Development of a zinc ion-selective luminescent lanthanide chemosensor for biological applications

Article abstract of DOI:10.1021/ja0469333

Detection of chelatable zinc (Zn²⁺) in biological studies has attracted much attention recently, because chelatable Zn²⁺ plays important roles in many biological systems. Lanthanide complexes (Eu ³⁺, Tb³⁺, etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn²⁺. This europium (Eu³⁺) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn²⁺. Upon addition of Zn²⁺ to a solution of [Eu-7], the luminescence of Eu³⁺ is strongly enhanced, with high selectivity for Zn²⁺ over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn²⁺-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn²⁺ changes in living HeLa cells was confirmed. This novel Zn²⁺-selective luminescent lanthanide chemosensor [Eu-7] should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications.

Full text of DOI:10.1021/ja0469333

Published on Web 09/10/2004
Development of a Zinc Ion-Selective Luminescent Lanthanide
Chemosensor for Biological Applications
Kenjiro Hanaoka,† Kazuya Kikuchi,*,†,‡ Hirotatsu Kojima,† Yasuteru Urano,† and
Tetsuo Nagano*,†
Contribution from the Graduate School of Pharmaceutical Sciences,
The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,
and Presto, JST Corporation, Kawaguchi, Saitama 332-0012, Japan
Received May 25, 2004; E-mail: tlong@mol.f.u-tokyo.ac.jp (T.N.); kkikuchi@mol.f.u-tokyo.ac.jp (K.K.)
Abstract: Detection of chelatable zinc (Zn2+) in biological studies has attracted much attention recently,
because chelatable Zn2+ plays important roles in many biological systems. Lanthanide complexes (Eu3+
,
Tb3+, etc.) have excellent spectroscopic properties for biological applications, such as long luminescence
lifetimes of the order of milliseconds, a large Stoke’s shift of >200 nm, and high water solubility. Herein,
we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn2+
.
This europium (Eu3+) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn2+
.
Upon addition of Zn2+ to a solution of [Eu-7], the luminescence of Eu3+ is strongly enhanced, with high
selectivity for Zn2+ over other biologically relevant metal cations. One of the important advantages of
[Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared
with previously reported Zn2+-sensitive luminescent lamthanide sensors, whose excitation wavelength is
at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn2+ changes
in living HeLa cells was confirmed. This novel Zn2+-selective luminescent lanthanide chemosensor [Eu-7]
should be an excellent lead compound for the development of a range of novel luminescent lanthanide
chemosensors for biological applications.
Introduction
the etiology of Alzheimer’s disease. Although many reports de-
scribe the significance of chelatable Zn2+ in biological systems,
its mechanisms of action are less well understood than those of
other cations, such as Ca2+, Na+, and K+. Therefore, there is
considerable interest in detecting chelatable Zn2+ in biological
systems.11
So far, several Zn2+-selective fluorescent sensor molecules
have been reported.12 However, novel types of Zn2+-sensitive
fluorescent sensor molecules are needed for studies on biological
phenomena, and several new types of sensors that are designed
for ratiometric measurement,13 or that are peptide- or protein-
based,14 have recently been introduced. Ratiometric measure-
Zinc (Zn2+) is the second most abundant heavy metal ion
after iron in the human body, and the total zinc ion concentration
in serum is of the order of 10 µM.1 Zn2+ is an essential com-
ponent of many enzymes (e.g., carbonic anhydrase), and also
plays critical roles in maintaining key structural features of gene
transcription proteins (e.g., zinc finger proteins).2 Moreover,
chelatable Zn2+ is present at especially high concentrations in
brain,3 pancreas,4 and spermatozoa.5 Chelatable Zn2+ regulates
neuronal transmission in excitatory nerve terminals,3 suppresses
apoptosis,6 contributes to neuronal injury in certain acute con-
ditions,7 epilepsy,8 and transient global ischemia,9 and induces
the formation of â-amyloid,10 which is reported to be related to
(10) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; Paradis, M. D.; Vonsattel, J.
P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi, R. E. Science
1994, 265, 1464-1467.
† The University of Tokyo.
‡ Presto, JST Corporation.
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182-191. (b) Jiang, P.; Guo, Z. Coord. Chem. ReV. 2004, 248, 205-229.
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Neurosci. Methods 1987, 20, 91-103. (b) Zalewski, P. D.; Forbes, I. J.;
Betts, W. H. Biochem. J. 1993, 296, 403-408. (c) Hirano, T.; Kikuchi,
K.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 6555-6562. (d)
Walkup, G. K.; Burdette, S. C.; Lippard, S. J.; Tsien, R. Y. J. Am. Chem.
Soc. 2000, 122, 5644-5645. (e) Haugland, R. P. Handbook of Fluorescent
Probes and Research Chemicals, 6th ed.; Molecular Probes, Inc.; Eugene,
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Lincoln, S. F.; Ward, A. D. J. Am. Chem. Soc. 2003, 125, 3889-3895. (g)
Gee, K. R.; Zhou, Z. L.; Qian, W. J.; Kennedy, R. J. Am. Chem. Soc.
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Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1778-1787.
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9
12470
J. AM. CHEM. SOC. 2004, 126, 12470-12476
10.1021/ja0469333 CCC: $27.50 © 2004 American Chemical Society

+
A Luminescent Lanthanide Chemosensor for Zn2
A R T I C L E S
ment in particular enables more precise analysis of Zn2+
concentrations. The other approach for precise analyses is time-
resolved fluorescence (TRF) measurement, which offers a better
signal-to-noise ratio. Luminescent lanthanide complexes are
suitable for TRF measurement. So, our interest in Zn2+-selective
fluorescent sensor molecules was directed toward luminescent
lanthanide complexes, in particular complexes of the europium
and terbium trivalent ions (Eu3+ and Tb3+). These complexes
have large Stoke’s shifts (>200 nm), long luminescence
lifetimes of the order of milliseconds, and high water solubility,15
whereas the typical organic fluorescent compounds possess small
Stoke’s shifts (Stoke’s shifts of fluorescein and rhodamine are
∼25 and ∼20 nm, respectively)12c-e,16 and short fluorescence
lifetimes in the nanosecond region. The long-lived luminescence
of the lanthanides has the advantage that short-lived background
fluorescence and scattered light decay to negligible levels when
a pulse of excitation light is applied and the emitted light is
collected after an appropriate delay time. For these reasons,
sensitization of lanthanide luminescence has been exploited for
a number of useful signaling systems for time-resolved assays
in the fields of medicine, biotechnology, and biological sci-
ence.17 The lanthanide f-f transitions have low absorbance, so
the ligand structure requires a sensitizing chromophore for high
luminescence.18 Absorption by the chromophore results in
effective population of its triplet level, and efficient intramo-
lecular energy transfer occurs from the excited chromophore to
the lanthanide metal, whereby the metal becomes excited to the
emission state (Scheme 1).15a,17g,18b By means of appropriate
chromophore design, it is possible to develop luminescent
lanthanide complexes which can be used to sense various
biological molecules.
