A sensitive and selective fluorescent probe for cysteine based on a new response-assisted electrostatic attraction strategy: The role of spatial charge configuration

Article abstract of DOI:10.1002/chem.201300078

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response-assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m-CP, a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response-assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys-preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m-CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria. Copyright

Full text of DOI:10.1002/chem.201300078

FULL PAPER
DOI: 10.1002/chem.201300078
A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New
Response-Assisted Electrostatic Attraction Strategy: The Role of Spatial
Charge Configuration
Xin Zhou, Xuejun Jin, Guangyan Sun, and Xue Wu*[a]
Abstract: A new strategy for fast fluo-
rescent detection of cysteine (Cys),
based on a response-assisted electro-
static attraction, is demonstrated. By
utilizing this strategy, we designed and
synthesized three fluorescent probes
for the specific detection of Cys under
actual physiological conditions. The
probe m-CP, a coumarin fluorophore
conjugated with a substituted methyl
pyridinium group through an unsatu-
rated ketone unit, showed highly selec-
tive and sensitive detection for cysteine
(Cys) over homocysteine (Hcy) and
glutathione (GSH). The kinetic analy-
sis indicated that the sensing process
was highly accelerated (a response
time less than 1 min) by the response-
assisted electrostatic attraction. More
importantly, control experiments with
isomeric probes first demonstrated that
the spatial charge configuration of the
probe played an important role in Cys-
preferred selectivity and kinetic rate
acceleration. Furthermore, the practical
utility of the probe m-CP in the fluo-
rescent labeling of Cys residues within
proteins was demonstrated. Finally,
these probes were employed in living
cell imaging with HeLa cells, in which
it displayed satisfactory cell permeabili-
ty and enabled us to distinguish active
thiols in the cytoplasm, nucleus, and
mitochondria.
Keywords: amino acids · cell imag-
ing · fluorescent probes · proteins ·
thiols
Introduction
under physiological conditions (pH 7.4), the thiol group of
Cys is presumably changed to the thiolate, which becomes a
better nucleophile. The other one is the steric shielding
effect. The thiol group of Cys is sterically less hindered than
those of Hcy and GSH.[6c] Considerable efforts have been
made on the basis of these two strategies.[4,5b,6d,7] However,
many of them are still associated with some common limita-
tions, including the use of a biotoxic organic or organic/
water medium, poor biocompatibility, and a long response
time in particular. These disadvantages restricted this probe
from further biological application. Thus, there still remains
a need to construct simple and effective fluorescent probes
for highly selective and sensitive detection of Cys, particular-
ly those based on novel alternative detection principles.
As we know, during the sensing processes, both thiols and
probe are maintained at very low concentrations, and effec-
tive collisions of them decrease relatively, which directly
leads to a low reaction rate and a long response time.[8]
Therefore, it is significant to consider accelerating the sens-
ing processes by some feasible means during probe design.
We note that biothiols have low pI values (5.02 for Cys, 5.27
for Hcy, and 5.93 for GSH).[9] Thus, under physiological con-
ditions (pH 7.4), biothiols presumably adopt their negatively
charged forms. Therefore it is reasonable to construct posi-
tively charged probes for those negatively charged thiols by
utilizing the electrostatic attraction between them to acceler-
ate sensing processes.
Among the twenty amino acids commonly found in proteins,
cysteine (Cys) is special in that it is present more often than
other residues in functionally important locations.[1] Abnor-
mal levels of Cys can cause several health problems such as
slowed growth, aging, edema, lethargy, liver damage, oxida-
tive damage, metabolic disorders, and AIDS.[2] Moreover,
the total cellular Cys concentration is significantly associat-
ed with a high risk of a wide variety of tumor progressions.[3]
Therefore, there currently exists significant interest in con-
structing small molecular probes (SMP) for the fluorescent
detection and real-time imaging of such species.
