An Efficient Synthesis of the Pentasaccharide Repeating Unit of Pseudomonas aeruginosa Psl Exopolysaccharide

Published as part of the Special Section 11^th EuCheMS Organic Division Young Investigator Workshop

Abstract

Pseudomonas aeruginosa is a biofilm-forming Gram-negative bacterium and a leading cause of life-threatening nosocomial infections. The polysaccharide synthesis locus (Psl) exopolysaccharide of P. aeruginosa is a key constituent of the defending bacterial biofilm layer and is a promising therapeutic target for resistant species. The Psl exopolysaccharide is built up from repeating pentasaccharide units which contain one α- and two β-mannosidic linkages, and one l-rhamnose and one d-glucose moieties. The preparation of this pentasaccharide was first described by Boons et al. in a 34-step synthesis. Based on their work, we have developed a new and effective pathway for the synthesis of the repeating pentasaccharide unit of the Psl exopolysaccharide. We have succeeded in simplifying the synthesis of the l-rhamnose and the α-selective d-mannose building blocks. Furthermore, taking advantage of a chemoselective pre-activation-based β-mannosylation, we directly prepare a thioglycoside disaccharide donor and use it in the next coupling reaction without further transformation. The pentasaccharide, in the form of a p-methoxyphenyl glycoside, is prepared in 26 steps, which is suitable for biological testing.

Key words

Psl exopolysaccharide - pentasaccharide - β-mannoside - glycosylation - lectin

• References and Notes

• 1 Li H, Mo K.-F, Wang Q, Stover CK, DiGiandomenico A, Boons G.-J. Chem. Eur. J. 2013; 19: 17425
• 2 Chiou ST, Chen YW, Chen SC, Chao CF, Liu TY. J. Biol. Chem. 2000; 275: 1630
• 3 Ng SK, Huang YT, Lee YC, Low EL, Chiu CH, Chen SL, Mao LC, Chang MD. PloS One 2014; 9: e115296
• 4 Lis H, Sharon N. Chem. Rev. 1998; 98: 637
• 5 Crich D, Sun S, Brunckova J. J. Org. Chem. 1996; 61: 605
• 6 Crich D, Li W, Li H. J. Am. Chem. Soc. 2004; 126: 15081
• 7 Budhadev D, Mukhopadhyay B. Carbohydr. Res. 2014; 384: 51
• 8 Komarova BS, Orekhova MV, Tsvetkov YE, Nifantiev NE. Carbohydr. Res. 2014; 384: 70
• 9 Crich D, Wu B, Jayalath P. J. Org. Chem. 2007; 72: 6806
• 10 Boltje TJ, Li C, Boons G.-J. Org. Lett. 2010; 12: 4636
• 11 Kametani T, Kawamura K, Honda T. J. Am. Chem. Soc. 1987; 109: 3010
• 12 Kerékgyártó J, Kamerling JP, Bouwstra JB, Vliegenthart JF. G, Lipták A. Carbohydr. Res. 1989; 186: 51
• 13 Pozsgay V. Carbohydr. Res. 1992; 235: 295
• 14 Herczeg M, Mező E, Molnár N, Ng S.-K, Lee Y.-C, Chang MD.-T, Borbás A. Chem. Asian J. 2016; 11: 3398
