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.

Abstract:The soluble catalytic domain of human N-acetylglucosaminyltransferase I was purified from Escherichia coli and utilized in the enzymecatalyzed conversion of high mannose N-linked oligosaccharide 1 into the rare hybrid oligosaccharide 2. Analysis of the reaction showed that the conversion of high mannose 1 into hybrid oligosaccharide 2 proceeded to 100% completion as assessed by MALDI-TOF-MS. Purification of the large polar oligosaccharide by gel filtration and silica gel chromatography afforded a 42% isolated yield of oligosaccharide 2. This enzyme-catalyzed reaction can be utilized to produce rare hybrid oligosaccharides for biochemical and structural studies.Keywords: enzyme catalysis; enzymes; glycosylation; oligosaccharidesThe surfaces of cells are covered with glycoproteins and glycolipids that mediate cell-cell interactions important in development, immune responses, and disease.[1] Increased interest in understanding these interactions has led to synthetic efforts aimed at producing pure oligosaccharides for biochemical and structural studies. N-linked oligosaccharides are large, branched sugar structures attached to the amino acid asparagine on proteins that range from 5 to several hundred sugar residues in size. Three broad classes of N-linked oligosaccharides are produced in humans: high mannose, hybrid, and complex N-linked oligosaccharides (Figure 1). These N-linked oligosaccharide types differ in the sugars attached to the non-reducing ends of the glycan, where high mannose oligosaccharides have all mannose residues on their non-reducing ends, complex oligosaccharides have sugar residues other than mannose terminating their non-reducing ends, and hybrid oligosaccharides have both mannose and other types of sugars terminating their chains.Although there have been many recent advances in chemical oligosaccharide synthesis, N-linked oligosaccharides still present large difficulties in synthesis due to their size, branched chains, and the wide variety of sugars and glycosidic linkages within their structures.[2] An alternative to total synthesis of N-linked oligosaccharides is to isolate them from natural sources. The difficulty with this strategy is that N-linked oligosaccharides are produced as multiple heterogeneous forms in biological systems. [3] This complicates the isolation of specific types of N-linked oligosaccharides from natural sources because the different types must be separated from one another chromatographically. Methods have been developed for the isolation of high mannose and complex type N-linked oligosaccharides using glycoproteins with a high percentage of those types of glycosylation. For instance, soybean agglutinin [4] and hen egg yolk sialylglycopeptide [5] are two glycoproteins that can be used to produce high mannose and complex type oligosaccharides, respectively. Unfortunately, no good method exists to produce hybrid N-linked oligosaccharides since they are generally present as a small percentage of the total oligosaccharides in glycoproteins.Here we report the use...

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.