- Cell- And Polymerase-Selective Metabolic Labeling of Cellular RNA with 2′-Azidocytidine
-
Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2′-azidocytidine (2′-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2′-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2′-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2′-AzCyd labeling. Further, 2′-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.
- Wang, Danyang,Zhang, Yu,Kleiner, Ralph E.
-
supporting information
p. 14417 - 14421
(2020/10/13)
-
- Improved synthesis of cytidine diphosphate choline (CDP-choline) via selective phosphorylation
-
An improved, three-step synthesis of cytidine diphosphate choline (CDP-choline) from cytidine was achieved in 68% overall yield. Selective phosphorylation of cytidine was accomplished by the use of morpholinophosphodichloridate to give cytidine-5′-phosphomorpholide, which was condensed with choline phosphate chloride in the presence of a catalytic amount of H2SO4 to give CDP-choline. The intermediates and products could be efficiently purified by recrystallisation, thus avoiding the use of chromatography at all stages. The reaction could be scaled up to 200 g in 64% overall yield, making this route attractive for industrial application.
- Xia, Ran,Sun, Li-Ping,Chen, Lei-Shan
-
p. 358 - 360
(2016/07/06)
-
- PDE7A1 hydrolyzes cCMP
-
The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC-MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP KM value of 135 ± 19 μM. The Vmax for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.
- Monzel, Maike,Kuhn, Maike,B?hre, Heike,Seifert, Roland,Schneider, Erich H.
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p. 3469 - 3474
(2015/03/31)
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- Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
-
The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
- Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
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p. 1575 - 1581
(2015/02/19)
-
- Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides
-
Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).
- Serra, Immacolata,Conti, Silvia,Piskur, Jure,Clausen, Anders R.,Munch-Petersen, Birgitte,Terreni, Marco,Ubiali, Daniela
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p. 563 - 570
(2014/05/20)
-
- The reaction of activated RNA species with aqueous fluoride ion: A convenient synthesis of nucleotide 5′-phosphorofluoridates and a note on the mechanism
-
The chemistry of 5′-phosphorimidazolides of ribonucleosides is extended to include their reaction with alkali metal fluorides in aqueous solution. High yields of 5′-phosphorofluoridates are formed, especially with potassium fluoride, but no detectable oligomerization products were formed. A combination of HPLC, mass spectrometry, synthesis, kinetics, and NMR confirms the identities of the products. Judicious control of pH leads to higher yields in shorter reaction times. This new methodology contrasts favorably with other synthetic routes involving non-aqueous chemistry or aqueous chemistry with a nucleotide triphosphate.
- Aldersley, Michael F.,Joshi, Prakash C.,Schwartz, Herbert M.,Kirby, Anthony J.
-
p. 1464 - 1466
(2014/03/21)
-
- Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics
-
Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an l-methionine:uridine-5′-aldehyde aminotransferase; (ii) LipP, a 5′-amino-5′-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-d-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.
- Chi, Xiuling,Pahari, Pallab,Nonaka, Koichi,Van Lanen, Steven G.
-
supporting information; experimental part
p. 14452 - 14459
(2011/11/04)
-
- Synthesis of oligoribonucleotides with phosphonate-modified linkages
-
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
-
supporting information; experimental part
p. 6120 - 6126
(2011/10/10)
-
- Deciphering the Late Biosynthetic Steps of Antimalarial Compound FR-900098
-
FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in Escherichia coli by reconstructing the entire biosynthetic pathway using a three-plasmid system. Based on this system, whole-cell feeding assays in combination with in vitro enzymatic activity assays reveal an unusual functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These studies also suggest a biosynthetic route to a second phosphonate antibiotic, FR-33289. A thorough understanding of the FR-900098 biosynthetic pathway now opens possibilities for metabolic engineering in E. coli to increase production of the antimalarial antibiotic and combinatorial biosynthesis to generate novel derivatives of FR-900098.
