Multiresponsive switchable diarylethene and its application in bioimaging

Article abstract of DOI:10.1021/ol9014267

Figur Presented A multiresponsive fluorescent switch based on diarylethene and terpyridine units was developed. It exhibits effective switchable fluorescence which can be controlled by UV/visible light or metal ion/EDTA in solution. More importantly, havi

Full text of DOI:10.1021/ol9014267

ORGANIC
LETTERS
2009
Vol. 11, No. 17
3818-3821
Multiresponsive Switchable Diarylethene
and Its Application in Bioimaging
Xijun Piao, Ying Zou, Junchen Wu, Chunyan Li, and Tao Yi*
Department of Chemistry and Laboratory of AdVanced Materials, Fudan UniVersity,
Shanghai 200433, P.R. China
yitao@fudan.edu.cn
Received June 23, 2009
ABSTRACT
A multiresponsive fluorescent switch based on diarylethene and terpyridine units was developed. It exhibits effective switchable fluorescence
which can be controlled by UV/visible light or metal ion/EDTA in solution. More importantly, having low toxicity, it can enter live cells as a
fluorescent probe and can also serve as a detector for the biological process of metal ion transmembrane transport.
Fluorescence labeling technology is important to the ad-
vancement of biological imaging, which is widely used for
the monitoring of internal cellular processes.1 In the past
few decades, much progress has been made in designing
fluorescent probes for bioimaging2 and some materials, such
as organic dyes,3 fluorescent proteins,4 quantum dots,5
metallic complexes,6 and up-conversion nanoparticles7 are
now standard and widely used. While very important to
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10.1021/ol9014267 CCC: $40.75
Published on Web 08/06/2009
 2009 American Chemical Society

cellular imaging, most of these conventional probes respond
irreversibly to a certain event8 or nondynamically to envi-
ronmental stimuli, so that the development of a novel
photocontrollable and multiresponsive fluorescent labeling
molecule would be a powerful tool in elucidating the
physiological dynamics in living cells.9
During the past decade, much effort has been focused on
designing and synthesizing fluorescence switches, since they
have potential applications in information storage and light-
controlled molecular data processing.10 Our previous work
focused on diarylethene-based switches.11 Diarylethene is
one of the most promising photoswitchable units within the
photochromic system because of its high fatigue resistance
and thermal stability.12 We have designed and synthesized
an amphiphilic diarylethene as a photoswitchable probe for
imaging living cells.13 However, static imaging may not meet
the need to have probes that possess more functions, not only
labeling the cells or components inside but also showing the
biological processes of the metal ion transmembrane trans-
port.
metal ions are essential elements for life, but changes in their
bioconcentration are associated with serious diseases.16 For
example, the disruption of the Zn2+ accumulation pattern is
relevant to some types of prostate cancer, diabetes, and
neurodegenerative disorders;17 alterations in the cellular
homeostasis of Cu2+ may cause serious neurodegenerative
diseases such as Menkes and Wilson diseases and Alzhe-
imer’s disease.18
Scheme 1. Chemical Structure Changes of Compound 1o
It is known that the terpyridine unit is able to coordinate
with several kinds of metal ions,14 especially Zn2+ and Cu2+,
causing fluorescence changes in the original ligands.15 These
Figure 1. (A) Absorption and (B) fluorescence emission changes
of 1o in THF (1 × 10-5 M) upon irradiation with 365 nm light (λex
) 330 nm).
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Herein, we designed a novel fluorescence switch combin-
ing diarylethene and terpyridine functional units (1o, Scheme
1). This compound 1o exhibits several clearly different and
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Org. Lett., Vol. 11, No. 17, 2009
3819

