Optical and relaxometric properties of monometallic (EuIII, TbIII, GdIII) and heterobimetallic (ReI/GdIII) systems based on a functionalized bipyridine-containing acyclic ligand

Article abstract of DOI:10.1039/c6dt00405a

A series of lanthanide complexes of [LnL(H₂O)]^2- composition where Ln = Eu^III, Tb^III or Gd^III has been studied for determining their photophysical and relaxometric properties in aqueous solution. The bifunctional ligand L (H₅BPMNTA) is an acyclic chelator based on a central functionalized 2,2′-bipyridine core and two iminodiacetate coordinating arms. The mono-aqua Eu^III and Tb^III complexes display attractive spectroscopic properties with an excitation wavelength at 316 nm, similar excited state lifetimes and overall quantum yields (in the ranges 0.5-0.6 ms and 10-13%, respectively) in Tris buffer (pH 7.4). The proton longitudinal relaxivity, r₁, of the Gd^III complex is 4.4 mM^-1 s^-1 at 20 MHz and 310 K, which is comparable to that of the clinically used Gd-DTPA (Magnevist). Interestingly, the water exchange rate between the coordination site and the bulk solvent is very fast (K_ex = 2.6 × 10⁸ s^-1 at 310 K). The ability of the complex to bind non-covalently to human serum albumin (HSA) was also examined by relaxometric measurements. We also report the synthesis and properties of a bimetallic complex based on Gd-BPMNTA and Re^I(bpy)(CO)₃ components. In this system, the Re core exhibits interesting photophysical properties (λ_em = 588 nm, Φ = 1.4%) and the Gd-BPMNTA core displays improved relaxivity (r₁ = 6.6 mM^-1 s^-1 at 20 MHz and 310 K), due to an increase of the rotational correlation time. Besides these appealing optical and relaxometric properties, the presence of a reactive function on the structure proposes this potential dual imaging probe for conjugation to biomolecules or nanomaterials.

Full text of DOI:10.1039/c6dt00405a

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Boulay, C. Galaup, E. Benoist, S. Laurent, L. Vander Elst, B. Mestre-Voegtlé and C. Picard, Dalton Trans.,
2016, DOI: 10.1039/C6DT00405A.
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Page 1 of 37
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DOI: 10.1039/C6DT00405A
Optical and relaxometric properties of monometallic (EuIII, TbIII, GdIII) and
heterobimetallic (ReI/GdIII) systems based on a functionalized bipyridine-containing
acyclic ligand
Nadine Leygue, Alexandre Boulay, Chantal Galaup, Eric Benoist, Sophie Laurent, Luce
Vander Elst, Béatrice Mestre-Voegtlé and Claude Picard
The photophysical and relaxometric properties in aqueous solutions of Ln-BPMNTA
complexes and a derived ReI/GdIII dinuclear complex are reported.
Fluorescent and
paramagnetic agent
H5BPMNTA
Fluorescent or
paramagnetic agent
1

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Page 2 of 37
III
Optical and relaxometric properties of monometallic (Eu , Tb , Gd ) and
III
III
View Article Online
DOI: 10.1039/C6DT00405A
heterobimetallic (ReI/GdIII) systems based on a functionalized bipyridine-containing
acyclic ligand†
Nadine Leygue,a,b Alexandre Boulay,a,b Chantal Galaup,a,b Eric Benoist,a,b Sophie Laurent,c,d
Luce Vander Elst,*c,d Béatrice Mestre-Voegtlé,* ‡a,b and Claude Picard*a,b
aCNRS; Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique,
SPCMIB, UMR-5068; 118 Route de Narbonne, F-31062 Toulouse cedex 9, France.
E-mail: picard@chimie.ups-tlse.fr (CP), beatrice.mestre@lcc-toulouse.fr (BMV)
bUniversité de Toulouse; UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules
d’Intérêt Biologique, SPCMIB; 118 route de Narbonne, F-31062 Toulouse cedex 9, France.
cNMR and Molecular Imaging Laboratory, Department of General, Organic and Biomedical
Chemistry, University of Mons, 23 Place du Parc, B-7000 Mons, Belgium.
E-mail: luce.vanderelst@umons.ac.be
dCenter for Microscopy and Molecular Imaging (CMMI), Rue Adrienne Bolland, 8, B-6041
Gosselies, Belgium
†Electronic supplementary information (ESI) available: Spectroscopic characterization of
ligands, Re and Gd complexes and dinuclear Re/Gd complex, influence of concentration and
temperature on the relaxation rate of Gd complexes, parameters obtained from the theoretical
fitting of the O-17 and 1H NMR data of dinuclear Re/Gd complex. See DOI:
‡Present Address: CNRS, Laboratoire de Chimie de coordination (LCC), UPR CNRS 8241,
205 route de Narbonne, 31077 Toulouse cedex 4, France.
2

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DOI: 10.1039/C6DT00405A
Abstract
A series of lanthanide complexes of [LnL(H2O)]2- composition with Ln = EuIII, TbIII or GdIII
has been studied for determining their photophysical and relaxometric properties in aqueous
solution. The bifunctional ligand L (H5BPMNTA) is an acyclic chelator based on a central
functionalized 2,2’-bipyridine core and two iminodiacetate coordinating arms. The mono-
aqua EuIII and TbIII complexes display attractive spectroscopic properties with excitation
wavelength at 316 nm, similar excited state lifetimes and overall quantum yields (in the range
0.5-0.6 ms and 10-13%, respectively) in Tris buffer (pH 7.4). The proton longitudinal
relaxivity, r1, of the GdIII complex is 4.4 mM-1 s-1 at 20 MHz and 310 K which is comparable
to that of the clinically used Gd-DTPA (Magnevist®). Interestingly, the water exchange rate
between the coordination site and the bulk solvent is very fast (Kex = 2.6 × 108 s-1 at 310 K).
The ability of the complex to bind noncovalently to human serum albumin (HSA) was also
examined by relaxometric measurements. We also report the synthesis and properties of a
bimetallic complex based on Gd-BPMNTA and ReI(bpy)(CO)3 components. In this system,
the Re core exhibits interesting photophysical properties (λem = 588 nm,
Φ = 1.4%) and the
Gd-BPMNTA core displays improved relaxivity (r1 = 6.6 mM-1 s-1 at 20 MHz and 310 K),
due to an increase of the rotational correlation time. Besides these appealing optical and
relaxometric properties, the presence of a reactive function on the structure nominates this
potential dual imaging probe for conjugation to biomolecules or nanomaterials.
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DOI: 10.1039/C6DT00405A
Introduction
Over the last three decades, the use of metallic ions has been fundamental in the development
of the major imaging techniques such as MRI (magnetic resonance imaging), Optical imaging
(fluorescence microscopy) or SPECT (single photon emission computed tomography) and
PET (positron emission tomography) nuclear imaging. As far as MIII ions are concerned, this
is due to the unique magnetic properties of GdIII ions (high magnetic moment coupled with a
long electronic relaxation time), luminescent properties of LnIII ions such as EuIII, TbIII, NdIII
or YbIII (long-lived excited states and emission in the visible or near-infrared regions) and
radiochemical properties of 111In (γ emitter), 86Y or 68Ga (β+ emitters).1-4
Interestingly, the above mentioned MIII ions can be chelated by a unique ligand, mainly a
poly(amino)poly(carboxylate) scaffold, leading the way to multimodal imaging based on the
use of metal complexes cocktails. In these systems, a simple exchange of the metal in the
complex allows multimodal imaging analyses to be undertaken, taking advantage of the
specific strengths of each imaging modality and overcoming their limitations (sensitivity,
spatial and temporal resolutions, depth penetration).5 Examples of ligands include the linear
diethylenetriaminepentaacetic
acid
(H5DTPA)
and
macrocyclic
1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid (H4DOTA). In particular, the GdIII-DTPA
complex (marketed as Magnevist) was introduced in 1988 as the first intravenous MRI
contrast agent authorized for clinical use, and is in widespread use around the world.6 DTPA
derivatives where a chromophoric unit is introduced through an acetate arm were employed
with other lanthanide metals (principally EuIII and TbIII) for an access to luminescent probes.7
In nuclear medicine, the 111In-DTPA-octreotide conjugate (commercially available under the
name OctreoscanTM) has been approved by FDA as a SPECT agent for imaging
neuroendocrine tumours.8 Radiocomplexes derived from 86Y or 68Ga and DTPA were also
used for PET imaging applications.9 H4OCTAPA (Figure 1) is another noticeable example of
acyclic poly(amino)poly(carboxylate) ligand which was assessed as a MIII chelator in various
metal complexes like radiopharmaceuticals (111In, 177Lu), luminescent probes (EuIII, TbIII) and
MRI contrast agent (GdIII).4,10
In the course of our research on the design of MIII ions chelating systems with potential
biomedical imaging applications, we were interested in the development of acyclic chelators
derived from the DTPA backbone where a functionalized pyridine or 2,2’-bipyridine unit is
substituted for the central dimethylene aminoacetate core (H5PMNTA, H5BPMNTA, Figure
1).11,12 The H5BPMNTA chelator is composed of (i) an octadentate N4O4 donor set adequate
4

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III
III
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for efficient complexation of lanthanides and related ions such as In or Y ionDsOaI:n10d.10(3i9i)/Ca6DnT00405A
aromatic carboxylic acid function, in position 4 of the bis-heterocycle, available to covalently
attach the ligand or the complex to a targeting moiety such as peptides, antibodies or
nanoparticles. We have recently synthesized an 111In-BPMNTA core conjugated to the C-
terminal function of a cholecystokinin tetrapeptide (CCK4) which targets tumours
overexpressing the cholecystokinin receptor subtype 2 (CCK2R).13 In this work, we have
successfully demonstrated that this bioconjugate may be used in vivo as a molecular nuclear
imaging agent for the visualization of CCK2R positive tumours on small animal. Moreover,
biodistribution and planar scintigraphic imaging studies showed that this radiometal ligand
has higher tumour uptake, lower background organ uptake, and higher tumour/muscle ratios
than the corresponding 111In-CHX-A″-DTPA conjugate, used as a reference in this study. This
highlights that the structure of the chelator may significantly modify the pharmacokinetic
profile of the conjugate, especially when the targeting moiety is a small peptide or molecule.
EuIII, TbIII-BPMNTA and GdIII-BPMNTA complexes are other candidates to act either as LnIII
luminescent probes or magnetic contrast agents, respectively. Most of EuIII, TbIII luminescent
probes of practical use incorporate a chromophoric unit with a suitable lowest triplet-state
energy level providing an efficient energy transfer to populate the LnIII ion excited states
(antenna effect).14 In this direction, the photophysical properties of the 2,2’-bipyridine
chromophore (3E00 = 23500 cm-1) are favourable for an efficient antenna effect for both EuIII
and TbIII luminescence.15 Concerning an MRI reporter, it is well established that a GdIII
complex should have at least one water molecule in the inner coordination sphere of the metal
allowing the proton nuclear relaxation of the lanthanide ion-bound water to be transferred to
the bulk aqueous solution. GdIII ion exists in aqueous solution below pH∼6 as a hydrated aqua
ion with ∼8-9 inner-sphere water molecules. Consequently, the octadentate ligand
H5BPMNTA might leave a vacant coordination position for a water molecule.
In this contribution, the scope of H5BPMNTA ligand is extended to the photophysical
characterization of its EuIII and TbIII complexes together with the relaxometric properties of its
GdIII complex. We aim at evaluating the parameters that control luminescence and proton
relaxometry in these systems (Figure 2). Moreover, we demonstrate here that a pyridine
derivative of the BPMNTA chelator is suitable for its direct coupling to a ReI(bpy)(CO)3 core
(bpy = 2,2’-bipyridine), hence affording a heterobimetallic ReI/GdIII complex (Figure 2). Such
heterobimetallic compounds were recently reported as potential dual imaging agents allowing
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simultaneous fluorescence live cell microscopy and whole body imaging.16 DPOrI:e10li.1m03i9n/Ca6rDyT00405A
results concerning our heterobimetallic system have been published previously.17
View Article Online
Results and discussion
Synthesis
The fully protected bifunctional chelating agent 1 (Scheme 1) was synthesized according to
our previously published procedures.18 Deprotection of the two kinds ester functions (aqueous
6N HCl, 90°C) in 1 afforded pentaacid 2, while selective deprotection of tert-butyl ester
groups (CF3COOH, CH2Cl2, rt) or methyl ester group (LiOH, THF/H2O, rt) gave tetraacid 3
and tetraester 4, respectively. In 4, the carboxylic acid function can be activated in situ by
PyBOP reagent and condensed with the amino group of 4-picolylamine, affording compound
5 in 79% isolated yield. Compound 5 was used to elaborate the heterobimetallic ReI/GdIII
architecture (see below). On the other hand, one can note that the methyl ester function in 1
and the carboxylic acid function in 4 can be readily converted toward amine or acetylenic
groups, respectively, affording other bifunctional derivatives.
The LnIII (Ln = Eu, Tb, Gd) chelates derived from ligand 2 were prepared at room
temperature in water solution (pH 5-6) with a slight molar excess (ca 10%) of the lanthanide
.
salt (LnCl3 6H2O) to avoid any presence of free ligand. After purification (Waters Sep-Pak
column C18), the absence of free lanthanide ions was checked by the Arsenazo test19 and no
peak ascribable to the free ligand was obtained in the ESI mass spectra and HPLC analyses of
these complexes. Yields were quantitative. It is noteworthy that the complexation of LnIII ions
by H5BPMNTA chelator is a relatively fast reaction which is completed within an hour at
room temperature, as evidenced by preliminary luminescent experiments on EuIII and TbIII
complexes (See ESI† section, Fig. S8).
Photophysical studies of Ln-BPMNTA complexes
Investigation of the photophysical properties of the Eu- and Tb-BPMNTA complexes was
carried out in Tris-buffered aqueous solutions at pH 7.4 and in various conditions. The most
important photophysical parameters are gathered in Table 1. Their UV-Vis spectra are similar,
displaying a strong absorption band in the low-energy region of the spectra tailing out to 340
nm (λmax = 316 nm,
transitions of the bpy core. When compared to the free ligand, the noticeable bathochromic
ε
> 12000 dm3 mol-1 cm-1) which corresponds to π→π* electronic
6

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shift of this absorption band (∆λ = 15 nm) may be associated with the cisoidDOtoI: 10tr.1a03n9s/Co6iDdT00405A
isomerization of the pyridine cycles and the induced perturbation by the coordinated
lanthanide ions. Photoexcitation in the lowest energy absorption band of the bpy chromophore
(316 nm) was used to obtain the emission spectra of EuIII and TbIII complexes (Figure 3).
Their emission profiles are typical of such LnIII ions: five narrow bands for the EuIII complex
5
arising from the D0 → FJ (J = 0 – 4) transitions are observed whereas bands arise from the
7
7
5D4 → FJ (J = 6 – 0) transitions for the TbIII complex. In these complexes, the strongest
5
7
emissions are located at 617 and 544 nm, corresponding to the EuIII-centred D0 → F2 and
5
7
TbIII-centred D4 → F5 hypersensitive transitions, respectively. The excitation spectra are all
located within the absorption range of the ligand and have similar shapes than the absorption
spectra, indicating that energy transfer from the ligand to the metal ion is operative.
For these two EuIII and TbIII complexes, luminescence decays are monoexponential functions,
suggesting the presence of a single lanthanide coordination environment. At room
temperature, the emission lifetimes of the Eu (5D0) and Tb (5D4) levels are 0.60 and 0.51 ms,
respectively, that make them compatible with the use of time-resolved luminescence
technique. As well reported in the literature, comparison of their lifetimes in water and
deuterated water allowed us to obtain an assessment of the hydration state of these complexes
(i.e. the number of water molecules coordinated to metal, q).20 Thus, q values were found at 1
and 0.9 for EuIII and TbIII complexes, respectively. Assuming ligand BPMNTA is octadentate,
these results are fitting in well with nine-coordinated LnIII complexes. The calculated rate
constants knr(OH) are 1200 s-1 (Eu complex) and 240 s-1 (Tb complex), signifying a
contribution of 600 or 120 s-1 per O-H oscillator, respectively. These values are in good
agreement with literature data.21 Nevertheless, and contrary to the EuIII complex, it is
noteworthy that the TbIII complex displays very short lifetime values at room temperature in
comparison with those usually found for mono-aqua TbIII complexes. Indeed, for such
complexes incorporating bpy or bpy-carboxylate chromophores, lifetimes within the 1.2 – 1.5
ms (H2O) and 2.0 - 2.5 ms (D2O) ranges were reported.22 These data imply the existence of an
additional non-radiative pathway in the case of TbIII complex, besides vibronic deactivation
5
via O-H oscillators. In D2O solution, the lifetime of the D0 level is temperature independent
5
between 298 K and 77 K, whereas that of the D4 level is increased by a factor of 4.6 upon
lowering the temperature at 77 K (Table 1). The corresponding high value (1350 s-1) of the
nonradiative temperature-dependent decay rate constant knr(T) suggests that a back energy
transfer from the metal-centred level to the ligand-centred level seems to be operative in Tb-
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BPMNTA. As a comparison, a knr(T) value of 2100 s-1 was reported for a Tb-trisbipyrVidiewinAreticle Online
DOI: 10.1039/C6DT00405A
cryptate in which equilibrium between chromophoric donor and TbIII acceptor occurs at room
temperature.23
To figure out whether or not such a thermally activated radiationless decay path might play a
5
key role in the decay of the D4-emitting state, we investigated as usual the behaviour of
ligand-centred (LC) levels in the corresponding GdIII complex. Room temperature GdIII
complex fluorescence and low-temperature GdIII complex phosphorescence led us to assess
that in Ln-BPMNTA complexes the singlet and triplet LC states lie at 30300 and 22100 cm-1,
1
respectively. The energy of the 0-phonon of the ππ* LC levels was determined by the
3
intercept of the absorption and fluorescence spectra and that of the ππ* LC levels by the
highest energy band maximum of the structured phosphorescence profile (Figure 4). The
energy gap ∆E (1ππ*-3ππ*) amounts to 8200 cm-1, predicting an efficient singlet-to-triplet
intersystem crossing process.24 We can also observe that the energy difference between the
3
5
first ππ* state of the ligand and the emissive D4 level of terbium is around 1600 cm-1. As
concluded in a recent Bünzli’s report, this energy gap is too low for preventing energy back-
transfer onto the ligand and a concomitant loss of sensitization efficiency at room
temperature.25
Overall quantum yields (Φ
ov) of these EuIII and TbIII complexes were determined
experimentally by excitation of the ligand (λexc = 316 nm) in aerated Tris buffer at room
temperature. The Φov values are similar for both complexes (10-13%) despite the presence of
a back-energy transfer observed for the TbIII complex. The
reported in aqueous solutions for EuIII complexes containing one or more 2,2’-bipyridine,
while the
ov(Tb) value lies in the medium range.22,26 Moreover, these values are markedly
Φov(Eu) value is among the largest
Φ
higher than those reported for the mono-aqua Eu-, Tb-OCTAPA complexes, based on
pyridine chromophores (Φov < 2%).10b In the case of the EuIII complex, it was possible to
estimate the two factors which contribute to the overall quantum yield: the sensitization
efficiency ηsens and the intrinsic luminescence quantum yield ΦEu
experimental data (spectrum, ov and lifetimes) and following the methodology developed by
(Φov = ηsens × Φ
Eu).27 From
Φ
Werts et al. and Beeby et al.,28 the calculated values of ΦEu and ηsens were 13.5 and 95.5%,
respectively. The value of ΦEu is comparable with other mono-hydrated EuIII complexes
derived from DTPA (12% - 13%)29 and that of ηsens suggests that a favourable geometric
effect (i.e., donor-acceptor distance) occurs in this complex. For excitation at 330 nm, the
brightness (B = ε × Φ
ov) are 920 and 650 M-1 cm-1 for EuIII and TbIII complexes, respectively.
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These brightness values are consistent with those required for emissive lanthanidDeOcI:o10m.10p3l9e/Cx6eDsT00405A
acting as tags for bioassays or optical probes.30 Moreover, similar lifetimes and overall
quantum yields of these complexes, differing in spectral output, allow their use for dual-
parametric analyses.
To evaluate the potential of these LnIII complexes in biological experiments, we investigated
the luminescence properties of the EuIII complex in buffers typically used in bioanalytical
chemistry, in the presence of human serum proteins, as well as of ethylenediaminetetraacetic
acid (EDTA), a strong competing ligand often used in biological experiments. The emission
properties of the Eu complex as recorded in Tris buffer remained unchanged in other assessed
buffers and pH range (50 mM HEPES buffer, pH 7.3; 50 mM phosphate buffer, pH 7.4 and 50
mM Borate buffer, pH 8.6). Emission spectrum, total intensity and lifetime of this complex
did not differ after 24 h incubations in media containing human serum albumin (HSA) and
transferrin (80 and 20 equiv., respectively). These results suggest that neither demetalation
nor replacement of the innersphere water molecule by metal chelating groups lying at the
surface of the proteins are occurring in the presence of an excess of these two serum protein
models. Finally, the Eu complex exhibited a great resistance in presence of EDTA, a chelator
which strongly binds EuIII (logKEu (EDTA) = 17.32).31 The dissociation kinetics of Eu
complex was studied at room temperature (Tris buffer, pH 7.4) in presence of a 1000-fold
5
7
excess of the trapping agent and by monitoring the disappearance of the D0 → F2 peak at
617 nm over time. In these experimental conditions, the Eu complex was only 12%
dissociated after 1.5 day. From these experiments and by using the Verhoeven analysis,
logKcond (pH 7.4) was found to be 19.0.32 This value is similar to that of Gd-OCTAPA
complex (logKcond (pH 7.4) = 19.2), an acyclic complex containing the same donor set but in a
different arrangement.10c
Relaxometric studies of Gd-BPMNTA complex
The ability of a GdIII complex to act as a MRI contrast agent (MRI CA) is generally described
in terms of its relaxivity r1 (r1 is the longitudinal relaxation rate enhancement induced by 1
millimole per liter of paramagnetic compound). This characteristic depends on several
experimental conditions: concentration of the paramagnetic complex, magnetic field strength,
temperature and pH. A 1 mM solution of Gd-BPMNTA complex at 20 MHz and neutral pH
(relevant conditions to compare with MRI CA references) exhibited a relaxivity r1 of 4.4 and
5.5 mM-1 s-1 at 310 and 298 K, respectively. This relaxivity is slightly higher than for mono-
aqua GdIII complexes of small size such as Gd-DTPA (3.8 mM-1 s-1 at 310 K) and Gd-
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OCTAPA (5.0 mM-1 s-1 at 298 K).10b,33 This result reflects perfectly the relationship between
View Article Online
DOI: 10.1039/C6DT00405A
molecular weights of complexes and associated relaxivity values (MW = 565.6, 617.6 and
662.6 g mol-1 for Gd-DTPA, Gd-OCTAPA and Gd-BPMNTA, respectively). The influence of
concentration and temperature on the relaxation rate of Gd-BPMNTA was also investigated at
20 MHz (See ESI† section, Figs. S14, S15). At 310 K, the study of R1 as a function of the
concentration of Gd-BPMNTA (0.5 to 3 mM) does not show any deviation from linearity,
suggesting the absence of aggregates by π-stacking of the bpy moiety in solution. On the
other hand, the continuous increase of the relaxivity between 318 K (r1 = 3.6 mM-1 s-1) and
278 K (r1 = 8.1 mM-1 s-1) is typical of a fast exchange between the coordinated water
molecule and bulk water.
According to the Solomon-Bloembergen-Morgan theory, a set of physico-chemical (structural
and dynamic) parameters of a GdIII chelate, with a rather complex interplay, governs the
observed r1 values. Among them, the most relevant ones are (i) the number of water
molecules (q) in the first coordination sphere of the metal, (ii) the residence time of these
water molecules at the metal site (
size (
R).3,34
τM), (iii) the tumbling motion of the complex, related to its
τ
In this relaxometry study, the number of coordinated water molecules for Gd-BPMNTA was
set to one in accordance with the hydration sphere of the corresponding EuIII complex (vide
supra). At 500 MHz and within the investigated temperature range (275 – 310 K), the
R
evolution of the bulk water transverse-relaxation rate of 17O (1/T2 ) for Gd-BPMNTA showed
a continuous increase with decreasing temperature, indicating a maximum at lower
temperature (Figure 5). This profile markedly differs from that of Gd-DTPA which exhibits a
classical bell shaped curve with a maximum around 310 K. A qualitative analysis of these
data suggests that Gd-BPMNTA exhibits a significantly lower value of τM compared to Gd-
310K
R
DTPA (τM
= 143 ns).33 The theoretical treatment of the experimental data (1/T2 ) was
performed as previously described and provided a residence time of the coordinated water
R
molecule of 3.9 ns.35 Although the absence of maximum in the 1/T2 data may lead to some
uncertainty in the calculated value, we can assume that Gd-BPMNTA exhibits a remarkably
short τ
M value in comparison with those usually observed in mono-aqua gadolinium chelates.1
Actually, this value is comparable to the τM reported for the TSAP isomer of a DOTA
derivative (6 ns).36 It should also be noted that this τM value is about three orders of
magnitude shorter than that reported for the corresponding neutral bpy macrocyclic chelate.22c
This result could be at least partly related to a more flexible coordination cage of the acyclic
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complex, leading probably to a larger steric crowding around the metal ion as well as to an
DOI: 10.1039/C6DT00405A
overall negative electric charge, another favourable factor.37 Finally, it should be pointed out
that Gd-BPMNTA τM value only marginally falls outside the optimal range of values (10 – 30
ns) for low imaging field (0.5 T). This is a favourable factor for molecular imaging if covalent
coupling of this bifunctional chelate to a vector is considered.38 On carrying out the fitting
procedure, other parameters were calculated: hyperfine coupling constant between the 17O
nucleus of bound water (A/h), activation enthalpy and entropy for the water-exchange process
(∆H≠ and ∆S≠, respectively), electron relaxation parameters (τv and B parameter) and Ev,
activation energy related to
τv. These data are reported in Table 2. The value of the A/h
parameter is typical of what is generally observed for GdIII complexes (- 3.6 × 10-6 0.3 rad s-
1), thus confirming the assumption of a mono-aqua complex. Besides, both the large
activation enthalpy, ∆H≠, and the positive activation entropy, ∆S≠, point to a dissociatively
activated mechanism for the water exchange, as commonly observed for nine-coordinated
complexes.39
1
The H NMRD profile of Gd-BPMNTA was recorded at 310 K in the proton Larmor
frequency range 10 kHz – 60 MHz and compared to the NMRD profiles of Gd-DTPA and
parent compound Gd-PMNTA (Figure 6).33,11 The three complexes display the typical s-shape
of low-molecular-weight and rapidly tumbling GdIII chelates. Compared to Gd-DTPA, the
profile of Gd-BPMNTA shows increased values over the entire investigated magnetic range
as a result of its slightly higher molecular weight. The higher r1 values observed for Gd-
PMNTA (r1 = 6.4 mM-1 s-1 at 20 MHz and 310 K) certainly arise from the higher number of
GdIII coordinated water molecules (q = 2 for this complex and q = 1 for the two other ones),
1
resulting in higher relaxation. H NMRD profile of Gd-BPMNTA was fitted using the usual
contributions from the inner-sphere (IS) water molecules (Solomon-Bloembergen-Morgan
theory) and from the outer-sphere (OS) water molecules, diffusing in the proximity of the
paramagnetic complex (Freed theory).40 In the fitting procedure some parameters were fixed:
the GdIII-H distance between the GdIII ion and the proton nuclei of the bound water (rGdH
=
0.31 nm), the distance of closest approach of an outer-sphere water proton to the GdIII ion (d =
0.36 nm) and the relative diffusion coefficient (D = 2.93 × 10-9 m2 s-1). The number of water
molecules in the first coordination sphere, q, was set to 1 and the water residence time, τM
,
17
was fixed to the value determined by O relaxometry. The parameters calculated from the
fitting were given in Table 3, together with analogous data of other relevant GdIII complexes
for comparison. The tumbling rate of Gd-BPMNTA (τR = 76 ps) agrees with the size of the
11

Dalton Transactions
Page 12 of 37
View Article Online
complex and is in concordance with reference complexes data. The correDlaOtIi:o10n.103t9im/C6eDsT00405A
modulating the electronic relaxation of GdIII (
V) are also similar while the longer electronic
SO) observed for Gd-PMNTA reflects its larger low-field
τ
relaxation time at zero field (
relaxivity.
τ
Gd-BPMNTA was also subjected to a relaxometry study for the evaluation of its possible
interaction with Human Serum Albumin (HSA). At 20 MHz and 310 K, the r1 value of the
complex at a concentration of 1 mM and in the presence of 4% HSA is significantly higher
than without HSA (r1 = 6.0 vs. 4.4 mM-1 s-1). Besides, the NMRD profile of the complex in
the presence of HSA (Figure 7) is characterized by a hump at high fields (between 20 and 60
MHz), as observed for slowly rotating molecules. It can be assigned to the presence of a non-
covalent interaction between the complex and HSA. To quantify this interaction, we used
established methods involving the analysis of the paramagnetic proton relaxation rate of
solutions containing 4% of HSA and various concentrations of the GdIII complex at 20 MHz
and 310 K (insert in Figure 7).41 By fitting these data with equation (4) given in experimental
section, we estimated a binding constant Ka of about 1170 290 M-1 corresponding to one
interaction site. Relaxivity values for free (r1f ) and bound (r1b ) complexes were 5.0 0.5 and
10.1 2.1 mM-1 s-1, respectively. Our complex shows similar binding compared with Gd-
BOPTA (Ka = 1.5 × 103 M-1), well known to interact with HSA and specifically developed as
a blood agent (Multihance, Bracco).42 However the enhancement of the relaxivity of our
HSA-bound complex with respect to the free complex is lower than anticipated (r1b /r1f = 2.0
vs. 8.2 for Gd-BOPTA). Clearly, the expected relaxation enhancement has been quenched by
a direct interference of residues on protein surface in close proximity of the binding site.
Luminescent experiments based on the corresponding Eu-BPMNTA complex in the presence
of HSA (vide supra), suggest that a partial replacement of the metal coordinated water
molecule by coordinating groups of the HSA protein does not occur. The relaxivity of the
adduct is likely quenched by a decrease of the water exchange rate, despite the very short τM
value measured for the free complex. Such a quenching has been previously observed in cases
of GdIII complexes with high exchange rates.42
Synthesis, photophysical and relaxometric properties of dinuclear ReI/GdIII complex
(13)
12

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Dalton Transactions
As pointed out previously,16 the association of GdIII and ReI cores into a heterobimeVtaiewllAircticle Online
DOI: 10.1039/C6DT00405A
complex may combine MRI and optical imaging modalities in a single architecture (dual-
imaging probe). Particularly, ReI polypyridyl tricarbonyl complexes are generally highly
luminescent, showing visible light excitation and emission, large Stokes shifts (λem
- λexc >
200 nm), as well as long-emission lifetimes (µs range) which make them serious candidates
for applications in cell imaging.43 Our approach was to bind the chelating moiety (BPMNTA)
as an ancillary ligand to a (bpyCOOMe)ReI(CO)3 core. Relying on our previous works about
functionalized bpy derivatives and a dinuclear ReI/99mTc complex, the incorporation of an
aromatic ester functionality on the bpy unit of the ReI core seemed a relevant way for
subsequent bioconjugation.18,44
Scheme 2 outlines the followed preparative strategy for accessing to the heterodinuclear
ReI/GdIII complex 13. The new ReI complexes 9 and 10 were obtained through a classical
pathway involving preparation of the (bpy-COOMe)Re(CO)3Cl complex by reacting 4-
carbomethoxy-2,2’-bipyridine with a slight excess of the Re(CO)5Cl precursor in refluxing
methanol (1.2:1 metal:ligand ratio) and its further activation with triflate silver salts in
acetonitrile. The reactive intermediate CH3CN adduct 10 was readily obtained in excellent
yield (91% for the two steps) by using simple filtration and recrystallization procedures.
Under microwave irradiation (155 W at 105°C in THF), the substitution of the Re-bound
acetonitrile molecule of 10 by the ancillary pyridinic ligand 5 took place within 1 h, thus
affording the desired complex 11 in 56% yield after purification by column chromatography
on alumina. It is worth noting that by using classical thermal activation (NaHCO3, CH3CN,
reflux, overnight), complex 11 was obtained in 40% isolated yield. Selective deprotection of
tert-butyl groups was performed in acidic conditions (CF3COOH) at room temperature and
the subsequent treatment of the tetraacid compound 12 with GdCl3 in water yielded the
neutral heterodinuclear complex 13. All complexes display metal carbonyl stretching
frequencies in the range 2050-1880 cm-1 which are consistent with a facial (fac) isomer
arrangement at the rhenium centre. The single and strong IR absorption at ~ 2030 cm-1 can be
≡
assigned to the axial C O group, whereas equatorial carbonyl ligands gave IR absorption in
the 1930-1900 cm-1 range, with a broad and intense band which splits into two peaks in the
13
case of complex 9. In C NMR spectra, three signals assigned to C O groups were also
≡
observed in the 190-195 ppm range. In addition, 1H, 13C NMR, UV-Vis and emission spectra
of the complexes provided evidence of the presence of the bpy and pyridine ligands within the
13

Dalton Transactions
Page 14 of 37
ReI coordination sphere while their MS spectra gave isotopic distribution in agrDeOeIm: 10e.n10t3V9wie/Cwi6tADhrtTic0le0O4n0li5nAe
the proposed structures.
The UV-Vis absorption and emission spectra of complexes 9-13 were recorded at room
temperature and in aerated CH3CN or aqueous solution (Tris buffer, pH 7.4). Relevant
photophysical data are given in Table 4. Unfortunately, the luminescence lifetime of these
complexes (
τ < 30 µs) could not be measured due to instrumental limitations. The electronic
spectra of these ReI species showed the typical characteristics of bis-imine ReI derivatives
displaying several absorption bands in the 240 – 450 nm range. The two bands at high
energies (λ < 320 nm) with high extinction coefficients (ε
> 104 dm3 mol-1 cm-1) are assigned
to intraligand (IL) transitions of the appended chromophores [π→π* (bpyCOOMe and py-
bpy)]. On the basis of previous spectroscopic studies of ReI(bpy)tricarbonyl complexes,43c,45
the lower-energy absorption band (340-450 nm) with extinction coefficient in the order of 103
dm3 mol-1 cm-1 is associated to spin-allowed metal-to-ligand charge-transfer (1MLCT,
1
formally, Re(dπ-bpyCOOMe (π*)). For all complexes, excitation into the MLCT absorption
band leads to a broad structureless emission band extending from 500 to 800 nm. This
emission band remains unchanged with the energy of excitation and is typical of the
phosphorescence derived from the triplet rhenium-to-ligand charge transfer (3MLCT) excited
state. By comparison with (bpy)ReI(CO)3Cl or (bpy)ReI(CO)3CH3CN complexes, substitution
of the bpy moiety by an electron withdrawing group in 9 and 10 leads to a red-shift for both
3
1MLCT absorption and MLCT emission bands.46 On the other hand, the coordination of the
BPMNTA moiety to GdIII induces in 13 a red-shift (13 nm) of the lowest energy of intraligand
1
3
transition but does not significantly influence both MLCT absorption and MLCT emission
bands as well as the emission quantum yield of the ReI core (Figure 8). Compared to its
precursor complex 12, the lack of quenching associated to the Re 3MLCT state in complex 13
is expected. Actually, the GdIII excited levels have far higher energies (> 31000 cm-1) than
those of Re chromophore MLCT states, thus preventing any Re → Gd energy transfer process.
1
As far as the target dinuclear complex 13 is concerned, it was characterized by a MLCT
3
absorption band at 367 nm, a MLCT emission band with a maximum at 588 nm and an
emission quantum yield of 1.4%. Although this latter value is rather low, it is large enough for
detection in fluorescence microscopy.47 Moreover, the emission intensity of the 3MLCT band
of 13 remained unchanged over a two-day period in Tris buffer (pH 7.4), in presence of 200
molecular excess of histidine (a strong competing ligand for a ReI core), EDTA (1000 equiv.)
or in a Tris buffer/serum (2/1) mixture.
14

Page 15 of 37
Dalton Transactions
The ReI/GdIII complex was subsequently investigated for its relaxometric prDoOpI:e1r0t.i1e03s9,/Cb6DyT00405A
View Article Online
assessment of relaxivity values r1, by analysis of the temperature dependence of the reduced
17
transverse relaxation rate of O and by recording the NMRD profile at 310 K (Figure 9) in
order to estimate the r1, τM and τR key parameters. Theoretical treatments of these
experimental data were performed as mentioned before by using the IS and OS model,
assuming the presence of a mono-aqua Gd-BPMNTA core (See ESI† section, Tables S1 and
S2). At 310 K, the experimental r1 values were 6.6 mM-1 s-1 at 20 MHz and 6.0 mM-1 s-1 at 60
MHz, i.e. significantly higher than those of Gd-BPMNTA parent complex. As observed for
Gd-BPMNTA, the influence of concentration and temperature on dinuclear complex suggests
the lack of aggregates and no limitation of relaxivity by the water residence time. We note
also a similar 17O NMR profile than for Gd-PMNTA, with a maximum of the curve located at
17
a temperature lower than 275 K. As expected, the theoretical analysis of the O NMR data
provides a short residence time of the coordinated water molecule (τM = 9.0 ns) in accordance
with what was observed for the Gd-BPMNTA complex (τM = 3.9 ns). The presence of the
extra ReI core poorly influences the value of the exchange rate, as expected for moieties that
do not contribute to the coordination sphere of GdIII ion. The NMRD profile of 13 had the
typical shape for a low-molecular weight complex, with no hump observed at high magnetic
fields (20 – 60 MHz) and revealed a higher relaxivity at all magnetic fields compared to Gd-
BPMNTA complex. This improved relaxivity is due to a slower tumbling rate as evidenced by
a higher calculated τR value (138 ps) than for Gd-BPMNTA (76 ps). This is in good
agreement with the increase by a factor 1.9 of the dinuclear complex molecular weight. In
order to compare with Gd-BPMNTA, the interaction of 13 with HSA was also investigated.
At 20 MHz and 310 K, the r1 relaxivity value of 13 in the presence of 4% HSA is 7.4 mM-1 s-1,
corresponding to an enhancement of 12% without HSA. The slight increase of the
longitudinal apparent relaxivity is minimal and can be rather explained by an increase in the
viscosity of medium compared to water solution than by a specific interaction between 13 and
HSA. This result is promising, because a molecular imaging agent must specifically bind to
its target and avoid non-specific interferences with blood macromolecules such HSA.
Conclusions
The mono-aqua LnIII complexes (Ln = Eu, Tb, Gd) of the bifunctional chelating agent
H5BPMNTA show promising photophysical and relaxometric properties. In aqueous media,
15

