Strategies to Stabilise Peptide Secondary Structure Using Unnatural Amino Acid Crosslinking

Abstract

The communication between proteins plays a pivotal role in both biological and pathological processes. Protein-protein interactions (PPIs) play crucial roles in transcription, signal transduction and apoptosis; as such, exploring modulators of PPIs is a promising strategy for the development of novel therapeutics. Due to the large, relatively featureless nature of PPIs developing small molecule inhibitors is highly challenging. However, peptides provide an alternative to small molecules, exhibiting remarkable capability in binding to these protein surfaces commonly involved in PPIs. PPIs are mediated by the cumulative binding energy of many amino acid residues, over an extended surface. Through analysis of PPIs, it has been determined that many of the residues involved adopt secondary structure elements such as a-helices and b-sheets which are crucial for effective binding. However, linear peptides possess great conformational flexibility in solution and as such modifications such a cyclisation are required to restrain these secondary structure elements. Within this work, peptides were conformationally restrained in order to stabilise their secondary structure. Particular focus was on the stabilisation of a b-hairpin that is involved in the PPI existing between the 5’ endonuclease XPF-ERCC1 and the XPA protein in the nuclear excision repair pathway. This pathway is one of the most versatile DNA repair pathways and upregulation of both the pathway and ERCC1 have been implicated in chemotherapy resistance. As such efforts toward the synthesis of a b- hairpin peptide to inhibit this interaction were investigated through cyclisation of the minimal binding motif using unnatural amino acids. In addition, methodology to develop a peptide staple to induce a-helicity is developed. This is shown through restraining a model pentapeptide incorporating an unnatural alkyne containing amino acid and a cysteine thiol using the thiol-yne coupling reaction. The methodology developed is shown to have promising results in its ability to nucleate an a-helix. Finally, the selective modification of lysine residues through functional group interconversion from a side chain amine to an azide is described on an amino acid and peptide system. This was later demonstrated on a model protein system. This provided a iii handle to undertake a copper(I)-catalysed alkyne-azide cycloaddition click reaction with an alkyne. Utilisation of an alkyne fluorophore could allow for this methodology to be used for imaging of proteins. Overall, the work presented in this thesis provide insights into methods to conformationally restrained peptides using a range of unnatural amino acids. In particular, significant progress in the development of a thiol-yne click methodology for stabilising a-helices which may, in the future, enable for the stabilisation of a-helices involved in PPIs.