Construction of long-wavelength fluorescein analogues and their application as fluorescent probes

Article abstract of DOI:10.1002/chem.201300418

Born to dye: Five fluorescein analogues were synthesised (see scheme). One analogue was found to emit in the NIR region with a high quantum yield, excellent photostability and good permeability. Three derivatives were found to specifically stain mitochondria and one dye responds to thiols with a strong turn-on NIR fluorescence signal and colorimetric change, in vitro and in vivo. Copyright

Full text of DOI:10.1002/chem.201300418

COMMUNICATION
DOI: 10.1002/chem.201300418
Construction of Long-Wavelength Fluorescein Analogues and Their
Application as Fluorescent Probes
Xiaoqing Xiong,[a] Fengling Song,*[a] Gengwen Chen,[a] Wen Sun,[a] Jingyun Wang,[b]
Pan Gao,[b] Yukang Zhang,[a] Bo Qiao,[a] Wenfang Li,[b] Shiguo Sun,[a] Jiangli Fan,[a] and
Xiaojun Peng*[a]
Chem. Eur. J. 2013, 00, 0 – 0
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Fluorescence imaging is one of the most powerful tech-
Herein, we present the synthesis of a new 2’,7’-dichloro-
fluorescein (DCF) analogue with red emission. The inter-
mediate aldehyde 3 was synthesised by the Vilsmeier reac-
tion without further purification and was used to prepare
the subsequent products by Knoevenagel condensation
(Scheme 1). We expect that this design strategy can be ex-
tended to the development of a wide variety of long-wave-
length functional dyes and then to further applications in bi-
ological studies.
ACHTUNGTRENNUNG
The most widely employed NIR fluorophores are cyanine
dyes, especially Cy5.5 and Cy7.[3] However, to the best of
our knowledge, this kind of dye suffers from severe disad-
vantages, like bad photostability and low fluorescence quan-
tum yield. Therefore, many groups have tried to develop
new fluorophores based on rhodamine[4] and BODIPY, the
spectra of which can extend to the red and even NIR region
through modification.[5] For example, Nagano and colleagues
designed and synthesised a novel NIR Si–rhodamine fluo-
rescence probe MMSiR.[4a] They have developed a series of
novel NIR wavelength-excited fluorescent dyes, SiR-NIRs,
by modifying the Si–rhodamine scaffold.[4b,6] They have also
developed 2-Me TeR as a reversible NIR fluorescence
probe.[4c] Other groups have also constructed red-emissive
or NIR BODIPY dyes.[5,7] However, very few red or NIR
fluorescein derivatives have been constructed.[8,9] As a
matter of fact, fluoresceins have predominant photostability,
which makes them suitable for biological application.
We first tested the optical properties of dye 4 in different
solvents, such as H2O, ethanol, DMSO and so on (Table S1
in the Supporting Information). The absorption and emis-
sion spectra of 4 in DMSO are illustrated in Figure 1. Dye 4
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Figure 1. The normalised absorbance ( ) and fluorescence ( ) spectra of
~
DCF (2.0mm in water, lex =490 nm). The normalised absorbance ( ) and
Fluorescein dyes have good photochemical properties,
such as high molar extinction coefficients, large fluorescence
quantum yields and tolerance to photobleaching.[10] Signifi-
cantly, alkylation of the hydroxyl of fluorescein can easily
tune the fluorescence property. So far, the hydroxyl-alkyla-
tion fluoresceins have become an effective platform for con-
struction of fluorescence turn-on sensors. Many probes for a
diverse array of targets including hydrogen peroxide,[11] per-
oxynitrite,[12] superoxide radical,[13] and cysteine[14] have been
reported based on the unique hydroxyl alkylation principle.
However, these probesꢁ absorption and emission wave-
lengths are in the visible region. To make full use of the hy-
droxyl alkylation principle, it is highly desirable to develop
stable fluorescein analogues with absorption and emission in
the red or NIR region.
!
fluorescence ( ) spectra of 4 (2.0mm in DMSO, lex =620 nm).
exhibited a high extinction coefficient (62505mÀ1 cmÀ1) and
with full width at half maximum (40 nm) in DMSO. Com-
pared with traditional DCF, the absorption and emission
spectra of 4 can extend to the NIR range. The fluorescence
quantum yield of the NIR fluorescein analogue 4 was mea-
AHCTUNGTREGUNsNN ured to be as high as 0.55 (rhodamine B was used as a
standard: F=0.97 in ethanol).
