Construction of pH sensitive smart glutathione peroxidase (GPx) mimics based on pH responsive pseudorotaxanes

Article abstract of DOI:10.1039/d0ob00122h

Two organoselenium compounds, both of which were modified with two primary amine groups, were designed and synthesized to mimic the catalytic properties of glutathione peroxidase (GPx). It was demonstrated that the catalytic mechanism of the diselenide organoselenium compound (compound 1) was a ping-pong mechanism while that of the selenide organoselenium compound (compound 2) was a sequential mechanism. The pH-controlled switching of the catalytic activities was achieved by controlling the formation and dissociation of the pseudorotaxanes based on the organoselenium compounds and cucurbit[6]uril (CB[6]). Moreover, the switching was reversible at pH between 7 and 9 for compound 1 or between 7 and 10 for compound 2.

Full text of DOI:10.1039/d0ob00122h

Organic &
Biomolecular Chemistry
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Construction of pH sensitive smart glutathione
peroxidase (GPx) mimics based on pH responsive
pseudorotaxanes†
Cite this: DOI: 10.1039/d0ob00122h
Jiaxi Li, Wenlong Jia, Ganghui Ma, Xiaoyin Zhang, Shaojie An, Tao Wang and
Shan Shi
*
Two organoselenium compounds, both of which were modified with two primary amine groups, were
designed and synthesized to mimic the catalytic properties of glutathione peroxidase (GPx). It was
demonstrated that the catalytic mechanism of the diselenide organoselenium compound (compound 1)
was a ping-pong mechanism while that of the selenide organoselenium compound (compound 2) was a
sequential mechanism. The pH-controlled switching of the catalytic activities was achieved by controlling
the formation and dissociation of the pseudorotaxanes based on the organoselenium compounds and
cucurbit[6]uril (CB[6]). Moreover, the switching was reversible at pH between 7 and 9 for compound 1 or
between 7 and 10 for compound 2.
Received 19th January 2020,
Accepted 30th March 2020
DOI: 10.1039/d0ob00122h
rsc.li/obc
modified by a selenium or tellurium catalytic centre, were
Introduction
2
0–24
studied.
Compared with organoselenium/tellurium com-
Enzymes play an irreplaceable role in vivo because of their pounds, these chemically operated host molecules possessed
high rate and good stereoselectivity in catalysing biochemical hydrophobic or hydrophilic cavities which could recognize
reactions. In order to further explore their catalytic mechanism different substrates. In this way, the concept of synergy of reco-
and extend their in vitro applications, much efforts have been gnition and catalysis was proposed to obtain GPx mimics with
devoted to the construction of various artificial enzymes over higher efficiency. Similarly, molecularly imprinted GPx mimics
1
–5
the past 50 years. In the design of artificial enzymes, gluta- were developed in view of transition state recognition by imi-
2
5,26
thione peroxidase (GPx), an antioxidant selenoenzyme, has tating the active site microenvironment of natural GPx.
attracted much attention.
In
6
–9
GPx catalyses the reduction of the recent two decades, with the booming development of
hydroperoxides (ROOH) at the expense of tripeptide gluta- supramolecular and nanoscientific strategies, various nanoen-
thione (GSH) and maintains the metabolic balance of reactive zyme models of GPx, including micelles, vesicles and nano-
oxygen species (ROS) in vivo to protect various organisms from tubes, have been demonstrated based on supramolecular
2
7–29
oxidative stress and to reduce the risk of a range of related nano-assemblies.
diseases.
Moreover, the molecular imprinting
1
0–12
strategy can also be used to mimic specific substrate binding
Pioneering works on GPx mimics were carried out by Sies sites of natural GPx at the surface of nanoenzyme models. The
et al. who designed ebselen (2-phenyl-1,2-benzoisoselenazol-3 catalytic efficiency thus has had a significant enhancement.
1
3,14
(2H)-one) as a potential antioxidant medicine.
Encouraged Furthermore, in order to obtain mimics that have a more
by the successful design of ebselen, a series of organosele- similar catalytic structure to natural GPx, the genetic engineer-
nium/tellurium compounds based on selenium/tellurium ing strategy was exploited to construct a series of artificial
1
5–19
30–32
ether bonds have been reported.
Afterwards, in an attempt seleno-proteins or telluro-proteins.
to simulate the cavity function of natural GPx, numerous host
Up to now, some of these GPx mimics have shown satisfac-
molecules, including cyclodextrin derivatives and antibodies tory enzymatic properties. Genetic engineering models even
displayed extraordinarily high activities that rival the native
3
2
ones. On the other hand, the design and preparation of
smart GPx mimics with controlled catalytic activity have also
attracted increasing attention. The smart GPx mimics can be
constructed using materials that are responsive to external
stimuli such as temperature, light and pH. Several pioneering
College of Materials Science and Engineering, Shenyang University of Chemical
Technology, Shenyang 110142, People’s Republic of China.
E-mail: sshi@syuct.edu.cn
†
Electronic supplementary information (ESI) available: Characterization of com-
pounds 1 and 2, and catalytic curves of compounds 1 and 2 in enzymatic kine-
3
3–38
tics tests. See DOI: 10.1039/d0ob00122h
works have been reported by Liu and co-workers.
For
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4
3–49
example, a photoresponsive GPx mimic was successfully two identical carbonyl-fringed portals.
CB[6] and di-proto-
designed by the supramolecular strategy using photocontrolled nated diaminoalkane can form a very stable 1 : 1 host–guest
5
6
−1
inclusion–exclusion reaction of azobenzene with telluride complex with a binding constant as high as 10 –10
M .
3
3
β-cyclodextrin (β-CD) dimers. Moreover, GPx active sites were However, the binding constant decreases significantly when
5
0
introduced into temperature responsive poly(N-isopropyl- the two nitrogen atoms are deprotonated.
acrylamide) scaffolds through the formation of microgels,
Inspired by the above works, we developed a pH sensitive
3
4–38
micelles and nanotubes.
As expected, these temperature GPx model by using a pseudorotaxane molecular switch
sensitive GPx mimics exhibited excellent GPx-like catalytic formed by CB[6] and an organoselenium compound. We
activity with typical saturation kinetics behaviours. designed organoselenium compounds 1 and 2 (Fig. 1a) as GPx
In the design of smart artificial enzymes, molecular models, which contain two primary amine groups and a disele-
machines which can be regulated by an external stimulus in nide catalytic center (compound 1) or a selenide catalytic
different chemical processes are ideal scaffolds due to their center (compound 2). In the presence of CB[6], the GPx
3
9–41
dynamic properties and particular topological structures.
mimics form 1 : 1 host–guest pseudorotaxane complexes when
The development of pH responsive molecular machines pro- the amine groups are di-protonated. In this case, the mimics
vides an opportunity to construct pH sensitive GPx mimics. cannot show GPx activity since the active sites are encapsu-
Such smart GPx mimics are promising in the future as poten- lated into the hydrophobic cavity of CB[6] (Fig. 1b). As the
tial medicines because the pH level in the human body varies primary amine groups are gradually deprotonated with the rise
4
2
in different cells and organs. One of the classical pH respon- of pH, the binding ability of CB[6] with compounds 1 and 2
sive molecular machines is a pseudorotaxane based on cucur- significantly decreases. As a result, the active sites are exposed
bit[6]uril (CB[6]). As a member of the cucurbit[n]uril (CB[n] n = to the solution, causing an increase of GPx activity. Thus, the
5
–8) family, CB[6] is a macrocyclic compound with 6-glycoluril catalytic activity of the GPx model can be switched on/off
units and has a hydrophobic cavity that is accessible through through changing the pH.
Results and discussion
Organoselenium compounds 1 and 2 were synthesized by the
1
procedures shown in Fig. 2. After the synthesis, H NMR titra-
tion experiments were performed at pH = 7 to test whether the
synthesized compound 1 can form pseudorotaxane with CB[6].
As shown in Fig. 3a, upon the addition of CB[6] into com-
pound 1 in D O, progressive disappearance of the signals
2
1 2
corresponding to H and H of compound 1 occurred, followed
by the growth of two new peaks. The appearance of the new
peaks can be attributed to the movement of H and H of com-
1
2
pound 1 due to its encapsulation inside CB[6]. After 1 equiv. of
CB[6] was introduced, the original peaks corresponding to H
1
and H disappeared completely. This indicates that CB[6] and
2
compound 1 formed 1 : 1 host–guest pseudorotaxane com-
plexes when the primary amine groups were protonated. It
should be noted that, due to the formation of pseudorotaxane
complexes, H
1
and H
in H NMR spectra (H
from 3.36 ppm to 3.16 ppm). After compound 1 was comple-
tely incorporated into CB[6], H was close to the internal part
of the cavity while H was close to the portal of the cavity,
hence leading to the chemical shift movement of H1 being
obviously larger than H . The binding constant between com-
2
showed different degrees of movement
1
1
from 3.11 ppm to 2.61 ppm and H
2
1
2
2
4
−1
pound 1 and CB[6] was calculated to be 1.19 ± 0.09 × 10 M
at pH = 7 (Fig. S15†).
1
H NMR titration experiments were also utilized to confirm
the formation of the pseudorotaxane based on CB[6] and com-
pound 2. The result was similar to that of compound 1
Fig. 1 (a) Structures of organoselenium compounds 1 and 2 and CB[6];
b) schematic representation of the pH sensitive smart GPx mimic based
(
1 2
Fig. 3b). After 1 equiv. of CB[6] was added, the H and H
(
peaks due to compound 2 completely moved from 2.75 to
on the formation and dissociation of pH responsive pseudorotaxanes
formed by CB[6] and compounds 1 and 2.
1.81 ppm and from 3.16 to 2.95 ppm, respectively. The binding
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Fig. 2 Synthesis procedures of organoselenium compounds 1 and 2.
constant between compound 2 and CB[6] was calculated to be
4
−1
2
.50 ± 0.10 × 10 M at pH = 7 (Fig. S16†), about twice that of
compound 1 with CB[6]. On the other hand, the binding con-
stant between diaminopentane and CB[6] can be as high as
6
−1
50
2
.40 × 10 M as reported in the literature. After the middle
carbon atom of the diaminopentane was replaced by a sulfur
atom to form H N(CH S(CH NH , the binding constant was
2
2
)
2
2
)
2
2
5
−1
found to reduce by an order of magnitude to 4.2 × 10 M . In
the present study, we synthesized compound 2 by substituting
a selenium atom, a same main group element as sulfur, for
the sulfur atom of H
2 2 2 2 2 2
N(CH ) S(CH ) NH . This resulted in the
4
decline of the binding constant further to 2.50 ± 0.10 × 10
−
1
M
. This is possibly because the size of methylene in diami-
nopentane matched the cavity of CB[6] best. As the middle
carbon atom was replaced by sulfur and selenium atoms, the
middle methylene changed to thioether and selenide, which
increased the molecular size and thus decreased the binding
constant with CB[6]. Moreover, when the selenide in com-
pound 2 was replaced by the diselenide in compound 1, the
molecular size was further increased. As a result, the binding
constant of compound 1 and CB[6] further decreased to 1.19 ±
4
−1
0
.09 × 10 M
.
