Photo-controlled Zn2+ release system with dual binding-sites and turn-on fluorescence

Article abstract of DOI:10.1039/b917948h

A phototriggered Zn-release system with dual binding-sites and turn-on fluorescence has been developed by employing a molecule with two Schiff base units as a ligand. It is found that the ligand with two Schiff base units can bind with two Zn²⁺

Full text of DOI:10.1039/b917948h

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PAPER
www.rsc.org/pccp | Physical Chemistry Chemical Physics
Photo-controlled Zn2+ release system with dual binding-sites and turn-on
fluorescencew
Xu Zhangab and Yi Chen*a
Received 2nd September 2009, Accepted 20th November 2009
First published as an Advance Article on the web 23rd December 2009
DOI: 10.1039/b917948h
A phototriggered Zn-release system with dual binding-sites and turn-on fluorescence has been
developed by employing a molecule with two Schiff base units as a ligand. It is found that the
ligand with two Schiff base units can bind with two Zn2+, and the bound Zn2+ can be released
completely when the Schiff base is converted into bi-benzoxazole when phototriggered. During the
conversion of the Schiff base into benzoxazole, the turn-on fluorescence was detected, which
provides a convenient method to monitor the process of phototrigger release. Besides, it is
also found that the amount of released Zn2+ depends on the exposure or irradiation time,
which enables the possibility to manipulate the amount of Zn2+-release via controlling
irradiation time.
experiments can be monitored to determine how much drug
the system is likely to release.20
1. Introduction
The photo-controlled delivery of chemical and/or biochemical
species offers great opportunities in photodynamic therapy,1,2
release system21 using a phenolic Schiff base derivative
We have recently developed a phototriggered metal-ion
chemical synthesis3–5 and the characterization of reactive
as ligand, in which the metal-ion is released when the
intermediates.6–9 The light-activated release of metal ions
phenolic Schiff base is converted into 2-arylbenzoxazole
has attracted considerable interest because some biologically
in the presence of NaOH by the phototrigger (Scheme 1).
active metals make an essential cofactor in numerous enzymes
In addition, the latter compound has a diminished affinity
that are critical for life.10–12 Zinc, for example, is an indispensable
for metal ions. In this paper, we expand our studies
element in biology and free Zn2+ has been shown to influence
for two purposes: one is to extend the phototriggered
a variety of receptors and ion channels present on cell surfaces,
release system from single binding-site to double binding-site,
including NMDA13 and GABA14 receptors. Recent advances
which enables metal-binding and metal-release in high
have shed light on the biological roles of zinc, particularly
concentration; and the other is to elucidate the fluorescence
on its functions related to neurobiology.15 It is believed
changes during phototrigger metal release, which may provide
that disorder of zinc homeostasis is implicated in a number
a convenient method to monitor the process of phototriggered
of diseases,16 such as Alzheimer’s disease. The regulation of
release. In addition, we also found that the photoconversion
concentration of Zn2+ may also play a role in the control of
of phenolic Schiff base to benzoxazole could be performed
Alzheimer’s disease and excitotoxicity.17,18
in the absence of base (NaOH), which is beneficial for
The ability to control drug delivery in terms of quantity,
applications in a physiological pH window. We employ
location, and time is a key goal for drug delivery science, as
a new phenolic Schiff base with dual binding-sites, 1, as
improved control maximizes therapeutic effect while minimizing
ligand (Scheme 2), and we found that the dual binding-sites
side effects.19 The level of control which can be exerted on light
bound to two Zn2+ to form a complex, and the bound
delivered to a molecule in terms of wavelength, duration,
Zn2+ could be released completely by a phototrigger in the
intensity, and location can be exploited through a light-
absence of base. Moreover, the turn-on fluorescence was
controlled drug liberation reaction to give control of the
detected during the process of Zn2+-release from the complex
quantity of drug released, the timing of the release event,
by a phototrigger. This probably provides a way for the
and its location. A fluorescent photo-controlled drug release
control and determination of metal release by monitoring
system is useful because the fluorescence during in vitro
the fluorescence.
a Key Laboratory of Photochemical Conversion and Optoelectronic
Materials, Technical Institute of Physics and Chemistry,
The Chinese Academy of Sciences, Beijing, 100190, China.