- Johannes, Tyler W.,DeSieno, Matthew A.,Griffin, Benjamin M.,Thomas, Paul M.,Kelleher, Neil L.,Metcalf, William W.,Zhao, Huimin
-
experimental part
p. 57 - 64
(2010/08/06)
-
- Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2′-C-methyl-cytidine to its 5′-monophosphate metabolite
-
Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2′-C-methyl-cytidine (2′-MeC), entered clinical development as NM283, a valine ester prodrug form of 2′-MeC possessing improved oral bioavailability. To be active against HCV, 2′-MeC must be converted to 2′-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase. Conversion of 2′-MeC to 2′-MeC monophosphate is the first step in 2′-MeC triphosphate production and is thought to be the rate-limiting step. Here we investigate which of three possible enzymes, deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK1), or uridine-cytidine kinase 2 (UCK2), mediate this first phosphorylation step. Purified recombinant enzymes UCK2 and dCK, but not UCK1, could phosphorylate 2′-MeC in vitro. However, siRNA knockdown experiments in three human cell lines (HeLa, Huh7 and HepG2) defined UCK2 and not dCK as the key kinase for the formation of 2′-MeC monophosphate in cultured human cells. These results underscore the importance of confirming enzymatic kinase data with appropriate cell-based assays. Finally, we present data suggesting that inefficient phosphorylation by UCK2 likely limits the antiviral activity of 2′-MeC against HCV. This paves the way for the use of a nucleotide prodrug approach to overcome this limitation.
- Golitsina, Nina L.,Danehy Jr., Francis T.,Fellows, Ross,Cretton-Scott, Erika,Standring, David N.
-
experimental part
p. 470 - 481
(2010/12/19)
-
- A reversed phase hplc method for the analysis of nucleotides to determine 5'-PDE enzyme activity
-
5'-Phosphodiesterase (5'-PDE) can be extracted from barley roots and used as a catalyst in the hydrolysis of RNA to produce 5'-nucleotides. The assay of enzyme activity is essential for the production of 5'PDE. To improve the conventional assays, we developed and validated a new method for the analysis of 5'-PDE enzyme activity using reversed phased high performance liquid chromatography (RP-HPLC). The method is based on the quantification of the four 5'-nucleotides namely cytidine 5'-monophosphate (5'-CMP), uridine 5'monophosphate (5'-UMP), guanosine 5'-mono-phosphate (5'-GMP) and adenosine 5'-mono-phosphate (5'AMP), produced in the enzymatic hydrolysis of yeast RNA. The optimal condition for the enzymatic hydrolysis of RNA to detect the enzyme activity was investigated. The results show that when the hydrolysis takes place at 70 °C for 30 min at pH 5.0, the hydrolysis reaction has highest yield for the four of the 5'-nucleotides. 5'-PDE demonstrated highest catalytic capability. These four 5'-nucleotides were utilized for the analysis of enzyme activity of 5'-PDE with our newly developed HPLC method. Excellent reproducibility, precision, and linearity were obtained for this HPLC method.
- Hua, Jie,Huang, Ke-Long
-
experimental part
p. 167 - 174
(2011/11/29)
-
- Staphylococcus aureus and bacillus subtilis W23 make polyribitol wall teichoic acids using different enzymatic pathways
-
Wall teichoic acids (WTAs) are anionic polymers that play key roles in bacterial cell shape, cell division, envelope integrity, biofilm formation, and pathogenesis. B. subtilis W23 and S. aureus both make polyribitol-phosphate (RboP) WTAs and contain similar sets of biosynthetic genes. We use in vitro reconstitution combined with genetics to show that the pathways for WTA biosynthesis in B. subtilis W23 and S. aureus are different. S. aureus requires a glycerol-phosphate primase called TarF in order to make RboP-WTAs; B. subtilis W23 contains a TarF homolog, but this enzyme makes glycerol-phosphate polymers and is not involved in RboP-WTA synthesis. Instead, B. subtilis TarK functions in place of TarF to prime the WTA intermediate for chain extension by TarL. This work highlights the enzymatic diversity of the poorly characterized family of phosphotransferases involved in WTA biosynthesis in Gram-positive organisms.
