- Mechanistic studies relevant to bromouridine-enhanced nucleoprotein photocrosslinking: Possible involvement of an excited tyrosine residue of the protein
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The results of mechanistic studies on formation of uridine (U) and N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) from irradiation of aqueous, pH 7 solutions of bromouridine (BrU) and N-acetyltyrosine N-ethylamide (1) are reported. Solutions were irradiated with monochromatic laser emission at 266, 308 and 325 nm. Quantum yield measurements as a function of excitation wave-length suggest that both products result from excitation of the tyrosine derivative followed by electron transfer to BrU, possibly with intermediacy of the hydrated electron. The BrU radical anion ejects bromide to form the uridinyl radical, which then abstracts a hydrogen atom from 1 or adds to the aromatic ring of 1. Formation of adduct 2 is a model for photocrosslinking of nucleic acids bearing the bromouracil chromophore to adjacent tyrosine residues of proteins in nucleoprotein complexes. The value of 325 nm excitation in photocrosslinking, where the tyrosine chromophore is more competitive for photons, was demonstrated with an RNA bound to the MS2 bacteriophage coat protein; more than a 60% increase in the yield of photocrosslinking relative to that obtained with 308 nm excitation was achieved.
- Norris, Christopher L.,Meisenheimer, Kristen M.,Koch, Tad H.
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- Hydrolytic Reactions of the Diastereomeric Phosphoromonothioate Analogs of Uridyl(3',5')uridine: Kinetics and Mechanisms for Desulfurization, Phosphoester Hydrolysis, and Transesterification to the 2',5'-Isomers
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Hydrolytic reactions of the RP and SP diastereomers of the phosphoromonothioate analog of uridylyl(3',5')uridine (3',5'-UpU), having a nonbridging oxygen replaced with sulfur, have been followed by HPLC over a wide range at 363.2 K.Under neutral and acidic conditions three reactions compete: (i) desulfurization to an equilibrium mixture of 3',5'- and 2',5'-UpU, (ii) hydrolysis to uridine 2'- and 3'-monophosphates with release of uridine (either via a 2',3'-cyclic phosphoromonothioate, or a desulfurized cyclic triester), and (iii) isomerization to the 2',5'-dinucleoside phosphoromonothioate.With both diastereomers, desulfurization predominates over hydrolysis and migration at pH 1-8.Migration proceeds by retention of configuration at phosphorus and is most pronounced in very acidic solutions (H0 > 0.5 mol L-1), representing 20-30percent of the total disappearance of the starting material.At pH 3-6, the proportion of this reaction is less than 10percent.In the latter pH range, all the reactions are pH-independent.At lower pH, first-order dependence on acidity is observed, but at H0 P diastereomer is at pH P isomer.Under alkaline conditions (pH > 9), only base-catalyzed hydrolysis to uridine 2'- and 3'-thiophosphates with release of uridine takes place.At pH 1, the thioate analogs are more than 1 order of magnitude more stable than UpU, while at higher pH the reactivities are comparable.
- Oivanen, Mikko,Ora, Mikko,Almer, Helena,Stroemberg, Roger,Loennberg, Harri
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- EM2487, a novel anti-HIV-1 antibiotic, produced by Streptomyces sp. Mer-2487: Taxonomy, fermentation, biological properties, isolation and structure elucidation
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For the purpose of discovering novel agents that inhibit HIV-1 replication at the transcriptional level, we have established cell lines reflecting the HIV-1 long terminal repeat-driven gene expression. Using these cell lines, we have screened approximately 10,000 microorganism products and found that the culture supernatant of Streptomyces sp. Mer-2487 suppresses the HIV-1 Tat-induced gene expression without affecting the basal or tumor necrosis factor-α-induced transcription. The purified active component has a unique structure. This compound has an inhibitory effect on HIV-1 replication in chronically infected cells as well as acutely infected cells, suggesting that the inhibition occurs at a postintegration step of HIV-1 proviral DNA in the HIV-1 replication cycle.
- Takeuchi, Hitoshi,Asai, Naoki,Tanabe, Kazunori,Kozaki, Teruya,Fujita, Masanori,Sakai, Takashi,Okuda, Akifumi,Naruse, Nobuaki,Yamamoto, Satoshi,Sameshima, Tomohiro,Heida, Naohiko,Dobashi, Kazuyuki,Baba, Masanori
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- Hydrolysis and desulfurization of the diastereomeric phosphoromonothioate analogs of uridine 2',3'-cyclic monophosphate
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Hydrolyses of the two diastereomeric phosphoromonothioate analogs of uridine 2',3'-cyclic monophosphate [(R(P))- and (S(P))-2',3'-cUMPS] at 363.2 K have been followed by HPLC over pH-range 0-12. In aqueous alkali (pH > 9) only base-catalyzed endocyclic phosphoester hydrolysis to a nearly equimolar mixture of uridine 2'- and 3'-phosphoromonothioates (2'- and 3'-UMPS) takes place, analogously to the hydrolysis of uridine 2',3'-cyclic monophosphate (2',3'-cUMP). The (R(P))- and (S(P))-2',3'-cUMPS are hydrolyzed 50 and 30%, respectively, more slowly than 2',3'-cUMP. Under neutral and acidic conditions, desulfurization of the cyclic thiophosphates to 2',3'-cUMP competes with the phosphoester hydrolysis, both reactions being acid-catalyzed at pH 0.1 mol L-1), where more than 90% of the starting material is degraded via this route. At pH 5 is with the R(P)-isomer 5-fold faster than with the S(P)-isomer. In contrast to 2',3'-cUMP, depyrimidination of the starting material (i.e., release of the uracil base) competes with the hydrolysis of the thiophosphate moiety under neutral conditions (pH 6-8).
- Ora,Oivanen,Lonnberg
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- Hydrolytic stability of a phosphate-branched oligonucleotide incorporating a ribonucleoside 3′-phosphotriester unit
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A phosphate-branched oligonucleotide has been prepared by using an appropriately protected trinucleoside phosphotriester building block in conventional solid-phase synthesis. Hydrolysis of the branched oligonucleotide has been followed over a wide pH range. Comparison of the present results with those previously obtained for simpler analogues indicates that a trinucleoside 3′,3′,5′-monophosphate, when embedded in an oligonucleotide structure, is stabilized toward hydroxide-ion catalyzed cleavage by more than one order of magnitude, lending some support to the feasibility of existence of phosphate-branched RNA X in biological systems. Copyright Taylor & Francis Group, LLC.
- Loennberg, Tuomas
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- Hydrolytic reactions of an RNA dinucleotide containing a 3'-S- phosphorothiolate linkage
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The pH-rate profiles (pH 0.2 to 9 at 90°C) for the competing hydrolytic reactions of (3'-deoxy-3'-thioinosylyl)-3',5'-uridine (IspU) have been determined by an HPLC method.
