A native-chemical-ligation-mechanism-based ratiometric fluorescent probe for aminothiols

Article abstract of DOI:10.1002/chem.201201606

Thiol-containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide-α-thioester and an N-terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Fo?rster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160-fold) in its fluorescence ratio (I ₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells. Copyright

Full text of DOI:10.1002/chem.201201606

FULL PAPER
DOI: 10.1002/chem.201201606
A Native-Chemical-Ligation-Mechanism-Based Ratiometric Fluorescent
Probe for Aminothiols
Lin Yuan, Weiying Lin,* Yinan Xie, Sasa Zhu, and Sheng Zhao[a]
Abstract: Thiol-containing amino acids
(aminothiols) such as cysteine (Cys)
and homocysteine (Hcy) play a key
role in various biological processes in-
cluding maintaining the homeostasis of
biological thiols. However, abnormal
levels of aminothiols are associated
with a variety of diseases. The native
chemical ligation (NCL) reaction has
attracted great attention in the fields of
chemistry and biology. NCL of peptide
segments involves cascade reactions
between a peptide-a-thioester and an
N-terminal cysteine peptide. In this
work, we employed the NCL reaction
mechanism to formulate a Fçrster reso-
nance energy transfer (FRET) strategy
for the design of ratiometric fluores-
cent probes that were selective toward
aminothiols. On the basis of this new
strategy, the ratiometric fluorescent
probe 1 for aminothiols was judiciously
designed. The new probe is highly se-
lective toward aminothiols over other
thiols and exhibits a very large varia-
tion (up to 160-fold) in its fluorescence
ratio (I458/I603). The new fluorescent
probe is capable of ratiometric detec-
tion of aminothiols in newborn calf and
human serum samples and is also suita-
ble for ratiometric fluorescent imaging
of aminothiols in living cells.
Keywords: aminothiols · biological
activity
·
fluorescent probes
·
FRET · native chemical ligation
Introduction
which provides a built-in correction for the environmental
effects.[5]
Thiol-containing amino acids (aminothiols) such as cysteine
(Cys) and homocysteine (Hcy) play a key role in various
bioACHTUNGTRENNUNGlogical processes including maintaining the homeostasis
The native chemical ligation (NCL) reaction has attracted
great attention in the fields of chemistry and biology.[6] Hun-
dreds of proteins have been prepared by total or semisyn-
thesis when using the NCL reaction. NCL of peptide seg-
ments involves cascade reactions between a peptide-a-thio-
ester and an N-terminal cysteine peptide. Prompted by the
chemoselective and in vivo compatible characters of the
NCL reaction, we have previously employed the reaction to
design a ratiometric fluorescent probe for thiols (Fig-
ure 1a)[7] on the basis of the Fçrster resonance energy trans-
fer (FRET) signaling mechanism.[8] However, the probe was
constructed by linking an energy donor to a rhodamine ac-
ceptor dye through a thioester linker (the interaction site).
of biological thiols, biocatalysis, metal binding, post-transla-
tional modifications, and detoxification of xenobiotics.[1]
However, abnormal levels of aminothiols are associated
with a variety of diseases such as liver damage, skin lesions,
slowed growth, edema, dementia, Alzheimerꢀs disease, coro-
nary heart disease, carotid therosclerosis, dystonia, psoriasis,
clinical stroke, lung damage, Parkinsonꢀs disease, and
asthma.[2] In light of the high sensitivity and easy operation
of fluorescece detection, a diverse array of fluorescent
probes selective toward aminothiols have been reported.[3,4]
However, the vast majority of them respond to aminothiols
with fluorescence signals that change only in intensity. The
main limitation of intensity-based probes is that variations
in the sample environment and probe distribution might be
problematic for their utilization in quantitative measure-
ments. By contrast, ratiometric fluorescent probes exhibit
spectral shifts upon interaction with the analytes of interest,
À
Thus, any compounds that contain a free SH group can
induce the cleavage of the FRET dyad and release the
energy donor. In other words, the probe is not capable of
distinguishing the aminothiols from other biological thiols
such as glutathione (GSH) and a-lipolic acid in a ratiometric
manner. To address this problem, herein we propose a novel
NCL-based FRET strategy to design ratiometric fluorescent
probes selective toward aminothiols over other biological
thiols (Figure 1b). In the new design scheme, unlike the pre-
vious strategy, the FRET donor is not connected to the thio-
ester interaction site but instead attached to a linker, which
is then bridged to the FRET acceptor. On the basis of the
reaction mechanism of the NCL reaction, we reasoned that
treatment of compound A with aminothiols (e.g., Cys) can
afford the intermediate thioester B1 by the reversible trans-
thioesterification reaction, which might further undergo irre-
versible intramolecular nucleophilic attack by the nearby
[a] L. Yuan, Prof. W. Lin, Y. Xie, S. Zhu, S. Zhao
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha, Hunan 410082 (P.R. China)
Fax: (+86)731-888-21464
E-mail: weiyinglin@hnu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201606.
