A new dual-function fluorescent probe of Fe3+for bioimaging and?probe-Fe3+complex for selective detection of CN?

Article abstract of DOI:10.1016/j.tet.2016.05.082

A rhodamine-based probe for Fe³⁺was prepared and confirmed by X-ray crystallography. The probe exhibited highly selective and sensitive recognition toward Fe³⁺with distinct color change. Interestingly, the probe-Fe³⁺comple

Full text of DOI:10.1016/j.tet.2016.05.082

Tetrahedron 72 (2016) 4361e4367
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
3þ
A new dual-function fluorescent probe of Fe for bioimaging
and probe-Fe3 complex for selective detection of CN
þ
ꢀ
Lirun Yan, Meipan Yang, Xin Leng, Ming Zhang, Ying Long, Bingqin Yang *
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of Education, College of Chemistry and Materials Science, Northwest
University, Xi’an 710127, PR China
a r t i c l e i n f o
a b s t r a c t
A rhodamine-based probe for Fe3 was prepared and confirmed by X-ray crystallography. The probe
þ
Article history:
Received 22 April 2016
Received in revised form 28 May 2016
Accepted 31 May 2016
Available online 2 June 2016
3þ
exhibited highly selective and sensitive recognition toward Fe with distinct color change. Interestingly,
the probe-Fe complex could detect CN according to the strong co-ordination ability of cyanide to Fe .
3þ
ꢀ
3þ
The whole recognition mechanism was explored through Job’s plot, MS data and theoretical calculations.
Importantly, the probe was further used for detecting Fe3 in living cells and mouse, which exhibited
þ
excellent fluorescence imaging.
Keywords:
Ó 2016 Elsevier Ltd. All rights reserved.
Fluorescent probe
3þ
Fe
ꢀ
CN
Bioimaging
1. Introduction
probes owing to its excellent photophysical properties. Asa result, the
excellent properties afford a potential for the construction of an ‘off-
3
þ 17e20
As one of the most essential trace element, iron is omnipresent
in the universe, from the core of the earth to living organisms, in-
on’ type fluorescent probes for Fe
.
Unfortunately, most of the
3þ
currently existing rhodamine-based probes for Fe suffered certain
shortcomings such as interferences from Cr
1
e3
3þ
2þ
cluding mammalian cells.
It provides the oxygen-carrying ca-
and Cu , worse bi-
pacity of heme and acts as a cofactor in many enzymatic reactions.
However, while iron is clearly an essential component of a healthy
diet, at levels that exceed the capacity of the organisms to safely use
it, iron can be toxic because of its ability to promote oxidation of
lipids, proteins and other cellular components. Recent research
ological compatibility and so on. At the same time, only few
ꢀ
21
rhodamine-based probes for CN have been reported. More re-
cently, a new design concept of ‘two-in-one’ has emerged, which is
recognizing two or more targets by one receptor using a single de-
2
2e26
tection method, or an array of detection methods.
The special
suggests that Fe3 could also be involved in the underlying mech-
þ
strategies lead to faster analytical processing times and potential cost
reduction. Without doubt, it is of challenge to rationally design sim-
ple, practical bifunctional probes in aqueous media.
anisms of many neurodegenerative diseases, such as Parkinson’s
4
,5
disease and Alzheimer’s disease. On the other hand, cyanide is
well known as one of the most toxic materials and as being ex-
tremely harmful to the environment and human health. It is
With these considerations in mind, we designed and synthesized
a novel colorimetric and fluorescent probe for Fe . The probe ach-
6
3þ
known cyanide is usually absorbed through the gastrointestinal
track, lungs or skin.7 Nevertheless, cyanide is widely applied in
many processes, such as plastics manufacturing, electroplating,
ieved high selectivityand sensitivity as well as ‘naked-eye’ detection.
At the same time, the probe was used for detecting Fe in living cells
3þ
and mouse, which exhibited excellent fluorescence imaging. More-
gold and silver extraction, tanning, and metallurgy.8
e13
So it is ur-
3þ ꢀ
over, the probe-Fe complex could detect CN according to the
3þ
gent to develop novel analytical methods with high sensitivity and
strong co-ordination ability of cyanide to Fe . As anticipated, the
selectivity to detect Fe3 ions as well as CN .
þ
ꢀ
complex displayed excellent performance for CN detection.
