Chelation of heavy group 2 (radio)metals by p-tert-butylcalix[4]arene-1,3-crown-6 and logK determination via NMR

Article abstract of DOI:10.1016/j.saa.2018.03.029

A crown-bridged calix[4]arene scaffold was investigated as lead compound for the ligation of heavy alkaline earth metals such as strontium and barium, which appear to be useful for radiopharmaceutical applications in diagnosis as well as in radiotherapy. In particular barium, due to its chemical similarities, could serve as a surrogate for radium, a metal of high radiopharmaceutical interest. The ability of p-tert-butylcalix[4]arene-1,3-crown-6 (1) in particular to chelate cations, such as group 1 and 2 metal ions or ammonium ions is well known. Also, the manifold possibilities of structural modification on the upper- and lower-rim as well as on the crown itself produce properties that may lead to a highly selective and effective chelating agent. In this work, titration experiments of the perchlorate salts of Ba²⁺, Sr²⁺ and Pb²⁺ with ligand 1 were performed to determine their stability constants (logK = 4.7, 4.3, and 3.3, respectively) by ¹H NMR measurements in acetonitrile-d₃.

Full text of DOI:10.1016/j.saa.2018.03.029

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 199 (2018) 50–56
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Chelation of heavy group 2 (radio)metals by p-tert-butylcalix[4]
arene-1,3-crown-6 and logK determination via NMR
David Bauer a,b, Matthew Gott a, Jörg Steinbach a,b, Constantin Mamat a,b,
⁎
a
Helmholtz-Zentrum Dresden-Rossendorf, Institut für Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400, D-01328 Dresden, Germany
b
Technische Universität Dresden, Fakultät Chemie und Lebensmittelchemie, D-01062 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A crown-bridged calix[4]arene scaffold was investigated as lead compound for the ligation of heavy alkaline earth
metals such as strontium and barium, which appear to be useful for radiopharmaceutical applications in diagno-
sis as well as in radiotherapy. In particular barium, due to its chemical similarities, could serve as a surrogate for
radium, a metal of high radiopharmaceutical interest. The ability of p-tert-butylcalix[4]arene-1,3-crown-6 (1) in
particular to chelate cations, such as group 1 and 2 metal ions or ammonium ions is well known. Also, the man-
ifold possibilities of structural modification on the upper- and lower-rim as well as on the crown itself produce
properties that may lead to a highly selective and effective chelating agent. In this work, titration experiments
of the perchlorate salts of Ba2+, Sr2+ and Pb2+ with ligand 1 were performed to determine their stability con-
stants (logK = 4.7, 4.3, and 3.3, respectively) by 1H NMR measurements in acetonitrile-d3.
Received 12 December 2017
Received in revised form 9 February 2018
Accepted 12 March 2018
Available online 13 March 2018
Keywords:
Calix[4]crown complex
Barium
Stability constant
207Pb NMR
© 2018 Elsevier B.V. All rights reserved.
Radium
1. Introduction
found regarding the application of the light alkaline earth metals beryl-
lium, magnesium, and calcium for therapy or diagnosis.
Radium is the heaviest known member of the alkaline earth metals
and all 33 of its isotopes are radioactive [1]. Four of these isotopes are
available from the decay of primordial nuclides [2]. Two of these,
radium-223 and radium-224, have suitable half-lives (223Ra: 11.4 d,
224Ra: 3.6 d) and nuclear decay properties that make them useful tools
for alpha particle therapy [3]. Alpha particles are highly energetic
(5–9 MeV) and create frequent ionization events (approx. 80 keV/μm)
across a short path length (40–100 μm), which leads to a high effective-
ness for tumor cell killing [4]. Both 223Ra and 224Ra rapidly decay via se-
ries of six daughter nuclides (four alpha and two beta particles) to stable
207Pb and 208Pb, respectively, which results in massive energetic emis-
sions of 28 and 27 MeV, respectively [1]. In 2013, [223Ra]RaCl2 (Xofigo®)
became the first and, until now, the only alpha-emitting radiopharma-
ceutical to receive FDA and EMEA approval for clinical use.
The homologous elements barium and strontium exist as stable nu-
clides, but offer radioisotopes for radiopharmaceutical applications [5].