- Elzagheid, Mohamed,Oivanen, Mikko,Colin,Jones,Cosstick, Richard,Loennberg, Harri
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- The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase
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The second order rate constant (kcat/Km) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping 14CO2 released during the reaction, is 2 × 10-4 M-1 s-1. This very low activity may be compared with a value of 3 × 107 M-1 s-1 for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (Ki = 7 × 10-4 M), imply an effective concentration of 1.1 × 109 M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 × 1011-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.
- Sievers, Annette,Wolfenden, Richard
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- Cytidine deaminase can deaminate fused pyrimidine ribonucleosides
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The tolerance of cytidine deaminase (CDA) to expanded heterocycles is exploredviathree fluorescent cytidine analogues, where the pyrimidine core is fused to three distinct five-membered heterocycles at the 5/6 positions. The reaction between CDA and each analogue is followed by absorption and emission spectroscopy, revealing shorter reaction times for all analogues than the native substrate. Pseudo-first order and Michaelis-Menten kinetic analyses provide insight into the enzymatic deamination reactions and assist in drawing comparison to established structure activity relationships. Finally, inhibitor screening modalities are created for each analogue and validated with zebularine and tetrahydrouridine, two known CDA inhibitors.
- Li, Yao,Ludford, Paul T.,Tor, Yitzhak,Yang, Shenghua
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- Kipukasins, nucleoside derivatives from Aspergillus versicolor
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Seven new aroyl uridine derivatives (kipukasins A-G; 1-7) were isolated from solid-substrate fermentation cultures of two different Hawaiian isolates of Aspergillus versicolor. The structures of compounds 1-7 were determined by analysis of NMR and MS data. The nucleoside portion of lead compound 1 was assigned as uracil-1-β-D-ribofuranoside by spectral comparison with an authentic standard. The bioactivity of the original A. versicolor extracts was accounted for mainly by the presence of the known metabolite sterigmatocystin, but kipukasins A and B showed modest activity in assays against Gram-positive bacteria.
- Jiao, Ping,Mudur, Sanjay V.,Gloer, James B.,Wicklow, Donald T.
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- Mechanistic studies of the 5-iodouracil chromophore relevant to its use in nucleoprotein photo-cross-linking
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The photoreactivity of the 5-iodouracil chromophore was investigated toward understanding photo-cross-linking of nucleic acids bearing the chromophore to functionality in associated proteins. Irradiation of 5-iodouridine (IU) in the presence of a 10-fold excess of N-acetyltyrosine N-ethylamide (1) at 308 nm with a XeCl excimer laser or at 325 nm with a HeCd laser yields uridine (U) and N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) in a 1:2 mole ratio. In the presence of N-acetylphenylalanine N-ethylamide, uridine and analogous ortho, meta, and para regioisomeric adducts (3o, 3m, and 3p) were formed in a similar U to adduct mole ratio. The primary photochemical process leading to products was established as carbon-iodine bond homolysis in the first excited singlet state from a deuterium labeling experiment, photoacoustic calorimetry, and quantum yield measurements. Photoreduction of IU in 2-propanol-d solvent gave U with no deuterium incorporation. Photoacoustic calorimetric measurements established that triplet benzophenone transferred energy to IU with a rate constant of 2 x 109 M-1 s-1. Further, the reaction of IU with 1 to form 2 was sensitized by benzophenone; however, comparison of quantum yields upon direct and sensitized excitation indicated that, at most, only a small portion of the reactions occurred via the triplet state. With direct excitation of IU, quantum yields as a function of the concentration of 1 showed that U and adduct 2 resulted from a common intermediate proposed to be the 5-uridinyl radical. Uridine formation was enhanced by the presence of hydrogen atom donors at the expense of formation of 2. Quantum yields were independent of excitation wavelength in the region 310-330 nm but not the reaction medium. The quantum yield of uridine formation but not adduct formation was approximately an order of magnitude higher in 90% acetonitrile - 10% water than in pH 7 water. The results are discussed in terms of high-yield cross-linking of nucleic acids bearing the 5-iodouracil chromophore to associated proteins in light of cocrystal X-ray structural data.
- Norris, Christopher L.,Meisenheimer, Poncho L.,Koch, Tad H.
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- TETRA-t-BUTOXYDISILOXANE-1,3-DIYL, A NEW TYPE OF BIFUNCTIONAL SILYL PROTECTIVE GROUP
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Tetra-t-butoxydisiloxane-1,3-diyl (TBDSi) group is introduced into nucleoside chemistry as an analogue of tetraisopropyldisiloxane-1,3-diyl (TIPDSi) and an example of a new type of bifunctional silyl protective group.
- Markiewicz, Wojciech T.,Nowakowska, Bozena,Adrych, Katarzyna
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- Pyrimidine nucleotidases/phosphotransferases from human erythrocyte
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Two cytoplasmic pyrimidine 5'-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5'- monophosphates and 3'-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5'-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.
- Amici,Emanuelli,Raffaelli,Ruggieri,Magni
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- An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir
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The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5′-acylation using Novozym 435.
- Birmingham, William R.,Burke, Ashleigh J.,Charnock, Simon J.,Crawshaw, Rebecca,Finnigan, James D.,Green, Anthony P.,Holgate, Gregory M.,Lovelock, Sarah L.,Muldowney, Mark P.,Rowles, Ian,Thorpe, Thomas W.,Turner, Nicholas J.,Young, Carl,Zhuo, Ying,Zucoloto Da Costa, Bruna
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supporting information
p. 3761 - 3765
(2022/03/15)
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- Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii
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Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/ reducing conditions) of nickel-affinity purified protein revealed the presence of a nearhomogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 × 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
- Smiley-Moreno, Elizabeth,Smith, Douglas,Yu, Jieh-Juen,Cao, Phuong,Arulanandam, Bernard P.,Chambers, James P.
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- Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19
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Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.
- Bade, Rachel,Bernardoni, Frank,Bothe, Jameson,Brito, Gilmar,Castro, Steve,Chang, Darryl,Diaz-Santana, Anthony,Diribe, Ike,Emerson, Khateeta M.,Fier, Patrick S.,Humphrey, Guy R.,Krishnamurthi, Bharath,Morris, William J.,Ouyand, Honggui,Poirier, Marc,Sirk, Kevin M.,Sirota, Eric,Stone, Kevin,Tan, Lushi,Taylor, Jerry,Ward, Michael,Xiao, Chengqian,Xu, Yingju,Zhan, Jianfeng,Zhang, Yongqian,Zhao, Ralph,Zheng, Michelle,Zompa, Michael A.
