Development of a cell permeable ratiometric chemosensor and biomarker for hydrogen sulphate ions in aqueous solution

Article abstract of DOI:10.1039/c4ra00291a

A newly designed organic moiety, 5H-5,7a,12-triaza-dibenzo[a,e]azulen-6-one (L) containing a seven membered ring behaves as a hydrogen sulphate ion selective ratiometric chemosensor. The formulation and detailed structural characterisation of L have been

Full text of DOI:10.1039/c4ra00291a

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Development of a cell permeable ratiometric
chemosensor and biomarker for hydrogen sulphate
Cite this: RSC Adv., 2014, 4, 15356
ions in aqueous solution†
Buddhadeb Sen,a Manjira Mukherjee,a Siddhartha Pal,a Sushil Kumar Mandal,b
Maninder S. Hundal, Anisur Rahman Khuda-Bukhsh and Pabitra Chattopadhyay
c
b
a
*
A newly designed organic moiety, 5H-5,7a,12-triaza-dibenzo[a,e]azulen-6-one (L) containing a seven
membered ring behaves as a hydrogen sulphate ion selective ratiometric chemosensor. The formulation
and detailed structural characterisation of
L have been established using physico-chemical,
spectroscopic tools and single crystal X-ray diffraction study. On additions of hydrogen sulphate ions to
the solution of L in HEPES buffer (1 mM; water : ethanol (v/v), 98 : 2) at 25 ꢀC at biological pH, a new
fluorescence peak generated at 483 nm was increased with concomitant decrease of the weak
fluorescence of L at 430 nm through an isoemissive point at 449 nm due to the selective binding of
HSO4 ions with L in a 1 : 1 ratio with a binding constant (K) of 4.13 ꢂ 106
M
ꢁ1, and detects HSO4 ions
ꢁ
ꢁ
as low as 5.5 ꢂ 10ꢁ7 M. The ratiometric enhancement of the fluorescence is based on intermolecular
hydrogen bonding assisted chelation enhanced fluorescence (CHEF) process which has been evidenced
by 1HNMR titration and supported by theoretical (DFT) calculations. The probe (L) having no cytotoxic
Received 11th January 2014
Accepted 7th March 2014
DOI: 10.1039/c4ra00291a
ꢁ
effect is also useful for the detection of intracellular HSO4 ion concentrations under a fluorescence
www.rsc.org/advances
microscope.
environmental effects and stability under illumination.5,6 So, it
is a challenge for a chemist to develop a ratiometric water
Introduction
soluble cell permeable uorescent probe for hydrogen sulfate
ions.
Anions play a vital role in a wide range of industrial, agricul-
tural, biological systems and environmental processes.1 Thus,
the design and development of selective optical chemosensors
for various anions have gained considerable attention.2 Among
the various anions, hydrogen sulfate (HSO4ꢁ) ions dissociate at
high pH to generate toxic sulfate (SO42ꢁ), causing irritation of
the skin and eyes and even respiratory paralysis. Despite its
crucial roles in biological processes, only few examples of
Chemosensors based on anion-induced changes in uores-
cence are particularly attractive because of the simplicity, high
spatial and temporal resolution of uorescence.7 Secondary
amides have been employed widely as hydrogen bond donor
groups to bind anionic species.8 The rst example of a synthetic
anion receptor containing secondary amides was reported by
Pascal and co-workers in 1986.9 Among the various types of
these receptors, imidazolium based receptors have also been
extensively investigated in recent years.
