Ribose Ring Research Articles

Synthesis of 3′-azido-4′-ethynyl-3′,5′-dideoxy-5′-norarabinouridine: a new anti-HIV nucleoside analogue

3′-Azido-4′-ethynyl-3′,5′dideoxy-5′-norarabinouridine 10 was synthesized from commercial uridine 1 in which the key step is the opening of protected 2′,3′-epoxyuridine derivative 7 by sodium azide and the hydroxymethyl at 4-position of the ribose ring are replaced by ethynyl group.

Computer-assisted combinatorial design of bicyclic thymidine analogs as inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase

Thymidine monophosphate kinase (TMPK(mt)) is an essential enzyme for nucleotide metabolism in Mycobacterium tuberculosis, and thus an attractive target for novel antituberculosis agents. In this work, we have explored the chemical space around the 2',3'-bicyclic thymidine nucleus by designing and in silico screening of a virtual focused library selected via structure based methods to identify more potent analogs endowed with favorable ADME-related properties. In all the library members we have exchanged the ribose ring of the template with a cyclopentane moiety that is less prone to enzymatic degradation. In addition, we have replaced the six-membered 2',3'-ring by a number of five-membered and six-membered heterocyclic rings containing alternative proton donor and acceptor groups, to exploit the interaction with the carboxylate groups of Asp9 and Asp163 as well as with several cationic residues present in the vicinity of the TMPK(mt) binding site. The three-dimensional structure of the TMPK(mt) complexed with 5-hydroxymethyl-dUMP, an analog of dTMP, was employed to develop a QSAR model, to parameterize a scoring function specific for the TMPK(mt) target and to select analogues which display the highest predicted binding to the target. As a result, we identified a small highly focused combinatorial subset of bicyclic thymidine analogues as virtual hits that are predicted to inhibit the mycobacterial TMPK in the submicromolar concentration range and to display favorable ADME-related properties.

The discovery and structure–activity relationships leading to CE-156811, a difluorophenyl cyclopropyl fluoroether: A novel potent antibacterial analog derived from hygromycin A

SAR studies and optimization of various modified Hygromycin A fluoroalkyl ethers, which led to the discovery of the highly potent 4′-(2-cyclopropyl-2-fluoroethyl ether) antibacterial CE-156811 (1) derived from truncation of the ribose ring and difluorination of the phenyl found in Hygromycin A, are discussed.

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.

Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4′)

Aminoglycosides are important antibiotics used against a wide range of pathogens. As a mechanism of defense, bacteria have evolved enzymes able to inactivate these drugs by regio-selectively adding a variety of functionalities (acetyl, phospho, and nucelotidyl groups) to their scaffolds. The aminoglycoside nucleotidyltransferase ANT(4′) is one of the most prevalent and unique modifying-enzymes. Here, by TLC, HRMS, and colorimetric assays, we demonstrate that the resistance enzyme ANT(4′) from Staphylococcus aureus is highly substrate and cosubstrate promiscuous. We show that deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than NTPs. We demonstrate that the position of the triphosphate group (5′ and not 3′) on the ribose/deoxyribose ring is important for recognition by ANT(4′), and that NTPs with larger substituents at the 3′-position of the ribose ring are not cosubstrates for ANT(4′). We confirm that for all aminoglycosides tested, the respective nucleotidylated products are completely inactive. These results provide valuable insights into the development of strategies to combat the ever-growing bacterial resistance problem.

Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.

ChemInform Abstract: An EPR Study of the Transfer of Radical-Induced Damage from the Base to Sugar in Nucleic Acid Components: Relevance to the Occurrence of Strand-Breakage.

EPR spectroscopy has been employed, in conjunction with a rapid-flow system and the TiIII–H2O2 and TiIII–HOOSO3– redox couples, to study the reactions of ˙OH and SO4˙– with pyrimidine nucleosides. From experiments which complement radiolytic and photolytic studies it is concluded that the formation of base-centred radical-cations (from SO4˙– and from ˙OH at low pH) is followed both by hydration and by loss of the C2′-H in the ribose (but not 2-deoxyribose) ring: further fragmentation follows the latter reaction. The crucial role of the C2′-OH group in the transfer of damage is discussed.

ChemInform Abstract: The Biosynthesis of Sinefungin: Investigations Using a Cell-Free System.

