Masuda Ryosuke, Goto Kei
Department of Chemistry, School of Science, Tokyo Institute of Technology, Tokyo, Japan.
Department of Chemistry, School of Science, Tokyo Institute of Technology, Tokyo, Japan.
Methods Enzymol. 2022;662:331-361. doi: 10.1016/bs.mie.2021.10.018. Epub 2021 Dec 7.
In the biological functions of selenoproteins, various highly reactive species formed by oxidative modification of selenocysteine residues have been postulated to play crucial roles. Representative examples of such species are selenocysteine selenenic acids (Sec-SeOHs) and selenocysteine selenenyl iodides (Sec-SeIs), which have been widely recognized as important intermediates in the catalytic cycle of glutathione peroxidase (GPx) and iodothyronine deiodinase, respectively. However, examples of even spectroscopic observation of Sec-SeOHs and Sec-SeIs in either protein or small-molecule model systems remain elusive so far, most likely due to their notorious instability. For the synthesis of small-molecule model compounds of these reactive species, it is essential to suppress their very facile bimolecular decomposition such as self-condensation and disproportionation. Here we outline a novel method for the synthesis of stable small-molecule model compounds of the selenocysteine-derived reactive species, in which a nano-sized molecular cavity is used as a protective cradle to accommodate the reactive selenocysteine unit. Stabilization by the molecular cradle led to the successful synthesis of Sec-SeOHs, which are stable in solution at low temperatures, and a Sec-SeI, which can be isolated as crystals. The catalytic cycle of GPx was investigated using the NMR-observable Sec-SeOH models, and all the chemical processes proposed for the catalytic cycle of GPx, including the bypass process from Sec-SeOH to the corresponding cyclic selenenyl amide, were experimentally confirmed. Detailed protocols for the syntheses of selenopeptide derivatives bearing the molecular cradle and for the spectroscopic monitoring of their reactions are provided.
在硒蛋白的生物学功能中，硒代半胱氨酸残基经氧化修饰形成的各种高反应活性物种被认为起着关键作用。这类物种的典型例子是硒代半胱氨酸亚硒酸（Sec-SeOHs）和硒代半胱氨酸亚硒酰碘（Sec-SeIs），它们分别被广泛认为是谷胱甘肽过氧化物酶（GPx）和碘甲状腺原氨酸脱碘酶催化循环中的重要中间体。然而，到目前为止，在蛋白质或小分子模型系统中，即使是对Sec-SeOHs和Sec-SeIs进行光谱观测的例子也仍然难以捉摸，这很可能是由于它们极不稳定。对于这些反应活性物种的小分子模型化合物的合成，抑制其极易发生的双分子分解，如自缩合和歧化反应至关重要。在此，我们概述了一种合成硒代半胱氨酸衍生的反应活性物种稳定小分子模型化合物的新方法，其中纳米尺寸的分子腔被用作保护支架来容纳反应性硒代半胱氨酸单元。分子支架的稳定作用导致成功合成了在低温溶液中稳定的Sec-SeOHs，以及一种可以结晶分离的Sec-SeI。使用可通过核磁共振观测的Sec-SeOH模型研究了GPx的催化循环，并且GPx催化循环中提出的所有化学过程，包括从Sec-SeOH到相应环状亚硒酰胺的旁路过程，都得到了实验证实。本文还提供了带有分子支架的硒肽衍生物的合成及其反应光谱监测的详细方案。
