Gd3+-based magnetic resonance imaging contrast agent responsive to Zn2+

Article abstract of DOI:10.1021/acs.inorgchem.5b01719

We report the heteroditopic ligand H₅L, which contains a DO3A unit for Gd³⁺ complexation connected to an NO2A moiety through a N-propylacetamide linker. The synthesis of the ligand followed a convergent route that involved the prepar

Full text of DOI:10.1021/acs.inorgchem.5b01719

Article
pubs.acs.org/IC
Gd3+-Based Magnetic Resonance Imaging Contrast Agent Responsive
to Zn2+
Martín Regueiro-Figueroa,† Serhat Gunduz,‡ Veronique Patinec,§ Nikos K. Logothetis,#,∥
́
̈
̈
David Esteban-Gomez,† Raphael Tripier,§ Goran Angelovski,*,‡ and Carlos Platas-Iglesias*,†
́
̈
†Grupo QUICOOR, Centro de Investigaciones Científicas Avanzadas (CICA) and Departamento de Química Fundamental,
Universidade da Coruna, Campus da Zapateira, Rua da Fraga 10, 15008 A Coruna, Spain
‡MR Neuroimaging Agents, Max Planck Institute for Biological Cybernetics, Spemannstr. 41, 72076 Tubingen, Germany
́
̃
̃
̈
§UFR des Sciences et Techniques, Universite
29238 BREST Cedex 3, France
́
de Bretagne Occidentale, UMR-CNRS 6521, 6 avenue Victor le Gorgeu, C.S. 93837,
#Physiology of Cognitive Processes, Max Planck Institute for Biological Cybernetics, Tubingen, Germany
∥Department of Imaging Science and Biomedical Engineering, University of Manchester, Manchester, U.K.
̈
S
* Supporting Information
ABSTRACT: We report the heteroditopic ligand H5L, which
contains a DO3A unit for Gd3+ complexation connected to an
NO2A moiety through a N-propylacetamide linker. The
synthesis of the ligand followed a convergent route that
involved the preparation of 1,4-bis(tert-butoxycarbonylmeth-
yl)-1,4,7-triazacyclononane following the orthoamide strategy.
The luminescence lifetimes of the Tb(5D4) excited state
measured for the TbL complex point to the absence of
coordinated water molecules. Density functional theory
calculations and 1H NMR studies indicate that the EuL
complex presents a square antiprismatic coordination in
aqueous solution, where eight coordination is provided by
the seven donor atoms of the DO3A unit and the amide
oxygen atom of the N-propylacetamide linker. Addition of Zn2+ to aqueous solutions of the TbL complex provokes a decrease of
the emission intensity as the emission lifetime becomes shorter, which is a consequence of the coordination of a water molecule
to the Tb3+ ion upon Zn2+ binding to the NO2A moiety. The relaxivity of the GdL complex recorded at 7 T (25 °C) increases by
almost 150% in the presence of 1 equiv of Zn2+, while Ca2+ and Mg2+ induced very small relaxivity changes. In vitro magnetic
resonance imaging experiments confirmed the ability of GdL to provide response to the presence of Zn2+.
which suggested that the amyloid pathology of Alzheimer’s
disease arises from Zn2+ release during glutamatergic neuro-
transmission events.6 Thus, the development of probes to
detect Zn2+ in biological systems is of great importance to shed
light on the biological processes (pathological or not) mediated
by this metal ion.7
INTRODUCTION
■
Zn2+ is one of the most abundant metal ions in the human body
that plays a critical role acting as a structural component of
many proteins or the active site of enzymes.1 Most of the Zn2+
present in the body is tightly bound in proteins, and thus the
concentration of free Zn2+ in serum is relatively low (∼15−20
μM).2 However, mobile Zn2+ is present at particularly high
concentrations in certain organs such as brain, pancreas and
prostate, where it plays important physiological roles.3 In the
case of brain, chelatable Zn2+ is involved in the regulation of
neuronal transmission, but both zinc overload and deficiency
induce susceptibility to apoptosis.4 Failures in homeostasis of
mobile Zn2+ have been also associated with different neuro-
logical diseases such as ischemia, epilepsy, Parkinson’s disease
or Alzheimer’s disease.5 A class of glutamatergic neurons found
mainly in the neo-cortex and limbic structures are susceptible to
accumulate Zn2+ at high concentrations (∼1 mM). High
concentrations of Zn2+ have been observed in the neo-cortex of
Alzheimer’s disease patients together with amyloid deposits,
Magnetic resonance imaging (MRI) is a technique widely
used by radiologists that is particularly well-suited for obtaining
1
high-resolution images of soft tissues. MRI uses the H NMR
signal of water molecules present in the body to generate
contrast taking advantage of differences in the density of
protons or the longitudinal (T1) and transverse (T2) relaxation
times.8 Image contrast may be improved with the admin-
istration of contrast agents (CAs), often Gd3+ complexes that
contain at least one water molecule coordinated to the metal
ion exchanging rapidly with the bulk water of the body.8,9
Received: July 30, 2015
© XXXX American Chemical Society
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a
Chart 1. Chemical Structure of the Ligands Discussed in This Work
a
The dashed rectangles indicate the structural motif responsible for relaxivity changes in responsive CAs, while the dashed ellipsoids highlight the
Ca2+- or Zn2+-binding motifs used in previous studies.
These paramagnetic compounds provide an efficient mecha-
nism for the longitudinal and transverse relaxation enhance-
ment (1/T1 and 1/T2) of water protons, which is translated
into image contrast. Responsive or smart CAs are a subclass of
these paramagnetic probes capable of changing the T1 and/or
T2 as a function of certain physiological parameters.10 Many
responsive agents have been reported in the past years that are
sensitive to temperature,11 pH,12 redox activity,13 enzymatic
activity,14 or biologically relevant ions.15 Among these, a
number of Zn-responsive agents with great structural variety
were studied. Most commonly, a well-known and selective
chelator N,N-bis(2-pyridyl-methyl) ethylene diamine (BPEN)
was coupled to DTPA,16 DOTA,17,18 or porphyrins19 to result
in potent Zn sensors. Alternatively, Zn2+ chelators consisting of
substituted diacetateamine20 or sulfonamidoquinoline21 frag-
ments, a bis(pyridinylmethyl)amine unit22 or an amidoquino-
line moiety23 coupled to DO3A or polyamino polycarboxylates
containing pyridine moieties as Gd3+-chelators were also used.
In spite of the variety of Zn2+-responsive Gd3+-based CAs
reported in the literature, some of their features are still to be
improved. First, the relatively fast dissociation kinetics of Gd3+-
DTPA complexes in vivo represents an important drawback of
the first generation of responsive probes based on DTPA,16 as
dissociation of the complex with Gd3+ release may result in
undesirable side effects.24,25 Second, MRI has the intrinsic
disadvantage of its low sensitivity compared to other techniques
such as fluorescence.17a The reported Zn2+-responsive MRI
probes provide responses that fall typically within the range of
20−70% in terms of the relaxivity changes induced by the
presence of Zn2+,16−23 although a relaxivity change of up to
165% has been reported for a Zn2+ probe in the presence of
human serum albumin (HSA).17c However, improving the
response of this kind of probes is highly desirable to improve
the sensitivity and/or reduce the doses of paramagnetic agent
to be injected. Third, several of the CAs responsive to Zn2+
present an optimal response at low fields (20−60
MHz),17,20b,22 while higher spatial resolution and better
sensitivity is currently stimulating the development of high-
field (3 T) and ultrahigh field (>3 T) MRI scanners.26
Our groups were also involved in the development of
responsive agents. A series of Gd3+ complexes with mono-
(H5L1, Chart 1) and bis-macrocyclic (H8L2−H8L3) ligands
providing selective and strong relaxivity response for Ca2+ over
Mg2+ were reported by Angelovski et al.28,27 Besides the
evaluation of the complex in vitro,29 their nanosized analogues
were recently tested in vivo, exhibiting very advantageous
properties.30,31 These systems contain one or two DO3A units
for Gd3+ complexation, which are coupled to a non-macrocyclic
Ca2+-binding moiety via an amide bond. This amide group is
also involved in the coordination to Gd3+,25 thus making the
whole system prone to easy coordination and thus the relaxivity
changes. Consequently, the number of water molecules
coordinated to Gd3+ increases in the presence of Ca2+,
provoking a relaxivity increase and thus MRI response.
Furthermore, the same structural motif was coupled to the
aza-crown containing Gd3+ complexes, resulting in the series of
neurotransmitter-sensitive contrast agents.32
However, a ligand H3L4, which contains a TACN moiety
introduced for Zn2+ sensing purposes, was recently reported by
Platas-Iglesias and Tripier et al.33 However, Zn2+ addition did
not provoke a change in the number of water molecules
coordinated to Gd3+, although turn-on behavior of the ligand-
centered luminescence was induced by Zn2+. Obviously, an
appropriate nature of the Gd3+ chelator (DO3A unit) and the
linker is needed to result in the potential contrast agent that
will respond to the external stimulus (e.g., change in Zn2+
concentration) by changing the relaxivity. Therefore, we sought
to combine the successfully used responsive part with a Zn2+
chelator. Thus, herein we report the heteroditopic ligand H5L,
which contains a DO3A unit for Gd3+ complexation linked by
the same N-propylacetamide moiety to a NO2A moiety for
Zn2+ binding. The GdL complex was assessed as a Zn2+-
responsive MRI probe by using different spectroscopic and
computational techniques.
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a
Scheme 1. Synthesis of H5L
a
Reagents and conditions: (i) toluene, RT, 12 h; (ii) PhCH2Br, THF, RT, 3 d; (iii) MeOH/HCl 12 M (1:1) reflux; HO−; (iv) 1.8 equiv of
BrCH2COOtBu, K2CO3, CH3CN, RT, 4 d; (v) H2, Pd/C (10%), EtOH, RT, 5 d; (vi) K2CO3, CH3CN, 70 °C, 16 h; (vii) TFA, CH2Cl2, RT, 16 h.
Cu2+ complex. Addition of an excess of metal ion causes a red
RESULTS AND DISCUSSION
■
shift of the absorption band to 742 nm due to the formation of
Ligand Synthesis. The synthesis of 1,7-bis(tert-butoxycar-
bonylmethyl)-1,4,7-triazacyclononane 5 was performed using a
the Cu2+ aqua ion.
