A photo-responsive catalytic vesicle with GPx activity

Article abstract of DOI:10.1002/cjoc.201300695

Glutathione peroxidase (GPx) is a vital antioxidant enzyme involved in the reduction of reactive oxygen species and protects cells from oxidative damage. Consequently enormous efforts have been devoted to developing artificial catalysts with GPx function. Besides the research on enhancing the catalytic activity of GPx mimics, the design and construction of smart GPx models has also inspired great interest. Herein, a novel photo-responsive selenium-containing vesicular GPx model was successfully constructed by supramolecular self-assembly of the cationic surfactant PyC₁₀AzoC₁₀Py with benzeneseleninic acid (PhSeO₂H) through hydrophobic and electrostatic interactions in aqueous media. This selenium-containing vesicular catalyst showed remarkable GPx-like activity, which is 692 times more effective than PhSeO₂H for the reduction of cumene hydroperoxide (CUOOH) by 4-nitrobenzenethiol (NBT). Interestingly, when an equimolar amount of α-CD was added, the GPx-like activity of the catalytic vesicle declines remarkably due to the vesicle disaggregation in the presence of α-CD. Whereas the biomimetic system was irradiated by UV light at 365 nm, the catalytic vesicle was formed again and the GPx-like activity recovered. Copyright

Full text of DOI:10.1002/cjoc.201300695

FULL PAPER
DOI: 10.1002/cjoc.201300695
A Photo-responsive Catalytic Vesicle with GPx Activity
Ruiqing Xiao, Lipeng Zhou, Zeyuan Dong, Yuzhou Gao, and Junqiu Liu*
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Chang-
chun, Jilin 130012, China
Glutathione peroxidase (GPx) is a vital antioxidant enzyme involved in the reduction of reactive oxygen species
and protects cells from oxidative damage. Consequently enormous efforts have been devoted to developing artificial
catalysts with GPx function. Besides the research on enhancing the catalytic activity of GPx mimics, the design and
construction of smart GPx models has also inspired great interest. Herein, a novel photo-responsive selenium-
containing vesicular GPx model was successfully constructed by supramolecular self-assembly of the cationic sur-
factant PyC10AzoC10Py with benzeneseleninic acid (PhSeO2H) through hydrophobic and electrostatic interactions in
aqueous media. This selenium-containing vesicular catalyst showed remarkable GPx-like activity, which is 692
times more effective than PhSeO2H for the reduction of cumene hydroperoxide (CUOOH) by 4-nitrobenzenethiol
(NBT). Interestingly, when an equimolar amount of α-CD was added, the GPx-like activity of the catalytic vesicle
declines remarkably due to the vesicle disaggregation in the presence of α-CD. Whereas the biomimetic system was
irradiated by UV light at 365 nm, the catalytic vesicle was formed again and the GPx-like activity recovered.
Keywords photo-responsive, self-assembly, vesicle, GPx mimics
Introduction
strate specificity and high GPx-like activity. Recently,
our group has managed to construct novel sele-
nium-containing micelle GPx models.[11] Taking advan-
tage of the specific microenvironment provided by the
CTAB micelle, this selenium-containing micelle exhib-
ited a remarkable GPx-like activity which is about 5
orders of magnitude more efficient than that of diphenyl
diselenide (PhSeSePh), a well-studied GPx mimic, for
the reduction of cumene hydroperoxide (CUOOH) by
3-carboxy-4-nitrobenzenethiol (TNB).
Many smart materials sensitive to the temperature,
pH, ion concentration and electric potential, such as
hydrogels, micelles, vesicles and nanoparticles, have
been reported.[12-17] Scientists have used these stimu-
lus-responsive materials to design and develop smart
enzyme models recently. According to this concept, our
group has constructed several smart enzyme models
with temperature-responsive GPx catalytic activity by
incorporating the catalytic center selenium or tellunium
into PNIPAM-based microgel,[18] micelle,[19] and vesi-
cle.[20] By changing temperature, we are able to regulate
the microenvironment of catalytic centers and the rec-
ognition between enzyme models and substrates, and
thus control the enzymatic activity. However, to our
knowledge, the construction of light-responsive GPx
model has rarely been reported.