ꢁ
sensors for HSO4 ions have been reported.3,4 Among all these
sensors, most of them performed through single point detec-
tion3 and only few acted upon ratiometric titration,4 but none of
them as a cell permeable sensor for hydrogen sulphate in
aqueous solvent has been reported. However, it is well known
that ratiometric responses are more attractive because the ratio
between the two emission intensities can be used to measure
the analyte concentration and provide a built-in correction for
ꢁ
In the search of a water soluble HSO4 ions selective che-
mosensor, we designed and synthesized a new compound (L) in
a facile route using imidazolium based secondary amides. L
behaves as a ratiometric amplied chemosensor highly selec-
ꢁ
tive and sensitive uorescent probe for HSO4 ions in HEPES
buffer (1 mM, pH 7.4; 2% EtOH) at 25 ꢀC upto a very low
concentration (5.5 ꢂ 10ꢁ7 M) of HSO4ꢁ ions. This probe (L) was
also useful to detect the presence of bisulphate ions by
acquiring image of HeLa cells under a uorescence microscope
and L has no cytotoxicity. To the best of our knowledge so far,
the 5H-5,7a,12-triaza-dibenzo[a,e]azulen-6-one (L) containing a
seven membered ring as a water soluble cell permeable ratio-
metric uorescent probe for hydrogen sulfate ions is still
unexplored.3,4
aDepartment of Chemistry, The University of Burdwan, Golapbag, Burdwan 713104,
India. E-mail: pabitracc@yahoo.com; Fax: +91-342-2530452; Tel: +91-342-2558554
extn 424
bMolecular Biology and Genetics Laboratory, Department of Zoology, Kalyani
University, India
cDepartment of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
† Electronic supplementary information (ESI) available. CCDC 972698. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ra00291a
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Experimental section
Materials and methods
High-purity
HEPES
[4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid], 2-(2-aminophenyl)benz-imidazole, 2-chloroacetyl
chloride, and tetra butyl ammonium hydrogen sulphate were
purchased from Sigma Aldrich (India), solvents used were spec-
troscopic grade. Other chemicals, tetrabutyl ammonium salts of
uoride, chloride, bromide, iodide, acetate, dihydrogen phos-
phate and sodium salt of monohydrogen phosphate, azide,
thiocyanate and nitrate were of analytical reagent grade and used
without further purication except when specied. Milli-Q, 18.2
MU cmꢁ1 water was used throughout all experiments. The
elemental analyses (C, H and N) were performed on a Perkin
Elmer 2400 CHN elemental analyzer. A Shimadzu (model UV-
1800) spectrophotometer was used for recording electronic
spectra. FTIR spectra were recorded using Prestige-21 SHI-
MADZU FTIR spectrometer preparing KBr disk. 1HNMR spec-
trum of organic moiety was obtained on a Bruker Avance DPX 500
MHz spectrometer using DMSO-d6 solution. Electrospray ioni-
zation (ESI) mass spectra were recorded on a Qtof Micro YA263
mass spectrometer. A Systronics digital pH meter (model 335)
was used to measure the pH of the solution and the adjustment
of pH was done using either 50 mM HCl or sodium hydroxide
solution. Steady-state uorescence emission and excitation
spectra were recorded with a Hitachi F-4500 FL Spectrophotom-
eter. Time-resolved uorescence lifetime measurements were
performed using a HORIBA JOBIN Yvon picosecond pulsed diode
laser-based time-correlated single-photon counting (TCSPC)
spectrometer from IBH (UK) at lex ¼ 377 nm and MCP-PMT as a
detector. Emission from the sample was collected at a right angle
to the direction of the excitation beam maintaining magic angle
polarization (54.71). The full width at half-maximum (FWHM) of
the instrument response function was 250 ps, and the resolution
was 28.6 ps per channel. Data were tted to multiexponential
functions aer deconvolution of the instrument response func-
tion by an iterative reconvolution technique using IBH DAS 6.2
data analysis soware in which reduced w2 and weighted resid-
uals serve as parameters for goodness of t.
Scheme 1 Synthetic strategy of L.
3205. (See ESI†); 1H NMR (400 MHz, DMSO-d6): 10.637 (s, 1H),
8.087 (dd, 1H), 7.824 (dd, 1H), 7.745 (dd, 1H), 7.574 (q, 1H),
7.377–7.266 (m, 4H), 4.946 (s, 2H, –CH2–CO); ESI-MS m/z 249.98
[M + H+, 100%]; calc.: 250.09; [M + H]+; 13C NMR (DMSO-d6):
166.332, 149.161, 137.348, 133.602, 131.465, 126.226, 126.041,
124.957, 118.106, 115.203, 46.215. Yield: 78%.
Synthesis of compound 2 (L–HSO4ꢁ). A 2.0 ml ethanolic
solution of tetra butyl ammonium hydrogen sulphate (67.906 mg,
0.20 mmol) was added slowly to the stirred 8.0 ml solution of L
(50 mg; ꢃ0.20 mmol) in EtOH and the stirring was continued for
4.0 h. The resulting greenish coloured reaction mixture was
ltered and kept aside for several days to get the solid product on
slow evaporation at room temperature. The solid was so obtained
and dried in vacuo for performing the characterization.
