Water soluble diaza crown ether derivative: Synthesis and barium complexation studies

Article abstract of DOI:10.1016/j.poly.2013.10.024

The combination of a mcrocyclic cavity with o-diaminobenzene derivative offers a new redox-active and water-soluble ligand for barium that incorporates 12 potential donors. The chelator was synthesized in four steps from diaza-18-crown-6-ether and fully characterized in solution by cyclic voltammetry, UV-Visible and NMR spectroscopies in the presence of barium. The corresponding complex exhibits a 1:1 stoichiometry in solution whereas it crystallizes as 3:2 (M:L) as evidenced by X-ray diffraction studies. The high affinity constant for barium estimated by NMR and electronic absorption techniques in aqueous medium, makes this ligand a promising candidate for redox-induced barium release.

Full text of DOI:10.1016/j.poly.2013.10.024

Polyhedron 68 (2014) 191–198
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Water soluble diaza crown ether derivative: Synthesis and barium
complexation studies
a
b
b
Leïla Boubekeur-Lecaque a, , Clément Souffrin , Geoffrey Gontard , Kamal Boubekeur ,
⇑
Christian Amatore a
a Department of Chemistry, UMR 8640 CNRS-ENS-UPMC, 24 rue Lhomond, 75005 Paris, France
b Institut Parisien de Chimie Moléculaire, UMR CNRS 7071, Université Pierre et Marie Curie, 4 Place Jussieu, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The combination of a mcrocyclic cavity with o-diaminobenzene derivative offers a new redox-active and
water-soluble ligand for barium that incorporates 12 potential donors. The chelator was synthesized in
four steps from diaza-18-crown-6-ether and fully characterized in solution by cyclic voltammetry,
UV–Visible and NMR spectroscopies in the presence of barium. The corresponding complex exhibits a
1:1 stoichiometry in solution whereas it crystallizes as 3:2 (M:L) as evidenced by X-ray diffraction
studies. The high affinity constant for barium estimated by NMR and electronic absorption techniques
in aqueous medium, makes this ligand a promising candidate for redox-induced barium release.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 10 September 2013
Accepted 22 October 2013
Available online 1 November 2013
Keywords:
Diaza-18-crown-6-ether
Barium
Affinity constant
Spectroscopy
Cation release
1. Introduction
under Ca2+-free conditions [5–7]. The protocol of stimulation with
barium is very simple and consists in incubating the cell in a mil-
Calcium serves as a key signaling messenger in various biolog-
ical processes such as intercellular communication [1,2]. The latter
is made possible by the release of chemical messengers from an
emitting cell to a target cell. For instance, the transmission in neu-
rons is operated by neurotransmitters located in vesicles inside the
emitting cell. The appropriate cell stimulation provoking a Ca2+ en-
try or increase, induces the docking of vesicles to the cell mem-
brane followed by the fusion of the vesicle with the cell
membrane and then the release and diffusion of the messenger
in extracellular medium toward the neighboring cells [3]. This cas-
cade of events known as vesicular exocytosis is biologically initi-
ated by a significant increase in cytosolic Ca2+ concentration
whose duration and concentration level are critical for the mecha-
nism of vesicular fusion [4]. In vitro studies of exocytosis cascade
require the stimulation of the emitting cells leading to an increase
in intracellular free Ca2+ concentration. Among the various secret-
agogues i.e. stimulating agents that trigger exocytosis (Ca2+/K+,
Ca2+/nicotine, Ca2+/digitonine, Ba2+. . .), barium stimlation has re-
tained our attention. Although not completely elucidated for all
cells, it is firmly established that barium can efficiently substitute
for Ca2+ and is able to induce secretion from a variety of cell types
limolar solution of Ba2+ in the absence of extracellular Ca2+. How-
ever, Ba2+-evoked release of neurotransmitters in such stimulation
protocol is not spatially resolved as all regions of the cell are
equally stimulated. Inspired by photo [8–10] and redox-induced
[11,12] Ca2+ release strategies, we envisaged designing a selective
redox-active barium chelator to deliver free barium as a secreta-
gogue with high spatial and temporal resolution in extracellular
medium. The oxidation or reduction of the redox reporter incorpo-
rated in the chelator provokes a charge and or/ a conformational
modification that may affect sufficiently the binding ability of the
ligand, thus favoring the release of a chelated ion.
The design of such selective chelator must fulfill strict require-
ments dictated primarily by biological constraints such as water
solubility, physiological pH, and remarkable selectivity for barium
over all other competing cations. Numerous exemples of selective
barium ligands for sensing applications are reported in the littera-
ture but most of them operate only in organic solvents [13,14]. The
combination of macrocyclic cavities with redox-active units has
been successfully exploited in organic solvents for sensing and
redox-switchable applications. In particular, pioneering results with
reducible subunits such as nitrobenzyl or quinone allowed the
detailed description both theoretically and experimentally of the
square-scheme mechansim that couples complexation and elec-
tron transfer reactions [14,15]. Oxidizable redox probes such as
ferrocene, tetrathiafulvalene and phenylenediamine, were also
combined to macrocycles for electrochemical cation sensing in or-
ganic solvents [16]. Few exemples of water-soluble chelating
⇑
Corresponding author. Address: Laboratoire Interfaces, Traitements, Organisa-
tion et Dynamique des Systèmes (ITODYS), CNRS UMR 7086, Université Paris
Diderot, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France. Tel.: +33
157278772; fax: +33 1 44323863.
E-mail address: leila.boubekeur@univ-paris-diderot.fr (L. Boubekeur-Lecaque).
0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.10.024

192
L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
agents for Ba2+ based on crown ether motif and featuring phospho-
nate or carboxylate groups are known but they do not feature a
redox probe [17,18]. Herein we describe the synthesis and charac-
an explicit expression of the absorbance versus the total concen-
tration cM can be derived without approximation from the second
order equation:
terization of
a
novel barium chelator combining diaza-
cL:x2 ꢀ ðcL þ cM þ KdÞ:x þ cM ¼ 0
ð1Þ
18-crown-6 ether as the binding pocket, o-diaminobenzene as
the redox probe and carboxylate side arms. Besides increasing
the solubility in water, carboxylate groups, when ionized, provide
strong electrostatic interaction with hard metals like alkali and
alkaline earth cations. This new water soluble platform features
12 potential donors which corresponds to the higher coordination
number encountered in barium coordination chemistry.
AꢀA0
where: x ¼
, A0 (Alim) is the absorbance at cM = 0 (at full satu-
A0ꢀAlim
ration respectively), A the absorbance at a given cM, cM = [M]free
+
[ML], cL = [L]free + [ML] and Kd the dissociation constant. The explicit
expression of the absorbance A versus cM is a solution of the second
order equation (Eq. 1). Kd can thus be obtained by a nonlinear least
square minimization.
2.4. Determination of binding constant by 1H NMR
2. Material and methods
For binding constant determination, 1H NMR spectra were re-
corded in deuterated water at constant ionic force of 0.1 set by
NaNO3. Titration experiments were carried out by mixing the li-
gand solution in D2O to a solution of barium chloride. Changes in
the chemical shift of the methylenic protons adjacent to the car-
boxylate groups were exploited for the detrmination of binding
constant. Data of
fitted to a 1:1 binding model using origin 8.1 software (see Fig. S9
in the ESI).
2.1. General considerations
All reactions were performed under argon if not stated other-
wise and all chemicals were purchased from Sigma/Aldrich
(France) or Alfa Aesar (France). Acetonitrile and DMF were distilled
from CaH2. 1H and 13C NMR spectra were recorded on Bruker
AC-250 spectrometer in the solvents indicated at 298 K. Chemical
shifts are reported using the deuterated (13C NMR) or the residual
monoprotonated (1H NMR) solvent signals as reference. All cou-
pling constants (J values) were measured in Hz. Elemental analysis
were conducted at the Service de Microanalyses- ICSN (Gif sur
Yvette, France). Thin layer chromatography (TLC) was performed
on aluminium sheets precoated with 60 F254 silica gel. Preparative
flash chromatography was performed on silica gel 60 (0.040–
0.063 mm, Merck).
D
d (chemical shift variation) against [Ba2+] were
½MLꢁ
D
d ¼ d ꢀ d0 ¼ ðdlim ꢀ d0Þ:
½Lꢁ0
Where
Dd is the measured change in chemical shift, dlim (and d0)
is the chemical shift of the barium complex (and of the free ligand
respectively). [L]0 is the total concentration of ligand (free and
complexed forms) and [ML] is the free barium concentration deter-
mined as a solution of the quadratic equation:
2.2. Electrochemistry
The electrochemical experiments were conducted in an air-
tight three electrodes glass cell under argon atmosphere,
1
Ka
2
½MLꢁ þ ðꢀ½Lꢁ0 ꢀ ½Mꢁ0 ꢀ Þ ꢂ ½MLꢁ þ ½Mꢁ0 ꢂ ½Lꢁ0 ¼ 0
controlled by
a
computer-controlled potentiostat
(l-Autolab,
Eco-Chemie, the Netherlands).
A
large platinum wire and
a
saturated calomel electrode were used as a counter electrode and
a reference electrode, respectively. A glassy carbon electrode with
a disk radius of 0.5 mm, was used as working electrode and pol-
ished to a mirror finish before each experiment. Electrochemical
studies in organic solvent were conducted in acetonitrile solutions
of 3 (1 mM) containing either 0.1 M of TBABF4 as a supporting
electrolyte. The aqueous supporting electrolyte employed with
the water soluble ligand 4 was prepared from ultra-pure water
Where [M]0 is the pre-equilibrium barium concentration. Non
linear curve-fitting of the experimental
Dd versus [M]0 at a known
[L]0, yielded the parameters dmax, Ka and [C].