With the increase of pH, the binding ability of CB[6] with
compound 1 or 2 significantly decreased followed by the dis-
sociation of the pseudorotaxane. These were also studied by
1
H NMR (Fig. 4). Compound 1 formed the pseudorotaxane
with 1 equiv. of CB[6] at pH = 7 as mentioned above. With the
rise of pH, the amine groups of compound 1 were deproto-
nated, causing the dissociation of the pseudorotaxane. As a
1
result, in H NMR spectra, the peaks attributed to the encapsu-
1
Fig. 3 (a) Partial H NMR spectra (500 MHz) of compound 1 and mix-
−1
lated compound 1 showed a significant reduction followed by
tures of compound 1 (1 mmol L ) with different amounts of CB[6] at pH
1
=
7. (b) Partial H NMR spectra (500 MHz) of compound 2 and mixtures the increase of the peaks of free compound 1 (Fig. 4a). It is
1
−
of compound 2 (1 mmol L ) with different amounts of CB[6] at pH = 7.
worth mentioning that the peaks of free compound 1 exhibited
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compound 1 exhibited a relatively high dissociation ratio of
the pseudorotaxane as compared with compound 2. This can
be attributed to the lower binding constant between CB[6] and
compound 1 as described above. The results indicate that the
formation and dissociation of the pseudorotaxane based on
CB[6] and compound 1 or 2 could be controlled by pH. That is,
when CB[6] and compound 1 or 2 formed the pseudorotaxane
at pH = 7, the active sites of compound 1 or 2 were encapsu-
lated into the hydrophobic cavity of CB[6] and were unable to
show their catalytic activities. In contrast, as part of the pseu-
dorotaxane was dissociated at higher pH, the active sites of the
free compound 1 or 2 were exposed to the solution and were
capable of catalysing the corresponding reactions. Thus the
GPx activity of compound 1 or 2 can be switched on/off by con-
trolling the pH of the solution.
The catalytic activities of compounds 1 and 2 were first
investigated in a 3-carboxy-4-nitrobenzenethiol (TNB) assay
system at 37 °C and pH = 7. Catalytic curves for different con-
centrations of compounds 1 and 2 were obtained. As shown in
Fig. 5, the slope of the catalytic curves displayed a linear
increase with the increase of the concentration of compounds
1
and 2, which was in accord with the typical enzymatic reac-
tion. The GPx activities of compounds 1 and 2 were calculated
−
3
−3
−1
to be 0.95 ± 0.01 × 10 and 2.69 ± 0.04 × 10 μmol min
μmol− , respectively. As compared to diphenyl diselenide
1
1
Fig. 4 Partial H NMR spectra (500 MHz) of mixtures of (a) compound 1
1 mmol L− ) and (b) compound 2 (1 mmol L ) with CB[6] (1 mmol L
1
−1
−1
)
(
at different pH values.
different chemical shifts at different pH values. At pH = 7, the
two primary amine groups of compound 1 were protonated. In
this case, the chemical shifts of H
.36 ppm, respectively (Fig. 3a). However, as the two primary
amine groups changed from the protonated to deprotonated
state at pH = 12, the chemical shifts of H and H had an
obvious upfield movement (H1 to 2.92 ppm and H2 to
.99 ppm). H exhibited a larger movement of the chemical
shift as compared to H because H was nearer to the amine
1 2
and H were 3.11 and
3
1
2
2
2
1
2
groups than H . Through comparing the integral of peaks for
1
the encapsulated and free compound 1, we found from calcu-
lations that 17% of the pseudorotaxane was dissociated when
the pH rose to 9. Moreover, the ratio of the dissociated pseu-
dorotaxane increased to 72% when the pH rose to 10. At pH =
1
2, the pseudorotaxane formed almost totally dissociated with
the ratio of the remaining pseudorotaxane less than 5%.
A similar result was observed for compound 2. Compound
2
7
5
also formed a pseudorotaxane with 1 equiv. of CB[6] at pH =
. The dissociation ratios of the pseudorotaxane were 15% and
7%, respectively, when the pH rose to 9 and 10. When the pH
went up to 12, the ratio of the remaining pseudorotaxane was
Fig. 5 Catalytic curves for different concentrations of (a) compound 1
also less than 5% (Fig. 4b). It is obvious that, at the same pH, and (b) compound 2 using the TNB assay system at pH = 7.
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(PhSeSePh), a typical GPx mimic, the efficiency of compounds amine groups. The amine groups can recognize the substrate
1
and 2 increased by about an order of magnitude. Different (TNB) by the electrostatic interactions with the carboxyl groups
from diphenyl diselenide, both compounds 1 and 2 have two of TNB, leading to the improvement of the catalytic activity.
The GPx activities of compounds 1 and 2 at different pH
values in the TNB assay system were also investigated. The GPx
−
3
activities of compound 1 were 0.57 ± 0.06 × 10 , 0.95 ± 0.01 ×
−
3
−3
−3
−1
1
0 , 0.85 ± 0.03 × 10 and 0.70 ± 0.02 × 10 μmol min
−
1
μmol at pH = 6, 7, 8 and 9, respectively (Fig. 6a). The result
indicates that the most suitable pH for compound 1 was 7.
The GPx activities of compound 2 were 2.37 ± 0.14 × 10 , 2.69
−
3
−
3
−3
−3
±
0
0.04 × 10 , 3.66 ± 0.07 × 10 , 4.31 ± 0.21 × 10 and 2.75 ±
.16 × 10− μmol min μmol−1 at pH = 6, 7, 8, 9 and 10,
3
−1
respectively (Fig. 6b). Compound 2 showed its highest GPx
activity at pH = 9. The GPx activity of compound 1 could not be
measured at pH = 10 since the slope of the catalytic curves at
Fig. 6 Catalytic activities of (a) compound 1 and (b) compound 2 at
different pH values using the TNB assay system.
1
mM was even smaller than that of the blank.
To fully understand the difference in the behaviour of GPx
activity between compounds 1 and 2, we further investigated
the enzyme kinetics with double-reciprocal plots using a GSH
reductase-reduced nicotinamide adenine dinucleotide phos-
phate (NADPH) coupled assay. The assay involved the use of
GSH and H
was fixed and the GPx activities of compounds 1 or 2 were
measured under different concentrations of GSH
Fig. S17–22†). The double-reciprocal plots were obtained from
the GPx activity reciprocal versus GSH concentration reciprocal
Fig. 7). Similarly, the concentration of GSH was kept constant
and the GPx activities of compound 1 or 2 were measured
versus 1/[GSH] (mM− ) at [H
]/V O ] = 0.5 mM ( ), under different concentrations of H O to obtain another
0 0 2 2
2 2 2 2
O as two substrates. The concentration of H O
(
(
Fig. 7 Double-reciprocal plots of the reduction of H
2
O
2
by GSH under
the catalysis of (a) compound 1 and (b) compound 2. [E
0
] = total enzyme
1
concentration; [E
.75 mM ( ) and 1.00 mM (■).
