ATP Induced Modulation in π-π Stacking Interactions in Pyrene Based Zinc Complexes: Chemosensor Study and Quantitative Investigation of Apyrase Activity

Article abstract of DOI:10.1021/acs.cgd.8b00165

Fluorescent zinc complexes of 1,2-disubstituted benzimidazole (R1-R3) have been synthesized and characterized using single crystal X-ray diffraction. The ligands L1-3 were found to be less emissive due to photoinduced electron transfer (PET) mechanism ori

Full text of DOI:10.1021/acs.cgd.8b00165

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Article
ATP Induced Modulation in ### Stacking Interactions
in Pyrene Based Zinc Complex: Chemosensor Study
and Quantitative Investigation of Apyrase Activity
Amanpreet Singh, Pushap Raj, Jan J Dubowski, and Narinder Singh
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00165 • Publication Date (Web): 20 Jun 2018
Downloaded from http://pubs.acs.org on June 21, 2018
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ATP Induced Modulation in π−π Stacking Interactions in Pyrene Based Zinc
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Complex: Chemosensor Study and Quantitative Investigation of Apyrase Activity
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Amanpreet Singha, Pushap Raja, Jan J. Dubowskib, Narinder Singha,*
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aDepartment of Chemistry, Indian Institute Technology Ropar, Punjab, 140001, India
bLaboratory for Quantum Semiconductors and PhotonꢀBased BioNanotechnology, Interdisciplinary
Institute for Technological Innovation (3IT), CNRS UMIꢀ3463, Department of Electrical and Computer
Engineering, Université de Sherbrooke, 2500 boul. de l’Université, Sherbrooke, Québec J1K
2R1, Canada
*Corresponding author (Narinder Singh) E-mail: nsingh@iitrpr.ac.in; Tel: +91-1881242176
Abstract: Fluorescent zinc complexes of 1,2ꢀdisubstituted benzimidazole (R1ꢀR3) have been
synthesized and characterized using single crystal XRD. The ligands L1ꢀ3 were found to be less
emissive due to photoinduced electron transfer (PET) mechanism originated from the electron pair of
benzimidazole nitrogen. The complexation of ligands with Zn(II) not only enhances the fluorescent
intensity, however also orient the ligands to an new packing. It was observed that the aromatic unit plays
a decisive role in the packing of the molecules. The complex R1 has extended the coordination through
CꢀHꢀπ interaction, whereas complex R2 involved CꢀHꢀπ interaction and CꢀHꢀBr interaction for
packing in supramolecular architecture. Among these complexes, R3 showed most interesting
noncovalent interaction pattern involving CꢀHꢀπ interaction, CꢀHꢀBr interaction and π ꢀ π stacking
between pyrene rings. These noncovalent interactions govern photophysical properties that are sensitive
toward microenvironment. Thus, by altering these interactions, the selective sensing for a particular
analyte can be achieved. The complexes R1 and R2 have shown enhanced emission intensity upon
interacting with ATP competitively in the presence of some other tested anions. A ratiometric change in
emission spectra of the complex R3 was observed upon binding with ATP in semiꢀaqueous medium
offering the lowest detection limit of 15 nM. Upon interaction with ATP the π ꢀ π stacking between
pyrene rings breaks and results in a decrease in excimer emission at 470 nm and increases in monomeric
emission intensity at 410 nm. The AFM images of receptor R3 show that upon addition of ATP to the
R3 solution, solvent mediated aggregation takes place, which results in the ratiometric detection. In the
DMF solvent system aggregates were formed, whereas in Water:THF solvent system clear solution was
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converted to a highly viscous gel. To investigate the applications of the prepared sensor, fluorescence
response of Hela cells enriched with ATP was observed using fluorescence microscopy. The
fluorescence modulation of sensor in living cells makes the receptor practically applicable in a
biological environment. Quantitative analysis of apyrase activity has shown that the presented sensor R3
is capable to monitor the hydrolysis process in the biological system.
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Key words: Fluorescent sensor, benzimidazole, pyrene, π ꢀ π stacking, ratiometric, adenosine
triphosphate
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INTRODUCTION
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Detection of anionic species in aqueous medium becomes an imperative goal for supramolecular
chemistry because these are pervasive in biological system and an play important role in environmental
monitoring and diagnosis.1–5 Unlike cations; anionic species have different shape and size, hence anionic
biomolecules require complementary receptors to encapsulate.6–9 The biologically important anionic
species such as phosphates, and nucleic bases; due to their diverse size and shape demand the receptor
design which is sometimes tedious to develop using synthetic skills. The literature reports reveal the
synthesis of organic receptors that can detect analyte through anion induced fluorescence
modulations.10,11 Most of these organic receptors are fabricated of urea/thiourea group or imidazolium
organic cation.12ꢀ15 The rationale of insertion of urea/thiourea in receptor design lies with the fact that
these moieties have a tendency to form hydrogen bonds with anionic analytes. However, on the other
hand, it has been observed that the receptors relying on purely hydrogen bonding have limited
application while working in aqueous medium due to the competition provided to binding site from
water, and moreover hydration of anionic species.16,17,18–20 Therefore, the design of a sensor for
biomolecules, particularly in an aqueous medium, is still a challenge to inorganicꢀanalytical chemistry.
Among these biomolecules, adenosine triphosphate (ATP) is one of the most important anions that
contain three negatively charged phosphates.21–23 It enzymatically hydrolyzes in the cell to give
adenosine diphosphate (ADP), adenosine monophosphate (AMP), and pyrophosphates.24,25 Apart from
these, it also plays significant role in DNA replication and transcription. Due to the similarity in
structure of various competing species, a selective sensor for ATP is the need of the hour. Selectivity
can be modulated (improved) by incorporation of hydrogen bonding interaction, ionic interaction, Van
der Waal interaction and π−π interaction into the receptor.26,27 Along with these interactions CꢀHꢀπ
interaction and CꢀHꢀhalide interactions also play a decisive role in the formation of the supramolecular
assembly through noncovalent interactions.28–31 Likewise, π−π stacking and hydrogen bonding
interaction causes the formation of supramolecular selfꢀassembly which results in aggregation formation
or gel formation.32–39 Not only supramolecular assembly of metal complex is engaged by noncovalent
interactions, their photophysical properties are also directed by these weak interactions in solution as
well as in solid state.40–44 Therefore, by taking advantages of noncovalent interactions sensitive and
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selective sensor can be developed. Recently Maji et al have developed a selective sensor for Al(III), that
responds toward Al(III) by fluctuating emission behavior due to change induced by noncovalent
interactions.45 ATP has adenosine subunit that can interact through π−π interaction with analyte.46–48
Similarly three negatively charged phosphate units have a tendency to interact through ionic interaction.
In view of this, Yoon at el had constructed a dipodal receptor that contains pyrene excimer as signaling
unit which upon stacking with adenine gave change in emission.49 The imidazolium unit conjugated to
pyrene act as a phosphate ion receptor. The sensor showed ratiometric sensing of ATP, however, some
analytes such as AMP and ADP also show some interference. Kataev at el have synthesized two copper
complexes labeled with fluorescent coumarin and anthracene units for detection of ATP in an aqueous
medium, which shows some interference due to other analytes.50 Also, some binuclear complexes have
been developed for sensing of ATP, AMP and pyrophosphates. The two phosphate units coordinate with
two different zinc ions, resulting in a change in photophysical properties of the receptor.
