Harnessing alcohol side-chain reactivity in peptides and proteins: serine- and threonine-specific cleavage and conjugation.

The side-chain hydroxyl groups of serine and threonine represent versatile and underexploited reactive handles for the selective chemical manipulation of peptides and proteins, offering complementary alternatives to conventional cysteine- and lysine-directed bioconjugation. Over the past decades, significant advances have been made in harnessing the unique reactivity of these alcohol-bearing amino acids for site-selective cleavage, functionalization, and bioconjugation. This review summarizes the fundamental principles governing alcohol side-chain reactivity and highlights emerging strategies that exploit serine and threonine residues for precise biomolecular modification. Particular emphasis is placed on serine- and threonine-specific peptide bond cleavage methodologies, including oxidative, dehydrative, acyl-transfer, and metal-mediated transformations that proceed through reactive intermediates such as dehydroalanine (Dha) and dehydrobutyrine (Dhb), oxazolines, and activated esters. These approaches have enabled controlled peptide fragmentation and protein editing under mild conditions. In parallel, recent developments in conjugation chemistry based on hydroxyl side-chain activation are discussed, encompassing glycosylation, phosphorylation-inspired modification, radical-mediated functionalization, and Dha/Dhb-based diversification. Such methods facilitate the site-selective installation of fluorophores, glycans, affinity tags, and therapeutic payloads onto complex biomolecular scaffolds. Collectively, the selective exploitation of serine and threonine side-chain reactivity has substantially expanded the toolbox of modern bioconjugate chemistry. These advances provide powerful opportunities in the areas of chemical biology, proteomics, biomaterials, and next-generation therapeutic development, while also offering new directions for selective peptide and protein engineering.