131Ba (Eγ = 496 keV, 48%) and 135mBa (Eγ = 268 keV, 16%) are both
possible γ-emitters for diagnostic use and were broadly discussed as
bone-scanning agents in scintigraphy [6–8]. 90Sr (Eβ,max = 546 keV,
100%) also finds extensive application as a strong beta-emitter for su-
perficial brachytherapy of some cancers [7,9,10]. No literature was
Searching for suitable ligands, a group of molecular baskets termed
calix[4]arenes was found to be a promising start. Calix[4]arenes are
metacyclophanes having a hydrophobic cavity between the lower and
upper rim, formed by four p-tert-butylphenol units bridged with meth-
ylene links [11]. This class of macromolecules is known for a wide range
of applications [12,13], most likely due to their numerous possibilities
for functionalization [14]. Calix[4]arenes act as biologically active com-
pounds and are used as antibacterial and even anti-malarial agents or
in cancer chemotherapy [15–18]. Additionally, they can be promising
enzyme inhibitors or interact with amino acids [19,20]. Their ability to
form inclusion compounds with neutral molecules or ions makes
them useful as sensors, catalysts, ligands or, when bound to a resin, as
effective separation agents for ions [21–26].
The calix[4]arene framework can also be seen as a platform for
building an optimized chelator. On the lower rim, there are four hy-
droxyl groups. Two can be functionalized as proton-ionizable groups
to form a neutral complex with divalent cations; the remaining two
can be bridged by a crown ether. With this concept, both the advantages
of the electrostatic, macrocyclic, and cryptate effect are combined. An-
other benefit of the calix[4]crown-6 scaffold is the easy access [27,28].
To provide the ideal cavity for heavy group 2 metals, it is essential to
choose a suitable crown size. The group around Bartsch focused on ex-
traction of alkaline earth metal cations using calixcrowns [29–31]. As a
result of their studies, calix[4]arene-1,3-crown-6 derivatives have
been found very effective for Ba2+. Barium, strontium and radium pos-
sess analogous chemical properties and their radii are of a similar range
⁎
Corresponding author at: Helmholtz-Zentrum Dresden-Rossendorf, Institut für
Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400, D-01328 Dresden,
Germany.
E-mail address: c.mamat@hzdr.de (C. Mamat).
https://doi.org/10.1016/j.saa.2018.03.029
1386-1425/© 2018 Elsevier B.V. All rights reserved.

D. Bauer et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 199 (2018) 50–56
51
[32], therefore, these studies were considered to be a useful starting
2.2. Synthesis of ligand 1
point. Additionally, these compounds showed a high selectivity
for barium over the lighter alkaline earth metals or alkali metals, how-
ever, Ra2+ was not investigated. Van Leeuwen et al. [33–35] compared
the efficiency of various ligands including calixarenes as ionophores
for Ra2+ extraction in the case of nuclear waste management. They
also investigated derivatives of p-tert-butylcalix[4]arene-1,3-crown-6
p-tert-Butylcalix[4]arene-1,3-crown-6 (1) was synthesized accord-
ing to the literature [29]. Briefly, a suspension of p-tert-butylcalix[4]
arene (1.00 g, 1.54 mmol), pentaethylene glycol di(p-toluenesulfonate)
(924 mg, 1.69 mmol) and K2CO3 (256 mg, 1.85 mmol) in acetonitrile
(100 mL) was refluxed under argon for 7 days. After cooling to rt, the
solvent was removed and CH2Cl2 (50 mL) was added. The suspension
was washed with 10% HCl (2 × 50 mL) and water (1 × 50 mL). The or-
ganic layer was dried over Na2SO4. After evaporation of CH2Cl2, the
crude mixture was purified by column chromatography (SiO2, petro-
leum ether/ethyl acetate, gradient: 0% → 60%). The product was ob-
tained as a colorless solid (580 mg, 50%). Analyses were in accordance
with the previously published literature [29].
(1) and showed that they have high extraction rates and
a
suitable selectivity in contrast to simple (aza-)crown ethers. In
radiopharmacy, a high stability constant of the complex is important
so that a radium release and accumulation in bone tissue is minimized.
