- Phosphodiester Cleavage of Ribonucleoside Monophosphates and Polyribonucleotides by Homo- and Heterodinuclear Metal Complexes of a Cyclohexane-Based Polyamino-Polyol Ligand
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The ability of the dinuclear complexes of tdci [1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol] to promote the cleavage of the phosphodiester bonds of nucleoside 2′,3′-cyclic monophosphates, dinucleoside monophosphates and polyribonucleotides has been studied. The homodinuclear copper(II) and zinc(II) complexes efficiently promote the hydrolysis of cyclic nucleotides. The second-order rate constant (k2≈0.44M-1S-1) estimated for the cleavage of 2′,3′-cAMP induced by dinuclear copper(II) complexes is about 107 times greater than that for the hydroxide-ion-catalysed reaction. The complex selectively cleaves the 2′O-P bond of 2′,3′-cUMP and forms the 3′-product in 91% yield. An equimolar mixture of copper(II), zinc(II) and tdci proved to be more efficient than either of the binary systems: a 7-20-fold rate enhancement was observed for the cleavage of 2′,3′-cNMP substrates. The half-life for the hydrolysis of 2′,3′-cAMP decreased from 300 days to five minutes at 25°C when the concentration of each of the three components was 2.5mM. In contrast to the copper(II) or zinc(II) complexes of tdci, the heterodinuclear species promoted the hydrolysis of several dinucleoside monophosphates. For two ApA isomers, cleavage of the 3′,5′-bond was about 6.5 times faster than cleavage of the 2′,5′-bond. On the basis of the kinetic data, a trifunctional mechanism is suggested for the heterodinuclear-complex-promoted cleavage of the phosphodiester bond. Double Lewis acid activation occurs when the metal ions bind to the phosphate oxygen atoms. In particular, a metal-bound hydroxide ion serves as a general base or a nucleophilic catalyst, and, presumably, a zinc(II)-bound aqua ligand behaves as a general acid and facilitates the departure of the leaving alkoxide group. The effect of the complexes on the hydrolysis of poly(U), poly(A) and type III native RNA was also investigated, and, for the first time, kinetic data on the cleavage of the phosphodiester bonds of polyribonucleotides by a dinuclear complex was obtained.
- Jancso, Attila,Mikkola, Satu,Loennberg, Harri,Hegetschweiler, Kaspar,Gajda, Tamas
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- The pKa of the internucleotidic 2′-hydroxyl group in diribonucleoside (3′→5′) monophosphates
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Ionization of the internucleotidic 2′-hydroxyl group in RNA facilitates transesterification reactions in Group I and II introns (splicing), hammerhead and hairpin ribozymes, self-cleavage in lariatRNA, and leadzymes and tRNA processing by RNase P RNA, as well as in some RNA cleavage reactions promoted by ribonucleases. Earlier, the pKa of 2′-OH in mono- and diribonucleoside (3′-5′) monophosphates had been measured under various nonuniform conditions, which make their comparison difficult. This work overcomes this limitation by measuring the pKa values for internucleotidic 2′-OH of eight different diribonucleoside (3′-5′) monophosphates under a set of uniform noninvasive conditions by 1H NMR. Thus the pKa is 12.31 (±0.02) for ApG and 12.41 (±0.04) for ApA, 12.73 (±0.04) for GpG and 12.71 (±0.08) for GpA, 12.77 (±0.03) for CpG and 12.88 (±0.02) for CpA, and 12.76 (±0.03) for UpG and 12.70 (±0.03) for UpA. By comparing the pKas of the respective 2′-OH of monomeric nucleoside 3′-ethyl phosphates with that of internucleotidic 2′-OH in corresponding diribonucleoside (3′→5′) monophosphates, it has been confirmed that the aglycons have no significant effect on the pKa values of their 2′-OH under our measurement condition, except for the internucleotidic 2′-OH of 9-adeninyl nucleotide at the 5′-end (ApA and ApG), which is more acidic by 0.3-0.4 pKα units.
- Acharya,Foeldesi,Chattopadhyaya
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- Rapid and highly base selective RNA cleavage by a dinuclear Cu(II) complex
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A bis-Cu(II) complex based on a covalently linked terpyridine and bipyridine ligand system is shown to rapidly cleave bis-ribonucleotides with remarkable selectivity for adenine bases.
