Engineering Reverse Turns: Implications in Conformation, Stability and Bioactivity of Peptides and Proteins

Abstract

The work presented in this thesis provides an overview of the potential role of N-methylation in inducing reverse turns. Backbone N-methylation is one of the common modifications designed by nature and exploited by humans to enhance the oral bioavailability of macrocyclic peptides. However, their predisposition in turns in cyclic peptides are undermined by the backbone cyclization restraint, where conformations are driven mainly by steric interactions. Therefore, the work sheds light on the intrinsic property of N-methylation in inducing turns and its influence in modulating the conformational stability of beta-hairpin containing peptides and proteins. The thesis is divided into five chapters. In the first chapter, we provide a brief introduction on a stable class of scaffolds known as peptidomimetics and the literature associated in designing of major secondary elements: alpha-helix and beta-sheet. Additionally, we highlight the strategies employed in engineering beta-turn mimetics and the challenges associated with the available repertoire of turn mimetics. In the second chapter, we largely focus on utilization of N-methylated heterochiral amino acids as beta-turn surrogate and their role in inducing beta-hairpin formation in both polar and apolar conditions, with inspiration derived from the conformations of naturally-occurring and designed cyclic peptides. Subsequently in the third chapter, we demonstrate the differential behavior of N-methylation at i+1 and i+2 site of a beta-turn and thereby, tune the steric restraints at the turn through non- covalent interaction. We also show the bio-compatibility of our engineered motif with different functional groups, present on the ribosomal-encoded amino acids. Finally, we validate our design in a protein model system and modulate the global conformational stability of the protein. In the fourth chapter, we venture into the applicability of our designed ‘chemical toolkit’, where we exploit our design in generating highly potent, low-cytotoxic and metabolically stable b-hairpin antimicrobial peptides that has the ability to kill bacteria in a very short period of time. Finally, in the last chapter, we summarize the overall work. The later part of the thesis has appendices, which consists of the materials and methods employed in the aforementioned chapters, characterization of all the compounds synthesized in the individual chapters using MALDI, HPLC and NMR and few supplementary data.