2.5. Synthesis
2.5.1. Bis(2-nitrophenyl)-diazacrown ether 1
(1,4,10,13)-Tetraoxa-7,16-diazacyclooctadecane (206 mg,
(<18 MX/cm) and contained 0.1 M NaNO3, pH 7. Cyclic Voltammo-
0.785 mmol), o-fluoronitrobenzene (175
lL, 1.65 mmol) and tri-
grams (CV) were recorded at room temperature (22 2 °C) at a po-
tential sweep rate of 0.1 V sꢀ1. Barium perchlorate was employed
in organic electrolyte and barium chloride in aqueous electrolyte.
ethylamine (241 L, 1.73 mmol) were dissolved in 2.5 mL of anhy-
l
drous DMF. The yellow solution was stirred for 6 h at 100 °C. The
rsulting orange solution is concentrated under vacuum and diluted
with dichloromethane and washed with aquous solution of
NaHCO3, water, then saturated solution of NaCl. The organic phase
was dried on anhydrous Na2SO4, filtered then evaporated to dry-
ness to give 1 yellow crystalline solid (696 mg, 90%).
2.3. UV–Vis studies
All spectra were recorded on a computer-controlled Lambda
45-Perkin Elmer spectrophotometer. Spectroscopic measurements
were performed in aqueous solutions at a constant ionic force set
1H NMR (250 MHz, CDCl3): d 7.65 (dd, 3JHH = 8.1 Hz, 4JHH = 1.2 Hz,
3
2H), 7.31–7.51 (m, 4H), 6.93–7.04 (m, 2H), 3.61 (t, JHH = 5.3 Hz,
3
8H, OCH2CH2N), 3.55 (s, 8H, OCH2CH2O), 3.47 (t, JHH = 5.3 Hz,
by 0.1 M NaNO3, pH 7. UV–Vis studies for 4 (100 lM) were con-
8H, OCH2CH2N). 13C NMR (62.5 MHz, CDCl3): d 145.1 (CAr–NO2),
136.6 (CAr–Nethercrown), 125.4, 123.4, 117.7 and 115.2 (CAr–H),
70.5, 54.7 and 69.0 (CH2O and CH2N). Elemental Anal. Calc. for
ducted by direct titration with sequential additions of a solution
of BaCl2 (or CaCl2, MgCl2, KCl) until full saturation is reached
([M]/[4] = 50 equivalents). After each concentration increment,
the solution was stirred for 5 min to reach equilibrium before
recording the UV–Vis spectrum. Data were analyzed at different
wavelengths (263 and 239 nm) and the normalized absorbance
((AꢀA0)/(A0ꢀAlim)) versus the ratio [M]/[4] plotted. Since the free
cation concentration [M] could not be approximated by the total
cation concentration cM, the Benesi–Hildebrand model is not
applicable. However, in the case of 1:1 complex stoichiometry,
C
24H32N4O8: C, 57.13; H, 6.39; N, 11.10. Found: C, 56.77; H, 6.54;
N, 11.48%.
2.5.2. Bis(2-aminophenyl)-diazacrown ether 2
2 (400 mg, 0.793 mmol) and Pd/C (10%, 75 mg) were placed in a
nitrogen flushed schlenk flask and suspended in 7 mL of ethanol. A
slow flow of H2 was bubbled in the reaction mixture until the solu-

L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
193
tion turned colorless. After purging with argon, the suspension was
3. Results and discussion
fltered through Celite and the pad washed with dichloromethane.
The filtrate was evaporated to dryness to give a colorless solid
residue.
3.1. Synthesis
1H NMR (250 MHz, CDCl3): d 7.05–7.08 (m, 1H), 6.85–7.00 (m,
The synthesis of the novel macrocyclic derivative 4 was carried
out in four steps from the commercially available diaza-18-crown-6.
The synthetic route depicted in Scheme 1 starts with nucleophilic
aromatic subsitution on o-fluoro-nitrobenzene which allowed
the direct bis-arylation of the parent macrocycle in 90% yield.
The nitro groups were reduced quantitatively to amino groups
by catalytic hydrogenation with H2 and Pd/C. Alkylation of
the amino derivative 2 with ethyl-2-iodoacetate provided the
desired proligand 3 in 57% yield. Subsequent alkaline hydrolysis
of the tetrakis ester 3 yielded the corresponding tetrapotassium
salt 4.
