Synthesis, characterization, and supramolecular architectures of two distinct classes of probes for the visualization of endogenously generated hypochlorite ions in response to cellular activity

Article abstract of DOI:10.1016/j.jphotobiol.2019.111594

Two distinct classes of compounds, (E)-2-(((3-amino-4-nitrophenyl) imino) methyl)-5-(diethylamino) phenol (SB) and 5-(diethylamino)-2-(5-nitro-1H-benzo[d]imidazol-2-yl) phenol (IM) were synthesized. SB, a bright red colored compound was crystallized in ac

Full text of DOI:10.1016/j.jphotobiol.2019.111594

JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, characterization, and supramolecular architectures of two distinct
classes of probes for the visualization of endogenously generated
hypochlorite ions in response to cellular activity
⁎
⁎
Richa Yadava,1, Keiko Oderab,1, Abhishek Raia,2, Ryoya Takahashib, , Lallan Mishraa,
a Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, India
b Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two distinct classes of compounds, (E)-2-(((3-amino-4-nitrophenyl) imino) methyl)-5-(diethylamino) phenol
(SB) and 5-(diethylamino)-2-(5-nitro-1H-benzo[d]imidazol-2-yl) phenol (IM) were synthesized. SB, a bright red
colored compound was crystallized in acetonitrile as a triclinic crystal system while IM, yellow colored com-
pound crystallized as a monoclinic crystal system in dimethylformamide by vapor diffusion of diethylether.
These compounds were characterized using spectroscopic techniques (IR, UV–visible, 1H, and 13C NMR), and X-
ray crystallography. SB and IM displayed classical and non-classical H-bonding involving C-H…O and π…π
interactions. These compounds detected hypochlorite ions in aqueous DMSO (1: 9, v/v, HEPES buffer, pH 7.4),
and detection was visible via color changes by naked eye. We also performed UV–visible and fluorescence
titrations, showing detection limits of 8.82 × 10−7 M for SB and 2.44 × 10−7 M for IM. The fluorometric re-
sponses from SB and IM were also studied against different ROS and anions. DFT calculations were performed to
strengthen the proposed sensing mechanisms of both SB and IM. Hypochlorite, which is endogenously generated
by myeloperoxidase in endosomes, was specifically visualized using SB and IM in lipopolysaccharide-treated
RAW264.7 cells. These probes were also used to image the generation of hypochlorite by RAW264.7 cells during
phagocytosis of non-fluorescent polystyrene beads.
Supramolecule
Hypochlorite
Endosomes
Phagosomes
1. Introduction
hypochlorite ion, ClO−, under relevant physiological conditions [6,7].
Hypochlorite ion is also induced by microbial products, such as lipo-
Molecular recognition is a process by which molecules select and
interact to each other governed by complementarity and forms an in-
tegral part of host–guest chemistry. To explore the treasure of mole-
cular recognition principles for the detection of reactive oxygen species
(ROS) is challenging at the cellular level [1]. Therefore, design and
synthesis of receptors for detection of ROS is a demanding area of
modern research. Among ROS, the hypochlorite ion targets multiple
biological components including DNA, lipids, cholesterol, and proteins
[2]. The oxidizing power of hypochlorite enables it to act as an anti-
microbial agent capable of killing a variety of pathogens in living or-
ganisms [3]. Hypochlorite is generated endogenously by peroxidation
of chloride ions; this process is catalyzed by the heme enzyme myelo-
peroxidase (MPO) via the MPO-H2O2-chloride system [4–6]. MPO is the
only mammalian enzyme that produces hypochlorous acid or
polysaccharide (LPS), which is one of the major bacterial virulence
factors with proinflammatory properties. However, abnormal over-
production of ClO− can cause tissue damage and lead to oxidative
stress, which is associated with cardiovascular diseases [8], in-
flammatory diseases, arthritis [9], kidney disorders [10] and several
other life-threatening conditions [11]. Nature maintains a balance in all
living organisms; endogenously-generated oxidative stress is counter-
acted by a low-molecular-weight antioxidant glutathione (GSH) [12].
GSH is abundantly generated in vivo to maintain the redox homeostasis
of cells and tissues and to protect the essential components of the cells.
This complex redox biology of the cell governs essential biological
processes and has broad implications in human health [13]. Never-
theless, the biological activities of ClO− have not been fully elucidated,
highlighting the necessity to design probes for the detection and
⁎ Corresponding authors.
E-mail addresses: takahasi@phar.toho-u.ac.jp (R. Takahashi), lmishrabhu@yahoo.co.in (L. Mishra).
1 Both authors contributed equally to the work.
2 Current address: Department of Chemistry, Kunwar Singh College (A constituent unit of L. N. Mithila University) Laheriasarai, Darbhanga, Bihar 846003, India.
https://doi.org/10.1016/j.jphotobiol.2019.111594
Received 19 March 2019; Received in revised form 23 July 2019; Accepted 13 August 2019
Availableonline14August2019
1011-1344/©2019ElsevierB.V.Allrightsreserved.

R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Scheme 1. Synthesis strategy for SB and IM.
squantification of ClO− under physiological conditions; such probes
would help us understand the biological redox cycle.
