Synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O117:K98:H4

  1. Pintu Kumar Mandal

Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram extension, Sitapur Road, Lucknow, 226 031, India

  1. Corresponding author email

Associate Editor: S. Flitsch
Beilstein J. Org. Chem. 2014, 10, 2724–2728. doi:10.3762/bjoc.10.287
Received 19 Aug 2014, Accepted 06 Nov 2014, Published 20 Nov 2014

Abstract

The pentasaccharide repeating unit of the O-antigen of E. coli O117:K98:H4 strain has been synthesized using a combination of sequential glycosylations and [3 + 2] block synthetic strategy from the suitably protected monosaccharide intermediates. Thioglycosides and glycosyl trichloroacetimidate derivatives have been used as glycosyl donors in the glycosylations.

Keywords: Escherichia coli; glycosylation; lipopolysaccharide; O-antigen; pentasaccharide

Introduction

Escherichia coli becomes an important human pathogen in recent years owing to the emergence of new pathogenic strains [1]. Several diseases, such as meningitis and sepsis [2], diarrhoeal outbreaks [3] and urinary tract infections [4] are associated with pathogenic Escherichia coli (E. coli) strains. E. coli strains have been found to produce the Shiga toxin (Stx), heat-labile (LT) or heat-stable (ST) enterotoxins, cytotoxic necrotizing factors (CNF1 and CNF2) and hemolysins (α-Hly and E-Hly) [5,6] and are responsible for hemorrhagic colitis and haemolytic-uremic syndroms in humans [7]. The different strains of E.coli as well as bacteria belonging to different genera, e.g., Shigella, Salmonella, and Klebsiella show serological cross-reactions within the species [8]. The E. coli O117 strain emerged as a significant cause for septicaemia, bovine diarrhoea in new born children and human [9]. Together with other E. coli strains E. coli 0117 strains are responsible for pyelonephritis which is sexually transmitted by a woman that spread up to 60 to 80% of community acquired urinary tract or travelled through the bloodstream to the kidneys [10,11]. The O-specific polysaccharide of E. coli O117:K98:H4 is a linear pentasaccharide repeating unit consisting of D-galactosamine, D-glucose, D-galactose, and L-rhamnose (Figure 1) [12].

[1860-5397-10-287-1]

Figure 1: Structure of the pentasaccharide repeating unit of the O-specific polysaccharide of E. coli O117:K98:H4.

Vaccination is the recent thrust in the drug discovery program to prevent bacterial infections. Several bacterial O-antigens have been chosen for the development of glycoconjugate vaccine candidates against infectious diseases [13-16]. As a consequence, a significant quantity of oligosaccharides is required to evaluate their immunological properties for detailed understanding of the role of O-antigens in the pathogenicity of the E. coli strains. Development of chemical synthetic strategies would be useful to get large quantities of the oligosaccharides. As a part of the ongoing studies on the synthesis of bacterial cell wall oligosaccharides [17-19], a straightforward synthesis of the pentasaccharide repeating unit of the O-specific polysaccharide of E. coli O117:K98:H4 as its 3-aminopropyl glycoside is presented herein (Figure 2). The 3-aminopropyl group would be suitable for attachment of the pentasaccharide to any surface or carrier proteins.

[1860-5397-10-287-2]

Figure 2: Structure of the synthesized pentasaccharide (1) and its precursor intermediates.

Results and Discussion

The target pentasaccharide 1 has been synthesized as its 3-aminopropyl glycoside using a combination of sequential and [3 + 2] block glycosylation strategy. A trisaccharide acceptor 11 and a disaccharide trichloroacetimidate donor 14 were synthesized from the appropriately protected monosaccharide intermediates 2 [20], 3 [21], 4 [22], 5 and 6 [23] (Figure 2) derived from the commercially available aldoses. Trisaccharide acceptor 11 was then glycosylated with disaccharide trichloroacetimidate donor 14 to form pentasaccharide derivative 15, which was finally deprotected to give target pentasaccharide 1 (see below Scheme 2). Some of the notable features of this synthetic strategy are (a) application of iodonium ion mediated general glycosylation conditions; (b) nitrosyl tetrafluoroborate (NOBF4) mediated activation of glycosyl trichloroacetimidate donor; (c) the attachment of an aminopropyl linker at the anomeric center; (d) glycosylation and removal of the p-methoxybenzyl (PMB) group in one-pot.