The GdL and TbL complexes were prepared by reacting
benzylation/dialkylation/debenzylation sequence (Scheme 1).
equimolar amounts of the H5L ligand and LnCl3·6H2O salts in
Reaction of 1,4,7-triazacyclononane (TACN) with N-dimethox-
aqueous solution at pH 5.8 and 80 °C for 24 h. The high-
ymethyl-N,N-dimethylamine gave rise to the formation of the
resolution mass spectra (ESI+) of the aqueous solutions of the
already known orthoamide 1,4,7-triazatricyclo[5.2.1.04,10]-
decane 1, which has been shown to be an efficient intermediary
GdL and TbL complexes show peaks due to the [GdNa2HL]+
and [NaTbH2L]+ entities, which indicates the formation of the
in the monoalkylation of TACN with halogenoalkane
derivatives.34 Addition of benzyl bromide to 1 resulted in the
mononuclear complexes (Figures S2 and S3, Supporting
Information). The emission spectrum of the TbL complex
precipitation of the corresponding ammonium salt 2 as a white
powder. Acidic hydrolysis with HCl 12 M/methanol (1/1) and
recorded upon excitation at 230.5 nm shows weak
5
7
luminescence due to the D4 → FJ transitions characteristic
of Tb3+ (488 nm, J = 6; 543 nm, J = 5; 583 nm, J = 4; 618 nm, J
= 3, Figure 1). The excited-state lifetimes of the TbL complex
were measured in water (τH2O = 2.22 ms) and deuterated water
(τD2O = 2.63 ms), which allows estimating the number of water
molecules in the first coordination sphere of the metal ion
further basic treatment gave the deprotected benzyl derivative 3
with 90% overall yield. Alkylation of the two remaining
secondary amine functions proceeded in acetonitrile in the
presence of an excess of K2CO3 by using 1.8 equiv of tert-butyl
bromoacetate in order to favor the dialkylated product 4
instead of the quaternarization of nitrogen atoms. Derivative 4
was obtained with 63% yield after purification by column
chromatography. Removal of the benzyl arm in the last step was
performed under H2 pressure with Pd/C (10%) in absolute
ethanol to produce compound 5 (NO2AtBu) in 76% yield.
This secondary amine was then alkylated with the bromo-
methyl derivative 628 that carries the responsive DO3A moiety
using K2CO3 as the base to give the bismacrocyclic compound
7. The desired chelator H5L was finally obtained by hydrolysis
of tert-butyl esters in trifluoroacetic acid at room temperature
(RT).
Complexation Studies. To avoid problems arising from
the formation of binuclear lanthanide complexes or the
presence of free ligand, the stock solution of the ligand used
for the preparation of the LnL complexes was standardized by
titration with CuSO4·5H2O in acetate buffer (pH 5.8, Figure
S1, Supporting Information). Addition of Cu2+ to a solution of
the ligand results in the formation of a broad and weak
absorption band at 725 nm attributed to d−d transitions. The
titration profile shows a sharp inflection point that signals a 1:2
stoichiometry (L/Cu), pointing to the formation of a binuclear
Figure 1. Emission spectra taken during the course of the titrations of
a 1 mM solution of TbL (λexc = 230.5 nm) with a standard solution of
Zn(CF3SO3)2 ca. 53 mM in aqueous solution at pH 7.4 (0.1 M MOPS
buffer).
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according to the methodology developed by Horrocks,35 using
the refined coefficients determined by Parker et al.36 These
emission lifetimes provide a hydration number q = 0.05, which
points to the absence of water molecules coordinated to the
Tb3+ ion in aqueous solution. Both the DO3A and NO2A
moieties of the ligand can in principle form stable complexes
with the Ln3+ ions in aqueous solutions. However, the stability
constant of the [Gd(NOTA)] complex (H3NOTA = 1,4,7-
triazacyclononane-1,4,7-triacetic acid, log KGdL = 14.4)37 is 7
orders of magnitude lower than that of [Gd(DO3A)]
(H3DO3A = 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid, log KGdL = 21.0),38 and thus the Ln3+ ions in LnL
complexes are expected to be coordinated to the DO3A unit,
provided that the thermodynamic equilibrium is attained. The
hydration number determined from luminescence lifetime
measurements suggests that the Ln3+ ion is coordinated by
the DO3A unit of the ligand, as [Ln(NOTA)] complexes were
reported to have 2−4 coordinated water molecules.39 The
absence of coordinated water molecules is in line with the
results obtained by Parker et al. for complexes with a DO3A
ligand containing a N-linked CH2CH2CH2NHCO−pyridyl
moiety.40 Furthermore, the absence of inner-sphere water
molecules points to the coordination of the amide oxygen atom
of the linker to the Ln3+ ion, as proposed by Parker and co-
workers for Ln3+ complexes containing the same linker.40
Coordination of the amide oxygen atoms was also proposed for
related Ca2+-sensitive probes on the basis of NMR data
recorded for the Y3+ complex.27 Indeed, the noncoordination of
the amide oxygen atom would result in a coordination
environment typical of DO3A derivatives, which contain at
least a coordinated water molecule.41
The EuL and GdL complexes were investigated by means of
density functional theory (DFT) calculations using the TPSSh
functional and the large-core relativistic ECP of Dolg et al.,
following the methodology reported in recent computational
studies (see Computational details below).29,43 Considering the
protonation constants reported for TACN derivatives, this
moiety is expected to be protonated at about neutral pH,34 and
therefore our calculations were performed on the LnHL+ model
systems (Ln = Eu or Gd). Geometry optimizations provided
the expected SAP and TSAP isomers as minimum energy
conformations. According to these computations the SAP
isomer is more stable than the TSAP one, the free energy
differences between the two forms being 10.3 (EuL) and 10.8
kJ mol−1 (GdL). Thus, most likely these complexes present an
SAP coordination in aqueous solution (Figure 3). As expected
The 1H NMR spectrum of the EuL complex presents a large
number of relatively broad paramagnetically shifted signals in
the range from +25 to −20 ppm (Figure 2). In Eu3+ complexes
Figure 3. Optimized geometry of the SAP isomer of GdHL+ obtained
from DFT calculations (TPSSh/6-31G(d,p)). H atoms are omitted for
simplicity, except those involved in hydrogen bonds.
