Sensitive near-infrared fluorescent probes for thiols based on Se-N bond cleavage: Imaging in living cells and tissues

Article abstract of DOI:10.1002/chem.201200671

Cy-NiSe and Cy-TfSe were designed and synthesized as sensitive near-infrared (NIR) fluorescent probes for detecting thiols on the basis of Se-N bond cleavage both in cells and in tissues. Since a donor-excited photoinduced electron transfer (d-PET) process occurs between the modulator and the fluorophore, Cy-NiSe and Cy-TfSe have weak fluorescence. On titration with glutathione, the free dye exhibits significant fluorescence enhancement. The two probes are sensitive and selective for thiols over other relevant biological species. They can function rapidly at pH 7.4, and their emission lies in the NIR region. Confocal imaging confirms that Cy-NiSe and Cy-TfSe can be used for detecting thiols in living cells and tissues. Turn-on fluorescent probes Cy-NiSe and Cy-TfSe, which show weak fluorescence due to donor-excited photoinduced electron transfer, release near-infrared fluorescent dye Cy7-MeAm on cleavage of the Se-N bond by thiols. Thus, they can be used for real-time imaging of thiols such as glutathione in living cells and tissue (see figure). Copyright

Full text of DOI:10.1002/chem.201200671

FULL PAPER
DOI: 10.1002/chem.201200671
À
Sensitive Near-Infrared Fluorescent Probes for Thiols Based on Se N Bond
Cleavage: Imaging in Living Cells and Tissues
Rui Wang,[a] Lingxin Chen,*[a] Ping Liu,[a] Qin Zhang,[b] and Yunqing Wang[a]
Abstract: Cy-NiSe and Cy-TfSe were
designed and synthesized as sensitive
near-infrared (NIR) fluorescent probes
have weak fluorescence. On titration
with glutathione, the free dye exhibits
significant fluorescence enhancement.
The two probes are sensitive and selec-
tive for thiols over other relevant bio-
logical species. They can function rap-
idly at pH 7.4, and their emission lies
in the NIR region. Confocal imaging
confirms that Cy-NiSe and Cy-TfSe
can be used for detecting thiols in
living cells and tissues.
À
for detecting thiols on the basis of Se
N bond cleavage both in cells and in
tissues. Since a donor-excited photoin-
duced electron transfer (d-PET) pro-
cess occurs between the modulator and
the fluorophore, Cy-NiSe and Cy-TfSe
Keywords: cleavage reactions · flu-
orescent probes · imaging agents ·
À
Se N bonds · thiols
Introduction
electrochemical assay,[10] UV/Vis assay,[11] and fluorescence
spectroscopy[12] have been used for detection of thiols. Com-
pared with other technologies, the fluorescence method pro-
vides greater sensitivity, less invasiveness, and the availabili-
ty of bioimaging in living cells and tissues.[13] In the past few
years, various mechanisms have been exploited, including
Michael addition,[14] cyclization with an aldehyde,[15] cleav-
age by thiols,[16] and so on.[17] Most of the reported fluores-
cent probes emit in the ultraviolet or visible region, which
can lead to cell autofluorescence.[18,19] In contrast, long-
wavelength probes with emission in the near-infrared (NIR)
region are optimal for biological imaging applications due to
minimal photodamage to biological samples and minimum
interference from background autofluorescence in living sys-
tems. Recently, fluorescent NIR imaging probes are being
increasingly used for optical imaging of live animals.[20]
Therefore, it is necessary to develop fluorescent probes that
can be used for rapid detection of thiols under physiological
conditions, preferably with emission in the NIR region.
