Terpyridine-based heteroditopic ligand for RuIILn3III metallostar architectures (Ln = Gd, Eu, Nd, Yb) with MRI/optical or dual-optical responses

Article abstract of DOI:10.1021/ic502342x

A new ditopic ligand (L) based on a 2,2′:5′,4″-terpyridine unit substituted in the 2″,6″ positions with iminodiacetate arms has been designed and synthesized for the construction of Ru^IIL₃Ln₃^III supramolecular architectures. The two components of this system, a 2,2′-bipyridine unit for Ru^II coordination and a pyridine-bis(iminodiacetate) core for Ln^III coordination, are tightly connected via a covalent C_arom(py)-C_arom(py) bond. The paramagnetic and photophysical properties of the corresponding tetrametallic Ru^IIL₃Gd₃^III complex have been evaluated, highlighting the potential of this metallostar structure to act as a bimodal MRI/optical imaging agent. Variable-temperature ¹⁷O NMR and proton nuclear magnetic relaxation dispersion (NMRD) measurements showed that this complex exhibits (i) a remarkable relaxivity per metallostar molecule, particularly at clinical and high magnetic fields (r₁^310K = 51.0 and 36.0 mM^-1 s^-1 at 20 and 300 MHz, respectively) and (ii) a near-optimal residence lifetime of Gd^III coordinated water molecule (τ_M^310K = 77.5 ns). This is the result of the presence of two inner-sphere water molecules in the Gd^III components of the metallostar and a slow tumbling rate of the molecule (τ_R^310K = 252 ps). Upon excitation in the visible domain (λ_exc = 472 nm), the Ru^II component of the complex exhibits a bright-red luminescence centered at 660 nm with a quantum yield of 2.6% in aqueous solutions at pH 7.4. Moreover, this Ru^IIL₃Gd₃^III assembly is also characterized by a high kinetic inertness in biological media (PBS and human serum solutions) and a high photostability (photobleaching). Finally, preliminary photophysical studies on RuL₃Nd₃ and RuL₃Yb₃ assemblies revealed that the Ru^II center acts as an effective sensitizer for Ln^III-based luminescence in the near-IR region. The Nd^III species was found to be the most effective at quenching the ³MLCT luminescence of the Ru center.

Full text of DOI:10.1021/ic502342x

Article
pubs.acs.org/IC
Terpyridine-Based Heteroditopic Ligand for RuIILn3 Metallostar
Architectures (Ln = Gd, Eu, Nd, Yb) with MRI/Optical or Dual-Optical
Responses
III
Alexandre Boulay,†,‡ Celine Deraeve,†,‡,# Luce Vander Elst,*,§,∥ Nadine Leygue,†,‡ Olivier Maury,⊥
́
Sophie Laurent,§,∥ Robert N. Muller,§,∥ Beatrice Mestre-Voegtle,*,†,‡ and Claude Picard*,†,‡
́
́
†CNRS, Laboratoire de Synthes
Narbonne, F-31062 Toulouse cedex 9, France
‡Universite
de Toulouse, UPS, Laboratoire de Synthes
Narbonne, F-31062 Toulouse cedex 9, France
̀
e et Physico-Chimie de Molec
́
ules d’Inter
́
et
̂
Biologique, SPCMIB, UMR-5068, 118 Route de
́
̀ ́ ́ ̂
e et Physico-Chimie de Molecules d’Interet Biologique, SPCMIB, 118 route de
§NMR and Molecular Imaging Laboratory, Department of General, Organic and Biomedical Chemistry, University of Mons, 23 Place
du Parc, B-7000 Mons, Belgium
∥Center for Microscopy and Molecular Imaging (CMMI), Rue Adrienne Bolland, 8, B-6041 Gosselies, Belgium
⊥Universite
de Lyon 1, Laboratoire de Chimie de l’ENS Lyon, CNRS UMR 5182, 46 allee d’Italie, 69364 Lyon, France
́ ́
S
* Supporting Information
ABSTRACT: A new ditopic ligand (L) based on a 2,2′:5′,4″-
terpyridine unit substituted in the 2″,6″ positions with iminodiace-
tate arms has been designed and synthesized for the construction of
RuIIL3Ln3 supramolecular architectures. The two components of
III
this system, a 2,2′-bipyridine unit for RuII coordination and a
pyridine-bis(iminodiacetate) core for LnIII coordination, are tightly
connected via a covalent Carom(py)−Carom(py) bond. The para-
magnetic and photophysical properties of the corresponding
tetrametallic RuIIL3Gd3III complex have been evaluated, highlighting
the potential of this metallostar structure to act as a bimodal MRI/
optical imaging agent. Variable-temperature 17O NMR and proton
nuclear magnetic relaxation dispersion (NMRD) measurements showed that this complex exhibits (i) a remarkable relaxivity per
310K
metallostar molecule, particularly at clinical and high magnetic fields (r1
= 51.0 and 36.0 mM−1 s−1 at 20 and 300 MHz,
respectively) and (ii) a near-optimal residence lifetime of GdIII coordinated water molecule (τM310K = 77.5 ns). This is the result
of the presence of two inner-sphere water molecules in the GdIII components of the metallostar and a slow tumbling rate of the
molecule (τR310K = 252 ps). Upon excitation in the visible domain (λexc = 472 nm), the RuII component of the complex exhibits a
bright-red luminescence centered at 660 nm with a quantum yield of 2.6% in aqueous solutions at pH 7.4. Moreover, this
RuIIL3Gd3III assembly is also characterized by a high kinetic inertness in biological media (PBS and human serum solutions) and a
high photostability (photobleaching). Finally, preliminary photophysical studies on RuL3Nd3 and RuL3Yb3 assemblies revealed
that the RuII center acts as an effective sensitizer for LnIII-based luminescence in the near-IR region. The NdIII species was found
3
to be the most effective at quenching the MLCT luminescence of the Ru center.
which does not limit them as bulk extracellular relaxation agents
but becomes a severe limitation for their use as molecular MRI
agents.7 In this latter emerging MRI domain, a high relaxivity
concentrated into a limited molecular volume is required for
selected biological targets or specific cellular events.
The more straightforward and simplest procedure to allow a
marked sensitivity enhancement deals with the use of
architectures containing several GdIII chelating units of
sufficient relaxivity. Such systems concentrate several MRI
probes in a small volume, but they may also enhance the
INTRODUCTION
■
In MRI domains, the use of gadolinium(III) contrast agents
(CAs), commonly called T1 or positive agents because they
increase signal intensity, is well-established, and, currently,
intensive research efforts are devoted to the design of more
efficient GdIII-based CAs.1−4 In addition to their main
applications in clinical diagnosis (∼30−40% MRI exams),
they are also used in medical research and pharmacogical
studies including in vitro and animal experiments.5 The
contrast efficiency, named relaxivity and symbolized by r1, of
GdIII-based CAs currently in clinical use is relatively low (r1 ≈ 4
mM−1 s−1 at 20 MHz and 37 °C).6 This implies the use of high
concentrations (0.1 mM kg−1 total body weight) of these CAs,
Received: September 30, 2014
© XXXX American Chemical Society
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Figure 1. Examples of reported ligands structures in the construction of Gd3M metallostars (M = Fe2+, Ru2+, Al3+, Ti4+) and bpy-PMNTA ligand
structure.
