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.