Enzymatic C-to-C Protein Ligation.

Abstract

Transpeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions.