Hydrazinopyrimidine derived novel Al3+ chemosensor: Molecular logic gate and biological applications

Article abstract of DOI:10.1039/c7nj05095j

Cost-effective and highly sensitive biocompatible probes for the detection of Al³⁺ have tremendously important practical applications. Herein, we report for the first time, the hydrazinopyrimidine based Al³⁺ chemosensor L (1-[(4,6-dimethyl-pyrimidin-2-yl)-hydrazonomethyl]-naphthalen-2-ol) prepared by the condensation of 2-hydroxy-1-naphthaldehyde and 4,6-dimethyl-2-hydrazino-pyrimidine. Our as-synthesized chemosensor L (Φ = 0.0066) shows ～15 fold fluorescence enhancement in the presence of Al³⁺ (Φ = 0.0955, K_a = 1.9 × 10⁴ M^-1) via chelation enhanced fluorescence (CHEF), excited state intramolecular proton transfer (ESIPT), and inhibited photo-induced electron transfer (PET) phenomena. The limit of detection (LOD) and limit of quantification (LOQ) were estimated to be 2.78 μM and 9.27 μM, respectively. Furthermore, for the first time, a hydrazino pyrimidine based 'INHIBIT' molecular logic gate for Al³⁺ was successfully developed using the fluorescence properties of L. The experimental sensing mechanisms of L for Al³⁺ were corroborated by theoretical calculations. Biocompatibility and good water-solubility properties of a pyrimidine moiety of L inspired us to investigate Al³⁺ imaging in human embryonic kidney cell lines, HEK293, and the results for practical applications are highly promising.

Full text of DOI:10.1039/c7nj05095j

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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3
+
Hydrazinopyrimidine derived novel Al chemosensor: molecular logic gate and biological
applications
a
*a
b
c
d
Bhriguram Das, Satyajit Dey, Guru Prasad Maiti, Ankita bhattacharjee, Anamika Dhara and
e*
Atanu Jana
a
Department of Chemistry, Tamralipta Mahavidyalaya, East Midnapore, West Bengal. Pin No.
7
21636, Email: satyajitdeyoc@gmail.com
b
Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City,
Okꢀ73104, USA.
c
Department of Ecological Studies, University of Kalyani, Kalyani, Nadiaꢀ741235, West
Bengal, India
d
Department of Chemistry, Jadavpur University, Raja S. C. Mallick Road, Kolkata 700032, India
e
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016,
India. Email: atanujanaic@gmail.com
Abstract
3
+
Costꢀeffective and high sensitive biocompatible probes for Al detection have tremendous
importance for practical applications. Herein, we report, for the first time, the hydrazino
3
+
pyrimidine based Al chemosensor L (1ꢀ[(4,6ꢀDimethylꢀpyrimidinꢀ2ꢀyl)ꢀhydrazonomethyl]ꢀ
naphthalenꢀ2ꢀol) prepared by the condensation of 2ꢀhydroxyꢀ1ꢀnaphthaldehyde and 4,6ꢀdimethyl
ꢀ2ꢀhydrazinoꢀpyrimidine. Our asꢀsynthesized chemosensor L (Φ = 0.0066) shows ~15 fold
3
+
4
ꢀ1
enhancement fluorescence enhancement in presence of Al (Φ = 0.0955, K = 1.9 × 10 M ) via
a
the chelation enhanced fluorescence (CHEF), excited state intramolecular proton transfer
(
ESIPT) and inhibited photoꢀinduced electron transfer (PET) phenomena. Limit of detection
LOD) and limit of quantification (LOQ) were estimated to be 2.78 µM and 9.27 µM,
(
respectively. Furthermore, for the first time, a hydrazino pyrimidine based ‘INHIBIT’ molecular
3
+
logic gate for Al was successfully developed using the fluorescence properties of L. The
3
+
experimental sensing mechanisms of L for Al were corroborated by theoretical calculations.
Furthermore, biocompatibility and good waterꢀsolubility of pyrimidine moiety of L inspired us
1

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3
+
for Al imaging in Human embryonic kidney cell lines, HEK293 and the results are highly
promising towards practical applications.
Keywords:
3
+
Pyrimidine derivative, Al ion, fluorescence, DFT, timeꢀresolved photoluminescence, cell
imaging
Highlights:
3
+
•
•
•
Pyrimidine based Al chemosensor.
Fluorescent enhancement was due to CHEF , PET and ESIPT
3
+
Bright greenishꢀblue fluorescent color of Al chemosensor under UVꢀlight
All the entities present in the nature exhibit its key role with respect to its place, time and
situation. However, excessive or dearth presence of these substances create misbalance in the
natural rule, and are harmful to living systems. Aluminum is the third most prevalent element
and the most abundant metal (8.3 % by weight) in the biosphere. It is found in natural waters and
3+
most biological tissues in its ionic form as Al . Due to acidic rain and several human activities
1
,2
accumulation of aluminum hampers plant growth and affects badly on aquatic life. Not only
that, its higher concentration causes microcytic hypochromic anemia, Alꢀrelated bone disease
(ARBD), Encephalopathy, Neurodementia, Myopathy, Alzheimer’s disease, Parkinson's disease,
gastrointestinal problems, extreme nervousness, anemia, headaches, decreased liver and kidney
3
–7
function. According to the WHO report, the average daily intake of aluminum by a human is
1
,8,9
approx. 3–10 mg and weekly tolerable dietary intake is 7 mg per kg of body weight.
3
+
Therefore determination and removal of Al came into prominence in most biological assays
and environmental processes. The proper architecture of small molecule fluorophores capable of
noticeable changes in their photophysical properties has drawn attention to the chemist for the
detection of analytes while interacting with fluorophore. There are various processes responsible
3
+
for the change in the photophysical response of these fluorophores for detection of Al ion, such
1
0–13
12–15
as chelationꢀenhanced fluorescence (CHEF),
photoꢀinduced electron transfer (PET),
1
6,17
12,13,18,19
internal charge transfer (ICT),
fluorescence resonance energy transfer (FRET),
2
0
15,21–23
resonance energy transfer (RET), excited state intramolecular proton transfer (ESIPT),
2
4
25
aggregationꢀinduced emission (AIE),
C=N isomerisation.
Recently, development of
2

