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devices, logic gate, display, chemical sensing [1–5]. In particular,
the development of photoswitchable fluorescent probe received
great attention because of their rapid and reversible, optically-
drive transitions between two distinct states that differ in their
absorption and emission spectroscopic properties [6]. Its biggest
advantage is that it can enhance the resolution and suppress inter-
ferences and autofluorescence for detecting the target in complex
background environment, especially in biological samples such as
live cells, tissues [7].
time to report fluorescent diarylethene appended rhodamine 6G
for the detection of Hg2+ both in solution and in living A549 lung
cancer cells.
Experimental
Materials and instruments
Most chemicals were purchased from Aldrich Chemical Co. and
TCI. Solvents were purified by normal procedures and handled un-
der moisture free atmosphere. The other materials were commer-
cial products and were used without further purification. Melting
points were determined using a Beijing Tech X-4 with a digital
thermometer and are uncorrected. UV–visible absorption spectra
were measured on a Shimadzu UV-2550 spectrometer. Fluores-
cence spectra were measured on a PerkinElmer LS-55 Fluorescence
spectrophotometer. Mass spectra were recorded on ESI Mass spec-
troscopy. 1H NMR and 13C NMR spectra were recorded using a
Varian Inova 400 MHz FT-NMR spectrometer with TMS as internal
standard. A UV lamp (365 nm, 1.5 mW cmÀ2) was used as a
UV-light source, and A Xenon lamp (k > 520 nm, 300 W) was a
visible-light source through a color filter. The live cells imaging
experiment was carried out by Leica TCS SP5 Laser Scanning
Confocal Microscopy.
Up to the present, some photoswitchable fluorescent probes
have been reported [8–12]. However, as the demand for high selec-
tivity and sensitivity photoswitchable fluorescent sensing and rec-
ognition of environmentally and biologically important ion species,
especially heavy metal ions are continuously increasing. Among of
heavy metal ions, Mercury is considered as one of the most danger-
ous heavy metal ions, because it can cause serious environmental
and health problems because of its toxic effects. It can easily pass
through skin and bio-accumulate in human body, which leads to
digestive, kidney, and especially neurological diseases [13–15].
For these reasons, there is an urgent need to design and synthesis
selective and sensitive photoswitchable fluorescent probes that are
capable of detecting the presence of Hg2+
.
Bearing those in mind, we hope to develop a new photoswitch-
able fluorescent molecular probe for Hg2+ to improve their selectiv-
ity and sensitivity. In molecular design strategy, we considered
that among all the fluorophores, rhodamine-based fluorescent
derivatives are excellent candidates for detecting metal ions due
to the excellent spectroscopic properties of large molar extinction
coefficients, high fluorescent quantum yields, and visible wave-
length excitation. The rhodamine derivatives can undergo equilib-
rium between spirocyclic (nonfluorescent) and ring-open form
(high fluorescent) [16,17]. Generally, these rhodamine-based fluo-
rescent probes bearing ‘soft’ chelators such as O, N and S atoms can
selectively coordinate with ‘soft’ Hg2+, which leads to the open-ring
form and significant signal output. Thus far, several rhodamine-
based derivatives have been developed for detecting Hg2+ with
moderate success [18]. These previous study have contributed to
a broad understanding of the mechanism of rhodamine-based
probes. So, if the photochromic molecular switches can be induced
to the rhodamine-based derivatives for constructing a photo-
switchable fluorescent probe, it has the potential for successfully
detect Hg2+ since the unique characters of rhodamine fluorophores,
and also, it may be a powerful tool to construct multifunction
molecular switches in solution and even in living cells. Among var-
ious types of photochromic compounds, bisthienylethene (BTE)
derivatives are one of the most promising compounds owing to
their excellent fatigue resistance and thermal stability. They
showed promising application in ultra high density optical mem-
ory media, logic gate, living cells fluorescence probe and so on
[19,20]. Until now, some bisthienylethene chemosensors have
been reported for detecting metal ions [21–25]. However, utilizing
photochromic bisthienylethenes and rhodamine to construct
probes for metal Hg2+ is rare. The unique physical and chemical
properties of bisthienylethenes and rhodamine make their combi-
nation compound maybe a promising compound for Hg2+ fluores-
cent probe and application in live cell imaging.
Synthesis
The design and synthesis of compound BTE–RD were illustrated
in Scheme 1. The compound 1 and compound 4 were synthesized
according to previously reported method [26] and [27], respec-
tively. The structure of BTE–RD and other intermediates were con-
firmed by 1H NMR, 13C NMR, and HRMS.
Synthesis of compound 2
The compound 1, 1,2-bis (5-chloro-2-methylthien-3-yl)cyclo-
pentene (2.0 g, 6 mmol) was dissolved in anhydrous THF (20 mL)
under nitrogen atmosphere at room temperature. The mixture
was cooled to À78 °C, into which n-BuLi (9 mL of 1.6 M in hexane,
14.4 mmol) was added dropwise. The solution was stirred at
À78 °C for 30 min, and then DMF (1.1 mL, 14.4 mmol) was added
and went on stirring for 1 h. The reaction mixture was warmed
to room temperature and quenched by 2 M HCl after reacting for
12 h. The crude product was extracted by dichloromethane, and
the combined organic layer was washed with water and brine,
and dried over MgSO4. After filtering and solvent evaporation,
the crude product was purified by silica gel column chromatogra-
phy using petroleum and dichloromethane (V/V = 1:2) as eluent to
obtain grey solid powder (0.8 g, yield: 52%). 1H NMR (400 MHz,
CDCl3) d = 9.74 (s, 2H), 7.43 (s, 2H), 2.84 (t, J = 7.5 Hz, 4H),
2.17–2.08 (m, 2H), 2.05 (s, 6H).
Synthesis of compound 4
Herein, we synthesis a new bisthienylethene bearing rhoda-
mine 6G dyad (abbreviated BTE–RD, shown in Scheme 1) as a
photoswitchable fluorescent probe for Hg2+. The BTE–RD showed
excellent reversibly switchable fluorescent changes when sensing
and binding Hg2+ by alternating irradiation with UV light and vis-
ible light. In addition, the BTE–RD fluorescent probe displayed a
high sensitivity and an excellent sensitivity towards Hg2+. More-
over, this probe is applied for the detection of Hg2+ in living cells,
showed good bioactive and can evidently enhance the fluorescence
in live cells imaging. To the best of our knowledge, this is the first
Rhodamine 6G hydrochloride (2.4 g, 5 mmol) was dissolved in
ethanol (60 mL), excess amount of hydrazine hydrate (5 mL,
82 mmol) was added dropwise. The reaction mixture was refluxed
for 3 h and then cooled to room temperature. The precipitate was
obtained by filtering and washing with water three times. After
drying under vacuum, the reaction afforded slightly pink powder
(1.96 g, yield: 92%).
1H NMR (400 MHz, CDCl3) d = 7.94–7.99 (m, 1H), 7.42–7.49 (m,
2H), 7.04–7.09 (m, 1H), 6.39 (s, 2H), 6.26 (s, 2H), 3.58 (s, 2H), 3.53
(s, 2H), 3.23 (m, 4H), 1.92 (s, 6H), 1.32 (t, J = 7.1 Hz, 6H).