- Characterization of Lhr-Core DNA helicase and manganese-dependent DNA nuclease components of a bacterial gene cluster encoding nucleic acid repair enzymes
-
Lhr is a large superfamily 2 helicase present in mycobacteria and a moderate range of other bacterial taxa. A shorter version of Lhr, here referred to as Lhr-Core, is distributed widely in bacteria, where it is often encoded in a gene cluster along with predicted binuclear metallo-phosphoesterase (MPE), ATP-dependent DNA ligase, and metallo-β-lactamase exonuclease enzymes. Here we characterized the Lhr-Core and MPE proteins from Pseudomonas putida. We report that P. putida Lhr-Core is an ssDNA-dependent ATPase/dATPase (Km, 0.37 mM ATP; kcat, 3.3 s–1), an ATP-dependent 3'-to-5' single-stranded DNA translocase, and an ATP-dependent 3'-to-5' helicase. Lhr-Core unwinds 3'-tailed duplexes in which the loading/tracking strand is DNA and the displaced strand is either DNA or RNA. We found that P. putida MPE is a manganese-dependent phosphodiesterase that releases p-nitrophenol from bis-p-nitrophenyl phosphate (kcat, 212 s–1) and p-nitrophenyl-5'-thymidylate (kcat, 34 s-1) but displays no detectable phosphomonoesterase activity against p-nitrophenyl phosphate. MPE is also a manganese-dependent DNA endonuclease that sequentially converts a closed-circle plasmid DNA to nicked circle and linear forms prior to degrading the linear DNA to produce progressively smaller fragments. The biochemical activities of MPE and a structure predicted in Phyre2 point to MPE as a new bacterial homolog of Mre11. Genetic linkage of a helicase and DNA nuclease with a ligase and a putative exonuclease (a predicted homolog of the SNM1/Apollo family of nucleases) suggests that these enzymes comprise or participate in a bacterial DNA repair pathway.
- Ejaz, Anam,Shuman, Stewart
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p. 17491 - 17504
(2018/11/23)
-
- Enzymatic Production of Non-Natural Nucleoside-5′-Monophosphates by a Thermostable Uracil Phosphoribosyltransferase
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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
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p. 439 - 448
(2017/12/13)
-
- Enzyme Activation with a Synthetic Catalytic Co-enzyme Intermediate: Nucleotide Methylation by Flavoenzymes
-
To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.
- Bou-Nader, Charles,Cornu, David,Guerineau, Vincent,Fogeron, Thibault,Fontecave, Marc,Hamdane, Djemel
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supporting information
p. 12523 - 12527
(2017/09/12)
-
- New phosphorylating reagents for deoxyribonucleosides and oligonucleotides
-
New phosphorylating reagents 1 and 2 were prepared in three steps from 4-hydroxybenzaldehyde. They showed good efficiency in the solid phase synthesis of 5′-phosphate monoester nucleosides. End-phosphate DNA sequence synthesis demonstrated the efficiency of the new reagents (1 and 2) according to the general procedure of automated DNA synthesis. The oxidation of P(III) to P(V) and the removal of benzyl protecting groups were achieved in a single step by treatment with a 0.02 M I2/pyridine/H2O solution. Due to this one-pot treatment, it is possible to use the phosphorylating reagents (1 and 2) for the synthesis of base-sensitive ODNs. The reagents 1 and 2 are unique among phosphorylating reagents.
- Romanucci, Valeria,Zarrelli, Armando,Guaragna, Annalisa,Di Marino, Cinzia,Di Fabio, Giovanni
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supporting information
p. 1227 - 1229
(2017/03/02)
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- Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-hydroxy-dCMP
-
Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy-dCMP was found to be strongly dependent on [Mg2+], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells.
