Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational features of 2′-O-methyl-adenosylyl-adenosine

In order to obtain information about the conformational features of a 2′-O-methylated polyribonucleotide at the nearest neighbor level, a detailed nuclear magnetic resonance study of AmpA was undertaken. AmpA was isolated from alkali hydrolysates of yeast RNA, and proton spectra were recorded at 100 MHz in the Fourier transform mode in D₂O solutions, 0.01 M, pH 5.4 and 1.5 at 25°C. ³¹P spectra were recorded at 40.48 MHz. Complete, accurate sets of nmr parameters derived for each nucleotidyl unit by simulation iteration methods. The nmr data were translated into conformational parameters for all the bonds using procedures developed in earlier studies from these laboratories. It is shown that AmpA exists in aqueous solution with a flexible molecular framework, which shows preferences for certain orientations. The ribose rings exist as a ²E ? ³E equilibrium with the —pA ribose showing a bias for the ³E pucker. The C(4′)—C(5′) bonds of both nucleotidyl units show significant preference (75–80%) to exist in gg conformation. The dominant conformer (80%) about C(5′)—O(5′) of the 5′-nucleotidyl unit is g′g′. Even though an unambiguous determination of the orientation of the 3′-phosphate group cannot be made, tentative evidence shows that it preferentially occupies g⁺ domains [O(3′)—P trans to C(3′)—C(2′)] in which the H(3′) —C(3′)—O(3′)—P(3′) dihedral angle is about 31°. There is reasonable evidence that the 2′-O-methyl preferentially occupies the domain in which the O(2′)—CH₃ bond is trans to C(2′)—C(1′). Lowering of pH to 1.5, which results in protonation of both the adenine moieties, causes destacking of AmpA. Such destacking is accompanied by small, but real, perturbations in the conformations about most of the bonds in the backbone. A detailed comparison of the solution conformations of ApA and AmpA clearly shows that 2′-O-methylation strongly influences the conformational preference about the C(3′)—O(3′) bond of the 3′-nucleotidyl unit, in addition to inducing small changes in the overall ribophosphate backbone conformational equilibria. The effect of 2′-O-methylation is such that the C(3′)—O(3′) is forced to occupy preferentially the g⁺ domain rather than the normally preferred g^? domain [O(3′)—P trans to C(3′)—C(4′)] in ApA. The data on ApA and AmpA further reveal that the extent of stacking interaction is less in AmpA compared to ApA. It is suggested that stacked species of AmpA exist as right-handed stacks where the magnitude of ω and ω′ about O(5′)—P and P—O(3′) is about 290°. The reason for the lesser degree of stacking in AmpA compared to ApA is intramolecular interaction between 2′-O-methyl and the flexible O(3′)—P—O(5′) bridge, the interaction causing some perturbation in the magnitudes of ω/ω′, causing destacking. The destacking will lead to an increase in _χCN by a few degrees, causing an increase in ²E populations; the latter in turn will shift the 3′ phosphate group from g^? to g⁺ domains. In short, a coupled series of conformational events is envisioned at the onset of destacking, made feasible by the interaction between the 2′-O-methyl group and the swivel O(3′)—P—O(5′) bridge.     

Substrate specificity of adenosine deaminase—Function of the 5′-hydroxyl group of adenosine

Nucleosides in which the adenine ring has been moved from the 1′ position to the 5′ position are resistant to degradation by the enzyme, adenosine deaminase. This study provides further evidence for the importance of the 5′-hydroxyl group as a structural requirement for significant substrate activity.     

