Synthesis and Conformational Analysis of Phosphate-Methylated RNA Dinucleotides

Article abstract of DOI:10.1021/jo00020a028

Synthesis of RNA dimers having a methyl phosphotriester group as the internucleoside linkage is reported; six pairs of diastereoisomerically pure systems were prepared, i.e., r(CpU) (15), r(ApU) (16), r(CpC) (17), r(ApC) (18), r(CpG) (19), and r(ApG) (20).Compounds 15-20 are stabilized by a 2'-O-methyl group in the 5'-terminal residue.The present systems represent the third class of backbone-modified RNA oligomers, following the 2'-O-methylribonucleotide phosphorothioates and the 2'-O-methylribonucleotide methyl phosphonates.Our synthetic approach comprises the use of 9-fluorenylmethoxycarbonyl (Fmoc) groups for transient protection of the exocyclic NH2 groups of the bases A, C, and G, levulinoyl (Lev) groups for the transient protection of the 2'- and 3'-OH groups of the 3'-terminal residue, methanolic K2CO3 for the simultaneous removal of Fmoc and Lev groups with full preservation of the methyl phosphotriester function, and finally reversed-phase HPLC separation of the S_P and R_P diastereoisomers.The availability of the six dimers in diastereoisomerically pure form enabled us to examine the molecular conformation using high-field NMR and circular dichroism (CD) spectroscopy.These studies led to the following conclusions: (i) NMR J-coupling analysis: the central C_4'-C_5' (γ) and C_5'-O_5' (β) bonds in 15-20 show less preference for the γ⁺ and β^t rotamers, in comparison with their natural analogues, i.e., base stacking is diminished upon introduction of the two methyl groups on O_2' and on the phosphate group; (ii) CD analysis: 15-20 show substantially reduced molecular ellipticities when compared to the natural counterparts, which also reveals that base stacking is reduced; (iii) UV and variable-temperature 1H NMR measurements: (S_P)- and (R_P)-19 show self-association, via the formation of a right-handed miniduplex with two C-G base pairs ((S_P)-19, T_m = 9.3 deg C, concn = 36.6 μM; (R_P)-19, T_m = 8.7 deg C, concn = 48.1 μM).The present conformational data on (R_P)- and (S_P)-15-20 are in agreement with literature data on other phosphate-triesterified oligonucleotides, e.g., the trimer d(T_POEtG_POEtG) and the tetramer d(T_POEtT_POEtC_POEtA).While the latter systems also showed little base-base stacking, it was estabished that they readily form a local duplex with a complementary natural RNA sequence.Hence we anticipate that phosphate-methylated 2'-O-methyl RNA oligomers, longer than the dimer systems described in the present work, will also hybridize easily with complementary natural RNA.