All compounds except 2 were characterized by NMR spectros-
copy and elemental analysis. In fact, the diamino derivative 2
which proved to be highly air-sensitive was characterized by 1H
NMR spectroscopy and then immediately subjected after workup
to the following alkylation step.
1H), 6.60–6.78 (m, 2H), 3.59 (s, 8H, OCH2CH2O), 3.49 (t, 3JHH = 5.4 Hz,
3
OCH2CH2N), 3.18 (t, JHH = 5.4 Hz, OCH2CH2N).
2.5.3. Bis(diethyl-2,20(phenylimino)diacetate)-diazacrown ether 3
To a solution of freshly prepared diamino derivative 2 (350 mg,
0.787 mmol) and proton sponge (843 mg, 5 eq) in 10 mL of aceto-
nitrile was added diethyl iminodiacetate (510 lL, 5.5 eq). The
resulting mixture was stirred at 80 °C overnight. The suspension
was filtered on a Celite pad. The filtrate was evaporated to dryness.
The residue dissolved in toluene was washed with HCl 0.1 M then
water. The organic phase was decanted, dried on anhydrous
Na2SO4 and evaporated to dryness. The desired compound 3
was isolated as a beige solid (355 mg, 57% yield) after purification
of the crude by column chromatography on silica gel using
dichloromethane/ethylacetate as eluent.
The reduction of the nitro into amino group was clearly evi-
denced by 1H NMR as significant highfield shiftings are measured
1H NMR (250 MHz, CDCl3): d 7.16–7.25 (m, 2H), 6.89–7.02 (m,
3
for the methylene protons CH2– (
matic protons (
sion of the pro-ligand 3 into the corresponding tetraacetate
ligand 4 was assessed by the disappearance of the triplet signal
associated to ethyl ester group on the 1H NMR spectrum and ele-
mental analysis further confirmed the formation of 4.
D
d = ꢀ0.33 ppm) and the aro-
6H), 4.27 (s, 8H, NCH2COOEt), 4.11 (q, JHH = 7.1 Hz, 8H, CH2CH3),
D
d = ꢀ0.65 ppm). The clean and complete conver-
3.45–3.67 (m, 16H, OCH2CH2N), 3.53 (s, 8H, OCH2CH2O), 1.21 (t,
3JHH = 7.1 Hz, 12H, CH2CH3). 13C NMR (62.5 MHz, CDCl3): d 171.1
(C = O), 142.8, 136.1, 123.7, 123.0, 120.5, 116.7, 70.4 and 69.6
(CH2O crown ether), 60.4 (NCH2CO2Et), 59.6 (NCH2 crown ether),
55.1 (CH2–CH3), 14.2 (CH2–CH3). Elemental Anal.: Calc. for C40H60-
N4O12ꢂ4 CH3CN: C, 60.49; H, 7.61; N, 11.76. Found: C, 60.30; H,
7.80; N, 11.68%.
3.2. X-ray crystal structures
Single crystal X-ray structures were obtained for the pro-ligand 1
(see ESI) and a barium complex 4-Ba. Representations of the
molecular structures are shown in Figs. 1 and 2, and selected
interatomic distances and angles for 4-Ba are collected in Table 1.
Crystal structure of 4-Ba shows an unusual complexation mode
in such a way that the apparent stoichiometry in the solid state is
2L4ꢀ:3Ba2+:2Na+. Considering that there is a good match between
Ba2+ ionic diameter and the size cavity of diaza-18-crown-6 ether,
it was unexpected to localize all Ba2+ ions in the asymmetric unit
out of the macrocylic cavity. The asymmetric unit (see Fig. 1) con-
2.5.4. Bis(phenyliminodiacetate)-diazacrown ether 4
An aqueous solution of NaOH was added to a solution of 3
(120 mg, 0.152 mmol) in 5 mL of THF and the resulting mixture
was stirred overnight at rt. The light-colored liquid phase above
was decanted, and the oily residue was dried to give an off-white
solid. 4 (rdt = 77%) 1H NMR (250 MHz, D2O): d 7.37 (d, JHH = 7.8 -
3
3
Hz, 2H), 7.03–7.09 (m, 2H), 6.95–7.01 (m, 2H), 6.91 (d, JHH = 7.3 -
Hz, 2H), 4.05 (s, 8H, CH2COOꢀ), 3.54–3.69 (s, 24H, crown ether). 13
C
NMR (62.5 MHz, CDCl3): d 179.9 (C carboxylate), 144.3, 139.3,
123.5, 123.1, 120.3, 118.6, 69.5 and 68.9 (CH2O), 55.4 (NCH2CO2ꢀ),
50.8 (NCH2CH2O). Elemental Anal. Calc. for C32H40N4Na4O12ꢂ4 H2O:
C, 45.94; H, 5.78; N, 6.70. Found: C, 46.01; H, 5.59; N, 7.06%.
tains four distincts complexes involving Na+ and Ba2+
.
