Synthesis and characterization of a smart contrast agent sensitive to calcium

Article abstract of DOI:10.1039/b801975d

A novel first-generation Ca²⁺ sensitive contrast agent, Gd-DOPTRA has been synthesized and characterized. The agent shows ～100% relaxivity enhancement upon addition of Ca²⁺. The agent is selective and sensitive to Ca²⁺ also in the presence of Mg²⁺ and Zn²⁺. The relaxivity studies carried out in physiological fluids prove the prospects of the agent for in vivo measurements. The Royal Society of Chemistry.

Full text of DOI:10.1039/b801975d

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Synthesis and characterization of a smart contrast agent sensitive to
calciumw
Kirti Dhingra,*a Martin E. Maier,b Michael Beyerlein,a Goran Angelovskia
and Nikos K. Logothetis*ac
Received (in Cambridge, UK) 4th February 2008, Accepted 25th April 2008
First published as an Advance Article on the web 4th June 2008
DOI: 10.1039/b801975d
A novel first-generation Ca2+ sensitive contrast agent, Gd-
DOPTRA has been synthesized and characterized. The agent
proposed a potential MRI contrast agent, Gd-DOPTA
which is sensitive to Ca2+ concentration in the 0.1–10 mM
range with an apparent dissociation constant of 0.96 mM.6
We recently reported a modification of Gd-DOPTA which
shows B100% relaxivity enhancement upon addition of Ca2+
.
The agent is selective and sensitive to Ca2+ also in the presence
of Mg2+ and Zn2+. The relaxivity studies carried out in
physiological fluids prove the prospects of the agent for in vivo
measurements.
is suitable for [Ca2+
] measurement, however the relaxivity
o
response of this probe to Ca2+ is too low for in vivo measure-
ments.7
With the objective of tracking the modulation in Ca2+ with
a high relaxivity response, we synthesized a novel calcium
sensitive MRI contrast agent, Gd-DOPTRA. Gd-DOPTRA
has proven to be both selective and sensitive to Ca2+ over its
competitor cation Mg2+, with a relaxivity response of B100%
on addition of Ca2+. Gd-DOPTRA has been designed by
exploiting the Ca2+ chelating properties of APTRA (o-amino-
phenol-N,N,O-triacetate) linked to a Gd3+ loaded DO3A
unit. The choice of a low affinity pentadentate Ca2+ chelator,
APTRA (Kd = 20–25 mM)8 was made because high affinity
chelators (such as BAPTA, Kd = 0.1–0.4 mM) would most
likely result in saturation of the indicator when the calcium
concentration ([Ca2+]) increases above 1 mM. Further, due to
their slow kinetics, they have limited ability to follow rapid
changes in [Ca2+] and significantly contribute to Ca2+ buffer-
ing capacity.9 Low affinity chelators have been reported to
show fewer problems with local saturation, Ca2+ binding
kinetics, and Ca2+ buffering.9 Another advantage of choosing
APTRA as chelator is to simplify the overall synthesis of the
contrast agent (CA) with just a simple seven-step synthesis
(Scheme 1).
After the first report of Ca2+ as a signaling molecule in
muscles, its role as a carrier of information has been recog-
nized and this fact has invaded all corners of biology from
biochemistry to cell biology and biophysics.1 Ca2+ plays an
important dual role as a carrier of electrical current and as a
second messenger in the brain. Since its actions are mediated
by a large array of proteins including protein kinases, the
effects are much more diverse than that of the other second
messengers such as cAMP (30,50-cyclic adenosine monophos-
phate) and DAG (diacylglycerol).2 The concentration of Ca2+
outside the cell ([Ca2+]o) is 1.5–2 mM while it is only 50–100
nM inside the cells resulting in an extreme Ca2+ gradient of
15 000–40 000:1, outside to inside.3 Studies done using ion
selective micropipettes have shown that during normal brain
activity, the [Ca2+]o decreases B15%. However, during max-
imal stimulation, [Ca2+
] drops 30% while under traumatic
o
events such as epileptic seizures and terminal anoxia it can
decrease up to 90%.4 The modulation in Ca2+ concentrations,
both inside and outside the cell is a significant factor in
determining nervous system function in both the normal
as well as in pathological conditions.5 Development of
fluorescent dyes has greatly added to our understanding of
this critical role played by Ca2+. However the depth penetra-
tion limit of optical imaging techniques and production of
toxic photobleaching byproducts of fluorescent dyes stimu-
lated the development of ‘smart contrast agents’ for
MRI which can provide information about physiological
signals and biochemical events noninvasively and with
high spatial resolution.6 Along these lines, Li et al. have
The synthesis started with 2-nitroresorcinol which was
monobenzylated using benzyl bromide giving 1 in 85% yield.
Alkylation of phenol 1 was done with 1,3-dibromopropane to
give the alkyl bromide 2 in 88% yield. This was then used for
alkylation of tris-tert-Bu-DO3A to give the macrocycle 4 in
66% yield. Thereafter, the NO2 group was reduced with
simultaneous removal of the benzyl group by hydrogenation
using Pd–C as the catalyst to obtain 5 in 85% yield. The tris-
tert-butylester 5 was then hydrolysed in neat TFA to give
triacid 6 in 68% yield. As the alkylation of aniline 5 with tert-
butylbromoacetate or with methylbromoacetate was not suc-
cessful, yielding a mixture of two products which were difficult
to separate, 7 was finally obtained in moderate yield by
alkylation of 6 with bromoacetic acid and NaOH. The ligand
7 was purified by RP-HPLC and loaded with Ln3+ (Gd3+ or
Eu3+) using LnCl3ꢀ6H2O in water at pH 7. The final concen-
tration of Gd3+ was determined by ICP-OES. Obtained
complexes once formed were stable, however very slow hydro-
lysis of one acetate arm was observed similarly to a previously
a Max Planck Institute for Biological Cybernetics, Department of
Physiology of Cognitive Processes, Tubingen, Germany.
¨
E-mail: Kirti.Dhingra@tuebingen.mpg.de,
Nikos.Logothetis@tuebingen.mpg.de; Fax: 00 49 7071 601 919;
Tel: 00 49 7071 601 917
b Institut fur Organische Chemie, University of Tubingen, Tubingen,
Germany
¨
¨
¨
c Imaging Science and Biomedical Engineering, University of
Manchester, Manchester, UK
w Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b801975d
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3444 | Chem. Commun., 2008, 3444–3446

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Scheme 1 Synthesis of Gd-DOPTRA. Reagents and conditions: (a)
benzyl bromide, K2CO3, MeCN; (b) 1,3-dibromopropane, K2CO3,
DMF; (c) tris-tert-Bu-DO3A, K2CO3, DMF; (d) H2, Pd–C; (e) TFA
(neat); (f) bromoacetic acid, NaOH, H2O; (g) GdCl3ꢀ6H2O.
reported molecule.10 The mechanism of hydrolysis has yet to
be explored and is under investigation.
The relaxivity response of the synthesized CA was checked
at 400 MHz in KMOPS buffer at pH 7.4. In the absence of
Ca2+ the relaxivity observed was 3.5 mMꢂ1 sꢂ1 which in-
creased by 97% to 6.9 mMꢂ1 sꢂ1 upon addition of 1 equiv. of
Ca2+ and leveled off with further addition (Fig. 1(a)), demon-
strating the high sensitivity of the CA toward Ca2+. Further-
more, the binding of CA to Ca2+ is reversible; addition of
EDTA to a solution of CA saturated with Ca2+ brings back
the increased relaxivity to the initial value (Fig. 1(a)).
Fig. 1 (a) Relaxivity enhancement profile of Gd-DOPTRA with
Ca2+ in KMOPS buffer at pH 7.4, 27 1C (E), relaxivity observed on
addition of EDTA after addition of 3.6 eq. of Ca2+ (’). (b)
Comparative relaxivity enhancement profile of Gd-DOPTRA in
KMOPS buffer, ACSF and AECM; rf and rb are the relaxivity value
corresponding to free form and bound form of the agent to Ca2+
.
The Mg2+ concentration inside the brain during neural
activity remains nearly constant. Thus we checked the selec-
tivity of Gd-DOPTRA by measuring its relaxivity response
toward Ca2+ in a Mg2+ containing buffer. At resting state the
cerebro spinal fluid (CSF) within the brain has 0.7 mM of
solution of CA resulted in 70% relaxivity enhancement. This
shows the selectivity of CA for Ca2+ over Zn2+ as well. As the
concentration of Zn2+ in the extracellular space of the brain is
much lower14 as compared to Ca2+ and Mg2+, the observed
weak Zn2+ binding to the CA should not interfere with its
response to large Ca2+ modulation observed during synaptic
transmission.
Mg2+ as compared to 1 mM of Ca2+ 11
(CSF is the fluid that
.
occupies the subarachnoid space and ventricular system
around and inside the brain. The extracellular space of the
brain freely communicates with the CSF compartment and
therefore the compositions of the two fluids are similar).11,12
When the CA was dissolved in a buffer containing more than
half an eq. of Mg2+ (0.62 equiv.), the relaxivity observed was
4.4 mMꢂ1 sꢂ1 which is only 25% higher than the relaxivity of
CA in Mg2+ free buffer, whereas the increase in relaxivity was
B60% with the same amount of Ca2+ added. When Ca2+ was
added to the above Mg2+ containing buffer, 70% relaxivity
enhancement was observed. This shows that the agent is
selective to [Ca2+] changes even in presence of a constant
[Mg2+]. We also checked the effect on relaxivity of CA due to
Zn2+ binding, as a similar ligand has been found to show a
Zn2+ binding effect.13 The Ca2+ titration was performed with
Zn2+ containing (0.5 equiv.) buffer. The initial relaxivity
We further checked the relaxivity response in Ca2+ free
artificial cerebro spinal fluid (ACSF) at 37 1C (see ESIw). The
relaxivity enhancement was observed to be 36%. This signifies
both the selectivity and sensitivity of the agent in a physiolo-
gical environment toward Ca2+. To further explore the
efficacy of CA, we performed relaxivity measurement in the
artificial extracellular matrix (AECM, for exact composition
see ESIw). ECM is a lattice of proteins, polysaccharides and
various compounds attached to the plasma membrane. ECM
materials are mostly present in intercellular spaces between
neurons and glia.15 The maximum changes observed were 27%
at 27 1C and 25% at 37 1C (Fig. 1(b)). The drop in relaxivity in
