Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, Zhejiang 310008, China.
Anal Chim Acta. 2013 Sep 17;795:15-24. doi: 10.1016/j.aca.2013.07.038. Epub 2013 Jul 22.
The O-methylation of active flavonoids can enhance their antiallergic, anticancerous, and cardioprotective effects depending on the methylation position. Thus, it is biologically and pharmacologically important to differentiate methylated flavonoid regioisomers. In this study, we examined the regioisomers of methylated kaempferol and quercetin using ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The methyl groups on the flavonoids can generally be cleaved as methyl radicals in a position-independent manner. We found that methyl groups can be cleaved as methane. If there are protons adjacent the methoxy on the flavonol rings, intra-molecule proton transfer can occur via collision-induced dissociation, and one molecule of methane can then be eliminated. The remaining charged fragment (M+H-CH4) reflects the adjacent structure and is specific to the methoxy position. Furthermore, the retro Diels-Alder (RDA) fragmentation of methylated flavonols can generate fragments with the methoxy at the original methylated ring. Combining the position-specific M+H-CH4 fragment with the RDA fragments provides a diagnostic pattern for rapidly identifying methylated regioisomeric flavonols. Along with their retention behaviour, we have successfully identified ten regioisomers of methylated kaempferol and quercetin, which include six compounds previously reported in plants and shown to be biologically active. The developed approach is sensitive, rapid, reliable, and requires few standard compounds. It is highly efficient for characterising the specificity of novel flavonoid O-methyltransferases and can help direct enzymatic or chemical syntheses during the early stages of drug discovery. This method also has potential for use in identifying other methylated isomeric flavonoids.
根据甲基化位置的不同，活性类黄酮的 O-甲基化可以增强其抗过敏、抗癌和心脏保护作用。因此，区分甲基化类黄酮的区域异构体在生物学和药理学上都很重要。在这项研究中，我们使用超高效液相色谱四极杆飞行时间串联质谱法研究了甲基化山柰酚和槲皮素的区域异构体。类黄酮上的甲基通常可以以位置无关的方式作为甲基自由基裂解。我们发现甲基可以裂解为甲烷。如果黄酮醇环上的甲氧基旁边有质子，通过碰撞诱导解离可以发生分子内质子转移，然后可以消除一个甲烷分子。剩余的带电碎片(M+H-CH4)反映了相邻的结构，并且特定于甲氧基的位置。此外，甲基化黄酮醇的逆 Diels-Alder（RDA）碎裂可以生成原始甲基化环上带有甲氧基的片段。将位置特异性的M+H-CH4片段与 RDA 片段相结合，可以为快速鉴定甲基化区域异构黄酮醇提供诊断模式。结合保留行为，我们成功鉴定了 10 种甲基化山柰酚和槲皮素的区域异构体，其中包括之前在植物中报道的 6 种具有生物活性的化合物。所开发的方法具有灵敏度高、快速、可靠、需要较少标准化合物的特点。它对鉴定新型黄酮类 O-甲基转移酶的特异性非常有效，并可以在药物发现的早期阶段指导酶或化学合成。该方法还有可能用于鉴定其他甲基化的异构黄酮类。