To rationalise the optical properties of the new functional
dye 4, representative optimised structures and molecular or-
bital plots (LUMO and HOMO) were obtained by density
functional theory (DFT) calculations with the B3LYP ex-
change functional employing 6-31GACTHNUTRGNEUNG(d,p) basis sets with a
suite of Gaussian 09 programs (Figure 2). The p electrons on
the HOMO of 4 are mainly located on the whole p-conju-
gated 7,8-dihydro-6H-xanthene-dicyanovinyl framework (in-
cluding the O and Cl atoms), but electrons of the LUMO
are mostly positioned at the centre of the conjugated 7,8-di-
hydro-6H-xanthene-vinyl bridge (Figure 2). Therefore, the
DFT calculation results (Tables S5–S7 in the Supporting In-
formation) are consistent with the absorption and emission
spectroscopic data of 4 (Table S1 in the Supporting Informa-
tion). In addition, time-dependent DFT (TDDFT) calcula-
tions indicated that the excitation HOMO–LUMO energy
gap between the S0 state and the S1 excited state of 4
(2.6616, 1.9924 eV based on the optimised S0 or S1 state ge-
ometry) is calculated by DFT calculation (Tables S5–S7 in
the Supporting Information).
[a] X. Xiong, Prof. F. Song, Dr. G. Chen, W. Sun, Y. Zhang, B. Qiao,
Dr. S. Sun, Dr. J. Fan, Prof. X. Peng
State Key Laboratory of Fine Chemicals
Dalian University of Technology, 2 Linggong Road
Dalian 116025 (P.R. China)
E-mail: songfl@dlut.edu.cn
pengxj@dlut.edu.cn
[b] J. Wang, P. Gao, W. Li
School of Life Science & Biotechnology
Dalian University of Technology, 2 Linggong Road
Dalian 116025 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300418.
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The photostability is of great
importance for long-wavelength
dyes. The stability measurement
of 4 was carried out in compari-
son with a commercial NIR dye
(Mitotracker Deep Red). Dye 4
was found to be reasonably
stable (Figure S1 in the Sup-
porting Information). The fluo-
rescence spectra of 4 at various
pH values (the pH ranges from
3.28 to 12.58) are recorded in
Figure S2a (in the Supporting
Information). The fluorescence
was significantly enhanced with
pH increasing together with a
63-fold increase of emission in-
tensity. As shown in Figure S2b
(in the Supporting Informa-
tion), the changes in the fluo-
rescence intensity as a function
of pH yielded a pKa of 6.41.
The absorption spectrum ap-
peared to have an altered ratio
(Figure S3 in the Supporting In-
formation). It is well-known
that the emission wavelength of
a D-p-A chromophore is sensi-
tive to the polarity of the sol-
Scheme 1. The synthetic routes to DCF analogue 4 and the derivatives 5, 6, 7 and 8.
vent.[15] Thus, we analysed the emission changes of dye 4 in
water/1,4-dioxane system with different polarity (Figure S4
in the Supporting Information). To our delight, the new dye
4 is not sensitive to polarity. This solvent-polarity independ-
ent emission wavelength will make this dye ideal for further
construction of fluorescent probes.
In order to explore the biological application of fluoro-
phore 4, we utilised it to stain HeLa cells; the results
showed that 4 does not performs well (Figure 3). Although
it can enter the cells, it does not exhibit obvious organelle
staining. We presume that the possible reason is that the
molecule contains more hydrophilic groups (such as hydrox-
yl, cyan, carboxyl); these groups may reduce the membrane
permeability. Therefore, we decided to broaden the new flu-
orophoreꢁs application and improve its lipid solubility.
Based on the above two requirements, we designed and syn-
Figure 2. Frontier molecular orbital plots of 4 in water; it is involved in
the vertical excitation (UV/Vis absorption, left column) and emission
(right column). The vertical-excitation-related calculations are based on
the optimised geometry of the ground state (S0), and the emission-related
calculations are based on the optimised geometry of the excited state
(S1). Excited and radioactive processes are marked as solid lines and the
non-radiative processes are marked by dotted lines.
Figure 3. Confocal fluorescence images of HeLa cells incubated with 4
(20mm) at 378C. After 150 min incubation: a) bright-field image; b) fluo-
rescent image with excitation at 635 nm for red emission of 4 (640Æ
20 nm); c) overlay of (a) and (b).
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thesised the control substances 5, 6, 7, 8, the structures of
which are similar to dye 4 (Scheme 1). We investigated the
optical properties of these compounds, and the results are
summarised in Tables S2–S4 and Figures S5, S6 in the Sup-
porting Information. Compound 5 shows almost no fluores-
cence in various solvents; weak background fluorescence is
useful for application as a fluorescent probe. From the ab-
sorption and emission spectra data in different solvents, it
was observed that the spectral changes of 6 and 7 are very
similar to 4. However, to our surprise, the fluorescence of
dye 6, the hydroxyl of which was modified by acetylation,
was not completely quenched.