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p. 2806 - 2815
(2021/12/30)
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- The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**
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The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4–10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 106 for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.
- Kaspar, Felix,Neubauer, Peter,Kurreck, Anke
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p. 1385 - 1390
(2021/01/29)
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- Innovative 2′- O-Imino-2-propanoate-Protecting Group for Effective Solid-Phase Synthesis and 2′- O-Deprotection of RNA Sequences
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The implementation of protecting groups for the 2′-hydroxyl function of ribonucleosides is still challenging, particularly when RNA sequences must be of the highest purity for therapeutic applications as nucleic acid-based drugs. A 2′-hydroxyl-protecting group should optimally (i) be easy to install; (ii) allow rapid and efficient incorporation of the 2′-O-protected ribonucleosides into RNA sequences to minimize, to the greatest extent possible, the formation of process-related impurities (e.g., shorter than full-length sequences) during solid-phase synthesis; and (iii) be completely cleaved from RNA sequences without the production of alkylating side products and/or formation of mutagenic nucleobase adducts. The reaction of 2′-O-aminoribonucleosides with ethyl pyruvate results in the formation of stable 2′-O-imino-2-methyl propanoic acid ethyl esters and, subsequently, of the fully protected ribonucleoside phosphoramidite monomers, which are required for the solid-phase synthesis of two chimeric RNA sequences (20-mers) containing the four canonical ribonucleosides. Upon treatment of the RNA sequences with a solution of sodium hydroxide, the 2′-O-imino-2-methyl propanoic acid ethyl ester-protecting groups are saponified to their sodium salts, which after ion exchange underwent quantitative intramolecular decarboxylation under neutral conditions at 65 °C to provide fully deprotected RNA sequences in marginally better yields than those obtained from commercial 2′-O-tert-butyldimethylsilyl ribonucleoside phosphoramidites under highly similar conditions.
- Takahashi, Mayumi,Grajkowski, Andrzej,Cawrse, Brian M.,Beaucage, Serge L.
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p. 4944 - 4956
(2021/04/06)
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- Meteorite-catalyzed intermoleculartrans-glycosylation produces nucleosides under proton beam irradiation
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Di-glycosylated adenines act as glycosyl donors in the intermoleculartrans-glycosylation of pyrimidine nucleobases under proton beam irradiation conditions. Formamide and chondrite meteorite NWA 1465 increased the yield and the selectivity of the reaction
- Bizzarri, Bruno Mattia,Fanelli, Angelica,Kapralov, Michail,Krasavin, Eugene,Saladino, Raffaele
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p. 19258 - 19264
(2021/06/03)
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- A short de novo synthesis of nucleoside analogs
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Nucleoside analogs are commonly used in the treatment of cancer and viral infections. Their syntheses benefit from decades of research but are often protracted, unamenable to diversification, and reliant on a limited pool of chiral carbohydrate starting materials. We present a process for rapidly constructing nucleoside analogs from simple achiral materials. Using only proline catalysis, heteroaryl-substituted acetaldehydes are fluorinated and then directly engaged in enantioselective aldol reactions in a one-pot reaction. A subsequent intramolecular fluoride displacement reaction provides a functionalized nucleoside analog. The versatility of this process is highlighted in multigram syntheses of D- or L-nucleoside analogs, locked nucleic acids, iminonucleosides, and C2′- and C4′-modified nucleoside analogs. This de novo synthesis creates opportunities for the preparation of diversity libraries and will support efforts in both drug discovery and development.
- Adluri, Bharanishashank,Britton, Robert,Campeau, Louis-Charles,Cohen, Ryan,Lehmann, Johannes,Meanwell, Michael,Silverman, Steven M.,Wang, Yang
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p. 725 - 730
(2020/09/02)
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- Thermodynamic Reaction Control of Nucleoside Phosphorolysis
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Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose-1-phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase-catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate-specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature-dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis. (Figure presented.).
- Kaspar, Felix,Giessmann, Robert T.,Neubauer, Peter,Wagner, Anke,Gimpel, Matthias
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p. 867 - 876
(2020/01/24)
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- Use of nucleoside phosphorylases for the preparation of 5-modified pyrimidine ribonucleosides
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Enzymatic transglycosylation, a transfer of the carbohydrate moiety from one heterocyclic base to another, is catalyzed by nucleoside phosphorylases (NPs) and is being actively developed and applied for the synthesis of biologically important nucleosides. Here, we report an efficient one-step synthesis of 5-substitited pyrimidine ribonucleosides starting from 7-methylguanosine hydroiodide in the presence of nucleoside phosphorylases (NPs).
- Alexeev, Cyril S.,Drenichev, Mikhail S.,Dorinova, Evgeniya O.,Esipov, Roman S.,Kulikova, Irina V.,Mikhailov, Sergey N.
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- DIVERSE AND FLEXIBLE CHEMICAL MODIFICATION OF NUCLEIC ACIDS
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The present invention provides a method for chemically modifying a nucleic acid molecule using sulfinate reagents to increase stability in vitro and in vivo. Screening methods for nucleobase modifications that reduce cleavage of a nucleic acid molecule by a nuclease are also provided.
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Paragraph 0124-0126; 0131; 0134
(2020/05/12)
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- Total synthesis of 2'-O-methyl-β-L-arabinosyluridine and reassignment the nucleoside from penicillium sp. as 2'-O-methyl-β-L-uridine
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In order to validate the structure of a rarely reported naturally occurring nucleoside isolated from the broth of Penicillium sp. (NO. 64), practical syntheses of 2′-O-methyl-β-L-arabinosyluridine, 2′-O-methyl-α-L-arabinosyluridine, and 2′-O-methyl-β-L-uridine were accomplished. Comparing their nuclear magnetic resonance (NMR) spectra and physical data, its structure was reassigned as 2′-O-methyl-β-L-uridine instead of former reported 2′-O-methyl-β-L-arabinosyluridine.
- Shen, Chunyang,Ding, Haixin,Tao, Xueping,Yang, Ruchun,Bai, Jiang,Cao, Ban-Peng,Peng, Yi-Yuan,Xiao, Qiang
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- Dehalogenation of Halogenated Nucleobases and Nucleosides by Organoselenium Compounds
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Halogenated nucleosides, such as 5-iodo-2′-deoxyuridine and 5-iodo-2′-deoxycytidine, are incorporated into the DNA of replicating cells to facilitate DNA single-strand breaks and intra- or interstrand crosslinks upon UV irradiation. In this work, it is shown that the naphthyl-based organoselenium compounds can mediate the dehalogenation of halogenated pyrimidine-based nucleosides, such as 5-X-2′-deoxyuridine and 5-X-2′-deoxycytidine (X=Br or I). The rate of deiodination was found to be significantly higher than that of the debromination for both nucleosides. Furthermore, the deiodination of iodo-cytidines was found to be faster than that of iodo-uridines. The initial rates of the deiodinations of 5-iodocytosine and 5-iodouracil indicated that the nature of the sugar moiety influences the kinetics of the deiodination. For both the nucleobases and nucleosides, the deiodination and debromination reactions follow a halogen-bond-mediated and addition/elimination pathway, respectively.
