Published on June 18, 2002 in J. Org. Chem volume pp 5352–5358.

PubMed ID: 12126427

DOI: 10.1021/jo025826t

Abstract:

The synthesis of a protected form of the hypermodified nucleoside, N-[(9-β-d-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNALys,3, the primer for HIV-1 reverse transcriptase. Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t6A containing E. coli tRNALys, despite the presence of the bulky methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA−RNA interactions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.