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The development of fluorescent opiate ligands
thesisposted on 2017-02-26, 22:50 authored by Schembri, Luke Steven
This thesis describes the design, synthesis, biological evaluation and application of fluorescent opiate ligands and traceless affinity labeling conjugates (Figure 1) for use in fluorescence-based assays and imaging applications. Each chapter has its own compound numbering scheme, references and experimental section. A brief summary of each chapter is described below: Figure 1. The four fluorescent ligands (top) and three traceless affinity labeling conjugates (bottom) synthesized and investigated in this thesis. Chapter 1: Introduction to Opioids and Fluorescent Ligands. This chapter serves as an introduction and literature review of the two main topics at the core of this thesis: opioid receptors and fluorescent ligands. It describes what is known about opioid receptor structure and pharmacology, with a focus on opioid receptor desensitization – a major issue with opioid analgesics. Fluorescent G protein-coupled receptor-binding ligands and traceless affinity labeling conjugates are then described, with an emphasis on critical design aspects and examples from the literature. Finally, the overall aims of the project are described. Chapter 2: Synthesis, Biological Evaluation and Utility of Fluorescent Ligands Targeting the µ-Opioid Receptor (Journal of Medicinal Chemistry Article). This paper describes the synthesis of four fluorescent ligands (1–4, Figure 1) based on the orvinol scaffold. Four different fluorophores (sulfonated cyanine-5, BODIPYTM 630/650, 4-((6-methoxy-1,2,4,5-tetrazin-3-yl)oxy)butanoic acid (tetrazine) and 3-(6-amino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoic acid) were connected to oripavine from the 7-position C-ring of oripavine via a 6-carbon spacer. These ligands were evaluated for their efficacy and affinity at the µ, κ and δ opioid receptors (MOR, KOR and DOR). They were then imaged in human embryonic kidney cells expressing either the MOR or a MOR-green fluorescent protein fusion protein. After further investigation of 2 and 3, 3 was found to be the most suitable probe for fluorescent binding assays and imaging applications. Chapter 3: Synthesis and Imaging of Traceless Affinity Labelling Conjugates for the µ-Opioid Receptor. This chapter describes the synthesis and attempted imaging of three TALCs (5–7), similar in design to the fluorescent ligands in the previous chapter but incorporating reactive spacers. These probes were designed to bind to the MOR and potentially be attacked by a lysine residue outside the binding domain, leading to covalent labelling of the receptor with the fluorophore initially attached to the TALC. Such a labelling strategy could allow further pharmacological interrogation of MOR with any ligand of choice. Synthesis of the TALCs involved attachment of a silicon-rhodamine fluorophore to an oripavine scaffold via a spacer containing a reactive acyl-imidazole group. Despite successful synthesis, the TALCs proved to be highly unstable, hampering efforts to employ them in imaging studies. Chapter 4: Conclusions and Future Work. This chapter presents a summary of the results of this thesis and discusses possible future work to extend upon the projects investigated in this thesis.