- Mondal, Santanu,Mugesh, Govindasamy
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p. 1773 - 1780
(2019/01/10)
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- Identification of Flavin Mononucleotide as a Cell-Active Artificial N6-Methyladenosine RNA Demethylase
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N6-Methyladenosine (m6A) represents a common and highly dynamic modification in eukaryotic RNA that affects various cellular pathways. Natural dioxygenases such as FTO and ALKBH5 are enzymes that demethylate m6A residues in mRNA. Herein, the first identification of a small-molecule modulator that functions as an artificial m6A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m6A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m6A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology.
- Xie, Li-Jun,Yang, Xiao-Ti,Wang, Rui-Li,Cheng, Hou-Ping,Li, Zhi-Yan,Liu, Li,Mao, Lanqun,Wang, Ming,Cheng, Liang
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supporting information
p. 5028 - 5032
(2019/03/17)
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- Effects of Pressure and pH on the Hydrolysis of Cytosine: Implications for Nucleotide Stability around Deep-Sea Black Smokers
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The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil remains unknown. Through in situ NMR spectroscopy measurements, it has been determined that the half-life of cytosine at 373.15 K decreases from (18.0±0.7) days at ambient pressure (0.1 MPa) to (8.64±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of (?11.8±0.5) cm3 mol?1; a decrease that is similar to the molar volume of water (18.0 cm3 mol?1) and consistent with a tetrahedral 3,3-hydroxyamine transition-state/intermediate species. Similar behaviour was also observed for cytidine. At both ambient and high pressures, the half-life of cytosine decreases significantly as the pH decreases from 7.0 to 6.0. These results provide scant support for the notion that RNA-based life forms originated in high-temperature, high-pressure, acidic environments.
- Lepper, Christopher P.,Williams, Martin A. K.,Penny, David,Edwards, Patrick J. B.,Jameson, Geoffrey B.
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p. 540 - 544
(2018/03/30)
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- NUCLEOSIDE OR DERIVATIVE OF THE NUCLEOTIDE, RNA DERIVATIVE CONTAINING THE SAME AS CONSTITUTIONAL UNIT, NUCLEIC ACID MEDICINE, AND METHOD FOR PRODUCING RNA DERIVATIVE OR RNA
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PROBLEM TO BE SOLVED: To provide a nucleoside in which 2'-position hydroxy group is protected or a nucleotide derivative thereof in which RNA synthesis efficiency is satisfactory and can be deprotect under a mild condition, and purification after deprotection is easy. SOLUTION: There is provide a nucleoside expressed by formula (1) or a derivative of the nucleotide. (X1 and X2 severally independently denote H, substituted/unsubstituted silyl group, 4-methoxytrityl group, 4,4'-dimethoxytrityl group or the like; Ba denotes modified/unmodified nucleic acid base; a formula (P1) is exemplified as Rp; R denotes substituent having electron-donating property such as methoxy group; R1 and R2 severally independently denote H or alkyl group). SELECTED DRAWING: Figure 2 COPYRIGHT: (C)2019,JPOandINPIT
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- SOLID-PHASE PURIFICATION OF SYNTHETIC NUCLEIC ACID SEQUENCES
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The invention provides a compound of the formula (I), and a capture support of the formula (9), wherein R1, R2, R3, R6, A, B, D, E, J, K, Q, W, and Z are as defined herein. The invention also provides a method of purifying an oligonucleotide or an oligonucleotide analog composed of "b" nucleotides from a mixture comprising the oligonucleotide or oligonucleotide analog and at least one oligonucleotide or oligonucleotide analog composed of "a" nucleotides, wherein b ≠ a, comprising use of the compound and the capture support.
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- Synthesis of Nucleosides through Direct Glycosylation of Nucleobases with 5-O-Monoprotected or 5-Modified Ribose: Improved Protocol, Scope, and Mechanism
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Simplifying access to synthetic nucleosides is of interest due to their widespread use as biochemical or anticancer and antiviral agents. Herein, a direct stereoselective method to access an expansive range of both natural and synthetic nucleosides up to a gram scale, through direct glycosylation of nucleobases with 5-O-tritylribose and other C5-modified ribose derivatives, is discussed in detail. The reaction proceeds through nucleophilic epoxide ring opening of an in situ formed 1,2-anhydrosugar (termed “anhydrose”) under modified Mitsunobu reaction conditions. The scope of the reaction in the synthesis of diverse nucleosides and other 1-substituted riboside derivatives is described. In addition, a mechanistic insight into the formation of this key glycosyl donor intermediate is provided.
- Downey, A. Michael,Pohl, Radek,Roithová, Jana,Hocek, Michal
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supporting information
p. 3910 - 3917
(2017/03/27)
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- Enzymatic synthesis of ribo- and 2′-deoxyribonucleosides from glycofuranosyl phosphates: An approach to facilitate isotopic labeling
-
Milligram quantities of α-D-ribofuranosyl 1-phosphate (sodium salt) (αR1P) were prepared by the phosphorolysis of inosine, catalyzed by purine nucleoside phosphorylase (PNPase). The αR1P was isolated by chromatography in >95% purity and characterized by 1H and 13C NMR spectroscopy. Aqueous solutions of αR1P were stable at pH 6.4 and 4 °C for several months. The isolated αR1P was N-glycosylated with different nitrogen bases (adenine, guanine and uracil) using PNPase or uridine phosphorylase (UPase) to give the corresponding ribonucleosides in high yield based on the glycosyl phosphate. This methodology is attractive for the preparation of stable isotopically labeled ribo- and 2′-deoxyribonucleosides because of the ease of product purification and convenient use and recycling of nitrogen bases. The approach eliminates the need for separate reactions to prepare individual furanose-labeled ribonucleosides, since only one ribonucleoside (inosine) needs to be labeled, if desired, in the furanose ring, the latter achieved by a high-yield chemical N-glycosylation. 2′-Deoxyribonucleosides were prepared from 2′-deoxyinosine using the same methodology with minor modifications.
- Zhang, Wenhui,Turney, Toby,Surjancev, Ivana,Serianni, Anthony S.
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p. 125 - 133
(2017/08/08)
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- Deprotection of silyl ethers by using SO3H silica gel: Application to sugar, nucleoside, and alkaloid derivatives
-
We applied a desilylation procedure using SO3H silica gel, with the surface modified by alkylsulfonic acid groups, to silylated sugar, nucleoside, and alkaloid derivatives. The treatment with SO3H silica gel provided desilylated products in good to excellent yield. In the reactions of sugar and nucleoside derivatives, no silyl residue was detected in the crude products, but the crude products of the reaction of alkaloids contained small amounts of silyl residues. Even though the sugar and nucleoside derivatives had a labile glycosyl and C–N bond, respectively, these bonds tolerated the reaction conditions. These outcomes suggested that the desilylation procedure using SO3H silica gel would be applicable to the deprotection of a variety of types of compounds protected by silyl groups. In a gram scale experiment, the desilylation procedure successfully proceeded without the observation of any silyl residue in the crude product.
- Fujii, Hideaki,Shimada, Naoyuki,Ohtawa, Masaki,Karaki, Fumika,Koshizuka, Masayoshi,Hayashida, Kohei,Kamimura, Mitsuhiro,Makino, Kazuishi,Nagamitsu, Tohru,Nagase, Hiroshi
-
p. 5425 - 5429
(2017/08/11)
-
- Cleavage of short oligoribonucleotides by a Zn2+binding multi-nucleating azacrown conjugate
-
A multi-nucleating azacrown conjugate (5a) consisting of two 3,5-bis(1,5,9-triazacyclododecan-3-yloxymethyl)benzyl groups attached to 1 and 7 sites of cyclen was prepared and tested as an artificial ribonuclease. The conjugate in the presence of five equivalents of zinc nitrate expectedly showed uridine selectivity comparable to that 1,3,5-tris(1,5,9-triazacyclododecan-3-yl)benzene (2), a compound known to bind to two adjacent uridine residues and cleave the intervening phosphodiester bond. 5a was, however, unable to discriminate between two and three adjacent uridine residues, but cleaved oligonucleotides containing a UpU and UpUpU site at a comparable rate, even when present at sub-saturating concentrations.
- Laine, Maarit,L?nnberg, Tuomas,Helkearo, Mia,L?nnberg, Harri
-
p. 111 - 117
(2016/10/04)
-
- DEAMINATION OF ORGANOPHOSPHORUS-NUCLEOSIDES
-
The invention relates to a new synthethic process for obtaining compounds of formula (I) from compounds of formula (II) by means of cytidine deaminase enzymes.
- -
-
Page/Page column 28; 29
(2016/10/24)
-
- 3-methyl uridine and 4-methyl cytidine nucleoside compound, synthetic method and its pharmaceutical use
-
The invention discloses a 3-methyluridine and 4-methylcytidine nucleosides compound and a synthesis method and pharmaceutical application thereof, belonging to the field of medicinal chemistry. The compound has a structural formula as shown in the specification. The compound has the effects of simultaneously modifying sugar rings and basic groups, increasing the activity of the compound and reducing the toxicity, provides a good application prospect for development of like medicines and can be applied to preparation of anti-HBV (Hepatitis B virus) medicines. The synthesis method is simple and feasible and provides conditions for mass synthesis of the compound.
- -
-
Paragraph 0021; 0033
(2016/10/08)
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- Chiral Nanozymes-Gold Nanoparticle-Based Transphosphorylation Catalysts Capable of Enantiomeric Discrimination
-
Enantioselectivity in RNA cleavage by a synthetic metalloenzyme has been demonstrated for the first time. Thiols containing chiral ZnII-binding head groups have been self-assembled on the surface of gold nanoparticles. This results in the spontaneous formation of chiral bimetallic catalytic sites that display different activities (kcat) towards the enantiomers of an RNA model substrate. Substrate selectivity is observed when the nanozyme is applied to the cleavage of the dinucleotides UpU, GpG, ApA, and CpC, and remarkable differences in reactivity are observed for the cleavage of the enantiomerically pure dinucleotide UpU.
- Chen, Jack L.-Y.,Pezzato, Cristian,Scrimin, Paolo,Prins, Leonard J.
-
supporting information
p. 7028 - 7032
(2016/05/19)
-
- Protection of the 2′-Hydroxy Function of Ribonucleosides as an Iminooxymethyl Propanoate and Its 2′-O-Deprotection through an Intramolecular Decarboxylative Elimination Process
-
The design and implementation of 2′-hydroxy protecting groups for ribonucleosides is still a daunting challenge to overcome when assembling RNA (ribonucleic acid) sequences for therapeutic applications. The reaction of 2′-O-aminooxymethylribonucleosides with ethyl pyruvate results in the formation of 2′-O-iminooxymethyl ethyl propanoates. The cleavage of this type of 2′-O-protecting groups is demonstrated through saponification of the esters to 2′-O-iminooxymethyl propanoate salts, which, when needed, decarboxylate quantitatively at 55 °C in the presence of tetra-n-butylammonium fluoride or chloride in dimethyl sulfoxide (DMSO) to produce all four native ribonucleosides.
- Cie?lak, Jacek,Grajkowski, Andrzej,Ausín, Cristina,Beaucage, Serge L.
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p. 5817 - 5821
(2016/12/18)
-
- PRODUCTION METHOD OF NUCLEOSIDE COMPOUND
-
PROBLEM TO BE SOLVED: To provide a production method of a nucleoside compound by which an isotopic labeled nucleoside compound can be produced efficiently. SOLUTION: A production method of a nucleoside compound comprises obtaining a target nucleoside compound by the base exchange reaction of a raw material nucleoside compound and a base in the solution containing a phosphoric acid ion by a nucleoside phosphorylase, wherein the target nucleoside compound is labeled with a stable isotope or a radioisotope. COPYRIGHT: (C)2015,JPOandINPIT
- -
-
Paragraph 0032-0033
(2017/03/24)
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- Catalytic isomerization of allyl functionalities in water by hexaaquaruthenium(II) tosylate
-
The water-soluble coordination compound hexaaquaruthenium(II) p-toluene sulfonate (1) catalyzes olefin isomerization that makes it a useful catalyst in unmasking allyl ether and ester protecting groups in water. Allyl ethers of alcohols and acetic acid allyl ester are readily converted to the corresponding alcohols and acid in a catalytic fashion with compound 1 in water (50°C). A mechanistic investigation on ethylene glycol monoallyl ether reveals the intermediate vinyl ether resulting from olefn isomerization (ΔH? = 19.0 (±0.4) kcal/mol). This is followed by hydrolysis to the final ethylene glycol that is promoted by 1. This "one-pot" reaction provides a new useful coordination compound as a deprotection reagent in synthetic organic chemistry.
- Kuo, Louis Y.,Delaney, Frances E.
