Nucleosides.  LXXIX.  Facile base-catalyzed hydrogen isotope labeling at position 6 of pyrimidine nucleosides

Rabi, Jaime A.; Fox, Jack J.

doi:10.1021/ja00786a044

Cited by 43 publications

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References 2 publications

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“…[5,6] Dideuterocytidine was synthesized by a modification of the procedure of Rabi and Fox. (18) NMR analysis revealed 83% and 96% incorporation at C-5 and C-6, respectively. For nucleoside chromatography, Merck kieselgel 60-F254 sheets were used for TLC and products were detected by UV or by development of color with Ce(SO 4 ) 2 /(NH 4 )6Mo 7 O 2 •4H 2 O/H 2 SO 4 /H 2 O. Merck kieselgel 60 (230−400 mesh) was used for column chromatography.…”

Section: Methodsmentioning

confidence: 97%

Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 ofE. coliRibonucleotide Reductase

et al. 2008

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The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-2H], [2′-2H], [4′-2H], [5′-2H], [U−13C, 15N], [U−15N], and [5,6 -2H] CDP and [β-2H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of 2H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.

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Section: Methodsmentioning

confidence: 97%

Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 ofE. coliRibonucleotide Reductase

et al. 2008

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“…Protonation at the C8 position of perdeuterated rGTP and rATP was achieved by incubation with ~5 equivalents of Triethylamine (TEA) in 1 H 2 O at 60 °C for 24 hours and for 5 days, respectively 103,104. Deuteration of the C8 carbon of fully-protonated GTP and ATP was achieved by incubation in 2 H 2 O (99.8 % deuteration; CIL).…”

Section: Methodsmentioning

confidence: 99%

Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Miyazaki

Irobalieva

Tolbert

et al. 2010

Journal of Molecular Biology

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The 5′-untranslated regions (5′-UTRs) of all gammaretroviruses contain a conserved “double hairpin motif” (ΨCD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming “kissing” interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney Murine Leukemia Virus (MoMuLV) ([ΨCD]2, 132-nucleotides, 42.8 kDaltons) using a 2H-edited NMR spectroscopy-based approach. This approach enabled the detection of 1H-1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H-1H correlated and 1H-13C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D’ and SLC’ to SL-D), and stack in an end-to-end manner (SL-C to SL-D and SL-C’ to SL-D’) that gives rise to an elongated overall shape (ca. 95 Å by 45 Å by 25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [ΨCD]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [ΨCD]2 functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

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“…I t change in both 1-methyl uracils and pyrimidine nucleosides [38]. To permit comparison of the deshielding effects of different substituents the proton resonance signals of a 5'-deoxynucleoside or nucleoside trialcohol could serve for reference; however, in this study such values are only available for the uracil derivatives.…”

Section: 4mentioning

confidence: 99%

Pyrimidine Nucleosides in Solution

Follmann¹,

Pfeil²,

Witzel³

1977

European Journal of Biochemistry

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Proton magnetic resonance (PMR) spectra of 45 nucleoside and nucleotide derivatives of uracil, thymine, cytosine, 5-methylcytosine, and other substituted pyrimidines in water or dimethyl sulfoxide solutions have been analysed in order to correlate structure variations with molecular conformations and to gain insight into the nature of intramolecular forces which are responsible for the 'rigidity' of the molecules. Irrespective of thc structure of pyrimidine base and the absolute chemical shift values (6) the C(6)-H and, to a lesser degree, the C(5)-H and C(5)-CH3 resonances are shifted downfield by very similar amounts ( A S ) in the regular nucleosides or in the presence of charged or polar C-4' substituents. In detail, the following conformation-determining effects are seen.1. Ribose vs 2-deoxyribose substitution of a pyrimidine makes little difference for the chemical environment of base hydrogens but an intact pentofuranose ring (4'-0) and the exocyclic 5'-OH group are essential for a nucleoside to assume a specific conformation.2. Profound shielding or deshielding of H-6 by exocyclic nitrate, halogen, hydroxyl, phosphate, and carboxylate substituents indicates preference for the anti and gauche,gauche conformation ranges in all compounds except in the case of C-6 substitution.3. Halogen atoms and the hydroxyl group ( A 6 = 0.30 -0.40 ppm) interact with pyrimidine bases by induced polarisation of the C(5)=C(6) double bond, phosphate anions ( A 6 % 0.50 ppm) interact by the electrostatic attraction mechanism previously described, and in nucleoside 5'-carboxylates ( A 6 > 0.90 ppm) a hydrogen bond between -COO-and H-6 is formed.4. The deshielding effects caused by a 5'-OH group and monoanionic 5'-phosphate are similar ( A 6 % 0.35 -0.45 ppm), supporting the view that intramolecular rigidity of nucleosides and the nucleotide unit is not fundamentally different.This systematic survey, together with our previous studies of adenosine derivatives, provides a rationale for why the majority of both pyrimidine and purine nucleosides and nucleotides in solution exhibit closely comparable conformations, which in turn explain the group specificity of certain key enzymes in nucleic acid metabolism.Enzymes which act upon nucleosides or nucleotides usually show stringent specificities with respect to the conformation of the heterocyclic base, furanose moiety, and exocyclic hydroxyl or phosphate group of their substrates. For example, modified ribonucleotides in which the base deviates from a regular anti position are poor substrates of ribonucleases [l], RNA polymerase [2], or ribonucleotide reductase (H. Follmann and W. Ludwig, unpublished). 'H and13C nuclear magnetic resonance spectra are excellent tools to study This paper is part 3 of a series entitled Forces Stabilizing the Conformation of Nucleosides in Solution.Abbreviations. PMR, proton magnetic resonance. The IUPAC-IUB abbreviations are used for nucleic acid constituents. these three-dimensional structures in solutions, and in recent years have provided description...

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Nucleosides. LXXIX. Facile base-catalyzed hydrogen isotope labeling at position 6 of pyrimidine nucleosides

Cited by 43 publications

References 2 publications

Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 ofE. coliRibonucleotide Reductase

Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 ofE. coliRibonucleotide Reductase

Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Pyrimidine Nucleosides in Solution

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