- Synthetic method of nucleoside tetraphosphate
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The invention discloses a synthetic method of nucleoside tetraphosphate. The synthetic method comprises the steps of carrying out selective phosphorylation reaction by virtue of nucleoside and a cyclic phosphorylation reagent, and carrying out oxidation and hydrolysis loop opening, so as to obtain nucleoside tetraphosphate. The structure of the cyclic phosphorylation reagent is represented by a formula I (shown in the description). According to the synthetic method, 5'-nucleoside tetraphosphate is selectively generated from nucleoside under the effect of the high-selectivity phosphorylation reagent, and 3'-OH (and 2'-OH) does not need to be protected in the process, namely that the generaiton of 3'(and 2'-)tetraphosphate can be effectively inhibited. Nucleoside tetraphosphate synthesized by virtue of the method has wide use ranges in the biology fields of DNA sequencing, labeling, extension and the like; currently, the selling prices is expensive, a synthetic method is complex, the reaction selectivity is poor; and the synthetic method provided by the invention is good in selectivity and easy in separation and purification, required experimental conditions are simple, and the synthetic processes are all conventional chemical reactions, so that the synthetic method is applicable to large-scale popularization and use.
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- Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: Implications for the design of better anti-HIV analogues
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The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the d-/l-configurations of the nucleotides, the l-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the l-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an l-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.
- Varga, Andrea,Chaloin, Laurent,Sagi, Gyula,Sendula, Robert,Graczer, Eva,Liliom, Karoly,Zavodszky, Peter,Lionne, Corinne,Vas, Maria
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experimental part
p. 1863 - 1873
(2012/04/17)
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- Identification and characterization of a strict and a promiscuous N-acetylglucosamine-1-P uridylyltransferase in Arabidopsis
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UDP-GlcNAc is an essential precursor for glycoprotein and glycolipid synthesis. In the present study, a functional nucleotidyltransferase gene from Arabidopsis encoding a 58.3 kDa GlcNAc1pUT-1 (N-acetylglucosamine-1-phosphate uridylyltransferase) was identified. In the forward reaction the enzyme catalyses the formation of UDP-N-acetylglucosamine and PPi from the respective monosaccharide 1-phosphate and UTP. The enzyme can utilize the 4-epimer UDP-GalNAc as a substrate as well. The enzyme requires divalent ions (Mg2+ or Mn2+) for activity and is highly active between pH 6.5 and 8.0, and at 30-37°C. The apparent Km values for the forward reaction were 337 μM (GlcNAc-1-P) and 295 μM (UTP) respectively. Another GlcNAc1pUT-2, which shares 86%amino acid sequence identity with GlcNAc1pUT-1, was found to convert, in addition to GlcNAc-1-P and GalNAc-1-P, Glc-1-P into corresponding UDP-sugars, suggesting that subtle changes in the UT family cause different substrate specificities. A three-dimensional protein structure model using the human AGX1 as template showed a conserved catalytic fold and helped identify key conserved motifs, despite the high sequence divergence. The identification of these strict and promiscuous gene products open a window to indentify new roles of amino sugar metabolism in plants and specifically their role as signalling molecules. The ability of GlcNAc1pUT-2 to utilize three different substrates may provide further understanding as to why biological systems have plasticity. The Authors.
- Yang, Ting,Echols, Merritt,Martin, Andy,Bar-Peled, Maor
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experimental part
p. 275 - 284
(2012/05/20)
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- An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
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Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.
- Gillerman, Irina,Fischer, Bilha
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p. 245 - 256
(2011/08/06)
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- A novel method for the preparation of nucleoside triphosphates from activated nucleoside phosphoramidates
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A novel method for the preparation of nucleoside triphosphates has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes efficient coupling with tris(tetra-n-butylammonium) hydrogen pyrophosphate to generate nucleoside triphosphate.
- Wu, Weidong,Meyers, Caren L. Freel,Borch, Richard F.
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p. 2257 - 2260
(2007/10/03)
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- Use of Escherichia coli Polyphosphate Kinase for Oligosaccharide Synthesis
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The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.
- Noguchi, Toshitada,Shiba, Toshikazu
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p. 1594 - 1596
(2007/10/03)
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- 4-Substituted uridine 5′-triphosphates as agonists of the P2y2 purinergic receptor
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Undine 5′-O-triphosphate (UTP) is a potent agonist of the purinergic receptor designated P2Y2. UTP is rapidly metabolized in human tissue. To find a compound with similar activity that may be more slowly metabolized, a series of 4-substituted uridine 5′-triphosphates were prepared and evaluated in a P2Y2 receptor second messenger assay. Copyright
- Shaver, Sammy R.,Pendergast, William,Siddiqi, Suhaib M.,Yerxa, Benjamin R.,Croom, Dallas K.,Dougherty, Robert W.,James, Michael K.,Neil Jones,Rideout, Janet L.
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p. 1099 - 1102
(2007/10/03)
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- Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: Enzymatic production of CDP-choline from orotic acid (Part II)
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A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. colt), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32°C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.
- Fujio,Teshiba,Maruyama
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p. 960 - 964
(2007/10/03)
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- Enzymatic production of pyrimidine nucleotides using corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: Enzymatic production of CDP-choline from orotic acid and choline chloride (Part I)
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Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Corynebacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY13505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CCTase; encoded by the CKIgene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.
- Fujio,Akihiko
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p. 956 - 959
(2007/10/03)
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- Mg(II) ION-MEDIATED CONVERSION OF MONO- AND OLIGONUCLEOTIDES TO 5'-POLYPHOSPHATES IN AQUEOUS SOLUTION
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5'-Polyphosphates of mono- and oligonucleotides were prepared from the corresponding 5'-monophosphates with phosphorotriimidazolide or phosphorotribenzimidazolide mediated by Mg(II) or Mn(II) ion in aqueous solution.
- Sawai, Hiroaki,Inaba, Yoshiko,Hirano, Atsushi,Wakai, Hiromichi,Shimazu, Masamitsu
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p. 4801 - 4804
(2007/10/02)
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- Applications of enzymes in the synthesis and hydrolytic study of 2-deoxy-α-D-glucopyranosyl phosphate
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The labile sugar phosphate 2-deoxy-α-D-glucopyranosyl phosphate has been synthesized enzymically in a two-step process from 2-deoxy-D-glucose-6-phosphate via an intermediate uridine 5'-diphospho-2-deoxy-D-glucose.Rate constants for acid-catalysed (1 M HClO4) hydrolysis at several temperatures were determined by using an enzymic assay to measure remaining substrate.The values obtained were consistent with that anticipated on the basis of known hydrolysis rates for alkyl- and aryl-2-deoxy-D-glucopyranosides.
- Percival, M. David,Withers, Stephen G.
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p. 1970 - 1972
(2007/10/02)
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- HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC CHARACTERIZATION OF THE ROLE OF INORGANIC PYROPHOSPHATASE IN REGULATING THE REACTION OF URIDINE 5'-TRIPHOSPHATE WITH GLUCOSE 1-MONOPHOSPHATE
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Inorganic pyrophosphatase (EC 3.6.1.1) drived the reaction of uridine 5'-triphosphate with glucose 1-monophosphate in the direction of uridine 5'-diphosphoglucose formation.
- Yoza, Norimasa,Hirano, Hisanobu,Baba, Yoshinobu,Ohashi, Shigeru
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p. 605 - 610
(2007/10/02)
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