Dalton Transactions
Page 16 of 37
III
the Eu complex displays an emission lifetime of 0.60 ms at room temperature and an overall
View Article Online
DOI: 10.1039/C6DT00405A
quantum yield of 12.9% under an excitation at 316 nm. The TbIII complex displays similar
lifetime and quantum yield values despite the presence of a back energy transfer process (5D4
Tb →3ππ* ligand) according to the energy-gap law (E(3ππ*) – E(5D4)). These complexes are
also characterized by a good kinetic stability both in aqueous buffers generally used in
bioanalytical analyses and in media containing blood proteins. The GdIII complex shows a r1
relaxivity at the upper limit of values observed for clinically used MRI contrast agents
combined to a much more appealing water residence time (τM = 3.9 ns), optimum for
vectorisation. In addition, we demonstrate that a pyridinic derivative of H5BPMNTA can self-
assemble with a (bpy)ReI(CO)3 core, giving rise to a neutral heterobimetallic ReI/GdIII
complex, highly soluble and stable in aqueous solution. This dinuclear complex leads to an
increase of the τR rotational tumbling time of the Gd-BPMNTA core, resulting in an
enhancement of the r1 relaxivity by a factor of 1.5. In addition, it exhibits orange broad-band
emission upon excitation in the visible range (367 nm) with a quantum yield of 1.4% in
aqueous solution, sufficiently large for detection in fluorescence microscopy. Finally, a
significant binding of Gd-BPMNTA complex with HSA was observed, but none with the
dinuclear ReI/GdIII complex. Taking into account both the luminescent and relaxometric
properties of Ln-BPMNTA complexes and our previous work on 111In-BPMNTA
radiocomplex,13 H5BPMNTA ligand can be further explored as an efficient platform to get
probes for a multimodal imaging purpose. Moreover, the presence of a reactive function in the
BPMNTA core offers an interesting opportunity for the construction of heteropolymetallic
complexes and conjugation to biomolecules or nanomaterials.
Experimental
General
Reactions utilizing microwave technology were conducted in a CEM Discover Benchmate
1 13
microwave reactor (model nr.908010). H and C spectra were recorded on Bruker Avance
300, 400 and 500 working at 300, 400 and 500 MHz respectively. For 1H chemical shifts (
δ)
are given in ppm relative to a residual protiated solvent and coupling constants (J) are given
in Hertz. IR spectra were recorded on a Perkin-Elmer FT-IR 1725x spectrometer using KBr
pellets (solid samples) and NaCl plates (liquid samples). Electrospray (ES) mass spectra were
obtained on a Q TRAP Applied Biosystems spectrometer and High-Resolution Mass Spectra
16

Page 17 of 37
Dalton Transactions
(HRMS) on a Xevo G2 QTof Waters spectrometer. DCI mass spectra were obtained Voienw Araticle Online
DOI: 10.1039/C6DT00405A
DSQ II Thermo Fisher. Elemental analyses were carried out by the “Service d’Analyse”,
Laboratoire de Chimie de Coordination (Toulouse). The analytical HPLC analyses were
performed on a Waters Alliance 2695 system with a PDA 2996 detector and using a reverse-
phase column (Phenomenex Luna C8, 5 µm, 100 Ǻ, 150 x 4.6 mm) with a flow rate of 1
mL/min. Linear gradient system was HCOONH4 10 mM pH 4/CH3CN 100/0 to 80/20 in 18
min (system A) or 90/10 to 10/90 in 18 min (system B). The analytical UPLC analyses were
performed on a Acquity UPLC Waters system with a PDA detector and using a reverse phase
column (Acquity BEH C18 column, 1.7 µm, 100 Ǻ, 50 x 2.1 mm) with a flow rate of 0.6
mL/min. Linear gradient system was HCOONH4 10 mM pH 4/CH3CN + 10% HCOONH4
100 mM pH 4 80/20 to 0/100 in 7 min
Pentaester compound 1 was synthesized according to our previously published procedures.18
Photophysical measurements
Absorption measurements were done with a Hewlett Packard 8453 temperature-controlled
spectrometer in 10 mm quartz cuvette. Fluorescence spectra at room temperature were
obtained using a Cary Eclipse spectrofluorimeter 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) or CH3CN 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 a LS-50B Perkin-Elmer
spectrofluorimeter equipped with a Xenon flash lamp source, a Hamamatsu R928
photomultiplier tube and the low-temperature accessory No. L2250136. Lifetimes
τ
(uncertainty ≤ 5%) were measured by monitoring the decay at a wavelength corresponding to
the maximum intensity of the emission spectrum, following pulsed excitation. They are the
average values from at least five separate measurements covering two or more lifetimes. The
luminescence decay curves were fitted by an equation of the form I(t) = I(0) exp(-t/τ) by
using a curve-fitting program. High correlation coefficients were observed in each cases
(higher than 0.999). The luminescence quantum yields (uncertainty 15%) were determined
by the method described by Haas and Stein 48 using as standards [Ru(bpy)3]2+ in aerated water
(Φ = 0.04)49 for the Eu(III) and Re(I) complexes or quinine sulfate in 1N sulfuric acid (Φ =
0.546)50 for the Tb(III) complex and corrected for the refractive index of the solvent. They
17

Dalton Transactions
Page 18 of 37
View Article Online
were measured according to conventional procedures with diluted solutions (optical density <
DOI: 10.1039/C6DT00405A
0.05).
The sensitization efficiency ηsens and the intrinsic luminescence quantum yield ΦEu were
calculated by using the following equations:28
ηsens
=
Φov
τobs
R = kr = 14.65 × n3 × (IΣFj / IF1)
where obs is the observed lifetime,
/
ΦEu
(1)
ΦEu
=
/
τR
(2)
1 /
τ
(3)
τ
τ
R the radiative lifetime, IΣFj the integrated intensity of the
entire emission spectrum, IF1 the integrated intensity of the purely magnetic dipole transition
5D0→7F1 and n the refractive index of the solution.
NMR measurements
Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded on a Stelar
Spin fast field cycling (FFC) NMR relaxometer (Mede, Italy) working between 0.24 mT and
1 T. The measurements were performed at 310 K with samples (0.6 mL) in 10 mm o.d. Pyrex
tubes. The additional relaxation rates at 0.47 and 1.41 T were measured on mq-20 and mq-60
1
Minispec systems (Bruker, Karlsruhe, Germany), respectively. Fitting of the H NMRD was
performed with data-processing software using different theoretical models describing the
nuclear-relaxation phenomena observed (Minuit, CERN Library).51,52 The exact Gd3+
concentration was determined by proton relaxivity measurements at 0.47 T and 37 °C after
complete hydrolysis in concentrated HNO3.
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 spectrometer. The temperature was
regulated by air or nitrogen flow controlled by a Bruker BVT 3200 unit.
17O diamagnetic transverse-relaxation times of water were measured using the Carr-Purcell-
Meiboom-Gill (CPMG) sequence (90° and 180° pulse lengths were 25 and 50 µs,
respectively). All 17O NMR spectra were proton decoupled. 17O transverse-relaxation times of
water in solutions containing the Gd complex were calculated from the spectral line width.
The data are presented as the reduced transverse relaxation rate (1/T 2R = 55.55/(q × [complex]
× 1/T 2p ) where q is the number of inner-sphere water molecules, [complex] is the molar
concentration of the Gd complex and T2p is the paramagnetic transverse relaxation rate.
For the study of noncovalent interaction with HSA, the proton paramagnetic relaxation rate
p
data (R1 ) were obtained at fixed field strength (0.47 T) and temperature (310 K) using a
18