Agilent Technologies 6530Accurate-Mass Q-TOF LC/MS unit, USA. X-
ray diffraction data were collected by mounting a single crystal of
sample on the glass fiber of an Agilent Technologies Oxford diffraction
XCALIBUR-EOS diffractometer, USA. Monochromated MoKα radiation
(λ = 0.71073 Å) was used for the measurements. The crystal structure
was solved by using structure solution by direct methods using SHELXS-
2014 program 7, and was refined using full matrix least squares
(SHELXL-2014) [40]. The interaction of ClO− with probes SB and IM
was supported by energy-optimized structures at B3LYP level with basis
sets 6-31G (d,p) and 6–31 (++) G (d,p) using Gaussian 09 program
[41]. RAW264.7 cells were obtained from DS Pharma Biomedical,
Japan. Fluorescent signals in cells were monitored using an inverted
fluorescent microscope (Carl Zeiss Axio Vert.A1 FL-LED, Germany).
Several methods, including colorimetry, fluorimetry, electro-
chemical approaches, and chromatography, allow for the detection of
hypochlorite ions [14–16]. Fluorescence spectrophotometry is used
most because of its enhanced sensitivity, applicability, high resolution,
and ability to conduct detection in real time [17,18]. Several dye-based
fluorescent receptors, including cyanine, fluorescein, rhodamine,
BODIPY, naphthalimide, and dicyanomethylene-4H-pyran, are also
used to detect hypochlorite ions [19–25]. However, these fluorescent
receptors show limited stability towards oxidants, are costly, require
multistep synthesis and are obtained in low yields; once more high-
lighting the need for new fluorescent probes that are economic cost
wise and easy to synthesize for hypochlorite ion detection [26,27].The
compounds SB and IM presented here overcome these limitations as
they give high yields in one pot reaction.
2.2. Synthesis of SB
In this report, we focused on two compounds for recognition of
hypochlorite ions; the structures of these compounds are illustrated in
Scheme 1. One is a Schiff base while the second one is an imidazole.
Schiff base are known for their wide applicability ranging from its use
as catalysts, stabilizers, dyes, organic intermediates [28,29] to its po-
tent biological role as antiviral, antimalarial, antifungal, antibacterial,
antiproliferative, antipyretic agents [30–33]. Similarly, imidazole is
also documented for their biological importance and sensing properties
towards anions but less work is done on studying its interaction with
ROS particularly hypochlorite ions [34–37]. Derivatives of 4-diethyla-
minosalicylaldehyde (DEA) show good spectral properties, biocompat-
ibility, and ability to penetrate cellular membrane, rendering these
compounds usable as fluorescent probes [38,39]. In this study, we in-
vestigated the use of SB and IM for the detection of hypochlorite ions
under physiologic conditions.
A solution of 4-diethylaminosalicylaldehyde (0.193 g, 1.0 × 10−3 mol)
in 10 mL methanol was refluxed together with a solution of 4-nitro-m-
phenylenediamine (0.153 g, 1.0 × 10−3 mol) in 10 mL methanol for 4 h in
the presence of two drops of glacial acetic acid. The bright red colored
precipitate thus obtained was filtered and washed with diethyl ether and
dried in vacuum. The product was recrystallized in acetonitrile. The crys-
tals of SB suitable for X-ray measurements were grown by slow evaporation
of a solution of SB in acetonitrile at room temperature. The obtained yield
for SB = 86% (0.282 g). The values for FT-IR (KBr; cm−1) were as follows:
3426, 3334, 2973, 1609, 1570, 1515, 1478, 1434, 1382, 1321, 1235, 1140,
1074, 1009, 960, 843, 818, 781, 749, 690, 571, 453. The values for 1H
NMR (500 MHz, DMSO-d6) were as follows: δ 13.30 (s, broad, 1H, OH),
8.66 (s, 1H, HC]N), 7.98 (d, 1H, J = 8.5 Hz, phenyl), 7.44 (broad, 2H,
NH2), 7.34 (d, 1H, J = 8.5 Hz, phenyl), 6.76 (d, 1H, J = 2 Hz, phenyl),
6.55 (q, 1H, Ar), 6.33 (d, 1H, J = 8 Hz, phenyl), 6.05 (d, 1H, J = 2 Hz,
phenyl), 3.39 (t, 4H), 1.10 (q, 6H). The values for 13C NMR (125 MHz,
DMSO‑d6) were as follows: 164.49 (-C-OH), 163.16 (-HC=N), 154.99,
128.43, 127.72, 110.19, 109.47, 109.04, 105.05, 97.20, 44.56 (-CH2),
13.07 (-CH3). The values for ESI-MS calculated for C17H20N4O3 [M + H]+
were as follows: 329.1614, found: 329.1615; crystal system, triclinic; space
group, P-1; a (Å),7.1633(3); b (Å), 8.1894(4); c (Å), 15.4615(9); α (°),
102.288(4); β (°), 94.259(5); γ (°), 111.069(3); volume (Å3), 815.61(7);
Z = 2.
2. Experimental Section
2.1. Materials and Methods
All the solvents were obtained from commercial sources and were
dried and distilled prior to their use. The chemicals (4-nitro-m-pheny-
lenediamine and 4-nitro-o-phenylenediamine) were purchased from
Alfa Aesar, India and 4-diethylaminosalicylaldehyde from Sigma
Aldrich, India. Infrared spectra were recorded at room temperature
(303 K) using KBr pellets on a Varian 3300 FT-IR spectrophotometer,
USA. UV–visible spectra were recorded on a Shimadzu UV-1601 spec-
trophotometer, Japan, and fluorescence spectra were measured on a
PerkinElmer LS 55 Fluorescence spectrophotometer, USA at room
temperature (303 K). The solvent system for all the absorbance and
emission experiments was an aqueous DMSO (1: 9, v/v, HEPES buffer,
1 × 10−3 M, pH 7.4). The NMR spectra (1H and 13C) were collected on
a JEOL AL500 FT-NMR spectrometer, USA, at room temperature
(303 K). The chemical shifts (δ, ppm) were normalized to that of tet-
ramethylsilane (Si(CH3)4) used as internal standard. Electro spray io-
nization mass spectrometry (ESI-MS) experiments were performed on
Bruker Daltonics amaZon SL Max ion trap mass spectrometer, USA and
2.3. Synthesis of IM
In our present study, IM was prepared as a control compound using
a slight modification to reported method [42]. IM was characterized
using X-ray crystallography. In this study, we used a solution of 4-
diethylaminosalicylaldehyde (0.289 g, 1.5 × 10−3 mol) in 10 mL me-
thanol; this was added under stirring to a solution of 4-nitro-o-pheny-
lenediamine (0.153 g, 1.0 × 10−3 mol) in methanol (10 mL) in the
presence of two drops of HCl. The resulting solution was refluxed for
8 h. The progress of the reaction was monitored by TLC. The reaction
mixture was then cooled to room temperature. The obtained yellow-
colored precipitate was filtered using a vacuum pump, washed twice
with 2 mL (each) of diethyl ether, and dried under vacuum. IM crystals
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R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 1. Molecular structure (ORTEP) of SB and IM drawn at 50% probability level.
H2O2 was determined from its absorbance at λmax 240 nm [46]. Per-
oxynitrite (ONOO−) was prepared using a method reported previously
[47]. The concentration of ONOO− was determined using an extinction
coefficient of 1670 M−1 cm−1 (λmax 302 nm). Singlet oxygen (1O2) was
obtained by the addition of an aqueous solution of NaClO to H2O2 using
a method reported previously [48]. The stock solutions of SB and IM
were then diluted separately to a concentration of 1.0 × 10−5 M with
the addition of aqueous DMSO (1: 9, v/v, HEPES buffer, 1 × 10−3 M,
pH 7.4). For absorption and fluorescence measurements, 3.0 mL of each
solution of SB and IM was placed into a quartz cell having 1 × 10−2
m
optical path length, and the spectra were recorded at room tempera-
ture. The excitation and emission slit widths were maintained at 10 nm.
The excitation wavelengths for the measurement of fluorescence
spectra were set to λexct = 438 nm and λexct = 415 nm for SB and IM,
respectively. For titrations, a solution of corresponding ion was gra-
dually added into the solution of SB or IM at a fixed fraction using a
micropipette, and was mixed thoroughly using micropipette before
recording the spectra.
Fig. 2. Packing diagram of SB generated via H-bonded interactions. Light
violet, red, and green represent carbon, nitrogen, and oxygen atoms, respec-
tively. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
2.5. 1H NMR Titrations
NMR titrations were carried out by incremental addition of ClO− (in
D2O) to the solution of SB (DMSO-d6) and to that of IM (DMSO-d6).
suitable for X-ray measurements were grown by slow vapor diffusion of
diethyl ether over a saturated solution of IM in dimethylformamide
(DMF) at room temperature. The obtained yield = 81% (0.264 g). The
values for FT-IR (KBr; cm−1) were: 3321, 1645, 1589, 1556, 1502,
1470, 1446, 1426, 1359, 1339, 1266, 1213, 1143, 1084, 818, 786, 732,
600. The values for 1H NMR (500 MHz, DMSO-d6) were: δ 13.12 (s,
broad, 1H, -OH), 12.36 (s, broad, 1H, -NH), 8.38 (s, 1H, -phenyl), 8.10
(d, 1H, J = 8.5 Hz, -phenyl), 7.84 (d, 1H, J = 8.5 Hz, −phenyl), 7.68
(d, 1H, J = 8.5 Hz, -phenyl), 6.42 (d, 1H, J = 9 Hz, -phenyl), 6.22 (s,
1H), 3.40 (q, 4H), 1.14 (t, 6H). The values for 13C NMR (125 MHz,
DMSO-d6) were: 13.44, 44.78, 98.47, 100.61, 104.96, 118.74, 129.06,
143.13, 151.96, 157.91, 160.92. The values for ESI-MS calculated for
2.6. Determination of Detection Limit
The limit of detection (LOD) for probes SB and IM with respect to
ClO− was calculated using the results of fluorometric titrations. To
ascertain the S/N ratio, emission intensity from SB and IM in the pre-
sence of ClO− was determined 10 times, and standard deviation of the
working curve was calculated. The LOD was then obtained using the
formula, LOD = 3δ/k, where δ refers to the standard deviation of blank
measurement, and k is the slope of the best fitted line [49].
C
17H18N4O3 [M + H]+ were: 327.1458, found: 327.1459; crystal
2.7. Cell Culture
system, monoclinic; space group, C 2/c; a (Å), 13.737(11); b (Å),
6.740(6); c (Å), 34.07(3); α (°), 90.00; β (°), 101.15(2); γ (°), 90.00;
volume (Å3), 3095(4); Z = 8.
RAW264.7 cells (DS Pharma Biomedical, Japan) were maintained in
DMEM (1.0 g/L glucose supplemented with sodium pyruvate, without
L-Gln or Phenol Red) (Nacalai Tesque Inc., Japan) containing 10% heat-
inactivated fetal bovine serum (Gibco, Australia), 2 mmol/L L-Ala-L-
Gln, 100 U/mL penicillin, and 100 U/mL streptomycin, under a humi-
dified atmosphere of 95% air and 5% CO2 at 37 °C.
2.4. UV–Visible and Fluorescence Spectra
For all the spectral studies, stock solutions of SB and IM were pre-
pared separately in DMSO (1.0 × 10−3 M). The stock solutions
(1.0 × 10−2 M) of F−, Cl−, Br−, CN−, N3−, OH−, NO3−, NO2−, AcO−
,
2.8. Hypochlorite Imaging in PMA- and LPS-Treated Cells
PO43−, and S2− were prepared separately by dissolving the corre-
sponding salts in double-distilled water for individual additions. The
concentration of hypochlorite (ClO−) was determined using an ex-
tinction coefficient of 350 M−1 cm−1 (λmax 292 nm) at pH 9.0 [43]. The
hydroxyl radical (•OH) was generated by Fenton reaction between H2O2
and aqueous solution of ferrous ammonium sulphate [44]. Superoxide
(O2•−) was generated from sodium hydroxide and hydrogen peroxide
[45]. The concentration of commercially available stock solution of
RAW264.7 cells were plated at 10,000 cells per well, in 100 μL
media per well, in a 96-well black-wall clear-bottom culture microplate
(EZVIEW glass bottom culture plate LB; IWAKI, Japan), and were al-
lowed to attach to the wells. The cells were then treated for 24 h with
1.0 × 10−6 M phorbol 12-myristate 13-acetate (PMA) or 1.0 × 10−7 g/
mL lipopolysaccharide (LPS from E. coli O111:B4, Sigma-Aldrich). For
fluorescence imaging of endogenous hypochlorite generation in PMA-
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R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 3. Packing of IM via: (a) H-bonding along crystallographic a-axis, (b) H-bonding along crystallographic c-axis, (c) π-π interactions of the two phenyl rings at a
centroid distance of 3.67 Å, (d) π-π interactions of imidazole rings at a centroid distance of 3.50 Å.
Fig. 4. UV–visible titrations of: (a) SB (1.0 × 10−5 M) and (b) IM (1.0 × 10−5 M) in aqueous DMSO (1: 9, v/v, HEPES buffer, 1 × 10−3 M, pH 7.4) using incremental
addition of aqueous solution of NaOCl. Inset: color change exhibited by the probes in the presence of ClO− under normal light.
4

R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 5. Fluorescence titrations of: (a) SB (1.0 × 10−5 M) and (b) IM (1.0 × 10−5 M) in aqueous DMSO (1: 9, v/v, HEPES buffer, 1 × 10−3 M, pH 7.4) using in-
cremental addition of aqueous solution of NaOCl. Inset: color changes in probes in the presence of ClO− under UV lamp.
Fig. 6. DFT optimized minimum energy structures of SB, SB′ (oxidized SB) and IM, IM′.
and LPS-stimulated cells, the cells were washed twice with phosphate
buffered saline (PBS) and incubated with 5.0 × 10−6 M hypochlorite
probe (SB or IM) in PBS.
inhibitor. Fluorescent signals in cells were monitored using an inverted
fluorescent microscope (Carl Zeiss) as described above.
Fluorescence signals in cells were monitored using an inverted
fluorescence microscope (Axio Vert.A1 FL-LED, Carl Zeiss Microscopy
GmbH, Germany) using a 40× EC Plan-Neofluar objective. Detection of
fluorescence using SB and IM was conducted using a Zeiss Filter Set 38
(excitation BP 470/40, beam splitter FT 495, emission BP 525/50).
Images were captured using a Zeiss AxioCam MRm camera and further
processed using AxioVision 4.8.2 (Carl Zeiss) and ImageQuant TL (GE
Healthcare Japan Corp., Japan) software.
2.10. Phagocytosis Assay
LPS-stimulated RAW264.7 cells were allowed to internalize 1-μm
non-fluorescent polystyrene beads (Polybead Polystyrene Microspheres,
Polysciences Inc., USA) for 30 min. The cells were then washed with
PBS and incubated with 5.0 × 10−6 M hypochlorite probe (SB or IM).
