Structural Features of Europium(II)-Containing Cryptates That Influence Relaxivity

Article abstract of DOI:10.1002/chem.201702158

Eu^II-containing complexes were studied with respect to properties relevant to their use as contrast agents for magnetic resonance imaging. The influences of molecular parameters and field strength on relaxivity were studied for a series of Eu^II-containing cryptates and their adducts with β-cyclodextrins, poly-β-cyclodextrins, and human serum albumin. Solid- and solution-phase characterization of Eu^II-containing complexes is presented that demonstrates the presence of inner-sphere molecules of water. Additionally, relaxivity, water-exchange rate, rotational correlation time, and electronic relaxation times were determined using variable-temperature ¹⁷O NMR, nuclear magnetic relaxation dispersion, and electron paramagnetic resonance spectroscopic techniques. These results are expected to be instrumental in the design of future Eu^II-based contrast agents.

Full text of DOI:10.1002/chem.201702158

A Journal of
Accepted Article
Title: Structural Features of Eu(II)-Containing Cryptates That Influence
Relaxivity
Authors: Chamika U Lenora, Fabio Carniato, Yimin Shen, Zahid Latif,
E. Mark Haacke, Philip D Martin, Mauro Botta, and Matthew
John Allen
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To be cited as: Chem. Eur. J. 10.1002/chem.201702158
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Structural Features of EuII-Containing Cryptates That Influence
Relaxivity
Chamika U. Lenora,[a] Fabio Carniato,[b] Yimin Shen,[c] Zahid Latif,[c][d] E. Mark Haacke,[c][d] Philip D.
Martin,[e] Mauro Botta,*[b] and Matthew J. Allen*[a][d]
Abstract: EuII-containing complexes were studied with respect to
properties relevant to their use as contrast agents for magnetic
resonance imaging. The influences of molecular parameters and
field strength on relaxivity were studied for a series of EuII-containing
applied magnetic field and molecular parameters, including
hydration number (q), water-exchange rate (kex), and rotational
correlation time (τR). An understanding of the relationships
among the structures of paramagnetic complexes, the molecular
cryptates and their adducts with β-cyclodextrins, poly-β-cyclodextrins, parameters that influence relaxivity, and magnetic field strength
and human serum albumin. Solid- and solution-phase
characterization of EuII-containing complexes is presented that
demonstrates the presence of inner-sphere molecules of water.
Additionally, relaxivity, water-exchange rate, rotational correlation
time, and electronic relaxation time were determined using variable-
temperature 17O-NMR, nuclear magnetic relaxation dispersion, and
electron paramagnetic resonance spectroscopic techniques. We
expect that our results will be instrumental in the design of future
EuII-based contrast agents.
is crucial to the ability to rationally design contrast agents. The
effects of paramagnetic metal ions on the relaxation of water
molecules
are
often
described
using
the
Solomon−Bloembergen−Morgan equations.[4] Relative to GdIII-
based contrast agents, the applicability of these equations to
EuII-containing complexes is understudied. To obtain
experimental validation of how molecular parameters influence
the relaxivity of EuII at different field strengths, we studied three
EuII-containing complexes and supramolecular adducts of one of
the complexes with cyclodextrins and albumin. These
complexes and inclusion complexes that differ in one or more
molecular parameters were studied over a range of field
strengths. Here, we report the findings from these studies in
terms of the relationships among the structures of EuII-
containing cryptates; molecular parameters including q, kex, and
τR; and field strengths from 0.00021 to 11.7 T.
Introduction
Magnetic resonance imaging (MRI) is a noninvasive technique
used to visualize the interior of objects with high spatial
resolution.[1] The contrast observed in most MR images can be
enhanced with contrast agents that modulate the attributes,
including longitudinal (T1) relaxation times, of protons on
proximal water molecules. Contrast agents for MRI are often
paramagnetic GdIII-based complexes,[2] but EuII-containing
complexes have been studied as redox-active alternatives to
GdIII-based complexes.[3] EuII is isoelectronic with GdIII and
shortens the T1 relaxation times of nearby water protons to a
similar extent as GdIII, leading to enhanced contrast in T1-
weighted images. The ability to shorten T1 relaxation times is
measured in terms of relaxivity (r1), and the relaxivity of a
paramagnetic contrast agent depends on factors such as the
Results and Discussion
Synthesis and characterization: To understand how molecular
parameters influence relaxivity, we studied EuII-containing
complexes 1−3 (Figure 1) and supramolecular inclusion
complexes of complex 3 with β-cyclodextrin (β-CD), poly-β-
cyclodextrin (poly-β-CD), and human serum albumin (HSA).
Ligands for EuII-containing complexes 1 and 2 are commercially
available, and the synthesis of ligand 7, which was used to form
complex 3, is shown in Scheme 1. Briefly, 3-(biphenyl-4-
yl)propane-1,2-diol was treated with methyl bromoacetate to
obtain diacetate derivative 4 that was saponified then acidified to
afford diacid 5. The diacid was converted to diamide 6 via
stepwise reactions with thionyl chloride followed by diaza-18-
crown-6. The diamide was reduced with borane-tetrahydrofuran
(BH3·THF) complex to afford ligand 7.
[a]
C. U. Lenora, Prof. M. J. Allen
Department of Chemistry
Wayne State University
5101 Cass Avenue, Detroit, Michigan 48202, USA
E-mail: mallen@chem.wayne.edu
[b]
Dr. F. Carniato, Prof. M. Botta
Dipartimento di Scienze e Innovazione Tecnologica
Universitâ del Piemonte Orientale "Amedeo Avogadro", Viale T.
Michel 11, I-15121 Alessandria, Italy
E-mail: mauro.botta@mfn.unipmn.it
[c]
[d]
[e]
Dr. Y. Shen, Dr. Z. Latif, Prof. E. M. Haacke
Department of Radiology, Wayne State University School of
Medicine 3990 John R Street, Detroit, MI 48201, USA
Dr. Z. Latif, Prof. E. M. Haacke, Prof. M. J. Allen
Barbara Ann Karmanos Cancer Institute
4100 John R Street, Detroit, MI 48201, USA
Dr. Philip D. Martin
Figure 1. EuII-containing cryptates used in this study. Complexes are drawn
with two coordinated molecules of water, and counter anions are not shown for
clarity.
Lumigen Instrument Center, Chemistry Department, Wayne State
University, 5101 Cass Avenue, Detroit, MI 48202, USA
Supporting information for this article is given via a link at the end of
the document.
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molar conductivities of metal complexes 1, 2, and 3 were 177 ±
3, 183 ± 6, and 199 ± 4 S cm2 mol−1, respectively. These values
are consistent with 2:1 dissociation in water,[7] indicating that, on
average, both chloride ions are dissociated from EuII in solution.
