A heterobimetallic ruthenium-gadolinium complex as a potential agent for bimodal imaging

Article abstract of DOI:10.1021/ic200726t

Trinuclear heterobimetallic Ln^III-Ru^II complexes (Ln = Eu, Gd) based on a 1,10-phenanthroline ligand bearing a diethylenetriaminepentaacetic acid (DTPA) core have been synthesized and fully characterized by a range of experimental te

Full text of DOI:10.1021/ic200726t

ARTICLE
pubs.acs.org/IC
A Heterobimetallic RutheniumꢀGadolinium Complex as a Potential
Agent for Bimodal Imaging
Geert Dehaen,† Peter Verwilst,† Svetlana V. Eliseeva,† Sophie Laurent,‡ Luce Vander Elst,‡ Robert N. Muller,‡
Wim M. De Borggraeve,† Koen Binnemans,† and Tatjana N. Parac-Vogt*,†
†Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, P.O. Box 2404, B-3001 Heverlee, Belgium
‡NMR and Molecular Imaging Laboratory, Department of General, Organic and Biomedical Chemistry, University of Mons-Hainaut,
7000 Mons, Belgium
S Supporting Information
b
ABSTRACT: Trinuclear heterobimetallic LnIIIꢀRuII com-
plexes (Ln = Eu, Gd) based on a 1,10-phenanthroline ligand
bearing a diethylenetriaminepentaacetic acid (DTPA) core
have been synthesized and fully characterized by a range of
experimental techniques. The 17O NMR and proton nuclear
magnetic relaxation dispersion (NMRD) measurements of
GdIIIꢀRuII show that, in comparison to the parent Gd-DTPA,
this complex exhibits improved relaxivity, which is the result of
an increase of the rotational correlation time. Relaxometry and
ultrafiltration experiments indicate that the 1,10-phenanthroline ligand has a high affinity for noncovalent binding to human serum
albumin, which results in a high relaxivity r1 of 14.3 sꢀ1 mMꢀ1 at 20 MHz and 37 °C. Furthermore, the LnIIIꢀRuII complexes (Ln =
Eu, Gd) show an intense light absorption in the visible spectral region due to metal-to-ligand charge transfer (MLCT) transitions.
Upon excitation into the MLCT band at 440 nm, the complexes exhibit a bright-red luminescence centered at 610 nm, with a
quantum yield of 4.7%. The luminescence lifetime equals 540 ns and is therefore long enough to exceed the fluorescent background.
Monometallic lanthanide complexes have also been synthesized, and the EuIII analogue shows a characteristic red luminescence with
a quantum yield of 0.8%. Taking into account the relaxometric and luminescent properties, the developed GdIIIꢀRuII complex can
be considered as a potential in vitro bimodal imaging agent.
’ INTRODUCTION
luminescence from lanthanide ions or transition metals makes them
ideal as probes for time-gated luminescence-based imaging.10ꢀ12
Several approaches to developing potential bimodal imaging
agents are already known. A mixture of a GdIII and EuIII
complex coordinated by a single ligand has been described
where the GdIII chelate acts as a T1 agent for MRI visualization,
while the EuIII chelate acts as a reporter in fluorescence
microscopy.13,14 To obtain a lower concentration of the ima-
ging agent in vivo, several complexes containing an optical
entity covalently bound to an MRI agent have been reported. In
the majority of the examples, the optical entity is a fluorescent
dye4,8,15,16 or a transition-metal complex.8,17 More recently,
functionalized nanoparticles based on iron oxide (Fe3O4),18
gadolinium oxide (Gd2O3) doped with rare earths,19 and
quantum dots20 have been used as bimodal agents.
In the search for the development of new, promising contrast
agents, multimodal imaging agents are gaining nowadays great
attention in the field of clinical and preclinical imaging applications.
Powerful in vivo techniques such as magnetic resonance imaging
(MRI), positron emission tomography, single-photon emission
tomography, and computed tomography are ubiquitous in clinical
diagnostics and research centers. Because each imaging technique
has its own strengths and weaknesses, the combination of different,
complementary techniques can overcome inherent limitations that
are associated with one individual technique.1ꢀ4 Whereas MRI is
ideal for whole body images because of its good spatial resolution,5
its sensitivity is rather low so that millimolar concentrations of GdIII-
based contrast agents are required. Luminescence-based imaging,
on the other hand, can provide high-resolution images, but this
technique is only suitable for thin tissue samples because of the low
optical transparency of biological tissue.6ꢀ8 Because biological
samples strongly absorb at UV and visible wavelengths, suitable
metal complexes for luminescence-based imaging are restricted to
those showing emission in the red end of the visible spectrum and in
the near-infrared (NIR) because radiation of these wavelengths
can more easily and effectively penetrate tissues.9 Long-lived
Because of their unique photochemical and photophysical
properties, RuII complexes (especially with polypyridine ligands)
have been extensively studied and widely used in a variety of
different applications.12,21ꢀ25 RuII complexes possess excellent light-
harvesting properties, have relatively long-lived metal-to-ligand
Received: April 8, 2011
Published: September 13, 2011
r
2011 American Chemical Society
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a
Scheme 1. Synthesis of Ligand DTPA(ph-phen)2
a Reaction conditions: (i) tert-butyl [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]carbamate, Cs2CO3, Pd(PPh3)4, reflux, 16 h; (ii) TFA,
room temperature, overnight; (iii) DTPA-BA, 60 °C, overnight.
while 1,10-phenanthroline as a soft base is perfectly suited for
coordination to RuII. The relaxometric and luminescent proper-
ties of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4, as well
as its ability to bind to human serum albumin (HSA), have been
studied in detail. In addition, monometallic complexes, [Ln{DT-
PA(ph-phen)2}(CH3OH)] (Ln = Gd, Eu), have been synthe-
sized, and the photophysical properties of the EuIII analogue have
been investigated in order to gain information about the co-
Figure 1. Framework molecular model of the [{Ru(bpy)2}2Gd{DTPA-
ordination environment and solvation numbers of the LnIII ions.
(ph-phen)2}(H2O)]Cl4 complex. Hydrogen atoms have been omitted for
clarity.