Fluorescent signals in the cells were monitored using an inverted
fluorescent microscope (Carl Zeiss) as described above.
2.9. Inhibition of Endogenous Hypochlorite Generation in Cells
3. Results and Discussion
Inhibition of endogenous hypochlorite generation in cells was per-
formed using a specific inhibitor of myeloperoxidase (4-aminobenzoic
acid hydrazide, ABAH) [50] and hypochlorite quenchers GSH [51] and
taurine [52]. PMA- and LPS-treated cells were pretreated with
0.1 × 10−3 M ABAH, 5.0 × 10−3 M GSH, and 5.0 × 10−3 M taurine for
2 h. The cells were then washed with PBS and incubated with
5.0 × 10−6 M hypochlorite probe (SB or IM) in PBS containing each
3.1. Synthesis and Characterization of SB and IM
The synthesized compounds SB and IM contain two components: an
electron-donating diethylamino group in a framework of a phenolic
ring, and an electron-withdrawing nitro group in a frame of aniline. In
SB, these two components are connected via an imino group. In IM, the
electron-donating group is directly connected to a nitrobenzimidazole
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JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
each compound. The IR spectrum of SB, displayed a prominent peak at
1609 cm−1
, attributed to υ (CH=N) vibration. Other peaks at
3426 cm−1 and 3334 cm−1 in the SB spectrum were assigned to υ
(NH2) vibrations, as shown in Fig. S1a†. The peaks observed at
3321 cm−1 and 1645 cm−1 in the IR spectrum of IM were assigned to υ
NH and υ C]N vibrations of the imidazolyl ring, respectively (Fig.
S1b†). In the 1H NMR spectrum of SB, the peaks observed at δ
13.30 ppm and δ 8.67 ppm were assigned to –OH and –CH=N protons,
respectively (Fig. S2†). The amino protons of SB were deshielded and
displayed at δ 7.44 ppm. The deshielding was due to the presence
of a neighbouring electron-withdrawing nitro group. A peak at δ
163.16 ppm, assigned to imine carbon, supported the formation of a
Schiff base (Fig. S3†). As shown in Fig. S4†, the peaks observed at δ
13.12, 12.36, and 8.38 ppm in the 1H NMR spectrum of IM were as-
signed to –OH, -NH protons, and the proton ortho to the nitro group,
respectively. The 13C NMR spectrum of IM is shown in Fig. S5†. The
ESI-MS spectra of SB and IM displayed major peaks at m/z 329.1614
and m/z 327.1459, attributed to [SB + H]+ and [IM + H]+ respec-
tively (Figs. S6-S7†). The molecular structures of SB and IM are shown
in Fig. 1.
3.2. Crystal Structures and Supramolecular Architectures of the Probe SB
and IM
The crystallographic parameters of SB and IM are provided in Table
S1. The compound SB crystallized in a triclinic system with space group
P-1. The interplanar angle between the two phenyl rings bearing an
electron-releasing group N-(C2H5)2 and an electron withdrawing nitro
group, connected via an imine bond in SB, was observed as 12.66o (Fig.
S11a). SB displays two intramolecular hydrogen bonds, one between
the hydroxyl and imino group (O3-H3…N3), and the other between the
amino group and nitro oxygen (N1-H1A…O1); this is shown in Fig.
S8a†. Additionally, a strong intermolecular H bond was displayed be-
tween the amino nitrogen and the hydroxyl hydrogen (Fig. S8b†). There
were two non-classical H-bonding interactions between C5-H5 of the
phenyl ring and O2 of the nitro group, and between C17-H17C of the N-
diethyl amino group and the O1 of the nitro group (Fig. S8c†).
Additionally, π…π interactions were observed at a centroid distance of
4.02 Å between the phenyl rings containing the nitro group and the
hydroxyl group. The parameters of these weaker interactions are pro-
vided in Table S2. The association of molecules via hydrogen bonding
generated stringed supramolecular architecture, as depicted in Fig. 2.
In IM, an intramolecular H-bond (O3-H3…N2) was formed at a
distance of 1.79 Å, whereas intermolecular H-bond formation (N1-H1…
O3) occurred at a distance of 2.29 Å (Figs. S9a-b†). In addition to these
classical H-bonds, a non-classical H-bond was also present between the
CH(CH3) of one molecule and the oxygen atom of NO2 in another
molecule at a distance of 2.56 Å (Fig. S9c†). Additionally, a strong π…π
interaction, at a distance of 3.67 Å, was observed between a phenyl ring
containing a nitro group belonging to one molecule and a phenyl ring
containing a diethylamino group belonging to another molecule (Fig.
S9d†). A π…π interaction was also observed between the imidazole
rings of two different molecules at a centroid distance of 3.50 Å (Fig.
S9e†). The supramolecular structures, formed via hydrogen bonding
along crystallographic axes a and c, as well as π…π interactions, are
shown in Fig. 3. The overall supramolecular architecture involving all
these non-covalent interactions generated a network of interconnected
rings (Fig. S10†). The bond lengths and bond angles of SB and IM are
provided in Table S3. The dihedral angles between the planes of SB and
IM are separately shown in Fig. S11†.