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In the present work, we have synthesized 1,2 disubstituted fluorescent labeled benzimidazole derivatives
(Figure S1) and their complexes with zinc ion. Particularly, zinc ion was chosen due to its d10
configuration, which resists the loss of fluorescence upon complexation due to nonꢀavailability of open
shell effect. The complexes R2 and R3 contain two halide ion and one solvent molecule which can be
easily replaced by some strong field ligand. The most interesting features of these complexes are their
differences in noncovalent interactions; resulting from a change in aromatic unit attached with
benzimidazole moiety. These complexes have the ability to interact with analyte through ionic
interaction as well as π−π stacking. Due to the dual interaction of analyte, ratiometric sensing can be
achieved. The ratiometric sensors are more reliable than simply “onꢀoff” fluorescence sensors because
the ratiometric behavior is independent from environmental factors, such as temperature, concentration,
slit width and scan rate.46 To the best of our knowledge, the prepared metal complexes of 1,2
disubstituted benzimidazole are the novel and there is no report on disubstituted benzimidazole based
sensor for biomolecules. Thus, this system provides a simpler and more effective way to detect ATP
than the methods reported in the existing literature.47ꢀ49 The interesting fact about this work is the
formation of metallogel upon interaction of R1 with ATP in THF: water (30:70).
RESULT AND DISCUSSION
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Synthesis and Characterization of Ligand and Metal Complex: The ligand L1 and L2 were prepared
by a reported method from our research group.51 The ligand L3 has been synthesized through a
condensation reaction between one equivalent of oꢀphenylenediamine and two equivalent of the
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corresponding aldehyde in methanol. Characterization of the ligand was achieved using HꢀNMR, Cꢀ
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NMR, and elemental analysis. HꢀNMR signal at 5.8 ppm (singlet) confirmed the formation of the
product, as this signal corresponds to aliphatic protons, showing intensity for two protons. Also, a
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number of protons in aromatic region of HꢀNMR spectra and number of carbons in CꢀNMR spectra
confirmed the structure of corresponding ligands. The complexes of ligand L1ꢀL3 with zinc ion were
prepared in tetrahydrofuran and dimethylformamide (90:10). For complex R1, 2.0 equivalent of L1 was
dissolved in THF along with one equivalent of Zinc bromide. The dark powder was separated out that
was dissolved by addition of 10% of dimethylformamide. The reaction mixture was heated for 3 hours
and then cool down to room temperature. The dark brown colored crystals were separated out after 6
hours. The complex R2 was prepared by reacting one equivalent of L2 and one equivalent of Zinc
chloride in THF: DMF (90:10). Similarly, complex R3 was prepared using similar method, the one
which was used for the synthesis of R2. All of the complexes were characterized using elemental
analysis and infrared spectroscopy. For the final validation of structures, single crystal structure was
obtained, showing 2:1, 1:1, and 1:1 composition of ligand: metal complexes, respectively for L1, L2 and
L3.
XꢀRay Structure Determination
Crystal suitable for Xꢀray diffraction studies were isolated by slow evaporation technique in THF: DMF.
The crystals [{L1}ZnBr2] (R1), [{L2(DMF)}ZnCl2] (R2) and [{L3(DMF)}ZnBr2] (R3) aree quite
stable, therefore data of all crystals were collected at room temperature. Data collection and unit cell
parameters are shown in table 1, whereas the list of bond length and bond angles is presented in table S1
to S9 respectively in the supporting information.52 The structure was solved with the olex2.solve52
structure solution program using Charge Flipping and refined with the olex2.refine refinement package
using GaussꢀNewton minimization. Interestingly, upon changing the number of rings from benzene to
naphthalene and pyrene, packing of the crystals changes dramatically. The angle of the plane between
benzimidazole ring and another aromatic subunit greatly affect the absorbance and emission profile of
complexes. Arguably, the most important feature of this paper concerns observation of the difference in
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nonꢀcovalent interaction resulting from a change in aromatic unit attached with benzimidazole moiety,
which plays a decisive role in supramolecular packing as well as photophysical properties.
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Figure 1: Crystal structure of complex (A) R1, (B) R2, and (C) R3 with 40 % probability thermal
ellipsoids.
Table 1: Crystal data and structure refinement
CCDC Number
Identification code
1526966
(R1)
1526967
(R2)
1526968
(R3)
C40H32Br2N4Zn
793.93
C31H27Cl2N3OZn
593.88
C43H31Br2N3OZn
830.95
Empirical formula
Formula weight
298.0
298.0
298.0
Temperature/K
monoclinic
C2/c
monoclinic
P21/c
Triclinic
Pꢀ1
Crystal system
Space group
19.0112(11)
10.6875(5)
18.8991(12)
90
116.307(6)
90
3442.3(4)
4
1.5318
11.6647(4)
13.5988(4)
17.5829(6)
90
94.2986(10)
90
2781.26(16)
4
1.4182
9.0618(3)
13.0320(5)
16.0651(6)
100.320(2)
105.531(2)
98.173(2)
1761.79(11)
2
a/Å
b/Å
c/Å
α/°
β/°
γ/°
Volume/Å3
Z
ρcalcg/cm3
1.5663
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1599.5
1226.8
835.8
F(000)
Crystal size/mm3
Radiation
0.5 × 0.4 × 0.4
Mo Kα (λ = 0.71073)
4.5 to 56.86
0.5 × 0.4 × 0.4
Mo Kα (λ = 0.71073)
4.6 to 56.54
0.28 × 0.2 × 0.16
Mo Kα (λ = 0.71073)
4.68 to 56.62
2Θ range for data
collection/°
Independent reflections
4308 [Rint = 0.1005,
6886 [Rint = 0.0430,
8730 [Rint = 0.0474,
Rsigma = 0.0467]
Rsigma = 0.0884]
Rsigma = 0.0415]
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4308/0/212
1.004
R1 = 0.0585, wR2 =
0.1419
R1 = 0.1516, wR2 =
0.1869
6886/0/344
0.966
R1 = 0.0416, wR2 =
0.1207
R1 = 0.0769, wR2 =
0.1453
8730/0/452
1.042
R1 = 0.0407, wR2 =
0.0748
R1 = 0.0855, wR2 =
0.0884
Data/restraints/parameters
Goodnessꢀofꢀfit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Effect of Aromatic Subunit Attached with Benzimidazole on Packing of Complexes
The complex R1 was crystallized in triclinic crystal system with space group Pꢀ1. As shown in the
crystal structure (Figure 1), two monodentate ligands form a mononuclear complex containing zinc in
a tetrahedral geometry. Another two coordination sites of tetrahedral geometry are occupied with
bromide ion, which also stabilizes the +2 oxidation state of Zn(II). Five different CꢀHꢀπ interactions
gripped different monomeric units into a supramolecular architecture. Only CꢀHꢀπ interactions were
found involved in the construction of the 3ꢀD supramolecular network. Upon replacement of benzene
ring into naphthalene ring in complex R2, packing of complex completely changed. Instead of two
ligands, only one ligand can coordinate with zinc ion in R2 and fourth coordination site was occupied
by solvent molecule (Dimethylformamide). As shown in Figure 2, the two monomeric units of
complex R2 hold together through CꢀHꢀH-C interaction, whereas chloride ion interacts with various
hydrogens (Methylene and aromatic) to extend this 3ꢀD supramolecular architecture. This kind of
noncovalent interactions highly depends upon the aromatic π surface, electron rich or electron
deficient aromatic system.53,54 The complex R3 contained highly conjugated pyrene ring system that
has completely different kind of noncovalent interactions involving π ꢀ π stacking between pyrene
rings along with CꢀHꢀπ and BrꢀH interactions (Figure 3). These weak noncovalent interactions are
highly sensitive toward microenvironment and play a decisive role in photophysical properties;55–61
therefore there are reasonable possibilities to develop the sensors for biomolecules. Till date, many
metalꢀorganic complexes have been reported with π ꢀ π stacking interactions between pyrene ring or
with another fluorophores.62–64 However few reports are available where such kind of complexes are
used for molecular recognition.65,66 Therefore to explore the possibility to interact these metal
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complexes with some particular analytes, molecular recognitions studies were performed with various
anionic species using fluorescence spectroscopy. We expected that these specific orientations of metal
complexes would introduce interesting photophysical properties that could be altered by the
interaction with specific analytes.
