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

Abstract

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-α-2,6-Gal-β-1,4-GlcNAc element to the biantennary glycan under semisynthetic conditions. The molecular weight of the newly added antennary element was ∼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.

Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin

“…Conversion of the glycopeptide hydrazide 25 to the corresponding thioester S14 required strict pH‐control, to avoid the formation of an unreactive lactam at the C‐terminal lysine. The lactamization can be avoided efficiently by use of side chain protecting groups [33] or by lowering the pH during thiolysis of the acyl azide [8d] . Cys‐peptide 26 was ligated with the glycopeptide thioester S14 furnishing the EPO glycopeptide 29–166 27 in high yield (73 %).…”

Optimizing the Semisynthesis towards glycosylated interferon-β-polypeptide by utilizing bacterial protein expression and chemical modification

“…23 This can be attributed to the incomplete cleavage of the peptide from the resin, the coupling inefficiency of the oligosaccharyl amino acid to the resin-bound peptide and the purity of SPPS peptides. An original experiment has been reported by the Unverzagt group 36 and our laboratory 41 has performed the coupling of segments of erythropoietin with a triantennary glycan at −20 °C. Here, we further optimized the liquid phase synthesis of glycopeptide thioesters.…”

Recent Advances in the Chemical Biology of N-Glycans

“…EPO with various glycoforms is used as a drug. Several groups have reported the synthesis of EPO with homogeneous glycoforms, and the effect of N-glycans on their biological activities has been investigated [112,115,116,[118][119][120]. In addition, various neoglycoprotein analogues of EPO have been reported [121][122][123][124].…”