Expeditious Synthesis of a Tetrasaccharide Repeating Unit of the O-Antigen of Escherichia coli O163

Abstract

The synthesis of the tetrasaccharide repeating unit of the O-antigen of Escherichia coli O163 as its p-methoxyphenyl (PMP) glycoside was achieved followed by sequential glycosylation strategy through thioglycoside activation using sulfuric acid immobilized on silica ­(H₂SO₄–silica) in conjunction with N-iodosuccinimide as a Brønsted acid catalyst. The application of one-pot reaction conditions for two glycosylations and in situ PMB-group removal reduced the number of reaction steps significantly. The l-QuipNAc building block was obtained from known carbohydrate l-rhamnose precursors. The stereoselective outcomes of all glycosylation reactions were found to be very good. A late-stage TEMPO-mediated oxidation was performed for the formation of required uronic acid moiety. An analogue of the target tetrasaccharide was also prepared by using one-pot glycosylation approach. Such synthetic oligosaccharides could later be effectively conjugated with an appropriate protein to furnish glycoconjugate derivatives for their use in immunochemical studies.

Keywords

polysaccharide - Escherichia coli - glycosylation - antigen - stereoselectivity

• References and Notes

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• 43 p-Methoxyphenyl (2-Acetamido-2,6-dideoxy-α-l-glucopyranosyl)-(1→3)-(2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→2)-(β-d-mannopyranosyl)-(1→4)-β-d-glucopyranosyluronic Acid (1) A solution of compound 14 (400 mg, 0.26 mmol) and thioacetic acid (0.2 mL) in pyridine (15 mL) was stirred at room temperature for 10 h. The solvent was removed under reduced pressure, and the crude product was passed through a short pad of SiO₂. To the solution of the N-acetylated product in THF (10 mL) was added Bu₄NF in THF (5 mL), and the reaction mixture was stirred at room temperature for 6 h. The solvents were removed, and the crude mass was dissolved in CH₂Cl₂ (40 mL). The organic layer was washed with sat. NaHCO₃ and water, dried, and concentrated. To a solution of the crude product in CH₂Cl₂ (20 mL) and H₂O (3.5 mL) were added aqueous solution of NaBr (1 mL, 1 M), aqueous solution of TBAB (2 mL; 1 M), TEMPO (80 mg, 0.4 mmol), sat. aqueous solution of NaHCO₃ (8 mL), and 4% NaOCl aq (10 mL) in succession, and the reaction mixture was stirred at 0–5 °C for 2 h. The reaction mixture was neutralized with 1 N HCl aq solution followed by addition of t-BuOH (25 mL), 2-methyl-but-2-ene (30 mL, 2 M solution in THF), aqueous solution of NaClO₂ (1 g in 5 mL), and aqueous solution of NaH₂PO₄ (1 g in 5 mL). The resultant mixture was allowed to stir at room temperature for 5 h and then diluted with sat. aqueous solution of NaH₂PO₄ and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layer was washed with water, dried (Na₂SO₄), and concentrated to dryness to give the oxidized product. To a solution of the oxidized product in MeOH (10 mL) was added 10% Pd/C (100 mg) and it was allowed to stir at room temperature for 24 h under a positive pressure of hydrogen. The reaction mixture was filtered through a Celite^® bed and the filtering bed was washed with MeOH (50 mL). The combined solution was concentrated under reduced pressure to give the crude product, which was dissolved in 0.1 M NaOMe in MeOH (20 mL), and the solution was stirred at room temperature for 1 h. The reaction mixture was neutralized with Dowex 50W-X8 (H⁺) resin, filtered, and concentrated to give compound 1, which was passed through a column of Sephadex LH-20 (25% MeOH–H₂O) to furnish pure compound 1 (121 mg, 54%) as white powder; [α]_D ²⁵ –28 (c 1.0, H₂O). IR (neat): 3419, 2910, 1598, 1327, 1142, 988, 679 cm^–1. ¹H NMR (400 MHz, D₂O): δ = 7.06 (d, J = 8.1 