Fig. 7. Proposed sensing mechanism of SB and IM with respect to ClO−
.
Fig. 8. Color changes of SB-loaded paper strips in the presence of ClO− under
daylight (a) and UV light (b) color changes of IM-loaded paper strips in the
presence of ClO− under daylight (c) and UV light (d).
ring system. The strategically designed frameworks of SB responded
robustly to the biologically relevant hypochlorite ions. To compara-
tively assess the recognition properties of SB, we synthesized another
distinct compound, IM, which bears a biologically relevant imidazolyl
group.
3.3. Colorimetric Response of Probes SB and IM to Hypochlorite Ion
The two compounds were fully characterized using IR, 1H and 13C
NMR, UV–visible, fluorescence spectra, and ESI-MS (Figs. S1-S7†), and
were authenticated by X-ray crystallography using a single crystal of
Visual inspection of probes SB and IM in aqueous DMSO (1: 9, v/v,
HEPES buffer, 1 × 10−3 M, pH 7.4), in the presence of different anions
and ROS (10 equivalents), showed color changes from yellow to dark
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JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 9. Fluorescence images of PMA- and LPS-stimulated RAW264.7 cells incubated with hypochlorite probes SB and IM. PMA-treated RAW264.7 cells were
incubated with 5 × 10−6 M SB (left) or IM (right). Bright field image (a), fluorescence image (b), and enlarged images of square area in b (c). Bars: 10 μm.
red and purple respectively, only with respect to hypochlorite ions
(Figs. S12 and S13†). This prompted us to record the UV–visible spectra
of SB and IM. SB displayed a strong band at λmax 438 nm and a weaker
band at λmax 331 nm. Upon incremental addition of ClO−, the peak
observed earlier at λmax 438 nm started to diminish with a red shift of
90 nm, and appeared at λmax 528 nm (Fig. S14a†). Additional peaks
were also observed at λmax 271 nm and 383 nm. SB produced a color
change from yellow to dark red in the presence of ClO− ions, as shown
in the inset of Fig. 4a. Other anions and ROS did not induce any ap-
preciable changes in the color of (Fig. S12†) or spectra of SB (Fig.
S14a†). Similarly, the peak observed at λmax 415 nm in the spectra of
free IM shifted to λmax 523 nm, showing a bathochromic shift of 108 nm
in the presence of ClO− (Figs. 4b and S14b†). The other IM peak was
retained at λmax 337 nm. The color of IM changed from yellow to purple
upon incremental addition of ClO−, as shown in the inset of Figs. 4b
and S13†.
ClO− in biological systems at physiological pH.
3.6. Response Time and Detection Limit
Response time is another important criterion for assessing the re-
liability of a fluorescent probe. As shown in Fig. S17†, an increase in
fluorescence occurred within 60 s of adding ClO− to SB, while the re-
sponse of IM was relatively more rapid at 40 s. The interaction of the
probe SB and IM with hypochlorite ions were also analysed quantita-
tively as depicted in the Figs. S18-S19†. A satisfactory linear relation-
ship was observed with an acceptable correlation coefficient. These
results indicate that SB and IM can be applied for the quantitative es-
timation of ClO− using fluorometric spectroscopy (Fig. S18†). The de-
tection limit of SB was 8.82 × 10−7 M (Fig. S19a†), and that of IM was
2.44 × 10−7 M (Fig. S19b†).
3.7. Interference and Competition Experiments
3.4. Fluorometric Responses of SB and IM to Hypochlorite Ions
To assess whether SB and IM showed specificity only towards ClO−
,
The fluorometric responses from SB and IM to various anions and
ROS are shown in Figs. S15a-d†. However, SB and IM selectively de-
tected only ClO− with enhanced intensity of emission. The emission
generated by free SB at λem 523 nm shifted to 491 nm, with intensity
increased 10-fold upon the addition of ClO−. The titrations of SB with
ClO− are depicted in Fig. 5a. The emissions generated by IM, initially
observed at λem 476 nm and λem 495 nm, finally coalesced at λem
518 nm, with intensity increasing upon gradual addition of ClO− as
shown in Fig. 5b. The final changes in the color are depicted as insets in
the corresponding figures.
we measured the fluorometric responses of each probe to other anions
and ROS; each probe was assessed in aqueous DMSO (1: 9, v/v, HEPES
buffer, 1 × 10−3 M, pH 7.4). Other anions and ROS failed to trigger the
increase in fluorescence intensity that was induced by ClO− in each
probe. The histograms in Figs. S20 and S21† show that ClO− increased
the emission of fluorescence notably more than did other analytes, in-
dicating the high selectivity of probes towards ClO−
.
3.8. Computational Analyses
To understand the sensing mechanisms involved in the reactivity of
SB and IM to ClO−, we performed DFT calculations using the B3LYP/6-
31G (d, p) or 6–31(++)G (d,p) method with the Gaussian 09 program.