Abstract The origin of the adenylyl moiety of sinefungin has been investigated by administration of doubly-labeled forms of ATP and adenosine to cell-free extracts of Streptomyces griseolus . The results demonstrated that both ATP and adenosine are significantly degraded by the extract before incorporation into sinefungin. However, the ribose ring of adenosine was shown to be incorporated into sinefungin intact, and without loss of tritium from C-5′. This observation rules out the intermediacy of A9145C in sinefungin biosynthesis. Additional experiments are described which suggest that CC bond formation between C-5 of arginine and C-5 of a ribose derivative may precede attachment of the adenine ring.

Pyrimidine Ribonucleotides with Enhanced Selectivity as P2Y6 Receptor Agonists: Novel 4-Alkyloxyimino, (S)-Methanocarba, and 5′-Triphosphate γ-Ester Modifications

The P2Y(6) receptor is a cytoprotective G-protein-coupled receptor (GPCR) activated by UDP (EC(50) = 0.30 microM). We compared and combined modifications to enhance P2Y(6) receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as alpha,beta-methylene and extension of the terminal phosphate group into gamma-esters of UTP analogues. The conformationally constrained (S)-methanocarba-UDP is a full agonist (EC(50) = 0.042 microM). 4-Methoxyimino modification of pyrimidine enhanced P2Y(6), preserved P2Y(2) and P2Y(4), and abolished P2Y(14) receptor potency, in the appropriate nucleotide. N(4)-Benzyloxy-CDP (15, MRS2964) and N(4)-methoxy-Cp(3)U (23, MRS2957) were potent, selective P2Y(6) receptor agonists (EC(50) of 0.026 and 0.012 microM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-gamma-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y(6) receptor potency but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N(4)-methoxycytidine 5'-triphospho-gamma-[1]glucose were active (EC(50) of 2.47 and 0.18 microM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y(6) receptor agonists may be enhanced by modest structural changes.

Conformations of ribonucleoside uridine in the low temperature Ar matrices

The conformational equilibrium of the ribonucleoside uridine molecules in low-temperature Ar matrices was investigated by the FTIR matrix isolation spectroscopy and quantum-chemical calculations at the DFT and MP2 levels of theory. It was established that conformers with a carbonyl group C2O, which takes part in the intramolecular hydrogen bonds, dominate in the low temperature matrices. Populations of syn- and anti- conformers with intramolecular hydrogen bonds O5′H–O2 and O2′–O2, respectively, are equal practically. Unlike 2′-deoxyuridine, the syn- conformers of Ur with C3′-endo structure of the ribose ring (South) have been found in Ar matrices. At the same time the conformations of C2′-endo (North) ribose ring prevail both in anti- and syn- conformers.

Oligonucleotide Analogues with Integrated Bases and Backbones. Part 25

AbstractThe ability of A*[s]U dinucleosides to gel organic solvents and water is modulated by changing the nature of the substituents at OC(2′) and OC(3′), as evidenced by comparing the gelation of the dinucleosides 7–9 and the properties of the gels. A mere extension of the hydrophobic moiety, by replacing the isopropylidene groups of 2 by cyclohexylidene groups, as in 7, has a small effect, while changing the conformation of the ribose ring and reducing the size of the hydrophobic moiety, as in 8, has a strong effect on the scope of gelation, the minimum gelation concentration, as low as 0.07% for pentanol and decanol, and the properties of the gel. The fully deprotected dinucleoside 9 gels water at a minimal gelation concentration of 0.6%. A TEM of the corresponding xerogel shows the formation of fibers with a diameter of ca. 30 to 90 nm.

Crystallization and preliminary X-ray study of the D-altritol oligonucleotide GTGTACAC.

In altritol nucleic acids (ANAs), the natural five-membered ribose ring of RNA is replaced by the six-membered D-altritol ring. ANAs are good candidates to act as siRNAs in the RNA-interference pathway. Crystals of the fully modified altritol self-complementary octamer GTGTACAC were grown by the hanging-drop vapour-diffusion technique at 289 K. Diffraction data were recorded on SLS beamline X06DA and processed to 3.0 A resolution. The crystals belonged to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = 25.05, c = 117.58 A.

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.

Direct Strand Scission from a Nucleobase Radical in RNA

RNA oxidation is important in the etiology of disease and as a tool for studying the structure and folding kinetics of this biopolymer. Nucleobase radicals are the major family of reactive intermediates produced in RNA exposed to diffusible species such as hydroxyl radical. The nucleobase radicals are believed to produce direct strand breaks by abstracting hydrogen atoms from their own and neighboring ribose rings. By independently generating the formal C5 hydrogen atom addition product of uridine in RNA, we provide the first chemical characterization of the pathway for direct strand scission from an RNA nucleobase radical. The process is more efficient under anaerobic conditions. The preference for strand scission in double-stranded RNA over single-stranded RNA suggests that this chemistry may be useful for analyzing the secondary structure of RNA in hydroxyl radical cleavage experiments if they are carried out under anaerobic conditions.