Glutathione peroxidase (GPx) is an enzyme family with
peroxidase activity whose main biological role is to protect
the organism from oxidative damage.[21] Ebselen (2-phenyl-
1,2-benzoisoselen-azol-3(2H)one), a well-known mimetic of
GPx with low toxicity, exhibits anti-inflammatory, anti-ather-
osclerotic, and cytoprotective properties.[22] According to
Thiol compounds in vivo, such as cysteine (Cys), homocys-
teine (Hcy), and glutathione (GSH), play key roles in phys-
iological and pathological processes. Abnormal levels of cel-
lular thiols have been linked to a number of diseases, such
as leucocyte loss, liver damage, cancer, and AIDS.[1] Specifi-
cally, cysteine is a nonessential amino acid, deficiency of
which is involved in many syndromes, such as slow growth
in children, liver damage, skin lesions, and loss of muscle
and fat.[2,3] At elevated levels in plasma, Hcy is a risk factor
for Alzheimerꢀs disease and cobalamin (vitamin B12) defi-
ciency.[4] More importantly, GSH is the most abundant intra-
cellular nonprotein thiol (1–10 mm).[5] It has vital roles in
maintenance of intracellular redox activity, intracellular
signal transduction, and gene regulation.[6] Glutathione
exists in reduced (GSH) and oxidized (GSSG) states. An in-
creased or decreased GSSG-to-GSH ratio is considered
indicative of diseases in vivo.
Accordingly, the detection of thiol levels in biological sys-
tems is very important. Several analytical techniques, includ-
ing HPLC,[7] capillary electrophoresis,[8] mass spectrometry,[9]
[a] R. Wang, Prof. L. X. Chen, P. Liu, Y. Wang
Key Laboratory of Coastal Zone Environmental Processes
Yantai Institute of Coastal Zone Research (YIC)
Chinese Academy of Sciences (CAS)
Shandong Provincial Key Laboratory of
Coastal Zone Environmental Processes
YICCAS, Yantai Shandong 264003, 17 Chunhui Road
Yantai 264003 (P.R. China)
previous studies,[23] the Se N bond in ebselen is readily
À
cleaved by thiols to produce the corresponding selenenyl
sulfide. This mechanism inspired us to design fluorescent
À
probes containing an Se N bond to detect thiols.
As shown in Scheme 1, we designed fluorescent NIR
Fax: (+86)535-2109130
À
probes Cy-NiSe and Cy-TfSe containing an Se N bond for
E-mail: lxchen@yic.ac.cn
detecting thiols via donor-excited photoinduced electron
transfer (d-PET). A cyanine dye, as one type of widely em-
ployed NIR fluorophores with high molar absorption coeffi-
cient, was selected as signal transducer, and strongly elec-
tron withdrawing 2-nitrophenylselane and 3-(trifluoro-
[b] Q. Zhang
Linshu Peopleꢀs Hospital of Shandong Province
Linshu 276700 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200671.
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room temperature, the reaction
mixture was filtered and evapo-
rated under reduced pressure.
Crude Cy-NiSe was purified by
silica gel chromatography. Next,
Cy-TfSe was synthesized by
a simple method. KSeCN and
3-bromobenzotrifluoride were
dissolved in DMF and stirred
under Ar atmosphere at room
temperature. Cy7-MeAm, tri-
ethylamine, and CuI were
added to the mixture in turn
under Ar. The solution was
Scheme 1. Structure and design concept of Cy-NiSe and Cy-TfSe probes for thiols.
stirred for 24 h at room temper-
methyl)phenylselane groups as modulators. The probes fea-
ture fast response, emission in the NIR region, and stable
fluorescence signal over a wide pH range in aqueous solu-
tion. Furthermore, Cy-NiSe and Cy-TfSe were used for
rapid sensing of thiols in living RAW 264.7 cells and fresh
rat liver tissue.
ature, and a blue powder was obtained after workup and re-
moval of the solvent under vacuum. NMR spectroscopy and
HRMS were employed to characterize the structures of the
intermediates and final products.
Results and Discussion
Design and synthesis of probes Cy-NiSe and Cy-TfSe: To
obtain thiol-selective fluorescent probes with fluorescence
emission in the NIR region under physiological conditions
for potential biological applications, we selected a hepta-
methine cyanine dye (Cy) as fluorophore, because of its
high molar absorption coefficient and NIR emission.[24] We
anticipated that the fluorescence properties of cyanine can
be modulated by a photoinduced electron transfer (PET)
process from the excited fluorophore to a strongly electron
withdrawing group (donor-excited PET, d-PET).[25] There-
fore, we chose electron-withdrawing 2-nitrophenylselane
and 3-(trifluoromethyl)phenylselane groups as modulators
for probes Cy-NiSe and Cy-TfSe.
