Lanthanide(iii) complexes of aminoethyl-DO3A as PARACEST contrast agents based on decoordination of the weakly bound amino group

Article abstract of DOI:10.1039/c3dt52031e

2-Aminoethyl DOTA analogues with unsubstituted (H₃L ¹), monomethylated (H₃L²) and dimethylated (H₃L³) amino groups were prepared by improved synthetic procedures. Their solid-state structures exhibit an extensive system of intramolecular hydrogen bonds, which is probably present in solution and leads to the rather high value of the last dissociation constant. The protonation sequence of H₃L¹ in solution corresponds to that found in the solid state. The stability constants of the H₃L¹ complexes with La³⁺ and Gd³⁺ (20.02 and 22.23, respectively) are similar to those of DO3A and the reduction of the pK _A value of the pendant amino group from 10.51 in the free ligand to 6.06 and 5.83 in the La³⁺ and Gd³⁺ complexes, respectively, points to coordination of the amino group. It was confirmed in the solid state structure of the [Yb(L¹)] complex, where disorder between the SA′ and TSA′ isomers was found. A similar situation is expected in solution, where a fast equilibration among the isomers hampers the unambiguous determination of the isomer ratio in solution. The PARACEST effect was observed in Eu(iii)-H₃L¹/H₃L² and Yb(iii)-H₃L¹/H₃L² complexes, being dependent on pH in the region of 4.5-7.5 and pH-independent in more alkaline solutions. The decrease of the PARACEST effect parallels with the increasing abundance of the complex protonated species, where the pendant amino group is not coordinating. Surprisingly, a small PARACEST effect was also observed in solutions of Eu(iii)/Yb(iii)-H₃L³ complexes, where the pendant amino group is dimethylated. The effect is detectable in a narrow pH region, where both protonated and deprotonated complex species are present in equilibrium. The data points to the new mechanism of the PARACEST effect, where the slow coordination-decoordination of the pendant amine is coupled with the fast proton exchange between the free amino group and bulk water mediates the magnetization transfer. The pH-dependence of the effect was proved to be measurable by MRI and, thus, the complexes extend the family of pH-sensitive probes. This journal is The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3dt52031e

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
Lanthanide(III) complexes of aminoethyl-DO3A as
PARACEST contrast agents based on decoordination
Cite this: Dalton Trans., 2013, 42, 15735
of the weakly bound amino group†
Tereza Krchová,a Jan Kotek,*a Daniel Jirák,b Jana Havlíčková,a Ivana Císařováa and
Petr Hermanna
2-Aminoethyl DOTA analogues with unsubstituted (H3L1), monomethylated (H3L2) and dimethylated
(H3L3) amino groups were prepared by improved synthetic procedures. Their solid-state structures exhibit
an extensive system of intramolecular hydrogen bonds, which is probably present in solution and leads
to the rather high value of the last dissociation constant. The protonation sequence of H3L1 in solution
corresponds to that found in the solid state. The stability constants of the H3L1 complexes with La3+ and
Gd3+ (20.02 and 22.23, respectively) are similar to those of DO3A and the reduction of the pKA value of
the pendant amino group from 10.51 in the free ligand to 6.06 and 5.83 in the La3+ and Gd3+ complexes,
respectively, points to coordination of the amino group. It was confirmed in the solid state structure of
the [Yb(L1)] complex, where disorder between the SA’ and TSA’ isomers was found. A similar situation is
expected in solution, where a fast equilibration among the isomers hampers the unambiguous determi-
nation of the isomer ratio in solution. The PARACEST effect was observed in Eu(III)–H3L1/H3L2 and Yb(III)–
H3L1/H3L2 complexes, being dependent on pH in the region of 4.5–7.5 and pH-independent in more
alkaline solutions. The decrease of the PARACEST effect parallels with the increasing abundance of the
complex protonated species, where the pendant amino group is not coordinating. Surprisingly, a small
PARACEST effect was also observed in solutions of Eu(III)/Yb(III)–H3L3 complexes, where the pendant
amino group is dimethylated. The effect is detectable in a narrow pH region, where both protonated and
deprotonated complex species are present in equilibrium. The data points to the new mechanism of the
PARACEST effect, where the slow coordination–decoordination of the pendant amine is coupled with the
fast proton exchange between the free amino group and bulk water mediates the magnetization trans-
fer. The pH-dependence of the effect was proved to be measurable by MRI and, thus, the complexes
extend the family of pH-sensitive probes.
Received 26th July 2013,
Accepted 27th August 2013
DOI: 10.1039/c3dt52031e
www.rsc.org/dalton
Introduction
aDepartment of Inorganic Chemistry, Universita Karlova (Charles University),
Hlavova 2030, 128 40 Prague 2, Czech Republic. E-mail: modrej@natur.cuni.cz;
Fax: +420-22195-1253; Tel: +420-22195-1261
Contrast agents (CAs) that alternate the properties of bulk
water have been used for a long time to improve the resolution
and utilization of magnetic resonance imaging (MRI) in medi-
cine and molecular biology. The classical molecular CAs are
based on complexes of highly paramagnetic metal ions,
mainly on trivalent gadolinium.1 The complexes change the
relaxation times of the bulk water protons most frequently
through the exchange of the whole coordinated water mole-
cule. Another class of CAs is based on a completely different
bDepartment of Radiodiagnostic and Interventional Radiology, Magnetic Resonance
Unit, Institute of Clinical and Experimental Medicine, Vídeňská 1958/9,
140 21 Prague 4, Czech Republic
†Electronic supplementary information (ESI) available: Crystal structures of
H3L2·6H2O and H3L3·3.5H2O, and selected geometric parameters. The distri-
bution diagram of H3L1. Chemical shift dependence of H3L1 on pH, and its pro-
tonation scheme. Distribution diagrams of the Ln3+:H3L1 systems. Temperature
dependence of the 1H NMR spectra of [Eu(H2O)(L1)] and [Yb(L1)]. Disorder of the
[Yb(L1)] molecule found in the solid-state structure of [Yb(L1)]·5H2O, and its principle, a chemical exchange saturation transfer (CEST), was
selected geometric parameters. Set of Z-spectra of the [Eu(H2O)(L1,2,3)] and
introduced some time ago.2 The method is based on
irradiation (saturation) of protons exchangeable with bulk
water protons leading, after the chemical exchange, to a
decrease of the bulk water proton signal. As signals of the bulk
water protons and the protons to be irradiated should be
[Yb(L1,2,3)] complexes at different temperatures and pH. MRI-CEST images of
phantoms of [Eu(H2O)(L1)] and [Yb(L1)], and the normalized intensity of the
CEST effect. 1H and 13C{1H}NMR spectra of H3L1, H3L2 and H3L3. CCDC 933967,
952384–952386. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c3dt52031e
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15735

View Article Online
Paper
Dalton Transactions
Scheme 2 Studied ligands.
Scheme 1 Ligands discussed in the paper.
In this work, we decided to investigate the PARACEST-
related properties of complexes, where the CEST-causing
amino group is directly coordinated to the central lanthanide(III)
ions. To ensure complex stability, the ligands are based
on a macrocyclic cyclen skeleton having three acetate and one
2-aminoethyl pendant arms, which contain primary (H3L1),
partially methylated secondary (H3L2) and dimethylated ter-
tiary (H3L3) amino groups, see Scheme 2. Some of these
ligands have already been investigated as bifunctional ligands
for gadolinium(III)-based targeted MRI CAs13 or radio-
nuclides,14 and, during work on this project, as pH-sensitive
gadolinium(III)-based positive MRI CAs.15
separated from each other as much as possible to reduce non-
specific proton irradiation, paramagnetic complexes causing
the extension of the NMR chemical shift scale were suggested
(PARACEST).2,3 Most of these CAs are based on lanthanide(III)
ions having suitable magnetic properties which are bound in
macrocyclic ligands derived from DOTA (e.g., DOTA-tetra-
amides, Scheme 1), ensuring the high thermodynamic stability
and kinetic inertness of the complexes. The exchangeable
protons in the complexes should be endowed with suitable
properties and the most important one is the rate of their
exchange with bulk water. There are two proton pools exhibit-
ing PARACEST effects in Ln(III)–DOTA-tetraamide complexes:
(i) protons of a coordinated water molecule if it is in a
slow-exchange region, typically in the microsecond range
(e.g. [Yb(H2O)(DOTA-4AmCE)] complex (τM = 3 μs)4 or [Eu-
Results and discussion
Synthesis
(H2O)(DOTAM)],5 [Eu(H2O)(DOTA-4AmCE)],6 and [Eu(H2O)- The ligands were synthesized (Schemes 3 and 4) by reaction of
(DOTA-4AmP)]4 complexes (τM = 55, 382 and 67 μs, respectively; t-Bu3DO3A with the appropriate amine-containing precursor.
