Novel Luminescent Tricarbonylrhenium(I) Polypyridine Tyramine

Organometallics 2009, 28, 4297–4307 DOI: 10.1021/om900311s

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Novel Luminescent Tricarbonylrhenium(I) Polypyridine TyramineDerived Dipicolylamine Complexes as Sensors for Zinc(II) and Cadmium(II) Ions Man-Wai Louie, Hua-Wei Liu, Marco Ho-Chuen Lam, Tai-Chu Lau, and Kenneth Kam-Wing Lo* Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, People’s Republic of China Received April 23, 2009

Three luminescent tricarbonylrhenium(I) polypyridine complexes containing a tyramine-derived 2,20 -dipicolylamine (DPAT) unit, [Re(N∧N)(CO)3(py-TU-DPAT)](CF3SO3) (py-TU-DPAT = 3-(2(4-hydroxy-3-(2,20 -dipicolylaminomethyl)phenyl)ethylthioureidyl)pyridine; N∧N=1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3a)), and their DPAT-free counterparts, [Re(N∧N)(CO)3(py-TU-Et)](CF3SO3) (py-TU-Et= 3-(ethylthioureidyl)pyridine; N∧N=phen (1b), Me4-phen (2b), Ph2-phen (3b)), have been synthesized and characterized. Their electrochemical and photophysical properties have been studied. Upon photoexcitation, all the complexes exhibited triplet metal-to-ligand charge-transfer (3MLCT) (dπ(Re) f π*(N∧N)) emission in fluid solutions at 298 K and in low-temperature alcohol glass. The DPAT complexes showed lower emission quantum yields and shorter emission lifetimes compared to those of the DPATfree analogues, indicative of the quenching properties of the appended DPAT unit. The DPAT complexes also exhibited pH-dependent emission, with their emission intensities at pH < 3 being ca. 40 fold higher than those at pH > 11. These complexes displayed emission enhancement and lifetime elongation in the presence of zinc(II) and cadmium(II) ions. The cellular uptake of all the complexes by human cervix epithelioid carcinoma (HeLa) cells has been examined by ICP-MS. We have investigated the cytotoxicity of the complexes by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the results revealed that all the complexes were more cytotoxic than cisplatin. Furthermore, the cellular uptake of complexes 3a and 3b and the intracellular ion-binding properties of the former complex have been studied by laser-scanning confocal microscopy. Introduction Zinc is an essential metal in many cellular processes.1 In particular, divalent zinc participates in many important biological controls such as gene expressions,2 neurotransmission,3 and bioinorganic catalysis.4 However, a too high level of zinc is cytotoxic and may lead to skin diseases,5 diabetes,6 and prostatic adenocarcinoma.7 The wide industrial applications of cadmium, for example, in batteries and pigment products, have become a potential threat to living organisms in the environment. Also, cadmium contamination has been reported to cause human lung, prostatic, and renal cancers.8

Thus, it is highly important to monitor the levels of these two ions in biological and environmental systems. The development of molecular probes for zinc(II)9 and cadmium(II)10 ions has relied on the use of fluorescent organic dyes9,10 and luminescent lanthanide chelates11 as the reporters. A number of sensors for these ions have utilized 2,20 -dipicolylamine (DPA) as

*To whom correspondence should be addressed. E-mail: bhkenlo @cityu.edu.hk. Fax: (852) 2788 7406. Tel: (852) 2788 7231. (1) Vallee, B. L.; Falchuk, K. H. Physiol. Rev. 1993, 73, 79–118. (2) Falchuk, K. H. Mol. Cell. Biochem. 1998, 188, 41–48. (3) (a) Cuajungco, M. P.; Lees, G. J. Neurobiol. Dis. 1997, 4, 137–169. (b) Choi, D. W.; Koh, J.-Y. Annu. Rev. Neurosci. 1998, 21, 347–375. (4) Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry; University Science Books: Mill Valley, 1994. (5) Bush, A. I. Trends Neurosci. 2003, 26, 207–214. (6) Chausmer, A. B. J. Am. Coll. Nutr. 1998, 17, 109–115. (7) Sorensen, M. B.; Stoltenberg, M.; Juhl, S.; Danscher, G.; Ernst, E. Prostate 1997, 31, 125–130. (8) (a) Goyer, R. A.; Liu, J.; Waalkes, M. P. BioMetals 2004, 17, 555– 558. (b) Satarug, S.; Baker, J. R.; Urbenjapol, S.; Haswell-Elkins, M.; Reilly, P. E. B.; Williams, D. J.; Moore, M. R. Toxicol. Lett. 2003, 137, 65–83.

(9) (a) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2000, 122, 12399–12400. (b) Walkup, G. K.; Burdette, S. C.; Lippard, S. J.; Tsien, R. Y. J. Am. Chem. Soc. 2000, 122, 5644–5645. (c) Burdette, S. C.; Walkup, G. K.; Spingler, B.; Tsien, R. Y.; Lippard, S. J. J. Am. Chem. Soc. 2001, 123, 7831–7841. (d) Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1778–1787. (e) Zhang, X.; Lovejoy, K. S.; Jasanoff, A.; Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10780–10785. (f) Dai, Z.; Canary, J. W. New J. Chem. 2007, 31, 1708–1718. (g) Nolan, E. M.; Lippard, S. J. Acc. Chem. Res. 2009, 42, 193–203. (10) (a) Lu, C.; Xu, Z.; Cui, J.; Zhang, R.; Qian, X. J. Org. Chem. 2007, 72, 3554–3557. (b) Peng, X.; Du, J.; Fan, J.; Wang, J.; Wu, Y.; Zhao, J.; Sun, S.; Xu, T. J. Am. Chem. Soc. 2007, 129, 1500–1501. (c) Cockrell, G. M.; Zhang, G.; VanDerveer, D. G.; Thummel, R. P.; Hancock, R. D. J. Am. Chem. Soc. 2008, 130, 1420–1430. (d) Taki, M.; Desaki, M.; Ojida, A.; Iyoshi, S.; Hirayama, T.; Hamachi, I.; Yamamoto, Y. J. Am. Chem. Soc. 2008, 130, 12564–12565. (e) Cheng, T.; Xu, Y.; Zhang, S.; Zhu, W.; Qian, X.; Duan, L. J. Am. Chem. Soc. 2008, 130, 16160–16161. (11) (a) Hanaoka, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 12470–12476. (b) Pope, S. J. A.; Laye, R. H. Dalton Trans. 2006, 3108–3113. (c) Song, X.; Dou, W.; Liu, W.; Yao, J.; Guo, Y.; Tang, X. Inorg. Chem. Commun. 2007, 10, 1058–1062.