Scheme 2. Synthesis of probes Cy-NiSe and Cy-TfSe. i) Methylamine hy-
drochloride, DMF, 408C, 24 h, 87.0%; ii) SO2Cl2, DMF, room tempera-
ture, 4 h, 86.0%; iii) a. Triethylamine, THF, 08C; b. room temperature,
4 h, 82.3%; iv) KSeCN, 3-bromobenzotrifluoride, triethylamine, CuI,
room temperature, 24 h, 56.9%.
Our strategy was based on using the strong nucleophilicity
À
of the sulfhydryl group to cleave the Se N bond of probes
Cy-NiSe and Cy-TfSe (Scheme 1). Owing to the presence of
electron-withdrawing groups, the electron densities of the
fluorophore moieties were lowered. The fluorescence of un-
cleaved probes Cy-NiSe and Cy-TfSe could be strongly
quenched by utilizing strongly electron withdrawing 2-nitro-
phenylselane and 3-(trifluoromethyl)phenylselane groups as
electron acceptors in a d-PET mechanism.[26] On cleavage
by thiols, free dye Cy7-MeAm exhibited significantly en-
hanced fluorescence in its fluorescence spectrum.
The synthesis of Cy-NiSe and Cy-TfSe is depicted in
Scheme 2. Intermediate Cy7-MeAm was readily prepared
by reaction of Cy7-Cl with methanamine hydrochloride in
87% yield. o-Nitrophenyl selenochloride was obtained from
o-nitroaniline by the classical method with some necessary
modifications. A THF solution of o-nitrophenyl selenochlor-
ide was added dropwise to a mixture of Cy7-MeAm and
triethylamine at 08C under Ar atmosphere. After stirring at
The yield of Cy-NiSe was much higher than that of Cy-
TfSe. This could be explained by the stability of the probes.
As previously proposed for other o-nitrophenylselenium
compounds,[27] a specific interaction between the nitro group
and the selenium atom possibly prevents bimolecular dis-
placement of selenium. The Se···O nonbonding interaction,
À
though expected to be weaker than the Se N bond, were
shown to increase the stability of probe Cy-NiSe. The stabil-
ity of Cy-NiSe is associated with intermolecular secondary
Se···O interactions with a CH2 group in the five-membered
heterocyclic ring,[28] while it is impossible to integrate such
a structure in Cy-TfSe. As a result, the stability of Cy-TfSe
may be much lower than that of Cy-NiSe.
Investigation of spectral properties of Cy-NiSe and Cy-TfSe
with GSH: We examined the spectral properties of Cy-NiSe
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Near-Infrared Fluorescent Probes for Thiols
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and Cy-TfSe in 15 mm phosphate buffered saline (PBS,
pH 7.4) under physiologically mimetic conditions. Cy-NiSe
and Cy-TfSe exhibited almost identical absorption peaks
with maxima at 609 nm, as shown for Cy-NiSe as represen-
tative of the two probes in Figure 1.
Figure 1. UV/Vis absorption of probe Cy-NiSe (5.0 mm) before (solid
line) and after (dashed line) addition of GSH (5.0 mm) at 258C for 3 min
in 15 mm PBS (pH 7.4). The maximum absorption wavelength was at
609 nm.
Figure 2. Fluorescence responses of a) Cy-NiSe (5.0 mm) and b) Cy-TfSe
(5.0 mm) toward different concentrations of GSH (final concentration:
0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mm) after incubation at 258C for
3 min in 15 mm PBS (pH 7.4). Spectra were acquired with excitation at
635 nm and emission ranging from 650 to 850 nm. Inset: Relationship be-
tween the fluorescence intensity at 750 nm of probe and [GSH].
On addition of 1 equivalent of GSH to a buffer solution
containing Cy-NiSe or Cy-TfSe, the absorbance was greatly
enhanced without wavelength shift. The enhancement of ab-
sorption intensity suggests formation of Cy-MeAm (e=
3.75ꢂ104 mÀ1 cmÀ1) when the strongly electron withdrawing
2-nitrophenylselane and 3-(trifluoromethyl)phenylselane
groups leave the probes.
As a typical biological thiol, GSH was used to further in-
vestigate the fluorescence response of Cy-NiSe and Cy-TfSe.
