Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles.

Here we present a synthetic route for solid phase synthesis of N-linked glycoconjugates containing high mannose oligosaccharides which allows the incorporation of useful functional handles on the N-terminus of asparagine. In this strategy, the C-terminus of an Fmoc protected aspartic acid residue is first attached to a solid phase support. The side chain of aspartic acid is protected by a 2-phenylisopropyl protecting group, which allows selective deprotection for the introduction of glycosylation. By using a convergent on-resin glycosylamine coupling strategy, an N-glycosidic linkage is successfully formed on the free side chain of the resin bound aspartic acid with a large high mannose oligosaccharide, Man8GlcNAc2, to yield N-linked high mannose glycosylated asparagine. The use of on-resin glycosylamine coupling provides excellent glycosylation yield, can be applied to couple other types of oligosaccharides, and also makes it possible to recover excess oligosaccharides conveniently after the on-resin coupling reaction. Useful functional handles including an alkene (p-vinylbenzoic acid), an alkyne (4-pentynoic acid), biotin, and 5-carboxyfluorescein are then conjugated onto the N-terminal amine of asparagine on-resin after the removal of the Fmoc protecting group. In this way, useful functional handles are introduced onto the glycosylated asparagine while maintaining the structural integrity of the reducing end of the oligosaccharide. The asparagine side chain also serves as a linker between the glycan and the functional group and preserves the native presentation of N-linked glycan which may aid in biochemical and structural studies. As an example of a biochemical study using functionalized high mannose glycosylated asparagine, a fluorescence polarization assay has been utilized to study the binding of the lectin Concanavalin A (ConA) using 5-carboxyfluorescein labeled high mannose glycosylated asparagine.

Targeting a homogeneously glycosylated antibody Fc to bind cancer cells using a synthetic receptor ligand.

The targeting of a glycosylated antibody Fc fragment to bind to cancer cells by site-selective incorporation of a synthetic ligand is described. Homogeneously glycosylated immunoglobulin G subclass 1 fragment crystallizable (IgG1 Fc) was produced by expression in a glycosylation-deficient yeast strain and subsequent treatment with mannosidase IA. A N-terminal cysteine was generated on the expressed IgG1 Fc by utilizing proteolytic processing enzymes in the yeast secretory pathway. A cyclic RGD peptide thioester 2 was synthesized and then site-selectively attached to the N-terminus of the IgG1 Fc glycoprotein using native chemical ligation. The resulting chemically modified antibody fragment, RGD-Man(5)-IgG1 Fc (5), retained biological activity similar to that of the free cyclic RGD peptide 1 when assayed for its ability to both promote and inhibit the adhesion of alpha(v)beta(3) integrin receptor-expressing WM-115 melanoma cells. In addition, fluorescent microscopy experiments were conducted using FITC-labeled 5 and confirmed binding of 5 to WM-115 melanoma cells. Site-selectively modified antibody fragments such as the one described here may be used to combine the beneficial properties of synthetic receptor ligands with antibody fragments to develop useful biochemical tools and improved therapeutics. The methods described here can also be used to produce glycoprotein fragments for the chemoenzymatic synthesis of homogeneous glycoproteins.