Combined study of anion recognition by a carbazole-based neutral tripodal receptor in a competitive environment

Article abstract of DOI:10.1039/c2ob06868k

Anion recognition studies have been carried out on a series of neutral synthetic receptors in which carbazole-2-carboxamide has been used as building block. Different ligands which include one to three carbazole units in their structure have been prepared

Full text of DOI:10.1039/c2ob06868k

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PAPER
Combined study of anion recognition by a carbazole-based neutral tripodal
receptor in a competitive environment†
David Curiel,*a Guzmán Sánchez,a Carmen Ramírez de Arellano,b Alberto Tárragaa and Pedro Molinaa
Received 6th November 2011, Accepted 23rd December 2011
DOI: 10.1039/c2ob06868k
Anion recognition studies have been carried out on a series of neutral synthetic receptors in which
carbazole-2-carboxamide has been used as building block. Different ligands which include one to three
carbazole units in their structure have been prepared. Binding experiments have been performed under
competitive conditions in DMSO and DMSO–water solutions. The tripodal receptor offered a better
host–guest association due to the synergistic effect of a well arranged set of hydrogen bonds. A selective
response towards the biologically important pyrophosphate anion has been achieved. This selectivity is
enhanced when studies are carried out with an increasing water content, which gets as high as 20% (v/v)
in NMR experiments. The significance of this result lies in the use of a neutral receptor which exclusively
interacts with the anionic guest through hydrogen bonding. The influence of multiple equilibria in the
studied system has been analysed. Several techniques (1H NMR, 31P NMR, mass spectrometry, diffusion-
NMR, ITC, absorption and emission spectroscopy) have been employed to get a better understanding of
the different processes taking place in solution for the evaluated receptors.
ability is exclusively based on much weaker interactions such as
Introduction
hydrogen bonds,8 especially in those cases in which aqueous
media are involved.9
In this context we have synthesised a new family of receptors
Anion recognition and sensing is becoming a well settled disci-
pline in supramolecular chemistry.1 The important roles that
anions play in biological systems and the environment make
them very interesting analytical targets.2 In this regard, the detec-
tion of pyrophosphate3 becomes specially relevant due to its
presence as a product of DNA amplification in PCR (polymerase
chain reaction) processes,4 as well as its relation to different
pathologies.5 The design of new synthetic receptors for anionic
species utilises a wide range of approaches depending on the
driving force for the anion recognition. In the case of pyropho-
sphate sensing in a highly competitive environment, receptors
based on metal coordination complexes, which interact with the
anion through Coulombic attractions and strong dative covalent
bonds, represent a very efficient approach.3,6 Additionally, cat-
ionic receptors which combine the electrostatic interaction with
hydrogen bonding are also quite common.7 Conversely, it is very
rare to find neutral receptors for pyrophosphate whose binding
which incorporate the carbazole unit with different topologies,
ranging from a simple 2-carbamoylcarbazole, to a tripodal carba-
zole-2-carboxamide (Fig. 1). This binding motif defines a cavity
where three different types of hydrogen bonding can be estab-
lished from an amide NH function, a pyrrole-like NH and an aro-
matic CH. The structural arrangement highlights the utility of the
carbazole system in anion supramolecular chemistry both as
binding and signalling unit, which has not been extensively used
in the design of synthetic receptors.10 Although several anions
were initially tested, our studies finally focused on fluoride,
acetate, dihydrogenphosphate and pyrophosphate.11 In this
regard, a stronger interaction has been determined for the pyro-
phosphate anion, resulting in a highly selective complexation.
Results and discussion
aDepartment of Organic Chemistry, Faculty of Chemistry, University of
Murcia, Campus of Espinardo, 30100-Murcia, Spain.
The synthesis of receptors 1, 2 and 3 was carried out as depicted
in Scheme 1. Initially, 4-iodobenzoic acid was nitrated to obtain
4-iodo-3-nitrobenzoic acid, 4. The corresponding ethyl ester 5
was then isolated by refluxing 4 in ethanol under acidic catalysis.
A Suzuki–Miyaura cross-coupling between compound 5 and
phenylboronic acid yielded the corresponding biphenyl deriva-
tive 6. The carbazole unit was synthesised through a Cadogan
E-mail: davidcc@um.es
bDepartment of Organic Chemistry, Faculty of Chemistry, University of
Valencia, c/Dr. Moliner, 50 E46100-Burjassot, Valencia, Spain
†Electronic supplementary information (ESI) available: 1H NMR
spectra, 13C NMR spectra, titration fitplots, 31P-titration isotherms, mass
spectrometry, NOESY spectra, diffusion NMR fitplots, emission spectra
and X-ray diffraction data. CCDC 853238. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c2ob06868k
reaction by refluxing
6 with triphenylphosphine in 1,2-
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dichlorobenzene.12 Hydrolysis of the ester function under basic
conditions led to the isolation of the desired carbazole 2-car-
boxylic acid, 8. The reaction of this acid with benzylamine,
m-xylylenediamine or 2,4,6-triethyl-1,3,5-tri(aminomethyl)
benzene,13 in the presence of PyBOP (benzotriazol-1-yl-oxytri-
pyrrolidinophosphonium hexafluorophosphate), enabled the iso-
lation of ligands 1, 2 and 3, respectively.
