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.