’ RESULTS AND DISCUSSION
charge transfer (MLCT) states, and show intense luminescence
at the red end of the visible spectrum. Taking advantage of these
properties, RuII complexes have been used as sensitizers of NIR
emission of Ln ions, such as YbIII and NdIII, in fꢀd hetero-
metallic diads.26ꢀ30 To explore the application of RuII com-
pounds in biology, they also have been tested as oxygen
sensors,31 DNA intercalators,32ꢀ34 anticancer agents35ꢀ38 and
cell imaging probes.12
Although multimodal imaging agents are developing at a
growing pace,3 reports dealing with the design of fꢀd hetero-
metallic systems for this purpose remain quite scarce. In 2008,
Faulkner and co-workers described a bimetallic complex contain-
ing a luminescent ReI chromophore bound to an MRI-active
GdIII-DOTA derivative.8 The discovery of a new class of fꢀd
metal systems, so-called metallostars,39,40 has led to the design of
a complex with six LnIII-DTTA moieties attached to a single RuII
center.17 The GdIIIꢀRuII analogues of such heptametallic species
have shown the capability to act as MRI contrast agents, while the
EuIIIꢀRuII complexes display interesting luminescent properties.
In this paper, we report the synthesis of a new fꢀd hetero-
polymetallic complex, [{Ru(bpy)2}2Ln{DTPA(ph-phen)2}-
(H2O)]Cl4 (Ln = Gd, Eu), built by a 1,10-phenanthroline
derivative coupled to diethylenetriaminepentaacetic acid bisan-
hydride (DTPA-BA) suitable for in vitro studies (Scheme 1 and
Figure 1). The DTPA-BA arms can coordinate to the LnIII ions,
Ligand and Complexes. Suzuki cross-coupling of 5-bromo-
1,10-phenanthroline with tert-butyl [4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl]carbamate in the presence of
Cs2CO3 and Pd(PPh3)4 in a water/toluene mixture (1:1, v/v)
yielded the Boc-protected aniline (1), which upon deprotection
by trifluoroacetic acid (TFA) gives compound 2. The reaction of
2 with diethylenetriaminepentaacetic acid bisanhydride (DTPA-
BA) in N,N-dimethylformamide (DMF) overnight at 60 °C
yielded the final ligand, DTPA(ph-phen)2. The free ligand was
1
characterized by H NMR (see Figure S1 in the Supporting
Information and Scheme 1 for labeling) and 13C NMR in
DMSO-d6, as well as by two-dimensional COSY NMR (Figure
S2 in the Supporting Information).
The electrospray ionization mass spectrometry (ESI-MS) spec-
trum of the ligand in the negative mode shows the molecular ion
peaks corresponding to [M ꢀ H]ꢀ at m/z 898.3. The lanthanide
complexes [Ln{DTPA(ph-phen)2}(CH3OH)] (Ln = Eu, Gd)
were obtained by reacting the corresponding lanthanide(III) triflate
with ligand DTPA(ph-phen)2 in methanol (MeOH) in an equi-
molar ratio. The removal of free Ln ions from the reaction mixture
was performed using Chelex 100. The solution was checked with an
arsenazo indicator solution, which confirmed the complete removal
of free Ln ions.41 Positive-mode ESI-MS of the complexes shows
molecular ion peaks for [M + Na]+ at m/z 1072.9 corresponding to
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Scheme 2. Synthesis of the LnIIIꢀRuII [{Ru(bpy)2}2Ln{DTPA(ph-phen)2}(H2O)]Cl4 (Ln = Eu, Gd) Complexes
the EuIII complex and those for [M + 2H]2+ at m/z 527.9 corres-
ponding to the GdIII complex.
seconds measured in a solution of GdIII with a concentration
of 1 mM, can be characterized by four parameters: the number
of water molecules in the first coordination sphere of GdIII (q),
the residence time of the coordinated water molecule (τM),
the relaxation behavior of the electron spin of GdIII (τS1 and
τS2), and the rotational diffusion of the complex in solution
(τR). The residence time of the coordinated water molecule
(τM) can be obtained by analysis of the temperature depen-
dence of the reduced transverse relaxation rate of the 17O
NMR resonance of water in solutions of the GdIII complex
(Figure 2).42ꢀ45 The data obtained in the temperature range
295ꢀ354 K show that the exchange rate is in the slow-to-
intermediate regime. The parameters related to the electronic
relaxation have to be considered with caution.
The final complex, [{Ru(bpy)2}2Ln{DTPA(ph-phen)2}-
(H2O)]Cl4, was obtained by reacting [Ln{DTPA(ph-phen)2}-
(CH3OH)] with cis-[RuCl2(bpy)2] in a water/ethanol (1:1, v/v)
mixture (Scheme 2). The RuIIꢀLnIII complexes were purified by
semipreparative reversed-phase high-performance liquid chroma-
tography (HPLC), resulting in a red solid. The formation of a
trinuclear complex, where two RuII ions are coordinated to 1,10-
phenanthroline moieties and one LnIII ion is bound to a DTPA
part, is confirmed by HPLC (Figure S3 in the Supporting Infor-
mation), and ESI-MS shows molecular peaks [M + 3H2O]4+ at m/z
483.2 and 483.3, corresponding to the EuIIIꢀRuII and GdIIIꢀRuII
complexes, respectively.
Relaxometric Studies. The water proton relaxivity, defined
as the paramagnetic longitudinal relaxation rate in reciprocal
A theoretical treatment of the experimental data was per-
formed assuming the presence of one water molecule in the first
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Figure 3. Proton relaxivity of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4 at 37 °C; circles, [Gd] = 0.795 mM; triangles, [Gd] =
0.397 mM. The dotted line corresponds to the profile of Gd-DTPA and
the dashed line to the profile of Gd-DTPA-BMA.
Figure 2. Evolution of the reduced 17O transverse relaxation rate (1/TR2 =
55.55/Tp2ara[Gd]) versus the reciprocal of the temperature for
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 ([Gd] = 6.81 mM).
The theoretical fitting of the NMRD profiles takes into
account the inner-49,50 and outer51-sphere contributions to the
paramagnetic relaxation rate. Some parameters were fixed during
the fitting procedure: the distance (d) of closest approach for the
outer-sphere contribution was set at 0.36 nm, τM was set to the
value obtained by 17O NMR (τM310 = 808 ns) as described above,
the number of coordinated water molecules was set to 1 (q = 1),
the relative diffusion constant D = 3.0 ꢁ 10ꢀ9 m2 sꢀ1, and r is the
distance between the GdIII ion and the proton nuclei of water (r =
0.31 nm). The results of the fittings are shown in Figure 3 and
Table 2. The NMRD profile of [{Ru(bpy)2}2Gd{DTPA-
(ph-phen)2}(H2O)]Cl4 was fitted three times. When τV was
fixed to the value used in 17O NMR (τV = 24.6 ps), no good
NMRD fitting was found according with the measured data.