Na+ cation, located out of the macrocyclic cavity, is in a dis-
torted octahedral environment where two acetate groups, two
nitrogen atoms of o-phenylenediamine ring, one oxygen donor
from the aza-crown ether are involved in the complexation (see
Fig. 2). The coordination sphere for Na+ is completed by one H2O
ligand that is further involved in hydrogen bonding to O8 in the
crownether and O2 from acetate donor of the second phenylenedi-
amine. The remaining three oxygen and nitrogen donors of the
macrocycle are interacting neither with Na+ nor with Ba2+. The
two nitrogen atoms N3 and N4 of the o-phenylenediamine interact
nearly equally to Na+ with bond lengths of 2.577(3) and 2.589(3) Å
respectively. The average C–N–C bond angle for the macrocyclic
amino group N3 (111°) and for the iminodiacetate group N4
(112°) points to a pyramidalization of both nitrogen atoms. This
sp3 hybridization originating from the perpendicular conformation
adopted by the phenylenediamine with respect to the macrocycle,
reinforces the basic character of the lone pairs allowing a stronger
coordination of Na+ cation. The average Na–O bond length is
2.355 Å with strong disparities between the various oxygen donors
(ether, water and acetate) as shown in Table 1.
2.6. X-ray structural determination
Slow diffusion of hexanes into a solution of 1 in dichlorometh-
ane gave yellow block crystals suitable for single-crystal X-ray dif-
fraction. Suitable colorless prism-like crystals of 4-Ba were grown
by slow diffusion of acetone into an aqueous solution of BaCl2:4
(1:1) at room temperature. The data for 1 and 4-Ba were collected
on a Bruker APEX-II CCD X-ray diffractometer by using graphite-
monochromated Mo
Ka radiation (wavelength = 0.71073 Å).
Unit-cell parameter determinations, data collection strategies,
and integrations were carried out with the Bruker SAINT software
[19]. The data were corrected from absorption by a multi-scan
method [20]. The structures were solved by direct methods and
2
refined by full-matrix least-squares on all F0 data using SHELXS-97
[21] and SHELXL-97 [21,22]. All non-hydrogen atoms were refined
anisotropically, and H atoms bonded to C atoms were placed at
calculated positions. Crystallographic data and refinement details
are presented in Table 3. Structural analyses and drawings were
made using ORTEP-3 [23] and POV-ray 3.6 software.
Three types of Ba2+ ions could be distinguished depending on
their coordination environment. Ba1 is coordinated by nine
donors arranged in a tricapped trigonal prism: four oxygen atoms

194
L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
Scheme 1. Synthesis of Ligand 4: (a) ethyl 2-iodoacetate, MeCN, reflux 24 h (57%); (b) NaOH, THF.
faces of the trigonal prism. The Ba3 cation is hepta-coordinated by
two oxygen atoms (O11, O12) from one acetate in g2 fashion and
five oxygen atoms (O57, O58, O59, O62, O63) from water molecule.
Although the bond distances Ba-O for 4-Ba are consistent with
those measured in previously reported azacrown ether crystal
structures, it is worth mentionning that the stoichiometry 3:2
and the coordination environment for barium ions are unusual.
[27–30] In fact, for crystals of diaza-18-crown-6-ether based com-
plex grown in organic solvants, the most frequently encountered
stoichiometry is 1:1 (Ba:L) where the barium cation is immersed
within the macrocyclic cavity. In our case, 4-Ba crystals were
grown from an aqueous solution, and the peculiar solid state struc-
ture observed could originate from the strongly competing charac-
ter of water ligand for hard metals like alkaline-earth.
3.3. UV–Vis spectrosopy
Affinity and selectivity of this new chelator were examined in
aqueous solution by UV–Vis spectroscopy. In the absence of added
cations, the ligand displays mainly three bands at 239, 263 and
300 nm. Spectral changes upon complexation provide the stoichi-
ometry of complexation and its affinity constant. The titration of
the tetra-anionic ligand with increasing amounts of Ba2+ ion is
shown in Fig. 3. Ba2+ has been added until the absorbance intensity
saturates. Similar trends are observed upon the addition of cations
such as Ca2+, Mg2+ or K+ leading to the progressive decrease in
intensity of the bands mentionned previously. Except for Ca2+, all
the studied cations follow the standard saturation behavior that
would be expected for a simple 1:1 ligand: metal complexation
(see Fig. S1–2 in the ESI). The stoichiometry of the barium complex
was further confirmed by the Job’s plot (see Fig. S3 in the ESI)
whose apex point approximates 0.5. The absorbance values at
two different wavelengths (263 and 239 nm) for various free cation
concentrations were fitted with a 1:1 ligand: metal association
model which allowed us to extract the affinity constants associated
to these cations (Table 2).
