Studies on the synthesis of Lewis-y oligosaccharides

doi:10.1016/j.carres.2011.07.002

Carbohydrate Research

Volume 346, Issue 14, 18 October 2011, Pages 2113-2120

https://doi.org/10.1016/j.carres.2011.07.002 Get rights and content

Abstract

Lewis-y histo-blood group oligosaccharides are tumour-associated antigens prevalent in several different types of cancer, and they may also be secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. The key step in the synthesis of these sterically congested oligosaccharides involves difucosylation of partially protected lactosamine derivatives. Existing methods require either prolonged reaction times or elaborate glycosyl donors to ensure high stereoselectivity. Herein we report an optimised procedure for using a simple thioglycoside donor that leads to the desired products in high yield and excellent stereoselectivity. It is found that initial glycosylation of the 3′-hydroxy group of lactosamine derivatives in dichloromethane solution can inhibit subsequent glycosylation at the 2-position; however, reaction in toluene solution leads to Lewis-y oligosaccharides in high yield.

Graphical abstract

Introduction

It is now half a century since the molecular features that distinguish blood groups were attributed to the histo-blood group oligosaccharides.¹ More recently, it has become apparent that specific members of this family of oligosaccharides can provide diagnostic and prognostic markers for a variety of diseases.2, 3 One such example is the type II Lewis-y oligosaccharide (1; Fig. 1) which is a tumour-associated antigen prevalent in several different types of cancer.⁴ Furthermore, blood group A oligosaccharides 2 based on the Lewis-y core tetrasaccharide have been identified as ligands for the B-subunit of E. coli heat-labile toxin (LTB)⁵ and also a hybrid protein derived from LTB and the analogous B-subunit of cholera toxin (CTB).⁶ This discovery could provide insight to the blood group dependence of diarrhoeal diseases, in which it has been observed that patients with blood group O are more susceptible to certain strains of cholera than those in blood groups A or B.7, 8

Therefore, as part of our on-going studies into the thermodynamics of bacterial toxin–ligand interactions,9, 10, 11 we sought an efficient synthetic route to oligosaccharides with the Lewis-y core structure. The key synthetic challenge is the difucosylaton reaction leading to the sterically congested tetrasaccharide. Not surprisingly, this important structure has attracted widespread attention from synthetic chemists who favour a range of glycosylation methods. While Lemieux’s bromide anomerisation protocol provides excellent stereoselectivity,12, 13, 14, 15, 16, 17 it typically demands large excesses of glycosyl donor¹⁴ and/or very prolonged reaction times.16, 17 Alternative, more reactive glycosyl donors often show modest stereoselectivity if all of the fucosyl hydroxy groups are benzyl-protected.18, 19 Flowers was the first to observe that introducing ester groups at the 3- and 4-positions of a fucosyl donor could improve stereoselectivity relative to a perbenzylated compound.²⁰ Therefore, virtually all modern donors for this type of difucosylation reaction (e.g., fucosyl fluorides,²¹ phosphates,¹⁸ trichloroacetimidates²² and thioglycosides¹⁹) incorporate ester groups at either the 4-, or 3- and 4-positions. Although this strategy leads to high stereoselectivity, it also significantly increases the length of synthesis to prepare the fucosyl donors. Our aim was to employ thioglycoside donors throughout the synthesis on account of their high stability prior to activation.²³ Therefore, we wondered if it would be possible to optimise the reaction conditions to allow a simple ethyl thioglycoside to provide rapid, efficient and highly stereoselective glycosylation without proceeding through the less reactive fucosyl bromide.²⁴

Section snippets

Results and discussion

Two model disaccharide acceptors were synthesised as outlined in Scheme 1. We chose to use a benzyloxycarbonyl-protected propyl amine as the aglycone as it would be suitable for subsequent conjugation of the oligosaccharides to other molecules for biochemical applications. Thus, glucosamine derivative 3²⁵ was glycosylated with thioglycoside donors 4²⁶ and 8²⁷ under standard conditions to provide disaccharides 5 and 9, respectively, in excellent yield. Disaccharide 5 was then deacetylated to

General methods

All solvents were dried prior to use, according to standard methods. All solvents used for flash chromatography were of GPR grade, except hexane and ethyl acetate, when HPLC grade was used. All concentrations were performed in vacuo, unless otherwise stated. All reactions were performed in oven dried glassware under a N₂(g) atmosphere, unless otherwise stated. Analytical TLC was performed on Silica Gel 60-F²⁵⁴ (Merck) with detection by fluorescence and/or charring following immersion in a 5% H₂

Acknowledgments

The authors thank the Royal Society for funding this research through a Newton International Fellowship awarded to P.K.M. and a Royal Society University Research Fellowship awarded to W.B.T.

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Analogues of cancer-associated Lewis Y (Le^y) antigen with varying structures at the reducing end were synthesized by a highly efficient strategy involving one-pot preactivation-based iterative glycosylation to obtain the key tetra-/pentasaccharide intermediates, which was followed by stereoselective fucosylation. After global deprotection, these oligosaccharides were coupled with carrier protein keyhole limpet hemocyanin. The resultant glycan–protein conjugates were subjected to immunological studies in mice. It was disclosed that the conjugate of the pentasaccharide analogue of Lewis Y antigen was more immunogenic than that of the hexasaccharide analogue, but the antisera of both conjugates could indiscriminately recognize each carbohydrate hapten. These results suggested that the short Lewis Y analogue may be utilized to develop functional conjugate cancer vaccines. More importantly, the results also proved that the reducing-end glucose residue in the hexasaccharide analogue of Lewis Y was probably not involved in its interaction with the immune system, whose discovery can have a broad impact on the design of new cancer vaccines.
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For this reason we propose here the enzymatic synthesis of a functionalized phenolic polymer to immobilize β–galactosidase from Bacillus circulans (ATCC 31382, β–Gal–3). This enzyme recognises N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc) as acceptors and is able to use them to synthesize β-(1 → 3)-galactooligosaccharides, such as Gal–β(1–3)–GlcNAc and Gal–β(1–3)–GalNAc, [2,12–19] which are involved in different biological functions such as metastasis [20–22]. The system described in this work brings to light the biosynthesis of interesting medical carbohydrates in a sustainable way, based on the efficient combination of two enzymes.
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Herein, we report on a highly efficient synthesis of a crystalline protected Lewis^X trisaccharide that was converted to Lewis^X following global deprotection. The trisaccharide was prepared in a highly convergent synthesis (seven steps, longest linear sequence) and in a 38% overall yield using a strategy that involved the regioselective glycosylation of a GlcNAc acceptor with a galactose thioglycoside donor, followed by fucosylation of the remaining free GlcNAc hydroxyl as key steps. The core trisaccharide also has the potential to be converted to other members of the Type-2 Lewis family of antigens due to the orthogonal nature of the protecting groups employed.
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Studies on the synthesis of Lewis-y oligosaccharides

Abstract

Graphical abstract

Introduction

Section snippets

Results and discussion

General methods

Acknowledgments

J. Mol. Biol.

Structure

FEBS Lett.

Carbohydr. Res.

Carbohydr. Res.

Mendeleev Commun.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Tetrahedron

Tetrahedron

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.