- Ludwiczak, Jan,Maj, Piotr,Wilk, Piotr,Fraczyk, Tomasz,Ruman, Tomasz,Kierdaszuk, Borys,Jarmu?a, Adam,Rode, Wojciech
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p. 1333 - 1341
(2016/04/09)
-
- Synthesis and application of isotopically labeled flavin nucleotides
-
Flavin nucleotides, i.e. flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are utilized as prosthetic groups and/or substrates by a myriad of proteins, ranging from metabolic enzymes to light receptors. Isotopically labeled flavins have served as invaluable tools in probing the structure and function of these flavoproteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides from commercially available labeled riboflavin and ATP. The synthetic procedure employs a bifunctional enzyme, Corynebacterium ammoniagenes FAD synthetase, that sequentially converts riboflavin to FMN and then to FAD. The final flavin product (FMN or FAD) is controlled by the concentration of ATP in the reaction. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors. Flavin nucleotides are utilized as prosthetic groups and/or substrates by a myriad of proteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors.
- Mishanina, Tatiana V.,Kohen, Amnon
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p. 370 - 375
(2015/08/06)
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- Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
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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)
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- Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides
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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)
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- Concerted versus stepwise mechanism in thymidylate synthase
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Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.
- Islam, Zahidul,Strutzenberg, Timothy S.,Gurevic, Ilya,Kohen, Amnon
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supporting information
p. 9850 - 9853
(2014/08/05)
-
- Synthesis of nucleoside 5′-tetraphosphates containing terminal fluorescent labels via activated cyclic trimetaphosphate
-
2′-Deoxynucleotide 5′-tetraphosphates in which a fluorescent label is attached to the terminal phosphate are used as key reagents in high-throughput DNA sequencing techniques and in single nucleotide polymorphism typing assays. We demonstrate that this class of compounds can be prepared by reacting fluorophores such as 7-hydroxy-4-methylcoumarin, methylfluorescein, fluorescein and resorufin with an activated form of cyclic trimetaphosphate to give intermediate 11. Reaction of 11 with 2′-deoxynucleoside 5′-monophosphates or a nucleoside 5′-monophosphate gave the target compounds in good yield.
- Mohamady, Samy,Taylor, Scott D.
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p. 2308 - 2313
(2014/04/03)
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- Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site
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Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e., the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 ? away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ~7-fold but does not affect the magnitude or temperature dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with polyglutamyl substitutes as species-specific antibiotic drugs.
- Wang, Zhen,Sapienza, Paul J.,Abeysinghe, Thelma,Luzum, Calvin,Lee, Andrew L.,Finer-Moore, Janet S.,Stroud, Robert M.,Kohen, Amnon
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supporting information
p. 7583 - 7592
(2013/07/11)
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- Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana
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Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development. 2012 The Authors Journal compilation
- Clausen, Anders R.,Girandon, Lenart,Ali, Ashfaq,Knecht, Wolfgang,Rozpedowska, Elzbieta,Sandrini, Michael P. B.,Andreasson, Erik,Munch-Petersen, Birgitte,Piskur, Jure
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p. 3889 - 3897
(2013/01/13)
-
- Synthesis of L-altrose and some derivatives
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A convenient approach to the chemical synthesis of L-altrose (1) and its 6-deoxy derivative 2 has been developed by starting from D-galactose (9) and D-fucose (10), respectively. The 5-epimerization by a Mitsunobu inversion of the open-chain D-hexoses was the key step for these routes. Furthermore, the conversion of 2 into peracetylated TDP-6-deoxy-α-L-altrose (3a) was achieved by the cycloSal approach. However, the final deacetylation led to an unexpected side-reaction resulting in the previously unknown 6-deoxy-α-L-altropyranose 1,3-cyclophosphate (4).
- Lunau, Nathalie,Meier, Chris
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p. 6260 - 6270
(2013/01/15)
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- Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase
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Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics.
- Mishanina, Tatiana V.,Koehn, Eric M.,Conrad, John A.,Palfey, Bruce A.,Lesley, Scott A.,Kohen, Amnon
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experimental part
p. 4442 - 4448
(2012/04/23)
-
- Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics
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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.
