pyrimidinones and 2-thiouridine

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.

Topics: Nucleosides; Pyrimidinones; RNA; RNA, Transfer; Solid-Phase Synthesis Techniques; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfuric Acids; Temperature; Thiouridine

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.

Topics: Humans; Models, Molecular; Oxidative Stress; Pyrimidinones; RNA, Transfer; Thiouridine

The 2-thiouridine (S2U) unit in the RNA strand is predominantly desulfured with H(2)O(2) to 4-pyrimidinone nucleoside (H2U). The resulting H2U-RNA exhibits significantly lower binding affinity to its complementary strand and in certain conditions undergoes strand scission. These results may explain the tRNA loss of biological function in oxidative stress conditions.

Topics: Base Pairing; Hydrogen Peroxide; Oxidation-Reduction; Oxidative Stress; Pyrimidinones; RNA, Transfer; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfur; Thiouridine; Transition Temperature