CHEMOENZYMATIC SYNTHESIS OF A HOMOGENEOUS GLYCOPROTEIN
John E. Schiel, Mark S. Lowenthal, Karen W. Phinney
Biologically relevant proteins often exist as a heterogeneous mixture of glycoforms due to glycosylation at numerous sites and with various glycans. The glycosylation pattern has significant implications for clinical diagnostics and biopharmaceutical development because it affects a glycoprotein’s stability, toxicity, and activity. Complete characterization of these complex analytes involves numerous multidisciplinary techniques, each of which provides complimentary information. A well defined glycoprotein containing a single glycan at a single glycosylation site would be of great value for the comparison and evaluation of glycoprotein analysis techniques.
The current project describes the chemoenzymatic rearrangement of Bovine Ribonuclease B (RNase B) to contain a single glycosylation site with a Man2GlcNAc glycan. The procedure involved enzymatic removal of native glycans from the protein, leaving only the penultimate N-Acetylglucosamine residue attached. A reactive disaccharide oxazoline derivative was then synthesized and added to the deglycosylated RNase through Endo-beta-N-Acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. The resulting product was characterized using mass spectrometry. This reaction signifies the first “one pot” synthesis of a homogeneous glycoprotein. The techniques described herein are not limited to this analyte or glycan, and should be amenable to the synthesis of other glycoproteins containing homogeneous glycan moieties.