The changes in the fluorescence emission spectra of the two
probes in the presence of GSH in 15 mm PBS (pH 7.4) are
displayed in Figure 2. The final concentrations of the probes
were maintained at 5 mm, while the concentrations of GSH
varied from 0 to 5.0 mm. Cy-NiSe and Cy-TfSe were essen-
tially nonfluorescent owing to d-PET between fluorophore
and modulator. The initial quantum yields of the probes
were determined to be 0.002 for Cy-NiSe, and 0.003 for Cy-
TfSe. However, the fluorescence intensity (lem =750 nm) in-
creases significantly on addition of GSH. On addition of
5.0 mm GSH, the quantum yield of Cy-MeAm was calculated
to be 0.13. When the concentration of GSH is increased to
5.0 mm, impressive fluorescence enhancement of the probes
provides evidence for a fluorescence turn-on response. The
aforementioned d-PET is turned off in the product because
tions are F750 nm =16236.17[GSH/mM]À17657.72 with a coef-
ficient of r=0.9814 for Cy-NiSe, and F750 nm =6599.80[GSH/
mM]À3179.94 with a coefficient of r=0.9867 for Cy-TfSe.
These results imply that Cy-NiSe and Cy-TfSe are potential-
ly useful for quantitative determination of thiol concentra-
tion.
Kinetic studies: Time courses of Cy-NiSe and Cy-TfSe were
tested by measuring the fluorescent response over 1 h. Fig-
ure S1 (Supporting Information) shows the time courses of
fluorescence intensity of the probes (5.0 mm) in 15 mm PBS
with pH 7.4 at room temperature. The fluorescence intensity
was measured simultaneously at lex/em =635/750 nm. On in-
troduction of GSH (1 equiv), the emission intensity essen-
tially reached a maximum within 3 min, and leveled off
thereafter for 1 h, which indicated that Cy-MeAm has good
stability to light and air, and that Cy-NiSe and Cy-TfSe
could rapidly detect thiols.
Effect of pH: To be useful in biological applications, it is
necessary for a probe to function over a suitable pH range
and particularly at physiological pH values.[29] Since our
probes contain an enamine group, ionization of the methyl-
amine moiety at different pH values may affect the fluores-
cence. Hence, we examined the influence of pH on the fluo-
rescence intensity of the probes over a wide pH range of
4.0–8.6 (Supporting Information Figure S2). Standard fluo-
rescence pH titrations were performed in 15 mm PBS at
À
of cleavage of the Se N bonds in the probes, which could
explain the fluorescence intensity enhancement. For conven-
ient and accurate determination of intracellular GSH levels,
a linear relationship is necessary. The fluorescence intensi-
ties at 750 nm were plotted as a function of GSH concentra-
tion to obtain a calibration graph (Figure 2, insets). The
fluorescence signal is linearly related to the concentration of
GSH in the given concentration range. The regression equa-
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probe concentration of 5.0 mm. On introduction of 5.0 mm
GSH, the fluorescent emission intensity showed no pro-
nounced change. As shown in Figure S2 (Supporting Infor-
mation), the pH of the medium has hardly effect on fluores-
cence intensity of probes within the given range. These re-
sults indicated that the fluorescence responses to GSH of
our probes are pH-independent over a wide pH range. Thus,
the probes can be employed to detect thiols in cells.
that Cy-NiSe and Cy-TfSe can be useful for selectively sens-
ing thiols at physiological pH values, even in the presence of
these biologically relevant analytes.
Reactivity of Cy-NiSe and Cy-TfSe with thiol-containing an-
alytes: Cy-NiSe and Cy-TfSe were designed to sensitively
detect thiols. We tested the reactivity of Cy-NiSe and Cy-
TfSe towards nonprotein and protein thiols by measuring
the increase of fluorescence intensity at 750 nm (Figure 4).
Nonprotein thiols detected included glutathione, cysteine,
N-acetylcysteine, and dithiothreitol with a concentration of
5 mm, as GSH has an intracellular level of 1–10 mm. Protein
thiols such as thioredoxin, glutathione reductase, and metal-
lothionein were detected at a concentration of 10.0 mm. Cy-
NiSe (5.0 mm) and Cy-TfSe (5.0 mm) showed high reactivity
to nonprotein and protein thiols.