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(D)
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Figure 2: (A) Structural details of R1, showing various non covalent interactions (B) Structural details
of R2, showing various non covalent interactions (C) C−H···Cl interaction leads to the formation of
polymeric structure in the R2 complex. (D) Fingerprint plots of the title complex: full and resolved into
all, C⋯H/H⋯C, X⋯H/H⋯X and Cπ⋯Cπ contacts showing the percentages of contacts contributed to
the total Hirschfeld surface area of molecules.
To clarify the nature of intermolecular interactions, the Hirschfeld surface of the molecular moiety was
analyzed which provides fingerprint of the interactions involved at intermolecular level. Hirshfeld
surface mapped with dnorm takes various parameters into consideration to calculate normalized contact
distance We have discovered that the major proportion of the Hirshfeld surface was compromised by
C···H/H···C interactions in each complex that were the highest in case of the R1 complex. The portion of the
Hirshfeld surface corresponding to Cπ··· Cπ increases as the number of aromatic rings increased from
benzene to naphthalene and further to pyrene. These results validate the ability of pyrene ring for the
formation of supramolecular selfꢀassembly through π·· π stacking.
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Photophysical Properties of Complexes R1 and R2: Initially, spectroscopic properties of ligand L1
(10 ꢀM) responding to Zn(II) were examined in HEPES buffer system (pH= 7.0, 1mM, DMF/Water
30:70). Absorbance spectra of L1 have shown two absorbance band at 225 nm and 290 nm
corresponding to π to π* and n to π* transitions, respectively. As displayed in Figure S2, absorbance
intensity of ligand L1 changes significantly upon addition of zinc ion.67,68 To find out the molar
excitation coefficient, absorbance was recorded by successive addition of Zn(II) to the solution of L1,
L2, and L3. The molar absorbance coefficient was measured and summarized in Table 2. The
modulation of molar absorbance coefficient can be seen upon substitution of a new aromatic ring to
ligand L1. The emission spectra of the ligand show emission maxima at around 433 nm (λex at 290 nm,
scan rate 200 and slit widths = 10 nm). As the concentration of zinc ion in L1 solution rises, the
emission intensity at 430 nm increases which is related to the cancellation of the PET mechanism caused
through the lone pair of the nitrogen donor (Figure S3). The solid state structure clarify that ligand L1
coordinates with zinc ion in 2:1 (L1: Zn2+) ratio. Further to find out the stoichiometry of complex
formation in a solution state, measurement were performed through Job’s plot method. A nonlinear
fitting showed a maximum value at 0.66 which allows us to conclude that alike solid state structure, in
solution state stoichiometry complex remain same 2:1 (L1: Zn2+). Further, binding constant of
complexation was calculated using the BenesiꢀHildebrand method. The inverse of the concentration of
zinc ion was plotted against the inverse of change in emission intensity. The slope and intercept of the
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curve were determined using a linear regression method, which allowed determining the binding
constant equal to 1.4 × 105 Mꢀ1. To check the working pH for binding studies, emission profiles were
recorded at different pH, suggesting that receptor stabilizes at pH 4 to 9 pH. At basic pH, zinc hydroxide
was formed and thus causing decomplexation and loss of fluorescence due to restore of PET channel.
Therefore all binding experiments were performed at pH 7.5.
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The absorbance spectra of ligand L2 has shown three absorbance maxima at 215nm, 245 nm and 295
nm (Figure S4), that correspond to the presence of two different chromophores in the system. Upon
addition of zinc chloride, the absorbance intensity of the receptor (~ 215 nm) changes significantly. The
emission spectra of ligand L2 were taken in DMF: water (30:70) (λex at 290 nm, scan rate 200 and slit
widths = 10 nm). The emission spectrum of the complex has shown two peaks at 425 nm and 550 nm
(Figure S5). As the concentration of zinc ion increases in a solution of complex R2, fluorescence
intensity at 425 nm increases significantly whereas little enhancement was observed at 550 nm. The loss
of emission due to photo induced electron transfer was inhibited through the coordination of lone pair of
benzimidazole with Zn(II). For R1, stoichiometry and binding constant of complex in solution state
were calculated using Job’s plot and BenesiꢀHildebrand curve. The stoichiometry of complex found to
be 1:1 which perfectly matches that of the solid state structure and the binding constant was found to be
6.89 x 104 Mꢀ1.
Binding Studies of Complexes R1 and R2 with Anionic Species: Due to the different electronic
arrangement and orientation, three metal complexes offer anion binding pattern; which is unique to the
structure of receptor with anionic species. The fluorescence intensity of each complex was recorded in
ꢀ
ꢀ
4ꢀ
2ꢀ
presence of anions (Fꢀ, Clꢀ, Brꢀ, Iꢀ, CH3COOꢀ, NO3 , H2PO4 , P2O7 , SO4 ) and ATP, AMP, and ADP.
The complex R1 showed enhancement in emission intensity upon interaction with ATP along with some
interference with AMP and phosphate (Figure S6ꢀS7). This enhancement in emission intensity attributes
toward replacement of bromide with phosphate ion. Zinc ion has a tendency to bind strongly with a
phosphate ion,69,70 while the orientation of a complex also provides the selectivity for a particular
analyte. Binding studies of complex R2 was performed using fluorescence spectroscopy with various
anionic species (Figure S8ꢀS9). Similarly for the complex R1, the studies also show fluorescence
enhancement with different phosphate species. As shown in Figure S10, ATP showed maximum
fluorescence enhancement among investigated analystes. The stoichiometries of R1 and R2 complexes
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with ATP were evaluated using Job’s Plot, which indicates the formation of a 1:1 complex between R2
and ATP. The major drawback of the R2 sensor was its nonꢀspecificity because it also shows emission
enhancement with other phosphate ions (Figure S11). Therefore it is not suitable for real time
determination of ATP.
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Table 2: Photophysical properties of ligands and metal complexes
ε0 (L mol−1 cm−1)
Quantum
Yield
τ (ns)
Kr (x108 sꢀ1)
Knr (x107 sꢀ1)
λ
abs (nm)
λ
em (nm)
λex (nm)
L1
L2
L3
220, 290
245, 290
4.42 × 104
4.81 ×104
433
220
290
245
290
0.31
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0.39
0.44
0.52
0.53
0.55
0.58
0.48
0.55
0.51
0.59
0.73
0.75
0.75
0.76
1.1569
1.2256
2.1256
3.45
3.67
2.67
5.64
4.67
2.10
427, 546
247, 277, 329, 5.62 ×104
346
410, 430, 472 247
277
329
346
225
390
242
290
R1
R2
R3
225, 290
242, 289
4.65 ×104
4.85 ×104
433
1.6856
1.9452
3.1436
3.38
3.11
2.45
2.88
2.16
7.74
427, 546
248, 277, 330, 6.61 ×104
345
410, 430, 472 247
277
329
346
Photophysical Properties of Ligand L3 and Complex R3: The absorption and emission spectra of all
complexes were recorded in DMF:H2O and the electronic absorption spectra of ligands and complexes
are shown in Figure S12. The ligand 3 exhibited strong absorption bands at 248 nm, 277 nm, 330 nm
and 345 nm (Figure S12). This is due to the presence of two pyrene subꢀgroups in ligand L3, four
different absorption bands appeared. One pyrene ring is directly conjugated with benzimidazole ring
whereas another ring is attached with benzimidazole through a methylene group. The conjugated pyrene
group should absorb at a higher wavelength, therefore peaks at 277 nm and 345 nm are due to πꢀπ* and
nꢀπ* transitions of conjugated pyrene ring. The peaks at 248 nm and 330 nm correspond, respectively to
πꢀπ* and nꢀπ* transitions of pyrene ring attached with a methylene group. The emission spectra of
ligand L3, taken in DMF:water (30:70) (λex at 345 nm, scan rate 200 and slit widths = 10 nm), has
shown four emission maxima at 380 nm, 410 nm, 440 nm and 470 nm. Here emission peaks at 410 nm
and 470 nm correspond to monomer and excimer state (π ꢀ π stacking) of the receptor, respectively. It is
interesting that upon addition of Zn(II) ion, the fluorescence intensity of receptor increases significantly.
