- Reactivity and DNA Damage by Independently Generated 2′-Deoxycytidin-N4-yl Radical
-
Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2′-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+?) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+?to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidicO6-methyl-2′-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5′-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5′-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.
- Peng, Haihui,Jie, Jialong,Mortimer, Ifor P.,Ma, Zehan,Su, Hongmei,Greenberg, Marc M.
-
p. 14738 - 14747
(2021/09/18)
-
- Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: A backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography
-
Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5′-deoxythymidine-5′-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 to-3.1 °C per phosphoroselenolate) when introduced at the 5′-Termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 ?. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.
- Conlon, Patrick F.,Eguaogie, Olga,Wilson, Jordan J.,Sweet, Jamie S. T.,Steinhoegl, Julian,Englert, Klaudia,Hancox, Oliver G. A.,Law, Christopher J.,Allman, Sarah A.,Tucker, James H. R.,Hall, James P.,Vyle, Joseph S.
-
p. 10948 - 10957
(2019/12/23)
-
- Identification of Flavin Mononucleotide as a Cell-Active Artificial N6-Methyladenosine RNA Demethylase
-
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
-
supporting information
p. 5028 - 5032
(2019/03/17)
-
- Dehalogenation of Halogenated Nucleobases and Nucleosides by Organoselenium Compounds
-
Halogenated nucleosides, such as 5-iodo-2′-deoxyuridine and 5-iodo-2′-deoxycytidine, are incorporated into the DNA of replicating cells to facilitate DNA single-strand breaks and intra- or interstrand crosslinks upon UV irradiation. In this work, it is shown that the naphthyl-based organoselenium compounds can mediate the dehalogenation of halogenated pyrimidine-based nucleosides, such as 5-X-2′-deoxyuridine and 5-X-2′-deoxycytidine (X=Br or I). The rate of deiodination was found to be significantly higher than that of the debromination for both nucleosides. Furthermore, the deiodination of iodo-cytidines was found to be faster than that of iodo-uridines. The initial rates of the deiodinations of 5-iodocytosine and 5-iodouracil indicated that the nature of the sugar moiety influences the kinetics of the deiodination. For both the nucleobases and nucleosides, the deiodination and debromination reactions follow a halogen-bond-mediated and addition/elimination pathway, respectively.
- Mondal, Santanu,Mugesh, Govindasamy
-
p. 1773 - 1780
(2019/01/10)
-
- Hydrogen peroxide-Triggered gene silencing in mammalian cells through boronated antisense oligonucleotides
-
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
-
p. 1112 - 1118
(2018/02/09)
-
- Novel nucleoside protective group and preparation thereof
-
The invention relates to a novel nucleoside protective group and a preparation thereof. Concretely, the invention provides a compound with a structure shown in a formula 1, wherein R1 is selected from C1-C6 alkyl or C6-C14 aryl, preferably C1-C4 alkyl or phenyl, such as methyl, ethyl or phenyl; R2 is selected from C1-C6 alkyl or C6-C14 aryl substituted C1-C6 alkyl, preferably C1-C4 alkyl or phenyl substituted C1-C4 alkyl, such as methyl, ethyl, benzyl or phenethyl; X is halogen, and preferably chlorine. In the acidic condition, compared with traditional 4,4'-dismethoxytriphenylmethyl nucleoside protective group, deprotection of the compound is easier.
- -
-
Paragraph 0181-0187
(2018/03/24)
-
- Copper-mediated arylsulfanylations and arylselanylations of pyrimidine or 7-deazapurine nucleosides and nucleotides
-
The syntheses of 5-arylsulfanyl- or 5-arylselanylpyrimidine and 7-arylsulfanyl- or 7-arylselanyl-7-deazapurine nucleosides and nucleotides were developed by the Cu-mediated sulfanylations or selanylations of the corresponding 5-iodopyrimidine or 7-iodo-7-
- Botha, Filip,Slaví?ková, Michaela,Pohl, Radek,Hocek, Michal
-
supporting information
p. 10018 - 10022
(2016/11/06)
-
- Mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from Enterococcus faecalis
-
EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPsregulatetheenzyme activity was not defined.Here, wepresent the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.
