Concise synthesis of a tetra- and a trisaccharide related to the repeating unit of the O-antigen from Providencia rustigianii O34 in the form of their p-methoxyphenyl glycosides

Abstract

Concise synthesis of a tetra- and a trisaccharide related to the repeating unit of the O-antigen from Providencia rustigianii O34 is reported. Synthesis of the target oligosaccharides was achieved by rational protecting group manipulations on the commercially available monosaccharides followed by stereoselective glycosylations, using H₂SO₄-silica for trichloroacetimidate activation and in conjunction with N-iodosuccinimide for thioglycoside activation.

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Introduction

Bacterial O-antigens are important for bacterial pathogenesis as they are exposed at the outer surface of the bacterial cell surface and are thus responsible for binding with the receptors of the host cells. Consequently, they become the target for host antibodies. Therefore, the repeating units of the bacterial O-antigens are good targets for designing synthetic carbohydrate vaccines against bacterial attack.¹ Since carbohydrates are more stable than proteins, they are used in various commonly used vaccines.² Synthetic oligosaccharide vaccines are also promising against diseases like influenza,³ malaria⁴ and cancer.⁵

Providencia, the genera belonging to the Proteeae tribe of the gram-negative bacterial family Enterobacteriaceae causes several types of enteric diseases, wound and urinary tract infections. Providencia rustigianii is one among the six species of the genus Providencia.⁶ Recently Knirel et al. reported the detailed structure of a heptasaccharide repeating unit of the O-antigen from P. rustigianii O34 from its lipopolysaccharide.⁷

Herein, we report the chemical synthesis of a tetra- and a trisaccharide related to the heptasaccharide repeating unit of the O-antigen from P. rustigianii O34 (Fig. 1). Syntheses of the tetra- and trisaccharides are planned to find the immunogenic part of the entire heptasaccharide repeating unit from P. rustigianni O34. The oligosaccharides were synthesized in the form of their p-methoxyphenyl glycosides. This type of glycoside may be cleaved selectively from the per-O-acetylated form of the target oligosaccharides and further glyco-conjugates can be prepared with suitable aglycons, as required for the vaccine candidates, by using well-established trichloroacetimidate chemistry. The advantage of using a p-methoxyphenyl glycoside is the fact that it remains stable during routine protecting group manipulations.

Fig. 1 Structures of the target tetrasaccharide (1) and the trisaccharide (2).

Results and discussion

The synthesis of the tetrasaccharide 1 commenced with the glycosylation between known glucosamine acceptor 3⁸ and the trichloroacetimidate donor 4⁹ in the presence of H₂SO₄-silica.¹⁰ Reaction in CH₂Cl₂ at 0–5 °C afforded the desired β-linked disaccharide 5 in 91% yield. Zemplén de-O-acetylation¹¹ of the disaccharide 5 gave the disaccharide acceptor 6 in 96% yield. Synthesis of the L-fucose donor started with known p-tolyl 1-thio-β-L-fucopyranoside 7.¹² Reaction of 7 with 2,2-dimethoxy propane (DMP) in the presence of 10-camphorsulfonic acid (CSA)¹³ resulted in the corresponding 3,4-O-isopropylidene derivative 8 in 90% yield. The 2-OH group was later protected by a 4-methoxybenzyl group by using 4-methoxybenzyl chloride in the presence of NaH in DMF¹⁴ to afford the suitably protected fucosyl donor 9 in 83% yield. Glycosylation of donor 9 with the disaccharide acceptor 6 was achieved by N-iodosuccinimide (NIS) in the presence of H₂SO₄-silica in CH₂Cl₂ at −40 °C. The desired 1,2-cis linked trisaccharide 10 was obtained in 83% yield. No trace of the 1,2-trans isomer was evident after chromatographic purification. Further, oxidative cleavage of the 4-methoxybenzyl group using DDQ¹⁵ gave the trisaccharide acceptor 11 in 81% yield. Final glycosylation of the trisaccharide acceptor 11 with the known mannosyl donor 12¹⁶ using NIS in the presence of H₂SO₄-silica in CH₂Cl₂ at 5–10 °C afforded the fully protected tetrasaccharide 13 in 84% yield. The N-phthalimido group was de-protected using ethylene diamine in n-butanol¹⁷ and sequentially acetylated using Ac₂O in pyridine. Hydrolysis of the acetals by 80% acetic acid at 80 °C¹⁸ followed by catalytic hydrogenation and Zemplén de-O-acetylation gave the target tetrasaccharide 1 in 63% yield over four steps (Scheme 1).

Scheme 1 Synthesis of the tetrasaccharide 1.

Synthesis of the trisaccharide 2 was started with p-methoxyphenyl β-L-fucopyranoside 14. Reaction of 14 with DMP in the presence of CSA in acetone furnished the corresponding 3,4-O-isopropylidene derivative 15 in 92% yield. The 2-OH group was benzylated using BnBr and NaH in DMF to afford fully protected compound 16 in 90% yield. Hydrolysis of the isopropylidene group using 80% AcOH at 80 °C afforded the diol 17 in 94% yield. Selective protection of the 3-OH group with the 4-methoxybenzyl group was achieved using stannylene chemistry¹⁹ to give the suitably protected fucosyl acceptor 18 in 83% yield. Glycosylation of the acceptor 18 with known glucosyl donor 19²⁰ with NIS in the presence of H₂SO₄-silica afforded the required 1,2-trans disaccharide 20 in 85% yield. Further, oxidative cleavage of the 4-methoxybenzyl group using DDQ gave the disaccharide acceptor 21 in 81% yield. Final glycosylation with the trichloroacetimidate donor 22 in the presence of H₂SO₄-silica afforded the fully protected trisaccharide 23 in 80% yield. Only the 1,2-cis glycoside was evident after chromatographic purification. The azido functionality was converted to the desired acetamido group by treating the trisaccharide 23 with thiolacetic acid for 48 h at room temperature.²¹ Hydrolysis of the benzylidene acetal using 80% AcOH at 80 °C followed by catalytic hydrogenation and de-O-acetylation afforded the target trisaccharide 2 in 67% yield over four steps (Scheme 2).

Scheme 2 Synthesis of the trisaccharide 2.

