Construction of an Artificial Glutathione Peroxidase Active Site on Copolymer Vesicles

Article abstract of DOI:10.1002/mabi.201000179

To construct an efficient GPx mimic, a novel method for preparing polymer-based vesicles carrying GPx-active sites was developed. A series of block copolymers loaded with recognition and catalytic sites were synthesized based on polystyrene-block-poly[tri(ethylene glycol) methyl ether acrylate]s (PS-PMEO₃MAs). By altering the molar ratio of the functional copolymers, vesicles with GPx activity were obtained by self-assembly of these functional copolymers through blending. The optimum GPx mimic constructed by the blending process exhibited high catalytic activity and acted as a real catalyst with typical saturation kinetics behavior. The method may be of benefit for designing other enzyme mimics and may cast a light on constructing other biologically related functional nanoparticles.Self-assembly of functional copolymers through blending is a novel and simple method to construct efficient glutathione peroxidase(GPx) mimics. The optimum blended GPx mimic is obtained by optimizing the structure of the functional block copolymers and altering the ratio of the functional block copolymers. The blended GPx mimic exhibits remarkable catalytic activity and acts as a real catalyst with typical saturation kinetics behavior.

Full text of DOI:10.1002/mabi.201000179

Full Paper
Construction of an Artificial Glutathione
Peroxidase Active Site on Copolymer Vesicles
a
Yanzhen Yin, Xin Huang, Chunyan Lv, Liang Wang, Shuangjiang Yu,
Quan Luo, Jiayun Xu, Junqiu Liu*
To construct an efficient GPx mimic, a novel method for preparing polymer-based vesicles
carrying GPx-active sites was developed. A series of block copolymers loaded with recognition
and catalytic sites were synthesized based on polystyrene-block-poly[tri(ethylene glycol)
methyl ether acrylate]s (PS-PMEO MAs). By altering the molar ratio of the functional copo-
3
lymers, vesicles with GPx activity were obtained by self-
assembly of these functional copolymers through blend-
ing. The optimum GPx mimic constructed by the blend-
ing process exhibited high catalytic activity and acted as
a real catalyst with typical saturation kinetics behavior.
The method may be of benefit for designing other
enzyme mimics and may cast a light on constructing
other biologically related functional nanoparticles.
Introduction
like hydrogen peroxides, organic hydroperoxides and
phospholipid hydroperoxides, by consuming reductive
[
2,3]
Enzymes, a class of highly efficient and specific biocatalysts
in nature, play an important role in human beings because
they participate in cellular metabolic processes by enhan-
cing the rates of reactions between biomolecules. Among
the family of enzymes, selenoenzyme glutathione perox-
idase (GPx, EC 1.11.1.9) functions to protect the cell
membrane and other cellular components from oxidative
substrate such as glutathione (GSH).
The catalytic
[
1,4]
moiety of GPx has been well studied.
In general, some
important catalytic factors contribute to maintaining an
efficient GPx catalytic activity: the catalytic selenium
center, recognition site and hydrophobic cavity. Firstly, the
selenocysteine located in a depression on the protein’s
surface is employed to protect various living organisms by
catalyzing the reduction of hydroperoxides (ROOHs) using
GSH as a reducing substrate. Secondly, the binding site is
formed between two arginine residues and the carboxylic
groups of GSH in natural GPx, which functions to fix the
substrate and catalytic center in a proper orientation to
complete a catalytic cycle efficiently. Lastly, the hydro-
phobic cavity of GPx is composed of some charged and
hydrophobic amino acid residues (Phe, Trp, Asp), which also
play an important role in strengthening the ability for
substrate binding. Due to its biologically crucial role,
considerable efforts have been devoted to producing
[
1]
damage as an important anti-oxidative enzyme. It can
scavenge numerous cellular reactive oxygen species (ROS),
Y. Z. Yin, X. Huang, C. Y. Lv, L. Wang, S. J. Yu, Q. Luo, J. Y. Xu, J. Q. Liu
State Key Laboratory of Supramolecular Structure and Materials,
Jilin Univerity, Changchun 130012, People’s Republic of China
Fax: þ86 431 8519 3421; E-mail: junqiuliu@jlu.edu.cn
a
:
Supporting information for this article is available at the bottom
of the article’s abstract page, which can be accessed from the
journal’s homepage at http://www.mbs-journal.de, or from the
author.
Macromol. Biosci. 2010, 10, 1505–1516
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
DOI: 10.1002/mabi.201000179 1505

Y. Yin et al.
organoselenium/tellurium compounds that mimic the
Experimental Part
[
5–10]
properties of GPx in recent years.
Recently, in our
Materials
group, based on the understanding of the structure of GPx,
some artificial GPx models have been designed in which a
catalytic center of selenium/tellurium was introduced into
existingorartificiallygeneratedsubstratebindingscaffolds
Sodium borohydride and 3-bromo-1-propanol were purchased
from Fluka and were used without further purification. Tellurium
powder and phenyl methanol were purchased from Sinopharm
Chemical Reagent Co. Ltd and were used without further
purification. Dimethylamine (33% aqueous solution) and 4-
toluenesulfonyl chloride were purchased from Shanghai Reagent
Co. Acryloyl chloride, propargyl alcohol and iodomethane were
purchased from Anhui Wotu Reagent Co. Triethylene glycol
monomethyl ether, teraethylene glycol and pentamethyldiethy-
lenetriamine (PMDETA) were purchased from Tokyo Chemical
Industry Co. Ltd. 2-bromopropanoyl bromide was purchased from
Lancaster. Triethylamine and tetrahydrofuran (THF) were rigor-
ously dried with sodium.
[
11–20]
by chemical or genetic strategies.
