Chemical Protein Synthesis by Hydroxylamine-Involved Ligation of Peptides

Chemical protein synthesis by native amide ligation between two unprotected peptides provides an effective protocol. It is well-known that the development of a ligation method that is not limited by the sites of specific amino acids is highly desired. Here, we report an efficient chemoselective ligation between C-terminal peptide thioesters and N-terminal aminoacyl-<i>N</i>-hydroxy peptides in water (pH 8) at room temperature. The ligation reaction undergoes sequential <i>S</i>,<i>O</i>-ester exchange of peptide thioesters with <i>N</i>-hydroxyl of aminoacyl-<i>N</i>-hydroxy peptides providing <i>O</i>-acyl isopeptides and <i>O</i>,<i>N</i>-acyl transfer across a six-membered ring yielding ligated <i>N</i>-hydroxyl peptides. Reduction of <i>N</i>-hydroxyl peptides with zinc in an aqueous medium (pH 3) affords long peptides with native amide bonds. This hydroxylamine-involved ligation (HAL) of peptides was successfully applied to the synthesis of ubiquitin.