Therefore, the upper limit of τV was set free, resulting in a proper
fitting but also a quite large value (τV = 50 ( 3 ps), so a third value
(τV = 35 ps) was fixed to check if any notable changes were found.
The agreement between these fitted results and the experimental
data slightly changed, but the value of the electronic relaxation
rate at very low field τSO [τSO = (5BτV)ꢀ1] was not affected and
the τR values changed by less than 12%. It is to be noted that
the model used to fit the data assumes a spherical shape of the
GdIII complex. This assumption is probably not fulfilled for the
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 complex.
At a frequency of 20 MHz, the NMRD profile of [{Ru-
(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 shows an in-
creased proton relaxivity compared with Gd-DTPA and
Gd-DTPA-BMA, which is the result of an increase of the rotational
correlation time (τR) of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4. The tumbling rate observed for [{Ru(bpy)2}2-
Gd{DTPA(ph-phen)2}(H2O)]Cl4 decreases by a factor of
about 4 compared to Gd-DTPA. This is in good agreement
with the increase of the molecular weight of the complex.
Finally, because contrast agents can show interaction with
proteins, interaction studies with HSA were performed. HSA
is one of the most important proteins in human plasma and has
a constitution of 4ꢀ4.5% to plasma. MRI contrast agents can
bind via a noncovalent interaction with HSA, reducing their
mobility. Figure 4 displays the longitudinal proton relaxation
rate in a solution containing 4% HSA, as a function of the
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 concentra-
tion. The increase of the paramagnetic relaxation rate in the
Table 1. Parameters Obtained by the Theoretical Adjustment
of the 17O Transverse Relaxation Rates versus the Reciprocal
of the Temperature
[{Ru(bpy)2}2Gd
{DTPA(ph-phen)2}(H2O)]Cl4 Gd-DTPA-BMA48
τ3M10 (ns)
ΔHq (kJ molꢀ1
ΔSq (J molꢀ1 Kꢀ1
A/p (106 rad sꢀ1
B (1020 sꢀ2
τ3V10 (ps)a
a τ3V10 = 0.985τ2V98
808 ( 34
37.8 ( 0.1
ꢀ6.4 ( 0.2
ꢀ3.80
5.7 ( 0.3
24.6
967 ( 36
)
48.0 ( 0.1
24.9 ( 0.2
ꢀ3.20 ( 0.04
2.04 ( 0.06
21.2 ( 0.6
)
)
)
.
coordination sphere. Some parameters related to the electronic
relaxation rate were fixed to usual values: the hyperfine coupling
constant between the oxygen nucleus of the bound water
molecule and the GdIII ion (A/p = ꢀ3.8 ꢁ 106 rad sꢀ1 ),46 the
correlation time modulating the electronic relaxation of GdIII
(τ2V98 = 25 ps),46 and the activation energy related to τV (EV = 1 kJ
molꢀ1 ).47 This results in the determination of the following
parameters: the residence time of the coordinated water mol-
ecules (τM); B, related to the mean-square of the zero-field-
splitting energy Δ (B = 2.4Δ 2); ΔHq and ΔSq, respectively, the
enthalpy and entropy of activation of the water exchange process.
The calculated parameters are shown in Table 1.
The calculated value of the water residence time at 37 °C for
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 is larger than that
of Gd-DTPA (τ3M10 = 143 ns) because of the presence of two amide
bonds but is smaller than that of Gd-DTPA-BMA (τM310
=
105046ꢀ967 ns;48 DTPA-BMA = 1,7-bis(methylcarbamoylmethyl)-
1,4,7-triazaheptane-1,4,7-triacetic acid). The [{Ru(bpy)2}2Gd{DTPA-
(ph-phen)2}(H2O)]Cl4 complex was investigated by proton nuclear
magnetic relaxation dispersion (NMRD) measurements in water at
37 °C from 0.24 ꢁ 10ꢀ3 to 1.41 T, and the NMRD profile is
shown in Figure 3. To exclude the possibility of stacking be-
tween the different molecules, the NMRD profile of [{Ru(bpy)2}2-
Gd{DTPA(ph-phen)2}(H2O)]Cl4 was measured at two concentra-
tions, 0.795 and 0.397 mM.
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Table 2. Parameters Obtained by the Theoretical Fitting of the Proton NMRD Data in Water at 310 K
parameter
τ3M10 (ns)
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4
Gd-DTPA-BMAa
Gd-DTPAa
808b
808b
808b
967
143
τ3R10 (ps)
τ3S1O0 (ps)d
τ3V10 (ps)
r1 (sꢀ1 mMꢀ1) at 20 MHz
206 ( 5
113 ( 2
50 ( 3
7.0
223 ( 5
112 ( 2
35c
231 ( 4
112 ( 1
24.6c
65 ( 2
95 ( 3
18 ( 3
3.8
54 ( 14
87 ( 3
25 ( 3
3.8
7.0
7.0
a From refs 48 and 52. b Fixed to the value obtained for τ3M10 by 17O relaxometry. c Fixed value. d τSO = (5BτV)ꢀ1
.
function of the total concentration. The results obtained by
relaxometry and ultrafiltration are in very good agreement.
The NMRD profile of a solution of [{Ru(bpy)2}2Gd-
{DTPA(ph-phen)2}(H2O)]Cl4 (0.79 mM) in 4% HSA is shown
in Figure 5 (left). It is the sum of the diamagnetic relaxation rate
of a 4% HSA solution, the paramagnetic relaxation rate of free
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 and the para-
magnetic relaxation rate of bound [{Ru(bpy)2}2Gd{DTPA(ph-
phen)2}(H2O)]Cl4. The free [Gd]f = 0.406 mM and complexed
[Gd]c = 0.388 mM concentrations of the [{Ru(bpy)2}2Gd-
{DTPA(ph-phen)2}(H2O)]Cl4 complex were calculated from
the Ka value obtained above. The paramagnetic relaxation rate of
free [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 was ob-
tained from its relaxivity in water and its concentration in the
HSA solution. The NMRD profile of the GdIII complex bound to
HSA was then calculated by subtracting the diamagnetic relaxation
rate and the paramagnetic relaxation rate of free [{Ru(bpy)2}2-
Gd{DTPA(ph-phen)2}(H2O)]Cl4 from the observed relaxation
rate and dividing the result by the concentration of bound
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 (Figure 5,
right). As expected, the profile shows a “hump” characteristic
of slowly rotating paramagnetic species at higher magnetic
fields. However, because of the slow water exchange rate, the
relaxivity of the bound complex is quite low.