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supporting information; experimental part
p. 14452 - 14459
(2011/11/04)
-
- 3-Acetyloxy-2-cyano-2-(alkylaminocarbamoyl)propyl groups as biodegradable protecting groups of nucleoside 5'-mono-phosphates
-
Thymidine 5'-bis[3-acetyloxy-2-cyano-2-(2-phenylethylcarbamoyl)propyl] phosphate (1) has been prepared and the removal of phosphate protecting groups by hog liver carboxyesterase (HLE) at pH 7.5 and 37 °C has been followed by HPLC. The first detectable intermediates are the (Rp)-and (S p)-diastereomers of the monodeacetylated triester 14, which subsequently undergo concurrent retro-aldol condensation to diester 4 and enzyme-catalyzed hydrolysis to the fully deacetylated triester 15. The former pathway predominates, representing 90% of the overall breakdown of 14. The diester 4 undergoes the enzymatic deacetylation 700 times less readily than the triester, but gives finally thymidine 5'-monophosphate as the desired main product. To elucidate the potential toxicity of the electrophilic 2-cyano-N-(2-phenylethyl)acrylamideby-product 17 released upon the deprotection, the hydrolysis of 1 has also been studied in the presence of glutathione (GSH).
- Ora, Mikko,Maentyvaara, Anne,Loennberg, Harri
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p. 552 - 566
(2011/03/22)
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- Prodrug activation by Cryptosporidium thymidine kinase
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Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of kcat= 0.28 s-1 and Km,ATP = 140 μM are similar to those of human TK1, the value of Km(thymidine) = 48 μM is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.
- Sun, Xin E.,Sharling, Lisa,Muthalagi, Mani,Mudeppa, Devaraja G.,Pankiewicz, Krzysztof W.,Felczak, Krzysztof,Rathod, Pradipsinh K.,Mead, Jan,Striepen, Boris,Hedstrom, Lizbeth
-
body text
p. 15916 - 15922
(2011/04/15)
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- Nonpolar nucleoside mimics as active substrates for human thymidine kinases
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We describe the use of nonpolar nucleoside analogues of systematically varied size and shape to probe the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate,thymidine. Comparison of polar thymidine with a nonpolar isostere, 2,4- difluorotoluene deoxyriboside, as substrates for the two enzymes establishes that TK1 requires electrostatic complementarity to recognize the thymine base with high efficiency. Conversely, TK2 does not and phosphorylates the hydrophobic shape mimic with efficiency nearly the same as the natural substrate. To test the response to nucleobase size, thymidine-like analogues were systematically varied by replacing the 2,4 substituents on toluene with hydrogen and the halogen series (H, F, Cl, Br, I). Both enzymes showed a distinct preference for substrates having the naturalsize. To examine the shape preference, we prepared four mono- and diflu orotoluene deoxyribosides with varying positions of substitutions. WhileTK1 did not accept these nonpolar analogues as substrates, TK2 did show varying levels of phosphorylation of the shape-varied set. This latter enzyme preferred toluene nucleoside analogues having steric projections at the 2 and 4 positions, as is found in thymine, and strongly disfavore d substitution at the 3-position. Steady-state kinetics measurements showed that the 4-fluoro compound (7) had an apparent Vmax/Kmax value within 14-fold of the natural substrate, and the 2,4-difluoro compound (1), which is the closest isostere of thymidine, had a value within 2.5-fold. The results establish that nucleoside recognition mechanisms for the two classes of enzymes are very different. On the basis of these data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for future bioactive molecules.
- Jarchow-Choy, Sarah K.,Sjuvarsson, Elena,Sintim, Herman O.,Eriksson, Staffan,Kool, Eric T.
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experimental part
p. 5488 - 5494
(2009/09/24)
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- Synthesis of 3'O-(2-cyanoethyl)-2'-deoxythymidine-5'-phosphate as a model compound for evaluation of Cyanoethyl Cleavage
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An essential and challenging task during the development of our sequencing-by-synthesis (SBS) technique is the evaluation of efficient cyanoethyl (CE) cleavage conditions. For this purpose 3'-O-(2-cyanoethyl)-2'- deoxythymidine-5'-phosphate as a model compound as well as a short DNA oligomer bearing the CE function as terminal group were synthesized and used for various deprotection experiments. As it is already known for 2'-O-CE-protected RNA oligonucleotides, the CE function can be cleaved with tetrabutylammonium fluoride (TBAF) in THF. Indeed, by using 3'-O-(2-cyanoethyl)-2'-deoxythymidine- 5'-phosphate as a simple model compound for cleavage tests, we found out that the 3'-O-CE function is quantitatively cleaved with 1 m TBAF in THF. However, the CE group is also cleaved by other small bases like hydroxy groups under alkaline conditions. The CE cleavage with TBAF in THF gives the fastest and quantitative removal of the CE group under mild conditions for our sequencing-by-synthesis (SBS) application. The efficient removal of the 3'-CE group is crucial for the proof-of-principle of our SBS approach using dye-labeled 3'-CE-blocked dNTPs, which is currently under investigation. Herein we describe the application of 3'-O-(2-cyanoethyl)-2'-deoxythymidine-5'- phosphate as model compound for the development of reversible terminators for the SBS technique. Furthermore we suggest that nucleoside phosphates bearing any removable 3'-modification might be suitable model compounds for cleavage studies in a heterogeneous environment comparable to an oligonucleotide/aprotic solvent system.
- Keller, Angelika C.,Serva, Saulius,Knapp, Diana C.,Kwiatkowski, Marek,Engels, Joachim W.
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experimental part
p. 515 - 534
(2010/02/28)
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- Biodegradable protections for nucleoside 5′-monophosphates: Comparative study on the removal of O-acetyl and O-acetyloxymethyl protected 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl groups
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(Chemical Equation Presented) The applicability of 3-acetyloxy-2,2- bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl) propyl groups as biodegradable phosphate protecting groups for nucleoside 5′-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2- bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5′-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl) propyl 5′-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5′-monophosphate (5′-TMP) is quantitative, while conversion of 1 to 5′-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5′-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.
- Ora, Mikko,Taherpour, Sharmin,Linna, Risto,Leisvuori, Anna,Hietamaeki, Emilia,Poijaervi-Virta, Paeivi,Beigelman, Leonid,Loennberg, Harri
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experimental part
p. 4992 - 5001
(2009/10/17)
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- Hydrolytic reactions of thymidine 5′-O-phenyl-N- alkylphosplioramidates, models of nucleoside 5′-monophosphate prodrugs
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To obtain detailed data on the kinetics of hydrolytic reactions of triester-like nucleoside 5′-O-aryl-N-alkylphosphoramidates, potential prodrugs of antiviral nucleoside monophosphates, the hydrolysis of diastereomeric (Rp/Sp) thymidine 5′-{O-phenyl-N- [(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate) (3), a phosphoramidate derived from the methyl ester of L-alanine, has been followed by reversed-phase HPLC over the range from H0 = 0 to pH 8 at 90°C. According to the time-dependent product distributions, the hydrolysis of 3 proceeds at pH 5, the predominant reaction is hydrolysis of the carboxylic ester linkage followed by intramolecular displacement of a phenoxide ion by the carboxylate ion and hydrolysis of the resulting cyclic mixed anhydride into an acyclic diester-like thymidine 5′-phosphoramidate. The latter product accumulated quantitatively without any indication of further decomposition. Hydroxide-ioncatalyzed P-OPh bond cleavage of the starting material 3 occurred as a side reaction. Comparative measurements with thymidine 5′-{N-[(1S)-2- oxo-2-methoxy-1-methylethyl]phosphoramidate( (4) revealed that, under acidic conditions, this diester-like compound is hydrolyzed by P-N bond cleavage three orders of magnitude more rapidly than the triester-like 3. At pH > 5, the stability order is reversed, with 3 being hydrolyzed six times as rapidly as 4. Mechanisms of the partial reactions are discussed.
- Ora, Mikko,Ojanperae, Jarno,Loennberg, Harri
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p. 8591 - 8599
(2008/09/16)
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- Purification and characterization of wild-type and mutant TK1 type kinases from Caenorhabditis elegans
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Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ~60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher kcat but unchanged Km. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 μg/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability. Copyright Taylor & Francis Group, LLC.
- Skovgaard,Munch-Petersen
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p. 1165 - 1169
(2007/10/03)
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- Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties
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Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5′-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication. The cell cycle specific activity of TK1 is regulated at the transcriptional level, but posttranslational mechanisms seem to play an important role for the level of functional TK1 protein as well. Thus, the C-terminal of TK1 is known to be essential for the specific degradation of the enzyme at the G2/M phase. In this work, we have studied the effect of deletion of the C-terminal 20, 40, and 44 amino acids of TK1 on in vitro stability, oligomerization, and enzyme kinetics. We found that deletion of the C-terminal fold markedly increased the stability as well as the catalytic activity. Copyright Taylor & Francis Group, LLC.
- Zhu,Harlow,Berenstein,Munch-Petersen,Munch-Petersen
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p. 1185 - 1188
(2007/10/03)
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- A lag-phase in the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mechanistic missing link
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An unexpected substrate-dependent lag-phase, found in the single turnover reduction of FDTS bound flavin, sheds light on the molecular mechanism of this alternative thymidylate synthase. The Royal Society of Chemistry 2006.
- Mason, Aaron,Agrawal, Nitish,Washington, M. Todd,Lesley, Scott A.,Kohen, Amnon
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p. 1781 - 1783
(2008/10/09)
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- Improved synthesis of oligonucleotides with an allylic backbone. Oligonucleotides containing acyclic, achiral nucleoside analogues: N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases
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An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (ΔTm per modification -1 to -5°C). The Royal Society of Chemistry 2006.
- Dahl, Britta M.,Henriksen, Ulla,Dahl, Otto
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p. 1115 - 1123
(2008/02/04)
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- Solid-phase reagents for selective monophosphorylation of carbohydrates and nucleosides
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(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
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p. 1100 - 1103
(2007/10/03)
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- An efficient reagent for the phosphorylation of deoxyribonucleosides, DNA oligonucleotides, and their thermolytic analogues
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(Chemical Equation Presented) The phosphoramidite 11 was prepared in three steps from methyl 2-mercaptoacetate and demonstrated efficiency in the synthesis of conventional 5′-/3′-phosphate/thiophosphate monoester derivatives of 2′-deoxyribonucleosides and DNA oligonucleotides. Moreover, the use of 11 has enabled the preparation of the dinucleoside phosphorothioate analogue 26 in high yields (>95%) with minimal cleavage (2%) of the thermolytic thiophosphate protecting group.
- Ausin, Cristina,Grajkowski, Andrzej,Cieslak, Jacek,Beaucage, Serge L.
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p. 4201 - 4204
(2007/10/03)
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- Polymer-bound oxathiaphospholane: A solid-phase reagent for regioselective monothiophosphorylation and monophosphorylation of unprotected nucleosides and carbohydrates
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(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
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p. 1955 - 1958
(2007/10/03)
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- Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases
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The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-β-D-ribofuranosyl)- isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were cat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1′-(2′-deoxy-β-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1′-(2′-deoxy-β-D-ribofuranosyl))-2,4-difluoro-5- iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-β-L-ribofuranosyl)]- aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.
- Al-Madhoun, Ashraf Said,Eriksson, Staffan,Wang, Zhi-Xian,Naimi, Ebrahim,Knaus, Edward E.,Wiebe, Leonard I.
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p. 1865 - 1874
(2007/10/03)
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- Novel method for the immobilization of nucleotides.
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[reaction: see text] A novel method for the immobilization of nucleotides has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes nucleophilic attack by long-chain alkylamine-controlled pore glass (LCAA-CPG) to generate an immobilized nucleotide. Quantification of nucleotide loading was accomplished by acidic hydrolysis of the P-N bond and subsequent HPLC analysis of TMP in the presence of an internal standard. Typical nucleotide loadings of 51-59 micromol/g of support were observed.
- Freel Meyers, Caren L,Borch, Richard F
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p. 341 - 344
(2007/10/03)
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- The Interaction of Trihalogenoacetic Anhydrides and Trihalogenoacetyl Chlorides with Thymidine 5′-Phosphate as an Approach to New Activating Agents in the Phosphorylation Reactions for Nucleotides
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The interaction of thymidine 5′-phosphate with trichloroacetic anhydride, trichloroacetyl chloride, and tribromoacetyl bromide was studied in dimethylformamide and acetonitrile in the presence of tertiary amines. The first two reactions gave the mixed anhydride of trichloroacetic and thymidylic acids (acyl phosphate) as the major product and P1,P 2-dithymidine 5′-pyrophosphate as the byproduct. The third reaction proceeded by a more complicated mechanism and mainly led to substituted polyphosphates. The subsequent treatment of the reaction mixtures with morpholine resulted in thymidine 5′-phosphoromorpholidate in a high yield. The phosphorylating activities of the trichloroacetyl and tribromoacetyl phosphates were 77 and 89%, respectively.
- Bogachev,Ulanov
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- Enzymatic phosphorylation of unnatural nucleosides
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In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA.
- Wu, Yiqin,Fa, Ming,Tae, Eunju Lee,Schultz, Peter G.,Romesberg, Floyd E.
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p. 14626 - 14630
(2007/10/03)
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- Evidence for direct attack by hydroxide in phosphodiester hydrolysis
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Phosphodiester hydrolysis has been the subject of intense study due to its importance in biology. Despite the numerous significant analyses of phosphodiester cleavage mechansim, comparatively little is known about the nucleophiles in these reactions. To determine whether hydroxide acts as a nucleophile or a general base in the hydrolysis of thymidine-5′-p-nitrophenyl phosphate,we determined solvent deuterium isotope effects (D2Ok), ionic strength effects, and 18O isotope effects on the solvent nucleophile (18knuc). The D2Ok for hydroxide-catalyzed phosphodiester hydrolysis is slightly inverse (0.9 ± 0.1), suggesting that a proton transfer does not occur in the transition state. A significant α effect is observed with hydroperoxide, demonstrating that oxyanions can act as nucleophiles in the reaction. Additionally, the ionic strength dependencies of hydroxide and hydroperoxide catalysis are indistinguishable, suggesting that they act by the same mechanism. Finally, the 18knuc for the hydroxide-catalyzed reaction is 1.068 ± 0.007, well in excess of the equilibrium 18O isotope effect between water and hydroxide (1.040 ± 0.003). Together, the data are most consistent with direct nucleophilic attack by hydroxide. From the observed 18knuc and the known equilibrium component, the kinetic component of the isotope effect was calculated to be 1.027 ± 0.010. This large kinetic component suggests that little bond order to the nucleophile occurs in the transition state. Copyright
- Cassano, Adam G.,Anderson, Vernon E.,Harris, Michael E.
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p. 10964 - 10965
(2007/10/03)
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- Synthesis and enzymatic characterization of P1-thio-P2-oxo trideoxynucleoside diphosphates having AZT, FdU, or dT at the 3'-position
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Model compounds for oligonucleotide-prodrugs, P1-thio-P2-oxo-tri-deoxyribonucleoside diphosphates: d[G5C0X] and d[TsA0X] (X = AZT, FdU or dT) have been prepared, and their hydrolyses by snake venom phosphodiesterase and nuclease S1 are described.
- Oetvoes, Laszlo,Bajor, Zoltan,Kraicsovits, Ferenc,Sagi, Gyula,Tegyey, Zsuzsanna
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- Mutation of Gln125 to Asn selectively abolishes the thymidylate kinase activity of herpes simplex virus type 1 thymidine kinase
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The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We have now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) residue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked contrast with wild-type HSV-1 TK, which displays both thymidine kinase and thymidylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phosphorylate pyrimidine nucleoside monophosphates but retained significant phosphorylation activity for thymidine and a series of antiherpetic pyrimidine and purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidylate kinase activity resulted in a 100-fold accumulation of the monophosphate form of (E)-5(2-bromovinyl)-2′-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise to a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q125N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteosarcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the presence of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was found to result in a multilog decrease in the cytostatic activity of those pyrimidine nucleoside analogs that in their monophosphate form do not have marked affinity for thymidylate synthase [i.e., 1-β-D-arabinofuranosylthymine and (E)-5-(2-bromovinyl)-1-β-D-arabinofuranosyluracil].
- Degreve, Bart,Esnouf, Robert,De Clercq, Erik,Balzarini, Jan
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p. 285 - 293
(2007/10/03)
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- Activation mechanisms of nucleoside phosphoramidate prodrugs
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A series of thymidine and tetrahydrofurfuryl phosphoramidates bearing haloethyl or piperidyl substituents was synthesized and used to investigate the activation mechanisms of nucleoside phosphoramidate prodrugs. Structure assignments for the tetrahydrofurfuryl reaction products were confirmed by comparison to authentic samples. Structural assignments for thymidine phosphoramidate reaction products were made by analogy to the tetrahydrofurfuryl products. Generation of the phosphoramidate anion leads to cyclization and subsequent nucleophilic attack at carbon and phosphorus of the resulting aziridinium ion intermediate to give the observed products. Nucleophilic attack by water at carbon and phosphorus occurs without selectivity, supporting a mechanism of action of haloethylamine nucleoside prodrugs involving intracellular release of the nucleotide. Activation of the benzotriazolyl piperidyl phosphoramidates is followed by P-N bond hydrolysis; this reaction is subject to specific acid catalysis and to nucleophilic catalysis by 1-hydroxybenzotriazole. These results suggest that the mechanism of action of the piperidyl nucleoside phosphoramidates involves the intracellular release of the active nucleotide following P-N bond cleavage, presumably by the action of an endogenous phosphoramidase.
- Freel Meyers,Borch
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p. 4319 - 4327
(2007/10/03)
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- A new method for the phosphorylation of nucleosides
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A new phosphorylating reagent, 2-O-(4,4'-dimethoxytrityl)ethylsulfonylethan-2'-yl-phosphate (1), was developed for the phosphorylation of primary and secondary alcohols of nucleosides. In the many examples studied, yields in the phosphorylation step were excellent (~80 to 95%). There is potential for wide applicability of this procedure, not only in nucleoside and nucleotide chemistry, but also in the phosphorylation of biomolecules such as carbohydrates and amino acids. (C) 2000 Elsevier Science Ltd.
- Taktakishvili,Nair
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p. 7173 - 7176
(2007/10/03)
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- Interactions between aminocalixarenes and nucleotides or nucleic acids
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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
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p. 1797 - 1803
(2007/10/03)
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- Interaction of the recombinant Herpes Simplex Virus type 1 thymidine kinase with thymidine and aciclovir: A kinetic study
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Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK) is a key target for antiviral therapy and it phosphorylates a broad spectrum of nucleosides and nucleotides. We report the results from kinetic and inhibition experiments with HSV 1 TK, and show that there is a preferred, but not exclusive, binding order of substrates, i.e. dT binds prior to ATP. Furthermore, the results provide new informations on the mechanism of binding suggesting that HSV1 TK undergoes conformational changes during the catalytic cycle.
- Kussmann-Gerber, Susanna,Wurth, Christine,Scapozza, Leonardo,Pilger, Beatrice D.,Pliska, Vladimir,Folkers, Gerd
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p. 311 - 330
(2007/10/03)
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- Pyridoxal-catalyzed release of nucleotides
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The synthesis of phosphoric esters linking the 5' alcohol of thymidine to the alcohol moiety of L-serine derivatives is reported, together with the pyridoxal-catalyzed β-elimination reaction releasing thymidylate from such compounds. The process was extended to the release of thymidine di- and tri- phosphate.
- Pochet, Sylvie,Beaussire, Jean-Jacques,Dugue, Laurence,Marliere, Philippe
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p. 1019 - 1020
(2007/10/03)
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- OLIGODEOXYNUCLEOSIDE METHYLPHOSPHONATES: SYNTHESIS AND ENZYMIC DEGRADATION
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The automated synthesis of oligodeoxynucleosides containing methylphosphonate linkages has been improved by using nucleoside methylphosphonamidites as starting materials.It was found that two adjacent methylphosphonate linkages protect an oligomer from degradation by snake venom phosphodiesterase or spleen phosphodiesterase.
- Agrawal, Sudhir,Goodchild, John
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p. 3539 - 3542
(2007/10/02)
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- PHOSPHORYLATING AGENT FOR THE SYNTHESIS OF OLIGONUCLEOTIDE WITH ALIPHATIC AMINO GROUP AT 5' END
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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)
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- Synthesis and Properties of Phosphoramidite Derivatives of Modified Nucleosides
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Protected N6-methyl-2'-deoxyadenosine (d-m6A), 2-amino-2'-deoxyadenosine (d-a2A), 2'-deoxyinosine (dI), 5-methyl-2'-deoxycytidine (d-m5C) and deoxyuridine (dU) were reacted with bis(diisopropylamino)methoxyphosphine in the presence of diisopropylammonium tetrazolide as the activating reagent to give the corresponding phosphoramidite derivatives in yields of 100, 65, 90, 78 and 65 percent respectively.The 31P-nuclear magnetic resonance spectra of the products were measured.Using these compounds, dinucleotides and trinucleotides were synthesized on a longchain alkylamine controlled pore glass (LCA-CPG) in quantitative yields.The stability of 6-methyldeoxyadenosine and N,N-diisobutyryl-2-aminodeoxyadenosine to acid was examined.When protected di- and trinucleotides (m6A-T, a2A-T, T-m6A-T, T-a2A-T) bound to the support (LCA-CPG) were treated with 3 percent trichloroacetic acid in dichloromethane, depurination was negligible within 10 min (dinucleotide) or 60 min (trinucleotide).Keywords - phosphoramidite; solid-phase synthesis; phosphite method; acid treatment; enzyme degradation
- Tanaka, Toshiki,Tamatsukuri, Sigeru,Ikehara, Morio
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p. 2044 - 2048
(2007/10/02)
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- A CHEMICAL 5'-PHOSPHORYLATION OF OLIGODEOXYRIBONUCLEOTIDES THAT CAN BE MONITORED BY TRITYL CATION RELEASE
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A new phosphoramidite-derived reagent, (2-cyanoethoxy)-2-(2'-O-4,4'-dimethoxytrityloxyethylsulfonyl)ethoxy-N,N-diisopropylaminophosphine, for the 5'-phosphorylation of oligodeoxyribonucleotides has been developed.Phosphorylation efficiency can be determined by the release of 4,4'-dimethoxytrityl cation in acid.
- Horn, Thomas,Urdea, Mickey S.
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p. 4705 - 4708
(2007/10/02)
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- S-4-METHYLPHENYL-O,O-BIS(1-BENZOTRIAZOLYL)PHOSPHOROTHIOATE: A VERSATILE PHOSPHORYLATING AGENT
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The phosphorylating agent obtained by treatment of S-4-methylphenyl phosphorodichloridothioate with 1-hydroxybenzotriazole can not only be applied for the introduction of polyphosphate functions at the terminal ends of nucleic acids, but also for the formation of 3'-5'-phosphotriester linkages.
- Wreesmann, C. T. J.,Fidder, A.,Veeneman, G. H.,van der Marel, G. A.,van Boom, J. H.
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p. 933 - 936
(2007/10/02)
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