Scheme 1. (a) Schematic View of a Chromophore Incorporated
into a Europium Emittera and (b) the General
Chromophore-to-Europium Ion Sensitization Processb
a The emission from Eu3+ after excitation of the chromophore is shown.
bLight absorption and lowest-lying singlet excited state (S1) formation at
the sensitizing chromophore are followed by intersystem crossing (ISC),
resulting in population of the lowest-lying triplet excited state (T1).
Subsequent chromophore-to-Eu3+ energy transfer leads to population of a
metal-centered level, which deactivates from Eu3+-emitting states to the
relevant ground states.
the novel lanthanide complexes obtained showed a large en-
hancement of luminescence upon Zn2+ addition with an apparent
dissociation constant Kd of 2.6 nM (295 K, pH 7.4).19b However,
these compounds are unsuitable for biological applications,
because of their short excitation wavelength, small emission
enhancement, inconvenient pH sensitivity, insufficient selectivity
for Zn2+, etc. So, it is necessary to develop sensors with a longer
excitation wavelength for biological applications without losing
the high selectivity and high affinity for Zn2+. Moreover,
development of a simple sensor switch for Zn2+, which would
serve as both chromophore and Zn2+ receptor, would be useful.
From this background, we set out to develop a novel lanthanide
complex which can detect Zn2+ in biological systems, in the
relevant concentration range.
There are only a few reports about lanthanide-based lumi-
nescent chemosensors for the detection of Zn2+ 19
Parker and
.
co-workers have developed a luminescent lanthanide agent
which binds Zn2+ with an apparent dissociation constant Kd of
0.6 µM (295 K, pH 7.3).19a,c We employed a different design
approach for a ligand and an antenna in a previous report, and
(14) (a) Thompson, R. B.; Cramer, M. L.; Bozym, R.; Fierke, C. A. J. Biomed.
Opt. 2002, 7, 555-560. (b) Shults, M. D.; Pearce, D. A.; Imperiali, B. J.
Am. Chem. Soc. 2003, 125, 10591-10597. (c) Barondeau, D. P.; Kassmann,
C. J.; Tainer, J. A.; Getzoff, E. D. J. Am. Chem. Soc. 2002, 124, 3522-
3524.
(15) (a) Parker, D.; Williams, J. A. G. J. Chem. Soc., Dalton Trans. 1996, 3613-
3628. (b) Li, M.; Selvin, P. R. J. Am. Chem. Soc. 1995, 117, 8132-8138.
(c) Beck, J. B.; Rowan, S. J. J. Am. Chem. Soc. 2003, 125, 13922-13923.
(d) Franz, K. J.; Nitz, M.; Imperiali, B. ChemBioChem 2003, 4, 265-271.
(e) Liu, W.; Jiao, T.; Li, Y.; Liu, Q.; Tan, M.; Wang, H.; Wang, L. J. Am.
Chem. Soc. 2004, 126, 2280-2281. (f) Weibel, N.; Charbonnie`re, L. J.;
Guardigli, M.; Roda, A.; Ziessel, R. J. Am. Chem. Soc. 2004, 126, 4888-
4896. (g) Alpha, B.; Lehn, J. M.; Mathis, G. Angew. Chem., Int. Ed. Engl.
1987, 26, 266-267. (h) Petoud, S.; Cohen, S. M.; Bu¨nzli, J. C. G.;
Raymond, K. N. J. Am. Chem. Soc. 2003, 125, 13324-13325. (i) Maffeo,
D.; Williams, J. A. G. Inorg. Chim. Acta 2003, 355, 127-136.
Here we report the design and synthesis of the novel Zn2+
-
sensitive luminescent lanthanide chemosensor [Eu-7]; upon
complexation with Zn2+, it exhibits strong, long-lived lumines-
cence (of the order of milliseconds), and it also offers a large
Stoke’s shift (>250 nm), high water-solubility, and high
selectivity for Zn2+ (Figure 1).
(16) Mizukami, S.; Kikuchi, K.; Higuchi, T.; Urano, Y.; Mashima, T.; Tsuruo,
T.; Nagano, T. FEBS Lett. 1999, 453, 356-360.
(17) (a) Kolb, A. J.; Kaplita, P. V.; Hayes, D. J.; Park, Y. W.; Pernell, C.; Major,
J. S.; Mathis, G Drug DiscoVery Today 1998, 3, 333-342. (b) Enomoto,
K.; Araki, A.; Nakajima, T.; Ohta, H.; Dohi, K.; Pre´audat, M.; Seguin, P.;
Mathis, G.; Suzuki, R.; Kominami, G.; Takemoto, H. J. Pharm. Biomed.
Anal. 2002, 28, 73-79. (c) Beeby, A.; Botchway, S. W.; Clarkson, I. M.;
Faulkner, S.; Parker, A. W.; Parker, D.; Williams, J. A. G. J. Photochem.
Photobiol. B: Biology 2000, 57, 83-89. (d) Frias, J. C.; Bobba, G.; Cann,
M. J.; Hutchison, C. J.; Parker, D. Org. Biomol. Chem. 2003, 1, 905-907.
(e) Barrios, A. M.; Craik, C. S. Bioorg. Med. Chem. Lett. 2002, 12, 3619-
3623. (f) Vereb, G.; Jares-Erijman, E.; Selvin, P. R.; Jovin, T. M. Biophys.
