Toward homogeneous erythropoietin: Chemical synthesis of the Ala ¹-Gly ²⁸ glycopeptide domain by "Alanine" ligation

The Ala ¹-Gly ²⁸ glycopeptide fragment (28) of EPO was prepared by chemical synthesis as a single glycoform. Key steps in the synthesis include attachment of a complex dodecasaccharide (7) to a seven amino acid peptide via Lansbury aspartylation, native chemical ligation to join peptide 19 with the glycopeptide domain 18, and a selective desulfurization at the ligation site to reveal the natural Ala ¹⁹. This glycopeptide fragment (28) contains both the requisite N-linked dodecasaccharide and a C-terminal ^αthioester handle, the latter feature permitting direct coupling with a glycopeptide fragment bearing N-terminal Cys ²⁹ without further functionalization.