Phosphotriester method synthesis

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. 

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). 

Polynucleotide synthesis. Narang and collaborators have reported the synthesis of a pentadecanucleotide of thymidine by a modified phosphotriester method. 

While a large number of improvements in procedures for solid phase synthesis of oligonucleotides have been described, the techniques have not altered fundamentally from those described in previous reports. Solid-phase synthesis using a continuous-flow phosphotriester method on a kieselguhr-polyamide support has been described. Other solid phases used as supports for synthesis include derivatized h.p.l.c.-grade silica gel, . derivatized cross-linked poly-... 

Sproat BS, Gait MJ. Solid-phase synthesis of oligodeoxyribonucleotides by the phosphotriester method. In Gait MJ, ed. Oligonucleotide Synthesis A Practical Approach. Oxford IRL Press Ltd, 1984 83-115. 

The phosphotriester method overcomes some of the disadvantages of the phosphodiester method by blocking each intermediate phosphodiester function during the synthesis of the required sequence. This method was used to synthesize 67 different oligonucleotides of chain length 10-20 which were then spliced to gen-... 

Fig. 2. DNA synthesis. The phosphotriester method of DNA synthesis. B = protected base. For other abbreviations and formulas see Fig. 5.

Fig. 3. DNA synthesis. Phosphotriester method of DNA synthesis, using a solid phase system. The brilliant orange color 498 nm) of the DMTr cation can be used to monitor coupling efficiency between synthetic cycles. Sa = spacer arm. B, B2 = protected bases. Condensation is activated by mesitylene-2-sulfonyl-3-nitro-1,2,3-triazoie (formula shown). R is a phosphoryl protecting group (see Fig. 2).

Synthesis of 01igodeo iQrribonucleotides by the Phosphotriester Method. l-(Mesitylene-2-sulfonyl)-3-nitro-l,2,4-triazole-(MSNT) has been widely used as a condensing agent for the synthesis of oligodeoxynucleotides by the phosphotriester method. ... 

MSNT has been employed as the condensing agent in a conti-nuous-fiow, solid-phase synthesis of oligodeoxyribonucleotides using the phosphotriester method (eq 2). ... 

Phenylsulfonyl-1//-tetrazole (1) has been used as a condensing reagent in deoxyoligonucleotide solution synthesis by the phosphotriester method. It activates 5 -dimethoxytrityl (DMT)... 

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