Oa AEP1-Mediated Ligation of Mirror-Image Peptides via Acyl Donor Substrate Engineering

AEP1, which is ligated to a d-peptide acyl acceptor bearing an N-terminal Gx- motif (x = l, i, v, c, G, etc.). This method enables efficient d-peptides ligation under nondenaturing conditions (PBS buffer, pH 7.0), and its effectiveness and practicality are exemplified by the synthesis of the d-cyclopeptides, d-mini-proteins, and mirror-image K6-ubiquitinated α-synuclein (1-17) comprising 102 amino acids. This work demonstrates that substrate engineering is a powerful strategy for harnessing the hidden potential of natural enzymes.