A coumarin-based turn-on fluorescent sensor for the determination of Al3+: Single crystal X-ray structure and cell staining properties

Article abstract of DOI:10.1039/c3dt51045j

An efficient Al³⁺ receptor, 6-(2-hydroxybenzylideneamino)-2H- chromen-2-one (HBC), has been synthesized by condensing salicylaldehyde with 6-aminocoumarin. The molecular structure of HBC has been determined by a single crystal X-ray analysis. It was established that in the presence of Al ³⁺, HBC shows 25 fold enhancement of fluorescence intensity which might be attributed to the chelation-enhanced fluorescence (CHEF) process. HBC binds Al(NO₃)₃ in a 1:1 stoichiometry with a binding constant (K) of 7.9 × 10⁴ M^-1. Fe³⁺ and Mn²⁺ quench the emission intensity of the [HBC + Al³⁺] system to an insignificant extent at a concentration 10 times higher compared to that of Al³⁺. HBC is highly efficient in the detection of intracellular Al³⁺ under a fluorescence microscope.

Full text of DOI:10.1039/c3dt51045j

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
A coumarin-based “turn-on” fluorescent sensor for the
determination of Al3+: single crystal X-ray structure and
cell staining properties†
Cite this: Dalton Trans., 2013, 42, 10198
Subarna Guha,a Sisir Lohar,a Animesh Sahana,a Arnab Banerjee,a Damir A. Safin,*b
Maria G. Babashkina,b Mariusz P. Mitoraj,*c Michael Bolte,d Yann Garcia,b
Subhra Kanti Mukhopadhyaye and Debasis Das*a
An efficient Al3+ receptor, 6-(2-hydroxybenzylideneamino)-2H-chromen-2-one (HBC), has been
synthesized by condensing salicylaldehyde with 6-aminocoumarin. The molecular structure of HBC has
been determined by a single crystal X-ray analysis. It was established that in the presence of Al3+, HBC
shows 25 fold enhancement of fluorescence intensity which might be attributed to the chelation-
enhanced fluorescence (CHEF) process. HBC binds Al(NO3)3 in a 1 : 1 stoichiometry with a binding
constant (K) of 7.9 × 104 M−1. Fe3+ and Mn2+ quench the emission intensity of the [HBC + Al3+] system to
an insignificant extent at a concentration 10 times higher compared to that of Al3+. HBC is highly
efficient in the detection of intracellular Al3+ under a fluorescence microscope.
Received 20th April 2013,
Accepted 8th May 2013
DOI: 10.1039/c3dt51045j
www.rsc.org/dalton
essential element for biological life and, hence, its high con-
centration in a body can cause harm to the brain and
Introduction
Aluminum (Al) is the third most prevalent and abundant kidneys.9,10 Al has widespread application in industry, pack-
metal in the earth’s crust. Leaching of Al from soil during acid aging materials, electrical equipment, food additives, clinical
rain increases free Al3+ which is deadly to growing plants.1 Al drugs, drinking water supplies, water purification, computers
has already been identified as a neurotoxin.2 According to the and many others.11–13 However, the detection of Al3+ has
World Health Organization (WHO) the average daily human always been problematic due to the lack of spectroscopic
intake of Al is ∼3–10 mg. Tolerable weekly Al dietary intake in characteristics and poor coordination ability compared to tran-
the human body is estimated to be 7 mg kg−1 body weight.3 sition metals.14,15 Recently, the fluorescent method has
Recently, evidence that ingested Al additives contained in pro- become popular due to its operational simplicity, high selecti-
cessed foods and alum-treated drinking water are a major risk vity, sensitivity, rapidity, nondestructive methodology and
factor for Alzheimer’s disease has been discussed.4,5 Further- direct visual perception.16 For an efficient fluorescent sensor,
more, Al might be responsible for Parkinson’s disease, in addition to a high selectivity towards an ion, a significant
osteomalacia6 and also for breast cancer.7 It is also responsible change in the fluorescence intensity in the presence of an ion
for the intoxication in hemodialysis patients.8 It is not an and/or a spectral change are required.17,18
On the other hand, several metal complexes with Schiff
bases19 have antitumor properties,20 antioxidative activities21
aDepartment of Chemistry, The University of Burdwan, Burdwan, 713104 West
and attractive electronic and photophysical properties.22 The
Bengal, India. E-mail: ddas100in@yahoo.com; Fax: +91 342 2530452;
use of a fluorescence technique for the trace level detection of
Al3+ sensor is becoming very popular and the volume of contri-
butions in this field is rising exponentially.23–28 Recently,
several Al3+ selective “turn-on” fluorescent probes derived
from hydrazone,29 Schiff bases,30 coumarin,31 pyrollidine,32
calixarene,33 boron dipyrromethene,34 hydroxyflavone,35
8-hydroxyquinoline,36 oxazoline and imidazoline,37 and bipyri-
dyl-dansyl38 have been reported. Most of them have suffered at
least from one of the following parameters: cost of synthesis,
the number of synthesis steps, selectivity, LOD, detection
medium, binding constant, crystal structure of a probe and
Tel: +91 342 2533913
bInstitute of Condensed Matter and Nanosciences, Molecules, Solids and Reactivity
(IMCN/MOST) – Université Catholique de Louvain, Place L. Pasteur 1, 1348 Louvain-
la-Neuve, Belgium. E-mail: damir.safin@ksu.ru; Fax: +32 (0) 1047 2330;
Tel: +32 (0) 1047 2831
cDepartment of Theoretical Chemistry, Faculty of Chemistry, Jagiellonian University,
R. Ingardena 3, 30-060 Cracow, Poland. E-mail: mitoraj@chemia.uj.edu.pl
dInstitut für Anorganische Chemie J.-W.-Goethe-Universität, Frankfurt/Main,
Germany
eDepartment of Microbiology, The University of Burdwan, Burdwan 713104, India
†Electronic supplementary information (ESI) available: Tables S2 and S3. CCDC
857625. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3dt51045j
10198 | Dalton Trans., 2013, 42, 10198–10207
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
applications.25,26,32,36,39–45 Recently, we have also reported on
In order to support the binding of Al3+ with the receptor
the ratiometric fluorescence sensing and intracellular imaging HBC, the 1H NMR titrations have been performed in a mixture
of Al3+ with a pyrimidine–pyrene scaffold.46 However, the of D2O–CD3OD = 7 : 3 (v/v) (Fig. 1). The H NMR spectrum of
1
described sensor also suffers from the cost of the starting HBC contains signals for the CHN and aryl fragments at 10.0
materials and requires coordination of the two ligands to Al3+
to enhance turn-on fluorescence.
