| 摘要: | 外切醣苷酶在醣穩態調控、細胞訊號傳導與廢物清除中具有關鍵作用，其功能異常可能導致底物累積、蛋白質錯誤摺疊或免疫反應異常，進而引發多種遺傳性、代謝性與腫瘤相關疾病。此外，外切醣苷酶在某些病理狀態下的上調或過度活躍，亦與動脈粥狀硬化、纖維化、神經退化及腫瘤進展密切相關。至今為止，與外切型醣苷酶相關的多項科學挑戰仍未被解決。例如，結合串聯質譜分析開發新的外切型醣苷酶酵素活性分析平台，仍有待探索。此外，針對與疾病相關的外切型醣苷酶，新的蛋白穩定劑或抑制劑也極為迫切需要。本論文可分為三個部分：
第一部分為建立α-甘露醣苷酶 (AM) 酵素活性平台: 設計α-甘露醣苷酶的新型酵素受質及內標，並利用流動注射串聯質譜分析（FIA-MSMS）成功地證明其操作使用可行性。與實驗室同仁合作下，在分子設計上我們提出以哌啶-酪胺酸為主混成結構之新型醣苷配基分子群，並檢測其個別的溶劑分配特性。經篩選後合格物再用於此α-甘露醣苷酶受質的合成及其酵素活性測驗。此成功開發出的新α-甘露醣苷酶受質及內標和其分析方法不僅可用於酵素或細胞實驗，未來也有潛力可用於臨床應用。
第二部分為開發可作為艾杜醣苷酸酶 (IDUA) 蛋白穩定劑的小分子，用於治療第一型黏多醣症疾病: 第一型黏多醣症（MPS I）由艾杜糖苷酸酶（IDUA）缺陷引起，導致醣胺聚醣累積。利用小分子穩定 IDUA 的藥理性伴護治療是一種具潛力的策略。第一代穩定劑能熱穩定重組 α-IDUA，但在細胞層面效果有限。第二代 IdoA-C 類糖 ACK600 能提高酵素活性並降低 MPS I 細胞中的肝素硫酸水平，但其效力及合成路徑的效率仍有限。本研究改良 ACK600 的合成途徑，克服 C-糖苷化及翻轉步驟的挑戰，成功優化合成路徑以利後續大量製備。開發合成轉化方法以利未來新穩定劑的找尋。同時啟動結晶實驗，為下一代 IDUA 穩定劑的合成與結構開發奠定基礎。
第三部分為高基氏體 α-甘露醣苷酶 II 抑制劑: 高基氏體α-甘露醣苷酶 II （α-hGMII）是 N-聚醣修飾過程中的關鍵酵素，也是調控醣化反應的重要治療標的。基於GMII選擇性抑制劑ACK900的發現，合成了其醣苷配基。同時建立 HPLC 純化與分析方法，進行溶劑溶解度測試，並製備血清校正曲線以應用於藥物動力學分析。本研究提供了合成與分析層面的基礎，為 ACK900 後續生物與動物實驗開發鋪路，展現其作為 α-hGMII 抑制劑的潛力。
;Exoglycosidases play a crucial role in regulating glycan homeostasis, cellular signaling, and waste clearance. Functional abnormalities of these enzymes may lead to substrate accumulation, protein misfolding, or altered immune responses, thereby contributing to various genetic, metabolic, and cancer-related diseases. In addition, upregulation or hyperactivation of exoglycosidases under certain pathological conditions is closely associated with atherosclerosis, fibrosis, neurodegeneration, and tumor progression.
Up to date, several exoglycosidases related scientific challenges have not been solved. For example, the new development of the exoglycosidases enzyme activity platform with the assistance of tandem mass analysis remains to be explore. Besides, new protein stabilizers or protein inhibitors toward disease associated exoglycosidases are urgently needed. This thesis can be divided into three parts:
Part 1 Establishment of an α-Mannosidase (AM) enzyme activity platform: A novel enzyme substrate and corresponding internal standard for α-mannosidase were developed, and their feasibility was successfully demonstrated using flow injection analysis tandem mass spectrometry (FIA-MSMS). In collaboration with colleagues in our laboratory, we proposed a novel group of glycosidic aglycone molecules based on a piperidine–tyrosine hybrid scaffold, followed by examination of their solvent partitioning. The qualified aglycones can be conjugated with a mannosidase to become a potential enzyme substrate for testing the corresponding enzymatic activity toward various enzymes and cells.
Part 2 Developing small molecules as α-L-iduronidase (IDUA) protein stabilizers toward MPSI disease: Mucopolysaccharidosis type I (MPS I) is caused by IDUA deficiency, leading to glycosaminoglycan accumulation. Using pharmacological chaperones to stabilize IDUA are a promising therapy. The first-generation stabilizers thermally stabilized recombinant α-IDUA but lacked cellular efficacy. The second-generation IdoA-C-glycoside ACK600 increased enzyme activity and decreased heparan sulfate levels in MPS I cells, though its potency and the efficiency of its synthetic route remained limited. Here, we improved the synthetic route for ACK600, overcoming C-glycosylation challenges. The synthetic route was successfully optimized to enable efficient large-scale production. Develop synthetic transformation methods to facilitate the future discovery of new stabilizers. Crystallization experiments were initiated, providing a synthetic and structural foundation for next-generation IDUA stabilizers.
Part 3 Golgi mannosidase II inhibitor: Golgi α-mannosidase II (α-hGMII) is a key enzyme in N-glycan maturation and an attractive target for regulating glycosylation. Based on the discovery of the GMII-selective inhibitor ACK900, its aglycone was synthesized. To ensure analytical reliability, we established an HPLC purification method, optimized formulation conditions for solubility, and developed a serum calibration curve for pharmacokinetic evaluation. This study provides a synthetic and analytical foundation for advancing ACK900 into biological and animal studies as a potential α-hGMII inhibitor. |
