Eu(III) Complex with DO3A-amino-phosphonate Ligand as a Concentration-Independent pH-Responsive Contrast Agent for Magnetic Resonance Spectroscopy (MRS)

Article abstract of DOI:10.1021/acs.inorgchem.6b02749

A new DOTA-like ligand H₅do3aNP with a 2-[amino(methylphosphonic acid)]ethyl-coordinating pendant arm was prepared, and its coordinating properties were studied by NMR spectroscopy and potentiometry. The study revealed a rare slow exchange (on

Full text of DOI:10.1021/acs.inorgchem.6b02749

Article
pubs.acs.org/IC
Eu(III) Complex with DO3A-amino-phosphonate Ligand as a
Concentration-Independent pH-Responsive Contrast Agent for
Magnetic Resonance Spectroscopy (MRS)
Tereza Krchova,† Vít Herynek,‡ Andrea Galisova,‡ Jan Blahut,† Petr Hermann,† and Jan Kotek*,†
́
́
́
†Department of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 43, Czech Republic
‡Department of Radiodiagnostic and Interventional Radiology, Magnetic Resonance Unit, Institute for Clinical and Experimental
̌ ́
Medicine, Vídenska 1958/9, Prague 4 140 21, Czech Republic
S
* Supporting Information
ABSTRACT: A new DOTA-like ligand H5do3aNP with a 2-
[amino(methylphosphonic acid)]ethyl-coordinating pendant
arm was prepared, and its coordinating properties were studied
by NMR spectroscopy and potentiometry. The study revealed a
1
rare slow exchange (on the H and 31P NMR time scale)
between protonated and unprotonated complex species with a
corresponding acidity constant pKA ∼ 8.0. This unusually slow
time scale associated with protonation is caused by a significant
geometric change from square-antiprismatic (SA) arrangement
observed for protonated complex SA-[Eu(Hdo3aNP)]− to
twisted-square-antiprismatic (TSA) arrangement found for
deprotonated complex TSA-[Eu(do3aNP)]2−. This behavior
results in simultaneous occurrence of the signals of both species in the 31P NMR spectra at approximately −118 and +70 ppm,
respectively. Such an unprecedented difference in the chemical shifts between species differing by a proton is caused by a
significant movement of the principal magnetic axis and by a change of phosphorus atom position in the coordination sphere of
the central Eu(III) ion (i.e., by relative movement of the phosphorus atom with respect to the principal magnetic axis). It changes
the sign of the paramagnetic contribution to the 31P NMR chemical shift. The properties discovered can be employed in the
measurement of pH by MRS techniques as presented by proof-of-principle experiments on phantoms.
presence of β-arylsulfonamide6 or p-nitrophenol7 groups on the
INTRODUCTION
■
chelate species. The deprotonation and coordination of these
groups is associated with removal of the inner-sphere water
molecule, and such a change in q leads to a change in relaxivity
r1. Similarly, in the case of Gd(III) complexes of H3do3a with
2-aminoethyl pendant arms, such as H3do3a-ae (Figure 1), a
change in the protonation state of the amino group causes a
(de)coordination of the pendant arm, and thus, it also leads to
changes in relaxivity.8 Interesting results have been obtained by
Hall et al., who studied the Gd(III) complex with terpyridine-
containing macrocycle.9 The authors demonstrated a significant
decrease of relaxivity with increasing pH and attributed it to a
decrease in q from 3 to 0 due to formation of the hydroxido
species and hydroxido-bridged dimer connected with a
negligible exchange rate of coordinated OH− ions.9 However,
for quantification of pH using these relaxation-based CAs,
knowledge of the actual agent concentration is needed.10 This
is the reason why new methods employing ratiometric
approaches are developed, as they remove dependence of the
observed signal on the local concentration of CAs. For example,
Over the past two decades, the ability to provide essential
information about variations in tissue pH has become
increasingly important. Therefore, it is not surprising that
magnetic resonance imaging (MRI), as one of the most widely
used diagnostic methods in clinical medicine,1 has been
proposed as a suitable technique for this purpose. The tumor
extracellular microenvironment is often slightly more acidic
than healthy tissue due to increased anaerobic glycolysis and a
related accumulation of lactic acid, reduced passive buffering
capacity, and poor tissue perfusion.2 For example, the
extracellular pH in solid tumors typically ranges between 6.5
and 7.2.3 Therefore, measurement of extracellular pH using
noninvasive techniques is important not only for early
detection of disease or metabolic disorder, but also for
suggesting the most efficient treatment and for monitoring
the effects of pH-altering therapies.2 Nowadays, several
strategies (and appropriate contrast agents, CAs) to measure
tissue pH are intensively investigated.4 Previous studies have
suggested relaxation-based MRI CAs with longitudinal
relaxivity (r1) dependent on pH.5 The Gd(III) complexes
investigated were based on a variation in the number of inner-
sphere water molecules (q), for example, caused by the
Received: November 17, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 1. Structural formulas of the ligands discussed in the text.
Gillies and co-workers developed a “dual-injection” relaxivity-
based method employing administration of two compounds
(pH-sensitive [Gd(dota-4AmP)]5− and pH-insensitive [Gd-
(dotp)]5−; for structures of both ligands, see Figure 1) with a
comparable biodistribution and pharmacokinetics, allowing
determination of the concentration of the first agent from the
concentration of the second one. It was shown that this
approach allowed pH mapping both in vitro and in vivo.5b,10,11
An alternative approach was introduced by Aime et al. They
investigated the possibility of measuring the ratio of transversal
and longitudinal relaxation rates of water protons (R2/R1) after
injecting a single paramagnetic Gd(III) complex of DOTA-
functionalized polypeptide (poly-L-ornithine).12 Recently, the
paramagnetic MRI contrast agents that are detected via the
chemical exchange saturation transfer (CEST) effect, so-called
PARACEST CAs, have also been used to ratiometrically
estimate in vitro/in vivo pH by utilizating a ratio of two different
CEST signals.3,13 These can originate from the exchangeable
protons of the same molecule, as in the case of paramagnetic
H4dotam-Gly (Figure 1) complexes with some Ln(III) ions
(Pr, Nd, and Eu) where the protons of a coordinated water
molecule and amide protons of the macrocyclic ligand
represent two CEST-active exchanging pools.14 Similarly, the
Yb(III) complex of H3do3aNN (Figure 1) with two proton-
exchanging pools of the primary and secondary amino groups
has been shown to be suitable for this purpose.15 Two different
CEST signals can also originate from two conformations of the
same molecule generating CEST signals at different chemical
shifts, as was shown for Yb(III)-analogue of the clinically used
[Gd(do3a-hp)(H2O)] (ProHance; Figure 1).13e,16
spectroscopic imaging (MRSI). Several methods for pH
mapping by MRS using different endogenous or exogenous
compounds have been used, and these are generally based on a
difference in chemical shifts between pH-dependent and pH-
independent resonances. Application of MRS to measure pH
began early on with the acquisition of the 31P MR spectra of
inorganic phosphate (Pi) because its resonance frequency is
pH-dependent. The majority of Pi resonance comes from
intracellular phosphate,18 and thus, the chemical shift of the Pi
generally reflects the intracellular pH.2,11,19 To determine the
extracellular pH of tumors in animal models, nontoxic
exogenous reporter 3-aminopropylphosphonic acid (3-APP;
Figure 1) with chemical shift dependence ∼1 ppm per pH unit
has been used.2,19 Tissue pH can also be detected in vivo using
diamagnetic 1H MRS probes with the pH-sensitive signal of 1H
nucleus. Exogenously administered 2-imidazol-1-yl-3-ethoxy-
carbonyl-propionic acid (IEPA; Figure 1) has been employed
for this purpose. The chemical shift of the imidazole ring C-2
hydrogen atom is in the 7−9 ppm range and is pH-sensitive.
This compound has been used for imaging extracellular pH in
breast or brain tumors.20 However, the disadvantage of using
diamagnetic compounds as pH probes is the fact that their
signal can be overlapped with the resonances of other
compounds present in vivo. Therefore, complexes of para-
magnetic metal ions are studied due to the expanded chemical
shift scale of NMR-active nuclei. In particular, Ln(III)
complexes with macrocyclic ligands have been used because
they are stable under in vivo conditions. The Yb(III) complex of
H8dotp (Figure 1) has been introduced as a prototype of a new
class of pH indicators because stepwise protonation of the
complex is accompanied by variations in the protons’ chemical
shifts (6 resonances in 1H NMR spectrum); this enables
creation of a calibration curve for pH determination.21
Simultaneous determination of pH and temperature by
Tm(III) complexes of H8dotp using three proton chemical
shifts was studied by a three-dimensional chemical shift method
called biosensor imaging of redundant deviation in shift
(BIRDS).22 The Ln(III) complexes of two metal ions (Tm
and Yb) with phosphonate ligand H8dota-4AmP (Figure 1)
An alternative approach to in vivo pH measurement is the
MRS (magnetic resonance spectroscopy) technique. In general,
MRS provides information about abundance and concentration
of tissue metabolites, and it can help to characterize many
pathologies, including neurological, psychiatric, and metabolic
diseases.17 In addition, spectral information on a metabolite/
compound with a particular chemical shift can be encoded into
the image, and a map of its spatial distribution can be
reconstructed by a method called magnetic resonance
B
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Luminescence spectra were acquired on a Luminescence Thermo
Spectronic spectrometer AMINCO Bowman Series 2. The lumines-
cence spectra were obtained after excitation at the Eu(III) L6 ← F0
also exhibit suitable pH-sensitivities for BIRDS detection using
chemical shift difference between two proton resonances.
Moreover, these agents are also CEST-active, and thus, the
CEST effect observed together with BIRDS opens the way
toward high-resolution and quantitative pH imaging.23
5
7
band (λ = 396 nm).
Syntheses. Synthesis of 1. Paraformaldehyde (4.00 g, 133 mmol)
was added to a well-stirred solution of N-benzylethanolamine (4.00 g,
26.5 mmol) and diethyl-phosphite (17.2 mL, 134 mmol) in dry MeCN
(30 mL). The reaction mixture was stirred at 60 °C for 2 days, filtered,
and then evaporated on a rotary evaporator. The oily residue was
dissolved in a small amount of EtOH, and then the solution was
loaded onto a strong cation exchanger column (Dowex 50, H+-form, 4
cm × 20 cm, in EtOH). Impurities were removed by elution with
EtOH, and product 1 was eluted with concentrated aqueous NH3/
EtOH (1/5). The fractions containing product 1 (TLC and 1H NMR
check) were combined, evaporated, redissolved in absolute EtOH, and
evaporated to give compound 1 (7.62 g, 95.6%) as a brownish oil.
