Glycoprotein synthesis and remodeling by enzymatic transglycosylation
A chemoenzymatic method for the preparation of a homogeneous glycoprotein or glycopeptide, including (a) providing an acceptor selected from the group consisting of GlcNAc-protein and GlcNAc-peptide; and (b) reacting the acceptor with a donor substrate including an activated oligosaccharide moiety, in the presence of a catalyst comprising endoglycosidase (ENGase), to transfer the oligosaccharide moiety to the acceptor and yield the homogeneous glycoprotein or glycopeptide. The donor substrate includes, in a specific implementation, a synthetic oligosaccharide oxazoline. A related method of glycoprotein or glycopeptide remodeling with a predetermined natural N-glycan or a tailor-made oligosaccharide moiety, and a method of remodeling an antibody including a heterogeneous sugar chain, are also described. The disclosed methodology enables glycoprotein drugs to be modified for prolonged half-life in vivo, reduced immunogenicity, and enhanced in vivo activity, and for targeting and drug delivery.
1. A method of generating remodeled and homogeneous glycopeptides or glycoproteins, the method comprising;
preparing an acceptor GlcNAc-containing peptide or protein that accepts an oligosaccharide; and
in the presence of an endoglycosidase (ENGase), enzymatically adding the oligosaccharide to the acceptor GlcNAc-containing peptide or protein, wherein the oligosaccharide is a synthetic oligosaccharide oxazoline comprising a sugar chain containing monosaccharide residues and glycosidic linkages, thereby generating the remodeled glycopeptide or glycoprotein.
2. The method of claim 1 , wherein the acceptor GlcNAc-containing peptide or protein is an antibody or fragment thereof.
3. The method of claim 1 , wherein the synthetic oligosaccharide oxazoline is a di-, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, deca-, or undeca-saccharide oxazoline.
4. The method of claim 1 , wherein the synthetic oligosaccharide oxazoline further comprises a biologically active agent.
5. The method of claim 2 , wherein the synthetic oligosaccharide oxazoline further comprises an azido group.
6. The method of claim 1 , wherein the step of preparing an acceptor GlcNAc-containing peptide or protein comprises
(a) providing a peptide or protein substrate comprising at least two GlcNAc residues and at least one GlcNAc-GlcNAc bond; and
(b) treating the peptide or protein substrate with an endo-enzyme to hydrolyze the bond between two GlcNAc residues positioned closest to the peptide or protein substrate thereby forming a peptide or protein substrate with a single GlcNAc-moiety.