- Brown, Stephanie,Meredith, Timothy,Swoboda, Jonathan,Walker, Suzanne
-
body text
p. 1101 - 1110
(2011/06/17)
-
- Mechanism of activation of β-D-2′-Deoxy-2′-fluoro- 2′-C-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase
-
β-D-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 μM and a kcat of 0.007 s -1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (Ki = 1.5 μM) and 2′-C-methylcytidine triphosphate (Ki = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C- methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination. Copyright
- Murakami, Eisuke,Bao, Haiying,Ramesh, Mangala,McBrayer, Tamara R.,Whitaker, Tony,Steuer, Holly M. Micolochick,Schinazi, Raymond F.,Stuyver, Lieven J.,Obikhod, Aleksandr,Otto, Michael J.,Furman, Phillip A.
-
p. 503 - 509
(2008/02/07)
-
- Selective synthesis of phosphate monoesters by dehydrative condensation of phosphoric acid and alcohols promoted by nucleophilic bases
-
(Chemical Equation Presented) Phosphate monoesters are synthesized from a mixture of phosphoric acid (1 or 2 equiv) and alcohols (1 equiv) in the presence of tributylamine. The reaction is promoted by nucleophilic bases such as N-alkylimidazole and 4-(N,N-dialkylamino)pyridine. 2′,3′-I- Isopropylidene ribonucleosides are selectively converted to their 5′-monophosphates without the protection of amino groups in nucleobases.
- Sakakura, Akira,Katsukawa, Mikimoto,Ishihara, Kazuaki
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p. 1999 - 2002
(2007/10/03)
-
- Borate-nucleotide complex formation depends on charge and phosphorylation state
-
Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 μM nucleotide and 500 μM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3
- Kim, Danny H.,Faull, Kym F.,Norris, Andrew J.,Eckhert, Curtis D.
-
p. 743 - 751
(2007/10/03)
-
- Studies on the nonmevalonate pathway: Formation of 2-C-methyl-D- erythritol 2,4-cyclodiphosphate from 2-phospho-4-(cytidine 5'-diphospho)-2-C- methyl-D-erythritol
-
2-Phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol was transformed to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate by a novel Escherichia coli enzyme involved in the nonmevalonate pathway. (C) 2000 Elsevier Science Ltd.
- Takagi, Motoki,Kuzuyama, Tomohisa,Kaneda, Kazuhide,Watanabe, Hiroyuki,Dairi, Tohru,Seto, Haruo
-
p. 3395 - 3398
(2007/10/03)
-
- Interactions between aminocalixarenes and nucleotides or nucleic acids
-
Four calixarenes with (trimethylammonium)methyl groups at the phenyl rings in the upper rim were prepared. Association constants K with mononucleotides were determined in D2O by NMR shift titration, partially also by fluorescence competition titration using ANS as dye. The complexation free energies ΔG obtained with the derivatives of the calix[4]cone (AC4c) and the calix[4]-1,3-alternate (AC4a) conformation were similar, but increased from AMP (18 ± 1 kJ mol-1) to ADP (20 ± 1 kJ mol-1), to ATP (22 ± 1 kJ mol-1]. With the calix[6] derivative (AC6) the corresponding values were 22, 24, 27 kJ mol-1, with the calix[8] host (AC8) 24, 26, 28 kJ mol-1, respectively. The large contribution of salt bridging to the complexation was obvious from the ΔG difference between adenosine and e.g. AMP (with the calix[4]cone derivative 5.6 and 17.7 kJ mol-1, respectively). Affinity differences between different nucleobases increased moderately with the size of the macrocyclic host, e.g. ΔΔG between AMP and TMP was 1 kJ mor-1 with calix[4]cone, 2 kJ mor-1 with calix[6], and 3 kJ mol-1 with calix[8] compounds. The results are in line with computer simulated complex structures in which the nucleobase or sugar parts are only partially inserted into the calix cavity. This agrees with the observed complexation induced NMR shifts (CIS), which are small but increase with the ring size of the host. Noticeably the CIS values are substantially larger for much weaker bound nucleosides. Affinities of the four aminocalixarenes with double-stranded calf thymus (CT) DNA, with polydA*polydT and with polydG*polydC were characterized by ΔTm of the double-strand denaturation temperature and by fluorimetric assays using ethidium bromide (C50 values). The calix[4]cone derivative AC4c shows, due to the four positive charges converging at one side, the strongest effects. They surpass spermine although this also bears four protonated ammonium groups, indicating additional binding contributions from the phenyl moieties. The larger, more flexible calix[6]- and calix[8]-derivatives AC6 and AC8 show only small affinity increases in spite of their 6 or 8 positive charges. Preliminary molecular modeling studies indicate that based on the distances between the ammonium centers only partial contact of all centers to the groove phosphates can materialize. The ligands AC4c, AC4a and AC6 exhibit a remarkable preference for DNA in comparison to RNA mimics.
