A novel dicyclodextrinyl diselenide compound with
glutathione peroxidase activity
Shao-Wu Lv
1
, Xiao-Guang Wang
1
, Ying Mu
1
, Tian-Zhu Zang
1
, Yue-Tong Ji
1
, Jun-Qiu Liu
2
,
Jia-Cong Shen
2
and Gui-Min Luo
1
1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, China
2 Key Laboratory for Supramolecular Structure and Materials of the Ministry of Education, Jilin University, Changchun, China
Glutathione peroxidase (GPX; EC 1.11.1.9) is a well-
known selenoenzyme that catalyzes the reduction
of harmful hydroperoxides by glutathione (GSH)
(Scheme 1) and protects lipid membranes and other
cellular components against oxidative damage [1–4]. It
is related to many diseases and is regarded as one of
the most important antioxidant enzymes in living
organisms. GPX enzyme activity is sometimes
increased in disease, possibly as a compensatory mech-
anism to try to counteract the oxidative stress associ-

27 May 2007, accepted 31 May 2007)
doi:10.1111/j.1742-4658.2007.05913.x
A 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin (6-CySeCD)
was designed and synthesized to imitate the antioxidant enzyme glutathione
peroxidase (GPX). In this novel GPX model, b-cyclodextrin provided a
hydrophobic environment for substrate binding within its cavity, and a
cyclohexylamine group was incorporated into cyclodextrin in proximity to
the catalytic selenium in order to increase the stability of the nucleophilic
intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6¢-di-
selenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-
3(2H)-one (Ebselen) in the reduction of H
2
O
2
, tert-butyl hydroperoxide and
cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism
was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reac-
tions. The enzymatic properties showed that there are two major factors for
improving the catalytic efficiency of GPX mimics. First, the substrate-bind-
ing site should match the size and shape of the substrate and second,
incorporation of an imido-group increases the stability of selenolate in the
catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and
Ebselen was also seen in the ferrous sulfate ⁄ ascorbate-induced mitochondria
damage system, and this implies its prospective therapeutic application.
Abbreviations
BHT, 2,6-di-tert-butyl-4-methylphenol; b-CD, b-cyclodextrin; 6-CySeCD, 6A,6A¢-dicyclohexylamine-6B,6B¢-diselenide-bis-b-cyclodextrin;
CumOOH, cumenyl hydroperoxide; Ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one; GPX, glutathione peroxidase; GSH, glutathione;
6-SeCD, 6,6¢-diselenide-bis-cyclodextrin; TBARS, thiobarbituric acid reactive substances; t-BuOOH, tert-butyl hydroperoxide.
3846 FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS
approach seems to help stabilize the selenolate and

6-CySeCD showed higher GPX activity than 6-SeCD
for the reduction of H
2
O
2
, tert-butyl hydroperoxide
(t-BuOOH) and cumenyl hydroperoxide (CumOOH)
by GSH, indicating that incorporation of the imido-
group in the proximity of the selenium atom may
increase the stability of the nucleophilic intermediate
selenolate and enhance GPX-like activity in selenium-
containing GPX mimics. We also studied the catalytic
mechanism using steady-state kinetics of 6-CySeCD
catalysis and investigated the antioxidant ability of
6-CySeCD using a mitochondria injury system.
Results and Discussion
Synthesis and characterization of 6-CySeCD
The synthetic routes of 6-CySeCD are shown in
Scheme 2. 6-CySeCD was analyzed using elemental
analysis, found (calculated for C
96
H
160
O
66
N
2
Se
2
Æ6H

change in NADPH absorption at 340 nm (Eqns 1,2).
Scheme 2. Synthetic route of 6-CySeCD.
Scheme 1. Catalytic cycle for GPX.
S W. Lv et al. 6-CySeCD displays GPX activity
FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS 3847
The GPX activities of 6-CySeCD and other GPX mim-
ics catalyzed the reduction of hydroperoxides by GSH
are listed in Table 1.
ROOH + 2GSH À!
GPX
ROH þ GSSG þ H
2
O ð1Þ
GSSG þ NADPH þ H
þ
ÀÀÀÀÀÀÀÀ!
GSH reductase
2GSH þ NADP
þ
ð2Þ
The GPX activities of 6-CySeCD and 6-SeCD for the
reduction of H
2
O
2
by GSH were 7.9 and 4.2 min
)1
,
respectively, indicating that 6-CySeCD and 6-SeCD
display higher GPX activity than Ebselen. This result

