Novel Methods for the Ribosomal Incorporation of β-Amino Acids

Protein-protein interactions (PPIs) dominate all cellular functions across every domain of life. If PPIs become aberrant, they may result in many human diseases, such as cancer or Alzheimer’s. Despite their clinical significance, modulating aberrant PPIs is a daunting task. Most PPI surfaces are long, hydrophilic and structurally complex. Thus, finding molecules that moderate specific aberrant PPIs is an important goal in drug discovery research. For example, PPIs have been modulated by peptidomimetics, synthetic peptides that assume three-dimensional structures similar to proteins, but unlike natural peptides, they are proteolytically stable. However, building libraries of peptidomimetics is challenging as current methods rely on solid phase peptide synthesis, which limits the size and diversity of peptidomimetic libraries. As such, using the translation machinery to synthesize peptidomimetics is an attractive approach. In Chapter 1, we begin by discussing bacterial protein synthesis. Then, we delve into a detailed discussion of the application of the bacterial translation machinery for the in vitro translation of synthetic peptides. In this discussion, we review the different technologies, their advantages and limitations with respect to the incorporation of amino acids with unnatural backbones. After reviewing the methods used to incorporate backbone analogs, and their compatibility with the bacterial translation machinery, we describe a novel approach for the ribosomal incorporation of -amino acids analogs containing an -substituent, -hydroxy--amino acids (Chapter 2). We demonstrate that the ribosome incorporates this new class of substrates through the formation of an intermediate ester bond that rapidly rearranges to form a native peptide bond. Using this approach, we show that -hydroxy--amino acid single incorporation efficiencies are comparable the incorporation efficiencies obtained with natural amino acids. In Chapter 3, we apply this approach to the synthesis of peptides containing multiple -hydroxy--amino acids. This chapter describes the results obtained with the in vitro synthesis of peptides containing two consecutive -hydroxy--amino acids, three consecutive -hydroxy--amino acids, and alternating -hydroxy--amino acids and -amino acids. Based on these results, we propose experiments to improve these incorporation yields for the application of this technology for the in vitro synthesis of diverse peptidomimetic libraries.