NEOGLYCOPROTEIN SYNTHESIS USING THE COPPER-CATALYZED AZIDE–ALKYNE CLICK REACTION AND NATIVE CHEMICAL LIGATION - Click Chemistry in Glycoscience: New Developments and Strategies - page 259

conjugation of different glycosyl azides. Using NCL and kinetically controlled liga-
tion, together with click chemistry, the successful synthesis of a full-length click EPO
polypeptide containing four different types of sugars will be carried out. The success
of this study will demonstrate the viability of our devised methodology [34, 40] as
a versatile chemical toolkit for the fully convergent synthesis of complex neoglyco-
proteins using click chemistry, native chemical ligation, and kinetically controlled
ligation. The synthesis of a neoglycoEPO will also provide a route to the systematic
exploration of the effect of glycan structure on the in vivo biological activity of ery-
thropoietin. This work is currently underway in our laboratories, and will be reported
in due course.
10.7 CONCLUSION
The synthesis of glycoprotein mimics, or neoglycoproteins, is emerging as an exciting
area of research as it allows the rapid and efficient production of glycosylated protein
constructs. By replacing the sensitive and synthetically difficult peptide N-glycosidic
linkage with an isosteric moiety, many challenges of current glycopeptide synthesis
can be overcome. In particular, the robust and reliable copper-catalyzed azide-alkyne
“click” reaction is an attractive choice to replace the glycosylation reaction. The
triazole linkage thus generated may in fact impart extra metabolic and chemical
stability to the neoglycopeptide.
By combining our synthetic organic chemistry, and peptide and protein synthesis
experience, our research groups embarked on a series of projects to develop tools
and methodologies for the synthesis of neoglycopeptides and neoglycoproteins. Both
the use of triazole-linked amino acid building blocks in SPPS, as well as performing
click chemistry on assembled peptides, were explored [30, 31]. The advantages of
using the denaturing 6 M GuHCl/0.2 M Na 2 HPO 4 buffer for the CuAAC reaction
on larger peptides [34], and the powerful combination of a one-pot native chemical
ligation and CuAAC click reaction were demonstrated [40]. These tools are currently
being applied to the synthesis of a large click neoglycoprotein mimic of EPO.
The click chemistry/native chemical ligation combination developed herein brings
chemists one step closer to being able to conjugate synthetic proteins, assembled using
chemical synthesis, with glycosides in a site-specific manner. The only difference
between the synthetic neoglycoprotein and the native glycoprotein is the nature of
the glycoside-peptide linkage. Such neoglycopeptides/neoglycoproteins provide a
valuable focused library of molecular probes of defined structure that provide useful
tools for biological evaluation.
REFERENCES
1. Haase, C.; Seitz, O. Top. Curr. Chem. 2007 , 267 , 1-36.
2. Specker, D.; Wittmann, V. Top. Curr. Chem. 2007 , 267 , 65-107.
3. (a) Lauc, G.; Rudan, I.; Campbell, H.; Rudd, P. M. Mol. BioSyst . 2010 , 6 , 329-335. (b)
Wittmann, V. In Glycoscience; Cote, G., Flitsch, S., Ito, Y., Kondo, H., Nishimura, S.I.,
Yu, B., Eds.; Springer: New York, 2008; pp 1771-1793.