2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Abstract

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

“…For instance, thus far, 2′-SeCH 3 or 2′-N 3 guanosine building block synthesis [26,27] relied on N 2 -acetyl-O 6 -(4-nitrophenyl)ethyl (NPE) or N 2 -dimethylform-amidine-O 6 -NPE functionalization to protect the guanine lactam moiety against electrophilic reagents. Introduction of the O 6 -NPE group was accomplished under Mitsunobu conditions.…”

“…This derivative can be integrated into a combined phosphotriester/phosphoramidite approach to synthesize 2′-azido RNA, which is a valuable tool for bioconjugation and siRNA technologies. [27] …”

Oligonucleotide Tagging for Copper-Free Click Conjugation

“…Most recently, Brown et al [25,26] further extended the latter approach by ON incorporation of aminoalkyl thymidine derivatives, followed by selective N-acylation with azide or cyclooctyne after cleavage from support. Alternative approaches for the preparation of azide-containing nucleotides-compromised by the incompatibility of azide with phosphoramidite chemistry-involve post-synthetic nucleophilic substitution [27][28][29] or selective diazotransfer reaction [30] or phosphonate-based coupling chemistry [31][32][33][34] However, a simple and general strategy for the on-support, automated synthesis of oligonucleotides with readily accessible building blocks, and suitable for introduction of any functional group (including cyclooctyne and azide), is still desirable.…”

Insight Into the Inhibition of Ribonucleotide Reductases by 2'-chloro-2'-deoxynucleotides and 2'-azido-2'-deoxynucleotides: Biomimetic Studies with Model Substrates

“…Fauster et al 105 synthesized the cytidine and guanosine 2′-azido modified analogues. Synthesis of oligonucleotides using phosphoramidite chemistry is generally not possible since azides are proven to be susceptible to Staudinger reduction in the presence of P (III).…”

“…If sulfenamide modified nucleosides will generate the aminyl radical successfully, they can have advantage over azido modified nucleosides as they should be stable under the conditions required for the standard solid phase synthesis of deoxynucleosides. 105,107,118 Thus the sulfenamides can serve as more convenient substrates for the synthesis of nucleotides and oligo nucleotides. Omeprazole, 75 is used for the treatment of gastric ulcers and shows its activity by inhibiting the gastric (H+/K+)-ATPase present in the acidic compartments of the parietal.…”