Peptidyl Asparaginyl Ligase-Mediated Orthogonal Ligation at Non-Asx Peptide Bonds for <scp>d</scp> -Peptide Cyclization and Antibody Dual Labeling

There is an increasing need to develop enzymatic methods for protein modification. PALs (peptidyl asparaginyl ligases) catalyze transpeptidation at the Asx (Asn or Asp) peptide bond with high efficiency and have been widely used for protein N- or C-terminal labeling. However, the stringent P1-Asx requirement and the inherent reversibility of the transpeptidation reaction limit the application scope of PAL-mediated ligation. Herein, we report the use of peptidyl-sulfaneylacetamides as the acyl donor substrates of PALs to allow irreversible ligation at a non-Asx junction. We show that PALs exhibit excellent catalytic activity toward the thioester substrates with a C-terminal Gly residue, enabling precise protein N-terminal labeling with functional tags such as fluorescent labels and cytotoxic agents. Notably, PALs can also process the thioester substrates for d-peptide cyclization, further highlighting their broad substrate scope. More importantly, that the newly formed non-Asx peptide bond is no longer recognized by PALs provides the needed orthogonality to conduct consecutive PAL-mediated orthogonal ligations at the N- and C-termini of the same protein. We used this sequential ligation scheme to prepare homogeneous dual-payload antibody-drug conjugates (ADCs). This advancement further enhances PALs' utility as powerful tools for the development of protein-based therapeutics.