Conclusion

In conclusion, we have reported the synthesis of a tetra- and a trisaccharide related to the repeating unit of the O-antigen from Providencia rustigianii O34. The synthesized oligosaccharides are in the form of their p-methoxyphenyl glycosides. After global de-protection and per-O-acetylation, the p-methoxyphenyl group can be removed selectively and further glyco-conjugate formation can be achieved through trichloroacetimidate chemistry.

Experimental section

General methods

All reagents and solvents were dried prior to use according to standard methods.²² Commercial reagents were used without further purification unless otherwise stated. Analytical TLC was performed on silica gel 60-F₂₅₄ (Merck) with detection by fluorescence and/or by charring following immersion in a 10% ethanolic solution of sulfuric acid. Flash chromatography was performed with silica gel 230–400 mesh (Qualigens, India). Optical rotations were measured at the sodium D-line at ambient temperature, with a Perkin Elmer 141 polarimeter. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance spectrometer at 500 and 125 MHz, respectively, using Me₄Si or CH₃OH as internal standards, as appropriate.

p-Methoxyphenyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (5). A mixture of the acceptor 3 (1.0 g, 2.0 mmol), donor 4 (2.5 g, 4.0 mmol) and MS 4 Å (2.0 g) in dry CH₂Cl₂ (25 mL) was stirred under a nitrogen atmosphere for 30 min. The mixture was cooled to 5–10 °C in a ice-water bath and H₂SO₄-silica (50 mg) was added. After stirring for 45 min, TLC (n-hexane–EtOAc, 2 : 1) showed complete consumption of the donor. The mixture was neutralized with Et₃N and filtered. The filtrate was evaporated in vacuo and the residue was purified by flash chromatography using n-hexane–EtOAc, 2 : 1 as eluent to afford pure disaccharide 5 (1.75 g, 91%) as a light yellow foam. [α]²⁵_D +101 (c 1.1, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.76–6.72 (m, 28H, ArH), 5.65 (d, 1H, J_1,2 = 8.5 Hz, H-1), 5.37 (s, 1H, CHPh), 4.80 (dd, 1H, J_1′,2′ = 8.0 Hz, J_2′,3′ 9.5 Hz, H-2′), 4.77 (dd, 1H, J_2,3 = 8.5 Hz, J_3,4 = 10.0 Hz, H-3), 4.65, 4.63 (2d, 2H, J = 11.0 Hz, CH₂Ph), 4.57 (dd, 1H, J_1,2 = 8.5 Hz, J_2,3 = 9.5 Hz, H-2), 4.46 (m, 5H, H-1′, 2 × CH₂Ph), 4.32 (dd, 1H, J_5,6a = 5.5 Hz, J_6a,6b = 11.0 Hz, H-6a), 3.91 (t, 1H, J_2′,3′, J_3′,4′ = 9.5 Hz, H-3′), 3.80 (t, 1H, J_5,6b, J_6a,6b = 11.0 Hz, H-6b), 3.71 (s, 3H, C₆H₄OCH₃), 3.68 (m, 1H, H-5), 3.59 (t, 1H, J_3′,4′, J_4′,5′ = 9.5 Hz, H-4′), 3.41 (m, 2H, H-6a′, H-6b′), 3.38 (t, 1H, J_3,4, J_4,5 = 9.5 Hz, H-4), 3.05 (m, 1H, H-5′), 1.35 (s, 3H, COCH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 168.8 (COCH₃), 155.5, 150.6, 138.2, 138.1, 137.8, 137.1, 134.2, 131.7, 129.2, 128.4(2), 128.3(2), 128.2(3), 128.0(2), 127.8(2), 127.7, 127.5(2), 126.2, 118.5(2), 114.5(2) (ArC), 101.5 (CHPh), 99.9 (C-1′), 97.9 (C-1), 82.9, 80.8, 77.6, 75.0, 74.9, 74.6, 74.5, 73.3, 73.2, 68.6, 68.1, 66.6, 55.6 (C₆H₄OCH₃), 55.3 (C-2), 20.1 (COCH₃). HRMS calcd. for C₅₇H₅₅O₁₄NNa (M+Na)⁺: 1000.3520; found: 1000.3518.

p-Methoxyphenyl 3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (6). To a solution of the disaccharide 5 (1.7 g, 1.7 mmol) in dry MeOH (30 mL), NaOMe (3 mL, 0.5M in MeOH) was added and the solution was stirred at room temperature for 2 h. After neutralizing the solution with DOWEX 50W H⁺ resin, the mixture was filtered and the filtrate was evaporated in vacuo. The syrupy residue thus obtained was purified by flash chromatography using n-hexane–EtOAc, 1 : 1 as the eluent to afford pure disaccharide 5 (1.55 g, 96%) as a colourless glass. [α]²⁵_D +153 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.66–6.71 (m, 28H, ArH), 5.86 (d, 1H, J_1,2 = 8.5 Hz, H-1), 5.61 (s, 1H, CHPh), 4.85 (t, 1H, J_2,3, J_3,4 = 8.5 Hz, H-3), 4.66, 4.64 (2d, 2H, J = 11.0 Hz, CH₂Ph), 4.54 (t, 1H, J_1,2, J_2,3 = 8.5 Hz, H-2), 4.51 (d, 1H, J = 11.0 Hz, CH₂Ph), 4.42 (dd, 1H, J_5,6a = 5.5 Hz, J_6a,6b = 10.5 Hz, H-6a), 4.35 (d, 1H, J = 11.0 Hz, CH₂Ph), 4.28 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.16, 4.04 (2d, 2H, J = 11.0 Hz, CH₂Ph),3.87 (m, 2H, H-4, H-6b), 3.78 (m, 1H, H-5), 3.71 (s, 3H, C₆H₄OCH₃), 3.49 (m, 2H, H-2′, H-4′), 3.31 (t, 1H, J_2′,3′, J_3′,4′ = 8.5 Hz, H-3′), 3.21 (dd, 1H, J_5′,6a′ = 4.0 Hz, J_6a′,6b′ = 10.5 Hz, H-6a′), 3.16 (m, 1H, H-5′), 2.96 (dd, 1H, J_5′,6b′ = 1.5 Hz, J_6a′,6b′ = 10.5 Hz, H-6b′), 2.94 (bs, 1H, OH). ¹³C NMR (CDCl₃, 125 MHz) δ: 155.6, 150.5, 138.6, 138.1(2), 136.4, 133.8, 131.8, 129.6, 128.4(2), 128.3(3), 128.2(2), 128.0, 127.5(2), 127.4(2), 126.4(2), 123.5, 118.6(2), 114.5(2) (ArC), 102.3 (C-1′), 102.1 (CHPh), 98.1 (C-1), 84.1, 80.7, 77.1, 76.9, 75.1, 74.8, 74.7, 72.8, 72.6(2), 68.7, 68.5, 66.2, 55.9 (C-2), 55.6 (C₆H₄OCH₃). HRMS calcd. for C₅₅H₅₃O₁₃NNa (M+Na)⁺: 958.3415; found: 958.3418.

