The “Speedy” Synthesis of Atom‐Specific ¹⁵N Imino/Amido‐Labeled RNA

Abstract

Although numerous reports on the synthesis of atom-specific (15)N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of (15)N(1) adenosine and (15)N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve (15)N(3) uridine and (15)N(3) cytidine amidites in order to tap full potential of (1)H/(15)N/(15)N-COSY experiments for directly monitoring individual Watson-Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…With the exception of imino signals the NMR resonance of the only four different RNA nucleotides resonate in a narrow spectral regime. The limited chemical shift dispersion has-in part-been overcome by multidimensional heteronuclear NMR in combination with uniformly labeling approaches using 13 C, 15 N and 2 H labeled nucleotides in T7 RNA polymerase mediated in vitro transcription (Alvarado et al 2014;Batey et al 1992;Dayie et al 1998;Furtig et al 2008;Nikonowicz et al 1992;Thakur and Dayie 2012) and position specific labeling using chemical synthesis (Kline and Serianni 1990;Neuner et al 2015;Quant et al 1994;Wenter et al 2006).…”

The synthesis of 15N(7)-Hoogsteen face-labeled adenosine phosphoramidite for solid-phase RNA synthesis

“…To achieve 15 N(7)-labeled adenosine amidite 12 , we conceived a strategy that employs a silyl-Hilbert-Johnson nucleosidation [9–11] and a recently introduced azido-to-acetamido purine transformation [4] as key steps. Therefore, 15 N(7)-hypoxanthine 5 was synthesized following the protocol by Jones and coworkers (Scheme 1) [12].…”

“…We have recently described our preferred synthetic routes for 15 N(1)-adenosine, 15 N(1)-guanosine, 15 N(3)-uridine, and 15 N(3)-cytidine phosphoramidites which allow base pair-specific labeling in RNA for direct monitoring of Watson–Crick base pairs by 1 H/ 15 N/ 15 N-COSY experiments [4]. The approach of individual Watson–Crick base pair labeling is particularly useful for the analysis of conformationally flexible RNAs when competing and interconverting secondary structures are encountered [5, 6].…”

Facile synthesis of a 3-deazaadenosine phosphoramidite for RNA solid-phase synthesis

“…Unfortunately, all our attempts to find appropriate conditions to reduce the 6-azido group to the corresponding amine failed. In short, these trials included i) hydrogenation under Pd/C catalysis at elevated pressure (30 psi) in ethanol or N , N -dimethylacetamide, ii) ammonium formiate, Pd/C, in methanol [28], iii) tin(II) chloride, in ethanol [29], iv) thioacetic acid, lutidine, in CH 2 Cl 2 [30], v) triphenylphosphine, in CH 2 Cl 2 , aqueous work-up, and finally vi) Mg 0 in methanol.…”