Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl into lymphocytes
Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl into lymphocytes
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DOI：
10.1039/b310467b
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发表时间：
2004-01-01
影响因子：
3.2
通讯作者：
Rosen, GM
中科院分区：
文献类型：
--
作者：
Kao, JPY;Rosen, GM
细胞内羟自由基（HO.）通过自旋捕获/电子顺磁共振（EPR）光谱学已经成为生物学中研究自由基的巨大挑战之一。而5-羧基-5-甲基-1-吡咯啉N-氧化物[3]可以特异性地自旋捕获HO。在均相溶液中，硝酮[3]在生理pH下的离子性质阻止其进入细胞。我们假设，羧基轴承自旋探针，如硝酮[3]转化为酯酶水解不稳定酯将允许细胞内定位和积累的自旋探针。为了测试这种方法的可行性，我们制备了模型化合物3-乙酰氧基甲氧羰基2，2，5，5-四甲基-1-吡咯烷氧基[ 7]。这种酯使自旋标记在淋巴细胞中容易积累到mM水平。我们认为它在这些细胞内的保留是细胞内水解为3-羧基-2，2，5，5-四甲基-1-吡咯烷氧基的结果[ 6]。此外，我们的研究表明氨氧基[ 6]在细胞内环境中是稳定的。这些模型研究提出了一个可行的策略，用于检测细胞内HO。使用5-羧基-5-甲基-1-吡咯啉N-氧化物的乙酰氧基甲酯[3]。
The intracellular detection of hydroxyl radical (HO.) through spin trapping/electron paramagnetic resonance (EPR) spectroscopy has been one of the great challenges in studying free radicals in biology. While 5-carboxy-5-methyl-1-pyrroline N-oxide, [3], can specifically spin trap HO. in homogeneous solutions, the ionic nature of nitrone [3] at physiologic pH prevents its entry into cells. We hypothesized that conversion of carboxyl-bearing spin probes such as nitrone [3] into an esterase-hydrolyzable labile ester would permit intracellular localization and accumulation of the spin probes. To test the feasibility of such an approach, we prepared the model compound, 3-acetoxymethoxycarbonyl2,2,5,5- tetramethyl-1-pyrrolidinyloxyl [ 7]. This ester enabled ready accumulation of spin label to mM levels in lymphocytes. We suggest that its retention within these cells was the result of intracellular hydrolysis to 3-carboxy2,2,5,5- tetramethyl- 1- pyrrolidinyloxyl [ 6]. Moreover, our studies show that aminoxyl [ 6] was stable in the intracellular environment. These model studies suggest a viable strategy for detecting intracellular HO. by using the acetoxymethyl ester of 5-carboxy-5-methyl-1-pyrroline N-oxide [3].