Covalent attachment of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to the ε-amino group of lysine residues in a target protein (including ubiquitin itself), a process termed ubiquitination, is one of the most prevalent mechanisms for regulating protein function and stability in eukaryotes. Indeed, sequence annotations suggest nearly 5% of the human genome is dedicated to the coupling and removal of Ub/Ubls to and from proteins. Given the central role of the ub network in cellular physiology, misregulation is often associated with numerous human diseases including. e.g., cancer, immune disorders neurodegenerative diseases and congestive heart failure.
Ubiquitination is unique among the ensemble of posttranslational modifications (PTMs), specifically from the standpoint of signal diversity. For example, in contrast to other prevalent PTMs such as phosphorylation, proteins can be modified with Ub on a single lysine residue (monoUb), multiple lysines (multi-monoUb), or a single lysine with a polymeric chain of Ub (polyUb). With regards to polyUb chain formation, Ub possesses seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), each of which may form an isopeptide linkage with the carboxy terminus of another Ub molecule. This feature adds significant complexity to intracellular Ub signaling networks as it permits the assembly of chains with many different types of linkages and lengths with the potential to control distinct biological processes.
A number of reports have recently emerged describing chemical approaches to the site-specific conjugation of Ub molecules through native Nε-Gly-L-Lys isopeptide linkages as well as various normative linkages. Indeed, some of the methods have elucidated important structural distinctions for Ub dimers linked through the different Ub lysines, and enabled studies that uncovered how the structure and function of target proteins is altered upon Ub modification. However, many of the chemical approaches designed to recapitulate the Nε-Gly-L-Lys linkage suffer from drawbacks such as instability, lengthy synthetic manipulations, and/or the use of specialized recombinant DNA technologies for incorporating unnatural amino acids. Moreover, branched Ub oligomers in which two or more Ub molecules are covalently attached to a single Ub through different lysines appear to be inaccessible using known methods.