cas 58-97-9, Uridine Monophosphate | Articles data

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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)
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
p. 960 - 964
(1997)
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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)
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
p. 956 - 959
(1997)
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Kinetic and NMR spectroscopic study of the chemical stability and reaction pathways of sugar nucleotides
The alkaline cleavage of two types of sugar nucleotides has been studied by 1H and 31P NMR in order to obtain information on the stability and decomposition pathways in aqueous solutions under alkaline conditions. The reaction of glucose 1-UDP is straightforward, and products are easy to identify. The results obtained with ribose 5-UDP and ribose 5-phosphate reveal, in contrast, a more complex reaction system than expected, and the identification of individual intermediate species was not possible. Even though definite proof for the mechanisms previously proposed could not be obtained, all the spectroscopic evidence is consistent with them. Results also emphasise the significant effect of conditions, pH, ionic strength, and temperature, on the reactivity under chemical conditions.
Jaakkola, Juho,Nieminen, Anu,Kivel?, Henri,Korhonen, Heidi,T?htinen, Petri,Mikkola, Satu
p. 178 - 193
(2020/12/21)

Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity
Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5′-monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi-dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase-like dimeric topology, with each monomer consisting of a five-stranded β-sheet that is sandwiched by five α-helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl-nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2′-hydroxyl of CMP and the ζ-OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.
Kang, Minhee,Doddapaneni, Kiran,Sarni, Samantha,Heppner, Zach,Wysocki, Vicki,Wu, Zhengrong
p. 1760 - 1773
(2020/07/27)

Practical preparation of UDP-apiose and its applications for studying apiosyltransferase
UDP-apiose, a donor substrate of apiosyltransferases, is labile because of its intramolecular self-cyclization ability, resulting in the formation of apiofuranosyl-1,2-cyclic phosphate. Therefore, stabilization of UDP-apiose is indispensable for its availability and identifying and characterizing the apiosyltransferases involved in the biosynthesis of apiosylated sugar chains and glycosides. Here, we established a method for stabilizing UDP-apiose using bulky cations as counter ions. Bulky cations such as triethylamine effectively suppressed the degradation of UDP-apiose in solution. The half-life of UDP-apiose was increased to 48.1 ± 2.4 h at pH 6.0 and 25 °C using triethylamine as a counter cation. UDP-apiose coordinated with a counter cation enabled long-term storage under freezing conditions. UDP-apiose was utilized as a donor substrate for apigenin 7-O-β-D-glucoside apiosyltransferase to produce the apiosylated glycoside apiin. This apiosyltransferase assay will be useful for identifying genes encoding apiosyltransferases.
Fujimori, Tae,Matsuda, Ryoko,Suzuki, Mami,Takenaka, Yuto,Kajiura, Hiroyuki,Takeda, Yoichi,Ishimizu, Takeshi
p. 20 - 25
(2019/04/01)

Enzymatic Production of Non-Natural Nucleoside-5′-Monophosphates by a Thermostable Uracil Phosphoribosyltransferase
The use of enzymes as biocatalysts applied to synthesis of modified nucleoside-5′-monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo-, regio-, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate-binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several 5 and 6-substituted pyrimidines as substrates. These experimental results suggest TtUPRT could be a valuable biocatalyst for the synthesis of modified NMPs.
del Arco, Jon,Acosta, Javier,Pereira, Humberto M.,Perona, Almudena,Lokanath, Neratur K.,Kunishima, Naoki,Fernández-Lucas, Jesús
p. 439 - 448
(2017/12/13)

Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs
Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.
Nakajima, Kazuki,Kizuka, Yasuhiko,Yamaguchi, Yoshiki,Hirabayashi, Yoshio,Takahashi, Kazuo,Yuzawa, Yukio,Taniguchi, Naoyuki
p. 401 - 407
(2017/11/17)

cUMP hydrolysis by PDE3A
As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a KM value of ~143?μM and a Vmax value of ~42?μmol/min/mg. PDE3A hydrolyzed cAMP with a KM value of ~0.7?μM and a Vmax of ~1.2?μmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (Ki?=?57?nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.
Berrisch, Stefan,Ostermeyer, Jessica,Kaever, Volkhard,K?lble, Solveig,Hilfiker-Kleiner, Denise,Seifert, Roland,Schneider, Erich H.
p. 269 - 280
(2017/02/18)

Chemical synthesis and isolation of UDP-2-deoxy glucose and galactose
2-Deoxy sugars are attractive compounds in synthetic chemistry with regard to reactivity and stereoselectivity. Moreover, their ability to inhibit enzymes and metabolism is significant in biology. In this study, uridine-5′-diphosphate (UDP)-2-deoxy glucose (11) and galactose (12) were synthesized chemically. These sugar donors for glycosyltransferases were obtained α-selectively via phosphorylation using thioglycosides, coupling reaction with uridine-5′-monophosphate (UMP)-morpholidate, and moderate deacetylation. Isolation was carried out by sequential silica-gel chromatography using two kinds of developing solvents in a refrigerator. The structures were elucidated from the NMR results. Investigation of stability showed that the synthesized UDP-2-deoxy sugars were hydrolyzed much faster in buffer (pH 4) than the natural UDP sugars.
Miyagawa, Atsushi,Takeuchi, Shunya,Itoda, Shinji,Toyama, Sanami,Kurimoto, Kenta,Yamamura, Hatsuo,Ito, Yukishige
supporting information
p. 1790 - 1795
(2016/11/18)

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
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
p. 563 - 570
(2014/05/20)

On the observation of discrete fluorine NMR spectra for uridine 5'-β,γ-fluoromethylenetriphosphate diastereomers at basic pH
Jakeman et al. recently reported the inability to distinguish the diastereomers of uridine 5'-β,γ-fluoromethylenetriphosphate (β,γ-CHF-UTP, 1) by 19F NMR under conditions we previously prescribed for the resolution of the corresponding β,γ-CHF-
Hwang, Candy S.,Kashemirov, Boris A.,McKenna, Charles E.
supporting information
p. 5315 - 5319
(2014/06/23)

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)

Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5′-phosphate decarboxylase-catalyzed reactions
The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5′-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d- erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number k ex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.
Goryanova, Bogdana,Goldman, Lawrence M.,Amyes, Tina L.,Gerlt, John A.,Richard, John P.
p. 7500 - 7511
(2013/11/19)

UDP made a highly promising stable, potent, and selective P2Y6-receptor agonist upon introduction of a boranophosphate moiety
P2Y6 nucleotide receptor (P2Y6-R) plays important physiological roles, such as insulin secretion and reduction of intraocular pressure. However, this receptor is still lacking potent and selective agonists to be used as potential drugs. Here, we synthesized uracil nucleotides and dinucleotides, substituted at the C5 and/or Pα position with methoxy and/or borano groups, 18-22. Compound 18A, Rp isomer of 5-OMe-UDP(α-B), is the most potent and P2Y6-R selective agonist currently known (EC50 0.008 μM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y2- and P2Y4-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t1/2 = 16.9 h). It was more stable to hydrolysis by nucleotide pyrophosphatases (NPP1,3) than UDP (15% and 28% hydrolysis by NPP1 and NPP3, respectively, vs 50% and 51% hydrolysis of UDP) and metabolically stable in blood serum (t1/2 = 17 vs 2.4, 11.9, and 21 h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y6-R agonist may be of future therapeutic potential.
Ginsburg-Shmuel, Tamar,Haas, Michael,Grbic, Djordje,Arguin, Guillaume,Nadel, Yael,Gendron, Fernand-Pierre,Reiser, Georg,Fischer, Bilha
p. 5483 - 5495
(2012/11/06)

