Development of Luminescent Coelenterazine Derivatives Activatable by β-Galactosidase for Monitoring Dual Gene Expression
Development of Luminescent Coelenterazine Derivatives Activatable by β-Galactosidase for Monitoring Dual Gene Expression
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DOI：
10.1002/chem.201302002
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发表时间：
2013-10-25
影响因子：
4.3
通讯作者：
Kikuchi, Kazuya
中科院分区：
文献类型：
--
作者：
Lindberg, Eric;Mizukami, Shin;Kikuchi, Kazuya
设计并合成了两种生物发光笼状腔肠素衍生物（bGalCoel和bGalNoCoel），以通过生物发光成像检测β-半乳糖苷酶活性和表达。我们的方法通过引入β-半乳糖笼化基团来阻断腔肠素的自氧化来解决腔肠素的不稳定性。两种探针均在咪唑并吡嗪酮部分的羰基处含有β-半乳糖苷酶可裂解的笼状基团。其中一种探针，特别是bGalNoCoel，显示出快速切割谱、高稳定性和对β-半乳糖苷酶的高特异性，优于其他糖苷水解酶。bGalN-oCoel可以检测表达突变型Gaussia荧光素酶的活HEK-293 T细胞培养物中的β-半乳糖苷酶活性。已确定腔肠素在通过β-半乳糖苷酶解除包裹后容易扩散进出细胞。我们发现，这种新的笼状腔肠素衍生物bGalNoCoel可以作为双酶底物，并检测两个不同细胞群的酶活性。
Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect beta-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing beta-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain beta-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for beta-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect beta-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by beta-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations.