A benzopyrylium-phenothiazine conjugate of a flavylium derivative as a fluorescent chemosensor for cyanide in aqueous media and its bioimaging

Article abstract of DOI:10.1039/c7nj02716h

A new benzopyrylium-phenothiazine conjugate (BP) of a flavylium derivative was synthesized and characterized. The probe BP was shown to be selective and sensitive for CN^- among the 17 anions and biothiols studied in HEPES buffer medium by fluorescence, absorption, and visual color change. The colorimetric and fluorescence response of the probe BP to cyanide ions is due to the Michael addition of cyanide to the activated Michael receptor of the probe which blocks an intramolecular charge transfer process. The probe displays a fast response to cyanide ions at room temperature, and a maximal fluorescence signal is achieved in the presence of only 2 equivalents of cyanide ions. Moreover, the probe BP could be used as a practical, visible colorimetric test strip for CN^- in an aqueous environment. TDDFT calculations were performed in order to demonstrate the electronic properties of the probe and its cyanide product. Density functional reactivity theory (DFRT) calculation also exhibits the characterisation of the most electrophilic and nucleophilic centres of the molecule. The probe could be applied for imaging cyanide in live cells by fluorescence imaging.

Full text of DOI:10.1039/c7nj02716h

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Ali, S. Manna, K. Maiti, Md. R. Uddin, S. Mandal, D. Mandal and A. K. Mahapatra, New J. Chem., 2017, DOI:
10.1039/C7NJ02716H.
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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
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ARTICLE TYPE
Benzopyryliumꢀphenothiazineꢀconjugate of flavylium derivative as fluorescent
chemosensor for cyanide in aqueous media and its bioimaging
Sanchita Mondal,a Syed Samim Ali,a Srimanta Manna,a Kalipada Maiti,a Md. Raihan Uddinb, Sukhendu
Mandalb, Debasish Mandalc and Ajit Kumar Mahapatra*a
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A new benzopyryliumꢀphenothizineꢀconjugate (BP) of flavylium derivative was synthesized and characterized. The probe BP was shown
to be selective and sensitive for CN− among the 17 anions and biothiols studied in HEPES buffer medium by fluorescence, absorption,
and visual color change. The colorimetric and fluorescent response of the probe BP to cyanide ion is due to the Michael addition of
10 cyanide to the activated Michael receptor of the probe which blocks an intramolecular charge transfer process. The probe displays a fast
response to cyanide ion at room temperature, and a maximal fluorescent signal is achieved in the presence of only 2 equivalents of
cyanide ion. Moreover, the probe BP could be used as a practical, visible colorimetric test strip for CN− in aqueous environment. TDDFT
calculations were performed in order to demonstrate the electronic properties of the probe and its cyanide product. Density functional
reactivity theory (DFRT) calculation also exhibit for the characterisation of the most electrophilic and nucleophilic centres of the
15 molecule. The probe could be applied for imaging the cyanide in live cells by fluorescence imaging.
A
variety of cyanideꢀselective colorimetric or fluorescent
Introduction
chemosensor probes have been developed over the past ten years
by making use of the coordination ability and the nucleophilic
reactivity of cyanide.11 There are two kinds of chemosensors, Hꢀ
Cyanide is well known as one of the extremely toxic rapidly
acting and fatal poisons1 due to its tendency to bind to the heme
20 iron in cytochrome c oxidase,2 and affects the oxygen supply to
the cell which resulted to cellular asphyxiation.3aꢀc However, even
very small amounts of the cyanide anion are extremely toxic to
living creatures. Cyanide has also been used as a chemical
warfare reagent, and even as terror material.3d Cyanide is a potent
25 inhibitor of some metalloenzymes and nonꢀmetalloꢀenzymes.
This leads to diseases of the vascular, cardiac, visual, endocrine,
45 bonding
/ coꢀordinationꢀbased sensors and reactionꢀbased
sensors, which differ in their signal output and time of response
types. Coꢀordinationꢀbased sensors appear to be particularly
attractive due to their fast, convenient and real time monitoring
through the formation of metal complexes and metal
50 displacement method.12 However, the change of signal intensity
is depend on several factors such as solvent system,
environmental conditions and the sensor concentration can
interfere with the signal output. A reactionꢀbased method can
overcome the drawbacks of coordinationꢀbased sensors since it
55 enables the measurement of emission intensities of new
compound generated from irreversible reaction with analytes,
thus providing a builtꢀin correction for environmental effects and
increasing the dynamic range of absorption/emission
measurements.13
central nervous and metabolic systems.4 Despite such
a
detrimental effect on human health, cyanide is a basic component
in the preparation of a wide variety of products5 ranging from
30 plastic, fibers, gold and silver extraction, tanning, and metallurgy,
dyes, chelating agents for water treatment, to pharmaceuticals.6ꢀ7
Cyanide is also released from biological processes of bacteria,
fungi and algae even from daily life of human and their activity
such as cigarette smoking.8ꢀ9 The seeds of several fruits including
35 apricots, apples and peaches contain substantial amounts of
biochemicals that release cyanide when metabolized.