Orbital analysis revealed that in the highest occupied molecular orbital
(HOMO), charge density in both SB and IM was mainly localized on the
diethylamino moiety, whereas in the lowest unoccupied molecular
3.5. pH Studies
pH-dependent variations in fluorescence, emitted by probes SB and
IM in the absence and presence of ClO−, are depicted in Fig. S16†.
These results show that these probes are suitable for the detection of
7

R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 10. Effects of myeloperoxidase inhibitor and hypochlorite scavengers on fluorescence intensity of SB and IM in PMA-treated RAW264.7 cells. PMA-untreated (a,
f) and PMA-treated RAW 264.7 cells (b-e, g-j) were cultured for 2 h in the presence of 0.1 × 10−3 M ABAH (c, h), 5 × 10−3 M GSH (d, i), and 5 × 10−3 M taurine (e,
j); this was followed by the addition of SB or IM. Bright-field images (a-e) and fluorescence images (f-j) of PMA-treated cells after incubation with 5 × 10−6 M SB or
IM.
orbital (LUMO), charge density was localized on the nitrophenyl group
(Fig. 6). Additionally, a decrease in molecular frontier orbitals energy
supported the observed bathochromic effect in the UV–visible spectra of
oxidized probes (SB′ and IM′).
assigned to [SB′ + H]+ [54]. While in case of IM, the addition of ClO−
,
leads to the deprotonation of the acidic protons (NH proton of the
benzimidazole ring and OH proton of the salicylal group) accompanied
by instant color change from yellow to purple and generates IM′. The
formation of IM′ has been supported by 1H NMR titrations (Fig. S25†)
and its mass spectrum (Fig. S26†) which showed a peak at m/
z = 325.1256 assigned to [IM′ + H]−. Thus, the two distinct classes of
compounds (SB and IM) discussed here trace two different pathways for
sensing hypochlorite i.e. oxidation and deprotonation respectively. The
bathochromic shift that occurred in the absorbance spectra of both
probes indicates intramolecular charge transfer (ICT) from the electron-
rich center (diethylamino moiety) to the electron-deficient center (nitro
group). Thus, based on above evidences the proposed sensing me-
chanism of probe SB and IM is shown in Fig. 7.
3.9. Proposed Mechanism
To investigate the sensing mechanism of ClO− interacting sepa-
rately with SB [53] and IM, 1H NMR titrations, ESI-mass analysis and
theoretical calculations were carried out. The 1H NMR titrations of SB
with incremental addition of ClO− has been carried out as depicted in
Fig. S22†). The phenolic OH proton of SB disappeared upon the addi-
tion of ClO−, while NH2 protons shifted from their original peak ob-
served at δ 7.41 ppm to that at δ 7.37 ppm. The phenyl protons shifted
upfield due to delocalization of negative charge throughout the organic
framework of SB. These observations suggest that ClO− oxidizes the
hydroxyl proton generating fluorogenic oxidized SB (SB′), as shown in
Fig. 7a. Further the change in chemical shift values of SB after addition
of excess of ClO− showing the emergence of a new proton signal at δ
2.92 ppm has been depicted in Fig. S23†. The mass spectrum of SB′,
shown in Fig. S24†, showed a peak at m/z = 329.1595, which was
3.10. Ready-to-Use Paper Strips
To show how SB and IM probes can be used on test-paper strips for
the detection of ClO−
, we used rectangular small paper strips
(Whatman 1440–125 Ashless Quantitative Filter Paper, 8 Micron,
Grade 40). These paper strips were dipped into a solution containing
8

R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 11. Effects of myeloperoxidase inhibitor and hypochlorite scavengers on fluorescence intensity of SB and IM in LPS-treated RAW264.7 cells. LPS-untreated (a, f)
and LPS-treated RAW 264.7 cells (b-e, g-j) were cultured for 2 h in the presence of 0.1 × 10−3 M ABAH (c, h), 5 × 10−3 M GSH (d, i), and 5 × 10−3 M taurine (e, j);
this was followed by the addition of SB or IM. Bright-field images (a-e) and fluorescence images (f-j) of LPS-treated cells after incubation with 5 × 10−6 M SB or IM.
each respective probe (1.0 × 10−3 M) in aqueous DMSO (1: 9, v/v,
HEPES buffer, 1 × 10−3 M, pH 7.4). The dried test-paper strips were
then dipped into a solution of ClO− (1 × 10−5 M in water) for 5 min,
and air dried, and then visualized under daylight and UV light
(365 nm). The color changes observed using these strips were similar to
those obtained using probe solutions. This indicates that the SB and IM
probes can be used on a solid cellulose surface to detect ClO− (Fig. 8).
increased cytoplasmic volume in RAW264.7 cells. In contrast, LPS in-
duced morphological changes in RAW264.7 cells, leading to increasing
polarization of these macrophages from an undifferentiated rounded
shape to an activated dendritic shape.
In PMA-treated cells, fluorescence signals emitted by SB and IM
were observed diffusely throughout the cytoplasm (Fig. 9). Conversely,
in LPS-treated cells, fluorescence signals emitted by SB and IM were
observed as granule-like structures with uniform reduced fluorescence
in the cytoplasm of the cells (Fig. 9). Previous studies [58–60] have
shown that granule-like structures found in the cytoplasm of the cells
are endosomes formed by stimulation with LPS. No significant fluor-
escence signals from SB or IM were detected in the nuclei of PMA- or
LPS-treated RAW264.7 cells.