biological media is likely due to anion binding to Gd in the
presence of Ca2+. Anion binding will also block water access
and this problem is well established for the DO3A class of
complexes.16,17 However these changes could be sufficient to
report dynamics of Ca2+ in the brain.
observed was 4.2 mMꢂ1 ꢂ1, which is 17% higher than the
s
relaxivity observed in Zn2+ free buffer, whereas the relaxivity
enhancement was B49% with the same amount of Ca2+
added. Further addition of Ca2+ to Zn2+ containing buffer
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We would like to thank Prof. A. J. Meixner (University of
Tubingen, Germany) for providing access to the fluorescence
¨
spectrometer. We would also like to thank Hertie foundation
and Max Planck Society for the financial support to carry out
this work.
Scheme 2 Equilibrium reaction with Ca2+
.
Notes and references
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N. C. Spitzer and M. J. Zigmond, Fundamental Neuroscience,
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3. D. M. Egelman and P. R. Montague, J. Neurosci., 1998, 18, 8580.
4. (a) C. Nicholson, Fed. Proc., 1980, 39, 1519; (b) M. Eresinsca and
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5. S. Zanotti and C. Andrew, J. Neurochem., 1997, 69(2), 594.
6. W.-H. Li, S. E. Fraser and T. J. Meade, J. Am. Chem. Soc., 1999,
121, 1413.
The dissociation constant (Kd) was determined using para-
magnetic relaxation enhancement (PRE) method. The relax-
ivity enhancement plot of CA vs. [Ca2+] was fitted to the
equation described in the ESI.w The Kd was found to be
B11 mM.
In order to elucidate the main parameter responsible for
relaxivity enhancement, we have performed luminescence life-
time measurements on Eu3+ loaded ligand (Eu-DOPTRA) in
H2O and D2O solutions. The hydration number, q was
calculated according to the revised equation of Beeby et al.18
In the absence of Ca2+, q was observed to be 0.17 while in the
presence of Ca2+, it increases to 0.88 (Scheme 2). This proves
that the relaxivity enhancement of Gd-DOPTRA in the pre-
sence of Ca2+ is largely determined by the changes in hydra-
tion number of the complex.7,10,16
´
7. K. Dhingra, P. Fouskova, G. Angelovski, M. E. Maier,
N. K. Logothetis and E. Toth, J. Biol. Inorg. Chem., 2008, 13, 35.
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8. K. Abdel-Hamid and M. Tyramianski, J. Neurosci., 1997, 17,
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Perkin Trans. 2, 2000, 1819; (b) O. Reany, T. Gunnlauggson and
D. Parker, Chem. Commun., 2000, 473.
In conclusion, we have reported the synthesis of a novel
first-generation calcium-sensitive MRI contrast agent, Gd-
DOPTRA. The probe showed B100% increase in relaxivity
upon addition of Ca2+. Relaxivity studies carried out in
physiological fluids such as ACSF and AECM prove the
prospects of the agent for in vivo measurements. The structural
design of the agent also offers the possibility for various
modifications that could be made to synthesize a series of
derivatives with a range of Ca2+ binding affinities. Further
investigations aim at modifying the present molecule in order
to achieve an even better selectivity toward Ca2+, particularly
in physiological fluids.
14. A. R. Kay, J. Neurosci., 2003, 6847.
15. Y. Yamaguchi, Cell. Mol. Life Sci., 2000, 57, 276.
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M. P. Lowe, J. M. Moloney, D. Parker and A. S. de Sousa, Chem.
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L. Royle, A. S. de Sousa, J. A. G. Williams and M. Woods,
J. Chem. Soc., Perkin Trans. 2, 1999, 493.
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This journal is The Royal Society of Chemistry 2008
3446 | Chem. Commun., 2008, 3444–3446