- Liu, Shanghao,Hamilton, Andrew D.
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- Rapid and highly selective cleavage of ribonucleoside 2',3'-cyclic monophosphates by dinuclear Cu(II) complexes
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Two characteristics that never before appeared together are combined in complexes 1 and 2, which show high activity and high selectivity in the hydrolysis of cyclic nucleoside 2',3'-monophosphates as model compounds for RNA. In the case of 1 the regioselectivity is exceptional, and, in the case of 2, the base selectivity.
- Liu,Luo,Hamilton
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- IMMUNOAFFINITY PURIFICATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE FROM LACTUCA COTYLEDONS
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To facilitate further study of a multifunctional phosphodiesterase, previously partially purified from Lactuca cotyledons, a new purification step has been devised.This uses an immunoaffinity column based upon polyclonal antibodies raised against the partially purified enzyme.Preparation of the immunoaffinity column, purufication of the enzyme using the new protocol, and analysis of the activity of the purified enzyme are described.The additional step produced an enzyme preparation with a significantly higher specific activity and free of nucleotidase and non-specific phosphatase activity.The observed properties of the enzyme confirm similarities with mammalian multifunctional phosphodiesterase but reaffirm the existence of two types of substrate binding site on the Lactuca phosphodiesterase.Key Word Index - Lactuca sativa; Compositae; lettuce; cotyledons; cyclic nucleotides; phosphodiesterase; immunoaffinity purification; 3',5'-cyclic AMP; 3',5'-cyclic GMP; 3',5'-cyclic CMP; 3',5'-cyclic UMP.
- Chiatante, Donato,Balconi, Carlotta,Newton, Russell P.,Brown, Eric G.
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- Dinuclear Zn2+ complexes in the hydrolysis of the phosphodiester linkage in a diribonucleoside monophosphate diester.
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Dizinc complexes that were formed from 2:1 mixtures of Zn(NO3)2 and dinucleating ligands TPHP (1), TPmX (2) or TPpX (3) in aqueous solutions efficiently hydrolyzed diribonucleoside monophosphate diesters (NpN) under mild conditions. The dinucleating ligand affected the structure of the aquo-hydroxo-dizinc core, resulting in different characteristics in the catalytic activities towards NpN cleavage. The pH-rate profile of ApA cleavage in the presence of (Zn2+)(2)-1 was sigmoidal, whereas those of (Zn2+)(2)-2 and (Zn2+)(2)-3 were bell-shaped. The pH titration study indicated that (Zn2+)(2)-1 dissociates only one aquo proton (up to pH 12), whereas (Zn2+)(2)-2 dissociates three aquo protons (up to pH 10.7). The observed differences in the pH-rate profile are attributable to the various distributions of the monohydroxo-dizinc species, which are responsible for NpN cleavage. As compared to that using (Zn2+)(2)-1, the NpN cleavage using (Zn2+)(2)-2 showed a greater rate constant, with a higher product ratio of 3'-NMP/2'-NMP. The saturation behaviors of the rate, with regard to the concentration of NpN, were analyzed by Michaelis-Menten type kinetics. Although the binding of (Zn2+)(2)-2 to ApA was weaker than that of (Zn2+)(2)-1, (Zn2+)(2)-2 showed a greater kcat value than (Zn2+)(2)-1, resulting in higher ApA cleavage activity of the former.
- Yashiro, Morio,Kaneiwa, Hideki,Onaka, Kenichi,Komiyama, Makoto
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- Synthesis of ribonucleotides from the corresponding ribonucleosides under plausible prebiotic conditions within self-assembled supramolecular structures
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Abiotic synthesis of ribonucleotides, mainly at the 5′ position, from the corresponding ribonucleosides within self-assembled supramolecular structures, based on guanosine:borate hydrogels, was carried out in the temperature range of 70-90 °C, using urea and a phosphate source (K2HPO4 or hydroxyapatite). Phosphorylation is possible at initial concentrations of guanosine lower than 20 mM and it is more efficient using wet/dry cycles. Monoamidophosphate (and, eventually, diamidophosphate), diamidodiphosphate and pyrophosphate are intermediates in the synthesis of ribonucleotides. These conclusions are supported by NMR spectroscopy and mass spectrometry analysis of samples. On the other hand, after reaction, hydrogels can produce globular aggregates by the addition of water and decreasing temperature, thus confirming that ribonucleotides, once activated under suitable conditions, could form polyribonucleotides.
