Ligation strategies such as Native Chemical Ligation (NCL) are widely used for the convergent synthesis of proteins.1 The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc-based solid-phase peptide synthesis.2
We have reported a route to peptide thioesters, following Fmoc solid-phase peptide synthesis, via an N→S acyl shift which is initiated by the presence of a C-terminal cysteine residue, under mildly acidic conditions. During the reaction peptides fragment across the Xaa-Cys motif to afford Xaa-thioester products. Interestingly a significant degree of selectivity can be achieved with Gly/His/Cys thioesters forming most rapidly, while β-branched amino acids are reluctant to participate in the reaction. Furthermore a terminal Xaa-Cys motif is converted to a thioester significantly more rapidly than the equivalent motif that is situated within a peptide sequence, facilitating the formation of cyclic peptides and proteins.
Major benefits of this approach include the often straightforward method of precursor assembly using Fmoc-based chemistry, and the potential for extension to biological systems. With the existing methodology we have found that several peptide thioesters can be readily prepared. However, there appears to be ample opportunity for further development and discovery.3-5