Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S‐Benzylidene Thioacetals

Abstract Although solid‐phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S‐ benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V‐like (PD‐L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.