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Figure 1. a) Known FRET approach to the development of ratiometric fluorescent probes for thiols based on the NCL reaction.[7] b) A proposed novel
NCL-based FRET strategy to design ratiometric fluorescent probes selective toward aminothiols over other biological thiols.
amino group to form amide C. As the FRET is on and off in
compounds A and C, respectively, we should observe a sig-
nificant change in the ratiometric fluorescent signal. On the
other hand, treatment of A with thiols might induce the for-
mation of the thioester B2, which cannot further undergo in-
tramolecular arrangement, because B2 lacks an amino
group in proximity. As the FRET is on in both A and B2,
no marked changes in the ratiometric fluorescent signal can
be noted. Thus, taken together, the new strategy (Figure 1b)
might be employed to construct ratiometric fluorescent
probes selective toward aminothiols over other biological
thiols.
Results and Discussion
The synthesis of probe 1 started with compound 2, which
was obtained by our previously reported method.[9] Reaction
of 2 with N-hydroxysuccinimide gave the succinimidyl ester
intermediate, which was further treated with 4-mercapto-
phenol to provide the target compound 1 (Scheme 1). Nota-
bly, compound 1 is stable as a solid, and it can be stored for
more than one year in a freezer.
The absorption spectrum of the FRET dyad 1 in phos-
phate buffer solution (PBS; pH 7.4, which contained 10%
DMF as a cosolvent) is shown in Figure S1 in the Supporting
Information. As designed, the absorption spectrum of the
FRET dyad 1 exhibits the typical absorption of rhodamines
at around 571 nm and the characteristic absorption of cou-
marins at around 395 nm. As shown in Figure S2 in the Sup-
porting Information, when
With this hypothesis in mind, in this work, we present
compound 1 (Scheme 1) as a new NCL-mechanism-based
ratiometric fluorescent probe with specificity toward amino-
thiols.
FRET dyad 1 was excited at
395 nm (the coumarin donor
absorption) in PBS (pH 7.4,
which contained 10% DMF as
a
cosolvent), the featured
emission of the coumarin
donor around 458 nm was no-
tably quenched when com-
pared with that of the refer-
ence
coumarin
donor
3
(Scheme 1). This suggests that
the excitation energy of the
coumarin donor is efficiently
transferred to the rhodamine
Scheme 1. Synthesis of the ratiometric fluorescent probe 1 and the structure of the reference energy donor 3.
Reagents and conditions: a) N,N’-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide, CH2Cl2; b) 4-mer-
captophenol, triethylamine (TEA).
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Native Chemical Ligation
FULL PAPER
acceptor. The energy-transfer efficiencies of dyad 1 were
calculated to be 95.3%.[10] The presence of highly efficient
energy transfer in the FRET dyad is further collaborated by
the excellent correspondence between the absorption and
excitation spectra of dyad 1 (Figure S1 in the Supporting In-
formation). Notably, the donor and acceptor emission bands
are essentially completely separated with a remarkable sepa-
ration (up to 145 nm, Figure S1 in the Supporting Informa-
tion) between the emission maxima. Thus, the FRET plat-
form appears to be promising for the construction of ratio-
metric fluorescent probes with highly favorable spectral
properties.
around 395 nm (Figure S5a in the Supporting Information),
which was attributed to the coumarin absorption, was
almost unchanged. This was further confirmed by a mass
spectrometry analysis of the probe treated with Cys (Fig-
ure S6 in the Supporting Information). A similar fluores-
cence response was also observed for other aminothiols such
as Hcy and cysteamine (Figure S7 and S8 in the Supporting
Information), although a higher amount of Hcy or cyste-
AHCTUNGTRENNUNG
only slight variations over a wide span of pH from 4.0 to
10.0 (Figure S9 in the Supporting Information). The ratio-
metric response of the probe to Cys is pH-dependent. When
The changes in the absorption and emission spectra of
probe 1 in the presence of Cys are exhibited in Figure 2.
the pH increased from 4.0 to 7.4, the emission ratio (I458/I603
)
significantly enhanced from 0.79 to 25, and then a plateau
was reached at higher pH values. In addition, probe 1 could
also work in 25 mm potassium phosphate buffer (pH 7.4,
which contained 5% DMF as a cosolvent) and physiological
conditions (e.g., pure newborn calf serum without any or-
ganic cosolvent) (Figure S10 and S11 in the Supporting In-
formation).
The time course of the emission ratios of probe 1 (3 mm)
at 458 and 603 nm (I458/I603) in the presence of Cys or Hcy
(0.5 mm) is shown in Figure 3. Upon introduction of Cys, a
Figure 2. Fluorescence spectral changes (with excitation at 395 nm) of 1
(3 mm) upon addition of increasing concentrations (0–250 mm) of Cys in
potassium phosphate buffer (25 mm; pH 7.4, containing 10% DMF as co-
solvent) for 30 min. The inset shows the changes to the fluorescent inten-
sity ratio at 458 and 603 nm (I458/I603) on increasing cthe oncentration of
Cys.
The free probe 1 displays an emission band at 603 nm (rho-
ACHTUNGTRENNUNGdamine moiety). However, treatment of Cys induces a
marked decrease in the emission intensity at 603 nm and
concurrently a dramatic enhancement of the emission inten-
sity at 458 nm (coumarin moiety). Thus, a significant emis-
sion blueshift (145 nm) was observed. Notably, the probe ex-
hibits two well-resolved emission peaks. This is favorable for
measurement of the emission intensity and signal ratios with
high precision. The emission ratio (I458/I603) shows a drastic
variation from 0.154 in the absence of Cys to 24.9 in the
presence of Cys (250 mm), a 160-fold enhancement (Figure 2,
inset). Furthermore, the emission ratios (I458/I603) are linearly
proportional to the concentrations of Cys (1–100 mm) (Fig-
ure S3 in the Supporting Information) with a detection limit
(signal/noise=3) of 6.4ꢂ10À7 m, which suggests that the
probe is potentially useful for quantitative determination of
aminothiol concentrations over a large dynamic range. In
addition, when excited at 550 nm, the fluorescent intensity
of the probe at 603 nm was gradually decreased with the ad-
dition of elevated concentrations of Cys (Figure S4 in the
Supporting Information). In accordance with the emission
changes, the addition of an increasing amount of Cys to
probe 1 triggered a gradual decrease in the absorption at
576 nm (Figure S5a in the Supporting Information), thus in-
dicating that the rhodamine acceptor is in the ring-closed
form in the presence of Cys, whereas the absorption band at
Figure 3. The time course of the emission ratios of 1 (3 mm) at 458 and
603 nm (I458/I603) in the presence of Cys (&) or Hcy (*; 0.5 mm) in potas-
sium phosphate buffer (25 mm; pH 7.4, containing 10% DMF as a cosol-
vent).
dramatic enhancement in the emission intensity was noted,
and the intensity essentially reached a maximum in 12 min.
A similar fluorescence increase was also observed for Hcy,
although it took up to 20 min for the maximum intensity to
be reached. Under pseudo-first-order kinetic conditions[4h,11]
(3 mm probe 1 and 0.5 mm Cys or Hcy), the observed rate
constant (kobs) was determined to be 0.419 minÀ1 for Cys or
0.335 minÀ1 for Hcy.
To examine the selectivity, probe 1 was treated with vari-
ous biologically relevant species including representative
aminothiols (e.g., Cys, Hcy, cysteamine), thiol-containing
species (e.g., GSH, 1,4-dithiothreitol (DTT), mercaptoetha-
nol, a-lipolic acid, 4-methylbenzenethiol, 4-chlorobenzene-
thiol), amino acids (e.g., Phe, Ala, Gly, Arg, Lys, Tyr, Leu,
Ser, Val), and glucose. As shown in Figure 4, introduction of
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Figure 5. The visual fluorescence emission of probe 1 (3 mm) toward vari-
ous species (16 equiv) for 30 min on excitation at 365 nm by using a UV
lamp at room temperature: a) 4-chlorobenzenethiol; b) 4-methylbenzene-
thiol; c) GSH; d) Cys; e) DTT; f) mercaptoethanol; g) a-lipolic acid;
h) blank; i) Phe; j) Arg; k) Leu; l) Ala; m) glucose; and n) Ser. For a
color version, see Figure S17 in the Supporting Information.
