Spectral libraries from nucleobases and deoxyribonucleosides facilitate the identification of ribonucleosides by nano-flow liquid chromatography-tandem mass spectrometry.

RATIONALE: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable. METHODS: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns. RESULTS: Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families. CONCLUSIONS: The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry.

1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties.

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.

Protection-Free, Two-step Synthesis of C5-C Functionalized Pyrimidine Nucleosides.

A simple, reliable, and efficient method for the gram-scale chemical synthesis of pyrimidine nucleosides functionalized with C5-carboxyl, nitrile, ester, amide, or amidine, starting from unprotected uridine and cytidine, is described. The protocol involves the synthesis of 5-trifluoromethyluridine and 5-trifluoromethylcytidine with Langlois reagent (CF3 SO2 Na) in the presence of tert-butyl hydroperoxide and subsequent transformation of the CF3 group to the C5-C 'carbon substituents' under alkaline conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of 5-trifluoromethyluridine (5-CF3 U) and 5-trifluoromethylcytidine (5-CF3 C) Basic Protocol 2: Conversion of 5-CF3 U and 5-CF3 C to several C5-substituted ribonucleosides.

Proliferação celular induzida por 8-oxoguanosina e 8-metilguanosina, dois produtos do ataque de radicais livres a ribonucleosídeos e RNA / Cell proliferation induced by 8-oxoguanosine and 8-methylguanosine, two products of free radical attack to ribonucleosides and RNA

Os efeitos de ribonucleosídeos de guanina substituídos na posição C-8 na proliferação de linfócitos B estão bem documentados na literatura. Esses compostos são análogos de adutos formados pela adição de radicais livres a ribonucleosídeos e a RNA. Neste trabalho, verificamos as propriedades proliferativas de dois desses adutos, 8-metilguanosina (8-MeGuo) e 8-oxo-7 ,8-di-hidroguanosina (8-OxoGuo) e comparamos com 8-bromoguanosina (8-BrGuo), o composto mais estudado como indutor da proliferação de linfócitos B. 8-MeGuo e 8-OxoGuo foram sintetisados em rendimentos de 28 e 55%, respectivamente, e foram caracterizados por UV, NMR e CG-massa. Seus efeitos sobre a incorporação de timidina radioativa ([3H] TdR) no DNA de células de baço, fibroblasto 3T3(A31) e melanoma B16F10 foram examinados. Os dois adutos foram mitogênicos para células de baço mas foram seletivos quanto as células imortalizadas. 8-MeGuo atuou sobre células 3T3(A31) e 8-OxoGuo sobre as células de melanoma B16F10. O análogo não fisiológico 8-BrGuo foi efetivo em todas as células testadas. Experimentos de contagem de células, citotoxicidade e citometria de fluxo, indicaram que a síntese de DNA induzida pelas guanosinas substituídas na posição C-8 refletia crescimento celular. Foi proposto que os compostos agem de dentro da célula uma vez que seus efeitos são bloqueados em presença de um inibidor de transporte de nucleosídeo, mas não foram inibidos por um antagonista de receptor purinérgico. Os resultados obtidos, junto com os descritos na literatura, sugerem que no caso dos fibroblastos 3T3(A31) e células de baço de camundongo os efeitos proliferativos dos compostos não são dependentes do metabolismo desses compostos via salvação das purinas. No caso das células de melanoma, entretanto, os compostos parecem fazer parte do "pool" de nucleosídeos. A demonstração de que adutos produzidos por ataques radicalares em ribonucleosídeos e RNA são capazes de induzir proliferação celular, abre novas perspectivas para a compreensão do papel de radicais livres em processos carcinogênicos

The ability of CS-substituted guanine ribonucleosides to induce B cell proliferation has been well documented in the literature. These compounds are analogues of adducts formed from free radical attack on ribonucleosides and RNA. Here we examined the proliferative properties of two of these radical adducts, 8-methylguanosine (8-MeGuo) and 8-oxo-7 ,8-dihydroguanosine (8-OxoGuo) and compared them with those of the well studied B cell mitogen, 8-bromoguanosine (8-BrGuo). 8-MeGuo and 8-OxoGuo were synthesized in yields of 28 and 55 %, respectively, and were characterized by UV, NMR and CG-MS. Their effects upon [3H] thymidine uptake by Swiss mice splenocytes, mouse embryo 3T3 (A31) fibroblasts and mouse B16F10 melanocytes were examined. Both guanosine radical adducts were shown to increase [3H] thymidine uptake by mice splenocytes but displayed selectivity in regard to continuous cell lines. 8-MeGuo acted upon 3T3(A31) fibroblasts whereas 8-OxoGuo acted upon B16F10 melanocytes. The non physiological analogue 8-BrGuo acted upon all tested cells. Parallel experiments of cell counting, cytotoxicity, and cell sorting indicated that DNA synthesis induced by the C8-substituted guanosines reflected cell growth. It is proposed that the compounds act intracellularly because their proliferative effects were blocked in the presence of a nucleoside transport inhibitor but were not inhibited by an antagonist of the A2 purine receptor. The obtained results, taken together with data from the literature suggest that in the case of 3T3 (A31) fibroblasts and mice splenocytes the proliferative effects of the compounds are not dependent on metabolism through purine salvage pathways. In the case of melanocytes, however, the compounds are likely to become part of the purine nucleoside pool. The demonstration that adducts produced by free radical attack on ribonucleosides and RNA are able to induce cell proliferation opens new perspectives for the understanding of free radical mediated carcinogenesis