Figure 4. Proton paramagnetic relaxation rate of solutions of
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 in water containing
4% HSA at 20 MHz and 37 °C. The dashed line represents the data
obtained in the absence of HSA.
presence of HSA confirms a noncovalent interaction. When we
fit these data by eq 1, an estimation of the association constant,
Ka, and the relaxivity of the noncovalently bound complex, rc1,
can be made.
Photophysical Properties. RuII-Centered Absorption and Lu-
minescence. The electronic absorption spectrum of
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 displays bands
due to spin-allowed d f π* metal-to-ligand charge-transfer
(1MLCT) transitions at 456 nm and a shoulder at 425 nm
(Figure 6 and Table S1 and Figure S5 in the Supporting
Information). In addition, the intense band at 287 nm corre-
sponds to the ligand-centered π f π* transitions. The bands at
254 and 244 nm might be attributed to either π f π* and/or d f
π* transitions, while the shoulder at 320 nm to RuII-centered d f
d transitions. The excitation spectrum of the [{Ru(bpy)2}2-
Gd{DTPA(ph-phen)2}(H2O)]Cl4 complex, recorded by mon-
itoring the emission at 610 nm, resembles the absorption
spectrum, displaying the bands at 285, 320sh, 425sh, and
455 nm (Figure 6, top). Upon excitation into the 1MLCT band
at 440 nm, the [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4
complex exhibits bright-red luminescence in the range
550ꢀ850 nm, centered at 620 nm, which is originating from
the 3MLCT excited states (Figure 6, bottom). The luminescence
lifetime is 0.54 μs, which is 1.2-fold longer than that for the
[Ru(bpy)2(phen)] complex with a nonderivatized 1,10-phenan-
throline ligand.54 The quantum yield of [{Ru(bpy)2}2Gd-
{DTPA(ph-phen)2}(H2O)]Cl4 was determined using Rhodamine
101 as a standard and was found to be 4.7%. In general, the
photophysical parameters of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4 are comparable with those of other luminescent RuII
complexes.21ꢀ23
R1p, obs ¼ 1000fðr1 s Þ þ 0:5ðr1 ꢀ r1 Þ½Np0 þ s0 þ Ka
f 0
c
f
ꢀ1
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ꢀ1
ꢀ
½ðNp0 þ s0 þ Ka Þ ꢀ 4Ns0p0ꢂꢂg
ð1Þ
In eq 1, p0 is the protein concentration, s0 is the concentration
of the paramagnetic complex, and N is the number of inde-
pendent and identical interaction sites. The data were fitted,
resulting in the following parameters: a value of 7.2 (
0.6 sꢀ1 mMꢀ1 for the relaxivity of the free contrast agent, a
Ka value equal to 4500 ( 638 Mꢀ1, and a r1c value of 14.3 (
1.7 sꢀ1 mMꢀ1. The number of equivalent and independent
sites was set to 1.
The interaction between [{Ru(bpy)2}2Gd{DTPA(ph-
phen)2}(H2O)]Cl4 and HSA is also confirmed by ultrafiltration
experiments.53 The unbound [{Ru(bpy)2}2Gd{DTPA(ph-
phen)2}(H2O)]Cl4 fractions were separated from the bound
fractions, and the bound [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4 concentration was calculated by subtracting the free
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 concentration,
measured by relaxometry, from the initial concentration. The
total concentration of the GdIII complex ranged from 0.20 to
0.78 mM, while the protein concentration was fixed at 4%
(Figure S4 in the Supporting Information). Subsequently, the
concentration of bound [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4 divided by its total concentration was plotted as a
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Figure 5. (Left) NMRD profile of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 (0.79 mM) in 4% HSA (closed circles) and that of 4% HSA (plain
line). (Right) Calculated NMRD profile of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 bound to HSA (open circles). The line through the data is
drawn to guide the eye. The relaxivity profile of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 in water is added for comparison (dotted line).
Figure 6. (Top) Excitation (λem = 610 nm, solid line), superimposed
Figure 7. (Top) Excitation (λem = 614 nm, solid line), superimposed
absorption (dashed line), and (bottom) emission (λex = 440 nm)
absorption (dashed line), and (bottom) emission (λex = 300 nm) spectra
spectra, as well as photographs of a 10 μM aqueous solution of
of a 10 μM MeOH solution of [Eu{DTPA(ph-phen)2}(CH3OH)].
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4.
[{Ru(bpy)2}2Eu{DTPA(ph-phen)2}(H2O)]Cl4 displays ab-
sorption, excitation, and emission spectra very similar to those of
its GdIII analogue (Figures S5 and S6 in the Supporting
Information). No characteristic EuIII emission was observed
under excitation into either the 1MLCT or the ππ* ligand band.
This points to the existence of very efficient nonradiative deactiva-
tion quenching of EuIII excited states.
EuIII-Centered Luminescence. It is known that luminescent
lanthanide ions can serve as probes of the coordination environ-
ment; moreover, solvation numbers can be determined by the
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Table 4. Photophysical Parameters of the [Eu{DTPA-
(ph-phen)2}(CH3OH)] Complex
significantly change the environment around the LnIII ion, the
solvation number of the lanthanide ions in the [{Ru(bpy)2}2Ln-
{DTPA(ph-phen)2}(H2O)]Cl4 complexes is considered to be 1.