p-Tolyl 3,4-O-isopropylidene-1-thio-β-L-fucopyranoside (8). To a mixture of compound 7 (3.0 g, 11.1 mmol) in acetone (30 mL), was added 2,2-DMP (2.7 mL, 22.2 mmol), followed by CSA (50 mg), and the mixture was stirred at room temperature for 3 h, after which TLC (n-hexane–EtOAc, 2 : 1) showed complete conversion of the starting material to a faster-moving spot. The solution was neutralized with Et₃N and evaporated in vacuo. The residue was purified by flash chromatography using n-hexane–EtOAc, 2 : 1, to afford pure compound 8 (3.1 g, 90%) as a light yellow glass. [α]²⁵_D +155 (c 1.1, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.44, 7.12 (2d, 4H, J = 6.5 Hz, ArH), 4.35 (d, 1H, J_1,2 = 9.5 Hz, H-1), 4.02 (m, 2H, H-3, H-4), 3.84 (m, 1H, H-5), 3.51 (dd, 1H, J_1,2 = 9.5 Hz, J_2,3 = 8.0 Hz, H-2), 2.53 (bs, 1H, OH), 2.32 (s, 3H, S-C₆H₄CH₃), 1.42, 1.33 (2s, 6H, 2 × isopropylidene-CH₃), 1.41 (d, 3H, J_5,6 = 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 138.2, 133.1(2), 129.7(2), 128.2, 109.8 (isopropylidene-C), 88.1 (C-1), 79.0, 76.3, 72.7, 71.3, 28.1, 26.3 (2 × isopropylidene-CH₃), 21.1 (SC₆H₄CH₃), 16.9 (C-6). HRMS calcd. for C₁₆H₂₂O₄SNa (M+Na)⁺: 333.1136; found: 333.1139.

p-Tolyl 3,4-O-isopropylidene-2-O-(4-methoxybenzyl)-1-thio-β-L-fucopyranoside (9). To a solution of compound 8 (3.0 g, 9.7 mmol) in dry DMF (20 mL) was added NaH (930 mg, 50% in mineral wax) followed by 4-methoxybenzyl chloride (1.7 mL, 12.6 mmol) and the mixture was stirred at room temperature for 4 h. Excess NaH was neutralized by careful addition of MeOH (2 mL) and the mixture was evaporated in vacuo. The residue was dissolved in diethyl ether (30 mL) and washed successively with H₂O (30 mL) and brine (30 mL). The organic layer was collected, dried (Na₂SO₄), filtered and evaporated. The residue was purified by flash chromatography using n-hexane–EtOAc, 4 : 1 to afford pure compound 9 (3.45 g, 83%) as a light yellow syrup. [α]²⁵_D +141 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.45–6.85 (m, 8H, ArH), 4.75, 4.60 (2d, 2H, J = 11.0 Hz, CH₂C₆H₄OCH₃), 4.50 (d, 1H, J_1,2 = 9.5 Hz, H-1), 4.19 (t, 1H, J_2,3, J_3,4 = 6.0 Hz, H-3), 4.01 (dd, 1H, J_3,4 = 6.0 Hz, J_4,5 = 2.0 Hz, H-4), 3.79 (s, 3H, C₆H₄OCH₃), 3.76 (dd, 1H, J_4,5 = 2.0 Hz, J_5,6 = 6.5 Hz, H-5), 3.46 (dd, 1H, J_1,2 = 9.5 Hz, J_2,3 = 6.0 Hz, H-2), 2.32 (s, 3H, S-C₆H₄CH₃), 1.39, 1.35 (2s, 6H, 2 × isopropylidene-CH₃), 1.37 (d, 3H, J_5,6 = 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 159.2, 137.4, 132.6(2), 130.1, 129.9(2), 129.8, 129.4(2), 113.6(2) (ArC), 109.6 (isopropylidene-C), 86.4 (C-1), 79.8, 77.8, 76.4, 73.1, 72.3, 55.2 (C₆H₄OCH₃), 27.9, 26.3 (2 × isopropylidene-CH₃), 21.1 (SC₆H₄CH₃), 16.8 (C-6). HRMS calcd. for C₂₄H₃₀O₅SNa (M+Na)⁺: 453.1712; found: 453.1716.

