NEOGLYCOPROTEIN SYNTHESIS USING THE COPPER-CATALYZED AZIDE–ALKYNE CLICK REACTION AND NATIVE CHEMICAL LIGATION - Click Chemistry in Glycoscience: New Developments and Strategies - page 255

SCHEME 10.7 Mechanism of the native chemical ligation reaction.

Previous work by Macmillan and Blanc [26c] has shown that the triazole linkage
is stable to NCL conditions. Other groups had also used a combination of NCL and
CuAAC in the synthesis of lipopeptides [48] and N -terminally-linked protein dimers
[37], but no comprehensive investigation of click conditions between sugar-azides
and alkyne-peptides containing all 20 amino acids found in proteins, has been
carried out.
We therefore began this investigation by designing and synthesizing three model
peptides ( 17 - 19 , Fig. 10.2) containing all 20 genetically encoded amino acids, as
well as the thiazolidine (Thz), thioester, and acetamidomethyl (Acm) moieties used
in the total chemical synthesis of proteins through NCL. The unprotected peptides
17 - 19 were then subjected to click conditions with GalNAc-N 3 ( 20 ) using a series
of conditions, with the best results obtained in 6 M GuHCl/0.2 M Na 2 HPO 4 (pH 7)
and using TCEP as the reducing agent [40]. The click reaction was compatible with
all unprotected amino acid side-chain functionalities, the peptide thioester and the
Acm protecting group. The Thz moiety (used as a masking group for cysteine during
the synthesis of large proteins by NCL) was not stable under these conditions—clean
conversion to the cysteine was observed in under 1 hour. Therefore, the click reaction
cannot be used directly to make a Thz-neoglycopeptide from a Thz-propargyl peptide.
However, a Cys to Thz conversion occurs readily in the presence of formaldehyde
[49], and hence may be applied to convert a Cys-neoglycopeptide to its corresponding
Thz-neoglycopeptide.
As the best solvent conditions for the click reaction were the same as those for
NCL, we postulated that consecutive native chemical ligation and click reactions