As illustrated in Figure 4, to estimate the potential use of
these derivatives for biological imaging, MCF-7 cells (breast
cancer cell line) were incubated with 6 (20 mm) and 7
(20 mm) at 378C for 120 min. As shown in Figure 4a–c and
d–f, both dyes proved to be membrane permeant. Their flu-
orescence was detected in the cytoplasmic rather than nucle-
ar regions. Based on morphological grounds, these regions
were judged to be the mitochondria.
Figure 5. Confocal images of 6 (20mm, top row) and 7 (20mm, bottom
row) at 378C with mitochondrial-specific dye in MCF-7 cells: a) bright-
field image; b) fluorescent image of MCF-7 cells stained with MitoTrack-
er Green FM; c) fluorescent image of MCF-7 cells stained with 6;
d) merged image of (a), (b) and (c); e) bright-field image; f) fluorescent
image of MCF-7 cells stained with MitoTracker Green FM; g) fluorescent
image of MCF-7 cells stained with 7; h) merged image of (e), (f) and (g),
excited at 488 nm for MitoTracker Green FM, green emission (520Æ
20 nm); excited at 488 nm for 6 and 7, red emission (590Æ50 nm).
ty of pathologies, from Alzheimerꢁs disease to cancers and
diabetes.[17] Mitochondrial morphological analysis, therefore,
attracts immense interest.
Figure 6, shows different damaged mitochondrial forms.
Figure 6h and k represent hollow spheres of extremely swol-
len mitochondria; in Figure 6g and j the mitochondria are
Figure 4. Confocal fluorescence images of MCF-7 cells incubated with 6
(20mm, top row) and 7 (20mm, bottom row) at 378C. After 2.0 h incuba-
tion: a), d) bright-field images; b), e) fluorescent images with excitation
at 488 nm for red emission of 6 and 7 (590Æ50 nm); c), f) overlays of (a)
and (b), and (d) and (e), respectively.
Figure 6. Confocal fluorescence images of MCF-7 cells incubated with
5 mm of dye 6 (top row) or 7 (bottom row). After incubation for 10 h: a),
d) Bright-field images; b), e) various damaged mitochondrial forms; c),
f) cells stained with 6 and 7, respectively; g), h) magnification of marked
areas in (b); j), k) magnification of marked areas in (e).
To further investigate the subcellular localisation of com-
pound 6 and 7, organelle-specific fluorescent dye (Mito-
Tracker Green FM) was employed for colocalisation studies.
Staining experiments of 6 and 7 were performed with MCF-
7 cells and show that both 6 and 7 colocalised in the mito-
chondria (Figure 5). With different incubation times (Mito-
Tracker Green FM, 30 min; 6 and 7, 120 min), both were
found to localise selectively in mitochondria with a bright
fluorescent signal. The Pearsonꢁs sample correlation factors
(Rr) of colocalisation with MitoTracker Green FM were
found to be 97 and 93% in MCF-7 cells, for 6 and 7, respec-
tively. Mitochondria supply the cellsꢁ energy, and are very
important but sensitive and variable organelles. So when mi-
tochondria are simulated by external factors, their morphol-
ogy, size, number and distribution can change significant-
ly.[16] Besides, mitochondria are crucially involved in a varie-
less swollen and appear as solid granules. The two dyes can
be used to monitor different degrees of mitochondrial
damage. The above results show that these derivatives have
good membrane permeability, and will provide a good plat-
form for further exploring mitochondrial fluorescent probes.
The probable reason for the good mitochondrial staining
of dyes 6 and 7 is that they are lipophilic weak acids. Ac-
cording to the reported literatures,[18] lipophilic weak acids
comprise equilibrium mixtures of lipophilic-free acids and
their more hydrophilic salts under physiological condition.
The entry of probes into cells mainly depends on their lipo-
philicity, which is modelled by their water/octanol partition
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Long-Wavelength Fluorescein Analogues
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coefficient (logP) values.[19] On the basis of simulations
(ChemDraw) the logP values for 6 and 7 were 3.57 and
3.64, respectively. Specifying mitochondria accumulation is
assigned numerically by the following criteria: +5>logP>
0.[18] This can explain why the new dyes can accumulate in
the mitochondria.
Equilibrium is re-established between free acids and hy-
drophilic salts in the aqueous environment in living cells.
The equilibrium is dependent on the pH of the compart-
ment. This effect controls the probesꢁ accumulation in the
mitochondria, because the internal pH of mitochondria (pH
ꢀ8.0) is higher than that of the cytosol (pHꢀ7.5).[20] This
somewhat alkaline environment results in the decrease of
the relative concentration of lipophilic free acid, but the
concentration of the ionised species is relatively increased.
Then, the corresponding weak acids become more hydro-
philic, and these anions will be less membrane permeant
and consequently accumulate inside the mitochondria.
Based on the literature,[21] the process for the accumulation
of 7 in mitochondria is illustrated in Scheme 2. The accumu-
lation of 6 can be attributed to the same mechanism.
