The Aqueous Phosphorylation and Ligation of Nucleoside Analogues and Aqueous Azide Reduction Methodology

Abstract

The research within this thesis is primarily concerned with the synthesis of modified nucleosides, their oxyphosphorylation and thiophosphorylation to form analogues of nucleoside monophosphates and phosphodiester linkages, and the chemistry of the thiophosphoryl group. These families of compounds may have potential in the areas of antisense oligonucleotide agents or nuclease inhibition. The work described here may also provide routes to new glycosyltransferase inhibitors.

This work builds upon previous work in the Hodgson research group, principally the thiophosphorylation and subsequent alkylation of organic amine fragments as a ligation strategy, and the optimisation of the thiophosphorylation procedure as applied to 5amino5deoxyguanosine. My role was to extend these techniques to the thiophosphorylation of other nucleoside derivatives and to use the thiophosphorylation procedure to produce potentially biologically active compounds.

This thesis is divided into a number of chapters and appendices, and commences by providing a review of the synthesis, properties, and applications of natural and unnatural phosphodiester compounds in the first chapter.

The second chapter details the work which has already been carried out within this research group on the synthesis, oxyphosphorylation, and thiophosphorylation of aminodeoxynucleosides; this forms the foundation to my own work on optimisation of the oxyphosphorylation procedure, and the application of both the oxyphosphorylation and the thiophosphorylation methodology to other aminodeoxynucleosides.

The third chapter describes the application of the thiophosphorylation procedure to the synthesis of a thiophosphoramidate mimic of a dinucleoside. The following chapter concerns the investigations into the hydrolytic stability of the thiophosphoramidate group, using the dinucleoside thiophosphoramidate analogue as a model.

The fifth chapter is on an aqueous method for the reduction of organic azides using the thiophosphate ion as the reducing agent. The reaction was tested on a number of alkyl and aryl susbstrates, and the mechanism of the reduction was investigated.

Appended to this thesis are some chapters on work related to the main project in their application of modified nucleosides and nucleotides; in the first appendix, modifications were made to the phosphate group of guanosine monophosphate to allow the role of the phosphate group in the formation of G-quadruplex structures to be studied. The second appendix concerns work done in support of a project to incorporate 5-deoxy-5-hydrazinoguanosine into the 5- terminus of RNA strands, which allowed the termini to be labelled with fluorescein isothiocyanate (FITC). Using 5-deoxy-5-hydrazinoguanosine as a model for the modified RNA strands, it was shown that within the limits of detection, each hydrazine group reacts with only one molecule of FITC.