A dichloro-substituted salicylimine as a bright yellow emissive probe for Al3+

Article abstract of DOI:10.1016/j.jphotochem.2016.06.023

A salicylimine based Al³⁺ specific fluorescent turn on probe (SB-1) was designed, characterized and evaluated through various spectroscopic techniques. The 1:1 chemical interaction between SB-1 and Al³⁺ takes place through O—N—O dono

Full text of DOI:10.1016/j.jphotochem.2016.06.023

Accepted Manuscript
Title: A dichloro-substituted salicylimine as a bright yellow
emissive probe for Al3+
Author: Neeraj Ajit Kumar Sharad Kumar Asthana Shweta
K.K. Upadhyay
PII:
S1010-6030(16)30289-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2016.06.023
JPC 10270
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Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
22-4-2016
4-6-2016
22-6-2016
Please cite this article as: Neeraj, Ajit Kumar, Sharad Kumar Asthana, Shweta,
K.K.Upadhyay, A dichloro-substituted salicylimine as a bright yellow emissive
probe for Al3+, Journal of Photochemistry and Photobiology A: Chemistry
http://dx.doi.org/10.1016/j.jphotochem.2016.06.023
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A dichloro-substituted salicylimine as a bright yellow emissive
probe for Al3+
Neeraj,a Ajit Kumar,b Sharad Kumar Asthana,a Shweta,a K. K. Upadhyaya*
aDepartment of Chemistry (Centre of Advanced Study), Institute of Science, Banaras Hindu
University, Varanasi, Uttar Pradesh-221005, India
bDepartment of Applied Sciences & Humanities, National Institute of Foundry & Forge Technology,
Ranchi-834003, Jharkhand, India.
*E-mail: drkaushalbhu@yahoo.co.in; kku@bhu.ac.in; Tel: +91 542-6702488

Graphical Abstract
A dichloro-substituted salicylimine (SB1) detected Al3+ down to nanomolar level
through bright yellow emission.
Highlights
 A new fluorescent probe SB-1 was explored for Al3+ with nanomolar detection
ability.
 X-ray crystal structure of SB-1 was also studied.
 This sensing ability was explained by theoretical calculations.
 The SB-1 was able to illuminate Al3+ inside the cells.

Abstract:
A salicylimine based Al3+ specific fluorescent turn on probe (SB-1) was designed,
characterized and evaluated through various spectroscopic techniques. The 1:1 chemical
interaction between SB-1 and Al3+ takes place through O-N-O donor sites leading to chelation
enhanced fluorescence (CHEF). The lowest detection limit for this measurement was found
to be 7.69×10-9 M. The binding interaction of SB-1 with Al3+ was studied through 1H & 13C
NMR studies, IR as well as through DFT calculations. The HeLa live cells were found to be
permeable enough for SB-1 and was able to detect Al3+ inside the cells through confocal
microscope.
Keywords: Fluorescent sensor, Al3+, CHEF, Cell Imaging, Density functional Theory

1. Introduction
Aluminum is well known for its widespread use in daily life because it is the
third most prevalent element in the earth crust [1]. It plays an important role in many
fields, including food packaging, drinking water supplies, cookware, manufacturing
industry, and so on [2]. According to the World Health Organization (WHO) report,
the average daily intake of aluminum in human is around 3–10 mg day-1 [3]. In spite
of its multiple roles in day to day life the aluminum has also been found to be toxic for
flora and fauna. A number of diseases associated with the central nervous system like
Alzheimer and Dementia etc. are supposed to involve some definite role of Al3+ [4]
.
The iron binding protein is known to be the main carrier of Al3+ in plasma, and by this
way the same crosses the blood-brain barrier and reaches to foetus through placenta
[5]. Release of free Al3+ by the leaching of Aluminum from soil through acid rain is
very harmful for growing plants [6]. Hence in context of widespread applications and
potential toxicity of aluminum, its detection is of great interest.
Most of the available techniques [7] for detection of aluminum involve costly
instruments, trained technicians and sample preparation. On the other hand the use of
optical sensors particularly the fluorescent ones for the detection of trace analytes have
come up as strong alternatives of these instrumental methods. A variety of fluorescent
sensors for Al3+ have been designed by various workers in recent past [5, 8-10] mainly
upon salicylimine [5, 8e-h], hydrazones [9] and acid hydrazine [10] frameworks.
These designs are in consonance with HSAB principle. The Al3+ being a hard acid
prefers hard donors like N and O, available through above mentioned compounds.
Nevertheless, many of these reported chemosensors have some limitations. On one
hand, mostly multiple step synthesis with costly chemicals raise the cost of sensors
[11], on the other hand the poor water solubility of the resulting sensors compel the