The objective of this research was to evaluate p-tert-butylcalix[4]
arene-1,3-crown-6 (1) as a possible leading compound that could,
upon further modification, yield a viable chelator for heavy group 2
metals in radiopharmaceutical application. Existing literature about
group 2 metal ligands specifically with radium are focused mainly on
extraction studies, while useful, this does not provide information
about comparable stability constants [30,33,36]. Therefore, a reliable
and constant method for the calculation of stability constants with bar-
ium and strontium via NMR spectroscopy was developed to determine
the efficiency of ligand 1. Since there is no stable radium isotope, barium
is additionally used as non-radioactive surrogate, due to its related
chemical behavior, in vivo distribution, and size [2,37,38]. To further
guarantee the size selectivity of the basic structure, sodium and
tetrabutylammonium cation were additionally investigated. Referring
to radiopharmaceutical application, the competitors of interest are the
ions, which primarily occur in blood plasma, such as sodium. This ion
is normally found in high concentrations (135–150 mM) and is there-
fore of particular relevance [39].
2.3. 1H NMR titration measurements
A solution of 1 was prepared in the appropriate deuterated solvent
(2.0 ∙ 10−3 M) and 1.0 mL was pipetted in a NMR tube. The sample
was referenced to the residual solvent signal. Then, the complexation
of cations with 1 was studied. A 0.1 M solution of the metal perchlorate
was prepared in the same solvent. Next, stepwise portions (2 μL) of the
respective perchlorate solution were added into the NMR tube contain-
ing the ligand, and after extensive mixing the complexation-induced
shifts were recorded. At a ligand:metal ratio of 2:3, 30 μL portions of a
1.0 M perchlorate salt solution were used, and stepwise additions
were continued until a ligand:metal ratio of 1:6 was reached to exclude
the formation of a complex with another stoichiometry. The displace-
ments of selected 1H NMR signals of ligand 1 upon addition of the per-
chlorate salt were used to calculate the complex stability constants.
The calculations were performed using the WinEQNMR2 software
[42]. The advised range for the data input covers the addition of metal
to ligand from 0.1 to 0.9 equivalents. This instruction was followed
and 9 points in this range were measured (steps of 0.1 equiv.) and
used for the calculation. The formation of a 1:1 complex was proven
by plotting the changes of selected signals against the cation to chelate
ratio, observing the change of the slope at a ligand:metal ratio of 1:1.
Furthermore, the interaction of ligand 1 and Pb2+ was studied by
using 1H and 207Pb NMR spectroscopy. For our research, lead is also a
metal of interest. On the one hand, it is the stable end product of both
radium decay chains. On the other hand, 212Pb is a promising β−
-
emitter (570 keV max β−, 12%), and a feasible candidate for radiophar-
maceutical applications, since it can also be used as an in vivo generator
for 212Bi, which is a strong alpha emitter [40,41].
2. Experimental Section
2.4. 207Pb NMR titration measurements
2.1. General
A solution of lead (II) perchlorate trihydrate in acetonitrile-d3
(5.0 ∙ 10−2 M) and a solution of ligand 1 in acetonitrile-d3 (1.0 ∙
10−1 M) were prepared. A 1.0 mL aliquot of the lead solution was pi-
petted in a NMR tube. The sample was measured and the 207Pb signal
determined. Next, stepwise portions of ligand 1 (40 μL, 0.08 equiv.)
were added into the NMR tube containing the lead solution, and
the spectra were recorded after extensive mixing.
1H NMR spectra were recorded on an Agilent DD2–400 MHz NMR
spectrometer with ProbeOne at 298 K. Chemical shifts of the spectra
were reported in parts per million (ppm) using TMS as internal stan-
dard. All 207Pb spectra were recorded at a frequency of 125.1 MHz on
an Agilent DD2–600 MHz NMR spectrometer with ProbeOne at 298 K
using a 90° pulse width of 6.0 μs, a 0.157 s acquisition time, and a 0.6 s
delay time. A 1.0 M Pb(NO3)2 solution (natural) was used as an external
standard (δ = −2965 ppm, D2O, 25 °C; relative to PbMe4). For the syn-
thesis of ligand 1, 4-tert-butylcalix[4]arene (abcr, 99%), pentaethylene
glycol di(p-toluenesulfonate) (Alfa Aesar, 95%), potassium carbonate
anhydrous (Acros, 99 + %), acetonitrile (Fisher Scientific, HPLC-
grade), dichloromethane (Fisher Scientific, HPLC-grade), hydrochloric
acid (Merck, 37%), and sodium sulfate anhydrous (Alfa Aesar, 99%)
were used as obtained. Preparative column chromatography was car-
ried out with silica gel 60 (Merck, particle size 0.040–0.063 mm), pe-
troleum ether (Fisher Scientific, bp 40–60 °C, analytical reagent
grade), and ethyl acetate (Fisher Scientific, HPLC-grade). For the
preparation of the complexes, barium perchlorate (Alfa Aesar,
99%), sodium perchlorate (Alfa Aesar, 98%), strontium perchlorate
(abcr, 99.9%), and tetrabutylammonium perchlorate (Acros, 98%)
were dried at room temperature under vacuum and used without
further purification. Lead (II) perchlorate trihydrate (abcr, 97%)
was used as obtained. The solvents used for NMR measurements
were purchased from Deutero GmbH.
