Synthesis and conformational analysis of phosphate-methylated RNA dinucleotides

Abstract

Synthesis of RNA dimers having a methyl phosphotriester group as the internucleoside linkage is reported; six pairs of diastereoisomerically pure system were prepared, Le., r(CpU) (151, r(ApU) (16), r(CpC) (17), r(ApC) (18), r(CpG) (19), and r(ApG) (20). Compounds 15-20 are stabilized by a 2’-0-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 NH, 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 residues, methanolic K2C03 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 Sp and Rp diastereoisomers. The availability of the six dimers in diastereoisomerically pure form enabled us to examine the molecular conformations using high-field NMR and circular dichroism (CD) spectroscopy. These studies led to the following conclusions: (i) NMR J-coupling analysis: the central C I A 5 $ (y) and C d 5 , (j3) bonds in 15-20 show lesa preference for the y+ and @ rotamers, in comparison with their natural analogues, Le., base stacking is diminished upon introduction of the two methyl groups on Or 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 ‘H NMR measurements: (Sp)and (Rp)-19 show self-association, via the formation of a right-handed miniduplex with two C-G base pairs ((Sp)-l9, T, = 9.3 O C , concn = 36.6 pM; (Rp)-19, T, = 8.7 O C , concn = 48.1 pM). The present conformatipnal data on (Rp)and (Sp)-15-20 are in agreement with literature data on other phosphate-triesterified oligonucleotides, e.g., the trimer d(TwmGpomG) and the tetramer d(TpoaTwmCp0mA). While the latter systems also showed little base-base stacking, it was established 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 svstems described in the Dresent work, will also hybridize easily with complementary natural RNA.