Lanthanide complexes of DOTA-nitroxide conjugates for redox imaging: spectroelectrochemistry, CEST, relaxivity, and cytotoxicity

Article abstract of DOI:10.1039/d1dt01628h

The lanthanide(iii) complexes (Gd, Eu, Dy, and Yb) of DOTA tris(amide) and bis(amide) derivatives (L₁andL₂) featuring one redox active TEMPO arm were prepared. LigandL₂harbours an alkyne fragment for further functionalization. The X-ray crystal structure of ligandL₂in complexation with Na⁺was solved. The complexes showed in their CV one oxidation wave (0.26-0. 34 Vvs.Fc⁺/Fc) due to an oxoammonium/nitroxide redox couple and a broad reduction corresponding to the nitroxide/hydroxylamine system. The Eu complexes demonstrated the presence of one water molecule in their coordination sphere. The nitroxide complexes were characterized by EPR spectroscopy, showing the typical 3-line pattern in the high temperature regime, which is quenched upon the addition of ascorbate (reduction into hydroxylamine). In their nitroxide form, the complexes show essentially no CEST peak. Conversely, the reduced complexes demonstrate a 12% CEST peak at 51 ppm, corresponding to the metal bound water molecule. Fast exchange precluded the CEST activity for the amide protons. All the complexes proved to be essentially non-toxic for M21 cells at concentrations up to 50 μM.

Full text of DOI:10.1039/d1dt01628h

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Lanthanide complexes of DOTA–nitroxide conjugates
for redox imaging: spectroelectrochemistry, CEST,
relaxivity, and cytotoxicity†
Cite this: Dalton Trans., 2021, 50,
10826
D. Mouchel Dit Leguerrier,a R. Barré,a Q. Ruet,b D. Imbert, c C. Philouze,a
a
P. H. Fries,c V. Martel-Frachet,b,d J. K. Molloy *a and F. Thomas
*
The lanthanide(III) complexes (Gd, Eu, Dy, and Yb) of DOTA tris(amide) and bis(amide) derivatives (L1 and
L2) featuring one redox active TEMPO arm were prepared. Ligand L2 harbours an alkyne fragment for
further functionalization. The X-ray crystal structure of ligand L2 in complexation with Na+ was solved.
The complexes showed in their CV one oxidation wave (0.26–0. 34 V vs. Fc+/Fc) due to an oxoammo-
nium/nitroxide redox couple and a broad reduction corresponding to the nitroxide/hydroxylamine
system. The Eu complexes demonstrated the presence of one water molecule in their coordination
sphere. The nitroxide complexes were characterized by EPR spectroscopy, showing the typical 3-line
pattern in the high temperature regime, which is quenched upon the addition of ascorbate (reduction
into hydroxylamine). In their nitroxide form, the complexes show essentially no CEST peak. Conversely,
the reduced complexes demonstrate a 12% CEST peak at 51 ppm, corresponding to the metal bound
water molecule. Fast exchange precluded the CEST activity for the amide protons. All the complexes
proved to be essentially non-toxic for M21 cells at concentrations up to 50 μM.
Received 19th May 2021,
Accepted 25th June 2021
DOI: 10.1039/d1dt01628h
rsc.li/dalton
redox environment of the extracellular space is dominated by
the cysteine disulfide/sulfhydryl redox couple CySSCy/CysSH,
1. Introduction
Medical imaging is an extremely important domain for current with higher concentrations (0.05–0.3 mM) than those of the
research due to the necessity of precocious detection, follow- GSSG/GSH couple (0.002–0.02 mM) and with less negative
up, and treatment of dangerous illnesses.1 In particular, mole- redox potential (−80 to −20 mV) than that of GSSG/GSH,
cular imaging can provide the details of biological processes giving a more oxidized compartment than the cytosol.4 The
in tissues of the human body, giving more information for the conditions of oxidative stress are encountered when the
diagnosis of diseased tissues.2 The development of molecular natural metabolism of oxygen is disrupted, leading to the over-
imaging requires the development of novel imaging probes production of reactive oxygen species (ROS) by incomplete
capable of giving an effective response to a stimulus. The reduction.5 Changes in the redox status (oxidative stress or
redox status of a tissue is an important marker of its activity. hypoxia) are believed to contribute to cellular signalling (differ-
The redox potential is defined by the sum of the redox couples entiation and apoptosis)6 but also the proliferation of diseases
weighted with the concentration of the respective reduced such as cardiovascular diseases,7,8 cancers9,10 or neurodegen-
species. The intracellular medium is buffered by several redox erative diseases.11
couples of which glutathione disulfide/sulfhydryl GSSG/GSH is
Among various non-invasive detection techniques, mag-
the main one (a redox potential of −120 to −70 mV).3 The netic resonance imaging (MRI) has proved to be a key tech-
nique for clinical imaging. It relies on the differential relax-
ation of protons of the bulk water in the human body.12 Its
success lies in its high spatial resolution, lack of hazardous
radiation, and the fact that it is painless and can be functional
in addition to being anatomical. Approximately one third of
the MRI scans are realized with a relaxation contrast agent
(CA), which is often a gadolinium complex. GdIII is the metal
ion of choice due to its quenched total electronic orbital
angular momentum L = 0, the highest possible total electronic
spin value S = 7/2, and slow longitudinal electronic relaxation,
aUniv. Grenoble Alpes, CNRS, DCM, 38000 Grenoble, France.
E-mail: jennifer.molloy@univ-grenoble-alpes.fr, fabrice.thomas@univ-grenoble-alpes.fr
bInstitute for Advanced Biosciences, INSERM U1209, UMR CNRS 5309, Grenoble
Alpes University, 38700 La Tronche, France
cUniv. Grenoble Alpes, CEA, CNRS, IRIG-LCBM, 38000 Grenoble, France
dEPHE, PSL Research University, 75014 Paris, France
†Electronic supplementary information (ESI) available. CCDC 1997062. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
d1dt01628h
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especially under a rather high magnetic field B0 ≥ 1 T of the
MRI scanners.13–15 The CA is injected into a vein before the
analysis to improve the details and clarity of the images. The
CA effectiveness is gauged by its relaxivity r1, which is the
increase in the longitudinal relaxation rate of the water
protons (hydrogen nuclei) per mM of the contrast agent in bio-
logical tissues.11,14 It was suggested almost ten years ago that
lanthanide-based CAs could also be used to sense the redox
environment.16,17 Two main strategies emerged that are based
on either a redox-reactive ligand or redox-active metal. In the
first case, the r1 value of the GdIII-based CA can be tuned by
the hydration state (the number of coordinated water mole-
cules q), which is itself modulated by a redox event at the
ligand. This first strategy is illustrated by DOTA-based Gd3+
complexes (DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid) appended by a merocyanine group. This group
cyclizes into spirooxazine upon reduction by NADH, resulting
in decoordination and further increase of q from 1 to 2 with
the change in r1.18–20 Several Gd3+ complexes functionalized by
a thiol-reactive 2-pyridyldithio group were also prepared and
shown to react with GSH with a change in the q number from
2 to 1.21,22 Alternatively, the slower rotational dynamics upon
the redox reaction of the ligand by reversible (or irreversible)
binding of low molecular weight Gd3+ complexes to macro-
molecules can be used to increase the r1 values at constant q.
Some Gd3+–DOTA complexes appended by terminal thiol
groups demonstrated such behaviour, whereby they react with
the Cys-34 residue of human serum albumin.23
The second strategy is exemplified by the cryptates of euro-
pium, the only lanthanide susceptible to adopting two
different oxidation states in vivo.24 The Eu2+ ion, isoelectronic
to Gd3+, is oxidized in vivo into Eu3+, which shows negligible
paramagnetic relaxation because of its very fast electronic
relaxation.25–27
Paramagnetic Chemical Exchange Saturation Transfer
(PARACEST) is an attractive contrast mechanism for MRI,
which often employs paramagnetic lanthanide complexes.29
Recently, this technique based on paramagnetic lanthanide
ions such as EuIII, YbIII, and DyIII which display very fast elec-
tronic relaxation thus lends itself to more sensitive specific
functional imaging. A pre-saturation pulse is applied to
exchangeable protons within the complex, resulting in a trans-
fer of saturated spins to the bulk. As a consequence, the water
signal intensity decreases and negative contrast is observed.
Chart 1 Structures of ligands whose Eu3+ complexes were reported as
redox-responsive PARACEST agents.28
Chart 2 Structures of ligands L1 and L2.
We recently reported LnIII complexes that are sensitive to
environmental redox changes. We monitored the changes in
the oxidation state by luminescence, wherein over 90% switch
could be achieved.34–36 We have also demonstrated through
EPR the spin trap of a major ROS, the hydroxyl radicals, by
lanthanide complexes of a DOTA-hydroxylamine derivative.37
Following the elegant strategy developed by Sherry et al. to
design PARACEST agents responsive to redox changes,32 we
herein report a series of lanthanide(III) complexes (Gd, Eu, Dy,
and Yb) based on a cyclen scaffold containing the redox active
TEMPO arm38 (Chart 2). Two different derivatives L1 and L2
have been designed containing three acetamides or with an
alkyne. The alkyne was chosen in order to allow future
functionalization and bioconjugation of these complexes via
click chemistry. We investigate their photophysical, redox and
relaxometry properties and their potential as redox-responsive
PARACEST agents.
2. Results and discussion
2.1 Synthesis
Very few lanthanide redox-responding complexes have been The synthesis of ligands L1 and L2 was performed using the
reported to date for PARACEST imaging.16 All are based on the successive functionalization of the cyclen macrocycle
Eu3+ complexes of DOTA tetraamide ligands as this lanthanide (Scheme 1). L1 was synthesised via substitution with the nitrox-
shows the slowest water exchange rates (Chart 1)28 and operate ide acetamide and further functionalization with three bromo-
according to two distinct mechanisms: the ligands can acetamide arms. L2 was synthesised by the protection of the
undergo reduction such as in a nitrobenzene (Chart 1a)30 or cyclen macrocycle in order to selectively functionalise 2 bromo-
quinolinium group,31 resulting in an alteration of the water acetamide arms, deprotection, and followed by the addition of
exchange rate. Alternatively, the nitroxide appended Eu3+– a dilute solution of propargyl bromide to perform the mono
DOTA complex (Chart 1b) exhibits a T1-shortening of bulk substitution. Purification and further addition of the nitroxide
water protons by the paramagnetic centers, which increases acetamide afforded the desired product (see experimental).
upon reduction.32 Interestingly, an Eu3+/Eu2+ switch was
recently proposed as a redox PARACEST agent.33
The GdIII, EuIII, YbIII and DyIII complexes were prepared
from L1 and L2 by reaction with the corresponding lanthanide
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The ligands demonstrate oxidation potentials of 0.25 V and
0.31 V vs. Fc+/Fc (0.88 to 0.94 V vs. NHE),39 respectively, for L1
and L2. The complexes exhibit a reversible oxidation wave in
the range of 0.26–0.34 V vs. Fc+/Fc (Table 1 and Fig. 2), which
is assigned to the oxidation of the TEMPO moiety into the per-
sistent oxoammonium species TEMPO+.40 These values are
slightly more positive than that reported for free TEMPO (0.21
V vs. Fc+/Fc)41 due to the presence of the amide function. An
irreversible reduction wave corresponding to the reduction of
the TEMPO moiety into the hydroxylamine TEMPOH is obser-
vable as a broad feature between −1 and −1.5 V vs. Fc+/Fc
(about −0.3 to −0.9 V vs. NHE, Fig. 2).40 The apparent irreversi-
bility of the redox wave is due to the very slow electron transfer
of the proton-coupled TEMPOH/TEMPO redox couple and an
exceedingly large anodic-to-cathodic peak separation, which
shifts the oxidation peak of TEMPOH above the TEMPO+/
TEMPO wave.40,42
Scheme 1 Synthetic procedures of L1, L2 and their lanthanide com-
plexes. Reaction conditions: (i) chloroacetyl chloride, Et3N, 0 °C to rt,
CH2Cl2, overnight; (ii) (1) 0, THF, 1 day, (2) o-PDA, MeOH, 1 week; (iii)
2-bromoacetamide, K2CO3, KI, MeCN, 50 °C, 5 days; (iv) Ln salt, H2O, pH
7, 50 °C, overnight; (v) (1) 2-bromoacetamide, MeCN, 1 day, (2) o-PDA,
MeOH, overnight, (3) CsCO3, MeOH, overnight; (vi) 2-propargyl
bromide, NaOAc, EtOH, 0 to 50 °C, 4 days; (vii) 0, K2CO3, KI, EtOH,
60 °C, 5 days; (vii) Ln salt, H2O, pH 6.5, 50 °C, 2 days.
Each complex, EuIII, DyIII, GdIII or YbIII, demonstrates
similar features with only a slight shift of the oxidation and
reduction waves, thus confirming that the metal ion had very
little effect on the redox behaviour of the TEMPO group. It
must be stressed that no oxidation or reduction of the LnIII
centre was observed in the investigated potential range.
salt in H2O at pH 7.4 and precipitation of the resulting
complex.
Ligand L2 was crystallised by slow evaporation of water solu-
tion to give single crystals of quality for X-ray diffraction.
Surprisingly, the structural analysis demonstrated the com-
plexation of a Na+ ion into the cyclen cavity, giving the species
[Na(L2)]+, 0.5 Br−, and 0.5 Cl−. The sodium ion is heptacoordi-
nated to four nitrogens of the macrocycle and three oxygens of
the acetamide arms (Fig. 1). The Na–N bond distances range
from 2.527(10) to 2.643(9) Å, while the Na–O bond distances
are between 2.398(3) and 2.436(3) Å. The C2–C3 bond distance
in one pending arm is 1.17(2) Å, which confirms its triple
bond character. The N–O bond distance in the opposite
pending arm is 1.289(4) Å, which is reminiscent of the TEMPO
radicals.
Table 1 Electrochemical data of the ligands and complexesa
ox
1=2
ox
1=2
Complex
E
Complex
E
[Gd(L1)]3+
[Eu(L1)]3+
[Dy(L1)]3+
[Yb(L1)]3+
L1
0.26
0.30
0.28
0.32
0.25
[Gd(L2)]3+
[Eu(L2)]3+
[Dy(L2)]3+
[Yb(L2)]3+
L2
0.33
0.34
0.33
0.33
0.31
a In 0.5 mM CH3CN solutions containing TBAP as the supporting elec-
trolyte. All the potential values are given in V and referred to the Fc+/Fc
redox couple. T = 298 K and the scan rate is 0.1 V s−1. Parameters for
ferrocene against the reference used (AgNO3 0.01 M): E1/2 = 0.090 V. In
order to convert the potentials from the Fc+/Fc reference to the NHE,
0.630 V must be added to the listed values.39
2.2 Electrochemistry
Electrochemical investigations were performed by cyclic vol-
tammetry (CV), differential pulse voltammetry (DPV) and rotat-
ing disc electrode (RDE) voltammetry in an acetonitrile solu-
tion containing 0.1 M tetra-n-butylammonium perchlorate
(TBAP) as the supporting electrolyte. All the potentials are
referred to the ferrocenium/ferrocene redox standard.
Fig. 2 Cyclic voltammetry curve of [Eu(L2)]3+ in 0.5 mM CH3CN solu-
tion containing 0.1 M TBAP. Scan rate, 0.1 V s−1; T = 298 K; carbon
electrode.
Fig. 1 ORTEP plot of [Na(L2)]+ (the H atoms are omitted for clarity).
10828 | Dalton Trans., 2021, 50, 10826–10837
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2.3 Absorption and luminescence spectroscopy
occurred via the accumulation of ligand and transfer towards
the EuIII metal.
The UV-vis absorption spectra of the ligands L1 and L2 in
Luminescence measurements were also used to determine
the lifetimes of the complexes via excitation at 396 nm in both
H2O and D2O. For [Eu(L1)]3+, we determined t = 0.43 s in H2O
and t = 1.32 in D2O, representing one water molecule in the
coordination sphere of the LnIII ion. This result is mostly as
expected due to the potential eight coordinate macrocyclic
ligand. Complex [Eu(L2)]3+ demonstrates t = 0.44 s and t = 1.36
s for H2O and D2O, respectively, again representing one co-
ordinated water molecule. Hence, the coordination number
changes upon the substitution of one amide with the alkyne,
but the number of coordinated water remains constant, q = 1.
This water molecule is crucial in the use of these complexes in
imaging.
CH3CN show two main bands at 246 nm (ε = 14 200 M−1 cm−1
)
and 286 nm (345 M−1 cm−1) for L1 and 246 nm (22 530 M−1
cm−1) and 286 nm (1590 M−1 cm−1) for L2.43 Upon complexa-
tion, these bands were slightly red shifted with respect to the
ligand, with the [Gd(L2)]3+ complex showing a λ value of
292 nm (4020 M−1 cm−1) and 363 nm (ε = 1760 M−1 cm−1).
The complexes also possess very weak epsilon absorption
corresponding to the f–f lanthanide transitions (Table 2).
The luminescence spectra were recorded for the europium
complexes since structural information can be extracted from
7
a F0–5 band shape analysis. They were recorded via excitation
of the ligand at 315 nm, providing the expected spectra of the
EuIII ions (Fig. 3). A sharp band is observed at 580 nm for [Eu
5
7
(L1)]3+ that corresponds to the transition D0 → F0. Its sharp-
ness suggests the high symmetry of the coordination sphere of
the complex and the presence of single site symmetry for EuIII.
