Adaptation of a protocol for the automated solid-phase phosphoramidite synthesis of oligodeoxyribonucleotides for the preparation of their N-unsubstituted phosphoramidate analogues (P-NH2)

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Abstract

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.

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About the authors

E. A. Malova

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: malova.ev.an@gmail.com
Russian Federation, prosp. Acad. Lavrentyeva 8, Novosibirsk, 630090

I. A. Pyshnaya

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: pyshnaya@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentyeva 8, Novosibirsk, 630090

M. I. Meschaninova

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: pyshnaya@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentyeva 8, Novosibirsk, 630090

D. V. Pyshnyi

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

Email: pyshnaya@niboch.nsc.ru
Russian Federation, prosp. Acad. Lavrentyeva 8, Novosibirsk, 630090

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Supplementary files

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3. Fig. 1. ESI-MS analysis of compound (V) (Scheme 1) in positive ion recording mode.

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4. Fig. 2. (a) – RP-HPLC analysis of reaction mixtures of hexathymidylates (T*T5) obtained under different oxidation conditions according to the Staudinger reaction and treated with concentrated aqueous ammonia for 30 min at 25°C. Acetonitrile concentration gradient 0–30% over 15 min. Peak assignment was performed based on the combined data of mass spectrometric analysis (Fig. S4 in the supplementary materials) and comparison with the control oligonucleotide T6: peak 1 – T5, peak 2 – T6, peak 3 – T(–P(V)–NH2–)T5, peak 4 – T(–P(V)=N–Fmoc–)T5; (b) – oxidation conditions and the degree of the Staudinger reaction without taking into account the degree of destruction of the modified unit, obtained as the ratio of the sum of the integral areas of peaks 3 and 4 to the sum of the integral areas of all components of the reaction mixture (according to rHPLC data).

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5. Fig. 3. PAGE electrophoresis under denaturing conditions (8 M urea, 89 mM Tris-borate buffer, pH 8.3) of reaction mixtures of T*T5 oligonucleotides obtained according to the protocol described in Table 1, using 0.25 M FmocN3 solution and a Staudinger oxidation time of 45 min depending on the sequential postsynthetic treatment: 1, 2 – control T5 and T6, respectively; 3, 5 – ammonolysis (concentrated aqueous ammonia, 30 min), detritylation (80% acetic acid, 10 min); 4 – detritylation on CPG (3% trichloroacetic acid in CH2Cl2, 2 min), removal of the Fmoc group on CPG (2% DBU solution in acetonitrile, 5 min), ammonolysis (methanol-ammonia mixture, 2 h); 6 – ammonolysis (concentrated aqueous ammonia, 30 min), detritylation (80% acetic acid, 10 min), additional ammonolysis (concentrated aqueous ammonia, 24 h); 7 – ammonolysis (concentrated aqueous ammonia, 30 min), detritylation (80% acetic acid, 10 min), additional ammonolysis (methanol-ammonia mixture, 24 h). The assignment of the main spots was made according to ESI-MS analysis data after rHPLC extraction (Fig. S5 in the supplementary materials).

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6. Fig. 4. PAGE electrophoresis under denaturing conditions (8 M urea, 89 mM Tris-borate buffer, pH 8.3) of reaction mixtures: 1 and 2 – control oligonucleotides T5 and T6 with natural phosphodiester bonds; 3 – T2*T4, obtained by method 1; 4 – T2*T4, obtained by method 2.

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7. Fig. 5. Resistance of P-NH2 oligonucleotides to deprotection by a methanol-ammonia mixture compared to standard removal of protecting groups using concentrated aqueous ammonia, determined by rPLC data.

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8. Fig. 6. Experimental scheme for assessing the effect of the stage of additional oxidation with a mixture of I2/Py/H2O on the ratio of by-products in the synthesis of P-NH2 oligonucleotide. The effect was assessed using rHPLC data.

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9. Fig. 7. Contribution of P-NH2 modification to the thermal stability (DT) of complementary complexes of bimodified oligodeoxyribonucleotides with the AGCTACCG matrix depending on the ionic strength of the solution relative to the unmodified complex.

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10. Scheme 1. The steps of transformation of thymidine phosphite amido during oxidation of FmocN3 by the Staudinger reaction with indication of 31P-NMR characteristics obtained during the experiment. Compound (VI) was not registered during the transformations and is indicated here as the expected product with the range of chemical shifts presented in the literature [41, 47].

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11. Scheme 2. Proposed mechanism of hydrolysis of the Fmoc-containing fragment at the stage of detritylation with trichloroacetic acid during the synthesis of P-NH2 oligonucleotides.

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