Butelase‐Mediated Macrocyclization of d ‐Amino‐Acid‐Containing Peptides

Abstract Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l‐peptides; their ability to cyclize d‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d‐amino acid residues at the P1′′ and P2′′ positions.