Although GPx
mimics constructed by chemical and genetic strategies
previously have demonstrated high catalytic activity, some
disadvantages still remain in such efficient GPx models. On
theonehand,catalyticfactorsarecommonlycombinedinto
one scaffold, such as cyclodextrin, cyclophanes, nanopar-
ticles, synthesized polymer, gel and catalytic antibodies,
through covalent chemical methods, as well as genetic
strategies, which have the limitation of complicated
synthetic routes, expensive cost and low productivity. On
the other hand, the molar ratio of the catalytic factors
combined into one scaffold is a fixed value. Consequently,
theachievementoftheoptimumGPxmimicviaalteringthe
molar ratio of the catalytic factors is more difficult and the
catalytic activity cannot be well controlled through
changing the composition of the catalytic factors. Thus,
the construction of GPx mimics using a simple and efficient
method is still a great challenge. To meet such a big
challenge, a blending process is employed to construct a
simpleandefficientGPxmimic,whichisaversatilestrategy
exploited for the development of new polymeric materials
with property profiles superior to those of the individual
Instrumentation
The characterization of the structure of the compounds was
performed with a Bruker 300 MHz spectrometer using a tetra-
methylsilane (TMS) proton signal as the internal standard.
Elemental analyses were completed on a Perkin-Elmer 240 DS
elemental analyzer. UV-vis spectra were obtained using
a
Shimadzu 2450 UV-vis/near infrared (NIR) spectrophotometer.
SEM observations were carried out on a JEOL JSM-6700F scanning
electron microscope with primary electron energy of 3 kV. TEM
observationswerecarriedoutonaJEOLJEM3010scanningelectron
microscope. The buffer pH values were determined with a Mettler
Toledo 320 pH meter. Static light scattering experiments were
performed using a Malvern Zetasizer Nanoseries instrument in
water solution. Molecular weights and molecular weight distribu-
tions were determined by gel permeation chromatography (GPC)
[
21–30]
components.
Furthermore, the novel GPx mimic
designed via this versatile strategy not only combines
three catalytic factors into one scaffold simply, but also
exhibits a special characteristic: the GPx mimic with high
catalytic activity can be easily obtained by altering the
molar ratio of the functional copolymers. It may also
overcome the disadvantages that exist in the traditional
construction method for GPx mimics. To the best of our
knowledge, this is the fist example in which the GPx mimic
is constructed by a simple and efficient blending process.
Herein, tellurium, quaternary ammonium salt and
cyclodextrin, as alternative catalytic factors were incorpo-
using THF or dimethylformamide (DMF) as the eluent at a flow rate
of 1.0 mL ꢀ minꢁ
1
.
[17]
Determination of GPx Activity
The catalytic activity was assayed according to a modified method
[31]
reportedbyHilvertetal. Thereactionwascarriedoutat25 8Cina
1
1
mL quartz cuvette. 700 mL of phosphate buffer (pH ¼ 7.0, 50 ꢂ
ꢁ3
M
ꢁ6
0
M) and 100 mL of the catalyst (1 ꢂ 10 M) were added, and
then 100 mL of the 3-carboxyl-4-nitrobenzenethiol (TNB) solution
ꢁ3
(
1.5 ꢂ 10 M) was added. The mixture in the quartz cuvette was
3
rated into the functional PS-PMEO MAs and a series of
pre-incubated at 25 8C for 3 min. Finally, the reaction was initiated
functional block copolymers (PP, PP-Te1, PP-Te2, PP-CD1, PP-
CD2, PP-q1, PP-q2) were obtained. Through optimizing the
structure ofthe functional blockcopolymers, PP-Te1, PP-CD2
andPP-q2wereselectedasfunctionalblockcopolymerswith
which to construct the optimum blended mimics. The self-
assembly behavior of the blended GPx mimic in aqueous
solutionwasinvestigatedbystaticlightscattering, scanning
and transmission electron microscopy (SEM and TEM,
respectively), and hollow vesicles were obtained. The
optimum blended GPx mimic with the structure of hollow
vesicles was achieved by altering the molar ratio of PP-Te1,
PP-CD2 and PP-q2, which exhibited significant catalytic
activity and strong substrates binding ability.
by the addition of 100 mL of cumene hydroperoxide (CUOOH)
ꢁ3
(2.5 ꢂ 10 M) and the absorption decrease of TNB at 410 nm
ꢁ
1
ꢁ1
(e410 ¼ 13 600 L ꢀ mol ꢀ cm , pH ¼ 7.0) was monitored using a
Shimadzu 2450 UV-vis/NIR spectrophotometer. Appropriate con-
trol of the non-enzymatic reaction was performed and was
subtracted from the catalyzed reaction.
Determination of the Content of the Catalytic Center
Tellurium in PP-Te1 (or PP-Te2)
2 2
Hydrogen peroxide (H O ) was added to the solution of PP-Te1(or
PP-Te2), and after stirring for 10 h at room temperature, the
2 2
solution was dialyzed against water for 1 d to remove H O . The
solid PP-Te1 polymer in which Te was in the form of Te (IV) was
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DOI: 10.1002/mabi.201000179

Construction of an Artificial Glutathione Peroxidase Active Site . . .
[
8]
1
obtained via freeze-drying. According to a report by Detty et al.,
HNMR(D
5.22 (s, 2 H, COOCH
6), 3.50–3.75 (br, 14 H, H-2, 4).
2
O):d ¼ 8.11(s, 1H, ꢁCH¼), 5.67–6.53(3H, CH
2
¼CHꢁ),
one molecule oxidized state Te(IV) can be reduced by two molecule
TNB, so the content of the catalytic center tellurium was obtained
by monitoring the decrease in the UV absorption of TNB at 410 nm.
2
ꢁ), 5.07 (d, 7 H, H-1), 3.86–4.06 (br, 28 H, H-3, 5,
Synthesis of Tetraethylene Glycol Monopropargyl
[
34]
Synthesis of Triethylene Glycol Monomethyl Ether
Ether (6b)
Acrylate (MEO MA)
3
Tetraethylene glycol monopropargyl ether was synthesized
according to a known procedure reported by S. Ramakrishnan
and co-workers with a slight alteration, using 4-toluene sulfonyl
chloride instead of mesyl chloride. There was a yield of 58% of the
Acryloyl chloride (3.24 mL, 4.0 ꢂ 10ꢁ mol) was dissolved in 30 mL
of anhydrous tetrahydrofuran and added dropwise to a stirred
solution of triethylene glycol monomethyl ether (6.57 g,
2
ꢁ
2
ꢁ2
4
.0 ꢂ 10 mol) and triethylamine (5.55 mL, 4.0 ꢂ 10 mol) in
target product after the crude product was purified.
