A Synthetically Simple, Click-Generated Cyclam-Based Zinc(II) Sensor Emiliano Tamanini,��� Arna Katewa,���,�� Lisa M. Sedger,���,�� Matthew H. Todd,*,| and Michael Watkinson*,��� The Walter Besant Building, School of Biological and Chemical Sciences, Queen Mary, UniVersity of London, Mile End Road, London, E1 4NS, United Kingdom, Institute for Immunology & Allergy Research, Westmead Millennium Institute, The UniVersity of Sydney, NSW 2006, Australia, Institute for the Biotechnology of Infectious Diseases and Department of Medical and Molecular Biosciences, The UniVersity of Technology, Sydney, Sydney, NSW 2007, Australia, and School of Chemistry, UniVersity of Sydney, NSW 2006, Australia Received September 15, 2008 A cyclam-based macrocyclic sensor has been prepared using synthetically simple ���click��� chemistry to link a fluorophore to the macrocyclic receptor. This sensor shows high selectivity for Zn(II) over a range of other metals, providing a significant enhancement of fluorescence intensity over a wide pH range. As such, this is the first cyclam-based sensor demonstrated to be selective for Zn(II) and is the first example of a triazole being used as a coordinating ligand on an azamacrocycle. The sensor can access biologically available zinc in mammalian cells, sensing the Zn(II) flux that exists during apoptotic cell death. Introduction Zinc is the second most abundant d-block metal in the human body, playing a critical role in enzyme regulation, structure and function, neural signal transmission, and gene expression.1 While most zinc is tightly bound in proteins, ���mobile��� pools of zinc exist in certain mammalian organs such as the brain and pancreas, which are carefully regu- lated,2 the disruption of which is associated with a number of disease states including types I and II diabetes, Parkinson���s disease, epilepsy, and certain cancers.2a,3 Moreover zinc is now recognized as an important factor in the regulation of apoptosis.4 Given that the d10 electronic configuration of Zn(II) renders it spectroscopically silent, the majority of recent efforts to develop effective small molecule sensors for Zn(II) have focused on the retardation of photoinduced electron transfer (PET) developed by de Silva, in which metal binding occurs at a site in close proximity to an appropriate fluorophore and switches on fluorescence.5 To date, a great many sensors have been reported,6 perhaps most notably by Lippard et al.7 There continues to be considerable interest * Authors to whom correspondence should be addressed. E-mail: m.todd@chem.usyd.edu.au (M.H.T.) m.watkinson@qmul.ac.uk (M.W.). ��� Queen Mary, University of London. ��� Westmead Millennium Institute, The University of Sydney. �� Institute for the Biotechnology of Infectious Diseases, The University of Technology, Sydney. | School of Chemistry, University of Sydney. (1) (a) O���Halloran, T. V. Science 1993, 261, 715���725. (b) Falchuk, K. H. Mol. Cell. Biochem. 1998, 188, 41���48. (c) Jiang, P. Coord. Chem. ReV. 2004, 248, 205���229. (d) Maret, W. Biometals 2001, 14, 187��� 190. whole issue. (e) Burdette, S. C. Lippard, S. J. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 3605���3610. (2) (a) Hambidge, M. Cousins, R. J. Costello, R. B J. Nutr. 2000, 130, 1341S���1343S whole supplement. (b) Ugarte, M. Osborne, N. N. Prog. Neurobiol. 2001, 64, 219���249. (c) Taylor, C. G. Biometals 2005, 18, 305���312. (d) Costello, L. C. Franklin, R. B. Feng, P. Tan, M. Bagasra, O. Cancer Cause Control 2005, 16, 901���915. (3) (a) Chausmer, A. B. J. Am. Coll. Nutr. 1998, 17, 109���115. (b) Smith, J. L. Xiong, S. Markesbery, W. R. Lovell, M. A. Neuroscience 2006, 140, 879���888. (c) Ho, E. J. Nutr. Biochem. 2004, 15, 572���578. (d) Sladek, R. Nature 2007, 445, 881���885. (e) Lu, M. Fu, D. Science 2007, 317, 1746���1748. (4) Kimura, E. Aoki, S. Kikuta, E. Koike, T. Proc. Natl. Acad. Sci., U. S. A. 2003, 100, 3731���3736, and references cited therein. (5) (a) de Silva, A. P. de Silva, S. A. Chem. Commun. 