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the recognition unit.9a-9e,10a,10d,11a,11b Despite these reports, the possibility of using luminescent transition metal complexes as both in vitro and in vivo zinc(II) and cadmium(II) ion sensors, to the best of our knowledge, has not been explored.12 Luminescent metal complexes are attractive candidates because of their intense, long-lived, and environment-sensitive emission with large Stokes’ shifts. Functionalization of luminescent tricarbonylrhenium(I) polypyridine complexes for biological applications has been attracting much attention because this type of complexes not only possess interesting emission properties13-26 but can also be effectively internalized by cancer cell lines,22-24,26j highlighting their potential as live-cell staining reagents and cellular probes for biological molecules and ions. This, together with our recent interest in designing luminescent transition metal polypyridine complexes as biological labels and (12) Luminescent supramolecular species composed of transition metal complexes and zinc(II) ions have been reported. See, for example: (a) Barigelletti, F.; Flamigni, L.; Calogero, G.; Hammarstr€ om, L.; Sauvage, J.-P.; Collin, J.-P. Chem. Commun. 1998, 2333–2334. (b) Loiseau, F.; Passalacqua, R.; Campagna, S.; Polson, M. I. J.; Fang, Y.; Hanan, G. S. Photochem. Photobiol. Sci. 2002, 1, 982–990. (13) (a) Wrighton, M. S.; Morse, D. L. J. Am. Chem. Soc. 1974, 96, 998–1003. (b) Fredericks, S. M.; Luong, J. C.; Wrighton, M. S. J. Am. Chem. Soc. 1979, 101, 7415–7417. (14) (a) Connick, W. B.; Di Bilio, A. J.; Hill, M. G.; Winkler, J. R.; Gray, H. B. Inorg. Chim. Acta 1995, 240, 169–173. (b) Di Bilio, A. J.; Crane, B. R.; Wehbi, W. A.; Kiser, C. N.; Abu-Omar, M. M.; Carlos, R. M.; Richards, J. H.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2001, 123, 3181–3182. (c) Wenger, O. S.; Henling, L. M.; Day, M. W.; Winkler, J. R.; Gray, H. B. Inorg. Chem. 2004, 43, 2043–2048. (d) Dunn, A. R.; BellistonBittner, W.; Winkler, J. R.; Getzoff, E. D.; Stuehr, D. J.; Gray, H. B. J. Am. Chem. Soc. 2005, 127, 5169–5173. (15) (a) Yam, V. W.-W.; Lau, V. C.-Y.; Wu, L.-X. J. Chem. Soc., Dalton Trans. 1998, 1461–1468. (b) Lam, S. C.-F.; Yam, V. W.-W.; Wong, K. M.-C.; Cheng, E. C.-C.; Zhu, N. Organometallics 2005, 24, 4298–4305. (16) (a) Shen, Y.; Maliwal, B. P.; Lakowicz, J. R. J. Fluoresc. 2001, 11, 315–318. (b) Kusba, J.; Li, L.; Gryczynski, I.; Piszczek, G.; Johnson, M.; Lakowicz, J. R. Biophys. J. 2002, 82, 1358–1372. (17) (a) Busby, M.; Gabrielsson, A.; Matousek, P.; Towrie, M.; Di Bilio, A. J.; Gray, H. B.; Vlcek, A., Jr. Inorg. Chem. 2004, 43, 4994–5002. (b) Gabrielsson, A.; Matousek, P.; Towrie, M.; Hartl, F.; Zali, S.; Vlcek, A., Jr. J. Phys. Chem. A 2005, 109, 6147–6153. (c) Blanco-Rodríguez, A. M.; Busby, M.; Grdinaru, C.; Crane, B. R.; Di Bilio, A. J.; Matousek, P.; Towrie, M.; Leigh, B. S.; Richards, J. H.; Vlcek, A., Jr.; Gray, H. B. J. Am. Chem. Soc. 2006, 128, 4365–4370. (18) (a) Wallace, L.; Rillema, D. P. Inorg. Chem. 1993, 32, 3836–3843. (b) Wallace, L.; Jackman, D. C.; Rillema, D. P.; Merkert, J. W. Inorg. Chem. 1995, 34, 5210–5214. (c) Villegas, J. M.; Stoyanov, S. R.; Huang, W.; Rillema, D. P. Dalton Trans. 2005, 1042–1051. (19) (a) Zipp, A. P.; Sacksteder, L.; Streich, J.; Cook, A.; Demas, J. N.; DeGraff, B. A. Inorg. Chem. 1993, 32, 5629–5632. (b) Sacksteder, L.; Lee, M.; Demas, J. N.; DeGraff, B. A. J. Am. Chem. Soc. 1993, 115, 8230– 8238. (c) Kneas, K. A.; Xu, W.; Demas, J. N.; Zipp, B. A.; DeGraff, A. P. J. Fluoresc. 1998, 8, 295–300. (20) (a) Shen, Y.; Sullivan, B. P. Inorg. Chem. 1995, 34, 6235–6236. (b) Smithback, J. L.; Helms, J. B.; Schutte, E.; Woessner, S. M.; Sullivan, B. P. Inorg. Chem. 2006, 45, 2163–2174. (21) (a) Lees, A. J. Chem. Rev. 1987, 87, 711–743. (b) Kotch, T. G.; Lees, A. J.; Fuerniss, S. J.; Papathomas, K. I. Chem. Mater. 1991, 3, 25–27. (c) Kotch, T. G.; Lees, A. J.; Fuerniss, S. J.; Papathomas, K. I. Inorg. Chem. 1991, 30, 4871–4874. (d) Kotch, T. G.; Lees, A. J.; Fuerniss, S. J.; Papathomas, K. I. Chem. Mater. 1992, 4, 675–683. (e) Kotch, T. G.; Lees, A. J.; Fuerniss, S. J.; Papathomas, K. I.; Snyder, R. W. Inorg. Chem. 1993, 32, 2570–2575. (f) Sun, S.-S.; Lees, A. J. Organometallics 2002, 21, 39–49. (22) Stephenson, K. A.; Banerjee, S. R.; Besanger, T.; Sogbein, O. O.; Levadala, M. K.; McFarlane, N.; Lemon, J. A.; Boreham, D. R.; Maresca, K. P.; Brennan, J. D.; Babich, J. W.; Zubieta, J.; Valliant, J. F. J. Am. Chem. Soc. 2004, 126, 8598–8599. (23) Amoroso, A. J.; Coogan, M. P.; Dunne, J. E.; FernandezMoreira, V.; Hess, J. B.; Hayes, A. J.; Lloyd, D.; Millet, C.; Pope, S. J. A.; Williams, C. Chem. Commun. 2007, 3066–3068. (24) Amoroso, A. J.; Arthur, R. J.; Coogan, M. P.; Court, J. B.; Fern andez-Moreira, V.; Hayes, A. J.; Lloyd, D.; Millet, C.; Pope, S. J. A. New J. Chem. 2008, 32, 1097–1102. (25) Reece, S. Y.; Nocera, D. G. J. Am. Chem. Soc. 2005, 127, 9448– 9458.

Louie et al.

cellular imaging reagents,26,27 has prompted us to develop luminescent cellular probes for metal ions. Herein, we report three luminescent tricarbonylrhenium(I) polypyridine complexes containing a tyramine-derived 2,20 -dipicolylamine (DPAT) unit, [Re(N∧N)(CO)3(py-TUDPAT)](CF3SO3) (py-TU-DPAT=3-(2-(4-hydroxy-3-(2,20 dipicolylaminomethyl)phenyl)ethylthioureidyl)pyridine; N∧N= 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3a)), and their DPAT-free counterparts [Re(N∧N)(CO)3(py-TU-Et)](CF3SO3) (py-TU-Et = 3-(ethylthioureidyl) pyridine; N∧N = phen (1b), Me4-phen (2b), Ph2-phen (3b)) (Chart 1). Their electrochemical and photophysical properties have been studied. We have investigated the proton- and ionbinding properties of the DPAT complexes by emission titrations. Additionally, the cellular uptake of all the complexes by human cervix epithelioid carcinoma (HeLa) cells has been examined by ICP-MS. The cytotoxicity of the complexes has been investigated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, we have studied the cellular uptake of complexes 3a and 3b and the intracellular ion-binding properties of the former complex by laser-scanning confocal microscopy.

Results and Discussion Synthesis. The synthetic route for DPA-Tyramine is shown in Scheme 1. The amine group of tyramine was protected using di-tert-butyl dicarbonate in MeOH to give Tyraminet BOC, which then underwent Mannich’s reaction with DPA and paraformaldehyde to give DPA-Tyramine-tBOC. This compound was then deprotected using TFA in CH2Cl2, yielding DPA-Tyramine (Scheme 1). The tricarbonylrhenium(I) polypyridine DPAT complexes 1a-3a were synthesized from the addition reaction of [Re(N∧N)(CO)3(py-3-NCS)] (CF3SO3)26a with DPA-Tyramine in acetone, whereas the DPAT-free complexes 1b-3b were prepared similarly using ethylamine instead of DPA-Tyramine. All the complexes were purified by column chromatography on alumina, followed by recrystallization from CH2Cl2/diethyl ether. They were characterized by 1H NMR spectroscopy, positive-ion ESI-MS, and IR spectroscopy and gave satisfactory microanalysis. Electrochemical Properties. The electrochemical properties of the complexes have been investigated by cyclic voltammetry, and the electrochemical data are summarized in (26) (a) Lo, K. K.-W.; Ng, D. C.-M.; Hui, W.-K.; Cheung, K.-K. J. Chem. Soc., Dalton Trans. 2001, 2634–2640. (b) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M.; Cheung, K.-K. Inorg. Chem. 2002, 41, 40–46. (c) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M. J. Am. Chem. Soc. 2002, 124, 9344–9345. (d) Lo, K. K.-W.; Tsang, K. H.-K.; Hui, W.-K.; Zhu, N. Chem. Commun. 2003, 21, 2704–2705. (e) Lo, K. K.-W.; Lau, J. S.-Y.; Fong, V. W.-Y. Organometallics 2004, 23, 1098–1106. (f) Lo, K. K.-W.; Hui, W.-K. Inorg. Chem. 2005, 44, 1992–2002. (g) Lo, K. K.-W.; Tsang, K. H.-K.; Hui, W.-K.; Zhu, N. Inorg. Chem. 2005, 44, 6100–6110. (h) Lo, K. K.-W.; Hui, W.-K.; Chung, C.-K.; Tsang, K. H.-K; Ng, D. C.-M.; Zhu, N.; Cheung, K.-K. Coord. Chem. Rev. 2005, 249, 1434–1450. (i) Lo, K. K.-W.; Tsang, K. H.-K.; Zhu, N. Organometallics 2006, 25, 3220–3227. (j) Lo, K. K.-W.; Louie, M.-W.; Sze, K.-S.; Lau, J. S.-Y. Inorg. Chem. 2008, 47, 602–611. (27) (a) Lo, K. K.-W.; Tsang, K. H.-K.; Sze, K.-S.; Chung, C.-K.; Lee, T. K.-M.; Zhang, K. Y.; Hui, W.-K.; Li, C.-K.; Lau, J. S.-Y.; Ng, D. C.-M.; Zhu, N. Coord. Chem. Rev. 2007, 251, 2292–2310. (b) Lo, K. K.-W.; Zhang, K. Y.; Leung, S.-K.; Tang, M.-C. Angew. Chem., Int. Ed. 2008, 47, 2213–2216. (c) Lo, K. K.-W.; Lee, P.-K.; Lau, J. S.-Y. Organometallics 2008, 27, 2998–3006. (d) Lau, J. S.-Y.; Lee, P.-K.; Tsang, K. H.-K.; Ng, C. H.-C.; Lam, Y.-W.; Cheng, S.-H.; Lo, K. K.-W. Inorg. Chem. 2009, 48, 708–719.