Up to now, numbers of thiol-specific probes have been de-
veloped on the basis of various strategies,[4] such as cleavage
reactions,[5] Michael addition reactions,[6] metal complexes/
displaced coordination, and others.[7] However, only a few
probes able to discriminate Cys from homocysteine (Hcy)
and glutathione (GSH) have been achieved to date. Gener-
ally, the conventional principle for designing Cys-specific
probes is mainly focused on two strategies. One is the diver-
sity of the nucleophilic ability of thiols. Cys has a lower pKa
(8.30) than those of Hcy (8.87) and GSH (9.20).[6a] Thus,
[a] X. Zhou, Dr. X. Jin, Dr. G. Sun, Prof. X. Wu
Department of Chemistry, Faculty of Science
Yanbian University
Yanji, Jilin, 133002 (P.R. China)
Fax : (+86)433-2732456
E-mail: wuxue@ybu.edu.cn
Taking these factors into account, we previously proposed
a novel strategy for the construction of a Cys-specific probe
by combining Michael addition and response-assisted elec-
trostatic attraction.[10] As shown in Scheme 1, we reported
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300078.
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more sensitive. 3) After the ad-
dition of thiols, the extended
conjugate is broken, accompa-
nied by the intramolecular
charge-transfer (ICT) process
turning off; therefore, a colori-
metric and turn-on fluorescent
dual response could be expect-
ed. 4) Moreover, the frequen-
cies of effective collisions be-
tween the cationic probe and
negative thiols will be promot-
ed through response-assisted
electrostatic interactions, thus,
the kinetic rates of the sensing
process will be accelerated and
the response time will be de-
creased. 5) The biocompatible
pyridinium group imbues the
probe with appreciable cell per-
meability for subcellular imag-
ing.
Scheme 1. Top: Structures of probes. Bottom: Design concept of probe m-CP for turn-on fluorescent sensing
of Cys based on the Michael addition promoted by a response-assisted electrostatic attraction strategy.
such a cationic probe, m-CP, a coumarin fluorophore conju-
gated with a substituted methyl pyridinium group through
an unsaturated ketone unit, which displayed a turn-on fluo-
rescent response to Cys in 100% aqueous solution, and in
particular enabled the sensitive and selective detection of
Cys over Hcy and GSH. We think that the spatial charge
configuration of the probe might play an important role in
this specific detection of Cys. To gain insight into the above-
mentioned hypotheses, we present here a further detailed
investigation of probe m-CP and its isomeric probes (o-CP
and p-CP) with different spatial charge configurations.
Moreover, the application of the probe m-CP in sensing Cys
within proteins and subcellular imaging is also investigated.
As expected, probe m-CP displayed appropriate water
solubility. At the same time, it also showed a typical polari-
ty-sensitive property. With increasing polarities of solvents
(polarity factor from 4.2 to 10.2), the main p–p* transition
absorption (peak at 475 nm) increased from 0.2 to 0.5, but
its fluorescence intensity decreased dramatically (Figure S1
in the Supporting Information). Probe m-CP was almost
nonfluorescent in water (fF =0.001). To further illustrate
these phenomena, DFT/time-dependent (TD)-DFT calcula-
tions were subsequently conducted at the B3LYP/6-31G(d)/
level using Gaussian 09.[11] Water was used as the solvent in
the calculations (polarizable continuum model (PCM)). As
confirmed (Figure S2 in the Supporting Information), the
electron-density distribution of the HOMO!LUMO transi-
tion (S0!S1) of probe m-CP was allowable (oscillator
strength f=0.4030), which indicated that a strong intramo-
lecular charge-transfer process took place from the electron-
donating diethyl amino group to the electron-deficient pyri-
dinium moiety. However, the S1!S0 transition is forbidden
(f=0.0000), which means that the probe is essentially non-
fluorescent. After the addition of thiols, the conjugated
system will be broken and the ICT process should be
blocked in the m-CP–Cys adduct. Thus, a turn-on fluores-
cent response might be reasonably predicted.
Results and Discussion
Design, synthesis, and properties of m-CP: The cationic
probe m-CP was conveniently synthesized from the conden-
sation of coumarin and m-pyridinecarboxaldehyde, and then
treated with iodomethane at room temperature (Scheme S1
in the Supporting Information). To better understand the
role of the spatial charge configuration during the thiol-sens-
ing processes, the isomeric probes o-CP and p-CP with dif-
ferent cationic pyridinium substitutes, as well as their neu-
tral precursors (P1, P2, and P3) were also prepared for con-
trol studies.