The controlled balance of reactive oxygen species in
biological system plays an important role in cell func-
tion, regulation, and adaptation to diverse growth condi-
tions.[1] As an important anti-oxidative enzyme, the se-
lenium-containing glutathione peroxidase (GPx) pos-
sesses unique functions to protect living organisms from
oxidative stresses by catalyzing the reduction of hy-
droperoxides (ROOHs) using glutathione (GSH) as a
reducing substrate.[2] In the active center of GPx, se-
lenocysteine residue, the catalytic moiety, is situated in
a hydrophobic pocket of the protein surface, which is
formed by some charged and hydrophobic amino acid
residues (Phe, Trp, Asp) and functions as the substrate
binding site.[3] In recent years, to study the structural
and functional relationship of GPx and to extend its po-
tential applications in the development of anti-oxidant
medicine, enormous effort has been made in the re-
search of artificial GPx simulation; one strategy of GPx
mimics is to introduce the catalytic center to artificial or
naturally existing scaffolds with binding sites for
susbstrates by chemical synthesis or genetic engineer-
ing.[4-6] For example, a series of selenium- or tellurium-
containing cyclodextrins, such as 2-SeCD, 2-TeCD,
2-Te-diCD, and 6-Te-diCD have been fabricated as GPx
mimics by taking advantage of the hydrophobic cavity
of cyclodextrin for binding substrates.[4,7-10] These
cyclodextrin-based GPx mimics displayed good sub-
Herein, we reported a novel photo-responsive sele-
nium-containing vesicular GPx model constructed by
incorporating benzeneseleninic acid (PhSeO2H) into the
*
E-mail: junqiuliu@jlu.edu.cn; Tel.: 0086-0431-85168452; Fax: 0086-0431-85168491
Received September 11, 2013; accepted November 14, 2013; published online December 11, 2013.
Chin. J. Chem. 2014, 32, 37—43
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
37

Xiao et al.
FULL PAPER
palisade layer of the vesicle formed from an azoben-
zene-containing bola amphiphile (PyC10AzoC10Py)
through supramolecular self-assembly (Scheme 1). In-
terestingly, this selenium-containing vesicle exhibited a
remarkable GPx-like activity (50.19 μmol•L−1•min−1),
which is about 692 times of that of PhSeO2H alone for
the reduction of CUOOH by 4-nitrobenzenethiol (NBT).
By using the photo-controllable inclusion and exclusion
reaction of PyC10AzoC10Py with α-CD, we are able to
manipulate the reversible assembly and disassembly of
this vesicle, and thus to regulate the GPx activity of this
enzyme model.
temperature for UV time course studies was controlled
within 0.5 ℃ by use of a LAUDA compact low-
temperature thermostat RC6 CP. The pH values of
buffers were determined with a METTLER TOLEDO
320 pH meter.
Synthesis of benzeneseleninic acid
PhSeO2H was synthesized according to the proce-
dure of McCullough and Gould[21] (Eq. 1). Diphenyl
diselenide (1.25 g, 4.0 mmol) was added to 5 mL of
1,4-dioxane, and the mixture was heated to become
clear. It was then cooled below 5 ℃, and hydrogen
peroxide (28% w/w) was added dropwise, stirring the
mixture and cooling externally to keep temperature un-
der 10 ℃. After the addition of a 3-fold excess of hy-
drogen peroxide, the ice (4.00 g) was added, and the
white precipitate was filtered and washed with 10 mL of
cooled water. Then the benzeneseleninic acid was ob-
Scheme 1 Schematic illustration of the change in the structure
of the vesicular catalyst with light
1
tained as a white product (0.87 g, 57.45% yield). H
NMR (300 MHz, D2O) δ: 7.84－7.94 (m, 2H), 7.64－
7.75 (m, 3H).
SeO2H
H2O2
Se
(1)
Se
Synthesis of 4,4'-dihydroxyazobenzene
4,4'-Dihydroxyazobenzene was prepared based on
the method of Willsätter[22] and Tomohiro (Scheme
2).[23] A mixture of KOH (100 g, 1.52 mol),
p-nitrophenol (20 g, 152 mmol), and water (25 mL) was
heated to 120 ℃ and left to stand for 1 h. Then the
mixture was slowly heated to 200 ℃, and left to react
for about 12 h, the mixture changed into a brown vis-
cous liquid with a large number of bubbles developing.
