A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans

Abstract

A systematic, efficient means of producing diverse libraries of asymmetrically branched N-glycans is needed to investigate the specificities and biology of glycan-binding proteins. To that end, we describe a core pentasaccharide that at potential branching positions is modified by orthogonal protecting groups to allow selective attachment of specific saccharide moieties by chemical glycosylation. The appendages were selected so that the antenna of the resulting deprotected compounds could be selectively extended by glycosyltransferases to give libraries of asymmetrical multi-antennary glycans. The power of the methodology was demonstrated by the preparation of a series of complex oligosaccharides that were printed as microarrays and screened for binding to lectins and influenza-virus hemagglutinins, which showed that recognition is modulated by presentation of minimal epitopes in the context of complex N-glycans.

Fig. 1

Orthogonally protected core pentasaccharide 1 and glycosyl donors 2-5 for extensions in a parallel combinatorial manner to give oligosaccharide precursors to enzyme substrates.

Fig. 2

Chemical synthesis of decasaccharide 15 for branch-specific enzymatic extensions. (A) Selective removal of temporary protecting groups. (B) Preparation of glycan precursor for enzymatic extension.

Fig. 3

Chemoenzymatic synthesis of (A) asymmetrically substituted multi-antennary glycan 22. (B) Structures of compounds (23-26) prepared by the chemoenzymatic approach. N-acetyl neuraminic acid (Neu5Ac, ); D-galactose (Gal, ); N-acetyl-D-glucosamine (GlcNAc, ); D-mannose (Man, ); L-fucose (Fuc, ).

Fig. 4

Glycan microarray binding analyses. Fluorescently labeled lectins (ECA, AAL, and SNA), and recombinant avian (VN/04) and human influenza A (KY07 and CA/05) HA were assessed for binding to the array. Shown is the mean signal and standard error calculated for six independent replicates on the array. Structures of each of the lettered glycans are found in Table S14.