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Organometallics 2010, 29, 3474–3476 DOI: 10.1021/om100597g
Cyclometalated Iridium(III) Bipyridine Complexes Functionalized with an N-Methylamino-oxy Group as Novel Phosphorescent Labeling Reagents for Reducing Sugars Hua-Wei Liu, Kenneth Yin Zhang, Wendell Ho-Tin Law, 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 June 19, 2010 Summary: Cyclometalated iridium(III) bipyridine complexes appended with an N-methylamino-oxy group have been developed as phosphorescent labeling reagents for reducing sugars; the emission behavior, reactivity, cytotoxicity, and cellular uptake of these iridium(III) complexes and their sugar conjugates have been investigated. Carbohydrates are commonly appended to pharmaceutically important natural products to develop new reagents with enhanced drug solubility, desired toxicity, and improved target recognition and pharmacological properties.1 Studies of carbohydrate-protein interactions, carbohydrateprocessing enzymes, and analysis of glycomes are of paramount importance.2 Thus, the development of labels, especially those with fluorescence properties, for carbohydrates and investigations on the resultant conjugates have received much attention.3 Recently, there has been a growing interest in using the N-methylamino-oxy group to couple unprotected carbohydrates chemoselectively to microarrays,4 peptides, and proteins.5 One important advantage is that this functional group reacts readily with reducing sugars at the reducing end, producing primarily the closed-chain form of the sugars, and thus the biological activity is usually retained (Scheme 1).6 Also, this conjugation method proceeds under mild reaction conditions that are amenable to parallel synthesis, rendering the N-methylamino-oxy group a very important and efficient tool for labeling reducing sugars. Owing to their interesting photophysical properties, phosphorescent iridium(III) complexes have been utilized as sensors *To whom correspondence should be addressed. E-mail: bhkenlo@ cityu.edu.hk. Fax: (852) 3442 0522. Tel: (852) 3442 7231. (1) See, for example: (a) Taylor, M. E.; Drickamer, K. Introduction to Glycobiology; Oxford University Press: Oxford, 2003. (b) Thorson, J. S.; Vogt, T. Carbohydrate-Based Drug Discovery; Wong, C.-H., Ed.; WileyVCH: Weinheim, Germany, 2003; pp 685-711. (c) Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046–1051. (2) See, for example: (a) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357–2364. (b) Ganem, B. Acc. Chem. Res. 1996, 29, 340–347. (c) Tissot, B.; Gasiunas, N.; Powell, A. K.; Ahmed, Y.; Zhi, Z.; Haslam, S. M.; Morris, H. R.; Turnbull, J. E.; Gallagher, J. T.; Dell, A. Glycobiology 2007, 17, 972–982. (3) See, for example: (a) Seyfried, N. T.; Blundell, C. D.; Day, A. J.; Almond, A. Glycobiology 2005, 15, 303–312. (b) Hagihara, S.; Miyazaki, A.; Matsuo, I.; Tatami, A.; Suzuki, T.; Ito, Y. Glycobiology 2007, 17, 1070– 1076. (4) See, for example: Lee, M.-R.; Shin, I. Org. Lett. 2005, 7, 4269– 4272. (5) See, for example: (a) Peri, F.; Nicotra, F. Chem. Commun. 2004, 623–627. (b) Jimenez-Castells, C.; de la Torre, B. G.; Andreu, D.; GutierrezGallego, R. Glycoconj. J. 2008, 25, 879–887. (6) Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269– 12278. pubs.acs.org/Organometallics
Published on Web 07/27/2010
Scheme 1. Reaction of an N-Methylamino-oxy Group with a Reducing Sugar Model
for various analytes including ions7 and biomolecules.8 Although both fluorescent tags for reducing sugars and transition metal-sugar conjugates have been reported,9 a systematic design of phosphorescent transition metal complexes as labels for reducing sugars, to the best of our knowledge, has not been explored. The advantages of using phosphorescent transition metal complexes as biological probes include their intense and long-lived emission, large Stokes shifts, environment-sensitive photophysical properties, and high photostability. With our interest in phosphorescent cyclometalated iridium(III) polypyridine complexes as biological labels and probes,8 we envisage that iridium(III) complexes functionalized with an N-methylamino-oxy group will have a high potential to serve as new phosphorescent labeling reagents for a range of reducing sugars. Herein, we report the design of three new cyclometalated iridium(III) bipyridine complexes appended with an N-methylamino-oxy group, [Ir(N∧C)2(N∧N)](PF6) (HN∧C = 2-phenylpyridine (Hppy), N∧N = 4-N-methylamino-oxy-methyl40 -methyl-2,20 -bipyridine (bpy-ONHCH3) (1), 4-(10-N-methylamino-oxy-2,5,8-trioxa-decyl)-40 -methyl-2,2 0 -bipyridine (bpy-TEG-ONHCH3) (2); HN∧C=2-phenylquinoline (Hpq), N∧N = bpy-TEG-ONHCH3 (3)) (Chart 1), and their emission (7) See, for example: (a) Licini, M.; Williams, J. A. G. Chem. Commun. 1999, 1943–1944. (b) Goodall, W.; Williams, J. A. G. J. Chem. Soc., Dalton Trans. 2000, 2893–2895. (c) Araya, J. C.; Gajardo, J.; Moya, S. A.; Aguirre, P.; Toupet, L.; Williams, J. A. G.; Escadeillas, M.; Le Bozec, H.; Guerchais, V. New J. Chem. 2010, 34, 21–24. (d) Zhao, Q.; Li, F.; Liu, S.; Yu, M.; Liu, Z.; Yi, T.; Huang, C. Inorg. Chem. 2008, 47, 9256–9264. (8) (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.; Lee, P.-K.; Lau, J. S.-Y. Organometallics 2008, 27, 2998–3006. (c) Lau, J. S.Y.; Lee, P.-K.; Tang, K. H.-K.; Ng, C. H.-C.; Lam, Y.-W.; Cheng, S.-H.; Lo, K. K.-W. Inorg. Chem. 2009, 48, 708–719. (d) Zhang, K. Y.; Li, S. P.-Y.; Zhu, N.; Or, I. W.-S.; Cheung, M. S.-H.; Lam, Y.-W.; Lo, K. K.-W. Inorg. Chem. 2010, 49, 2530–2540. (e) Li, S. P.-Y.; Liu, H.-W.; Zhang, K. Y.; Lo, K. K.-W. Chem.;Eur. J. 2010, 16, 8329–8339. (9) See, for example: (a) Ma, D.-L.; Shum, T. Y.-T.; Zhang, F.; Che, C.-M.; Yang, M. Chem. Commun. 2005, 4675–4677. (b) Kikkeri, R.; García-Rubio, I.; Seeberger, P. H. Chem. Commun. 2009, 235–237. (c) Tian, Y. S.; Lee, H. Y.; Lim, C. S.; Park, J.; Kim, H. M.; Shin, Y. N.; Kim, E. S.; Jeon, H. J.; Park, S. B.; Cho, B. R. Angew. Chem., Int. Ed. 2009, 48, 1–6. r 2010 American Chemical Society
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Table 2. Product Yieldsa and Diastereoselectivities for the Conjugation of Reducing Sugars with Complexes 2 and 3
Chart 1. Structures of Complexes 1-3
complex 2 3
D-glucose b
59 66b
D-galactose c
57 60d
D-lactose b
43 42b
D-maltose
58b 60b
a
Yields were calculated from the integration ratio of the methyl group of the N-methylamino-oxy moiety to that of the C40 of bpy. b Anomer ratio (β:R) = 100:0. c Anomer ratio (β:R) = 60:40. d Anomer ratio (β:R) = 67:33.
Table 1. Photophysical Data of Complexes 1-3 complex
medium (T/K)
λem/nm
το/μs
Φem
1
CH2Cl2 (298) CH3CN (298) buffera (298) glassb (77) CH2Cl2 (298) CH3CN (298) buffera (298) glassb (77) CH2Cl2 (298) CH3CN (298) buffera (298) glassb (77)
569 578 581 472, 506 (max), 535 sh 568 577 577 473, 508 (max), 537 sh 554, 595 sh 554, 604 sh 557, 594 sh 543 (max), 583, 630 sh
0.65 0.4 0.059 4.91 0.65 0.4 0.066 4.8 2.5 2.8 2.11 4.75
0.21 0.12 0.017
2
3
0.22 0.14 0.016 0.42 0.39 0.16
a Potassium phosphate (50 mM, pH 7.4)/MeOH (9:1, v/v). b EtOH/ MeOH (4:1, v/v).
