As mitochondrial superoxide is becoming an attractive metabolic and pharmacological target, there is an important need for developing analytical tools able to detect superoxide with high sensitivity and specificity. Among EPR-based methods, it has been recently reported that cyclic hydroxylamines offer a high sensitivity to measure superoxide production. Here, we report the synthesis and evaluation of mitoCPH, in which a 5-membered ring hydroxylamine was coupled to a triphenylphosphonium moiety to allow mitochondrial accumulation. MitoCPH efficiently reacted with superoxide with a bimolecular rate constant of 1.5 × 104 M-1 s-1. We assessed the ability of this compound to detect superoxide in PBS buffer, lysates, and in paraquat-stimulated cells. We compared its performance with CMH, a nontargeted 5-membered ring hydroxylamine, and mitoTEMPO-H, a classically used 6-membered ring hydroxylamine targeted to mitochondria. MitoCPH presented a higher sensitivity for superoxide anion detection than commonly used mitoTEMPO-H, both in buffer and in cell lysates. While we have described the ability of mitoCPH to detect superoxide in different cellular media, we cannot exclude other potential contributors to the nitroxide production from this probe. Therefore, mitoCPH should be considered as a mitochondria-targeted probe and its use as selective superoxide probe should be used cautiously.

中文翻译：

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结合三苯基的五元环环状羟胺的合成和表征，通过EPR光谱法检测线粒体超氧化物。

随着线粒体超氧化物成为有吸引力的代谢和药理学目标，迫切需要开发能够以高灵敏度和特异性检测超氧化物的分析工具。在基于EPR的方法中，最近有报道说，环状羟胺对测量超氧化物的产生具有很高的灵敏度。在这里，我们报告了mitoCPH的合成和评估，其中5元环羟胺与三苯基phosph部分偶联以允许线粒体积累。MitoCPH与超氧化物有效反应，其双分子速率常数为1.5×104 M-1 s-1。我们评估了该化合物检测PBS缓冲液，裂解物和百草枯刺激的细胞中超氧化物的能力。我们将其性能与非靶向5元环羟胺CMH和mitoTEMPO-H的性能进行了比较，靶向线粒体的经典使用的六元环羟胺。在缓冲液和细胞裂解物中，MitoCPH对超氧阴离子的检测灵敏度均高于常用的mitoTEMPO-H。尽管我们已经描述了mitoCPH在不同细胞介质中检测超氧化物的能力，但我们不能排除该探针产生氮氧化物的其他潜在贡献者。因此，应将mitoCPH视为针对线粒体的探针，应谨慎使用其作为选择性超氧化物探针。我们不能从该探针中排除其他可能导致氮氧化物产生的因素。因此，应将mitoCPH视为针对线粒体的探针，应谨慎使用其作为选择性超氧化物探针。我们不能从该探针中排除其他可能导致氮氧化物产生的因素。因此，应将mitoCPH视为针对线粒体的探针，应谨慎使用其作为选择性超氧化物探针。