Two-stage aminooxy and ester ligation enables programmable Serine/Threonine editing in unprotected peptides

Serine and threonine residues are critical for numerous regulatory post-translational modifications in nature, yet their chemical modification in unprotected peptides remains a significant challenge. The inherent weak hydroxyl nucleophilicity and the presence of multiple indistinguishable sites within proteins have historically prevented the development of general, site-selective strategies. Current methods often lack the precision or mild conditions required for complex peptide engineering, limiting the ability to create novel therapeutic peptide analogues or study specific protein functions dependent on Ser/Thr modifications.

Researchers developed a two-stage chemical strategy for site-specific Serine/Threonine editing. The first stage involved using aminooxy serine or aminooxy threonine to mediate an effective aminooxy ligation (AOL) in aqueous solution. This reaction proceeds through a transient 1,2,4-oxadiazinane intermediate, enabling chemoselective peptide ligation at the N-terminal position. Subsequently, the embedded aminooxy functionality underwent a second chemoselective ester ligation (CEL) with keto acids. This second stage served to restore the aminooxy group into native Ser/Thr, while simultaneously installing specific O-acylation.

The study successfully demonstrated that aminooxy ligation (AOL) mediated by aminooxy serine or aminooxy threonine provides a highly chemoselective method for peptide ligation at the N-terminal position. This initial step forms a transient 1,2,4-oxadiazinane intermediate, crucial for the reaction's specificity. The subsequent chemoselective ester ligation (CEL) with keto acids proved effective in restoring the aminooxy functionality back into native Ser/Thr residues. Simultaneously, this two-stage process allowed for the precise installation of O-acylation. This versatile strategy permits various modifications, including reductive restoration, neoglycosylation, or O-acylation, under mild conditions. > Applications range from the late-stage modification of therapeutic peptide analogues to the convergent chemical synthesis of histone H2B bearing site-specific O-neoglycosylation and O-acylation, showcasing its broad utility in protein chemical synthesis and engineering.