Zinc(II)-selective ratiometric fluorescent probe based on perylene bisimide derivative

Article abstract of DOI:10.1002/bio.1214

A fluorescent probe of N,N′-biscyclohexyl-1,7-di(3-pyridoxy)- perylene-3,4:9,10-tetracarboxylic acid diimide has been synthesized, and exhibited excellent selectivity and sensitivity for Zn²⁺ over other competing biological cations. The Zn

Full text of DOI:10.1002/bio.1214

Research article
Received: 02 October 2009,
Revised: 06 January 2010,
Accepted: 05 March 2010,
Published online in Wiley Online Library: 21 May 2010
(wileyonlinelibrary.com) DOI 10.1002/bio.1214
Zinc(II)-selective ratiometric fluorescent probe
based on perylene bisimide derivative
Yingjie Zhao, Juanjuan Sun, Zhiqiang Shi*, Cuicui Pan and Maoyou Xu
ABSTRACT: A fluorescent probe of N,NЈ-biscyclohexyl-1,7-di(3-pyridoxy)-perylene-3,4:9,10-tetracarboxylic acid diimide has
been synthesized, and exhibited excellent selectivity and sensitivity for Zn2+ over other competing biological cations. The
Zn2+-selective fluorescence blue-shift and enhancing property in conjunction with a visible colorimetric change from orange to
green could be observed. With favorable photophysical properties in the visible region, the perylene bisimide derivatives
remarkably improved the performance of the probe. Copyright © 2010 John Wiley & Sons, Ltd.
Keywords: fluorescent probe; perylene derivative; zinc ion; ratiometric probe
the sensitivity for Zn2+ detection were increased and could be
detected by the naked eye owing to change of colors.
Introduction
Zinc plays an important role in the human body (1–4). This has
been a concern of chemists and has resulted in considerable
development of Zn2+-specific molecular probes (5,6). The estima-
tion of free zinc in extremely low concentrations is difficult with
classical methods. Fluorescence techniques stand out as the
method of choice. The main mechanism of fluorescent molecular
probe includes charge transfer (7), electron transfer (8,9), energy
transfer (10), excimer formation and conformational change
(11,12). A fluorescent probe is advantageous due to its high sen-
sitivity; however, fluorescence measurement can be influenced
by many factors, such as the localization of the probe, changes of
environment around the probe and changes in the excitation
intensity. To reduce the influence of such factors, a ratiometric
measurement is to be utilized (13,14). This technique provides
greater precision than measurement at a single wavelength, and
is suitable for cellular imaging studies. Recently, more and more
probes have used for the fluorescent detection and imaging of
target biological molecules in living systems (15,16).
Experimental
N,N′-Bis(2,6-cyclohexyl)-1,7-dibromoperylene-3,4:9,10-tetracar-
boxylic acid diimide (712 mg, 1 mmol), 3-hydroxypyridine
(475 mg, 5 mmol) and anhydrous K2CO3 (276 mg, 2 mmol) were
added to N-methylpyrrolidone (20 mL). The solution was stirred
at 80°C under argon for 16 h. The reaction mixture was allowed to
reach room temperature and poured into aqueous hydrochloride
acid (200 mL, 1 M). The precipitated product was filtered and was
then washed thoroughly with water and dried at 75°C under
vacuum. The product was purified by column chromatography to
give bcdp–PDI (451 mg, 61%) as a red solid; m.p. > 300°C. 1H-NMR
(300 MHz, CDCl3, ppm): d = 9.48 (d, 2H), 8.61 (d, 2H), 8.55 (m, 4H),
8.27 (s, 2H), 7.47 (m, 4H), 4.96 (m, 2H), 2.48 (m, 4H), 1.89 (m, 4H),
1.75–1.62 (m, 6H), 1.32–1.46 (m, 6H). 13C-NMR (75 MHz, CDCl3,
ppm): d = 163.34, 162.82, 153.94, 132.61, 131.06, 130.52, 129.07,
128.90, 127.91, 125.62, 124.95, 124.19, 123.91, 123.22, 54.21,
29.06, 26.46, 25.37. MS (MALDI-TOF): m/z = 740 [M]+.
Perylene bisimide derivatives are characterized by strong
absorption in the visible region of spectra and exhibit a similarly
strong fluorescence. The fluorescence quantum yield is near 1
and the lifetime of the single excited state is approximately 4 ns
(17–19). Also the perylene bisimide dyes have excellent photo-
stability, thermal stability and chemical inertness. Recent years
have witnessed an ever-increasing interest in this class of chro-
mophores because of their favorable properties. Thus, perylene
bisimide dyes have been proved to be one of the best fluoro-
phores available for single molecule spectroscopy (20,21). Adams
and coworkers showed the suitability of perylene bisimide dyes
for sensors with specificity at the single molecule level if appro-
priate receptor units are attached to the perylene bisimide
fluorophore (22). In this paper, perylene bisimide derivative
was used as a fluorophore, which was attached to a recogni-
tion group, 3-hydroxypyridine, to obtain the fluorescent
probe of N,N′-biscyclohexyl-1,7-di(3-pyridoxy)-perylene-3,4:9,10-
tetracarboxylic acid diimide (bcdp–PDI; Scheme 1). A similar
compound has been reported by Müllen and co-workers (23).
With ratiometric fluorescence measurements, the selectivity and
UV–vis and fluorescence titration were carried out by introduc-
ing the metal ions dissolved in methanol to a solution of 10-5
M
bcdp–PDI in chloroform. Metal chlorides were used as source of
Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Ru3+, Hg2+, Ca2+, Cd2+, Mn2+ and Pb2+. The
influence of the small amount of the methanol could be
neglected.
Results and discussion
Figure 1 shows the absorption spectral changes of bcdp–PDI
upon addition of different equivalents of Zn2+ in chloroform at
* Correspondence to: Zhiqiang Shi, Department of Chemistry, Shandong
Normal University, Jinan, 250014, People’s Republic of China. E-mail:
fatal911@163.com
Department of Chemistry, Shandong Normal University, Jinan, 250014,
People’s Republic of China
Copyright © 2010 John Wiley & Sons, Ltd.
Luminescence 2011; 26: 214–217