Selectivity studies: To test for fluorescent response to other
biological analytes, Cy-NiSe and Cy-TfSe (5.0 mm) were
treated with various biologically relevant analytes, such as
representative amino acids, metal ions, anions, reactive
oxygen species, reducing agents, and small-molecular thiols
in 15 mm PBS (pH 7.4). As shown in Figure 3, no noticeable
Figure 3. Fluorescence responses (lex/em =635/750 nm) of a) Cy-NiSe
(5.0 mm) and b) Cy-TfSe (5.0 mm) to diverse bioanalytes in 15 mm PBS
(pH 7.4). 1) Na+ (3.0 mm), 2) K+ (3.0 mm), 3) Ca2+ (50 mm), 4) Mg2+
(50 mm), 5) Fe2+ (25 mm), 6) Fe3+ (25 mm), 7) Zn2+ (50 mm), 8) Cu2+
Figure 4. Fluorescence responses of a) Cy-NiSe (5.0 mM) and b) Cy-TfSe
(5.0 mM) toward nonprotein (5.0 mm) and protein thiols (10.0 mm) in
15 mm PBS (pH 7.40). 1) Glutathione, 2) cysteine, 3) N-acetylcysteine,
4) homocysteine, 5) dithiothreitol, 6) thioredoxin, 7) glutathione reduc-
tase, 8) metallothionein. All data were obtained after incubation at 258C
for 3 min (lex/em =635/750 nm).
À
(0.5 mm), 9) ClÀ, 10) SO42À, 11) CO32À, 12) H2PO4À, 13) NO3 (all 20 mm);
14) histidine, 15) glycine, 16) l-adrenaline (all 100 mm); 17) uric acid,
18) ascorbic acid, 19) dopamine, 20) mannitol (all 100 mm); 21) H2O2
À
(2 mm); 22) hydroquinone (2.5 mm); 23) HOCl, 24) HOBr, 25) O2
,
26) NO (all 0.4 mm). Bars represent fluorescence responses to various
bioanalytes. In each group, the black bars represent the fluorescence in-
tensity after addition of analytes, and the gray bars represent that after
the subsequent addition of 5.0 mm GSH.
MTT assay: To evaluate the cytotoxicity of Cy-NiSe and Cy-
TfSe, we performed an MTT assay on RAW 264.7 cells with
probe concentrations from 10À7 to 10À3 m. The results
showed IC50 values of 405 mm for Cy-NiSe and 327 mm for
Cy-TfSe, that is, Cy-NiSe and Cy-TfSe are of low toxicity to-
wards cell cultures under experimental conditions.
changes in fluorescence intensity were observed on addition
of amino acids (His, Gly), metal ions (Na+, K+, Ca2+, Mg2+
2À
, Fe2+, Fe3+, Zn2+, Cu2+), anions (ClÀ, SO42À, CO3
,
Cell and tissue imaging: Owing to the chemical and spectro-
scopic properties of the probes, Cy-NiSe and Cy-TfSe
should be suited to detect thiols in living cells and tissues.
We assessed the function of Cy-NiSe and Cy-TfSe within
living cells by exposing RAW 264.7 cells to GSH. The RAW
264.7 cells were incubated with solution of Cy-NiSe and Cy-
TfSe (5.0 mm in 1/100 DMSO/PBS v/v, pH 7.4) for 5 min at
À
À
H2PO4 , NO3 ), reactive oxygen species (H2O2, HOCl,
HOBr, O2 ), reactive nitrogen species (NO), reducing
À
agents (uric acid, dopamine, hydroquinone, ascorbic acid)
and other analytes (histidine, l-adrenaline, mannitol) in PBS
at pH 7.4. An error of Æ5.0% in the relative fluorescence
intensity was considered tolerable. These results indicated
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We also investigated the utility of Cy-NiSe and Cy-TfSe
to image thiols in fresh rat liver tissue. After incubation
with 5.0 mm Cy-NiSe or Cy-TfSe for 10 min, a fresh rat liver
slice was washed with 15 mm PBS (pH 7.4). The dye-stained
liver slice showed strong fluorescence due to intracellular
thiols (Figure 6a, c). These results reveal that our probes
have good permeability in tissues. Next, another sample was
treated with 5 mm of NEM in PBS bubbled with 95% O2
and 5% CO2 at 378C for 30 min before probe was added,[31]
and we observed faint fluorescence intensity (Figure 6b, d).