2
2
0
double-reciprocal plot (Fig. S23 and S24†). The double-recipro-
cal plots of compound 1 yielded a series of parallel linear plots
for both substrates (Fig. 7a and S23†), suggesting that the cata-
lytic mechanism of compound 1 was a ping-pong mechanism
5
3–58
with at least one covalent intermediate.
According to the
5
6
detailed kinetic studies carried out by Engman
and
5
7
Mugesh, the catalytic intermediate of diselenide organosele-
nium compounds was a catalyst–substrate complex (RSeSR’).
Herein, the catalytic reaction of compound 1 might proceed
via the mechanism shown in Fig. 8. Saturation kinetics were
observed for each of the enzymatic peroxidase reactions at all
the individual concentrations of GSH and H O investigated.
Fig. 8 Proposed catalytic mechanism of compound 1.
2
2
Table 1 Apparent kinetic parameters for H
2
O
2
reduction by GSH catalyzed by compounds 1 and 2
Compound 2
Compound 1
cat (min−
1
)
KGSH (M)
k
cat/KGSH (M−1 min−1
)
k
cat (min−1
)
KGSH (M)
k
cat/KGSH (M−1 min−1
)
[
2 2
H O ] (mM)
k
3
3
3
2
−3
−3
−3
−4
−4
−4
0
0
1
.5
.75
0.66 ± 0.01
0.97 ± 0.02
1.23 ± 0.02
1.19 ± 0.01 × 10−
5.52 ± 0.07 × 10
5.32 ± 0.05 × 10
5.55 ± 0.03 × 10
4.16 ± 0.09 × 10
4.33 ± 0.09 × 10
4.52 ± 0.03 × 10
5.24 ± 0.05 × 10
3.66 ± 0.09 × 10
1.70 ± 0.04 × 10
7.94 ± 0.24
11.83 ± 0.53
26.55 ± 0.81
−
2
2
1.82 ± 0.02 × 10
2.21 ± 0.03 × 10−
Compound 1
Compound 2
cat (min−
1
)
K
H O (M)
k
cat/KH O (M−1 min−1
)
k
cat (min−1
)
K
H O (M)
k
cat/KH O (M−1 min−1
2 2
)
[
GSH] (mM)
k
2
2
2
2
2
2
4
3
3
3
−3
−3
−3
−3
−4
−4
0
1
2
.5
0.26 ± 0.01
0.52 ± 0.03
0.94 ± 0.07
1.72 ± 0.17 × 10−
1.51 ± 0.19 × 10
1.43 ± 0.14 × 10
1.48 ± 0.12 × 10
7.68 ± 3.97 × 10
6.05 ± 1.45 × 10
5.32 ± 0.58 × 10
1.41 ± 0.51 × 10
6.34 ± 0.10 × 10
3.05 ± 0.51 × 10
5.43 ± 1.32
9.54 ± 2.40
17.44 ± 4.13
−
4
4
3.64 ± 0.16 × 10
6.35 ± 0.05 × 10−
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The relevant steady-state equation for the enzymatic peroxi-
dase reaction is as follows.
0 0
Here, v is the initial reaction rate, [E ] is the initial enzyme
mimic concentration, kcat is a pseudo-first-order rate constant
and KmH O and KmGSH are the Michaelis–Menten constants for
2
2
v0
kcat½GSHꢀ½H2O2ꢀ
¼
hydrogen peroxide and glutathione, respectively. The kinetic
parameters of the enzymatic reactions of compound 1 were cal-
culated according to the steady-state equation and double-reci-
procal plots and are shown in Table 1.
½
E0ꢀ KmGSH½H2O2ꢀ þ KmH O ½GSHꢀ þ ½GSHꢀ½H2O2ꢀ
2
2
In contrast to the double-reciprocal plots of compound 1,
those of compound 2 yielded a series of intersecting linear
plots for both substrates (Fig. 7b and S24†), which indicates
that the catalytic mechanism of compound 2 was a sequential
5
9
mechanism. According to the studies by Carsol et al. the
single product (GSSG) was necessarily released in the last step
in the sequential mechanism. Before the release of the
product, both of the substrates formed an enzyme–substrate
complex with the enzyme. Engman et al. and others systemati-
cally analysed the catalytic process of selenide organoselenium
compounds and conjectured that the selenide organoselenium
compounds were in their oxidized state (RSe(OH)
2
R) in the
6
0–62
Fig. 9 Proposed catalytic mechanism of compound 2.
catalytic cycle.
Combining the studies carried out by
Fig. 10 Catalytic curves of compound 1 (1 mmol L−1) and mixtures of compound 1 (1 mmol L−1) with different amounts of CB[6] at (a) pH = 7, (b)
pH = 8 and (c) pH = 9; catalytic curves of compound 2 (0.5 mmol L 1) and mixtures of compound 2 (0.5 mmol L−1) with different amounts of CB[6]
at (d) pH = 7, (e) pH = 8, (f) pH = 9 and (g) pH = 10; decrease of relative activity versus mixtures of (h) compound 1 and (i) compound 2 with different
amounts of CB[6] at different pH values. The activity of compound 1 or compound 2 without any CB[6] was defined as 100%.
−
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Carsol and Engman, the catalytic reaction of compound 2
might proceed via the mechanism shown in Fig. 9. The rele-
vant steady-state equation for compound 2 is identical to that
for compound 1. The kinetic parameters of the enzymatic reac-
tions of compound 2 were calculated according to the steady-
state equation and double-reciprocal plots and are also shown
in Table 1.
The GPx activities of compounds 1 and 2, after the for-
mation of the pseudorotaxane with CB[6], were then evaluated
using the TNB assay system. At pH = 7, compound 1 was dipro- Fig. 12 (a) The ON–OFF switch of the catalytic activity of the pseudor-
otaxane formed by CB[6] and compound 1 by a change in pH between 7
and 9. (b) The ON–OFF switch of the catalytic activity of the pseudoro-
taxane formed by CB[6] and compound 2 by a change in pH between 7
and 10.
tonated and had strong host–guest interactions with CB[6] to
form the pseudorotaxane. Upon the addition of CB[6], the
active sites of compound 1 were progressively enclosed by the
hydrophobic cavity of CB[6], which led to a progressive
decrease of GPx activity (Fig. 10a and h). After 1 equiv. CB[6]
was added, the active sites of compound 1 were fully incorpor-
ated into CB[6] and could not come in contact with any sub-
μmol min−1 μmol−1) relative to compound 1 without CB[6]
(
Fig. 11a and b). As a result, the GPx activity of the pseudoro-
strate. As a result, compound 1 showed a very small activity of
taxane formed by CB[6] and compound 1 can be switched on/
off through changing the pH between 7 and 9. A similar result
was also found when adding CB[6] to compound 2 at pH = 9
5
−1
−1
1
.40 ± 0.83 × 10− μmol min μmol (Fig. 11a and b). A
similar result was found upon the addition of CB[6] to com-
pound 2 at pH = 7 (Fig. 11d and i). After the addition of 1
equiv. CB[6], compound 2 also hardly showed any activity (4.68
(
Fig. 10f and i). After 1 equiv. CB[6] was added, there was still
−3
−1
−1
about 15% activity (0.59 ± 0.07 × 10 μmol min μmol ) as
compared to that of compound 2 without CB[6] (Fig. 11c and
d). Moreover, when the pH was increased to 10, the GPx
activity of compound 2 remained more that 50% after 1 equiv.
5
−1
−1
±
1.86 × 10− μmol min μmol , Fig. 11c and d). These
results indicate that the formation of the pseudorotaxane
between CB[6] and compound 1 or 2 could switch off the GPx
activity, irrespective of the catalytic mechanism of compound
−3
−1
−1
CB[6] was added (1.45 ± 0.02 × 10 μmol min μmol
,
1
or 2. However, when the solution was adjusted to a pH above
Fig. 10g, 11c and d). As a result, the GPx activity of the pseu-
dorotaxane formed by CB[6] and compound 2 could be
switched on/off to a greater extent through changing the pH
between 7 and 10.
The above investigations demonstrate that the GPx activity
of the pseudorotaxane formed by CB[6] and compound 1 or 2
can be switched on/off by changing the pH. As switchable GPx
models, the reversibility of the catalytic activity of the pseudor-
otaxanes was also tested. The results reveal that the catalytic
activities of two enzymes were completely reversible after mul-
tiple changes in pH between 7 and 9 for compound 1
7
, the pseudorotaxane started to partly dissociate because of
the deprotonation of amine groups. At pH = 9, a decrease of
the GPx activity of compound 1 occurred upon the addition of
CB[6] (Fig. 10c and h). However, after 1 equiv. CB[6] was
added, there was still about 20% activity (0.14 ± 0.01 × 10−
3
(
Fig. 12a) or between 7 and 10 for compound 2 (Fig. 12b).
Conclusions
In conclusion, we designed and synthesized two organosele-
nium compounds to mimic the catalytic properties of gluta-
thione peroxidase (GPx). Organoselenium compound 1 con-
tained a diselenide active centre while organoselenium com-
pound 2 contained a selenide active centre. The catalytic
mechanisms of the two organoselenium compounds were
investigated in detail. The results indicate that compound 1
catalysed the reaction with a ping-pong mechanism while com-
pound 2 with a sequential mechanism. Moreover, the GPx
activity can be switched on and off by controlling the for-
mation and dissociation of the pseudorotaxane based on CB[6]
and the organoselenium compounds. Thus the reversible pH-
controlled switching of catalytic activities was achieved even
after multiple changes in pH between 7 and 9 (compound 1)
Fig. 11 (a) Relative catalytic activities and (b) catalytic activities of the
pseudorotaxane formed by CB[6] and compound 1 at a range of pH
from 7 to 9; (c) relative catalytic activities and (d) catalytic activities of
the pseudorotaxane formed by CB[6] and compound 2 at a range of pH
from 7 to 10.
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or between 7 and 10 (compound 2). We believe that the suc-
cessful construction of such pH sensitive smart GPx mimics be 247.9931 and found to be 248.9427 (M + H ) (Fig. S7†). The
High resolution ESI-TOF mass spectrum (m/z) calculated to
+
will provide a novel route to the design of smart artificial mass spectrum contained about thirteen isotopic peaks (from
enzymes.
240.9 to 252.9) which were typical isotopic peaks of the disele-
nide organoselenium compound.
Synthesis of compound 9
Experimental
Synthesis of compound 5
Compound 5 (3.0 g, 6.7 mmol) was dissolved to 50 mL methyl
alcohol in a 100 mL round bottom flask and deoxygenated
with nitrogen. Sodium borohydride (1.2 g, 31.7 mmol) was
added into a 250 mL round bottom flask under the protection
of nitrogen. 50 mL deoxygenated methyl alcohol solution of
compound 5 was injected into the 250 mL round bottom flask.
After 30 min the solution completely changed from yellow to
colourless and transparent, indicating that compound 5 had
totally reacted, giving compound 8. Compound 4 (3.0 g,
Selenium (1737.1 mg, 22.0 mmol) and sodium borohydride
(756.6 mg, 20.0 mmol) were added into a 250 mL round
bottom flask under the protection of nitrogen, followed by the
injection of 20 mL deoxygenated water. After 30 min selenium
reacted completely to give Na
2 2
Se . Compound 4 (4.48 g,
2
0.0 mmol; see the ESI for its synthesis, Fig. S1†) was dissolved
in 100 mL methyl alcohol and deoxygenated with nitrogen.
The deoxygenated solution of compound 4 was injected into
the round bottom flask and stirred for 12 hours at room temp-
erature. The volatiles were removed under reduced pressure
and the residue was transferred to a 500 mL separating funnel
using 200 mL dichloromethane. The dichloromethane solu-
tion was extracted with sodium chloride solution (3 × 100 mL)
and then the organic phase was dried with anhydrous sodium
sulfate. The volatiles were removed and the residue was puri-
fied by column chromatography (silica gel, dichloromethane/
ethyl acetate: 15/1 as eluent) to give a yellow solid 5 (4.0 g, 45%
yield).
1
3.5 mmol) was dissolved in 50 mL methyl alcohol and deoxy-
genated with nitrogen. The deoxygenated solution of com-
pound 4 was injected into the methyl alcohol solution of com-
pound 8 and stirred for 12 hours at room temperature. The
volatiles were removed under reduced pressure and the residue
was transferred to a 500 mL separating funnel using 200 mL
dichloromethane. The dichloromethane solution was extracted
with sodium chloride solution (3 × 100 mL) and then the
organic phase was dried with anhydrous sodium sulfate. The
volatiles were removed and the residue was purified by column
chromatography (silica gel, dichloromethane/ethyl acetate: 15/
1 as eluent) to give a transparent liquid 9 (3.7 g, 75% yield).
1
H NMR (500 MHz; CDCl ; Me Si) δ 1.46 (18H, s, 2C(Me) )
3
4
3
1
H NMR (500 MHz; CDCl ; Me Si) δ 1.46 (18H, s, 2C(Me) )
3
4
3
3
.02 (4H, t, J = 7.0 Hz, SeCH
2
) 3.48 (4H, t, J = 7.0 Hz, NCH
2
)
2.70 (4H, t, J = 7.0 Hz, SeCH
2
) 3.37 (4H, t, J = 7.0 Hz, NCH
2
)
(
Fig. S2†).
1
3
(Fig. S8†).
C NMR (500 MHz; CDCl ; Me Si) δ 153.33, 76.95, 74.85,
3
4
1
3
C NMR (500 MHz; CDCl ; Me Si) δ 153.28, 76.82, 74.87,
3
4
7
4.60, 74.35, 38.40, 26.84, 25.90 (Fig. S3†).
74.61, 74.36, 37.97, 25.85, 21.54 (Fig. S9†).
High resolution ESI-TOF mass spectrum (m/z) calculated to
+
High resolution ESI-TOF mass spectrum (m/z) calculated to
be 448.0380 and found to be 449.0392 (M + H ) and 471.0210
+
+
be 368.1214 and found to be 369.1316 (M + H ) and 391.1137
(
M + Na ) (Fig. S4†). The mass spectrum contained about thir-
+
(
M + Na ) (Fig. S10†). The mass spectrum contained seven iso-
teen isotopic peaks (from 441.0 to 453.0 and 463.0 to 475.0)
which were typical isotopic peaks of the diselenide organosele-
nium compound.
topic peaks (from 365.1 to 371.1 and 387.1 to 393.1) which
were typical isotopic peaks of the selenide organoselenium
compound.
Synthesis of compound 1
Synthesis of compound 2
Compound 5 (4.0 g, 8.9 mmol) was added to a mixture of Compound 9 (3.7 g, 10.1 mmol) was added to a mixture of
1
1
0 mL dichloromethane and 10 mL trifluoroacetic acid in a 10 mL dichloromethane and 10 mL trifluoroacetic acid in a
00 mL round bottom flask and stirred for 12 hours at room 100 mL round bottom flask and stirred for 12 hours at room
temperature. Most of the volatiles were removed under temperature. Most of the volatiles were removed under
reduced pressure and the residue was deposited with 500 mL reduced pressure and the residue was deposited with 500 mL
cold ethyl ether. A primrose yellow precipitate (compound 6, cold ethyl ether. The white precipitate (compound 10, 3.7 g,
4
.1 g, 96% yield) was obtained by filtration under reduced 93% yield) was obtained by filtration under reduced pressure
pressure and was dissolved in 50 mL saturated sodium carbon- and dissolved in 50 mL saturated sodium carbonate solution.
ate solution. After extracting with ethyl acetate (3 × 30 mL), the After extracting with ethyl acetate (3 × 30 mL), the organic
organic phase was dried with anhydrous sodium sulfate. The phase was dried with anhydrous sodium sulfate. The volatiles
volatiles were removed under reduced pressure to give a trans- were removed under reduced pressure to give a transparent
parent primrose yellow liquid 1 (1.5 g, 68% yield).
liquid 2 (1.2 g, 71% yield).
1
1
H NMR (500 MHz; D
2
O) δ 3.11 (4H, t, J = 7.0 Hz, SeCH
) (Fig. S5†).
C NMR (500 MHz; D O) δ 39.60, 23.51 (Fig. S6†).
2
)
H NMR (500 MHz; D
2
O) δ 2.75 (4H, t, J = 7.0 Hz, SeCH
) (Fig. S11†).
2
)
3
.37 (4H, t, J = 7.0 Hz, NCH
2
3.16 (4H, t, J = 7.0 Hz, NCH
2
1
3
13
C NMR (500 MHz; D O) δ 38.98, 19.10 (Fig. S12†).
2
2
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High resolution ESI-TOF mass spectrum (m/z) calculated to is μM min− μM−1. All the experiments were performed in
1
+
be 247.9931 and found to be 169.0218 (M + H ) (Fig. S13†). triplicate.
The mass spectrum contained seven isotopic peaks (from
1
65.0 to 171.0) which were typical isotopic peaks of the sele-
nide organoselenium compound.
Conflicts of interest
1
There are no conflicts to declare.
H NMR titration experiment
Compound 1 was dissolved in D O at a concentration of
2
1
−1
2
.0 mmol L− . CB[6] was dissolved in 1 mol L KCl aqueous
Acknowledgements
(D O) solution at the concentrations of 0.5, 1.0, 1.5 and
2
.0 mmol L− , respectively. The solutions of compound 1 and
1
2
This work was supported by the Natural Science Foundation of
China (no. 51573100), Educational Commission of Liaoning
Province, China (no. LQ2019006) and Doctoral Scientific
Research Foundation of Liaoning Science and Technology
Department, China (no. 201601203).
CB[6] were mixed at a ratio of 1 : 1 to give a mixture of com-
pound 1 and 0.25, 0.50, 0.75 and 1.00 equiv. CB[6] in D O. In
order to investigate the change of the interaction between
CB[6] and compound 1 at different pH values, CF COOD and
NaOD were utilized to control the pH of the mixture in a range
2
3
1
of 7–12. H NMR titration experiment of compound 2 was the
same as that of compound 1. All the experiments were per-
formed in triplicate.