Further, 17O-NMR line-broadening (Tables S2−S4) in the
presence of EuII-containing complexes is consistent with the
presence of inner-sphere water, suggesting that chloride
dissociation enables the coordination of water. Both conductivity
and 17O-NMR data are consistent with the presence of EuII-
containing complexes that exist in solution as nine-coordinate
complexes with one inner-sphere water molecule, ten-coordinate
complexes with two inner-sphere water molecules, or a mixture
of nine- and ten-coordinate complexes. After studying the
coordination environment of EuII-containing cryptates 1−3 in
solution, we focused on studying the ability of the complexes to
influence relaxation enhancement. Because we expected EuII-
containing complexes to behave similarly to GdIII-containing
complexes with respect to relaxivity and because aggregation of
small GdIII-containing complexes results in increased positive
relaxation enhancement,[8] we studied the influence of
concentration on relaxation-rate enhancement to determine if,
and at what concentration, aggregates form with complexes 1−3.
Scheme 1. Synthesis of Ligand 7.
EuII-containing complexes 1−3 were prepared by mixing ligands
and EuCl2 in water followed by adjustment of pH to 7.4 with
phosphate-buffered saline. Formation of complexes 1 and 2
were monitored using water-proton-relaxation-rate enhancement.
In this experiment, water-proton-relaxation rate was measured
as a function of the amount of metal added to a solution of
ligand. Relaxation rate decreased with the addition of ligand up
to a 1:1 ligand-to-metal ratio (Figures S1 and S2), and after that
point, the rate did not change [analysis of variance (ANOVA
single factor analysis, 95% confidence interval)]. Because EuCl2
and complexes 1 and 2 have different relaxivity values, these
data suggest that in solution, complexes 1 and 2 exist as 1:1
ligand-to-metal complexes. However, the relaxivity of complex 3
is similar to that of EuCl2, preventing the monitoring of the
formation of complex 3 via proton relaxation rate. Also, the
UV−visible absorption profiles of complex 3 and EuCl2 overlap,
indicating that UV−visible spectroscopy does not provide useful
information regarding the formation of EuII-containing complex 3.
Therefore, we used inductively coupled plasma mass
spectrometry (ICP−MS) to investigate the complexation of EuII
by cryptand 7 to form complex 3. We prepared two samples of 7
and EuCl2 with ligand-to-metal ratios of 1:2 and 1:1. Metal not
bound to cryptand was precipitated with phosphate-buffered
saline,[5] and the concentrations of Eu in the supernatants were
measured using ICP−MS. The metal concentrations after
precipitation were consistent with the formation of 1:1 ligand-to-
metal complexes, even in the presence of excess metal (Table
S1). Once complex formation was verified, we characterized the
complexes in the solid state and in solution.
To study coordination environments in the solid state, we
obtained X-ray crystal structures of 1 and 2. We were unable to
grow X-ray quality crystals of complex 3. Complex 1 displays a
coordination number of nine in an eclipsed hula-hoop geometry
(Figure 2a).[6] Eight coordination sites are occupied by the six
oxygen atoms and two nitrogen atoms of the cryptand, and the
ninth coordination site is occupied by chloride. Similarly, the
Figure 2. Crystal structures of (a) cryptate 1 (R-factor = 0.0259, resolution =
0.75 Å) and (b) cryptate 2 (R-factor = 0.0203, resolution = 0.55 Å). Thermal
ellipsoids are drawn at the 50% probability level. Hydrogen atoms and outer-
sphere chloride counter anions are omitted for clarity.
crystal structure of complex
2 features a nine-coordinate
eclipsed hula-hoop geometry (Figure 2b), where EuII is
coordinated by six oxygen atoms and two nitrogen atoms from
the cryptand and one chloride ion. These data indicate that, in
the solid state, 1 and 2 exist as 1:1 metal-to-ligand complexes,
Aggregation Studies: To determine if relaxometric properties
allude to the aggregation of complexes 1−3, longitudinal water-
consistent
with
proton-relaxation-rate-enhancement
measurements in solution. Additionally, the crystal structures
provide valuable bond distances for modeling relaxometric
properties in solution. Accordingly, we turned our attention to
further studying the coordination environments of 1−3 in solution.
To investigate the coordination of chloride in solution, we
measured the molar conductivity of complexes 1−3 in water. The
proton-relaxation rates were measured as
a function of
concentration at 37 oC. For complexes 1 and 2, plots of
relaxation rate versus concentration afforded linear regression
lines (Figure 3). The slopes of the lines afford relaxivities (2.1
and 3.7 mM−1 s−1 for complexes 1 and 2, respectively) that
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match reported values.[9] These values are also consistent with
the relaxivities expected for low molecular weight chelates.
Because the data in Figure 3 can be well-described by one line,
it is unlikely that complexes 1 and 2 aggregate at concentrations
below 10 mM. Unlike complexes 1 and 2, complex 3 showed an
inflection point in the relaxation-rate profile around 3.8 mM
(Figure 4). Below 3.8 mM, a linear relationship was observed
with a relaxivity value of 4.3 mM−1 s−1, expected for complex 3.[9]
A change in the slope of the line was observed above 3.8 mM,
consistent with a higher relaxivity value of 12.1 mM−1 s−1. This
change in slope is likely due to the formation of aggregates
occurring through intermolecular hydrophobic interactions
between biphenyl moieties. Aggregation increases the molecular
tumbling time resulting in larger relaxivities, and these types of
hydrophobic intermolecular interactions have been observed
with similarly functionalized GdIII-containing complexes.[8a,b] To
further study the aggregation of complex 3, dynamic light
scattering was performed at two different concentrations (1 and
8 mM). The data (Figure S4) indicate that complex 3 aggregates
at the higher concentration (8 mM) but not at the lower
concentration (1 mM). This data is consistent with and supports
the relaxometric data. It has also been reported that aggregate
formation leads to high field bumps in nuclear magnetic
relaxation dispersion (NMRD) profiles and affects electronic
relaxation parameters.[8b] We used the results of these
aggregation studies to avoid aggregation by performing all
relaxometric characterizations, including binding-interaction
studies of complex 3 with cyclodextrins and HSA, below the self-
aggregation concentration of complex 3.
Figure 4. Water-proton-relaxation rates (60 MHz, pH = 7.4, 37 °C) versus
concentration of complex 3. The solid lines are linear best fits (R2 = 0.99 for
both lines).
Formation of inclusion compounds of complex 3 with β-
cyclodextrin (β-CD) and poly-β-cyclodextrin (poly-β-CD): The
formation of host–guest non-covalent interactions between
suitably functionalized complexes and slowly tumbling
substrates represents a well-established approach to increase
relaxivity.[10] The presence of the biphenyl moiety in 3 leads to
an interaction of the complex with the hydrophobic cavity of β-
CD. The binding interaction was investigated using the well-
established proton-relaxation-enhancement technique.[11] This
technique consists in measuring the changes of R1 of a solution
of a complex as a function of increasing concentrations of the
host. With this method, the binding parameters KA, n (the
b
number of equivalent and independent binding sites), and r1
(the relaxivity of the final supramolecular adducts) can be
evaluated. A dilute aqueous solution of 3 (0.9 mM) was titrated
with β-CD, at 60 MHz and 310 K, and the least-squares fit of R1
versus the binding isotherm of β-CD concentration (Figure 5)
provided the value of the affinity constant [KA = (4.0 ± 0.2) × 103
M–1] and the relaxivity of the adduct (r1 = 8.7 ± 0.2 mM–1 s–1).
b
Upon binding of complex 3 with β-CD, a relaxivity enhancement
of 2.1× was observed (4.2 ± 0.1 and 8.7 ± 0.2 mM−1 s−1 for 3 and
3/β-CD, respectively). This enhancement of relaxivity (+107%) is
likely an effect of the increase in rotational correlation time (τR)
associated with the increase in molecular weight.[10] This data
led us to use 2.5 equivalents of β-CD for all further relaxometric
studies of 3/β-CD to favor (>99% macromolecule-bound
complex based on KA) complex 3 being associated with β-CD.