1
anhydrous THF (90 mL) at 0 8C. After complete addition, the
mixture was stirred for 3 h at room temperature. After the reaction
was complete, the precipitated triethylamine hydrochloride was
filtered and the filtrate was concentrated. The crude product was
purified by column chromatography (silica gel, petroleum ether/
H NMR (CDCl
3
): d ¼ 4.21 (d, 2 H, ꢁCH
2
C CH), 3.60–3.74 (m, 16 H,
glycol), 2.43 (t, 1 H, ꢁC CH).
The syntheses of acryloyloxypropyl-3-hydroxypropyltelluride
(1), tetraethylene glycol monodimethylamine (4b) and triazol-CD
monomer 2 (6) are described in the Supporting Information.
ethyl acetate, 8:1) and there was a yield of 81.5%.
1
H NMR (CDCl
3
): d ¼ 5.82–6.46 (3 H, CH
2
¼CHꢁ), 4.32 (t, 2 H,
).
Synthesis of PS-Br
COOCH
2
ꢁ), 3.55–3.76 (m, 10 H, glycol), 3.38 (s, 3 H, ꢁCH
3
The syntheses of benzyl 2-bromopropanoate (2), 3-(dimethyla-
mino)propyl acrylate (3), tetraethylene glycol mono(4-methylben-
zenesulfonate) (4a), tetraethylene glycol monodimethylamine
acrylate (4), 2-propynyl acrylate (5a), propargyl-4-methylbenzene-
sulfonate (6a) and tetraethylene glycol monopropargyl ether
The synthesis of PS-Br and functional block copolymers followed
[35–37]
polymerization procedures reported previously.
The typical
polymerization procedure was as follows. A Schlenk flask equipped
with a magnetic stirrer was flamed and dried under a vacuum, and
ꢁ4
then charged with CuBr (0.0143 g, 1.0 ꢂ 10 mol). The flask was
then sealed under a vacuum and backfilled with nitrogen
three times. Deoxygenated acetone (2 mL), styrene (2.3 mL,
3
acrylate (6c) were similar to the synthesis of MEO MA. The detailed
procedure is given in the Supporting Information.
ꢁ2
ꢁ4
2
.0 ꢂ 10 mol) and PMDETA (0.0173 g, 1.0 ꢂ 10 mol) were added
sequentially and stirred until the Cu complex was formed. The
Synthesis of 3-(Dimethylamino)propan-1-ol (3a)
ꢁ4
initiator benzyl 2-bromopropanoate (0.0243 g, 1.0 ꢂ 10 mol) was
ꢁ
2
3
-Bromopropan-1-ol (18 mL, 2.0 ꢂ 10 mol) and dimethylamine
added to the Schlenk tube via a syringe. The tube was kept in a
thermostaticallycontrolled oilbath at 100 8C. After 8 h, thereaction
flask was quenchedwith liquid nitrogen, exposed to air and diluted
with 10 mL of THF. After passing though a column of activated
alumina to remove the copper catalysts, the solution was
precipitated in 10-fold excess methanol. The resulting polymer
was filtered and dried under a vacuum at 60 8C overnight. GPC
analysis of PS-Br revealed an Mn of 10 480 and a polydispersity,
Mw=Mn, of 1.20. The degree of polymerization (DPs) of PS was
estimated to be 98. The macroinitiator was denoted as PS-Br.
Thesynthesesofthefunctionalblockcopolymers,PP, PP-Te1, PP-
Te2, PP-CD1, PP-CD2, PP-q1 and PP-q2 were similar to the
preparation of PS-Br. The syntheses and characterization of these
functional block copolymers are given in the Supporting Informa-
tion.
ꢁ2
(
aq) (82 mL, 6.0 ꢂ 10 mol) were dissolved in tetrahydrofuran
300 mL). The mixture was stirred for 24 h at 50 8C under a nitrogen
(
atmosphere. After the mixture was cooled, the solvent and
dimethylamine (aq) was removed by a vacuum. The crude product
was purified by column chromatography (silica gel, ethanol), and
obtained in a yield of 73.5%.
1
H NMR (CDCl
3
): d ¼ 3.83 (t, 3 H, ꢁCH
], 1.82 (m, 2 H, ꢁCH
2
OH), 2.80 (t, 2 H, ꢁCH
ꢁ).
2
N),
2
.49 [s, 6H, –N(CH
)
3 2
2
[
33]
Synthesis of Triazol-CD Monomer 1 (5)
Thecompoundtriazol-CDmonomer1wassynthesizedaccordingto
a known procedure reported by Ritter et al. with a modification.
ꢁ3
DMF(10 mL),2-propynylacrylate(0.192 g,2.0 ꢂ 10 mol),PMDETA
ꢁ
3
(
0.347 g, 2.0 ꢂ 10 mol), mono-(6-azido-6-desoxy)-b-cyclodextrin
ꢁ3
(
1.16 g, 1.0 ꢂ 10 mol) were added to a 50 mL Schlenk tube. After
ꢁ3
Results and Discussion
three brief freeze/thaw cycles, CuBr (0.287 g, 1.0 ꢂ 10 mol) was
introduced under the protection of a nitrogen flow. The reaction
tube was carefully degassed using five freeze/pump/thaw cycles
and sealed under a vacuum, and then placed in a thermostatically
controlled oil bath at 40 8C. After stirring for 40 h, the flask was
exposed to air and cooled to room temperature. The mixture was
diluted with DMF and passed through a basic alumina column to
remove the copper catalyst. After the solvents was concentrated
and precipitated with acetone, the product was separated by
simple filtration. The product was dried under a vacuum for 24 h at
Preparation and Characterization of the Blended GPx
Mimic
The crystal structure of bovine erythrocyte glutathione
[
4]
peroxidase was reported by Epp et al. in 1983. The
selenocysteine residue, as the catalytic active center of GPx,
is located at the N-terminal ends of a-helices and some
aromatic amino acid side chains are present in the vicinity
of the reactive group. Functional residues such as Arg-40,
5
0 8C, to give a brown solid (1.1 g, yield of 87.6%).