Conformational changes in orotidine 5′-monophosphate decarboxylase: A structure-based explanation for how the 5′-phosphate group activates the enzyme
The binding of a ligand to orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E c?S? complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5′-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5′-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (β/α)8-barrel structure. Our data support a model in which the IBE provided by the 5′-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec?S and Ec?S? complexes relative to the Eo?S complex. The conclusion that the IBE of the 5′-phosphate group provides stabilization to both the E c?S and Ec?S? complexes, not just the Ec?S? complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.
Desai, Bijoy J.,Wood, B. McKay,Fedorov, Alexander A.,Fedorov, Elena V.,Goryanova, Bogdana,Amyes, Tina L.,Richard, John P.,Almo, Steven C.,Gerlt, John A.
p. 8665 - 8678
(2013/01/15)

A nucleotide dimer synthesis without protecting groups using montmorillonite as catalyst
A synthesis has been developed providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately 30% of the imidazolide is converted into each 2-5 dimer and 3-5 dimer with the rest hydrolyzed to the 5-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5-monophosphates.
Joshi, Prakash C.,Aldersley, Michael F.,Zagorevskii, Dmitri V.,Ferris, James P.
experimental part
p. 536 - 566
(2012/10/08)

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; scheme or table
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)

Structural basis for the exceptional stability of bisaminoacylated nucleotides and transfer RNAs
At least one bisaminoacyl-tRNA is synthesized in nature (by Thermus thermophilus phenylalanyl-tRNA synthetase), and many disubstituted tRNAs have been prepared in vitro. Such misacylated tRNAs are able to participate in protein synthesis, even though they lack the free 2-OH group of the 3-terminal adenosine moiety. Their ready participation in protein synthesis implies significant chemical reactivity. The basis for this reactivity has been documented previously. Surprisingly, the aminoacyl moieties of these tRNAs also exhibit exceptional chemical stability. In the present report, bisaminoacylated nucleotides are investigated computationally and experimentally to define the basis for the stability of such species. Molecular modeling of bisalanyl-AMP in the absence of solvent and in the presence of a limited number of water molecules revealed two common features among the low-energy structures. The first was the presence of H-bonding interactions between the two aminoacyl moieties. The second was the presence of a H-bonding interaction between the 2-O-alanyl moiety and the N-3 atom of the adenine nucleobase, typically mediated through a water molecule. The prediction of an interaction between an aminoacyl moiety and the adenine nucleobase was confirmed experimentally by comparing the behavior of bisalanyl-AMP and bisalanyl-UMP in the presence of model nucleophiles. This study suggests a possible role for the adenosine moiety at the 3-end of aminoanyl-tRNAs in controlling the stability and reactivity of the aminoacyl moiety and has important implications for the reactivity and stability of normal aminoacyl-tRNAs.
Duca, Maria,Trindle, Carl O.,Hecht, Sidney M.
experimental part
p. 11368 - 11377
(2011/09/16)

Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction: Importance of residues in the orotate binding site
The reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for rate enhancement (k cat/knon = 7.1 ± 1016) and catalytic proficiency [(kcat/KM)/knon = 4.8 ± 1022 M-1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that "substrate destabilization" by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ～5 kcal/mol (contributing ～2.7 ± 103 to the rate enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker, H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry 48, 5518-5531]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of kcat by as much as 400-fold but have a minimal effect on the value of kex for exchange of H6 of the FUMP product analogue with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions of Ser 127 that is proximal to O4 of the orotate ring decrease the value of k cat/KM, with the S127P substitution that eliminates hydrogen bonding interactions with O4 producing a 2.5 ± 10 6-fold reduction; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 that produces an anionic carbene intermediate and thereby provides a structural strategy for stabilization of the intermediate. These observations provide additional information about the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis.
Iiams, Vanessa,Desai, Bijoy J.,Fedorov, Alexander A.,Fedorov, Elena V.,Almo, Steven C.,Gerlt, John A.
experimental part
p. 8497 - 8507
(2012/05/20)

