Solid-phase peptide synthesis (SPPS) has been widely used for the preparation of many of biologically significant peptides. Since its inception, the methodologies have been the subject of continuous refinement, which has lead to a better understanding of side reactions, hence improved quality and efficiency of syntheses. Furthermore, preparation of peptides by chemical means allows modifications that are otherwise inaccessible using recombinant DNA technology, including structural changes, such as backbone cyclisation and modifications to internal disulfide bonds, which provide further insight into the role of structure and activity.
Of particular interest are the peptides isolated from the venom of Conus species, or conotoxins. This diverse class of molecules provides a diverse source of ion channel neurotoxins. Their high potency and selectivity for different ion channel sub-types makes them excellent
pharmacological tools for studying the nervous system. Furthermore, they have generated much interest as potential drug leads for the treatment of pain and other neurological disease states. The high potency and selectivity exhibited by conotoxins is owed to their multiply disulfide bonded frameworks, which stabilises their conformation in solution.
While modification to the structural framework of peptides by cyclisation and replacement of disulfide bonds with a similar chemical moiety have been demonstrated to improve physical and biological properties, the consequence of these structural modifications on conotoxins is unknown. Therefore, the broad objective of this thesis is to explore the consequences of modifying the structural framework of conotoxin molecules, particularly with respect to the cysteine frameworks.
The specific aims and outcomes of this thesis include:
• Characterisation and chemical synthesis
of a previously unidentified peptide from the venom of Conus textile, designated as Tx1473. The disulfide bond pattern was determined by chemical synthesis, using directed disulfide bond formation, revealing a unique cysteine framework and loop structure.
• Investigation of the impact of N-to-C cyclisation of α-conotoxin Iml through the use of a variable length spacer unit on the formation of the disulfide bond framework. A directed disulfide bond formation strategy and random oxidation strategy demonstrated that formation of different ratios of disulfide bond isomers in solution was dependent on the size of the spacer. Additionally, it was shown that although the activity of the cyclic analogues decreased significantly from that of native α-conotoxin Iml, a greater resistance to enzymatic degradation was exhibited by selected analogues, indicating that cyclisation can improve in vivo stability of
• Examination of the effects of substitution of one or both of the disulfide bonds of a-conotoxin Iml with highly oxidisable diselenide bonds. Incorporation of diselenide bonds was achieved using modified Boc SPPS procedures. Systematic replacement of disulfide bonds resulted in a higher stability in a reducing environment, with little change in activity at the α7 nicotinic acetylcholine receptor. It was also shown that replacement of disulfide bonds with diselenide bonds in conotoxins has very little impact on the three-dimensional structure as demonstrated by nuclear magnetic resonance (NMR) spectroscopy.
• Synthesis of an N-to-C cyclic and diselenide containing analogue of χ-conotoxin MrIA was achieved using strategies based on methods developed during this thesis. Three-dimensional structural studies by circular dichroism (CD) and NMR spectroscopy demonstrated a structure similar to the native structure of
MrIA. The use of intramolecular selenocysteine mediated chemical ligation also allowed synthetic access to an N-to-C cyclic diselenide-containing analogue. Radioligand binding experiments showed that the affinity to the noradrenaline transporter (NET) was not significantly altered by these structural modifications.
The results presented in this thesis demonstrate that modifications to the structural framework can improve the physical and chemical properties of conotoxins.