Synthesis of N3-SFO-dUrd 21

A solution of either ()-SFO 3 was reacted with 2’-deoxycytidine 20 at a 2:1 molar ratio in 0.2 N K2HPO4 (pH 7.4) solution and incubated at 37 ^oC for 72 h. Products were purified and further desalted using reverse phase HPLC. The solutions containing pure adducts were dried under vacuum.

Each pure adduct was subjected to spectroscopic and spectrometric characterization. 100.5 (C-g), 100.9 (C-5), 107.8 (C-d), 109.53; 109.56 (C-a), 121.93; 121.95

82

(C-e), 132.67; 132.71 (C-f), 138.68; 138.74 (C-6), 145.3 (C-c), 146.8 (C-b), 150.71; 150.73 (C-2), 162.21; 162.23 (C-4). (附圖四十).

DEPT spectra（附圖四十一）、H,H-COSY spectrum（附圖四十二）與 HMQC spectrum（附圖四十三）。

1.4.10 Synthesis of N3-SFO-Thd 23.

A solution of either ()-SFO 3 was reacted with thymidine 22 at a 2:1 molar ratio in 0.2 N K2HPO4 (pH 7.4) solution and incubated at 37 ^oC for 72 h. Products were purified and further desalted using reverse phase HPLC.

The solutions containing pure adducts were dried under vacuum. Each pure adduct was subjected to spectroscopic and spectrometric characterization.

N3-SFO-Thd 23. The ESI

⁺/MS/MS of adducts 23 showed the fragments at

m/z 421 ([M + H]

⁺), 305 ([M - dR + H]⁺), and 287 ([M - dR – H2O+ H]⁺).

83

(CH₃)



40.4 (C-2’), 40.90; 40.96 (C-), 46.24; 46.30 (C-), 61.2 (C-5’), 67.9 (C-), 70.15; 70.21 (C-3’), 84.63; 84.73 (C-1’), 87.3 (C-4’), 100.5 (C-g), 107.8 (C-d), 108.4 (C-5), 109.46; 109.49 (C-a), 121.86; 121.88 (C-e), 132.62;

132.67 (C-f), 134.48; 134.53 (C-6), 145.20; 145.22 (C-c), 146.8 (C-b), 150.61; 150.64 (C-2), 162.91; 162.93 (C-4), 163.1 (HCOO^) (附圖四十五).

DEPT spectra（附圖四十六）、H,H-COSY spectrum （附圖四十七）與 HMQC spectrum （附圖四十八）。

1.4.11 Synthesis of [¹⁵N5]-N1-SFO-dAdo 11, [¹⁵N5]-N⁶

-SFO-dAdo 12,

and [¹⁵N5]-N7-SFO-Gua 19.

[¹⁵N5]-2’-deoxyadenosine 10 and [¹⁵N5]-2’-deoxy- guanosine 18 (5 mg) was dissolved into H2O (1 mL), respectively, to serve as the stock soultion.

For synthesis of [¹⁵N5]-N1-SFO-dAdo 11 and [¹⁵N5]-N⁶

-SFO-dAdo 12,

solution without further purification.

84

1.4.12 High Performance Liquid ChromatographyElectrospray Ionization-Tandem Mass Spectrometry (HPLCESI-MS/MS) for Quanitification of DNA Adducts in Calf Thymus DNA.

HPLC-ESI-MS/MS analyses were performed on an API 3000TM spectrometer (Applied Biosystems/ MDS SCIEX, Foster City, CA) together with Hitachi L-7000 pump and L-7200 autosampler (Hitachi Ltd., Tokyo).

Electrospray ionization source was used in the positive mode (ESI⁺-MS/MS) throughout the study. A Prodigy ODS (3) column, 150

 mm, 5 m

(Phenomenex, Torrance, CA) was utilized. Total ion chromatograms and mass spectra were recorded on a personal computer with the Analyst software version 1.1 (Applied Biosystems). The mobile phase consisted of a linear gradient from 0% to 42% acetonitrile in 50 mM ammonium formate SFO-Gua 19 (m/z 335), with the collision energy set at 29, 27, 35 and 39 V, respectively. The dwell time for MRM experiments was set at 150 ms.

Nitrogen was used as the turbo gas with temperature set at 450 °C; it was further used as the nebulizer, curtain, and collision gas with pressure settings of 8, 8, and 12 psi, respectively. Calibration curves were established in the concentration range 5 to 250 ng/mL for adducts 11, 12, 16, and 19.