Treatment of p-methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (7) [24] (prepared from D-galactosamine hydrochloride in six steps) with acetic anhydride in pyridine followed by regioselective reductive opening of the benzylidene acetal on treatment with sodium cyanoborohydride in the presence of HCl/Et2O [25] furnished p-methoxyphenyl 3-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (5) in 77% yield over two steps (Scheme 1).

[1860-5397-10-287-i1]

Scheme 1: Reagents and conditions: (a) (i) acetic anhydride, pyridine, room temperature, 2 h; (ii) NaBH3CN, HCl/Et2O, 5 °C, 2 h, 77% overall yield.

Trisaccharide acceptor 11 could be synthesized following the reaction pathway depicted in Scheme 2. Glycosylation of 3-azidopropyl 2,3,6-tri-O-benzyl-β-D-galactopyranoside (2) with the thioglycoside donor 3 in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) [26,27] gave disaccharide derivative 8 in 72% yield. NMR spectroscopy confirmed the formation of compound 8 [δ 5.04 (d, J = 3.6 Hz, 1H, H-1B), 4.38 (d, J = 7.6 Hz, 1H, H-1A) in 1H NMR and at δ 103.9 (C-1A), 100.5 (C-1B) in 13C NMR spectra]. Following an earlier report [28], cleavage of the benzylidene acetal from compound 8 catalyzed by perchloric acid on silica (HClO4/SiO2) [28,29] afforded 3-azidopropyl (2,3-di-O-benzyl-β-D-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-galactopyranoside (9) in 85% yield. Selective 6-O-benzoylation of compound 9 was accomplished with benzoyl cyanide [30] to furnish disaccharide acceptor 10 in 80% yield. Compound 10 was reacted with 3-O-PMB protected L-rhamnosylthioglycoside donor 4 and NIS/TfOH [26,27] to yield the trisaccharide derivative by an iodonium ion catalyzed glycosylation. Participation of the 2-O-acetyl group in donor 4 ensured the α-selectivity of the glycosylation. Following an earlier report [19], raising the temperature of the reaction mixture after the glycosylation led to the removal of the PMB group in the same pot [31] to furnish trisaccharide acceptor 11 in 77% yield. The formation of compound 11 was supported by NMR spectral analysis [signals at δ 4.85 (d, J = 2.4 Hz, 1H, H-1B), 4.84 (brs, 1H, H-1C), 4.18 (d, J = 7.4 Hz, 1H, H-1A) and at δ 104.1 (C-1A), 98.7 (C-1B), 97.6 (C-1C) in the 1H and 13C NMR spectra respectively]. In another experiment, coupling of 2-azido-α-D-galactosyl trichloroacetimidate derivative 6 and compound 5 in the presence of NOBF4 [32] in Et2O/CH2Cl2 gave disaccharide derivative 12 in 75% yield. The formation of compound 12 was confirmed by NMR spectroscopic analysis [signals at δ 5.92 (d, J = 8.1 Hz, 1H, H-1D), 5.06 (d, J = 2.7 Hz, 1H, H-1E) in the 1H NMR and δ 99.1 (C-1E), 97.7 (C-1D) in the 13C NMR, respectively]. Reduction of the azido groups were carried out by treatment with triphenylphosphine [33], then the product was acetylated using acetic anhydride and pyridine to give disaccharide derivative 13 in 84% overall yield in two steps. The anomeric PMP group of compound 13 was oxidatively cleaved using ceric(IV) ammonium nitrate (CAN) [15] and the hemeacetal thus obtained was reacted with trichloroacetonitrile in the presence of DBU [34] to afford the desired disaccharide trichloroacetimidate derivative 14 in 77% yield. It was used directly without further purification [17] (Scheme 2). Finally, glycosylation of trisaccharide acceptor 11 with the trichloroacetimidate donor 14 in the presence of NOBF4 [32] in CH2Cl2 furnished pentasaccharide derivative 15 in 70% yield. Formation of compound 15 was supported by NMR spectral analysis [signals at δ 5.10 (d, J = 3.3 Hz, 1H, H-1E), 5.00 (d, J = 2.4 Hz, 1H, H-1B), 4.92 (brs, 1H, H-1C), 4.69 (d, J = 7.7 Hz, 1H, H-1D),4.35 (d, J = 7.8 Hz, 1H, H-1A) and at δ 104.0 (C-1A), 101.2 (C-1D), 98.9 (C-1B), 98.3 (C-1C), 97.4 (C-1E) in the 1H and 13C NMR spectra, respectively]. The N-phthaloyl group was removed using hydrazine hydrate and the free amine thus formed was acetylated using acetic anhydride in pyridine [35]. Then the removal of the benzyl group was accomplished by hydrogenation using Pearlman’s catalyst [36]. Finally the acetates were removed by Zemplén de-O-acetylation [37] using sodium methoxide to afford the target pentasaccharide 1 in 58% overall yield (Scheme 2).

