a Department of Medicine, Vanderbilt University Medical Center , Nashville , TN , USA.
b Laboratory of Nitrogen Compounds, Novosibirsk Institute of Organic Chemistry , Novosibirsk , Russia.
Free Radic Res. 2018 Mar;52(3):339-350. doi: 10.1080/10715762.2017.1390744. Epub 2017 Nov 3.
Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50 nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4 mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.
氮氧自由基广泛应用于生物学领域，包括作为抗氧化剂、自旋标记物、用于 pH 值、氧和巯基水平的功能性自旋探针，以及使用电子顺磁共振（EPR）进行组织氧化还原状态成像；然而，氮氧自由基的生物学应用受到快速生物还原为 EPR 静默的羟胺和快速清除的限制。在这项工作中，我们研究了带有乙酰氧基甲氧基羰基的吡咯烷氮氧自由基，它们可以通过细胞酯酶水解为亲水性羧酸衍生物，从而抵抗生物还原。含有乙酰氧基甲氧基羰基的氮氧自由基可以从培养基中被细胞快速吸收，3,4-双（乙酰氧基甲氧基羰基）-2,2,5,5-四甲基-1-吡咯啉-1-氧基（DCP-AM2）和 3-(2-(双（2-(乙酰氧基甲氧基）-2-氧代乙基)氨基)乙酰胺基）-2,2,5,5-四甲基-1-吡咯啉-1-氧基（DCAP-AM2）显示出细胞部分最强的 EPR 信号。值得注意的是，3,4-二羧酸基-2,2,5,5-四甲基-1-吡咯啉-1-氧基（DCP）及其单和二乙酰氧基甲酯的 EPR 参数不同，并且随后可以通过 EPR 跟踪 DCP-AM2 中乙酰氧基甲氧基羰基的细胞内水解。为了阐明所得 DCP 的细胞内位置，已经分离出线粒体部分。EPR 测量表明，线粒体是 DCP 最终积累的主要场所。与吡咯烷氮氧自由基相比，TEMPO 衍生物在细胞部分的 EPR 信号衰减明显更快。发现向内皮细胞补充 50 nM 的 DCP-AM2 可完全使线粒体中超氧阴离子水平正常化。此外，向小鼠（1.4 mg/kg/天）给予 DCP-AM2 可导致组织中大量氮氧自由基积累，并显著降低高血压。我们发现，二羧酸基吡咯烷氮氧自由基 DCP-AM-H 的羟胺衍生物可用于体内检测高血压的血管紧张素 II 模型中的超氧阴离子。在血管紧张素 II 输注的野生型小鼠中，DCP-AM-H 的输注导致 EPR 信号的持久氮氧自由基信号在血液和血管组织中的积累，但在 SOD2 过表达的小鼠中则没有。我们的数据表明，含有乙酰氧基甲氧基羰基的氮氧自由基在细胞内积累并表现出特定于部位的抗氧化活性。