ꢀ
Fluorescent probes of various structures have been synthesized
and used,1
4e16
rhodamine B framework is one of the most frequently
2
2
. Results and discussion
employed functions as a signaling device for constructing fluorescent
.1. Structural properties
The rhodamine derivative YG was prepared in high yield and
confirmed by H NMR, C NMR, MS data and X-ray analysis. The
*
Corresponding author. Tel.: þ86 29 88302217; fax: þ86 29 88302617; e-mail
1
13
addresses: yangbq@nwu.edu.cn, hsylr1992@163.com (B. Yang).
http://dx.doi.org/10.1016/j.tet.2016.05.082
0
040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

4362
L. Yan et al. / Tetrahedron 72 (2016) 4361e4367
single crystal of YG was grown in dichloromethane and methanol
solutions, and a unique spirolactam structure ring formation was
observed (Fig. 1).
Selectivity is a very important parameter to evaluate the per-
formance of a fluorescence probe. The fluorescence response of YG
to different metal ions was investigated. Fig. 3 illustrated that no
obvious changes were noted upon addition of the representative
ions, indicating that our proposed probe YG exhibited high selec-
3
þ
tivity to Fe over other metal ions. At the same time, YG could be
3
þ
served as a sensitive ‘naked-eye’ probe for Fe (Fig. S4). To test
3
þ
practical applicability of our fluorescent probe for Fe , competi-
tion experiments were also carried out. As expected, the recogni-
3
þ
tion performance of YG to Fe was unaffected by the presence of
the other coexisting metal ions. These observations indicated that
3þ
YG had good selectivity for Fe and could be used as an excellent
3
þ
fluorescent probe for Fe determination.
Fig. 1. X-ray crystal structure of YG.
.2. Spectral characteristics of YG to Fe3
D
2
As shown in Fig. S1, the absorbance spectra of YG at varied
concentrations of Fe3 were investigated in MeOHeTriseHCl (4:6,
V: V, pH¼7.4) solutions. As expected, YG itself displayed colorless
and exhibited almost no characteristic absorption peak at above
þ
00 nm. Addition of Fe3 , however, immediately produced a pink
þ
5
color with an absorption maximum at 563 nm, which can be
explained by the opening of the spirolactam ring in the rhodamine
3
þ
unit. Upon gradual addition of Fe to the solution of YG, the ab-
sorbance peak at 563 nm increased gradually. Similarly, a new in-
tense peak at 583 nm in the emission spectra was observed,
exhibiting the ring-opening mechanism of the rhodamine lactam
3
þ
ring when YG coordinated with Fe (Fig. 2). These suggested that
the probe effectively avoided the fluorescence quenching caused by
3þ
Fig. 3. The selectivity and competition of YG for Fe . The pillars in the front row
represent fluorescence response of (20
pillars in the back row represent the subsequent addition of 20
containing YG and the metal ions, respectively (
mM) probe for the examined metal ions. The
3
þ
m
M Fe3þ to the solution
the paramagnetic nature of Fe . The binding association constant
þ
4
l
ex¼555 nm). From 1 to 16: blank, Na ,
K, was also estimated to be 3.0ꢁ10 M (Fig. S2) by the Bene-
þ
2þ
2þ
2þ
2þ
2þ
2þ
3þ
2þ
2þ
2þ
2þ
3þ
3þ
K , Ca , Cd , Mg , Co , Mn , Cu , Al , Zn , Ni , Fe , Hg , Cr and Fe .
sieHildebrand method.2 Base on the definition of detection limit,
7
3
þ
we studied the detection limit of YG for Fe (Fig. S3), which was
.5 M.
2
m
L
.3. Spectral characteristics of YG to CN in presence of Fe3D
2
2
.3.1. Kinetic studies. Because the optical signal changes relied on
2
2
1
1
500
3
þ
ꢀ
the chemical reaction between complex YG-Fe and CN . The ef-
fect of reaction time on the spectra of the detecting system was
investigated and the results were shown in Fig. 4. The fluorescence
intensity decreased greatly with the time increasing and a plateau
was achieved after 10 min, indicating that a reaction time of 10 min
may be used for this system.
1
0
.1 equiv.
000
500
000
3+
Fe
equiv.
