Toward homogeneous erythropoietin: Fine tuning of the C-terminal acyl donor in the chemical synthesis of the Cys ²⁹-Gly ⁷⁷ 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.