- Vorontsov, Ivan I.,Wu, Ying,Delucia, Maria,Minasov, George,Mehrens, Jennifer,Shuvalova, Ludmilla,Anderson, Wayne F.,Ahn, Jinwoo
-
p. 2815 - 2824
(2014/02/14)
-
- Mapping structurally defined guanine oxidation products along DNA duplexes: Influence of local sequence context and endogenous cytosine methylation
-
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
-
p. 4223 - 4235
(2014/04/03)
-
- Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine
-
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
-
p. 14593 - 14599
(2013/10/22)
-
- RADIOLABELED NUCLEOSIDE ANALOGUE, AND PREPARATION METHOD AND USE THEREOF
-
A radiolabeled nucleoside analogue is provided, which includes radioactive iodine 123I/131I, and a nucleoside analogue selected from a group consisting of cytidine, thymidine, uridine, and a derivative thereof. A method for preparing the radiolabeled nucleoside analogue, and a use thereof are further provided. The nucleoside analogue, prepared through the preparation method with a short synthesis time and a high radiochemical yield, has a long in vivo physiological half life and a high stability in serum, and, as a radiopharmaceutical composition, is useful in development of tumor proliferation diagnosis or therapy prognosis evaluation, and further assists in observation of long-time in vivo metabolism of a drug.
- -
-
Page/Page column 3-4
(2012/07/27)
-
- A single nuclease-resistant linkage in DNA as a versatile tool for the characterization of DNA lesions: Application to the guanine oxidative lesion g+34 generated by metalloporphyrin/KHSO5 reagent
-
The oxidation of an oligonucleotide containing a single nuclease-resistant phosphodiester link, a stereoisomerically pure methylphosphonate, by manganese (Mn-TMPyP) or iron (Fe-TMPyP) porphyrin associated to KHSO5 allowed the isolation and characterization of a guanine lesion corresponding to an increase of mass of 34 amu as compared to guanine ( G+34 ), namely, 5-carboxamido-5-formamido-2-iminohydantoin. Enzymatic digestion of the damaged oligonucleotide afforded, apart from the undamaged nucleotide monomer pool, a unique dinucleotide doubly modified with a methylphosphonate and an oxidized guanine base that is suitable for NMR analysis. The method can be applied to the study of any DNA lesion. More importantly, the method can be extended to the analysis of DNA damage in a sequence context. Any preselected residue in a DNA sequence may be individually analyzed by the easy introduction of a single nuclease-resistant link at the 3′- or 5′-position.
- Tomaszewska, Agnieszka,Mourgues, Sophie,Guga, Piotr,Nawrot, Barbara,Pratviel, Genevieve
-
p. 2505 - 2512
(2013/01/15)
-
- Enzymatic synthesis of 2-deoxy-β-d-ribonucleosides of 8-azapurines and 8-aza-7-deazapurines
-
The enzymatic synthesis of 8-azapurine and 8-aza-7-deazapurine 2-deoxyribonucleosides has been studied. Two methods have been used: (i) transglycosylation employing 2-deoxyguanosine, 2-deoxycytidine, 2-deoxyuridine, and 2-deoxythymidine as 2-deoxy-d-ribofuranose donors and recombinant E. coli purine nucleoside phosphorylase (PNP) as biocatalyst, and (ii) one-pot synthesis from 2-deoxy-d-ribose and nucleobases employing recombinant E. coli ribokinase (RK), phosphopentomutase (PPM) and PNP as biocatalysts. Good substrate activity was observed for all bases studied except 2-amino-8-aza-6-chloro-7-deazapurine, which afforded the desired N9-nucleoside in moderate yield due to very low solubility of the base and partial replacement of C6-chloro atom of the base and formed nucleoside with a hydroxy group. The participation of Ser90 Oγ of E. coli PNP in the binding of 8-aza-7-deazapurines in the catalytic center of PNP followed by the formation of a productive complex and glycosidic bond is suggested. Georg Thieme Verlag Stuttgart · New York.
- Stepchenko, Vladimir A.,Seela, Frank,Esipov, Roman S.,Miroshnikov, Anatoly I.,Sokolov, Yuri A.,Mikhailopulo, Igor A.
-
experimental part
p. 1541 - 1545
(2012/09/08)
-
- Phosphorylating reagent-free synthesis of 5′-phosphate oligonucleotides by controlled oxidative degradation of their 5′-end
-
The 5′-phosphorylated oligonucleotides (5′-pONs) are currently synthesized using expensive and sensitive modified phosphoramidite reagents. In this work, a simple, cost-effective, efficient, and automatable method is presented, based on the controlled oxidation of the 5′-terminal alcohol followed by a β-elimination reaction. The latter reaction leads to the removal of the terminal 5′-nucleoside and subsequent formation of the 5′-phosphate moiety. Thus, chemical phosphorylation of oligonucleotides (DNA or RNA) is achieved without using modified phosphoramidites.