for ligand structures see Scheme 1). The water molecule is Alkylation of t-Bu3DO3A with N-(2-bromoethyl)-phthalimide
bound directly to the metal ion and is close to the magnetic followed by sequential deprotection by trifluoroacetic acid and
axis of the complexes and, thus, its protons exhibit a maxi- hydrazine led, after chromatography on anion exchanger, to
mized difference in their chemical shift from the bulk water zwitterionic H3L1 in 59% overall yield (Scheme 3). The syn-
protons. (ii) Protons of O-coordinated amide group(s); thesis is more simple compared to the previously published
however, these protons have their chemical shift much closer procedure,14 and is easily scalable and avoids HPLC
to the bulk water resonance. Nevertheless, their exchange purification.
depends on a number of external factors (e.g., τM for [Yb-
To obtain ligand H3L2, 2-[N-(ethyloxycarbonyl)-N-methyl-
(DOTAM)]3+ lays, with a dependence on the pH, in the amino]bromoethane was used as the alkylation agent reacting
400–5000 μs range7) and, therefore, the Ln(III) complexes of with t-Bu3DO3A and, after deprotection using trifluoroacetic
DOTA amide derivatives can be used as probes for various acid and 10% aq. NaOH and chromatography on ion exchange
physiological parameters, such as pH, temperature, metabolite resins, the ligand was isolated as a zwitterion in 30% overall
or metal ion concentrations, etc.2c,3
yield (Scheme 4). Direct methylation of H3L1 with a form-
More recently, other PARACEST CAs based on different aldehyde–formic acid mixture easily produced H3L3 in high
ligands (and, thus, on different pools of exchangeable protons) yield (Scheme 3); it is a significant improvement compared
and/or metal ions have been suggested. Protons of the with the previous synthesis.15
hydroxyl group are also labile and lanthanide(III) complexes of
All three studied ligands were structurally characterized in
cyclen derivatives with alcohol pendant arms have been shown their zwitterionic forms by single-crystal X-ray diffraction ana-
to exhibit a PARACEST effect.8 Some transition metal ions can lysis. Single-crystals of sufficient quality were obtained from
also present suitable magnetic properties and complexes of solutions in water or aqueous ethanol. Title ligands were iso-
high-spin iron(II) or nickel(II) with derivatives of cyclam, cyclen lated in the form of hydrates (H3L1·5H2O, H3L2·6H2O and
or 1,4,7-triazacyclononane having 2-hydroxypropyl, acetamide H3L3·3.5H2O, respectively). All three molecular structures are
(e.g., DOTAM and NOTAM, see Scheme 1) or (5-aminopyridine- very similar, and therefore, only the molecular structure of
2-yl)methyl pendant arms have been shown to have a pro- H3L1 is shown here in Fig. 1; other structures are shown in the
nounced PARACEST effect.9–11 Another pool of exchangeable ESI (Fig. S1 and S2†). In all three cases, the protonation
protons, a distant non-coordinated amino group, has also scheme is the same – two protons are bound to the macrocycle
been explored in some complexes of lanthanide(III)12 as well as amino groups bearing acetate moieties and are located
transition metal9 ions.
mutually trans, and the third one is bound to the pendant
15736 | Dalton Trans., 2013, 42, 15735–15747
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
Scheme 3 Synthesis of studied ligands H3L1 and H3L3: (i) DMF, K2CO3, TBAB, 40 °C; (ii) t-Bu3DO3A·HBr, MeCN, K2CO3, 60 °C; (iii) CF3COOH–CHCl3 (1 : 1), reflux
24 h; (iv) aq. NH2NH2, 90 °C, 18 h; (v) (CH2O)n, HCOOH, reflux 24 h.
Scheme 4 Synthesis of studied ligand H3L2: (i) CH3CH2OC(O)Cl, dioxan–H2O (1 : 1), RT, 2 h; (ii) CBr4, PPh3, THF, RT, 1 h; (iii) t-Bu3DO3A·HBr, K2CO3, MeCN, 60 °C; (iv)
CF3COOH–CHCl3 (1 : 1), reflux 24 h; (v) 10% aq. NaOH, 90 °C, 24 h.
rings (Table S1†).16 Such a conformation is stabilized by intra-
molecular hydrogen bonds between the protonated and
non-protonated macrocycle amino groups. Beside these inter-
actions, the molecular structure is stabilized by further intra-
molecular hydrogen bonds, including protonated amines and
deprotonated oxygen atoms of the carboxylate pendant arms,
and the whole crystal structure is stabilized by extended hydro-
gen bond networks with water solvate molecules.
Stability of the complexes
The complexing properties of the most important ligand,
H3L1, having a primary amino group, were investigated in
detail. First, the conditions for the pendant amino group
deprotonation and coordination were investigated by potentio-
metry. The ligand’s overall protonation constants βh10 were
Fig. 1 Molecular structure of H3L1 found in the crystal structure of H3L1·5H2O
determined by potentiometry and are compiled together with
corresponding pKA values in Table 1; the ligand distribution
showing an intramolecular hydrogen bond network. The carbon-bound hydro-
gen atoms were omitted for clarity.
diagram is shown in Fig. S3.† As the highest protonation con-
stant β110 is slightly behind the −log[H+] range suitable for
aminoethyl group. Such a protonation scheme is consistent potentiometric titrations, we also performed a 1H NMR
1
with the form suggested on the basis of H NMR titration (see titration in the pH range 1.2–13.5 to confirm such a high value
below, Scheme S1†). The conformation of the macrocyclic unit of the first protonation constant and to assign the individual
is (3,3,3,3)-B, as usually observed for double-protonated cyclen protonation sites. The values of the dissociation constants
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15737

View Article Online
Paper
Dalton Transactions
Table 1 Overall protonation constants (log βh10) of H3L1 and overall stability
constants (log βh11) of its lanthanide(III) complexes, and corresponding pKA
values (I = 0.1 m (Me4N)Cl, 25 °C)
amino group as it is the most basic site of the in-cage complex
molecule and a difference of ∼4–5 orders of magnitude
between the constants, corresponding to its protonation in the
complexes and analogous protonation in the free ligand,
points to an easier deprotonation induced by coordination of
the group to the metal ion. Thus, one can conclude, that
studied ligands are coordinated in an octadentate fashion
involving the pendant amino group in the [Ln(L1)] species,
and that the amino group is decoordinated upon protonation
in the weakly acidic region. The maximum abundance of the
[Gd(HL1)]+ complex species in solution is observed at pH ∼5.
Stoichiometric coefficients
Constant
a
h
l
M
log βhlm
pKA
1
2
3
4
1
1
1
1
1
0
0
0
0
0
13.19(2)
23.70(2)
32.60(2)
36.47(2)
37.75(2)
13.19
10.51
8.90
3.87
1.27
5
La
0
1
1
1
1
1
20.02(3)
26.08(2)
—
6.06
Structure of the complexes
Gd
0
1
To study the structure and properties of the lanthanide(III)
complexes of H3L1, sample solutions were prepared by dissol-
ving LnCl3 and the ligand in a Ln : L = 1 : 1.1 molar ratio,
adjusting the pH with aq. LiOH to 6.0, heating the solution at
60 °C overnight, re-adjusting pH to 6.5 and heating at 60 °C
1
1
1
1
22.23(4)
28.06(1)
—
5.83
a βhlm = [HhLlMm]/{[H]h·[L]l·[M]m}.
obtained by NMR (pKA = 13.3(3), 11.0(3), 9.3(2), 3.8(1) and overnight again.
1.3(3)) are in a good agreement with those obtained by poten-
The complexes of the DOTA-like ligands are usually nona-
tiometry (Table 1); the differences can be attributed to the coordinated (eight donor atoms coming from macrocyclic
different and non-controlled ionic strength during the NMR ligands and one site is occupied by a water molecule) and
experiment and inaccurate (non-linear) potential–pH relation- form two possible diastereomers – square-antiprismatic (SA)
ship in highly acidic/alkaline regions. However, the high value and twisted-square-antiprismatic (TSA) ones.1 These diaster-
of the first protonation constant is confirmed and is clearly eoisomers differ in the relative screwing of the macrocyclic
seen from the chemical shift dependence in the pH region (δ/λ) and pendant (Δ/Λ) chelate rings, giving rise to Δλλλλ/
12.0–13.5 (Fig. S4†). In addition, it is possible, from pH-depen- Λδδδδ (SA) and Δδδδδ/Λλλλλ (TSA) combinations. In the
1
dent changes of individual H NMR signals, to determine also 1H NMR spectra, the signals showing best the isomer ratio
the sites of consecutive protonations (Scheme S1†): the first belongs to those of the “axial” protons of the macrocyclic
proton is distributed over all ring nitrogen atoms; the rather chelate rings, which are the closest ones to the lanthanide(III)
high value of the corresponding protonation constant is a con- ion and to the magnetic axis of the complexes.18 In the Ln(III)–
sequence of intramolecular hydrogen bonds involving the ring DOTA-like complexes, the TSA isomer with a larger coordi-
nitrogen atoms as well as the pendant arms. Two pKA values nation cage is strongly preferred at the beginning of the
can be assigned to the partly simultaneous protonation of the lanthanide series, with the SA one becoming dominant for
pendant amino group and the second nitrogen atom of the heavier lanthanides.18 Although the situation is sometimes
macrocyclic ring, associated with re-location of the protons complicated by the possible presence of an octa-coordinated
bound to the ring nitrogen atoms. The same proton distri- species (i.e., without a coordinated water molecule, usually
bution was also found in the zwitterionic forms of all studied denoted as SA′/TSA′), which have a similar chemical shift
1
ligands in the solid state (see above). The last two measured range in the H NMR spectra to the nona-coordinated SA/TSA
constants correspond to the protonation of the pendant carb- species. Due to this fact, the abundance of twisted-square-anti-
oxylate groups. Further protonations of the ring nitrogen atoms prismatic isomers can slightly increase towards the end of the
take place below pH 1. Such a protonation sequence is consist- lanthanide series.18,19
ent with those commonly found for all DOTA-like ligands.
The solution structures of the studied complexes were
The stability constants, log β011, determined for La(III) and investigated by variable-temperature 1H NMR. To assure the
Gd(III) ions were found to be 20.02 and 22.23, respectively coordination of the pendant amino group, the pH of the
(Table 1). Corresponding distribution diagrams are shown in samples was adjusted to the weakly alkaline region. However,
Fig. S5 and S6.† The stability constants are lower than those the spectrum of the Eu(III) complex at pH 9 (Fig. S7†) acquired
reported for Ln(III)–DOTA (La(III) 22.9, Gd(III) 24.6) complexes at 25 °C was not resolved enough to observe the required
and similar to those of Ln(III)–DO3A (Gd(III) 21.0) complexes.17 signals and to determine the isomer ratio; only very broad
In both studied systems, protonated species with pKA 6.06 and signals in the 17–27 ppm region (after correction for bulk mag-
5.83 for the La(III) and Gd(III) complexes, respectively, are netic susceptibility shift) were observed. Such chemical shifts
formed. The value for the protonation of the Gd(III)–H3L1 are just between the regions typical for SA and TSA species,
complex is in good agreement with the reported value (5.95) although slightly closer to the TSA one.18 Both heating up to
determined by relaxometric measurements.15 The distribution 90 °C or cooling to 0 °C led to a significant broadening and
diagrams show that free lanthanide(III) ions are not present visual disappearance of these signals. It points to the fact that
above pH 5.5. The protonation takes place on the pendant two relatively independent fluxional processes occur, affecting
15738 | Dalton Trans., 2013, 42, 15735–15747
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
the position of the axial hydrogen atoms with respect to the
lanthanide(III) ion. Therefore, we can only speculate that a
mixture of the isomers is present, as it is common for most of
Eu(III) complexes with DOTA-like ligands.18,19 A similar situ-
ation occurs for the Yb(III) complex. The 1H NMR spectrum
acquired at 25 °C and pH 8.5 (Fig. S8†) shows signals of “axial”
protons as broad peaks in the 86–112 ppm region. The chemi-
cal shifts,18,19 signal broadening and their virtual disappear-
ance, observed either at lower and higher temperatures, point
to the presence of a SA(′)/TSA(′) exchange process.
The hypothesis of ongoing SA(′)/TSA(′) isomerism is sup-
ported by the solid-state structure of the [Yb(L1)]·5H2O
complex (Fig. 2). The central ion is octacoordinated between
mutually parallel macrocycle N4- and pendant-arm O3N-
planes, but closer to the pendant-arm one. As a result of
smaller values of the trans (“opening”) angles in the O3N-
plane (120° and 125° for O311–Yb1–N52 and O211–Yb1–O411
angles, respectively), comparing to the limiting value (∼135°)
for water coordination,20 no water molecule is directly bound
to the metal centre. During the refining of the crystal structure,
the set of difference maxima in the electron density map were
located close to the nitrogen and carbon atoms of the macrocycle,
and pointed to some disorder in the macrocyclic unit. It was
successfully modelled, leading to a calculated ratio of the
SA′ : TSA′ isomers of 15 : 85% (Fig. 2 and S9†). The structural
parameters of both coordination spheres fall into regions
typical for the given isomers,20 with mean torsion angles
between N4- and O3N-planes of 37.5 and 22.5°, and separation
of the N4- and O3N-planes of 2.50 and 2.59 Å for the SA′ and
TSA′ species, respectively. Further selected geometric para-
meters are compiled in Table S2.†
Chemical exchange saturation transfer
PARACEST experiments (measurements of so-called Z-spectra)
produced the clean saturation of bulk water after irradiation of
the broad regions with maxima at +19.5 and +34 ppm for the
Eu(III)–H3L1 complex (pH = 7.67, t = 25 °C), and +42 ppm and
+89 ppm for the Yb(III)–H3L1 complex (pH = 7.40, t = 25 °C);
chemical shifts are given with respect to the signal of the bulk
water protons. In the 1H NMR spectra of the Eu(III) complex
acquired in aqueous solution, a broad signal at 34 ppm is
observable, and disappears on selective water pre-saturation or
when acquiring the spectra in D2O (Fig. S10†). The other
signal is overlapped with C–H proton signals and, therefore,
cannot be distinguished in the 1H NMR spectra. Also in the
case of the Yb(III) complex, there are C–H proton signals in the
Fig. 2 Molecular structure of [Yb(L1)] found in the crystal structure of [Yb
(L1)]·5H2O; TSA’ (Λλλλλ, 85%) species (A) and SA’ (Λδδδδ, 15%) species (B).
Carbon-bound H-atoms are omitted for clarity.
regions corresponding to the CEST effect and, thus, it prevents complex of the slightly larger Eu(III) ion (but it cannot be easily
the direct observation of the signals.
confirmed by the normally used luminescence measurements
For both complexes, the two observed CEST signals have due to the presence of two types of quenchers, O–H and N–H,
equal intensities. With respect to the chemical structure of the which complicates the data evaluation). However, in the case
complexes, the observed effect can be theoretically caused of the complex with the significantly smaller Yb(III), an anhy-
by two exchangeable pools of protons, i.e., by protons of drous species might be expected on the basis of the solid-state
the amino group of the pendant arm and/or by protons of the structure (see above). In such cases, three possible elucidations
coordinated water molecule. From relaxometric data, it is can be considered: (i) both signals come from the amino
known that one water molecule is coordinated in the Gd(III) group, (ii) one signal belongs to the coordinated water mole-
complex,15 and this fact is also undoubtedly valid for the cule and other to the amine, and (iii) both signals belong to
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15739

View Article Online
Paper
Dalton Transactions
Fig. 3 Z-spectra of a 80 mM aqueous solution (H2O–D2O 1 : 10) of Eu(III)–H3L1 complex (B0 = 7.05 T, satpwr = 29 dB ∼1000 Hz, satdly = 2 s) at T = 298 K (A) and
pH = 7.40 (C). Z-spectra of a 100 mM aqueous solution (H2O–D2O 1 : 10) of Yb(III)–H3L1 complex (B0 = 7.05 T, satpwr = 29 dB ∼1000 Hz, satdly = 2 s) at T = 298 K
(B) and pH = 7.40 (D).
the water molecule. To distinguish between these possibilities,
Thus, the observed CEST signals are assigned to hydrogen
more detailed studies were carried out. The most interesting atoms of the coordinated pendant amino group. Two observed
CEST behaviour was observed while changing the solution pH. signals rise from the fact that two amine hydrogen atoms
The CEST effect started to be observable at pH ∼6 for both of become magnetically non-equivalent after the group coordi-
the Eu(III) and Yb(III)–H3L1 complexes and reached a maximum nation. A similar situation has been observed for the amide
at pH ∼8, and the saturation transfer efficiency was not hydrogen atoms of DOTAM (Scheme 1).7 The lanthanide-
changed with further pH increases up to ∼12; the mutual induced shift of the amine hydrogen atoms is higher than that
intensity ratio of the CEST peaks is not changed with pH. The of the amide hydrogen atoms in analogous complexes, as the
results are shown in Fig. 3A and 3B and strongly support the coordinated amine group is much closer to the metal ion and
first possibility, as when the CEST effect is caused by the co- to the magnetic axis of the complexes. The possibility that two
ordinated water molecule (or by exchangeable protons of the signals arise from the presence of two (SA and TSA) isomers
amide7 or hydroxy8 moieties), the effect drops with increasing could be excluded as both signals have an equal intensity and
pH due to the faster prototropic (base-catalyzed) exchange of it would imply that the SA and TSA isomers have the same
the corresponding protons (of the coordinated water molecule abundance for both complexes, which is very improbable due
or the other mentioned pools). The behaviour reported in this to the general trends along the lanthanide series. In addition,
work has not yet been reported in the literature. The magni- as both isomers should have a very similar geometry of the
tude of the CEST effect at different pH values follows an abun- N3O-plane, the NMR signals associated with the protons of the
dance of the fully deprotonated species – the CEST effect amino pendant arm should have similar chemical shifts in
consistently disappears at lower pH with protonation and, both the SA and TSA species; however, there is a significant
thus, decoordination of the amino group (see potentiometric difference in the chemical shifts between the signals (Δδ ∼15
results, Table 1 and Fig. S5 and S6†). The observations men- and 50 ppm for Eu(III) and Yb(III) complexes, respectively).
tioned above are fully consistent with the low “acidity” of the With increasing temperature, the saturation transfer becomes
N–H bond in the studied amino derivative if compared to the more effective and two amine hydrogen signals converge and,
generally higher acidity of the N–H proton in carboxylic finally, coalescence around 55 °C and 65 °C for the Eu(III) and
amides; therefore, in the case of DOTAM complexes, base-cata- Yb(III) complexes, respectively (Fig. 3C and 3D). On further
lysed proton exchange significantly decreases the CEST effect heating (up to 95 °C, Fig. S11†), the coalesced CEST signals
in alkaline solutions but, in the case of our complexes, the approach slightly closer to signal of the bulk water but are still
CEST effect remains unchanged even at very high pH.
observable. Such a behaviour points to the dynamic averaging
15740 | Dalton Trans., 2013, 42, 15735–15747
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
of the non-equivalent amine hydrogen signals; it can originate explanation is consistent with the fact that the observed CEST
from a faster coordination–decoordination equilibrium or effect disappears at a relatively low temperature ∼65 °C, which
from a faster geometry change.
is probably due to the much faster chemical exchange process
Due to the insensitivity of the CEST effect to pH changes in (Fig. S13†), contrary to the complexes of H3L1 where the ana-
the alkaline region, the process described above cannot be logous signal disappearance cannot be reached even at 95 °C
caused by neither hydroxide nor proton attack on the co- (see above). In the case of the Eu(III)–H3L3 complex, a slight
ordinated amino group. Therefore, the mechanism of the CEST effect was also observed, causing some asymmetric
observed CEST effect is very probably associated with the broadening of the water signal in the Z-spectra (Fig. S13†).
(semi)labile coordination of the 2-aminoethyl pendant arm. Its This feature can be explained in a similar way to the above.
coordination–decoordination is the rate-limiting step – when
To test the applicability of the complexes as MRI pH
the pendant arm is coordinated, no exchange occurs but in probes, the CEST effect was measured in phantoms containing
the moment of the pendant arm decoordination, the exchange Eu(III)–H3L1 and Yb(III)–H3L1 complex solutions having
of protons of the free amino group with those of bulk water different pH and concentrations. The final CEST images were
proceeds very quickly.
obtained after irradiation at the CEST and symmetrical nega-
To support the hypothesis presented above, we prepared tive frequencies and subtracting the images (Fig. 5 and S14†).
and studied complexes of mono- and dimethylated analogues, Normalized signal intensities are shown in Fig. S15 and S16.†
i.e., with ligands H3L2 and H3L3. The complexes of H3L2 The results clearly show that the prepared complexes can be
showed significant CEST effects, observed as two close peaks successfully employed as CEST probes in the pH region rele-
with unequal intensity at 43 and 49.5 ppm for Eu(III) and one vant to living systems.
peak at 57 ppm for Yb(III), respectively (Fig. S12†). The two
signals in the Z-spectra, observed in the case of the Eu(III)
complex, can be attributed to two modes of coordination of
Experimental
the methylamino group, in which the methyl and hydrogen
Materials and methods
occupy equatorial/axial or reverse positions. In the case of the
Commercially available chemicals had synthetic purity and
were used as received. Deionized water, used for the synthesis
of the ligands and their complexes, was prepared using a de-
ionization water system ROWAPUR 200/100. Water used for
potentiometric titrations was prepared by a Milli-Q (Millipore).
t-Bu3DO3A·HBr [1,4,7-tris(t-butylcarboxymethyl)-1,4,7,10-
tetraazacyclododecane hydrobromide] was prepared according
to the published procedure.21 Dry solvents were prepared by
the standard purification procedures22 and stored over mole-
cular sieves under an argon atmosphere: dry MeCN was
obtained by distillation from P2O5, THF was dried by refluxing
with potassium and distilled under an argon atmosphere.
The ESI-MS spectra were acquired on a Bruker ESQUIRE
3000 spectrometer equipped with an electrospray ion source
and ion-trap detection. Measurements were carried out in the
positive and negative modes. NMR characterization data (1D:
1H, 13C; 2D: HSQC, HMBC, 1H–1H COSY) were recorded on
VNMRS300, VarianUNITY INOVA 400 or Bruker Avance III 600,
using 5-mm sample tubes. Chemical shifts δ are given in ppm
and coupling constants J are reported in Hz. For the 1H and
13C measurements in D2O, t-BuOH was used as the internal
standard (δH = 1.25, δC = 30.29). For the measurements in
complex with the smaller Yb(III) ion, one of these possibilities
is probably strongly preferred due to the larger steric strain
around the methyl group, leading to only one observable
signal in the Z-spectra. However, to our surprise, the Yb(III)–
H3L3 complex also exhibits a CEST effect at 26 ppm, although
the CEST peak has a small intensity and is observable only in
a narrow pH range 6–8.5 (Fig. 4 and S13†). In this pH region,
the protonation–deprotonation of the –NMe2 group is sup-
posed (accordingly to the corresponding log KA ∼7.8 reported
previously for its Gd(III) complex15). Thus, an equilibrium
between the deprotonated (and mostly coordinated) and proto-
nated (and uncoordinated) amino group in the complex is
present. Therefore, the observation of the CEST effect can be
explained by the protonation–deprotonation of the uncoordi-
nated dimethylamino group, which is still located sufficiently
close to the highly paramagnetic centre to mediate the satur-
ation transfer to the bulk water through this process. Such an
CDCl3, TMS was used as the internal standard (δH = 0.00, δC
=
0.00). Abbreviations s (singlet), t (triplet), q (quartet), m (multiplet)
and br (broad) are used in order to express the signal multipli-
cities. Elemental analysis was performed at the Institute of
Macromolecular Chemistry of the Academy of Science of the
Czech Republic (Prague).
Synthesis
N-(2-Bromoethyl)phthalimide (3). Compound 3 was pre-
Fig. 4 Z-spectra of a 50 mM aqueous solution (H2O–D2O 1 : 10) of the Yb(III)–
H3L3 complex (B0 = 7.05 T, T = 298 K, satpwr = 29 dB ∼1000 Hz, satdly = 2 s).
pared according to a modified published procedure.23 To a
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15741

View Article Online
Paper
Dalton Transactions
Fig. 5 MRI-CEST images of phantoms consisting of one vial containing 80 mM aq. solution of [La(H2O)(L1)] as a diamagnetic standard and eight vials containing
80 mM aq. solutions of [Eu(H2O)(L1)] and [Yb(L1)] with different pH values; pH is shown in figures A. Experimental conditions: MSME pulse sequence, B0 = 4.7 T, B1
=
20 μT, T = 293 K, satdly = 2 s, TR = 5 s, TE = 8.9 ms, scan time = 8 min. I.A: T1-weighted image, satfrq = 34 ppm from the bulk water signal. I.B: T1-weighted image,
satfrq = −34 ppm from the bulk water signal. I.C: The difference between images I.A and I.B in false colours. II.A: T1-weighted image, satfrq = 89 ppm from the bulk
water signal. II.B: T1-weighted image, satfrq = −89 ppm from the bulk water signal. II.C: The difference between images II.A and II.B in false colours.
well-stirred suspension of K2CO3 (9.40 g, 68 mmol), 1,2-dibromo- organic portion was dried over anhydrous Na2SO4 and concen-
ethane 2 (11.7 ml, 136 mmol) and tetrabutylammonium trated in vacuo to give 3.40 g of yellow oil containing compound
bromide (TBAB, 0.70 g) in DMF (20 ml), was added phthali- 4 contaminated with phthalhydrazide and an excess of alkylat-
mide 1 (5.00 g, 34 mmol) and the reaction mixture was stirred ing reagent 3. The by-product and alkylating reagent were not
for 18 h at 40 °C. Next, the mixture was poured into water removed, and the crude product 4 was used in the next step
(150 ml) and the product 3 was extracted with ethylacetate without purification (only a small amount of the crude product
(4 × 25 ml). The organic portion was concentrated in vacuo. 4 was purified for characterization purposes using HPLC).