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2
6–30
fluorescence chemosensors,
for recognition and sensing of biologically and environmentally
important species have flourished as significant goals that is expedients in the field of hostꢀguest
chemistry over other sophisticated methods such as atomic absorption or inductively coupled
plasma mass/atomic emission spectrometric methods because their operational simplicity, high
selectivity, sensitivity, low cost, facile sample preparation and easy detection by both visual and
2
7
instrumental methods. In the last decades, attention has been grown fast to the synthesis of
’
Schiff s base as chemosensor due to its attractive electronic and photophysical properties. Owing
3
+
to weak coordination ability and strong hydration ability of Al , the formation of a stable
3
+
receptor for Al is difficult compared to other biologically significant metal ions. Therefore,
3
+
15,29–31
design and the synthesis of highly selective Al chemosensor remains highly challenging.
3
2
Schiff bases type metal complexes have been widely studied in the literature, due to their
3
3,34
35,36
37–39
antimicrobial activity,
antitumor properties,
antioxidant activities,
catalytic
4
0,41
42,43
activities,
attractive electronic and photophysical properties.
Naphthalene moiety is one
of the promising components of a fluorescent chemosensor due to its low fluorescence quantum
4
4
25,45
46,47
yield, short fluorescence lifetime,
and ability to act as a donor as well as an acceptor.
Our present investigation entails the synthesis of a 2ꢀnaphtholꢀbased Schiff’s base (E)ꢀ1ꢀ((2ꢀ(4,6ꢀ
dimethylpyrimidinꢀ2ꢀyl)hydrazono)methyl)naphthalenꢀ2ꢀol (L) as chemosensor which shows high
3
+
specificity and sensitivity towards Al over coexisting metal cations in ethanol medium via “turn
on” signaling response. (Scheme 1).
Scheme 1. Synthesis of 1ꢀ[(4,6ꢀDimethylꢀpyrimidinꢀ2ꢀyl)ꢀhydrazonomethyl]ꢀnaphthalenꢀ2ꢀol (L)
To the best of our knowledge, our asꢀsynthesized chemosensor L is the first example of
3
+
hydrazino pyrimidineꢀbased Al chemosensor which can be used to build ‘INHIBIT’ molecular
3

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logic gate. It is also noteworthy that there is no report on hydrazino pyrimidine derived
3
+
3+
molecular logic gate for Al . The UVꢀvis electronic transitions of L and its Al ꢀcomplex were
corroborated by theoretical calculations. Finally, cell imaging study was done using the L to
explore the real applications of our asꢀsynthesized chemosensor L.
Experimental section
Chemicals, instruments and methods
2
ꢀNaphthol (≥99.0 %) (a/a), chloroform (≥99.0 %), NaOH (≥97.0 %), ethyl acetate (98.0 %),
silica gel (60ꢀ120 mess), methanol (≥99.0 %), highꢀpurity HEPES (99.0 %), Na EDTA (98.0 %),
2
metal perchlorate salts and related solvent were purchased from Merck (India)Ltd. and were used
4
8
without further purifications. 2ꢀhydroxyꢀnaphlaldehyde
and 2,6ꢀdimethyl pyrimidine
3
6
hydrazine were prepared by using literature method described elsewhere. Solvents used were of
the spectroscopic grade for spectral measurements. Other chemicals were of analytical reagent
grade and have been used without further purification except when specified. Elemental analyses
1
were done in a CHN analyzer (Perkin Elmer). H NMR was recorded on Brucker 400 MHz
instruments using TMS as an internal standard. Chemical shifts δ were expressed in ppm unit
1
1
and H– H coupling constants in Hz. Mass spectra were recorded on Qt of Micro YA263 mass
spectrometer. Absorbance data obtained from a UVꢀVisible spectrometer (modelꢀevolution 201).
Fluorescence data were recorded using a xenoflash (PTI) fluorescence spectrophotometer with
excitation wavelength 380 nm for ligand and complex. Anthracene (φ = 0.27 in ethanol) had
been taken as reference compound to measure the fluorescence quantum yield.
Synthesis
(a) Preparation of Chemosensor (L)
A solution of 4,6ꢀdimethylꢀ2ꢀhydrazinoꢀpyrimidine (100 mg, 0.72 mmol) in ethanol(~15
mL) was added dropwise to a solution containing 2ꢀhydroxyꢀ1ꢀnaphthaldehyde (125 mg, 0.72
mmol) in ethanol(~15 mL) with stirring. The resulting solution was then refluxed for 4 hours.
After that volume of the solution was reduced to oneꢀthird of its initial volume to get a faintꢀ
yellow precipitate which was collected by filtration followed by washing thoroughly with cold
methanol. The product was purified by column chromatography separation using ethyl acetate:
petroleum ether (8:2; v/v) as eluent (Scheme 1). The yield of the product was found to be 76 %.
o
m.pt: 196ꢀ198 C, Anal. Calc. For C H N O: C, 69.85; H, 5.52; N, 19.17 %. Found: C, 69.87;
1
7
16
4
4