- Sallamand, Corinne,Miscioscia, Audrey,Lartia, Remy,Defrancq, Eric
-
supporting information; body text
p. 2030 - 2033
(2012/06/18)
-
- 5-Fluoro-4-thiouridine phosphoramidite: New synthon for introducing photoaffinity label into oligodeoxynucleotides
-
The synthesis of phosphoramidite of 5-fluoro-4-thio-2′-O- methyluridine is described. An appropriate set of protecting groups was optimized including the 4-thio function introduced via 4-triazolyl as the 4-(2-cyanoethyl)thio derivative, and the t-butyldimethyl silyl for 2′ and 3′ hydroxyl protection, enabling efficient synthesis of the phosphoramidite. These protecting groups prevented unwanted side reactions during oligonucleotide synthesis. The utility of the proposed synthetic route was proven by the preparation of several oligonucleotides via automated synthesis. Photochemical experiments confirmed the utility of the synthon.
- Milecki, Jan,Nowak, Joanna,Skalski, Bohdan,Franzen, Stefan
-
experimental part
p. 6098 - 6106
(2011/11/07)
-
- Unprecedented gas-phase chiroselective logic gates
-
The gas-phase encounters between 2-aminobutane and proton-bound chiral resorcin[4]arene/nucleoside complexes behave in the gas phase as supramolecular "chiroselective logic gates" by releasing the nucleoside depending on the resorcin[4]arene and the 2-aminobutane configurations.
- Botta, Bruno,Fraschetti, Caterina,D'Acquarica, Ilaria,Sacco, Fabiola,Mattay, Jochen,Letzel, Matthias C.,Speranza, Maurizio
-
supporting information; experimental part
p. 1717 - 1719
(2011/05/03)
-
- Synthesis of oligodeoxynucleotides using fully protected Deoxynucleoside 3c-Phosphoramidite building blocks and base recognition of Oligodeoxynucleotides incorporating N3-Cyano-Ethylthymine
-
Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side products, is of great importance to biochemistry-based technology development. One side reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this reaction by synthesizing ODNs using fully protected deoxynucleoside 3c-phosphoramidite building blocks, where the remaining reactive nucleobase residues were completely protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of cyanoethylation at the nucleobase site showed that N3-protection of the thymine base efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating N3-cyanoethylthymine was synthesized using the phosphoramidite method, and primer extension reactions involving this ODN template were examined. As a result, the modified thymine produced has been proven to serve as a chain terminator.
- Tsunoda, Hirosuke,Kudo, Tomomi,Ohkubo, Akihiro,Seio, Kohji,Sekine, Mitsuo
-
experimental part
p. 7509 - 7531
(2011/02/28)
-
- Photolysis and thermolysis of platinum(IV) 2,2′-bipyridine complexes lead to identical platinum(II)-DNA adducts
-
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
-
experimental part
p. 11420 - 11431
(2010/11/24)
-
- Radiation-induced formation of purine 5′,8-cyclonucleosides in isolated and cellular DNA: High stereospecificity and modulating effect of oxygen
-
The present work is aimed at gaining conclusive mechanistic insights into the radiation-induced formation of the 5′R and 5′S diastereomers of both adenine and guanine 5′,8-cyclo-2′-deoxyribonucleosides, with emphasis on the delineation of the inhibitory effect of O2 in isolated and cellular DNA. The levels of purine 5′,8-cyclo-2′- deoxyribonucleosides as assessed by HPLC-MS/MS were found to decrease steadily with the increase of O2 concentration, the 5′,8-cyclo-2′- deoxyguanosine being produced more efficiently than the 5′,8-cyclo- 2′-deoxyadenosine for low O2 concentrations. A high stereoselectivity was observed in the intramolecular addition of the C5′ radical to the C8 of the purine leading, after the creation of the C5′-C8 bond and a subsequent oxidation step, to the predominant formation of the 5′R diastereomer for both purine 5′,8-cyclonucleosides. The reduced formation yield of the 4 tandem lesions in the presence of O2 explains, at least partly, the low efficiency of radiation-induced yields of the purine 5′,8-cyclo-2′-deoxyribonucleosides in cellular DNA, which are about two orders of magnitude lower than the previously reported data obtained from HPLC-MS analysis. The Royal Society of Chemistry 2010.