After the reaction was completed, products were dis-
solved in water. Then the dark-red solution was acidi-
fied with 2.0 mol•L−1 HCl and extracted with ethyl ace-
tate. Combined ethyl acetate extracts were dried over
Na2SO4 overnight and then the solvent was removed to
dryness under reduced pressure. The residue was puri-
fied by silica gel column chromatography eluting suc-
cessively with dichloro- methane and ethyl acetate. The
evaporation of the solvent gave 7.6 g (38.0% yield)
Experimental
Materials
p-Nitrophenol and diphenyl diselenide were pur-
chased from Fluka and were used without further puri-
fication. 1,10-Dibromodecane and α-cyclodextrin were
purchased from Aldrich and were used directly. Pyri-
dine was obtained from Shanghai Chem. Reagent Co.
and was redistilled under low-pressure before use. The
characterization of the compounds was performed with
a Bruker Advance 500 (500 MHz) 1H NMR spectrome-
ter. UV-Vis spectra were obtained using a Shima-
dzu2450 UV-Vis-NIR spectrophotometer. The size of
the vesicle was determined in the Malven Zetasizer
NanoSeries instrument. Data was acquired and analyzed
by using ultraviolet spectroscopy software. The
1
tawny solid. H NMR (300 MHz, DMSO) δ: 7.71 (d,
J＝8.7 Hz, 4H), 6.90 (d, J＝8.7 Hz, 4H).
Scheme 2 The synthetic route of PyC10AzoC10Py
Br
Br
KOH
HO
8
N
HO
Br
NO2
△
200 °C, 12 h
N
OH
N
O
N
N
△
Br
O
N+
Br-
Br-
O
N
+
N
O
N
38
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© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2014, 32, 37—43

A Photo-responsive Catalytic Vesicle with GPx Activity
Synthesis of 4,4'-di(10-bromo-decyloxy)-azobenzene
was also prepared in the same way.
BrC10AzoC10Br was prepared based on the previous
method (Scheme 2).[24] 4,4'-Dihydroxyazobenzene (0.86
g, 4.0 mmol), 1,10-dibromodecane (7.20 g, 24.0 mmol),
and K2CO3 (4.14 g, 30.0 mmol) were added to anhy-
drous acetone under magnetic stirring. The solution was
then stirred refluxing for 24 h under N2 protection. Fi-
nally the solvent was removed and the residue was ex-
Characterization of the vesicular catalyst
UV spectra were obtained on a Shimadzu 2450
UV-Vis spectrophotometer equipped with a temperature
controller.
The morphology of the vesicular catalyst was char-
acterized via atomic force microscope (AFM). The ve-
－
1
sicular catalyst solution (0.10 mmol•L ) and the silica
wafer were preincubated in a constant temperature oven
at 37 ℃ before use, then the vesicular catalyst solution
was added onto the silica wafer dropwise, and was
carefully dried in vacuum at 37 ℃; finally the sample
characterization was carried out on an atomic force mi-
croscopy. The particle size distribution was determined
by dynamic light scattering in 1 mL of the vesicular
catalyst solution (0.2 mmol•L−1) in a plastic cuvette and
preincubated at the appointed temperature for 2 min
before the start of the detection. The size distribution
was shown in Figure 2D, the average size is about 38.0
nm at 25 ℃.
tracted
with
dichloromethane.
Combined
dichloromethane extracts were dried over Na2SO4
overnight, and then the solvent was removed to dryness
under reduced pressure. The residue was purified by
silica gel column chromatography eluting successively
with dichloromethane∶petroleum ether (1∶1, volume
ratio) and dichloromethane. The evaporation of the
1
solvent gave 1.36 g (58.0% yield) yellow powder. H
NMR (300 MHz, CDCl3) δ: 7.94 (d, J＝8.7 Hz, 4H),
6.99 (d, J＝8.7 Hz, 4H), 4.04 (t, J＝6.4 Hz, 4H), 3.41 (t,
J＝6.8 Hz, 4H), 1.60－1.90 (m, 8H), 1.30－1.60 (m,
24H). MALDI-TOF MS m/z ([M＋H]＋ ): calculated
653.21, found 653.72.
Determination of GPx activity
Synthesis of PyC10AzoC10Py
GPx mimic
PyC10AzoC10Py was prepared according to the pre-
vious method.[24] BrC10AzoC10Br (0.65 g, 1.0 mmol)
was added to 50 mL pyridine under magnetic stirring.