[1860-5397-10-287-i2]

Scheme 2: Reagents: (a) N-iodosuccinimide (NIS), TMSOTf, CH2Cl2, MS 4 Å, −30 °C, 1 h, 72%; (b) HClO4/SiO2, CH3CN, rt, 20 min, 85%; (c) benzoyl cyanide, DCM/pyridine, rt, 2 h, 80%; (d) N-iodosuccinimide (NIS), TfOH, CH2Cl2, MS 4 Å, −30 °C, 1 h, then 0 °C, 1 h, 77%; (e) NOBF4, Et2O/CH2Cl2 (3:1), −15 °C, 1 h, 75%; (f) PPh3, THF, 6 h, then Ac2O, pyridine, rt, 1 h, 84%;(g) CAN, CH3CN, H2O, rt, 1.5 h; (h) CCl3CN, DBU, CH2Cl2, −10 °C, 1 h; (i) NOBF4, CH2Cl2, −15 °C, 1 h, 70%; (j) i) NH2NH2·H2O, CH3CH2OH, 80 °C, 8 h, ii) acetic anhydride, pyridine, rt, 1 h; (k) H2, 20% Pd(OH)2/C, CH3OH, rt, 24 h; (l) 0.1 M CH3ONa, CH3OH, rt, 3 h, 58% overall yield.

Conclusion

In summary, a [3 + 2] block glycosylation strategy has been developed to synthesize a pentasaccharide 3-aminopropyl glycoside (1) corresponding to the O-antigen of E. coli O117:K98:H4 strain. The in situ removal of the PMB ether in one-pot following the glycosylation reaction reduced the overall number of steps.

Supporting Information

Supporting Information File 1: Experimental part.
Format: PDF Size: 2.4 MB Download

Acknowledgements

The author gratefully acknowledges financial support by the Department of Science and Technology (DST), India under Fast Track Proposal Scheme for Young Scientists (CS-127/2012) and SAIF Division of CSIR-CDRI for providing the spectroscopic and analytical data. CDRI communication no. 8849.

