Immobilized Thiol Catalysts for Native Chemical Ligation in Flow

Abstract Native chemical ligation (NCL) is a well-established research tool used to access long and complex peptides that cannot be efficiently synthesized by other means such as linear solid-phase peptide synthesis (SPPS). However, its broader application beyond the research laboratory scale has been limited by low-volume yields, slow reaction kinetics, and challenges associated with homogeneous thiol catalysts, as these catalysts can be incompatible with desulfurization methods and difficult to remove in subsequent chromatographic purification. In this study, we report a continuous-flow NCL platform enabled by the development of a novel immobilized version of the aromatic thiol catalyst 4-mercaptophenylacetic acid (MPAA). This strategy employs shelf-stable peptide hydrazides that are activated in situ to acyl pyrazoles and subsequently converted to reactive thioesters in flow by the immobilized catalyst, thereby facilitating ligation with cysteine-containing peptides. The platform is directly compatible with inline desulfurization and supports high-throughput synthesis of complex, structurally diverse, and therapeutically relevant peptides up to 84 amino acids in length. This work establishes immobilized aryl thiols as viable catalysts for NCL in continuous flow, identifies involved key challenges, and provides a foundation for its prospects toward industrial peptide manufacturing.