2
O
2
by GSH were determined as a function of the substrate
Table 1. Comparison between GPX activities of the 6-CySeCD-cata-
lyzed reduction of hydroperoxides by GSH and other species. One
unit of enzyme activity is defined as amount of mimic that utilizes
of 1 lmol of NADPH per minute. All data are presented as
means ± SD.
Mimics Hydroperoxide Activity (min
)1
)
Ebselen H
2
O
2
0.99
b
6-SeCD H
2
O
2
4.20 ± 0.15
b
t-BuOOH 6.3 ± 0.2
CumOOH 10.7 ± 0.4
6-CySeCD
a
H
2

2
O
2

ðKH
2
O
2
½GSHþK
GSH
Á½H
2
O
2
þ½GSHÁ½H
2
O
2
Þ
ð3Þ
Where v
0
is the initial reaction rate, [E]
0
is the initial
enzyme mimic concentration, k
max
is a pseudo-first-
order rate constant K
H

results were found for the 6-CySeCD-catalyzed reduc-
tion of H
2
O
2
by GSH. BHT inhibited the spontaneous
reaction, but not the 6-CySeCD-catalyzed reduction
(Fig. 2). This suggested that 6-CySeCD also catalyzes
the reduction of hydroperoxide by GSH via a nonradi-
cal mechanism.
Protection of mitochondria against oxidative
damage by 6-CySeCD
The swelling and shrinking of mitochondria are nor-
mal physiological phenomena during respiration. How-
ever, abnormal swelling disrupts the mitochondrial
membrane resulting in cell death. Mitochondrial swell-
ing therefore characterizes its integrity. Figure 3A
shows that the mitochondrial swelling is greatly
increased by ferrous sulfate ⁄ ascorbate-induced mito-
chondrial damage and the swelling is decreased by
addition of 6-CySeCD.
The absorbance at 520 nm for the control group
was basically constant, whereas that for the damage
group decreasede considerably over time, indicating
that mitochondrial swelling was increased. However,
the swelling in the protection group, which contained a
certain concentration of 6-CySeCD, was apparently
decreased compared with the damage group, and
the mitochondrial swelling decreased with increasing
Fig. 1. Double-reciprocal plots for the reduction of H

Table 2. Kinetic parameters of the 6-CySeCD. Reactions were carried out in 50 mM potassium phosphate buffer, pH 7.0, at 37 °C,
0.5–3.0 m
M GSH, 0.5–2.0 mM H
2
O
2
. All data are presented as means ± SD.
GPX mimic k
max
(min
-1
) K
GSH
(mM) k
max
⁄ K
GSH
(M
)1Æ
min
-1
) K
H2O2
(lM) k
max
⁄ K
H2O2
(M
)1Æ
min

the damage group without 6-CySeCD, indicating that
76% of TBARS production was inhibited. In order to
gauge the ability of the three GPX mimics (6-CySeCD,
6-SeCD, and Ebselen) to inhibit TBARS production,
their antioxidant activities were compared under iden-
tical conditions. As shown in Fig. 4B, the ability of
6-CySeCD to decrease the accumulation of TBARS
was greater than that of 6-SeCD and Ebselen. In addi-
tion, we also tested the effect of 20 lm 6-CySeCD in
the absence of damage (data no shown) and the result
shows that 20 lm 6-CySeCD did not have any effect
Fig. 2. Plots of v
0
versus [H
2
O
2
] for 1 mM GSH in 50 mM potas-
sium phosphate buffer, pH 7.4, and 37 °C, at [BHT] ¼ 0 l
M (a) and
50 l
M (b). (A) [6-CySeCD], 0 lM; (B) [6-CySeCD], 5 lM.
Fig. 3. (A) Effect of concentration of 6-CySeCD on the swelling of
mitochondria. (a) Control; (b) damage + 20 l
M 6-CySeCD; (c) dam-
age + 10 l
M 6-CySeCD; (d) damage + 4 lM of 6-CySeCD; (e) dam-
age. (B) Effect of different GPX mimics on mitochondrial swelling.
(a) Control; (b) damage + 10 l
M 6-CySeCD; (c) damage + 10 lM