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I
H, 5.49; N, 19.16 % H NMR (ESIꢀFig. S2) [400 MHz, DMSOꢀd ] δ (ppm):13.16 (1H, s), 11.49
6
(1H, s),9.15 (1H, s), 8.06 (1H, d, J = 8.4 Hz), 7.84ꢀ7.88 (2H, m), 7.57 (1H, d, J = 7.6 Hz), 7.40
(1H, d, J = 7.2 Hz), 7.21(1H, d, J = 8.8 Hz), 6.69 (1H, s), 2.33 (6H, s). MS (ESIꢀFig. S3): m/z =
+
ꢀ1
2
92.13(calculated), 293.13 [M+H] (observed). IR (KBr, cm ): (ESIꢀFig. S4) 3350, 2921, 2851,
1
620, 1598, 1566, 1483, 1401, 1383, 1364, 1315, 1282, 1187, 1158, 1125 and 1028.
3
+
(
b) Preparation of Al ꢀComplex
To the stirring solution of the ligand (80 mg, 0.27 mmol) in methanol was added
Al(NO ) .9H O (102 mg, 0.27 mmol) taken in methanol, and the solution was refluxed for 4
3
3
2
hours. The resulting solution was kept for slow evaporation. After 5 days, yellow thin scale type
solid appeared. It was collected and washed with methanol several times and dried under vaccuo.
o
1
m.pt = 218ꢀ220 C, H NMR (ESIꢀFig. S5) [ 400 MHz, DMSOꢀd ] δ (ppm): 13.11 (1H, s), 11.51
6
(
(
(
1H, s), 9.14 (1H, s), 8.06 (1H, d, J = 8.8 Hz), 7.87ꢀ7.83 (2H, m), 7.58ꢀ7.54 (1H, m), 7.39ꢀ7.35
1H, m), 7.21 (1H,d,J = 9.2Hz), 6.69 (1H,s), 2.33 (6H,s). MS (ESIꢀFig. S6): m/z = 336.10
3
+
+
3+
ꢀ
+
calculated), observed 335.4090 (L+Al +H Oꢀ2H ) and 380.3245 (L+Al +NO ꢀH ), IR (KBr,
2
3
ꢀ1
cm ): (ESIꢀFig. S7) 3211, 2919, 2350, 1595, 1560, 1464, 1382, 1360, 1341, 1276, 1215, 1185,
and 1021.
UVꢀVis absorbance and fluorescence measurements
−
3
−1
For absorbance and fluorescence study, stock solution of L (c = 1×10 mol L )
−
5
−1
was prepared in acetonitrile. The solution of L was then diluted to 2.0 × 10 mol L with
ꢀ3
ꢀ1
ethanol. Solutions of the guest metal cations (c = 1×10 mol L ) were prepared in deꢀionized
water (20 µM HEPES buffer, pH 7.2) for metal selectivity and titration experiment. Solutions of
desired concentrations of the L and increasing concentrations of the cations were prepared
separately. For investigating metal ions selectivity of the sensor L and in titration experiments,
quartz optical cell of 1 cm optical path length was filled with 2 mL solution of L (20 µM) to
which stock solutions of the metal ions were gradually added using a micropipette. Each spectral
data were recorded after 1 min of addition of the ions. For fluorescence measurements, excitation
was provided at 380 nm, and emission was acquired from 400 nm to 650 nm.
Calculations of detection limit (LOD)
4
9
The detection limit was calculated on the basis of the fluorescence titration using the equation
5

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Detection limit = 3σ/k
Where σ is the standard deviation of blank measurement, and k is the slope between the
3
+
fluorescence emission intensity versus [Al ]. To obtain the slope, the fluorescence emission
3
+.
intensity at wavelength 450 nm was plotted as a function of the concentration of Al
Binding ratio measurements (Job’s plot)
3
+
The Job’s plot was done by fluorescence titrations of the receptor L with Al keeping the total
5
0
volume of the solution constant (2 mL) and changing the molar ratio of ions from 0.1 to 0.9.
Binding constant (K ) measurement
a
3
+
The binding constant (k ) for L–Al complex was determined using the Benesi–Hildebrand
a
equations from fluorescence titration data and also from UVꢀVis titration data separately as
51–53
follows:
n+ m
1
/(IꢀI )=1/(I ꢀI )+1/(I ꢀI )K [M ]
….……..equation (i)
…………equation (ii)
0
1
O
1
0
a
n+ m
1
/(AꢀA )=1/(A ꢀA )+1/(A ꢀA )K [M ]
0 1 O 1 0 a
n+
where [M ] is the metal ion concentration; I , I and I indicate emission intensities in the
0
1
absence of, at intermediate and at infinite concentrations of metal ions, respectively. Similarly,
A , A, and A are absorbance intensities in the absence of, at intermediate and at infinite
0
1
concentrations of metal ions, respectively. A stock solution of Al(NO ) .9H O with a
3
3
2
−
3
−1
concentration of 1.0 × 10 mol L , in an aqueous solution was used. Receptor L with an
−
5
−1
effective concentration of 2.0 ×10 mol L in ethanol (20 µM HEPES buffer, pH 7.2) was used
for the emission titration studies.
Determination of the fluorescence quantum yield
3
6
The fluorescence quantum yield (φ) was calculated using the following equation
ODstandard× Αsample
Astandard×ODsample
Φsample =
×Φstandard
6

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where A represents the area under the fluorescence spectral curve and OD symbolizes the optical
density of the compound at the excitation wavelength. Anthracene (φ = 0.32 in ethanol) was used
as a reference compound to measure the fluorescence quantum yield.
NMR titration
1
3+
For H NMR titration of sensor L with Al , NMR tubes were prepared by dissolving the sensor
L (10 mg) in DMSOꢀd , and three different solution of Al(NO ) .9H O (0.5, 1.0 and 1.5
6
3 3
2
equivalent) were also prepared in DMSOꢀd and added to each solution of sensor L. NMR
6
spectra were then recorded after shaking the mixtures for two minutes.
Fluorescence lifetime measurements
Fluorescence lifetimes were measured by the method of TimeꢀCorrelated Singleꢀ Photon
Counting (TCSPC) using a HORIBA Jobin Yvon Fluorocubeꢀ01ꢀNL fluorescence lifetime
spectrometer. The sample was excited using a laser diode at 375 nm and the signals were
collected at the magic angle of 54.7° to eliminate any considerable contribution from
5
4
fluorescence anisotropy decay. The typical time resolution of our experimental setup is ~ 100
ps. The decays were deconvoluted using DASꢀ6 decay analysis software. The acceptability of the
2
fits was judged by χ criteria and visual inspection of the residuals of the fitted function to the
5
5,56
data. Mean (average) fluorescence lifetimes were calculated using the following equation:
2
i
α iτ
∑
∑
τ av
=
α iτ
i
th
in which α is the preꢀexponential factor corresponding to the i decay time constant, τ .
i
i
Computational study
3
+
To clarify the mode of interaction between receptors and Al , DFT calculations were performed
5
7
using at CAMꢀB3LYP/6ꢀ31g(d) level in Gaussion09 software. CPCM model is used for TDꢀ
DFT calculation in ethanol solvent.
Cell culture
Human embryonic kidney cell lines, HEK293 were collected from National Center for Cell
Science, Pune, India, and used throughout the experiments. Cells were grown in DMEM growth
medium supplemented with 10 % FBS and an antibiotic mixture (1 %) containing PSN at 37º C
in a humidified incubator with 5 % CO and cells were grown to 80ꢀ90 % confluence, harvested
2
7

