Structure-related variable responses of calcium sensitive MRI probes

Article abstract of DOI:10.1039/c1ob05463e

A new series of Gd³⁺ complexes based on DO3A (GdL ¹-GdL⁴) was synthesized and investigated. They possess side chains with different structures which determine their varying binding properties and response towards endogenou

Full text of DOI:10.1039/c1ob05463e

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Organic &
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PAPER
Structure-related variable responses of calcium sensitive MRI probes
Ilgar Mamedov,a Nikos K. Logothetisa,b and Goran Angelovski*a
Received 23rd March 2011, Accepted 13th May 2011
DOI: 10.1039/c1ob05463e
A new series of Gd3+ complexes based on DO3A (GdL1–GdL4) was synthesized and investigated. They
possess side chains with different structures which determine their varying binding properties and
response towards endogenous metal ions, measured by changes in the longitudinal relaxivity (r1). GdL4
exhibits the highest selectivity toward Ca2+ in comparison to the other complexes, with up to a 63%
increase of the r1. GdL2 and GdL3 also respond to different Ca2+ concentration ranges, however with a
lower selectivity since the r1 changes are also observed in the presence of other cations such as Mg2+,
Zn2+ or Cu2+. Assessment of the hydration number (q) via luminescence lifetime measurements
confirmed that the change in q is responsible for the r1 response for all the complexes.
extracellular Ca2+ acts as “secondary messenger” and plays an
important role in regulating a variety of neuronal processes. The
Introduction
The use of Gd3+ based contrast agents for magnetic resonance
ability to non-invasively observe its fluctuations in concentration
imaging (MRI) has enhanced the potential of this technique and
would be of obvious importance for understanding brain function.
its application in clinical trials and biological research.1 Recent
developments in the chemistry of contrast agents have focused on
the design of new categories which improve the contrast observed
in the MR image.2 One category, responsive or smart MR agents
(SCAs), has become very important in this field of molecular
imaging. These agents are designed to follow extra- or intracellular
activities in vivo, and they are tailored in such a way to increase
or decrease the MR signal intensity only in the presence of a
given ion or molecule or upon the occurrence of a specific event.
The choice of the biochemical target, the chelating moiety and the
physiological/pathological environments are critical deterministic
parameters for the design, development and application of SCAs
in imaging the biological functions.
To this end various structures, mostly Gd3+–DO3A (1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid) macrocyclic moieties,
have been combined with acyclic chelators that can form com-
plexes with the target metal ion, Ca2+. A series of such complexes
which can act as a Ca2+ sensors were developed in our group.8–10
These studies show that the proper choice of the Ca2+ chelator
and binding mode are crucial to achieve the desired selectivity
of the chelators towards Ca2+ against competing cations such as
Mg2+, Zn2+ or Cu2+. Similarly, other groups that focused on the
development of SCA sensitive to Zn2+ or Cu2+, concluded that
even a minor change in the structure of the ligand could have a
dramatic influence on the selectivity and sensitivity of the contrast
agents towards the target metal ion.11,12
Several extensive studies on metal sensitive SCAs have been
reported recently and are the topic of extensive review articles.3,4
The alteration of the MR signal intensity in these molecules
occurs through structural changes induced by a fluctuation in
the concentration of a specific metal ion (e.g. Na+, K+, Ca2+,
Fe2+/Fe3+, Cu2+ or Zn2+) that has a critical functional role in
organisms. Methods that enable the detection of concentration
changes of such molecules in vivo have therefore become of great
importance over the last few years.5,6 For example, developments
of responsive biochemical functional markers that detect neuronal
activity promise direct visualization of neural activation, with
a spatiotemporal resolution and specificity that is likely to be
much greater than that afforded by hemodynamics.7 Specifically,
Consequently, an investigation that explores the different
types of Ca2+ chelators and the various coupling modes to the
paramagnetic centre, would be most useful in furthering the
progress of Ca2+ sensitive SCA. Here we report four Gd3+–
DO3A based molecules that bear polyaminocarboxylates and
polyetheraminocarboxylates as Ca2+ chelators. These two units
are conjugated with side chains of varying length and rigidity
(Scheme 1). Their synthesis and relaxivities as a function of
added endogenous cations and chelator type are investigated and
reported.
Results and discussion
Synthesis of ligands and complexes
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
Several polyaminocarboxylates, such as EDTA (2-[2-[bis(car-
boxymethyl)amino]ethyl-(carboxymethyl)amino]acetic
acid),
EGTA (ethyleneglycol-bis(2-aminoethylether)-N,N,N¢,N¢-tetra-
acetic acid) and pyro-EGTA (1,2-bis(2-aminoethoxy)benzene-
N,N,N¢,N¢-tetraacetic acid) are known to be very efficient Ca2+
bImaging Science and Biomedical Engineering, University of Manchester,
Manchester, UK
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Scheme 1 The structures of studied ligand L1–4
.
chelators as they form complexes with logKCaL ª 10.13 However, in
order to couple these types of chelators to a DO3A reporting unit,
a derivative of these chelators must be used where specific
functional groups are excluded or replaced to enable the coupling
of this type of chelator to the DO3A moiety.8–10 Furthermore,
an alteration of the EDTA, EGTA or pyro-EGTA is required to
reduce the affinity of the chelator towards Ca2+, making it more
sensitive to extracellular Ca2+ concentration ranges (low mM).14
Unfortunately, an intentional decrease in the binding affinity and
simplification of the synthesis can lead to a loss of selectivity
towards Ca2+ over other ions, or a weaker relaxometric response
due to a lower number of functional groups that can both interact
with Ca2+ and Gd3+.
water/acetone, the final ligand L2 was obtained as a colorless
hygroscopic powder.