Compound 2. Bu4N[C15H12N3O5S]: C31H48N4O5S. Anal.
found: C, 63.53; H, 8.08; N, 9.74; S, 5.19; calc.: C, 63.24; H, 8.22;
N, 9.52; S, 5.45; IR (KBr, cmꢁ1): nC]O, 1643, nO–H, 3422; nS]O
1
1113. H NMR (400 MHz, DMSO-d6): 11.060 (s, 1H), 8.212 (dd,
1H), 8.146 (dd, 1H), 7.948–7.916 (m, 1H), 7.798 (q, 1H), 7.716
(m, 2H), 7.520 (q, 1H), 7.448 (dd, 1H), 5.195 (s, 2H, –CH2–CO),
and for Bu4N:11 3.209 (m, 4CH2) 1.607 (m, 4CH2) 1.340 (m,
4CH2), 0.970 (t, 4CH3) (see ESI†); ESI-MS: [M ꢁ Bu4N+ +H+
+
Na+]+, m/z, found, 369.97, calc.: 370.05; 13C NMR (DMSO-d6):
166.346, 149.201, 137.506, 133.612, 131.474, 126.003, 125.667,
125.057, 117.960, 115.223, 46.325.
X-ray crystallography
Synthetic procedures
X-ray data of the suitable crystal of L was collected on a Bruker's
Apex-II CCD diffractometer using Mo Ka (l ¼ 0.71069). The data
were corrected for Lorentz and polarization effects and empir-
ical absorption corrections were applied using SADABS from
Bruker. A total of 12449 reections were measured out of which
3426 were independent and 2801 were observed [I > 2s(I)] for
theta (q) 1.66 to 28.19ꢀ. The structure was solved by direct
methods using SIR-92 and rened by full-matrix least squares
renement methods based on F2, using SHELX-97.12 All calcu-
lations were performed using Wingx package.13,14 Important
crystallographic parameters are given in Table S1.†
Synthesis of [5H-5,7a,12-triaza-dibenzo[a,e]azulen-6-one]
(L). 2-chloroacetyl chloride (5.31 ml) was dissolved in chloroform
(5 ml) and then added dropwise to an ice cold solution of 2-(2-
aminophenyl)benzimidazole (4.1848 g, 20 mmol) and Et3N
(3.0 ml) in chloroform (10 ml) very slowly with stirring. Aer being
stirred for 4 h at room temperature, the mixture was removed
under reduced pressure to obtain a white solid, which was ltered
out and extracted with dichloromethane to afford compound 1.10
In the next step, compound 1 was taken in dry acetonitrile having
anhydrous K2CO3 and reuxed for 4 h. The volume of the solution
was reduced to obtain a solid and then extracted with dichloro-
methane and nally puried by silica gel column chromatog-
raphy using dichloromethane as the eluent (Scheme 1).
General method of UV-vis and uorescence titration
L. C15H11N3O. Anal. found: C, 72.52; H, 4.39; N, 17.01; calc.: Path length of the cells used for absorption and emission studies
C, 72.28; H, 4.45; N, 16.86; IR (KBr, cmꢁ1): nC]O, 1687, nN–H
,
was 1 cm. For UV-vis and uorescence titrations, stock solution
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of L was prepared in HEPES buffer (1 mM, pH 7.4; 2% EtOH) at methanolic solution of L in 1 : 1 mole ratio at stirring condition.
25 ꢀC. Fluorescence measurements were performed using 5 nm The ESI mass spectrum of 2 in methanol shows a molecular-ion
ꢂ 5 nm slit width. All the uorescence and absorbance spectra peak at m/z 369.97 with ꢃ14% abundance, which can be
ꢁ
were taken aer 30 minutes of mixing of HSO4 and L.
assigned to [M ꢁ Bu4N+ + H+ + Na+]+ (calculated value at m/z,
370.05). A characteristic peak for nS]O at 1113 cmꢁ1 in the FTIR
spectrum of 2 conrms the existence of sulphate ion attached to
2.16 The 1HNMR spectrum obtained in DMSO-d6 conrmed the
presence of the L bound to HSO4 ions (Fig. S2a–d†).
