Synthesis of Structural and Fluorescently Labelled Peptidic Ligands for Optical Imaging of G Protein-Coupled Receptors

Fluorescence imaging is capable of facilitating highly specific and non-invasive investigation of cellular and molecular events both in vitro and in vivo. Fluorescently labelled peptidic ligands are useful in studying the location, distribution, trafficking and functions of G protein-coupled receptors (GPCRs). Although GPCRs are popular targets in modern target-oriented drug design, many of them have not been therapeutically exploited. An effective strategy in preparing such ligands is to conjugate fluorophores to high-affinity and selective peptide analogues that are derived from the endogenous peptides. This thesis describes the synthesis and pharmacological properties of structural and fluorescent peptide analogues that target the GPCRs of interest. Their usefulness as receptor optical imaging agents has been verified in selected analogues.
   
   Chapter 1 describes the advantages of using fluorescently labelled peptides as optical imaging agents in studying GPCRs, and provides an overview of the key steps and considerations in designing such peptides. To support our ideas, a table containing representative examples of literature-documented fluorescently labelled peptides that target GPCRs is included.
   
   Chapter 2 demonstrates the application of various synthesis strategies in preparing structural and fluorescent peptide analogues derived from the two endogenous neuropeptides: ghrelin and kisspeptin. Specifically, we have verified the effectiveness of standard Fmoc-based solid phase synthesis, use of orthogonal protecting groups and fluorophore conjugation in both solid and solution phase, which have resulted in fluorescently labelled ghrelin and kisspeptin analogues useful in visualising their corresponding receptors.
   
   Chapter 3 and 4 describe utility of the verified synthesis strategies in preparing fluorescently labelled peptides that target neuropeptide Y (NPY) receptors. Chapter 3 presents synthesis and pharmacological evaluation of ligands derived from the modified NPY C-terminal 9-amino acid fragment BVD-15 scaffold (Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-NH₂). Fluorescence labelling was attempted at the 3-position via propargyloxyproline, and the 2- and 4-position via Lys or Lys(azide). We have found that the 2-position labelled analogue [Lys(sCy5)², Arg⁴]BVD-15 exhibited Y₁R antagonism and Y₄R agonism, and it represents a novel ligand useful in imaging studies of these receptors. Chapter 4 presents synthesis and pharmacological evaluation of Y₄ receptor ligands derived from the Y₄R agonist BVD-74D, a dimeric peptide comprised of two Tyr-Arg-Leu-Arg-Tyr-NH₂ monomers cross-linked by a 2,7-diaminosuberoyl group. We have shown the synthesis strategies towards the two optically pure BVD-74D stereoisomers and their structural and mono-labelled fluorescent analogues, by exploiting cross metathesis between suitably protected allylglycine residues with the desired stereo configuration. We have found that the (R,R)-stereoisomer exhibited stronger Y₄R affinity and agonism. Importantly, the fluorescent analogue mono-sCy5-(R,R)-BVD-74D retained the pharmacological profiles of the unlabelled parent compound, and represents a novel ligand useful in imaging studies of Y₄R.
   
   Chapter 5 summarises the achievement presented in this thesis, and provides future directions in the relevant areas.