3. Results and Discussion
3.1. Initial 1H NMR Studies
To determine the stability constant for the complexation of Ba2+
,
Sr2+, and Pb2+, a reliable method was developed using 1H NMR spec-
troscopy. To study the influence of the solvent on the complexation
and to optimize shift characterization, NMR measurements involving
ligand 1 were carried out in various solvents: CDCl3, DMSO-d6,
acetone-d6, acetonitrile-d3, and methanol-d4. Since ligand 1 is not solu-
ble in aqueous solutions, D2O was not used. Afterward, stability con-
stant measurements were performed using Ba2+, Sr2+, Pb2+, Na+
,
and Bu4N+ as their perchlorate salts in acetonitrile-d3.
First, the protons of ligand 1 were assigned to their signals. The 1H
NMR spectra of 1 in different solvents are shown in Fig. 1 and the assign-
ment with the chemical shifts is listed in Table 1. A C2-symmetry is
found for ligand 1 which results in the number of 12 1H signals.

52
D. Bauer et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 199 (2018) 50–56
4
5
3
2
H
6
endo
OH 1
O
O
OH
O
O
H
7
exo
8
9
10
11
O
O
12
Ligand 1
Fig. 1. 1H NMR spectra of ligand 1 in different solvents (solvent peaks have been removed for clarity).
Next, the titration method had to be developed, which was repre-
Consequently, all NMR titration experiments were performed in
acetonitrile-d3. Therefore, 1H NMR spectra of a 2.0 ∙ 10−3 M solution
(1 mL) of 1 were measured upon stepwise addition (2 μL) of a 0.1 M so-
lution of the perchlorate (prepared in acetonitrile-d3). Each step repre-
sents the addition of 0.1 equiv. of the metal perchlorate to ligand 1. In
Fig. 2, the titration experiment with Ba2+ is shown. Shifts for all proton
signals are observed.
The differences of the chemical shifts ΔδH2-H3, ΔδH7-H6 and ΔδH5-H4
upon Ba2+ addition indicate a significant alteration in the orientation
of the aromatic rings. ΔδH7-H6 decreased from 0.99 ppm (ligand) to
0.47 ppm (1:1 complex). The shifting of these doublets is related to a
twisting of the aromatic rings [46]. The general presence of the doublets
proves the fixed cone conformation, since a free rotation of the aryl
rings would cause only a singlet [47]. The distance between the doublets
H6 and H7 results from the orientation of the protons into or out of the
aromatic ring current and gives information about the cone flattening
[48]. The difference in the chemical shift between the axial (H7) and
equatorial (H6) protons (ΔδH7-H6) gets smaller when the aromatic
rings rotate (Fig. 3). It can be deduced from Fig. 2 that the two non-
sentatively executed with Ba2+ as guest ion. For the calculation of stabil-
ity constants, the chemical shifts had to be exactly determined [43]. To
ensure this, the changes of the chemical shifts in the 1H signals between
ligand 1 and the Ba2+ complex must be sufficiently pronounced. For this
reason, it had to be ascertained which solvent is most advantageous
[44]. Since the solubility of the perchlorate salts is very low in CDCl3
and the solubility of the formed complexes is also insufficient, this sol-
vent could not be used. Remarkably, upon addition of barium perchlo-
rate to a solution of 1 in DMSO-d6 there was no change visual in the
1H spectra. This can be attributed to the formation of Ba-DMSO-
complexes which are described in the literature [45]. Once formed,
these complexes are not interacting with the calix[4]crown
anymore. The DMSO-interaction is discussed in more detail later.
The use of acetone-d6 and methanol-d4 showed clear changes in
the spectra comparing the ligand and the 1:1 complex. However,
the strongest difference was obtained in acetonitrile-d3 which
makes it the favorable solvent for the titration experiment (see SI
for spectra).
Table 1
1H NMR (400 MHz,) shifts of compound 1.
1H Signala
Multiplicity [Hz]
Integral
Shift [ppm]
CDCl3
Assignment
Acetone-d6
DMSO-d6
Acetonitrile-d3
Methanol-d4
H1
s
2
6.96
8.22
8.19
8.02
–
OH
H2
H3
H4
H5
H6
H7
H8
H9
s
s
s
s
d
d
m
m
m
m
s
4
4
18
18
4
4
4
4
4
7.07
6.74
0.91
1.31
3.30
4.36
4.12–4.08
4.02–3.98
3.95–3.92
3.86–3.82
3.77
7.19
7.13j
1.03
1.25
3.43
4.45
4.19–4.15
4.11–4.07
3.92–3.88
3.82–3.79
3.67
7.11
7.09
1.10
1.18
3.38
4.26
4.10–4.06
3.98–3.94
3.81–3.77
3.74–3.70
3.60
7.21
7.16
1.15
1.21
3.37
4.36
4.17–4.14
4.00–3.96
3.84–3.81
3.78–3.75
3.65
7.14
7.00
1.06
1.29
3.37
4.45
4.19–4.15
4.05–4.01
3.96–3.91
3.89–3.84
3.75
Ar-H
Ar-H
tBu
tBu
Hendo
Hexo
CH2O
CH2O
CH2O
CH2O
CH2O
H10
H11
H12
4
4
a
Is not representing the IUPAC assignment.

D. Bauer et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 199 (2018) 50–56
53
4
5
3
2
H
6
endo
OH 1
O
O
OH
O
O
H
7
exo
8
9
10
11
O
O
12
Ligand 1
Fig. 2. 1H NMR spectra of ligand 1 at different Ba(ClO4)2 concentrations measured in acetonitrile-d3.
bridged aromatic rings turn, so that the hydroxyl groups point towards
the Ba2+ ion which is located on the lower rim. This is explained by
Fig. 4. When ring B rotates at a certain angle, the meta-aryl proton will
face ring A frontally. The proton will perceive a lower magnetic field
and this will be noticed as a high field shift in NMR measurements. If
the proton moves to the outside of ring A, it will perceive a higher mag-
netic field and this will be noticed as a down field shift. The tert-butyl
group behaves contrary. When the meta-aryl proton of ring B faces
ring A, the tert-butyl group directs to the outside of ring A and will per-
ceive an increase of the magnetic field strength this results in a displace-
ment of the 1H signal to down field. For this reason, H2 and H4 are high
field but H3 and H5 down field shifted.
Not only an interaction with the calix cone can be determined but
also clear changes in chemical shifts of the crown signals (H8-H12)
are observed due to the coordination of Ba2+ to the ether oxygen
atoms. The strongest movements occurred for the chemical shifts of
the crown protons H8, H9 and H10, whereas only a slight movement
was found for the signals H11 and H12. This indicates that the Ba2+ is
bound more closely to the lower calix rim, strongly interacting
with the aromatic ether oxygen atoms but hardly with the upper
ether oxygen atoms of the crown. However, using Fig. 4, another ef-
fect can be explained. In the lowest energy conformers of ligand 1 the
OH groups form hydrogen bonds with neighboring aromatic ether
oxygen atoms and the lower rim is pushed together [11]. As a result
of this interaction, the OH signals are located down field (δ =
8.02 ppm, acetonitrile-d3) [49]. The incorporation of the barium
ions lead to a conformational change, which is causing a decrease
of the hydrogen bonds by opening the lower rim, and therefore a sig-
nificant shift of the OH groups to the high field is observed (δ =
7.54 ppm, acetonitrile-d3, Ba2+-1:1-complex). This is noteworthy,
since otherwise, it would be expected that the interaction of barium
with the OH groups of ligand 1 would result in a shift to the down
field, due to the electron withdrawing effect of the ion.
For evaluation of the data, the four 1H signals of H2, H3, H4 and H5
were used since these singlets were easy to follow upon Ba2+ addition.
When the 1H shifts are plotted against the equivalents of Ba2+, Fig. 5 is
Fig. 3. Conformational dependence of ΔδH6-H7 [11].

54
D. Bauer et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 199 (2018) 50–56
Fig. 4. Conformational dependence of ΔδH2-H3 and ΔδH4-H5
.
obtained. All curves show a change of the slope at the ratio of 1:1, indi-
cating the formation of a 1:1 complex.
caused by the ring current effect, but by the diminishing of the hydrogen
bonds between the hydroxyl groups and the aromatic ether oxygens.
In contrast, Sr2+ shows a different interaction with 1. It can be
looked up in the titration experiment data (SI) that the 1H signal H2,
which shifts strongly for Ba2+ (Fig. 2), only shifts slightly upon Sr2+ ad-
dition. Δδ's listed in Table 3 reveal that the Sr2+-1 complex is not com-
parably pinched to the Ba2+-1 complex. The H2 signal refers to the
meta-protons of the non-bridged aromatic rings. This indicates that
Sr2+ is not situated directly at the lower rim of the calix cone, and not
actively influencing the orientation of the aromatic rings, like Ba2+ is.
Additionally, there only is a small shift of the OH signals to the high
field compared to barium (δ = 7.86 ppm, Sr2+-1:1-complex / δ =
7.54 ppm, Ba2+-1:1-complex), showing, that there is only a minor
opening of the lower rim. It is supposed to sit lower in the crown and
the wrapping of the crown around Sr2+ leads to a pinch of exclusively
the two attached aromatic rings. As a result, the meta-aryl protons refer-
ring to the H3 signal are facing the other two aromatic rings which is
confirmed by the down field shift of signal H3. The rotation of the
rings can be confirmed by the ΔδH7-H6 value. Other than barium, stron-
tium induces significant shifts for the 1H crown signals H11 and H12,
proving that it is situated in the lower crown. These facts confirm a dif-
ferent binding mode. The reason why Sr2+ is not incorporated into the
calix cone might be the significant smaller ionic radius. For a similar in-
teraction as Ba2+, Sr2+ would demand a greater pinch of the cone. Con-
sidering the necessary tension of the cone, it is comprehensible that
interaction of the strontium and the aromatic ether oxygens is not ad-
vantageous. This is resulting in a stronger interaction with the ether ox-
ygens in the middle of the crown. It is notable that only the smaller
radius, considering similar chemical properties of both ions, results in
a decrease of the stability constant. Since the radius of Ra2+ is slightly
bigger than the radius of Ba2+ it might result in a small increase of the
complex stability.
The same titration method was applied to the perchlorate salts of
Sr2+, Pb2+, Na+ and Bu4N+. For the small Na+ and the bulky
tetrabutylammonium ion, no changes in the spectra were observed, in-
dicating that 1 shows size selectivity for the cations of interest. Further-
more, these two experiments prove that there is no interaction
between the perchlorate and ligand 1. For the divalent metals Sr2+
and Pb2+ incorporation into the calix[4]arene-crown cavity was ob-
served as well, and the formation of a 1:1 complexes was confirmed
(see SI).
After the 1H NMR shifts upon Ba2+, Sr2+ and Pb2+ addition were
measured, the stability constants were calculated [42]. The logK values
for the 1:1 complex of ligand 1 with Ba2+, Sr2+and Pb2+ calculated for
different 1H signals are listed in Table 2.
For the calculation of the stability constant of the Pb2+-1 complex,
the signal H2 did not move significantly enough and signal H3 was too
broad to be used. The calculation was performed with the chemical
shifts of signals H4 and H5 only, but due to the shape of these signals
with high inaccuracy.
Stability constants for all three metal ions were calculated, where-
upon the choice of a specific 1H signal plays no significant role for the re-
sult. Overall, the highest stability constant was calculated for Ba2+
(approx. 4.7) followed by Sr2+ (approx. 4.3) and Pb2+ (approx. 3.3).