Article


Table 1. All the complexes displayed a quasi-reversible couple at ca. þ1.6 to þ1.8 V vs SCE, which has been assigned to a rhenium(II/I) oxidation couple.15,18a,18c,20b,26d,26e,26g,26i The irreversible waves of the DPAT complexes at ca. þ0.9 and þ1.1 V have been assigned to the oxidation of the DPAT moiety because DPA-Tyramine-tBOC and tyrosine derivatives also exhibited similar waves at comparable potentials.25 With reference to previous electrochemical studies of related tricarbonylrhenium(I) polypyridine systems,15,18a,18c,20b,26d,26e,26g,26i we have ascribed the first reduction waves of all the complexes (from -1.14 to -1.47 V) to the reduction of the diimine ligands. The first reduction of the Me4-phen complexes 2a and 2b occurred at a more negative potential (-1.47 and -1.42 V, respectively) than those of the phen complexes 1a and 1b and Ph2-phen complexes 3a and 3b (from -1.14 to -1.20 V), which is a consequence of the four electron-donating methyl groups that destabilize the π* orbitals of the Me4-phen ligand. Electronic Absorption Properties. The electronic absorption spectral data of the complexes are summarized in Table 2, and the electronic absorption spectrum of complex 1a in CH3CN at 298 K is shown in Figure 1. All the Chart 1. Structures of the Tricarbonylrhenium(I) Polypyridine Complexes

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complexes showed intense absorption bands at ca. 251330 nm with extinction coefficients on the order of 104 dm3 mol-1 cm-1. With reference to previous spectroscopic studies on related tricarbonylrhenium(I) polypyridine complexes,13,14a,14c,15-17,18a,18c,19,20b,21a,21b,21d-21f,22,26a-26g,26i,26j we have assigned these bands to spin-allowed intraligand (1IL) (π f π*) (diimine and pyridine ligands) transitions. The lower-energy absorption shoulders at ca. 368-397 nm, with smaller extinction coefficients, have been assigned to spinallowed metal-to-ligand charge-transfer (1MLCT) (dπ(Re) f π*(N∧N)) transitions. The absorption shoulders at ca. 328-342 nm of complexes 3a and 3b should have substantial 1 IL character because of the electron-withdrawing phenyl substituents that stabilize the π* orbitals of the Ph2-phen ligand. Photophysical Properties. Excitation of the complexes in fluid solutions at 298 K and in low-temperature alcohol glass gave rise to green to orange-yellow emission. The photophysical data are listed in Table 3. The emission spectra of complex 1a in CH3CN at 298 K and in EtOH/MeOH (4:1 v/v) at 77 K are shown in Figure 1. In fluid solutions at 298 K, all the complexes showed decreasing emission energy, quantum yields, and lifetimes from CH 2Cl2 to more polar CH3CN and MeOH (Table 3). This, together with the blue-shifts of the emission maxima upon cooling the samples to low temperature (Table 3), indicates that the emissive state is 3MLCT (dπ(Re) f π*(N∧N)) in nature.13,14,15a,16-18,19a,19b,20-24,26 Noteworthily, the structural features and long emission lifetimes of the Me4-phen complexes 2a and 2b in solutions at room temperature and in lowtemperature alcohol glass indicated that the emissive state of these complexes exhibited substantial 3IL (π f π*) (Me4-phen) character.18,19b,26e-26g,26j The unique photophysical properties of other Me4-phen complexes have been reported; for example, the emission lifetime of [Re(Me4-phen)(CO)3(py)]þ is insensitive to the solvent systems, and the temperature-dependent emission properties of this complex are very different from those of other Table 1. Electrochemical Data of the Tricarbonylrhenium(I) Polypyridine Complexesa complex

oxidation E1/2 or Ea/V

reduction E1/2 or Ec/V

þ0.85, þ1.05, þ1.77 -1.14,c -1.89,b -2.15,b -2.27b b c þ1.35, þ1.68 -1.15,c -1.94,c -2.26,c -2.37c þ0.85,b þ1.07,b þ1.71c -1.47,c -1.68,c -1.76,b -2.30c þ1.32,b þ1.63c -1.42,c -1.56,b -1.70,c -2.30b þ0.86,b þ1.10,b þ1.74c -1.20,c -1.37,c -1.41,c -2.10b þ1.42,b þ1.73c -1.16,c -1.32,c -1.51,c -2.02c a In CH3CN (0.1 mol dm-3 nBu4NPF6) at 298 K, glassy carbon electrode, sweep rate 100 mV s-1, all potentials versus SCE. b Irreversible waves. c Quasi-reversible waves. 1a 1b 2a 2b 3a 3b

Scheme 1. Synthesis of DPA-Tyramine

b

b

c

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Table 2. Electronic Absorption Spectral Data of the Tricarbonylrhenium(I) Polypyridine Complexes at 298 K complex

solvent

λabs/nm (ε/dm3 mol-1 cm-1)

1a

CH2Cl2

260 (47 335), 268 sh (45 565), 276 sh (41 218), 322 sh (8675), 374 sh (3775), 385 sh (3295) 256 (43 980), 269 sh (41 530), 270 sh (41 125), 319 sh (9125), 370 sh (3360), 380 sh (2880) 256 (50 765), 275 sh (48 790), 324 sh (10 030), 385 sh (4155) 253 (36 905), 273 sh (34 005), 327 sh (9240), 380 sh (3135) 254 (47 930), 280 (43 635), 324 sh (12 180), 372 sh (3450) 252 (46 105), 280 (39 755), 316 sh (14 450), 368 sh (3305) 254 (39 430), 281 (40 090), 329 sh (12 140), 374 sh (3420) 251 (34 960), 279 (33 220), 330 sh (9140), 371 sh (3090) 263 (45 690), 269 (44 460), 287 sh (43 215), 334 sh (14 675), 380 sh (7010) 259 (38 185), 266 (36 295), 287 sh (37 575), 328 sh (14 720), 382 sh (5790) 258 (37 475), 293 (40 465), 342 sh (13 545), 397 sh (5845) 256 (32 365), 290 (36 980), 338 sh (13 560), 393 sh (5370)

CH3CN 1b

CH2Cl2 CH3CN

2a

CH2Cl2 CH3CN

2b

CH2Cl2 CH3CN

3a

CH2Cl2 CH3CN

3b

CH2Cl2 CH3CN

Figure 1. Electronic absorption (solid line) and emission spectra of complex 1a in CH3CN at 298 K (dashed line) and in EtOH/MeOH (4:1 v/v) at 77 K (dotted line).

tricarbonylrhenium(I) polypyridine complexes.18b These have been ascribed to the contribution of a 3IL emissive state. In another study, theoretical calculations on the complexes [M(N∧N)(CO)4] (M=Cr, W; N∧N=phen, Me4-phen) showed that the LUMOs of the phen and Me4-phen complexes are of b1 and a2 symmetry, respectively.28 The exceptionally long emission lifetime of [W(Me4-phen)(CO)4] as compared to [W(phen)(CO)4] was attributed to the different contributions of individual orbital excitations to the excited states involved. The excitation spectra of all the complexes in CH2Cl2 were characterized by high-energy bands at ca. 300 nm and shoulders at ca. 375400 nm. The shoulders of the Ph2-phen complexes 3a and 3b occurred at slightly lower energy, ca. 400-420 nm. These features are in accordance with the electronic absorption spectra of the complexes. (28) Farrell, I. R.; Hartl, F.; Zalis, S.; Mahabiersing, T.; Vlcek, A., Jr. J. Chem. Soc., Dalton Trans. 2000, 4323–4331.