85

1.4.13 Reaction of ()-SFO 3 with Calf Thymus DNA.

Calf thymus DNA (1 mg) in Tris-HCl buffer (pH 7.58.5, 1 mL) containing 1 mM EDTA was kept at 4 ^oC for overnight as a stock solution. A solution of 100

L of DNA (100 g) was hydrolyzed with a mixture of

DNase I (4 U), phosphodiesterase I (32 mU), phosphodiesterase II (80 mU), and acid phosphatase (1 U), and incubated at 37 ^oC for 8–10 h (Matter et al., 2006; Pang et al., 2007). The amounts of reagents were adjusted according to the amounts of DNA in the sample. To evaluate the efficiency of enzymatic hydrolysis, calibration curves of dAdo, dGuo, dCyd, and dThd were established using HPLC analysis. The retention time of each 2’-deoxyribonucleosides was at 9.9 min (dCyd), 12.4 min (dGuo), 14.6 min (dThd), and 16.7 min (dAdo) (data not shown), respectively. The hydrolysis efficiency of double strand calf thymus DNA was estimated to be 97.8%.

Calf thymus DNA (10 mg) was reacted with ()-SFO 3 (60 mol) in 10 mL of 0.2 N K2HPO4 buffer at pH 7.4 and incubated at 37 ^oC for 72 h. All samples were then kept in an ice bath for a few hours to vaporize the Et2O and then analyzed using two different methods modified from Goggin et al.(Goggin et al., 2007) Method 1: The solution was spiked with 100

L of

the isotope enriched [¹⁵N5]-N1-SFO-dAdo 11 (4 ng), [¹⁵N5]-N⁶

-SFO-dAdo 12 (9 ng), N9-SFO-Ade 16 (5 ng), and

[¹⁵N5]-N7-SFO-Gua 19 (4 ng) to serve as internal standards and then filtered with a 0.22

m PVDF membrane to remove the DNA backbone for

HPLC-ESI-MS/MS analysis. Method 2: The reaction mixture was subjected to neutral thermal hydrolysis at 70 ^oC for 1 h follow by hydrolysis

86

of the biopolymer using an enzymatic method described above (final volume 1 mL), and then analyzed after removal of the enzymes by filtration.

Calibration curve of each DNA adduct with added internal standards was established for quantitative analysis by HPLC-ESI-MS/MS method.

1.4.14 Animal Experiments.

The animal experiment was approved by and conducted in accordance with the China Medical University Animal Ethics Committee guidelines on animal care. Sixteen male FVB mice aged 6 to 7 weeks and weighing 2025 g were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Animals were acclimatized for seven days prior to (±)-SFO 3 treatment. (±)-SFO 3 was dissolved in olive oil and the final concentration was 9 mg/mL of (±)-SFO 3. Mice were divided into control (5 mice) and exposed groups (four groups with a toal of 11 mice). For the exposed group, mice were intraperitoneally injected with 30 mg/kg body weight of (±)-SFO 3 in olive oil and then housed in 4 metabolic cages. There were 2, 3, 3, and 3 mice in each group. For the control group, mice were intraperitoneally injected with the same volume of olive oil as the exposed group. Urine samples were collected at 24, 48, and 72 h after treatment. The collected urine samples were mixed with 10 μL of sodium azide (final concentration 0.05%) and centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was collected and stored at 80 ^oC until used for analysis.

87

1.4.15 High Perfromance Liquid Chromatographylectrospray Ionization-Tandem Mass (LCESI-MS/MS) Spectrometric Method for Mice Urine Analysis. spectrometer (TSQ Quantum ACCESS, Thermo Scientific, MA) comprising a heated-electrospray ionization (H-ESI) source was operated in the positive ion mode. Quantification of N7-SFO-Gua 19 was carried out by monitoring the ion pairs m/z 330→152 and [¹⁵N5]-N7-SFO-Gua 19 was monitored by the ion pairs m/z 335→157 in multiple reaction monitoring (MRM) mode.