As mentioned before, Ba2+ appears to be located close to the lower
rim of the calix, strongly interacting with the aromatic ether oxygens
and barely with the top of the crown. Upon complexation, the 1H signals
for the hydroxyl protons H1 are high field shifted (Fig. 2). This is not
Pb2+ has a similar size to Sr2+, but different chemical properties.
Upon addition of lead perchlorate to ligand 1 the proton signals of the
measured spectra appeared very broad and almost vanished (for titra-
tion experiment data see SI). Interestingly, 1H signal H2 was the only
one, which was barley affected upon the titration, like it is for Sr2+. As
soon as reaching the 1:1 ratio, the signals are again sharply defined.
This behavior indicates a fast exchange of Pb2+ between the free ligands
and the complex during the timescale of a 1H measurement. The
Table 2
Stability constants for ligand
1
determined by 1H NMR titration experiment in
acetonitrile-d3.
Cation
Stability constants, determined by selected 1H signals
H2
H3
H4
H5
Ba2+
Sr2+
Pb2+
4.6 0.1
–
–
4.9 0.2
4.4 0.1
–
4.6 0.1
4.3 0.1
4.8 0.2
4.3 0.2
Fig. 5. Shifts of four selected 1H NMR signals of 1 at different Ba(ClO4)2 concentrations
measured in acetonitrile-d3. ○: shift of H2, ■: shift of H3, △: shift of H5, ◆: shift of H4.
3.3
1
3.2
1

D. Bauer et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 199 (2018) 50–56
55
Table 3
Comparison of the Δδ of ligand 1 and its 1:1 complexes (negative values represent the
crossing of the two peaks).
Compound
ΔδH2-H3
ΔδH7-H6
ΔδH5-H4
Ligand 1
0.05
0.99
0.47
0.43
0.49
0.06
0.22
0.15
0.14
Ba2+-1:1 Complex
Sr2+-1:1 Complex
Pb2+-1:1 Complex
−0.19
−0.11
−0.08
titration also reveals a strong interaction with the crown, indicating that
Pb2+ is situated only in the crown, and the wrapping of the crown
around Pb2+ leads to the pinch of the cone, comparable to Sr2+. Com-
paring the Δδ's in Table 1 the pinch is least distinct for this guest ion.
Furthermore, there is no significant shift of the OH signals to the high
field, showing intact hydrogen bonds and no opening of the lower
rim. Pb2+ shows a low stability constant in this study, the calix scaffold
seems to have no significant effect on this ion.
Fig. 7. Shift of the 207Pb NMR signal upon DMSO addition to a 1 M Pb(ClO4)2 solution in
acetonitrile-d3.
3.2. Pb 207Pb NMR Studies
Since 207Pb (natural abundance: 22.6%) has a medium sensitivity
NMR spin-½ nucleus [50,51], 207Pb NMR is a valuable and helpful tool
to describe the chemical situation in the Pb-calix complex. Thus, 207Pb
spectra were recorded upon ligand 1 addition to a 1 M Pb(ClO4)2 solu-
tion. In contrast to the 1H nucleus, the relaxation time for the 207Pb nu-
cleus is very short. During this short time range no exchange process of
the lead ion between ligand 1 and the complex Pb2+-1 was observed. As
a result, the received spectra (Fig. 6) show two signals: a decreasing sig-
nal for the free Pb2+at δ = −3380 ppm and an increasing signal for the
Pb-calix-complex downfield shifted at δ = −3100 ppm. A slight shift for
the free Pb2+ signal is noticed and is dependent on the free metal ion
concentration (see SI and [52]). This data confirms the formation of a
1:1 complex, but it cannot be used for the calculation of the stability
constant.
To confirm the previously mentioned complexation of Pb2+ with
DMSO, an additional experiment was examined. The 207Pb NMR titra-
tion was repeated with stepwise addition of DMSO to a 1 M Pb(ClO4)2
solution instead of ligand 1 (Fig. 7, see also SI). A strong shift of the
Pb2+ signal upon DMSO addition was observed. This proves the forma-
tion of Pb2+-DMSO complexes which are also expected for barium and
strontium [45]. This fact makes it impossible to perform the titration ex-
periments in DMSO-d6, as discussed previously.
Fig. 6. 207Pb NMR spectra at different ligand concentrations measured in acetonitrile-d3.

56
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