Conformational and stacking properties of 3′–5′ and 2′–5′ linked oligoribonucleotides studied by CD

Comparative CD studies have been carried out to characterize the properties of 2′–5′ and 3′–5′ oligoriboadenylates and oligoribouridylates from dimer to decamer. The CD band of the 3′–5′ oligoribonucleotides was larger than that of the 2′–5′ oligoribonucleotides and increased with the increase in chain length, while the CD band of the 2′–5′ oligoribonucleotides increased little beyond the dimer level. The CD analysis of the chain length dependency revealed that the 3′–5′ oligoribonucleotides adopt mainly the base-base stacking interaction, while the base-sugar interaction is predominant in the 2′–5′ oligoribonucleotides. The CD intensity of 3′–5′ oligoribonucleotides decreased to a larger extent at elevated temperatures or in the presence of ethanol compared to that of the 2′–5′ counterparts. Mg²⁺ or Mn²⁺ ion enhanced the magnitude of the CD of 3′–5′ octariboadenylate, while a small decrease in the CD was observed by the presence of Mg²⁺ or Mn²⁺ ion to the 2′–5′ octariboadenylate. The 3′–5′ oligoribonucleotide is likely conformationally flexible and can form helical ordered structure with strong base-base stacking depending on changes in the environment such as temperature, the presence of Mg²⁺ ion, or hydrophobicity of the solution. © 1996 John Wiley & Sons, Inc.     

Preferred phosphodiester conformations in nucleic acids. A virtual bond torsion potential to estimate lone-pair interactions in a phosphodiester

The lone-pair orbital interactions arising in a phosphodiester are incorporated into semiempirical conformational energy calculations using a unifold “torsional potential” around the virtual bond linking the ester oxygen atoms. The results explain the observed experimental data better than other methods.     

Conformational studies on polynucleotide chains. IV. Backbone structure of ribonucleic acids.

In preceding papers the energies associated with the internal rotations in the sugar–phosphate–sugar complex were described with an analytical potential consisting of a Lennard-Jones 6–12 term and an intrinsic torsional term and representing the best fit to a large number of energies computed with a quantum mechanical ab initio technique. The complex considered there (of 37 atoms and with the chemical formula C₁₀H₁₈O₈P) is repesentative of deoxyribonucleic acids. In this paper we apply our potential to evaluating the intramolecular energies of the 39-atom complex C₁₀H₁₈O₁₀P, representative of the ribonucleic acids. The potential energies for the internal rotations (considered independent from one another) and the energy maps for rotations about consecutive bonds of the backbone chain are critically compared, both with those obtained for the deoxy system and with those obtained from different theoretical approaches as available from literature. It is shown that, at least for certain combinations of the internal rotation angles, the choice of the starting geometry for the sugarphosphate–sugar molecule (bond lengths and valence angles) strongly affects the value of the computed energy. If a proper geometry is used, very low energies are predicted by our potential in correspondence of the sets of torsional angles found in various RNAs by x-ray crystallography.     

Conformational studies of linear β-D-1,4′-mannan and galactan

The nonbonded interaction energy of disaccharides, mannobiose and galactobiose and polysaccharides mannan and galactan have been computed as a function of dihedral angles (?,ψ). The conformation (40°, ?20°) has been preferred for the mannan chain from nonbonded interaction energy considerations. The O₅…O₃′ type of intramolecular hydrogen bond has been found to be possible in the above conformation. Comparison of the allowed region of mannan with those of cellulose and xylan indicates that the monomer unit, in mannan chain has slightly higher freedom of rotation than that of cellulose and less than that of xylan. As in cellulose and mannan, the freedom of rotation of the monomer units in β-1,4′ galactan is highly restricted. Unlike mannan (which prefers an extended conformation) the β-1,4′ galactan prefers a helical conformation similar to amylose. Just as in amylose the O₂…O₃′ type hydrogen bond between contiguous residues is also possible in β-1,4′ galactan.     

The synthesis of the internucleotide (phosphodiester) bond by a base-catalysed reaction.

Potassium tert-butoxide in hexamethyl phosphoramide and dimethyl formamide provides an excellent catalyst for the reaction of a 3'-hydroxyl or a 5'-hydroxyl group of a nucleoside with an appropriate nucleoside phosphorofluoridate to yield the dinucleoside phosphate. This paper describes the experiments leading to the development of this reaction together with the synthesis of thymidylyl-(5' leads to 3')-thymidine (dT-dT).     