• 15 Compounds 14 and 15 A mixture of thiomannosyl donor 6 (270 mg, 0.457 mmol), BSP (96 mg, 0.4575 mmol, 1 equiv), TTBP (227 mg, 0.915 mmol, 2 equiv) and 4 Å molecular sieves (0.6 g) in CH₂Cl₂ (6.2 mL) was stirred under an atmosphere of argon for 1 h. The reaction was cooled to –60 °C and Tf₂O (92 μL, 0.549 mmol, 1.2 equiv) was added. After 30 min of stirring at –60 °C, a solution of the acceptor 13 (218 mg, 0.869 mmol, 1.4 equiv) in CH₂Cl₂ (3.3 mL) was added. The reaction mixture was stirred for a further 1 h at –60 °C and then quenched by the addition of triethyl phosphite (149 μL, 0.869 mmol). The mixture was filtered and the filtrate (100 mL) was washed with saturated NaHCO₃ (70 mL) and brine (60 mL). The organic phase was dried (MgSO₄), filtered and the filtrate concentrated under reduced pressure. The crude product was purified by silica gel chromatography (n-hexane/EtOAc, 7:3 to 55:45) to give 14 (200 mg, 54%) as a colorless syrup and 15 (32 mg, 8.5%) as a colorless syrup. Compound 14 [α]_D ²⁵ –34.5 (c 0.20, CHCl₃); R_f = 0.44 (n-hexane/EtOAc, 7:3). ¹H NMR (400 MHz, CDCl₃): δ = 7.82–7.25 (m, 22 H, arom), 5.63 (s, 1 H, H_ac), 5.48 (d, J ₁,₂ = 1.3 Hz, 1 H, H-1), 5.39 (dd, J ₂,₃ = 3.3 Hz, J ₁,₂ = 1.6 Hz, 1 H, H-2), 5.21 (t, J ₃,₄ = J ₄,₅ = 9.8 Hz, 1 H, H-4), 4.95–4.70 (m, 4 H, NAP-CH ₂, Bn-CH ₂), 4.54 (s, 1 H, H-1′), 4.32 (dd, J = 10.5 Hz, J = 4.9 Hz, 1 H, H-6′a), 4.26–4.09 (m, 2 H, H-4′, H-5), 4.03 (dd, J ₃,₄ = 9.8 Hz, J ₂,₃ = 3.4 Hz, 1 H, H-3), 3.90 (t, J = 10.3 Hz, 1 H, H-6′b), 3.75 (d, J _1′,_2′ = J _2′,_3′ = 3.1 Hz, 1 H, H-2′), 3.55 (dd, J _2′,_3′ = 3.2 Hz, J _3′,_4′ = 9.9 Hz, 1 H, H-3′), 3.32 (td, J = 9.8 Hz, J = 4.9 Hz, 1 H, H-5′), 2.00, 1.85 (2 × s, 6 H, 2 × Ac-CH ₃), 1.21 (d, J = 6.2 Hz, 3 H, CH ₃). ¹³C NMR (100 MHz, CDCl₃): δ = 170.2, 169.8 (2 C, 2 × Ac-CO), 138.6, 137.7, 135.8, 133.5, 133.3, 133.1 (6 C, 6 × C_q arom), 131.8–125.8 (22 C, arom), 103.6 (1 C, J _{C1′–H1′} = 152.5 Hz, C-1′), 101.6 (1 C, C_ac), 85.5 (1 C, C-1), 78.3 (1 C, C-4′), 77.1 (1 C, C-3′), 76.2 (1 C, C-2′), 76.1 (1 C, C-3), 74.7 (1 C, Bn-CH₂), 73.9 (1 C, C-2), 73.0 (1 C, C-4), 72.3 (1 C, Bn-CH₂), 68.6 (1 C, C-6′), 67.9 (1 C, C-5′), 67.8 (1 C, C-5), 21.0, 20.8 (2 C, 2 × Ac-CH₃), 17.5 (1 C, 1 × CH₃). HRMS (UHR ESI-QTOF): m/z [M + Na]⁺ calcd for C₄₇H₄₈NaO₁₁S: 843.2810; found: 843.2810. Compound 15 [α]_D ²⁵ –15.0 (c 0.20, CHCl₃); R _f = 0.53 (n-hexane/EtOAc, 7:3). ¹H NMR (400 MHz, CDCl₃): δ = 7.82–7.25 (m, 22 H, arom), 5.64 (s, 1 H, H_ac), 5.55 (dd, J ₂,₃ = 2.9 Hz, J ₁,₂ = 1.3 Hz, 1 H, H-2), 5.40 (s, 1 H, H-1), 5.07 (s, 1 H, H-1′), 5.02 (t, J ₃,₄ = J ₄,₅ = 9.8 Hz, 1 H, H-4), 4.82–4.65 (m, 4 H, NAP-CH ₂, Bn-CH ₂), 4.32–4.27 (m, 2 H, H-5, H-6′a), 4.25 (t, J _3′,_4′ = J _4′,_5′ = 9.7 Hz, 1 H, H-4′), 4.08 (dd, J ₃,₄ = 9.8 Hz, J ₂,₃ = 3.3 Hz, 1 H, H-3), 3.90 (t, J = 10.3 Hz, 1 H, H-6′b), 3.81–3.75 (m, 2 H, H-3′, H-5′), 3.58–3.57 (m, 1 H, H-2′), 2.02, 1.94 (2 × s, 6 H, 2 × Ac-CH ₃), 1.22 (d, J = 6.2 Hz, 3 H, CH ₃). ¹³C NMR (100 MHz, CDCl₃): δ = 170.2, 170.1 (2 C, 2 × Ac-CO), 138.2, 137.8, 136.0, 133.4, 133.3, 133.0 (6 C, 6 × C_q arom), 131.8–125.8 (22 C, arom), 101.9 (1 C, C_ac), 95.2 (1 C, J _{C1′–H1′} = 171.4 Hz, C-1′), 86.1 (1 C, C-1), 78.6 (1 C, C-4′), 75.9 (1 C, C-3′), 75.7 (1 C, C-2′), 73.1, 73.0 (2 C, 2 × Bn-CH₂), 71.7 (1 C, C-4), 70.6 (1 C, C-3), 69.0 (1 C, C-2), 68.9 (1 C, C-6′), 67.9 (1 C, C-5), 64.8 (1 C, C-5′), 21.1, 20.8 (2 C, 2 × Ac-CH₃), 17.5 (1 C, 1 × CH₃). HRMS (UHR ESI-QTOF): m/z [M + Na]⁺ calcd for C₄₇H₄₈NaO₁₁S: 843.2810; found: 843.2805.
