Phosphite and Solid-Phase Methods

The chemical basis for the phosphite triester approach is the observation, that dialkyl phos-phorochloridites such as (CjHjOJjPCI react very rapidly at the 3 -OH of nucleosides in pyridine even at low temperatures. In contrast, the reactions of analogous chloridates, e.g. (C2HjO)2POCIi require several hours at room temperature. It was later found that phosphite esters can be oxidized quantitatively to the phosphates by using iodine in water and that clean condensation of phosphorochloridites with nucleosides can be achieved in THF at -78 °C. To develop this chemistry into a useful synthetic procedure it was necessary to establish which 

In step 1 of each oligonucleotide-synthesis cycle the S -terminal 4,4 -dimethoxytrityl protecting group is removed with trichloroacetic acid, and the support is washed with acetonitrile to prevent detritylation of the next incoming phosphoramidite. The 4,4 -dimethoxy- 

The acid treatment in each detritylation step may remove purines from deoxyriboses. Purine residues near the 3 -end will suffer the longest cumulative times of exposure to acid and therefore have the greatest chance for depurination . Thus each acid treatment should be as brief as possible. 

Because coupling is not always quantitative, the non-reacted terminal deoxynucleoside must be excluded from the following synthesis cycles. Otherwise deletion sequences will render the isolation of the pure final product difficult. Therefore a capping step (step 3) follows, e.g., acetylation with acetic anhydride and N,N-dimethyl-4-pyridinamine (DMAP) in dioxane. Capping times should be as short as possible, especially with 2-cyanoethyl phosphite tri-esters, which are sensitive to bases such as DMAP. 

The newly formed phosphite triester linkage is unstable to acids and bases and is immediately oxidized to a stable phosphate triester (step 4). A solution of iodine, water, 2,6-dimethylpyridine, and tetrahydrofuran is commonly used. The oxidation is usually complete within 30 seconds. 

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