Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems.
Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems.
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DOI：
10.1021/ar200126t
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发表时间：
2011-09-20
影响因子：
18.3
通讯作者：
Chang, Christopher J.
中科院分区：
文献类型：
--
作者：
Lippert, Alexander R.;De Bittner, Genevieve C. Van;Chang, Christopher J.
活性氧（ROS），例如过氧化氢，是氧代谢的重要产物，如果调节不当，会积聚并在细胞内引起氧化应激。因此，生物体已经进化出分子系统，包括抗氧化金属酶（例如超氧化物歧化酶和过氧化氢酶）和一系列基于硫醇的氧化还原对，以中和这种对细胞的威胁。另一方面，新出现的证据表明，ROS（尤其是 H2O2）的受控生成对于维持细胞健康是必要的。 NADPH 氧化酶的鉴定就是一个很好的例子，NADPH 氧化酶可产生特定的 ROS，并且存在于全身几乎所有细胞类型中。事实上，越来越多的研究表明，H2O2 和其他 ROS 在健康的生理信号通路中具有重要功能。 H2O2 的信号-应激二分法是区分其对生命系统的有益影响和有害影响的动机来源。使用基于反应的探针对这种氧代谢物进行分子成像是一种实时、无创监测生物样本中 H2O2 化学的强大方法，但以这种方式研究 H2O2 的两个关键挑战是探针分子的化学选择性和生物正交性。化学选择性存在问题，因为传统的 ROS 检测方法与其他 ROS 存在非特异性反应。此外，一些方法需要与活细胞或活体动物标本不相容的酶添加剂。此外，生物正交性要求反应不得与内在的细胞化学竞争或扰乱；对于介导细胞内主要氧化还原事件的硫醇或金属对来说，这一要求尤其重要。化学选择性生物正交反应——例如炔烃-叠氮化物环加成和相关的点击反应、Staudinger-Bertozzi 连接以及各种基于反应的分子探针中使用的转化——已在生物分子和过程的修饰、标记和检测中得到广泛应用。在本报告中，我们总结了我们实验室的 H2O2 研究，使用 H2O2 介导的芳基硼酸酯氧化为酚作为生物正交方法来检测生命系统中这种重要的 ROS 通量。我们已将这种多功能开关安装到有机和无机支架上，作为“开启”探针，用于可见光和近红外 (NIR) 荧光、比率荧光、时间门控镧系元素发光以及活细胞和动物中 H2O2 的体内生物发光检测。进一步的化学和遗传操作将这些探针靶向特定的细胞器和其他亚细胞区域，也可以让它们被捕获在细胞内，从而增强它们的敏感性。这些新颖的化学工具揭示了关于 H2O2 在各种生命系统（包括免疫、癌症、干细胞和神经细胞模型）中的产生、定位、运输和体内作用的基本新生物学见解。
Reactive oxygen species (ROS), such as hydrogen peroxide, are important products of oxygen metabolism that, when misregulated, can accumulate and cause oxidative stress inside cells. Accordingly, organisms have evolved molecular systems, including antioxidant metalloenzymes (such as superoxide dismutase and catalase) and an array of thiol-based redox couples, to neutralize this threat to the cell when it occurs. On the other hand, emerging evidence shows that the controlled generation of ROS, particularly H2O2, is necessary to maintain cellular fitness. The identification of NADPH oxidase enzymes, which generate specific ROS and reside in virtually all cell types throughout the body, is a prime example. Indeed, a growing body of work shows that H2O2 and other ROS have essential functions in healthy, physiological signaling pathways. The signal–stress dichotomy of H2O2 serves as a source of motivation for disentangling its beneficial from its detrimental effects on living systems. Molecular imaging of this oxygen metabolite with reaction-based probes is a powerful approach for real-time, noninvasive monitoring of H2O2 chemistry in biological specimens, but two key challenges to studying H2O2 in this way are chemoselectivity and bioorthogonality of probe molecules. Chemoselectivity is problematic because traditional methods for ROS detection suffer from nonspecific reactivity with other ROS. Moreover, some methods require enzymatic additives not compatible with live-cell or live-animal specimens. Additionally, bioorthogonality requires that the reactions must not compete with or disturb intrinsic cellular chemistry; this requirement is particularly critical with thiol- or metal-based couples mediating the major redox events within the cell. Chemoselective bioorthogonal reactions—such as alkyne–azide cycloadditions and related click reactions, the Staudinger–Bertozzi ligation, and the transformations used in various reaction-based molecular probes—have found widespread application in the modification, labeling, and detection of biological molecules and processes. In this Account, we summarize H2O2 studies from our laboratory using the H2O2-mediated oxidation of aryl boronates to phenols as a bioorthogonal approach to detect fluxes of this important ROS in living systems. We have installed this versatile switch onto organic and inorganic scaffolds to serve as ‘turn-on’ probes for visible and near-infrared (NIR) fluorescence, ratiometric fluorescence, time-gated lanthanide luminescence, and in vivo bioluminescence detection of H2O2 in living cells and animals. Further chemical and genetic manipulations target these probes to specific organelles and other subcellular locales and can also allow them to be trapped intracellularly, enhancing their sensitivity. These novel chemical tools have revealed fundamental new biological insights into the production, localization, trafficking, and in vivo roles of H2O2 in a wide variety of living systems, including immune, cancer, stem, and neural cell models.
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