These findings demonstrated that Cy-NiSe and Cy-TfSe are
capable of sensing thiols in tissues.
Figure 6. Detection of GSH in fresh rat liver tissue. Confocal fluores-
cence images of a fresh rat liver slice stained with a) 5.0 mm Cy-NiSe and
c) 5.0 mm Cy-TfSe for 10 min at a depth of about 120 mm with magnifica-
tion at ꢂ20. Scale bar is 100 mm. b) Confocal image of a) pretreated with
NEM (5.0 mm) before labeling with 5.0 mm Cy-NiSe. d) Confocal image
of c) pretreated with NEM (5.0 mm) before labeling with 5.0 mm Cy-TfSe.
e)–h) Bright-field images. Fluorescence was collected at 650–800 nm on
excitation at 633 nm.
Figure 5. Confocal fluorescence images of living RAW 264.7 cells. RAW
264.7 cells incubated with a) Cy-NiSe (5.0 mm) and g) Cy-TfSe (5.0 mm)
for 5 min after preincubation with NEM (5.0 mm) for 30 min. b) RAW
264.7 cells incubated with Cy-NiSe (5.0 mm) for 5 min. c) and i) RAW
264.7 cells incubated with SYTO-16 for 5 min. d) and e) Bright-field
images of a) and b); (f) overlay of images of b) and c); h) RAW 264.7
cells incubated with Cy-TfSe (5.0 mm) for 5 min; j) and k) are bright-field
images of g) and h). l) Overlay of images h) and i). Scale bar is 10 mm. In-
cubation was performed at 378C under a humidified atmosphere contain-
ing 5% CO2. The fluorescence was collected at 650–800 nm on excitation
at 633 nm.
Conclusion
We have developed two fluorescent probes for thiols, Cy-
À
378C and then washed three times with PBS. A strong fluo-
rescence signal was observed from the RAW 264.7 cells
(Figure 5b, h). In the control experiment, RAW 264.7 cells
were pretreated with an excess of the thiol-reactive N-ethyl-
maleimide (NEM),[30] which consumed all of the free thiols
within the cells, and then incubated with Cy-NiSe and Cy-
TfSe. No significant fluorescence signal was observed (Fig-
ure 5a, g). This established that Cy-NiSe and Cy-TfSe are
membrane-permeable, and the fluorescence changes in cells
were indeed due to the changes in intracellular thiol levels.
The bright-field images confirmed that the cells were viable
throughout the imaging experiments (Figure 5d, e, j, k). To
confirm whether Cy-NiSe and Cy-TfSe can specifically stain
the cytoplasm, co-localization experiments were conducted
with the RAW 264.7 cells co-stained with Cy-NiSe or Cy-
TfSe and SYTO. SYTO dyes are cell-permeant nucleic acid
stains that show a large fluorescence enhancement on bind-
ing to nucleic acids. Very strong green fluorescence in the
nucleus of RAW 264.7 cells was observed when the cells
were loaded with SYTO-16 (0.5 mm) for 5 min (Figure 5c, i).
By co-staining with SYTO-16 dye, we confirmed that the
probes were retained in the cytoplasm. (Figure 5 f, l).
NiSe and Cy-TfSe, on the basis of Se N bond cleavage in
cells and tissues. In addition, the probes are not only highly
sensitive and selective for sulfhydryl-containing molecules in
a wide pH range, but also emit in the NIR region (650–
900 nm). Furthermore, the probes also show fast signal re-
sponse and good linearity. Confocal fluorescence images in-
dicate that the probes can be used for detecting different
levels of thiols within living cells and tissues. We anticipate
that the fluorescent probes will be of great benefit for bio-
medical researchers investigating the effects of thiols in bio-
logical systems.
Experimental Section
Instruments: Fluorescence spectra were obtained on
a FluoroMax-4
Spectrofluorometer with a xenon lamp and 1.0 cm quartz cells. Absorp-
tion spectra were measured on NANO Drop 2000c UV/Vis spectropho-
tometer (Thermo Fisher Scientific). All pH measurements were per-
formed with a pH-3c digital pH meter (Shanghai Lei Ci Device Works,
Shanghai, China) with a combined glass–calomel electrode. Mass spectra
were taken on LCQ Fleet LC-MS System (Thermo Fisher Scientific). El-
emental analyses were obtained with a Vario MACRO cube element ana-
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L. X. Chen et al.
lyzer. 1H and 13C NMR spectra were taken on a Bruker 500 MHz spec-
trometer. The fluorescence images of cells were taken by using a confocal
laser scanning microscope (Japan Olympus Co., Ltd) with ꢂ20 and ꢂ40
objective lenses.
