--Di- and oligonucleotide synthesis using H-phosphonate chemistry ..

Di- and Oligonucleotide Synthesis Using H-Phosphonate ..
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Di- and Oligonucleotide Synthesis using H-Phosphonate Chemistry.

DNA synthesis can be achieved by using O‐selective methods for internucleotide bond formation. This greatly simplifies the synthesis of oligodeoxyribonucleotides by eliminating the need for nucleobase protection and deprotection steps. This unit describes strategies that can be used for DNA synthesis without base protection. The discussion includes synthesis of phosphoramidite and ‐phosphonate monomers, solid‐phase assembly by the phosphoramidite and ‐phosphonate methods, and future prospects for DNA synthesis using N‐unprotected approaches.

Di- and Oligonucleotide Synthesis Using H-Phosphonate Chemistry ..
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Di and Oligonucleotide Synthesis Using H ..

Relatively high molecular weight DNA sequences have been prepared successfully by the phosphotriester approach in solution by following essentially the procedure indicated in outline in Figure 1a. However, solution-phase synthesis is relatively laborious in that chromatographic purification steps are usually necessary after each coupling step. Nevertheless, if a very large quantity of a specific sequence is required (see text below), solution-phase synthesis may very well prove to be the method of choice. If, on the other hand, relatively small (i.e., milligram to gram) quantities of material are required for biological or biophysical studies, there is little doubt that solid-phase synthesis is to be preferred. While all three of the above phosphorylation methods (Fig. 1) have been used in solid-phase synthesis, the phosphoramidite approach (9) has emerged as the method of choice. This is mainly because its use leads to high coupling efficiencies and no significant side reactions. Furthermore, most commercial automatic synthesizers have been designed specifically to accommodate phosphoramidite chemistry. The main advantages of solid-phase synthesis, particularly by the phosphoramidite approach, are: (1) that it is very rapid and a DNA sequence containing, say, 50 nucleotide residues can easily be assembled and unblocked within one day; (2) only one purification step is required at the end of a synthesis as the growing DNA sequence is attached to a solid support (such as controlled pore glass [CPG] or polystyrene), and the excesses of all reagents are washed away; (3) all chemical reactions can be made to proceed in very high yield by using large excesses of reagents; and (4) the whole process may be fully automated in a DNA synthesizer. Solid-phase DNA synthesis has been developed to such an extent that the whole process can be carried out by a competent technician with no specialist knowledge of nucleotide chemistry. Automatic synthesizers, some of which are capable of assembling several different specific DNA sequences simultaneously, are readily available, and all the necessary building blocks [particularly phosphoramidites 17] and other reagents and solvents may be purchased in containers that are designed to be attached directly to the synthesizer.

“ Di and oligonucleotide synthesis using H-phosphonate chemistry ”
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It is anticipated that success in the design of oligonucleotide drugs will stimulate further research and consequent improvements in solution-phase synthesis. Indeed, a modified phosphotriester approach involving low temperature H-phosphonate coupling (Fig. 3, step i) has recently been proposed (15).

Di- and oligonucleotide synthesis using H-phosphonate chemistry ..
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