Hz, 2 H, ArH), 6.92 (d, J = 8.1 Hz, 2 H, ArH), 5.18 (br s, 1 H, H-1_C), 5.04 (d, J = 7.6 Hz, 1 H, H-1_A), 4.83 (br s, 1 H, H-1_D), 4.69 (br s, 1 H, H-1_B), 4.59–4.57 (m, 1 H, H-2_D), 4.22–4.20 (m, 1 H, H-2_C), 4.11–4.09 (m, 3 H, H-5_A, H-2_B, H-5_C), 3.95–3.90 (m, 4 H, H-4_B, H-5_D, H-6_aB, H-6_aC), 3.88–3.82 (m, 3 H, H-4_A, H-6_bB, H-3_C), 3.76 (s, 3 H, OCH₃), 3.72–3.61 (m, 6 H, H-2_A, H-3_A, H-3_B, H-4_C, H-6_bC, H-3_D), 3.42–3.33 (m, 2 H, H-5_B, H-4_D), 2.02 (s, 6 H, NHCOCH₃), 1.25 (d, J = 6.0 Hz, 3 H, CH₃). ¹³C NMR (100 MHz, D₂O, CD₃OD internal standard at δ = 49.5 ppm): δ = 175.5(2 C, C-6_A, NHCOCH₃), 174.9 (NHCOCH₃), 155.5–115.6 (ArC), 101.6 (C-1_A), 100.7 (C-1_B), 100.4 (C-1_C), 95.9 (C-1_D), 81.1 (C-4_A), 77.5 (C-5_B), 76.3 (C-2_B), 74.3 (2 C, C-5_A, C 3_C), 74.1 (C-4_D), 73.3 (C-3_B), 72.9 (2 C, C-5_C, C-3_D), 70.0 (C-3_A), 69.5 (C-5_D), 69.2 (C-4_B), 67.2 (C-4_C), 65.2 (C-2_A), 61.4 (C-6_C), 60.7 (C-6_B), 56.4 (OCH₃), 53.3 (C-2_C), 49.0 (C-2_D), 22.6 (NHCOCH₃), 22.3 (NHCOCH₃), 17.2 (CH₃). HRMS (ESI-TOF): m/z calcd for C₃₅H₅₁N₂O₂₂ [M + Na]: 874.2831; found: 874.2843
• 44 p-Methoxyphenyl (2-Acetamido-2,6-dideoxy-α-l-glucopyranosyl)-(1→3)-(2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→2)-(β-d-mannopyranosyl)-(1→4)-β-d-glucopyranoside (2) A solution of compound 14 (600 mg, 0.39 mmol) and thioacetic acid (0.2 mL) in pyridine (20 mL) was stirred at room temperature for 10 h. The solvent was removed under reduced pressure, and the crude product was passed through a short pad of SiO₂. To the solution of the N-acetylated product in THF (10 mL) was added Bu₄NF in THF (5 mL), and the reaction mixture was stirred at room temperature for 6 h. The solvents were removed, and the crude product was passed through a short pad of SiO₂. To a solution of the desilylated product in MeOH (10 mL) was added 10% Pd/C (150 mg), and it was allowed to stir at room temperature for 24 h under a positive pressure of hydrogen. The reaction mixture was filtered through a Celite^® bed, and the filtering bed was washed with MeOH (60 mL). The combined solution was concentrated under reduced pressure to give the crude product, which was dissolved in 0.1 M MeONa in MeOH (30 mL), and the solution was stirred at room temperature for 1 h. The reaction mixture was neutralized with Dowex 50W-X8 (H⁺) resin, filtered, and concentrated to give compound 1, which was passed through a column of Sephadex LH-20 (25% MeOH–H₂O) to furnish pure compound 1 (142 mg, 62%) as white powder;[α]_D ²⁵ –12 (c 1.0, H₂O). IR (neat): 3445, 2936, 1546, 1377, 1030, 988, 669 cm^–1. ¹H (400 MHz, D₂O): δ = 7.12 (d, J = 8.1 Hz, 2 H, ArH), 6.98 (d, J = 8.1 Hz, 2 H, ArH), 5.20 (br s, 1 H, H-1_C), 5.03 (d, J = 7.4 Hz, 1 H, H-1_A), 4.87 (br s, 1 H, H-1_D), 4.81 (br s, 1 H, H-1_B), 4.63–4.62 (m, 1 H, H-2_D), 4.26–4.25 (m, 1 H, H-2_C), 4.19 (br s, 1 H, H-2_B), 4.15–4.13 (m, 2 H, H-5_A, H-5_C), 3.99–3.90 (m, 6 H, H-4_B, H-5_D, H-6_abA, H-6_aB, H-6_aC), 3.82 (s, 3 H, OCH₃), 3.79–3.71 (m, 7 H, H-2_A, H-4_A, H-3_B, H-3_C, H-4_C, H-6_bB, H-6_bC), 3.70–3.58 (m, 2 H, H-3_A, H-3_D), 3.51–3.48 (m, 1 H, H-5_B), 3.38–3.36 (m, 1 H, H-4_D), 2.07 (s, 3 H, NHCOCH ₃), 2.06 (s, 3 H, NHCOCH ₃), 1.31 (d, J = 6.0 Hz, 3 H, CH₃). ¹³C NMR (100 MHz, D₂O, CH₃OD internal standard at δ = 49.5 ppm): δ = 175.2 (NHCOCH₃), 174.6 (NHCOCH₃), 155.3–115.6 (ArC), 101.6 (C-1_A), 100.6 (2 C, C-1_B, C-1_C), 95.9 (C-1_D), 79.9 (C-4_A), 77.5 (C-2_B), 76.8 (C-5_B), 75.4 (C-3_D), 74.7 (C-3_C), 74.1 (C-3_B), 73.4 (C-5_C), 73.0 (C-5_A), 72.6 (C-3_A), 72.3 (C-4_D), 69.5 (C-5_D), 69.2 (C-4_B), 67.3 (C-4_C), 65.2 (C-2_A), 61.5 (C-6_C), 60.8 (C-6_B), 60.6 (C-6_A), 56.4 (OCH₃), 53.3 (C-2_C), 49.1 (C-2_D), 22.6 (NHCOCH₃), 22.3 (NHCOCH₃), 17.2 (CH₃). HRMS (ESI-TOF): m/z calcd for C₃₅H₅₄N₂O₂₁ [M + Na]: 861.3219; found: 861.3158