AN IMPROVED SYNTHESIS OF OLIGODEOXYNUCLEOTIDE N3'-]P5' PHOSPHORAMIDATES AND THEIR CHIMERA USING HINDERED PHOSPHORAMIDITE MONOMERS AND A NOVEL HANDLE FOR REVERSE-PHASE PURIFICATION

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidat es for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonuc leotide analogs which makes use of a phosphoramidite amine-exchange re action in the key coupling step. We report herein an improved set of m onomers that utilize a more reactive, hindered phosphoramidite to prod uce optimal yields in a single coupling step followed by oxidation, th ereby eliminating the need for the previously reported couple-oxidize- couple-oxidize approach. On the 10 mu mol scale, the synthesis is perf ormed using only 3.6 equivalents (equiv,) of monomer, An improved oxid ation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide puri fication handle that is removed under non-acidic conditions, in contra st to the conventional dimethoxy-trityl group. The synthesis and purif ication of uniformly modified N3'-->P5' phosphoramidate oligodeoxynucl eotides, as well as their chimera containing phosphodiester and/or pho sphorothioate linkages at predefined positions, using these new method ologies are included herein. The results of P-31 NMR studies that led to this improved amine-exchange methodology are also described.