Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: β-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N′-triacetylchitobiose and 6′-O,N,N′-triacetylchitobiose

doi:10.1016/S0008-6215(01)00027-1

Carbohydrate Research

Volume 331, Issue 2, 22 March 2001, Pages 143-148

https://doi.org/10.1016/S0008-6215(01)00027-1 Get rights and content

Abstract

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-β-d-glucopyranoside (5a) was used as the glycosyl donor in a β-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6′-O,N,N′-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-β-d-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N′-triacetylchitobiose (4).

Introduction

In the last decade dynamic development of glycosciences has brought new fundamental findings from molecular glycobiology, molecular glycoimmunology and related frontier areas, while, in the meantime, demands for new glycostructures have stimulated the development of efficient syntheses of these compounds. Enzymatic methods are widely exploited in carbohydrate chemistry nowadays.¹ Besides glycosyltransferases and glycosidases, other enzymes such as lipases, proteases and amidases are also used in this area, and the synthetic diversity of these enzymes can be broadened by the use of non-natural substrates (xenosubstrates). For instance, glycosyltransferases are able to accept synthetically modified sugar nucleoside donors, e.g., various deoxysugars and thio analogues,¹ as well as completely different nucleosides, e.g., UDP-glucose with β-(1→4)-galactosyltransferase.² Glycosyltransferases keep their main synthetic advantage with these xenosubstrates that is to combine regioselectivity and absolute stereoselectivity. However, these enzymes have certain disadvantages, such as high price (both the enzymes and substrates), low number of different biocatalysts available, sensitivity to environmental stress (typical for intracellular enzymes, moreover membrane bound) and last, but not least, limitations in respect of the acceptors: they are rather stringent towards the distal one to two saccharide moieties of the acceptor, so that non-sugar acceptors can hardly be glycosylated by these enzymes (with the obvious exception of glycosyltransferases specific for a particular aglycon).

Contrary to glycosyltransferases, glycosidases, especially the extracellular ones, are quite robust enzymes, resistant to environmental stress (cosolvents, salts, pH, temperature, etc.,) and easier to handle. Glycosidases of microbial origin can be prepared in a relatively easy way from different sources, so that, libraries of these biocatalysts can be made available. Additionally, these enzymes can often be used for the glycosylation of non-sugar acceptors. Main drawbacks of glycosidases are lower yields and poorer regioselectivity. Specifically, using pyranosidic acceptors, quite often glycosylation, takes place at the primary OH group besides at one (or more) of the secondary OHs, thus lowering the yield of the desired product and producing complex and hardly separable reaction mixtures. To overcome this problem we have developed an approach that exploits selectively enzymatic 6-O-acylated glycosyl acceptors, and we have demonstrated its usability by accomplishing the enzymatic synthesis of the trisaccharide iso-globotriose from 6′-O-acylated lactose.³

Chitooligomers and some of their derivatives have been identified as strong activating ligands of the natural killer cell-activating receptor (NKR-P1),² making the preparation of these compounds and of their derivatives important. In this paper we report, for the first time, the use of 6-O-acylated-β-d-2-deoxy-2-acetamidoglucopyranosides, both as acceptors and as donors, in the synthesis of selectively acylated chitobioses catalysed by β-N-acetylhexosaminidases.

Section snippets

General methods

Reactions were monitored by thin-layer chromatography (TLC) on Silica Gel F₂₅₄ plates (Merck) using the solvent system 7:2:1 (v/v) isopropanol–water–concd NH₃. The spots were visualised by UV light and by charring with 10% H₂SO₄ in EtOH.

¹H- and ¹³C NMR spectra were recorded on a Varian INOVA-400 spectrometer (399.91 and 100.57 MHz, respectively) in D₂O at 30 °C. Carbon signal multiplicity was determined by an attached proton test (APT) experiment. Manufacturer's software was used for 2D NMR

Results and discussion

Transglycosylation of N-acetylglucosamine (GlcNAc, 1) as the acceptor using p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside (p-NP-GlcNAc, 2) as the glycosyl donor in a reaction catalysed by, e.g., a β-N-acetylhexosaminidase from A. oryzae, gives mostly di-N,N′-acetylchitobiose and about 10% of its (1→6) isomer.⁶ Separation of these compounds, either by selective cleavage of the (1→6) isomer by a β-N-acetylhexosaminidase from jack beans on by tedious carbon-Celite chromatography, is rather

Acknowledgements

Supports from AVČR-CNR bilateral project (V.K.+S.R.), and Grant Agency of the Czech Republic, grant No. 303/99/1382 are acknowledged. We thank P. Halada (Institute of Microbiology, Prague) for recording the MS spectra.

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Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: β-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N′-triacetylchitobiose and 6′-O,N,N′-triacetylchitobiose

Abstract

Introduction

Section snippets

General methods

Results and discussion

Acknowledgements

Tetrahedron Lett.

J. Mol. Cat. B: Enzym.

Carbohydr. Res.

Chem. Soc. Rev.

Carbohydr. Res.