Figure 5. Water-proton-relaxation rates (60 MHz, pH = 7.4, 37 °C) of solutions
of 3 (0.9 mM) as a function of the amount of β-CD. Error bars represent the
standard error of the mean of three independently prepared samples.
Figure 3. Water-proton-relaxation rates (60 MHz, pH = 7.4, 37 °C) versus
concentration of complexes 1 () and 2 (). The solid lines are the linear best
fit (R2 = 0.99).
A similar titration was performed to investigate the larger
supramolecular assemblies formed by complex 3 with poly-β-CD.
A solution of complex 3 (1 mM) in phosphate-buffered saline
was titrated with poly-β-CD, and relaxation-rate enhancement
was measured (Figure 6). The poly-β-CD that was used had an
average molecular weight of 19 kDa, corresponding to
approximately 16 β-CD units, each of which can bind in principle
one unit of complex 3. The polymer is characterized by nearly
equivalent binding sites and, therefore, is expected to influence
the rotational dynamics of each bound complex in an
approximately equivalent manner. Data were fit to a simple
model that considers the presence of
n equivalent and
independent binding sites, affording association constants and
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bound relaxivities. The best-fit of the data was obtained with n =
9 and a KA value of 2.4(±0.2) × 103 M–1, similar to the value
found for the interaction with β-CD. Association of complex 3
with poly-β-CD increased relaxivity from 4.2 to 12.5 mM−1 s−1,
which represents an enhancement of +198%, consistent with a
pronounced decrease in molecular tumbling.[8a,10a,10b,12] This data
led us to use one equivalent (with the assumption of nine
binding sites per equivalent) of poly-β-CD for all further
relaxometric studies of 3/poly-β-CD to favor (~95%
macromolecule-bound complex based on KA) complex 3 being
associated with poly-β-CD.
Figure 7. Water-proton-relaxation rates (60 MHz, pH = 7.4, 37 °C) of solutions
of 3 (1.0 mM) as a function of the concentration of HSA. Error bars represent
the standard error of the mean of three independently prepared samples.
Relaxometric characterization: To evaluate the molecular
parameters that determine the efficiency of the EuII-containing
complexes as relaxation agents, variable-field (0.00021 to 11.7
T) longitudinal 1H-NMR relaxation rates (NMRD profiles) were
measured via fast-field-cycling relaxometry and variable-
temperature 17O-NMR spectroscopy was performed. The NMRD
profiles of complexes 1−3 showed that relaxivity was in the order
3 > 2 > 1 at every field studied (Figure 8). The relaxivities of
complexes 1−3 are highest at low field strengths (<0.02 T), and
there is a dispersion between 0.05 and 0.2 T. Around ultra-high
fields (≥7 T), the relaxivities of complexes 1−3 increase from 3 to
7 T and then decrease with increasing field strengths above 7 T.
When complex 3 forms inclusion complexes with cyclodextrins
or HSA, relaxivity changes. The NMRD profiles of inclusion
complexes 3/β-CD and 3/poly-β-CD indicate that these
complexes possess higher relaxivities than complex 3 at all
fields studied (Figure 8). Broad bumps in relaxivity peaking
around 1 T were observed, and these relaxivities decreased at
higher field strengths. When complex 3 interacted with HSA,
relaxivity increased relative to cyclodextrin-bound complexes
below 7 T, with a narrow peak around 0.7 T. In the ultra-high
field region, the relaxivity of 3/HSA was lower than that of 3/β-
CD and 3/poly-β-CD.
Figure 6. Water-proton-relaxation rates (60 MHz, pH = 7.4, 37 °C) of solutions
of 3 (1 mM) as a function of the amount of poly-β-CD. Error bars represent the
standard error of the mean of three independently prepared samples.
Interaction of complex 3 with HSA: In addition to forming
inclusion complexes with cyclodextrins, the biphenyl group can
promote non-covalent interactions with hydrophobic binding
sites on HSA. Binding parameters for the interaction of 3 with
HSA were determined using the proton-relaxation-enhancement
method by measuring R1 of a solution of complex 3 (1 mM) in
phosphate-buffered saline as a function of protein concentration
(Figure 7). The binding interactions of complex 3 with HSA result
in a large relaxivity gain (from 4.2 to 16.6 mM−1 s−1) that we
primarily attribute to the decrease of the rotational dynamics of
the complex in the adduct with the protein.[13] Analysis of the
experimental data was carried out assuming the presence of
one class of equivalent and independent binding sites (n = 1),
even though the presence of multiple sites with lower affinity on
HSA cannot be excluded.[14] In this way, we could estimate the
value of the thermodynamic association constant (KA = 2.7(±0.4)
× 103 M–1), which is of the same order of magnitude as found
with β-CD and polyβ-CD. Consequently, this data led us to use
eight equivalents of HSA for all further relaxometric studies of
3/HSA to favor (~91% macromolecule-bound complex based on
KA) complex 3 being associated with HSA.
After characterizing EuII-containing complexes 1−3 and the
interactions of complex 3 with β-CD, poly-β-CD, and HSA, we
studied the relaxometric behavior of those complexes and
macromolecular systems at different field strengths.
Figure 8. NMRD profiles of solutions of 1 (●), 2 (■), 3 (♦), 3/β-CD (○),
3/poly-β-CD (Δ), and 3/HSA (◊) at pH 7.4 and 298 K. The solid lines were
fitted assuming r = 3.2 Å, q = 2, a = 4.5 Å, and D = 23.0 × 10− m2 s−
.
10
1
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The analysis of these NMRD profiles using the theory of
paramagnetic relaxation enables evaluation of the molecular
parameters and elucidation of the relationship between these
parameters and relaxivity. The field-dependence of r1 arises
from modulation of magnetic dipolar interactions between metal
ions and solvent nuclei. Modulation occurs either through
chemical exchange between bound and bulk water molecules
(inner-sphere)[4a] or through long-range interactions with outer-
sphere water molecules that diffuse near the paramagnetic ions
(outer-sphere).[4a] Experimentally acquired data were analyzed in
terms of the Solomon–Bloembergen–Morgan equations and
Freed’s model for the inner- and outer-sphere contributions,
respectively, to proton relaxivity.[4] In the relaxation equations,
some parameters were set to known or reasonable values:
water-coordination number (q), distance between EuII ions and
the nuclei of inner-sphere (r) and outer-sphere water protons (a),
Table 1. Parameters relevant to the relaxivity of 1−3 and macromolecular
adduct of 3.
