Late‐Stage Intermolecular <i>O</i>‐Peptidylation Protocol Enabled by Sequential Acyl Transfer on Thiol‐Incorporated Threonine Followed by Desulfurization

The thiol functionality on the methyl group of a threonine derivative [Thr(SH)] facilitates O-acylation of the Thr hydroxy group with a thioester. We previously showed that a Thr(SH)-incorporated peptide thioester can be converted to the corresponding Thr-containing cyclic depsipeptide through intramolecular thioester exchange (S─S acyl transfer) and subsequent desulfurization of the O-acyl peptide resulting from intramolecular S─O acyl transfer. Based on our success in depsipeptide synthesis, we applied the Thr(SH)-facilitated protocol to the synthesis of branched O-acyl isopeptides. Initial attempts identified two issues. First, the S-acylation step with a thioester proceeds in an entropically preferential manner in cyclic depsipeptide synthesis, but not in the case of branched isopeptides. Using a highly volatile thiol component in the thioester solved this issue. Second, the intermolecular thioester change step was accompanied by the formation of an S,O-diacyl intermediate as a major component; this issue was solved by using thioester-selective activation of the diacyl species with silver(I) salt followed by desulfurization. Ultimately, the optimized Thr(SH)-mediated protocol facilitated the late-stage O-acylation of a Thr residue in peptide sequences. We show that the protocol has wide substrate scope and demonstrate its application to ubiquitination of the Thr residue of HOIL-1 peptide.