due to their negative charge, the Gd−O distances involving
oxygen atoms of carboxylate groups (2.33−2.35 Å) are shorter
than the Gd−O amide distance (2.42 Å). Overall, the distances
between the Gd3+ ion and the ligand donor atoms are in good
agreement with those observed for complexes of this ion with
different cyclen-based ligands.9a The protonated NO2A unit is
placed above the GdDO3A entity, which likely prevents the
coordination of a water molecule to the metal ion. This
conformation appears to be stabilized by the presence of a
hydrogen-bond involving the amide NH group and one of the
nitrogen atoms of the NO2A moiety (Figure 3).
Zn2+ Binding Studies. The high-resolution mass spec-
trometry (HR-MS) of an aqueous solution of TbL in the
presence of equimolar Zn2+ presents a peak due to the
[NaTbZnL]+ entity, which demonstrates the formation of the
heterodinuclear complex (Figure S4, Supporting Information).
Addition of Zn2+ to an aqueous solution of TbL at pH 7.4 (0.1
M 3-(N-morpholino)propanesulfonic acid (MOPS) buffer)
causes a decrease of the emission intensity due to the Tb3+-
1
Figure 2. H NMR spectra of EuL recorded in D2O solution (pD =
7.0, 300 MHz, 25 °C) in the absence and in the presence of 1 equiv of
Zn2+.
of DOTA, DOTA−tetraamide, and DO3A derivatives the
signals of the pseudo axial protons on the cyclen rings are
usually found between 24 and 45 ppm in the square
antiprismatic (SAP) isomer and below 20 ppm in the
twisted-square antiprismatic (TSAP) isomer.42 These protons
are observed in the range from +15 to +25 ppm for EuL, and
therefore an unequivocal assignment of the structure of the
complex in solution is not possible in this case.
centered 5D4
→
7FJ transitions (Figure 1), with a sharp
inflection point being observed upon addition of 1 equiv of
Zn2+. Addition of an excess of the divalent metal ion does not
cause further changes in the emission intensity nor in the
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emission lifetime of the 5D4 excited state. The emission lifetime
decreases from 2.22 to 1.37 ms upon addition of 1 equiv of
Zn2+ in H2O solution. The emission lifetime recorded in D2O
solution in the presence of Zn2+ was τD2O = 2.18 ms, which
corresponds to a hydration number q = 1.07 as estimated by the
relationship proposed by Parker.33 This result points to an
increase of the number of coordinated water molecules from 0
to 1 upon Zn2+ binding. This suggests that the amide oxygen
atom of the linker connecting the cyclen and TACN fragments
of the ligand remains coordinated to the Ln3+ ion, as Ln3+
DO3A derivatives often have hydration numbers close to two.44
The 1H NMR spectrum of the EuL complex (Figure 2)
experiences only slight changes upon Zn2+ addition, which also
supports that the coordination environment of the Ln3+ ion
remains very similar. DFT calculations support this hypothesis,
as they show that coordination of the amide group to the
lanthanide ion and pentadentate coordination of Zn2+ to the
NO2A moiety are feasible (Figure 4). Both metal ions are likely
Given that the amide oxygen atom is likely to be coordinated to
the Gd3+ ion both in the absence and in the presence of Zn2+,
the driven force behind the hydration number increase
observed upon Zn2+ binding is not obvious. We hypothesize
that the increased positive charge of the system upon Zn2+
coordination is mainly responsible for this effect. The absence
of coordinated water molecules in the case of the charge neutral
DO3A complex of Parker and co-workers, which contains an
amide group with the same linker as L,40 together with the
presence of a coordinated water molecule in a very similar Gd3+
complex that presents a positive charge,32 are in line with this
hypothesis.