intrinsic relaxivity properties of the GdIII chelating unit. In such
polymetallic architectures, the increase in relaxation per
gadolinium is realized by slowing the rotational motion of the
GdIII chelating agent as a result of increased molecular weight
of the system.8
In this context, we designed a novel d−f metallostar contrast
agent in which three gadolinium(III) complexes are assembled
around a ruthenium(II) ion to a tetrametallic species. The
ditopic ligand contains a PMNTA (abbreviated from [2,6-
pyridinediylbis(methylene nitrilo)] tetraacetic acid) as the
complexing unit for the LnIII ion and a 2,2′-bipyridine core
for the coordination of RuII (Figure 1). These two complexing
units are tightly connected via a covalent Carom(py)−Carom(py)
bond, minimizing local flexibility arising from the linker that
binds the two components and that might reduce the relaxivity
gain achieved by the increased molecular size.34 In Gd-
PMNTA, the presence of two inner-sphere water molecules
and an overall negative charge of the complex contribute to an
efficient water exchange (τM310K = 35.2 ns) and a high relaxivity
(r1 = 5.7 mM−1 s−1 at 20 MHz and 310 K).35 Moreover, the
replacement of the metal-bound water molecules by bioactive
bidentate anions such as L-lactate, citrate, carbonate, or
bicarbonate has not been observed for Ln-PMMTA com-
plexes.36 The formation of such ternary complexes, which is
common for DO3A-based heptatendate LnIII complexes,37 can
be an important limitation to the MRI use of bishydrated GdIII
chelates. With respect to bisaqua GdIII complexes, the stability
constant of Gd-PMNTA (log K = 18.6) is comparable to those
of Gd-DTTA derivatives (log K = 18.2−18.6) and about 2.5
orders of magnitude lower than that of Gd-DO3A (log K =
21.1).38−40 We have chosen Ru2+ vs Fe2+ as the central d-
transition metal ion for the formation of the supramolecular
entity because it was reported that metallostars based on a
(Bpy)3Fe2+ core do not have sufficient long-term stability in
biological media.18 Moreover, (Bpy)3Ru2+ can be utilized as an
efficient luminescent probe. As a matter of fact, polypyridine
complexes of ruthenium(II) display emission in the visible
region with lifetimes in microsecond range that allows their use
in time-resolved applications, large Stokes shift (>5000 cm−1)
that minimizes self-quenching, and high photostability that
permits continuous monitoring of biological events by
fluorescence spectroscopy and microscopy.41,42 Thus, this
heterometallic Gd3Ru supramolecular assembly could serve as
a potential MRI/optical bimodal agent, owing to it minimizing
the differences in sensitivity of these two imaging modalities.
Finally, considering such a RuII moiety, it can also act as a
visible-light-excited antenna for sensitizing near-infrared (NIR)
emitting NdIII and YbIII lanthanide ions. In this direction,
several d−f and d−f3 complexes based on these ions were
reported as dual visible−NIR emitting probes.43−45
In this direction, several works reported the covalent
attachment of GdIII chelates to macromolecules such as
polyamino acids, polysaccharides, and dendrimers and to
nanoparticles.9−11 Another approach concerned midsize
molecules and was based on the use of several GdIII small
chelates covalently attached to an organic platform (a single
atom such as nitrogen or a small aromatic ring such as
benzene)12−14 or assembled in a rigid and compact space
around a central d- or p-block metal ion (FeII, RuII, AlIII,
TiIV).15−27 As can be seen in Figure 1, these latter
supramolecular heteropolymetallic compounds (also called
metallostars) are based on polyfunctional ligands containing
two different complexing moieties, each with structural features
proper to the coordination mode of transition metal ion or
lanthanide ion. For FeII or RuII chelation, 2,2′-bipyridine,
phenanthroline, and 2,2′:6′,2″-terpyridine derivatives were
employed, whereas bidentate ligands 8-hydroxyquinoline and
catechol derivatives were utilized for AlIII and TiIV chelation,
respectively. The complexing moiety for GdIII binding was
based on standard poly(aminocarboxylate) ligands such as
DTPA (dietylenetriaminepentaacetate), DTTA (diethylene-
triamine-N,N,N″,N″-tetraacetate), or DO3A (2,2′,2″-(1,4,7,10-
tetraazacyclododecane-1,4,7-tryl)triacetate). At 20 MHz and 37
°C, the highest per Gd relaxivity enhancement (r1 ≈ 25 mM−1
s−1) for the metallostars described in the literature was reported
by Moriggi et al. for the RuII tris(adduct) of the [Gd2{bpy-
(dtta)2}(H2O)4]2− complex.19 It is worth noting that
pharmacokinetics and in vivo MRI feasibility studies of
metallostars were also reported.28,29 Additionally, it is
important to note that most of the d- or p-metal ion centered
heterometallic compounds offer photophysical properties of
interest for their applications in the optical imaging
field.16,19,21−25 Yet, in recent years, growing attention has also
been focused on the design of dual-modality probes possessing
two signaling components for two imaging systems into a single
structure.30,31 In this direction, dual MRI−optical imaging
contrast agents combine the high spatial resolution (micro-
meter), good temporal resolution (millisecond), and deep
tissue penetration of MRI with the sensitivity (nano- to
picomolar) of optical imaging probes.32,33
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Here, we report the synthesis and the paramagnetic and
photophysical properties of this new tetrametallic Gd3Ru
supramolecular complex (Figure 2). The paramagnetic proper-
Scheme 1. Synthesis of Fully Protected bpy-PMNTA
Compound 8
a
Figure 2. Ln3Ru metallostars studied in this work.
a
ties were investigated by oxygen-17 relaxometry and by proton
relaxometry at various temperatures, magnetic fields, and media
(pure water, phosphate buffer, and HSA-containing solutions).
Luminescence properties (λem, Φ) of the RuII center and
resistance against photobleaching of the Gd3Ru assembly were
investigated in aqueous solution at neutral pH. Stability tests on
this metallostar were also realized in biological media (PBS and
human serum). Finally, preliminary results related to the ability
of NIR-emissive lanthanide ions (NdIII and YbIII) to act as
energy acceptors from the Ru(II) core in the corresponding
Nd3Ru and Yb3Ru assemblies are presented.
Conditions: (i) Pd(PPh3)4, toluene, 48 h, reflux; (ii) Me3SnSnMe3,
Pd(PPh3)4, dioxane, 20 h, reflux; (iii) see ref 48; (iv) CuBr, Pd(PPh3)4,
toluene, 130 °C (microwave 260W), 90 min.
The ruthenium(II) complex 9 (Scheme 2) was obtained
from the reaction of Ru(DMSO)4Cl2 with the bpy ligand 8 in
refluxing ethanol followed by chromatography on silica gel and
anion exchange with KPF6. The red complex, isolated in 90%
yield, was characterized by elemental analysis, MS, and 1H, 13C,
and 19F NMR spectroscopies. Its absorption spectrum in
CH3CN is typical for polypyridyl RuII complexes, with a band
at 450 nm due to a spin-allowed d → π* metal-to-ligand
charge-transfer (1MLCT) transition. In ESI+-MS experiments,
we observed several doubly or triply charged ions, e.g., [M −
2PF6]2+ at m/z 1172.1 or [M − 2PF6+H]3+ at m/z 781.7. It is
worth noting that the fragmentation of this later triply charged
ion is governed by the usual rupture of the tert-butyl ester bond,
which leads to the observation of 10 successive losses of 18.7
Da (C4H8 fragment), confirming the presence of three ligands
RESULTS AND DISCUSSION
■
Ligand and Complexes Syntheses. Metallostar com-
pounds studied in this work were obtained as depicted in
Schemes 1 and 2. We used an uncommon approach, that is, by
first preparing the d-block metal component and subsequently
inserting LnIII ions; the reverse order is usually reported for the
construction of such d−f or p−f metallostar assemblies.16−26
The fully protected ligand 8 (Scheme 1) was synthesized by
Pd(0)-catalyzed Stille cross-coupling protocols. In the first step,
we took advantage of the differential reactivity of the two
bromine substituents in 2,5-dibromopyridine toward oxidative
addition in a Stille palladium-promoted coupling reaction.
Indeed, 2-tributylstannylpyridine underwent Stille-type cou-
pling with 2,5-dibromopyridine to give 5-bromo-2,2′-bipyridine
in good yield (77%) by modification of the literature
procedure.46 In the subsequent step, our first attempts to
obtain a trialkylstannylpyridine by lithiation and transmetala-
tion by Me3SnCl or nBu3SnCl remained unsuccessful, and we
turned to direct stannylation of 6 with hexaalkylditin.47 With
hexamethylditin and Pd(PPh3)4 as catalyst, the 5-stannylated
product 6 was obtained in 89% isolated yield. The tetraester
ditopic ligand 8 results from the reaction of trimethylstannyl
1
around the Ru ion. The H NMR spectrum of 9 proved to be
more complex than that of the free ligand due to the presence
of fac- and mer-isomers. It is well-known that tris-unsymmetric
diimine complexes of ruthenium(II) give rise to a mixture of
fac-isomer (C3 symmetry with the three ligands being
chemically equivalent) and mer-isomer (C1 symmetry with all
three ligands in different chemical environments).49−51 In the
1H NMR spectrum of 9, all of the aromatic protons are
reported as multiplets, as the large number overlapping signals
prevented detailed analysis. The methylene protons adjacent to
the pyridine ring (3.83−3.89 ppm) and tert-butyl protons
(1.33−1.38 ppm) each gave four sets of singlets. This seems to
indicate the existence of a nearly statistical mixture of three mer
to one fac and that this system does not rearrange to a preferred
conformation.
derivati ve
6 and 4-bromo-2, 6-bis[N , N- bi s(t-
butoxycarbonylmethyl)aminomethyl]pyridine 7. This last com-
pound was conveniently prepared in four steps from
commercially available chelidamic acid as described by Takalo
et al.48 Finally, the Stille coupling reaction involving
compounds 6 and 7 afforded the desired compound 8 in a
good 71% yield after chromatographic purification.