Product deuterium isotope effects for orotidine 5′-monophosphate decarboxylase: Effect of changing substrate and enzyme structure on the partitioning of the vinyl carbanion reaction intermediate
A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-1H]-uridine 5′-monophosphate (50%) and [6-2H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO2 in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH2-NL3+ group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5′-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.
Toth, Krisztina,Amyes, Tina L.,Wood, Bryant M.,Chan, Kui,Gerlt, John A.,Richard, John P.
experimental part
p. 7018 - 7024
(2010/07/09)

An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
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
p. 245 - 256
(2011/08/06)

Conformational changes in orotidine 5′-monophosphate decarboxylase: "remote" residues that stabilize the active conformation
The structural factors responsible for the extraordinary rate enhancement (～1017) of the reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and "clamps" the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a "remote" structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases kcat/Km with a minimal effect on kcat, providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5′-phosphate group of orotidine 5′-monophosphate for the mutant enzymes are similar to that for the wild type, supporting this conclusion.
Wood, B. McKay,Amyes, Tina L.,Fedorov, Alexander A.,Fedorov, Elena V.,Shabila, Andrew,Almo, Steven C.,Richard, John P.,Gerlt, John A.
scheme or table
p. 3514 - 3516
(2011/03/22)

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)

Bimetallic Cu2+ complexes of bis-terpyridine ligands as catalysts of the cleavage of mRNA 5′-cap models. the effect of linker length and base moiety
Ligands, where two terpyridine units are linked via an alkyl chain of three to five methylene units, have been synthesized. Their Cu2+ complexes have been studied as catalysts for the hydrolysis of the triphosphate bridge of three different dinucleoside triphosphates. The results show that the bimetallic complexes are up to 600 times more efficient catalysts than monomeric Cu2+-TerPy, and up to 5 × 105-fold rate enhancement in comparison to the uncatalysed reaction, is achieved. However, the catalytic activity strongly depends on the length of the linker and the base composition of the substrate. The differences can be attributed to interactions between the Cu2+-TerPy and nucleic acid base moieties as well as steric factors that may hinder the productive interaction between the substrate and the catalyst.
Maanpaeae, Leena,Luzet, Vincent,Guillaume, Glemence,Taherpour, Sharmin,Maeki, Esa,Mikkola, Satu
experimental part
p. 1853 - 1858
(2009/12/04)

Structure-activity relationships of C6-uridine derivatives targeting Plasmodia orotidine monophosphate decarboxylase
Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5′-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5′-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6-N-methylamino, and 6-N,N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5′-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6-N-Methylamino and 6-N,N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.
Bello, Angelica M.,Poduch, Ewa,Liu, Yan,Wei, Lianhu,Crandall, Ian,Wang, Xiaoyang,Dyanand, Christopher,Kain, Kevin C.,Pai, Emil F.,Kotra, Lakshmi P.
p. 439 - 448
(2008/09/19)

A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: Mechanistic implications
Orotidine-5′-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the decarboxylation of orotidine-5′-monophosphate (OMP) to uridine-5′-monophosphate (UMP). Despite extensive enzymological, structural, and computational studies, the mechanism of ODCase remains incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 ± 0.0007, compared to the value of 1.0289 ± 0.0009 for natural abundance OMP, revealing an 18O2 isotope effect of 0.991 ± 0.001. This value equates to an intrinsic isotope effect of approximately 0.983, using a calculated commitment factor derived from previous isotope effect data. The measured 18O2 isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or chemistry, that contribute to this substantial inverse isotope effect. 18O2 kinetic isotope effects were calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4; and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse 18O2 effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation at O2, O4, or C5 are all calculated to proceed with inverse 18O2 effects, and could contribute to the experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.
Wepukhulu, Wickliffe O.,Smiley, Vanessa L.,Vemulapalli, Bhargavi,Smiley, Jeffrey A.,Phillips, Linda M.,Lee, Jeehiun K.
experimental part
p. 4533 - 4541
(2009/03/12)