Louie et al. Table 3. Photophysical Data of the Tricarbonylrhenium(I) Polypyridine Complexes complex

medium (T/K)

λem/nma

τo/μsa

Φemb

530 1.29 0.023 545 1.03 0.015 543 0.54 0.010 475 sh, 493 16.67 534 1.99 0.23 1b 550 1.13 0.12 546 0.77 0.045 473 sh, 489 16.15 489 sh, 512 3.15 0.015 2a 490 sh, 515 2.51 0.011 491 sh, 518 1.57 0.0093 467 (max), 499, 196.49 (53%), 536 sh 56.25 (47%) 491 sh, 514 5.01 0.10 2b CH2Cl2 (298) 492 sh, 519 3.11 0.054 CH3CN (298) MeOH (298) 492 sh, 521 1.71 0.050 466 (max), 498, 231.90 (43%), glassc (77) 537 sh 83.74 (57%) 545 2.88 0.028 3a CH2Cl2 (298) 559 2.18 0.013 CH3CN (298) MeOH (298) 554 1.21 0.0085 504, 536 sh 36.24 glassc (77) 549 6.06 0.16 3b CH2Cl2 (298) 554 3.45 0.070 CH3CN (298) MeOH (298) 553 1.56 0.046 501, 544 sh 35.74 glassc (77) a λex = 355 nm, [Re] = 50 μM. b λex = 455 nm, A455 nm = 0.1. c EtOH/ MeOH (4:1 v/v). 1a

CH2Cl2 (298) CH3CN (298) MeOH (298) glassc (77) CH2Cl2 (298) CH3CN (298) MeOH (298) glassc (77) CH2Cl2 (298) CH3CN (298) MeOH (298) glassc (77)

It is interesting to note that the DPAT complexes 1a-3a showed lower emission quantum yields and shorter lifetimes compared to their DPAT-free counterparts 1b-3b (Table 3). In addition, the lifetimes of the DPAT complexes decreased with increasing complex concentrations, suggestive of a selfquenching process in which the emission of the rhenium(I) polypyridine unit was quenched by the DPAT moiety. The self-quenching rate constants (ksq) and emission lifetimes at infinite dilution (τi.d.) of the DPAT complexes in CH3CN have been determined using the equation 1/τ = 1/τi.d. þ ksq[Re], where τ is the emission lifetime of the complex at concentration [Re]. The slope and y-intercept of the linear fit of a plot of τ-1 vs [Re] gave ksq and τi.d.-1, respectively. The results showed that the self-quenching rate constants ksq are on the order of 108 to 109 dm3 mol-1 s-1 (Table 4), indicative of a very efficient quenching pathway. The lifetimes at infinite dilution (Table 4) are comparable to those of the DPAT-free complexes 1b-3b (Table 3), reflecting that the self-quenching of the DPAT complexes is dominated by an intermolecular process. We have studied the emission quenching of the DPAT-free complexes by DPA-Tyraminet BOC in CH3CN using Stern-Volmer analysis. The SternVolmer plot for complex 3b is shown in Figure 2. The bimolecular quenching rate constants of the DPATfree complexes by DPA-Tyramine-tBOC are 6.7 108, 3.0 109, and 8.4 108 dm3 mol-1 s-1, respectively, which are comparable to the self-quenching rate constants of the DPAT complexes 1a-3a (Table 4). These results illustrate that intermolecular quenching plays a key role in the selfquenching of the DPAT complexes. From the potentials of the diimine-based reduction of the DPAT-free complexes (from -1.15 to -1.42 V vs SCE, Table 1) and their lowtemperature emission energy (E00=2.48 to 2.66 eV, Table 3), the excited-state reduction potentials of the DPAT-free complexes, E[Reþ*/0], have been estimated to be ca. þ1.24 to þ1.47 V vs SCE. On the basis of these potentials and the

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Table 4. Self-Quenching Rate Constants and Emission Lifetimes at Infinite Dilution of the Tricarbonylrhenium(I) Polypyridine DPAT Complexes 1a-3a in CH3CN at 298 K complex

ksq/dm3 mol-1 s-1

τi.d./μs

1a 2a 3a

5.5 10 1.3 109 1.1 109

1.07 3.17 3.37

8

Figure 3. pH titration curve for complex 1a in aerated 100 mM KCl(aq)/MeOH (7:3 v/v) at 298 K. The inset shows the emission spectral traces upon increasing pH.

Figure 2. Stern-Volmer plot for the emission quenching of complex 3b by DPA-Tyramine-tBOC in degassed CH3CN at 298 K.

redox potential of DPA-Tyramine-tBOC (ca. þ1.05 V vs SCE), reductive quenching of the excited complexes is favored by ca. 0.19 to 0.42 eV. Thus, it is conceivable that the emission quenching mechanism of the DPAT complexes is electron-transfer in nature. pH-Dependent Emission. Incorporation of a DPAT moiety into complexes 1a-3a resulted in pH-dependent emission of these complexes. At pH < 3, the DPAT complexes showed relatively intense emission under ambient conditions. Upon increasing the pH, the emission intensity decreased substantially, and at pH > 11, the emission was almost completely quenched. The emission of the complexes at pH < 3 was ca. 40 fold higher than that at pH > 11. The pH titration curve for complex 1a is shown in Figure 3 (the emission spectral traces are shown in the inset). The change in emission intensity in the acidic region fits an apparent pKa value of 5.0, 5.0, and 5.2 for the DPAT complexes 1a-3a, respectively. We have assigned these values to the deprotonation of the ammonium group of DPAT, yielding the tertiary amine form, which quenches the emission of the complexes.29 The decrease of emission intensity at pH=ca. 9 to 11 may be due to the deprotonation of the tyrosyl group since phenolate is an effective quencher for luminescent tricarbonylrhenium(I) polypyridine systems; for example, the deprotonated tyrosine group of [Re(bpy-Y)(CO)3(CN)] (bpy-Y = 40 -methyl2,20 -bipyridine-4-tyrosine) quenches the excited complex by electron-transfer.25 However, we found that the DPAT-free complexes 1b-3b also displayed a drop of emission intensity at a similar pH, whereas the unmodified pyridine analogues [Re(N∧N)(CO)3(pyridine)](CF3SO3) did not give such an observation. Thus, it appears that the emission quenching was related to the thiourea unit. Since thiourea has a very high pKa value (ca. 21),30 and it is well known that thiourea exhibits hydrogen bonding with anions such as fluoride, (29) Chen, P.; Westmoreland, T. D.; Danielson, E.; Schanze, K. S.; Anthon, D.; Neveux, P. E., Jr.; Meyer, T. J. Inorg. Chem. 1987, 26, 1116– 1126. (30) Bordwell, F. G.; Algrim, D. J.; Harrelson, J. A., Jr. J. Am. Chem. Soc. 1988, 110, 5904–5906.

dihydrogen phosphate, and acetate, we have tentatively assigned the reduction of emission intensity at pH = ca. 9 to 11 to the interaction of the thiourea unit of the pyridine ligand with hydroxide ions.31 The emission of the DPAT complexes was recovered by addition of acid. This acid-base switching process can be repeated for at least six cycles without significant deterioration in emission intensity; this is illustrated in Figure 4 with complex 1a as an example. Ion-Dependent Emission. The ion-binding properties of the complexes have been studied by emission titrations in aerated 100 mM KCl(aq)/MeOH (7:3 v/v) at 298 K.32 Upon addition of zinc(II) or cadmium(II) ions, the DPAT complexes 1a-3a showed emission enhancement (I/Io=ca. 1.8 to 3.9) and lifetime extension (τ/τo = ca. 1.4 to 1.6) (Table 5). The emission profiles and the electronic absorption spectra did not display notable changes. Interestingly, similar changes were absent for the DPAT-free complexes 1b-3b. As an example, the titration results for complexes 3a and 3b with zinc(II) and cadmium(II) ions are illustrated in Figure 5. Job plots indicated that the DPAT complexes formed 1:1 complexes with the metal ions. Since the DPAT-free analogues did not display similar emission changes, we have attributed the increase of emission intensities and lifetimes to the specific binding of the zinc(II) and cadmium(II) ions to the DPAT unit. It is likely that the coordination of the amine of the DPAT unit to the metal cations will substantially suppress the self-quenching of the complexes, leading to the observed emission enhancement and lifetime extension. This (31) The interaction of the thiourea moiety of the complexes with hydroxide ions has been confirmed by 1H NMR experiments. In a solvent mixture of D2O/CD3OD (v/v 7:3) containing 100 mM KCl, the chemical shifts of Hj, Hk, Hl, and Hm of the py-TU-Et ligand of [Re (phen)(CO)3(py-TU-Et)](CF3SO3) (1b) were 8.56 (br), 8.42 (d, J=4.8 Hz), 7.24-7.20 (m), and 7.39-7.37 (m) ppm, respectively. Upon adjusting the pH of the solution to ca. 12 with KOH, these protons resonated as multiplets at 8.13-8.07, 8.13-8.07, 7.05-7.01, and 7.12-7.10 ppm, respectively, while the protons on the phen ligand did not display any shifts. We have assigned these upfield shifts of the pyridine protons to the interaction of the thiourea moiety of the complex and hydroxide ions. This has been confirmed by the observations that the unmodified pyridine complex [Re(phen)(CO)3(pyridine)](CF3SO3) did not show any shifts in the resonances of all the protons when the pH was raised. (32) The use of other buffers including borate, PIPES, and phosphate buffers has been attempted. However, both borate and PIPES buffers reduced the emission intensities of the complexes. In phosphate buffer, the complexes showed little change in emission intensity upon addition of zinc(II) ion, probably due to the interaction of phosphate ions with the thiourea moiety.