The solution was then stirred refluxing for 72 h under
N2 protection. After being cooled to room temperature,
the solvent was removed and the residue was dissolved
in 5.0 mL methane. Then the methane solution was
added dropwise to 500 mL anhydrous diethyl ether un-
der magnetic stirring to give lots of yellow precipitate.
The yellow precipitate was collected by centrifugation
and washed with anhydrous diethyl ether to yield crude
product. The crude product was then purified by recrys-
tallization from methyl cyanide to yield 0.43 g of de-
sired product as a yellow solid. (52.5% yield). 1H NMR
(300 MHz, DMSO) δ: 9.08 (d, J＝6.3 Hz, 4H), 8.60 (t,
J＝7.8 Hz, 2H), 8.16 (t, J＝6.7 Hz, 4H), 7.81 (d, J＝8.6
Hz, 4H), 7.09 (d, J＝8.7 Hz, 4H), 4.59 (t, J＝7.4 Hz,
4H), 4.05 (d, J＝6.1 Hz, 4H), 1.82－1.98 (m, 4H), 1.76
RSSH
2RSH + R'OOH
+ H O
(2)
+ R'OH
2
The GPx-like catalytic ability of the selenium-
containing vesicular catalyst was tested according to a
modified method reported by Hilvert et al.[26-28]. The
reaction was carried out at 37 ℃ in 1.0 mL of phos-
phate buffer (pH 7.0) containing 0.75 μmol•L−1 sele-
nium-containing vesicular catalyst and 100 μmol•L−1
NBT. The reaction was initiated by the addition of 2.50
mmol•L−1 CUOOH (100 μL). The initial rates for the
reduction of CUOOH by RSH were determined by
monitoring the disappearance of NBT at 410 nm (ε410
＝
14500 L•mol−1•cm−1, pH＝7.0) with a Shimadzu 3100
UV-vis-NIR spectrophotometer. Appropriate control of
the non-enzymatic reaction was performed and sub-
tracted from the catalyzed reaction.
Photoisomerization
－1.80 (m, 4H), 1.20－1.50 (m, 24H). ESI MS m/z ([M
The enzyme sample solutions were irradiated in a
quartz cell in the dark by UV light at 365 nm with an
ultraviolet lamp (ZF-7A 16 W, Shanghai Gucun Elec-
tron Optic Instrument Factory) and in a glass cell by
visible light with a fluorescent bulb (30 W) at ambient
temperature.
＋
－2Br]2 ): calculated 325.23, found 325.2.
Preparation of selenium-containing vesicular catalyst
As known, a surfactant molecule containing a long
hydrophobic alkyl chain and a hydrophilic head-group
can aggregate to form a spherical micelle or vesicle in
water when it exceeds the critical micelle concentration
(cmc). PyC10AzoC10Py with PhSeO2H can spontane-
ously aggregate to form a co-vesicle in water. The sele-
nium-containing vesicular catalyst was prepared[25] by
ultrasonic mixing of PyC10AzoC10Py (16.20 mg, 0.020
mmol) and PhSeO2H in aqueous solution ([PhSeO2H]＝
15.0 μmol•L−1, 2.0 mL) until a clear solution was ob-
tained. Thus this vesicular catalyst contains
PyC10AzoC10Py (10.0 mmol•L−1) and PhSeO2H (15.0
μmol•L−1). The vesicle without benzeneseleninic acid
Results and Discussion
Design and preparation of selenium-containing ve-
sicular catalyst
The design of the selenium-containing vesicular
catalyst could address some major features of active site
of natural GPx.[3] The PhSeO2H moiety is designated as
the catalytic group, just like the selenocysteine in the
GPx active site, and the hydrophobic layer of the vesicle
Chin. J. Chem. 2014, 32, 37—43
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39

Xiao et al.
FULL PAPER
consisted of the azobenzene moiety and the long hy-
drophobic alkyl chain is similar to the hydrophobic
pocket of GPx. In order to make this GPx model photo-
responsive, we took advantage of the photo-controllable
inclusion and exclusion reaction between azobenzene
and α-CD. When α-CD was added to the selenium-
containing vesicular catalyst, the azobenzene moiety of
PyC10AzoC10Py would be included into the cavity of
cyclodextrin, and as a result, the vesicle would disas-
semble and the GPx activity would decline as the hy-
drophobic layer of the vesicle which was responsible for
substrates recognition disappeared. When the above
system was irradiated by UV light, the vesicle would
form again, and the GPx activity recovered. The sele-
nium-containing vesicular catalyst was prepared by ul-
trasonic mixing of PyC10AzoC10Py with benzeneselen-
inic acid aqueous solution ([PhSeO2H]＝15.0 μmol•L−1)
until a clear solution was obtained.[26] It was character-
ized by UV spectroscopy, dynamic light scattering and
atomic force spectroscopy.
