Pyrophosphate Prompted Aggregation-Induced Emission: Chemosensor Studies, Cell Imaging, Cytotoxicity, and Hydrolysis of the Phosphoester Bond with Alkaline Phosphatase

Article abstract of DOI:10.1002/ejic.201801173

Two benzimidazole-based zinc complexes [(Zn)₂(L2)₂Cl₂(DMSO)₂] (R1) and [Zn(L2)₂(NO₃)₂(H₂O)₂] (R2) were synthesized and characterized with various spectrosco

Full text of DOI:10.1002/ejic.201801173

DOI: 10.1002/ejic.201801173
Full Paper
Fluorescence Bioimaging
Pyrophosphate Prompted Aggregation-Induced Emission:
Chemosensor Studies, Cell Imaging, Cytotoxicity, and Hydrolysis
of the Phosphoester Bond with Alkaline Phosphatase
Pushap Raj,[a] Amanpreet Singh,[a] Ajnesh Singh,[b] Ashutosh Singh,[d] Neha Garg,[d]
Navneet Kaur,*[c] and Narinder Singh*[a]
Abstract: Two
benzimidazole-based
zinc
complexes sion intensity due to aggregation-induced emission. However,
[(Zn)2(L2)2Cl2(DMSO)2] (R1) and [Zn(L2)2(NO3)2(H2O)2] (R2) were
synthesized and characterized with various spectroscopic data.
The single X-ray structure determination reveals that complex
R1 is dinuclear and tetrahedral in geometry, while complex R2
is mononuclear and octahedral in geometry. Further, both zinc
complexes were investigated for pyrophosphate sensing in an
aqueous medium. Complex R1 is found to be selective towards
pyrophosphate; it leads to 5.5-fold enhancement in the emis-
complex R2 has shown binding with all, ATP, AMP, ADP, and
pyrophosphate, which is attributed to the chelate effect. Conse-
quently, complex R1 was utilized for the intracellular detection
of pyrophosphate in HeLa cells. Furthermore, the PPi based zinc
complex R1 is also used as a bio-analytical tool to construct a
real-time fluorescence assay for the enzymatic activity of
alkaline phosphatases (ALP).
Introduction
ceptors make use of binding sites such as urea/thiourea, amide,
pyrazole and imidazolium derivative; and hence only operative
in a purely organic or semi-organic solvent.[5,21,22] Pyrophos-
phate recognition in physiological condition is still a challeng-
ing task due to competition for the binding site between
solvent and the target analyte.[23,24] Moreover, to explore the
application of sensor for bio-analytical measurements; the sen-
sor system should be operative in an aqueous medium. Conse-
quently, most of the sensors which are fabricated with organic
moieties exhibit a restricted solubility in aqueous medium and
impede the application of sensor in bio-analytical tech-
niques.[25,26]
The detection and quantification of inorganic phosphates are
crucial because of their role in many environmental and biologi-
cal processes.[1–4] Pyrophosphate is a dimeric form of inorganic
phosphate which has a decisive role in the cellular metabolic
process, real-time DNA sequencing, energy storage, signal
transduction, ATP hydrolysis, and calcium pyrophosphate depo-
sition etc.[4–8] Moreover, pyrophosphate has become an indis-
pensable biomarker for diagnosis of numerous diseases like
chondrocalcinosis, arthritis, and cancer.[9–12] Therefore, the de-
tection of PPi is an important criterion to establish the disease
state under physiological conditions.
To overcome these constraints, aggregation-induced emis-
sion (AIE) is an important phenomenon for sensing of pyro-
phosphate in an aqueous medium, even using the organic moi-
eties for sensors.[27] The AIE phenomenon was first invented
with the pioneering work of Tang et al. in 2001, for 1,1-di-
methyl-2,3,4,5-tetraphenylsilole and tetraphenylethene (TPE)
molecules.[28] The tetraphenylsilole molecules are non-emissive
or weakly fluorescent in molecular state. However, in the aggre-
gate state, these compounds offer strong emission; most
probably through the restriction of non-radiative decay from
the excited state.[29] The success of AIE encouraged the re-
searchers to synthesized various probe based on AIE phenome-
non and used for the application like organic light-emitting
diodes, chemo/bio-sensors and biological cell imaging.[30–33]
Owing to the simplicity and efficiency, AIE becomes one of the
promising strategies for sensing of environmentally and biologi-
cally important analytes in the aqueous medium.[34] To the best
of our understanding, there are only a few reports available for
sensing of pyrophosphate based on AIE approach. Rissanen et
al. have employed the self-assembled terpyridine-Zn2+complex
The most predominant strategies reported for the detection
of pyrophosphate are based on hydrogen bonding interaction,
charged transfer, pyrophosphate metal complex formation and
metal displacement assay.[13–17] The binding sites available in
these approaches are directly or indirectly linked to fluorophore
unit through covalent bonding and afford a direct signal re-
sponse on interaction with PPi.[18–20] The available organic re-
[a] Department of Chemistry, Indian Institute Technology Ropar,
Punjab, 140001, India
E-mail: nsingh@iitrpr.ac.in
[b] Department of Applied Sciences and Humanities, Jawaharlal Nehru Govt.
Engineering College,
Sundernagar, Mandi, Himachal Pradesh 175018, India
[c] Department of Chemistry, Panjab University Chandigarh, Chandigarh,
160014, India
E-mail: navneetkaur@pu.ac.in
[d] School of Basic Sciences, Indian Institute of Technology Mandi,
Kamand, Mandi, Himachal Pradesh 175005, India
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejic.201801173.
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for nanomolar detection of pyrophosphate in water.[35] Ni et al. 1H NMR spectrum of L2 indicates the formation of benzimid-
have also used the terpyridine-Zn2+ complex for selective and azole moiety. Further, the mass spectra of ligands L1 and L2
sensitive detection of pyrophosphate under a physiological have exhibited m/z peak at 198.04 and 196.02 respectively,
condition which is also based on AIE phenomenon.[36]
which confirmed the molecular ion peak [M + H] of the struc-
tures of L1-2.
In the present manuscript, we have explored AIE approach
for pyrophosphate sensing under physiological condition using
benzimidazole-based zinc complexes R1 and R2, respectively.