The efficiency of a given paramagnetic complex as an MRI
contrast agent is often assessed in vitro in terms of relaxivity,
which is defined as the longitudinal relaxation rate enhance-
ment of water proton nuclei per millimolar concentration of the
paramagnetic probe. The relaxivity of GdL measured at 25 °C
and 7 T amounts to 3.08
0.08 mM−1 s−1, a value that is
compatible with the absence of inner sphere water
molecules.27,28 As a result, the observed relaxivity arises from
the outer-sphere mechanism, which is related to the close
diffusion of outer-sphere water molecules in the vicinity of the
paramagnetic center. Addition of Zn2+ induces a dramatic
increase of the relaxivity, which reaches a value of 7.50 0.08
mM−1 s−1 upon addition of 1 equiv of Zn2+ (Figure 5). This
represents almost a 150% relaxivity increase, which can only be
explained by the coordination of a water molecule to the Gd3+
ion, although a contribution of second-sphere water molecules
to the overall relaxivity cannot be ruled out.45 The relaxivity
measured in the presence of 1 equiv of Zn2+ is very similar to
those reported for related Ca2+- and neurotransmitter-sensitive
probes.27,32 Furthermore, it is lower than those observed for
Ca2+-sensitive probes exhibiting second-sphere contribution to
relaxivity.45 In those cases, the hydration numbers were
confirmed with luminescence measurements (using both Eu3+
and Tb3+ complexes) and extensive NMRD and 17O NMR
studies, which provided 17O hyperfine coupling constants in
agreement with monohydrated complexes.
The steep curvature of the titration profile around the 1:1
(GdL/Zn2+) stoichiometry ratio is characteristic of an especially
high association constant, preventing the determination of an
accurate equilibrium constant.46 Interestingly, addition of Ca2+
or Mg2+ instead of Zn2+ causes only slight relaxivity
enhancements (16 and 6%, respectively), which shows that
the GdL probe provides selective response for Zn2+ over the
main competitors in vivo. In line with these results addition of
Ca2+ or Mg2+ to the TbL complex provokes very minor changes
Figure 4. Optimized geometry of the SAP isomer of GdZnL2+
obtained from DFT calculations (TPSSh/6-31G(d,p)). H atoms are
omitted for simplicity, except those involved in hydrogen bonds.
to complete coordination numbers 6 (for Zn2+) and 9 (for
Gd3+) with the presence of a coordinated water molecule.
Figure 5. Relaxometric studies of GdL with endogenous cations. (left) Titration with Zn2+. The r1 values are represented as mean SD of three
independent experiments and are normalized to [Gd3+] = 1 mM. (right) Competition experiments of Ca2+, Mg2+, or Cu2+ with Zn2+ in the presence
of GdL (3 mM). Small SD values prevent visualization of error bars. In all cases the experiments were performed at 7 T magnetic field strength and
pH 7.4 (HEPES).
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Figure 6. T1-weighted MR images obtained from the imaging scanner operating at 7 T magnetic field. (left) Solutions of GdL (1 mM) mixed with
different concentrations of Zn2+ at pH 7.4 (HEPES). (right) Control experiments with GdDOTA (1 mM, pH 7.4, HEPES) or water in absence or
presence of Zn2+ (1 mM). The graphs placed below the MR images show the SNR calculated for each of the solutions.
in the luminescence emission intensity (Figure S5, Supporting
Information). Furthermore, the stability constants reported for
complexes of the TACN derivative NOTA show that the
complex formed with Zn2+ is 8−9 orders of magnitude more
stable than those formed with Ca2+ and Mg2+, and thus the
presence of these metal ions even in large excess is not expected
to interfere with Zn2+ binding.47 As expected, relaxivity studies
show that Cu2+ induces a relaxivity response comparable to that
of Zn2+ (Figure 5), while the [Cu(NOTA)]− complex was
shown to be more stable than the Zn2+ analogue.47 However,
this is likely not an important drawback for Zn2+ sensing in
vivo, given the very low concentration of Cu2+ ion the body.48
The relaxivity enhancement observed for GdL in the
presence of Zn2+ was further investigated by performing MRI
experiments with tube phantoms (Figure 6) under the same
conditions as in the relaxometric studies (7 T, pH 7.4, HEPES).
For the sake of comparison, MRI experiments were also
conducted using identical acquisition parameters and tubes
with solution of GdDOTA and water as controls. In the
absence of Zn2+, GdL provides less contrast enhancement,
almost comparable to GdDOTA (r1 ≈ 3.9 mM−1 s−1).49
Addition of Zn2+ to a solution of GdL provides a clear contrast
enhancement, while the presence of Zn2+ does not provoke any
contrast change in the solution of GdDOTA. Finally, no
contrast enhancement was observed in tubes that did not
contain any of the Gd3+ complexes but only water, either in
absence or presence of Zn2+. The signal-to-noise ratios (SNR)
calculated for the solutions of GdL in the absence and in the
presence of Zn2+ confirm the contrast enhancement (Figure 6).
CONCLUSIONS
■
In this work we have reported the synthesis of a heteroditopic
ligand that yields a MRI contrast agent responsive to Zn2+ upon
Gd3+ complexation. A combined study including luminescence
lifetime measurements, 1H NMR spectroscopy, and DFT
calculations indicates octadentate coordination of the ligand to
Gd3+ in GdL, which lacks inner-sphere water molecules.
Coordination of Zn2+ to the TACN unit of the ligand triggers
the coordination of a water molecule to Gd3+, resulting in a
150% relaxivity enhancement at 7 T and 25 °C. This
represents, to the best of our knowledge, the highest relaxivity
response to Zn2+ of a Gd3+ probe at high field, although
relaxivity changes as high as 200% have been reported at lower
fields.20b Furthermore, Ca2+ and Mg2+ provoke very small
relaxivity changes, showing that GdL provides a selective
response toward Zn2+ over their main competitors. MRI studies
in vitro confirmed the ability of GdL to provide remarkable
response to Zn2+ in terms of an enhanced contrast.
EXPERIMENTAL AND COMPUTATIONAL SECTION
■
General Methods. Reagents were purchased from commercial
sources unless otherwise specified. 1,4,7-Triazacyclononane was
purchased from CheMatech (Dijon, France). Acetonitrile, tetrahy-
drofuran (THF), toluene, and chloroform were distilled before use.
Compound 6 was synthesized according to previously published
procedure.28 ESI-TOF mass spectra were recorded using a LC-Q-q-
TOF Applied Biosystems QSTAR Elite spectrometer or a MAXis 4G,
Bruker Daltonics Inc, Germany, using both the positive and negative
modes. High-resolution mass spectra were recorded on a Bruker
Daltonics APEX II (FT-ICR-MS) with an ESI source. UV−vis spectra
were recorded on a PerkinElmer Lambda 900 spectrophotometer in
1.0 cm path quartz cells. Excitation and emission spectra were
recorded on a PerkinElmer LS-50B spectrometer. Luminescence
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lifetimes were calculated from the monoexponential fitting of the
average decay data, and they are averages of at least 3−5 independent
(4-{3-[2-(4,7-Bis-carboxymethyl-[1,4,7]triazonan-1-yl)-acetylami-
no]-propyl}-7,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-
1-yl)-acetic acid (L). Compound 7 (0.33 mmol, 300 mg) was dissolved
in anhydrous dichloromethane (5 mL), and the solution was cooled to
0 °C. Trifluoroacetic acid (TFA; 5 mL) was slowly added, and the
solution was stirred at RT for 16 h. The reaction mixture was
concentrated under reduced pressure, and the residue was dissolved in
a small portion of methanol. The product was precipitated from
diethyl ether, isolated by decantation, and dried under reduced
pressure to give H5L·5CF3COOH as a pale brown hygroscopic solid
1
determinations. H and 13C NMR spectra of organic molecules were
recorded with a Bruker AMX-3 300 (300 MHz) or a Bruker Avance III
1
300 MHz spectrometer. H NMR spectra of the EuL complex were
recorded at 25 °C on a Bruker Avance 300 MHz spectrometer.
Relaxometric titrations were performed using a Bruker Avance III 300
MHz spectrometer. Magnetic resonance imaging experiments at field
strength of 7 T were performed on a BioSpec 70/30 USR magnet
using Bruker quadrature volume coil (RF RES 300 1H 112/86 QSN
TD AD).
1
(300 mg, 73%). H NMR (300 MHz, D2O): δ 1.84−2.03 (br, 2H,
1-Benzyl-[1,4,7]-triazacyclononane (3). N-Dimethoxymethyl-N,N-
dimethylamine (670 mg, 5.60 mmol, 1.1 equiv) was added to a
solution of TACN (660 mg, 5.10 mmol) in chloroform (1 mL) and
toluene (10 mL). The solution was stirred at RT for 12 h. The solvent
was then evaporated under reduced pressure to yield an oily product.
Benzyl bromide (1.1 equiv, 5.62 mmol, 670 μL) in solution in 15 mL
of freshly distilled THF was added to the previous crude product. The
precipitated white solid was filtered after the mixture was stirred at RT
for 3 d, washed with THF, and dried under vacuum. The white solid
was dissolved in 10 mL of a solution of methanol/HCl 12 M (1:1) and
stirred at reflux for 12 h. After it cooled, the pH was raised to 12 by
addition of NaOH pellets. Extraction with chloroform (3 × 15 mL),
drying with MgSO4, and evaporation of the solvent under reduced
pressure gave the 1-benzyl-1,4,7-triazacyclononane 3 as a yellow oil
CH2), 2.73−4.13 (br, 44H, CH2). 13C NMR (75 MHz, D2O): δ 23.2,
35.9, 36.6, 48.6, 48.9, 49.5, 49.9, 50.7, 51.3, 51.9, 53.3, 53.5, 55.7, 56.6,
58.9, 60.9, 116.3 (TFA), 162.9 (TFA), 169.8, 172.7, 172.8, 174.1,
+
175.1. ESI-HRMS (m/z): [M + H]+ calcd. for C29H53N8O11
,
689.3828, found 689.3830.
Computational Details. All calculations reported in this work
were performed employing the Gaussian 09 package (Revision
D.01).50 Full geometry optimizations of the LnHL+ and LnZnL2+
systems were performed in aqueous solution employing DFT within
the hybrid meta-generalized gradient approximation (hybrid meta-
GGA), with the TPSSh exchange-correlation functional.51 Geometry
optimizations were performed by using the large-core quasirelativistic
effective core potential of Dolg and coworkers and its associated
[5s4p3d]-GTO valence basis set,52 while the ligand atoms and Zn
were described by using the standard 6-31G(d,p) basis set. No
symmetry constraints were imposed during the optimizations. The
stationary points found on the potential energy surfaces as a result of
geometry optimizations were tested to represent energy minima rather
than saddle points via frequency analysis. The default values for the
integration grid (75 radial shells and 302 angular points) and the self-
consistent field energy convergence criteria (1 × 10−8) were used in all
calculations. Solvent effects were included by using the integral
equation formalism of the polarizable continuum model as
implemented in Gaussian 09.53
1
(1.01g, 4.60 mmol, 90%). H NMR (300 MHz, CDCl3): δ 2.53 (m,
8H, CH2TACN), 2.80 (s, 4H, CH2TACN), 3.07 (s, 2H, NH), 3.60 (s, 2H,
CH2Ph), 7.12−7.23 (m, 5H, C6H5). 13C NMR (75 MHz, CDCl3): δ
46.1, 46.2, 52.3, 61.2, 126.7, 127.9, 128.6, 139.2.