Accumulation of cyanide in humans could also happen through
consumption of such foods and plants.10 Thus, the development of
cyanideꢀselective colorimetric or fluorescent probes is in high
40 demand.
60 In recent years, some reactionꢀbased chemosensor probes for
cyanide have been reported. For example, Kim’s group14 used a
α, βꢀ unsaturated carbonyl group containing chemodosimeter for
the rapid detection of cyanide in analytical sample based on the
fluorescence intensity. By combining indole and coumarin
65 moieties Lee and Kim et al. able to develop new hybrid probe15
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which selectively sense cyanide by not only chromogenic but also
The sensitivity of probe BP toward different anions and their
fluorogenic way. The response time was very short (<1 s) in 50 preferential selectivity toward CN− over the other anions has been
acetonitrile–water (95:5) solvent medium. Chen et al. introduced
dicyanoꢀvinyl moiety,16 which showed a large blue shift (96 nm)
in the absorption spectra upon addition of cyanide to the probe.
The resulted colour change could clearly be observe by the naked
eye. They developed, the test strips based on this probe for
practical and efficient detection of cyanide. As a part of our onꢀ
going research on ion sensing17 and all these research studies18
10 inspire us to develop a probe which could be used in reactionꢀ
based analytes sensing with a fast and sensitive fluorescence
response.
studied by using UVꢀvis, fluorescence, and 1H NMR
spectroscopic techniques.
5
UVꢀvis and Fluorescence Spectral Behaviour of BP: The
sensitivity of probe BP toward different anions (CN−, AcO−, F−,
−
−
−
2−
55 Cl−, Br−, I−, S2−, H2PO4 , SO42−, NO2 , ClO4 , SCN−, SO3 and
CO32−) and biothios (Cys, Hcy and GSH) and their preferential
selectivity toward CN− over the other ions has been studied by
absorption and fluorescence titrations. The UVꢀvis spectrum of
BP exhibited an absorption maximum at 552 nm with a small
60 hump in the region at 450 nm in aqueous acetonitrile (3/7 v/v)
HEPES buffer (pH 7.4). The band at 552 nm could be attributed
to the intramolecular charge transfer transition from the donating
phenothiazine ring to the diethylaminobenzopyrylium moiety
which acts as an acceptor. Upon addition of increasing amounts
Hence, reactionꢀbased sensors displaying both colorimetric and
fluorometric dualꢀchannel responses to cyanide are able to offer
15 quantitative information via their internal calibration of
absorption and simultaneously to show the presence of cyanide
via the altered color. With these considerations in mind, we 65 of cyanide (CN−) anion (as its nꢀBu4N+ salt) (0–2 equiv.), the λmax
designed and synthesized a novel reactionꢀbased fluorometric and
colorimetric sensor BP for cyanide anion based on a hybrid
20 benzopyryliumꢀphenothizineꢀliked flavylium dye (Scheme 1).
Such a molecular design makes probe BP possess the expanded
πꢀconjugation as well as the strong ICT from the phenothizine to
the diethylaminobenzopyrylium moiety, which will lead to the
higher region absorption. Importantly, this type of compound can
25 achieve high selectivity as well as nakedꢀeye detection in a very
short time because it possesses an electronꢀwithdrawing
benzopyrylium group that is highly reactive for cyanide.
Therefore, the nucleophilic attack of cyanide toward the
benzopyrylium group of probe BP will block the π ꢀconjugation
30 between phenothizine and diethylaminobenzopyrylium moiety,
by which a hypsochromic shift of absorption and emission of
phenothizine should be induced, resulting in the spectral changes
as well as the change in color. These signal changes indicate that
probe BP can serve as a selective chemosensor for cyanide anion.
of probe BP at 552 nm was decreased its intensity and another
band at 450 nm was showed almost similar structural patterns.
The same was recognized by the nakedꢀeye in the form of color
change from purple to light yellow (Fig. 1a, inset).
70
(a)
(b)
BP+
CNꢀ
BP
75
Fig. 1 (a) Absorption titration curve of BP (c = 1×10−5 M) in aq.
CH3CN (CH3CN/H2O = 7:3 v/v, 20 mM HEPES buffer, pH = 7.4)
with NBu4CN (c = 1×10−4 M). Inset shows naked eye color
change before (purple) and after (light yellow) addition of CNꢀ to
35 RESULTS AND DISCUSSION
80 BP. (b) Competitive absorption spectra of BP (c = 1×10−5 M) in
the presence of different anions (c = 1×10−4 M) (nꢀBu4N+ salts of
Probe BP could be conveniently synthesized via the condensation
of 4ꢀ(diethylamino)ꢀ salicylaldehyde
with Nꢀmethylꢀ2ꢀ
−
2─
CN−, AcO−, F─, Cl−, Br−, I− and Na+ salts of S2−, H2PO4 , SO4
,
acetylthiophenazine in conc. H2SO4. Structural identification of
1
the compound was confirmed by H NMR, 13C NMR, and ESIꢀ
NO2 , ClO4 , SCN−, SO32− and CO32−) and different biothios (c =
1×10−4 M) (Cys, Hcy and GSH) in aq. CH3CN (CH3CN:H2O =
85 7:3 v/v, 20 mM HEPES buffer, pH = 7.4).
−
−
40 MS spectroscopy (ESI†).
O
CH3
N
O
C
H
N
C
CH3
a
CH3
S
S
Furthermore, the selectivity of BP for cyanide over other anions
was examined. The results revealed that all potentially
competitive anions exerted no or little influence (for S2−) on the
UV–vis detection of cyanide in aqueous acetonitrile (3/7 v/v)
90 (Fig. 1b).
1
2
CHO
OH
b, c
N
NC
H
CH3
CH3
N
CN ꢀ
N
N
O
N
O
The most remarkable changes were seen in the fluorescence
titration studies. The free probe BP exhibited weak emission at
∼442 nm when excited at 350 nm in aqueous acetonitrile (3/7
v/v) mixture at pH 7.4 to have an effective 20 mM HEPES buffer
95 solution. Titration of probe BP with CN− results in the
enhancement of fluorescence intensity as a function of the added
S
45
S
ꢀ
ClO4
BPꢀCN
BP
Scheme 1 Synthesis of BP. Reagents and conditions: (a) MeI,
K2CO3, KI, TBAB, Acetonitrile, (b) Conc. H2SO4, heat at 90oc , 6
h; (c) 70% HClO4.
2
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CN− concentration (Fig. 2a). With the addition of CN−, the
emission at 442 nm increased sharply, followed by the red shifted
emission from 442 nm to 512 nm was observed (only Nꢀ
methylphenothiazine moiety shows at 526 nm), indicating that
the Michael addition reaction interrupted the πꢀconjugation and
blocked the ICT process, after which the fluorescence of Nꢀ
methylphenothiazine recovered. This is in good agreement with
the aforementioned design concept. The fluorescence intensity
increased linearly with added cyanide concentration (Fig. 2b).
(b)
50
(a)
5
10 Addition of only 2 equiv of cyanide anion saturated the system,
and no more change was seen with further addition of cyanide
anion, suggesting 1:1 stoichiometric reaction of BP with cyanide
anion; the corresponding Jobs plot provided additional support to
the stoichiometric ratio (Fig. S6†). The fluorescence detection
15 limit was 0.13 µM in HEPES solution, which was also much
lower than that of the most reported fluorometric and colorimetric
cyanide probes (Fig. S9†).19
–
–
–
–
ꢀ
2
2
4
O
O
4
O
l
C
P
H
S
55
Fig. 3 (a) Competitive fluorescence emission spectra of BP (c =
1×10−5 M) in the presence of different anions (c = 1×10ꢀ4 M) (nꢀ
Bu4N+ salts of CN−, AcO−, F─, Cl−, Br−, I− and Na+ salts of S2−,
H2PO4 , SO42─, NO2 , ClO4 , SCN−, SO3 and CO32−) and
60 different biothios (c = 1×10ꢀ4 M) (Cys, Hcy and GSH) in aq.
CH3CN (CH3CN:H2O = 7:3 v/v, 20 mM HEPES buffer, pH =
7.4). (b) Fluorescence response of BP (c = 1×10ꢀ5 M) to addition
of 2 equiv. CN− (c = 1× 10ꢀ4 M) and 12 equiv. of other anions (c
= 1×10ꢀ4 M) [the black bar portion] and to the mixture of 12
65 equiv. of other anions (c = 1×10ꢀ4 M) with addition of 2 equiv.
CN− (c = 1× 10ꢀ4 M) [the red bar portion].
−
−
−
2−
(a)
(b)
BP+
BP
ꢀ
CN
20
These observations suggest that compound BP is highly selective
toward CN− even in the presence of the complex mixture of
anions.
Fig. 2 (a) Emission spectra of BP (c = 1×10−5 M) (λex = 350 nm)
70 Time Response Experiments: Timeꢀdependent fluorescence
spectra of BP were monitored in the presence of 2 equiv. cyanide
(Fig. 4). The result revealed that the Michael type addition
reaction can be finished within 30 s, indicating that the probe BP
would provide a rapid detection of cyanide.
25 in aq. CH3CN (CH3CN/H2O = 7:3 v/v, 20 mM HEPES buffer, pH
= 7.4) with NBu4CN (c = 1×10−4 M). Inset shows fluorescent
color change before (low intense blue) and after (bright green)
addition of CN− to BP. (b) Change of emission intensity at 512
nm with incremental addition of CN− ions [λext = 350 nm].
75
30 The cyanide sensing process was also clearly seen not only by
color change but also by bright green fluorescence under UV
lamp. The color change from low intense blue to bright green
fluorescence could be distinguished by the naked eyes as shown
in Fig. 2a, inset.
35 Interference of Other Anions Towards BP: To evaluate the
selectivity of BP, we measured the fluorescence intensity of BP
in the presence of various anions (CN−, AcO−, F−, Cl−, Br−, I−,
80 Fig. 4 Variation of the fluorescence intensity at 512 nm vs. the
reaction time of BP (1×10−5 M) in aq. CH3CN (CH3CN/H2O =
7:3 v/v, 20 mM HEPES buffer, pH = 7.4) in the presence of
different equiv. amount of CN− (c = 1× 10ꢀ4 M).
