- Synthesis of Phosphoramidite Monomers Equipped with Complementary Bases for Solid-Phase DNA Oligomerization
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We describe the preparation of two monomers that bear complementary nucleobases at the edges (guanine-2′-deoxycytidine and 2-aminoadenine-2′-deoxyuridine) and that are conveniently protected and activated for solid-phase automated DNA synthesis. We report the optimized synthetic routes leading to the four nucleobase derivatives involved, their cross-coupling reactions into dinucleobase-containing monomers, and their oligomerization in the DNA synthesizer.
- Romero-Pérez, Sonia,López-Martín, Isabel,Martos-Maldonado, Manuel C.,Somoza, álvaro,González-Rodríguez, David
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supporting information
p. 41 - 45
(2020/01/03)
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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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supporting information
p. 867 - 876
(2020/01/24)
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- Site-specific incorporation of multiple units of functional nucleotides into DNA using a step-wise approach with polymerase and its application to monitoring DNA structural changes
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We have developed a new method, a step-wise approach with polymerase, for site-specific incorporation of multiple units of functional nucleotides into DNA to form hairpin secondary structures. The fluorescence of the resulting DNA incorporating the functional nucleotides varied upon transitioning from single-strand to hairpin and duplex structures.
- Huy Le, Binh,Nguyen, Van Thang,Seo, Young Jun
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supporting information
p. 2158 - 2161
(2019/02/20)
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- 5-iodo-4-thio-2′-deoxyuridine as a sensitizer of X-ray induced cancer cell killing
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Nucleosides, especially pyrimidines modified in the C5-position, can act as radiosensitizers via a mechanism that involves their enzymatic triphosphorylation, incorporation into DNA, and a subsequent dissociative electron attachment (DEA) process. In this paper, we report 5-iodo-4-thio-2′-deoxyuridine (ISdU) as a compound that can effectively lead to ionizing radiation (IR)-induced cellular death, which is proven by a clonogenic assay. The test revealed that the survival of cells, pre-treated with 10 or 100 μM solution of ISdU and exposed to 0.5 Gy of IR, was reduced from 78.4% (for non-treated culture) to 67.7% and to 59.8%, respectively. For a somewhat higher dose of 1 Gy, the surviving fraction was reduced from 68.2% to 54.9% and to 40.8% for incubation with 10 or 100 μM ISdU, respectively. The cytometric analysis of histone H2A.X phosphorylation showed that the radiosensitizing effect of ISdU was associated, at least in part, with the formation of double-strand breaks. Moreover, the cytotoxic test against the MCF-7 breast cancer cell line and human dermal fibroblasts (HDFa line) confirmed low cytotoxic activity of ISdU. Based on the results of steady state radiolysis of ISdU with a dose of 140 Gy and quantum chemical calculations explaining the origin of the MS detected radioproducts, the molecular mechanism of sensitization by ISdU was proposed. In conclusion, we found ISdU to be a potential radiosensitizer that could improve anticancer radiotherapy.
- Makurat, Samanta,Spisz, Paulina,Kozak, Witold,Rak, Janusz,Zdrowowicz, Magdalena
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- Bio-catalytic synthesis of unnatural nucleosides possessing a large functional group such as a fluorescent molecule by purine nucleoside phosphorylase
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Unnatural nucleosides are attracting interest as potential diagnostic tools, medicines, and functional molecules. However, it is difficult to couple unnatural nucleobases to the 1′-position of ribose in high yield and with β-regioselectivity. Purine nucleoside phosphorylase (PNP, EC2.4.2.1) is a metabolic enzyme that catalyses the conversion of inosine to ribose-1α-phosphate and free hypoxanthine in phosphate buffer with 100% α-selectivity. We explored whether PNP can be used to synthesize unnatural nucleosides. PNP catalysed the reaction of thymidine as a ribose donor with purine to produce 2′-deoxynebularine (3, β form) in high conversion (80%). It also catalysed the phosphorolysis of thymidine and introduced a pyrimidine base with a halogen atom substituted at the 5-position into the 1′-position of ribose in moderate yield (52-73%), suggesting that it exhibits loose selectivity. For a bulky purine substrate [e.g., 6-(N,N-di-propylamino)], the yield was lower, but addition of a polar solvent such as dimethyl sulfoxide (DMSO) increased the yield to 74%. PNP also catalysed the reaction between thymidine and uracil possessing a large functional fluorescent group, 5-(coumarin-7-oxyhex-5-yn) uracil (C4U). Conversion to 2′-deoxy-[5-(coumarin-7-oxyhex-5-yn)] uridine (dRC4U) was drastically enhanced by DMSO addition. Docking simulations between dRC4U and E. coli PNP (PDB 3UT6) showed the uracil moiety in the active-site pocket of PNP with the fluorescent moiety at the entrance of the pocket. Thus, the bulky fluorescent moiety has little influence on the coupling reaction. In summary, we have developed an efficient method for producing unnatural nucleosides, including purine derivatives and modified uracil, using PNP.
- Hatano, Akihiko,Wakana, Hiroyuki,Terado, Nanae,Kojima, Aoi,Nishioka, Chisato,Iizuka, Yu,Imaizumi, Takuya,Uehara, Sanae
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p. 5122 - 5129
(2019/10/05)
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- Direct incorporation and extension of a fluorescent nucleotide through rolling circle DNA amplification for the detection of microRNA 24-3P
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We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene, which have different sizes, and screened their incorporation and extension capability during the rolling circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incorporate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP, and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA 24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P. This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P and also exhibited highly sensitive and selective detection properties.
- Le, Binh Huy,Seo, Young Jun
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supporting information
p. 2035 - 2038
(2018/05/04)
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- Diverse size approach to incorporate and extend highly fluorescent unnatural nucleotides into DNA
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We have prepared a series of size-diverse unnatural nucleotides containing fluorescent (dApyrTP, dUpyrTP, dUantTP, dUthiTP) and quencher (dUazoTP) units, as well as nucleotides presenting small functional groups (dAethTP, dAoctTP, dUethTP, dUiodTP), all based on deoxyadenosine and deoxyuridine, and examined their suitability for use in enzymatic incorporation and extension into DNA. We observed a size-dependence of the incorporation and extension capability (following the order dUiodTP?=?dUethTP?=?dUthiTP?>?dUazoTP?>?dUpyrTP?>?dUantTP) during primer extension. This result was supported by circular dichroism (CD) spectra, which revealed a trend in the different B-form DNA structures depending on the size of the unit at the 5-position of the deoxyuridine (dUiodTP?>?dUethTP?>?dUthiTP?>?dUpyrTP), obtained from the PCR products. Interestingly, dUthiTP could be incorporated and extended into long DNA strands during primer extension and even PCR amplification, with CD spectroscopy confirming a stable secondary B-form duplex DNA structure. We observed full-length extension products even when combining dUthiTP with a template containing 24 continuous dA units during the primer extension. Thus, we believe that dUthiTP is a promising fluorescent nucleotide for a diverse range of biological applications requiring multiple incorporation and extension directly without disruption of B-form DNA structures.
- Le, Binh Huy,Koo, Ja Choon,Joo, Han Na,Seo, Young Jun
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supporting information
p. 3591 - 3596
(2017/06/13)
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- A comparison between immobilized pyrimidine nucleoside phosphorylase from Bacillus subtilis and thymidine phosphorylase from Escherichia coli in the synthesis of 5-substituted pyrimidine 2′-deoxyribonucleosides
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Pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli (EcTP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2′-deoxyribonucleosides (14-18) by transglycosylation in fully aqueous medium. From the comparative study of the two biocatalysts, no remarkable differences emerged about their substrate specificity, bioconversion yield, stability in organic cosolvents (DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2′,3′- dideoxynucleosides as substrates, whereas they catalyze bioconversions involving 5′-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14-18 proceeded at a similar conversion (33-68% for BsPyNP and 25-62% for EcTP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2′-deoxyuridine (14) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% (EcTP) or 76% (BsPyNP) conversion (about 9 g/L).
- Serra, Immacolata,Bavaro, Teodora,Cecchini, Davide A.,Daly, Simona,Albertini, Alessandra M.,Terreni, Marco,Ubiali, Daniela
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- Synthesis of novel nucleoside 5′-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids
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A series of novel nucleoside 5′-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative functional capabilities: an amino group for the reaction of amino-alkynyl- modified oligonucleotides with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the new linker can be used to synthesize a diversity of modified pyrimidine nucleosides. Copyright Taylor and Francis Group, LLC.
- Vasilyeva, Svetlana V.,Budilkin, Boris I.,Konevetz, Dmitrii A.,Silnikov, Vladimir N.
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body text
p. 753 - 767
(2012/07/28)
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- Ionic liquid mediated synthesis of 5-halouracil nucleosides: Key precursors for potential antiviral drugs
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Synthesis of antiviral 5-halouracil nucleosides, also used as key precursors for the synthesis of other potential antiviral drugs, has been demonstrated using ionic liquids as convenient and efficient reaction medium.
- Kumar, Vineet,Malhotra, Sanjay V.