reversible fluorescence states that can be controlled by
varying light frequency and metal ion concentration. Besides
functioning well in solution, it also has potential for effective
application in biological systems. More importantly, it
exhibits an easily detectable change in fluorescence when
stimulated by metal ions and thus would serve well as a
photoswitchable probe for imaging live cells and previously
nonvisualizable biological processes.
absorption and fluorescence intensity changes with the
alternate irradiation with ultraviolet and visible light. The
UV-vis absorption spectrum of 1o showed a single absorp-
tion band in the ultraviolet range ascribed to π-π* transition
(300 nm for 1o, Figure 1A). 1o exhibited blue fluorescence
centered at 440 nm with a fluorescent quantum yield of 0.041
when using Rhodamine B as reference (Figure 1B). Upon
irradiation with ultraviolet light (λ ) 365 nm), the colorless
solution turned slightly purple within 4 min and a new
absorption maximum appeared at 550 nm (ε ) 1.7 × 104
dm3mol-1cm-1). This was ascribed to the closed isomer form
with photocyclic quantum yield (Φo-c) of 23% (365 nm),
accompanied by quenching of 95% of the fluorescence in
the photostationary state (PSS) caused by an increase of
π-electron delocalization.12,19 The photocyclic quantum yield
was dependent on the irradiation wavelength, for example
Φo-c ) 21% upon irradiation with 380 nm (Table S1 in the
Supporting Information).
Under visible light irradiation (549 nm) in the PSS,
absorption in the visible range ceased and fluorescence
intensity was almost restored to the original state (before
irradiation with UV), demonstrating diarylethene’s classical
switching characteristic. The quantum yield of photocyclo-
reversion (Φc-o) was 22% upon irradiation with 549 nm and
decreased to 2% upon irradiation with 650 nm. The differ-
ence in absorbance at 550 nm for the open isomer and the
PSS was less than 2% and 8%, respectively, after the open-
close cycle was repeated 5 times by alternating irradiation
with UV and visible light (Supporting Information). The low
rate of fatigue apparent in the cycle repetition indicated that
1 possessed good fatigue-resistance. Thus, compound 1,
having changeable states that can be reversed multiple times
with little fatigue, could be used in a repeated “write-erase”
process.
1o was synthesized by a coupling of 1-(5-chloro-2-
methylthien-3-yl)-2-[2-methyl-5-(4-(dodecyloxy) benzene-
4-yl) thien-3-yl] cyclopentene (2) with 4′-(4-bromophenyl)-
2, 2′:6′, 2′′-terpyridine (3) (Scheme S1 in the Supporting
Information). To 6.0 mL anhydrous THF solution of 2 (0.3
g, 0.54 mmol), n-BuLi (0.4 mL of 1.6 M solution in hexane)
was added under Ar atmosphere at -5 °C. After stirred for
45 min at room temperature, B(OBu)3 (0.40 mL, 1.15 mmol)
was added in one portion to the mixture. The resulting
reddish solution was then stirred for 6 h at room temperature
and was added to a flask containing 3 (0.23 g, 0.59 mmol),
Pd(PPh3)4 and 3 mL Na2CO3 solution (20 wt %) at 50 °C.
The reaction was refluxed under Ar atmosphere for 19 h.
The pure product was obtained as a white solid by column
chromatograph (petroleum ether: triethylamine ) 18:1) and
washing with petroleum ether/ CH3OH in a yield around
1
35%. The molecular structure of 1o was confirmed by H
and 13C NMR spectroscopy, MALDI-TOF mass spectrometry
and elemental analysis (Supporting Information). Compound
1o is easily dissolved in a variety of organic solvents, such
as THF, DMSO, CH2Cl2, and CHCl3.
1o Can coordinate with both Zn2+ and Cu2+, resulting in
the decrease of the fluorescent intensity. Alkali and alkaline-
earth metal cations such as Na+, K+, Mg2+, Ca2+ gave no
interference at a 3.5-fold excess concentration, and transition-
metal and heavy-metal ions such as Fe2+, Cd2+, Hg2+ gave
a weak response (Supporting Information). Here, Zn2+ was
chosen as the metal ion in the investigations into the
quenching of the fluorescence of 1o because aqueous
solutions of Zn2+ are usually colorless and it is the strongest
Lewis acid among divalent metal ions.20 As shown in Figure
2 inset, fluorescence of 1o could be gradually quenched by
the addition of aqueous solution of Zn(NO3)2, which may
be attributed to the formation of a 1o-Zn complex, changing
the charge density of the terpyridine unit. Nonlinear fitting
of the fluorescence titration curve exhibited a 1:1 stoichi-
ometry for 1o and Zn2+, with the association constant (K)
of 1.76 × 106 M-1 (Figure 2).21 When EDTA solution (1.0
eq to Zn2+) was added, the fluorescence intensity was
Figure 2. Fluorescence intensity changes at 440 nm with different
molar ratio of Zn2+ and 1o in THF/ water solution (100:1, v/v) (1o
from 2 × 10-5 to 1 × 10-6 M, Zn2+ with the fixed concentration
of 1 × 10-5 M). (Inset) Fluorescence spectral changes of 1o (1 ×
10-5 M) by the addition of Zn2+ from 0 to 3.5 equiv.
The photochromic behavior of 1 was studied in THF
solution. As expected, compound 1 showed reversible
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Org. Lett., Vol. 11, No. 17, 2009

