Iminocoumarin based fluorophores: Indispensable scaffolds for rapid, selective and sensitive detection of thiophenol

Article abstract of DOI:10.1016/j.dyepig.2014.02.001

Off-on fluorescence probes for the detection of thiophenol were designed based on the reaction mechanism to ensure rapid sensing process. 2,4-Dinitrophenylsulfonyl based probes with iminocoumarin fluorophores were validated by theoretical calculations for their off-fluorescence states. Reaction of these probes with thiophenol released strong fluorescent iminocoumarin species with good response times. These results, upon comparing with reported probes, confirmed that higher pK_aH values of the imino nitrogen of iminocoumarins are important for rapid sensing process. Reactivity of these probes was limited to only thiophenol and no reaction was observed with aliphatic thiols. The benzothiazole substituted probe displayed up to 260-fold fluorescence enhancement upon reaction with thiophenol. Sensing of the analyte by the probe was characterized by change in color from red to yellow and with appearance of the green fluorescence. A detection limit up to 6.9 × 10^-9 M was also determined for this probe.

Full text of DOI:10.1016/j.dyepig.2014.02.001

Dyes and Pigments 106 (2014) 25e31
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Iminocoumarin based fluorophores: Indispensable scaffolds for rapid,
selective and sensitive detection of thiophenol
,
Dnyaneshwar Kand a, Prashant Sahebrao Mandal b, Avdhoot Datar a, Pinaki Talukdar a
*
a Department of Chemistry, Indian Institute of Science Education and Research Pune, Pune, Maharashtra 411008, India
b Department of Chemistry, Institute of Chemical Technology (ICT), Mumbai 400019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Off-on fluorescence probes for the detection of thiophenol were designed based on the reaction mech-
anism to ensure rapid sensing process. 2,4-Dinitrophenylsulfonyl based probes with iminocoumarin
fluorophores were validated by theoretical calculations for their off-fluorescence states. Reaction of these
probes with thiophenol released strong fluorescent iminocoumarin species with good response times.
These results, upon comparing with reported probes, confirmed that higher pKaH values of the imino
nitrogen of iminocoumarins are important for rapid sensing process. Reactivity of these probes was
limited to only thiophenol and no reaction was observed with aliphatic thiols. The benzothiazole
substituted probe displayed up to 260-fold fluorescence enhancement upon reaction with thiophenol.
Sensing of the analyte by the probe was characterized by change in color from red to yellow and with
appearance of the green fluorescence. A detection limit up to 6.9 ꢀ 10ꢁ9 M was also determined for this
probe.
Received 23 October 2013
Received in revised form
29 January 2014
Accepted 2 February 2014
Available online 28 February 2014
Keywords:
Iminocoumarin
Thiophenol sensing
Off-on probe
Fast responsive
Selectivity
Ó 2014 Elsevier Ltd. All rights reserved.
Benzothiazole
1. Introduction
an increased tR ¼ 20 min was determined, when studied under
comparable conditions. The fastest responsive DNs-based probe 3
Thiophenols, widely used in the production of polymers, pesti-
cides and pharmaceuticals [1e3], are highly toxic and polluting
chemicals. These aromatic thiols target central nervous system,
kidney and liver [4e6] resulting in burning sensation, wheezing,
nausea, vomiting, etc. [4,7] Presence of thiophenols in water and soil
are also reported to cause damage to natural habitats [8]. Since, the
first report of 2,4-dinitrophenylsulfonyl (DNs) based fluorescent
thiophenol probe 1 by Jiang et al.,[9] considerable number of DNs-
based [10e13] and other thiophenol probes [14] have been devel-
oped [15]. DNs-based thiophenol probes undergo thiolate mediated
SNAr reaction on the DNs-ring to release the fluorophore (Fig. 1(a)).
Either an intramolecular charge transfer (ICT) or photoinduced
electron transfer (PET) pathway from the fluorophore to the DNs
moiety is responsible for the off-fluorescence state of these probes.
Selectivity of these probes toward aromatic thiols by discriminating
aliphatic ones, has paved their significance in sensing applications
e.g. detection of thiophenol in water, living cells. However, slow
reactivity of these probes with PhSH has resulted in relatively long
response times (tRs). For example, with a 1:10 molar ratio of probe
versus PhSH, probe 1 displayed a tR ¼ 17 min (Fig. 1(b)). For probe 2,
which provided a tR ¼ 12 minwas reported byour group [13]. To date
the best tR ¼ 2 min was reported by Lin et al. for a probe in which
a coumarin fluorophore was linked to the 2,4-dinitrophenol
quencher. However, a molar ratio of 1:20 was used during the
sensing experiment and a decrease in molar ratio to 1:5 resulted in
an increase in tR value to 7 min [14]. High reactivity of the analyte
towards various Michael acceptors is expected to result in fluctua-
tions of actual concentration of the species. Hence, it is essential to
develop fast responsive off-on thiophenol probes for quantitative
detection of the species particularly applicable in biological and
natural systems.