For dye 8 (Figures S5 and S6 in the Supporting Informa-
tion), the absorption and emission spectra can extend to the
NIR region. We utilised dye 8 in biological tests to expand
its applications. The results show that dye 8 can also stain
mitochondria (Figure 7). This can be explained in terms of
the positive charge of dye 8. As with the common mitochon-
drial dyes, in order to balance the potential of the mitochon-
drial membrane, the molecule can enter the mitochondria.
Figure 7. Confocal fluorescence images of HeLa cells incubated with 8
(5mm) at 378C. After 30 min incubation: a) bright-field image; b) fluores-
cent image with excitation at 635 nm for 8, red emission (705Æ50 nm);
c) overlay of (a) and (b).
As illustrated in Figure S7 (in the Supporting Information),
the colocalisation results suggest that 8 can selectively stain
mitochondria in living cells.
Compared with the above-mentioned compounds, dye 5
exhibited almost no fluorescence in different solvents; so
the drastic difference in the fluorescence intensity between
compound 5 and 4 suggests that 5 can be used as a fluores-
cent turn-on probe. Recently, Chenꢁs group constructed a
fluorescence turn-on sensor for cysteine.[14a] The construc-
tion mechanism was employed in our case. Furthermore, to
test the robust nature of the approach for novel NIR fluo-
rescein analogues, we utilised the nonfluorescent dye 5 as a
new NIR fluorescent turn-on thiol sensor.
Upon addition of cysteine (Cys), homocysteine (Hcy) and
glutathione (GSH) to a solution of probe 5, the fluorescence
intensity of the detection system was recorded. The fluores-
cence intensity increased rapidly with prolonged reaction
times, and the reaction of 5 with Cys was nearly complete
within 10 min (Figure 8). At the same time, the free probe 5
exhibited no noticeable changes in solution. A similar fluo-
rescence increase was observed for Hcy and GSH, but both
Figure 8. Time course of the fluorescence response (lem =650 nm) of
probe 5 (5.0mm) in aqueous solution (40 mm HEPES buffer, pH 7.37) in
the presence of 34 equiv Cys, Hcy or GSH, with excitation at 620 nm.
took more than 60 min to reach the maximal intensity.
Therefore, to obtain highly sensitive and reproducible re-
sults, all experiments were completer within 10 min.
The typical biological thiol, Cys, was examined by the
sensing response of probe 5. After the immediate addition
of Cys, the emission colour of probe 5 changed to red (Fig-
ure S8 in the Supporting Information). Free 5 is weakly fluo-
rescent perhaps because the acryloyl substituent of 5 re-
Scheme 2. Schematic illustration of the mechanism for ion-trapping of 7.
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F. Song, X. Peng et al.
duces the electron-donating ability of the oxygen atom and
thus forbids the formation of the zwitterionic resonance
form. But upon addition of Cys (26 equiv), the fluorescence
emission at 650 nm increased about 20-fold within 10 min
(Figure S8 in the Supporting Information). The fluorescence
intensity of 5 at 650 nm shows a good linear relationship
with the concentration of Cys ranging from 0 to 40 mm (Fig-
ure S9 in the Supporting Information), and the detection
limit for Cys is 0.0309 mm. Increasing the amount of Cys (0–
130 mm) added to a solution of probe 5 (5.0 mm) caused the
absorption bands of 5 at 474 and 520 nm to gradually de-
crease, whereas new absorption bands at 576 and 625 nm ap-
peared with a distinct isosbestic point at 525 nm; this result-
ed in an apparent colour change from red to hyacinthine
(Figure 9). This shows that 5 can allow colorimetric detec-
tion of Cys with the naked-eye. The combination of the low
detection limit and the large fluorescence dynamic range in-
dicates that the probe is highly sensitive to Cys.
Scheme 3. A plausible mechanism for the reaction between 5 and Cys.
To further establish the utility of 5 for the determination
of Cys in a biological sample, we evaluated 5 (50 mm) with
commercially available newborn-calf serum (Figure S17 in
the Supporting Information). The detection of Cys was ach-
ieved successfully in the samples. An excellent linear corre-
lation between the added Cys concentrations and the fluo-
rescence emission responses at lem =650 nm was observed in
the serum (Figure S18 in the Supporting Information). The
fluorescence intensity response in serum was lower than
that observed with the detection system used in Figures S8
and S9 in the Supporting Information. However, these data
show that physiologically relevant sensitivity and limits of
detection can be obtained in detecting Cys in blood plasma.
To assess permeability and ability to monitor thiols in
living cells, confocal microscopy experiments were carried
out. When MCF-7 cells were incubated with 5 (20 mm)
bright fluorescence was exhibited inside the cells (Fig-
ure 10b). There was no intracellular background fluores-
Figure 9. Absorption spectral changes of 5 (5.0mm in DMSO/HEPES
buffer, 7:3) after incubation with different concentrations of cysteine (0,
0.66, 1.32, 2.0, 4.0, 8.0, 10, 14, 18, 22 and 26 equiv). Each spectrum was re-
corded after 10 min. Inset: probe 5 (5.0 mm) in the absence of Cys (left)
and in the presence of 130mm Cys (right).
