Figure 3. Traceless Staudinger ligation for isopeptide bond formation.

Genetic incorporation of a lysine derivative with Nε azido functionality (Anl) into recombinant protein and synthesis of a Ub C-terminal phosphothioester (via intein technology) generates the mutually reactive functional ligation handles. Staudinger ligation involves the liberation of nitrogen, an S to N acyl shift and hydrolytic oxidation of phosphorus resulting in the formation of a native isopeptide bond.