p-Methoxyphenyl 3,4-O-isopropylidene-2-O-(4-methoxybenzyl)-α-L-fucopyranosyl-(1 → 2)-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (10). A mixture of the disaccharide acceptor 6 (1.2 g, 1.3 mmol), donor 9 (730 mg, 1.7 mmol) and MS 4 Å (1.5 g) in dry CH₂Cl₂ (20 mL) was stirred under nitrogen for 30 min. The mixture was cooled to −40 °C after adding NIS (500 mg, 2.2 mmol). H₂SO₄-silica (50 mg) was added and the mixture was stirred for 90 min. The mixture was filtered and washed with CH₂Cl₂ (10 mL). The filtrate was washed successively with aq. Na₂S₂O₃ (2 ×30 mL), aq. NaHCO₃ (2 ×30 mL) and brine (30 mL). The organic layer was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by flash chromatography using n-hexane–EtOAc, 2 : 1, as the eluent to afford pure trisaccharide 10 (1.7 g, 83%) as a colourless foam. [α]²⁵_D +76 (c 1.1, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.38–6.72 (m, 32H, ArH), 5.82 (d, 1H, J_1,2 = 8.5 Hz, H-1), 5.42 (s, 1H, CHPh), 4.93 (d, 1H, J = 11.0 Hz, CH₂Ph), 4.86 (d, 1H, J_{1′′,2′′} = 3.0 Hz, H-1′′), 4.85 (t, 1H, J_2,3, J_3,4 = 8.5 Hz, H-3), 4.65 (t, 1H, J_1,2, J_2,3 = 8.5 Hz, H-2), 4.63, 4.59 (2d, 2H, J = 11.0 Hz, CH₂Ph), 4.49 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.47, 4.45, 4.41 (3d, 3H, J = 11.0 Hz, CH₂Ph), 4.36, 4.32 (2d, 2H, J = 11.0 Hz, CH₂Ph), 4.30 (m, 1H, H-5′′), 4.08 (dd, 1H, J_{2′′,3′′} = 8.5 Hz, J_{3′′,4′′} = 5.5 Hz, H-3′′), 3.98 (t, 1H, J_3′,4′, J_4′,5′ = 8.5 Hz, H-4′), 3.91 (dd, 1H, J_{3′′,4′′} = 5.5 Hz, J_{4′′,5′′} = 3.0 Hz, H-4′′), 3.79 (s, 3H, CH₂C₆H₄OCH₃), 3.75 (m, 1H, H-2′), 3.71 (s, 3H, OC₆H₄OCH₃), 3.69 (m, 1H, H-2′′), 3.55 (t, 1H, J_3,4, J_4,5 = 8.5 Hz, H-4), 3.53-3.43 (m, 4H, H-6ª, H-6b, H-6a′, H-6b′), 3.16 (m, 1H, H-5), 1.29, 1.23 (2s, 6H, 2 × isopropylidene-CH₃), 1.27 (d, 3H, J = 6.5 Hz, C-CH₃). ¹³C NMR (CDCl₃, 125 MHz) δ: 158.9, 155.5, 150.7, 138.8, 138.2, 138.1, 137.3, 133.9, 131.8, 130.5, 129.7, 129.0, 128.9(2), 128.4(2), 128.3(2), 128.2(3), 128.1(2), 128.0(2), 127.9(2), 127.7(2), 127.6, 127.5(2), 127.1, 126.1(2), 123.4, 118.5(2), 114.5(2), 113.6(2) (ArC), 108.3 (isopropylidene-C), 101.2 (CHPh), 100.3 (C-1′), 98.0 (C-1), 97.2 (C-1′′), 84.5, 81.4, 80.1, 77.5, 76.6, 76.1, 75.9, 74.7(2), 74.6, 74.1, 73.4, 71.2, 68.9, 68.6, 66.5, 63.6, 56.0 (C-2), 55.6 (C₆H₄OCH₃), 55.3 (CH₂C₆H₄OCH₃), 28.4, 26.4 (2 × isopropylidene-CH₃), 16.2 (C-6). HRMS calcd. for C₇₂H₇₅O₁₈NNa (M+Na)⁺: 1264.4882; found: 1264.4885.

p-Methoxyphenyl 3,4-O-isopropylidene-α-L-fucopyranosyl-(1→2)-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (11). To a solution of compound 10 (1.3 g, 1.0 mmol) in CH₂Cl₂–H₂O (4 : 1, 20 mL), DDQ (450 mg, 2.0 mmol) was added and the mixture was allowed to stir at room temperature for 3 h, after which TLC (n-hexane–EtOAc, 1 : 1) showed complete conversion of the starting material to a slower moving spot. The mixture was diluted with CH₂Cl₂ (15 mL) and washed successively with H₂O (3×30 mL). The organic layer was collected, dried (Na₂SO₄), filtered and evaporated in vacuo. The residue was purified by flash chromatography using n-hexane–EtOAc, 2 : 1, as the eluent to afford the pure trisaccharide acceptor 11 (950 mg, 81%) as a light yellow syrup. [α]²⁵_D +81 (c 1.1, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.83–6.71 (m, 28H, ArH), 5.81 (d, 1H, J_1′,2′ = 8.5 Hz, H-1′), 5.38 (s, 1H, CHPh), 4.60 (d, 1H, J_1,2 = 9.0 Hz, H-1), 4.47 (d, 1H, J_{1′′,2′′} = 2.5 Hz, H-1′′), 3.70 (s, 3H, C₆H₄OCH₃), 1.21 (d, 3H, J_{5′′,6′′} = 6.0 Hz, H-6′′). ¹³C NMR (CDCl₃, 125 MHz) δ: 155.5, 150.6, 139.2, 138.0, 137.7, 137.6, 137.2, 134.0, 131.7, 129.1, 128.5(3), 128.4(2), 128.3(2), 128.2(2), 128.1(2), 127.9(2), 127.8(2), 127.8, 127.7, 127.5(2), 126.1(2), 123.5, 118.6(2), 114.5(2) (ArC), 108.7 (isopropylidene-C), 101.3 (CHPh), 100.1 (C-1′), 98.7 (C-1), 97.9 (C-1′′), 83.7, 80.3, 79.8, 78.3, 77.6, 76.6, 75.6, 74.9, 74.7, 74.4, 74.3, 73.5, 70.4, 68.6, 66.5, 64.0, 56.0 (C-2), 55.6 (C₆H₄OCH₃), 28.0, 26.1 (2 × isopropylidene-CH₃), 16.1 (C-6). HRMS calcd. for C₆₄H₆₇O₁₇NNa (M+Na)⁺: 1144.4307; found: 1144.4311.

p-Methoxyphenyl 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1 → 2)-3,4-O-isopropylidene-α-L-fucopyranosyl-(1 → 2)-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside (13). A mixture of the trisaccharide acceptor 11 (900 mg, 0.8 mmol), donor 12 (600 mg, 1.0 mmol) and MS 4 Å (1.0 g) in dry CH₂Cl₂ (15 mL) was stirred under nitrogen for 30 min. The mixture was cooled to 5–10 °C in an ice-water bath after adding NIS (290 mg, 1.3 mmol). H₂SO₄-silica (30 mg) was added and the mixture was stirred for 45 min. The mixture was filtered and washed with CH₂Cl₂ (20 mL). The combined filtrate was washed successively with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2×30 mL) and brine (30 mL). The organic layer was separated, dried (Na₂SO₄), filtered and evaporated. The residue thus obtained was purified by flash chromatography using n-hexane–EtOAc, 2 : 1, as the eluent to afford pure tetrasaccharide 13 (1.1 g, 84%) as a colourless foam. [α]²⁵_D +91 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.72–6.79 (m, 43H, ArH), 5.92 (d, 1H, J_1,2 = 8.5 Hz, H-1), 5.42 (s, 1H, CHPh), 4.92 (d, 1H, J_{1′′,2′′} = 2.0 Hz, H-1′′), 4.89 (d, 1H, J_{1′′′,2′′′} = 1.5 Hz, H-1′′′), 4.54 (d, 1H, J_1′,2′ = 8.5 Hz, H-1′), 3.77 (s, 3H, C₆H₄OCH₃), 2.20 (s, 3H, COCH₃), 1.35, 1.33 (2s, 6H, 2 × isopropylidene-CH₃), 1.29 (d, 3H, J_{5′′,6′′} = 6.5 Hz, H-6′′). ¹³C NMR (CDCl₃, 125 MHz) δ: 170.3 (COCH₃), 167.9, 167.8 (2 × phthalimido CO), 155.5, 150.7, 138.9,138.6, 138.4, 138.2, 138.1, 137.9, 137.3, 133.9, 131.8(2), 129.0, 128.5(2), 128.2(9), 128.1(6), 128.0(4), 127.9(4), 127.8(3), 127.6(2), 127.5, 127.4(2), 127.3, 127.2(2), 126.1(2), 118.6(2), 114.4(2) (ArC), 108.6 (isopropylidene-C), 101.2 (CHPh), 100.3 (C-1′), 98.9 (C-1′′′), 97.9 (C-1), 97.6 (C-1′′), 84.3, 81.3, 80.3, 78.0, 77.3, 77.2, 76.0, 75.3, 74.9, 74.8, 74.6, 74.0, 73.8, 73.4, 73.3, 72.0, 71.4, 69.0, 68.9, 68.6, 68.4, 66.4, 63.6, 55.8, 55.6(C₆H₄OCH₃), 28.5, 26.4 (2 × isopropylidene-CH₃), 21.2 (COCH₃), 16.1 (C-6). HRMS calcd. for C₉₃H₉₇O₂₃NNa (M+Na)⁺: 1618.6349; found: 1618.6352.

p-Methoxyphenyl α-D-mannopyranosyl-(1 → 2)-α-L-fucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranoside (1). To a solution of the compound 13 (1.0 g, 0.6 mmol) in n-butanol (15 mL) was added ethylene diamine (80 μL, 1.2 mmol) and the solution was stirred at 110 °C for 24 h. After evaporating the solvents in vacuo, the residue was dissolved in pyridine (10 mL) followed by Ac₂O (10 mL) and the solution was stirred at room temperature for 2 h. The solvents were evaporated in vacuo and the residue was dissolved in 80% aq. AcOH (20 mL) and the solution was then stirred at 80 °C for 2 h, after which TLC (n-hexane–EtOAc, 1 : 1) showed complete conversion of the starting material to a slower moving spot. After evaporating the solvents the residue was dissolved in MeOH (100 mL) and the solution was passed through a H-cube flow hydrogenation assembly using a 10% Pd-C cartridge with a flow rate of 1 mL min⁻¹. Complete removal of the benzyl groups was achieved after two cycles, as determined by the mass spectra. The solvents were evaporated and the residue was re-dissolved in MeOH (10 mL). NaOMe (1 mL, 0.5M in MeOH) was added and the solution was stirred at room temperature for 2 h. Excess NaOMe was neutralized by DOWEX 50W H⁺ resin, filtered and evaporated. The residue was passed through a short flash column using CH₂Cl₂-MeOH (3 : 1) to get pure target tetrasaccharide 1 (315 mg, 63% over four steps). [α]²⁵_D +62 (c 0.8, MeOH). ¹H NMR (CD₃OD, 500 MHz) δ: 6.94, 6.81 (2d, 4H, J 7.5 Hz, C₆H₄OCH₃), 5.37 (d, 1H, J_{1′′,2′′} = 3.5 Hz, H-1′′), 5.16 (bs, 1H, H-1′′′), 4.75 (d, 1H, J_1,2 = 8.5 Hz, H-1), 4.59 (d, 1H, J_1′,2′ = 8.5 Hz, H-1′), 3.73 (s, 3H, C₆H₄OCH₃), 2.03 (s, 3H, NHCOCH₃), 1.20 (d, 3H, J = 6.5 Hz, C–CH₃). ¹³C NMR (CD₃OD, 125 MHz) δ: 173.6 (NHCOCH₃), 156.8, 153.3, 119.1(2), 115.6(2) (ArC), 102.9 (C-1), 102.8 (C-1′′′), 102.2 (C-1′), 99.8 (C-1′′), 80.8, 79.1, 78.2, 78.0(2), 74.9, 74.4, 74.1, 72.4, 72.0(2), 70.9, 70.1, 68.8, 67.4, 62.9, 62.5, 56.1 (C₆H₄OCH₃),22.7 (NHCOCH₃), 16.4 (C–CH₃). HRMS calcd. for C₃₃H₅₁O₂₁NNa (M+Na)⁺: 820.2851; found: 820.2854.