3
þ
titrated with
2.3.2. UVevis and fluorescence spectra of YG-Fe
ꢀ
3þ
ꢀ
CN . The interaction of YG-Fe complex with CN of different
concentrations was firstly investigated through UVevis and fluo-
rescence spectra and the results were shown in Fig. 5. The solution
3
þ
of YG-Fe complex in MeOHeTriseHCl (4:6, V:V, pH¼7.4) was
5
00
0
pink with an absorption maximum at 563 nm. However, upon
ꢀ
gradual addition of increasing concentrations of CN (0e1.5 equiv)
to the above solution, a decrease of absorbance at 563 nm could be
observed (Fig. 5a). The phenomenon indicated that CN could
capture Fe from the YG-Fe complex according to the strong co-
ordination ability of cyanide to Fe . Significantly, as designed, the
system exhibited drastic fluorescence decrease with the
ꢀ
5
60
580
600
620
640
660
3
þ
3þ
wavelength (nm)
3þ
Fig. 2. Fluorescence titration spectra of YG (20
pH¼7.4) solutions upon addition of Fe
mM) in MeOHeTriseHCl (4:6, V:V,
a
3
þ
(l
ex¼555 nm).

L. Yan et al. / Tetrahedron 72 (2016) 4361e4367
4363
ꢀ
2
2
2
1
1
1
700
400
100
800
500
200
A linear relationship for CN detection under the optimum condi-
tion was obtained at 583 nm with a regression equation as
ꢀ
Y¼ꢀ0.70Xþ21.53 (Fig. S5). Further, the detection limit of CN was
ꢀ5
around 3.9ꢁ10 M (Fig. S6). In a word, the significant spectra in-
dicated that the spirocycle form of YG was recovered via interaction
ꢀ
3þ
.
of CN and YG-Fe
3
þ
ꢀ
2
.3.3. Selectivity and tolerance of YG-Fe system to CN . The se-
lectivity experiments of YG-Fe to CN were extended to various
anions, which was similar to the work for Fe detection just now.
As shown in Fig. 6, the addition of CN resulted in remarkably
3þ
ꢀ
3þ
ꢀ
9
6
3
00
00
00
0
decreased absorbance and fluorescence intensity. Under the same
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
condition, additions of other anions including F , Cl , Br , I , SO
4
,
,
ꢀ
2ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
2ꢀ
3ꢀ
SCN , S , HSO
NO
3
, HSO
4
, ClO
4
, ClO
3
, BrO
3
, IO
3
, CO
3
, C
2
O
4
, PO
4
-
ꢀ
ꢀ
3
, NO
2
and Ac did not cause any discernible changes. These
observations indicated that probe YG had a high sensitivity and
ꢀ
excellent selectivity for CN in MeOHeTriseHCl (4:6, V:V, pH¼7.4)
0
5
10
15
20
25
30
3
þ
.
in the presence of Fe
Time/min
ꢀ
3þ
(l
Fig. 4. Effect of time on the reaction of CN with probe YG-Fe
ex¼555 nm).
(a)
(a)
0
0
0
0
0
.20
.15
.10
.05
.00
0.20
F , Cl , Br , I , SO , SCN , S , HSO ,
HSO , ClO , ClO , BrO , IO , CO
C O , PO , NO , NO , Ac , blank
,
-
0
equiv. CN
0.15
0.10
0.05
0.00
-
1.5 equiv.
CN
5
00
525
550
575
600
4
50
500
550
600
wavelength (nm)
wavelength (nm)
(b)
(b)
2500
2000
1500
1000
3000
2500
0
equiv.
-
-
-
-
2-
-
2-
-
-
F , Cl , Br , I , SO , SCN , S , HSO ,
4
3
CN
-
-
-
-
-
2-
HSO , ClO , ClO , BrO , IO , CO
,
4
4
3
3
-
3
3
2
-
3-
-
-
C O , PO4 , NO , NO , Ac , blank
2
4
3
2
2000
1500
1000
1
.5 equiv.
-
CN
5
00
0
5
00
0
560
580
600
620
640
660
560
580
600
620
640
660
wavelength (nm)
wavelength (nm)
3
þ
3þ
Fig. 5. Changes in UVevis absorption (a) and fluorescence (b) spectra of YG-Fe
Fig. 6. Absorption (a) and fluorescence intensity (b) changes of YG-Fe (20 mM) upon
different anions.
ꢀ
(
20
m
M) with various amounts of CN in MeOHeTriseHCl (4:6, V:V, pH¼7.4) solutions.
l
ex¼555 nm.
ꢀ
introduction of CN . As depicted in Fig. 5b, a linear decrease of
In addition, the other important feature of the probe is its high
selectivity toward the analyte over other competitive species. As
shown in Fig. 7, the addition of CN still resulted in large fluores-
fluorescence intensity centered at 583 nm could be observed with
increasing CN concentrations over the range of 0e30
the fluorescence emission was nearly quenched after 1.5 equiv CN .
ꢀ
ꢀ
mM. Even,
ꢀ
cence changes even in the presence of 10 equiv miscellaneous

4364
L. Yan et al. / Tetrahedron 72 (2016) 4361e4367
competitive anions. These observations indicated that YG could be
ꢀ
served as potential CN probe with excellent properties.
3+
probe-Fe +competing anions
3000
2500
2000
1500
1000
3+
-
probe-Fe +competing anions+CN
500
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19
Fig. 7. Fluorescence intensity of probe YG-Fe3þ (20
m
M) upon addition of CN in the
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
presence of 10 equiv other anion.
l
ex¼555 nm. From 1 to 19: F , Cl , Br , I , SO
4
ꢀ
,
ꢀ
2ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
2ꢀ
3ꢀ
ꢀ
ꢀ
SCN , S , HSO
3
, HSO
4
, ClO
4
, ClO
3
, BrO
3
, IO
3
, CO
3
, C
2
O
4
, PO
4
, NO
3
, NO
2
and Ac .
2
.4. Mechanism
In order to confirm why the significant absorbance and fluo-
rescence of YG changed, we studied the recognition mechanism.
According to the Job’s plot, it can be observed that the absorbance
went through a maximum at a molar fraction of about 1/2, in-
dicating that a 1:1 stoichiometry was most possible for the binding
Fig. 8. The 3D representation and condensed Fukui function for YG.
Fe3 , and probe reverted to the spirolactam form (fluorescence-off).
As a result, the probable recognition process was shown in Scheme
þ
3
þ
mode of YG and Fe (Fig. S7).
1
Then, H NMR experiment was carried out in CDCl
3
after adding
Fe3 into the solution of YG to investigate the coordination process.
þ
2.
Compared the data, there were some obvious differences. Addition
3
þ
of Fe
d
resulted in shifting and broadening of the peak at
2
.5. Fluorescence imaging
6.2e6.5 ppm to 5.7e6.6 ppm (Fig. S8), which revealed opening of
the spirolactam ring on coordination to Fe in the aromatic rings
of the xanthenes moiety.
Further, DFT calculations were carried out with the Gaussian 09
program to study the binding site. Basis set of double-z quality
3
þ
To evaluate cytotoxicity of the fluorescent probe, YG was taken
to perform an MTT assay on L929 cells with different concentra-
tions (100, 50.0, 20.0, 12.5, 6.25 M). The result (Fig. S10) indicated
the cell viability was more than 86% under the treatment of 20
YG, exhibiting low toxicity to cultured cells at the application dose.
Due to its hypotoxicity and excellent spectroscopic properties,
the probe was used for living cell fluorescence imaging to monitor
Fe ions. The L929 cells were incubated in confocal dishes to make
the adherent growth. To be attached in good condition, L929 cells
were incubated with probe YG (20
Fig. 10a1, no fluorescence was observed inside the living cells. After
the cells were treated with Fe (20 mM) for another 1.5 h, a pro-
nounced increase in fluorescence was observed from the in-
tracellular area (Fig. 10b1). The results confirmed that the probe
would have a good recognition to Fe
environment.
Encouraged by the results of fluorescence imaging in living cells,
we wondered whether probe YG may be potentially useful for
imaging applications in living animals. To this end, the properties of
the probe in Kunming mouse were evaluated. The mouse was given
an ip injection with probe YG and imaged. Then the mouse was
followed by ip injection with Fe and then imaged in vivo imaging
system. As shown in Fig. 11a, there is little fluorescence when the
mouse was only treated with probe. Obviously, a large fluorescence
m
m
M
(
6e31 þ G* for O and N, 6e31G** for C and H) was applied for the
geometry optimization and the following single point energy cal-
culation. According to the results of vibration analysis, the opti-
mized structure was proven to be the local minimum. Based on
NBO analysis,28 the carbonyl O2 (0.028), N4 (0.009) were larger
than that of N5 (ꢀ0.001) and amide N3 (ꢀ0.003), which indicated
3
þ
mM) for 1.0 h. As shown in
3þ
that the probe was most likely to chelate Fe via O2 and N4 atoms
3
þ
(
Fig. 8).
At the same time, we investigated the recognition mecha-
nism of probe toward CN in the presence of Fe through the
ESI-MS spectra of the complex of YG-Fe -CN . As shown in
Fig. 9, the peaks at m/z 636.2215 and 658.2038 were charac-
ꢀ
3þ
3
þ
ꢀ
3
þ
ions in the biological
þ
þ
ꢀ
terized to be [YGþH] and [YGþNa] upon addition of CN to
the solution of YG-Fe3 . The ESI-MS spectrum was also done
þ
ꢀ
after adding some anions including CN into the solution of
3
þ
YG-Fe . As shown in Fig. S9, the peaks at m/z 636.2195 and
þ
þ
6
58.2014 were characterized to be [YGþH] and [YGþNa] as
3
þ
3þ
well as showing in Fig. 9, which revealed the probe YG-Fe
ꢀ
could also detect CN in the presence of other anions.
Taken together, the spirolactam moiety of the rhodamine group
acted as a signal switcher, which was envisioned to turn on when
Fe was binded with the O2 and N4 atoms of probe YG (fluores-
cence-on). Then, CN could take Fe away from the complex YG-
3
þ
signal was noted after the mouse was injected with probe and Fe
Fig. 11b). The result established that YG was a reasonable probe for
3
þ
(
ꢀ
3þ
3
þ
imaging Fe in the mouse.

L. Yan et al. / Tetrahedron 72 (2016) 4361e4367
4365
Fig. 9. Mass spectrum of YG-Fe3 in the presence of CN .
þ
ꢀ
exhibited good fluorescence imaging. The design strategy can be
extensive used to construct other multifunctional fluorescent
probes with kind of potential applications in environmental and
biological analysis.
4
4
. Experimental
.1. Apparatus reagents and chemicals
The absorption spectra were recorded on a Shimadzu UV-1700
spectrophotometer. The fluorescence spectra were measured on
a HITACHI F-4500 fluorescence spectrophotometer. NMR spectra
were recorded on a Varian INOVA-400 MHz spectrometer (at
1
13
4
00 MHz for H NMR and 100 MHz for C NMR). HRMS was recorded
on a microTOFQ II ESI-Q-TOF LC/MS/spectrometer. Single crystal X-
ray diffraction data were collected on a Bruker SMARTAPEX II CCDX-
ray crystallography. Bioimaging of the probe was performed on an
Olympus FV1000 confocal microscopy. Imaging of living mouse was
conducted employing Xenogen IVIS spectrum imaging system. The
solvents and chemicals were commercially available and were used
without further purification unless otherwise noted. Double distilled
water was used throughout the experiment.
Scheme 1. Synthesis of probe YG.
3
. Conclusion
In summary, we have developed a colorimetric and fluorescent
probe for Fe3 based on rhodamine. The probe YG could detect
þ
selectively and sensitively accompanied by obvious color change.
4.2. Preparation of the test solution
3
þ
Moreover, the ensemble YG-Fe can be an good sensory system for
ꢀ
detecting CN , indicating that the metal-based complex was
Stock solution of probe (200
mM) was prepared in methanol.
þ
2þ
þ
2þ
a promising tool to detect anions. Importantly, probe YG was fur-
Stock solutions of metal ions (200
Cd , Mg , Co , Mn , Cu , Al , Zn , Ni , Fe , Hg , Cr and
m
M) including Na , K , Ca ,
3
þ
2þ
2þ
2þ
2þ
2þ
3þ
2þ
2þ
2þ
3þ
ther applied for detecting Fe in living cells and mouse, which
Scheme 2. Proposed recognition mechanism of probe for Fe3 or CN .
þ
ꢀ

4366
L. Yan et al. / Tetrahedron 72 (2016) 4361e4367
Fig. 10. Bioimaging application of probe in L929 cells. Representative fluorescence images of cells treated with probe (20 mM) in either the absence (a) or the presence (c) of 20 mM
3þ
ꢃ
Fe for 1.5 h at 37 C. (b) Bright-field image of cells shown in panel. (d) Overlay image of (b) and (c).
Fig. 11. Representative fluorescent images of probe and Fe3þ in vivo: (a) Probe was injected in the intraperitoneal (ip) cavity of mouse; (c) then followed by ip injection of Fe3þ
0 min after probe injection, the mouse was imaged.
.