Notes and references
enzyme
1
2
3
4
A. J. Kirby, Angew. Chem., Int. Ed. Engl., 1996, 35, 707.
R. Breslow and S. Dong, Chem. Rev., 1998, 98, 1997.
G. Wulff, Chem. Rev., 2002, 102, 1.
M. Raynal, P. Ballester, A. Vidal-Ferran and P. W. N. M. van
Leeuwen, Chem. Soc. Rev., 2014, 43, 1660.
M. Raynal, P. Ballester, A. Vidal-Ferran and P. W. N. M. van
Leeuwen, Chem. Soc. Rev., 2014, 43, 1734.
G. Mugesh, W. W. du Mont and H. Sies, Chem. Rev., 2001,
2
TNB þ H2O2 ꢁ! DTNB þ 2H2O
TNB catalytic activity assay system
A TNB (3-carboxy-4-nitrobenzenethiol) assay system, proposed
5
1
5
6
by Hilvert et al., is an improved GPx catalytic activity assay
method using TNB as a glutathione (GSH) alternative. The
reaction equation has been shown above. The assay mixture
contained 50 mM phosphate buffer (pH = 7–10), 100 μM TNB,
101, 2125.
7
8
G. Mugesh and H. Singh, Chem. Soc. Rev., 2000, 29, 347.
X. Huang, X. M. Liu, Q. Luo, J. Q. Liu and J. C. Shen, Chem.
Soc. Rev., 2011, 40, 1171.
2
(
50 μM hydrogen peroxide (H
2 2
O ) and 250–1000 μM enzyme
compound 1 or 2). The initial rate (v ) for the reduction of
H O by TNB was determined by monitoring the UV absorp-
2 2
0
9
Z. Y. Dong, Q. Luo and J. Q. Liu, Chem. Soc. Rev., 2012, 41,
tion at 410 nm due to the thiolate (TNB). The definition of
catalytic activity is the required enzyme quantity to oxidize
7890.
1
1
1
0 J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson,
D. G. Hafeman and W. G. Hoekstra, Science, 1973, 179, 588.
1 L. Flohe, W. A. Gunzler and H. H. Schock, FEBS Lett., 1973,
1
μM TNB per minute. The unit of catalytic activity is μM
−
1
−1
min μM . All the experiments were performed in triplicate.
enzyme
2
GSH þ H2O2 ꢁ! GSSG þ 2H2O
32, 132.
2 L. Flohe, G. Loschen, W. A. Gunzler and E. Eichele, Hoppe-
Seyler’s Z. Physiol. Chem., 1972, 352, 987.
GSSG reductase
GSSG þ NADPH þ Hþ
ꢁ!
2GSH þ NADPþ
1
1
1
3 H. Sies, Free Radical Biol. Med., 1993, 14, 313.
4 H. Sies and H. Masumoto, Adv. Pharmacol., 1997, 38, 229.
5 T. G. Back and Z. Moussa, J. Am. Chem. Soc., 2003, 125,
GSH reductase-reduced NADPH coupled catalytic activity assay
system
13455.
The GSH reductase-reduced nicotinamide adenine dinucleo- 16 Y. You, K. Ahsan and M. R. Detty, J. Am. Chem. Soc., 2003,
tide phosphate (NADPH) coupled assay system is a classical 125, 4918.
GPx catalytic activity assay method using GSH as the substrate, 17 S. S. Zade, H. B. Singh and R. J. Butcher, Angew. Chem., Int.
5
2
and is the same with natural GPx. The reaction equation has
been shown above. The assay mixture contained 50 mM phos- 18 S. Tomoda and M. Iwaoka, J. Am. Chem. Soc., 1994, 116,
phate buffer (pH = 7.0), 1 mM GSH, 500 μM hydrogen peroxide 2557.
), 1 U glutathione reductase (GR), 300 μM NADPH and 19 M. McNaughton, L. Engman, A. Birmingham, G. Powis and
50–1000 μM enzyme (compound 1 or 2). The initial rate (υ I. A. Cotgreave, J. Med. Chem., 2004, 47, 233.
of the reduction of H O by TNB was determined by monitor- 20 Z. Wu and D. Hilvert, J. Am. Chem. Soc., 1990, 112, 5647.
Ed., 2004, 43, 4513.
2 2
(H O
2
0
)
2
2
ing the UV absorption at 340 nm due to the NADPH. The defi- 21 G. M. Luo, Z. Q. Zhu, L. Ding, G. Gao, Q. A. Sun, Z. Liu,
nition of catalytic activity is the required enzyme quantity to
oxidize 1 μM NADPH per minute. The unit of catalytic activity
T. S. Yang and J. C. Shen, Biochem. Biophys. Res. Commun.,
1994, 198, 1240.
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
2
2
2
2
2
2
2
2
2 J. Q. Liu, S. J. Gao, G. M. Luo, G. L. Yan and J. C. Shen, 42 J. X. Li, C. Y. Si, H. C. Sun, J. Y. Zhu, T. Z. Pan, S. D. Liu,
Biochem. Biophys. Res. Commun., 1998, 247, 397.
3 G. M. Luo, X. J. Ren, J. Q. Liu, Y. Mu and J. C. Shen, Curr.
Med. Chem., 2003, 10, 1151.
Z. Y. Dong, J. Y. Xu, Q. Luo and J. Q. Liu, Chem. Commun.,
2015, 51, 9987.
43 K. Kim, Chem. Soc. Rev., 2002, 31, 96.
4 X. Huang, Y. Z. Yin and J. Q. Liu, Macromol. Biosci., 2010, 44 H. Cong, X. L. Ni, X. Xiao, Y. Huang, Q. J. Zhu, S. F. Xue,
1
0, 1385.
Z. L. F. Lindoy and G. Wei, Org. Biomol. Chem., 2016, 14,
4335.
5 X. Huang, Y. Liu, K. Liang, Y. Tang and J. Q. Liu,
Biomacromolecules, 2008, 9, 1467.