Dynamic light scattering (DLS) was employed to
measure the size and size distributions of the particles.
As shown in Figure 2D, the catalytic vesicle composed
of trans-PyC10AzoC10Py has an average diameter of 38
nm. When equimolar α-CD is added to an aqueous solu-
tion of trans-PyC10AzoC10Py, the particle size is de-
clined to 6 nm. Interestingly, the particles do not disap-
pear upon the addition of α-CD, one possible reason for
this is that the long hydrophobic alkyl chains of the in-
clusion complex of PyC10AzoC10Py with α-CD inter-
twine with each other and form some hydrophobic areas.
Upon irradiation by UV light at 365 nm, the particle size
of the enzyme sample recovers to 34 nm, which sug-
gests the re-formation of catalytic vesicle composed of
the inclusion complex of cis-PyC10AzoC10Py with α-CD.
And the AFM test result (Figure 2) is in accordance with
that of DLS study.
As shown in Figure 1, we can find that most of
PyC10AzoC10Py and the inclusion complex of
PyC10AzoC10Py with α-CD could undergo trans-cis
photoisomerization easily. Upon irradiation by UV light,
the absorption at 362 nm attributable to the π-π* transi-
tion of trans-PyC10AzoC10Py decreases dramatically,
and meanwhile an absorption peak at 443 nm attribut-
able to the n-π* transition of cis-PyC10AzoC10Py ap-
pears. Comparing to the absorption of trans-PyC10Azo-
C10Py, the absorption of trans-PyC10AzoC10Py in the
presence of α-CD is enhanced and blue shifted to 364
nm, which suggests that the trans- azobenzene moiety
of PyC10AzoC10Py is included in the cavity of α-CD.
Meanwhile, the absorption of cis-PyC10AzoC10Py in the
presence of α-CD is nearly the same as that of pure
cis-PyC10AzoC10Py, which suggests that the cis-azo-
benzene moiety of PyC10AzoC10Py hardly interacts with
α-CD.
Figure 2 AFM images of (A) 0.20 mmol•L−1 PyC10AzoC10Py;
(B) PyC10AzoC10Py/α-CD; (C) PyC10AzoC10Py/α-CD after irra-
diation of UV light (37 ℃); (D) the hydrodynamic diameters of
0.20 mmol•L−1 PyC10AzoC10Py (red), PyC10AzoC10Py/α-CD
(blue) and PyC10AzoC10Py/α-CD after irradiation of UV light
(green) in H2O (25 ℃).
Catalytic behavior
The relative activities of the mimics were summa-
rized in Table 1. For the GPx-like activity, the enzy-
matic rates were corrected for the background (non-
enzymatic) reaction between hydroperoxide and thiol.
The initial rate of the background reaction between
CUOOH and NBT was very slow, and a slight en-
hancement of the reaction rate was observed when
PhSeO2H (6.0 μmol•L−1) was added (0.580 μmol•
L−1•min−1) to the solution (Figure 3). However, under
identical conditions, the selenium-mediated vesicular
catalyst (0.75 μmol•L−1 PhSeO2H and 0.20 mmol•L−1
PyC10AzoC10Py) exhibits a remarkable rate enhance-
ment (50.19 μmol•L−1•min−1). Assuming that the rate
had a first-order dependence on the concentration of
catalyst, it is suggested that the selenium-mediated ve-
sicular catalyst was about 692 times more efficient than
that of PhSeO2H.
Figure 1 UV spectra of PyC10AzoC10Py and PyC10AzoC10Py/
α-CD in PBS (pH＝7.0): (a) trans-PyC10AzoC10Py; (b) trans-
PyC10AzoC10Py/α-CD; (c) cis-PyC10AzoC10Py; (d) trans-
PyC10AzoC10Py/α-CD.
Typical saturation kinetics was observed for the
40
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Chin. J. Chem. 2014, 32, 37—43

A Photo-responsive Catalytic Vesicle with GPx Activity
Table 1 The initial rate ν0 and activity for the reduction of
ROOH (250 μmol•L−1) by thiol (100 μmol•L−1)
L•mol−1•min−1 ([CUOOH]＝0.250 mmol•L−1) (Figure
5).