J. 1998, 74, 2210-2222. (g) Matsumoto, K.; Nojima, T.; Sano, H.; Majima,
K. Macromol. Symp. 2002, 186, 117-121. (h) Karvinen, J.; Hurskainen,
P.; Gopalakrishnan, S.; Burns, D.; Warrior, U.; Hemmila¨, I. J. Biomol.
Screen. 2002, 7, 223-231. (i) Pre´audat, M.; Ouled-Diaf, J.; Alpha-Bazin,
B.; Mathis, G.; Mitsugi, T.; Aono, Y.; Takahashi, K.; Takemoto, H. J.
Biomol. Screen. 2002, 7, 267-274. (j) Lin, Z.; Wu, M.; Scha¨ferling, M.;
Wolfbeis, O. S. Angew. Chem., Int. Ed. 2004, 43, 1735-1738.
Results and Discussion
Design and Synthesis of [Eu-7] and [Gd-7]. N,N,N′,N′-
Tetrakis(2-pyridylmethyl)ethylenediamine20 (TPEN) shows high
selectivity for Zn2+ over other metal ions found under physi-
ological conditions, such as Ca2+ and Mg2+. Accordingly, we
(18) (a) Parker, D.; Dickins, R. S.; Puschmann, H.; Crossland, C.; Howard, J.
A. K. Chem. ReV. 2002, 102, 1977-2010. (b) Quici, S.; Marzanni, G.;
Carazzini, M.; Anelli, P. L.; Botta, M.; Gianolio, E.; Accorsi, G.; Armaroli,
N.; Barigelletti, F. Inorg. Chem. 2002, 41, 2777-2784. (c) Latva, M.;
Takalo, H.; Mukkala, V. M.; Matachescu, C.; Rodr´ıguez-Ubis, J. C.;
Kankare, J. J. Lumin. 1997, 75, 149-169. (d) Dadabhoy, A.; Faulkner, S.;
Sammes, P. G. J. Chem. Soc., Perkin Trans. 2 2002, 348-357.
9
J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004 12471

A R T I C L E S
Hanaoka et al.
Figure 1. Structures of Eu3+ and Gd3+ N-{N-[2-N,N-bis(2-pyridylmethyl)-
aminomethylquinolin-6-yl]carbamoylmethyl}-N,N′,N′′,N′′-diethylenetri-
aminetetraacetic acid complexes: [Eu-7] and [Gd-7].
designed a novel sensitive luminescent sensor for Zn2+ [Eu-7]
by the combination of Eu3+-diethylenetriaminepentaacetic acid
(DTPA) complex and a quinoline-containing TPEN-based ligand
for Zn2+. The quinoline chromophore was selected as an antenna
and a ligand, because quinoline has a longer excitation
wavelength (>300 nm)21 than pyridine and can coordinate to
metal ions.
Figure 2. Time-delayed emission spectra (excitation at 320 nm) of
[Eu-7] (50 µM) in the presence of various concentrations of Zn2+: 0, 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 2.0, 3.0, 4.0, 5.0, and 10.0
equiv of Zn2+ with respect to [Eu-7]. These spectra were measured at pH
7.4 (100 mM HEPES buffer) and 22 °C using a delay time of 0.05 ms and
a gate time of 1.00 ms. The inset shows the changes of the luminescence
5
7
intensity at λ ) 614 nm. The bands arise from D0 f FJ transitions; the
J values of the bands are labeled.
In the structure of [Eu-7], the quinolyl chromophore is fixed
close to the Eu3+ ion, allowing it to function as an antenna.
The synthetic schemes for the lanthanide complexes, [Eu-7]
and [Gd-7], and details of the chemical characterization of
compounds are provided in the Supporting Information.
Spectroscopic Characteristics of a Solution of [Eu-7] upon
Addition of Zn2+. The complex [Eu-7] in aqueous solution
was characterized by a time-delayed luminescence spectrum
with a delay time of 0.05 ms. The time-resolved luminescence
emission intensity of [Eu-7] (50 µM) increased significantly
(8.5-fold) upon addition of 1.0 equiv of Zn2+, with a large
Stoke’s shift of >250 nm. The emission intensity remained at
a plateau in the presence of an excess of Zn2+ (Figure 2). The
luminescence emission displayed three bands at 579, 593, and
614 nm, corresponding to the deactivation from the 5D0 excited
state to F0, F1, and F2 ground state, respectively.18b,21b The
UV-vis absorption spectral change was monitored during Zn2+
addition. The absorption spectrum of [Eu-7] without Zn2+ had
λmax ) 249 nm and broad bands at 330 and 318 nm, tailing out
to 350 nm (Figure 3). The absorption spectrum of [Eu-7]
changed upon addition of Zn2+ (0-1.0 equiv), with three
isosbestic points at 334, 268, and 248 nm, and then remained
at a plateau upon further addition of Zn2+ (Figure 3 and Figure
S4), in accordance with the time-resolved luminescence spectra
(Figure 2). The three peaks (330, 318, and 249 nm) of absorption
changed to two peaks (320 and 253 nm) upon Zn2+ addition.
The absorption wavelength changes between 300 and 350 nm
were supposed to be due to the photophysical property change
of the quinolyl moiety with Zn2+ chelation, whereas pyridine-
Zn2+ coordination resulted in changes below 300 nm.19b Thus,
1:1 complex stoichiometry was observed in both the absorption
and the luminescence emission spectra of [Eu-7]. The Job’s
plot using the luminescence emission intensity of Eu3+ at 614
Figure 3. Absorbance spectra of 50 µM aqueous solution (100 mM HEPES
buffer; pH 7.4) of [Eu-7] at 22 °C upon addition of aliquots of Zn2+, which
was added as ZnSO4: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0
equiv of Zn2+ with respect to [Eu-7]. The inset shows the spectra magnified
between 290 and 360 nm.
