Cysteine-Free Native Peptide Ligation for the Assembly of Glycoproteins
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Date
2007Author
Anderson, David W
Metadata
Abstract
Homogeneous, structurally defined glycoproteins can be assembled by coupling synthetic
glycopeptides to synthetic or bacterially-derived protein fragments using the native
chemical ligation (NCL) reaction. A limitation of NCL is the requirement for an Nterminal
cysteine residue in one of the peptide fragments. One method for cysteine-free
peptide ligation utilizes thiol acyl transfer auxiliaries which effect ligation and can then
be removed under mild conditions.
We developed new, rapid routes to 1-phenyl-2-mercaptoethyl and 2-mercaptobenzyl
auxiliaries. The key steps involved: 1) introduction of a suitably protected thiol to
auxiliary precursors; 2) direct reductive amination of the auxiliary aldehyde or ketone to
afford the auxiliary-amine, which can be conjugated to a peptide via the “sub-monomer”
approach, or the auxiliary-amino acid “cassette” for use in conventional solid phase
peptide synthesis. Overall yields are 53-83 %. A glycopeptide was then assembled via
auxiliary-mediated ligation at a Gly-Gly junction, which was complete within 48 hours.
Thioester- and auxiliary-peptides were assembled to investigate the scope and limitations
of auxiliary-mediated ligation for non Gly-Gly junctions. The 1-phenyl-2-mercaptoethyl
auxiliary effected ligation at Ala-Gly, Lys-Gly and Gly-Ala junctions in 24-70 % yield,
whereas the 2-mercaptobenzyl auxiliary effected ligation at a Gly-Ala junction in 42 %
yield. Excess thiol was found to inhibit ligation, indicating a change in rate-determining step
relative to cysteine ligation.