The rational design concepts of the probe are shown in
Scheme 1 and are described as follows: 1) The cationic
probe will provide appropriate water solubility, which allows
the sensing processes to be performed under 100% physio-
logical conditions. 2) Owing to the strong electron-withdraw-
ing character of the pyridinium moiety, the b-carbon atom
of the unsaturated ketone will be more reactive for the Mi-
chael reaction with thiols, which causes the probe to become
Colorimetric and fluorescent dual-response of probe m-CP
for Cys: Consequently, the sensing response of probe m-CP
to Cys was first investigated in phosphate-buffered saline
(PBS; pH 7.4; Figure S12 in the Supporting Information).
Upon addition of an increasing amount of Cys, a new emis-
sion peak around 500 nm emerged, and a dramatic enhance-
ment in the fluorescence intensity was observed (Figure 1a).
Correspondingly, the visual emission color of m-CP turned
bright green immediately (Figure 1b, inset).
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A Fluorescent Probe for Cysteine
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Figure 2. 1H NMR spectral changes of probe m-CP (10 mg) upon addi-
tion of Cys (1 equiv) in [D6]DMSO at room temperature. a) Probe m-CP
only; b) m-CP–Cys adduct.
(calcd 484.1), which was consistent with the formation of the
expected [m-CP–Cys]+ adduct.
Fluorescent selectivity: The sensing selectivity of m-CP
toward thiols over other biological species was investigated
in PBS (pH 7.4). After addition of various analytes, only
thiols (Cys, Hcy, and GSH) caused turn-on responses in the
fluorescence spectra (Figure S4 in the Supporting Informa-
tion); others, including common amino acids (Ala, Arg,
Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr, Val), as well as common biological metal ions
(Zn2+and Fe3+), reactive oxygen species (H2O2), adenosine
triphosphate (ATP), and glucose, caused no significant
changes. The similar change was also observed in the ab-
sorption spectra (Figure S4 in the Supporting Information).
Only thiols induced clear blueshifts; others induced no clear
alterations. It is worth noting that probe m-CP exhibits high
selectivity toward Cys over Hcy and GSH. Addition of Cys
caused a 148-fold increase in the emission intensity, whereas
the analogous addition of Hcy and GSH resulted in only a
13- and 9-fold enhancement, respectively (Figure 3).
Figure 1. a) Fluorescence response spectra and b) UV/Vis spectra of
probe m-CP (10 mm) upon gradual addition of Cys (0–10 mm) in PBS
(pH 7.4, lex =450 nm). Inset (a): Normalized response of the fluorescence
signal versus Cys concentrations. Inset (b): Colorimetric and fluorescent
responses of probe m-CP (0.1 mm) towards Cys (0.1 mm) in PBS
(pH 7.4). A represents the probe m-CP and B represents the m-CP–Cys
adduct.
Furthermore, the fluorescence intensity is proportional to
the amount of Cys added; a coefficient of R=0.9934 and a
detection limit of 25 nm were obtained from fitting the
curves (Figure 1a, inset).[12] As shown in the absorbance
spectra (Figure 1b), addition of Cys caused a clear blueshift
(about 23 nm) from 475 to 452 nm along with a dramatic
color change from red to yellow (Figure 1b, inset). The col-
orimetric and fluorescent dual response is attributed to the
formation of the m-CP–Cys adduct through 1,4-Michael ad-
dition, thereby resulting in the prohibition of the aforemen-
tioned ICT process of probe m-CP.[6a,13] This hypothesis was
1
Figure 3. Fluorescent enhancement factors (FEF) of probe m-CP (10 mm)
upon exposure to different biological analytes (0.1 mm) in PBS (pH 7.4,
lex =450 nm). Inset: FEF of probe m-CP toward Cys (10 equiv) in the
presence of different competing analytes (100 equiv) in PBS (pH 7.4).
confirmed by H NMR spectroscopy (Figure 2). Upon addi-
tion of Cys, the vinylic protons at d=8.18 (Hb) and
7.71 ppm (Ha) dramatically disappear with the concomitant
appearance of two new peaks at d=4.60 and 3.77 ppm that
correspond to the saturated protons, which is consistent with
the formation of the m-CP–Cys adduct. This formation was
further confirmed by a TOF-MS spectrometric analysis (Fig-
ure S3 in the Supporting Information). Probe m-CP treated
with Cys in PBS (pH 7.4) revealed a signal at m/z: 484.1
Kinetic analysis and electrostatic attrition effect: To further
investigate the comparison of the reactivity of probe m-CP
toward Cys, Hcy, or GSH, the time-dependent fluorescence-
response spectra were analyzed by monitoring the fluores-
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X. Wu et al.
