Synthesis and characterization of dinuclear heterometallic lanthanide complexes exhibiting MRI and luminescence response

Article abstract of DOI:10.1039/b925556g

A molecule bearing a macrocyclic DOTA-type chelator and an acyclic chelator based on the 5-aminoisophthalamide diethylenediaminetetraacid (5A-PADDTA) was synthesized by linking these two moieties via an amide bond. The ligand has the possibility to complex two identical or different lanthanide ions, depending on the desire for its potential application. Luminescence studies involving titrations of the Eu³⁺ or Gd³⁺ complex with Tb ³⁺ confirm the formation of heterometallic complexes, as well as the presence of different species in the solution. Comparative ¹H NMR spectra of the ligand, its Eu³⁺ complex, and that containing both Eu³⁺ and Tb³⁺ proves the existence of respective monometallic or bimetallic species. NMR diffusion measurements on 5A-PADDTA as a model compound indicate the formation of aggregates upon the addition of Y ³⁺ (chosen as a diamagnetic analogue of lanthanide ions). Hydration values were calculated from the respective luminescence lifetime values. They show the dominance of a q = 1 species for both ions in monometallic complexes, or q = 1 and q = 2 species of ions in aggregated complexes, for DOTA and 5A-PADDTA chelators, respectively.

Full text of DOI:10.1039/b925556g

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PAPER
www.rsc.org/dalton | Dalton Transactions
Synthesis and characterization of dinuclear heterometallic lanthanide
complexes exhibiting MRI and luminescence response
Ilgar Mamedov,a Tatjana N. Parac-Vogt,b Nikos K. Logothetisa,c and Goran Angelovski*a
Received 3rd December 2009, Accepted 20th April 2010
First published as an Advance Article on the web 19th May 2010
DOI: 10.1039/b925556g
A molecule bearing a macrocyclic DOTA-type chelator and an acyclic chelator based on the
5-aminoisophthalamide diethylenediaminetetraacid (5A-PADDTA) was synthesized by linking these
two moieties via an amide bond. The ligand has the possibility to complex two identical or different
lanthanide ions, depending on the desire for its potential application. Luminescence studies involving
titrations of the Eu3+ or Gd3+ complex with Tb3+ confirm the formation of heterometallic complexes, as
well as the presence of different species in the solution. Comparative 1H NMR spectra of the ligand, its
Eu3+ complex, and that containing both Eu3+ and Tb3+ proves the existence of respective monometallic
or bimetallic species. NMR diffusion measurements on 5A-PADDTA as a model compound indicate
the formation of aggregates upon the addition of Y3+ (chosen as a diamagnetic analogue of lanthanide
ions). Hydration values were calculated from the respective luminescence lifetime values. They show the
dominance of a q = 1 species for both ions in monometallic complexes, or q = 1 and q = 2 species of
ions in aggregated complexes, for DOTA and 5A-PADDTA chelators, respectively.
for the lanthanide ion,11,12 or consisted of two chelating units of
Introduction
the same or different type, consequently bearing same or different
Ln3+.13-18 Alternatively, some bimetallic lanthanide complexes were
used as luminescent sensors for the recognition of cationic or
anionic analytes.19-22
Heteronuclear lanthanide complexes have gained an increased
level of interest recently, due to their potential for application
in various molecular imaging techniques. They appear the most
rational choice to be employed as agents in multimodal imaging
approaches.1-2 Namely, due to their versatile physicochemical
properties, they are widely used in MRI3 or luminescence imaging.4
Gd3+ complexes are very efficient MRI agents,3,5 though only the
ligands that form kinetically stable complexes with Gd3+ can be
employed.6 Luminescent lanthanide complexes based on Eu3+ and
Tb3+ are well suited for optical imaging in the visible region,7
whereas Nd3+, Yb3+ or Er3+ emit in the near infrared region allow-
ing for better sensitivity, reduced light scattering and increased
tissue depth crossing.8 However, the luminescent ions require
a sensitising unit (also called an antenna) to be incorporated
into the ligand which will enable the indirect excitation of the
ion and produce the desired emission.7 The potential application
of these agents varies with the different types of dual assays in
biochemistry, like a lab-on-a-chip assay, detection of two or more
analytes, or to carry out simultaneous microscopy and whole body
imaging.9,10
Following the principles described above, we sought a ligand
containing two different chelators where the antenna acts not only
as a linker between these two chelators, but also as an integral
component in one of their structures. This complex containing
two different lanthanide ions could display interesting ‘hybrid’
properties of the separate ions. Depending on the choice of the
ion (Gd3+, Tb3+, Eu3+, Nd3+, Yb3+ or Er3+), the system could act as
a potential dual-modal (MRI/Vis or NIR luminescence imaging)
or dual-emissive (luminescence imaging at various wavelengths in
Vis/NIR region) contrast agent.