The above spectral studies and the sensing response of
the probe to Cys indicate that most probably a new com-
pound is formed. So we conducted further experiments to
corroborate this. The plausible reaction mechanism for the
formation 10 and 4 is proposed in Scheme 3. Mass spectrom-
etry analysis showed the formation of 4 and a cyclised prod-
uct 10 was obtained from the reaction of 5 with Cys. The
peak at 430.0 corresponding to the fluorescent 4 and anoth-
er peak at 174.0 corresponding to the cyclisation product 10
were clearly observed (Figure S10 in the Supporting Infor-
mation).
To investigate its selectivity, probe 5 (5.0 mm) was treated
with various biologically relevant analytes (such as represen-
tative amino acids, metal ions, anions) and monitored by ab-
sorption and emission spectroscopy (Figures S11–S16 in the
Supporting Information). No noticeable changes in the ab-
sorption were observed upon addition of amino acids (ala-
nine, valine, leucine, proline, tyrosine, asparamide and tau-
Figure 10. Fluorescence images of MCF-7 cells incubated with (a, b) or
without (d, e) 5 (20mm) at 378C. After 90 min incubation: a) bright-field
image; b) fluorescent image with excitation at 635 nm; c) overlay of (a)
and (b); d) bright-field image; e) fluorescent image with excitation at
635 nm, no fluorescence was observed; f) overlay of (d) and (e).
A
,
,
,
+
Mn2+, Zn2+, Fe3+, Na+, Pd2+, Ca2+, Ag+, Hg2+, NH4
À
À
2À
Ni2+), and anions (BrÀ, AcÀ, NO3 , ClÀ, ClO4 , ClOÀ, CO3
À
À
IÀ, H2PO4 , HPO42À, HCO3 ).
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Centre. 1H and 13C NMR spectra were recorded on a Varian Inova-400
spectrometer with chemical shifts reported as ppm (in DMSO or metha-
nol, TMS as internal standard). The following abbreviations are used to
indicate the multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiple; br, broad. Mass spectrometric data were obtained on a Q-TOF
cence in untreated control cells (Figure 10e). This confirmed
that 5 could be used to monitor thiols in living cells. To fur-
ther test the robust nature of this probe, we examined its ap-
plication for visualising Cys in living animals. Different con-
centrations of probe 5 (0, 10, 20 and 50 mm) in 4-(2-hy
ACHTUNGTRENNUNG
droxy-
Micro mass spectrometer. UV–visible spectra were collected on a
Perkin–Elmer Lambda 35 UV/Vis spectrophotometer. Fluorescence
measurements were performed on a Varian Cary Eclipse fluorescence
spectrophotometer (serial No.: FL1109M018).
HEPES buffer, pH 7.37) were injected into mice. Different
fluorescence intensities were observed after 10 min, whereas
no fluorescence was noted in uninjected regions (Figure 11).
The results show that probe 5 also can be exploited for the
development of NIR fluorescent sensors for in vivo imaging
applications.
Photostability experiment: Compound 4 and Mitotracker Deep Red
were dissolved in aqueous solution (DMSO/40 mm HEPES buffer, 7:3,
pH 7.37) at a concentration of 2.0 mm. These solutions were irradiated
under a 500 W iodine-tungsten lamp for 250 min at a distance of 30 cm
away from the lamp. A saturated sodium nitrite aqueous solution was
placed between the samples and the lamp as a light filter (to cut off
wavelengths shorter than 400 nm) and heat filter.
Calculating pKa: Dye 4 (15 mm) was dissolved in phosphate-buffered
saline (PBS, 50 mL). Then the solution was divided into two equivalent
parts. Hydrochloric acid (1.0m) was added to one part to adjust the pH
in the acidic range. Sodium hydroxide (1.0m) was added to the second
part to adjust the pH in the basic range. The fluorescence intensity was
recorded at the maximum emission wavelength every 0.2 pH interval.
The pKa value was obtained after sigmoidal fitting.
Cell incubation and fluorescence imaging: MCF-7 and HeLa cells were
seeded onto cover slips at a concentration of 2ꢂ104 cellsmLÀ1 and cul-
tured in Dulbeccoꢁs modified Eagle medium (DMEM) in an incubator
(378C, 5% CO2, 20% O2). After 24 h, the cover slips were rinsed three
times with PBS to remove the media and then cultured in DMEM for
later use. For the verification procedure, dyes 4 (20 mm), 5 (20 mm), 6
(20 mm), and 7 (20 mm) were added to the above cellular samples and in-
cubated for 120 min, and 8 (5.0 mm) was incubated for 30 min; then the
samples were rinsed three times with PBS and observed under an Olym-
pus FV1000-IX81 confocal fluorescence microscope; for the confocal flu-
orescence imaging the 100ꢂ objective lens was used.