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Y. Yin et al.
polystyrene macroinitiator was synthesized using benzyl
2
-bromopropanoate as an initiator. On the other hand,
various functional block copolymers (PP, PP-Te1, PP-Te2,
PP-CD1, PP-CD2, PP-q1 and PP-q2) were obtained according
totheprotocolsshown inScheme3. GPCindicatedthatboth
the polystyrene macroinitiator and the functional block
copolymer were well controlled (see Supporting Informa-
tion). From elemental analyses, the content of N in the
functional block copolymers (PP-CD1, PP -CD2, PP-q1 and
PP-q2)weremeasured, andfurthermore, thecontentsofthe
catalytic factors (cyclodextrin, quaternary ammonium salt)
were calculated. Also, the content of the catalytic center
tellurium was determined according to the measure
[
17]
previously reported by our group
Information).
(see Supporting
Considering that a blending process is able to yield
materials with superior properties compared to those of the
individual components, chemists have paid much more
attentions to the development of new polymeric materials
usingablendingprocessasaconvenientroute. Thismethod
is commonly simpler, cheaper and less time consuming for
thepreparationofpolymericmaterialswithnewproperties
than traditional protocols. An additional advantage of
polymerblendsisthattheproperties ofthematerialscanbe
well controlled via changing the blend composition.
Figure 1. Structures of the functional monomers 1–6 and sub-
strates (NBT, TNB).
Arg-167 and Gln-130 play an important contribution in
binding substrates, and could form salt bridges and a
hydrogen bond with the substrate GSH molecule. It has
been clearly shown that three important catalytic factors
contribute to maintaining a high GPx catalytic activity: the
catalytic center, binding site and hydrophobic cavity.
Herein, based on our previous studies, tellurium, quatern-
ary ammonium salt and cyclodextrin were selected as
efficient and novel alternative catalytic factors to selenium,
arginine residues as binding sites, and a hydrophobic
Consequently, a blending process was employed to
construct the GPx mimic, since it provides the advantage
that the properties of the materials can be controlled by
changing the blend composition. Accordingly, three func-
tional copolymers, PP-Te1, PP-CD2 and PP-q2, modified
with tellurium, quaternary ammonium salt and cyclodex-
trin, respectively, were selected as individual copolymers.
Theblended GPx mimicbased onthese functional polymers
was prepared with the aim of obtaining higher catalytic
activity. As is well known, the stability and uniform
dimensions are significantly important for the nanoen-
zyme scaffold of GPx mimics. Therefore, static light
scattering and SEM were employed to confirm whether
the polymer blend could assemble into similar nanopar-
ticles with the individual copolymers. As shown in
Figure 2(A), the hydrodynamic diameters of the individual
copolymers (PP-Te1, PP-CD2 and PP-q2) in aqueous solution
were typically about 90 nm (curve a, b and c). It is noticeable
thatthefunctionalindividualcopolymerscouldformstable
nanoparticles with similar hydrodynamic diameters,
although each copolymer was modified with different
catalytic factors. Significantly, the blended GPx mimic
constructed bytheblending process, denoted asPP-q2-CD2-
Te1max, could equally assemble into nanoparticles with a
hydrodynamic diameter about 90 nm, as shown in
Figure 2(B) (curve d). This means that the self-assembly
behavior of the blended GPx mimic was similar to that of
the individual functional copolymers. To further confirm
this conclusion, the actual morphology of the nanoparticles
[
11–18]
cavity, respectively.
GPx mimics constructed using
these alternative catalytic factors demonstrated high
catalytic activity and substrate specificity. Consequently,
based on the structure of GPx and our previous research, the
corresponding functional monomers 1–6 were designed
(
Figure 1). Monomer 1, with a similar function to
selenocysteine, was prepared as a catalytic center. Mono-
mer 2 was employed to enhance the hydrophilicity and
biocompatibility of the GPx mimic. Monomer 3 and 5 were
responsible for the complexation of carboxyl groups of
substrates, and monomer 4 and 6 were contributed to
provide a hydrophobic microenvironment similar to the
hydrophobic cavity in the native GPx. The synthetic routes
to functional monomers 3–6 are depicted in Scheme 1.
Subsequently, functional block copolymers modified
with three catalytic factors (catalytic center, binding site
and hydrophobic cavity) were synthesized via ATRP, which
is one of the significant controlled/living radical polymer-
izations employed to design versatile functional materials
and polymers with complex architectures and composi-
[
17,32,38,40–44]
tions.
On one hand, as Scheme 2 displays, the
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DOI: 10.1002/mabi.201000179

Construction of an Artificial Glutathione Peroxidase Active Site . . .
Scheme 1. Synthesis of functional monomers 3–6. (1), (4) THF/50 8C/24 h; (2), (3), (5), (6), (8), (10) THF/triethylamine/0 8C; (7), (11) CuBr/
PMDETA/DMF/40 8C/40 h; (9) THF/Na/0 8C.
the individual copolymers and the blended GPx mimic
could assemble into spherical nanoparticles with similar
diameters, which is in agreement with the phenomenon
observed from the static light scattering experiments. To
Scheme 2. Synthesis of polystyrene macroinitiator PS-Br.
further investigate the structure of the spherical nanopar-
ticles formed by the blended GPx mimic, their detailed
morphology was observed by TEM (see Figure 4). From the
nanoparticles observed in the TEM image, we could see a
thinnerregionsurroundedbyathickerregionandwereable
to clearly confirm the boundary between the thinner and
the thicker ones. This observation indicated that the
spherical nanoparticles formed by the GPx mimic were
vesicle-like particles with a hollow structure. Similar
was observed by SEM (Figure 3). SEM clearly revealed the
presence of spherical nanoparticles with average diameters
of 80 nm for the functional individual copolymers [PP-Te1
(
image a), PP-CD2 (image b) and PP-q2 (image c)]. As
anticipated, the diameter of the blended GPx mimic [PP-q2-
CD2-Te1max (image d)] was also about 80 nm on average. As
determined by SEM, the diameters of the spherical
nanoparticles in the dry state were reasonably smaller
than those detected by static light scattering, since the
hydrophobic diameters of nanoparticles in solution
reported from the Malven Zetasizer Nanoseries instrument,
which contains the contribution from the swollen corona.
Similar behavior has been observed in previous
[
39]
behavior has been reported by Chen and coworkers.
Consequently, based on the above measurements, we can
draw the conclusion that the blended GPx mimic can
assemble into uniform and stable vesicles that are
analogous to the individual copolymers, which makes
the blending process a feasible method to construct GPx
mimics. A graphical representation of the GPx mimic
constructed by the blending process is displayed in
Scheme 4.
[
17,45]
reports.
It was found from SEM images that both
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Y. Yin et al.
ꢁ
1
ꢁ1
(
e
410 ¼ 13 600 L ꢀ mol ꢀ cm , pH ¼ 7.0)
was recorded for a few minutes to
calculate the reaction rate. The relative
activities are summarized in Table 1.
Considering that the catalytic activity is
crucially important for a successful GPx
mimic, we chose the data on the catalytic
activities as an effective value to reflect
the optimization of the GPx mimic.
First, the influence of the structure of
the polymer on the catalytic activity was
evaluated. Consequently, twoblockcopo-
lymers modified with the catalytic center
tellurium (denoted as PP-Te1 and PP-Te2)
were synthesized, which have different
ratios of polystyrene to poly[tri(ethylene
glycol) methyl ether acrylate] (the ratios
in PP-Te1 and PP-Te2 were 98:16 and
9
8:30, respectively). The GPx catalytic
activities of PP-Te1 and PP-Te2
are summarized in Table 1. The
catalytic reaction rate of PP-Te1 was
ꢁ
6
ꢁ1
ꢁ1
1
.32 ꢂ 10 M ꢀ min
,
and it was
ꢁ6
1
.27 ꢂ 10 M ꢀ min
for PP-Te2. We
noted that PP-Te2 exhibited a similar
catalytic reaction rate to PP-Te1,
although there is much more difference
in their structure. We speculate that the
reason for the interesting phenomena is
that the microenvironments of the cat-
alytic center tellurium in PP-Te1 and PP-
Te2 are similar. As an amphiphilic block
3
copolymer, PS-PMEO MAcanself-assem-
ble into vesicles with the hydrophobic PS
block as the core and the hydrophilic
3
PMEO MA block as the shell in aqueous
solution. Accordingly, the catalytic tell-
urium centers are in a hydrophilic
microenvironment when they are incor-
Scheme 3. Synthesis of functional polystyrene-block-poly(triethylene glycol methyl
ether acrylate)s (PS-PMEO3MAs) modified with catalytic factors.
3
porated into the PMEO MA block in both
PP-Te1 and PP-Te2. As is known, the hydrophobic micro-
environment takes a positive role for enhancing the
catalytic activities of the GPx mimics. Considering that
the catalytic center tellurium is located in the hydrophilic
microenvironment in PP-Te1 and PP-Te2, the hydrophobic
environment endowed from the PS block may play less of a
contribution to enhancing the catalytic activities. Conse-
quently, the catalytic reaction rate of PP-Te2 is similar to
that of PP-Te1, although the structure of PP-Te2 is different
to that of PP-Te1 to some degree. Considering that catalytic
activities of PP-Te1 and PP-Te2 were slight affected by their
structure, PP-Te1 was selected as the functional copolymer
modified with catalytic center tellurium to construct the
blended GPx mimic.
Optimizing the Structure of the GPx Mimic by the
Blending Process
The catalytic activity of the GPx mimic for the reduction of
cumene hydroperoxide (CUOOH) by TNB was evaluated
according to the modified method reported by Hilvert
[
31]
et al.
using TNB as a GSH alternative (see Figure 5). The
assayed mixture contained 0.05 M phosphate buffer,
pH ¼ 7.0, 0.15 M TNB,0.25 M CUOOHandamoderateamount
of GPx mimic. The activity was given assuming one
molecule catalytic center (Te-monomer) in the
enzyme model as one active site of the enzyme. The
reaction was initiated by the subsequent addition of
hydroperoxides, and the absorbance at 410 nm
Macromol. Biosci. 2010, 10, 1505–1516
1510 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.201000179

Construction of an Artificial Glutathione Peroxidase Active Site . . .
through an efficient combination of PP-
CD2 with PP-Te1 by a blending process.
By plotting the catalytic reaction rate
against the molar ratio of PP-CD2 to PP-
Te1, as shown in Figure 6(B), we found
that the catalytic reaction rate increased
tosomeextentwiththe molarratiogoing
up, and it reached the highest value (PP-
ꢁ
6
ꢁ1
CD2-Te1max ¼ 5.12 ꢂ 10 M ꢀ min ) when
the molar ratio is 7:1, and it goes down
when the molar ratio increases further.
This is not surprising, as we know, for a
native enzyme, that the slight change in
the structure would result in a dramatic
change in catalytic activity. For this GPx
mimic, the better match between the
catalytic center tellurium and the hydro-
phobic microenvironment of cyclodex-
trin would result in the higher catalytic
activity. Thus, the catalytic reaction rate
increases when the binding ability
endowed from the molar ratio of PP-
CD2 to PP-Te1 going up is enhanced.
However, stronger binding does not
imply higher catalytic activity, so the
catalytic reaction rate goes down when
the molar ratio increases further despite
the binding ability being enhanced. A
possible reason for this is that the
hydrophobic microenvironment endo-
wed from the cyclodextrins in PP-CD2-
Te1increasestoahigherdegreewhenthe
molar ratio is higher, which could bind
much more substrate in a distribution
away from the catalytic center and make
the completion of the catalytic cycle not
efficient. Similar results were also
Figure 2. Hydrodynamic diameters of the functional block copolymers: PP-Te1 (a), PP-
CD2 (b), PP-q2 (c) and the blended GPx mimic: PP-q2-CD2-Te1max. (d) Determined by
static light scattering.
