SPECTROSCOPIC AND ENZYMATIC CHARACTERIZATION OF 2'-5' AND 3'-5' RNA HEXAMERS AACCUU SYNTHESIZED BY PHOSPHOTRIESTER APPROACH IN SOLUTION USING 2'-TERT-BUTYLDIMETHYLSILYL PROTECTION

Comparative H-1, P-31 NMR and CD spectroscopic results and enzymatic cleavage of single stranded RNA hexamers which have identical base sequence (AACCUU) but are regioisomeric at the internucleotide phosphate linkages (2'-5', 3'-5' and their covalent hybrid) are presented. The P-31 NMR results revealed significant differences in local phosphate backbone conformation among these isomers, with 2'-5' isomer exhibiting maximum heterogeneity as compared to the 3'-5' isomer, the analogous DNA hexamer and the covalent hybrid hexamer. In contrast to this, there are no appreciable differences in the overall base-base stacking as seen in the CD spectra of 2'-5' and 3'-5' isomers. All RNA hexamers were synthesised by solution phase phosphotriester chemistry with t-butyldimethylsilyl (TBDMS) as 2'-0-protecting group. The fully protected oligorobomers were deprotected in two steps: (i) saturated anhydrous MeOH-NH3 for phosphate and amino deprotections and (ii) TBAF for removal of 2'-0-TBDMS group. Use of sat. MeOH-NH3 (instead of aq. NH3) prevents 2'-3' internucleotide phosphate migrations, chain fragmentations and 5'-terminal modifications by neighbouring group participation (NGP). The retention of isomeric integrity and absence of 5'-terminal modification in the final products was established by digestion with several nucleases.