- Desulfuration of 2-thiouridine with hydrogen peroxide in the physiological pH range 6.6-7.6 is pH-dependent and results in two distinct products
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The 2-thiomodified nucleosides, located at first position of tRNAs anticodon, may constitute a primary target for oxidative attack under conditions of oxidative stress. Desulfuration of 2-thiouridine (S2U) was investigated in the 1H NMR scale in the presence of 100 mM H2O2 and phosphate buffer in the physiological pH range, from pH 6.6 to 7.6. The obtained data demonstrate an intriguing result that within one unit of the pH range uridine is the major product of the S2U desulfuration in the pH 7.6, while the 4-pyrimidinone nucleoside (H2U) is dominant in pH 6.6. The possible desulfuration pathway and the biological importance of the transformation of S2U either to U or H2U are discussed in the context of the tRNA oxidative damage.
- Sochacka, Elzbieta,Bartos, Paulina,Kraszewska, Karina,Nawrot, Barbara
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- An efficient approach for conversion of 5-substituted 2-thiouridines built in RNA oligomers into corresponding desulfured 4-pyrimidinone products
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Abstract An efficient approach for the desulfuration of C5-substituted 2-thiouridines (R5S2U) bound in the RNA chain exclusively to 4-pyrimidinone nucleoside (R5H2U)-containing RNA products is proposed. This post-synthetic transformation avoids the preparation of a suitably protected H2U phosphoramidite, which otherwise would be necessary for solid-phase synthesis of the modified RNA. Optimization of the desulfuration, which included reaction stoichiometry, time and temperature, allowed to transform a set of ten R5S2U-RNAs into their R5H2U-RNA congeners in ca. 90% yield.
- Chwialkowska, Anna,Wielgus, Ewelina,Leszczynska, Grazyna,Sobczak, Milena,Mikolajczyk, Barbara,Sochacka, Elzbieta,Nawrot, Barbara
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supporting information
p. 3100 - 3104
(2015/07/08)
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- The influence of the C5 substituent on the 2-thiouridine desulfuration pathway and the conformational analysis of the resulting 4-pyrimidinone products
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In recent years, increasing attention has been focused on the posttranscriptional modifications present in transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which are located in the wobble position of the anticodon and play a fundamental role in the tuning of the translation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-dependent. In this study, for the first time, we demonstrate that the substituent at the C5 position of the 2-thiouracil ring of R5S2Us influences the desulfuration pathway, and thus the products ratio. As the substituent R changes, the amount of R5H2U increases in the order H- > CH3O- > CH3OC(O)CH2- > HOC(O)CH2NHCH2- ≈ CH3NHCH2-, and this effect is more pronounced at lower pH. The conformational analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent, the R5H2U nucleosides predominantly adopt a C2′-endo sugar ring conformation, as opposed to the preferred C3′-endo conformation of the parent R5S2Us.
- Bartos, Paulina,Ebenryter-Olbinska, Katarzyna,Sochacka, Elzbieta,Nawrot, Barbara
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supporting information
p. 5587 - 5594
(2015/11/11)
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- Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: Characterization of the oxidation and oxidative desulfurization of 2-thiouridine
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In order to efficiently assess the chemical stability of modified nucleosides to the reagents and conditions of automated oligonucleotide synthesis, we designed, developed and tested a scheme in which the modified nucleoside, directly attached to a solid support, is exposed to the cyclic chemistry of the instrument. Stability of 2-thiouridine against different oxidizers was investigated. Tertbutyl hydroperoxide (1 M) in anhydrous acetonitrile was a more effective oxidizer for the incorporation of 2-thiouridine into oligonucleotide chains than the same oxidizer in methylene chloride. Carbon tetrachloride/water in the presence of a basic catalyst was superior in maintaining the thiocarbonyl function, but its utility for RNA synthesis has yet to be fully tested, whereas 2-phenylsulfonyloxaziridine was a very efficient reagent for oxidative desulfurization of 2-thiouridine.
- Sochacka
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p. 1871 - 1879
(2007/10/03)
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