[
13,14]
reported in our previous work.
Furthermore, for examining the influ-
ence of the structure of the cyclodextrin
monomer on the catalytic activity, the
catalytic reaction rate of the blended GPx
mimic (PP-CD1-Te1) was also investi-
gated [Figure 6(A)].The catalytic behavior
of PP-CD1-Te1 is similar to that of PP-
CD2-Te1 but the catalytic reaction rate of
Figure 3. SEM images for PP-Te1 (a), PP-CD2 (b), PP-q2 (c) and the blended GPx mimic: PP-
q2-CD2-Te1max (d).
ꢁ6
ꢁ1
Subsequently, the optimization of the functional copo-
lymer modified with quaternary ammonium salt or
cyclodextrin was accomplished. On the one hand, for
examining the influence of the structure of the cyclodextrin
monomer on the catalytic activity, two functional mono-
mers 5 and 6 were synthesized and employed to synthesize
two functional copolymers (PP-CD1 and PP-CD2). The
blended GPx mimic, denoted PP-CD2-Te1, was constructed
the PP-CD1-Te1max (v
0
¼ 3.92 ꢂ 10 M ꢀ min ) is much
lower than that of PP-CD2-Te1max. We deduced that the
differenceincatalyticreaction rateispossiblya resultofthe
difference in the functional monomers 5 and 6. Considering
thatmonomer5incorporatedintoPP-CD1-Te1hasashorter
carbon chain than that of monomer 6 in PP-CD2-Te1, the
cyclodextrin of monomer 5 may be buried into the
PMEO MA block when the blended GPx mimic self-
3
Macromol. Biosci. 2010, 10, 1505–1516
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mbs-journal.de 1511

Y. Yin et al.
Figure 5. Determination of GPx activity using TNB as a substrate.
0
Table 1. The initial rates (v ) and activities for the reduction of
ꢁ4
hydroperoxides (2.50 ꢂ 10 M) by TNB assay system
ꢁ4
(
1.50 ꢂ 10 M) in the presence of the GPx mimics at pH ¼ 7.0
0.05 M PBS) and 25 8C.
(
a)
Catalyst
Thiol
v
0
ꢁ6
mol ꢀ Lꢁ1 ꢀ minꢁ1
1
0
PP-Te1
TNB
TNB
TNB
TNB
TNB
TNB
TNB
1.32
1.27
3.92
5.12
2.55
3.66
9.04
PP-Te2
Figure 4. The TEM image of the blended GPx mimic: PP-q2-CD2-
Te1max
.
PP-CD1-Te1max
PP-CD2-Te1max
PP-q1-Te1max
PP-q2-Te1max
PP-q2-CD2-Te1max
a)
The initial rate of reaction was corrected for the spontaneous
ꢁ6
oxidation, the concentration of catalyst is 10 M and assuming
one molecule catalytic center (tellurium moiety) is one active site
of enzyme.
Te1, PP-CD2 and PP-q2. The plots of the catalytic reaction
rates against different molar ratio of three copolymers are
shown in Figure 7. To obtain the optimum blended GPx
mimic, denoted PP-q2-CD2-Te1, we kept the molar ratio of
PP-CD2toPP-Te1constant(7:1)andchangedthemolarratio
of PP-q2 to PP-Te1, and curve c was obtained. It was
noticeable that the catalytic reaction rate increased with
the molar ratio going up and reached the highest value (PP-
Scheme 4. A graphical representation of the GPx mimic con-
structed by blending.
assembles into vesicles. Consequently, the match between
the catalytic center tellurium and hydrophobic microenvir-
onment of cyclodextrin in PP-CD1-Te1 may be not as good
asthatinPP-CD2-Te1,whichmayfunctiontomakePP-CD1-
Te1 exhibit a lower catalytic reaction rate than PP-CD2-Te1.
Therefore, we chose PP-CD2 as the functional copolymer
modified with cyclodextrin to construct the blended GPx
mimic. On the other hand, the functional copolymers
modified with quaternary ammonium salt (PP-q1 and PP-
q2) were optimized using the same protocol, as mentioned
above. Through comparing the catalytic reaction rates of
ꢁ
6
ꢁ1
q2-CD2-Te1max ¼ 9.04 ꢂ 10 M ꢀ min ) when the molar
ratio was 9:7:1 and went down when the molar ratio
increased further. Typically, such striking a catalytic
reaction rate of PP-q2-CD2-Te1max is ascribed to the
presence of three catalytic factors and the good match
among them. Our previous research proves that a good
match of the catalytic center and the binding site is of
[
12–17]
importance when developing the GPx mimics.
In this
work, although the three catalytic factors in the vesicles
constructed by the blending process are distributed in a
random manner, a good match of the catalytic factors still
could be achieved by changing the molar ratio of the three
functional copolymers, PP-Te1, PP-CD2 and PP-q2. The
catalytic behavior of PP-q2-CD2 -Te1 depicted in Figure 7(c)
has directly confirmed this conclusion. In short, based on
the experiments mentioned above, we could draw the
ꢁ
6
ꢁ1
the PP-q1-Te1max (v
0
¼ 2.55 ꢂ 10 M ꢀ min ) and PP-q2-
ꢁ6 ꢁ1
Te1max (v
0
¼ 3.92 ꢂ 10 M ꢀ min ), weselectedPP-q2asthe
functional copolymer to provide the quaternary ammo-
nium salt to bind substrates.
Finally, the optimum blended GPx mimic was con-
structedbytheblendingofthreefunctionalcopolymers, PP-
Macromol. Biosci. 2010, 10, 1505–1516
1512 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.201000179

Construction of an Artificial Glutathione Peroxidase Active Site . . .
GPx mimic with high catalytic activity
could be easily obtained by changing the
molar ratio of the functional copolymers.