Zinc(II)-selective ratiometric fluorescent probe
Scheme 1. Synthesis
of
N,N′-biscyclohexyl-1,7-di(3-pyridoxy)-perylene-3,4:9,10-
tetracarboxylic acid diimide (bcdp–PDI).
Figure 2. Changes in the emission spectra of bcdp–PDI upon addition of Zn2+
Figure 1. Absorbance spectra of bcdp–PDI in the presence of increasing amounts
of Zn2+ (0, 0.5, 1, 1.5, 2, 2.5 and 3 equiv.) in chloroform. The concentration of the
bcdp–PDI was 10-5 M.
(left) and Cu2+ (right) in chloroform. The concentration of the bcdp–PDI was 10-5
and the excitation wavelength was 518 nm.
M
room temperature. The UV–vis spectrum of bcdp–PDI is charac-
terized by a very intense band centered at 531 nm (e = 48
000 M-1 cm-1) which is responsible for the orange color of the
solution. The absorption maximum of bcdp–PDI has about a
10 nm red shift in comparison to that of the starting PDI dye.
Upon addition of Zn2+, the intensity of the absorption maximum
of bcdp–PDI at 531 nm decreases following the formation of a
new band centered at 519 nm (e = 52 700 M-1 cm-1), and this
change is very small after addition of 1.5 equiv. Zn2+. Three isos-
bestic points at 490, 505 and 525 nm are observed in spectra of
UV–vis titration. In other words, the blue shift in absorption spec-
trum is observed upon Zn2+ binding. More importantly, the Zn2+
sensing and the concomitant absorption changes are clearly
visible to the naked eye because the orange solution of bcdp–PDI
becomes green upon titration with Zn2+ ions.
Fig. 2. bcdp–PDI has a high degree of fluorescence in chloroform
(F = 90%). Upon addition of Zn2+ ion, the fluorescence intensity of
bcdp–PDI increases obviously accompanied by a large blue shift
in the emission spectra. The free bcdp–PDI displays a maximum
emission wavelength at 561 nm. Upon addition of Zn2+, the emis-
sion intensity of bcdp–PDI decreases at 561 nm and increases
significantly at 535 nm. This trend is not obvious until 1.5 equiv.
Zn2+ is added. Different from the Zn2+, when Cu2+ is introduced
into the bcdp–PDI solution, the emission intensity at 561 nm
decreases gradually and is quenched almost totally after addition
of 2 equiv. of Cu2+. From a mechanistic viewpoint, we speculate
that the fluorescence intensity enhancement and the blue-shift
of the maximum emission wavelength can be assigned to the
formation of new complex between metal ions and bcdp–PDI.
The diamagnetic Zn2+ with d10 electronic configuration having
flexible coordination geometry is advantageous for the specific
binding. Second, coordination of the Zn2+ to the ligand con-
nected with the PDI core leads to the distortion of the PDI plane
Compared with the UV–vis spectrum, more significant change
happened in the fluorescence spectra. A comparison of the emis-
sion behavior of bcdp–PDI against Zn2+ and Cu2+ is shown in
Luminescence 2011; 26: 214–217
Copyright © 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence

Y. Zhao et al.
that, although a range of metal ions can bind to the sensor, most
lose out when in competition with Zn2+. bcdp–PDI exhibits excel-
lent selectivity for Zn2+ over other competing biological cations,
thus making bcdp–PDI a suitable probe for Zn2+.
Conclusion
In conclusion, we have developed a new class of colorimetric and
fluorometric probe based on perylene bisimide derivatives for
Zn2+ detection with high selectivity and sensitivity. A highly Zn2+-
selective fluorescence blue-shift and enhancing property in con-
junction with a visible colorimetric change from orange to green
can be observed. Significantly, the perylene bisimide derivatives
were used as the fluorophore, which improved the performance
of the probe.
Figure 3. Emission spectra of 10-5 M bcdp–PDI, 10-5 M bcdp–PDI + 2 ¥ 10-5 M Cu2+
and 10-5 M bcdp–PDI + 2 ¥ 10-5 M Cu2+ + 4 ¥ 10-5 M Zn2+ in chloroform.
Supporting information
Supporting information can be found in the online version of this
article.
Table 1. Photophysical data and binding constants of
10-5 M bcdp–PDI in chloroform after adding 2 equiv. different
metal ions
Acknowledgment
This work was supported by the National Natural Science Foun-
dation of China (20771067) and Natural Science Foundation of
Shandong Province (2008BS02004).
Metal
ions
UV–vis
(lmax/e)
Emission Emission
Binding
(lEx/lEm
)
(I/I0)
constant (M-1)
Zn2+
Cu2+
Fe3+
Hg2+
Co2+
Ni2+
519/52165
523/53750
520/44683
526/45417
522/47350
523/51160
522/47895
518/535
535/561
535/561
535/561
535/561
535/561
535/561
1.55
0.01
0.35
0.80
0.48
0.98
0.24
53800
20000
8900
3440
1480
References
1. Berg J, Shi Y. The galvanization of biology: a growing appreciation for
the roles of zinc. Science 1996;271:1081–5.
2. Jiang P, Guo Z. Fluorescent detection of zinc in biological systems:
recent development on the design of chemosensors and biosensors.
Coord Chem Rev 2004;248:205–29.
15700
6950
Ru3+
3. Lim N, Freake H, Brückner C. Illuminating zinc in biological systems.
Chem Eur J 2005;11:38–49.
4. Que E, Domaille D, Chang C. Metals in neurobiology: probing their
chemistry and biology with molecular imaging. Chem Rev 2008;
108:4328.
5. Kimura E, Koike T. Recent development of zinc-fluorophores. Chem
Soc Rev 1998;27:179–84.
6. Carol P, Sreejith S, Ajayaghosh A. Ratiometric and near-infrared
molecular probes for the detection and imaging of zinc ion. Chem
Asian J 2007;2:338–48.
7. Maruyama S, Kikuchi K, Hirano T, Urano Y, Nagano T. A novel, cell-
permeable, fluorescent probe for ratiometric imaging of zinc ion.
J Am Chem Soc 2002;124:10650–51.
8. de Silva A, Gunaratne H, Gunnlaugsson T, Huxley A, McCoy C,
Rademacher J, Rice T. Signaling recognition events with fluorescent
sensors and switches. Chem Rev 1997;97:1515–66.
9. Jiang W, Fu Q, Fan H, Wang W. An NBD fluorophore-based sensitive
and selective fluorescent probe for zinc ion. Chem Commun
2008;259–61.
10. Van Dongen E, Dekkers L, Spijker K, Meijer E, Klomp L, Merkx M.
Ratiometric fluorescent sensor proteins with subnanomolar affinity
for Zn(II) based on copper chaperone domains. J Am Chem Soc
2006;128:10754–62.
which can also be a reason for the blue-shift of the maximum
UV–vis and emission wavelength.
An important criterion for a selective cation probe is the ability
to detect a specific cation in the vicinity of other competing ions.
To explore the utility of bcdp–PDI as an ion-selective fluorescence
probe for Zn2+, the competition experiments were conducted.
Besides Cu2+ and Zn2+, bcdp–PDI interacts with several metal ions
such as Fe3+, Co2+, Ni2+, Ru3+ and Hg2+, which showed a blue-shift
and significant increase in the absorption band with three isos-
bestic points. Ca2+, Cd2+, Mn2+ and Pb2+ had no significant inter-
action with bcdp–PDI, which showed no change in the
absorption spectra. Most importantly, the emission of bcdp–PDI
could be significantly quenched by Cu2+, Fe3+, Co2+, Ni2+, Ru3+ and
Hg2+ and showed almost no change when Ca2+, Cd2+, Mn2+ or Pb2+
was introduced, whereas the titration of Zn2+ resulted in a strong
emission at 535 nm with DEem = 26 nm. The color of bcdp–PDI
changed from orange to green. However, addition of Zn2+
into the nonfluorescent solution of the bcdp–PDI–Cu2+ com-
plex showed the green fluorescence of bcdp–PDI–Zn2+ again
(Fig. 3).
11. Ajayaghosh A, Carol P, Sreejith S. A ratiometric fluorescence probe for
selective visual sensing of Zn2+.
14962–3.
J Am Chem Soc 2005;127:
12. Sreejith S, Divya K, Ajayaghosh A. Detection of zinc ions under
aqueous conditions using chirality assisted solid-state fluorescence
of a bipyridyl based fluorophore. Chem Commun 2008;2903–5.
13. Xiang Y, Tong A. Ratiometric and selective fluorescent chemodosi-
meter for Cu(II) by Cu(II)-induced oxidation. Luminescence 2008;23:
28–31.
The binding constants of these metal ions with bcdp–PDI were
calculated by Benesi–Hildebrand-type analyses (Table 1). These
data show that the Zn2+ has the strongest binding force with
bcdp–PDI. These data and observations unambiguously prove
wileyonlinelibrary.com/journal/luminescence
Copyright © 2010 John Wiley & Sons, Ltd.
Luminescence 2011; 26: 214–217