and sugar-conjugation properties. The synthesis of the two diimine ligands bpy-ONHCH3 and bpy-TEG-ONHCH3 is illustrated in Scheme S1. The iridium(III) complexes 1-3 were synthesized from the reaction of the dimer [Ir2(N∧C)4Cl2] with bpy-ONHCH3 or bpy-TEG-ONHCH3 in refluxing MeOH/ CH2Cl2 (1:1, v/v), followed by anion exchange to the PF6salt. They were characterized by 1H NMR spectroscopy, positive-ion ESI-MS, and IR spectroscopy and gave satisfactory microanalysis (Supporting Information). Upon photoexcitation, complexes 1-3 showed intense and longlived yellow to orange emission (Table 1). The solvent dependence of the emission energies of the ppy complexes 1 and 2 suggests that the emissive origin is a triplet metal-to-ligand charge-transfer (3MLCT) (dπ(Ir) f π*(N∧N)) state. This is supported by the observation of significant blue-shifts of their emission maxima upon cooling to 77 K. In contrast, the pq complex 3 exhibited structured emission bands (Figure S1) and much longer emission lifetimes (τo > 2 μs) (Table 1). The emission properties of this complex were much less sensitive to
the polarity of the solvents, and the emission maxima underwent a much smaller blue-shift upon cooling to 77 K compared to the ppy complexes 1 and 2 (Table 1), indicative of a high parentage of triplet intraligand (3IL) (π f π*) (pq) character in the emissive state of complex 3. We have studied the conjugation reactions of complexes 1-3 with reducing sugars including monosaccharides (D-glucose and D-galactose) and disaccharides (D-lactose and D-maltose). The reactions were performed in a solvent mixture of DMF and AcOH (1:1, v/v) at 298 K for 8 h.6 After the organic solvent was removed using high vacuum, the reaction mixtures were directly analyzed by 1H NMR. The spectra revealed that the reactions resulted in conjugation products except complex 1, whose conjugation yields were too low to be determined accurately. This indicates that in the absence of a TEG spacer arm the steric hindrance of [Ir(ppy)2(bpy)]þ substantially suppressed the reactivity of the N-methylamino-oxy moiety toward the reducing sugars. The diastereoselectivity of the sugar-conjugation reactions of complexes 2 and 3 has been examined (Table 2). All the products were obtained exclusively as the pyranose form. On the basis of analysis of the JH1-H2 coupling constants (ca. 9.0 Hz), the reactions with D-glucose, D-lactose, and D-maltose, which all contain D-glucose at the reducing end, proceeded with complete diastereoselectivity, and only the β-pyranose forms were produced (Table 2). For D-galactose, however, two pyranose forms with an anomer ratio (β:R) of ca. 3:2 and 2:1 were obtained for complexes 2 and 3, respectively (Table 2). Since complex 3 showed higher emission quantum yields and longer emission lifetimes (Table 1), we have prepared the sugar conjugates of this complex in a larger scale using a different synthetic approach and studied their photophysical and biological properties. The ligand bpy-TEGONHCH3 was reacted with the four reducing sugars under the same conditions as those used for the complex conjugation (Scheme S2). All the bipyridine-sugar ligands were isolated as the pyranose forms. Similar to the case of the complex conjugation, the reactions with D-glucose, D-lactose, and D-maltose afforded exclusively the β-forms (bpy-TEGONCH3-β-D-glucose, bpy-TEG-ONCH3-β-D-lactose, and bpy-TEG-ONCH3-β-D-maltose, respectively). For D-galactose, however, two pyranose forms (bpy-TEG-ONCH3β-D-galactose and bpy-TEG-ONCH3-R-D-galactose) in an anomer ratio (β:R) of 2:1 were obtained after column chromatographic purification. We then reacted these five bipyridinesugar ligands with the iridium dimer [Ir2(pq)4Cl2] (and KPF6 for anion exchange) to afford the conjugates [Ir(pq)2(N∧N)](PF6) (N∧N = bpy-TEG-ONCH3-β-D-glucose (3-β-glu), bpy-TEG-ONCH3-β-D-galactose (3-β-gal), bpy-TEG-ONCH3R-D-galactose (3-r-gal), bpy-TEG-ONCH3-β-D-lactose (3-β-lac), bpy-TEG-ONCH3-β-D-maltose (3-β-mal)) (Scheme S2), whose identities and diastereochemistry have been confirmed (Supporting Information). Upon photoexcitation, all the sugar conjugates 3-β-glu, 3-β-gal, 3-r-gal, 3-β-lac, and 3-β-mal exhibited
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Organometallics, Vol. 29, No. 16, 2010
Figure 1. Emission spectra of conjugate 3-β-glu in potassium phosphate (50 mM, pH 7.4)/MeOH (9:1, v/v) at 298 K (solid line) and in EtOH/MeOH (4:1, v/v) at 77 K (dashed line).
