Peptides and proteins with N-terminal amino acid sequences NH -Xxx-His (XH) and NH -Xxx-Zzz-His (XZH) form well-established high-affinity Cu -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity Cu -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of Cu in XZH, like PET imaging (with Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong Cu binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.

Total chemical synthesis of proteins is usually achieved by assembling unprotected peptide segments using site-specific and chemoselective native peptide ligation methods. Access to large proteins often requires the assembly of at least three segments due to the current limits of solid phase synthesis of individual peptide segments. The aim of this tutorial review is to present the basic concepts and challenges underlying the design of sequential peptide ligation strategies using solution or solid phase chemistry. A special emphasis is given to C-to-N and N-to-C three-segment assembly strategies, which potentially give access to proteins composed of up to 150 amino acid residues.