Page 19 of 37
Dalton Transactions
Minispec mq-20 (Bruker, Karlsruhe, Germany). The HSA concentration was seDtOtIo: 100.1.063V9ime/Cw6MADrtTic0le0O4n0li5nAe
(4%) and the concentration of the GdIII agent ranged from 0 to 1.5 mM.
The proton data obtained in HSA solution were fitted using equation (4):41
p
R1 = 1000
f
b
×{(r1 ×L0) + 0.5×(r1 - r1 )
f
-1
× [(N×P0) + Lo + Ka
-1 2
[(N×P0) + L0 + Ka ) - 4×N×L0×P0]]}
-
(4)
where Ka is the association constant, P0 is the protein concentration, L0 is the concentration of
the paramagnetic complex, N is the number of independent and identical interaction sites, and
r 1b and r 1 (mM-1 s-1) are the relaxivities of the HSA–contrast agent adduct and the free
f
contrast agent, respectively.
2,2′,2′′,2′′′-[(4-carboxy-2,2′-bipyridine-6,6′-diyl)bis(methylenenitrilo)]-tetrakis
(acetic
acid) (2). The pentaester 1 (590 mg, 0.81 mmol) was stirred in a 6M HCl solution (30 mL) at
90 °C for 4 days. After cooling to room temperature, the solution was washed with
diethylether (20 mL), and the aqueous layer was evaporated under reduced pressure to give 2
in the form of its trishydrochloride salt as a white solid (515 mg, 100%). IR
3009, 1723br, 1591, 1572, 1510. 1H NMR (300 MHz; DMSO-d6):
3.53 (s, 4H), 3.55 (s, 4H),
ν
/cm-1 3385, 3185,
δ
4.08 (s, 2H), 4.13 (s, 2H), 7.65 (dd, J = 7.7, 1.1 Hz, 1H), 7.96 (t, J = 7.7 Hz, 1H), 8.05 (d, J =
13
1.5 Hz, 1H), 8.26 (dd, J = 7.7, 1.1 Hz, 1H), 8.66 (d, J = 1.5 Hz, 1H), 12.60 (br s, 4H).
NMR (75 MHz; DMSO-d6):
C
δ
54.3 (CH2), 54.4 (CH2), 59.1 (CH2), 59.5 (CH2), 118.0 (CH),
119.1 (CH), 122.1 (CH), 123.4 (CH), 137.9 (CH), 140.0 (Cq), 153.8 (Cq), 155.6 (Cq), 159.2
(Cq), 160.3 (Cq), 166.4 (CO), 172.4 (CO), 172.45 (CO). ES+/MS: m/z 491.4 ([M+H]+, 100%),
513.4 ([M+Na]+, 75%), 529.3 ([M+K]+, 65%). ES+/HRMS Calcd for C21H23N4O10 [M+H]+:
m/z = 491.1414, found: m/z = 491.1421. UV/Vis λmax (50 mM Tris buffer, pH 7.4)/nm: 238
(ε/dm3 mol-1 cm-1 9000), 301 (8800). Anal. Found: C, 39.37; H, 4.64; N, 8.74. Calcd for
.
C21H22N4O10 3HCl 2H2O: C, 39.67; H, 4.60; N, 8.81.
.
Ln-BPMNTA chelates. General procedure: To a solution of compound 2 in H2O was added
LnCl3,6H2O (1.1 equiv.). After stirring at room temperature for 1h, pH was adjusted to 5-6
with NaOH 0.1M and the mixture was then stirred overnight at room temperature. The solvent
was evaporated to a minimum and the solution was loaded on a Waters Sep-Pak column (C18,
10 g). Column was rinsed with 5x8 mL of H2O to remove salts and the product was eluted
19

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Page 20 of 37
with a H2O/MeOH mixture (1/1, 5x1 mL). The solvents were removed in vacuo to givVeiewthAreticle Online
DOI: 10.1039/C6DT00405A
expected complexes with quantitative yields. The absence of free Ln(III) ion in these
complexes was verified using a classic test with an arsenazo indicator solution.
Eu-BPMNTA complex. HPLC analysis (system A); tR = 4.7 min. ES-/MS: m/z 639.2 ([M-
2Na+H]-, 100%). UV/Vis λmax (50 mM Tris buffer, pH 7.4)/nm: 246 (ε/dm3 mol-1 cm-110500),
316 (12200). Luminescence λem (50 mM Tris buffer, pH 7.4; λexc = 316 nm)/nm: 580 (relative
intensity, corrected spectrum 2), 592 (32), 617 (100), 652 (5), 676-714 (70).
Tb-BPMNTA complex. HPLC analysis (system A); tR = 4.7 min. ES-/MS: m/z 645.2 ([M-
2Na+H]-, 100%). UV/Vis λmax (50 mM Tris buffer, pH 7.4)/nm: 246 (ε/dm3 mol-1 cm-1 9600),
316 (12100). Luminescence λem (50 mM Tris buffer, pH 7.4; λexc = 316 nm)/nm: 490 (relative
intensity, corrected spectrum 43), 544 (100), 584 (33), 623 (15), 636-693 (7).
Gd-BPMNTA complex. HPLC analysis (system A); tR = 4.7 min. ES-/MS: m/z 644.0 ([M-
2Na+H]-, 100%). ES-/HRMS Calcd for C21H18N4O10Gd [M-2Na+H]-: m/z = 644.0269, found:
m/z = 644.0265. UV/Vis λmax (50 mM Tris buffer, pH 7.4)/nm: 246 (ε/dm3 mol-1 cm-1 9900),
316 (12100).
2,2′,2′′,2′′′-{[4-(methoxycarbonyl)-2,2′-bipyridine-6,6′-diyl]bis(methylenenitrilo)}-
tetrakis (acetic acid) (3). To a solution of pentaester 1 (175 mg, 0.24 mmol) in CH2Cl2 (7
mL) was added 7 mL of CF3CO2H, and the resulting solution was stirred at rt for 18 h. The
reaction mixture was evaporated to dryness, and the residue was taken up in 2 mL of MeOH.
14 mL of Et2O were added, resulting in the precipitation of a fine white solid, which was
collected by filtration, washed with Et2O, and dried, to yield tetraacid 3 as a pale grey solid
1
(105 mg, 87%). mp ≈ 180 ºC (dec.). IR
DMSO-d6):
7.96 (m, 1H), 8.07 (s, 1H), 8.27 (d, J = 6.5 Hz, 1H), 8.66 (s, 1H). 13C NMR (75 MHz; DMSO-
d6): 52.8 (CH3), 54.35 (CH2), 54.4 (CH2), 59.0 (CH2), 59.3 (CH2), 117.6 (CH), 119.1 (CH),
ν
/cm-1 3436, 1728, 1635, 1405. H NMR (300 MHz;
δ
3.53 (s, 8H), 3.94 (s, 3H), 4.08 (s, 2H), 4.13 (s, 2H), 7.64 (d, J = 6.8 Hz, 1H),
δ
121.8 (CH), 123.4 (CH), 137.9 (CH), 138.6 (Cq), 153.5 (Cq), 155.6 (Cq), 159.1 (Cq), 160.5
(Cq), 165.3 (CO), 172.4 (CO). ES+/MS: m/z 505.1 ([M+H]+, 100%). ES-/HRMS Calcd for
C22H23N4O10 [M-H]-: m/z = 503.1414, found: m/z = 503.1418.
Tetra (tert-butyl) 2,2′,2′′,2′′′-[(4-carboxy-2,2′-bipyridine-6,6′-diyl)bis(methylenenitrilo)]-
tetrakis (acetate) (4). To a solution of pentaester 1 (59 mg, 0.08 mmol) in THF (1 mL) was
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Dalton Transactions
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added lithium hydroxide monohydrate (4 mg, 0.09 mmol) in H2O (1 mL). The DmOiI:x1t0u.1r0e39/wC6aDsT00405A
vigorously stirred at rt for 2 h and then H2O (10 mL) and CH2Cl2 (10 mL) were added. The
pH of the aqueous phase was adjusted to 6 with HCl 1M. The organic phase was dried
(MgSO4) and the solvent was removed under reduced pressure. The crude product was
purified by chromatography over silica gel (CH2Cl2/MeOH 97:3) to give tetraester 4 as a
colourless oil (51 mg, 89%). Rf (silicagel, CH2Cl2/MeOH 90:10): 0.3. IR
1732, 1613, 1574, 1456. 1H NMR (300 MHz; CDCl3):
1.39 (s, 18H), 1.40 (s, 18H), 3.39 (s,
4H), 3.47 (s, 4H), 3.92 (s, 2H), 4.07 (s, 2H), 7.20 (d, J = 7.5 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H),
7.89 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 8.50 (s, 1H). 13C NMR (75 MHz; CDCl3):
28.0
ν
/cm-1 2978, 2931,
δ
δ
(CH3), 56.6 (CH2), 57.0 (CH2), 60.2 (CH2), 60.7 (CH2), 82.1 (Cq), 82.2 (Cq), 120.5 (CH),
121.0 (CH), 122.8 (CH), 124.0 (CH), 138.2 (CH), 151.2 (Cq), 153.9 (Cq), 155.8 (Cq), 156.1
(Cq), 156.7 (Cq), 168.6 (CO), 171.0 (CO), 171.4 (CO). ES+/MS: m/z 715.6 ([M+H]+, 100%),
737.6 ([M+Na]+, 15%). ES+/HRMS Calcd for C37H55N4O10 [M+H]+: m/z = 715.3918, found:
m/z = 715.3928.
Tetra
(tert-butyl)
2,2′,2′′,2′′′-{[4-(pyridin-4-ylmethylcarbamoyl)-2,2′-bipyridine-6,6′-
diyl]bis(methylenenitrilo)}-tetrakis (acetate) (5). To a solution of carboxylic acid compound
4 (230 mg, 0.322 mmol) in CH2Cl2 (18 mL) were added PyBoP (276 mg, 0.530 mmol), N,N-
diisopropylethylamine (208 mg, 1.61 mmol) and 4-(aminomethyl)pyridine (45.3 mg, 0.419
mmol). The mixture was stirred at rt for 48 h, washed with 0.1N HCl aqueous solution (3 × 5
mL), then 0.1N NaOH aqueous solution (3 × 5 mL) and water (3 × 5 mL). The organic layer
was dried over MgSO4 and evaporated under reduced pressure. The oily residue was purified
by column chromatography over alumina (petroleum ether/ethyl acetate 70:30) to give 5 as a
white solid (204 mg, 79%). UPLC analysis: tr = 4.93 min. 1H NMR (300 MHz; CDCl3):
δ 1.36
(s, 36H), 3.44 (s, 8H), 4.00 (s, 2H), 4.10 (s, 2H), 4.62 (d, J = 5.9 Hz, 2H), 7.21 (d, J = 6.0 Hz,
2H), 7.47 (dd, J = 7.8, 0.5 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H), 8.07 (d, J = 1.4 Hz, 1H), 8.13 (t, J
= 5.9 Hz, 1H), 8.24 (dd, J = 7.8, 0.5 Hz, 1H), 8.43 (d, J = 6.0 Hz, 2H), 8.71 (d, J = 1.4 Hz, 1H).
13C NMR (75 MHz; CDCl3):
δ 27.93 (CH3), 27.94 (CH3), 42.5 (CH2), 55.74 (CH2), 55.77
(CH2), 59.1 (CH2), 59.6 (CH2), 80.9 (Cq), 81.0 (Cq), 116.6 (CH), 119.4 (CH), 120.7 (CH),
122.3 (CH), 123.4 (CH), 137.3 (CH), 142.2 (Cq), 147.4 (Cq), 149.5 (CH), 154.5 (Cq), 156.2
(Cq), 157.8 (Cq), 159.8 (Cq), 165.9 (CO), 170.4 (CO), 170.6 (CO). ES+/MS: m/z 805.5
([M+H]+, 100%), 827.5 ([M+Na]+, 15%). ES+/HRMS Calcd for C43H61N6O9 [M+H]+: m/z =
805.4500, found: m/z = 805.4509; Calcd for C43H60N6O9Na [M+Na]+: m/z = 827.4319, found:
21