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Figure 4. Emission responses of complex 1a in aerated 100 mM KCl(aq)/MeOH (7:3 v/v) at 298 K upon successive pH switching between acidic (pH < 3) (squares) and basic (pH > 11) (circles) conditions. Table 5. Relative Emission Intensities and Lifetimes of the Tricarbonylrhenium(I) Polypyridine DPAT Complexes 1a-3a (50 μM) in the Absence and Presence of Zinc(II) or Cadmium(II) Ions in Aerated 100 mM KCl(aq)/MeOH (7:3 v/v) at 298 K, and the Dissociation Constants of the Re-Zn and Re-Cd Adducts complex

I (τ/μs)a

I (τ/μs)b

I (τ/μs)c

Kd/Md

Kd/Me

1.00 (0.44) 1.76 (0.65) 2.44 (0.64) 5.9 10-5 5.9 10-5 1.00 (0.66) 2.13 (0.96) 3.90 (0.94) 6.6 10-5 2.6 10-5 1.00 (0.79) 2.90 (1.30) 2.16 (1.21) 4.8 10-6 3.0 10-6 2þ a [Zn ] = [Cd2þ] = 0 M. b [Zn2þ] = 100 μM. c [Cd2þ] = 100 μM. d Dissociation constants of the Re-Zn adducts. e Dissociation constants of the Re-Cd adducts. 1a 2a 3a

is in agreement with the pH titration results. The dissociation constants of the adducts formed from the DPAT complexes and the cations, determined from the emission titrations, are on the order of 10-5 to 10-6 M (Table 5). These are about 3 to 4 orders of magnitude larger than those of related organic fluorophore-DPA systems,9a-9e,10c,10d which should be a consequence of the formal cationic charge of the tricarbonylrhenium(I) complexes. The ion-binding selectivity of complex 1a has been examined in detail, and the results are shown in Figure 6. We found that only zinc(II) or cadmium (II) ions induced significant emission enhancement even in the presence of a range of other metal cations. The first-row transition metal divalent cations themselves did not bring significant changes to the emission intensity of the complex (except that the emission was quenched by iron(II) and copper(II) ions, for which I/Io=0.58 and 0.65, respectively). Addition of lead(II) ions slightly enhanced the emission intensity of the complex by 1.07-fold, and the copresence of zinc(II) and cadmium(II) ions, respectively, could give I/Io = 1.50 and 1.56 only, which means lead(II) may cause some interference in the detection of these two ions. However, upon addition of 2 equiv (100 μM) of sodium, magnesium, or calcium ions, which exist in high concentrations under physiological conditions, the emission intensity of complex 1a did not change substantially (Figure 6). This illustrates that the DPAT complexes can selectively sense zinc(II) and cadmium (II) ions among almost all the ions we studied. Cellular Uptake Studies. Although internalization of tricarbonylrhenium(I) polypyridine complexes by mammalian cells has been studied,22-24,26j quantitative data on their cellular uptake have not been reported. In this work, the cellular uptake efficiency of all the tricarbonylrhenium(I)

Figure 5. Emission titration curves for the titrations of complexes 3a (solid squares) and 3b (hollow circles) with zinc(II) (top) and cadmium(II) (bottom) ions in aerated 100 mM KCl (aq)/MeOH (7:3 v/v) at 298 K.

Figure 6. Relative emission intensities of (i) complex 1a (50 μM), (ii) complex 1a (50 μM) and zinc(II) ion (100 μM), and (iii) complex 1a (50 μM) and cadmium(II) ion (100 μM) in aerated 100 mM KCl(aq)/MeOH (7:3 v/v) containing different cations (100 μM) at 298 K. Io and I are the emission intensities of the complex in the absence and presence of cations, respectively.

polypyridine complexes by HeLa cells has been determined by ICP-MS measurements. Upon incubation with the complex (5 μM) at 37 C for 1 h, a typical cell (average volume= 3.4 pL) contained 1.1 10-15 to 6.9 10-14 mol of rhenium (Table 6). This cellular uptake efficiency is higher than that of other inorganic complexes such as [Ru(Ph2phen)2(dppz)]2þ (dppz = dipyrido[3,2-a:20 ,30 -c]phenazine)

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Table 6. Numbers of Moles and Concentrations of Rhenium Associated with a Typical HeLa Cell (average volume = 3.4 pL) upon Incubation at 37 C for 1 h ([Re] = 5 μM in the incubation medium) complex

no. of moles

concentration/mM

1a 1b 2a 2b 3a 3b

2.8 10-15 1.1 10-15 3.2 10-14 2.2 10-15 6.9 10-14 1.8 10-14

0.82 0.33 9.6 0.64 20.4 5.3

(2.6 10-17 to 6.7 10-16 mol of ruthenium),33 [Eu.L1]3þ (L1 = (S,S)-1,7-bis(ethoxycarbonyl-2-ethylcarbamoylmethyl)4-[7-(methylcarbamoylmethyl)-1-aza-thioxanthone]-1,4,7,10tetraazacyclododecane) (1.1 10-16 mol of europium),35 and [Eu2(LC2)3] (LC2=6,60 -[methylenebis(1-methyl-1H-benzimidazole-5,2-diyl)]bis(4-{2-[2-(2-methoxyethoxy)ethoxy] ethoxy}pyridine-2-carboxylate) (7.9 10-16 mol of europium).34 Importantly, HeLa cells loaded with the DPAT complexes 1a-3a revealed higher rhenium concentrations compared to those treated with the DPAT-free complexes 1b-3b, respectively (Table 6). Also, the Me4-phen complexes 2a and 2b and the Ph2-phen complexes 3a and 3b exhibited more effective uptake than the phen complexes 1a and 1b. This indicates that the presence of the DPAT moiety and the methyl and phenyl substituents increased the cellular uptake efficiency, probably due to enhanced lipophilicity of the complexes. It is interesting to note that the smaller molecular size of the DPATfree and the phen complexes did not lead to higher efficiency,33 which suggests that the uptake is not due to passive diffusion. Also, the rhenium concentration of all the complexes associated with the cells was much higher than that in the medium (5 μM), suggestive of cellular accumulation of complexes. However, the measured concentrations only indicate rhenium associated with the cells, which is not necessarily all in the interior. Cytotoxicity. We have studied the cytotoxicity of the tricarbonylrhenium(I) polypyridine complexes by the MTT assay using HeLa cells as a model cell line. The IC50 values have been determined from the dependence of the survival of HeLa cells on the dose of the complexes for the incubation period of 48 h (Table 7).36 The results reveal that all the complexes are more cytotoxic than cisplatin and related tricarbonylrhenium(I) polypyridine complexes such as [Re(CO)3(2-appt)Cl] (2-appt=2-amino-4-phenylamino-6-(2pyridyl)-1,3,5-triazine) (IC50 = ca. 50 μM)37 and [Re(N∧N)(CO)3(py-biotin-TU-Et)](PF6) (py-biotin-TU-Et=3-ethylthioureidyl-5-((2-(biotinamido)ethyl)aminocarbonyl)pyridine, N∧N=phen, Me4-phen, Ph2-phen) (IC50=17.5 to 28.5 μM).26j The cytotoxicity of the Me4-phen complexes 2a and 2b and the Ph2-phen complexes 3a and 3b is higher than that of the phen (33) Puckett, C. A.; Barton, J. K. Biochemistry 2008, 47, 11711– 11716. (35) Chauvin, A.-S.; Comby, S.; Song, B.; Vandevyver, C. D. B.; B€ unzli, J.-C. G. Chem.;Eur. J. 2008, 14, 1726–1739. (34) Yu, J.; Parker, D.; Pal, R.; Poole, R. A.; Cann, M. J. J. Am. Chem. Soc. 2006, 128, 2294–2299. (36) The IC50 values of cisplatin in culture medium with and without 1% MeOH were 19.7 ( 1.9 and 19.7 ( 2.1 μM, respectively, which were very similar to that of the same drug in culture medium with 1% DMSO (19.0 ( 0.4 μM) (Table 7). The presence of DMSO did not reduce the activity of cisplatin in our studies. (37) Ma, D.-L.; Che, C.-M.; Siu, F.-M.; Yang, M.; Wong, K.-Y. Inorg. Chem. 2007, 46, 740–749. (38) Zhang, J.; Vittal, J. J.; Henderson, W.; Wheaton, J. R.; Hall, I. H.; Hor, T. S. A.; Yan, Y.-K. J. Organomet. Chem. 2002, 650, 123–132.

