利用唾液酸醛縮脢的定向演化增進L-唾液酸合成效率

Directed Evolution of N-acetylneuraminic Acid Aldolase for Synthesis of L-sialic Acid

楊禮州(Li-Jhou Yang)

指導教授 ： 翁啟惠

國立臺灣大學/理學院/化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01864

摘要

能夠與天然界醣分子作用的胜肽 (peptide)，藉由高親和力相結合，間接地阻止蛋白質與細胞表面的醣分子反應，可以當作藥物開發的方法之一。當有專一性的L-peptide 被篩選出，其相對應的鏡像異構物D-peptide即可利用化學合成製造，作用於細胞表面的D-sugar，阻止蛋白質與醣分子反應以作為藥物，因此合成L-sugar (為了篩選L-peptide)在此方法中便成了先決條件；這些非天然界的D-peptide之所以可能發展為新的藥物，最重要的原因就是因為其在生物體中相較於L-peptide，對蛋白水解酶抵抗性較好，在人體及動物體內不易被水解酶影響。
在本論文研究中，著手於利用酵素去合成非天然界的醣分子；首先利用L-ManNAc先合成L-sialic acid，之後再以N5B2醛縮酶為模板，利用隨機突變進行酵素的演化，並以L-sialic acid為基質進行篩選以找出對L-sialic acid立體選擇性較佳的酵素。在第一階段的實驗，合成L-sialic acid的過程中，發現利用乾填法的純化方式可以有效的將非鏡像異構物純化，包括L-glucose 上保護基後alpha/ beta的anomer，以及L-sialic acid不必策略性地上保護基團便可將C-4 hydroxy非鏡像異構物成功純化。在第二階段，實驗企圖進一步提升酵素 (N5B2醛縮酶)對L-sialic acid的專一性以合成L-sialic acid；演化的酵素經由蛋白質純化後，進行動力學參數測量。實驗的結果發現並未篩選出較好的酵素，其理由以及解決的方法會進一步的進行討論。

關鍵字

唾液酸 ； 醛縮酶

並列摘要

Peptides that can bind to natural D-sugar with high affinity and specificity can act as functional inhibitors to block glycan-protein interaction, and may have the potentials to be developed as therapeutics. When high affinity L-peptides that bind to unnatural L-sugar was identified, corresponding unnatural D-peptides can then be artificially synthesized by chemical methods to bind to natural D-sugars to interfere protein-carbohydrate interactions and used as potential inhibitors. Hence, preparation of L-sugar is a prerequisite in this approach. The advantages of D-peptides compared to natural L-peptides include being resistant to protease and increased serum half-lives.
In this study, enzymatic synthesis was attempted to produce unnatural L-sugar. L-sialic acid was chemically prepared from L-ManNAc and then N5B2 sialic acid aldolase was improved to increase its stereoselectivity toward L-sialic acid by directed evolution. For the synthesis of L-sialic acid, it was found that diastereomer with partial protection groups can be successfully purified by using a dry loading method to facilitate diastereomer separation during chromatography. Furthermore, evolution of N5B2 sialic acid aldolase resulted in no successful improvements in reactivity towards L-sialic acid. The reasons and possible resolutions will be discussed.

並列關鍵字

sialic acid ； aldolase

參考文獻

1. Funke SA & Willbold D (2009) Mirror image phage display—A method to generate D-peptide ligands for use in diagnostic or therapeutical applications. Molecular BioSystems 5 (8): 783-786.

2. Sun N, Funke SA, & Willbold D (2012) Mirror image phage display—Generating stable therapeutically and diagnostically active peptides with biotechnological means. Journal of Biotechnology 161 (2): 121-125.

3. Faillard H (1989) The early history of sialic acids. Trends in Biochemical Sciences 14 (6): 237-241.

4. Kozlov IA, Mao S, et al. (2001) Synthesis of solid-supported mirror-image sugars: a novel method for selecting receptors for cellular-surface carbohydrates. Chembiochem 2 (10): 741-746.

5. Choi SK, Lee S, & Whitesides GM (1996) Synthesis of C-5 analogs of N-acetylneuraminic acid via indium-mediated allylation of N-substituted 2-amino-2-deoxymannoses. The Journal of Organic Chemistry 61 (25): 8739-8745.