The sensitized luminescence quantum yield of [Eu{DTPA-
(ph-phen)2}(CH3OH)] in MeOH is 0.77%, and it increases up
to 2.3% in MeOD being in agreement with the above discussion
on nonradiative transitions. Because of the low absorption
coefficients of f-f transitions of lanthanide ions the intrinsic
quantum yield could not be measured and thus was estimated
using eq 2 and the following relation for the radiative lifetime:
Eub
Eu
L a
Eu
solvent
τobs/msa
τrad/msb
Q
Q
ηsens/%b
MeOH
MeOD
0.80(1)
2.18(2)
4.35
4.42
18
49
0.77(7)
2.3(2)
4.3(6)
4.7(7)
a 2σ values within parentheses. b The refractive indexes for solutions
taken as equal to these of neat solvents, n(MeOH) = 1.329 and
n(MeOD) = 1.326; for the definition, see eqs 2 and 4.
5
lifetime method.55,56 The nondegenerate D0 emission state
ꢀ
ꢁ
1
Itot
¼ AMD, 0 n3
ð4Þ
simplifies the interpretation of the luminescence spectra, so that
the EuIII ion is the most appropriate ion for use as a luminescent
structural probe.
3
3
τrad
IMD
where AMD,0 is the Einstein coefficient and equals 14.65 sꢀ1, n is
the refractive index, Itot the total integrated intensity of 5D0f7FJ
(J = 0ꢀ4) and IMD the integrated intensity of magnetic-dipole
transition 5D0 f 7F1. The values of 18 and 49% were obtained
for [Eu{DTPA(ph-phen)2}(CH3OH)] in MeOH and MeOD
Because of the strong quenching of the fꢀf transitions by
the MLCT states involving RuII in [{Ru(bpy)2}2Eu{DTPA-
(ph-phen)2}(H2O)]Cl4, the photophysical properties of [Eu-
{DTPA(ph-phen)2}(CH3OH)] in a MeOH solution have been
investigated to gain insight into the coordination environment
around the Ln ion offered by the DTPA(ph-phen)2 ligand.
[Eu{DTPA(ph-phen)2}(CH3OH)] exhibits ligand-centered ab-
sorption bands in the range 200ꢀ350 nm with maxima at 230 nm
(ε = 56 805 Mꢀ1 cmꢀ1) and 268 nm (ε = 57 270 Mꢀ1 cmꢀ1) and
a shoulder at 310 nm (ε = 16 370 Mꢀ1 cmꢀ1) (Figure 7, top).
When the EuIII 5D0 f 7F2 transition at 614 nm is monitored, the
excitation spectrum resembles the absorption spectrum. Upon
excitation into the ligand levels, [Eu{DTPA(ph-phen)2}-
(CH3OH)] exhibits characteristic bright-red luminescence due
to the 5D0 f 7FJ transitions (J = 0ꢀ4) (Figure7, bottom;Table S2
in the Supporting Information). The hypersensitive 5D0 f 7F2
transition is dominating the spectrum with I(5D0f5F2)/
(Table 4) following the same trend as τobs and QLEu. Although the
Eu
Eu
Q
value is reaching almost 50% for a MeOD solution, the
overall luminescence quantum yield remains quite low as a result
of the inefficient energy transfer from the organic ligand to the
EuIII ion. Indeed, the sensitization efficiency calculated as a ratio
of quantum yields using eq 2 lies in the range 4.3ꢀ4.7%.
’ CONCLUSIONS
In this paper, the design of a new trinuclear heterobimetallic
complex, [{Ru(bpy)2}2Ln{DTPA(ph-phen)2}(H2O)]Cl4 (Ln =
Eu, Gd), is presented. The formation of [{Ru(bpy)2}2Gd{DTPA-
(ph-phen)2}(H2O)]Cl4 results in a decrease of the tumbling rate by
5
7
I(5D0f5F1) = 2.9, while the D0 f F4 transition also has a
sizable intensity with I(5D0f5F2)/I(5D0f5F1) = 2.5 (Table S3
in the Supporting Information). The highly forbidden 5D0 f 7F0
transition has a quite large intensity (16% of the magnetic dipole
5D0 f 7F1 transition), so the EuIII ion is occupying a site with a
Cs, Cn, or Cnv symmetry.
1
a factor of 3.8 in comparison with Gd-DTPA (Magnevist). H
NMRD measurements of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4 in water show a r1 relaxivity at 20 MHz and 37 °C of
7.0 sꢀ1 mMꢀ1. Furthermore, relaxometry and ultrafiltration experi-
ments indicate that the DTPA(ph-phen)2 ligand has a high affinity
to noncovalently bind to HSA, resulting in a r1 relaxivity of 14.3
Quantitatively, the EuIII luminescence can be analyzed using
s
ꢀ1 mMꢀ1 at 20 MHz and 37 °C. Unfortunately, the slow water
Eu
Eu
eq 2, where QLEu and Q are the overall and intrinsic quantum
exchange rate of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4
restrains the increase of the longitudinal relaxation rate, resulting in a
rather low relaxivity. [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]-
Cl4 also shows interesting photophysical properties, exhibiting
bright-red emission with a quantum yield of 4.7%. The lumines-
cence lifetime of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]-
Cl4 is long enough to exceed any fluorescent background.
Considering both the relaxometric and photophysical properties
of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4, it can act as
a potential probe for time-gated luminescence-based imaging as
well as a contrast agent for MRI.
yields, ηsens is the sensitization efficiency, and τobs and τrad are the
observed and radiative lifetimes of the EuIII 5D0 level:
ꢀ
ꢁ
τobs
τrad
QELu ¼ ηsensQEEuu ¼ ηsens
ð2Þ
The luminescence lifetimes of [Eu{DTPA(ph-phen)2}-
(CH3OH)] in a MeOH solution (τMeOH) is equal to 0.80 ms,
and it increases by 2.7 times when going to a deuterated MeOH
(MeOD) solution (τMeOD), being in line with less nonradiative
deactivation induced by OꢀD vibrations in comparison with
OꢀH vibrations (Table 4). To estimate the number of solvent
molecules coordinated to a EuIII ion, Beeby’s equation was
used,57 taking into account that an alcohol molecule has one
OꢀH oscillator and thus causes twice less the effect than water:
’ EXPERIMENTAL SECTION
Materials. All reagents were obtained from Aldrich Chemical
(Bornem, Belgium) and Acros Organics (Geel, Belgium) and were used
without further purification. Europium(III) triflate was synthesized
according to the literature procedure, starting from Eu2O3 (99.995%).58
Synthesis. Synthesis of 5-Bromo-1,10-phenanthroline. 5-Bromo-
1,10-phenanthroline was prepared according to a literature procedure.59
Yield: 68%. Mp: 113 °C. 1H NMR (300 MHz, CDCl3, ppm): δ 9.20
(m, 2H), 8.66 (d, J = 7.8 Hz, 1H), 8.17 (dd, J = 1.8 and 8.2 Hz, 1H), 8.13
(s, 1 H), 7.74 (dd, J = 4.1 and 8.3 Hz, 1H), 7.65 (dd, J = 4.5 and 8.2 Hz,
1H). 13C NMR (150 MHz, CDCl3, ppm): δ 151.33, 151.10, 147.01,
qMeOH ¼ 2:4ðΔk ꢀ 0:25 ꢀ 0:075qNÞ
ð3Þ
where Δk = 1/τMeOH ꢀ 1/τMeOD and qN is the number of
coordinated amide groups. This resulted in qMeOH = 0.9. So,
the organic ligand in the [Eu{DTPA(ph-phen)2}(CH3OH)]
complex arranges around the EuIII ion in such a way that
coordination of one solvent molecule is possible. Assuming that
coordination of RuII to the 1,10-phenanthroline moieties will not
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146.03, 136.35, 135.52, 130.08, 129.20, 128.31, 124.23, 124.07, 121.22.
