An organometallic derivative of a BAPTA ligand: Towards electrochemically controlled cation release in biocompatible media

Article abstract of DOI:10.1039/c1cc11081k

The combination of a ferrocenyl moiety with BAPTA provides a novel, water-soluble, redox-active chelator. This chelator behaving as a conformational sensor exhibits an unexpected electrochemical response with high affinity and selectivity for calcium.

Full text of DOI:10.1039/c1cc11081k

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COMMUNICATION
An organometallic derivative of a BAPTA ligand: towards
electrochemically controlled cation release in biocompatible mediaw
Koyel X. Bhattacharyya,a Leı
¨
la Boubekeur-Lecaque,a Issa Tapsoba,a
ac
Emmanuel Maisonhaute,ab Bernd Schollhorn and Christian Amatore*a
¨
Received 23rd February 2011, Accepted 8th March 2011
DOI: 10.1039/c1cc11081k
The combination of a ferrocenyl moiety with BAPTA provides a
novel, water-soluble, redox-active chelator. This chelator behaving
as a conformational sensor exhibits an unexpected electrochemical
response with high affinity and selectivity for calcium.
unprecedented in the literature, though it is typically seen in
the framework of sensor applications and is conducted in
organic solvents.5 Recently, our group has demonstrated that
caged calcium in redox-active aza-crown ethers is released in
organic solvents with high spatial and temporal resolution
using electrons as an external stimulus.6 However, this system
proved to be ineffective in water, preventing studies in biological
media. In order to overcome this drawback, we envisioned the
union of BAPTA with the redox properties of ferrocene to
design a new water-soluble, redox-active ligand with high
affinity and excellent selectivity towards calcium. The efficient
interplay between the binding and the redox subunits is based
on through bond communication via an ethynyle linker. Herein
we describe the design, synthesis, and characterization of a
new organometallic chelator aimed at developing an electro-
chemical approach for the selective capture and release of
calcium ions under physiological conditions.
Calcium behaves as a chemical messenger in a wide variety
of cells, and local variations in its concentration play a key role
in many biological processes.1,2 The understanding of how
calcium affects cellular functions necessitates the precise
modulation of calcium concentration with high spatial and
temporal resolution. Tremendous efforts have led to the
development of molecules designed to encapsulate calcium,
and more importantly, to capture and release calcium under
external physicochemical stimuli such as photons. The design
of such molecules must fulfill strict requirements dictated by
biological constraints (water solubility, physiological pH,
remarkable selectivity for calcium over all other competing
cations, and biological inertness). Tsien’s group has developed
and thoroughly studied a series of calcium chelators based
on BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,-N0,N0-tetra-
acetic acid)-type ligands, which coordinate Ca2+ with high
affinity and sufficient selectivity over the most likely competing
ions, such as Na+, K+, and Mg2+.3 This family of molecules
opened new possibilities of Ca2+ study, such as calcium
buffering and mapping inside cells or the triggering of calcium
release/uptake using light as an external command.4 Although
it is a very powerful approach, the main drawback of the
photo-induced calcium release remains the irreversibility
since the chelation ability is definitively lost upon photolysis.
Alternatively, electrochemistry is an attractive approach to
reversibly switch the cation affinity. Moreover the peculiar
properties of diffusion at ultramicroelectrodes would a priori
allow the cation release with high spatial and temporal resolution.
The use of electrochemistry to probe cation affinity is not
Among the large variety of BAPTA derivatives described in
the literature,1,7 bromo-substituted BAPTA has been selected
as a building block for the synthesis. The mono-halogenated
arene 1 reported by Grynkiewicz et al. was conveniently used
to form C–C bonds by palladium catalyzed coupling reactions.8
The synthesis of the novel organometallic derivative 3
was carried out in six steps from 2-nitrophenol and 2-nitro-
5-methylphenol (see Scheme 1 and the ESIw). Sonogashira
coupling of derivative 1 with ethynylferrocene provided the
desired proligand 2 in 38% yield, which is satisfactory
a Department of Chemistry, UMR 8640 CNRS-ENS-UPMC,
´
Ecole Normale Superieure, 24 rue Lhomond, 75005 Paris, France.