References

  1. Johnson, J. R.; Russo, T. A. J. Lab.Clin. Med. 2002, 139, 155–162. doi:10.1067/mlc.2002.121550
    Return to citation in text: [1]
  2. Robbins, J. B.; McCracken, G. H.; Gotschlich, E. C.; Ørskov, F.; Ørskov, I.; Hanson, L. A. N. Engl. J. Med. 1974, 290, 1216–1220. doi:10.1056/NEJM197405302902202
    Return to citation in text: [1]
  3. Johnson, J. R. Clin. Microbiol. Rev. 1991, 4, 80–128.
    Return to citation in text: [1]
  4. Besser, R. E.; Lett, S. M.; Weber, J. T.; Doyle, M. P.; Barrett, T. J.; Wells, J. G.; Griffin, P. M. JAMA, J. Am. Med. Assoc. 1993, 269, 2217–2220. doi:10.1001/jama.1993.03500170047032
    Return to citation in text: [1]
  5. Gay, C. C.; Besser, T. E. Escherichia coli septicaemia in calves, 1st ed.; CAB International: Wallingford, UK, 1994; p 75.
    Return to citation in text: [1]
  6. Salvadori, M. R.; Valadares, G. F.; da Silva Leite, D.; Blanco, J.; Yano, T. Braz. J. Microbiol. 2003, 34, 230–235. doi:10.1590/S1517-83822003000300009
    Return to citation in text: [1]
  7. Eklund, M. F.; Scheutz, F.; Siitonen, A. J. Clin. Microbiol. 2001, 39, 2829–2834. doi:10.1128/JCM.39.8.2829-2834.2001
    Return to citation in text: [1]
  8. Ørskov, F.; Ørskov, I. Methods Microbiol. 1984, 14, 43–112. doi:10.1016/S0580-9517(08)70447-1
    Return to citation in text: [1]
  9. Blanco, J.; González, E. A.; Blanco, M.; Garabal, J. I.; Alonso, M. P.; Fernández, S.; Villanueva, R.; Aguilera, A.; García, M. A.; Torres, J.; Rey, A.; Jansen, W. H.; Guinée, P. A. M. J. Med. Microbiol. 1991, 35, 162–167. doi:10.1099/00222615-35-3-162
    Return to citation in text: [1]
  10. Hebelka, M.; Lincoln, K.; Sandberg, T. Scand. J. Infect. Dis. 1993, 25, 141–143.
    Return to citation in text: [1]
  11. Chang, S. L.; Shortliffe, L. D. Pediatr. Clin. North Am. 2006, 53, 379–400. doi:10.1016/j.pcl.2006.02.011
    Return to citation in text: [1]
  12. Leslie, M. R.; Parolis, H.; Parolis, L. A. S. Carbohydr. Res. 1999, 323, 103–110. doi:10.1016/S0008-6215(99)00232-3
    Return to citation in text: [1]
  13. Verez-Bencomo, V.; Fernández-Santana, V.; Hardy, E.; Toledo, M. E.; Rodríguez, M. C.; Heynngnezz, L.; Rodriguez, A.; Baly, A.; Herrera, L.; Izquierdo, M.; Villar, A.; Valdés, Y.; Cosme, K.; Deler, M. L.; Montane, M.; Garcia, E.; Ramos, A.; Aguilar, A.; Medina, E.; Toraño, G.; Sosa, I.; Hernandez, I.; Martínez, R.; Muzachio, A.; Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.; Diaz, M.; Diaz, R. Science 2004, 305, 522–525. doi:10.1126/science.1095209
    Return to citation in text: [1]
  14. Roy, R. Drug Discovery Today: Technol. 2004, 1, 327–336. doi:10.1016/j.ddtec.2004.10.005
    Return to citation in text: [1]
  15. Werz, D. B.; Seeberger, P. H. Angew. Chem., Int. Ed. 2005, 44, 6315–6318. doi:10.1002/anie.200502615
    Return to citation in text: [1] [2]
  16. Meeks, M. D.; Saksena, R.; Ma, X.; Wade, T. K.; Taylor, R. K.; Kovác, P.; Wade, W. F. Infect. Immun. 2004, 72, 4090–4101. doi:10.1128/IAI.72.7.4090-4101.2004
    Return to citation in text: [1]
  17. Mandal, P. K.; Misra, A. K. Glycoconjugate J. 2008, 25, 713–722. doi:10.1007/s10719-008-9120-1
    Return to citation in text: [1] [2]
  18. Mandal, P. K.; Branson, T. R.; Hayes, E. D.; Ross, J. F.; Gavín, J. A.; Daranas, A. H.; Turnbull, W. B. Angew. Chem., Int. Ed. 2012, 51, 5143–5146. doi:10.1002/anie.201109068
