Expanding the Scope of 2′‐SCF3 Modified RNA

Lukas Jud,Markus Hartl,Klaus Bister,Christoph Kreutz,Ronald Micura,Marija Košutić,Veronika Schwarz

doi:10.1002/chem.201500415

Abstract

The 2′-trifluoromethylthio (2′-SCF3) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2′-SCF3 adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc-protecting group strategy, are reported. RNA solid-phase synthesis provided site-specifically 2′-SCF3-modified oligoribonucleotides that were investigated intensively. Their excellent behavior in 19F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule–RNA interaction. Moreover, comparably to the 2′-SCF3 pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off-target effects and their performance in silencing of the BASP1 gene was demonstrated.

Highlights

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and equip them with new fascinating properties.[1,2,3,4,5,6,7,8,9] Recently, we have reported the original synthesis of 2’-SCF3-modified RNA.[10,11] This modification has considerable potential for broad NMR spectroscopic applications in the nucleic acids field, for probing of RNA–ligand interactions and for monitoring structural rearrangements, both at the secondary and tertiary structure level.[12,13,14,15,16,17,18,19,20,21] The main reason for this alluring prospect originates from the very high sensitivity compared to the commonly used single-fluorine labeling patterns that involve mostly 5fluoro or 2’-fluoro uridines.[13]
We hypothesize that possible oxidation products of the protected RNA were reduced by this additive, and follow-up side-products that otherwise dominated during RNA deprotection at high pH values could not form any more. This observation is reminiscent of the chemical synthesis of 2’-SeCH3 RNA that we investigated several years ago;[33] for 2’-SeCH3 guanosine-modified RNA, the corresponding oxidation products were analyzed in detail by mass spectrometry, and isolated and quantitatively reduced by DTT.[26]
The syntheses of the novel 2’-SCF3 purine nucleoside phosphoramidites and the corresponding RNAs has been demonstrated in this work and significantly expands the scope of applications for this modification

Summary

Introduction

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and equip them with new fascinating properties.[1,2,3,4,5,6,7,8,9] Recently, we have reported the original synthesis of 2’-SCF3-modified RNA.[10,11] This modification has considerable potential for broad NMR spectroscopic applications in the nucleic acids field, for probing of RNA–ligand interactions and for monitoring structural rearrangements, both at the secondary and tertiary structure level.[12,13,14,15,16,17,18,19,20,21] The main reason for this alluring prospect originates from the very high sensitivity compared to the commonly used single-fluorine labeling patterns that involve mostly 5fluoro or 2’-fluoro uridines.[13]. We used the 2’-O-TOM approach for the solid-phase synthesis of RNA with site-specific 2’-SCF3 adenosine and guanosine www.chemeurj.org

Results

Conclusion