Figure 11. Fluorescent images of mice (pseudocolour) without probe 5
(left) and with injection of different concentrations of probe 5 (right) in
HEPES (0.1 mL; 40 mm buffer, pH 7.37). A) 0mm, B) 10mm, C) 20mm,
D) 50mm. Images were taken after incubation for 10 min.
Fluorescent imaging in vivo: ICR mice (25 g) were given an sp (skin-
pop) injection of a solution of 5 (0, 10, 20 and 50 mm in 0.1 mL of 40 mm
HEPES buffer, pH 7.37). Images were taken after incubation for 10 min
by using a NightOWL II LB983 small animal in vivo imaging system con-
taining a sensitive Charge Coupled Device (CCD) camera, with an exci-
tation filter of 630 nm and an emission filter of (655Æ20) nm. Adult
male ICR mice (25 g) were provided by the Specific Pathogen Free
Animal Laboratory at Dalian Medical University. All experimental pro-
cedures were conducted in conformity with institutional guidelines for
the care and use of laboratory animals in Dalian Medical University
(Dalian, China) and conformed to the National Institutes of Health
Guide for Care and Use of Laboratory Animals (publication no. 85–23,
revised 1996).
In summary, we have successfully developed a new NIR
2’,7’-dichlorofluorescein analogue 4, which exhibits high
quantum yield, an excellent photostability and cellular per-
meability. All of these properties are very desirable for NIR
fluorophores. Through modification, it was found that the
derivatives 6, 7, 8 can localise in mitochondria specifically.
Moreover, we have demonstrated that the approach can be
easily employed to develop NIR thiol fluorescent sensor 5,
which can be used to colorimetrically detect Cys, in vitro.
The NIR thiol sensor can be utilised in biological imaging
applications. We anticipate that such red-emissive DCF ana-
logues can be very useful as a fluorescence sensing platform.
Acknowledgements
This work was supported financially by the NSF of China (21222605,
21006009, 21136002, 2176032 and 20923006), the Fundamental Research
Funds for the Central Universities of China, 973 program of China
(2009CD724700 and 2012CB733702) and 863 program of China
(2011AA02A105).
Experimental Section
Materials and instruments: Common reagents used in the experiments
were all of analytical grade. Cyclohexanone, POC13 and DMF were pur-
chased from Tianjin Bodi Co., Ltd. Et3N was purchased from Tianjin
Damao Chemical Reagent. Malononitrile, acryloyl chloride, acetic anhy-
dride, ethyl cyanoacetate and 2-methyl benzothiazole were purchased
from Alading. Cys, Hcy were purchased from Solarbio. GSH was pur-
chased from Tokyo Chemical Industry Co., Ltd. 2’,7’-Dichlorofluores-
cein-6-carboxylic acid was synthesised according to the literature.[22] Rho-
damine B was purchased from Tianjin Fine Chemicals Development
Keywords: colorimetric analysis · fluorescence · fluorescent
probes · mitochondria · thiol probes · imaging
[1] a) X. Qian, Y. Xiao, Y. Xu, X. Guo, J. Qian, W. Zhu, Chem.
Commun. 2010, 46, 6418–6436; b) A. Loudet, K. Burgess, Chem.
Rev. 2007, 107, 4891–4932; c) M. S. T. GonÅalves, Chem. Rev. 2008,
108, 190–212; d) S. Hapuarachchige, G. Montao, C. Ramesh, D. Ro-
Chem. Eur. J. 2013, 00, 0 – 0
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driguez, L. H. Henson, C. C. Williams, S. Kadavakkollu, D. L. John-
son, C. B. Shuster, J. B. Arterburn, J. Am. Chem. Soc. 2011, 133,
6780–6790; e) X. Peng, Z. Yang, J. Wang, J. Fan, Y. He, F. Song, B.
Wang, S. Sun, J. Qu, J. Qi, M. Yan, J. Am. Chem. Soc. 2011, 133,
6626–6635; f) E. Kim, M. Koh, J. Ryu, S. B. Park, J. Am. Chem.
Soc. 2008, 130, 12206–12207; g) T. Peng, D. Yang, Org. Lett. 2010,
12, 496–499; h) G. M. Fischer, C. Jungst, M. Isomaki-Krondahl, D.
Gauss, H. M. Moller, E. Daltrozzo, A. Zumbusch, Chem. Commun.
2010, 46, 5289–5291.