Full deprotection of tert-butyl esters in 9 to reach the
targeted complex 10 was achieved with a TFA−CH2Cl2
mixture at room temperature for 24 h. It is an easy and high-
yield procedure and takes advantage of the very high stability of
the Ru(bpy)3 core in acidic media.52 The lanthanide
2+
complexes were easily obtained by treating an aqueous solution
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a
Scheme 2. Synthetic Route for Ln3Ru Complexes 1−4
a
Conditions: (i) (a) Ru(DMSO)4Cl2, EtOH, 48 h, reflux, (b) KPF6, acetone; (ii) TFA, CH2Cl2, 24 h, rt; (iii) LnCl3·6H2O, H2O.
of Ru complex 10 with an excess of the lanthanide salt (LnCl3·
6H2O) to avoid any presence of mono- and di-lanthanide
species. All complexes were purified by a Waters Sep-Pak
column (C18), and the absence of free lanthanide ions was
checked by the Arsenazo test.53 The identity of the complexes
was determined by mass spectrometric analyses and UV−visible
absorption, and their purity was established by HPLC analyses.
In MS spectra (ESI−, ESI+), all complexes exhibit peaks due to
doubly charged species, with a good match between predicted
and observed isotope patterns, thereby confirming the
formation of the heterometallic compounds (Figure 3).
Interestingly, the LnIII (Ln = Gd, Eu, Nd, Yb) complexes
showed similar retention times, suggesting that the LnIII ions
share the same coordination sphere in these metallostar
architectures.
Relaxometric Studies. The ability of a GdIII complex to act
as a MRI contrast agent is generally described in terms of its
relaxivity, that is, the longitudinal relaxation rate (r1 = 1/T1)
increase of the water protons induced by 1 mM solution of the
paramagnetic complex at a given temperature and magnetic
field strength. At 20 MHz (a relevant field strength to compare
MRI CAs), the proton relaxivity per metallostar molecule
Gd3Ru 1 in H2O was measured to be 61.8 mM−1 s−1 at 298 K
(51.0 mM−1 s−1 at 310 K). The corresponding r1 value per GdIII
ion (20.6 and 17.0 mM−1 s−1 at 298 and 310 K, respectively) is
significantly higher than that of parent complex Gd-PMNTA
(5.7 mM−1 s−1 at 310 K) and those typical for bis-aqua GdIII
complexes of small size (relaxivities ranging from 6.0 to 10.2
mM−1 s−1 at 298 K).54,55 The enhancement of relaxivity may be
mainly attributed to the slower rotational correlation time (τR)
due to the increase in molecular weight. Under these
experimental conditions (20 MHz, 310 K), Gd3Ru complex 1
Figure 3. Observed (upper) and calculated (lower) isotope
distributions for the doubly charged ion obtained by fragmentation
of Gd3Ru complex 1 (HRMS ESI−).
showed also an increased proton relaxivity compared with that
of Gd3M metallostars (M = Fe, Ru, Al, Ti) of similar size
reported in the literature (relaxivities r1310K ranging from 9.5 to
12.3 mM−1 s−1).15,21,22,24,25,29 The higher number of GdIII-
coordinated water molecules (q = 2 for our compound and q =
1 for these Gd3M metallostars), resulting in higher relaxation,
may account for this difference. Compared to the r1 value of
D
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Gd6Ru metallostar reported by Morrigi et al., that is
{Ru[Gd2bpy-DTTA2(H2O)4]3}4−, the r1 value per GdIII ion
of our compound is 1.5 times smaller, as expected from the
ratio of their molecular weight (1.7).19 Metallostar 1 exhibits
high relaxivity, but it also has a relaxivity that is concentrated in
a small molecular volume. In terms of efficiency per mass unit, a
concept introduced by Livramento et al., metallostar 1 exhibits
a density of relaxivity ρ(r1) of 27.6 and 22.8 (g/L)−1 s−1 at 298
and 310 K, respectively.56 These values compare favorably with
those of GdIII-based macromolecular MRI agents, such as Gd-
chelate-loaded dendrimers or polysaccharides. As examples, a
dendrimer loaded with 1157 GdIII ions per molecule [G9-
(GdEPTA)1157] is characterized by a ρ(r1) value of 20.7 (g/
L)−1 s−1 at 20 MHz and 310 K, and a cyclodextrin loaded with
∼7 GdIII ions per molecule [β-CD-(Gd-DOTA derivative)6.9],
by a ρ(r1) of 24.0 (g/L)−1 s−1 at 20 MHz and 298 K.34,57
In order to determine the parameters that characterize water
exchange (τM) and rotational dynamic (τR) for Gd3Ru complex
1, we performed a variable-temperature 17O NMR study and
proton relaxation rate measurements at different magnetic
fields. The transverse 17O relaxation rate was measured between
275 and 347 K at 500 MHz on an 8 mM solution of the
complex. The 1H nuclear magnetic relaxation dispersion profile
(1H NMRD) was determined at 310 K in the proton Larmor
frequency range 10 kHz and 300 MHz, corresponding to
magnetic field strengths varying between 2.35 × 10−4 and 7 T.
These data are shown in Figures 4 and 5.
1
Figure 5. H NMRD profile of Gd3Ru complex 1 in water at 310 K
(open circles) and Gd-PMNTA (dashed line) in water at 310 K. The
solid and the dotted lines correspond to the Lipari−Szabo and classical
theoretical fittings of the data points, respectively.
(ΔH⧧) and entropy (ΔS⧧) for the water-exchange process, the
hyperfine coupling constant between the oxygen-17 and the
electronic spin of GdIII (A/ℏ), the B-parameter, which is related
to the mean-square of the zero-field-splitting energy (B =
2.4Δ2), and τv, the correlation time modulating the electronic
relaxation of GdIII (Table 1). The inclusion of Gd-PMNTA
Table 1. Parameters Obtained from the Theoretical Fittings
of the 17O Data of the Gd3Ru Complex 1 in Water at 11.75 T
parameter
value
77.5 6
310K
τM
ΔH⧧ [kJ mol−1
ΔS⧧ [J mol−1 K−1
A/ℏ [106 rad s−1
[ns]
]
42.2 0.1
27.0 0.3
−3.63 0.03
8.31 0.3
18.5 0.6
18.8 2.6
]
]
B [1020 s−2
τv298K [ps]
]
Ev [kJ mol−1
]
units in a supramolecular structure has only a minor influence
on the lability of the first-sphere water molecule. The calculated
value of the water residence time at 310 K on the Gd3Ru
310K
complex 1 (τM
= 77.5 ns) is about two times larger than
R
Figure 4. 17O reduced transverse relaxation rate (1/T2 ) as a function
310K
that for Gd-PMNTA (τM
= 35.2 ns). This τM value
of the reciprocal of the temperature for an aqueous solution of Gd3Ru
complex 1 at 11.75 MHz. The solid line corresponds to the theoretical
fitting of the data points. The dotted and dashed lines correspond to
the fitting of Gd-DTPA and Gd-PMNTA data, respectively.
compares favorably with those reported for d-Gd or p-Gd
metallostars and does not limit the use of complex 1 for the
design of magnetic tracers for molecular imaging. The large
activation enthalpy, ΔH⧧, and the positive activation entropy,
ΔS⧧, suggest a dissociative mechanism for the water
exchange.60 On the other hand, the assumption of bis-aqua
Gd components in 1 was confirmed by the value of the A/ℏ
parameter, characteristic of q = 2 complexes of the Gd-PMNTA
family.61,62
The maximum of the curve of the evolution of the reduced
transverse relaxation rate of 17O with temperature corresponds
to a temperature close to 295 K. This value is between those of
Gd-DTPA (310 K) and Gd-PMNTA (290 K), qualitatively
1
indicating an intermediate value of the residence time of
As can be seen from Figure 5, the H NMRD profile of Gd-
310K
coordinated H2O molecules (Gd-DTPA, τM
PMNTA, τM
= 143 ns; Gd-
PMNTA displays the typical s-shape of low-molecular-weight
and rapidly tumbling gadolinium(III) chelates: a plateau at low
magnetic fields followed by a drop around 10 MHz leading to a
lower level plateau in the high-field region (>60 MHz).