E-mail: yichencas@yahoo.com.cn; Fax: +86 10 6256 4049;
Tel: +86 10 8254 3595
b Graduate School of ChineseAcademy of Sciences, Beijing, 100049,
People’s Republic of China
w Electronic supplementary information (ESI) available: Absorption
changes of the photoreaction process of 2 and both absorption and
fluorescence spectra for the control experiments of 2 binding with
Scheme 1 Outline of the phototriggered metal-release system with
Zn2+. See DOI: 10.1039/b917948h
single binding-site.
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Scheme 3 Synthetic routes for the preparation of 3 and 4.
Scheme 2 Outline of the phototriggered Zn2+-release system with
J1 = 4.9 Hz, J2 = 4.1 Hz), 7.63 (t, 2H, J1 = 4.2 Hz,
J2 = 4.9 Hz), 7.41–7.39 (m, 4H).
dual binding-sites.
A
mixture of 4-(benzo[d]oxazol-2-yl) benzaldehyde25
(223 mg, 1.0 mmol) and 2-aminophenol (120 mg, 1.1 mmol)
dissolved in anhydrous EtOH (20 ml) was stirred at room
temperature for 0.5 h. During this time, the color of the
solution gradually changed from colorless to yellow-green,
and a yellow precipitate was produced. The yellow product
was filtered off and washed with EtOH. The crude product was
purified by recrystallisation from hexane to give 4 (2-((4-
(benzo[d]oxazol-2-yl)methyleneamino)phenol) in 78% yield
(245 mg). 1H NMR (CDCl3): 8.77 (s, 1H), 8.39 (d, 2H,
J = 8.24 Hz), 8.08 (d, 2H, J = 8.3 Hz), 7.81 (t, 1H,
J1 = 4.9 Hz, J2 = 4.0 Hz), 7.62 (t, 1H, J1 = 4.1 Hz, J2 =
4.9 Hz), 7.41–7.33 (m, 3H), 7.24 (t, 2H, J1 = 7.2 Hz, J2 =
7.4 Hz), 7.06 (d, 1H, J = 7.9 Hz), 6.93 (t, 1H, (t, 1H, J1 = 7.9 Hz,
J2 = 7.3 Hz).
2. Experimental
General
1H NMR spectra were recorded at 400 MHz with TMS as an
internal reference and CDCl3 as solvent. UV absorption
spectra were measured with an absorption spectrophotometer
(Hitachi U-3010). All chemicals for synthesis were purchased
from commercial suppliers, and solvents were purified according
to standard procedures. Reactions were monitored by TLC
silica gel plates (60F-254). Column chromatography was
performed on silica gel (Merck, 70–230 mesh). A lamp
with 365 nm (30 W) light was used as the light source for
photoreactions.
Material
3. Results and discussion
Ligand 1 was prepared by the general method described in the
literature,22 and the detailed procedure is presented as follows:
terephthalaldehyde (6.7 g, 50 mmol) in ethanol (30 ml) was
added to a solution of 2-aminophenol (11 g, 100 mmol) in
ethanol (30 ml). The solution mixture was refluxed. When no
starting material (terephthalaldehyde) was detectable by TLC
plate, the mixture was cooled, and the crude product removed
by filtration. Recrystallisation from n-butanol gave ligand 1 in
78% yield (12.3 g). mp = 221–223 1C (lit.22 220–221 1C).
1H NMR (CDCl3): 8.76 (s, 2H), 8.03 (s, 4H), 7.36 (d, J =
8.0 Hz, 2H), 7.26–7.21 (m, 4H), 7.05 (d, J = 8.1, 2H), 6.93
(t, J1 = 8.0 Hz, J2 = 8.0 Hz, 2H).
The absorption changes of ligand 1 (lmax = 387 nm, e = 3.1 ꢁ
104 L molꢂ1 cmꢂ1, in CH3CN) with the addition of Zn2+ were
presented in Fig. 1. The absorption at 387 nm decreased,
whilst a new band at 465 nm appeared, which corresponded to
the absorption of the complex of ligand 1 binding to
Zn(OAc)2. The optical density of the complex is not increased
until a stoichiometric amount of Zn2+ (1.0 equiv.) is added to
the solution, which reveals the formation of a 1 : 1 complex.
