Phosphite triester method

A common method for DNA synthesis is the phosphite triester method (Fig. 7-6). A single-stranded oligonucleotide is formed by the sequential creation of diester bonds between the 5 -hydroxyl of one residue and the 3 -phosphate of the next. The 3 -phosphate is activated by substitution by dialkyl phosphoamidite (DPA) and reacts readily with the free 5 -OH of the first nucleotide. To prevent the formation of unwanted linkages the first nucleotide is linked, by the 3 -OH, to a solid support (often silica gel) in a column or funnel the 5 -OH of the second nucleotide is prevented from reacting by a dimethoxytrityl group (DMT). 

Figure 6.7. Solid-Phase Synthesis of a DNA Chain by the Phosphite Triester Method. The activated monomer added to the growing chain is a deoxyribonucleoside 3 -phosphoramidite containing a DMT protecting group on its 5 oxygen atom, a p -cyanoethyl ( p CE) protecting group on its 3 phosphoryl oxygen, and a protecting group on the base.

Figure 5.6 Solid-phase synthesis of a DNA chain, by the phosphite triester method.

The phosphotriester approach solves some of the problems of the phosphodi-ester method. More recently, the phosphite-triester method has been introduced this makes use of the extreme reactivity of the phosphite reagent, i.e., the two building blocks are joined in a few minutes compared with the hours required by the phosphodiester approach. 

Atkinson A, Smith M. Solid-phase synthesis of oligodeoxyribonucleotides by the phosphite-triester method. In Gait MJ, ed. Oligonucleotide Synthesis A Practical Approach. Oxford IRL Press Ltd, 1984 35-82. 

The most important strategy for the linear synthesis of oligonucleotides has been developed by M. Caruthers. Nowadays, the phosphite triester method is widely used in the automated synthesis of oligonucleotides on silica. The principle is shown in Scheme 25.1. 

The phosphite triester method represents a further improvement and allows successive condensations to be carried out at greater speed than with other methods. The 5 OH protecting groups can be the same as in the phosphotriester method, but the reactive 3 component is a dialkylphosphoa-midite which is activated by a suitable weak acid such as tetrazole. The product is a phosphite triester which must be oxidised to a stable phosphate triester before further condensations can be carried out (10.85). Various modifications of this method have now been developed [1,35,38]. 

Finnan, J. L., Varshney, A., and Letsinger, R. L, (1980) Developments in the phosphite-triester method of synthesis of oligonucleotides Nucl Acids Res, Symp. No. 7, 133-145 and references therein cited. 

Atkinson, T. and Smith, T (1984) Solid-phase synthesis of oligodeoxy-ribonucleotides by the phosphite-triester method, in Oligonucleotide Synthesis- A Practical Approach (Gait, M. J., ed ). IRL Press, Oxford, pp. 35-81... 

---