Sortase-mediated ligation (SML) is a powerful tool of protein chemistry allowing the ligation of peptides containing LPxTG sorting motifs and N-terminal glycine nucleophiles. The installation of a sorting motif into the product prohibits the assembly of multiple fragments by SML. Here we report multi-fragment SML based on switchable sortase substrates. Substitution of the Leu residue by disulfide-containing Cys(StBu) results in active sorting motifs, which are inactivatable by reduction. In combination with a photo-protected N-Gly nucleophile, multi-fragment SML is enabled by repetitive cycles of SML and ligation site switching. The feasibility of this approach was demonstrated by a proof-of-concept four-fragment ligation, the assembly of peptide probes for bivalent chromatin binding proteins and oligomerization of peptide antigens. Biochemical and immuno-assays demonstrated functionality of these probes rendering them promising tools for immunology and chromatin biochemistry.