Chem. Soc. 2010, 132, 5906–5915; c) E. W. Miller, O. Tulyanthan,
E. Y. Isacoff, C. J. Chang, Nat. Chem. Biol. 2007, 3, 263–267;
d) E. W. Miller, O. Tulyathan, E. Y. Isacoff, C. J. Chang, Nat. Chem.
Biol. 2007, 3, 349–349; e) B. Heyne, V. Maurel, J. C. Scaiano, Org.
Biomol. Chem. 2006, 4, 802–807.
[12] D. Yang, H. L. Wang, Z. N. Sun, N. W. Chung, J. G. Shen, J. Am.
Chem. Soc. 2006, 128, 6004–6005.
[13] a) F. He, Y. L. Tang, M. H. Yu, S. Wang, Y. L. Li, D. B. Zhu, Adv.
Funct. Mater. 2006, 16, 91–94; b) K. H. Xu, X. Liu, B. Tang, Chem-
BioChem 2007, 8, 453–458; c) H. Maeda, K. Yamamoto, I. Kohno,
L. Hafsi, N. Itoh, S. Nakagawa, N. Kanagawa, K. Suzuki, T. Uno,
Chem. Eur. J. 2007, 13, 1946–1954; d) K. H. Xu, X. Liu, B. Tang,
G. W. Yang, Y. Yang, L. G. An, Chem. Eur. J. 2007, 13, 1411–1416;
e) H. Maeda, K. Yamamoto, Y. Nomura, I. Kohno, L. Hafsi, N.
Ueda, S. Yoshida, M. Fukuda, Y. Fukuyasu, Y. Yamauchi, N. Itoh, J.
Am. Chem. Soc. 2005, 127, 68–69.
[2] a) L. Yuan, W. Y. Lin, K. B. Zheng, L. W. He, W. M. Huang, Chem.
Soc. Rev. 2013, 42, 622–661; b) K. Kiyose, H. Kojima, T. Nagano,
Chem. Asian J. 2008, 3, 506–515; c) L. Yuan, W. Lin, S. Zhao, W.
Gao, B. Chen, L. He, S. Zhu, J. Am. Chem. Soc. 2012, 134, 13510–
13523; d) L. Yuan, W. Lin, Y. Yang, H. Chen, J. Am. Chem. Soc.
2012, 134, 1200–1211; e) G. Qian, Z. Y. Wang, Chem. Asian J. 2010,
5, 1006–1029.
[3] a) B. Tang, F. Yu, P. Li, L. Tong, X. Duan, T. Xie, X. Wang, J. Am.
Chem. Soc. 2009, 131, 3016–3023; b) T. Myochin, K. Kiyose, K. Ha-
naoka, H. Kojima, T. Terai, T. Nagano, J. Am. Chem. Soc. 2011, 133,
3401–3409; c) Y. Lin, R. Weissleder, C.-H. Tung, Bioconjugate
Chem. 2002, 13, 605–610; d) D. Oushiki, H. Kojima, T. Terai, M.
Arita, K. Hanaoka, Y. Urano, T. Nagano, J. Am. Chem. Soc. 2010,
132, 2795–2801.
[4] a) Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, J. Am.
Chem. Soc. 2011, 133, 5680–5682; b) Y. Koide, Y. Urano, K. Hanao-
ka, W. Piao, M. Kusakabe, N. Saito, T. Terai, T. Okabe, T. Nagano, J.
Am. Chem. Soc. 2012, 134, 5029–5031; c) Y. Koide, M. Kawaguchi,
Y. Urano, K. Hanaoka, T. Komatsu, M. Abo, T. Terai, T. Nagano,
Chem. Commun. 2012, 48, 3091–3093.
[5] a) X.-D. Jiang, R. Gao, Y. Yue, G.-T. Sun, W. Zhao, Org. Biomol.
Chem. 2012, 10, 6861–6865; b) M. Nakamura, H. Tahara, K. Taka-
hashi, T. Nagata, H. Uoyama, D. Kuzuhara, S. Mori, T. Okujima, H.
Yamada, H. Uno, Org. Biomol. Chem. 2012, 10, 6840–6849.
[6] Y.-Q. Sun, J. Liu, X. Lv, Y. Liu, Y. Zhao, W. Guo, Angew. Chem.
2012, 124, 7752–7754; Angew. Chem. Int. Ed. 2012, 51, 7634–7636.
[7] T. P. Gustafson, Y. Yan, P. Newton, D. A. Hunter, S. Achilefu, W. J.
Akers, S. E. Mackinnon, P. J. Johnson, M. Y. Berezin, MedChem-
Comm 2012, 3, 685–690.
[14] a) H. Wang, G. Zhou, H. Gai, X. Chen, Chem. Commun. 2012, 48,
8341–8343; b) X. Chen, S. K. Ko, M. J. Kim, I. Shin, J. Yoon, Chem.
Commun. 2010, 46, 2751–2753; c) H. Wang, G. Zhou, C. Mao, X.