Whatever the applied magnetic field, the relaxivity of Gd3Ru
310K
= 35.2 ns). The theoretical treatment of the
experimental data (Figure 4) was performed as previously
described.58,59 In the fitting, the number of inner-sphere water
molecules was assumed to be q = 2, analogous to that of the
parent Gd-PMNTA complex and in accordance with the data
derived from the luminescence lifetime measurements on the
corresponding Eu3Ru metallostar assembly 2 (vide infra). From
this fitting, the following parameters have been adjusted
simultaneously: the residence time of the water molecules
coordinated to the GdIII ions (τM), the activation enthalpy
complex 1 was higher and displayed a hump between 10 and
310K
100 MHz with a maximum at 60 MHz (r1
= 18.5 mM−1
s−1), characteristic for slowly rotating molecules and assigned to
the presence of a supramolecular structure. It is interesting to
note that the relaxivity of Gd3Ru complex 1 remains remarkably
high at 300 MHz (11.9 mM−1 s−1 per GdIII ion at 310 K). If
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most MRI machines work at 0.2−3 T for clinical exams, then
higher fields (up to 9.4 T, 400 MHz) become commonly used
for experimental animal studies.63 A first theoretical adjustment
of the NMRD profile was performed with the usual inner-
sphere (IS) and outer-sphere (OS) contributions to the
paramagnetic relaxation rate. The inner-sphere contribution is
linked to water molecules bound to the GdIII ion, whereas the
outer-sphere contribution is referring to water molecules
diffusing near the complex.64 In the fitting procedure,
parameters were fixed to common values in order to decrease
the number of variables. The GdIII−H distance (rGdH) between
the GdIII ion and the proton nuclei of the bound water was
fixed to 3.10 Å, the distance (d) of closest approach of an outer-
sphere water proton to the GdIII ion was set to 3.60 Å, and the
relative diffusion coefficient (D), to 2.93 × 10−9 m2 s−1. The
water residence time was fixed to the value determined by 17O
relaxometry. The parameters describing the rotational
correlation time τR and the electronic relaxation times, τV and
τSO, were optimized simultaneously and are reported in Table
2. The electronic relaxation time at zero field (τSO) of
Figure 6. Kinetic stability of Gd3Ru complex 1 in phosphate buffer (10
mM, pH 7.4) at 310 K. Evolution of the relative water proton
p
p
paramagnetic relaxation rate R1 (t)/R1 (0) at 20 MHz versus time.
= 0.128 min−1), indicating a weak interaction of the complex
with phosphate. This suggests that, under these experimental
conditions, metallostar 1 is resistant toward dissociation of the
(bpy)3Ru core and that the GdIII inner-sphere water molecules
are replaced by phosphate ions. In contrast, larger effects of
phosphate on the relaxivity (∼70% decreasing) of the Gd6Fe
metallostar, that is, {Fe[Gd2bpy-DTTA2(H2O)4]3}4−, have
been observed.18 Finally, no significant binding of Gd3Ru
complex 1 with human serum albumin (HSA) was detected. In
the presence of 4% HSA and at 310 K, the proton relaxation
rates are slightly greater over the whole magnetic field range in
Table 2. Relaxivities Values at 20 and 60 MHz (T = 310 K)
and Parameters Obtained from the Theoretical Fittings of
the 17O Data and of the Proton NMRD Profile (T = 310 K)
of the Gd3Ru Complex 1 and Comparison with the Gd-
PMNTA Complex
1
the H NMRD profile, and a r1 relaxivity value of 18.7 mM−1
a
s−1 was observed at 20 MHz. Such a weak increase (∼10%) of
the r1 value does not indicate a significant interaction of the
complex with HSA. As reported in the literature, a significant
interaction between a Gd(III) complex and HSA is
characterized by a greater than 60% increase in the relaxation
rate.65
Gd3Ru complex 1
Gd-PMNTA
IS + OS fit
35.2 3.0
IS + OS fit
Lipari−Szabo fit
b
τM [ns]
τR [ps]
77.5
70 9.8
241 3.0
56
1
c
τRG [ps]
788 164
237 14
d
τRL [ps]
Photophysical Properties of Ru Complex 10 and
Gd3Ru Complex 1. The electronic absorption and emission
properties of ruthenium complex 10 and Gd3Ru metallostar 1
were recorded in nondegassed 50 mM Tris-buffered aqueous
solutions (pH 7.4). The spectroscopic data of these complexes
are summarized in Table 3, together with analogous data for the
[Ru(bpy)3]2+ complex for comparison. Absorption spectra of
solutions of 10 display an intense ligand-centered (LC) π → π*
τSO [ps]
204 3.2
50.4 3.4
225 5.6
45.3 2.9
0.12 0.05
205 15
τv [ps]
S2
r1 [mM−1 s−1
12.9 3.8
e
]
17.0 (18.5)
5.7 (5.0)
a
b
310K
From ref 36. Fixed to the value obtained for τM
relaxometry. Global rotational correlation time. Local rotational
correlation time. At 20 (60) MHz.
by 17O
c
d
e
metallostar 1 is similar to that of the Gd-PMNTA complex,
and, as expected, the τR value of 1 (241 ps) is larger by a factor
of 4.3. This longer rotational correlation time is associated with
the larger dimension (molecular weight) of 1 and is mainly
responsible for the 300% increase in r1 of metallostar 1 as
compared to that of the small contrast agent, Gd-PMNTA. In
order to increase the agreement between the fitted and
experimental data, a theoretical adjustment using the Lipari−
Szabo model was applied. This approach has been previously
used for the evaluation of NMR relaxation data for similar Gd
systems.13,18,24 The agreement between experimental and fitted
data is slightly improved, but the results show a low coupling
between global and local motions (S2 = 0.12), whereas the
other parameters are similar to those obtained with the classical
inner-sphere and outer-sphere model.
Table 3. Photophysical Properties of Ru Complex 10 and
Ln3Ru Complexes 1−4 in Tris Buffer (50 mM, pH 7.4)
a
b
298K
λabs (nm)
[ε (M−1 cm−1)]
λem
(nm)
ΦRu
a
77K
compd
[λem (nm)]
(%)
c
[Ru(bpy)3]2+
285 [87 000]
452 [14 500]
299 [73 100]
468 [7100]
300 [74 500]
472 [6900]
300 [74 900]
472 [6900]
300 [88 900]
472 [8300]
300 [89 500]
472 [9500]
625 [584, 630]
656 [615, 668]
660 [621, 671]
4.0
10
1
2.7
2.6
2.6
0.3
0.9
d
2
616 , 660
3
660, 894, 1059
660, 978
We also evaluated the kinetic inertness of metallostar 1 by
monitoring proton relaxivity at 20 MHz over a 58 h period in
phosphate-buffered saline (PBS). The time dependence of the
4
p
p
p
a
b
Excitation at 472 nm (1MLCT absorption band). Quantum yield of
ratio R1 (t)/R1 (0), where R1 (t) is the relaxation rate at time t
p
c
d
RuII-centered luminescence. From ref 69. Excitation at 300 nm (LC
absorption band). The emission was recorded in time-resolved mode
(delay time 50 μs).