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m/z = 827.4327. Anal. Found: C, 63.91; H, 7.47; N, 10.12. Calcd for C43H60N6O9: C, 64.16; H,
DOI: 10.1039/C6DT00405A
7.51; N, 10.44.
Tetra
(tert-butyl)
2,2′,2′′,2′′′-{[4-(but-3-yn-1-ylcarbamoyl)-2,2′-bipyridine-6,6′-
diyl]bis(methylenenitrilo)} -tetrakis (acetate) (6). To a stirred solution of carboxylic acid
compound 4 (260 mg, 0.364 mmol) and but-3-yn-1-amine hydrochloride53 (50 mg, 0.476
mmol) in CH2Cl2 (20 mL) were added, at rt under argon, diisopropylethylamine (236 mg, 1.83
mmol) and PyBOP (312 mg, 0.600 mmol). The resulting solution was stirred at rt for 24 h,
then sequentially washed with aq. HCl 0.1M (3×20 mL), aq. NaOH 1M (2×20 mL) and H2O
(20 mL). The organic layer was dried (Na2SO4), and the solvent was removed under reduced
pressure. The crude product was purified by flash chromatography over alumina (petroleum
ether/ethylacetate 70:30), to give the corresponding acetylenic compound (200 mg, 72%) as a
colorless oil. 1H NMR (300 MHz; CDCl3):
δ 1.46 (s, 18H), 1.47 (s, 18H), 2.06 (t, J = 2.6 Hz,
1H), 2.56 (td, J = 6.8, 2.6 Hz, 2H), 3.52 (s, 8H), 3.65 (q, J = 6.0 Hz, 2H), 4.11 (s, 2H), 4.17 (s,
2H), 7.39 (t, J = 5.9 Hz, 1H), 7.57 (dd, J = 7.7, 1.1 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 8.05 (d,
J = 1.6 Hz, 1H), 8.31 (dd, J = 7.8, 1.1 Hz, 1H), 8.69 (d, J = 1.6 Hz, 1H). 13C NMR (75 MHz;
CDCl3):
δ 19.5 (CH2), 28.26 (CH3), 28.29 (CH3), 38.9 (CH2), 55.97 (CH2), 56.03 (CH2), 59.6
(CH2), 59.8 (CH2), 70.2 (CH), 81.2 (Cq), 81.3 (Cq), 81.5 (Cq), 116.8 (CH), 119.7 (CH), 120.8
(CH), 123.6 (CH), 137.6 (CH), 143.1 (Cq), 154.9 (Cq), 156.5 (Cq), 158.3 (Cq), 160.0 (Cq),
166.1 (CO), 170.7 (CO), 170.8 (CO). ES+/MS: m/z 766.2 ([M+H]+, 20%), 788.1 ([M+Na]+,
100%).
Tetra
(tert-butyl)
2,2′,2′′,2′′′-{[4-(-2-aminoethylcarbamoyl)-2,2′-bipyridine-6,6′-
diyl]bis(methylenenitrilo)} -tetrakis (acetate) (7). To ethylenediamine (140 mg, 2.33 mmol)
was added pentaester 1 (52 mg, 0.071 mmol) and the mixture was stirred at rt for 24 h.
Elimination of diamine under reduced pressure afforded compound 7 as a tan oil (53 mg,
1
100%). IR
CDCl3):
ν
/cm-1 3364, 3300, 3065, 2978, 2932, 1733, 1661, 1558. H NMR (300 MHz;
δ
1.43 (s, 36H), 2.94 (m, 3H), 3.48 (s, 4H), 3.49 (s, 4H), 3.54 (m, 2H), 4.09 (s, 2H),
4.13 (s, 2H), 7.51 (d, J = 7.7 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 8.05 (s ,1H), 8.28 (d, J = 7.7Hz,
13
1H), 8.70 (s, 1H). C NMR (75 MHz; CDCl3):
δ 28.10 (CH3), 28.15 (CH3), 41.4 (CH2), 42.9
(CH2), 55.7 (CH2), 55.9 (CH2), 59.5 (CH2), 59.7 (CH2), 81.1 (Cq), 81.2 (Cq), 116.9 (CH),
119.7 (CH), 120.8 (CH), 123.5 (CH), 137.4 (CH), 143.1 (Cq), 155.0 (Cq), 156.2 (Cq), 158.1
(Cq), 159.7 (Cq), 166.2 (CO), 170.6 (CO), 170.7 (CO). ES+/MS: m/z 757.8 ([M+H]+, 100%),
22

Page 23 of 37
Dalton Transactions
+
+
+
View Article Online
779.8 ([M+Na] , 72%). ES /HRMS Calcd for C39H61N6O9 [M+H] : m/z = 757D.4O5I:0100.1,03f9o/Cu6nDdT:00405A
m/z = 757.4503.
4-Carbomethoxy-2,2’-bipyridine (8). To a solution of methyl 2-bromopyridine-4-
carboxylate (317 mg, 1.47 mmol) in anhydrous toluene (15 mL) were added Pd(PPh3)4 (342
mg, 0.30 mmol), CuBr (34 mg, 0.24 mmol) and 2-(tributylstannyl)pyridine (654 mg, 1.78
mmol). The reaction mixture was then degassed in vacuo and heated at 140°C under
microwave irradiation (260 W) for 2 hours. The solution was filtered through a pad of Celite,
concentrated and loaded on a alumina column (eluent: petroleum ether/CH2Cl2 50:50) to
provide 8 as a white solid (227 mg, 72%). mp 82-83°C. Rf (alumina, petroleum ether/CH2Cl2
50:50): 0.38. IR ν δ 3.99 (s, 3H), 7.34 (ddd, J = 1.2,
/cm-1 1723. 1H NMR (300 MHz; CDCl3):
4.8, 7.5 Hz, 1H), 7.84 (td, J = 1.6, 8.0 Hz, 1H), 7.86 (dd, J = 1.6, 5.0 Hz, 1H), 8.42 (td, J = 1.0,
8.0 Hz, 1H), 8.72 (ddd, J = 0.9, 1.8, 4.8 Hz, 1H), 8.82 (dd, J = 0.8, 5.0 Hz, 1H), 8.94 (dd, J =
13
0.9, 1.6 Hz, 1H). C NMR (75 MHz; CDCl3):
δ 52.6 (CH3), 120.4 (CH), 121.2 (CH), 122.8
(CH), 124.1 (CH), 137.1 (CH), 138.4 (Cq), 149.3 (CH), 149.9 (CH), 155.3 (Cq), 157.3 (Cq),
165.7 (CO).
(bpy-COOMe)Re(CO)3Cl (9). To a solution of 8 (50 mg, 0.233 mmol) in MeOH (10 mL)
was added Re(CO)5Cl (101.3 mg, 0.28 mmol) and the reaction mixture was refluxed
overnight at 65°C. After cooling down to room temperature, the solvent was evaporated to the
third and the resulting precipitate was filtered and washed with cold MeOH to obtain 9 as a
yellow solid (110 mg, 91%). IR
4.07 (s, 3H), 7.60 (ddd, J = 1.3, 5.5, 7.7 Hz, 1H), 8.04 (dd, J = 1.6, 5.7 Hz, 1H), 8.13 (ddd, J
ν
/cm-1 2018, 1914, 1877, 1729. 1H NMR (300 MHz; CDCl3):
δ
= 1.6, 7.7, 8.2 Hz, 1H), 8.34 (ddd, J = 0.7, 1.2, 8.3 Hz, 1H), 8.73 (dd, J = 0.8, 1.7 Hz, 1H),
9.09 (ddd, J = 0.8, 1.6, 5.5 Hz, 1H), 9.21 (dd, J = 0.8, 5.7 Hz, 1H). 13C NMR (125 MHz;
CDCl3):
δ 53.7 (CH3), 122.6 (CH), 123.6 (CH), 126.2 (CH), 127.6 (CH), 139.1 (CH), 139.8
≡
(Cq), 153.3 (CH), 154.0 (CH), 155.0 (Cq), 156.8 (Cq), 163.4 (C=O), 188.8 (C O), 196.7
+
≡
≡
(C O), 196.8 (C O). DCI-NH3/MS: m/z 537.9 ([M+NH4] , 100%). DCI-CH4/HRMS Calcd
for C15H10ClN2O5187Re: m/z = 519.9827, found: m/z = 519.9822. UV/Vis λmax (CH3CN)/nm:
241 (ε/dm3 mol-1 cm-1 20000), 303 (13000), 392 (3800). Fluorescence λem (CH3CN; λexc = 392
nm)/nm: 652.
23