Receptor regulatory properties evident in the molecular similarity of dopamine receptor ligands and purine nucleotides.

Computational studies have revealed similarities in the relative configurations of purine nucleotides and ligands for histamine, acetylcholine and adrenergic receptors. In common with other G-protein-regulated receptors, dopamine receptors are associated with specific changes in nucleotide levels during signal transduction processes. The purpose of this study was to investigate molecular similarity in dopamine receptor ligands and purine nucleotides. Molecular superimposition and fitting data for D1-like receptor ligands identified a pharmacophore in the adenine and ribose rings of ATP. D2-like agonists and antagonists related to a pharmacophore in the guanine and ribose rings of GTP. The results are consistent with the hypothesis that the dopamine receptor family may have evolved from receptors for the ATP and GTP nucleotides.

Visualizing the Molecular Interactions of a Nucleotide Analog, GS-9148, with HIV-1 Reverse Transcriptase–DNA Complex

GS-9148 ([5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid) is a dAMP (2′-deoxyadenosine monophosphate) analog that maintains its antiviral activity against drug-resistant HIV. Crystal structures for HIV-1 reverse transcriptase (RT) bound to double-stranded DNA, ternary complexes with either GS-9148-diphosphate or 2′-deoxyadenosine triphosphate (dATP), and a post-incorporation structure with GS-9148 translocated to the priming site were obtained to gain insight into the mechanism of RT inhibition. The binding of either GS-9148-diphosphate or dATP to the binary RT–DNA complex resulted in the fingers subdomain closing around the incoming substrate. This produced up to a 9 Å shift in the tips of the fingers subdomain as it closed toward the palm and thumb subdomains. GS-9148-diphosphate shows a similar binding mode as dATP in the nucleotide-binding site. Residues whose mutations confer resistance to nucleotide/nucleoside RT inhibitors, such as M184, Y115, L74, and K65, show little to no shift in orientation whether GS-9148-diphosphate or dATP is bound. One difference observed in binding is the position of the central ring. The dihydrofuran ring of GS-9148-diphosphate interacts with the aromatic side chain of Y115 more than does the ribose ring of dATP, possibly picking up a favorable π–π interaction. The ability of GS-9148-diphosphate to mimic the active-site contacts of dATP may explain its effective inhibition of RT and maintained activity against resistance mutations. Interestingly, the 2′-fluoro moiety of GS-9148-diphosphate was found in close proximity to the Q151 side chain, potentially explaining the observed moderately reduced susceptibly to GS-9148 conferred by Q151M mutation.

The hydration and unusual hydrogen bonding in the crystal structure of an RNA duplex containing alternating CG base pairs

The X-ray structures of two crystal forms of the RNA duplex [CGCG(5-FC)G]2 are the first crystal structures for an RNA duplex containing alternating CG base pairs with unmodified 2′-hydroxyl groups. The triclinic and the rhombohedral crystal forms (PDB ids 3jxq, 3jxr) differ in the molecular packing, the amount of ordered structure and in molecular details, especially the hydration. The P1 data extend to 1.45 Å resolution, while the R32 data reach 1.25 Å. In both structures the RNA oligomers take the form of a right-handed half-turn with conformational parameters close to the canonical A-RNA. The duplexes stack coaxially in the crystal lattice. The geometry of C:G vs. 5-FC:G pairing is similar in terms of H-bond geometry. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. Both structures contain ordered water molecules, forming characteristic hydration network in the major and the minor grooves. In addition to water, the triclinic structure contains four magnesium cations, one of them forming an inner complex with a phosphate. Two types of unusual interactions are observed. In the major groove, the 5-fluorocytidine residues form C–F⋯H–O–H hydrogen bonds with water molecules. In the minor groove, intermolecular contacts include C–H hydrogen bonds (C1′–H1⋯O2′ and C4′–H4′⋯O4′) between two ribose rings.