and R1 (0) is the relaxation rate at time zero for complex 1 in
10 mM PBS (pH 7.4 and 310 K), is shown in Figure 6. We
observed a slight decrease in R1p of ∼10% in the first 30 min (kd
F
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Inorganic Chemistry
Article
band at 299 nm (log ε = 4.86) as well as a broad and less
intense band in the visible region around 468 nm (log ε = 3.85)
attributed to the classical spin-allowed metal-to-ligand charge-
transfer (1MLCT) d → π* transitions. Shoulders around 265
No changes of absorption and emission spectra of Gd3Ru
complex 1 were observed in aerated solutions after several days
at room temperature in Tris buffer (pH 7.4). Moreover, no
3
decrease in the emission intensity of the MLCT state and no
1
and 360 nm might be attributed to either LC or MLCT
changes in the shapes of emission and excitation spectra over a
58 h period were observed when metallostar 1 (25 μM) was
incubated in PBS (10 mM) or a mixture of human serum/Tris
buffer (2:1) at pH 7.4 and 37 °C (Figure 8a). Under these
transitions and to metal-centered (MC) d → d transitions,
respectively.66 When compared to [Ru(bpy)3]2+, the main
1
difference is that the LC and MLCT bands are significantly
red-shifted (ca. 15 nm), owing to the extension of conjugation
in the bpy-PMNTA moieties. Excitation into the 1MLCT
absorption band at 468 nm leads to a broad emission extending
from 560 to 830 nm with a maximum at 656 nm at room
temperature. Upon cooling the sample at 77 K, the emission
maximum of the complex was blue-shifted (λmax = 615 nm)
compared to the emission at room temperature. With reference
to related photophysical studies of [Ru(bpy)3]2+ and RuII
polypyridine systems, the emission has been assigned to a
3MLCT (dπ(Ru) → π*(bpy)) excited state.67,68 When
monitoring the emission at 656 nm, the excitation spectrum
follows the absorption spectrum and presents two bands with
maxima at ≈300 and 468 nm, showing that Ru(II) emission
occurs from LC and 1MLCT transitions. The quantum yield of
the RuII-centered luminescence was determined by excitation
69
1
into the MLCT state, using [Ru(bpy)3]Cl2 (Φ = 4.0%) on
nondegassed water as a reference, and was found to be 2.7%.
The coordination of the PMN moieties to GdIII in metallostar 1
does not significantly influence the absorption and emission
properties of the RuII center (Figure 7). Upon GdIII
Figure 8. (a) Evolution of the relative total fluorescence intensity I(t)/
I(0) (λexc = 472 nm, λem = 660 nm) versus time in phosphate buffer
(10 mM, pH 7.4) at 310 K of Gd3Ru complex 1. (b) Relative
photobleaching profiles of Gd3Ru complex 1, fluorescein, or Alexa
fluor 665 in Tris buffer (50 mM, pH 7.4) during continuous
illumination with a xenon lamp excitation source. Fluorescence values
were normalized to the initial intensity.
experimental conditions, these results confirm the robustness of
the (bpy)3Ru core vs dissociation processes. We have also
investigated the degree of photostability (photobleaching) of
metallostar 1 and compared it to conventional fluorescent dyes,
such as fluorescein and Alexa Fluor 665. The photostability was
determined via measurements of the relative change in
fluorescence intensity upon exposure to a white light from a
xenon lamp in Tris buffer solutions at pH 7.4. Figure 8b clearly
demonstrates that organic dyes showed a complete photofading
after 1 h of constant illumination, whereas the luminescent
emission of the metallostar retained 80% of its initial
luminescence after 4 h of illumination. This demonstrates
again that RuII complexes are less sensitive to photobleaching
than are singlet-state fluorescent dyes. This good photostability
is advantageous for its use in applications, allowing for a longer
exposure time to excitation light and repeatability of experi-
ments.
Figure 7. Normalized absorption, excitation, and luminescence spectra
of 15 μM solutions of Gd3Ru complex 1 in Tris buffer (50 mM, pH
7.4). A 298 K steady-state excitation spectrum was monitored at 660
nm. The 298 and 77 K steady-state emission spectra were monitored
upon excitation at 472 nm.
1
3
complexation, the MLCT absorption and MLCT emission
are slightly red-shifted (4 nm), and the emission quantum yield
was found to be 2.6%. In Gd3Ru complex 1, the triplet energy
level of the RuII center was calculated by reference to the lower
wavelength emission edge (580 nm, 17 250 cm−1) from the
low-temperature phosphorescence spectrum. The lack of
quenching of the RuII 3MLCT state in complex 1 is expected
since the GdIII excited levels have energies (>31 000 cm−1)
much higher than those of MLCT states of RuII chromophore,
preventing any Ru → Gd energy transfer process. The
luminescence lifetime of these complexes could not be
measured due to instrumental limitations.
Photophysical Properties of Ln3Ru Complexes (Ln =
Eu, Nd, Yb). The absorption spectra of LnIII complexes 2−4
are identical in terms of band shapes and positions to that of
Gd3Ru complex 1, taken as a reference (Table 3). However, the
G
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Inorganic Chemistry
Article
relative intensity of the RuII 3MLCT emission spectra of these
three complexes differs greatly, as demonstrated in Figure 9a.
Figure 9. (a) Fluorescence emission spectra (λexc = 472 nm) of Gd3Ru
complex 1 (solid line), Nd3Ru complex 3 (dotted line), and Yb3Ru
complex 4 (dashed line) showing the characteristic MLCT emission
from the RuII center in Tris buffer solutions (50 mM, pH 7.4). (b)
Evolution of the fluorescence spectrum of Ru complex 10 upon
addition of NdCl3 solutions in Tris buffer solutions (50 mM, pH 7.4).
Figure 10. Visible emission at 298 K of Eu3Ru complex 2 (a) under
300 nm (LC) excitation and recorded with a 50 μs delay and NIR
emission spectra at 298 K of Nd3Ru complex 3 (b) and Yb3Ru
complex 4 (c) under 472 nm (MLCT) excitation. Solutions are in Tris
buffer (50 mM, pH 7.4).
As far as Eu3Ru complex 2 is concerned, no quenching of the
RuII 3MLCT emission was observed under excitation into the
1MLCT transition (λexc = 472 nm). At this excitation
wavelength, no EuIII emission was observed, neither in
steady-state nor time-gated emission spectroscopies. On the
contrary, upon excitation in the LC transition band (λexc = 300
nm), the typical emission pattern characteristic of the EuIII ions
(5D0 →7FJ, J = 0−4 transitions) can be clearly observed in the
time-resolved luminescence mode with a 50 μs delay (Figure
10a). The hypersensitive 5D0 →7F2 and the sensitive 5D0 →7F4
transitions are dominant and account for ∼80% of the total
emitted intensity. On the other hand, the excitation spectrum,
indicating that the local coordination environment around EuIII
remains similar in these two complexes. The hydration state q
of the europium component in Eu3Ru complex 2 was evaluated
by comparison of the luminescence lifetimes of the europium-
centered emission in H2O (τH) and D2O (τD) solutions and by
using the empirical equation proposed by Supkowski and
Horrocks.70 At 298 K, the lifetimes τH (0.270 ms) and τD
(0.700 ms) point to the presence of two metal-bound water
molecules per EuIII ion (q = 2.18). These emission lifetimes are
somewhat surprising since bis-hydrated Eu complexes usually
5
obtained by recording the intensity of the D0 →7F2 transition
5
at 616 nm as a function of excitation wavelength, resembles the
have longer lifetime values. The D0 lifetime in deuterated
1
water increases up to 2 ms at 77 K, indicating that the EuIII
luminescence is quenched at room temperature by a temper-
ature-dependent mechanism.
absorption spectrum except for the MLCT band at 472 nm.
These results indicate that sensitization of the EuIII
luminescence at room temperature is possible only through
the organic ligand and not by the MLCT states of RuII
chromophore. This is in accordance with the highest energy
3MLCT level of the RuII center, as determined at 77 K in the
corresponding Gd3Ru complex (17 250 cm−1), which lies close
to the Eu(5D0) emitting level located at 17 300 cm−1. With
respect to the EuPMNTA complex, the crystal-field splitting of
5D0 →7FJ transitions of complex 2 is the same, and no changes
in emission probabilities to different J levels are observed,
The emission spectra (λexc = 472 nm) of Nd3Ru complex 3
and Yb3Ru complex 4 showed that the emission intensity of the
RuII center is quenched compared to that of the Gd3Ru
complex (Figure 9a), with a quenching more pronounced in the
case of the Nd3Ru complex. The emission intensity arising from
the RuII 3MLCT state is reduced by 90% (complex 3) and 65%
(complex 4). This is indicative of effective energy migration
between the RuII chromophore to the NdIII and YbIII moieties
H
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Inorganic Chemistry
Article
with a solvent composition of 50% A/50% B and following a linear
gradient up to 0% A/100% B from 0 to 18 min. For system B, the
HPLC gradient system used begins with a solvent composition of 90%
A/10% B and follows a linear gradient up to 10% A/90% B from 0 to
18 min. System C: solvents were 10 mM, pH 4, ammonium formate
buffer (solvent A) and acetonitrile (solvent B); analyses were
performed using the HPLC gradient system beginning with a solvent
composition of 100% A and following a linear gradient up to 50% A/
50% B from 0 to 18 min.
in these heterometallic structures. These results are corrobo-
rated by the luminescence intensity titration of RuII complex 10
with NdCl3 and YbCl3. In these experiments, the intensity of
3
the MLCT state emission decreases until a LnIII/complex 10
concentration ratio of 1.8, and then a plateau is observed at
higher concentration ratios (Figure 9b in the case of the
titration with NdCl3). The excitation of these complexes at 472
nm produces NIR emission that is characteristic of NdIII and
YbIII ions (Figure 10b,c). Specifically, the NdIII complex
displayed emission within the 850−1400 nm range dominated
by a broad band at 1059 nm (4F3/2 → 4I11/2) with weak features
at ca. 894 and 1330 nm, assigned to the 4F3/2 → 4I9/2 and 4F3/2
Photophysical Measurements. Fluorescence spectra in the
visible region of the spectrum were obtained using LS-50B
PerkinElmer and Cary Eclipse spectrofluorimeters equipped with a
Xenon flash lamp source and a Hamamatsu R928 photomultiplier
tube. The measurements were carried out at pH 7.4 in Tris buffer (50
mM), and all samples were prepared with an absorbance between 0.01
and 0.05 at the excitation wavelength in order to prevent the inner-
filter effect. Phosphorescence spectra at 77 K were recorded with the
LS-50B PerkinElmer spectrofluorimeter equipped with the low-
temperature accessory no. L2250136. Spectra were corrected for
both the excitation light source variation and the emission spectral
response. The fluorescence quantum yields (uncertainty 15%) were
determined by using [Ru(bpy)3]2+ in aerated water as standard (Φ =
0.04)69 and corrected for the refractive index of the solvent. The
luminescence decay curves of the EuIII complex were fitted by an
equation of the form I(t) = I(0) exp(−t/τ) by using a curve-fitting
4
→ I11/2 transitions, respectively. The steady-state emission
spectrum of Yb3Ru complex gave an emission within the 900−
1100 nm range, assigned to the 2F5/2 → 2F7/2 transition, with a
sharp band at 978 nm. For RuYb3 and RuNd3 structures based
on a Ru(Phen)3 core and Ln-cyclen-based ligands, Nonat et al.
reported NIR emissions with quantum yields in the range
0.040−0.075%.43 In our case, one can anticipate the quantum
yields of complexes 3 and 4 to be rather lower, as their YbIII and
NdIII components present two water molecules in their
coordination spheres.
program. The number of metal-coordinated water molecules at 298 K
CONCLUSIONS
−1
■
was calculated using the following equation: qH O(Eu) = 1.11[(τH O
)
2
2
− 0.31].70
−1
In this article, the design of new tetranuclear heterobimetallic
complexes Ln3Ru (Ln = Gd, Eu, Nd, Yb) is presented. These
metallostar architectures were based on an original ditopic
ligand with a bipyridine moiety and a PMNTA chelating unit
that are closely connected. The Gd3Ru assembly exhibits, at
310 K, a high r1 relaxivity per GdIII ion at clinical and high
magnetic fields (r1 = 17.0 and 11.9 mM−1 s−1 at 20 and 300
MHz, respectively). This complex is characterized by a fast
− (τD O
)
2
Fluorescence measurements in the NIR region were conducted on a
Horiba-Jobin Yvon Fluorolog-3 spectrofluorimeter, equipped with a
three slit double grating excitation and emission monochromator with
dispersions of 2.1 nm/mm (1200 grooves/mm). The steady-state
fluorescence was excited by unpolarized light from a 450 W xenon CW
lamp and detected by a red-sensitive Hamamatsu R928 photo-
multiplier tube. Spectra were reference corrected for both the
excitation source light intensity variation (lamp and grating) and the
emission spectral response (detector and grating). Uncorrected near-
infrared spectra were recorded using a liquid nitrogen cooled, solid
indium/gallium/arsenic detector (850−1600 nm). An RG 830 filter
was inserted in the emission pathway to suppress all visible light. The
experimental data were treated using Origin software (normalization
and baseline correction).
water-exchange rate (τM310K = 77.5 ns) and a slow tumbling rate
310K
(τR
∼ 240 ps). It also shows interesting photophysical
properties, with excitation and emission in the visible domain
(472/660 nm, respectively), and a fluorescence quantum yield
of 2.6% in aqueous solutions at neutral pH. These luminescent
and relaxometric properties are retained under biologically
relevant conditions. This opens the way for the development of
Gd3Ru metallostar 1 or derivatives as bimodal contrast agents
for MRI and optical imaging. Furthermore, preliminary
photophysical data determined for Nd3Ru and Yb3Ru
assemblies demonstrate the feasibility of a polypyridine-Ru
center to act as a sensitizer for the NIR emission of NdIII and
YbIII bis-hydrated complexes.
NMR Measurements. 17O NMR measurements of solutions were
performed at 11.75 T on 0.35 mL samples contained in 5 mm o.d.
tubes on a Bruker Avance 500 MHz spectrometer. The temperature
was regulated by air or nitrogen flow controlled by a Bruker BVT 3200
unit. Longitudinal proton relaxation rates were measured at 0.47 T and
310 K on a Minispec mq-20 (Bruker, Karlsruhe, Germany) instrument
with a standard inversion−recovery sequence. Proton nuclear
magnetic relaxation dispersion (NMRD) profile was recorded on a
Stelar relaxometer (Mede, Italy) working between 0.24 mT and 1 T.
The additional relaxation rates at 0.47, 1.41, and 7.05 T were measured
on mq-20 and md-60 minispec systems and on a AMX300
spectrometer (all from Bruker, Karlsruhe, Germany). Fitting of the
1H NMRD was performed with data-processing software using
different theoretical models describing the nuclear relaxation
phenomena observed (Minuit, CERN Library).71,72 The exact Gd3+
concentration was determined by proton relaxivity measurements at
0.47 T and 37 °C after complete hydrolysis in concentrated HNO3.
Synthesis of 5-Bromo-2,2′-bipyridine (5). To a solution of 2,5-
dibromopyridine (0.712 g, 3.01 mmol) in anhydrous toluene (8 mL)
was added 2-tributylstannylpyridine (0.88 mL, 2.72 mmol), and the
reaction mixture was degassed by bubbling argon through the solution
for 1 h. Pd(PPh3)4 (0.031 g, 0.027 mmol) was then added, and the
mixture was refluxed for 48 h. After cooling to room temperature, the
solids were removed by filtration, and the filtrate was concentrated in
vacuo. Aqueous HCl (1 M) was added to the crude product, and the
aqueous layer was washed with dichloromethane, neutralized with
solid NaHCO3, and extracted with dichloromethane. The resulting
EXPERIMENTAL SECTION
■
Instruments. 1H and 13C spectra were recorded on Bruker Avance
300 and 500 MHz spectrometers; chemical shifts are given in ppm
according to the solvent peak, and coupling constants (J) are given in
Hertz (Hz). Multiplicities were recorded as s (singlet), d (doublet), t
(triplet), q (quadruplet), and m (multiplet). Electrospray (ES) mass
spectra were obtained on a Q TRAP Applied Biosystems spectrometer,
and high-resolution mass spectra (HRMS), on a Xevo G2 QToF
Waters spectrometer. Elemental analyses were carried out by the
Service d’Analyse, Laboratoire de Chimie de Coordination (Tou-
louse). Absorption measurements were recorded with a Hewlett-
Packard 8453 temperature-controlled spectrometer in a 10 mm quartz
cuvette. Analytical HPLC was performed on a Waters Alliance 2695
system with a PDA 2996 detector using a reverse-phase column
(Phenomenex Luna C8, 5 μM, 100 Å, 150 × 4.6 mm) with a flow rate
of 1 mL/min. For systems A and B, solvents were H2O containing
0.1% TFA (solvent A) and acetonitrile (solvent B). For system A,
analyses were performed using the HPLC gradient system beginning
I
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organic phase was dried (MgSO4) and evaporated in vacuo.
