Phosphotriester, synthesis

Treatment of methoxyphosphorodichloridite with li/-1,2,4-triazole in THF at —78 °C affords methoxyphosphorobis(triazolidite) which reacts with 5 -0-dimethoxytrityl-2 -deoxynucIeosides to give (24), which in turn affords (25) and (26) on treatment with excess A -trimethylsilyl-dimethylamine or -morpholine, respectively. This preparation of deoxynucleoside phosphoramidites avoids the use of chlorodialkyl aminomethoxyphosphines, which are unstable and difficult to prepare. Little or no 3 -3 dinucleoside phosphite is formed in this case, although if the methyl group is replaced by 2-chlorophenyl, some 3 -3 -linked by-product is found. Species of type (24)—(26) are of central importance in the phosphite method of oligonucleotide phosphotriester synthesis. 

In the phosphotriester synthesis, 5 -0-MMTr-((V-acylated)-deoxyribonucleotide-3 -0-(CP phosphate) is coupled to a 3 -hydroxyl protected deoxyribonucleotide or an existing chain using mesitylenesulfonyl chloride as the coupling agent. 

Large-Scale Preparations Combined with Blockwise Phosphotriester Synthesis... 

OUgoribonucleotides modified in the 2 position (120-122) as well as ohgomers of cyclonucleoside phosphates (123,124) have been of interest for conformational studies. The first preparations of a-anomeric oligonucleotides were also done by solution phosphotriester synthesis (125), although solid-phase phosphoramidite chemistry was used for longer sequences. 

Fig, 6. Construction of a lariat triribonucleoside diphosphate by solution-phase phosphotriester synthesis (from 137a)... 

Phosphotriester synthesis needs somewhat longer condensation times, although yields have been improved to 90% and beyond. High selectivity is shown even in additions to nucleosides with unprotected hydroxyl functions (see discussion in Section 3.), but the avoidance of irreversible base modifications (20) may require additional protection. Last, but not least, there is enormous experience in the preparation of protected blocks, which may facilitate the design of routes for scale-up. 

A rigid polyamide/silica gel composite (Fig. 5) was eventually developed for continuous-flow synthesis, which produced quite good results with phosphotriester synthesis chemistry. This support produced quite good results, but it was not widely used despite its commercial availability. 

Kohli, V., Balland, A., Wintzerith, M., Sauerwald, R, Staub, A., and Lecocq, J. P. (1982) Silica gel - an improved support for the solid-phase phosphotriester synthesis of oligonucleotides Nucl. Acids Res 10,7439-7448. 

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