Anthracene-BODIPY Dyads as Fluorescent Sensors for Biocatalytic Diels-Alder Reactions Alexander Nierth, † Andrei Yu. Kobitski, ‡ G. Ulrich Nienhaus, ‡,§ and Andres Ja ¨ schke* ,† Institute of Pharmacy and Molecular Biotechnology, Heidelberg UniVersity, Im Neuenheimer Feld 364, Heidelberg 69120, Germany, Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology, Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany, and Department of Physics, UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received October 4, 2009; E-mail: jaeschke@uni-hd.de Abstract: 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 ∼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. Introduction Fluorescent reporter molecules have become indispensable tools in all areas of science and have contributed significantly to our understanding of chemical and biological systems. Ideally, the fluorescence emission properties of the sensor change upon binding, reaction, or environmental changes in a characteristic manner, thereby generating an optical output signal. Different chemosensors have been developed that are sensitive to pH, 1,2 solvent polarity, 3 heavy-metal ions, 4 and small molecules. 5 In biology, fluorescent probes enable a vast range of experimental techniques, from live cell imaging to mechanistic studies at the single-molecule level. 6-8 Fluorescent probes have also permitted investigations of the catalytic mechanisms of various enzymes. 9,10 Ideally, the probe’s fluorescence properties should allow one to distinguish between free substrate, bound substrate, bound product, and free product to observe the fundamental species of a catalytic cycle. Progress in single-molecule enzymology is, however, restricted by the paucity of substrate-analogue probes with suitable properties. 11 Whereas fluorescence labeling is frequently straightforward for macromolecular substrates of enzymes such as proteases or nucleases, it is challenging or even entirely impossible for many small-molecule substrates. Our laboratory has a long-standing interest in catalysis by ribonucleic acids (ribozymes). We have discoveredsby com- binatorial chemistrysribozymes that catalyze C-C bond forma- tion by Diels-Alder reaction between two small-molecule substrates, namely, anthracenes as dienes and N-substituted maleimides as dienophiles (Figure 1A). 12 These ribozymes show multiple turnovers and saturation behavior and have been † Heidelberg University. ‡ Karlsruhe Institute of Technology. § University of Illinois at Urbana-Champaign. (1) Han, J.; Loudet, A.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. J. Am. Chem. Soc. 2009, 131, 1642–1643. (2) Baruah, M.; Qin, W. W.; Basaric, N.; De Borggraeve, W. M.; Boens, N. J. Org. Chem. 2005, 70, 4152–4157. (3) Sunahara, H.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 5597–5604. (4) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447–13454. (5) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. 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