Substituent effect on fluorescence signaling of the cell permeable HSO 4- receptors through single point to ratiometric response in green solvent

Article abstract of DOI:10.1039/c4ra03551h

Two new 2-(2-aminophenyl)benzimidazole-based HSO₄^- ion selective receptors, 6-(4-nitrophenyl)-5,6-dihydrobenzo[4,5]imidazo[1,2-c] quinazoline (L₁H) and 6-(4-methoxyphenyl)-5,6-dihydrobenzo[4,5] imidazo[1,2-c]quinazoline (L₂H), and their 1:1 molecular complexes with HSO₄^- were prepared in a facile synthetic method and characterized by physicochemical and spectroscopic techniques along with the detailed structural analysis of L₁H by single crystal X-ray crystallography. Both receptors (L₁H and L₂H) behave as highly selective chemosensor for HSO₄^- ions at biological pH in ethanol-water HEPES buffer (1/5) (v/v) medium over other anions such as F^-, CI^-, Br^-, I^-, AcO^-, H₂PO₄^-, N₃^- and ClO ₄^-. Theoretical and experimental studies showed that the emission efficiency of the receptors (L₁H and L₂H) was tuned successfully through single point to ratiometric detection by employing the substituent effects. Using 3σ method the LOD for HSO₄ ^- ions were found to be 18.08 nM and 14.11 nM for L₁H and L₂H, respectively, within a very short responsive time (15-20 s) in 100 mM HEPES buffer (ethanol-water: 1/5, v/v). Comparison of the utility of the probes (L₁H and L₂H) as biomarkers for the detection of intracellular HSO₄^- ions concentrations under a fluorescence microscope has also been included and both probes showed no cytotoxic effect. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra03551h

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PAPER
Substituent effect onꢀfluorescence signaling of the
cell permeable HSO4 receptors through single
point to ratiometric response in green solvent†
Cite this: RSC Adv., 2014, 4, 27665
Manjira Mukherjee,a Siddhartha Pal,a Buddhadeb Sen,a Somenath Lohar,a
b
c
a
*
Samya Banerjee, Snehasis Banerjee and Pabitra Chattopadhyay
ꢀ
Two new 2-(2-aminophenyl)benzimidazole-based HSO4 ion selective receptors, 6-(4-nitrophenyl)-5,6-
dihydrobenzo[4,5]imidazo[1,2-c]quinazoline (L1H) and 6-(4-methoxyphenyl)-5,6-dihydrobenzo[4,5]
ꢀ
imidazo[1,2-c]quinazoline (L2H), and their 1 : 1 molecular complexes with HSO4 were prepared in a
facile synthetic method and characterized by physicochemical and spectroscopic techniques along with
the detailed structural analysis of L1H by single crystal X-ray crystallography. Both receptors (L1H and
ꢀ
L2H) behave as highly selective chemosensor for HSO4 ions at biological pH in ethanol–water HEPES
ꢀ
ꢀ
buffer (1/5) (v/v) medium over other anions such as Fꢀ, CIꢀ, Brꢀ, Iꢀ, AcOꢀ, H2PO4ꢀ, N3 and ClO4
.
Theoretical and experimental studies showed that the emission efficiency of the receptors (L1H and L2H)
was tuned successfully through single point to ratiometric detection by employing the substituent
ꢀ
effects. Using 3s method the LOD for HSO4 ions were found to be 18.08 nM and 14.11 nM for L1H and
Received 18th April 2014
Accepted 21st May 2014
L2H, respectively, within a very short responsive time (15–20 s) in 100 mM HEPES buffer (ethanol–water:
1/5, v/v). Comparison of the utility of the probes (L1H and L2H) as biomarkers for the detection of
DOI: 10.1039/c4ra03551h
ꢀ
intracellular HSO4 ions concentrations under a fluorescence microscope has also been included and
www.rsc.org/advances
both probes showed no cytotoxic effect.
ꢀ
HSO4 have been reported.6 Thus, the design of anion sensors
Introduction
for the hydrogen sulfate ion is important and desirable.
Sensors based on anion-induced changes in uorescence are
particularly attractive due to their simplicity, high degree of
specicity and low detection limits.7 However, from the exper-
imental point of view, it is well known that the ratiometric
responses are more attractive because the ratio between the two
emission intensities can be used to measure the analyte
concentration and to provide a built-in correction for environ-
mental effects and stability under illumination.8 Although there
are some reports on single point sensor and ratiometric
response, there is no report on the tuning of single point to
ratiometric response while keeping the same receptor envi-
ronment, except for the change in electronic effect, using
substituents.
