The synthesis, detection and repair of nucleotides containing the 8-nitroguanine modification

There is accumulating evidence that reactive nitrogen species derived from nitric oxide metabolism are involved in cancer as they are able to damage DNA largely through oxidation or nitration of the guanine base. The 8-nitroguanine lesion is increasingly associated with cancers that result from chronic inflammation; however due to its instability, very little is known about this base modification. Consequently this thesis focuses upon establishing methods to detect and quantify the lesion and investigate enzymes potentially involved in repair systems directed against 8-nitroguanine in DNA. The approach outlined in this thesis utilises ribonucleoside analogues of the lesion which sufficiently stabilised the labile glycosidic bond. The 8-nitroguanine nucleosides were prepared prior to incorporation into the oligodeoxynucleotide sequences using the traditional 3’- to 5’-solid-phase phosphoramidite chemistry. A number of oligodeoxynucleotides of varying lengths containing a single modification, and dinucleotides containing two modifications were prepared. A variety of reactions of the 8-nitroguanine base both in nucleosides and oligodeoxynucleotides have been investigated. Studies revealed a different pattern of alkylation for the modified base when compared to results reported in the literature for the natural nucleoside. Thus demonstrating the dramatic effect that nitration has on the intrinsic reactivity of the nucleoside. In view of the susceptibility of nitro group to reaction with thiol nucleophiles, displacement of the nitro group from within nucleosides and oligodeoxynucleotides has been achieved. In particular a fluorescent nucleophile has been developed which stabilises the lesion and could enable direct detection of the levels of 8-nitroguanine in DNA. Using a variety of substrates prepared in this thesis, detection of the 8-nitroguanine base in oligodeoxynucleotides has been investigated using surface enhanced Raman spectroscopy in collaboration with Professor Steven Bell at Queen’s University, Belfast. The unique absorption profile of the 8-nitroguanine derivatives allows for signals exclusively associated with the lesion to be identified using this highly sensitive technique. The synthesis of 8-nitroguanosine triphosphate was investigated using a number of different approaches. Although the initial aim was not successful, the principles for the phosphorylation of a nucleoside have been shown. The problems encountered were attributed to the conformational constraints of the molecule