cence changes of the reaction mixture in PBS (pH 7.4). As
shown in Figure 4a, the emission intensity immediately
reached a maximum within 1 min after addition of Cys
(10 equiv). This addition reaction was also rapidly complet-
ed even at trace concentration levels of Cys (1-10 mm; Fig-
ure S6 in the Supporting Information). Hcy and GSH pre-
sented similar and slight fluorescent increases, but at much
lower rates, and they were more time-consuming. A reason-
able explanation of this high sensitivity could be due to the
electrostatic attractions between the probe and Cys. Under
experimental conditions (pH 7.4), Cys became more nega-
tive than Hcy and GSH because of its lower pI values than
those of Hcy and GSH. Therefore, the frequencies of effec-
tive collisions between negative Cys and positive probe m-
CP are highly promoted by electrostatic attractions, thus
leading to an acceleration of the reaction rate.
To confirm the hypothesis that the response-assisted elec-
trostatic interaction plays an important role during the sens-
ing process, the neutral probes P1, P2, and P3 were also in-
vestigated under the same conditions. It is interesting that
all the probes displayed similar turn-on fluorescent respons-
es to thiols based on the aforementioned mechanism. How-
ever, compared to cationic probe m-CP, neutral probe P1
displayed poor sensitivity and selectivity. In the case of
probe P1, the sensing processes were more time-consuming.
The response lifetime of compound P1 for Cys was 52.8 min
(Table 1), which was much longer than that of probe m-CP
Table 1. Observed lifetimes (t) through the kinetic analysis on the basis
of a single-exponential decay model.
t [min]
Cys
Hcy
GSH
P1
52.82
0.33
0.47
0.41
21.35
35.26
61.94
18.83
277.01
12.07
10.45
34.12
m-CP
o-CP
p-CP
(0.33 min). Moreover, probe P1 displayed poorer selectivity
for Cys over Hcy and GSH than that of m-CP. The ratio of
the kinetic rate of probe P1 towards Cys/Hcy was just 1.5,
but it became 23.6 in the case of probe m-CP. The sensing
process of probe P2 for Cys was also time-consuming, and it
could not differentiate Cys from Hcy.[6c] This evidence sup-
ports the hypothesis that the electrostatic attraction plays a
significant role in the kinetic rate acceleration during the
sensing process.
Spatial charge configuration effect: Kinetic analysis on the
basis of a single-exponential decay model[14] showed that
probe m-CP reacted with Cys 115 times faster than Hcy, and
36 times faster than GSH, respectively. The reactivity in the
order Cys@Hcy>GSH can be partly attributed to the
lower steric hindrance effect and the lower pKa value of Cys
relative to that of the other thiols.[15] However, this explana-
tion might be applicable to many other thiol probes and
might not be unique to probe m-CP. Another possible
reason for this preference could be attributed to the spatial
charge configuration of the probe.
To gain further insight into this hypothesis, isomeric
probes o-CP and p-CP were investigated under the same
conditions. Probes o-CP and p-CP displayed similar fluores-
cent responses to m-CP. Both of them displayed high selec-
tivity and sensitivity toward Cys within a short response
time (0.47 min for probe o-CP, and 0.41 for p-CP, respec-
tively). However, unexpectedly, probes o-CP and p-CP
showed unsatisfactory fluorescent stability with their corre-
sponding Cys adducts. As shown in Figure 4b and c, their
fluorescence intensity reached a maximum within 1 min, and
then decreased dramatically over a period of time. After
30 min, their fluorescence was almost quenched; only probe
m-CP still maintained a satisfactory fluorescent stability and
showed a high selectivity ratio for Cys over Hcy and GSH.
Figure 4. Time-course fluorescence response spectra of the probes
(10 mm) towards Cys, Hcy, or GSH (10 equiv, respectively) in PBS
(pH 7.4, lex =450 nm). The fluorescence intensity was recorded near
500 nm at room temperature. a) Spectra of probe m-CP; b) spectra of
probe o-CP; c) spectra of probe p-CP. The times were recorded after
3 min.
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A Fluorescent Probe for Cysteine
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This result was also confirmed by time-dependent absorb-
ance spectra (Figure S9 in the Supporting Information); the
absorption peak at 450 nm for the o-CP–Cys adduct gradu-
ally decreased over 30 min. Similar changes were observed
with the p-CP–Cys adduct. However, no significant change
was observed in the absorption of the m-CP–Cys adduct.
