- 學位論文
設計與開發以麥可加成應用於蛋白質修飾之功能性連接段
Design and Development of Functional Linkers for Protein Modification via Michael Addition
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摘要
甘露醣-6-磷酸 (Mannose-6-Phosphate, M6P) 是一種生物體內存在的重要辨識標記物，當其與甘露醣-6-磷酸受體結合後，酸性水解酶會被運輸至溶酶體，並將細胞內多餘的大分子分解進行回收利用；然而，當其中一種水解酶突變導致溶酶體功能異常時，會使大分子堆積在細胞中造成細胞死亡，這一種罕見遺傳性疾病稱為溶酶體儲積症 (Lysosomal Storage Disorders, LSDs)。目前針對此類疾病的主要治療方法為酵素替代療法 (Enzyme Replacement Therapy, ERT)，意即將缺乏的酵素透過外部生產的方式送至細胞內，以維持細胞正常運作。因此，通過化學修飾將 M6P 結合至酵素，以提升療效，已成為相關研究的熱門主題。 本研究首先嘗試合成具有疊氮基團 (N3) 的 M6P 衍生物，將天然物 M6P 中 6 號位的磷酸根改為較不易水解的丙二酸根 (malonate)，據文獻指出其不但能提升與受體的結合能力，同時具有較不易水解的特性；另外在 1 號位引入疊氮基團，能再透過點擊化學連接具炔鍵的結構，大幅提升其後續發展性。 接著則著重於設計能進行點擊反應之多種連接段，希望透過點擊反應快速合成單價及三價且具有不同麥可受體 (michael acceptor) 之分子工具，並初步測試其與硫醇的反應性，以挑選最適合與酵素結合的化合物。期望未來能將這些化合物結合目標水解酶，在不影響水解酶活性的前提下，提升酵素中 M6P 之含量，進一步改善酵素替代療法之效能。
並列摘要
Mannose-6-Phosphate (M6P) is an essential recognition marker found in biological systems. When M6P binds to the Mannose-6-Phosphate Receptor (M6PR), acidic hydrolases will be transported to the lysosome, where they degrade excess macromolecules within the cell for recycling. However, when one of these hydrolases is defective due to mutations, it will lead to the accumulation of macromolecules within the cell and ultimately resulting in cell death. This rare disease is known as Lysosomal Storage Disorder (LSDs). The current primary treatment for LSDs is Enzyme Replacement Therapy (ERT), which involves delivering externally produced enzymes into cells to maintain normal cellular functions. Consequently, chemically modifying M6P to enhance its binding to enzymes and improve therapeutic efficacy has become a prominent research focus. This study aims to synthesize M6P derivatives containing an azido group (N₃). Specifically, the phosphate group at the 6-position of natural M6P is replaced with a more hydrolysis-resistant malonate group. According to the literature, this modification not only enhances the binding affinity to the receptor but also improves stability. Additionally, introducing an azido group at the 1-position enables further functionalization through click chemistry with alkyne-containing compounds, significantly broadening its potential applications. This research then focuses on designing various linkers compatible with click reactions to rapidly synthesize monovalent and trivalent molecular tools containing different michael acceptors. These tools are subjected to preliminary reactivity tests with thiols to identify the most suitable compound for enzyme conjugation. It is anticipated that these compounds can be conjugated to target hydrolases, increasing the M6P content in enzymes while preserving their catalytic activity. This approach aims to further enhance the efficacy of enzyme replacement therapy.
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