Amide-forming chemical ligation via <i>O</i> -acyl hydroxamic acids

Abstract

The facile rearrangement of "<i>S</i>-acyl isopeptides" to native peptide bonds via <i>S</i>,<i>N</i>-acyl shift is central to the success of native chemical ligation, the widely used approach for protein total synthesis. Proximity-driven amide bond formation via acyl transfer reactions in other contexts has proven generally less effective. Here, we show that under neutral aqueous conditions, "<i>O</i>-acyl isopeptides" derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid; Asp(β-HA)] rearrange to form native peptide bonds via an <i>O</i>,<i>N</i>-acyl shift. This process constitutes a rare example of an <i>O</i>,<i>N</i>-acyl shift that proceeds rapidly across a medium-size ring (t_1/2 ∼ 15 min), and takes place in water with minimal interference from hydrolysis. In contrast to serine/threonine or tyrosine, which form <i>O</i>-acyl isopeptides only by the use of highly activated acyl donors and appropriate protecting groups in organic solvent, Asp(β-HA) is sufficiently reactive to form <i>O</i>-acyl isopeptides by treatment with an unprotected peptide-^αthioester, at low mM concentration, in water. These findings were applied to an acyl transfer-based chemical ligation strategy, in which an unprotected <i>N</i>-terminal Asp(β-HA)-peptide and peptide-^αthioester react under aqueous conditions to give a ligation product ultimately linked by a native peptide bond.