Peptidomimetic techniques and their application in the design and synthesis of Kisspeptin analogues

Abstract

Peptidomimetic strategies play an important role in the development of peptides as therapeutics and biological probes. Strategies such as cyclisation and the introduction of unusual amino acids are central to the tailoring of peptide‐based compounds which capture the molecular features important for binding, whilst improving stability to proteases. This has the potential to lead to potent and stable peptides which may be used as biological probes or drug candidates. Kisspeptin is a novel peptide hormone which displays anti‐metastatic properties in several tumour types as well as an important role in reproduction. While the study of linear peptide analogues has revealed important features of Kisspeptin, the bioactive conformation is not known. 2D‐NMR structures of Kisspeptins and close Kisspeptin analogues have suggested two preferred conformations, helical and unstructured. This thesis describes the use of peptidomimetic strategies such as cyclisation and the incorporation of unusual amino acids to perform a peptide conformational analysis of Kisspeptin. This is separated into the exploration of the two potential Kisspeptin peptide binding modes – where the active conformation of Kisspeptin is either helical (Chapters 2 and 3) or linear (Chapter 4). Chapter 1 covers the prior literature considered relevant to this thesis. The reader is introduced to the pre‐eminent techniques of peptidomimetic design and the discovery, biological importance and previous work carried out in the field of Kisspeptin‐GPR54. The first body of work presented in this thesis investigates the helix hypothesis by the design of helix stabilised Kisspeptin mimetics. Chapter 2 presents an evaluation of current modelling techniques in the peptidomimetic design of lactam‐bridged peptides is presented and the usefulness and limitations of these methods is described. Chapter 3 presents the synthesis and conformational evaluation of a series of lactam‐bridged Kisspeptin analogues designed to stabilise a helical conformation. With stabilised Kisspeptin analogues in hand, they were evaluated for biological activity at GPR54, giving some useful insights to the bioactive conformation of Kisspeptin. The second body of work presented in this thesis explores the potential use of β-amino acids as a means of increasing stability in the linear analogues. Chapter 4 presents the synthesis and biological evaluation of a β‐amino acid scan of Kisspeptin‐10 and the downsized GPR54 agonist Amb‐Phe‐Gly‐Leu‐Arg‐Phe‐NH2. A series of Boc‐protected β2‐amino acids were synthesised and used for the synthesis of a series of mixed α/β peptides. Peptides were tested GPR54 activity as either a mixture of diastereomers or as separated diastereomers in a functional luciferase assay. The third body of work presented in this thesis describes a new method to facilitate the use of unusual amino acids (such as β2‐amino acids). Chapter 5 investigates the synthesis and application of the chiral pre‐column derivatisation reagent FLEC as a preparative scale separating agent and protecting group in standard Fmoc solid phase peptide synthesis. An efficient synthesis of enantiopure FLEC was devised, employing chiral reduction. Prepared unusual amino acids were protected using (R)‐FLEC and conditions reported for the separation of diastereomers under RP‐HPLC conditions. The use of FLEC as an amine protecting group was demonstrated by the incorporation of FLEC derivatised amino acids in linear peptide sequences under standard Fmoc solid phase synthesis conditions. The final chapter of this thesis (Chapter 6) summarises the achievements that were presented in this body of work.