In this work, we report the synthesis and characterization
of a novel ligand L (Fig. 1) which possesses two chelating
moieties, a cyclic chelator DOTA and an aryl-containing acyclic
chelator 5A-PADDTA (abbreviated from 5-aminoisophthalamide
diethylenediaminetetraacid) linked via an amide bond to the 5-
amino position of the isophthalamide moiety. L will form a
kinetically stable complex with Ln3+ via the macrocylic moiety,
whereas 5A-PADDTA can serve as an additional chelator (for the
second Ln3+), and simultaneously as an antenna for both Ln3+. The
Several lanthanide complexes have been reported recently with
the potential to be used as multimodal agents. Depending on the
approach, the final ligand structure contained a single chelator
aDepartment for Physiology of Cognitive Processes, Max Planck Institute
for Biological Cybernetics, Spemannstr. 38, 72076, Tu¨bingen, Germany.
E-mail: goran.angelovski@tuebingen.mpg.de; Fax: +49 7071 601 919;
Tel: +49 7071 601 916
bDepartment of Chemistry, Laboratory of Molecular Design and Synthesis,
Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001, Leuven,
Belgium
cImaging Science and Biomedical Engineering, University of Manchester,
Manchester, U.K
Fig. 1 The structure of the studied ligand L.
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Scheme 1 Reagents and conditions: a) Cbz-Cl, K2CO3, CH2Cl2–H2O, 88%; b) ethylenediamine, CH3OH, 91%; c) tert-butyl bromoacetate, K2CO3,
CH3CN, 87%; d) H2, Pd/C (10%), C2H5OH, 92%; e) Bromoacetyl bromide, K2CO3, CH2Cl2–H2O; f) TFA:CH2Cl2 (1 : 1); g) DO3A-t-Bu ester, K2CO3,
CH3CN, 70%.
choice of Ln3+ ions which are incorporated in the final bimetallic
complex will in turn determine its final spectroscopic and potential
imaging properties.
Physico-chemical characterization
Two types of complexes were prepared and their properties were
initially investigated by means of luminescence spectroscopy. EuL
was prepared and titrated with Tb3+ in order to explore the
potential for EuLTb to be used as a dual-emissive agent. GdL
was titrated with Tb3+ and the luminescence emission properties
of this complex were also studied. The longitudinal relaxivity (r1)
before and after the addition of Y3+ was recorded in parallel to
the luminescence spectroscopy in order to assess the relaxometric
properties of GdL for its potential application in MRI.
Luminescence steady state emission spectra were recorded using
excitation wavelengths of 267 and 297 nm. Typical emission
maxima of Eu3+ were observed for EuL at 580, 589, 594, 617,
689 and 701 nm showing that the Eu3+ emission is sensitised by
the 5A-PADDTA antenna.
Addition of Tb3+ resulted in the appearance of emission peaks
characteristic of this ion, namely at 489 and 545 nm with the
other two maxima being unresolvable due to their overlap with
the emission maxima of Eu3+. Whilst the Tb3+ centered emission
increased with the addition of its ions, the intensity of Eu3+
centered emission remained constant (Fig. 2). This suggests that
the antenna is able to sensitise both ions in parallel.
The saturation point appears after the addition of approxi-
mately 1.3–1.4 equiv. of Tb3+, which is followed by a decrease
in the Tb3+ centered emission intensity, indicating relatively weak
binding of the Tb3+ with 5A-PADDTA moiety (see below).
The factor analysis revealed the presence of at least two
complexes in solution. Because a satisfactory fit was obtained
for the presence of two species in the solution, further models
assuming the presence of higher number of species were not
considered. According to the analysis, the data were fitted on
the assumption that these two complexes contained one and two
Results and discussion
Synthesis
The ligand L was synthesized in a 7-step procedure starting
from the commercially available dimethyl 5-aminoisophthalate,
1 (Scheme 1). The amino group was protected using benzyl
chloroformate resulting in 2. This diester was converted into the
diamide by treatment with an excess of ethylendiamine to give 3.
Alkylation of 3 with tert-butyl bromoacetate in the presence of
K2CO3 resulted in 4. The Cbz protecting group was reductively
cleaved by hydrogenation using 10% Pd on charcoal as a catalyst
producing 5. Tetraacid 5a was obtained by the hydrolysis of the
tert-butyl esters in a solution of TFA:CH2Cl2 (1 : 1) to give the
model compound for the NMR diffusion experiments (see bellow).
5 was also treated with bromoacetyl bromide to give 6. Tris-tert-
butyl-DO3A was alkylated with 6 in the presence of K2CO3 and
compound 7 was obtained. All tert-butyl ester protecting groups
were hydrolyzed in one step using a solution of TFA:CH2Cl2 (1 : 1).
The final ligand L was recrystallised from a mixture of methanol–
diethyl ether followed by recrystallization with water/acetone and
was obtained as a light brown hygroscopic powder.
Complexation of L with Eu3+ and Gd3+ was performed by
the addition of an equimolar amount of EuCl3 or GdCl3 to
the aqueous solution of L. Formation of a white precipitate
immediately followed the addition of lanthanide ion. The solution
was neutralized with NaOH and the precipitate was re-dissolved
after stirring for several hours at 60 ◦C.
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intensity due to the quenching effect of the coordinated water
molecules.
Similar behavior in the luminescence spectra of GdL was
observed upon the titration with Tb3+. Peaks at 489, 545, 584
and 621 nm typical for Tb3+ centered emission were observed
along with similar changes in the emission intensity with the
addition of Tb3+ (Fig. 3). Given that no interaction of the Eu3+
or Gd3+ (positioned in the macrocycle) could be observed with the
acyclic 5A-PADDTA chelator (see above), it is expected that the
interaction of Tb3+ with EuL or GdL occurs in the same manner,
leading to the formation of the same types of species in solution
with comparable stabilities of the respective complexes. One can
note that the only difference in the luminescence of EuL and GdL
is the stronger emission of Tb3+ in GdL. This can be explained
either by the sensitisation of two lanthanide ions in the case of
EuL, compared to only one emissive ion — Tb3+ — as in the case
of GdL, or by transfer of energy from the antenna to Eu3+ via Tb3+
(see below).
Fig. 2 (a) Changes in the luminescence emission spectrum of EuL (35 mM,
HEPES buffer, pH 7.4, lex = 297 nm) and (b) changes of the peak intensity
at 545 nm (right) upon the addition of Tb3+
.
Tb3+ ions, and their formation is characterized by the equilibrium
constants b11 and b12 respectively. The evolution of the emission
spectra can be fitted with a relatively high degree of accuracy to
the proposed model, leading to the affinity constants log K11
5.88 and log K12 = 4.79 where b11 = K11 and b12 = K11K12.
=
The obtained affinity constants confirmed the relatively weak
binding of Tb3+ with 5A-PADDTA moiety in both types of
complexes. A 1 : 1 complex EuLTb has slightly higher affinity
constant than the other type of formed species which is probably
due to the more preferred interactions of the two aliphatic chains
coupled to the single aromatic moiety. However, the presence of
additional Tb3+ ions in the solution moves the equilibrium towards
a species where one Tb3+ ion could also interact with aliphatic
chains of two different aromatic moieties. Such interaction results
in formation of additional species where various ratios could be
envisaged. The first consequence is certainly an increase in the
size of these species in comparison to EuLTb, which is indeed
confirmed on the model compound in NMR diffusion experiments
(see bellow). It is also notable that the emission intensity reduces
on further addition of Tb3+ beyond the saturation point. This effect
may be explained by a stronger emission of EuLTb in comparison
to a newly formed species where Tb3+ presumably has higher
hydration number (see below), which in turn reduces the emission
Fig. 3 (a) Changes in the luminescence emission spectrum of GdL (35 mM,
HEPES buffer, pH 7.4, lex = 297 nm) and (b) changes of the peak intensity
at 545 nm upon the addition of Tb3+
.
Luminescence lifetimes were determined to assess the hydration
states of the emissive ions and provide additional information
about their coordination environment. The emissive lifetimes (t)
were obtained in water and deuterium oxide, and the number of
inner sphere water molecules (q) was calculated from a general
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Table 1 Emission lifetimes and estimated q values of EuL (35 mM) upon
the addition of Tb3+ (lex = 297 nm, lem = 700 nm, HEPES buffer, pH 7.4)
broad with multiple peaks observed over the range -20 to 35 ppm.