p-Methoxyphenyl 3,4-O-isopropylidene-β-L-fucopyranoside (15). To a mixture of compound 14 (3.0 g, 11.1 mmol) and 2,2-DMP (2.7 mL, 22.2 mmol) in dry acetone (20 mL) was added CSA (50 mg), and the mixture was stirred at room temperature for 2 h. The solution was neutralized with Et₃N and the solvents were evaporated. The residue was purified by flash chromatography using n-hexane–EtOAc (3 : 1) to afford pure compound 15 (3.2 g, 92%) as a colourless gel. [α]²⁵_D +170 (c 1.1, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 6.99, 6.82 (2d, 4H, J = 9.0 Hz, ArH), 4.63 (d, 1H, J_1,2 = 8.0 Hz, H-1), 4.12 (dd, 1H, J_2,3 = 8.0 Hz, J_3,4 = 6.0 Hz, H-3), 4.04 (dd, 1H, J_3,4 = 6.0 Hz, J_4,5 = 1.6 Hz, H-4), 3.94 (m, 1H, H-5), 3.78 (t, 1H, J_1,2, J_2,3 = 8.0 Hz, H-2), 3.76 (s, 3H, C₆H₄OCH₃), 1.56, 1.37 (2s, 6H, 2 × isopropylidene-CH₃), 1.43 (d, 3H, J_5,6 = 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 155.5, 151.2, 118.7(2), 114.5(3) (ArC), 109.9 (isopropylidene-C), 101.7 (C-1), 78.7, 76.2, 73.3, 69.3, 55.6 (C₆H₄OCH₃), 28.2, 26.3 (2 × isopropylidene-CH₃), 16.6 (C-6). HRMS calcd. for C₁₆H₂₂O₆Na (M+Na)⁺: 333.1314; found: 333.1318.

p-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-β-L-fucopyranoside (16). To a solution of compound 15 (3.0 g, 9.7 mmol) in dry DMF (30 mL) was added NaH (930 mg, 19.4 mmol, 50% in mineral wax) followed by BnBr (1.7 mL, 14.6 mmol) and the mixture was stirred at room temperature for 3 h. Excess NaH was neutralized by careful addition of MeOH (2.0 mL) and H₂O (50 mL) was added and stirred for 45 min. Then the solution was extracted by Et₂O (3 × 40 mL) and the combined organic layer was dried (Na₂SO₄), filtered and evaporated. The crude material thus obtained was purified by flash chromatography using n-hexane–EtOAc (4 : 1) to give pure compound 16 (3.5 g, 90%) as a light yellow syrup. [α]²⁵_D +148 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.44–6.83 (m, 9H, ArH), 4.93, 4.88 (2d, 2H, J = 11.5 Hz, CH₂Ph), 4.78 (d, 1H, J_1,2 = 8.0 Hz, H-1), 4.21 (dd, 1H, J_2,3 = 7.0 Hz, J_3,4 = 5.5 Hz, H-3), 4.03 (dd, 1H, J_3,4 = 5.5 Hz, J_4,5 = 2.5 Hz, H-4), 3.91 (m, 1H, H-5), 3.78 (s, 3H, C₆H₄OCH₃), 3.63 (dd, 1H, J_1,2 = 8.0 Hz, J_2,3 = 7.0 Hz, H-2), 1.43, 1.37 (2s, 6H, 2 × isopropylidene-CH₃), 1.42 (d, 3H, J_5,6 = 6.0 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 155.2, 151.5, 138.2, 128.2(3), 127.6, 118.6(2), 114.4(3) (ArC), 109.7 (isopropylidene-C), 102.1 (C-1), 79.2, 79.1, 76.2, 73.7, 68.8, 55.6 (C₆H₄OCH₃), 27.8, 26.3 (2 × isopropylidene-CH₃), 16.6 (C-6). HRMS calcd. for C₂₃H₂₈O₆Na (M+Na)⁺: 423.1784; found: 423.1788.

p-Methoxyphenyl 2-O-benzyl-3-O-(4-methoxybenzyl)-β-L-fucopyranoside (18). A solution of compound 16 (3.2 g, 8.0 mmol) in 80% aq. AcOH (30 mL) was stirred at 80 °C for 2 h. After evaporating the solvents the residue was purified by flash chromatography using n-hexane–EtOAc (2 : 1) to afford the diol 17 (2.7 g, 94%) as a white amorphous solid. The slurry of compound 17 (2.5 g, 6.9 mmol) and Bu₂SnO (1.9 g, 7.6 mmol) in dry MeOH (30 mL) was refluxed for 2 h. After evaporating the solvents, the residue was re-dissolved in dry DMF (25 mL), followed by the addition of 4-methoxybenzyl chloride (1.2 mL, 9.0 mmol) and Bu₄NI (2.5 g, 6.9 mmol). The solution was stirred at 60 °C for 12 h. After removal of the solvents, the residue was dissolved in CH₂Cl₂ and washed successively with Na₂S₂O₃ (30 mL), NaHCO₃ (30 mL) and brine (30 mL). The organic layer was collected, dried (Na₂SO₄), filtered and evaporated. The residue was purified by flash chromatography using n-hexane–EtOAc (4 : 1) to afford pure compound 18 (2.8 g, 83%) as a light yellow syrup. [α]²⁵_D +143 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.39–6.82 (m, 13H, ArH), 4.99, 4.81 (2d, 2H, J = 11.0 Hz, CH₂Ph), 4.80 (d, 1H, J_1,2 = 8.0 Hz, H-1), 4.68 (s, 2H, CH₂C₆H₄OCH₃), 3.82 (m, 1H, H-2), 3.81 (s, 3H, CH₂C₆H₄OCH₃), 3.78 (s, 3H, OC₆H₄OCH₃), 3.73 (m, 1H, H-3), 3.61 (m, 1H, H-5), 3.55 (m, 1H, H-4), 2.47 (bs, 1H, OH), 1.38 (d, 3H, J_5,6 = 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 159.4, 155.2, 151.6, 138.5, 129.9, 129.5(2), 128.3(2), 128.1(2), 127.6, 118.6(2), 114.5(2), 113.9(2) (ArC), 102.9 (C-1), 80.5, 78.5, 75.3, 72.1, 70.2, 69.3, 55.6 (OC₆H₄OCH₃), 55.3 (CH₂C₆H₄OCH₃), 16.4 (C-6). HRMS calcd. for C₂₈H₃₂O₇Na (M+Na)⁺: 503.2046; found: 503.2049.