- Franco,Ascenso,Ilharco,Da Silva
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- Guanidine-based polymer brushes grafted onto silica nanoparticles as efficient artificial phosphodiesterases
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Polymer brushes grafted to the surface of silica nanoparticles were fabricated by atom-transfer radical polymerization (ATRP) and investigated as catalysts in the cleavage of phosphodiesters. The surfaces of silica nanoparticles were functionalized with an ATRP initiator. Surface-initiated ATRP reactions, in varying proportions, of a methacrylate moiety functionalized with a phenylguanidine moiety and an inert hydrophilic methacrylate species afforded hybrid nanoparticles that were characterized with potentiometric titrations, thermogravimetric analysis, and SEM. The activity of the hybrid nanoparticles was tested in the transesterification of the RNA model compound 2-hydroxypropyl para-nitrophenylphosphate (HPNP) and diribonucleoside monophosphates. A high catalytic efficiency and a remarkable effective molarity, thus overcoming the effective molarities previously observed for comparable systems, indicate the existence of an effective cooperation of the guanidine/guanidinium units and a high level of preorganization in the nanostructure. The investigated system also exhibits a marked and unprecedented selectivity for the diribonucleoside sequence CpA. The results presented open up the way for a novel and straightforward strategy for the preparation of supramolecular catalysts.
- Savelli, Claudia,Salvio, Riccardo
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p. 5856 - 5863
(2015/03/31)
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- Synthesis of oligoribonucleotides with phosphonate-modified linkages
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Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2′-O-benzoyloxymethoxymethyl protected monomers is presented in both 3′→5′ and 5′→3′ directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3′- and 5′-phosphonate linkages are evaluated. The introduction of the 5′-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔTm -1.8 °C/mod.), whereas the introduction of the 3′-phosphonate units resulted in considerable destabilisation of the duplexes (ΔTm -5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.
- Pav, Ondej,Koiova, Ivana,Barvik, Ivan,Pohl, Radek,Budinsky, Milo,Rosenberg, Ivan
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supporting information; experimental part
p. 6120 - 6126
(2011/10/10)
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- RETRACTED ARTICLE: First prebiotic generation of a ribonucleotide from adenine, d-ribose and trimetaphosphate
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Adenosine monophosphate isomers are obtained by self-assembling of adenine, d-ribose and trimetaphosphate in aqueous solution in good yields. This generation of a ribonucleotide from its three molecular components occurs in a one-pot reaction at room temperature for about 30-40 days and with high chemio-, regio-, and stereo-selectivity. Similar results are obtained with guanine. A mechanism is also proposed.
- Baccolini, Graziano,Boga, Carla,Micheletti, Gabriele
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supporting information; experimental part
p. 3640 - 3642
(2011/05/04)
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- Efficient cleavage of ribonucleoside 3′,5′-cyclic phosphates and deoxyribonucleoside 3′,5′-cyclic phosphate by lanthanides
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The cleavage of 3′,5′-cAMP, 3′,5′-cGMP and 3′,5′-dcAMP by lanthanides has been investigated by HPLC and 1H NMR. Rapid cleavage of cAMP, cGMP and dcAMP by Ce(III) under air at pH 8 and 37°C has been observed. Regioselective cleavage of P.O(5′) bond in cAMP, cGMP and dcAMP to give the corresponding 3′-AMP, 3′-GMP and 3′-dAMP by lanthanide chlorides has been achieved, and 3′-AMP and 3′-GMP are cleaved to adenosine(A) and guanosine(G) more slowly, respectively. The notable difference in reactivity between Ce(III) and the other lanthanide ions under air has also been studied. The cleavage is enhanced with the increase in the molar fraction of Ce(IV). The fast cleavage of cAMP by Ce(III) under air at pH 8 is ascribed to the resultant Ce(IV) in the reaction mixture.
- Li, Xinmin,Zhu, Bing,Zhao, Daqing,Ni, Jiazuan
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p. 1144 - 1147
(2007/10/03)
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- Chemical synthesis of a pentaribonucleoside tetraphosphate constituting the 3'-acceptor stem sequence of E. coli tRNAIle using 2'-O-(3-methoxy-1,5-dicarbomethoxypentan-3-yl)-ribonucleoside building blocks. Application of a new achiral and acid-labile 2'-hydroxyl protecting group in tRNA synthesis.
