Phosphotriester methods

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

A phosphotriester method has been developed for rapid synthesis of oligodeoxynucleoside phosphorodithioates in solution. Couplings are performed by the chemoselective oxygen activation of protected nucleoside dithiophosphate anions (56) with 4-nitro-6-trifluoromethylbenzotriazol-l-yl-oxy-tris(pyrrolidine)-phosphonium hexafluorophosphate (PyFNOP) (57) or 6-nitrotriazol-l-yl-oxy-tris(pyrrolidine)-phosphonium hexafluorophosphate (PyNOP) (58). Under optimised conditions, coupling yields above 95% were achieved in between 10 and 20... 

While Merrifield experimented with the polystyrene cross-linked with 2% and later 1% DVB [55], Letsinger adopted the popcorn polystyrene [54] which relies on less cross-linking agent (0.05-0.2%) to achieve complete insolubility in common organic solvents. Ah of his early ohgodeoxyribonucleohde syntheses by the phos-photriester method were performed on this type of support [53, 58, 59], Other researchers have used popcorn PS as well in the syntheses by the phosphodiester method [144] and, later, more advanced phosphotriester method [145], However, with the widespread use of low cross-linked PS and the advent of polyacrylamide and especially silica gel supports (Section 19.2.3), use of popcorn polystyrene in oligonucleotide synthesis has practically ceased. 

With the advent of the phosphotriester method, gel-type PS resins were evaluated for stepwise and block syntheses of short- and medium-length oligonucle-... 

However, the best results were obtained when the first nucleoside was attached via the succinyl linker onto the ethylenediamine-treated polydimethylacrylamide resin (Figure 19.4) [178,179] or Enzacryl Gel K-2 [177]. The polydimethylacrylamide support has been utilized by Gait et al. [180] in the automated solid phase oligonucleotide synthesis by the phosphotriester method on a peptide synthesizer. 

The most common trityl anchor was monomethoxytrityl (Figure 19.5), employed in the phosphodiester method on PS-1% DVB by Melby and Strobach [146], on macroporous PS by Roster and Cramer [159] and on PTFE grafted polystyrene by Potapov d. al. [167] A dimethoxytrityl linker was used by Roster and Cramer for the phosphodiester chemistry on popcorn PS [144] and by Belagaje and Brush [155] for their original adaptation of the phosphotriester method for the synthesis in 5 -to-3 direction (Figure 19.5). Roster has also described a trityl anchor linked to a silica gel support [185]. Similar linkers have been exploited for liquid-phase oligonucleotide synthesis (Section 19.4). 

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