Substrate-Selective Supramolecular Tandem Assays: Monitoring Enzyme Inhibition of Arginase and Diamine Oxidase by Fluorescent Dye Displacement from Calixarene and Cucurbituril Macrocycles Werner M. Nau,* Garima Ghale, Andreas Hennig, Hu ¨ seyin Bakirci, and David M. Bailey School of Engineering and Science, Jacobs UniVersity Bremen, Campus Ring 1, D-28759 Bremen, Germany Received May 22, 2009; E-mail: w.nau@jacobs-university.de Abstract: A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays (“supramolecular tandem assays”) exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products (“substrate-selective assays”). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M -1 with arginine, 550 M -1 with ornithine, and 60 000 M -1 with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 × 10 6 M -1 with cadaverine, 1.1 × 10 5 M -1 with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 × 10 5 M -1 with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a “product-selective” with a “substrate-selective” assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This “domino tandem assay” was performed in the same solution with a single reporter pair (CB7/AO). Introduction The monitoring of enzymatic processes is of fundamental importance for the understanding of biological phenomena. 1 Inspired by indicator displacement and synthetic pore strate- gies, 2-13 we have recently introduced a label-free method based on the competitive encapsulation of a fluorescent dye and an enzymatic product by macrocyclic hosts to monitor enzymatic reactions (Scheme 1). 14-16 In our previous examples, the investigated enzymes transformed a weak competitor (substrate) into a strong competitor (product) which displaced the fluores- (1) Reymond, J.-L.; Fluxa `, V. S.; Maillard, N. Chem. Commun. 2009, 34–46. (2) Inouye, M.; Hashimoto, K.; Isagawa, K. J. Am. Chem. Soc. 1994, 116, 5517–5518. (3) Koh, K. N.; Araki, K.; Ikeda, A.; Otsuka, H.; Shinkai, S. J. Am. Chem. Soc. 1996, 118, 755–758. (4) Niikura, K.; Anslyn, E. V. J. Chem. Soc., Perkin Trans. 2 1999, 2769– 2775. (5) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963–972. (6) Nguyen, B. T.; Anslyn, E. V. Coord. Chem. ReV. 2006, 250, 3118– 3127. (7) Zhang, T.; Anslyn, E. V. Org. Lett. 2007, 9, 1627–1629. (8) Zhu, L.; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, 3676–3677. (9) Das, G.; Talukdar, P.; Matile, S. Science 2002, 298, 1600–1602. (10) Sorde, N.; Das, G.; Matile, S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11964–11969. (11) Litvinchuk, S.; Tanaka, H.; Miyatake, T.; Pasini, D.; Tanaka, T.; Bollot, G.; Mareda, J.; Matile, S. Nat. Mater. 2007, 6, 576–580. (12) Mora, F.; Tran, D.-H.; Oudry, N.; Hopfgartner, G.; Jeannerat, D.; Sakai, N.; Matile, S. Chem.sEur. J. 2008, 14, 1947–1953. (13) Sakai, N.; Mareda, J.; Matile, S. Acc. Chem. Res. 2008, 41, 1354– 1365. (14) Hennig, A.; Bakirci, H.; Nau, W. M. Nat. Methods 2007, 4, 629– 632. (15) Bailey, D. M.; Hennig, A.; Uzunova, V. D.; Nau, W. M. Chem.sEur. J. 2008, 14, 6069–6077. (16) Praetorius, A.; Bailey, D. M.; Schwarzlose, T.; Nau, W. M. Org. Lett. 2008, 10, 4089–4092. Published on Web 07/23/2009 10.1021/ja904165c CCC: $40.75  2009 American Chemical Society 11558 9 J. AM. CHEM. SOC. 2009, 131, 11558–11570