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Table 7. Cytotoxicity (IC50, 48 h) of the Tricarbonylrhenium(I) Polypyridine Complexes and Cisplatin toward the HeLa Cell Line complex

IC50/μM

1a 1b 2a 2b 3a 3b cisplatin

4.0 ( 0.5 16.7 ( 4.0 2.3 ( 0.2 5.3 ( 0.3 0.7 ( 0.1 1.6 ( 0.4 19.0 ( 0.4

complexes 1a and 1b (Table 7), but comparable to that of the rhenium(I) diphosphine complexes [Re(CO)3(diphosphine) Br].38 Also, the DPAT complexes are more cytotoxic than their DPAT-free counterparts (Table 7). These two results suggest that the cytotoxicity of the complexes is related to their cellular uptake efficiency and lipophilicity. Although we did not perform assays on other mammalian cells, it is very likely that these complexes will exhibit higher cytotoxicity toward noncancer cells. Live-Cell Confocal Microscopy. Exploitation of luminescent tricarbonylrhenium(I) polypyridine complexes in in vivo biological applications has attracted much attention recently.22-24,26j We have studied the cellular internalization of the Ph2-phen complexes 3a and 3b using laser-scanning confocal microscopy. Incubation of HeLa cells with the complexes at 37 C under a 5% CO2 atmosphere for 1 h resulted in efficient cellular uptake, and the emission properties were retained within the cells (Figure 7). Treatment of the cells with the complexes led to almost full cytoplasmic staining, and the complex distribution patterns were similar. The nucleoplasm and nucleoli of the cells showed negligible emission, indicating that the complexes were excluded from the nucleus. It is noteworthy that the emission of the cells treated with the DPAT complex 3a was much less intense than those loaded with the DPAT-free complex 3b. From the ICP-MS results, the cellular uptake of the DPAT complexes is much higher than that of the DPAT-free analogues (Table 6). This shows that the weaker emission of HeLa cells loaded with the DPAT complex 3a is due to the intrinsic selfquenching properties of the complex. Since complex 3b displayed more intense emission, its cellular uptake properties have been studied in more detail. Incubation of HeLa cells at 4 C before treatment with this complex led to negligible cellular uptake of the complex (Figure 8, left), excluding the possibility of passive diffusion. Also, when the cells were incubated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (20 μM) at 37 C in a glucose-free medium before complex 3b was loaded, a significant decrease of cellular uptake resulted (Figure 8, middle). Since CCCP is an oxidative phosphorylation inhibitor that decreases ATP production and metabolic rates, the observed decrease of cellular uptake indicates that internalization of complex 3b occurs via an energy-requiring pathway such as endocytosis. It is interesting to note that incubation of HeLa cells with N-ethylmaleimide (NEM) (0.1 mM), before complex 3b was added, gave rise to nuclear uptake of the complex (Figure 8, right). It is possible that the nuclear accumulation of this compound is caused by an increased nuclear entry as a result of NEM-induced nuclear pore malformation39 or a reduced nuclear export due to the inhibitory effect of NEM on nuclear export pathways.40 (39) Macaulay, C.; Forbes, D. J. J. Cell Biol. 1996, 132, 5–20. (40) Kudo, N.; Matsumori, N.; Taoka, H.; Fujiwara, D.; Schreiner, E. P.; Wolff, B.; Yoshida, M.; Horinouchi, S. Proc. Natl. Acad. Sci. U.S. A. 1999, 96, 9112–9117.

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Figure 7. Bright-field (left), overlaid (middle), and fluorescence (right) laser-scanning confocal microscopy images of HeLa cells incubated with complexes 3a (top row) and 3b (bottom row) (5 μM) at 37 C for 1 h.

Figure 8. Laser-scanning confocal microscopy images of HeLa cells incubated with complex 3b (5 μM) at 4 C (left) and at 37 C after the cells were preincubated with CCCP (20 μM) (middle) and NEM (0.1 mM) (right).

Figure 9. Laser-scanning confocal microscopy images of HeLa cells incubated with complex 3a (5 μM) at 37 C for 1 h followed by incubation of zinc(II) chloride/MPO (25 μM) (left), cadmium(II) chloride/MPO (25 μM) (middle), and MPO (25 μM) only (right) for 5 min.

Finally, the interesting ion-binding properties of the DPAT complexes and their effective uptake by HeLa cells have prompted us to study their potential as luminescent intracellular zinc(II) and cadmium(II) ion probes. A typical confocal microscopy image of HeLa cells treated with complex 3a and a mixture of 25 μM zinc(II) chloride/2-mercaptopyridine-N-oxide (MPO) and cadmium(II) chloride/MPO, respectively, revealed much higher emission intensity compared to those treated with MPO only (Figure 9). In an average cell, the increase of emission intensities was ca. 2.2and 1.8-fold for zinc(II) and cadmium(II) ions, respectively. This emission enhancement resulted from binding of the intracellular ions to the DPAT moiety of the complex because the DPAT-free complex 3b did not give a similar

change of emission properties in the presence of the ions and MPO. Unfortunately, N,N,N0 ,N0 -tetrakis(2-picolyl)ethylenediamine (TPEN), a cell-permeable metal ion chelator commonly used for reversibility studies, cannot be applied here since the emission of these tricarbonylrhenium(I) polypyridine complexes was quenched by this reagent. Under our experimental conditions, a high degree of cell death was observed when the concentration of zinc(II) or cadmium(II) ions was higher than ca. 50 μM. At a lower concentration of either ion (between ca. 5 and 25 μM), the difference in emission intensity was small but discernible. The ion concentration range (ca. 5 to 50 μM) is somewhat narrow for these complexes, in their current forms, to be ideal in vivo ion sensors. However, these rhenium(I) DPAT complexes are

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the first examples of luminescent transition metal complexes that are responsive toward zinc(II) and cadmium(II) ions in both in vitro and in vivo systems. With the use of other metal-ligand systems (for example, more strongly emissive complexes with higher sensitivity toward quenching by the DPA or related ion-binding units), we anticipate that luminescent organometallic complexes that can detect zinc(II) and cadmium(II) ions, and probably other ions, in an intracellular environment with low detection limits and high sensitivity can be developed.

Summary Three luminescent tricarbonylrhenium(I) polypyridine DPAT complexes and their DPAT-free counterparts have been synthesized and characterized, and their electrochemical and photophysical properties have been investigated. The DPAT complexes showed lower emission quantum yields and shorter lifetimes compared to their DPAT-free counterparts, which have been attributed to their self-quenching properties. The emission titration results showed that the DPAT complexes exhibited emission enhancement and lifetime extension upon binding of zinc(II) and cadmium(II) ions. Incubation of the complexes with HeLa cells led to efficient cellular uptake. ICP-MS results indicate that the cellular uptake of complexes containing the DPAT moiety or the more hydrophobic Me4-phen and Ph2-phen ligands was more effective. The DPAT complexes were more cytotoxic toward HeLa cells than the DPAT-free complexes in a 48 h incubation period, as revealed by the MTT assay. Confocal microscopy studies showed that HeLa cells stained with complex 3a displayed weaker emission compared to those loaded with complex 3b, despite the higher cellular uptake of the former complex, pointing to the fact that self-quenching of this complex occurred within the cells. Interestingly, the DPAT complex exhibited emission enhancement when zinc(II) and cadmium(II) ions were loaded exogenously to the cells, reflecting its potential use as a luminescent intracellular probe for these two ions. Although many fluorescent organic compounds have been designed as sensors for zinc(II) and cadmium(II) ions, these tricarbonylrhenium(I) polypyridine DPAT complexes, to the best of our knowledge, are the first luminescent transition metal complexes that function as in vitro and in vivo probes for these two biologically and environmentally important metal ions.

Experimental Section Materials and Synthesis. All solvents were of analytical reagent grade and purified according to standard procedures. Tyramine, phen, Me4-phen, Ph2-phen, AgCF3SO3, ethylamine, thiophosgene, cisplatin, MPO, cadmium(II) chloride, CCCP, and NEM were purchased from Acros. MTT and paraformaldehyde were purchased from Sigma. DPA, Re(CO)5Cl, zinc(II) chloride, di-tert-butyl dicarbonate, and TPEN were purchased from Aldrich. All these chemicals were used without further purification. [Re(N∧N)(CO)3(py-3-NCS)](CF3SO3) were prepared as described previously.26a Autoclaved Milli-Q water was used for the preparation of the aqueous solutions. Tetran-butylammonium hexafluorophosphate was obtained from Aldrich and was recrystallized from hot ethanol and dried in vacuo at 110 C. HeLa cells were obtained from American Type Culture Collection. Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin were

