AbstractThe work of this thesis is primarily based on the development of novel probes for peroxynitrite (ONOO-) detection and their applications in vitro.
Chapter 1 introduces peroxyresorufin-1 (PR1) as an ONOO- sensitive and selective probe. Confocal imaging in J774.2 macrophages confirmed PR1’s ability to detect exogenous and endogenous ONOO-. Subsequent studies using a plate reader focused on validating PR1 as an effective tool to study pathways involved in generating ONOO-. Stimulation with LPS, IFN-γ, PMA and ATP, scavenging effects with O2∙- and NOS inhibitors as well as hypoxic environments were investigated.
Following on from validating PR1 as an effective fluorescent tool in macrophages, Chapter 2 discusses the synthesis and in vitro studies of PR1 derivatives. Two classes of PR1 derivatives are explored in this chapter: organelle targeting probes and alkyne and alkane based probes with increasing lipophilicity and in situ click chemistry applications. The organelle targeting PR1 derivatives showed poor cell permeability in contrast to the alkyne and alkane derived probes. The latter were shown to detect exogenous and endogenous ONOO- in RAW264.7 macrophages with one showing good applicability for in vivo studies. Subsequently, this led to the development of theranostics probes: detection of ONOO- and release of a drug in situ.
Chapter 3 outlines the development of three ONOO- coumarin probes that target specific organelles in the cell: mitochondria, lysosome and endoplasmic reticulum. All three ratiometric probes displayed excellent selectivity and sensitivity towards ONOO- over other ROS. In vitro studies with RAW264.7 macrophages were carried out using exogenous and endogenous ONOO- generation. The lysosome targeting coumarin probe was the only one found to be cell permeable and able to detect exogenous and endogenous ONOO-.
Chapter 4 introduces a series of ONOO- probes based on fluorescein, rhodamine, and rhodol fluorophores with benzyl Bpin target groups for ONOO-. Rhodamine and rhodol should allow for targeting the mitochondria and endoplasmic reticulum respectively, whereas fluorescein is of interest since it has been approved by the FDA. All three probes were selective and sensitive for in situ detection of ONOO- but showed poor properties in vitro.
Finally, Chapter 5 describes a rhodamine based probe for the selective and sensitive detection of glutathione (GSH). The unusual reactivity of the azide to preferentially detect GSH over hydrogen sulfide (H2S) led to comprehensive fluorescence studies to validate the GSH probe in situ. Azides are known to be reduced by H2S into the corresponding amine. To our surprise, the binding of GSH to the azide group is preferred and releases a fluorescence signal.
|Date of Award||15 Jan 2020|
|Supervisor||Tony James (Supervisor), Steven Bull (Supervisor) & Amanda Mackenzie (Supervisor)|