Purification by chromatography (alumina, petroleum ether/EtOAc
98:2) provided 5 as a white solid (0.489 g, 77%). mp 73−74 °C (lit.46
(73) [M − 2PF6-C4H8 + H]3+, 744.4 (87) [M − 2PF6-2C4H8 + H]3+,
725.7 (70) [M − 2PF6 − 3C4H8 + H]3+, 707.0 (49) [M − 2PF6-4C4H8
+ H]3+, 688.3 (33) [M − 2PF6-5C4H8 + H]3+, 669.6 (19) [M − 2PF6-
6C4H8 + H]3+, 650.9 (14) [M − 2PF6-7C4H8 + H]3+, 632.2 (7) [M −
2PF6-8C4H8 + H]3+, 613.5 (4) [M − 2PF6-9C4H8 + H]3+, 595.0 (3)
1
73 °C). TLC (alumina, petroleum ether/EtOAc 98:2) Rf = 0.58. H
and 13C NMR data are in accordance with those in ref 46.
[M
− 2PF6-10C4H8 + .
H]3+ HRMS (ESI+) calcd for
Synthesis of 5-Trimethylstannyl-2,2′-bipyridine (6). A
solution of 5-bromo-2,2′-bipyridine 5 (200 mg, 0.851 mmol) in
anhydrous dioxane (16 mL) was degassed by argon bubbling for 30
min. Hexamethylditin (554 mg, 1.69 mmol) in degassed dioxane (4
mL) and Pd(PPh3)4 (98 mg, 0.085 mmol) were then added, and the
mixture was refluxed for 20 h. After cooling to room temperature, the
solvents were evaporated, and the crude product was purified by
chromatography (alumina, petroleum ether/EtOAc 100:0 to 98:2) to
provide 6 as a colorless oil (242 mg, 89%). TLC (alumina, petroleum
ether/EtOAc 98:2) Rf = 0.48. 1H NMR (300 MHz, CDCl3) δ 8.70 (s,
1H), 8.68 (dd, J = 0.9, 4.8 Hz, 1H), 8.40 (d, J = 7.8 Hz, 1H), 8.33 (dd,
J = 0.9, 7.8 Hz, 1H), 7.92 (dd, J = 1.5, 7.8 Hz, 1H), 7.81 (td, J = 1.8,
7.5 Hz, 1H), 7.30 (ddd, J = 1.5, 4.8, 7.5 Hz, 1H), 0.37 (s, J Sn−H =
54/56 Hz, 9H). 13C NMR (75 MHz, CDCl3) δ 156.5 (Cq), 155.7
(Cq), 155.0 (CH), 149.2 (CH), 144.5 (CH), 137.7 (Cq), 136.9 (CH),
123.7 (CH), 121.0 (CH), 120.9 (CH), −9.5 (CH3, J Sn−C = 341/357
Hz). MS (ESI+) m/z (%) = 317/319/321 (12) [M + H]+, 339/341/
343 (100) [M + Na]+. HRMS (ESI+) calcd for C13H16N2NaSn [M +
Na]+, m/z 343.0233; found, m/z 343.0219.
C123H171N15O24Ru [M − 2PF6]2+, m/z 1172.0850; found, m/z
1172.0887. Calcd for C123H172N15O24Ru [M − 2PF6 + H]3+, m/z
781.7260; found, m/z 781.7283. Anal. Calcd for
C123H171F12N15O24P2Ru: C, 56.07; H, 6.54; N, 7.97. Found: C,
55.79; H, 6.65; N, 7.83.
Synthesis of Ruthenium(II) Complex [10] (CF3COO)2. To a
stirring solution of 9 (53 mg, 20.1 μmol) in CH2Cl2 (6 mL) was added
TFA (1.4 mL, 18.1 mmol) at 0 °C. The mixture was then stirred 24 h
at room temperature. The solvent was coevaporated several times in
vacuo with CH3CN to give 10 as a red powder (38 mg, 100%). HPLC
(system B): tR = 3.9 min. 1H NMR (300 MHz, D2O) δ 8.72−8.58 (m,
6H), 8.37−8.30 (m, 3H), 8.12−7.79 (m, 9H), 7.51−7.35 (m, 9H),
4.85−4.59 (m, partially masked by H2O signal), 4.14−3.97 (m, 24H).
13C NMR (125 MHz, D2O) δ 169.7−169.3 (3 signals, Cq), 163.1 and
162.8 (Cq), 157.9−157.7 (4 signals, Cq), 156.3 and 156.2 (Cq),
152.0−151.7 (3 signals, CH), 151.0−150.7 (4 signals, Cq), 149.7−
149.1 (3 signals, CH), 146.2−146.0 (4 signals, Cq), 138.3−138.0 (3
signals, CH), 136.9 and 136.8 (CH), 136.0−135.9 (3 signals, Cq),
127.9 and 127.7 (CH), 125.0−124.9 (3 signals, CH), 123.5−123.1 (4
signals, CH), 119.8, 117.4, 115.1, and 112.8 (CF3), 58.6−58.4 (4
signals, CH2), 56.3 and 56.2 (CH2). 19F NMR (300 MHz, D2O) δ
−75.6 (CF3). HRMS (ESI+) calcd for C75H75N15O24Ru [M −
2CF3COO]2+, m/z 835.7087; found, m/z = 835.7102. UV−vis (50
mM Tris buffer, pH 7.4): λmax/nm (ε/M−1 cm−1) = 299 (73 100), 468
(7100).
Synthesis of Lanthanide(III)−Ruthenium(II) Complexes. To a
solution of complex 10 (78 mg, 41 μmol) in H2O (7 mL) was added
LnCl3·6H2O (139 μmol). After stirring at room temperature for 1 h,
pH was adjusted to 5−6 with NaOH 0.1 M, and the mixture was then
stirred 18 h at room temperature. The solvent was evaporated to a
minimum, and the solution was loaded on a Waters Sep-Pak column
(C18, 10 g). The column was rinsed with 5 × 8 mL of H2O to remove
salts, and the product was eluted with a H2O/MeOH mixture (1:1, 5 ×
1 mL). The absence of free lanthanide ions was checked by the
Arsenazo test. Yields were quantitative.
Synthesis of 4-(2,2′-Bipyridine-5-yl)-2,6-bis[N,N-bis(t-
butoxycarbonylmethyl)aminomethyl]pyridine (8). To a solution
of compounds 7 (176 mg, 0.262 mmol) and 6 (100 mg, 0.313 mmol)
in anhydrous toluene (5 mL) were added copper(I) bromide (6 mg,
0.042 mmol) and Pd(PPh3)4 (60 mg, 0.052 mmol). The solution was
degassed in vacuo and transferred under argon to a microwave reactor.
The mixture was heated at 130 °C under microwave irradiation (300
W) for 90 min. After cooling to room temperature, the solvents were
evaporated, and the crude product was purified by chromatography
(alumina, petroleum ether/EtOAc 90:10 to 80:20) to provide 8 as
colorless oil (140 mg, 71%). TLC (alumina, petroleum ether/EtOAc
1
85:15) Rf = 0.30. H NMR (300 MHz, CDCl3) δ 9.03 (d, J = 1.8 Hz,
1H), 8.70 (dd, J = 0.9, 4.8 Hz, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.47 (d, J
= 8.1 Hz, 1H), 8.15 (dd, J = 2.4, 8.4 Hz, 1H), 7.86 (s, 2H), 7.83 (dd, J
= 1.8, 7.8 Hz, 1H), 7.33 (ddd, J = 0.9, 4.8, 7.5, 1H), 4.11 (s, 4H), 3.52
(s, 8H), 1.45 (s, 36H). 13C NMR (75 MHz, CDCl3) δ 170.6 (Cq),
159.9 (Cq), 156.2 (Cq), 155.7 (Cq), 149.3 (CH), 147.8 (CH), 146.1
(Cq), 137.0 (CH), 135.5 (CH), 134.2 (Cq), 123.9 (CH), 121.4 (CH),
120.9 (CH), 118.7 (CH), 81.1 (Cq), 59.9 (CH2), 55.9 (CH2), 28.2
(CH3). MS (ESI+) m/z (%) = 748.4 (37) [M + H]+, 770.3 (100) [M
+ Na]+, 786.4 (6) [M + K]+. HRMS (ESI+) calcd for C41H58N5O8 [M
+ H]+, m/z 748.4285; found, m/z 748.4261.