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purchased from Invitrogen. Unless specified, the growth medium for cell culture contained DMEM with 10% FBS and 1% penicillin/streptomycin. Tyramine-tBOC. A solution of di-tert-butyl dicarbonate (3 g, 13.8 mmol) and tyramine (1.67 g, 12.2 mmol) in MeOH (30 mL) was stirred at room temperature under an inert atmosphere of nitrogen for 2 h. It was evaporated to dryness, and the crude product was dissolved in ethyl acetate. The solution was washed with water and dried over MgSO4. The solvent was removed by rotary evaporation, and the residual red oil was then purified by column chromatography on silica gel. The desired product was eluted with hexane/ethyl acetate (2:1 v/v). DPA-Tyramine-tBOC. A mixture of DPA (3.51 g, 17.6 mmol) and paraformaldehyde (0.76 g) was heated to reflux in MeOH (30 mL). The mixture became a clear yellow solution upon reflux. Tyramine-tBOC (7.88 g, 33.2 mmol) was added to the solution, and then the solution was refluxed for 48 h. The solvent was removed by rotary evaporation, and the residual yellow oil was purified by column chromatography on silica gel with the eluent changing from hexane/ethyl acetate (3:7 v/v) to CHCl3/ MeOH (49:1 v/v). The solvent was removed by rotary evaporation to give a white solid. Yield: 6.31 g (80%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 8.53 (d, 2H; J = 7.8 Hz, Hi), 7.73 (t, 2H; J=8.7 Hz, Hg), 7.41 (d, 2H; J=8.1 Hz, Hh), 7.24 (t, 2H; J=5.4 Hz, Hf), 6.99-6.96 (m, 2H; Ha, Hb), 6.73 (d, 1H; J= 9.0 Hz, Hc), 5.96 (br, 1H; OH), 3.85 (s, 4H; He), 3.75 (s, 2H; Hd), 3.23 (q, 2H; J=6.9 Hz, phenol-CH2CH2NH), 2.66 (t, 2H; J=7.8 Hz, phenol-CH2CH2NH), 1.36 ppm (s, 9H; CH3). Positive-ion ESI-MS ion clusters at m/z 448 [M þ H]þ. DPA-Tyramine. DPA-Tyramine-tBOC in a TFA/CH2Cl2 mixture (1:1 v/v) was stirred at room temperature under an inert atmosphere of nitrogen for 2 h. The solvent was removed by rotary evaporation. The residual yellow oil was dissolved in CH2Cl2, and the solution was washed with 10% ammonium hydroxide, water, and brine. The CH2Cl2 solution was dried over MgSO4, and the solvent was removed by rotary evaporation. The white solid was used without further purification. [Re(N∧N)(CO)3(py-TU-DPAT)](CF3SO3) (N∧N=phen (1a), Me4-phen (2a), Ph2-phen (3a)). A mixture of [Re(N∧N)(CO)3(py3-NCS)](CF3SO3) (0.17 mmol) and DPA-Tyramine (56 mg, 0.16 mmol) was stirred in acetone (30 mL) at room temperature under an inert atmosphere of nitrogen for 12 h. The solvent was removed by rotary evaporation. The residual brownish-yellow oil was then purified by column chromatography on alumina. The desired product was eluted with CH3CN/MeOH/H2O (3:3:1 v/v). Recrystallization of the crude product from CH2Cl2/diethyl ether afforded the complex as brownish-yellow crystals. Complex 1a. Yield: 82 mg (44%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.83 (d, 2H; J=5.4 Hz, H2, H9 phen), 9.31 (br, 1H; py-3-NH(CS)NH), 9.13 (s, 1H; Hj), 9.06 (d, 2H; J=8.4 Hz, H4, H7 phen), 8.53-8.50 (m, 3H; py-3-NH(CS)NH, Hi), 8.39 (d, 1H; J=5.7 Hz, Hm), 8.35-8.29 (m, 4H; H3, H5, H6, H8 phen), 7.75-7.69 (m, 3H; Hg, Hk), 7.40 (d, 2H; J= 7.8 Hz, Hh), 7.29-7.20 (m, 3H; Hf, Hl), 6.99-6.96 (m, 2H; Ha, Hb), 6.70 (d, 1H; J=8.7 Hz, Hc), 3.85 (s, 4H; He), 3.75 (s, 2H; Hd), 3.62 (br, 2H; phenol-CH2CH2NH), 2.72 ppm (t, 2H; J=6.9 Hz, phenol-CH2CH2NH). IR (KBr): ν 3447 (br, N-H), 3293 (br, OH), 2909 (w, C-H), 2032 (s, CtO), 1923 (m, CtO), 1253 (m, CdS), 1163 (m, CF3SO3 ), 1030 (m, CF3SO3 ). Positive-ion ESIþ MS ion clusters at m/z 935 [M] . Anal. Calcd (%) for C43H36N8O7S2F3Re 3 CH3CN: C 48.26, H 3.63, N 10.95. Found: C 48.04, H 3.49, N 11.20. Complex 2a. Yield: 89 mg (45%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.58 (s, 2H; H2, H9 Me4-phen), 9.36 (br, 1H; py-3-NH(CS)NH), 9.25 (s, 1H; Hj), 8.56-8.50 (m, 3H; py-3-NH(CS)NH, Hi), 8.44-8.42 (m, 3H; Hm, H5, H6 Me4-phen), 7.73 (t, 2H; J=7.8 Hz, Hg), 7.69-7.61 (m, 1H; Hk), 7.42 (d, 2H; J=8.1 Hz, Hh), 7.27-7.23 (m, 3H; Hf, Hl), 7.02-7.00 (m, 2H; Ha, Hb), 6.72 (d, 1H; J=9.0 Hz, Hc), 3.85 (s, 4H; He), 3.75 (s, 2H; Hd), 3.62 (br, 2H; phenol-CH2CH2NH), 2.81-2.72 ppm (m, 8H; phenol-CH2CH2NH, CH3 at C3, C8

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Me4-phen). IR (KBr): ν 3453 (br, N-H), 3293 (br, O-H), 2919 (w, C-H), 2029 (s, CtO), 1919 (s, CtO), 1251 (m, CdS), 1159 (m, CF3SO3 ), 1028 (m, CF3SO3 ). Positive-ion ESI-MS ion þ clusters at m/z 992 [M] . Anal. Calcd (%) for C47H44N8O7S2F3Re 3 CH3CN: C 49.82, H 4.01, N 10.67. Found: C 50.12, H 4.16, N 10.45. Complex 3a. Yield: 121 mg (58%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.87 (d, 2H; J=5.4 Hz, H2, H9 Ph2-phen), 9.34 (br, 1H; py-3-NH(CS)NH), 9.06 (s, 1H; Hj), 8.60 (d, 2H; J=5.1 Hz, Hm), 8.54 (br, 1H; py-3-NH(CS)NH), 8.49 (d, 2H; J = 5.7 Hz, Hi), 8.25-8.22 (m, 4H; H3, H5, H6, H8 Ph2phen), 7.76-7.62 (m, 13H; Hg, Hk, Ph Ph2-phen), 7.38 (d, 2H; J= 8.3 Hz, Hh), 7.28-7.20 (m, 3H; Hf, Hl), 6.99-6.96 (m, 2H; Ha, Hb), 6.70 (d, 1H; J=2.9 Hz, Hc), 3.85 (s, 4H; He), 3.75 (s, 2H; Hd), 3.62 (br, 2H; phenol-CH2CH2NH), 2.72 ppm (t, 2H; J=6.9 Hz, phenol-CH2CH2NH). IR (KBr): ν 3436 (br, N-H), 3293 (br, O-H), 2914 (w, C-H), 2030 (s, CtO), 1923 (m, CtO), 1253 (m, CdS), 1160 (m, CF3SO3 ), 1029 (m, CF3SO3 ). Positiveion ESI-MS ion clusters at m/z 1087 [M]þ. Anal. Calcd (%) for C55H44N8O7S2F3Re 3 CH3CN: C 53.60, H 3.71, N 9.87. Found: C 53.71, H 3.83, N 9.65. [Re(N∧N)(CO)3(py-TU-Et)](CF3SO3) (N∧N=phen (1b), Me4phen (2b), Ph2-phen (3b)). A mixture of [Re(N∧N)(CO)3(py-3NCS)](CF3SO3) (0.15 mmol) and ethylamine (10 μL, 0.15 mmol) was stirred in acetone (20 mL) at room temperature under an inert atmosphere of nitrogen for 12 h. The solution was evaporated to dryness to give a yellow solid. Recrystallization of the crude product from CH2Cl2/diethyl ether afforded the complex as brownishyellow crystals. Complex 1b. Yield: 72 mg (66%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.85 (d, 2H; J=5.1 Hz, H2, H9 phen), 9.70 (br, 1H; py-3-NH(CS)NH), 9.17 (s, 1H; Hj), 9.06 (d, 2H; J=8.1 Hz, H4, H7 phen), 8.40-8.30 (m, 5H; Hm, H3, H5, H6, H8 phen), 7.90-7.88 (m, 2H; Hk, py-3-NH(CS)NH), 7.31-7.23 (m, 1H; Hl), 3.49-3.39 (m, 2H; CH3CH2NH), 1.09 ppm (t, 3H; CH3CH2NH). IR (KBr): ν 3440 (br, N-H), 2975 (w, C-H), 2032 (s, CtO), 1917 (m, CtO), 1248 (m, CdS), 1164 (m, CF3SO3 ), 1030 (m, CF3SO3 ). Positive-ion ESI-MS ion clusters at m/z 631 [M]þ. Anal. Calcd (%) for C24H19N5O6S2F3Re 3 1/2CH3CN: C 37.43, H 2.58, N 9.61. Found: C 37.63, H 2.64, N 9.54. Complex 2b. Yield: 58 mg (49%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.53 (s, 2H; H2, H9 Me4-phen), 9.30 (s, 1H; Hj), 8.67 (br, 1H; py-3-NH(CS)NH), 8.39-8.34 (m, 3H; py-3-NH(CS)NH, H5, H6 Me4-phen), 8.32 (d, 1H; J = 5.7 Hz, Hm), 7.97 (d, 1H; J = 5.8 Hz, Hk), 7.22-7.15 (m, 1H; Hl), 3.48-3.38 (m, 2H; CH3CH2NH), 2.75 (s, 6H; CH3 at C3, C8 Me4-phen), 1.05 ppm (t, 3H; J = 7.5 Hz, CH3CH2NH). IR (KBr): ν 3429 (br, N-H), 2985 (w, C-H), 2030 (s, CtO), 1916 (m, CtO), 1246 (m, CdS), 1163 (m, CF3SO3 ), 1029 þ (m, CF3SO3 ). Positive-ion ESI-MS ion clusters at m/z 687 [M] . Anal. Calcd (%) for C28H27N5O6S2F3Re 3 H2O 3 CH3CN: C 40.21, H 3.60, N 9.38. Found: C 40.02, H 3.51, N 9.21. Complex 3b. Yield: 75 mg (57%). 1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.90 (d, 2H; J=7.5 Hz, H2, H9 Ph2-phen), 9.29 (br, 1H; py-3-NH(CS) NH), 9.12 (s, 1H; Hj), 8.58 (d, 1H; Hm), 8.27-8.22 (m, 4H; H3, H5, H6, H8 Ph2-phen), 7.79-7.66 (m, 12H; Hk, py-3-NH(CS)NH, Ph Ph2-phen), 7.40-7.33 (m, 1H; Hl), 3.51-3.38 (m, 2H; CH3CH2NH), 1.10 ppm (t, 3H; J = 7.2 Hz, CH3CH2NH). IR (KBr): ν 3440 (br, N-H), 2974 (w, C-H), 2032 (s, CtO), 1912 (m, CtO), 1243 (m, CdS), 1159 (m, CF3SO3 ), 1028 (m, CF3SO3 ). þ Positive-ion ESI-MS ion clusters at m/z 783 [M] . Anal. Calcd (%) for C36H27N5O6S2F3Re 3 (CH3)2CO: C 47.27, H 3.36, N 7.07. Found: C 47.45, H 3.57, N 7.29. Instrumentation and Electrochemical and Photophysical Measurements. 1H NMR spectra were recorded on a Varian Mercury 300 MHz NMR spectrometer at 298 K. Positive-ion ESI mass spectra were recorded on a Perkin-Elmer Sciex API 365 mass spectrometer. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrophotometer. Elemental analyses were carried out on a Vario EL III CHN elemental analyzer. Electrochemical measurements were performed on a CH Instruments Electrochemical Workstation CHI750A and were carried out at room temperature using a two-compartment glass cell

