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We first prepared a cyanoethyl-protected dodecathymidine (T12*) on solid support using well established phosphoramidite chemistry (Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-1862). Then three H-phosphonate diester linkages were introduced using H-phosphonate monoester 1 to yield the modified supported oligonucleotide 2 (FIG. 8). An amidative oxidation with CCl4 in the presence of propargylamine afforded the alkyne-functionalized oligonucleotide 3 with three propargyl phosphoramidate linkages. An aliquot was treated with aqueous ammonia and analyzed by HPLC/MS to determine the efficiency of these synthetic steps (i.e. three H-phosphonate couplings and amidative oxidation). The first H-phosphonate coupling was not complete (86-94%) while the two subsequent couplings were higher yielding. The resulting mixture was composed of 3 (˜75%), unreacted T12 (˜14%) and intermediates with one (˜3%) and two propargyl groups (˜4%) as determined by MALDI-TOF MS analyses.

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