Diels-Alder ligation of peptides and proteins

Abstract

In light of the emerging area of protein chemistry, we demonstrated that the Diels-Alder ligation offers an advantageous new way for the site-selective functionalization and labeling of proteins and peptides. The first studies have shown that peptides containing a diene (2,4-hexadiene) and a dienophile (maleimide) functions can be ligated chemoselectivety in aqueous solution with high efficiency. The scope of the Diels-Alder ligation approach for protein ligation was further investigated using streptavidin, avidin and, in a more biologically relevant example, Rab7 proteins as model systems. The hexadiene moiety revealed to be stable and bioorthogonal to all functionalities found in peptides and could be incorporated into proteins by different methods: non-covalently by formation of a protein-hexadienyl ligand complex; covalently by standard bioconjugation techniques; or covalently and site-specifically employing the expressed protein ligation (EPL) approach. The resulting protein diene derivative was further functionalized by means of the Diels-Alder reactions with different maleimides under very mild conditions. Because the maleimide group can potentially react with cysteine residues found in proteins via Michael conjugated addition, the mercapto group of these amino acids must be masked as disulfides during the course of the Diels-Alder ligation. This procedure is technically very simple and showed not to impair the biological function of the model proteins. The developed methodology opens a new alternative approach to equip a given protein (or polypeptide) with appropriate functionalities that can be further explored for biochemical and biological investigations.