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A synthesis of a pentaribonucleotide fragment constituting the residues 59-63 of 3'-terminus of E. coli tRNAIle, 5'-ApGpUpCpC-3', has been carried out using a new, easily accessible and achiral 2'-ketal protecting group. The new 2'-ketal group has an additional advantage in that it is easily functionalized to the diamide with aqueous ammonia in the penultimate step of deblocking of fully protected oligoribonucleotides. Such a functionalization of the 2'-ketal group at the penultimate step of deblocking of the fully protected tRNA molecule enhances its relative rate of removal under an acidic condition with a minimum of damage of the target tRNA molecule.
- Sandstr?m,Kwiatkowski,Chattopadhyaya
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p. 273 - 290
(2007/10/02)
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- Synthesis of a Dodecaribonucleotide, GUAUCAAUAAUG, by Use of "Fully" Protected Ribonucleotide Building Blocks
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The fully protected ribonucleotide monomer units (17, 19, 26, and 32) have been synthesized in excellent overall yields from unprotected ribonucleosides.Several carbamoyl groups were tested for protection of the guanosine base moiety.Finally, the diphenylcarbamoyl group was chosen and O6-(diphenylcarbamoyl)-N2-propionylguanosine was readily prepared in high yield and converted to the guanosine units 12 and 17.The uridine unit 19 was prepared by the acylation of the previous unit 18 with anisoyl chloride in the presence of i-Pr2EtN.In the case of the adenosine and cytidine units (26 and 32) , the regioselective 2'-O-tetrahydropyranylation was involved in their syntheses.These "perfectly" protected monomer units have succesfully been utilized in the synthesis of GUAUCAAUAAUG, a modified 5'-terminal structure, of brome mosic virus (BMV) mRNA no. 4 filament.The dodecamer chain was elongated by fragment condensation from the 3'-5' direction.The yields of the oligomer blocks have proved to be dramatically high because no side reactions occurred during the condensation reactions.Indeed, the final coupling to give the target 12-mer was achieved in 91percent yield.The deprotection of the fully protected in the usual manner gave GUAUCAAUAAUG in ca. 30percent yield.
- Kamimura, Takashi,Tsuchiya, Masahiko,Urakami, Ken-ichi,Koura, Koji,Sekine, Mitsuo,et al.
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p. 4552 - 4557
(2007/10/02)
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- DECYCLIZING PHOSPHODIESTERASES ASSOCIATED WITH PLASMA MEMBRANES OF THE CHICK CHORIOALANTOIS
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Two different enzymatic activities hydrolyzing 2',3'-cyclic phosphates of purine and pyrimidine ribonucleosides to give 2'- or 3'-ribonucleotides have been detected in plasma membranes of the chick chorioalantois.The enzymes differ in their temperature optima: At lower temperatures, cleavage to the 2'-isomers predominates.
- Pristasova, Svetlana,Rosenbergova, Marta,Holy, Antonin
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p. 520 - 524
(2007/10/02)
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- AFFINITY CHROMATOGRAPHY OF GUANYLORIBONUCLEASE FROM Streptomyces aureofaciens
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2',3'-O-2-Carboxyethylideneinosine 5'-phosphate (Ia), 5'-phosphorothioate (IIa), 5'-O-carboxymethylinosine 2'(3')-phosphate (IIIa) and 2'(3')-phosphorothioate (IVa) were bound to 6-aminohexyl-Sepharose 4B by the method of mixed anhydrides with ethyl chloroformate. 5'-O-(2-Aminoethylamidocarbonylmethyl)inosine 2'(3')-phosphorothioate (Va), O-(4-aminophenyl)-inosine 5'-phosphorothioate (VIa) and O-(4-aminophenyl)-O-phenyl phosphorothioate (VIIa) were bound to Sepharose 4B activated with cyanogen bromide.The resulting modified supports IIa-VIb bind the ribonuclease of Streptomyces aureofaciens within the pH range 6.5-7.5.The specific affinity bond manifests itself in derivatives with ligands of type IVb and Vb, on which a 50-70fold purification degree can be achieved, with respect to the crude preparation.
- Kois, Pavol,Rosenberg, Ivan,Holy, Antonin
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p. 2839 - 2846
(2007/10/02)
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