The product was purified by crystallization from hot EtOH to
A portion (3.35 g) of this mixture containing compound 4
give 3 (7.61 g, 88%).
was dissolved in CF3COOH and CHCl3 (30 ml, 1 : 1); the result-
3
1H NMR (299.9 MHz, CDCl3): δ 3.59 (2H, t, JHH = 6.6, ing solution was refluxed for 24 h. After evaporation to dryness
3
NCH2CH2Br); 4.09 (2H, t, JHH = 6.6, NCH2CH2Br); 7.70–7.73 the oily residue was dissolved in a small amount of distilled
(2H, m, arom.); 7.84–7.86 (2H, m, arom.).
water and then loaded onto a strong cation exchange column
13C{1H} NMR (75.4 MHz, CDCl3): δ 28.55 (1C, NCH2CH2Br); (Dowex 50, 50–100 mesh, H+-form, 2 × 9 cm). Acidic impurities
39.70 (1C, NCH2CH2Br); 123.93 (2C, arom.); 132.23 (2C, arom. were removed by elution with water and the product 5 was
quaternary); 134.63 (2C, arom.); 168.22 (2C, CO).
eluted with 5% aq. NH3. Fractions containing the product
Elemental analysis: found (calcd for C10H8BrNO2, Mr = 254.1) (TLC check) were combined and evaporated to give 2.82 g of
C: 47.68 (47.27), H: 3.07 (3.17), N: 5.32 (5.51), Br: 31.15 (31.45). yellow oil containing compound 5. The crude product 5 was
1,4,7-Tris(carboxymethyl)-10-(2-aminoethyl)-1,4,7,10-tetra- used in the next step without purification.
azacyclododecane (H3L1·3H2O). Compound 4 was prepared
The portion (2.80 g) of the crude product 5 was dissolved in
in situ according to a modified published procedure.14 To 80% aq. NH2NH2·H2O (15 ml); the resulting mixture was then
a well-stirred suspension of K2CO3 (2.40 g, 17.4 mmol) and heated for 18 h at 90 °C and concentrated in vacuo. The
t-Bu3DO3A·HBr (2.59 g, 4.37 mmol) in dry MeCN (40 ml), alky- residue was dissolved in a small amount of distilled water.
lating reagent 3 (1.22 g, 4.80 mmol) was gradually added over Then the solution was filtered and loaded onto a strong anion
30 min and the reaction mixture was stirred for 24 h at 60 °C. exchange column (Dowex 1, OH−-form, 1.25 × 8 cm). Impuri-
The solids were filtered off and the filtrate was evaporated on a ties were removed by elution with water and the product H3L1
rotary evaporator. The oily residue was dissolved in CHCl3 was eluted with 5% aq. CH3COOH. Fractions containing the
(20 ml) and extracted with distilled water (4 × 10 ml). The pure product (TLC and 1H NMR check) were combined and
15742 | Dalton Trans., 2013, 42, 15735–15747
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
evaporated to give H3L1 (1.90 g) as an yellow oil. The crude The reaction mixture was stirred for 1 h at room temperature,
product was purified by crystallization from hot EtOH to give filtered and then evaporated on a rotary evaporator. The oily
H3L1·3H2O (1.15 g, 59%) as a white powder.
residue was dissolved in small amount of CH2Cl2 and purified
by chromatography (SiO2, 18 × 3.5 cm). Impurities were
removed by elution with CH2Cl2, the pure product was eluted
using a mixture of acetone and CH2Cl2 (1 : 9). Fractions con-
taining the product 8 (TLC check) were combined and evapo-
rated to give 8 (1.32 g) as a colourless oil.
Compound 4. HPLC: Solvent: A = CH3CN, B = 0.1% of
CF3COOH in H2O, C = H2O. Gradient: A: 20–50%, 0–24 min;
50%, 24–40 min; 50–20%, 40–41 min; 20%, 41–61 min. B:
20%, 0–61 min. tR = 17.0 min.
1H NMR (299.9 MHz, CDCl3):
δ 1.45–1.48 (27H, m,
3
1H NMR (299.9 MHz, CDCl3): δ 1.26 (3H, t, JHH = 6.9,
(C(CH3)3); 3.15–3.43 (18H, m, CH2); 3.53 (4H, s, CH2CO2); 3.86
(2H, s, CH2CO2); 4.02 (2H, br, CH2); 7.73–7.76 (2H, m, arom.);
7.84–7.87 (2H, m, arom.).
CH2CH3); 2.97 (3H, s, NCH3); 3.45 (2H, br, NCH2); 3.62 (2H, br,
CH2Br); 4.14 (2H, q, 3JHH = 6.9, CH2CH3).
13C{1H} NMR (150.9 MHz, CDCl3): δ 27.92 (9C, C(CH3)3);
32.58, 48.97, 49.92, 50.13, 51.17, 51.86, 54.77 (13C, CH2); 82.96
(2C, C(CH3)3); 84.28 (1C, C(CH3)3); 123.58 (2C, arom.); 131.65
(2C, arom. quaternary); 134.37 (2C, arom.); 167.85 (3C,
CH2CO2); 169.22 (2C, NCO).
13C{1H} NMR (75.4 MHz, CDCl3): δ 14.89 (1C, CH2CH3);
29.50 (1C, CH2Br); 35.60 (1C, NCH3); 51.30 (1C, CH2CH2Br);
61.76 (1C, CH2CH3); 156.31 (1C, CO).
1,4,7-Tris(carboxymethyl)-10-[2-(N-methylamino)ethyl)]-
1,4,7,10-tetraazacyclododecane (H3L2·5.5H2O). To a well-stirred
suspension of K2CO3 (3.30 g, 23.9 mmol) and t-Bu3DO3A·HBr
(2.89 g, 4.85 mmol) in dry MeCN (30 ml), a solution of alkylat-
ing reagent 8 (1.32 g, 6.28 mmol) in dry MeCN (10 ml) was
added dropwise. The reaction mixture was stirred for 24 h at
60 °C, filtered, and the filtrate was evaporated on a rotary evapo-
rator. The oily residue was dissolved in CHCl3 (20 ml) and
extracted with distilled water (4 × 10 ml). The organic portion
was dried over Na2SO4 and concentrated in vacuo to give 3.82 g
of yellow oil containing compound 9 contaminated with an
excess of alkylating reagent 8. The alkylating reagent was not
removed, and the crude product 9 was used in the next step
without purification.
A portion (3.80 g) of the mixture containing compound 9
was dissolved in a mixture of CF3COOH and CHCl3 (30 ml,
1 : 1); the resulting solution was refluxed for 24 h and evapo-
rated on a rotary evaporator. The oily residue was dissolved in a
small amount of distilled water and evaporated (this procedure
was then repeated three more times) to give 5.00 g of yellow oil
containing compound 10 (it was used in the next step without
purification).
MS-ESI: (+): 688.5 ([M + H]+, calcd 688.4); 710.4 ([M + Na]+;
calcd 710.4).
Compound 5. MS-ESI: (−): 518.0 ([M − H]−, calcd 518.2).
H3L1·3H2O. 1H NMR (600.2 MHz, D2O, pD = 5.4, Fig. S17†):
2.85–2.89 (4H, m, CH2NCH2CO); 2.94 (2H, br, CH2CH2NH2);
3.02–3.04 (4H, m, CH2N(CH2)2NH2); 3.06 (2H, t, JHH
5.1, CH2NH2); 3.13 (2H, s, CH2CO); 3.33–3.37 (2H, m,
CH2NCH2CO); 3.40–3.45 (2H, m, CH2NCH2CO); 3.54–3.58 (2H,
m, CH2NCH2CO); 3.71–3.77 (2H, m, CH2NCH2CO); 3.90 (4H,
AB-multiplet, CH2CO).
3
=
13C{1H} NMR (150.9 MHz, D2O, pD = 5.4, Fig. S18†):
δ 37.01 (1C, CH2NH2); 48.90 (2C, CH2N(CH2)2NH2); 48.99
(2C, CH2NCH2CO); 50.82 (1C, CH2CH2NH2); 51.00 (2C, CH2-
NCH2CO); 53.06 (2C, CH2NCH2CO); 55.85 (1C, CH2CO); 58.07
(2C, CH2CO); 171.02 (2C, CH2CO); 178.77 (1C, CH2CO).
MS-ESI: (+): 412.0 ([M + Na]+, calcd 412.2); 428.0 ([M + K]+,
calcd 428.2). (−): 425.8 ([M + K − 2H]−, calcd 426.2).
Elemental analysis: found (calcd for C16H37N5O9, Mr = 443.5)
C: 43.17 (43.33), H: 8.40 (8.41), N: 15.39 (15.79).
2-[N-(Ethyloxycarbonyl)-N-methylamino]ethanol (7). To
a
solution of 6 (2.64 g, 35.2 mmol) in a mixture of dioxane
and H2O (30 ml, 1 : 1), ethyloxycarbonylchloride (0.96 g,
8.85 mmol) was added dropwise. The solution was stirred at
room temperature for 2 h and concentrated in vacuo. The oily
residue was dissolved in CH2Cl2 (30 ml) and extracted with
H2O (3 × 15 ml) and 3% aq. HCl (1 × 15 ml). The organic
portion was dried over Na2SO4 and concentrated in vacuo to
give 1.10 g of 7 as a colourless oil.
The crude product 10 (5.00 g) was dissolved in 10% aq.
NaOH (50 ml) and stirred for 24 h at 90 °C. Then the solution
was loaded onto a strong anion exchange column (Dowex 1,
OH−-form, 1.25 × 20 cm). Impurities were removed by elution
with water and the product H3L2 was eluted with 5% aq.
CH3COOH. Fractions containing the product (TLC and
1H NMR check) were combined and evaporated to give H3L2
(1.97 g) as a brownish oil. The crude product was dissolved in
small amount of distilled water and anhydrous EtOH was
added dropwise. Crystallization started after a few minutes,
and the suspension was left overnight. The white crystalline
solid was filtered off and dried under a vacuum to give
H3L2·5.5H2O (0.66 g, 30%) as a white powder.
3
1H NMR (299.9 MHz, CDCl3): δ 1.24 (3H, t, JHH = 7.2,
CH2CH3); 2.94 (3H, s, NCH3); 3.41 (2H, br, NCH2); 3.73 (2H, br,
CH2OH); 4.11 (2H, q, 3JHH = 7.2, CH2CH3).
13C{1H} NMR (75.4 MHz, CDCl3): δ 14.85 (1C, CH2CH3);
35.42 (1C, NCH3); 51.92 (1C, CH2CH2OH); 61.40 (1C, CH2OH);
61.74 (1C, CH2CH3); 158.17 (1C, CO).
Compound 9. MS-ESI: (+): 644.5 ([M + H]+, calcd 644.5);
666.4 ([M + Na]+; calcd 666.5).
MS-ESI: (+): 148.5 ([M + H]+, calcd 148.1); 170.4 ([M + Na]+,
calcd 170.1).
Compound 10. MS-ESI: (−): 474.1 ([M − H]−, calcd 474.3);
512.0 ([M + K − 2H]−, calcd 512.0).
2-[N-(Ethyloxycarbonyl)-N-methylamino]bromoethane (8).
To a well-stirred solution of 7 (1.09 g, 7.41 mmol) in dry THF
H3L2·5.5H2O. 1H NMR (299.9 MHz, D2O, pD = 1.15): δ 2.63
(40 ml), in a flask equipped with a drying tube, CBr4 (3.68 g, (3H, s, NCH3); 2.80–3.15 (12H, m, CH2); 3.20–3.63 (10H, m, CH2);
11.1 mmol) and PPh3 (2.92 g, 11.1 mmol) were added. 3.97 (4H, br, CH2CO2).
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15743

View Article Online
Paper
1H NMR (299.9 MHz, D2O, pD = 5.3, Fig. S19†): δ 2.77 (3H,
Dalton Transactions
Eu3+–H3L2. MS-ESI: (+): 553.6 ([M + H]+, calcd 554.2). (−):
s, NCH3); 2.80–2.91 (4H, m, CH2); 2.94–3.08 (8H, m, CH2); 3.13 587.7 ([M + Cl]−, calcd 588.1).
Yb3+–H3L2. MS-ESI: (+): 581.8 ([M + Li]+, calcd 581.2). (−):
(2H, s, CH2CO2); 3.31–3.46 (4H, m, CH2); 3.52–3.60 (2H, m,
CH2); 3.70–3.76 (2H, m, CH2); 3.89 (4H, AB-multiplet,
CH2CO2).
609.6 ([M + Cl]−, calcd 609.1).
Eu3+–H3L3. MS-ESI: (+): 574.7 ([M + Li]+, calcd 574.2). (−):
602.6 ([M + Cl]−, calcd 602.1).
13C{1H} NMR (150.9 MHz, D2O, pD = 5.6, Fig. S20†): δ 34.93
(1C, NCH3); 46.89 (1C, CH2NCH3); 48.89 (2C, CH2); 49.03 (2C,
CH2); 50.42 (1C, CH2CH2NCH3); 51.04 (2C, CH2); 53.10
(2C, CH2); 55.68 (1C, CH2CO2); 58.16 (2C, CH2CO2); 171.08
(2C, CH2CO2); 178.63 (1C, CH2CO2).
Yb3+–H3L3. MS-ESI: (+): 595.8 ([M + Li]+, calcd 595.2). (−):
623.6 ([M + Cl]−, calcd 623.1).
X-Ray diffraction
Single-crystals of H3L1·5H2O were prepared by slow evaporation
of the aqueous solution. The crystals of H3L2·6H2O were pre-
pared by evaporation of the ethanolic solution, and the crystals
of H3L3·3.5H2O were prepared by the slow cooling of a satu-
rated hot ethanolic solution. The Yb3+–H3L1 complex for crys-
tallization was prepared by mixing the ligand with 1.1 equiv. of
the YbCl3 in a small amount of distilled water. The pH was
adjusted to ∼6 with 1 M NaOH, and the mixture was stirred
overnight at 60 °C. Then the pH was re-adjusted to 6.5 and the
solution was stirred overnight at 60 °C. The complex was puri-
fied on a Al2O3 column by chromatography. The pure product
was eluted using a mixture of EtOH–H2O–conc. aq. NH3
(10 : 8 : 1). Single crystals of [Yb(L1)]·5H2O were prepared from
a concentrated aqueous solution of the complex by slow
diffusion of the THF vapours.
The diffraction data were collected by employing a ApexII
CCD diffractometer using Mo-Kα radiation (λ = 0.71073 Å) at
150(1) K and analyzed using the SAINT V8.27B (Bruker AXS
Inc., 2012) program package. The structures were solved by
direct methods (SHELXS9724) and refined by full-matrix least-
squares techniques (SHELXL9725). The relevant data for the
structures have been provided.†
MS-ESI: (+): 404.3 ([M + H]+, calcd 404.2); 426.9 ([M + Na]+,
calcd 426.2); 442.8 ([M + K]+, calcd 442.2).
Elemental analysis: found (calcd for C17H44N5O11.5, Mr
=
502.6) C: 40.84 (40.63), H: 9.27 (9.22), N: 13.80 (13.84).
1,4,7-Tris(carboxymethyl)-10-[2-(N,N-dimethylamino)ethyl)]-
1,4,7,10-tetraazacyclododecane (H3L3·4H2O). To a well-stirred
solution of H3L1·3H2O (100 mg, 0.225 mmol) in 10% aq.
HCOOH (10 ml), paraformaldehyde (26.5 mg, 0.88 mmol) was
added; the reaction mixture was refluxed for 5 h and analysed
1
by H NMR. To the reaction mixture, more paraformaldehyde
(26.5 mg, 0.88 mmol) was added and the reaction mixture
was refluxed for 5 h (this procedure was then repeated once
more). After the reaction was completed, the solution was
filtered and the filtrate was concentrated in vacuo. The
residue was dissolved in a small amount of distilled water
and evaporated under reduced pressure (this procedure was
then repeated three more times) to give H3L3 (95 mg) as a
yellow oil. The crude product was purified by crystallization
from hot EtOH to give H3L3·4H2O (78 mg, 71%) as a white
powder.
1H NMR (600.2 MHz, D2O, pD = 4.8, Fig. S21†): δ 2.87–2.90
3
(2H, m, CH2); 2.93 (6H, s, N(CH3)2); 2.96 (2H, t, JHH = 6.0 Hz,
NCH2CH2N(CH3)2); 3.05–3.08 (4H, m, CH2); 3.13–3.17 (2H, m,
3
Crystal data
CH2); 3.27 (2H, t, JHH = 6.0 Hz, CH2N(CH3)2); 3.36 (2H, s,
CH2CO2); 3.38–3.40 (2H, m, CH2); 3.47–3.56 (6H, m, CH2); 3.88 H3L1·5H2O: C16H41N5O11, M = 479.54, monoclinic, a = 7.80380(10),
(4H, AB-multiplet, CH2CO2).
b = 16.7236(3), c = 17.8081(3) Å, β = 92.1431(12)°, U =
13C{1H} NMR (150.9 MHz, D2O, pD = 4.8, Fig. S22†): δ 44.32 2322.47(6) Å3, space group P21/c, Z = 4, 5358 total reflections,
(2C, N(CH3)2); 48.08 (1C, CH2, NCH2CH2N(CH3)2); 48.70 (2C, 4408 intense reflections, R1[I > 2σ(I)] = 0.0344, wR2(all data) =
CH2); 49.14 (2C, CH2); 51.32 (2C, CH2); 52.68 (2C, CH2); 55.63 0.0873. CCDC 952386.† All non-hydrogen atoms were refined
(1C, CH2CO2); 56.08 (1C, CH2N(CH3)2); 57.49 (2C, CH2CO2); anisotropically. Although all hydrogen atoms could be found
170.51 (2C, CH2CO2); 177.96 (1C, CH2CO2).
in the electron difference map, those bound to carbon atom
were fixed in theoretical positions using a riding model with
Ueq(H) = 1.2Ueq(C) to keep the number of refined parameters
low. Hydrogen atoms bound to nitrogen or oxygen atoms were
MS-ESI: (+): 456.9 ([M + K]+, calcd 456.2).
Elemental analysis: found (calcd for C18H43N5O10, Mr
489.6) C: 44.29 (44.16), H: 8.86 (8.85), N: 14.14 (14.31).
=
Syntheses of Ln(III) complexes. The Ln(III) complexes of fully refined.
H3L1, H3L2 and H3L3 for NMR, NMR CEST and MRI CEST
H3L2·6H2O: C17H45N5O12, M = 511.58, monoclinic, a =
experiments were prepared by mixing the lanthanide(III) chlor- 9.4479(7), b = 17.7199(14), c = 15.5379(9) Å, β = 106.573(2)°, U =
ide (Eu3+, Yb3+, La3+) with 1.1 equiv. of the ligand in a small 2493.2(3) Å3, space group P21/n, Z = 4, 5723 total reflections,
amount of distilled water, adjusting to pH 6 with 1 M aq. 4765 intense reflections, R1[I > 2σ(I)] = 0.0415, wR2(all data) =
LiOH, and stirring overnight at 60 °C. Then the pH was re- 0.1129. CCDC 952384.† The strategy of refinement was the
adjusted to 6.5 with 1 M aq. LiOH and the solution was stirred same as that described in the previous case. Beside this,
overnight at 60 °C.
several solvate water molecules were refined as twisted in two
positions with some disordered hydrogen atoms in two posi-
tions with half-occupancy.
, M = 480.57, monoclinic,
a = 7.4798(2), b = 15.9561(4), c = 20.0951(6) Å, β = 91.5670(10)°,
Eu3+–H3L1. MS-ESI: (+): 540.1 ([M + H]+, calcd 540.2); 562.1
([M + Na]+; calcd 562.1). (−): 573.9 ([M + Cl]−, calcd 574.1).
Yb3+–H3L1. MS-ESI: (+): 561.1 ([M + H]+, calcd 561.2); 683.1
([M + Na]+; calcd 683.2). (−): 594.9 ([M + Cl]−, calcd 595.1).
H3L3·3.5H2O: C18H42N5O9.50
15744 | Dalton Trans., 2013, 42, 15735–15747
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
U = 2397.42(11) Å3, space group P21/n, Z = 4, 5503 total reflec- Titrations were performed with a (Me4N)OH solution. Titra-
tions, 4686 intense reflections, R1[I > 2σ(I)] = 0.0370, wR2- tions with metal ions were performed at metal-to-ligand molar
(all data) = 0.0952. CCDC 952385.† The refining strategy was ratios of 1 : 1. Ligand titrations consisted of about 40 points
the same as described above.
per titration and were run in triplicate. As the complexation
[Yb(L1)]·5H2O: C16H38N5O11Yb, M = 649.55, monoclinic, was too slow for conventional titration, the “out-of-cell”
a = 16.8708(7), b = 7.5238(4), c = 18.4880(9) Å, β = 90.4230(10)°, method was used;26 about 25 points per titration, two parallel
U = 2346.7(2) Å3, space group P21/c, Z = 4, 5374 total reflec- titrations, three weeks at room temperature for equilibration.
tions, 5160 intense reflections, R1[I > 2σ(I)] = 0.0137, wR2(all To calculate the protonation constants of the ligand and the
data)
revealed a number of different maxima of the electronic package was used.
=
0.0306. CCDC 933967.† Preliminary refinement stability constants of the complexes, the OPIUM27 software
density, which were located close to the carbon and nitrogen
The overall protonation constants βh10 are concentration
atoms of the macrocyclic part of the molecule. It pointed to a constants defined as βh10 = [HhL]/([H]h·[L]) (they can be trans-
disorder of the macrocycle in a similar way as already reported ferred into stepwise dissociation constants as pKA(HhL) = log
in the literature (see, e.g., ref. 20c, Fig. S9†). It was successfully βh − log βh−1). The concentration stability constants βhlm are
modelled using the anisotropic refinement of atoms occupying generally defined by βhlm = [HhLlMm]/([H]h·[L]l·[M]m). The value
positions with a higher occupancy, whilst the less occupied of pKw used was 13.81.
part was refined in isotropic mode. All other non-hydrogen
atoms were refined anisotropically. Although hydrogen atoms
1H NMR titrations
could be found in the electron difference map, they were fixed
The 1H NMR titration over the whole pH region (31 points), for
in theoretical (C–H) or original (N–H, O–H) positions using the
determination of the protonation constants, was performed at
riding model with Ueq(H) = 1.2Ueq(X) to keep the number of
25 °C (ligand concentration: 0.05 M; no ionic strength control)
refined parameters low.
in H2O on a Brucker Avance (III) 600 (B0 = 14.1 T), using 5 mm
sample tubes. 1H NMR spectra were measured without pre-
HPLC
saturation of the water signal. A coaxial capillary with D2O (for
The analytical HPLC system consisted of a gradient pump Beta
the lock) and t-BuOH (as external standard; δH = 1.25 ppm)
10 (ECOM) equipped with a mixer Knauer A0285 and dual UV-
was used. The solution pH (0.5–13.5) was adjusted with
detector (ECOM). Analysis was performed on a LunaPHC8
aqueous HCl or NMe4OH solutions and measured with a com-
column (150 × 4.6 mm, Phenomenex, the flow rate 1 ml
bined glass electrode (Mettler Toledo) and pH-meter (3505 pH
min−1). The mobile phase was continuously vacuum degassed
Meter, JENWAY) calibrated with standard buffers. Protonation
in a DG 3014 degasser (ECOM) and it was mixed in the gradi-
constants were calculated with the OPIUM software package.27
ent pump from the stock solutions. The detector wavelengths
were set to 210 and 254 nm. Injection volumes were 20 μl (con-
CEST experiments
centrations of the samples were 1 mg ml−1).
The preparative HPLC system was composed of a gradient All Z-spectra were recorded on a VNMRS300 operating at
pump LCD 50 K (ECOM) and UV-Vis detector LCD 2083 299.9 MHz (B0 = 7.05 T), using 5 mm sample tubes and a
(ECOM). Preparation was performed on a LunaPHC8 (250 × coaxial capillary with D2O and t-BuOH as the external stan-
21.1 mm, Phenomenex, the flow rate was maintained at 20 ml dard. Samples were of 25–100 mM concentration in the
min−1). The detector wavelengths were set to 210 nm. The mixture of H2O and D2O (1 : 10) or in H2O; pH was adjusted
mobile phase was prepared separately and degassed using an with aqueous HCl or LiOH solutions. Standard pulse
ultrasound probe (Cole-Parmer 750-Watt Ultrasonic Homogen- sequences for presaturation experiments were used. Saturation
izer). Injection volumes were 1 ml. The collection and proces- offsets were set using the array-function (increment 200–250
sing of the data was performed using Clarity (DataApex) Hz). Other measurement parameters are listed below each
software.
figure.
MRI CEST images were measured with phantoms consist-
ing of one vial containing an aqueous solution of the La3+
Potentiometry
-
Potentiometric titrations26 were carried out in a vessel thermo- complex as a control and eight (resp. five) vials containing
statted at 25.0 0.1 °C, at a constant ionic strength (I = 0.1 M aqueous solution of Eu3+ or Yb3+-complexes with different pH
(Me4N)Cl) using a PHM 240 pH-meter, a GK 2401B combined values or concentrations. All MRI CEST images were acquired
glass electrode and a 2 ml ABU 900 automatic piston burette on a 4.7 T scanner (Bruker BioSpec, Germany) using RARE
(all Radiometer). The initial volume was ca. 5 ml and the (Rapid Acquisition with Refocused Echoes) or MSME (Multi
ligand concentration in the titration vessel was ca. 0.004 M. An Slice Multi Echo) pulse sequences with presaturation pulse.
inert atmosphere was provided by the constant passage of Experimental conditions: TR = 5000 ms, TE = 8.9 ms, resolu-
argon saturated with the vapour of the solvent [0.1 M (Me4N) tion 0.35 × 0.35 × 2 mm, turbo factor = 4 (in RARE sequence).
Cl]. Extra HCl was added to the starting solution to run titra- Other measurement parameters are listed below each figure.
tions in the −log[H+] range 2.0–12.0 (for protonation con- Parameters of presaturation pulse: B1 = 20 μT, satdly = 2 s. For
stants) and 1.8–6.0 for the Gd3+ and La3+ stability constants. all MR experiments, the resonator coil was used.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15745

View Article Online
Paper
All MR images were processed using an in-house program,
written in Matlab (The Mathworks Inc, USA), where the signal
intensity was normalized to unit slope and receiver gain. Then
the difference image between the images acquired with nega-
tive and positive frequency offset of the saturation frequency
was calculated. Difference images were analysed using ImageJ
software (NIH, USA), regions of interest were outlined manually.
Dalton Transactions
4 S. Zhang and A. D. Sherry, J. Solid State Chem., 2003, 171,
38–43.
5 M. Woods, A. Pasha, P. Zhao, G. Tireso, S. Chowdhury,
G. Kiefer, D. E. Woessner and A. D. Sherry, Dalton Trans.,
2011, 40, 6759–6764.
6 S. Zhang, K. Wu, M. C. Biewer and A. D. Sherry, Inorg.
Chem., 2001, 40, 4284–4290.
7 S. Zhang, L. Michaudet, S. Burgess and A. D. Sherry, Angew.
Chem., Int. Ed., 2002, 41, 1919–1921.
Conclusions
8 (a) C.-H. Huang and J. R. Morrow, Inorg. Chem., 2009, 48,
7237–7243; (b) C.-H. Huang and J. R. Morrow, J. Am. Chem.
Soc., 2009, 131, 4206–4207; (c) C.-H. Huang, J. Hammell,
S. J. Ratnakar, A. D. Sherry and J. R. Morrow, Inorg.
Chem., 2010, 49, 5963–5970; (d) J. Hammell, L. Buttarazzi,
C.-H. Huang and J. R. Morrow, Inorg. Chem., 2011, 50,
4857–4867; (e) D. D. Castelli, E. Terreno and S. Aime,
Angew. Chem., Int. Ed., 2011, 50, 1798–1800.
9 S. J. Dorazio, P. B. Tsitovich, K. E. Siters, J. A. Spernyak and
J. R. Morrow, J. Am. Chem. Soc., 2011, 133, 14154–14156.
10 (a) S. J. Dorazio and J. R. Morrow, Inorg. Chem., 2012, 51,
7448–7450; (b) S. J. Dorazio, P. B. Tsitovich, S. A. Gardina
and J. R. Morrow, J. Inorg. Biochem., 2012, 117, 212–219;
(c) P. B. Tsitovich and J. R. Morrow, Inorg. Chim. Acta, 2012,
393, 3–11.
We prepared 2-aminoethyl derivatives of DO3A and showed
that the coordinated amino group mediates NMR saturation
transfer to the bulk water. We suggest a new mechanism for
the PARACEST effect, consisting of the coordination–decoordi-
nation of the amino group having an optimal rate, combined
with the very fast protonation–deprotonation of the group
while the group is non-bound to the paramagnetic metal ion.
The pH-dependence of the saturation transfer parallels with
the abundance of the species with the coordinated amino
group. This novel type of PARACEST agent broadens the
arsenal of pH-sensitive probes for possible in vivo pH measure-
ments by MRI.
11 A. O. Olatunde, S. J. Dorazio, J. A. Spernyak and
J. R. Morrow, J. Am. Chem. Soc., 2012, 134, 18503–18505.
12 (a) B. Yoo and M. D. Pagel, J. Am. Chem. Soc., 2006,
128, 14032–14033; (b) G. Liu, Y. Li and M. D. Pagel,
Magn. Reson. Med., 2007, 58, 1249–1256; (c) T. Chauvin,
P. Durand, M. Bernier, H. Meudal, B.-T. Doan, F. Noury,
B. Badet, J.-C. Beloeil and É. Tóth, Angew. Chem., Int. Ed.,
2008, 47, 4370–4372; (d) T. Chauvin, S. Torres, R. Rosseto,
J. Kotek, B. Badet, P. Durand and É. Tóth, Chem.–Eur. J.,
2012, 18, 1408–1418; (e) V. R. Sheth, G. Liu, Y. Li and
M. D. Pagel, Contrast Media Mol. Imaging, 2012, 7, 26–34;
(f) V. R. Sheth, Y. Li, L. Q. Chen, C. M. Howison, C. A. Flask
and M. D. Pagel, Magn. Reson. Med., 2012, 67, 760–768.
Acknowledgements
We thank J. Plutnar and Z. Tošner for their help with the
setting of the CEST pulse sequences. The financial support of
the Ministry of Education of the Czech Republic (no.
MSM0021620857), the Grant Agency of the Czech Republic
(no. 207/11/1437) and the Grant Agency of the Charles Univer-
sity (no. 110213) is acknowledged.
Notes and references
1 (a) P. Caravan, J. J. Ellison, T. J. McMurry and R. B. Lauffer,
Chem. Rev., 1999, 99, 2293–2352; (b) P. Hermann, J. Kotek, 13 (a) J. A. Duimstra, F. J. Femia and T. J. Meade, J. Am.
V. Kubíček and I. Lukeš, Dalton Trans., 2008, 3027–3047;
(c) C. F. G. C. Geraldes and S. Laurent, Contrast Media Mol.
Imaging, 2009, 4, 1–23.
Chem. Soc., 2005, 127, 12847–12855; (b) A. Mishra, J. Pfeuffer,
R. Mishra, J. Engelmann, A. K. Mishra, K. Ugurbil and
N. K. Logothetis, Bioconjugate Chem., 2006, 17, 773–780.
2 (a) J. Zhou and P. C. M. van Zijl, Prog. Nucl. Magn. Reson. 14 A. K. Mishra and J.-F. Chatal, New J. Chem., 2001, 25,
Spectrosc., 2006, 48, 109–136; (b) Y. Wu, M. Evbuomwan, 336–339.
M. Melendez, A. Opina and A. D. Sherry, Future Med. 15 G. B. Giovenzana, R. Negri, G. A. Rolla and L. Tei,
Chem., 2010, 2, 351–366; (c) E. Terreno, D. D. Castelli and Eur. J. Inorg. Chem., 2012, 2035–2039.
S. Aime, Contrast Media Mol. Imaging, 2010, 5, 78–98; 16 M. Meyer, V. Dahaoui-Gindrey, C. Lecomte and R. Guilard,
(d) P. C. M. van Zijl and N. N. Yadav, Magn. Reson. Med., Coord. Chem. Rev., 1998, 178–180, 1313–1405.
2011, 65, 927–948; (e) G. Liu, X. Song, K. W. Y. Chan and 17 K. Kumar, C. A. Chang, L. C. Francesconi, D. D. Dischino,
M. T. McMahon, NMR Biomed., 2013, 26, 810–828.
3 (a) S. Zhang, M. Merritt, D. E. Woessner, R. E. Lenkinski
M. F. Malley, J. Z. Gougoutas and M. F. Tweedle, Inorg.
Chem., 1994, 33, 3567–3575.
and A. D. Sherry, Acc. Chem. Res., 2003, 36, 783–790; 18 S. Aime, M. Botta, M. Fasano, M. P. M. Marques,
(b) M. Woods, D. E. Woessner and A. D. Sherry, Chem. Soc.
Rev., 2006, 35, 500–511; (c) S. Viswanathan, Z. Kovács,
C. F. G. C. Geraldes, D. Pubanz and A. E. Merbach, Inorg.
Chem., 1997, 36, 2059–2068.
K. N. Green, S. J. Ratnakar and A. D. Sherry, Chem. Rev., 19 (a) J. Rudovský, P. Cígler, J. Kotek, P. Hermann, P. Vojtíšek,
2010, 110, 2960–3018; (d) T. C. Soesbe, Y. Wu and
A. D. Sherry, NMR Biomed., 2013, 26, 829–838.
I. Lukeš, J. A. Peters, L. Vander Elst and R. N. Muller,
Chem.–Eur. J., 2005, 11, 2373–2384; (b) P. Lebdušková,
15746 | Dalton Trans., 2013, 42, 15735–15747
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
P. Hermann, L. Helm, É. Tóth, J. Kotek, K. Binnemans, 23 N. Cesari, C. Biancalani, C. Vergelli, V. Dal Piaz, A. Graziano,
J. Rudovský, I. Lukeš and A. E. Merbach, Dalton
Trans., 2007, 493–501; (c) T. Vitha, V. Kubíček, J. Kotek,
P. Biagini, C. Ghelardini, N. Galeotti and M. P. Giovannoni,
J. Med. Chem., 2006, 49, 7826–7835.
P. Hermann, L. Vander Elst, R. N. Muller, I. Lukeš and 24 G. M. Sheldrick, SHELXS97, Program for Crystal Structure
J. A. Peters, Dalton Trans., 2009, 3201–3214; (d) D. D. Castelli,
M. C. Caligara, M. Botta, E. Terreno and S. Aime, Inorg.
Chem., 2013, 52, 7130–7138.
Solution from Diffraction Data, University of Göottingen,
Göottingen, 1997.
25 G. M. Sheldrick, SHELXL97, Program for Crystal Structure
Refinement from Diffraction Data, University of Göottingen,
Göottingen, 1997.
20 (a) I. Lukeš, J. Kotek, P. Vojtíšek and P. Hermann, Coord.
Chem. Rev., 2001, 216–217, 287–312; (b) P. Vojtíšek,
P. Cígler, J. Kotek, J. Rudovský, P. Hermann and I. Lukeš, 26 (a) P. Táborský, P. Lubal, J. Havel, J. Kotek, P. Hermann
Inorg. Chem., 2005, 44, 5591–5599; (c) J. Kotek, J. Rudovský,
P. Hermann and I. Lukeš, Inorg. Chem., 2006, 45, 3097–
3102.
and I. Lukeš, Collect. Czech. Chem. Commun., 2005, 70,
1909–1942; (b) M. Försterová, I. Svobodová, P. Lubal,
P. Táborský, J. Kotek, P. Hermann and I. Lukeš, Dalton
Trans., 2007, 535–549.
21 (a) A. Dadabhoy, S. Faulkner and P. G. Sammes, J. Chem.
Soc., Perkin Trans. 2, 2002, 348–357; (b) D. A. Moore, Org. 27 M. Kývala and I. Lukeš, International Conference Chemo-
Synth., 2008, 85, 10–14.
22 D. D. Perrin and W. L. F. Armarego, Purification of Labora-
tory Chemicals, Pergamon Press, Oxford, 3rd edn, 1988.
metrics 95, p. 63, Pardubice, Czech Republic, 1995. Full
version of “OPIUM” available (free of charge) on: http://
web.natur.cuni.cz/~kyvala/opium.html.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 15735–15747 | 15747