The design and development of selective receptors for the
anionic analytes have gained considerable attention in recent
years because of the biological signicance, potential applica-
tions in sensors and for the development of phase transfer
reagents.1,2 Critical physiological processes are being operated
through negative ion gradients across lipid bilayer membranes
maintained by anion channels.3 The malfunction of this
process leads to severe diseases such as cystic brosis, neph-
rolithiasis, osteopetrosis, Angelman syndrome and Bartter's
syndrome type III.4 Among the various anions, hydrogen sulfate
(HSO4ꢀ) ions dissociate at high pH to generate toxic sulfate
(SO42ꢀ), which causes irritation of the skin and eyes, and even
respiratory paralysis.5 Despite its crucial roles in biological
processes, only few examples of cell permeable sensors for
ꢀ
Herein, two newly designed efficient HSO4 ion-selective
receptors, 6-(4-nitrophenyl)-5,6-dihydrobenzo[4,5]imidazo[1,2-
c]quinazoline (L1H) and 6-(4-methoxyphenyl)-5,6-dihydrobenzo-
[4,5]imidazo[1,2-c]quinazoline (L2H) from 2-(2-aminophenyl)-
benzimidazol (viz. Scheme 1) while keeping same receptor
environments for the guest (HSO4ꢀ ions) have been employed to
tune the emission efficiency of these new receptors (L1H and
L2H) through single point to ratiometric detection in a green
solvent by exploiting the effects of the substituents within the
receptors. The organic moieties (L1H and L2H) and the resulting
compounds (K[L1H–HSO4] and K[L2H–HSO4]) have been
aDepartment of Chemistry, Burdwan University, Golapbag, Burdwan-713104, West
Bengal, India. E-mail: pabitracc@yahoo.com
bDepartment of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore, 560012, India
cGovt. College of Engineering and Leather Technology, Salt Lake Sector-III, Kolkata 9,
India
† Electronic supplementary information (ESI) available. CCDC 968396. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ra03551h
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(2.09 g, 10.0 mmol) and 4-nitrobenzaldehyde (1.51 g, 10.0 mmol)
(for L1H) or 4-methoxybenzaldehyde (1.36 g, 10.0 mmol) (for L2H)
were mixed in dry ethanol (25.0 mL) at room temperature. Then,
the reaction mixture was reuxed for 6.0 h. The yellow (L1H) or
brown (L2H) precipitate of the compounds were obtained from the
solution through slow evaporation of the solvent. The two pure
recrystallized compounds were then isolated from methanol.
L1H$C20H14N4O2. Anal. found: C, 70.49; H, 4.24; N, 16.51;
calc.: C, 70.17; H, 4.12; N, 16.37. IR (cmꢀ1): nNH ¼ 3190.26, nC]N
¼ 1612.49; ESI-MS: [M + H]+, m/z, 343.1480 (100%) (calcd: m/z,
342.11; where M ¼ molecular weight of [L1H]); 1H NMR (d, ppm
in DMSO-d6): 8.201 (d–d, 2H, J1 ¼ 7, J2 ¼ 2); 7.986 (d–d, 1H, J1 ¼
7.75, J2 ¼ 1.5); 7.8 (d, 1H, J ¼ 2.5); 7.7 (d, 1H, J ¼ 8); 7.448 (d–d,
2H, J1 ¼ 7, J2 ¼ 2); 7.361–7.337 (m, 2H); 7.281–7.165 (m, 3H);
6.884–6.856 (m, 2H). Yield: 90%.
L2H$C21H17N3O. Anal. found: C, 76.81; H, 5.15; N, 13.07;
calc.: C, 77.03; H, 5.24; N, 12.84. ESI-MS: [M + H]+, m/z, 328.1246
(100%) (calcd: m/z, 328.14; where M ¼ molecular weight of
[L2H]); IR (cmꢀ1): nNH ¼ 3209.6, nC]N ¼ 1608.63 1H NMR (d,
ppm in DMSO-d6): 7.96 (d–d, 1H, J1 ¼ 7.6, J2 ¼ 2); 7.651 (d, 1H, J
¼ 8); 7.307–7.077 (m, 7H); 6.866–6.734 (m, 3H); 6.604 (d–d, 1H,
J1 ¼ 7.6, J2 ¼ 2); 4.401 (s, 1H); 3.601 (s, 1H). Yield: 90%.
Scheme 1 Schematic representation of the synthesis of the probes
L1H and L2H, and their corresponding complexes.