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This large enhancement in emission intensity has been rationalized by the cancellation of the
Photoinduced Electron Transfer mechanism. On complexation, the lone pair of benzimidazole ring
causes the fluorescence quenching due to the PET phenomenon transferred to the zinc ion. However, the
emission intensity at 470 nm enhanced due to the formation of excimers. To confirm the binding of
Zn(II) with ligand L3, the titration was performed by successive addition of zinc ion into 10 ꢀM
solution of complex L3. As shown in figure 2a, the orientation of ligand L3 changes on complexation
with zinc ion in such way that all the pyrene rings got stacked and gave strong excimer emission band at
470 nm. The binding constant for L3ꢀZn(II) complex calculated using Benesi–Hildebrand method. A
graph has plotted between inverse of concentration of analyte and inverse of change in fluorescence
(Figure S12). The binding constant has calculated from intercept and slope of the curve, which has
found to be 7.34 x 104.
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Figure 3: Structural details of R3: (a) Interact between two monomers showing in space fill model. (b)
Intramolecular π···π stacking interaction between pyrene rings in the R3 complex (c) 2D supramolecular
layer formed in the ab plane.
Binding Studies of Complex R3 with Anionic Species
The binding behavior of complex R3 was examined using fluorescence spectroscopy towards various
anionic species such as iodide, bromide, chloride, fluoride, nitrate, dihydrogen phosphate, acetate, ATP,
AMP, ADP, PPi (Pyrophosphate), NADH (Nicotinamide adenine dinucleotide), etc. Emission spectra of
complex R3 were recorded after the addition of 16 ꢀM of each analyte (Figure 4) and it was observed
that only ATP has shown a significant change in the emission profile of R3, whereas all other tested
anionic species did not affect fluorescence profile of the complex. Interestingly, the emission at 410 nm
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(monomeric state) enhanced whereas emission due to the excimer state (470 nm) quenched as the
concentration of ATP increases in the solution. This change in emission intensity can be explained on
the basis of formation of a R3ꢀATP complex. Zinc ion coordinate with two bromide ions and one DMF
molecule, which can easily replace by the phosphate ion of ATP. Upon replacement of two bromide
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ions, the fluorescence intensity at 410 nm increases, however R3ꢀATP complex alters the π ꢀ π stacking
interaction in such a way that luminescence due to the excimer state disappears. To analyze the binding
behavior, emission spectra of R3 were recorded by successive addition of ATP solution (0ꢀ16 ꢀM). This
allowed the ratiometric detection of ATP. The ratio of fluorescence intensity was plotted against the
concentration of ATP added. The calibration plot for ATP exhibited a nonlinear regression up to 16 ꢀM
with an R2 value of 0.9993 (Figure S13) and detection limit of 15 nM. To analyze the effect of another
ion on detection of ATP, the competitive binding experiments were performed, which showed that none
of the interfering ions obstruct the detection of ATP ion. Also, the UVꢀvisible absorbance spectra of the R3
complex were recorded with the addition of ATP in the THF:Water (30:70) solvent system (Fig S15).
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Figure 4: (A) Change in emission profile of ligand L3 (10 ꢀM) upon addition of 15 ꢀM of Zinc
bromide in DMF: water (30:70) HEPES buffer (B) Packing of complex 3, showing πꢀπ stacking
interaction between pyrene rings. (C) Change in emission profile of complex 3 (10 ꢀM) upon addition of
16 ꢀM of various anions in HEPES buffer (DMF: water) (d) Change in emission profile of complex R3
with successive addition of ATP (0ꢀ16 ꢀM).
Binding Pattern of ATP with Receptor R3: To find out the stoichiometry of complexation, the Job’s
plot has constructed. The plot between mole fraction of ATP and [HG], shows maximum value for [HG]
at a mole fraction of 0.5 which indicates the 1:1 complexation between R3 and ATP. The changes
induced in excimer and the monomeric emission peak of the receptor R3 indicate the breakdown of π ꢀ π
stacking (Figure 5); also the clear solution converts into a highly viscous liquid which formed a
luminescent metallogel upon cooling. Earlier Yoon, et. al. have studied the Sandwich Stacking of
PyreneꢀAdenineꢀPyrene for determination of ATP. Here adenine ring breaks the π ꢀ π stacking in
receptor and penetrate between two pyrene rings.49 Similar results were obtained from fluorescence
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spectroscopy. Upon addition of ATP, two types of interactions were involved between receptor R3 and
ATP. One is ionic interaction and another is π ꢀ π stacking between PyreneꢀAdenineꢀPyrene sandwiches.
In this way, polymeric structure extended through hydrophobic π ꢀ π stacking interaction was formed
causing turbidity in the solution. The ratiometric change in emission (Figure S14) and increase in
turbidity indicate that the clear solution changes into the aggregates. To find out the morphology of
aggregates formed upon addition of ATP, atomic force microscopic images (AFM) of receptor R3 were
taken in the presence of ATP. AFM imaging confirmed the formation of aggregates (Figure 5BꢀC).
Initially, the receptor R3 was purely crystalline in nature. On the addition of 1.5 equivalent of ATP,
aggregates were formed as also confirmed using DLS (Dynamic Light Scattering) experiments. The size
of aggregates formed was determined using DLS which showed the particles size distribution in the
range of 35 nm to 40 ꢀm (Figure S16). To determine the stoichiometry of complex formation between
ATP and complex R3, fluorescence data were utilized to construct Job’s plots. The xꢀaxis of the Job’s
plot is labeled as the mole fraction of ATP (Figure S17). The intersection of the two linear portions of
the Job’s plot gives the mole ratio corresponding to the binding stoichiometry between ATP and R3,
which confirmed formation of the 1:1 complex between ATP and R3. The binding constant (Ka) of R3
for ATP was also determined by constructing the Benesi Hildebrand plot; a Ka value of 2.57 (± 0.04) ×
104 Mꢀ1 was obtained from the slope and intercept of the line (Figure S18).
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Gel Formation with the interaction of ATP: All the binding studies were performed in Water: DMF
(70:30), while the solution of receptor R3 turned turbid upon interaction with ATP. Further, we have
examined the behavior of receptor R3 with ATP in another solvent system. It was observed that in
tetrahydrofuran:water (30:70), the more viscous liquid was formed which turned to metallogel upon
cooling for 6 hours. To investigate the morphology of gel, scanning tunneling microscopy images were
recorded and it was observed that a polymeric network was formed which quench the excimer
fluorescence. As shown below in cartoon diagram, ATP interacts with probe R3 in such a way that
adenosine unit penetrate between two pyrene rings which abolish the πꢀ π interaction between two
pyrene rings and induced new sandwich complex having πꢀ π stacking between pyreneꢀadenosineꢀ
pyrene. Such kind of planar conjugated systems are well known for formation of πꢀ π stacking induced
gel.71–73 This supramolecular assembly highly depends upon anionic species, nature of interaction and
solvent system.74–77 It is interesting that metallogel is formed in THF:Water (30:70), whereas aggregates
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are formed in DMF:Water. Therefore the gel formation is not only selective for anionic species (ATP)
but also solvent decide the morphology formed in the solution.
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Selectivity of Receptor R3 for ATP: To investigate the interference caused by competing analytes,
such as ADP, AMP, PPi, NADH and other anions, we recorded the corresponding fluorescence spectra.
It was find out that ADP and AMP show negligible change in monomeric and excimer emission peaks.
Similarly, other analytes did not show any significant change in emission profile. As shown in Figure 6,
the ratio (Imonomer/Iexcimer) for ATP is much larger as compared to other analytes. The above selectivity for
ATP can be explained on the basis of the binding pattern. ATP bind with zinc ions through ionic bond
by replacing two bromide ions and, similarly, AMP and ADP bind with receptor R3 through ionic bond.
However hydrophobic interaction (π ꢀ π stacking) plays a crucial role in emission change. Therefore due
to the difference in chain length in ATP, AMP and ADP, these analytes behave differently while
interacting with receptor R3. For perfect binding or ratiometric sensing, it is necessary that both
interactions (ionic and hydrophobic) occur simultaneously, which is possible only in case of ATP. Thus,
neither AMP nor ADP shows response to receptor R3 (Figure S19). Further to validate the observation,
the powder XRD spectra of complex R3 has recorded and compared with the simulated XRD pattern,
which was perfectly superimposed (Figure S24). Then the complex was reacted with ATP and ADP in
THF: water, after removing the solvent with the vacuum, the prepared compound has washed with
water, to remove the free phosphate species. The broad XRD pattern of R3ꢀATP revealed the formation
of polymeric aggregates,80 whereas solid state XRD spectra of R3ꢀADP was showing sharp peaks, which
also have similarity with XRD spectra of R3 complex. To confirm the formation of R3ꢀATP complex,
Raman spectra has recorded. To record the Raman spectra, the crude obtained from the reaction of Zinc
complex R3 with ATP, has washed with distilled water to remove the unreacted Adenosine triphosphate
ions. The Raman spectra of this crude expressively different pure R3 complex and showing peaks
corresponds to Zincꢀphosphate bond and Phosphorousꢀoxygen bonds (725 cmꢀ1, 1005 cmꢀ1, 1122 cmꢀ1)
which confirm the formation of R3ꢀATP complex (Figure S25). However, ADP ion was showing
similar spectra as the complex R3, reveals that complex R3 is not reactive toward ADP. To confirm the
complexation of ATP with complex R3, the chemical characterization of R3 complex and R3ꢀATP has
performed using energyꢀdispersive Xꢀray spectroscopy (Figure S26). It revealed that the complex R3
has Zn, Br, C, N and O; however, the bromide ion disappears upon complexation with ATP. The
presence of peak at 2.1 corresponds to phosphorous, which confirmed the replacement of Brꢀ ion with
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phosphate unit of ATP. Whereas, in crude of R3ꢀADP, the peak corresponds to Br still appear and
negligible presence of phosphorous has seen.
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Figure 5: (A) Cartoon diagram of ATP induced gel formation. (B) AFM images of R3 upon interaction
with ATP in THF: water (C) AFM images of R3 upon interaction with ATP in DMF: water (D) The
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reaction showing the hydrolysis of ATP in presence of apyrase enzyme. (E) Change in turbidity of R3 +
ATP solution in DMF: water in presence of apyrase with time. (F) Change in turbidity of R3 + ATP
solution in DMF: water in presence of apyrase (0 to 250 mU) with time.
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Biological Applications of Receptor R3: To analyze the response of the receptor to intercellular
investigation of ATP, HeLa cells were investigated under a fluorescence microscope for an excitation
wavelength of 340 nm ꢀ 380 nm and an emission wavelength of 435nm ꢀ 485nm for blue fluorescence.
For the fluorescence microscopic experiments, HeLa cells were grown in Dulbecco's Modified Eagle's
medium (DMEM) using reported protocol.78 The cells were divided into two parts. Among these, one
part was incubated with 10 ꢀM of oligomycin that is well known for its ATP syntheses inhibition
activity. Then both parts were exposed to receptor R3 (20 ꢀM) for 3 hours. Before the fluorescence
measurement, the cells were washed with phosphate buffer solution (PBS). The receptor R3 was causing
a loss in fluorescence in oligomycin part, whereas HeLa cell incubated with only probe R3 show
emission in HeLa cells (Figure S20). These experiments showed that receptor R3 is capable to
determine ATP in presence of another inorganic phosphate such as AMP and ADP in the intercellular
system. Further, the ratiometric emission conversion with ATP was used to explore the probe R3 to
study the activity of apyrase enzyme that is well known for hydrolysis of nucleotide triphosphates. Our
probe R3 show ratiometric response only with ATP, therefore upon hydrolysis signature emission
profile of R3ꢀATP complex should disappear. With this in mind, a solution of R3 (20 ꢀM), ATP (20
ꢀM) and apyrase was prepared at pH 6 and emission profile was recorded after a short interval of time.
The curve (Figure 5) shows that in the absence of apyrase, fluorescence intensity (F410nm/F470nm) remains
almost unchanged, however in the presence of two different concentrations of apyrase it decreases
dramatically, which validate the conversion of ATP into AMP and ADP and finally into phosphates. To
explore the mechanism more deeply, the turbidity of the solution was measured with time, which also
shows a continuous decreas with time (up to 60 minutes) (Figure 5). Thus, the prepared probe can be
used for monitoring the activity of apyrase enzyme.
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2.5
2.0
1.5
1.0
0.5
(A)
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10000
8000
6000
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0
6.0x10-9
5.0x10-9
4.0x10-9
3.0x10-9
2.5x108
2.0x108
1.5x108
1.0x108
(B)
(C)
Kr
Life time
ATP
5.0x107
0.0
Knr
5.0x10-6
1.0x10-5
1.5x10-5
2.0x10-5
0.0
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14
Conc of ATP (in ꢀM)
τ (ns)
Figure 6: (A) Competiting binding of complex R3 with various anionic species. (B) Change in
fluorescence life time of R3 upon interaction with ATP.
Fluorescence decay time and radiative constant
Fluorescence is perfect nano scale probe as its decay could take place on the nanosecond level.
Therefore it is a promising technique to evaluate molecular interactions and changes in a nanoꢀ
environment. To analyze the effect of aggregation on photophysical properties and decay time, time
resolved fluorescence spectra were recorded at the wavelength of 472 nm, which enlightens the
mechanism of sensing of ATP. Fluorescence quantum yield and life time were determined using time
resolved fluorescence and radiative decay constant using the following formulas:
ꢁꢂ
ꢆ
ᶲꢀ = ꢁꢂꢃꢁꢄꢂ
and ꢅ = ꢁꢂꢃꢁꢄꢂ
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where ᶲf is fluorescence quantum yield, τ is a life time, Kr is radiative decay constant and Knr is nonꢀ
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radiative decay constant.
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The rate of radiative decay of the excited state decreases as the concentration of ATP increases in
solution, whereas life time of excited state increases. This transformation is due to the formation of
aggregates where properties of the excited state are different and lead to an increased life time.79
Whereas a decreasing radiative decay constant is attributed to quenching of the excimer emission. We
observed a minor change in the nonradiative decay constant upon addition of ATP to the R3 solution.
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Conclusion
In summary, we have synthesized three zinc complexes tagged with fluorescence moieties that can form
a tertiary complex with biologically important phosphates. Upon addition of ATP, the complex R1
showed distinct spectroscopic behavior due to the replacement of bromide ion. The R1 and R2 interact
only through ionic interaction with phosphates. Therefore, these complexes show emission
enhancements with ATP along with some interference of AMP and phosphate. The complex R3 has a
tendency to interact through by both π ꢀ π stacking and ionic interaction. It selectively binds with ATP
even in the presence of other phosphates. Two types of interactions act differently on emission
properties of the complex. Ionic interactions cause enhancement in fluorescence intensity at 410 nm,
whereas the π ꢀ π stacking interaction between pyrene and adenosine ring quenches the emission at 470
nm. Thus, the pyrene conjugated complex of Zinc (R3) is capable of detecting ATP ratiometrically with
a low detection limit. Furthermore, the complex R3 was explored for monitoring the activity of Apyrase
enzyme during (?) hydrolysis of ATP. The outcome of this work is important for the development of a
highly selective ATP sensor using ionic and other nonꢀcovalent interactions.
Experimental
General Information: Orthoꢀphenylenediamine, aldehydes, and biomolecules (Analytical grade) were
procured from Sigma Aldrich and used without further purification. Oligomycin (from Streptomyces
diastatochromogenes) and Apyrase enzyme (From Potato) was purchased from Sigma Aldrich.
Methanol and tetrahydrofuran were purchased from Merck. The photoluminescence experiments were
performed on Perkin Elmer LSꢀ55 instrument at a constant scan speed of 200. For all experiments,
excitation and emission slit width was kept 10 nm. 1HꢀNMR and the 13CꢀNMR spectrum were recorded
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on Jeol instrument which was operated at 400 MHz for HꢀNMR and 100 MHz for CꢀNMR. The
Single crystal data for three complexes were measured on Bruker X8 APEXIII KAPPA CCD
diffractometer at room temperature using graphite monochromatized MoꢀKα radiation (λ = 0.71073
Å). Elemental analyses were carried out using Fisons CHNS analyzers. The morphology of gel was
found out on a scanning electron microscope (SEM JEOL JSMꢀ6610LV) using a voltage of 15 KV and
Atomic force microscopy (AFM). The particle size of nanoparticles was determined by dynamic light
scattering (DLS) using external probe feature of Metrohm Microtrac Ultra Nanotrac particle size
analyzers. Fluorescence microscopy of HeLa cells was recorded on Leica fluorescence microscope.
Fluorescence life time was calculated from time resolved fluorescence spectroscopy using PicoQuant
fluorescence spectrophotometer.
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Detail of UVꢀvisible absorbance and Fluorescence Measurements: The stock solution of each ligand
(10 ꢀM) was prepared in DMF: water (30:70). Similarly, a stock solution of zinc bromide and zinc
chloride (each 1mM) was prepared in hot distilled water and diluted further as a requirement of the
experiment. The solution of various anions as tetra butyl ammonium salt (Fꢀ, Clꢀ, Brꢀ, Iꢀ, CH3COOꢀ, NO3
ꢀ
ꢀ
4ꢀ
2ꢀ
, H2PO4 , P2O7 , SO4 ) (1mM) and ATP (Adenosine triphosphate), AMP (Adenosine monophosphate),
and ADP (Adenosine diphosphate) (1mM) were prepared in distilled water. The pH of the solution was
maintained at 7.0 using 1mM HEPES buffer solution.
Synthesis of L1 (1ꢀBenzylꢀ2ꢀphenylꢀ1Hꢀbenzo[d]imidazole):51 In a round bottom flask, the
benzaldehyde (2.44 gm, 10 mmol) and oꢀphenylenediamine (1.08 gm, 10 mmol) was dissolved in 50 mL
of methanol along with (0.185 gm, 0.5 mmol) of zinc perchlorate. The reaction mixture was refluxed for
8 hours; and the progress of the reaction was monitor using TLC. After completion of the reaction, the
mixture was allowed to cool to room temperature. The orange crystalline solid was separated out which
constituted pure compound L1 with following properties. Yield 94%, Elemental analysis: Calculated: C,
84.48; H, 5.67; N, 9.85; Found: C, 84.42; H, 5.61; N, 9.78.
Synthesis of L2 (2ꢀ(naphthaleneꢀ1ꢀyl)ꢀ1ꢀ(naphthaleneꢀ1ꢀylmethyl)ꢀ1Hꢀbenzo[d]imidazole): The
compound L2 was synthesized using the same method as L1, naphthaldehyde (3.44 gm, 10 mmoles)
was used instead of benzaldehyde and reaction mixture was refluxed for 10 hours. The orange solid was
separated out after 10 hours of refluxing which constituted pure compound L2 with following
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properties. Yield 91%, Elemental analysis: Calculated: C, 87.47; H, 5.24; N, 7.29; Found: C, 87.41; H,
5.17; N, 9.27.
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Synthesis of L3 (1ꢀ((4,5a1ꢀdihydropyrenꢀ1ꢀyl)methyl)ꢀ2ꢀ(pyrenꢀ1ꢀyl)ꢀ1Hꢀbenzo[d]imidazole): The
compound L3 was synthesized using same method as L1, 1ꢀPyrenecarboxaldehyde (4.96 gm, 10 mmole)
was used instead of benzaldehyde and reaction mixture was refluxed for 10 hours. The orange solid was
separated out after 10 hours of refluxing which constituted pure compound L3 with following
properties. Yield 91%, Elemental analysis: Calculated: C, 89.86; H, 4.90; N, 5.24; Found: C, 89.82; H,
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4.85; N, 5.18. H NMR (400 MHz, DMSOꢀD6) δ 8.01 ꢀ 7.94 (t, 4H, J = 8Hz), 7.86 ꢀ 7.79 (t, 4H, J =
8Hz), 7.69 (t, 1H, J = 8Hz), 7.61 (t, 2H, J = 8Hz), 7.57 (t, 2H, J = 8Hz), 7.51 (t, 2H, J = 8Hz), 7.45 (t,
3H, J = 8Hz), 7.38 (t, 2H, J = 8Hz), 7.28 (t, 1H, J = 8Hz), 7.22 (t, 3H, J = 8Hz), 6.60 (d, 1H, J = 8Hz),
5.83 (s, 2H).13C NMR (400 MHz, DMSOꢀD6) δ 175.70, 152.80, 151.86, 144.40, 143.35, 135.87, 134.94,
134.13, 133.71, 133.59, 132.46, 132.04, 131.00, 130.69, 130.29, 129.11, 128.92, 128.85, 128.73, 128.39,
128.27, 127.84, 127.61, 126.99, 126.94, 126.87, 126.59, 125.91, 125.83, 125.53, 123.63, 123.38, 123.19,
122.79, 122.13, 120.03, 119.60, 111.88, 111.79, 46.12.
Synthesis of complex R1: Zinc(II)bromide (100 mmol) was dissolved in methanol – tetrahydrofuran
(20 mL) along with ligand L1 (200 mmol). The reaction mixture was heated and stirred for 20 minutes.
The color of the solution becomes more intense. The solution was kept undisturbed for 24 hours for slow
evaporation. Dark brown needle shaped crystals were formed that were suitable for single crystal data
collection. Elemental analysis: Calculated: C, 60.52; H, 4.06; N, 7.06; Found: C, 60.47; H, 4.00; N, 7.01.
Synthesis of complex R2: Zinc(II)chloride (100 mmol) was dissolved in methanol – tetrahydrofuran
(20 mL) along with ligand L1 (200 mmol) The reaction mixture was heated and stirred for 20 minutes.
The color of the solution becomes more intense. The solution was kept undisturbed for 24 hours for slow
evaporation. Dark brown needle shaped crystals were formed that were suitable for single crystal data
collection. Elemental analysis: Calculated: C, 62.70; H, 4.58; N, 7.08; Found: C, 62.67; H, 4.55; N, 7.02.
Synthesis of complex R3: Zinc(II)bromide (100 mmol) was dissolved in methanol – tetrahydrofuran
(20 mL) along with ligand L1 (200 mmol). The reaction mixture was heated and stirred for 20 minutes.
The color of the solution becomes more intense. The solution was kept undisturbed for 24 hours for slow
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evaporation. Dark brown needle shaped crystals were formed that were suitable for single crystal data
collection. Elemental analysis: Calculated: C, 62.01; H, 3.99; N, 5.04; Found: C, 61.95; H, 3.95; N, 5.00.
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI.
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Absorption and emission properties, HꢀNMR, CꢀNMR, Bond angles and bond length of complex,
DLS, Fluorescence microscopy images.
AUTHOR INFORMATION
Corresponding Author
Dr. Narinder Singh, aDepartment of Chemistry, Indian Institute Technology Ropar, Punjab, 140001,
India E-mail: nsingh@iitrpr.ac.in; Tel: +91-1881242176
Acknowledgement
This work was supported by research grant from IndoꢀCanada project sponsored by DBTꢀNew Delhi
and ICꢀIMPACTS Canada. A. S. and P. R. are thankful to CSIR−New Delhi (9/1005(0010)/2014ꢀEMRꢀ
1) and UGCꢀNew Delhi respectively for their fellowship.
Reference
(1)
Gale, P. A.; Caltagirone, C. Anion Sensing by Small Molecules and Molecular Ensembles. Chem.
Soc. Rev. 2015, 44 (13), 4212–4227 DOI: 10.1039/C4CS00179F.
(2)
Gong, Z.ꢀL.; Zhong, Y.ꢀW. H 2 PO 4 – ꢀ and SolventꢀInduced Polymorphism of an Amideꢀ
Functionalized [Pt(N^C^N)Cl] Complex. Inorg. Chem. 2016, 55 (20), 10143–10151 DOI:
10.1021/acs.inorgchem.6b01059.
(3)
Patra, C.; Bhanja, A. K.; Mahapatra, A.; Mishra, S.; Saha, K. Das; Sinha, C. Coumarinyl
Thioether Schiff Base as a Turnꢀon Fluorescent Zn(II) Sensor and the Complex as Chemosensor
for the Selective Recognition of ATP, along with Its Application in Whole Cell Imaging. RSC
Adv. 2016, 6 (80), 76505–76513 DOI: 10.1039/C6RA12369D.
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Page 26 of 37
1
2
3
4
5
6
7
8
9
(4)
(5)
(6)
(7)
(8)
Raj, P.; Singh, A.; Kaur, K.; Aree, T.; Singh, A.; Singh, N. Fluorescent Chemosensors for
Selective and Sensitive Detection of Phosmet/Chlorpyrifos with Octahedral Ni 2+ Complexes.
Inorg. Chem. 2016, 55, 4874–4883 DOI: 10.1021/acs.inorgchem.6b00332.
Singh, A.; Singh, A.; Singh, N.; Jang, D. O. A 2ꢀMercaptobenzimidazoleꢀBased Emissive Cu(I)
Complex for Selective Determination of Iodide with Large Stokes Shift. Sensors Actuators B
Chem. 2017, 243, 372–379 DOI: 10.1016/j.snb.2016.11.157.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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34
35
36
37
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Lu, Q.ꢀS.; Dong, L.; Zhang, J.; Li, J.; Jiang, L.; Huang, Y.; Qin, S.; Hu, C.ꢀW.; Yu, X.ꢀQ.
ImidazoliumꢀFunctionalized BINOL as a Multifunctional Receptor for Chromogenic and Chiral
Anion Recognition. Org. Lett. 2009, 11 (3), 669–672 DOI: 10.1021/ol8027303.
Fu, Y.; Mu, L.; Zeng, X.; Zhao, J.ꢀL.; Redshaw, C.; Ni, X.ꢀL.; Yamato, T. An NBDꢀArmed
thiacalix[4]areneꢀDerived Colorimetric and Fluorometric Chemosensor for Ag+: A MetalꢀLigand
Receptor of Anions. Dalton Trans. 2013, 42 (10), 3552–3560 DOI: 10.1039/c2dt32115g.
Kundu, T.; Chowdhury, A. D.; De, D.; Mobin, S. M.; Puranik, V. G.; Datta, A.; Lahiri, G. K.
Selective Recognition of Fluoride and Acetate by a Newly Designed Ruthenium Framework:
Experimental and Theoretical Investigations. Dalt. Trans. 2012, 41 (15), 4484 DOI:
10.1039/c2dt12126c.
(9)
Singh, A.; Raj, P.; Singh, N. BenzimidazoliumꢀBased SelfꢀAssembled Fluorescent Aggregates for
Sensing and Catalytic Degradation of Diethylchlorophosphate. ACS Appl. Mater. Interfaces 2016,
8, 28641–28651 DOI: 10.1021/acsami.6b09983.
(10) Yang, Y.; Chen, S.; Ni, X. Fluorescent Chemosensor for Nitrate in Acidic Aqueous Solution and.
Anal. Chem. 2015, 87 (14), 7461–7466 DOI: 10.1021/acs.analchem.5b01774.
(11) Zhao, J.; Yang, D.; Zhao, Y.; Cao, L.; Zhang, Z.; Yang, X.ꢀJ.; Wu, B. PhosphateꢀInduced
Fluorescence of a TetraphenyletheneꢀSubstituted Tripodal Tris(urea) Receptor. Dalt. Trans. 2016,
45 (17), 7360–7365 DOI: 10.1039/C6DT00672H.
(12) Jia, C.; Zuo, W.; Zhang, D.; Yang, X.; Wu, B. Anion Recognition by Oligoꢀ(Thio)ureaꢀBased
Receptors. Chem. Commun. 2016, 52, 9614–9627 DOI: 10.1039/C6CC03761E.
ACS Paragon Plus Environment

Page 27 of 37
Crystal Growth & Design
1
2
3
(13) Singh, J.; Singh, A.; Singh, N. Urea Based Organic Nanoparticles for Selective Determination of
4
5
6
NADH. RSC Adv. 2014, 4 (106), 61841–61846 DOI: 10.1039/C4RA10209F.
7
8
9
(14) Singh, A.; Singh, A.; Singh, N.; Jang, D. O. A BenzimidazoliumꢀBased Organic Trication: A
Selective Fluorescent Sensor for Detecting Cysteine in Water. RSC Adv. 2015, 5 (88), 72084–
72089 DOI: 10.1039/C5RA14501E.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(15) Wang, S.; Chang, Y.ꢀT. Combinatorial Synthesis of Benzimidazolium Dyes and Its Diversity
Directed Application toward GTPꢀSelective Fluorescent Chemosensors. J. Am. Chem. Soc. 2006,
128 (32), 10380–10381 DOI: 10.1021/ja063733d.
(16) Hargrove, A. E.; Nieto, S.; Zhang, T.; Sessler, J. L.; Anslyn, E. V. Artificial Receptors for the
Recognition of Phosphorylated Molecules. Chem. Rev. 2011, 111, 6603–6782 DOI:
10.1021/cr100242s.
(17) Rommel, S. A.; Sorsche, D.; Rau, S. A Supramolecular HꢀBond Driven Light Switch Sensor for
Small Anions. Dalt. Trans. 2016, 45 (1), 74–77 DOI: 10.1039/C5DT04152J.
(18) Singh, A.; Singh, A.; Singh, N.; Ok, D. Selective Detection of Hg ( II ) with BenzothiazoleꢀBased
Fl Uorescent Organic Cation and the Resultant Complex as a Ratiometric Sensor for Bromide in
Water. Tetrahedron 2016, 72 (24), 3535–3541 DOI: 10.1016/j.tet.2016.04.082.