The first precursor 8 for the synthesis of L3 was prepared
according to a literature procedure.19 The alkylation of tris-tBu-
DO3A with 8 yielded the macrocycle 9 (Scheme 2C). Cleavage
of the phthalimide was achieved by refluxing 9 with a solution
of ethylenediamine in isopropanol. The alkylation of resulting
amine with tert-butyl bromoacetate yielded 10. The hydrolysis of
the tert-butyl esters with TFA resulted in L3 as a yellow oil. All
attempts to recrystallize L3 failed due to its high hygroscopicity.
The product was therefore purified by reverse phase HPLC and
upon the evaporation of the solvents, L3 was obtained as a yellow
hygroscopic powder.
Four different ligands were prepared, and in each case a
unique synthetic strategy (A–D, Scheme 2) was applied for their
preparation, to allow each side chain to be effectively coupled to
the macrocyclic moiety.
The first molecule in the synthetic route of L4 was
the aryl-containing compound 11 (Scheme 2D). It was ob-
tained through the alkylation of the commercially avail-
able 2-(2-hydroxyethoxy)phenol with N-benzyloxycarbonyl-2-
bromoethylamine, according to a literature procedure.20 The
alcohol in 11 was brominated in a biphasic solution of thionyl
bromide in cyclohexane and the resulting product was used in
the alkylation of tris-tBu-DO3A to give 12. Reductive cleavage
of the Cbz protective group by catalytic hydrogenation and
further alkylation of this intermediate product with tert-butyl
bromoacetate yielded 13. The final ligand L4 was obtained after
the hydrolysis of the tert-butyl esters with formic acid, followed
by recrystallization from water/acetone, as a brown hygroscopic
powder.
The corresponding Gd3+ and Eu3+ complexes of L1–L4 were
formed by mixing stock solutions of the metal chlorides with
the corresponding 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 to
remove the excess of the lanthanide ions, and the resulting
solutions were filtered and lyophilized. The xylenol orange
test was performed to ensure no free metal ions were present
in solution.21 The formation of the complexes was confirmed
by ESI-MS in negative and positive modes. All the ana-
lyzed spectra contained the appropriate molecular ion peaks
with the isotope pattern characteristic of the Gd3+ and Eu3+
complexes.
The ligands were synthesized in
a series of multi-
step reactions commencing with the commonly used pre-
cursor molecules tris-tBu-DO3A15 (tri-tert-butyl 1,4,7,10-
tetraazacyclododecane-1,4,7-triacetate) for the synthesis of L1,
L3 and L4, and tris-Boc-cyclen16 (1,4,7-tris-tert-butoxycarbonyl-
1,4,7,10-tetraazacyclododecane) for the synthesis of L2.
The synthesis of ligand L1 started with the bromide 117
(Scheme 2A). The alkylation of tris-tBu-DO3A resulted in the
macrocycle 2. After the selective deprotection of the Boc protected
amino groups using 2 M HCl solution in MeOH at 0 ◦C and
further alkylation of the obtained product with the tert-butyl
bromoacetate, the resulting hexaester 3 was isolated. Hydrolysis of
the tert-butyl esters in the presence of TFA and precipitation from
methanol/diethyl ether yielded the final ligand L1 as a colorless
hygroscopic powder.
L2 was synthesized by the alkylation of tris-Boc-cyclen with 4,18
in MeCN at 60 ◦C using K3CO3 as a base (Scheme 2B). After the
complete deprotection of the Boc protected amines using 2 M
HCl solution in MeOH, the amide bond in this intermediate
product was reduced with LiAlH4 in THF, resulting in 6. The
hexaamine was alkylated with the tert-butyl bromoacetate to
give the hexaester 7. After the hydrolysis of the tert-butyl esters
using formic acid and recrystallization of the product from
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Scheme 2 Synthesis of the ligands L1–4. Reagents and conditions: A: (i) tris-tBu-DO3A, K2CO3, MeCN, 70 ◦C, 12 h; (ii) 2 M HCl in MeOH, 0 ◦C, 30 min;
(iii) tert-butyl bromoacetate, K2CO3, MeCN, 70 ◦C, 12 h; (iv) TFA, CH2Cl2, 12 h. B: (i) tris-Boc-cyclen, K2CO3, MeCN, 70 ◦C, 18 h; (ii) 2 M HCl in
MeOH, 2 h; (iii) LiAlH4, THF, reflux, 18 h; (iv) tert-butyl bromoacetate, K2CO3, MeCN, 70 ◦C, 18 h; (v) HCOOH, 60 ◦C, 12 h. C: (i) tris-tBu-DO3A,
K2CO3, MeCN, 70 ◦C, 18 h; (ii) EDA in i-PrOH, reflux, 24 h; (iii) tert-butyl bromoacetate, K2CO3, MeCN, 60 ◦C, 12 h; (iv) TFA, CH2Cl2, 12 h. D:
(i) thionyl bromide, cyclohexane, DMF, rt; (ii) tris-tBu-DO3A, K2CO3, MeCN, 70 ◦C, 12 h; (iii) H2, Pd/C, MeOH, 12 h; (iv) tert-butyl bromoacetate,
K2CO3, MeCN, 18 h; (v) HCOOH, 60 ◦C, 12 h.
Relaxivity experiments
Ca2+ titration curves resulted in the apparent association constants
of logKA = 3.1 0.4, 3.4 0.5 and 2.2 0.1 for GdL2, GdL3
and GdL4, respectively, which demonstrates the sensitivity of the
investigated complexes towards Ca2+ in the desired, low millimolar
range, observed in the extracellular region in vivo.
The results from the relaxometric titration reveal how a change
in the overall structure can produce a significantly different MR
response towards Ca2+. To further investigate their behaviour and
obtain a better understanding on the responses of these complexes
towards other endogenous metal ions (Zn2+, Cu2+, Na+, K+, Mg2+),
we performed a relaxometric screening where the r1 values of
GdL1–GdL4 were determined upon the addition of at least one
equivalent of metal ion, before and after the addition of an amount
of Ca2+ (that causes the maximal change in r1 for each complex,
Fig. 2).