ν0/(μmol•L−1•min−1)
Activity
TNB NBT TNB
0.870
Catalyst Hydroperoxide
PhSeO2Ha CUOOH
NBT
0.580
16.06
50.19
1
1
H2O2
2.647 221.5 24.34
Vesicle
Catalystb
CUOOH
26.16 692.3 240.6
a [PhSeO2H]＝6.0 μmol•L−1; b Vesicle catalyst: [PyC10AzoC10Py]
＝0.20 mmol•L−1, [PhSeO2H]＝0.75 μmol•L−1.
Figure
5
Lineweaver-Burk plots of the artificial en-
zyme-catalyzed reduction of CUOOH by NBT.
For this selenium-containing vesicular catalyst, the
catalytic activity in CUOOH-NBT system (50.19
μmol•L−1•min−1) is much greater than that in the
H2O2-NBT system (16.06 μmol•L−1•min−1) (Table 1).
The large difference in GPx-like activity in the two as-
say systems suggests that hydrophobic interaction plays
an important role in catalytic process and the vesicle
seems to be a better scaffold for the hydrophobic sub-
strate CUOOH than the hydrophilic substrate H2O2.
Herein, we ascribe this difference in activity to distinct
substrate enrichment. The vesicle allows both NBT and
CUOOH to enter the hydrophobic layer and prompts the
reaction rate enormously. For the hydrophilic substrate
H2O2, it is apt to locate on the vesicle surface and cannot
be enriched into the hydrophobic layer. Meanwhile, we
also found that the catalytic activity in CUOOH-NBT
system (50.19 μmol•L−1•min−1) is much greater than that
in the CUOOH-TNB system (26.16 μmol•L−1•min−1),
which means the electrostatic attraction between the
vesicle and substrate TNB does not have any positive
impact on activity enhancement. Thus, it is reasonable
to believe that the hydrophobic layer of the vesicle plays
an important role in catalysis.
Figure 3 Plots of initial reduction rates ν0 (μmol•L−1•min−1) in
CUOOH-NBT system: (a) blank; (b) PhSeO2H (6.0 μmol•L−1); (c)
vesicle ([trans-PyC10AzoC10Py]＝0.20 mmol•L−1); (d) vesicle
catalyst ([PyC10AzoC10Py]＝0.20 mmol•L−1, [PhSeO2H]＝0.75
μmol•L−1).
peroxidase-like reactions, indicating that this model is a
real GPx mimic (Figure 4). For the NBT assay system,
in the presence of selenium-mediated vesicular catalyst
(0.75 μmol•L−1 PhSeO2H and 0.20 mmol•L−1
PyC10AzoC10Py) at pH 7.0 (20 mmol•L−1 PBS) and 37
℃, the apparent kinetic parameters of the sele-
nium-mediated vesicular catalyst were obtained Vmax
＝
497.51 μmol•L−1•min−1, kcat(app)＝663.35 min−1,
Km(NBT)＝0.950 mmol•L−1, and kcat/Km＝6.983×105
In order to confirm the mechanism of this vesicular
GPx model, the initial rate of the non-enzymatic
reaction between CUOOH and NBT in the presence of
PyC10AzoC10Py vesicle (without PhSeO2H) was tested
(Figure 6).
As was shown in Figure 6, the vesicle without cata-
lytic center selenium acts as a good catalyst for catalyz-
ing the reduction of CUOOH by NBT (28.01
μmol•L−1•min−1). This is mainly because the hydropho-
bic layer of the vesicle can greatly enrich NBT and
CUOOH and thus prompt the reaction rate. When α-CD
was added into the vesicle solution, the non-enzymatic
reaction became much slower (15.18 μmol•L−1•min−1).
One plausible reason for this reduction in GPx activity
is that the inclusion of the azobenzene group by α-CD
Figure 4 Plots of initial rates ν0 (μmol•L−1•min−1) at different
concentrations of NBT in the presence of the selenium-containing
vesicular catalyst.
Chin. J. Chem. 2014, 32, 37—43
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41

Xiao et al.