7
7
7
nm for the complex of [Eu-7] and Zn2+ also indicated the
presence of the 1:1 complex (see Supporting Information).22
For [Eu-7] (20 µM), the fluorescence emission spectra were
measured without a delay time, with excitation at 320 nm
(Figure 4). In the fluorescence emission of [Eu-7], one band
with a short lifetime appeared at 397 nm with a concomitant
linear fluorescence increase following addition of Zn2+ at
between 0 and 1.0 equiv to [Eu-7], and it remained at a plateau
with further Zn2+ addition (Figure S6). The short-lived fluo-
rescence at 397 nm can be ascribed to direct emission from the
quinolyl moiety of [Eu-7].21a,b All these results can be rational-
ized in terms of 1:1 complex formation of [Eu-7] with Zn2+
,
via the quinolyl ligand.
Luminescence and Chemical Properties of [Eu-7] and
[Gd-7]. The luminescence and chemical properties of [Eu-7]
and [Gd-7] are listed in Table 1. [Eu-7] and [Gd-7], even at
10 mM, were highly water-soluble. The phosphorescence spectra
of [Gd-7] were measured in MeOH:EtOH ) 1:1 at 77 K in the
absence and in the presence of Zn2+. The triplet energy levels
for free and Zn2+-bound [Gd-7] were around 20 790 and 20 576
(19) (a) Reany, O.; Gunnlaugsson, T.; Parker, D. J. Chem. Soc., Perkin Trans.
2 2000, 1819-1831. (b) Hanaoka, K.; Kikuchi, K.; Kojima, H.; Urano,
Y.; Nagano, T. Angew. Chem., Int. Ed. 2003, 42, 2996-2999. (c) Reany,
O.; Gunnlaugsson, T.; Parker, D. Chem. Commun. 2000, 473-474.
(20) Sille´n, L. G.; Martell, A. E. Stability Constants of Metal-Ion Complexes;
Special Publication No. 17; The Chemical Society: Burlington House,
London, 1964.
(21) (a) Gunnlaugsson, T. Tetrahedron Lett. 2001, 42, 8901-8905. (b)
Gunnlaugsson, T.; MacDo´naill, D. A.; Parker, D. J. Am. Chem. Soc. 2001,
123, 12866-12876. (c) Manning, H. C.; Goebel, T.; Marx, J. N.; Bornhop,
D. J. Org. Lett. 2002, 4, 1075-1078. (d) Jotterand, N.; Pearce, D. A.;
Imperiali, B. J. Org. Chem. 2001, 66, 3224-3228.
(22) Nabeshima, T.; Tsukada, N.; Nishijima, K.; Ohshiro, H.; Yano, Y. J. Org.
Chem. 1996, 61, 4342-4350.
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+
A Luminescent Lanthanide Chemosensor for Zn2
A R T I C L E S
presence of Zn2+ (see Supporting Information). The lumines-
cence lifetimes of [Eu-7] were found to be 0.52 (without Zn2+
)
)
and 0.58 (with Zn2+) ms in H2O (τH O), and 2.03 (without Zn2+
2
and 2.23 (with Zn2+) ms in D2O (τD O). These values indicated
2
that the numbers of coordinated water molecules (q values) at
the metal center were 1.42 and 1.22, respectively, according to
eq 1.26 Thus, the lanthanide hydration state was hardly affected
no. of water molecules:
q
Eu ) 1.2(τH O-1 - τD O-1 - 0.25) (1)
2
2
by the addition of Zn2+. Therefore, it can be considered that
the increase of the emission intensity caused by Zn2+ addition
was not due to a change in the direct interaction of water
Figure 4. Fluorescence spectra of [Eu-7] (20 µM) in 100 mM HEPES
buffer at pH 7.4 without a delay time upon the addition of increasing
amounts of Zn2+: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0
equiv of Zn2+ with respect to [Eu-7]. Excitation at 320 nm, 22 °C. The
inset shows the spectra magnified between 330 and 575 nm.
molecules with Eu3+
.
Next, the spatial arrangement of [Eu-7] was further assessed
by measuring the relaxometric properties. The water proton
relaxivity R1 of Gd3+ complexes is routinely used as an
important parameter for MRI contrast agents.27 The relaxation
enhancement is modulated by the electron-nucleus dipolar
interaction, which can be changed by rotation of the complex,
by electron spin relaxation of the metal ion, and by the co-
ordinated exchange of water molecules.28 The R1 relaxivity of
[Gd-7] at 20 MHz showed similar values in the absence and in
the presence of Zn2+, i.e., 6.05 and 5.81 mM-1 s-1, respectively.
This result indicates that the environment around the Eu3+ ion
of [Eu-7] was hardly changed by Zn2+ binding.
Table 1. Luminescence and Chemical Properties
φ
(%)b
τH O (ms)c
τD O (ms)d
qe
2
2
+
+
+
+
apparent Kd
Zn2
Zn2
Zn2
Zn2
for Zn2+ (nM)a free complex free complex free compex free complex
[Eu-7]
59
0.9 7.4 0.52 0.58 2.03 2.23 1.42 1.22
1
f
1 g
triplet state energy (cm-
)
r )
1 (mM-1 s-
free
Zn2+ complex
free
Zn2+ complex
[Gd-7]
20790
20576
6.05
5.81
a [Zn2+] was controlled by using Zn2+/NTA systems below 398 nM free
Zn2+ and unbuffered Zn2+ above 200 µM free Zn2+. The buffer contained
100 mM HEPES, pH 7.4, I ) 0.1 (NaNO3). b Quantum yields were
calculated using [Ru(bipy)3]Cl2 (bipy ) 2,2′-bipyridine; φ ) 0.028 in water)
as a standard, and measured in 100 mM HEPES buffer at pH 7.4, 25 °C.
c In H2O-based buffer (100 mM HEPES buffer; pH 7.4). d In D2O-based
buffer (100 mM HEPES buffer; pD 7.4). e q values were estimated using
Effect of pH and Other Cations on the Long-Lived
Luminescence Intensity. The luminescence emission intensity
of [Eu-7] at 614 nm was examined at various pH values, with
excitation at 320 nm. There was almost no effect of H+ on the
emission spectrum of [Eu-7] between pH 3.6 and 8.8 either in
the presence or in the absence of Zn2+ (see Supporting Infor-
mation). Thus, the luminescence emission intensity of [Eu-7]
is stable at around physiological pH. Parker et al. reported a
Zn2+-sensitive luminescent lanthanide probe based on photo-
induced electron transfer (PeT) from the benzylic nitrogen to
the chromophore’s singlet excited state.19a,c This approach has
the disadvantage that the pivotal nitrogen for Zn2+ binding can
also be protonated at lower pH, resulting in strong luminescence
owing to inhibition of PeT. However, our compound [Eu-7]
was not affected by lowering of the pH. The tertiary amine of
[Eu-7] can be protonated at around pH 3.6, because TPA has
pKa values of 6.10, 4.28, and 2.49.24 Thus, the finding of
insensitivity to lower pH means that the luminescence augmen-
tation was not due to cessation of PeT from tertiary amine. We
think that this pH stability of the luminescence is one of the
key advantages of [Eu-7].
the equation qEu ) 1.2(τH O -τD O - 0.25), which allows for the
-1
-1
2
contribution of unbound water molec2ules.23 f In MeOH:EtOH ) 1:1 at 77
K. g The R1 relaxivity as measured at 20 MHz and 25 °C in 100 mM HEPES
buffer (pH 7.4).
cm-1, respectively, and both were sufficiently close to the 5D0
level, the excited state, of Eu3+ (E ) 17 250 cm-1).23 The
luminescence quantum yield (φ) was 0.9% before addition of
Zn2+ and was increased 8.2-fold to 7.4% by Zn2+ addition, under
air-equilibrated conditions. This luminescence quantum yield
is sufficiently large for luminescence detection. Further, the
binding affinity for Zn2+ was assessed by using the lumines-
cence intensity. The affinity of [Eu-7] for Zn2+ ions was
measured at pH 7.4, 22 °C, in a high salt background (100 mM
HEPES buffer, I ) 0.1 (NaNO3)). The apparent dissociation
constant Kd for Zn2+ was calculated to be 59 nM. This Kd value
for Zn2+ was larger than that reported for tris(2-pyridylmethyl)-
amine (TPA) (Kd ) 0.014 nM; pH ) 7.4, 298 K, I ) 0.1),24
probably due to a larger steric repulsion in the case of the
quinolyl substituent than the pyridyl substituent, but the value
is still sufficiently small for biological applications.25 Measure-
ments of the decay rate constants of the Eu3+ excited state were
carried out in both H2O and D2O, in the absence and in the
The effect of adding Na+, K+, Ca2+, Mg2+, and various heavy
metal ions on the luminescence emission intensity of [Eu-7]
was also examined. Luminescence emission enhancement of
(26) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle,
L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin
Trans. 2 1999, 493-503.
(27) (a) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. ReV.
1999, 99, 2293-2352. (b) Lowe, M. P. Aust. J. Chem. 2002, 55, 551-
556. (c) Li, W. H.; Fraser, S. E.; Meade, T. J. J. Am. Chem. Soc. 1999,
121, 1413-1414. (d) Li, W. H.; Parigi, G.; Fragai, M.; Luchinat, C.; Meade,
T. J. Inorg. Chem. 2002, 41, 4018-4024. (e) Moats, R. A.; Fraser, S. E.;
Meade, T. J. Angew. Chem., Int. Ed. Engl. 1997, 36, 726-728. (f) Louie,
A. Y.; Hu¨ber, M. M.; Ahrens, E. T.; Rothba¨cher, U.; Moats, R.; Jacobs, R.
E.; Fraser, S. E.; Meade, T. J. Nat. Biotechnol. 2000, 18, 321-325.
(28) Merbach, A. E.; To´th, EÄ . The Chemistry of Contrast Agents in Medical
Magnetic Resonance Imaging; John Wiley & Sons, Ltd.: Chichester, 2001.
(23) (a) Crosby, G. A.; Whan, R. E.; Alire, R. M. J. Chem. Phys. 1961, 34,
743-748. (b) Stein, G.; Wu¨rzberg, E. J. Chem. Phys. 1975, 62, 208-213.
(24) Martell, A. E.; Smith, R. M. NIST Critically Selected Stability Constants
of Metal Complexes; NIST Standard Reference Database 46, Version 6.0;
NIST: Gaithersburg, MD, 2001.
(25) (a) Weiss, J. H.; Sensi, S. L.; Koh, J. Y. Trends Pharmacol. Sci. 2000, 21,
395-401. (b) Li, Y.; Hough, C. J.; Suh, S. W.; Sarvey, J. M.; Frederickson,
C. J. J. Neurophysiol. 2001, 86, 2597-2604. (c) Li, Y.; Hough, C. J.;
Frederickson, C. J.; Sarvey, J. M. J. Neurosci. 2001, 21, 8015-8025.
9
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A R T I C L E S
Hanaoka et al.
Figure 5. Luminescence intensity change profiles of [Eu-7] (50 µM) in
the presence of various cations in 100 mM HEPES buffer at pH 7.4, 22 °C
(excitation 320 nm, emission 614 nm). Heavy metal ions (1 equiv relative
to [Eu-7]) were added as Fe2(SO4)3, CuSO4, NiSO4, CoSO4, CdSO4, FeSO4,
and MnSO4. Other cations were added as ZnSO4 (50 µM), NaNO3 (100
mM), KNO3 (100 mM), CaCl2 (5 mM), and MgSO4 (5 mM).
[Eu-7] (50 µM) was not observed upon the addition of 100
mM Na+ or K+, 5 mM Ca2+ or Mg2+, or 50 µM of various
heavy metal ions except for Cd2+ (Figure 5). Thus, Na+, K+,
Ca2+, and Mg2+, which exist at high concentrations in biological
systems, did not enhance the luminescence intensity of [Eu-7].
The luminescence emission intensity was weakened or quenched
upon the addition of several cations (particularly Cu2+, Ni2+
,
and Co2+) together with Zn2+, in comparison with that upon
adding Zn2+ alone, as shown in Figure S8. However, Zn2+ can
be distinguished from these heavy metals, since Zn2+ chelation
selectively enhances the luminescence intensity.
Figure 6. Emission spectra of [Eu-7] (50 µM) without (a) and with (b)
time resolution in the presence of various concentrations of Zn2+: 0, 0.2,
0.4, 0.6, 0.8, and 1.0 equiv of Zn2+ with respect to [Eu-7] (excitation at
300 nm, respectively). Both spectra were measured in 100 mM HEPES
buffer containing 1 µM rhodamine 6G as a chromophore to provide artificial
background fluorescence at pH 7.4 and 22 °C. The emission spectra (a)
were measured with a Hitachi F4500, and the time-resolved emission spec-
tra (b) were measured using a delay time of 0.05 ms and a gate time
of 1.00 ms with a Perkin-Elmer LS-50B. Bands can be assigned to (i)
scattered excitation light, (ii) the fluorescence of the quinolyl chromophore
of [Eu-7], (iii) the fluorescence of rhodamine 6G, and (iv) the long-lived
luminescence of [Eu-7].
Utility of the Long-Lived Luminescence. To explore further
the utility of the long luminescence lifetime of [Eu-7], we tested
whether it could be well distinguished from the short-lived
background fluorescence and scattered light. Figure 6 presents
the emission spectra of [Eu-7] solution, with 1 µM rhodamine
6G as an artificial short-lived background, without (Figure 6a)
or with (Figure 6b) a time resolution process. There are three
fluorescent peaks in Figure 6a due to scattered light (300 nm),
the increased fluorescence of the quinoline moiety (around 400
nm), and the fluorescence of rhodamine 6G (around 550 nm).
Among them, rhodamine 6G fluorescence can directly interfere
with Eu3+ luminescence. However, the long-lived luminescence
detection of [Eu-7] solution was not affected at all by these
three peaks (Figure 6b). Thus, the luminescence of [Eu-7]
should be little influenced by experimental artifacts when
measured with the aid of a time-resolution process.
addition of the cell-membrane-permeable chelator TPEN (150
µM) at 7.5 min. Clear images were obtained, because [Eu-7]
has long-wavelength emission with a large Stoke’s shift, which
minimizes the influence of the excitation light. These results
demonstrate that [Eu-7] can be used to monitor changes of
intracellular ionic Zn2+ reversibly, and has potential for biologi-
cal applications.
Conclusions
Biological Applications of [Eu-7]. We next examined the
application of [Eu-7] to cultured living cells (HeLa cells) by
fluorescence microscopy (Figure 7). Since [Eu-7] can be excited
with a relatively long excitation wavelength, this compound is
suitable for cellular applications, in contrast to previously
reported Zn2+-sensitive luminescent lanthanide sensors. The
fluorescence microscope had an optical window centered at 617
( 37 nm for the emission due to Eu3+-based luminescence upon
excitation at 360 ( 40 nm (the Stoke’s shift is >250 nm).
Compound [Eu-7] was injected only into the single cultured
HeLa cell in the bottom left-hand part of the field of view in
Figure 7. We then added Zn2+ (150 µM) and a zinc-selective
ionophore, pyrithione (2-mercaptopyridine N-oxide, 50 µM), to
the medium at 3 min, inducing a prompt increase of intracellular
luminescence. This luminescence was decreased by extracellular
We have designed and synthesized a novel lanthanide-based
luminescent sensor molecule for Zn2+, [Eu-7], by using Eu3+
as the fluorophore and a quinolyl moiety as the antenna. This
compound [Eu-7] showed pronounced long-lived luminescence
enhancement upon Zn2+ addition. Previously reported Zn2+
-
sensitive lanthanide sensors are not suitable for biological
applications, because of their short excitation wavelength, small
enhancement of emission, inconvenient pH sensitivity, etc. In
contrast, the properties with [Eu-7] are favorable for biological
applications. It is noteworthy that [Eu-7] has a longer excitation
wavelength than previously reported Zn2+-sensitive lanthanide
sensors, and this permits fluorescence microscopy measurements
with [Eu-7] to monitor Zn2+ concentrations in living cells. We
expect that a larger signal-to-noise ratio in cellular imaging
9
12474 J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004

+
A Luminescent Lanthanide Chemosensor for Zn2
A R T I C L E S
Figure 7. Bright-field transmission and luminescence images of Zn2+ in [Eu-7]-injected HeLa cells in HBSS buffer. The luminescence at 580-654 nm,
excited at 320-400 nm, was measured at 30 s intervals. The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and
1% streptomycin at 37 °C in a 5% CO2/95% air incubator. The cells were then washed with HBSS buffer and injected with [Eu-7] solution. (a) Bright-field
transmission image (0 min). (b) Luminescence image of (a) (0 min). (c) Luminescence image (5 min) following an addition of 50 µM pyrithione (zinc
ionophore) and 150 µM ZnSO4 to the medium at 3 min. (d) Luminescence image (10 min) following an addition of 150 µM TPEN to the medium at 7.5
min. Luminescence images (b-d) correspond to the luminescence intensity data in (e), which shows the average intensity of the corresponding area or cell
area (1, extracellular region; 2, intracellular region of the injected cell; 3, 4, intracellular regions of noninjected cells).
Measurements of relaxation times T1 were made using an NMR analyzer
operating at 20 MHz (Minispec mq20, Bruker).
would be achievable by using time-resolved imaging with
[Eu-7]. Furthermore, we confirmed the advantage of the long-
lived luminescence of [Eu-7] for eliminating fluorescence
background interference. This complex [Eu-7] is the first
Zn2+-sensitive luminescent lanthanide chemosensor that can be
used for studies on the biological functions of Zn2+, and our
design strategy should yield a range of long-lived luminescent
lanthanide probes for sensing Zn2+ or, after appropriate modi-
fication of the acceptor moiety, other molecules of interest in
biological applications.
Time-Delayed Luminescence Spectral Measurements. The time-
delayed luminescence spectra of [Eu-7] (50 µM) were measured in
100 mM HEPES buffer at pH 7.4, 22 °C (excitation at 320 nm), with
addition of various amounts of Zn2+ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 2.0, 3.0, 4.0, 5.0, and 10.0 equiv of Zn2+) to
[Eu-7]. The slit width was 5 nm for both excitation and emission. A
delay time of 0.05 ms and a gate time of 1.00 ms were used.
UV-Visible Absorption Spectral Measurements. The absorption
spectral changes of [Eu-7] (50 µM) upon addition of Zn2+ were
measured at 22 °C in an aqueous solution buffered to pH 7.4 (100 mM
HEPES buffer). Zn2+ was added as ZnSO4 at 0, 0.2, 0.4, 0.6, 0.8, 1.0,
2.0, 3.0, 4.0, 5.0, and 10.0 equiv of Zn2+ with respect to [Eu-7].
Fluorescence Emission Spectral Measurements. The fluorescence
emission spectra of [Eu-7] (20 µM) without a delay time were measured
in 100 mM HEPES buffer (pH 7.4) at 22 °C, following excitation at
320 nm. The amounts of added Zn2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0,
2.0, 3.0, 4.0, 5.0, and 10.0 equiv with respect to [Eu-7].
Quantum Yield Measurements. The luminescence spectra were
measured with a Hitachi F4500 spectrofluorometer. The slit width was
2.5 nm for both excitation and emission. The photomultiplier voltage
was 950 V. The luminescence spectra of [Eu-7] were measured in 100
mM HEPES buffer at pH 7.4, 25 °C, with irradiation at 300 nm. The
quantum yields of Eu3+ complexes were evaluated using a relative
method with reference to a luminescence standard, [Ru(bpy)3]Cl2 (φ
) 0.028 in air-equilibrated water).29 The quantum yields of Eu3+
complexes can be expressed by eq 2,30 where Φ is the quantum yield
(subscript “st” stands for the reference and “x” for the sample), A is
Experimental Section
Materials. DTPA bisanhydride was purchased from Aldrich Chemi-
cal Co. Inc. (St. Louis, MO). All other reagents were purchased from
either Tokyo Kasei Kogyo Co., Ltd. (Japan) or Wako Pure Chemical
Industries, Ltd. (Japan). All solvents were used after distillation. Silica
gel column chromatography was performed using BW-300, Chroma-
torex-NH, and Chromatorex-ODS (all from Fuji Silysia Chemical Ltd.).
1
Instruments. H and 13C NMR spectra were recorded on a JEOL
JNM-LA300. Mass spectra were measured with a JEOL JMS-DX 300
mass spectrometer (EI+) or a JEOL JMS-700 mass spectrometer
(FAB+). HPLC purification was performed on a reverse-phase column
(GL Sciences (Tokyo, Japan), Inertsil Prep-ODS 30 mm × 250 mm)
fitted on a Jasco PU-1587 System. Time-resolved fluorescence spectra
were recorded on a Perkin-Elmer LS-50B (Beaconsfield, Buckingham-
shire, England). The slit width was 5 nm for both excitation and
emission. UV-visible spectra were obtained on a Shimadzu UV-1600
(Tokyo, Japan). Fluorescence spectroscopic studies were performed with
a Hitachi F4500 (Tokyo, Japan). The slit width was 2.5 nm for both
excitation and emission. The photomultiplier voltage was 700 V.
(29) Nakamura, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697-2705.
9
J. AM. CHEM. SOC. VOL. 126, NO. 39, 2004 12475

A R T I C L E S
Hanaoka et al.
2
Φx/Φst ) [Ast/Ax] [nx /nst2] [Dx/Dst]
(2)
320 nm, emission 614 nm) of each sample of [Eu-7] (50 µM) was
plotted.
Metal Ion Selectivity Measurements. The luminescence emission
enhancement of [Eu-7] was measured in 100 mM HEPES buffer (pH
7.4) at 22 °C (excitation 320 nm, emission 614 nm). Heavy metal ions
(50 µM) were added as Fe2(SO4)3, CuSO4, NiSO4, CoSO4, CdSO4,
FeSO4, and MnSO4. Other cations were added as ZnSO4 (50 µM),
NaNO3 (100 mM), KNO3 (100 mM), CaCl2 (5 mM), and MgSO4
(5 mM).
Confirmation of the Utility of the Long Luminescence Lifetime
of [Eu-7]. The emission spectra without a time-resolution process were
measured with a Hitachi F4500. The time-resolved emission spectra
were measured with a Perkin-Elmer LS-50B using a delay time of 0.05
ms and a gate time of 1.00 ms. Spectra of [Eu-7] (50 µM) with various
concentrations of Zn2+ were measured in 100 mM HEPES buffer (pH
7.4) at 22 °C containing 1 µM rhodamine 6G as an artificial provider
of short-lived background fluorescence (excitation at 300 nm). The
amounts of added Zn2+ were 0, 0.2, 0.4, 0.6, 0.8, and 1.0 equiv of
Zn2+ with respect to [Eu-7].
the absorbance at the excitation wavelength, n is the refractive index,
and D is the area (on an energy scale) of the luminescence spectra.
The samples and the reference were excited at the same wavelength.
The sample absorbance at the excitation wavelength was kept as low
as possible to avoid fluorescence errors (Aexc < 0.06).
Luminescence Lifetime Measurements. The luminescence lifetimes
of the complexes were recorded on a Perkin-Elmer LS-50B. The data
were collected with 10-µs resolution in H2O (100 mM HEPES buffer
at pH 7.4) and D2O (100 mM HEPES buffer at pD 7.4, based on the
equation pD ) pH + 0.4031), and fitted to a single-exponential curve
obeying eq 3, where I0 and I are the luminescence intensities at the
I ) I0 exp(-t/τ)
(3)
time t ) 0 and time t, respectively, and τ is the luminescence emission
lifetime. Lifetimes were obtained by monitoring the emission intensity
at 614 nm (λex ) 320 nm).
Relaxation Time Measurements. The relaxation time, T1, of
aqueous solutions of the Gd3+ complex [Gd-7] was measured in 100
mM HEPES buffer (pH 7.4) at 20 MHz and 25 °C (Minispec mq20,
Bruker). The value of T1 was measured from 10 data points generated
by using the standard inversion-recovery procedure. The relaxivity,
R1 (mM-1 s-1), of [Gd-7] was determined from the slope of the plot
of 1/T1 vs [[Gd-7]] (0, 0.4, 0.6, 0.8, and 1.0 mM). The buffered Gd3+
complex ([Gd-7]) solution was allowed to equilibrate for at least 10
min after addition of ZnSO4 aqueous stock solution.
Preparation of Cells. HeLa cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA),
supplemented with 10% fetal bovine serum (Invitrogen Corp.), 1%
penicillin, and 1% streptomycin (Invitrogen Corp.) at 37 °C in a 5%
CO2/95% air incubator. The cells were grown on an uncoated 35-mm-
diameter glass-bottomed dish (MatTek, Ashland, MA), and washed
twice with Hanks’ balanced salt solution (HBSS) buffer (Invitrogen
Corp.), and then the medium was replaced with HBSS buffer before
imaging. The compound [Eu-7] (2 mM) was dissolved in microinjection
buffer (HBSS buffer), and injected into the cells with an Eppendorf
injection system (Transjector 5246).
Microscopy and Imaging Methods. The imaging system comprised
an inverted microscope (IX71; Olympus) and a cooled CCD camera
(Cool Snap HQ; Roper Scientific, Tucson, AZ). The microscope was
equipped with a xenon lamp (AH2-RX; Olympus), a 40× objective
lens (Uapo/340, N.A. 1.35; Olympus), a dichroic mirror (420DCLP;
OMEGA), an excitation filter (S360/40×; Chroma), and an emission
filter (S617/73m; Chroma). The whole system was controlled using
MetaFluor 6.1 software (Universal Imaging, Media, PA). The lumi-
nescence images were measured every 30 s. Additions of zinc sulfate
(150 µM) with pyrithione (50 µM) or N,N,N′,N′-tetra(2-picolyl)-
ethylenediamine (TPEN) (150 µM) to cell samples were performed on
the microscope stage.
Apparent Dissociation Constant (Kd) Measurements. Upon ad-
dition of various concentrations of Zn2+, the luminescence intensity
and the absorbance of [Eu-7] linearly changed up to a 1:1 [Zn2+]/
[[Eu-7]] molar ratio, and the luminescence and absorption spectra
remained at a plateau with further addition of Zn2+. Furthermore, the
Job’s plot analysis revealed that maximum luminescence intensity was
obtained at a 1:1 ratio. These data suggested that [Eu-7] should form
a 1:1 complex with Zn2+. So, the apparent dissociation constant, Kd,
was determined from the luminescence intensity in 100 mM HEPES
buffer (pH 7.4, I ) 0.1 (NaNO3)) at 22 °C (λexc ) 320 nm). [Zn2+
]
was controlled by using 0-9 mM ZnSO4/10 mM NTA (nitrilotriacetic
acid) systems19b,32 below 398 nM free Zn2+ and unbuffered Zn2+ above
200 µM free Zn2+. The luminescence intensity data were fitted to eq
4, and Kd was calculated, where F is the luminescence intensity, Fmax
F ) F0 + (Fmax - F0) ([Zn2+]f)/(Kd + [Zn2+]f)
(4)
Acknowledgment. This work was supported in part by the
Ministry of Education, Culture, Sports, Science and Technology
of Japan (Grants for The Advanced and Innovational Research
Program in Life Sciences to T.N., 15681012 and 16048206
to K.K.). K.K. was also supported by the Sankyo Foundation,
by the Kanagawa Academy of Science, and by the Suzuken
Memorial Foundation. K.H. is the recipient of Research Fel-
lowships of the Japan Society for the Promotion of Science for
Young Scientists.
is the maximum luminescence intensity, F0 is the luminescence intensity
with no addition of Zn2+, and [Zn2+]f is the free Zn2+ concentration.
The value of Kd was determined from the fittings for the luminescence
intensity data shown in the Supporting Information.
Phosphorescence Spectral Measurements. Phosphorescence spec-
tra were obtained with a Hitachi F4500. The phosphorescence spectra
of [Gd-7] (20 µM) in the absence and in the presence of Zn2+ (1 equiv
relative to [Gd-7]) were measured at 77 K in MeOH:EtOH ) 1:1
(excitation at 320 nm) (see Supporting Information). The slit width
was 10.0 nm for excitation and 20.0 nm for emission. The photomul-
tiplier voltage was 950 V.
Effect of pH on the Luminescence Intensity. The following buffers
were used: 100 mM ClCH2COOH-ClCH2COONa buffer (pH 3.6),
100 mM AcOH-AcONa buffer (pH 4.0-5.5), 100 mM morpholino-
ethanesulfonic acid (MES) buffer (pH 5.5-6.5), 100 mM HEPES buffer
(pH 7.0-8.0), and 100 mM N-cyclohexyl-2-aminoethanesulfonic acid
(CHES) buffer (pH 8.5-9.0). The luminescence intensity (excitation
Supporting Information Available: Detailed descriptions of
synthetic procedures for [Eu-7] and [Gd-7]; data on the
measurements of the emission lifetime of [Eu-7] and on the
measurement of the apparent dissociation constant of [Eu-7]
with Zn2+; phosphorescence spectra of [Eu-7]; plot of the
absorbance intensity changes detected at 340 nm, shown in
Figure 3; Job’s plot for complexation between [Eu-7] and
Zn2+; plot of the changes in the fluorescence intensity detected
at 397 and 614 nm, shown in Figure 4; and pH profile of the
luminescence intensity of [Eu-7]. This material is available free
of charge via the Internet at http://pubs.acs.org.
(30) Chen, Q. Y.; Feng, C. J.; Luo, Q. H.; Duan, C. Y.; Yu, X. S.; Liu, D. J.
Eur. J. Inorg. Chem. 2001, 1063-1069.
(31) Wang, Y. M.; Wang, Y. J.; Wu, Y. L. Polyhedron 1998, 18, 109-117.
(32) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem.
Soc. 2000, 122, 12399-12400.
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