intermolecular non-covalent interactions. In this regard two unu-
sually short intramolecular C–H⋯H–C contacts (1.830 and
1.946 Å) between the amide N-methylene group and both
methylene groups of the ethyl substituents on the benzene ring
could be detected. Furthermore, all three carbazole-2-carboxa-
mide units are not planar, but carboxamide–carbazole dihedral
angles of 29.4(1), 31.9(3) and 15.5(3)° have been found. This
fact could be due to the formation of receptor dimers through
intermolecular NH(carboxamide)⋯OvC hydrogen bonds with
two amide groups of one molecule acting as hydrogen bond
donors to a OvC bifurcated acceptor of an inversely related
molecule (Fig. 2).
Initially, anion binding studies were carried out in DMSO-d6
without any drying treatment.14 As expected, ligand 1 displayed
weaker responses than receptors 2 and 3. In general, it is worth
highlighting that no response was detected for chloride, bromide,
hydrogensulfate, nitrate, azide and cyanide anions; carboxylate
anions such as acetate and benzoate offered a very weak
All the compounds were characterised by the usual tech-
1
niques, i.e., H NMR, 13C NMR and mass spectrometry. In the
case of compound 3, single crystals could be grown, by slow
evaporation from a DMSO solution, suitable for X-ray diffrac-
tion analysis. In the crystal, the molecular structure of the recep-
tor shows a conformation where the three bulky carbazole-2-
carboxamide groups present different dispositions, with two of
them pointing to one side of the plane defined by the benzene
core and the third one pointing to the opposite side of that plane
(Fig. 2). This unexpected conformation, might be due to a solid
state packing pattern influenced by several intramolecular and
response in the NMR spectra; the more relevant results were
obtained for F−, H2PO4− and HP2O7
.
3−
Studies on compound 1 were performed as a useful set of
experiments to evaluate the validity of the carbazole-2-carboxa-
mide binding structure. Thus, when 1 was titrated with fluoride
anion the carbazole NH proton shifted downfield, corresponding
to a hydrogen bond to the anion, and disappeared after the
addition of 1 equivalent of anion. However, no interaction was
initially detected for the peaks corresponding to the CH on pos-
ition 1 of the carbazole and the amide NH, which was displaced
to lower chemical shifts from one equivalent of added anion
onwards (Fig. 3). The small size of the fluoride anion would
explain the absence of bonding to this proton and the sufficiency
of the interaction with the carbazole NH. Besides, the observed
upfield shift should be due to a shielding effect provoked by the
deprotonation of the carbazole by fluoride anion, as has been
argued by some other authors.15,16 Similar upfield shift could
1
also be observed in the H NMR spectrum for other protons on
the carbazole unit. The deprotonation by fluoride anion could be
confirmed by the detection of the HF2− triplet at 16.2 ppm. This
acid–base interaction was further supported by the titration of 1
with TBAOH, which displays a similar chemical shift pattern for
most of the monitored nuclei (see the ESI†).17
The fluoride titrations of 2 and 3 (Fig. 3) showed a different
evolution. In both cases the carbazole NH disappeared after
Fig. 1 Structures of carbazole-2-carboxamide receptors.
Scheme 1 Synthesis of carbazole-2-carboxamides: (a) HNO3, H2SO4; (b) EtOH, H2SO4; (c) PhB(OH)2, Pd(PPh3)4, Na2CO3, DME; (d) PPh3,
o-DCB; (e) NaOH, H2O, EtOH; (f) PyBOP, Et3N, CH2Cl2, benzylamine (1), m-xylylenediamine (2) or 2,4,6-triethyl-1,3,5-tri(aminomethyl)benzene
(3).
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Fig. 2 X-ray structure of receptor 3 (left) and its dimer (right). Solvent molecules have been omitted for the sake of clarity.
Fig. 3 Evolution of the chemical shifts of the carbazole CH(1) proton
of 1 (▲), 2 (■) and 3 (●), 2 × 10−3 M in DMSO-d6, upon titration
with TBAF.
adding the first aliquot of anion. Nevertheless the peak corre-
sponding to CH(1) experienced a downfield shift which reverted
to an upfield shift after a certain anion concentration (2–3
equivalents).18 This behaviour could be related to an initial
binding process favoured by the assistance of multiple branches
in 2 and 3, followed by the deprotonation of the carbazole
unit.16 A similar titration profile was also detected for the titra-
tion of 2 and 3 with TBAOH (see the ESI†), which would
confirm the influence of an acid–base equilibrium during the
titration with fluoride anion.
Fig. 4 Evolution of the chemical shifts of the amide NH proton of 1
(▲), 2 (■) and 3 (●), 2 × 10−3 M in DMSO-d6, upon titration with
TBAH2PO4.
The anion binding equilibrium was also followed by 31P
−
NMR titration for the more stable complex 3·H2PO4 (see the
ESI†). In this case, aliquots of the receptor were added into a
−
solution of the H2PO4 anion, whose chemical shift decreased
upon complexation. Reasonably good agreement was found
between the binding constants determined by 1H NMR (K = 440
M−1) and 31P NMR (K = 670 M−1) when fitted to a 1 : 1
stoichiometry.
