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.