Ubiquitination of both peptides and proteins using a novel water-soluble acylation reagent, based on a 2,4-dichloro-6-sulfonic acid phenol ester, is presented. At alkaline pH, fast lysine ubiquitination is observed on model peptide FLYRANK, and its regioselectivity is confirmed using controls. The same selective lysine ubiquitination is shown on the Fau gene encoded Ubiquitin-like protein (FUBI) protein, which is confirmed using liquid chromatography tandem mass-spectrometry analysis/mass spectrometry analysis.

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.