restored (Supporting Information), which can be explained
by the difference in the association constant between EDTA-
Zn and 1o-Zn. The KEDTA-Zn (3.16 × 1016 M-1)22 is much
larger than that of 1o-Zn. Therefore, Zn2+ acts as a trigger
for fluorescence switch-OFF, while EDTA acts as a trigger
for fluorescence switch-ON. The fluorescence intensity of
1o can be reversibly controlled by UV/visible light or Zn/
EDTA, and 1o acts as a double-controlled molecular
fluorescence switch reacting to light and chemical stimuli.
The luminescence of 1o could be controlled as readily in
living cells as in solution by using UV/ visible light as the
switching trigger, and this photoswitchable labeling was
demonstrated in one selected cell (shown in red circle, Figure
3C). After irradiation with 405 nm light (2 mW) for 3 min,
the brightness of the selected cell noticeably decreased
compared to the brightness of surrounding cells which
remained almost unchanged (Figure 3D). Upon irradiation
with 633 nm light (0.7 mW) the brightness of the selected
cell was recovered within 40 min (Figure 3E). In contrast,
when the 1o labeled cells were treated with different
concentrations of Zn2+, an obvious change in fluorescence
was observed with CLSM (Figure 3G-I). Luminescence
intensity decreased with increasing Zn2+ concentration
(Figure 3K-M). This effect provides a good means for
visualizing the process of M2+ (M ) Zn, Cu) uptake from
the outside into the cell. As expected, the fluorescence was
almost restored to the original state upon the addition of
EDTA solution (Figure 3J and N). The cytotoxicity of 1o is
important for its use as a bioprobe, so the effect of 1 on cell
proliferation was determined by means of an MTT assay
(Suppporting Information). The cellular viabilities were
estimated to be greater than 85% after 24 h in the presence
of 1-100 µM 1 (Supporting Information). This indicates that
1 has low cytotoxicity.
Figure 3. CLSM images of KB cells incubated with 1o for 20 min
at 25 °C (1 × 10-5 M in PBS/ DMSO, 100:2, v/v). (A, F) Bright-
field transmission image of KB cells. (B) Overlay image of A and
C. Confocal fluorescence image of (C) original state, (D) irradiated
by 405 nm light (2 mW, 3 min) for one selected cell and (E)
recovered by 633 nm light (0.7 mW, 40 min). Confocal fluorescence
image of (G) original state of F, and incubation by Zn2+ solution
with the concentrations of (H) 5 × 10-5 M, (I) 1 × 10-4 M. (J)
Recovered by 5 × 10-4 M EDTA solution. (K), (L), (M), (N) were
the distribution of fluorescence indensity of (G), (H), (I), (J),
respectively.
In conclusion, we have developed a new multiresponsive
fluorescence switch based on diarylethene and terpyridine
units. It exhibits effective switchable fluorescence controlled
by UV/visible light or metal ions/EDTA in solution. More
importantly, having low cytotoxicity, it can enter live cells
as a fluorescence probe and can act as a detector for the
biological process of the metal ion transmembrane transport.
We expect that this material will be of great benefit to
biomedical research.
Most diarylethene fluorescence switches have only been
studied in solution23 and little research has been carried out
using a biological system. Our research shows that 1o can
be effectively applied as a fluorescent probe in living cells.
Using confocal laser scanning microscopy (CLSM), we
observed a blue luminescence in the cytoplasm of KB cells
(human nasopharyngeal epidermal carcinoma cell) after
incubation with a PBS/ DMSO (100:2, v/v) solution of 1o
(1 × 10-5 M) for 20 min at 25 °C (Figure 3A, B, F, and G).
Acknowledgment. This work was supported by National
Science Foundation of China (20771027, 30890141), Na-
tional Basic Research Program of China (2009CB930400),
Specialized Research Fund for the Doctoral Program of
Higher Education (200802460007), Shanghai Sci. Tech.
Comm. (08JC1402400) and Shanghai Leading Academic
Discipline Project (B108).
Supporting Information Available: Synthetic and ex-
perimental details. This material is available free of charge
via the Internet at http://pubs.acs.org.
(22) Bell, P. F.; Chaney, R. L.; Angle, J. S. Plant Soil 1991, 130, 51–
62.
(23) Xiao, S.; Yi, T.; Zhou, Y.; Zhao, Q.; Li, F.; Huang, C. Tetrahedron
2006, 62, 10072–10078.
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