- Belmadoui, Nourreddine,Boussicault, Fabien,Guerra, Maurizio,Ravanat, Jean-Luc,Chatgilialoglu, Chryssostomos,Cadet, Jean
-
experimental part
p. 3211 - 3219
(2010/08/21)
-
- New thermolytic carbamoyl groups for the protection of nucleobases
-
It was found that N-arylcarbamoyl and N-(phenylsulfonyl)carbamoyl (psc) groups could be effectively introduced onto the amino groups of deoxycytidine and deoxyadenosine derivatives and could be removed thermolytically. We succeeded in synthesizing DNA pro
- Ohkubo, Akihiro,Kasuya, Rintaro,Miyata, Kenichi,Tsunoda, Hirosuke,Seio, Kohji,Sekine, Mitsuo
-
experimental part
p. 687 - 694
(2009/06/20)
-
- DNA interstrand cross-link formation by the 1,4-dioxobutane abasic lesion
-
The oxidized abasic lesion 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly with a single-strand break by a variety of DNA-damaging agents that abstract a hydrogen atom from the C5′-position. Independent generation of the DOB lesion in DN
- Guan, Lirui,Greenberg, Marc M.
-
supporting information; experimental part
p. 15225 - 15231
(2010/01/29)
-
- Lactobacillus Fermentum N-Desoxyribosyl Transferases and the Use Thereof for Enzymatic Synthesis of 2', 3' - Didesoxynucleosides and 2',3'- Didehydro-2',3'- Didesoxynucleosides
-
The inventive method for evaluating an X protein encoded by an Lactobacillus fermentum (L. fermentum ) ntd gene in such a way that the characteristics thereof are modified consists a) in obtaining the Lactobacillus fermentum (L. fermentum ) ntd gene mutants by random mutagenesis, b) in transforming cells containing a [P-] phenotype provided with vectors containing mutated nucleic acids obtained at the stage a) coding for the thus modified X* proteins, wherein P- means that said cells are auxotrophic for a substance P produced by the action of X on a natural substrate S, c) in culturing said cells in a medium comprising a substrate S*, wherein S* is an analog to the natural substrate S of the protein X and d) in selecting the cells [P-::X*] which survived at the stage c) and ijn which the proteins X* are capable of carrying out the biosynthesis of the product P based on the substrate S*. The mutated L. fermentum N-desoxyribosyl transferases have an N-didesoxyribosyl transferase activity, corresponding nucleic acids, expression vectors, host cells containing said vectors and an application for the enzymatic synthesis of 2′,3′-didesoxynucleosides and 2′,3′-didehydro-2′,3′-didesoxynucleosides.
- -
-
Page/Page column 6-8
(2008/06/13)
-
- Flow injection amperometric detection of 2′-deoxyguanosine at a ruthenium oxide hexacyanoferrate modified electrode
-
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
-
p. 5392 - 5398
(2008/02/10)
-
- A new protecting group for the exocyclic amino groups of nucleosides
-
A new protecting group has been developed for the exocyclic amino groups of nucleosides that occur in DNA. 3-Phenyl-[{N-(2-trimethylsilyl-ethoxycarbonyl)- 2-amino}]-propanoic acid used as the protective agent.
- Varaprasad, Chamakura V.N.S.,Johnson, Francis
-
p. 2163 - 2165
(2007/10/03)
-
- Process for producing 2'-deoxyguanosine
-
The invention provides a process for producing 2′-deoxyguanosine, characterized in that the process includes reacting one compound selected from the group consisting of guanosine, guanosine 5′-monophosphate, and 2-amino-6-substituted purine with 2′-deoxynucleoside in the presence of nucleoside deoxyribosyl transferase and a hydrolase. According to the process of the present invention, 2′-deoxyguanosine can be synthesized efficiently from inexpensive and easily available starting materials. Since no guanosine, which disturbs purification, is virtually present in a reaction mixture, isolation and purification of 2′-deoxyguanosine can be performed in a very simple manner. Thus, the process for producing 2′-deoxyguanosine is practical.
- -
-
Page/Page column 5
(2008/06/13)
-
- Preparation of deoxynucleosides
-
Methods for preparing deoxynucleosides from their corresponding ribonucleosides by forming 3-tert-butylphenoxythiocarbonylderivatives of the ribonucleosides and subsequently effecting radical deoxygenation reactions at the carbon atoms to be deoxygenated.