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p. 335 - 339
(2015/08/18)
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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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- Ribonuclease activity of an artificial catalyst that combines a ligated CuII ion and a guanidinium group at the upper rim of a cone -Calix[4]arene platform
-
A cone-calix[4]arene derivative, featuring a guanidinium group and a CuII ion ligated to a 1,4,7-triazacyclononane (TACN) ligand at the 1,3-distal positions of the upper rim, effectively catalyzes the cleavage of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) and a number of diribonucleoside 3′,5′-monophosphates (NpN′). Kinetic and potentiometric measurements support the operation of a general-base/general-acid mechanism and demonstrate that the hydroxo form of the ligated CuII ion is the sole catalytically active species. Rate enhancements relative to the background hydrolysis reaction at 1 mM catalyst concentration are 6 × 105-fold for HPNP and cluster around 107-fold with the most favorable catalyst-NpN′ combinations.
- Salvio, Riccardo,Volpi, Stefano,Cacciapaglia, Roberta,Casnati, Alessandro,Mandolini, Luigi,Sansone, Francesco
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p. 5887 - 5893
(2015/06/16)
-
- METHOD FOR PROTECTING HYDROXYL OR AMINE OR THIOL FUNCTIONS, NOVEL COMPOUNDS WITH PROTECTED HYDROXYL OR AMINE OR THIOL GROUPS, AS WELL NOVEL COMPOUNDS FOR THE IMPLEMENTATION OF THIS METHOD
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A novel method of simultaneously protecting two functions which are same or different, namely hydroxyl, amine, or thiol ones, particularly in sugars, polyalcohols, nucleosides, nucleotides, peptides, and nucleic acids during an organic synthesis, and to novel compounds for implementing this method, as well as to the method of obtaining these compounds. Method of simultaneously protecting two hydroxyl, amine, or thiol functions according to the invention by carrying out a protecting reaction between a compound having at least two free hydroxyl, amine, or thiol groups, and the disilane of formula 1, where R stands for Cl or Br, or I, or a substituent of formula 2, where X1, X2, X3, X4 are the same or different.
- -
-
Paragraph 0112
(2015/04/15)
-
- The influence of the C5 substituent on the 2-thiouridine desulfuration pathway and the conformational analysis of the resulting 4-pyrimidinone products
-
In recent years, increasing attention has been focused on the posttranscriptional modifications present in transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which are located in the wobble position of the anticodon and play a fundamental role in the tuning of the translation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-dependent. In this study, for the first time, we demonstrate that the substituent at the C5 position of the 2-thiouracil ring of R5S2Us influences the desulfuration pathway, and thus the products ratio. As the substituent R changes, the amount of R5H2U increases in the order H- > CH3O- > CH3OC(O)CH2- > HOC(O)CH2NHCH2- ≈ CH3NHCH2-, and this effect is more pronounced at lower pH. The conformational analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent, the R5H2U nucleosides predominantly adopt a C2′-endo sugar ring conformation, as opposed to the preferred C3′-endo conformation of the parent R5S2Us.
- Bartos, Paulina,Ebenryter-Olbinska, Katarzyna,Sochacka, Elzbieta,Nawrot, Barbara
-
supporting information
p. 5587 - 5594
(2015/11/11)
-
- Direct One-Pot Synthesis of Nucleosides from Unprotected or 5-O-Monoprotected d -Ribose
-
New, improved methods to access nucleosides are of general interest not only to organic chemists but to the greater scientific community as a whole due their key implications in life and disease. Current synthetic methods involve multistep procedures employing protected sugars in the glycosylation of nucleobases. Using modified Mitsunobu conditions, we report on the first direct glycosylation of purine and pyrimidine nucleobases with unprotected d-ribose to provide β-pyranosyl nucleosides and a one-pot strategy to yield β-furanosides from the heterocycle and 5-O-monoprotected d-ribose.
- Downey, A. Michael,Richter, Celin,Pohl, Radek,Mahrwald, Rainer,Hocek, Michal
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supporting information
p. 4604 - 4607
(2015/09/28)
-
- Buffer catalyzed cleavage of uridylyl-3′,5′-uridine in aqueous DMSO: Comparison to its activated analog, 2-hydroxypropyl 4-nitrophenyl phosphate
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Buffer catalysis of the cleavage and isomerization of uridylyl-3′,5′-uridine (UpU) has been studied over a wide pH range in 80% aq. DMSO. The diminished hydroxide ion concentration in this solvent system made catalysis by amine buffers (morpholine, 4-hydroxypiperidine and piperidine) visible even at relatively low buffer concentrations (10-200 mmol L-1). The observed catalysis was, however, much weaker than what has been previously reported for the activated RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) in the same solvent system. In the case of morpholine, contribution of both the acidic and the basic buffer constituent was significant, whereas with 4-hydroxypiperidine and piperidine participation of the acidic constituent could not be established unambiguously. The results underline the importance of using realistic model compounds, along with activated ones, in the study of the general acid/base catalysis of RNA cleavage.
- Lain,L?nnberg,L?nnberg
-
p. 3484 - 3492
(2015/03/18)
-
- The activating oxydianion binding domain for enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation: Specificity and enzyme architecture
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The kinetic parameters for activation of yeast triosephosphate isomerase (ScTIM), yeast orotidine monophosphate decarboxylase (ScOMPDC), and human liver glycerol 3-phosphate dehydrogenase (hlGPDH) for catalysis of reactions of their respective phosphodianion truncated substrates are reported for the following oxydianions: HPO32-, FPO32-, S2O32-, SO42- and HOPO32-. Oxydianions bind weakly to these unliganded enzymes and tightly to the transition state complex (E?S?), with intrinsic oxydianion Gibbs binding free energies that range from -8.4 kcal/mol for activation of hlGPDH-catalyzed reduction of glycolaldehyde by FPO32- to -3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-β-D-erythrofuranosyl)orotic acid by HOPO32-. Small differences in the specificity of the different oxydianion binding domains are observed. We propose that the large -8.4 kcal/mol and small -3.8 kcal/mol intrinsic oxydianion binding energy for activation of hlGPDH by FPO32- and S2O32-, respectively, compared with activation of ScTIM and ScOMPDC reflect stabilizing and destabilizing interactions between the oxydianion -F and -S with the cationic side chain of R269 for hlGPDH. These results are consistent with a cryptic function for the similarly structured oxydianion binding domains of ScTIM, ScOMPDC and hlGPDH. Each enzyme utilizes the interactions with tetrahedral inorganic oxydianions to drive a conformational change that locks the substrate in a caged Michaelis complex that provides optimal stabilization of the different enzymatic transition states. The observation of dianion activation by stabilization of active caged Michaelis complexes may be generalized to the many other enzymes that utilize substrate binding energy to drive changes in enzyme conformation, which induce tight substrate fits. (Table Presented).
