仿生酵素 : 銅鋅錯合物系列模擬超氧歧化酵素的合成及其鑑定

Abstract

銅鋅超氧歧化酵素(superoxide dismutase)是一種抗氧化金屬媒，其普遍存在人體和動、植物細胞裏。一般生理上會引起氧化壓力(oxidative stress)，進而傷害細胞、破壞DNA，甚至造成細胞凋亡(apoptosis)。銅鋅超氧歧化酵素在此扮演著重要的清除超氧離子自由基的角色，使超氧離子自由基轉化形成毒性較小的二氧化氫產物。 我們經由模擬銅鋅超氧歧化酵素的活性中心結構，設計合成一系列同核(Cu(II)-Cu(II))或異核(Cu(II)-Zn(II))雙金屬錯合物，並藉由不同合成方法將錯合物附載到規則性孔洞材料的內表面，用來了解催化超氧離子自由基的活性與機制。另外，藉由嫁接帶有正電荷的官能基(TMAC)與不同孔洞大小的載體更進一步地觀察促進超氧歧化反應的因素。我們利用不同鑑定方法：UV-Vis, EPR and EXAFS 了解活性中心金屬銅的幾何結構與活性之間的關聯。並且透過NBT assay 活性測試實驗尋找最佳的模擬超氧歧化酵素的仿生材料。 在應用上，藉由本實驗室之前所開發出來的具有FITC 螢光物的中孔洞奈米粒子(FITC-MSN)，由於之前已確定它具有進入細胞的能力，於是將生物模擬銅鋅金屬錯合物附載在材料內表面上，進一步地研究它們在細胞中的毒性測試與活性表現。

Copper zinc superoxide dismutase (CuZnSOD) is an antioxidant metalloenzyme to catalyze the dismutation of superoxide anion radical (O2－․) into hydrogen peroxide and oxygen to reduce the steady-state concentration of O2－․. A series of imidazolate-bridged homo- and hetero- dinuclear Cu(II)-Cu(II) and Cu(II)-Zn(II) model compounds were synthesized and encapsulated into various mesoporous silica/aluminosilicates (MPS/Al-MPS) to mimic the structure and functionalities of CuZnSOD enzyme. Inspired by the local environments of guanidinium group in the native CuZnSOD enzyme in facilitating the superoxide dismutation, we carefully modified the silanol surface of MPS with N-trimethoxysilylpropyl-N,N,N-trimethyl-ammonium chloride (TMAC) to prepare MPS-N+ to yield the needed local environment for the active site of model compounds and to facilitate the disproportionation of O2－․. We employed the following spectroscopic techniques: UV-Vis, EPR and EXAFS to characterize the active site-Cu(II) ion of the immobilized mimic complexes, and to obtain the structural information of Cu(II) and Zn(II) centers confined in MPSs. The O2－․ disproportionation activity of mimic complexes was measured by nitro blue tetrazolium (NBT) assay. The encapsulated complexes yielded good stability against hydrothermal treatment and enhanced the O2－․ disproportionation activity in various MPS solids. The spectroscopic studies further allow us to establish the structure-reactivity relationship between the nanostructure of MPS and the efficacy of the superoxide dismutation in MPS, and to elucidate the mechanism of the SOD-like activity of immobilized model compounds in MPS. Furthermore, we utilized the oxidative damage model of HeLa cells to study the protective effect of mimic materials in the presence of paraquat (PQ) oxidant.