Synthesis and bio-pharmacological activity of antisense phosphatidyl-oligonucleotides

Lipid conjugated oligonucleotides are of great interest in the field of antisense oligonucleotides used for functional genomics, gene target validation and therapeutic pourpose. Although various lipid conjugates of oligonucleotides have already been prepared, it was not possible until now to prepare phosphatidyl conjugates owing to an actual difficulty encountered in a direct attachment of the phosphatidyl group to oligonucleotides elongated on the solid phase by standard phosphoramidite chemistry procedures. Now, appropriately exploiting some synthetic opportunities, available in the recent literature for the preparation of oligonucleotides bearing residual base-labile side groups, we designed a synthetic path to obtain 5 -O-phosphatidyloligonucleotides. By applying this synthetic route we prepared some phosphatidyltetradecanucleotides all having the antisense sequence against the Vascular Endothelial Growth Factor (VEGF) gene, but differing from each other in their phosphatidyl moiety. This consisting of different fatty acyl residues, such as myristoyl, palmitoyl and stearoyl as well. The newly synthesized phosphatidyloligonucleotides have been analyzed for their spectroscopic (NMR, MS) and chromatographic (HPLC) properties which confirmed the expected structure. The annealing features of these compounds have been investigated by Differential Scanning Calorimetry analysis . As a preliminary experiment, the antisense effectiveness of 1,2-O-dimyristoyl- and 1,2-O-dipalmitoyl-sn-glycero-3-O-phosphoryl-tetradeca-mers has been assessed by observing their effect on the expression of VEGF at mRNA level in human Neuroblastoma cells. Both the phosphatidyl-tetradecamers were able to inhibit the expression of VEGF mRNA with an effective concentration significantly lower than was found, in parallel experiments, for the corresponding unmodified antisense oligonucleotide.