Selective Lysine Ubiquitination Using Activated Phenol Esters

Ubiquitination of target proteins is an essential post-translational modification influencing a wide variety of cellular processes. Herein, the use of a novel water-soluble acylation reagent based on the 2,4-dichloro-6-sulfonic acid phenol ester of ubiquitin is described for efficient and selective ubiquitin modification of peptides. Under alkaline conditions, this reagent is swift and regioselective toward lysine acylation, while at neutral pH it shows loss of regioselectivity and is able to acylate both lysine and N-terminal modification at reduced speeds. As proof of concept, a model peptide is utilized to demonstrate this strategy, proving to be successful. Then the ubiquitination of a synthetic protein called Fau gene encoded Ubiquitin-like protein (FUBI) is performed under alkaline conditions followed by tandem MS analysis, proving that the selective lysine ubiquitination works to prepare protein-protein conjugates.