1986, 1709���1710. (b) de Silva, A. P. Gunaratne, H. Q. N. Gunnlaugsson, T. Huxley, A. J. M. McCoy, C. P. Riademacher, J. T. Rice, T. E. Chem. ReV. 1997, 97, 1515���1566. (c) Valeur, B. Leray, I. Coord. Chem. ReV. 2000, 205, 3���40. (d) Kimura, E. Koike, T. Chem. Soc. ReV 1998, 27, 179���184. (e) Moore, E. G. Bernhardt, P. V. Fu ��rstenberg, A. Riley, M. J. Smith, T. A. Vauthey, E. J. Phys. Chem. A 2005, 109, 3788��� 3796. (f) Bergonzi, R. Fabbrizzi, L. Licchelli, M. Mangano, C. Coord. Chem. ReV. 1998, 170, 31���46. (6) Reviews: (a) Gunnlaugsson, T. Glynn, M. Tocci, G. M. Kruger, P. E. Pfeffer, F. M. Coord. Chem. ReV. 2006, 250, 3094���3117. (b) Callan, J. F. de Silva, A. P. Magri, D. C. Tetrahedron 2005, 61, 8551���8588. (c) Carol, P. Sreejith, S. Ajayaghosh, A. Chem. Asian J. 2007, 2, 338���348. (d) Kikuchi, K. Komatsu, K. Nagano, T. Curr. Opin. Chem. Biol. 2004, 8, 182���191. (e) Lim, N. C. Freake, H. C. Bru ��ckner, C. Chem.sEur. J. 2005, 11, 38���49. Selected examples: (f) Parkesh, R. Lee, T. C. Gunnlaugsson, T. Org. Biomol. Chem. 2007, 5, 310���317. (g) Wang, J. Xiao, Y. Zhang, Z. Qian, X. Yang, Y. Xu, Q. J. Mater. Chem. 2005, 15, 2836���2839. (h) Huang, S. Clark, R. J. Zhu, L. Org. Lett. 2007, 9, 4999���5002. (i) Liu, Y. Zhang, N. Chen, Y. Wang, L.-H. Org. Lett. 2007, 9, 315���318. (j) Bencini, A. Dalton Trans. 2004, 2180���2187. Inorg. Chem. 2009, 48, 319-324 10.1021/ic8017634 CCC: $40.75 ��� 2009 American Chemical Society Inorganic Chemistry, Vol. 48, No. 1, 2009 319 Published on Web 12/03/2008

in the development of more effective analogues to further our understanding of the plethora of in vivo settings in which zinc is known to play a crucial role.8 Despite the extensive literature of cyclen-based sensor agents,4,6c,9 rather surprisingly, there are very few examples of analogous small molecule cyclam-based fluorescent sen- sors, and these have not been shown to display selectivity for Zn(II).5e,10 We have recently become interested in the incorporation of biological binding motifs into azamacro- cyclic scaffolds,11 in particular, the use of ���click��� chemistry12 to construct sensors based on this design. We have demon- strated that the triazole resulting from the click reaction is a competent coordinating motif for ���scorpion-like��� com- plexes,10a,13 and that perturbation of the interaction between the triazole donor and the metal can act as a novel means of detecting analyte binding spectroscopically.11b This approach seemed promising for the development of a PET Zn(II) sensor. The triazole here would resemble histidine ligands that strongly bind zinc ions in a range of enzymes such as carbonic anhydrase.14 Experimental Section General Information. All reagents were purchased from Sigma- Aldrich and used without further purification. All reactions were carried out using commercial-grade solvents. 1,4,8-Triboc-cyclam and compound 2 were prepared as described in the literature.15-17 Silica gel chromatography was carried out using BDH silica gel for flash chromatography as the stationary phase and EtOAc/Pet. Spirit 30-40 ��C as eluents. 1H and 13C NMR spectra were recorded in CDCl3, CD3CN, or DMSO-d6 with a Jeol 270 MHz spectrometer. 1H chemical shifts are reported in parts per million relative to the residual proton signal of the deuterated solvents. Coupling constants (J) are reported in Hertz. 13C chemical shifts are reported in parts per million relative to the carbon signal of the solvents. IR spectra were recorded with a Shimadzu FTIR-8300. Fluorescence emission spectra were recorded with a Jobin Yvon Horiba FluoroMax-3 in a 1-cm-path- length cell. Electrospray ionization mass spectra were obtained from the EPSRC National Mass Spectrometry Service, University of Wales, Swansea, using either a Waters ZQ400 or a Micromass Quattro II. Accurate masses were recorded with either a Finnigan MAT900 or MAT 95 using polyethylenimine as the reference. 