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with 0.025 % trypsin and in phosphateꢀbuffered saline (PBS), plated at the desired cell
concentration and allowed to grow overnight before any treatment.
Cell cytotoxicity assay
In order to determine the cytotoxicity of the receptor L, yellow colored tetrazolium salt 3ꢀ(4, 5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,Sꢀdiphenyltetrazolium bromide (MTT) assay was performed in HEK293
5
8
cells according to standard procedure. Briefly, after treatment of overnight culture of HEK293
3
cells (10 cells in each well of 96ꢀwell plate) with L (10, 20, 50 and 100 ꢁM) for 6 h, 10 ꢁL of a
MTT solution (1mg/ml in PBS) was added in each well and incubated at 37ºC continuously for 3
h. All media were removed from wells and 100 ꢁL of acidic isopropyl alcohol was added to each
well. The intracellular formazan crystals (blueꢀviolet) formed were solubilized with 0.04N acidic
isopropyl alcohol and the absorbance of the solution was measured at 595 nm wavelength with a
microplate reader (Model: THERMO MULTI SCAN EX). The cell viability was expressed as
the optical density ratio of the treatment to control. Values are mean ± standard deviation of
three independent experiments. The cell cytotoxicity was calculated as % cell cytotoxicity = 100
%
ꢀ % cell viability.
Cell imaging study
The overnight subꢀconfluent culture of HEK293 cells were used for the experiment. Cells were
rinsed with Phosphate buffer saline (PBS) and incubated with DMEM containing the receptor L
making the final concentration up to 10 ꢁM in DMEM for 10 min at 37º C. After incubation,
fluorescence images of HEK293 cells were taken by a fluorescence microscope (Model:
Olympus, UꢀLH100HG, Japan) with an objective lens of 20X magnification. Similarly,
fluorescence images of HEK293 cells (preꢀincubated with 10 ꢁM L) were taken with addition of
3+
1
0 ꢁM Al ions. Fluorescence images were taken with excitation max 380 nm.
Results and discussion
The receptor L was synthesized following a simple Schiff base condensation reaction between
4
3
31
4
,6ꢀdimethylꢀ2ꢀhydrazinopyrimidine and 2ꢀhydroxyꢀ1ꢀnaphthaldehyde in ethanol medium.
1
The structure of L was characterized and confirmed by elemental analysis, H NMR, IR and ESꢀ
MS+ spectra.
Spectral properties and binding behavior
8

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Optical characterizations of L and its metal complexes were performed using UVꢀVis and
fluorescence spectroscopy. We first checked the pH dependency of our asꢀsynthesized
3
+
chemosensor L. The fluorescence intensity of LꢀAl ꢀcomplex did not vary as much with wide
pH range from 6.5 to 8.5 (Fig. S8) but beyond this range the fluorescence intensity noticeably get
diminished. So we choose ethanol as solvent medium (20 µM HEPES buffer, pH 7.2) for our
present investigation.
To examine the UVꢀVis and fluorescence selectivity of the probe L, we restricted our
+
+
2+
investigation on biologically and environmentally significant metal cations viz., Na , K , Ca ,
2
+
3+
2+
2+
2+
2+
2+
2+
2+
2+
3+
3+
2+
Mg , Fe , Co , Ni , Cu , Zn , Pb , Cd , Hg , Ni , Al , Cr and Mn in ethanol (20
µM HEPES buffer, pH 7.2). Initially, binding selectivity of L towards different metal ions was
investigated by means of UVꢀVisible spectra, recorded by adding 50 equiv. stock solution of
aforementioned metal ions to the 20 µM of the probe L in ethanol (20 µM HEPES buffer, pH
7
.2) separately. From the UVꢀVis absorption spectra it was observed that several metal ions
showed characteristic peaks in the region between 350 nm to 500 nm (Fig. S9). Therefore, we
were unable to discriminate the metal ion detection by the absorption study. However, the
preliminary naked eye observation under UVꢀcabinet (λ = 366 nm) of mentioned metal ions (50
3
+
equiv.) with ligand L (20 µM) in ethanol (20 µM HEPES buffer, pH 7.2) showed only Al ions
made a distinguished bluish fluorescence (Fig. 2, inset). This result prompted us to focus our
3
+
study on spectrometric behavior (absorbance and fluorescence) in detail for Al .
9

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0
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
.0
25
20
15
10
5
0
4
ꢀ1
)
K
a
=
1.77 X10 (M
Decreasing
Increasing
0
50 100 150 200 250 300 350 400
3+
3
ꢀ1
[
1/Al ] X10 M
Increasing
3
00 325 350 375 400 425 450 475 500
Wavelength(nm)
3
+
Fig. 1 UVꢀVis absorption spectra of sensor L (20 µM) upon titration with Al (0ꢀ2.5 equiv.) in
ethanol (20 µM HEPES buffer, pH 7.2). inset: BenesiꢀHildebrand plot from absorbance spectra.
3
+
The chelating ability of the probe L with Al was examined by means of UV–Vis titration
experiment in ethanol (20 µM HEPES buffer, pH7.2). As shown in Fig. 1, in the absence of
n+
metal ions (M ), the receptor L exhibited characteristic absorption bands at 326 nm and 361 nm
3
+
which was assigned to πꢀπ* transitions of naphthalene ring. On successive addition of Al to the
solution of the probe L, the absorption bands initially appeared at wavelength 326 nm and 361
nm gradually decreased with the appearance of new absorption bands centered at wavelength
3
36 nm and 407 nm (Fig. 1), respectively. Moreover, the appearance of three distinct isosbestic
3
+
points at 332 nm, 344 nm, and 386 nm clearly indicate the formation of the new LꢀAl complex
in equilibrium with the uncomplexed receptor L. Next we explored the fluorescence phenomena
of L towards aforementioned metal ions in ethanol (20 µM HEPES buffer, pH 7.2). The sensor
(20 µM) showed no characteristic emission band on addition of the aforesaid metal ions (50
3+
3+
equiv.) except Al , upon excitation at wavelength 380 nm (Fig. 2). In presence of Al , the
probe L showed a sharp emission band at λmax 475 nm, and emitted a strong bluishꢀgreen
fluorescence which was visible in naked eye under UV lamp (λ=366 nm) (Fig. 3, inset). This
1
0

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observation demonstrates that the probe possesses high selective fluorescence response towards
3
+
Al .
3
2
2
1
1
00
50
00
50
00
3
+
L
L + Al
2
+
+
Ba
2
Ca
Cd
Co
Cr
2
+
2+
3+
3+
Fe
2
+
+
Cu
Hg
2
+
K
2
+
Mg
2+
L+ other metal ions
Mn
+
Na
Ni
2+
5
0
2+
Pb
Zn
2+
3+
Al
0
4
00
450
500
550
600
650
Wave length (nm)
Fig. 2 Fluorescence spectra of sensor L (20 µM) upon addition of different metal (50 equiv.
+
+
2+
2+
3+
2+
2+
2+
2+
2+
2+
2+
2+
3+
)
ions viz. Na , K , Ca , Mg , Fe , Co , Ni , Cu , Zn , Pb , Cd , Hg , Ni , Al ,
3
+
2 +
Cr and Mn in ethanol (20 µM HEPES buffer, pH 7.2)
1
1

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300
250
200
150
100
50
L
+
+
+
+
+
+
3
+
+
+
K
+
+
+
+
+
2
b
P
+
2
+
2
2
2
2
3
2
2
2
2
n
a
N
2
3
i
N
l
A
a
B
a
C
d
C
o
C
r
C
e
F
u
C
g
H
g
n
Z
M
M
Fig. 3 Relative FL intensity of sensor L (20 µM) in presence of different metal cations (50
equiv.) in ethanol (20 µM HEPES buffer, pH 7.2). λex = 380 nm. inset: corresponding
photographic image under UVꢀcabinet (λ = 366 nm).
In order to test the practical applicability of the probe L, the competitive experiment was carried
3
+
out by mixing of Al (50 µM) in 2 mL ethanol solution of the probe (20 µM) and interfering
ions (50 µM) mentioned previously. The experimental results showed that in presence of various
3
+
competing metal ions, the fluorescence spectral behavior of the LꢀAl ꢀcomplex remained
remarkably unaltered (Fig. 4). Furthermore, we performed the interference studies with higher
3+
equivalent of other metal ions by adding 5 equiv. of interfering metals ions as that of Al . The
3
+
fluorescence properties of the probe LꢀAl moiety remained almost same as aluminum metal
1
2

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ions for most of the metal ions. However, there occurred fluorescence quenching to the large
2+
3+
extent for Cu and Fe when added excess amount (5 equivalents), while the remaining metal
3+
ions did not show any remarkable changes in the fluorescence behavior of the complex LꢀAl
Fig. 4). Therefore, our probe can be used as selective fluorescence probe for aluminum in
(
presence of same equivalents of several metal ions without interferences. The effects of different
counterꢀanions of aluminum salt were also examined under the same conditions using different
Alꢀsalts like Al(NO ) , Al SO , AlCl . The investigation revealed that the counterꢀanions did not
3
3
2
4
3
3
+
influence the recognition process of Al by L. This observation further reveals the high
3
+
selectivity and specificity of the probe L towards Al .
(a)
3
+
350
300
250
200
150
100
L+Al +Metal ions (1:5)
3+
L+Al +Metal ions (1:1)
50
0
+
+
+
+
+
+
+
+
+
+
K
+
+
+
+
+
+
3
2
2
2
2
3
3
2
2
2
2
a
N
2
2
b
2
l
A
i
N
a
B
a
C
d
C
o
C
r
C
e
F
u
C
g
H
g
n
n
Z
P
M
M
Al3+ Ba2+ Ca2+ Cd2+ Co2+ Cr3+ Fe3+ Cu2+ Hg2+ K+ Mg2+ Mn2+ Na Ni Pb2+ Zn2+
+
2+
(b)
Al3+ Ba2+ Ca2+ Cd2+ Co2+ Cr3+ Fe3+ Cu2+ Hg2+ K+ Mg2+ Mn2+ Na Ni Pb2+ Zn2+
+
2+
3
+
Fig. 4 (a) Competitive fluorescence intensities of L (20 µM) toward Al in the presence of
1
3

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1
equiv. various competing metal ions (olive bars) and 5 equiv. various competing metal ions
(
purple bars) in ethanol (20 µM HEPES buffer, pH 7.2) λ = 380 nm; (b) corresponding
ex
photographic image under UVꢀcabinet, λ = 366 nm
3
+
To gain more insights into the sensing behavior of the probe L towards Al ions, fluorometric
3
+
titration was performed with Al in ethanol (20 µM HEPES buffer, pH 7.2). Upon successive
3
+
addition (0ꢀ50 µM) of Al to the solution of the probe L (20 µM), the fluorescence intensity of
the emission band at λmax = 475 nm was gradually enhanced with the increase in the
3
+
3+
concentration of Al (Fig. 5) and reached to a maxima with 2.5 equiv. of Al addition.
1
1
1
1
6
4
2
0
8
6
4
2
0
Increasing
3
2
2
1
1
00
50
00
50
00
4
-1
K
= 1.9 x 10 M
a
0
50 100 150 200 250 300 350 400 450
ꢀ1
1
/[Al3+] x 10
3
M
5
0
0
4
00
450
500
550
600
650
Wavelength (nm)
3+
Fig. 5 Fluorescence spectra of sensor L (20 µM) upon titration with Al (0ꢀ2.5 equiv.) in ethanol
(
20µM HEPES buffer, pH 7.2). λ = 380 nm; inset: BenesiꢀHildebrand plot from emission
ex
3
+
spectra and color changes after addition of Al to the sensor L under UV lamp (λ = 366 nm).
3
+
This increase in the fluorescence intensity of the emission band indicates that addition of Al to
3
+
the sensor solution forms a stable LꢀAl complex by chelation, through –OH, imine (ꢀCH=N)
3
+
nitrogen and Nꢀatom of the pyrimidine ring of the probe L. In the absence of Al , the nitrogen
atom of imine could transfer an electron to the naphthalene ring (PET ON), and the probe L
could transfer a phenolic proton to a neighboring imine nitrogen along with the formation of
1
4

