Toward Homogeneous Erythropoietin: Fine Tuning of the C-Terminal Acyl Donor in the Chemical Synthesis of the Cys29-Gly77 Glycopeptide Domain

Described herein is the chemical synthesis of the Cys(29)-Gly(77) glycopeptide domain (22) of erythropoietin. Our initial ligation strategy targeted a C -> N termini condensation between glycopeptide 3 and peptide 4. However, the reaction was hindered by the unattainable reactivity, mismatched polarity, and severe aggregation of the (glyco)peptide substrates. In contrast, by tuning the C-terminal acyl donor and using smaller peptide fragments, the Cys(29)-Gly(77) glycopeptide domain of erythropoietin was prepared through unconventional N -> C termini condensation reactions. The use of a p-cyanonitrophenyl ester and the development of a masked thiophenyl ester as acyl donors enabled us to promptly access glycopeptides bearing complex carbohydrates and offer potential synthetic applications beyond our current work.