When compound 1 was titrated with the H2PO4− anion a weak
binding could be determined. The evolution of the 1H NMR
spectra revealed that carbazole NH, CH(1) and amide proton
shifted downfield due to hydrogen bonding to the anionic guest
(Fig. 4), which contrasts with the result previously discussed for
3−
Titration experiments carried out with the HP2O7 anion evi-
denced a remarkably high affinity towards the carbazole-2-car-
boxamide unit (Fig. 5). Once again, the studies performed for
compound 1 showed an early vanishing of the carbazole NH.
The amide NH experienced a downfield shift before disappear-
ing in the proximity of one equivalent of added anion. Interest-
ingly, the peak corresponding to CH(1) did not initially displace
much, but between half an equivalent and one equivalent of
−
the fluoride anion. The larger size of the H2PO4 anion causes
the involvement of all the available hydrogen bond donor func-
tions in the structure of the receptors. A similar titration profile
was confirmed for both receptors 2 and 3, with a stronger
binding observed for the tripodal ligand, which has more anchor-
ing points and a better geometrical correspondence with the tet-
rahedral anionic guest. Regarding the rest of the carbazole
signals, they did not experience any significant displacement
during the titration experiment.
3−
HP2O7 it moved to a higher chemical shift, describing a sig-
moidal titration profile. This result is indicative of CH(1) hydro-
gen bonding the anion. In agreement with this evolution we
3−
Experimental data could be fitted to a 1 : 1 model by nonlinear
regression. The determined binding constants are summarised in
Table 1.19
could postulate that, due to the size of the HP2O7 anion, this
may induce the formation of a 2 : 1 host : guest complex, when
working at substoichiometric concentration. Then, as the
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Table 1 Binding constants for a 1 : 1 host : guest model in DMSO-d6
1
2
3
AcO−
55
14
—
65
20
>10 000
165
440
—
−
H2PO43−
a
a
HP2O7
[Host] = 2 × 10−3 M, T = 25 °C. Nonlinear regression analysis of the
CH(1) signal gave errors <10% in all cases.a Experimental data could
not be fitted.
Fig. 6 Evolution of the 1H NMR spectra of receptor 3 upon titration
with TBA3HP2O7. 1 : 1 complex signals (a, b, c, d). 2 : 1 (H : G)
complex signals (b′, c′).
carbazole NH could be observed for some of the intermediate
points of the titration experiment. Additionally, it could be
noticed that even proton CH(8) on the carbazole unit experi-
enced a downfield shift caused by hydrogen bonding to the large
HP2O73− anion.
Although it is not common to resort to CH bonds as hydrogen
bond donors, it is evident that a correct orientation of these
groups, as is the case for the carbazole system, can render useful
and thermodynamically favourable interactions which will con-
tribute to gain efficiency in the molecular recognition processes.
The graphical display of these data (Fig. 5) led us to an
interpretation based on the coexistence of two possible com-
plexes with 2 : 1 and 1 : 1 host : guest stoichiometries. Neverthe-
less, the sigmoidal titration isotherm abruptly reaches the
saturation zone and thwarts any possibility of calculating a
binding constant. The fact that a mixture of species can be
detected, along with the apparent strength of the anion binding,
makes the elucidation of the system difficult. However, a quali-
tative analysis of the titration profile unequivocally correlates to
a stronger complexation than the other studied receptors. As
expected, the increasing number of carbazole units enhances the
Fig. 5 Evolution of the chemical shifts of the carbazole CH(1) proton
of 1 (▲), 2 (■) and 3 (●), 2 × 10−3 M in DMSO-d6, upon titration
with TBA3HP2O7.
concentration of the anion increases, the complex rearranges to a
1 : 1 stoichiometry, which would demand more participation
from CH(1) proton.
As far as receptor 2 is concerned, the signals of the carbazole
and amide NHs disappeared after the addition of 0.2 equivalents
3−
of HP2O7 anion. Nevertheless, the latter appeared again after
the addition of 0.6 equivalents of anion, at much higher chemical
shift, as did the CH(1) proton. Accordingly, the CH(2) proton on
the m-xylylene spacer also shifts downfield, which confirms its
contribution to the anion binding as well as the optimum confor-
1
mation of the receptor. In general, all the peaks in the H NMR
3−
spectrum broadened at the initial stage of the titration but shar-
pened as the titration progressed as a consequence of the displa-
cement of the equilibrium towards the more stable anionic
complex. The titration profile could be fitted to a 1 : 1 model.
The calculated binding constant shows an extraordinary enhance-
ment of three orders of magnitude for the complexation of
HP2O7 anion recognition due to a better size correspondence
between the receptor and the guest, as well as the higher charge
of this anionic guest.
In order to get further evidence of the processes taking place
in solution, the system (3 + HP2O73−) was studied in more detail
by isothermal titration calorimetry (Fig. 7).
3−
HP2O7 anion with respect to the other tested anions, which
The plot of the thermodynamic data displays a complex
pattern, which cannot be fitted to any binding model but
reinforces our previous observation, which denotes the occur-
rence of multiple equilibria. The initial part of the experiment
shows an endothermic drop20 which is followed by two exother-
mic processes. This titration profile presents two inflection points
at 0.5 and 1 guest–host molar ratio, respectively, which would
confirm the evolution from a 2 : 1 to a 1 : 1 host : guest complex.
Additionally, NOESY experiments performed in the absence
reinforces the suitability of the carbazole-2-carboxamide binding
unit.
In this regard, when receptor 3 was titrated with a solution of
3−
HP2O7 anion a different behaviour could be detected (Fig. 6).
During the initial part of the titration all the signals broadened
and some of them were difficult to follow. Nevertheless, for
those detectable signals defining the binding cavity, namely
CH(1) and amide NHs, two set of peaks could be detected
between 0.3 and 0.7 equivalents. During the course of the titra-
tion, one of these groups of signals faded out as the other set of
peaks got better defined. With this particular receptor, even the
3−
and the presence of HP2O7 anion unveiled the induced fit
effect of the anion (see the ESI†). At this point, it is worth men-
tioning that the NMR spectra of the free receptor 3 showed a
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3−
Fig. 9 Evolution of the diffusion coefficient with increasing HP2O7
anion concentration.
Fig. 7 ITC enthalpogram of receptor 3, 2 × 10−3 M in DMSO, upon
addition of TBA3HP2O7. The heats of dilution were subtracted from the
raw data after a blank experiment.
Fig. 10 Absorption spectra of receptor 3, 2 × 10−5 M in DMSO, upon
titration with HP2O73− anion. Inset: titration profile at 370 nm.
titration profile clearly describes a sigmoidal curve, which again
should be interpreted as a result of a multiple equilibria system,
and is sharply differentiated from the result previously obtained
for the 31P NMR titration with H2PO4 anion, which fitted well
−
to a 1 : 1 binding model (see the ESI†).
When the addition of aliquots of HP2O7 anion to a solution
3−
of 3 was monitored by mass spectrometry, a peak corresponding
Fig. 8 NOEs detected in receptor 3 for the amide NH proton in the
to a 1 : 1 complex was detected (see the ESI†).21
absence (left) and the presence (right) of HP2O73− anion.
Furthermore, diffusion NMR studies were performed to gain a
better understanding of the speciation of the system. The diffu-
sion coefficient of 3 (0.16 × 10−9 m2 s−1) was calculated by fol-
lowing the attenuation of the carbazole signals. A decrease in
this coefficient was determined after the addition of aliquots of
single set of signals, which proves the magnetic equivalence of
all the appended carbazole units in solution at room temperature.
This differs from what was observed in the X-ray analysis of 3.
Interestingly, 2D-NOE spectra of 3 showed how the amide NH
proton presented a NOE with the protons H(1) and H(3) on the
carbazole. This confirms the rotation of the carbazole unit
around the C(2)–CO sigma bond. When one equivalent of
3−
the HP2O7 anion, as a result of the receptor 3 swelling up to
accommodate the anionic guest (Fig. 9). The evolution of the
experiment showed a drop in the value of the diffusion coeffi-
cient which did not cease until the addition of one equivalent of
anion. This result, in agreement with those obtained previously
from the different techniques discussed above, indicates that,
within the concentration range of 10−3 M, the species in equili-
brium evolve towards the formation of a 1 : 1 complex.22
3−
HP2O7 anion was added the most significant change corre-
sponded to the amide NH showing a NOE with proton H(1) of
the carbazole only (Fig. 8).
3−
Thus, we could conclude that upon HP2O7 complexation
The presence of carbazole as part of the tripodal receptor
allows the analysis of the anion complexation by following the
changes by absorption and emission spectroscopy.23 The absorp-
tion spectrum of 3 resembled that of the carbazole unit in
DMSO solution, showing an intense band at 307 nm and a
weaker band at 341 nm with a shoulder at 358 nm (Fig. 10).
the anion sitting in the cavity of the receptor blocks its confor-
mational freedom and makes the carbazole units point inwards,
which improves the binding ability of the tripodal receptor 3.
A
31P NMR titration was carried out, where a solution of
receptor 3 was added over a solution of TBA3HP2O7. Only one
phosphorus signal could be followed during the experiment. The
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The addition of anion caused a general decrease in the absor-
bance accompanied by a red shift of the bands. These changes in
3−
the spectra were evident for the HP2O7 anion. However, the
changes induced by H2PO4−, F− or AcO− anions were irrelevant.
Therefore, the dilution of the host enhanced the selectivity of 3
3−
towards HP2O7 anion. Under these diluted conditions the
possible 2 : 1 host : guest complex does not seem to manifest
itself since its concentration is negligible. Moreover, the presence
of four clearly resolved isosbestic points at 280, 312, 322 and
351 nm supports the prevalence of a 1 : 1 host : guest equili-
brium. Accordingly, the titration profile could be fitted to a
1 : 1 model with a binding constant value K = 1.8 × 104 M−1 24
.
The inherently higher sensitivity of emission spectroscopy
represents an important advantage of this technique within the
area of molecular recognition. Consequently, fluorescence titra-
tions were performed for receptor 3 with the above mentioned
anions. As occurred in previous experiments only the pyropho-
sphate anion offered a good response, with the rest of the tested
anions causing marginal changes in the emission spectrum
(Fig. 11) (see the ESI†).
Fig. 12 Emission spectra of receptor 3, 2 × 10−5 M in DMSO, upon
titration with HP2O7 anion. λexc = 300 nm. Inset: titration profile at
3−
391 nm.
The fluorescence spectrum has a band at 389 nm with a subtle
shoulder at 380 nm. A quenching in the emission intensity could
3−
be clearly observed for HP2O7 (Fig. 12) which, in agreement
with the UV-vis results, fitted well to a 1 : 1 binding model (K =
1.5 × 104 M−1). Curiously, an incipient growth of the fluor-
escence could be detected in the range of 450–550 nm which is
responsible for an analytically useful colour change in the fluor-
escence of the receptor.
This made us question the possibility of having a deprotona-
tion process interfering with the anion complexation equilibrium,
perhaps facilitated by the diluted conditions of the exper-
iment.25,26 Thus, control experiments were then carried out by
titrating TBAOH into a solution of 3. The effect of the addition
of a strong base was a fluorescence quenching during the initial
stage of the titration followed by the complete disappearance of
the original spectrum and the emergence of the emission from a
new species. This new spectrum has an emission maximum at
Fig. 13 Emission spectra of receptor 3, 2 × 10−5 M in DMSO, upon
3−
titration with (left) HP2O7 (0–50 equivalents) and (right) OH− (0–20
equivalents).
3−
After having checked the selectivity of 3 towards HP2O7
anion in DMSO solution we performed experiments under more
competitive conditions. Initially, the addition of five equivalents
of dihydrogenphosphate, acetate and benzoate to a solution of 3
493 nm which differs from the spectrum resulting from the pres-
1
3−
in DMSO did not produce important changes in the H NMR
ence of HP2O7
anion in large excess, whose emission
spectrum as has been previously discussed. Upon titration of this
mixture with HP2O7 anion a noticeable modification of the
maximum is at 463 nm (Fig. 13). Therefore, this rules out the
possibility of interpreting the response towards HP2O7 as a
3−
3−
chemical shifts was recorded (see the ESI†). The resulting titra-
tion curve was interpreted as a piece of evidence of the selective
recognition of HP2O73− anion.
mere deprotonation process and indicates that the recognition of
the anion is the dominant process taking place in solution.27
Additionally, an evaluation of the increase in the water content
of a solution of the tripodal host was performed (Fig. 14). As the
concentration of water goes from 0.1 to 20% a perfectly readable
3−
response is obtained upon titration with HP2O7 anion. The
abrupt change, previously observed for the titration isotherm,
became more rounded as the percentage of water increased. Sim-
ultaneously, the initial slope decreases its angle and the isotherm
tends to reach a plateau at a higher guest–host ratio. All these
changes indicate that upon increasing the amount of highly com-
petitive water it is more difficult to displace the complexation
equilibrium towards the formation of the anionic complex.
However, even in such a competitive medium (DMSO–water,
20% v/v), the experimental data still reveal a binding process.
Similar studies were performed by fluorescence spectroscopy
(Fig. 15). An appreciable emission quenching was still detected
Fig. 11 Ratio of emission quenching of receptor 3, 2 × 10−5 M in
DMSO, in the presence of 10 equivalents of different anions.
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3−
HP2O7 anion is exclusive of receptor 3 when studies are per-
formed under more competitive conditions such as the presence
of other interfering anions or the study in aqueous environment.
To conclude, the tripodal receptor 3 represents one of the few
neutral receptors with selectivity towards HP2O73− anion.
Experimental
General remarks
Reagents used as starting materials were commercially available
and were used without further purification. Solvents were dried
following the usual protocols (THF, Et2O and toluene were dis-
tilled from sodium wire with benzophenone indicator; CH3CN
and CH2Cl2 were distilled from CaCl2; EtOH and MeOH were
distilled from magnesium and stored with molecular sieves).
Unless stated otherwise, all reactions were carried out under
nitrogen atmosphere. Column chromatography was run with
silica gel 60 A CC 70–200 μm as stationary phase and using
HPLC grade solvents. Melting points were measured in a Reich-
Fig. 14 Evolution of the chemical shifts of the carbazole CH(1) proton
of 3 upon titration with TBA3HP2O7 in DMSO-d6 with 0.1% (○), 1%
(□), 5% (4), 10% (●), 15% (■) and 20% (▲) water content (v/v).
1
ert instrument and are not corrected. H NMR, 13C NMR, NOE
and NOESY experiments were recorded on either a Bruker
AV300 instrument, or a Bruker AV400 instrument. Chemical
shifts are referred to the residual peak of the solvent. In the
experimental data “bp” stands for broad peak and “Cq” for qua-
ternary carbon atom. Mass spectrometry was recorded on a
HPLC-MS TOF 6220 instrument. Absorption spectra were
recorded on a Cary 500 UV-vis-NIR spectrophotometer. Emis-
sion spectra were recorded on a Cary Eclipse spectrophotometer.
Isothermal titration calorimetry experiments were run in a Micro-
cal VP-ITC microcalorimeter and data were analysed using
Origin software. X-ray structure determination was measured at
120(2) K using graphite-monochromated Mo-Kα radiation (λ =
0.71073 Å) and ω scan mode on a Oxford Diffraction Super-
Nova diffractometer. 33 146 reflections were collected of which
10 672 were independent. The structure was solved by direct
methods and all non-hydrogen atoms refined anisotropically on
F2 (SHELXS-97 and SHELXL-97, G.M. Sheldrick, University
of Göttingen, 1997). Pulse gradient spin echo (PGSE) diffusion
measurements were performed on a Bruker AV600 MHz spec-
trometer equipped with a HR Z-gradient BBO probe and using
the standard ledbpgp2s pulse program from Bruker Topspin soft-
ware (using a stimulated echo and longitudinal eddy current
delay, with bipolar gradient pulses and two spoiling gradients).
Sine shaped gradients were used and the measurements were
recorded with 16k time domain data points in t2 dimension and
16 t1 increments, 24 transients for each t1 increment and a relax-
ation delay of 5 s. The gradient length (δ = 2*P30) was 2.5 ms
with diffusion time Δ (D20) = 70 ms. The D-values were deter-
mined following the integral decay of the carbazole signals.
Fig. 15 Emission spectra of receptor 3, 2 × 10−5 M in DMSO–water
3−
5%, upon titration with HP2O7 anion. λexc = 300 nm. Inset: fluor-
escence colour change upon complexation of HP2O73− anion.
3−
for the titration of 3 with HP2O7 anion in DMSO–water 5%
(v/v).
The higher dilution of the experiment increases the competi-
tiveness of water which weakens the interaction with the anion
to obtain a 1 : 1 complex (K = 145 M−1). Nevertheless, the
change in the colour of the fluorescence, from the pink free
receptor to the blue anionic complex, makes the response of 3 a
good analytical signal towards pyrophosphate anion in aqueous
environment.
Conclusions
In summary, we have synthesised a new family of carbazole-2-
carboxamide derivatives. Receptors bearing two and three carba-
zole units display a high selectivity towards HP2O7 anion in
4-Iodo-3-nitrobenzoic acid (4)
3−
DMSO solution. Receptor 3 shows a multiple equilibria behav-
iour under more concentrated conditions (10−3 M) which has
been demonstrated by 1H NMR, 31P NMR and ITC experiments.
The binding model simplifies to a 1 : 1 model and enhances its
selectivity when diluted (10−5 M) as has been demonstrated by
absorption and emission spectroscopy. The selective response for
To a round bottomed flask containing 4-iodobenzoic acid (6 g,
24.19 mmol) was sequentially added HNO3 65% (50 mL) and
H2SO4 96% (50 mL). Orange vapours immediately evolved. The
reaction was stirred at room temperature for 5 h. Afterwards, the
crude was poured onto crushed ice to obtain a yellow precipitate.
The solid was filtered, washed with water (3 × 20 mL) and dried
1902 | Org. Biomol. Chem., 2012, 10, 1896–1904
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in air to isolate the expected pure product (7 g, 98%). M.
(CH), 110.9 (CH), 61.0 (CH2), 14.4 (CH3); HR-MS m/z: Calcd
(C15H14NO2, [M + H]): 240.1024, Found: 240.1021.
1
p. 214–215 °C; H NMR (300 MHz, DMSO-d6); δ (ppm) 8.31
(s, 1H), 8.25 (d, 1H, J = 8.1 Hz), 7.85 (d, 1H, J = 8.1 Hz); 13C
NMR (75 MHz, DMSO-d6); δ (ppm) 165.1 (CvO), 153.2 (Cq),
141.8 (CH), 133.3 (CH), 132.0 (Cq), 125.0 (CH), 94.0 (Cq).
Carbazole-2-carboxylic acid (8)
Ethyl carbazole-2-carboxylate (0.50 g, 2.1 mmol) was placed in
a round bottomed flask with 50 mL of ethanol. A solution of
NaOH (0.72 g, 31.3 mmol) in water (10 mL) was added to
obtain a clear solution whose colour changed from yellow to
orange. The reaction was refluxed for 16 h. During this time a
precipitate formed. The reaction was quenched by the addition
of HCl 15% (150 mL) and stirred for 1 h. A pale yellow precipi-
tate formed. This solid was filtered, washed with water (3 ×
15 mL) and dried in the air to isolate the pure product (0.44 g,
Ethyl 4-iodo-3-nitrobenzoate (5)
4-Iodo-3-nitrobenzoic acid, 4 (7.0 g, 23.9 mmol), was dissolved
in ethanol (150 mL) and 1 mL of concentrated sulfuric acid was
added. The solution was refluxed overnight. Then the reaction
was allowed to cool down and the solvent was removed by
rotary evaporation. The crude was dissolved in ethyl acetate
(80 mL) and washed with a saturated solution of NaHCO3 (3 ×
50 mL). The organic extracts were dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. A
yellow solid was obtained which corresponds to the analytically
1
99%). M.p. >300 °C; H NMR (400 MHz, DMSO-d6); δ (ppm)
12.78 (s, 1H, br), 11.50 (s, 1H), 8.20–8.17 (m, 2H), 8.08 (s, 1H),
7.75 (d, 1H, J = 8 Hz), 7.467–7.431 (m, 2H), 7.19 (t, 1H, J =
7.2 Hz); 13C NMR (100 MHz, DMSO-d6); δ (ppm): 168.0
(CvO), 140.9 (Cq), 139.0 (Cq), 127.5 (Cq), 126.8 (CH), 125.8
(Cq), 121.6 (Cq), 121.0 (CH), 119.9 (CH), 119.4 (CH), 119.0
(CH), 112.4 (CH), 111.3 (CH); HR-MS m/z: Calcd (C13H10NO2,
[M + 1]): 212.0711, Found: 212.0697.
1
pure ester (6.7 g, 87%). M.p. 88–89 °C; H NMR (200 MHz,
CDCl3); δ (ppm) 8.44 (d, 1H, J = 1.8 Hz), 8.14 (d, 1H, J = 8.2
Hz), 7.88 (dd, 1H, J1 = 2 Hz, J2 = 8.2 Hz), 4.42 (q, 2H, J = 7.2
Hz), 1.41 (t, 3H, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3); δ
(ppm): 164.1 (CvO), 153.0 (Cq), 142.3 (CH), 133.4 (CH),
132.0 (Cq), 126.0 (CH), 92.0 (Cq), 62.1 (CH2), 14.2 (CH3).
N-Benzyl carbazole-2-carboxamide (1)
Ethyl 4-phenyl-3-nitrobenzoate (6)
To a slurry of carbazole-2-carboxylic acid (0.20 g, 1 mmol) and
PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hex-
afluorophosphate) (1.56 g, 3 mmol) in CH2Cl2 (35 mL), triethyl-
amine (0.4 mL, 3 mmol) was added to get a clear solution. This
mixture was stirred at room temperature for 2 h. Then a solution
of benzylamine (0.4 mL, 3 mmol) in CH2Cl2 (10 mL) was
added dropwise and the reaction was stirred overnight. A white
precipitate was formed during the course of the reaction. This
was filtered, washed with water (3 × 15 mL) and dried in the air
A solution of ethyl 4-iodo-3-nitrobenzoate (1.00 g, 4.120 mmol)
in dimethoxyethane (40 mL) was prepared in a 100 mL round
bottomed flask equipped with a reflux condenser. This solution
was deoxygenated by bubbling dry nitrogen for 15 minutes.
Pd(PPh3)4 (0.11 g, 0.095 mmol), phenylboronic acid (0.57 g,
4.670 mmol) and aqueous Na2CO3 2 M were sequentially
added. The mixture was refluxed for 18 h. After this time the
reaction was allowed to cool to room temperature and was
quenched by adding 50 mL of HCl (10%). Then the mixture was
extracted with CH2Cl2 (4 × 50 mL). The organic extracts were
combined, dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure to obtain a yellow oil. This crude was
purified by column chromatography (CH2Cl2 : n-hexane, 1 : 1) to
isolate the desired product as a yellow solid (0.335 g, 83%). M.
p. 63–64 °C; 1H NMR (300 MHz, CDCl3); δ (ppm) 8.28 (d, 1H,
J = 0.6 Hz), 8.26 (dt, 1H, J1 = 0.6 Hz, J2 = 7.9 Hz), 7.54 (d, 1H,
J = 8.1 Hz), 7.46–7.44 (m, 3H), 7.35–7.32 (m, 2H), 4.45 (q, 2H,
J = 6.9 Hz), 1.44 (t, 3H, J = 7 Hz). 13C NMR (75 MHz, CDCl3);
δ (ppm) 164.4 (CvO), 149.2 (Cq), 140.2 (Cq), 136.4 (Cq),
132.7 (CH), 132.1 (CH), 130.7 (Cq), 128.8 (CH), 127.7 (CH),
125.1 (CH), 61.8 (CH2), 14.2 (CH3); HR-MS m/z: Calcd
(C15H15NO5, [M + 18]): 289.0950, Found: 289.1185.
1
to obtain the pure product (0.18 g, 60%). M.p. 210–211 °C. H
NMR (200 MHz, DMSO-d6): δ (ppm) 11.46 (s, 1H), 9.09
(t, 1H, J = 5.8 Hz), 8.19–8.14 (m, 2H), 8.03 (d, 1H, J = 0.8 Hz),
7.71 (dd, 1H, J1 = 1.4 Hz, J2 = 8.2 Hz), 7.54–7.10 (m, 8H), 4.52
(d, 2H, J = 6 Hz); 13C NMR (75 MHz, DMSO-d6): δ (ppm)
166.9 (CvO), 140.8 (Cq), 140.0 (Cq), 139.2 (Cq), 131.6 (Cq),
128.5 (CH), 128.3 (2 × CH), 127.2 (CH), 126.7 (CH), 126.4
(CH), 124.7 (Cq), 121.8 (Cq), 120.8 (CH), 119.8 (CH), 118.9
(CH), 117.7 (CH), 111.2 (CH), 110.5 (CH), 42.8 (CH2);
HR-MS-ES m/z Calcd (C20H17N2O, [M + 1]): 301.1341, Found:
301.1342.
N,N′-(1,3-Phenylenebis(methylene))bis(carbazole-
2-carboxamide) (2)
This compound was synthesised in a 40% yield using the same
methodology as described for 1. M.p. >300 °C; 1H NMR
(600 MHz, DMSO-d6); δ (ppm) 11.43 (s, 2H), 9.11 (m, 2H),
8.10–8.07 (m, 4H), 8.02 (s, 2H), 7.68 (dd, 2H, J1 = 1.2 Hz, J2 =
8.4 Hz), 7.51 (d, 2H, J = 8.4 Hz), 7.42 (td, 2H, J1 = 0.6 Hz, J2 =
7.2 Hz), 7.36 (s, 1H), 7.316–7.291 (m, 1H), 7.24 (d, 2H, J = 7.8
Hz), 7.17 (t, 2H, J = 7.2 Hz), 4.52 (d, 4H, J = 5.4 Hz); 13C
NMR (50 MHz, DMSO-d6); δ (ppm) 166.8 (CvO), 140.8 (Cq),
140.0 (Cq), 139.2 (Cq), 131.6 (Cq), 128.3 (CH), 126.4 (CH),
Ethyl carbazole-2-carboxylate (7)
(0.323 g, 91%). M.p. 182–183 °C; 1H NMR (200 MHz, CDCl3);
δ (ppm) 8.27 (s, 1H, br), 8.18–8.09 (m, 3H), 7.95 (dd, 1H, J1 =
1.2 Hz, J2 = 8.2 Hz), 7.49–7.47 (m, 2H), 7.34–7.23 (m, 1H,
overlapped with solvent), 4.44 (q, 2H, J = 7.2 Hz), 1.45 (t, 3H, J
= 7 Hz); 13C NMR (75 MHz, CDCl3); δ (ppm) 167.3 (CvO),
140.7 (Cq), 138.8 (Cq), 127.6 (Cq), 127.2 (CH), 127.0 (Cq),
122.5 (Cq), 121.1 (CH), 120.6 (CH), 119.9 (2 × CH), 112.4
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1896–1904 | 1903

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126.0 (CH), 125.7 (CH), 124.6 (Cq), 121.8 (Cq), 120.8 (CH),
119.7 (CH), 118.9 (CH), 117.6 (CH), 111.2 (CH), 110.4 (CH),
42.7 (CH2); HR-MS m/z: Calcd (C34H27N4O2, [M + 1]):
523.2134, Found: 523.2190.
N,N′,N′′-(2,4,6-Triethylbenzene-1,3,5-triyl)tris(methylene)-tris-
(carbazole-2-carboxamide) (3)
This compound was synthesised in a 12% yield using the same
methodology as described for 1. M.p. >300 °C; 1H NMR
(400 MHz, DMSO-d6); δ (ppm) 11.38 (s, 3H), 8.37 (bt, 3H),
8.14–8.11 (m, 6H), 8.00 (s, 3H), 7.69 (dd, 3H, J1 = 1.6 Hz, J2 =
8.6 Hz), 7.49 (d, 3H, J = 8 Hz), 7.41 (td, 3H, J1 = 1.2 Hz, J2 = 8
Hz), 7.16 (t, 3H, J = 7 Hz), 4.64 (d, 6H, J = 3.6 Hz), 2.94 (q,
6H, J = 7.2 Hz), 1.19 (t, 9H, J = 6.8 Hz); 13C NMR (100 MHz,
DMSO-d6); δ (ppm) 166.8 (CvO), 143.8 (Cq), 140.7 (Cq),
139.1 (Cq), 132.2 (Cq), 131.6 (Cq), 126.4 (CH), 124.5 (Cq),
121.8 (Cq), 120.8 (CH), 119.6 (CH), 118.8 (CH), 117.9 (CH),
111.2 (CH), 110.6 (CH), 38.1 (CH2), 22.8 (CH2), 16.3 (CH3);.
HR-MS m/z: Calcd (C54H49N6O3, [M + 1]): 829.3866, Found:
829.3858.
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Acknowledgements
Authors gratefully acknowledge the financial support from
MICINN project CTQ2008-01402, and Fundación Séneca
(Agencia de Ciencia y Tecnología de la Región de Murcia)
04509/GERM/06. G. S. is thankful to the Spanish Ministry of
Science and Innovation for the fellowship awarded trough the
project CTQ2008-01402.
14 An estimated water content ≥0.15% was determined for the commercial
deuterated solvent used in these experiments.
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17 The initial downfield shift observed during the titration with TBAOH is
due to the polarization of the C–H bond prior to deprotonation of the
adjacent NH group.
18 In the case of receptor 3, the signals suffered an extreme broadening
during the titration after 3 equivalents making impossible its assignment.
However, it was possible to observe the upfield reversion in the signal
corresponding to CH(1).
19 M. J. Hynes, J. Chem. Soc., Dalton Trans., 1993 (2), 311–312.
20 The initial endothermic evolution in the calorimetry experiment might be
due to a preorganisation of the tripodal receptor to accommodate the
anion afterwards.
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could be detected (Fig. 6).
1904 | Org. Biomol. Chem., 2012, 10, 1896–1904
This journal is © The Royal Society of Chemistry 2012