To contribute to the fields described above, we prepared a
new macrocyclic ligand H5do3aNP (Figure 1). The amino-
phosphonate-coordinating pendant arm was chosen because, in
general, protonation constants of amino and phosphonate
groups are close to the physiological pH; thus, the complexes of
the new ligand were expected to change their properties in the
pH region relevant for living systems. In addition, the presence
of a phosphorus atom could be potentially employed in 31P
NMR-based applications. In this work, we report on ligand
1
synthesis and the unusual H and 31P NMR properties of its
3
1H NMR (299.9 MHz, CD3OD): δ 1.34 (6H, t, JHH = 7.0,
europium(III) complex which are employable in 31P MRS/
MRSI for determination of pH. In addition, potentiometric and
NMR studies found high stability for the studied complex in
solution, which is promising for potential in vivo use of related
compounds.
3
CH2CH3); 2.80 (2H, t, JHH = 6.0, CH2CH2OH); 3.07 (2H, d, 2JHP
=
3
10.0, PCH2N); 3.67 (2H, t, JHH = 6.0, CH2CH2OH); 3.85 (2H, s,
3
3
CH2Ph); 4.13 (4H, dq, JHH = JPH = 7.0, CH2CH3); 7.25−7.41 (5H,
m, arom). 13C{1H} NMR (75.4 MHz, CD3OD): δ 17.64 (2C, d, 3JCP
=
6.0, OCH2CH3); 51.09 (1C, d, 1JCP = 162, PCH2N); 58.89 (1C, d, 3JCP
= 8.0, CH2); 61.9 (1C, CH2); 62.24 (1C, 3JCP = 7.5, CH2); 64.39 (2C,
2JCP = 7.0, OCH2CH3); 129.16 (1C, arom); 130.18 (2C, arom);
131.02 (2C, arom); 140.83 (1C, arom quaternary). 31P{1H} NMR
(121.4 MHz, CD3OD): δ 27.17 (1P, s). 31P NMR (121.4 MHz,
CD3OD): δ 27.17 (1P, m). MS-ESI: (+) 323.7 ([M + Na]+, calcd
324.1).
EXPERIMENTAL SECTION
■
Materials and Methods. Unless stated otherwise, commercially
available chemicals and solvents were used without further purification.
Water used for potentiometric titrations was deionized by the Milli-Q
device (Millipore).
Synthesis of 2. A solution of CH3SO2Cl (1.14 g, 9.96 mmol, 1.2
equiv) in dry CH2Cl2 (10 mL) was dropwise added to a well-stirred
solution of 1 (2.50 g, 8.30 mmol) and Et3N (1.68 g, 16.6 mmol, 2
equiv) in dry CH2Cl2 (70 mL) with cooling (ice bath). The reaction
mixture was then stirred for 20 min. Next, saturated aqueous NaHCO3
(50 mL) was added, and the mixture was stirred for 10 min. The
organic layer was separated, washed with saturated aqueous NaHCO3
(2 × 30 mL) and H2O (2 × 30 mL), dried over Na2SO4, and
concentrated in vacuo to give 3.05 g (97%) of 2 as a brownish oil.
Compound 2 is not stable and must be immediately used for further
synthesis.
1,4,7-Tris(tert-butylcarboxymethyl)-1,4,7,10-tetraazacyclododecane
hydrobromide (tBu3do3a·HBr)24 and N-benzylethanolamine25 were
prepared according to the published procedures. Dichloromethane
(DCM) was dried by the standard procedure26 and stored over
molecular sieves under an argon atmosphere.
NMR characterization data (1D: 1H, 13C; 2D: HSQC, HMBC,
1H−1H COSY, 1H−1H EXSY, 31P−31P EXSY) were recorded on
VNMRS300, Bruker Avance III 400, or Bruker Avance III 600
spectrometers, using 5 mm sample tubes. The longitudinal relaxation
times T1’s of 31P were measured on the VNMRS300 by inversion
recovery pulse sequence (15 or 10 increments on d2 exponentially
sampled) with the spectrometer offset identical to the compound
3
1H NMR (299.9 MHz, CDCl3): δ 1.31 (6H, t, JHH = 7.0,
CH2CH3); 2.98 (2H, d, 2JHP = 11.0, PCH2N); 2.99 (3H, s, CH3); 3.08
(2H, t, 3JHH = 6.0, OCH2CH2N); 3.85 (2H, s, CH2Ph); 4.09 (4H, dq,
1
signal and properly calibrated pulse length. Selectively H-decoupled
3
3JHH
=
3JPH = 7.0, CH2CH3); 4.29 (2H, t, JHH = 6.0,
13C NMR spectra were obtained on the Bruker Avance III 600
spectrometer by standard one-pulse 13C measurement with low-power
continuous-wave 1H decoupling at the desired frequency offset.
Exchange rates were measured using a selective 31P−31P EXSY pulse
sequence with the mixing-time changing from 20 μs to 500 ms
exponentially. Exchange rates of isomer interchange were determined
by numerical fitting of the mixing-time-dependent 31P NMR integral
value with the Bloch−McConnell equation using Matlab.27 Unless
stated otherwise, NMR experiments were performed at 25 °C. The pD
value means a reading of the freshly calibrated pH electrode of the
sample dissolved in D2O corrected by +0.4. Chemical shifts δ are given
S(O)2CH2CH2N); 7.23−7.35 (5H, m, arom). 13C{1H} NMR (75.4
MHz, CDCl3): δ 16.60 (2C, d, JCP = 6.0, OCH2CH3); 37.50 (1C,
S(O)2CH3); 49.67 (1C, d, JCP = 159, PCH2N); 53.62 (1C, d, JCP
6.0, CH2); 60.40 (1C, JCP = 9.0, CH2); 62.03 (2C, JCP = 7.0,
OCH2CH3); 68.19 (1C, CH2); 127.56 (1C, arom); 128.49 (2C,
arom); 129.11 (2C, arom); 138.17 (1C, arom quaternary). 31P{1H}
NMR (121.4 MHz, CDCl3): δ 24.80 (1P, s). 31P NMR (121.4 MHz,
CDCl3): δ 24.80 (1P, m).
3
1
3
=
3
2
Synthesis of 3. A solution of alkylating reagent 2 (2.71 g, 7.14
mmol, 1.70 equiv) in dry MeCN (30 mL) was dropwise added to a
well-stirred suspension of tBu3do3a·HBr (2.50 g, 4.20 mmol) and
K2CO3 (1.15 g, 8.3 mmol, 2 equiv) in dry MeCN (45 mL). The
reaction mixture was stirred at room temperature for 1 day and
filtered, and the filtrate was evaporated on a rotary evaporator. The
oily residue was dissolved in CHCl3 (30 mL) and extracted with
distilled water (4 × 15 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo to give 4.50 g of a yellow oil containing
crude compound 3 contaminated with an excess of the alkylating
reagent 2 and the products of its degradation. Crude product 3 was
used in the next step without purification.
1
in ppm, and coupling constants J are reported in Hz. For H and
13C{1H} measurements of diamagnetic compounds in D2O, tBuOH
was used as the internal standard (δH = 1.25, δC = 30.29). For the
measurements in CDCl3, TMS was used as the internal standard (δH =
0.00, δC = 0.00). For 31P measurements, 85% H3PO4 in H2O was used
as the external standard (δP = 0.00). In the case of paramagnetic
complexes, 1H chemical shifts were referenced to the tBuOH signal of
the sample (δH = 1.25). Abbreviations [s (singlet), d (doublet), t
(triplet), m (multiplet), and br (broad)] are used in order to express
the signal multiplicities. Lanthanide(III) concentrations in solutions
were determined by measuring bulk magnetic susceptibility (BMS)
shifts.28
31P{1H} NMR (121.4 MHz, CDCl3, 25 °C): δ 25.89 (1P, s). 31P
NMR (121.4 MHz, CDCl3): δ 25.89 (1P, m). MS-ESI: (+) 797.8 ([M
+ H]+, calcd 798.5); 819.9 ([M + Na]+, calcd 820.5).
The ESI-MS spectra were run on the Bruker ESQUIRE 3000
spectrometer equipped with an electrospray ion source and ion-trap
detection. The measurements were carried out in both positive and
negative modes.
Synthesis of 4. The total amount of crude compound 3 obtained
above (4.50 g) was dissolved in a mixture of CF3CO2H and CHCl3
(40 mL, 1:1 v:v). The resulting solution was refluxed for 18 h and then
evaporated on a rotary evaporator. The oily residue was dissolved in a
C
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
small amount of distilled water and evaporated, and this procedure was
repeated three more times. The oily residue was dissolved in a small
amount of distilled water, and the solution was loaded onto a strong
cation exchanger column (Dowex 50, H+-form, 4 cm × 15 cm). The
impurities were removed by elution with water, and product 4 was
eluted with aq NH3 (5%). The fractions containing product 4 (1H
NMR check) were combined. After evaporation of volatiles, the oily
residue was dissolved in water and poured onto a column of a weak
cation exchanger (Amberlite CG50, 200−400 mesh, 4 cm × 15 cm).
The impurities were eluted off with water, and product 4 was collected
by 20% aq CH3COOH. The fractions containing product 4 were
combined and evaporated to give 2.30 g of brownish oil, which was
used in the next step without further purification.
macrocyclic CH2); 3.07−3.10 (2H, br m, macrocyclic CH2); 3.20 (2H,
br s, CH2CO2); 3.23 (2H, d, 2JHP = 12.0, NCH2P); 3.30 (2H, br t, 3JHH
= 5.0, NCH2CH2NH); 3.30−3.37 (2H, br m, macrocyclic CH2);
3.42−3.45 (2H, br m, macrocyclic CH2); 3.53−3.56 (2H, br m,
macrocyclic CH2); 3.69−3.72 (2H, br m, macrocyclic CH2); 3.88 (4H,
AB-multiplet, CH2CO2). 13C{1H} NMR (150.9 MHz, D2O, pD =
1
5.81, Figure S6): δ 45.78 (1C, d, JCP = 136, NCH2P); 46.54 (1C, d,
3JCP = 3.5, CH2NHCH2P); 48.97 (2C, macrocyclic CH2); 49.00 (2C,
macrocyclic CH2); 50.36 (1C, CH2CH2NCH2P); 51.08 (2C, macro-
cyclic CH2); 53.02 (2C, macrocyclic CH2); 55.71 (1C, CH2CO); 57.83
(2C, CH2CO); 170.92 (2C, CH2CO); 178.98 (1C, CH2CO). 31P{1H}
NMR (121.4 MHz, D2O, pD = 5.81, Figure S7): δ 9.37 (1P, s). 31P
NMR (121.4 MHz, D2O, pD = 5.81, Figure S8): δ 9.37 (1P, t, 2JHP
=
12.0). MS-ESI: (+) 483.6 ([M + H]+, calcd 484.2); 505.6 ([M + Na]+,
calcd 506.2); (−) 481.3 ([M−H]−, calcd 482.2); 519.1 ([M + Cl]−,
calcd 518.2). Elemental analysis found (calcd for H5do3aNP·4.5H2O,
C17H43N5O13.5P, Mr = 564.4): C, 36.22 (36.17); H, 8.09 (7.68); N,
12.17 (12.41); P, 5.61 (5.49).
When crude product 4 prepared in another batch was left to stand
for several days, it solidified on standing. The sample for elemental
analysis was isolated by washing this solid with ethanol, filtering, and
then equilibrating the solid in ambient air.
1H NMR (600 MHz, D2O, 50 °C, pD = 6.74, Figure S1): δ 1.33
3
3
(6H, t, JHH = 7.0, CH2CH3); 2.94 (2H, t, JHH = 6.0, CH2NCH2Ph);
3.01−3.85 (6H, br, CH2CH2NCH2Ph + macrocyclic CH2); 3.11−3.16
(6H, br, NCH2P + macrocyclic CH2); 3.18−3.22 (4H, br m,
macrocyclic CH2); 3.29−3.34 (4H, br m, macrocyclic CH2); 3.45
(4H, br s, CH2CO); 3.58 (2H, br s, CH2CO); 3.81 (2H, s, NCH2Ph);
4.14 (4H, m, CH2CH3); 7.38−7.47 (5H, m, arom). 13C{1H} NMR
(150.9 MHz, D2O, 50 °C, pD = 6.74, Figure S2): δ 16.30 (2C,
Synthesis of Eu(III)−H5do3aNP Complex. Three procedures for
preparation of the Eu(III) complex of H5do3aNP for NMR and MRI
experiments were used. In the first one, EuCl3·6H2O was mixed with
1.05 equiv of the ligand in a small amount of distilled water. The pH
was adjusted to 7 with 1 M aq NaOH, and the solution was stirred
overnight at 60 °C. Then, the pH was readjusted to 7 with 1 M aq
NaOH, and the solution was again stirred overnight at 60 °C.
In the second case, the Eu(III) complex was prepared by mixing the
ligand with 1 equiv of Eu(III) acetate stock solution (concentration
was determined by measuring BMS shifts)28 in a small amount of
distilled water. The pH was adjusted to ∼8 with concentrated aq NH3,
and the mixture was stirred overnight at 60 °C. Then, the solution was
filtered and evaporated to dryness leaving a glassy solid, which was
dissolved in distilled water and evaporated on a rotary evaporator at 90
°C to remove ammonium acetate (this procedure was then repeated
five more times).
1
3
OCH2CH3); 49.69 (1C, d, JCP = 160, PCH2N); 49.69 (1C, d, JCP
=
8.0, CH2NCH2Ph); 49.91 (2C, macrocyclic CH2); 50.71 (2C,
macrocyclic CH2); 50.86 (1C, CH2CH2NCH2Ph); 51.11 (4C,
macrocyclic CH2); 56.40 (1C, CH2CO); 57.47 (2C, CH2CO); 60.66
3
2
(1C, d, JCP = 8.5, CH2Ph); 64.22 (1C, d, JCP = 6.5, OCH2CH3);
128.52 (1C, arom); 129.34 (2C, arom); 13.21 (2C, arom); 138.44
(1C, arom quaternary); 173.73 (1C, CH2CO); 174.94 (2C, CH2CO).
31P{1H} NMR (121.4 MHz, D2O, pD = 6.74, Figure S3): δ 28.39 (1P,
s). 31P NMR (121.4 MHz, D2O, pD = 6.74, Figure S4): δ 28.39 (1P,
m). MS-ESI: (+) 629.3 ([M + H]+, calcd 630.0); 652.0 ([M + Na]+,
calcd 652.3); (−) 627.8 ([M − H]−, calcd 628.3). Elemental analysis
found (calcd for 4·4H2O, C28H56N5O13P, Mr = 701.8): C, 47.90
(47.92); H, 7.54 (8.04); N, 9.76 (9.98); P, 3.94 (4.41).
In the third case, the Eu(III) complex was prepared by mixing
EuCl3·6H2O with 1.05 equiv of the ligand in a small amount of
distilled water. The pH was adjusted to ∼8 with 0.8 M aq (NMe4)OH,
and the mixture was stirred overnight at 60 °C.
Synthesis of 5. The total amount of crude product 4 obtained
above (2.30 g) was dissolved in concentrated aqueous HCl (20 mL)
and stirred at 95 °C for 24 h. Then, the solution was evaporated on a
rotary evaporator, dissolved in a small amount of water, and loaded
onto a strong cation exchanger column (Dowex 50, H+-form, 4 cm ×
15 cm). The impurities were removed by elution with water, and
product 5 was eluted with 5% aq NH3. The fractions containing the
product were combined, filtered, and evaporated to give compound 5
(1.35 g) as a brownish oil which was used in the next step without
further purification.
All prepared samples were checked using the xylenol orange test
(acetate buffer, pH = 5.7) to exclude the presence of free Eu(III) ions.
The exact concentration of Eu(III) complex in solution was
determined using Evans’ method.28
Eu(III)−H5do3aNP MS-ESI: (−) 631.6 with appropriate isotopic
pattern ([M − H]−, calcd 632.1).
X-ray Diffraction Study. Single crystals of H5do3aNP·4.75H2O
were prepared by slow evaporation of the aqueous solution of
H5do3aNP. Diffraction data were collected by employing an 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
(SHELXS97)29 and refined by full-matrix least-squares techniques
(SHELXL97).30 Relevant data for the structures have been deposited
at the Cambridge Crystallographic Data Centre. The structure was
31P{1H} NMR (121.4 MHz, D2O, pD = 3.9): 7.68 (1P, s). 31P
NMR (121.4 MHz, D2O, pD = 3.9): 7.68 (1P, t, 2JHP = 12.5). MS-ESI:
(+) 574.0 ([M + H]+, calcd 574.3); 611.9 ([M + K]+, calcd 612.2);
633.9 ([M + Na + K − H]+, calcd 634.2); (−) 571.8 ([M − H]−, calcd
572.3); 609.8 ([M + K − 2H]−, calcd 610.2).
Synthesis of H5do3aNP. Crude product 5 (1.30 g) was dissolved in
20% aq CH3CO2H (50 mL). Next, Pd/C catalyst (10%, 0.26 g) was
added, and the flask was evacuated and filled by H2. The mixture was
stirred under a hydrogen atmosphere (using a rubber balloon) for 48 h
at room temperature (RT). Then, the catalyst was removed by
filtration, and all volatiles were evaporated in vacuo. The product
H5do3aNP was purified by chromatography on a strong cation
exchanger column (Dowex 50, H+-form, 3 cm × 15 cm). Impurities
were removed by elution with water, and the product H5do3aNP was
eluted with 5% aq pyridine. The fractions containing the product were
combined, filtered, and evaporated to dryness leaving a glassy solid
which was dissolved in a small amount of water and crystallized by
standing for 2 h. The white crystalline solid was isolated by filtration,
washed with EtOH, and air-dried to give a white powder of hydrate
H5do3aNP·4.5H2O (1050 mg, 44% based on tBu3do3a·HBr).
1H NMR (600 MHz, D2O, pD = 5.81, Figure S5): δ 2.86−2.94
(4H, br m, macrocyclic CH2); 3.00−3.05 (4H, br, NCH2CH2NH +
solved in space group P1, where the independent unit consists of two
̅
ligand molecules which have very similar conformations. However,
higher symmetry (c/2) is prevented by disorder of the phosphonate
moiety of one of the units. The 10 highest isolated electronic maxima
were attributed to the water solvate molecules, one of them half-
occupied due to collision with one position of disordered phosphonate
oxygen atoms. All non-hydrogen atoms were refined anisotropically,
and hydrogen atoms were fixed in theoretical (C−H, N−H) or
original (O−H) positions using a riding model UH = 1.2UX.
Crystal data follow: H5do3aNP·4.75H2O, C17H43.5N5O13.75P, M =
569.04, triclinic, a = 11.0215(3) Å, b = 12.3502(3) Å, c = 19.0688(6)
Å, α = 84.311(1)°, β = 80.106(1)°, γ = 81.467(1)°, U = 2521.46(12)
Å3, space group P1, z = 4, 11576 total reflections, 8615 intense
̅
reflections, R1[I > 2σ(I)] = 0.0535, wR2(all data) = 0.1543. CCDC-
1500120.
Potentiometry. Potentiometric titrations were carried out in a
thermostated (25.0 0.1 °C) vessel at a constant ionic strength [I =
D
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
a
Scheme 1. Synthesis of H5do3aNP
a
(i) HP(O)(OEt)2, (CH2O)n, dry MeCN, 60 °C, 2 days; (ii) CH3SO2Cl, Et3N, dry CH2Cl2, 0 °C−RT, 20 min; (iii) tBu3do3a·HBr, K2CO3, dry
MeCN, RT, 24 h; (iv) CF3COOH:CHCl3 (1:1), reflux 18 h; (v) conc aq HCl, 95 °C, 24 h; (vi) H2, Pd/C, 20% aq CH3COOH, RT, 2 days.
0.1 M (Me4N)Cl]. Measurements were taken with an excess of HCl
stock solution added to the initial mixture, and the mixtures were
titrated with stock solution of (NMe4)OH. The ligand concentration
in the titration vessel was ca. 0.004 M. An inert atmosphere was
provided by a constant passage of argon saturated with water vapor.
Before and after each titration, calibrations [titrations of HCl stock
solution with (NMe4)OH stock solution] were performed for
determination of electrode calibration parameters as described
previously.31
For determination of the ligand protonation constants, a standard
technique was used,31 with the initial volume ca. 5 cm3 and in the pH
range 1.9−12.1. Four titrations were carried out with ∼70 points per
titration.
Titrations of the Eu(III)−H5do3aNP system were performed by an
out-of-cell technique31 as the equilibrium was established slowly. In
the experiments, the metal-to-ligand molar ratio was 1.00:1.04, and the
starting volume of each titration point [before addition of (NMe4)-
OH] was ca. 1 cm3. In total, two titration sets in the pH range 1.8−9.0
were prepared with 30 points per titration, and ampules with solutions
were flame-sealed. After 4 weeks (waiting time for equilibration), the
potential of each solution was determined with a freshly calibrated
electrode, with measurement of odd and even data points separately
affording four independent data series of 15 data points.
A preformed Eu(III) complex for determination of its protonation
constants was prepared in the following way: in the ampule, equimolar
amounts of the ligand and metal stock solutions were mixed, and a
calculated amount (based on out-of-cell titration data) of (NMe4)OH
stock solution was gradually added to reach pH ca. 6, which
corresponds to full complexation according to the out-of-cell titration.
Ampules were flame-sealed and left at 55 °C for 3 days. From the final
solution, aliquot volumes were pipetted, a defined amount of HCl
stock solution was added into these samples, and the mixtures were
immediately titrated by (NMe4)OH stock solution in an analogous
way as described above for the determination of the ligand protonation
constants; the concentration of the complex in the titration vessel was
ca. 0.003 M. The pH range used in these titrations was 1.9−9.5, and
the titrations were carried out four times with 30 points per titration.
The OPIUM program package32 was used for calculation of the
ligand protonation constants and stability/protonation constants of
the Eu(III) complex. All constants are concentration constants.
Calculated protonation constants are defined as βh = [HhL]/
{[H]h[L]}, and they can be transferred to consecutive protonation
constants log K by log K(HhL) = log βh − log βh−1. Calculated stability
constants βhlm are defined by βhlm = [HhLlMm]/{[H]h[L]l[M]m}.
Calculated constants with their standard deviations are compiled in
Tables S8−S10, and the fits of titration data are shown in Figures S27,
S31, and S32. The water ion product used in all calculations was pKw =
13.81.33
31P and 1H NMR Titration of H5do3aNP and Its Eu(III)
Complex. 31P and 1H NMR spectra were performed on a
VNMRS300 (B0 = 7.05 T) using 5 mm sample tubes. A coaxial
capillary with 85% H3PO4 in H2O (δP = 0) or with tBuOH in D2O (δH
= 1.25) as the external standard was used. Solution pH was measured
with a combined glass electrode (Spintrode Hamilton) employing a
pH meter (3505 pH Meter, JENWAY) calibrated with standard
buffers. Protonation constants were calculated with the OPIUM
software package.32
31P NMR titration (46 points) for determination of protonation
constants and sites of H5do3aNP was performed at 25 °C (0.05 M
ligand concentration; no ionic strength control) in H2O. The solution
pH (0.1−13.6) was adjusted with aq HCl or aq (NMe4)OH solutions.
The fit is shown in Figure S28.
1H NMR titration of H5do3aNP in the alkaline region (14 points)
was performed at 25 °C (0.05 M ligand concentration; no ionic
strength control) in H2O. Solution pH (7.9−12.0) was adjusted with
aq HCl or aq KOH solutions. The fit is shown in Figure S29.
31P NMR titration in acidic region (14 points) of preformed
Eu(III)−H5do3aNP complex was performed at 25 °C (0.038 M
complex concentration; ∼ 150 mM NaCl) in H2O. Solution pH (from
7.0 to 2.0) was adjusted with aq HCl solutions. The fit is shown in
Figure S20.
MRS Experiments. A phantom consisting of one vial containing an
aq solution of H3PO4 as a reference and three vials containing ∼50
mM aq solutions of the Eu(III)−H5do3aNP complex (∼150 mM
NaCl) with different pH values (6.4, 8.0, and 9.0) was prepared for
MRS experiments. At these pH values, the once protonated complex
[Eu(Hdo3aNP)]− and the fully deprotonated species [Eu-
(do3aNP)]2− are present, with the former predominant at pH = 6.4
and the latter at pH = 9.0. At pH = 8.0, both species are present in
similar abundance (see distribution diagram in Figure 6).
All MRS experiments were performed on a 4.7 T MR scanner
1
BioSpec (Bruker BioSpin, Germany) using a dual H/31P surface coil
with a diameter of 5 cm (Bruker BioSpin, Germany). Nonlocalized 31P
MRS spectra of the phantom were acquired by using a single
rectangular pulse with 0.064 ms duration and 20000 Hz bandwidth
(BW); the other measurement parameters were the following:
acquisition delay 0.05 ms, repetition time (TR) = 1000 ms, number
1
of acquisitions (NA) = 512, and scan time 8.5 min. For H reference
E
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
images, a turbo spin echo sequence was used with the following
parameters: TR/TE = 3000 ms/12 ms, turbo factor = 8, matrix size of
256 × 256, field of view (FOV) = 35 mm × 35 mm, slice thickness 1
mm, and scan time 5 min. 31P MRSI spectra localized from 4 × 4
voxels covering the whole phantom volume were acquired by chemical
shift imaging sequence using the same rectangular pulse as for the
nonlocalized 31P MR spectra. Spectral width 20 kHz (246.44 ppm)
was sampled by 8192 points. The spectra were acquired by 16384
accumulations from the volume with field of view = 32 mm × 32 mm
and slice thickness 10 mm; the other parameters were TR = 1000 ms,
effective echo time (TEeff) = 15 ms, and scan time 4.5 h. Raw data
were interpolated to a matrix 32 × 32 × 8192 by zero-filling, and
Fourier transform provided a three-dimensional matrix of numbers
containing information about signal amplitude spatially encoded in x-
and y-axis; frequency chemical shift was encoded in z-axis.
31P MRSI data were processed using ImageJ software (version 1.46r,
National Institutes of Health). First, the data were interpolated into a
matrix size of 256 × 256 × 256. Then, the images acquired within an
interval of 70 4 ppm were summed (to obtain an integral signal) as
well as the images in the interval of 0 4 ppm and −118 4 ppm,
and were finally false color coded.
RESULTS AND DISCUSSION
■
Synthesis. The target ligand H5do3aNP was prepared
following the multistep synthesis outlined in Scheme 1. The
key phosphorus-containing intermediate 1 was obtained by
Mannich-type reaction of N-benzyl-ethanolamine with diethyl-
phosphite and paraformaldehyde and was isolated after cation-
exchange chromatography. It was converted to alkylation agent
2 by reaction with methanesulfonyl chloride, and compound 2
was used in the reaction with tBu3do3a, affording intermediate
3. After sequential deprotection of tBu-ester and ethyl-ester
groups using trifluoroacetic acid and aq hydrochloric acid,
respectively, the de-esterified compound 5 was hydrogenated
using the Pd/C catalyst to give the target ligand H5do3aNP that
was purified by cation-exchange chromatography and was
isolated in the zwitterionic form in the overall 44% yield (based
on tBu3do3a·HBr).
The target ligand H5do3aNP was structurally characterized
by single-crystal X-ray diffraction analysis. In the crystal
structure of H5do3aNP·4.75H2O, two independent ligand
molecules are present, but both adopt a very similar
conformation. Therefore, only one of them is shown in Figure
2. A zwitterionic molecule is protonated on two nitrogen atoms
of the macrocycle (those bearing the “trans” acetate groups)
and the nitrogen atom of the side arm. The remaining protons
are located on the phosphonate and on the “odd” acetate
groups. Molecular conformation is stabilized by medium-
strength intramolecular hydrogen bonds (Table S1), and the
whole structure is stabilized through a wide system of
intermolecular hydrogen bonds involving water molecules.
Solution Structure of the Eu(III)−H5do3aNP Complex.
Ln(III) complexes of the DOTA-like ligands are typically
present in a solution as a mixture of square-antiprismatic (SA)
and twisted-square-antiprismatic (TSA) isomers.34 These
isomers differ in conformation of the five-membered chelate
rings of the macrocycle (δ/λ) and course of rotation of the
pendant arms (Δ/Λ). Combination of the macrocycle and
pendant conformations results in two diastereomeric pairs of
the enantiomers (Figure S9). The pair Δλλλλ/Λδδδδ forms the
SA arrangement while the Δδδδδ/Λλλλλ enantiomers
correspond to the TSA structure.35 Typically, two diaster-
eomers are in a slow exchange with respect to the time scale of
an NMR experiment, giving rise to two sets of 1H NMR signals.
Therefore, both isomers can be distinguished, and their relative
Figure 2. Molecular structure of H5do3aNP found in the crystal
structure of H5do3aNP·4.75H2O. Intramolecular hydrogen bonds are
dashed (Table S1).
abundance in solution can be easily determined. Isomerism
evaluation mostly uses signals of “axial” protons of the
macrocyclic ring (Figure S9) because these are the closest
ones to the Ln(III) ion and to the principal magnetic axis; thus,
1
they are shifted away from the other signals in the H NMR
spectra. Typically, “axial” signals of the TSA species of Eu(III)
complexes with DOTA-like ligands are found in the ∼7−28
ppm range whereas those of the SA isomers appear in the
∼25−40 ppm region.36
Solution structure of the Eu(III) complex species was
1
investigated by H NMR spectroscopy in the pD range 6.8−
9.9 (Figure 3 and Figure S10). At pD < 7.3, only one set of
signals was observed in the 1H NMR spectra, where the signals
of the most positively shifted “axial” protons of the complex
species appear in the range typical for the SA isomer (20−49
1
ppm). At higher temperatures, the H NMR signals somewhat
shift and broaden due to faster conformational changes of the
complex molecule (Figure S11). With increasing pD, 1H NMR
signals of the SA isomer gradually disappear with a
simultaneous appearance of peaks attributable to the TSA
isomer with the most shifted signals in the 7−14 ppm range
(Figure 3 and Figure S10). Thus, one can suggest that both SA
and TSA species are present in the pD range 7.5−9.0 (Figures
S10, S12, and S13). At pD > 9.0, only signals of pure TSA
species were detected. Heating of the alkaline solution also led
to a significant broadening of signals of the TSA isomer,
pointing to conformational change of the complex molecule
(Figures S12 and S14). On the basis of these observations, one
can suggest that two differently protonated species are present
in the pD range 7.5−9.0; so, the pKA of the deprotonation
process lies in this range, and thus, the deprotonation obviously
occurs on the pendant amino group. The protonated form of
the complex exists exclusively in the SA isomer, whereas the
pure TSA species is formed upon deprotonation.
F
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
Figure 3. H NMR spectra of SA-[Eu(Hdo3aNP)]−/TSA-[Eu(do3aNP)]2− (∼0.08 M solution in D2O, B0 = 7.05 T) at different pD values.
Figure 4. 31P{1H} NMR spectra of SA-[Eu(Hdo3aNP)]−/TSA-[Eu(do3aNP)]2− complex species at various pH values and corresponding pH
dependence of an integral intensity of 31P NMR signals (50 mM solution in H2O, ∼150 mM NaCl, B0 = 7.05 T, 25 °C).
31P NMR spectroscopic study also revealed a slow chemical
exchange between both complex species with respect to the
time scale of the NMR experiment (Figure 4 and Figures S16
and S18) as the signals of both species are present in the 31P
NMR spectra acquired in the pH range 7.5−8.9; their chemical
shifts remain nearly constant with pH change. 31P NMR 1D-
EXSY revealed a rate for isomer interconversions on a
millisecond scale (rate constants 191(8) s−1 for SA → TSA
and 156(7) s−1 for TSA → SA rearrangements, respectively, 25
°C, pD = 8.6). In addition, an extreme chemical shift difference
(by ∼190 ppm) between signals of the SA and TSA species was
observed. In both species, the phosphonate group is obviously
coordinated, as evidenced by a significantly influenced 31P
NMR chemical shift (+70 ppm for the TSA and −118 ppm for
G
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
the SA species, respectively, Figure 4) compared to that for the
diamagnetic La(III) complex (δ = 25.3 ppm at pD = 7.0 and δ
= 22.7 ppm at pD = 10.1). The coordination of the
phosphonate group was also confirmed by measurement of
T1 relaxation times of 31P NMR signal, which are T1 = 0.207(4)
s for the SA species (measured at pD = 6.8) and T1 = 0.202(11)
s for the TSA species (pD = 9.6), respectively, whereas the
La(III) complex relaxes much slower with T1 = 1.84(8) s (pD =
7.0) and T1 = 3.09(10) s (pD = 10.1).
