Elsevier

Tetrahedron

Volume 66, Issue 39, 25 September 2010, Pages 7850-7855
Tetrahedron

Synthesis of the Lewis b pentasaccharide and a HSA-conjugate thereof

https://doi.org/10.1016/j.tet.2010.07.036Get rights and content

Abstract

Helicobacter pylori, a gastric pathogen, binds to various blood group antigens, including the Lewis types, present in the gastric tissue and a relation between the presentation of the ligands and the overall strength of binding has been assumed. Synthetic Lewis b tetra- and hexasaccharide conjugates are available but not the analogous pentasaccharide. An efficient synthesis of the amino spacer equipped Lewis b pentasaccharide, 3-aminopropyl α-l-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→3)-[α-l-fucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→3)-β-d-galactopyranoside, is presented to enable further investigation of the carbohydrate recognition process of H. pylori.

Introduction

The spiral-shaped Gram-negative bacterium Helicobacter pylori, a gastric pathogen, binds to various blood group antigens, including the Lewis types, present in the gastric tissue.1, 2 In developed countries, approximately 50% of the population over the age of 50 are infected with this bacterium, while, in contrast, such infection is uncommon in children. In developing nations, 70–90% of the population are carrying H. pylori and virtually everyone gets infected during childhood.3, 4

The adhesion process of H. pylori bacteria to Lewis b structures is mediated by a membrane lectin, the blood group antigen binding adhesion protein (BabA).5 Synthetic glycoconjugates containing the Lewis b hexasaccharide ligand are used in affinity chromatography for purification of the BabA, but analogous Lewis b tetrasaccharide conjugates are not effective for this purpose, although the free tetrasaccharide can inhibit the adhesion process. Reductive amination, the conjugation technique preferably used for preparing glycoconjugates from native Lewis b hexasaccharide, destroys the reducing end glucose unit. These glycoconjugates are feasible for purification purposes, although only a pentasaccharide unit is available for recognition. Kojima et al.6 investigated the adhesion properties of Lewis b oligosaccharide conjugates linked to Bovine serum albumin (BSA), and polyacrylamide (PAA) and palmitoylphosphatidylethanolamine (DPPE), concluding that the recognition is affected by the carrier. The DPPE conjugate was synthesised via reductive amination from the hexasaccharide, but no further information was given for the purchased conjugates (Lewis b hexasaccharide BSA-conjugate, Funakoshi, Tokyo, Japan; Lewis b tetrasaccharide PAA-conjugate, Seikagaku Kogyuo Co., Tokyo, Japan). All conjugates were recognised by H. pylori, but the BSA-conjugate was clearly the best recognised conjugate.

Syntheses of both the Lewis b tetrasaccharide and the hexasaccharide have been published. The synthesis of the Lewis b tetrasaccharide has been elaborated many times, e.g., as the free reducing oligosaccharide by Matta et al.7 and as methyl glycoside, first by Lemieux and Spohr8 and later by Kahne and Yan.9 Solid phase synthesis has been employed by Danishefsky’s group10 and a large scale approach, reducing chromatography to a bare minimum, was achieved by Norberg et al.11 Recently an enzymatic approach has been established.12 Also for the Lewis b hexasaccharide several approaches have been investigated and human serum albumin (HSA) conjugates have been prepared.13, 14, 15, 16 The pentasaccharide structure (Fig. 1) has been prepared earlier as a protected intermediate, e.g., in Danishefsky’s glycal approach, but has never been deprotected and used for synthetic conjugates. To further investigate the binding specificity of BabA, we envisaged using synthetic Lewis pentasaccharide glycoconjugates. To obtain comparable parameters, we intended to use the same spacer and conjugation techniques as in earlier studies with the hexa- and tetrasaccharides.

Section snippets

Results and discussion

Attempts to adapt the synthetic approach used for our previous synthesis of the Lewis b hexasaccharide,16 where a suitable protected lacto-N-tetraose unit was assembled by a 2+2 approach to gain quick access to the desired target pentasaccharide 1 were hampered by the loss of regioselectivity during the coupling of the disaccharide donor to the analogous 3-azidopropyl 2,6-di-O-benzyl galactoside acceptor. Glycosylations, using the 4-O-benzyl protected derivative 4 as glycosyl acceptor instead,

General methods

Organic solutions were dried over Na2SO4 before concentration, which was performed under reduced pressure at <40 °C (bath). NMR spectra were recorded at 25 °C at 300 or 400 MHz (Varian) or 500 MHz (Bruker) (1H) or 75, 100 or 125 MHz (13C), respectively, CDCl3, D2O or at ambient temperature if not otherwise stated. All proton and carbon NMR spectra in CDCl3 were referenced to the chloroform signal (1H δ 7.27 ppm, 13C δ 77.17 ppm) if not otherwise stated. No reference was used for the spectrum in D2O.

Acknowledgements

Financial support from the Swedish Research Council (Vetenskapsrådet), The Royal Society (RG081255), the European Commission (MRTN-CT-2004-005645 GlycoGold), and the Science Foundation Ireland (Grant Number 08/IN.1/B2067) are gratefully acknowledged.

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