Devereaux Zachary J, Zhu Y, Rodgers M T
Department of Chemistry, Wayne State University, Detroit, USA.
Eur J Mass Spectrom (Chichester). 2019 Feb;25(1):16-29. doi: 10.1177/1469066718798097. Epub 2018 Sep 6.
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.
转录后修饰的频率和多样性在经典核酸序列之外增加了一层额外的化学复杂性。甲基化尤其频繁发生，并且在整个生命王国中通常高度保守。然而，这些修饰的核酸成分的复杂功能往往尚未完全被理解。将系统简化为其最小成分的系统性基础研究可能有助于揭示核酸生物化学的复杂性。在这里，我们通过能量分辨碰撞诱导解离串联质谱研究并辅以量子化学计算，考察了鸟苷和五种天然存在的甲基鸟苷（O2'-、1-、7-、N2,N2-二甲基和N2,N2,O2'-三甲基鸟苷）的相对内在N-糖苷键稳定性。通常发现表观糖苷键稳定性随着甲基取代的增加而增加（经典型＜单甲基化＜双甲基化＜三甲基化）。许多生物化学转化，包括碱基切除修复机制，都涉及质子化和/或非共价相互作用以增加核碱基的离去基团能力。质子化的气相甲基鸟苷糖苷键断裂所需的活化能比其钠阳离子化形式少。然而，N7位的甲基化比随后的阳离子化更显著地内在削弱了7-甲基鸟苷的糖苷键，因此7-甲基鸟苷被认为处于永久活化状态。N7甲基化还改变了相对于其他系统的核苷几何偏好，包括中性形式下的核碱基取向、质子化形式下的糖环构象以及优选的质子化和钠阳离子结合位点。这里研究的所有甲基化鸟苷预计具有超过经典鸟苷的质子亲和力和气相碱度。此外，质子亲和力和气相碱度趋势与表观糖苷键稳定性大致呈负相关。