- Reyes, Archie C.,Zhai, Xiang,Morgan, Kelsey T.,Reinhardt, Christopher J.,Amyes, Tina L.,Richard, John P.
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p. 1372 - 1382
(2015/02/19)
-
- An efficient approach for conversion of 5-substituted 2-thiouridines built in RNA oligomers into corresponding desulfured 4-pyrimidinone products
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Abstract An efficient approach for the desulfuration of C5-substituted 2-thiouridines (R5S2U) bound in the RNA chain exclusively to 4-pyrimidinone nucleoside (R5H2U)-containing RNA products is proposed. This post-synthetic transformation avoids the preparation of a suitably protected H2U phosphoramidite, which otherwise would be necessary for solid-phase synthesis of the modified RNA. Optimization of the desulfuration, which included reaction stoichiometry, time and temperature, allowed to transform a set of ten R5S2U-RNAs into their R5H2U-RNA congeners in ca. 90% yield.
- Chwialkowska, Anna,Wielgus, Ewelina,Leszczynska, Grazyna,Sobczak, Milena,Mikolajczyk, Barbara,Sochacka, Elzbieta,Nawrot, Barbara
-
supporting information
p. 3100 - 3104
(2015/07/08)
-
- Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway
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Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA. Saying goodbye to I: Selenium compounds are employed to mediate the dehalogenation of halogenated nucleobases and nucleosides in aqueous media under physiological conditions. These results may be important for the development of novel reagents for DNA modification and repair and suggest that Se derivatives may play a broader role in the metabolism of halogenated organic compounds in biology.
- Mondal, Santanu,Manna, Debasish,Mugesh, Govindasamy
-
supporting information
p. 9298 - 9302
(2015/08/06)
-
- Guanidine based self-assembled monolayers on Au nanoparticles as artificial phosphodiesterases
-
Gold nanoparticles passivated with a long chain alkanethiol decorated with a phenoxyguanidine moiety were prepared and investigated as catalysts in the cleavage of the RNA model compound HPNP and diribonucleoside monophosphates. The catalytic efficiency and the high effective molarity value of the Au monolayer protected colloids points to a high level of cooperation between the catalytic groups.
- Salvio, Riccardo,Cincotti, Antonio
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p. 28678 - 28682
(2014/07/22)
-
- Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata
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Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2′-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2′-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-d-arabinofuranoside than in deaminating 2′-deoxycytinde to 2′-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2′-deoxycytidine and 63 μM and 2.072 mM for cytosine β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.
- Shibu, Marthandam Asokan,Yang, Hsueh-Hui,Lo, Chaur-Tsuen,Lin, Hong-Shin,Liu, Shu-Ying,Peng, Kou-Cheng
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p. 1796 - 1801
(2014/03/21)
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- METHOD FOR INCORPORATING PROTECTING ACETAL AND ACETAL ESTER GROUPS, AND ITS APPLICATION FOR THE PROTECTION OF HYDROXYL FUNCTION
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The present invention is the method for incorporation of acetal and acetal ester groups for protection of hydroxyl function. The method is applied in particular in the processes of RNA synthesis. The method can be employed in the synthesis of nucleosides with acetal and acetal ester groups for the protection of hydroxyl functions. The method according to the invention consists of the reaction of an organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1, R1 -S-CH2 -O-R2(1) in the presence of SnCl4, in an aprotic solvent. In the second aspect, the present invention is the method of protecting the hydroxyl function, particularly in position 2', in nucleoside derivatives, based on the incorporation of an acetal or acetal ester group.
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Page/Page column 31
(2014/10/04)
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- In(III) triflate-catalyzed detritylation and glycosylation by solvent-free ball milling
-
A highly efficient In(III) triflate-assisted method for the detritylation of O-trityl derivatives of carbohydrates, phenols, and alcohols using solvent-free mechanochemical method is described. In the case of carbohydrates, further reaction in the presence of an acceptor sugar leads to highly efficient glycosylation in the same pot resulting in the formation of the desired glycoside-product in very high yields. The method was applied successfully to the synthesis of a combinatorial library of galactose-based (1,6)-linked cyclohexa-, hepta-, and octasaccharides on gram scale.
- Kumar, Vajinder,Yadav, Narender,Kartha, K.P. Ravindranathan
-
-
- Diguanidinocalix[4]arenes as effective and selective catalysts of the cleavage of diribonucleoside monophosphates
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Calix[4]arenes derivatives 1 and 2, featuring two guanidine units at the upper rim, catalyze the transesterification of diribonucleoside monophosphates much more effectively than that of HPNP. Rate accelerations relative to the background range from 10su
- Salvio, Riccardo,Cacciapaglia, Roberta,Mandolini, Luigi,Sansone, Francesco,Casnati, Alessandro
-
p. 34412 - 34416
(2014/11/12)
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- H-Bond activated glycosylation of nucleobases: Implications for prebiotic nucleoside synthesis
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Glycosylation of nucleobases is achieved by heating metal free aqueous solution of nucleobase and sugar. It seems that abstraction of N 9/N1 H by C1′-OH promotes N 9/N1(nucleobase)-C1′ (sug
- Singh, Palwinder,Singh, Amrinder,Kaur, Jagroop,Holzer, Wolfgang
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p. 3158 - 3161
(2014/01/06)
-
- METHOD FOR PROTECTING HYDROXYL, AMINE OR THIOL FUNCTIONAL GROUPS WITH TETRAISOPROPYDISILANE AND THE CORRESPONDING PROTECTED COMPOUNDS
-
The invention relates to a method of simultaneously protecting two functions which are the same or different, namely hydroxyl, amine, or thiol groups, particularly in sugars, polyalcohols, nucleosides, nucleotides, peptides, and nucleic acids and to compounds protected using this method. The method of simultaneously protecting two hydroxyl, amine, or thiol functions according to the invention is carried out by reacting the two functional groups with the disilane of formula 1, thereby forming a disilane-1,2-diyl residue, where R stands for CI or Br, or I, or a substituent of formula 2, where X1, X 2, X 3, X 4 are the same or different and are N, CH or C-R1.
- -
-
Page/Page column 20; 21
(2013/11/19)
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- Desulfuration of 2-thiouridine with hydrogen peroxide in the physiological pH range 6.6-7.6 is pH-dependent and results in two distinct products
-
The 2-thiomodified nucleosides, located at first position of tRNAs anticodon, may constitute a primary target for oxidative attack under conditions of oxidative stress. Desulfuration of 2-thiouridine (S2U) was investigated in the 1H NMR scale in the presence of 100 mM H2O2 and phosphate buffer in the physiological pH range, from pH 6.6 to 7.6. The obtained data demonstrate an intriguing result that within one unit of the pH range uridine is the major product of the S2U desulfuration in the pH 7.6, while the 4-pyrimidinone nucleoside (H2U) is dominant in pH 6.6. The possible desulfuration pathway and the biological importance of the transformation of S2U either to U or H2U are discussed in the context of the tRNA oxidative damage.