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5
9
intraꢀmolecular hydrogen bond (OH ┈ N) (ESIPT) causing fluorescence quenching in it. In the
3
+
presence of Al , the Oꢀatom of phenolic group and Nꢀatom of the imine group as well as the Nꢀ
3
+
atom of the pyrimidine ring of the probe L engaged in the coordination with Al , which
inhibited the process of PET and ESIPT accompanied with remarkable fluorescence
enhancement. This type of chelation inhibits the free rotation in the molecule and impedes the
photoꢀinduced electron transfer (PET) as well as ESIPT to set up CHEF process. The binding
3
+
mechanism and formation of stable LꢀAl complex were further supported by several
3
+
1
spectroscopic techniques. The interaction between sensor L and Al was further studied by H
NMR titration (Fig. 6).
L+1.5 equiv. Al3+
L+1.0 equiv. Al3+
L+0.5 equiv. Al3+
L only
1
Fig 6. H NMR titration of L with Al(NO ) in DMSOꢀd6
3
3
The appearance of a very downfield proton at δ 13.16 ppm (Fig. S2) in the free sensor is
attributed to the existence of intraꢀmolecular Hꢀbonding between imine nitrogen and hydroxylic
1
5

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2
1,60
3+
proton of naphthol unit.
Upon gradual addition of Al , the signal for the hydroxyl proton at
1
3.16 ppm disappeared, and the aldimine proton (–CH=N–) close to the naphthyl ring
experienced a downfield shift by 0.043 ppm (Fig. 6) indicated that the direct involvement of the
3
+ 61
oxygen atom of the –OH group coordinate with Al . The FTꢀIR spectrum of the bare ligand
ꢀ1
ꢀ1
showed characteristic peaks at 3350 cm (ꢀOH), and at 1598 cm (ꢀC=Nꢀ) (Fig. S4). In complex
ꢀ1
molecule, these peaks were shifted to a broad signal around at 3256 cm and sharp peak at 1595
ꢀ1
62
3+
cm , respectively (Fig. S7). These results suggested that probe L coordinates to Al ion via
the imine nitrogen, the pyrimidine Nꢀatom and the hydroxyl oxygen as proposed in Scheme 2.
3
+
To find out the possibility of ESIPT mechanism operated for binding of Al with L we have
carried out excited state lifetime measurement (Fig. 7). The relative population of various species
in the excited state was calculated using the standard protocol.
Prompt
Ligand L
Ligand L+Al3+
1
000
1
00
10
30
40
50
60
70
Lifetime(ns)
3
+
Fig. 7 Timeꢀresolved fluorescence decay of L (red), L–Al (blue) and prompt (black) (λex =
80 nm)
3
In protic solvent, the probe L showed biexponential decay with time constants of 0.35 ns and
4
.87 ns where the faster and slower components are due to the decays of enol and keto
1
6

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emission of the probe, respectively (Table 1).
3
+
Table 1 Fluorescence lifetimes of L and LꢀAl complex in ethanol solvent (20 µM HEPES
buffer, pH 7.2)
2
EtOH
L
τ1(ns)
τ2(ns)
α1
α2
χ
τav
0.35
0.67
4.87
4.29
0.73
0.27
0.79
1.2
7.87
3
+
LꢀAl
ꢀ0.21
1.12
18.03
3
+
Upon addition of Al , the lifeꢀtime of the first component increased but its relative abundance
decreased. For the second component the lifetime decreases but its relative abundance increases.
By averaging these values it has been found that the average lifetime of L increases with the
3
+
addition of Al . On the fast time scales, a slight growth was observed as the hydrogen bond of
6
3, 64
the protic solvents impedes the ESIPT process.
Enhancement of fluorescence intensity and
the corresponding lifetime is quite obvious due to lowering of nonꢀradiative channels generated
by the rigid structure of the complex than the comparatively more flexible structure of the bare
ligand.
3
+
To obtain the binding stoichiometry of L with Al , the Job’s plot experiment using fluorescence
3+
titrations of the receptor L with Al was carried out.
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7

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1
1
1
1
60
40
20
00
8
6
4
2
0
0
0
0
0
.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
3
+
3+
[
Al
]/([L] + [Al ])
3
+
Fig. 8 Job's plot for complexation of L with Al
plotted with emission data at
4
75 nm. The total volume of the ligand and the metal was kept constant (2 mL) for each of the
data sets.
From the Job’s plot (Fig. 8), it can be seen that in ethanolꢀHEPES buffer medium, the
fluorescence intensity of L increased first and then decreased with increase of the molar fraction
3
+
of Al ions and the maximum fluorescence intensity was observed when the molar fraction of
3
+
Al was 0.5, which reveals that the receptor L form complex in 1: l ratio. This conclusion was
further ascertained from the positiveꢀion mass spectrum (Fig. S6), which exhibited peaks at m/z
3
+
3
35.4090 and 380.3245 were assigned to the coordination structures of (receptor L+Al +H Oꢀ
2
+
3+
ꢀ
+
2
H ,) and (receptor L+Al +NO ꢀH ), for 1:1 complexation, respectively. Using fluorescence
3
3
+
and UVꢀVis titrations, we determined the binding constant for LꢀAl complexation from BꢀH
4
ꢀ1
4
ꢀ1
plot, and it was found to be 1.9×10 M and 1.77×10 M , respectively [Fig. 3, inset: Fig. 1,
inset]. The close proximity of these two results on binding constant value further strengthened
experimental accuracy.
Reversibility is prerequisite in developing novel chemosensors for its practical
3+
applications. The reversible binding nature of the probe L, i.e.; the formation of LꢀAl complex,
and dissociation of the complexes investigated by UVꢀVis and PL titrations experiments in
1
8

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3
+
identical conditions. In this regards, titration experiments of the probe with Al ion and the
3
+
resulting LꢀAl complex with hexadentate chelating ligand Na EDTA were carried out.
2
0.5
Increasing
0
equiv.EDTA
3
00
50
200
50
00
(a)
(b)
0.4
0equiv. EDTA
Decreasing
2
0.3
1
0.2
2.5equiv. EDTA
1
2.5equiv. EDTA
0.1
50
0.0
0
400
450
500
550
600
650
300 325 350 375 400 425 450 475
Wavelength(nm)
Wavelength(nm)
Fig. 9 (a) Absorbance spectra and (b) Fluorescence spectra of complex formed upon titration
with Na EDTA (0ꢀ2.5 equiv.) in ethanol (20 µM HEPES buffer, pH 7.2). λ = 380 nm.
2
ex
As shown in the Figure 9a and Figure 9b, it is observed that upon successive addition of 2.5
3
+
equivalents of Na EDTA to the LꢀAl complex solution, the bluishꢀgreen fluorescence
2
disappeared as the metal ion completely strip away from the complex. The experimental outcome
indicates the probe L could be regenerated for repeated use. For practical issue, the limit of
detection for metal ions also an important parameter. By using aboveꢀmentioned fluorometric
3
+
titration results and based on 3σ method, the detection limit (LOD) for Al was estimated as
3
+
2
.78 µM (Fig. S10). This sufficiently low limit of detection is capable of detecting Al in subꢀ
micromolar concentration level. Therefore, our receptor can be used as a unique reversible
3
+
fluorescent “onꢀoff” sensor for Al ion. All these aboveꢀmentioned evidences help us to explain
3
+
the mechanistic insight into the formation of complex from the receptor. Binding of Al causes a
very large enhancement of the luminescence quantum yield, as expect from cationꢀinduced
reduction of the ESIPT and PET efficiency. Fluorescence enhancement caused by CHEF process
1
9

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with the inhibition of PET process, is shown in the following proposed pathway (Scheme2)
3
+
Scheme 2: Proposed ESIPT and PET onꢀoff mechanism during Al sensing. PETꢀoff/CHEFꢀon
3
+
phenomena enhance the fluorescence intensity upon binding with Al . ESIPT process in L was
3
+
inhibited upon binding with Al and consequently, fluorescence intensity was increased.
Molecular Logic Gate
Logic gates are the fundamental components of any kind of electronic and
6
5–67
digital devices
. These logic gates carry out logical output signals with one or more
logical input signals. The system is generally represented with Boolean algebra dealing with
binary code 0 and 1 for an inactive and active input/output respectively. Mimicking
the functions of semiconductor logic gates used in modern chip level electronic devices,
luminescence is one of the most important techniques to monitor the operation at the molecular
level. The relationship between two input and single output may be described by 16 different
6
8
logic gate with its truth tables . From this viewpoint, our experimental displayed result on the
3
+
way of double input/single output from two active chemical input of Al
and EDTA to the
probe L, and deciphering the output emission intensity follow a truth table (Table 2) of an
INHIBIT logic gate (Fig. 10). 0 and 1 represent the fluorescence properties (output i.e. 1 for
‘YES’/‘ONꢀstate’ and 0 for ‘NO’/‘OFFꢀstate’) of L caused by chemical inputs. In the absence of
2
0

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any chemical input, the chemosensor L didn’t exhibit prominent emission band around 475 nm,
3
+
i.e. output 0 (OFFꢀstate). Addition of Al gives the output 1 i.e. ‘ONꢀstate’ but the presence of
3
+
both Al and EDTA showed the output 0 i.e. ‘OFFꢀstate’. The corresponding circuit diagram for
L may be envisioned as following
Table 2 Truth table for ‘INHIBIT’ logic gate
Input1
Input2
(EDTA)
Output(emission
intensity at 475
nm)
3
+
(
Al )
0
1
0
1
0
0
1
1
0
1
0
0
Fig. 10 General representation of the symbol of an ‘INHIBIT’ gate
DFT study:
3
+
To investigate the electronic transitions of L and its Al complex, DFT calculations have been
carried out at ground state (gas phase). All the UV electronic transitions are wellꢀcorroborated
3
+
with the theoretical values. The optimized geometry of L and its Al complex are shown in Fig.
1. Main optimized geometrical parameters of the complex 1 are listed in Table 3. In case of L at
1
ground state, the electron density at LUMO orbital mainly resides on the naphthalene moiety. On
the other hand, electron density at HOMO spread over the naphthalene as well as in pyrimidine
3
+
moiety. There occurs a dramatic change in electron density upon binding of L with Al ion
2
1