- -
-
Page column 14
(2008/06/13)
-
- Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds
-
Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2′-deoxycytidylyl- (3′>5′)-2′-deoxyuridine (dCpdU) and 2′-deoxyadenylyl- (3′→5′)-2′-deoxycytidine (dApdC)] and isomeric dinucleotides
- Nair, Vasu,Pal, Suresh
-
p. 289 - 291
(2007/10/03)
-
- Lewis acid deprotection of silyl-protected oligonucleotides and base-sensitive oligonucleotide analogues
-
a- a- a- Oligonucleotides protected with N-(trimethylsilylethoxycarbonyl) (Teoc) and P-(trimethylsilylethanol) (Tse) groups were synthesized and deprotected by a single ZnBr2 treatment. Teoc group stabilized dA against depurination. This strategy was applied to the synthesis of base-sensitive oligonucleotide prodrugs bearing S-acetyl-2-thioethyl (Sate) phosphotriesters.
- Ferreira, Fernando,Vasseur, Jean-Jacques,Morvan, Fran?ois
-
p. 6287 - 6290
(2007/10/03)
-
- Facile synthesis of oligonucleotides on solid support using pyridine derivatives for amino and phosphate protection
-
The picolinoyl group has been used for amino protection in the case of all the three deoxynucleosides-dC, dA and dG, and good yields of N-protected nucleosides are obtained. This pyridine derivative along with another pyridine derivative, viz. (α-pyridyl) methyl, an autocatalytic phosphate protecting group, has been used successfully in the synthesis of two oligonucleotides, d(TACGTTTTGCT) and d(ACCGATATCGT) following solid phase methodology. The good yields of these 11-mers (48 and 45%, respectively) are attributed to the greater solubilising effect generated due to the combination of these two groups. The structures of these oligomers have been confirmed by enzymatic hydrolysis with snake venom phosphodiesterase followed by alkaline phosphatase.
- Sinha, Sarika,Singh, Ramendra K.
-
p. 1696 - 1700
(2007/10/03)
-
- Enzymatic synthesis of 2'-deoxyguanosine with nucleoside deoxyribosyltransferase-II.
-
Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2'-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2'-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.
- Okuyama, Kiyoshi,Shibuya, Susumu,Hamamoto, Tomoki,Noguchi, Toshitada
-
p. 989 - 995
(2007/10/03)
-
- Stereospecific synthesis of oligonucleotides containing crotonaldehyde adducts of deoxyguanosine
-
Crotonaldehyde reacts with DNA to form two diastereomeric 1,N2 cyclic adducts of deoxyguanosine. A synthesis of the two diastereomeric deoxynucleosides has been achieved by reaction of mixed diastereomers of 4-amino-1,2-pentanediol with 2-fluoro-O6-(trimethylsilylethyl)-deoxyinosine. The resulting N2-(1-methyl-3,4-dihydroxybutyl)-deoxyguanosine was treated with NaIO4, cleaving the vicinal diol to the aldehyde. Spontaneous cyclization gave the two diastereomers of the crotonaldehyde-adducted nucleoside that were readily separated by HPLC. The absolute configurations were assigned by an enantiospecific synthesis of one diastereomer from (S)-3-aminobutanoic acid. The synthetic strategy has been extended to preparation of a site-specifically adducted oligonucleotide by reaction of the mixed diastereomers of 4-amino-1,2-pentanediol with an 8-mer oligonucleotide containing 2-fluoro-O6-(trimethylsilylethyl)-deoxyinosine. The diastereomeric oligonucleotides were separated by HPLC and absolute configurations of the adducts were established by enzymatic digestion to the adducted nucleosides.
- Nechev,Kozekov,Harris,Harris
-
p. 1506 - 1512
(2007/10/03)
-
- Synthesis and characterization of nucleosides and oligonucleotides with a benzo[a]pyren-6-ylmethyl adduct at adenine N6 or guanine N2
-
Benzo[a]pyrene (1) can be converted to reactive electrophilic species by a number of metabolic pathways, of which the route to the mutagenic and carcinogenic diol epoxide(s) is the best studied. An alternative and interesting pathway to a highly genotoxic electrophile is through alkylation at the 6 position to 6-methylbenzo[a]pyrene (2) followed by oxidation of the methyl group to give 6-hydroxymethylbenzo[a]pyrene (3). Esterification of 3, especially to sulfate ester 4, gives compounds which are both mutagenic and carcinogenic. The major DNA adduct identified from exposure of rats and mice to 4 is the guanine N2 adduct [2′-deoxy-N2-(benzo-[a]pyren-6-ylmethyl)guanosine,5] which is also formed via activation of 2 to a radical cation species by horseradish peroxidase/H2O2 or iodine. To study the biological and structural properties of this adduct and the analogous adenine N6 adduct (6), a nonbiomimetic synthesis of the adducted nucleosides 5 and 6 has been developed and has been extended to preparation of oligonucleotides containing 5 or 6 at a single site.
- Kim,Cooper,Nechev,Harris,Harris
-
p. 1306 - 1314
(2007/10/03)
-
- Large-scale manufacturing of all four 2′-deoxynucleosides via novel strategies including a chemo-enzymatic process
-
A chemical synthesis of 2-deoxyribose-1-phosphate 2 and its enzymatic conversion into purine 2′-deoxynucleosides (dNus) are shown. Besides the chemoenzymatic process for purine dNus, a modified process for practical dC preparation is also established. Consequently, a series of practical manufacturing processes of all four dNus have been realized via novel strategies.
- Komatsu,Awano,Tanikawa,Itou,Ikeda
-
p. 1291 - 1293
(2007/10/03)
-
- Ceric ammonium nitrate on silica gel for efficient and selective removal of trityl and silyl groups
-
Silicna gel-supported ceric ammonium nitrate (CAN-SiO2) was found effective for rapid and selective cleavage of trityl (Tr), monomethoxytrityl (MMTr), and dimethoxytrityl (DMTr) groups from protected nucleosides and nucleotides under mild conditions. Efficiency of deprotections depended upon the stability of th resultant carbocationic species: DMTr+ > MMTr+ > Tr+. Use of a catalytic amount of this solid-supported reagent can also efficiently and selectively remove the tert butyldimethylsilyl or the triisopropylsilyl group from a primary hydroxyl functionality in di- or trisilyl ethers of ribonucleosides. A comparative stud of deprotection reactions by utilization of CAN alone or CAN-SiO2 indicates a remarkable increase in the rate of the reactions involving a solid support. The mechanism of electron-transfer processes is proposed for the use of CAN-SiO2 in the removal of these protective groups from organic molecules.
- Hwu, Jih Ru,Jain, Moti L.,Tsai, Fu-Yuan,Tsay, Shwu-Chen,Balakumar, Arumugham,Hakimelahi, Gholam H.
-
p. 5077 - 5088
(2007/10/03)
-
- Synthesis and deprotection of oligonucleotides under aprotic conditions
-
(matrix presented) Oligonucleotides containing an alkali-labile nucleotide are synthesized and deprotected using a synthetic method that eliminates the use of Bronsted acid and base. The development of a new family of exocyclic amine-protecting groups is an integral component of this approach.
- Chen, Tongqian,Fu, Jiasheng,Greenberg, Marc M.
-
p. 3691 - 3694
(2007/10/03)
-
- Stereocontrolled Syntheses of Deoxyribonucleosides via Photoinduced Electron-Transfer Deoxygenation of Benzoyl-Protected Ribo- and Arabinonucleosides
-
The stereocontrolled, de novo syntheses of β-2′-deoxy-, α-2′-deoxy-, β-3′-deoxy-, and β-2′,3′-dideoxyribonucleosides are described. Strategically protected ribose, arabinose, and xylose glycosylation precursors were synthesized bearing C2-esters capable of directing Vorbrueggen glycosylation. The key step is the regioselective deoxygenation of the desired hydroxyl group as either the benzoyl- or 3-(trifluoromethyl)benzoyl derivative. This deoxygenation is accomplished via a photoinduced electron-transfer (PET) mechanism using carbazole derivatives as the photosensitizer. The syntheses of the desired deoxynucleoside generally proceed in three steps from a common, readily available precursor.
- Wang, Zhiwei,Prudhomme, Daniel R.,Buck, Jason R.,Park, Minnie,Rizzo, Carmelo J.
-
p. 5969 - 5985
(2007/10/03)
-
- Direct strand cleavage via furanyladenine formation in anaerobic photoirradiation of 5-bromouracil-containing oligonucleotides
-
The mechanism of direct strand cleavage induced by anaerobic irradiation of 5-bromouracil ((Br)U)-containing DNA was investigated. The formation of an oligonucleotide fragment containing 3'-furanyladenine end like 5 was observed as a major photoproduct for the direct strand breaks. (C) 2000 Elsevier Science Ltd.