characterized by physicochemical and spectroscopic techniques
along with crystallographic analysis of L1H by single crystal
X-ray diffraction. Both L1H and L2H behave as highly selective
ꢀ
uorescent and colorimetric sensors for HSO4 ions at biolog-
ical pH in ethanol–water HEPES buffer (1/5; v/v) compared to
other anions such as Fꢀ, CIꢀ, Brꢀ, Iꢀ, AcOꢀ, H2PO4ꢀ, N3 and
ꢀ
Preparation of compounds K[L1H–HSO4] and K[L2H–HSO4]
ClO4ꢀ. The receptor L1H behaves as a single point uorosensor, The preparation of solid complexes was carried out following a
whereas the receptor L2H behaves as a ratiometric uorosensor common procedure. To a methanolic solution of L1H (342 mg,
in identical conditions. Both probes (L1H and L2H) were also 1.0 mmol) or L2H (327 mg, 1.0 mmol) (for K[L1H–HSO4] or for K
employed to detect the presence of intracellular bisulphate ions
by acquiring the images of HeLa cells under a uorescence
microscope. Comparison of these acquired images showed that
[L2H–HSO4]), solid potassium hydrogen sulphate (136 mg, 1.0
mmol) was added and the reaction mixture was stirred at
ambient temperature for 6.0 h. The solution thus obtained was
the image through ratiometric signaling using L2H is the better then kept aside for slow evaporation at room temperature. Aer
one for cell staining, although both probes have no cytotoxic a few days, deep yellow crystalline complexes were collected by
effect.
washing with water and methanol, and then dried in vacuo.
K[L1H–HSO4]$C20H15KN4O6S. Anal. found: C, 50.04; H, 3.25;
N, 11.91; calc.: C, 50.19; H, 3.16; N, 11.71. ESI-MS in methanol:
[I2ꢀ + 2H + Na]+, m/z, 463.16 (obsd with 6% abundance) (calcd:
m/z, 463.06); [I2ꢀ + K + Na + H]+, m/z, 505.0080 (obsd with 12%
abundance) (calcd: m/z, 505.06); where I2ꢀ ¼ L1 + HSO4ꢀ. IR
(cmꢀ1): nS]O ¼ 1114.86; 1H NMR (d, ppm in DMSO-d6): 8.201
(d–d, 2H, J1 ¼ 7, J2 ¼ 2); 8.0 (d–d, 1H, J1 ¼ 7.75, J2 ¼ 1.5); 7.82 (d,
1H, J ¼ 2.5); 7.72 (d, 1H, J ¼ 7.5); 7.480–7.458 (m, 2H); 7.39–
7.175 (m, 5H); 6.906–6.875 (m, 2H). Yield: 75%.
K[L2H–HSO4]$C21H18KN3O5S. Anal. found: C, 54.15; H, 3.99;
N, 9.25; calc.: C, 54.41; H, 3.92; N, 9.07. ESI-MS in methanol: [I2ꢀ
+ 2H + Na]+, m/z, 447.92 (obsd with 11% abundance) (calcd: m/z,
448.089); [I2ꢀ + K + Na + H]+, m/z, 486.2086 (obsd with 35%
abundance) (calcd: m/z, 486.09; where I2ꢀ ¼ L2 + HSO4ꢀ; IR
(cmꢀ1)): nS]O ¼ 1111.00, 1H NMR (d, ppm in DMSO-d6): 7.96 (d–
d, 1H, J1 ¼ 7.6, J2 ¼ 2); 7.645 (d, 1H, J ¼ 8); 7.306–7.077 (m, 7H);
6.867–6.731 (m, 3H); 6.65 (d–d, 1H, J1 ¼ 7.6, J2 ¼ 1.6); 4.401
(s, 1H); 3.601 (s, 1H). Yield: 75%.
Experimental section
Physical measurements
The uorescence property of the sensor was investigated in
water–ethanol (5 : 1, v/v) solvent. The pH study was performed
in 100 mM HEPES buffer solution by adjusting pH with HCl or
NaOH. The stock solutions (ꢁ10ꢀ2 M) for the selectivity study of
the receptors (L1H and L2H) towards different anions were
prepared with sodium salt of azide, perchlorate, disodium
hydrogen arsenate; tetra butyl ammonium salt of chloride,
bromide, iodide, acetate, uoride; dihydrogen phosphate and
potassium hydrogen sulphate in water–ethanol (5 : 1, v/v)
solvent. In this selectivity study, the amount of these anions was
a hundred times greater than that of the receptor used. Fluo-
rescence titration was performed with potassium hydrogen
sulphate in water–ethanol (5 : 1, v/v) solvent varying the anion
concentration from 0 to 100 mM and concentration of the
receptors were xed at 25 mM throughout the experiment.
X-ray data collection and structural determination
Preparation of L1H and L2H
Single crystals suitable for single crystal X-ray crystallography
Preparation of two receptors, i.e., L1H and L2H, was carried out were obtained from the methanolic solution of L1H on slow
following a common procedure. 2-(2-Aminophenyl)-benzimidazole evaporation at room temperature. X-ray single crystal data were
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˚
collected using Mo Ka (l ¼ 0.7107 A) radiation on a SMART
APEX II diffractometer equipped with a CCD area detector. Data
collection, data reduction and structure solution/renement
were carried out using the soware package of SMART APEX II.