With mononuclearity of the complex species taken into
account, electrostatic repulsion of the central metal ion and the
protonated pendant amino group, as well as general steric
hindrances, the phosphonate group should be in the protonated
SA-[Eu(Hdo3aNP)]− species coordinated in the O4-plane
(Scheme 2). In such a case, it opens a space for coordination of
Scheme 2. Suggested Geometry of the SA-[Eu(Hdo3aNP)]−
a
and TSA-[Eu(do3aNP)]2− Species
The dependence of integral intensities of these signals on pH
is sigmoidal and, in accordance with the suggestion stated
above, could be successfully modeled by an equilibrium
involving just one (de)protonation step (Figure 4 and Figures
S16−S18), affording the protonation constant log K(=pKA) =
7.4−8.5 with dependence on ionic strength, see Table S2. The
31P chemical shifts of both species (Figures S16 and S18) and
derived protonation constants (Table S2) are independent of
complex concentration; therefore, intermolecular coordination
of the phosphonate to the metal ion belonging to a different
complex molecule and the formation of polynuclear complexes
can be excluded. Furthermore, the protonated complex was
found to be fully stable at pD = 5.9 (Figure S19). The value of
the protonation constant log K is dependent on the quality and
concentration of a background electrolyte: in the presence of
NaCl, the value drops with increasing concentration of the salt
because the presence of a coordinating metal ion generally
facilitates a deprotonation of the donor atoms. In contrast,
when 31P NMR titration was performed with the use of
(NMe4)Cl as the background electrolyte, the value of the
protonation constant was significantly higher (Table S2). In the
presence of CaCl2 and MgCl2, i.e., in the presence of stronger
a
A water molecule in the protonated species is expected to be
coordinated in the apical position.
a water molecule in an O4-capping apical position.
Luminescence lifetimes of Eu(III) ion were measured, giving
τ = 697 μs for SA-[Eu(Hdo3aNP)]− species in H2O (pH = 6.4)
and τ = 2.21 ms when this sample was evaporated and dissolved
in D2O, consistent with the assumption of water coordina-
tion.38 After deprotonation, coordination of the secondary
amino group occurs and forms an O3N-coordination plane in
the TSA-[Eu(do3aNP)]2− species. The phosphonate group
very probably moves to the position capping this O3N-plane
(Scheme 2). However, although the geometry discussed above
is the sole possibility making chemical sense, it is a bit
contradictory in a comparison of the 31P NMR shift of SA-
[Eu(Hdo3aNP)]− species with that of Eu(III) complexes of
DOTA-like ligands having methylenephosphonate/phosphinate
pendant moieties, which have a phosphorus atom in a similar
spatial position as that expected for SA-[Eu(Hdo3aNP)]−. To
the best of our knowledge, in all Eu(III) complexes of
phosphinate/phosphonate ligands reported previously, the
paramagnetic contribution to the 31P NMR shift is positive,
giving signals with 15−70 ppm higher chemical shift compared
to that of the free ligands or diamagnetic La(III) complexes
(Table S3).36b,c,e,f,39,40 Contrary to these values, when
comparing 31P NMR shift differences between Eu(III) and
La(III) complexes of H5do3aNP in acid media, the para-
magnetic contribution to chemical shift of SA-[Eu-
(Hdo3aNP)]− is Δδ = −143 ppm.
The origin of this contradiction probably lies in the low
symmetry of the ligand field of the SA-[Eu(Hdo3aNP)]−
complex, which can lead to a discrepancy between principal
magnetic and pseudo-C4 axes and to significant anisotropy of
magnetically induced shifts.41 Typically, the complexes of
DOTA-like ligands have (pseudo)-C4 symmetry, and it is
usually suggested that the principal magnetic axis is identical
with (or very close to) the (pseudo)symmetry C4 axis. In such
axially symmetric systems, the phosphorus atoms lie outside the
McConnell cone, which defines a sign of paramagnetic
contribution to the chemical shift [in relevant crystal structures,
angle between (pseudo)-C4 axis and Ln−P vector is 73−89°
(Figure S22 and Table S4),36e,b,40,42 whereas the McConnell
critical angle is 54.7°]. It should be noted that, for historical
reasons, the direction of upfield−downfield shifts differs for 1H
coordinating metal ions (Figure S18), the chemical shifts in 31
P
NMR spectra remain unaffected, and log K’s are lower than
those calculated in the case of NaCl-containing solutions
(Table S2).
On the basis of the value of log K, the protonation process
obviously occurs on the pendant arm amino group, and thus,
one can conclude that the equilibrium involves monodeproto-
nated and fully deprotonated complex species; thus, the
complex species are SA-[Eu(Hdo3aNP)]− and TSA-[Eu-
1
(do3aNP)]2−. To confirm such an assignment, the H NMR
spectra were also measured in H2O solution. In a slightly acid
region, no difference between the spectra of SA-[Eu-
(Hdo3aNP)]− acquired in D2O and H2O was found (Figure
S15A,B). At this pH, the side amino group is protonated (and
thus uncoordinated), and the hydrogen atoms are exchanged
with the solvent (D2O) or overlaid with others; on the basis of
chemical exchange saturation transfer experiments, chemical
shift of these hydrogen atoms is −17 ppm if referenced as
δ(H2O) = 0.37 In contrast, in a slightly alkaline region, a new
signal at 36.4 ppm (if referenced as δ(H2O) = 0) appeared in
the spectra of TSA-[Eu(do3aNP)]2− acquired in H2O (Figure
S15C,D). This peak can be attributed to a hydrogen atom
bound in the coordinated pendant secondary amino group,
where this hydrogen atom is not in fast exchange with the
solvent. This assignment is also supported by a similarity in the
chemical shift of this signal to the values observed for
structurally related compounds [Eu(do3a-ae)] and [Eu-
1
(do3aNN)] (Figure 1), respectively; one of the H NMR
signals of the coordinated primary amino group in [Eu(do3a-
ae)] lies at 34 ppm,44 and the signal of one of the isomers of
[Eu(do3aNN)] with a coordinated secondary amino group is
located at 35 ppm.15
H
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 5. Assignment of the 1H NMR spectra of SA-[Eu(Hdo3aNP)]−/TSA-[Eu(do3aNP)]2− complex species. Geometry of the complex species is
tentative and was not optimized. Atoms are labeled by order numbers from the spectra going from left to right according to Figure S24. For better
visualization, protons with positive paramagnetic contribution are red, those with small negative contribution are pale blue, those with large negative
contribution dark blue, and those which are not influenced are green. For phosphorus atom, the red color indicates a negative 31P NMR shift
1
contribution (i.e., position in space with positive H NMR shift contribution), and blue indicates a positive 31P NMR shift contribution (i.e., space
1
with negative H NMR shift contribution).
a
Table 1. Equilibrium Constants (log K and log KML) of H5do3aNP (0.1 M NMe4Cl, 25 °C) and Its Eu(III) Complexes and
Comparison with Corresponding Constants Reported for Related Ligands
b
c
equilibrium
H5do3aNP potentiometry
H5do3aNP 31P NMR
H3do3aNN
H3do3a-ae
H4dota
e
H+ + Ln− ↔ HL1−n
12.56(1)
10.37(1)
9.23(1)
6.17(1)
4.19(1)
1.90(1)
23.49(3)
8.00(3)
3.75(4)
4.35(4)
3.31(4)
12.52(17)
10.59(3)
9.37(8)
12.62
10.28
9.67
8.30
3.30
1.58
23.16
6.03
5.09
13.19
10.51
8.90
12.9
e
H+ + HL1−n ↔ H2L2−n
9.72
e
H+ + H2L2−n ↔ H3L3−n
4.60
e
H+ + H3L3−n ↔ H4L4−n
5.96(8)
3.87
4.15
e
H+ + H4L4−n ↔ H5L5−n
1.27
2.29
e
H+ + H5L5−n ↔ H6L6−n
2.05(9)
1.34
f
d
Eu3+ + Ln− ↔ [Eu(L)]3−n
22.23
24.2
d
H+ + [Eu(L)]3−n ↔ [Eu(HL)]4−n
H+ + [Eu(HL)]4−n ↔ [Eu(H2L)]5−n
H+ + [Eu(H2L)]5−n ↔ [Eu(H3L)]6−n
H+ + [Eu(H3L)]6−n ↔ [Eu(H4L)]7−n
5.83
a
b
c
d
e
K = [HhL]/{[H][Hh−1L]} or [HhLM]/{[H][Hh−1LM]}; KML = [ML]/{[L]·[M]}. Ref 15. Ref 44. Data reported for Gd(III) complex. Ref 45.
f
Ref 33.
1
and 31P NMR scales; therefore, in the same space, the 31P NMR
paramagnetic induced shift contribution is positive whereas that
of 1H NMR is negative (and vice versa). However, in the case of
the SA-[Eu(Hdo3aNP)]− complex, there is a large difference in
chemical shifts of “axial” macrocycle protons [these protons
have a similar position with respect to pseudo-C4 symmetry
defined by the central ion and the barycenter of the N4-plane,
and thus, they should have a similar chemical shift when the
magnetic axis is identical to the (pseudo)symmetry axis]; this
points to some difference between positions of pseudosym-
metric-C4 and principal magnetic axes. Therefore, the
phosphorus atom could fall into a space close to the principal
COSY and H EXSY experiments and 13C NMR spectra with
1
selectively tuned H decoupling (Figure S23−S26 and Tables
S5−S7). The assignment is shown in Figure 5 (for detailed
description see SI). This assignment confirmed that the
position of the phosphorus atom moves from a space with
induced positive 1H/negative 31P NMR shifts in SA-[Eu-
(Hdo3aNP)]− to an area with negative H/positive 31P NMR
1
shifts in TSA-[Eu(do3aNP)]2− complexes, respectively. Anoth-
er surprising finding is the fact that the most shifted signals in
1H NMR spectra of TSA-[Eu(do3aNP)]2− species are not the
1
“axial” ones. In this case, the direction of the most positive H
induced shift probably lies almost parallel in respect to the N4-
plane, so the principal magnetic axis should be almost
perpendicular to the pseudo-C4 axis. The phosphorus atom
falls outside this direction, and so, its induced 31P paramagnetic
shift is positive in the 31P NMR scale.
1
magnetic axis direction with induced positive H/negative 31P
NMR shifts. To confirm this assumption, the signals found in
1H NMR spectra of both SA-[Eu(Hdo3aNP)]− and TSA-
1
[Eu(do3aNP)]2− complexes were assigned on the basis of H
I
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Further acidification of the solution of the SA-[Eu-
(Hdo3aNP)]− species leads to a gradual change of the 31P
chemical shift from −118 ppm at pH = 7 to −156 ppm at pH =
2 (Figure S20). Obviously, as the chemical shift is very affected
in the whole of this pH region, the phosphonate group is still
coordinated, and the gradual change of the chemical shift
corresponds to formation of the SA-[Eu(H2do3aNP)] species.
From the 31P NMR data, log K = 3.40(1) was calculated which
confirms this assignment because the protonation constant of
the free phosphonate group (log K ∼ 5−7) drops to the log K
∼ 3−4 range upon coordination to the metal ion.43 Formation
of the SA-[Eu(H2do3aNP)] species was also supported by the
1H NMR spectra of the complex in acidic solution (Figure
S21).
signal due to a long distance between these groups, so this
constant cannot be calculated from 31P NMR data. The last
(sixth) protonation occurs on one of the remaining acetate
arms or macrocycle amino groups. The suggested protonation
scheme is fully consistent with the molecular structure found in
the solid state (see above).
The stability constant of the [Eu(do3aNP)]2− complex (log
KML = 23.49, Table 1) was obtained by an out-of-cell titration
technique. The value falls into the expected range, as can be
seen from a comparison with the stability constants reported
for the related ligands (Table 1). According to the distribution
diagrams of the Eu(III)−H5do3aNP system shown in Figure 6,
the metal ion complexation is quantitative at pH ∼ 4.5.
Thermodynamic Behavior of H5do3aNP and Its Eu(III)
Complex. To obtain information on thermodynamic stability
of the studied Eu(III) complex and confirm the conclusions
stated above, potentiometric studies of the H5do3aNP,
equilibrated Eu(III)−H5do3aNP system and preformed Eu-
(III)−H5do3aNP complex were performed.
The potentiometric titrations of H5do3aNP performed in the
pH range 1.9−12.1 revealed six consecutive protonation
processes (Table 1 and Table S8; fit shown in Figure S27);
the ligand distribution diagram is shown in Figure S30. Five of
these constants could also be calculated from the pH
dependence of the 31P NMR spectra (Table 1 and Table S8,
fit shown in Figure S28). On the basis of comparison with the
literature data,43 the first protonation step (log K(HL) = 12.56)
can be attributed to a proton shared by the macrocycle amino
groups. This assignment of the protonation site is consistent
with a relatively small change (+0.5 ppm) of the 31P NMR shift
Figure 6. Distribution diagram of metal-containing species in the
Eu(III)−H5do3aNP system (black solid lines, cM = cL = 0.004 M) and
of species present in acid−base titration of the preformed [Eu-
(do3aNP)]2− complex (red dashed lines) Conditions: 25 °C, I = 0.1
M NMe4Cl.
1
and a large change of the H chemical shift of the α-acetate
protons (+0.21 and +0.28 ppm for “double” and “odd” acetates,
respectively) compared to that of the NCH2P group (+0.05
ppm), see Table S8 and Figure S29. According to the highest
change (∼6 ppm) in 31P chemical shift during the NMR
titration, the second protonation step (log K(H2L) = 10.37)
obviously occurs on the amino group belonging to the pendant
moiety. It is also consistent with changes observed in 1H NMR
spectra; change of δ(1H) of NCH2P is 0.25 ppm, much higher
than the δ(1H) changes of the acetate groups (0.11 and 0.08
ppm, respectively, see Table S8). The value of the protonation
constant is higher than the value reported for the analogous
protonation of the 2-aminoethyl arm for H3do3a-ae (log K =
8.90)44 due to the positive induction effect of the phosphonate
moiety.43 Analogous protonation of H3do3aNN occurs in this
pH region, although in that case it was not possible to assign an
exact value due to a number of protonation steps occurring
with similar log K’s.15 The third protonation (log K(H3L) =
9.23) corresponds to the second protonation of the macrocycle
amino groups. The log K(H3L) is somewhat lower than the
second ring-amine protonation of related ligands due to
electrostatic repulsion with a protonated pendant amino
group. The third protonation locks two protons on the
macrocycle nitrogen atoms bearing “trans” acetate groups due
to mutual electrostatic repulsion, as evidenced by change of
δ(1H) (+0.38 and −0.26 ppm for “double” and “odd” acetates,
respectively, see Figure S29). The fourth protonation with log
K(H4L) = 6.17 occurs on the phosphonate moiety, as
evidenced by a reverse change of the 31P NMR shift (Figure
S28). The next one (log K(H5L) = 4.19) is associated with the
protonation of the acetate trans to the aminophosphonate arm,
and it has no influence on the chemical shift of the 31P NMR
The first protonation of the fully deprotonated species,
[Eu(do3aNP)]2−, proceeds with log K(HLM) = 8.0. In the
analysis of the NMR spectra (see above), this protonation was
suggested to occur on the side arm secondary amino group.
The protonation constant of this group in the free ligand has
the value log K(H2L) = 10.37, and so a significant metal-
induced decrease of this value confirms coordination of the
deprotonated amino group to the central Eu(III) ion.
A further three protonation steps in the equilibrated
solutions occur with log K’s in a narrow range ∼3−4 (Table
1). These processes occur at pH higher than that corresponding
to protonation of the coordinated acetate group and, thus, can
be attributed to simultaneous protonations of the phosphonate
group and the macrocycle nitrogen atoms. Therefore, the
equilibrated species H3LM and H4LM are expected to be out-
of-cage complexes with double-protonated macrocycle amino
groups similar to those suggested for other systems with
DOTA-like ligands.40
To support the conclusions stated above, an acid−base
titration of the preformed complex was performed. In this case,
only three stepwise protonation constants were found: log
K(HLM) = 8.29(2), log K(H2LM) = 4.07(3), and log
K(H3LM) = 1.75(3) (see Table S10 and distribution diagram
in Figure 6). According to the interpretation above, the first
constant corresponds to a protonation of the pendant amino
group, and this value is consistent with the results of 31P NMR
and out-of-cell potentiometric studies. The second constant is
assigned to protonation of the coordinated phosphonate group,
consistent with the 31P NMR data presented above. The
J
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
differences between values obtained by the 31P NMR and
potentiometric titrations can be attributed to a difference in
ionic strength, different methods of pH electrode calibration,
and temperature of the pH measurement. The third constant
calculated for the preformed complex is significantly lower
compared to constants observed in the equilibrated solutions
(out-of-cell titration). One can assume that during the short
time scale of the potentiometric titration only the in-cage
complex is present (i.e., no dissociation of macrocycle−Eu(III)
bonds occurs) due to relative kinetic inertness of the complexes
with DOTA-like ligands,1 and therefore, this constant
corresponds very probably to the protonation of a coordinated
carboxylate group.
Overall, the results of the potentiometric study are fully
consistent with the results of 31P NMR studies discussed above
and support the interpretation shown in Scheme 2.
pH Mapping Using MRS. To address possible pH
determination from 31P NMR data, the logarithm-weighted
ratio of integral intensity of the 31P NMR signals of TSA-
[Eu(do3aNP)]2− and SA-[Eu(Hdo3aNP)]− was calculated.
This function is linear in the pH range 6.5−9.5, as shown in
Figure 7, proving the principle of the suggested method (the
data were observed at various experimental conditions).
Figure 8. Left: localized 31P MR spectra. Right: pH map and legend
(1H MR reference image) of phantom consisting of one vial containing
aqueous H3PO4 as a reference and three vials containing aqueous
solutions of the Eu(III)−H5do3aNP complex with different pH values.
Postprocessing included interpolation to a spatial 256 × 256 matrix,
integration over the selected signals, and false color coding. Color
scheme: red, signal at +70 ppm; blue, −118 ppm; purple color results
from red/blue superposition; green, 0 ppm.
CONCLUSIONS
■
New ligand H5do3aNP with a 2-[amino(methylphosphonic
acid)]ethyl pendant arm was synthesized, and its coordination
properties were studied by potentiometry and NMR spectros-
copy. Full in-cage complexation of Eu(III) is finished at pH >
4.5, forming the protonated SA-[Eu(Hdo3aNP)]− complex
with square-antiprismatic (SA) geometry. Upon deprotonation
with pKA ∼ 8, the twisted-square-antiprismatic TSA-[Eu-
(do3aNP)]2− complex is formed. Both complexes are in a
slow equilibrium on the NMR time scale, and their 31P NMR
shifts differ by ∼190 ppm due to significant movement of the
principal magnetic axis leading to different paramagnetic
contributions to the 31P NMR shift. To the best of our
knowledge, such a strict change of the coordination geometry
with (de)protonation has not been observed before.
Figure 7. Dependence of y = log{Int(−118 ppm)/Int(70 ppm)} ∼
log{[SA]/[TSA]} on pH in solutions with different concentrations of
the complex probe and at different ionic strengths relevant for
physiologic conditions.
Finally, we tested the applicability of the [Eu(do3aNP)]2−/
[Eu(Hdo3aNP)]− system as a pH probe for MRI. However,
this experiment had some limitations due to the accessible
hardware. The broad spectral width (20 kHz/246 ppm)
necessary for the experiment required a short excitation
pulse. Due to hardware limitations, a block pulse (instead of
modulated one) had to be used for excitation to reach sufficient
pulse intensity. As the excitation profile of the block pulse is
inhomogeneous, intensities at different frequencies may not
exactly correspond to the concentrations. The space-localized
31P MR spectra were measured in the phantom consisting of
four vials: a vial containing an aqueous solution of H3PO4 as a
reference and three vials containing the Eu(III)−H5do3aNP
complex solutions having different pH values (6.4, 8.0, and 9.0).
The localized spectra and intensity of individual signals from
different parts of the phantom are shown in Figure 8, and the
results clearly show a spatial resolution of areas with different
pH values, proving the suggested concept.
The existence of two separate signals in the 31P NMR spectra
allows ratiometric determination of pH by MRS. As both
signals belong to the same compound (differing just by its
protonation state), this ensures the same biodistribution of
both species, and the ratiometric approach brings the advantage
of independence of the measured data on the probe
concentration. Unfortunately, the pKA of the studied complex
lies somewhat outside the region optimal for in vivo use;
nevertheless, this parameter can be altered by a suitably
designed ligand, and thus, the discovered principle shows
promising potential for in vivo applications.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b02749.
■
S
K
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
NMR spectra and studies, hydrogen bond lengths, and
Article
playing a high relaxivity with a pronounced pH dependence. New J.
Chem. 1998, 22, 627−631.
potentiometric results (PDF)
Crystal data (CIF)
(10) de Leon-Rodriguez, L. M.; Lubag, A. J. M.; Malloy, C. R.;
Martinez, G. V.; Gillies, R. J.; Sherry, A. D. Responsive MRI agents for
sensing metabolism in vivo. Acc. Chem. Res. 2009, 42, 948−957.
(11) Garcia-Martin, M. L.; Martinez, G. V.; Raghunand, N.; Sherry,
A. D.; Zhang, S.; Gillies, R. J. High resolution pH(e) imaging of rat
glioma using pH-dependent relaxivity. Magn. Reson. Med. 2006, 55,
309−315.
AUTHOR INFORMATION
Corresponding Author
*E-mail: modrej@natur.cuni.cz. Phone: +420-221951261. Fax:
+420-221951253.