Fig. 1. Asymmetric unit in 4-Ba showing the atom numbering scheme with the
ellipsoids drawn at the 50% probability level. For the sake of clarity, hydrogen
bonded to carbon atoms have been omitted.
(O1, O1_h, O12, O12_i) from four different acetate anions, five oxygen
atoms (O51, O52, O53, O54, O54_h) from water molecules amongst
which three are bridged with Ba2 (O53, O54, O54_h). Ba1-O bond
distances range from 2.694 to 2.951 Å in accordance with reported
barium complex structures [24–26]. Ba2 is octa-coordinated and
located in bicapped trigonal prism. Two oxygen atoms (O4, O4_h)
from acetate anions and four water molecules (O54, O54_e, O55,
O55_h) occupy the corners of the trigonal prism. Two oxygen
atoms (O53, O56) from water molecules cap two of the rectangular
The relative change in absorbance at 239 nm (Fig. 4) shows
qualitatively that the affinity of the chelator for the studied cations
decreases in the following order: Ca2+ ꢃ Ba2+ > Mg2+ ꢃ K+. There is
no clear correlation between the size of the cation and the affinity

L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
195
Fig. 2. Local coordination environment of Na+ ion in 4-Ba showing the atom numbering scheme with the ellipsoids drawn at the 50% probability level. For the sake of clarity,
hydrogen atoms bonded to carbon atoms have been omitted.
Table 1
Table 2
Selected bond lengths (Å) and angles (°) for 4-Ba.
Stability constants of Metal-ligand 4 complexes in water.
Na1–O6
Na1–O9
Na1–O11
O50–O8
O50–O2
2.485(2)
2.315(3)
Na1–O50
Na1–N3
Na1–N4
O50–H50A–O8
O50–H50B–2
O6–Na1–O11
2.286(3)
2.577(3)
2.589(3)
178(5)
168(3)
179.89(10)
Cation
Ionic diameter* (Å)
Charge density* q (Åꢀ1
)
LogK
Ba2+
Ca2+
K+
2.68
1.98
2.66
1.32
1.49
2.02
0.75
3.03
4.9
N.D
3.9
4.5
2.333(3)
2.819(4)
2.847(4)
162.34(10)
126.85(10)
2.951(2)
2.860(5)
2.907(4)
2.951(2)
2.694(3)
2.751(2)
2.852(4)
2.775(3)
2.812(2)
2.762(3)
2.815(7)
2.807(7)
2.728(7)
2.782(4)
Mg2+
O50–Na1–N4
O9–Na1–N3
Ba1–O1
Ba1–O52
Ba1–O53
Ba1–O1_h
Ba1–O12_i
Ba2–O4
Ba2–O56
Ba2–O53_c
Ba2–O54_e
Ba2–O55_h
Ba3–O62
Ba3–O63
Ba3–O58
Ba3–O57
*
See Ref. [31].
Ba1–O51
Ba1–O54
Ba1–O12_b
Ba1–O54_h
2.872(6)
2.963(2)
2.694(3)
2.963(2)
Table 3
Crystallographic data and refinement details for 1 and 4-Ba.
Ba2–O55
Ba2–O65
Ba2–O54_c
Ba2–O4–h
2.762(3)
3.094(15)
2.812(2)
2.751(2)
1
4-Ba
Formula
C
24H32N4O8
504.54
monoclinic
P 21/c
C64H111Ba3N8Na2O42
2122.58
orthorhombic
Pnma
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
Ba3–O12
Ba3–O11
Ba3–O59
2.561(3)
2.787(3)
2.764(5)
7.8791(6)
8.7413(7)
18.0281(14)
90
19.1396(6)
36.2618(12)
12.3022(4)
90
a
(°)
Symmetry transformation codes: (b) x, y,1 + z; (c) ꢀ1/2 + x, 1/2 ꢀ y, 5/2 ꢀ z;
(e) ꢀ1/2 + x, y, 5/2 ꢀ z; (h) x, 1/2 ꢀ y, z; (i) x, 1/2 ꢀ y, 1 + z.
b (°)
91.436(3)
90
90
90
c
(°)
V (Å3)
Z
1241.27(17)
2
8538.2(5)
4
T (K)
200(2)
200(1)
k (Å)
0.71073
1.350
0.102
0.71073
1.651
1.476
Dcalc (g cmꢀ3
)
l
(mmꢀ1
)
Reflections collected
9619
50031
Independent reflections (Rint
)
3804 (0.0194)
1.022
13313 (0.0323)
1.117
Goodness-of-fit (GOF) on F2
Final R, wR2 [I > 2
All data
r(I)]
0.0431, 0.1213
0.0623, 0.1330
0.0463, 0.0969
0.0614, 0.1019
(see Table 2). In fact based on the ionic diameter, the size of potas-
sium cation is almost the same as barium. Although being the
smallest cation in the studied series, magnesium proved to be
more strongly complexed by the ligand than potassium. The car-
boxylates when ionized provide a strong electrostatic field which
greatly enhances the complex stability for alkali and alkaline earth
metals. If we consider only the columbic forces, it becomes clear
that a ligand with four anionic charges has a stronger interaction
with divalent than with monovalent cations.
The titration curves for Ca2+ obtained by UV–Vis spectroscopy
show clean isobestic points (at 202 and 215 nm) indicative of the
co-existence of only two species in solution (the free ligand and
the complex). However, the data points at a given wavelength
Fig. 3. UV–Vis spectra of compound 4 (100 lM) in water with increasing amount of
barium (from 0 to 5 mM); (inset) Variation of the absorbance at 263 nm as a
function of Ba2+ concentration. The solid line corresponds to the best fit of
experimental data with the complexation model 1:1.

196
L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
Fig. 6. Effect of Ba2+ concentration on the electrochemical response of 3 (1 mM) in
acetonitrile solution (0.1 M nBu4NBF4). Scan rate: 100 mV sꢀ1 on glassy carbon
electrode (diameter = 1 mm).
Fig. 4. Evolution of the normalized absorbance of ligand 4 in water with increasing
amount of metal ion (Ba2+, Ca2+, K+ or Mg2+).
follow a complex behavior (see inset Fig. 5) rather than the stan-
dard saturation curve that would be expected for a simple 1:1 stoi-
chiometry. All attempts to fit the data points at a given wavelength
with a 1:1 stoichiometry model (nonlinear least square analysis of
the absorbance A versus the pre-equilibrium concentration cM of
Ca2+) failed. The method of continuous variations applied for Ca2+
showed that the absorbance reaches a maximum at the mole frac-
tion of 0.4 (see Job plot: Fig. S3 in the ESI). This value points to
higher order association reaction such as 2L + 3M M L2M3. Despite
being not very common, this stoichiometry is reasonable when
considering the X-ray structure of the corresponding barium com-
plex. From a quantitative point of view, calculation of the binding
constant for stoichiometry other than 1:1 or 1:2, is more compli-
cated because there is no explicit expression of the absorbance ver-
sus the total Ca2+ concentration cM if the approximation [Ca2+] ꢄ cM
is not valid [31]. Therefore, we were not able to treat the data
the redox probe in 3 derives from an o-tetramethylphenylenedi-
amine (o-TMPD), it is interesting to compare the electrochemical
response of 3 to the parent o-TMPD whose redox behavior is well
established in terms of reversibility and oxidation peak potentials
(0.59 and 0.84 V versus SCE corresponding to the radical cation and
dication formation respectively) [32].
The tetra-ester derivative 3 exhibits two irreversible oxidation
peaks at 595 and 900 mV vs SCE at a scan rate of 100 mV/s. The
first oxidation process assigned to the formation of the radical cat-
ion occurs at a comparable potential to o-TMPD but remains irre-
versible at all explored scan rates (0.1 to 5 V/s). Therefore, the
replacement of two methyl groups of o-TMPD by a crown ether
group has no effect on the oxidation potential but alters the stabil-
ity of the o-phenylenediamine radical cation. This result differ from
the electrochemical behavior of o-TMPD substituted azacrown
ether reported by Sibert [33]. In that case, due to rapid conforma-
tional change the electron transfer is irreversible at low scan rate
and reversible at faster scan rates (v P 3 V/s).
quantitatively to extract the affinity constant of 4 for Ca2+
.
In the presence of increasing amounts of Ba2+, a progressive de-
crease of the first redox peak of the free ligand is observed until a
complete disparearence of this signal beyond one equivalent of
Ba2+. The electrochemical responses of chelators where complexa-
tion/decomplexation reactions are coupled to redox processes
(square scheme mechanism) have been thoroughly studied by
3.4. Electrochemistry
The electrochemical properties of ligands 3 (see Fig. 6) and 4
(see Fig. S10 in ESI) were explored by cyclic voltammetry in aceto-
nitrile and water respectively. The tetraester 3 has been success-
fully studied in the absence and in the presence of barium. Since
Fig. 5. (left). UV–Vis spectra of compound 4 (100
lM) in water with increasing amount of calcium (from 0 to 5 mM); (right) Variation of the absorbance at 263 nm as a
function of Ca2+ concentration. Inset: Enlargement of the variation of the absorbance at low ratio [Ca2+]/[4].

L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
197
Fig. 7. 1H NMR studies of barium complexation in D2O.
means of theoretical and experimental tools. Two limiting electro-
chemical behaviors (‘‘two-wave’’ versus ‘‘anodic shift’’) are de-
scribed in the literature in the presence of sub-stoichiometric
amounts of cations. The electrochemical response of 3 observed
in the presence of sub-stoichiometric amount of Ba2+ differs widely
from the reported electrochemical behavior of redox-active aza-
crown ether in the presence of substoichiometric amount of cation.
