Synlett 2020; 31(05): 469-474
DOI: 10.1055/s-0039-1690747
letter
© Georg Thieme Verlag Stuttgart · New York

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

Fruzsina Demeter
a   Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary
b   MTA-DE Molecular Recognition and Interaction Research Group, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary
c   Doctoral School of Chemistry, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary
,
Margaret Dah-Tsyr Chang
d   Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
,
Yuan-Chuan Lee
d   Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
e   Department of Biology, Johns Hopkins University, Baltimore, ML 21218, USA
,
Anikó Borbás
a   Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary
,
Mihály Herczeg
a   Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary
f   Research Group for Oligosaccharide Chemistry of HAS, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary   eMail: herczeg.mihaly@science.unideb.hu
› Institutsangaben
This research was supported by the New National Excellence Program of the Ministry of Human Capacities (ÚNKP-19-3/4), the Ministry of Science and Technology (MOST) (MOST 107-0210-01-19-04), the Industrial Value Creation Program for Academia (108B7016V1), the Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis (AS-KPQ-108-TSPA). The authors gratefully acknowledge financial support for this research from the Premium Postdoctoral Program of HAS (PPD 461038), Nemzeti Kutatási Fejlesztési és Innovációs Hivatal (National Research, Development and Innovation Office of Hungary) (K 128368) and from the European Union co-financed by the European Regional Development Fund (GINOP-2.3.2-15-2016-00008 and GINOP-2.3.3-15-2016-00004).
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Publikationsverlauf

Received: 09. Oktober 2019

Accepted after revision: 30. Oktober 2019

Publikationsdatum:
19. November 2019 (online)


Published as part of the Special Section 11th 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.

Supporting Information

 
  • 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); Rf = 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, Hac), 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 × Cq arom), 131.8–125.8 (22 C, arom), 103.6 (1 C, J C1′–H1′ = 152.5 Hz, C-1′), 101.6 (1 C, Cac), 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, Hac), 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 × Cq arom), 131.8–125.8 (22 C, arom), 101.9 (1 C, Cac), 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); Rf = 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, Hac), 5.51 (s, 1 H, Hac), 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 × Cq 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 × Cac), 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); Rf = 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 × Hac), 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 × Cq 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 × Cac), 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); Rf = 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); Rf = 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