    Return to citation in text: [1]
  19. Mandal, P. K.; Dhara, D.; Misra, A. K. Beilstein J. Org. Chem. 2014, 10, 293–299. doi:10.3762/bjoc.10.26
    Return to citation in text: [1] [2]
  20. Joosten, J. A. F.; Loimaranta, V.; Appeldoorn, C. C. M.; Haataja, S.; El Maate, F. A.; Liskamp, R. M. J.; Finne, J.; Pieters, R. J. J. Med. Chem. 2004, 47, 6499–6508. doi:10.1021/jm049476+
    Return to citation in text: [1]
  21. Crich, D.; Bowers, A. A. J. Org. Chem. 2006, 71, 3452–3463. doi:10.1021/jo0526688
    Return to citation in text: [1]
  22. Mukherjee, C.; Misra, A. K. Glycoconjugate J. 2008, 25, 611–624. doi:10.1007/s10719-008-9107-y
    Return to citation in text: [1]
  23. Kalidasan, K.; Su, Y.; Wu, X.; Yao, S. Q.; Uttamchandani, M. Chem. Commun. 2013, 49, 7237–7239. doi:10.1039/c3cc42836b
    Return to citation in text: [1]
  24. Zhang, Z.; Magnusson, G. Carbohydr. Res. 1996, 295, 41–55. doi:10.1016/S0008-6215(96)90118-4
    Return to citation in text: [1]
  25. Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108, 97–101. doi:10.1016/S0008-6215(00)81894-7
    Return to citation in text: [1]
  26. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 1331–1334. doi:10.1016/S0040-4039(00)88799-7
    Return to citation in text: [1] [2]
  27. Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313–4316. doi:10.1016/S0040-4039(00)97609-3
    Return to citation in text: [1] [2]
  28. Agnihotri, G.; Misra, A. K. Tetrahedron Lett. 2006, 47, 3653–3658. doi:10.1016/j.tetlet.2006.03.133
    Return to citation in text: [1] [2]
  29. Chakraborti, A. K.; Gulhane, R. Chem. Commun. 2003, 1896–1897. doi:10.1039/b304178f
    See for a preparation of HClO4-SiO2.
    Return to citation in text: [1]
  30. Abbas, S. A.; Haines, A. H. Carbohydr. Res. 1975, 39, 358–363. doi:10.1016/S0008-6215(00)86148-0
    Return to citation in text: [1]
  31. Bhattacharyya, S.; Magnusson, B. G.; Wellmar, U.; Nilsson, U. J. J. Chem. Soc., Perkin Trans. 1 2001, 886–890. doi:10.1039/b009448j
    Return to citation in text: [1]
  32. Sau, A.; Santra, A.; Misra, A. K. Synlett 2012, 23, 2341–2348. doi:10.1055/s-0032-1317135
    Return to citation in text: [1] [2]
  33. Zou, W.; Sandbhor, M.; Bhasin, M. J. Org. Chem. 2007, 72, 1226–1234. doi:10.1021/jo062057v
    Return to citation in text: [1]
  34. Schmidt, R. R.; Jung, K.-H. In Preparative Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York, NY, 1997; pp 283–312.
    Return to citation in text: [1]
  35. Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O. Carbohydr. Res. 1993, 243, 139–164. doi:10.1016/0008-6215(93)84087-M
    Return to citation in text: [1]
  36. Pearlman, W. M. Tetrahedron Lett. 1967, 8, 1663–1664. doi:10.1016/S0040-4039(00)70335-2
    Return to citation in text: [1]
  37. Zemplén, G. Ber. Dtsch. Chem. Ges. 1926, 59, 1254–1266. doi:10.1002/cber.19260590626
    Return to citation in text: [1]

News

Logo OA

Registration is open for the Beilstein Organic Chemistry Symposium 2018 from 24 to 26 April 2018 in Potsdam, Germany

Logo OA

According to the 2016 Journal Citation Reports (Clarivate Analytics, 2017), the 2016 Journal Impact Factor is 2.337.

© 2014 Mandal; licensee Beilstein-Institut.
This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (http://www.beilstein-journals.org/bjoc)

 
Back to Article List