THF (10 mL) at 08C under Ar atmosphere. After stirring at room tem-
perature for 4 h, the reaction mixture was filtered and evaporated under
reduced pressure. The crude product was purified by chromatography on
silica gel with ethyl acetate/methanol (6/1, v/v) to give a deep blue
solid.[35] Yield: 186 mg (82.3%). 1H NMR (500 MHz, CD3OD): d=8.91
(d, 1H), 8.49–7.04 (m, 11H), 6.56 (t, 2H), 5.60 (d 2H), 3.28–3.36 (m,
9H), 2.48 (t, 4H), 1.62–1.65 (m, 2H), 1.51 ppm (s, 16H); 13C NMR
(100 MHz, CD3OD): d=168.5, 166.8, 144.7, 143.2, 139.3, 135.8, 134.2,
132.8, 128.5, 127.6, 122.3, 122.1, 120.1, 109.1, 92.4, 45.7, 35.9, 29.7, 28.1,
24.6, 18.7, 14.5 ppm; 77Se NMR (CD3OD-D4, 95 MHz): d=998.5 ppm; el-
emental analysis calcd (%) for C41H47N4O2Se: C 69.67, H 6.70, N 7.93, O
4.53, Se 11.17; found: C 69.65, H 6.71, N 7.92, O 4.54, Se 11.18; LC-MS
(ESI+): m/z calcd for C41H47N4O2Se+: 707.2859, found: 707.2847.
Materials:
2-[4-Chloro-7-(1-ethyl-3,3-dimethyl(indolin-2-ylidene)]-3,5-
(propane-1,3-diyl)-1,3,5-heptatrien-1-yl)-1-ethyl-3,3-dimethyl-3H-indoli-
um (Cy7-Cl) was synthesized by us. 3-(4,5-Dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT), KSeCN, and 3-bromobenzotrifluoride
were purchased from Sigma-Aldrich. Mouse leukaemic monocyte macro-
phage cell line (RAW 264.7) was obtained from the cell bank of the
Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China).
All other reagents and chemicals were from commercial sources, of ana-
lytical-reagent grade, and dried by standard procedures before use. Sol-
vents used for spectroscopic studies were purified and dried by standard
procedures before use. Ultrapure water (Millipore, Bedford, MA, USA)
was used throughout.
Synthesis of Cy-TfSe: We synthesized probe Cy-TfSe according to the
synthesis of SeN heterocycles by the classical method with some modifi-
cation.[36] 3-Bromobenzotrifluoride (237 mL, 2 mmol) was added to a solu-
tion of KSeCN (0.288 g, 2 mmol) in DMF (10 mL) under vacuum at
room temperature. After stirring for 1 h, Cy7-MeAm (31.7 mg,
0.05 mmol), triethylamine (Et3N, 4.5 mL, 8.0 mmol), and copper(I) iodide
(CuI, 0.38 g, 2 mmol) were added to the mixture in turn under Ar. The
solution was stirred for 24 h at room temperature. Then the solution was
diluted with Et2OAc (50 mL), washed with water (25 mL) and aqueous
NaOH solution (1.0m, 25 mL), and the organic phase dried over MgSO4,
concentrated, and purified by chromatography on a silica gel column
with ethyl acetate/methanol (9/1, v/v) to give a blue powder after solvent
removal under vacuum. Yield: 20.76 mg (56.9%). 1H NMR (500 MHz,
CD3OD): d=8.87 (d, 1H), 8.50–7.01 (m, 11H), 6.52 (t, 2H), 5.62 (d 2H),
3.30–3.55 (m, 9H), 2.50 (t, 4H), 1.60–1.66 (m, 2H), 1.50 ppm (s, 16H);
13C NMR (100 MHz, CD3OD): d=169.5, 167.8, 142.2, 139.1, 134.8, 135.2,
131.4, 128.1, 128.6, 125.6, 124.2, 122.6, 122.3, 121.1, 108.4, 92.6, 45.2, 36.1,
28.9, 28.6, 25.6, 18.9, 13.8 ppm; 77Se NMR (CD3OD, 95 MHz) d=720.07;
elemental analysis calcd (%) for C42H47F3N3Se: C 69.12, H 6.49, F 7.81, N
5.76, Se 10.82; found: C 69.11, H 6.48, F 7.82, N 5.76, Se 10.83; LC-MS
(ESI+): m/z calcd for C42H47F3N3Se+: 730.2882, found: 730.2893.