emission bands with structural features in fluid solutions under ambient conditions and in low-temperature glass (Table S1). The emission spectra of conjugate 3-β-glu in phosphate buffer at 298 K and in alcohol glass at 77 K are shown in Figure 1. Although the emission energies and lifetimes are similar to those of complex 3, the emission quantum yields are substantially enhanced (Φem = 0.28-0.85) (Table S1). The emission has been assigned to a 3IL (π f π*) (pq) excited state. The cytotoxicity of complex 3 and its five sugar conjugates toward HeLa cells has been evaluated by the MTT assay,10 in which the dose dependence of surviving cells after exposure to the samples for 48 h has been evaluated (Table S2). The IC50 values of complex 3 (2.9 μM) and its glucose conjugate 3-β-glu (4.0 μM) were comparable to those of related iridium(III)-pq complexes [Ir(pq)2(N∧N)](PF6) (N∧N = 4-ethylaminocarbonyl-40 -methyl-2,20 -bipyridine, IC50 = 3.8 μM;8b N∧N = 4-((2(indol-3-yl)ethyl)aminocarbonyl)-40 -methyl-2,20 -bipyridine, IC50 = 2.4 μM;8c N∧N = dipyrido[3,2-f:20 ,30 -h]quinoxaline, IC50 = 1.3 μM)8d but much smaller than those of some iridium(III) poly(ethylene glycol) (PEG) complexes (IC50 = 287 to 1180 μM)8e and cisplatin (IC50 = 28.8 μM) under the same experimental conditions. Interestingly, the galactose, lactose, and maltose conjugates showed larger IC50 values (from 9.8 to 12.0 μM) compared to their glucose counterpart. It is likely that the lower cytotoxicity of these non-glucose conjugates originates from their less efficient cellular uptake (see below) since glucose transporters present on the cell membrane can specifically transport glucose and its analogues.11 ICP-MS analysis and laser-scanning confocal microscopy experiments have been performed to further understand the cellular uptake properties of complex 3 and its sugar conjugates. The ICP-MS analysis showed that an average HeLa cell treated with complex 3 and conjugate 3-β-glu (5 μM, 37 °C, 2 h) contained 8.6 and 5.2 fmol of iridium, respectively, which are higher than those of conjugates 3-β-gal, 3-r-gal, 3-β-lac, and 3-β-mal (from 1.6 to 2.6 fmol) under the same experimental conditions (Table S3). These findings are in accordance with the confocal microscopy images, which showed that both complex 3 and conjugate 3-β-glu were localized in the perinuclear region of HeLa cells, displaying very intense emission (Figure 2). Although the non-glucose conjugates such (10) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63. (11) (a) Zhao, F.-Q.; Keating, A. Curr. Genomics 2007, 8, 113–128. (b) Park, J.; Lee, H. Y.; Cho, M.-H.; Park, S. B. Angew. Chem., Int. Ed. 2007, 46, 2018–2022.
Liu et al.
Figure 2. Laser-scanning confocal microscopy images of HeLa cells incubated with complex 3 (left column) and conjugates 3-β-glu (middle column) and 3-r-gal (right column) (5 μM) for 2 h at 37 °C (top row) and 4 °C (bottom row).
as 3-r-gal also showed emissive perinuclear rings (Figures 2 and S2), the intensity was far lower. Upon lowering the incubation temperature to 4 °C, the emission intensities of complex 3 and conjugate 3-β-glu were reduced and no emission was observed for the non-glucose conjugates (Figures 2 and S2). The significant decrease in the emission intensities of complex 3 and conjugate 3-β-glu at low temperature supports the involvement of an energy-requiring uptake pathway. In summary, we have designed cyclometalated iridium(III) bipyridine complexes containing an N-methylaminooxy group as the first phosphorescent labeling reagents for reducing sugars. These complexes allow reducing sugars to exhibit interesting emission behavior. Also, the biological properties of the resultant conjugates can be studied by fluorescence spectroscopy and microscopy. Despite the high cytotoxicity of complex 3 and its sugar conjugates, by using substituents such as PEG that can substantially increase the biocompatibility of iridium(III) polypyridine complexes,8e we anticipate that these complexes will become useful molecular tools for a range of glycobiological studies.
Acknowledgment. K.K.-W.L. acknowledges support from the Hong Kong Research Grants Council (Project No. CityU 102109) and City University of Hong Kong (Project No. 7002575). The work described in this paper was also supported by a Special Equipment Grant from the Hong Kong University Grants Committee (SEG_CityU02). K.Y.Z. acknowledges the receipt of a Postgraduate Studentship, Research Tuition Scholarship, and Outstanding Academic Performance Award, and W.H.-T.L. acknowledges the receipt of a Postgraduate Studentship, all administered by City University of Hong Kong. Supporting Information Available: Experimental details, 1H NMR spectra of the diimine ligands, complexes 1-3, and conjugates 3-β-glu, 3-β-gal, 3-r-gal, 3-β-lac, and 3-β-mal, photophysical data of the sugar conjugates, cytotoxicity data and cellular uptake efficiencies of complex 3 and its sugar conjugates, emission spectra of complex 3, and laser-scanning confocal microscopy images of HeLa cells stained with conjugates 3-β-gal, 3-β-lac, and 3-β-mal. This material is available free of charge via the Internet at http://pubs.acs.org.