Zinc(II)-selective ratiometric fluorescent probe
14. Li N, Tang W, Xiang Y, Tong A, Jin P, Ju Y. Fluorescent salicylaldehyde
hydrazone as selective chemosensor for Zn2+ in aqueous ethanol: a
ratiometric approach. Luminescence; doi: 10.1002/bio.1175.
15. Xue L, Liu C, Jiang H. A ratiometric fluorescent sensor with a large
Stokes shift for imaging zinc ions in living cells. Chem Commun
2009;1061–3.
16. Tomat E, Nolan E, Jaworski J, Lippard S. Organelle-specific zinc detec-
tion using zinpyr-labeled fusion proteins in live cells. J Am Chem Soc
2008;130:15776–7.
17. Xiao S, El-Khouly M, Li Y, Gan Z, Liu H, Jiang L, Araki Y, Ito O, Zhu D.
Dyads and triads containing perylenetetracarboxylic diimide and
porphyrin: efficient photoinduced electron transfer elicited via both
excited singlet states. J Phys Chem B 2005;109:3658–67.
18. Kaletas B, Dobrawa R, Sautter A, Würthner F, Zimine M, De Cola L,
Williams R. Photoinduced electron and energy transfer processes in a
bichromophoric pyrene–perylene bisimide system. J Phys Chem A
2004;108:1900–09.
19. Lee S, Zu Y, Herrmann A, Geerts Y, Müllen K, Bard A. Electrochemistry,
spectroscopy and electrogenerated chemiluminescence of perylene,
terrylene, and quaterrylene diimides in aprotic solution.
J Am Chem Soc 1999;121:3513–20.
20. Mais S, Tittel J, Basché T, Bräuchle C, Göhde W, Fuchs H, Müller G,
Müllen K. Terrylenediimide: a novel fluorophore for single-molecule
spectroscopy and microscopy from 1.4 K to room temperature. J Phys
Chem A 1997;101:8435–40.
21. Langhals H, Jaschke H, Ring U, Von Unold P. Peryleneimidazoloim-
ides: highly fluorescent and stable replacements of terrylene. Angew
Chem Int Ed 1999;38:201–3.
22. Zang L, Liu R, Holman M, Nguyen K, Adams D. A single-molecule
probe based on intramolecular electron transfer. J Am Chem Soc
2002;124:10640–41.
23. Kohl C, Weil T, Qu J, Müllen K, Towards highly fluorescent and water-
soluble perylene dyes. Chem Eur J 2004;10:5297–310.
Luminescence 2011; 26: 214–217
Copyright © 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence