Chemical Synthesis of Homogeneous Glycoprotein Mimetics

Abstract

The therapeutic glycoprotein, erythropoietin (EPO) is generally produced by human recombinant protein expression, which renders a complex mixture of glycoforms that share the same peptide backbone but differ in the chemical structures of the pendent glycans. The separation of these glycoforms with current chromatographic techniques poses a great challenge, and this problem has led to an increased demand to produce EPO as a single glycoform (homogeneous) for structural and bioactivity studies. Chemical protein synthesis has developed into an elegant approach to preparing homogeneous glycoproteins from a readily accessible pool of amino acids and carbohydrates. The development of native chemical ligation (NCL) overcomes the obstacle of using conventional solid phase peptide synthesis (SPPS) to prepare long polypeptide backbones. Most importantly, the chemical synthesis of the polypeptide by NCL strategy enables atomic-level installation of the requisite mutation at the site of interest. In addition, neo-glycosylation has been regarded as a novel approach to incorporate sugar moieties to peptides or proteins through non-natural chemical linkages.1 In this thesis, studies towards the total chemical synthesis of homogeneous neo-glycosylated EPO protein were undertaken. A robust synthetic route to EPO peptideα thioester fragments to access full-length EPO polypeptide using NCL strategy has been established. A facile and efficient approach using a thiol-bromo alkylation reaction to enable the incorporation of fully unprotected sialic acid, Neu5Ac, onto EPO peptide fragments has been developed by evaluating three types of conjugation chemistry using the EPO peptide fragments and Neu5Ac chemically modified at C-9 position. These conjugation strategies include copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) as described in chapter III, radical-mediated thiol-ene reaction in chapter IV, and a thiol-bromo alkylation reaction in chapter V. Furthermore, ligation intermediates EPO (98 - 165) and EPO (30 - 67) with an unprotected sialic acid incorporated have been assembled towards the preparation of a fully assembled neo-glycosylated EPO polypeptide.