(4-Benzyl-7-tert-butoxycarbonylmethyl-[1,4,7]-triazonan-1-yl)-
acetic acid tert-butyl ester (4). Compound 3 (1.01g, 4.60 mmol) was
dissolved in 15 mL of freshly distilled acetonitrile, and 1.8 equiv of tert-
butyl bromoacetate (1.61 g, 8.28 mmol) and K2CO3 (excess) were
added. After it was stirred at RT for 4 d, the solution was filtered.
Evaporation of the solvent gave an oily crude product. Purification was
performed by chromatography on silica gel (elution with hexanes/
CHCl3 2:1, Rf = 0.7). Compound 4 was obtained as a clear oil (1.30 g,
1
2.90 mmol, 63%). H NMR (300 MHz, CDCl3): δ 1.42 (s, 18H,
Relaxometric Experiments. For titration experiments, a solution
of ZnCl2 of known concentration was added stepwise to a solution of
GdL (starting concentration 3.0 mM), and the longitudinal proton
relaxation time T1 was measured after each addition of the analyte.
The 1H relaxivity r1 was calculated from the eq 1/T1,obs = 1/T1,d + r1 ×
[Gd3+], where T1,obs is the observed longitudinal relaxation time, T1,d is
the diamagnetic contribution in the absence of the paramagnetic
substance, and [Gd3+] is the actual concentration of Gd3+ at each point
of the titration. The initial Gd3+ concentrations were determined by
measuring the bulk magnetic susceptibility shifts.54 The concentration
of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
was 50 mM. The reported values are mean values of three independent
experiments.
Competition experiments were performed in a similar fashion. The
longitudinal proton relaxation time T1 was measured for the solution
of GdL (starting concentration 3.0 mM) alone, and then after addition
of the following solutions: CaCl2, MgCl2, or CuSO4 (2 × 1 equiv).
The final values were measured upon addition of ZnCl2 (2 equiv) to
these mixtures.
C(CH3)3), 2.78 (bs, 4H, CH2TACN), 2.81 (bs, 4H, CH2TACN), 2.92 (s,
4H, CH2TACN), 3.28 (s, 4H, CH2CO2tBu), 3.65 (s, 2H, CH2Ph), 7.14−
7.32 (m, 5H, C6H5). 13C NMR (75 MHz, CDCl3): δ 28.2, 55.2, 55.3,
55.6, 59.9, 62.6, 80.4, 126.6, 128.0, 128.9, 140.2, 171.4.
(4-tert-Butoxycarbonylmethyl-[1,4,7]-triazonan-1-yl)-acetic acid
tert-butyl ester (5): NO2AtBu. Compound 4 (1.30 g, 2.90 mmol)
was dissolved in absolute ethanol, and 10% Pd/C activated was added.
The reaction mixture was left under hydrogen atmosphere at RT
under stirring for 4 d. After filtration through Celite, ethanol was
removed by evaporation at reduced pressure to produce 5 as a yellow
1
oil (790 mg, 2.21 mmol, 76%). H NMR (300 MHz, CDCl3): δ 1.40
(s, 18H, C(CH3)3), 2.71 (s, 4H, CH2TACN), 3.01 (m, 4H, CH2TACN),
3.19 (m, 4H, CH2TACN), 3.32 (s, 4H, CH2CO2tBu). 13C NMR (75
MHz, CDCl3): δ 28.1, 44.5, 49.2, 51.9, 56.7, 81.8, 170.7.
(4-{3-[2-(4,7-Bis-tert-butoxycarbonylmethyl-[1,4,7]-triazonan-1-
yl)-acetylamino]-propyl}-7,10-bis-tert-butoxycarbonylmethyl-
1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid tert-butyl ester (7).
The bromide 6 (750 mg, 1.05 mmol) was dissolved in acetonitrile (5
mL) and added slowly to the suspension of the azamacrocycle 5 (320
mg, 0.90 mmol) and potassium carbonate (371 mg, 2.69 mmol) in
acetonitrile (20 mL). The reaction mixture was stirred at 70 °C for 16
h. After it cooled, the mixture was filtered, and the solvent was
evaporated under reduced pressure. The residue was purified using
column chromotography (silica gel, 5% methanol/dichloromethane, Rf
= 0.3) to give compound 7 as a light brown solid (330 mg, 0.34 mmol,
Magnetic Resonance Imaging Experiments. 1H MRI was
performed on two sets of probes: (1) A solution of GdL (1 mM) to
which 0, 0.33, 0.66, and 1 equiv of Zn2+ were added; (2) solutions of
Dotarem (1 mM) in absence and presence of Zn2+ (1 equiv), or water
without or with Zn2+ (1 mM). These sets were placed in 4 × 200 μL
plastic tubes and inserted in the 20 mL syringe filled with water and
1
38%). H NMR (300 MHz, CDCl3): δ 1.41 (s, 9H, CH3), 1.42 (s,
18H, CH3), 1.48 (s, 18H, CH3), 1.77 (br, 2H, CH2), 1.95−3.73 (br,
42H, CH2), 4.10 (s, 2H, CH2), 9.02 (s, 1H, NH). 13C NMR (75 MHz,
CDCl3): δ 24.3, 27.3, 27.5, 27.7, 37.2, 48.3, 50.0, 51.4, 51.7, 52.9, 55.2,
56.0, 56.4, 81.5, 82.1, 82.4, 169.3, 169.5, 171.0, 173.0. ESI-HRMS (m/
z): [M + H]+ calcd. for C49H93N8O11+, 969.6958, found 969.6950, [M
+ Na]+ calcd. for C49H92N8NaO11+, 991.6778, found 991.6768.