Synthesis and Preliminary Biological Activity of Some New Pyrazole Derivatives as Acyclonucleoside Analogues

Some novel pyrazole acyclonucleosides were prepared through binding of pyrazole-3,5-substituted derivatives with acyclic substituents mimetizing individual fragments of the ribose ring such as: 1-[(2-hydroxyethoxy) methyl], 1-[4-(hydroxybutyl)], 1-[3- (hydroxypropyl)] and 1-[(2-hydroxyethyl amino) methyl]. Their syntheses were performed from easily accessible starting materials. The prepared compounds were evaluated for their antibacterial activity. Structure-activity relationship studies (SAR) indicate that introduction of modified arm in N1 position of pyrazole moieties has not any impact on the biolgical activity. Keywords: Pyrazole, Acyclonucleoside, Synthesis, Biological activity

RNA structural motifs that entail hydrogen bonds involving sugar–phosphate backbone atoms of RNA

The growing number of high-resolution crystal structures of large RNA molecules provides much information for understanding the principles of structural organization of these complex molecules. Several in-depth analyses of nucleobase-centered RNA structural motifs and backbone conformations have been published based on this information, including a systematic classification of base pairs by Leontis and Westhof. However, hydrogen bonds involving sugar-phosphate backbone atoms of RNA have not been analyzed systematically until recently, although such hydrogen bonds appear to be common both in local and tertiary interactions. Here we review some backbone structural motifs discussed in the literature and analyze a set of eight high-resolution multi-domain RNA structures. The analyzed RNAs are highly structured: among 5372 nucleotides in this set, 89% are involved in at least one "long-range" RNA-RNA hydrogen bond, i.e., hydrogen bonds between atoms in the same residue or sequential residues are ignored. These long-range hydrogen bonds frequently use backbone atoms as hydrogen bond acceptors, i.e., OP1, OP2, O2', O3', O4', or O5', or as a donor (2'OH). A surprisingly large number of such hydrogen bonds are found, considering that neither single-stranded nor double-stranded regions will contain such hydrogen bonds unless additional interactions with other residues exist. Among 8327 long-range hydrogen bonds found in this set of structures, 2811, or about one-third, are hydrogen bonds entailing RNA backbone atoms; they involve 39% of all nucleotides in the structures. The majority of them (2111) are hydrogen bonds entailing ribose hydroxyl groups, which can be used either as a donor or an acceptor; they constitute 25% of all hydrogen bonds and involve 31% of all nucleotides. The phosphate oxygens OP1 or OP2 are used as hydrogen bond acceptors in 12% of all nucleotides, and the ribose ring oxygen O4' and phosphodiester oxygens O3' and O5' are used in 4%, 4%, and 1% of all nucleotides, respectively. Distributions of geometric parameters and some examples of such hydrogen bonds are presented in this report. A novel motif involving backbone hydrogen bonds, the ribose-phosphate zipper, is also identified.

What is the conformation of physiologically-active dinucleoside polyphosphates in solution? Conformational analysis of free dinucleoside polyphosphates by NMR and molecular dynamics simulations

Dinucleoside polyphosphates, or dinucleotides (Np(n)N'; N, N' = A, U, G, C; n = 2-7), are naturally occurring ubiquitous physiologically active compounds. Despite the interest in dinucleotides, and the relevance of their conformation to their biological function, the conformation of dinucleotides has been insufficiently studied. Therefore, here we performed conformational analysis of a series of Np(n)N' Na(+) salts (N = A, G, U, C; N' = A, G, U, C; n = 2-5) by various NMR techniques. All studied dinucleotides, except for Up(4/5)U, formed intramolecular base stacking interactions in aqueous solutions as indicated by NMR. The conformation around the glycosidic angle in Np(n)N's was found to be anti/high anti and the preferred conformation around the C4'-C5', C5'-O5' bonds was found to be gauche-gauche (gg). The ribose moiety in Np(n)N's showed a small preference for the S conformation, but when attached to cytosine the ribose ring preferred the N conformation. However, no predominant conformation was observed for the ribose moiety in any of the dinucleotides. Molecular dynamics simulations of Ap(2)A and Ap(4)A Na(+) salts supported the experimental results. In addition, three modes of base-stacking were found for Ap(2/4)A: α-α, β-β and α-β, which exist in equilibrium, while none is dominant. We conclude that natural, free Np(n)N's (n = 2-5) at physiological pH exist mostly in a folded (stacked), rather than extended conformation, in several interconverting stacking modes. Intramolecular base stacking of Np(n)N's does not alter the conformation of each of the nucleotide moieties, which remains the same as that of the mononucleotides in solution.