For instance, ferrocenyl or o-TMPD substituted azacrown ether li-
gands exhibit an anodic-shift in the presence of increasing amount
of cation. [33–36] The second oxidation wave of 3 in the presence
of Ba2+ is barely affected suggesting a decomplexation of Ba2+ after
the first redox process. Such barium decomplexation is the key
step to release caged barium ions under electrochemical stimution
as mentioned in the introduction of this paper.
This behavior which could seem unsual, is attributed to conforma-
tional changes in the crownether upon complex formation
[26,29,37–39]. Depending on the relative conformation adopted by
the diaminobenzene ring with respect to the diazacrown ether, the
lone pair of N-donor will be more or less involved in the aromatic
system thus modulating the Lewis basicity of the macrocyclic nitro-
gen lone pair. As a consequence, the nitrogen donor in perpendicular
orientation with respect to the macrocycle, behaves more as a ter-
tiary aliphatic amine than an aꢀniline.
The methylenes NCH2–CO2 protons are significantly highfield
shifted by 0.25 ppm suggesting that the second amino substituent
(aminodiacetate) is also in a perpendicular orientation with re-
spect to the phenyl ring. The substantial conformational rearrange-
ment in the course of Ba2+ complexation due to the twisting of both
nitrogen donors of the phenyl ring induces coherently the down-
field shift of the aromatic protons.
3.5. NMR studies
The shifting of resonance peaks with increasing amount of Ba2+
in the medium was succesfully exploited to extract the affinity
constant of ligand 4 towards Ba2+ in water (logK ꢄ 4.4) and to con-
firm the expected 1:1 stoichiometry (see Fig. S9 in ESI). The affinity
constants for Ba2+ determined by NMR and UV–Vis techniques are
of the same order of magnitude and most importantly prove that
the ligand 4 is well suited to operate in the millimolar concentra-
tion range required for the in vitro barium stimulation of cells.
To probe the effective participation of the potential donors in
the cation coordination, 1H NMR studies in deuterated water were
conducted (see Fig. 7). The free ligand is characterized in the ali-
phatic region by overlapping multiplets at 3.55 ppm (CH2–N and
CH2–O) and a singlet at 3.98 ppm (NCH2–CO2-). A gradual increase
in Ba2+ concentration causes significant changes in the 1H NMR
spectrum with a progressive shifting of all resonance signals1
.
The magnitude of the shifting reaches a maximum at one equivalent
of barium per ligand. Further increase in Ba2+ concentration has no
significant effect on the chemical shifts, which is fully consistent
with the 1:1 stoichiometry, as evidenced by UV–Vis. Upon addition
of 0.1 equivalent of Ba2+ all the signals except one doublet in the aro-
matic region are shifted and the overlapping multiplets in the ali-
phatic region split into 2 signals. The resonance of the macrocyclic
methylenes CH2O protons are downfield shifted as a consequence
of the chelation of barium ion. Such low field shift is due to the de-
crease of electron density on carbon adjacent to O-donor involved in
Ba2+ complexation. Conversly, the macrocyclic methylenes CH2N are
highfield shifted in the presence of increasing amount of barium.
4. Conclusions
In summary, a novel water soluble chelating agent intended to
anther application than cation sensing has been designed and fully
characterized. The affinity of this ligand towards commonly
encountered metal ions in biological medium has been evaluated.
Except for calcium, all other complexes have been characterized
in solution as 1:1/M:L stoichiometry complexes. X-ray studies of
the resulting crystalline barium complex showed that the apparent
stoichiometry of the diazacrown ether in the solid state is not nec-
essarly a reliable measure of the actual metal–ligand binding in
solution. NMR and electrochemical studies revealed conforma-
tional rearrangement of the ligand upon complexation of barium
ion and upon oxidation. The redox-induced barium release is cur-
rently investigated in our group.
1
In the presence of, Ca2+, the 1H NMR spectrum proved to be hardly exploitable as
all signals are broaden which points to a slow equilibrium compared to the time scale
of, NMR experiment.

198
L. Boubekeur-Lecaque et al. / Polyhedron 68 (2014) 191–198
[12] K.X. Bhattacharyya, L. Boubekeur-Lecaque, I. Tapsoba, E. Maisonhaute,
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XiamENS), ENS, the Université Pierre et Marie Curie. We thank
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fruitful discussions about exocytosis cascade and cell stimulation.
Appendix A. Supplementary data
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CCDC 940233–940234 contains the supplementary crystallo-
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from the Cambridge Crystallographic Data Centre, 12 Union Road,
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