Ascorbic acid þ 2Fe
3þ
! dehydroascorbic acid þ 2Fe
2þ
þ 2H
þ
ð6Þ
where H
2
O
2
was produced by oxidation of ascorbic
acid to dehydroascorbic acid (Eqn 4) [22], in addition,
mitochondria can produce superoxide by Fe(II), which
could be dismutated by mitochondrial superoxide
dismutase to hydrogen peroxide. A hydroxyl radical
was produced via the Fenton reaction (Eqns 5,6)
[25–27]. The biological molecules in mitochondria are
easily attacked by hydroxyl radicals, when changes in
composition, morphology, structure, integrity, and
function of the mitochondria take place. GPX mimics
can scavenge hydroperoxides and block hydroxyl
radical production, therefore protecting mitochondria
against oxidative damage.
In the ferrous sulfate ⁄ ascorbate-induced mitochond-
rial damage model system, swelling and TBARS con-
tent were chosen according to the standard, which was
used to determine the injury and extent of protection
in mitochondria. 6-CySeCD reduced the mitochondrial
swelling during damage and decreased the maximal

centration of 6-CySeCD. (a) Control; (b) damage + 20 l
M 6-CySeCD;
(c) damage + 10 l
M 6-CySeCD; (d) damage + 4 lM 6-CySeCD; (e)
damage. (B) Effect of different GPX mimics on TBARS accumulated
during mitochondrial damage. (a) Control; (b) damage + 10 l
M
6-CySeCD; (c) damage + 10 lM 6-SeCD; (d) damage + 10 lM Ebse-
len; (e) damage. Relative TBARS content calculated based on
amount of TBARS for 50 min with damage group ¼ 1.
S W. Lv et al. 6-CySeCD displays GPX activity
FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS 3851
Inc, Palo Alto, CA) and a Perkin-Elmer 240 DS elemental
analyzer (Wellesley, MA). The content and valence of
selenium in the 6-CySeCD were determined by using an
ESCALAB MKII X-ray photoelectron spectrometer
(VG Scientific, Sussex, UK). Spectrometric measurements
were carried out by using a Shimadzu UV-2550 spectropho-
tometer (Kyoto, Japan).
b-CD was purchased from Shanghai Sanpu Chemical
Plant (Shanghai, China), recrystallized three times from
water and dried for 12 h at 120 °C in vacuum. Sodium
borohydride, selenium, BHT and 1,3-benzene-disulfonyl
chloride were obtained from Sigma (St Louis, MO). GSH,
glutathione reductase, t-BuOOH, CumOOH, and NADPH
were also obtained from Sigma. Sephadex G-25 was pur-
chased from Pharmacia (Uppsala, Sweden). All the other
materials were of analytical grade and obtained from
Beijing Chemical Plant (Beijing, China).
Synthesis of 6-CySeCD

phosphate buffer, 1 mm EDTA, 1 mm sodium azide, 1 mm
GSH, 0.25 mm NADPH, 1 unit glutathione reductase,
5 lm 6-SeCD and 6-CySeCD. The reaction was initiated by
addition of 0.5 mm hydroperoxide. Organic hydroperoxides
(t-BuOOH, CumOOH) were dissolved in 0.2% (v ⁄ v) Tri-
ton X-100, which was the cosolvent and did not affect the
GPX-like activity assay. Activity was determined by the
decrease of NADPH absorption at 340 nm (e
NADPH
¼
6220 m
)1
cm
)1
). Background absorption of the noncatalytic
reaction was run without mimic and was subtracted. The
activity unit of enzyme mimic was defined as the amount of
enzyme mimic, which utilizes 1 lmol NADPH per min.
The assay of 6-CySeCD kinetics was similar to that for
native GPX [30]. Initial reduction rates of H
2
O
2
by GSH
were determined by observing the change in NADPH
absorption at 340 nm at 37 °C and pH 7.0, varying one
substrate concentration while another is fixed. All kinetic
experiments were performed at 37 °Cin700lL of reaction
solution containing 0.5–3.0 mm GSH, 0.5–2.0 mm H
2

