马兜铃酸Ⅳ_a与2′-脱氧核苷体外反应形成加合物的LC-MS/MS研究

A Liquid Chromatography Tandem-Mass Spectrometry (Lc-Ms/Ms) Study of the in Vitro Formation of Presumptive Adducts of Aristolochic Acid Ⅳ_a and 2′-Deoxynucleoside

目的 利用液相色谱-串联质谱法(LC-MS/MS)研究马兜铃酸Ⅳ_a(aristolochic acid Ⅳ_a,AA Ⅳ_a)与2′-脱氧核苷在溶液中的反应,检测可能的加合物,为进一步研究奠定基础。方法 马兜铃酸Ⅳ_a与2′-脱氧腺嘌呤核苷或2′-脱氧鸟嘌呤核苷在锌尘存在下37℃孵育,通过LC-MS/MS中性丢失模式筛选反应产物,再通过质谱碎裂方式和高分辨质谱进行验证。结果 马兜铃酸Ⅳ_a与2′-脱氧腺嘌呤核苷或2′-脱氧鸟嘌呤核苷的反应较为复杂,产生多种产物。根据预期的加合物分子量(分别为558和574)以及是否具有明显的[MH-116]+子离子,分别筛选到3种和4种可能的2′-脱氧腺嘌呤核苷和2′-脱氧鸟嘌呤核苷加合物。同时,还发现了非“常规”的加合物。在马兜铃酸Ⅳ_a与2′-脱氧腺嘌呤核苷反应混合物中,发现了1种分子量572的可能加合物,而在马兜铃酸Ⅳ_a与2′-脱氧鸟嘌呤核苷反应混合物中则发现了3种分子量590的可能加合物。结论 马兜铃酸Ⅳ_a与2′-脱氧腺嘌呤核苷或2′-脱氧鸟嘌呤核苷体外条件下能够发生反应形成可能的加合物,但加合物结构尚需通过核磁共振光谱确定。

Objective To study the reaction of aristolochic acid IVa(AA IVa)with 2-deoxynucleoside in solution,to detect possible adducts,and to lay foundations for further research by using liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods AA IVa and 2′-deoxyadenosine(dA)or 2′-deoxyguanosine(dG)were incubated at 37℃in the presence of zinc dust,and possible adducts were screened from the reaction mixtures by LC-MS/MS under the neutral loss mode.Possible adducts were subjected to further examinations by their fragmentation patterns and highresolution mass spectrometry.Results It was observed that the reactions of AA IVa and dA or dG were complex with multiple products being formed.3 possible dA adducts and 4 dG ones were identified,which exhibited expected molecular weights(558 for dA adducts and 574 for dG ones)and loss of the 2′-deoxyribose moieties(the presence of the[MH-116]+fragments in their mass spectra),a mass spectrometric feature of 2′-deoxynucleoside adducts.Meanwhile,other possible adducts were also discovered.A presumptive dA-AA IVa adduct with the molecular weight of 572 was identified,while 3 possible dG-AA IVa ones with the molecular weights of 590 were detected.Conclusion AA IVa could react with dA/dG to form presumptive adducts in vitro.However,the structures of the adducts need to be determined by NMR.