• 16a Xia J, Abbas SA, Locke RD, Piskorz CF, Alderfer JL, Matta KL. Tetrahedron Lett. 2000; 41: 169
• 16b Wright JA, Yu J, Spencer JB. Tetrahedron Lett. 2001; 42: 4033
• 17 Compound 17 Compound 16 (111 mg, 0.094 mmol) was dissolved in a mixture of CH₂Cl₂ (1.46 mL) and water (161 μL), DDQ was added (32 mg, 0.1413 mmol) and the resulting mixture was stirred for 1.5 h at room temperature. The mixture was then diluted with CH₂Cl₂ (50 mL) and washed with a saturated aqueous solution of NaHCO₃ (2 × 20 mL) and water (3 × 20 mL) until neutral pH. The organic phase was dried over MgSO₄, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel chromatography (n-hexane/EtOAc, 6:4) to give 17 (80 mg, 83%) as a colorless syrup. [α]_D ²⁵ +59.6 (c 0.15, CHCl₃); R_f = 0.36 (n-hexane/EtOAc, 6:4). ¹H NMR (400 MHz, CDCl₃): δ = 7.49–6.82 (m, 24 H, arom), 5.57 (s, 1 H, H_ac), 5.51 (s, 1 H, H_ac), 5.39 (d, J ₂,₃ = 3.3 Hz, 1 H, H-2′), 5.27 (s, 1 H, H-1′), 5.06 (t, J _3′,_4′ = J _4′,_5′ = 9.9 Hz, 1 H, H-4′), 5.01 (d, J ₁,₂ = 7.6 Hz, 1 H, H-1), 4.97–4.88 (m, 3 H, NAP-CH ₂, Bn-CH _2a), 4.65 (s, 1 H, H-1′′), 4.53 (d, J = 11.6 Hz, 1 H, Bn-CH _2b), 4.39–4.30 (m, 2 H, H-6a, H-6′′a), 4.17 (dd, J _4′,_5′ = 10.0 Hz, J _5′,_CH3 = 6.2 Hz, 1 H, H-5′), 4.05 (dd, J _3′,_4′ = 10.2 Hz, J _2′,_3′ = 3.0 Hz, 1 H, H-3′), 4.02 (t, J ₂,₃ = J ₃,₄ = 9.2 Hz, 1 H, H-3), 3.86–3.71 (m, 9 H, H-2, H-2′′, H-3′′, H-4′′, H-6b, H-6′′b, OCH ₃), 3.67 (t, J ₃,₄ = J ₄,₅ = 9.5 Hz, 1 H, H-4), 3.52 (td, J = 9.7 Hz, J = 4.9 Hz, 1 H, H-5), 3.32 (td, J = 9.6 Hz, J = 4.9 Hz, 1 H, H-5′′), 2.51 (d, J = 8.2 Hz, 1 H, H-3′′-OH), 1.98, 1.94 (2 × s, 6 H, 2 × Ac-CH ₃), 0.80 (d, J = 6.2 Hz, 3 H, CH ₃). ¹³C NMR (100 MHz, CDCl₃): δ = 169.8, 169.7 (2 C, 2 × Ac-CO), 155.7, 150.9, 138.2, 137.6, 137.3, 137.2 (6 C, 6 × C_q arom), 129.2–114.7 (24 C, arom), 103.4 (1 C, C-1′′), 103.1 (1 C, C-1), 102.0, 101.8 (2 C, 2 × C_ac), 97.6 (1 C, C-1′), 82.2 (1 C, C-2), 79.3 (1 C, C-4′′), 79.0 (1 C, C-4), 78.0 (1 C, C-2′′), 76.2 (1 C, C-3′), 76.1 (1 C, C-3), 75.1, 75.0 (2 C, 2 × Bn-CH₂), 72.9 (1 C, C-4′), 71.8 (1 C, C-2′), 70.4 (1 C, C-3′′), 68.8 (1 C, C-6), 68.6 (1 C, C-6′), 67.1 (1 C, C-5′′), 66.4 (1 C, C-5), 66.0 (1 C, C-5′), 55.7 (1 C, OCH₃), 21.0, 20.9 (2 C, 2 × Ac-CH₃), 16.8 (1 C, 1 × CH₃). HRMS (UHR ESI-QTOF): m/z [M + Na]⁺ calcd for C₅₇H₆₂NaO₁₈: 1057.3828; found: 1057.3829.
