We herein developed a racemization-free segment condensation based on the O-acyl isopeptide method. This method allows the use of an N-segment possessing a C-terminal Ser/Thr residue for segment condensation, without any racemization, as a result of the C-terminal O-acyl isopeptide structure with a urethane-protected Ser/Thr residue. Thus, segment condensation becomes possible at not only the C-terminal Gly/Pro but also Ser/Thr residues without epimerization. Additionally, final deprotected peptides/proteins synthesized using the O-acyl isopeptide method-based segment condensation are effectively purified by HPLC, because a simple isomerization to an O-acyl isopeptide remarkably and temporarily changes the physicochemical properties of the native peptide, and an O-N intramolecular acyl migration triggers the native amide bond formation under physiological conditions.