2.4 Electron paramagnetic resonance spectroscopy
5
7
The D0 → F1 transition is a magnetic dipole transition inde-
pendent of the coordination sphere. The hypersensitive elec-
tric dipole transition 5D0 → 7F2 is most influenced by the local
symmetry. The distinct ratio of the integrated bands corres-
Both the lanthanide ions and the ligands harbour unpaired
electrons and hence can be analysed by electron paramagnetic
resonance (EPR).
We first recorded the fluid solution spectra of the free
ligands L1 and L2 at T = 293 K. They show a typical isotropic
3-line pattern centered around g = 2.006 (Fig. 4). The hyperfine
coupling constant AN is 1.7 mT, typical for TEMPO radicals. In
the absence of metal ions, the broadening of the high field
component is solely due to the Brownian rotational motion of
the radical. In the fast tumbling regime, the rotational corre-
lation time τc can be calculated by using Kivelson’s formula
(eqn (1)), where C is a constant calculated from the principal
values of g and A tensors of the nitroxide radical (6.6 10−10 for
di-tert-butylnitroxide; this value is not expected to change
much between nitroxide radicals),44,45 ΔH(+1) is the peak-to-
peak linewidth of the low field line and h(+1) and h(−1) are the
heights of the low and high field resonances.
ponding to the transitions 5D0
→ →
7F2 over 5D0 7F1 in [Eu
(L1)]3+ and [Eu(L2)]3+ suggests a different coordination environ-
ment. In particular, the higher ratio of [Eu(L2)]3+ (Fig. 3) sup-
ports a lower symmetry around the first coordination sphere.
Finally, the excitation spectra confirmed that the excitation
Table 2 UV/vis data of the nitroxide complexesa
Complex
λ (nm) /ε (M−1 cm−1
)
[Gd(L1)]3+
[Eu(L1)]3+
[Dy(L1)]3+
[Yb(L1)]3+
[Gd(L2)]3+
[Eu(L2)]3+
[Dy(L2)]3+
[Yb(L2)]3+
245 (30 040), 292 (5770), 363 (3000)
245 (26 970), 292 (8110), 363 (4270)
245 (34 190), 292 (3620), 363 (1800)
246 (22 720), 292 (5220), 363 (2700)
292 (4020), 363 (1760)
292 (3900), 363(1860)
288 (7090), 363 (1840)
1=2
τc ¼ CΔH
½ðh =hðꢀ1ÞÞ ꢀ 1ꢁ
ð1Þ
ðþ1Þ
ðþ1Þ
The rotational correlation time τc is 1.6 × 10−10 s for both L1
and L2, in agreement with their low chemical weight and com-
parable hydrodynamic diameters. The spectrum shown in
Fig. 4 was simulated accordingly.
279 (8780), 363 (918)
a In 0.5 mM CH3CN solutions containing TBAP as the supporting elec-
trolyte (0.1 M). T = 298 K. In order to ensure full nitroxide radical for-
mation, the complexes were subjected to electrolysis at a potential
intermediate between the TEMPOH/TEMPO and TEMPO+/TEMPO
redox couples.
The spectra of the gadolinium complexes [Gd(L1)]3+ and
[Gd(L2)]3+ at T = 293 K show noticeable differences. Both
display the sharp 3-line nitroxide pattern (Γ = 0.28 mT) with
weaker intensity compared to the free ligands. In the case of
[Gd(L1)]3+, this 3-line pattern overlaps with an intense and
broad resonance (Γ = 3.24 mT) centered at g = 1.99, which is
assigned to the gadolinium signal (Fig. 4). Upon the addition
of ascorbic acid, the TEMPO unit is reduced to TEMPOH,
resulting in the quenching of the 3-line pattern without
affecting the broad resonance. Strikingly, the gadolinium reso-
nance cannot be observed in the case of [Gd(L2)]3+. This
suggests faster relaxation and hence the distinct influence of
the nitroxide and coordination environment on the gadoli-
nium relaxation rate.
Fig. 3 Luminescence spectra of [Eu(L1)]3+ (left) and [Eu(L2)]3+ (right) in
D2O upon excitation at 395 nm.
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Fig. 5 X-band EPR spectra of [Yb(L1)]3+ in 0.5 mM water solution. Black
lines, experimental spectra; red lines, simulation by using the parameters
(S = 1), |D| = 300 MHz, giso = 2.00. Microwave freq., 9.636 GHz; power,
0.2 mW; mod. amp., 0.4 mT; and freq., 100 kHz.
nature but differs markedly from the 3-line pattern of isolated
nitroxides. The spectrum of the free ligand L1 was indeed
recorded at this low temperature and shows exclusively the an-
isotropic 3-line pattern of the nitroxide radical. Hence this
behaviour strongly suggests that the paramagnetic lanthanide
interacts magnetically with the nitroxide moiety. From the
+
X-ray crystal structure of NaL1 , the N(nitroxide)–metal dis-
Fig. 4 Fluid solution X-band EPR spectra in water solution. (a) 0.5 mM
L1; black lines, experimental spectra; red lines, simulation by using the
parameters given in the text. Microwave freq., 9.42 GHz; power, 3.2 mW;
mod. amp., 0.2 mT; and freq., 100 kHz. (b) 2 mM [Gd(L1)]3+; green line.
2 mM [Gd(L1)]3+ in the presence of 2 molar equiv. of ascorbate; blue
line. Microwave freq., 9.42 GHz; power, 1.7 mW; mod. amp., 0.2 mT, and
freq., 100 kHz.
tance could be estimated at ca. 7.8 Å, confirming that a triplet
state may be accessible. Upon considering the feature at 4 K as
a spin triplet resonance and using a simple model with an iso-
tropic g tensor, the simulation gives |D| = 300 MHz, which is
consistent with the above interspin distance. Note that simu-
lation of this feature by considering an isolated nitroxide an-
isotropic signal leads to unreasonable 14N hyperfine coupling
constants. The addition of ascorbate to [Yb(L1)]3+ results in the
We also investigated the reversibility of the reduction by full quenching of this feature around g = 2, confirming its
adding the oxidant NaIO4 to an ascorbate-reduced sample of assignment. The EPR spectrum of reduced [Yb(L1)]3+ shows
[Gd(L2)]3+ (Fig. S15†). The initial radical signal of [Gd(L2)]3+ resonances at g = 8.6, 6.41 and 5.2, which are assigned to the
was restored to more than 80% after 30 min. The ascorbate- parallel features of an isolated Yb3+ ion (Fig. 6). The main line
reduced sample could be alternatively reoxidized by O2 bub- at g = 6.41 is assigned to the even isotope, while the satellite
bling under basic conditions but the reaction proceeds less ones (approx. 15% in intensity) correspond to the 171Yb3+ and
efficiently (only 20% recovery after 30 min).
173Yb3+ isotopes. The g∥ value of the even isotope compares
Low temperature measurements were conducted (4–50 K) in well with that reported for the [Yb·DTMA·OH2]3+ complex
order to characterize the Yb, Eu and Dy complexes, wherein (6.45),46 congruent with a similar environment of the metal
fast relaxation prevents the observation of the lanthanides at center.
higher temperatures. At T = 40 K, the complexes exhibit the
For [Dy(L1)]3+, the sharp nitroxide lines decrease in inten-
typical signature of immobilized nitroxide radicals, with the sity upon decreasing the temperature, at the expense of a
broadening and shift of the mI = +1 and −1 transitions due to broader signal centered around g = 2 that we assign to the
anisotropy. Accordingly, similar spectra were obtained for the interacting nitroxide, as observed for [Yb(L1)]3+. It is distribu-
free ligands in frozen solutions (see the ESI†). Upon cooling ted over a larger window compared to [Yb(L1)]3+, indicative of a
down to 5 K, drastic changes were evidenced in the EPR distinct influence of the lanthanide ion on the radical moiety.
spectra for [Yb(L1)]3+, [Dy(L1)]3+ and their alkyne counterparts. This is also revealed by the spectrum at T = 8 K, which still
For a given Ln ion, the EPR spectra of the alkyne derivative [Ln contains both the sharp nitroxide lines and the broader line
(L2)]3+ show essentially the same temperature dependence as (see the ESI†). After the addition of ascorbate (reduction of the
[Ln(L1)]3+ and hence will not be commented in detail.
nitroxide unit), the signal at g = 2 completely disappears,
The spectrum of [Yb(L1)]3+ taken at 4 K shows an intense affording a spectrum with an intense resonance at geff ≈ 11.6,
doublet at g = 2.00 (Fig. 5), which corresponds to a radical sig- which is reminiscent of isolated Dy3+ complexes (Fig. 6).
