非核醣體肽的化學及生物合成之研究

Chemical Synthesis and Biosynthetic Studies of Nonribosomal Peptide

吳承翰(Cheng-Han Wu)

指導教授 ： 朱忠瀚

國立臺灣大學/理學院/化學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103857

摘要

天然物為生物的次級代謝物，往往具有多樣的生物活性，為生物帶來生存演化上的優勢。然而傳統天然物的發現方式十分繁瑣，需經過發酵、萃取層析分離、活性篩選、結構鑑定等步驟。不僅如此，現有技術僅有1%的微生物易於在實驗室培養取得，且許多的生合成基因在實驗室培養條件下不表現，更加限制了科學家能探索的天然物。因此我們期望發展一套技術平台，結合生物資訊結構預測以及化學合成，快速有效地找尋具有生物活性的天然物，而本論文即是針對此兩項技術奠定基礎。
論文第一部分為與荷蘭瓦赫寧恩大學M. Medema教授的生物資訊團隊合作，針對他們提供的6個非核醣體肽合成酶中的腺苷酸化區進行活性測試，希望藉此增加生物資訊演算法的樣本數，以提高演算法對胺基酸受質預測的準確度。我們透過基因工程方式克隆得到了這6個重組蛋白酵素，提供它們三磷酸腺苷，量測其與20種胺基酸能否反應釋出焦磷酸分子，屆此了解這些非核醣體肽合成酶所相對應的胺基酸受質。
論文第二部分為開發新穎胜肽固相噻唑(thiazole)合成法。噻唑五員雜環為胜肽天然物中常見的特殊骨架，我們希望藉此增加胜肽固相合成法用於合成此類型天然物的廣度。在本論文中我們以Hantzsch法合成完全氧化態噻唑構築體並以胜肽固相合成法合成出我們選定的目標天然物：於2019年由W. Gerwick團隊於海洋綠藻德式藻屬(Derbesia sp.)分離的天然物Pagoamide A。

關鍵字

非核醣體肽 ； 非核醣體肽結構預測 ； 胜肽固相合成法 ； 噻唑合成法

並列摘要

Natural products are secondary metabolites. They bring evolutionary advantages to the organism that produces them through the various biological activities they possess. However, the traditional pipeline for natural product discovery is a complicated multistep process, which requires culture, fermentation, extraction, fractionation, bioactivity screening, and structure determination. Moreover, only about 1% of microbes are easily cultured, and among them, most of their biosynthetic gene clusters remain silent under laboratory fermentation conditions, which precludes their discovery by natural product researchers. Therefore, our laboratory aims to develop a novel platform that combines bioinformatics structure prediction and chemical synthesis to quickly and effectively search for bioactive natural products. My thesis has two parts and each establishes the foundation for the above two aspects.
The first part of my thesis describes setting up an activity assay for six nonribosomal peptide synthetase adenylation domains (AD) provided by the Medema group at Wageningen University, who are experts in bioinformatics based natural product structure prediction. They were unable to predict the substrate specificity of these six AD and hoped that we can find out via experimentation, which will increase the size of the training set for their bioinformatics algorithm, so as to improve the accuracy of future substrate predictions. We cloned these six AD as recombinant proteins, provided them with adenosine triphosphate and the 20 canonical amino acids, and measured which amino acid resulted in amino acid activation, which was observed as pyrophosphate release. This assay will help us identify the substrates corresponding to these non-ribosomal peptide synthetase (NRPS) ADs.
The second part of my thesis entails the development of a novel methodology for solid phase peptide-thiazole synthesis. Thiazole is a five-member heterocycle that is commonly seen in the skeleton of peptide natural products. We hope to demonstrate on-resin thiazole synthesis to expand the scope of solid-phase peptide synthesis (SPPS) and its application in natural product synthesis. Our strategy is to synthesize the thiazole-containing building blocks via Hantzch method and then use these building blocks on solid-phase to synthesize our model target pagoamide A isolated from Derbesia sp. culture and reported by the Gerwick group in 2020.

並列關鍵字

Nonribosomal peptide ； NRPS adenylation domain ； SPPS ； Thiazole

參考文獻

1. Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D., Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 1997, 97 (7), 2651-2674.

2.Sieber, S. A.; Marahiel, M. A., Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. Chem. Rev. 2005, 105 (2), 715-738.

3. Sussmuth, R. D.; Mainz, A., Nonribosomal Peptide Synthesis-Principles and Prospects. Angew. Chem. Int. Ed. 2017, 56 (14), 3770-3821.

4. Conti, E.; Stachelhaus, T.; Marahiel, M. A.; Brick, P., Structural basis for the activation of phenylalanine in the non‐ribosomal biosynthesis of gramicidin S. EMBO J. 1997, 16 (14), 4174-4183.

5. Stachelhaus, T.; Mootz, H. D.; Marahiel, M. A., The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 1999, 6 (8), 493-505.