A rational approach for controlling the rates of decomposition of
DNs-amides was reported recently by Malwal et al [16]. A structure
activity relationship (SAR) study provided crucial insight into the
reaction mechanism of cysteine-activated SO2 (a gaseous anti-
mycobacterial agent) release from these pro-drugs. During the thiol
mediated decomposition of DNs-linked arylamines 4e6 to free
aniline derivatives 4ae6a, a reactivity order 6 > 5 > 4 was observed
based on half-lives (t1/2) of these DNs-derivatives (Fig. 2). A corre-
lation of pKaH values of 6a, 5a and 4a with determined t1/2 values
confirmed a linear relation between these parameters i.e. a faster
cleavage of DNs group was achieved with higher pKaH value of the
released aniline derivative. This structure activity relationship
(SAR) study suggests that the protonation of aniline N-center is the
* Corresponding author. Tel.: þ91 20 2590 8098; fax: þ91 20 2589 9790.
E-mail address: ptalukdar@iiserpune.ac.in (P. Talukdar).
http://dx.doi.org/10.1016/j.dyepig.2014.02.001
0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.

26
D. Kand et al. / Dyes and Pigments 106 (2014) 25e31
Fig. 3. Structures of iminocoumarin based thiophenol probes 7-8 and active fluo-
rophores 7a and 8a.
(Aldrich, Spectrochem, and Sdfine Chemical Limited) and used as
received unless stated otherwise. Solvents: petroleum ether and
ethyl acetate (EtOAc) were distilled prior to thin layer and column
chromatography. Column chromatography was performed on
Merck silica gel (100e200 mesh). TLC was carried out with E. Merck
silica gel 60-F-254 plates.
2.2. Physical measurements
1H and 13C spectra were recorded on 400 MHz Jeol ECS-400 (or
100 MHz for 13C) spectrometers using either residual solvent
signals as an internal reference or from internal tetramethylsilane
Fig. 1. The off-on mechanism for DNs appended fluorescent probes (a), Structures,
response times of reported DNs-based thiophenol probes 1-3 and pKaH values released
fluorophores 1a-3a (b).
on the
d
scale (CDCl3 d H, 7.24 ppm,
d
C 77.0 ppm and for DMSO-d6
) are reported
d
H, 2.52 ppm,
d
C 41.23 ppm). The chemical shifts (d
in ppm and coupling constants (J) in Hz. The following abbrevia-
tions are used: m (multiplet), s (singlet), br s (broad singlet),
d (doublet), t (triplet) dd (doublet of doublet). High-resolution
mass spectra were obtained from Micro Mass ESI-TOF MS spec-
trometer. Absorption spectra were recorded on a Thermo Scien-
tific, Evolution 300 UV-VIS spectrophotometer. Steady State
fluorescence experiments were carried out in a micro fluorescence
cuvette (Hellma, path length 1.0 cm) on a FluoroMax-4 (Horiba
JobinYvon). FT-IR spectra were obtained using NICOLET 6700 FT-IR
spectrophotometer as KBr disc and reported in cmꢁ1. Melting
points were measured using a VEEGO Melting point apparatus. All
melting points were measured in open glass capillary and values
are uncorrected. Crystal structures were recorded on a Bruker
single crystal X-Ray diffractometer.
rate determining step (rds) and this step occurs after a thiol
mediated ipso attack (for eSH group of Cys, pKa ¼ 8.0) on the DNs
ring. This study has provided a simple and effective methodology to
alter onset times of SO2 releasing prodrugs by adjusting the basicity
(pKaH) of the released amine. We realized the importance of this
work in the design of new thiophenol probes to ensure faster
response times compared to reported probes 1-3.
Herein, we report two DNs-derivatized iminocoumarin probes 7
and 8 for rapid, selective and sensitive detection of thiophenol
(Fig. 3). We speculate that an intramolecular charge transfer (ICT)
pathway from iminocoumarin to DNs moiety is responsible for the
fluorescence off-state in these probes. Free iminocoumarins 7a and
8a can be released as strongly fluorescent species from these probes
via the thiolate (PhSꢁ) mediated cleavage of DNs moiety. Particularly
in 8a, an electron donating diethylamino group at the 7-position and
electron withdrawing benzothiazole group at 3-position favor
strong fluorescence properties at longer wavelength compared to 7a
via the intramolecular electron transfer process [17].
2.3. Theoretical calculations
Geometry optimization of 7 and 8 were carried out in the gas
phase using density functional theory (DFT) at the B3LYP/6-311G
(d,p) level using Gaussian 09 software [18,19]. Time-dependent
DFT (TDDFT) calculations were carried out on the geometry opti-
mized structures of 7 and 8 at the B3LYP/6-311G(d,p) level to pre-
dict excitation and emission properties.
2. Materials and methods
2.1. General methods
2.4. Synthesis
All reactions were conducted under the nitrogen atmosphere.
All the chemicals were purchased from commercial sources
2.4.1. 2,4-Dinitro-N-(3-phenyl-2H-chromen-2-ylidene) benzene
sulfonamide (C21H13N3O7S) 7
To a solution of 3-phenyl-2H-chromen-2-imine 7a (200 mg,
0.9 mmol) in pyridine (10 mL) placed in a 100 mL two-neck round
bottomed flask were added 2,4-dinitrobenzene-1-sulfonyl chloride
(362 mg, 1.35 mmol) at 0 ꢂC slowly with stirring. The reaction
mixture was placed under nitrogen atmosphere and stirred at room
temperature for 1 h. After completion of the reaction, the solvent
was removed under reduced pressure. The residue was poured into
H2O (10 mL) and extracted with CH2Cl2 (15 mL ꢀ 3). The combined
organic layer was washed with water (10 mL ꢀ 3), brine (10 mL)
and dried over Na2SO4. The solvent was removed under reduced
pressure to obtain a yellow residue which was purified by column
Fig. 2. Correlation of calculated half lives (t1/2) of prodrugs 4-6 with and pKaH values of
released amines 4a-6a.

D. Kand et al. / Dyes and Pigments 106 (2014) 25e31
27
ꢀ
chromatography over silica gel (Eluent: 45% EtOAc in petroleum
ether) to furnish the pure compound 7 (169 mg, 35%) as yellow
solid. M.p.: 242e243 ꢂC; IR (KBr): nmax/cmꢁ1 3367, 1624, 1607, 1557,
1535, 1481, 1446, 1384, 1366, 1333, 1313, 1159, 1105; 1H NMR
Z ¼ 2; rcalc ¼ 1.308 g cmꢁ3; 2qmax ¼ 56.48; MoKal ¼ 0.71073 A. Fine-
focus sealed tube source with graphite monochromator. R ¼ 0.0408
(for 1746 reflection I > 2
s
(I)), wR ¼ 0.1040 which was refined ÐF2Ð
and S ¼ 1.056 for 158 parameters and 2212 unique reflections. The
structure was obtained by direct methods using SHELXS-97 [20,21].
All non-hydrogen atoms were refined anisotropically. The
hydrogen atoms were fixed geometrically in the idealized position
and refined in the final cycle of refinement as riding over the atoms
(400 MHz, DMSO-d6):
8.36 (s, 1H), 7.97e7.76 (m, 1H), 7.68 (dd, J ¼ 7.9, 1.5 Hz, 3H), 7.56e
7.19 (m, 5H); 13C NMR (100 MHz, DMSO-d6):
159.0, 152.0, 150.6,
d
8.95e8.75 (m, 1H), 8.67 (d, J ¼ 1.6 Hz, 2H),
d
148.4, 143.8, 139.0, 134.5, 133.6, 133.2, 129.9, 129.7 (2C), 129.5, 128.8
(2C), 128.5, 127.7, 127.1, 120.7, 120.4, 116.2; HRMS (ESI): Calc. for
to which they are bonded
2.6. Preparation of samples
2.6.1. Preparation of the medium
m
¼ 0.082 mmꢁ1
.
C
21H14N3O7S [M þ H]þ: 452.0553; Found: 452.0554.
2.4.2. (Z)-N-(3-(benzo[d]thiazol-2-yl)-7-(diethylamino)-2H-
chromen-2-ylidene)-2,4-dinitrobenzenesulfonamide
(C26H21N5O7S2) 8
HEPES buffer (0.01 M, pH 7.4) was prepared by dissolving HEPES
(free acid) in deionized water and then pH was adjusted to the
desired value with 0.1 N NaOH.
To a solution of 3-(benzo[d]thiazol-2-yl)-N,N-diethyl-2-imino-
2H-chromen-7-amine 8a (125 mg, 0.357 mmol) in pyridine (10 mL)
placed in a 100 mL two-neck round bottomed flask were added 2,4-
dinitrobenzene-1-sulfonyl chloride (362 mg, 1.35 mmol) at 0 ꢂC
slowly with stirring. The reaction mixture was placed under ni-
trogen atmosphere and stirred at room temperature for 1 h. After
completion of the reaction, the solvent was removed under reduced
pressure. The residue was poured into H2O (10 mL) and extracted
with CH2Cl2 (15 mL ꢀ 3). The combined organic layer was washed
with water (10 mL ꢀ 3), brine (10 mL) and dried over Na2SO4. The
solvent was removed under reduced pressure to obtain a yellow
residue which was purified by column chromatography over silica
gel (Eluent: 2% MeOH in Chloroform) to furnish the pure compound
8 (232 mg, 72%) as red solid. M.p.: 261e262 ꢂC; IR (KBr): nmax/cmꢁ1
3565, 1643, 1532, 1484, 1466, 1366, 1346, 1315, 1128, 1082; 1H NMR
2.6.2. Preparation of the solutions of 7 and 8
A stock solutions of 7 (2437
in DMSO. Final concentration of each of 7 and 8 during each assay
was 10 M.
mM) and 8 (1138 mM) were prepared
m
2.6.3. Preparation of solutions of analytes
Stock solutions of aniline, phenol, thioacetic acid, 4-
Chlorothiophenol, 4-Methylthiophenol, 2-Naphthalenethiol and
thiophenol were prepared in DMSO (concentrations ranging from
9076
amino acids were prepared in deionized H2O with varied concen-
trations ranging from 2391 M to 13,076 M. Calculated volumes of
analytes were added from respective stock solutions to fluores-
cence each cuvette to provide 100 M.
mM to 21,475 mM). Stock solutions of other analytes including
m
m
(400 MHz, DMSO-d6)
d 9.20 (s, 1H), 8.92e8.76 (m, 2H), 8.60 (dd,
J ¼ 8.7, 2.3 Hz, 1H), 8.13 (d, J ¼ 7.9 Hz, 1H), 7.96 (d, J ¼ 8.1 Hz, 1H),
7.85 (d, J ¼ 9.1 Hz, 1H), 7.50 (t, J ¼ 8.2 Hz, 1H), 7.39 (t, J ¼ 7.5 Hz, 1H),
6.96 (dd, J ¼ 9.1, 2.2 Hz, 1H), 6.45 (d, J ¼ 1.7 Hz, 1H), 3.52 (d,
J ¼ 7.1 Hz, 4H), 1.11 (t, J ¼ 7.0 Hz, 6H); 13C NMR (100 MHz, DMSO-
m
2.7. UVevisible and fluorescence spectra of 7, 7a, 8 and 8a
UV-visible and fluorescence spectra (Fig. S4) of probes 7
d6):
d 159.3 (2C), 156.1, 155.1, 152.6, 151.2, 149.4, 147.4, 143.7, 143.7,
138.2,135.5,132.5,132.0,126.1,125.9,124.3,121.5,121.4,119.4,112.3,
109.9, 108.8, 94.4, 44.3 (2C), 11.7 (2C); HRMS (ESI): Calc. for
(10 mM), 7a (10 mM), 8 (10 mM) and 8a (10 mM) were recorded in
C
26H21N5O7S2 [M þ H]þ: 580.0961; Found: 580.0952.
HEPES buffer (10 mM, pH 7.4). From absorption spectra molar
extinction coefficient, ε of species 7, 7a, 8 and 8a were determined
using BeereLambert law (Eq. (1)):
2.5. Crystal structure parameters of 7 and 7a
A
c ꢀ l
2.5.1. 2,4-Dinitro-N-(3-phenyl-2H-chromen-2-ylidene) benzene
sulfonamide 7 (CCDC 940800)
ε ¼
(1)
C
21H13N3O7S; Compound 7 was crystallized from dimethyl
where,
A
¼
absorbance,
ε
¼
molar extinction coefficient,
sulfoxide at room temperature. An yellow needle shaped crystal
with approximate dimensions 0.286 ꢀ 0.090 ꢀ 0.037 mm gave an
c ¼ concentration of the solution and l ¼ path length of UV cuvette
(l ¼ 1 cm).
Orthorhombic with space group P212121;
a
b
¼
7.2532 (7)
g
Fluorescence spectra of 7, 7a, 8 and 8a were recorded by exciting
at respective lmax values (333 nm for 7 and 7a, 487 nm for 8 and 8a).
The excitation and emission slit width were 5 nm/5 nm for 7 and
2 nm/5 nm for 8.
ꢀ
b ¼ 14.6345 (14) c ¼ 18.2027 (18) A,
a
¼ 90ꢂ
¼ 90ꢂ
¼ 90ꢂ;
3
V ¼ 1932.2 (3) A ; T ¼ 296 (2) K; Z ¼ 4; rcalc ¼ 1.504 g cmꢁ3
;
ꢀ
ꢀ
2
qmax ¼ 48.24; MoKal ¼ 0.71073 A. Fine-focus sealed tube source
with graphite monochromator. R ¼ 0.0300 (for 2826 reflection
I > 2
s
(I)), wR ¼ 0.0749 which was refined ÐF2Ð and S ¼ 1.027 for
289 parameters and 3071 unique reflections. The structure was
obtained by direct methods using SHELXS-97 [20,21]. All non-
hydrogen atoms were refined anisotropically. The hydrogen
atoms were fixed geometrically in the idealized position and
refined in the final cycle of refinement as riding over the atoms to
2.8. Thiol sensing
2.8.1. Determination of reaction rate (k), half-life (t1/2) and response
time (tR) for probes 7 and 8
Reaction rate (k), half-life (t1/2) and response time (tR) for probes
7 and 8 were determined by fluorescence kinetics experiments. In
which they are bonded
m
¼ 0.215 mmꢁ1
.
fluorescence kinetics experiment, a solution of either 7 (10
(10
M) in HEPES buffer (10 mM, pH ¼ 7.4) was placed in a fluo-
rescence cuvette (under stirring conditions) and fluorescence
intensity (at
¼ 435 nm for 7 upon lex ¼ 333 nm and at 524 nm for
mM) or 8
2.5.2. 3-phenyl-2H-chromen-2-imine 7a (CCDC 940801)
m
C15H11NO; Compound 7a was crystallized from toluene at room
temperature. A colorless cube shaped crystal with approximate
dimensions 0.210 ꢀ 0.117 ꢀ 0.065 mm gave an Monoclinic with
l
8 upon lex ¼ 487 nm) was monitored. At t ¼ 30 s an analyte (100
mM
ꢀ
space group P21; a ¼ 6.247 (5ꢂ) b ¼ 7.475 (7) c ¼ 12.177 (10) A,
of either of PhSH, Cys and Cys) was added. Subsequently, the time
3
ꢀ
a
¼ 90ꢂ
b
¼ 98.78 (2)o
g
¼ 90 ; V ¼ 561.9 (9) A ; T ¼ 296 (2) K;
was normalized with t ¼ tꢁ30 s and then converted to minute

28
D. Kand et al. / Dyes and Pigments 106 (2014) 25e31
Table 1
Wavelengths (
l), oscillator strengths (f) and CI coefficients for the vertical excitation (UVevis absorptions) of probes 7 and 8 obtained from the TDDFT/B3LYP/6-311G (d, p)
calculations, based on the optimized ground state geometries.
Excitation
Transition
E (eV)
l
(nm)a
fb
Main configurations
CIc
7
S
S
S
S
0 / S1
0 / S2
0 / S3
0 / S4
2.7838
3.2571
3.4038
3.5347
445.38
380.66
364.26
350.77
0.0006
0.0202
0.0015
0.2044
HOMO / LUMO
0.69683
0.69287
0.69974
0.30261
0.60935
0.68577
0.11714
0.69515
0.10475
0.46727
0.18796
0.48701
0.45951
0.11113
0.48419
0.16175
HOMO / LUMO þ 1
HOMO ꢁ 1 / LUMO
HOMO ꢁ 2 / LUMO
HOMO / LUMO þ 2
HOMO / LUMO
HOMO ꢁ 2 / LUMO
HOMO ꢁ 1 / LUMO
HOMO / LUMO
HOMO ꢁ 1 / LUMO þ 1
HOMO ꢁ 1 / LUMO þ 2
HOMO / LUMO þ 2
HOMO ꢁ 1 / LUMO þ 1
HOMO ꢁ 1 / LUMO þ 2
HOMO / LUMO þ 2
HOMO / LUMO þ 3
8
S
S
S
0 / S1
0 / S2
0 / S3
2.8477
3.0458
3.2452
435.38
407.07
382.06
0.0004
0.0004
0.0610
S
0 / S4
3.2843
377.5
0.1257
a
l
values were calculated from respective energies of transition using formula
l
¼ hc/E.
b
c
Oscillator strength.
CI coefficient.
scale. Fluorescence intensity values were normalized according to
the Eq. (2):
2.8.2. Determination of sensitivity for probes 7 and 8
The reactivity of probes 7 and 8 with various analytes was exam-
ined. 10 M of each probe was separately treated with 100 M of an-
m
m
ðIt ꢁ I Þ
alyte (either of thiophenol, 4-chlorothiophenol, 4-methylthiophenol,
2-naphthalenethiol, PhNH2, PhOH, NaN3, KI, Ala, Lys, Arg, His, Tyr,
Ser, GSH, Cys and thioacetic acid) in HEPES buffer (10 mM, pH ¼ 7.4)
and fluorescence was recorded after 5 min. After the treatment with
eitheraliphaticthiolornon-thiolspecies,eachsolutionwasseparately
treated with PhSH. Relative fluorescence for probes 7 and 8 was
calculated by using Eq. (5):
ðImax ꢁ0I Þ
Y ¼
(2)
0
where, Y ¼ fractional fluorescence intensity, It ¼ fluorescence in-
tensity recorded at time ¼ t min; I0 ¼ fluorescence intensity
recorded at time ¼ 0 min; Imax ¼ fluorescence intensity recorded at
either t ¼ 9.0 min (for probe 7 in response to PhSH) or t ¼ 2.5 min
(for probe 8 in response to PhSH). Slit values: 5 nm/5 nm (for 7) and
2 nm/5 nm (for 8).
Irel ¼ I=I0
(5)
Rate constant, k for each reaction was calculated according to
the following Eq. (3):
where, Irel ¼ relative fluorescence intensity, I ¼ fluorescence in-
tensity recorded at either lem ¼ 435 nm (for probe 7) or
lem ¼ 524 nm (for probe 8) after addition of an analyte and
I0 ¼ fluorescence intensity of the probe (either 7 or 8) at either
lem ¼ 435 nm (for probe 7) or lem ¼ 524 nm (for probe 8).
h
i
Y ¼ a ꢀ 1 ꢁ eðꢁktÞ
(3)
where, Y ¼ fractional fluorescence intensity, a ¼ arbitrary constant,
k ¼ pseudo first order rate constant, t ¼ time.
2.8.3. Quantitative response of probes 7 and 8 towards PhSH
Half-life of the reaction (t1/2) was calculated using Eq. (4)
Fluorometric titrations for probe 8 (10
mM) was carried out with
increasing concentrations (0e100 M) of PhSH after 5 min of addi-
tion. In each case, increase in fluorescence intensity was observed.
m
t1=2 ¼ 0:693=k
(4)
where k ¼ pseudo first order rate constant.
2.8.4. Determination of quantum yields of probes 7 and 8
The quantum yields of probe 7 and 8 were determined accord-
ing to Eq. (6):
Response time (tR) in each case was determined from corre-
sponding each fluorescence kinetics plot.
I1 ꢀ AB ꢀ lexB ꢀ ðh1Þ2
Table 2
F1
¼
FB
ꢀ
(6)
Wavelengths (l), oscillator strengths (f) and CI coefficients for the emission of probes
7 and 8 obtained from the TDDFT/B3LYP/6-311G (d, p) calculations, based on the
optimized S1 state geometries.
IB ꢀ A1 ꢀ lex1 ꢀ ðhBÞ2
where,
fluorescence spectra; A is absorbance at the excitation wavelength;
lex is the excitation wavelength; is the refractive index of the
solution; the subscripts 1 and B refer to the unknown and the
standard, respectively.
F is quantum yield; I is integrated area under the corrected
Emission Transition E (eV)
l
(nm)a fb
Main configurations CIc
0.70356
7
S1 / S0
S2 / S0
S3 / S0
S4 / S0
S1 / S0
S2 / S0
S3 / S0
S4 / S0
1.2259 1011.37 0.0001 HOMO / LUMO
h
2.0434 606.75 0.0006 HOMO / LUMO þ 1 0.70400
2.2911 541.15 0.0010 HOMO ꢁ 1 / LUMO 0.70355
2.3662 523.98 0.0003 HOMO ꢁ 2 / LUMO 0.69540
8
1.4534 853.06 0.0000 HOMO / LUMO
0.70047
2.1583 574.45 0.0008 HOMO ꢁ 1 / LUMO 0.70357
2.1991 563.79 0.0005 HOMO / LUMO þ 2 0.69988
2.4388 508.39 0.0003 HOMO ꢁ 2 / LUMO 0.69355
2.8.5. Determination of detection limits of probes 7 and 8
The detection limits were determined based on the fluorescence
titrations. Each probe was separately employed at 10
mM. To
a
l
values were calculated from respective energies of transition using formula
determine the S/N ratio, the fluorescence intensity of these probes
was separately measured without PhSH by 6-times and the stan-
dard deviations of blank measurements were determined. Under
l
¼ hc/E.
b
Oscillator strength.
CI coefficient.
c

D. Kand et al. / Dyes and Pigments 106 (2014) 25e31
29
these conditions, a good linear relationship between the fluores-
cence intensity and the thiophenol concentration could be obtained
optimized and the excitation based on the optimized S1 state ge-
ometry were calculated. S1 / S0 transition (HOMO / LUMO) for 7
(f ¼ 0.0001) and 8 (f ¼ 0.0000) was not allowed. Absence of an
emissive S1 state (S1 / S0 transition) suggests that the radiative
S1 / S0 decay is not possible for both probes 7 and 8. It is clear from
the distributions of the MOs, that the HOMO and LUMO are localized
at different locations for 7 and 8 (Fig. S1). HOMOs are located on the
iminocoumarin moieties, whereas, LUMOs are mainly localized on
the DNs group [28]. Therefore it is expected that S1 / S0 transitions
for 7 and 8 involve an intramolecular charge transfer to iminocou-
marin fluorophore from DNs group.
in the 2e10
m
M
for probe
7
(R
¼
0.99736) and probe 8
(R ¼ 0.99397), as shown in Fig. S7. The detection limit (DL), is then
calculated according to Eq. (7):
3 ꢀ sbi
DL ¼
(7)
m
where, sbi is the standard deviation of six blank measurements, m is
the slope between intensity versus sample concentration.
The detection limits of 7 and 8 towards PhSH were calculated to
be 1.87 ꢀ 10ꢁ8 M and 6.90 ꢀ 10ꢁ9 M, respectively at S/N ¼ 3 (signal-
to-noise ratio of 3:1).
3.2. Synthesis of probes
Syntheses of probes 7 and 8 were carried out from 7a and 8a,
respectively. Free iminocoumarins 7a [29,30] and 8a [25] were
synthesized following reported procedures. Treatment of 7a with
2,4-dinitrophenylsulfonyl chloride (DNs-Cl) in the presence of pyr-
idine at room temperature provided 7 in 35% yield (Scheme 1(a)).
Similarly, probe 8 was synthesized from 8a under comparable
conditions in 72% yield (Scheme 1(b)). Single crystal X-ray diffrac-
tion analysis of 7 (Scheme 1(c)) was useful to confirm the relative
spatial arrangement between iminocoumarin and DNs moieties.
Similar spatial arrangement between DNs and iminocoumarin is
also anticipated in 8 although its crystal structure was not available.
3. Results and discussions
3.1. Theoretical calculations
As iminocoumarins are well known fluorophores [17,22e24] and
fluorescent properties of iminocoumarin 8a were also reported [25],
theoretical calculations were carried out only for probes 7 and 8 to
investigate their off-states and understand the mechanism of fluo-
rescence quenching. Selected parameters for the vertical excitation
(UVevis absorption) and the fluorescence emission are shown in
Tables 1and 2 respectively. Gas phase geometry optimizations were
carried out using density functional theory (DFT) at the B3LYP/6-
311G (d,p) level followed by time-dependent DFT (TDDFT) [26,27]
calculations at the same level. Calculation based on the optimized
ground state (S0) geometry showed that S0 / S1 electronic transi-
tion was not allowed for 7 (i.e. HOMO / LUMO, f ¼ 0.0006). First
allowed transition for 7 is S0 / S4 with f ¼ 0.2044. Similarly, for 8
electronic transition S0 / S1 (i.e. HOMO / LUMO oscillator
strength, f ¼ 0.0004) was not allowed (Table 1). First allowed tran-
sition for 8 was S0 / S4 with f ¼ 0.1257. In order to investigate the
emission of 7 and 8, the geometry of the singlet excited state (S1) was
3.3. Photophysical properties of probes 7 and 8
Photophysical studies of probe 7 (10
mM) in HEPES buffer
(10 mM, pH ¼ 7.4) displayed a strong visible band centered at
lmax
¼
368 nm (Fig. S4) with molar extinction coefficient,
ε ¼ 1.7 ꢀ 104 Mꢁ1 cmꢁ1 (Table 3). When excited at lex ¼ 368 nm, the
probe 7 displayed negligible fluorescence. On the other hand, 7a
(10
mM) exhibited an intense UV-absorption band with
lmax ¼ 333 nm (ε ¼ 1.4 ꢀ 104 Mꢁ1 cmꢁ1) and a strong fluorescence
with lem ¼ 435 nm (lex ¼ 333 nm). The UVevisible spectrum of
probe
8
provided
a
red-shifted lmax
¼
498 nm
(ε ¼ 2.5 ꢀ 104 Mꢁ1 cmꢁ1), when compared to probe 7. Upon exci-
tation at lex ¼ 498 nm, 8 exhibited negligible fluorescence. The free
iminocoumarin 8a displayed strong UV-absorption band at
lmax ¼ 487 nm (ε ¼ 2.8 ꢀ 104 Mꢁ1 cmꢁ1) and a strong fluorescence
emission band centered at lem ¼ 524 nm (lex ¼ 478 nm). These
results corroborate with our predictions from theoretical calcula-
tions that DNS-derivatives 7 and 8 are non-fluorescent.
3.4. Determination of response time of thiophenol sensing by
fluorescence spectroscopy
After confirming the off-fluorescence states in 7 and 8, tR values
of these probes were determined in HEPES buffer (10 mM,
pH
¼
7.4). When fluorescence intensity at
l
¼
435 nm
M) to
(
lex ¼ 333 nm) was monitored upon addition of PhSH (100
m
probe 7 (10 mM), a pseudo first order kinetics was observed with
rate constant, k ¼ 0.0069 sꢁ1 and half-life, t1/2 ¼ 100.4 s. From this
plot, a tR ¼ 5.5 min was determined for probe 7 (Fig. 4(a)).
Table 3
Photophysical properties of 7, 7a, 8 and 8a in HEPES buffer (10 mM, pH ¼ 7.4).
Probe
lmax (nm)
ε (Mꢁ1 cmꢁ1
)
lem (nm)a
7
7a
8
368
333
498
478
1.7 ꢀ 104
1.4 ꢀ 104
2.5 ꢀ 104
2.8 ꢀ 104
e
435
e
8a
524
a
Scheme 1. Synthesis of probes 7 (a). Synthesis of probes 8 (b). Crystal structures of 7
and 7a (c).
Fluorescence spectra were recorded by exciting at lmax values of respective
compounds.

30
D. Kand et al. / Dyes and Pigments 106 (2014) 25e31
software from ChemAxon [31] was used for predicting pKaH values
fluorophores 1a (pKaH ¼ ꢁ1.62), 2a (pKaH ¼ ꢁ5.71) and 3a
(pKaH ¼ 2.84), 7a (pKaH ¼ 5.75) and 8a (pKaH ¼ 5.22). The correla-
tion diagram demonstrates a decrease in tR value of probe toward
PhSH upon increase in pKaH of released fluorophore. The mecha-
nism via a thiolate (for eSH group of PhSH, pKa ¼ 6.62) mediated
ipso attack on the DNs-ring, indicates that the protonation of the
heteroatom, (X ¼ O or NH) of a fluorophore is the rds of the overall
cleavage process (Scheme S3).
3.5. Selectivity of thiol detection determined
To evaluate the selectivity and sensitivity of 8 toward PhSH and
other aryl thiols, various analytes were examined under the com-
parable conditions. As shown in Fig. 6, the reaction of 8 with PhSH
and other substituted aryl thiols (4-chlorothiophenol, 4-
methylthiophenol and 2-naphthalenethiol) resulted in w260-fold
increase of the fluorescence intensity (back row, red bar), whereas
Ala, Lys, Arg, His, Tyr, Ser, GSH, Cys, MeCOSH, PhNH2, PhOH, NaN3
and KI had negligible influence (front row, green bars). Fluorescence
spectra profiles also confirm the sensitivity of the probe 8 toward
PhSH in the presence of other analytes (back row, blue bars). A 220-
Fig. 4. Determination of tR values for probe 7 (10
m
M) in HEPES (10 mM, pH ¼ 7.4)
toward various sulfhydryls (100
upon excitation at lex ¼ 333 nm (a). Determination of tR for probe 8 (10
various sulfhydryls (100
M of PhSH, Cys and GSH) recorded at lem ¼ 524 nm upon
excitation at lex ¼ 487 nm (b).
mM of PhSH, Cys and GSH) recorded at lem ¼ 435 nm
m
M) toward
m
fold enhancement in quantum yield,
dard: fluorescein in 0.1 N NaOH,
during the sensing. Similar selectivity for probe 8 toward PhSH was
also observed for upon thiophenol sensing. However, with signifi-
cantly lower sensitivity of w65-fold (Fig. S5). A 16-fold enhance-
F (from 0.003 to 0.66; stan-
¼ 0.95) [32] was also calculated
F
ment in quantum yield,
F (from 0.002 to 0.033; standard: 2-
Aminopyridine in 0.1 M H2SO4,
calculated during the sensing of thiophenol.
PhSH sensing by probe 8 was also characterized by a visible
color change from red to yellow within 1.5 min confirming a fast
direct visual detection of the process (Fig. 7(a) and supporting
video). When placed under the hand-held UV lamp
F
¼ 0.60) [32,33] _ENREF_34 was
Fig. 5. Correlation diagram of tR values of probes 1-3, 7 and 8 with pKaH of 1a-3a, 7a
and 8a.
Similarly, when probe 8 (10
mM) was subjected PhSH (100 mM)
addition and fluorescence intensity was monitored at
(
l
¼ 524 nm
(
lex ¼ 365 nm), probe 8 exhibited no fluorescence while a strong
lex ¼ 487 nm), a pseudo first order rate constant, k ¼ 0.0290 sꢁ1
green fluorescence was observed for cuvette containing 8 and
and t1/2 ¼ 23.9 s were obtained. The fluorescence kinetics plot of
the probe provided an excellent tR ¼ 1.5 min (Fig. 4(b)). These data
confirm faster response of the probes 7 and 8 toward PhSH
compared to DNs-based thiophenol probes 1e3 (Fig. 2(b)). Under
the applied assay conditions, the reaction of either probe 7 or 8
was limited to only PhSH and substituted aryl thiols while other
aliphatic thiols (e.g. Cys and GSH) did not show any noticeable
reactivity.
PhSH (Fig. 7(b)). We further examined the quantitative response of
the probe 8 toward PhSH. Fluorometric titrations of 8 (10
varied concentrations (0e100 M) of PhSH displayed sharp in-
crease in fluorescence intensity (Fig. 7(c)). Based on these results a
linear correlation for fluorescence intensity and PhSH concentra-
tion was observed only up to one equivalent of the analyte
(Fig. S7B). A detection limit of 6.9 ꢀ 10ꢁ9 M for 8 toward PhSH was
calculated based on a signal-noise ratio, S/N ¼ 3. These data
established the usefulness of 8 as selective thiophenol sensing
probe.
mM) with
m
Based on these outcome, a correlation between experimentally
determined tR values of DNs-based PhSH probes 1e3 (reported in
references [9,10,13]), 7-8 versus pKaH values of respective free flu-
orophores 1a-3a, 7a and 8a was established (Fig. 5). Marvin
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.dyepig.2014.02.001.
Fig. 6. Relative fluorescence intensity (at lem ¼ 524 nm upon lex ¼ 487 nm) of 8 (10
m
M) in HEPES (10 mM, pH ¼ 7.4) with various analytes after 5 min of mixing. Front row
represents either only probe or addition of an analyte (100 mM of Ala, Lys, Arg, His, Tyr, Ser, GSH, Cys, MeCOSH, PhNH2, PhOH, NaN3 and KI (from left to right); back row represents
the addition of either an arylthiol (100
and each competitive analyte.
mM of PhSH, 4-chlorothiophenol, 4-methylthiophenol and 2-naphthalenethiol) to the probe or PhSH (100 mM) to solution containing probe

D. Kand et al. / Dyes and Pigments 106 (2014) 25e31
31
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(c). (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
4. Conclusions
In summary, we have successfully developed two 2,4-
dinitrophenylsulfonyl-derivatized iminocoumarin probes 7 and 8
for rapid, selective and sensitive detection of thiophenol. These
probes were designed based on the understanding of the reaction
mechanism to improve response time (tR) of thiophenol sensing.
Off-on response of these probes were predicted by theoretical cal-
culations. A pseudo first order kinetics observed during the reaction
of the probe 7 with thiophenol with k ¼ 0.0069 sꢁ1 and t1/
¼ 100.4 s. The kinetics also afforded a tR ¼ 5.5 min. A similar
2
pseudo first order kinetics with k ¼ 0.0290 sꢁ1 was determined for
probe 8. The probe exhibited a faster t1/2 ¼ 23.9 s and tR ¼ 1.5 min.
The significant improvement of tR compared to reported probes is
attributed to higher basicities of generated fluorophores 7a
(pKaH ¼ 5.75) and 8a (pKaH ¼ 5.22). Reactivities of probes 7 and 8
were specific to only thiophenol and aliphatic thiols (cysteine and
glutathione) were inert to these probes. In response to thiophenol,
the probe 8 exhibited 260-fold off-on fluorescence. Direct visual
detection of the sensing was marked by the color change from red
to yellow and turn-on green fluorescence. The probe 8 also pro-
vided a detection limit of 6.9 ꢀ 10ꢁ9 M during the sensing of the
analyte.
Acknowledgments
P.T. is grateful to the Director, IISER Pune, and DAE (Grant No.
2010/20/37C/6/BRNS/2480) for financial supports. We are also
thankful to Dr. Anirban Hazra of IISER Pune for his valuable sug-
gestions on theoretical calculations. D.K. thanks CSIR, P.S.M. thanks
DAE and A.D. thanks IISER Pune for research fellowships.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.02.001.
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