Catalytic Behavior of the Optimum
GPx Mimic
The catalytic activity of the optimum GPx
mimic, PP-q2-CD2-Te1max, was studied
using TNB as a GSH alternative. The
reaction between CUOOH and TNB is very
slow under the spontaneous oxidation
conditions. However, under identical con-
ditions except that the PP-q2-CD2-Te1max
was added, a remarkable rate enhance-
ꢁ
6
ꢁ1
ment (v
0
¼ 9.04 ꢂ 10 M ꢀ min
)
was
observed. As Figure 8 displays, the satura-
tion kinetics of PP-q2-CD2-Te1max for the
peroxidase reaction was studied at the
individual concentrations of TNB and
CUOOH, which indicates that PP-q2-
CD2-Te1max exhibits typical saturation
kinetics and acts as a real catalyst for
the peroxidase reaction. In the TNB assay
system, in the presence of PP-q2-CD2-
Figure 6. Plots of catalytic reaction rate v
0
against molar ratio of functional copolymer
[
PP-CD1 (A), PP-CD2 (B), PP-q1 (C), PP-q2 (D)] to PP-Te1.
ꢁ6
Te1max (1 ꢂ 10 M catalytic center tellurium) at pH ¼ 7.0
and 25 8C, the apparent kinetic parameters of PP-q2-CD2-
ꢁ
6
ꢁ1 app
Te1max were obtained, Vmax ¼ 31.95 ꢂ 10 M ꢀ min , kcat
¼
ꢁ
ꢁ1
1
ꢁ6
app
cat
3
1.95 min , K
¼ 419 ꢂ 10 M, k /K
¼ 7.63 ꢂ
mCUOOH
ꢁ1
m CUOOH
4
1
0 L ꢀ mol ꢀ min , and the turnover number per catalytic
ꢁ
1
center tellurium was calculated to be 32 min
.
For an efficient enzyme mimic, the recognition of
substrates is one of the most important characteris-
[
46,47]
tics.
In nature for GPx, the hydrophobic cavity, which
is composed of some hydrophobic amino residues and the
0
Figure 7. Plot of catalytic reaction rate v against molar ratio of
functional block copolymer. (a) PP-q2-Te1: ratio of PP-q2 to PP-
Te1;(b) PP-CD2-Te1: ratio of PP-CD2 to PP-Te1;(c) PP-q2-CD2-
Te1:ratio of PP-q2 to PP-Te1 (the ratio of PP-CD2 to PP-Te1
was fixed at 7:1).
conclusion that the construction of a simple and efficient
GPx mimic (PP-q2-CD2-Te1max) has been achieved through
altering the molar ratio of three functional copolymers by
the blending process. Compared with the complicated
preparation process, expensive cost and low productivity of
the traditional preparation methods, an efficient GPx
mimic can be obtained using a blend of three functional
copolymers in a simple and efficient method. The optimum
Figure 8. Plots of initial rates at different concentrations of
CUOOH in the presence of PP-q2-CD2-Te1max. The initial concen-
ꢁ3
tration of TNB was fixed at 0.15 ꢂ 10 M, The concentration of
ꢁ3
CUOOH was 0.05, 0.10, 0.25, 0.5, 1, 2.5 and 5 ꢂ 10 M, respectively.
Macromol. Biosci. 2010, 10, 1505–1516
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mbs-journal.de 1513

Y. Yin et al.
binding site formed between two arginines and the
carboxylic group of GSH, plays an important role in binding
substrates and maintaining a high catalytic activity. For
this blended GPx mimic, the high catalytic activity is
ascribed to the similarity between the native GPx and the
designed GPx mimic. Firstly, as an alternative to the
selenocysteine of native GPx, a tellurium-containing
monomer 1 was introduced into the blended GPx mimic.
Secondly, to mimic the binding site formed between the
two arginine residues and carboxylic groups of GSH in
natural GPx, a functional copolymer (PP-q2) modified with
quaternary ammonium salt was employed. Finally, to
imitate the hydrophobic cavity composed of some charged
and hydrophobic amino acid residues (Phe, Trp, Asp) in
natural GPx, cyclodextrin witha hydrophobic cavityto bind
the aromatic substrate TNB was incorporated into a
functional copolymer (PP-CD2). Consequently, both the
catalytic center tellurium and the recognition ability
derived from the binding site and hydrophobic cavity play
an important role in maintaining such high catalytic
activity. To confirm the interaction between the substrate
TNB and the blended GPx mimic, UV spectroscopy was
employed. As is known, TNB has a maximum absorbance
wavelength at 410 nm in aqueous solution (pH ¼ 7.0), and
its maximum absorbance will appear as a red shift when it
is in a hydrophobic microenvironment. In Figure 9(A), the
UV spectrum of the assayed mixture of various amounts of
PP-q2-CD2-Te1max is given, and it presented an apparent
red shift of the maximum absorbance wavelength, which
indicated that a strong hydrophobic interaction existed
between the aryl moietyof TNB and the hydrophobic cavity
of PP-q2-CD2-Te1max. To further confirm the substrate
binding of PP-q2-CD2-Te1max, the catalytic reaction rate
was measured in different assay systems (shown in
Figure 10). The catalytic reaction rates were as follows:
Figure 10. The initial rates (v
0
) and activities for the reduction of
ꢁ4
hydroperoxides (2.50 ꢂ 10 M) by thiol TNB and NBT
ꢁ
4
(
1.50 ꢂ 10 M) in the presence of the PP-q2-CD2-Te1max at
pH ¼ 7.0 (0.05 M PBS) and 25 8C. (A) CUOOH, TNB; (B) H
TNB; (C) CUOOH, NBT; and (D) H , NBT.
O
2 2
,
2 2
O
ꢁ
6
ꢁ1
assay system [Figure 10(D)], v ¼ 0.24 ꢂ 10 M ꢀ min
.
0
Comparing the catalytic reaction rates determined in the
NBT and TNB assay system, we found that a much lower
catalytic reaction rate was observed when NBT was used as
the substrate (C < A; D < B). Considering that the same
blended GPx mimic was used in the two systems, the
dramatic change in the catalytic activity in the two systems
should be mainly due to the fact that an electrostatic
interaction could be formed in the TNB assay system
between the carboxyl in TNB and the quaternary ammo-
nium salt in the blended GPx mimic. Furthermore, the
catalytic reaction rates were measured under the same
conditions, except the ROOH was changed. The rate
constants of the spontaneous reaction between hydroper-
CUOOH, TNB assay system [Figure 10(A)],
v
0
¼
oxide and thiol vary in magnitude in the order k(H
[11]
2
O
2
) > K(-
.04 ꢂ 10ꢁ M ꢀ min
6
ꢁ1
;
H O ,
TNB
assay
system
CUOOH).
However, comparing the different assay
9
2
2
ꢁ
6
ꢁ1
[Figure 10(B)], v 1.62 ꢂ 10 M ꢀ min ; CUOOH, NBT assay
systems A and B, we noted that the catalytic activity of
the blended GPx mimic has a significant enhancement in
the TNB assay system when using
0
¼ 5.64 ꢂ 10 M ꢀ minꢁ1; H
ꢁ
6
system [Figure 10(C)], v
0
2 2
O , NBT
2 2
CUOOH instead of H O as a substrate.
The dramatic increase in the catalytic
activity when using hydrophobic
CUOOH as an alternative substrate
instead of H O2 is mainly because
2
the hydrophobic microenvironment
endowed from the blended GPx mimic
makes the hydrophobic substrate
CUOOH approach the GPx mimic more
easily and complete the catalytic cycle
more preferentially. Consequently,
based on the above experiments, we
can see that the blended GPx mimic has
a stronger substrate binding ability
Figure 9. (A) UV spectra with varying concentrations of PP-q2-CD2-Te1max in water; the
ꢁ5
concentration of TNB is 9.5 ꢂ 10 M, and from a to e the concentrations of PP-q2-CD2-
ꢁ3
Te1max are 0, 0.2, 0.4, 1.5, 2.5 and 4 ꢂ 10 M, respectively. (B) PP-PP-Te1: the condition is
as same as (A) except that PP-q2 and PP-CD2 are replaced by PP.
Macromol. Biosci. 2010, 10, 1505–1516
1514 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.201000179

Construction of an Artificial Glutathione Peroxidase Active Site . . .
factors plays an important role in enhancing the catalytic
efficiencyoftheGPxmimic. PP-q2-CD2-Te1max, constructed
by the blending process in this study, exhibits both strong
substrate binding ability and high catalytic activity.
Conclusion
A series of functional block copolymers modified with
tellurium, quaternary ammonium salt and cyclodextrin
were synthesized via atom-transfer radical polymerization
and click chemistry. A simply and efficient GPx mimic was
obtained using the blending process for the first time. A
study of the assembly behavior of the blended GPx mimic
and the individual copolymers indicated that the blended
GPx mimic can assemble into uniform and stable vesicles
that are analogous to the individual copolymers, which
makestheblendingprocessafeasibleandexcellentmethod
to construct blended GPx mimics. In particular, the
optimum GPx mimic was achieved through optimizing
the structure of the functional block copolymers and
changing the molar ratio of three functional block
copolymers, PP-Te1, PP-CD2 and PP-q2. Although the
specific substrate binding plays an important role in
designing a desirable GPx mimic, the match degree among
the catalytic factors also makes a great contribution to
obtaining high GPx activity. As a new GPx mimic,
considering its high catalytic activity, simple preparation
process and better match of catalytic factors, the blended
GPx mimic may make the preparation of efficient GPx
mimic more simple and the application of it more
achievable. We anticipate that this study will open up a
new field in artificial enzyme construction, and we also
hope this preparation process could be used in the
preparation of other biologically related functional nano-
particles.
Figure 11. Plots of catalytic reaction rates v
0
against molar ratio of
functional copolymer. (a) PP-q2-CD2-Te1: ratio of PP-q2 to PP-Te1
(
the ratio of PP-CD2 to PP-Te1 was fixed to 7:1); (b) PP-PP-Te1: the
conditions are the same as (a) except that PP-q2 and PP-CD2 are
replaced by PP.
through both electrostatic interactions and hydrophobic
interactions.
However, strong substrate binding does not imply better
[
12,14]
catalytic activity.
To confirm this in this study, a
contrast experiment was carried out. Another blended GPx
mimic, denoted PP-PP-Te1, was constructed using the same
method except that PP-q2 and PP-CD2 were replaced by
copolymer modified with no catalytic factors (PP). The
interaction between the substrate TNB and PP-PP-Te1 was
also determined by UV spectroscopy [see Figure 9(B)]. It was
shown that a red shift of the maximum absorbance
wavelength was also observed, which implied that the
hydrophobic interactions also existed between TNB and PP-
PP-Te1. This is possibly because the hydrophobic micro-
environment endowed from the PS block in self-assembled
vesicles could bind the substrate TNB to some degree.
However, as Figure 11 displays, the catalytic activity of PP-
PP-Te1isstrikingly lowerthanthatof PP-q2-CD2-Te1under
various molar ratios of the functional copolymers. The
absence of catalytic factors in PP-PP-Te1 and the better
match among the catalytic factors in PP-q2-CD2-Te1
possibly provides the remarkable difference between the
two blended GPx mimics. For PP-PP-Te1, a good match
between the hydrophobic microenvironment and the
catalytic center tellurium may be difficult because the
hydrophobicmicroenvironmentofthePSblockexistsinthe
core of the micelles and the catalytic center tellurium exists
in the shell. Compared with PP-PP-Te1, the catalytic factors
of the PP-q2-CD2-Te1 are all incorporated into the shell of
the micelles, which makes the match among the catalytic
factors much easier. In short, the above evidence allows us
to draw the conclusion that not only the strong substrate
binding but also the good match amongst the catalytic
Acknowledgements: Financial support from the Natural Science
Foundation of China (No: 20874036, 20921003), the NSFC for
Outstanding Younger Scientist (No. 20725415), the 111 project
(
B06009) and the National Basic Research Program
(2007CB808006) is gratefully acknowledged.
Received: April 29, 2010; Published online: September 20, 2010;
DOI: 10.1002/mabi.201000179
Keywords: atom transfer radical polymerization (ATRP); block
copolymers; enzymes; self-assembly; synthesis
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1516 ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.201000179