NEOGLYCOPROTEIN SYNTHESIS USING THE COPPER-CATALYZED AZIDE–ALKYNE CLICK REACTION AND NATIVE CHEMICAL LIGATION - Click Chemistry in Glycoscience: New Developments and Strategies - page 257

OH

OH OH

SCHEME 10.8 Ligation of peptide thioester and Cys-peptide, followed by one-pot CuAAC
reaction with GalNAc-N 3 [40].

sequence, which have been incorporated through site-specific genetic mutations, with
glycosyl partners (such as glycosyl azides). However, global, simultaneous conjuga-
tion can lack control, with different residues having varying levels of reactivity that
ultimately leads to a complex mixture of fully and partially clicked neoglycoproteins
which may or may not be separable. Most importantly, this method is limited to a
single type of sugar to be conjugated, and thus does not permit a diversity of glycans
to be installed onto the protein of interest.
In contrast to this work, total chemical synthesis was used to make a “synthetic
erythropoiesis protein” with full biological activity, in which the complex glycan was
replaced by a branched olig(ethyleneoxide-amide) moiety of defined chemical struc-
ture [54]. Our goal is to undertake the total chemical synthesis of neoglycoproteins.
The main advantage of total chemical synthesis is that the type and extent of glycocon-
jugation can be controlled in a site-specific manner. We wished to take advantage of
this and utilize the methodology developed within our research groups in order to syn-
thesize a large and complex neoglycoprotein in a fully convergent manner. Our initial
target was the synthesis of a click neoglycoprotein analog of erythropoietin (EPO).
Erythropoietin is the principal hormone that regulates mammalian erythrocyte
differentiation [55]. The EPO polypeptide chain contains 165 amino acids, and
has four glycosylation sites—three N-linked, and an O-linked; this glycosylation
is important for its in vivo activity [56], although non-glycosylated EPO retains