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with the two acetamide arms. Hence this conformation allows
minimization of the steric clash between the coordinating
3+
water molecule and the alkyne group, while stabilizing YL2
through H-bonding interactions. The distinct orientation of
3+
3+
the water hydrogens in YL1 and YL2 prompted us to
perform an additional relaxed PES scan, whereby the water
molecule was rotated along the M–O axis (ϕ angle in Fig. 7).
For YL13+, the variation of energy is monotonic and sinusoidal
with alternating minima and maxima every ca. 120° and a
3+
total change that does not exceed 0.3 kcal mol−1. Model YL2
exhibits a strikingly different behaviour: the total energy
change during the scan is one order of magnitude higher, with
the lowest energy conformation corresponding to the water
hydrogens orthogonal to the alkyne. These results show that
the barrier for the rotation of the water molecule is very small
3+
for YL1 but significantly higher (immobilized) for YL23+. The
functionalization of the macrocycle via an amide linker
(TEMPO arm) hence appears to weakly affect the rotational
dynamic, in contrast to alkyl linkers (alkyne arm), which do
not allow the formation of stabilizing H-bonds with the coordi-
nating water molecule and prevent rotation. Furthermore, the
coordination bonds are shorter in YL23+, which is not surpris-
ing owing to the lower expected coordination number in this
complex.
2.6 CEST activity and relaxivity
Fig. 6 X-band EPR spectra of (a) [Yb(L1)]3+ and (b) [Dy(L1)]3+ in 4 mM
water solution after the addition of 2 equiv. of ascorbate. Black lines,
experimental spectra; red lines, simulation by using the parameters
given in the text. Microwave freq., 9.636 GHz; power, 2 mW; mod. amp.,
1 mT; and freq., 100 kHz. T = 5 K.
The ligand design incorporated exchangeable NH2 protons,
which can confer potential CEST activity. In addition, these
complexes are 8 or 9-coordinate, thus containing a metal
bound water molecule that can confer CEST activity via an
exchangeable H2O on the metal centre. The CEST spectra,
plotted as a percentage decrease in water proton magnetisa-
tion as a function of the presaturation pulse frequency, were
recorded in 0.1 M HEPES buffer in H2O : acetonitrile (2 : 1) at
The EPR spectrum of [Eu(L1)]3+ at 40 K shows the same nitr-
oxide feature as [Yb(L1)]3+ and [Dy(L1)]3+. At 4 K, the nitroxide
signal broadens slightly, but does not show any distinct evol-
ution of its shape, which is likely the consequence of the small
magnetic moment of trivalent europium.
2.5 DFT calculations on model compounds
The structure of the lanthanide complexes was investigated by
DFT calculations. In order to overcome the prohibitive compu-
tational cost of complexes of f-metal ions, the optimizations
were performed on two models wherein the lanthanide ion
was replaced by Y3+, namely YL13+ and YL23+. Yttrium is indeed
a common substitute for Gd3+ in imaging. The number of
coordinating water molecules was set to one, in agreement
with the luminescence data (see above). For YL13+, the opti-
mized structure shows a pendent TEMPO arm, with the hydro-
gens of the water molecule pointing along but opposite to the
TEMPO group. For YL23+, we first performed a relaxed poten-
tial energy surface (PES) scan along the C–N axis of the alkyne
arm to determine its preferred orientation. The lowest energy
conformation corresponds to the alkyne pointing away from
the metal. It is also interesting to note that the water hydro-
3+
3+
Fig. 7 Relaxed PES scans of YL1 and YL2 (B3LYP/6-31g* (C, H, N,
and O) ECP (Y)). ϕ is the dihedral angle between one water hydrogen,
the water oxygen, yttrium and the O2 oxygen of the ligand (amide con-
necting the TEMPO arm). The O1, O3 and O4 oxygens are from the acet-
gens point orthogonal to the alkyne and establish H-bonds amide groups.
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500 MHz for both the nitroxide and hydroxylamine forms of 2.7 Relaxometry
complexes [Ln(L1)]3+ and [Ln(L2)]3+ (Ln = EuIII, DyIII, and YbIII).
The slow electronic relaxation of the f7 GdIII ion makes this
metal ion inefficient as a PARACEST agent but ensures its
importance as a relaxation CA. The relaxivity r1 has been
studied by Fast Field Cycling (FFC)-NMR (Fig. 9). It shows
interesting behaviour. Under a high field (30 MHz), [Gd(L1)]3+
provides a T1 relaxation time of 0.26 s corresponding to an r1
of 1.92 mM−1 s−1 and [Gd(L2)]3+ yields a T1 relaxation time of
0.16 s corresponding to an r1 of 3.18 mM−1 s−1. A very small
change in relaxivity was observed between the oxidised and
reduced species of 2% and 4% for [Gd(L1)]3+ and [Gd(L2)]3+,
respectively.
The activity of [Eu(L1)]3+ in its TEMPO form shows a weak
CEST spectrum (6% in intensity, Fig. 8) as the relaxation of the
water protons near the TEMPO unit is accelerated by the mag-
netic moment of the free electron spin of that unit.32 Upon the
addition of ascorbic acid, the TEMPO unit is reduced,
affording a closed-shell ligand. There is no more acceleration
of the relaxation of the vicinal water protons. As a conse-
quence, the complex [Eu(L1)]3+ in the reduced form demon-
strated a significant CEST peak at +51 ppm with respect to the
bulk water proton resonance. The exchangeable water mole-
cule on the metal centre presents a larger CEST effect of 12%.
The CEST effect demonstrates small pH dependence with
values varying from 9% to more than 13% between pH 5 and
8. This small pH dependence demonstrates that the metal
bound water molecule is little affected by pH changes. The
CEST effect in [Eu(L1)]3+ is within the same range, albeit
slightly weaker than that for the Eu3+ complex of the ligand
depicted in Chart 1b in aqueous medium.
Under a low field (30 kHz), [Gd(L1)]3+ provides a T1 relax-
ation time of 0.15 s corresponding to an r1 of 3.33 mM−1 s−1
and [Gd(L2)]3+ yields a T1 relaxation time of 0.09 s corres-
ponding to an r1 of 5.28 mM−1 s−1. Upon reduction of nitrox-
ide into hydroxylamine, the relaxivity decreases by 13% and
44% for [Gd(L1)]3+ and [Gd(L2)]3+, respectively.
In the high-field region, it is known that the low exchange
rate of the water molecules in the first coordination sphere
and fast rotational correlation time of the complex limit the
relaxivity of a GdIII-based CA. Besides, in the low-field region,
fast electronic relaxation limits the relaxivity under 0.1 T
(3–4 MHz).13–15,47 The high relaxivity values in this region are
typical of the highly symmetric GdIII complexes with long elec-
tronic relaxation times.48 Complex [Gd(L1)]3+ exhibits a higher
symmetry than [Gd(L2)]3+ and consequently presents a longer
electronic relaxation time, allowing the visualization of the
gadolinium signal in the EPR spectrum (0.34 T). Surprisingly,
[Gd(L2)]3+ in its reduced form shows similar relaxivity as [Gd
(L1)]3+ under a low field but a significantly higher value under
a high field. Much more research beyond the objectives of this
article is yet needed to unravel the physical origins of the
observed effects.14 It is worth noting that the influence of the
redox status on relaxivity is weak for [Gd(L1)]3+, ranging from a
20% increase in the low field region to less than 5% in the
Calculation of the rate of exchange via Omega plot calcu-
lation and confirmation by RL QUEST plots gave a kex value of
approximately 4200 s−1 for the complex [Eu(L1)]3+, which
corresponds to a proton residence time of 238 μs.
Not surprisingly, both [Dy(L1)]3+ and [Yb(L1)]3+ did not
show any significant activity in the PARACEST spectrum, pre-
sumably due to the faster exchange of the NH2 protons
mediated by these metal ions.
Surprisingly, the PARACEST spectra for [Eu(L2)]3+ in both
its TEMPO and TEMPOH forms demonstrated no CEST effect
for the NH exchangeable protons. This result is in line with
the distinct coordination number and conformation of the
complex due to the presence of the alkyne. The present data
do not yet allow for further rationalizing this behaviour. Not
surprisingly, complexes [Dy(L2)]3+ and [Yb(L2)]3+ demonstrated
no activity like that of the [Dy(L1)]3+ and [Yb(L1)]3+ (Fig. S18
and S20†).
Fig. 8 CEST spectrum of 14 mM solution of [Eu(L1)]3+ in its nitroxide
(black) and hydroxylamine (red) forms (reduced using sodium ascor-
bate). Recorded in a water : CD3CN (2 : 1) mixture at pH 7.4 (0.1 M HEPES
buffer) at 11.7 T. T = 298 K, B1 = 25 μT, and irradiation time of 3 s.
Fig. 9 FFC-NMR profiles of 2 mM solution of [Gd(L1)]3+ in its nitroxide
(black) and hydroxylamine (red) forms and [Gd(L2)]3+ in its nitroxide
(blue) and hydroxylamine (green) forms (reduced using sodium ascor-
bate). Recorded in water at pH 7.4 (0.1 M HEPES buffer) at 298 K.
10832 | Dalton Trans., 2021, 50, 10826–10837
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high field region upon the oxidation of the hydroxylamine either the cell pellets or cell lysate. The complex was found to
moiety. Complex [Gd(L2)]3+ shows a better response to the be localized in the supernatant, as demonstrated by a similar
redox stimulus with up to 75% increase in the low field region integration of the nitroxide resonances of the supernatant and
upon the hydroxylamine to nitroxide conversion. Under the a blank containing the complex at similar concentrations in a
highest field, the difference in relaxivity between both complex culture medium lacking cells. Both the polar nature of the
oxidation states remains very weak.
complex and its tripositive nature most likely prevent cell pene-
The T1 relaxation time of the water protons is modulated by tration, which may be an advantage for use as an MRI agent. It
the redox state without influencing the exchange rate value as must be stressed that the alkyne moiety does not affect cell
demonstrated by D. Sherry and coworkers.32 This is an inter- penetration, but offers a possibility for vectorising the complex
esting trend whereby the formation of a paramagnetic radical by adequate functionalization.
center in the vicinity of Gd3+ alters significantly the relaxation
of the protons of the coordinated water. Hence, complexes [Gd
(L1)]3+ and [Gd(L2)]3+ could be considered as gadolinated redox
probes though their relaxivity values are still too low to envi-
3. Conclusions
sage their use as functional MRI probes.
In summary, we have prepared two series of lanthanide(III)
complexes (Gd, Eu, Dy, and Yb) from two DOTA acetamide
derivatives appended by one TEMPO arm. One features three
acetamide groups ([Ln(L1)]3+) while the other ([Ln(L2)]3+) har-
bours two acetamides and one alkyne group for further
functionalization or vectorization through click chemistry. By
luminescence measurements, we demonstrate that both [Eu
(L1)]3+ and [Eu(L2)]3+ possess one water molecule in their first
coordination sphere, inferring that the metal ion is 9- or 8-co-
ordinated, respectively. The distinct coordination polyhedron
between the series is confirmed by EPR spectroscopy. The
lanthanide complexes are redox-active in a physiological
window, with the TEMPO being, for instance, readily reduced
into hydroxylamine by sodium ascorbate. Complex [Eu(L1)]3+ is
CEST-active in its hydroxylamine form (CEST effect of 12%). In
its nitroxide form, the paramagnetism of the ligand accelerates
spin relaxation, resulting in a significant quenching of the
CEST effect. Interestingly, [Eu(L2)]3+ does not show CEST
activity, which might be the consequence of the different
environment and mobility of the coordinated water molecule.
The relaxivity of the gadolinium complexes is affected by
changes in the oxidation state of the TEMPO arm, whereby r1
decreases by about 15% at 10 MHz upon reduction. It must be
stressed that the influence of the nitroxide’s redox state is
about 10 times greater under a low field than under a high
field.
2.8 Biological studies
The toxicity of complexes [Ln(L1)]3+ and [Ln(L2)]3+ has been
investigated by MTT assays on M21 cell lines. The range of
concentration tested was 1–40 μM, with incubation times up to
72 h. The cell viability upon treatment with [Dy(L1)]3+ and [Dy
(L2)]3+ is depicted in Fig. 10 (see the ESI† for the other com-
pounds). Even after the longest incubation time, the viability
of the cells treated by up to 40 μM [Ln(L1)]3+ was similar to
that of the control sample without complex. Hence, all the
compounds of this series show negligible cytotoxicity, which is
a prerequisite for their use as imaging agents. Complexes of
the [Ln(L2)]3+ series demonstrate slight toxicity above 1 μM,
with invariably 60–70% of cell viability upon treatments with
up to 40 μM of the compounds. The difference in toxicity is
clearly inferred to the substitution of one acetamide arm by
the alkyne group, but it is yet unclear why this modification
has such an effect.
One possible explanation for the absence of toxicity in the
[Ln(L1)]3+ series is the lack of cellular penetration, in line with
the clinically approved MRI CAs that are injected into the cir-
culating system. We quantified the cellular penetration of the
representative complex [Dy(L1)]3+ by using EPR spectroscopy.
The complex was incubated with M21 cells at 200 µM, which
was a minimal concentration for straightforward detection.
The supernatant, cell pellets and cell lysate were next analysed
by EPR and the intensity of the nitroxide lines was quantified
under non-saturating conditions. No signal was detected
within the detection limit of EPR (<0.1% penetration) for
Finally, the complexes show only weak or no toxicity at all
at concentrations up to 40 μM against M21 cells, which is
inferred to disfavoured cellular penetration. These results
demonstrate that the original concept of redox-active lantha-
nide complexes has large potentialities for biological imaging
(CEST, MRI). Further works are currently in progress in our
laboratory to increase the CEST effect and incorporate sensitiz-
ing units for bimodal magnetic/optical imaging.
4. Experimental section
4.1 Materials and methods
All chemicals were of reagent grade and were used without
purification. The high resolution mass spectra were recorded
using a Waters Xevo G2-S QTof apparatus. The UV/Vis spectra
Fig. 10 Cell viability after 48 h treatment of M21 cells with increasing
amounts of (left) [Dy(L1)]3+ and (right) [Dy(L2)]3+
.
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were recorded using a Cary Varian 50 spectrophotometer. 0 °C. After the addition was complete, the reaction mixture
NMR spectra were recorded using a Bruker AM 400 (1H at was allowed to warm to room temperature and stirred for
400 MHz) spectrometer. Chemical shifts are quoted relative to 6 hours. The reaction mixture was quenched with 3 mL of
tetramethylsilane (TMS). The X-band EPR spectra were water. The organic phase was washed with HCl (1 M) (2 ×
recorded using a Bruker EMX Plus spectrometer equipped 10 mL) and NaHCO3 sat (2 × 20 mL). The organic phase was
with a Bruker Helium flow cryostat and a dual mode cavity. dried using MgSO4 and the solvent was removed under
The relaxivity values r1 were derived from the experimental reduced pressure. The product was purified by column chrom-
longitudinal relaxation times T1 of the water protons.14 The T1 atography (CH2Cl2/MeOH; 95/5; v/v). The solvent was removed
values were measured from 20 kHz to 30 MHz using a com- under reduced pressure to obtain the title compound as a red
mercial Spinmaster FFC 2000 Stelar relaxometer49,50 (Stelar s.r. solid (85%). MS-ESI m/z [M + H]+: 247.08.
l., Mede PV, Italy). The prepolarized (PP) and non-polarized
1-(2′,2′,6′,6′,-Tetramethyl-1′-oxyl-4′-piperidyl)-amide-1,4,7,10-
(NP) sequences (see Fig. 1 of ref. 49) were used below and tetraazacyclododecane (1). Under an argon atmosphere, to a
above about 12 MHz, respectively. A high polarization field solution of cyclen glyoxal (0.536 g, 2.8 mmol) in THF (10 mL)
Bpol corresponding to a proton resonance frequency of 28 MHz was added 0 (0.750 g, 3.0 mmol, 1.1 eq.) in THF (5 mL). The
was employed in the PP experiments. For each magnetic field, reaction mixture was stirred at room temperature for 24 hours.
r1 was calculated according to the formula r1 = (R1 − R10)/ A precipitate was formed, isolated by filtration and washed
[complex], where R1 is the relaxation rate measured in the pres- with diethyl ether. The resulting solid was dissolved in THF
ence of the complex, R10 the relaxation rate measured in the (20 mL) and o-phenylenediamine (0.108 g, 1.0 mmol, 1.1 eq.)
absence of complex (typically 0.4 s−1). The complex concen- was added. The mixture was stirred at room temperature for 7
tration is expressed in mM. Cyclic voltammetry curves as well days. The residue was then filtered and rinsed with diethyl
1
as differential pulse voltammetry curves were recorded using a ether and the desired product was obtained in 33% yield. H
CHI 620 potentiostat. The measurements were performed in NMR (400 MHz, MeOD) δ = 3.10–2.80 (m br, 16H), 1.80 (d br, J
0.5 mM CH3CN solution containing 0.1 M tetra-n-butyl = 12.5 Hz, 2H), 1.46 (t, J = 13.3 Hz, 2H), 1.17 (d, J = 5.6 Hz,
ammonium perchlorate (TBAP) as the supporting electrolyte. 12H); 13C NMR (100 MHz, MeOD) δ = 78.5, 62.7, 58.9, 50.3,
The experiments were performed in a standard three-electrode 44.7, 31.0, 19.2; IR: 3275, 3038, 2975, 2930, 2822, 1653, 1534,
cell under an argon atmosphere. A glassy carbon disc electrode 1458, 1362, 1243, 1179, 1112, 929, 731; HR-MS m/z calcd for
(3 mm diameter) that was polished with 1 mm diamond paste C19H40N6O2, [M + H]+: 384.321; found, 384.321.