Single crystals of the probe (L) were obtained as HL$NO3 from
the methanolic solution of L in presence of aq. sodium nitrate.
Preparation of cell and in vitro cellular imaging with L
Human cervical cancer cell, HeLa cell line was purchased from
National Center for Cell Science (NCCS), Pune, India and was
used throughout the study. Cell were cultured in Dulbecco's
modied Eagle's medium (DMEM, Gibco BRL) supplemented
with 10% FBS (Gibco BRL), and 1% antibiotic mixture con-
taining penicillin, streptomycin and neomycin (PSN, Gibco
BRL), at 37 ꢀC in a humidied incubator with 5% CO2. For
experimental study, cells were grown to 80–90% conuence,
harvested with 0.025% trypsin (Gibco BRL) and 0.52 mM EDTA
(Gibco BRL) in PBS (phosphate-buffered saline, Sigma Diag-
nostics) and plated at desire cell concentration and allowed to
re-equilibrate for 24 h before any treatment. Cells were rinsed
with PBS and incubated with DMEM-containing L (10 mM, 1%
DMSO) for 30 min at 37 ꢀC. All experiments were conducted in
DMEM containing 10% FBS and 1% PSN antibiotic. The
imaging system was composed of a uorescence microscope
(ZEISS Axioskop 2 plus) with an objective lens [10ꢂ].
ꢁ
ꢀ
The HL$NO3 crystallizes in the triclinic space group P1. An
ORTEP view of the probe with atom labelling scheme is illus-
trated in Fig. 1, and a selection of bond distances and angles is
˚
listed in Table S2.† The bond distance C1–N1 (1.3557(18) A) or
˚
C11–N1 (1.4114(17) A) is somewhat longer than that of any bond
˚
˚
of C9–N3 (1.3351(18) A) or C9–N2 (1.3455(17) A) due to the single
bond character of C1–N1 or C11–N1 and sp3 hybridisation of N1.
˚
In the same way, the longer bond distance (1.4602(18) A) is also
indicative of the single bond character of C2–N2 bond. The
crystal structure of L is stabilized by the presence of the nitrate
anion as counter anion for balancing the produced charge of
HL+ formed due to the protonation of N(3) atom.
UV-vis spectroscopic studies of L
Cell cytotoxicity assay
UV-vis spectra of L was recorded in HEPES buffer (1 mM, pH 7.4;
2% ethanol) at 25 ꢀC shows an absorption maximum at 383 nm
To test the cytotoxicity of L, MTT [3-(4,5-dimethyl-thiazol-2-yl)-
2,S-diphenyl tetrazolium bromide] assay was performed with
the help of the procedure described earlier.15 Aer treatments of
the probe (5, 10, 20, and 50 mM), 10 ml of MTT solution (10 mg
mlꢁ1 PBS) was added in each well of a 96-well culture plate and
incubated continuously at 37 ꢀC for 6 h. All mediums were
removed from wells and replaced with 100 ml of acidic iso-
propanol. 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 wavelength
with a microplate reader. Values are means ꢄ S.D. of three
independent experiments. The cell cytotoxicity was calculated
as percent cell cytotoxicity ¼ 100% cell viability.
Fig. 1 A molecular view with atom numbering scheme of HL$NO3
(50% ellipsoid probability).
Result and discussion
Synthesis and structural characterisation
Organic moiety was synthesized by the reaction of 2-(2-amino-
phenyl)benzimidazole with 2-chloroacetyl chloride in chloro-
form solvent followed by ring closer step (Scheme 1) and
characterised by physico-chemical and spectroscopic tools
(Fig. S1a–d†). The FTIR spectrum of L contains bands for the
C]O and NH groups at 1687 and 3205 cmꢁ1, respectively along
with other characteristic peaks for benzimidazole unit (Fig. S1a
in ESI†). The QTOF-ESI-MS of L contains molecular ion peak at
m/z 249.98 corresponding to [L + H+] (Fig. S1c, ESI†). The well-
resolved 1H NMR spectrum of L is in support of the formulation
and the structure established by single crystal X-ray crystallo-
graphic analysis of HL$NO3.
Fig. 2 UV-vis titration spectra of L (10 mM) upon incremental addition
of HSO4ꢁ ions (0–20 mM) in HEPES buffer (1 mM, pH 7.4; 2% EtOH) at
The solid L–HSO4ꢁ ensembled species (2) was obtained from
the reaction of tetra butyl ammonium hydrogen sulphate with 25 ꢀC.
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which may possibly be attributed to the intramolecular charge ions indicates that the uorescence intensity ratio of wavelength
transfer (CT) transition. The absorption intensity of L at 383 nm 483 nm and 430 nm (I483/I430) gradually increases with increase
gradually decreased, accompanied by the formation of a new of the concentration of HSO4ꢁ ions (Fig. S7†) and aer a certain
absorption peak at 425 nm (Fig. 2) as the addition of HSO4ꢁ ions time it level up producing a sigmoid curve. L exhibited a near
was increased stepwise (0–20 mM) with an isosbestic point at about 14-fold increase of its uoꢁrescence intensity upon addition
402 nm and the solution turned from colourless to faint of only 2.0 equivalent of HSO4 ions. The detection limit was
greenish yellow (Fig. S3†).
determined and was found to be 5.5 ꢂ 10ꢁ7 Mꢁ1 (Fig. S8†),
which is signicantly low. Interestingly, the introduction of
other anions does not interfere the uorescence enhancement.
The uorescence characteristics of L (10 mM) were observed
Emission study
Organic moiety (L) showed a weak emission at 430 nm excited at
383 nm in HEPES buffer (1 mM, pH 7.4; 2% ethanol) at 25 C
considering the absorption at 383 nm (Fig. S4†). Fluorescence
quantum yields (F) were estimated by integrating the area
under the uorescence curves with the equation:
upon the addition of excess 50 equivalents of different anions i.e.
ꢀ
3ꢁ
Fꢁ, Clꢁ, Brꢁ, Iꢁ, CNꢁ, N3– NO3ꢁ, ClO4ꢁ, H2PO4ꢁ, HPO42ꢁ, PꢁO4
,
H2AsO4ꢁ, HAsO42ꢁ, AsO33ꢁ, OAcꢁ, SO42ꢁ, S2ꢁ, and HSO4 but
only HSO4ꢁ ions selectively enhance the uorescence intensity of
L system (Fig. 4). In presence of 10 times excess of various tested
anions together with L and HSO4ꢁ, almost no adverse effect on
intensity was observed (Fig. S9†).
2
ODref ꢂ Asample ꢂ h
ODsample ꢂ Aref ꢂ sharmefple
Fsample ¼ Fref
2
The compound formed between L and HSO4ꢁ is found to be
1 : 1 in stoichiometry, which was established with the help of
Job's plot (Fig. S10†) using the uorescence study. This 1 : 1
stoichiometric ratio is also supported by the physico-chemical
where A is the area under the uorescence spectral curve and
OD is optical density of the compound at the excitation wave-
length, 383 nm, h is the refractive index of the solvent used. The
standard used for the measurement of uorescence quantum
yield was anthracene (F ¼ 0.29 in ethanol). This emission
property of the probe (L) has not been affected over the pH
range 4.0–10.0 (Fig. S5†).
ꢁ
and spectroscopic data of the L–HSO4 ensemble species iso-
lated in the solid form.
To ensure the formation of the L–HSO4ꢁ species in solution
1
state, H NMR titration was also performed in dmso-d6. Fig. 5
clearly indicates the interaction of amide N–H proton with
HSO4ꢁ ions as the signal due to this proton shied to downeld
(d value from 10.637 ppm to 11.060 ppm) and the protons of
–CH2 of the seven membered ring also shied to higher d value
from 4.946 ppm to 5.195 ppm along with a signicant shiing
of Hc towards downeld by Dd of 0.388 ppm (Dd ¼ 8.212–
Fluorescence studies of L
The emission property of L in presence of various concentrations
ꢁ
of HSO4 ions was veried. On addition of various concentra-
tions of HSO4ꢁ ions (0–20 mM), uorescence intensity at 430 nm
was signicantly decreased with a concomitant increase of
intensity at 483 nm through an isoemissive point at 449 nm
(Fig. 3). The uorescence quantum yield has also been calculated
ꢁ
7.824 ppm, Fig. S11†) on gradual addition of HSO4 ions.
The binding constant value was determined from the emis-
sion intensity data following the modied Benesi–Hildebrand
equation.17,18
ꢁ
in absence and presence of HSO4 ions. And from this
measurement it is clear that the uorescence quantum yield in
presence of HSO4ꢁ ions (F ¼ 0.45) increases ꢃ4 times than that
of free L (F ¼ 0.12). This spectral feature for the addition of
HSO4ꢁ ions was also supported by the uorescence color change
from blue to green in presence of UV light (Fig. S6†).
1/(Fx ꢁ F0) ¼ 1/(Fmax ꢁ F0) + (1/K[C])(1/(Fmax ꢁ F0))
Ratiometric signaling of uorescence output at two different
ꢁ
wavelengths plotted as a function of concentration of HSO4
Fig. 4 Anion selectivity of L in presence of different anions in HEPES
buffer (1 mM, pH 7.4; 2% EtOH) at 25 ꢀC where (a) L, (b) Fꢁ, (c) Clꢁ, (d)
Brꢁ, (e) Iꢁ, (f) CNꢁ, (g) N3ꢁ, (h) NO3ꢁ, (i) ClO4ꢁ, (j) H2PO4ꢁ, (k) HPO4
,
2ꢁ
Fig. 3 Emission spectra of L in presence of HSO4ꢁ ions (0–20 mM) at (l) H2AsO4ꢁ, (m) HAsO42ꢁ, (n) AsO33ꢁ, (o) OAcꢁ, (p) SO42ꢁ, (q) S2ꢁ and
lex ¼ 383 nm in HEPES buffer (1 mM, pH 7.4; 2% EtOH) at 25 ꢀC.
(r) HSO4ꢁ at lem ¼ 483 nm.
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Fig. 6 Time resolved fluorescence decay of L (10 mM) only and in
presence of added HSO4ꢁ in HEPES buffer (1 mM, pH 7.4; 2% EtOH) at
25 ꢀC using a nano LED of 377 nm as the light source at lem ¼ 483 nm.
Fig. 5 Partial 1H NMR spectra for L (10 mM) in presence of varying
[HSO4ꢁ] [(A) 0 mM, (B) 3.33 mM, (C) 6.67 mM, and (D) 10 mM] in DMSO-
1
d6 [for clarity, expanded region of 7.2–8.3 ppm of HNMR spectra in
Fig. S11†].
Scheme 2 Proposed mechanism of fluorescence enhancement of
ꢁ
receptor (L) in presence of HSO4 ions.
where F0, Fx, and FN are the emission intꢁensities of organic
moiety consideꢁred in the absence of HSO4 ions, at an inter-
mediate HSO4 concentration, and at a concentration of
complete interaction, respectively, and where K is the associa-
tion constant and [C] is the concentration of HSO4ꢁ. K value
(4.13 ꢂ 106 Mꢁ1) was calculated from the slope/intercept using
the plot of (FN ꢁ F0)/(Fx ꢁ F0) against 1/[HSO4ꢁ] (Fig. S12†). This
value of binding constant is the indication of strong binding
Geometry optimization
To clarify the congurations and H-bonding feature of the host
(L) and guest–host species (2; L–HSO4ꢁ), DFT calculations were
performed using Gaussian-09 soware over a Red Hat Linux
IBM cluster. Molecular level interactions between L and 2 have
been studied using density functional theory (DFT) with the
B3LYP/6-31 G(d) functional model and basis set.
ꢁ
affinity of the organic moiety towards the HSO4 ions. In the
uorescence average life time measurement the life time of L
was found to be 5.77 ns at lem ¼ 483 nm. Aer gradual addition
of HSO4ꢁ ions to the solution of L, the average lifetime of the L–
HSO4ꢁ species (at lem ¼ 483 nm) increased from 11.79 ns (when
L : HSO4ꢁ; 1 : 0.5) to 12.15 ns (when L : HSO4ꢁ; 1 : 1), and it is
clearly ascribed by the intermolecular hydrogen bonding
assistꢁed CHEF process (Table S3†)(Fig. 6). The strong binding of
HSO4 ion with organic moiety (L), is evidenced by the signi-
cant binding constant value (4.13 ꢂ 106 Mꢁ1) and this
phenomenon played a key role to deter the PET process in
ꢁ
To assure the mode of interaction of the probe (L) with HSO4
ions either in ring or chain fashion, the optimised geometries in
both fashions were obtained by theoretical calculation
(Fig. S13†). From the calculation of energy of HOMO and LUMO
in this study, it is indicated that the binding mode of the guest
ꢁ
(HSO4 ion) with the host (L) through ring is more preferable
than that through chain. In case of ring structure, the energy gap
between HOMO-LUMO is 3.77 eV which is lower than that of free
L (4.53 eV) (Table S4†). Here, charge distribution of HOMO in
ꢁ
ꢁ
compound 2 conrms that the most of the charge is on HSO4
support of the selective detection of HSO4 ions through uo-
ion attached with N–H through H-bonding (Fig. 7).
