Adaptation of the Protocol of the Automated Solid-Phase Phosphoramidite Synthesis of Oligodeoxyribonucleotides for Preparing Their N-Unsubstituted Phosphoramidate Analogs (P–NH2)

Abstract

Objective: The systematic use of P–NH₂ analogs of nucleic acids as objects and/or tools in molecular biology and biomedicine is limited by the complexity of their synthesis. For almost 40 years, researchers have been looking for effective synthetic approaches to P–NH₂ oligonucleotides. These analogs, which are isostructurally identical to natural oligonucleotides, have not been further developed even though a lot of publications on their synthesis and characteristics. To develop a more straightforward and cost-effective method than those being practiced today, our group set out to modify the phosphoramidite protocol of oligonucleotide (ON) synthesis for preparing the P–NH₂ analogs of oligodeoxyribonucleotides. Methods: For the synthesis of the P–NH₂ analogs, a standard, presently widely used protocol of the phosphoramidite synthesis of oligonucleotides was taken as a basis. The P–NH₂ modification was introduced at the oxidation step via the Staudinger reaction, using (9-fluorenyl)methoxycarbonyl azide (FmocN₃). The subsequent formation of an N-unsubstituted phosphoramidate moiety in the oligonucleotide was accomplished by the removal of the Fmoc group by treatment with a strong base. The thermodynamic properties of the P–NH₂ analogs as part of complementary nucleic acid complexes formed in low-ionic-strength solutions were studied by thermal denaturation analysis with optical signal registration. Results and Discussion: It was found that to increase the efficiency of synthesis of electroneutral P–NH₂ oligonucleotides additional Fmoc cleavage step should be introduced to the protocol of automated synthesis. This step should be added after each step of oxidation of the growing oligomer chain via the Staudinger reaction. It was shown that the yield of the P–NH₂ oligonucleotide was almost entirely independent of the type of the dinucleotide fragment being modified, as well as of the localization of the P–NH₂ linkage in the chain. The attenuation of the destabilizing effect of the introduction of a single P–NH₂ linkage with decreasing ionic strength of the solution provided additional evidence for the electroneutral state of the inserted phosphoramidate linkage. Conclusions: A new approach to the automated synthesis of partially modified oligonucleotide derivatives bearing uncharged N-unsubstituted phosphoramidate linkages isostructural to native P–O linkages by an optimized solid-phase phosphoramidite protocol using the Staudinger reaction has been proposed.