FULL PAPER
renders this amphiphilic PyC10AzoC10Py hydrophilic.
with each other to form a weak hydrophobic microenvi-
ronment as enzymatic binding site. When the above
system was irradiated by UV light at 365 nm, we found
that the GPx activity restored, but its activity was a little
lower than that of the initial vesicular GPx model owing
to the weakening of the binding site. On alternating ir-
radiation of the catalyst sample with UV and visible
light, this reversible photoregulation of GPx activity
process can be recycled many times as shown in Figure
8.
Figure 6 Plots of initial reduction rates ν0 (μmol•L−1•min−1) of
non-catalytic reactions (CUOOH-NBT): (a) blank; (b) vesicle
([trans-PyC10AzoC10Py]＝0.20 mmol•L−1); (c) vesicle catalyst
([trans-PyC10AzoC10Py] ＝ 0.20 mmol•L−1, [PhSeO2H] ＝ 0.75
μmol•L−1); (d) [trans-PyC10AzoC10Py/α-CD]＝0.20 mmol•L−1; (e)
[trans-PyC10AzoC10Py/α-CD] ＝ 0.20 mmol•L−1, [PhSeO2H] ＝
0.75 μmol•L−1.
Photoregulation of the GPx-like activity of sele-
nium-containing vesicular catalyst
Figure 8 The catalytic activity changes of the inclusion of
PyC10AzoC10Py/α-CD upon alternating irradiation with UV light
at 365 nm and visible light.
As we have mentioned above, the selenium-
containing vesicular GPx model exhibits the highest
catalytic activity in the CUOOH-NBT system as the
enrichment of substrates in the hydrophobic layer of the
vesicle. We also tested the influence of light stimuli on
the catalytic behavior. As we anticipated, this vesicular
GPx model lost its activity upon addition of α-CD, ow-
ing to the collapse of binding site. However, in the assay
we found that this GPx model maintained part of its
catalytic activity (16.50 μmol•L−1•min−1, Figure 6e and
Figure 7c) possibly for the reason that the long hydro-
phobic alkyl chains of PyC10AzoC10Py could intertwist
Conclusions
In summary, we have designed a photo-responsive
vesicular GPx model by means of supramolecular
self-assembly. This selenium-containing vesicular cata-
lyst exhibited a remarkable GPx-like activity in the
CUOOH-NBT assay system. Compared with other GPx
models, the selenium-containing vesicular catalyst has
obvious advantages: simple preparation process, re-
markable rate enhancement and obvious regulation of
enzyme activity. Considering the hydrophobic recogni-
tion for both CUOOH and thiol substrates, the vesicular
catalyst is a good scaffold for constructing GPx mimics.
The work on constructing more efficient photo-respon-
sive GPx models is undergoing in our lab. It is antici-
pated that this work opens a new field for the design of
smart antioxidative enzyme models as well as for the
mechanistic understanding and practical application of
natural selenoenzymes.
Acknowledgement
We acknowledge financial support from the National
Natural Science Foundation of China (Nos. 21234004,
91027023, 21221063, and 21004028) and the 111 pro-
ject (No. B06009).
Figure 7 Plots of initial reduction rates ν0 (μmol•L−1•min−1) in
CUOOH-NBT system before or after irradiation of UV and visi-
ble light: (a) blank; (b) vesicle catalyst ([trans-PyC10AzoC10Py]＝
0.20 mmol•L−1, [PhSeO2H] ＝ 0.75 μmol•L−1); (c) [trans-
PyC10AzoC10Py/α-CD] ＝ 0.20 mmol•L−1, [PhSeO2H] ＝ 0.75
μmol•L−1; (d) vesicle catalyst: ([cis-PyC10AzoC10Py/α-CD]＝0.20
mmol•L−1, [PhSeO2H]＝0.75 μmol•L−1).
References
[1] Nordberg, J.; Arner, E. S. J. Free Radic Biol. Med. 2000, 31, 1287.
[2] Flohé, L.; Loschen, G.; Günzler, W. A.; Eichele, E. Hoppe-Seyler’s
42
www.cjc.wiley-vch.de
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2014, 32, 37—43

A Photo-responsive Catalytic Vesicle with GPx Activity
Z. Physiol. Chem. 1972, 353, 987 .
[15] Yan, H.; Frielinghaus, H.; Nykanen, A.; Ruokolainen, J.; Saianid, A.;
Miller, A. F. Soft Matter 2008, 4, 1313.
[16] Jonathan, V. M. W.; Richard, T. W.; Brodyck, J. L. R.; Paul, H. F.;
Andrew, I. C.; Steven, P. R. Soft Matter 2008, 4, 985.