Product deuterium isotope effect for orotidine 5′-monophosphate decarboxylase: Evidence for the existence of a short-lived carbanion intermediate
A product isotope effect (PIE) of 1.0 was obtained from the ratio of the yields of [6-1H]-uridine 5′-monophosphate (50%) and [6-2H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) H2O/D2O catalyzed by orotidine 5′-monophosphate decarboxylase. This unitary product isotope effect eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO2 in a concerted reaction. The complete lack of selectivity for the reaction of solvent H and D that is implied by the value of PIE = 1.0 may be enforced by restricted motion of the NL3+ group of the side-chain of Lys-93 that has been proposed to protonate a vinyl carbanion intermediate. Copyright
Toth, Krisztina,Amyes, Tina L.,Wood, Bryant M.,Chan, Kui,Gerlt, John A.,Richard, John P.
p. 12946 - 12947
(2008/04/04)

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
p. 1999 - 2002
(2007/10/03)

Polymer-bound oxathiaphospholane: A solid-phase reagent for regioselective monothiophosphorylation and monophosphorylation of unprotected nucleosides and carbohydrates
(Chemical Equation Presented) Two polymers bound to N,N-diisopropylamino-1, 3,2-oxathiaphospholane were reacted with unprotected carbohydrates and nucleosides in the presence of 1H-tetrazole, followed by oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent. The 1,3,2-oxathiaphospholane ring-opening with 3-hydroxypropionitrile, followed by treatment with DBU, afforded the corresponding monophosphate and monothiophosphate derivatives, respectively, through the elimination of polymer-bound ethylene episulfide. Reactions using this strategy offer the advantages of high regioselectivity, monosubstitution, and facile isolation and recovery of products.
Ahmadibeni, Yousef,Parang, Keykavous
p. 1955 - 1958
(2007/10/03)

Solid-phase reagents for selective monophosphorylation of carbohydrates and nucleosides
(Chemical Equation Presented) Two classes of aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to produce the corresponding polymer-bound phosphitylating reagents. These were reacted with a number of unprotected nucleosides and carbohydrates in the presence of 1H-tetrazole. Oxidation with tert-butyl hydroperoxide followed by removal of the cyanoethoxy group with 1,8-diazabicyclo-[5.4.0]undec-7-ene afforded the corresponding polymerbound phosphate diesters. Acidic cleavage of the p-acetoxybenzyl alcohol linker yielded monophosphorylated products with high regioselectivity and trapped linkers on the resins that can be reused.
Ahmadibeni, Yousef,Parang, Keykavous
p. 1100 - 1103
(2007/10/03)

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3
We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca 2+>Mg2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis-Menten kinetics with apparent Kms of 11, 10, 19 and 27 μM, respectively. In agreement with the Km values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.
Lavoie, Elise G.,Kukulski, Filip,Levesque, Sebastien A.,Lecka, Joanna,Sevigny, Jean
p. 1917 - 1926
(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)

Structure-activity studies of glucose transfer: Determination of the spontaneous rates of hydrolysis of uridine 5′-diphospho-α-D-glucose (UDPG) and uridine 5′-diphospho-α-D-glucuronic acid (UDPGA)
The pH-rate profiles for the hydrolysis of uridine 5′-diphospho-α-D-glucose (UDPG) and uridine 5′-diphospho-α-D-glucuronic acid (UDPGA) in aqueous solution have been measured. The results obtained and a comparison with other data suggests that the mechanism of hydrolysis of each activated glycosyl-donor at pH 1-4 probably involves the slow ionisation, via an SN1 process, of the neutral molecule to a glycosyl ion and UDP. From these data, the catalytic power (kcat/kuncat) of the glycosyltransferases has been estimated for the first time to be in the order of 1011-13.
Bedford, Colin T.,Hickman, Alan D.,Logan, Christopher J.
p. 2339 - 2345
(2007/10/03)

First synthesis of enantio-uracil dinucleotide, comparison of physicochemical properties of their enantiomers, and separation by chiral column chromatography
Enantio-uracil dinucleotide 5, which consists of two L-uridylic acids and one pyrophosphate, was synthesized for the first time in our laboratory. Benzolyated L-uridine was prepared by a stereoselective glycosylation of silylated uracil with L-1-O-acetyl-2,3,5-tri-O-benzoylribose (L-ABR 7). After deprotection, L-uridine 9 was converted to P1,P4-di(L- uridine 5′-) tetraphosphate tetrasodium salt (L-UP4U 5) by treatment of L-UMP morpholidate 10c with triethylammonium pyrophosphate (TEA-PPi 11b). Spectral data of synthesized L-UP4U 5 are given in the references. All spectral data were identical with those of UP4U 3 except the specific rotation, which showed a positive value compared to UP4U 3 having a negative value. Furthermore, the separation by chiral column chromatography was investigated.
Miyashita, Takanori,Sakata, Shinji,Hayakawa, Hiroyuki
p. 8605 - 8607
(2007/10/03)

Hydrolytic reactions of diribonucleoside 3′,5′-(3′-N-phosphoramidates): Kinetics and mechanisms for the P-O and P-N bond cleavage of 3′-amino-3′-deoxyuridylyl-3′,5′-uridine
Hydrolytic reactions of 3′-amino-3′-deoxyuridylyl-3′,5′-uridine (2a), an analogue of uridylyl-3′,5′-uridine having the 3′-bridging oxygen replaced with nitrogen, have been followed by RP HPLC over a wide pH range. The only reaction taking place under alkaline conditions (pH > 9) is hydroxide ion-catalyzed hydrolysis (first-order in [OH-]) to a mixture of 3′-amino-3′-deoxyuridine 3′-phosphoramidate (7) and uridine (4). The reaction proceeds without detectable accumulation of any intermediates. At pH 6-8, a pH-independent formation of 3′-amino-3′-deoxyuridine 2′-phosphate (3) competes with the base-catalyzed cleavage. Both 3 and in particular 7 are, however, rather rapidly dephosphorylated under these conditions to 3′-amino-3′-deoxyuridine (5). In all likelihood, both 3 and 7 are formed by an intramolecular nucleophilic attack of the 2′-hydroxy function on the phosphorus atom, giving a phosphorane-like intermediate or transition state. Under moderately acidic conditions (pH 2-6), the predominant reaction is acid-catalyzed cleavage of the P-N3′ bond (first-order in [H+]) that yields an equimolar mixture of 5 and uridine 5′-phosphate (6). The reaction is proposed to proceed without intramolecular participation of the neighboring 2′-hydroxyl group. Under more acidic conditions (pH 2), hydrolysis to 3 and 4 starts to compete with the cleavage of the P-N bond, and this reaction is even the fastest one at pH 1. Formation of 2′-O,3′-N-cyclic phosphoramidate as an intermediate appears probable, although its appearance cannot be experimentally verified. The rate constants for various partial reactions have been determined. The reaction mechanisms and the effect that replacing the 3′-oxygen with nitrogen has on the behavior of the phosphorane intermediate are discussed.
Ora, Mikko,Mattila, Kati,Loennberg, Tuomas,Oivanen, Mikko,Loennberg, Harri
p. 14364 - 14372
(2007/10/03)

