃₁ (1,045.4): C, 44.78; H, 6.46; found: C, 44.60; H, 6.80.

Methyl (β-d-galactofuranosyl)-(1→6)-(β-d-glucopyranosyl)-(1→6)-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-(β-d-galactopyranosyl)-(1→4)-β-d-glucopyranoside (2)

To a solution of pentasaccharide derivative 28 (500 mg, 0.2 mmol) in EtOH (5.0 ml) was added hydrazine hydrate (1.0 ml) and the reaction mixture was stirred at 80°C for 8 h. The solvents were removed and the crude mass was treated with pyridine (2 ml) and acetic anhydride (2 ml) at room temperature for 2 h. The reaction mixture was concentrated and purified over SiO₂ using toluene-EtOAc (1:1) as eluant. To a solution of the N-acetylated pentasaccharide derivative in CH₃OH (10 ml) was added solid CH₃ONa until the pH of the solution reached ∼10. The reaction mixture was allowed to stir at room temperature for 5 h and neutralized with Amberlite IR-120 (H⁺) resin. The reaction mixture was filtered and evaporated to dryness. To a solution of the crude product in CH₃OH (10 ml) was added 20% Pd(OH)₂-C (100 mg) and the reaction mixture was allowed to stir at room temperature under a positive pressure of hydrogen for 12 h. After completion (TLC; CH₃CN-AcOH-H₂O 10:5:1), the reaction mixture was filtered through a Celite® bed and concentrated to a white mass, which was further purified through a Sephadex LH-20 column using CH₃OH-H₂O (4:1) as eluant to furnish pure pentasaccharide 2 (110 mg, 66%) as an amorphous powder; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 32.3{\left( {c\,1.0,\,{\text{H}}_{2} {\text{O}}} \right)}$); IR (KBr): 3,018, 2,926, 1,740, 1,366, 1,218, 1,067, 769 cm⁻¹; ¹H NMR (D₂O, 400 MHz): δ 4.98 (d, J = 1.2 Hz, 1 H, H-1_E), 4.77 (d, J = 8.4 Hz, 1 H, H-1_C), 4.46 (d, J = 8.0 Hz, 1 H, H-1_D), 4.44 (d, J = 8.0 Hz, 1 H, H-1_B), 4.38 (d, J = 7.6 Hz, 1 H, H-1_A), 4.07–3.96 (m, 2 H, H-2_E and H-3_C), 3.90–3.87 (m, 1 H, H-3_E), 3.85–3.77 (m, 3 H, H-4_D and H-6_abE), 3.76–3.70 (m, 2 H, H-2_D and H-6_aA), 3.68–3.60 (m, 9 H, H-3_A, H-3_B, H-4_A, H-4_B, H-4_C, H-4_E, H-6_bA and H-6_abC ), 3.58–3.45 (m, 11 H, H-2_A, H-3_D, H-5_A, H-5_B, H-5_C, H-5_D, H-5_E, H-6_abB and H-6_abD), 3.29 (s, 3 H, OCH ₃), 3.20-3.12 (m, 1 H, H-2_C), 3.10-3.02 (m, 1 H, H-2_B), 1.77 (s, 3 H, NHCOCH ₃); ¹³C NMR (D₂O, 100 MHz): δ 172.5 (NHCOCH₃), 107.0 (C-1_E), 102.2 (2 C, C-1_A and C-1_B), 101.8 (C-1_C), 100.3 (C-1_D), 82.2, 80.3, 77.6, 76.1, 75.6, 75.3, 75.0, 74.3, 74.2, 74.0, 73.5, 73.4, 72.5 (2 C), 72.0, 71.5, 71.4, 70.9, 69.9, 69.1, 68.9, 68.8, 62.2, 61.9, 55.3 (C-2_C), 55.2 (OCH₃), 19.6 (NHCOCH₃); ESI–MS: m/z = 906.3 [M+Na]⁺; Anal. Calcd. for C₃₃H₅₇NO₂₆ (883.3): C, 44.85; H, 6.50; found: C, 44.65; H, 6.88.

Methyl [(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)]-(α-d-glucopyranosyl)-(1→4)-(β-d-galactopyranosyl)-(1→4)-β-d-glucopyranoside (3)

To a solution of tetrasaccharide derivative 30 (220 mg, 0.1 mmol) in EtOH (5.0 ml) was added hydrazine hydrate (700 μl) and the reaction mixture was stirred at 80°C for 8 h. The solvents were removed and the crude mass was treated with pyridine (2.0 ml) and acetic anhydride (2.0 ml) at room temperature for 2 h, concentrated and purified over SiO₂ using toluene-EtOAc (1:1) as eluant. To a solution of the N-acetylated tetrasaccharide derivative in CH₃OH (5 ml) was added solid CH₃ONa until the pH of the solution reached ∼10. The reaction mixture was allowed to stir at room temperature for 5 h, and neutralized with Amberlite IR-120 (H⁺) resin. The reaction mixture was filtered and evaporated to dryness. To a solution of the crude product in CH₃OH (5 ml) was added 20% Pd(OH)₂-C (80 mg) and the reaction mixture was allowed to stir at room temperature under a positive pressure of hydrogen for 12 h. After completion (TLC; CH₃CN-AcOH-H₂O 10:5:1), the reaction mixture was filtered through a Celite® bed and concentrated to a white powder, which was further purified through a Sephadex LH-20 column using CH₃OH-H₂O (4:1) as eluant to furnish pure tetrasaccharide 3 (52 mg, 70%) as amorphous powder; ${\left[ \alpha \right]}^{{25}}_{{\text{D}}} + 13.5{\left( {c\,1.0,\,{\text{H}}_{2} {\text{O}}} \right)}$; IR (KBr): 3,018, 2,926, 1,740, 1,366, 1,218, 1,067, 769 cm⁻¹; ¹H NMR (D₂O, 300 MHz): δ 4.80 (d, J = 8.1 Hz, 1 H, H-1_C), 4.73 (d, J = 4.0 Hz, 1 H, H-1_D), 4.62 (d, J = 8.2 Hz, 1 H, H-1_B), 4.32 (d, J = 7.5 Hz, 1 H, H-1_A), 3.79–3.72 (m, 4 H), 3.70–3.52 (m, 14 H), 3.37–3.35 (m, 4 H), 3.33 (s, 3 H, OCH ₃), 3.32–3.30 (m, 2 H), 1.87 (s, 3 H, NHCOCH ₃); ¹³C NMR (D₂O, 75 MHz): δ 170.9, 101.9 (C-1_B), 101.7 (C-1_A), 100.8 (C-1_C), 100.0 (C-1_D), 82.3, 79.2, 76.9, 76.5 (2 C), 76.3, 75.6, 75.1, 73.6, 72.4, 71.3, 71.2, 70.8, 70.5, 70.2, 61.6, 61.2, 60.9, 60.8, 56.9 (OCH₃), 56.5 (C-2″), 21.6 (NHCOCH₃); ESI–MS: m/z = 744.2 [M+Na]⁺; Anal. Calcd. for C₂₇H₄₇NO₂₁ (721.3): C, 44.94; H, 6.56; found: C, 44.81; H, 6.72.