HRMS (EI). Calcd for C12H7BrN2 [M]: m/z 257.9793. Found: m/z
257.9792.
concentrated under reduced pressure, redissolved in a small amount of
MeOH, and precipitated by diethyl ether. The purity was checked by
HPLC with MeOH/H2O and 0.1% TFA as eluents, resulting in a single
peak. Free Ln ions were removed by Chelex 100, and their absence was
checked by a test with an arsenazo indicator solution. A shift of
approximately 65 cmꢀ1 to lower energy is observed for the carbonyl
stretching in IR spectra of [Ln{DTPA(ph-phen)2}(CH3OH)], thus
confirming complexation of the Ln ion by the ligand.
Synthesis of tert-Butyl [4-(1,10-Phenanthrolin-5-yl)phenyl]carbamate
(1). To a solution of 5-bromo-1,10-phenanthroline (399 mg, 1.54 mmol),
tert-butyl [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]carbamate
(538 mg, 1.69 mmol, 1.1 equiv), and Cs2CO3 (1.01 g, 3.10 mmol, 2 equiv)
in a 1:1 H2O/toluene biphasic mixture (32 mL) was added 10 mol % of
Pd(PPh3)4 (178 mg), and the resulting solution was refluxed for 16 h. The
reaction mixture was added to CH2Cl2 (100 mL) and water (100 mL) and
thoroughly stirred, and the organic layer was separated. The aqueous layer
was extracted twice with CH2Cl2 (100 mL), the combined organic layer was
dried over Na2SO4 and evaporated in vacuo, and the residue was purified by
column chromatography [silica gel, CH2Cl2 (saturated NH3)]. After
evaporation, the product was recrystallized from hot toluene (25 mL) and
collected as a white solid (387 mg, 68%).
EuIII complex [Eu{DTPA(ph-phen)2}(CH3OH)]. Yield: 45.7 mg
(78%). IR (KBr): 1595 (COOꢀ asym stretch), 1544 (amide II), 1400
(COOꢀ sym stretch) cmꢀ1. ESI-MS. Calcd for C50H42EuN9O8: m/z
1072.2 ([M + Na]+), 547.4 ([M + 2Na]2+). Found: m/z 1072.9 ([M +
Na]+), 548.3 ([M + 2Na]2+). HPLC (solvent A, H2O + 0.1% HCOOH;
solvent B, MeOH, 20% B f 100% B, 20 min): tr 20.65 min.
GdIII complex [Gd{DTPA(ph-phen)2}(CH3OH)]. Yield: 45.2 mg
(77%). IR (KBr): 1598 (COOꢀ asym stretch), 1548 (amide II), 1400
(COOꢀ sym stretch) cmꢀ1. ESI-MS. Calcd for (C50H42GdN9O8 [M]:
m/z 528.1 ([M + 2H]2+), 352.3 ([M + 3H]3+). Found: m/z 527.9 ([M +
2H]2+), 352.2 ([M + 3H]3+). HPLC (solvent A, H2O + 0.1% HCOOH;
solvent B, MeOH, 20% B f 100% B, 20 min): tr 20.27 min.
Mp: >250 °C, product starts to decompose at 258 °C. 1H NMR (300
MHz, CDCl3, ppm): δ 9.20 (s, 2H), 8.30 (dd, J = 1.4 and 8.2 Hz, 1H),
8.24 (d, J = 8.3 Hz, 1H), 7.71 (s, 1H), 7.65 (dd, J = 4.5 and 8.1 Hz, 1H),
7.60ꢀ7.55 (m, 3H), 7.46 (d, J = 8.3, 2H), 6.71 (s, 1H), 1.56 (s, 9H). 13C
NMR (150 MHz, CDCl3, ppm): δ 153.19, 150.61, 150.45, 146.85,
146.14, 138.82, 138.73, 136.32, 134.96, 133.82, 130.99, 129.45, 128.63,
126.76, 123.74, 123.19, 119.01, 81.29, 28.75. HRMS (EI). Calcd for
C23H21N3O2 [M]: m/z 371.1634. Found: m/z 371.1632.
Synthesis of 4-(1,10-Phenanthrolin-5-yl)aniline (2). To a solution of
tert-butyl [4-(1,10-phenanthrolin-5-yl)phenyl]carbamate (387 mg, 1.04
mmol) in CH2Cl2 (10 mL) was added TFA (10 mL) dropwise, and the
solution was stirred overnight at room temperature. The solvent was
removed in vacuo, and the residue was purified by column chromatog-
raphy [silica gel, CH2Cl2 (saturated NH3)] and collected as a yellow
solid (229 mg, 81%).
Synthesis of LnIIIꢀRuII Complexes. cis-[RuCl2(bpy)2] was synthe-
sized following a literature procedure.60
A solution of cis-[RuCl2(bpy)2] (17.2 mg, 0.036 mmol, 2 equiv) in a
1:1 H2O/EtOH mixture (2 mL) was refluxed under an argon atmo-
sphere for 5 h, the corresponding [Ln{DTPA(ph-phen)2}(CH3OH)]
complex was added (20 mg, 0.02 mmol, 1 equiv), and the mixture was
refluxed overnight. The solution was concentrated under reduced
pressure at 50 °C, and the resulting dark-violet product was purified
by HPLC. The collected fractions were concentrated under reduced
pressure and dried overnight under vacuum at 50 °C. The purity of the
compound was checked again by HPLC with ACN/H2O and 0.1% TFA
as eluents, resulting in a single peak. The HPLC analysis of
[{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 is shown in the Sup-
porting Information (Figure S3).