Parameters
1
2
3
3/β-CD
3/poly-β-CD 3/HSA
r1 / mM−1 s−1[a]
Δ2 / 1020 s−2[a]
τv298 / ps[a]
τRL298 / ps[a]
τRG298 / ps[a]
S2[a]
3.85
4.61
5.64
13.1
17.8
27.9
1.35 ± 0.08 1.35 ± 0.11 1.23 ± 0.13 0.16 ± 0.01 0.15 ± 0.02
0.09 ± 0.01
64 ± 7
276 ± 18
50,000[c]
0.14 ± 0.01
0.5[c]
3.0 ± 0.3
NA
4.2 ± 0.2
NA
4.1 ± 0.3
NA
23 ± 2
30 ± 2
150 ± 9
454 ± 25
259 ± 13
1920 ± 124
46 ± 2
NA
63 ± 3
NA
93 ± 5
NA
0.43 ± 0.02 0.17 ± 0.02
0.5[c]
kex298 / 108 s–1[b]
ΔHM / kJ mol−1[b]
2.5 ± 0.2
0.5 ± 0.1
0.5 ± 0.1
0.5[c]
33.4 ± 0.9 39.9 ± 1.1 31.1 ± 0.6 NA
NA
NA
A0/ħ / 106 rad s−1[b] −3.9 ± 0.2 −4.2 ± 0.2 −4.2 ± 0.2 NA
NA
NA
r / Å[c]
q[c]
a / Å[c]
3.2
2.0
4.5
3.2
3.2
3.2
3.2
3.2
2.0
2.0
2.0
2.0
2.0
and the diffusion coefficient (D). Moreover, as
a first
4.5
4.5
4.5
4.5
4.5
D298 /10−10 m2 s−1[c] 23
23
23
23
23
23
approximation, the exchange lifetime τM was set to the value
previously determined for 1 by Tóth, i.e. 298τM = 3.2 ns.[15] Water-
coordination numbers of complexes 1−3 were assumed to be
two (q = 2) based on a ten-coordinate EuII-containing cryptate
that was recently reported with two non-adjacent water
molecules.[16] In addition, the isomorphic SrII analog of complex 1,
provides support of a water-coordination number of two.[15]
Another key parameter that is related to coordinated water is the
Eu−H distance between the EuII ions and the proton of
coordinated water molecules. This value was fixed to 3.2 Å,
which was obtained from a crystal structure of a ten-coordinate
EuII-containing cryptate with two non-adjacent inner-sphere
water molecules.[16] The Eu−H distance of 3.2 Å matches the
value reported for another EuII-containing complex (3.2 Å for
ΔHpp / G[d]
1/T2e / 109 s−1[d]
τM310 / ns[b]
178.8
2.70[e]
225.3
3.40[e]
10
265.0
4.00[e]
12
198.7
3.00
12[c]
152.3
2.30
12[c]
217.3
3.28[e]
12[c]
2
[a] Parameters obtained from NMRD fitting. [b] Parameters obtained from
17O-NMR spectroscopy. [c] Parameters fixed during the fitting procedure. [d]
Parameters obtained from X-band EPR spectroscopy at 110 K. [e] Values
obtained from reference 18. NA = not applicable
Water exchange: Variable-temperature 17O-NMR relaxation
rates, R2p, for complexes 1−3 were measured at 9.4 T and pH =
7.4. The data are often reported as reduced transverse
relaxation rates, R2r, defined as 1/T2r = R2r = R2p/pM, where pM is
the molar fraction of inner-sphere water molecules. The reduced
transverse 17O-relaxation rates measured for EuII-containing
complexes 1, 2, and 3 are presented in Figure 9. The sign of the
temperature dependence of 1/T2r depends on whether the
transverse relaxation is dominated by τM, which increases with
decreasing temperature, or by the transverse relaxation time of
the inner-sphere water molecules, T2m, which increases with
increasing temperature. For the complexes 1, 2, and 3, 1/T2r
increases with decreasing temperature over the temperature
range studied, indicating short residence times for inner-sphere
water molecules in these complexes. Thus, all three complexes
can be considered to be in the fast-exchange regime for water
exchange.
[EuDTPA(H2O)]3− (DTPA
= diethylenetriamine-N,N,N',N'',N''-
pentaacetate).[17] In addition, two other parameters affecting the
outer-sphere contributions to relaxivity are the distance between
EuII ions and the nuclei of outer-sphere water protons, a, and the
relative diffusion coefficient, D. The value of a was set to 4.5 Å
based on the distance from EuII to the furthest ligand proton of
crystal structure of complex 1. The diffusion coefficient D was
fixed to the value for [EuDTPA(H2O)]3−.[17] The parameters q, r, a,
and D were fixed to the values mentioned above during the
least-squares analyses of the NMRD profiles. In the analyses,
the parameters of electron relaxation, Δ2 and τV, and the
rotational correlation time, τR, were used as adjustable
parameters. The results of the best-fit procedure are reported in
Table 1.
More detailed information on the kinetics of water exchange are
obtained by measuring 17O-NMR transverse relaxation rate (R2)
as a function of temperature, typically obtained at a relatively
high magnetic field strength (>4.7 T). The temperature-
dependence of R2 is described by the Swift–Connick
equations.[19] These equations involve the bound-water
residency lifetime τM, electronic relaxation times T1e and T2e, the
hyperfine coupling constant A0/ħ, and q.
Figure 9: Temperature dependence of reduced transverse 17O-NMR
relaxation rates of 1 (●), 2 (■), and 3 (♦) at 9.4 T and pH 7.4. The solid lines
are the best fits of the experimental data points.
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The kex value of 1 is consistent with the previously reported
value, obtained from a variable-temperature and pressure 17O-
NMR study at 9.4 T.[15] The rates of water exchange of
complexes 2 and 3 are quite comparable with each other, with
differences of the order of experimental error. Compared to the
value of 1, the rates for complexes 2 and 3 are slower by a
factor of 5. Although the parameters are affected by a non-
negligible uncertainty due to the accuracy of the measurements
in the case of low-concentration solutions, the increase in the
residency times of the inner-sphere water molecules for
298
298
complexes 2 (τM = 20 ns) and 3 (τM = 20 ns) relative to 1
298
(τM = 4 ns) appears to be significant. A well-known complex,
investigated in detail, is [Gd(EGTA)(H2O)]– (EGTA = 3,12-
bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioate) for
which it has been reported a τM of 30 ns (at 298 K), a
surprisingly short value for a q = 1 GdIII-containing complex of a
Figure 10: Water-exchange rate of anionic, neutral, and cationic EuII-
containing complexes. Data for EuII-containing complexes of DOTA,[3b]
DTPA,[25] and ODDA[17] are from published reports.
poly(aminocarboxylic)
ligand.[20]
Two
derivatives
of
[Gd(EGTA)(H2O)]– functionalized with benzene or naphthalene
moieties fused to the oxyethylene bridges were synthesized and
their relaxometric properties studied.[21] The kex values were
found to be roughly twice as high as the corresponding value of
[Gd(EGTA)(H2O)]–,[22] an opposite trend to that measured for
complexes 1–3. In the absence of variable-pressure 17O-NMR
data that can provide information on the mechanism of the
exchange process, we can only suggest a plausible hypothesis
of this trend. For [Gd(EGTA)(H2O)]–, the mechanism of
exchange is dissociative: The faster rate of water exchange
measured for the functionalized derivatives was rationalized in
terms of a higher steric crowding of the aromatic moiety relative
to the ethylene group, inducing a destabilization of the bound
water molecule and thus a decrease in its mean residency
lifetime. On the other hand, water exchange for 1 was found to
proceed via an interchange mechanism,[15] which implies that the
incoming water molecule also participates in the rate-
determining step. Therefore, the introduction of hydrophobic
functional groups can perturb the solvation shell of the complex
close to the water coordination sites and make the exchange
process slower (longer τM).