The presence of multiple peaks is consistent with the occurrence
of interconverting isomers, characteristic of the Ln3+ complexes of
DOTA derivatives.24,25 Titration of Eu3+ into a solution of L was
Compound
t/ms H2O
t/ms D2O
q
1
also followed by H NMR spectroscopy and the results showed
EuL
0.63
0.67
0.70
0.71
2.24
2.28
2.23
2.20
1.1
1.0
0.9
0.8
EuL + 25 mM Tb3+
EuL + 50 mM Tb3+
EuL + 75 mM Tb3+
a gradual broadening of aliphatic resonances of the ligand with
increasing concentrations of Eu3+, as a result of its complexation.
The 1H NMR spectrum of the EuLTb complex contained few weak
resonances that were very broad, due to the high paramagnetic
relaxation caused by the presence of the Tb3+ ion. Although,
due to the strong paramagnetic nature of the compound, no
structural data could be extracted from the NMR spectra, the
large broadening of all resonances in EuLTb as compared to EuL
is consistent with complexation of Tb3+ to the EuL moiety.
relation expressed in eqn (1), where A = 1.2 and B = 0.25 for
Eu3+.23
q = A× t−1O −tD−1 −B
(1)
(
)
H
O
2
2
Two different sets of data were collected when EuL is titrated with
Tb3+, sensitising the emission of both Eu3+ and Tb3+ at 297 nm.
Tb3+ centered emision was recorded at 545 nm, whereas Eu3+
centered emission was recorded at 700 nm in order to avoid any
1H pulsed gradient spin echo (PGSE) NMR diffusion
measurements
7
7
interference between the Eu3+ (5D0→ F1, 7F2) and Tb3+ (5D4→ F3,
7F4) emission bands. Data was collected in the absence of Tb3+,
and at Tb3+ concentrations of 25, 50 and 75 mM, these correspond
to EuL:Tb3+ ratios at half the intensity, the maximum intensity and
after the decrease in the luminescence intensity of Tb3+ (Fig. 2).
The calculated value of q = 1 for the Eu3+ species is consistent
with that frequently observed for eight coordinate DOTA-type
complexes (where one carboxylate group is replaced with an
amide). Furthermore, the q value did not change upon addition
of Tb3+ (Table 1). Unlike Eu3+, the lifetimes measured for the Tb3+
centered emission exhibited differences dependent on whether it
was titrated with EuL or GdL. The obtained Tb3+ lifetimes in both
H2O and D2O solutions of EuL decrease upon its addition. For
GdL, the Tb3+ lifetimes decreased in H2O but remained constant
in D2O. Furthermore, the respective Tb3+ lifetime values in H2O or
D2O for EuL were smaller than those obtained for GdL suggesting
the existence of a quenching mechanism other than that induced
by O–H oscillators, which could be attributed to the existence of
energy transfer between Tb3+ and Eu3+. Therefore the assessment
of the hydration state of Tb3+ would lead to unreliable values.
However, the reduction of the emission lifetime in H2O for Tb3+
on its increasing addition to GdL, would suggest the formation
of a species with a greater degree of hydration. This is in good
agreement with the previous observation that more than one type
of species exists in solution (see above) and that addition of Tb3+
beyond one equivalent of EuL/GdL should lead to the formation
of less stable (and more hydrated) species.
As mentioned previously in the synthesis section, addition of
increasing amounts of lanthanide ion resulted in the formation of
a precipitate. Fitting of the luminescence steady-state and lifetime
data indicated the existence of a second type of species. The
occurrence of both effects can be attributed to the formation of
dimers and further types of aggregates as they i) will most likely
have lower solubilities than the monomers, ii) will probably be a
significant component in the luminescence emission properties
of EuL/GdL. In order to prove that the properties described
above are the result of the formation of aggregates between the
lanthanide ions and the 5A-PADDTA moiety, we have performed
PGSE NMR experiments.
⎛
⎜
⎝
⎞
⎟
⎠
I
d
3
2
2
⎟
⎜
ln = − gd D Δ−
G
⎟
(2)
(
)
⎜
⎟
I0
The diffusion coefficient D is known to be very sensitive to
alterations in the molecular shape and size, thus any changes
to GdL in solution will be clearly reflected in its value. It is
well known that D decreases in the case of the agglomeration of
complexes.26-28 Therefore, to simplify the determination of D and
prove the possibility of the agglomeration processes occurring,
the ligand 5a was used as a model compound for the PGSE NMR
diffusion measurements. The titration was performed using Y3+ (as
a diamagnetic substitution for the lanthanides) and the diffusion
coefficient D was determined during the addition of up to one
equivalent of Y3+ using eqn (2).
Finally, the determination of longitudinal relaxivity r1 of GdL
was performed in the absence and the presence of Y3+, (a
diamagnetic analogue of the lanthanide ions). The r1 value of
GdL was calculated to be 5.48 mM-1s-1 which does not change
upon the addition of an equimolar amount of Y3+. This result is
in good agreement with the previous finding that no change in q
of Eu3+ in EuL occurs upon the addition of Tb3+.
As anticipated, any further addition of Y3+ beyond this point
leads to the formation of a precipitate which can negatively affect
the results obtained in the experimental measurements.
The D values were obtained by plotting the logarithmic ratios of
the resonance intensities in the presence and absence of the pulsed-
field-gradient versus the square value of the pulsed-field-gradient.
The slope of the fitted line obtained is directly proportional to
D (Fig. 4). The hydrodynamic radius rH can be determined using
the D value which, for spherical molecules is related to D via the
Stokes-Einstein equation (eqn (3)), where kB is the Boltzmann
constant, T is the absolute temperature, and h is the viscosity.
Finally, the viscosity independent hydrodynamic volume VH is
often used as a parameter to quantify the aggregation processes
(Table 2).29
1H NMR spectroscopy of L, EuL and EuLTb
1
The H NMR spectrum of the ligand L, was recorded in D2O
and the resonances were assigned with the aid of 2D COSY
experiments. Complexation of L to Eu3+ caused significant changes
in the 1H NMR spectrum. In comparison to the resonances of the
ligand (sharp and well separated), those of the Eu3+ complex were
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Table 2 Values of diffusion coefficients D of 5a at various concentrations
also forms complexes with lanthanides and their existence is
confirmed by the means of luminescence and NMR spectroscopy.
Studies show that structural improvements are required to develop
an analogous agent that will be able to bear two different
lanthanide ions with a sufficient kinetic and thermodynamic
stability for the potential for application in vivo. The investigated
structure, however, demonstrates one possible route towards the
development of efficient dual-emissive and dual modal agents.
of Y3+ (300 MHz, 25 ◦C, pH 7.3, HEPES)
Compound
D
rH/Aa
VH/A3b
5a
3.50 ¥ 10-10
3.16 ¥ 10-10
2.78 ¥ 10-10
2.37 ¥ 10-10
2.25 ¥ 10-10
5.6
6.2
7.0
8.2
8.6
736
998
1437
2310
2664
5a + 0.25 equiv. Y3+
5a + 0.50 equiv. Y3+
5a + 0.75 equiv. Y3+
5a + 1.00 equiv. Y3+
a Calculated from eqn (3) using h = 1.13 ¥ 10-3 Pa s for D2O. b Calculated
Experimental
4
from rH whereV = prH3
H
3
General remarks
All chemicals were purchased from commercial sources and were
used without further purification. Dimethyl 5-aminoisophthalate
was purchased from Aldrich (Cat. No. D142158). Column chro-
matography was performed using silica gel 60 (70-230 mesh
1
ASTM) from Merck, Germany. H and 13C NMR spectra were
recorded on a Bruker DRX400 spectrometer at room temper-
1
ature. The relaxometric and the H pulsed gradient spin echo
(PGSE) NMR diffusion measurements were performed on a
Bruker Avance II 300 MHz ‘Microbay’ spectrometer. Lumines-
cence steady-state and lifetime measurements were performed on
QuantaMasterTM 3 PH fluorescence spectrometer from Photon
Technology International, Inc., USA. ESI-LRMS were performed
on a ion trap SL 1100 system (Agilent, Germany). FT-ICR-MS
were performed on a Bruker FT-ICR Apex II spectrometer.
N1,N3-bis(2-aminoethyl)-5-(phenethoxycarbonyloxyamino)-
isophthalamide (2)
Fig. 4 Normalized signal decay as a function of the gradient square for
5a (25 mM) before addition of Y3+ (ꢀ) and upon addition of 0.25 equiv.
Diester 1 (2.09 g, 10 mmol), was dissolved in a solution of
CH2Cl2 (20 mL) and water (40 mL). The solution was treated
with an alternating drop wise addition of a solution of benzyl
chloroformate (1.90 g, 12 mmol) in CH2Cl2 (20 mL), and 3.5 M
K2CO3 aqueous solution with vigorous stirring at rt to maintain
the pH at 6–7. After benzyl chloroformate was added over 0.5 h,
the reaction mixture was stirred for 2 h at pH 7–8. The white
precipitate was isolated by filtration and recrystallized from hot
ethylacetate to give 3.00 g (88%) of 2 as white powder, m.p. 203–
208 ◦C.
1H NMR (DMSO): d 3.80 (s, 6H, CH3), 5.19 (s, 2H, CH2), 7.32–
7.46 (m, 5H, C6H5), 8.05 (s, 1H, C6H3), 8.32 (s, 2H, C6H3), 10.20
(s, 1H, NH). 13C NMR (DMSO): d 52.4 (CH3), 66.1 (CH2), 122.4,
123.3, 128.1, 128.4, 130.6, 136.3, 138.3, 140.1 (C6H3), 153.3, 165.2
(CONH). FT-ICR-MS: for C18H17NO6 calcd. 366.0948 [M + Na]+,
found 366.0950.
(ꢁ), 0.50 equiv. (᭹) and 0.75 equiv. (ꢂ) of Y3+
.
kBT
D =
(3)
6phrH
Indeed, by taking into account the possible formation of
various types of aggregates, and that the formed complexes and
aggregates are not spherical, the presented calculations are only
approximations of the real values and thus can be used only to
verify the trend of 5a to form aggregates when mixed with Y3+.
The obtained values for D and calculated values for rH (and
consequently VH) clearly show the tendency of the formed
complex Y5a to agglomerate with the further addition of Y3+.
The continuous decrease of D and increase of rH and VH with
the increasing concentrations of Y3+ indicates that formation of
aggregates certainly takes place in the case of 5a, which in turn
should be also valid for systems containing the 5A-PADDTA
moiety, namely EuL and GdL.
Benzyl 3,5-bis(2-aminoethylcarbamoyl)phenylcarbamate (3)
A suspension of 2 (3.00 g, 8.75 mmol) in dry methanol (15 mL)
was added drop wise to ethylenediamine (50 mL) at rt. Vigorous
stirring was continued for a further 12 h. Removal of the solvent
left an off-brown solid, which was dried at 50 ◦C in vacuo for 6 h
and left at rt in vacuo for another 24 h. Finally it was recrystallized
from hot ethylacetate to give 3.18 g of 3 (91%) as white solid, m.p.
136–140 ◦C.
1H NMR (CDCl3): d 3.80–4.60 (m, 8H, CH2), 5.19 (s, 2H,
CH2), 7.32–7.46 (m, 5H, C6H5), 8.05 (s, 2H, C6H3), 8.15 (s,
1H, C6H3), 8.32 (s, 2H, NH), 10.15 (s, 1H, NH). 13C NMR
Conclusions
A novel ligand bearing two different chelating moieties was
prepared and heterometallic lanthanide complexes were synthe-
sized and investigated. The macrocyclic, DOTA-type moiety of
this ligand forms a stable complex with Eu3+ and Gd3+ which
exhibit the expected luminescence emission and relaxometric
characteristics, respectively. The acyclic chelator of this ligand
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(CDCl3): d 40.1, 43.5, 67.1 (CH2), 122.4, 123.3, 128.1, 128.4,
130.6, 136.3 138.3, 140.1 (C6H3, C6H5), 153.3, 165.2 (CONH).
FT-ICR-MS: for C20H25N5O4 calcd. 400.1979 [M + H]+, found:
400.1980.
({2-[3-[2-(Bis-tert-butoxycarbonylmethyl-amino)-ethylcarbamoyl]-
5-(2-bromo-acetylamino)-benzoylamino]-ethyl}-tert-
butoxycarbonylmethyl-amino)-acetic acid tert-butyl ester (6)
K2CO3 (0.48 g, 3.5 mmol) was added to a solution of 5 (1.65 g,
2.29 mmol) in CH2Cl2 (50 mL). The reaction mixture was stirred
vigorously and cooled to 0 ◦C in an ice bath. Bromoacetyl bromide
(0.7 g, 3.5 mmol) in dichloromethane (50 mL) was then added
drop wise over a period of 1 h. The reaction mixture was then
allowed to warm to room temperature and stirred for a further
6 h. The reaction was then quenched by the cautious addition
of water (30 mL). Methanol (60 mL) was added to afford a
homogeneous solution and the N-aryl bromoacetamide isolated
by slow evaporation of the solvents at room temperature. The dark
brown viscous oil was used for the next step without purification.
1H NMR (CDCl3): d 1.32 (s, 36H, CH3), 2.72–3.63 (m, 16H,
CH2) 5.06 (s, 2H, CH2), 7.99 (s, 2H, C6H3), 8.33 (m, 1H, C6H3,
2H, NHCO) 10.00 (s, 1H, NHCO). 13C NMR (CDCl3): d 29.8
(CH3), 40.0, 54.0, 57.7, 68.3 (CH2), 82.6 (C(CH3)3), 122.3, 129.9,
130.3, 137.4 (C6H3), 167.9, 168.8 (CONH), 172.9 (COOtBu). ESI-
MS: for C38H60BrN5O11: calcd. 843.3 [M + H]+, found 843.5.