11-Prop-2-ynyl-1,4,8,11-tetraaza-cyclotetradecane-1,4,8-tri- carboxylic Acid Tri-tert-butyl Ester (1). To a solution of 1,4,8- triboc-cyclam15 (250 mg, 0.5 mmol) in CH3CN (15 mL) were added Na2CO3 (1 mmol, 212 mg) and propargyl bromide (0.6 mmol, 67 ��L), and the mixture was stirred at reflux (85 ��C) overnight. The insoluble salts were filtered and the solvent removed in vacuo. The crude material was purified by flash chromatography on silica gel (EtOAc/Pet. Spirit 7:3) to give 1 as a white solid (211 mg, 78% yield): mp 47-49 ��C. ��max (CH2Cl2)/cm-1: 3301, 2135, 1685. 1H NMR (CDCl3, 270 MHz): �� 3.48-3.15 (m, 14H), 2.71-2.58 (m, 2H), 2.49 (t, J ) 5.4 Hz, 2H), 2.14 (s, 1H), 1.98-1.77 (m, 2H), 1.76-1.60 (m, 2H), 1.44 (s, 27H). 13C NMR (CDCl3, 67.5 MHz): �� 155.8, 155.5, 79.6, 79.5, 77.9, 73.2, 53.0, 51.9, 50.7, 48.0, 47.5, 46.9, 46.7, 44.8, 41.9, 28.5, 25.5. MS (ESI): m/z 539 [(M + H)+]. HRMS (ES) calcd. for C28H51N4O6 (M + H)+: 539.3803. Found: 539.3800. 6-Azido-2-ethyl-benzo[de]isoquinoline-1,3-dione (2).16,17 4-Bro- mo-1,8-naphthalic anhydride (500 mg, 1.8 mmol) and ethyl amine (70% solution in H2O, 172 ��L, 2.2 mmol) were refluxed in dioxane (30 mL) for 7 h. The solution was cooled to room temperature and poured into water to precipitate out a solid, which was collected by filtration, washed with water, and dried in vacuo to give a cream- colored solid (450 mg, 82%). A mixture of the solid so obtained (420 mg, 1.4 mmol) and sodium azide (449 mg, 6.9 mmol) in N-methylpyrrolidinone (6 mL) was heated at 110 ��C for 1.5 h. The reaction mixture was diluted with water and extracted with EtOAc (3 �� 20 mL). The organic phase was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (EtOAc/Pet. Spirit 4:6) to give the desired product (2) as a dark brown solid (182 mg, 50%). Spectroscopic data were identical to those reported in the literature.16,17 11-[1-(2-Ethyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquino- lin-6-yl)-1H-[1,2,3]triazol-4-ylmethyl]-1,4,8,11tetraaza-cyclotet- radecane-1,4,8-tricarboxylic Acid Tri-tert-butyl Ester (3). To a solution of 1 (269 mg, 0.5 mmol) in THF/H2O (7/3, 10 mL) were added 2 (133 mg, 0.5 mmol), CuSO4 �� 5H2O (5 mol%, 6.2 mg, 0.025 mmol), and sodium ascorbate (10 mol %, 9.9 mg, 0.05 mmol) under N2. The solution was stirred at room temperature overnight. Saturated NH4Cl was added (20 mL), and THF was evaporated in vacuo. The aqueous phase was extracted with DCM (3 �� 20 mL) the organic phase was dried (MgSO4), filtered, and concentrated in vacuo. The crude material was filtered through a short plug of silica (EtOAc/Pet. Spirit 1:1) to give 3 as a yellow solid (195 mg, 82%): mp ) 91-93 ��C. ��max (CH2Cl2)/cm-1: 3055, 1662, 1647. 1H NMR (CDCl3, 270 MHz): �� 8.76-8.66 (m, 2H), 8.30-8.22 (m, 1H), 8.02-7.80 (m, 3H), 4.27 (q, J ) 7.0 Hz, 2H), 3.94 (s, 2H), 3.48-3.24 (m, 12H), 2.78-2.68 (m, 2H), 2.62-2.52 (m, 2H), 1.96-1.84 (m, 2H), 1.84-1.72 (m, 2H), 1.48-1.30 (m, 30H). 13C NMR (CDCl3, 67.5 MHz): �� 163.3, 162.8, 155.8, 155.5, 144.5, 138.1, 131.9, 130.6, 129.5, 128.9, 128.5, 126.3, 125.1, 123.7, 123.5, (7) See for example: (a) Zhang, X. Lovejoy, K. S. Jasanoff, A. Lippard, S. J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 10780���10785. (b) Nolan, E. M. Jaworski, J. Okamoto, K. I. Hayashi, Y. Sheng, M. Lippard, S. J. J. Am. Chem. Soc. 2005, 127, 16812���16823. (8) Domaille, D. W. Que, E. L. Chang, C. J. Nat. Chem. Biol. 2008, 4, 168���175. (9) (a) Akkaya, E. U. Huston, M. E. Czarnik, A. W. J. Am. Chem. Soc. 1990, 112, 3590���3593. (b) Koike, T. Watanabe, T. Aoki, S. Kimura, E. Shiro, M. J. Am. Chem. 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