The crystallographic data, selected bond lengths and bond
angles are tabulated in Tables 1 and 2. A total of 26 495 reec-
tions were measured, out of which 3542 were independent, and
3202 were observed [I > 2s(I)]. The structure was solved by direct
methods using SHELXS-97,9 and rened by full-matrix least-
squares renement methods based on F2 using SHELXL-97. All
non-hydrogen atoms were rened anisotropically, and all
calculations were performed using WingX Package.10 The
important crystal and renement parameters are given in Table
1. The resulting crystals were found suitable for structural
studies.
Cell preparation and in vitro cellular imaging
Human cervical cancer cells, i.e., HeLa cell lines, were used
throughout the study. Cells were cultured in Dulbecco's modi-
ed Eagle's medium (DMEM, Gibco BRL) supplemented with
10% FBS (Gibco BRL) and 1% antibiotic mixture containing
penicillin, streptomycin and neomycin (PSN, Gibco BRL) at 37 ꢂC
in a humidied incubator with 5% CO2. For the experimental
study, cells were grown to 80–90% conuence, harvested with
0.025% trypsin (Gibco BRL) and 0.52 mM EDTA (Gibco BRL) in
PBS (phosphate-buffered saline, Sigma Diagnostics) and then
plated at the desire cell concentration and allowed to re-equili-
brate for 24 h before any treatment. Cells were rinsed with PBS
and incubated with DMEM-containing L1H or L2H (10 mM, 1%
ꢂ
DMSO) for 15 min at 37 C. All experiments were conducted in
DMEM containing 10% FBS and 1% PSN antibiotic mixture. The
imaging system was composed of a uorescence microscope
(ZEISS Axioskop 2 plus) with an objective lens [10ꢃ].
Table 1 Crystal data and refinement details for L1H
Empirical Formula
Formula weight
Crystal system
Space group
C20H15N4O2
Cell cytotoxicity assay
343.36
Orthorhombic
Pca21
12.0792(5)
9.1171(4)
15.1341(7)
90ꢂ
1666.68(13)
296(2)
4
1.368
0.092
716
2.80–26.78
26495
To test the cytotoxicity of L1H and L2H, MTT [3-(4,5-dime-
thylthiazol-2-yl)-2,S-diphenyl tetrazolium bromide] assay was
performed as per a previously reported procedure.11 Aer
treatments of the probe (5, 10, 25, 50, and 100 mM), 10 ml of MTT
solution (10 mg mLꢀ1 PBS) was added in each well of a 96-well
culture plate, and incubated at 37 ꢂC for 8 h. All of the mediums
were removed from the wells and replaced with 100 ml of acidic
isopropanol. The intracellular formazan crystals (blue-violet)
formed were solubilized with 0.04 N acidic isopropanol, and the
absorbance of the solution was measured at 595 nm with a
microplate reader. The data obtained are shown as means ꢄ
S.D. of three independent experiments. The cell cytotoxicity was
calculated based on a cell viability of 100%.
˚
a (A)
˚
b (A)
˚
c (A)
a ¼ b ¼ g
3
˚
Volume (A )
Temperature (K)
Z
rcalc (g cmꢀ3
)
m (mmꢀ1
F(000)
)
q range (deg)
Reections collected
Reections independent
Final R indices [I > 2s(I)]
R indices (all data)
Goodness-of-t on F2
3542
3202
0.0302
1.033
Theoretical calculation
The gradient-corrected DFT level, involving the hybrid 3-
parameter t of exchange and correlation functionals of Becke
(B3LYP), which includes the correlation functional of Lee, Yang,
and Parr (LYP), was used. The standard split valence basis sets
6-31G(d) and 6-31G(d) were applied for other atoms. Natural
population analysis (NPA) (implemented in Gaussian 09
program) at B3LYP/6-31G(d) level was carried out to compute
the charge on each atom.
Table 2 Selected bond distances (A˚) and bond angles (ꢂ) for L1H
˚
Bond length (A)
C22–N3
C23–N3
C23–N2
N2–C11
N2–C4
1.389(2)
1.3220(18)
1.3697(19)
1.386(2)
1.4570(17)
1.471(2)
C7–N1
Results and discussion
Bond angles (ꢂ)
Synthesis and characterization
N3–C23–N2
N3–C23–C3
N2–C23–C3
C23–N2–C11
C23–N2–C4
C11–N2–C4
N3–C22–C12
N3–C22–C11
N4–C4–N2
112.68(13)
128.21(13)
119.10(12)
107.34(11)
126.13(13)
126.27(13)
130.12(14)
110.30(13)
108.20(12)
112.33(12)
111.87(11)
The organic moieties (L1H and L2H) were synthesized by
condensing an ethanolic solution of 2-(2-aminophenyl)-benz-
imidazole with benzaldehyde derivatives (for L1H, 4-nitro-
benzaldehyde and for L2H, 4-methoxybenzaldehyde) in 1 : 1
mole ratio (Scheme 1). They were characterized by physico-
chemical and spectroscopic techniques. In addition, the solid
state structure of L1H was conrmed by single crystal X-ray
crystallography aer collecting the single crystals of L1H from
the methanolic solution. The molecular view of L1H with atom
labeling scheme is shown in Fig. 1, which shows that L1H
N4–C4–C21
N2–C4–C21
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crystallizes in the orthorhombic space group Pca21. The crys-
tallographic data and bond parameters are tabulated in Tables 1
˚
and 2. The bond distance of C23–N2 (1.3697 A) is longer than
˚
that of C23–N3 (1.3220 A); however, both these values are
˚
signicantly shorter than that of C4–N2 (1.4570 A) or C4–N4
˚
(1.4530 A).