The vaporizer temperature was set at 300 °C, nitrogen sheath gas was set at 35, auxiliary gas was set at 15, and the heated capillary temperature was set at 300 °C. Collision energy of 23 eV was applied. The argon gas pressure was set at 0.2 Pa for collision-induced dissociation (CID), and the discharge current was set at 5

A. Total ion chromatograms and mass spectra were

recorded on a personal computer with Xcalibur software (Version 2.0.7, Thermo Fisher Scientific Inc., MA).

88

1.4.16 Analysis of N7-SFO-Gua 19 in Urines of Mouse Treated with ()-SFO 3.

Urine samples (200 µl) of mice were spiked with 17

L of

[¹⁵N₅]-N7-SFO-Gua 19 (13 pg/L) with a final concentration of 1.1 ng/mL.

These samples were filtered through 0.22

m PVDF membrane filter, and

then analyzed using the newly-developed isotope-dilution HPLC-ESI-MS/MS method. Creatinine in each urine sample was analyzed at a local hospital (Taipei, Taiwan). The concentration of N7-SFO-Gua 19 in urine was adjusted to the level of creatinine and expressed as ng/mg creatinine.

N7-SFO-Gua 19 standard solutions (0.25, 0.5, 1, 1.5, 2, 4, and 5 ng/mL)

were prepared in urine from control mice. Each standard solution was spiked with 17 L of [¹⁵N5]-N7-SFO-Gua 19 (13 pg/L) with a final concentration of 1.1 ng/mL, follow by filtration through a 0.22 m PVDF membrane filter.

Linear calibration curve were constructed by plotting the ratios of the peak areas for N7-SFO-Gua 19 and [¹⁵N5]-N7-SFO-Gua 19 versus the concentrations of the standards, the sample was ready for analysis with our newly developed isotope-dilution HPLC-ESI-MS/MS method. The precision was evaluated by repeatedly analyzing the calibration standard solutions 5 times on three different days.

89

第二章 製備 2’-去氧腺嘌呤模版高分子 2.1 緒論

2.1.1 分子模版的發展歷史

早期的分子模版 (molecular imprinting)是以酵素的功能(enzyme function)與抗體的形成 (antibody formation) 為理論基礎所發展出來。

1894 年德國科學家 Emil Fischer 提出了“lock and key”的理論描述酵素 的專一性 (specificity) (圖三十八)，酵素除了形狀與受質 (substrate) 互補 (complementrary) 之外也必須具有與受質作用之功能性基團 (functional group)，而這些作用包括：氫鍵作用力 (hydrogen bonding)、靜電作用力 (electrostatic bonding)和疏水作用力(hydrophobic force)。而 Mudd 和 Pauling 提出抗體的形成是氨基酸或胜肽在抗原 (antigen) 表面進行合成 所得，而抗體的專一性便是來自於其具有與抗原互補結構的立體化學，

這種以目標物分子形成自己的辨識基座的理論對接下來發展分子模版 的影響非常大 (Mudd, 1932; Pauling, 1940)。

圖三十八、Emil Fischer 所提出“lock and key”的理論示意圖(Spivak and Shea, 2001)。

90

1940 年開始，Pauling 與 Dickey 利用酵素和抗體為理論基礎，將不 同取代之 alkylorange 染料與矽酸鈉一起聚合（圖三十九），之後利用溶 劑將這些染料洗去，得到對這些染料具有選擇性之矽膠凝體，正式開啟 了分子模版研究的大門 ( Pauling, 1940; Dickey, 1949)。

圖三十九、可選擇性吸附不同取代基的染料之矽高分子(silica polymer) (Dickey, 1949; Spivak and Shea, 2001)。

2.1.2 分子模版高分子 (molecularly imprinted polymer, MIP) 設計 概念

分子模版高分子設計概念如下圖四十所示 (Spivak et al., 1997)，首 先 將 想 要 辨 識 之 目 標 分 子 當 成 模 版 (template) ， 利 用 高 分 子 單 體 (monomer) 將模版分子圍繞聚合起來輔助高分子孔洞的形成，接著加入 大量的交聯單體 (cross-linking monomer) 與等體積之不具反應性之溶劑 做 為 致 孔 劑 (porogen) ， 最 後 再 加 入 自 由 基 的 起 始 劑 利 用 熱 化 學

91

(thermalchemical)或光化學 (photochemical) 方式進行分子模版高分子的 反應，形成不可溶之網狀高分子，最後在不破壞分子模版高分子共價鍵 結的情況下利用溶劑將模版分子移除，形成具專一性辨識之分子模版高 分子 (macroporous polymer)。