• 18 Compound 19 Compound 18 (805 mg, 0.523 mmol) was dissolved in dry THF (11 mL) and the reaction mixture was cooled to 0 °C. TBAF (1.046 mL, 1 M in dry THF, 1.046 mmol, 2.0 equiv) was added and the reaction mixture was stirred for 24 h at room temperature. The reaction mixture was diluted with EtOAc (300 mL) and washed with water (75 mL) and brine (75 mL). The organic layer was dried (MgSO₄), filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel chromatography (n-hexane/acetone, 6:4) to give 19 (718 mg, 96%) as a white foam. [α]_D ²⁵ –55.8 (c 0.12, CHCl₃); R_f = 0.34 (n-hexane/acetone, 6:4). ¹H NMR (400 MHz, CDCl₃): δ = 7.90–6.87 (m, 36 H, arom), 5.61, 5.59, 5.48 (3 × s, 3 H, 3 × H_ac), 5.42 (d, J _2′,_3′ = 3.4 Hz, 1 H, H-2′), 5.32 (s, 1 H, H-1′), 5.13 (t, J _3′,_4′ = J _4′,_5′ = 9.8 Hz, 1 H, H-4′), 5.06 (d, J ₁,₂ = 7.7 Hz, 1 H, H-1), 5.01–4.84 (m, 5 H, NAP-CH ₂, Bn-CH ₂, Bn-CH _2a), 4.65 (s, 1 H, H-1′′), 4.63 (d, J = 12.8 Hz, 1 H, Bn-CH _2b), 4.44–4.38 (m, 2 H, H-6a, H-6′′′a), 4.22–3.76 (m, 17 H), 3.72 (t, J ₃,₄ = J ₄,₅ = 9.4 Hz, 1 H, H-4), 3.57 (td, J = 9.7 Hz, J = 5.0 Hz, 1 H), 3.46–3.38 (m, 2 H), 3.04 (td, J = 9.8 Hz, J = 4.9 Hz, 1 H), 2.86 (s, 1 H, H-2′′′-OH), 2.05, 2.01 (2 × s, 6 H, 2 × Ac-CH ₃), 0.85 (d, J = 6.1 Hz, 3 H, CH ₃). ¹³C NMR (100 MHz, CDCl₃): δ = 169.8, 169.6 (2 C, 2 × Ac-CO), 155.7, 151.0, 138.1, 137.7, 137.5, 137.3, 137.2, 135.7, 133.4, 133.2 (10 C, 10 × C_q arom), 129.2–114.8 (36 C, arom), 103.4 (1 C, C-1′′), 103.2 (1 C, C-1), 101.9, 101.8, 101.5 (3 C, 3 × C_ac), 97.6 (1C, C-1′), 96.0 (1C, C-1′′′), 82.2, 79.1, 78.4, 76.5, 76.2, 76.0, 75.6, 73.6, 73.3, 71.8, 69.6, 67.9, 67.0, 66.4, 66.0 (16 C, skeleton carbons), 75.2, 74.0, 72.1 (3 C, 2 × Bn-CH₂, NAP-CH₂), 68.9, 68.7 (3 C, 3 × C-6), 55.8 (1 C, OCH₃), 21.0 (2 C, 2 × Ac-CH₃), 16.9 (1 C, 1 × CH₃). HRMS (UHR ESI-QTOF): m/z [M + Na]⁺ calcd for C₈₁H₈₄NaO₂₃: 1447.5296; found: 1447.5298.