Absorption analysis: Absorption spectra were obtained with 1.0 cm glass
cells. The probe (DMSO, 0.1 mL, 1.0 mm) was added to a 10.0 mL color-
comparison tube. After dilution to 5.0 mm with 15 mm PBS, GSH was
added. The mixture was equilibrated for 5 min before measurement.
Fluorescence analysis: Fluorescence spectra were obtained with a xenon
lamp and 1.0 cm quartz cells. The probe (DMSO, 0.1 mL, 1.0 mm) was
added to a 10.0 mL color-comparison tube. After dilution to 5.0 mm with
15 mm PBS, GSH was added. The mixture was equilibrated for 5 min
before measurement.
Cell culture: RAW 264.7 cells were cultured in RPMI 1640 Medium sup-
plemented with 10% fetal bovine serum (FBS) at 378C in a humidified
atmosphere containing 5% CO2.
MTT assay: Cytotoxicity in vitro was measured by MTT assay in RAW
264.7 cells. Cells were seeded into 96-well cell culture plates at 4000/well,
cultured at 378C and 5% CO2 for 48 h, and then different concentrations
of chemosensor Cy-NiSe or Cy-TfSe (0, 10À3, 10À4, 10À5, 10À6, 10À7 m)
were added to the wells. The cells were then incubated for 48 h at 378C
under 5% CO2. Subsequently, MTT (20 mL, 5 mgmLÀ1) was added to
each well and incubated for an additional 4 h at 378C under 5% CO2.
Cells were lysed in triple liquid (10% SDS, 0.012m HCl, 5% 2-propanol),
and the amount of MTT formazan was qualified by determining the ab-
sorbance at 570 nm with a microplate reader (Tecan, Austria). IC50 values
were calculated according to Huber and Koella.[32] The following formula
was used to calculate the inhibition of cell growth: Cell viability (%)=
(mean of absolute value of treatment group/mean absolute value of con-
trol)ꢂ100%.
Acknowledgements
This work was financially supported by the National Natural Science
Foundation of China (20975089, 81102415), the Chinese Academy of Sci-
ences (KZCX2-EW-206), and the 100 Talents Program of the Chinese
Academy of Sciences.
Confocal imaging: Fluorescence images were acquired on an Olympus
Fluo View FV1000 confocal laser-scanning microscope (Japan) with ꢂ20
and ꢂ40 objective lenses. The excitation wavelength was 635 nm. Cell
imaging was carried out after washing three times with PBS.
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Preparation and staining of fresh rat liver slices: Slices were prepared
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thickness by using a vibrating-blade microtome in 15 mm PBS (pH 7.4).
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was 635 nm. To assess the effect of NEM, the slices were treated with
5 mm of NEM for 30 min before the probe was added. Following this in-
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A solution of o-nitrophenyl selenochloride
(0.142 g, 0.6 mmol) in THF (5 mL) was added dropwise to a solution of
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Near-Infrared Fluorescent Probes for Thiols
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Received: February 29, 2012
Revised: April 15, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
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L. X. Chen et al.
Fluorescent Probes
R. Wang, L. X. Chen,* P. Liu,
Q. Zhang, Y. Wang .......... &&&& — &&&&
Sensitive Near-Infrared Fluorescent
À
Probes for Thiols Based on Se N
Bond Cleavage: Imaging in Living
Cells and Tissues
À
Turn-on fluorescent probes Cy-NiSe
and Cy-TfSe, which show weak fluo-
rescence due to donor-excited photoin-
duced electron transfer, release near-
infrared fluorescent dye Cy7-MeAm
on cleavage of the Se N bond by
thiols. Thus, they can be used for real-
time imaging of thiols such as gluta-
thione in living cells and tissue (see
figure).
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