Chemical Synthesis of an Erythropoietin Glycoform Having a Triantennary N-Glycan: Significant Change of Biological Activity of Glycoprotein by Addition of a Small Molecular Weight Trisaccharide

The glycosylation of proteins contributes to the modulation of the structure and biological activity of glycoproteins. Asparagine-linked glycans (N-glycans) of glycoproteins naturally exhibit diverse antennary patterns, such as bi-, tri-, and tetra-antennary forms. However, there are no chemical or biological methods to obtain homogeneous glycoproteins via the intentional alteration of the antennary form of N-glycans. Thus, the functions of the individual antennary form of N-glycan at a molecular level remain unclear. Herein, we report the chemical synthesis of an erythropoietin (EPO) glycoform having a triantennary sialylglycan at position 83, as well as two biantennary sialylglycans at both positions 24 and 38. We demonstrated efficient liquid-phase condensation reactions to prepare a sialylglycopeptide having a triantennary N-glycan prepared by the addition of a Neu5Ac-alpha-2,6-Gal-beta-1,4-GlcNAc element to the biantennary glycan under semisynthetic conditions. The molecular weight of the newly added antennary element was similar to 3% of the EPO glycoform, and the introduced position was the most distant from the bioactive protein. However, in vivo assays using mice revealed that the additional antennary element at position 83 dramatically increased the hematopoietic activity compared to a commercially available native EPO. These unprecedented data clearly indicate that the antennary pattern of N-glycans inherently plays a critical role in the modulation of protein functions.