user for inappropriate organic solvents [12]. This further reduces the biocompatibility
of the sensor making them non suitable for bioassay of Al3+ in living cells.
The salicylimine-based optical probes have been an active area of current
research as the same offers advantages such as low cost, easier synthetic protocols and
better optical responses [8]. Recently, our group reported a few fluorescent
chemosensors for Al3+ based on salicylimine [13]. The chemosensors developed for
this purpose mostly involves ‘O–N–O’ coordination site to detect Al3+ selectively and
with high sensitivity [8, 13-14]. Although significant progress has been made in
detection of Al3+, still there is a demand for a fluororeceptor of new architecture with
improved fluorescent properties. A number of characteristic properties of fluorescent
chemosensors viz. sensitivity, selectivity, water compatibility, binding constant,
biological and environmental applications, sensing mechanisms, are known to be
readily influenced by substituents on the benzene ring of salicylaldehyde as well as on
amine part.
Although 3,5-dichloro salicylaldehyde is itself already reported as chemosensor
for Mg2+ ion [15] but its Schiff bases with variety of amines were further utilised by
different workers for sensing of different ionic and neutral analytes [16]. In the same
line present report deals with synthesis, characterization and evaluation of a
salicylimine based turn-on chemosensor SB-1 for the fluorescent detection of Al3+ by
exploiting CHEF process. The nano molar sensitivity and high selectivity of SB-1
towards Al3+ as well as the good permeability in living cells makes the SB-1 further
lucrative.
2. Experimental
2.1 Apparatus:
The IR Spectra for the receptors were recorded on FTIR Spectrophotometer (Model
name: spectrum Two; Manufacturer: Perkin Elmer) while 1H NMR and 13C NMR spectra for

the same were recorded on a FT NMR Spectrometer (Model Name: JEOL AL 300,
Manufacturer: JEOL Japan); Mass spectrometric analysis was carried out on a MDS Sciex
API 2000 LCMS/Brukar Compass data analysis spectrometer. UV-vis. absorption spectra
were recorded at 25°C using UV-Visible spectrophotometer (Model Name: UV-1800,
Manufacturer: SHIMADZU CORPORATION, ANALYTICAL & MEASURING
INSTRUMENTS DIVISION) while the emission spectra were recorded on Fluorolog R -3
spectrofluorometer (Model Name: FL3-11, Manufacturer: JY HORIBA Scientific). Mass
spectrometric analysis was carried out on Bruker amaZon SL spectrometer using ultrascan
mode (Bruker Daltonics, Bremen, Germany).
2.2. General Methods:
All titration experiments were carried at room temperature. All the metal ions (Al3+,
Na+, K+, Ca2+, Mg2+, Ba2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+)
were used as their chloride salts. The 1H NMR spectra were recorded by using
tetramethylsilane (TMS) as an internal reference standard. For 1H NMR titration spectra,
5x10-3 M solution of SB-1 was prepared in DMSO-d6 while the stock solution of Al3+ was
prepared in D2O.
For UV-visible/fluorescence titration experiments, the solutions of metal ions were
prepared in methanol, except Al3+ which was prepared in aqueous medium. Chloride salt of
metal ions was used for solution preparation. Due to insufficient solubility of receptors in
pure ethanol, its stock solution of 1.0 mM was prepared in DMSO which was used at 10μM
and 1μM concentration in ethanol for UV-Visible and fluorescence titration experiment
respectively through dilution.
2.3. X-ray diffraction studies
Single crystals of the receptor SB-1 was grown by slow evaporation of saturated
solution of receptor SB-1 in DMF over a period of few weeks. The single crystal X-ray

