Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg 69120, Germany.
J Am Chem Soc. 2010 Mar 3;132(8):2646-54. doi: 10.1021/ja9084397.
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 approximately 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.
荧光光谱学是一种强大的、极其灵敏的技术，可用于研究酶和核酶的机制。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 荧光团通过共轭的苯乙炔基桥融合而成。完整的蒽能有效地猝灭 BODIPY 荧光，可能是通过光诱导电子转移。当芳香体系被 Diels-Alder 反应破坏时，荧光发射强度增加 20 倍。Diels-Alderase 核酶在催化口袋中的结合导致两种探针（蒽-BODIPY 和 Diels-Alder-产物-BODIPY）的荧光强度进一步增加约 2 倍。因此，可以通过荧光来区分游离底物、结合底物、结合产物和游离产物。使用这些一体式报告分子，我们在单轮和多轮条件下监测了 RNA 催化的 Diels-Alder 反应，浓度低至纳摩尔范围。在单分子水平上的爆发分析显示，二联体在开态和关态之间发生闪烁，可能是由于苯乙炔基桥的旋转。与核酶结合不会增加单个荧光爆发的强度，而是增加处于开态的平均时间。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很好地相关。
