Toward homogeneous erythropoietin: fine tuning of the C-terminal acyl donor in the chemical synthesis of the Cys29-Gly77 glycopeptide domain

Abstract

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.

Scheme 1

Retrosynthetic analysis of Epo and fragment Cys²⁹-Gly⁷⁷.

Scheme 2

Initial construction of fragment Epo 29–77.

Scheme 3

Successful fragment condensation to deliver EPO(29–77) with disaccharide.

Scheme 4

Lansbury aspartylation between dodecasaccharide and reactive acyl donors

Scheme 5

Revised retrosynthetic analysis for Epo 29–77

Scheme 6

Potential problem associated with hydrolysis of the masked thioester.

Scheme 7

Preparation of masked thiophenol 18

Scheme 8

Completion of synthesis EPO(29–77)