- Fujimoto,Ikeda,Saito
-
p. 6455 - 6459
(2007/10/03)
-
- Recognition and inhibition of HIV integrase by novel dinucleotides
-
HIV integrase is involved in the integration of viral DNA into chromosomal DNA, a biological process that occurs by a sequence involving HIV DNA splicing and subsequent integration steps. In the quest for small nucleotide systems with nuclease stability of the internucleotide phosphate bond and critical structural features for recognition and inhibition of HIV-1 integrase, we have discovered novel, nuclease-resistant dinucleotides with defined base sequences that are inhibitors of this key viral enzyme. Synthetic methodologies utilized for the syntheses of the novel dinucleotides include an excellent new phosphorylating agent.
- Taktakishvili, Michael,Neamati, Nouri,Pommier, Yves,Pal, Suresh,Nair, Vasu
-
p. 5671 - 5677
(2007/10/03)
-
- Photocleavable protecting groups as nucleobase protections allowed the solid-phase synthesis of base-sensitive SATE-prooligonucleotides
-
The first synthesis of oligodeoxynucleotide heteropolymers carrying base-sensitive S-pivaloylthioethyl (t-Bu-SATE) phosphotriester linkages has been performed. It is based on the use of 6-nitroveratryloxycarbonyl (NVOC) and 2,2'-bis(2-nitrophenyl)ethoxycarbonyl (diNPEOC) groups as nucleobase protections in combination with photolysis deprotection. The synthesis was realized using the phosphoramidite approach on solid support bearing a 1-(o- nitrophenyl)-1,3-propanediol linker. The removal of the protecting groups and the cleavage of the oligonucleotides from the solid support were accomplished in a single photolysis procedure upon UV irradiation at wavelengths > 300 nm. Faster deprotection rates were observed for diNPEOC-protected nucleosides and oligomers than with NVOC-protected ones. The synthesis of pentanucleoside t- Bu-SATE-phosphotriesters d((5')TpCpCpCpTp(3')), d((5')TpApApApAp(3')), and d((5')TpGpGpGpTp(3')) and of dodecanucleoside t-Bu-SATE-phosphotriesters and -phosphorothioate d((5')ApCpApCpCpCpApApTpTpCpTp(3')) and d((5')ApGpApApTpTpGpGpGpTpGpTp(3')) demonstrated the efficiency of the method.
- Alvarez, Karine,Vasseur, Jean-Jacques,Beltran, Thierry,Imbach, Jean-Louis
-
p. 6319 - 6328
(2007/10/03)
-
- Selective hydrolysis of nucleotides to nucleosides and free bases
-
The kinetics of the hydrolysis of 2'-deoxyadenosine-5'-monophosphoric acid (dAMP), 2'-deoxycytidine-5'-monophosphoric acid (dCMP), 2'-deoxyguanosine-5'-monophosphoric acid (dGMP) and tymidine-5'-monophosphoric acid (dTMP) was studied in the presence of Xanthomonas maltophilia [1]. The reaction products are nucleosides: 2'-deoxyadenosine (dA), 2'-deoxycytidine (dC), 2'-deoxyguanosine (dG) and tymidine (dT), respectively, or the respective free bases. Hydrolysis of dTMP and dGMP proceeded stepwise according to the sequence: nucleotide→nucleoside→free base, whereas no accumulation of the free base was observed during the hydrolysis of dAMP and dCMP. Copyright (C) 1999 Elsevier Science S.A.
- Chmielowiec, Urszula,Kruszewska, Hanna,Cybulski, Jacek
-
p. 611 - 614
(2007/10/03)
-
- Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo.
-
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
-
p. 335 - 340
(2007/10/03)
-
- Cleavage of oligodeoxyribonucleotides from polymer supports and their rapid deprotection under microwaves
-
Novel conditions for the cleavage of oligodeoxynucleotides from polymer supports and their complete deprotection under microwaves have been developed. The oligonucleotides synthesized using phosphoramidite synthons carrying base labile (Pac, Dmf and t-Bpac) and conventional (Bz for A and C and Pac for G) protecting groups for nucleic bases were deprotected using 0.2M sodium hydroxide (MeOH : H2O :: 1:1, v/v) = Reagent A and 1M sodium hydroxide (MeOH : H2O :: 1:1, v/v) = Reagent B, respectively under microwaves. The deprotected oligonucleotides were found to be comparable with the corresponding oligonucleotides deprotected under standard conditions (aq. ammonia at 55°C).