■
(12) Aime, S.; Fedeli, F.; Sanino, A.; Terreno, E. R2/R1 Ratiometric
Procedure for a Concentration-Independent, pH-Responsive, Gd(III)-
Based MRI Agent. J. Am. Chem. Soc. 2006, 128, 11326−11327.
(13) (a) Soesbe, T. C.; Wu, Y.; Sherry, A. D. Advantages of
paramagnetic CEST complexes having slow-to-intermediate water
exchange properties as responsive MRI agents. NMR Biomed. 2013, 26,
829−838. (b) Dorazio, S. J.; Olatunde, A. O.; Spernyak, J. A.; Morrow,
J. R. CoCEST: cobalt(II) amide-appended paraCEST MRI contrast
agents. Chem. Commun. 2013, 49, 10025−10027. (c) Sheth, V. R.; Li,
Y.; Chen, L. Q.; Howison, C. M.; Flask, C. A.; Pagel, M. D. Measuring
in vivo tumor pHe with CEST-FISP MRI. Magn. Reson. Med. 2012, 67,
760−768. (d) Liu, G.; Li, Y.; Sheth, V. R.; Pagel, M. D. Imaging in vivo
extracellular pH with a single paramagnetic chemical exchange
saturation transfer magnetic resonance imaging contrast agent. Mol.
Imaging 2012, 11, 47−57. (e) Castelli, D. D.; Ferrauto, G.; Cutrin, J.
C.; Terreno, E.; Aime, S. In vivo maps of extracellular pH in murine
melanoma by CEST-MRI. Magn. Reson. Med. 2014, 71, 326−332.
(14) Aime, S.; Castelli, D. D.; Terreno, E. Novel pH-reporter MRI
contrast agents. Angew. Chem., Int. Ed. 2002, 41, 4334−4336.
ORCID
Jan Kotek: 0000-0003-1777-729X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Z. Bohmova
and Dr. I. Cısaro
■
́
for the potentiometric measurements
for the X-ray diffraction study. Support
̈
́
̌
va
́
from the Grant Agency of the Czech Republic (Project 16-
03156S) is acknowledged. We really appreciate remarks from
the referees regarding the spatial dependence of the para-
magnetic contribution to chemical shift.
REFERENCES
■
(1) The Chemistry of Contrast Agents in Medical Magnetic Resonance
Imaging, 2nd ed.; Merbach, A., Helm, L., Tot
́
́
h, E., Eds.; Wiley:
Chichester, United Kingdom, 2013.
(2) Gillies, J. R.; Raghunand, N.; Garcia-Martin, M. L.; Gatenby, R. A.
pH imaging: A review of pH measurement methods and applications
in cancers. IEEE Eng. Med. Biol. 2004, 23, 57−64.
(3) Hingorani, D. V.; Bernstein, A. S.; Pagel, M. D. A review of
responsive MRI contrast agents: 2005−2014. Contrast Media Mol.
Imaging 2015, 10, 245−265.
(4) Zhu, L.; Yang, Y.; Farquhar, K.; Wang, J.; Tian, C.; Ranville, J.;
Boyes, S. G. Surface Modification of Gd Nanoparticles with pH-
Responsive Block Copolymers for Use As Smart MRI Contrast
Agents. ACS Appl. Mater. Interfaces 2016, 8, 5040−5050.
(5) (a) Zhang, S.; Wu, K.; Sherry, A. D. A Novel pH-Sensitive MRI
Contrast Agent. Angew. Chem., Int. Ed. 1999, 38, 3192−3194.
(b) Raghunand, N.; Howison, C.; Sherry, A. D.; Zhang, S.; Gillies,
R. J. Renal and systemic pH imaging by contrast-enhanced MRI. Magn.
́ ́ ́ ́
(15) Krchova, T.; Galisova, A.; Jirak, D.; Hermann, P.; Kotek, J.
Ln(III)-complexes of a DOTA analogue with an ethylenediamine
pendant arm as pH-responsive PARACEST contrast agents. Dalton
Trans. 2016, 45, 3486−3496.
(16) Castelli, D. D.; Terreno, E.; Aime, S. YbIII-HPDO3A: A Dual
pH- and Temperature-Responsive CEST Agent. Angew. Chem. 2011,
123, 1838−1840.
(17) Posse, S.; Otazo, R.; Dager, S. R.; Alger, J. MR spectroscopic
imaging: principles and recent advances. J. Magn. Reson. Imaging. 2013,
37, 1301−1325.
(18) Griffiths, R. J. Are cancer cells acidic? Br. J. Cancer 1991, 64,
425−427.
(19) Gillies, R. J.; Morse, D. L. In vivo magnetic resonance
spectroscopy in cancer. Annu. Rev. Biomed. Eng. 2005, 7, 287−326.
(20) (a) van Sluis, R.; Bhujwalla, Z. M.; Raghunand, N.; Ballesteros,
P.; Alvarez, J.; Cerdan, S.; Galons, J. P.; Gillies, R. J. In vivo imaging of
Reson. Med. 2003, 49, 249−257. (c) Kal
Caravan, P.; Jurek, P.; Spiller, M.; Tircso, G.; Kiraly, R.; Brucher, E.;
́
man
́
, F. K.; Woods, M.;
̈
Sherry, A. D. Potentiometric and Relaxometric Properties of
Gadolinium-based MRI Contrast Agent for Sensing Tissue pH.
Inorg. Chem. 2007, 46, 5260−5270.
(6) Lowe, M. P.; Parker, D.; Reany, O.; Aime, S.; Botta, M.;
Castellano, G.; Gianolio, E.; Pagliarin, R. pH-dependent modulation of
relaxivity and luminescence in macrocyclic gadolinium and europium
complexes based on reversible intramolecular sulfonamide ligation. J.
Am. Chem. Soc. 2001, 123, 7601−7609.
1
extracellular pH using H MRSI. Magn. Reson. Med. 1999, 41, 743−
750. (b) Garcia-Martin, M. L.; Herigault, G.; Remy, C.; Farion, R.;
Ballesteros, P.; Coles, J. A.; Cerdan, S.; Ziegler, A. Mapping
Extracellular pH in Rat Brain Gliomas in Vivo by H Magnetic
Resonance Spectroscopic Imaging: Comparison with Maps of
Metabolites. Cancer Res. 2001, 61, 6524−6531.
(21) Aime, S.; Botta, M.; Milone, L.; Terreno, E. Paramagnetic
complexes as novel NMR pH indicators. Chem. Commun. 1996, 1265−
1266.
(22) Coman, D.; Trubel, H. K.; Rycyna, R. E.; Hyder, F. Brain
temperature and pH measured by (1)H chemical shift imaging of a
thulium agent. NMR Biomed. 2009, 22, 229−239.
(23) Huang, Y.; Coman, D.; Ali, M. M.; Hyder, F. Lanthanide ion
(III) complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraami-
nophosphonate (DOTA-4AmP8−) for dual biosensing of pH with
CEST (chemical exchange saturation transfer) and BIRDS (biosensor
imaging of redundant deviation in shifts). Contrast Media Mol. Imaging
2015, 10, 51−58.
(24) Jagadish, B.; Brickert-Albrecht, G. L.; Nichol, G. S.; Mash, E. A.;
Raghunand, N. On the Synthesis of 1,4,7-Tris(tert-butoxycarbonyl-
methyl)-1,4,7,10-tetraazacyclododecane. Tetrahedron Lett. 2011, 52,
2058−2061.
(25) Jiang, X.-J.; Lo, P.-Ch; Tsang, Y.-M.; Yeung, S.-L.; Fong, W.-P.;
Ng, D. K. P. Phthalocyanine−Polyamine Conjugates as pH-Controlled
(7) Woods, M.; Kiefer, G. E.; Bott, S.; Castillo-Muzquiz, A.;
Eshelbrenner, C.; Michaudet, L.; McMillan, K.; Mudigunda, S. D. K.;
Ogrin, D.; Tircso, G.; Zhang, S.; Zhao, P.; Sherry, A. D. Synthesis,
Relaxometric and Photophysical Properties of a New pH-Responsive
MRI Contrast Agent: The Effect of Other Ligating Groups on
Dissociation of a p-Nitrophenolic Pendant Arm. J. Am. Chem. Soc.
2004, 126, 9248−9256.
(8) (a) Giovenzana, G. B.; Negri, R.; Rolla, G. A.; Tei, L. Gd-
Aminoethyl-DO3A Complexes: A Novel Class of pH-Sensitive MRI
Contrast Agents. Eur. J. Inorg. Chem. 2012, 2012, 2035−2039.
́
(b) Baranyai, Z.; Rolla, G. A.; Negri, R.; Forgacs, A.; Giovenzana, G.
B.; Tei, L. Comprehensive Evaluation of the Physicochemical
Properties of LnIII Complexes of Aminoethyl-DO3A as pH-Responsive
T1-MRI Contrast Agents. Chem. - Eur. J. 2014, 20, 2933−2944.
(9) Hall, J.; Haner, R.; Aime, S.; Botta, M.; Faulkner, S.; Parker, D.;
de Sousa, A. S. Relaxometric and luminescence behaviour of
triaquahexaazamacrocyclic complexes, the gadolinium complex dis-
L
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Photosensitizers for Photodynamic Therapy. Chem. - Eur. J. 2010, 16,
4777−4783.
Monophosphinic Acid DOTA Analogue Ligand and Its Lanthanide-
(III) Complexes. A Gadolinium(III) Complex Endowed with an
Optimal Water Exchange Rate for MRI Applications. Org. Biomol.
Chem. 2005, 3, 112−117.
(26) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pargamon Press: Oxford, 1988.
(27) MATLAB R2015b; The MathWorks Inc.: Natick, MA, 2000.
(28) Corsi, D. M.; Platas-Iglesias, C.; van Bekkum, H.; Peters, J. A.
Determination of paramagnetic lanthanide (III) concentrations from
bulk magnetic susceptibility shifts in NMR spectra. Magn. Reson. Chem.
2001, 39, 723−726.
́
(37) Krchova, T.; Kotek, J. Unpublished results.
(38) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker,
D.; Royle, L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. Non-
radiative deactivation of the excited states of europium, terbium and
ytterbium complexes by proximate energy-matched OH, NH and CH
oscillators: an improved luminescence method for establishing solution
hydration states. J. Chem. Soc., Perkin Trans. 2 1999, 493−504.
(29) Sheldrick, G. M. SHELXS97, Program for Crystal Structure
Solution from Diffraction Data; University of Gottingen: Gottingen,
̈
̈
Germany, 1997.
(30) Sheldrick, G. M. SHELXL97, Program for Crystal Structure
Refinement from Diffraction Data; University of Gottingen: Gottingen,
(39) (a) Rudovsky, J.; Botta, M.; Hermann, P.; Koridze, A.; Aime, S.
́
Relaxometric and solution NMR structural studies on ditopic
lanthanide(III) complexes of a phosphinate analogue of DOTA with
a fast rate of water exchange. Dalton Trans. 2006, 2323−2333.
̈
̈
Germany, 1997.