1
Fe3 were prepared by dissolving corresponding nitrate or chloride
þ
128.13, 127.80, 124.97, 123.58, 107.23, 105.30, 97.88, 77.36, 77.05,
ꢀ
ꢀ
ꢀ ꢀ
salts in water. The anions stock solutions (200
m
M) of F , Cl , Br , I ,
76.73, 68.59, 53.47, 44.23, 12.67. HRMS (ESI) calcd for
2
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
2ꢀ
þ
SO
PO
4
, SCN , S , HSO
, NO
3
, HSO
4
, ClO
4
, ClO
3
, BrO
3
, IO
3
, CO
3
, C
2
O
4
,
C
36
H34ClN
5
O
2
S [MþH] : 636.2195, found: 636.2217.
3
ꢀ
ꢀ
ꢀ
4
3
, NO
2
, Ac and CN were obtained by dissolving their
sodium salts in H
2
O. Before spectroscopic measurements, the stock
4.4. Cytotoxicity
solutions of probe and ions were then diluted to the corresponding
concentration with MeOHeTriseHCl (4:6, V: V, pH¼7.4) solution.
Then the mixture was equilibrated for 5 min and then the fluo-
rescence intensity was recorded at 583 nm. The excitation and
emission slits were both set to 2.5 nm.
YG was prepared by dissolving in 10 mL methanol with
ꢀ
3
concentration of 8ꢁ10 M, then 5.0 mL of this solution was placed
in a 10-mL tube and diluted by water, which got a solution of YG
ꢀ
3
with concentration of 4ꢁ10 M. In turn, the solution was diluted to
a small concentrations, and the concentrations of the probe were
ꢀ
3
ꢀ3
ꢀ3
ꢀ3
ꢀ4
4
.3. Synthesis of YG
8ꢁ10 M, 4ꢁ10 M, 2ꢁ10 M,1ꢁ10 M, 5ꢁ10 M, respectively.
Cytotoxicity of probe YG on cells was determined by conven-
Probe YG was readily prepared according to Scheme 1.
tional MTT assay. The L929 cells were seeded into 96-well plates at
3
Compound 1a was synthesized according to the literature pro-
cell density of 3.0ꢁ10 cells/well in 180
mL DMEM and maintained
mL different concentrations of probe were added to
2
9
cedure. Rhodamine B (1.59 g, 3 mmol) was dissolved in dry 1, 2-
dichloroethane, followed by addition of phosphorus oxychloride
for 24 h. Then 2
the wells, and incubated for another 24 h. Following that, the me-
dium was removed and washed three times with PBS. Finally, 180
of fresh culture medium and 20 L MTT solutions (5 mg/mL) were
added to each well. After incubation for 4 h, the supernatant was
removed and 150 L of DMSO was added to each well. The working
concentration gradient of the probe was 100.0, 50.0, 20.0, 12.5,
6.25 M. The trays were then vigorously shaken to solubilize the
(
5 mL). After being reflux for 4 h, the solvent and excess amount of
mL
phosphorus oxychloride was removed by evaporation to give the
corresponding acid chloride, and then dissolved in acetonitrile
without further purification. Then the solution of 1a (0.63 g,
m
m
3
mmol), triethylamine (2 mL) in acetonitrile was added. After
refluxing for 12 h, the solvent was removed under reduced pressure
to give a violent-oil. Water was then added to the mixture and the
aqueous was extracted with dichloromethane (15 mLꢁ3). The or-
m
formazan product and the absorbance at a wavelength of 490 nm
was read on Microplat Reader and analyzed. All experiments were
conducted in triplicate. Data were expressed as meanꢂSD.
4
ganic layer was dried over anhydrous MgSO and filtered. The crude
product was purified by silica gel column chromatography to give
1
YG (yellow solid) in 70.8% yield. H NMR (400 MHz, CDCl
.07 (d, J¼8.0 Hz, 1H), 7.81 (d, J¼8.0 Hz, 2H), 7.65e7.55 (m, 2H), 7.35
d, J¼8.0 Hz, 2H), 7.24 (d, J¼8.0 Hz, 1H), 6.48 (s, 2H), 6.38 (d,
J¼8.0 Hz, 2H), 6.16 (, J¼12.0 Hz, 2H), 3.30 (q, J¼8.0 Hz, 8H), 1.13 (t,
3
)
d
(ppm)
4.5. Fluorescence imaging
8
(
The mouse fibroblasts cells (L929 cells) were cultured in DMEM.
