2',3'-環氧黃樟素所形成DNA加成產物之體外與體內研究

Abstract

本論文第一部份首先合成黃樟素代謝產物3分別與2’-去氧腺嘌呤(2’-deoxyadenosine, 2’-dAdo 10)、腺嘌呤 (adenine, Ade 15)、2’-去氧鳥糞嘌呤 (2’-dexoyguanosine, 2’-dGuo 18)、2’-去氧胞嘧啶(2’-de oxycytidine, 2’-dCyt 20)和胸腺嘧啶 (2’-thymidine, 2’-dThd 22)以及[15N5]-10和 [15N5]-18進行反應之加成產物的單離與鑑定。分離且鑑定的產物分別有N1□-SFO-dAdo 11 (4.2%)、N6□-SFO-dAdo 12 (4.5%)、N3□-SFO-Ade 16 (1.0%)、N9□-SFO-Ade 17 (2.4%)、N7□-SFO-Gua 19 (16%)、N3□-SFO-dUrd 21 (3.8%) 和 N3□-SFO-dThd 23 (1.2%)以及內標準品[15N5]-11、[15N5]-12和[15N5]-19。

為了探討黃樟素代謝產物3是否會攻擊DNA形成DNA加成產物，選擇文獻上較先探討的嘌呤加成產物11、12、16和19當成生物指標，利用同位素稀釋(isotope dilution)高效液相層析電噴灑游離串聯質譜法(liquid chromatography electrospray ionization tandem mass spectrometry, LC□ESI-MS/MS)定量這些加成產物體內與體外的生成量。體外實驗，將黃樟素代謝產物3與小牛胸腺DNA (Calf thymus DNA)反應，得到每106個核苷酸 (nucleotides) 中有2000個加成產物11、170個加成產物12、660個加成產物16以及2670個加成產物19。接著以靜脈注射方式給予老鼠單一劑量之黃樟素代謝產物3 (30 mg/kg body weight)並成功分析到尿液中加成產物19，證實了黃樟素代謝產物3在體內會攻擊DNA產生DNA加成產物。 本論文第二部份是製備2’-去氧腺嘌呤模版高分子。本研究將模版分子10、甲基丙烯酸 27單體、二甲基丙烯酸乙二醇酯28交聯劑與偶氮二異丁腈29在乙氰/水 (4/1, v/v)之混合液，在0 oC下利用波長365 nm 之紫外光燈源照射，進行聚合反應並利用索氏萃取(Soxhlet extraction)將模版分子移除得到具吸附2’-去氧腺嘌呤專一性之模版高分子。

In the first part of this thesis, we synthesized SFO 3 and characterized the DNA adducts from the reactions of 3 with 2’-deoxyadenine (2’-dAdo, 10), adenine (Ade, 15), 2’-deoxyguanosine (2’-dGuo, 18), 2’-deoxycytidine (2’-dCyt, 20), and thymidine (Thd, 22) to investigate the SFO-DNA adducts formed in vivo and in vitro. The reactions of 3 with 10, 15, 18, 20, and 22 were carried out under physiological conditions (pH 7.4, 37 oC) which gave five adducts N1□-SFO-dAdo (11) (4.2%), N6□-SFO-dAdo (12) (4.5%), N3□-SFO-Ade (16) (1.0%), N9□-SFO-Ade (17) (2.4%), N7□-SFO-Gua (19) (16%), N3□-SFO-dUrd (21) (3.8%), and N3□-SFO-Thd (23) (1.6%). Moreover, 15N labeled analogs of adducts [15N5]-11, [15N5]-12, and [15N5]-19 were synthesized to serve as internal standards.

A newly-developed isotope-dilution high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC□ESI-MS/MS) was developed to investigate the SFO-DNA adducts in vitro and in vivo. In vitro study, the reaction of 3 with Calf thymus DNA showed that the levels of 11, 12, 16, and 19 were determined to be 2000, 170, 660, and 2670 adducts per 106 nucleotides. Subsequently, we measured the amount of adduct 19 in the urine of mice after intraperitoneally treatment with a single dose of 3 (30 mg/kg body weight). These results suggested that 3 can cause in vivo formation of DNA adducts. In the second part of this thesis, we attemped to prepare a molecularly imprinted polymer (MIP) for 2’-deoxyadenosine 10. A mixture of 2’-deoxyadenosine 10, methacrylic acid (MAA, 27), ethylene glycol dimethacrylate (EGDMA, 28), and 2, 2’-azoisobutyonitrile (AIBN, 29) in acetonitrile/water (4/1) was initiated by UV light (365 nm) at 0 oC for 16 houres to obtain a polymer. The polymer after Soxhlet extraction to remove the 2’-deoyxadenosine was found to be a specific receptor for 2’-deoxyadenosine.