• 19 Compound 8 Compound 20 (396 mg, 0.226 mmol) was dissolved in MeOH (10 mL) then NaOMe was added (pH = 10–12) and the reaction mixture was stirred for 24 h at room temperature. The reaction mixture was neutralized with Amberlite IR-120 (H⁺) ion-exchange resin and then filtered, the resin was washed with MeOH and the filtrate was concentrated. [α]_D ²⁵ –36.7 (c 0.25, CHCl₃); R_f = 0.51 (CH₂Cl₂/MeOH, 9:1). HRMS (UHR ESI-QTOF): m/z calcd for [M + Na]⁺ C₈₃H₉₀NaO₂₆: 1525.5613; found: 1525.5622. The crude product was reacted further without purification. A mixture of the crude product (339 mg, 0.226 mmol) in EtOH (17 mL)/AcOH (96%, 1 mL) and Pd/C (10%, 235 mg) was stirred in an autoclave under a H₂ atmosphere (10 bar) for 24 h. The reaction mixture was diluted with MeOH, filtered through a pad of Celite^® and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (CH₂Cl₂/MeOH/H₂O, 7:6:1) to give compound 8 (145 mg, 70% over two steps) as a white solid. [α]_D ²⁵ –13.5 (c 0.14, MeOH); R_f = 0.29 (CH₂Cl₂/MeOH/H₂O, 7:6:1). 1H NMR (400 MHz, D2O) δ = 7.01–6.85 (m, 4 H, arom), 5.08 (s, 2 H, H-1-B, H-1-E), 4.90 (d, J1,2 = 7.7 Hz, 1 H, H-1-A), 4.76 (s, 2 H, H-1-C, H-1-D), 4.21–4.19 (m, 2 H, H-2-C, H-2-E), 4.11–4.08 (m, 2 H, H-2-D, H-5-E), 3.99–3.96 (m, 2 H, H-2-B, H-5-B), 3.87–3.83 (m, 2H, H-3-E, H-3-B), 3.80–3.76 (m, 5 H, H-3-C, 4 x H-6a), 3.69 (s, 3 H, OCH3), 3.66–3.44 (m, 13 H, H-2-A, H-3-A, H-4-A, H-5-A, H-4-B, H-4-C, H-3-D, H-4-D, H-4-E, 4 x H-6b), 3.32–3.27 (m, 2 H, H-5-C, H-5-D), 1.16 (d, J = 6.2 Hz, 3 H, CH3). 13C NMR (100 MHz, D2O) δ = 154.7, 150.8 (2 C, 2 x Cq arom), 118.2, 115.0 (4C, arom), 101.2 (1 C, C-1-C), 101.1 (1 C, C-1-E), 101.0 (1 C, C-1-A), 100.6 (1 C, C-1-B), 96.6 (1 C, C-1-D), 81.8 (1 C, C-3-A), 79.4 (1 C, C-3-E), 79.1 (1 C, C-3-C), 76.8 (1 C, C-5-C), 76.2 (1 C, C-5-D), 76.0 (1 C, C-5-A), 75.6 (1 C, C-2-D), 73.6 (1 C, C-3-D), 73.5 (1 C, C-2-A), 72.3 (1 C, C-5-E), 71.0 (1 C, C-4-E), 70.3 (1 C, C-2-E), 70.2 (1 C, C-3-C), 70.0 (1 C, C-2-B), 68.8 (1 C, C-5-B), 67.8 (1 C, C-4-A), 67.6 (1 C, C-2-C), 66.8 (1 C, C-4-D), 66.5 (1 C, C-4-B), 65.0 (1 C, C-4-C), 61.0, 60.9, 60.7, 60.5 (4 C, 4 x C-6), 55.7 (1 C, OCH3), 16.5 (1 C, 1 x CH3). HRMS (UHR ESI-QTOF): m/z [M + Na]⁺ calcd for C₃₇H₅₈NaO₂₆: 941.3109; found: 941.3112.
• 20 Fu T.-K, Ng S.-K, Chen Y.-E, Lee Y.-C, Demeter F, Herczeg M, Borbás A, Chiu C.-H, Lan C.-Y, Chen C.-L, Chang MD.-T. Mar. Drugs 2019; 17: 355