(19) Kumar, S.; Singh, P.; Mahajan, A.; Kumar, S. Aggregation Induced Emission Enhancement in
Ionic SelfꢀAssembled Aggregates of Benzimidazolium Based Cyclophane and Sodium
Dodecylbenzenesulfonate. Org. Lett. 2013, 15 (13), 3400–3403 DOI: 10.1021/ol401452t.
(20) Yao, J.; Fu, Y.; Xu, W.; Fan, T.; Gao, Y.; He, Q.; Zhu, D.; Cao, H.; Cheng, J. Concise and
Efficient Fluorescent Probe via an Intromolecular Charge Transfer for the Chemical Warfare
Agent Mimic Diethylchlorophosphate Vapor Detection. Anal. Chem. 2016, 88 (4), 2497–2501
DOI: 10.1021/acs.analchem.5b04777.
(21) Liu, X.; Xu, J.; Lv, Y.; Wu, W.; Liu, W.; Tang, Y. An ATPꢀSelective, Lanthanide Complex
Luminescent Probe. Dalton Trans. 2013, 42 (27), 9840–9846 DOI: 10.1039/c3dt50986a.
(22) Kumar, A.; Pandey, R.; Kumar, A.; Gupta, R. K.; Dubey, M.; Mohammed, A.; Mobin, S. M.;
ACS Paragon Plus Environment

Crystal Growth & Design
Page 28 of 37
1
2
3
4
Pandey, D. S. SelfꢀAssembled copper(II) Metallacycles Derived from Asymmetric Schiff Base
5
6
7
Ligands: Efficient Hosts for ADP/ATP in Phosphate Buffer. Dalt. Trans. 2015, 44, 17152–17165
DOI: 10.1039/c5dt01433f.
8
9
(23) Minami, T.; Emami, F.; Nishiyabu, R.; Kubo, Y.; Anzenbacher, P. Quantitative Analysis of
Modeled ATP Hydrolysis in Water by a Colorimetric Sensor Array. Chem. Commun. 2016, 52
(10), 2–5 DOI: 10.1039/C6CC02923J.
10
11
12
13
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16
17
18
19
20
21
22
23
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25
26
27
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29
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44
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46
47
48
49
50
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52
53
54
55
56
57
58
59
60
(24) Vancraenenbroeck, R.; Webb, M. R. A Fluorescent, Reagentless Biosensor for ATP, Based on
MalonylꢀCoenzyme A Synthetase. ACS Chem. Biol. 2015, 10 (11), 2650–2657 DOI:
10.1021/acschembio.5b00346.
(25) Liu, Z.; Chen, S.; Liu, B.; Wu, J.; Zhou, Y.; He, L. Intracellular Detection of ATP Using an
Aptamer Beacon Covalently Linked to Graphene Oxide Resisting Nonꢀ Specific Probe
Displacement. Anal. Chem. 2014, 86 (519), 1–10.
(26) Asha, K. S.; Bhattacharyya, K.; Mandal, S. Discriminative Detection of Nitro Aromatic
Explosives by a Luminescent Metal–organic Framework. J. Mater. Chem. C 2014, 2 (47), 10073–
10081 DOI: 10.1039/C4TC01982B.
(27) Au, V. K. M.; Zhu, N.; Yam, V. W. W. Luminescent Metallogels of BisꢀCyclometalated
alkynylgold(III) Complexes. Inorg. Chem. 2013, 52 (2), 558–567 DOI: 10.1021/ic3007519.
(28) Ghosh, S.; Mishra, M. K.; Ganguly, S.; Desiraju, G. R. Dual Stress and Thermally Driven
Mechanical Properties of the Same Organic Crystal: 2,6ꢀDichlorobenzylideneꢀ4ꢀFluoroꢀ3ꢀ
Nitroaniline. J. Am. Chem. Soc. 2015, 137 (31), 9912–9921 DOI: 10.1021/jacs.5b05324.
(29) Banik, M.; Gopi, S. P.; Ganguly, S.; Desiraju, G. R. Cocrystal and Salt Forms of Furosemide:
Solubility and Diffusion Variations. Cryst. Growth Des. 2016, 16, 5418–5428 DOI:
10.1021/acs.cgd.6b00902.
(30) Rana, L. K.; Sharma, S.; Hundal, G. First Report on Crystal Engineering of Hg(II) Halides with
Fully Substituted 3,4ꢀPyridinedicarboxamides: Generation of TwoꢀDimensional Coordination
Polymers and Linear ZigꢀZag Chains of Mercury Metal Ions. Cryst. Growth Des. 2016, 16 (1),
ACS Paragon Plus Environment

Page 29 of 37
Crystal Growth & Design
1
2
3
4
5
6
7
92–107 DOI: 10.1021/acs.cgd.5b00989.
(31) Jassal, A. K.; Sharma, S.; Hundal, G.; Hundal, M. S. Structural Diversity, Thermal Studies, and
Luminescent Properties of Metal Complexes of Dinitrobenzoates: A Single Crystal to Single
Crystal Transformation from Dimeric to Polymeric Complex of copper(II). Cryst. Growth Des.
2015, 15 (1), 79–93 DOI: 10.1021/cg500883w.
8
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
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41
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44
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49
50
51
52
53
54
55
56
57
58
59
60
(32) Chung, C. Y. S.; Li, S. P. Y.; Lo, K. K. W.; Yam, V. W. W. Synthesis and Electrochemical,
Photophysical, and SelfꢀAssembly Studies on WaterꢀSoluble pHꢀResponsive Alkynylplatinum(II)
Terpyridine Complexes. Inorg. Chem. 2016, 55 (9), 4650–4663 DOI:
10.1021/acs.inorgchem.6b00513.
(33) Ghosh, S.; Mukherjee, P. S. SelfꢀAssembly of a Nanoscopic Prism via an Organometallic Pt 3
Acceptor and Its Fluorescenct Deetection of Nitroaromatics. 2008, 3 (5), 9–11.
(34) Ghosh, S.; Mukherjee, P. S. SelfꢀAssembly of Molecular Nanoball: Design, Synthesis, and
Characterization. J. Org. Chem. 2006, 71 (22), 8412–8416 DOI: 10.1021/jo061311g.
(35) Lee, J. H. Y. H. Y.; Kang, S.; Lee, J. H. Y. H. Y.; Jung, J. H. A TetrazoleꢀBased Metallogel
Induced with Ag+ Ion and Its Silver Nanoparticle in Catalysis. Soft Matter 2012, 8, 6557–6563
DOI: 10.1039/c2sm25316j.
(36) Aiyappa, H. B.; Saha, S.; Wadge, P.; Banerjee, R.; Kurungot, S. Fe( III ) Phytate Metallogel as a
Prototype Anhydrous, Intermediate Temperature Proton Conductor. Chem. Sci. 2015, 6 (1), 603–
607 DOI: 10.1039/C4SC02294G.
(37) Tam, A. Y.ꢀY.; Yam, V. W.ꢀW. Recent Advances in Metallogels. Chem. Soc. Rev. 2013, 42 (4),
1540–1567 DOI: 10.1039/c2cs35354g.
(38) Feldner, T.; Hring, M.; Saha, S.; Esquena, J.; Banerjee, R.; Daz, D. D. Supramolecular Metallogel
That Imparts SelfꢀHealing Properties to Other Gel Networks. Chem. Mater. 2016, 28 (9), 3210–
3217 DOI: 10.1021/acs.chemmater.6b01144.
(39) Sarmah, K.; Pandit, G.; Das, A. B.; Sarma, B.; Pratihar, S. Steric Environment Triggered Selfꢀ
Healing CuII/HgII Bimetallic Gel with Old CuII–Schiff Base Complex as a New Metalloligand.
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