5-Aminoisophthalamide-bis(ethylamine,N,N’bis-methylacetic acid
t-butyl ester) (4)
The bisamide 3 (3.50 g, 8.8 mmol) was dissolved in 50 mL
of acetonitrile. After addition of K2CO3 (5.52 g, 40 mmol) the
solution was stirred for 30 min at rt. t-butyl 2-bromoacetate
(7.72 g, 39.6 mmol) in 50 mL of acetonitrile was added drop
wise and the mixture was stirred for 18 h at 70 ◦C. The inorganic
salts were removed by filtration and the solvent was evaporated
under reduced pressure. The light brown viscous oil was purified
by column chromatography (silica gel, 20% EtOAc in CH2Cl2) to
yield 6.53 g (87%) of 4 as a brown viscous oil.
1H NMR (DMSO): d 1.47 (s, 36H, CH3), 2.87–3.00 (m 8H,
CH2), 3.38–3.43 (m 8H, CH2) 5.22 (s, 2H, CH2), 7.33–7.46 (m, 5H,
C6H5), 8.01 (s, 2H, C6H3), 8.07 (s, 2H, C6H3), 8.15 (s, 2H, NH). 13
C
NMR (DMSO): d 28.5 (CH3), 31.7, 54.1, 57.0, 67.7 (CH2), 82.6
(C(CH3)3), 121.7, 129.1, 129.3, 129.6, 137.1, 138.0, 141.0, 155.6
(C6H3, C6H5), 164.9, 169.3 (CONH), 172.8 (COOtBu). FT-ICR-
MS: for C44H65N5O12 calcd. 858.4241 [M + H]+, found: 858.4243.
[4-({3,5-Bis-[2-(bis-tert-butoxycarbonylmethyl-amino)-
ethylcarbamoyl]-phenylcarbamoyl}-methyl)-7,10-bis-tert-
butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl]-acetic
acid tert-butyl ester (7)
5-Aminoisophthalamide-bis(ethylamine,N,N’bis-methylacetic acid
t-butyl ester) (5)
To the solution of 1,4,7-tri(t-butoxycarbonylmethyl)cyclen (1.00 g,
1.94 mmol) and K2CO3 (0.40 g, 2.9 mmol) in acetonitrile (30 mL) a
solution of 6 (1.96 g, 2.32 mmol) in acetonitrile (20 mL) was added.
After the reaction mixture had been stirred for 16 h at 70 ◦C, the
solution was allowed to cool to rt, filtered and concentrated in
vacuo to give a brown viscous oil. The compound was purified by
column chromatography (silica gel, 5% MeOH in CH2Cl2) to yield
1.70 g (70%) of 7 as yellow oil.
4 (2.13 g, 2.5 mmol) was dissolved in ethanol (50 mL) and
hydrogenation at 30 psi was performed in the presence of 200 mg
of Pd/C (10%) for 24 h. The catalyst was removed by filtration
through celite and the filtrate was evaporated to dryness. A light
brown viscous oil of 5 (1.65 g, 92%) was obtained which was used
for the next step without further purification.
1H NMR (CDCl3): d 1.38 (s, 36H, CH3), 3.37 (br. s, 16H,
CH2), 8.00 (s, 2H, C6H3), 8.07 (s, 2H, C6H3), 8.14 (s, 2H, NH).
13C NMR (CDCl3): d 28.1 (CH3), 38.1 52.3, 55.9 (CH2), 81.5
(C(CH3)3), 115.8, 116.4, 136.0, 146.8 (C6H3), 167.1 (CONH), 171.2
(COOtBu). FT-ICR-MS: for C36H59N5O10 calcd. 722.4334 [M +
H]+, found: 722.4329.
1H NMR (CDCl3): d 1.19–1.23 (m, 63H, CH3), 1.95–2.73 (m
26H, CH2), 3.25–3.48 (m, 16H, CH2), 7.53 (s, 2H, NHCO), 7.90
(s, 1H, C6H3), 8.09 (s, 2H, C6H3), 10.13 (s, 1H, NHCO). 13C
NMR (CDCl3): d 27.7, 27.8, 28.0 (CH3), 38.1, 51.8, 52.7, 53.5,
54.4, 55.4, 55.5, 55.7, 55.8, 56.9 (CH2), 80.9, 81.5, 81.9 (C(CH3)3),
120.9, 122.6, 135.5, 138.7 (C6H3), 166.8 (CONH), 170.8, 172.2
(COOtBu), 172.7 (CONH), 172.8 (COOtBu). FT-ICR-MS: for
C64H109N9O17 calcd. 1276.8013 [M + H]+, found: 1276.7997.
