Quinolinium-based fluorescent probes for the detection of thiophenols in environmental samples and living cells

Article abstract of DOI:10.1002/asia.201200594

A new type of fluorescent probes for thiophenols, 6HQM-DNP and 7HQM-DNP, containing 6- or 7-hydroxy quinonlinium as fluorophore and 2,4-dinitrophenoxy (DNP) as nucleophilic recognition unit were constructed. As ethers, these non-fluorescent probe molecules can release the corresponding fluorescent quinolinium (6HQM and 7HQM) through aromatic nucleophilic substitution (S _NAr) by thiolate anions from thiophenols. The sensing reaction is highly sensitive (detection limit of 8 nM for 7HQM-DNP) and highly selective to thiophenols over aliphatic thiols and other nucleophiles under neutral conditions (pH 7.3). The probes respond rapidly to thiophenols, with second-order rate constants k=45 M^-1 s^-1 for 7HQM-DNP and 24 M^-1 s^-1 for 6HQM-DNP. Furthermore, the selective detection of thiophenols in living cells by 7HQM-DNP was demonstrated by confocal fluorescence imaging. In addition, these quinolinium salts show excellent chemical and thermal stability. In conclusion, this type of probes may find use in the detection of thiophenols in environmental samples and biosystems. Copyright

Full text of DOI:10.1002/asia.201200594

DOI: 10.1002/asia.201200594
Quinolinium-Based Fluorescent Probes for the Detection of Thiophenols in
Environmental Samples and Living Cells
Xiu-Ling Liu,[a] Xue-You Duan,[a] Xiao-Jiao Du,[b] and Qin-Hua Song*[a]
Abstract: A new type of fluorescent
probes for thiophenols, 6HQM-DNP
and 7HQM-DNP, containing 6- or 7-
hydroxy quinonlinium as fluorophore
and 2,4-dinitrophenoxy (DNP) as nu-
cleophilic recognition unit were con-
structed. As ethers, these non-fluores-
cent probe molecules can release the
corresponding fluorescent quinolinium
(6HQM and 7HQM) through aromatic
nucleophilic substitution (SNAr) by thi-
olate anions from thiophenols. The
sensing reaction is highly sensitive (de-
tection limit of 8 nm for 7HQM-DNP)
and highly selective to thiophenols
over aliphatic thiols and other nucleo-
constants k=45mꢀ1 sꢀ1 for 7HQM-
DNP and 24mꢀ1 sꢀ1 for 6HQM-DNP.
Furthermore, the selective detection of
thiophenols in living cells by 7HQM-
DNP was demonstrated by confocal
fluorescence imaging. In addition,
these quinolinium salts show excellent
chemical and thermal stability. In con-
clusion, this type of probes may find
use in the detection of thiophenols in
environmental samples and biosystems.
philes
under
neutral
conditions
(pH 7.3). The probes respond rapidly
to thiophenols, with second-order rate
Keywords: 7-hydroxy quinolinium ·
cellular imaging · charge transfer ·
fluorescent probes · thiophenols
Introduction
Significant efforts have been directed toward the develop-
ment of fluorescent probes for thiols, that is, mainly Cys,
Hcy, and GSH, in the past decade,[5]. A variety of colorimet-
ric and fluorescent probes for thiols have been constructed
by exploiting the high nucleophilic reactivity or transition
metal-affinity of the thiol group,[6] which involve specific re-
actions between probes and thiols, such as Michael addition,
cleavage reactions by thiols, cyclization with aldehyde, and
metal coordination. The nucleophilicity of most probes is
generally dependent on the deprotonated thiolate anions,
which are more reactive than free thiols. The pKa value of
aliphatic thiols is about 8.5, whereas that of thiophenols is
around 6.5. Hence, under neutral aqueous conditions, the
fluorescent probes for aliphatic thiols usually sense thiophe-
nols as well.
For this reason, the selectivity toward thiophenols over
aliphatic thiols could be realized by decreasing the nucleo-
philicity of thiol probes and selecting neutral aqueous condi-
tions. So far, several fluorescent probes that can discriminate
thiophenols against aliphatic thiols based on nucleophilic ar-
omatic substitution (SNAr) have been reported.[7] In general,
the manipulation of the electronic nature of the substituents
of a fluorophore would change the fluorescence emission
profile through the alteration of the intramolecular charge-
transfer (ICT) or photoinduced electron-transfer (PET) pro-
cesses. On the basis of design strategy, a pioneering work
that can discriminate thiophenols over aliphatic thiols
through an ICT pathway was reported by Wang and co-
workers.[7a] The strongly electron-withdrawing 2,4-dinitro-
benzenesulfonyl group as the protecting group of an amino
group blocks an ICT process, and the resulting sulfonamide
can be cleaved by thiophenols through an SNAr process to
release fluorescence. Subsequently, the same group reported
The development of highly sensitive and selective detection
techniques for the discrimination of relevant biologically
active and toxic molecules is of considerable importance in
the fields of chemical, biological, and environmental scien-
ces. Thiols are an important class of molecules in biological
systems; for instance, cysteine (Cys), homocysteine (Hcy),
and glutathione (GSH) play crucial roles in many physiolog-
ical processes.[1] On the other hand, thiophenols, in spite of
their broad synthetic utility, are a class of highly toxic and
pollutant compounds. Generally, thiophenols are more toxic
than aliphatic thiols. Symptoms of exposure include a burn-
ing sensation, coughing, wheezing, laryngitis, shortness of
breath, and headache.[2] The median lethal dose (LC50) of
thiophenols ranges from 0.01 to 0.4 mm for fish,[3] and thio-
phenol has been added to the priority lists of pollutants by
the United States Environmental Protection Agency (EPA
waste code: P014).[4]
[a] X.-L. Liu, X.-Y. Duan, Prof. Dr. Q.-H. Song
Department of Chemistry, Joint Laboratory of Green Synthetic
Chemistry
University of Science and Technology of China
Hefei 230026 (P. R. China)
Fax : (+86)551-3601592
E-mail: qhsong@ustc.edu.cn
[b] X.-J. Du
School of Life Sciences
University of Science and Technology of China
Hefei, 230027 (P. R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200594.
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another fluorescent probe that functions through a PET
mechanism and possessed an increased fluorescence en-
hancement and higher sensitivity towards thiophenols in
comparison to the prior reported probe.[7b] Meanwhile, a flu-
orescent probe for thiophenols based on the thiolysis of di-
nitrophenyl 7-coumarin ether was reported by Lin et al.[7c]
The ICT emission of the coumarin derivative was restored
through thiolysis, which converts the ether functionality into
a phenolic hydroxy group. Recently, an indole-based Bodipy
fluorescent probe for thiophenols was reported, in which the
2,4-dinitrobenzenesulfonyl group was used to block an ICT
process.[7d] The Bodipy fluorescence was selectively released
through an SNAr mechanism by thiolate anions from thio-
phenols. Another sensory system based on an organic–inor-
ganic hybrid enables the selective detection of thiophenols
over aliphatic thiols at pH 2–5 through cleavage of the disul-
fide bond by thiophenols.[7e] However, these probes have
some limitations, including instability at high pH values,
high detection limits, and low sensitivity, especially a long
response time. Furthermore, in only one case,[7c] thiophenol
was detected in a biological system through cellular imaging.
Therefore, the development of new better sensors toward
thiophenols is warranted.
lined in Scheme 2. The condensation of 2 with 1 in the pres-
ence of base in DMF (1008C) afforded compound 3, which
was then reacted with ethyl iodide in toluene (1008C)
through a Menshutkin reaction to give the target product,
7HQM-DNP. 6HQM-DNP was prepared similarly, as shown
in Scheme 2.
Scheme 2. Synthetic procedure for 7HQM-DNP and 6HQM-DNP.
In this work, we have constructed a new type of fluores-
cent probe with a quinolinium as a fluorophore and a 2,4-di-
nitrophenolic group (DNP) as a nucleophilic recognition
unit (Figure 1). These molecules, 6HQM-DNP and 7HQM-
Thiophenol Sensing Properties of Probes
Probes were designed based on N-ethyl-7-hydroxy- or N-
ethyl-6-hydroxy quinolinium as fluorophores and the strong-
ly electron-withdrawing 2,4-dinitrophenoxy group as the rec-
ognition unit. We reasoned that the fluorescence of these
probe molecules would be quenched due to the inhibition of
intramolecular charge transfer (ICT) from the hydroxy
group to the positively charged pyridinium ring by the
strong electron-withdrawing ability of the 2,4-dinitrophenyl
group. After thiolysis of 2,4-dinitrophenyl ethers to form the
hydroxy group, fluorescence emission of HQM would be re-
stored. The measurements of the fluorescence quantum
yields (Ff) of probes and their thiolysis products support the
above idea (see Table 1). The data imply that these probes
could respond to thiophenol as a fluorescent “turn-on”
sensor.
Next, the fluorescence response of 7HQM-DNP to thio-
phenol was investigated. Addition of thiophenol resulted in
a rapid increase in fluorescence intensity with a peak at
498 nm, and the enhancement of fluorescence intensity
reached a maximum after about 15 min (Figure 2). An un-
precedented signal-to-background ratio of about 330 was
observed (photographs of solutions containing various ana-
lytes and 7HQM-DNP are shown in Figure S1 in the Sup-
Figure 1. Molecular structure of the probes used in this study.
DNP, can quickly release the corresponding hydroxyquinoli-
nium (HQM) cation in the presence of thiophenol as shown
in Scheme 1. For comparison, the photophysical and sensing
properties of both probes were investigated. Furthermore,
the detection of thiophenol inside cells was demonstrated by
using 7HQM-DNP.
Scheme 1. Release of fluorescent 7HQM by thiolysis of 7HQM-DNP
with thiophenols.
Table 1. Properties of the two probe compounds used in this study.
[b]
Compd.
Ff[a]
I/I0
k/Mꢀ1 sꢀ1
Results and Discussion
7HQM-DNP
6HQM-DNP
0.002
0.008
N
>330
>8
45
24
Synthesis of Probe Molecules
[a] Values in parentheses are fluorescence quantum yields of their thioly-
sis products, 7HQM and 6HQM. [b] Fluorescence enhancement from
25 mm probe solution (pH 7.3) in the presence and absence of thiophenol
(4 equiv).
Probe molecules were synthesized by a two-step procedure
from readily available starting materials, 2,4-dinitrochloro-
benzene (1) and methyl-7-hydroxy-quinoline (2),[8] as out-
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Quinolinium-based Fluorescent Probes
Figure 2. Fluorescence spectra (lex =400 nm) of 7HQM-DNP (25 mm)
upon addition of thiophenol (4 equiv) in 0.01m PBS (pH 7.3) recorded at
different times from 0 to 20 min. Inset: Fluorescence intensity at 498 nm
as a function of the reaction time.
Figure 4. Partial 1H NMR spectra of (1) 7HQM-DNP in [D6]DMSO,
(2) 7HQM-DNP+thiophenol in [D6]DMSO, (3) 7HQM in [D6]DMSO,
and (4) 2,4-dinitrophenyl phenylsulfide in CDCl3.
porting Information). The sensing reaction was also moni-
tored by UV/Vis spectroscopy, as shown in Figure 3. The ad-
dition of thiophenol leads to the disappearance of the ab-
2,4-dinitrophenyl phenylsulfide shows that the peaks a’, b’,
c’, d’, and e’ of the mixture correspond to those of 7HQM,
while f’’, g’’, and h’’ and Ph are in agreement with those of
2,4-dinitrophenyl phenylsulfide. Obviously, 7HQM-DNP in
the reaction mixture converted completely into the thiolysis
products. Analysis of the reaction mixture by mass spectros-
copy further confirmed the single thiolysis reaction, as the
spectrum displayed two peaks at m/z 188.1068 and 277.0281,
corresponding to molecular masses of 7HQM and 2,4-dini-
trophenyl phenylsulfide, respectively (see Figure S3 in the
Supporting Information).
Kinetic Analysis and pH Effects
The kinetics of the reaction of 7HQM-DNP with thiophenol
were determined in PBS solution (pH 7.3) at room tempera-
ture. The time-dependent absorption response of 7HQM-
DNP at diffrent excess of thiophenol followed pseudo-first-
order kinetics kobs. The plot of kobs versus the concentration
of thiophenol yielded a second-order rate constant k=
45mꢀ1 sꢀ1 for the reaction of 7HQM-DNP with thiophenol.
Hence, the thiolysis reaction of 7HQM-DNP is much faster
Figure 3. Absorption spectra of 7HQM-DNP (20 mm) upon the addition
of thiophenol (4 equiv) in 0.01m PBS (pH 7.3) recorded at different reac-
tion times from 0 to 16 min. Inset: Absorbance at 398 nm as a function of
the reaction time.
than those of reported sensors for aliphatic thiols.[6c,9]
.
sorption peak at 328 nm accompanied by the appearance of
a new peak at 398 nm. The absorbance of the new peak in-
creased rapidly and reached a maximum after about 15 min.
The presence of an isosbestic point at 342 nm indicates that
7HQM-DNP reacts with thiophenol in 1:1 stochiometry, re-
sulting in a perceived color change from colorless to orange
(see Figure S1 in the Supporting Information).
As a comparison, the photophysical and sensing proper-
ties of 6HQM-DNP were investigated. Similar to 7HQM-
DNP, 6HQM-DNP emits a very weak fluorescence (Ff
0.008, Table 1). Time-dependent fluorescence spectra of the
6HQM-DNP solution (pH 7.3) taken after the addition of
thiophenol revealed an 8-fold enhancement in fluorescence;
notable changes were also observed in the UV/Vis absorp-
tion spectra (Figure S4 in the Supporting Information).
Based on the time-dependent absorption spectra, a rate con-
stant of k=24mꢀ1 sꢀ1 was determined, which is significantly
lower than that with 7HQM-DNP. Further investigation re-
vealed that 6HQM is weakly fluorescent (Ff 0.046) in the
buffer due to the formation of a twisted ICT (TICT) state.
The excited state of 6HQM has larger dipole moment than
To further confirm the validity of the proposed sensing
mechanism, a solution of 7HQM-DNP in [D6]DMSO was
1
analyzed by H NMR spectroscopy in the presence or ab-
sence of thiophenol (partial spectra are shown in Figure 4
and entire spectra are given in Figure S2 in the Supporting
1
Information). Comparison of the H NMR spectrum of the
reaction mixture with those of 7HQM-DNP, 7HQM, and
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7HQM, and 6HQM more easily forms the TICT state with
a very low fluorescence efficiency. This is the reason the flu-
orescent response of 6HQM-DNP to thiophenol is low.
Next, the effects of pH on the reactivity of 7HQM-DNP
with thiophenol were investigated by monitoring its fluores-
cence emission and absorption (Figure 5). The probe
7HQM-DNP displayed a fluorescence response towards thi-
ophenol starting from pH 6.0. Hence, 7HQM-DNP could be
employed to detect thiophenols under neutral physiological
condition.
Figure 7. Plot of the fluorescence enhancement ratio ((IꢀI0)/I0) at 498 nm
as a function of the concentration of thiophenol in the range of 0–35 mm.
was determined, thereby indicating that 7HQM-DNP is
highly sensitive for the detection of thiophenols.
For an excellent sensor, a high selectivity is required. To
investigate the selectivity of 7HQM-DNP for thiophenols
over aliphatic thiols and other common nucleophiles, fluo-
rescence spectra were determined in the presence of various
analytes. As shown in Figure 8, a pronounced increase in
Figure 5. Dependence of the absorbance (left) and fluorescence intensity
~
at 498 nm (right) of 7HQM-DNP (25 mm) on pH in the absence ( ) and
~
presence ( ) of thiophenol (4 equiv).
Sensitivity and Selectivity of 7HQM-DNP to Thiophenols
To investigate the sensitivity of 7HQM-DNP as a fluores-
cence sensor of thiophenol, fluorescence spectra of 7HQM-
DNP at various amounts of thiophenol (0–4.5 equiv) were
determined. The fluorescence intensity at 498 nm increased
in a concentration-dependent manner, saturating at about 3
equivalents of thiophenol (Figure 6). At the concentration
range of 0–35 mm of thiophenol, a linear relationship was ob-
tained between the observed fluorescent signal and the thio-
phenol concentration (Figure 7), thus enabling a quantitative
analysis of thiophenol in this range. Furthermore, based on
a signal-to-background ratio of 3, a detection limit of 8 nm
Figure 8. Fluorescence responses of 7HQM-DNP (25 mm) in the presence
of various analytes (4 equiv each) in 0.01m PBS buffer (pH 7.3) at room
temperature.
fluorescence was only observed in the presence of thiophe-
nols, whereas the change in fluorescence in the presence of
aliphatic thiols (Gly, Cys, mercaptoacetic acid, and cyclohex-
anethiol) and nucleophilic reagents (such as amino acids
and phenol) in PBS buffer was negligible. Moreover,
7HQM-DNP can readily detect thiophenols at pH 7.3 in the
presence of aliphatic thiols and other nucleophiles
(Figure 9), thereby indicating that these nucleophiles do not
interfere with sensing of thiophenols. However, in alkaline
solutions such as alkaline buffer (pH 10.0), aliphatic thiols
such as Cys that exist as deprotonated thiolate anions can
react with 7HQM-DNP (Figure S5 in the Supporting Infor-
mation). Hence, under these conditions, 7HQM-DNP
cannot discriminate thiophenols against aliphatic thiols.
These data clearly confirm that the selectivity of fluorescent
Figure 6. Fluorescence spectra (lex =400 nm) of 7HQM-DNP (25 mm)
upon addition of increasing concentrations (0–4.5 equiv) of thiophenol in
0.01m PBS buffer (pH 7.3). Inset: Fluorescence intensity at 498 nm as
a function of the concentration of thiophenol.
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Quinolinium-based Fluorescent Probes
Figure 9. Fluorescence response of 7HQM-DNP (25 mm, pH 7.3) to thio-
phenol/4-methoxylbenzenethiol in the presence of various reagents
(4 equiv): 1, thiophenol; 2, 4-methoxylbenzenethiol; 3, Gly+thiophenol;
4, Cys+thiophenol; 5, mercaptoacetic acid+thiophenol; 6, cyclohexane-
thiol+thiophenol; 7, aniline+thiophenol; 8, phenol+thiophenol; 9, 4-
methoxylbenzenethiol+thiophenol.
probes based on SNAr is pH-dependent and that a careful
selection of the pH value in the reaction is critical for the
selective detection of thiophenols.
Figure 10. Confocal fluorescence images of MDA-MB-231 cells. a–c)
Cells treated with 7HQM-DNP (5 mm). d–f) Cells incubated with 7HQM-
DNP and then treated with thiophenol (5 mm). g–i) Cells pre-treated with
thiophenol and then successively treated with N-ethylmaleimide and
7HQM-DNP. Fluorescence images (a, d, g), brightfield images (b, e, h),
and overlay images of fluorescence images and the corresponding bright
field images (c, f, i).
Stability of 7HQM-DNP
The stability of 7HQM-DNP under acidic and basic solu-
tions was examined by means of absorption and fluores-
cence spectrometry (Figure 5). No significant changes in
both absorption and fluorescence were observed in wide pH
range (pH 1 to 11), thus showing that 7HQM-DNP is con-
siderably stable at various pH.
Conclusions
7-Dialkyamino-N-alkylquinolinium salts were demonstrat-
ed to have superior stability compared to a number of hemi-
cyanine dyes and rigid charge-transfer probes.[10] The two
probes studied in this work are highly thermostable based
on their melting points. Hence, the probe 7HQM-DNP has
excellent chemical and thermal stability.
In summary, we have prepared two fluorescent probes,
7HQM-DNP and 6HQM-DNP, containing a quinolinium
unit as a fluorophore and the strongly electron-withdrawing
2,4-dinitrophenoxy group as a recognition unit. The results
reveal that 7HQM-DNP has a high sensitivity (detection
limit: 8 nm) and high selectivity with regard to detection of
thiophenols over aliphatic thiols and other nucleophiles. The
sensing reaction undergoes an SNAr process to yield turn-on
fluorescence from an almost non-fluorescent probe to
a strongly fluorescent thiolysis product. The selectivity
under physiological conditions (pH 7.3) derives from the dif-
ference in pKa values of thiophenols (ca. 6.5) and aliphatic
thiols (ca. 8.5). Although the two probes have a similar reac-
tivity with thiophenols, the sensitivity of 7HQM-DNP is
much higher than that of 6HQM-DNP due to the formation
of a TICT state in 6HQM-DNP, which results in a low fluo-
rescence efficiency. We furthermore showed that 7HQM-
DNP can be used for the intracellular detection of thiophe-
nol. As the probes have an excellent chemical stability over
a wide pH range and high thermostability, they may find use
for the quantitative analysis of thiophenols in environmental
samples and biological systems.
Cellular Detection of Thiophenol
To examine whether thiophenol can be detected intracellu-
larly by using 7HQM-DNP, MDA-MB-231 cells were incu-
bated with 7HQM-DNP in a growth medium for 30 min and
washed thrice with PBS buffer, followed by treatment with
thiophenol for 30 min and imaging by confocal fluorescence
microscopy (Figure 10). The bright fluorescence signals of
the cells shows that 7HQM-DNP is cell-permeable and can
react with thiophenol in living cells (Figure 10d). Cells incu-
bated with only 7HQM-DNP in one control experiment did
not result in a fluorescent signal (Figure 10a).In another
control experiment, the MDA-MB-231 cells were pre-treat-
ed with thiophenol (5 mm), then incubated with N-ethylma-
leimide (1 mm), which consumes completely the thiophenol,
and at last incubated with 7HQM-DNP (5 mm). These cells
also did not yield a fluorescent signal (Figure 10 g). Thus,
cellular imaging indicates a good specificity of 7HQM-DNP
for thiophenol over biothiols and other biomolecules in
living cells.
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Synthesis
Experimental Section
7-(2,4-Dintrophenoxy)-2-methylquinoline (3)
Materials and Methods
Compound 1 (202 mg, 1 mmol), 7-hydroxyquinoline (159 mg, 1 mmol),
potassium carbonate (472 mg, 4 mmol), and 10 mL of DMF were mixed
in a 50 mL flask and stirred for 2 h at 1008C. The reaction mixture was
then cooled at room temperature, concentrated to dryness, and the resi-
due was purified by flash chromatography (EtOAc/petroleum ether 1:4),
affording 3 as a brown solid (250 mg, 77%). M.p. 134–1358C; Rf =0.32
(EtOAc/petroleum ether 1:2); 1H NMR (300 Hz, CDCl3, 258C, TMS):
d=8.90 (d, J=8.2 Hz, 1H), 8.37 (dd, J=2.2 Hz, J=2.7 Hz, 1H), 8.14 (d,
J=2.7 Hz, 1H), 7.92 (d, J=8.9 Hz, 1H), 7.68 (d, J=2.2 Hz, 1H), 7.36 (d,
J=8.4 Hz, 1H), 7.35 (dd, J=8.4 Hz, J=2.2 Hz, 1H), 7.24 (d, J=8.8 Hz,
1H), 2.77 ppm (s, 3H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=
160.6, 155.1, 155.0, 148.2, 142.2, 136.4, 130.3, 128.9, 124.5, 122.3, 122.2,
120.3, 119.4, 116.8, 25.2 ppm; IR (Nujol): n˜ =1601 (s), 1532 (m), 1347
(m), 1272 (m), 1153 (s), 834 (s), 743 cmꢀ1 (s); FTMS (ESI) calcd for
C16H12N3O5: 326.0771 [M+H+], found 326.0773.
Solvents for organic synthesis were reagent grade, and were dried prior
to use. 2,4-Dinitrochlorobenzene, 2-methyl-7-hydroxy-quinoline,[8] and
2,4-dinitrophenyl phenylsulfide[11] were prepared according to reported
methods. Other chemicals were purchased from commercial sources and
used as received. Double distilled water was used throughout the experi-
ments.
1H and 13C NMR spectra were recorded in CDCl3 or [D6]DMSO on
a Bruker AV spectrometer operated at 300 MHz and 75 MHz, respective-
ly, and chemical shifts are reported in ppm using tetramethylsilane
(TMS) as an internal standard. FT-IR spectra were measured with
a Bruker Vector22 Infrared Spectrometer. Mass spectra were obtained
using a Micromass GCF TOF mass spectrometer and a Thermo LTQ Or-
bitrap mass spectrometer (ESI). UV/Vis absorption and fluorescence
emission spectra were measured at room temperature with a Shimadzu
UV-2450 UV/Vis spectrometer and an F-4600 Fluorescence Spectropho-
tometer, respectively.
7HQM-DNP
Spectral Measurements
Compound 3 (144 mg, 0.4 mmol) and ethyl iodide (128 mg, 0.8 mmol)
were dissolved in 15 mL toluene, and the mixture was stirred for 48 h at
1008C under nitrogen atmosphere. The reaction was then cooled to room
temperature, and concentrated to dryness. Recrystallization of the crude
product in methanol/CH2Cl2/petroleum ether gave the target product as
black crystals (60 mg, 30%). M.p. 203–2058C; 1H NMR ((300 MHz,
[D6]DMSO): d=9.11 (d, J=8.5 Hz, 1H), 9.01 (d, J=2.8 Hz, 1H), 8.56
(dd, J=9.2 Hz, J=2.8 Hz, 1H), 8.54 (d, J=9.0 Hz, 1H), 8.41 (d, J=
2.1 Hz, 1H), 8.10 (d, J=8.5 Hz, 1H), 7.88 (d, J=9.1 Hz, J=2.1 Hz, 1H),
7.56(d, J=9.2 Hz, 1H), 4.94 (m, 2H), 3.12 (s, 3H), 1.46 ppm (t, 3H);
13C NMR (75 MHz, [D6]DMSO): d=161.2, 159.3, 152.8, 145.4, 143.1,
140.4, 139.7, 133.7, 123.0, 126.0, 125.0, 122.2, 121.8, 108.4, 47.3, 22.3,
13.3 ppm; IR (Nujol): n˜ =1610 (s), 1528 (m), 1344 (m), 1328 (m), 1238
(s), 833 (s), 739 cmꢀ1 (s); FTMS (ESI) calcd for C18H16N3O5+: 354.1084
[M], found 354.1084.
A stock solution of the sensor (2.5ꢁ10ꢀ3 m) was prepared in DMSO, and
then sample solutions of 7HQM-DNP (25 mm) were prepared by adding
25 mL of the sensor stock solution using a microsyringe to 2.5 mL of
0.01m sodium phosphate buffer (pH 7.3) in a quartz cuvette (1 cmꢁ
1 cm). After addition of 10 mL of an analyte (cysteine, PhNH2, PhOH,
glucine, PhCH2SH, or CH3PhSH) in ethanol (25 mm), the resulting solu-
tion was kept for 20 min at room temperature and the emission of the so-
lution was recorded. Fluorescence quantum yield was determined using
quinine sulfate (Fr =0.546 in 1n H2SO4)[12] as a reference.
Cell Culture and Fluorescence Imaging
MDA-MB-231 cells were grown in Dulbeccoꢂs modified Eagle Medium
supplemented with 10% fetal calf serum, 1% penicillin, and 10000
unitsmLꢀ1 of streptomycin at 378C under humidified air containing 5%
CO2. Cells (20000) were located and stabilized in a single well of a 24-
well plate. The cells were incubated with 7HQM-DNP (5 mm) for 30 min
at 378C, and used directly for fluorescence imaging in a control experi-
ment. Other cells were washed with PBS three times to remove the re-
maining 7HQM-DNP. The cells were further incubated with thiophenol
(5 mm) for 30 min at 378C. At last, after washing with PBS three times to
remove remaining thiophenol, fluorescence imaging was undertaken by
using a confocal microscope (Zeiss LSM 510 Meta NLO), at an excita-
tion wavelength of 405 nm. For another control experiment, MDA-MB-
231 cells were incubated with thiophenol (5 mm) for 30 min at 378C, then
washed with PBS three times to remove remaining thiophenol, and incu-
bated with N-ethylmaleimide (1 mm) for 30 min at 378C. Subsequntly,
after washing three times with PBS, the cells were incubated with
7HQM-DNP (5 mm) for 30 min at 378C for fluorescence imaging.
1-Ethyl-2-methyl-7-hydroxyquinolinium iodide (7HQM)
Compound 2 (0.9 g, 6 mmol) and ethyl iodide (1.5 g, 9 mmol) was dis-
solved in toluene (25 mL). Under nitrogen atmosphere, the mixture was
stirred for 48 h at 1008C. Then the reaction was cooled at room tempera-
ture, concentrated to dryness. The crude product was obtained. The resi-
due was purified using flash chromatography, isopropanol as fluent
phase. 7HQM as a black crystals was obtained by recrystallization from
methanol/CH2Cl2 (1.2 g, 62%). M.p.> 2458C cabonization; 1H NMR
(300 MHz, [D6]DMSO): d=11.71 (s, 1H), 8.90 (d, J=6.3 Hz, 1H), 8.25
(d, J=6.9 Hz, 1H), 7.81 (d, J=6.3 Hz, 1H), 7.59 (d, J=1.5 Hz, 1H), 7.51
(dd, J=6.9 Hz, J=1.5 Hz, 1H), 4.74 (m, 2H), 3.23 (s, 3H), 1.43 ppm (t,
3H); 13C NMR (75 Hz, [D6]DMSO): d=157.2, 156.4, 143.7, 132.5, 130.3,
126.6, 125.4, 120.6, 111.0, 47.0, 21.6, 13.5 ppm; IR (Nujol): n˜ =3405 (m),
1628 (m), 1602 m, 1526 (s), 1321 (s), 1240 (s), 1161 (s), 856 cmꢀ1 (s);
FTMS (ESI) calcd for C12H14NO+: 188.1070 [MꢀIꢀ], found 188.1065.
Kinetic Analysis
The reaction of probes (25 mm) with thiophenol in 0.01m PBS (pH 7.3) at
room temperature was monitored by measuring the absorbance at
398 nm. The pseudo-first-order rate constant, kobs, for the reaction was
determined by using Equation (1),
2,4-Dinitrophenyl phenylsulfide
It was prepared according to published procedures.[11] 1H NMR
(300 MHz, CDCl3, 258C, TMS): d=8.89 (d, J=2.7 Hz, 1H), 8.35 (dd,
J1 =J2 =9.0 Hz, 1H), 7.68 (m, 5H), 7.02 ppm (d, J=9.0 Hz, 1H).
0
0
ln½ðA0ꢀA0 Þ=ðAꢀA0 Þꢁ ¼ ꢀkobs
t
ð1Þ
6-(2,4-Dinitrophenoxy)-2-methylquinoline (4)
Compound 1 (202 mg, 1 mmol), 6-hydroxyquinoline (159 mg, 1 mmol),
potassium carbonate (472 mg, 4 mmol), and 10 mL of DMF were mixed
in a 50 mL flask. After stirring for 2 h at 1008C, the reaction mixture was
cooled at room temperature, concentrated to dryness, and the residue
was purified by flash chromatography, affording 4 as a yellow solid
(280 mg, 86%). M.p. 108–1108C; Rf =0.30 (EtOAc/petroleum ether 1:6);
1H NMR (300 MHz, CDCl3, 258C, TMS): d=8.89 (d, J=2.7 Hz, 1H),
8.36 (dd, J=9. 2 Hz, J=2.7 Hz, 1H), 8.18 (d, J=8.9 Hz, 1H), 8.05 (d, J=
8.2 Hz, 1H), 7.52 (m, 2H), 7.39 (d, J=8.5 Hz, 1H), 7.10 (d, J=9.2 Hz,
1H), 2.79 ppm (s, 3H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=
where A0 and A are the absorbance of the reaction solution before and
after the addition of thiophenol, and A0’ is the absorbance of the corre-
sponding quinolinium at complete conversion of probes. The second-
order rate constant k (Mꢀ1 sꢀ1), was obtained from Equation (2),
kobs ¼ k ½Mꢁ
ð2Þ
where [M] is the concentration of thiophenol.
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Quinolinium-based Fluorescent Probes
158.7, 154.8, 149.8, 144.8, 140.7, 138.8,134.7, 130.8, 127.8, 126.2, 122.2,
122.9, 121.1, 117.9, 115.9, 24.2 ppm; FTMS (ESI) calcd for C16H12N3O5:
326.0771 [M+H+], found 326.0767.
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6HQM-DNP
Compound 4 (80 mg, 0.22 mmol) and ethyl iodide (64 mg, 0.4 mmol)
were dissolved in toluene (15 mL) under a nitrogen atmosphere, and the
mixture was stirred for 48 h at 1008C. The reaction mixture was then
cooled at room temperature and concentrated to dryness. The obtained
crude product was recrystallized from methanol/CH2Cl2/petroleum ether
to yield the target product as yellow crystals (65 mg, 62%). M.p.>1608C
(carbonization); 1H NMR (300 MHz, [D6]DMSO): d=9.01 (d, J=2.7 Hz,
1H), 8.98 (d, J=8.6 Hz, 1H), 8.75 (d, J=10.0 Hz, 1H), 8.62 (dd, J=
9.2 Hz, J=3.0 Hz 1H), 8.19–8.15 (m, 3H), 7.57 (d, J=9.0 Hz, 1H), 5.05
(m, 2H), 3.11 (s, 3H), 1.55 ppm (t, 3H); 13C NMR (75 MHz,
[D6]DMSO): d=160.2, 153.9, 152.9, 144.8, 143.1, 140.5, 135.6, 129.9,
129.8, 127.7, 126.6, 122.2, 122.1, 122.0, 117.4, 47.6, 22.2, 13.5 ppm; FTMS
(ESI) calcd for C18H16N3O5+: 354.1084 [M], found 354.1078.
1-Ethyl-2-methyl-6-hydroxyquinolinium iodide (6HQM)
6-hydoxyquinoline (100 g, 0.6 mmol) and ethyl iodide (0.15 g, 0.9 mmol)
were dissolved in toluene (15 mL) under a nitrogen atmosphere, and the
mixture was stirred for 48 h at 1008C. The reaction was then cooled at
room temperature and concentrated to dryness. The obtained crude
product was recrystallized in methanol/CH2Cl2 to yield the target product
as a puce crystal (140 mg, 74%). M.p.>2208C (carbonization); 1H NMR
(300 MHz, [D6]DMSO): d=10.96 (s, 1H), 8.89 (d, J=8.7 Hz, 1H), 8.48
(d, J=9.6 Hz, 1H), 7.99 (d, J=8.7 Hz, 1H), 7.72 (dd, J=2.4 Hz, J=
9.4 Hz, 1H), 7.54 (d, J=2.5 Hz, 1H), 4.95 (m, 2H), 3.02 (s, 3H),
1.52 ppm (t, 3H); 13C NMR (75 MHz, [D6]DMSO): d=163.9, 159.3,
144.6, 141.9, 132.6, 122.5, 121.0, 120.9, 101.3, 47.0, 22.7, 13.5 ppm; FTMS
(ESI) calcd for C12H14NO+: 188.1070 [M-Iꢀ], found 188.1065.
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Acknowledgements
We are grateful for financial support from the National Natural Science
Foundation of China (Grant No. 20972149) and the Natural Science
Foundation of Anhui Province (1208085MB19). We thank Dr. Jun Wang
for access to cell culture facilities.
[12] J. N. Demas, G. A. Crosby, J. Phys. Chem. 1971, 75, 991–1024.
Received: July 4, 2012
Published online: && &&, 0000
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Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
7
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FULL PAPER
Biosensors
Xiu-Ling Liu, Xue-You Duan,
Xiao-Jiao Du,
Qin-Hua Song*
&&&& — &&&&
Quinolinium-Based Fluorescent
Probes for the Detection of Thiophe-
nols in Environmental Samples and
Living Cells
Receiving high recognition: Two fluo-
rescent probes were designed and syn-
thesized for detection of thiophenols.
Weakly fluorescent probe molecules
are rapidly converted into strongly flu-
orescent molecules through thiolysis,
thus giving a fluorescent turn-on
response. The sensing reaction reveals
high sensitivity (detection limit of
8 nm) and high selectivity for thiophe-
nols over aliphatic thiols and other
nucleophiles under neutral conditions
(pH 7.3).
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!