Figure 4. a) Fluorescence spectra (with excitation at 395 nm) of 1 (3 mm)
in the presence of various analytes (250 mm) in potassium phosphate
buffer (25 mm; pH 7.4, containing 10% DMF as cosolvent) for 30 min.
b) The fluorescent ratio of 1 (3 mm) at 458 and 603 nm (I458/I603) in the
presence of various biologically relevant analytes (250 mm): A) free;
B) Cys; C) Hcy; D) cysteamine; E) 4-methylbenzenethiol; F) 4-chloro-
benzenethiol G) GSH; H) mercaptoethanol; I) DTT; J) a-lipolic acid;
K) Ala; L) Gly; M) Tyr; N) Arg; O) Lys; P) Leu; Q) Phe; R) glucose;
S); Ser; and T) Val.
aminothiols (Cys, Hcy, cysteamine) to the probe induced a
dramatic ratiometric response. By contrast, no noticeable
changes in the emission were observed upon addition of the
other thiol-containing species, thus indicating that the probe
is specific for the detection of aminothiols over other thiols.
In good accordance with the fluorescence changes, the intro-
duction of aminothiols (Cys, Hcy, cysteamine) to the probe
induced a gradual decrease of the absorption at 576 nm
(Figure S5b in the Supporting Information). By contrast, no
noticeable changes in the absorption at 576 nm were ob-
served upon addition of the other thiol-containing species
(Figure S5b in the Supporting Information), thus indicating
that the probe is specific toward the detection of amino-
thiols over other thiols. Furthermore, the emission color of
probe 1 is analyte-dependent (Figure 5), which indicates
that the probe could be employed for convenient visual
sensing of Cys. Notably, probe 1 can respond to Cys even in
the presence of a high concentration of GSH (Figure 6 and
Figure S12 in the Supporting Information), thereby further
supporting the evidence for high selectivity toward amino-
thiols over other thiols. It is known that some transition-
metal ions might influence the fluorescence of rhodamine.[8a]
Thus, we also investigated the potential fluorescence re-
sponse of probe 1 to the representative transition-metal ions
such as Cu2+, Hg2+, Fe3+, Cr3+, Pb2+, Zn2+, and Ag+ (Fig-
ure S13 in the Supporting Information), and the results indi-
Figure 6. Fluorescence spectral changes (excitation at 395 nm) of 1 (3 mm)
upon addition of increasing concentrations (0–1500 mm) of Cys in potassi-
um phosphate buffer (25 mm; pH 7.4, containing 10% DMF as cosolvent)
containing GSH (1 mm) for 30 min.
cated that these transition-metal ions only showed a mini-
mal effect on the fluorescence of the probe.
The high selectivity of probe 1 based on the NCL reaction
mechanism was further corroborated by mass spectrometry
studies. Treatment of probe 1 with mercaptoethanol (as a
representative thiol) only gave the ring-opened thioester
product (Figure S14b in the Supporting Information). By
contrast, incubation of probe 1 with Cys provided the cy-
clized amide product (Figure S6 in the Supporting Informa-
tion). These results are in good agreement with the design
strategy shown in Figure 1.
The above studies demonstrate that the novel ratiometric
fluorescent probe has key features that include high sensitiv-
ity, a large ratio signal variation, and high selectivity even in
the presence of 1 mm GSH. Thus, the probe seems to be
useful for the detection of aminothiol in real biological sam-
ples. To test this possibility, probe 1 was employed to detect
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Native Chemical Ligation
FULL PAPER
aminothiols in newborn calf and human serum samples. Ali-
quots of a reduced newborn calf serum sample were added
to a solution of probe 1, and a linear enhancement in the
emission ratios (I458/I603) was noted (Figure 7a), thus indicat-
experiments were also conducted. The cells were pretreated
with N-ethylmaleimide (NEM, as an aminothiol/thiol-reac-
tive reagent[13]) for 60 min, and further incubated with the
probe for 30 min, thereby providing significant red fluores-
cence (Figure 8d) but almost no green fluorescence (Fig-
ure 8c), in accordance with the emission profile of the free
probe. The cells pretreated with N-acetylcysteine (NAC, a
membrane-permeable precursor for cysteine),[14] and then
incubated with probe 1 showed a marked decrease in the
red emission (Figure 8f) and a significant increase in the
green emission (Figure 8e), which is consistent with the ami-
nothiol-induced ratiometric fluorescent response. The cells
pretreated with a-lipolic acid (a cell-permeable biological
thiol without an amino group), and then treated with probe
1 showed very slight changes both in the green (Figure 8g)
and red emission windows (Figure 8h), which is in good
agreement with the above observation that other thiols are
not capable of inducing a ratiometric response (Figure 4).
