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Title: Synthetic nucleotides and their biological applications
Author: Ademola, John I.
ISNI:       0000 0001 3398 2862
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1980
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The chemical synthesis and characterisation of the nucleotides, 2-5A and 8-azidoadenosine-5'-monophosphate was accomplished. The activity of both nucleotides was tested in appropriate biological systems. (1) Synthesis and biological properties of 2-5A. (a) The interferon messenger, pppA2'p5'A2'p5’A(2-5A) was synthesised in three stages. First, the 'core' A2'p5'A2,p5'A was chemically synthesised by Khorana's diester method. The core was then monophosphorylated first by wheat shoot phosphotransferase in the presence of ATP. This was followed by chemical phosphorylations, to yield 2-5A with a terminal triphosphate. (b) During the synthesis of 2-5A, the rates of acid hydrolysis of nucleoside and nucleotide protecting groups were studied. Substituted trityl- and isopropylidene adenosine derivatives were found to have optimum properties for use during oligonucleotide synthesis. The efficiencies of various condensing agents were investigated and it was concluded that for the phosphodiester method, the use of sulphonyl imidazoles offered the best method for linking together alcohol function of nucleotides and nucleoside phosphomonoesters. The synthetic 2-5A and related nucleotides were characterised by both chemical and enzymatic methods. In preliminary studies, 2-5A at mM concentrations was shown to inhibit protein synthesis in permeabilised MG63 cells, the replication of Semliki Forest virus in permeabilised MG63 cells, and the growth of MG63 and HFF cells. In one experiment 2-5A produced 50Z inhibition of protein synthesis, but with other preparations of permeabilised cells much higher concentrations were required. It was also observed that MG63 cells yielded high amounts of interferon following incubation with poly I.C. (2) The properties of wheat shoot phosphotransferase were investigated, following its purification by several steps including affinity chromatography on Matrex Gel Blue A. The use of the enzyme in nucleo tide synthesis was investigated. The enzyme was immobilised on various insoluble supports and these forms of the enzyme were also used for the synthesis of nucleotides. Benzoquinone- immobilised phosphotransferase attached to Sepharose gave the highest yield of nucleotides under the conditions investigated. (3) Finally, in the final chapter of the thesis, a study was made of the interaction between a photoactivated affinity labelling reagent, 8-azidoadenosine-5'-monophosphate and the enzyme staphylococcal nuclease. The kinetics of the inhibition of the enzyme by the reagent were studied and were found to be competitive with substrate treatment. Following cleavage of the enzyme into five peptides by cyanogen bromide treatment, it was observed that 80Z of the radioactive photolabel was recovered in one peptide, while a second peptide contained the remaining 20Z. This later study was undertaken as a model for the type of work that would be required to identify the biological receptors for 2-5A in the cell. However, one receptor, endonuclease, which cleaves mRNA, is already known.
Supervisor: Not available Sponsor: Association of Commonwealth Universities
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology