Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex

The isomerization of phosphodiester functionality of nucleic acids from 3′,5′- to a less common 2′,5′-linkage influences the complex interplay of stereoelectronic effects that drive pseudorotational equilibrium of sugar rings and thus affect the conformational propensities for compact or more extended structures. The present study highlights the subtle balance of non-covalent forces at play in structural equilibrium of 2′,5′-linked RNA analogue, 3′-O-(2-methoxyethyl) substituted dodecamer *CG*CGAA*U*U*CG*CG, 3′-MOE-2′,5′-RNA, where all cytosines and uracils are methylated at C5. The NMR and UV spectroscopic studies have shown that 3′-MOE-2′,5′-RNA adopts both hairpin and duplex secondary structures, which are involved in a dynamic exchange that is slow on the NMR timescale and exhibits strand and salt concentration as well as pH dependence. Unusual effect of pH over a narrow physiological range is observed for imino proton resonances with exchange broadening observed at lower pH and relatively sharp lines observed at higher pH. The solution structure of 3′-MOE-2′,5′-RNA hairpin displays a unique and well-defined loop, which is stabilized by Watson–Crick A5·*U8 base pair and by n → π* stacking interactions of O4′ lone-pair electrons of A6 and *U8 with aromatic rings of A5 and *U7, respectively. In contrast, the stem region of 3′-MOE-2′,5′-RNA hairpin is more flexible. Our data highlight the important feature of backbone modifications that can have pronounced effects on interstrand association of nucleic acids

Identification of new process-related impurity in the key intermediate in the synthesis of TCV-116

Development of safe and effective drugs requires complete impurity evaluation and, therefore, knowledge about the formation and elimination of impurities is necessary. During impurity profiling of a key intermediate during synthesis of candesartan cilexetil (1-(((cyclohexyloxy)carbonyl)oxy)ethyl 1-((2\u27-(2H-tetrazol-5-yl)-[1,1\u27-biphenyl]-4-yl)methyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxylate, TCV-116), a novel compound, which had not been reported previously, was observed. Structural elucidation of impurity was achieved by liquid chromatography hyphenated to different high resolution mass analyzers. Based on exact mass measurements and fragmentation pattern, a chloroalkyl carbonate ester analogue of the intermediate was identified. Structure of the impurity was confirmed by mass spectrometric and NMR analyses of the target substance. Identified impurity could represent a hazard if it is transferred to the final API stage and its presence should be kept below allowed limits. Further investigation could reveal whether bis(1-chloroethyl) carbonate is a precursor to impurity formation. Therefore, synthesis should be regulated so as to minimize impurity production. Analysis of the final product indicated that the amount of impurity did not exceed 50 mg L-1, which represents the detection limit, determined according to the signal/noise ratio

UV melting profiles of 3′-MOE-2′,5′-RNA at various oligomer and salt concentrations and pH

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The calculated fits of the first derivatives of a melting curves are presented as a function of temperature at the following solution conditions: () 1 M NaCl and pH of 6.0 over a concentration range of 8 μM (magenta), 17 μM (cyan) and 123 μM (green). The of high temperature transition is 48°C and low temperature transition is melted in the range of 15–23°C. () The 1 M NaCl and pH 7.5 over a concentration range of 9 μM (magenta), 39 μM (blue), 101 μM (green) and 266 μM (orange). The of high temperature transition is 45°C, whereas the of low temperature transition is in the range of 15–26°C. () The 50 mM NaCl and pH of 6.0. Concentrations are 9 μM (magenta), 23 μM (cyan), 39 μM (blue) and 169 μM (green). The of high temperature transition form is 45°C whereas the of low temperature transition appears under the broad transition profile of high temperature form. () The 50 mM NaCl and pH of 7.4. Concentrations are 17 μM (cyan) and 270 μM (orange). The of high temperature transition is near 42°C and low temperature transition melts in 16–20°C range

() Imino proton spectra of 3′-MOE-2′,5-RNA at 5°C as a function of pH

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The sample was 9 mM strand concentration, 50 mM NaCl in 90% HO/10% HO. Imino protons involved in Watson–Crick base pairs of duplex (D) are shown. () Schematic representation of the duplex–hairpin equilibrium of 3′-MOE-2′,5-RNA. Closed lines represent Watson–Crick base pairs

Non-terminal building blocks of 2′,5′- and 3′,5′-linked RNAs and imino proton spectra of 3′-MOE-2′,5′ RNA

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Shematic representation of 3′-MOE-2′,5′-RNA monomer (left) and 2′-MOE-3′,5′-RNA monomer (right). () Imino proton spectrum recorded in 90% HO/10% HO at 9.0 mM strand concentration, 50 mM NaCl, pH 6.9 and 20°C. Duplex (D) and hairpin (H) resonances are denoted

H NMR spectra of () imino and aromatic proton region and () methyl proton region of 3′-MOE-2′,5′-RNA as a function of pH and temperature

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The vertical scale of imino and aromatic region is doubled. The sample was 0.9 mM strand concentration, 50 mM NaCl in 90% HO/10% HO. Stars indicate C7(H7) resonances of duplex form whose intensities increase upon lowering pH and temperature

() Methyl–methyl and () aromatic–aromatic region of ROESY spectrum (100 ms mixing time) recorded at 25°C in 100% HO

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The assignments of the resolved exchange signals for hairpin (H) and duplex (D) forms are indicated. Sample conditions were 3.0 mM strand concentration, 350 mM NaCl and pH* 7.4

Comparison of () aromatic and () methyl region of H NMR spectra of 3′-MOE-2′,5′-RNA at various strand concentrations (0

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>2, 0.4, 2.6 and 3.0 mM from bottom to top). Arrows indicate signals of the duplex that are separated from signals of the dominant hairpin form. Spectra were recorded in 100% HO at 30°C, 50 mM NaCl and pH* 7.4

H NMR signal intensity ln(′) as a function of gradient field strength (g) at 25°C

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of a modified 2′,5′-linked RNA hairpin involved in an equilibrium with duplex"</p><p>Nucleic Acids Research 2005;33(6):1749-1759.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069515.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The filled squares represent A6 H2 signal intensity of 3′-MOE-2′,5′-RNA hairpin, while the open triangles represent A6 H2 of the duplex form. The open circles represent the average signal intensity of aromatic resonances of 2′-MOE-3′,5′ RNA duplex. The sample conditions were 3.0 mM strand concentration, 350 mM NaCl and pH* 7.4 in 100% HO