Rate enhancements brought about by uridine nucleotides in the reduction of NAD+ at the active site of UDP-galactose 4-epimerase
UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP glucose. In the course of the reaction, the galacto- and glucopyranosyl rings undergo reversible oxidation to the 4-keto- glucopyranosyl ring by reaction with the enzyme-bound NAD+. The UDP-moiety of a substrate participates in catalysis by inducing a conformational change in the enzyme that enhances the chemical reactivity of NAD+ toward reducing agents. This is modeled by UMP-dependent reductive inactivation of the epimerase-NAD+ complex by various sugars as well as by borohydrides. The present work shows that UDP also activates the reduction of epimerase-bound NAD+. Furthermore, the reduction of epimerase-NAD+ by glucose at a very slow rate can be observed under anaerobic conditions in the absence of a uridine nucleotide. Comparisons of the second order rate constants for reduction of epimerase-NAD+ by glucose in the presence and absence of uridine nucleotides have allowed the magnitude of the rate enhancements brought about by UMP and UDP to be estimated. The rate enhancements by UMP and UDP correspond to decreases of 5.7 and 4.1 kcal mol-1, respectively, in the activation energy. A decrease of 4.0 kcal mol-1 in the activation energy for reduction by NaBH3CN was brought about by UMP-binding. The maximum increases in the reduction potential of epimerase-NAD+ induced by UMP- and UDP-binding are estimated to be 120 and 90 mV, respectively. The results are well correlated with the perturbations of the nicotinamide-13C NMR chemical shifts brought about by uridine nucleotides (Burke, J. R., and Frey, P. A. (1993) Biochemistry 32, 13220-12230). (C) 2000 Academic Press.
Liu, Yijeng,Arabshahi, Abolfazl,Frey, Perry A.
p. 29 - 37
(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)

Evidence for a stepwise mechanism of OMP decarboxylase [16]
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Ehrlich, Joel I.,Hwang, Chi-Ching,Cook, Paul F.,Blanchard, John S.
p. 6966 - 6967
(2007/10/03)

The synthesis of 2'-homouridine, its incorporation into a dinucleoside monophosphate and hydrolytic behaviour of the dimer
An efficient route to 2'-homouridine (1), a new nucleoside analogue, is reported that is based on an one reaction. This nucleoside has been incorporated into a dinucleotide monophosphate and hydrolytic studies on the dimer show that it does not behave like a ribonucleotide.
Pavey, John B. J.,Lawrence, Anthony J.,Potter, Andrew J.,Cosstick, Richard,O'Neil, Ian A.
p. 6967 - 6970
(2007/10/03)

Hydrolytic dethiophosphorylation and desulfurization of the monothioate analogues of uridine monophosphates under acidic conditions
The hydrolytic reactions of uridine 2′-, 3′- and 5′-phosphoromonothioates (2′-, 3′- and 5′-UMPS) under acidic and neutral conditions have been followed by HPLC. Under slightly acidic conditions (pH 2-5), only pH-independent dethiophosphorylation to uridine takes place. This reaction is 200- to 300-fold as fast as dephosphorylation of the corresponding uridine monophosphates (UMP), presumably due to higher stability of the thiometaphosphate monoanion compared to metaphosphate anion. At pH > 5, i.e. at pH > pKa2 of the thiophosphate moiety, the dethiophosphorylation is retarded with increasing basicity of the solution. At pH 1, acid-catalysed desulfurization of 2′- and 3′-UMPS to an isomeric mixture of 2′/3′-UMP competes with their dethiophosphorylation. This reaction is suggested to proceed by a nucleophilic attack of the neighbouring hydroxy group on phosphorus. No such reaction occurs with 5′-UMPS. In contrast to 2′- and 3′-UMP, no sign of interconversion of 2′- and 3′-UMPS is detected.
Ora, Mikko,Oivanen, Mikko,Loennberg, Harri
p. 771 - 774
(2007/10/03)

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)

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)

Oxidation of nucleic acid related compounds by the peroxodisulfate ion
The treatment of nucleic acid bases, nucleosides, and nucleotides with peroxodisulfate ion in a phosphate buffer solution at pH 7.0 or water at 70-75°C was investigated. The reaction of thymine and 5-methylcytosine nucleosides and nucleotides resulted in the oxidation of the 5-methyl groups. The oxidation products from 1,3-dimethyluracils and the time-course of the reaction of uracils led to two plausible reaction mechanisms for the oxidation of uracils.
Itahara,Yoshitake,Koga,Nishino
p. 2257 - 2264
(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
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)