圖四十、分子模版高分子設計概念 (Spivak and Shea, 2001)。

2.1.3 模版與單體間的作用力

模版與單體之間的作用是主要決定模版高分子孔洞形成的關鍵因 素 ， 而 這 個 作 用 力 可 以 是 共 價 鍵 (covalent bond) 或 是 非 共 價 鍵 (noncovalent bond) 來輔助孔洞的形成，不論哪一種作用力，都必須可以 在不破壞分子模版高分子的結構下，將模版移除形成具專一性辨識之孔 洞。

共價鍵結

利用共價鍵結來輔助孔洞的形成，最早是由 Wulff 研究團隊 1977 年

92

(Wulff et al., 1977a; Wulff et al., 1977b)提出，將模版分子 phenyl--D-mannopyranoside (24a) 與兩分子的 4-vinylphenylboronic acid 單體 (24b) 反應形成共價鍵結後，再加入大量的交聯試劑二甲基丙烯酸乙二醇酯 (ethylene glycol dimeth acrylate, EGDMA) 在 惰 性 溶 劑 中 共 同 聚 合 (copolymerization) 製備出分子模版高分子（圖四十一），接著利用水或 甲醇可以移除 95% 的模版 (Wulff and Schauhoff, 1991)。

圖四十一、Phenyl--D-mannopyranoside 24 之分子模版高分子(Wulff et al., 1977b)。

非共價鍵結

利用非共價鍵結來輔助孔洞的形成，最早是由 Mosbach 與 Shea 的

93

研究團隊將 L-phenylalanine anilide (25) 做為模版分子，methacrylic acid 為單體（圖四十二），利用模版分子與單體之間存在靜電與氫鍵的非共 價鍵結作用力形成分子孔洞，讓分子模版應用更廣。

圖四十二、L-Phenylaniline anilide 25 模版高分子(Sellergren et al., 1988)。

2.1.4 研究動機

核苷酸鹼基 (nucleotide bases) 分子模版高分子的研究，最早是由 Shea 研究團隊於 1993 年合成 9-ethyladenine 模版高分子開啟了核苷酸鹼 基分子模版高分子的研究 (Shea et al., 1993; Spivak et al., 1997; Spivak and Shea, 1998)。2010 年 Scorrano 等人利用甲基丙烯酸 (methacrylic acid, MAA 27) 當成單體 (monomer) 、二甲基丙烯酸乙二醇酯 28 當成交聯 劑 (crosslinker) (圖四十三) 以及乙氰/水(4/1, v/v)混合溶劑當成致孔劑，

製備辨識 1-methyladenosine 26 的 1-methyladenosine 模版高分子。實驗結

94

果顯示所製備的分子模版高分子在 adenosine 29、 2’-deoxyadenosine 10、cytidine 30、inosine 31 同時存在下僅對 1-methyladenosine 26 具有專 一選擇性 (圖四十四)。

圖四十三、模版分子 1-methyladenosine 26、甲基丙烯酸 27 單體與二甲 基丙烯酸乙二醇酯 28 交聯劑之構造。

圖四十四、1-methyladenosine 26 分子模版高分子與不同核苷酸鹼基之結 合常數。

95

除此之外，作者也將此 1-methyladenosine 26 分子模版高分子做為固 相萃取（MIP-SPE）的填充材料，並將加入 1-methyladenosine 26 的尿液 樣本進行固相萃取並以乙氰/水/醋酸 (4/1/1, v/v/v) 之混合液沖提。利用 高效液相層析分析（圖四十五），可看到經過固相萃取可將尿液中的間 質去除。

圖四十五、高效液相層析圖譜。(A) 尿液樣品、(B) 加入 1-methyladenosine 26 的尿液樣品以及(C)將加入 1-methyladenosine 26 的尿液樣品經過分子 模版高分子萃取 (Scorrano et al., 2010)。

間質影響 (matrix effect) 是分析生物樣品時最大的干擾，因此可以 發展對分析物具專一性鍵結之分子模版高分子作為固相萃取或是層析 管柱填充材料，提高分析之靈敏度。在本論文第一部份已經由動物實驗