To explore the influence of charge on water-exchange rate, the
water-exchange rates of complexes 1−3 were compared with
other EuII-containing complexes (Figure 10). Two complexes are
anionic and have in common the presence of one coordinated
water molecule. One is neutral (EuODDA; q = 1) (ODDA =
1,14,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diacetate),
and 1–3 are cationic with q = 2. The number and the type of EuII-
containing complexes are limited, making it impossible to draw
firm conclusions; however, the impact of the global electric
charge appears to have a predominant role on water-exchange
rate, as observed with GdIII-containing complexes.[23] Anionic
complexes exhibit significantly faster rates of exchange relative
to that of the neutral complex, and the cationic chelates have
even slower values of kex. On the other hand, the kex values of
the EuII-containing complexes cover a much more limited range
than that characteristic of GdIII chelates, i.e. two orders of
magnitude instead of five. At least partially, the faster rates of
exchange of the EuII-containing complexes compared to those of
GdIII can be attributed to the lower charge density and longer
metal–coordinated water distance. Both parameters are more
favorable for fast water exchange with EuII relative to GdIII.[24]
Because of the short τM values, complexes 1–3 are in the fast-
exchange regime, where this parameter does not influence or
limit relaxivity. The same condition occurs for the
macromolecular adducts of 3. Plots of the temperature-
1
dependence of H-relaxivity, determined at 9.4 T in the range of
288 to 318 K (Figure S7), show that r1 decreases with increasing
temperature for all the systems. These results indicate that the
inclusion complexes of 3 are in the fast water-exchange regime
over the entire temperature range studied.
Electronic relaxation time: Electronic relaxation is
characterized by two parameters: the trace of the square of the
transient zero field splitting (ZFS) tensor, Δ2, and the correlation
time that describes the modulation of the ZFS, τV. In the least-
squares fitting procedure, these parameters are essentially
determined from NMRD profiles. The values of Δ2 and τV of
complexes 1–3 are similar to those reported for [EuII(H2O)8]2+
and to each other.[26] As noted by Tóth and coworkers, this is a
different behavior from GdIII-containing complexes for which,
typically, electron spin relaxation is slower after the aqua ion is
chelated.[27]
Unlike GdIII-containing chelates, 1/T1e cannot be considered
negligible compared to 1/τR. Therefore, electronic relaxation can
limit relaxivity at typical imaging fields, even for low-molecular-
weight EuII-containing complexes. For example, in the case of
complex 3, 1/T1e contributes to the value of 1/τC about 40% at
0.5 T and 4.5% at 3 T (Figure S8). The contribution becomes
negligible in the ultra-high field range (<1% at 7 T). This
influence, albeit limited, of the electronic relaxation time on
relaxivity might explain the small bump near 7 T, observable in
NMRD profiles of 1–3. The X-band EPR spectra of 1–3 were
recorded at 110 K.[18] The X-band (0.34 T) line width (ΔHpp, G)
values show a slight increase from 1 to 3, reflecting small
difference in their electronic relaxation times. From the
bandwidth, the T2e values were calculated (Table 1), using
simplifications introduced by Reuben and adapted by
Merbach.[28] TheT2e values compare favorably to the values
estimated from fitting the NMRD profiles using the Morgan
equation.[4]
In the analysis of 3/β-CD, 3/poly-β-CD, and 3/HSA, the
parameters for electronic relaxation were used as empirical
fitting parameters and do not have precise physical meanings for
these slowly tumbling systems.[29]
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10.1002/chem.201702158
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(at 1.5 T) observed upon formation of inclusion complexes
between complex 3 and β-CD further supports the presence of a
rigid adduct with restricted internal rotation in 3/β-CD. As
expected, an even larger increase in rotational correlation time
was observed when complex 3 was mixed with poly-β-CD. The
larger difference between local and global rotations of the
macrocyclic complex 3/poly-β-CD and the lower value of the
order parameter S2 indicate a higher degree of rotational
flexibility than 3/β-CD. Because of internal rotation, the relaxivity
enhancement observed in 3/poly-β-CD at 1.4 T is only 3.5
despite the global rotational correlation time of 3/poly-β-CD
being 20× longer than that of complex 3.
The biphenyl moiety in complex 3 also interacts with HSA to
yield an increase in global rotational correlation time. However,
this slowdown in molecular motion is partly offset by the high
degree of local motion: The relaxivity enhancement of complex 3
obtained upon binding with HSA (20.9 mM−1 s−1, 1.4 T) is only
3.9× that of complex 3. Therefore, the relaxivity of the adduct is
primarily limited by the poor correlation between global and local
motions. A more restricted local rotation of the complex at the
binding site of the protein would favor a larger enhancement of
relaxivity. It has to be noted that the fitting (continuous lines in
Figure 8) was restricted to frequencies above 3 MHz because of
the inadequacy of Solomon−Bloembergen−Morgan theory for
the description of the rotational dynamics of slow-rotating
systems at low magnetic field strengths.
Rotational correlation time: In the clinically relevant region of
proton Larmor frequencies (>10 MHz), rotational correlation
represents the limiting factor of relaxivity. This relationship
implies that the overall correlation time, τC, is largely dominated
by the rotational correlation time and that the contribution of both
the exchange lifetime and the electronic relaxation can be safely
neglected. Because τR is roughly proportional to the molecular
weight of EuII-containing chelates, a linear dependence of τR on
the molecular masses of the complexes should exist. A plot of
the τR values of 1–3 (Table 1) as a function of molecular mass
yields a straight line (R2 = 0.987). This linear behavior implies
that the outer-sphere contribution to r1 is nearly identical for the
three complexes. In comparison to low-molecular-weight GdIII-
containing complexes, the τR values are slightly shorter, even
accounting for the larger size of EuII relative to GdIII.[4a,17] This
discrepancy might arise, in part, from the choice of the distance
value r. For example, if the value of r is fixed to 3.3 instead of
3.2, then the τR value of 2, which has the same molecular mass
as [Gd(DOTA)(H2O)]– (τR = 66 ps),[30] increases from 63 to 75 ps.
Additionally, a different solvation sphere due to the different
charge densities of the two ions might contribute to the
differences in rotational correlation times.
