Pyridium-π interaction preserves N,S-benzylidene thioacetals in acidolysis enabling efficient protein chemical synthesis.

Chemical protein synthesis provides the atomic-level precision to access peptides and proteins, yet the synthesis of aggregation-prone one remains a major challenge. Here, we report the picolinoylated N,S-benzylidene thioacetal dipeptides (NTDs) strategy to facilitate the chemical synthesis of difficult peptides and proteins. NTDs can serve as an effective aggregation disruptor and be robustly prepared in high yield, which can be rapidly introduced through standard solid-phase peptide synthesis (SPPS). Furthermore, we discover a picolinoylation-based strategy to address the acid lability of NTDs, which preserves the scaffold during global deprotection through pyridium-π interaction and can be selectively removed on demand. This approach effectively suppresses aggregation both on-resin and in-solution, facilitates macrocyclization, and permits selective manipulation of cysteine residues during multi-fragment assembly. As a demonstration, we achieve the chemical synthesis of the hydrophobic protein human erythropoietin (hEPO) via a convergent route with native chemical ligation and serine/threonine ligation incorporating the NTD chemistry.