Both the zinc complexes were prepared by reaction of zinc
chloride and zinc nitrate to the respective ligand L1 and L2,
which yielded dinuclear (R1) and mononuclear zinc complex
(R2). The prepared dinuclear and mononuclear benzimidazole-
based zinc complexes are utilized for selective sensing of pyro-
phosphate in an aqueous medium. The pyrophosphate has
strong tendency to coordinate with zinc metal ions by replacing
labile anions/solvent and shown modulation in the emission
spectrum of zinc complexes (R1-2).[37–39] The zinc complex R1
was further utilized for pyrophosphate sensing in the HeLa cells
imaging and also evaluated for their cytotoxic activity. More-
over, we have developed pyrophosphate based zinc complex
for bio-analytical tool to extend the real-time activities for alka-
line phosphatase enzyme.[40,41]
Alkaline phosphatase is a homodimeric metalloenzyme con-
taining two zinc atom in its structural unit, which are decisive
for its catalytical role.[42] The enzyme is active in the basic envi-
ronment and regulates the hydrolysis of monophosphate ester
into inorganic phosphate and alcohol.[43–45]It is accounted that
the deprotonated hydroxyl group of serine moiety of ALP en-
zyme undergo nucleophilic attack to a phosphate group and
hydrolyze the phosphate ester bond.[46] The ALP enzyme hydro-
lytically promotes the breakdown of phosphate ester bond of
various molecules like alkaloids, nucleotides, and protein
through the nucleophilic attack.[47] Some of the recent reports
have suggested that the interaction between Zn2+and non-
bridging phosphorus-oxygen group lead to enhancement of
ALP catalyzed phosphate ester bond cleavage. Although, the
role of zinc ion for the binding of phosphorus-oxygen bond
and cleavage of phosphate ester bond through ALP was not
well recognized.[42]However, there are few reports available in
the literature; which explored the enzymatic role of ALP for
hydrolyzing the phosphate ester bond by monitoring the mod-
ulation in the emission intensity of the molecular sensor.[48]
Therefore, the present sensor is used for monitoring of ALP ac-
tivities through modulation in the fluorescence intensity.
Scheme 1. Synthesis of ligands L1-2.
Finally, our attempts remained successful to grow the crys-
tals of ligand L1 (in ethanol); which confirmed the structure of
L1 through single X-ray crystallography (Figure 1A). The com-
plex R1 was synthesized from L1 in methanol/DMSO solvent
system, while complex R2 was synthesized from L2 in MeOH:
THF solvent system as shown in Scheme 2. Both the complexes
were characterized with spectroscopic method of analysis (Fig-
ure S9–S12) and the structures were authenticated with single
1
X-ray crystallography. The comparison of H NMR spectra of L1
and R1 (both recorded in [D6]DMSO) revealed that the imine-
linked structure of L1 has been converted into the benzimid-
azole moiety. We have already reported the mechanism for the
formation of benzimidazole moiety from similar type of sub-
strates. Further, the sp2 nitrogen of benzimidazole moiety
seems to coordinate with Zn(II) leading to locking of benzimid-
azole tautomerism. Consequently, this directed the splitting in
aromatic signals of benzimidazole as evident from 1H NMR
spectrum of R1 (Figure S9).[49,50] Similarly, the 1H NMR spectrum
of complex R2 in [D6]DMSO displayed the shift in aromatic sig-
nals of pyridine moiety by Δδ = 0.04-0.06 ppm and also con-
firmed the formation of zinc complex (Figure S10). The crystalli-
nity of the bulk samples of R1-2 was established from the com-
parison of powder XRD pattern and the simulated data ob-
tained from mercury software as shown in Figure S16–S17. The
result of powder XRD pattern was similar to the simulated data,
which confirm the phase purity of complexes in the solid state.
The photophysical properties of R1-2 complexes were recorded
through UV/Visible absorption and emission spectroscopy in
the aqueous solvent system. The 10 μ
M
solution of R1 shows
Result and Discussion
absorption maxima at 310 nm with molar absorption value of
ε0 = 2.8 × 104 L·mol–1 cm–1and assigned to π–π* transition of
ligand L1. Similarly, the complex R2 demonstrates absorption
maxima at 315 nm (ε0 = 1.2 × 104 L·mol–1 cm–1) and again due
Synthesis and Characterization of Ligands and Metal
Complexes
Ligands L1-2 were synthesized through the condensation reac- to π–π* transition of the ligand as shown in Figure S18A–B.[51]
tion between o-phenylenediamine and 4-pyridinecarboxalde- Both the complexes R1 and R2 were excited at 310 and 315
hyde as shown in Scheme 1. It is important to mention here nm respectively and exhibit emission at 487 and 485 nm with
that the reactants under dilute conditions afford the formation low quantum yield (Φ = 0.10 and 0.08) respectively.[51] The
of L1. The structural formulae of both the ligands L1-2 were weak fluorescence emission of complex R2 is due to free rota-
1
elucidated with spectroscopic data such as H NMR, 13C NMR, tion across single bond connecting the pyridyl and benzimid-
1
FTIR and mass spectrometry (Figure S1–S8). The H NMR spec- azole segments of the molecules and thus, leads to nonradia-
trum of L1 has shown the formation of imine linkage; while the tive decay.
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Figure 1. ORTEP diagram along with atom numbering scheme with 40 % probability thermal ellipsoids: (A) L1, (B) R1, (C) R2.
hydrogen bonds. Two such chains are packed in double helical
type as shown in Figure S13.
The complex R1 crystallizes in monoclinic crystal system with
P21/c space group. The asymmetric unit consists of one moiety
of complex [(C14H15N3)2Cl2Zn2] and two molecules of dimethyl-
sulfoxide (DMSO) as a solvent of crystallization. The Zn(II) has
tetrahedral coordination which is satisfied by two chloride ions
and two nitrogen atoms originating from benzimidazole and
pyridyl moieties. The ORTEP diagram along with atom number-
ing scheme is shown in Figure 1(B). The selected bond lengths
and bond angles are given in Table 1. In the crystal lattice, the
molecules are interacting with each other by different type of
hydrogen bonding interactions (N-H···O, C-H···O, and C-H···Cl).
The solvent molecules are acting as linkers between different
moieties of complex R1. This arrangement of molecules in the
crystal lattice along c-axis is given in Figure S14.