GdL1 was found to be unresponsive to the presence of any of
the above mentioned cations, with its relaxivity remaining almost
constant. This behaviour is expected, since the amide group in
vicinity of DO3A moiety will most probably coordinate to the
lanthanide ion, preventing any type of interaction/coordination of
the additional functional groups. The relaxivity enhancement for
GdL2 was observed almost to the same extent upon the addition of
Mg2+ and Zn2+ (45% and 67% increase in r1, respectively) as seen on
the addition of Ca2+. This clearly demonstrates the low selectivity
Relaxivity titrations with Ca2+ were performed on GdL1–GdL4
(~2.5 mM) at a magnetic field of 7 T and 25 ◦C and at neutral
pH (HEPES buffer). The longitudinal relaxivity, r1, was calculated
from eqn (1), whereT1,obs is the measured T1, T1,d is the diamagnetic
contribution of the solvent and [Gd] is the concentration of the
appropriate Gd3+ complex.
1
1
=
+ r ×[Gd]
1
(1)
T1,obs T1,d
The initial r1 values were found to be similar for three of the
complexes (4.44, 4.35 and 4.42 mM-1s-1 for GdL1, GdL3 and GdL4
respectively) whilst a slightly lower r1 value of 3.67 mM-1s-1 was
found for GdL2. The addition of Ca2+ to the buffered solutions of
GdL1–GdL4 revealed the very different relaxometric responses of
these complexes (Fig. 1). GdL1 has a very weak response upon its
saturation with Ca2+ which does not exceed 10% when compared
to the initial r1 value. The r1 relaxivity of GdL2 is saturated upon
the addition of less than 3 equiv. of Ca2+ and increases to 55%
above the initial r1 value. Similarly, the addition of a comparable
amount of Ca2+ increases the r1 of GdL3 to 30%. However, the
highest increase in r1 (63%) is observed for GdL4 which is saturated
with the addition of almost 5 equiv. of Ca2+. The fitting of the
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Fig. 1 Relaxometric titration of GdL1–4 with Ca2+ at 7 T, 25 ◦C, pH 7.3 (HEPES). The curves correspond to the fit as explained in the text.
of GdL2, which is probably caused by the absence of additional
coordination sites in the side chain of GdL2. Its ethylenediamine
core appended with three carboxylates can interact with several
metal ions, however it does not show selectivity over a particular
ion.
in this complex is very similar to that of GdL3, except that aliphatic
dimethylene group is replaced with the two aromatic carbons of
the catechol group. This obviously contributes to the increase of
the selectivity of GdL4 versus GdL3 towards Ca2+, as well as in
higher r1 changes upon the addition of this ion.
A similar behavior was demonstrated in the comparative
analysis of the r1 responses of GdL3. Namely, the addition of
Mg2+ and Cu2+ increases the r1 for 20% and 24%, respectively. The
level of these changes is of a similar order as observed for Ca2+
(30%). A slightly higher r1 enhancement upon the addition of Ca2+
is presumably due to the presence of the two ether oxygens and the
size of the binding site, which can coordinate more effectively to
Ca2+ over the other two cations. However, the high flexibility of the
side chain and the longer distance of the iminodiacetate moiety
from the reporting moiety may also contribute to the weaker r1
response.
GdL4 exhibits the highest and the most selective response
towards Ca2+ in the presence of investigated competing cations.
The total r1 enhancement is 63%, as in the initial relaxometric
titration experiment. The addition of Mg2+ (10 equiv.) to the
buffered solution of GdL4 leads to a slight increase in r1 (17%). The
presence of the other metal ions in the system has no noticeable
effect on the r1 changes. Interestingly, the structure of the side chain
Luminescence emission lifetimes
Luminescence lifetime measurements for the complexes EuL1–
EuL4 were recorded in H2O and D2O in the absence and presence
of Ca2+. The number of the inner sphere water molecules (q) was
calculated from the empirical equation expressed in the eqn (2),
where A = 1.2 and B = 0.25 for Eu3+.22
q = A ×(tH−1O −tD−1O −B)
(2)
2
2
The data obtained provided a useful insight into the europium
coordination environment and the number of coordinated water
molecules (Table 1). EuL1 exists in solution as a mono-aqua
complex which is rather to be expected, considering the number of
donor atoms available. This hydration state does not change also
after addition of 3 equivalents of Ca2+ to the solution of EuL1 in
buffer, which supports the results from the relaxometric titrations
where minor r1 changes were observed (see above). The hydration
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Fig. 2 Comparison of the r1 response of GdL1–4 in presence of Na+, K+, Mg2+, Zn2+ and Cu2+, before and after the addition of Ca2+ (7 T, 25 ◦C, pH 7.4,
HEPES buffer).
Table 1 Emission lifetimes and estimated q values of Eu3+ complexes in the presence and absence of Ca2+ (25 ◦C, pH 7.4, HEPES buffer)
Compound
t (ms) H2O
t (ms) D2O
q
Compound
t (ms) H2O
t (ms) D2O
q
EuL1
EuL2
EuL3
EuL4
0.54
0.70
0.51
0.80
1.27
0.99
0.65
1.17
1.0
0.2
0.2
0.2
EuL1+ 3 equiv. Ca2+
EuL2+ 3 equiv. Ca2+
EuL3+ 3 equiv. Ca2+
EuL4+ 6 equiv. Ca2+
0.55
0.57
0.47
0.38
1.29
1.16
0.64
1.29
0.9
0.8
0.4
1.9
state of other three complexes EuL2–EuL4 is close to zero in the
absence of Ca2+, and it increases upon the addition of Ca2+. The
amplitude of changes in q correspond to those observed in r1. The
smallest change in q (for EuL3) is associated with the smallest r1
enhancement of GdL3 and the highest changes in q for EuL4 are
followed with the highest r1 changes in GdL4. However a more
detailed physicochemical study would be required to fully explain
the r1 behaviour of the bishydrated EuL4 + Ca2+ species.