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experimental part
p. 821 - 834
(2010/08/20)
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- Highly efficient method for C-5 halogenation of pyrimidine-based nucleosides in ionic liquids
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A novel, highly efficient, convenient, and benign methodology for C-5 halogenation of pyrimidine-based nucleosides has been developed using N-halosuccinimides as halogenating reagents without using any catalyst in ionic liquid medium. The ionic liquids were successfully recovered and reused for all the reactions. Georg Thieme Verlag Stuttgart.
- Kumar, Vineet,Yap, Jeremy,Muroyama, Andrew,Malhotra, Sanjay V.
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experimental part
p. 3957 - 3962
(2010/03/26)
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- Importance of 3′-hydroxyl group of the nucleosides for the reactivity of thymidine phosphorylase from Escherichia coli
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Thymidine phosphorylase in phosphate buffer catalyzed the conversion of thymidine to unnatural nucleosides. The 3′-OH, but not the 5′-OH of ribosyl moiety is necessary to be recognized as a substrate. Thus 3′-deoxythymidine could not convert to 5-fluorouracil-2′,3′- dideoxyribose. However, 5′-deoxythymidine was converted to 5-fluorouracil-2′,5′-dideoxyribose. Copyright
- Hatano, Akihiko,Harano, Aiko,Kirihara, Masayuki
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p. 232 - 233
(2007/10/03)
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- Antiviral agents
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Nucleoside compounds of the formula STR1 wherein: B is a purine or a pyrimidine; X and X' are H, OH or F, provided that at least one is H; Y and Y' are H, OH, OCH3 or F, provided that at least one is H; Y' and Z together form a cyclic phosphate ester, provided that Y is H; or Z is STR2 where n is zero, one, two or three; and Z' is N3 or OCH3 ; provided that when X' and Y' are OH and Z' is N3, B is not cytosine, and when X' and Y' are OH and Z' is OCH3, B is not uracil, adenine or cytosine; and the pharmaceutically acceptable esters, ethers and salts thereof, have been found to have potent antiviral activity with a high therapeutic ratio.
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- SYNTHESIS OF 2-DEOXY-β-D-RIBONUCLEOSIDES AND2,3-DIDEOXY.β-D-PENTOFURANOSIDES ON IMMOBILIZED BACTERIAL CELLS
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Alginate gel-entrapped cells of auxotrophic thymine-dependent strain of E. coli catalyze the transfer of 2-deoxy-D-ribofuranosyl moiety of 2'-deoxyuridine to purine and pyrimidine bases as well as their aza and deaza analogs.All experiments invariably gave β-anomers; in most cases, the reaction was regiospecific, affording N9-isomers in the purine and N1-isomers in the pyrimidine series.Also a 2,3-dideoxynucleoside can serve as donor of the glycosyl moiety.The acceptor activity of purine bases depends only little on substitution, the only condition being the presence of N7-nitrogen atom.On the other hand, in the pyrimidine series the activity is limited to only a narrow choice of mostly short 5-alkyl and 5-halogeno uracil derivatives.Heterocyclic bases containing amino groups are deaminated; this can be avoided by conversion of the base to the corresponding N-dimethylaminomethylene derivative which is then ammonolyzed.The method was verified by isolation of 9-(2-deoxy-β-D-ribofuranosyl) derivatives of adenine, guanine, 2-chloroadenine, 6-methylpurine, 8-azaadenine, 8-azaguanine, 1-deazaadenine, 3-deazaadenine, 1-(2-deoxy-β-D-ribofuranosyl) derivatives of 5-ethyluracil, 5-fluorouracil, and 9-(2,3-deoxy-β-D-pentofuranosyl)hypoxanthine, 9-(2,3-deoxy-β-D-pentofuranosyl)-6-methylpurine, and other nucleosides.
- Votruba, Ivan,Holy, Antonin,Dvorakova, Hana,Guenter, Jaroslav,Hockova, Dana,et al.
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p. 2303 - 2330
(2007/10/02)
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- A mild and efficient methodology for the synthesis of 5-halogeno uracil nucleosides that occurs via a 5-halogeno-6-azido-5,6-dihydro intermediate
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A mild and efficient methodology for the synthesis of 5-halogeno (iodo, bromo, or chloro) uracil nucleosides has been developed. 5-Halo-2'-deoxyuridines 4a-c (84-95%), 5-halouridines 7a-c (45-95%), and 5-haloarabinouridines 8a-c (65-95%) were synthesized in good to excellent yields by the reaction of 2'-deoxyuridine (2), uridine (5) and arabinouridine (6), respectively with iodine monochloride, or N-bromo (or chloro)succinimide, and sodium azide at 25-45°C. These C-5 halogenation reactions proceed via a 5-halo-6-azido-5,6-dihydro intermediate (3), from which HN3 is eliminated, to yield the 5-halogeno uracil nucleoside. The 5-halo-6-azido-5,6-dihydro intermediate products (10a, 10b) could be isolated from the reaction of 3',5'-di-O-acetyl-2'-deoxyuridine (9) with iodine monochloride or N-bromosuccinimide and sodium azide at 0°C. The isolation of 10a, 10b indicates that the C-5 halogenation reaction proceeds via a 5-halo-6-azido-5,6-dihydro intermediate.
