Site-Specific and Programmable Editing of Serine and Threonine in Unprotected Peptides

Serine and threonine residues dominate regulatory post-translational modifications in nature, yet remain largely inaccessible to programmable chemical editing. Their weak hydroxyl nucleophilicity and the presence of multiple indistinguishable sites within proteins have prevented the development of general, site-selective strategies for modification in unprotected polypeptides. Here, we report that aminooxy serine or aminooxy threonine can mediate an effective aminooxy ligation (AOL) in aqueous solution through a transient 1,2,4-oxadiazinane intermediate, enabling chemoselective peptide ligation at the N-terminal position. Furthermore, the embedded aminooxy functionality after AOL subsequently undergoes a chemoselective ester ligation (CEL) with keto acids to restore aminooxy into native Ser/Thr and install O-acylation. This two-stage strategy offers site-specific Ser/Thr modification under mild conditions, permitting reductive restoration, neoglycosylation, or O-acylation. Applications range from late-stage modification of therapeutic peptide analogues to the convergent chemical synthesis of histone H2B bearing site-specific O-neoglycosylation and O-acylation. Together, this work establishes a general chemical framework for serine and threonine editing for protein chemical synthesis and engineering.