1H-nmr studies on the dinucleoside monophosphates containing 2′-halogeno-2′-deoxypurinenucleosides: Effects of 2′-substitutes on conformation

Seven dinucleoside monophosphates containing 2′-halogeno-2′-deoxypurine nucleoside residue, dAfl-U, dAcl-U, dAbr-U, dAio-U, dGfl-U, and dIfl-C, were chemically synthesized and investigated by ¹H-nmr spectroscopy at 300 MHz. The sugar and backbone conformations of these compounds were analyzed by the spectral pattern of furanose proton resonances; and the extents of base-base interaction were estimated from chemical shifts and their temperature-dependent changes of base-proton resonances. It is found that the population of C_3′-endo conformer and the extent of base-base interaction decrease as the electronegativity of 2′-substituent decreases in dAx-U (x = fl, cl, br, and io) series. The C_3′-endo (³E) population and the base-base interaction in Nfl-U (N = A,G)-type dimers as well as dIfl-C are relatively higher than the corresponding natural ribo-dimers but can be recognized as grossly similar to the conformation of regular RNA dimers.     

Rapid synthesis of 2′,3′-dideoxy-3′β-fluoro-pyrimidine nucleosides from 2′-deoxypyrimidine nucleosides

A rapid synthesis of 2′,3′-dideoxy-3′-fluoro-β-d-threo-nucleosides bearing the pyrimidine canonical bases of nucleic acids has been developed in order to discover new nucleoside derivatives as potential antiviral drugs. However, when evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Conformational studies of adenosine 5′-phosphorodiamidates in aqueous solution

CD and nmr studies show that the conformational equilibrium of N(P)-alkylated adenosine 5′-phosphorodiamidates 2 – 4 and, to a much lesser extent, adenosine 5′-phosphorodiamidate 1 differ at two parts from that of adenosine 5′-phosphate in water. The glycosidic bond conformation is slightly shifted into the direction of the syn range, and the ribose ring N-conformer population increases. The conformational changes increase with increasing degree of N(P)-alkylation and cause a change of the sign of the long-wavelength CD band for 2 – 4 . The new conformation may be characterized by a stacked arrangement for the adenine ring and the 5′-substituted phosphate group of 2 – 4 . Hydrophobic attraction between the head and the tail of the molecules, as well as the entropy effect (which promotes the clustering of apolar faces in water), may be jointly responsible for this stacked arrangement. The intramolecular attractive forces stabilizing the stacked conformation may act at the expense of the solubility decreasing intermolecular attractive forces, and may thus be responsible for the increased water solubility of 2 – 4 , which is higher than that of 1 by more than two orders of magnitude.     

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates inhibit the repair of DNA in rat liver chromatin

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates are shown to be strong inhibitors of repair DNA synthesis in γ-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase β-catalyzed repair DNA synthesis.     

Crystal structure of the C‐terminal 2′,5′‐phosphodiesterase domain of group a rotavirus protein VP3

In response to viral infections, the mammalian innate immune system induces the production of the second messenger 2′–5′ oligoadenylate (2–5A) to activate latent ribonuclease L (RNase L) that restricts viral replication and promotes apoptosis. A subset of rotaviruses and coronaviruses encode 2′,5′‐phosphodiesterase enzymes that hydrolyze 2–5A, thereby inhibiting RNase L activation. We report the crystal structure of the 2′,5′‐phosphodiesterase domain of group A rotavirus protein VP3 at 1.39 Å resolution. The structure exhibits a 2H phosphoesterase fold and reveals conserved active site residues, providing insights into the mechanism of 2–5A degradation in viral evasion of host innate immunity. Proteins 2015; 83:997–1002. © 2015 Wiley Periodicals, Inc.