| 摘要: | β-甲基胺基酸為組裝具有生物活性天然產物的重要組成部件，天然產物使用β-甲基色胺酸（β-methyltryptophan, β-MeTrp）作為組合單元（building block）已被發現，本論文首先介紹了數種以β-MeTrp作為前驅體的天然產物，如streptonigrin和maremycin，它們具有抗癌、抗菌、抗病毒和抗真菌等多種生物活性。本論文進一步探討β-MeTrp在各領域的應用，以及天然產物的開發和多樣化。天然產物一直是藥物發現的最佳來源，其中異戊二烯化吲哚生物鹼（Prenylated indole alkaloids, PIAs）是一種多樣性的天然產物，長期以來一直被認為是富含藥性的天然產物。在此基礎上，本研究嘗試以天然的PIAs含色胺酸、脯胺酸雙胜肽組成作為架構，利用多樣性導向的生物合成概念來合成一系列化學上難以合成的相關衍生物，結合C-甲基轉移酶、鹵化物甲基轉移酶、轉胺酶與異構酶等，經由一鍋法酵素催化創造多種非對映異構體之組合單元，再搭配化學合成的組合方式來生產具結構多樣性和立體特異性的生物鹼，以產生類似天然物PIAs且具結構複雜性的生物活性分子。而本論文主要根據D-丙胺酸轉胺酶（D-alanine aminotransferase, DAAT）活性位點上的殘基，以蛋白質工程改良得到突變型DAAT，其中突變型DAAT針對(2R,3S)-β-methyltryptophan會導致產物消旋化，我們因此對其進行結構與機制的探討。本研究旨在利用所獲得的知識及新的生物合成途徑產生新生物鹼衍生物，作為新藥物發現開發的一種策略，透過結構多樣性及合成生物學，期許結構多樣性能對新藥開發有所貢獻，以實現量產具有增益藥理和生物活性的新化合物。;β-Methyl amino acids are crucial building blocks existing in many biologically active natural products. For example, natural products streptonigrin and maremycin each contains a characteric β-methyltryptophan (β-MeTrp), which is derived from tryptophan, featuring such phenomenal biological effects as anticancer, antibacterial, antiviral, and antifungal. In this thesis, I first went over β-MeTrp reported with various applications, then I came to discuss its roles in the development and improvement of natural products for new medicines. Natural products are the origin of many clinically used drugs, in which desipte their synthetic difficulty prenylated indole alkaloids (PIAs) fraught with numerous favorable medicinal properties arouse great interest recently. This study was aimed to synthesize chemically/structurally challenging PIA-related derivatives by taking advantage of the diversity-oriented biosynthesis approach on the framework of tryptophan and proline. By virtue of the biosynthetic utilizations of C-methyltransferase, halide methyltransferase, aminotransferase, and epimerase, enatiospecific building blocks were made from given one-pot enzymatic conditions. These building blocks will be employed to form new complex PIAs, that are dissimilar to natural PIAs with structural diversity and stereochemical specificity but enhanced biological activity instead. The present work investigated D-alanine aminotransferase (DAAT) mutants, of which selected residues at the active site were subjected to protein engineering to bring up steroselectivity. The rationale of the reaction steroselectivity for each individual DAAT mutant was established through thorough structural and biochemical interogations. The final goal of this study is to establish a sound methodology to rewire biosynthetic pathways to produce novel bioactive alkaloids with new functionalities. Hopefully, this semial study has paved a solid foundation for the goal described above. |