45 L. Isaacs, Acc. Chem. Res., 2014, 47, 2052.
6 X. Huang, Y. Z. Yin, Y. Liu, X. L. Bai, Z. M. Zhang, J. Y. Xu, 46 L. Liu, N. Nouvel and O. A. Scherman, Chem. Commun.,
J. C. Shen and J. Q. Liu, Biosens. Bioelectron., 2009, 25, 657. 2009, 3243.
7 X. Zhang, H. P. Xu, Z. Y. Dong, Y. P. Wang, J. Q. Liu and 47 S. Gürbüz, M. Idris and D. Tuncel, Org. Biomol. Chem.,
J. C. Shen, J. Am. Chem. Soc., 2004, 126(34), 10556. 2015, 13, 330.
8 H. P. Xu, J. Gao, Y. P. Wang, Z. Q. Wang, M. Smet, 48 Q. W. Zhang, X. Y. Yao, D. H. Qu and X. Ma, Chem.
W. Dehaenb and X. Zhang, Chem. Commun., 2006, 796. Commun., 2014, 50, 1567.
9 Y. Tang, L. P. Zhou, J. X. Li, Q. Luo, X. Huang, P. Wu, 49 H. Cong, X. L. Ni, X. Xiao, Y. Huang, Q. J. Zhu, S. F. Xue,
Y. G. Wang, J. Y. Xu, J. C. Shen and J. Q. Liu, Angew. Chem.,
Int. Ed., 2010, 49, 3920.
Z. Tao, L. F. Lindoy and G. Wei, Org. Biomol. Chem., 2016,
14, 4335.
3
3
3
0 S. Boschi-Muller, S. Muller, A. V. Dorsselaer, A. Bock and 50 J. Lagona, P. Mukhopadhyay, S. Chakrabarti and L. Isaacs,
G. Branlant, FEBS Lett., 1998, 439, 241. Angew. Chem., Int. Ed., 2005, 44, 4844.
1 S. Z. Mao, Z. Y. Dong, J. Q. Liu, X. Q. Li, X. M. Liu, G. M. Luo 51 I. M. Bell and D. Hilvert, Biochemistry, 1993, 32, 13969.
and J. C. Shen, J. Am. Chem. Soc., 2005, 127, 115883.
52 H. J. Yu, J. Q. Liu, A. Bock, J. Li, G. M. Luo and J. C. Shen,
2 X. M. Liu, L. A. Silks, C. P. Liu, M. Ollivault-Shiflett,
J. Biol. Chem., 2005, 280, 11930.
X. Huang, J. Li, G. M. Luo, Y. M. Hou, J. Q. Liu and 53 K. Dalziel, Biochem. J., 1969, 114, 547.
J. C. Shen, Angew. Chem., Int. Ed., 2009, 48, 2020.
3 P. Wu, R. Q. Xiao, C. Q. Zhang, L. P. Zhou, Q. Luo, J. Y. Xu
and J. Q. Liu, Catal. Lett., 2010, 138, 62.
4 X. Huang, Y. Z. Yin, X. Jiang, Y. Tang, J. Y. Xu, J. Q. Liu and
J. C. Shen, Macromol. Biosci., 2009, 9, 1202.
5 X. Huang, Y. Z. Yin, Y. Tang, X. L. Bai, Z. M. Zhang, 56 L. Engman, D. Stern, I. A. Cotgreave and C. M. Andersson,
J. Y. Xu, J. Q. Liu and J. C. Shen, Soft Matter, 2009, 5, 1905. J. Am. Chem. Soc., 1992, 114, 9737.
6 Y. Z. Yin, L. Wang, H. Y. Jin, C. Y. Lv, S. J. Yu, X. Huang, 57 G. Mugesh, A. Panda, H. B. Singh, N. S. Punekar and
Q. Luo, J. Y. Xu and J. Q. Liu, Soft Matter, 2011, 7, 2521. R. J. Butcher, J. Am. Chem. Soc., 2001, 123, 839.
7 L. Wang, H. X. Zou, Z. Y. Dong, L. P. Zhou, J. X. Li, Q. Luo, 58 F. Ursini, M. Maiorini and C. Gregolin, Biochim. Biophys.
J. Y. Zhu, J. Y. Xu and J. Q. Liu, Langmuir, 2014, 30, 4013. Acta, 1982, 710, 197.
8 H. X. Zou, H. C. Sun, L. Wang, L. L. Zhao, J. X. Li, 59 M. A. Carsol, I. Pouliquen-Sonaglia, G. Lesgards,
54 L. Flohe, G. Loschen, W. A. Gunzler and E. Eichele, Hoppe-
3
3
3
3
3
3
Seyler’s Z. Physiol. Chem., 1972, 353, 987.
55 Z. Y. Dong, J. Q. Liu, S. Z. Mao, X. Huang, B. Yang,
X. J. Ren, G. M. Luo and J. C. Shen, J. Am. Chem. Soc., 2004,
126, 16395.
Z. Y. Dong, Q. Luo, J. Y. Xu and J. Liu, Soft Matter, 2016, 12,
192.
G. Marchis-Mouren, A. Puigserver and M. Santimone,
Eur. J. Biochem., 1997, 247, 248.
1
39 E. R. Kay, D. A. Leigh and F. Zerbetto, Angew. Chem., Int. 60 S. R. Wilson, P. A. Zucker, R. R. C. Huang and A. Spector,
Ed., 2007, 46, 72. J. Am. Chem. Soc., 1989, 111, 5936.
40 M. Schmittel, S. De and S. Pramanik, Angew. Chem., Int. 61 L. Engman, D. Stern, I. A. Cotgrware and C. M. Andersson,
Ed., 2012, 51, 3832. J. Am. Chem. Soc., 1992, 114, 9737.
41 V. Blanco, A. Carlone, K. D. Hanni, D. A. Leigh and 62 M. Iwaoka and S. Tomoda, J. Am. Chem. Soc., 1994, 116,
B. Lewandowski, Angew. Chem., Int. Ed., 2012, 51, 5166. 2557.
Org. Biomol. Chem.
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