Chen, Dyes Pigm. 2013, 96, 232–236; d) H. Wang, G. Zhou, X.
Chen, Sensor. Actuat. B-Chem. 2013, 176, 698–703; e) H. Maeda, H.
Matsuno, M. Ushida, K. Katayama, K. Saeki, N. Itoh, Angew. Chem.
2005, 117, 2982–2985; Angew. Chem. Int. Ed. 2005, 44, 2922–2925;
f) O. Rusin, N. N. St. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian, R. M. Strongin, J. Am. Chem.
Soc. 2004, 126, 438–439.
[15] M. R. Wasielewski, D. G. Johnson, M. P. Niemczyk, G. L. Gaines,
M. P. OꢁNeil, W. A. Svec, J. Am. Chem. Soc. 1990, 112, 6482–6488.
[16] J. S. Lee, Y. K. Kim, M. Vendrell, Y. T. Chang, Mol. BioSyst. 2009, 5,
411–421.
[17] a) A. T. Hoye, J. E. Davoren, P. Wipf, M. P. Fink, V. E. Kagan, Acc.
Chem. Res. 2008, 41, 87–97; b) L. F. Yousif, K. M. Stewart, S. O.
Kelley, ChemBioChem 2009, 10, 1939–1950; c) J. Nunnari, A. Suo-
malainen, Cell 2012, 148, 1145–1159; d) J. Bereiter-Hahn, M. Voth,
S. Mai, M. Jendrach, Biotechnol. J. 2008, 3, 765–780; e) S. Zhang, T.
Wu, J. L. Fan, Z. Y. Li, N. Jiang, J. Y. Wang, B. R. Dou, S. G. Sun,
F. L. Song, X. J. Peng, Org. Biomol. Chem. 2013, 11, 555–558.
[18] F. Rashid, R. W. Horobin, J. Microsc. 1991, 163, 233–241.
[19] H. Zhu, J. L. Fan, Q. L. Xu, H. L. Li, J. Y. Wang, P. Gao, X. J. Peng,
Chem. Commun. 2012, 48, 11766–11768.
[8] E. Azuma, N. Nakamura, K. Kuramochi, T. Sasamori, N. Tokitoh, I.
Sagami, K. Tsubaki, J. Org. Chem. 2012, 77, 3492–3500.
[9] X. Xiong, F. Song, S. Sun, J. Fan, X. Peng, Asian J. Org. Chem. 2013,
2, 145–149.
[20] A. Roos, W. F. Boron, Physiol. Rev. 1981, 61, 296–433.
[21] E. P. Serjeant, B. Dempsey, Biochim. Biophys. Acta 1979, 977, 266–
277.
[10] B. A. Sparano, K. Koide, J. Am. Chem. Soc. 2007, 129, 4785–4794.
[11] a) H. Maeda, Y. Futkuyasu, S. Yoshida, M. Fukuda, K. Saeki, H.
Matsuno, Y. Yamauchi, K. Yoshida, K. Hirata, K. Miyamoto,
Angew. Chem. 2004, 116, 2443–2445; Angew. Chem. Int. Ed. 2004,
43, 2389–2391; b) B. C. Dickinson, C. Huynh, C. J. Chang, J. Am.
[22] J. C. Castro, A. Malakhov, K. Burgess, Synthesis 2009, 7, 1224–1226.
Received: February 2, 2013
Published online: && &&, 0000
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www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Long-Wavelength Fluorescein Analogues
COMMUNICATION
Born to dye: Five fluorescein ana-
logues were synthesised (see scheme).
One analogue was found to emit in the
NIR region with a high quantum yield,
excellent photostability and good per-
meability. Three derivatives were
found to specifically stain mitochon-
dria and one dye responds to thiols
with a strong turn-on NIR fluores-
cence signal and colorimetric change,
in vitro and in vivo.
Fluorescent Probes
X. Xiong, F. Song,* G. Chen, W. Sun,
J. Wang, P. Gao, Y. Zhang, B. Qiao,
W. Li, S. Sun, J. Fan,
X. Peng* ....................... &&&& — &&&&
Construction of Long-Wavelength
Fluorescein Analogues and Their
Application as Fluorescent Probes
Long Wavelength Fluorophores
Biological imaging in the red and near-infrared (NIR)
region is desirable due to its deep tissue penetration and
minimum background autofluorescence from biomolecules
in living systems. However, photostable NIR small-mole-
cule fluorophores are limited. In their Communication on
page && ff., X. Xiong et al. report five photostable fluo-
rescein analogues obtained through a Vilsmeier reaction
and Knoevenagel condensation. The new NIR fluoro-
phores retain the particular characteristics of fluoresceins,
but exhibit excellent bioimaging capabilities in both living
cells and mice.
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
9
&
ÞÞ
These are not the final page numbers!