1
The peaks obtained in H NMR spectrum of L1H and L2H
have been identied, and they are in accordance with the
structural formula of L1H and L2H in the solution state (Fig. S1
and S2†). The ESI mass spectrum of the compound L1H in
methanol shows a peak at m/z 443.1480 with 100% abundance,
which is assignable to [M + H]+ (calculated value at m/z, 443.12),
where M is the molecular weight of L1H (Fig. S3†). The ESI mass
spectrum of the compound L2H in methanol shows a peak at
m/z 328.1246 with 100% abundance, which is assignable to [M +
H]+ (calculated value at m/z, 328.14), where M is the molecular
weight of L2H (Fig. S4,†). The IR spectra of L1H and L2H show
the characteristic stretching of the N–H and C]N bonds
(Fig. S5 and S6†). L1H and L2H undergo non-covalent hydrogen
Scheme 2 Schematic representation of the plausible mechanism of
hydrogen sulfate sensing.
ꢀ
bonding interaction with HSO4 ions, which results in
enhancement of the uorescence intensity (Scheme 2). To
establish the mechanism of the formation of the adduct with
ꢀ
HSO4 ions, the species, formulated as K[L1H–HSO4] and K
[L2H–HSO4], were isolated in solid state from the reaction of one
mole potassium hydrogen sulphate with one mole of the
organic moiety (L1H or L2H) in methanol under stirring
condition. The complexes are soluble in methanol, DMSO, and
acetonitrile. The peaks obtained in 1H NMR spectrum of K[L1H–
HSO4] and K[L2H–HSO4] have been identied, and they are in
accordance with the structural formula of L1H and L2H in the
solution state (Fig. S7 and S8†). The ESI mass spectrum of the
compound K[L1H–HSO4] in methanol shows a peak at m/z
463.16 and 505.0080 (Fig. S9†), which is assignable to [I2ꢀ + 2H +
ꢀ
Na]+ and [I2ꢀ + K + Na + H]+, respectively, where I2ꢀ ¼ L1
+
HSO4ꢀ. The ESI mass spectrum of the K[L2H–HSO4] in meth-
anol shows a peak at m/z, 447.9254 and 486.2086 (Fig. S10†),
which is assignable to [I2 + 2H + Na]+ and [I2ꢀ + K + Na + H]+,
respectively, where I2ꢀ ¼ L2ꢀ + HSO4ꢀ. The IR spectra of K[L1H–
HSO4] and K[L2H–HSO4] show the characteristic stretching of
S]O at 1114 and 1111 cmꢀ1, respectively (Fig. S11 and S12†). All
Fig. 2 (a) Fluorescence spectra of L1H (25 mM) as a function of
externally added HSO4ꢀ [0–30 mM] in ethanol–water (1 : 5, v/v) HEPES
buffer (0.1 M, pH ¼ 7.4) at 25 ꢂC [lem ¼ 485 nm, lex ¼ 400 nm]. (b)
Fluorescence spectra of L2H (25 mM) as a function of externally added
ꢀ
HSO4 [0–30 mM] in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M,
pH ¼ 7.4) at 25 ꢂC [lem: 485 nm, lex ¼ 390 nm].
Fig. 1 Molecular view of L1H with atom numbering scheme.
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these data conrm the composition of K[L1H–HSO4] and K
[L2H–HSO4].
ODstandard ꢃ Asample
ODsample ꢃ Astandard
fsample
¼
ꢃ fsample
where A is the area under the uorescence spectral curve and
OD is the optical density of the compound at the excitation
wavelength. The standard used for the measurement of uo-
rescence quantum yield was anthracene (f ¼ 0.29 in ethanol).
The emission intensities of the organicꢀmolecule in the pres-
ence of various concentrations of HSO4 ions were measured.
The uorescence spectrum properties of L1H (25 mM) and L2H
1H NMR titration
ꢀ
In order to strengthen the above pathway of bonding of HSO4
ions with the receptors, 1H NMR titration was performed by
concomitant addition of HSO4ꢀ ions to the DMSO-d6 solution of
L1H and L2H (Fig. S13 and S14, ESI†). Signicant spectral
ꢀ
changes for L1H and L2H were observed upon addition of HSO4
ions. In case of L1H, 5 min aer the addition of HSO4ꢀ ions, the
peaks due to the proton in N–Hi appeared with the peak of Hh
proton at d ¼ 6.884–6.856 ppm (2H, m). It is noteworthy that they
were remarkably affected and became equivalent to one
hydrogen of Hh due to the disappearance of Hi from N–Hi.
