Mechanistic studies are reported for the detritylation, coupling and sulphurisation reactions involved in oligonucleotide synthesis by the phosphoramidite method. Detritylation is the acid catalysed removal of a 4,4-dimethoxytrityl protecting group from the 5' protected nucleotide to give the 5' deprotected nucleotide and the 4,4- dimethoxytrityl carbocation. In the absence of water and at high acid concentrations the equilibrium favours carbocation formation. Equilibrium profiles show a sigmoidal shape rather than the expected hyperbolic curves and it is likely that residual water in the system reacts with the carbocation to form the 4,4-dimethoxytrityl alcohol. Results of kinetic studies of the detritylation reaction show that the detritylation reaction proceeds by a concerted general acid catalysis mechanism. The coupling step is the reaction between an alcohol and a phosphoramidite in the presence of an acidic activator, in this research the salt of saccharin and Nmethylimidazole. 31P NMR studies have shown that initial activation of the phosphoramidite forms a reactive saccharin adduct bonded through its carbonyl oxygen to phosphorus. Reaction of the alcohol and phosphoramidite in the presence of saccharin/N-methylimidazole salt shows second order kinetics. However, at high alcohol concentrations the reaction becomes independent of alcohol. This indicates a change in rate limiting step from the final alcoholysis step to the activation step. Phosphite sulphurisation was performed with the sulphur transfer reagent 3-amino- 1,2,4-dithiazole-5-thione (xanthane hydride). Contrary to the previously reported mechanism of sulphurisation, nucleophilic attack by the phosphorus upon xanthane hydride occurs on the sulphur adjacent to the thiocarbonyl group and not on the sulphur adjacent to the amino group. Kinetic measurements of the sulphurisation reaction show second order kinetics. Reaction constants determined from Hammett and Taft constants for triaryl and trialkylphosphites are -1.09 and -1.20 respectively. These reaction constants indicate partial formation of a positive charge in the transition state. The effect of solvent polarity on the rate of sulphurisation has shown a decrease in rate on increasing the polarity of the solvent. This has been attributed to an increase in the xanthane hydride stability in more polar solvents.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:445064 |
| Date | January 2007 |
| Creators | Russell, Mark A. |
| Publisher | University of Huddersfield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.hud.ac.uk/id/eprint/731/ |
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