Enhancing Auxiliary‐Mediated Native Chemical Ligation at Challenging Junctions with Pyridine Scaffolds

To expand the scope of native chemical ligation (NCL) beyond reactions at cysteine, ligation auxiliaries are appended to the peptide N‐terminus. After the introduction of a pyridine‐containing auxiliary, which provided access to challenging junctions (proline or β‐branched amino acids), we herein probe the role of the pyridine‐ring nitrogen. We observed side reactions leading to preliminary auxiliary loss. We describe a new easy to attach β‐mercapto‐β‐(4‐methoxy‐2‐pyridinyl)‐ethyl (MMPyE) auxiliary, which 1) has increased stability; 2) enables NCL at sterically encumbered junctions (e. g., Leu‐Val); and 3) allows removal under mildly basic (pH 8.5) conditions was introduced. The synthesis of a 120 aa long peptide containing eight MUC5AC tandem repeats via ligation of two 60mers demonstrates the usefulness. Making use of hitherto unexplored NCL to tyrosine, the MMPyE auxiliary provided access to a head‐to‐tail‐cyclized 21‐mer peptide and a His(6)‐tagged hexaphosphorylated peptide comprising 6 heptapeptide repeats of the RNA polymerase II C‐terminal domain.