was used as the working electrode. The auxiliary electrode is a
compartmentalized platinum wire, while an Ag/AgNO3 0.01 M tetraazacyclododecane-4,7,10-bisacetamide (L1). The monosub-
electrode was used as a reference. stituted cyclen 1 (0.334 g, 0.87 mmol, 1 eq) was dissolved in
1-(2′,2′,6′,6′,-Tetramethyl-1′-oxyl-4′-piperidyl)-amide-1,4,7,10-
The luminescence spectra of the lanthanide complexes 20 mL of MeCN. 2-Bromoacetamide (0.419 g, 3.0 mmol, 3.5
were recorded using a modular Fluorolog FL3-22 spectrometer eq.), potassium carbonate (0.420 g, 3.0 mmol, 3.5 eq.) and pot-
Horiba-Jobin Yvon-Spex equipped with a double grating exci- assium iodide (0.540 g, 3.3 mmol, 3.8 eq.) was added. The
tation monochromator and an iHR320 imaging spectrometer. reaction was stirred at 50 °C for 5 days. The solution was fil-
Hamamatsu R928P and Hamamatsu R5509 photomultipliers tered and the filtrate was evaporated under reduced pressure.
were used for visible and NIR measurements, respectively. All The product was purified by an alumina column chromato-
spectra were corrected for the detection and optical spectral graphy (EtOH/NH4OH) to afford the title compound as an
responses (instrumental functions) of the spectrofluorimeters. orange solid (0.587 g, 99%). 1H NMR (400 MHz, D2O) δ =
Quartz capillaries of diameter 4 mm were used. For the acqui- 3.26–2.10 (m br, 24H), 1.82 (d, J = 11.3 Hz, 2H), 1.42 (m, 2H),
sition of the excitation and emission spectra in the NIR, a long 1.16 (m, 12H); 13C NMR (125 MHz, D2O) δ = 177.0, 174.8,
pass coloured filter was always used at 870 nm to block the 174.7, 145.2, 130.3, 69.8, 69.1, 61.0, 57.6, 56.9, 55.9, 55.7, 17.0;
signals of the 2nd harmonics. The q value was determined by IR: 3375, 3175, 2966, 2836, 1661, 1549, 1457, 1384, 1328, 1284,
using the formula q = 1.2 [Δkobs − 0.25 − 0.075qCONHR],51 1110; HR-MS m/z calcd for C25H49N9O5 [M]+: 555.385; found,
which gives for the [Ln(L1)]3+ series q = 1.2 [Δkobs − 0.55] and 555.385.
for the [Ln(L2)]3+ series q = 1.2 [Δkobs − 0.48].
General procedure for the synthesis of the complex [Ln
(L1)]3+. The ligand L1 was dissolved in water (10 mL) and the
lanthanide salt was added to the solution. The pH was
4.2 Synthesis
4-Amino-2,2,6,6-tetramethylpiperidine-1-oxyl, free radical was adjusted to 7 by the addition of sodium hydroxide (1 M). The
purchased from Sigma-Aldrich; 1,4,7,10-tetraazacyclodode- reaction mixture was stirred and heated to 50 °C for 1 day.
cane-1,7-bisacetamide (2) was synthesized according to litera- Water was evaporated under reduced pressure. The yellow
ture procedures.52
4-(2-Chloroacetamido)-2′,2′,6′,6′,-tetramethylpiperidine-1′-
solid was dissolved in minimum of methanol and precipitated
in diethyl ether. It was then dissolved in acetonitrile, filtered
oxyl, free radical (0). To a solution of 4-amino-tempo, free and the solvent was evaporated to give the desired compound
radical (0.200 g, 1.17 mmol) in CH2Cl2 (7 mL) was added Et3N [Ln(L1)]3+ as an orange powder. [Yb(L1)]3+: from L1 (100 mg,
(310 µL, 2.2 mmol, 1.9 eq.). Chloroacetyl chloride (140 µL, 0.15 mmol) and ytterbium triflate (93 mg, 0.15 mmol). Yield:
1
1.8 mmol, 1.5 eq.) in CH2Cl2 (5 mL) was added dropwise at 100%. H NMR (400 MHz, D2O) δ = 97.71, 94.42, 92.95, 19.51,
10834 | Dalton Trans., 2021, 50, 10826–10837
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18.21, 16.86, 16.65, 15.97, 15.80, 15.24, 14.86, 12.31, 8.56, 4.12, nide salt was added to the solution. The pH was adjusted to
3.73, 1.57, 1.48, 0.13, −1.71, −24.16, −25.80, −26.42, −29.00, 6.5 by the addition of sodium hydroxide (1 M). The reaction
−31.27, −34.80, −53.92, −57.43, −58.00; IR (cm−1): 3361, 2981, mixture was stirred and heated to 50 °C for 2 days. Water was
2927, 2867, 1667, 1626, 1458, 1323, 1246, 1161, 1083, 1030, evaporated in reduced pressure. Purification by precipitation
924, 668, 639; MS-ESI m/z an appropriate isotope pattern was in diethyl ether gives the desired compound [Ln(L2)]3+ as an
observed for C26H49F3O8N9SYb [M + CF3SO3]2+: 439.14; found, orange powder. [Eu(L2)]3+: from L2 (300 mg, 0.45 mmol) and
440.77. [Eu(L1)]3+: from L1 (187 mg, 0.27 mmol) and europium europium triflate (272 mg, 0.45 mmol). Yield: 83%. 1H NMR
triflate (164 mg, 0.27 mmol). Yield: 49%. IR (cm−1): 3354, (400 MHz, MeOD) δ = 18.1, −2.1, −6.3, −7.3, −9.0, −10.9,
3197, 2987, 1658, 1628, 1590, 1459, 1391, 1242, 1225, 1173, −12.7, −17.0; IR (cm−1): 3369, 3244, 2958, 1667, 1606, 1454,
1079, 1027, 639. [Gd(L1)]3+: from L1 (30 mg, 0.06 mmol) and 1233, 1172, 1029, 647; ESI-MS m/z an appropriate isotope
gadolinium triflate (34 mg, 0.06 mmol). Yield: 37%. IR (cm−1): pattern was observed for [M]3+: C26H47N8O4Eu, 229.43; found,
3367, 3291, 3208, 2984, 2939, 2876, 1661, 1627, 1458, 1245, 229.39.
Elemental
analysis:
calculated
for
1169, 1027, 634; HR-MS m/z calcd for C26H49O8N9F3GdS [M + C26H47N8O4EuI2Cl·4H2O; C: 29.76, H: 5.30 N: 10.68, observed
CF3SO3]2+, 431.130; found 431.131. [Dy(L1)]3+: from L1 C: 29.71 H: 5.30 N: 10.62. [Yb(L2)]3+: from L2 (300 mg,
(100 mg, 0.15 mmol) and dysprosium triflate (92 mg, 0.45 mmol) and ytterbium triflate (281 mg, 0.45 mmol). Yield:
0.15 mmol). Yield: 27%. 1H NMR (400 MHz, D2O) δ = 18.1, 96%. 1H NMR (500 MHz, MeOD) δ = 77.8, 65.8, 57.9, 46.5,
−2.6, −18.3; IR (cm−1): 3331, 3172, 2982, 1655, 1621, 1588, 36.9, 22.4, −5.3, −15.7, −17.4, −32.5, −53.2, −83.1; IR (cm−1):
1454, 1403, 1322, 1272, 1244, 1080, 632.
4-Propargyl-1,4,7,10-tetraazacyclododecane-1,7-bisacetamide 1081, 1028; ESI-MS m/z an appropriate isotope pattern was
(3). To a solution of 2 (1.31 g, 4.6 mmol), NaOAc (0.804 g, observed for C26H47N8O4Yb(CF3SO3)4, [M
4CF3SO3]−:
3376, 3270, 2975, 2945, 2876, 1670, 1627, 1463, 1243, 1150,
+
9.9 mmol, 1.2 eq.) in EtOH (200 mL) was added and cooled at 1305.12; found, 1304.89. [Gd(L2)]3+: from L2 (300 mg,
0 °C. Propargyl bromide (580 µL, 5.2 mmol, 1.2 eq.) in EtOH 0.45 mmol) and gadolinium triflate (274 mg, 0.45 mmol).