S2−, H2PO4 , SO42−, NO2 , ClO4 , SCN−, SO3 and CO32−) and
biothios (Cys, Hcy and GSH). The fluorescence maximum
40 intensity of BP was dramatically increased almost 6 times after
the addition of cyanide, whereas the fluorescence intensity
changes were not observed by other anions but were just
comparable to BP itself. Competitive assay also showed
consistent evidence that BP only was transformed by cyanide
45 anion. The fluorescence intensity of BP and other anions were
restored as much as that of BP by addition of cyanide to the
mixture of BP and other anions (Fig. 3b).
−
−
−
2−
Fluorescence Response of BP at Various pH. The titrations
85 were carried out at different pH of the medium exhibited
maximum fluorescence intensity in the pH ~ 7.4. However, at
highly acidic and basic pH, the fluorescence of the BP during
reaction with CN− shows a gradual decrease in intensity and the
instability of the nucleophilic CN− under these conditions (Fig.
90 S10†). This result indicates that the probe may be suitable for
bioꢀapplications at the physiological pH.
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Proposed Sensing Mechanism: Such a molecular design makes
probe BP possess the expanded πꢀconjugation as well as the 45 ppm (Hi) went nearly disappeared and a new peak was appeared
produce the neutral molecule. In presence of CN−, peak at 8.02
strong
ICT
from
the
phenothizine
to
the
at 3.78 ppm and peak at 7.92 ppm (Hj) of benzopyrylium moiety
shifted upꢀfield to 7.75 ppm. This result clearly indicate that Cꢀ4
carbon lost its unsaturation, supports the formation of BPꢀCN
adduct.
diethylaminobenzopyrylium moiety, which will lead to the higher
region absorption. Importantly, this type of compound can
achieve high selectivity as well as nakedꢀeye detection in a very
5
short time because it possesses an electronꢀwithdrawing 50 Theoretical Studies: To investigate the electronic structure and
benzopyrylium group that is highly reactive for cyanide.
Therefore, the nucleophilic attack of cyanide toward the
10 benzopyrylium group of probe BP will block the πꢀconjugation
the observed spectroscopic properties of the probe BP and its
cyanide adduct BPꢀCN, DFT geometry optimizations followed
by TDꢀDFT calculations using the Gaussian 09W20 software
package at the B3LYP/6ꢀ31G(d,p) + solv(PCM) level of theory
between phenothizine and diethylaminobenzopyrylium moiety,
by which a decrease in intensity of absorption and emission of 55 were carried out. As well as to explain the highly reactive nature
phenothizine should be induced, resulting in the spectral changes
as well as the change in color (Scheme 2). These signal changes
15 indicate that probe BP can serve as a selective chemosensor for
cyanide anion.
towards nucleophile of probe BP, the electrophilic Fukui function
from density functional reactivity theory (DFRT)21 was
performed to estimate the electrophilicity of the reactive carbon
centres such as Cꢀ2, Cꢀ4 and Cꢀ5 of chromene moiety in probe
60 BP using the B3LYP exchange functional and the NBO charge.
In general, electrophiles are molecules able to accept an amount
of electron density along the reaction, nucleophiles are molecules
able to donate an amount of electron density during the reaction.
NC
H
CH3
N
CH3
N
N
O
CN ꢀ
O
N
S
ꢀ
ClO4
S
(b)
(a)
ICT On
ICT Off
20
65
Scheme 2 Schematic presentation of nucleophilic attack of
cyanide to BP.
1HꢀNMR Titration: In addition, the binding pattern between
1
probe BP and CN− was first examined by a H NMR titration
Fig. 6 Calculated energyꢀminimized structure of (a) BP and (b)
BPꢀCN.
1
25 experiment (Fig. 5). The H NMR titration studies showed that
almost all aromatic proton signals shifted to upꢀfield upon
addition of CN− to the solution of BP; this is consistent with the 70 From
a theoretical point of view, the electrophilic and
proposed
product
in
which
the
cationic
nucleophilic behaviours of organic molecules can be
characterized by using the reactivity indices defined within the
conceptual density functional theory (DFT) framework.22 The
calculations provide the electrophilicity of the carbon centers
75 such as Cꢀ2, Cꢀ4 and Cꢀ5 as 3.0741, 4.4663 and 1.3922 au,
respectively.
diethylaminobenzopyrylium moiety is changed into a neutral one.
Ha
H
Hi
30
NC
Hj
CH3
N
HK
CH3
N
ꢀ
CN
Hl
N
O
O
N
S
ꢀ
ClO4
S
BPꢀCN
BPꢀCN
BP
LUMO
BP
35
LUMO
80
4.021 eV
HO MO
2.269 eV
HO MO
1
Fig. 5 H NMR titration chart of BP (10.0 mg in 0.5 ml DMSO)
without CN─ and with addition of 2 equiv. of CN─ in DMSOꢀd6
40 medium.
