Total Synthesis of Erythropoietin through the Development and Exploitation of Enabling Synthetic Technologies

摘要

Glycosylation is the most common co- or posttranslational modification of proteins, with 50-70% of all human proteins estimated to bear covalently linked glycans. Glycosylation of proteins is implicated in a diverse number of processes including fertilization, immune surveillance, hormone activity, neuronal development, and protein folding. Despite the unquestionable importance of glycosylation, progress towards understanding the role of this modification on both the structure and function of proteins has been hampered by difficulties in accessing them in pure form. Specifically, these issues arise from the fact that, unlike protein synthesis, the glycosylation process is not templated. Rather, it is controlled by the activities of glycosyltransferases that give rise to mixtures of glycoforms. As these glycoforms differ only in the nature of covalently appended glycan(s), they are often impossible to separate using chromatographic methods. Recombinant human erythropoietin (rhEPO) a multi-billion dollar drug for the treatment of anemia is a 166 amino acid protein bearing four glycosylation sites, three highly variable N-linked (at Asn-24, Asn-38, and Asn-83) and one more conserved O-linked (at Ser-126). As a consequence of its production in mammalian cells, rhEPO is sold as a complex mixture of glycoforms, and the glycosylation state of EPO that exhibits optimal activity remains unknown.