5-Aminoisophthalamide-bis(ethylamine,N,N’bis-methylacetic acid)
(5a)
[4-({3,5-Bis-[2-(bis-carboxymethyl-amino)-ethylcarbamoyl]-
phenylcarbamoyl}-methyl)-7,10-bis-carboxymethyl-1,4,7,10-
tetraaza-cyclododec-1-yl]-acetic acid (L)
Compound 5 (1.65 g) was dissolved in CH2Cl2 (5 mL) and TFA
(10 mL) was added to the solution. After mixture had been stirred
at rt for 12 h, the solvents were removed under reduced pressure.
The excess of TFA was removed by the addition of CH2Cl2 (10 mL)
and its evaporation. This procedure was repeated twice for CH2Cl2
and also twice with methanol. The viscous residues were taken up
in a minimum amount of methanol and cold ether was added drop
wise. The formed precipitate was recrystallized from hot methanol
Compound 7 (2.5 g, 2 mmol) was dissolved in CH2Cl2 (20 mL)
and TFA (20 mL) was added carefully. After the mixture was
stirred at rt for 24 h, the solvent was removed under reduced
pressure. CH2Cl2 (40 mL) was added and evaporated twice to
remove the excess of trifluoroacetic acid. The same procedure was
repeated twice with methanol. The viscous residue was dissolved
in a minimum amount of methanol and added drop wise to cold
ether. The formed precipitates were filtered, washed with cold
ether and dried in vacuo. A light brown hygroscopic powder was
obtained (0.7 g, 68%).
1
to yield 0.931 g (73.5%) of 5a. H NMR (D2O): d 3.44 (m, 4H,
CH2NCO), 3.65 (m, 4H, CH2NCO), 3.96 (s, 8H, CH2COOH), 7.76
(s, 2H, C6H3), 8.00 (s, 1H, C6H3). 13C NMR (D2O): d 35.7 (CH2),
56.4 (CH2COOH), 56.6 (CH2), 125.1, 125.8, 133.2, 135.5 (C6H3),
169.9 (COOH). ESI-MS: for C20H27N5O10 calcd. 498.5 [M + H]+,
found: 498.5.
1H NMR (D2O): d 2.69–3.77 (m, 32H, CH2), 3.92 (s, 8H, CH2),
7.77 (s, 1H, C6H3), 7.85 (s, 2H, C6H3). 13C NMR (D2O): d 29.6,
5726 | Dalton Trans., 2010, 39, 5721–5727
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30.2, 35.3, 48.5, 50.8, 53.6, 55.1, 56.1, 56.4 (CH2), 121.9, 122.8,
134.0, 138.3 (C6H3), 165.7 (CONH), 169.5, 169.8 (COOH), 173.2
(CONH). FT-ICR-MS: for C36H53N9O17 calcd. 440.6707 [M -
2H]2-, found: 440.6706.
Acknowledgements
The authors are thankful to Dr L Charbonnie`re for the help in
the fitting of the data obtained from the steady state emission,
and Prof. H. A. Mayer, Dr M. Placidi, Dr P. Kadjane and Dipl.
Chem. J. Henig for helpful discussions. Financial support of the
Max-Planck Society, the Hertie Foundation, German Research
Foundation (DFG, grant AN 716/2-1) and German Ministry for
Education and Research (BMBF, FKZ: 01EZ0813) is gratefully
acknowledged. This work has been performed within the frame
of the European COST Action D38 “Metal-Based Systems for
Molecular Imaging Applications”.
Preparation of Ln3+ complexes
Complexation with lanthanides was performed by stirring
1.0 equiv. of L with the corresponding lanthanide chlorides (1.0–
1.1 equiv.) in aqueous solutions at 60 ◦C for 24 h at pH 7.0–7.5. The
complexes were finally treated with Chelex 100, filtered through
a 0.2 mm syringe filter and lyophilized. The xylenol orange test
was performed to demonstrate the absence free metal ions in the
solution. The formation of the complexes was confirmed by ESI-
MS spectrometry, where the appropriate molecular ion peaks with
the characteristic isotope pattern of the complexes were present in
the analyzed spectra.
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Dalton Trans., 2010, 39, 5721–5727 | 5727
©