Single crystal X-ray structure determination of complex R2
was undertaken to establish the structure unambiguously. X-ray
structure determination revealed that complex R2 crystallizes
in triclinic crystal system with space group P1¯ and consists of
one molecule of complex R2.
Scheme 2. Synthesis of zinc complexes R1-2.
Structure Description
Ligand L1 crystallizes in orthorhombic crystal system with Fdd2
The central Zn(II) metal ion is attained slightly distorted octa-
space group, and the asymmetric unit contains one moiety of hedral geometry. Octahedral coordination around central metal
Ligand L1. Figure 1 (A) shows the ORTEP diagram along with ion completed by two nitrogen atoms originating from ligand
atom numbering scheme of the ligand L1. The 2-aminobenzene L2 and four oxygen atoms, two each originating from nitrate
and pyridyl moieties are not coplanar; both planes have a dihe- ions and water molecules. The ORTEP diagram along with atom
dral angle of 46.63°. The selected bond lengths and bond an- numbering scheme of the complex is shown in Figure 1C. The
gles are given in Table S2. The moieties of ligand L1 arranged selected bond lengths and bond angles are given in Table 2. In
in the form of one-dimensional chain running along c-axis. Dif- the crystal lattice, different moieties are interacting with each
ferent moieties in these chains are joined together by N-H·N other through O/N-H···O, H/O-H···N and C-H···O hydrogen
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Table 1. Selected bond lengths and angles (Å (°)) for complex R1.
Bond lengths (Å)
Zn(1)–N(1)
2.037(2)
2.061(2)
2.2233(9)
Zn(1)–Cl(1)
N(1)–C(1)
N(1)–C(7)
2.2237(9)
1.326(3)
1.399(4)
N(2)–C(1)
N(2)–C(2)
N(3)–C(11)
1.340(4)
1.376(4)
1.331(4)
Zn(1)–N(3)
Zn(1)–Cl(2)
Bond angles (°)
N(1)–Zn(1)–N(3)
N(1)–Zn(1)–Cl(2)
N(3)–Zn(1)–Cl(2)
N(1)–Zn(1)–Cl(1)
111.08(9)
108.80(7)
103.93(7)
108.78(7)
N(3)–Zn(1)–Cl(1)
Cl(2)–Zn(1)–Cl(1)
C(1)–N(1)–C(7)
C(1)–N(1)–Zn(1)
107.66(7)
116.50(4)
104.8(2)
C(7)–N(1)–Zn(1)
C(1)–N(2)–C(2)
C(1)–N(2)–H(2A)
C(2)–N(2)–H(2A)
119.67(18)
107.5(2)
126.2
133.96(19)
126.2
Table 2. Selected bond lengths and angles (Å (°)) for complex R2.
Bond lengths (Å)
Zn(1)–N(1)
Zn(1)–O(4)
Zn(1)–O(1)
2.0991(12)
2.1217(10)
2.2025(11)
O(1)–N(4)
O(2)–N(4)
O(3)–N(4)
N(1)–C(1)
1.2473(15)
1.2312(17)
1.2387(17)
1.3389(19)
N(1)–C(5)
N(2)–C(6)
N(2)–C(7)
N(3)–C(6)
1.3428(19)
1.3576(18)
1.3708(19)
1.3207(18)
Bond angles (°)
N(1)#1-Zn(1)–N(1)
N(1)#1-Zn(1)–O(4)
N(1)–Zn(1)–O(4)
N(1)–Zn(1)–O(4)#1
180.0
O(4)–Zn(1)–O(4)#1
N(1)#1-Zn(1)–O(1)
N(1)–Zn(1)–O(1)
O(4)–Zn(1)–O(1)
180.0
O(4)#1-Zn(1)–O(1)
78.93(4)
88.63(5)
91.37(4)
88.63(5)
86.79(4)
93.21(4)
101.07(4)
bonding which results in the formation of chains. These chains analyzed through TGA in the temperature range of 25 to 700 °C
are held together in crystal lattice through some C-H···O at a heating rate of 10 °C per minute under nitrogen environ-
hydrogen bonds. The coordinated nitrate ions and water are ment. The complex R1 is stable up to ca 220 °C, after this tem-
acting as linkers between different moieties and resulting in a perature the progressive decomposition of the complex oc-
robust 3-D crystal structure. The packing diagram of compound curred. The organic ligand completely decomposes at 450 °C
R2 is shown in Figure S15. The hydrogen bonding parameters with the loss of 60 % weight of the complex. The 40 % black
are given in Table S5.
color residue remains at the end of the heating process which
contains ZnO and carbonaceous matter. Similarly, the R2 com-
plex start decomposing from 200 °C and complete decomposi-
tion of ligand take place at 360 °C with the loss of 55 % of the
mass of the complex. The remained black residue contains ZnO
and carbonaceous materials. Thus, TGA studies confirm that
both zinc complexes are crystalline and display thermal stability
at the room temperature (Figure S21).
Aqueous and Thermal Stability of the Complexes
1H NMR spectroscopy and XRD analysis were employed to eval-
uate the aqueous stability of the zinc complexes R1-2. The H
NMR spectrum of R1 recorded in D2O show signals at δ = 8.63,
7.96, 7.56 (split), 7.28 ppm corresponding to aromatic protons
of R1 (Figure S19). It has been envisaged that the coordination
of Zn(II) with benzimidazole nitrogen restrict the rapid tautom-
erism of hydrogen atom across N-1 and N-3. The locking of
1
Chemosensor Activity of Zinc Complexes R1 and R2
tautomer in particular state split the aromatic signals of benz- The emission spectroscopy was employed to study the chemo-
imidazole moiety. The complex R2 shows proton signals at δ = sensor activity of both zinc complexes with the library of an-
8.51, 7.80, 7.49 and 7.20 ppm corresponding to aromatic pro- ions. The fixed concentration of zinc complexes (10 μ
M
) was
tons of the R2 as shown in Figure S20. Further, the H NMR of prepared in an aqueous medium (HEPES buffer at pH = 7.4) and
both the complexes was recorded on alternative days till ten used for recognition studies. The 10 μ solution of R1 was ex-
1
M
days and no significant changes were observed. Hence, it is cited at 310 nm, and emission was obtained at 487 nm with
evident from1H NMR spectrum that both the complexes are low quantum yield (Φ = 0.10). To investigate the anion binding
stable in an aqueous medium. Moreover, both zinc complexes behavior of complex the emission spectra of R1 was recorded
were suspended in water for ten days and after that the powder on the addition of various anions (30 μM) namely as ATP, ADP,
XRD pattern was recorded (Figure S16–S17) which shows that AMP, NADP, NAD, Phosphate, fluoride, carbonate, sulfate, nitrate,
the complexes remain stable after suspending them in aqueous hydrogen phosphate, pyrophosphate, bromide, iodide, cyanide,
medium for long duration. The thermal studies of R1-2 were hydroxide, chlorate and sulfide ions. The significant change in
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the emission intensity of R1 was occurred in the presence of centration of the pyrophosphate (Figure 2B). Further, to find
pyrophosphate. However, the other anions used did not out the practical utility of the sensor system, the interference
showed any significant shift in the emission spectrum of R1. experiment was conducted (Figure 2D). In this typical study, the
Upon addition of 30 μ of pyrophosphate, the emission inten- library of anion solutions were added to R1 along with the
M
sity of R1 was enhanced 5.5-fold at 487 nm with high quantum pyrophosphate solution and emission was noted. The competi-
yield (Φ = 0.29) as shown in Figure 2A. The enhancement in tive binding assay demonstrates that no interference was ob-
emission intensity confirms the selective binding of pyrophos- served for detection of pyrophosphate. HyperSpec Software
phate with the coordination sphere of zinc complex. The modu- was used to determine the stability constant between R1 and
lation in emission intensity was due to “aggregation-induced PPi. The fluorescence titration data was fitted into HyperSpec
emission”, which provides structural rigidity in the com- software, multiple binding models was applied and a good fit
plex.[52,53] Furthermore, the titration experiment was performed of data was obtained in Figure 2C. In the species distribution
to confirm the reproducibility of the experiment. In titration curve, the 1:2 species was dominant in a solution state. The
experiment; the successive addition of PPi (0-30 μ ) to the R1 fitting data provided log K values of 3.21 0.02, 8.74 0.04 and
M
solution was carried out, and emission intensity was recorded. 11.21 0.05 for 1:1, 1:2 and 2:1 complex, respectively.[54,55] The
The fluorescence titration experiment shows the linear en- detection limit of PPi sensing was calculated through IUPAC
[54]
hancement in fluorescence intensity with increase in the con- reported 3σ method, and it came out to be 25 nM.
Figure 2. (A) The emission spectra of R1(10 μM, λex = 310 nm, HEPES buffer, pH = 7.4 at room temperature) at λem = 487 nm on treatment with various anions
(30 μ ) in aqueous solution. (B) Fluorescence titration of R1(10 μ
M
M
, HEPES buffer, pH = 7.4) in the presence of PPi (0 -30 μ ) at room temperature. (C) Fitting
M
of fluorescence titration data to determine the stability constant between R1 and PPi with HyperSpec software. (D) The interference studies of complex R1
for selective detection of PPi in the presence of various anions.
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Similarly, the complex R2 was excited at 315 nm and exhibit PPi the average excited lifetime of (τ) was detected at 4.53 ns.
emission intensity at 485 nm with low quantum yield (Φ = This large increase in the value of τ was due to aggregation in
0.08). The chemosensor study of R2 complex was tested with a the complex and lifetime of R1+ PPi species become longer.[56]
similar type of analyte as utilized for the complex R1. The result Analogously, the TRPL spectra of 10 μ
of emission experiment shows that R2 binds with ATP, ADP, age excited lifetime at 3.05 ns, whereas on the addition of
AMP,and pyrophosphate. However, the binding pattern toward 30 μ PPi the fluorescence lifetime has shifted to 3.34 ns. This
M
R2complex exhibit aver-
M
PPi was more significant than the other nucleotides (Figure small change in the lifetime of R2+ PPi species confirms the
S22A). The fluorescence binding mechanism seems to be gov- chelation of PPi to R2 complex (Figure S23B).[57] Further, the
erned by the chelate effect; as all nucleotide and PPi form che- radiative and nonradiative rate constant of both zinc complexes
lation with complex R2. Upon addition of 0-30 μ
M of PPi to and their pyrophosphate complexes were calculated by using
complex, the emission intensity was enhanced with an increase Equation (1).
in the concentration of the analyte (Figure S22B). The interfer-
ence experiment was performed, and the result showed that
the nucleotides interfere with the recognition of PPi (Figure
S22D). The stability constant between R2 and PPi was deter-
mined by the similar method as used in complex R1. The fitting
data showed that 1:2 species was dominant in the complex
equilibria having log K value of 6.34 0.05 (Figure S22C). The
different binding behavior of these two complexes towards PPi
may entirely depend upon their distinct geometry, i.e., tetrahe-
dral and octahedral. The R1 complex possesses tetrahedral ge-
ometry (stearic hindered) and coordination sphere can easily
accommodate the pyrophosphate anion over the ATP, ADP and
AMP. Hence, such binding pattern of R1 with PPi may lead to
aggregate formation as shown in Figure S30. However, the R2
complex is octahedral in geometry and Zn(II) is easily approach-
able from PPi as well as other phophorylated biomolecules
through solvent or anion exchange leading to non-selective
binding pattern.
Whereas φ is the quantum yield, kr and knr are the radiative
and non-radiative rate constant, and τ is the lifetime of the
complex. The value of the radiative rate constant (kr) for pyro-
phosphate zinc complexes was more than of its pure zinc com-
plex, and interestingly these results are well sustained with the
static fluorescence intensity of pyrophosphate sensor system.