(100 mL) was added dropwise at 0 °C. After the addition was Yield: 84%. IR (cm−1): 3379, 3257, 2972, 2868, 1664, 1615,
complete, the reaction mixture was allowed to stir for 4 days at 1455, 1167, 1080, 1019, 632. [Dy(L2)]3+: from L2 (300 mg,
50 °C. The solvent was removed under reduced pressure. The 0.45 mmol) and gadolinium triflate (276 mg, 0.45 mmol).
residue was taken up in MeCN and filtered. The solvent was Yield: 87%. IR (cm−1): 3364, 3274, 2969, 2874, 1667, 1618,
removed under reduced pressure to obtain the title compound 1459, 1246, 1174, 1079, 1024, 635.
1
as a brown oil (93%). H NMR (400 MHz, MeOD) δ = 3.45 (d, J
= 2.2 Hz, 1H), 3.38–3.36 (m, 2H), 3.30 (s, 2H), 3.19 (s, 2H),
2.76–2.56 (m, 16H); 13C NMR (100 MHz, MeOD) δ = 176.4,
168.2, 77.9, 75.6, 59.2, 53.4, 53.0, 51.3, 46.8, 39.7, 30.7; IR:
3283, 2962, 2842, 2689, 2464, 2062, 1654, 1586, 1457, 1372,
1341, 1118, 973, 758; HRMS (m/z): [M + H]+ calcd for
C15H29N6O2, 325.235; found, 325.235.
1-(2′,2′,6′,6′,-Tetramethyl-1′-oxyl-4′-piperidyl)-amide-4-propar-
gyl-1,4,7,10-tetraazacyclododecane-1,7-bisacetamide (L2). To a
solution of 3 (1.39 g, 4.3 mmol, 1 eq.) in EtOH (100 mL),
K2CO3 (1.18 g, 8.6 mmol, 2 eq.), KI (1.46 g, 8.8 mmol, 2.1 eq.)
and 1 (1.18 g, 4.8 mmol, 1.1 eq.) were added. The reaction
mixture was stirred for 5 days at 60 °C. The solution was fil-
tered with Celite, rinsed with EtOH, and the solvent was
removed under reduced pressure. The product was purified by
an alumina column chromatography (AcOEt then EtOH/
NH4OH). The solvent was removed under reduced pressure to
obtain the title compound as an orange solid (88%). Slow evap-
oration of water led to orange crystals which were determined
4.3 X-ray diffraction
Single crystals were coated with perfluoropolyether, picked up
with nylon loops and mounted in the nitrogen cold stream of
the diffractometer. Mo-Kα radiation (λ = 0.71073 Å) from a Mo-
target rotating-anode X-ray source equipped with INCOATEC
Helios mirror optics was used. Final cell constants were
obtained from least squares fits of several thousand strong
reflections. Intensity data were corrected for absorption using
the intensities of redundant reflections with the SADABS
program. The structures were solved by Patterson methods and
subsequent difference Fourier techniques. The OLEX software
was used for the refinement.53 All non-hydrogen atoms were
anisotropically refined and hydrogen atoms were placed at cal-
culated positions and refined as riding atoms with isotropic
displacement parameters. CCDC 1997062† contains the crys-
tallographic data for [Na(L2)]+(Cl−).
4.4 DFT calculations
by X-ray crystallography. Reduction with sodium ascorbate was
1
used to characterize the ligand. H NMR (400 MHz, D2O) δ = Full geometric optimizations were performed with the
4.53 (s, 2H), 4.22 (t, J = 10.5 Hz, 1H), 3.56 (br s, 8H), 3.02 (s, Gaussian 9.0 program.54 The B3LYP functional55,56 was used
16H), 1.94 (d, J = 13.0 Hz, 2H), 1.60 (t, J = 13.0 Hz, 2H), 1.28 (d, together with the 6-31g* basis set for the C, H, and N atoms57
J = 16.4 Hz, 12H); 13C NMR (100 MHz, D2O) δ = 78.4, 69.6, and a pseudopotential (LanL2DZ)58 was used for the central
62.5, 43.1, 29.9, 19.6; IR: 3360, 3283, 3194, 3123, 2977, 2941, metal ion. Frequency calculations were systematically per-
2825, 1653, 1568, 1456, 1363, 1320, 1108, 647; HRMS (m/z): [M formed on the optimized structures in order to ensure that
+ H]+ calcd for C26H48N8O4, 536.379; found, 536.379.
they correspond to a real energy minimum and not a saddle
General procedure for the synthesis of complexes [Ln(L2)]3+. point. The relaxed potential energy surface scans were per-
The ligand L2 was dissolved in water (20 mL) and the lantha- formed by varying dihedral angles.
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4.5 Cell culture
lations and the Centre de Calcul Intensif en Chimie de
Grenoble (CECIC) for providing the computational resources.
The ICMG Platform (FR 2607) is acknowledged for the analyti-
cal support.
Human melanoma cell line M21 was purchased from ATCC
(Molshein, France). Cells were cultured in a DMEM medium
supplemented with 10% (v/v) fetal calf serum (FCS) and 2 mM
glutamine (Thermo Fisher Scientific, Courtaboeuf, France).
−
The cells were maintained at 37 °C in a 5% CO2 humidified
atmosphere and tested to ensure freedom from mycoplasma Notes and references
contamination. All cell lines were used within 5–50 passages of
thawing the original stocks.
1 G. Maulucci, G. Bačić, L. Bridal, B. Tavitian, T. Viel,
H. Utsumi, A. S. Yalçın and M. De Spirito, Antioxid. Redox
Signal., 2016, 24, 939–958.
4.6 MTT assay
2 E. Terreno, D. D. Castelli, A. Viale and S. Aime, Chem. Rev.,
2010, 110, 3019–3042.
3 C. Jacob, M. Doering and T. Burkholz, in Redox Signaling
and Regulation in Biology and Medicine, ed. C. Jacob and
P. G. Winyard, Wiley-VCH, Weinheim, Germany, 2009, pp.
63–122.
4 F. Q. Schafer and G. R. Buettner, Free Radical Biol. Med.,
2001, 30, 1191–1212.
5 B. D’Autréaux and M. B. Toledano, Nat. Rev. Mol. Cell Biol.,
2007, 8, 813–824.
M21 cells were seeded into 96-well plates (1.5 × 103 cells per
well) with 100 μl of culture medium. After 24 h, the cells were
treated with the lanthanide(III) complexes at various concen-
trations. Following incubation for 24 h, 48 h or 72 h, 10 μl of
an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) stock solution (Euromedex, Mundolsheim, France)
in PBS at 5 mg ml−1 was added to each well and the plates
were incubated at 37 °C for 2 h. To solubilize water-insoluble
purple formazan crystals, SDS 10%/HCl 0.1% solution was
used. After 24 h, absorbance was measured using an ELISA
reader (Tecan, Männedorf, Switzerland) at a test wavelength of
570 nm and a reference wavelength of 650 nm.
6 T. R. Hurd and M. P. Murphy, in Redox Signaling and
Regulation in Biology and Medicine, ed. C. Jacob and
P. G. Winyard, Wiley-VCH, Weinheim, Germany, 2009, pp.
13–43.
4.7 EPR spectroscopy
7 G. Csányi and F. J. Miller Jr., Int. J. Mol. Sci., 2014, 15,
6002–6008.
8 H. Abe, H. Semba and N. Takeda, J. Atheroscler. Thromb.,
2017, 24, 884–894.
9 D. M. Gilkes, G. L. Semenza and D. Wirtz, Nat. Rev. Cancer,
2014, 14, 430.
10 R. Bakalova, Z. Zhelev, I. Aoki and T. Saga, Clin. Cancer
Res., 2013, 19, 2503–2517.
M21 cells were seeded in 4-well plates (1 × 105 cells per well) in
1 mL of culture medium. After 24 h, the cells were treated with
the complexes of concentration 200 μM for 5 min, 10 min,
30 min, and 1 h. The cell culture medium was recovered and
the M21 cells were harvested. A fraction of M21 pellets was
then washed twice in cold PBS and incubated in lysis buffer
(10 mmol L−1 Tris-HCl pH 7.5, 120 mmol L−1 NaCl, 1 mmol
L−1 EDTA, 1 mmol L−1 dithiothreitol, 0.5% Nonidet P-40,
0.05% sodium dodecyl sulfate, and supplementation with pro-
tease inhibitors (Na3VO4 and NaF). After 10 min on ice, the
lysates were centrifugated at 20 000g for 15 minutes and the
soluble fraction was recovered. The cell culture medium,
pellets of intact M21 cells and M21 cell lysates were then ana-
lyzed by EPR spectroscopy.
11 F. Zhang, R. Zhong, H. Qi, S. Li, C. Cheng, X. Liu, Y. Liu
and W. Le, Front. Neurosci., 2018, 12, 314.
