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

Geeta Karki; Harikesh Kumar; Remya Rajan; Pintu Kumar Mandal

doi:10.1055/s-0035-1562606

Synlett, Table of Contents

Synlett 2016; 27(18): 2581-2586
DOI: 10.1055/s-0035-1562606

letter

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

Authors

Geeta Karki

^aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow, 226 031, India Email: pintuchem06@gmail.com
Harikesh Kumar

^aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow, 226 031, India Email: pintuchem06@gmail.com
Remya Rajan

^aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow, 226 031, India Email: pintuchem06@gmail.com
Pintu Kumar Mandal*

^aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow, 226 031, India Email: pintuchem06@gmail.com

^bAcademy of Scientific and Innovative Research, New Delhi, 11000, India

Abstract

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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

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References

References and Notes
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38 p-Methoxyphenyl (3,4-di-O-Acetyl 2-azido-2,6-dideoxy-α-l-glucopyranosyl)-(1→3)-(2-azido-4,6-O-benzylidene-2-deoxy-α-d-glucopyranosyl)-(1→2)-(3-O-benzyl-4,6-O-benzylidene-β-d-mannopyranosyl)-(1→4)-6-O-tert-butyldiphenylsilyl-2,3-di-O-benzyl-β-d-glucopyranoside (14) To a solution of compound 13 (1.5 g, 1.50 mmol) and compound 5 (730 mg, 1.65 mmol) in anhydrous CH₂Cl₂–Et₂O (1:2 v/v, 30 mL) MS 4 Å (3 g) was added and cooled to –20 °C under argon. To the cooled reaction mixture, NIS (410 mg, 1.82 mmol) and H₂SO₄–SiO₂ (100 mg) were added, and it was allowed to stir at the same temperature for 30 min. After consumption of the starting materials (TLC; hexane–EtOAc, 3:1), the temperature of the reaction mixture was raised to 10 °C and stirred for 30 min. After the formation of a new spot which was confirmed by TLC (hexane–EtOAc, 3:1), again the reaction mixture was cooled to –20 °C. To the cooled reaction mixture, a solution of compound 6 (689 mg, 1.81 mmol) in CH₂Cl₂–Et₂O (1:2 v/v, 5 mL) and NIS (448 mg, 1.99 mmol) were added, and the reaction mixture was then again stirred at –20 °C for another 30 min. The reaction mixture was filtered through a Celite bed, and the filtering bed was washed with CH₂Cl₂ (100 mL). The combined organic layer was successively washed with 5% Na₂S₂O₃, sat. NaHCO₃ and water, dried (Na₂SO₄), and concentrated. The crude product was purified over SiO₂ using hexane–EtOAc (4:1) as the eluent to give pure compound 14 (1.4 g, 61%); colorless oil; [α]_D ²⁵ +18 (c 1.0, CHCl₃). IR (neat): 3089, 2866, 1849, 1732, 1435, 1236, 1022, 988, 698 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 7.66–7.64 (m, 4 H, ArH), 7.52–7.50 (m, 2 H, ArH), 7.40–7.20 (m, 26 H, ArH), 7.15–7.11 (m, 1 H, ArH), 7.01–6.97 (m, 2 H, ArH), 6.96 (d, J = 9.0 Hz, 2 H, ArH), 6.80 (d, J = 9.0 Hz, 2 H, ArH), 5.62 (s, 1 H, PhCH), 5.55 (s, 1 H, PhCH), 5.32 (dd, J = 3.8, 9.8 Hz, 1 H, H-3_D), 5.25 (br s, 1 H, H-1_C), 5.10 (d, J = 10.4 Hz, 1 H, PhCH₂), 5.02 (d, J = 11.0 Hz, 1 H, PhCH₂), 4.89 (d, J = 7.8 Hz, 1 H, H-1_A), 4.87 (br s, 1 H, H-1_B), 4.86–4.83 (m, 2 H, H-4_D, PhCH₂),4.82 (d, J = 11.7 Hz, 1 H, PhCH₂), 4.77 (d, J = 10.8 Hz, 1 H, PhCH₂), 4.65 (d, J =11.8 Hz, 1 H, PhCH₂), 4.64–4.59 (m, 1 H, H-2_C), 4.56 (br s, 1 H, H-1_D), 4.27–4.22 (m, 3 H, H-3A, H-3_C, H-6_aB), 4.15–4.13 (m, 1 H, H-2_D), 4.09–4.05 (m, 3 H, H-2_B, H-3_B, H-6_bB), 4.01–3.96 (m, 2 H, H-6_aA, H-4_C), 3.94–3.88 (m, 2 H, H-5_C, H-5_D), 3.83–3.76 (m, 3 H, H-2_A, H-6_bA, H-6_aC), 3.78 (s, 3 H, OCH₃), 3.72–3.67 (m, 2 H, H-4_B, H-6_bC), 3.52–3.49 (m, 1 H, H-5_A), 3.41–3.78 (m, 1 H, H-4_A), 3.28–3.22 (m, 1 H, H-5_B), 2.04 (s, 3 H, COCH₃), 1.92 (s, 3 H, COCH₃), 1.06 [s, 9 H, SiC(CH₃)₃], 0.35 (d, J = 6.1 Hz, 3 H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 169.9 (2 C, COCH₃), 155.4–114.6 (ArC), 103.2 (C-1_A), 102.2 (PhCH), 101.5 (PhCH), 99.7 (2 C, C-1_B, C-1_C), 95.7 (C-1_D), 83.5 (C-2_A), 82.0 (C-4_B), 79.1 (C-3_B), 78.7 (C-5_A), 77.4 (C-4_C), 76.8 (C-3_A), 76.4 (PhCH₂), 75.7 (C-4_A), 75.1 (PhCH₂), 74.6 (C-2_B), 73.8 (C-3_C), 73.6 (PhCH₂), 70.9 (2 C, C-3_D, C-4_D), 68.7 (C-6_B), 68.5 (C-6_C), 67.2 (C-5_B), 66.3 (C-5_C), 63.8 (C-2_C), 62.2 (C-6_A), 61.6 (C-2_D), 61.4 (C-5_D), 55.7 (OCH₃), 26.8 [SiC(CH₃)₃], 20.7 (COCH₃), 20.6 (COCH₃), 19.4 [SiC(CH₃)₃], 16.1 (CH₃). HRMS (ESI-TOF): m/z calcd for C₈₆H₉₄N₆O₂₁Si [M + Na]: 1597.6241; found: 1597.6259
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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