This formation is confirmed by the plot of the optical density
of the complex against the amount of Zn2+ (inset in Fig. 1)
according to the literature method,26,27 and a binding constant
(Kb = 3.3 ꢁ 103 Mꢂ1) was estimated from the changes in the
spectral intensities by using the Excel program.28
3
and
4
(2-((4-(benzo[d]oxazol-2-yl)methyleneamino)-
phenol) were prepared according to the literature23 and are
illustrated in Scheme 3, and the detailed procedures and
spectra data were as follows: a mixture of 2-((E)-((E)-4-((E)-
(2-hydroxyphenylimino)methyl)benzyli-dene)amino)phenol
(158 mg, 0.5 mmol) and lead acetate, Pb(OAc)4 (244 mg,
0.55 mmol), dissolved in 20 ml of CHCl3 was refluxed for
2 h. The solution was filtered and the filtrate was extracted with
CH2Cl2–H2O (v/v = 1/1, 10 ml ꢁ 3). The combined organic
solution was dried over Na2SO4. The resulting solution was
concentrated and the crude product was purified by recrystal-
lisation from ethyl benzoate to give 3 (bis-p-phenylene-20-
benzoxazole) in 10% yield (16.2 mg). mp = 354–356 1C
Upon 365 nm light irradiation, the absorption of the
complex at 465 nm and 387 nm decreased and disappeared
with increase of irradiation time and, accompanying this
process, three new bands at 353, 336, 322 nm, respectively,
which were attributed to the absorption of 2, appeared
(Fig. 2). Comparing the absorption of 2 with that of 3, which
was prepared according to the literature23 and was illustrated
in the experimental, it was found that the absorption spectra
of 2 was similar to that of 3 (inset in Fig. 2), and absorption
bands for both were exactly at 353, 336, 322 nm, respectively.
As presented in Fig. 2, the absorption at 465 nm and 387 nm
disappeared completely when the solution of the complex was
1
(lit.24 354 1C). H NMR (CDCl3): 8.43 (s, 4H), 7.82 (t, 2H,
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Fig. 1 Titration of 25 mM of ligand 1 with Zn(OAc)2 in MeCN
(periods: 0, 5, 10, 15, 20, 25, 30 mM). The inset describes a 1 : 1 binding
isotherm with error bars calculated from three trials.
Fig.
3
Plot of fluorescence changes of
1
and the complex
[1 : Zn2+ (1 : 1 equiv.); 1 : Zn2+ (1 : 2 equiv.); 1 : Zn2+ (2 : 1 equiv.);
and 1 : Zn2+ (1 : 10 equiv.)] against irradiation time (1 : 25 mM,
MeCN), lex = 353 nm.
irradiated for 42 min. This suggested that Zn2+ was completely
released during the conversion of the complex to 2. It worth
noting that this current photo-triggered release system could
not be performed in pure water because ligand 1 did not
dissolve in water, and it has not been carried out yet in other
organic solvents, such as DMSO and EtOH or the mixtures of
water with organic solvents.
process is a result of the direct conversion of the complex
instead of free ligand 1 only.
The fluorescence of 2 during the process of Zn2+-photo-
release was investigated. It is found that no fluorescence of the
complex was detected in MeCN when using 353 nm as the
excitation wavelength. The fluorescence of 2 was, however,
observed when the complex was converted to 2 by a
phototrigger. As presented in Fig. 4, the fluorescence of 2
appeared and increased with increase of irradiation time, and
the largest fluorescence intensity was obtained with 42 min
irradiation. By that time, the conversion of the complex to 2
had completed. Comparing the fluorescence of 2 with that of 3
(quantum yield jf = 0.42, life time tf = 1.14 ns, in CH3CN,
using quinine sulfate in 1 M H2SO4 as a standard), we found
that the profile of the emission spectra of 2 was similar to that
of 3 (inset in Fig. 4), and both emission bands were at the same
position (lem = 370, 387, 408 nm) with the 353 nm excitation
wavelength. Control experiments showed that no binding
between 2 and Zn2+ was detected (see Fig. 2 and 3 in ESIw).
To explore the mechanism of the Zn2+-release process, 4
(Scheme 3) with a benzoxazole unit and one Schiff base unit
was prepared as a model for control experiments. First, it
is found that the absorption at 360 nm with a shoulder at
The photoreaction process of 2 probably occurred via two
routes. One is from both ligand 1 and the complex. Upon
irradiation with 365 nm light, both absorption at 465 nm
(absorption of the complex) and 387 nm (absorption of
ligand 1) decreased simultaneously, and both absorptions
disappeared with increase of irradiation time. Another is from
ligand 1 only. When ligand 1 photo-converted to 2, the
equilibrium between ligand 1 and the complex was disturbed,
and the complex had to release Zn2+ to rebuild the equilibrium,
which resulted in complete Zn2+-release from the complex
when 1 converted completely to 2 (for absorption changes of 1
when photo-converted to 2, see Fig. 1 in the ESIw). To confirm
the photoreaction process of Zn release, a competition experiment
was carried out. As presented in Fig. 3, it took about 48 min
for free ligand 1 to be completely converted to 2, which is
longer than that of the complex; the latter took 42 min to
complete conversion. It is thought that the main photoconversion
Fig. 4 Fluorescence changes of the complex upon 365 nm light
irradiation (in CH3CN, lex = 353 nm, periods: 0, 7, 14, 21, 28, 35,
42 min). The inset shows the fluorescence of 3 (25 mM, in MeCN,
lex = 353 nm).
