8-methylguanosine and 7-methylguanosine

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5' cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn

Topics: Endoribonucleases; Eukaryotic Initiation Factor-4E; Genetic Variation; Guanine; Guanosine; Humans; Models, Molecular; Nerve Tissue Proteins; Nucleotide Motifs; Nucleotides; Phosphates; Protein Biosynthesis; RNA Cap Analogs; RNA, Messenger; Tryptamines

The frequency and diversity of posttranscriptional modifications add an additional layer of chemical complexity beyond canonical nucleic acid sequence. Methylations are particularly frequently occurring and often highly conserved throughout the kingdoms of life. However, the intricate functions of these modified nucleic acid constituents are often not fully understood. Systematic foundational research that reduces systems to their minimum constituents may aid in unraveling the complexities of nucleic acid biochemistry. Here, we examine the relative intrinsic N-glycosidic bond stabilities of guanosine and five naturally occurring methylguanosines (O2'-, 1-, 7-, N2,N2-di-, and N2,N2,O2'-trimethylguanosine) probed by energy-resolved collision-induced dissociation tandem mass spectrometry and complemented with quantum chemical calculations. Apparent glycosidic bond stability is generally found to increase with increasing methyl substitution (canonical < mono- < di- < trimethylated). Many biochemical transformations, including base excision repair mechanisms, involve protonation and/or noncovalent interactions to increase nucleobase leaving-group ability. The protonated gas-phase methylguanosines require less activation energy for glycosidic bond cleavage than their sodium cationized forms. However, methylation at the N7 position intrinsically weakens the glycosidic bond of 7-methylguanosine more significantly than subsequent cationization, and thus 7-methylguanosine is suggested to be under perpetually activated conditions. N7 methylation also alters the nucleoside geometric preferences relative to the other systems, including the nucleobase orientation in the neutral form, sugar puckering in the protonated form, and the preferred protonation and sodium cation binding sites. All of the methylated guanosines examined here are predicted to have proton affinities and gas-phase basicities that exceed that of canonical guanosine. Additionally, the proton affinity and gas-phase basicity trends exhibit a roughly inverse correlation with the apparent glycosidic bond stabilities.

Topics: Binding Sites; Cations; Glycosides; Guanosine; Mass Spectrometry; Methylation; Models, Molecular; Sodium

A conventional, rapid and high throughput method for tissue extraction and accurate and selective LC-MS/MS quantification of 2'-C-methylguanosine triphosphate (2'-MeGTP) in mouse liver was developed and qualified. Trichloroacetic acid (TCA) was used as the tissue homogenization reagent that overcomes instability challenges of liver tissue nucleotide triphosphates due to instant ischemic degradation to mono- and diphosphate nucleotides. Degradation of 2'-MeGTP was also minimized by harvesting livers using in situ clamp-freezing or snap-freezing techniques. The assay also included a sample clean-up procedure using weak anion exchange solid phase extraction followed by ion exchange chromatography and tandem mass spectrometry detection. The linear assay range was from 50 to 10000 pmol/mL concentration in liver homogenate (250-50000 pmol/g in liver tissue). The method was qualified over three intraday batches for accuracy, precision, selectivity and specificity. The assay was successfully applied to pharmacokinetic studies of 2'-MeGTP in liver tissue samples after single oral doses of IDX184, a nucleotide prodrug inhibitor of the viral polymerase for the treatment of hepatitis C, to mice. The study results suggested that the clamp-freezing liver collection method was marginally more effective in preventing 2'-MeGTP degradation during liver tissue collection compared to the snap-freezing method.

Topics: Animals; Antiviral Agents; Chromatography, Ion Exchange; Chromatography, Liquid; Freezing; Guanosine; Guanosine Monophosphate; Guanosine Triphosphate; Hepatitis C; Liver; Male; Mice; Nucleotides; Prodrugs; Solid Phase Extraction; Tandem Mass Spectrometry; Trichloroacetic Acid

Metabolic activation of peroxides and hydroperoxides to free radicals is associated with the tumor promoting activity of these compounds. tert-Butyl hydroperoxide (t-BOOH) metabolism has been extensively studied as a model of peroxide biotransformation. In vivo studies are limited, and the hemoglobin-thiyl radical was the only species thus far identified in the blood of treated rats. Here we further examine t-BOOH metabolism in vivo with regard to free radical and DNA adduct production. Spin-trapping experiments with phenyl-N-tert-butylnitrone (PBN) led to the detection of EPR signals in the blood, bile, and organic extracts of the liver and stomach of rats treated with t-BOOH. Analysis of these signals demonstrated that t-BOOH metabolism in vivo produces alkyl radicals, detected in the bile and organic extracts of liver and stomach, in addition to the previously identified hemoglobin-thiyl radical. To characterize the produced alkyl radicals, experiments were performed with (13)C-labeled t-BOOH and two spin traps, PBN and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The latter was used because the EPR signals obtained with PBN were too weak to be unambiguous. Nevertheless, the EPR signals present in the bile of animals treated with (13)C-labeled t-BOOH and PBN or POBN were consistent with adducts of (13)C-labeled methyl radical and an unidentified alkyl radical. The latter is probably derived from lipids oxidized by the metabolically produced primary radicals, methyl and its precursor, tert-butoxyl. The presence of 8-methylguanine and 7-methylguanine in hydrolysates of DNA from liver and stomach of rats treated with t-BOOH was also examined. 8-Methylguanine, a typical product of methyl radical attack on DNA, was detectable in both the liver and stomach of treated rats. The results may be relevant to the understanding of the genotoxic properties of other peroxides, particularly of cumene hydroperoxide.

Topics: Animals; Cyclic N-Oxides; DNA; DNA Methylation; Free Radicals; Gastric Mucosa; Guanosine; Indicators and Reagents; Liver; Male; Nitrogen Oxides; Rats; Rats, Wistar; Spin Trapping; Stomach; tert-Butylhydroperoxide