The presence of a biphenyl group in complex 3 facilitates the
formation of adducts with cyclodextrins and HSA, and the
rotational correlation time of those adducts was also studied. To
obtain information regarding the rotational dynamics of the
inclusion complexes of 3, the Lipari−Szabo model was used.[31]
This model-free approach considers the occurrence of an
Conclusions
internal rotation, characterized by
a correlation time τRL,
superimposed on a global motion described in terms of the
correlation time τRG. The degree of correlation between the two
types of motion is described by the parameter S2 that assumes
the value of zero when the two motions are completely
independent; in the absence of local fluctuations, the complex is
immobilized, the motion is isotropic, and S2 = 1. In complex 3,
the biphenyl moiety is connected to the cryptand through a
methylene group that can freely rotate causing local rotation
superimposed to global tumbling motion of the complex.
Therefore, a five-parameter (Δ2, τV, τRL, τRG, and S2) least-
squares fit of the data was performed (Table 1).
The global rotational correlation times of complexes 1−3 and of
cyclodextrins and HSA bound to complex 3 (Table 1) correlate
with the molecular weights of the molecules, where increasing
molecular weight roughly correlates with increasing rotational
correlation time.
The marked decrease in molecular tumbling following the
formation of the supramolecular adducts is signaled by the
formation of broad relaxivity peaks in the high field region of the
NMRD profiles, distinctive of slowly rotating systems. However,
an increase of the local rotational flexibility of the metal complex
at the binding site is highlighted by the increasing difference
between the local and global values of the rotational correlation
times, which limits the relaxivity. This local rotational flexibility is
also shown by the decrease of S2 from 0.43 for 3/β-CD to 0.14
for 3/HSA. The S2 obtained for complex 3/β-CD indicates that
the inclusion complex prevents internal rotation to some extent.
This observation is further evidenced by global rotational
correlation time of 3/β-CD being only 3.0× longer than the local
rotational correlation time. The relaxivity enhancement of 2.1×
The present study describes
a detailed analysis of the
contribution of molecular parameters (water-exchange rate,
electronic relaxation time, and rotational correlation time) to the
relaxivity of EuII-containing cryptates over a wide range of field
strengths using 1H-NMR, 17O-NMR, and EPR spectroscopies.
The results reported here demonstrate for the first time that it is
possible to achieve a high relaxivity enhancement through non-
covalent interactions between suitably functionalized EuII-
containing complexes and slowly tumbling substrates like β-CD,
poly-β-CD, and HSA. Similarly to the case of well-known GdIII-
based complexes, the relaxivities of rapidly rotating EuII chelates
are essentially limited by the value of the rotational correlation
time. A lengthening of τR causes a considerable increase in the
longitudinal relaxation rate of water protons. However, unlike
what is often observed in the case of GdIII-based complexes, the
slow exchange process of the bound water molecules does not
hamper the increase in relaxivity at the imaging fields of the
supramolecular adducts. This is because the EuII complexes
investigated to date have solvent exchange rates values in the
range necessary to achieve high relaxivity. This property, in the
case of macromolecular systems, largely compensates for the
negative effect on r1 of a greater distance between the water
protons and the paramagnetic ion, thus enabling relaxivity
values comparable to those of the analogous GdIII-based
systems.
Despite these interesting and encouraging results, the number
and type of complexes studied is limited and there is a need to
expand the available library of EuII-based systems to obtain
more in-depth information on the correlation between solution
structure and molecular relaxation parameters. However, these
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10.1002/chem.201702158
Chemistry - A European Journal
FULL PAPER
buffered saline (pH = 7.4) with 1% 17O-enriched water (diluted from 20%
enriched H217O). Transverse relaxation rates were calculated from line
widths at half height following a reported procedure.[9]
preliminary results clearly indicate that in the case of EuII-
containing complexes, it is possible to design and develop highly
sensitive macromolecular or nano-sized probes for advanced
MRI applications.
EPR spectroscopy was performed on
a
Bruker EMX X-band
spectrometer equipped with an Oxford variable-temperature cryostat.
EPR samples were prepared and sealed under an atmosphere of N2 in
Norell SEPR250S EPR tubes and were frozen in liquid N2 before
insertion into the sample cavity. EPR spectra of 3/β-CD and 3/poly-β-CD
were acquired at 110 K (Figures S5 and S6). Acquisition parameters
included a microwave frequency of 9.38 GHz, a microwave power of 1.99
mW, a modulation frequency of 100 kHz, a receiver gain of 30 dB, and a
modulation amplitude of 4.0 G.
Experimental Section
Commercially available chemicals were of reagent-grade purity or better
and were used without further purification unless otherwise stated. Water
was purified using a PURELAB Ultra Mk2 water purification system
(ELGA) and degassed under reduced pressure prior to use. 3-(Biphenyl-
4-yl)propane-1,2-diol was synthesized according to
a
reported
procedure.[9] Analytical thin-layer chromatography (TLC) was carried out
Molar conductivity measurements were carried out using an Omega CDH
280 portable conductivity meter that was calibrated with aqueous KCl
(0.01 M, 1.413 mS cm−1). Conductivity values were calculated from three
independently prepared solutions of metal complexes (1.00 mM, 4.00
mL) measured in water under an atmosphere of N2 at ambient
temperature. Results are reported as mean ± standard error of three
independently prepared samples.
on ASTM TLC plates precoated with silica gel 60 F254 (250 µm layer
thickness). TLC visualization was accomplished using
followed by charring with potassium permanganate stain (3 g KMnO4, 20
K2CO3, mL 5% w/v aqueous NaOH, 300 mL H2O). Flash
a UV lamp
g
5
chromatography was performed using silica gel 60, 230–400 mesh (EMD
chemicals). 1H-NMR, correlation spectroscopy (COSY), 13C-NMR,
distortionless enhancement by polarization transfer (DEPT), and
heteronuclear multiple quantum coherence (HMQC) spectra were
obtained using an MR400 (9.4 T) or V500 (11.7 T) spectrometers. Peaks
were assigned using DEPT, COSY, and HMQC spectra or by
comparison with reported spectra.[9] Chemical shifts are reported relative
to residual solvent signals (CD3CN: 1H: δ 1.94; 13C: δ 1.32). 1H-NMR data
are assumed to be first order, and the apparent multiplicity is reported as
"s" = singlet, "d" = doublet, "dd" = doublet of doublet, "ddd" = doublet of
doublet of doublets, "m" = multiplet, and "brs" = broad singlet. Italicized
elements are those that are responsible for the shifts. High-resolution
electrospray ionization mass spectra (HRESIMS) were obtained using an
electrospray time-of-flight high-resolution Waters Micromass LCT
Premier XE mass spectrometer.
Dynamic light scattering data were obtained using a Malvern Zetasizer
Nano-ZS instrument (ZEN3600) operating with a 633 nm wavelength
laser. Dust was removed from samples by filtering through 0.2 µm
hydrophilic filters. Samples of complex 3 (1 and 8 mM in phosphate-
buffered saline) were prepared in air-tight cuvettes inside a wet glove box
under an atmosphere of N2.
