Influence of calcium-induced aggregation on the sensitivity of aminobis(methylenephosphonate)-containing potential MRI contrast agents

Article abstract of DOI:10.1021/ic1024235

A novel class of 1,4,7,10-tetraazacyclododecane-1,4,7- tris(methylenecarboxylic) acid (DO3A)-based lanthanide complexes with relaxometric response to Ca²⁺ was synthesized, and their physicochemical properties were investigated. Four macrocyclic ligands containing an alkyl-aminobis(methylenephosphonate) side chain for Ca ²⁺-chelation have been studied (alkyl is propyl, butyl, pentyl, and hexyl for L¹, L², L³, and L⁴, respectively). Upon addition of Ca²⁺, the r₁ relaxivity of their Gd³⁺ complexes decreased up to 61% of the initial value for the best compounds GdL³ and GdL⁴. The relaxivity of the complexes was concentration dependent (it decreases with increasing concentration). Diffusion NMR studies on the Y³⁺ analogues evidenced the formation of agglomerates at higher concentrations; the aggregation becomes even more important in the presence of Ca²⁺. ³¹P NMR experiments on EuL¹ and EuL⁴ indicated the coordination of a phosphonate to the Ln³⁺ for the ligand with a propyl chain, while phosphonate coordination was not observed for the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd ³⁺ complexes. log K_H1 values for all complexes lie between 6.12 and 7.11 whereas log K_H2 values are between 4.61 and 5.87. Luminescence emission spectra recorded on the Eu³⁺ complexes confirmed the coordination of a phosphonate group to the Ln³⁺ center in EuL¹. Luminescence lifetime measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r₁. Variable temperature ¹⁷O NMR experiments evidenced high water exchange rates on GdL¹, GdL², and GdL³ comparable to that of the aqua ion.

Full text of DOI:10.1021/ic1024235

ARTICLE
pubs.acs.org/IC
Influence of Calcium-Induced Aggregation on the Sensitivity
of Aminobis(methylenephosphonate)-Containing Potential
MRI Contrast Agents
§
‡,||
§ ꢀ
J€org Henig,†,^ Ilgar Mamedov,‡,^ Petra Fouskova, Eva Tꢀoth, Nikos K. Logothetis,
Goran Angelovski,*,‡ and Hermann A. Mayer*,†
†Institut f€ur Anorganische Chemie, Eberhard Karls Universit€at T€ubingen, Auf der Morgenstelle 18, 72076 T€ubingen, Germany
‡Abteilung f€ur Physiologie kognitiver Prozesse, Max-Planck-Institut f€ur biologische Kybernetik, Spemannstrasse 38,
72076 T€ubingen, Germany
§Le Centre de Biophysique Molꢀeculaire, Centre National de la Recherche Scientifique, CNRS, Rue Charles Sadron,
45071 Orlꢀeans, France
Imaging Science and Biomedical Engineering, The University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
S Supporting Information
b
ABSTRACT: A novel class of 1,4,7,10-tetraazacyclododecane-
1,4,7-tris(methylenecarboxylic) acid (DO3A)-based lanthanide
complexes with relaxometric response to Ca2þ was synthesized,
and their physicochemical properties were investigated. Four
macrocyclic ligands containing an alkyl-aminobis(methylene-
phosphonate) side chain for Ca2þ-chelation have been studied
(alkyl is propyl, butyl, pentyl, and hexyl for L1, L2, L3, and L4,
respectively). Upon addition of Ca2þ, the r1 relaxivity of their
Gd3þ complexes decreased up to 61% of the initial value for the
best compounds GdL3 and GdL4. The relaxivity of the complexes was concentration dependent (it decreases with increasing
concentration). Diffusion NMR studies on the Y3þ analogues evidenced the formation of agglomerates at higher concentrations; the
aggregation becomes even more important in the presence of Ca2þ 31P NMR experiments on EuL1 and EuL4 indicated the
.
coordination of a phosphonate to the Ln3þ for the ligand with a propyl chain, while phosphonate coordination was not observed for
the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd3þ complexes. log KH1 values for
allcomplexeslie between6.12 and 7.11whereaslog KH2 values are between 4.61and 5.87. Luminescence emissionspectra recorded on
the Eu3þ complexes confirmed the coordination of a phosphonate group to the Ln3þ center in EuL1. Luminescence lifetime
measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r1.
Variabletemperature17O NMR experiments evidenced highwater exchangerates on GdL1, GdL2, and GdL3 comparabletothatofthe
aqua ion.
’ INTRODUCTION
ion sensing, most of the efforts have been directed to design
gadolinium complexes that are able to report changes in con-
centrations of the physiologically important Ca2þ and Zn2þ
The evolution of magnetic resonance imaging (MRI) into one
of the most powerful tools in medical diagnosis is strongly related
to the development of paramagnetic contrast agents (CAs). Most
commonly, these are based on trivalent gadolinium which has the
strongest influence on the relaxation of surrounding water
protons.1,2 Lately, efforts to develop “smart” contrast agents
(SCA) have become an important field in MRI research.3,4 The
relaxivity, r1, of these CAs is responsive to changes in the
chemical microenvironment of the Gd3þ complex, such as
variations in the pH or the concentration of inorganic ions and
organic molecules, and so forth (r1 is the paramagnetic long-
itudinal proton relaxation rate enhancement referred to one
millimolar concentration of Gd3þ).
cations.9ꢀ16 Many CAs developed for the detection of Ca2þ
,
consist of a macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-
tris(methylenecarboxylic) acid (DO3A) based Gd3þ complex
which is linked to an additional aminocarboxylate moiety for
selective Ca2þ coordination. It can be assumed that at least one
donor group of this moiety is able to coordinate to the Gd3þ
center in the absence of Ca2þ, whereas in the presence of the
divalent metal ion, the donor group dissociates from the Gd3þ to
complex the sensed metal ion. This reorganization of the
complex exposes the gadolinium ion to bulk water, indicated
Several pH responsive CAs have been reported,5ꢀ7 some of
them already finding in vivo application in the determination of
the pH in tumor tissue and the generation of pH maps.8 In metal
Received: December 4, 2010
Published: June 14, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ic1024235 Inorg. Chem. 2011, 50, 6472–6481
|

Inorganic Chemistry
ARTICLE
at ꢀ20 ꢀC for 16 h. The products were separated from the solvent,
washed with cold n-hexane, and dried in vacuo.
tert-Butyl 3-Bromopropylcarbamate (2a). Colorless crystals, yield
68%. 1H NMR (CDCl3): δ 5.25 (s, 1H, NH), 3.38 (t, 2H, 3JHH = 6.5 Hz,
BrCH2), 3.25 (t, 2H, 3JHH = 6.5 Hz, NHCH2), 1.95 (tt, 2H, 3JHH = 6.5 Hz,
3JHH = 6.5 Hz, CH2), 1.37 (s, 9H, CH3). 13C {1H} NMR (CDCl3): δ
155.6 (CO), 79.8 (C(CH3)3), 38.6, 32.4, 30.4 (CH2), 27.9 (CH3). EA
Calcd: C 40.35%, H 6.77%, N 5.88%. Found: C 40.29%, H 7.23%, N 5.88%.
tert-Butyl 5-Bromobutylcarbamate (2b). Yellow crystals, yield 67%.
Figure 1. Structures of the investigated DO3A alkylaminobis-
(methylenephosphonates), L1, n = 1; L2, n = 2; L3, n = 3; L4, n = 4.
3
1H NMR (CDCl3): δ 4.81 (s, 1H, NH), 3.52 (t, 2H, JHH = 6.6 Hz,
by an increase of the number of coordinated water molecules q
and resulting in positive change in relaxivity.15,17
BrCH2), 3.18 (m, 2H, NHCH2), 1.72 (m, 2H, CH2), 1.32 (s, 9H, CH3),
1.25ꢀ1.40 (m, 2H, CH2). 13C {1H} NMR (CDCl3): δ 156.3 (CO), 79.4
(C(CH3)3), 41.1, 32.1, 28.9 (CH2), 27.7 (CH3), 24.3 (CH2). EA Calcd:
C 42.87%, H 7.20%, N 5.5%. Found: C 44.18%, H 7.48%, N 5.69%.
tert-Butyl 5-Bromopentylcarbamate (2c). Yellow oil, yield 65%. 1H
NMR (CDCl3): δ 4.92 (s, 1H, NH), 3.52 (t, 2H, 3JHH = 6.7 Hz, BrCH2),
3.25 (m, 2H, NHCH2), 1.94 (tt, 2H, 3JHH = 6.7 Hz, 3JHH = 6.9 Hz, CH2),
1.32 (s, 9H, CH3), 1.25ꢀ1.40 (m, 4H, CH2). 13C {1H} NMR (CDCl3):
δ 158.3 (CO), 79.8 (C(CH3)3), 40.5, 33.8, 32.6, 29.4 (CH2), 28.7
(CH3), 25.5 (CH2). EA Calcd: C 45.12%, H 7.57%, N 5.26%. Found: C
45.16%, H 7.20%, N 5.49%.
General Procedure for 3aꢀc. To the solution of 1,4,7-tri(t-butox-
ycarbonylmethyl)cyclen (1 equiv) and K2CO3 (1.2 equiv) in acetonitrile
(30 mL) was added a solution of 2aꢀc (1.2 equiv) in acetonitrile
(20 mL). After the reaction mixture had been stirred for 16 h at 70 ꢀC,
the solution was allowed to cool to room temperature (r. t.), filtered, and
concentrated in vacuo to give a yellow to brown oil. The compounds
were purified by column chromatography (5% CH3OH in CH2Cl2).
tri-tert-Butyl 2,20,200-(10-(3-(tert-butoxycarbonylamino)propyl)-
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (3a). Yellow
oil, yield 85%. 1H NMR (CDCl3): δ 5.90 (s,1H, NH), 3.60ꢀ2.03 (m,
26H, CH2), 1.29 (m, 2H, CH2NH), 1.11ꢀ1.05 (m, 36H, C(CH3)3).
13C {1H} NMR (CDCl3): δ 173.6, 172.6 (COOt-Bu), 156.3 (COBoc),
82.7, 82.4, 81.7 (C(CH3)3), 57.4, 56.4, 55.7, 51.7, 50.2, 49.7, 38.7 (CH2),
28.4, 28.0, 27.8 (C(CH3)3), 26.6, 18.3 (CH2). ESI-MS: for
C34H65N5O8: calcd. [MþH]þ 672.5, found 672.4.
Very recently we have reported the synthesis and the in vivo
characterization of a novel Ca2þ sensitive potential MRI CA
based on DO3A, which bears an hexylaminobis(methylene-
phosphonate) group instead of an aminocarboxylate chelator
as the Ca2þ sensitive binding site (LnL4 in Figure 1).18 The
aminobis(methylenephosphonate) unit was chosen since the
introduction of negatively charged phosphonate groups into
common Gd3þ-based CAs usually increases their r1 because of
an acceleration of the water exchange rate kex and their ability to
form a second sphere water network around the Gd3þ com-
plexes, both factors being important to improve the MR signal,
especially in high field magnets.19ꢀ21Bis(phosphonates) at-
tached to the DO3A core or to superparamagnetic iron oxide
(SPIO) particles were already proposed as potential bone
targeting CAs.22,23 Moreover, aminopolyphosphonic acids are
well-known chelators for transition metal ions,24,25 but they also
exhibit high complexation efficiency toward biologically impor-
tant metal ions such as Ca2þ, Mg2þ, and Zn2þ 26
. Initial in vitro
physicochemical studies of GdL4 showed a very untypical Ca2þ
sensitivity. Namely, the addition of Ca2þ ions resulted in a
negative change in relaxivity.18 To investigate this interesting
effect more thoroughly, we have prepared three additional
analogous compounds which bear a propyl (L1), butyl (L2), or
pentyl (L3) linker instead of a hexyl (L4) one between the
aminobis(methylenephosphonates) and DO3A. Here we pre-
sent an extensive mechanistic investigation of LnL1ꢀLnL4.
tri-tert-Butyl 2,20,200-(10-(3-(tert-butoxycarbonylamino)butyl)-
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (3b). Yellow
oil, yield 83%. 1H NMR (CDCl3): δ 5.98 (s,1H, NH), 3.50ꢀ2.15 (m,
26H, CH2), 1.33 (m, 4H, CH2NH), 1.21ꢀ1.18 (m, 36H, C(CH3)3).
13C {1H}w NMR (CDCl3): δ 174.3, 172.9 (COOt-Bu), 157.0
(COBoc), 83.4, 82.9, 81.6 (C(CH3)3), 59.0, 57.9, 56.7, 52.3, 51.7,
40.1, 49.9, 39.1 (CH2), 29.1, 28.7, 28.1 (C(CH3)3), 26.4, 19.3 (CH2).
ESI-MS: for C35H67N5O8: calcd. [MþH]þ 686.5, found: 686.7.
tri-tert-Butyl 2,20,200-(10-(3-(tert-butoxycarbonylamino)pentyl)-
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (3c). Yellow
oil, yield 85%. 1H NMR (CDCl3): δ 5.22 (s,1H, NH), 3.42ꢀ1.64 (m,
26H, CH2), 1.36ꢀ1.32 (m, 36H, C(CH3)3), 1.14ꢀ1.30 (m, 6H CH2).
13C {1H} NMR (CDCl3): δ 171.6, 170.7 (COt-Bu), 155.7 (COBoc),
81.6, 80.2, 78.3 (C(CH3)3), 56.1, 55.9, 53.1, 51.9, 51.6, 51.3, 39.9, 33.3,
31.8 (CH2) 28.0, 27.7, 27.5 (C(CH3)3), 24.3, 22.8 (CH2). ESI-MS: for
C36H69N5O8: calcd 700.5 [MþH]þ, found: 700.6.
’ EXPERIMENTAL SECTION
General Remarks. 1H NMR, 13C{1H} and 31P{1H} NMR spectra
were recorded, and the relaxometric measurements were performed on a
Bruker DRX400 spectrometer (9.4 T) at room temperature, unless
otherwise specified. The 1H PGSE NMR diffusion measurements were
performed on a Bruker Avance IIþ 500 spectrometer (11.75 T)
equipped with a gradient unit and a multinuclear inverse probe with a
Z-gradient coil. 17O NMR experiments were performed on a Bruker
Avance 500 spectrometer (11.75 T, 67.8 MHz). Luminescence steady-
state and lifetime measurements were performed on QuantaMaster 3
PH fluorescence spectrometer from Photon Technology International,
Inc., U.S.A. ESI-LRMS were performed on an ion trap SL 1100 system
(Agilent, Germany). Column chromatography was performed using
silicagel 60 (70ꢀ230 mesh ASTM) from Merck, Germany. Experimen-
tal details on the synthesis and characterization data of compounds 2d,
3d, 4d, L4, GdL4, and EuL4 have been previously reported.18
Synthesis of Ligands and Complexes. General Procedure for
2aꢀc. A solution of NaOH (3 equiv) in water (30 mL) was added
dropwise to a vigorously stirring biphasic mixture consisting of 1aꢀc
(1.5 equiv) in water (50 mL) and Boc2O (1 equiv) in 100 mL of
dichloromethane. After 3 h the organic phase was separated and washed
with a 2 N HCl (50 mL) and a saturated NaCl solution (50 mL).
The organic phase was dried over MgSO4 and concentrated in vacuo.
n-Hexane (20 mL) was added to the oil and the solution was stored
General Procedure for 4aꢀc. Compounds 3aꢀc (2 mmol) were
taken in dichloromethane (20 mL), and trifluoroacetic acid (20 mL) was
added carefully. After the mixture had been stirred at r. t. for 24 h, the
solvents were removed under reduced pressure. Dichloromethane
(40 mL) was added and evaporated off twice to take out the excess of
trifluoracetic acid. The same procedure was repeated twice with
methanol. The viscous residues were taken up in a minimum amount
of methanol and cold ether was added dropwise. The formed precipi-
tates were filtered and resuspended in 3 mL of water. A large excess of
acetone (100 mL) was added, and the cloudy solutions were stored at
ꢀ20 ꢀC for 16 h. Colorless crystalline powders were filtered off, washed
with acetone, and dried in vacuo.
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Inorganic Chemistry
ARTICLE
2,20,200-(10-(3-Aminopropyl)-1,4,7,10-tetraazacyclododecane-
Relaxometric Ca2þ Titrations. The titrations were performed at
9.4 T, 25 ꢀC, and pH 7.3ꢀ7.4 (maintained by HEPES buffer). A solution
of CaCl2 of known concentration was added stepwise to the complex
solution, and the longitudinal proton relaxation time T1 was measured
after each addition of the analyte. The relaxivity r1 was calculated from
eq 1, using the actual Gd3þ concentration at each point of the titration.
1,4,7-triyl)triacetic Acid (4a). Colorless crystalline powder, yield 64%
1H NMR (D2O): δ 3.69ꢀ2.66 (m, 26H, CH2), 1.73 (m, 2H, CH2). 13
C
{1H} NMR (D2O): 174.2, 171.2 (COOH), 56.5, 53.8, 51.6, 50.6, 49.7,
48.7, 37.4, 24.9, 24.9 (CH2). HRMS (EI): for C17H33N5O6: calcd:
402.3265 [M-H]ꢀ; found: 402.3266.
2,20,200-(10-(3-Aminobutyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl)triacetic Acid (4b). Colorless crystalline powder, yield 66%.
1H NMR (D2O): δ 3.94 (m, 2H, CH2NH2), 3.45ꢀ2.52 (m, 24H, CH2),
1.60ꢀ1.32 (m, 4H, CH2). 13C {1H} NMR (D2O): δ 176.3, 169.8
(COOH), 56.7, 55.3, 54.2, 52.7, 50.4, 49.5, 39.9, 26.9, 23.7, 23.2 (CH2).
ESI-MS: for C18H35N5O6: calcd: 418.3 [MþH]þ; found: 418.0.
2,20,200-(10-(3-Aminopentyl)-1,4,7,10-tetraazacyclododecane-1,4,
7-triyl)triacetic Acid (4c). Colorless crystalline powder, yield 62%.
1H NMR (D2O): δ 3.74 (m, 2H, CH2NH2), 3.50ꢀ2.64 (m, 24H,
CH2), 1.57ꢀ1.21 (m, 6H, CH2). 13C {1H} NMR (D2O): δ 174.4, 169.1
(COOH), 55.9, 54.1, 53.2, 51.6, 49.9, 48.5, 48.3, 39.2, 26.3, 22.9, 22.7
(CH2). HRMS (EI): for C19H37N5O6: calcd: 430.2671 [M-H]ꢀ; found:
430.2664.
1
1
¼
þ r1 ꢁ ½Gdꢂ
ð1Þ
T1, obs
T1, d
where T1,obs is the observed longitudinal relaxation time, T1,d is the
diamagnetic contribution in the absence of the paramagnetic substance,
and [Gd] is the concentration of Gd3þ
.
NMR Diffusion Experiments. Appropriate amounts of YL1 and
YL4 were dissolved in D2O and in a solution of 3 equiv of CaCl2,
respectively. The samples were diluted stepwise while keeping the pD at
7.2ꢀ7.4. The measurements were performed by using the standard
stimulated echo pulse sequence. The temperature was 298 K, and the
sample was not spun. The shape of the gradient pulses was rectangular,
their duration (δ) was between 1.4 and 1.8 ms, depending on the sample,
and their strength (G) was varied during the experiments. The delay
between the midpoints of the gradients (Δ) was set to 200 ms. The
spectra were recorded using 32ꢀ1k scans, 32K points, a relaxation delay
of 1.4ꢀ3.8 s, a spectral width of 7500 Hz, and were processed with a line
broadening of 2. The intensity of the 1H resonances of the alkyl groups
of the side chain was determined in all spectra. The plots of ln(I/I0)
versus G2 were fitted using a linear regression algorithm to obtain the D
values according to eq 2, where I = intensity of the observed spin-echo,
I0 = intensity of the spin-echo without gradients, γ = magnetogyric ratio,
δ = length of the gradient pulse, D = diffusion coefficient, Δ = delay
between the midpoints of the gradients, and G = gradient strength.
General Procedure for L1ꢀL3. Compounds 4aꢀc (1 equiv) were
dissolvedin 6 M HCl (10 mL). Phosphoric acid (2 equiv) inwater (5 mL)
was added to these solutions, and the mixtures were heated to reflux.
Paraformaldehyde (4 equiv) was added portionwise within 1 h, and
heating under reflux was continued for further 24 h. The reaction mixtures
were concentrated under reduced pressure and cold ethanol was added
slowly under stirring. The solutions were cooled to ꢀ20 ꢀC for 12 h. The
solid product was separated by filtration and dried by prolonged standing
in vacuo.
2,20,200-(10-(3-(Bis(phosphonomethyl)amino)propyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic Acid (L1). Colorless pow-
der, yield 54%. 1H NMR (D2O): δ 2.20ꢀ3.35 (m, 30H), 1.29ꢀ1.39 (m,
2H). 31P {1H} NMR (D2O): δ 10.02. 13C {1H} NMR (D2O): δ 173.7,
167.7 (COOH), 54.0 (d, 1JPC = 33.6 Hz), 52.9, 52.2, 51.5, 51.4, 50.9,
50.7, 50.1, 48.3, 19.4. HRMS (EI): for C19H39N5O12P2: calcd: 590.2000
[M-H]ꢀ; found: 590.1985.
ꢀ
ꢁ
I
δ
2
ln ¼ ꢀ ðγδÞ D Δ ꢀ G2
ð2Þ
I0
3
Potentiometric titrations were carried out in a cell thermostatted at
25 ꢀC, at ionic strength I(KCl) = 0.1 mol dmꢀ3 and in the presence of
2,20,200-(10-(3-(Bis(phosphonomethyl)amino)butyl)-1,4,7,10-tet-
raazacyclododecane-1,4,7-triyl)triacetic Acid (L2). Colorless powder,
3
extra HCl in the ꢀlog[Hþ] range 3.8ꢀ11, using a combined glass
electrode (LL Biotrode, Metrohm) connected to a Metrohm 827 pH/
ion-meter, equipped with a Metrohm Dosimat 765 automatic buret. The
initial volume was 3 mL, the concentration of the Gd3þ complex was
1
yield 56%. H NMR (D2O): δ 4.01 (m, 2H, CH2N(CH2PO3H2)2),
2.95ꢀ3.70 (m, 28H), 1.35ꢀ1.72 (m, 4H). 13C {1H} NMR (D2O): δ
174.2, 168.0 (COOH), 55.4, 54.4, 53.6, 52.3, 51.7, 50.1, 49.5, 47.3(d,
1JPC = 33.8 Hz), 22.1, 21.9, 21.7. 31P {1H} NMR (D2O): δ 9.74. HRMS
(EI): for C20H41N5O12P2: calcd: 604.3252 [M-H]ꢀ; found: 604.3249.
2,20,200-(10-(3-(Bis(phosphonomethyl)amino)pentyl)-1,4,7,10-tetra-
azacyclododecane-1,4,7-triyl)triacetic Acid (L3). Colorless powder,
0.001 mol dmꢀ3. An inert atmosphere was ensured by constant passage
3
of N2 through the solution. The Hþ concentration was obtained from
the measured pH values according to the method proposed by Irving
et al.28 The protonation and stability constants were calculated with the
program PSEQUAD.29 The errors given correspond to one standard
1
yield 52%. H NMR (D2O): δ 4.01 (m, 2H, CH2N(CH2PO3H2)2),
deviation. The protonation constants βn are defined by βn
=
2.85ꢀ3.60 (m, 28H), 1.25ꢀ1.66 (m, 6H). 13C {1H} NMR (D2O):
δ 174.3, 168.4 (COOH), 55.9, 54.1, 53.8, 52.8, 51.7, 50.5, 49.9, 48.7,
48.1(d, 1JPC = 33.8 Hz), 22.6, 22.5, 22.3. 31P {1H} NMR (D2O): δ 7.44.
HRMS (EI): for C21H43N5O12P2: calcd: 618.2311 [M-H]ꢀ; found:
618.2310.
[HnL]/([H]n ꢁ [L]), therefore log KH1 = log β1, log KH2 = log β2 ꢀ
log β1. The stability constant is defined by βpqr = [MpHqLr]/([M]p ꢁ
[H]q ꢁ [L]r).
Luminescence Lifetime Experiments. The decay experiments
were performed in H2O and D2O (25 ꢀC, pH 7.3, HEPES) on
EuL1ꢀEuL3 at concentrations ranging from 5 to 50 mM. The Eu3þ
ion was directly excited at 395 nm and emission intensity at 615 nm was
recorded with 10 μs resolution. Excitation and emission slits were set to
15 and 5 nm bandpass, respectively. Data sets are the average of 25 scans,
and each reported value is the mean of three independent measure-
ments. Obtained curves are fitted to the first order exponential decay
with r2 = 0.99. q values are calculated using eq 3.
General Procedure for the Synthesis of Ln-Complexes. To the water
solutions the ligands L1ꢀL4 were mixed with the equimolar (referred to
the calculated molecular weight of the ligand) amount of the corre-
sponding LnCl3. The mixtures were heated to 60 ꢀC for 24 h. The pH
was periodically checked and adjusted to 6.5ꢀ7.5 using a 1 M solution of
NaOH. After 24 h the reaction mixture was cooled to room temperature.
An anion exchange resin (Chelex 100) was added to the stirring solution,
the suspensions were filtered after 1 h, and the solvent evaporated. The
absence of the noncoordinated metal ions in the systems was confirmed
by the xylenol test. The remaining residue was dissolved in a defined
amount of water, and for the gadolinium and europium complexes the
metal concentration was determined by measuring the bulk magnetic
susceptibility shifts (BMS).27 From these results the concentrations of
the analogously prepared yttrium complex solutions were estimated.
ꢀ1
ꢀ1
q ¼ 1:2 ꢁ ðτ
ꢀ τ
ꢀ 0:25Þ
ð3Þ
H2O
D2O
17O NMR Spectroscopy. The longitudinal (1/T1) and transverse
(1/T2) 17O NMR relaxation rates were measured for compounds
GdL1ꢀ4 in the temperature range of 275ꢀ350 K. The concentration
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ARTICLE
Scheme 1a
a (i) Boc2O, H2O/CH2Cl2, 62-65%; (ii) tris-t-Bu-DO3A, K2CO3,
CH3CN, 80 ꢀC, 83-85%; (iii) TFA, CH2Cl2, 62-66%; (iv) H3PO3,
formaldehyde, H2O, 100 ꢀC, 52-56%.
Figure 2. Relaxometric Ca2þ titration curves of GdL1 (9), GdL2 (O),
GdL3 (Δ) and GdL4 (1)18 at [GdL] = 2.5 mM, 9.4 T, 25 ꢀC, pH 7.3
(HEPES buffer). The lines correspond to the fitted curves.
of the samples was 50 mM and the pH of the solutions was adjusted to 6
using KOH/HCl. The exact Gd3þ concentration was determined using
the BMS method. The samples were sealed in glass spheres to eliminate
the influence of the bulk magnetic susceptibility.30,31 The exact tem-
perature was calculated according to previous calibration with ethylene
glycol and methanol.32 The 1/T1-data were obtained by the inversion
recovery method, while the 1/T2-data were measured by the
CarrꢀPurcellꢀMeiboomꢀGill spin-echo technique. Acidified water
(HClO4, pH 3.3) was used as external reference. The least-squares fit
of the 17O NMR data was performed by using Micromath Scientist
version 2.0 (Salt Lake City, UT, U.S.A.). The reported errors correspond
to one standard deviation obtained by the statistical analysis.
ESI-MS spectrometry in negative and positive mode. The
appropriate molecular ion peaks with the characteristic isotope
pattern of the complexes were present in the analyzed spectra.
Relaxivity Experiments. Initially, the paramagnetic response
of the complexes GdL1ꢀGdL3 to physiologically relevant metal
ions was studied by means of relaxometric titrations at complex
concentrations of 2.5 mM (9.4 T, 25 ꢀC). The experimental data
were compared with the experimental results for the complex
GdL4.18 Already in the absence of any divalent metal ion, the
relaxivities of the complexes vary as a function of the length of the
pendant arm. r1 values of 6.92, 7.43, 6.70, and 5.76 mMꢀ1 sꢀ1
were determined for GdL1, GdL2, GdL3, and GdL4, respectively.
Significant differences were observed in the r1 response of
GdL1ꢀGdL4 to Ca2þ (Figure 2). Namely, the sensitivity of the
complexes toward Ca2þ increases with the extension of the
aliphatic side chain. No significant changes in r1 of GdL1 were
found over the whole span of Ca2þ concentration. In the case of
GdL2, a moderate decrease of r1 was observed on addition of
Ca2þ, whereas r1 of the GdL3 and GdL4 solutions showed a
strong dependence on the calcium concentration resulting in a
decrease to 66% and 61% of the initial r1 values, respectively. The
fitting of the curves gave the apparent association constants of log
KA = 1.6 ( 0.3, log KA = 1.5 ( 0.2, and log KA = 2.0 ( 0.2 for
GdL2, GdL3, and GdL4, respectively. Note that these apparent
equilibrium constants are only valid for the specific GdLn
concentration. The constants give information about the calcium
concentration range where the GdLn complexes (in millimolar
concentration) have a relaxometric response. Upon addition of
equimolar amounts of EDTA after the titration of GdL3 and
GdL4 with Ca2þ, the relaxivity of both complexes returned to the
initial values obtained in Ca2þ-free solutions, indicating that r1
’ RESULTS AND DISCUSSION
Synthesis of Ligands and Complexes. The ligands L1ꢀL3
were synthesized according to Scheme 1, and L4 according to the
protocol described previously.18 Aminoalkyl alcohols were bro-
minated with HBr/acetic acid solutions using a modified procedure
previously published to give 1aꢀc.33 Amino groups of 1aꢀc were
protected using Boc2O at basic conditions to yield 2aꢀc. The
tert-butyl ester of DO3A was alkylated with the corresponding
Boc-protected bromoalkylamines to give 3aꢀc. All protective
groups on acids and the amine were removed in a one step reaction
using trifluoroacetic acid in dichloromethane. Compounds 4aꢀc
were obtained after recrystallization from diethyl ether followed by
recrystallization with acetone. The methylenephosphonates were
attached to the amines on the side chains by a Mannich reaction
using phosphorous acid and formaldehyde at 100 ꢀC.34 After
recrystallization from water/ethanol or water/isopropanol, the final
ligands L1ꢀL3 were obtained as colorless powders. The excess of
phosphoric acid was removed by several recrystallizations, and the
process was followed with 31P NMR spectroscopy. The obtained
ligands were characterized by 1H, 13C, 31P NMR spectroscopy and
HRMS spectrometry. All final compounds contained a characteristic
doublet in the 13C{1H} NMR spectrum which is due to the
interaction of the carbon atom next to the phosphonate group with
the corresponding phosphorus nucleus.
changes are reversible and exclusively induced by Ca2þ
.
Relaxometric titrations of GdL3 and GdL4 with Mg2þ did not
show any change in r1. These complexes, however, exhibit a
certain sensitivity to Zn2þ ions, though the Zn2þconcentrations
needed to reach saturation of r1 are much higher than those of
Ca2þ (>8 equiv of Zn2þ vs <3 equiv of Ca2þ) and the addition of
EDTA did not lead to a recovery of the initial r1.
The metal complexes of L1ꢀL3 with Gd3þ, Eu3þ, or Y3þ and
YL4 were formed by mixing the corresponding stock solutions of
the metal ions with the respective ligands in a 1:1 ratio. The
solutions were heated to 60 ꢀC for 24 h while the pH was
maintained at ∼7. The complexes were finally treated with
Chelex 100, filtered and lyophilized. The xylenol orange test
was performed to ensure that there are no free metal ions in the
solution. The formation of the complexes was confirmed by
Concentration Dependent Relaxivities. The relaxivity de-
crease observed for GdL2ꢀGdL4 upon addition of Ca2þ is
unusual and not explainable with the mechanism assumed for
the previously reported Ca2þ responsive SCAs. Phosphonates,
diphosphonates, and aminobis(methylenephosphonates) are
able to form aggregates in solution, either hydrogen bonded
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Figure 3. Concentration-dependence of r1 for GdL1 (9), GdL2 (O),
GdL3 (Δ), and GdL4 (1)18 in (a) the absence of Ca2þ and (b) the
presence of 3 equiv of Ca2þ at 9.4 T, 25 ꢀC, pH 7.3 (HEPES buffer).
Figure 4. Concentration-dependence of the diffusion coefficient for
YL1 (top), and YL4 (bottom) in the absence of Ca2þ (full symbols) and
the presence of 3 equiv of Ca2þ (empty symbols) at 11.75 T, 25 ꢀC, pH
7.3 (HEPES buffer).
(units with two or more molecules) or metal-assisted,25,35 which
might have an influence on the relaxivity. As the ratio between
aggregated and non aggregated complexes should be concentra-
tion dependent, the relaxivity was studied as a function of the
GdL1ꢀGdL4 concentration, in the presence and absence of
Ca2þ. In Ca2þ-free solutions only GdL1 showed concentration
independent relaxivities, while for GdL2ꢀGdL4, the r1 values
were found to be concentration dependent (Figure 3a). The r1 of
GdL2, GdL3, and GdL4 dropped by 14, 38, and 17%, respec-
tively, when the complex concentration was increased from 2.5 to
20 mM. However, when the same experiment was performed in
the presence of 3 equiv of Ca2þ, the change in r1 for GdL3 and
GdL4 was low, while r1 decreased by 19 and 20% for GdL1 and
GdL2, respectively (Figure 3b).
highly concentrated samples D corresponds to that of the
aggregate. In samples with concentrations between those two
extremes only an average D is determined corresponding to
mixtures of aggregated, partly aggregated, and monomeric
species. For the quantification of aggregation processes usually
the viscosity independent hydrodynamic volume VH is used.38
VH can be calculated from the hydrodynamic radius rH which is,
for spherical molecules, related to the diffusion coefficient D via
the StokesꢀEinstein equation (eq 4), where kB is the Boltzmann
constant, T is the absolute temperature, η is the viscosity, and rH
is the hydrodynamic radius. Additionally, the global rotational
correlation time τRg can be calculated from rH using eq 5.
kBT
PGSE Measurements. Pulsed gradient spin echo (PGSE) 1H
NMR diffusion measurements were performed to confirm that the
changes in relaxivity are the result of an aggregation/disaggrega-
tion process. Over the past years, this method has shown to be a
powerful tool in the elucidation of aggregation processes36ꢀ40 as
well as for the determination of global rotational correlation times
τRg.41,42 Since complexes of the paramagnetic Gd3þ are not
suitable for such measurements, the corresponding diamagnetic
Y3þ complexes were applied. The diffusion coefficients D of the
complexes YL1 and YL4 were determined in the same range of
concentration as were used for the concentration dependent
relaxivity measurements, both in the absence and presence of
Ca2þ ions at 25 ꢀC (Figure 4). For comparison, D of the Y3þ
complex of compound 4d was also ascertained.
D ¼
ð4Þ
6πηrH
4πηrH3
3kBT
τRg
¼
ð5Þ
Assuming dilute solutions where ηsolution = ηsolvent, values for
rH, τRg, and VH were calculated from the obtained diffusion
constants (Table 1).
Here, these values should only be regarded as approximations
and serve mainly for visualization purposes, as not all complexes
and possible aggregates can be considered as spherical. However,
it is unambiguous that in the absence of Ca2þ the diffusion
constants of YL1 are significantly higher than those of YL4. The
calculated hydrodynamic volume VH of YL4 is about twice the
As mentioned above, aggregation is a concentration depen-
dent process. Hence, for an infinitely diluted sample the deter-
mined diffusion coefficient D is that of the monomer, whereas in
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Table 1. Diffusion Coefficients D of Y-4d, YL1, and YL4 at Various Concentrations in the Absence of Ca2þ and the Presence of 3
equiv of Ca2þ at 9.4 T, 25 ꢀC, pH 7.3 (HEPES)
concentration
complex
2.5 mM
5 mM
10 mM
20 mM
D [m2s1]
Y-4d
YL1
3.85 ꢁ 10ꢀ10
3.53 ꢁ 10ꢀ10
3.45 ꢁ 10ꢀ10
2.85 ꢁ 10ꢀ10
2.49 ꢁ 10ꢀ10
5.01
3.85 ꢁ 10ꢀ10
3.50 ꢁ 10ꢀ10
3.11 ꢁ 10ꢀ10
2.74 ꢁ 10ꢀ10
1.95 ꢁ 10ꢀ10
5.01
3.85 ꢁ 10ꢀ10
3.46 ꢁ 10ꢀ10
2.93 ꢁ 10ꢀ10
2.71 ꢁ 10ꢀ10
1.84 ꢁ 10ꢀ10
5.01
3.85 ꢁ 10ꢀ10
3.30 ꢁ 10ꢀ10
2.38 ꢁ 10ꢀ10
2.60 ꢁ 10ꢀ10
1.59 ꢁ 10ꢀ10
5.01
YL1 þ 3 equiv of Ca2þ
YL4
YL4 þ 3 equiv of Ca2þ
Y-4d
rH [Å]a
YL1
5.47
5.52
5.57
5.84
YL1 þ 3 equiv of Ca2þ
5.60
6.21
6.58
8.10
YL4
6.76
7.05
7.12
7.42
YL4 þ 3 equiv of Ca2þ
Y-4d
7.75
9.87
10.51
12.12
VH [Å3]b
528
528
528
528
YL1
684
702
725
835
YL1 þ 3 equiv of Ca2þ
736
1003
1192
2228
YL4
1295
1465
1511
1711
YL4 þ 3 equiv of Ca2þ
Y-4d
1953
4031
4867
7454
τRg [ps]a
145
145
145
145
YL1
188
193
199
230
YL1 þ 3 equiv of Ca2þ
202
276
328
613
YL4
356
403
416
471
YL4 þ 3 equiv of Ca2þ
537
1109
1339
2050
a Calculated from eq 1 using η = 1.13 ꢁ 10ꢀ3 Pa s for D2O. b Calculated from rH with VH = 4/3πr3H.
volume of YL1, whereas VH of YL1 is only slightly larger than that
of the monomeric Y-4d. This confirms the presence of aggregates
for LnL4 already in the absence of Ca2þ. D of YL4 and all related
parameters are therefore only average values over all forms of YL4
in solution. The aggregation process is also shown by the increase
in VH of YL4 with higher complex concentrations, at which the
equilibrium between aggregated and monomeric species is
shifted toward the aggregated complexes. As, on the other hand,
the relaxivity of GdL4 is decreasing with increasing complex
concentration, r1 of these aggregates must be smaller than r1 of
the monomeric complex.
of YL1. The VH of YL4 at c = 2.5 mM in the presence of Ca2þ is
1.5 times bigger than without Ca2þ. As for LnL1, the increased
formation of aggregates seems to be responsible for the decrease
in relaxivity of LnL4. With increasing complex concentration the
average size of the species in solution seems to further increase.
However, no influence on r1 was observed for GdL4 in the
concentration dependent experiments (Figure 3b).
The results indicate that Ca2þ generally amplifies the forma-
tion of aggregates of these systems. Up to a certain point, the
formation of bigger aggregates results in the observed decrease in
relaxivity. Even though the formation of aggregates leads to a
slower rotation (see τRg in Table 1), its relaxivity enhancing effect
should not be too strong at this high field (9.4 T),43 and in those
cases where r1 decreases an opposite effect must be also operative.
31P NMR Spectroscopy. To understand whether the different
tendencies to form aggregates are related to differences in the
interaction of the phosphonate-containing side arms with the
paramagnetic center, 31P{1H} NMR experiments on EuL1 and
EuL4 solutions (50 mM in D2O) were performed (see Support-
ing Information). The europium complexes are usually used to
obtain information on the coordination behavior of the gadoli-
nium analogues by NMR spectroscopy because of their favorable
ratio between paramagnetic induced shift and line broadening.
The 31P{1H} NMR spectrum of EuL4 (pD 7.4, 298 K) shows
only one signal at 6.8 ppm. As this is nearly at the same position as
the 31P signal of the free ligand (6.6 ppm at pD 7.4), both
phosphonate groups of LnL4 are most likely not coordinated to
the lanthanide ion. The addition of Ca2þ results in a broadening
of the signal but not in a change of the chemical shift. As for LnL4
none of the phosphonate groups interact with the lanthanide ion,
both groups can interact with calcium. This allows the formation
of aggregates involving one or even more Ca2þ ions.
The tendency of LnL1 to form aggregates is much lower
resulting in a significantly lower hydrodynamic volume. How-
ever, in contrast to the relaxivity measurements, VH of YL1 shows
a small dependence on complex concentration especially at
higher concentrations. Therefore, it might be possible that a
certain amount of weak aggregates is formed at higher concen-
trations of LnL1, but they do not seem to influence the relaxivity.
The addition of 3 equiv of Ca2þ to LnL1 then clearly induces
aggregation. VH of YL1 at c = 20 mM in the presence of Ca2þ
(3 equiv) is about three-times higher than VH of YL1 in the
absence of Ca2þ. However, at c = 2.5 mM the hydrodynamic
volume of YL1 with Ca2þ is only slightly larger than without Ca2þ
.
This implies that Ca2þ does not significantly increase the amount of
aggregates at this concentration, which can be an explanation for the
independence of r1 on the Ca2þ concentration in the initial r1
measurements at lower Gd3þ complex concentrations (2.5 mM).
The ratio of the aggregated to nonaggregated complexes increases at
higher concentrations of Ca2þ which in turn results in the observed
decrease in relaxivity.
The addition of Ca2þ ions to solutions containing YL4
increases the hydrodynamic volume even more than in the case
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Table 2. Protonation Constants of GdL1ꢀGdL4 Complexes
GdL1
GdL2
GdL3
GdL4
log KH1
log KH2
6.12 ( 0.01
4.61 ( 0.01
6.60 ( 0.01
5.17 ( 0.01
6.96 ( 0.01
4.80 ( 0.01
7.11 ( 0.03
5.87 ( 0.03
In the case of EuL1, the 31P{1H} NMR spectrum measured at
298 K displays three resonances, two major signals at 3.1 and
102.3 as well as a minor one at 109.6 ppm. The lanthanide
induced shift decreases rapidly with the number of bonds and the
distance between the Ln3þ ion and the nucleus under study,
respectively. Thus the presence of the two strongly upfield
shifted resonances suggests an interaction of phosphonate
groups with the paramagnetic Eu3þ while the signal at 3.1
ppm indicates the existence of phosphonate species which are
not coordinated to the paramagnetic center. This allows the
conclusion that one of the two phosphonate groups of the
aminobis(methylenephosphonate) arm is interacting with the
Eu3þ center in EuL1.
The two upfield shifted signals merge into one broadened peak
when the temperature is increased to 320 K, which can be
explained by the existence of two different coordination isomers
at lower temperatures.22,44 Moreover, when the temperature is
further increased, the signals of the free and the coordinated
phosphonates keep broadening and move closer to each other
demonstrating an equilibrium between coordinated and non
coordinated phosphonates. Unfortunately, from these data it
cannot be concluded whether there is only one species in solution
having one coordinated and one uncoordinated phosphonate
group which interchange or if there is also a certain amount of
complexes whose phosphonates are both non coordinated. Since
the diffusion measurements indicated a weak tendency to form
aggregates at higher complex concentrations, as are required for
the 31P{1H} NMR measurements, an intermolecular interaction
cannot be excluded, as well. However, after the addition of 3 equiv
of Ca2þ, the 31P{1H} NMR spectrum of EuL1 shows a signal at
3.9 ppm and still two upfield shifted resonances, now at ꢀ104.3
and ꢀ131.6 ppm, respectively. Since the addition of Ca2þ affects
the chemical shift of the signals corresponding to coordinated
phosphonate groups, the aminobis(methylenephosphonate)
group of LnL1 must be able to interact with Ca2þ while one
Figure 5. Luminescence emission spectra of EuL1 (a) and EuL4 (b) in
the presence and absence of 3 equiv of Ca2þ (5 mM, λex = 395 nm,
25 ꢀC, pH 7.3, HEPES).
the phosphonate (see above). Corresponding titration curves are
depicted in the Supporting Information. According to these
protonation constants, the complexes of GdL1ꢀ4 are partially
protonated at physiological pH on one of the phosphonate
groups, with the molar fraction of the protonated species varying
from a few percent for GdL1 to ∼30% for GdL4. It should be
noted that the nitrogen of the aminobis(phosphonate) group
likely remains protonated until a pH above 12, as previously
reported for other aminobis(methylenephosphonic acids).26
Luminescence Spectroscopy. Luminescence emission spec-
tra of EuL1ꢀEuL4 in aqueous media give further details about
their coordination environment. An advantage of this technique
is the possibility to measure at much lower complex concentra-
tions than that required for 31P{1H} NMR spectroscopy. This
allows observing concentration dependent changes. For EuL1,
the coordination of a phosphonate group in the absence and
presence of Ca2þ could be confirmed for all measured complex
concentrations (5 mMꢀ50 mM). The appearance of a distinctive
band at 625 nm and the form and splitting of the ΔJ = 1 manifold
suggest coordination of the europium with the phosphonate
group45,46 (Figure 5a). Since the coordination can be observed
also at low complex concentrations where the diffusion measure-
ments indicated no significant aggregation, it is much more
likely that the coordination to the lanthanide(III) ion is an
phosphonate group is still coordinated to the Ln3þ
.