To overcome most of these limitations, herein we report a
very cheap Al3+ selective “turn-on” fluorescent probe 6-(2-
hydroxybenzylideneamino)-2H-chromen-2-one (HBC) derived
from 6-aminocoumarin and salicylaldehyde in a facile single
step. HBC can detect intra-cellular Al3+ in the contaminated
living cell under a fluorescence microscope.
Results and discussion
The compound HBC was synthesized by reacting 6-amino-
coumarin with the salicylaldehyde in methanol to afford a
yellow crystalline solid (Scheme 1). Reaction of HBC with
Al(NO3)3 in methanol leads to the formation of the deep yellow
[Al(HBC)-(MeOH)(NO3)3] complex (Scheme 1).
The FTIR spectrum of HBC contains bands for the CvN,
CvO and OH groups at 1624, 1724, 3427 cm−1, respectively
(Fig. S1 in ESI†). The same bands in the FTIR spectrum of
[Al(HBC)(MeOH)(NO3)3] were observed at 1615, 1724 and
3423 cm−1, respectively (Fig. S2 in the ESI†). While the band
for the CvO group remains constant, the bands for the CvN
and OH groups slightly shift to low frequencies. This might
testify to a coordination of the metal cation through the imine
nitrogen and hydroxyl oxygen atoms of the parent ligand HBC
in the solid state. Besides, the FTIR spectrum of [Al(HBC)-
(MeOH)(NO3)3] also contains an intense band for the NO3
group at 1385 cm−1
.
Fig. 1 1H NMR titration of HBC by Al(NO3)3·9H2O in D2O–CD3OD = 7 : 3 (v/v):
(A) HBC; (B) HBC + 0.5 equivalent of Al3+; (C) HBC + 1 equivalent of Al3+
;
(D) HBC + 2 equivalents of Al3+; (E) HBC + 3 equivalents of Al3+. Signals for the
free HBC are in black, while the signals for the complex are in red.
Scheme 1 Preparation of HBC and [Al(HBC)(MeOH)(NO3)3].
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 10198–10207 | 10199

View Article Online
Paper
Dalton Transactions
and 6.2–8.0 ppm, respectively (Fig. 1, A). It should be noted
that the spectrum of HBC contains no signals for the OH
proton due to a fast exchange with the solvent. Addition of 0.5
equivalent of Al(NO3)3·9H2O leads to the appearance of the OH
proton signal at about 13 ppm as well as the signal for the
CHN proton is 0.2 ppm low-field shifted compared to that in
the 1H NMR spectrum of the parent ligand HBC (Fig. 1, B).
This testifies to a binding of Al3+ by the imine nitrogen and
OH oxygen atoms of HBC. Furthermore, the signals for the
aryl protons are also shifted compared to those in the spec-
trum of free ligand. Addition of 1 and 2 equivalents of
Al(NO3)3·9H2O leads to a further gradual increase of the inte-
gral intensities of the signals corresponding to the complex
(Fig. 1, C and D). At the same time a gradual decrease of the
integral intensities of the signals corresponding to the free
ligand HBC has been observed. Addition of 3 equivalents of Al
(NO3)3·9H2O results in the exclusive presence of the signals of
the complex (Fig. 1, E).
Fig. 2 UV-Vis titration of HBC (1 μM) by Al3+ in H2O–MeOH = 7 : 3 (v/v).
the overall shape of the calculated UV-Vis spectrum of
[Al(HBC)(MeOH)(NO3)3] is similar to the experimental one
(Fig. 3, A). The most intense band at 274.3 nm involves pre-
dominantly two transitions. The former transition HOMO − 4
→ LUMO with the oscillator strength f = 0.4055 a.u. involves a
π → π* charge transfer predominantly within the salicylalde-
hyde ring, whereas the latter one HOMO − 2 → LUMO + 2 con-
sists of a similar π → π* transfer but at the coumarin unit
(Fig. 3). A less intense peak (f = 0.2380 a.u.) at 367.7 nm is
based on the HOMO → LUMO transfer, arising from the
In order to further confirm binding of HBC to Al3+ we have
calculated, based on the DFT/BP86/TZP method, the structure
of [Al(HBC)(NO3)3] and its 1H NMR spectrum based on the
GIAO approach as implemented in the Amsterdam Density
Functional (ADF) package (Fig. S3 in ESI†).47,48 We have
omitted methanol from the coordination sphere of the
complex because a mixture of D2O and CD3OD has been used
in the experiment. Analysis of the [Al(HBC)(NO3)3] structure
leads to the conclusion that the OH proton in HBC has been
transferred to the adjacent NO3 fragment, forming the O2NO–
H⋯O(HBC) intramolecular hydrogen bond (Fig. S3 in the
ESI†). This explains the appearance of the OH proton signal at
∼13 ppm (Fig. 1). In addition, the theoretical 1H NMR spec-
trum of [Al(HBC)(NO3)3] confirms that the OH proton signal is
located at the lowest-field region (Fig. S3 in the ESI†).
The UV-Vis absorption spectrum of HBC in aqueous MeOH
contains four bands in the UV region at 240, 277, 318 and
347 nm (Fig. S4 in the ESI†). The former two bands are signifi-
cantly more intense compared to the latter two bands. The
UV-Vis absorption spectrum of [Al(HBC)(MeOH)(NO3)3] in the
same solvent also exhibits four bands, where the first three
bands were observed almost at the same wavelengths (240, 275
and 321 nm) as was observed for HBC (Fig. S4 in the ESI†). It
should be noted that while the intensity of the band at 240 nm
remains constant, intensities of the latter two bands decrease.
The absorption band at 347 nm disappeared in the UV-Vis
absorption spectrum of [Al(HBC)(MeOH)(NO3)3], while a new
band appeared at 375 nm. It is worth noting that further
UV-Vis titration of HBC by Al3+ in aqueous MeOH leads to a
gradual increase of the absorption bands at 270, 310 and
386 nm with increasing [Al3+]. The former band exhibits a
slightly hypsochromic shift compared to the corresponding
band in the spectrum of [HBC + Al3+] (Δλ = 5 nm), while the
latter band exhibits a bathochromic shift (Δλ = 11 nm) (Fig. 2).
In order to shed light on the qualitative picture of the
absorption in HBC and [Al(HBC)(MeOH)(NO3)3] species, we
have performed TD-DFT calculations (at the B3LYP/TZP level
Fig. 3 Simulated TD-DFT absorption spectrum (B3LYP/TZP, gas phase) of [Al-
(HBC)(MeOH)(NO3)3] (A) together with the contours of molecular orbitals
of theory) using the ADF program.47,48 It can be noticed that involved in the dominant transitions (B).
10200 | Dalton Trans., 2013, 42, 10198–10207
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
occupied π orbitals of both fragments (coumarin and salicylal-
dehyde) to the empty π*, mostly of the salicylaldehyde unit.
Similar π → π* based transitions were obtained for HBC
(Fig. S7 in the ESI†).
The QTOF-MS ES+ spectrum of HBC contains the [M + Na]+
molecular ion peak (Fig. S5 in the ESI†), while the QTOF-MS
ES+ spectrum of [Al(HBC)(MeOH)(NO3)3] contains two mole-
cular ion peaks of the following structures: [M − NO3]+ and
[M + Na]+ (Fig. S6 in the ESI†). Furthermore, the mass spectrum
of the complex also exhibits peaks for the parent ligand: [HBC +
H]+, [HBC + Na]+ and [HBC + K]+. The former two peaks are the
most intense in the mass spectrum of the complex. There are
also two peaks corresponding to the 6-aminocoumarin: [6-ami-
nocoumarin + H]+ and [6-aminocoumarin + Na]+.
It is well known that the performance of fluorescence
sensors based on the electron donor/acceptor mechanism
depends largely on the concentration of proton in the medium
as it competes with the metal ion of interest for binding the
probe. Thus, optimization of pH on the efficiency of the sensor
is essential. Fluorescence pH titrations have been carried out
for this purpose in aqueous MeOH. Equivalent amounts of
HBC and Al3+ have been taken in different sets of pH
(pH 2.2–11.0) (Fig. 4). The fluorescence emission intensity of
HBC remains unchanged on addition of Al3+ at pH below
6.2 and above 9. Most presumably, protonation of HBC at
pH below 6.2 inhibited it to coordinate Al3+. Over pH 9, compe-
tition of OH− with HBC for binding to Al3+ succeeded.
Interaction of HBC with Al3+ has resulted in the fluore-
scence enhancement at 488 nm (λex = 400 nm), which is attrib-
uted as chelation-enhanced fluorescence (CHEF). Fig. 5 (top)
shows changes in the emission intensities upon gradual
addition of Al3+ to the HBC solution. The plot shown in Fig. 5
(bottom) reveals no further change in the emission intensity of
the system over a certain amount of externally added Al3+. We
have observed linearity up to 18 equivalents of Al3+ (R2 = 0.9763).
This linear part of the plot may be used for the determination
of unknown [Al3+] in a sample. Moreover, Al3+ has induced a
blue shift (about 70 nm) of the emission maximum of HBC.
Fig. 5 Fluorescence spectra i of HBC (1 μM) upon addition of different equi-
valents of Al3+ (top). The plot of emission intensities of HBC as a function of
added [Al3+] (bottom).
In order to characterize chelation from the qualitative and
quantitative points of view, we have analyzed the bonding
between HBC and the rest species [Al(MeOH)(NO3)3] based on
the recently proposed charge and energy decomposition
scheme ETS–NOCV (Fig. 6).49
First of all, an analysis of the most stable geometry of
[Al(HBC)(MeOH)(NO3)3], obtained from the BP86/TZP cal-
culations, leads to the following conclusions: (i) the OH hydro-
gen of HBC has been transferred to the adjacent NO3 unit
(Fig. 6, A); (ii) HBC adopts a twisted conformation, which is
similar to the isolated molecule (Fig. S8 in the ESI†). The
former process makes the oxygen atom of the salicylaldehyde
ring of HBC more nucleophilic, which facilitates donation
from HBC to Al3+ and, accordingly, can result in the fluo-
rescence enhancement due to the reduction of the intramole-
cular photoinduced electron transfer (PET).50,51 Indeed, ETS–
NOCV data allowed us to observe that donation of the lone
electron pairs of oxygen and nitrogen atoms of HBC to the Al3+
center (characterized by Δρ2 and Δρ3) leads to a very high ener-
getic stabilization ΔEorb(2) + ΔEorb(3) = −74.4 kcal mol−1
(Fig. 6, B). It is of comparable importance to the strength of a
newly formed covalent O–H connection of the hydrogen
bonding O2NO–H⋯O(HBC), Δρ1 with the corresponding
Fig. 4 pH-dependent titration of HBC (black) and [HBC + Al3+] (red) in H2O–
MeOH = 7 : 3 (v/v).
