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Luminescent biological probes derived from ruthenium(II) estradiol polypyridine complexes.
Inorg. Chem. 2008, 47, 200−208
Luminescent Biological Probes Derived from Ruthenium(II) Estradiol
Polypyridine Complexes
Kenneth Kam-Wing Lo,* Terence Kwok-Ming Lee, Jason Shing-Yip Lau, Wing-Lin Poon, and
Shuk-Han Cheng
Department of Biology and Chemistry, City UniVersity of Hong Kong, Tat Chee AVenue,
Kowloon, Hong Kong, People’s Republic of China
Received September 4, 2007
Four luminescent ruthenium(II) polypyridine estradiol complexes [Ru(N∧N)2(bpy-estradiol)](PF6)2 (N∧N ) 2,2′-bipyridine
(bpy), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen); bpy-estradiol ) 5-(4-(17R-ethynylestradiolyl)phenyl)-2,2′-bipyridine
(bpy-ph-est), 4-(N-(6-(4-(17R-ethynylestradiolyl)benzoylamino)hexyl)aminomethyl)-4′-methyl-2,2′-bipyridine (mbpyC6-est)) have been designed as new luminescent biological probes. The lipophilicity and photophysical and
electrochemical properties of these complexes have been investigated. Upon photoexcitation, all the complexes
exhibited intense and long-lived triplet metal-to-ligand charge-transfer (3MLCT) (dπ(Ru) f π*(diimine)) emission in
fluid solutions at 298 K and in low-temperature glass. The binding of the complexes to estrogen receptor-R (ERR)
has been studied by emission titrations. The Ph2-phen complexes showed emission enhancement and increased
lifetimes upon binding to the protein. Additionally, the cytotoxicity of the complexes toward the HeLa cell line has
been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay and the IC50 values
ranged from 83.1 to 166.6 µM (cisplatin showed an IC50 value of 34.3 µM under the same experimental conditions).
Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser-scanning
confocal microscopy.
Introduction
Estrogens are responsible for the development of female
secondary sexual characteristics, stimulation of endometrial
and uterine growth, and regulation of the menstrual cycle.1
They are also involved in bone resorption and building,2
coagulation of blood,3 and control of the levels of lipoproteins, triglyceride, and fat deposit.4 Most of the biological
effects of estrogens are mediated through an interaction with
estrogen receptors (ERs).5 ERs (ERR and ERβ) belong to
the superfamily of nuclear receptor proteins, which are
membrane or intracellular proteins6,7 existing in a variety of
tissues.8 They are not only vital in regulating the differentiation and maintenance of neural, skeletal, cardiovascular, and
reproductive tissues9 but also participate in the development
of estrogen-dependent cancer such as breast, ovarian, colon,
prostate, and endometrial cancers.10 Many clinical studies
have concluded that the receptor content gives the most
* To whom correspondence should be addressed. E-mail: bhkenlo@
cityu.edu.hk. Fax: (+852) 2788 7406. Phone: (+852) 2788 7231.
(1) See, for example: (a) Punyadeera, C.; Dunselman, G.; Marbaix, E.;
Kamps, R.; Galant, C.; Nap, A.; de Goeij, A.; Ederveen, A.; Groothuis,
P. J. Steroid Biochem. Mol. Biol. 2004, 92, 175. (b) Clifton, V. L.;
Crompton, R.; Read, M. A.; Gibson, P. G.; Smith, R.; Wright, I. M.
R. J. Endocrinol. 2005, 186, 69.
(2) (a) Fiorelli, G.; Brandi, M. L. J. Endocrinol. InVest. 1999, 22, 589.
(b) Zallone, A. Ann. N.Y. Acad. Sci. 2006, 1068, 173.
(3) Owens, M. R.; Cimino, C. D. Blood 1985, 66, 402.
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H.; Jauhiainen, M.; Tikkanen, M. J. J. Clin. Endocrinol. Metab. 2001,
86, 1294.
(5) (a) Ying, C.; Hsu, W.-L.; Hong, W.-F.; Cheng, W. T. K.; Yang,
Y.-C. Mol. Reprod. DeV. 2000, 55, 83. (b) Dimitrova, K. R.; DeGroot,
K.; Myers, A. K.; Kim, Y. D. CardioVasc. Res. 2002, 53, 577. (c) Li,
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Kampa, M.; Nifli, A.-P.; Charalampopoulos, I.; Alexaki, V.-I.;
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E. Exp. Cell Res. 2005, 307, 41.
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2003, 16, 587. (c) Alvaro, D.; Mancino, M.; Onori, P.; Franchitto,
A.; Alpini, G.; Francis, H.; Glaser, S.; Gaudio, E. World J. Gastroenterol. 2006, 12, 3537.
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200 Inorganic Chemistry, Vol. 47, No. 1, 2008
10.1021/ic701735q CCC: $40.75
© 2008 American Chemical Society
Published on Web 12/08/2007
Ruthenium(II) Estradiol Polypyridine Complexes
accurate index of the cancer.11 Since the binding affinity of
estradiol to ERs is the highest among all estrogens,12 various
therapeutic and diagnostic units modified with estradiol have
been used to study estrogen binding and develop site-specific
drugs for ER-related diseases.12-19 These units include
radioactive labels,12,14 radiopharmaceuticals,13 IR-active
organometallic complexes,15 and organic fluorophores.16 In
addition, biotinylated estradiol has been used to develop an
enzyme immunoassay for estradiol in human plasma.17 We
have recently reported luminescent tricarbonylrhenium(I)18
and cyclometalated iridium(III)19 polypyridine estradiol
conjugates that can recognize ERs. In view of the high
photostability, low-energy absorption, and relatively longlived luminescence of ruthenium(II) polypyridine complexes,20 we anticipate that they are promising candidates
as luminescent biological probes.
Here we report the synthesis, characterization, and photophysical and electrochemical properties of four luminescent
ruthenium(II) polypyridine estradiol complexes [Ru(N∧N)2(bpy-estradiol)](PF6)2 (N∧N ) 2,2′-bipyridine (bpy), 4,7(11) (a) McGuire, W. L. Proc. Soc. Exp. Biol. Med. 1979, 162, 22. (b)
McCarty, K. S., Jr.; Reintgen, D. S.; Seigler, H. F. Br. Cancer Res.
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Chamness, G. C.; Huff, K.; McGuire, W. L. Steroids 1975, 25, 627.
(c) Zava, D. T.; Harrington, N. Y.; McGuire, W. L. Biochemistry 1976,
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J. A.; Welch, M. J. J. Med. Chem. 1993, 36, 1619. (e) Katzenellenbogen, J. A. J. Fluor. Chem. 2001, 109, 49. (f) Luyt, L. G.; Bigott, H.
M.; Welch, M. J.; Katzenellenbogen, J. A. Bioorg. Med. Chem. 2003,
11, 4977.
(15) (a) Vessières, A.; Top, S.; Ismail, A. A.; Butler, I. S.; Louer, M.;
Jaouen, G. Biochemistry 1988, 27, 6659. (b) Jaouen, G.; Vessières,
A.; Butler, I. S. Acc. Chem. Res. 1993, 26, 361.
(16) (a) Katzenellenbogen, J. A.; Johnson, H. J., Jr.; Myers, H. N.
Biochemistry 1973, 12, 4085. (b) Anstead, G. M.; Carlson, K. E.;
Katzenellenbogen, J. A. Steroids 1997, 62, 268. (c) Adamczyk, M.;
Johnson, D. D.; Reddy, R. E. Bioconjugate Chem. 1998, 9, 403. (d)
Adamczyk, M.; Chen, Y.-Y.; Moore, J. A.; Mattingly, P. G. Bioorg.
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(17) Mares, A.; DeBoever, J.; Stans, G.; Bosmans, E.; Kohen, F. J.
Immunol. Methods 1995, 183, 211.
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Eur. J. 2007, 13, 7110.
Chart 1. Structures of the Ruthenium Estradiol Complexes
diphenyl-1,10-phenanthroline (Ph2-phen); bpy-estradiol )
5-(4-(17R-ethynylestradiolyl)phenyl)-2,2′-bipyridine (bpy-phest), 4-(N-(6-(4-(17R-ethynylestradiolyl)benzoylamino)hexyl)aminomethyl)-4′-methyl-2,2′-bipyridine (mbpy-C6-est)) (Chart
1). The lipophilicity of these complexes has been determined
by reversed-phase HPLC. The binding of the complexes to
estrogen receptor-R (ERR) has been studied by emission
titrations. The results have been compared to control experiments involving the estradiol-free analogues [Ru(bpy)3](PF6)2
and [Ru(Ph2-phen)2(bpy)](PF6)2. Additionally, the cytotoxicity of the ruthenium(II) estradiol complexes toward the
HeLa cell line has been examined by the 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
The cellular uptake of the complexes has been investigated
by flow cytometry and laser-scanning confocal microscopy.
Experimental Section
Materials and Synthesis. All solvents were of analytical grade.
All buffer components were of biological grade and used as
received. Diethylamine (Sigma) and DMF (Lab-Scan) were freshly
(20) (a) Kalyanasundaram, K. Photochemistry of Polypyridine and Porphyrin Complexes; Academic Press: San Diego, CA, 1992. (b)
Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer
Academic and Plenum Publishers: New York, 2007. (c) Kalyanasundaram, K. Coord. Chem. ReV. 1982, 46, 159. (d) Watts, R. J. J.
Chem. Educ. 1983, 60, 834. (e) Juris, A.; Balzani, V.; Barigelletti, F.;
Campagna, S.; Belser, P.; von Zelewsky, A. Coord. Chem. ReV. 1988,
84, 85. (f) Terpetschnig, E.; Szmacinski, H.; Lakowicz, J. R. Anal.
Biochem. 1995, 227, 140. (g) Lo, K. K.-W.; Lee, T. K.-M. Inorg.
Chem. 2004, 43, 5275. (h) Lo, K. K.-W.; Lee, T. K.-M.; Zhang, K.
Y. Inorg. Chim. Acta 2006, 359, 1845. (i) Lo, K. K.-W.; Lee, T. K.M. Inorg. Chim. Acta 2007, 360, 293.
Inorganic Chemistry, Vol. 47, No. 1, 2008
201
Lo et al.
distilled over KOH and MgSO4, respectively, under nitrogen before
use. Ruthenium(III) chloride hydrate (Arcos), bpy (Acros), Ph2phen (Aldrich), 17R-ethynylestradiol (Aldrich), triphenylphosphine
(Aldrich), palladium(II) chloride (Acros), copper(I) iodide (Acros),
5-(4-bromophenyl)-2,2′-bipyridine (Wako), 4-iodobenzoic acid
(Acros), N-hydroxysuccinimide (Acros), N,N′-dicyclohexylcarbodiimide (Acros), 1,6-hexanediamine (Acros), cisplatin (Acros), MTT
(Sigma), NaBH4 (Acros), and KPF6 (Acros) were used without
purification. 4′-Methyl-2,2′-bipyridyl-4-carboxaldehyde,21 cis-[Ru(N∧N)2Cl2]‚2H2O,22 and bpy-ph-est19 were prepared by reported
methods.
Lamb uteri cytosol was used as a source of ERR, which was
purified and quantitated according to reported procedures.15a,23 Lamb
uteri tissues obtained from the HKSAR slaughterhouse in Sheung
Shui, New Territories, Hong Kong, were immediately frozen after
isolation and stored at -70 °C prior to purification. Before
purification, they were thawed and minced. The resulting tissues
were ground using mortar and pestle in 50 mM Tris-Cl with 0.25
M sucrose and 0.1% 2-mercaptoethanol, pH 7.4, at 25 °C. The
homogenate was centrifuged at 14 000 rpm for 1 h at 4 °C to
remove the solid residue. Uterine cytosol was made 30% saturated
with ammonium sulfate and centrifuged at 14 000 rpm for 30 min
at 4 °C. After removal of the supernatant, the pellets were stored
at -70 °C. Before use, the receptor pellets were thawed on ice and
then dissolved in ice-cold 50 mM potassium phosphate buffer. The
concentration of ERR was determined by the Bradford method.23
Human cervix epithelioid carcinoma (HeLa) cells were obtained
from the American Type Culture Collection. Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsinEDTA, and penicillin/streptomysin were purchased from Invitrogen.
The growth medium for cell culture contained DMEM with 10%
FBS and 1% penicillin/streptomysin.
4-(N-(6-(4-Iodobenzoylamino)hexyl)aminomethyl)-4′-methyl2,2′-bipyridine. A mixture of 4-iodobenzoic acid (1.00 g, 4.03
mmol), N-hydroxysuccinimide (0.56 g, 4.87 mmol), and N,N′dicyclohexylcarbodiimide (1.00 g, 4.87 mmol) in 100 mL of
anhydrous THF was stirred under nitrogen at room temperature
for 12 h. The white solid precipitated was removed by filtration.
The filtrate was evaporated to dryness to give a white solid. The
solid and 1,6-hexanediamine (4.67 g, 40.26 mmol) were then
dissolved in 150 mL of CH2Cl2, and the solution was stirred at
room temperature for 12 h. The white solid precipitated was
removed by filtration. The filtrate was washed with H2O (300 mL
× 3). The organic layer was dried over MgSO4 and evaporated to
dryness to give a white solid. The solid and 4′-methyl-2,2′-bipyridyl4-carboxaldehyde (0.80 g, 4.03 mmol) were dissolved in 30 mL of
MeOH, and the mixture was stirred at room temperature for 12 h.
The white solid precipitated and was collected by filtration. The
residue was washed with 30 mL of cold MeOH and was then
suspended in 150 mL of MeOH. Addition of NaBH4 solid (0.76 g,
20.11 mmol) led to a colorless solution, which was stirred at room
temperature for 5 h. The solution was evaporated to dryness to
give a white solid. The crude product was washed with water and
then dried in a vacuum desiccator. Yield ) 0.63 g (30%). Positiveion ESI-MS ion cluster: m/z ) 529, {M + H+}+.
Mbpy-C6-est. The procedure was similar to that of bpy-ph-est,19
except that 4-(N-(6-(4-iodobenzoylamino)hexyl)aminomethyl)-4′methyl-2,2′-bipyridine (396 mg, 0.75 mmol) was used instead of
(21) Peek, B. M.; Ross, G. T.; Edwards, S. W.; Meyer, G. J.; Meyer, T. J.;
Erickson, B. W. Int. J. Peptide Protein Res. 1991, 38, 114.
(22) Sullivan, B. P.; Salmon, D. J.; Meyer, T. J. Inorg. Chem. 1978, 17,
3334.
(23) Bradford, M. M. Anal. Biochem. 1976, 72, 248.
202 Inorganic Chemistry, Vol. 47, No. 1, 2008
5-(4-bromophenyl)-2,2′-bipyridine. The crude product was washed
with cold DMF and diethyl ether. The ligand mbpy-C6-est was
isolated as a pale brown solid. Yield ) 236 mg (45%). 1H NMR
(500 MHz, DMSO-d6, 298 K, TMS): δ ) 9.03 (s, 1H, 3-OH of
estradiol), 8.61-8.39 (m, 3H, bpy-4-CH2NHC6H12NH and H6 and
H6′ of pyridyl rings), 8.34 (s, 1H, H3 of pyridyl ring), 8.20 (s, 1H,
H3′ of pyridyl ring), 7.80 (d, 2H, J ) 6.7 Hz, H2 and H6 of phenyl
ring), 7.45 (d, 2H, J ) 6.5 Hz, H3 and H5 of phenyl ring), 7.35 (d,
1H, J ) 4.5 Hz, H5 of pyridyl ring), 7.23 (d, 1H, J ) 4.7 Hz, H5′
of pyridyl ring), 7.02 (d, 1H, J ) 7.6 Hz, H1 of estradiol), 6.556.39 (m, 2H, H2 and H4 of estradiol), 5.54 (s, 1H, 17-OH of
estradiol), 3.75 (s, 2H, bpy-4-CH2NH), 3.28-3.11 (m, 3H, bpy4-CH2NHC5H10CH2 and bpy-4-CH2NH), 2.78-2.49 (m, 2H, H6
of estradiol), 2.49-2.34 (m, 5H, bpy-4-CH2NHCH2, and CH3 on
C4′ of pyridyl ring), 2.31-2.02 (m, 4H, H9R, H11R, H12β, and
H16R of estradiol), 2.01-1.59 (m, 5H, H7β, H8β, H11β, H15R, and
H16β of estradiol), 1.58-1.11 (m, 12H, H7R, H12R, H14β, and H15β
of estradiol, and bpy-4-CH2NHCH2C4H8), 0.78 (s, 3H, CH3 of
estradiol). IR (KBr) (ν/cm-1): 3429 (br, O-H and N-H), 2196
(w, CtC), 1669 (s, CdO). Positive-ion ESI-MS ion cluster: m/z
) 697, {M + H+}+.
[Ru(bpy)2(bpy-ph-est)](PF6)2. A mixture of cis-[Ru(bpy)2Cl2]‚
2H2O (52 mg, 0.10 mmol) and bpy-ph-est (63 mg, 0.12 mmol) in
20 mL of 50% aqueous ethanol was heated at reflux for 12 h. The
color of the solution turned from purple to deep red. The volume
of the mixture was reduced to ca. 10 mL, and the solution was
then filtered. Excess KPF6 was added to the solution to precipitate
a deep red solid. The solid was collected by filtration, washed with
water, a small amount of cold MeOH, and diethyl ether. Recrystallization of the product from acetone/diethyl ether afforded the
target complex as red crystals. Yield ) 71 mg (58%). 1H NMR
(300 MHz, acetone-d6, 298 K, TMS): δ ) 8.94-8.77 (m, 6H, H3
and H3′ of pyridyl rings of bpy and bpy-ph-est), 8.49 (d, 1H, J )
8.2 Hz, H4 of pyridyl ring of bpy-ph-est), 8.32-8.01 (m, 14H,
3-OH of estradiol, H4, H4′, H6, and H6′ of bpy, H3 and H5 of
phenyl ring of bpy-ph-est, and H4′, H6, and H6′ of pyridyl rings
of bpy-ph-est), 7.65-7.51 (m, 7H, H5 and H5′ of bpy, H2 and H6
of phenyl ring of bpy-ph-est, and H5′ of pyridyl ring of bpy-phest), 7.12 (d, 1H, J ) 8.5 Hz, H1 of estradiol), 6.71 (dd, 1H, J )
8.5 and 5.6 Hz, H2 of estradiol), 6.53 (s, 1H, H4 of estradiol),
4.56 (s, 1H, 17-OH of estradiol), 2.83-2.72 (m, 2H, H6 of
estradiol), 2.41-2.23 (m, 4H, H9R, H11R, H12β, and H16R of
estradiol), 1.95-1.72 (m, 5H, H7β, H8β, H11β, H15R, and H16β of
estradiol), 1.54-1.21 (m, 4H, H7R, H12R, H14β, and H15β of
estradiol), 0.95 (s, 3H, CH3 of estradiol). IR (KBr) (ν/cm-1): 3418
(m, O-H), 2207 (w, CtC), 838 (s, P-F). Positive-ion ESI-MS
ion cluster: m/z 1085, {M - PF6-}+, 470, {M - 2PF6-}2+. Anal.
Calcd for C56H50N6O2F12P2Ru‚2H2O: C, 53.13; H, 4.30; N 6.64.
Found: C, 53.38; H, 4.29; N, 6.77.
[Ru(bpy)2(mbpy-C6-est)](PF6)2. The procedure was similar to
that described for the preparation of complex [Ru(bpy)2(bpy-phest)](PF6)2, except that mbpy-C6-est (84 mg, 0.12 mmol) was used
instead of bpy-ph-est. Recrystallization of the crude product from
acetone/diethyl ether afforded the target complex as red crystals.
Yield ) 57 mg (41%). 1H NMR (300 MHz, acetone-d6, 298 K,
TMS): δ ) 8.84-8.74 (m, 5H, H3 of pyridyl ring of mbpy-C6est and H3 and H3′ of bpy), 8.68 (s, 1H, H3′ of pyridyl ring of
mbpy-C6-est), 8.26-8.13 (m, 5H, 3-OH of estradiol and H4 and
H4′ of bpy), 8.10-8.01 (m, 4H, H6 and H6′ of bpy), 7.96-7.80
(m, 4H, H6 and H6′ of pyridyl rings of mbpy-C6-est and H2 and
H6 of phenyl ring of mbpy-C6-est), 7.64-7.36 (m, 9H, bpy-4CH2NH, H5 and H5′ of pyridyl rings of bpy and mbpy-C6-est,
and H3 and H5 of phenyl ring of mbpy-C6-est), 7.12 (d, 1H, J )
Ruthenium(II) Estradiol Polypyridine Complexes
8.6 Hz, H1 of estradiol), 6.66-6.51 (m, 2H, H2 and H4 of
estradiol), 4.53 (s, 1H, 17-OH of estradiol), 4.02 (s, 2H, bpy-4CH2NH), 3.49-3.31 (m, 2H, bpy-4-CH2NHC5H10CH2), 2.95-2.85
(m, 2H, H6 of estradiol), 2.78 (br, 1H, bpy-4-CH2NH), 2.65-2.51
(m, 5H, bpy-4-CH2NHCH2 and CH3 on C4′ of pyridyl ring of mbpyC6-est), 2.41-2.21 (m, 4H, H9R, H11R, H12β, and H16R of
estradiol), 1.94-1.74 (m, 5H, H7β, H8β, H11β, H15R, and H16β of
estradiol), 1.68-1.16 (m, 12H, H7R, H12R, H14β, and H15β of
estradiol and bpy-4-CH2NHCH2C4H8), 0.96 (s, 3H, CH3 of estradiol). IR (KBr) (ν/cm-1): 3428 (br, O-H and N-H), 2208 (w,
CtC), 1673 (s, CdO), 836 (s, P-F). Positive-ion ESI-MS ion
cluster: m/z 1255, {M - PF6-}+, 555, {M - 2PF6-}2+. Anal. Calcd
for C65H68N8O3F12P2Ru‚H2O: C, 55.05; H, 4.97; N, 7.90. Found:
C, 55.14; H, 5.12; N, 7.70.
[Ru(Ph2-phen)2(bpy-ph-est)](PF6)2. The procedure was similar
to that described for the preparation of complex [Ru(bpy)2(bpyph-est)](PF6)2, except that cis-[Ru(Ph2-phen)2Cl2]‚2H2O (87 mg,
0.10 mmol) was used instead of cis-[Ru(bpy)2Cl2]‚2H2O. Recrystallization of the crude product from acetone/diethyl ether afforded
the target complex as red crystals. Yield ) 52 mg (33%). 1H NMR
(300 MHz, acetone-d6, 298 K, TMS): δ ) 9.03-8.90 (m, 2H, H3
and H3′ of pyridyl rings of bpy-ph-est), 8.84 (d, 1H, J ) 5.1 Hz,
H2 of Ph2-phen), 8.71 (d, 1H, J ) 5.6 Hz, H2 of Ph2-phen), 8.63
(d, 1H, J ) 5.6 Hz, H9 of Ph2-phen), 8.59-8.47 (m, 2H, H9 of
Ph2-phen and H4 of pyridyl ring of bpy-ph-est), 8.38-8.12 (m,
9H, H5 and H6 of Ph2-phen, H3 and H5 of phenyl ring of bpyph-est, and H4′, H6, and H6′ of pyridyl rings of bpy-ph-est), 8.03
(s, 1H, 3-OH of estradiol), 7.97 (d, 1H, J ) 5.6 Hz, H3 of Ph2phen), 7.94 (d, 1H, J ) 5.1 Hz, H3 of Ph2-phen), 7.79 (d, 1H, J )
5.9 Hz, H8 of Ph2-phen), 7.76 (d, 1H, J ) 5.1 Hz, H8 of Ph2phen), 7.71-7.46 (m, 23H, phenyl rings of Ph2-phen, H2 and H6
of phenyl ring of bpy-ph-est, and H5′ of pyridyl ring of bpy-phest), 7.11 (d, 1H, J ) 8.5 Hz, H1 of estradiol), 6.62 (dd, 1H, J )
8.5 and 5.5 Hz, H2 of estradiol), 6.56 (s, 1H, H4 of estradiol),
4.59 (s, 1H, 17-OH of estradiol), 2.82-2.73 (m, 2H, H6 of
estradiol), 2.41-2.21 (m, 4H, H9R, H11R, H12β, and H16R of
estradiol), 2.03-1.68 (m, 5H, H7β, H8β, H11β, H15R, and H16β of
estradiol), 1.58-1.18 (m, 4H, H7R, H12R, H14β, and H15β of
estradiol), 0.93 (s, 3H, CH3 of estradiol). IR (KBr) (ν/cm-1): 3421
(m, O-H), 2213 (w, CtC), 831 (s, P-F). Positive-ion ESI-MS
ion cluster: m/z 1437, {M - PF6-}+, 646, {M - 2PF6-}2+. Anal.
Calcd for C84H66N6O2F12P2Ru: C, 63.76; H, 4.20; N, 5.31. Found:
C, 63.64; H, 4.02; N, 5.60.
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2. The procedure was similar to that described for the preparation of complex [Ru(bpy)2(mbpyC6-est)](PF6)2, except that cis-[Ru(Ph2-phen)2Cl2]‚2H2O (87 mg,
0.10 mmol) was used instead of cis-[Ru(bpy)2Cl2]‚2H2O. Recrystallization of the crude product from acetone/diethyl ether afforded
the complex as red crystals. Yield ) 53 mg (30%). 1H NMR (300
MHz, acetone-d6, 298 K, TMS): δ ) 9.32 (s, 1H, H3 of pyridyl
ring of mbpy-C6-est), 8.91 (s, 1H, H3′ of pyridyl ring of mbpyC6-est), 8.77-8.58 (m, 2H, H2 of Ph2-phen), 8.54-8.41 (m, 2H,
H9 of Ph2-phen), 8.31 (s, 4H, H5 and H6 of Ph2-phen), 8.16 (s,
1H, 3-OH of estradiol), 8.06-7.82 (m, 4H, H2 and H6 of phenyl
ring and H6 and H6′ of pyridyl rings of mbpy-C6-est), 7.81-7.19
(m, 29H, phenyl rings of Ph2-phen, H3 and H5 of phenyl ring of
mbpy-C6-est, H5 and H5′ of pyridyl rings of mbpy-C6-est, and
H3 and H8 of Ph2-phen), 7.11 (d, 1H, J ) 8.3 Hz, H1 of estradiol),
6.61-6.48 (m, 2H, H2 and H4 of estradiol), 4.55 (s, 1H, 17-OH of
estradiol), 3.51-3.35 (m, 4H, bpy-4-CH2NH and bpy-4-CH2NHC5H10CH2), 3.01-2.93 (m, 2H, H6 of estradiol), 2.86 (br, 1H,
bpy-4-CH2NH), 2.65-2.39 (m, 5H, bpy-4-CH2NHCH2 and CH3
on C4′ of pyridyl ring of mbpy-C6-est), 2.38-2.21 (m, 4H, H9R,
H11R, H12β, and H16R of estradiol), 1.96-1.62 (m, 5H, H7β, H8β,
H11β, H15R, and H16β of estradiol), 1.61-1.02 (m, 12H, H7R, H12R,
H14β, and H15β of estradiol and bpy-4-CH2NHCH2C4H8), 0.92 (s,
3H, CH3 of estradiol). IR (KBr) (ν/cm-1): 3436 (br, O-H and
N-H), 2210 (w, CtC), 1677 (s, CdO), 838 (s, P-F). Positiveion ESI-MS ion cluster: m/z 1607, {M - PF6-}+, 731, {M 2PF6-}2+. Anal. Calcd for C93H84N8O3F12P2Ru: C, 63.73; H, 4.83;
N, 6.39. Found: C, 63.62; H, 4.54; N, 6.21.
Instrumentation and Methods. The instruments used for
characterization and photophysical and electrochemical studies have
been described previously.20g Luminescence quantum yields were
measured using the optically dilute method24 with an aerated
aqueous solution of [Ru(bpy)3]Cl2 (Φem ) 0.028, λex ) 455 nm)25
as the standard solution.
Determination of Lipophilicity. The lipophilicity of the complexes, which is referred to as log Po/w (Po/w ) n-octan-1-ol/water
partition coefficient), was determined from the log k′w values (k′w
) chromatographic capacity factor at 100% aqueous solution).
Detailed procedures for the determination of lipophilicity have been
described previously.18
Emission Titrations. Aliquots (25 µL) of an ERR solution (6.4
µM) in 50 mM potassium phosphate buffer at pH 7.4 at 298 K
were added to the ruthenium(II) estradiol complex or estradiolfree complex [Ru(N∧N)2(bpy)](PF6)2 (N∧N ) bpy, Ph2-phen) (0.92
µM) in 2 mL of 50 mM potassium phosphate buffer at pH 7.4/
DMSO (8:2, v/v) at 1-min intervals. The emission spectrum of the
solution was measured after each addition.
The Hill equation was used to determine the binding parameters
(Ka) of the complexes to ERR:26
log
(1 -Y Y) ) n log [ERR] + n log K
H
H
a
Here Y ) (Iobs - Imin)/(Imax - Imin), Iobs, Imin, and Imax are the
emission intensities of the apparent, free, and bound forms of the
ruthenium(II) complex, respectively, and nH and Ka are the Hill
coefficient and binding constant, respectively.
Cytotoxicity Assays.27 HeLa cells were seeded in a 96-well flatbottomed microplate (10 000 cells/well) in growth medium (100
µL) and incubated at 37 °C under a 5% CO2 atmosphere for 24 h.
The ruthenium(II) estradiol complexes and cisplatin (positive
control) were then added to the wells with concentrations ranging
from 10-6 to 10-4 M in a mixture of growth medium/DMSO (99:
1). Wells containing growth medium without cells were used as
blank controls. The microplate was incubated at 37 °C under a 5%
CO2 atmosphere for 48 h. Then, 10 µL of MTT in PBS (5 mg
mL-1) was added to each well. The microplate was incubated at
37 °C under a 5% CO2 atmosphere for another 3 h. Solubilization
solution (100 µL) containing 10% SDS in 2-propanol/0.04 M
hydrochloric acid (1:1, v/v) was then added to each well, and the
microplate was further incubated for 24 h. The absorbance of the
solutions at 570 nm was measured with a SPECTRAmax 340
microplate reader (Molecular Devices Corp., Sunnyvale, CA). The
IC50 values of the complexes were determined from dose dependence of surviving cells after exposure to the complexes for 48 h.
Flow Cytometry. HeLa cells in growth medium (100 000 cells
mL-1) were seeded in a 35-mm tissue culture dish and incubated
at 37 °C under a 5% CO2 atmosphere for 48 h. The culture medium
was removed and replaced with medium/DMSO (99:1, v/v)
(24) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991.
(25) Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697.
(26) Yamada, Y.; Matsuura, K.; Kobayashi, K. Bioorg. Med. Chem. 2005,
13, 1913.
(27) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
Inorganic Chemistry, Vol. 47, No. 1, 2008
203
Lo et al.
Table 1. Electronic Absorption Spectral Data of the Ruthenium Estradiol Complexes at 298 K
complex
[Ru(bpy)2(bpy-ph-est)](PF6)2
[Ru(bpy)2(mbpy-C6-est)](PF6) 2
solvent
λabs/nm (/dm3 mol-1 cm-1)
CH2Cl2
CH3CN
CH2Cl2
256 (33 520), 288 (80 435), 325 (41 665), 422 sh (10 815), 456 (14 710)
254 (34 675), 288 (81 095), 325 (49 315), 425 sh (11 985), 454 (15 010)
257 (36 560), 287 (74 320), 327 sh (9220), 358 sh (5250), 398 sh (5120),
423 sh (9470), 456 (12 115)
256 (37 780), 286 (69 680), 326 sh (10 010), 360 sh (5960), 397 sh (5925),
424 sh (10 225), 454 (12 565)
279 (97 005), 317 sh (46 560), 344 sh (32 600), 433 sh (20 110),
463 sh (21 820)
278 (96 365), 318 sh (46 720), 344 sh (30 325), 436 sh (20 700),
462 sh (21 895)
280 (117 725), 327 sh (21 075), 434 sh (23 575), 465 (24 320)
279 (112 320), 326 sh (19 500), 439 sh (23 390), 467 (22 905)
CH3CN
[Ru(Ph2-phen)2(bpy-ph-est)](PF6)2
CH2Cl2
CH3CN
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
CH2Cl2
CH3CN
containing the ruthenium(II) estradiol complexes at a concentration
of 5 µM. After incubation for 24 h, the medium was removed and
the cell layer was washed gently with PBS (1 mL × 3). The cell
layer was then trypsinized and added up to a final volume of 3 mL
with PBS. The samples were analyzed by a FACSCalibur flow
cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ). The
cell samples were excited with an argon laser at 488 nm, and the
emission was monitored at 585 ( 21 nm. The number of cells
analyzed for each sample was between ca. 9000 and 10 000.
Live-Cell Confocal Imaging. HeLa cells were grown on sterile
glass coverslips in a 35-mm tissue culture dish. The sample
preparation procedure was similar to that of the flow cytometry.
After washing with PBS, the coverslips were mounted onto slides
for measurements. Imaging was performed using a confocal
microscope (Carl Zeiss, LSM510) with an excitation wavelength
at 488 nm. The emission was measured using a long-pass filter at
505 nm.
Results and Discussion
Synthesis. 17R-Ethynylestradiol was chosen as the starting
material because the rigid ethynyl group at position 17R of
estradiol directs the substituent away from the 17β-hydroxyl
group without conformational flexibility and thus allows the
probe to have strong binding affinity to ERs.13b,c The diimine
ligands bpy-ph-est and mbpy-C6-est were prepared from
Sonogashira coupling of 17R-ethynylestradiol with the aryl
halides 5-(4-bromophenyl)-2,2′-bipyridine and 4-(N-(6-(4iodobenzoylamino)hexyl)aminomethyl)-4′-methyl-2,2′-bipyridine, respectively, in diethylamine in the presence of a
palladium(II) catalyst and a copper(I) cocatalyst. The ruthenium(II) polypyridine estradiol complexes were obtained from
the reactions of cis-[Ru(N∧N)2Cl2]‚2H2O22 with the corresponding bpy-estradiol ligands in refluxing aqueous ethanol,
followed by anion exchange with KPF6 and recrystallization
from a mixture of acetone and diethyl ether. All the
complexes were characterized by 1H NMR spectroscopy,
positive-ion ESI-MS, and IR spectroscopy and gave satisfactory microanalysis.
Electronic Absorption and Emission Properties. The
electronic absorption spectral data of all the complexes are
listed in Table 1. The electronic absorption spectra of the
complexes in CH3CN at 298 K are shown in Figure 1. All
the spectra featured intense absorption bands at ca. 254288 nm ( on the order of 104 dm3 mol-1 cm-1), which have
been assigned to spin-allowed intraligand (1IL) (π f π*)
204 Inorganic Chemistry, Vol. 47, No. 1, 2008
Figure 1. Electronic absorption spectra of [Ru(bpy)2(bpy-ph-est)](PF6)2
(blue), [Ru(bpy)2(mbpy-C6-est)](PF6)2 (green), [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2 (red), and [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 (black) in CH3CN
at 298 K.
(diimine) transitions.20a,c,e,28-33 The bpy-ph-est complexes
revealed an intense absorption band at ca. 317-325 nm
(Table 1 and Figure 1), which is associated with 1IL (π f
π*) (bpy-ph-est) transitions because a similar absorption band
has been observed in ruthenium(II) complexes containing
5-phenyl-substituted bpy ligands.34 The moderately intense
bands of all the complexes in the visible region (ca. 422467 nm) have been attributed to spin-allowed metal-to-ligand
(28) Paris, J. P.; Brandt, W. W. J. Am. Chem. Soc. 1959, 81, 5001.
(29) Staniewicz, R. J.; Sympson, R. F.; Hendricker, D. G. Inorg. Chem.
1977, 16, 2166.
(30) (a) Belser, P.; Zelewsky, A. V. HelV. Chim. Acta 1980, 63, 1675. (b)
Cook, M. J.; Lewis, A. P.; McAuliffe, G. S. G.; Skarda, V.; Thomson,
A. J.; Glasper, J. L.; Robbins, D. J. J. Chem. Soc., Perkin Trans. 2
1984, 1293. (c) Ackermann, M. N.; Interrante, L. V. Inorg. Chem.
1984, 23, 3904. (d) Ross, H. B.; Boldaji, M.; Rillema, D. P.; Blanton,
C. B.; White, R. P. Inorg. Chem. 1989, 28, 1013. (e) Kawanishi, Y.;
Kitamura, N.; Tazuke, S. Inorg. Chem. 1989, 28, 2968. (f) Mecklenburg, S. L.; Peek, B. M.; Schoonover, J. R.; McCafferty, D. G.; Wall,
C. G.; Erickson, B. W.; Meyer, T. J. J. Am. Chem. Soc. 1993, 115,
5479. (g) Mecklenburg, S. L.; McCafferty, D. C.; Schoonover, J. R.;
Peek, B. M.; Erickson, B. W.; Meyer, T. J. Inorg. Chem. 1994, 33,
2974. (h) de Carvalho, I. M. M.; de Sousa Moreira, IÄ.; Gehlen, M. H.
Inorg. Chem. 2003, 42, 1525.
(31) Kozlov, D. V.; Castellano, F. N. J. Phys. Chem. A 2004, 108, 10619.
(32) Baggott, J. E.; Gregory, G. K.; Pilling, M. J.; Andersonk, S.; Seddon,
K. R.; Turp, J. E. J. Chem. Soc., Faraday Trans. 2 1983, 79, 195.
(33) (a) Watts, R. J.; Crosby, G. A. J. Am. Chem. Soc. 1971, 93, 3184. (b)
Watts, R. J.; Crosby, G. A. J. Am. Chem. Soc. 1972, 94, 2606. (c)
Hager, G. D.; Watts, R. J.; Crosby, G. A. J. Am. Chem. Soc. 1975,
97, 7037. (d) Lin, C.-T.; Böttcher, W.; Chou, M.; Creutz, C.; Sutin,
N. J. Am. Chem. Soc. 1976, 98, 6536. (e) Alford, P. C.; Cook, M. J.;
Lewis, A. P.; McAuliffe, G. S. G.; Skarda, V.; Thomson, A. J.; Glasper,
J. L.; Robbins, D. J. J. Chem. Soc., Perkin Trans. 2 1985, 705.
(34) (a) Ghirotti, M.; Schwab, P. F. H.; Indelli, M. T.; Chiorboli, C.;
Scandola, F. Inorg. Chem. 2006, 45, 4331. (b) Ott, S.; Borgström,
M.; Hammarström, L.; Johansson, O. Dalton Trans. 2006, 1434.
Ruthenium(II) Estradiol Polypyridine Complexes
Table 2. Photophysical Data of the Ruthenium Estradiol Complexes
complex
[Ru(bpy)2(bpy-ph-est)]
(PF6)2
medium (T/K)
CH2Cl2 (298)
CH3CN (298)
bufferc (298)
glassd (77)
λem/nma
605
619
622
590 (max),
637
[Ru(bpy)2(mbpy-C6-est)]
CH2Cl2 (298) 603
CH3CN (298) 614
(PF6)2
bufferc (298) 615
glassd (77)
583 (max),
627
[Ru(Ph2-phen)2(bpy-ph-est)]
CH2Cl2 (298) 609
CH3CN (298) 615
(PF6)2
bufferc (298) 620
glassd (77)
590 (max),
639
[Ru(Ph2-phen)2(mbpy-C6-est)] CH2Cl2 (298) 605
CH3CN (298) 616
(PF6)2
bufferc (298) 619
glassd (77)
594 (max),
641
τo/µsb
Φa
1.05 0.081
1.30 0.067
0.81 0.051
5.23
0.96 0.070
0.97 0.084
0.71 0.046
5.01
2.14 0.17
2.35 0.13
2.11 0.074
7.01
3.79 0.23
5.12 0.18
2.88 0.10
8.82
a Excitation wavelength ) 455 nm. b Excitation wavelength ) 355 nm.
c 30% DMSO in 50 mM potassium phosphate buffer pH 7.4. d EtOH/MeOH
(4:1 v/v).
Figure 2. Emission spectra of [Ru(bpy)2(bpy-ph-est)](PF6)2 in CH3CN at
298 K (s) and in EtOH/MeOH (4:1, v/v) at 77 K (- - -).
charge-transfer 1MLCT (dπ(Ru) f π*(diimine)) transitions.20a,c,e,28-33 The 1MLCT bands of the bpy complexes
occurred at higher energy than those of Ph2-phen complexes
owing to the lower lying π* orbitals of the Ph2-phen ligand.
Upon irradiation, all the complexes displayed intense and
long-lived orange-red luminescence in fluid solutions under
ambient conditions and in low-temperature alcohol glass. The
photophysical data are summarized in Table 2. The emission
spectra of [Ru(bpy)2(bpy-ph-est)](PF6)2 in CH3CN at 298 K
and in alcohol glass at 77 K are shown in Figure 2. The
emission maxima of all the complexes occurred at ca. 603609 nm in CH2Cl2, ca. 614-619 nm in CH3CN, and ca. 615622 nm in aqueous buffer solution at 298 K. Upon cooling
of the samples to 77 K, the emission maxima of all the
complexes were blue-shifted to ca. 583-594 nm. With
reference to related photophysical studies of ruthenium(II)
polypyridine systems,20a,c-e,28,30-33,35,36 the emission has been
assigned to a 3MLCT (dπ(Ru) f π*(diimine)) excited state.
The emission energy of [Ru(bpy)2(bpy-ph-est)](PF6)2 is
slightly lower than its mbpy-C6-est counterpart [Ru(bpy)2(mbpy-C6-est)](PF6)2 (Table 2) and [Ru(bpy)3]2+,20a,c-e,28,30,35
suggesting that its emissive state possesses predominant
Table 3. Electrochemical Data of the Ruthenium Estradiol Complexes
in CH3CN (0.1 M TBAP) at 298 K (Glassy Carbon Working Electrode,
Sweep Rate ) 100 mV s-1, All Potentials vs SCE)
complex
oxidn, E1/2/V
redn, E1/2 or Ec/V
[Ru(bpy)2(bpy-ph-est)](PF6)2
+1.25a
[Ru(bpy)2(mbpy-C6-est)](PF6)2
+1.26a
[Ru(Ph2-phen)2(bpy-ph-est)](PF6)2
+1.22a
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
+1.22a
-1.26, -1.49,a
-1.66,b -1.79b
-1.35, -1.56,b
-1.66,b -1.88b
-1.26, -1.43,b
-1.64,b -1.79b
-1.30, -1.51,b
-1.68,b -1.87b
a Quasi-reversible couples. b Irreversible waves.
3
MLCT (dπ(Ru) f π*(bpy-ph-est)) character. The reason
is that the π* orbitals of bpy-ph-est are lower lying in energy
than those of bpy owing to the electron-withdrawing ethynylphenyl substituent. The assignment is supported by the
fact that the emission lifetime of [Ru(bpy)2(bpy-ph-est)](PF6)2
is slightly longer than those of [Ru(bpy)2(mbpy-C6-est)](PF6)2 (Table 2) and [Ru(bpy)3]2+,30b,e-h which is consistent
with the previous finding that aryl substitutions of polypyridine ligands generally increase the emission lifetimes of
ruthenium(II) polypyridine complexes.33b,c For the same
reason, the emission lifetimes and quantum yields of the Ph2phen complexes are longer and higher than those of the bpy
complexes (Table 2), suggesting that the Ph2-phen ligand is
significantly involved in the 3MLCT emissive state of these
complexes.31,33,36 Interestingly, [Ru(Ph2-phen)2(mbpy-C6est)](PF6)2 showed more intense and longer lived emission
than [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2. It is likely that the
emissive state of the latter complex has mixed contributions
from the Ph2-phen and bpy-ph-est ligands because of the
similar energies of their π* orbitals. In contrast, the electrondonating methyl and aminomethyl substituents of the mbpyC6-est ligand destabilize its π* orbitals, resulting in a lower
degree of involvement of this ligand in the emissive state of
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2. This is in agreement
with the fact that this complex shares similar emission
properties with its homoleptic counterpart [Ru(Ph2phen)3]2+.31,33,36
Electrochemical Properties. The electrochemical properties of the ruthenium(II) polypyridine estradiol complexes
have been studied by cyclic voltammetry, and the electrochemical data are listed in Table 3. All the complexes
displayed a quasi-reversible ruthenium(III/II) couple at ca.
+1.22 to +1.26 V vs SCE (ianodic/icathodic ) ca. 3.5-5.0). Since
common ruthenium(III/II) couples are reversible in
nature,20e,29,30a,c-e,g,35a,d,g,37 the reduced reversibility of these
(35) (a) Tokel-Takvoryan, N. E.; Hemingway, R. E.; Bard, A. J. J. Am.
Chem. Soc. 1973, 95, 6582. (b) Elfring, W. H., Jr.; Crosby, G. A. J.
Am. Chem. Soc. 1981, 103, 2683. (c) Braterman, P. S.; Harriman, A.;
Heath, G. A.; Yellowlees, L. J. J. Chem. Soc., Dalton Trans. 1983,
1801. (d) Caspar, J. V.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444.
(e) Caspar, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583. (f)
Cook, M. J.; Lewis, A. P.; McAuliffe, G. S. G.; Skarda, V.; Thomson,
A. J. J. Chem. Soc., Perkin Trans. 2 1984, 1303. (g) Mabrouk, P. A.;
Wrighton, M. S. Inorg. Chem. 1986, 25, 526. (h) Kumar, C. V.; Barton,
J. K.; Gould, I. R.; Turro, N. J.; Van Houten, J. Inorg. Chem. 1988,
27, 648.
(36) Demas, J. N.; Harris, E. W.; McBride, R. P. J. Am. Chem. Soc. 1977,
99, 3547.
(37) Guarr, T. F.; Anson, F. C. J. Phys. Chem. 1987, 91, 4037.
Inorganic Chemistry, Vol. 47, No. 1, 2008
205
Lo et al.
Table 4. Lipophilicity (log Po/w Values) of the Ruthenium Estradiol
Complexes, Estradiol, and 17R-Ethynylestradiol
compd
log Po/w
[Ru(bpy)2(bpy-ph-est)](PF6)2
[Ru(bpy)2(mbpy-C6-est)](PF6)2
[Ru(Ph2-phen)2(bpy-ph-est)](PF6)2
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
estradiol
17R-ethynylestradiol
1.33
2.18
7.71
8.58
3.26
3.42
couples could be a consequence of the involvement of
oxidation of the estradiol moiety of all the complexes and
the amine group of the mbpy-C6-est complexes.38 Also,
similar irreversible waves have been observed at comparable
potentials for the free ligands (ca. +1.21 V for bpy-ph-est
and ca. +0.94 and +1.56 V for mbpy-C6-est). Both bpyph-est complexes exhibited the first reversible reduction
couple at -1.26 V, which has been ascribed to the reduction
of the bpy-ph-est ligand. This assignment is supported by
the fact that this couple occurred at a slightly less negative
potential than that of the first bpy-based reduction of [Ru(bpy)3]2+ (ca. -1.33 V)30c,f,g,35a due to the electron-withdrawing ethynylphenyl group of the bpy-ph-est ligand. The first
reversible reduction couple of [Ru(bpy)2(mbpy-C6-est)](PF6)2
at -1.35 V has been assigned to the reduction of the ancillary
bpy ligand because it is more difficult to reduce the mbpyC6-est ligand due to its electron-donating methyl and
aminomethyl substituents. Owing to the lower lying π*
orbitals of Ph2-phen compared to those of mbpy-C6-est, the
first reduction couple of [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
at ca. -1.30 V has been assigned to the reduction of the
ancillary Ph2-phen ligand.30a,37
Lipophilicity. The cellular13d,g,39 and in vivo tissue13a,b,d,15a,14d,40 uptake selectivity characteristics of probes
and therapeutic reagents can be estimated by their lipophilicity. This is commonly referred to as the n-octan-1-ol/water
partition coefficients (expressed in log Po/w) of the compounds, which can be determined by reversed-phase HPLC.
The log Po/w values of the ruthenium(II) estradiol complexes,
estradiol, and 17R-ethynylestradiol are listed in Table 4.
Owing to their 2+ cationic charge, the bpy complexes are
less lipophilic than the natural hormone estradiol (ca. 3.26)
and 17R-ethynylestradiol (ca. 3.42) (Table 4).14d However,
the lipophilicity of the complexes can be substantially
increased by incorporating more hydrophobic ligands such
as Ph2-phen, as revealed by the much larger log Po/w values
of the Ph2-phen complexes than those of their bpy counterparts and the neutral estrogens (Table 4). The presence of a
spacer arm in the mbpy-C6-est complexes increased the log
Po/w values by ca. 0.9. The high lipophilicity of the Ph2phen complexes is anticipated to facilitate the tissue and
cellular uptake of these complexes.
(38) Cyclic voltammetric analysis of an equimolar solution of [Ru(Ph2phen)2(bpy-estradiol)](PF6)2 and ferrocene revealed that the anodic
current of the complexes at ca. +1.22 V vs SCE was ca. 1.6-1.7
times that of the ferrocene oxidation. This indicates that the wave is
associated with the transfer of more than one electron, reflecting the
possible involvement of the oxidation of the estradiol and amine
moieties in addition to the ruthenium(III/II) oxidation.
(39) Puckett, C. A.; Barton, J. K. J. Am. Chem. Soc. 2007, 129, 46.
(40) Vanbrocklin, H. F.; Liu, A.; Welch, M. J.; O’Neil, J. P.; Katzenellenbogen, J. A. Steroids 1994, 59, 34.
206 Inorganic Chemistry, Vol. 47, No. 1, 2008
Figure 3. Emission titration curves for the titrations of [Ru(bpy)2(bpyph-est)](PF6)2 (solid circles), [Ru(bpy)2(mbpy-C6-est)](PF6)2 (triangles), [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2 (diamonds), [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 (open circles), [Ru(bpy)3](PF6)2 (crosses), and [Ru(Ph2-phen)2(bpy)](PF6)2 (squares) with ERR in 50 mM potassium phosphate buffer
pH 7.4 at 298 K. Io and I are the emission intensities of the complexes in
the absence and presence of ERR, respectively.
Table 5. Relative Emission Intensities and Emission Lifetimes of the
Ruthenium Estradiol Complexes, [Ru(bpy)3](PF6)2, and
[Ru(Ph2-phen)2(bpy)](PF6)2 in the Absence and Presence of ERR in
Aerated 50 mM Potassium Phosphate Buffer pH 7.4 at 298 K
complex
I/Ioa
τob/µs
τb/µs
[Ru(bpy)2(bpy-ph-est)](PF6)2
[Ru(bpy)2(mbpy-C6-est)](PF6)2
[Ru(Ph2-phen)2(bpy-ph-est)](PF6)2
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
[Ru(bpy)3](PF6)2
[Ru(Ph2-phen)2(bpy)](PF6)2
0.93
0.90
2.25
1.78
0.89
1.30
0.44
0.40
0.74
0.72
0.37
0.71
0.45
0.37
1.96
1.10
0.36
0.92
a I and I are the emission intensities of the complexes in the absence
o
and presence of ERR, respectively. b τo and τ are the emission lifetimes of
the complexes in the absence and presence of ERR, respectively.
Emission Titrations. The ERR-binding properties of the
ruthenium(II) polypyridine estradiol complexes have been
investigated by emission titrations. The emission titration
curves are shown in Figure 3. While the bpy complexes did
not show significant changes, the emission intensities of the
Ph2-phen complexes were increased by ca. 2.3- and 1.8-fold
in the presence of ERR, and their lifetimes were also
elongated (Table 5). The emission spectra of [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2 in the absence and presence of ERR are
displayed in Figure 4. Interestingly, the control complex [Ru(Ph2-phen)2(bpy)](PF6)2 also revealed emission enhancement
and lifetime elongation in the presence of ERR but the
enhancement factors are relatively small (ca. 1.3). The higher
amplification factors of the Ph2-phen complexes have been
attributed to the specific binding of the estradiol moieties of
these complexes to ERR because similar changes were not
observed when unmodified estradiol was present from the
outset. The ERR-induced emission enhancement is a consequence of the increase in hydrophobicity and rigidity of
the local environment of the metal complexes upon the
binding event.18,19 Unfortunately, the bpy complexes, similar
to the estradiol-free complex [Ru(bpy)3](PF6)2, did not show
noticeable emission changes in the presence of ERR. This
is probably due to the fact that the photophysical properties
of ruthenium(II) bipyridine complexes are much less sensitive
to different media compared to their substituted phenanthro-
Ruthenium(II) Estradiol Polypyridine Complexes
Figure 4. Emission spectra of [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2 in the
absence (- - -) and presence (s) of ERR in aerated 50 mM potassium
phosphate buffer pH 7.4 at 298 K.
Figure 6. Results of flow cytometry of HeLa cells incubated with blank
medium (black), [Ru(bpy)2(bpy-ph-est)](PF6)2 (red), [Ru(bpy)2(mbpy-C6est)](PF6)2 (green), [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2 (blue), and [Ru(Ph2phen)2(mbpy-C6-est)](PF6)2 (orange) (5 µM) for 24 h.
Table 6. Cytotoxicity (IC50, 48 h) of the Ruthenium Estradiol
Complexes and Cisplatin toward the HeLa Cell Line
Figure 5. Hill plot for the binding of [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
to ERR.
line counterparts.20g-i,35e,41 From the titration data, the binding
constants (Ka) of [Ru(Ph2-phen)2(bpy-ph-est)](PF6)2 and [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 to ERR have been determined to be ca. 6.3 × 106 and 6.8 × 106 M-1, respectively.26
The Hill plot for [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 is
shown in Figure 5. The binding constants of the complexes
are smaller than that of unmodified estradiol (Ka ) 5 × 109
M-1)16a but are similar to common organometallic estradiol
complexes such as 17R-[(L)Re(CO)3]-estradiol (L ) 4′,4′bis(ethanethio)-4′-carboxybutyn-1′-yl, 6′,6′-bis(ethanethio)6′-carboxyhexyn-1′-yl; Ka ) 1.3 × 107 and 1.1 × 107 M-1,
respectively),14f 17R-[(CtCCH2N(CH3)C2H4N(CH3)2)Pt(X)]estradiol (X ) diiodide, malonato; Ka ) 1.0 × 107 and 2.5
× 106 M-1, respectively),42 [Re(N∧N)(CO)3(L)](CF3SO3)
(N∧N ) diimines; L ) pyridine-estradiol; Ka ) 1.5 to 2.0
× 107 M-1),18 and [Ir(N∧C)2(N∧N)](PF6) (N∧C- ) cyclometalating ligands; N∧N ) diimine-estradiol; Ka ) 1.0 to
2.1 × 107 M-1).19 The Hill coefficients (nH) of both [Ru(Ph2phen)2(bpy-ph-est)](PF6)2 and [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 (3.2 and 2.4, respectively) are >1, suggestive of
cooperative binding.16d,e,43
(41) (a) Meyer, T. J. Pure Appl. Chem. 1990, 62, 1003. (b) Fox, M. A.;
Channon Photoinduced Electron Transfer; Elsevier: Amsterdam, 1988.
(c) Hartshorn, R. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 5919.
(42) Cassino, C.; Gabano, E.; Ravera, M.; Cravotto, G.; Palmisano, G.;
Vessières, A.; Jaouen, G.; Mundwiler, S.; Alberto, R.; Osella, D. Inorg.
Chim. Acta 2004, 357, 2157.
(43) (a) Schwartz, J. A.; Skafar, D. F. Biochemistry 1993, 32, 10109. (b)
Schwartz, J. A.; Skafar, D. F. Biochemistry 1994, 33, 13267.
complex
IC50/µM
[Ru(bpy)2(bpy-ph-est)](PF6)2
[Ru(bpy)2(mbpy-C6-est)](PF6)2
[Ru(Ph2-phen)2(bpy-ph-est)](PF6)2
[Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2
cisplatin
133.4 ( 2.1
166.6 ( 4.5
141.6 ( 4.0
83.1 ( 2.2
34.3 ( 2.9
Cytotoxicity and Cellular Uptake Studies. The cytotoxicity of the ruthenium(II) estradiol complexes has been
studied by the MTT assay using HeLa cells as the model
cell line.27 The IC50 values have been determined from the
dose dependence of surviving cells after exposure to the
complexes for 48 h. The IC50 values of the ruthenium(II)
complexes ranged from 83.1 to 166.6 µM (Table 6), which
are substantially larger than that of cisplatin (34.3 µM) under
the same experimental conditions. The cytotoxicity of these
complexes is also lower than that of [Ru(tBu2-bpy)2(2appt)]2+ (tBu2-bpy ) 4,4′-di-tert-butyl-2,2′-bipyridine, 2-appt
) 2-amino-4-(phenylamino)-6-(2-pyridyl)-1,3,5-triazine) (59.7
µM), which has been identified to be a double-stranded DNA
groove-binder.44 In general, these complexes are much less
cytotoxic compared to the organometallic ruthenium arene
complexes [(η6-arene)Ru(ethylenediamine)(X)](PF6)n (X )
substituted pyridines and halides), some of which exhibit fast
hydrolysis kinetics and high cytotoxicity toward the human
ovarian cancer cell line A2780.45 Since our results indicate
that the ruthenium(II) estradiol complexes are relatively
noncytotoxic, they are promising candidates as luminescent
probes for live-cell imaging. The cellular uptake characteristics of the complexes have been investigated using flow
cytometry and laser-scanning confocal microscopy. The
results of the flow cytometric studies are shown in Figure
6. Upon excitation at 488 nm, all the cell samples incubated
with the ruthenium(II) estradiol complexes displayed higher
emission intensities compared to the autofluorescence of
(44) Ma, D.-L.; Che, C.-M.; Siu, F.-M.; Yang, M.; Wong, K.-Y. Inorg.
Chem. 2007, 46, 740.
(45) Wang, F.; Habtemariam, A.; van der Geer, E. P. L.; Fernández, R.;
Melchart, M.; Deeth, R. J.; Aird, R.; Guichard, S.; Fabbiani, F. P. A.;
Lozano-Casal, P.; Oswald, I. D. H.; Jodrell, D. I.; Parsons, S.; Sadler,
P. J. Proc. Nat. Aca. Sci. U.S.A. 2005, 102, 18269.
Inorganic Chemistry, Vol. 47, No. 1, 2008
207
Lo et al.
Figure 7. Fluorescence (left), brightfield (middle), and overlaid (right) images of HeLa cells incubated with [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 (5 µM)
at 37 °C for 24 h.
untreated HeLa cells, reflecting the efficient internalization
of the complexes by the cells. The emission intensities of
the cells treated with the Ph2-phen complexes are higher than
those treated with the bpy complexes, which is in accordance
with the relative emission quantum yields of the free
complexes (Table 2). It is conceivable that the efficient
internalization of the complexes by the cells is assisted by
their high lipophilicity, especially in the cases of the Ph2phen complexes (Table 4).39
The possibility of the ruthenium(II) estradiol complexes
as luminescent probes for live-cell imaging has been
examined using [Ru(Ph2-phen)2(mbpy-C6-est)](PF6)2 as an
example. Incubation of HeLa cells with the complex at
37 °C under a 5% CO2 atmosphere for 24 h led to efficient
interiorization of the complex as observed by laser-scanning
confocal microscopy with an excitation wavelength at 488
nm (Figure 7). It is interesting to note that most of the
complex molecules were distributed inside the cytoplasm
with a lower extent of nuclear uptake, as revealed by the
much weaker luminescence intensity of the nucleus.46
Importantly, a higher degree of localization of the complexes
in the perinuclear region suggests that the complex molecules
interact with hydrophobic organelles such as endoplasmic
reticulum and Golgi apparatus. No interiorization was
(46) A possible reason for the minimal nuclear uptake is that HeLa cells
are ER-negative in nature. We expect that the use of cell lines such
as ER-positive MCF-7 would lead to more significant interiorization
of the complexes in the nucleus. Related work on other cell lines is in
progress.
(47) Reaven, E.; Tsai, L.; Azhar, S. J. Biol. Chem. 1996, 271, 16208.
208 Inorganic Chemistry, Vol. 47, No. 1, 2008
observed when the cells were incubated at 4 °C, implying
that the uptake of the complex and its subsequent localization
are due to energy-requiring processes such as endocytosis.47
Investigations on the detailed internalization mechanism are
underway.
Conclusions
Four ruthenium(II) polypyridine estradiol complexes have
been designed as luminescent biological probes. The photophysical and electrochemical properties and lipophilicity
of these complexes have been examined. The highly lipophilic Ph2-phen estradiol complexes revealed enhanced
emission intensities and extended lifetimes upon binding to
ERR, rendering these complexes new homogeneous probes
for the receptor. The cytotoxicity of all the complexes toward
HeLa cells was relatively low compared to cisplatin.
Importantly, flow cytometry and live-cell confocal imaging
studies showed that these complexes were readily interiorized
by HeLa cells and the emission of the complexes was
maintained after the uptake. All these findings indicate that
these complexes are very promising candidates as live-cell
imaging reagents that could contribute to the understanding
of cellular uptake of transition metal complexes.
Acknowledgment. We thank the Hong Kong Research
Grants Council (Project Nos. CityU 101605 and 101606) for
financial support. We also thank Mr. Kenneth King-Kwan
Lau and Mr. Michael Wai-Lun Chiang for their assistance
on the cellular and imaging studies.
IC701735Q