The DLS measurements were also carried out to understand the
aggregation of R1 in the presence of an excess of pyrophos-
phate. The DLS result revealed that on the addition of 5-30 μ
M
of PPi in 10 μ of R1; the size of aggregate increases from 200
M
nm to 1200 nm (Figure 3A). The detailed investigation of the
role of pyrophosphate induced aggregation of the R1 complex
was further employed with TEM analysis. The results of TEM
analysis provide evidence for aggregate formation in the R1
The time-resolved photoluminescence (TRPL) measurements complex as in Figure S24. The TEM analysis predicted that the
were carried out to realize the mechanism of fluorescence en- PPi induced aggregations of R1 are spherical to oval in shape
hancement of R1 in the presence of PPi (Figure S23A). The ex- and possess a size of 1.2 μm. The AFM investigation was also
cited state decay of both zinc complexes and their pyrophos- performed to confirm the aggregations of R1 by PPi. The AFM
phate complexes were best tail fitted with bi-exponential func- studies showed that the nano-aggregates are spherical and
tion. Lifetimes of the individual components, average lifetime have a size distribution of 1.0 μm to 1.5 μm as depicted in
and radiative (kr) and nonradiative (knr) rate constant of both Figure 3B and Figure S25. All three-techniques used for charac-
zinc complexes and the pyrophosphate sensing are summa- terization of aggregates correlate with each other and exhibit
rized in Table S6. The average excited lifetime (τ) of R1 in the evidence for the role of pyrophosphate to induce aggregation
aqueous medium is 3.49 ns; however, on the addition of 30 μ
M
of R1.
Figure 3. (A) Dynamic light scattering histogram of R1+ PPi aggregates showing effect of concentration on the average size of aggregate. (B) AFM measure-
ments of R1+ PPi, showing size distribution from 1.0 to 1.5 μm.
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The PPi prompted aggregation phenomenon possesses re- Detection of Intracellular PPi and Imaging
versibility of R1+PPi aggregate, as depicted in Figure S26. The
reversibility is the one of the important domains to gratify the
demand of a novel chemosensor for reusability of the complex.
To scrutinize the reversible binding of R1 with PPi, the calcium
ions have been employed. This is because the Ca(II) has strong
affinity to form interaction with pyrophosphate.[58] On addition
Pyrophosphate is one of the important biological anions, as it
demonstrates an intense role in many cellular processes.[60,61]
Therefore, it has led to the remarkable interest of analytical
chemist to synthesize such sensor which can detect PPi in the
biological system. Herein, we report the intracellular detection
of pyrophosphate with R1 in HeLa cells using fluorescence mi-
of 0 - 10 μ
M
of Ca2+ to R1+ PPi aggregates, the quenching in
croscopy. The HeLa cells were incubated with 10 μ
M
of R1 for
the fluorescence intensity was observed, this was due to disrup-
tion of R1+ PPi aggregates and formation of calcium pyrophos-
phate complex. Thus, it was concluded from the reversibility
experiment that the PPi triggered the aggregation-induced
emission of R1 complex and caused enhancement in the emis-
sion spectrum. Further turbidimetry method was employed to
check whether the aggregates were destroyed or re-dissolved,
and the investigation confirmed that the turbid solution of PPi
+ zinc complex become semi-transparent after addition of 0-
2 hours followed by three times washing with phosphate-buff-
ered saline (PBS) and fixed with 4 % paraformaldehyde. The
cover-slip was further mounted over microscopic slides, and im-
ages of the cells were recorded as shown in Figure 4 The investi-
gations corroborate that HeLa cells shows blue emission on
treatment with R1, which attributed to intracellular detection
of pyrophosphate. Fascinatingly, as revealed in Figure 4, the
nucleus of HeLa cell is deeply blue stained as compared to the
cytoplasm. This means that the R1, complex principally stain
the nucleus of HeLa cell. However, we cannot rule out the stain-
ing of cytoplasm of HeLa cell by R1. Thus, the cell imaging
studies confirm that the complex R1 could be used for pyro-
phosphate sensor in living cells. The cytotoxicity is among one
of major concern which is related to developing of sensor sys-
tem for biological relevance. It is well familiar that most transi-
tion metal complex based sensors have shown toxicity to cells
over some interval of time. Therefore, the metal complexes
used for cell tracking and cell imaging must have minimum cell
toxicity. The cell viability assay for zinc complex R1 and R1+
PPi were evaluated using MTT assay with HeLa and MCF-7 cell
cultured in growth medium. The HeLa and MCF-7 cell were cul-
tured in MEM media nutrient (Antimycotic) with 10 % fetal bo-
vine serum at 37 °C for 24 hours in 5 % carbon dioxide environ-
10 μM of Ca(II). Further addition of calcium ions did not cause
any physical change in the solution. It may be due to the break-
down of PPi+ zinc complex to calcium pyrophosphate and free
zinc complex. The DLS studies were further utilized to check
the effect of calcium ion on the size of nano-aggregates. The
result exhibits that the size of nano-aggregates decreases from
1200 nm to 367 nm (Figure S26B). These studies confirm the
reversibility of the sensor system.
Plausible Sensing Mechanism
To study the possible binding mechanism of PPi with R1 com-
plex, 31P NMR spectroscopy was employed (Figure S27). The 31
P
ment. The zinc complex R1 (10 μM) along with cell (HeLa and
NMR spectrum of PPi shows phosphorus signal at -5.83 ppm in
D2O. Upon binding with R1 complex, the chemical shift value
of phosphorus signal got perturbed from -5.83 to -5.34 ppm.
This small downfield change in chemical shift value was due to
the binding of PPi with the zinc complex, which causes reduc-
tion in electron density on phosphorus center. Furthermore, to
confirm the binding events of PPi with R1complex; the FTIR
spectra of pure pyrophosphate and R1+ PPi aggregates were
recorded (Figure S28). FTIR spectra of pure pyrophosphate ex-
hibit stretching vibration frequency at 1093and 914 cm–1 corre-
sponding to P=O and O-P–O bond. The FTIR spectra of aggre-
gates exhibit vibration frequency at 3440, 1625, 1436, 1062,
880 cm–1attributed to -NH, -C=N, C=C, P=O and O-P–O bonds,
respectively. Thus, it is observed that on the binding of pyro-
phosphate to R1 complex, the stretching vibration frequency
of P=O bond and O-P–O bond shifted to lower frequency side.
It is due to the P=O, and O-P–O bond binds with the zinc metal
ion and cause a decrease in the strength of P=O and O-P–O
bonds. The powder XRD pattern of aggregates was also re-
corded to check the effect of pyrophosphate binding on the
crystallinity of zinc complex (Figure S29). The powder XRD pat-
MCF-7 cell) incubated at 37 °C for 4 hours and observation
shows 80 - 85 % cell viability. Also, to ensure the cytotoxicity of
tested complexes the dose-dependent studies were carried out.
The investigation affirms 75- 80 % of cells keep on viable in the
concentration range of 25-80 μM as shown in Figure S31A–B.
Figure 4. Fluorescence images of Hela cells showing nuclear staining (blue
emission) on treatment with complex R1.
Alkaline Phosphatase (ALP) Activity
tern of aggregates had shown broad peak which is due to semi- Phosphorylation reaction plays an essential role in many biolog-
crystalline or amorphous state. These changes in the physical ical processes like energy transduction, gene expression, and
state indicate that the aggregation reduces the crystallinity of cell signaling.[62] Most of the phosphorylation reaction are very
zinc complex.[59] Thus, based on all characterization data the slow and enzyme can accelerate such reaction under relevant
possible sensing mechanism of PPi is shown in Figure S30.
biological condition.[63] The enzymatic reactions are proceeded
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Figure 5. (A) The 3D ribbon diagram of real-time ALP activity having 10 μΜ of R1+30 μ
M
of PPi (λext = 310 nm, λemi = 487 nm); (B) Lineweaver–Burk plot for
the hydrolysis of R1+PPi complex by ALP enzyme.
through stable transition state and thus fast. Therefore, the ba- tration the rate is diverted from the straight line. Further, initial
sic understanding of such reaction is important which are stabi- rate law data was fitted into GraphPad Prism 7 program and
lized by transition state.[64] Alkaline phosphatase (ALP) is the Lineweaver–Burk fitting model was applied (Figure 5B). The fit-
enzyme that containing two zinc atom for its catalytic center ting data provided the value of Vmax = 3.696 μ
M =
min–1, Km
and active at basic pH.[65] ALP is regularly used enzyme for 17.43 × 10–4 and Kcat = 38.4 min–1, respectively. Hence, it is
M
diagnosis of numerous diseases especially related to liver and depicted from the Michaelis–Menten parameter that the hydrol-
bone.[66] It is also employed as a biomarker for liver disease in ysis reaction proceeded by the single enzyme-substrate inter-
various pathological laboratory.[67]Here, we report the PPi based mediate. This has also been reported that the active unit of ALP
zinc complex as a bio-analytical tool to construct real-time fluo- consists of two dinuclear zinc ions and the arginine group of
rescence “OFF“ assay for ALP. In the control experiment, a solu- ALP can undergo orientation change and interact with sub-
tion of 10 μ
M
R1, 30 μM of PPi and 10 μM of Tris-HCl buffer strate to provide serine alkoxide residue for hydrolysis of pyro-
(pH = 8.3) was prepared and incubated for 30 minutes at 37 °C. phosphate through covalent enzyme-phosphate intermediate.
The varying concentration of ALP was added to above solution, Further, this intermediate was hydrolyzed in the second step of
and emission response was observed at 487 nm with respect the reaction.[42,68] Hence, it is concluded from this studies that
to time (0-20 minutes). It could be observed from Figure 5A that the R1 + PPi solution could be used for real-time fluorescence
the emission intensity of R1+ PPi complex at 487 nm, starts turn- OFF sensor for ALP.
decreasing with increase in the time interval. However, ALP has
a very little effect on the emission intensity of the R1 complex
as in Figure S32. This examination indicates that the fluores-
cence intensity of R1 decline due to consumption of pyrophos-
Conclusions
phate by ALP enzyme. More prominently, the extent of de- We have synthesized and utilized two benzimidazole-based
crease in the emission intensity indicates that ALP concentra- zinc complexes for sensing of pyrophosphate at physiological
tion increases with an increase in the time interval. The turbid- pH. Complex R1 shows selective detection of pyrophosphate,
ity of R1 + PPi solution in the presence of ALP was checked which was corroborated by the emission spectrum. The emis-
with different time interval. The investigation shows that the sion spectra of R1 exhibit 5.5-fold enhancement on binding
turbid solution become transparent after 40 minute that also with pyrophosphate, which is attributed to aggregation-in-
confirms the consumption of pyrophosphate by ALP enzyme in duced emission. The pyrophosphate induced aggregation of
the solution (Figure S33). Time-dependent emission spectra was complex R1 was further investigated with microscopic studies.
used to scrutinize the kinetics of the hydrolysis reaction under Facilitating sensing in the aqueous phase, complex R1 was also
fixed ALP concentration (20 nM). The initial rate of ALP catalyzed utilized for PPi detection in HeLa cells imaging and cytotoxicity
reaction was determined from the fluorescence titration experi- studies. The cell imaging experiments demonstrate that the R1
ment. Further, a non-linear regression plot between initial rates complex senses pyrophosphate in the intracellular system.
(Vo) vs. different concentration of pyrophosphate (5 to 30 μ
M
)
Complex R2 shows binding with PPi and other nucleotides,
was made in GraphPad Prism7 window software (Figure S34). which seems to be due to the chelate effect. The real sample
The plot indicates that the straight line is observed at initial analysis of complex R1 was executed for the enzymatic activity
enzyme concentration. However, at saturated enzyme concen- of ALP. The result of ALP activity illustrates the decrease in fluo-
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pure product. Yield 90 %, m.p. ≥ 196 °C. FTIR (cm–1) ν: 3084, 1608
and 1432. 1H NMR; (400 MHz, [D6]DMSO): δ = 8.71 (d, 2 H, Ar-H),
8.06 (d, 2 H, Ar-H), 7.62 (d, 2 H, Ar-H), 7.22 (d, 2H Ar-H);13C NMR
(100 MHz, [D6]DMSO): δ = 151.0, 149.3, 137.7, 123.4, 120.8, 117.8
and 115.0; CHN analysis; Calcd. for C12H9N3: C = 73.83, H = 4.65,
rescence intensity of R1 + PPi at 487 nm related to time. It is
due to the progressive consumption of PPi in the reaction mix-
ture by ALP. Thus, complex R1 acts as a bio-analytical tool to
construct real-time fluorescence “OFF” assay for ALP.
N = 21.52, found in (%) C = 73.93, H = 4.63, N = 21.43, ESI-MS;
+
(m/z) = 196.02 [M + H]
.