- Shi, Youheng,Schneider, Hans-Joerg
-
p. 1797 - 1803
(2007/10/03)
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- The N-acetyl neuraminyl oxecarbenium ion is an intermediate in the presence of anionic nucleophiles
-
Solvolysis of CMP N-acetyl neuraminate (CMP-NeuAc) in 1.8 M acetate buffer at pH 5 containing 0.9 M azide results in the formation of both anomers of 2-deoxy-2-azido N-acetyl neuraminic acid in addition to N-acetyl neuraminic acid as determined by 1H-NMR product analysis. A rate dependence on [azide] was observed with an apparent bimolecular rate constant of (2.1 ± 0.30) x 10-3 M-1 min-1 which could only account for half of the azido- NeuAc formed. Comparison of rate, product ratio, and stereochemical data indicate that concurrent pathways for formation of N3-NeuAc are operative, with 17% of product forming from reaction of azide and the tight ion pair. 12% via the solvent separated ion pair, and 6% from the free NeuAc oxocarbenium ion. From the corrected product ratio data, the lifetime of the oxocarbenium ion was estimated to be ≤ 3 x 10-11 s. Solvolysis of CMP- NeuAc at pL. = 5.0 afforded an observed solvent deuterium isotope effect (SDIE) k(H2O)/k(D2O) = 0.45, consistent with specific acid catalysis of glycosidic bond cleavage. A SDIE of 0.66 for the apparent bimolecular azide trapping pathway was also observed. An apparent isotope effect of ~1.1 for trapping of the N-acetyl neuraminyl oxocarbenium ion by water was determined by product analysis of azide trapping in H2O and D2O. An ab initio transition state for attack of water on an N-acetyl neuraminyl oxocarbenium ion model was located which featured a hydrogen bond between the oxocarbenium ion carboxylate and water; proton transfer was not part of the reaction coordinate. It is proposed that the N-acetyl neuraminate carboxylate group stabilizes an intermediate oxocarbenium ion, but the barrier for capture by water is lowered by a transition state hydrogen bond.
- Horenstein, Benjamin A.,Bruner, Michael
-
p. 1357 - 1362
(2007/10/03)
-
- Phosphorylation of nucleosides with phosphorus oxychloride in trialkyl phosphate
-
The reaction of guanosine and triethyl phosphate at 50°C for 15 min produced the guanosine-triethyl phosphate complex in which triethyl phosphate is coordinated to guanosine of high-anti form in a 1:1 molar ratio. During the conversion of guanosine to guanosine 5'-monophosphate with phosphorus oxychloride, the guanosine-triethyl phosphate complex showed excellent selectivity and high reactivity toward phosphorus oxychloride compared with those of guanosine. The rate of selective phosphorylation of guanosine into guanosine 5'-monophosphate was markedly improved by preheating the mixture of guanosine and triethyl phosphate at 50°C, followed by adding phosphorus oxychloride to the mixture at 0°C. Thus, the 5'-phosphorylation of guanosine with phosphorus oxychloride in triethyl phosphate is considered to progress via the guanosine-triethyl phosphate complex as the reaction intermediate.
