| Synthetic oligonucleotides have become important tools for studying molecular biology. Several new chemical methods have facilitated the techniques required to prepare DNA fragments of defined sequence. The current work describes recent modifications to the phosphite-triester scheme which permit the rapid synthesis of oligodeoxyribonucleotides directly from protected nucleoside precursors using manual and automated methods.; The basis of the method involves the use of a new group, 2,2,2-trichloro-1,1-dimethylethyl for the protection of the P-O of the internucleotide bond. The group is more stable than existing alternatives and is readily removed by a new reaction of tributylphosphine with oligodeoxyribonucleotide phosphotriesters. The use of the sterically hindered group permits the selective preparation of deoxyribonucleoside phosphoromonochloridite reagents which give high yields for the nucleotide addition step. The use of scavengers which selectively inactivate small amounts of unreacted dichloridite reagent facilitates the preparation of useful active nucleoside phosphitylating agents. In addition, the use of scavengers permits the preparation of the reagents directly on an automated machine using protected nucleosides as precursors. The synthesis and characterization of several oligodeoxyribonucleotides is described. |