[17] Nerapusri, V.; Keddie, J. L.; Vincent, B.; Bushnak, I. A. Langmuir
2006, 22, 5036.
[3] Epp, O.; Ladenstein, R.; Wendel, A. Eur. J. Biochem. 1983, 133, 51.
[4] Dong, Z. Y.; Liu, J. Q.; Mao, S. Z.; Huang, X.; Yang, B.; Luo, G. M.;
Shen, J. C. J. Am. Chem. Soc. 2004, 126, 16395.
[5] Mao, S. Z.; Dong, Z. Y.; Liu, J. Q.; Li, X. Q.; Liu, X. M.; Luo, G.
M.; Shen, J. C. J. Am. Chem. Soc. 2005, 127, 11588.
[6] Yu, H. J.; Liu, J. Q.; August, B.; Li, J.; Luo, G. M.; Shen, J. C. J.
Biol. Chem. 2005, 280, 11930.
[18] Huang, X.; Yin, Y. Z.; Tang, Y.; Bai, X. L.; Zhang, Z. M.; Xu, J. Y.;
Liu, J. Q.; Shen, J. C. Soft Matter 2009, 5, 1905.
[7] Ren, X. J.; Xue, Y.; Zhang, K.; Liu, J. Q.; Luo, G. M.; Zheng, J.; Mu,
Y.; Shen, J. C. FEBS Lett. 2001, 507, 377.
[19] Huang, X.; Yin, Y. Z.; Jiang, X.; Tang, Y.; Xu, J. Y.; Liu, J. Q.;
Shen, J. C. Macromol. Biosci. 2009, 9, 1202.
[8] Dong, Z. Y.; Liang, K.; Wang, C. Y.; Huang, X.; Mao, S. Z.; Li, X.
Q.; Xu, J. Y.; Liu, J. Q.; Luo, G. M.; Shen, J. C. J. Mol. Catal. A:
Chem. 2007, 277, 193.
[9] Liu, J. Q.; Luo, G. M.; Ren, X. J.; Mu, Y.; Bai, Y.; Shen, J. C.
Biochim. Biophys. Acta 2000, 1481, 222.
[10] McNaughton, M.; Engman, L.; Birmingham, A.; Powis, G.; Cot-
greave, I. A. J. Med. Chem. 2004, 47, 233.
[11] Huang, X.; Dong, Z. Y.; Liu, J. Q.; Mao, S. Z.; Xu, J. Y.; Luo, G. M.;
Shen, J. C. Langmuir 2007, 23, 1518.
[12] Kim, J.; Nayak, S.; Lyon, L. A. J. Am. Chem. Soc. 2005, 127, 9588.
[13] Shen, Y.; Kuang, M.; Shen, Z.; Nieberle, J.; Duan, H.; Frey, H.
Angew. Chem., Int. Ed. 2008, 47, 2227.
[14] Chiu, H. C.; Lin, Y. W.; Huang, Y. F.; Chuang, C. K.; Chern, C. S.
Angew. Chem., Int. Ed. 2008, 47, 1875.
[20] Yin, Y. Z.; Wang, L.; Jin, H. Y.; Lv, C. Y.; Yu, S. J.; Huang, X.;
Luo, Q.; Xu, J. Y.; Liu, J. Q. Soft Matter 2011, 7, 2521.
[21] McCullough, J. D.; Gould, E. S. J. Am. Chem. Soc. 1949, 71, 674.
[22] Willstätter, R.; Benz, M. Ber. Dtsch. Chem. Ges. 1906, 339, 3492.
[23] Wei, W. H.; Tomohiro, T.; Kodaka, M.; Okuno, H. J. Org. Chem.
2000, 65, 8979.
[24] Wang, Y. P.; Ma, N.; Wang, Z. Q.; Zhang, X. Angew. Chem. 2007,
119, 2881.
[25] Tang, S. S.; Chang, G. G. J. Org. Chem. 1995, 60, 6183.
[26] Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1990, 112, 5647.
[27] Bell, I. M.; Hilvert, D. Biochemistry 1993, 32, 13969.
[28] Bell, I. M.; Fisher, M. L.; Wu, Z. P.; Hilvert, D. Biochemistry 1993,
32, 3754.
(Cheng, F.)
Chin. J. Chem. 2014, 32, 37—43
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