Louie et al. with a working volume of 500 μL. A platinum gauze counter electrode was accommodated in the working electrode compartment. The working and reference electrodes were a glassy carbon electrode and an Ag/AgNO3 (0.1 mol dm-3 nBu4NPF6 in CH3CN) electrode, respectively. The reference electrode compartment was connected to the working electrode compartment via a Luggin capillary. Solutions for electrochemical measurements were degassed with prepurified nitrogen gas. All potentials were referred to SCE. Electronic absorption and steady-state emission spectra were recorded on a HewlettPackard 8453 diode array spectrophotometer and a SPEX FluoroLog 3-TCSPC spectrophotometer, respectively. Emission lifetimes were measured in the Fast MCS or a TCSPC lifetime mode with a NanoLED N-340 or NanoLED N-375 as the excitation source, respectively. Unless specified, all the solutions for photophysical studies were degassed with no fewer than four successive freeze-pump-thaw cycles and stored in a 10 cm3 round-bottomed flask equipped with a side arm 1 cm fluorescence cuvette and sealed from the atmosphere by a Rotaflo HP6/6 quick-release Teflon stopper. Emission quantum yields were measured by the optically dilute method41 with an aerated aqueous solution of [Ru(bpy)3]Cl2 (Φem =0.028, λex = 455 nm) as the standard solution.42 pH- and Ion-Dependent Emission Studies. In the pH-dependence studies, a 20 mL stock solution of the DPAT complex (50 μM) in a mixture of 10 mM HCl and 100 mM KCl(aq)/MeOH (7:3 v/v) was prepared. The pH of the stock solution was increased gradually by addition of appropriate amounts of 1, 0.5, 0.25, 0.13, 0.07, 0.04, or 0.02 N KOH(aq). After a desired pH was obtained, 2 mL of the stock solution was transferred to a quartz cuvette and the emission spectrum was measured. The solution was then returned to the stock, and the pH was further adjusted. The overall increase in the volume of the stock solution due to pH adjustment was kept below 2% (i.e., approximately 400 μL). In the emission titrations, the tricarbonylrhenium(I) polypyridine complex (50 μM) in 100 mM KCl(aq)/MeOH (7:3 v/v) solution was titrated with zinc(II) chloride or cadmium(II) chloride (2 mM), which was dissolved in the same solvent mixture. The emission spectrum of the solution was measured after successive additions (5 μL aliquots) of the ion solution at 1 min intervals. Determination of Dissociation Constants. Steady-state emission titrations were used to determine the dissociation constants. The dissociation constant, Kd, of the cation M2þ from the DPAT complex as described in the equilibrium Reþ-M2þ = Reþ þ M2þ was obtained using the following equation:43

Io ¼ Io -Ix

Io Io -I¥

Kd þ1 ½M2þ

!

where Io is the emission intensity of the DPAT complex only, Ix is the emission intensity of the DPAT complex in the presence of the cation at a concentration [M2þ], and I¥ is the limiting emission intensity. Kd was determined as the ratio of the slope to the y-intercept of the linear fit of a plot of Io/(Io Ix) vs [M2þ]-1. ICP-MS. HeLa cells in growth medium were incubated with the tricarbonylrhenium(I) polypyridine complex (5 μM) in a 60 mm tissue culture dish for 1 h. The culture medium was then removed and washed thoroughly with PBS (1 mL 5). The cells were trypsinized and harvested. The culture dish was further washed with PBS (100 μL 10), and the PBS was collected. The harvested cells, together with the collected PBS, were digested (41) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991–1024. (42) Nakamura, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697–2705. (43) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703–2707.

Article with 65% HNO3 at 60 C. The digested solution was filtered, and the concentration of rhenium in the filtrate was measured using an Elan 6100 DRC-ICP-MS system (PerkinElmer SCIEX Instruments, USA) equipped with a peristaltic pump, Meinhard quartz nebulizer, cyclonic spray chamber, nickel skimmer, and sample cones. MTT Assays. Cytotoxicity assays were conducted in 96-well, flat-bottomed microtiter plates. The supplemented culture medium (100 μL) with ca. 10 000 cells per well was incubated at 37 C under a 5% CO2 atmosphere for 24 h. The tricarbonylrhenium(I) polypyridine complex was dissolved in the culture medium with 1% DMSO, and the solutions were added to the wells. After the microtiter plate was incubated for 48 h, MTT in PBS (5 mg/mL, 10 μL) was added to each well. The microplate was incubated for another 3 h. The medium was removed carefully, and 2-propanol (200 μL) was added to each well. All the assays were run in parallel with a positive control, in which cisplatin was used as a cytotoxic agent. The absorbance of all the solutions at 570 nm was measured with a SPECTRAmax 340 microplate reader (Molecular Devices Corporation, Sunnyvale, CA). The IC50 values of the complexes were evaluated on the basis of the percentage cell survival in a dose-dependent manner relative to the controls. Live-Cell Confocal Microscopy. HeLa cells in growth medium were seeded on a sterilized coverslip in a 60 mm tissue culture dish and grown at 37 C under a 5% CO2 atmosphere for 48 h. The culture medium was then removed and replaced with


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medium/DMSO (99:1 v/v) containing the tricarbonylrhenium(I) polypyridine complex (5 μM). After incubation for 1 h, the medium was removed and the cell layer was washed with PBS (1 mL 5). The coverslip was mounted onto a sterilized glass slide and then imaged using a Leica TCS SPE confocal microscope. The excitation wavelength was 405 nm and the emission was measured using a 532 nm long-pass filter. HeLa cells for in vivo zinc(II) and cadmium(II) ion-sensing experiments were grown under the same conditions as described above. The cells were washed with PBS and incubated with protein-free medium/DMSO (99:1 v/v) containing zinc(II) chloride or cadmium(II) chloride (25 μM) and MPO (25 μM) for 5 min. The medium was then removed, and the cell layer was washed thoroughly with PBS (1 mL 5). The coverslip was mounted onto a sterilized glass slide and then imaged as described above.

Acknowledgment. We thank City University of Hong Kong (Project No. 7002483) and Hong Kong Research Grants Council (CityU 2/06C) for financial support. M.W.L. acknowledges receipt of a Postgraduate Studentship administered by City University of Hong Kong. We are grateful to Mr. Kenneth King-Kwan Lau, Mr. Michael Wai-Lun Chiang, and Mr. Ho-Hang Chan for their assistance with the cellular experiments. We thank Dr. Yun-Wah Lam for helpful discussions.

Novel Luminescent Tricarbonylrhenium(I) Polypyridine Tyramine

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