E-mail: christian.amatore@ens.fr; Fax: +33 1-44323863
b Laboratoire Interfaces et Syste`mes Electrochimiques-UPR 15,
Universite Pierre et Marie Curie-Paris 06, 75005 Paris, France
c
´
Laboratoire d’Electrochimie Moleculaire, Universite Paris Diderot,
UMR CNRS 7591, 15 rue Jean de Baıf, 75013 Paris, France
¨
´
w Electronic supplementary information (ESI) available: Experimental
details for the synthesis, UV-Vis and electrochemical studies. See DOI:
10.1039/c1cc11081k
Scheme 1 Synthesis of Ligand 3: (a) PdCl2(PPh3)2, ethynylferrocene,
DMF, iPr2NH, 110 1C (38%); (b) KOH, THF/water (>98%)
c
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considering the highly deactivated nature of the bromoarene.
Subsequent alkaline hydrolysis of the tetrakis ester 2 yielded
the corresponding tetrapotassium salt 3.
UV-Visible spectroscopy (UV-Vis) and cyclic voltammetry
(CV) studies were conducted in buffered aqueous solutions to
evaluate the calcium affinity of ligand 3 and to investigate
redox-induced cation release. In the absence of calcium,
chelator 3 mainly exhibits two absorption maxima at 319 nm
(e = 3.10+4
M M
ꢀ1 cmꢀ1) and 430 nm (e = 2.10+3 ꢀ1 cmꢀ1).
The large absorbance band in the 270–380 nm range shifts to
shorter wavelengths upon calcium binding. Conversely, the
weaker band around 430 nm is barely affected by the increase
in the calcium concentration.
Changes in UV-Vis spectra upon complexation (Fig. 1)
provide the stoichiometry of complexation and its affinity
constant. For this purpose, the absorbance values at two
different wavelengths (266 and 326 nm) for various free
calcium concentrations set by EGTA buffer have been processed
in Hill plots. The linear behavior with an absolute value of the
slope equal to 1 is fully consistent with the expected 1 : 1
complex of calcium (see inset, Fig. 1). Furthermore, identical
x-axis intercepts for the two selected wavelengths allow us to
estimate a dissociation constant KdCa of 320 ꢁ 8 nM (at 22 ꢁ 2 1C,
pH = 7.2) for ligand 3. A similar study conducted with
magnesium showed that the UV-Vis properties of 3 require a
millimolar scale concentration of Mg2+ to be significantly
perturbed (KdMg = 6.0 ꢁ 0.5 mM at 20 ꢁ 2 1C; details given in
the ESIw). The values of the dissociation constants determined
for Ca2+ and Mg2+ differ by four orders of magnitude,
ensuring that chelator 3 is able to discriminate between the
cation of interest, namely, Ca2+, and its serious competitors,
such as Mg2+, that are present in biological media at much
higher concentrations.
Fig. 2 Effect of Ca2+ concentration on the electrochemical response
of 3 (1 mM) in aqueous solutions (0.1 M KCl, 30 mM MOPS
pH = 7.4). Scan rate: 100 mV sꢀ1 on a glassy carbon electrode.
A gradual increase in Ca2+ concentration leads to an anodic
potential shift of the Fc/Fc+ redox couple, but does not affect
the reversible shape of the peak (Fig. 2). The magnitude of the
potential shift reaches 80 mV for 1 equivalent of Ca2+ per
ligand. Further increases in Ca2+ concentration or the addi-
tion of a large excess of Mg2+ have no significant effect on the
oxidation peak potential, which is fully consistent with a 1 : 1
stoichiometry, as evidenced by UV-Vis.