Dimethyl-2,2'-([1,1'-biphenyl]-4-yl)propane-1,2-dily)bis(oxy) diacetate (4):
To a solution of 3-(biphenyl-4-yl)propane-1,2-diol (2.00 g, 8.76 mmol, 1
equiv) in anhydrous tetrahydrofuran (15 mL) under an atmosphere of Ar
at ambient temperature was added NaH (58% in oil, 1.45 g, 35.0 mmol, 4
equiv) followed by the dropwise addition of methyl bromoacetate (6.60
mL, 70.1 mmol, 8 equiv). The resulting reaction mixture was stirred at
ambient temperature for 22 h, and excess NaH was quenched with
methanol (3 mL). Solvents were removed under reduced pressure, and
the resulting residue was extracted with diethyl ether (5 × 30 mL).
Purification was performed using silica gel chromatography (5:1
hexanes/ethyl acetate) to yield 2.31 g (71%) of 4 as a colorless oil. TLC:
Rf = 0.20 (3:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CD3CN): δ =
2.90 (d, 3J (H,H) = 4.0 Hz, 2H; CH2), 3.56 (ddd, 3J (H,H) = 4.0, 12.0, 40.0
Hz, 2H; CH2CH), 3.66 (s, 3H; CH3), 3.68 (s, 3H; CH3), 3.80−3.87 (m, 1H;
CHCH2), 4.09 (s, 2H; CH2C=O), 4.18 (s, 2H; CH2C=O), 7.31−7.66 ppm
(m, 9H; CHCH); 13C NMR (101 MHz, CD3CN): δ = 37.8 (CH2), 52.1 (CH3),
68.0 (CH2C=O), 69.1 (CH2C=O), 73.5 (CH2CH), 81.0 (CHCH2), 127.6
(CHCH), 127.7 (CHCH), 128.2 (CHCH), 129.8 (CHCH), 131.0 (CHCH),
131.4 (CHCH), 138.6, 139.7, 141.6, 171.6, 171.8 ppm; HRESIMS (m/z):
[M + Na]+ calcd for C21H24O6Na, 395.1471; found, 395.1477.
The concentrations of Eu were determined using ICP−MS or energy-
dispersive X-ray fluorescence (EDXF) spectroscopy. Samples for
ICP−MS were diluted using aqueous nitric acid (2%). Standard solutions
were prepared by serial dilution of a commercially available standard
(Eu2O3 in aqueous nitric acid (5%) 1000 ppm, Alfa Aesar). Calibration
curves were generated using the 153Eu isotope ion counts for a 10–100
ppb concentration range. ICP−MS analyses were performed using an
Agilent Technologies 7700x ICP–MS instrument in the Lumigen
Instrument Center of Wayne State University. EDXF measurements were
performed with a Shimadzu EDX-7000 spectrometer. Calibration curves
were generated using fluorescence intensity at 5.845 keV for a 250−1000
ppm concentration range.
Longitudinal proton relaxation times were measured using six different
instruments. A Bruker minispec was used at 1.4 T and 37 οC using an
inversion recovery technique. NMRD profiles were measured at 25 οC
over a continuum of magnetic fields from 0.0002 to 0.2 T on a fast field-
cycling Stelar SmartTracer relaxometer, and data points at high field
strengths were measured on a Stelar Spinmaster relaxometer (0.5–1.6 T)
equipped with a BrukerWP80 magnet. Susceptibility weighted imaging
(SWI) was performed to acquire relaxivity data at 3 (Siemens TRIO) and
7 T (Clinscan). Multiple flip angles (5, 10, 15, 20, 25, and 30°) were used
in the SWI experiment to allow for the determination of T1 following a
reported procedure.[32] MR images were processed using SPIN (SVN
Revision 1757), and Matlab (T.12.0.635 R2011a) was used to calculate
2,2'-((3-([1,1'-Biphenyl]-4-yl)propane-1,2-dily)bis(oxy) diacetic acid (5): To
a solution of 4 (2.00 g, 5.38 mmol, 1 equiv) in methanol (9 mL) and water
(3 mL) was added LiOH (1.03 g, 43.0 mmol, 8 equiv), and the reaction
mixture was stirred at ambient temperature for 6 h. The resulting yellow
solution was concentrated under reduced pressure, and the residue was
dissolved in water (8 mL) and acidified with HCl (6 M, 3 mL). The
resulting white solid was collected by filtering, washed with water (15 mL),
and dried under reduced pressure to yield 1.85 g (quantitative) of 5 as a
white solid. 1H NMR (400 MHz, CD3CN): δ = 2.88 (dd, 3J (H,H) = 2.0, 6.4
Hz, 2H; CH2), 3.47−3.68 (m, 2H; CH2), 3.83−3.91 (m, 1H; CH), 4.03−4.19
(m, 4H; CH2), 7.29−7.69 (m, 9H; CHCH), 9.60 ppm (brs, 2H; OH); 13C
NMR (101 MHz, CD3CN): δ = 37.7 (CH2), 68.2 (CH2), 68.7 (CH2), 73.9
(CH2), 82.0 (CH), 127.7 (CHCH), 127.8 (CHCH), 128.3 (CHCH), 129.9
(CHCH), 131.1 (CHCH), 138.0, 140.0, 141.5, 171.8, 172.2 ppm;
HERESIMS (m/z): [M + Na]+ calcd for C19H20O6Na, 367.1158; found,
367.1148.
*
effective transverse relaxation times, T2 , and corrected T1 maps. Data
points at 9.4 and 11.7 T were measured using inverse recovery methods
with Varian unity 400 and Varian 500S instruments.
Variable-temperature 17O-NMR measurements were performed using a
Varian-500S (9.4 T) spectrometer for samples of 1 (5 mM), 2 (5 mM),
and 3 (1.2 mM) in phosphate-buffered saline, with a blank of phosphate-
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10.1002/chem.201702158
Chemistry - A European Journal
FULL PAPER
5-([1,1'-Biphenyl]-4-ylmethyl)-4,7,13,16,21,24-hexaoxa-1,10-
To study the stability of complex 2 in phosphate-buffered saline, an
aliquot (300 µL) of a solution of complex 2 in phosphate-buffered saline
was flame sealed in an NMR tube under an atmosphere of N2. The
diazabicyclo[8.8.8]hexacosane-2,9-dione (6): Thionyl chloride (3.60 mL,
49.4 mmol, 10 equiv) was added to 5 (1.70 g, 4.94 mmol, 1 equiv), and
the resulting solution was heated at reflux for 3 h under an atmosphere of
Ar. The reaction mixture was concentrated under reduced pressure to
obtain a yellow oil that was dissolved in anhydrous toluene (150 mL)
under an atmosphere Ar. To a separate solution of 4,13-diaza-18-crown-
6-ether (1.30 g, 4.94 mmol, 1 equiv) in anhydrous toluene (150 mL) was
added triethylamine (2.00 mL, 14.8 mmol, 3 equiv), and the resulting
solution was stirred for 15 min. The two solutions were added
simultaneously over 60 min to anhydrous toluene (300 mL) at 0−5 oC.