Moreover, the multiplet peaks appeared at d ¼ 7.361–7.337 ppm
(2H, m), which is assignable to split up of He and He' protons, aꢀs
the peak for He shied downeld due to the bonding of HSO4
ions with He. In case of L2H titration, the peak at 4.401 ppm, due
(25 mM) were investigated in ethanol–water (1 : 5, v/v) HEPES
ꢀ
buffer (0.1 M, pH ¼ 7.4) at 25 ꢂC as a function of added [HSO4
]
(Fig. 2a and b). L2H showed a uorꢀescence of higher intensity at
430 nm. Aer addition of HSO4 the uorescence intensity
band shows a ratiometric enhancement at 485 nm (Fig. 2b). The
ratiometric signaling of uorescence output at two different
ꢀ
wavelengths plotted as a function of concentration of HSO4
indicates that the uorescence intensity ratio at wavelengths
485 nm and 430 nm (I485/I430) gꢀradually increases with increase
in the concentration of HSO4 ions (Fig. S17†), and aer a
certain time it levels up to produce a sigmoidal curve. Note that
ꢀ
to the proton of N–H, disappeared aer HSO4 addition. All
other protons of L1H and L2H remained unaffected aer inter-
ꢀ
action with HSO4 ions (Tables S1 and S2, ESI†).
Spectral characteristics
Emission study. On excitation of L1H at 400 nm and L2H at
390 nm, a weak emission at 485 nm in both was observed in
water–ethanol (5 : 1) solvent mixture (Fig. S15 and S16†). Fluo-
rescence quantum yields (F) were estimated by integrating the
area under the uorescence curves with the equation:
Fig. 4 (a) Binding constant (K) value of 3.25 ꢃ 105 Mꢀ1 for L1H
determined from the intercept/slope of the plots. (b) Binding constant
(K) value of 1.48 ꢃ 105 Mꢀ1 for L2H determined from the intercept/
Fig. 3 (a) Job's plot of L1H and (b) L2H showing maxima at 1:1 molar ratio. slope of the plots.
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the uorescence emission band of L1H at 485 nm is very weak at of chloride, bromide, iodide and acetate; sodium salt of azide,
room temperature. Addition of HSO4ꢀ (25 mM) to L1H (25 mM) in sulphide, and cyanide; dihydrogen phosphate; dihydrogen
HEPES buffer solution at pH 7.4 afforded a 120 times single arsenate; and potassium salt of nitrate and sulphate. Job's plot
point enhancement in uorescence intensity (Fig. 2a). In the analysis (Fig. 3a and b) revealed that L1H and L2H both bonded
ꢀ
ꢀ
absence of HSO4 anion, the uorescence intensity of L1H is with HSO4 ions to form the adducts in 1 : 1 molar ratio. The
very low. However, in presence of HSO4ꢀ, uorescence intensity binding constant values, calculated from the emission intensity
greatly increased due to bonding interaction between the data, were found to be 3.25 ꢃ 105 Mꢀ1/2 for L1H and 1.48 ꢃ 105
deprotonated Nꢀ atom (as pKa of NH z 6.9, resulting experi- for L2H (Fig. 4a and b) using the modied Benesi–Hildebrand
mental pH of the medium) of imidazole moiety with the proton equation:12,13
ꢀ
of the added HSO4 ion. This is also supported by the compu-
1/(Fx ꢀ F0) ¼ 1/(Fmax ꢀ F0) + (1/K[C])(1/(Fmax ꢀ F0)
tational study of the probes (Lꢀ1H and L2H) and their corre-
sponding adducts with HSO4 (Fig. S18 and S19†). Thꢀe
where F0, Fx, and FN are the emission intꢀensities of organic
computational study indicates that the closeness of the HSO4
moiety considered in the absence of HSO4 ions, at an inter-
ion with L1H is to a great extent comparable to that of the
ꢀ
mediate HSO4 concentration, and at a concentration causing
HSO4ꢀ ioꢀn with L2H as the theoretical bond distances of CH/O
ꢀ
complete interaction, respectively; K is the association constant
and [C] is [HSO4ꢀ]. The average uorescence lifetime
measurements of L1H and L2H, in presence and absence of
˚
˚
(of HSO4 ) (2.258 A) and N/H (of HSO4 ) (1.898 A) in [L1H–
ꢀ
HSO4]ꢀ adducts are shorter than those of CH/O (of HSO4
)
(2.371 A) and N/H (of HSO4) (1.958 A) in [L2H–HSO4]ꢀ adducts.