metal ions was also investigated. The r1 of GdL2–GdL4 increases
after addition of Ca2+, whereas no analogous changes are observed
for the amide containing GdL1. The levels of the r1 response, as
well as the selectivity of GdL2–GdL4 towards Ca2+ and/or Mg2+,
Zn2+, Cu2+, depend on the structure of the side chain, the number
of coordination sites and its flexibility. The advantage gained by
the easy synthesis of L1–L4 by selecting simple precursors for
chelators is apparently outweighed by the loss of binding sites that
could optimise both the r1 response and the selectivity towards
the investigated metal ions. EuL2–EuL4 have a low hydration
number as determined by luminescence lifetime experiments,
similar to that observed previously for monomacrocylic SCA with
similar structures.9,11,12 The hydration number of these complexes
increases upon the addition of Ca2+ and Mg2+, Zn2+ or Cu2+,
which corresponds with the r1 changes observed for the analogous
Conclusions
Four novel DO3A-based macrocyclic ligands appended with
different side chains and their Gd3+/Eu3+ complexes were syn-
thesized via four convenient routes. The systems display varying
interactions with Ca2+, and their response to several endogenous
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Gd3+ complexes. The exception to this is EuL1, where q remains
at a constant value of one throughout the experiments, which
is explained by the presence of a strong coordination between
the amide and paramagnetic ion in this complex. This study has
revealed much useful information for the future design of metal
sensitive SCA and potential improvements to their structure.
was dissolved in 30 ml of acetonitrile. K2CO3 (0.276 g, 2.0 mmol)
was added to the solution and the reaction mixture was stirred at
r.t. for 30 min. tert-Butyl bromoacetic acid (0.347 g, 1.75 mmol)
was added dropwise into the mixture which was stirred at 70 ◦C for
12 h. The reaction mixture was allowed to cool to r.t., the inorganic
salts were removed by filtration and the solvent concentrated under
reduced pressure to give the crude product as an brown oil which
was purified by column chromatography (5% of MeOH in CH2Cl2)
to give the final product 3 as a yellow oil (0.49 g, 70%). 1H NMR
(CDCl3): d 1.09–1.15 (br, 54H, CH3), 1.14–3.92 (br, 24H, CH2),
3.38 (br, 6H, CH2), 3.92 (br, 4H, CH2). 13C NMR (CDCl3): d
27.5, 27.6, 27.7, 27.8 (CH3), 49.2, 50.0, 50.7, 52.7, 53.6, 55.3,
56.2, 57.8, 60.8, 65.4, 65.7 (CH2), 81.9, 82.1, 82.2, 82.6 (C(CH3)3),
154.9 (CONH), 172.1, 172.4, 172.8, 173.3 (COOtBu). ESI-MS: for
C48H88N6O13 calcd. 957.6 [M + H]+, found: 957.6.
Experimental section
General
All chemicals were purchased from commercial sources and
were used without further purification. All dried solvents were
stored under argon. The lanthanide metal ion solutions with
known concentrations were prepared by dissolving an accu-
rately weighed amount of the chloride salt in the appropriate
volume of doubly distilled water, and standardized with the
2,2¢,2¢¢-(10-(2-((2-(Bis(carboxymethyl)amino)ethyl)(carboxy-
methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
triyl)triacetic acid (L1). 3 (2.01 g, 2.1 mmol) was dissolved in
dichloromethane (20 ml) and trifluoroacetic acid (20 ml) was
added slowly. After the mixture had been stirred at r.t. for 24 h, the
solvent was removed under reduced pressure. Dichloromethane
(40 ml) was added and evaporated twice in order 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 cold diethyl ether was added
dropwise. The resulting precipitate was isolated by filtration and
resuspended in water (3 ml). A large excess of acetone (100 ml)
was added and the cloudy solutions were stored at -20 ◦C for
16 h. The colorless crystalline powder was isolated by filtration,
washed with acetone and dried under reduced pressure. (0.93 g,
71%). 1H NMR (D2O): d 1.22 (bs, 2H, CH2NCO), 1.49–1.57 (br,
6H, CH2), 2.78–3.05 (br, 12H, CH2COOH), 3.18–3.43 (br, 12H,
CH2), 3.75 (s, 2H, CH2). 13C NMR (D2O): d 22.7, 22.9, 28.4, 39.2,
48.3, 48.5, 51.4, 51.6, 53.2, 54.1, 56.0 (CH2), 162.8, 163.0, 169.8,
174.4 (COOH). FT-ICR-MS: for C24H40N6O13 calcd. 619.2581 [M
- H]+, found: 619.2656.
1
complexometric titration with Na2EDTA. 1H NMR, and 13C{ H}
NMR spectra were recorded on Bruker DRX300 spectrometer
at room temperature. Mass spectra (ESI-LRMS in positive and
negative ion mode) were performed on an ion trap SL 1100
system (Agilent, Germany). FT-ICR-MS were performed on
a Bruker FT-ICR Apex II spectrometer. Reversed-phase high-
performance liquid chromatography (RP-HPLC) was performed
at room temperature on a Varian PrepStar Instrument, Australia,
equipped with PrepStar SD-1 pump heads. UV absorbance was
measured using a ProStar 335 photodiode array detector at 214
and 254 nm. Analytical RP-HPLC was performed in a stainless
steel Chromsep (length 250 mm, internal diameter 41.4 mm,
outside diameter 2 inch and particle size 8 mm) C18 column (Varian,
Advanced Chromatographic Solutions). Luminescence lifetime
measurements were performed on a QuantaMasterTM 3 PH
fluorescence spectrometer from Photon Technology International,
Inc., USA. Column chromatography was performed using silica
gel 60 (70–230 mesh ASTM) from Fluka, Germany.
Tri-tert-butyl-2,2¢,2¢¢-(10-(2-((2-tert-butoxy-2-oxoethyl)(2-(tert-
butoxycarbonylamino)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraaza-
Tri-tert-butyl 10-(2-(2-(tert-butoxycarbonylamino)ethylamino)-
2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate
(5). A solution of 4 (2.53 g, 9.0 mmol) in acetonitrile (30 ml)
was added to a solution of tris-Boc-cyclen (3.78 g, 8.0 mmol) and
K2CO3 (1.4 g, 10 mmol) in acetonitrile (80 ml). After the reaction
mixture was stirred for 12 h at 70 ◦C, the solution was allowed
to cool to r.t., the inorganic salts were removed by filtration and
the solvent concentrated under reduced pressure to give a yellow
oil. The oil was purified by column chromatography (15% of ethyl
cyclododecane-1,4,7-triyl)triacetate
(2). Tris-tBu-DO3A
(0.514 g, 1.0 mmol) and K2CO3 (0.138 g, 1.0 mmol) were
dissolved in acetonitrile (30 ml); to this a solution of 1 (0.473 g,
1.2 mmol) in acetonitrile (10 ml) was added. After the reaction
mixture was stirred for 12 h at 70 ◦C, the solution was allowed
to cool to r.t., the inorganic salts were removed by filtration and
the solvent was concentrated under reduced pressure to give
a crude product as a yellow oil. This was purified by column
chromatography (silica gel, 5% of MeOH in CH2Cl2) to give
1
1
acetate in DCM) to give 4.20 g (78%) of 5 as a white solid. H
0.605 g (73%) of 2 as yellow viscous oil. H NMR (CDCl3): d
NMR (CDCl3): d 1.40–1.42 (br, 36H, CH3), 1.97 (bs, 4H, CH2),
2.79 (s, 2H, CH2), 3.21–3.42 (br, 12H, CH2), 3.58 (s, 4H, CH2).
13C NMR (CDCl3): d 28.2, 28.3, 28.5 (CH3), 30.7, 32.6, 38.8, 39.7,
45.8, 49.3, 50.8 (CH2), 79.0, 79.1, 79.2 (C(CH3)3), 155.2, 155.4,
155.8 (CONH). ESI-MS: for C32H60N6O9 calcd. 672.4 [M + H]+,
found: 672.3.
1.22–1.29 (br, 45H, CH3), 1.85 (bs, 6H, CH2), 3.03–3.39 (br, 16H,
CH2), 3.62–3.94 (bs, 6H, CH2), 5.66 (bs, 2H, CH2). 13C NMR
(CDCl3): d 25.8, 25.9, 26.4, 26.5 (CH3), 36.7, 46.4, 47.1, 47.7,
48.0, 50.0, 50.3, 53.4, 54.3, 54.7 (CH2), 78.6, 80.1, 81.3, 81.5
(C(CH3)3), 155.6, 155.7 (CONH), 168.2, 171.7, 171.6 (COOtBu).
ESI-MS: for C41H76N6O11 calcd. 829.6 [M + H]+, found: 829.6.
Tri-tert-butyl 2,2¢,2¢¢-(10-(2-((2-(bis(2-tert-butoxy-2-oxoethyl)-
amino)ethyl)(2-tert-butoxy-2-oxoethyl)amino)-2-oxoethyl)-1,4,7,
10-tetraazacyclododecane-1,4,7-triyl)triacetate (3). A methano-
lic solution of 2 in 4 M HCl was stirred at r.t. for 20 min. The
methanol was evaporated under reduced pressure and the residue
N1-(2-(1,4,7,10-Tetraazacyclododecan-1-yl)ethyl)ethane-1,2-
diamine (6). 4 N HCl in methanol (50 ml) was added to the
compound 5 (4.20 g, 6.2 mmol) and the solution was stirred for
1 h at r.t. The solvent was evaporated and the resulting white
solid was resuspended in dry THF (20 ml) and added slowly to a
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stirring suspension of LiAlH4 (0.23 g, 6.2 mmol) in dry THF (50
ml) under N2 at 0 ◦C. Following the complete addition, the reaction
mixture was heated to reflux temperature for 6 h. The mixture was
then cooled in an ice-bath and the reaction product and excess of
hydride precipitated from solution. The hydride was quenched by
the dropwise addition of water followed by 15% sodium hydroxide.
After vigorous stirring for another 20 min, further impurities were
removed by filtration. The filtrate was concentrated and dried
overnight under reduced pressure, and the yellow oil (6, 0.9 g,
(CDCl3): d 1.38 (s, 9H, CH3), 1.40 (s, 18H, CH3), 2.24–3.80 (br,
34H, CH2), 7.72–7.78 (br, 4H, C6H4). 13C NMR (CDCl3): d 27.8,
27.9 (CH3), 37.1, 42.8, 49.4, 50.2, 52.0, 53.5, 55.5, 56.2, 67.0,
67.4, 69.5, 69.9 (CH2), 82.0, 82.6 (C(CH3)3), 123.1, 131.7, 134.3
(C6H4), 168.0 (NCO), 172.4, 172.8 (COOtBu). FT-ICR-MS: for
C40H65N5O10 calcd. 776.4804 [M + H]+, found: 776.4799.
Tri-tert-butyl 2,2¢,2¢¢-(10-(9-(2-tert-butoxy-2-oxoethyl)-13,13-
dimethyl-11-oxo-3,6,12-trioxa-9-azatetradecyl)-1,4,7,10-tetraa-
zacyclododecane-1,4,7-triyl)triacetate
(10). Compound
9
1
70%) was used in the next step without purification. H NMR
(1.00 g, 1.29 mmol) was dissolved in 10 ml of a mixture of
ethylenediamine and isopropanol (1 : 1). The solution was heated
to reflux temperature for 16 h, and then was allowed to cool to
r.t. The solvent was removed under reduced pressure and the
remainingethylenediaminee was removed using the bulb-to-bulb
distillation apparatus. The resulting viscous oil was dissolved in
the 30 ml of acetonitrile. Following the addition of K2CO3 (0.43 g,
3.00 mmol) the solution was stirred for 30 min at r.t. tert-Butyl
bromoacetate (0.61 g, 3.01 mmol) in acetonitrile (50 ml) was
added dropwise 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, 5% MeOH
(D2O): d 2.67–2.96 (br, 10H, CH2), 3.19–3.40 (br, 10H, CH2), 3.7
(br, 4H, CH2). 13C NMR (D2O): d 37.4, 48.7, 49.8, 50.6, 51.6, 53.8,
56.5 (CH2). ESI-MS: for C12H30N6 calcd. 259.2 [M + H]+, found:
259.3.
Tri-tert-butyl 2,2¢,2¢¢-(10-(2-((2-(bis(2-tert-butoxy-2-oxoethyl)-
amino)ethyl)(2-tert-butoxy-2-oxoethyl)amino)ethyl)-1,4,7,10-tet-
raazacyclododecane-1,4,7-triyl)triacetate (7). 6 (0.9 g, 3.6 mmol)
and K2CO3 (2.5 g, 18 mmol) were dissolved in acetonitrile (70
ml); to this a solution of 4.9 g (25 mmol) tert-butyl bromoacetate
in acetonitrile (30 ml) was added. After the reaction mixture was
stirred for 12 h at 70 ◦C, the solution was allowed to cool to r.t.,
the inorganic salts were removed by filtration and the solvent was
concentrated under reduced pressure to give the crude product as
a brown oil. The oil was purified by column chromatography (5%
of MeOH in CH2Cl2) to give 2.5 g (76%) of 7 as a yellow oil. 1H
NMR (CDCl3): d 1.31–1.34 (2 ¥ bs, 63H, CH3), 2.12–3.32 (br, 36H,
CH2). 13C NMR (CDCl3): d 27.7, 27.8, 27.9, 28.0 (CH3), 50.0, 50.1,
50.7, 51.8, 51.9, 52.3, 53.5, 55.5, 55.7, 55.9, 56.1, 56.3, (CH2), 80.7,
81.8, 82.3, 82.6 (C(CH3)3), 170.3, 170.4, 172.5, 172.8 (COOtBu).
ESI-MS: for C48H90N6O12 calcd. 943.7 [M + H]+, found: 943.8.
1
in CH2Cl2) to yield 0.85 g (75%) of 10 as brown viscous oil. H
NMR (CDCl3): d 1.38 (br, 27H, CH3), 1.43 (s, 18H, CH3), 2.64 (s,
4H, CH2), 3.01–4.39 (br, 34H, CH2). 13C NMR (CDCl3): d 27.9,
28.0, 28.1 (CH3), 35.0, 37.8, 39.1, 45.3, 45.4, 49.2, 49.5, 50.7, 51.5,
53.2, 56.0, 56.8, 61.8 (CH2), 81.8, 82.2, 83.8 (C(CH3)3), 163.8,
166.1, 171.8 (COOtBu). FT-ICR-MS: for C44H83N5NaO12 calcd.
896.5930 [M + Na]+, found: 896.5930.
2,2¢,2¢¢-(10-(2-(2-(2-(Bis(carboxymethyl)amino)ethoxy)ethoxy)-
2,2¢,2¢¢-(10-(2-((2-(Bis(carboxymethyl)amino)ethyl) (carboxy-
methyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-
triacetic acid (L2). 7 (2.1 g, 2.2 mmol) was dissolved in formic
ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
acid
(L3). Compound 10 (0.85 g, 0.98 mmol) was dissolved in
CH2Cl2 (5 ml) and TFA (3 ml) was added slowly. 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 and volatiles. The
same procedure was repeated twice with methanol. The viscous
residue was dissolved in a minimum amount of methanol and
cold diethyl ether was added dropwise. The formed precipitates
were filtered, washed with cold ether and dried in vacuo. A light
brown hygroscopic powder was obtained (L3, 0.4 g, 69%).
1H NMR (D2O): d 3.03 (br, 4H, CH2), 3.21 (s, 6H, CH2), 3.36–
3.56 (br, 18H CH2), 3.78 (bs, 2H CH2), 3.91–3.94 (br, 8H CH2).
13C NMR (D2O): d 37.1, 48.3, 48.8, 50.8, 51.3, 52.2, 53.0, 55.2,
56.5, 57.2, 64.6, 69.6, 70.0 (CH2), 164.9, 168.5, 169.2 (COOH).
FT-ICR-MS: for C28H43N5O12 calcd. 594.2978 [M + H]+, found:
594.2980.
◦
acid (20 ml) and the mixture was stirred at 60 C for 24 h. The
solvent was removed under reduced pressure and the viscous
residue was dissolved in a minimum amount of methanol and
added dropwise to cold diethyl ether. The resulting precipitate was
isolated by filtration and resuspended in 3 ml of water. A large
excess of acetone (100 ml) was added and the cloudy solution
was stored at -20 ◦C for 16 h. A colorless crystalline powder
was isolated by filtration, washed with acetone and dried under
1
reduced pressure to give L2 (0.225 g, 71%). H NMR (D2O): d
2.99–4.07 (br, 36H). 13C NMR (D2O): d 45.7, 46.5, 47.5, 49.1,
49.3, 49.6, 50.6, 52.9, 54.0, 56.5, 56.6, 57.1 (CH2), 169.9, 173.9,
174.1, 174.2 (COOH) ESI-MS: for C24H42N6O12 calcd. 605.2788
[M + H]-, found: 605.2790.
Tri-tert-butyl
2,2¢,2¢¢-(10-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)-
ethoxy)ethoxy)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-
triacetate (9). Tris-tBu-DO3A (1.00 g, 1.94 mmol) and K2CO3
(0.40 g, 2.9 mmol) were dissolved in acetonitrile (30 ml); to this a
solution of 8 (0.79 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 r.t., the inorganic salts were
removed by filtration and the solvent was concentrated under
reduced pressure to give a brown viscous oil. The compound
was purified by column chromatography (silica gel, 5% MeOH
Benzyl 2-(2-(2-hydroxyethoxy)phenoxy)ethylcarbamate (11).
A solution of N-benzyloxycarbonyl-2-bromoethylamine (4.0 g,
16 mmol) in DMF (20 ml) was added to a mixture of 2-
(2-hydroxyethoxy)phenol (2.0 g, 13 mmol) and K2CO3 (2.2 g,
16 mmol) in DMF (30 ml). The reaction mixture was stirred for
16 h at reflux temperature. The solution was allowed to cool to r.t.,
the inorganic salts were removed by filtration and the solvent was
concentrated under reduced pressure to give brown viscous oil.
The compound was purified by column chromatography (silica
gel, 20% EtOAc in CH2Cl2) to yield 2.66 g (63%) of 11 as a brown
1
in CH2Cl2) to yield 1.05 g (70%) of 9 as yellow oil. H NMR
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1
oil. H NMR (CDCl3): d 3.46 (bs, 2H, CH2), 3.79 (s, 2H, CH2),
172.2, 173.0, 173.1 (COOtBu). FT-ICR-MS: for C44H83N5O12
3.95 (s, 4H, CH2), 5.00 (s, 2H, CH2), 6.00 (s, 1H, NH), 6.82 (s, 4H,
C6H4), 7.23 (s, 5H, C6H5), 7.88 (s, 1H, C6H5). 13C NMR (CDCl3):
d 21.0, 40.7, 61.0, 66.7, 68.9, 71.2, (CH2), 115.1, 122.1, 128.1,
128.5, 136.7, 148.7, 148.9 (C6H4 and C6H5), 158.6 (NHCOO). FT-
ICR-MS: for C18H21NNaO5 calcd. 354.1312 [M + Na]+, found:
354.1309.
calcd. 922.4749 [M + H]+, found: 922.4749.
2,2¢,2¢¢-(10-(2-(2-(2-(Bis(carboxymethyl)amino)ethoxy)-pheno-
xy)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid
(L4). Compound 13 (0.81 g, 0.88 mmol) was di◦ssolved in formic
acid (20 ml) and the mixture was stirred at 60 C for 24 h. The
solvent was removed under reduced pressure and the viscous
residue was dissolved in a minimum amount of methanol and
added dropwise to cold diethyl ether. The resulting precipitate was
isolated by filtration and resuspended in 3 ml of water. A large
excess of acetone (100 ml) was added and the cloudy solution
was stored at -20 ◦C for 16 h. The white crystalline powder
was isolated by filtration, washed with acetone and dried under
reduced pressure resulting in 0.39 g (70%) of the final compound
Tri-tert-butyl 2,2¢,2¢¢-(10-(2-(2-(2-(benzyloxycarbonylamino)-
ethoxy)phenoxy)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-
triacetate (12). Compound 11 (1.0 g, 3.0 mmol) was dissolved
in dry cyclohexane (10 ml) under a nitrogen atmosphere. Dry
DMF (0.5 ml) was added to the solution followed by a dropwise
addition of the thionyl bromide (1.2 g, 6.0 mmol). The reaction
mixture was stirred at r.t. for 3 h, after which dichloromethane (30
ml) was added to the mixture and the excess thionyl bromide was
quenched by the addition of a saturated solution of NaHCO3.
The organic phase was washed with brine (2 ¥ 50 ml), water (1
¥ 50 ml), dried over sodium sulphate and concentrated in vacuo.
The resulting brown oil was dissolved in acetonitrile (20 ml)
and added dropwise to the mixture of tris-tBu-DO3A (1.03 g,
2.0 mmol) and K2CO3 (0.40 g, 2.9 mmol) in acetonitrile (30 ml).
After the reaction mixture had been stirred for 16 h at 70 ◦C,
the solution was allowed to cool to r.t., the inorganic salts were
removed by filtration and the solvent was concentrated under
reduced pressure to give a brown viscous oil. The compound
was purified by column chromatography (silica gel, 5% MeOH
1
L4. H NMR (D2O): d 3.01–3.64 (br, 26H, CH2), 3.90 (bs, 4H,
CH2), 4.29 (br, 4H, CH2), 6.91 (br, 4H, C6H4). 13C NMR (D2O): d
47.7, 48.1, 49.1, 49.5, 51.4, 51.6, 52.9, 53.3, 53.8, 55.7, 61.7 (CH2),
112.6, 112.7, 121.1, 121.5, 145.4, 145.9 (C6H4), 164.7, 168.3, 171.3
(COOH). FT-ICR-MS: for C28H43N5O12 calcd. 640.2782 [M - H]+,
found: 640.2777.
General procedure for preparation of GdL1–4 and EuL1–4
L1–4 (~50–100 mmol) was dissolved in water (5 ml) and a stock
solution with known concentration of GdCl3 or EuCl3 (1 equiv.)
was added. The solutions were heated to 60 ◦C for 24 h and the pH
was maintained at ~7 using NaOH. The complexes were treated
with Chelex 100 to remove the excess lanthanide ions. The resulting
1
in CH2Cl2) to yield 1.05 g (70%) of 12 as yellow oil. H NMR
(CDCl3): d 1.31 (br, 27H, CH3), 2.09–3.61 (br, 26H, CH2), 4.08
(s, 2H, CH2), 5.03 (s, 2H, CH2), 5.25 (s, 2H, CH2), 6.20 (bs,
1H, C6H4), 6.71 (bs, 1H, C6H4), 6.84 (br, 2H, C6H4), 7.27 (br,
5H, C6H5). 13C NMR (CDCl3): d 27.6, 27.7 (CH3), 40.3, 49.7,
50.2, 51.7, 51.7, 53.6, 55.3, 56.0, 66.1, 67.1, 68.1 (CH2), 81.9,
82.1 (C(CH3)3), 113.2, 118.6, 121.1, 123.4, 127.7, 128.3, 136.7,
148.1, 149.8, 156.6, (C6H5 and C6H4), 172.3, 173.0, (COOtBu).
FT-ICR-MS: for C44H69N5O10 calcd. 828.5234 [M + H]+, found:
828.5234.
solutions were filtered and lyophilized to give GdL1–4 or EuL1–4
.
