Adaptation of a protocol for the automated solid-phase phosphoramidite synthesis of oligodeoxyribonucleotides for the preparation of their <i>N</i>-unsubstituted phosphoramidate analogues (P-NH<sub>2</sub>)

A new approach to the automated synthesis of N-unsubstituted phosphoramidate oligodeoxyribonucleotides (P-NH2) based on an optimized solid-phase phosphoramidite protocol using the Staudinger reaction has been proposed. The rapid and efficient oxidation of model P(III)-containing phosphite triethers by the organic azide (9H-fluoren-9-yl)methylcarbonylazide (FmocN3) to the corresponding phosphamides –(OPO(OR)(NFmoc))–, where R is a residue of nucleoside or alkyl nature, has been demonstrated. Removal of the alkaline-labile fluorenyl group from the modified internucleoside linkage allows the production of electroneutral, under physiological conditions of pH ~7, N-unsubstituted phosphoramidate (–(OPO(O)(NH2))– or (P-NH2)) residues in the oligonucleotide chain instead of the classical negatively charged phosphodiester (–(OPO(O)(O)(O¯))–) or (P-O)) residues. In optimizing the synthetic protocol, it has been demonstrated that to improve the efficiency of P-NH2-oligonucleotide synthesis, it is necessary to include an additional Fmoc-group cleavage step in the automatic synthesis protocol after each oxidation step of the growing oligomer chain via the Staudinger reaction. An almost complete absence of dependence of the P-NH2-oligonucleotide yield on both the localization of the P-NH2-strand in the chain and the type of dinucleotide fragment being modified was shown. A set of mono- and bis-modified octadeoxyribonucleotides was obtained, and a detailed study of the thermal stability of complementary DNA/DNA complexes under different buffer conditions was performed. It was shown that under high ionic strength conditions (1 M NaCl, pH 7.2), the introduction of a single P-NH2 strand reduced the thermostability of the DNA complex by an average of 1.3°C. When the ionic strength of the solution decreases, the destabilizing effect of the P-NH2-modification decreases significantly, which further confirms the electroneutral status of the introduced phosphoramidate linkage. Thus, we have developed a protocol for the preparation of partially modified oligonucleotide derivatives bearing uncharged but isostructured to native P-O-strands – phosphoramidate residues P-NH2.