3.11. Imaging of Endogenously Produced Hypochlorite in PMA- and LPS-
Treated RAW264.7 Cells
Macrophages stimulated with PMA and LPS increase their produc-
tion of reactive oxygen species including that of ClO−; however, the
mechanisms of hypochlorite generation, stimulated by treatment with
PMA or LPS, are different [55–58]. PMA activates NADPH oxidase and
generate the production of ClO− coupled with that of ROS [59]. LPS
generates the production of ClO− via MPO, other inducible gene pro-
ducts, and morphological alterations [60].
To assess the specificity of SB and IM probes in living cells, we in-
vestigated the effects of a myeloperoxidase inhibitor and hypochlorite
scavengers in PMA- and LPS-treated RAW264.7 cells. As shown in Fig. 10
(left), the cells untreated with PMA showed barely detectable back-
ground-fluorescence signals. Pretreating PMA-stimulated RAW264.7
cells with ABAH (which inhibits production of hypochlorous acid by
myeloperoxidase) significantly decreased the fluorescent signal emitted
by the SB probe compared with that in corresponding PMA-stimulated
control RAW264.7 cells not treated with the inhibitor. Treating PMA-
To investigate whether our hypochlorite-sensing probes SB and IM
are usable in living cells, we imaged fluorescence triggered by the
generation of ClO− in RAW264.7 cells stimulated with PMA or LPS.
As shown in Fig. S27†, PMA induced squamous morphology with
9

R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
Fig. 12. Fluorescence images of hypochlorite-sensing probes in LPS-treated RAW264.7 cells phagocytosing non-fluorescent polystyrene beads. LPS-treated
RAW264.7 cells were allowed to phagocytose polystyrene beads 1 μm in diameter for 30 min. The cells were then washed with PBS and incubated with 5 × 10−6
M
SB or IM. Bright-field images (a, c); fluorescence images of RAW264.7 cells incubated with SB (left) and IM (right) (b, d). Arrowheads in (a-d) indicate: beads located
outside of the cells; arrows in (c, d) indicate: beads located inside of the cells. Dashed line in (c, d) indicates locale of the cell membrane. Bars: 10 μm (a, b), 1 μm (c,
d).
stimulated RAW264.7 cells with GSH (a scavenger of active oxygen
species including hypochlorous acid) or taurine (a hypochlorite sca-
venger) strongly inhibited the fluorescence signal generated by SB.
However, ABAH, GSH, and taurine were less effective in reducing
fluorescence emission from the IM probe in PMA-stimulated cells com-
pared with their effects on the SB probe in PMA-stimulated cells (Fig. 10,
right).
In LPS-treated cells, the fluorescence signals emitted by SB, ob-
served throughout the cytoplasm and as granule-like cellular structures
(endosomes), were quenched by the addition of ABAH, GSH, and
taurine (Fig. 11, left). With the IM probe, however, the inhibitory ef-
fects of ABAH, GSH, and taurine were restricted to the fluorescence
signals detected as granule-like structures. Our results indicate that
ABAH, GSH, and taurine did not inhibit the fluorescence signal
10

R. Yadav, et al.
JournalofPhotochemistry&Photobiology,B:Biology198(2019)111594
intensity of IM, which was observed throughout the cytoplasm (Fig. 11,
right).
Appendix A. Supplementary data
Our results show that SB is a fluorescent probe that is capable of
visualizing hypochlorite endogenously generated by MPO in PMA- and
LPS-treated RAW264.7 cells. IM is also suitable for the detection of
hypochlorite generated in the endosomes formed by treatment with
LPS. However, IM fluorescence, observed throughout the cytoplasm,
was retained in PMA- and LPS-treated cells even after the addition of
ABAH, GSH, and taurine. The mechanisms involved in the generation of
IM fluorescence, observed in the cytoplasm of RAW264.7 cells, are still
unclear; these mechanisms may be part of the sensing profile of the IM
probe towards different ROS and anions. As shown in Fig. S15†, the IM
probe showed relatively high background fluorescence and a notice-
able, but not strong, fluorescent signal in the presence of peroxynitrate,
hydroxyl radicals, and acetate. LPS can stimulate the production of
various reactive chemical species such as NO•, O2•−, ONOO−, •OH, and
HOBr (Fig. S28†) [61]. The IM probe may have been emitting fluor-
escence via reaction with certain reactive chemical species such as
hydroxyl radicals; such species are generated by various mechanisms,
including the Fenton reaction, and are not completely inhibited by GSH
[62] and taurine [63].
Supplementary data CCDC reference no. 1482721 and 1583949
contains the supplementary crystallographic data for SB and
IM respectively. This data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK, fax: (+44) 1223–336-033, Supplementary data to this
article can be found online at https://doi.org/10.1016/j.jphotobiol.
2019.111594.
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In summary, we have developed the two probes, SB and IM, for
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