diffraction measurements were carried out on an Oxford Diffraction Xcalibur system with a
Ruby CCD detector as well as on a Bruker SMART APEX CCD diffractometer using
graphite-monochromated MoKa radiation (k = 0.71073 Å). All the determinations of unit cell
and intensity data were performed with graphite-monochromated Mo-Kα radiation
(λ=0.71073 A°). The structures were solved by direct methods, using Fourier techniques and
refined by full-matrix least-squares on F2 using the SHELXTL-97 program package [17].
Crystal data and details of the structure determination for receptor SB-1 are summarized in
ESI, Table S1.
2.4. Synthesis of (E)-2,4-dichloro-6-(((2-hydroxyphenyl)imino)methyl)phenol (SB-1):
The SB-1 was synthesized by a simple Schiff base condensation was
characterized by various spectrometric techniques; like FT-IR, 1H & 13C NMR along
with ESI-MS studies (ESI; Fig. S1-S4). 2.0 mM methanolic solution of 3,5-dichloro-
2-hydroxybenzaldehyde was added to a 2.0 mM methanolic solution of 2-aminophenol
followed by constant stirring for ~ 2 hours at the room temperature. A red crystalline
solid was precipitated which was filtered and washed several times with methanol and
finally dried under vacuum over anhydrous CaCl2.
Yield : 80 %; IR/cm−1: 3047, 1628, 1499, 1467, 1370, 1301, 1206, 1134, 1041, 747,
567, 489, 450; 1H NMR : (300 MHz, DMSO-d6, TMS): δ = 15.52 (s, 1H, -OH2),
10.21 (s, 1H -OH1), 9.06 (s, 1H, -CH=N), 7.65 (s, 1H, Ar-H), 7.61 (s, 1H, Ar-H),
7.50-7.47 (d, 1H, Ar-H), 7.20-7.15 (m, 1H, Ar-H), 6.94-6.89 (m, 2H, Ar-H); 13
C
NMR (75 MHz, DMSO-d6, TMS): δ = 159.54, 158.99, 150.90, 132.36, 131.08,
130.33, 129.13, 122.98, 119.87, 119.74, 119.40, 118.99, 116.65; ESI-MS: m/z (100
%) Calc. for C13H9Cl2NO2 [M] = 281.0, found (M-H+) =279.9.
2.5. Synthesis of Al3+ complex of SB-1

A 2 mL aqueous solution of AlCl3 (0.067g, 0.5 m mol) was added slowly to a
magnetically stirred 10 mL methanolic solution of the SB-1 (0.141 g, 0.5 mmol). The
reaction mixture was stirred at room temperature for overnightfollowed by its
evaporation under vacuum yielding yellowish ppt. which was finally dried over
anhydrous CaCl2. Yield: 85%; IR/cm-1 : 3195, 1625, 1480, 1456, 1385, 1289, 1267,
1233, 1180, 881, 835; 1H NMR (300 MHz, DMSO-d6, TMS): δ ppm = 9.38 (s, 1H, –
OH1), 8.86 (s, 1H, –CH=N), 7.60-6.58 (7H, Ar–H); 13C NMR (75 MHz, DMSO-d6
TMS): δ ppm = 190.50, 158.32, 154.42, 149.40, 147.94, 132.92, 132.65, 131.80,
130.91, 125.52, 121.84, 118.57, 115.61; ESI MS: m/z (100 %), Calc. for [SB1-
H++Al3++2Cl-+CH3OH]+ = 410.9, found m/z = 410.9.
,
3. Results and discussion
3.1. Crystallographic details for SB1:
The structure of SB-1 was confirmed through its single crystal X-ray analysis.
Crystals of SB-1 were obtained by slow evaporation of its saturated DMF solution.
The SB-1 crystallizes into a monoclinic lattice with space group P1. The SB-1
incorporates two benzene rings and azomethine groups in a coplanar fashion. The
molecule adopts an (E)-configuration about the C=N bond (C7–N1) having bond
distance 1.296 Å, which is appreciably close to the reported value (1.300 Å). The
hydrogen atom particularly N1H is localized at aldimine N atom and thus the SB-1
takes tautomeric keto form with the strong intra and intermolecular hydrogen bond
interaction N–H----O and O–H----O respectively (Fig. 2b) resulting into a nice
supramolecular architectures (Fig. 3a & b). The above structure is further supported
by the shortening of the bond C13—O2 to 1.275 Å. On the other hand the bond
lengths between C-atom and phenolic O-atom i.e., C5–O1 was found to be 1.336 Å.
These observations clearly indicated the presence of only one phenolic –OH group in

solid state. While in solution state 1H NMR spectrum of SB-1 in DMSO-d6 enabled
the visibility of both phenolic protons.
3.2. Colorimetric and fluorescent properties of SB-1
The sensing ability of SB-1 was tested primarily through UV-visible studies in
its 30 μM ethanolic solution. As illustrated in Fig. 4, free SB-1 exhibited strong
absorption bands centred at ~ 358 and ~ 467 nm due to π – π* and n – π* transitions
respectively. The band at ~ 467 nm underwent blue shifting by ~ 42 nm upon
concomitant additions of Al3+ ion (0–15 equiv.). At this stage slight change in the
colour of the solution was observed from light yellow to olive green (Inset, Fig. 4).
The isosbestic points at ~ 445 nm further confirmed the formation of the SB-1.Al3+
complex. The separate additions of 10 equiv. each of a number of metal ions viz., Na+,
K+, Ca2+, Mg2+, Ba2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+ as
their chloride salts to SB-1 did not show selective colour change for any particular
metal ion. Thus SB-1 was not specific for any particular ion either by naked-eye or by
UV-visible spectral changes (ESI; Fig. S5a & b)
.
The above observations prompted us to check emission spectral pattern of SB-1
against above mentioned cations. Single crystal X-ray diffraction revealed that SB-1
exists in keto-enamine form in solid state while 1H NMR spectral studies indicated its
enol-imine form in solution state. This enol imine - keto enamine tautomerism through
intramolecular proton transfer, PET and allowed C=N isomerization are the major
reasons behind weak fluorescent nature of SB-1 [13, 18]. However, weakly fluorescent
ethanolic solution of SB-1 showed a bright yellow fluorescence emission upon
addition of Al3+ only, enabling its naked-eye detection under illumination with a UV
lamp (Fig 5). The above sensing studies were also performed in EtOH/H2O mixture up
to 2:3; v/v. However, the higher ratio of water affected the fluorescence intensity

indicating the dominance of solvent effect over Chelation Enhanced Fluorescence
(ESI; Fig. S6)
.
In order to further understand the chemosensing properties of SB-1, the
fluorimetric titration was performed with the Al3+ ion. The emission spectrum of free
SB-1 showed a very weak single emission band with emission maximum positioned at
~ 530 nm upon its excitation at ~ 420 nm. The fluorescence titration experiments were
performed by gradual additions of Al3+ into 1.0 μM ethanolic solution of SB-1. Upon
concomitant additions of Al3+ (0-200 equiv.) to the above SB-1 solution fully
developed emission band at ~ 530 nm with nearly 53 fold fluorescence enhancement
was observed (Fig. 6).
3.3. Selectivity study
Upon separate respective additions of 40 equivalents of above mentioned metal
ions to the ethanolic solution of SB-1 (1.0 μM), bright yellow fluorescence emission
was observed in case of Al3+ only (Fig. 7). The fluorescence response of SB-1.Al3+
ensemble was also studied in the presence of the same metal ions. Upon their
respective separate additions only Cu2+ and Fe3+ showed complete quenching of bright
yellow fluorescence of SB-1.Al3+ ensemble, while metal ions like Ni2+, Co2+ showed
partial quenching only due to their paramagnetic behaviour (ESI; Fig. S7a & b)
.
Nevertheless negligible effects were noticed upon change of counter anions of Al3+
(ESI; Fig. S8)
.
For the selective fluorescent enhancement of SB-1 in the presence of Al3+
several factors can be listed to rationalize. (i) The chelation of SB-1 by Al3+ leads
signal transduction through CHEF. (ii) The binding of Al3+ with donor atoms of SB-1
ultimately lead to effective locking of PET and cis-trans isomerization around C=N
bond. (iii) The hydroxyl group seems to play an important role in the recognition of
the Al3+ by strengthening the CHEF and hence restricting the C=N isomerization and
,

PET completely leading to fluorescence enhancement in the form of single emission
band at 530 nm. The detection limit of SB-1 towards Al3+ was calculated using
fluorescence titration data according to the IUPAC definition [19] and the same was
found to be 7.68 × 10-9 M with a linearity range of 3.00 × 10−6 to 1.00 × 10−5 M for
Al3+ (ESI; Fig. S9). This nano molar sensitivity of SB-1 towards Al3+ as well as the
high selectivity makes the SB-1 further lucrative.
3.4. Job’s plot analyses and binding stoichiometry
The 1:1 binding stoichiometry of SB-1 with the Al3+ was obtained by the
careful analysis of the Job’s plot derived from the fluorescence titration experiment
(
ESI; Fig. S10). The corresponding binding constant was determined by the non-
linear fitting of fluorescence titration data into 1:1 binding equation [20]. The value of
the binding constant was found to be (1.23 ± 0.19) × 106 with a satisfactory correlation
coefficient value, R2 = 0.9965 (ESI; Fig. S11). The same was further confirmed by the
ESI-MS of the SB-1.Al3+ complex which showed a molecular ion peak, at m/z 410.9
for [M] = 410.9, calculated for [SB1-H++Al3++2Cl-+CH3OH] (ESI; Fig. S12)
.
3.5. Binding behaviour of SB-1 towards Al3+
The FTIR measurements indicated binding of imine and hydroxyl groups of
SB-1 with the Al3+. The aldimine absorption was lowered in terms of wavenumber
(from 1628 to 1625 cm−1) upon the interaction of Al3+ with SB-1 (ESI; Fig. 13). For
having further insight into the binding event of SB-1 with Al3+, 1H NMR titration
experiment was performed. Significant spectral changes were observed upon the
addition of Al3+ (as its chloride salt) to the DMSO-d6 solution (5 × 10-3 M) of SB-1
The peaks for –OH1 at 10.17 δ ppm shifted marginally upfield to 10.04 δ ppm,
.
nevertheless the peak for –OH2 proton at 15.48 δ ppm underwent downfield shift and

finally disappeared upon addition of different aliquots of Al3+. The aromatic protons
also exhibited marginal upfield shifting (Fig. 8). The same conclusion was further
supported by 1H NMR spectrum of isolated Al3+ complex of SB-1 (ESI; Fig. S14)
The corresponding 13C NMR spectrum of the SB-1.Al3+ ensemble showed peak at
.
190.50 for partial double bond character in C-O bond due to deprotonation of phenolic
proton OH2 upon binding with Al3+ (ESI; Fig. S15). The same further supported our
above speculation regarding the binding of SB-1 and Al3+.
Thus, on the basis of aforementioned experimental supports from various
spectroscopic techniques, the most probable binding mode of Al3+ with SB-1 can be
proposed as follows (Fig. 9).
3.6. Density functional theory (DFT) studies
To get further insight into the binding mechanism of the SB-1 and Al3+ and
corresponding blue shift in absorption band of SB-1 in presence of Al3+, we carried
out density functional theory (DFT) calculations with 6-31G** basis sets using
Gaussian 03 package [21]. According to the above discussion, we proposed a complex
with 1:1 stoichiometry and hexa coordinated mode of Al3+ with the tridentate ligand
SB-1, two monodentate chloride ions and one monodentate methanol. The optimized
configuration of the Al3+ complex indicated that the bond length between Al3+ and O2
of SB-1 was 1.825 while Al3+ and O1 distance observed was 2.127 Å , very close to
Al–N distance (2.065 Å) (Fig. 10).
The optimized structure of SB-1 shows almost homogeneous electron density
pattern in highest occupied molecular orbitals (HOMO) and lowest unoccupied
molecular orbitals (LUMO). The SB-1.Al3+ ensemble exhibited slight heterogeneous
electron density pattern in their HOMO. The HOMO-LUMO energy gap also
underwent change from 3.0797 to 3.4068 eV. Hence weakening of ICT of SB-1 in

presence of Al3+ seems to be responsible for blue shift in the UV-vis. absorption
pattern of SB-1 (Fig. 11).
3.7. Practical applicability of SB-1
3.7.1. Paper test strips
The practical applicability of chemosensor SB-1 was verified by its
incorporation in paper strips. We prepared paper strips by immerging whatman filter
paper into the solution of SB-1 (50 µM) followed by their drying in air. As shown in
Fig. 12, SB-1 strips adopted brownish yellow colour which changed to yellow upon
their interaction with Al3+. We also checked fluorescence response of the same under
UV light. The SB-1 strip was non-fluorescent but became bright yellow fluorescent
upon its interaction with Al3+. Hence instant qualitative informations regarding Al3+
can be obtained through SB-1 without resorting to instrumental analysis.
3.7.2. Cell imaging Studies
To further validate the feasibility of SB-1 for the detection of Al3+ in vivo,
confocal microscope images of HeLa cells incubated with SB-1 were obtained. As
shown in Fig. 13, no fluorescence was observed in HeLa cells upon their incubation
with 40 µg mL-1 of SB-1 for 30 minutes. The cells were washed five times with PBS
(Phosphate Buffered Saline) buffer solution and 250 µM Al3+ was added. The same is
well within the range shown to elicit physiological responses (10-500 µM). After
further incubation for 5 minutes, an enhancement of the fluorescence in the cells was
observed. This is a demonstration of principle test where a significant increase in the
intracellular fluorescence intensity was also observed upon increasing the reaction
time as the consequence of CHEF as mentioned above also.
4. Conclusion
A salicylimine based ligand SB-1 was established as a turn “on” fluorescent
probe for the selective identification of Al3+ in ethanol/ethanol-water mixture. SB-1

exhibited a fluorescence enhancement upon binding to Al3+ due to the combined effect
of the CHEF and inhibition of C=N isomerization.The nanomolar detection limit,
water compatibility up to 50 % as well as its good permeability in living cells leads to
in vivo determination of Al3+ successfully.
Acknowledgements
KKU acknowledges the financial support from UGC, New Delhi [41-
330/2012(SR)]. Neeraj gratefully thanks UPE-JRF for research fellowship
(chem./2012-13/154). AK thanks SERB, New Delhi for young Scientist award
[YSS/2015/000057]. SKA acknowledges CSIR, New Delhi for JRF/SRF
(Aw.No.09/013 (0475)/2012-EMR-I). Shweta acknowledges the financial support
from UGC, New Delhi for JRF/SRF (Aw.No.19-12/2010 (i) EU-IV).

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Fig. 1: Synthetic route for receptor SB-1

Fig. 2 (a) Single crystal of SB-1 (b) Intra and intermolecular hydrogen bonding in SB-
1
.

Fig. 3 (a) & (b) Different modes of supramolecular architecture of SB-1 through
hydrogen bonding.

Fig. 4 UV-visible spectral change of 30 μM SB-1 after addition of Al3+ in Ethanol.
[Inset] Visual colour response of SB-1 and SB-1.Al3+ ensemble.

Fig. 5 Visual changes in SB-1 (10 μM) fluorescence after the addition of 10
equivalents of various metal ions (Under UV-light).

Fig. 6 Fluorescence titration spectra of SB-1 (1.0 μM) upon incremental addition of
Al3+ in ethanol. [Inset] Visual response of SB-1 in the presence of Al3+ under UV-
light.

Fig. 7 Graphical representation of fluorescence response of SB-1 in presence of
various metal ions. [Inset] Emission spectra of SB-1 (1.0 μM) in the presence of
various metal ions.

Fig.8 Partial 1H NMR titration experiment of SB-1 with Al3+ ion.