Study of On-Resin Convergent Synthesis of N-Linked Glycopeptides Containing a Large High Mannose N-Linked Oligosaccharide

Abstract

Here we present a convergent on-resin glycosylamine coupling strategy for solid phase N-linked glycopeptide synthesis, and apply it to the synthesis of high mannose containing glycopeptides. In this strategy, the 2-phenylisopropyl protecting group is used as an orthogonal handle to create glycosylation sites on-resin after synthesis of nonglycosylated peptides. In addition to allowing selective deprotection of aspartic acid residues for creation of glycosylation sites, the 2-phenylisopropyl protecting group also efficiently suppresses aspartimide formation during peptide synthesis. The key step of on-resin glycosylamine coupling to an aspartic acid residue was first optimized for a small sugar, N-acetylglucosamine, and then applied to a much larger high mannose oligosaccharide, Man(8)GlcNAc(2). Satisfying coupling yields were obtained for both small and large sugars. The use of on-resin glycosylamine coupling simplifies purification of N-linked glycopeptides, and also allows convenient recovery of unreacted valuable large oligosaccharides. This approach was applied to the solid phase synthesis of glycosylated forms of the 34 amino acid HIV-1 gp41 C34 glycopeptide, which is an HIV-1 entry inhibitor. The HIV-1 entry inhibition assay of synthesized glycopeptides showed the retention of bioactivity of high mannose Man(8)GlcNAc(2)-C34.