Design, Synthesis, and Biological Evaluation of Chemical Probes for Visualising DNA Synthesis in Proliferating Cells

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Poulsen, Sally-Ann

Avery, Vicky

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Bornaghi, Laurent

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DNA biolabels are chemical probe compounds used detect and visualise DNA synthesis in replicating cells. These compounds are tools that enable biologists to answer fundamental questions regarding the proliferation of cells in diverse fields ranging from developmental biology, infectious disease, and cancer biology. This thesis describes the design and synthesis of new and synthetically challenging compounds as prospective DNA biolabels. These compounds were then evaluated for DNA biolabelling capacity and cytotoxicity against T. cruzi parasites, the causative agent of Chagas disease. Chapter One serves as a general introduction and literature review of the history and current state of the art of DNA biolabels, in particular, DNA metabolism of nucleoside analogues and development of new strategies for detection of biolabels incorporated into DNA. This area of research had culminated in the discovery of 5-Ethynyl-2ʹ-deoxyuridine-uridine derivatives for detection and visualisation of DNA synthesis employing click chemistry and fluorescent azides. Chapter Two describes the design and synthesis of 2ʹ-halo-arabinosyl EdU derivatives as prospective DNA biolabels. This chapter also reviews the state of the art of synthesis of 2ʹ-halo-arabinosyl pyrimidine nucleosides. The synthesis of these new 2ʹ-halo-arabinosyl EdU analogues required a challenging late-stage halogenation strategy. In the development of this strategy, new synthetic methodology evolved employing the unusual N-nitro protecting group. Optimised conditions were developed for quick and gentle installation and removal of the N-nitro protecting group in the presence of sensitive functional groups. Furthermore, the techniques developed for the removal this protecting group allowed for its removal in the presence of functional groups sensitive to reducing conditions. Chapter Three describes synthesis of cycloSaligenyl 5ʹ-phosphotriester of EdU analogues and a literature review of the synthesis and synthetic strategies used to synthesise cycloSaligenyl phosphotriester of nucleoside analogues. New conditions were explored and developed for the synthesis of the cycloSal phosphotriesters from unprotected nucleoside analogues. These methodologies were successfully applied to multiple nucleoside substrates. A new synthesis and purification of cyclosaligenyl phosphoramidites was developed and successfully applied to four different saligenyl alcohols. This methodology allows for simple and straight forward synthesis of both the cycloSal phosphotriesters and their phosphoramidite precursors. Chapter Four describes the attempted synthesis of 2ʹ-fluoro-arabinosyl EdU by direct fluorination of the 2ʹ-triflyl precursor and serves to review the particularly difficult synthetic transformation of 2ʹ-fluorination of preformed pyrimidine nucleosides. Towards this end we successfully achieved 2ʹ nucleophilic fluorination of uridine by employing 3-N-nitro stabilised 2ʹ-triflates. This required extensive exploration of conditions and fluoride sources to find appropriate conditions for successful reactions. It was found that basicity of the amine base of the HF-amine complexes was integral to the success of fluorination reactions. Attempts to increase the yields of the fluorination reaction through alternative hydroxyl protecting groups of the uridine-N-nitro-triflate failed. An alternative non-reducing deprotection strategy of the the N-nitro group of the 2ʹ-fluro-3-N-nitro-uridine was developed employing a two-step process. This relied on first forming an N-amino intermediate which was then deaminated to give the free 2ʹ-fluoro-arabinosyl uridine nucleoside. This synthesis afforded 2ʹ-fluoro-arabinosyl uridine in yields 17 times greater than previous reports by direct fluorination of preformed uridine nucleosides. Chapter Five briefly describes the differences of nucleoside and DNA metabolism of T. cruzi parasite to that of mammalian cells. This chapter also describes the biological evaluation of the EdU and its 2ʹ-halo analogues, and the cycloSal phosphotriester derivatives. The findings revealed that the T. cruzi parasite is extremely sensitive both labelling with EdU and its associated cytotoxicity. Labelling of the parasite with any of the EdU analogues was associated with corresponding cytotoxicity. Improvement of labelling efficiency of some the EdU analogues by functionalisation with cycloSal phosphotriesters demonstrates that thymidine kinase blockade occurs for some nucleoside analogues. Thus the parasite expresses a putative thymidine kinase with restricted substrate specificity comparable to that of higher eukaryotes. The high cytotoxicity of EdU and its analogues may indicate the presence of an alternative cytotoxic pathway not present in mammalian systems, possibly resulting from interference with the base J metabolism.

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Thesis (PhD Doctorate)

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Doctor of Philosophy (PhD)


School of Environment and Sc

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Biological evaluation

Chemical probes

Visualising DNA synthesis

Proliferating cells

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