However, our results differ widely from the predictions based
on the square scheme mechanism often relevant for such
chelators with high affinity constants. In this framework, two
limiting electrochemical behaviors (‘‘two-wave’’ vs. ‘‘anodic shift’’)
are described in the literature in the presence of sub-stoichiometric
amounts of cations.9,10 According to this commonly accepted
reversible square scheme mechanism (see ESIw), the dissociation
constant would be predicted in our case to be Kdtheo(OX) E
24ꢂKdCa E 7.7 mM for the oxidized form of the chelator 3+.11
This predicted value was compared to the affinity constant
obtained experimentally by UV-Vis for the chemically oxidized
chelator Kdexp(OX) = 460 ꢁ 35 nM. The discrepancy between
these values led us to test the validity of the reversible square
scheme in our case. Digital CV simulations of the square
scheme mechanism based upon the set of parameters obtained
The electrochemical behavior of free ligand 3 is charac-
terized by a first reversible process at 205 mV vs. SCE involving
the iron center, and a second irreversible process at 495 mV
from the oxidation of the amino groups on the benzene rings.
Fig. 1 UV-Vis absorption spectra of 3 for different free Ca2+
concentrations set by EGTA buffer (0.1 M KCl, 30 mM MOPS
pH = 7.2, 10 mM EGTA). Inset: Hill plot for absorbance at 266
and 326 nm. Amin (Amax) is the absorbance of the free ligand 3
(Ca2+ complex, respectively).
Fig. 3 Simulated CV of ligand 3 (E10 = 0.2 V for the free ligand,
E10 = 0.28 V for the complex) with different amounts of Ca2+
.
[Ligand 3] = 1.0 mM, scan rate = 100 mV sꢀ1, Kred = 3.10+6
M = 1/KdCa(RED).
c
5200 Chem. Commun., 2011, 47, 5199–5201
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p system of the phenyl ring, but instead takes part in calcium
binding. Therefore, the anodic shift of the ferrocenyl redox
response is not directly linked to the decrease of electronic
density on the nitrogen atom upon coordination of Ca2+, but
is more likely due to the rotation of the amino substituent
with respect to the phenyl plane. In the complexed ligand, the
electron donating amino group is much less involved in the
molecular conjugation, leading to a decrease in electronic
density in the phenyl p system, and thus a fortiori on the
remote ferrocenyl group.
In conclusion, a novel, water-soluble, redox-active, and selective
calcium chelator was designed. Joint theoretical and experi-
mental studies reveal unexpected electrochemical behavior for this
chelator with high affinity for Ca2+, behaving as a conformational
sensor. Work is thus currently underway to explore other synthetic
approaches which may lead to a larger cationic charge localization
within the chelating moiety of the ligand.13
Fig. 4 HOMO orbital (top) and spin-density isosurface (bottom) of
the oxidized form of the model system. Contour values are plotted
at ꢁ0.04 (e bohrꢀ3 1/2
for molecular orbitals and at ꢁ0.003 a.u. for
)
spin density.
experimentally clearly show (Fig. 3) that even for our rather
small potential separation, a two-wave behavior should be
observed, such that two separate redox signals (free and
complexed ligands) appear at potentials that are independent
of cation concentration. Therefore, a more complex mecha-
nism coupling complexation to electron tranfer governs the
unexpected anodic shift behavior of our system.
This work was supported by the CNRS (UMR 8640
and LIA XiamENS), ENS, the Universite Pierre et Marie
´
Curie, the ANR through REEL Blan06-2_136291 and the
Franco-American Commission through the Fulbright advanced
student fellowship (KXB).
Notes and references
The measured affinity for the oxidized form seeming barely
affected by the oxidation prompted us to reexamine the mutual
influence between Ca2+ and the ferrocenyl moiety. Indeed the
effective electronic communication between the redox and the
coordinating subunits of the ligand is a stringent requirement
to observe a significant cation affinity modulation.9 On the
molecular level, DFT calculations were carried out on model
systems of the free ligand and the calcium complex, to examine
whether the oxidation of ligand 3 significantly impacts the
cation binding.
1 G. C. R. Ellis-Davies, Chem. Rev., 2008, 108, 1603–1613.
2 R. Y. Tsien, Fluorescent Chemosensors for Ion and Molecule
Recognition, ACS Symp. Ser., 1993, 538, 130–146.
3 R. Y. Tsien, Biochemistry, 1980, 19, 2396–2404.
4 S. R. Adams, J. P. Y. Kao, G. Grynkiewicz, A. Minta and
R. Y. Tsien, J. Am. Chem. Soc., 1988, 110, 3212–3220;
S. R. Adams, J. P. Y. Kao and R. Y. Tsien, J. Am. Chem. Soc.,
1989, 111, 7957–7968.
5 P. D. Beer, P. A. Gale and G. Z. Chen, Coord. Chem. Rev., 1999,
186, 3–36; P. D. Beer, P. A. Gale and G. Z. Chen, J. Chem. Soc.,
Dalton Trans., 1999, 1897–1909; P. D. Beer, C. Blackburn,
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378–381; H. Plenio and D. Burth, Organometallics, 1996, 15,
4054–4062; H. Plenio, D. Burth and R. Vogler, Chem. Ber.,
1997, 130, 1405–1409.
The long-range electronic communication between the iron
and the nitrogen atom implies that the removal of one electron
(oxidation process) from the HOMO (Fig. 4) should affect the
chelation ability of the nitrogen atom. The spin density of the
oxidized ligand (Fig. 4) reveals a non-negligible contribution
of the nitrogen atom, though the iron center carries the largest
positive spin density. Thus, this simplified electronic structure
calculation confirms that the oxidation of the iron centre is
associated with a weakening of the donating ability of the
nitrogen atom towards the coordinated cation (Ca2+, etc.).
The interaction of Ca2+ with the BAPTA ligand in the gas
phase reveals structural distortions from planarity with rotation
around the C(aryl)–N bond as required for the coordination of
Ca2+ (Scheme 2).12 Whether this structural reorganization is
concerted or sequential cannot be decided presently. However
in either case these results strongly support the inadequacy of
simple square scheme formulation. Along with the conformational
change, the nitrogen lone pair is no longer involved in the
6 C. Amatore, D. Genovese, E. Maisonhaute, N. Raouafi and
B. Schollhorn, Angew. Chem., Int. Ed., 2008, 47, 5211–5214.
¨
7 P. S. Mohan, C. S. Lim, Y. S. Tian, W. Y. Roh, J. H. Lee and
B. R. Cho, Chem. Commun., 2009, 5365–5367; R. Pethig,
M. Kuhn, R. Payne, E. Adler, T. H. Chen and L. F. Jaffe,
Cell Calcium, 1989, 10, 491–498; X. H. Dong, Y. Y. Yang,
J. Sun, Z. H. Liu and B. F. Liu, Chem. Commun., 2009, 3883–3885.
8 G. Grynkiewicz, M. Poenie and R. Y. Tsien, J. Biol. Chem., 1985,
260, 3440–3450.
9 A. E. Kaifer and S. Mendoza, in Comprehensive Supramolecular
Chemistry, ed. G. W. Gokel, Elsevier, New York, 1996, vol. 1,
pp. 701–731.
10 A. E. Kaifer, Acc. Chem. Res., 1999, 32, 62–71.
11 In square schemes, the voltammetric behavior is governed by the
equilibrium constants Kred and Kox corresponding to the com-
plexation of Ca2+ by the reduced (3) and oxidized (3+) forms of
ꢀ
ꢁ
nF
RT
00
ligand
00
00
ligand
Kox
Kred
the ligand and related by: ln
00
complex
¼
ðE
ꢀ E
Þ, E
complex
and E
being the formal potentials for the oxidation of the
free ligand and complex. n is the number of electrons involved in
the redox process (here equal to 1).
12 J. T. Gerig, P. Singh, L. A. Levy and R. E. London, J. Inorg.
Biochem., 1987, 31, 113–121; C. K. Schauer and O. P. Anderson,
J. Am. Chem. Soc., 1987, 109, 3646–3656.
13 The screened positive charge created on the ferrocenyl appears to
have a low influence on the electrostatic repulsion force on the
Ca2+ in agreement with the use of Fc+/Fc as a reference redox
couple.
Scheme 2 Structural distortion upon complexation of Ca2+
.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 5199–5201 5201