(31) (a) Taborsky, P.; Lubal, P.; Havel, J.; Kotek, J.; Hermann, P.;
Lukes, I. Thermodynamic and Kinetic Studies of Lanthanide(III)
́
́
́
́
(b) Kotkova, Z.; Pereira, G. A.; Djanashvili, K.; Kotek, J.; Rudovsky, J.;
̌
Hermann, P.; Vander Elst, L.; Muller, R. N.; Geraldes, C. F. G. C.;
Lukes, I.; Peters, J. A. Lanthanide(III) Complexes of Phosphorus Acid
Complexes with H5do3ap (1,4,7,10-Tetraazacyclododecane-1,4,7-
triacetic-10-(methylphosphonic Acid)), a Monophosphonate Ana-
logue of H4dota. Collect. Czech. Chem. Commun. 2005, 70, 1909−
̌
Analogues of H4DOTA as Model Compounds for the Evaluation of
the Second-Sphere Hydration. Eur. J. Inorg. Chem. 2009, 2009, 119−
136. (c) Pereira, G. A.; Ball, L.; Sherry, A. D.; Peters, J. A.; Geraldes, C.
F. G. C. NMR Characterization of Lanthanide(3+) Complexes of
Tetraazatetrakisphosphinato and Tetraazatetrakisphosphonato Li-
gands. Helv. Chim. Acta 2009, 92, 2532−2551. (d) Rohovec, J.;
1942. (b) Forsterova,
Kotek, J.; Hermann, P.; Lukes, I. Thermodynamic study of
́
M.; Svobodova,
́ ́
́
I.; Lubal, P.; Taborsky, P.;
̈
̌
lanthanide(III) complexes with bifunctional monophosphinic acid
analogues of H4dota and comparative kinetic study of yttrium(III)
complexes. Dalton Trans. 2007, 535−549.
̌
Lukes, I.; Hermann, P. Lanthanide complexes of a cyclen derivative
1
(32) Kyvala, M.; Lukes,
̌
I. International Conference Chemometrics 95;
with phenylphosphinic pendant arms for possible H and 31P MRI
́
Pardubice, Czech Republic, 1995; p 63. Full version of OPIUM
available (free of charge) on http://web.natur.cuni.cz/~kyvala/opium.
html.
temperature sensitive probes. New J. Chem. 1999, 23, 1129−1132.
(e) Rohovec, J.; Kyvala, M.; Vojtísek, P.; Hermann, P.; Lukes, I.
́
̌ ̌
Synthesis, Crystal Structures, and Solution Properties of N-Methylene-
(phenyl)phosphinic Acid Derivatives of Cyclen and Cyclam. Eur. J.
Inorg. Chem. 2000, 2000, 195−203. (f) Aime, S.; Batsanov, A. S.; Botta,
M.; Howard, J. A. K.; Parker, D.; Senanayake, K.; Williams, J. A. G.
Solution and Solid-state Characterization of Highly Rigid, Eight-
Coordinate Lanthanide(III) Complexes of a Macrocyclic Tetrabenzyl-
phosphinate. Inorg. Chem. 1994, 33, 4696−4706. (g) Aime, S.;
Batsanov, A. S.; Botta, M.; Dickins, R. S.; Faulkner, S.; Foster, C. E.;
Harrison, A.; Howard, J. A. K.; Moloney, J. M.; Norman, T. J.; Parker,
D.; Royle, L.; Williams, J. A. G. Nuclear magnetic resonance,
luminescence and structural studies of lanthanide complexes with
octadentate macrocyclic ligands bearing benzylphosphinate groups. J.
Chem. Soc., Dalton Trans. 1997, 3623−3636.
(33) Martell, A. E.; Smith, R. M.; Motekaitis, R. J. NIST Critically
Selected Stability Constants of Metal Complexes (Version 7); Texas A&M
University: College Station, TX.
(34) Aime, S.; Botta, M.; Ermondi, G. NMR study of solution
structures and dynamics of lanthanide(III) complexes of DOTA. Inorg.
Chem. 1992, 31, 4291−4299.
(35) (a) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B.
Gadolinium(III) Chelates as MRI Contrast Agents: Structure,
Dynamics, and Applications. Chem. Rev. 1999, 99, 2293−2352.
̌ ̌
(b) Hermann, P.; Kotek, J.; Kubícek, V.; Lukes, I. Gadolinium(III)
complexes as MRI contrast agents: ligand design and properties of the
complexes. Dalton Trans. 2008, 3027−3047. (c) Geraldes, C. F. G. C.;
Laurent, S. Classification and basic properties of contrast agents for
magnetic resonance imaging. Contrast Media Mol. Imaging 2009, 4, 1−
23.
(40) Campello, M. P. C.; Lacerda, S.; Santos, I. C.; Pereira, G. A.;
Geraldes, C. F. G. C.; Kotek, J.; Hermann, P.; Vanek
̌
, J.; Lubal, P.;
Kubícek, V.; Toth, E.; Santos, I. Lanthanide(III) complexes of 4,10-
̌
́
(36) (a) Aime, S.; Botta, M.; Fasano, M.; Marques, M. P. M.;
Geraldes, C. F. G. C.; Pubanz, D.; Merbach, A. E. Conformational and
Coordination Equilibria on DOTA Complexes of Lanthanide Metal
Ions in Aqueous Solution Studied by (1)H-NMR Spectroscopy. Inorg.
bis(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diacetic
acid (trans-H6do2a2p) in solution and in the solid state: structural
studies along the series. Chem. - Eur. J. 2010, 16, 8446−8465.
(41) (a) Blackburn, O. A.; Edkins, R. M.; Faulkner, S.; Kenwright, A.
M.; Parker, D.; Rogers, N. J.; Shuvaev, S. Electromagnetic
susceptibility anisotropy and its importance for paramagnetic NMR
and optical spectroscopy in lanthanide coordination chemistry. Dalton
Trans. 2016, 45, 6782−6800. (b) Cucinotta, G.; Perfetti, M.; Luzon, J.;
Etienne, M.; Car, P.-E.; Caneschi, A.; Calvez, G.; Bernot, K.; Sessoli, R.
Magnetic Anisotropy in a Dysprosium/DOTA Single-Molecule
Magnet: Beyond Simple Magneto-Structural Correlations. Angew.
Chem., Int. Ed. 2012, 51, 1606−1610.
Chem. 1997, 36, 2059−2068. (b) Lebdusk
̌
ova,
́
P.; Hermann, P.; Helm,
I.;
́
L.; Tot
́
h, E.; Kotek, J.; Binnemans, K.; Rudovsky, J.; Lukes,
̌
́
Merbach, A. E. Gadolinium(III) Complexes of Mono- and Diethyl
Esters of Monophosphonic Acid Analogue of DOTA as Potential MRI
Contrast Agents: Solution Structures and Relaxometric Studies. Dalton
Trans. 2007, 493−501. (c) Vitha, T.; Kubíce
̌
k, V.; Kotek, J.; Hermann,
I.; Peters, J. A. Gd(III)
P.; Vander Elst, L.; Muller, R. N.; Lukes,
̌
complex of a monophosphinate-bis(phosphonate) DOTA analogue
with a high relaxivity; Lanthanide(III) complexes for imaging and
radiotherapy of calcified tissues. Dalton Trans. 2009, 3204−3214.
(d) Castelli, D. D.; Caligara, M. C.; Botta, M.; Terreno, E.; Aime, S.
Combined high resolution NMR and H-1 and O-17 relaxometric study
sheds light on the solution structure and dynamics of the
Lanthanide(III) complexes of HPDO3A. Inorg. Chem. 2013, 52,
̌
̌ ́
k, P.; Hermann, P.; Mosinger, J.; Zak, Z.;
(42) (a) Rohovec, J.; Vojtíse
Lukes, I. Synthesis, crystal structures and NMR and luminescence
̌
spectra of lanthanide complexes of 1,4,7,10-tetraazacyclododecane
with N-methylene(phenyl)phosphinic acid pendant arms. J. Chem. Soc.,
Dalton Trans. 1999, 3585−3592. (b) Avecilla, F.; Peters, J. A.;
Geraldes, C. F. G. C. X-ray Crystal Structure of a Sodium Salt of
[Gd(DOTP)]5−: Implications for Its Second-Sphere Relaxivity and the
23Na NMR Hyperfine Shift Effects of [Tm(DOTP)]5−. Eur. J. Inorg.
7130−7138. (e) Rudovsky, J.; Cígler, P.; Kotek, J.; Hermann, P.;
́
̌ ̌
Vojtísek, P.; Lukes, I.; Peters, J. A.; Vander Elst, L.; Muller, R. N.
Lanthanide (III) complexes of a mono (methylphosphonate) analogue
of H4dota: The influence of protonation of the phosphonate moiety
on the TSAP/SAP isomer ratio and the water exchange rate. Chem. -
̌
Chem. 2003, 2003, 4179−4186. (c) Vojtísek, P.; Cígler, P.; Kotek, J.;
Rudovsky, J.; Hermann, P.; Lukes, I. Crystal Structures of Lanthanide-
̌
́
(III) Complexes with Cyclen Derivative Bearing Three Acetate and
One Methylphosphonate Pendants. Inorg. Chem. 2005, 44, 5591−
5599. (d) Luck, R. L.; Maupin, C. L.; Parker, D.; Riehl, J. P.; Williams,
Eur. J. 2005, 11, 2373−2384. (f) Rudovsky, J.; Kotek, J.; Hermann, P.;
́
̌
Lukes, I.; Mainero, V.; Aime, S. Synthesis of a Bifunctional
M
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
J. A. G. Circularly polarized luminescence and structural studies of a
dysprosium(III) complex with an octadentate macrocyclic ligand
bearing benzylphosphinate groups. Inorg. Chim. Acta 2001, 317, 331−
337.
̌ ̌
(43) Lukes, I.; Kotek, J.; Vojtísek, P.; Hermann, P. Tetraazacycles
with Methylphosphinic/phosphonic Acid Pendant Arms. A Compar-
ison with their Acetic Acid Analogues. Coord. Chem. Rev. 2001, 216−
217, 287−312.
́ ́ ̌ ́ ̌ ́
(44) Krchova, T.; Kotek, J.; Jirak, D.; Havlíckova, J.; Císarova, I.;
Hermann, P. Lanthanide(III) complexes of aminoethyl-DO3A as
PARACEST contrast agents based on decoordination of the weakly
bound amino group. Dalton Trans. 2013, 42, 15735−15747.
̌ ̌ ́
(45) Pniok, M.; Kubícek, V.; Havlíckova, J.; Kotek, J.; Sabatie-
Gogova, A.; Plutnar, J.; Huclier-Markai, S.; Hermann, P. Thermody-
namic and kinetic study of scandium(III) complexes of DTPA and
DOTA: a step toward scandium radiopharmaceuticals. Chem. - Eur. J.
2014, 20, 7944−7955.
N
DOI: 10.1021/acs.inorgchem.6b02749
Inorg. Chem. XXXX, XXX, XXX−XXX