Gd3Ru complex 1: HPLC analysis (system C): tR = 5.9 min. Anal.
Calcd for C75H63Gd3N15NaO24Ru·10H2O: C, 38.59; H, 3.58; N, 9.00.
Found: C, 38.10; H, 3.49; N, 8.74. HRMS (ESI−) calcd for
C75H63Gd3N15O24Ru [M − Na]2−, m/z 1065.5482; found, m/z
1065.5452. The ratio Gd/Ru determined by ICP-MS technique was
2.95:1.00.
Synthesis of Ruthenium(II) Complex [9] (PF6)2. To a stirring
solution of 8 (50 mg, 66.9 μmol) in EtOH (10 mL) was added
Ru(DMSO)4Cl2 (11 mg, 22.3 μmol), and the mixture was degassed
Eu3Ru complex 2: HPLC analysis (system C): tR = 6.0 min. HRMS
(ESI+) calcd for C75H66Eu3N15O24Ru [M − Na + 3H]2+, m/z
1059.5548; found, m/z 1059.5511. HRMS (ESI−) calcd for
C75H63Eu3N15O24Ru [M − Na]2−, m/z 1058.5470; found, m/z
73
by bubbling argon for 30 min. The solution was then allowed to reflux
for 48 h. After cooling to room temperature, the solvents were
removed in vacuo, and the crude product was purified by
chromatography (silica gel, acetonitrile/H2O/KNO3 sat 100:7:1).
The resulting product was dissolved in a minimum amount of acetone,
precipitated with saturated aqueous solution of KPF6, and filtered to
1058.5448. Luminescence: λem (50 mM Tris buffer, pH 7.4, λexc
=
299 nm, td = 50 μs)/nm 580 (relative intensity, corrected spectrum 2),
593 (28), 616 (100), 686, 699 (59).
Nd3Ru complex 3. HPLC analysis (system C): tR = 6.0 min. MS
(ESI−) m/z 2091.1 [M − Na]−.
provide 9 as a red powder (53 mg, 90%). HPLC (system A): tR
=
13.24 min. 1H NMR (500 MHz, CD3CN) δ 8.74−8.58 (m,6H), 8.38−
8.33 (m, 3H), 8.15−8.10 (m, 3H), 7.93−7.86 (m, 6H), 7.49−7.37 (m,
9H), 3.89, 3.88, 3.84, and 3.83 (4 s, 12H), 3.42−3.26 (m, 24H), 1.38,
1.36, 1.35, and 1.33 (4 s, 108 H). 13C NMR (125 MHz, CD3CN) δ
171.5 and 171.4 (Cq), 161.0−160.7 (4 signals, Cq), 158.1−157.3 (3
signals, Cq), 153.2−152.9 (4 signals, CH), 150.4−150.2 (4 signals,
CH), 144.2−144.0 (3 signals, Cq), 139.1 and 139.0 (CH), 136.9−
136.8 (3 signals, CH), 129.1−128.7 (3 signals, CH),126.0−125.5 (5
signals, CH), 120.1−119.7 (3 signals, CH), 81.8 (Cq), 60.7−60.5 (3
signals, CH2), 56.85−56.7 (3 signals, CH2), 28.2 (CH3). 19F NMR
(300 MHz, CD3CN) δ −72.87 (1JPF = 749 Hz). MS (ESI+) m/z (%) =
1391.0 (14) [M + PF6 + 3H]2+, 1318.1 (15) [M + 2H]2+, 1245.1 (19)
[M − PF6 + H]2+, 1172.1 (31) [M − 2PF6]2+, 879.0 (78) [M + 3H]3+,
830.4 (100) [M − PF6 + 2H]3+, 781.7 (83) [M − 2PF6 + H]3+, 763.0
Yb3Ru complex 4. HPLC analysis (system C): tR = 5.8 min. MS
(ESI−) m/z 2178.2 [M − Na]−.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compound 8 (Figure S1) and
Ru(II) complex 9 (Figure S2); 13C NMR spectrum of Ru(II)
complex 10 (Figure S3); HPLC chromatogram of Gd3Ru
complex 1 (Figure S4); HRMS of Eu3Ru complex 2 (Figure
S5); R1p relaxivity as a function of the concentration in complex
1 (Figure S6); excitation and emission spectra of Ru(II)
complex 10 (Figure S7); excitation spectrum of Eu3Ru complex
J
DOI: 10.1021/ic502342x
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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Chem.Eur. J. 2006, 12, 989−1003.
2 (Figure S8); evolution of the fluorescence spectrum of Ru(II)
complex 10 upon addition of YbCl3 solutions (Figure S9). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Bunzli, J.-C. G.; Helm, L. Dalton Trans. 2009, 2088−2095.
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1898−1900.
AUTHOR INFORMATION
Corresponding Authors
■
(21) Verwilst, P.; Eliseeva, S. V.; Vander Elst, L.; Burtea, C.; Laurent,
S.; Petoud, S.; Muller, R. N.; Parac-Vogt, T. N.; De Borggraeve, W. M.
Inorg. Chem. 2012, 51, 6405−6411.
(22) Dehaen, G.; Eliseeva, S. V.; Verwilst, P.; Laurent, S.; Vander
Elst, L.; Muller, R. N.; De Borggraeve, W.; Binnemans, K.; Parac-Vogt,
T. N. Inorg. Chem. 2012, 51, 8775−8783.
(23) Li, W.-S.; Luo, J.; Jiang, F.; Chen, Z.-N. Dalton Trans. 2012, 41,
9405−9410.
(24) Debroye, E.; Dehaen, G.; Eliseeva, S. V.; Laurent, S.; Vander
Elst, L.; Muller, R. N.; Binnemans, K.; Parac-Vogt, T. N. Dalton Trans.
2012, 41, 10549−10556.
*(L.V.E.) E-mail: luce.vanderelst@umons.ac.be.
*(B.M.-V.) E-mail: mestre@chimie.ups-tlse.fr.
*(C.P.) E-mail: picard@chimie.ups-tlse.fr.
Present Address
#(C.D.) CNRS, Laboratoire de Chimie de coordination (LCC),
UPR 8241, 205 route de Narbonne, 31077 Toulouse cedex 4,
France.
Author Contributions
A.B. and C.D. contributed equally to this work and should be
considered as first authors.
(25) Dehaen, G.; Eliseeva, S. V.; Kimpe, K.; Laurent, S.; Vander Elst,
L.; Muller, R. N.; Dehaen, W.; Binnemans, K.; Parac-Vogt, T. N.
Chem.Eur. J. 2012, 18, 293−302.
Notes
The authors declare no competing financial interest.
(26) Debroye, E.; Parac-Vogt, T. N. Chem. Soc. Rev. 2014, 43, 8178−
8192.
ACKNOWLEDGMENTS
■
(27) Sung, S.; Holmes, H.; Wainwright, L.; Toscani, A.; Stasiuk, G. J.;
White, A. J. P.; Bell, J. D.; Wilton-Ely, J. D. E. T. Inorg. Chem. 2014, 53,
1989−2005.
The authors thank Dr. Step
́
hane Balayssac and Prof. Alain
Vigroux for fruitful discussions. This work was supported by the
̀
French Ministere de la Recherche (Ph.D. grant for A.B.) and
(28) Livramento, J. B.; Weidensteiner, C.; Prata, M. I. M.; Allegrini,
P. R.; Geraldes, C. F. G. C.; Helm, L.; Kneuer, R.; Merbach, A. E.;
Santos, A. C.; Schmidt, P.; Toth, E. Contrast Media Mol. Imaging 2006,
1, 30−39.
the CNRS. The ARC (research contract AUWB-2010-10/15-
UMONS-5), the FNRS, the Walloon Region, the COST
TD1004 (Theranostics imaging and therapy: an action to
develop novel nanosized systems for imaging-guided drug
delivery), the UIAP VII program, the European Network of
Excellence EMIL (European Molecular Imaging Laboratories)
program LSCH-2004-503569, and the Center for Microscopy
and Molecular Imaging (CMMI, supported by the European
Regional Development Fund and the Walloon Region) are
thanked for their support.
(29) Parac-Vogt, T. N.; Vander Elst, L.; Kimpe, K.; Laurent, S.;
́
Burtea, C.; Chen, F.; Van Deun, R.; Ni, Y.; Muller, R. N.; Binnemans,
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