Mp: >250 °C, product starts to decompose at 258 °C. 1H NMR (300
MHz, CDCl3, ppm): δ 9.18 (m, 2H), 8.37 (d, J = 8.3 Hz, 1H), 8.21 (d,
J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.62 (dd, J = 8.3 and 4.6 Hz, 1H), 7.57 (dd,
J = 8.3 and 4.6 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H),
3.87 (br s, 2H). 13C NMR (150 MHz, CDCl3, ppm): δ 150.29, 146.89,
146.76, 145.95, 139.40, 136.19, 135.13, 131.35, 129.19, 128.66, 126.39,
123.65, 123.07, 115.41. HRMS (EI). Calcd for C18H13N3 [M]: m/z
271.1109. Found: m/z 271.1110.
Synthesis of Bis(1,10-phenanthroline) Derivative [DTPA(ph-
phen)2]. A mixture of 4-(1,10-phenanthrolin-5-yl)aniline (100 mg,
0.36 mmol, 2 equiv) and diethylenetriaminepentaacetic acid bisanhy-
dride (64 mg, 0.18 mmol, 1 equiv) in DMF (7 mL) was stirred overnight
at 60 °C. Diethyl ether (100 mL) was added to the reaction mixture at
0 °C, and DTPA(ph-phen)2 (148 mg, 89%) was collected as a
precipitate and used without further purification. IR spectral data show
strong absorption at 1661 cmꢀ1 due to the CdO stretching mode of the
free acid. The lower energy of the CdO vibrations can be explained by
intramolecular hydrogen bonding.
EuIII complex [{Ru(bpy)2}2Eu{DTPA(ph-phen)2}(H2O)]Cl4. Yield:
59%. IR (KBr): 1596 (COOꢀ asym stretch), 1544 (amide II), 1417
(COOꢀ sym stretch) cmꢀ1. ESI-MS. Calcd for C90H74EuN17O8Ru2 [M]:
m/z 643.1 ([M + H2O + Cl]3+), 482.4 ([M + 3H2O]4+), 386.2 ([M + H +
3H2O]5+), 240.5 ([M + 2H + 2Na]8+). Found: m/z 644.0 ([M + H2O +
Cl]3+), 483.2 ([M + 3H2O]4+), 386.8 ([M + H + 3H2O]5+), 240.1 ([M +
2H + 2Na]8+). HPLC (solvent A, H2O + 0.1% HCOOH; solvent B, ACN,
10% B f 50% B, 30 min): tr 17.24 min.
GdIII complex [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4. Yield:
36%. IR (KBr): 1598 (COOꢀ asym stretch), 1548 (amide II), 1419
(COOꢀ sym stretch) cmꢀ1. ESI-MS. Calcd for C90H74GdN17O8Ru2 [M]:
m/z 644.8 ([M + H2O + Cl]3+), 483.7 ([M + 3H2O]4+), 387.2 ([M + H +
3H2O]5+), 241.1 ([M + 2H + 2Na]8+). Found: m/z 644.2 ([M + H2O +
Cl]3+), 483.3 ([M + 3H2O]4+), 386.7 ([M + H + 3H2O]5+), 240.1 ([M +
2H + 2Na]8+). HPLC (solvent A, H2O + 0.1% HCOOH; solvent B, ACN,
10% B f 50% B, 30 min): tr 15.81 min.
Mp: 189 °C. 1H NMR (400 MHz, DMSO-d6, ppm): δ 9.12 (d, J = 4.5
Hz, 2H), 9.02 (d, J = 4.2 Hz, 2 H), 8.41 (d, J = 8.4 Hz, 2H), 8.33 (d, J =
8.5 Hz, 2H), 7.89 (dd, J = 8.4 and 4.5 Hz, 2H), 7.79 (d, J = 8.2 Hz, 4H),
7.72 (dd, J = 8.5 and 4.2 Hz, 2H), 7.58 (s, 2H), 7.27 (d, J = 8.2 Hz, 4H),
4.20 (s, 2H), 4.08 (s, 4H), 3.87ꢀ3.81 (m, 8H), 3.63 (t, J = 6.1 Hz, 4H).
13C NMR (100 MHz, DMSO-d6, ppm): δ 173.58, 170.10, 150.31,
150.06, 146.19, 145.31, 139.10, 138.18, 136.57, 134.46, 133.52, 130.58,
128.29, 127.67, 126, 87, 123.98, 123.58, 119.91, 58.94, 55.90, 52.91,
51.58. IR (KBr): 1661 (CO free acid), 1603 (amide I), 1519 (amide
II) cmꢀ1. ESI-MS. Calcd for C50H45N9O8 [M ꢀ H]ꢀ: m/z 898.9.
Found: m/z 898.3.
Instruments. 1H and 13C NMR spectra were recorded by using a
Bruker Avance 300 spectrometer (Bruker, Karlsruhe, Germany), oper-
1
ating at 300 MHz for H and 75 MHz for 13C, a Bruker Avance 400
spectrometer, operating at 400 MHz for 1H and 100 MHz for 13C, and a
Bruker Avance 600 spectrometer operating at 150 MHz for 13C. IR
spectra were measured by using a Bruker Alpha-T FT-IR spectrometer
(Bruker, Ettlingen, Germany), and data were processed with OPUS 6.5
software. The LC/MS data were collected using an Agilent 1100 system
coupled to an Agilent 6110 single-quadrupole MS system. The LC/MS
method used a Grace Prevail RP-C18 column (150 mm ꢁ 2.1 mm;
particle size 3 μm). ESI-MS spectra were obtained by using a Thermo
Finnigan LCQ Advantage mass spectrometer. Preparative HPLC was
performed using a Waters Delta 600 system equipped with a Waters
996 photodiode-array detector. The preparative HPLC method used a
Synthesis of LnIII Complexes. To a solution of DTPA(ph-phen)2 (50
mg, 0.06 mmol, 1 equiv) in MeOH (5 mL) was added the correspond-
ing lanthanide(III) triflate (0.06 mmol, 1 equiv), and the mixture was
stirred at 50 °C for 24 h under an argon atmosphere. The solution was
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Inorganic Chemistry
ARTICLE
Phenomenex Luna C18 column (150 mm ꢁ 21.20 mm; particle size
5 μm). Melting points were determined using a Reichert-Jung Thermo-
var apparatus and were uncorrected.
