Extending Peptide Ligation to Acidic pH and Junctions Comprising Glycosylated and Phosphorylated Amino Acids

The native chemical ligation (NCL) is a cornerstone of protein synthesis, allowing the assembly of peptide fragments into larger proteins. Ligation auxiliaries extend the scope of NCL beyond cysteine, but fragment ligation at sterically encumbered junctions remained inaccessible. Herein, we introduce a pyridine-containing ligation auxiliary that allows NCL to proceed at virtually any junction, including reactions at β-branched amino acids, as well as O-glycosylated or O-phosphorylated amino acids. Through iteration of the auxiliary scaffold, we revealed a fundamental relationship between the basicity of the pyridine and ligation speed. Surprisingly, the 2-mercapto-2-(4,5-dimethoxypyridin-2-yl)ethyl (MDPE) auxiliary operates over a broad pH range, enabling ligations even in highly acidic conditions. This property was leveraged in the synthesis of dodeca-phosphorylated peptides from the large RNA polymerase II CTD domain. Additionally, we utilized the MDPE auxiliary for the synthesis of heavily glycosylated segments derived from mucin MUC5AC.