Thus, these data establish that probe 1 is cell-membrane-per-
meable and therefore suitable for ratiometric imaging of
aminothiols in living cells.
Figure 7. a) Linear relationship between the emission ratios and the
volume of the reduced newborn calf serum that was added. The data
were obtained by the addition of different amounts of the reduced new-
born calf serum sample (0–500 mL) to probe 1 (3 mm) for 30 min. b) Plot
showing the variation in the amount of total aminothiol levels in human
serum sample (300 mL) from an individual 1) before and 2) after break-
fast.
Conclusion
In summary, we have employed the NCL reaction mecha-
nism to formulate a FRET strategy for the design of a ratio-
metric fluorescent probe that is selective toward amino-
thiols. Notably, unlike the previous NCL-based ratiometric
fluorescent probe,[7] the new probe 1 is highly selective
toward aminothiols over other thiols. The novel probe ex-
hibits a very large variation (up to 160-fold) in the fluores-
cence ratio (I458/I603). Importantly, we have demonstrated
that probe 1 can detect aminothiols in real biological sam-
ples including newborn calf and human serum samples in a
ratiometric fashion. Furthermore, we have confirmed that
probe 1 is suitable for the ratiometric fluorescent imaging of
aminothiols in living cells.
ing that the probe is capable of detecting aminothiols in the
plasma sample. Furthermore, probe 1 displays a larger emis-
sion ratio (I458/I603) in the presence of a human serum
sample from an individual after breakfast than one before
breakfast (Figure 7b), thereby suggesting that the level of
aminothiols in human serum sample after breakfast is
higher than before breakfast, which is consistent with previ-
ous reports.[12]
To demonstrate the capability of the novel probe for ra-
tiometric fluorescence imaging of aminothiols in living cells,
the living HepG2 cells were incubated with probe 1, and
strong green fluorescence in the coumarin emission window
(Figure 8a) and red fluorescence in the rhodamine emission
window (Figure 8b) could be observed. A series of control
Experimental Section
Materials and instruments: Unless otherwise stated, all reagents were
purchased from commercial suppliers and used without further purifica-
tion. Solvents used were purified by standard methods prior to use.
Twice-distilled water was used throughout all experiments. Human serum
sample was obtained from the first hospital of Changsha City. Melting
points of compounds were measured using a Beijing Taike XT-4 micro-
scopy melting-point apparatus, and all melting points were uncorrected.
Mass spectra were performed using an LCQ Advantage ion-trap mass
spectrometer from Thermo Finnigan or an Agilent 1100 HPLC/MSD
spectrometer. NMR spectra were recorded using a Bruker AV-500 spec-
trometer, with TMS as an internal standard. Electronic absorption spec-
tra were obtained using a LabTech UV Power spectrometer. Photolumi-
nescent spectra were recorded using a Hitachi F4600 fluorescence spec-
trophotometer. Cell imaging was performed using an Olympus fluores-
cence microscope. TLC analysis was performed on silica gel plates, and
column chromatography was conducted over silica gel (mesh 200–300),
both of which were obtained from Qingdao Ocean Chemicals.
Figure 8. Fluorescence images of the living HepG2 cells treated with
probe 1. a,b) Cells incubated with only 1 (3 mm) for 30 min. c,d) Cells
pretreated with NEM (1 mm) for 60 min, and then incubated with 1
(3 mm) for 30 min. e,f) Cells pretreated with NAC (1 mm) for 60 min, and
then incubated with 1 (3 mm) for 30 min. g,h) Cells pretreated with a-lip-
olic acid (1 mm) for 60 min, and then incubated with 1 (3 mm) for 30 min.
For a color version, see Figure S18 in the Supporting Information.
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W. Lin et al.
