U360302.pdf (8.53 MB)
A new route to oligonucleotides.
thesisposted on 2015-11-19, 08:48 authored by John. Weetman
The application of the Arbusov reaction to generate a nucleoside-containing phosphoryl halide for use in oligonucleotide synthesis has been investigated. In Arbusov reactions on mixed trialkyl phosphites, t-butyl halide is always lost in preference to methyl, ethyl or cyclo-pentyl halide. Exploitation of this apparent SNI nature of the second stage of the reaction was proposed. The phosphitylating agent t-butyl methyl phosphorochloridite was found not to be isolable and t-butyl methyl N, N-dimethyl phosphoramidite upon phosphitylation of a 5'-protected monomer using tetrazole as acid catalyst was prone to loss of isobutylene The use of alternative groupings, namely, 2-methoxyethyl, 1-methoxypropan 2-yl, allyl, 1-methoxy-2-methyl propan-2-yl and 1-methylcyclopentyl to exploit the SNI nature of the reaction proved unsuccessful. Dimethyl phosphorochloridite was found to phosphitylate a 5'- protected monomer efficiently. Reaction with bromine proceeded with exclusive loss of methyl bromide. SN2 attack at methyl is thought to occur preferentially since bromide ion is sterically prevented from attacking the 3'-position by the nucleobase and the bulky trityl protecting group. The resulting phosphorobromidates were found to phosphorylate the 5'-hydroxyl of a 3'-protected monomer efficiently in solution in the presence of l-methylimidazole. DMF, DMPU, pyridine and tetrazole have also been investigated as activating agents. Using o-chlorophenyl as the internucleotide protecting group this procedure was unsuccessful but this led to a series of interesting acid catalysed ligand exchange reactions on aryl diethyl phosphites containing various substituted aryl groups, those with the most electron-withdrawing substituents generally exchanging most rapidly. The route beginning from dimethyl phosphorochloridite was investigated on a CPG polymer support. A thymidine residue bearing an acid labile 5'-0-protecting group and attached through its 3'-position via a succinate linkage and a LCAA spacer to the polymer support was deprotected with trichloroacetic acid. Subsequent phosphorylation of the free 5'-hydroxyl was found to be inefficient, the reason suggested being that the longer phosphorylation reaction times required on the solid support allowed demethylation of the active phosphorylating agent by halide ion to occur.
Date of award1985-01-01
Awarding institutionUniversity of Leicester