Precision Protein Modification via Ligation and Bioconjugation at Selenocysteine

Conspectus Selenocysteine (Sec), often referred to as the 21st amino acid, is a rare yet essential amino acid found in a small number of naturally occurring selenoproteins that play critical roles in redox regulation, antioxidant defence, and hormone metabolism. Owing to its distinct chemical properties, namely enhanced nucleophilicity, lower reduction potential, and versatile redox behaviour of the side chain selenol/diselenide moiety relative to the thiol/disulfide of Cys, Sec has recently been leveraged for a range of applications in protein science. In this account, we summarize our endeavors towards the development of selenium-mediated ligation methodologies for the rapid and highly efficient assembly of proteins, including those bearing native post-translational modifications (PTMs) or “designer” modifications. Key innovations include the diselenide–selenoester ligation (DSL) and expressed protein selenoester ligation (EPSL). DSL enables the rapid fusion of unprotected peptides through a chemoselective reaction between peptide selenoesters and selenopeptides. Notably, in the presence of suitable additives, DSL can be performed at nanomolar concentrations, providing access to challenging classes of modified proteins, such as lipoproteins, that are difficult to access via other techniques. We also discuss the extension of DSL chemistry to larger proteins through the generation of protein selenoesters from recombinantly expressed intein fusion proteins. By combining the advantages of recombinant bacterial expression and the rapid kinetics of DSL at Sec, we highlight the use of the EPSL technology for the generation of large, aggregation-prone proteins, including the lipidated GTPase YPT6 and mycobacterial lipoproteins as novel vaccine antigens. Beyond protein synthesis and semi-synthesis, we have also demonstrated the utility of selenocysteine (Sec) as a powerful bioconjugation handle. Specifically, we highlight two novel Sec-selective bioconjugation reaction manifolds in this account; the photocatalytic diselenide contraction (PDC) and electrochemical selenoetherification (e-SE). These transformations enable dimerization and site-specific functionalization of peptide and protein diselenides with exceptional regio- and chemoselectivity, offering significant advantages over other bioconjugation methods. Overall, we believe that these technologies represent a major advance in the generation of site-selectively modified peptides and proteins, unlocking new possibilities for applications in fundamental biology but also the generation of next-generation diagnostics and therapeutics bearing precise modifications at defined sites on proteins.