ment carried out without the mimic, ascorbate, and ferrous
sulfate was known as the control group.
Biological analysis of mimics against
mitochondrial damage
Mitochondrial swelling was assayed as described by Hunter
et al. [33]. Mitochondrial swelling was measured by the
decrease in the turbidity of the reaction mixture at 520 nm.
The decrease in absorbance indicated an increase in mito-
chondrial swelling and a decrease in mitochondria integrity.
TBARS content in ferrous sulfate ⁄ ascorbate-treated
mitochondria was analyzed by thiobarbituric acid assay
[34]. In this assay, thiobarbituric acid reacts with malonal-
dehyde and ⁄ or other carbonyl by-products of free-radical-
mediated lipid peroxidation to give 2 : 1 (mol ⁄ mol) colored
conjugates, which have an A
532
value.
6-CySeCD displays GPX activity S W. Lv et al.
3852 FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS
Acknowledgements
This research was supported by Natural Science Foun-
dation of China (Project no. 20572035 and 20534030)
and Jilin University, Changchun, China.
References
1 Flohe
´
L (1985) The glutathione peroxidase reaction:
molecular basis of the antioxidant function of selenium
in mammals. Curr Top Cell Regul 27, 473–478.
2 Tappel AL (1984) Selenium–glutathione peroxidase:

11 Wilson SR, Zucker PA, Huang RRC & Spectror A
(1989) Development of synthetic compounds with gluta-
thione peroxidase activity. J Am Chem Soc 111, 5936–
5939.
12 Iwaoka M & Tomoda S (1994) A model study on the
effect of an amino group on the antioxidant activity of
glutathione peroxidase. J Am Chem Soc 116, 2557–2561.
13 Mugesh G, Panda A, Singh HB, Punekar NS & Butcher
RJ (2001) Glutathione peroxidase-like antioxidant activ-
ity of diaryl diselenides: a mechanistic study. JAm
Chem Soc 123, 839–850.
14 Luo G, Ren X, Liu J, Mu Y & Shen J (2003) Towards
more efficient glutathione peroxidase mimics: substrate
recognition and catalytic group assembly. Curr Med
Chem 10, 1151–1183.
15 Ren X, Gao S, You D, Huang H, Liu Z, Mu Y, Liu J,
Zhang Y, Yan G, Luo G et al. (2001) Cloning and
expression of a single-chain catalytic antibody that acts
as a glutathione peroxidase mimic with high catalytic
efficiency. Biochem J 359, 369–374.
16 Ding L, Liu Z, Zhu Z, Luo G, Zhao D & Ni J (1998)
Biochemical characterization of selenium-containing cat-
alytic antibody as a cytosolic glutathione peroxidase
mimic. Biochem J 332, 251–255.
17 Liu J, Luo G, Gao S, Zhang K, Chen X & Shen J
(1999) Generation of a glutathione peroxidase-like
mimic using bioimprinting and chemical mutation.
Chem Commun 20, 199–200.
18 Ren X, Jemth P, Board PG, Luo G, Mannervik B, Liu
J, Zhang K & Shen J (2002) A semisynthetic glutathione

antioxidant activity. Eur J Biochem 270, 4326–4331.
28 Iwao T, Yasuhisa K & Tadashi M (1986) Artificial
receptors for amino acids in water. Local environmental
effect on polar recognition by 6A-amino-6B-carboxy-
and 6B-amino-6A-carboxy-beta-cyclodextrins. JAm
Chem Soc 108, 4514–4518.
29 Klayman DL & Griffin TS (1973) Reaction of selenium
with sodium borohydride in protic solvents. A facile
S W. Lv et al. 6-CySeCD displays GPX activity
FEBS Journal 274 (2007) 3846–3854 ª 2007 The Authors Journal compilation ª 2007 FEBS 3853
method for the introduction of selenium into organic
molecules. J Am Chem Soc 95, 197–199.
30 Flohe
´
L, Loschen G, Gu
¨
nzler WA & Eichele E (1972)
Glutathione peroxidase. V. The kinetic mechanism.
Hoppe Seylers Z Physiol Chem 353, 987–999.
31 Lansman RA, Shade RO, Shapiro JF & Avise JC
(1981) The use of restriction endonucleases to measure
mitochondrial DNA sequence relatedness in natural
populations. III. Techniques and potential applications.
J Mol Evol 17, 214–226.
32 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein–dye binding. Anal
Biochem 72, 248–254.
33 Hunte FE, Scott JA, Hoffsten PE, Guerra F, Weinstein
J, Schneider A, Schutz B, Fink J, Ford L & Smith E