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.     

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 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.     

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.     

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.     

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.     

Enzymatic alcoholysis of 3′,5′-di-O-acetyl-2′-deoxynucleosides

Candida antarctica-B (CAL-B) lipase-catalysed alcoholysis of a set of 3′,5′-di-O-acetyl-2′-deoxynucleosides (1a–e) gave the corresponding 3′-O-acetyl-2′-deoxy-nucleosides (2a–e) in yields ranging from 50 to 96%. The alcohol employed in the biotransformation affected the rate of the enzymatic reaction and the yield of the 3′-O-acetylated product, but in all cases only this regioisomer was formed. The obtained results are in agreement with the regioselectivity displayed by CAL-B lipase in previously reported biotransformations of nucleosides. CAL-B catalysed alcoholysis of 2′,3′,5′-tri-O-acetyl-cytidine and 4-N-acetyl-2′,3′,5′-tri-O-acetylcytidine was also studied, affording with the same regioselectivity the corresponding free 5′-hydroxyl nucleosides.     

Localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes

The location of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes was determined. This was accomplished by comparing the enzyme's accessibility with that of glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic surface marker) and acetylcholinesterase (external marker) in sealed and unsealed ghosts and normal and inverted membrane vesicles. The results showed that 2′,3′-cyclic nucleotide 3′-phosphodiesterase, like glyceraldehyde-3-phosphate dehydrogenase, meets several criteria for an inner (cytoplasmic) membrane location: (1) the enzyme was accessible to substrate in unsealed ghosts and inside-out vesicles but not in sealed or right-side-out vesicles, (2) latent activity in sealed ghosts could be exposed with detergent (Triton X-100), (3) activity in unsealed ghosts was gradually sequestered during resealing and could be re-exposed with detergent, and (4) the enzyme was susceptible to trypsin proteolysis only in unsealed ghosts. These results demonstrate that the active site of 2′,3′-cyclic nucleotide 3′-phosphodiesterase faces the cytoplasm of erythrocytes and that the enzyme may not span the lipid bilayer of the membrane. The localization of the phosphodiesterase on the inner membrane surface of erythrocytes suggests that the similar enzyme of myelin may be embedded within the major dense line of the compact lamellae.     

Stereoselective synthesis of 3′-C-methylene- and 2′-methyl-3′-C-methylene-3′-deoxythymidine

Stereoselective synthesis of 3′-C-methylene- and 2′-methyl-3′-C-methylene-3′-deoxythymidine is described, the key reaction being the formation of 3-C-methylene function by catalytic isomerization of a chiral epoxyalcohol, prepared from commercially available 3-methyl-2-butenal and 3-methyl-2-pentenal.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

The role of 5′-methylthioadenosine phosphorylase in 5′-methylthioadenosine-mediated inhibition of lymphocyte transformation

To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation.     

Synthesis and anti-HIV activity of novel 2′,3′-dideoxy-3′-thiacytidine prodrugs

We report here the synthesis of a novel series of 5′-O-carbonates of 3TC, using different aliphatic alcohols and N,N-carbonyldiimidazol. Its antiviral activity was determined in peripheral blood mononuclear cells (PBMCs) showing some carbonate derivatives with an activity similar to or better than 3TC, except 3TC-Metha and 3TC-2Pro with less activity. In vitro assays in PBMCs have demonstrated that cytotoxicity increases as the carbon chain length of the alcohol moiety increases, showing compounds with a normal chain length of n = 2–5 good selective index, compared to the parent drug. Thus, this work is an important contribution leading to the suppression of HIV replication.