rescence enhancement (Scheme 2).
According to the equations:19 sꢁ1 ¼ kr + knr and kr ¼ Ff/s, the
radiative rate constant kr and total nonradiative rate constant
ꢁ
ꢁ
Biological studies of L in presence of HSO4
knr of organic moiety, L and HSO4 complex with L were listed
in Table S3.† The data suggest that kr has just slightly changed To examine the utility of the probe in biological systems, it was
but the factor that induces uorescent enhancement is mainly applied to human cervical cancer HeLa cell. In these experi-
ascribed to the decrease of knr.
ments both the L and HSO4ꢁ ions was allowed to uptake by the
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added HSO4ꢁ ions are clearly evident from the cellular imaging.
In addition, the in vitro study showed that 10 mM of L did not
show no cytotoxic effect to cell upto 6 h (Fig. S14†). These results
indicate that the probe has a huꢁge potentiality for both in vitro
and in vivo application as HSO4 sensor as well as imaging in
different ways as same manner for live cell imaging.
Conclusions
In conclusion, a water soluble dual receptor for HSO4ꢁ ions was
synthesized by a facile two-step process. The formulation and
detailed structural characterisations has been established using
physico-chemical and spectroscopic tools along with detailed
structural analyses by single crystal X-ray crystallography. The
uorimetric and titrimetric titration of L by adding HSO4ꢁ ions
ꢀ
in HEPES buffer (1 mM, pH 7.4; 2% ethanol) at 25 C showed
ꢁ
that L is very specic and sensitive chemosensor for HSO4
ꢁ
ions. On addition of HSO4 ions, the emission intensity at
430 nm of free L decreases gradually with the generation of a
new peak at 483 nm through an isoemissive point at 449 nm due
to the hydrogen bonding formation between the amide
hydrogen and bisulphate ion. This interaction was also clearly
supported by 1HNMR titration and theoretical DFT calculation.
Interestingly, this probe could be considered as a potent probe
ꢁ
Fig. 7 Optimized structures of L and L–HSO4 species.
for HSO4ꢁ ions because the uorescence enhancement at lem
¼
483 nm of bio-friendly visible region is taken place in a more
attractive ratiometric response fashion with almost no inter-
ference by the biologically relevant anions in aqueous solvent.
This probe has been successfully used as biomarker of bisul-
phate ions as L is signicantly efficient to detect the distribution
of hydrogen sulphate ions in vitro in living cells (HeLa) in
aqueous medium at biological pH by developing the good
image.
cells of interest and the images of the cells were recorded by the
uorescence microscopy following excitation at ꢃ383 nm. Aer
incubation with L (10 mM) for 30 min, the cells displayed very
faint intracellular uorescence. Howeverꢁ, cells exhibited inten-
sive uorescence when exogenous HSO4 ions was introduced
into the cell via incubation with Bu4NHSO4 (Fig. 8). The uo-
rescence responses of the probe with various concentrations of
Acknowledgements
Financial assistance from CSIR, New Delhi, India is gratefully
acknowledged. B. Sen wishes to thank to UGC, New Delhi, India
for offering him the fellowship. The authors sincerely
acknowledge Prof. Samita Basu and Mr Ajay Das, Chemical
Science Division, SINP, Kolkata for enabling TCSPC instrument,
and Prof. B. Mukhopadhyay, IISER, Kolkata for providing the
1
facility to record some H NMR spectra and ESI Mass Spectra
and also thankful to Mr Samya Banerjee, IISC, Bangalore for
recording the 13C NMR spectra.
Notes and references
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were excited at l¼ 383 nm.
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15362 | RSC Adv., 2014, 4, 15356–15362
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