The bound ligand concentration was calculated by subtracting the free
ligand concentration measured by relaxometry from the initial concen-
tration. The relative error on the T1 measurements was 3%.
Photophysical Measurements. All photophysical measure-
ments were performed on 10 μM solutions in degassed solvents.
Absorption spectra were recorded with a Varian Cary 5000 spectro-
photometer using quartz Suprasil cells (115F-QS; path length 0.2 cm).
Excitation and luminescence spectra were recorded on Edinburgh
Instruments FS900 or FS920 steady-state spectrofluorimeters using
the same cells and measuring the emitted light at the right angle and
along the long (1 cm) path length. The spectrofluorimeters are equipped
with a 450 W xenon arc lamp, a microsecond flashlamp (pulse length
2 μs), and a red-sensitive (300ꢀ850 nm, Hamamatsu R928P on FS920)
or an extended red-sensitive (185ꢀ1010 nm, Hamamatsu R2658P on
FS920) photomultiplier. All spectra were corrected for the instrumental
functions. Luminescence lifetimes of the 5D0 level for EuIII-containing
solutions were measured on an Edinburgh Instruments FS920 steady-
state spectrofluorimeter under 280ꢀ300 nm excitation. Lifetimes are
averages of at least three independent measurements. Quantum yields
were determined by a comparative method using Rhodamine 101
(Aldrich) in ethanol (Q = 100%) as a standard.61 Luminescence lifetimes
of RuII emission in [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4
under excitation into the MLCT band were determined using the
time-correlated single-photon-counting technique described in detail
previously.62 All measurements were performed at room temperature.
Framework Molecular Model. The model was built using the
Avogadro software package. The geometry of the GdIIIꢀRuII complex
was optimized with the universal force field.63
’ ASSOCIATED CONTENT
Supporting Information. 1H and 2D COSY NMR spec-
S
b
tra of ligand DTPA(ph-phen)2 in DMSO-d6 (Figures S1 and S2),
HPLC analysis of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]-
Cl4 (Figure S3), ultrafiltration and relaxometry results of [{Ru-
(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4 (Figure S4), absor-
ption spectra of [{Ru(bpy)2}2Ln{DTPA(ph-phen)2}(H2O)]-
Cl4 (Figures S5 and S6), excitation and emission spectra of
[{Ru(bpy)2}2Eu{DTPA(ph-phen)2}(H2O)]Cl4 (Figure S7),
mass spectra of all synthesized compounds (Figures S8ꢀS15),
as well as (i) the assignment of the bands in the absorption
spectrum of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}(H2O)]Cl4
(Table S1), (ii) the main peaks in the emission spectrum of
[Eu{DTPA(ph-phen)2}(CH3OH)] (Table S2), and (iii) rela-
tive integral intensities of fꢀf transitions for [Eu{DTPA-
(ph-phen)2}(CH3OH)] (Table S3). This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: tatjana.vogt@chem.kuleuven.be.
17O NMR. 17O NMR measurements of solutions were performed on
350 μL samples placed in 5-mm-external-diameter tubes on a Bruker
Avance-500 spectrometer (Bruker, Karlsruhe, Germany). The tempera-
ture was regulated by air or nitrogen-flow-controlled by a Bruker BVT
3200 unit. 17O transverse relaxation times of distilled water (pH =
6.5ꢀ7) were measured using a CPMG sequence and a subsequent two-
parameter fit of the data points. The 90° and 180° pulse lengths were
27.5 and 55 μs, respectively. 17O T2 of water in a complex solution was
obtained from line-width measurements. All spectra were proton-
decoupled. The concentration of [{Ru(bpy)2}2Gd{DTPA(ph-phen)2}-
(H2O)]Cl4 was equal to 6.81 mM. The data are presented as the reduced
transverse relaxation rate (1/TR2 = 55.55/T2para[Gd], where [Gd] is the
molar concentration of the complexed GdIII, q is the number of
coordinated water molecules, and Tp2ara is the paramagnetic transverse
relaxation rate). The treatment of the experimental data was performed
as described previously.42,43
Proton NMRD. Proton NMRD profiles were measured on a Stelar
Spinmaster FFC, fast-field-cycling NMR relaxometer (Stelar, Mede
(PV), Italy) over a magnetic field strength range extending from 0.24 mT
to 1.4 T. Measurements were performed on 0.6 mL samples contained
in 10-mm-o.d. Pyrex tubes. Additional relaxation rates at 20 and
60 MHz were respectively obtained on a Minispec mq20 and a
Minispec mq60 (Bruker, Karlsruhe, Germany). The proton NMRD
curves were fitted using data-processing software,64 including different
theoretical models describing the nuclear relaxation phenomena
(Minuit, CERN Library).49ꢀ51
’ ACKNOWLEDGMENT
G.D. and P.V. acknowledge the IWT Flanders (Belgium) for
financial support. T.N.P.-V. and K.B. thank the FWO-Flanders (for
Projects G.0412.09 and KAN2008 1.5.157.08). S.V.E. is a visiting
postdoctoral fellow of the FWO-Flanders (Project G.0412.09). S.L.,
L.V.E., and R.N.M. thank the FNRS, the ARC program (Contract
05/10 335), the ENCITE program, and the EMIL program, the
Interuniversity Attraction Poles P6/29 of the Belgian Federal
Science Policy Office, and the COST Action D38. CHN micro-
analysis was performed by Dirk Henot. ESI-MS measurements were
made by Dirk Henot and Bert Demarsin, and inductively coupled
plasma mass spectrometry measurements were performed by
Michꢀele Vanroelen (Department of Chemical Engineering). Karel
Duerinckx is acknowledged for his help in the NMR measurements
and Dr. Lesley Pandey for lifetime determination on {Gd[Ru-
(bpy)2]2DTPA(ph-phen)2}4+. We also thank Servaas Michielssens
for his development of the framework molecular model.
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