Hydrogen-Deuterium Addition and Exchange in N-Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.
Hydrogen-Deuterium Addition and Exchange in N-Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.
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核磁共振波谱法研究N-乙基马来酰亚胺与谷氨酸反应中的氢氘加成和交换。
DOI：
10.1021/acsomega.2c03482
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发表时间：
2022-08-02
期刊：
影响因子：
4.1
通讯作者：
Raftery, Daniel
中科院分区：
文献类型：
--
作者：
Gowda, G. A. Nagana;Pascua, Vadim;Neto, Fausto Carnevale;Raftery, Daniel
谷胱甘肽（GSH）是一种重要的、普遍存在的巯基化合物，大量存在于几乎每一个活细胞中。它是一种强大的抗氧化剂，保护细胞免受氧化损伤和自由基损伤。其在离体分析中的定量仍然是一个主要挑战，因为它自发氧化形成谷胱甘肽二硫化物。N-乙基马来酰亚胺（NEM）是一种常见的迈克尔受体，它与硫醇反应迅速且不可逆，并阻止二硫键的形成。基于硫醇与NEM的缀合，最近，使用NMR光谱法测定人血液中GSH的浓度[Anal. Chem.，2021，93（44）：14844-14850]。发现在硫醇-马来酰亚胺反应以及NMR分析期间发生氢-氘加成和交换，产生一系列探索性较差的非对映异构体/同位素异构体。在这里，我们建立了一个通用的NMR方法来确定来自硫醇-马来酰亚胺反应的硫代琥珀酰亚胺非对映异构体/同位素异构体。分别用D_2O和H_2O对GSH和另一种巯基化合物半胱氨酸进行巯基-迈克尔加成反应。在不同的溶剂、缓冲液和pH条件下，通过1H/13 C 1D/2D NMR表征缀合物。迈克尔加成与H/D交换相结合形成了12种独特的非对映异构体/同位素异构体。NMR测量允许溶液中所有结构的不同分配和H/D加成和交换的定量。有趣的是，氘交换速率取决于结构、pH和缓冲液。巯基-马来酰亚胺反应和H/D交换机制的阐明可能会影响代谢组学、小分子合成和生物缀合化学等领域。
Glutathione (GSH) is an important and ubiquitous thiol compound abundantly present in virtually every living cell. It is a powerful antioxidant critically required to protect cells from oxidative damage and free radical injury. Its quantification in ex vivo analysis remains a major challenge because it spontaneously oxidizes to form glutathione disulfide. N-Ethylmaleimide (NEM) is a well-known Michael acceptor, which reacts rapidly and irreversibly with thiol and prevents disulfide bond formation. Based on thiol conjugation to NEM, recently, the concentration of GSH was determined in human blood using NMR spectroscopy [Anal. Chem, 2021, 93(44): 14844–14850]. It was found that hydrogen–deuterium addition and exchange occur during the thiol–maleimide reaction as well as NMR analysis, generating a series of poorly explored diastereomers/isotopomers. Here, we establish a general NMR approach to identify the thiosuccinimide diastereomers/isotopomers derived from the thiol–maleimide reaction. The thiol-Michael addition reaction was conducted for GSH and another thiol compound, cysteine, separately, using D2O and H2O. The conjugates were characterized by 1H/13C 1D/2D NMR under different solvent, buffer, and pH conditions. The Michael addition combined with the H/D exchange formed twelve unique diastereomers/isotopomers. NMR measurements allowed the distinct assignment of all structures in solutions and quantification of H/D addition and exchange. Interestingly, the deuterium exchange rate was dependent on structure, pH, and buffer. The elucidation of the thiol–maleimide reaction and H/D exchange mechanism can potentially impact areas including metabolomics, small molecule synthesis, and bioconjugation chemistry.
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影响因子：
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作者：
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