The site-selective modification of peptide backbones allows an outstanding fine-tuning of peptide conformation, folding ability, and physico-chemical and biological properties. However, to achieve selectivity in the core of these biopolymers is challenging. In the last few years, many advances towards this goal have been developed. This review addresses the selective modification of Cα- and N-positions, from the use of “customizable units” to the residue-directed introduction of substituents. The site-selective modification of the peptide bond, i.e. the formation of thioamides or heterocycles, which alters backbone rigidity and ability to form hydrogen bonds or recognition by enzymes, is also described. Moreover, not only the modifications in internal backbone positions, but also in the N- and C-termini are discussed. In addition to chemical methodologies, the review addresses some reactions, catalyzed by natural or engineered enzymes, that afford unprecedent regio- and stereoselectivity in backbone modifications.

The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes but still is a difficult synthetic challenge. Herein, we introduce natural hydroxyproline as a convertible unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, homoserine lactones, and Agl lactams, and to achieve the mild, efficient, and site-selective modification of peptides. A domino process is used to cleave the customizable Hyp unit under mild, metal-free conditions. Both terminal and internal positions can be modified, and similar customizable units can be differentiated. The resulting products possess two reactive chains which can be manipulated independently. The versatility and scope of this process is highlighted by its application to the ligation of two peptide chains, and the generation of peptides with several chains and peptides with conformational restrictions.