Protein-protein interactions play important roles at all levels of cellular organization and thus are therapeutically relevant targets for the treatment of a wide array of diseases. Modulators of protein-protein interactions are therefore highly valuable molecules sought after in many drug discovery endeavors. Peptides that emulate protein domains at protein-protein interfaces represent a promising strategy for drug development. As the functions and binding affinities of proteins are directly related to their three-dimensional structure, peptidomimetic approaches seek to recreate folded elements of proteins with high-fidelity, independent of the larger folded context from which they are derived. This can be achieved by using macrocyclization to constrain peptides into their unique and bioactive conformations. Macrocyclic-based designs of biologically relevant peptides may be informed by the patterns of hydrogen bonding observed in X-ray crystal structures. Covalent surrogates of hydrogen bonds reduce entropy by destabilizing unfolded states and support the natural tendency of the encoded polymer chain to fold into complex structures.As unmodified peptides tend to be disordered in solution, macrocyclization is routinely employed to constrain native peptide epitopes in their bioactive conformation. Macrocyclization involving internal backbone amides is an underexplored mode of connectivity and the methodology to functionalize these amides with reactive handles for cross-linking has mostly been limited to N-alkylation. Herein, we demonstrate a late-stage bis-alkylation reaction of N-amino peptides with haloaldehydes to afford ethylene bridged N-amino peptide macrocycles (NAProcycles). Our approach is based on the utilization of backbone N-amino groups as reactive handles in bioorthogonal reactions to synthesize macrocyclic peptides. Using this approach, we prepare a series of macrocycles with varying ring size from crude, unprotected linear substrates under aqueous conditions. We further incorporate our covalent constraint into a model loop-helix motif derived from the viral matrix protein VP40. We demonstrate that NAProcycle constraint of specific residues involved in a sidechain-to-backbone hydrogen-bond observed in the X-ray crystal structure of dimeric VP40 significantly enhances helicity.To complement our NAProcycle constraint methodology, we present an informatics analysis of sidechain-to-backbone hydrogen bonds observed in X-ray crystal structures. We detail the compilation of databases of proteins containing sidechain-to-backbone hydrogen bonds that may serve as test cases for our covalent surrogate approach. Our work offers insight into the nature and extent of interresidue sidechain-to-backbone hydrogen bonding in proteins and suggests that several of these interactions may play an important role in protein folding as they are evolutionarily conserved. We also describe a structure-based workflow for the identification of sidechain-to-backbone hydrogen bonds that reside at protein-protein interfaces. This work lays the foundation for future synthetic endeavors to study the impact NAProcycle constraint has on tertiary folding in miniproteins and to construct minimized binding domains that target biologically relevant protein-protein interactions.In continuation of our efforts toward conformationally defined and proteolytically stable peptidomimetics, we explore the effect N-amination has on the structure and activity of a β-sheet model system. We demonstrate an efficient protocol for the synthesis of the antimicrobial Gramicidin S and N-aminated analogues thereof. We describe the parallel synthesis of N-aminated analogues of Gramicidin S via the incorporation of N-amino dipeptide units on solid support. Compared to Gramicidin S, the N-aminated analogues show reduced toxicity to human red blood cells and many exhibit similar or enhanced activity against a panel of ESKAPE pathogens. Our work suggests that relative to Gramicidin S, the improved therapeutic profile of N-aminated analogues may be due to the enhanced stability of the β-sheet like character afforded by the conformational constraints imposed by N-amination.