A Threonine-Forming Oxazetidine Amino Acid for the Chemical Synthesis of Proteins through KAHA Ligation

alpha-Ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments through the chemoselective formation of an amide bond. Currently, the most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. In order to directly form amide-linked threonine residues at the ligation site, we prepared a new 4-membered cyclic hydroxylamine building block. This monomer was applied to the synthesis of wild-type ubiquitin-conjugating enzyme UbcH5a (146 residues) and Titin protein domain TI I27 (89 residues). Both the resulting UbcH5a and the variant with two homoserine residues showed identical activity to a recombinant variant in a ubiquitination assay.