12 L. Helm, J. R. Morrow, C. J. Bond, F. Carniato, M. Botta,
M. Braun, Z. Baranyai, R. Pujales-Paradela, M. Regueiro-
Figueroa, D. Esteban-Gómez, C. Platas-Iglesias and
T. J. Scholl, in Contrast Agents for MRI: Experimental
Methods, The Royal Society of Chemistry, 2018, pp.
121–242. DOI: 10.1039/9781788010146-00121.
13 E. Belorizky, P. H. Fries, L. Helm, J. Kowalewski, D. Kruk,
R. R. Sharp and P.-O. Westlund, J. Chem. Phys., 2008, 128,
052315.
Conflicts of interest
There are no conflicts to declare.
14 C. S. Bonnet, P. H. Fries, A. Gadelle, S. Gambarelli and
P. Delangle, J. Am. Chem. Soc., 2008, 130, 10401–
10413.
15 C. S. Bonnet, P. H. Fries, S. Crouzy and P. Delangle, J. Phys.
Chem. B, 2010, 114, 8770–8781.
Acknowledgements
The authors thank the French National Research Agency in the
framework of the “Investissements d’avenir” program (ANR-15- 16 Q. N. Do, J. S. Ratnakar, Z. Kovács and A. D. Sherry,
IDEX-02), Labex ARCANE and CBH-EUR-GS (ANR-17- ChemMedChem, 2014, 9, 1116–1129.
EURE-0003) and the French National Agency for Research, 17 S. M. Pinto, V. Tomé, M. J. F. Calvete, M. M. C. A. Castro,
program Co-Lantha (ANR-17-CE07-0034) for financial support.
This work was performed under the auspices of the COST
É. Tóth and C. F. G. C. Geraldes, Coord. Chem. Rev., 2019,
390, 1–31.
Action AC15209, EURELAX. The authors are grateful to 18 C. Tu and A. Y. Louie, Chem. Commun., 2007, 1331–1333,
Dr Pierre Girard for technical assistance for the DFT calcu-
DOI: 10.1039/B616991K.
10836 | Dalton Trans., 2021, 50, 10826–10837
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Paper
19 C. Tu, E. A. Osborne and A. Y. Louie, Tetrahedron, 2009, 65, 42 Y. Kato, Y. Shimizu, L. Yijing, K. Unoura, H. Utsumi and
1241–1246. T. Ogata, Electrochim. Acta, 1995, 40, 2799–2802.
20 C. Tu, R. Nagao and A. Y. Louie, Angew. Chem., Int. Ed., 43 The difference in molar absortivity was not due to the dis-
2009, 48, 6547–6551.
tinct oxidation state or purity of the samples, as demon-
strated by a similar intensity in the EPR resonances for
both L1 and L2.
21 C. Carrera, G. Digilio, S. Baroni, D. Burgio, S. Consol,
F. Fedeli, D. Longo, A. Mortillaro and S. Aime, Dalton
Trans., 2007, 4980–4987, DOI: 10.1039/B705088G.
22 G. Digilio, V. Menchise, E. Gianolio, V. Catanzaro,
44 L. J. Libertini and O. H. Griffith, J. Chem. Phys., 1970, 53,
1359–1367.
C. Carrera, R. Napolitano, F. Fedeli and S. Aime, J. Med. 45 F. Thomas, J. Michon and J. Lhomme, Biochemistry, 1999,
Chem., 2010, 53, 4877–4890. 38, 1930–1937.
23 N. Raghunand, G. P. Guntle, V. Gokhale, G. S. Nichol, 46 O. A. Blackburn, N. F. Chilton, K. Keller, C. E. Tait,
E. A. Mash and B. Jagadish, J. Med. Chem., 2010, 53, 6747–
6757.
24 N.-D. H. Gamage, Y. Mei, J. Garcia and M. J. Allen, Angew.
Chem., Int. Ed., 2010, 49, 8923–8925.
25 J. Garcia, J. Neelavalli, E. M. Haacke and M. J. Allen, Chem.
Commun., 2011, 47, 12858–12860.
26 J. Garcia, A. N. W. Kuda-Wedagedara and M. J. Allen,
Eur. J. Inorg. Chem., 2012, 2012, 2135–2140.
W. K. Myers, E. J. L. McInnes, A. M. Kenwright, P. D. Beer,
C. R. Timmel and S. Faulkner, Angew. Chem., Int. Ed., 2015,
54, 10783–10786.
47 J. Wahsner, E. M. Gale, A. Rodríguez-Rodríguez and
P. Caravan, Chem. Rev., 2019, 119, 957–1057.
48 S. Aime, A. Barge, J. I. Bruce, M. Botta, J. A. K. Howard,
J. M. Moloney, D. Parker, A. S. de Sousa and M. Woods,
J. Am. Chem. Soc., 1999, 121, 5762–5771.
27 L. Burai, É. Tóth, S. Seibig, R. Scopelliti and A. E. Merbach, 49 P. H. Fries, G. Ferrante, E. Belorizky and S. Rast, J. Chem.
Chem. – Eur. J., 2000, 6, 3761–3770. Phys., 2003, 119, 8636–8644.
28 M. Woods, A. Pasha, P. Zhao, G. Tircso, S. Chowdhury, 50 G. Ferrante and S. Sykora, Adv. Inorg. Chem., 2005, 57, 405–
G. Kiefer, D. E. Woessner and A. D. Sherry, Dalton Trans.,
2011, 40, 6759–6764.
29 S. Zhang, M. Merritt, D. E. Woessner, R. E. Lenkinski and
A. D. Sherry, Acc. Chem. Res., 2003, 36, 783–790.
30 S. J. Ratnakar, M. Woods, A. J. M. Lubag, Z. Kovács and
A. D. Sherry, J. Am. Chem. Soc., 2008, 130, 6–7.
31 S. J. Ratnakar, S. Viswanathan, Z. Kovacs, A. K. Jindal,
470.
51 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner,
D. Parker, L. Royle, A. S. de Sousa, J. A. Gareth Williams
and M. Woods, J. Chem. Soc., Perkin Trans. 2, 1999, 493–
504, DOI: 10.1039/A808692C.
52 F. Oukhatar, M. Beyler and R. Tripier, Tetrahedron, 2015,
71, 3857–3862.
K. N. Green and A. D. Sherry, J. Am. Chem. Soc., 2012, 134, 53 O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard
5798–5800. and H. Puschmann, J. Appl. Crystallogr., 2009, 42, 339–340.
32 S. J. Ratnakar, T. C. Soesbe, L. L. Lumata, Q. N. Do, 54 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
S. Viswanathan, C.-Y. Lin, A. D. Sherry and Z. Kovacs, J. Am.
Chem. Soc., 2013, 135, 14904–14907.
33 A. M. Funk, V. Clavijo Jordan, A. D. Sherry, S. J. Ratnakar
and Z. Kovacs, Angew. Chem., Int. Ed., 2016, 55, 5024–
5027.
34 J. Molloy, K. O. Jarjayes, C. Philouze, L. Fedele, D. Imbert
and F. Thomas, Chem. Commun., 2017, 53, 605–608.
35 J. K. Molloy, L. Fedele, O. Jarjayes, C. Philouze, D. Imbert
and F. Thomas, Inorg. Chim. Acta, 2018, 483, 609–617.
36 J. K. Molloy, C. Philouze, L. Fedele, D. Imbert,
O. Jarjayes and F. Thomas, Dalton Trans., 2018, 47, 10742–
10751.
37 R. Barré, D. Mouchel dit Leguerrier, L. Fedele, D. Imbert,
J. K. Molloy and F. Thomas, Chem. Commun., 2020, 56,
435–438.
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J. J. A. Montgomery, J. E. Peralta,
F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers,
K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand,
K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar,
J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene,
J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin,
R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador,
J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas,
J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox,
Gaussian 09, Revision D.01, Gaussian, Inc., Wallingford CT,
2009.
38 Z. Zhelev, V. Gadjeva, I. Aoki, R. Bakalova and T. Saga, Mol.
Biosyst., 2012, 8, 2733–2740.
39 V. V. Pavlishchuk and A. W. Addison, Inorg. Chim. Acta,
2000, 298, 97–102.
40 J. E. Nutting, M. Rafiee and S. S. Stahl, Chem. Rev., 2018,
118, 4834–4885.
55 C. Lee, W. Yang and R. G. Parr, Phys. Rev. B: Condens.
Matter Mater. Phys., 1988, 37, 785–789.
56 A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652.
41 M. Shibuya, F. Pichierri, M. Tomizawa, S. Nagasawa, 57 G. A. Petersson and M. A. Al-Laham, J. Chem. Phys., 1991,
I. Suzuki and Y. Iwabuchi, Tetrahedron Lett., 2012, 53,
2070–2073.
94, 6081–6090.
58 P. J. Hay and W. R. Wadt, J. Chem. Phys., 1985, 82, 299–310.
This journal is © The Royal Society of Chemistry 2021
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