- Gupta,Kumar
-
p. 1761 - 1766
(2007/10/03)
-
- Hydrolysis of phosphomonoesters in nucleotides by cerium(IV) ions. Highly selective hydrolysis of monoester over diester in concentrated buffers
-
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
-
p. 1685 - 1688
(2007/10/03)
-
- Transformations of thiopyrimidine and thiopurine nucleosides following oxidation with dimethyldioxirane
-
A general and convenient method for the synthesis of several pyrimidine and purine nucleosides by selective oxidation of thionucleosides with dimethyldioxirane is reported. Thioketo moieties in the C-4 position of the pyrimidine ring, and in the C-6, and C-8 positions of the purine ring are the domain of oxidative nucleophilic substitution. Thioketo moieties in the C-2 position of both purine and pyrimidine rings are the domain of desulfurization or formation of disulfides.
- Saladino, Raffaele,Mincione, Enrico,Crestini, Claudia,Mezzetti, Maurizio
-
p. 6759 - 6780
(2007/10/03)
-
- Novel synthetic route to 1-substituted cytosines
-
1-Alkylcytosines, cytidine and 2'-deoxy-5-methylcytidine were synthesized in good yields from appropriate 1-alkyluracils, uridine or thymidine derivatives by hydrogenation of 6-substituted tetrazolo[1,5-c]pyrimidin-5(6H)-ones 2 over 10% palladium on charcoal in methanol. Compounds 2 were obtained by the reaction of 1-substituted uracils 1 with sodium azide and phosphorus oxychloride in acetonitrile.
- Ciszewski,Celewicz,Golankiewicz
-
p. 777 - 779
(2007/10/02)
-
- 1,1,1,3,3,3-Hexafluoro-2-propanol for the Removal of the 4,4'-Dimethoxytrityl Protecting Group from the 5'-Hydroxyl of Acid-Sensitive Nucleosides and Nucleotides
-
1,1,1,3,3,3-Hexafluoro-2-propanol is introduced as a suitable reagent and solvent for the detritylation of 5'-O-(4,4'-dimethoxytrityl)-nucleosides and -deoxy- nucleosides, especially those that are susceptible to N-glycosyl cleavage under more strongly acidic conditions.
- Leonard, Nelson J.,Neelima
-
p. 7833 - 7836
(2007/10/02)
-
- Fast Cleavage and Deprotection of Oligonucleotides
-
We have developed methylamine/ammonia as a fast cleavage and deprotection reagent which effects complete cleavage of oligonucleotides from the solid support in 5 min at room temperature and complete deprotection in 5 min at 65 deg C.The problem of transamination side products formation, faced with the commonly used dCbz (10percent side product) upon treatment with methylamine/ammonia, has been sucessfully solved by the use of dCac (0.0percent side product).DMT dCac phosphoramidite-methylamine/ammonia system furnished oligonucleotides in equal or superior quality as compared to the other systems.
- Reddy, M. P.,Hanna, N. B.,Farooqui, Firdous
-
p. 4311 - 4314
(2007/10/02)
-
- A New and Efficient Synthesis of Cytidine and Adenosine Derivatives by Dimethyldioxirane Oxidation of Thiopyrimidine and Thiopurine Nucleosides
-
Dimethyldioxirane oxidation of thiopyrimidine and thiopurine nucleosides, in the presence of amines in stoichiometric amount, afforded selectively and under mild experimental conditions cytidine and adenosine nucleosides.
- Saladino, Raffaele,Crestini, Claudia,Bernini, Roberta,Frachey, Giuseppe,Mincione, Enrico
-
p. 3053 - 3054
(2007/10/02)
-
- Electron transfer in di(deoxy)nucleoside phosphates in aqueous solution: Rapid migration of oxidative damage (via adenine) to guanine
-
In aqueous solution, the one-electron loss centers created statistically by the oxidant SO4.- or by photoionization in di(2′-deoxy)nucleoside phosphates (DNPs) containing the base guanine (G) become localized at G, as concluded from pulse radiolysis and 193-nm laser photolysis experiments. From the latter it is evident that, in the case of adenylyl(3′→5′)guanosine (ApG), the charge-transfer process is complete in ≤50 ns. With DNPs containing a pyrimidine and the purine base adenine, the oxidative damage is collected by the adenine moiety (k ≥ 2 × 105 s-1).
- Candeias, Luis Pedro,Steenken, Steen
-
p. 2437 - 2440
(2007/10/02)
-