Potentiometric Titrations. Generally, phosphonic acids first
protonate at slightly acidic pH (log KH1 = 5ꢀ7) while the second
protonation occurs at considerably lower pH (log KH2 e 2). The
studied Gd3þ complexes possess four protonation sites on the
two phosphonate groups. Because of the low stability of Gd3þ
complexes at very acidic pH, the titrationscould notbe carried out
at pH < 4; therefore, the two more acidic protonation constants
could not be determined (Table 2). Both protonation constants
determined for each system lie in the pH range 4.6ꢀ7.1. It can
therefore be expected that one protonation step occurs on each of
the phosphonates. A general trend is an increase of the proton-
ation constant with the increasing length of the linker between the
macrocycle and the bis(phosphonate) group. This can be ex-
plained by a changeof the electron densityon the tertiary amine in
the proximity of the phosphonates, which influencestheir basicity.
Note that the increase of the protonation constant is more
pronounced from GdL1 to GdL2 than for the further steps. This
can be related to the fact that the metal coordination of the
phosphonate in GdL1 also contributes to the increased acidity of
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Table 3. Luminescence Emission Lifetimes and Estimated q
Values of EuL1ꢀEuL4 (4ꢀ5 mM) in the Absence and Pre-
sence of Ca2þ (3 equiv)
complex
q
complex þ Ca2þ
q
EuL1
EuL2
EuL3
EuL4
1.7
1.0
1.4
0.8a
EuL1 þ 3 equiv of Ca2þ
EuL2 þ 3 equiv of Ca2þ
EuL3 þ 3 equiv of Ca2þ
EuL4 þ 3 equiv of Ca2þ
1.2
0.7
0.4
0.4a
a ref. 18
intramolecular rather than an intermolecular process. In this case
only one of the phosphonates is available to interact with Ca2þ
which might explain the smaller and less stable aggregates. None
of the luminescence emission spectra of EuL2ꢀEuL4 exhibit
such a pattern, neither in the absence nor in the presence of Ca2þ
indicating that the phosphonate groups of these complexes are
not coordinated to the lanthanide (Figure 5b).
Figure 6. Variable temperature reduced 17O transverse (9) and long-
itudinal (2) relaxation rates for (a) GdL1, (b) GdL2, (c) GdL3, (d)
GdL4. B = 11.75 T. q = 1 in all cases.
The hydration number q of EuL1ꢀEuL3 was calculated from
the luminescence lifetimes in H2O and D2O solutions using the
equation reported (Table 3),47 and the resulting values were
compared to those previously obtained for EuL4.18 For EuL1, the
only complex with a coordinated phosphonate group, a q value of
1.7 is obtained at 5 mM complex concentration in the absence of
Ca2þ, suggesting the presence of dihydrated species. Since a
coordination number of 10 is unlikely for a Eu3þ complex, it is
indeed possible that a certain amount of species with two
noncoordinated phosphonates exists in solution. However, the
applied equation is empirical, working already for systems with
no second sphere only with an error of (0.5. Furthermore, the
obtained q value might be increased by the influence of additional
oscillator(s) brought into the proximity to Eu3þ by the coordina-
tion of the aminobis(methylenephosphonate) group which is not
considered in the calculation of q.48 Unfortunately it is difficult to
estimate the amount of the different influences in this system. By
contrast, for EuL2ꢀEuL4, the q values are 1 rather than 2 in the
absence of Ca2þ ions. This does not disagree with the fact that no
experimental evidence of a coordination of phosphonates to the
paramagnetic center is found, since it is known that nonpolar
moieties attached to the fourth cyclen nitrogen can reduce the
overall coordination number in water.49
the obtained q values explains the observed decrease in r1 for
GdL2ꢀ GdL4, although the relation between q and r1 is not linear.
This is most obvious in the case of LnL1 were a decrease of 0.5 in q
results in hardly any r1 change at a complex concentration of
5 mM. In contrast to this, for EuL4 q decreases by only 0.4 whereas
the r1 of GdL4 for the same concentration decreases from
5.54 mMꢀ1 sꢀ1 to 3.86 mMꢀ1 sꢀ1 (ꢀ30%).
The hydration number was also determined as a function of
the complex concentration (see Supporting Information), and
similar trends were observed as for the concentration-dependence
of the relaxivities (Figure 3). In the systems where r1 was
independent of the Gd3þ complex concentration, q also re-
mained unchanged. However, in all cases were r1 decreased with
increasing complex concentration, the hydration number also
dropped for the respective Eu3þ complexes. Since the decrease in
relaxivity is accompanied with the formation of aggregates, it is
assumed that in the so formed structures the water access to the
inner and/or second sphere of the lanthanide ion is significantly
hindered. The lower number of water molecules being able to interact
with the paramagnetic center, is then reflected in the reduced
relaxivity. Similarly, Rojas-Quijano et al. reported a decrease of q with
increasing complex concentration for the phosphonate containing
LnPCTA-(ampOBu)3 which was related to oligomer formation.50
17O NMR Spectroscopy. It is a common approach to assess
parameters characterizing the water exchange and rotational
dynamics on Gd3þ complexes by determining their transverse
and longitudinal 17O NMR relaxation rates as well as chemical
shifts at variable temperatures. We performed the measurements
at 11.7 T on the aqueous solutions of GdL1ꢀ4 at concentrations
of about 50 mM. However, the formation of aggregates is likely
temperature dependent, thereby possibly altering the investi-
gated system during the measurements at different temperatures.
This is obvious in the case of GdL4 where the 1/T2r values at high
temperature start to increase (see Figure 6d). Since here the
temperature influence on the aggregation state is already re-
flected in this unusual behavior, we did not perform any further
analysis of the 17O NMR data on GdL4. For complexes GdL1,
GdL2, and GdL3 (Figure 6a, 6b, 6c, respectively) no unusual
behavior was observed. In all three cases the reduced 17O
The addition of 3 equiv of Ca2þ to solutions of EuL1ꢀEuL4
reduce q for all four complexes (Table 3). Since the amino
nitrogen of the aminobis(methylenephosphonate) groups is pro-
tonated at the given conditions, this NꢀH or NꢀD moiety is one
of these additional oscillators which can influence the luminescene
lifetimes and the derived q values. However, it has been shown by
pKa measurements for severalaminobis(methylenephosphonates)
that the amino nitrogen is not involved in the coordination of
Ca2þ ions (deprotonation of NꢀH in the calcium complex occurs
only at pH above 10).26 It is therefore very unlikely that the
observed change in q is due to a change in the NꢀH oscillator. On
the other hand, this means that even though the apparent q value
might not in all cases represent the exact number of coordinated
water molecules in the inner-sphere of these systems, the reduc-
tion of the apparent q values must be related to a decreasing
number of OꢀH and OꢀD oscillators, respectively. This can arise
from a lower number of inner-sphere water molecules, as well as
from a reduction of second sphere water on the aminobis-
(methylenephosphonate) groups or a deprotonation of partly
protonated phosphonate groups. In all cases the reduction in
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transverse relaxation rates (1/T2r) decrease with increasing
temperature in the whole temperature range investigated, in-
dicating that they are in the fast exchange region. The experi-
mental data have been fitted to the SolomonꢀBloembergenꢀ
Morgan theory of paramagnetic relaxation using the equations
presented in the Supporting Information. However, since the
temperature influence on the aggregation state remains unclear
and also the calculation of the exact hydration number is difficult
in these systems, the reliability of the calculated parameters has to
be challenged. Therefore, the parameters obtained from these
measurements are not discussed here in detail, but are given in
the Supporting Information.
displays no calcium dependence. Concentration-dependent re-
laxivity and diffusion measurements show that calcium-induced
aggregation is responsible for the observed changes in relaxivity.51
GdL1 has a lower tendency to form aggregates in the presence of
calcium than GdL2ꢀGdL4, resulting in a calcium dependent
relaxivity change only at high complex concentrations. This is
attributed to a different coordination behavior of the phosphonate
groups in GdL1 as compared to GdL2ꢀGdL4: 31P NMR
spectroscopy and luminescence measurements on the europium
analogues demonstrated a coordination of one phosphonate
group to the lanthanide ion for GdL1, but not for GdL2ꢀGdL4.
For GdL2ꢀGdL4, this likely allows the involvement of both
phosphonate groups in the calcium-induced aggregate formation,
resulting in larger and more stable aggregates. The formation of
dimers and the metal-induced formation of larger aggregates is a
well-described phenomenon for aminobisphosphonates.26,35
Such aggregation has an impact on the water access to the inner
and/or the second sphere of the lanthanide complexes, which is
reflected by a reduction in the apparent hydration number q.
Structural models have been proposed for these interactions
involving intermolecular metal bridges between phosphonate
oxygens and amino groups.26 We should note that the increase
of the rotational correlation time upon Ca2þ addition is likely not
enough to lead to any detectable change in the proton relaxivities
at the high magnetic field used in this study.
Independent from the assumed q value (i.e., q = 1 or q = 2 for
GdL1 and q = 1 or q = 0.7 for GdL2 and GdL3) the calculatedwater
exchange rates obtained from the fittings are very high. They are in
the same order of magnitude as for the [Gd(H2O)8]3þ aqua ion
298
(kex
= 8 ꢁ 108 sꢀ1). In general, DOTA- or DO3A-type
complexes have much slower water exchange rates, though one
example of a very fast water exchange has been reported on the
Gd3þ complex of a DO3A ligand bearing an N-linked
CH2CH2NHCO-pyridyl moiety (kex298 = 1.1 ꢁ 108 sꢀ1).49 For
LnL1 the phosphorus NMR data points to a main fraction of
molecules with a coordination of one phosphonate group to the
lanthanide, which implies an overall coordination number of nine.
For that nine-coordinate species, the fast exchange can be related
to the presence of the coordinated phosphonate group. Indeed, a
coordinated phosphonate group has been often recognized to
induce fast water exchange on Gd3þ complexes.21 Here, the water
exchange might be further accelerated by the equilibrium between
species with a coordinated and species with a noncoordinated
phosphonate group. If more than one species is present, the water
exchange rate determined from the 17O T2 data is an averaged
value from the two species. In GdL2 and GdL3, we assume an
overall coordination number of eight with the participation of
seven donor groups of the ligand and one inner sphere water
molecule for q = 1 or even partly 7-fold coordination in the case of
q = 0.7 (as obtained for higher complex concentrations). Since the
common coordination numbers for Gd3þ are eight or nine, this
would allow for a strongly associative water exchange.
Despite the uncertainty accompanied with a likely tempera-
ture-dependence of the aggregate formation, variable tempera-
ture 17O NMR measurements indicate an extremely high water
exchange rate kex298 for these complexes, being in the same order
of magnitude as that of the [Gd(H2O)8]3þ aqua ion. Both GdL3
and GdL4 show a relaxivity increase upon decrease in Ca2þ
concentration (at low GdLn concentration), which has not been
reported so far for a gadolinium-based Ca2þ responsive SCA. In
terms of potential MRI applications, this behavior would be
favorable for tracking the changes in the extracellular Ca2þ
.
Namely, its concentration drops during the neuronal activity,52
and such changes could be followed by the increase in the
relaxivity of GdL3ꢀ4 and thus the increase in the resulting MR
signal. However, the previous in vivo study on GdL4 indicated
that this intricate mechanism which is responsible for the
relaxivity change is impeded in the living rat brain.18 The
performed studies and their results demonstrate that structurally
relatively simple lanthanide complexes might have unexpected
and complicated solution chemistry behavior. Nevertheless, the
quest for responsive SCAs can be further enriched with such
novel approaches and the resulting complexes.
The rotational correlation time τRO298, as obtained from the
analysis of the longitudinal 17O relaxation rates for GdL1, is
similar to that calculated from the NMR diffusion experiments
for the Y3þ analogue. It is longer than τRO298 of GdDOTA which
can be explained in terms of a less compact structure brought by
the side-chain, a high number of water molecules hydrogen-
bound to the bisphosphonate group and a certain amount of
aggregated species at this complex concentration. The rotational
correlation times of GdL2 and GdL3 depend on the q assumed,
but in any case, they are longer than that for GdL1, which is
indicative of stronger aggregation. For GdL1, we have also
measured the longitudinal 17O relaxation rates in the presence
of 3 equiv of calcium. The 1/T1r values were higher than in the
absence of Ca2þ, in accordance with a longer rotational correla-
tion time affirming the calcium-induced aggregation.
’ ASSOCIATED CONTENT
Supporting Information. Relaxometric Ca2þ titration
S
b
data of GdL1ꢀGdL4; 31P NMR spectra of EuL1 and EuL4;
luminescence emission spectra of EuL2 and EuL3; concentration
dependent emission lifetimes and estimated q values of Eu-
L1ꢀEuL4 in the absence and presence of 3 equiv of Ca2þ
;
potentiometric titration curves of GdL1ꢀGdL4; temperature
dependent 1H T1 relaxation rates of GdL4; temperature depen-
dent 17O T2 relaxation times of GdL4; equations used in the
’ CONCLUSION
We have synthesized a series of novel DO3A-type gadolinium
complexes, provided with alkylaminobis(methylene-phos-
phonate) moieties for interaction with Ca2þ. At low complex
concentrations (2.5 mM), the longitudinal relaxivity of
GdL2ꢀGdL4 decreases after addition of Ca2þ, whereas GdL1
analysis of the 17O NMR data; parameters obtained from the 17
O
NMR data of GdL1ꢀ3; variable temperature reduced longitudinal
and transverse 17O relaxation rate and chemical shift data of
GdL1ꢀGdL4. This material is available free of charge via the
Internet at http://pubs.acs.org.
6480
dx.doi.org/10.1021/ic1024235 |Inorg. Chem. 2011, 50, 6472–6481

Inorganic Chemistry
ARTICLE
’ AUTHOR INFORMATION
(22) Kubicek, V.; Rudovsky, J.; Kotek, J.; Hermann, P.; Elst, L. V.;
Muller, R. N.; Kolar, Z. I.; Wolterbeek, H. T.; Peters, J. A.; Lukes, I. J. Am.
Chem. Soc. 2005, 127, 16477–16485.
Corresponding Author
*Phone:þ497071601 916(G.A.), þ49707129 76229 (H.A.M.).
Fax: þ49 7071 29 2436 (H.A.M.). E-mail: goran.angelovski@
tuebingen.mpg.de (G.A.), hermann.mayer@uni-tuebingen.de
(H.A.M.).
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Author Contributions
^These authors equally contributed to this work.
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