Kinetic and Mechanistic\nStudies of Native Chemical\nLigation with Phenyl α‑Selenoester Peptides

Native chemical ligation\n(NCL) ligates two unprotected peptides\nin an aqueous buffer. One of the fragments features a C-terminal α-thioester\nfunctional group, and the second bears an N-terminal cysteine. The\nreaction mechanism depicts two steps: an intermolecular thiol–thioester\nexchange resulting in a transient thioester, followed by an intramolecular <i>S-to-N</i> acyl shift to yield the final native peptide bond.\nAlthough this mechanism is well established, the direct observation\nof the transient thioester has been elusive because the fast intramolecular\nrearrangement prevents its accumulation. Here, the use of α-selenoester\npeptides allows a faster first reaction and an early buildup of the\nintermediate, enabling its quantification and the kinetic monitoring\nof the first and second steps. The results show a correlation between\nthe steric hindrance in the α-thioester residue and the rearrangement\nrate. In bulky residues, the <i>S-to-N</i> acyl shift has\na significant contribution to the overall reaction rate. This is particularly\nnotable for valine and likely for other similar β-branched amino\nacids.