- Kumar,Wiebe,Knaus
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p. 2005 - 2010
(2007/10/02)
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- Radiolabeling kit/generator for 5-radiohalogenated uridines
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A rapid, simple and inexpensive synthesis of 5-radiohalogenated-2'- deoxyuridine from 5-trimethylstannyl-2'-deoxyuridine is described. The total reaction and purification time including thin layer chromatography (tlc) for quality control is less than 30 min. This method produces excellent yields (>95%) of 123I-, 125I-, 131I-UdR. The radiochemical purity of all tested preparations (>20) was determined to be greater than 99%. This new method is the basis of a radiolabeling kit/generator for preparation of radiohalogenated nucleosides. 2'-Deoxyuridine (UdR) halogenated with a stable isotope of bromine was also synthesized indicating that the method can be applied to the preparation of 5-radiobromo-2'-deoxyuridine (BUdR).
- Baranowska-Kortylewicz,Helseth,Lai,Schneiderman,Schneiderman,Dalrymple
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p. 513 - 522
(2007/10/02)
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- In-cell Indirect Electrochemical Halogenation of Pyrimidine Bases and their Nucleosides to 5-Haloderivatives
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Reaction of anodically generated "halonium" species (LiX or Bu4NX, LiClO4, MeCN, Pt/Pt; I2, LiClO4, MeCN) with pyrimidine bases and their nucleosides leads to 5-halo compounds in good yields.
- Palmisano, G.,Danieli, B.,Santagostino, M.,Vodopivec, B.,Fiori, G.
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p. 7779 - 7782
(2007/10/02)
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- Cerium(IV)-Mediated Halogenation at C-5 of Uracil Derivatives
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Treatment of protected uracil nucleosides 1 or 2 with elemental iodine or metal halogenides and ceric ammonium nitrate (CAN) at 80 deg C gave the corresponding protected 5-halouracil nucleosides 3a-f in excellent yields.Treatment of the resulting crude 3a-f with 0.1 M NaOMe/MeOH at ambient temperature gave the corresponding 5-halouridines 4a-f in high overall yields from 1 or 2.Further, 5-halouraciles 9a-f were prepared in good yields by treatment of 1,3-dimethyluracil (7) or uracil (8) with elemental iodine, metal halogenides, or hydrochloric acid and CAN.Halouridines 4a-e also were obtained in good yields by treatment of unprotected uracil nucleosides 5 or 6 with halogen sources as above and CAN.
- Asakura, Jun-ichi,Robins, Morris J.
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p. 4928 - 4933
(2007/10/02)
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- Iodination with Silver Sulfate and Iodine. II. Uridines
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Iodination of uridines with iodine and silver sulfate at room temperature gives iodinated uridines in excellent yield.
- Sy, Wing-Wah
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p. 3391 - 3394
(2007/10/02)
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- Method of making radioiodinated pyrimidine nucleoside or nucleotide
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The method of making radioiodinated pyrimidine nucleoside or nucleotide which comprises contacting a water-insoluble halomercuri pyrimidine nucleoside or nucleotide with an aqueous medium containing a dissolved radioactive iodide ion and an oxidizing agent, the molar amounts of said nucleoside or nucleotide and said oxidizing agent being in excess of the molar amount of said iodide, whereby water-soluble radioactive iodinated pyrimidine nucleoside or nucleotide is formed in solution, and separating residual water-insoluble halomercuri pyrimidine nucleoside or nucleotide from said solution.
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- Nucleic acid related compounds. 38. Smooth and high-yield iodination and chlorination at C-5 of uracil bases and p-toluyl-protected nucleosides
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Treatment of uracil bases and protected nucleosides with iodine monochloride (ICl) gave the corresponding 5-iodouracil products in over 95percent purified yields.Analogously facile chlorination was effected with iodobenzene dichloride (PhICl2).Protection of the nucleosides as p-toluyl esters provided reactants that were soluble in organic solvents and crystallized readily in high yields.
- Robins, Morris, J.,Barr, Philip J.,Giziewicz, Jerzy
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p. 554 - 557
(2007/10/02)
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