Fig. 2 Absorption changes of the complex upon 365 nm light
irradiation (periods: 0, 7, 14, 21, 28, 35, 42 min). The inset shows
the absorption of 3 (25 mM, in MeCN).
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Fig. 7 Fluorescence changes of compound 4–Zn2+ upon 365 nm
light irradiation (periods: 0, 5, 10, 15, 20, 25 min, in CH3CN,
lex = 353 nm).
Fig. 5 Titration of 25 mM of 4 with Zn(OAc)2 in MeCN. The inset
describes a 2 : 1 binding isotherm with error bars calculated from three
trials.
370 nm, which is attributed to 4, decreases with the addition of
Zn2+ and a new absorption at 455 nm, which corresponded to
a 2 : 1 complex of 4 and Zn2+, appeared (Fig. 5). Upon
irradiation of the 4–Zn2+ with 365 nm light, 4–Zn2+ was
completely converted to 2. As shown in Fig. 6, the absorption
of 2 from 4–Zn2+ is same as the absorption of 3 (inset in
Fig. 2). Second, 4–Zn2+ showed no fluorescence emission
with a 353 nm excitation wavelength, and a fluorescence
(lem = 370, 387, 408 nm) was obtained only when the complex
4–Zn2+ was converted completely to 2 (Fig. 7). Results
indicated that two Zn2+ was released from the complex,
instead of one during the process of Zn2+-photorelease.
The kinetics of Zn2+-photorelease with different irradiation
times were also investigated by monitoring the change of the
fluorescence of 2. As presented in Fig. 8, the fluorescence
intensity of 2 increased slowly for the first 20 min, and
Fig. 8 Kinetics of Zn2+-photorelease from the system (25 mM, in
MeCN) with 365 nm light (30 W) at different irradiation times. The
y-axis represents the initial integrated fluorescence response (Fi) over
the final integrated emission (Ff).
increased faster afterwards, during the process of Zn2+
-
photorelease. It is likely that the emitted photons of 2 were
This study represents a platform for the development of
phototriggered metal release systems with dual binding-sites
and fluorescence monitoring, which allow metal release in high
concentrations and the process of metal release monitoring by
fluorescence. Although the system presented herein has some
shortcomings at present, such as the system not being able to
be performed in aqueous solution, modification of the system
is under way by covalently joining the Schiff base ligand to a
water-soluble polymer or incorporating them in the interior of
amphiphilic polymeric micelles. From a practical viewpoint, it
is desirable for the system to have some merits, such as
being performed in buffer solution and with visible light
(l Z 400 nm) irradiation. This can be achieved by further
modifying the molecular structure or environment and will be
the subject of future studies.
re-absorbed by the complex due to considerable overlap
between the absorption spectrum of the complex (lmax
=
387 nm) and fluorescence spectrum of 2 (lem = 387 nm). As
the absorption band decreased and disappeared with irradiation
time, this effect cancelled out, and the Zn2+-release increased
with irradiation time. The result shown in Fig. 8 indicated that
the amount of Zn2+-photorelease depends on irradiation time,
and the quantity of Zn2+-photorelease can be manipulated by
controlling irradiation time.
4. Conclusion
In conclusion, the example presented herein clearly demon-
strates that the metal release system with two Schiff base units
can be converted into bis-benzoxazole by a phototrigger,
which allows metal-binding and metal-release in high
concentration. The results also demonstrated that the process
of release can be monitored by fluorescence and the
amount of metal-release can be manipulated by controlling
Fig. 6 Absorption changes of complex 4–Zn2+ upon 365 nm light
irradiation (periods: 0, 5, 10, 15, 20, 25 min).
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the irradiation time. Future studies will take advantage of the
demonstrated system with photo-controlled release and
turn-on fluorescence, and explore photorelease systems with
multiplex binding-sites and nonlinear optical properties, which
enable the possibilities of metal release in larger concentra-
tions and three-dimensional distribution of release.
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