Synthesis of compound 1: Compound 2 (45.0 mg, 0.06 mmol), N-hydroxy-
succinimide (21.0 mg, 0.18 mmol), and DCC (37.1 mg, 0.18 mmol) were
dissolved in CH2Cl2 (5.0 mL) with stirring at room temperature. After
30 min, triethylamine (0.05 mL) and 4-mercaptophenol (11.5 mg,
0.09 mmol) were added. The reaction solution was stirred for another
30 min at room temperature, and the mixture was then concentrated
under vacuum. The resulting residue was subjected to preparative thin-
layer chromatography (CH2Cl2/CH3OH=5:1) to provide compound 1 as
a carmine powder (31.5 mg, 61.0% yield). 1H NMR (CD3OD, 500 MHz):
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d=1.31 (12H; CH3CH2), 3.58–3.93 (16H; CH3CH2 and N
ACHTNUGERTN(NUGN CH2CH2)2N),
6.71 (d, J=8.0 Hz, 2H; xanthene-H), 6.74 (s, 1H; coumarin-H), 6.85 (d,
À
À
J=8.0 Hz, 1H; Ar H), 6.95 (4H; xanthene-H and Ar H), 7.05 (s, 2H;
À
À
xanthene-H), 7.23 (2H; Ar H), 7.55 (d, J=8.5 Hz, 1H; Ar H), 7.63 (d,
J=6.0 Hz, 1H; coumarin-H), 8.00 (d, J=5.5 Hz, 1H; coumarin-H), 8.07
(s, 1H; coumarin-H), 8.41 ppm (s, 1H; Ar H); 13C NMR (CD3OD,
À
À
À
125 MHz): d=12.7 (CH3CH2), 46.8 (CH3CH2), 97.3 (Ar C), 103.3 (Ar
C), 112.3 (Ar C), 115. 1 (Ar C), 115.5 (Ar C), 116.2 (Ar C), 117.3
À
À
À
À
À
À
À
À
À
(Ar C), 120.1 (Ar C), 128.9 (Ar C), 131.6 (Ar C), 132.2 (Ar C), 132.3
À
À
À
À
À
(Ar C), 132.6 (Ar C), 133.6 (Ar C), 137.2 (Ar C), 138.9 (Ar C), 139.1
À
À
À
À
À
(Ar C), 145.8 (Ar C), 157.0 (Ar C), 157.5 (Ar C),157.6 (Ar C), 159.0
À
À
(Ar C), 160.5 (Ar C), 164.3 (C=O), 166.6 (C=O), 170.1 (C=O),
+
À
192.1 ppm (S C=O); ESI-MS: m/z: 851.2. [M] ; HRMS (ESI): m/z calcd
for: C49H47N4O8S1 [M]+: 851.3148; found: 851.3109; elemental analysis
calcd (%) for C49H47N4O8S+·H2PO4À·4H2O: C 57.64, H 5.63, N 5.49;
found: C 57.39, H 5.99, N 5.21. The presence of water molecules is con-
sistent with the hygroscopic nature of the compound.
Cell culture and living cell imaging studies with probe 1: HepG2 cells
were obtained from the third hospital of Xiangya and cultured in Dulbec-
co’s modified eagle medium (DMEM) supplemented with 10% fetal
bovine serum and 1% penicillin–streptomycin at 378C in a 5% CO2/
95% air environment. The cells were seeded on 12-well plates and stabi-
lized for 24 h. Before imaging, the cells were washed with PBS three
times and then incubated with probe 1 (3 mm) in PBS for 30 min under an
atmosphere of 5% CO2/95% air at 378C. For the control experiment, the
cells were pretreated with 1 mm NEM (or NAC or a-lipolic acid) for
60 min, and then incubated with probe 1 (3 mm) in PBS for 30 min in an
atmosphere of 5% CO2/95% air at 378C. After washing with PBS three
times to remove the remaining probe, the fluorescence images were ac-
[7] L. Long, W. Lin, B. Chen, W. Gao, L. Yuan, Chem. Commun. 2011,
47, 893.
quired using an Olympus fluorescence microscope equipped with
cooled CCD camera.
a
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Acknowledgements
Funding was partially provided by NSFC (20872032, 20972044,
21172063), NCET (08–0175), the Doctoral Fund of Chinese Ministry of
Education (20100161110008), and the Fundamental Research Funds for
the Central Universities, Hunan University.
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Native Chemical Ligation
FULL PAPER
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Received: May 8, 2012
Revised: July 17, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!

W. Lin et al.
Fluorescent Probes
L. Yuan, W. Lin,* Y. Xie, S. Zhu,
S. Zhao ........................ &&&& — &&&&
A Native-Chemical-Ligation-Mecha-
nism-Based Ratiometric Fluorescent
Probe for Aminothiols
Going native: A ratiometric fluores-
cent probe for aminothiols has been
designed based on the native chemical
ligation (NCL) reaction mechanism
(see scheme). The probe is highly
selective toward aminothiols over
other thiols and exhibits a very large
variation (up to 160-fold) in the fluo-
rescence ratio (I458/I603).
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www.chemeurj.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!