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where electron cloud resides mainly in HOMO. The energy difference between HOMO and
LUMO is 3.30 eV of ligand L. In case of 1, all the electronic transitions originate from ligand π
and π* orbital contribution. The energy difference between HOMO and LUMO is 3.24 eV of 1
(Fig. 12). The absorption energies along with their oscillator strengths, the main configurations
and their assignments calculated using TDDFT methods using the ground state geometry of the
complex are tabulated in Table 4. The TDDFT calculated absorption bands are located at 406,
3
40 and 292 nm for 1, which are in good agreement with experimental results of 382, 332 and
2
87 nm (Fig. 12).
4
5
2
4
3
2
5
3
6
6
7
1
1
8
3
+
(a) Ligand L
(b) LꢀAl complex 1
3
+
Fig.11 Optimized structure of the (a) ligand L and (b) Al complex 1 using CAMꢀB3LYP/6ꢀ
1g(d) level in Gaussion09 software
3
Table 3 Selected optimized geometrical parameters for ligand L and the complex in the ground
state calculated at B LYP Levels.
3
Bond length (angstrom)
d2ꢀ3 = 1.277, d3ꢀ4 = 1.346
d4ꢀ5 = 1.374, d5ꢀ6 = 1.330
Bond angle ( º)
Ligand (L)
Complex 1
∠ 3ꢀ4ꢀ5 = 123.6, ∠ 4ꢀ5ꢀ6 = 119.3
d2ꢀ3 = 1.331, d3ꢀ4 = 1.386
∠ 3ꢀ4ꢀ5 = 115.7, ∠ 4ꢀ5ꢀ6 = 115.9
∠ 1ꢀ7ꢀ3= 98.7, ∠ 3ꢀ7ꢀ6 =86.4
∠ 1ꢀ7ꢀ8= 97.5, ∠ 6ꢀ7ꢀ8= 105.2
d4 = 1.351, d5ꢀ6 = 1.373
ꢀ5
d1 = 1.710, d3ꢀ7 = 1.846,
ꢀ7
d6ꢀ7 = 1.882, d8ꢀ7 = 1.880
2
2

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Fig. 12 Frontier molecular orbital of ligand as well as complex optimized.
Table 4 Vertical electronic transition calculated by TDDFT/CPCM method
Eexcitation (eV)
λexcitation
nm)
Osc. Strength
(f)
Key transitions
Character
(
3
3
.2434
.7298
382.26
332.41
0.5547
0.1703
HOMO → LUMO
HOMOꢀ1→ LUMO
HOMOꢀ1→ LUMO+1
HOMOꢀ2→ LUMO
HOMOꢀ2→ LUMO
HOMOꢀ2→ LUMO+1
HOMO → LUMO+2
HOMO → LUMO+1
HOMO → LUMO+2
HOMOꢀ2→ LUMO+1
HOMOꢀ1→ LUMO+1
HOMO→ LUMO+2
L(π) → L(π*)
L(π) → L(π*)
4
4
.3154
.7355
287.3
0.1154
0.1332
L(π) → L(π*)
L(π) → L(π*)
261.82
5
5
.0795
.5704
244.09
222.58
0.0872
0.1009
L(π) → L(π*)
L(π) → L(π*)
2
3

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Cell studies
3
+
To reflect the intention of the fluorescenceꢀbased probe for intracellular imaging of Al , it was
foremost initially to verify the cytotoxic effect of L on cells. To ascertain their suitability in
cellular environment, the performed standard MTT assay revealed that HEK293 cells showed
9
7
1.78 ±0.471 % viability in presence of L compared to the control i.e. the probe L showed only
.92±0.471 % cytotoxicity at 100 µM concentrations and only 1.4±0.321 % cytotoxicity at 10
µM concentration which was very negligible as compared to control cells and thus the probe L
didn’t influence the viability of cultured human embryonic kidney cell lines for at least up to 6 h
of its treatment at concentrations up to 100 ꢁM (Fig. 13).
Fig. 13 represents % cell viability of HEK293 cells treated with different concentrations (10 µMꢀ
1
00 µM) of receptor L for 6 hrs determined by MTT assay. Results are expressed as mean ± S.D
of three independent experiments.
In fluorescence cell imaging studies, after incubation with L (10 ꢁM) at 37ºC for 10 min, the
cells displayed no intracellular fluorescence (Fig. 14A). However, cells displayed intensive blue
3
+
fluorescence when exogenous Al ion (10 ꢁM) was introduced into the cell via incubation with
3
+
an aluminum salt solution (Fig. 14B). The fluorescence image of L with Al salt presents the
confirmatory evidence of the synthesized chemosensor L to penetrate the cell membrane and
2
4

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3
+
3+
bind with intracellular Al forming the LꢀAl complex. The study suggests that the probe L can
3
+
be readily used as an indelible signature of selective sensor for bioꢀimaging Al ions at the
indicated doses and incubation time without cytotoxic effects.
Fig. 14 (A) Fluorescence image of HEK293 cells incubated with 10 ꢁM receptor L alone for 10
min at 37º C. L (10 ꢁM) incubated cells were washed with PBS and were exposed to added
3
+
extracellular Al ion (10 ꢁM) represented by (B).
Conclusion
In summary, we developed a new hydrazinopyrimidineꢀbased chemosensor capable of
3
+
detecting Al via “turnꢀon” fluorescence response through 1:1 complex formation. The
3
+
1
complexation of receptor L to Al was confirmed by H NMR, FTꢀIR, and ESꢀMS+ studies.
3
+
Fluorescence enhancement of L after addition of Al is attributed to inhibition of PET and
ESIPT process, restricted rotation and introduction of CHEF process. Computational study of L
3
+
and L–Al ensemble also support our proposed mechanism. This receptor offers to detect Al
selectively in the presence of several other metal ions up to micromolar limits, even in the
presence of higher concentration of competing metal ions. Furthermore, the present study
3
+
demonstrates the detection of intracellular Al concentrations through immensely intense turnꢀ
on blue fluorescence by incubating cultured human cell lines with the chemosensor L in
exclusively physiological medium without cytotoxicity.
2
5

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Acknowledgements
S. Dey is grateful to the University Grants Commission, New Delhi, India, for financial support
by sanctioning a minor research project (Sanction letter No. PSWꢀ155/14ꢀ15(ERO) ID No.
WV6ꢀ027, S. No. 223832 dated 03ꢀ02ꢀ2015). We thank Dr. Asim Kr Bera, Principal, Mahisadal
Raj College for providing laboratory and instrumental facilities and also Dr. Atanu Bhaumik,
CBMR, Dr. Milan kr. Barman, NISER for spectral data acquire. A. D. acknowledges the
financial support provided by University Grants Commission, India (Award letter No. F.4ꢀ
2
/2006(BSR)/CH/14ꢀ15/0163, dated May 2015) through the Dr. D. S. Kothari PostꢀDoctoral
Fellowship (DSKPDF).
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