96

證實黃樟素代謝產物 SFO 3 在體內會攻擊 DNA 產生 SFO-DNA 加成產 物，為了進一步探討 SFO 的基因毒性，我們將分析給予黃樟素代謝產 物 SFO 3 的老鼠不同器官之組織 DNA，並經由酵素將組織 DNA 水解 後 分 析 N1-SFO-dAdo 11 與 N⁶

-SFO-dAdo 12 的 生 成 量 。 由 於 N1-SFO-dAdo 11 會經由 Dimroth rearrangement 重排成 N

⁶

-SFO-dAdo

12，所以我們會在鹼性條件下將 N1-SFO-dAdo 11 重排成 N⁶

-SFO-dAdo

12 並在分析之前利用 C18 之固相萃取匣進行樣品的純化以及預濃縮，

因此我們想發展可辨識 N⁶

-SFO-dAdo 12 分子模版高分子（圖四十六）

取代傳統固相萃取，做為組織 DNA 酵素水解後純化與預濃縮固相萃取 材料。

圖四十六、DNA 加成產物 N⁶

-SFO-dAdo 12 分子模版高分子。

97

2.2 結果與討論

由於所合成之 N⁶

-SFO-dAdo 12 量非常的少，我們先依照文獻的實

驗方法先合成 2’-去氧腺嘌呤 10 模版高分子，建立分子模版高分子技術。

2.2.1 2’-去氧腺嘌呤 (2’-deoxyadenosine, 2’-dAdo 10) 與甲基丙烯酸 (methacrylic acid, MAA 27) 結合之變溫 NMR 量測

本實驗開始之初參照文獻 (Scorrano et al., 2010) 在 60 ^oC 下加熱進 行分子模版高分子之聚合反應，並利用乙氰/水/醋酸 (4/1/1, v/v/v) 之混 合液將模版分子洗去，但得到之分子模版高分子經測試並不具任何分子 鍵結能力，同時掃瞄式電子顯微鏡 (scanning electron microsope, SEM) 拍攝所得到的 MIP 形態 (morphology) (圖四十七) 也與文獻上所報導這

類分子模版高分子會有花椰菜的形態不同，因此我們推測在 60 ^oC 下，

模版分子與單體 27 之間的氫鍵作用力已經不存在因此無法得到具辨識 效果孔洞之分子模版高分子。

98

圖四十七、利用熱化學法 (60 ^oC) 製備 2’-去氧腺嘌呤模版高分子之掃瞄 式電子顯微鏡圖。

由於文獻上報導，當 2’-去氧腺嘌呤上的鹼基與醋酸間有氫鍵作用力 存在時，2-H 與 8-H 隨著溫度降低時會分別往高磁場區 (upfield) 與低磁 場(downfield) 區位移 (附圖三十九) ( Rao et al., 1999; Basilio Janke et al.,

2004) 為了找尋最佳化反應條件，我們將2’-去氧腺嘌呤與甲基丙烯酸配

製在合成分子模版高分子 (molecular imprinting polymer, MIP) 時的 CD3CN/D2O = 4:1 混合溶劑，利用¹H NMR 進行變溫實驗，探討 2’-去氧 腺嘌呤與甲基丙烯酸之間的氫鍵作用。

根據文獻上的研究我們將 25 ^oC 所測得之 ¹H NMR 光譜，化學位移

 8.17 ppm 與 

8.20 ppm 的訊號分別定為 2’-去氧腺嘌呤上 2-H 與 8-H 的訊號（圖四十八）(Narukulla et al., 2008)。從變溫實驗中，我們可以看 到在 55 ^oC 所測得之¹H NMR 光譜，化學位移



8.19 ppm 為 2-H 與 8-H 重疊在一起之訊號隨著溫度降低氫鍵作用越強，2-H 會朝高磁場區移動

99

而 8-H 會朝低磁場區移動，可確定一開始所使用加熱 (60 ^oC) 進行模版 高分子聚合反應已經破壞了模版分子與甲基丙烯酸單體 27 之間的氫 鍵，因此我們修改反應方法利用照光裂解產生自由基進行分子模版高分

而 8-H 會朝低磁場區移動，可確定一開始所使用加熱 (60 ^oC) 進行模版 高分子聚合反應已經破壞了模版分子與甲基丙烯酸單體 27 之間的氫 鍵，因此我們修改反應方法利用照光裂解產生自由基進行分子模版高分

在文檔中 2',3'-環氧黃樟素所形成DNA加成產物之體外與體內研究 (頁 110-0)