Synthesis of Erythropoietins Site-Specifically Conjugated with Complex-Type N-Glycans.

The biological activity of the glycoprotein hormone erythropoietin (EPO) is dependent mainly on the structure of its N-linked glycans. We aimed to readily attach defined N-glycans to EPO through copper-catalyzed azide alkyne cycloaddition. EPO variants with an alkyne-bearing non-natural amino acid (Plk) at the N-glycosylation sites 24, 38, and 83 were obtained by amber suppression followed by protein purification and refolding.

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO.