- Tri-O-acyl 2-deoxy-D-ribofuranose: An effective enzyme-assisted one-pot synthesis from 2-deoxy-D-ribose and transformation into 2′-deoxynucleosides
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Regioselective lipase-catalyzed (Novozym 435 from Candida antartica) acylations of unprotected 2-deoxy-D-ribose and D-ribose, by use of propionic anhydride, afforded the novel 5-0-monoacylated derivatives 1 and 4, respectively. Subsequent addition of acetic anhydride and pyridine into the reaction mixtures gave the corresponding per-0-acylated pentofuranoses 2 and 5 in almost quantitative overall yields from the unprotected carbohydrates. The versatility of compound 2 as an universal synthon for synthesis of anomeric mixtures of 2′-deoxynucleosides is demonstrated.
- Prasad, Ashok K.,Sorensen, Mads D.,Parmar, Virinder S.,Wengel, Jesper
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- Active species for Ce(IV)-induced hydrolysis of phosphodiester linkage in cAMP and DNA
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The hydrolysis of cyclic adenosine 3′,5′-monophosphate and 2′-deoxythymidylyl(3′-5′)2′-deoxythymidine by Ce(NH 4)2(NO3)6 was kinetically studied. The rate of hydrolysis was fairly proportional to the concentration of [Ce 2IV (OH)4]4+, showing that this is the catalytically active species. According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce2IV (OH)4] 4+ cluster are bridged by two OH residues. Upon the complex formation with H2PO4- (a model compound for the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of phosphodiesters has been proposed on the basis these results. Copyright Taylor & Francis Group, LLC.
- Sumaoka, Jun,Furuki, Kenichiro,Kojima, Yuki,Shibata, Masahiko,Hirao, Kimihiko,Takeda, Naoya,Komiyama, Makoto
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- THE USE OF ZINC BROMIDE FOR REMOVAL OF DIMETHOXYTRITYL ETHERS FROM DEOXYNUCLEOSIDES
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Zinc bromide has been observed to detritylate specifically 5'-trityldeoxy-deoxynucleosides whereas 3'-trityl ethers are not cleaved.Of additional practical consideration and in contrast to protic acids, no depurination was observed with this reagent.
- Matteucci, M. D.,Caruthers, M. H.
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- Sensitized two-photon photochemical deprotection
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The photoremovable protecting group NPPoc has little sensitivity to two-photon excitation, limiting its use in applications requiring high spatial control of its photochemistry. In the presence of a triplet sensitizer with a large two-photon absorption cross-section, however, the two-photon uncaging action cross-section is improved to levels useful in a variety of applications.
- Pirrung, Michael C.,Dore, Timothy M.,Zhu, Yue,Rana, Vipul S.
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- Synthesis of thymidine from 5-iodo-2'-deoxyuridine
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A simple, high yield synthesis of thymidine from 5-iodo-2'-deoxyuridine is described, using tetramethyltin and tetrakis(triphenylphosphine)palladium(0) in hexamethylphosphoric triamide. Likewise, 5-phenyl- and 5-vinyl-2'-deoxyuridine can be obtained, and through reduction of the latter the 5-ethyl analogue is also at hand.
- Herdewijn,Kerremans,Wigerinck,Vandendriessche,Van Aerschot
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- Synthesis and stereochemical assignment of DNA spore photoproduct analogues
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Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosyl-methionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1a and 1b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2a and 2b with closed backbones were prepared. The key step of the synthesis of 2a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transfer red to the data for isomers 1a and 1b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis,[1] in that only the 5 isomer la is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.
- Friedel, Marcus G.,Pieck, J. Carsten,Klages, Jochen,Dauth, Christina,Kessler, Horst,Carell, Thomas
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- 1:1 and 1:2 complexes of Bu4NF and BF3·Et2O: Unique properties as reagents for cleavage of silyl ethers
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A new method for removal of trialkylsilyl groups from silyl ethers using Complex A and Complex B, generated from tetrabutylammonium fluoride (TBAF) and BF3·Et2O has been developed. The desilylation by use of these boron complexes is significantly affected by the steric factor on the Si atom and the reagent.
- Kawahara,Wada,Sekine
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- New acid photogenerators based on thioxanthen-9-one sulfonium derivatives for detritylation in the oligonucleotide synthesis
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Photochemical activity of a number of cationic thioxanthen-9-one derivatives for the formation of the trityl cation was studied, which resulted in the selection of compounds suitable for photodetritylation. 10-(4-Heptyloxyphenyl)-9-oxo-2-(N,N,N-triethylammonio)methyl-9H-thioxanthenium bis(hexafluorophosphate) and 2-methyl-, 2-(2-methyl-1-propanoyl-2-tosyl)-, 1-chloro-4-propoxy-, and 2,4-diethyl-10-(4-heptyloxyphenyl)-9-oxo-9H- thioxanthenium hexafluorophosphates were found to be photoactivators of detritylation of 5′-O-(4,4′-dimethoxytrityl)thymidine. The detritylation reaction is the most efficient in dichloromethane. 2,4-Diethyl-10-(4-heptyloxyphenyl)-9-oxo-9H-thioxanthenium hexafluorophosphate was used as a detritylation photoactivator in the oligonucleotide synthesis using an automated DNA synthesizer. The yield in the elongation step of the oligonucleotide chain was 98%.
- Shelkovnikov,Loskutov,Vasil'Ev,Shekleina,Ryabinin,Sinyakov
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- Tuning the stability of alkoxyisopropyl protection groups
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Five different 2-alkoxypropan-2-yl groups are introduced as acid-labile protecting groups for the 5’- and 3’-hydroxy groups of a 2’-deoxynucleoside. All studied protecting groups were readily introduced with good to excellent yields using the appropriate enol ether as a reagent and 0.5 to 1 mol % p-toluenesulfonic acid as a catalyst. The protected compounds could be purified by silica gel column chromatography without degradation. The compatibility of these protecting groups in parallel use with benzoyl and silyl groups was verified. The stabilities of the different alkoxy acetal protecting groups were compared by following the kinetics of their hydrolysis at 25.0 °C in buffered solutions through an HPLC method. In the pH range 4.94 to 6.82 the hydrolysis reactions are of first order in the hydronium ion. The rate of hydrolysis correlates with the electron-donating or electron-withdrawing ability of the corresponding alkoxy group. The studied 2-alkoxypropan-2-yl groups and the relative rate constants for their cleavage from the 5’-hydroxy group of 2’-deoxythymidine were: cyclohexyloxy (krel = 7.7), isopropoxy (7.4), methoxy (1), benzyloxy (0.6) and 2,2,2-trifluoroethyloxy (0.04). The attachment of the same groups to the 3’-hydroxy group are from 1.3 to 1.9-fold more stable. The most reactive of these acetone-based acetal groups are faster removed than a dimethoxytrityl group, and they are easier to cleave completely in solution. The structural variation allows steering of the stability and lipophilicity of the compounds in some range.
- Liang, Zehong,Koivikko, Henna,Oivanen, Mikko,Heinonen, Petri
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- DIALKYL ALUMINIUM CHLORIDE: A REAGENT FOR REMOVAL OF TRITYL GROUP FROM TRITYL ETHERS OF DEOXYNUCLEOSIDES, DEOXYNUCLEOTIDES, AND OLIGODEOXYNUCLEOTIDES
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Detritylation of N-acyl-5'-O-tritylated deoxynucleosides, deoxynucleotides, and oligodeoxynucleotides have been quantitatively achieved in minutes at room temperature by using diethylaluminium chloride or diisobutylaluminium chloride.Reactions take place in unpolar solvents in homogeneous phase under completely aprotic conditions.
- Koester, H.,Sinha, N. D.
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- Synthesis and hydrolytic properties of thymidine boranomonophosphate
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Detailed synthetic procedures of thymidine 5′-O-(P-borano) monophosphate (TMPB) via an H-phosphonate approach are reported. The hydrolytic properties of TMPB in pH 1.8 and 6.76 buffers at 22 or 37 C were studied using LC-MS. One nucleoside product, thymid
- Xu, Zhihong,Sergueeva, Zinaida A.,Shaw, Barbara Ramsay
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- Photochemical Control of DNA Structure through Radical Disproportionation
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Photolysis of an aryl sulfide-containing 5,6-dihydropyrimidine (1) at 350 nm produces high yields of thymidine and products resulting from trapping of a 5,6-dihydrothymidin-5-yl radical by O2 or thiols. Thymidine is believed to result from disproportionation of the radical pair originally generated from C-S bond homolysis of 1 on the microsecond timescale, which is significantly shorter than other photochemical transformations of modified nucleotides into their native forms. Duplex DNA containing 1 is destabilized, presumably due to disruption of π-stacking. Incorporation of 1 within the binding site of the restriction endonuclease EcoRV provides a photochemical switch for turning on the enzyme's activity. In contrast, 1 is a substrate for endonuclease VIII and serves as a photochemical off switch for this base excision repair enzyme. Modification 1 also modulates the activity of the 10-23 DNAzyme, despite its incorporation into a nonduplex region. Overall, dihydropyrimidine 1 shows promise as a tool to provide spatiotemporal control over DNA structure on the miscrosecond timescale.
- San Pedro, Joanna Maria N.,Greenberg, Marc M.
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- Potential of 4-thiothymidine as a molecular probe for H2O2 in systems related to PhotoDynamic therapy: A structuristic and mechanistic insight by UV–visible and FTIR-ATR spectroscopies and by ElectroSpray ionization mass spectrometry
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An investigation, based on the synergy between UV–Vis and FTIR-ATR spectroscopies and ElectroSpray Ionization Mass Spectrometry (ESI-MS), on the reactivity of 4-thiothymidine (S4-TdR) with H2O2, selected as a Reactive Oxyg
- Rizzi, Vito,Losito, Ilario,Abbattista, Ramona,Fini, Paola,Agostiano, Angela,Cataldi, Tommaso R.I.,Cosma, Pinalysa
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- 4-Acetylthio-2,2-dimethyl-3-oxobutyl group as an esterase- And thermo-labile protecting group for oligomeric phosphodiesters
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(4-Acetylthio-2,2-dimethyl-3-oxobutyl)-protected oligomeric phosphodiesters 1 and 2 were synthesized and removal of the protecting groups in the presence and absence of hog liver esterase was followed at pH 7.5 and 37 °C. Phosphotriesters 1 and 2 were successfully converted into the desired fully deprotected phosphodiesters 3 and 4, respectively. Some cleavage of internucleosidic P-O bonds took place, which reduced the yield of 3 and 4. Non-enzymatic removal of the protecting group was only modestly retarded by accumulation of negative charge on the molecule. With 1, the half-lives for the departure of the first and second protecting groups were 7.8 and 10.7 h, respectively, and with 2, 6.2 and 7.2 h, respectively. After 4 d, 70% of both starting materials 1 and 2 were converted into the unprotected phosphodiester. The presence of hog liver esterase (2.6 units mL-1) resulted in fast removal of the first protecting group (τ1/2 2.7 min and 36 min with 1 and 2, respectively), but the appearance of fully deprotected 3 and 4 was accelerated only by a factor of 2, consistent with dramatic retardation of the enzymatic reaction upon accumulation of the negative charge.
- Leisvuori, Anna,L?nnberg, Harri,Ora, Mikko
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- Flow injection amperometric detection of 2′-deoxyguanosine at a ruthenium oxide hexacyanoferrate modified electrode
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A ruthenium oxide hexacyanoferrate (RuOHCF) modified electrode was developed. Hydrodynamic voltammetry was employed to demonstrate the remarkable electrocatalytic activity toward the oxidation of 2′-deoxyguanosine. The RuOHCF modified electrode was used as amperometric detector for 2′-deoxyguanosine determination in a FIA apparatus. The influence of various experimental conditions was explored for optimum analytical performance, and at these experimental conditions, the method exhibited a linear response range to 2′-deoxyguanosine extending from 3.8 to 252 μmol L -1 with detection limit of 94 nmol L-1. Applications in DNA samples were examined, and the results for determination of 2′-deoxyguanosine were in good agreement with those obtained by HPLC analysis. Studies on the kinetics of the in vitro consumption of 2′-deoxyguanosine by acetaldehyde were also performed.
- Paixao, Thiago R. L. C.,Garcia, Camila C. M.,Medeiros, Marisa H. G.,Bertotti, Mauro
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- Selectivity in the catalytic transfer hydrogenolysis of silyl ether protecting groups
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Catalytic transfer hydrogenolysis has been used to selectively cleave silyl protecting groups from primary and secondary alcohols, including from thymidine. Ease of cleavage is in the order triethyl >> t-butyldimethyl > triisopropyl > t-butyldiphenyl.
- Cormier,Isaac,Chen
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- DNA hydrolysis by cerium(IV)-saccharide complexes
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Homogeneous solutions are prepared by mixing Ce(NH4)2(NO3)6 with either lyxose, ribose, xylose, gentiobiose, isomaltose, or palatinose at pH 7, under the conditions that [monomeric residue of saccharide]0/[Ce(IV)]0>1. Melibiose and dextran give homogeneous solutions of Ce(IV) when the ratio is greater than 2. Formation of Ce(IV) hydroxide gel is efficiently suppressed by these saccharides. Sugar alcohols (arabinitol, galactitol, mannitol, ribitol, glucitol, and xylitol), glucamine (1-amino-1-deoxy-d-glucitol), lyxosylamine, and N-methylglucamine (1-deoxy-1-methylamino-d-glucitol) are also effective for the solubilization of Ce(IV). In contrast, other monosaccharides (fructose, galactose, and glucose), disaccharides (cellobiose, lactose, maltose, sucrose, and trehalose), cyclodextrins [α-cyclodextrin (cyclomaltohexaose), β-cyclodextrin (cyclomaotoheptaose), and γ-cyclodextrin (cyclomaltooctaose)], and amyloses (as well as galacturonic acid and glucosamine) are poor for the solubilization. The activities of the homogeneous solutions for DNA hydrolysis are in the following order: glucamine>>gentiobiose, isomaltose, ribose, lyxosylamine>lyxose, xylose, arabinitol, galactitol, mannitol, ribitol, glucitol, xylitol, N-methylglucamine. The pseudo-first-order rate constant (5.0x10-3h-1) for the hydrolysis of thymidylyl(3'-5') thymidine by the Ce(IV)-glucamine system at pH 7.0 and 50 °C ([Ce(IV)]0=[glucamine]0=10mmolL-1) is far greater than those of the Ce(IV) complexes of iminodiacetate and ethylenediaminetetraacetate. Copyright (C) 1998 Elsevier Science Ltd.
- Kajimura, Ayako,Sumaoka, Jun,Komiyama, Makoto
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- Photogenerator of trichloroacetic acid as a promising detritylation agent for oligonucleotide microarray synthesis
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[[4-(4-Methoxyphenyl)-2,6-dinitro-phenyl](phenyl)methyl]-2,2,2-trichloroacetate, which generates trichloroacetic acid when irradiated with light of 405 nm wavelength, has been studied as a detritylation agent in the oligonucleotide microarray synthesis. This agent was shown to be suitable for the deprotection of the 4,4'-dimethoxytrityl group during the solid phase oligonucleotide synthesis.
- Sinyakov, A. N.,Nikolaenkova, E. B.,Os'Kina, I. A.,Savel'Ev, V. A.,Samsonov, V. A.,Tikhonov, A. Y.,Palatkina, M. Yu.,Zaytsev, D. E.
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- Sulfonium derivatives of thioxanthenone, a new class of photodetritylating agents for microarray oligonucleotide synthesis
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The usability of a new class of photo acids, namely, sulfonium hexaphosphates based on thioxanthenone, for the removal of the dimethoxytrityl protective group in the process of oligonucleotide synthesis has been studied in order to search for new detritylating agents for microarray oligodeoxyribonucleotide synthesis. 2,4-Diethyl-9-oxo-10-(4-heptyloxyphenyl)-9H- thioxanthenium hexafluorophosphate has been successfully used for the solid-phase synthesis of (dT)10.
- Sinyakov,Ryabinin,Maksakova,Shelkovnikov,Loskutov,Vasil'Ev,Shekleina
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- Participation of an additional 4′-hydroxymethyl group in the cleavage and isomerization of ribonucleoside 3′-phosphodiesters
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4′-(Hydroxymethyl)uridylyl-3′,5′-thymidine, an RNA model bearing an extra hydroxymethyl group at the 4′-position of the 3′-linked nucleoside, has been prepared and its cleavage and isomerization reactions studied over a wide pH range (from 0 to 12). Overa
- Lain, Luigi,L?nnberg, Harri,L?nnberg, Tuomas
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- Triplet-Sensitized Photodeprotection of Oligonucleotides in solution and on Microarray Chips
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Conditions and kinetics of triplet sensitization as a method for increasing the light sensitivity of photolabile protecting groups used for the photolithographic synthesis of oligonucleotide microarrays were quantitatively studied with the photolabile 2-(2-nitrophenyl)propyl protecting group in homogeneous solutions and on glass substrates by using laser flash photolysis, continuous illumination with HPLC analysis, fluorescence dye labelling, and hybridization. In terms of effifiency and avoidance of chemical side reactions, 9H-thioxanthene-9-one was the most-suitable sensitizer. Both in solution and on a glass substrate, the photostationary kinetics were quantitatively modelled and the relevant kinetic parameters determined. While the sensitization kinetics was quantitatively modelled and the relevenat kinetic parameters determined. While the sensitization kinetics were diffusion-controlled both in solution and on the chip, the photostationary kinetics was essentially of zero order only on the chip because here the triplet-quenching effect of the released photoproduct 2-(2-nitrophenyl)propene was suppreseed as a consequence of the inhomogeneous reaction that took place in a narrow diffusion zone above the surface from where the photoproducts could quickly escape. The kinetic simulation allowed quantitative estimate of the density of reactive groups on the surface. It was further demonstarted that, with 9H-thioxanthen-9-one as a sensitizer, high-density oligonucleotide microarrays of high quality can be produced with one-third of the normal exposure time.
- Woell, Dominik,Walbert, Stefan,Stengele, Klaus-Peter,Albert, Tom J.,Richmond, Todd,Norton, Jason,Singer, Michael,Green, Roland D.,Pfleiderer, Wolfgang,Steiner, Ulrich E.
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- Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo.
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2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.
- Schuler,Budiawan,Eder
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- Independent generation of C5′-nucleosidyl radicals in thymidine and 2′-deoxyguanosine
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(Chemical Equation Presented) The synthesis of the C5′ tert-butyl ketone of thymidine 1a and 2′-deoxyguanosine 2 is achieved by reaction of 5′-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5′R configuration is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The (5′S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction medium. The photochemistry of 1a and 2 effectively produced the thymidin-5′-yl radical and the 2′-deoxyguanosin-5′-yl radical, respectively. In the thymidine system, the C5′ radical is fully quenched in the presence of a physiological concentration of thiols. In the 2′-deoxyguanosine system, the C5′ radical undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5′,8-cyclo-2′-deoxyguanosine derivative. The cyclization of the 2′-deoxyguanosin-5′-yl radical occurs with a rate constant of ca. 1 × 106 s-1 and is highly stereoselective affording only the (5′S)-diastereomer.
- Manette, Antonio,Georganakis, Dimitris,Leondiadis, Leondios,Gimisis, Thanasis,Mayer, Peter,Carell, Thomas,Chatgilialoglu, Chryssostomos
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- Highly efficient photolabile protecting groups with intramolecular energy transfer
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(Chemical Equation Presented) Brought to light: The light sensitivity of photolabile protecting groups of the nitrophenylpropoxycarbonyl (NPPOC) type was enhanced by up to a factor of 25. This was achieved by covalently linking the protecting group to a sensitizer with a much better absorptivity and the ability to transfer its electronic excitation to the protecting group (see scheme).
- Woell, Dominik,Smirnova, Julia,Pfleiderer, Wolfgang,Steiner, Ulrich E.
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- Nucleoside 2′-deoxyribosyltransferase from psychrophilic bacterium Bacillus psychrosaccharolyticus - Preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides
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Nucleoside 2′-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2′-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2′-deoxyadenosine from 2′-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2′-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5.
- Fresco-Taboada, Alba,Serra, Immacolata,Fernandez-Lucas, Jesus,Acebal, Carmen,Arroyo, Miguel,Terreni, Marco,De La Mata, Isabel
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- THE TRIPHENYLMETHYL (TRITYL) GROUP AND ITS USES IN NUCLEOTIDE CHEMISTRY
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The triphenylmethyl (trityl) group can be removed from 5'-trityl 2'deoxynucleosides (and their N-acyl derivatives) under aprotic neutral conditions without causing any side reactions.An efficient method for tritylation of N-acyl 2'-deoxynucleosides is described.Potential use of such derivatives for stepwise synthesis of deoxyoligonucleotides is discussed.
- Kohli, V.,Bloecker, H.,Koester, H.
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- Flavin-sensitized photoreduction of thymidine glycol
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Photochemical reactivity of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) toward thymidine glycol (dTg) has been investigated. Fluorescence intensity of FAD was enhanced as increasing the concentration of dTg, suggesting that adenosine
- Ito, Takeo,Kondo, Akiko,Terada, Satoru,Nishimoto, Sei-ichi
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- Unprecedentedly fast DNA hydrolysis by the synergism of the cerium(IV)-praseodymium(III) and the cerium(IV)-neodymium(III) combinations
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The most active catalysts for DNA hydrolysis so far have been obtained by using the synergetic cooperation of the cerium(IV) ion with the praseodymium(III) ion. The pseudo first-order rate constant for the hydrolysis of thymidylyl(3'-5')thymidine by the 2:1 combination is 10 times as great as that by the cerium(IV) ion.
- Takeda, Naoya,Imai, Takamitsu,Irisawa, Makoto,Sumaoka, Jun,Yashiro, Mono,Shigekawa, Hidemi,Komiyama, Makoto
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- Formation of a fairly stable diazoate intermediate of 5-methyl-2′-deoxycytidine by HNO2 and NO, and its implication to a novel mutation mechanism in CpG site
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The intermediate produced from 5-methyl-2′-deoxycytidine (5medCyd) by HNO2 and NO treatments was isolated and characterized. When 10 mM 5medCyd was incubated with 100 mM NaNO2 at pH 3.7 and 37°C, a previously unidentified product was formed. The product was identified as a diazoate derivative of 5medCyd, 1-(β-D-2′-deoxyribofuranosyl)-5-methyl-2-oxopyrimidine-4-diazoate (5medCyd-diazoate), on the bases of several measurements including LC/MS. The time course of the concentration change of the diazoate showed a characteristic profile of a reaction intermediate, and the steady state concentration was 2.3 μM (0.023% yield). When an aqueous solution of 10mM 5medCyd (10 mL) was bubbled by NO at 37°C under aerobic conditions holding the pH around 7.4, the diazoate was also generated. The yield of the diazoate was 0.041 μmol (0.041% yield) at 20 mmol of NO absorption. At physiological pH and temperature (pH 7.4, 37°C), the diazoate was converted to dThd exclusively with a first order rate constant κ 9.1 x 10-6 s-1 (t1/2 = 21 h). These results show that the diazoate is generated as a relatively stable intermediate in the reactions of 5medCyd with HNO2 and NO and further suggest that the diazoate can be formed in cellular DNA with biologically relevant doses of HNO2 and NO. Copyright
- Suzuki, Toshinori,Yamada, Masaki,Nakamura, Takanori,Ide, Hiroshi,Kanaori, Kenji,Tajima, Kunihiko,Morii, Takashi,Makino, Keisuke
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- Fast repair of deoxythymidine radical anions by two polyphenols: Rutin and quercetin
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The effects of rutin and quercetin on the repair of the deoxythemindine radical anion (dT?-) were studied using the technique of pulse radiolysis. The radical anion of dT was formed by the reaction of hydrated electron with dT. After pulse irradiation of nitrogen-saturated aqueous solutions containing dT, 0.2M t-BuOH and either rutin or quercetin, the initially formed dT?-, detected spectrophotometrically, rapidly decayed with the concurrent formation of the radical anion of rutin or quercetin. The results indicated that dT?- can be rapidly repaired by rutin or quercetin. The rate constants of the repair reactions were determined to be 3.1 and 4.1×109M-1s-1 for rutin and quercetin, respectively. With substitution by glycosyl groups at C3-OH being neighbor to C4 keto group, which is the active site for electron transfer, rutin has a lower repair reaction rate constant toward dT?- than quercetin. Together with findings from our previous studies, the present results demonstrated that nonenzymatic fast repair may be a universal form of repair involving phenolic antioxidants.
- Zhao, Chenyang,Shi, Yimin,Wang, Wenfeng,Jia, Zhongjian,Yao, Side,Fan, Botao,Zheng, Rongliang
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- Photolysis and thermolysis of platinum(IV) 2,2′-bipyridine complexes lead to identical platinum(II)-DNA adducts
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Two PtIV and two Pt11 complexes containing a 2,2′-bipyridine ligand were treated with a short DNA oligonucleotide under light irradiation at 37 °C or in the dark at 37 and 50 °C. Photolysis and thermolysis of the PtIV complexes led to spontaneous reduction of the PtIV to the corresponding PtII complexes and to binding of PtII 2,2′-bipyridine complexes to N7 of guanine. When the reduction product was [Pt(bpy)Cl2], formation of bis-oligonucleotide adducts was observed, whereas [Pt(bpy)(MeNH 2)Cl]+ gave monoad- ducts, with chloride ligands substituted in both cases. Neither in the dark nor under light irradiation was the reductive elimination process of these PtIV complexes accompanied by oxidative DNA damage. This work raises the question of the stability of photoacti- vatable PtIV complexes toward moderate heating conditions.
- Loup, Christophe,Tesouro Vallina, Ana,Coppel, Yannick,Letinois, Ulla,Nakabayashi, Yasuo,Meunier, Bernard,Lippert, Bernhard,Pratviel, Genevieve
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- Hydrolysis of phosphomonoesters in nucleotides by cerium(IV) ions. Highly selective hydrolysis of monoester over diester in concentrated buffers
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Phosphomonoesters in nucleotides are efficiently hydrolysed by CeIV ions under physiological conditions. The half-lives of the residues at pH 7.2 and 50 °C ([CeIV] = 10 mM) are around 10 min. Phosphomonoester hydrolysis by CeIV ions is faster than the hydrolysis of phosphodiesters. Significantly, the selectivity for monoester hydrolysis over diester hydrolysis is remarkably increased by using concentrated buffer solutions (TRIS and HEPES). In 500 mM TRIS buffer, pdA and dAp are hydrolysed 500- and 580-fold faster than is d(ApA), whereas the corresponding ratios in 50 mM TRIS buffer are 85 and 90 respectively. Selective removal of the terminal monophosphate from d(pApA) is achieved by CeIV in these concentrated buffers.
- Miyama, Sachiko,Asanuma, Hiroyuki,Komiyama, Makoto
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Read Online
- REMOVAL OF CYCLIC DI-T-BUTYLSILANEDIYL PROTECTING GROUPS USING TRIBUTYLAMINE HYDROFLUORIDE (TBAHF) REAGENT
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Cyclic di-t-butylsilanediyl protecting groups were rapidly and cleanly removed from 3',5'-cyclic silanediyl nucleosides by a mild and convenient reagent, tributylamine hydrofluoride in tetrahydrofuran.
- Furusawa, Kiyotaka
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Read Online
- An Improved Approach for Practical Synthesis of 5-Hydroxymethyl-2′-deoxycytidine (5hmdC) Phosphoramidite and Triphosphate
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5-Hydroxymethyl-2′-deoxycytidine (5hmdC) phosphoramidite and triphosphate are important building blocks in 5hmdC-containing DNA synthesis for epigenetic studies. However, efficient and practical methods for the synthesis of these compounds are
- Chen, Zhen-Zhen,Chi, Mei,Dong, Ying-Ying,Pu, Shou-Zhi,Sun, Qi,Yang, Dong-Zhao
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- Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii
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Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/ reducing conditions) of nickel-affinity purified protein revealed the presence of a nearhomogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 × 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
- Smiley-Moreno, Elizabeth,Smith, Douglas,Yu, Jieh-Juen,Cao, Phuong,Arulanandam, Bernard P.,Chambers, James P.
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- Meteorite-catalyzed intermoleculartrans-glycosylation produces nucleosides under proton beam irradiation
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Di-glycosylated adenines act as glycosyl donors in the intermoleculartrans-glycosylation of pyrimidine nucleobases under proton beam irradiation conditions. Formamide and chondrite meteorite NWA 1465 increased the yield and the selectivity of the reaction
- Bizzarri, Bruno Mattia,Fanelli, Angelica,Kapralov, Michail,Krasavin, Eugene,Saladino, Raffaele
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p. 19258 - 19264
(2021/06/03)
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- SYNTHESIS AND IMPROVEMENT OF A NUCLEOSIDE ANALOGUE AS AN ANTI-CANCER AND ANTI-VIRAL DRUG
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The invention is a drug for anticancer and antiviral therapy, comprising a nucleoside analogue (7) comprising a furan ring irreversibly bound to the RNA/DNA synthesis chain by phosphodiester bonds and having SP3 hybridization, and folic acid (A) bound to the nucleoside analogue (7) comprising furan ring. The synthesis method of the said nucleoside analogue is also contained within the scope of the invention. In this work, a nucleoside-analogue was transformed after converting the furan-ring hybridization from Sp2 to Sp3 to make it more selectivity with different enzymes and linking it via site 5 with the effective folic acid towards entering the substances inside the cells and to become the final compound possessing anti-cancer and anti- virus properties after controlling the replication and reproduction process in DNA.
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Page/Page column 7; 13
(2021/05/29)
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- Synthesis and evaluation of 3′-[18F]fluorothymidine-5′-squaryl as a bioisostere of 3′-[18F]fluorothymidine-5′-monophosphate
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The squaryl moiety has emerged as an important phosphate bioisostere with reportedly greater cell permeability. It has been used in the synthesis of several therapeutic drug molecules including nucleoside and nucleotide analogues but is yet to be evaluated in the context of positron emission tomography (PET) imaging. We have designed, synthesised and evaluated 3′-[18F]fluorothymidine-5′-squaryl ([18F]SqFLT) as a bioisostere to 3′-[18F]fluorothymidine-5′-monophosphate ([18F]FLTMP) for imaging thymidylate kinase (TMPK) activity. The overall radiochemical yield (RCY) was 6.7 ± 2.5% and radiochemical purity (RCP) was >90%. Biological evaluationin vitroshowed low tracer uptake (?1) but significantly discriminated between wildtype HCT116 and CRISPR/Cas9 generated TMPK knockdown HCT116shTMPK?. Evaluation of [18F]SqFLT in HCT116 and HCT116shTMPK?xenograft mouse models showed statistically significant differences in tumour uptake, but lacked an effective tissue retention mechanism, making the radiotracer in its current form unsuitable for PET imaging of proliferation.
- Brickute,Beckley,Allott,Braga,Barnes,Thorley,Aboagye
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p. 12423 - 12433
(2021/04/07)
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- MODIFIED OLIGOMERIC COMPOUNDS AND USES THEREOF
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The present disclosure provides oligomeric compounds comprising a modified oligonucleotide having at least one stereo-non-standard nucleoside. An oligomeric compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein at least one nucleoside of the modified oligonucleotide is a stereo-non-standard nucleoside; and wherein the oligomeric compound is selected from among an RNAi compound, a modified CRISPR compound, and an artificial mRNA compound.
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Page/Page column 77; 146-147; 152
(2021/02/19)
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- Spacer-Mediated Control of Coumarin Uncaging for Photocaged Thymidine
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Despite its importance in the design of photocaged molecules, less attention is focused on linker chemistry than the cage itself. Here, we describe unique uncaging properties displayed by two coumarin-caged thymidine compounds, each conjugated with (2) or without (1) an extended, self-immolative spacer. Photolysis of 1 using long-wavelength UVA (365 nm) or visible (420, 455 nm) light led to the release of free thymidine along with the competitive generation of a thymidine-bearing recombination product. The occurrence of this undesired side reaction, which is previously unreported, was not present with the photolysis of 2, which released thymidine exclusively with higher quantum efficiency. We propose that the spatial separation between the cage and the substrate molecule conferred by the extended linker can play a critical role in circumventing this unproductive reaction. This report reinforces the importance of linker selection in the design of coumarin-caged oligonucleosides and other conjugates.
- Baker, James R.,Cannon, Jayme,Choi, Seok Ki,Krummel, Matthew F.,Tang, Shengzhuang,Yang, Kelly
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- RETRACTED ARTICLE: Divergent synthesis of 5-substituted pyrimidine 2′-deoxynucleosides and their incorporation into oligodeoxynucleotides for the survey of uracil DNA glycosylases
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Recent studies have indicated that 5-methylcytosine (5mC) residues in DNA can be oxidized and potentially deaminated to the corresponding thymine analogs. Some of these oxidative DNA damages have been implicated as new epigenetic markers that could have profound influences on chromatin function as well as disease pathology. In response to oxidative damage, the cells have a complex network of repair systems that recognize, remove and rebuild the lesions. However, how the modified nucleobases are detected and repaired remains elusive, largely due to the limited availability of synthetic oligodeoxynucleotides (ODNs) containing these novel DNA modifications. A concise and divergent synthetic strategy to 5mC derivatives has been developed. These derivatives were further elaborated to the corresponding phosphoramidites to enable the site-specific incorporation of modified nucleobases into ODNs using standard solid-phase DNA synthesis. The synthetic methodology, along with the panel of ODNs, is of great value to investigate the biological functions of epigenetically important nucleobases, and to elucidate the diversity in chemical lesion repair.
- Tran, Ai,Zheng, Song,White, Dawanna S.,Curry, Alyson M.,Cen, Yana
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p. 11818 - 11826
(2020/11/18)
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- MODIFIED OLIGOMERIC COMPOUNDS AND USES THEREOF
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The present disclosure provides oligomeric compounds comprising a modified oligonucleotide having at least one stereo-non-standard nucleoside.
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Page/Page column 71; 73
(2020/05/15)
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- Thermodynamic Reaction Control of Nucleoside Phosphorolysis
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Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose-1-phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase-catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate-specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature-dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis. (Figure presented.).
- Kaspar, Felix,Giessmann, Robert T.,Neubauer, Peter,Wagner, Anke,Gimpel, Matthias
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p. 867 - 876
(2020/01/24)
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- Method for preparing beta-thymidine by adopting solid acid catalysis
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The invention relates to a method for preparing beta-thymidine by adopting solid acid catalysis. The method includes the following steps: the toluene feed liquid of intermediate bromide is obtained by5-methyluridine and [1] under the action of a solid acid and a phase transfer catalyst; and reduction reaction is performed on the toluene feed liquid of the intermediate bromide by using a palladium-carbon catalyst under characteristic environments, and the beta-thymidine is generated through post-treatment crystallization. The method is controllable in reaction condition, high in yield, low incost and suitable for industrial production, and has industrial application value.
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Paragraph 0016-0017
(2020/07/15)
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- RETRACTED ARTICLE: Convenient synthesis of pyrimidine 2′-deoxyribonucleoside monophosphates with important epigenetic marks at the 5-position
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Methyl groups of thymine and 5-methylcytosine (5mC) bases in DNA undergo endogenous oxidation damage. Additionally, 5mC residues can be enzymatically deaminated or oxidized through either genetic alterations or the newly identified epigenetic reprogramming pathway. Several methods have been developed to measure the formation of modified DNA nucleobases including 32P-postlabeling. However, the postlabeling method is often limited by the absence of authentic chemical standards. The synthesis of monophosphate standards of nucleotide oxidation products is complicated by the presence of additional functional groups on the modified bases that require complex protection and deprotection strategies. Due to the emerging interest in the pyrimidine oxidation products, the corresponding protected 3′-phosphoramidites needed for solid-phase oligonucleotide synthesis have been reported, and several are commercially available. We report here an efficient synthesis of 3′-monophosphates from 3′-phosphoramidites and the subsequent enzymatic conversion of 3′-monophosphates to the corresponding 5′-monophosphates using commercially available enzymes. This journal is
- Zheng, Song,Tran, Ai,Curry, Alyson M.,White, Dawanna S.,Cen, Yana
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p. 5164 - 5173
(2020/07/23)
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- Radiosynthesis of [18F]-labelled pro-nucleotides (ProtIDes)
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Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of P
- Cavaliere, Alessandra,Probst, Katrin C.,Paisey, Stephen J.,Marshall, Christopher,Dheere, Abdul K.H.,Aigbirhio, Franklin,McGuigan, Christopher,Westwell, Andrew D.
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- ORTHOESTER COMPOSITIONS FOR AFFINITY PURIFICATION OF OLIGONUCLEOTIDES
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Compounds and methods for purifying oligonucleotides such as RNA and DNA. A target oligonucleotide is reacted with an orthoester linker comprising an affinity tag to form an orthoester oligonucleotide-orthoester linker conjugate which is subjected to a purification technique to separate the target oligonucleotide from impurities such as truncated oligonucleotides. The orthoester linker can be then removed under mild conditions to generate the target oligonucleotide in high purity.
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Paragraph 00298
(2019/03/05)
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- Identification of Flavin Mononucleotide as a Cell-Active Artificial N6-Methyladenosine RNA Demethylase
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N6-Methyladenosine (m6A) represents a common and highly dynamic modification in eukaryotic RNA that affects various cellular pathways. Natural dioxygenases such as FTO and ALKBH5 are enzymes that demethylate m6A residues in mRNA. Herein, the first identification of a small-molecule modulator that functions as an artificial m6A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m6A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m6A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology.
- Xie, Li-Jun,Yang, Xiao-Ti,Wang, Rui-Li,Cheng, Hou-Ping,Li, Zhi-Yan,Liu, Li,Mao, Lanqun,Wang, Ming,Cheng, Liang
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supporting information
p. 5028 - 5032
(2019/03/17)
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- Interaction of α-Thymidine Inhibitors with Thymidylate Kinase from Plasmodium falciparum
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Plasmodium falciparum thymidylate kinase (PfTMK) is a critical enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. N-(5′-Deoxy-α-thymidin-5′-yl)-N′-[4-(2-chlorobenzyloxy)phenyl]urea was developed as an inhibitor of PfTMK and has been reported as an effective inhibitor of P. falciparum growth with an EC50 of 28 nM [Cui, H., et al. (2012) J. Med. Chem. 55, 10948-10957]. Using this compound as a scaffold, a number of derivatives were developed and, along with the original compound, were characterized in terms of their enzyme inhibition (Ki) and binding affinity (KD). Furthermore, the binding site of the synthesized compounds was investigated by a combination of mutagenesis and docking simulations. Although the reported compound is indicated to be highly effective in its inhibition of parasite growth, we observed significantly lower binding affinity and weaker inhibition of PfTMK than expected from the reported EC50. This suggests that significant structural optimization will be required for the use of this scaffold as an effective PfTMK inhibitor and that the inhibition of parasite growth is due to an off-target effect.
- Chen, Mengshen,Sinha, Kaustubh,Rule, Gordon S.,Ly, Danith H.
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p. 2868 - 2875
(2018/05/14)
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- A Practical Method for Regioselective 5′-O-tert-Butyldimethylsilyl Deprotection of Persilylated Nucleosides by Methanolic Phosphomolybdic Acid
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In nucleoside/nucleotide chemistry, the regioselective cleavage of 5′-O-TBS groups of persilylated nucleosides is a desired approach for structural functionalization at the 5′-position. However, efficient and practical methods for this purpose are still limited. In our research, we found that homogeneous methanolic phosphomolybdic acid (PMA) efficiently catalyzes the regioselective deprotection of 5′- O -TBS groups of a diversity of persilylated nucleoside substrates and can be applied in practical synthesis at scales of up to 15 g. 31 P NMR results indicated that an anion cluster forms and the Lewis acidity of homogeneous PMA is organic-solvent dependent. The efficacy and pronounced regioselectivity of methanolic PMA occurs as a result of a lowering of the Lewis acid strength upon solvation of the molybdophosphate anions.
- Huang, Hua-Shan,Kong, Rui,Zheng, Xiu-An,Chen, Wei-Jie,Han, Shuai-Bo,Zeng, De-Yun,Gong, Shan-Shan,Sun, Qi
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supporting information
p. 2437 - 2443
(2018/11/23)
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- Hydrogen peroxide-Triggered gene silencing in mammalian cells through boronated antisense oligonucleotides
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Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) involved in various diseases, including neurodegeneration, diabetes, and cancer. Here, we introduce a new approach to use H2O2 to modulate specific gene expression in mammalian cells. H2O2-responsive nucleoside analogues, in which the Watson-Crick faces of the nucleobases are caged by arylboronate moieties, were synthesized. One of these analogues, boronated thymidine (dTB), was incorporated into oligodeoxynucleotides (ODNs) using an automated DNA synthesizer. The hybridization ability of this boronated ODN to complementary RNA was clearly switched in the off-To-on direction upon H2O2 addition. Furthermore, we demonstrated H2O2-Triggered gene silencing in mammalian cells using antisense oligonucleotides (ASOs) modified with dTB. Our approach can be used for the regulation of any gene of interest by the sequence design of boronated ASOs and will contribute to the development of targeted disease therapeutics.
- Mori, Shohei,Morihiro, Kunihiko,Okuda, Takumi,Kasahara, Yuuya,Obika, Satoshi
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p. 1112 - 1118
(2018/02/09)
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- Photoredox-Catalyzed Deformylative 1,4-Addition of 2′-Deoxy-5′- O-phthalimidonucleosides for Synthesis of 5′-Carba Analogs of Nucleoside 5′-Phosphates
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A concise approach for the synthesis of the 5′-carba analogs of nucleoside 5′-phosphates from 2′-deoxy-5′-O-phthalimidonucleosides by a visible-light-mediated deformylative 1,4-addition was developed. This method enabled rapid and facile generation of 4′-carbon radicals of nucleosides. Moreover, this synthetic strategy was applicable to the 5′-carba analogs of nucleoside 5′-phosphates as well as other 5′-carba nucleosides bearing methoxycarbonyl, cyano, and N-methylsulfamoyl groups.
- Ito, Yuta,Kimura, Airi,Osawa, Takashi,Hari, Yoshiyuki
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p. 10701 - 10708
(2018/09/21)
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- REVERSIBLY BLOCKED NUCLEOSIDE ANALOGUES AND THEIR USE
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Reversibly blocked nucleoside analogues and methods of using such nucleoside analogues for sequencing of nucleic acids are provided.
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- AZIDOMETHYL ETHER DEPROTECTION METHOD
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The invention relates to a method of converting an azidomethyl ether substituent to a free hydroxyl group. The invention also relates tomethods of nucleic acid synthesis and sequencing comprising the use of nucleotide triphosphates having a 3'-O-azidomethyl substituent, to kitscomprising nucleotide triphosphates having a 3'-O-azidomethyl substituent and photoactivatable transition metal complex and to the use of said kits in methods of nucleic acid synthesis and sequencing.
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Page/Page column 15
(2017/02/09)
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- Enzymatic synthesis of ribo- and 2′-deoxyribonucleosides from glycofuranosyl phosphates: An approach to facilitate isotopic labeling
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Milligram quantities of α-D-ribofuranosyl 1-phosphate (sodium salt) (αR1P) were prepared by the phosphorolysis of inosine, catalyzed by purine nucleoside phosphorylase (PNPase). The αR1P was isolated by chromatography in >95% purity and characterized by 1H and 13C NMR spectroscopy. Aqueous solutions of αR1P were stable at pH 6.4 and 4 °C for several months. The isolated αR1P was N-glycosylated with different nitrogen bases (adenine, guanine and uracil) using PNPase or uridine phosphorylase (UPase) to give the corresponding ribonucleosides in high yield based on the glycosyl phosphate. This methodology is attractive for the preparation of stable isotopically labeled ribo- and 2′-deoxyribonucleosides because of the ease of product purification and convenient use and recycling of nitrogen bases. The approach eliminates the need for separate reactions to prepare individual furanose-labeled ribonucleosides, since only one ribonucleoside (inosine) needs to be labeled, if desired, in the furanose ring, the latter achieved by a high-yield chemical N-glycosylation. 2′-Deoxyribonucleosides were prepared from 2′-deoxyinosine using the same methodology with minor modifications.
- Zhang, Wenhui,Turney, Toby,Surjancev, Ivana,Serianni, Anthony S.
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p. 125 - 133
(2017/08/08)
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- Methyl ketone derivative, and preparation method and applications thereof
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The invention discloses a methyl ketone derivative, and a preparation method and applications thereof. The preparation method comprises following steps: a ketone derivative and an organic peroxide are dissolved in a solvent, and reaction is carried out at 80 to 130 DEG C so as to obtain methyl pyrimidone and a methyl pyrimidone derivative. According to the preparation method, the ketone derivative is taken as a starting material; the raw materials are easily and widely available; products of different kinds can be obtained via the preparation method, and can be used directly or used in other further reaction. No metal is involved, so that the preparation method is suitable to be applied in pharmaceutical preparation technology. The preparation method is short in route, mild in reaction conditions, simple in reaction operation and postprocessing process, and high in yield, and is suitable for large-scale production.
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Paragraph 0046; 0047
(2017/08/28)
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- Synthesis method of beta-thymidine
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The invention discloses a synthesis method of beta-thymidine. The synthesis method takes trimethylchlorosilane and 5-methyluracil as raw materials to react; in a reaction line process, tetraacetylribose, trifluoromethanesulfonic acid, N,N-dimethylformamide and acetylchloride are introduced; then hydrogenation reaction and hydrolysis reaction are carried out to finally obtain a beta-thymidine finished product and the yield is 89 percent. Compared with an existing synthesis method, the synthesis method of the beta-thymidine has the advantages that the price of raw materials is low, the content of the beta-thymidine in the final product is high, and pollution to the environment in a production process is small; in a synthesis process, the content of generated impurities is less. According to the synthesis method disclosed by the invention, an obtained result is stable and the operation is simple; demands on equipment and preparation environments are not strict so that large-scale popularization is facilitated.
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- Method for preparing β-thymidine
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The invention relates to a preparation method of beta-thymidine, and specifically discloses a method of preparing beta-thymidine from the formula II compound. The method comprises following steps: subjecting the compound represented as the formula II to carry out dehalogenation reactions, in a hydrogen atmosphere, in the presence of a catalyst, and in a buffer system with a pH value of 6.5 to 8.0, so as to obtain beta-thymidine represented as the formula I, wherein X represents Cl or Br. The dehalogenation reactions are carried out under atmospheric pressure, and the preparation method has the advantages of environment-friendliness, economy, high product yield, good product purity, and suitability for industrial production.
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Paragraph 0102-0107
(2018/02/04)
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- Method for preparing Thymidine
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The invention discloses a method for preparing Thymidine as shown in a formula (IV). The method comprises the following step of performing a three-step reaction of dehydration, halogenation and reduction on ribothymidine as shown in a formula (I) according to the following chemical equation so as to obtain the Thymidine as shown in a formula (IV), wherein the three-step reaction of dehydration, halogenation and reduction is completed by a one-pot method. The method for preparing the Thymidine disclosed by the invention is simple to operate, low in cost of human resources and low in equipment investment, the production cycle is substantially shortened, and the method is suitable for industrialized mass production and has wide application prospects.
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Paragraph 0025; 0026; 0027; 0028; 0029; 0030; 0031-0037
(2017/02/24)
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- Efficient and green approach for the complete deprotection of O-acetylated biomolecules
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A simple, efficient and mild strategy for the complete O-deacetylation of different per-acetylated biomolecules in aqueous media has been described. Different lipases were tested but only the commercial Amano lipase A from Aspergillus Niger catalyzed the complete deprotection of peracetylated α-glucose to glucose in excellent yield. The experimental conditions were tested, in particular the pH effect. The reaction was performed at different pHs considering the only enzymatic process was evaluated at pH 5 and the combination of enzymatic and chemical migration process was evaluated at higher pHs. Finally pH 7 and 25 °C were selected as best conditions. Thus this lipase fully hydrolyzed different peracetylated α-glycopyranosides (glucose, mannose, glucal, galactal) with >99% yields, whereas very good deprotecting yields (75-80%) were achieved for different acetylated β-glycopyranosides (galactose, ribofuranose) under these mild conditions. This strategy was successfully extended to the fully O-selective deprotection of acetylated nucleosides where >99% yield was rapidly obtained. No selectivity was observed for the N-deacetylation in amino acids and peptides.
- Dunne, Anthony,Palomo, Jose M.
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p. 88974 - 88978
(2016/10/03)
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- METHODS FOR THE TREATMENT OF HEPATITIS B AND HEPATITIS D VIRUS INFECTIONS
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It is disclosed a method for treating hepatitis B virus infection or hepatitis B virus/hepatits delta virus co-infection, the method comprising administering to a subject in need of such treatment a first pharmaceutically acceptable agent that comprises at least one phosphorothioated nucleic acid polymer and a second pharmaceutically acceptable agent that comprises at least one nucleoside/nucleotide analog HBV polymerase inhibitor.
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- Method for preparing high-purity telbivudine compound
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The invention belongs to the technical field of medicine and provides a method for preparing a high-purity telbivudine compound. The method includes the steps that an LTD-4 compound serves as the raw material and reacts with thymine subjected to silicification protection, and an intermediate, namely an LTD-5 compound, can be obtained; then, through deprotection reaction, the telbivudine compound is obtained after post-processing, wherein MeONa serves as an alkaline reagent of the deprotection reaction, and strong-acidity resin serves as a dealkalization reagent. The method simplifies the production process, the yield of each step is high, and a target product high in purity and yield is obtained. Please see the structural formula in the description.
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Paragraph 0046; 0047
(2017/01/02)
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- Method for preparing telbivudine
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The invention relates to a method for preparing telbivudine. The reaction mechanism is as shown in the specification specifically. Compared with a method of the prior art, the method provided by the invention has the advantages that firstly, all raw materials are low in cost and can be easily obtained from the market; secondly, reaction of different steps is normal reaction, and the reaction steps are simple and easy to implement; and thirdly, production requirements can be met by using normal preparation equipment, the production is easy to control, and industrial production can be achieved.
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Paragraph 0052; 0053; 0054
(2016/10/09)
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- PRODUCTION METHOD OF NUCLEOSIDE COMPOUND
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PROBLEM TO BE SOLVED: To provide a production method of a nucleoside compound by which an isotopic labeled nucleoside compound can be produced efficiently. SOLUTION: A production method of a nucleoside compound comprises obtaining a target nucleoside compound by the base exchange reaction of a raw material nucleoside compound and a base in the solution containing a phosphoric acid ion by a nucleoside phosphorylase, wherein the target nucleoside compound is labeled with a stable isotope or a radioisotope. COPYRIGHT: (C)2015,JPOandINPIT
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Paragraph 0032-0033
(2017/03/24)
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- Efficacy and site specificity of hydrogen abstraction from DNA 2-deoxyribose by carbonate radicals
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The carbonate radical anion CO3?- is a potent reactive oxygen species (ROS) produced in vivo through enzymatic one-electron oxidation of bicarbonate or, mostly, via the reaction of CO2 with peroxynitrite. Due to the vitally essential role of the carbon dioxide/bicarbonate buffer system in regulation of physiological pH, CO3?- is arguably one of the most important ROS in biological systems. So far, the studies of reactions of CO3?- with DNA have been focused on the pathways initiated by oxidation of guanines in DNA. In this study, low-molecular products of attack of CO3?- on the sugar-phosphate backbone in vitro were analyzed by reversed phase HPLC. The selectivity of damage in double-stranded DNA (dsDNA) was found to follow the same pattern C4′ > C1′ > C5′ for both CO3?- and the hydroxyl radical, though the relative contribution of the C1′ damage induced by CO3?- is substantially higher. In single-stranded DNA (ssDNA) oxidation at C1′ by CO3?- prevails over all other sugar damages. An approximately 2000-fold preference for 8-oxoguanine (8oxoG) formation over sugar damage found in our study identifies CO3?- primarily as a one-electron oxidant with fairly low reactivity toward the sugar-phosphate backbone.
- Roginskaya, Marina,Moore,Ampadu-Boateng,Razskazovskiy
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p. 1431 - 1437
(2015/11/09)
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- Nucleotides and nucleosides and methods for their use in DNA sequencing
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The present invention relates generally to labeled and unlabled cleavable terminating groups and methods for DNA sequencing and other types of DNA analysis. More particularly, the invention relates in part to nucleotides and nucleosides with chemically cleavable, photocleavable, enzymatically cleavable, or non-photocleavable groups and methods for their use in DNA sequencing and its application in biomedical research.
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Page/Page column 149; 150
(2015/12/18)
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- Rose Bengal-photosensitized oxidation of 4-thiothymidine in aqueous medium: evidence for the reaction of the nucleoside with singlet state oxygen
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The photoreactivity of 4-thiothymidine (S4TdR) under visible light in the presence of Rose Bengal (RB), acting as a photosensitizer, was investigated in aqueous solutions at pH 7 and 12, using UV-vis, FTIR-ATR and 1H-NMR spectroscopic techniques, time resolved absorption spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Evidence for the generation of thymidine (TdR) as the main product, after one hour of irradiation, was obtained from UV-Vis data, that suggested 4-thiothymidine photodegradation to be faster at basic pH, and confirmed by FTIR-ATR and 1H-NMR data. Clues for the presence of a further product, likely corresponding to a dimeric form of S4TdR, were obtained from the latter techniques. Besides indicating the presence of thymidine, the ESI-MS and MS/MS spectra of the reaction mixtures enabled the identification of the additional product as a S-S bridged covalent dimer of 4-thiothymidine. The concentration of the dimeric species could be estimated with the aid of 1H-NMR data and was found to be lower than that of thymidine in pH 7 reaction mixtures and almost negligible in the pH 12 ones. From a mechanistic point of view, time-resolved absorption spectroscopy measurements provided direct evidence that the formation of the two products cannot be ascribed to a photoinduced electron transfer involving S4TdR and the excited triplet state of RB. Rather, their generation can be interpreted as the result of a bimolecular reaction occurring between singlet state oxygen (1O2), photogenerated by RB, and S4TdR, as demonstrated by the direct detection of 1O2 through IR luminescence spectroscopy. More specifically, a sequential reaction pathway, consisting in the generation of an electrophilic hydroxylated form of S4TdR and its subsequent, rapid reaction with S4TdR, was hypothesized to explain the presence of the S-S bridged covalent dimer of 4-thiothymidine in the reaction mixtures. The described processes make S4TdR an interesting candidate in the role of molecular probe for the detection of 1O2 under different pH conditions.
- Rizzi, Vito,Losito, Ilario,Ventrella, Andrea,Fini, Paola,Fraix, Aurore,Sortino, Salvatore,Agostiano, Angela,Longobardi, Francesco,Cosma, Pinalysa
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p. 26307 - 26319
(2015/10/12)
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- Reductive removal of methoxyacetyl protective group using sodium borohydride
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Herein, we have developed a mild and selective reductive deprotection method for the MAc protected alcohols using sodium borohydride. The new deprotection conditions provide a complete orthogonality between O-MAc and other protecting groups such as tert-butyl ester, N-Boc, Fmoc, Cbz, O-TBDMS, N-benzyl, O-benzyl, O-acetyl, N-acetyl, N-MAc, etc. In addition to O-MAc deprotection, this method is also applicable for S-MAc deprotection.
- Gadekar, Pradip K.,Hoermann, Maryann,Corbo, Faith,Sharma, Rajiv,Sarveswari,Roychowdhury, Abhijit
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p. 503 - 506
(2014/01/06)
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- Mapping structurally defined guanine oxidation products along DNA duplexes: Influence of local sequence context and endogenous cytosine methylation
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DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along p53 gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2H-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2′- deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated MeCG dinucleotides and at 5′ Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of MeCG sequences may be caused by a lowered ionization potential of guanine bases paired with MeC and the preferential intercalation of riboflavin photosensitizer adjacent to MeC:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known p53 lung cancer mutational hotspots at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer.
- Ming, Xun,Matter, Brock,Song, Matthew,Veliath, Elizabeth,Shanley, Ryan,Jones, Roger,Tretyakova, Natalia
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p. 4223 - 4235
(2014/04/03)
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- Regioselective mono-deprotection of di-ferf-butylsilylene acetal derived from 1,3-diol with ammonium fluoride
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Here we report a novel and efficient method for the regioselective mono-deprotection of di-terf-butylsilylene acetals derived from 1,3-diols consisting of primary and secondary alcohols. The ammonium fluoride-mediated reactions of pyripyropene A derivative, thymidine and uridine derivatives, methyl β-D-glucofuranoside, and pyranoside derivatives each gave the corresponding primary alcohol with high regioselectivity.
- Ohtawa, Masaki,Tomoda, Hiroshi,Nagamitsu, Tohru
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p. 113 - 118
(2014/02/14)
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- DERIVATIVES OF PHENYL (THIO) UREA DEOXYTHYMIDINE AND USE THEREOF AS ANTIMALARIALS
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Deoxythymidine derivatives according to formula (I) are disclosed. wherein: X may be O or S; and R1, R2, R3, R4 and R5 may each be independently selected from H, halo, C1-C6 alkyl, C1-C6 haloalkyl, nitro, phenyl, heteroaryl, substituted heteroaryl wherein the substituents may be C1-C6 alkyl or C1-C6 haloalkyl, benzyl, -CH2OAr, -OR6 and six-membered ring heterocyclic groups containing 1 or more O and/or N heteroatoms wherein any N heteroatom may be C1-C6 alkyl-substituted; and R6 may be selected from C1-C6 alkyl, phenyl, six-membered ring heterocyclic groups containing at least one O heteroatom, benzyl and substituted benzyl wherein the substituents may be halo, C1-C6 alkyl or C1-C6 alkoxy; R7 may be H or C1-C6 alkyl; and the stereochemistry of the bond depicted as ? is either α or β. Such derivatives have shown good inhibitory activity against malaria-causing parasites, e.g. Plasmodium falciparum, but have shown low levels of toxicity to human cells.
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Page/Page column 13; 18
(2013/03/26)
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- Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine
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Three new cytosine derived DNA modifications, 5-hydroxymethyl-2′- deoxycytidine (hmdC), 5-formyl-2′-deoxycytidine (fdC) and 5-carboxy-2′-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C-C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2′- deoxyuridine (hmdU). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C-C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C-C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C-C bond cleavage.
- Schiesser, Stefan,Pfaffeneder, Toni,Sadeghian, Keyarash,Hackner, Benjamin,Steigenberger, Barbara,Schroeder, Arne S.,Steinbacher, Jessica,Kashiwazaki, Gengo,Hoefner, Georg,Wanner, Klaus T.,Ochsenfeld, Christian,Carell, Thomas
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supporting information
p. 14593 - 14599
(2013/10/22)
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- Continuous flow photochemistry for the rapid and selective synthesis of 2′-deoxy and 2′,3′-dideoxynucleosides
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A new photochemical flow reactor has been developed for the photo-induced electron-transfer deoxygenation reaction to produce 2′-deoxy and 2′,3′-dideoxynucleosides. The continuous flow format significantly improved both the efficiency and selectivity of the reaction, with the streamlined multi-step sequence directly furnishing the highly desired unprotected deoxynucleosides.
- Shen, Bo,Jamison, Timothy F.
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p. 157 - 164
(2013/04/10)
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- Developing a collection of immobilized nucleoside phosphorylases for the preparation of nucleoside analogues: Enzymatic synthesis of arabinosyladenine and 2',3'-dideoxyinosine
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The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly expressed nucleoside phosphorylases from the bacterial pathogens Citrobacter koseri, Clostridium perfringens, and Streptococcus pyogenes (CkPNPI, CkPNPII, CpUP, SpUP) and their substrate specificity was investigated towards either natural pyrimidine or purine nucleosides and some analogues, namely, arabinosyladenine (araA) and 2',3'-dideoxyinosine (ddI). A 2-3 % activity towards these latter compounds (compared to the natural substrates) was observed. Enzyme activities were compared to the specificities obtained for the enzymes pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP) and purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNPII) previously reported by some of the authors. The enzymes displaying the suitable specificity for the synthesis of araA and ddI were immobilized on aldehyde-agarose. The immobilized preparations were highly stable at alkaline pH and in the presence of methanol or acetonitrile as cosolvent. They were used in the synthesis of araA and ddI by a one-pot, bienzymatic transglycosylation achieving 74 and 44 % conversion, respectively. Something different: Nucleoside phosphorylases are a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. Four new nucleoside phosphorylases have been prepared, characterized, and tested for their use in biocatalyzed syntheses of araA and ddI (see scheme). A generally applicable immobilization technique has been found to provide active and stable biocatalysts.
- Serra, Immacolata,Ubiali, Daniela,Piskur, Jure,Christoffersen, Stig,Lewkowicz, Elizabeth S.,Iribarren, Adolfo M.,Albertini, Alessandra M.,Terreni, Marco
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p. 157 - 165
(2013/04/24)
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- Oxidation and reduction of the 5-(2-Deoxyuridinyl)methyl radical
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Sleeping beauty: The 5-(2-Deoxyuridinyl)methyl radical 1 is a key intermediate in the thymine oxidative reaction mediated by reactive oxygen species. Evidence is presented that 1 is prone to both oxidation and reduction reactions at the absence of O2. These results question the current paradigm and suggest that the redox chemistry of 1, which has been largely overlooked in the past, may play a major role in determining the fate of 1. Copyright
- Lin, Gengjie,Li, Lei
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p. 5594 - 5598
(2013/06/27)
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- A straightforward diphenylmethyl protection method and deprotection of some pyrimidine nucleosides
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Benzhydryl protection of primary and secondary alcohols has been reported previously via reaction with metal alcoholates. Our aim was to find generally useful and very mild conditions for the alcoholic protection and deprotection of nucleosides with the diphenylmethyl group. This was accomplished for some pyrimidine nucleosides using PdCl2 as the transition metal catalyst, and with optimization yields of 70-90% have been achieved. A lack of solubility of other nucleosides hampers its more general use.
- Saudi, Milind,Van Aerschot, Arthur
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p. 8524 - 8534
(2013/08/23)
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