- Sochacka, Elzbieta,Bartos, Paulina,Kraszewska, Karina,Nawrot, Barbara
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supporting information
p. 5803 - 5805
(2013/10/22)
-
- An RNA modification with remarkable resistance to RNase A
-
A 3′-deoxy-3′-C-methylenephosphonate modified diribonucleotide is highly resistant to degradation by spleen phosphodiesterase and not cleaved at all by snake venom phosphodiesterase. The most remarkable finding is that, despite the fact that both the vicinal 2-hydroxy nucleophile and the 5′-oxyanion leaving group are intact, the 3′-methylenephosponate RNA modification is also highly resistant towards the action of RNase A.
- Ghidini, Alice,Ander, Charlotte,Winqvist, Anna,Stroemberg, Roger
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supporting information
p. 9036 - 9038
(2013/09/24)
-
- Developing a collection of immobilized nucleoside phosphorylases for the preparation of nucleoside analogues: Enzymatic synthesis of arabinosyladenine and 2',3'-dideoxyinosine
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The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly expressed nucleoside phosphorylases from the bacterial pathogens Citrobacter koseri, Clostridium perfringens, and Streptococcus pyogenes (CkPNPI, CkPNPII, CpUP, SpUP) and their substrate specificity was investigated towards either natural pyrimidine or purine nucleosides and some analogues, namely, arabinosyladenine (araA) and 2',3'-dideoxyinosine (ddI). A 2-3 % activity towards these latter compounds (compared to the natural substrates) was observed. Enzyme activities were compared to the specificities obtained for the enzymes pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP) and purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNPII) previously reported by some of the authors. The enzymes displaying the suitable specificity for the synthesis of araA and ddI were immobilized on aldehyde-agarose. The immobilized preparations were highly stable at alkaline pH and in the presence of methanol or acetonitrile as cosolvent. They were used in the synthesis of araA and ddI by a one-pot, bienzymatic transglycosylation achieving 74 and 44 % conversion, respectively. Something different: Nucleoside phosphorylases are a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. Four new nucleoside phosphorylases have been prepared, characterized, and tested for their use in biocatalyzed syntheses of araA and ddI (see scheme). A generally applicable immobilization technique has been found to provide active and stable biocatalysts.
- Serra, Immacolata,Ubiali, Daniela,Piskur, Jure,Christoffersen, Stig,Lewkowicz, Elizabeth S.,Iribarren, Adolfo M.,Albertini, Alessandra M.,Terreni, Marco
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p. 157 - 165
(2013/04/24)
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- The 2-cyano-2,2-dimethylethanimine-N-oxymethyl group for the 2′-hydroxyl protection of ribonucleosides in the solid-phase synthesis of RNA sequences
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The reaction of 2-cyano-2-methyl propanal with 2′-O- aminooxymethylribonucleosides leads to stable and yet reversible 2′-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides. Following N-protection of the nucleobases, 5′-dimethoxytritylation and 3′-phosphitylation, the resulting 2′-protected ribonucleoside phosphoramidite monomers are employed in the solid-phase synthesis of three chimeric RNA sequences, each differing in their ratios of purine/pyrimidine. When the activation of phosphoramidite monomers is performed in the presence of 5-benzylthio-1H-tetrazole, coupling efficiencies averaging 99 % are obtained within 180 s. Upon completion of the RNA-chain assemblies, removal of the nucleobase and phosphate protecting groups and release of the sequences from the solid support are carried out under standard basic conditions, whereas the cleavage of 2′-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl) protective groups is effected (without releasing RNA alkylating side-products) by treatment with tetra-n-butylammonium fluoride (0.5 m) in dry DMSO over a period of 24-48 h at 55 °C. Characterization of the fully deprotected RNA sequences by polyacrylamide gel electrophoresis (PAGE), enzymatic hydrolysis, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry confirmed the identity and quality of these sequences. Thus, the use of 2′-O-aminooxymethylribonucleosides in the design of new 2′-hydroxyl protecting groups is a powerful approach to the development of a straightforward, efficient, and cost-effective method for the chemical synthesis of high-quality RNA sequences in the framework of RNA interference applications. Copyright
- Cie?lak, Jacek,Ausín, Cristina,Grajkowski, Andrzej,Beaucage, Serge L.
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supporting information
p. 4623 - 4632
(2013/04/24)
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- Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis, biochemical, and structural evaluation
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Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low μM inhibition constant (Ki) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(β-d-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with Ki = 1.6 μM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B1 subsite while the triazole moiety binds at the main subsite P1, where P-O5′ bond cleavage occurs, and the ribose at the interface between subsites P1 and P0 exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A - ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential.
- Parmenopoulou, Vanessa,Chatzileontiadou, Demetra S.M.,Manta, Stella,Bougiatioti, Stamatina,Maragozidis, Panagiotis,Gkaragkouni, Dimitra-Niki,Kaffesaki, Eleni,Kantsadi, Anastassia L.,Skamnaki, Vassiliki T.,Zographos, Spyridon E.,Zounpoulakis, Panagiotis,Balatsos, Nikolaos A.A.,Komiotis, Dimitris,Leonidas, Demetres D.
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p. 7184 - 7193
(2013/01/15)
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- 2'-O-AMINOOXYMETHYL NUCLEOSIDE DERIVATIVES FOR USE IN THE SYNTHESIS AND MODIFICATION OF NUCLEOSIDES, NUCLEOTIDES AND OLIGONUCLEOTIDES
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Disclosed are O-protected compounds of the formula (I):wherein B is an optionally protected nucleobase, and R1-R3 are as described herein, a method of preparing such compounds, and a method of preparing oligonucleotides such as RNA starting from such compounds. The O-protected compounds have one or more advantages, for example, the 2'-O-protected compound is stable during the various reaction steps involved in oligonucleotide synthesis; the protecting group can be easily removed after the synthesis of the oligonucleotide, for example, by reaction with tetrabutylammonium fluoride; and/or the O-protected groups do not generate DNA/RNA alkylating side products, which have been reported during removal of 2'-O-(2-cyanoethyl)oxymethyl or 2'-O-[2-(4-tolylsulfonyl)ethoxymethyl groups under similar conditions.
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Page/Page column 8/13
(2012/10/18)
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- Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase
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UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH2p) yielding uridine 5′-(β, γ-methylenetriphosphate) (UppCH2p). The f
- Guenther Sillero, Maria Antonia,De Diego, Anabel,Sillero, Antonio
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experimental part
p. 1871 - 1875
(2012/09/22)
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