Especially, ꢀNCH2 (HK) proton of diethylaminobenzopyrylium
moiety shifted upꢀfield from 3.46 ppm to 3.29 ppm, indicate the
blocking of ICT process after the attack of CN− and hence
85 Fig. 7 HOMO and LUMO distributions of BP and BPꢀCN,
calculated at the B3LYP/6ꢀ31G(d) level.
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Thus, the Cꢀ4 carbon of probe BP is much more electrophilic
human cells were incubated with probe BP (2 ꢁM) for 30 min at
than that of other centers Cꢀ2, and Cꢀ5, in good agreement with 50 37°C, a very weak fluorescence was observed. However, when
the observation that probe BP is much more reactive towards
nucleophilic cyanide attack at Cꢀ4 centre instead of other centers.
The optimized geometries of BP and BPꢀCN are shown in Fig. 6.
The spatial distributions of frontier orbitals and orbital energies
of HOMO and LUMO of BP and BPꢀCN were also determined
(Fig. 7). The calculated HOMOꢀLUMO energy gaps of BP and
BPꢀCN adduct is 2.269 and 4.021 eV respectively (Table S2†).
10 There is a considerable difference in the energy minimisation
structure in BP and BPꢀCN adduct, which can shed light on the
change in spectral intensity in absorption spectra due to blockage
of πꢀconjugation on CN− addition and the corresponding change
in color. In BP, FMOs are mostly separated, HOMO is positioned
15 along the electronꢀricher phenothiazine moiety, while the LUMO
chiefly resides along the positive part of chromene moiety. This
orbital distribution suggested that πꢀconjugation occurs at excited
state and the cyanideꢀadduct of BP should impact HOMO and
LUMO unevenly and thus constitute the basis of an optical
20 response of BP to cyanide addition.
A459 human cells were treated with probe BP followed by
cyanide salts, we observed a bright green fluorescent from the
intracellular area (in the green channel).
5
55
(a)
(c)
(b)
(d)
60
Fig. 9 Confocal microscopic images of the probe BP in A549
human cells:(a) bright field image of the cells treated with probe
BP, (b) confocal fluorescence image of probe BP at 2.0 × 10−6
M
Practical Application: To investigate the practical application of
probe BP, test strips were prepared by immersing filter papers
into aqueous acetonitrile (3/7 v/v) mixture at pH 7.4 solutions of
BP (1.0× 10−2 M) and then drying in air. The test strips
25 containing BP were utilized to sense cyanide.
concentration, (c) bright field image of the cells treated with
65 probe BP and NBu4CN (1.0 × 10−5 M) (d) confocal fluorescence
image of probe BP and NBu4CN. all images were acquired with a
40× objective lens.
Appearance of such type of green fluorescence indicated the
formation of BPꢀCN complex in the A549 human cells, which is
70 also in good agreement with the fluorescence turnꢀon report of
the probe in presence of cyanide ions in solution. Further, brightꢀ
field images of cells confirmed that the A549 human cells were
viable throughout the imaging experiments. Thus, these results
revealed that probe BP possesses good membrane permeability
75 and is proficient for monitoring cyanide ions in living cells. In
addition, we also studied MTT assay to know the cytotoxicity in
A549 human cells and it is found that probe BP does not show
noticeable cytotoxicity to A549 human cells (Fig. S11†).
(a)
(b)
30
(1)
(2)
(3)
(4)
(5)
(6)
Fig. 8 (a) Naked eye in visible light and (b) Fluorescence color
changes visualized on filter paper strips of (1) BP (c = 1.0× 10−2
M) and during addition of CN− at (2) 1.0×10−5 M; (3) 1.0×10−4
35 M; (4) 1.0 ×10−3 M; (5) 1.0 × 10−2 M ; (6) 4.0×10−2 M in aq.
CH3CN (CH3CN/H2O = 7:3 v/v).
Conclusion: In conclusion, we have synthesized and
80 characterized a new colorimetric and fluorometric probe BP for
CN− based on
a hybrid benzopyryliumꢀphenothizineꢀliked
flavylium moiety. Their selectivity and sensitivity were
demonstrated on the basis of fluorescence, absorption, and 1H
NMR spectroscopy, and visual fluorescent color changes. The
85 probe BP can detect CN− up to 0.13 µM, by switchꢀon
fluorescence, suggesting its applicability to detect CN− ions in
aqueous HEPES buffer medium. This work highlights a simple
reaction mechanism between BP and cyanide anion. Furthermore,
the test strips could conveniently and rapidly (within a short time
90 of 30 s) detect cyanide in solutions. The probe BP was
demonstrated as potential liveꢀcell fluorescence imaging agents
under microscopy using A549 human cells. Potential practical
applications of the method are under intensive investigation in
our laboratories.
As shown in Fig. 8, when cyanide was added on the test kits at
various concentrations, the obvious color changes from burgundy
to light yellow in visible light and from low intense blue to bright
40 green under the 365 nm UV–lamp was observed. Therefore, the
test strips coated with the probe BP solution would be convenient
for detecting CN− according to guideline of WHO. These results
showed that probe BP could have a practical application for
detecting CN− in environmental samples.
45 Live Cell Imaging: To demonstrate the practical application of
probe BP in biological sample, we performed a cellular imaging
experiment of BP using A549 human cells under the confocal
laser scanning microscope. As shown in Fig. 9, when A459
95
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Computational studies: All geometries for BP and BPꢀCN were
optimized by density functional theory (DFT) calculations as well
as timeꢀdependent density function theory (TDDFT) calculations
55 were performed with Gaussian09 program package, with the aid
of the GaussView visualization program.
Experimental Section
General information and materials: Unless stated otherwise,
commercial grade chemicals were used for the synthesis of
probes were purchased from Sigma Aldrich Chemical Co. (USA)
and used without further purification. Salts of different anions
viz. CN−, AcO−, F−, Cl−, Br− and I− as nꢀBu4N+ salts and S2−,
5
Cell incubation studies: A549 human cell (ATCC: CCLꢀ185)
lines were prepared from continuous culture in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma Chemical Co., St.
60 Louis, MO) supplemented with 10% fetal bovine serum
(Invitrogen), penicillin (100 ꢁg/mL), and streptomycin (100
ꢁg/mL). The A549 human cells were obtained from the American
Type Culture Collection (Rockville, MD) and maintained in
DMEM containing 10% (v/v) fetal bovine serum and antibiotics
65 in a CO2 incubator. Cells were initially propagated in 75 cm2
polystyrene, filterꢀcapped tissue culture flask in an atmosphere of
5% CO2 and 95% air at 37°C in CO2 incubator. When the cells
reached the logarithmic phase, the cell density was adjusted to
1.0×105 per/well in culture media. The cells were then used to
70 inoculate in a glass bottom dish, with 1.0 mL (1.0×104 cells) of
cell suspension in each dish. After cell adhesion, culture medium
was removed. The cell layer was rinsed twice with phosphate
buffered saline (PBS), and then treated according to the
experimental need.
−
−
H2PO4 , SO42─, NO2 , ClO4 and SCN− as Na+ salts were
purchased from Spectrochem Pvt Ltd. (India), stored in a
desiccators under vacuum containing selfꢀindicating silica, and
10 used without any further purification. All the solvents were of
analytic grade. Solvents were dried according to standard
procedures. Elix Millipore water was used throughout all
experiments. All reactions were magnetically stirred and
monitored by thinꢀlayer chromatography (TLC) using
15 Spectrochem GF254 silica gel coated plates. Column
1
chromatography was performed with 60ꢀ120 silica gel. H and
13C NMR spectra were recorded on a Brucker 400 MHz, 300
MHz instrument. Mass spectra were carried out using a Waters
QTOF Micro YA 263 mass spectrometer. For NMR spectra,
20 DMSOꢀd6 and CDCl3 were used as solvent using TMS as an
internal standard. Chemical shifts are expressed in δ ppm units
1
1
and H–1H and H–C coupling constants in Hz. UV–visible and
fluorescence spectra measurements were performed on
SHIMADZU UVꢀ1800 and PerkinElmer LSꢀ55
a
a
75 Cellular imaging methodology: For confocal imaging studies
A549 human cells, 1×104 cells in 1000 ꢁL of medium, were
seeded on sterile 35 mm covered Petridis, glass bottom culture
dish (ibidi GmbH, Germany), and incubated at 37°C in a CO2
incubator for 10 hours. Then cells were washed with 500 ꢁL
80 DMEM followed by incubation with 1.0 x 10ꢀ5 M NBu4CN
dissolved in 500 ꢁL DMEM at 37°C for 1 h in a CO2 incubator
and observed under an Olympus IX81 microscope equipped with
a FV1000 confocal system using 1003 oil immersion Plan Apo
(N.A. 1.45) objectives. Images obtained through section scanning
85 were analyzed by Olympus Fluoview (version 3.1a; Tokyo,
Japan) with excitation at 395 nm monochromatic laser beam, and
emission spectra were integrated over the range 350ꢀ650 nm
(single channel). The cells were again washed thrice with
phosphate buffered saline PBS (pH 7.4) to remove any free
90 NBu4CN and incubated in PBS containing probe BP to a final
concentrations of 2.0 × 10ꢀ6 M, incubated for 10 min followed by
washing with PBS three times to remove excess probe outside the
cells and images were captured. In separate culture dish the cells
were similarly treated with 2.0×10ꢀ6 M probe BP incubated for 10
95 min, washed thrice with PBS and the image was captured to get
any possible background fluorescence.
25 spectrofluorimeter respectively. The following abbreviations are
used to describe spin multiplicities in 1H NMR spectra: s =
singlet; d = doublet; t = triplet; m = multiplet.
Preparation of test solution for UV−vis and fluorescence
spectral studies: A stock solution of the probes BP (1.0×10−5 M)
30 were prepared in CH3CN/H2O (7:3 v/ v). Solutions of 1.0×10−4
M
salts of the respective anions were prepared in Millipore water.
All experiments were carried out in CH3CN/H2O solution
(CH3CN/H2O = 7:3 v/v, 20 mM HEPES buffer, pH = 7.4). In
titration experiments, each time a 1×10−5 M solution of BP were
35 filled in a quartz optical cell of 1 cm optical path length, and the
ion stock solutions were added into the quartz optical cell
gradually by using a micropipet. Spectral data were recorded at 1
min after the addition of the ions. In selectivity experiments, the
test samples were prepared by placing appropriate amounts of the
40 anions (1.0×10−4 M) stock into 2 mL of solution of BP (1×10−5
M).
Detection limit studies : The detection limit was calculated on
the basis of the fluorescence titration. The fluorescence emission
spectrum of BP was measured 10 times, and the standard
45 deviation of blank measurement was achieved. To gain the slope,
the ratio of the fluorescence intensity at 512 nm was plotted as a
concentration of CN˗. So the detection limit was calculated with
the following equation.
Preparation of compound 2: 2ꢀacetylthiophenazine (300 mg,
1.24 mmol) was dissolved in dry CH3CN. K2CO3 (257 mg, 1.86
mmol), a pinch of KI and catalytic amount TBAB were added to
100 the solution. Drop wise methyl iodide (265 mg, 1.86 mmol) was
o
Detection limit =3Sb1/S
(1)
added into it and the reaction mixture was stirred at 82 C for
overnight. After reflux, the solvent was evaporated in vacuum.
Afterwards, the mixture was extracted with dichloromethane. The
extractions were combined, washed with distilled water, saturated
105 brine, and then dried over anhydrous sodium sulfate. After
50 where Sb1 is the standard deviation of blank measurement and S
is the slope of the calibration curve.
6
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New Journal of Chemistry
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DOI: 10.1039/C7NJ02716H
evaporation of solvent, the crude compound was purified over
silica gel column and desired compound was eluted by PE: EA
(10:1 v/v) to obtain 210 mg pure product as yellow liquid. Yield:
J. Withey, Toxicology Profile for Cyanide, U.S. Department of
Health and Human Services, Washington, DC, 2006; (c) M. A.
Cherian and I. J. Richmond, Clin. Pathol., 2000, 53, 794; (d) M.
Laforge, H. Gourlain, D. Fompeydie, F. Buneaux, S. W. Borron
1
66%. Mp above 250°C. HꢀNMR (CDCl3, 400 MHz): δ (ppm)
5
7.35 (d, 1H, J=7.8 Hz), 7.11 (s, 1H), 6.96 (q, 3H), 6.81 (1H, t, 55 and M. J. GalliotꢀGuilley, J. Toxicol. Clin. Toxicol., 1999, 37,
J=7.16 Hz), 6.53 (d, 1H, J=7.64 Hz), 5.95 (s, 1H), 3.29 (s, 3H),
2.51 (s, 3H). MS (ESIꢀMS): (m/z, %): 256.40 [M+H]+;
Calculated for C14H11NSO: 255.25.
337.
2. S. I. Baskin, T. G. Brewer, F. Sidell, E. T. Takafuji and D. R.
Frenz, Medicinal Aspects of Chemical and Biological Warfare,
TMM, Washington, DC, 1997, ch. 10, pp. 271.
Preparation of BP: 4ꢀdiethylaminoꢀsalicylaldehyde (0.151 g,
10 0.784 mmol) and compound 2 (0.2 g, 0.784 mmol) were
dissolved in conc. H2SO4 (7 ml). The reaction mixture was stirred
at 80ꢀ90oC temperature for 6 h. After the completion of the
reaction (monitored by TLC), the reaction mixture was poured
into iceꢀwater, and then 70 % perchloric acid (2 ml) were added;
15 then deep purple solid precipiteted was filtered through suction,
washed several times with milipore water and dried in air. The
residue was purified via chromatography with silica gel (CH3Cl :
CH3OH = 95.5 : 0.5) and solid purple product was obtained
(0.342 g, 93 % yield); M.P. > 250˚C. 1H NMR (DMSOꢀ d6, 400
20 MHz) δ(ppm): 8.9475 (s, 1H), 8.7801 (d, 1H, J= 8.0 Hz), 8.02985
(d, 1H, J= 9.48 Hz), 7.92595 (d, 1H, J= 7.8 Hz), 7.6764 (t, 1H, J=
7.32 Hz), 7.5443 (d, 1H, J= 9.6), 7.3555 (d, 1H, J= 8.72 Hz),
7.1625 (s, 1H), 7.033 (q, 1H), 6.94 (d, 1H, J= 7.32 Hz), 6.79 (t,
1H, J= 7.28 Hz), 3.467325 (q, 4H), 3.2588 (s, 3H), 1.193 (t, 6H,
25 J= 8.8 Hz),. 13C NMR (DMSOꢀd6, 75 MHz) δ(ppm): 159.79,
142.39, 142.07, 141.72, 141.14, 138.64, 131.64, 129.07, 128.27,
127.47, 124.54, 123.16, 122.39, 122.08, 115.78, 114.93, 114.52,
113.30, 110.14, 104.74, 97.71, 44.42, 42.49, 12.93. TOF MS
60 3. (a) S. I. Baskin, J. B. Kelly, B. I. Maliner, G. A. Rockwood, C.
K. Zoltani and D. T. Shirley, Medicinal Aspects of Chemical
Warfare, TMM, Washington, DC, 2008, ch. 11, pp. 331; (b) J. L.
Way, Annu. Rev. Pharmacol. Toxicol., 1984, 24, 451; (c) C. E.
Cooper and G. C. Brown, J. Bioenerg. Biomembr., 2008, 40, 533;
65 (d) J. D. Johnson, T. L. Meisenheimer and G. E. Isom, Toxicol.
Appl. Pharmacol., 1986, 84, 464.
4. (a) K. W. Kulig, Cyanide toxicity., Atlanta, GA: U.S.
Department of Health and Human Services; 1991; (b) C. Baird
and M. Cann, Environmental chemistry, New York: Freeman;
70 2005.
5. (a) R. Koenig, Science, 2000, 287, 1737; (b) Ullmann’s
Encyclopedia of Industrial Chemistry, WileyꢀVCH, New York,
6th edn, 1999; (c) F. Huo, J. Kang, C. Yin, J. Chao and Y. Zhang,
Sens. Actuators B Chem., 2015, 215, 93; (d) X. Lou, D. Ou, Q. Li
75 and Z. Li, Chem. Commun., 2012, 48, 8462.
ES+, m/z = 414.4 [M+H]+; calculated for C26H25N2SO
30 413.168.
=
6. (a) K. Grosssmann, Pest Manag Sci., 2010, 66, 113; (b) P. A.
Gale, Coord. Chem. Rev., 2013, 213, 79.
Acknowledgements
7. T. Z. Sadyrbaeva, Sep Purif Technol., 2012, 86, 262.
We
gratefully
acknowledge
DSTꢀSERB
[File
No.
8. C. Young, L. Tidwell and C. Anderson, Minerals, Metals and
80 Materials Society, Warrendale, PA 2001.
EMR/2016/006640] for financial support. SM and SSA thanks to
the UGC and UGCꢀMANF, New Delhi for the financial and
35 research support.
9. (a) H. Sun, Y. Y. Zhang, S. H. Si, D. R. Zhu and Y. S. Fung,
Sens. Actuators, B Chem., 2005, 108, 925.
Notes and references
10. (a) G. Qian, X. Z. Li and Z. Y. Wang, J. Mater. Chem., 2009,
19, 522; (b) L. H. Peng, M. Wang, G. X. Zhang, D. Q. Zhang and
85 D. B. Zhu, Org. Lett., 2009, 11, 1943; (c) X. Lou, L. Zhang, J.
Qin and Z. Li, Chem. Commun., 2008, 5848.
aDepartment of Chemistry, Indian Institute of Engineering
Science and Technology, Shibpur, Howrahꢀ711103, West Bengal,
India,
Email:
akmahapatra@rediffmail.com,
Fax:
40 +913326684564
bDepartment of Microbiology, University of Calcutta, Kolkataꢀ
700019.
11. (a) Z. Xu, X. Chen, H. Kim and J. Yoon, Chem. Soc. Rev.,
2010, 39, 127; (b) F. Wang, L. Wang, X. Chen and J. Yoon,
Chem. Soc. Rev., 2014, 43, 4312; (c) E. P. Randviir and C. E.
90 Banks, TrAc. Trends Anal. Chem., 2015, 64, 75; (d) S.ꢀW. Nam,
X. Chen, J. Lim, S. H. Kim, S.ꢀT. Kim, Y.ꢀH. Cho, J. Yoon, S.
Park, PLos ONE, 2011, 6, e21387; (e) J. Lv, F. Wang, J. Qiang,
X. Ren, Y. Chen, Z. Zhang, Y. Wang, W. Zhang and X. Chen,
Biosensors and Bioelectronics 2017, 87, 96; (f) X. Chen, S.ꢀW.
95 Nam, G.ꢀHee Kim, N. Song, Y. Jeong, I. Shin, S. K. Kim, J. Kim,
S. Park and J. Yoon, Chem. Commun., 2010, 46, 8953; (g) B.
Garg and Y.ꢀC. Ling, Chem. Commun., 2015, 51, 8809; (h) B.
cInstitute of Chemistry, The Hebrew University of Jerusalem,
Israel
45 †Electronic Supplementary Information (ESI) available: [details
of any supplementary information available should be included
here]. See DOI: 10.1039/b000000x/
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DOI: 10.1039/C7NJ02716H
Table of Contents
Benzopyrylium-phenothiazine-conjugate of flavylium derivative as
fluorescent chemosensor for cyanide in aqueous media and its bioimaging
Sanchita Mondal,a Syed Samim Ali,a Srimanta Manna,a Kalipada Maiti,a Md. Raihan Uddinb,
Sukhendu Mandalb, Debasish Mandalc and Ajit Kumar Mahapatra*a
aDepartment of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur,
Howrah – 711103, India.
bDepartment of Microbiology, Ballygunge Science College, Kolkata- 700019.
cInstitute of Chemistry, The Hebrew University of Jerusalem, Israel
*Corresponding author: Tel.: +91 33 2668 4561; fax: +91 33 26684564;
E-mail: mahapatra574@gmail.com
NC
H
CH3
N
CH3
N
CN -
N
O
O
N
S
-
ClO4
S
BP
BP-CN