Experimental Section
Synthesis of Complex [Zn2 (L2)2 Cl2 (DMSO)2] or R1: The complex
was synthesized by preparing the solutions of ZnCl2 (13.6 mg,
0.1 mmol) in 1 mL of methanol and L1(19.7 mg, 0.1 mmol) dissolved
in 1 mL of methanol/DMSO (99:1; v/v) solvent system. Both the
solutions were mixed together and stirred for one hour at room
temperature. The dark brown colored solution was obtained after
one hour of reaction, and the reaction mixture was filtered. The
filtrate was kept for slow evaporation at room temperature and
after three days, the brown colored crystals were separated out.
These crystals were washed with methanol and found to be suitable
for X-ray crystallographic analysis. Yield: 40 %, FTIR (cm–1) ν: 3406,
General Information: All the chemicals used for this manuscript
are of analytical grade and purchased from Sigma Aldrich co., Avra,
1
and SD Fine India. H and 13C NMR spectra were recorded on JEOL
instrument operated at 400 MHz for 1H NMR, 100 MHz for 13C NMR
and 160 MHz for 31P NMR spectroscopy. FTIR spectra of a dried
sample of both ligands (L1-2) and their zinc complexes R1-2 were
measured on a Bruker Tensor 27 spectrophotometer using a solid
cell. Elemental analysis was monitored through a Fisons instrument
(Model EA 1108 CHN). Powder XRD pattern was measured on a
PAN analytical X′PERT PRO diffractometer using Cu-Kα radiation (λ =
0.1542 nm, 40 kV, 40 mA) and a proportional counter detector in
the 2θ range of 5°–80° with a scan speed of 2°min–1. TGA was
performed on a Mettler Toledo thermogravimetric analyzer under
a nitrogen atmosphere in the temperature range 25–700 °C (heat-
ing rate = 10 °C min–1). UV/Visible absorption and Fluorescence
emission spectra were recorded on a Shimadzu UV-2600 and Perkin
L55 instrument, respectively. The absorption and emission spectra
were measured in the fixed wavelength range of 200-700 nm at
room temperature using quartz cuvette with a 1 cm path length.
Dynamic Light Scattering (DLS) was used to measure the particle
size of nanoaggregates with external probe feature of Metrohm
Microtrac Ultra Nanotrac Particle Size Analyzer. TEM studies were
performed on a Hitachi (H-7500) instrument working at 120 kV and
a 400-mesh; carbon-coated copper grid was used for sample prepa-
ration. The surface morphology and size of aggregates were meas-
ured with Bruker scanning probe microscope. Fluorescence lifetimes
of all complexes were recorded with Pico Quant FluoTime 300 High-
Performance Fluorescence Lifetime Spectrometer using time-corre-
lated single photon counting (TCSPC) technique. The multiexpo-
nential function was used to measure the fitted decay profile. The
response function of the instrument was determined with LUDOX
as an internal reference standard.
1
1618, 1548 and 1441. H NMR: (400 MHz, [D6]DMSO): δ = 8.71 (d, 2
H, Ar-H), 8.06 (d, 2 H, Ar-H),7.67-7.57 (m, 2 H, Ar-H), 7.23 (br., 2 H,
Ar-H);13C NMR (100 MHz, [D6]DMSO): δ = 152.5, 149.5, 133.1, 128.1,
124.0, 123.9, 122.7, 121.9, 119.3 and 112.4; Anal Calcd for C28 H30
Cl4 N6 O2 S2 Zn2: C = 43.04, H = 3.69, N = 10.25 found in (%): C =
43.34, H = 3.25, N = 10.41.
Synthesis of Complex [Zn(L2)2(NO3)2(H2O)2] or R2: The complex
was synthesized by mixing the solutions of ZnNO3·6H2O (29.7 mg,
0.01 mmol) in methanol (1 mL) and ligand L2 (19.5 mg, 0.01 mmol)
in THF (1 mL). The solution was allowed to stir for one hour at room
temperature. The obtained solution was kept for slow evaporation
for two days; which generated crystals suitable for X-ray studies.
Yield: 35 %, FTIR (cm–1) ν: 3057, 1620, 1558 and 1456. 1H NMR:
(400 MHz, [D6]DMSO): δ = 8.67 (d, 2 H, Ar-H), 8.12 (d, 2 H, Ar-H),
7.67-7.56 (m, 2 H, Ar-H), 7.22 (br., 2 H, Ar-H). 13C NMR (100 MHz,
[D6]DMSO): δ = 150.7, 148.8, 135.3, 123.5, 121.2, 119.0 and 116.4;
Anal Calcd for C24H22N8O8Zn: C = 46.81, H = 3.60, N = 18.19 found:
in (%) C = 46.83, H = 3.56, N = 18.23.
X-ray Structure Determination
The X-ray diffraction data for the ligand L1 and complexes R1-2
were collected on a Bruker X8 APEX II KAPPA CCD diffractometer
at 293(2) K using graphite monochromatized Mo-Kα radiation (λ =
0.71073 Å). The crystals positioned at 50 mm from the CCD, and
the diffraction spots were measured using a counting time of 15 s.
Data reduction and multi scan absorption were performed using
the APEX II program suite (Bruker, 2007). The structures were solved
by direct methods with the SIR97 program [S1] and refined using
full-matrix least-squares with SHELXL-97[S2]. Anisotropic thermal
parameters were used for all non-H-atoms. The hydrogen atoms of
C–H groups were with isotropic parameters equivalent to 1.2 times
those of the atom to which they were attached. All other calcula-
tions were performed using the programs WinGX [S3] and PARST
[S4]. The molecular diagrams of both complexes were drawn with
DIAMOND [S5] software. Final R-values along with selected refine-
ment parameters is given in Table S1.