On the basis of a previous report on the formation of a
seven-membered heterocycle by treatment of a,b-unsaturat-
ed aldehydes with Cys,[16] a possible mechanism is proposed
in Scheme 2. Initially, the thiol group of Cys reacts with the
a,b-unsaturated ketone moiety by Michael addition, which
induces such turn-on fluorescent responses. And then the
amino group of Cys can undergo a ring-closure reaction to
afford a more stable seven-membered heterocycle, which fi-
nally quenches the fluorescence of the adducts. In the case
of m-CP, the appropriate spatial charge configuration makes
its Cys adduct adopt a more stable conformation (the posi-
tive pyridinium moiety of the probe allows the formation of
an effective electrostatic attraction with the carboxyl group
of Cys), which prevents ring closure. On the other hand, the
mismatched spatial charge configuration of o-CP and p-CP
make their Cys adducts much more unstable (electrostatic
interaction between the positive pyridinium group and the
negative carboxyl group is not effective in maintaining their
stable conformations). As a result, they underwent a cycliza-
tion reaction and quenched their fluorescence. Moreover,
this proposed mechanism was also confirmed by TOF-MS
spectra, in which a peak at 466.0 that corresponds to the
cyclic adduct was found. On the basis of the above data and
the proposed mechanism, we can confirm that the high se-
lectivity for Cys over Hcy and GSH is significantly depend-
ent on the spatial charge configuration of the probes. This
could be considered an important principle for the further
design of Cys-specific probes.
Fluorescent labeling of protein through covalent attachment
of free Cys residue: With these desirable properties of
probe m-CP taken into account, it was further employed to
detect free Cys residues within proteins. In this study,
bovine serum albumin (BSA) was used as a template pro-
tein because it has only one free cysteine residue at position
34 (Cys34), which is necessary to maintain its biological func-
tions such as antioxidant properties, and binding of a variety
of drugs and physiological ligands.[17] The sensing processes
were monitored with fluorescence spectra.
As shown in Figure 5a, with addition of BSA, a gradual
fluorescent enhancement emerged. Meanwhile, remarkable
colorimetric and fluorescent color changes were also ob-
served. As shown in Figure 5b, BSA was colorless and ini-
tially gave a blue emission. After the addition of probe m-
CP, a bright bluish–green emission emerged along with a
visible color change to yellow. This indicated that the probe
m-CP conferred selective covalent binding to the Cys34 resi-
due within BSA. However, compared to the fast response of
probe m-CP toward free Cys, this sensing process was more
time-consuming (about 2 h). As we know, the Cys34 residue
of BSA has an unusually low pKa (<6.7) and exists primari-
ly in the highly nucleophilic thiolate form. Thus, such a long
Scheme 2. Proposed mechanisms for different fluorescent response of Cys adducts
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X. Wu et al.
lin was used because it contains three disulfide bonds and
no free Cys residue (Figure 5d).[20] The results indicated that
insulin initially induced no fluorescent response. However,
after reduction of the disulfide bonds of insulin with dl-di-
thiothreitol (DTT), a remarkable fluorescence enhancement
was observed (Figure 4e). A similar response was also found
with DTT-treated BSA, whereby its 17 pairs of disulfide
bonds were reduced to free thiols. This evidence indicated
that the probe m-CP could be considered an effective tool
for labeling free Cys residues within proteins.
Fluorescent confocal imaging in HeLa cells: Cellular thiol-
containing biomolecules are generally active in a wide vari-
AHCTUNGTREGeNNNU ty of malignant cancers. They play multiple roles in tumor
invasion and metastasis, such as cellular signal transductions,
cancer-cell proliferation, and tumor growth.[3a,b,6b,21] Thus, it
is of great significance to monitor these active thiols in
cancer cells. Owing to the highly sensitive, selective, and
turn-on fluorescent responses for thiols, probe m-CP can be
considered a promising tool for cellular thiol imaging. Con-
sequently, the actual utility of the probe in confocal fluores-
cent imaging was conducted with HeLa cells. As shown in
Figure 6a, probe m-CP gave a bright green emission, which
revealed the actual distributions of intracellular active
thiols. Unfortunately, probes o-CP and p-CP showed no flu-
orescent signal under the same experimental conditions
(Figure 6b,c), which might be due to their unsatisfactory flu-
orescent stability. To further confirm the staining region of
probe m-CP, clearer pictures at the single-cell level were
captured. As shown in Figure 6d, probe m-CP mainly
stained not only in cytoplasm (around endoplasmic reticu-
lum) but also in nucleoli, which indicated that there were
more active thiols located in these regions. Moreover, cells
in different cell cycles with different nucleoli were easily dis-
tinguished. As shown in Figure 6d, cell A is in the inter-
Figure 5. a) Fluorescence titrations of BSA (10 mm) towards probe m-CP
(0–2 equiv) in PBS (pH 7.4). Inset: The stoichiometric ratio between
BSA and probe m-CP. b) Colorimetric and fluorescent responses of
probe m-CP (0.1 mm) after the addition of native BSA (0.1 mm) in PBS
(pH 7.4). A represents the native BSA, B represents labeled BSA with
probe m-CP, and C represents the probe m-CP only. c) The partially
modeled structure of BSA containing the Cys34 residue obtained from
molecular docking, in which Cys34 is covalently conjugated with probe m-
CP. c) The structure of insulin (1a7f). d) The fluorescence response of
probe m-CP (1 mm) towards insulin (without free Cys), DTT-treated in-
sulin, native BSA (with only one free cys34), and DTT-treated BSA. All
the concentrations of proteins were maintained at 0.1 mm in PBS
(pH 7.4). The times were recorded after 30 min.
response time might be mainly attributed to the conserved
microenvironment around Cys34. To confirm this possibility,
molecular modeling[18] was performed. As shown in Fig-
ure 5c, the Cys34 residue was buried in a crevice (6 ꢁ) within
domain I, in which it stayed in a unique microenvironment
close to three ionizable residues: Asp38, His39, and Tyr84.[19]
Such a conserved motif prevents Cys34 from reacting with
probe m-CP efficiently.
Figure 6. Confocal fluorescence images of HeLa cells stained with probes
(5 mm) a) m-CP, b) o-CP, and c) p-CP, respectively. These cells were incu-
bated with probes for 1 h at 378C before imaging. Individual Hela cells
were also co-stained with probe m-CP (5 mm) and Mito tracker (2 mm):
c) green emission for probe m-CP, d) red emission for Mito-tracker, and
f) merged. These cells were incubated with probes for 1 h at 378C before
imaging.
In addition, the fluorescent response towards protein
without free Cys residues was also investigated. Here, insu-
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Preparation of probe m-CP: Compound P1 (100.0 mg, 0.28 mmol) and
an excess amount of iodomethane were mixed with acetonitrile (15 mL),
and then the mixture was stirred in darkness at room temperature for
10 h. Then, a bright brick-red precipitate was gained by addition of cold
absolute ethanol (10 mL). The precipitate was collected by filtration and
recrystallization from absolute ethanol gave probe m-CP (80 mg,
phase with one nucleolus, and cell B is in the telophase with
double nucleoli.[22] To further confirm the stain region of
probe m-CP in cytoplasm, HeLa cells were co-stained with
MitoTracker (Figure 6e). The merged picture (Figure 6f) in-
dicates that probe m-CP also revealed those active thiols in
mitochondria (yellow regions).[23] This indicated that probe
m-CP was able to permeate through multiple membranes,
such as cytoplasmic membrane, the nuclear envelope, and
the mitochondrial membrane. This evidence proves that
probe m-CP should be considered an ideal probe for subcel-
lular imaging of active thiols.
1
0.22 mmol, 82%). H NMR (300 MHz, DMSO): d=9.39 (s, 1H), 8.96 (d,
J=6.0 Hz, 1H), 8.86 (d, J=8.2 Hz, 1H), 8.65 (s, 1H), 8.17 (dd, J=15.0,
5.4 Hz, 2H), 7.71 (dd, J=12.6, 3.2 Hz, 2H), 6.84 (dd, J=9.0, 2.0 Hz, 1H),
6.63 (s, 1H), 4.36 (s, 3H), 3.50 (q, J=7.1 Hz, 4H), 1.48 ppm (t, J=7.1 Hz,
6H); 13C NMR (75 MHz, DMSO): d=185.16, 159.99, 158.58, 153.53,
149.09, 145.48, 145.23, 142.94, 134.88, 133.99, 132.80, 131.07, 127.81,
114.31, 110.64, 108.10, 96.16, 48.14, 44.47, 12.61 ppm; MS: m/z calcd:
363.4 [M+]; found: 363.43.
DFT/TD-DFT calculations: The UV/Vis absorption and emission proper-
ties of probe m-CP were studied by DFT/TD-DFT calculations at the
B3LYP/6-31G (d)/level of theory by using Gaussian 09. Water was used
as the solvent in the calculations (PCM). First, the optimized ground-
state geometry of probe m-CP was obtained with several possible con-
formers. By using the lowest-energy conformer of probe m-CP, the struc-
ture of m-CP–Cys was obtained. The UV/Vis absorption was calculated
by the TD-DFT method on the basis of the ground-state geometry (verti-
cal excitation, Franck–Condon principle). The emission was calculated
with the TD-DFT method (usually the excited state is responsible for the
fluorescence; Kashaꢂs rule). The vertical excitation and the emission-re-
lated calculations were based on the optimized excited state.
Conclusion
We have designed and developed a new Cys-specific fluores-
cent probe m-CP based on a novel response-assisted electro-
static attraction strategy. The probe showed a rapid response
and selectivity for detection of Cys over Hcy and GSH.
Control studies of neutral precursors (P1, P2, and P3) and
isomers (o-CP and p-CP) first revealed that both electro-
static attraction and spatial charge configuration played im-
portant roles in the sensitivity and selectivity of the probe
toward Cys. Furthermore, the practical utility of the probe
in fluorescent detection in the microenvironment of Cys34
within BSA was demonstrated. In particular, nonreactive
protein without free Cys but with disulfide bonds showed a
remarkable fluorescent response after treatment with DTT.
Finally, the ability of the probe to determine the subcellular
distribution of active thiols was investigated with HeLa
cells. The probe displayed satisfactory cell permeability and
enabled us to distinguish active thiols in cytoplasm, nucleus,
and mitochondria. Encouraged by these desirable attributes
for bioassays, we expect that it could be employed in further
biological applications, including single-molecule spectrosco-
py and fluorescence-based protein-interaction studies. More-
over, the design strategy employed here should be applica-
ble to other fluorophores, such as rhodamines, boron–dipyr-
romethene (bodipys), and cyanines, which would allow con-
trol of excitation and emission wavelengths, leakage from
cells, and localization to organelles. Efforts toward this end
are underway in our laboratory.
Cell-culture and confocal microscopy experiments: The HeLa cells were
cultured on the surface of a glass slide in SPP medium (1% proteose
peptone, 0.2% glucose, 0.1% yeast extract, 0.003% EDTA ferric sodium
salt) at 378C. For confocal microscopy experiments, HeLa cells were
stained with probes (5 mm) for 1 h at 378C and washed three times with
PBS, and then confocal fluorescent images were captured with an excita-
tion light at 450 nm. In addition, HeLa cells were co-stained with probe
m-CP (5 mm) and Moti tracker (2 mm) for 1 h at 378C, washed three times
with PBS, and then individual excitation light (l450 (nm) for m-CP and
l540 (nm) for Mito tracker, respectively) was used to gain distinct fluores-
cent pictures at the single-cell level.
Acknowledgements
We thank the Natural Science Foundation of China (NSFC) (grant nos.
21065013 and 21165019) for support of this work.
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Received: January 9, 2013
Revised: March 11, 2013
Published online: && &&, 0000
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ÝÝ
These are not the final page numbers!

A Fluorescent Probe for Cysteine
FULL PAPER
Cysteine grapple: A new strategy for
the development of a cysteine (Cys)-
specific probe, based on a response-
assisted electrostatic attraction, is dem-
onstrated. We constructed three fluo-
rescent probes with isomeric structures
(see scheme). We demonstrate that the
spatial charge configuration plays an
important role in the Cys-preferred
selectivity and sensitivity.
Fluorescent Probes
X. Zhou, X. Jin, G. Sun,
X. Wu* ......................... &&&& — &&&&
A Sensitive and Selective Fluorescent
Probe for Cysteine Based on a New
Response-Assisted Electrostatic
Attraction Strategy: The Role of
Spatial Charge Configuration
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
9
&
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These are not the final page numbers!