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 10198–10207 | 10201

View Article Online
Paper
Dalton Transactions
Fig. 7 Job’s plot for the determination of the stoichiometry of [HBC-Al3+] in
H2O–MeOH = 7 : 3 (v/v).
of the complex [Al(HBC)(MeOH)(NO3)3] (see the description
above) also supports the found stoichiometry.
The fluorescence quantum yields of HBC and [Al(HBC)-
(MeOH)(NO3)3] were determined as Φ = 0.1% and 11.4%,
respectively.
The binding constant of HBC with Al3+ has been deter-
mined using the Benesi–Hildebrand eqn (1) by the fluore-
scence method (Fig. 8).53
n
1=ΔF ¼ 1=ΔFmax þ ð1=K½Cꢀ Þ ꢁ ð1=ΔFmax
Þ
ð1Þ
here ΔF = (Fx − F0) and ΔFmax = F∞ − F0, where F0, Fx, and F∞
are the emission intensities of HBC in the absence of Al3+, at
an intermediate Al3+ concentration, and at a concentration of
the complete interaction, respectively. K is the binding con-
stant, C is the concentration of Al3+ and n is the number of
Al3+ ions bound to each HBC (here n = 1).
The value of K, as obtained from the slope, is 7.9 × 104 M−1
.
A linear regression curve was then fitted to the normalized
Fig. 6 ETS–NOCV based deformation density channels, Δρ1, Δρ2, Δρ3 (A)
together with the corresponding energies, ΔEorb(1), ΔEorb(2), ΔEorb(3), charac-
terizing the chelation interaction in the [Al(HBC)(MeOH)(NO3)3] complex (B).
Red and green shadows in part A show the way of fragmentation used in the
ETS–NOCV analysis. The contour values are 0.003 a.u.
ΔEorb(1) = −95.2 kcal mol−1 (Fig. 6, B). One could also add that
quenching of the fluorescence in non-complexed HBC can also
be related to the deactivation of the excited state due to intra-
molecular proton transfer (Fig. S9 in the ESI†).52
Fig. 7 shows the Job’s plot for the determination of the sto-
ichiometry of [HBC-Al3+] in aqueous MeOH. It is clearly
observed that the Al3+ : HBC ratio is 1 : 1. The mass spectrum
Fig. 8 Determination of the binding constant of HBC (10 μM) with Al3+
(10 μM) in H2O–MeOH = 7 : 3 (v/v) using the Benesi–Hildebrand equation by the
fluorescence method.
10202 | Dalton Trans., 2013, 42, 10198–10207
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
Fig. 10 Interference of different metal ions in the determination of [Al3+
]
(10 μM) with HBC (1 μM) and foreign alkali, alkaline earth and transition metal
ions (100 μM).
Fig. 9 Emission intensities of HBC (1 μM) in the presence of different metal
ions (10 μM) in H2O–MeOH = 7 : 3 (v/v).
fluorescence intensity data and the point at which this line
crossed the ordinate axis has been considered as the detection
limit. HBC could detect as low as 0.5 μM of Al3+ in aqueous
MeOH.
The selectivity of HBC towards Al3+ over other common
accompanying metal ions was examined in aqueous MeOH
solutions. Fig. 9 clearly indicates that only Al3+ enhances the
fluorescence intensity of HBC.
This unique selectivity of HBC towards Al3+ can be inter-
preted in terms of the absolute hardness (η), defined as η =
(I + A)/2, where I and A are the ionization potential and proton
affinity, respectively. Namely, it was reported by Paar and
Pearson that Al3+ is the hardest acid among all of the cations
considered in this study.54 This allows a strong coordination
ability between HBC and Al3+, which can lead to the effective
(HBC)N,O → Al3+ donation and accordingly to fluorescence
enhancement. We have further calculated, based on the DFT/
BP86/TZP method, the total interaction energies between the
selected cations and HBC (Table S1 in ESI†). It has been
found, in line with the picture originating from the hardness
parameter, that HBC is by far the strongest bonded to Al3+
(Table S1 in the ESI†). The ETS–NOCV method allowed us to
observe that it is indeed predominantly due to the lowest
orbital interaction term, determined by the (HBC)N,O → Al3+
donation (Fig. S10 and Table S1 in the ESI†).
Studies on the interferences of common alkali, alkaline
earth and transition metal ions in the emission intensity of
the [HBC-Al3+] system are presented in Fig. 10. An increase in
fluorescence intensity of the [HBC-Al3+] system upon addition
of a foreign ion was designated as positive interference,
whereas the reverse phenomenon was termed as negative inter-
ference. Fe3+ and Mn2+ show insignificant negative inter-
ference.
Fig. 11 Fluorescence microscopic images of Bacillus sp. and Candida sp.
(Candida albicans) cells treated with HBC (1 μM): images in the absence (A and C)
and presence (B and D) of Al3+ (incubation temperature is 40 °C).
bio-pesticide for controlling looper killer at tea plantations,
have been grown from 24 h culture medium and treated with
an aqueous solution of Al3+ (1 mg mL−1) for 1 h, washed with
normal saline and observed under a fluorescence microscope
after adding HBC. HBC treated cells without any externally
added Al3+ have been used as a control.
Crystals of HBC were obtained by slow evaporation of the
solvent from its MeOH solution. The molecular structure is
shown in Fig. 12, while the crystal and structure refinement
data are given in the Experimental section.
The compound HBC crystallizes in the triclinic space group
ˉ
P1 and contains two independent molecules in the asymmetric
unit cell. Each molecule is found in the enolimine form
(Fig. 12). The bond lengths of C(12)–O(1) and C(12A)–O(1A) are
1.354(4)–1.364(4) Å and those of N(1)–C(2) and N(1A)–C(2A) are
1.426(4)–1.440(4) Å, which indicate single bonds, whereas a
double bond is revealed from N(1)vC(1) and N(1A)vC(1A)
being 1.264(5)–1.289(4) Å (Table S2 in the ESI†). Furthermore,
Al3+ treated and untreated cells have been mixed with HBC
and observed under a fluorescence microscope. Fig. 11 shows
that HBC can easily permeate to the tested living cells with no
harm as the cells remain alive even after a 0.5 h exposure
to HBC (1 μM). Bacillus sp., which is widely used as a
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 10198–10207 | 10203

View Article Online
Paper
Dalton Transactions
interaction energy is −11.42 kcal mol−1) predominantly by the
dispersion forces (Table S4 in the ESI†).
Conclusions
In summary, a comparison of the present probe with other
existing Al3+ sensitive “turn-on” fluorescent probes reveals that
it is very competitive and somewhat better than others in
respect of all the parameters. The present probe is less expen-
sive as it involves a facile one step reaction with commercially
available much cheaper chemicals. Its structure has been
confirmed by a single crystal X-ray analysis. Coumarin being a
bioactive molecule, the use of its derivative (HBC) for living
cell imaging will be more effective and justified.
Fig. 12 Thermal ellipsoid (50%) plot of molecule A (top) and molecule B
(bottom) of HBC. H-atoms, not involved in hydrogen bonding, are omitted.
the bond angles C(1)vN(1)–C(2) and C(1A)vN(1A)–C(2A)
are 120.9(3)–121.0(3)°, indicating the sp2-hybridization of the
nitrogen atoms (Table S2 in the ESI†). Molecules of HBC are
found to be completely planar with torsion angles N(1)–C(1)–
C(11)–C(12) and N(1A)–C(1A)–C(11A)–C(12A) being −179.9(3)
and 0.3(5)°, respectively, and dihedral angles Φ between aro-
matic rings of 5.48(17) and 2.73(2)°, respectively. The crystal
structure of HBC is stabilized by the intramolecular hydrogen
bond of the type O–H⋯N, which is formed between the hydro-
gen atom of the OH group and the nitrogen atom of the imine
group (Fig. 12, Table S2 in the ESI†). Moreover, molecules of
HBC are perfectly stacked in the crystal packing (Fig. 13). The
energy decomposition analysis (ETS)55 indicated that HBC
units in the crystal structure are weakly bonded (the total
Experimental
Materials and methods
Salicylaldehyde and coumarin have been purchased from
Aldrich (USA) and S. D. Fine Chem. Ltd (India), respectively.
6-Aminocoumarin is synthesized starting from coumarin by
following a published procedure.56 Analytical grade chemicals
and spectroscopy grade solvents are used. Milli-Q 18.2 MΩ
cm−1 water is used throughout all the experiments. The
sources of Mn2+, Cr3+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+
and Pb2+ ions are either their chloride, nitrate or perchlorate
salts.
Physical measurements
IR spectra in KBr are recorded on a Perkin Elmer FTIR spectro-
meter (model: RX-1). 1H NMR spectra in D2O–CD3OD = 7 : 3
(v/v) are recorded with a Bruker Avance 300 MHz using tetra-
methylsilane as an internal standard. Absorption and fluore-
scence spectra in aqueous MeOH (H2O–MeOH = 7 : 3, v/v) are
recorded with a Shimadzu Multi Spec 1501 spectrophotometer
and a Hitachi F-4500 fluorescence spectrometer, respectively.
Mass spectra are recorded with a QTOF Micro YA 263 mass
spectrometer in the ESI positive mode. pH measurements are
performed with a Systronics digital pH meter (model 335). The
fluorescence imaging system is comprised of an inverted fluore-
scence microscope (Leica DM 1000 LED), a digital compact
camera (Leica DFC 420C) and an image processor (Leica Appli-
cation Suite v3.3.0). The microscope is equipped with a 50 W
mercury arc lamp. Elemental analyses are performed on a
Perkin Elmer 2400 CHNS/O elemental analyzer.
Quantum yield measurements
The fluorescence quantum yields have been determined using
anthracene as a reference with a known ϕR value of 0.2 in
MeOH.57 The sample and the reference dye are excited at the
same wavelength (λex = 350 nm), maintaining nearly equal
absorbance (0.1) and emission spectra. The area of the emis-
sion spectrum is integrated using the software available in the
instrument and the quantum yield is calculated according to
Fig. 13 Thermal ellipsoid (50%) plot of the crystal packing of HBC along the
0a (A), 0b (B) and 0c (C) axes. Molecule A and molecule B are given in red and
green, respectively. H-atoms are omitted.
10204 | Dalton Trans., 2013, 42, 10198–10207
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
TS
i,i
the following equation:
where F are diagonal Kohn–Sham matrix elements defined
over NOCV with respect to the transition state (TS) density (at
the midpoint between the density of the molecule and the
sum of fragment densities). Components ΔEorb(k) provide the
energetic estimation of Δρk that may be related to the impor-
tance of a particular electron flow channel for the bonding
between the considered molecular fragments. ETS–NOCV ana-
lysis was done based on the Amsterdam Density Functional
(ADF) package in which this scheme was implemented.47,48
Synthesis of HBC. 6-Aminocoumarine (0.5 g, 3.1 mmol) and
salicylaldehyde (0.378 g, 3.1 mmol) in MeOH (15 mL) have
been refluxed for 8 h. Slow evaporation of the solvent has
yielded a yellow crystalline compound. Yield: 0.740 g (90%).
m.p. 140 2 °C. FTIR, ν: 1624 (CvN), 1724 (CvO), 3427 (OH)
2
ϕS=ϕR ¼ ½AS=ARꢀ ꢁ ½ðAbsÞR=ðAbsÞSꢀ ꢁ ½ηS2=ηR
ꢀ
where ϕS and ϕR are the fluorescence quantum yields of the
sample and the reference, respectively; AS and AR are the area
under the fluorescence spectra of the sample and the refer-
ence, respectively; (Abs)S and (Abs)R are the corresponding
optical densities of the sample and the reference solution at
the wavelength of excitation; ηS and ηR are the refractive
indexes of the sample and the reference, respectively.