GdL1 ESI-MS: for C24H35GdN6NaO13 calcd. 796.1 [M +
-
Na - 2H]-, found: 796.2. EuL1 ESI-MS: for C24H36EuN6O13
-
calcd. 769.2 [M - H]-, found: 768.2. GdL2 ESI-MS: for
C24H37GdN6NaO12 calcd. 782.2 [M + Na - 2H]-, found: 782.2.
-
-
EuL2 ESI-MS: for C24H37EuN6NaO12 calcd. 777.2 [M + Na
- 2H]-, found: 777.1. GdL3 ESI-MS: for C24H38GdN5NaO12
-
calcd. 769.2 [M + Na - 2H]-, found: 769.2. EuL3 ESI-MS: for
C24H38EuN5NaO12 calcd. 764.2 [M + Na - 2H]-, found: 764.2.
-
GdL4 ESI-MS: for C28H38GdN5NaO12 calcd. 817.2 [M + Na -
-
Tri-tert-butyl
2,2¢,2¢¢-(10-(2-(2-(2-(bis(2-tert-butoxy-2-oxo-
-
2H]-, found: 817.2. EuL4 ESI-MS: for C28H38EuN5NaO12 calcd.
ethyl)amino)ethoxy)phenoxy)ethyl)-1,4,7,10-tetraaza-cyclodode-
cane-1,4,7-triyl)triacetate (13). Compound 12 (1.00 g,
1.21 mmol) was dissolved in EtOH (30 ml). Pd/C was added and
mixture was placed into the Parr hydrogenator under 3 bar of H2
pressure for 12 h. The reaction mixture was filtered through celite,
concentrated under reduced pressure and dissolved in acetonitrile
(30 ml). K2CO3 (0.37 g, 2.6 mmol) was added and the mixture
was stirred for 20 min at r.t. followed by the dropwise addition
of the solution of tert-butyl bromoacetate (0.52 g, 2.6 mmol)
in acetonitrile (20 ml). The reaction mixture was stirred for
16 h at reflux temperature. The solution was allowed to cool
to r.t., filtered and concentrated under reduced pressure to give
a brown viscous oil. The compound was purified by column
chromatography (silica gel, 5% MeOH in CH2Cl2) to yield 0.81 g
(73%) of 13 as light brown oil. 1H NMR (CDCl3): d 1.02 (s,
18H, CH3), 1.10 (s, 18H, CH3), 1.18 (s, 9H, CH3), 1.96–2.91 (br,
22H, CH2), 3.09 (bs, 12H, CH2), 6.51 (s, 1H, C6H4), 6.63 (br, 3H,
C6H4). 13C NMR (CDCl3): d 27.5, 27.6, 27.7 (CH3), 49.6, 49.8,
50.1, 50.3, 51.7, 55.2, 55.4, 55.6, 55.9, 59.2, 67.9 (CH2), 81.8, 81.9,
82.1 (C(CH3)3), 117.2, 121.1, 122.9, 128.5, 129.8, 148.0 (C6H4),
812.2 [M + Na - 2H]-, found: 812.0.
Relaxometric titrations
The titrations were performed at 7 T, 25 ◦C and pH 7.3–7.4
(maintained by HEPES buffer). A solution of metal ion salt
(CaCl2, MgCl2, ZnCl2, NaCl, KCl, CuSO4) of known concentra-
tion was added in stepwise portions 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 eqn (1), using the actual Gd3+ concentration at each point of
the titration. The initial Gd3+ concentrations were determined by
measuring the bulk magnetic susceptibility shifts.23 The titration
curves were fitted according to the modified equation previously
reported to obtain the apparent association constants.24
Luminescence lifetime experiments
The luminescence decay experiments were performed in H2O
◦
and D2O (25 C, pH 7.3, HEPES) on EuL1–EuL4 at 5 mM
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concentrations. The Eu3+ ion was directly excited at 395 nm and
emission intensity at 615 nm was recorded with 10 ms resolution.
Excitation and emission slits were set to 15 and 5 nm bandpass
respectively. Datasets are an average of 25 scans and each reported
value is the mean of three independent measurements. Obtained
curves are fitted to the first order exponential decay with r2 = 0.99.
q values are calculated using eqn (2).
9 K. Dhingra, M. E. Maier, M. Beyerlein, G. Angelovski and N. K.
Logothetis, Chem. Commun., 2008, 3444–3446.
10 A. Mishra, P. Fouskova, G. Angelovski, E. Balogh, A. K. Mishra, N. K.
Logothetis and E. Toth, Inorg. Chem., 2008, 47, 1370–1381.
11 J. L. Major, R. M. Boiteau and T. J. Meade, Inorg. Chem., 2008, 47,
10788–10795.
12 E. L. Que, E. Gianolio, S. L. Baker, A. P. Wong, S. Aime and C. J.
Chang, J. Am. Chem. Soc., 2009, 131, 8527–8536.
13 L. Tei, Z. Baranyai, M. Botta, L. Piscopo, S. Aime and G. B.
Giovenzana, Org. Biomol. Chem., 2008, 6, 2361–2368.
14 G. G. Somjen, Ions in the brain: normal function, seizures, and strokes,
Oxford University Press, New York, Oxford, 2004.
Acknowledgements
15 A. Dadabhoy, S. Faulkner and P. G. Sammes, J. Chem. Soc., Perkin
The authors are thankful to Dr M. Placidi for helpful discussions.
Financial support from the Max-Planck Society, the Hertie Foun-
dation, German Research Foundation (DFG, grant AN 716/2-
1) and German Ministry for Education and Research (BMBF,
FKZ: 01EZ0813) is gratefully acknowledged. The European
COST Action D38 “Metal-Based Systems for Molecular Imaging
Applications” is also acknowledged.
Trans. 2, 2002, 348–357.
16 M. Woods, G. E. Kiefer, S. Bott, A. Castillo-Muzquiz, C. Eshelbrenner,
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