Synthesis of Amine- and Thiol-Modified Nucleoside Phosphoramidites for Site-Specific Introduction of Biophysical Probes into RNA

Abstract

For studies of RNA structure, folding, and catalysis, site-specific modifications are typically introduced by solid-phase synthesis of RNA oligonucleotides using nucleoside phosphoramidites. Here, we report the preparation of two complete series of RNA nucleoside phosphoramidites; each has an appropriately protected amine or thiol functional group. The first series includes each of the four common RNA nucleotides, U, C, A, and G, with a 2'-(2-aminoethoxy)-2'-deoxy substitution (i.e., a primary amino group tethered to the 2'-oxygen by a two-carbon linker). The second series encompasses the four common RNA nucleotides, each with the analogous 2'-(2-mercaptoethoxy)-2'-deoxy substitution (i.e., a tethered 2'-thiol). The amines are useful for acylation and reductive amination reactions, and the thiols participate in displacement and oxidative cross-linking reactions, among other likely applications. The new phosphoramidites will be particularly valuable for enabling site-specific introduction of biophysical probes and constraints into RNA.

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

“…Following the isolation of 1, a second Mitsunobu reaction was performed with triphenylmethanethiol to obtain the orthogonally protected heterobifunctional linker 2 in 78 % yield. The Mitsunobu reaction with triphenylmethanethiol, reported initially by Jin et al [44] in 2005, is a highly convenient method for the "thiolation" of OEGs that obviates the use of volatile sulfur compounds typically used in alkylation protocols. Purification of 1 and 2 was simplified by the use of polymer-supported triphenylphosphine that could be removed by simple filtration.…”

Effective isomerization of 3', 5'-O-(tetraisopropyldisiloxane-1,3-diyl)nucleosides in the presence of trimethylsilyl trifluoromethanesulfonate

“…Tetraisopropyldisiloxane-1,3-diyl (TIPDS) group developed by Markiewicz 1,2 is widely used for simultaneous protection of 3′,5′-hydroxyl groups in ribonucleosides and subsequent manipulation with 2'-OH group of ribonucleosides: deoxygenation, 3,4 oxidation, 5 alkylation, [6][7][8][9][10] glycosylation, [11][12][13][14][15][16] protection, 17-21 preparation of 2'-amino-2'-deoxynuclesides, 22,23 and so on. This group may be considered as one of the most popular and useful protecting group in the field of nucleoside chemistry.…”

Chemical RNA Modifications for Studies of RNA Structure and Dynamics

“…[51] For example, the fluorescent reporter group pyrene was attached via flexible tethers to 2'-amino and 2'-aminoethyl-modified RNA, and was successfully used for the investigation of Mg 2 + -induced folding of the P4-P6 domain of the group I intron ribozyme. [52] Important applications of linker-attached donor (e.g., Cy3) and acceptor (e.g., Cy5) fluorophores at the termini or internal positions involve single-molecule fluorescence resonance energy transfer (smFRET) experiments.…”