Environmentally Robust Rhodamine Reporters for Probe-based Cellular Detection of the Cancer-linked Oxidoreductase hNQO1
Environmentally Robust Rhodamine Reporters for Probe-based Cellular Detection of the Cancer-linked Oxidoreductase hNQO1
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DOI：
10.1021/acschembio.5b00792
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发表时间：
2016-01-01
影响因子：
4
通讯作者：
McCarley, Robin L.
中科院分区：
文献类型：
--
作者：
Best, Quinn A.;Johnson, Amanda E.;McCarley, Robin L.
我们成功地合成了一种荧光探针，能够检测人类细胞内的癌症相关的NAD（P）H：醌氧化还原酶同工酶-1，根据从各种罗丹明和α-萘酚罗丹明对生物还原剂NADH的稳定性的研究结果，目前在细胞内类似于100-200 μ M。虽然罗丹明通常以其化学稳定性而闻名，但我们观察到NADH会导致许多罗丹明类似物发生显着且有时快速的修饰，包括那些经常用于成像应用的类似物。从机理研究的结果使我们排除了自由基为基础的还原途径，这表明罗丹明还原NADH收益的氢化物转移过程中产生的还原无色形式的罗丹明和氧化NAD+。罗丹明的结构特征和它们与NADH的反应性之间的关系进行了观察。在N3-和N6-氮上具有增加的烷基化的罗丹明，以及氧杂蒽核，与NADH反应最少;而具有吸电子取代基的非烷基化变体或类似物具有最快的反应速率。这些结果使我们能够明智地构建一种基于萘若丹明的开启荧光探针，该探针能够选择性地检测人类癌细胞中的癌症相关的、NADH依赖性酶NAD（P）H：醌氧化还原酶同工酶-1，而不会产生NADH诱导的萘若丹明报告基因失活的问题。
We successfully synthesized a fluorescent probe capable of detecting the cancer-associated NAD(P)H:quinoneoxidoreductase isozyme-1 within human cells, based on results from an investigation of the stability of various rhodamines and seminaphthorhodamines toward the biological reductant NADH, present at similar to 100-200 mu M within cells. While rhodamines are generally known for their chemical stability, we observe that NADH causes significant and sometimes rapid modification of numerous rhodamine analogues, including those oftentimes used in imaging applications. Results from mechanistic studies lead us to rule out a radical-based reduction pathway, suggesting rhodamine reduction by NADH proceeds by a hydride transfer process to yield the reduced leuco form of the rhodamine and oxidized NAD+. A relationship between the structural features of the rhodamines and their reactivity with NADH is observed. Rhodamines with increased alkylation on the N3- and N6-nitrogens, as well as the xanthene core, react the least with NADH; whereas, nonalkylated variants or analogues with electron-withdrawing substituents have the fastest rates of reaction. These outcomes allowed us to judiciously construct a seminaphthorhodamine-based, turn-on fluorescent probe that is capable of selectively detecting the cancer-associated, NADH-dependent enzyme NAD(P)H:quinoneoxidoreductase isozyme-1 in human cancer cells, without the issue of NADH-induced deactivation of the seminaphthorhodamine reporter.