A thioamide, a single atom substitution on the peptide backbone, can be used as a minimally perturbing probe for various applications.  Previously, the incorporation of thioamides has been limited to synthetic small peptides. Here, we demonstrate that thioamides in both C-terminal thioesters and N-terminal Cys fragments are compatible with native chemical ligation conditions.  Multiple ligations in the presence of thioamides are possible by utilizing thiazolidine protection of the N-terminal Cys.  We have also devised a method to generate thioamide peptides with a C-terminal linker which exposes a thioester in situ.  To expand the scope of our method, we show that thioamides can be incorporated into a protein by ligating expressed protein fragments and chemically synthesized thioamide peptides.  As a proof of principle, we have generated thioamide labeled versions of the amyloid protein a-synuclein and monitored its conformational changes during aggregation by fluorescence quenching.

Native chemical ligation of thioamide-containing peptides

244th National Meeting (2012)

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