1
162 mM of Dotarem to avoid susceptibility artifacts. H T1 images
were acquired using the fast low angle shot pulse sequence: field-of-
view = 40 × 40 mm2, matrix size = 512 × 512, one slice, slice thickness
1 mm, flip angle = 90 o, echo time = 3.0 ms, repetition time = 60.0 ms,
number of averages = 4. The total acquisition time was 1m 1s 440 ms.
G
DOI: 10.1021/acs.inorgchem.5b01719
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b01719.
■
S
UV−vis spectra from titration experiments, HR-MS
spectra, excitation and emission spectra of TbL, NMR
spectra, and optimized Cartesian coordinates obtained
with DFT calculations. (PDF)
(14) (a) Chauvin, T.; Durand, P.; Bernier, M.; Meudal, H.; Doan, B.-
́
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T.; Noury, F.; Badet, B.; Beloeil, J.-C.; Toth, E. Angew. Chem., Int. Ed.
2008, 47, 4370−4372. (b) Duimstra, J. A.; Femia, F. J.; Meade, T. J. J.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: carlos.platas.iglesias@udc.es. (C.P.-I.)
*E-mail: goran.angelovski@tuebingen.mpg.de. (G.A.)
Notes
■
(15) (a) Que, E. L.; Chang, C. J. Chem. Soc. Rev. 2010, 39, 51−60.
(b) Bonnet, C. S.; Toth, E. Future Med. Chem. 2010, 2, 367−384.
́
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T.; Sakamoto, S.; Yamaguchi, K.; Nagano, T. Chem. Biol. 2002, 9,
1027−1032. (b) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Nagano, T. J.
Chem. Soc., Perkin Trans. 2 2001, 1840−1843.
The authors declare no competing financial interest.
(17) (a) Lubag, A. J. M.; De Leon
Sherry, A. D. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 18400−18405.
(b) De Leon-Rodríguez, L. M.; Lubag, A. J. M.; Lopez, J. A.; Andreu-
de-Riquer, G.; Alvarado-Monzon, J. C.; Sherry, A. D. MedChemComm
2012, 3, 480−483. (c) Esqueda, A. C.; Lopez, J. A.; Andreu-de-Riquer,
G.; Alvarado-Monzon, J. C.; Ratnakar, J.; Lubag, A. J. M.; Sherry, A.
D.; De Leon-Rodríguez, L. M. J. Am. Chem. Soc. 2009, 131, 11387−
11391.
́
-Rodríguez, L. M.; Burgess, S. C.;
ACKNOWLEDGMENTS
We are thankful to T. Savic
■
́
́
́
for performing the MRI
́
experiments. C.P.-I., D.E.-G., and M.R.-F. thank Ministerio de
́
́
Economia y Competitividad (CTQ2013-43243-P) for generous
financial support and Centro de Supercomputacion de Galicia
(CESGA) for providing the computer facilities. R.T. and V.P.
acknowledge the Ministere de l’Enseignement Superieur et de
́
́
́
̀
́
(18) Rivas, C.; Stasiuk, G. J.; Sae-Heng, M.; Long, N. J. Dalton Trans.
la Recherche, the Centre National de la Recherche Scientifique.
The authors gratefully acknowledge financial support from the
Max Planck Society, the Turkish Ministry of Education (Ph.D.
fellowship to S.G.), and the COST CM1006 Action “European
F-Element Network (EUFEN)”. This work is dedicated to
2015, 44, 4976−4985.
(19) Zhang, X.-a.; Lovejoy, K. S.; Jasanoff, A.; Lippard, S. J. Proc. Natl.
Acad. Sci. U. S. A. 2007, 104, 10780−10785.
(20) (a) Major, J. L.; Parigi, G.; Luchinat, C.; Meade, T. J. Proc. Natl.
Acad. Sci. U. S. A. 2007, 104, 13881−13886. (b) Matosziuk, L. M.;
Leibowitz, J. H.; Heffern, M. C.; MacRenaris, K. W.; Ratner, M. A.;
Meade, T. J. Inorg. Chem. 2013, 52, 12250−12261. (c) Major, J. L.;
Boiteau, R. M.; Meade, T. J. Inorg. Chem. 2008, 47, 10788−10795.
(21) Luo, J.; Li, W.-S.; Xu, P.; Zhang, L.-Y.; Chen, Z.-N. Inorg. Chem.
2012, 51, 9508−9516.
́
Professor Imre Toth on occasion of his 65th birthday.
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