Synthesis of Ligand L1: Ligand L1 was synthesized via condensa-
tion reaction between o-phenylenediamine (540 mg, 5 mmol) and
4-pyridinecarboxaldehyde (535 mg, 5 mmol) under dilute condition
maintained in ethanol as solvent (30 mL). The reaction mixture was
stirred for three hours at room temperature. The dark brown col-
ored solid product was obtained after 3hrs of reaction. The brown
color product was filtered and recrystallized from ethanol to afford
pure product in the form of crystals. Yield 85 %, m.p. ≥ 200 °C. FTIR
(cm–1) ν: 3246, 1607, 1500 and 1451;1H NMR; (400 MHz, [D6]DMSO):
δ = 8.66 (m, 2 H, Ar-H, and 1 H, CH = N), 7.88 (d, 2 H, Ar-H), 7.17
(d, 1 H, Ar-H), 6.97 (t, 1 H, Ar-H), 6.70 (d, 1 H, Ar-H), 6.52 (t, 1 H, Ar-
H), 5.30 (br. s, 2 H, NH2);13C NMR (100 MHz, [D6]DMSO): δ = 154.3,
150.7, 145.2, 143.8, 134.4, 129.3, 122.6, 117.6, 116.5 and 115.4;CHN
analysis: Calcd for C12H11N3: C = 73.07, H = 5.62, N = 21.30, found
CCDC 1408280 (for L1), 1578371 (for R1), and 1552809 (for R2)
contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre.
in (%) C = 73.09, H = 5.51, N = 21.38, ESI-MS; (m/z) = 198.04 [M +
+
H]
.
Synthesis of Ligand L2: Ligand L2 was synthesized via condensa-
tion reaction between o-phenylenediamine (540 mg, 5 mmol) and
4-pyridinecarboxaldehyde (535 mg, 5 mmol) in methanol solvent
Evaluation of the Photophysical Properties
(5 mL). The reaction mixture was stirred for 3hrs at room tempera- Photophysical properties of complex R1 and R2 were measured
ture. After 3 hrs, a dark brownish colored solid product was ob-
through UV/Visible absorption and emission spectroscopy. Before
tained, which was filtered and recrystallized from CH3OH to afford
recording the absorption and emission spectra; the standard solu-
Eur. J. Inorg. Chem. 0000, 0–0
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
tion of zinc complexes (10 μM, HEPES buffer, pH = 7.4) were pre-
Fluorescence Assay for ALP Activity: A purified ALP from calf in-
pared in an aqueous medium at room temperature and used for
testinal mucosa used for ALP activity. The enzyme was store at
recognition studies. All the photophysical studies were recorded in -20 °C before use in the experiment. The R1+ PPi complex act as a
the absorption and emission wavelength range of 200 -700 nm at
room temperature using 1 cm quartz cuvette.
substrate, and the stock solution was stored at 4 °C. The ALP and
R1+ PPi stock solution were incubated 37 °C for 30 minutes before
measuring the emission experiment. The catalytical activity of ALP
(5 to 20 nM) was monitored by a decrease in the emission intensity
at 487 nm for 0 to 20 minutes of time interval. All the hydrolytic
reaction was studied with the effect of different substrate concen-
The fluorescence quantum yield of both complexes and R1+PPi ag-
gregates were measured by using the relative method. The optically
matching solution of standard 2-Aminopyridine in 0.1
M H2SO4 (ex-
tration (1 μ
M
to 30 μM) under Tries buffer (8.3 pH), by fixed ALP
cited wavelength of 300 - 340 nm) used as a reference sample for
quantum yield calculation. The fluorescence quantum yield of the
complex was determined using Equation (2).
concentration (20 nM). The initial rate of reaction was determined
from time-dependent emission spectrum data. The Kinetic data was
fitted into GraphPad Prism 7 window software and Lineweaver–
Burk fitting model was applied to determine the Michaelis–Menten
parameter. Fitting data gives the value of (Km), Vmax and Kcat
.
Whereas Φs and Φr the radiative quantum yield of sample and
reference and ODr, OD is the absorbance of reference and sample
respectively. Ir and I are the integrated fluorescence intensity of
reference and sample, and ή and ήr are the refractive index of sam-
ple and reference solution respectively.
GraphPad Prism 7 window software is freely available online
(https://www.graphpad.com) program. All the kinetic experiments
were repeated for three times, and the average data was used for
kinetic parameter determination.
Acknowledgments
Chemosensor Studies: The chemosensor studies of both the com-
plexes were performed with library of anion solutions. The10 μ
solutions of complexes were made in HEPES buffer, at pH = 7.4 in
aqueous system at room temperature. The 10 μ solution of com-
plex was mixed with 30 μ of analyte in 5 mL volumetric flasks.
Fourteen such solutions of zinc complexes with different anions
were made and used for recognition studies. Before recording the
fluorescence experiment, the solution was allowed to stand for one
hour to prevent any fluctuation in the emission spectrum. The fluo-
rescence titration experiment of zinc complexes towards pyrophos-
phate were performed in 1 mL cuvette. The successive addition of
M
This work was supported with a research grant from SERB-DST
India, Project number EMR/2014/000613. P. R. is thankful to
UGC-New Delhi for a Senior Research Fellowship. Dr. Neha Garg
acknowledges financial assistance from SERB, India for Ramanu-
jan grant (SB/S2/RJN-072/2015). Ashutosh Singh is grateful to
UGC for Senior Research Fellowship.
M
M
Keywords: Zinc · Fluorescence · Sensors · Intracellular
detection · ALP activity · Pyrophosphate
pyrophosphate (0- 30 μM) to R1 complex (10 μM, HEPES buffer, pH =
7.4) was carried out and emission intensity was recorded at each
addition of analyte. In order to evaluate the possible interference,
the interfering analyte was added to zinc complexes in the presence
of PPi and emission spectra was recorded.
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Received: September 24, 2018
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Fluorescence Bioimaging
P. Raj, A. Singh, A. Singh,
A. Singh, N. Garg, N. Kaur,*
N. Singh* ............................................ 1–12
Pyrophosphate Prompted Aggrega-
tion-Induced Emission: Chemosen-
sor Studies, Cell Imaging, Cytotoxic-
ity, and Hydrolysis of the Phospho-
ester Bond with Alkaline Phospha-
tase
We have designed and synthesized Hela cells. It is also used as a bio-
two benzimidazole-based zinc com- analytical tool to construct a real-time
plexes for pyrophosphate sensing in fluorescence assay for the enzymatic
aqueous media. One of the complexes activity of alkaline phosphatases (ALP).
also shows pyrophosphate sensing in
DOI: 10.1002/ejic.201801173
Eur. J. Inorg. Chem. 0000, 0–0
www.eurjic.org
12
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim