Anthracene-BODIPY Dyads as Fluorescent Sensors for Biocatalytic Diels-Alder Reactions
Anthracene-BODIPY Dyads as Fluorescent Sensors for Biocatalytic Diels-Alder Reactions
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DOI：
10.1021/ja9084397
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发表时间：
2010-03-03
影响因子：
15
通讯作者：
Jaeschke, Andres
中科院分区：
文献类型：
--
作者：
Nierth, Alexander;Kobitski, Andrei Yu.;Jaeschke, Andres
荧光光谱是一种功能强大、灵敏度极高的技术，可用于研究酶和核酶的作用机制。在此，我们描述了用于研究生物催化Diels-Alder反应的水溶性荧光探针的合成和表征。这些探针由蒽和磺化BODIPY荧光团通过共轭苯乙炔桥融合组成。完整的蒽有效地淬灭BODIPY荧光，可能是通过光诱导的电子转移。在通过Diels-Alder反应破坏芳族体系时，荧光发射增加20倍。在Diels-Alderase核酶的催化口袋中的结合产生蒽-BODIPY和Diels-Alder-产物-BODIPY探针两者的荧光强度的进一步类似于2倍的增加。因此，基于荧光区分游离底物、结合底物、结合产物和游离产物是可能的。有了这些一体化的报告，我们监测RNA催化的狄尔斯-阿尔德反应在单一和多个营业额的条件下下降到纳摩尔浓度范围。在单分子水平上的爆发分析揭示了在开状态和关状态之间的二元体的闪烁，推测是由于围绕苯乙炔基桥的旋转。与核酶结合不会增加单个荧光猝发的强度，而是增加了处于开启状态的平均时间。不同探针的量子产率的变化与蒽和苯乙炔基桥之间的共轭程度密切相关。
Fluorescence spectroscopy is a powerful, extremely sensitive technique for the investigation of enzyme and ribozyme mechanisms. Herein, we describe the synthesis and characterization of water-soluble fluorescence probes for studying biocatalytic Diels-Alder reactions. These probes consist of anthracene and sulfonated BODIPY fluorophores fused by conjugated phenylacetylenyl bridges. Intact anthracene efficiently quenches BODIPY fluorescence, likely by photoinduced electron transfer. Upon destruction of the aromatic system by the Diels-Alder reaction, the fluorescence emission increases 20-fold. Binding in the catalytic pocket of a Diels-Alderase ribozyme yields a further similar to 2-fold increase in the fluorescence intensity of both the anthracene-BODIPY and the Diels-Alder-product-BODIPY probes. Therefore, a fluorescence-based distinction of free substrate, bound substrate, bound product, and free product is possible. With these all-in-one reporters, we monitored RNA-catalyzed Diels-Alder reactions under both single- and multiple-turnover conditions down to the nanomolar concentration range. Burst analysis at the single-molecule level revealed blinking of the dyads between an on state and an off state, presumably due to rotation around the phenylacetylenyl bridge. Binding to the ribozyme does not increase the intensity of the individual fluorescence bursts, but rather increases the average time spent in the on state. Variations in the quantum yields of the different probes correlate well with the degree of conjugation between anthracene and the phenylacetylenyl bridge.