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

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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