Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin
Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin
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DOI：
10.1016/j.peptides.2012.06.016
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发表时间：
2012-09-01
期刊：
影响因子：
3
通讯作者：
Conde-Frieboes, Kilian W.
中科院分区：
文献类型：
--
作者：
Eliasen, Rasmus;Andresen, Thomas L.;Conde-Frieboes, Kilian W.
Gurmarin是一种35个氨基酸的肽，在抑制剂胱氨酸结中具有三个二硫键。在植物匙羹藤中发现，并已被确定为啮齿动物的甜味抑制剂。在这篇文章中，我们提供了一个有效的路线，通过控制的随机氧化策略合成的gurmarin。我们比较了形成三个二硫键的两种氧化方法。首先，基于无规氧化反应，采用三苯甲基半胱氨酸保护法合成还原型香豆素，并在含有胱胺和还原型谷胱甘肽的Tris-HCl缓冲液中氧化48 h，以促进二硫键错配。第二个是基于逐步脱保护，然后氧化，其中半胱氨酸对正交保护叔丁基硫基，三苯甲基和乙酰胺基甲基。为了验证获得天然的gurmarin氧化产物，使用嗜热菌蛋白酶裂解。随机氧化的香豆素裂解显示两种可能的二硫键组合;天然和非天然的香豆素二硫键异构体。因此，使用正交脱保护-氧化策略合成非天然异构体，并使用UPLC分析天然和非天然香豆素异构体。结果发现，无规氧化过程导致高收率的天然gurmarin。因此，合成路线简单，并且比先前报道的合成gurmarin和其它富含半胱氨酸的肽的方法显著更有效。重要的是，天然gurmarin是通过随机氧化获得的，这是首次通过合成方法证实。(C)2012 Elsevier Inc. All rights reserved.
Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48 h in a Tris-HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time. (C) 2012 Elsevier Inc. All rights reserved.