The ETS–NOCV method
Historically, the natural orbitals for chemical valence (NOCV)
have been derived from the Nalewajski–Mrozek valence theory
as eigenvectors that diagonalize the deformation density
matrix. It is shown that the natural orbitals for chemical
valence pairs (ψ−k, ψk) decompose the differential density Δρ
into NOCV-contributions (Δρk):
1
3
cm−1. H NMR, δ: 6.44 (d, JH,H = 9.9 Hz, 1H, aryl), 6.93–7.13
3
3
(m, 4H, aryl), 7.25 (d, JH,H = 9.2 Hz, 1H, aryl), 7.58 (t, JH,H
=
=
3
3
8.4 Hz, 1H, aryl), 7.71 (d, JH,H = 7.7 Hz, 1H), 7.92 (d, JH,H
9.5 Hz, 1H), 9.97 (s, 1H, CHN) ppm. UV-Vis, λmax (ε × 103, M−1
cm−1): 240 (425), 277 (310), 318 (sh., 84), 347 (99) nm.
QTOF-MS ES+, m/z (I, %): 266.05 (100) [M + Na]+. Anal. Calc.
for C16H11NO3 (265.27): C 72.45, H 4.18, N 5.28. Found:
C 72.18, H 4.13, N 5.26%.
M=2
M=2
X
X
ΔρðrÞ ¼
νk½ꢂψ2ꢂkðrÞ þ ψ2kðrÞꢀ ¼
ΔρkðrÞ
k¼1
k¼1
where νk and M stand for the NOCV eigenvalues and the
number of basis functions, respectively. Visual inspection of
deformation density plots (Δρk) helps us to attribute the sym-
metry and the direction of the charge flow. In addition, these
pictures are enriched by providing the energetic estimations,
ΔEorb(k), for each Δρk within the ETS–NOCV scheme. The exact
formula which links the ETS and NOCV methods will be given
in the next paragraph after we briefly present the basic concept
of the ETS scheme. In this method, the total bonding energy
ΔEtotal between interacting fragments exhibiting the geometry
as in the combined complex is divided into three components:
Synthesis of [Al(HBC)(MeOH)(NO3)3]. Al(NO3)3·9H2O (0.025 g,
0.067 mmol) in MeOH (10 mL) is slowly added to a magneti-
cally stirred solution of HBC (0.035 g, 0.067 mmol) in the
same solvent (10 mL). The mixture is stirred for 0.5 h. Then
the solvent is evaporated to obtain a yellow compound. FTIR,
ν: 1385 (NO3), 1615 (CvN), 1724 (CvO), 3072 (OH, MeOH),
1
3
3423 (OH) cm−1. H NMR, δ: 6.32 (d, JH,H = 9.8 Hz, 1H, aryl),
3
6.88–7.12 (m, 4H, aryl), 7.31 (d, JH,H = 9.0 Hz, 1H, aryl), 7.59
(t, 3JH,H = 8.3 Hz, 1H, aryl), 7.74 (d, 3JH,H = 7.8 Hz, 1H), 8.02 (d,
3JH,H = 9.6 Hz, 1H), 10.32 (s, 1H, CHN), 13.17 (s, 1H, OH) ppm.
UV-Vis, λmax (ε × 103, M−1 cm−1): 240 (425), 275 (276), 321 (50),
375 (53) nm. QTOF-MS ES+, m/z (I, %): 162.09 (15) [6-amino-
ΔEtotal = ΔEelstat + ΔEPauli + ΔEorb
.
coumarin
+ +
H]+, 184.08 (13) [6-aminocoumarin Na]+,
The first term, ΔEelstat, corresponds to the classical electro-
static interaction between the promoted fragments as they are
brought to their positions in the final complex. The second
term, ΔEPauli, accounts for the repulsive Pauli interaction
between occupied orbitals on the two fragments in the com-
266.12 (93) [HBC + H]+, 288.10 (100) [HBC + Na]+, 304.08 (9)
[HBC + K]+, 449.20 (4) [M − NO3]+, 533.19 (50) [M + Na]+. Anal.
Calc. for C17H15AlN4O13 (510.31): C 40.01, H 2.96, N 10.98.
Found: C 40.00, H 3.08, N 10.16%.
bined molecule. Finally, the last stabilizing term, ΔEorb
,
Measurement procedures
represents the interactions between the occupied molecular
orbitals of one fragment and the unoccupied molecular orbi-
tals of the other fragment as well as the mixing of occupied
and virtual orbitals within the same fragment (inner-fragment
polarization). This energy term may be linked to the electronic
bonding effect coming from the formation of a chemical
bond. The three last terms (ΔEelstat, ΔEPauli, and ΔEorb) very
often are combined into the instantaneous interaction energy,
ΔEint, as it describes the interaction between the fragments in
the geometry of the complex.
Solutions of Al3+ and HBC are prepared by dissolving the
corresponding amounts of Al(NO3)3 and HBC in aqueous
MeOH (H2O–MeOH = 7 : 3, v/v). Solutions of Al3+ and HBC are
mixed in different ratios in different sets for subsequent
UV-Vis and fluorescence measurements using a 1 cm quartz
cell.
X-ray crystallography
The X-ray data of HBC are collected at 173(2) K on a STOE
IPDS-II diffractometer with graphite-monochromatised Mo-Kα
radiation generated by a fine-focus X-ray tube operated at
50 kV and 40 mA. The reflections of the images are indexed,
integrated and scaled using the X-Area data reduction
package.58 Data are corrected for absorption using the
PLATON program.59 The structure is solved by a direct method
In the combined ETS–NOCV scheme,49 the orbital inter-
action term (ΔEorb) is expressed in terms of NOCV’s eigen-
values (νk) as:
M=2
X
X
TS
k;k
ΔEorb
¼
ΔEorbðkÞ ¼
νk½ꢂFꢂTSk;ꢂk þ F
ꢀ
k
k¼1
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 10198–10207 | 10205

View Article Online
Paper
Dalton Transactions
using the SHELXS-97 program60 and refined first isotropically 16 A. W. Czarnik, Fluorescent Chemosensors for Ion and Mole-
and then anisotropically using SHELXL-97.60 Hydrogen atoms
cule Recognition, ACS Symposium Series No. 538. American
are revealed from Δρ maps and those bonded to C are refined
Chemical Society, Washington, 1992.
using appropriate riding models. The hydrogen atom bonded 17 Chemosensors for Ion and Molecule Recognition, ed.
to N is freely refined. Figures are generated using the Mercury
J. P. Desvergne and A. W. Czarnik, Kluwer, Boston, 1997.
triclinic, 18 E. Bakker, P. Buhlmann and E. Pretsch, Chem. Rev., 1997,
97, 3083.
program.61 C16H11NO3, Mr
= 265.26 g ,
mol−1
ˉ
space group P1, a = 5.7162(10), b = 15.008(3), c = 15.078(2) Å,
α = 74.238(12), β = 83.545(13), γ = 80.741(14)°, V = 1225.4(4) Å3, 19 L. Salmon, P. Thuery, E. Riviere and M. Ephritikhine, Inorg.
Z = 4, ρ = 1.438 g cm−3, μ(Mo-Kα) = 0.101 mm−1, reflections:
Chem., 2006, 45, 83.
16 166 collected, 16 166 unique, Rint = 0.0000, R1(all) = 0.1470, 20 V. C. Da Silveira, J. S. Luz, C. C. Oliveira, I. Graziani,
wR2(all) = 0.2091.
M. R. Ciriolo and A. M. Ferreira, J. Inorg. Biochem., 2008,
CCDC 857625 contains the supplementary crystallographic
data.
102, 1090.
21 S. Padhye and G. B. Kauffman, Coord. Chem. Rev., 1985, 63,
127.
22 S. Kasselouri, A. Garoufis, A. Katehanakis, G. Kalkanis,
S. P. Perlepes and N. Hadjiliadis, Inorg. Chim. Acta, 1993,
207, 255.
Acknowledgements
Financial support from UGC-DAE-Kolkata Center is gratefully 23 J. M. Cano-pavón, M. L. Trujillo and A. García De Torres,
acknowledged. A. Sahana and S. Lohar are thankful to CSIR, Anal. Chim. Acta, 1980, 117, 319.
New Delhi for providing fellowships. We convey our sincere 24 C. Jiang, B. Tang, R. Wang and J. Yen, Talanta, 1997, 44,
thanks to USIC, Burdwan University for extending the facility 197.
of a fluorescence microscope. This work was also funded by 25 D. Maity and T. Govindaraju, Chem. Commun., 2012, 48,
the Fonds National de la Recherche Scientifique-FNRS (FRFC 1039.
No 2.4508.08). D. A. Safin and M. B. Babashkina thank WBI 26 D. Maity and T. Govindaraju, Eur. J. Inorg. Chem., 2011,
(Belgium) for post-doctoral positions. M. P. Mitoraj acknow- 5479.
ledges the financial support from the Polish Ministry of 27 F. Pablos, J. L. G. Ariza and F. Pino, Analyst, 1986, 111,
Science and Higher Education (“Outstanding Young Research- 1159.
ers” scholarship, 2011–2014), the National Science Center in 28 D. Karak, S. Lohar, A. Sahana, S. Guha, A. Banerjee and
Poland (grant no. N204 198040) and the computational
support from PL-Grid Infrastructure.
D. Das, Anal. Methods, 2012, 4, 1906.
29 M. P. Manuel-Vez and M. Garcia-Vargas, Talanta, 1994, 41,
1553.
30 M. S. J. Briggs, J. S. Fossey, C. J. Richards, B. Scotta and
J. Whately, Tetrahedron Lett., 2002, 43, 5169.
31 M. Arduini, F. Felluga, F. Mancin, P. Rossi, P. Tecilla,
U. Tonellato and N. Valentinuzzi, Chem. Commun., 2003,
1606.
32 D. Maity and T. Govindaraju, Chem. Commun., 2010, 46,
4499.
33 A. B. Othman, J. W. Lee, Y. D. Huh, R. Abidi, J. S. Kimd and
J. Vicens, Tetrahedron, 2007, 63, 10793.
34 Y. W. Wang, M. X. Yu, Y. H. Yu, Z. P. Bai, Z. Shen, F. Y. Li
and X. Z. You, Tetrahedron Lett., 2009, 50, 6169.
35 R. S. Sathish, A. G. Raju, G. N. Rao and C. Janardhana,
Spectrochim. Acta, Part A, 2008, 69, 282.
36 Y. Zhao, Z. Lin, H. Liao, C. Duan and Q. Meng, Inorg.
Chem. Commun., 2006, 9, 966.
37 A. Jeanson and V. Bereau, Inorg. Chem. Commun., 2006, 9,
13.
38 D. Maity and T. Govindaraju, Inorg. Chem., 2010, 49, 7229.
Notes and references
1 E. Delhaize and P. R. Ryan, Plant Physiol., 1995, 107, 315.
2 D. R. Crapper McLachlan, W. J. Lukiw and T. P. A. Kruck,
Environ. Geochem. Health, 1990, 12, 103.
3 B. Valeur and I. Leray, Coord. Chem. Rev., 2000, 205, 3.
4 T. P. Flaten, Brain Res. Bull., 2001, 55, 187.
5 J. R. Walton, Curr. Inorg. Chem., 2012, 2, 19.
6 G. C. Woodson, Bone, 1998, 22, 695.
7 P. D. Darbre, J. Inorg. Biochem., 2005, 99, 1912.
8 G. D. Fasman, Coord. Chem. Rev., 1996, 149, 125.
9 M. Sargazi, N. B. Roberts and A. J. Shenkin, Inorg. Biochem.,
2001, 87, 37.
10 M. I. Yousef, A. M. EI-Morsy and M. S. Hassan, Toxicology,
2005, 215, 97.
11 M. G. Sont, S. M. White, W. G. Flamm and G. A. Burdock,
Regul. Toxicol. Pharmacol., 2001, 33, 66.
12 N. W. Bavlor, W. Egan and P. Richman, Vaccine, 2002, 20, 39 Y. Lu, S. Huang, Y. Liu, S. He, L. Zhao and X. Zeng, Org.
S18.
Lett., 2011, 13, 5274.
13 C. J. Exley, Inorg. Biochem., 2005, 99, 1747.
14 K. Soroka, R. S. Vithanage, D. A. Phillips, B. Walker and
P. K. Dasgupta, Anal. Chem., 1987, 59, 629.
40 A. Sahana, A. Banerjee, S. Das, S. Lohar, D. Karak,
B. Sarkar, S. K. Mukhopadhyay, A. K. Mukherjee and
D. Das, Org. Biomol. Chem., 2011, 9, 5523.
15 F. Launay, V. Alain, E. Destandau, N. Ramos, E. Bardez, 41 L. Wang, W. Qin, X. Tang, W. Dou, W. Liu, Q. Teng and
P. Baret and J. L. Pierre, New J. Chem., 2001, 25, 1269.
X. Yao, Org. Biomol. Chem., 2010, 8, 3751.
10206 | Dalton Trans., 2013, 42, 10198–10207
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
42 C. Gou, S.-H. Qin, H.-Q. Wu, Y. Wang, J. Luo and X.-Y. Liu,
Inorg. Chem. Commun., 2011, 14, 1622.
43 E. Oliveira, H. M. Santos, J. L. Capelo and C. Lodeiro, Inorg.
Chim. Acta, 2012, 381, 203.
L. Versluis, L. Visscher, O. Visser, F. Wang,
T. A. Wesolowski, E. M. van Wezenbeek, G. Wiesenekker,
S. K. Wolff, T. K. Woo, A. L. Yakovlev and T. Ziegler, Theore-
tical Chemistry, Vrije Universiteit, Amsterdam, ADF2009.01.
44 X.-H. Jiang, B.-D. Wang, Z.-Y. Yang, Y.-C. Liu, T.-R. Li and 49 M. Mitoraj, A. Michalak and T. Ziegler, J. Chem. Theory
Z.-C. Liu, Inorg. Chem. Commun., 2011, 14, 1224. Comput., 2009, 5, 962.
45 A. Banerjee, A. Sahana, S. Das, S. Lohar, S. Guha, B. Sarkar, 50 A. Prasanna de Silva, T. S. Moody and G. D. Wright, Analyst,
S. K. Mukhopadhyay, A. K. Mukherjee and D. Das, Analyst,
2012, 137, 2166.
46 S. Das, A. Sahana, A. Banerjee, S. Lohar, D. A. Safin,
2009, 134, 2385.
51 K. Sasamoto, T. Ushijima, M. Sato and Y. Ohkura, Anal.
Sci., 1996, 12, 189.
M. G. Babashkina, M. Bolte, Y. Garcia, I. Hauli, 52 A. Maliakal, G. Lem, N. J. Turro, R. Ravichandran,
S. K. Mukhopadhyayd and D. Das, Dalton Trans., 2013, 42,
J. C. Suhadolnik, A. D. DeBellis, M. G. Wood and J. Lau,
4757.
J. Phys. Chem. A, 2002, 106, 7680 and references therein.
47 G. te Velde, F. M. Bickelhaupt, E. J. Baerends, C. Fonseca 53 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949,
Guerra, S. J. A. van Gisbergen, J. G. Snijders and T. Ziegler,
J. Comput. Chem., 2001, 22, 931 and references therein.
71, 2703.
54 R. G. Paar and R. G. Pearson, J. Am. Chem. Soc., 1983, 105,
48 E. J. Baerends, J. Autschbach, D. Bashford, A. Bérces,
7512.
F. M. Bickelhaupt, C. Bo, P. M. Boerrigter, L. Cavallo, 55 T. Ziegler and A. Rauk, Theor. Chim. Acta, 1977, 46, 1.
D. P. Chong, L. Deng, R. M. Dickson, D. E. Ellis, M. van 56 S. Guha, S. Lohar, M. Bolte, D. A. Safin and D. Das,
Faassen, L. Fan, T. H. Fischer, C. Fonseca Guerra,
Spectrosc. Lett., 2012, 45, 225.
A. Ghysels, A. Giammona, S. J. A. van Gisbergen, 57 W. H. Melhuish, J. Phys. Chem., 1961, 65, 229.
A. W. Götz, J. A. Groeneveld, O. V. Gritsenko, M. Grüning, 58 Stoe & Cie, X-AREA. Area-Detector Control and Integration
F. E. Harris, P. van den Hoek, C. R. Jacob, H. Jacobsen,
L. Jensen, G. van Kessel, F. Kootstra, M. V. Krykunov, E. van 59 A. L. Spek, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2009,
Lenthe, D. A. McCormack, A. Michalak, M. Mitoraj, 65, 148.
J. Neugebauer, V. P. Nicu, L. Noodleman, V. P. Osinga, 60 G. M. Sheldrick, Acta Crystallogr., Sect. A: Fundam. Crystal-
S. Patchkovskii, P. H. T. Philipsen, D. Post, C. C. Pye, logr., 2008, 64, 112.
W. Ravenek, J. I. Rodríguez, P. Ros, P. R. T. Schipper, 61 I. J. Bruno, J. C. Cole, P. R. Edgington, M. Kessler,
Software, Stoe & Cie, Darmstadt, Germany, 2001.
G. Schreckenbach, M. Seth, J. G. Snijders, M. Solà,
M. Swart, D. Swerhone, G. te Velde, P. Vernooijs,
C. F. Macrae, P. McCabe, J. Pearson and R. Taylor, Acta
Crystallogr., Sect. B: Struct. Sci., 2002, 58, 389.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 10198–10207 | 10207