Immediately before the experiments, cells were pretreated with
13
ꢃ
J¼6.0 Hz, 12H) C NMR (100 MHz, CDCl
3
)
d
166.30, 161.57, 154.80,
probe YG (20
m
M) for 1 h at 37 C in humidified air and 5% CO
2
,
1
54.09, 153.81, 148.98, 136.09, 134.85, 129.28, 129.16, 128.77, 128.47,
washed three times with PBS and imaged. After incubation with

L. Yan et al. / Tetrahedron 72 (2016) 4361e4367
4367
ꢃ
FeCl
times with PBS to remove remaining FeCl
Kunming mouse (20e25 g) was given an intraperitoneal (ip)
injection of probe (400 M). The mouse was anesthetized
Lꢁ400
by inhalation of isoflurane and imaged. Then Fe (40
was injected to the same region. The mouse was then imaged
3
(20
m
M) for another 1.5 h at 37 C, cells were washed three
6. Kulig, K. W. Cyanide Toxicity; U.S. Department of Health and Human Services:
Atlanta, GA, 1991.
3
and imaged.
7. Bhattacharya, P. K., Alpha Science International Ltd., Harrow, U.K., 2005.
8. Qian, G.; Li, X. Z.; Wang, Z. Y. J. Mater. Chem. 2009, 19, 522.
m
m
9. Peng, L. H.; Wang, M.; Zhang, G. X.; Zhang, D. Q.; Zhu, D. B. Org. Lett. 2009, 11,
1943.
3þ
mLꢁ40
mM)
10. Xu, Z. C.; Chen, X. Q.; Kim, H. N.; Yoon, J. Y. Chem. Soc. Rev. 2010, 39, 127.
11. Zhang, J.; Zhu, S.; Valenzano, L. RSC Adv. 2013, 3, 68.
3þ
(
10 min after the injection of Fe ) by using an IVIS imaging system.
12. Kim, D. S.; Chung, Y. M.; Jun, M.; Ahn, K. H. J. Org. Chem. 2009, 74, 4849.
1
3. Vallejos, S.; Est eꢀ vez, P.; García, F. C.; Serna, F.; Pe n~ a, J. L.; García, J. M. Chem.
Commun. 2010, 7951.
14. Huang, J.; Tang, M.; Liu, M.; Zhou, M.; Liu, Z. G.; Cao, Y.; Zhu, M. Y.; Liu, S. G.;
Acknowledgements
Zeng, W. B. Dyes Pigm. 2014, 107, 1.
1
5. Huang, J.; Liu, M.; Ma, X. Q.; Dong, Q.; Ye, B.; Wang, W.; Zeng, W. B. RSC Adv.
014, 4, 22964.
16. Hong, J.; Zheng, W. B.; Zhong, M.; Gao, F. Acta Biophy. Sin. 2011, 27, 301.
7. Xiang, Y.; Tong, A. Org. Lett. 2006, 8, 1549.
8. Dong, L.; Wu, C.; Zeng, X.; Mu, L.; Xue, S. F.; Tao, Z.; Zhang, J. X. Sens. Actuators, B
2010, 145, 433.
This work was supported by the National Natural Science
Foundation of China (No. 21172178).
2
1
1
Supplementary data
19. Yin, W.; Cui, H.; Yang, Z.; Li, C.; She, M.; Yin, B.; Li, J.; Zhao, G.; Shi, Z. Sens.
Actuators, B 2011, 157, 675.
0. Yang, Z.; She, M.; Yin, B.; Cui, J.; Zhang, Y.; Sun, W.; Li, J.; Shi, Z. J. Org. Chem.
2012, 77, 1143.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2016.05.082.
This data includes MOL file and InChiKey of the most important
compounds described in this article
2
21. Liu, Y.; Lv, X.; Zhao, Y.; Liu, J.; Sun, Y.-Q.; Wang, P.; Guo, W. J. Mater. Chem. 2012,
22, 1747.
2
2
2
2
2
2. Schmittel, M.; Lin, H. W. Angew. Chem., Int. Ed. 2007, 46, 893.
3. Singh, N.; Mulrooney, R. C.; Kaur, N.; Callan, J. F. Chem. Commun. 2008, 4900.
4. Jung, H. J.; Singh, N.; Lee, D. Y.; Jang, D. O. Tetrahedron Lett. 2010, 51, 3962.
5. Singh, N.; Kaur, N.; Choitir, C. N.; Callan, J. F. Tetrahedron Lett. 2009, 50, 4201.
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