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Kinetics and mechanisms of steps in anti-sense oligonucleotide synthesis

Russell, Mark A. (2007) Kinetics and mechanisms of steps in anti-sense oligonucleotide synthesis. Doctoral thesis, University of Huddersfield.

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    Abstract

    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.

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    Item Type: Thesis (Doctoral)
    Additional Information: © The Author 2007
    Uncontrolled Keywords: kinetics mechanics; anti-sense oligonucleotide synthesis
    Subjects: Q Science > Q Science (General)
    Q Science > QD Chemistry
    Schools: School of Applied Sciences
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    Depositing User: Sara Taylor
    Date Deposited: 09 May 2008 11:46
    Last Modified: 28 Jul 2010 19:23
    URI: http://eprints.hud.ac.uk/id/eprint/731

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