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Page 24 of 37
[(bpy-COOMe)Re(CO)3(CH3CN)][OTf] (10). To a solution of 9 (50 mg, 96.2 µmol)Viienw A1rti0cle Online
DOI: 10.1039/C6DT00405A
mL of fresh distilled acetonitrile in the dark, under N2, was added a solution of silver
trifluoromethanesulfonate (29.5 mg, 0.115 mmol) in 1 mL of THF and the mixture was
allowed to reflux overnight. The reaction mixture was filtered through a pad of Celite to
remove any residual AgCl. All volatiles were removed under reduced pressure and the
recrystallization was performed by layering Et2O on a CH2Cl2 solution of the crude product at -
18°C yielding to the desired product 10 as yellow needles (65 mg, 100%). IR
1924 (br), 1734. 1H NMR (300 MHz; CDCl3):
2.26 (s, 3H), 4.07 (s, 3H), 7.71 (ddd, J = 1.1,
ν
/cm-1 2039,
δ
5.5, 7.6 Hz, 1H), 8.15 (dd, J = 1.6, 5.7, 1H), ), 8.30 (td, J = 1.5, 8.0 Hz, 1H), 8.66 (d, J = 8.2
Hz, 1H), 8.96 (dd, J = 0.8, 5.5 Hz, 1H), 8.99 (d, J = 0.9 Hz, 1H), 9.08 (dd, J = 0.5, 5.7 Hz, 1H).
13
δ -78.3. C NMR (75 MHz; CDCl3): δ 3.7 (CH3), 53.7 (CH3),
19F NMR (300 MHz; CDCl3):
120.3 (q, CF3, J = 319 Hz), 122.7 (CN), 124.5 (CH), 125.7 (CH), 127.3 (CH), 128.7 (CH),
141.3 (CH), 141.4 (Cq), 153.2 (CH), 154.0 (CH), 155.4 (Cq), 157.4 (Cq), 163.3 (C=O), 189.5
185
≡
≡
≡
(C O), 193.0 (C O), 195.3 (C O). DCI-CH4/HRMS Calcd for C16H10F3N2O8S Re [M-
CH3CN]+: m/z = 631.9640, found: m/z = 631.9697. Anal. Found: C, 32.17; H, 2.04; N, 6.10.
Calcd for C18H13F3N3O8SRe: C, 32.05; H, 1.94; N, 6.23. UV/Vis λmax (CH3CN)/nm: 248
(ε/dm3 mol-1 cm-1 18000), 275 (13000), 317 (11400), 328 (11600), 359 (4600). Fluorescence
λem (CH3CN; λexc = 359 nm)/nm: 583.
[(bpy-COOMe)Re(CO)3(ligand 5)][OTf] (11). To a solution of 10 (34 mg, 50.3 µmol) in
THF (4 mL) in a microwave reactor was added 5 (100 mg, 124 µmol) and the mixture was
heated at 105°C under microwave irradiation (155 W) for one hour. The solvent was removed
in vacuo and the crude product was purified by chromatography over alumina (CH2Cl2 to
CH2Cl2 /MeOH 95:05) to provide 11 as a yellow thick oil (41 mg, 56 %). UPLC analysis: tR =
5.07 min. IR ν δ 1.43 (s,
/cm-1 2032, 1926 (br), 1734, 1690 (br). 1H NMR (300 MHz; CDCl3):
18H), 1.44 (s, 18H), 3.51 (s, 4H), 3.52 (s, 4H), 4.04 (s, 3H), 4.10 (s, 2H), 4.13 (s, 2H), 4.59
(bs, 2H), 7.35 (d, J = 6.3 Hz, 2H), 7.58 (d, J = 7.5 Hz, 1H), 7.73 – 7.83 (m, 2H), 7.99 – 8.05
(m, 3H), 8.22 (dd, J = 1.5, 5.7 Hz, 1H), 8.27 (d, J = 7.5 Hz, 1H), 8.32 (d, J = 7.8 Hz, 1H),
8.63 (d, J = 8.1 Hz, 1H), 8.70 (d, J =1.3 Hz, 1H), 8.94 (d, J = 0.7 Hz, 1H), 9.11 (dd, J = 0.8,
19
13
5.4 Hz, 1H), 9.23 (d, J = 5.4 Hz, 1H). F NMR (300 MHz; CDCl3) δ -78.3. C NMR (125
MHz; CDCl3): δ 28.13 (CH3), 28.14 (CH3), 42.3 (CH2), 53.8 (CH3), 55.7 (CH2), 55.8 (CH2),
59.43 (CH2), 59.45 (CH2), 81.1 (Cq), 81.3 (Cq), 117.3 (CH), 119.9 (CH), 120.5 (q, CF3, J =
319 Hz), 120.9 (CH), 122.7 (CH), 123.4 (CH), 124.8 (CH), 126.0 (CH), 126.1 (CH), 127.8
24

Page 25 of 37
Dalton Transactions
(CH), 129.2 (CH), 137.8 (CH), 141.5 (CH), 141.6 (Cq), 151.1(CH), 153.2 (Cq),D1O5I:310.4.10(3VC9ie/CwH6A)Drt,Tic0le0O4n0li5nAe
155.2 (Cq), 157.0 (Cq), 159.78 (Cq), 159.80 (Cq), 162.9 (Cq), 165.4 (CO), 166.4 (CO), 170.4
+
≡
≡
≡
(CO), 170.6 (CO), 190.4 (C O), 195.2 (C O), 195.3 (C O). ES /HRMS Calcd for
C58H70N8O14185Re [M-CF3SO3]+: m/z = 1287.4541, found: m/z = 1287.4591. UV/Vis λmax
(CH3CN)/nm: 245 (ε/dm3 mol-1 cm-1 17500), 303 (12000), 366 (3200). Fluorescence λem
(CH3CN; λexc = 366 nm)/nm: 580.
[(Bpy-COOMe)Re(CO)3(pyBPMNTA)][OTf] (12). To a stirred solution of complex 11 (40
mg, 27.8 µmol) in CH2Cl2 (4 mL) was added TFA (0.640 mL, 8.34 mmol) at 0°C. The
mixture was then stirred 24h at room temperature. The solvent was co-evaporated several
times in vacuo with CH3CN to give 12 as a yellow powder (34 mg, 100 %). UPLC analysis: tR
= 1.35 min. 1H NMR (400 MHz; D2O):
δ 3.32-4.00 (m, 15H), 4.48 (s, 2H), 7.23 (d, J = 5.8 Hz,
2H), 7.43 (d, J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.70-7.73 (m, 1H), 7.93-7.97 (m, 1H), 8.09 (d, J =
5.6 Hz, 1H), 8.17 (d, J = 6.0 Hz, 3H), 8.32-8.45 (m, 2H), 8.69 (s, 1H), 8.77 (s, 1H), 9.23 (d, J
= 5.4 Hz, 1H), 9.38 (d, J = 5.6 Hz, 1H). ES+/HRMS Calcd for C42H38N8O14185Re [M-
CF3SO3]+: m/z = 1063.2037, found: m/z = 1063.2039. UV/Vis λmax (50 mM Tris buffer, pH
7.4)/nm: 244 (ε/dm3 mol-1 cm-1 16400), 304 (10900), 368 (2500). Fluorescence λem (50 mM
Tris buffer, pH 7.4) λexc = 368 nm)/nm: 600.
Dinuclear Re/Gd complex 13. For preparation, see general procedure for Ln-BPMNTA
complexes. Yield 36 %. The absence of free Gd(III) ion in 13 was verified using a classic test
with an arsenazo indicator solution. HPLC analysis (system B); tR = 6.9 min. UPLC analysis:
tR = 1.59 min. IR
ν
/cm-1 2027, 1915, 1686, 1614, 1416, 1203. ES+/HRMS Calcd for
C42H35N8O14ReGd [M+H]+: m/z = 1218.1058 and 1220.1080, found: m/z = 1218.1069 and
1220.1099, respectively. UV/Vis λmax (50 mM Tris buffer, pH 7.4))/nm: 247 (ε/dm3 mol-1 cm-
1
15800), 317 (11600), 367 (2400). Fluorescence λem (50 mM Tris buffer, pH 7.4) λexc = 367
nm)/nm: 588.
Acknowledgements
The contributions of Mélanie Godeau to photophysical study of Ln-BPMNTA chelates and
Sophie Laine to the structural determination of some Re complexes are gratefully
acknowledged. We are grateful to Prof. R. N. Muller for his interest and support for this
25

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current scientific work. This work was supported by the French Ministère de lDaORI: 1e0c.1h03e9r/cCh6DeT00405A
(Ph.D. grant for A.B.) and the CNRS. S.L., R.N.M. and L.V.E. thank the FNRS (Fonds
National de la recherche scientifique), and ARC Programs (AUWB-2010-10/15-UMONS-5)
of the French Community of Belgium, the UIAP VII program of Belgium and the Center for
Microscopy and Molecular Imaging (CMMI, supported by the European Regional
Development Fund and the Walloon Region).
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