Abstract
The α-ketoacid-hydroxylamine (KAHA) ligation enables the chemoselective coupling of unprotected peptide segments. The most commonly used hydroxylamine building block, (S)-5-oxaproline, yields homoserine residues at ligation sites, limiting applications where the native sequence is essential. To overcome this limitation, we developed cyclic dipeptide-derived hydroxylamine building blocks that enable the formation of canonical amino acids directly under modified KAHA ligation conditions. These building blocks are prepared from dipeptides and are applicable at nonobvious peptide ligation junctions, including Leu-Ile and Lys-Ile. We applied this approach to the synthesis of K48/K63 selectively protected ubiquitin monomers for chemoenzymatic ubiquitin chain formation and the total synthesis of tirzepatide, a GLP-1 receptor agonist peptide therapeutic containing amino-isobutyric acid (Aib) residues and a fatty acid side chain modification. This work establishes a practical approach for KAHA ligation at fully native sites and expands its applicability to the practical synthesis of challenging peptide targets.