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

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 of 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.