The resulting mixture was stirred for 24 h while allowing to warm to
ambient temperature. Solvent was removed under reduced pressure, and
the residue was extracted with CH2Cl2 (5 × 10 mL). Purification was
performed using silica gel chromatography (10:1 CH2Cl2/methanol) to
o
relaxivity of this sample was measured (60 MHz, 37 C) over the course
of one month and was triplicated with independently prepared samples
(Figure S3). No change in relaxivity was observed, indicating that any
competition between phosphate and the cryptand in 2 was below the
detection limit of the technique.
X-ray quality crystals of EuII-containing complexes were obtained by
vapor diffusion. A solution of metal complex was prepared by dissolving
EuCl2 and ligand in methanol under an inert atmosphere. The resulting
clear solution was stirred for 1 h then filtered through a 0.2 µm
hydrophilic filter. Tetrahydrofuran was diffused into the filtrate to obtain
needle-like crystals. Crystal structure analyses were performed on a
Bruker APEX-II Kappa geometry diffractometer with Mo radiation and a
graphite monochromator using a Bruker-charge-coupled-device-based
diffractometer equipped with an Oxford Cryostream low-temperature
apparatus. Data were acquired at 100 K. Structures were solved by the
direct method using the SHELXS-97 program that is part of APEX II2 and
refined by the least squares method, SHELXL 2012 incorporated into
ShelXle.[33] Single crystals of 1 contained one cation of 1 with one
chloride, and no water molecules bound, one non-covalent chloride
counter ion, and one non-covalent molecule of water in the asymmetric
unit. The structure of 1 was solved with a resolution of 0.75 Å in space
group P121/c1. Single crystals of 2 contained one cation of 2 with one
bound chloride, no water molecules bound, and one non-covalent
molecule of water in the asymmetric unit. The structure of 2 was solved
with a resolution of 0.55 Å in space group P121/c1. All non-hydrogen
atoms were refined anisotropically. CCDC 1549045 and CCDC 1549046
contain the supplementary crystallographic data for this paper. These
data are provided free of charge by The Cambridge Crystallographic
Data Centre.
yield 1.46
CH2Cl2/methanol); 1H-NMR (400 MHz, CD3CN): δ = 2.60−3.10 (m, 5H;
CH2), 3.20−4.80 (m, 28H, CH and CH2), 7.20−7.70 ppm (m, 9H; CH); 13
g (52%) of 6 as a yellow oil. TLC: Rf = 0.32 (10:1
C
NMR (101 MHz, CD3CN): δ = 37.2 (CH2), 37.4 (CH2), 37.7 (CH2), 38.0
(CH2), 47.3 (CH2), 47.7 (CH2), 48.2 (CH2), 48.3 (CH2), 48.4 (CH2), 49.0
(CH2), 49.1(CH2), 49.3 (CH2), 49.35 (CH2) 49.4 (CH2), 49.6 (CH2), 49.65
(CH2) 49.7 (CH2), 49.8 (CH2), 49.9 (CH2), 68.7 (CH2), 69.2 (CH2), 69.3
(CH2), 69.7 (CH2), 69.8 (CH2), 69.9 (CH2), 70.0 (CH2), 70.1 (CH2), 70.2
(CH2), 70.3 (CH2), 70.4 (CH2), 70.9 (CH2), 71.0 (CH2),71.1 (CH2), 71.28
(CH2), 71.3 (CH2), 71.4 (CH2), 71.6 (CH2), 71.7 (CH2), 71.8 (CH2), 71.9
(CH2), 72.0 (CH2), 72.1 (CH2), 72.2 (CH2), 72.4 (CH2), 73.6 (CH2), 73.7
(CH2), 74.9 (CH2), 78.6 (CH), 79.7 (CH), 81.6 (CH), 82.7 (CH), 127.5
(CHCH), 128.0 (CHCH), 129.7 (CHCH), 130.7 (CHCH), 130.8 (CHCH),
138.5, 139.6, 141.4, 169.5, 169.6 ppm; HRESIMS (m/z): [M + H]+ calcd
for C31H43N2O8, 571.3019; found 571.3012.
5-([1,1'-Biphenyl]-4-ylmethyl)-4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane (7): To a solution of 6 (0.800 g, 1.40
mmol, 1 equiv) in anhydrous tetrahydrofuran under an atmosphere of Ar
at 0 oC was added borane–tetrahydrofuran complex (14 mL, 14 mmol, 1
M in tetrahydrofuran, 10 equiv) in a drop-wise manner, and the resulting
mixture was heated at reflux for 24 h before the reaction mixture was
allowed to cool to ambient temperature. To the reaction mixture was
added aqueous HCl (6 M, 14 mL), and the mixture was heated at reflux
for 4 h. The pH of the reaction solution was adjusted to 11 by adding
concentrated ammonium hydroxide (20 mL). The resulting solution was
concentrated under reduced pressure to obtain white solid that was
purified using silica gel chromatography (20:1 CH2Cl2 / methanol) to yield
0.5 g (66%) of 7 as a yellow oil. TLC: Rf = 0.23 (10:1 CH2Cl2/methanol);
1H-NMR (400 MHz, CD3CN): δ = 2.55−2.88 (m, 10H; CH2), 3.03−3.11 (m,
1H; CH2), 3.32−3.82 (m, 26H; CH and CH2), 7.30−7.66 ppm (m, 9H; CH);
13C NMR (101 MHz, CD3CN): δ = 38.4 (CH2), 53.4 (CH2), 53.5 (CH2),
53.7 (CH2), 53.8 (CH2), 54.1 (CH2), 54.3 (CH2), 66.1 (CH2), 68.0 (CH2),
68.1 (CH2), 68.57 (CH2), 68.59 (CH2), 68.8 (CH2), 68.9 (CH2), 69.2 (CH2),
69.6 (CH2), 71.4 (CH2), 79.4 (CH), 126.7 (CH), 127.7 (CH), 127.8 (CH),
128.3 (CH), 129.2 (CH), 129.9 (CH), 131.0 (CH), 138.4, 139.6, 141.5
ppm; HRESIMS (m/z): [M + H]+ calcd for C31H47N2O6, 543.3434; found
543.3114.
Acknowledgements
This work was supported by the National Institutes of Health
(R01EB013663). M.B. and F.C. are grateful to Università del
Piemonte Orientale for financial support (Ricerca locale 2016).
The authors thank Dr. Derek J. Averill from Shimadzu Scientific
Instruments for use of the EDXF spectrometer, Prof. Jeremy
Kodanko for use of the conductivity meter, and Levi Ekanger for
assistance acquiring EPR spectra.
Keywords: cryptands • cyclodextrins • europium • lanthanides •
magnetic resonance imaging
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EuII-containing cryptates as contrast
agents: Relationship between
relaxivity, field strength and molecular
parameters for EuII-containing
cryptates are discussed using
Solomon–Bloembergen–Morgan
theory.
Chamika U. Lenora, Fabio Carniato,
Yimin Shen, Zahid Latif, E. Mark
Haacke, Philip D. Martin, Mauro Botta,*
and Matthew J. Allen*
Page No. – Page No.
Structural Features of EuII-Containing
Cryptates That Influence Relaxivity
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