Spectroscopic and enzymatic characterization of 2'-5' and 3'-5' RNA hexamers AACCUU synthesised by phosphotriester approach in solution using 2'-t-butyldimethylsilyl protection

Abstract

Comparative ¹H, ³¹P 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'-t', 3'-5' and their covalent hybrid) are presented. The ³¹P 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'-O-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'-O-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. ¹H, ³¹P NMR and CD spectroscopic studios of titled RNA compounds and an isomeric covalent hybrid indicates conformational differences among these, arising due to different nature of phosphodiester linkages. The compounds, synthesised by solution phase phosphotriester chemistry and 2'-TBDMS protecting group, were shown to be isomerically pure by their specificities towards digestion with the enzyme pancreatic ribonuclease.