The electronic effect of the substituents (–NO2 in L1H and –OMe
in L2H) present in the para-position of the phenyl ring plays the
key role in the tuning of the uorescence signaling from single
point response to ratiometric response.
˚
˚
ꢀ
HSO4 ion in water–ethanol (5 : 1) medium indicates the
gradual increase with increase in [HSO4ꢀ] (Fig. 5a and b). The
average lifetimes are calculated to be 8.32 ns for L1H, 9.42 ns for
the mixture of L1H : HSO4ꢀ (1 : 0.5) and 10.75 ns for the mixture
of L1H : HSO4ꢀ in 1 : 1 molar ratio. The average lifetime for L2H
ꢀ
There was almost no interference for the detection of HSO4
ꢀ
is 8.26 ns for L2H : HSO4 (1 : 0.5) is 8.58 ns and in case of
in the presence of 100 equivalents of tetrabutylammonium salt
Fig. 6 (a) Changes in the absoꢀrption spectrum of L1H (25 mM) upon
Fig. 5 (a) Time resolved fluorescence decay of L1H (10 mM) and (b) addition of 0–30 mM of HSO4 in ethanol–water (1 : 5, v/v) HEPES
ꢀ
L2H (10 mM) in the absence and presence of added HSO4 ions buffer (0.1 M, pH 7.4) at 25 ꢂC. (b) Changes in the absorption spectra of
ꢀ
(5 mM and 10 mM) in 100 mM HEPES buffer (ethanol/water: 1/5, v/v) L2H (25 mM) upon addition of 0–30 mM of HSO4 in ethanol–water
[lem: 485 nm].
(1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 25 ꢂC.
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L2H : HSO4 (1 : 1), is 11.79 ns. The strong binding of HSO4
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ꢀ
ꢀ
390 nm (Fig. 6b) through an isosbestic point at 365 nm due to
ions with organic moiety is also due to the binding constant the formation of an adduct of [L2H–HSO4]ꢀ in the solution
value. According to the equations: s ꢀ1 ¼ kr + knr and kr ¼ Ff/s,14 state.
the radiative rate constant kr and total non-radiative rate
Selectivity. The uorescence response of organic moiety
constant knr of the organic moieties (L1H, L2H), K[L1H–HSO4], towards the different anions were investigated with 100 times
and K[L2H–HSO4] were listed in Tables S3 and S4.† The data concentrations of Clꢀ, ꢀBrꢀ, Iꢀ, Fꢀ, CNꢀ, OAcꢀ, NO3ꢀ, S2ꢀ
,
suggest that the uorescent enhancement is ascribed to the SO42ꢀ, H2PO4ꢀ, H2AsO4 (Fig. S20 and S23†). This study indi-
ꢀ
decrease in ratio of knr/kr from 181.40 for L1H to 1.8 for K[L1H– cates that both L1H and L2H have excellent selectivity for HSO4
HSO4], and from 14.4 for L2H to 1.0839 for K[L2H–HSO4].
Absorption study. The UV-Vis spectrum of L1H showed the
ions over other anions.
Effect of pH. The uorescence intensity of the organic
characteristic absorption bands at ca. 226 nm, 262 nm, 290 nm, moieties L1H and L2H was measured at various pH values in
300 nm, and 346 nm, which are attributable to intramolecular HEPES buffer (0.1 M) at 25 ꢂC by adjusting the pH using HCl or
ꢀ
p–p* and n–p* trꢀansitions. During the titration, by adding the NaOH in the presence and absence of HSO4 ions (Fig. 7a and
solution of HSO4 ions to the colourless solution of L1H in b†). Here, we demonstrate that the uorescence intensity of
ethanol–water (1 : 5, v/v) in HEPES buffer (0.1 M, pH 7.4) at both organic compounds does not vary in the pH range of 5.0ꢀ–
ꢀ
25 ꢂC, the peak at 346 nm gradually decreased, and a new peak 12.0 in absence of HSO4 ions; however, in presence of HSO4
around 390 nm was generated through an isosbestic point at ions, both moieties exhibited a pH dependency in the uores-
364 nm (Fig. 6a) due to the formation of a complex of the cence intensity over the pH range 5.0 to 8.0. It is also noteworthy
receptor with HSO4ꢀ ion in the solution state. Similarly, UV-Vis that the uorescence intensity of the organic moiety in presence
spectrum of L2H showed the characteristic absorption bands at of HSO4ꢀ ions is higher than that in the absence of HSO4ꢀ ions
ꢀ
225 nm, 291 nm, and 350 nm. In a similar type of titration, by due to the formation of adducts of HSO4 ions with the
addition of the solution of HSO4ꢀ ion to the colourless solution deprotonated receptors (L1ꢀ and L2ꢀ; aer deprotonation of the
of L2H in ethanol–water (1 : 5, v/v) in HEPES buffer (0.1 M, pH
7.4) at 25 ꢂC, the peak at 350 nm was red-shied to a new peak at
Fig. 7 (a) Fluorescencꢀe response to pH of L1H (25 mM) in absence and
in presence of HSO4 (one equivalent) at different pH in 100 mM Fig. 8 (a) Calibration curve in the nanomolar range with error bars for
ꢀ
HEPES buffer (ethanol–water: 1/5) at 25 ꢂC. (b) Fluorescence response calculating the LOD of HSO4 by L1H in 100 mM HEPES buffer
ꢀ
to pH of L2H (25 mM) in the absence and presence of HSO4 (one (ethanol–water: 1 : 5) at 25 ꢂC. (b) Calibration curve in the nanomolar
equivalent) at different pH values in 100 mM HEPES buffer (ethanol– range with error bars for calculating the LOD of HSO4ꢀ by L2H in 100
water: 1 : 5) at 25 ꢂC.
mM HEPES buffer (ethanol–water: 1 : 5) at 25 ꢂC.
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nitrogen atom of –NH in the imidazole ring) through signicaꢀnt potentiality for both in vitro and in vivo application
H-bonding. At a higher pH range (pH 8.0–12.0), the gradual as HSO4 sensors as well as imaging in different ways as same
decrease in the uorescence intensity is due to the decreasing manner for live cell imaging can be followed instead of xed
ꢀ
formation probability of the adducts of HSO4 ions with the cells. In this study, it is also observed that the clarity of the
ꢀ
deprotonated receptors (L1 and L2ꢀ) as there is a tendency of image is signicantly better by employing L2H than L1H due to
HSO4ꢀ ions to be deprotonated. As a result of this observation, the ratiometric signaling.
both probes are very effective as sensors in analytical and bio-
analytical studies, which were carried out at biological pH 7.4 in
ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M) at 25 C.
Conclusion
ꢂ
Analytical gure of merit. To calculate the detection limit, In coꢀnclusion, two new 2-(2-aminophenyl)benzimidazol-based
calibration curves (Fig. 8a and b) in the lower concentration HSO4 ion-selective receptorsꢀ(L1H and L2H), and their 1 : 1
region (0–5 mM) were obtained. From the slope of the curve(s) molecular adducts with HSO4 were synthesized and charac-
and the standard deviation of seven replicate measurements of terized using physicochemical and spectroscopic techniques
the zero level (szero) the detection limit was estimated using the along with single crystal X-ray crystallography of L1H for detailed
equation, 3s/Sꢀ.15 From this study, the detection limit of L1H and structural analysis. Both receptors (L1H and L2H) behave as
ꢀ
L2H for HSO4 ions were calculated to be 18.8 nM and 14.11 highly selective uorescent sensor for HSO4 ions at biological
nM, respectively.
pH in green solvent than other anions by the naked eye. Here,
Cell imaging. To examine the utility of the probes in bio- the new receptors (L1H and L2H) have the same environments to
logical systems, they were applied to human cervical cancer accept the guest (HSO4ꢀ ions), but the emission efficiency of the
HeLa cell lines. Here, HSO4ꢀ ions and L1H or L2H were allowed receptors has been tuned successfully through single point to
to uptake by the cells of interest and the images of the cells were ratiometric detection in green solvent by exploiting substituent
recorded by uorescence microscopy aer excitation at ꢁ400 effects (ꢀR effect of –NO2 group, and +R effect of OMe group)
ꢀ
and 405 nm (Fig. 9). In addition, the in vitro study showed that within the receptors. The limit of detection for HSO4 ions (by
50 mM of L1H and L2H was not cytotoxic to the cell for up to 8.0 h 3s method) were calculated to be 18.08 nM and 14.11 nM for
(Fig. S24 and S25†). The results indicate that both probes have L1H and L2H, respectively. Both probes could be used as
biomarkers for the detection of intracellular HSO4ꢀ ions in HeLa
cells as they are both non-cytotoxic agents. However, L2H is a
better candidate compared to L1H for acquiring the uorescence
image (Fig. 9), although they have the same receptor environ-
ments for the HSO4ꢀ ions. From this study, it may be concluded
that substituents present in the appropriate position of the
receptor control the mode of signaling (ratiometric or single
point) of the sensor, and the ratiometric signaling is better than
the single point signaling as expected.
Acknowledgements
Financial assistance from CSIR, New Delhi, India is gratefully
acknowledged. M. Mukherjee wishes to thank to UGC, New
Delhi, India for offering the fellowship. The authors are grateful
to USIC, The University of Burdwan, for the single crystal X-ray
diffractometer facility under PURSE program. We sincerely
acknowledge Prof. Samita Basu and Mr Ajay Das, Chemical
Science Division, SINP, Kolkata for enabling TCSPC instrument
1
and Prof. B. Mukhopadhyay, IISER, Kolkata to record some H
NMR spectra and ESI Mass Spectra.
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