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easily form hydrogen-bonded secondary structures such as
-sheets.
Even sequences as short as ten amino acids can fold to adopt a well-defined three-
dimensional structure [41]. This phenomenon inevitably means that some residues
may be internal and away from the surface, making them less accessible than others,
and hence drastically reducing their reactivity [42]. In 2007, van Kasteren et al. [43]
reported the incorporation, through site-directed mutagenesis, of the unnatural amino
acids azidohomoalanine (Aha) and homopropargylglycine (Hpg) into two large pro-
teins, both over 50 kDa in size. The researchers were then able to utilize the CuAAC
reaction to quantitatively attach several azido- and propargyl-containing sugars to
the proteins, with no loss of biological activity of the neoglycoprotein. However,
they observed that reactivity at specific sites was correlated to the accessibility of the
residue in question, to the extent that they were able to effect complete transforma-
tion at one residue, with completely no reaction observed under the same conditions
at a different position. We suspect that longer polypeptides may encounter similar
problems and hence the denaturing action of the GuHCl may be critical to the success
of the click reaction.
Interestingly, the GuHCl appeared to have additional effects beyond just a dena-
turing function: the reduction of CuSO
4
by TCEP was accelerated, and the Cu(I)
species formed were less susceptible to air oxidation. Cu(I) species are generally
thermodynamically unstable, easily oxidizing to Cu(II) and/or disproportionating to
Cu(0). To maintain sufficient Cu(I) ion concentrations usually requires either inert
atmospheres and anhydrous solvents or large quantities of reducing agent [44], which
(as mentioned above) may be detrimental to the reaction; although more recently the
use of specific tris(heterocyclemethyl)amine ligands to stabilize Cu(I) in aerobic,
aqueous conditions has also been described [38, 44, 45]. Therefore the Cu(I) sta-
bilizing property of the 6 M GuHCl solution is a great advantage in addition to its
peptide-denaturing ability, making it an attractive choice of solvent for the CuAAC
reaction on longer peptides and proteins.
-helices or
10.5 APPLICATION OF NCL TO THE SYNTHESIS
OF NEOGLYCOPEPTIDES
10.5.1 A One-Pot Approach
The abovementioned reports of neoglycopeptide synthesis still face the inherent lim-
itation of SPPS: the practically achievable peptide size is usually no more than 30-40
amino acids. Peptide chains larger than that are difficult to obtain due to the accumu-
lation of byproducts and poor yield [46]. To access chemically synthesized protein
constructs for study, different techniques must be applied, such as NCL developed by
Dawson and Kent [12]. NCL involves the chemoselective, thiol-catalysed reaction of
a
C
-terminal thioester with an
N
-terminal cysteine residue to afford a native amide
linkage through an
S
to
N
acyl shift, as depicted in Scheme 10.7. This reaction has dra-
matically increased the size of proteins accessible by chemical synthesis, the largest
reported currently being the 304 amino acid K48-linked tetraubiquitin protein [47].
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