p-Methoxyphenyl 2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1 → 4)-2-O-benzyl-3-O-(4-methoxybenzyl)-β-L-fucopyranoside (20). A mixture of the acceptor 18 (1.5 g, 3.1 mmol), donor 19 (2.4 g, 4.1 mmol) and MS 4 Å (2.0 g) in dry CH₂Cl₂ (25 mL) was stirred under nitrogen for 30 min. The mixture was cooled to 5–10 °C in an ice-water bath after adding NIS (1.2 g, 5.3 mmol). H₂SO₄-silica (50 mg) was added and the mixture was stirred for 90 min. The mixture was filtered and washed with CH₂Cl₂ (10 mL). The filtrate was washed successively with aq. Na₂S₂O₃ (2 × 30 mL), aq. NaHCO₃ (2 × 30 mL) and brine (30 mL). The organic layer was separated, dried (Na₂SO₄), filtered and evaporated. The residue was purified by flash chromatography using n-hexane–EtOAc, 2 : 1, as the eluent to afford pure disaccharide 20 (2.1 g, 85%) as a colourless foam. [α]²⁵_D +109 (c 1.1, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.02–6.78 (m, 28H, ArH), 5.75 (t, 1H, J_2′,3′, J_3′,4′ = 9.0 Hz, H-3′), 5.63 (dd, 1H, J_1′,2′ = 8.0 Hz, J_2′,3′ = 9.0 Hz, H-2′), 5.48 (s, 1H, CHPh), 5.07 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.96, 4.86 (2d, 2H, J = 11.0 Hz, CH₂Ph), 4.76 (d, 1H, J_1,2 = 7.5 Hz, H-1), 4.71 (s, 2H, CH₂C₆H₄OCH₃), 4.30 (dd, 1H, J_5′,6a′ = 5.0 Hz, J_6a′,6b′ = 10.5 Hz, H-6a′), 4.12 (t, 1H, J_3′,4′ = 9.0 Hz, J_4′,5′ = 9.0 Hz, H-4′), 4.03 (dd, 1H, J_1,2 = 7.5 Hz, J_2,3 = 8.5 Hz, H-2), 3.86 (m, 2H, H-4, H-6b′), 3.78 (s, 3H, CH₂C₆H₄OCH₃), 3.76 (s, 3H, OC₆H₄OCH₃), 3.64 (m, 1H, H-5′), 3.50 (dd, 1H, J_2,3 = 8.5 Hz, J_3,4 = 3.0 Hz, H-3), 3.48 (m, 1H, H-5), 1.15 (d, 1H, J_5,6 = 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 165.7, 164.9 (2 × COPh), 159.3, 155.1, 151.5, 138.7, 136.9, 133.0, 132.9, 130.4, 129.8(2), 129.7(2), 129.5, 129.4, 129.3(2), 128.9, 128.3(3), 128.2(3), 128.1(4), 127.6, 126.1(2), 118.9(2), 114.3(2), 113.8(2) (ArC), 103.1 (C-1), 101.7 (C-1′), 101.3 (CHPh), 80.4, 78.4, 78.3, 75.2, 73.1, 72.5, 72.4, 70.1, 68.6, 66.5 (C-6′), 55.6 (CH₂C₆H₄OCH₃), 55.2 (OC₆H₄OCH₃), 16.6 (C-6). HRMS calcd. for C₅₅H₅₄O₁₄Na (M+Na)⁺: 961.3411; found: 961.3417.

p-Methoxyphenyl 2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1 → 4)-2-O-benzyl-β-L-fucopyranoside (21). To a solution of compound 20 (2.0 g, 2.1 mmol) in CH₂Cl₂–H₂O (4 : 1, 20 mL), DDQ (950 mg, 4.2 mmol) was added and the solution was stirred at room temperature for 2 h when TLC (n-hexane–EtOAc, 2 : 1) showed complete conversion of the starting material to a slower running spot. The solution was diluted with CH₂Cl₂ (mL) and washed with H₂O (2 × 40 mL). The organic layer was collected, dried (Na₂SO₄), filtered and evaporated. The residue was purified by flash chromatography using n-hexane–EtOAc (3 : 1) to afford pure compound 21 (1.4 g, 81%) as a light yellow syrup. [α]²⁵_D +123 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 7.98–6.79 (m, 24H, ArH), 5.79 (t, 1H, J_2′,3′, J_3′,4′ = 9.0 Hz, H-3′), 5.61 (dd, 1H, J_1′,2′ = 8.0 Hz, J_2′,3′ = 9.0 Hz, H-2′), 5.56 (s, 1H, CHPh), 4.97, 4.88 (2d, 2H, J = 11.5 Hz, CH₂Ph), 4.96 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.76 (d, 1H, J_1,2 = 7.5 Hz, H-1), 4.42 (dd, 1H, J_5′,6a′ = 4.5 Hz, J_6a′,6b′ = 10.0 Hz, H-6a′), 4.03 (t, 1H, J_3′,4′ = 9.0 Hz, J_4′,5′ = 9.0 Hz, H-4′), 3.87 (t, 1H, J_5′,6b′, J_6a′,6b′ = 10.0 Hz, H-6b′), 3.82 (m, 2H, H-2, H-5′), 3.76 (s, 3H, OC₆H₄OCH₃), 3.72 (m, 2H, H-3, H-4), 3.61 (m, 1H, H-5), 3.37 (bs, 1H, OH), 1.08 (d, 1H, J_5,6 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 165.6, 164.9 (2 × COPh), 155.2, 151.4, 138.6, 136.5, 133.2, 133.1, 129.8(2), 129.7(2), 129.2, 129.1, 129.0, 128.3(6), 128.2(2), 128.0(2), 127.6, 126.0(2), 118.6(2), 114.4(2) (ArC), 102.8 (2C, C-1, C-1′), 101.5 (CHPh), 82.8, 79.1, 78.3, 74.8, 72.8, 72.7, 72.2, 69.9, 68.3, 66.8, 55.6 (OC₆H₄OCH₃), 16.3 (C-6). HRMS calcd. for C₄₇H₄₆O₁₃Na (M+Na)⁺: 841.2836; found: 841.2840.

p-Methoxyphenyl 2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1 → 4)-2-O-benzyl-3-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl)-β-L-fucopyranoside (23). A mixture of the disaccharide acceptor 21 (1.0 g, 1.2 mmol), donor 22 (1.1 g, 2.4 mmol) and MS 4 Å (1.5 g) in dry CH₂Cl₂ (20 mL) was stirred under a nitrogen atmosphere for 30 min. The mixture was cooled to −40 °C and H₂SO₄-silica (30 mg) was added. After stirring for 2 h, TLC (n-hexane–EtOAc, 2 : 1) showed complete consumption of the donor, the mixture was neutralized with Et₃N and filtered. The filtrate was evaporated in vacuo and the residue was purified by flash chromatography using n-hexane–EtOAc, 2 : 1, as the eluent to afford pure trisaccharide 23 (1.1 g, 80%) as a light yellow foam. [α]²⁵_D +103 (c 0.9, CHCl₃). ¹H NMR (CDCl₃, 500 MHz) δ: 8.00–6.72 (m, 24H, ArH), 5.83 (t, 1H, J_{2′′,3′′}, J_{3′′,4′′} = 9.5 Hz, H-3′′), 5.71 (s, 1H, CHPh), 5.61 (dd, 1H, J_{1′′,2′′} = 8.0 Hz, J_{2′′,3′′} = 9.5 Hz, H-2′′), 5.54 (m, 2H, H-1′, H-3′), 5.46 (bs, 1H, H-4′), 5.08, 4.84 (2d, 2H, J = 10.5 Hz, CH₂Ph), 4.97 (d, 1H, J_{1′′,2′′} = 8.0 Hz, H-1′′), 4.67 (d, 1H, J_1,2 = 7.5 Hz, H-1), 4.52 (m, 1H, H-6a′′), 4.31 (m, 2H, H-6a′, H-6b′), 4.14 (t, 1H, J_3′,4′ = 9.5 Hz, J_4′,5′ = 9.5 Hz, H-4′′), 4.06 (m, 2H, H-2, H-3), 3.99 (m, 1H, H-6b′′), 3.86 (d, 1H, J_4,5 = 3.0 Hz, H-4), 3.74 (s, 3H, OC₆H₄OCH₃), 3.72 (m, 1H, H-5′), 3.63 (m, 1H, H-5′′), 3.61 (dd, 1H, J_1′,2′ = 3.0 Hz, J_2′,3′ = 9.5 Hz, H-2′), 3.52 (m, 1H, H-5), 2.16, 2.13, 2.05 (3s, 9H, 3 × COCH₃), 1.23 (d, 1H, J_5,6 = 6.5 Hz, H-6). ¹³C NMR (CDCl₃, 125 MHz) δ: 170.3, 170.1, 169.8 (3 × COCH₃), 165.8, 165.2 (2 × COPh), 155.2, 151.3, 138.6, 137.0, 133.0, 132.6, 129.9(2), 129.8(2), 129.5, 128.9, 128.3(2), 128.2(2), 128.1(7), 127.6, 126.2(2), 119.0(2), 114.3(2) (ArC), 103.0 (C-1), 101.9 (C-1′′), 101.1 (CHPh), 99.1 (C-1′), 78.1, 77.3, 76.5, 76.0, 74.9, 72.9, 72.2, 70.1, 68.2, 67.7, 67.6, 67.2, 66.2, 61.5, 57.4 (C-2′), 55.6 (OC₆H₄OCH₃), 20.7, 20.6, 20.5 (3×COCH₃), 16.6 (C-6). HRMS calcd. for C₅₉H₆₁O₂₀N₃Na (M+Na)⁺: 1154.3746; found: 1154.3750.

p-Methoxyphenyl β-D-glucopyranosyl-(1 → 4)-3-O-(2-acetamido-2-deoxy-α-D-galactopyranosyl)-β-L-fucopyranoside (2). Compound 23 (1.0 g, 0.9 mmol) was dissolved in thiolacetic acid (10 mL) and the solution was allowed to stir in the dark for 48 h. After evaporating the solvents in vacuo and co-evaporating with toluene, the residue was dissolved in 80% aq. AcOH (15 mL) and the solution was stirred at 80 °C for 3 h, after which TLC (n-hexane–EtOAc, 1 : 1) showed complete conversion of the starting material to a slower moving spot. The solvents were evaporated and the residue was dissolved in MeOH (80 mL) and the solution was passed through a H-cube Flow Hydrogenation assembly using a 10% Pd–C cartridge with a flow rate of 1 mL per minute. The hydrogenation was complete in two cycles, as confirmed by mass spectrometry. The volume of the methanolic solution was reduced to 10 mL and NaOMe (1 mL, 0.5M in MeOH) was added. The solution was stirred at room temperature for 6 h. Excess NaOMe was neutralized by DOWEX 50W H⁺ resin, filtered and evaporated. The residue was purified by passing through a short silica column using CH₂Cl₂–MeOH (3 : 1) to get pure target trisaccharide 2 (375 mg, 67% over four steps). [α]²⁵_D +54 (c 0.8, MeOH). ¹H NMR (D₂O, 500 MHz) δ: 7.13, 7.00 (2d, 4H, J = 7.5 Hz, C₆H₄OCH₃), 5.23 (d, 1H, J_1′,2′ = 4.0 Hz, H-1′), 4.99 (d, 1H, J_{1′′,2′′} = 6.5 Hz, H-1′′), 4.65 (d, 1H, J_1,2 = 7.5 Hz, H-1), 3.83 (s, 3H, C₆H₄OCH₃), 2.08 (s, 3H, NHCOCH₃), 1.38 (d, 3H, J = 6.5 Hz, C–CH₃). ¹³C NMR (D₂O, 125 MHz) δ: 174.6 (NHCOCH₃), 154.8, 150.9, 118.5(2), 115.1(2) (ArC), 103.9 (C-1), 101.9 (C-1′′), 99.3 (C-1′), 79.4, 77.0, 76.5, 74.9, 73.4, 71.7, 71.4, 71.3, 70.9, 68.7, 67.5, 61.7, 55.9 (OC₆H₄OCH₃), 22.1 (NHCOCH₃), 15.3 (C-6). HRMS calcd. for C₂₇H₄₁O₁₆NNa (M+Na)⁺: 658.2323; found: 658.2327.

Acknowledgements

PRV is thankful to IISER-Kolkata for Senior Research Fellowship. This work is supported by CSIR, New Delhi through research grant 01(2370)/10/EMR-II to BM.

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra of all new compounds. See DOI: 10.1039/c2ra22407k

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