- Ikemoto,Haze,Hatano,Kitamoto,Ishida,Nara
-
p. 210 - 215
(2007/10/02)
-
- Regiodefined synthesis and conformational properties of adenyldiyl trimers with unsymmetrical 2'-5' and 3'-5' internucleotide linkages
-
Adenyldiyl trimers with different kinds of substituents on 2'-5' and 3'-5' phosphate linkages have been synthesized in a general, regiodefined manner. Examination of the 2D NMR spectra reveals that the trimers with adenyl(2'-5')adenosine linkage make a syn-anti as well as syn-syn base stack between the two adenyl bases and exist as a mixture of the two conformers.
- Hayakawa,Hirose,Noyori
-
p. 9899 - 9916
(2007/10/02)
-
- Additional Evidence for the Exceptional Mechanism of the Acid-catalysed Hydrolysis of 4-Oxopyrimidine Nucleosides: Hydrolysis of 1-(1-Alkoxyalkyl)uracils, Seconucleosides, 3'-C-Alkyl Nucleosides and Nucleoside 3',5'-Cyclic Monophosphates
-
The rate constants for the acid-catalysed hydrolysis of 1-(1-alkoxyethyl)uracils and 1-alkoxymethyluracils have been determined.With both series of compounds, the hydrolysis rate is rather insensitive to the polar nature of the alkoxy group, in striking contrast with the hydrolysis of the corresponding analogues of adenine and cytosine nucleosides, which react via rate-limiting formation of an oxocarbenium ion intermediate.Furthermore, it has been shown that 3',5'-cyclic monophosphates of thymidine and uridine undergo hydrolysis of the N-glycosidic bond 760 and 260 times as fast as their parent nucleosides, while the cyclic monophosphates of 2'-deoxyadenosine and adenosine are depurinated much more slowly than the corresponding nucleosides.On this basis it is suggested that 4-oxopyrimidine nucleosides are hydrolysed by opening of the sugar ring.To obtain further evidence for this exceptional mechanism, comparative kinetic measurements with some seco- and 3'-C-alkyl nucleosides of uracil and adenine have been carried out.
- Oivanen, Mikko,Rajamaeki, Markku,Varila, Jaana,Hovinen, Jari,Mikhailov, Sergey,Loennberg, Harri
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p. 309 - 314
(2007/10/02)
-
- One-electron-reduction potentials of pyrimidine bases, nucleosides, and nucleotides in aqueous solution. Consequences for DNA redox chemistry
-
The reduction potentials in aqueous solution of the pyrimidine bases, nucleosides, and nucleotides of uracil (U) and thymine (T) were determined using the technique of pulse radiolysis with time-resolved spectrophotometric detection. The electron adducts of U and T were found to undergo reversible electron exchange with a series of ring-substituted N-methylpyridinium cations with known reduction potential. From the concentrations of the pyrimidine electron adducts and the reduced N-methylpyridinium compounds at electron-transfer equilibrium, the thermodynamical equilibrium constants were obtained and from these the reduction potentials. The results show U and T and their nucleosides and nucleotides to have very similar reduction potentials, ～ -1.1 V/NHE at pH 8, i.e., the effect of methylation at C5, C6, or of substitution at N1 is small, ≤0.1 V. In the case of cytosine (C) the electron adduct is protonated (probably at N3), even up to pH 13. The protonated adduct (C(H)?) undergoes a reversible electron transfer with the N-methylpyridinium cations. This is accompanied in one direction by transfer of a proton but by that of a water molecule in the other direction. As a result of the protonation of the electron adduct, the effective ease of reduction of C in aqueous solution is similar to that of U and T. It is suggested that in DNA the tendency for C?- to be protonated (by its complementary base G) is larger by ≥10 orders of magnitude than that for protonation of T?- by its complementary base A. This results in C and not T being the most easily reduced base in DNA. A further consequence is that lack of neutralization by intrapair proton transfer of T?- enables the irreversible extra-pair protonation on C6 of the radical anion to take place.
- Steenken,Telo,Novais,Candeias
-
p. 4701 - 4709
(2007/10/02)
-
- Catalysis of Hydrolysis and Nucleophilic Substitution at the P-N Bond of Phosphoimidazolide-Activated Nucleotides in Phosphate Buffers
-
Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 (*) pH (*) 8.6.This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phophate.The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate.Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities.The results show that both H2PO4(-) and HPO4(2-) participate in both hydrolysis and nucleophilic substitution.Statistically corrected bimolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile.The low Bronsted value β = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before.The βnuc = 0.35 calculated for water, H2PO4(-), HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR(-) with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (βnuc = 0.25).With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phophate, assuming the concentrations of phosphate, Pj, on prebiotic Earth were similar to those in the present oceans (j> ca. 2.25 μM).
- Kanavarioti, Anastassia,Rosenbach, Morgan T.
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p. 1513 - 1521
(2007/10/02)
-
- 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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p. 2477 - 2484
(2007/10/02)
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- PHOSPHORYLATING AGENT FOR THE SYNTHESIS OF OLIGONUCLEOTIDE WITH ALIPHATIC AMINO GROUP AT 5' END
-
A lipophilic phosphorylating agent was prepared and used for the synthesis of pentadeoxyribonucleotide with aminoethyl group at 5' end on a polymer support by the phosphotriester method.
- Tanaka, Toshiki,Tamatsukuri, Shigeru,Ikehara, Morio
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p. 2611 - 2614
(2007/10/02)
-
- A GENERAL APPROACH TO NUCLEOSIDE 3'- AND 5'-MONOPHOSPHATES
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Diallyloxyphosphorylation of nucleoside hydroxyls followed by palladium(0)-catalyzed deallylation provides a new, general method for the preparation of the 3'- and 5'-monophosphates.
- Hayakawa, Y.,Wakabayashi, S.,Nobori, T.,Noyori, R.
-
p. 2259 - 2262
(2007/10/02)
-
- Bis(2,2,2-trichloroethyl) Phosphorochloridite as a Reagent for the Phosphorylation of Oligonucleotides: Preparation of 5'-Phosphorylated 2',5'-Oligoadenylates
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Bis(2,2,2-trichloroethyl) phosphorochloridite was found to be a useful reagent for the phosphorylation of protected nucleosides and oligonucleotides especially when the phosphate blocking group was 2,2,2-trichloroethyl and the hydroxyl protecting group was tert-butyldimethylsilyl.Thus compounds 2a-c,f,g could be phosphorylated to the corresponding 5'-phosphotriesters 4a-c,f,g in yields of 75-99percent.Removal of the protecting groups (to give the 5'-monophosphates 6a-c,f,g) was achieved by zinc-copper couple/2,4-pentanedione/DMF treatment to remove the 2,2,2-trichloroethyl group and tetrabutylammonium fluoride/THF treatment to remove the tert-butyldimethylsilyl groups.A method was found that permits preparation of reproducibly active zinc-copper couple.As a hydroxyl protecting group, the isopropylidene moiety was somewhat less useful in conjunction with the use of bis(2,2,2-trichloroethyl) phosphorochloridite.Thus, upon phosphorylation of 2d and 2e, the phosphotriesters 4d and 4e were obtained in yields of 83percent and 61percent, respectively.Deblocking of the isopropylidene groups was accomplished with formic acid at room temperature to give 6a and 6b in yields of 74percent and 70percent, respectively.The 5'-phosphorylated 2'-5'-linked oligonucleotides 6f and 6g were converted to the corresponding 5'-triphosphates to give compounds 1a and 1b which are found in extracts of interferon-treated cells upon incubation with double-stranded RNA.
- Imai, Jiro,Torrence, Paul F.
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p. 4015 - 4021
(2007/10/02)
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